TGF-β1/FGF-2 signaling mediates the 15-HETE-induced differentiation of adventitial fibroblasts into myofibroblasts.

PubMed

Zhang, Li; Chen, Yan; Li, Guixia; Chen, Minggang; Huang, Wei; Liu, Yanrui; Li, Yumei

2016-01-05

Pulmonary adventitial fibroblasts (PAFs) are activated under stress stimuli leading to their differentiation into myofibroblasts, which is involved in vessel remodeling. 15-HETE is known as an important factor in vessel remodeling under hypoxia; however, the role of 15-HETE in PAF phenotypic alteration is not clear. The effect of 15-HETE on PAF phenotypic alterations was investigated in the present study. PAFs were treated with 15-HETE (0.5 μM) for 24 h, and the myofibroblast marker α-smooth muscle actin (α-SMA) was analyzed. The 15-HETE induced α-SMA expression and cell morphology. 15-HETE upregulated FGF-2 levels in PAFs, and knockdown FGF-2 by siRNAs blocked the enhanced α-SMA expression induced by 15-HETE. p38 kinase was activated, and blocked depressed 15-HETE-induced FGF-2 expression. The downstream of p38 pathway, Egr-1 activation, was also raised by 15-HETE treatment, and silenced Egr-1 suppressed the 15-HETE-induced upregulation of FGF-2. TGF-β1 was upregulated with FGF-2 treatment, and α-SMA expression induced by FGF-2 was inhibited after the cell was transferred with TGF-β1 siRNA. Meanwhile, FGF-2 increased α-SMA expression and improved proliferation, which was associated with p27(kip1) and cyclin E variation. The above results suggest that p38/Egr-1 pathway-mediated FGF-2 is involved in 15-HETE-induced differentiation of PAFs into myofibroblasts and cell proliferation.

Role of Alpha-Smooth Muscle Actin and Fibroblast Activation Protein Alpha in Ovarian Neoplasms.

PubMed

da Silva, Ana Carolinne; Jammal, Millena Prata; Etchebehere, Renata Margarida; Murta, Eddie Fernando Candido; Nomelini, Rosekeila Simões

2018-04-05

Studies show that tumor growth is not just determined by the presence of malignant cells, since interactions between cancer cells and stromal microenvironment have important impacts on the cancer growth and progression. Cancer-associated fibroblasts play a prominent role in this process. The aims of the study were to investigate 2 cancer-associated fibroblasts markers, alpha-smooth muscle actin (α-SMA), and fibroblast activation protein alpha (FAP) in the stromal microenvironment of benign and malignant ovarian epithelial neoplasms, and to relate their tissue expression with prognostic factors in ovarian cancer. α-SMA and FAP were evaluated by immunohistochemistry in malignant (n = 28) and benign (n = 28) ovarian neoplasms. Fisher's exact test was used with a significance level lower than 0.05. FAP immunostaining was stronger in ovarian cancer when compared to benign neoplasms (p = 0.0366). There was no significant difference in relation to α-SMA expression between malignant and benign ovarian neoplasms as well as prognostic factors. In ovarian cancer, FAP stainings 2/3 was significantly related to histological grades 2 and 3 (p = 0.0183). FAP immunostaining is more intense in malignant neoplasms than in benign ovarian neoplasms, as well as in moderately differentiated and undifferentiated ovarian carcinomas compared to well-differentiated neoplasms, thus indicating that it can be used as a marker of worse prognosis. © 2018 S. Karger AG, Basel.

Tetramethylpyrazine Effects on the Expression of Scar-Related Genes in Rabbit Benign Biliary Stricture Fibroblasts.

PubMed

Li, Ke Yue; Shi, Cheng Xian; Huang, Jian Zhao; Tang, Ke Li

2016-10-01

To investigate the effects of tetramethylpyrazine (TMP) on transforming growth factor-β1 (TGF- β1), α-smooth muscle actin (α-SMA), and neuronal regeneration related protein (P311) in benign biliary stricture fibroblasts of rabbit. An experimental study. Guizhou Medical University, Guiyang, Guizhou, China, from April to December 2015. Fibroblasts isolated from rabbits following benign biliary stricture were cultured and treated with different concentrations of TMP(0.08, 0.4, and 2.0 mg/ml). TMP-treated cells and non-treated control groups were incubated for 48-hours, and proliferation was assessed using the cell counting kit-8 assay. The mRNAexpressions of TGF-β1, α-SMA, and P311 were assessed by quantitative RT-PCR. Protein expressions of TGF-β1 and α-SMAwere investigated by Western blotting. Treatment with TMPsignificantly reduced the proliferation of benign biliary stricture fibroblasts, and significantly attenuated both the mRNAand protein expressions of TGF-β1, α-SMA, and P311 (p < 0.05) in a dose-dependent manner. TMPsignificantly reduced the proliferation of benign biliary stricture fibroblasts, and significantly downregulated the mRNA/protein expression of TGF-β1, α-SMA, and P311. Therefore, TMPmay be a therapeutic option for the prevention of benign biliary stricture.

Fractal heterogeneity in minimal matrix models of scars modulates stiff-niche stem-cell responses via nuclear exit of a mechanorepressor

NASA Astrophysics Data System (ADS)

Dingal, P. C. Dave P.; Bradshaw, Andrew M.; Cho, Sangkyun; Raab, Matthew; Buxboim, Amnon; Swift, Joe; Discher, Dennis E.

2015-09-01

Scarring is a long-lasting problem in higher animals, and reductionist approaches could aid in developing treatments. Here, we show that copolymerization of collagen I with polyacrylamide produces minimal matrix models of scars (MMMS), in which fractal-fibre bundles segregate heterogeneously to the hydrogel subsurface. Matrix stiffens locally--as in scars--while allowing separate control over adhesive-ligand density. The MMMS elicits scar-like phenotypes from mesenchymal stem cells (MSCs): cells spread and polarize quickly, increasing nucleoskeletal lamin-A yet expressing the `scar marker' smooth muscle actin (SMA) more slowly. Surprisingly, expression responses to MMMS exhibit less cell-to-cell noise than homogeneously stiff gels. Such differences from bulk-average responses arise because a strong SMA repressor, NKX2.5, slowly exits the nucleus on rigid matrices. NKX2.5 overexpression overrides rigid phenotypes, inhibiting SMA and cell spreading, whereas cytoplasm-localized NKX2.5 mutants degrade in well-spread cells. MSCs thus form a `mechanical memory' of rigidity by progressively suppressing NKX2.5, thereby elevating SMA in a scar-like state.

Human pericytes adopt myofibroblast properties in the microenvironment of the IPF lung.

PubMed

Sava, Parid; Ramanathan, Anand; Dobronyi, Amelia; Peng, Xueyan; Sun, Huanxing; Ledesma-Mendoza, Adrian; Herzog, Erica L; Gonzalez, Anjelica L

2017-12-21

Idiopathic pulmonary fibrosis (IPF) is a fatal disease of unknown etiology characterized by a compositionally and mechanically altered extracellular matrix. Poor understanding of the origin of α-smooth muscle actin (α-SMA) expressing myofibroblasts has hindered curative therapies. Though proposed as a source of myofibroblasts in mammalian tissues, identification of microvascular pericytes (PC) as contributors to α-SMA-expressing populations in human IPF and the mechanisms driving this accumulation remain unexplored. Here, we demonstrate enhanced detection of α-SMA+ cells coexpressing the PC marker neural/glial antigen 2 in the human IPF lung. Isolated human PC cultured on decellularized IPF lung matrices adopt expression of α-SMA, demonstrating that these cells undergo phenotypic transition in response to direct contact with the extracellular matrix (ECM) of the fibrotic human lung. Using potentially novel human lung-conjugated hydrogels with tunable mechanical properties, we decoupled PC responses to matrix composition and stiffness to show that α-SMA+ PC accumulate in a mechanosensitive manner independent of matrix composition. PC activated with TGF-β1 remodel the normal lung matrix, increasing tissue stiffness to facilitate the emergence of α-SMA+ PC via MKL-1/MTRFA mechanotranduction. Nintedanib, a tyrosine-kinase inhibitor approved for IPF treatment, restores the elastic modulus of fibrotic lung matrices to reverse the α-SMA+ phenotype. This work furthers our understanding of the role that microvascular PC play in the evolution of IPF, describes the creation of an ex vivo platform that advances the study of fibrosis, and presents a potentially novel mode of action for a commonly used antifibrotic therapy that has great relevance for human disease.

Phenotypic differences between oral and skin fibroblasts in wound contraction and growth factor expression.

PubMed

Shannon, Diane B; McKeown, Scott T W; Lundy, Fionnuala T; Irwin, Chris R

2006-01-01

Wounds of the oral mucosa heal in an accelerated fashion with reduced scarring compared with cutaneous wounds. The differences in healing outcome between oral mucosa and skin could be because of phenotypic differences between the respective fibroblast populations. This study compared paired mucosal and dermal fibroblasts in terms of collagen gel contraction, alpha-smooth muscle actin expression (alpha-SMA), and production of the epithelial growth factors: keratinocyte growth factor (KGF) and hepatocyte growth factor/scatter factor (HGF). The effects of transforming growth factor -beta1 and -beta3 on each parameter were also determined. Gel contraction in floating collagen lattices was determined over a 7-day period. alpha-SMA expression by fibroblasts was determined by Western blotting. KGF and HGF expression were determined by an enzyme-linked immunosorbent assay. Oral fibroblasts induced accelerated collagen gel contraction, yet surprisingly expressed lower levels of alpha-SMA. Oral cells also produced significantly greater levels of both KGF and HGF than their dermal counterparts. Transforming growth factor-beta1 and -beta3, over the concentration range of 0.1-10 ng/mL, had similar effects on cell function, stimulating both gel contraction and alpha-SMA production, but inhibiting KGF and HGF production by both cell types. These data indicate phenotypic differences between oral and dermal fibroblasts that may well contribute to the differences in healing outcome between these two tissues.

Limitations of commonly used internal controls for real-time RT-PCR analysis of renal epithelial-mesenchymal cell transition.

PubMed

Elberg, Gerard; Elberg, Dorit; Logan, Charlotte J; Chen, Lijuan; Turman, Martin A

2006-01-01

Progressive renal fibrotic disease is accompanied by the massive accumulation of myofibroblasts as defined by alpha smooth muscle actin (alphaSMA) expression. We quantitated gene expression using real-time RT-PCR analysis during conversion of primary cultured human renal tubular cells (RTC) to myofibroblasts after treatment with transforming growth factor-beta1 (TGF-beta1). We report herein the limitations of commonly used reference genes for mRNA quantitation. We determined the expression of alphaSMA and megakaryoblastic leukemia-1 (MKL1), a transcriptional regulator of alphaSMA, by quantitative real-time PCR using three common internal controls, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin A and 18S rRNA. Expression of GAPDH mRNA and cyclophilin A mRNA, and to a lesser extent, 18S rRNA levels varied over time in culture and with exposure to TGF-beta1. Thus, depending on which reference gene was used, TGF-beta1 appeared to have different effects on expression of MKL1 and alphaSMA. RTC converting to myofibroblasts in primary culture is a valuable system to study renal fibrosis in humans. However, variability in expression of reference genes with TGF-beta1 treatment illustrates the need to validate mRNA quantitation with multiple reference genes to provide accurate interpretation of fibrosis studies in the absence of a universal internal standard for mRNA expression. 2006 S. Karger AG, Basel.

Lipoxin A4 Attenuates Constitutive and TGF-β1–Dependent Profibrotic Activity in Human Lung Myofibroblasts

PubMed Central

Feghali-Bostwick, Carol A.; Amrani, Yassine; Bradding, Peter

2015-01-01

Idiopathic pulmonary fibrosis (IPF) is a common, progressive, and invariably lethal interstitial lung disease with no effective therapy. The key cell driving the development of fibrosis is the myofibroblast. Lipoxin A4 (LXA4) is an anti-inflammatory lipid, important in the resolution of inflammation, and it has potential antifibrotic activity. However, the effects of LXA4 on primary human lung myofibroblasts (HLMFs) have not previously been investigated. Therefore, the aim of this study was to examine the effects of LXA4 on TGF-β1–dependent responses in IPF- and nonfibrotic control (NFC)–derived HLMFs. HLMFs were isolated from IPF and NFC patients and grown in vitro. The effects of LXA4 on HLMF proliferation, collagen secretion, α-smooth muscle actin (αSMA) expression, and Smad2/3 activation were examined constitutively and following TGF-β1 stimulation. The LXA4 receptor (ALXR) was expressed in both NFC- and IPF-derived HLMFs. LXA4 (10−10 and 10−8 mol) reduced constitutive αSMA expression, actin stress fiber formation, contraction, and nuclear Smad2/3, indicating regression from a myofibroblast to fibroblast phenotype. LXA4 also significantly inhibited FBS-dependent proliferation and TGF-β1–dependent collagen secretion, αSMA expression, and Smad2/3 nuclear translocation in IPF-derived HLMFs. LXA4 did not inhibit Smad2/3 phosphorylation. In summary, LXA4 attenuated profibrotic HLMF activity and promoted HLMF regression to a quiescent fibroblast phenotype. LXA4 or its stable analogs delivered by aerosol may offer a novel approach to the treatment of IPF. PMID:26276873

Suppression of α Smooth Muscle Actin Accumulation by Bovine Fetal Dermal Collagen Matrix in Full Thickness Skin Wounds

PubMed Central

Lineaweaver, William; Bush, Katie; James, Kenneth

2015-01-01

Abstract The suppression of elements associated with wound contracture and unfavorable scarring is a potentially important strategy in clinical wound management. In this study, the presence of α smooth muscle actin (αSMA), a protein involved in wound contraction, was analyzed in a series of wounds in which bovine fetal collagen (BFC) acellular dermal matrix (PriMatrix) was used in staged split thickness skin graft procedures. The results obtained through histological and quantitative image analyses of incidental biopsies from these wounds demonstrated a suppression of αSMA in the wound regions occupied by assimilated BFC relative to increased levels of αSMA found in other areas of the wound. The αSMA levels found in assimilated BFC were similar to αSMA levels in uninjured human dermis. These findings suggest a mechanism by which application of BFC could decrease contraction of full thickness skin wounds. PMID:25695450

Suppression of α Smooth Muscle Actin Accumulation by Bovine Fetal Dermal Collagen Matrix in Full Thickness Skin Wounds.

PubMed

Lineaweaver, William; Bush, Katie; James, Kenneth

2015-06-01

The suppression of elements associated with wound contracture and unfavorable scarring is a potentially important strategy in clinical wound management. In this study, the presence of α smooth muscle actin (αSMA), a protein involved in wound contraction, was analyzed in a series of wounds in which bovine fetal collagen (BFC) acellular dermal matrix (PriMatrix) was used in staged split thickness skin graft procedures. The results obtained through histological and quantitative image analyses of incidental biopsies from these wounds demonstrated a suppression of αSMA in the wound regions occupied by assimilated BFC relative to increased levels of αSMA found in other areas of the wound. The αSMA levels found in assimilated BFC were similar to αSMA levels in uninjured human dermis. These findings suggest a mechanism by which application of BFC could decrease contraction of full thickness skin wounds.

Antioxidants and NOX1/NOX4 inhibition blocks TGFβ1-induced CCN2 and α-SMA expression in dermal and gingival fibroblasts

PubMed Central

Murphy-Marshman, Hannah; Quensel, Katherine; Shi-wen, Xu; Barnfield, Rebecca; Kelly, Jacalyn; Peidl, Alex; Stratton, Richard J.

2017-01-01

TGFbeta induces fibrogenic responses in fibroblasts. Reactive oxygen species (ROS)/nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) may contribute to fibrogenic responses. Here, we examine if the antioxidant N-acetylcysteine (NAC), the NOX inhibitor diphenyleneiodonium (DPI) and the selective NOX1/NOX4 inhibitor GKT-137831 impairs the ability of TGFbeta to induce profibrotic gene expression in human gingival (HGF) and dermal (HDF) fibroblasts. We also assess if GKT-137831 can block the persistent fibrotic phenotype of lesional scleroderma (SSc) fibroblasts. We use real-time polymerase chain reaction and Western blot analysis to evaluate whether NAC and DPI impair the ability of TGFbeta1 to induce expression of fibrogenic genes in fibroblasts. The effects of GKT-137831 on TGFbeta-induced protein expression and the persistent fibrotic phenotype of lesional scleroderma (SSc) fibroblasts were tested using Western blot and collagen gel contraction analyses. In HDF and HGF, TGFbeta1 induces CCN2, CCN1, endothelin-1 and alpha-smooth muscle actin (SMA) in a fashion sensitive to NAC. Induction of COL1A1 mRNA was unaffected. Similar results were seen with DPI. NAC and DPI impaired the ability of TGFbeta1 to induce protein expression of CCN2 and alpha-SMA in HDF and HGF. GKT-137831 impaired TGFbeta-induced CCN2 and alpha-SMA protein expression in HGF and HDF. In lesional SSc dermal fibroblasts, GKT-137831 reduced alpha-SMA and CCN2 protein overexpression and collagen gel contraction. These results are consistent with the hypothesis that antioxidants or NOX1/4 inhibition may be useful in blocking profibrotic effects of TGFbeta on dermal and gingival fibroblasts and warrant consideration for further development as potential antifibrotic agents. PMID:29049376

Novel Adult Stem Cells for Peripheral Nerve Regeneration

DTIC Science & Technology

2013-09-01

conduit (cross sections). (a) Neurofilamen (NFM) staining shows that the implanted MVSC (GFP+) formed circular structure wrapping the newly...F-actin (g–i) (nuclei were stained with DAPI) or used for qPCR to measure the gene expression of smA and Cnn1 (j–k). 18s ribosomal RnA was used to...Publishers Limited. All rights reserved. To determine whether MVSCs could differentiate into mature SMCs and turn on EGFP expression, we activated Notch

Basic Fibroblast Growth Factor Influences Epidermal Homeostasis of Living Skin Equivalents through Affecting Fibroblast Phenotypes and Functions.

PubMed

Yang, Lujun; Zhang, Dangui; Wu, Hongjuan; Xie, Sitian; Zhang, Mingjun; Zhang, Bingna; Tang, Shijie

2018-05-30

To elucidate the possible mechanisms of how basic fibroblast growth factor (bFGF) influences epidermal homeostasis in a living skin equivalent (LSE) model. Several wound healing-related growth factors were analyzed at protein and mRNA levels for dermal fibroblasts of induced alpha-smooth muscle actin (α-SMA)-positive or α-SMA-negative phenotypes. During culturing an LSE model by seeding normal human keratinocytes on a fibroblast-populated type I collagen gel, bFGF or neutralizing antibody for keratinocyte growth factor (KGF) was added to investigate its effects on fibroblast phenotypes and, subsequently, epidermal homeostasis by histology and immunohistochemistry. The α-SMA-positive phenotype of fibroblasts induced by transforming growth factor beta-1 (TGF-β1) markedly suppressed the expression of KGF and hepatocyte growth factor (HGF), and slightly upregulated vascular endothelial growth factor (VEGF) and TGF-β1 at mRNA and protein levels, compared with α-SMA-negative fibroblasts treated with bFGF. α-SMA expression of fibroblasts at the epidermal-mesenchymal junction of the LSEs was suppressed by the addition of bFGF, and a better-differentiated epidermis was presented. The abrogation of KGF from fibroblasts by the addition of the KGF neutralizing antibody disenabled the LSE culturing system to develop an epidermis. bFGF, through affecting the phenotypes and functions of fibroblasts, especially KGF expression, influenced epidermal homeostasis in an LSE model. © 2018 S. Karger AG, Basel.

Up-regulation of Pim-3 in Chronic Obstructive Pulmonary Disease (COPD) patients and its potential therapeutic role in COPD rat modeling.

PubMed

Yang, Cheng; Li, Li; Guo, Junhua; Zhang, Weiqiang; Zhu, Wenbiao; Rao, Xinhui; Huang, Wenjie

2017-04-01

Pim-3 belongs to the PIM kinase family and plays an important role in promoting inflammation, which is essential in the pathogenesis of Chronic Obstructive Pulmonary Disease (COPD). Immunohistochemistry (IHC), western blot, and RT-PCR analyses were performed to assess the expression of Pim-3 in both COPD and healthy lung tissue samples. SMA (Smooth Muscle Actin) and Cyclin D1 expression were detected by IHC. We also constructed animal models for the control, COPD, and Pim-3 inhibition groups, in order to analyze the effects of Pim-3 inhibition on COPD, and the role of Pim-3 in the p38 pathway. Compared with normal lung tissue, Pim-3 mRNA and protein were up-regulated in COPD tissue. Expression of Cyclin D1 and SMA were also up-regulated in the COPD group. In the animal model experiment, we found that suppression of Pim-3 decreased Pim-3, Cyclin D1, and SMA expression, as well as ameliorated lung damage in COPD patients. The inhibition of Pim-3 also resulted in the suppression of the p38 pathway. Our study suggests that up-regulation of Pim-3 successfully accelerated COPD development, and aggravated lung damage. The molecular mechanism of Pim-3 in COPD might be related to the p38 pathway, and is correlated with Cyclin D1 and SMA expression. Copyright © 2017 Elsevier GmbH. All rights reserved.

Molecular and functional analyses of the contractile apparatus in lymphatic muscle

NASA Technical Reports Server (NTRS)

Muthuchamy, Mariappan; Gashev, Anatoliy; Boswell, Niven; Dawson, Nancy; Zawieja, David; Delp, Z. (Principal Investigator)

2003-01-01

Lymphatics are necessary for the generation and regulation of lymph flow. Lymphatics use phasic contractions and extrinsic compressions to generate flow; tonic contractions alter resistance. Lymphatic muscle exhibits important differences from typical vascular smooth muscle. In this study, the thoracic duct exhibited significant functional differences from mesenteric lymphatics. To understand the molecular basis for these differences, we examined the profiles of contractile proteins and their messages in mesenteric lymphatics, thoracic duct, and arterioles. Results demonstrated that mesenteric lymphatics express only SMB smooth muscle myosin heavy chain (SM-MHC), whereas thoracic duct and arterioles expressed both SMA and SMB isoforms. Both SM1 and SM2 isoforms of SM-MHC were detected in arterioles and mesenteric and thoracic lymphatics. In addition, the fetal cardiac/skeletal slow-twitch muscle-specific beta-MHC message was detected only in mesenteric lymphatics. All four actin messages, cardiac alpha-actin, vascular alpha-actin, enteric gamma-actin, and skeletal alpha-actin, were present in both mesenteric lymphatics and arterioles. However, in thoracic duct, predominantly cardiac alpha-actin and vascular alpha-actin were found. Western blot and immunohistochemical analyses corroborated the mRNA studies. However, in arterioles only vascular alpha-actin protein was detected. These data indicate that lymphatics display genotypic and phenotypic characteristics of vascular, cardiac, and visceral myocytes, which are needed to fulfill the unique roles of the lymphatic system.

The Significance of the Stromal Response in Breast Cancer: An Immunohistochemical Study of Myofibroblasts in Primary and Metastatic Breast Cancer.

PubMed

Roozdar, Alale; Hayes, Malcolm M; Pourseyedei, Bahram; Zeinalinejad, Hamid; Shamsi Meymandi, Manzumeh; Dabiri, Bahram; Dabiri, Shahriar

2018-05-01

Gene expression profiling of breast cancer has demonstrated the importance of stromal response in determining the prognosis of invasive breast cancer. The host response to breast cancer is of increasing interest to pathologists and may be a future focus for novel pharmacological treatments. This study describes the pattern of distribution of stromal myofibroblasts using immunostains for CD10 and smooth muscle actin (SMA) in 50 primary breast cancers and their matched nodal metastases (68.6% nodes positive and 31.4% nodes negative). The stroma within the tumor (intratumoral) and at the advancing tumor edge (peri-tumoral) was studied in both primary and nodal sites. A simple quantitative scoring system was employed for both immunostains. The correlation between expression of these markers by stromal cells and standard pathological prognostic factors of stage, grade, hormone receptor and Her-2 status was analysed. SMA-positive stromal cells were more abundant in peri-tumoral stroma compared with intratumoral stroma in both primary and metastatic lesions. SMA expression in the lymph node metastases showed a significant correlation with tumor stage. SMA expression in peri-tumoral stroma correlated with Her-2 status. The results of this study suggest that myofibroblasts, particularly those expressing SMA, might potentiate the progression of the carcinomatous process especially in nodal metastases. Thus these cells may be a potential therapeutic target. © 2018 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Expression of transforming growth factor-beta1, -beta2 and -beta3 in normal and diseased canine mitral valves.

PubMed

Aupperle, H; März, I; Thielebein, J; Schoon, H-A

2008-01-01

The pathogenesis of chronic valvular disease (CVD) in dogs remains unclear, but activation and proliferation of valvular stromal cells (VSC) and their transdifferentiation into myofibroblast-like cells has been described. These alterations may be influenced by transforming growth factor-beta (TGF-beta), a cytokine involved in extracellular matrix (ECM) regulation and mesenchymal cell differentiation. The present study investigates immunohistochemically the expression of TGF-beta1, -beta2, -beta3 and smooth muscle alpha actin (alpha-SMA) in normal canine mitral valves (MVs) (n=10) and in the valves of dogs with mild (n=7), moderate (n=14) and severe (n=9) CVD. In normal mitral valves there was no expression of alpha-SMA but VSC displayed variable expression of TGF-beta1 (10% of VSC labelled), TGF-beta2 (1-5% labelled) and TGF-beta3 (50% labelled). In mild CVD the affected atrialis contain activated and proliferating alpha-SMA-positive VSC, which strongly expressed TGF-beta1 and -beta3, but only 10% of these cells expressed TGF-beta2. In unaffected areas of the leaflet there was selective increase in expression of TGF-beta1 and -beta3. In advanced CVD the activated subendothelial VSC strongly expressed alpha-SMA, TGF-beta1 and -beta3. Inactive VSC within the centre of the nodules had much less labelling for TGF-beta1 and -beta3. TGF-beta1 labelling was strong within the ECM. These data suggest that TGF-beta plays a role in the pathogenesis of CVD by inducing myofibroblast-like differentiation of VSC and ECM secretion. Changed haemodynamic forces and expression of matrix metalloproteinases (MMPs) may in turn regulate TGF-beta expression.

Diversity of Histologic Patterns and Expression of Cytoskeletal Proteins in Canine Skeletal Osteosarcoma.

PubMed

Nagamine, E; Hirayama, K; Matsuda, K; Okamoto, M; Ohmachi, T; Kadosawa, T; Taniyama, H

2015-09-01

Osteosarcoma (OS), the most common bone tumor, includes OS of the head (OSH) and appendicular OS (OSA). In dogs, it is classified into 6 histologic subtypes: osteoblastic, chondroblastic, fibroblastic, telangiectatic, giant cell, and poorly differentiated. This study investigated the significance of the histologic classification relevant to clinical outcome and the histologic and immunohistochemical relationships between pleomorphism and expression of cytoskeletal proteins in 60 cases each of OSH and OSA. Most neoplasms exhibited histologic diversity, and 64% of OS contained multiple subtypes. In addition to the above 6 subtypes, myxoid, round cell, and epithelioid subtypes were observed. Although the epithelioid subtypes were observed in only OSH, no significant difference in the frequency of other subtypes was observed. Also, no significant relevance was observed between the clinical outcome and histologic subtypes. Cytokeratin (CK) was expressed in both epithelioid and sarcomatoid tumor cells in various subtypes, and all CK-positive tumor cells also expressed vimentin. Vimentin and α-smooth muscle actin (SMA) were expressed in all subtypes. A few SMA-positive spindle-shaped tumor cells exhibited desmin expression. Glial fibrillary acidic protein-positive tumor cells were observed in many subtypes, and some of these cells showed neurofilament expression. Although OSH exhibited significantly stronger immunoreactivity for SMA than OSA, no significant difference in other cytoskeletal proteins was observed. Some tumor cells had cytoskeletal protein expression compatible with the corresponding histologic subtypes, such as CK in the epithelioid subtype and SMA in the fibroblastic subtype. Thus, canine skeletal OS is composed of pleomorphic and heterogenous tumor cells as is reflected in the diversity of histologic patterns and expression of cytoskeletal proteins. © The Author(s) 2015.

Inhibition mechanism of compound ethanol extracts from wuweizi (fructus schisandrae chinensis) on renal interstitial fibrosis in diabetic nephropathy model mice.

PubMed

Zhang, Yanqiu; Zhang, Daning; Zhang, Mianzhi

2012-12-01

To evaluate inhibition effect and mechanism of compound ethanol extracts from Wuweizi (Fructus Schisandrae Chinensis), Chuanxiong (Rhizoma Chuanxiong) and Muli (Cocha Ostreae) (FRC) on glomerular and tubular interstitial fibrosis in streptozocin (STZ)-induced diabetic nephropathy (ND) model mice. Twenty-seven male C57BL/6 mice were divided randomly into 3 groups: nondibetic (ND), STZ-induced diabetic (D), and STZ-induced diabetic that were treated with 5 g x kg(-1) x day(-1) of FRC by oral gavage (D(FRC)), with 9 in each group. The protein expressions of E-cadherin, alpha-smooth muscle actin (alpha-SMA), Plasminogen Activator Inhibitor-1 (PAL-1) in renal tissues were investigated by Western blotting. The expressions of fibronectin (FN) and alpha-SMA were detected by immunohistochemical method. The morphological changes of renal tissues were observed under a microscope. Renal tissues in the D(FRC) group showed a lessened degree of fibrosis. Meanwhile, the expressions of FN, alpha-SMA and PAI-1 were significantly lower in the D(FRC) group than those in the D group (all P < 0.05). FRC can ameliorate the DN in the C57BL/6 mice, and its mechanism may relate to inhibition on the epithelial to mesenchymal transdifferentiation, endothelial-myofibroblast transition and PAL-1 expression.

Specialized mouse embryonic stem cells for studying vascular development.

PubMed

Glaser, Drew E; Burns, Andrew B; Hatano, Rachel; Medrzycki, Magdalena; Fan, Yuhong; McCloskey, Kara E

2014-01-01

Vascular progenitor cells are desirable in a variety of therapeutic strategies; however, the lineage commitment of endothelial and smooth muscle cell from a common progenitor is not well-understood. Here, we report the generation of the first dual reporter mouse embryonic stem cell (mESC) lines designed to facilitate the study of vascular endothelial and smooth muscle development in vitro. These mESC lines express green fluorescent protein (GFP) under the endothelial promoter, Tie-2, and Discomsoma sp. red fluorescent protein (RFP) under the promoter for alpha-smooth muscle actin (α-SMA). The lines were then characterized for morphology, marker expression, and pluripotency. The mESC colonies were found to exhibit dome-shaped morphology, alkaline phosphotase activity, as well as expression of Oct 3/4 and stage-specific embryonic antigen-1. The mESC colonies were also found to display normal karyotypes and are able to generate cells from all three germ layers, verifying pluripotency. Tissue staining confirmed the coexpression of VE (vascular endothelial)-cadherin with the Tie-2 GFP+ expression on endothelial structures and smooth muscle myosin heavy chain with the α-SMA RFP+ smooth muscle cells. Lastly, it was verified that the developing mESC do express Tie-2 GFP+ and α-SMA RFP+ cells during differentiation and that the GFP+ cells colocalize with the vascular-like structures surrounded by α-SMA-RFP cells. These dual reporter vascular-specific mESC permit visualization and cell tracking of individual endothelial and smooth muscle cells over time and in multiple dimensions, a powerful new tool for studying vascular development in real time.

Yin yang 1 is a novel regulator of pulmonary fibrosis.

PubMed

Lin, Xin; Sime, Patricia J; Xu, Haodong; Williams, Marc A; LaRussa, Larry; Georas, Steve N; Guo, Jia

2011-06-15

The differentiation of fibroblasts into myofibroblasts is a cardinal feature of idiopathic pulmonary fibrosis (IPF). The transcription factor Yin Yang 1 (YY1) plays a role in the proliferation and differentiation of diverse cell types, but its role in fibrotic lung diseases is not known. To elucidate the mechanism by which YY1 regulates fibroblast differentiation and lung fibrosis. Lung fibroblasts were cultured with transforming growth factor (TGF)-β or tumor necrosis factor-α. Nuclear factor (NF)-κB, YY1, and α-smooth muscle actin (SMA) were determined in protein, mRNA, and promoter reporter level. Lung fibroblasts and lung fibrosis were assessed in a partial YY1-deficient mouse and a YY1(f/f) conditional knockout mouse after being exposed to silica or bleomycin. TGF-β and tumor necrosis factor-α up-regulated YY1 expression in lung fibroblasts. TGF-β-induced YY1 expression was dramatically decreased by an inhibitor of NF-κB, which blocked I-κB degradation. YY1 is significantly overexpressed in both human IPF and murine models of lung fibrosis, including in the aggregated pulmonary fibroblasts of fibrotic foci. Furthermore, the mechanism of fibrogenesis is that YY1 can up-regulate α-SMA expression in pulmonary fibroblasts. YY1-deficient (YY1(+/-)) mice were significantly protected from lung fibrosis, which was associated with attenuated α-SMA and collagen expression. Finally, decreasing YY1 expression through instilled adenovirus-cre in floxed-YY1(f/f) mice reduced lung fibrosis. YY1 is overexpressed in fibroblasts in both human IPF and murine models in a NF-κB-dependent manner, and YY1 regulates fibrogenesis at least in part by increasing α-SMA and collagen expression. Decreasing YY1 expression may provide a new therapeutic strategy for pulmonary fibrosis.

Adipogenesis of the mesenchymal stromal cells and bone oedema in rheumatoid arthritis.

PubMed

Okada, A; Yamasaki, S; Koga, T; Kawashiri, S Y; Tamai, M; Origuchi, T; Nakamura, H; Eguchi, K; Kawakami, A

2012-01-01

Bone oedema is a pathological change in rheumatoid arthritis (RA) that is detectable by magnetic resonance imaging (MRI). Recent histological analyses revealed that a prominent feature of bone oedema is the replacement of adipose tissue with inflammatory cells. Here, we demonstrate the possible roles of mesenchymal stromal cells (MSCs) in bone oedema formation and the pathogenic potential of the cells in RA. Adipogenesis of bone marrow-derived human MSCs was induced by a standard adipogenic induction medium in the presence or absence of cytokines. The cytokine productions from MSCs were screened by an antibody array system and confirmed by ELISA. The migration assay was performed to determine the locomotive abilities of undifferentiated MSCs or MSCs after adipogenesis. The expression of α smooth muscle actin (SMA) and F-actin was examined by immunostaining and phalloidin staining, respectively. TNF-α, interleukin (IL)-1β, IL-6, and TGF-β clearly inhibited the adipogenesis of MSCs. Production of IL-6 was markedly reduced, and IL-8 secretion was augmented in MSCs after adipogenesis. The mobility of MSCs after adipogenesis was clearly reduced in migration assays compared to that of undifferentiated MSCs. Consistent with these findings, SMA and F-actin expressions were clearly suppressed in MSCs committed to adipogenesis. Our data suggest that the inflammatory milieu promotes bone oedema by blocking adipogenesis of MSCs. In bone oedema, the enhanced IL-6 production and the increased mobility of MSCs may contribute to the progression of RA. Therefore, bone oedema may be an important target lesion in the treatment of RA.

Expression of HSP47 in Usual Interstitial Pneumonia and Nonspecific Interstitial Pneumonia

PubMed Central

Kakugawa, Tomoyuki; Mukae, Hiroshi; Hayashi, Tomayoshi; Ishii, Hiroshi; Nakayama, Seiko; Sakamoto, Noriho; Yoshioka, Sumako; Sugiyama, Kanako; Mine, Mariko; Mizuta, Yohei; Kohno, Shigeru

2005-01-01

Background Heat shock protein (HSP) 47, a collagen-specific molecular chaperone, is involved in the processing and/or secretion of procollagens, and its expression is increased in various fibrotic diseases. The aim of this study was to determine whether quantitative immunohistochemical evaluation of the expression levels of HSP47, type I procollagen and α-smooth muscle actin (SMA) allows the differentiation of idiopathic usual interstitial pneumonia (UIP) from UIP associated with collagen vascular disease (CVD) and idiopathic nonspecific interstitial pneumonia (NSIP). Methods We reviewed surgical lung biopsy specimens of 19 patients with idiopathic UIP, 7 with CVD-associated UIP and 16 with idiopathic NSIP and assigned a score for the expression of HSP47, type I procollagen and α-SMA in type II pneumocytes and/or lung fibroblasts (score 0 = no; 1 = weak; 2 = moderate; 3 = strong staining). Results The expression level of HSP47 in type II pneumocytes of idiopathic UIP was significantly higher than in CVD-associated UIP and idiopathic NSIP. The expression of HSP47 in fibroblasts was significantly higher in idiopathic UIP and idiopathic NSIP than in CVD-associated UIP. The expression of type I procollagen in type II pneumocytes was significantly higher in idiopathic UIP than in idiopathic NSIP. The expression of type I procollagen in fibroblasts was not different in the three groups, while the expression of α-SMA in fibroblasts was significantly higher in idiopathic UIP than in idiopathic NSIP. Conclusion Our results suggest the existence of different fibrotic pathways among these groups involved in the expression of HSP47 and type I procollagen. PMID:15955241

Endothelial-Mesenchymal Transition of Brain Endothelial Cells: Possible Role during Metastatic Extravasation

PubMed Central

Krizbai, István A.; Gasparics, Ákos; Nagyőszi, Péter; Fazakas, Csilla; Molnár, Judit; Wilhelm, Imola; Bencs, Rita; Rosivall, László; Sebe, Attila

2015-01-01

Cancer progression towards metastasis follows a defined sequence of events described as the metastatic cascade. For extravasation and transendothelial migration metastatic cells interact first with endothelial cells. Yet the role of endothelial cells during the process of metastasis formation and extravasation is still unclear, and the interaction between metastatic and endothelial cells during transendothelial migration is poorly understood. Since tumor cells are well known to express TGF-β, and the compact endothelial layer undergoes a series of changes during metastatic extravasation (cell contact disruption, cytoskeletal reorganization, enhanced contractility), we hypothesized that an EndMT may be necessary for metastatic extravasation. We demonstrate that primary cultured rat brain endothelial cells (BEC) undergo EndMT upon TGF-β1 treatment, characterized by the loss of tight and adherens junction proteins, expression of fibronectin, β1-integrin, calponin and α-smooth muscle actin (SMA). B16/F10 cell line conditioned and activated medium (ACM) had similar effects: claudin-5 down-regulation, fibronectin and SMA expression. Inhibition of TGF-β signaling during B16/F10 ACM stimulation using SB-431542 maintained claudin-5 levels and mitigated fibronectin and SMA expression. B16/F10 ACM stimulation of BECs led to phosphorylation of Smad2 and Smad3. SB-431542 prevented SMA up-regulation upon stimulation of BECs with A2058, MCF-7 and MDA-MB231 ACM as well. Moreover, B16/F10 ACM caused a reduction in transendothelial electrical resistance, enhanced the number of melanoma cells adhering to and transmigrating through the endothelial layer, in a TGF-β-dependent manner. These effects were not confined to BECs: HUVECs showed TGF-β-dependent SMA expression when stimulated with breast cancer cell line ACM. Our results indicate that an EndMT may be necessary for metastatic transendothelial migration, and this transition may be one of the potential mechanisms occurring during the complex phenomenon known as metastatic extravasation. PMID:25742314

Novel mechanism of regulation of fibrosis in kidney tumor with tuberous sclerosis

PubMed Central

2013-01-01

Background Deficiency in tuberin results in activation the mTOR pathway and leads to accumulation of cell matrix proteins. The mechanisms by which tuberin regulates fibrosis in kidney angiomyolipomas (AMLs) of tuberous sclerosis patients are not fully known. Method In the present study, we investigated the potential role of tuberin/mTOR pathway in the regulation of cell fibrosis in AML cells and kidney tumor tissue from tuberous sclerosis complex (TSC) patients. Results AML cells treated with rapamycin shows a significant decrease in mRNA and protein expression as well as in promoter transcriptional activity of alpha-smooth muscle actin (α-SMA) compared to untreated cells. In addition, cells treated with rapamycin significantly decreased the protein expression of the transcription factor YY1. Rapamycin treatment also results in the redistribution of YY1 from the nucleus to cytoplasm in AML cells. Moreover, cells treated with rapamycin resulted in a significant reduce of binding of YY1 to the αSMA promoter element in nuclear extracts of AML cells. Kidney angiomyolipoma tissues from TSC patients showed lower levels of tuberin and higher levels of phospho-p70S6K that resulted in higher levels of mRNA and protein of αSMA expression compared to control kidney tissues. In addition, most of the α-SMA staining was identified in the smooth muscle cells of AML tissues. YY1 was also significantly increased in tumor tissue of AMLs compared to control kidney tissue suggesting that YY1 plays a major role in the regulation of αSMA. Conclusions These data comprise the first report to provide one mechanism whereby rapamycin might inhibit the cell fibrosis in kidney tumor of TSC patients. PMID:23705901

Rapamycin inhibits epithelial-to-mesenchymal transition of peritoneal mesothelium cells through regulation of Rho GTPases.

PubMed

Xiang, Shilong; Li, Meng; Xie, Xishao; Xie, Zhoutao; Zhou, Qin; Tian, Yuanshi; Lin, Weiqiang; Zhang, Xiaohui; Jiang, Hong; Shou, Zhangfei; Chen, Jianghua

2016-06-01

Epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) is a key process of peritoneal fibrosis. Rapamycin has been previously shown to inhibit EMT of PMCs and prevent peritoneal fibrosis. In this study, we investigated the undefined molecular mechanisms by which rapamycin inhibits EMT of PMCs. To define the protective effect of rapamycin, we initially used a rat PD model which was daily infused with 20 mL of 4.25% high glucose (HG) dialysis solution for 6 weeks to induce fibrosis. The HG rats showed decreased ultrafiltration volume and obvious fibroproliferative response, with markedly increased peritoneal thickness and higher expression of α-smooth muscle actin (α-SMA) and transforming growth factor-β1. Rapamycin significantly ameliorated those pathological changes. Next, we treated rat PMCs with HG to induce EMT and/or rapamycin for indicated time. Rapamycin significantly inhibited HG-induced EMT, which manifests as increased expression of α-SMA, fibronectin, and collagen I, decreased expression of E-cadherin, and increased mobility. HG increased the phosphorylation of PI3K, Akt, and mTOR. Importantly, rapamycin inhibits the RhoA, Rac1, and Cdc42 activated by HG. Moreover, rapamycin repaired the pattern of F-actin distribution induced by HG, reducing the formation of stress fiber, focal adhesion, lamellipodia, and filopodia. Thus, rapamycin shows an obvious protective effect on HG-induced EMT, by inhibiting the activation of Rho GTPases (RhoA, Rac1, and Cdc42). © 2016 Federation of European Biochemical Societies.

The RhoA Activator GEF-H1/Lfc Is a Transforming Growth Factor-β Target Gene and Effector That Regulates α-Smooth Muscle Actin Expression and Cell Migration

PubMed Central

Tsapara, Anna; Luthert, Phillip; Greenwood, John; Hill, Caroline S.

2010-01-01

Maintenance of the epithelial phenotype is crucial for tissue homeostasis. In the retina, dedifferentiation and loss of integrity of the retinal pigment epithelium (RPE) leads to retinal dysfunction and fibrosis. Transforming growth factor (TGF)-β critically contributes to RPE dedifferentiation and induces various responses, including increased Rho signaling, up-regulation of α-smooth muscle actin (SMA), and cell migration and dedifferentiation. Cellular TGF-β responses are stimulated by different signal transduction pathways: some are Smad dependent and others Smad independent. Alterations in Rho signaling are crucial to both types of TGF-β signaling, but how TGF-β-stimulates Rho signaling is poorly understood. Here, we show that primary RPE cells up-regulated GEF-H1 in response to TGF-β. GEF-H1 was the only detectable Rho exchange factor increased by TGF-β1 in a genome-wide expression analysis. GEF-H1 induction was Smad4-dependant and led to Rho activation. GEF-H1 inhibition counteracted α-SMA up-regulation and cell migration. In patients with retinal detachments and fibrosis, migratory RPE cells exhibited increased GEF-H1 expression, indicating that induction occurs in diseased RPE in vivo. Our data indicate that GEF-H1 is a target and functional effector of TGF-β by orchestrating Rho signaling to regulate gene expression and cell migration, suggesting that it represents a new marker and possible therapeutic target for degenerative and fibrotic diseases. PMID:20089843

The Water Fraction of Calendula officinalis Hydroethanol Extract Stimulates In Vitro and In Vivo Proliferation of Dermal Fibroblasts in Wound Healing.

PubMed

Dinda, Manikarna; Mazumdar, Swagata; Das, Saurabh; Ganguly, Durba; Dasgupta, Uma B; Dutta, Ananya; Jana, Kuladip; Karmakar, Parimal

2016-10-01

The active fraction and/or compounds of Calendula officinalis responsible for wound healing are not known yet. In this work we studied the molecular target of C. officinalis hydroethanol extract (CEE) and its active fraction (water fraction of hydroethanol extract, WCEE) on primary human dermal fibroblasts (HDF). In vivo, CEE or WCEE were topically applied on excisional wounds of BALB/c mice and the rate of wound contraction and immunohistological studies were carried out. We found that CEE and only its WCEE significantly stimulated the proliferation as well as the migration of HDF cells. Also they up-regulate the expression of connective tissue growth factor (CTGF) and α-smooth muscle actin (α-SMA) in vitro. In vivo, CEE or WCEE treated mice groups showed faster wound healing and increased expression of CTGF and α-SMA compared to placebo control group. The increased expression of both the proteins during granulation phase of wound repair demonstrated the potential role of C. officinalis in wound healing. In addition, HPLC-ESI MS analysis of the active water fraction revealed the presence of two major compounds, rutin and quercetin-3-O-glucoside. Thus, our results showed that C. officinalis potentiated wound healing by stimulating the expression of CTGF and α-SMA and further we identified active compounds. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

DTU I isolates of Trypanosoma cruzi induce upregulation of Galectin-3 in murine myocarditis and fibrosis.

PubMed

Ferrer, María F; Pascuale, Carla A; Gomez, Ricardo M; Leguizamón, María S

2014-05-01

Chagas heart disease is a major public concern since 30% of infected patients develop cardiac alterations. The relationship between Trypanosoma cruzi discrete typing units (DTUs) and the biological properties exhibited by the parasite population has yet to be elucidated. In this study, we analysed the expression of α-smooth muscle actin (α-SMA) and galectin-3 (Gal-3) associated with cardiac extracellular matrix (ECM) remodelling a murine chronic cardiomyopathy induced by Tc I genotypes. We found the induction of myocarditis was associated with the upregulation of Col I, α-SMA, Gal-3, IFN-γ and IL-13, as analysed by q-PCR. In myocardial areas of fibrosis, the intensity of myocarditis and significant ECM remodelling correlated with the presence of Col I-, Gal-3- and α-SMA-positive cells. These results are promising for the further efforts to evaluate the relevance of Gal-3 in Chagas heart disease, since this galectin was proposed as a prognosis marker in heart failure patients.

Analysis of cytoskeletal proteins in posterior capsule opacification after implantation of acrylic and hydrogel intraocular lenses.

PubMed

Matsushima, Hiroyuki; Mukai, Kouichiro; Obara, Yoshitaka; Yoshida, Shinichiro; Clark, John I

2004-01-01

To analyze selected lens cytoskeletal proteins in posterior capsule opacification (PCO) 2 weeks after intraocular lens (IOL) implantation in rabbits. Department of Ophthalmology, Dokkyo University School of Medicine, Tochigi, Japan. Eight 10-week-old albino rabbits were prepared and anesthetized for phacoemulsification and aspiration of the crystalline lens and implantation of an acrylic or a hydrogel IOL. Two weeks postoperatively, the rabbits were killed and the IOLs removed for immunohistochemistry. Deparaffinized tissue sections were processed with antibodies against alpha-smooth muscle actin (alpha-SMA) and beta-crystallin to observe the types of PCO with the 2 IOL types. The proteins in the PCO tissue and the normal lens were homogenized, centrifuged, and analyzed using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) densitometric analysis and Western immunoblotting for actin and vimentin. Immunohistochemistry demonstrated a fibroblastic cell type expressing alpha-SMA and partial regeneration of epithelial cells, resulting in a lenticular structure that stained irregularly for beta-crystallin. The immunoreactivity of fibroblast-like cells to beta-crystallin appeared weaker than that of the regenerated lenticular structure. SDS-PAGE showed variability in the content of cytoskeletal proteins in the insoluble fractions of the PCO. Degradation of the cytoskeletal components was greater with the acrylic IOL than with the hydrogel IOL. Cytoskeletal proteins expressed during the formation of PCO and IOL implantation may have potential as therapeutic target proteins to improve the biocompatibility of IOLs.

Dynamic Regulation of Platelet-Derived Growth Factor Receptor α Expression in Alveolar Fibroblasts during Realveolarization

PubMed Central

Chen, Leiling; Acciani, Thomas; Le Cras, Tim; Lutzko, Carolyn

2012-01-01

Although the importance of platelet-derived growth factor receptor (PDGFR)-α signaling during normal alveogenesis is known, it is unclear whether this signaling pathway can regulate realveolarization in the adult lung. During alveolar development, PDGFR-α–expressing cells induce α smooth muscle actin (α-SMA) and differentiate to interstitial myofibroblasts. Fibroblast growth factor (FGF) signaling regulates myofibroblast differentiation during alveolarization, whereas peroxisome proliferator-activated receptor (PPAR)-γ activation antagonizes myofibroblast differentiation in lung fibrosis. Using left lung pneumonectomy, the roles of FGF and PPAR-γ signaling in differentiation of myofibroblasts from PDGFR-α–positive precursors during compensatory lung growth were assessed. FGF receptor (FGFR) signaling was inhibited by conditionally activating a soluble dominant-negative FGFR2 transgene. PPAR-γ signaling was activated by administration of rosiglitazone. Changes in α-SMA and PDGFR-α protein expression were assessed in PDGFR-α–green fluorescent protein (GFP) reporter mice using immunohistochemistry, flow cytometry, and real-time PCR. Immunohistochemistry and flow cytometry demonstrated that the cell ratio and expression levels of PDGFR-α–GFP changed dynamically during alveolar regeneration and that α-SMA expression was induced in a subset of PDGFR-α–GFP cells. Expression of a dominant-negative FGFR2 and administration of rosiglitazone inhibited induction of α-SMA in PDGFR-α–positive fibroblasts and formation of new septae. Changes in gene expression of epithelial and mesenchymal signaling molecules were assessed after left lobe pneumonectomy, and results demonstrated that inhibition of FGFR2 signaling and increase in PPAR-γ signaling altered the expression of Shh, FGF, Wnt, and Bmp4, genes that are also important for epithelial–mesenchymal crosstalk during early lung development. Our data demonstrate for the first time that a comparable epithelial–mesenchymal crosstalk regulates fibroblast phenotypes during alveolar septation. PMID:22652199

Syndecan-2 Attenuates Radiation-induced Pulmonary Fibrosis and Inhibits Fibroblast Activation by Regulating PI3K/Akt/ROCK Pathway via CD148.

PubMed

Tsoyi, Konstantin; Chu, Sarah G; Patino-Jaramillo, Nasly G; Wilder, Julie; Villalba, Julian; Doyle-Eisele, Melanie; McDonald, Jacob; Liu, Xiaoli; El-Chemaly, Souheil; Perrella, Mark A; Rosas, Ivan O

2018-02-01

Radiation-induced pulmonary fibrosis is a severe complication of patients treated with thoracic irradiation. We have previously shown that syndecan-2 reduces fibrosis by exerting alveolar epithelial cytoprotective effects. Here, we investigate whether syndecan-2 attenuates radiation-induced pulmonary fibrosis by inhibiting fibroblast activation. C57BL/6 wild-type mice and transgenic mice that overexpress human syndecan-2 in alveolar macrophages were exposed to 14 Gy whole-thoracic radiation. At 24 weeks after irradiation, lungs were collected for histological, protein, and mRNA evaluation of pulmonary fibrosis, profibrotic gene expression, and α-smooth muscle actin (α-SMA) expression. Mouse lung fibroblasts were activated with transforming growth factor (TGF)-β1 in the presence or absence of syndecan-2. Cell proliferation, migration, and gel contraction were assessed at different time points. Irradiation resulted in significantly increased mortality and pulmonary fibrosis in wild-type mice that was associated with elevated lung expression of TGF-β1 downstream target genes and cell death compared with irradiated syndecan-2 transgenic mice. In mouse lung fibroblasts, syndecan-2 inhibited α-SMA expression, cell contraction, proliferation, and migration induced by TGF-β1. Syndecan-2 attenuated phosphoinositide 3-kinase/serine/threonine kinase/Rho-associated coiled-coil kinase signaling and serum response factor binding to the α-SMA promoter. Syndecan-2 attenuates pulmonary fibrosis in mice exposed to radiation and inhibits TGF-β1-induced fibroblast-myofibroblast differentiation, migration, and proliferation by down-regulating phosphoinositide 3-kinase/serine/threonine kinase/Rho-associated coiled-coil kinase signaling and blocking serum response factor binding to the α-SMA promoter via CD148. These findings suggest that syndecan-2 has potential as an antifibrotic therapy in radiation-induced lung fibrosis.

Fibrocyte migration, differentiation and apoptosis during the corneal wound healing response to injury.

PubMed

Lassance, Luciana; Marino, Gustavo K; Medeiros, Carla S; Thangavadivel, Shanmugapriya; Wilson, Steven E

2018-05-01

The aim of this study was to determine whether bone marrow-derived fibrocytes migrate into the cornea after stromal scar-producing injury and differentiate into alpha-smooth muscle actin (αSMA) + myofibroblasts. Chimeric mice expressing green fluorescent protein (GFP) bone marrow cells had fibrosis (haze)-generating irregular phototherapeutic keratectomy (PTK). Multiplex immunohistochemistry (IHC) for GFP and fibrocyte markers (CD34, CD45, and vimentin) was used to detect fibrocyte infiltration into the corneal stroma and the development of GFP+ αSMA+ myofibroblasts. IHC for activated caspase-3, GFP and CD45 was used to detect fibrocyte and other hematopoietic cells undergoing apoptosis. Moderate haze developed in PTK-treated mouse corneas at 14 days after surgery and worsened, and persisted, at 21 days after surgery. GFP+ CD34+ CD45+ fibrocytes, likely in addition to other CD34+ and/or CD45+ hematopoietic and stem/progenitor cells, infiltrated the cornea and were present in the stroma in high numbers by one day after PTK. The fibrocytes and other bone marrow-derived cells progressively decreased at four days and seven days after surgery. At four days after PTK, 5% of the GFP+ cells expressed activated caspase-3. At 14 days after PTK, more than 50% of GFP+ CD45+ cells were also αSMA+ myofibroblasts. At 21 days after PTK, few GFP+ αSMA+ cells persisted in the stroma and more than 95% of those remaining expressed activated caspase-3, indicating they were undergoing apoptosis. GFP+ CD45+ SMA+ cells that developed from 4 to 21 days after irregular PTK were likely developed from fibrocytes. After irregular PTK in the strain of C57BL/6-C57/BL/6-Tg(UBC-GFP)30Scha/J chimeric mice, however, more than 95% of fibrocytes and other hematopoietic cells underwent apoptosis prior to the development of mature αSMA+ myofibroblasts. Most GFP+ CD45+ αSMA+ myofibroblasts that did develop subsequently underwent apoptosis-likely due to epithelial basement membrane regeneration and deprivation of epithelium-derived TGFβ requisite for myofibroblast survival. Copyright © 2018 Elsevier Ltd. All rights reserved.

Development of wound healing models to study TGFβ3's effect on SMA.

PubMed

Sriram, Sriniwas; Tran, Jennifer A; Guo, Xiaoqing; Hutcheon, Audrey E K; Kazlauskas, Andrius; Zieske, James D

2017-08-01

The goal of this study was to test the efficacy of transforming growth factor beta 3 (TGFβ3) in reducing α-smooth muscle actin (SMA) expression in two models-an ex vivo organ culture and an in vitro 3D cell construct-both of which closely mimic an in vivo environment. For the ex vivo organ culture system, a central 6.0 mm corneal keratectomy was performed on freshly excised rabbit globes The corneas were then excised, segregated into groups treated with 1.0 ng/ml TGFβ1 or β3 (T1 or T3, respectively), and cultured for 2 weeks. The corneas were assessed for levels of haze and analyzed for SMA mRNA levels. For the 3D in vitro model, rabbit corneal fibroblasts (RbCFs) were cultured for 4 weeks on poly-transwell membranes in Eagle's minimum essential media (EMEM) + 10% FBS + 0.5 mM vitamin C ± 0.1 ng/ml T1 or T3. At the end of 4 weeks, the constructs were processed for analysis by indirect-immunofluorescence (IF) and RT-qPCR. The RT-qPCR data showed that SMA mRNA expression in T3 samples for both models was significantly lower (p < 0.05) than T1 treatment (around 3-fold in ex vivo and 2-fold in constructs). T3 also reduced the amount of scarring in ex vivo corneas as compared with the T1 samples. IF data from RbCF constructs confirmed that T3-treated samples had up to 4-fold (p < 0.05) lower levels of SMA protein expression than samples treated with T1. These results show that T3 when compared to T1 decreases the expression of SMA in both ex vivo organ culture and in vitro 3D cell construct models. Understanding the mechanism of T3's action in these systems and how they differ from simple cell culture models, may potentially help in developing T3 as an anti-scarring therapy. Copyright © 2017. Published by Elsevier Ltd.

Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer. | Office of Cancer Genomics

Cancer.gov

Pancreatic stellate cells (PSCs) differentiate into cancer-associated fibroblasts (CAFs) that produce desmoplastic stroma, thereby modulating disease progression and therapeutic response in pancreatic ductal adenocarcinoma (PDA). However, it is unknown whether CAFs uniformly carry out these tasks or if subtypes of CAFs with distinct phenotypes in PDA exist. We identified a CAF subpopulation with elevated expression of α-smooth muscle actin (αSMA) located immediately adjacent to neoplastic cells in mouse and human PDA tissue.

Vitamin K1 attenuates bile duct ligation-induced liver fibrosis in rats.

PubMed

Jiao, Kun; Sun, Quan; Chen, Baian; Li, Shengli; Lu, Jing

2014-06-01

Vitamin K1 is used as a liver protection drug for cholestasis-induced liver fibrosis in China, but the mechanism of vitamin K1's action in liver fibrosis is unclear. In this study, a model of liver fibrosis was achieved via bile duct ligation in rats. The rats were then injected with vitamin K1, and the levels of serum aspartate aminotransferase, alanine transaminase, total bilirubin and the fibrotic grade score, collagen content, the expressions of α-smooth muscle actin (SMA) and cytokeratin 19 (CK19) were measured on day 28 after ligation. The levels of the biochemical parameters, fibrotic score and collagen content were significantly reduced by treatment with vitamin K1 in bile duct-ligated rats. In addition, α-SMA and CK19 expression was significantly reduced by vitamin K1 treatment in bile duct-ligated rats. These results suggested that vitamin K1 may attenuate liver fibrosis by inhibiting hepatic stellate cell activation in bile duct-ligated rats.

In vitro comparison of human fibroblasts from intact and ruptured ACL for use in tissue engineering.

PubMed

Brune, T; Borel, A; Gilbert, T W; Franceschi, J P; Badylak, S F; Sommer, P

2007-12-17

The present study compares fibroblasts extracted from intact and ruptured human anterior cruciate ligaments (ACL) for creation of a tissue engineered ACL-construct, made of porcine small intestinal submucosal extracellular matrix (SIS-ECM) seeded with these ACL cells. The comparison is based on histological, immunohistochemical and RT-PCR analyses. Differences were observed between cells in a ruptured ACL (rACL) and cells in an intact ACL (iACL), particularly with regard to the expression of integrin subunits and smooth muscle actin (SMA). Despite these differences in the cell source, both cell populations behaved similarly when seeded on an SIS-ECM scaffold, with similar cell morphology, connective tissue organization and composition, SMA and integrin expression. This study shows the usefulness of naturally occurring scaffolds such as SIS-ECM for the study of cell behaviour in vitro, and illustrates the possibility to use autologous cells extracted from ruptured ACL biopsies as a source for tissue engineered ACL constructs.

PPARgamma agonists inhibit TGF-beta-PKA signaling in glomerulosclerosis.

PubMed

Zou, Rong; Xu, Gang; Liu, Xiao-cheng; Han, Min; Jiang, Jing-jing; Huang, Qian; He, Yong; Yao, Ying

2010-01-01

To study the probable mechanisms of the anti-glomerulosclerosis effects induced by peroxisome proliferator-activated receptor gamma (PPARgamma) agonists in rat intraglomerular mesangial cells (MCs). Cells were transfected with the pTAL-PPRE-tk-Luc(+) plasmid and then treated with different concentrations of PPARgamma agonist, either troglitazone or telmisartan, for the indicated times. Promega luciferase assays were subsequently used for the detection of PPARgamma activation. Protein expression levels were assessed by Western blot, and PepTag assays were used for the non-radioactive detection of protein kinase A (PKA) activity. The deposition of alpha-smooth muscle actin (alpha-SMA) and p-cyclic AMP responsive element binding protein (pCREB) were analyzed by confocal laser scanning. Both troglitazone and telmisartan remarkably inhibit the PKA activation and pCREB expression that is stimulated by TGF-beta. The PPARgamma agonists also inhibited alpha-SMA and collagen IV protein expression by blocking PKA activation. PPARgamma ligands effectively suppress the activation of MCs and the accumulation of collagen IV stimulated by TGF-beta in vitro. The renal protection provided by PPARgamma agonists is partly mediated via their blockade of TGF-beta/PKA signaling.

Novel Omega-3 Fatty Acid Epoxygenase Metabolite Reduces Kidney Fibrosis

PubMed Central

Sharma, Amit; Khan, Md. Abdul Hye; Levick, Scott P.; Lee, Kin Sing Stephen; Hammock, Bruce D.; Imig, John D.

2016-01-01

Cytochrome P450 (CYP) monooxygenases epoxidize the omega-3 polyunsaturated fatty acid (PUFA) docosahexaenoic acid into novel epoxydocosapentaenoic acids (EDPs) that have multiple biological actions. The present study determined the ability of the most abundant EDP regioisomer, 19,20-EDP to reduce kidney injury in an experimental unilateral ureteral obstruction (UUO) renal fibrosis mouse model. Mice with UUO developed kidney tubular injury and interstitial fibrosis. UUO mice had elevated kidney hydroxyproline content and five-times greater collagen positive fibrotic area than sham control mice. 19,20-EDP treatment to UUO mice for 10 days reduced renal fibrosis with a 40%–50% reduction in collagen positive area and hydroxyproline content. There was a six-fold increase in kidney α-smooth muscle actin (α-SMA) positive area in UUO mice compared to sham control mice, and 19,20-EDP treatment to UUO mice decreased α-SMA immunopositive area by 60%. UUO mice demonstrated renal epithelial-to-mesenchymal transition (EMT) with reduced expression of the epithelial marker E-cadherin and elevated expression of multiple mesenchymal markers (FSP-1, α-SMA, and desmin). Interestingly, 19,20-EDP treatment reduced renal EMT in UUO by decreasing mesenchymal and increasing epithelial marker expression. Overall, we demonstrate that a novel omega-3 fatty acid metabolite 19,20-EDP, prevents UUO-induced renal fibrosis in mice by reducing renal EMT. PMID:27213332

Downregulated Smad4 Affects Extracellular Matrix Remodeling in Ventilator-induced Lung Injury.

PubMed

Huang, Xiaofang; Zhou, Wei; Ding, Shifang

2016-09-01

To explore the effect of Smad4 on the extracellular matrix remodeling in ventilator-induced lung injury (VILI). We randomized 24 C57BL/6 mice to 4 groups for treatment (n=6/group): control, ventilation, non-targeted (scramble) lentivirus transfection plus ventilation, and Smad4 small interfering RNA (siRNA) lentivirus transfection plus ventilation. Lentivirus was delivered by intranasal instillation. Four weeks later, the 3 ventilated groups underwent high tidal volume (VT 40mL/kg) ventilation to induce lung injury. After 72 hours, lungs were collected from the anesthetized live mice. Histological changes in lungs were evaluated by hematoxylin and eosin and Masson's staining. The expression of α-smooth muscle actin (α-SMA) was determined by immunohistochemistry, and the mRNA and protein levels of Smad4, α-SMA, and collagen I and III were detected by quantitative real-time PCR and western blotting analysis. Smad4 siRNAs significantly knocked down Smad4 expression (P<.05), which was increased with ventilation, thereby alleviating inflammatory cell infiltration. It also inhibited accumulation of α-SMA-positive myofibroblasts and pulmonary fibrosis, as seen by reduced collagen I and III expression (P<.05), induced by ventilation. Scramble siRNA treatment had no effect (P>.05). Smad4 gene silencing may be a therapeutic target for treating ventilator-induced lung injury and pulmonary fibrosis. © 2016 by the Association of Clinical Scientists, Inc.

The kampo medicine Daikenchuto inhibits peritoneal fibrosis in mice.

PubMed

Kitamura, Mineaki; Nishino, Tomoya; Obata, Yoko; Oka, Satoru; Abe, Shinichi; Muta, Kumiko; Ozono, Yoshiyuki; Koji, Takehiko; Kohno, Shigeru

2015-01-01

Long-term peritoneal dialysis therapy causes inflammation and histological changes in the peritoneal membrane. Inflammation generally activates fibroblasts and results in fibroblast-myofibroblast differentiation. Heat-shock protein 47 (HSP 47), a collagen-specific molecular chaperone, is localized in myofibroblasts and is involved in the progression of peritoneal fibrosis. Daikenchuto (DKT), a Kampo medicine, is used to prevent postoperative colon adhesion. It inhibits inflammation and HSP 47 expression in the gastrointestinal tract. We examined the effect of DKT on chlorhexidine gluconate (CG)-induced peritoneal fibrosis in mice injected with 0.1% CG dissolved in 15% ethanol. DKT was dissolved in the drinking water. Histological changes were assessed using Masson trichrome staining. Cells expressing α-smooth muscle actin (α-SMA), HSP 47, phospho-Smad 2/3, F4/80, and monocyte chemotactic protein-1 were examined immunohistochemically. Compared with the control group, the peritoneal tissues of the CG group were markedly thickened, and the number of cells expressing α-SMA, HSP 47, phospho-Smad 2/3, F4/80, and monocyte chemotactic protein-1 was significantly increased. However, these changes were inhibited in the DKT-treated group. These results indicate that DKT can prevent peritoneal fibrosis by inhibiting inflammation and HSP 47 expression.

Chlorogenic Acid Inhibits Liver Fibrosis by Blocking the miR-21-Regulated TGF-β1/Smad7 Signaling Pathway in Vitro and in Vivo.

PubMed

Yang, Fan; Luo, Lei; Zhu, Zhi-De; Zhou, Xuan; Wang, Yao; Xue, Juan; Zhang, Juan; Cai, Xin; Chen, Zhi-Lin; Ma, Qian; Chen, Yun-Fei; Wang, Yu-Jie; Luo, Ying-Ying; Liu, Pan; Zhao, Lei

2017-01-01

Aims: Chlorogenic acid (CGA) is a phenolic acid that has a wide range of pharmacological effects. However, the protective effects and mechanisms of CGA on liver fibrosis are not clear. This study explored the effects of CGA on miR-21-regulated TGF-β1/Smad7 liver fibrosis in the hepatic stellate LX2 cell line and in CCl4-induced liver fibrosis in Sprague-Dawley rats. Methods: The mRNA expression of miR-21, Smad7, connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), tissue inhibitor of metalloproteinase 1 (TIMP-1), matrix metalloproteinase-9 (MMP-9), and transforming growth factor-β1 (TGF-β1) and the protein levels of Smad2, p-Smad2, Smad3, p-Smad3, Smad2/3, p-Smad2/3, Smad7, CTGF, α-SMA, TIMP-1, MMP-9 and TGF-β1 were assayed in LX2 cells and liver tissue. The effects of CGA after miR-21 knockdown or overexpression were analyzed in LX2 cells. The liver tissue and serum were collected for histopathological examination, immunohistochemistry (IHC) and ELISA. Results: The mRNA expression of miR-21, CTGF, α-SMA, TIMP-1, and TGF-β1 and the protein expression of p-Smad2, p-Smad3, p-Smad2/3, CTGF, α-SMA, TIMP-1, and TGF-β1 were inhibited by CGA both in vitro and in vivo . Meanwhile, CGA elevated the mRNA and protein expression of Smad7 and MMP-9. After miR-21 knockdown and overexpression, the downstream molecules also changed accordingly. CGA also lessened the degree of liver fibrosis in the pathological manifestation and reduced α-SMA and collagen I expression in liver tissue and TGF-β1 in serum. Conclusion: CGA might relieve liver fibrosis through the miR-21-regulated TGF-β1/Smad7 signaling pathway, which suggests that CGA might be a new anti-fibrosis agent that improves liver fibrosis.

Silibinin prevents prostate cancer cell-mediated differentiation of naïve fibroblasts into cancer-associated fibroblast phenotype by targeting TGF β2.

PubMed

Ting, Harold J; Deep, Gagan; Jain, Anil K; Cimic, Adela; Sirintrapun, Joseph; Romero, Lina M; Cramer, Scott D; Agarwal, Chapla; Agarwal, Rajesh

2015-09-01

Tumor microenvironment (TM) is an essential element in prostate cancer (PCA), offering unique opportunities for its prevention. TM includes naïve fibroblasts that are recruited by nascent neoplastic lesion and altered into 'cancer-associated fibroblasts' (CAFs) that promote PCA. A better understanding and targeting of interaction between PCA cells and fibroblasts and inhibiting CAF phenotype through non-toxic agents are novel approaches to prevent PCA progression. One well-studied cancer chemopreventive agent is silibinin, and thus, we examined its efficacy against PCA cells-mediated differentiation of naïve fibroblasts into a myofibroblastic-phenotype similar to that found in CAFs. Silibinin's direct inhibitory effect on the phenotype of CAFs derived directly from PCA patients was also assessed. Human prostate stromal cells (PrSCs) exposed to control conditioned media (CCM) from human PCA PC3 cells showed more invasiveness, with increased alpha-smooth muscle actin (α-SMA) and vimentin expression, and differentiation into a phenotype we identified in CAFs. Importantly, silibinin (at physiologically achievable concentrations) inhibited α-SMA expression and invasiveness in differentiated fibroblasts and prostate CAFs directly, as well as indirectly by targeting PCA cells. The observed increase in α-SMA and CAF-like phenotype was transforming growth factor (TGF) β2 dependent, which was strongly inhibited by silibinin. Furthermore, induction of α-SMA and CAF phenotype by CCM were also strongly inhibited by a TGFβ2-neutralizing antibody. The inhibitory effect of silibinin on TGFβ2 expression and CAF-like biomarkers was also observed in PC3 tumors. Together, these findings highlight the potential usefulness of silibinin in PCA prevention through targeting the CAF phenotype in the prostate TM. © 2014 Wiley Periodicals, Inc.

Topical Rosiglitazone Is an Effective Anti-Scarring Agent in the Cornea

PubMed Central

Huxlin, Krystel R.; Hindman, Holly B.; Jeon, Kye-Im; Bühren, Jens; MacRae, Scott; DeMagistris, Margaret; Ciufo, David; Sime, Patricia J.; Phipps, Richard P.

2013-01-01

Corneal scarring remains a major cause of blindness world-wide, with limited treatment options, all of which have side-effects. Here, we tested the hypothesis that topical application of Rosiglitazone, a Thiazolidinedione and ligand of peroxisome proliferator activated receptor gamma (PPARγ), can effectively block scar formation in a cat model of corneal damage. Adult cats underwent bilateral epithelial debridement followed by excimer laser ablation of the central corneal stroma to a depth of ∼160 µm as a means of experimentally inducing a reproducible wound. Eyes were then left untreated, or received 50 µl of either 10 µM Rosiglitazone in DMSO/Celluvisc, DMSO/Celluvisc vehicle or Celluvisc vehicle twice daily for 2 weeks. Cellular aspects of corneal wound healing were evaluated with in vivo confocal imaging and post-mortem immunohistochemistry for alpha smooth muscle actin (αSMA). Impacts of the wound and treatments on optical quality were assessed using wavefront sensing and optical coherence tomography at 2, 4, 8 and 12 weeks post-operatively. In parallel, cat corneal fibroblasts were cultured to assess the effects of Rosiglitazone on TGFβ-induced αSMA expression. Topical application of Rosiglitazone to cat eyes after injury decreased αSMA expression and haze, as well as the induction of lower-order and residual, higher-order wavefront aberrations compared to vehicle-treated eyes. Rosiglitazone also inhibited TGFβ-induced αSMA expression in cultured corneal fibroblasts. In conclusion, Rosiglitazone effectively controlled corneal fibrosis in vivo and in vitro, while restoring corneal thickness and optics. Its topical application may represent an effective, new avenue for the prevention of corneal scarring with distinct advantages for pathologically thin corneas. PMID:23940641

[Effect of fluorofenidone on renal interstitial fibrosis in rats with unilateral ureteral obstruction].

PubMed

Tan, Wenqing; Wang, Wei; Zheng, Xuan; Chen, Jiying; Yuan, Xiangning; Zhang, Fangfang; Wang, Shuting; Tao, Lijian

2018-05-28

To investigate the effect of fluorofenidone on renal interstitial fibrosis in rats with unilateral ureteral obstruction (UUO) and to observe the effect of fluorofenidone on the expressions of collagen type I (Col I), collagen type III (Col III), α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), platelet derived growth factor (PDGF) in the renal tissues of UUO rats.
 Methods: Male Sprague-Dawley (SD) rats were randomly divided into a sham-operated group, a UUO group, and a flurofenidone group (n=5). UUO model was induced by ligating the left ureter in rats. The rats were treated with 125 mg/(kg.d) fluorofenidone by gastric gavage in the fluorofenidone group at 24 h before the operation, and the rats were treated with the identical dose of 0.5% sodium carboxyl methyl cellulose (CMC-Na) in the other 2 groups. The rats were sacrificed at 14 days after UUO. Pathological changes of the renal tissue were observed by HE and Masson staining, the mRNA expressions of Col I, Col III, α-SMA, PDGF and CTGF were detected by real-time PCR, and the protein expressions of Col I, Col III, PDGF and CTGF were detected by immunohistochemical staining.
 Results: The renal interstitial damage index, relative collagen area and mRNA and protein expressions of Col I and Col III in the renal tissues of the rats in the UUO group significantly increased (P<0.05), and fluorofenidone could reduce these indexes (P<0.05). Compared with the sham-operated group, the protein expressions of α-SMA, PDGF, CTGF and the mRNA expressions of PDGF and CTGF in the renal tissues of the rats in the UUO group were increased, but fluorofenidone could decrease the protein expressions of α-SMA, PDGF, CTGF and the mRNA expressions of PDGF and CTGF (P<0.05).
 Conclusion: Fluorofenidone (125 mg/kg.d) could attenuate renal interstitial fibrosis through inhibition of fibroblast proliferation, myofibroblastic activation, PDGF and CTGF expression.

Latrunculin B and substratum stiffness regulate corneal fibroblast to myofibroblast transformation.

PubMed

Thomasy, Sara M; Raghunathan, Vijay Krishna; Miyagi, Hidetaka; Evashenk, Alexander T; Sermeno, Jasmyne C; Tripp, Geneva K; Morgan, Joshua T; Murphy, Christopher J

2018-05-01

The transformation of keratocytes and fibroblasts to myofibroblasts is important to corneal wound healing as well as formation of stromal haze. The purpose of this study was to determine the effect of latrunculin B, an actin cytoskeleton disruptor in conjunction with a fundamental biophysical cue, substrate stiffness, on myofibroblast transformation in vitro and in vivo. Rabbit corneal fibroblasts were cultured on substrates of differing compliance (1.5, 22, and 71 kPa) and tissue culture plastic (TCP; > 1 GPa) in media containing 0 or 10 ng/ml TGFβ1 for 72 h. Cells were treated with 0.4 μM Lat-B or DMSO for 30 min every 24 h for 72 h. RNA was collected from cells and expression of alpha-smooth muscle actin (α-SMA), keratocan, and ALDH1A1 determined using qPCR; immunocytochemistry was used to assess α-SMA protein expression. A rabbit phototherapeutic keratectomy (PTK) model was used to assess the impact of 0.1% Lat-B (n = 3) or 25% DMSO (vehicle control, n = 3) on corneal wound healing by assessment of epithelial wound size with fluorescein stain and semi-quantitative stromal haze scoring by an observer masked to treatment group as well as Fourier-domain optical coherence tomography (FD-OCT) at set time points. Statistical analysis was completed using one-way or two-way analysis of variance. Treatment with Lat-B versus DMSO resulted in significantly less αSMA mRNA (P ≤ 0.007) for RCF cells grown on 22 and 71 kPa substrates as well as TCP without or with TGFβ1, and significantly decreased α-SMA protein expression in RCFs cultured on the intermediate (22 kPa) stiffness in the absence (P = 0.028) or presence (P = 0.018) of TGFβ1. Treatment with Lat-B versus DMSO but did not significantly alter expression of keratocan or ALDH1A1 mRNA in RCFs (P > 0.05) in the absence or presence of TGFβ1, but RCFs grown on stiff hydrogels (71 kPa) had significantly more keratocan mRNA expression versus the 22 kPa hydrogel or TCP (P < 0.001) without TGFβ1. Administration of topical Lat-B BID was well tolerated by rabbits post-PTK but did not significantly alter epithelial wound closure, stromal haze score, stromal haze thickness as measured by FD-OCT in comparison to DMSO-treated rabbits. When corneal stromal cells are cultured on substrates possessing biologically relevant substratum stiffnesses, Lat-B modulates mRNA and protein expression of α-SMA and thus modulates myofibroblast transformation. At a dose and dose-frequency that reduced IOP in human glaucoma patients, Lat-B treatment did not substantially impact corneal epithelial or stromal wound healing in a rabbit PTK model. While a significant impact on wound healing was observed at the concentration and dose frequency reported here was not found, encouraging in vitro data support further investigations of topically applied Lat-B to determine if this compound can reduce stromal fibrosis. Copyright © 2018 Elsevier Ltd. All rights reserved.

A subset of myofibroblastic cancer-associated fibroblasts regulate collagen fiber elongation, which is prognostic in multiple cancers.

PubMed

Hanley, Christopher J; Noble, Fergus; Ward, Matthew; Bullock, Marc; Drifka, Cole; Mellone, Massimiliano; Manousopoulou, Antigoni; Johnston, Harvey E; Hayden, Annette; Thirdborough, Steve; Liu, Yuming; Smith, David M; Mellows, Toby; Kao, W John; Garbis, Spiros D; Mirnezami, Alex; Underwood, Tim J; Eliceiri, Kevin W; Thomas, Gareth J

2016-02-02

Collagen structure has been shown to influence tumor cell invasion, metastasis and clinical outcome in breast cancer. However, it remains unclear how it affects other solid cancers. Here we utilized multi-photon laser scanning microscopy and Second Harmonic Generation to identify alterations to collagen fiber structure within the tumor stroma of head & neck, esophageal and colorectal cancers. Image segmentation algorithms were then applied to quantitatively characterize these morphological changes, showing that elongated collagen fibers significantly correlated with poor clinical outcome (Log Rank p < 0.05). We used TGF-β treatment to model fibroblast conversion to smooth muscle actin SMA-positive cancer associated fibroblasts (CAFs) and found that these cells induce the formation of elongated collagen fibers in vivo. However, proteomic/transcriptomic analysis of SMA-positive CAFs cultured ex-vivo showed significant heterogeneity in the expression of genes with collagen fibril organizing gene ontology. Notably, stratifying patients according to stromal SMA-positivity and collagen fiber elongation was found to provide a highly significant correlation with poor survival in all 3 cancer types (Log Rank p ≤ 0.003). In summary, we show that increased collagen fiber length correlates with poor patient survival in multiple tumor types and that only a sub-set of SMA-positive CAFs can mediate the formation of this collagen structure.

Migration of mononuclear cells expressing β-actin through the adventitia into media and intima in coronary arteriogenesis and venogenesis in ischemic myocardium.

PubMed

Uchida, Yasuto; Uchida, Yasumi; Maezawa, Yoshiro; Maezawa, Yuko; Tabata, Tsuyoshi

2012-01-01

It was previously thought that arteriogenesis and venogenesis are induced not only by proliferation of vessel-resident smooth muscle cells (SMCs) and endothelial cells (ECs) but also by migration of their precursors. However, it is not well understood through what route(s) the precursors migrate into the existing vessels.We examined through what route or routes circulating mononuclear cells expressing β-actin (β-MNCs), which we identified in canine coronary vessels, migrate into coronary vessel walls and cause arteriogenesis and venogenesis at 1, 2, 4 and 8 weeks after induction of myocardial infarction.The following changes were observed: (1) The β-MNCs migrated via coronary microvessels to the interstitial space at one week; (2) β-MNCs traversed the adventitia into the media and settled in parallel with pre-existing smooth muscle cells (SMCs) in arterioles and arteries and lost β-actin and acquired α-smooth muscle actin (α-SMA) to become mature SMCs at 2-4 weeks; (3) at the same time, other β-MNCs migrated across the adventitia and media into the intima and settled in parallel with pre-existing endothelial cells (ECs) and lost β-actin, while acquiring CD(31), to become mature ECs, resulting in arteriogenesis; (4) Similarly, β-MNCs migrated into venular and venous walls and became SMCs or ECs, resulting in venogenesis.β-MNCs in the interstitial space expressed CD(34) but not other major vascular cell markers.β-MNCs, possibly a vascular progenitor, migrate not from the lumen but across the adventitia into the media or intima of coronary vessels and transit to SMCs or ECs, and participate in arteriogenesis and venogenesis in ischemic myocardium.

By Different Cellular Mechanisms, Lymphatic Vessels Sprout by Endothelial Cell Recruitment Whereas Blood Vessels Grow by Vascular Expansion

NASA Technical Reports Server (NTRS)

Parsons-Wingerter, Patricia; McKay, Terri L.; Leontiev, Dmitry; Condrich, Terence K.; DiCorleto, Paul E.

2005-01-01

The development of effective vascular therapies requires the understanding of all modes of vessel formation contributing to vasculogenesis, angiogenesis (here termed hemangiogenesis) and lymphangiogenesis. We show that lymphangiogenesis proceeds by blind-ended vessel sprouting via recruitment of isolated endothelial progenitor cells to the tips of growing vessels, whereas hemangiogenesis occurs by non-sprouting vessel expansion from the capillary network, during middevelopment in the quail chorioallantoic membrane (CAM). Blood vessels expanded out of capillaries that displayed transient expression of alpha smooth muscle actin (alphaSMA), accompanied by mural recruitment of migratory progenitor cells expressing SMA. Lymphatics and blood vessels were identified by confocal/fluorescence microscopy of vascular endothelial growth factor (VEGF) receptors VEGFR-1 and VEGFR-2, alphaSMA (expressed on CAM blood vessels but not on lymphatics), homeobox transcription factor Prox-1 (specific to CAM lymphatic endothelium), and the quail hematopoetic/vascular marker, QH-1. Expression of VEGFR-1 was highly restricted to blood vessels (primarily capillaries). VEGFR-2 was expressed intensely in isolated hematopoietic cells, lymphatic vessels and moderately in blood vessels. Prox-1 was absent from endothelial progenitor cells prior to lymphatic recruitment. Although vascular endothelial growth factor-165 (VEGF(sub 165)) is a key regulator of numerous cellular processes in hemangiogenesis and vasculogenesis, the role of VEGF(sub 165) in lymphangiogenesis is less clear. Exogenous VEGF(sub 165) increased blood vessel density without changing endogenous modes of vascular/lymphatic vessel formation or marker expression patterns. However, VEGF(sub 165) did increase the frequency of blood vascular anastomoses and strongly induced the antimaturational dissociation of lymphatics from blood vessels, with frequent formation of homogeneous lymphatic networks.

The platelet-derived growth factor receptor/STAT3 signaling pathway regulates the phenotypic transition of corpus cavernosum smooth muscle in rats.

PubMed

Yan, Jun-Feng; Huang, Wen-Jie; Zhao, Jian-Feng; Fu, Hui-Ying; Zhang, Gao-Yue; Huang, Xiao-Jun; Lv, Bo-Dong

2017-01-01

Erectile dysfunction (ED) is a common clinical disease that is difficult to treat. We previously found that hypoxia modulates the phenotype of primary corpus cavernosum smooth muscle cells (CCSMCs) in rats, but the underlying molecular mechanism is still unknown. Platelet-derived growth factor receptor (PDGFR)-related signaling pathways are correlated with cell phenotypic transition, but research has been focused more on vascular smooth muscle and tracheal smooth muscle and less on CCSMCs. Here, we investigated the role of PDGFR-related signaling pathways in penile CCSMCs, which were successfully isolated from rats and cultured in vitro. PDGF-BB at 5, 10, or 20 ng/ml altered CCSMC morphology from the original elongated, spindle shape to a broader shape and promoted the synthetic phenotype and expression of the related proteins vimentin and collagen-I, while inhibiting the contractile phenotype and expression of the related proteins smooth muscle (SM) α-actin (α-SMA) and desmin. Inhibition of PDGFR activity via siRNA or the PDGFR inhibitor crenolanib inhibited vimentin and collagen-I expression, increased α-SMA and desmin expression, and considerably inhibited serine-threonine protein kinase (AKT) and signal transducer and activator of transcription 3 (STAT3) phosphorylation. STAT3 knockdown promoted the contractile phenotype, inhibited vimentin and collagen-I expression, and increased α-SMA and desmin expression, whereas AKT knockdown did not affect phenotype-associated proteins. STAT3 overexpression in CCSMC cells weakened the suppressive effect of PDGFR inhibition on the morphology and phenotypic transformation induced by PDGF-BB. Through activation of the PDGFR/STAT3 signaling pathway, PDGF promoted the synthetic phenotype transition; thus, regulation of this pathway might contribute to ED therapy.

Chitosan and thiolated chitosan: Novel therapeutic approach for preventing corneal haze after chemical injuries.

PubMed

Zahir-Jouzdani, Forouhe; Mahbod, Mirgholamreza; Soleimani, Masoud; Vakhshiteh, Faezeh; Arefian, Ehsan; Shahosseini, Saeed; Dinarvand, Rasoul; Atyabi, Fatemeh

2018-01-01

Corneal haze, commonly caused by deep physical and chemical injuries, can greatly impair vision. Growth factors facilitate fibroblast proliferation and differentiation, which leads to haze intensity. In this study, the potential effect of chitosan (CS) and thiolated-chitosan (TCS) nanoparticles and solutions on inhibition of fibroblast proliferation, fibroblast to myofibroblast differentiation, neovascularization, extracellular matrix (ECM) deposition, and pro-fibrotic cytokine expression was examined. Transforming growth factor beta-1 (TGFβ 1 ) was induced by interleukin-6 (IL6) in human corneal fibroblasts and expression levels of TGFβ 1 , Platelet-derived growth factor (PDGF), α-smooth muscle actins (α-SMA), collagen type I (Col I), fibronectin (Fn) and vascular endothelial growth factor (VEGF) were quantified using qRT-PCR. To assess wound-healing capacity, TCS-treated mice were examined for α-SMA positive cells, collagen deposition, inflammatory cells and neovascularization through pathological immunohistochemistry. The results revealed that CS and TCS could down-regulate the expression levels of TGFβ 1 and PDGF comparable to that of TGFβ 1 knockdown experiment. However, down-regulation of TGFβ 1 was not regulated through miR29b induction. Neovascularization along with α-SMA and ECM deposition were significantly diminished. According to these findings, CS and TCS can be considered as potential anti-fibrotic and anti-angiogenic therapeutics. Furthermore, TCS, thiolated derivative of CS, will increase mucoadhesion of the polymer at the corneal surface which makes the polymer efficient and non-toxic therapeutic approach for corneal injuries. Copyright © 2017 Elsevier Ltd. All rights reserved.

Immunohistochemical characterisation of the hepatic stem cell niche in feline hepatic lipidosis: a preliminary morphological study.

PubMed

Valtolina, Chiara; Robben, Joris H; Favier, Robert P; Rothuizen, Jan; Grinwis, Guy Cm; Schotanus, Baukje A; Penning, Louis C

2018-05-01

Objectives The aim of this study was to describe the cellular and stromal components of the hepatic progenitor cell niche in feline hepatic lipidosis (FHL). Methods Immunohistochemical staining for the progenitor/bile duct marker (K19), activated Kupffer cells (MAC387), myofibroblasts (alpha-smooth muscle actin [α-SMA]) and the extracellular matrix component laminin were used on seven liver biopsies of cats with FHL and three healthy cats. Double immunofluorescence stainings were performed to investigate co-localisation of different cell types in the hepatic progenitor cell (HPC) niche. Results HPCs, Kupffer cells, myofibroblasts and laminin deposition were observed in the liver samples of FHL, although with variability in the expression and positivity of the different immunostainings between different samples. When compared with the unaffected cats where K19 positivity and minimal α-SMA and laminin positivity were seen mainly in the portal area, in the majority of FHL samples K19 and α-SMA-positive cells and laminin positivity were seen also in the periportal and parenchymatous area. MAC387-positive cells were present throughout the parenchyma. Conclusions and relevance This is a preliminary morphological study to describe the activation and co-localisation of components of the HPC niche in FHL. Although the HPC niche in FHL resembles that described in hepatopathies in dogs and in feline lymphocytic cholangitis, the expression of K19, α-SMA, MAC387 and lamin is more variable in FHL, and a common pattern of activation could not be established. Nevertheless, when HPCs were activated, a spatial association between HPCs and their niche could be demonstrated.

VEGF165 Stimulates Vessel Density and Vessel Diameter Differently in Angiogenesis and Lymphangiogenesis

NASA Technical Reports Server (NTRS)

Parsons-Wingerter, Patricia; Radhakrishnan, Krishnan; DiCorleto, Paul E.; Leontiev, Dmitry; Anand-Apte, Bela; Albarran, Brian; Farr, Andrew G.

2005-01-01

Vascular endothelial growth factor-165 (VEGF(sub 165)) stimulated angiogenesis in the quail chorioallantoic membrane (CAM) by vessel expansion from the capillary network. However, lymphangiogenesis was stimulated by the filopodial guidance of tip cells located on blind-ended lymphatic sprouts. As quantified by fractal/generational branching analysis using the computer code VESGEN, vascular density increased maximally at low VEGF concentrations, and vascular diameter increased most at high VEGF concentrations. Increased vascular density and diameter were statistically independent events (r(sub s), -0.06). By fluorescence immunohistochemistry of VEGF receptors VEGFR-1 and VEGFR-2, alpha smooth muscle actin ((alpha) SMA) and a vascular/lymphatic marker, VEGF(sub 165) increased the density and diameter of sprouting lymphatic vessels guided by tip cells (accompanied by the dissociation of lymphatics from blood vessels). Isolated migratory cells expressing (alpha)SMA were recruited to blood vessels, whereas isolated cells expressing VEGFR-2 were recruited primarily to lymphatics. In conclusion, VEGF(sub 165) increased lymphatic vessel density by lymphatic sprouting, but increased blood vessel density by vascular expansion from the capillary network.

Resveratrol attenuates the progress of liver fibrosis via the Akt/nuclear factor-κB pathways.

PubMed

Zhang, Hui; Sun, Qingfeng; Xu, Tingyan; Hong, Liang; Fu, Rongquan; Wu, Jinguo; Ding, Jiguang

2016-01-01

Liver fibrosis is a wound-healing response to chronic liver injury that results in the accumulation of extracellular matrix proteins. It eventually leads to cirrhosis of the liver and liver failure, and it is a critical threat to the health and lives of patients with chronic liver diseases. No effective treatment is currently available. Resveratrol is a polyphenol with antioxidant, anti‑cancer and anti‑inflammatory properties. It has been reported that resveratrol prevents liver fibrosis, possibly by inhibiting NF‑κB activation. The present study investigated the mechanisms by which resveratrol prevented liver fibrosis, focusing on the possible involvement of the NF‑κB pathway. Mice with carbon tetrachloride (CCl4)‑induced liver fibrosis were treated with various concentrations of resveratrol. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and tumor necrosis factor (TNF)‑α were detected by ELISAs. Expression of α‑smooth muscle actin (α‑SMA), collagen I, inhibitor of NF‑κB (IκB) and NF‑κB were detected by western blot analysis. In addition, the present study examined the effects of resveratrol on the expression of fibrosis markers in LX‑2 cells. Western blot analysis was further used to detect the levels of Akt and phosphorylated Akt, as well as the nuclear levels of IκB, phosphorylated IκB and NF‑κB p65. The expression of α‑SMA in resveratrol‑treated LX‑2 cells was detected by immunofluorescence and flow cytometry, which demonstrated that resveratrol decreased the expression of α‑SMA in LX‑2 cells. Resveratrol also decreased CCl4‑induced upregulation of serum AST, ALT, TNF‑α, α‑SMA and collagen I. Finally, resveratrol prevented the activation of NF‑κB and Akt. The results of the present study therefore indicated that resveratrol attenuates liver fibrosis via the Akt/NF-κB pathways.

Ac-SDKP suppresses epithelial-mesenchymal transition in A549 cells via HSP27 signaling.

PubMed

Deng, Haijing; Yang, Fang; Xu, Hong; Sun, Yue; Xue, Xinxin; Du, Shipu; Wang, Xiaojun; Li, Shifeng; Liu, Yan; Wang, Ruimin

2014-08-01

The synthetic tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) has been shown to be a modulator of molecular aspects of the fibrosis pathway. This study reveals that Ac-SDKP exerts an anti-fibrotic effect on human type II alveolar epithelial cells (A549), which are a source of myofibroblasts once exposed to TGF-β1, by decreasing the expression of heat shock protein 27 (HSP27). We used A549 cells in vitro to detect morphological evidence of epithelial-mesenchymal transition (EMT) by phase-contrast microscopy. Immunocytochemical and western blot analysis determined the distributions of cytokeratin 8 (CK8), α-smooth muscle actin (α-SMA), and SNAI1. Confocal laser scanning microscopy revealed a colocalization of HSP27 and SNAI1 on TGF-β1-induced A549 cells. These results also demonstrated that A549 cells became spindle-like when exposed to TGF-β1. Coincident with these morphological changes, expression levels of CK8 and E-cad decreased, while those of vimentin and α-SMA increased. This process was accompanied by increases in levels of HSP27, SNAI1, and type I and type III collagen. In vitro transfection experiments demonstrated that the inhibition of HSP27 in cultured A549 cells could decrease the expression of SNAI1 and α-SMA while increasing the expression of E-cad. A noticeable reduction in collagen types I and III was also evident. Our results found that Ac-SDKP inhibited the transition of cultured A549 cells to myofibroblasts and attenuated collagen synthesis through modulating the expression of HSP27. Copyright © 2014 Elsevier Inc. All rights reserved.

Rho/Rock cross-talks with transforming growth factor-β/Smad pathway participates in lung fibroblast-myofibroblast differentiation.

PubMed

Ji, Hong; Tang, Haiying; Lin, Hongli; Mao, Jingwei; Gao, Lili; Liu, Jia; Wu, Taihua

2014-11-01

The differentiation of fibroblasts, which are promoted by transforming growth factor-β (TGF-β)/Smad, is involved in the process of pulmonary fibrosis. The Rho/Rho-associated coiled-coil-forming protein kinase (Rock) pathway may regulate the fibroblast differentiation and myofibroblast expression of α-smooth muscle actin (α-SMA), however, the mechanism is not clear. The aim of the present study was to evaluate the role of Rho/Rock and TGF-β/Smad in TGF-β1-induced lung fibroblasts differentiation. Human embryonic lung fibroblasts were stimulated by TGF-β1, Y-27632 (inhibitor of Rho/Rock signaling) and staurosporine (inhibitor of TGF-β/Smad signaling). The α-SMA expression, cell cycle progression, content of the extracellular matrix (ECM) in cell culture supernatants and the expression of RhoA, RhoC, Rock1 and Smad2 were detected. The results demonstrated that α-SMA-positive cells significantly increased following TGF-β1 stimulation. Rho/Rock and TGF-β/Smad inhibitors suppressed TGF-β1-induced lung fibroblast differentiation. The inhibitors increased G 0 /G 1 and decreased S and G 2 /M percentages. The concentrations of the ECM proteins in the supernatant were significantly increased by TGF-β1 stimulation, whereas they were decreased by inhibitor stimulation. RhoA, RhoC, Rock1, Smad2 and tissue inhibitor of metalloproteinase-1 were upregulated by TGF-β1 stimulation. The Rho/Rock inhibitor downregulated Smad2 expression and the TGF-β/Smad inhibitor downregulated RhoA, RhoC and Rock1 expression. Therefore, the Rho/Rock pathway and Smad signaling were involved in the process of lung fibroblasts transformation, induced by TGF-β1, to myofibroblasts. The two pathways may undergo cross-talk in the lung fibroblasts differentiation in vitro .

Salt-induced epithelial-to-mesenchymal transition in Dahl salt-sensitive rats is dependent on elevated blood pressure.

PubMed

Wang, Y; Mu, J J; Liu, F Q; Ren, K Y; Xiao, H Y; Yang, Z; Yuan, Z Y

2014-02-01

Dietary salt intake has been linked to hypertension and cardiovascular disease. Accumulating evidence has indicated that salt-sensitive individuals on high salt intake are more likely to develop renal fibrosis. Epithelial-to-mesenchymal transition (EMT) participates in the development and progression of renal fibrosis in humans and animals. The objective of this study was to investigate the impact of a high-salt diet on EMT in Dahl salt-sensitive (SS) rats. Twenty-four male SS and consomic SS-13(BN) rats were randomized to a normal diet or a high-salt diet. After 4 weeks, systolic blood pressure (SBP) and albuminuria were analyzed, and renal fibrosis was histopathologically evaluated. Tubular EMT was evaluated using immunohistochemistry and real-time PCR with E-cadherin and alpha smooth muscle actin (α-SMA). After 4 weeks, SBP and albuminuria were significantly increased in the SS high-salt group compared with the normal diet group. Dietary salt intake induced renal fibrosis and tubular EMT as identified by reduced expression of E-cadherin and enhanced expression of α-SMA in SS rats. Both blood pressure and renal interstitial fibrosis were negatively correlated with E-cadherin but positively correlated with α-SMA. Salt intake induced tubular EMT and renal injury in SS rats, and this relationship might depend on the increase in blood pressure.

Curcumin Suppresses Intestinal Fibrosis by Inhibition of PPARγ-Mediated Epithelial-Mesenchymal Transition

PubMed Central

Jiang, Bin; Wang, Hui; Shen, Cunsi; Chen, Hao

2017-01-01

Intestinal fibrotic stricture is a major complication of Crohn's disease (CD) and epithelial-to-mesenchymal transition (EMT) is considered as an important contributor to the formation of intestinal fibrosis by increasing extracellular matrix (ECM) proteins. Curcumin, a compound derived from rhizomes of Curcuma, has been demonstrated with a potent antifibrotic effect. However, its effect on intestinal fibrosis and the potential mechanism is not completely understood. Here we found that curcumin pretreatment significantly represses TGF-β1-induced Smad pathway and decreases its downstream α-smooth muscle actin (α-SMA) gene expression in intestinal epithelial cells (IEC-6); in contrast, curcumin increases expression of E-cadherin and peroxisome proliferator-activated receptor γ (PPARγ) in IEC-6. Moreover, curcumin promotes nuclear translocation of PPARγ and the inhibitory effect of curcumin on EMT could be reversed by PPARγ antagonist GW9662. Consistently, in the rat model of intestinal fibrosis induced by 2,4,5-trinitrobenzene sulphonic acid (TNBS), oral curcumin attenuates intestinal fibrosis by increasing the expression of PPARγ and E-cadherin and decreasing the expression of α-SMA, FN, and CTGF in colon tissue. Collectively, these results indicated that curcumin is able to prevent EMT progress in intestinal fibrosis by PPARγ-mediated repression of TGF-β1/Smad pathway. PMID:28203261

[Intramuscular injection of lentivirus-mediated EPAS1 gene improves hind limb ischemia and its mechanism in a rat model of peripheral artery vascular disease].

PubMed

Wang, Zhihong; Gu, Hongbin; Yang, Fan; Xie, Huajie; Sheng, Lei; Li, Mingfei

2017-11-01

Objective To investigate the effect of over-expressed endothelial Per-Arnt-Sim domain protein 1 (EPAS1) on peripheral arterial disease (PAD) in a rat model. Methods PAD rat model was established by external iliac artery ligation followed by lentivirus-mediated EPAS1 gene injection into rat right adductor magnus. The models were evaluated by quantitative analysis of gait disturbance. The changes of blood flow in the posterior extremity of the rats were detected using laser Doppler. The expressions of EPAS1, hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) mRNAs were tested by real-time quantitative PCR. The expression of α-smooth muscle actin (αSMA) was detected by immunohistochemical staining. Results Compared with lenti-EGFP group, rat hind limb function and circulation got recovered obviously 7 days after lenti-EPAS1 injection. The mRNA expressions of EPAS1, HGF, bFGF, and VEGF were up-regulated in the lenti-EPAS1-treated sites.The expression of αSMA showed an obvious increase in the lenti-EPAS1-treated muscles. Conclusion Over-expressed lenti-EPAS1 can promote angiogenesis via the up-regulation of EPAS1-related angiogenic factors in the muscles of the affected hind limb and reduce gait disturbance.

alpha-Smooth muscle actin immunoreactivity may change in nature in interlobular fibrosis of the pancreas in patients with congenital biliary dilatation.

PubMed

Matsubara, Kenro; Suda, Koichi; Suzuki, Fujihiko; Kumasaka, Toshio; Shiotsu, Hidetoshi; Miyano, Takeshi

2004-07-01

Pancreatic fibrosis in patients with congenital biliary dilatation (CBD) or choledochal cyst was studied to determine why biliary pancreatitis seldom progresses to chronic pancreatitis/more progressive state. Pancreatic collagenization in eight patients (three adults with pancreatoduodenectomy and five children with biopsy of the pancreas performed when excising the cyst) with CBD was evaluated histopathologically and immunohistochemically. Interlobular and periductal fibrosis with both collagen Type I and Type III immunoreactivities was found in six out of eight cases and in all four cases in which the pancreatic duct was included, respectively. The interlobular area was seldom immunoreactive for alpha-smooth muscle actin (alpha-SMA), a marker for myofibroblasts, but was usually positive for CD34, a human progenitor cell antigen. In contrast, the periductal area was usually immunoreactive for alpha-SMA, but usually negative for CD34 and immunopositive for bcl-2, indicating a continuously progressive state of fibrosis, in which 'pre-existing'alpha-SMA immunoreactivity in the interlobular area may change in nature and lead to CD34-positive fibrosis or apoptosis. In conclusion, biliary pancreatitis is not likely to evolve into chronic pancreatitis/more progressive state because 'pre-existing'alpha-SMA immunoreactivity in the interlobular area may change in nature.

Interleukin-6 downregulated vascular smooth muscle cell contractile proteins via ATG4B-mediated autophagy in thoracic aortic dissection.

PubMed

An, Zhao; Qiao, Fan; Lu, Qijue; Ma, Ye; Liu, Yang; Lu, Fanglin; Xu, Zhiyun

2017-12-01

Interleukin-6 (IL-6) overexpression played an important role in the pathogenesis of thoracic aortic dissection (TAD). Our previous study found enhanced autophagy accompanying with contractile proteins α smooth muscle actin (α-SMA) and smooth muscle 22α (SM22α) degradation in TAD aortic vascular smooth muscle cells (VSMCs). Autophagy is an important way for intracellular proteins degradation, while IL-6 has been found as a contributing factor of autophagy in some cancers. These indicated IL-6 might contribute to the occurrence of TAD by promoting autophagy-induced contractile proteins degradation, which has not been investigated. The aim of the present study is to verify this hypothesis and investigate the mechanism of it. We collected 10 TAD and 10 control aortic specimens from patients underwent TAD surgical repair and coronary artery bypass grafting, respectively. Quantitative real-time polymerase chain reaction was used to detect mRNA expression. Protein expression level was assessed by enzyme-linked immunosorbent assay, western blot, and immunohistochemistry. Microtubule-associated protein 1 light chain 3 beta overexpression adenovirus with green and red fluorescent protein tags and transmission electron microscopy were used to detect autophagy level in VSMCs. 3-Methyladenine (3-MA) and chloroquine were used to block autophagy in human VSMCs. Experiment results showed that the expression of IL-6 was significantly increased accompanying with up-regulated autophagy in TAD aortic wall compared with controls. In vitro results showed that IL-6 stimulation decreased the expression of VSMCs contractile proteins α-SMA and SM22α accompanying with up-regulated autophagy. Blocking autophagy with 3-MA or chloroquine inhibited IL-6 induced α-SMA and SM22α degradation. Further investigation showed that autophagy-related 4B cysteine peptidase (ATG4B) was significantly overexpressed in TAD aortic wall and played important role in IL-6 induced autophagy up-regulation. ATG4B knockdown blocked IL-6-induced autophagy and α-SMA and SM22α degradation, while ATG4B overexpression partly replaced the function of IL-6 in human VSMCs. In conclusion, our study demonstrated that IL-6 downregulated expression of VSMCs contractile proteins α-SMA and SM22α via enhancing ATG4B-mediated autophagy in TAD.

Establishment of dermal sheath cell line from Cashmere goat and characterizing cytokeratin 13 as its novel biomarker.

PubMed

Zhu, Bing; Guo, Zhili; Jin, Muzi; Bai, Yujuan; Yang, Wenliang; Hui, Lihua

2018-05-01

To establish a dermal sheath cell line, a dermal papilla cell line and a outer root sheath cell line from Cashmere goat and clarify the similarities and differences among them. We established a dermal sheath cell line, a dermal papilla cell line and a outer root sheath cell line from the pelage skin hair follicles of Cashmere goat. The growth rate of dermal sheath cells was intermediate between that of dermal papilla cells and outer root sheath cells. Immunofluorescence experiments and reverse transcription-polymerase chain reaction analysis showed that at both the transcriptional and translational levels, the dermal sheath cells were alpha-smooth muscle actin (α-SMA) + /cytokeratin 13 + , while the dermal papilla cells were α-SMA + /cytokeratin 13 - and the outer root sheath cells were α-SMA - /cytokeratin 13 + . Patterns of cytokeratin 13 expression could distinguish the dermal sheath cells from the dermal papilla cells. These results suggest that cytokeratin 13 could serve as a novel biomarker for dermal sheath cells of Cashmere goat, and should prove useful for researchers investigating dermal stem cells or interaction of different types of cells during hair cycle.

Histological and immunohistochemical effects of Curcuma longa on activation of rat hepatic stellate cells after cadmium induced hepatotoxicity.

PubMed

El-Mansy, A A; Mazroa, S A; Hamed, W S; Yaseen, A H; El-Mohandes, E A

2016-01-01

The liver is a target for toxic chemicals such as cadmium (Cd). When the liver is damaged, hepatic stellate cells (HSC) are activated and transformed into myofibroblast-like cells, which are responsible for liver fibrosis. Curcuma longa has been reported to exert a hepato-protective effect under various pathological conditions. We investigated the effects of C. longa administration on HSC activation in response to Cd induced hepatotoxicity. Forty adult male albino rats were divided into: group 1 (control), group 2 (Cd treated), group 3 (C. longa treated) and group 4 (Cd and C. longa treated). After 6 weeks, liver specimens were prepared for light and electron microscopy examination of histological changes and immunohistochemical localization of alpha smooth muscle actin (αSMA) as a specific marker for activated HSC. Activated HSC with a positive αSMA immune reaction were not detected in groups 1 and 3. Large numbers of activated HSC with αSMA immune reactions were observed in group 2 in addition to Cd induced hepatotoxic changes including excess collagen deposition in thickened portal triads, interlobular septa with hepatic lobulation, inflammatory cell infiltration, a significant increase in Kupffer cells and degenerated hepatocytes. In group 4, we observed a significant decrease in HSC that expressed αSMA with amelioration of the hepatotoxic changes. C. longa administration decreased HSC activation and ameliorated hepatotoxic changes caused by Cd in adult rats.

Characterisation of myofibroblasts in fibrovascular tissues of primary and recurrent pterygia

PubMed Central

Touhami, A; Di Pascuale, M A; Kawatika, T; Valle, M Del; Rosa, R H; Dubovy, S; Tseng, S C G

2005-01-01

Aims: To determine the presence and origin of myofibroblasts in pterygia. Methods: 86 specimens including head, body, and fibrovascular tissue from 52 primary and 34 recurrent pterygia and five exenterated eyes without pterygia were searched for the origin of myofibroblasts. All tissues were subjected to haematoxylin and eosin staining, immunohistochemistry using antibodies against alpha smooth muscle actin (α-SMA), desmin, vimentin, and caldesmon, and transmission electron microscopy (TEM). The phenotype of fibroblasts subcultured in a serum free medium from pterygium fibrovascular tissues was characterised by the above antibodies. Bundles of dense fibrous tissues were noted in 86% of the fibrovascular tissue specimens evaluated. Cells within these bundles were characterised as myofibroblasts based on positive staining to α-SMA, but negative to desmin and caldesmon, markers for smooth muscle cells. Interestingly, positive α-SMA staining was also found in the periorbital fibroadipose tissue posterior to Tenon’s capsule near the nasal conjunctiva in all exenterated specimens. All first passage fibroblasts expressed vimentin, some were positive to α-SMA, but all were negative to desmin or caldesmon. Cells in pterygium fibrovascular tissues showed ultrastructural features of intracytoplasmic bundles of microfilaments, consistent with myofibroblastic differentiation. Conclusion: These studies collectively demonstrate the presence of contractile myofibroblasts bundle in pterygia and in the periorbital fibroadipose tissue posterior to Tenon’s capsule of exenterated eyes without pterygium. PMID:15722301

Tongxinluo ameliorates renal structure and function by regulating miR-21-induced epithelial-to-mesenchymal transition in diabetic nephropathy.

PubMed

Wang, Jin-yang; Gao, Yan-bin; Zhang, Na; Zou, Da-wei; Xu, Li-ping; Zhu, Zhi-yao; Li, Jiao-yang; Zhou, Sheng-nan; Cui, Fang-qiang; Zeng, Xiang-jun; Geng, Jian-guo; Yang, Jin-kui

2014-03-01

Diabetic nephropathy (DN) is one of the most important diabetic microangiopathies. The epithelial-to-mesenchymal transition (EMT) plays an important role in DN. The physiological role of microRNA-21 (miR-21) was closely linked to EMT. However, it remained elusive whether tongxinluo (TXL) ameliorated renal structure and function by regulating miR-21-induced EMT in DN. This study aimed to determine the effect of TXL on miR-21-induced renal tubular EMT and to explore the relationship between miR-21 and TGF-β1/smads signals. Real-time RT-PCR, cell transfection, in situ hybridization (ISH), and laser confocal microscopy were used, respectively. Here, we revealed that TXL dose dependently lowered miR-21 expression in tissue, serum, and cells. Overexpression of miR-21 can enhance α-smooth muscle actin (SMA) expression and decrease E-cadherin expression by upregulating smad3/p-smad3 expression and downregulating smad7 expression. Interestingly, TXL also increased E-cadherin expression and decreased α-SMA expression by regulating miR-21 expression. More importantly, TXL decreased collagen IV, fibronectin, glomerular basement membrane, glomerular area, and the albumin/creatinine ratio, whereas it increased the creatinine clearance ratio. The results demonstrated that TXL ameliorated renal structure and function by regulating miR-21-induced EMT, which was one of the mechanisms to protect against DN, and that miR-21 may be one of the therapeutic targets for TXL in DN.

Inhibition of liver fibrosis by solubilized coenzyme Q10: Role of Nrf2 activation in inhibiting transforming growth factor-beta1 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Hoo-Kyun; Pokharel, Yuba Raj; Lim, Sung Chul

2009-11-01

Coenzyme Q10 (CoQ10), an endogenous antioxidant, is important in oxidative phosphorylation in mitochondria. It has anti-diabetic and anti-cardiovascular disease effects, but its ability to protect against liver fibrosis has not been studied. Here, we assessed the ability of solubilized CoQ10 to improve dimethylnitrosamine (DMN)-induced liver fibrogenesis in mice. DMN treatments for 3 weeks produced a marked liver fibrosis as assessed by histopathological examination and tissue 4-hydroxyproline content. Solubilized CoQ10 (10 and 30 mg/kg) significantly inhibited both the increases in fibrosis score and 4-hydroxyproline content induced by DMN. Reverse transcription-polymerase chain reaction and Western blot analyses revealed that solubilized CoQ10 inhibitedmore » increases in the transforming growth factor-beta1 (TGF-beta1) mRNA and alpha-smooth muscle actin (alpha-SMA) protein by DMN. Interestingly, hepatic glutamate-cysteine ligase (GCL) and glutathione S-transferase A2 (GSTA2) were up-regulated in mice treated with CoQ10. Solubilized CoQ10 also up-regulated antioxidant enzymes such as catalytic subunits of GCL and GSTA2 via activating NF-E2 related factor2 (Nrf2)/antioxidant response element (ARE) in H4IIE hepatoma cells. Moreover, CoQ10's inhibition of alpha-SMA and TGF-beta1 expressions disappeared in Nrf2-null MEF cells. In contrast, Nrf2 overexpression significantly decreased the basal expression levels of alpha-SMA and TGF-beta1 in Nrf2-null MEF cells. These results demonstrated that solubilized CoQ10 inhibited DMN-induced liver fibrosis through suppression of TGF-beta1 expression via Nrf2/ARE activation.« less

Dietary Supplementation of Blueberry Juice Enhances Hepatic Expression of Metallothionein and Attenuates Liver Fibrosis in Rats

PubMed Central

Wang, Yuping; Cheng, Mingliang; Zhang, Baofang; Nie, Fei; Jiang, Hongmei

2013-01-01

Aim To investigate the effect of blueberry juice intake on rat liver fibrosis and its influence on hepatic antioxidant defense. Methods Rabbiteye blueberry was used to prepare fresh juice to feed rats by daily gastric gavage. Dan-shao-hua-xian capsule (DSHX) was used as a positive control for liver fibrosis protection. Liver fibrosis was induced in male Sprague-Dawley rats by subcutaneous injection of CCl4 and feeding a high-lipid/low-protein diet for 8 weeks. Hepatic fibrosis was evaluated by Masson staining. The expression of α-smooth muscle actin (α-SMA) and collagen III (Col III) were determined by immunohistochemical techniques. The activities of superoxide dismutase (SOD) and malondialdehyde (MDA) in liver homogenates were determined. Metallothionein (MT) expression was detected by real-time RT-PCR and immunohistochemical techniques. Results Blueberry juice consumption significantly attenuates CCl4-induced rat hepatic fibrosis, which was associated with elevated expression of metallothionein (MT), increased SOD activity, reduced oxidative stress, and decreased levels of α-SMA and Col III in the liver. Conclusion Our study suggests that dietary supplementation of blueberry juice can augment antioxidative capability of the liver presumably via stimulating MT expression and SOD activity, which in turn promotes HSC inactivation and thus decreases extracellular matrix collagen accumulation in the liver, and thereby alleviating hepatic fibrosis. PMID:23554912

Increased CCT-eta expression is a marker of latent and active disease and a modulator of fibroblast contractility in Dupuytren's contracture.

PubMed

Satish, Latha; O'Gorman, David B; Johnson, Sandra; Raykha, Christina; Gan, Bing Siang; Wang, James H-C; Kathju, Sandeep

2013-07-01

Dupuytren's contracture (DC) is a fibroproliferative disorder of unknown etiology characterized by a scar-like contracture that develops in the palm and/or digits. We have previously reported that the eta subunit of the chaperonin containing T-complex polypeptide (CCT-eta) is increased in fibrotic wound healing, and is essential for the accumulation of α-smooth muscle actin (α-SMA) in fibroblasts. The purpose of this study was to determine if CCT-eta is similarly implicated in the aberrant fibrosis seen in DC and to investigate the role of CCT-eta in the behavior of myo/fibroblasts in DC. Fibroblasts were obtained from DC-affected palmar fascia, from adjacent phenotypically normal palmar fascia in the same DC patients (PF), and from non-DC palmar fascial tissues in patients undergoing carpal tunnel (CT) release. Inherent contractility in these three populations was examined using fibroblast-populated collagen lattices (FPCLs) and by cell traction force microscopy. Expression of CCT-eta and α-SMA protein was determined by Western blot. The effect of CCT-eta inhibition on the contractility of DC cells was determined by deploying an siRNA versus CCT-eta. DC cells were significantly more contractile than both matching palmar fascial (PF) cells and CT cells in both assays, with PF cells demonstrating an intermediate contractility in the FPCL assay. Whereas α-SMA protein was significantly increased only in DC cells compared to PF and CT cells, CCT-eta protein was significantly increased in both PF and DC cells compared to CT cells. siRNA-mediated depletion of CCT-eta inhibited the accumulation of both CCT-eta and α-SMA protein in DC cells, and also significantly decreased the contractility of treated DC cells. These observations suggest that increased expression of CCT-eta appears to be a marker for latent and active disease in these patients and to be essential for the increased contractility exhibited by these fibroblasts.

[Emodin inhibits the proliferation, transdifferentiation and collagen synthesis of pulmonary fibroblasts].

PubMed

Liu, Lijing; Yin, Huiming; He, Jianbin; Xie, Maofeng; Wang, Zaiyan; Xiao, Hua

2016-07-01

Objective To explore the effect of emodin on the proliferation, differentiation into myofibroblasts and collagen synthesis of pulmonary fibroblasts and the underlying mechanisms. Methods Human pulmonary fibroblasts MRC-5 were cultured in vitro, then the cells were inoculated with dimethyl sulfoxide (DMSO) added with 0, 10, 20, 40, 80 and 160 μmol/L emodin for 24, 48 and 72 hours. Inhibitory rate of cell proliferation was analyzed by MTT assay. Based on the results of cell proliferation experiment, MRC-5 cells were treated with DMSO (control group) and 40, 80 μmol/L emodin (in DMSO) for 48 hours. Fluorescence real-time quantitative PCR was then used to measure the mRNA expressions of α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), a disintegrin-like and metalloproteinase with thrombospondin type 1 motif (ADAMTS-1), collagen type 1 (Col1) and collagen type 3 (Col3). The protein expressions of the above mentioned factors were also measured by Western blotting. Results In a concentration- and time-dependent manner, emodin inhibited MRC-5 cell proliferation. After 48 hours of co-culture, in comparison with control group, the mRNA and protein expression levels of α-SMA, TGF-β1, Col1 and Col3 significantly decreased, while the mRNA and protein expression levels of ADAMTS-1 significantly increased in 40 and 80 μmol/L emodin-treated groups. Moreover, in comparison with 40 μmol/L emodin-treated group, the mRNA and protein expressions of α-SMA, TGF-β1, Col1 and Col3 were significantly downregulated, but ADAMTS-1 mRNA and protein expressions were significantly upregulated in 80 μmol/L emodin-treated group. Conclusion Emodin can block pulmonary fibroblast proliferation and differentiation into myofibroblasts, and reduce the synthesis of Col1 and Col3 by inhibiting TGF-β1/ADAMTS-1 signaling pathway.

Analysis of MUC4 expression in human pancreatic cancer xenografts in immunodeficient mice.

PubMed

Ansari, Daniel; Bauden, Monika P; Sasor, Agata; Gundewar, Chinmay; Andersson, Roland

2014-08-01

Mucin 4 (MUC4) is a cell surface glycoprotein that is overexpressed in most pancreatic tumors. The aim of the present study was to characterize MUC4 expression in experimental pancreatic cancer in order to clarify the correlation between MUC4 and pancreatic cancer histology in vivo. Pancreatic xenograft tumors were generated in immunodeficient mice (n=15) by subcutaneous injection of MUC4(+) human pancreatic cancer cell lines Capan-1, HPAF-II or CD18/HPAF. MUC4 immunoreactivity was compared between the cancer models. Alpha-smooth muscle actin (α-SMA) was used to identify cancer-associated fibroblasts and the amount of collagen fibers was quantified with sirius red. Tumor incidence was 100%. Tumor size showed no difference across groups (p=0.796). The median MUC4 count was highest in Capan-1 tumors (p=0.002). α-SMA and collagen extent were also highest in Capan-1 tumors (p=0.018). The Capan-1 xenograft model could serve as a valuable resource to test new therapeutic strategies targeting MUC4 in pancreatic cancer. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Subepithelial corneal fibrosis partially due to epithelial-mesenchymal transition of ocular surface epithelium

PubMed Central

Kawashima, Motoko; Higa, Kazunari; Satake, Yoshiyuki; Omoto, Masahiro; Tsubota, Kazuo; Shimmura, Shigeto; Shimazaki, Jun

2010-01-01

Purpose To determine whether epithelial-mesenchymal transition is involved in the development of corneal subepithelial fibrosis (pannus). Methods Frozen samples of pannus tissue removed from human corneas with a diagnosis of total limbal stem cell deficiency were characterized by immunostaining for both epithelial and mesenchymal markers. We selected transformation-related protein 63 (p63) and pancytokeratin as epithelial markers and vimentin and α-smooth muscle actin (α-SMA) as mesenchymal markers. Immunostaining for β-catenin and E-cadherin was performed to determine wingless-Int (Wnt)-pathway activation. RT–PCR analysis was also performed on epithelial tissue obtained from pannus samples after dispase digestion. Results Immunohistochemistry revealed strong nuclear expression of p63 and weak intercellular expression of E-cadherin in epithelial basal cells of pannus tissue. Furthermore, translocation of β-catenin from intercellular junctions to the nucleus and cytoplasm was also observed. Double-positive cells for both p63 and α-SMA were observed in the subepithelial stroma of pannus tissue, which was supported by RT–PCR and cytospin analysis. Conclusions Epithelial-mesenchymal transition may be partially involved in the development of subepithelial corneal fibrosis due to total limbal stem cell deficiency. PMID:21179238

Tumour endothelial marker-1 is expressed in canine Haemangiopericytomas.

PubMed

Fujii, Y; Tsuchiya, T; Morita, R; Kimura, M; Suzuki, K; Machida, N; Mitsumori, K; Shibutani, M

2013-01-01

The aim of this study was to characterize immunohistochemically 18 cases of canine haemangiopericytoma (CHP) using two new candidate markers for pericytes, tumour endothelial marker (TEM)-1 and new glue (NG)-2, as well as the conventional mesenchymal cellular markers, vimentin, α-smooth muscle actin (α-SMA), desmin and von Willebrand factor (vWF). Because pericytes may have the same origin as endothelial or smooth muscle cells or the same differentiation potential as myofibroblasts, 17 cases of leiomyosarcoma (LMS), 20 cases of haemangiosarcoma (HS) and three cases of myofibroblastic sarcoma (MFS) were also examined. Expression of TEM-1 by >10% of the neoplastic population was observed in 94.4% (17/18) of haemangiopericytomas, 23.5% (4/17) of LMSs, 30.0% (6/20) of HSs and 66.7% (2/3) of MFSs. NG-2 expression by >10% of the neoplastic population was observed in 16.7% (3/18) of haemangiopericytomas, 52.9% (9/17) of LMSs, 0% (0/20) of HSs and 33.3% (1/3) of MFSs. Vimentin was expressed by all of tumours. In haemangiopericytoma, the incidence of positive immunoreactivity in >10% of the neoplastic population was 5.6% (1/18) for both α-SMA and desmin and 0% (0/18) for vWF. Considering the phenotypic features of cells expressing TEM-1, CHPs are thought to originate from immature vascular mural cells sharing their phenotype with myofibroblasts. NG-2 expression may be a phenotype of smooth muscle cells rather than pericytes in dogs. Copyright © 2012 Elsevier Ltd. All rights reserved.

Progesterone receptor expression during prostate cancer progression suggests a role of this receptor in stromal cell differentiation.

PubMed

Yu, Yue; Yang, Ou; Fazli, Ladan; Rennie, Paul S; Gleave, Martin E; Dong, Xuesen

2015-07-01

The progesterone receptor, like the androgen receptor, belongs to the steroid receptor superfamily. Our previous studies have reported that the PR is expressed specifically in prostate stroma. PR inhibits proliferation of, and regulates cytokine secretion by stromal cells. However, PR protein expression in cancer-associated stroma during prostate cancer progression has not been profiled. Since the phenotypes of prostate stromal cells change dynamically as tumors progress, whether the PR plays a role in regulating stromal cell differentiation needs to be investigated. Immunohistochemistry assays measured PR protein levels on human prostate tissue microarrays containing 367 tissue cores from benign prostate, prostate tumors with different Gleason scores, tumors under various durations of castration therapy, and tumors at the castration-resistant stage. Immunoblotting assays determined whether PR regulated the expression of alpha smooth muscle actin (α-SMA), vimentin, and fibroblast specific protein (FSP) in human prostate stromal cells. PR protein levels decreased in cancer-associated stroma when compared with that in benign prostate stroma. This reduction in PR expression was not correlated with Gleason scores. PR protein levels were elevated by castration therapy, but reduced to pre-castration levels when tumors progressed to the castration-resistant stage. Enhanced PR expression in human prostate stromal cells increased α-SMA, but decreased vimentin and FSP protein levels ligand-independently. These results suggest that PR plays an active role in regulating stromal cell phenotypes during prostate cancer progression. © 2015 Wiley Periodicals, Inc.

Curcumin suppresses transforming growth factor-β1-induced cardiac fibroblast differentiation via inhibition of Smad-2 and p38 MAPK signaling pathways

PubMed Central

LIU, HUZI; LIU, AIJUN; SHI, CHUNLI; LI, BAO

2016-01-01

The differentiation of cardiac fibroblasts (CFs) into myofibroblasts and the subsequent deposition of the extracellular matrix is associated with myocardial fibrosis following various types of myocardial injury. In the present study, the effect of curcumin, which is a pharmacologically-safe natural compound from the Curcuma longa herb, on transforming growth factor (TGF)-β1-induced CFs was investigated, and the underlying molecular mechanisms were examined. The expression levels of α-smooth muscle actin (SMA) stress fibers were investigated using western blotting and immunofluorescence in cultured neonatal rat CFs. Protein and mRNA expression levels of α-SMA and collagen type I (ColI) were determined by western blotting and reverse transcription-quantitative polymerase chain reaction. In addition, the activation of Smad2 and p38 was examined using western blotting. Curcumin, SB431542 (a TGF-βR-Smad2 inhibitor) and SB203580 (a p38 inhibitor) were used to inhibit the stimulation by TGF-β1. The results demonstrated that the TGF-β1-induced expression of α-SMA and ColI was suppressed by curcumin at the mRNA and protein levels, while SB431542 and SB203580 induced similar effects. Furthermore, phosphorylated Smad-2 and p38 were upregulated in TGF-β1-induced CFs, and these effects were substantially inhibited by curcumin administration. In conclusion, the results of the present study demonstrated that treatment with curcumin effectively suppresses TGF-β1-induced CF differentiation via Smad-2 and p38 signaling pathways. Thus, curcumin may be a potential therapeutic agent for the treatment of cardiac fibrosis. PMID:26998027

Protective Effects of Hydrogen-Rich Saline Against Lipopolysaccharide-Induced Alveolar Epithelial-to-Mesenchymal Transition and Pulmonary Fibrosis.

PubMed

Dong, Wen-Wen; Zhang, Yun-Qian; Zhu, Xiao-Yan; Mao, Yan-Fei; Sun, Xue-Jun; Liu, Yu-Jian; Jiang, Lai

2017-05-19

BACKGROUND Fibrotic change is one of the important reasons for the poor prognosis of patients with acute respiratory distress syndrome (ARDS). The present study investigated the effects of hydrogen-rich saline, a selective hydroxyl radical scavenger, on lipopolysaccharide (LPS)-induced pulmonary fibrosis. MATERIAL AND METHODS Male ICR mice were divided randomly into 5 groups: Control, LPS-treated plus vehicle treatment, and LPS-treated plus hydrogen-rich saline (2.5, 5, or 10 ml/kg) treatment. Twenty-eight days later, fibrosis was assessed by determination of collagen deposition, hydroxyproline, and type I collagen levels. Development of epithelial-to-mesenchymal transition (EMT) was identified by examining protein expressions of E-cadherin and α-smooth muscle actin (α-SMA). Transforming growth factor (TGF)-β1 content, total antioxidant capacity (T-AOC), malondialdehyde (MDA) content, catalase (CAT), and superoxide dismutase (SOD) activity were determined. RESULTS Mice exhibited increases in collagen deposition, hydroxyproline, type I collagen contents, and TGF-β1 production in lung tissues after LPS treatment. LPS-induced lung fibrosis was associated with increased expression of α-SMA, as well as decreased expression of E-cadherin. In addition, LPS treatment increased MDA levels but decreased T-AOC, CAT, and SOD activities in lung tissues, indicating that LPS induced pulmonary oxidative stress. Hydrogen-rich saline treatment at doses of 2.5, 5, or 10 ml/kg significantly attenuated LPS-induced pulmonary fibrosis. LPS-induced loss of E-cadherin in lung tissues was largely reversed, whereas the acquisition of α-SMA was dramatically decreased by hydrogen-rich saline treatment. In addition, hydrogen-rich saline treatment significantly attenuated LPS-induced oxidative stress. CONCLUSIONS Hydrogen-rich saline may protect against LPS-induced EMT and pulmonary fibrosis through suppressing oxidative stress.

Frequent β-catenin gene mutations in atypical polypoid adenomyoma of the uterus.

PubMed

Takahashi, Hiroyuki; Yoshida, Tsutomu; Matsumoto, Toshihide; Kameda, Yoichi; Takano, Yasuo; Tazo, Yuki; Inoue, Hisako; Saegusa, Makoto

2014-01-01

Atypical polypoid adenomyoma (APA) is an uncommon polypoid lesion of the uterus. To clarify the mechanism of its histogenesis, we examined the functional role of β-catenin, with reference to expression of p21(waf1), cyclin D1, cyclin E, CD10, and α-smooth muscle actin (SMA), as well as cell proliferation, in 7 lesions. In the epithelial components, expression of nuclear β-catenin, p21(waf1), and cyclin D1 was increased in a stepwise fashion from normal tissue through complex atypical hyperplasia and adenomyoma to APA lesions, particularly in squamous morular areas, whereas cell proliferation, as well as cyclin E expression, was significantly decreased in the latter. Similar findings were evident in the stromal lesions, with the exception of a case of nuclear β-catenin. In addition, coexpression of CD10 and α-SMA markers was observed in the stromal components in 3 APA cases, in line with the results of normal secretory endometrial and adenomyoma samples, suggesting that cells progress to myofibromatous cells in response to differentiation-promoting events. Finally, β-catenin gene (CTNNB1) mutations were detected in all APA cases, the single nucleotide substitutions being in the epithelial but not the stromal components. These findings suggest that activation of β-catenin signaling, probably secondary to the gene abnormalities, plays an important role in the formation of the complex epithelial architecture in APAs, leading to inhibition of cell proliferation through overexpression of p21(waf1). In contrast, changes in the stromal cell phenotype may occur through a shift from CD10 to α-SMA immunopositivity, independent of CTNNB1 status. © 2013.

Comparative marker analysis after isolation and culture of testicular cells from the immature marmoset.

PubMed

Albert, Silvia; Wistuba, Joachim; Eildermann, Katja; Ehmcke, Jens; Schlatt, Stefan; Gromoll, Joerg; Kossack, Nina

2012-01-01

The marmoset monkey is a valuable model in reproductive medicine. While previous studies have evaluated germ cell dynamics in the postnatal marmoset, the features of testicular somatic cells remain largely unknown. Therefore, the aim of this study was to establish marmoset-specific markers for Sertoli and peritubular cells (PTCs) and to compare protocols for the enrichment and culture of testicular cell types. Immunohistochemistry of Sertoli and PTC-specific markers - anti-müllerian hormone (AMH), vimentin (VIM), α-smooth muscle actin (SMA) - was performed and corresponding RNA expression profiles were established by quantitative PCR analysis (SOX9,AMH, FSHR,VIM, and SMA). For these analyses, testicular tissue from newborn (n = 4), 8-week-old (n = 4) and adult (n = 3) marmoset monkeys was used. Protocols for the enrichment and culture of testicular cell fractions from the 8-week-old marmoset monkeys (n = 3) were evaluated and cells were analyzed using germ cell- and somatic cell-specific markers. The expression of AMH, VIM and SMA reflects the proportion and differentiation status of Sertoli and PTCs at the RNA and the protein levels. While applied protocols did not support the propagation of germ cells in vitro, our analyses revealed that PTCs maintain their proliferative potential and constitute the dominant cell type after short- and long-term culture. Expression of functionally meaningful testicular somatic markers is similar in the human and the marmoset monkey, indicating that this primate can indeed be used as model for human testicular development. The PTC culture system established in this study will facilitate the identification of factors influencing male sex differentiation and spermatogenesis. Copyright © 2012 S. Karger AG, Basel.

Moesin as a key cytoskeleton regulator in corneal fibrosis.

PubMed

Zhu, Hong-Yuan; Yeo, Sia-Wey; Ng, Jennifer; Htoon, Hla Myint; Beuerman, R W

2013-04-01

: Corneal fibrosis is the third leading cause of blindness worldwide. α-Smooth muscle actin (SMA), a marker of fibrosis, is closely regulated through an intermediate group of submembrane molecules - cytoskeleton regulators. The purpose of this study was to elucidate the role of specific cytoskeleton regulators in a mouse model of corneal fibrosis. : A mouse model of corneal fibrosis was developed using anterior keratectomy (AK) and the topical application of transforming growth factor (TGF)-β1 (1 μg/ml). The RT² Profiler™ PCR Array for cytoskeleton regulators was used to assay changes in levels of specific members of this class of proteins. Moesin siRNA was delivered into the corneal stroma by iontophoresis in vivo. Transformation of the corneal keratocyte-to-myofibroblast in corneal fibrosis, as defined by the expression of α-SMA, was determined by Western blot. : After AK and topical application of TGF-β1, moesin was the most highly upregulated gene among 84 cytoskeleton regulator genes; iontophoresing moesin siRNA into the corneal stroma reduced the expression of α-SMA to 0.22-, 0.52-, and 0.31-fold of control at postoperative (PO) day 1, 3, and 5, respectively; also, upregulation of phospho-Smad 2 induced by TGF-β1 was reduced by moesin siRNA to 0.59-, 0.56-, and 0.31-fold of control and expression of phospho-Smad 3 was reduced to 0.58-, 0.53-, and 0.47-fold of control at the same PO days. : Moesin may be a potential drug target for inhibiting corneal fibrosis, and the details of moesin-related signaling pathways would be critical for understanding corneal fibrosis. Copyright © 2013 Elsevier Inc. All rights reserved.

Intestinal organoids: A model of intestinal fibrosis for evaluating anti-fibrotic drugs

PubMed Central

Rodansky, Eva S.; Johnson, Laura A.; Huang, Sha; Spence, Jason R.; Higgins, Peter D. R.

2016-01-01

Background & Aims Intestinal fibrosis is a critical complication of Crohn’s disease (CD). Current in vitro models of intestinal fibrosis cannot model the complex intestinal architecture, while in vivo rodent models do not fully recapitulate human disease and have limited utility for large-scale screening. Here, we exploit recent advances in stem cell derived human intestinal organoids (HIOs) as a new human model of fibrosis in CD. Methods Human pluripotent stem cells were differentiated into HIOs. We identified myofibroblasts, the key effector cells of fibrosis, by immunofluorescence staining for alpha-smooth muscle actin (αSMA), vimentin, and desmin. We examined the fibrogenic response of HIOs by treatment with transforming growth factor beta (TGFβ) in the presence or absence of the anti-fibrotic drug spironolactone. Fibrotic response was assayed by expression of fibrogenic genes (COL1A1 (collagen, type I, alpha 1), ACTA2 (alpha smooth muscle actin), FN1 (fibronectin 1), MYLK (myosin light chain kinase), and MKL1 (megakaryoblastic leukemia (translocation) 1)) and proteins (αSMA). Results Immunofluorescent staining of organoids identified a population of myofibroblasts within the HIO mesenchyme. TGFβ stimulation of HIOs produced a dose-dependent pro-fibrotic response. Spironolactone treatment blocked the fibrogenic response of HIOs to TGFβ. Conclusions HIOs contain myofibroblasts and respond to a pro-fibrotic stimulus in a manner that is consistent with isolated human myofibroblasts. HIOs are a promising model system that might bridge the gap between current in vitro and in vivo models of intestinal fibrosis in IBD. PMID:25828392

Serum levels of alpha-smooth muscle actin and c-Met as biomarkers of the degree of severity of Henoch-Schonlein purpura nephritis.

PubMed

Zhang, Lei; Han, Changsong; Sun, Chuanhui; Meng, Hongxue; Ye, Fei; Na, Shiping; Chen, Fulai; Zhang, Duo; Jin, Xiaoming

2013-01-01

Approximately 40% of patients with Henoch-Schonlein purpura (HSP) develop Henoch-Schonlein purpura nephritis (HSPN) after 4 to 6 weeks of subcutaneous hemorrhaging. Immunoglobulin-A nephropathy (IgAN) and HSPN have numerous similarities, which can cause difficulty in correctly diagnosing the disorder during a differential diagnosis. The pathogenesis of the 2 diseases is not clear. We enrolled 137 patients with HSPN, 107 patients with IgAN, and 28 healthy (control) patients in our study. The levels of alpha-smooth muscle actin (α-SMA), c-Met, and Gal-deficient IgA1 (Gd-IgA1) in the 3 patient groups were determined and compared. The α-SMA, c-Met, and Gd-IgA1 levels and the clinical data from the patients with HSPN were analyzed for any correlations. The α-SMA and c-Met levels of the HSPN group were significantly higher than those of the IgAN and healthy control groups (P < 0.01). The Gd-IgA1 levels of the HSPN and IgAN groups were significantly different from the Gd-IgA1 level of the healthy control group (P < 0.01). The α-SMA levels of the HSPN group were positively correlated with blood urea nitrogen levels, serum creatinine levels, hematuria index, and proteinuria levels (P < 0.01). The c-Met levels of the HSPN group were positively correlated with the blood urea nitrogen and serum creatinine levels (P < 0.01). There were no significant differences among the α-SMA, c-Met, and Gd-IgA1 levels or the clinical data for the child and adult patients with HSPN. The serum levels of α-SMA and c-Met in patients with HSPN may be associated with the degree of disease severity. Gd-IgA1 is involved in the common immunologic pathogenesis of HSPN and IgAN. Copyright © 2013 Mosby, Inc. All rights reserved.

Immunohistochemical expression of matrix metalloproteinase-1, matrix metalloproteinase-2 and matrix metalloproteinase-9, myofibroblasts and Ki-67 in actinic cheilitis and lip squamous cell carcinoma.

PubMed

Bianco, Bianca C; Scotti, Fernanda M; Vieira, Daniella S C; Biz, Michelle T; Castro, Renata G; Modolo, Filipe

2015-10-01

Matrix metalloproteinases (MMPs), myofibroblasts (MFs) and epithelial proliferation have key roles in neoplastic progression. In this study immunoexpression of MMP-1, MMP-2 and MMP-9, presence of MFs and the epithelial proliferation index were investigated in actinic cheilitis (AC), lip squamous cell carcinoma (LSCC) and mucocele (MUC). Thirty cases of AC, thirty cases of LSCC and twenty cases of MUC were selected for immunohistochemical investigation of the proteins MMP-1, MMP-2, MMP-9, α-smooth muscle actin (α-SMA) and Ki-67. The MMP-1 expression in the epithelial component was higher in the AC than the MUC and LSCC. In the connective tissue, the expression was higher in the LSCC. MMP-2 showed lower epithelial and stromal immunostaining in the LSCC when compared to the AC and MUC. The epithelial staining for MMP-9 was higher in the AC when compared to the LSCC. However, in the connective tissue, the expression was lower in the AC compared to other lesions. The cell proliferation rate was increased in proportion to the severity of dysplasia in the AC, while in the LSCC it was higher in well-differentiated lesions compared to moderately differentiated. There were no statistically significant differences in number of MFs present in the lesions studied. The results suggest that MMPs could affect the biological behaviour of ACs and LSCCs inasmuch as they could participate in the development and progression from premalignant lesions to malignant lesions. © 2015 The Authors. International Journal of Experimental Pathology © 2015 International Journal of Experimental Pathology.

Markers for human brain pericytes and smooth muscle cells.

PubMed

Smyth, Leon C D; Rustenhoven, Justin; Scotter, Emma L; Schweder, Patrick; Faull, Richard L M; Park, Thomas I H; Dragunow, Mike

2018-06-07

Brain pericytes and vascular smooth muscle cells (vSMCs) are a critical component of the neurovascular unit and are important in regulating cerebral blood flow and blood-brain barrier integrity. Identification of subtypes of mural cells in tissue and in vitro is important to any study of their function, therefore we identified distinct mural cell morphologies in neurologically normal post-mortem human brain. Further, the distribution of mural cell markers platelet-derived growth factor receptor-β (PDGFRβ), α-smooth muscle actin (αSMA), CD13, neural/glial antigen-2 (NG2), CD146 and desmin was examined. We determined that PDGFRβ, NG2, CD13, and CD146 were expressed in capillary-associated pericytes. NG2, and CD13 were also present on vSMCs in large vessels, however abundant CD146 and desmin staining was also detected in vSMCs on large vessels, co-labelling with αSMA. To determine whether cultures recapitulated observations from tissue, primary human brain pericytes derived from neurologically normal autopsies were analysed for the presence of pericyte markers by immunocytochemistry, western blotting and qPCR. The proteins observed in brain pericytes in tissue (PDGFRβ, αSMA, desmin, CD146, CD13, and NG2) were present in vitro, validating a panel of proteins that can be used to label brain pericytes and vSMCs in tissue and in vitro. Finally, we showed that the proteins CD146 and desmin that are expressed on large vessels in situ, are also selective markers of a smooth muscle cell phenotype in vitro. Copyright © 2018 Elsevier B.V. All rights reserved.

The Favorable Effect of Mesenchymal Stem Cell Treatment on the Antioxidant Protective Mechanism in the Corneal Epithelium and Renewal of Corneal Optical Properties Changed after Alkali Burns.

PubMed

Cejka, Cestmir; Holan, Vladimir; Trosan, Peter; Zajicova, Alena; Javorkova, Eliska; Cejkova, Jitka

2016-01-01

The aim of this study was to examine whether mesenchymal stem cells (MSCs) and/or corneal limbal epithelial stem cells (LSCs) influence restoration of an antioxidant protective mechanism in the corneal epithelium and renewal of corneal optical properties changed after alkali burns. The injured rabbit corneas (with 0.25 N NaOH) were untreated or treated with nanofiber scaffolds free of stem cells, with nanofiber scaffolds seeded with bone marrow MSCs (BM-MSCs), with adipose tissue MSCs (Ad-MSCs), or with LSCs. On day 15 following the injury, after BM-MSCs or LSCs nanofiber treatment (less after Ad-MSCs treatment) the expression of antioxidant enzymes was restored in the regenerated corneal epithelium and the expressions of matrix metalloproteinase 9 (MMP9), inducible nitric oxide synthase (iNOS), α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), and vascular endothelial factor (VEGF) were low. The central corneal thickness (taken as an index of corneal hydration) increased after the injury and returned to levels before the injury. In injured untreated corneas the epithelium was absent and numerous cells revealed the expressions of iNOS, MMP9, α-SMA, TGF-β1, and VEGF. In conclusion, stem cell treatment accelerated regeneration of the corneal epithelium, restored the antioxidant protective mechanism, and renewed corneal optical properties.

The interplay between hepatic stellate cells and hepatocytes in an in vitro model of NASH.

PubMed

Barbero-Becerra, Varenka J; Giraudi, Pablo J; Chávez-Tapia, Norberto C; Uribe, Misael; Tiribelli, Claudio; Rosso, Natalia

2015-10-01

A complex interplay exists between hepatocytes and hepatic stellate cells (HSC) in hepatic fibrogenesis. The activation of HSCs after liver injury leads to production of extracellular matrix (ECM). Co-culture models could be useful to mimic the liver microenvironment. This study evaluates the effect of free fatty acids (FFA) on HSC cells and the interplay with hepatocytes via both soluble-mediator and cell-cell contact. The human hepatocyte cell line (HuH7) and HSC cells (LX2) were exposed to FFA for 24 h in 3 different experimental set-ups: (A) monoculture of HSC; (B) Transwell® system (effect of soluble mediators); and (C) Simultaneous Co-Culture (SCC) (cell-to-cell connections). Intracellular FFA accumulation was assessed qualitatively (microscopy) and quantitatively (flow cytometry); the activation of HSC (alpha smooth muscle actin, α-SMA) expression of ECM components were quantified by RT-PCR. FFA exposure induces intracellular fat accumulation in all the experimental set-up but the expression of α-SMA was significantly increased only in SCC. On the contrary, the expression of ECM was substantially decreased in the transwell® system. The HSC activation is independent of FFA accumulation but requires cell-to-cell interaction with hepatocyte. On the contrary, the gene regulation of ECM components seems to occur through paracrine mediators. Copyright © 2015 Elsevier Ltd. All rights reserved.

Prolonged use of alendronate alters the biology of cranial repair in estrogen-deficient rats' associated simultaneous immunohistochemical expression of TGF-β1+, α-ER+, and BMPR1B.

PubMed

Giovanini, Allan Fernando; de Sousa Passoni, Giuliene Nunes; Göhringer, Isabella; Deliberador, Tatiana Miranda; Zielak, João Cesar; Storrer, Carmem Lucia Muller; Costa-Casagrande, Thais Andrade; Scariot, Rafaela

2018-06-01

TGF-β1 is a cytokine that may induce both osteoneogenesis through Runx-2 or fibrosis via the transcription of α-smooth muscle actin (α-SMA). Because it has been previously known that alendronate increases the level of TGF-β1 and that under the usual condition of bone metabolism the estrogen may prevent the fibrotic effect of TGF-β1, the aim of this study was to evaluate if alendronate alters the cellular differentiation process post calvarial surgery in estrogen-deficient specimens. A transosseous defect that was 5 mm in diameter was created on the calvarium of each of 32 female rats with previous ovarian-salpingo-oophorectomy. All defects were treated with autografts, and 16 rats received the administration of 1 mg/kg of alendronate three times a week until euthanasia on the 15th and 60th day post surgery. Histomorphometric and immunohistochemical analyses of the expression of TGF-β1, estrogen receptor alpha nuclear (α-ER), α-SMA, BMPR1B, and Runx-2 were performed, and ELISA was used to measure the level of estrogen. All animals demonstrated low levels of estrogen post ovarian-salpingo-oophorectomy. The histological results demonstrated larger bone matrix deposition in specimens treated with alendronate on the 15th day post surgery. The result was associated with a higher co-expression of TGF-β1, BMPR1B, and Runx-2 when compared with the control group. In addition, on the 60th day post surgery, the increase of bone matrix deposition from 15th to 60th day was discrete in specimens treated with alendronate compared with the control group. This result coincided with the intense simultaneous expression of TGF-β1, α-ER, and α-SMA, whereas the expression of BMPR1B and Runx-2 decreased. The prolonged administration of alendronate altered the cranial repair in ovarian-salpingo-oophorectomized specimens due to the simultaneous occurrence of low estrogen and the presence of TGF-β1+/α-ER+ inducing the presence of α-SMA + , whereas BMPR1B and Runx-2 were suppressed. The prolonged administration of alendronate alters osteoneogenesis and induces an unusual microenvironment in the bone that seems to imitate the physiological tissue damage that culminates in the loss of the functional layer of endometrium.

Bilateral crosstalk of rho- and extracellular-signal-regulated-kinase (ERK) pathways is confined to an unidirectional mode in spinal muscular atrophy (SMA).

PubMed

Hensel, Niko; Stockbrügger, Inga; Rademacher, Sebastian; Broughton, Natasha; Brinkmann, Hella; Grothe, Claudia; Claus, Peter

2014-03-01

Rho-kinase (ROCK) as well as extracellular signal regulated kinase (ERK) control actin cytoskeletal organization thereby regulating dynamic changes of cellular morphology. In neurons, motility processes such as axonal guidance and neurite outgrowth demand a fine regulation of upstream pathways. Here we demonstrate a bilateral ROCK-ERK information flow in neurons. This process is shifted towards an unidirectional crosstalk in a model of the neurodegenerative disease Spinal Muscular Atrophy (SMA), ultimately leading to neurite outgrowth dysregulations. As both pathways are of therapeutic relevance for SMA, our results argue for a combinatorial ROCK/ERK-targeting as a future treatment strategy. Copyright © 2013 Elsevier Inc. All rights reserved.

Mechanisms of smooth muscle antibody production: a clinical study in children with infections, haemolytic syndromes, and idiopathic thrombocytopenic purpura.

PubMed Central

Kanakoudi-Tsakalidis, F; Cassimos, C; Papastavrou-Mavroudi, T; Akoglu, T; Toh, B H; Yildiz, A; Osung, O; Holborow, E J; Sotelo, J

1979-01-01

Sera from 530 children suffering from various diseases and from 64 controls were tested for smooth muscle autoantibodies (SMA) by indirect immunofluorescence. A high incidence of SMA (51-86%) was found in patients with viral and bacterial infections (viral hepatitis, infectious mononucleosis, measles, mumps, chickenpox, typhoid fever, and brucellosis), independently of liver invovlvement, and in patients with acute haemolytic anaemia due to G-6-PD deficiency (48%). By contrast, the incidence of SMA from patients with beta-thalassaemia major and idiopathic thrombocytopenic purpura was no higher than in the controls. The discrepancy in incidence in haemolytic anaemias due to different causes may reflect the effect of endogenous and extrinsic agents. In the viral infections, SMA were mainly of the IgM class and gave an 'SMA-V' staining pattern. In bacterial infections (typhoid fever and brucellosis), SMA were either IgG only or IgM and IgG, and the staining pattern was also mainly 'SMA-V'. In infections which affect or may affect the liver (viral hepatitis, infectious mononucleosis, typhoid fever, and brucellosis), SMA was present at high titres (1:80-1:320), whereas in infections not affecting the liver (measles, mumps, and chickenpox) the titres were lower (less than or equal to 1:80). In most patients SMA occurred transiently and without apparent pathogenetic significance. The antigen against which infection-induced SMA is directed is not actin; its nature has yet to be identified. PMID:575362

Renalase Protects against Renal Fibrosis by Inhibiting the Activation of the ERK Signaling Pathways

PubMed Central

Wu, Yiru; Wang, Liyan; Deng, Dai; Zhang, Qidong; Liu, Wenhu

2017-01-01

Renal interstitial fibrosis is a common pathway for the progression of chronic kidney disease (CKD) to end-stage renal disease. Renalase, acting as a signaling molecule, has been reported to have cardiovascular and renal protective effects. However, its role in renal fibrosis remains unknown. In this study, we evaluated the therapeutic efficacy of renalase in rats with complete unilateral ureteral obstruction (UUO) and examined the inhibitory effects of renalase on transforming growth factor-β1 (TGF-β1)-induced epithelial–mesenchymal transition (EMT) in human proximal renal tubular epithelial (HK-2) cells. We found that in the UUO model, the expression of renalase was markedly downregulated and adenoviral-mediated expression of renalase significantly attenuated renal interstitial fibrosis, as evidenced by the maintenance of E-cadherin expression and suppressed expression of α-smooth muscle actin (α-SMA), fibronectin and collagen-I. In vitro, renalase inhibited TGF-β1-mediated upregulation of α-SMA and downregulation of E-cadherin. Increased levels of Phospho-extracellular regulated protein kinases (p-ERK1/2) in TGF-β1-stimulated cells were reversed by renalase cotreatment. When ERK1 was overexpressed, the inhibition of TGF-β1-induced EMT and fibrosis mediated by renalase was attenuated. Our study provides the first evidence that renalase can ameliorate renal interstitial fibrosis by suppression of tubular EMT through inhibition of the ERK pathway. These results suggest that renalase has potential renoprotective effects in renal interstitial fibrosis and may be an effective agent for slowing CKD progression. PMID:28448446

[Influence of bear bile on rat hepatocarcinoma induced by diethylnitrosamine].

PubMed

Zhou, Jian-Yin; Yin, Zhen-Yu; Wang, Sheng-Yu; Yan, Jiang-Hua; Zhao, Yi-Lin; Wu, Duan; Liu, Zheng-Jin; Zhang, Sheng; Wang, Xiao-Min

2012-11-01

To investigate the influence of bear bile on rat hepatocarcinoma induced by diethylnitrosamine (DEN), a total of 40 rats were randomly divided into 4 groups: normal control group, model group, and two bear bile treatment groups. The rat liver cancer model was induced by breeding with water containing 100 mg x L(-1) DEN for 14 weeks. The rats of the bear bile groups received bear bile powder (200 or 400 mg x kg(-1)) orally 5 times per week for 18 weeks. The general condition and the body weight of rats were examined every day. After 18 weeks the activities of serum alanine transaminase (ALT), aspartate transaminase (AST) and total bilirubin (TBIL) were detected. Meanwhile, the pathological changes of liver tissues were observed after H&E staining. The expression of proliferative cell nuclear antigen (PCNA) and a-smooth muscle actin (alpha-SMA) in liver tissue were detected by immunohistochemical method. After 4 weeks the body weights of rats in normal group were significantly more than that in other groups (P < 0.05); and that in the two bile groups was significantly more than that in the model group. Compared with normal group, the level of serum glutamic-pyruvic transaminase and total bilirubin increased significantly in other groups; compared with model group, these two indexes decreased significantly in two bile groups. Hepatocellular carcinoma occurred in all rats except for normal group; there were classic cirrhosis and cancer in model group while there were mild cirrhosis and high differentiation in two bile groups. There were almost no expressions of PCNA and alpha-SMA in normal group while there were high expressions in model group; the two bile groups had some expressions but were inferior to the model group, and alpha-SMA reduced markedly. It indicated that bear bile restrained the development of liver cancer during DEN inducing rat hepatocarcinoma, which may be related to its depressing hepatic stellate cell activation and relieving hepatic lesion and cirrhosis.

Subsets of telocytes: Myocardial telocytes.

PubMed

Rusu, M C; Hostiuc, S; Vrapciu, A D; Mogoantă, L; Mănoiu, V S; Grigoriu, F

2017-01-01

Telocytes (TCs) are morphologically defined as small-sized cells with long, thin, moniliform processes called telopodes (Tps). Numerous papers imply that TCs are a distinctive cell type, and that transmission electron microscopy (TEM) is the gold standard tool for their identification. We aimed to reproduce previous studies on myocardial TCs to check their validity. For this purpose we performed an immunohistochemical study on human cardiac samples from six autopsied donor cadavers, using antibodies against CD10, CD31, CD34, CD146, Ki67, alpha-smooth muscle actin (α-SMA), Platelet-Derived Growth Factor Receptor-alpha (PDGFRα) and laminin. Additionally we performed a TEM study on cardiac samples from three human autopsied donor cadavers and five adult Sprague-Dawley rats. We found endothelial cells (ECs), cords, and filopodia-projecting endothelial tip cells (ETCs) that expressed CD10, CD31, CD34, CD146, and PDGFR-α. Often, endothelial cells closely neighbored the sarcolemmal basal laminae. Endothelial progenitor cells, as well as nascent capillaries, were CD31+/CD34+. Proliferative endothelial cells expressed Ki67. In larger vessels we found pericytes that expressed CD146 and α-SMA; scarce α-SMA-expressing spindle-shaped cells lining cardiomyocytes were suggestive of a pericytic role in angiogenic sprout guidance. The TEM study showed that endothelial tubes are almost exclusively found in the narrow myocardial interstitia. ECs that built them up appeared identical to the cells that previous TEM studies have suggested to be myocardial telocytes. A subset of stromal cells with TC-like phenotype and telopodes-like processes actually seem to configure blood vessels, and therefore belong to the endothelial lineage. This study shows that data presented in previous studies on myocardial telocytes is not enough to allow the reproducibility of the results. At least a subset of cells considered to be TCs might belong to the endothelial lineage. Copyright © 2016 Elsevier GmbH. All rights reserved.

Suberoylanilide hydroxamic acid (vorinostat): its role on equine corneal fibrosis and matrix metalloproteinase activity.

PubMed

Donnelly, Kevin S; Giuliano, Elizabeth A; Sharm, Ajay; Mohan, Rajiv R

2014-07-01

To explore the effect of suberoylanilide hydroxamic acid (SAHA) (i) on corneal fibroblast differentiation, morphology, and viability; and (ii) on the expression levels of matrix metalloproteinases (MMPs) 2 and 9 using an in vitro model of equine corneal fibrosis. Healthy donor corneas were used to generate primary cultures of equine corneal fibroblasts. The fibroblasts were exposed to 5 ng/mL TGFβ1 to induce myofibroblast formation. The cultures were treated with either 5 μm or 10 μm SAHA for 72 h in the presence of TGFβ1. Real-time PCR and immunocytochemistry were used to determine the antifibrotic efficacy of SAHA by quantifying α-smooth muscle actin (αSMA), a marker of myofibroblast formation and fibrosis. Real-time PCR was used to determine the effects of SAHA on MMP2 and MMP9 expression. Cytotoxicity of SAHA was evaluated with phase contrast microscopy and trypan blue exclusion assays. Suberoylanilide hydroxamic acid (SAHA) significantly attenuated TGFβ1-induced differentiation of equine fibroblasts to myofibroblasts as indicated by 3- to 3.5-fold (P < 0.001) decrease in αSMA mRNA and 86-88% (P < 0.001) decrease in αSMA+ immunocytochemical staining. SAHA treatment also resulted in 4.5- to 5.5-fold (P < 0.01) decrease in MMP9 expression. A dose-dependent bimodal effect of SAHA on MMP2 expression was noted (3.5-fold increase with 5 μm dose; 0.5-fold decrease with 10 μm dose). No change in fibroblast viability was observed with a 5 μm SAHA dose, whereas a 10 μm dose resulted in a moderate 17% decrease in cell viability. Suberoylanilide hydroxamic acid (SAHA) can effectively inhibit TGFβ-induced differentiation of equine corneal fibroblasts to myofibroblasts and modulates MMP production in vitro. © 2013 American College of Veterinary Ophthalmologists.

Inhibition of α-SMA by the Ectodomain of FGFR2c Attenuates Lung Fibrosis

PubMed Central

Ju, Wang; Zhihong, Yu; Zhiyou, Zhou; Qin, Huang; Dingding, Wang; Li, Sun; Baowei, Zhu; Xing, Wei; Ying, He; An, Hong

2012-01-01

The soluble ectodomain of fibroblast growth factor receptor-IIIc (sFGFR2c) is able to bind to fibroblast growth factor (FGF) ligands and block the activation of the FGF-signaling pathway. In this study, sFGFR2c inhibited lung fibrosis dramatically in vitro and in vivo. The upregulation of α-smooth muscle actin (α-SMA) in fibroblasts by transforming growth factor-β1 (TGF-β1) is an important step in the process of lung fibrosis, in which FGF-2, released by TGF-β1, is involved. sFGFR2c inhibited α-SMA induction by TGF-β1 via both the extracellular signal-regulated kinase 1/2 (ERK1/2) and Smad3 pathways in primary mouse lung fibroblasts and the proliferation of mouse lung fibroblasts. In a mouse model of bleomycin (BLM)-induced lung fibrosis, mice were treated with sFGFR2c from d 3 or d 10 to 31 after BLM administration. Then we used hematoxylin and eosin staining, Masson staining and immunohistochemical staining to evaluate the inhibitory effects of sFGFR2c on lung fibrosis. The treatment with sFGFR2c resulted in significant attenuation of the lung fibrosis score and collagen deposition. The expression levels of α-SMA, p-FGFRs, p-ERK1/2 and p-Smad3 in the lungs of sFGFR2c-treated mice were markedly lower. sFGFR2c may have potential for the treatment of lung fibrosis as an FGF-2 antagonist. PMID:22451267

Antifibrotic effects of pirfenidone on Tenon's fibroblasts in glaucomatous eyes: comparison with mitomycin C and 5-fluorouracil.

PubMed

Na, Jung Hwa; Sung, Kyung Rim; Shin, Jin A; Moon, Jung Il

2015-09-01

The purpose of this study was to evaluate the antifibrotic effects of pirfenidone (PFD) on primary cultured human Tenon's fibroblasts (HTFs) from primary open-angle glaucoma (POAG) eyes, compared to mitomicin C (MMC) and 5-fluorouracil (5-FU). Samples of human Tenon's capsule were obtained during respective surgeries from three groups of patients: patients with cataract (CAT group), patients with POAG who underwent glaucoma filtration surgery (GFS) (POAG1 group), and patients with POAG who underwent GFS due to failed bleb of previous GFS (POAG2 group). Cell toxicity, cell migration, and the expression level of α-smooth muscle actin (α-SMA) protein were evaluated in primary cultured HTFs from the three patient groups after treatment (PFD, MMC, or 5-FU). Overall, cell viability after PFD treatment was higher compared to MMC treatment (82.3 ± 5.1 % vs 56.7 ± 3.8 %; p = 0.001) and comparable to 5-FU treatment (82.3 ± 5.1 % vs 85.7 ± 10.7 %, p = 0.214) at the same concentration (0.4 mg/ml). Both 0.3 mg/ml PFD and 0.1 mg/ml MMC inhibited cell migration compared to control (without treatment) cells (p = 0.014 and 0.005, respectively), while 0.2 mg/ml 5-FU showed the highest degree of cell migration among the three agents in the POAG1 group (PFD vs MMC vs 5-FU; 29.5 ± 2.1 % vs 34.5 ± 0.7 % vs 76.0 ± 8.5 %, PFD vs MMC; p = 1.000, PFD vs 5-FU; p = 0.008, MMC vs 5-FU; p = 0.011). PFD (0.1 or 0.3 mg/ml) and MMC (0.05 and 0.1 mg/ml) treatment significantly reduced the protein expression level of α-SMA in the POAG 1 group (all p < 0.05), and the α-SMA protein level following treatment with 0.3 mg/ml PFD was lower than that of 0.1 mg/ml MMC (p = 0.040). PFD showed less cytotoxicity compared to MMC. PFD and MMC inhibited cell migration and reduced α-SMA protein expression levels, while 5-FU showed neither inhibition of cell migration nor reduction in α-SMA expression level. These findings indicate PFD as a potential adjunctive antifibrotic agent to prevent bleb failure during GFS.

Inhibitory effects of trehalose on fibroblast proliferation and implications for ocular surgery.

PubMed

Takeuchi, Kimio; Nakazawa, Mitsuru; Ebina, Yuichi; Sato, Kota; Metoki, Tomomi; Miyagawa, Yasuhiro; Ito, Tadashi

2010-11-01

Trehalose is a disaccharide which plays an important role in preserving cells from completely dehydrated circumstances. In this study, we investigated effects of trehalose on proliferative activity of fibroblasts and epithelial cells both in vitro and in vivo. As in vitro assessment, normal human dermal fibroblasts and normal human epidermal keratinocytes were cultured in media containing various concentrations of trehalose. Growth activities of cells were evaluated with MTT assay and diff-quick™ staining. Expressions of vimentin and α smooth muscle actin (α-SMA) changed by trehalose were semiquantitatively measured by Western blot. As an in vivo study, 5% or 10% trehalose was topically instilled onto rabbit eyes after simple conjunctival incision or trabeculectomy. Condition of the surgical wound was evaluated by morphologically and immunohistochemically using isolectin B4 and antibodies specific for vimentin and α-SMA. Intraocular pressures (IOPs) after trabeculectomy were compared between eyes treated with trehalose and 0.04% mitomycin C (MMC). Results obtained by in vitro experiments showed that growth activities of cultured fibroblasts and keratinocytes were inhibited by trehalose in a dose-dependent manner. Fibroblasts were strongly inhibited by trehalose concentrations ≧ 5% of trehalose, whereas keratinocytes were less inhibited compared to fibroblasts. Expressions of vimentin and α-SMA were reduced by trehalose. With in vivo experiments, postoperative application of trehalose resulted in less firm adhesion between conjunctiva and sclera compared to controls. Immunohistochemical studies showed reduced staining of isolectin B4, vimentin and α-SMA in conjunctival wounds treated by topical trehalose. Also, after trabeculectomy, IOP remained in a low range during instillation of topical trehalose solution. We concluded that trehalose has inhibitory effects on proliferation of fibroblasts and vascular tissues, partially due to inhibition of transformation of fibroblasts into myofibroblasts in wound tissues. The present results imply that trehalose can be a potential agent for preventing postoperative fibrous scar formation after ocular surgery such as glaucoma filtration surgery. Copyright © 2010 Elsevier Ltd. All rights reserved.

Prophylactic role of coenzyme Q10 and Cynara scolymus L on doxorubicin-induced toxicity in rats: Biochemical and immunohistochemical study.

PubMed

Mustafa, Hesham N; El Awdan, Sally A; Hegazy, Gehan A; Abdel Jaleel, Gehad A

2015-01-01

The study aims to evaluate the protective effects of coenzyme Q10 (CoQ10) and Cynara scolymus L (CS) on doxorubicin (dox)-induced toxicity. Sixty male rats were divided into six groups. Group 1 as a control. Group 2 received dox (10 mg/kg) intraperitoneally. Group 3 received CoQ10 (200 mg/kg). Group 4 received CS (500 mg/kg). Group 5 received CoQ10 (200 mg/kg) and dox (10 mg/kg). Group 6 received CS (500 mg/kg) and dox (10 mg/kg). The rats were then evaluated biochemically and immunohistochemically. Dox produced a significant deterioration of hepatic and renal functional parameters. Moreover, an upsurge of oxidative stress and nitrosative stress markers. The expression of alpha-smooth muscle actin (α-SMA) was increased and proliferating cell nuclear antigen (PCNA) expression was decreased. Administration of CoQ10 and CS resulted in a significant improvement of hepatic and renal functional parameters, and an improvement of both α-SMA and PCNA. It is concluded that pretreatment with CoQ10 and CS is associated with up-regulation of favorable protective enzymes and down-regulation of oxidative stress. That can be advised as a supplement to dox-treated patients.

Prophylactic role of coenzyme Q10 and Cynara scolymus L on doxorubicin-induced toxicity in rats: Biochemical and immunohistochemical study

PubMed Central

Mustafa, Hesham N.; El Awdan, Sally A.; Hegazy, Gehan A.; Abdel Jaleel, Gehad A.

2015-01-01

Objective: The study aims to evaluate the protective effects of coenzyme Q10 (CoQ10) and Cynara scolymus L (CS) on doxorubicin (dox)-induced toxicity. Materials and Methods: Sixty male rats were divided into six groups. Group 1 as a control. Group 2 received dox (10 mg/kg) intraperitoneally. Group 3 received CoQ10 (200 mg/kg). Group 4 received CS (500 mg/kg). Group 5 received CoQ10 (200 mg/kg) and dox (10 mg/kg). Group 6 received CS (500 mg/kg) and dox (10 mg/kg). The rats were then evaluated biochemically and immunohistochemically. Results: Dox produced a significant deterioration of hepatic and renal functional parameters. Moreover, an upsurge of oxidative stress and nitrosative stress markers. The expression of alpha-smooth muscle actin (α-SMA) was increased and proliferating cell nuclear antigen (PCNA) expression was decreased. Administration of CoQ10 and CS resulted in a significant improvement of hepatic and renal functional parameters, and an improvement of both α-SMA and PCNA. Conclusion: It is concluded that pretreatment with CoQ10 and CS is associated with up-regulation of favorable protective enzymes and down-regulation of oxidative stress. That can be advised as a supplement to dox-treated patients. PMID:26729958

Evidence for pleural epithelial-mesenchymal transition in murine compensatory lung growth

PubMed Central

Ysasi, Alexandra B.; Wagner, Willi L.; Valenzuela, Cristian D.; Kienzle, Arne; Servais, Andrew B.; Bennett, Robert D.; Tsuda, Akira; Ackermann, Maximilian; Mentzer, Steven J.

2017-01-01

In many mammals, including rodents and humans, removal of one lung results in the compensatory growth of the remaining lung; however, the mechanism of compensatory lung growth is unknown. Here, we investigated the changes in morphology and phenotype of pleural cells after pneumonectomy. Between days 1 and 3 after pneumonectomy, cells expressing α-smooth muscle actin (SMA), a cytoplasmic marker of myofibroblasts, were significantly increased in the pleura compared to surgical controls (p < .01). Scanning electron microscopy of the pleural surface 3 days post-pneumonectomy demonstrated regions of the pleura with morphologic features consistent with epithelial-mesenchymal transition (EMT); namely, cells with disrupted intercellular junctions and an acquired mesenchymal (rounded and fusiform) morphotype. To detect the migration of the transitional pleural cells into the lung, a biotin tracer was used to label the pleural mesothelial cells at the time of surgery. By post-operative day 3, image cytometry of post-pneumonectomy subpleural alveoli demonstrated a 40-fold increase in biotin+ cells relative to pneumonectomy-plus-plombage controls (p < .01). Suggesting a similar origin in space and time, the distribution of cells expressing biotin, SMA, or vimentin demonstrated a strong spatial autocorrelation in the subpleural lung (p < .001). We conclude that post-pneumonectomy compensatory lung growth involves EMT with the migration of transitional mesothelial cells into subpleural alveoli. PMID:28542402

Glutamate control of pulpal blood flow in the incisor dental pulp of the rat.

PubMed

Zerari-Mailly, Fouzia; Braud, Adeline; Davido, Nicolas; Touré, Babacar; Azérad, Jean; Boucher, Yves

2012-10-01

Glutamate is present in primary sensory afferents innervating the dental pulp and is known to exert vasoactive effects. The aims of this study were (i) to assess pulpal blood flow (PBF) after glutamate infusion in the dental pulp and (ii) to observe the distribution of glutamatergic nerve fibers expressing the vesicular transporters of glutamate (VGluT). The PBF was monitored with laser Doppler flowmetry before and after glutamate (0.5 M) infusion in the dental pulp vs. saline infusion. Immunochemistry for VGluT1, 2, and 3 was performed in addition to immunochemistry for the vascular and neuronal markers smooth-muscle actin (SMA), isolectin B4 (IB4), and calcitonin gene-related peptide (CGRP). Glutamate infusion resulted in a PBF increase that lasted for 60 s. Positive immunolabeling was observed for the three glutamate transporters, but was more pronounced for VGluT3. Moreover, VGluT3 immunoreactivity was observed within nerve fibers entering the dental pulp and terminating at the periphery and at the vicinity of odontoblasts. Also, VGluT3 was colocalized with the vascular marker SMA, and in some nerve fibers with IB4, but not with CGRP. This study provides support for a control of dental pulp microcirculation by neurons expressing VGluT3. © 2012 Eur J Oral Sci.

Withaferin A effectively targets soluble vimentin in the glaucoma filtration surgical model of fibrosis.

PubMed

Bargagna-Mohan, Paola; Deokule, Sunil P; Thompson, Kyle; Wizeman, John; Srinivasan, Cidambi; Vooturi, Sunil; Kompella, Uday B; Mohan, Royce

2013-01-01

Withaferin A (WFA) is a natural product that binds to soluble forms of the type III intermediate filament (IF) vimentin. Currently, it is unknown under what pathophysiological contexts vimentin is druggable, as cytoskeltal vimentin-IFs are abundantly expressed. To investigate druggability of vimentin, we exploited rabbit Tenon's capsule fibroblast (RbTCF) cell cultures and the rabbit glaucoma filtration surgical (GFS) model of fibrosis. WFA potently caused G₀/G₁ cell cycle inhibition (IC₅₀ 25 nM) in RbTCFs, downregulating ubiquitin E3 ligase skp2 and inducing p27(Kip1) expression. Transforming growth factor (TGF)-ß-induced myofibroblast transformation caused development of cell spheroids with numerous elongated invadopodia, which WFA blocked potently by downregulating soluble vimentin and α-smooth muscle actin (SMA) expression. In the pilot proof-of-concept study using the GFS model, subconjunctival injections of a low WFA dose reduced skp2 expression in Tenon's capsule and increased p27(Kip1) expression without significant alteration to vimentin-IFs. This treatment maintains significant nanomolar WFA concentrations in anterior segment tissues that correspond to WFA's cell cycle targeting activity. A ten-fold higher WFA dose caused potent downregulation of soluble vimentin and skp2 expression, but as found in cell cultures, no further increase in p27(Kip1) expression was observed. Instead, this high WFA dose potently induced vimentin-IF disruption and downregulated α-SMA expression that mimicked WFA activity in TGF-ß-treated RbTCFs that blocked cell contractile activity at submicromolar concentrations. These findings illuminate that localized WFA injection to ocular tissues exerts pharmacological control over the skp2-p27(Kip1) pathway by targeting of soluble vimentin in a model of surgical fibrosis.

The role of miR-370 in fibrosis after myocardial infarction

PubMed Central

Yuan, Hui; Gao, Jie

2017-01-01

In the present study, we investigated the expression of miR-370 in the border area of infarction after myocardial infarction and its role in the process of post-infarction fibrosis. A myocardial infarction model in Sprague-Dawley rats was established. After two weeks, the mRNA levels of transforming growth factor-β1 (TGFβ1), TGFβRII, ColIa1, ColIIIa1 and miR-370 and the expression of TGFβ1, TGFβRII and α-smooth muscle actin (α-SMA) proteins in the border area of infarction were detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) and western blot analysis. Cardiac fibroblasts in neonatal rat were isolated and cultured, and the changes in the above indicators were detected after AngII and miR-370 intervention. Luciferase reporter gene assay was conducted to verify whether TGFβRII was a target gene of miR-370. In the border area after myocardial infarction, the expression of miR-370 decreased, while mRNA levels of TGFβ1, TGFβRII, ColIa1 and ColIIIa1 and levels of TGFβ1, TGFβRII and α-SMA proteins were all increased. Luciferase reporter gene assay confirmed that TGFβRII was the target gene of miR-370. miR-370 reduced the expression of TGFβRII and inhibited the increased expression of TGFβRII and collagen protein caused by AngII. As well, its inhibited the differentiation effect of muscle fibroblasts while it did not inhibit the expression of TGFβ1. miR-370 inhibited the expression of TGFβRII protein by combining with TGFβRII mRNA. miR-370 also partially blocked TGFβ1-TGFβRII and induced the downstream signal transduction pathways, thus exerting anti-fibrotic effects. PMID:28350072

Human trabecular meshwork cells exhibit several characteristics of, but are distinct from, adipose-derived mesenchymal stem cells.

PubMed

Morgan, Joshua T; Wood, Joshua A; Walker, Naomi J; Raghunathan, Vijay Krishna; Borjesson, Dori L; Murphy, Christopher J; Russell, Paul

2014-01-01

To support the growing promise of regenerative medicine in glaucoma, we characterized the similarities and differences between human trabecular meshwork (HTM) cells and human mesenchymal stem cells (hMSCs). HTM cells and hMSCs were phenotypically characterized by flow cytometry. Using quantitative polymerase chain reaction, the expression of myoc, angptl7, sox2, pou5f1, and notch1 was determined in both cell types with and without dexamethasone (Dex). Immunosuppressive behavior of HTM cells and hMSCs was determined using T cells activated with phytohemagglutinin. T-cell proliferation was determined using BrdU incorporation and flow cytometry. Multipotency of HTM cells and hMSCs was determined using adipogenic and osteogenic differentiation media as well as aqueous humor (AH). Alpha-smooth muscle actin (αSMA) expression was determined in HTM cells, hMSCs, and HTM tissue. Phenotypically, HTM and hMSCs expressed CD73, CD90, CD105, and CD146 but not CD31, CD34, and CD45 and similar sox2, pou5f1, and notch1 expression. Both cell types suppressed T-cell proliferation. However, HTM cells, but not hMSCs, upregulated myoc and angptl7 in response to Dex. Additionally, HTM cells did not differentiate into adipocytes or osteocytes. Culture of hMSCs in 20%, but not 100%, AH potently induced alkaline phosphatase activity. HTM cells in culture possessed uniformly strong expression of αSMA, which contrasted with the limited expression in hMSCs and spatially discrete expression in HTM tissue. HTM cells possess a number of important similarities with hMSCs but lack multipotency, one of the defining characteristics of stem cells. Further work is needed to explore the molecular mechanisms and functional implications underlying the phenotypic similarities.

Human Trabecular Meshwork Cells Exhibit Several Characteristics of, but Are Distinct from, Adipose-Derived Mesenchymal Stem Cells

PubMed Central

Morgan, Joshua T.; Wood, Joshua A.; Walker, Naomi J.; Raghunathan, Vijay Krishna; Borjesson, Dori L.; Murphy, Christopher J.

2014-01-01

Abstract Purpose: To support the growing promise of regenerative medicine in glaucoma, we characterized the similarities and differences between human trabecular meshwork (HTM) cells and human mesenchymal stem cells (hMSCs). Methods: HTM cells and hMSCs were phenotypically characterized by flow cytometry. Using quantitative polymerase chain reaction, the expression of myoc, angptl7, sox2, pou5f1, and notch1 was determined in both cell types with and without dexamethasone (Dex). Immunosuppressive behavior of HTM cells and hMSCs was determined using T cells activated with phytohemagglutinin. T-cell proliferation was determined using BrdU incorporation and flow cytometry. Multipotency of HTM cells and hMSCs was determined using adipogenic and osteogenic differentiation media as well as aqueous humor (AH). Alpha-smooth muscle actin (αSMA) expression was determined in HTM cells, hMSCs, and HTM tissue. Results: Phenotypically, HTM and hMSCs expressed CD73, CD90, CD105, and CD146 but not CD31, CD34, and CD45 and similar sox2, pou5f1, and notch1 expression. Both cell types suppressed T-cell proliferation. However, HTM cells, but not hMSCs, upregulated myoc and angptl7 in response to Dex. Additionally, HTM cells did not differentiate into adipocytes or osteocytes. Culture of hMSCs in 20%, but not 100%, AH potently induced alkaline phosphatase activity. HTM cells in culture possessed uniformly strong expression of αSMA, which contrasted with the limited expression in hMSCs and spatially discrete expression in HTM tissue. Conclusions: HTM cells possess a number of important similarities with hMSCs but lack multipotency, one of the defining characteristics of stem cells. Further work is needed to explore the molecular mechanisms and functional implications underlying the phenotypic similarities. PMID:24456002

Silibinin inhibits myofibroblast transdifferentiation in human tenon fibroblasts and reduces fibrosis in a rabbit trabeculectomy model.

PubMed

Chen, Yi-Hao; Liang, Chang-Min; Chen, Ching-Long; Chen, Jiann-Torng; Chang, Yun-Hsiang; Lu, Da-Wen; Chien, Ke-Hung; Tai, Ming-Cheng

2013-11-01

To investigate the effect of silibinin in myofibroblast transdifferentiation and in animal trabeculectomy models. The effect of silibinin on the expression of α-smooth muscle actin (α-SMA) and vimentin in response to transforming growth factor-β1 (TGF-β1) was determined in human tenon fibroblasts (HTFs). Cell migration and collagen contraction arrays were used to demonstrate the functionality of silibinin-modulated HTFs. ELISA analysis was used to determine the effect of silibinin on the release of type 1 collagen and connective tissue growth factor (CTGF). The effect of silibinin on the activation of the TGF-β receptor-related pathway was evaluated by Western blotting. A rabbit model of trabeculectomy was established to assess the effect of silibinin in vivo. TGF-β1 elevated the expression of α-SMA and vimentin in HTFs; this elevation was inhibited by silibinin. TGF-β1 increased cell migration and collagen contraction of HTFs, which were also suppressed by silibinin. The production of both CTGF and type 1 collagen in TGF-β1-treated HTFs was inhibited by silibinin. The effects of silibinin on TGF-β1-stimulated HTFs were mediated via the down-regulation of TGF-β receptor-related SMAD signalling pathways. In the rabbit model of trabeculectomy, silibinin increased the period of decreasing intraocular pressure after trabeculectomy and reduced the production of collagen and α-SMA at the site of blebs in vivo. Silibinin inhibited the TGF-β receptor-related signalling pathway in TGF-β-treated HTFs and several of the downstream events associated with myofibroblast transdifferentiation. Silibinin also improved the outcome of trabeculectomies by reducing the fibrotic response in the bleb tissue in vivo. © 2013 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

Pirfenidone inhibits transforming growth factor β1-induced extracellular matrix production in nasal polyp-derived fibroblasts.

PubMed

Shin, Jae-Min; Park, Joo-Hoo; Park, Il-Ho; Lee, Heung-Man

2015-01-01

Pirfenidone has been shown to have antifibrotic and anti-inflammatory effects in the lungs. The purpose of this study was to evaluate the inhibitory effects of pirfenidone on transforming growth factor (TGF)-β1-induced myofibroblast differentiation and extracellular matrix accumulation. We also determined the molecular mechanisms of pirfenidone in nasal polyp-derived fibroblasts (NPDF). NPDFs were isolated from nasal polyps from eight patients who had chronic rhinosinusitis with nasal polyp. Pirfenidone was used to treat TGF-β1-induced NPDFs. Cytotoxicity was evaluated by using a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay. Fibroblast migration was evaluated with scratch assays. Expression levels of α-smooth muscle actin (SMA), fibronectin, and phosphorylated Smad2/3 were determined by Western blot and/or reverse transcription-polymerase chain reaction and immunofluorescent staining. Total collagen production was analyzed with the Sircol collagen assay and contractile activity was measured by a collagen gel contraction assay. Pirfenidone (0-2 mg/mL) has no significant cytotoxic effects in TGF-β1-induced NPDFs. Migration of NPDFs was significantly inhibited by pirfenidone treatment. The expression levels of α-SMA and fibronectin were significantly reduced in pirfenidone-treated NPDFs. Collagen contraction and production were also significantly decreased by pirfenidone treatment. Finally, pirfenidone significantly inhibited phosphorylation of the Smad2/3 pathway in TGF-β1-induced NPDFs. Pirfenidone has an inhibitory effect on TGF-β1-induced migration, myofibroblast differentiation (α-SMA), extracellular matrix accumulation, and collagen contraction by blocking the phosphorylation of Smad2/3 pathways in NPDFs. Thus, pirfenidone may inhibit TGF-β1-induced extracellular matrix by regulating Smad2/3.

High glucose induces podocyte epithelial-to-mesenchymal transition by demethylation-mediated enhancement of MMP9 expression

PubMed Central

Ling, Li; Chen, Libo; Zhang, Changning; Gui, Shuyan; Zhao, Haiyan; Li, Zhengzhang

2018-01-01

Abnormal expression of matrix metalloproteinase 9 (MMP9) is correlated with podocyte epithelial-to-mesenchymal transition (EMT) in diabetic nephropathy (DN). However, the mechanisms underlying this process are not well defined. Site-specific demethylation may sustain high expression levels of target genes. In the present study, in order to investigate the association between DNA demethylation of MMP9 promoter and podocyte EMT in DN, human podocytes were cultured in high-glucose (HG) medium and a rat model of DN was established by intraperitoneal injection of streptozotocin (STZ) to determine whether site-specific demethylation of the MMP9 promoter was involved in regulating podocyte EMT in DN. The MTT assay was used to assess the effects of HG culture on the growth of podocytes, and the demethylation status of the MMP9 promoter was assessed by bisulfite sequencing polymerase chain reaction. mRNA and protein expression levels of MMP9, α-smooth muscle actin (α-SMA), podocalyxin and fibronectin-1 in podocytes were assessed by reverse transcription-quantitative PCR (RT-qPCR) and western blot analyses. The results demonstrated that HG treatment up regulated the expression of MMP9, α-SMA and fibronectin-1, but down regulated the expression of podocalyxin in podocytes. The MMP9 promoter region was revealed to contain a variety of demethylated CpG sites, and HG treatment reduced the rate of MMP9 promotermethylation, which, in turn, enhanced its promoter activity. In summary, these data suggested that demethylation of the MMP9 promoter may serve an important role in podocyte EMT in DN. The demethylation status of the MMP9 promoter maybe used as an important prognostic marker of DN in clinic. PMID:29436620

Emodin alleviates bleomycin-induced pulmonary fibrosis in rats.

PubMed

Guan, Ruijuan; Zhao, Xiaomei; Wang, Xia; Song, Nana; Guo, Yuhong; Yan, Xianxia; Jiang, Liping; Cheng, Wenjing; Shen, Linlin

2016-11-16

Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease with few treatment options and poor prognosis. Emodin, extracted from Chinese rhubarb, was found to be able to alleviate bleomycin (BLM)-induced pulmonary fibrosis, yet the underlying mechanism remains largely unknown. This study aimed to further investigate the effects of emodin on the inflammation and fibrosis of BLM-induced pulmonary fibrosis and the mechanism involved in rats. Our results showed that emodin improved pulmonary function, reduced weight loss and prevented death in BLM-treated rats. Emodin significantly relieved lung edema and fibrotic changes, decreased collagen deposition, and suppressed the infiltration of myofibroblasts [characterized by expression of α-smooth muscle actin (α-SMA)] and inflammatory cells (mainly macrophages and lymphocytes). Moreover, emodin reduced levels of TNF-α, IL-6, TGF-β1 and heat shock protein (HSP)-47 in the lungs of BLM-treated rats. In vitro, emodin profoundly inhibited TGF-β1-induced α-SMA, collagen IV and fibronectin expression in human embryo lung fibroblasts (HELFs). Emodin also inhibited TGF-β1-induced Smad2/3 and STAT3 activation, indicating that Smad2/3 and STAT3 inactivation mediates emodin-induced effects on TGF-β1-induced myofibroblast differentiation. These results suggest that emodin can exert its anti-fibrotic effect via suppression of TGF-β1 signaling and subsequently inhibition of inflammation, HSP-47 expression, myofibroblast differentiation and extracellular matrix (ECM) deposition. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Inhibition of integrin-linked kinase blocks podocyte epithelial-mesenchymal transition and ameliorates proteinuria.

PubMed

Kang, Young Sun; Li, Yingjian; Dai, Chunsun; Kiss, Lawrence P; Wu, Chuanyue; Liu, Youhua

2010-08-01

Proteinuria is a primary clinical symptom of a large number of glomerular diseases that progress to end-stage renal failure. Podocyte dysfunctions play a fundamental role in defective glomerular filtration in many common forms of proteinuric kidney disorders. Since binding of these cells to the basement membrane is mediated by integrins, we determined the role of integrin-linked kinase (ILK) in podocyte dysfunction and proteinuria. ILK expression was induced in mouse podocytes by various injurious stimuli known to cause proteinuria including TGF-beta1, adriamycin, puromycin, and high ambient glucose. Podocyte ILK was also found to be upregulated in human proteinuric glomerular diseases. Ectopic expression of ILK in podocytes decreased levels of the epithelial markers nephrin and ZO-1, induced mesenchymal markers such as desmin, fibronectin, matrix metalloproteinase-9 (MMP-9), and alpha-smooth muscle actin (alpha-SMA), promoted cell migration, and increased the paracellular albumin flux across podocyte monolayers. ILK also induced Snail, a key transcription factor mediating epithelial-mesenchymal transition (EMT). Blockade of ILK activity with a highly selective small molecule inhibitor reduced Snail induction and preserved podocyte phenotypes following TGF-beta1 or adriamycin stimulation. In vivo, this ILK inhibitor ameliorated albuminuria, repressed glomerular induction of MMP-9 and alpha-SMA, and preserved nephrin expression in murine adriamycin nephropathy. Our results show that upregulation of ILK is a convergent pathway leading to podocyte EMT, migration, and dysfunction. ILK may be an attractive target for therapeutic intervention of proteinuric kidney diseases.

Mefunidone Attenuates Tubulointerstitial Fibrosis in a Rat Model of Unilateral Ureteral Obstruction

PubMed Central

Liu, Chunyan; Mei, Wenjuan; Tang, Juan; Yuan, Qiongjing; Huang, Ling; Lu, Miaomiao; Wu, Lin; Peng, Zhangzhe; Meng, Jie; Yang, Huixiang; Shen, Hong; Lv, Ben; Hu, Gaoyun; Tao, Lijian

2015-01-01

Background Inflammation has a crucial role in renal interstitial fibrosis, which is the common pathway of chronic kidney diseases. Mefunidone (MFD) is a new compound which could effectively inhibit the proliferation of renal fibroblasts in vitro. However, the overall effect of Mefunidone in renal fibrosis remains unknown. Methods Sprague-Dawley rats were randomly divided intro 6 groups: sham operation, unilateral ureteral obstruction (UUO), UUO/Mefunidone (25, 50, 100mg/kg/day) and UUO/PFD (500mg/kg/day). The rats were sacrificed respectively on days 3, 7, and 14 after the operation. Tubulointerstitial injury index, interstitial collagen deposition, expression of fibronectin (FN), α-smooth muscle actin (α-SMA), type I and III collagen and the number of CD3+ and CD68+ cells were determined. The expressions of proinflammatory cytokines, p-ERK, p-IκB, and p-STAT3 were measured in human renal proximal tubular epithelial cells of HK-2 or macrophages. Results Mefunidone treatment significantly attenuated tubulointerstitial injury, interstitial collagen deposition, expression of FN, α-SMA, type I and III collagen in the obstructive kidneys, which correlated with significantly reduced the number of T cells and macrophages in the obstructive kidneys. Mechanistically, Mefunidone significantly inhibited tumor necrosis factor-α (TNF-α-) or lipopolysaccharide (LPS)-induced production of proinflammatory cytokines. This effect is possibly due to the inhibition of phosphorylation of ERK, IκB, and STAT3. Conclusion Mefunidone treatment attenuated tubulointerstitial fibrosis in a rat model of UUO, at least in part, through inhibition of inflammation. PMID:26042668

Losartan Attenuates Scar Formation in Filtering Bleb After Trabeculectomy.

PubMed

Shi, Huimin; Wang, Huiying; Fu, Shuhao; Xu, Kang; Zhang, Xiaoyan; Xiao, Yiqin; Ye, Wen

2017-03-01

To examine the effects of losartan on scar formation after trabeculectomy and on fibrotic changes of human Tenon's fibroblasts (HTFs). Trabeculectomy was performed on New Zealand rabbits. They were randomized to receive one of the following treatments: 0.9% normal saline, mitomycin-C, or one of the three doses of losartan. Bleb morphology, IOP, and histopathology examination were performed. Primary cultured HTFs were treated with losartan or vehicle, with or without angiotensin II (Ang II). Cell proliferation was assessed by Cell Counting Kit-8 assay, and cell migration was detected by scratch wound and transwell assay. Transdifferentiation was evaluated through the expression of α-smooth muscle actin (α-SMA) by immunofluorescence, real-time PCR, and Western blot. The expression of fibronectin (FN) was evaluated by real-time PCR and Western blot. An amount of 5 mg/mL of losartan subconjunctival injection significantly decreased IOP postoperatively and attenuated wound healing of the filtering bleb in the rabbit model. Immunostaining results showed less myofibroblast and collagen deposition around the bleb area in the losartan-treated eyes. Losartan (10-5 M) in vitro significantly attenuated Ang II's stimulatory effects on proliferation and migration of HTFs. Expressions of α-SMA and FN in these cells were also decreased by losartan pretreatment. Losartan attenuates scar formation of filtering bleb after trabeculectomy likely via decreasing proliferation, migration, transdifferentiation, and extracellular matrix deposition of Tenon's fibroblasts. These results indicate that losartan may be an effective therapeutic agent in preventing bleb scar formation and in improving surgical outcome after trabeculectomy.

Potassium and ANO1/TMEM16A chloride channel profiles distinguish atypical and typical smooth muscle cells from interstitial cells in the mouse renal pelvis

PubMed Central

Iqbal, Javed; Tonta, Mary A; Mitsui, Retsu; Li, Qun; Kett, Michelle; Li, Jinhua; Parkington, Helena C; Hashitani, Hikaru; Lang, Richard J

2012-01-01

BACKGROUND AND PURPOSE Although atypical smooth muscle cells (SMCs) in the proximal renal pelvis are thought to generate the pacemaker signals that drive pyeloureteric peristalsis, their location and electrical properties remain obscure. EXPERIMENTAL APPROACH Standard patch clamp, intracellular microelectrode and immunohistochemistry techniques were used. To unequivocally identify SMCs, transgenic mice with enhanced yellow fluorescent protein (eYFP) expressed in cells containing α-smooth muscle actin (α-SMA) were sometimes used. KEY RESULTS Atypical SMCs were distinguished from typical SMCs by the absence of both a transient 4-aminopyridine-sensitive K+ current (IKA) and spontaneous transient outward currents (STOCs) upon the opening of large-conductance Ca2+-activated K+ (BK) channels. Many typical SMCs displayed a slowly activating, slowly decaying Cl- current blocked by niflumic acid (NFA). Immunostaining for KV4.3 and ANO1/ TMEM16A Cl- channel subunits co-localized with α-SMA immunoreactive product predominately in the distal renal pelvis. Atypical SMCs fired spontaneous inward currents that were either selective for Cl- and blocked by NFA, or cation-selective and blocked by La3+. α-SMA- interstitial cells (ICs) were distinguished by the presence of a Xe991-sensitive KV7 current, BK channel STOCs and Cl- selective, NFA-sensitive spontaneous transient inward currents (STICs). Intense ANO1/ TMEM16A and KV7.5 immunostaining was present in Kit-α-SMA- ICs in the suburothelial and adventitial regions of the renal pelvis. CONCLUSIONS AND IMPLICATIONS We conclude that KV4.3+α-SMA+ SMCs are typical SMCs that facilitate muscle wall contraction, that ANO1/ TMEM16A and KV7.5 immunoreactivity may be selective markers of Kit- ICs and that atypical SMCs which discharge spontaneous inward currents are the pelviureteric pacemakers. PMID:22014103

Gastroschisis in the rat model is associated with a delayed maturation of intestinal pacemaker cells and smooth muscle cells.

PubMed

Midrio, P; Faussone-Pellegrini, M S; Vannucchi, M G; Flake, A W

2004-10-01

A pacemaker system is required for peristalsis generation. The interstitial cells of Cajal (ICC) are considered the intestinal pacemaker, and are identified by expression of the c-kit gene--encoded protein. Gastroschisis is characterized by a severe gastrointestinal dysmotility in newborns. In spite of this clinical picture, few studies have focused on smooth muscle cells (SMC) morphology and none on ICC. Therefore, their morphology has been studied in fetuses at term in the rat model of gastroschisis. At 18.5 day's gestation (E18.5), 10 rat fetuses were killed, 10 underwent surgical creation of gastroschisis, and 10 underwent manipulation only. The small intestine of the latter 2 groups was harvested at E21.5. Specimens were processed for H&E, c-kit and actin (alpha smooth muscle antibody [alpha-SMA]) immunohistochemistry, and transmission electron microscopy (TEM). In the controls, SMC were c-kit+ and alpha-SMA+, with labeling intensity increasing by age. At E21.5, some cells around the Auerbach's plexus were more intensely c-kit+, and differentiating ICC were seen under TEM at this level. Gastroschisis fetuses had no c-kit+ cells referable to ICC. In the more damaged loops, SMC were very faintly c-kit+ and alpha-SMA+. Under TEM, there were few differentiated SMC and no presumptive ICC. In the less-damaged loops, SMC were faintly c-kit+ and alpha-SMA+ and had ultrastructural features intermediate between those of E18.5 and E21.5 controls; ICC were very immature. ICC and SMC differentiation is delayed in gastroschisis with the most damaged loops showing the most incomplete picture. These findings might help in understanding the delayed onset of peristalsis and the variable time-course of the recover seen in babies affected by gastroschisis.

Connective tissue cells expressing fibro/adipogenic progenitor markers increase under chronic damage: relevance in fibroblast-myofibroblast differentiation and skeletal muscle fibrosis.

PubMed

Contreras, Osvaldo; Rebolledo, Daniela L; Oyarzún, Juan Esteban; Olguín, Hugo C; Brandan, Enrique

2016-06-01

Fibrosis occurs in skeletal muscle under various pathophysiological conditions such as Duchenne muscular dystrophy (DMD), a devastating disease characterized by fiber degeneration that results in progressive loss of muscle mass, weakness and increased extracellular matrix (ECM) accumulation. Fibrosis is also observed after skeletal muscle denervation and repeated cycles of damage followed by regeneration. The ECM is synthesized largely by fibroblasts in the muscle connective tissue under normal conditions. Myofibroblasts, cells that express α-smooth muscle actin (α-SMA), play a role in many tissues affected by fibrosis. In skeletal muscle, fibro/adipogenic progenitors (FAPs) that express cell-surface platelet-derived growth factor receptor-α (PDGFR-α) and the transcription factor Tcf4 seem to be responsible for connective tissue synthesis and are good candidates for the origin of myofibroblasts. We show that cells positive for Tcf4 and PDGFR-α are expressed in skeletal muscle under normal conditions and are increased in various skeletal muscles of mdx mice, a murine model for DMD, wild type muscle after sciatic denervation and muscle subjected to chronic damage. These cells co-label with the myofibroblast marker α-SMA in dystrophic muscle but not in normal tissue. The Tcf4-positive cells lie near macrophages mainly concentrated in dystrophic necrotic-regenerating foci. The close proximity of Tcf4-positive cells to inflammatory cells and their previously described role in muscle regeneration might reflect an active interaction between these cell types and growth factors, possibly resulting in a muscular regenerative or fibrotic condition.

Intracrine sex steroid synthesis and signaling in human epidermal keratinocytes and dermal fibroblasts.

PubMed

Pomari, Elena; Dalla Valle, Luisa; Pertile, Paolo; Colombo, Lorenzo; Thornton, M Julie

2015-02-01

Peripheral intracrine sex steroid synthesis from adrenal precursors dehydroepiandrosterone (DHEA) and DHEA-sulfate has evolved in humans. We sought to establish if there are differences in intracrine, paracrine, and endocrine regulation of sex steroids by primary cultures of human skin epidermal keratinocytes and dermal fibroblasts. Microarray analysis identified multifunctional genes modulated by steroids, quantitative RT-PCR (qRT-PCR) mRNA expression, enzymatic assay aromatase activity, scratch assay cell migration, immunocytochemistry α-smooth muscle actin (α-SMA), and collagen gel fibroblast contraction. All steroidogenic components were present, although only keratinocytes expressed the organic anion organic anion transporter protein (OATP) 2B1 transporter. Both expressed the G-protein-coupled estrogen receptor (GPER1). Steroids modulated multifunctional genes, up-regulating genes important in repair and aging [angiopoietin-like 4 (ANGPTL4), chemokine (C-X-C motif) ligand 1 (CXCL1), lamin B1 (LMNB1), and thioredoxin interacting protein (TXNIP)]. DHEA-sulfate (DHEA-S), DHEA, and 17β-estradiol stimulated keratinocyte and fibroblast migration at early (4 h) and late (24-48 h) time points, suggesting involvement of genomic and nongenomic signaling. Migration was blocked by aromatase and steroid sulfatase (STS) inhibitors confirming intracrine synthesis to estrogen. Testosterone had little effect, implying it is not an intermediate. Steroids stimulated fibroblast contraction but not α-SMA expression. Mechanical wounding reduced fibroblast aromatase activity but increased keratinocyte activity, amplifying the bioavailability of intracellular estrogen. Cultured fibroblasts and keratinocytes provide a biologically relevant model system to investigate the complex pathways of sex steroid intracrinology in human skin. © FASEB.

Dual effect of silymarin on experimental non-alcoholic steatohepatitis induced by irinotecan.

PubMed

Marcolino Assis-Júnior, Eudmar; Melo, Anielle Torres; Pereira, Venúcia Bruna Magalhães; Wong, Deysi Viviana Tenazoa; Sousa, Nathalia Ribeiro Pinho; Oliveira, Christiane Mendes Gonçalves; Malveira, Lara Raissa Cavalcante; Moreira, Leonardo Silva; Souza, Marcellus Henrique Loiola Ponte; Almeida, Paulo Roberto Carvalho; Lima-Júnior, Roberto César Pereira

2017-07-15

Irinotecan-based regimens are commonly used for treatment of colorectal cancer, which is limited by mucositis and non-alcoholic steatohepatitis (NASH). Silymarin (SIL) prevents fatty liver disease in the clinical setting and in models of liver damage induced chemically. This study investigated the possible effect of SIL on irinotecan (IRI)-induced NASH. Swiss female mice were injected with saline (SAL 5ml/kg i.p.), IRI (50mg/kg i.p.), SIL (150mg/kg p.o.) or IRI (50mg/kg i.p.)+(SIL 1.5, 15 or 150mg/kg p.o.) thrice/week/7weeks. On the seventh week, blood samples were collected for transaminases assay and livers were collected for histopathology, measurement of the total lipids, malondyadehyde (MDA), non-protein sulfhydryl groups (NPSH), cytokines (IL-1β, IL 6 and IL-10), 3-nitrotyrosine (N-Tyr) and toll-like receptor 4 (TLR4) immunoexpression, quantification of NF-kB, α-smooth muscle actin (α-SMA), and Escherichia coli 16S rRNA gene (RRS) expression. IRI increased liver transaminases, neutrophil infiltration, lipid accumulation, MDA, IL-1β and IL-6 levels, N-Tyr and TLR4 immunostaining, NF-kB, α-SMA expression and RRS versus the SAL group (p<0.05). Additionally, SIL (1.5mg/kg) improved these parameters (p<0.05), except neutrophil infiltration and RSS versus the IRI group. Furthermore, the SIL (15mg/kg) only improved the inflammatory parameters, the expression of α-SMA and RRS versus the IRI group (p<0.05). The higher dose of SIL (150mg/kg) was even more deleterious than the intermediate dose. Therefore, silymarin showed a dual effect on liver damage induced by IRI. Hepatoprotection seems to involve the inhibition of oxidative stress and protein nitrosylation, preventing activation of hepatic fibrosis mechanisms. Copyright © 2017 Elsevier Inc. All rights reserved.

Induction of fibroblast senescence generates a non-fibrogenic myofibroblast phenotype that differentially impacts on cancer prognosis

PubMed Central

Thirdborough, Steve; Mellows, Toby; Garcia, Edwin; Woo, Jeongmin; Tod, Joanne; Frampton, Steve; Jenei, Veronika; Moutasim, Karwan A.; Kabir, Tasnuva D.; Brennan, Peter A; Venturi, Giulia; Ford, Kirsty; Herranz, Nicolas; Lim, Kue Peng; Clarke, James; Lambert, Daniel W.; Prime, Stephen S.; Underwood, Timothy J.; Vijayanand, Pandurangan; Eliceiri, Kevin W.; Woelk, Christopher; King, Emma V.; Gil, Jesus; Ottensmeier, Christian H.; Thomas, Gareth J.

2017-01-01

Cancer-associated fibroblasts (CAF) remain a poorly characterized, heterogeneous cell population. Here we characterized two previously described tumor-promoting CAF sub-types, smooth muscle actin (SMA)-positive myofibroblasts and senescent fibroblasts, identifying a novel link between the two. Analysis of CAF cultured ex vivo, showed that senescent CAF are predominantly SMA-positive; this was confirmed by immunochemistry in head & neck (HNSCC) and esophageal (EAC) cancers. In vitro, we found that fibroblasts induced to senesce develop molecular, ultrastructural and contractile features typical of myofibroblasts and this is dependent on canonical TGF-β signaling. Similar to TGF-β1-generated myofibroblasts, these cells secrete soluble factors that promote tumor cell motility. However, RNA-sequencing revealed significant transcriptomic differences between the two SMA-positive CAF groups, particularly in genes associated with extracellular matrix (ECM) deposition and organization, which differentially promote tumor cell invasion. Notably, second harmonic generation imaging and bioinformatic analysis of SMA-positive human HNSCC and EAC showed that collagen fiber organization correlates with poor prognosis, indicating that heterogeneity within the SMA-positive CAF population differentially impacts on survival. These results show that non-fibrogenic, SMA-positive myofibroblasts can be directly generated through induction of fibroblast senescence and suggest that senescence and myofibroblast differentiation are closely linked processes. PMID:27992856

Induction of fibroblast senescence generates a non-fibrogenic myofibroblast phenotype that differentially impacts on cancer prognosis.

PubMed

Mellone, Massimiliano; Hanley, Christopher J; Thirdborough, Steve; Mellows, Toby; Garcia, Edwin; Woo, Jeongmin; Tod, Joanne; Frampton, Steve; Jenei, Veronika; Moutasim, Karwan A; Kabir, Tasnuva D; Brennan, Peter A; Venturi, Giulia; Ford, Kirsty; Herranz, Nicolas; Lim, Kue Peng; Clarke, James; Lambert, Daniel W; Prime, Stephen S; Underwood, Timothy J; Vijayanand, Pandurangan; Eliceiri, Kevin W; Woelk, Christopher; King, Emma V; Gil, Jesus; Ottensmeier, Christian H; Thomas, Gareth J

2016-12-15

Cancer-associated fibroblasts (CAF) remain a poorly characterized, heterogeneous cell population. Here we characterized two previously described tumor-promoting CAF sub-types, smooth muscle actin (SMA)-positive myofibroblasts and senescent fibroblasts, identifying a novel link between the two. Analysis of CAF cultured ex vivo , showed that senescent CAF are predominantly SMA-positive; this was confirmed by immunochemistry in head & neck (HNSCC) and esophageal (EAC) cancers. In vitro , we found that fibroblasts induced to senesce develop molecular, ultrastructural and contractile features typical of myofibroblasts and this is dependent on canonical TGF-β signaling. Similar to TGF-β1-generated myofibroblasts, these cells secrete soluble factors that promote tumor cell motility. However, RNA-sequencing revealed significant transcriptomic differences between the two SMA-positive CAF groups, particularly in genes associated with extracellular matrix (ECM) deposition and organization, which differentially promote tumor cell invasion. Notably, second harmonic generation imaging and bioinformatic analysis of SMA-positive human HNSCC and EAC showed that collagen fiber organization correlates with poor prognosis, indicating that heterogeneity within the SMA-positive CAF population differentially impacts on survival. These results show that non-fibrogenic, SMA-positive myofibroblasts can be directly generated through induction of fibroblast senescence and suggest that senescence and myofibroblast differentiation are closely linked processes.

The tumour-associated glycoprotein podoplanin is expressed in fibroblast-like synoviocytes of the hyperplastic synovial lining layer in rheumatoid arthritis

PubMed Central

2011-01-01

Introduction Activated fibroblast-like synoviocytes (FLSs) in rheumatoid arthritis (RA) share many characteristics with tumour cells and are key mediators of synovial tissue transformation and joint destruction. The glycoprotein podoplanin is upregulated in the invasive front of several human cancers and has been associated with epithelial-mesenchymal transition, increased cell migration and tissue invasion. The aim of this study was to investigate whether podoplanin is expressed in areas of synovial transformation in RA and especially in promigratory RA-FLS. Methods Podoplanin expression in human synovial tissue from 18 RA patients and nine osteoarthritis (OA) patients was assessed by immunohistochemistry and confirmed by Western blot analysis. The expression was related to markers of synoviocytes and myofibroblasts detected by using confocal immunofluoresence microscopy. Expression of podoplanin, with or without the addition of proinflammatory cytokines and growth factors, in primary human FLS was evaluated by using flow cytometry. Results Podoplanin was highly expressed in cadherin-11-positive cells throughout the synovial lining layer in RA. The expression was most pronounced in areas with lining layer hyperplasia and high matrix metalloproteinase 9 expression, where it coincided with upregulation of α-smooth muscle actin (α-sma). The synovium in OA was predominantly podoplanin-negative. Podoplanin was expressed in 50% of cultured primary FLSs, and the expression was increased by interleukin 1β, tumour necrosis factor α and transforming growth factor β receptor 1. Conclusions Here we show that podoplanin is highly expressed in FLSs of the invading synovial tissue in RA. The concomitant upregulation of α-sma and podoplanin in a subpopulation of FLSs indicates a myofibroblast phenotype. Proinflammatory mediators increased the podoplanin expression in cultured RA-FLS. We conclude that podoplanin might be involved in the synovial tissue transformation and increased migratory potential of activated FLSs in RA. PMID:21385358

LXA{sub 4} actions direct fibroblast function and wound closure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Herrera, Bruno S.; Microbiology Branch, US Army Dental and Trauma Research Detachment, Institute of Surgical Research, JBSA Fort Sam Houston, TX; Kantarci, Alpdogan

Timely resolution of inflammation is crucial for normal wound healing. Resolution of inflammation is an active biological process regulated by specialized lipid mediators including the lipoxins and resolvins. Failure of resolution activity has a major negative impact on wound healing in chronic inflammatory diseases that is manifest as excess fibrosis and scarring. Lipoxins, including Lipoxin A{sub 4} (LXA{sub 4}), have known anti-fibrotic and anti-scarring properties. The goal of this study was to elucidate the impact of LXA{sub 4} on fibroblast function. Mouse fibroblasts (3T3 Mus musculus Swiss) were cultured for 72 h in the presence of TGF-β1, to induce fibroblast activation.more » The impact of exogenous TGF-β1 (1 ng/mL) on LXA{sub 4} receptor expression (ALX/FPR2) was determined by flow cytometry. Fibroblast proliferation was measured by bromodeoxyuridine (BrdU) labeling and migration in a “scratch” assay wound model. Expression of α-smooth muscle actin (α-SMA), and collagen types I and III were measured by Western blot. We observed that TGF-β1 up-regulates LXA{sub 4} receptor expression, enhances fibroblast proliferation, migration and scratch wound closure. α-SMA levels and Collagen type I and III deposition were also enhanced. LXA{sub 4} slowed fibroblast migration and scratch wound closure at early time points (24 h), but wound closure was equal to TGF-β1 alone at 48 and 72 h. LXA{sub 4} tended to slow fibroblast proliferation at both concentrations, but had no impact on α-SMA or collagen production by TGF-β1 stimulated fibroblasts. The generalizability of the actions of resolution molecules was examined in experiments repeated with resolvin D2 (RvD2) as the agonist. The activity of RvD2 mimicked the actions of LXA{sub 4} in all assays, through an as yet unidentified receptor. The results suggest that mediators of resolution of inflammation enhance wound healing and limit fibrosis in part by modulating fibroblast function. - Highlights: • TGF-β1 up-regulates LXA{sub 4} receptor (ALX/FPR2) expression on fibroblast. • LXA{sub 4} regulates fibroblast migration and proliferation induced by TGF-β1. • SPMs have no impact on α-SMA, collagen type-I and III expression by fibroblast. • RvD2 regulates TGF-β1-induced fibroblast proliferation and scratch wound closure.« less

Expression pattern and function of tyrosine receptor kinase B isoforms in rat mesenteric arterial smooth muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Otani, Kosuke; Okada, Muneyoshi; Yamawaki, Hideyuki, E-mail: yamawaki@vmas.kitasato-u.ac.jp

Tyrosine receptor kinaseB (TrkB) is a high affinity receptor for brain-derived neurotrophic factor (BDNF). TrkB isoforms involve full length TrkB (TrkB FL) and truncated TrkB type1 (TrkB T1) and type 2 (TrkB T2) in rats. The aim of present study was to explore their expression pattern and function in mesenteric arterial smooth muscle cells (MASMCs). The expression of TrkB isoform protein and mRNA was examined by Western blotting, immunofluorescence and quantitative RT-PCR analyses. Cell proliferation was measured by a bromodeoxyuridine (BrdU) incorporation assay. Cell migration was measured by a Boyden chamber assay. Cell morphology was observed with a phase-contrast microscope.more » Protein and mRNA expression of BDNF and TrkB isoforms was confirmed in MASMCs. Expression level of TrkB FL was less, while that of TrkB T1 was the highest in MASMCs. Although BDNF increased phosphorylation of ERK, it had no influence on migration and proliferation of MASMCs. TrkB T1 gene knockdown by a RNA interference induced morphological changes and reduced expression level of α-smooth muscle actin (α-SMA) in MASMCs. Similar morphological changes and reduced α-SMA expression were induced in MASMCs by a Rho kinase inhibitor, Y-27632. In conclusion, we for the first time demonstrate that TrkB T1 expressed highly in MASMCs contributes to maintain normal cell morphology possibly via regulation of Rho activity. This study firstly defined expression level of TrkB isoforms and partly revealed their functions in peripheral vascular cells. - Highlights: • BDNF-TrkB axis mediates neurogenesis, growth, differentiation and survival. • Expression pattern and function of TrkB in vascular smooth muscle remain unclear. • Expression of TrkB FL is low, while that of TrkB T1 is the highest. • TrkB T1 contributes to maintain normal morphology possibly via activating Rho.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Horie, Masafumi; Saito, Akira, E-mail: asaitou-tky@umin.ac.jp; Mikami, Yu

Highlights: Black-Right-Pointing-Pointer We established three patient-paired sets of CAFs and NFs. Black-Right-Pointing-Pointer CAFs and NFs were analyzed using three-dimensional co-culture experiments. Black-Right-Pointing-Pointer CAFs clearly enhanced collagen gel contraction. Black-Right-Pointing-Pointer CAFs showed higher {alpha}-SMA expression than NFs. Black-Right-Pointing-Pointer CAFs were implicated in invasion and differentiation of lung cancer cells. -- Abstract: Lung cancer is the most common cause of cancer-related death worldwide. Stromal cancer-associated fibroblasts (CAFs) play crucial roles in carcinogenesis, proliferation, invasion, and metastasis of non-small cell lung carcinoma, and targeting of CAFs could be a novel strategy for cancer treatment. However, the characteristics of human CAFs still remain tomore » be better defined. In this study, we established patient-matched CAFs and normal fibroblasts (NFs), from tumoral and non-tumoral portions of resected lung tissue from lung cancer patients. CAFs showed higher {alpha}-smooth muscle actin ({alpha}-SMA) expression than NFs, and CAFs clearly enhanced collagen gel contraction. Furthermore, we employed three-dimensional co-culture assay with A549 lung cancer cells, where CAFs were more potent in inducing collagen gel contraction. Hematoxylin and eosin staining of co-cultured collagen gel revealed that CAFs had the potential to increase invasion of A549 cells compared to NFs. These observations provide evidence that lung CAFs have the tumor-promoting capacity distinct from NFs.« less

Diethylcarbamazine attenuates the expression of pro-fibrogenic markers and hepatic stellate cells activation in carbon tetrachloride-induced liver fibrosis.

PubMed

França, Maria Eduarda Rocha de; Rocha, Sura Wanessa Santos; Oliveira, Wilma Helena; Santos, Laise Aline; de Oliveira, Anne Gabrielle Vasconcelos; Barbosa, Karla Patrícia Sousa; Nunes, Ana Karolina Santana; Rodrigues, Gabriel Barros; Lós, Deniele Bezerra; Peixoto, Christina Alves

2018-04-01

While diethylcarbamazine citrate (DEC) displays important anti-inflammatory effects in experimental models of liver injury, the mechanisms of its action remain poorly understood. The aim of the present study was to investigate the fibrolytic potential of DEC. Mice receive two injections of carbon tetrachloride (CCl 4 ) per week for 8 weeks. DEC 50 mg/kg body weight was administered through drinking water during the last 12 days of liver injury. The expression of hepatic stellate cells (HSCs) activation markers, including smooth muscle α-actin (α-SMA), collagen I, transforming growth factor-β 1 (TGF-β1), matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1) was assessed. The influence of DEC on the intracellular MAPK pathways of the HSCs (JNK and p38 MAPK) was also estimated. DEC inhibited HSCs activation measured as the production of α-SMA and collagen I. In addition, it down regulated the production of TGF-β1 and TIMP-1, and concomitantly increased MMP-2 activity. Furthermore, DEC significantly inhibited the activation of the JNK and p38 MAPK signaling pathways. In conclusion, DEC significantly attenuated the severity of CCl 4 -induced liver injury and the progression of liver fibrosis, exerting a potential fibrolytic effect in the CCl 4 -induced fibrosis model.

Baicalin Ameliorates Experimental Liver Cholestasis in Mice by Modulation of Oxidative Stress, Inflammation, and NRF2 Transcription Factor

PubMed Central

Feng, Xiaowen; Zhang, Feng; Xie, Haiyang

2017-01-01

Experimental cholestatic liver fibrosis was performed by bile duct ligation (BDL) in mice, and significant liver injury was observed in 15 days. Administration of baicalin in mice significantly ameliorates liver fibrosis. Experimental cholestatic liver fibrosis was associated with induced gene expression of fibrotic markers such as collagen I, fibronectin, alpha smooth muscle actin (SMA), and connective tissue growth factor (CTGF); increased inflammatory cytokines (TNFα, MIP1α, IL1β, and MIP2); increased oxidative stress and reactive oxygen species- (ROS-) inducing enzymes (NOX2 and iNOS); dysfunctional mitochondrial electron chain complexes; and apoptotic/necrotic cell death markers (DNA fragmentation, caspase 3 activity, and PARP activity). Baicalin administration on alternate day reduced fibrosis along with profibrotic gene expression, proinflammatory cytokines, oxidative stress, and cell death whereas improving the function of mitochondrial electron transport chain. We observed baicalin enhanced NRF2 activation by nuclear translocation and induced its target genes HO-1 and GCLM, thus enhancing antioxidant defense. Interplay of oxidative stress/inflammation and NRF2 were key players for baicalin-mediated protection. Stellate cell activation is crucial for initiation of fibrosis. Baicalin alleviated stellate cell activation and modulated TIMP1, SMA, collagen 1, and fibronectin in vitro. This study indicates that baicalin might be beneficial for reducing inflammation and fibrosis in liver injury models. PMID:28757911

Nephroprotective effect of bee honey and royal jelly against subchronic cisplatin toxicity in rats.

PubMed

Ibrahim, Abdelazim; Eldaim, Mabrouk A Abd; Abdel-Daim, Mohamed M

2016-08-01

Cisplatin is one of the most potent and effective chemotherapeutic agents. However, its antineoplastic use is limited due to its cumulative nephrotoxic side effects. Therefore, the present study was undertaken to examine the nephroprotective potential of dietary bee honey and royal jelly against subchronic cisplatin toxicity in rats. Male Wistar rats were randomly divided into controls, cisplatin-treated, bee honey-pretreated cisplatin-treated and royal jelly-pretreated cisplatin-treated groups. Bee honey and royal jelly were given orally at doses of 20 and 100 mg/kg, respectively. Subchronic toxicity was induced by cisplatin (1 mg/kg bw, ip), twice weekly for 10 weeks. Cisplatin treated animals revealed a significant increase in serum level of renal injury products (urea, creatinine and uric acid). Histopathologically, cisplatin produced pronounced tubulointerstitial injuries, upregulated the fibrogenic factors, α-smooth muscle actin (α-SMA) and transforming growth factor β1(TGF-β1), and downregulated the cell proliferation marker, bromodeoxyuridine (Brdu). Dietary bee honey and royal jelly normalized the elevated serum renal injury product biomarkers, improved the histopathologic changes, reduced the expression of α-SMA and TGF-β1 and increased the expression of Brdu. Therefore, it could be concluded that bee honey, and royal jelly could be used as dietary preventive natural products against subchronic cisplatin-induced renal injury.

PASS-Predicted Hepatoprotective Activity of Caesalpinia sappan in Thioacetamide-Induced Liver Fibrosis in Rats

PubMed Central

Kadir, Farkaad A.; Kassim, Normadiah M.; Abdulla, Mahmood Ameen; Ahmadipour, Fatemeh; Yehye, Wageeh A.

2014-01-01

The antifibrotic effects of traditional medicinal herb Caesalpinia sappan (CS) extract on liver fibrosis induced by thioacetamide (TAA) and the expression of transforming growth factor β1 (TGF-β1), α-smooth muscle actin (αSMA), and proliferating cell nuclear antigen (PCNA) in rats were studied. A computer-aided prediction of antioxidant and hepatoprotective activities was primarily performed with the Prediction Activity Spectra of the Substance (PASS) Program. Liver fibrosis was induced in male Sprague Dawley rats by TAA administration (0.03% w/v) in drinking water for a period of 12 weeks. Rats were divided into seven groups: control, TAA, Silymarin (SY), and CS 300 mg/kg body weight and 100 mg/kg groups. The effect of CS on liver fibrogenesis was determined by Masson's trichrome staining, immunohistochemical analysis, and western blotting. In vivo determination of hepatic antioxidant activities, cytochrome P450 2E1 (CYP2E1), and matrix metalloproteinases (MPPS) was employed. CS treatment had significantly increased hepatic antioxidant enzymes activity in the TAA-treated rats. Liver fibrosis was greatly alleviated in rats when treated with CS extract. CS treatment was noted to normalize the expression of TGF-β1, αSMA, PCNA, MMPs, and TIMP1 proteins. PASS-predicted plant activity could efficiently guide in selecting a promising pharmaceutical lead with high accuracy and required antioxidant and hepatoprotective properties. PMID:24701154

Contractility in type III cochlear fibrocytes is dependent on non-muscle myosin II and intercellular gap junctional coupling.

PubMed

Kelly, John J; Forge, Andrew; Jagger, Daniel J

2012-08-01

The cochlear spiral ligament is a connective tissue that plays diverse roles in normal hearing. Spiral ligament fibrocytes are classified into functional sub-types that are proposed to carry out specialized roles in fluid homeostasis, the mediation of inflammatory responses to trauma, and the fine tuning of cochlear mechanics. We derived a secondary sub-culture from guinea pig spiral ligament, in which the cells expressed protein markers of type III or "tension" fibrocytes, including non-muscle myosin II (nmII), α-smooth muscle actin (αsma), vimentin, connexin43 (cx43), and aquaporin-1. The cells formed extensive stress fibers containing αsma, which were also associated intimately with nmII expression, and the cells displayed the mechanically contractile phenotype predicted by earlier modeling studies. cx43 immunofluorescence was evident within intercellular plaques, and the cells were coupled via dye-permeable gap junctions. Coupling was blocked by meclofenamic acid (MFA), an inhibitor of cx43-containing channels. The contraction of collagen lattice gels mediated by the cells could be prevented reversibly by blebbistatin, an inhibitor of nmII function. MFA also reduced the gel contraction, suggesting that intercellular coupling modulates contractility. The results demonstrate that these cells can impart nmII-dependent contractile force on a collagenous substrate, and support the hypothesis that type III fibrocytes regulate tension in the spiral ligament-basilar membrane complex, thereby determining auditory sensitivity.

Silymarin and caffeine combination ameliorates experimentally-induced hepatic fibrosis through down-regulation of LPAR1 expression.

PubMed

Eraky, Salma M; El-Mesery, Mohamed; El-Karef, Amro; Eissa, Laila A; El-Gayar, Amal M

2018-05-01

Lysophosphatidic acid is a lipid mediator that is supposed to be implicated in hepatic fibrosis. Silymarin and caffeine are natural compounds known for their anti-inflammatory and antioxidant effects. Our study aimed to explore the effect of silymarin, caffeine, and their combination on lysophosphatidic acid receptor 1 (LPAR1) pathway in thioacetamide (TAA)-induced hepatic fibrosis. Hepatic fibrosis was induced in male Sprague-Dawley rats by intraperitoneal injection of 200 mg/kg of TAA twice a week for 8 weeks. Silymarin (50 mg/kg), caffeine (50 mg/kg), and their combination (50 mg/kg silymarin + 50 mg/kg caffeine) were orally given to rats every day for 8 weeks along with TAA injection. Liver functions were measured. Histopathological examination of liver tissues was performed using hematoxylin and eosin and Masson's trichrome staining. mRNA expressions of LPAR1, transforming growth factor beta 1 (TGF-β1), connective tissue growth factor (CTGF), and alpha smooth muscle actin (α-SMA) were measured using RT-PCR. LPAR1 tissue expression was scored using immunohistochemistry. Silymarin, caffeine, and their combination significantly improved liver function. They caused significant decrease in fibrosis and necro-inflammatory scores. Combination of silymain and caffeine caused a significant decrease in the necro-inflammatory score than the single treatment with silymarin or caffeine. In addition, silymarin, caffeine, and their combination significantly decreased hepatic LPAR1, TGF-β1, CTGF, and α-SMA gene expressions and LPAR1 tissue expression. Silymarin, caffeine, and their combination protect against liver fibrosis through down-regulation of LPAR1, TGF-β1, and CTGF. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

[Primary culture and characteristics of colorectal cancer-associated fibroblasts].

PubMed

Wen, Huan; Nie, Qianqian; Jiang, Zhinong; Deng, Hong

2015-10-01

To compare the biological characteristics of colorectal cancer associated fibroblasts (CAFs) with normal fibroblasts (NFs). CAFs and NFs were isolated from fresh specimens of colorectal cancer and their paired normal colon tissue and cultured by tissue explant method. Light microscopy, quantitative polymerase chain reaction (qPCR), Western blot, immunofluorescence microscopy, electron microscopy and flow cytometry were used to identify isolated fibroblasts and to explore their characteristics of activation and growth. Primary colorectal CAFs and NFs were isolated and cultured successfully. NFs showed spindled morphology and were arranged in interlacing or spiral bundles. CAFs were polygonal or spindle, but were fatter than NFs. They were distributed randomly and arranged irregularly, and had obvious actin expression. CAFs and NFs both expressed fibronectin, but not E-cadherin, CD31 and caldesmon. qPCR showed that CAFs expressed more fibroblast activation protein (FAP) and less fibroblast specific protein 1 (FSP1) than that of NFs. There was no difference in the expression of α-SMA between NFs and CAFs by Western blot. α-SMA was bundled in parallel to the long axis of the cell by immunofluorescence. By electron microscopy, CAFs but not NFs showed dense myofilament that was arranged regularly. Flow cytometry showed that the percentage of S- and G2-phase in CAFs were significantly lower than that in NFs. mRNA expression of transforming growth factor β1, stromal derived factor 1 (SDF-1) and platelet derived growth factor (PDGF)-D in CAFs were lower while that for PDGFC was higher than that in NFs. That indicated the proliferation of CAFs was inhibited and the secretion of some cytokines was different when compared with NFs. CAFs show differences with NFs in morphology, characteristics of activation and secretion of some cytokines. The proliferation of CAFs is down regulated as compared with NFs.

Reactive stroma in the prostate during late life: The role of microvasculature and antiangiogenic therapy influences.

PubMed

Montico, Fabio; Kido, Larissa Akemi; San Martin, Rebeca; Rowley, David R; Cagnon, Valéria H A

2015-10-01

Prostate cancer is associated to a reactive stroma microenvironment characterized by angiogenic processes that are favorable for tumor progression. Senescence has been identified as a predisposing factor for prostate malignancies. In turn, the relationships between aging, reactive stroma, and the mechanisms that induce this phenotype are largely unknown. Thus, we investigated the occurrence of reactive stroma in the mouse prostate during advanced age as well as the effects of antiangiogenic and androgen ablation therapies on reactive stroma recruitment. Male mice (52-week-old FVB) were treated with two classes of angiogenesis inhibitors: direct (TNP-470; 15 mg/kg; s.c.) and/or indirect (SU5416; 6 mg/kg; i.p.). Androgen ablation was carried out by finasteride administration (20 mg/kg; s.c.), alone or in association to both inhibitors. The Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model was used as a paradigm of cancer-associated reactive stroma. The dorsolateral prostate was collected for α-actin (αSMA), vimentin (VIM), and transforming growth factor-beta (TGF-β) immunohistochemical and Western blotting analyses as well as for CD34/αSMA and CD34/VIM colocalization. Senescence was associated with increased αSMA, VIM, and TGF-β expression as well as with the recruitment of CD34/αSMA and CD34/VIM dual-positive fibroblasts. These observations were similar to those verified in TRAMP mice. Antiangiogenic treatment promoted the recovery of senescence-associated stromal changes. Hormonal ablation, despite having led to impaired CD34/αSMA and CD34/VIM dual-positive cell recruitment, did not result in decreased stimulus to reactive stroma development, due to enhanced TGF-β expression in relation to the aged controls. Reactive stroma develops in the prostate of non-transgenic mice as a result of aging. The periacinar microvasculature is a candidate source for the recruitment of reactive stroma-associated cells, which may be derived either from perivascular-resident mesenchymal stem cells (MSCs) or from an endothelial-to-mesenchymal transition (EndMT) process. Thus, antiangiogenic therapy is a promising approach for preventing age-associated prostate malignancies by means of its negative interference in the development of reactive stroma phenotype from the vascular wall. © 2015 Wiley Periodicals, Inc.

Different protective actions of losartan and tempol on the renal inflammatory response to acute sodium overload.

PubMed

Rosón, María I; Della Penna, Silvana L; Cao, Gabriel; Gorzalczany, Susana; Pandolfo, Marcela; Toblli, Jorge E; Fernández, Belisario E

2010-07-01

The aim of this work was to study the role of local intrarenal angiotensin II (Ang II) and the oxidative stress in the up-regulation of pro-inflammatory cytokines expression observed in rats submitted to an acute sodium overload. Sprague-Dawley rats were infused for 2 h with isotonic saline solution (Control group) and with hypertonic saline solution alone (Na group), plus the AT1 receptor antagonist losartan (10 mg kg(-1) in bolus) (Na-Los group), or plus the superoxide dismutase mimetic tempol (0.5 mg min(-1) kg(-1)) (Na-Temp group). Mean arterial pressure, glomerular filtration rate, and fractional sodium excretion (FE(Na)) were measured. Ang II, NF-kappaB, hypoxia inducible factor-1 alpha (HIF-1 alpha), transforming growth factor beta1 (TGF-beta1), smooth muscle actin (alpha-SMA), endothelial nitric oxide synthase (eNOS), and RANTES renal expression was evaluated by immunohistochemistry. Ang II, NF-kappaB, and TGF-beta1 and RANTES early inflammatory markers were overexpressed in Na group, accompanied by enhanced HIF-1 alpha immunostaining, lower eNOS expression, and unmodified alpha-SMA. Losartan and tempol increased FE(Na) in sodium overload group. Although losartan reduced Ang II and NF-kappaB staining and increased eNOS expression, it did not restore HIF-1 alpha expression and did not prevent inflammation. Conversely, tempol increased eNOS and natriuresis, restored HIF-1 alpha expression, and prevented inflammation. Early inflammatory markers observed in rats with acute sodium overload is associated with the imbalance between HIF-1 alpha and eNOS expression. While both losartan and tempol increased natriuresis and eNOS expression, only tempol was effective in restoring HIF-1 alpha expression and down-regulating TGF-beta1 and RANTES expression. The protective role of tempol, but not of losartan, in the inflammatory response may be associated with its greater antioxidant effects. (c) 2010 Wiley-Liss, Inc.

Arousal of cancer-associated stroma: overexpression of palladin activates fibroblasts to promote tumor invasion.

PubMed

Brentnall, Teresa A; Lai, Lisa A; Coleman, Joshua; Bronner, Mary P; Pan, Sheng; Chen, Ru

2012-01-01

Cancer-associated fibroblasts, comprised of activated fibroblasts or myofibroblasts, are found in the stroma surrounding solid tumors. These myofibroblasts promote invasion and metastasis of cancer cells. Mechanisms regulating the activation of the fibroblasts and the initiation of invasive tumorigenesis are of great interest. Upregulation of the cytoskeletal protein, palladin, has been detected in the stromal myofibroblasts surrounding many solid cancers and in expression screens for genes involved in invasion. Using a pancreatic cancer model, we investigated the functional consequence of overexpression of exogenous palladin in normal fibroblasts in vitro and its effect on the early stages of tumor invasion. Palladin expression in stromal fibroblasts occurs very early in tumorigenesis. In vivo, concordant expression of palladin and the myofibroblast marker, alpha smooth muscle actin (α-SMA), occurs early at the dysplastic stages in peri-tumoral stroma and progressively increases in pancreatic tumorigenesis. In vitro introduction of exogenous 90 kD palladin into normal human dermal fibroblasts (HDFs) induces activation of stromal fibroblasts into myofibroblasts as marked by induction of α-SMA and vimentin, and through the physical change of cell morphology. Moreover, palladin expression in the fibroblasts enhances cellular migration, invasion through the extracellular matrix, and creation of tunnels through which cancer cells can follow. The fibroblast invasion and creation of tunnels results from the development of invadopodia-like cellular protrusions which express invadopodia proteins and proteolytic enzymes. Palladin expression in fibroblasts is triggered by the co-culture of normal fibroblasts with k-ras-expressing epithelial cells. Overall, palladin expression can impart myofibroblast properties, in turn promoting the invasive potential of these peri-tumoral cells with invadopodia-driven degradation of extracellular matrix. Palladin expression in fibroblasts can be triggered by k-ras expression in adjacent epithelial cells. This data supports a model whereby palladin-activated fibroblasts facilitate stromal-dependent metastasis and outgrowth of tumorigenic epithelium.

Zinc supplementation suppresses the progression of bile duct ligation-induced liver fibrosis in mice.

PubMed

Shi, Fang; Sheng, Qin; Xu, Xinhua; Huang, Wenli; Kang, Y James

2015-09-01

Metallothionein (MT) gene therapy leads to resolution of liver fibrosis in mouse model, in which the activation of collagenases is involved in the regression of liver fibrosis. MT plays a critical role in zinc sequestration in the liver suggesting its therapeutic effect would be mediated by zinc. The present study was undertaken to test the hypothesis that zinc supplementation suppresses liver fibrosis. Male Kunming mice subjected to bile duct ligation (BDL) resulted in liver fibrosis as assessed by increased α-smooth muscle actin (α-SMA) and collagen I production/deposition in the liver. Zinc supplementation was introduced 4 weeks after BDL surgery via intragastric administration once daily for 2 weeks resulting in a significant reduction in the collagen deposition in the liver and an increase in the survival rate. Furthermore, zinc suppressed gene expression of α-SMA and collagen I and enhanced the capacity of collagen degradation, as determined by the increased activity of total collagenases and elevated mRNA and protein levels of MMP13. Therefore, the results demonstrate that zinc supplementation suppresses BDL-induced liver fibrosis through both inhibiting collagen production and enhancing collagen degradation. © 2014 by the Society for Experimental Biology and Medicine.

CD248/endosialin critically regulates hepatic stellate cell proliferation during chronic liver injury via a PDGF-regulated mechanism.

PubMed

Wilhelm, Annika; Aldridge, Victoria; Haldar, Debashis; Naylor, Amy J; Weston, Christopher J; Hedegaard, Ditte; Garg, Abhilok; Fear, Janine; Reynolds, Gary M; Croft, Adam P; Henderson, Neil C; Buckley, Christopher D; Newsome, Philip N

2016-07-01

CD248 (endosialin) is a stromal cell marker expressed on fibroblasts and pericytes. During liver injury, myofibroblasts are the main source of fibrotic matrix. To determine the role of CD248 in the development of liver fibrosis in the rodent and human setting. CD248 expression was studied by immunostaining and quantitative PCR in both normal and diseased human and murine liver tissue and isolated hepatic stellate cells (HSCs). Hepatic fibrosis was induced in CD248(-/-) and wild-type controls with carbon tetrachloride (CCl4) treatment. Expression of CD248 was seen in normal liver of humans and mice but was significantly increased in liver injury using both immunostaining and gene expression assays. CD248 was co-expressed with a range of fibroblast/HSC markers including desmin, vimentin and α-smooth muscle actin (α-SMA) in murine and human liver sections. CD248 expression was restricted to isolated primary murine and human HSC. Collagen deposition and α-SMA expression, but not inflammation and neoangiogenesis, was reduced in CD248(-/-) mice compared with wild-type mice after CCl4 treatment. Isolated HSC from wild-type and CD248(-/-) mice expressed platelet-derived growth factor receptor α (PDGFR-α) and PDGFR-β at similar levels. As expected, PDGF-BB stimulation induced proliferation of wild-type HSC, whereas CD248(-/-) HSC did not demonstrate a proliferative response to PDGF-BB. Abrogated PDGF signalling in CD248(-/-) HSC was confirmed by significantly reduced c-fos expression in CD248(-/-) HSC compared with wild-type HSC. Our data show that deletion of CD248 reduces susceptibility to liver fibrosis via an effect on PDGF signalling, making it an attractive clinical target for the treatment of liver injury. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Anti-fibrotic effects of thalidomide on hepatic stellate cells and dimethylnitrosamine-intoxicated rats.

PubMed

Chong, Lee-Won; Hsu, Yi-Chao; Chiu, Yung-Tsung; Yang, Kuo-Ching; Huang, Yi-Tsau

2006-05-01

Tumor necrosis factor-alpha (TNF-alpha) plays a central role in cellular necrosis, apoptosis, organ failure, tissue damage, inflammation and fibrosis. These processes, occurring in liver injury, may lead to cirrhosis. Thalidomide, alpha-N-phthalidoglutarimide, (C(13)H(10)N(2))(4), has been shown to have immunomodulatory and anti-inflammatory properties, possibly mediated through its anti-TNF-alpha effect. In this study, we investigated the in vitro and in vivo effects of thalidomide on hepatic fibrosis. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with transforming growth factor-beta1 (TGF-beta1) or TNF-alpha. The inhibitory effects of thalidomide on the NFkappaB signaling cascade and fibrosis markers including alpha-smooth muscle actin (alpha-SMA) and collagen, were assessed. An in vivo therapeutic study was conducted in dimethylnitrosamine (DMN)-treated rats, which were randomly assigned to 1 of 4 groups: vehicle (0.7% carboxyl methyl cellulose, CMC), thalidomide (40 mg/kg), thalidomide (200 mg/kg), or silymarin (50 mg/kg), each given by gavage twice daily for 3 weeks starting after 1 week of DMN administration. Thalidomide (100-800 nM) concentration-dependently inhibited NFkappaB transcriptional activity induced by TNF-alpha, including IKKalpha expression and IkappaBalpha phosphorylation in HSC-T6 cells. In addition, thalidomide also suppressed TGF-beta1-induced alpha-SMA expression and collagen deposition in HSC-T6 cells. Fibrosis scores of livers from DMN-treated rats receiving high dose of thalidomide (0.89 +/- 0.20) were significantly reduced in comparison with those of DMN-treated rats receiving vehicle (1.56 +/- 0.18). Hepatic collagen contents of DMN rats were also significantly reduced by either thalidomide or silymarin treatment. Immunohistochemical double staining results showed that alpha-SMA- and NFkappaB-positive cells were decreased in the livers from DMN rats receiving either thalidomide or silymarin treatment. In addition, real-time PCR analysis indicated that hepatic mRNA expressions of TGF-beta1, alpha-SMA, collagen 1alpha2, TNF-alpha and iNOS genes were attenuated by thalidomide treatment. In conclusion, our results showed that thalidomide inhibited activation of HSC-T6 cells by TNF-alpha and ameliorated liver fibrosis in DMN-intoxicated rats.

Extracorporeal shock wave therapy with low-energy flux density inhibits hypertrophic scar formation in an animal model.

PubMed

Zhao, Jing-Chun; Zhang, Bo-Ru; Hong, Lei; Shi, Kai; Wu, Wei-Wei; Yu, Jia-Ao

2018-04-01

Hypertrophic scar is characterized by excessive deposits of collagen during skin wound healing, which could become a challenge to clinicians. This study assessed the effects of the extracorporeal shock wave therapy (ESWT) on hypertrophic scar formation and the underlying gene regu-lation. A rabbit ear hypertrophic scar model was generated and randomly divided into three groups: L-ESWT group to receive L-ESWT (energy flux density of 0.1 mJ/mm2), H-ESWT (energy flux density of 0.2 mJ/mm2) and sham ESWT group (S-ESWT). Hypertrophic scar tissues were then collected and stained with hematoxylin and eosin (H&E) and Masson's trichrome staining, respectively, to assess scar elevation index (SEI), fibroblast density and collagen fiber arrangement. Expression of cell proliferation marker proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (α-SMA) were assessed using RT-PCR and immunohistochemistry in hypertrophic scar tissues. H&E staining sections showed significant reduction of SEI and fibroblast density in both ESWT treatment groups compared to S-ESWT, but there was no dramatic difference between L-ESWT and H-ESWT groups. Masson's trichrome staining showed that collagen fibers were more slender and broader and oriented in parallel to skin surface after administration of ESWT compared to control tissues. At the gene level, PCNA‑positive fibroblasts and α-SMA-positive myofibroblasts were significantly decreased after L-ESWT or H-ESWT compared to the controls. Furthermore, there was no significant difference in expression of PCNA mRNA between L-ESWT or H-ESWT and S-ESWT, whereas expression of α-SMA mRNA significantly decreased in L-ESWT compared to that of H-ESWT and S-ESWT (P=0.002 and P=0.030, respectively). In conclusion, L-ESWT could be effective on suppression of hypertrophic scar formation by inhibition of scar elevation index and fibroblast density as well as α-SMA expression in hypertrophic scar tissues of the rabbit model.

Activin B regulates adipose-derived mesenchymal stem cells to promote skin wound healing via activation of the MAPK signaling pathway.

PubMed

Zhang, Lei; Xu, Pengcheng; Wang, Xueer; Zhang, Min; Yan, Yuan; Chen, Yinghua; Zhang, Lu; Zhang, Lin

2017-06-01

Adipose-derived stem cells (ADSCs) are multipotent stromal cells that can differentiate into a variety of cell types, including skin cells, and they can provide an abundant source of cells for skin tissue engineering and skin wound healing. The purpose of this study is to explore the therapeutic effects of activin B in combination with ADSCs and the possible signaling mechanism. In this study, we found that activin B was able to promote ADSC migration by inducing actin stress fiber formation in vitro. In vivo, activin B in combination with ADSCs was capable of enhancing α-SMA expression and wound closure. This combined treatment also promoted fibroblast and keratinocyte proliferation and accelerated re-epithelialization and collagen deposition. Moreover, activin B in combination with ADSCs boosted angiogenesis in the wound area. Further study of the mechanism revealed that activation of JNK and ERK signaling, but not p38 signaling, were required for activin B-induced ADSC actin stress fiber formation and cell migration. These results showed that activin B was able to activate JNK and ERK signaling pathways to induce actin stress fiber formation and ADSC migration to promote wound healing. These results suggest that combined treatment with activin B and ADSCs is a promising therapeutic strategy for the management of serious skin wounds. Copyright © 2017. Published by Elsevier Ltd.

Abnormalities in early markers of muscle involvement support a delay in myogenesis in spinal muscular atrophy.

PubMed

Martínez-Hernández, Rebeca; Bernal, Sara; Alias, Laura; Tizzano, Eduardo F

2014-06-01

Spinal muscular atrophy (SMA) is characterized by loss of motor neurons in the spinal cord that results in muscle denervation and profound weakness in affected patients. We sought evidence for primary muscle involvement in the disease during human development by analyzing the expression of several muscle cytoskeletal components (i.e. slow, fast, and developmental myosin, desmin, and vimentin) in fetal or postnatal skeletal muscle samples from 5 SMA cases and 6 controls. At 14 weeks' gestation, SMA samples had higher percentages of myotubes expressing fast myosin and lower percentages of myotubes expressing slow myosin versus control samples. Desmin and vimentin were highly expressed at prenatal stages without notable differences between control and SMA samples, although both proteins showed persistent immunostaining in atrophic fibers in postnatal SMA samples. We also studied the expression of Pax7-positive nuclei as a marker of satellite cells and found no differences between control and SMA prenatal samples. There was, however, a significant increase in satellite cells in postnatal atrophic SMA fibers, suggesting an abnormal myogenic process. Together, these results support the hypothesis of a delay in muscle maturation as one of the primary pathologic components of SMA. Furthermore, myosins and Pax7 may be useful research markers of muscle involvement in this disease.

Effects of increased Kindlin-2 expression in bladder cancer stromal fibroblasts.

PubMed

Wu, Jitao; Yu, Cuicui; Cai, Li; Lu, Youyi; Jiang, Lei; Liu, Chu; Li, Yongwei; Feng, Fan; Gao, Zhenli; Zhu, Zhe; Yu, Shengqiang; Yuan, Hejia; Cui, Yuanshan

2017-08-01

Kindlin-2 is a focal adhesion protein highly expressed in bladder cancer stromal fibroblasts. We investigated the prognostic significance of Kindlin-2 in bladder cancer stromal fibroblasts and evaluated the effects of Kindlin-2 on the malignant behaviors of tumor cells. Immunohistochemical staining of 203 paraffin-embedded bladder cancer tissues showed that Kindlin-2 expression correlated with advanced stage, high grade, and relapse of bladder cancer. Kaplan-Meier survival analysis demonstrated that patients exhibiting high Kindlin-2 expression had shorter survival times than those with low Kindlin-2 expression ( p < 0.01). Multivariate analysis revealed that high Kindlin-2 expression leads to poor prognosis in bladder cancer. Using cancer-associated fibroblasts (CAFs) isolated from human bladder cancer tissue, we observed that Kindlin-2 knockdown decreased CAFs activation, resulting in decreased expression of α-smooth muscle actin (α-SMA) and the extracellular matrix protein fibronectin. Kindlin-2 suppression also reduced CAF-induced bladder cancer cell migration and invasion. Moreover, we found that Kindlin-2 activates CAFs and promotes the invasiveness of bladder cancer cells by stimulating TGF-β-induced epithelial-mesenchymal transition. These results support targeting Kindlin-2 and the corresponding activated CAFs in bladder cancer therapy.

Effects of vitamin E on mercuric chloride-induced renal interstitial fibrosis in rats and the antioxidative mechanism.

PubMed

Tao, Yan-yan; Wang, Qing-lan; Yuan, Ji-li; Shen, Li; Liu, Cheng-hai

2011-02-01

To observe the effects of vitamin E (Vit E) on mercuric chloride (HgCl2)-induced renal interstitial fibrosis (RIF) in rats and discuss its antioxidative mechanism. A total of 32 Sprague-Dawley rats were randomly assigned to three groups: normal group, model group and Vit E group. RIF was induced by oral administration of HgCl(2) at a dose of 8 mg/kg body weight once a day for 9 weeks. Rats in Vit E group were administered with Vit E capsule at 100 mg/kg body weight, and rats in normal and model groups were treated with normal saline. At the end of the 9th week, rats were sacrificed and renal hydroxyproline (Hyp)'s trichrome and periodic acid-silver methenamine (PASM) staining. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and contents of glutathione (GSH) and malondialdehyde (MDA) in kidney tissue were tested with commercial kits. The expressions of nuclear factor-κB (NF-κB), inhibitor-κB (IκB), phospho-IκB (p-IκB) and tumor necrosis factor-α (TNF-α) were determined by Western blot. The expression of α-smooth muscle actin (α-SMA) was assayed by Western blot and immunofluorescent staining. Renal Hyp content, HE, Masson's trichrome and PASM staining results and α-SMA expression confirmed development of HgCl2-induced RIF in rats. Oxidative stress markers GSH, GSH-Px and MDA confirmed oxidative stress in RIF rats. Compared with model rats, rats in Vit E group had lower kidney Hyp content (P<0.01). GSH and MDA contents decreased significantly in Vit E group compared with model group (P<0.01). The expressions of NF-κB and IκB had no significant difference among all groups (P>0.05). In Vit E group, the expressions of p-IκB and TNF-α decreased significantly compared with model group (P<0.01). The expression of α-SMA in Vit E group was also decreased significantly compared with model group (P<0.01). Vit E has a protective effect on experimental RIF induced by HgCl(2) in rats and it is related to inhibition of lipid peroxidation, which involves blocking of NF-κB signaling pathway and the activation of cells producing extracellular matrix.

Dataset on transcriptional profiles and the developmental characteristics of PDGFRα expressing lung fibroblasts.

PubMed

Endale, Mehari; Ahlfeld, Shawn; Bao, Erik; Chen, Xiaoting; Green, Jenna; Bess, Zach; Weirauch, Matthew; Xu, Yan; Perl, Anne Karina

2017-08-01

The following data are derived from key stages of acinar lung development and define the developmental role of lung interstitial fibroblasts expressing platelet-derived growth factor alpha (PDGFRα). This dataset is related to the research article entitled "Temporal, spatial, and phenotypical changes of PDGFRα expressing fibroblasts during late lung development" (Endale et al., 2017) [1]. At E16.5 (canalicular), E18.5 (saccular), P7 (early alveolar) and P28 (late alveolar), PDGFRα GFP mice, in conjunction with immunohistochemical markers, were utilized to define the spatiotemporal relationship of PDGFRα + fibroblasts to endothelial, stromal and epithelial cells in both the proximal and distal acinar lung. Complimentary analysis with flow cytometry was employed to determine changes in cellular proliferation, define lipofibroblast and myofibroblast populations via the presence of intracellular lipid or alpha smooth muscle actin (αSMA), and evaluate the expression of CD34, CD29, and Sca-1. Finally, PDGFRα + cells isolated at each stage of acinar lung development were subjected to RNA-Seq analysis, data was subjected to Bayesian timeline analysis and transcriptional factor promoter enrichment analysis.

Effect of Yiguanjian decoction on cell differentiation and proliferation in CCl4-treated mice

PubMed Central

Wang, Xiao-Ling; Jia, Dong-Wei; Liu, Hui-Yang; Yan, Xiao-Feng; Ye, Ting-Jie; Hu, Xu-Dong; Li, Bo-Qin; Chen, Yong-Liang; Liu, Ping

2012-01-01

AIM: To investigate the cellular mechanisms of action of Yiguanjian (YGJ) decoction in treatment of chronic hepatic injury. METHODS: One group of mice was irradiated, and received enhanced green fluorescent protein (EGFP)-positive bone marrow transplants followed by 13 wk of CCl4 injection and 6 wk of oral YGJ administration. A second group of Institute for Cancer Research mice was treated with 13 wk of CCl4 injection and 6 wk of oral YGJ administration. Liver function, histological changes in the liver, and Hyp content were analyzed. The expression of α-smooth muscle actin (α-SMA), F4/80, albumin (Alb), EGFP, mitogen-activated protein kinase-2 (PKM2), Ki-67, α fetoprotein (AFP), monocyte chemotaxis protein-1 and CC chemokine receptor 2 were assayed. RESULTS: As hepatic damage progressed, EGFP-positive marrow cells migrated into the liver and were mainly distributed along the fibrous septa. They showed a conspicuous coexpression of EGFP with α-SMA and F4/80 but no coexpression with Alb. Moreover, the expression of PKM2, AFP and Ki-67 was enhanced dynamically and steadily over the course of liver injury. YGJ abrogated the increases in the number of bone marrow-derived fibrogenic cells in the liver, inhibited expression of both progenitor and mature hepatocyte markers, and reduced fibrogenesis. CONCLUSION: YGJ decoction improves liver fibrosis by inhibiting the migration of bone marrow cells into the liver as well as inhibiting their differentiation and suppressing the proliferation of both progenitors and hepatocytes in the injured liver. PMID:22783047

Bone marrow-derived mesenchymal stem cells effectively regenerate fibrotic liver in bile duct ligation rat model

PubMed Central

Elswefy, Sahar E; Rashed, Laila A; Younis, Nahla N; Shaheen, Mohamed A; Ghanim, Amal MH

2016-01-01

Mesenchymal stem cells (MSCs) have attracted lots of attention for the treatment of acute liver failure and end-stage liver diseases. This study aimed at investigating the fundamental mechanism by which bone marrow-derived MSCs (BM-MSCs) induce liver regeneration of fibrotic liver in rats. Rats underwent bile duct ligation (BDL) surgery and four weeks later they were treated with either BM-MSCs (3 × 106 cells /rat, once, tail vein injection) or silymarin (100 mg/kg, daily, orally) for four weeks. Liver function tests and hepatic oxidative stress were determined. Hepatic injury and fibrosis were assessed by H and E, Sirus red staining and immunohistochemical expression of α-smooth muscle actin (α-SMA). Hepatocyte growth factor (HGF) and the gene expression of cytokeratin-19 (CK-19) and matrix metalloproteinase-2 (MMP-2) in liver tissue were determined. BDL induced cholestatic liver injury characterized by elevated ALT and AST activities, bilirubin and decreased albumin. The architecture damage was staged as Metavir score: F3, A3. Fibrosis increased around proliferating bile duct as indicated by sirus red staining and α-SMA immunostaining. Fibrogenesis was favored over fibrolysis and confirmed by decreased HGF with increased expression of CK-19, but decreased MMP-2 expression. BM-MSCs treatment restored deteriorated liver functions and restored the histological changes, resolved fibrosis by improving liver regenerative capabilities (P < 0.001), increases in HGF and MMP-2 mRNA and downregulating CK-19 mRNA. Sliymarin, however, induced similar but less prominent effects compared to BM-MSCs. In conclusion, liver regenerative capabilities can be stimulated by BM-MSCs via augmentation of HGF that subsequently up-regulate MMP-2 mRNA while downregulating CK-19 mRNA. PMID:26811102

Glycogen synthase kinase-3 inhibition attenuates fibroblast activation and development of fibrosis following renal ischemia-reperfusion in mice

PubMed Central

Singh, Shailendra P.; Tao, Shixin; Fields, Timothy A.; Webb, Sydney; Harris, Raymond C.; Rao, Reena

2015-01-01

ABSTRACT Glycogen synthase kinase-3β (GSK3β) is a serine/threonine protein kinase that plays an important role in renal tubular injury and regeneration in acute kidney injury. However, its role in the development of renal fibrosis, often a long-term consequence of acute kidney injury, is unknown. Using a mouse model of renal fibrosis induced by ischemia-reperfusion injury, we demonstrate increased GSK3β expression and activity in fibrotic kidneys, and its presence in myofibroblasts in addition to tubular epithelial cells. Pharmacological inhibition of GSK3 using TDZD-8 starting before or after ischemia-reperfusion significantly suppressed renal fibrosis by reducing the myofibroblast population, collagen-1 and fibronectin deposition, inflammatory cytokines, and macrophage infiltration. GSK3 inhibition in vivo reduced TGF-β1, SMAD3 activation and plasminogen activator inhibitor-1 levels. Consistently in vitro, TGF-β1 treatment increased GSK3β expression and GSK3 inhibition abolished TGF-β1-induced SMAD3 activation and α-smooth muscle actin (α-SMA) expression in cultured renal fibroblasts. Importantly, overexpression of constitutively active GSK3β stimulated α-SMA expression even in the absence of TGF-β1 treatment. These results suggest that TGF-β regulates GSK3β, which in turn is important for TGF-β–SMAD3 signaling and fibroblast-to-myofibroblast differentiation. Overall, these studies demonstrate that GSK3 could promote renal fibrosis by activation of TGF-β signaling and the use of GSK3 inhibitors might represent a novel therapeutic approach for progressive renal fibrosis that develops as a consequence of acute kidney injury. PMID:26092126

Simultaneous application of bevacizumab and anti-CTGF antibody effectively suppresses proangiogenic and profibrotic factors in human RPE cells.

PubMed

Bagheri, Abouzar; Soheili, Zahra-Soheila; Ahmadieh, Hamid; Samiei, Shahram; Sheibani, Nader; Astaneh, Shamila Darvishalipour; Kanavi, Mozhgan Rezaei; Mohammadian, Azam

2015-01-01

Retinal pigment epithelial (RPE) cells play key roles in the development of choroidal neovascularization and subsequent fibrosis. We investigated the impact of bevacizumab, antihuman vascular endothelial growth factor (VEGF) antibody, and anticonnective tissue growth factor (anti-CTGF) neutralizing antibody, individually or in combination, on proangiogenic and profibrotic properties of RPE cells. Primary cultures of human RPE cells were incubated with different concentrations of bevacizumab (0.25, 0.5, and 0.8 mg/ml) and/or anti-CTGF (10 μg/ml), and cell proliferation and apoptosis were determined. Expression and activity of proangiogenic and profibrotic genes including matrix metalloproteinases (MMP)-2 and 9, VEGFA, CTGF, vascular endothelial growth factor receptor-1 (VEGFR-1), cathepsin D, tissue inhibitor of metalloproteinases (TIMP) -1 and -2, and alpha smooth muscle actin (α-SMA) were assessed with slot blot, real-time RT-PCR, and zymography. Bevacizumab alone inhibited proliferation of RPE cells while anti-CTGF or bevacizumab and anti-CTGF combined had no inhibitory effect in this regard. Bevacizumab increased MMP-2, MMP-9, and cathepsin D but decreased VEGFA and VEGFR-1 expression. The CTGF level was increased by using 0.25 mg/ml bevacizumab but decreased at the 0.8 mg/ml concentration of bevacizumab. Treatment with anti-CTGF antibody decreased MMP-2 expression whereas combined treatment with bevacizumab and anti-CTGF resulted in decreased expression of MMP-2, TIMP-1, cathepsin D, VEGFA, CTGF, and α-SMA in the treated cultures. Treatment of RPE cells with the combination of bevacizumab and anti-CTGF could effectively suppress the proangiogenic and profibrotic activity of RPE cells.

Simultaneous application of bevacizumab and anti-CTGF antibody effectively suppresses proangiogenic and profibrotic factors in human RPE cells

PubMed Central

Bagheri, Abouzar; Ahmadieh, Hamid; Samiei, Shahram; Sheibani, Nader; Astaneh, Shamila Darvishalipour; Kanavi, Mozhgan Rezaei; Mohammadian, Azam

2015-01-01

Purpose Retinal pigment epithelial (RPE) cells play key roles in the development of choroidal neovascularization and subsequent fibrosis. We investigated the impact of bevacizumab, antihuman vascular endothelial growth factor (VEGF) antibody, and anticonnective tissue growth factor (anti-CTGF) neutralizing antibody, individually or in combination, on proangiogenic and profibrotic properties of RPE cells. Methods Primary cultures of human RPE cells were incubated with different concentrations of bevacizumab (0.25, 0.5, and 0.8 mg/ml) and/or anti-CTGF (10 μg/ml), and cell proliferation and apoptosis were determined. Expression and activity of proangiogenic and profibrotic genes including matrix metalloproteinases (MMP)-2 and 9, VEGFA, CTGF, vascular endothelial growth factor receptor-1 (VEGFR-1), cathepsin D, tissue inhibitor of metalloproteinases (TIMP) −1 and −2, and alpha smooth muscle actin (α-SMA) were assessed with slot blot, real-time RT–PCR, and zymography. Results Bevacizumab alone inhibited proliferation of RPE cells while anti-CTGF or bevacizumab and anti-CTGF combined had no inhibitory effect in this regard. Bevacizumab increased MMP-2, MMP-9, and cathepsin D but decreased VEGFA and VEGFR-1 expression. The CTGF level was increased by using 0.25 mg/ml bevacizumab but decreased at the 0.8 mg/ml concentration of bevacizumab. Treatment with anti-CTGF antibody decreased MMP-2 expression whereas combined treatment with bevacizumab and anti-CTGF resulted in decreased expression of MMP-2, TIMP-1, cathepsin D, VEGFA, CTGF, and α-SMA in the treated cultures. Conclusions Treatment of RPE cells with the combination of bevacizumab and anti-CTGF could effectively suppress the proangiogenic and profibrotic activity of RPE cells. PMID:25883524

Fibroblast growth factor 21 attenuates hepatic fibrogenesis through TGF-β/smad2/3 and NF-κB signaling pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Pengfei; Zhang, Yingjie; Liu, Yunye

2016-01-01

Fibroblast growth factor 21 (FGF-21) is a secreted protein, which has anti-diabetic and lipocaic effects, but its ability to protect against hepatic fibrosis has not been studied. In this study, we investigated the ability of FGF-21 to attenuate dimethylnitrosamine (DMN)-induced hepatic fibrogenesis in mice and the mechanism of its action. Hepatic fibrosis was induced by injection of DMN, FGF-21 was administered to the mice once daily in association with DMN injection till the end of the experiment. Histopathological examination, tissue 4-hydroxyproline content and expressions of smooth muscle α-actin (α-SMA) and collagen I were measured to assess hepatic fibrosis. Ethanol/PDGF-BB-activated hepaticmore » stellate cells (HSCs) were used to understand the mechanisms of FGF-21 inhibited hepatic fibrogenesis. Results showed that FGF-21 treatment attenuated hepatic fibrogenesis and was associated with a significant decrease in intrahepatic fibrogenesis, 4-hydroxyproline accumulation, α-SMA expression and collagen I deposition. FGF-21 treatment inhibited the activation of HSCs via down-regulating the expression of TGF-β, NF-κB nuclear translocation, phosphorylation levels of smad2/3 and IκBα. Besides, FGF-21 treatment caused activated HSC apoptosis with increasing expression of Caspase-3, and decreased the ratio of Bcl-2 to Bax. In conclusion, FGF-21 attenuates hepatic fibrogenesis and inhibits the activation of HSC warranting the use of FGF-21 as a potential therapeutic agent in the treatment of hepatic fibrosis. - Highlights: • Fibroblast growth factor 21 attenuates hepatic fibrogenesis. • Fibroblast growth factor 21 attenuates hepatic fibrogenesis via TGF-β/smad2/3 signaling pathways. • Fibroblast growth factor 21 attenuates hepatic fibrogenesis via NF-κB signaling pathways.« less

Matrix Stiffness Corresponding to Strictured Bowel Induces a Fibrogenic Response in Human Colonic Fibroblasts

PubMed Central

Johnson, Laura A.; Rodansky, Eva S.; Sauder, Kay L.; Horowitz, Jeffrey C.; Mih, Justin D.; Tschumperlin, Daniel J.; Higgins, Peter D.

2013-01-01

Background Crohn’s disease is characterized by repeated cycles of inflammation and mucosal healing which ultimately progress to intestinal fibrosis. This inexorable progression towards fibrosis suggests that fibrosis becomes inflammation-independent and auto-propagative. We hypothesized that matrix stiffness regulates this auto-propagation of intestinal fibrosis. Methods The stiffness of fresh ex vivo samples from normal human small intestine, Crohn’s disease strictures, and the unaffected margin were measured with a microelastometer. Normal human colonic fibroblasts were cultured on physiologically normal or pathologically stiff matrices corresponding to the physiological stiffness of normal or fibrotic bowel. Cellular response was assayed for changes in cell morphology, α-smooth muscle actin (αSMA) staining, and gene expression. Results Microelastometer measurements revealed a significant increase in colonic tissue stiffness between normal human colon and Crohn’s strictures as well as between the stricture and adjacent tissue margin. In Ccd-18co cells grown on stiff matrices corresponding to Crohn’s strictures, cellular proliferation increased. Pathologic stiffness induced a marked change in cell morphology and increased αSMA protein expression. Growth on a stiff matrix induced fibrogenic gene expression, decreased matrix metalloproteinase and pro-inflammatory gene expression, and was associated with nuclear localization of the transcriptional cofactor MRTF-A. Conclusions Matrix stiffness, representative of the pathological stiffness of Crohn’s strictures, activates human colonic fibroblasts to a fibrogenic phenotype. Matrix stiffness affects multiple pathways suggesting the mechanical properties of the cellular environment are critical to fibroblast function and may contribute to autopropagation of intestinal fibrosis in the absence of inflammation, thereby contributing to the intractable intestinal fibrosis characteristic of Crohn’s disease. PMID:23502354

Bone marrow-derived mesenchymal stem cells effectively regenerate fibrotic liver in bile duct ligation rat model.

PubMed

Mohamed, Hoda E; Elswefy, Sahar E; Rashed, Laila A; Younis, Nahla N; Shaheen, Mohamed A; Ghanim, Amal M H

2016-03-01

Mesenchymal stem cells (MSCs) have attracted lots of attention for the treatment of acute liver failure and end-stage liver diseases. This study aimed at investigating the fundamental mechanism by which bone marrow-derived MSCs (BM-MSCs) induce liver regeneration of fibrotic liver in rats. Rats underwent bile duct ligation (BDL) surgery and four weeks later they were treated with either BM-MSCs (3 × 10(6) cells /rat, once, tail vein injection) or silymarin (100 mg/kg, daily, orally) for four weeks. Liver function tests and hepatic oxidative stress were determined. Hepatic injury and fibrosis were assessed by H and E, Sirus red staining and immunohistochemical expression of α-smooth muscle actin (α-SMA). Hepatocyte growth factor (HGF) and the gene expression of cytokeratin-19 (CK-19) and matrix metalloproteinase-2 (MMP-2) in liver tissue were determined. BDL induced cholestatic liver injury characterized by elevated ALT and AST activities, bilirubin and decreased albumin. The architecture damage was staged as Metavir score: F3, A3. Fibrosis increased around proliferating bile duct as indicated by sirus red staining and α-SMA immunostaining. Fibrogenesis was favored over fibrolysis and confirmed by decreased HGF with increased expression of CK-19, but decreased MMP-2 expression. BM-MSCs treatment restored deteriorated liver functions and restored the histological changes, resolved fibrosis by improving liver regenerative capabilities (P < 0.001), increases in HGF and MMP-2 mRNA and downregulating CK-19 mRNA. Sliymarin, however, induced similar but less prominent effects compared to BM-MSCs. In conclusion, liver regenerative capabilities can be stimulated by BM-MSCs via augmentation of HGF that subsequently up-regulate MMP-2 mRNA while downregulating CK-19 mRNA. © 2016 by the Society for Experimental Biology and Medicine.

Arterially Delivered Mesenchymal Stem Cells Prevent Obstruction-Induced Renal Fibrosis

PubMed Central

Asanuma, Hiroshi; Vanderbrink, Brian A.; Campbell, Matthew T.; Hile, Karen L.; Zhang, Hongji; Meldrum, Daniel R.; Meldrum, Kirstan K.

2010-01-01

Purpose Mesenchymal stem cells (MSCs) hold promise for the treatment of renal disease. While MSCs have been shown to accelerate recovery and prevent acute renal failure in multiple disease models, the effect of MSC therapy on chronic obstruction-induced renal fibrosis has not previously been evaluated. Materials and Methods Male Sprague-Dawley rats underwent renal artery injection of vehicle or fluorescent-labeled human bone marrow-derived MSCs immediately prior to sham operation or induction of left ureteral obstruction (UUO). One or 4 weeks later, the kidneys were harvested and the renal cortex analyzed for evidence of stem cell infiltration, epithelial-mesenchymal transition (EMT) as evidenced by E-cadherin/α-smooth muscle actin (α-SMA) expression and fibroblast specific protein (FSP+) staining, renal fibrosis (collagen content, Masson’s trichrome staining), and cytokine and growth factor activity (ELISA and real time RT-PCR). Results Fluorescent-labeled MSCs were detected in the interstitium of the kidney up to 4 weeks post-obstruction. Arterially delivered MSCs significantly reduced obstruction-induced α-SMA expression, FSP+ cell accumulation, total collagen content, and tubulointerstitial fibrosis, while simultaneously preserving E-cadherin expression, suggesting that MSCs prevent obstruction-induced EMT and renal fibrosis. Exogenous MSCs reduced obstruction-induced tumor necrosis factor-α (TNF-α) levels, but did not alter transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF), interleukin-10 (IL-10), fibroblast growth factor (FGF), or hepatocyte growth factor (HGF) expression. Conclusions Human bone marrow-derived MSCs remain viable several weeks after delivery into the kidney and provide protection against obstruction-induced EMT and chronic renal fibrosis. While the mechanism of MSCs-induced renal protection during obstruction remains unclear, our results demonstrate that alterations in TNF-α production may be involved. PMID:20850784

Collagen triple helix repeat containing 1 is a new promigratory marker of arthritic pannus.

PubMed

Shekhani, Mohammed Talha; Forde, Toni S; Adilbayeva, Altynai; Ramez, Mohamed; Myngbay, Askhat; Bexeitov, Yergali; Lindner, Volkhard; Adarichev, Vyacheslav A

2016-07-19

The formation of destructive hypercellular pannus is critical to joint damage in rheumatoid arthritis (RA). The collagen triple helix repeat containing 1 (CTHRC1) protein expressed by activated stromal cells of diverse origin has previously been implicated in tissue remodeling and carcinogenesis. We recently discovered that the synovial Cthrc1 mRNA directly correlates with arthritis severity in mice. This study characterizes the role of CTHRC1 in arthritic pannus formation. Synovial joints of mice with collagen antibody-induced arthritis (CAIA) and human RA-fibroblast-like synoviocytes (FLS) were immunostained for CTHRC1, FLS and macrophage-specific markers. CTHRC1 levels in plasma from patients with RA were measured using sandwich ELISA. The migratory response of fibroblasts was studied with a transwell migration assay and time-lapse microscopy. Velocity and directness of cell migration was analyzed by recording the trajectories of cells treated with rhCTHRC1. Immunohistochemical analysis of normal and inflamed synovium revealed highly inducible expression of CTHRC1 in arthritis (10.9-fold). At the tissue level, CTHRC1-expressing cells occupied the same niche as large fibroblast-like cells positive for α-smooth muscle actin (α-SMA) and cadherin 11 (CDH11). CTHRC1 was produced by activated FLS predominantly located at the synovial intimal lining and at the bone-pannus interface. Cultured RA-FLS expressed CDH11, α-SMA, and CTHRC1. Upon treatment with exogenous rhCTHRC1, embryonic fibroblasts and RA-FLS significantly increased migration velocity, directness, and cell length along the front-tail axis (1.4-fold, p < 0.01). CTHRC1 was established as a novel marker of activated synoviocytes in murine experimental arthritis and RA. The pro-migratory effect of CTHRC1 on synoviocytes is considered one of the mechanisms promoting hypercellularity of the arthritic pannus.

Overexpression of protein kinase C ɛ improves retention and survival of transplanted mesenchymal stem cells in rat acute myocardial infarction.

PubMed

He, H; Zhao, Z-H; Han, F-S; Liu, X-H; Wang, R; Zeng, Y-J

2016-01-21

We assessed the effects of protein kinase C ɛ (PKCɛ) for improving stem cell therapy for acute myocardial infarction (AMI). Primary mesenchymal stem cells (MSCs) were harvested from rat bone marrow. PKCɛ-overexpressed MSCs and control MSCs were transplanted into infarct border zones in a rat AMI model. MSCs and PKCɛ distribution and expression of principal proteins involved in PKCɛ signaling through the stromal cell-derived factor 1 (SDF-1)/CXC chemokine receptor type 4 (CXCR4) axis and the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) pathway were analyzed by immunofluorescence and western blot 1 day after transplantation. Echocardiographic measurements and histologic studies were performed at 4 weeks after transplantation, and MSC survival, expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor β (TGFβ), cardiac troponin I (cTnI), von Willebrand factor (vWF), smooth muscle actin (SMA) and factor VIII and apoptosis in infarct border zones were assessed. Rat heart muscles retained more MSCs and SDF-1, CXCR4, PI3K and phosphorylated AKT increased with PKCɛ overexpression 1 day after transplantation. MSC survival and VEGF, bFGF, TGFβ, cTnI, vWF, SMA and factor VIII expression increased in animals with PKCɛ-overexpressed MSCs at 4 weeks after transplantation and cardiac dysfunction and remodeling improved. Infarct size and apoptosis decreased as well. Inhibitory actions of CXCR4 or PI3K partly attenuated the effects of PKCɛ. Activation of PKCɛ may improve retention, survival and differentiation of transplanted MSCs in myocardia. Augmentation of PKCɛ expression may enhance the therapeutic effects of stem cell therapy for AMI.

Lung tissue remodelling in MCT-induced pulmonary hypertension: a proposal for a novel scoring system and changes in extracellular matrix and fibrosis associated gene expression.

PubMed

Franz, Marcus; Grün, Katja; Betge, Stefan; Rohm, Ilonka; Ndongson-Dongmo, Bernadin; Bauer, Reinhard; Schulze, P Christian; Lichtenauer, Michael; Petersen, Iver; Neri, Dario; Berndt, Alexander; Jung, Christian

2016-12-06

Pulmonary hypertension (PH) is associated with vasoconstriction and remodelling. We studied lung tissue remodelling in a rat model of PH with special focus on histology and extracellular matrix (ECM) remodelling. After induction of PH by monocrotaline, lung tissue was analysed histologically, by gene expression analysis and immunofluorescence labelling of ED-A domain containing fibronectin (ED-A+ Fn), B domain containing tenascin-C (B+ Tn-C) as well as alpha-smooth muscle actin (α-SMA). Serum concentrations of ED-A+ Fn were determined by ELISA. Systolic right ventricular pressure (RVPsys) values were significantly elevated in PH (n = 18; 75 ± 26.4 mmHg) compared to controls (n = 10; 29 ± 19.3 mmHg; p = 0.015). The histological sum-score was significantly increased in PH (8.0 ± 2.2) compared to controls (2.5 ± 1.6; p < 0.001). Gene expression analysis revealed relevant induction of several key genes of extracellular matrix remodelling. Increased protein deposition of ED-A+ Fn but not of B+ Tn-C and α-SMA in lung tissue was found in PH (2.88 ± 3.19 area%) compared to controls (1.32 ± 0.16 area%; p = 0.030). Serum levels of ED-A+ Fn were significantly higher in PH (p = 0.007) positively correlating with RVPsys (r = 0.618, p = 0.019). We here present a novel histological scoring system to assess lung tissue remodelling in PH. Gene expression analysis revealed induction of candidate genes involved in collagen matrix turnover, fibrosis and vascular remodelling. The stable increased tissue deposition of ED-A+ Fn in PH as well as its dynamics in serum suggests a role as a promising novel biomarker and potential therapeutic target.

Activation of cardiac fibroblasts by ethanol is blocked by TGF-β inhibition.

PubMed

Law, Brittany A; Carver, Wayne E

2013-08-01

Alcohol abuse is the second leading cause of dilated cardiomyopathy, a disorder specifically referred to as alcoholic cardiomyopathy (ACM). Rodent and human studies have revealed cardiac fibrosis to be a consequence of ACM, and prior studies by this laboratory have associated this occurrence with elevated transforming growth factor-beta (TGF-β) and activated fibroblasts (myofibroblasts). To date, there have been no other studies to investigate the direct effect of alcohol on the cardiac fibroblast. Primary rat cardiac fibroblasts were cultured in the presence of ethanol (EtOH) and assayed for fibroblast activation by collagen gel contraction, alpha-smooth muscle actin (α-SMA) expression, migration, proliferation, apoptosis, collagen I and III, and TGF-β expression. The TGF-β receptor type 1 inhibitor compound SB 431542 and a soluble recombinant TGF-βII receptor (RbII) were used to assess the role of TGF-β in the response of cardiac fibroblasts to EtOH. Treatment for cardiac fibroblasts with EtOH at concentrations of 100 mg/dl or higher resulted in fibroblast activation and fibrogenic activity after 24 hours including an increase in contraction, α-SMA expression, migration, and expression of collagen I and TGF-β. No changes in fibroblast proliferation or apoptosis were observed. Inhibition of TGF-β by SB 431542 and RbII attenuated the EtOH-induced fibroblast activation. EtOH treatment directly promotes cardiac fibroblast activation by stimulating TGF-β release from fibroblasts. Inhibiting the action of TGF-β decreases the fibrogenic effect induced by EtOH treatment. The results of this study support TGF-β to be an important component in cardiac fibrosis induced by exposure to EtOH. Copyright © 2013 by the Research Society on Alcoholism.

Activation of cardiac fibroblasts by ethanol is blocked by TGF-β inhibition

PubMed Central

Law, Brittany A.; Carver, Wayne E.

2013-01-01

Background Alcohol abuse is the second leading cause of dilated cardiomyopathy, a disorder specifically referred to as Alcoholic Cardiomyopathy (ACM). Rodent and human studies have revealed cardiac fibrosis to be a consequence of ACM and prior studies by this lab have associated this occurrence with elevated transforming growth factor-beta (TGF-β) and activated fibroblasts (myofibroblasts). To date there have been no other studies to investigate the direct effect of alcohol on the cardiac fibroblast. Methods Primary rat cardiac fibroblasts were cultured in the presence of ethanol and assayed for fibroblast activation by collagen gel contraction, alpha smooth muscle- actin (α-SMA) expression, migration, proliferation, apoptosis, collagen I & III and TGF-β expression. The TGF-β receptor type 1 inhibitor compound SB 431542 and a soluble recombinant TGF-βII receptor (RbII) were used to assess the role of of TGF-β in the response of cardiac fibroblasts to ethanol. Results Treatment of cardiac fibroblasts with ethanol at concentrations of 100 mg/dl or higher resulted in fibroblast activation and fibrogenic activity after 24 hours including an increase in contraction, α-SMA expression, migration, and expression of collagen I and TGF-β. No changes in fibroblast proliferation or apoptosis were observed. Inhibition of TGF-β by SB 431542 and RbII attenuated the ethanol-induced fibroblast activation. Conclusions Ethanol treatment directly promotes cardiac fibroblast activation by stimulating TGF-β release from fibroblasts. Inhibiting the action of TGF-β decreases the fibrogenic effect induced by ethanol treatment. The results of this study support TGF-β to be an important component in cardiac fibrosis induced by exposure to ethanol. PMID:23528014

Morphology and contractile gene expression of adipose-derived mesenchymal stem cells in response to short-term cyclic uniaxial strain and TGF-β1.

PubMed

Rashidi, Neda; Tafazzoli-Shadpour, Mohammad; Haghighipour, Nooshin; Khani, Mohammad-Mehdi

2018-06-27

Previous studies have shown smooth muscle induction in adipose-derived mesenchymal stem cells (ASCs) caused by long-term cyclic stretch. Here we examined the capability of the short-term straining with time steps of 4, 8, 16 and 24 h alone or combined with TGF-β1 on smooth muscle induction of rabbit ASCs. Alterations in cell morphology were quantified through the cell shape index and orientation angle, and expression levels of α-SMA, SM22-α, h-caldesmon and calponin3 markers were examined using the real-time polymerase chain reaction (PCR) method. Moreover, F-actin cytoskeleton organization was observed by fluorescence staining. Mechanical strain either alone or combined with growth factor treatment caused significant up-regulation of both early and intermediate smooth muscle cells (SMCs) specific markers during the initial hours of stimulation peaking in 8 to 16 h. Furthermore, gradual alignment of cells perpendicular to the strain direction during loading time, and cell elongation resembling contractile SMC phenotype, together with alignment and reorganization of F-actin fibers were observed. Considering previously reported protein up-regulation in following days of straining, the effects of short-term cyclic stretch on smooth muscle induction of ASCs were revealed which can be helpful in achieving functional contractile SMCs through synergistic mechano-chemical regulation of ASCs as an appealing cell source for vascular tissue engineering.

Asbestos exposure induces alveolar epithelial cell plasticity through MAPK/Erk signaling.

PubMed

Tamminen, Jenni A; Myllärniemi, Marjukka; Hyytiäinen, Marko; Keski-Oja, Jorma; Koli, Katri

2012-07-01

The inhalation of asbestos fibers is considered to be highly harmful, and lead to fibrotic and/or malignant disease. Epithelial-to-mesenchymal transition (EMT) is a common pathogenic mechanism in asbestos associated fibrotic (asbestosis) and malignant lung diseases. The characterization of molecular pathways contributing to EMT may provide new possibilities for prognostic and therapeutic applications. The role of asbestos as an inducer of EMT has not been previously characterized. We exposed cultured human lung epithelial cells to crocidolite asbestos and analyzed alterations in the expression of epithelial and mesenchymal marker proteins and cell morphology. Asbestos was found to induce downregulation of E-cadherin protein levels in A549 lung carcinoma cells in 2-dimensional (2D) and 3D cultures. Similar findings were made in primary small airway epithelial cells cultured in 3D conditions where the cells retained alveolar type II cell phenotype. A549 cells also exhibited loss of cell-cell contacts, actin reorganization and expression of α-smooth muscle actin (α-SMA) in 2D cultures. These phenotypic changes were not associated with increased transforming growth factor (TGF)-β signaling activity. MAPK/Erk signaling pathway was found to mediate asbestos-induced downregulation of E-cadherin and alterations in cell morphology. Our results suggest that asbestos can induce epithelial plasticity, which can be interfered by blocking the MAPK/Erk kinase activity. Copyright © 2012 Wiley Periodicals, Inc.

Notch as a Diagnostic Marker and Therapeutic Target in Human Breast Cancer

DTIC Science & Technology

2009-09-01

PCR. Mm5MT transfectants were cultured 7 d with vehicle or 1 Amol /L PD166866 [inhibitor of FGF receptor (FGFR) kinase], total RNA isolated (RNeasy mini...analysis. Paraffin-embedded sections (5 Amol /L) were immunostained for CD-31/PECAM and a-smooth muscle actin (aSMA). To detect apoptosis [terminal

[Protective effect of pretreatment of Salvia miltiorrhiza Bunge. f. alba plasma against oxygen-glucose deprivation-induced injury of cultured rat hippocampal neurons by inhibiting apoptosis].

PubMed

Li, Mei-Yi; Zhang, Yan-Bo; Zuo, Huan; Liu, Li-Li; Niu, Jing-Zhong

2012-02-25

The present study was to investigate the effect of Salvia miltiorrhiza Bunge. f. alba (SMA) pharmacological pretreatment on apoptosis of cultured hippocampal neurons from neonate rats under oxygen-glucose deprivation (OGD). Cultured hippocampal neurons were randomly divided into five groups (n = 6): normal plasma group, low dose SMA plasma (2.5%) group, middle dose SMA plasma (5%) group, high dose SMA plasma (10%) group and control group. The hippocampal neurons were cultured and treated with plasma from adult Wistar rats intragastrically administered with saline or aqueous extract of SMA. The apoptosis of neurons was induced by glucose-free Earle's solution containing 1 mmol/L Na2S2O4 and labeled by MTT and Annexin V/PI double staining. Moreover, protein expressions of Bcl-2 and Bax were detected by immunofluorescence. The results showed that few apoptotic cells were observed in control group, whereas the number of apoptotic cells was greatly increased in normal plasma group and low dose SMA plasma group. Both middle and high dose SMA plasma could protect cultured hippocampal neurons from apoptosis induced by OGD (P < 0.05). The protective effect of high dose SMA plasma was stronger than that of middle one (P < 0.05). Compared to control, normal plasma and low dose SMA plasma groups, middle and high dose SMA plasma groups both showed significantly higher levels of Bcl-2 (P < 0.05 or 0.01), whereas expressions of Bax was opposite. There were no significant differences of Bcl-2 and Bax expressions between middle and high dose SMA plasma groups. Number of Bcl-2- and Bax-positive cells had similar tendency. Bcl-2/Bax (number of positive cells) ratio was higher in high dose SMA plasma group than those of all the other groups (P < 0.05 or 0.01). These results suggest that pharmacological pretreatment of blood plasma containing middle and high dose SMA could raise viability and inhibit apoptosis of OGD-injured hippocampal neurons by up-regulating the expression of Bcl-2 and down-regulating the expression of Bax.

Thalidomide in rat liver cirrhosis: blockade of tumor necrosis factor-alpha via inhibition of degradation of an inhibitor of nuclear factor-kappaB.

PubMed

Paul, Shelley Chireyath; Lv, Peng; Xiao, Yan-Jv; An, Ping; Liu, Shi-Quan; Luo, He-Sheng

2006-01-01

Thalidomide inhibited tumor necrosis factor-alpha (TNF-alpha) effectively in many trials. The aim of this study was to investigate the effect of thalidomide on the expression of nuclear factor-kappaB (NF-kappaB), inhibitor of NF-kappaB (IkappaB) and TNF-alpha in a rat model of liver cirrhosis. Liver cirrhosis was achieved by intraperitoneal injection of carbon tetrachloride thrice weekly, and thalidomide (10 or 100 mg/kg/day) was given daily by intragastric route for 8 weeks. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), prealbumin (PA), hyaluronic acid (HA) and laminin (LN), and hydroxyproline (HYP), NF-kappaBp65, alpha-smooth muscle actin (alpha-SMA) protein and TNF-alpha mRNA were studied in the liver, IkappaBalpha and TNF-alpha protein in the cytoplasm and NF-kappaBp65 protein in the nucleus. Compared with nontreated cirrhotic rats, the histopathology of rats given thalidomide (100 mg/kg) was significantly better. Serum ALT, AST, HA and LN and HYP content in the liver were significantly decreased and PA was elevated (p < 0.01) in this group; the expression of TNF-alpha mRNA and protein, NF-kappaBp65 and alpha-SMA were significantly decreased and IkappaBalpha protein was also elevated (p < 0.01). Thalidomide downregulates NF-kappaB-induced TNF-alpha and activates hepatic stellate cells (HSC) via inhibition of IkappaB degradation to prevent liver cirrhosis. Copyright 2006 S. Karger AG, Basel.

Myofibroblasts and colonic anastomosis healing in Wistar rats.

PubMed

Kosmidis, Christophoros; Efthimiadis, Christoforos; Anthimidis, Georgios; Basdanis, George; Apostolidis, Stylianos; Hytiroglou, Prodromos; Vasiliadou, Kalliopi; Prousalidis, John; Fahantidis, Epameinondas

2011-03-02

The myofibroblasts play a central role in wound healing throughout the body. The process of wound healing in the colon was evaluated with emphasis on the role of myofibroblasts. One hundred male Wistar rats weighing 274 ± 9.1 g (mean age: 3.5 months) were used. A left colonic segment was transected and the colon was re-anastomosed. Animals were randomly divided into two groups. The first group experimental animals (n = 50) were sacrificed on postoperative day 3, while the second group rats (n = 50) were sacrificed on postoperative day 7. Healing of colonic anastomosis was studied in terms of anastomotic bursting pressure, as well as myofibroblastic reaction and expression of α-smooth muscle actin (α-SMA), adhesion formation, inflammatory reaction and neovascularization. The mean anastomotic bursting pressure increased from 20.6 ± 3.5 mmHg on the 3rd postoperative day to 148.8 ± 9.6 Hg on the 7th postoperative day. Adhesion formation was increased on the 7th day, as compared to the 3rd day. In addition, the myofibroblastic reaction was more profound on the 7th postoperative day in comparison with the 3rd postoperative day. The staining intensity for α-SMA was progressive from the 3rd to the 7th postoperative day. On the 7th day the α-SMA staining in the myofibroblats reached the level of muscular layer cells. Our study emphasizes the pivotal role of myofibroblasts in the process of colonic anastomosis healing. The findings provide an explanation for the reduction in the incidence of wound dehiscence after the 7th postoperative day.

Synergistic effects of BAY 60-4552 and vardenafil on relaxation of corpus cavernosum tissue of patients with erectile dysfunction and clinical phosphodiesterase type 5 inhibitor failure.

PubMed

Albersen, Maarten; Linsen, Loes; Tinel, Hanna; Sandner, Peter; Van Renterghem, Koenraad

2013-05-01

Overall efficacy rates of phosphodiesterase type 5 inhibitors (PDE5-i) for erectile dysfunction (ED) are 60-70%. PDE5-i treatment failures currently have to resort to invasive treatment options for restoration of erectile function. AIMS.: To assess changes in the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP)/protein kinase (PKG) pathway in human corpus cavernosum (HCC) of PDE5-i nonresponders compared with healthy controls. To evaluate the effects of BAY 60-4552, a stimulator of soluble guanylate cyclase (sGC), and vardenafil on relaxation of HCC strips from PDE5-i nonresponders. mRNA expression, morphological localization of the NO/cGMP/PKG pathway, and relaxant capacity of both compounds alone or combined. Analysis of variance, t-test or Mann-Whitney test based upon number of groups and normality of data. HCC tissues were harvested after consent from individuals undergoing penile prosthesis implantation (patients) and potent patients undergoing transurethral surgery (healthy controls, needle biopsy). HCC tissues of patients were compared with those of healthy controls for the expression of mRNA coding for PDE5A, eNOS, PKGα1, PKG2, sGCα1, sGCα2, sGCβ1, sGCβ2, α-smooth muscle actin (aSMA) and β-actin by quantitative polymerase chain reaction (qPCR). The respective proteins were localized using immunofluorescence. Tissue strips of patients were precontracted with phenylepinephrine followed by incubation with 1 μM of either vardenafil or BAY 60-4552, or both simultaneously. The main targets in the NO/cGMP/sGC pathway were downregulated in PDE5-i nonresponders. The pathway was morphologically located to HCC smooth muscle, of which the overall content was preserved in ED patients based on aSMA expression. BAY 60-4552 and vardenafil have synergistic effects on relaxation of HCC of PDE5-i nonresponders. The main limitation is the small amount of control tissue precluding functional testing on these samples. Despite downregulation of the NO/cGMP/PKG pathway, combining BAY 60-4552 with vardenafil significantly enhanced relaxation HCC strips of PDE5-i nonresponders. © 2013 International Society for Sexual Medicine.

Melatonin suppresses activation of hepatic stellate cells through RORα-mediated inhibition of 5-lipoxygenase.

PubMed

Shajari, Shiva; Laliena, Almudena; Heegsma, Janette; Tuñón, María Jesús; Moshage, Han; Faber, Klaas Nico

2015-10-01

Liver fibrosis is scar tissue resulting from an uncontrolled wound-healing process in response to chronic liver injury. Liver damage generates an inflammatory reaction that activates hepatic stellate cells (HSC) that transdifferentiate from quiescent cells that control retinol metabolism to proliferative and migratory myofibroblasts that produce excessive amounts of extracellular matrix proteins, in particular collagen 1a1 (COL1A1). Although liver fibrosis is reversible, no effective drug therapy is available to prevent or reverse HSC activation. Melatonin has potent hepatoprotective properties in a variety of acute and chronic liver injury models and suppresses liver fibrosis. However, it remains unclear whether melatonin acts indirectly or directly on HSC to prevent liver fibrosis. Here, we studied the effect of melatonin on culture-activated rat HSC. Melatonin dose-dependently suppressed the expression of HSC activation markers Col1a1 and alpha-smooth muscle actin (αSMA, Acta2), as well as HSC proliferation and loss of lipid droplets. The nuclear melatonin sensor retinoic acid receptor-related orphan receptor-alpha (RORα/Nr1f1) was expressed in quiescent and activated HSC, while the membranous melatonin receptors (Mtrn1a and Mtrn1b) were not. The synthetic RORα agonist SR1078 more potently suppressed Col1a1 and αSma expression, HSC proliferation, and lipid droplet loss, while the RORα antagonist SR1001 blocked the antifibrotic features of melatonin. Melatonin and SR1078 inhibited the expression of Alox5, encoding 5-lipoxygenase (5-LO). The pharmacological 5-LO inhibitor AA861 reduced Acta2 and Col1a1 expression in activated HSC. We conclude that melatonin directly suppresses HSC activation via RORα-mediated inhibition of Alox5 expression, which provides novel drug targets to treat liver fibrosis. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Epoxyeicosatrienoic Acid Analog Decreases Renal Fibrosis by Reducing Epithelial-to-Mesenchymal Transition

PubMed Central

Skibba, Melissa; Hye Khan, Md. Abdul; Kolb, Lauren L.; Yeboah, Michael M.; Falck, John R.; Amaradhi, Radhika; Imig, John D.

2017-01-01

Renal fibrosis, which is a critical pathophysiological event in chronic kidney diseases, is associated with renal epithelial-to-mesenchymal transition (EMT). Epoxyeicosatrienoic acids (EETs) are Cyp epoxygenase arachidonic acid metabolites that demonstrate biological actions that result in kidney protection. Herein, we investigated the ability of 14,15-EET and its synthetic analog, EET-A, to reduce kidney fibrosis induced by unilateral ureter obstruction (UUO). C57/BL6 male mice underwent sham or UUO surgical procedures and were treated with 14,15-EET or EET-A in osmotic pump (i.p.) for 10 days following UUO surgery. UUO mice demonstrated renal fibrosis with an 80% higher kidney-collagen positive area and 70% higher α-smooth muscle actin (SMA) positive renal areas compared to the sham group. As a measure of collagen content, kidney hydroxyproline content was also higher in UUO (6.4 ± 0.5 μg/10 mg) compared to sham group (2.5 ± 0.1 μg/10 mg). Along with marked renal fibrosis, UUO mice had reduced renal expression of EET producing Cyp epoxygenase enzymes. Endogenous 14,15-EET or EET-A demonstrated anti-fibrotic action in UUO by reducing kidney-collagen positive area (50–60%), hydroxyproline content (50%), and renal α-SMA positive area (85%). In UUO mice, renal expression of EMT inducers, Snail1 and ZEB1 were higher compared to sham group. Accordingly, renal epithelial marker E-cadherin expression was reduced and mesenchymal marker expression was elevated in the UUO compared to sham mice. Interestingly, EET-A reduced EMT in UUO mice by deceasing renal Snail1 and ZEB1 expression. EET-A treatment also opposed the decrease in renal E-cadherin expression and markedly reduced several prominent renal mesenchymal/myofibroblast markers in UUO mice. Overall, our results demonstrate that EET-A is a novel anti-fibrotic agent that reduces renal fibrosis by decreasing renal EMT. PMID:28713267

Inhibitory effects of hepatocyte growth factor and interleukin-6 on transforming growth factor-beta1 mediated vocal fold fibroblast-myofibroblast differentiation.

PubMed

Vyas, Bimal; Ishikawa, Keiko; Duflo, Suzy; Chen, Xia; Thibeault, Susan L

2010-05-01

The role of myofibroblasts in vocal fold scarring has not been extensively studied, partly because of the lack of a robust in vitro model. The objective of this investigation was to develop and characterize a myofibroblast in vitro model that could be utilized to investigate the molecular mechanism of myofibroblast differentiation and function in injured vocal fold tissue. Differentiation of human primary vocal fold fibroblasts (hVFFs) to myofibroblasts was stimulated with 5, 10, or 20 ng/mL of recombinant transforming growth factor-beta1 (TGF-beta1). Cultures were analyzed by immunofluorescence and Western blotting, with an alpha-smooth muscle actin (alpha-SMA) antibody used as a myofibroblast marker. Normal rabbit vocal folds were treated with 10 ng/mL of TGF-beta1 for 7 days for in vivo corroboration. The effects of interleukin-6 (IL-6) and hepatocyte growth factor (HGF) on myofibroblast differentiation were studied with Western blots. The hVFFs demonstrated positive alpha-SMA labeling in cells stimulated by 10 and 20 ng/mL TGF-beta1, indicating that hVFFs were capable of differentiation to myofibroblasts. Transforming growth factor-beta1 induced the largest increase in alpha-SMA at 10 ng/mL on day 5 of treatment. Both HGF and IL-6 suppressed the expression of TGF-beta1-induced alpha-SMA. Our work characterizes a useful in vitro model of TGF-beta1-mediated vocal fold fibroblast-myofibroblast differentiation. The extent of differentiation appears to be attenuated by HGF, suggesting a potential mechanism to support prior work indicating that HGF plays a protective role in reducing scar formation in vocal fold injuries. Paradoxically, IL-6, which has been shown to play a profibrotic role in dermal studies, also attenuated the TGF-beta1 response.

Fibrous hamartoma of infancy: a clinical pathological analysis of seventeen cases

PubMed Central

Yu, Guojun; Wang, Yibing; Wang, Guangjun; Zhang, Dawei; Sun, Yong

2015-01-01

To discuss the clinical and pathological features, differential diagnosis and prognosis of fibrous hamartoma of infancy (FHI), seventeen FHI specimens were analyzed with H&E staining and strepavidin peroxidase (SP) immunohistochemistry to detect distinguishing tissue markers. The long-term outcomes of select cases were also obtained. Among the 17 patients (13 males, 4 females, average age 16 months), FHI manifested as a subcutaneous painless mass, primarily on the back of the neck, the upper arms and buttocks. One recurrence was noted among six follow-up cases. The tumors consisted of three main components: fibrous connective tissue; mature fat; and undifferentiated mesenchymal tissue. Immunohistochemistry revealed that fibrous connective tissue was positive for SMA and actin, mature fat tissue was positive for S-100 protein, and undifferentiated mesenchymal tissue was positive for CD34 and was partially positive for actin and SMA. The tumors were negative for desmin, NSE, bcl-2, β-catenin and Ki-67. In brief, FHI is a benign, fibroblastic/myofibroblastic proliferative lesion. Defined histologic features of FHI as presented here would distinguish FHI from similar invasive tumors including infant fibromatosis, calcifying aponeurotic fibroma, fibrous fatty tumor and embryonal rhabdomyosarcoma. Once clearly identified, FHI is curable with complete resection. PMID:26045872

Fibrous hamartoma of infancy: a clinical pathological analysis of seventeen cases.

PubMed

Yu, Guojun; Wang, Yibing; Wang, Guangjun; Zhang, Dawei; Sun, Yong

2015-01-01

To discuss the clinical and pathological features, differential diagnosis and prognosis of fibrous hamartoma of infancy (FHI), seventeen FHI specimens were analyzed with H&E staining and strepavidin peroxidase (SP) immunohistochemistry to detect distinguishing tissue markers. The long-term outcomes of select cases were also obtained. Among the 17 patients (13 males, 4 females, average age 16 months), FHI manifested as a subcutaneous painless mass, primarily on the back of the neck, the upper arms and buttocks. One recurrence was noted among six follow-up cases. The tumors consisted of three main components: fibrous connective tissue; mature fat; and undifferentiated mesenchymal tissue. Immunohistochemistry revealed that fibrous connective tissue was positive for SMA and actin, mature fat tissue was positive for S-100 protein, and undifferentiated mesenchymal tissue was positive for CD34 and was partially positive for actin and SMA. The tumors were negative for desmin, NSE, bcl-2, β-catenin and Ki-67. In brief, FHI is a benign, fibroblastic/myofibroblastic proliferative lesion. Defined histologic features of FHI as presented here would distinguish FHI from similar invasive tumors including infant fibromatosis, calcifying aponeurotic fibroma, fibrous fatty tumor and embryonal rhabdomyosarcoma. Once clearly identified, FHI is curable with complete resection.

Pirfenidone inhibits migration, differentiation, and proliferation of human retinal pigment epithelial cells in vitro

PubMed Central

Wang, Jing; Yang, Yangfan; Xu, Jiangang; Lin, Xianchai; Wu, Kaili

2013-01-01

Purpose To investigate the effects of pirfenidone (PFD) on the migration, differentiation, and proliferation of retinal pigment epithelial (RPE) cells and demonstrate whether the drug induces cytotoxicity. Methods Human RPE cells (line D407) were treated with various concentrations of PFD. Cell migration was measured with scratch assay. The protein levels of fibronectin (FN), connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), transforming growth factor beta (TGFβS), and Smads were assessed with western blot analyses. Levels of mRNA of TGFβS, FN, and Snail1 were analyzed using reverse transcriptase–polymerase chain reaction. Cell apoptosis was detected with flow cytometry using the Annexin V/PI apoptosis kit, and the percentages of cells labeled in different apoptotic stage were compared. A Trypan Blue assay was used to assess cell viability. Results PFD inhibited RPE cell migration. Western blot analyses showed that PFD inhibited the expression of FN, α-SMA, CTGF, TGFβ1, TGFβ2, Smad2/3, and Smad4. Similarly, PFD also downregulated mRNA levels of Snail1, FN, TGFβ1, and TGFβ2. No significant differences in cell apoptosis or viability were observed between the control and PFD-treated groups. Conclusions PFD inhibited RPE cell migration, differentiation, and proliferation in vitro and caused no significant cytotoxicity. PMID:24415895

Silymarin suppresses hepatic stellate cell activation in a dietary rat model of non-alcoholic steatohepatitis: Analysis of isolated hepatic stellate cells

PubMed Central

KIM, MINA; YANG, SU-GEUN; KIM, JOON MI; LEE, JIN-WOO; KIM, YOUNG SOO; LEE, JUNG IL

2012-01-01

Non-alcoholic steatohepatitis (NASH) is characterized by hepatocellular injury and initial fibrosis severity has been suggested as an important prognostic factor of NASH. Silymarin was reported to improve carbon tetrachloride-induced liver fibrosis and reduce the activation of hepatic stellate cells (HSC). We investigated whether silymarin could suppress the activation of HSCs in NASH induced by methionine- and choline-deficient (MCD) diet fed to insulin-resistant rats. NASH was induced by feeding MCD diet to obese diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Non-diabetic Long-Evans Tokushima Otsuka (LETO) rats were fed with standard chow and served as the control. OLETF rats were fed on either standard laboratory chow, or MCD diet or MCD diet mixed with silymarin. Histological analysis of the liver showed improved non-alcoholic fatty liver disease (NAFLD) activity score in silymarin-fed MCD-induced NASH. Silymarin reduced the activation of HSCs, evaluated by counting α-smooth muscle actin (SMA)-positive cells and measuring α-SMA mRNA expression in the liver lysates as well as in HSCs isolated from the experimental animals. Although silymarin decreased α1-procollagen mRNA expression in isolated HSCs, the anti-fibrogenic effect of silymarin was not prominent so as to show significant difference under histological analysis. Silymarin increased the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and decreased tumor necrosis factor (TNF)-α mRNA expression in the liver. Our study suggested that the possible protective effect of silymarin in diet induced NASH by suppressing the activation of HSCs and disturbing the role of the inflammatory cytokine TNF-α. PMID:22710359

Early Activation of Growth Pathways in Mitral Leaflets Exposed to Aortic Regurgitation: New Insights from an Animal Model.

PubMed

Marsit, Ons; Royer, Olivier; Drolet, Marie-Claude; Arsenault, Marie; Couet, Jacques; Morin, Stéphane; Levine, Robert A; Pibarot, Philippe; Beaudoin, Jonathan

2017-05-01

Mitral leaflet enlargement in patients with chronic aortic regurgitation (AR) has been identified as an adaptive mechanism potentially able to prevent functional mitral regurgitation (FMR) in response to left ventricular (LV) dilatation. The timing of valve enlargement is not known, and the related mechanisms are largely unexplored. AR was induced in 58 rats, and another 54 were used as sham controls. Animals were euthanized at different time points after AR creation (48 h, one week, and three months), and AR severity, FMR and LV dilatation were assessed using echocardiography. Mitral valves were harvested to document the reactivation of embryonic growth pathways. AR animals had increased LV dimensions and mitral annulus size. No animal developed FMR. No change in leaflet length or thickness was seen at 48 h; however, anterior mitral leaflets were longer and thicker in AR animals at one week and three months. Molecular changes were present early (at 48 h and at one week), with positive staining for transforming growth factor-b1 (TGF-b1), Alpha-smooth muscle actin (α-SMA) and matrix metalloproteinase-2 (MMP-2), which suggested active matrix remodeling. Increased gene expression for collagen 1, TGF-β1, α-SMA and MMP-2 was found in the mitral valve at 48 h and at one week, but after three months their expression had returned to normal. This model of AR induces active expansion and thickening of the mitral leaflets. Growth signals are expressed acutely, but not at three months, which suggests that most of this enlargement occurs at an early stage. The stimulation of valvular growth could represent a new strategy for the prevention of FMR.

Notch3 Ameliorates Cardiac Fibrosis After Myocardial Infarction by Inhibiting the TGF-β1/Smad3 Pathway.

PubMed

Zhang, Mingming; Pan, Xietian; Zou, Qian; Xia, Yuesheng; Chen, Jiangwei; Hao, Qimeng; Wang, Haichang; Sun, Dongdong

2016-10-01

Notch3 and TGF-β1 signaling play a key role in the pathogenesis and progression of chronic cardiovascular disease. However, whether Notch3 protects against myocardial infarction (MI) and the underlying mechanisms remains unknown. C57BL/6 mice were randomized to be treated with Notch3 siRNA (siNotch3) or lentivirus carrying Notch3 cDNA (Notch3) before coronary artery ligation. Four weeks after constructing MI model, cardiac function and fibrosis were compared between groups. The cardiac fibroblast cells (CFs) were isolated from newborn C57BL/6 mice (1-3 days old) and transfected with lentivirus carrying Notch3 cDNA. TGF-β1 (5 ng/ml), a well-known pro-fibrotic factor, was administered 72 h after Notch3 cDNA administration in CFs. The related proteins of fibrosis such as a-smooth muscle actin (a-SMA), Type I collagen, metalloprotease (MMP)-9 and the tissue inhibitor of metalloproteinases (TIMP)-2 were examined by western blot analysis. Notch3 cDNA treatment attenuated cardiac damage and inhibited fibrosis in mice with MI. Meanwhile, Notch3 siRNA administration aggravated cardiac function damage and markedly enhanced cardiac fibrosis in mice with MI. Overexpression of Notch3 inhibited TGF-β1-induced fibroblast-myofibroblast transition of mouse cardiac fibroblast cells, as evidenced by down-regulating a-SMA and Type I collagen expression. Notch3 cDNA treatment also increased MMP-9 expression and decreased TIMP-2 expression in the TGF-β1-stimulated cells. This study indicates that Notch3 is an important protective factor for cardiac fibrosis in a MI model, and the protective effect of Notch3 is attributable to its action on TGF-β1/Smad3 signaling.

Sipi soup inhibits cancer‑associated fibroblast activation and the inflammatory process by downregulating long non‑coding RNA HIPK1‑AS.

PubMed

Zhou, Bingxiu; Yu, Yuanyuan; Yu, Lixia; Que, Binfu; Qiu, Rui

2018-06-06

Sipi soup (SPS), the aqueous extract derived from the root bark of Sophora japonical L, Salix babylonica L., Morus alba L., as well as Amygdalus davidiana (Carr.) C. de Vos, is a traditional Chinese medicine frequently used to prevent and treat infection and inflammation. However, the role of SPS in cancer‑associated fibroblasts (CAFs) require further investigation. In the present study, the effects of SPS on fibroblast inactivation and the underlying mechanism were investigated. Reverse transcription‑quantitative polymerase chain reaction was used to analyze the mRNA expression levels of fibroblast activation protein (FAP), interleukin (IL)‑6, α‑smooth muscle actin (α‑SMA) and programmed cell death 4 (PDCD4). Flow cytometry was used to evaluate cell apoptosis. Immunofluorescence was used to determine the number of activated fibroblasts. The present study reported that SPS treatment did not affect the proliferative apoptotic potential of fibroblasts. Treatment with HeLa cell culture medium (CM) induced a significant increase in the expression levels of FAP, IL‑6 and α‑SMA, but reduced the expression of PDCD4. SPS reversed the effects of HeLa CM on the expression of these genes. Analysis with a long non‑coding (lnc)RNA array of numerous differentially expressed lncRNAs revealed that the expression levels of the lncRNA homeodomain‑interacting protein kinase 1 antisense RNA (HIPK1‑AS) were increased in cervicitis tissues and cervical squamous cell carcinoma tissues compared with in normal cervical tissues. HIPK1‑AS expression levels were upregulated in response to HeLa CM, but were decreased under SPS treatment. The downregulation of HIPK1‑AS expression via short hairpin RNA abolished the effects of HeLa CM on the expression of inflammation‑associated genes. The findings of the present study suggested that SPS may prevent the progression of cervical cancer by inhibiting the activation of CAF and the inflammatory process by reducing HIPK1‑AS expression.

Scleroderma keratinocytes promote fibroblast activation independent of transforming growth factor beta.

PubMed

McCoy, Sara S; Reed, Tamra J; Berthier, Celine C; Tsou, Pei-Suen; Liu, Jianhua; Gudjonsson, Johann E; Khanna, Dinesh; Kahlenberg, J Michelle

2017-11-01

SSc is a devastating disease that results in fibrosis of the skin and other organs. Fibroblasts are a key driver of the fibrotic process through deposition of extracellular matrix. The mechanisms by which fibroblasts are induced to become pro-fibrotic remain unclear. Thus, we examined the ability of SSc keratinocytes to promote fibroblast activation and the source of this effect. Keratinocytes were isolated from skin biopsies of 9 lcSSc, 10 dcSSc and 13 control patients. Conditioned media was saved from the cultures. Normal fresh primary fibroblasts were exposed to healthy control and SSc keratinocyte conditioned media in the presence or absence of neutralizing antibodies for TGF-β. Gene expression was assessed by microarrays and real-time PCR. Immunocytochemistry was performed for α-smooth muscle actin (α-SMA), collagen type 1 (COL1A1) and CCL5 expression. SSc keratinocyte conditioned media promoted fibroblast activation, characterized by increased α-SMA and COL1A1 mRNA and protein expression. This effect was independent of TGF-β. Microarray analysis identified upregulation of nuclear factor κB (NF-κB) and downregulation of peroxisome proliferator-activated receptor γ (PPAR-γ) pathways in both SSc subtypes. Scleroderma keratinocytes exhibited increased expression of NF-κB-regulated cytokines and chemokines and lesional skin staining confirmed upregulation of CCL5 in basal keratinocytes. Scleroderma keratinocytes promote the activation of fibroblasts in a TGF-β-independent manner and demonstrate an imbalance in NF-κB1 and PPAR-γ expression leading to increased cytokine and CCL5 production. Further study of keratinocyte mediators of fibrosis, including CCL5, may provide novel targets for skin fibrosis therapy. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Role of epithelial-mesenchymal transition (EMT) and fibroblast function in cerium oxide nanoparticles-induced lung fibrosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, Jane

The emission of cerium oxide nanoparticles (CeO{sub 2}) from diesel engines, using cerium compounds as a catalyst to lower the diesel exhaust particles, is a health concern. We have previously shown that CeO{sub 2} induced pulmonary inflammation and lung fibrosis. The objective of the present study was to investigate the modification of fibroblast function and the role of epithelial-mesenchymal transition (EMT) in CeO{sub 2}-induced fibrosis. Male Sprague-Dawley rats were exposed to CeO{sub 2} (0.15 to 7 mg/kg) by a single intratracheal instillation and sacrificed at various times post-exposure. The results show that at 28 days after CeO{sub 2} (3.5 mg/kg)more » exposure, lung fibrosis was evidenced by increased soluble collagen in bronchoalveolar lavage fluid, elevated hydroxyproline content in lung tissues, and enhanced sirius red staining for collagen in the lung tissue. Lung fibroblasts and alveolar type II (ATII) cells isolated from CeO{sub 2}-exposed rats at 28 days post-exposure demonstrated decreasing proliferation rate when compare to the controls. CeO{sub 2} exposure was cytotoxic and altered cell function as demonstrated by fibroblast apoptosis and aggregation, and ATII cell hypertrophy and hyperplasia with increased surfactant. The presence of stress fibers, expressed as α-smooth muscle actin (SMA), in CeO{sub 2}-exposed fibroblasts and ATII cells was significantly increased compared to the control. Immunohistofluorescence analysis demonstrated co-localization of TGF-β or α-SMA with prosurfactant protein C (SPC)-stained ATII cells. These results demonstrate that CeO{sub 2} exposure affects fibroblast function and induces EMT in ATII cells that play a role in lung fibrosis. These findings suggest potential adverse health effects in response to CeO{sub 2} nanoparticle exposure. - Highlights: • CeO{sub 2} exposure induced lung fibrosis. • CeO{sub 2} were detected in lung tissue, alveolar type II (ATII) cells and fibroblasts. • CeO{sub 2} caused ATII cell hypertrophy and hyperplasia and altered fibroblast function. • Increased α-SMA in CeO{sub 2}-exposed lung fibroblasts indicating myofibroblast formation. • CeO{sub 2} induced EMT in ATII cells demonstrated as increased α-SMA expression.« less

Antifibrotic effect of aloe vera in viral infection-induced hepatic periportal fibrosis

PubMed Central

Hegazy, Sahar K; El-Bedewy, Mohamed; Yagi, Akira

2012-01-01

AIM: To investigate the anti-oxidative and anti-fibrotic effects of aloe vera in patients with liver fibrosis. METHODS: Aloe vera high molecular weight fractions (AHM) were processed by patented hyper-dry system in combination of freeze-dry technique with microwave and far infrared-ray radiation. Fifteen healthy volunteers as the control group and 40 patients were included. The patients were randomly subdivided into two equal groups: the conventional group was treated with placebo (starch), and AHM group was treated with 0.15 gm/d AHM, both for 12 consecutive weeks. The patients were investigated before and after treatment. Serum activity of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), hyaluronic acid (HA), transforming growth factor-β (TGF-β) and matrixmetalloproteinase-2 (MMP-2) were determined. The reduced glutathione (GSH) and malondialdehyde (MDA) levels in liver were assayed and the expression of hepatic α-smooth muscle actin (α-SMA) was identified by immunohistochemistry. RESULTS: At the start of the study, the hematoxylin and eosin staining revealed fibro-proliferated bile ductules, thick fibrous septa and dense inflammatory cellular infiltration in the patients before treatment. The use of AHM for 12 wk significantly ameliorated the fibrosis, inhibited the inflammation, and resulted in minimal infiltration and minimal fibrosis compared to the conventional group. The enzyme activities of the liver (ALT, AST and ALP) were attenuated after treatment in both groups, and the decrease in the AHM group was more significant as compared with the conventional group. Similar to the AST, the MDA levels were significantly higher before treatment, and were attenuated after treatment in both groups. In contrast, the hepatic glutathione content in the patients were decreased significantly in the AHM group compared to the controls. The serum levels of the fibrosis markers (HA, TGF-β and MMP-2) were also reduced significantly after treatment. The expression of α-SMA was modified in patients before and after treatment as compared with the normal controls. In the conventional group, there was only thin and incomplete parenchymal α-SMA positive septum joining the thickened centrilobular veins, while in the AHM group, few α-SMA positive cells were present in sinusoid and lobule after treatment. CONCLUSION: Oral supplementation with AHM could be helpful in alleviating the fibrosis and inflammation of hepatic fibrosis patients. PMID:22563189

Contractile markers distinguish structures of the mouse aqueous drainage tract

PubMed Central

Ko, MinHee K.

2013-01-01

Purpose Structures of the aqueous humor drainage tract are contractile, although the tract is not entirely composed of muscle. We characterized the mouse aqueous drainage tract by immunolabeling contractile markers and determined whether profiling these markers within the tract distinguished its key structures of the trabecular meshwork (TM) and ciliary muscle (CM). Methods Enucleated eyes from pigmented C57BL/6 (n=8 mice) and albino BALB/c (n=6 mice) mice were processed for cryo- and formalin-fixed paraffin-embedded sectioning. Immunofluorescence labeling was performed for the following: (a) filamentous actin (using fluorescence-conjugated phalloidin), representing a global contractile marker; (b) α-smooth muscle actin (α-SMA), caldesmon, and calponin, representing classic smooth muscle epitopes; and (c) nonmuscle myosin heavy chain, representing a nonmuscle contractile protein. Tissue labeling was identified by confocal microscopy and analyzed quantitatively. Hematoxylin and eosin staining provided structural orientation. Results A small portion of the TM faced the anterior chamber; the rest extended posteriorly alongside Schlemm’s canal (SC) within the inner sclera. Within the drainage tract, filamentous actin labeling was positive in TM and CM. α-SMA and caldesmon labeling was seen primarily along the CM, which extended from the anterior chamber angle to its posterior termination beyond the SC near the retina. Low intensity, patchy α-SMA and caldesmon labeling was seen in the TM. Myosin heavy chain immunoreactivity was primarily found in the TM and calponin was primarily observed in the CM. C57BL/6 and BALB/c comparison showed that pigment obscured fluorescence in the ciliary body. Conclusions Our strategy of profiling contractile markers distinguished mouse aqueous drainage tract structures that were otherwise indistinguishable by hematoxylin and eosin staining. The mouse TM was seen as an intervening structure between SC, a part of the conventional drainage tract, and CM, a part of the unconventional drainage tract. Our findings provide important insights into the structural and functional organization of the mouse aqueous drainage tract and a basis for exploring the role of contractility in modulating aqueous outflow. PMID:24357924

Hydroxyurea enhances SMN2 gene expression through nitric oxide release.

PubMed

Xu, Cheng; Chen, Xin; Grzeschik, Susanna M; Ganta, Madhuri; Wang, Ching H

2011-02-01

Small molecules that increase full-length survivor motor neuron (SMN) gene transcript are promising therapeutic candidates for spinal muscular atrophy (SMA). Hydroxyurea (HU) has recently been shown to increase full-length SMN transcript in cultured lymphocytes from patients with SMA. We investigate the mechanism by which HU enhances full-length SMN2 gene expression in SMA lymphocytes. Nitric oxide (NO) is a major intracellular metabolite of HU. We test whether NO donors can themselves enhance full-length SMN2 expression. Eighteen cell lines (five type I, five type II, six type III SMA, and two non-SMA controls) were treated with or without NO donors for 48 h. SMA cells treated with HU and three NO donors: two long-acting donors, Deta-NONOate and S-nitrosoglutathione, and one short-acting donor, 3-ethyl-3-(ethylaminoethyl)-1-hydroxy-2-oxo-1-triazene, resulted in significant increase in full-length SMN2 mRNA. These effects were abolished by co-treatment with an NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide. One short-acting NO donor, S-nitroso-N-acetyl-DL-penicillamine, failed to show significant effect on full-length SMN2 expression, possibly due to high degree of cytotoxicity. These results were observed using both densitometry and quantitative PCR methods. We conclude that HU enhances SMN2 expression through the release of NO. NO donors may themselves be considered as new therapeutic candidates for SMA.

Effect of mitomycin-C on contraction and migration of human nasal mucosa fibroblasts: implications in dacryocystorhinostomy.

PubMed

Kumar, Vinay; Ali, Mohammad Javed; Ramachandran, Charanya

2015-09-01

To determine the effect of mitomycin-C (MMC) on the contraction and migration of human nasal mucosal fibroblasts (HNMFs) in vitro in order to identify the least concentration of MMC required to prevent cicatrix development following dacryocystorhinostomy (DCR). Primary cultures of HNMFs were established from nasal mucosal tissues of patients undergoing DCR. Myofibroblast transformation of HNMFs was induced using transforming growth factor-β (TGF-β1) and confirmed by immunostaining for α-smooth muscle actin (α-SMA). Collagen gel contraction assay was employed to study contraction in the presence or absence of TGF-β1 (5 and 10 ng/mL) and MMC (0.2 and 0.4 mg/mL). Scratch wound assay was employed to determine the influence of MMC treatment on cell migration. Quantification of gel contraction and wound closure was done using Image J software. α-SMA expression increased with TGF-β1 treatment in a time- and dose-dependent manner indicating myofibroblast transformation of HNMFs. MMC inhibited TGF-β1- induced collagen gel contraction in a dose-dependent manner (0.4 mg/mL>0.2 mg/mL). Further, there was a decrease in the migration of MMC-treated HNMFs, resulting in delayed wound closure that corroborated with the loss of actin stress fibres. MMC successfully inhibited TGF-β1-induced myofibroblast transformation, collagen gel contraction and significantly reduced the migration of HNMFs to cover the wound even at a low concentration of 0.2 mg/mL. This study provides evidence that low concentration and short duration of MMC treatment is efficient in reducing increased contraction and migration of HMNFs in response to injury. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Gelam honey potentiates ex vivo corneal keratocytes proliferation with desirable phenotype expression.

PubMed

Yusof, Alia Md; Abd Ghafar, Norzana; Kamarudin, Taty Anna; Hui, Chua Kien; Yusof, Yasmin Anum Mohd

2016-02-24

This study aimed to evaluate the effects of Gelam honey on corneal keratocytes proliferative capacity and phenotypic characterization via MTT assay, gene expression and immunocytochemistry. Corneal keratocytes from New Zealand white rabbits were cultured in basal medium (BM) and serum enriched medium (BMS). Serial dilutions of Gelam honey (GH) were added to both media and cells were cultured until passage 1. MTT assay was performed on corneal keratocytes in both media to ascertain the optimal dose of GH that produced maximum proliferation. Gelam honey at the concentration of 0.0015% in both media showed the highest proliferative capacity with no morphological changes compared to their respective controls. The gene expression of aldehyde dehydrogenase (ALDH), a marker for quiescent keratocytes and vimentin, a marker for fibroblast, were higher in the GH enriched groups. The alpha smooth muscle actin (α-SMA) expression, marker for myofibroblast, was lower in GH treated groups compared to the controls. Immunocytochemistry results were in accordance to the gene expression analyses. Gelam honey at a concentration of 0.0015% promotes ex vivo corneal keratocytes proliferation while retaining desirable phenotype expression. The results serve as a basis for the development of Gelam honey as a potential natural product in promoting corneal wound healing.

Glutathione and catalase suppress TGFβ-induced cataract-related changes in cultured rat lenses and lens epithelial explants

PubMed Central

Chamberlain, Coral G.; Cerra, Anna

2009-01-01

Purpose The damaging effects of oxidative stress and transforming growth factor-β (TGFβ)-induced transdifferentiation of lens epithelial cells have both been implicated independently in the etiology of cataract. The aim of this study was to investigate whether the presence of antioxidant systems in the lens influences the ability of lens epithelial cells to respond to TGFβ. Methods Whole lenses from young rats were cultured with or without TGFβ in the presence or absence of reduced glutathione (GSH). Lens epithelial explants from weanling rats were used to investigate the effects of GSH and catalase on TGFβ-induced cataract-related changes. Lenses were monitored for opacification for three to four days, photographed, and then processed for routine histology. Explants were assessed by phase contrast microscopy, enzyme-linked immunosorbent assay (ELISA) of α-smooth muscle actin (αSMA), and/or immunolocalization of αSMA and Pax6, markers for transdifferentiation and normal lens epithelial phenotype, respectively. Results In cultured lenses, GSH strongly suppressed TGFβ-induced opacification and subcapsular plaque formation. In explants, both GSH and catalase suppressed changes typically associated with TGFβ-induced transdifferentiation including wrinkling of the lens capsule, cell-surface blebbing, apoptotic cell loss, induction of αSMA, and loss of Pax6 expression. Conclusions This study suggests that antioxidant systems present in the normal lens, which protect the epithelium against the damaging effects of reactive oxygen species, may also serve to protect it against the potentially cataractogenic effects of TGFβ. Taken together with other recent studies, it also raises the possibility that TGFβ may induce cataract-related changes in lens epithelial cells via release of hydrogen peroxide. PMID:19421408

Human esophageal myofibroblasts secrete proinflammatory cytokines in response to acid and Toll-like receptor 4 ligands

PubMed Central

Gargus, Matthew; Niu, Chao; Vallone, John G.; Binkley, Jana; Rubin, Deborah C.

2015-01-01

The pathophysiology of esophageal injury, repair, and inflammation in gastroesophageal reflux-disease (GERD) is complex. Whereas most studies have focused on the epithelial response to GERD injury, we are interested in the stromal response. We hypothesized that subepithelial esophageal myofibroblasts in GERD secrete proinflammatory cytokines in response to injurious agents encountered via epithelial barrier breaches or through dilated epithelial intercellular spaces. We determined the percentage of myofibroblasts [α-smooth muscle actin (α-SMA)+vimentin+CD31−] in the subepithelial GERD and normal esophageal stroma by immunomorphologic analysis. We performed α-SMA coimmunostaining with IL-6 and p65. We established and characterized primary cultures of α-SMA+vimentin+CD31−CD45− human esophageal myofibroblasts (HuEso MFs). We modeled GERD by treatment with pH 4.5-acidified media and Toll-like receptor 4 (TLR4) ligands, LPS and high-mobility group box 1 protein (HMGB1), and determined myofibroblast cytokine secretion in response to GERD injury. We demonstrate that spindle-shaped cell myofibroblasts are located near the basement membrane of stratified squamous epithelium in normal esophagus. We identify an increase in subepithelial myofibroblasts and activation of proinflammatory pathways in patients with GERD. Primary cultures of stromal cells obtained from normal esophagus retain myofibroblast morphology and express the acid receptor transient receptor potential channel vanilloid subfamily 1 (TRPV1) and TLR4. HuEso MFs stimulated with acid and TLR4 agonists LPS and HMGB1 increase IL-6 and IL-8 secretion via TRPV1 and NF-κB activation. Our work implicates a role for human subepithelial stromal cells in the pathogenesis of GERD-related esophageal injury. Findings of this study can be extended to the investigation of epithelial-stromal interactions in inflammatory esophageal mucosal disorders. PMID:25882613

Sirtuin 7 is decreased in pulmonary fibrosis and regulates the fibrotic phenotype of lung fibroblasts.

PubMed

Wyman, Anne E; Noor, Zahid; Fishelevich, Rita; Lockatell, Virginia; Shah, Nirav G; Todd, Nevins W; Atamas, Sergei P

2017-06-01

Pulmonary fibrosis is a severe condition with no cure and limited therapeutic options. A better understanding of its pathophysiology is needed. Recent studies have suggested that pulmonary fibrosis may be driven by accelerated aging-related mechanisms. Sirtuins (SIRTs), particularly SIRT1, SIRT3, and SIRT6, are well-known mediators of aging; however, limited data exist on the contribution of sirtuins to lung fibrosis. We assessed the mRNA and protein levels of all seven known sirtuins in primary lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) and systemic sclerosis-associated interstitial lung disease (SSc-ILD) in comparison with lung fibroblasts from healthy controls. These unbiased tests revealed a tendency for all sirtuins to be expressed at lower levels in fibroblasts from patients compared with controls, but the greatest decrease was observed with SIRT7. Similarly, SIRT7 was decreased in lung tissues of bleomycin-challenged mice. Inhibition of SIRT7 with siRNA in cultured lung fibroblasts resulted in an increase in collagen and α-smooth muscle actin (α-SMA). Reciprocally, overexpression of SIRT7 resulted in lower basal and TGF-β-induced levels of COL1A1, COL1A2, COL3A1, and α-SMA mRNAs, as well as collagen and α-SMA proteins. Induced changes in SIRT7 had no effect on endogenous TGF-β mRNA levels or latent TGF-β activation, but overexpression of SIRT7 reduced the levels of Smad3 mRNA and protein. In conclusion, the decline in SIRT7 in lung fibroblasts has a profibrotic effect, which is mediated by changes in Smad3 levels.

Immunohistochemical evaluation of canine peripheral nerve sheath tumors and other soft tissue sarcomas.

PubMed

Chijiwa, K; Uchida, K; Tateyama, S

2004-07-01

Seventeen cases of canine peripheral nerve sheath tumors (PNSTs), 11 malignant PNSTs (MPNSTs), and six benign PNSTs (BPNSTs) were examined. The prognosis in five of six dogs with BPNSTs was excellent, whereas all dogs with MPNSTs died within 2 years after the last surgical resection. One BPNST formed a recurrent mass with features of a MPNST. Histopathologically, the predominant tumor cell of MPNSTs was either spindle or round in shape with epithelioid characteristics. Other atypical cells had abundant granular cytoplasm or were multinucleated giant cells with periodic acid-Schiff-positive cytoplasmic globules. Furthermore, two MPNSTs contained cartilaginous and osseous metaplasia. On the contrary, most BPNSTs exhibited typical features of schwannoma or neurofibroma, whereas two BPNSTs had atypical morphology. One BPNST consisted of epithelioid cell proliferation with some tumor cells revealing nuclear atypia. Immunohistochemically, the expression of vimentin (100%), S-100 (73%), nerve growth factor receptor (NGFR, 64%), and myoglobin (64%) was commonly found in MPNSTs. The two BPNSTs with atypical histologic appearances were positive for vimentin, S-100, NGFR, and neuron-specific enolase, and one of these had moderate immunoreactivity for cytokeratin. Most BPNSTs were positive for glial fibrillary acidic protein, as well as S-100 and NGFR. Although most rhabdomyosarcomas (RMSs) and canine hemangiopericytomas (CHPs) also showed focal immunoreactivity for S-100, most RMSs were intensely positive for myoglobin and negative for NGFR. Most CHPs (80%) exhibited focal alpha-smooth muscle actin (alpha-SMA) expression, whereas all PNSTs were negative. These results indicate that immunohistochemistry for NGFR and alpha-SMA might be useful for differentiating canine PNSTs from RMSs or CHPs, respectively.

Hepatoprotective effect of Matrine salvianolic acid B salt on Carbon Tetrachloride-Induced Hepatic Fibrosis

PubMed Central

2012-01-01

The aim of this study was to investigate the hepatoprotective effect of Matrine salvianolic acid B salt on carbon tetrachloride (CCl4)-induced hepatic fibrosis in rats. Salvianolic acid B and Matrine has long been used to treat liver fibrosis. Matrine salvianolic acid B salt is a new compound containing Salvianolic acid B and Matrine. Hepatic fibrosis induced by CCl4 was studied in animal models using Wistar rats. Organ coefficient, serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), hexadecenoic acid (HA), laminin (LN), hydroxyproline (Hyp), and glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD) in liver tissues were measured, respectively. Histopathological changes in the livers were studied by hematoxylin-eosin (H&E) staining and Masson Trichrome (MT) examination. The expression of transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) was observed by immunohistochemical analysis. A significant reduction in serum levels of AST, ALT, HA, LN and Hyp was observed in the Matrine salvianolic acid B salt treated groups, suggesting that the salt had hepatoprotective effects. The depletion of GSH and SOD, as well as MDA accumulation in liver tissues was suppressed by Matrine salvianolic acid B salt too. The expression of TGF-β1 and α-SMA measured by immunohistology was significantly reduced by Matrine salvianolic acid B salt in a dose-dependent manner. Matrine salvianolic acid B salt treatment attenuated the necro-inflammation and fibrogenesis induced by CCl4 injection, and thus it is promising as a therapeutic anti-fibrotic agent against hepatic fibrosis. PMID:22559721

Hepatoprotective effect of Matrine salvianolic acid B salt on Carbon Tetrachloride-Induced Hepatic Fibrosis.

PubMed

Gao, Hong-Ying; Li, Guo-Yu; Lou, Meng-Meng; Li, Xiao-Yu; Wei, Xiu-Yan; Wang, Jin-Hui

2012-05-04

The aim of this study was to investigate the hepatoprotective effect of Matrine salvianolic acid B salt on carbon tetrachloride (CCl4)-induced hepatic fibrosis in rats. Salvianolic acid B and Matrine has long been used to treat liver fibrosis. Matrine salvianolic acid B salt is a new compound containing Salvianolic acid B and Matrine. Hepatic fibrosis induced by CCl4 was studied in animal models using Wistar rats. Organ coefficient, serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), hexadecenoic acid (HA), laminin (LN), hydroxyproline (Hyp), and glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD) in liver tissues were measured, respectively. Histopathological changes in the livers were studied by hematoxylin-eosin (H&E) staining and Masson Trichrome (MT) examination. The expression of transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) was observed by immunohistochemical analysis. A significant reduction in serum levels of AST, ALT, HA, LN and Hyp was observed in the Matrine salvianolic acid B salt treated groups, suggesting that the salt had hepatoprotective effects. The depletion of GSH and SOD, as well as MDA accumulation in liver tissues was suppressed by Matrine salvianolic acid B salt too. The expression of TGF-β1 and α-SMA measured by immunohistology was significantly reduced by Matrine salvianolic acid B salt in a dose-dependent manner. Matrine salvianolic acid B salt treatment attenuated the necro-inflammation and fibrogenesis induced by CCl4 injection, and thus it is promising as a therapeutic anti-fibrotic agent against hepatic fibrosis.

Ursolic acid suppresses TGF-β1-induced quiescent HSC activation and transformation by inhibiting NADPH oxidase expression and Hedgehog signaling

PubMed Central

Yu, Shan-Shan; Chen, Biao; Huang, Chen-Kai; Zhou, Juan-Juan; Huang, Xin; Wang, An-Jiang; Li, Bi-Min; He, Wen-Hua; Zhu, Xuan

2017-01-01

Activation of quiescent hepatic stellate cells (q-HSCs) and their transformation to myofibroblasts (MFBs) is a key event in liver fibrosis. Hedgehog (Hh) signaling stimulates q-HSCs to differentiate into MFBs, and NADPH oxidase (NOX) may be involved in regulating Hh signaling. The author's preliminary study demonstrated that ursolic acid (UA) selectively induces apoptosis in activated HSCs and inhibits their proliferation in vitro via negative regulation of NOX activity and expression. However, the effect of UA on q-HSCs remains to be elucidated. The present study aimed to investigate the effect of UA on q-HSC activation and HSC transformation and to observe alterations in the NOX and Hh signaling pathways during q-HSC activation. q-HSC were isolated from adult male Sprague-Dawley rats. Following culture for 3 days, the cells were treated with or without transforming growth factor-β1 (TGF-β1; 5 µg/l); intervention groups were pretreated with UA (40 µM) or diphenyleneiodonium chloride (DPI; 10 µM) for 30 min prior to addition of TGF-β1. mRNA and protein expression of NOX and Hh signaling components and markers of q-HSC activation were examined by western blotting and reverse transcription-polymerase chain reaction. TGF-β1 induced activation of q-HSCs, with increased expression of α-smooth muscle actin (α-SMA) and type I collagen. In addition, expression of NOX subunits (gp91phox, p67phox, p22phox, and Rac1) and Hh signaling components, including sonic Hh, sterol-4-alpha-methyl oxidase, and Gli family zinc finger 2, were upregulated in activated HSCs. Pretreatment of q-HSCs with UA or DPI prior to TGF-β1 significantly downregulated expression of NOX subunits and Hh signaling components and additionally inhibited expression of α-SMA and type I collagen, thereby preventing transformation to MFBs. UA inhibited TGF-β1-induced activation of q-HSCs and their transformation by inhibiting expression of NOX subunits and the downstream Hh pathway. PMID:29042951

ERα inhibited myocardin-induced differentiation in uterine fibroids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liao, Xing-Hua, E-mail: xinghualiao@hotmail.com; Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education and Tianjin, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457; Li, Jun-Yan

Uterine fibroids, also known as uterine leiomyomas, are a benign tumor of the human uterus and the commonest estrogen-dependent benign tumor found in women. Myocardin is an important transcriptional regulator in smooth and cardiac muscle development. The role of myocardin and its relationship with ERα in uterine fibroids have barely been addressed. We noticed that the expression of myocardin was markedly reduced in human uterine fibroid tissue compared with corresponding normal or adjacent myometrium tissue. Here we reported that myocardin induced the transcription and expression of differentiation markers SM22α and alpha smooth muscle actin (α-SMA) in rat primary uterine smoothmore » muscle cells (USMCs) and this effect was inhibited by ERα. Notably, we showed that, ERα induced expression of proliferation markers PCNA and ki-67 in rat primary USMCs. We also found ERα interacted with myocardin and formed complex to bind to CArG box and inhibit the SM22α promoter activity. Furthermore, ERα inhibited the transcription and expression of myocardin, and reduced the levels of transcription and expression of downstream target SM22α, a SMC differentiation marker. Our data thus provided important and novel insights into how ERα and myocardin interact to control the cell differentiation and proliferation of USMCs. Thus, it may provide potential therapeutic target for uterine fibroids.« less

A Comparative Immunohistochemical Study of Anal Canal Epithelium in Humans and Swine, Focusing on the Anal Transitional Zone Epithelium and the Anal Glands.

PubMed

Muranaka, Futoshi; Nakajima, Tomoyuki; Iwaya, Mai; Ishii, Keiko; Higuchi, Kayoko; Ogiwara, Naoko; Miyagawa, Shinichi; Ota, Hiroyoshi

2018-05-01

To better understand the cellular origins and differentiation of anal canal epithelial neoplasms, the immunohistochemical profiles of the anal canal epithelium in humans and swine were evaluated. Formalin-fixed tissue sections were immunostained for mucin (MUC: MUC2, MUC5AC, MUC5B), desmoglein 3 (DGS3), p63, CDX2, SOX2, and α-smooth muscle actin (α-SMA). The anal transitional zone (ATZ) epithelium covered the anal sinus and consisted of a stratified epithelium with mucous cells interspersed within the surface lining. Anal glands opened into the anal sinus. Ducts and acini of intraepithelial or periepithelial mucous type were the main structures of human anal glands, whereas those of swine were compound tubuloacinar mixed glands. Distal to the ATZ epithelium, non-keratinized stratified squamous epithelium merged with the keratinized stratified squamous epithelium of the perianal skin. MUC5AC expression predominated over MUC5B expression in the ATZ epithelium, while MUC5B expression was higher in the anal glands. SOX2 was positive in the ATZ epithelium, anal glands, and squamous epithelium except in the perianal skin. In humans, DGS3 was expressed in the ATZ epithelium, anal gland ducts, and squamous epithelium. p63 was detected in the ATZ epithelium, anal glands, and squamous epithelium. Myoepithelial cells positive for α-SMA and p63 were present in the anal glands of swine. Colorectal columnar cells were MUC5B + /MUC2 + /CDX2 + /MUC5AC - /SOX2 - . The ATZ epithelium seems to be a distinctive epithelium, with morphological and functional features allowing smooth defecation. The MUC5AC + /SOX2 + /MUC2 - /CDX2 - profile of the ATZ epithelium and anal glands is a useful feature for diagnosing adenocarcinoma arising from these regions. Anat Rec, 301:796-805, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

MicroRNA-143-3p inhibits hyperplastic scar formation by targeting connective tissue growth factor CTGF/CCN2 via the Akt/mTOR pathway.

PubMed

Mu, Shengzhi; Kang, Bei; Zeng, Weihui; Sun, Yaowen; Yang, Fan

2016-05-01

Post-traumatic hypertrophic scar (HS) is a fibrotic disease with excessive extracellular matrix (ECM) production, which is a response to tissue injury by fibroblasts. Although emerging evidence has indicated that miRNA contributes to hypertrophic scarring, the role of miRNA in HS formation remains unclear. In this study, we found that miR-143-3p was markedly downregulated in HS tissues and fibroblasts (HSFs) using qRT-PCR. The expression of connective tissue growth factor (CTGF/CCN2) was upregulated both in HS tissues and HSFs, which is proposed to play a key role in ECM deposition in HS. The protein expression of collagen I (Col I), collagen III (Col III), and α-smooth muscle actin (α-SMA) was obviously inhibited after treatment with miR-143-3p in HSFs. The CCK-8 assay showed that miR-143-3p transfection reduced the proliferation ability of HSFs, and flow cytometry showed that either early or late apoptosis of HSFs was upregulated by miR-143-3p. In addition, the activity of caspase 3 and caspase 9 was increased after miR-143-3p transfection. On the contrary, the miR-143-3p inhibitor was demonstrated to increase cell proliferation and inhibit apoptosis of HSFs. Moreover, miR-143-3p targeted the 3'-UTR of CTGF and caused a significant decrease of CTGF. Western blot demonstrated that Akt/mTOR phosphorylation and the expression of CTGF, Col I, Col III, and α-SMA were inhibited by miR-143-3p, but increased by CTGF overexpression. In conclusion, we found that miR-143-3p inhibits hypertrophic scarring by regulating the proliferation and apoptosis of human HSFs, inhibiting ECM production-associated protein expression by targeting CTGF, and restraining the Akt/mTOR pathway.

Age-Dependent Decline in Mouse Lung Regeneration with Loss of Lung Fibroblast Clonogenicity and Increased Myofibroblastic Differentiation

PubMed Central

Paxson, Julia A.; Gruntman, Alisha; Parkin, Christopher D.; Mazan, Melissa R.; Davis, Airiel; Ingenito, Edward P.; Hoffman, Andrew M.

2011-01-01

While aging leads to a reduction in the capacity for regeneration after pneumonectomy (PNX) in most mammals, this biological phenomenon has not been characterized over the lifetime of mice. We measured the age-specific (3, 9, 24 month) effects of PNX on physiology, morphometry, cell proliferation and apoptosis, global gene expression, and lung fibroblast phenotype and clonogenicity in female C57BL6 mice. The data show that only 3 month old mice were fully capable of restoring lung volumes by day 7 and total alveolar surface area by 21 days. By 9 months, the rate of regeneration was slower (with incomplete regeneration by 21 days), and by 24 months there was no regrowth 21 days post-PNX. The early decline in regeneration rate was not associated with changes in alveolar epithelial cell type II (AECII) proliferation or apoptosis rate. However, significant apoptosis and lack of cell proliferation was evident after PNX in both total cells and AECII cells in 24 mo mice. Analysis of gene expression at several time points (1, 3 and 7 days) post-PNX in 9 versus 3 month mice was consistent with a myofibroblast signature (increased Tnc, Lox1, Col3A1, Eln and Tnfrsf12a) and more alpha smooth muscle actin (αSMA) positive myofibroblasts were present after PNX in 9 month than 3 month mice. Isolated lung fibroblasts showed a significant age-dependent loss of clonogenicity. Moreover, lung fibroblasts isolated from 9 and 17 month mice exhibited higher αSMA, Col3A1, Fn1 and S100A expression, and lower expression of the survival gene Mdk consistent with terminal differentiation. These data show that concomitant loss of clonogenicity and progressive myofibroblastic differentiation contributes to the age-dependent decline in the rate of lung regeneration. PMID:21912590

Myofibroblast distribution in Dupuytren's cords: correlation with digital contracture.

PubMed

Verjee, Liaquat Suleman; Midwood, Kim; Davidson, Dominique; Essex, David; Sandison, Ann; Nanchahal, Jagdeep

2009-12-01

Dupuytren's tissue has typically been described as being composed of myofibroblast-rich palmar nodules and relatively acellular tendon-like cords. We aimed to determine myofibroblast distribution (alpha-smooth muscle actin [alpha-SMA] positive cells) within Dupuytren's tissue and to correlate histologically defined alpha-SMA-positive nodules with digital contracture and recurrent disease. One hundred and three digital Dupuytren's cords (72 fasciectomy, 31 dermofasciectomy) were stained with anti-alpha-SMA antibody. The presence of alpha-SMA-positive nodules, their surface area, and alpha-SMA-positive cells were quantified throughout excised Dupuytren's tissue. Clinical data on diathesis, flexion deformity, and previous surgeries were collected. Cords were nodular (66%) or non-nodular (34%). Nodular cords contained 1 (55%), 2 (33%), or 3 or more nodules (12%) composed of localized collections of cells. The mean total nodule surface area was 23 mm(2) (range, 1.3-105 mm(2)). Nodules contained the highest number of alpha-SMA-positive cells (mean 97%, 2374 cells/mm(2)) compared to peri-nodular areas (mean 32%, 763 cells/mm(2)), and more distant cord (mean 8%, 495 cells/mm(2)). Non-nodular cords contained 9% to 17% alpha-SMA-positive cells (mean 475-663 cells/mm(2)), with higher numbers distally. There was greater digital contracture in patients with non-nodular cords. Thirty-six of 38 proximal interphalangeal (PIP) joint-marked samples had a nodule that co-localized with the PIP joint. Nodule size did not correlate with flexion deformity or with primary or recurrent disease. We found that two thirds of digital cords were nodular. Nodules were hypercellular, the majority being alpha-SMA-positive cells. Nodules varied in size and co-localized with the PIP joint. Cord was relatively cellular throughout; a proportion of these cells were alpha-SMA-positive and cells aligned with collagen fibers. Non-nodular cords correlated with significantly greater digital flexion contracture. We propose that cells in nodular cords contract and deposit extracellular matrix components. The matrix is then remodeled in shortened configuration, and as fixed flexion deformity develops, stress shielding eventually leads to myofibroblast apoptosis, and cord becomes less cellular.

Preventive effects of Ophiocordyceps sinensis mycelium on the liver fibrosis induced by thioacetamide.

PubMed

Wu, Yi-Hsieng Samuel; Tseng, Jung-Kai; Chou, Chung-Hsi; Chiu, Chih-Hsien; Lin, Yi-Ling; Chen, Yi-Chen

2017-06-01

Thioacetamide (TAA), usually used as a fungicide to control the decay of citrus products, itself is not toxic to the liver, but its intermediates are able to increase oxidative stress in livers and further cause fibrosis. Ophiocordyceps sinensis mycelium (OSM) which contains 10% polysaccharides and 0.25% adenosine decreased (P < 0.05) the lipid accumulation and increased (P < 0.05) antioxidative capacity in livers of thioacetamide (TAA) injected rats. Meanwhile, the increased (P < 0.05) liver sizes, serum alanine transaminase (AST) and aspartate transaminase (ALT) values in thioacetamide (TAA)-injected rats were ameliorated (P < 0.05) by OSM supplementation. Moreover, the levels of proinflammatory cytokines, such as the tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), were also reduced (P < 0.05). The fibrosis phenomena in pathological (Masson's trichrome and H&E stainings) and immunohistochemical [α-smooth actin (αSMA) and CD86/ED1] observations in TAA-treated rats were reduced (P < 0.05) by OSM cotreatment. The protective effect of OSM against TAA-induced liver inflammation/fibrosis may be via downregulations (P < 0.05) of TGF-β pathways and NFκB which further influenced (P < 0.05) the expressions of fibrotic and inflammatory genes (i. e., αSMA, Col1α, COX2). Therefore, OSM shows preventive effects on the development of TAA-induced hepatic fibrosis. © 2017 Wiley Periodicals, Inc.

Modified citrus pectin stops progression of liver fibrosis by inhibiting galectin-3 and inducing apoptosis of stellate cells.

PubMed

Abu-Elsaad, Nashwa M; Elkashef, Wagdi Fawzi

2016-05-01

Modified citrus pectin (MCP) is a pH modified form of the dietary soluble citrus peel fiber known as pectin. The current study aims at testing its effect on liver fibrosis progression. Rats were injected with CCl4 (1 mL/kg, 40% v/v, i.p., twice a week for 8 weeks). Concurrently, MCP (400 or 1200 mg/kg) was administered daily in drinking water from the first week in groups I and II (prophylactic model) and in the beginning of week 5 in groups III and IV (therapeutic model). Liver function biomarkers (ATL, AST, and ALP), fibrosis markers (laminin and hyaluronic acid), and antioxidant biomarkers (reduced glutathione (GSH) and superoxide dismutase (SOD)) were measured. Stained liver sections were scored for fibrosis and necroinflammation. Additionally, expression of galectin-3 (Gal-3), α-smooth muscle actin (SMA), tissue inhibitor metalloproteinase (TIMP)-1, collagen (Col)1A1, caspase (Cas)-3, and apoptosis related factor (FAS) were assigned. Modified pectin late administration significantly (p < 0.05) decreased malondialdehyde (MDA), TIMP-1, Col1A1, α-SMA, and Gal-3 levels and increased levels of FAS, Cas-3, GSH, and SOD. It also decreased percentage of fibrosis and necroinflammation significantly (p < 0.05). It can be concluded that MCP can attenuate liver fibrosis through an antioxidant effect, inhibition of Gal-3 mediated hepatic stellate cells activation, and induction of apoptosis.

Microhemorrhage is an Early Event in the Pulmonary Fibrotic Disease of PECAM-1 Deficient FVB/n Mice

PubMed Central

Young, Lena C.; Woods, Steven J.; Groshong, Steven D.; Basaraba, Randall J.; Gilchrist, John M.; Higgins, David M.; Gonzalez-Juarrero, Mercedes; Bass, Todd A.; Muller, William A.; Schenkel, Alan R.

2014-01-01

Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1) deficient mice in the FVB/n strain exhibit fatal chronic pulmonary fibrotic disease. The illness occurs in the absence of a detectable pro-inflammatory event. PECAM-1 is vital to the stability of vascular permeability, leukocyte extravasation, clotting of platelets, and clearance of apoptotic cells. We show here that the spontaneous development of fibrotic disease in PECAM-1 deficient FVB/n mice is characterized by early loss of vascular integrity in pulmonary capillaries, resulting in spontaneous microbleeds. Hemosiderin-positive macrophages were found in interstitial spaces and bronchoalveolar lavage (BAL) fluid in relatively healthy animals. We also observed a gradually increasing presence of hemosiderin-positive macrophages and fibrin deposition in the advanced stages of disease, corresponding to the accumulation of collagen, IL-10 expression, and myofibroblasts expressing alpha smooth muscle actin (SMA). Together with the growing evidence that pulmonary microbleeds and coagulation play an active part in human pulmonary fibrosis, this data further supports our hypothesis that PECAM-1 expression is necessary for vascular barrier function control and regulation of homeostasis specifically, in the pulmonary environment. PMID:24972347

Tributyltin chloride induces renal dysfunction by inflammation and oxidative stress in female rats.

PubMed

Coutinho, João V S; Freitas-Lima, Leandro C; Freitas, Frederico F C T; Freitas, Flávia P S; Podratz, Priscila L; Magnago, Rafaella P L; Porto, Marcella L; Meyrelles, Silvana S; Vasquez, Elisardo C; Brandão, Poliane A A; Carneiro, Maria T W D; Paiva-Melo, Francisca D; Miranda-Alves, Leandro; Silva, Ian V; Gava, Agata L; Graceli, Jones B

2016-10-17

Tributyltin chloride (TBT) is an organometallic pollutant that is used as a biocide in antifouling paints. TBT induces several toxic and endocrine-disrupting effects. However, studies evaluating the effects of TBT on renal function are rare. This study demonstrates that TBT exposure is responsible for improper renal function as well as the development of abnormal morphophysiology in mammalian kidneys. Female rats were treated with TBT, and their renal morphophysiology was assessed. Morphophysiological abnormalities such as decreased glomerular filtration rate and increased proteinuria levels were observed in TBT rats. In addition, increases in inflammation, collagen deposition and α-smooth muscle actin (α-SMA) protein expression were observed in TBT kidneys. A disrupted cellular redox balance and apoptosis in kidney tissue were also observed in TBT rats. TBT rats demonstrated reduced serum estrogen levels and estrogen receptor-α (ERα) protein expression in renal cortex. Together, these data provide in vivo evidence that TBT is toxic to normal renal function and that these effects may be associated with renal histopathology complications, such as inflammation and fibrosis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Aberrant SSEA-4 upregulation mediates myofibroblast activity to promote pre-cancerous oral submucous fibrosis

NASA Astrophysics Data System (ADS)

Yu, Cheng-Chia; Yu, Chuan-Hang; Chang, Yu-Chao

2016-11-01

Oral submucous fibrosis (OSF), regarded as a precancerous condition, is characterized by juxta-epithelial inflammatory reaction followed by fibro-elastic change in the lamina properia and epithelial atrophy. The pathologic mechanisms of OSF still need to be further clarified. In the study, we investigated the functional expression of SSEA-4, which is a well-known stemness marker, in myofibroblast activity and the clinical significance in OSF tissues. The expression of SSEA-4 in OSF was evaluated by immunohistochemical staining. Functional analysis of SSEA-4 on myofibroblast activity of OSF was achieved by lentiviral silencing ST3GAL2. Immunohisitochemistry demonstrated that SSEA-4 expression was significantly higher expression in areca quid chewing-associated OSF tissues than those of normal oral mucosa tissues. From flow cytometry analysis, arecoline dose-dependently activated SSEA-4 expression in primary human normal buccal mucosal fibroblasts (BMFs). Sorted SSEA-4-positive cells from fibrotic BMFs (fBMFs) have higher colony-forming unit, collagen gel contraction, and α-smooth muscle actin (α-SMA) expression than SSEA-4-negative subset. Knockdown of ST3GAL2 in fBMFs suppressed SSEA-4 expression, collagen contraction, migration, invasiveness, and wound healing capability. Consistently, silencing ST3GAL2 was found to repress arecoline-induced myofibroblast activity in BMFs. The study highlights SSEA-4 as a critical marker for therapeutic intervention to mediate myofibroblast transdifferentiation in areca quid chewing-associated OSF.

Combined Effects of Brain-Derived Neurotrophic Factor Immobilized Poly-Lactic-Co-Glycolic Acid Membrane with Human Adipose-Derived Stem Cells and Basic Fibroblast Growth Factor Hydrogel on Recovery of Erectile Dysfunction

PubMed Central

Lee, Seung Hwan; Kim, In Gul; Jung, Ae Ryang; Shrestha, Kshitiz Raj; Lee, Jin Ho; Park, Ki Dong; Chung, Byung Ha; Kim, Sae Woong; Kim, Ki Hean

2014-01-01

Erectile dysfunction (ED) is the most frequent long-term problem after radical prostatectomy. We aimed to evaluate whether the use of combination therapy with basic fibroblast growth factor (bFGF)-hydrogel on corpus cavernosum and with adipose-derived stem cells (ADSCs) and brain-derived neurotrophic factor (BDNF)-immobilized poly-lactic-co-glycolic acid (PLGA) membrane on the cavernous nerve (CN) could improve erectile function in a rat model of bilateral cavernous nerve crush injury (BCNI). Rats were randomly divided into five groups (n=15 per group): a normal group (N group), a group receiving saline application after bilateral cavernous nerve crush injury (BCNI), a group undergoing bFGF-hydrogel injection in the corpus cavernosum after BCNI (bFGF), a group receiving ADSC application covered with BDNF-membrane after BCNI (ADSC/BDNF), and a group undergoing coadministration of bFGF-hydrogel injection and BDNF-membrane with ADSCs after BDNF (bFGF+ADSC/BDNF). Four weeks postoperatively, the erectile function was assessed by detecting the ratio of intracavernous pressure (ICP) to mean arterial pressure (MAP). Smooth muscle and collagen contents were measured using Masson's trichrome staining. Neuronal nitric oxide synthase (nNOS) expression in the dorsal penile nerve was detected by immunostaining. The protein expression of the α-smooth muscle actin (α-SMA) and the cyclic guanosine monophosphate (cGMP) level of the corpus cavernosum were quantified by western blot and cGMP assay, respectively. In the bFGF+ADSC/BDNF group, the erectile function was significantly elevated compared with the BCNI and other treated groups and showed a significantly increased smooth muscle/collagen ratio, nNOS content, α-SMA expression, and cGMP level. In particular, there were no statistical differences in the ICP/MAP ratio, smooth muscle/collagen ratio, and α-SMA and cGMP levels between the bFGF+ADSC/BDNF group and normal group. Application of the BDNF-immobilized PLGA membrane with human ADSC into the CN and bFGF-incorporated hydrogel into the corpus carvernosum improved nearly normal erectile function in a rat model of postprostatectomy ED. This result suggests that a combined application of bFGF+ADSC/BDNF might be a promising treatment for postprostatectomy ED. PMID:24673637

OPC-13013, a cyclic nucleotide phosphodiesterase type III, inhibitor, inhibits cell proliferation and transdifferentiation of cultured rat hepatic stellate cells.

PubMed

Shimizu, E; Kobayashi, Y; Oki, Y; Kawasaki, T; Yoshimi, T; Nakamura, H

1999-01-01

Activated hepatic stellate cells (HSC; lipocytes; Ito cells) proliferate and are responsible for extracellular matrix synthesis during hepatic fibrogenesis. During activation, HSC undergo transdifferentiation into myofibroblasts expressing alpha-smooth muscle actin (alpha-SMA). Adenosine 3', 5'-cyclic monophosphate (cyclic AMP) is an ubiquitous intracellular signaling molecule, and is upregulated by the activation of adenylate cyclase and downregulated via hydrolysis by cyclic nucleotide phosphodiesterases (PDEs). Recently, increased intracellular cyclic AMP has been shown to inhibit HSC activation. The aim of the current study was to determine the effects of inhibition of PDEs on cell proliferation and transdifferentiation in cultured rat HSC. Cell proliferation was determined by [3H]thymidine incorporation, and Western blot analysis was performed for detection of alpha-SMA, a phenotypic marker of transdifferentiation into myofibroblast. When the cells were exposed to 3-isobutyl-1-methylxanthine (IBMX; 50-1000 microM), a nonselective PDE inhibitor, serum-stimulated [3H]thymidine incorporation was suppressed in a dose-dependent manner with a maximum inhibition of 66% at a concentration of 500 microM OPC-13013 (1-60 microM), a selective PDE III isoenzyme inhibitor, induced a dose-dependent inhibitory effect on serum-stimulated DNA synthesis that reached a maximum inhibition of 95% at a concentration of 60 microM, while neither 8-methoxymethyl-3-isobutyl-1-methylxanthine (8-MMX), a PDE I isoenzyme inhibitor, nor Ro-20-1724, a PDE IV isoenzyme inhibitor, had an inhibitory effect. Western blot analysis revealed that IBMX or OPC-13013 decreased alpha-SMA expression, while other selective PDE isoenzyme inhibitors did not have a suppressive effect. IBMX, OPC-13013 or Ro-20-1724, but not 8-MMX augmented forskolin-induced increase in intracellular cyclic AMP levels although cyclic AMP levels were not affected by treatment with any of these PDE inhibitors alone. These data indicate that inhibition of PDEs, especially PDE III isoenzyme, can produce an inhibitory effect on HSC activation. The PDE III isoenzyme may contribute to the regulation of HSC activation during fibrogenesis. In addition, OPC-13013 may have the potential to inhibit initiation and progression of hepatic fibrosis by interfering with HSC activation.

Human Embryonic Stem Cell-Derived Cardiomyocytes Self-Arrange with Areas of Different Subtypes During Differentiation.

PubMed

Vestergaard, Maj Linea; Grubb, Søren; Koefoed, Karen; Anderson-Jenkins, Zoe; Grunnet-Lauridsen, Kristina; Calloe, Kirstine; Clausen, Christian; Christensen, Søren Tvorup; Møllgård, Kjeld; Andersen, Claus Yding

2017-11-01

The derivation of functional cardiomyocytes (CMs) from human embryonic stem cells (hESCs) represents a unique way of studying human cardiogenesis, including the development of CM subtypes. In this study, we investigated the development and organization of hESC-derived cardiomyocytes (hESC-CMs) and examined how the expression levels of CM subtypes correspond to human in vivo cardiogenesis. Beating clusters were used to determine cardiac differentiation, which was evaluated by the expression of cardiac genes GATA4 and TNNT2 and subcellular localization of GATA4 and NKX2.5. Sharp electrode recordings to determine action potentials (APs) further revealed spatial organization of intracluster CM subtypes (ie, complex clusters). Nodal-, atrial-, and ventricular-like AP morphologies were detected within distinct regions of complex clusters. The ability of different CM subtypes to self-organize was documented by immunohistochemical analyses and a differential spatial expression of β-III tubulin, myosin light chain 2v (MLC-2V), and α-smooth muscle actin (α-SMA). Furthermore, all hESC-CM subtypes formed expressed primary cilia, which are known to coordinate cellular signaling pathways during cardiomyogenesis and heart development. This study expands the foundation for studying regulatory pathways for spatial and temporal CM differentiation during human cardiogenesis.

Hypoxia-inducible vascular endothelial growth factor gene therapy using the oxygen-dependent degradation domain in myocardial ischemia.

PubMed

Kim, Hyun Ah; Lim, Soyeon; Moon, Hyung-Ho; Kim, Sung Wan; Hwang, Ki-Chul; Lee, Minhyung; Kim, Sun Hwa; Choi, Donghoon

2010-10-01

A hypoxia-inducible VEGF expression system with the oxygen-dependent degradation (ODD) domain was constructed and tested to be used in gene therapy for ischemic myocardial disease. Luciferase and VEGF expression vector systems were constructed with or without the ODD domain: pEpo-SV-Luc (or pEpo-SV-VEGF) and pEpo-SV-Luc-ODD (or pEpo-SV-VEGF-ODD). In vitro gene expression efficiency of each vector type was evaluated in HEK 293 cells under both hypoxic and normoxic conditions. The amount of VEGF protein was estimated by ELISA. The VEGF expression vectors with or without the ODD domain were injected into ischemic rat myocardium. Fibrosis, neovascularization, and cardiomyocyte apoptosis were assessed using Masson's trichrome staining, α-smooth muscle actin (α-SMA) immunostaining, and the TUNEL assay, respectively. The plasmid vectors containing ODD significantly improved the expression level of VEGF protein in hypoxic conditions. The enhancement of VEGF protein production was attributed to increased protein stability due to oxygen deficiency. In a rat model of myocardial ischemia, the pEpo-SV-VEGF-ODD group exhibited less myocardial fibrosis, higher microvessel density, and less cardiomyocyte apoptosis compared to the control groups (saline and pEpo-SV-VEGF treatments). An ODD-mediated VEGF expression system that facilitates VEGF-production under hypoxia may be useful in the treatment of ischemic heart disease.

Chaperonin containing T-complex polypeptide subunit eta is a potential marker of joint contracture: an experimental study in the rat.

PubMed

He, Ronghan; Wang, Zhe; Lu, Yunxiang; Huang, Junqi; Ren, Jianhua; Wang, Kun

2015-11-01

Joint contracture is a fibroproliferative disorder that restricts joint mobility, resulting in tissue degeneration and deformity. However, the etiology of joint contracture is still unknown. Chaperonin containing T-complex polypeptide subunit eta (CCT-eta) is reported to increase in fibrotic diseases. The purpose of this study was to investigate whether CCT-eta is implicated in joint contracture and to determine the role of CCT-eta in the progression of joint contracture by analyzing a rat model. We immobilized the left knee joint of rat by internal fixation for 8 weeks. The non-immobilized right leg served as a control. The range of motion (ROM) of the knee was investigated. Fibroblasts were obtained from the posterior joint capsule of the joints. The outcome was followed by quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, fibroblast migration assay, and collagen assay. The effect of CCT-eta on the functions of fibroblasts was observed by utilizing a short inhibitory RNA (siRNA) targeting CCT-eta. The ROM of the immobilized joints was significantly limited compared to the contralateral joints (p < 0.05). Fibroblasts derived from the contractive joints showed higher mRNA and protein expressions of CCT-eta in parallel with alpha-smooth muscle actin (α-SMA) compared to the cells from the contralateral knees (p < 0.05). siRNA-mediated downregulation of CCT-eta inhibited the expressions of both CCT-eta and α-SMA. Moreover, the reduction of CCT-eta also significantly decreased fibroblast functions such as cell mobility and collagen synthesis (all p < 0.05). Our findings indicate that CCT-eta appears to be a potential marker of joint contracture disease.

Human Umbilical Cord-Derived Mesenchymal Stem Cells Conditioned Medium Attenuate Interstitial Fibrosis and Stimulate the Repair of Tubular Epithelial Cells in an Irreversible Model of Unilateral Ureteral Obstruction.

PubMed

Liu, Bo; Ding, Feng-Xia; Liu, Yang; Xiong, Geng; Lin, Tao; He, Da-Wei; Zhang, Yuan-Yuan; Zhang, De-Ying; Wei, Guang-Hui

2017-07-01

The growing number of patients suffering from chronic renal disease (CKD) is a challenge for the development of innovative therapies. Researchers have studied the therapeutic effects of cell therapy in acute kidney injury (AKI). However, the therapeutical effect of conditional medium (CM) in the CKD models have been rarely reported. Here, we examined the effects of umbilical cord derived-mesenchymal stem cells (hUC-MSCs) CM on renal fibrosis in a rat model of unilateral ureteral obstruction (UUO). We randomly divided the animals into three groups: sham-operated, UUO, UUO + CM. CM was administered via the left renal artery after total ligation of the left ureter. Rats were killed after 14 days of obstruction. Histological changes and oxidative stress parameters were assessed. Western blotting and immunohistochemistry analysis were used to measure epithelial-mesenchymal transition (EMT) markers, including epithelial cadherin (E-cadherin), α-smooth muscle actin (α-SMA), tumor necrosis factor-α (TNF-α), Collagen-I, and transforming growth factor β1 (TGF-β1). Proliferation and apoptosis of renal tubular epithelial cells (RTEs) were also measured. HucMSC-CM significantly reduced the levels of malondialdehyde (MDA) and reactive oxygen species (ROS), and increased the activity of glutathione (GSH) induced by UUO. Moreover, CM significantly reduced the expression of TGF-β1, α-SMA, TNF-α and Collagen-I in UUO kidney, promoted the proliferation of RTEs and inhibited its apoptosis. In addition, the increased expression of E-cadherin also reflects the effective improvement of renal interstitial fibrosis. This study shows that CM protects UUO-induced kidney damage and therefore could be a potential tool to prevent CKD progression. This article is protected by copyright. All rights reserved.

Silymarin inhibits the progression of fibrosis in the early stages of liver injury in CCl₄-treated rats.

PubMed

Clichici, Simona; Olteanu, Diana; Nagy, Andras-Laszlo; Oros, Adrian; Filip, Adriana; Mircea, Petru A

2015-03-01

Liver fibrosis, a common condition occurring during the evolution of almost all chronic liver diseases, is the consequence of hepatocyte injury that leads to the activation of Kupffer cells and hepatic stellate cells (HSC). Silymarin (Si) is a herbal product widely used for its hepatoprotective potential. Our study aims to investigate the effects of two different doses of Silymarin on a CCl4-induced model of liver fibrosis with a focus on the early stages of liver injury. Fifty Wistar rats were randomly divided into five groups (n=10): control group (sunflower oil twice a week); CMC group (carboxymethyl cellulose five times a week, sunflower oil twice a week); CCl4 group (CCl4 in sunflower oil, by gavage, twice a week); CCl4+Si 50 group (CCl4 twice a week, Silymarin 50 mg/b.w. in CMC five times a week); and CCl4+Si 200 group (similar to the previous group, with Si 200 mg/b.w.). One month after the experiment began we explored hepato-cytolysis (aminotransferases and lactate dehydrogenase), oxidative stress, fibrosis (histological score, hyaluronic acid), markers of HSC activation (transforming growth factor β1 [TGF-β1], and α-smooth muscle actin [α-SMA] expression by western blot) and activation of Kupffer cells by immunohistochemistry. Our data showed that Si 50 mg/b.w. had the capacity of reducing oxidative stress, hepato-cytolysis, fibrosis, activation of Kupffer cells, and the expression of α-SMA and TGF-β1 with better results than Si 200 mg/b.w. Thus, the usual therapeutic dose of Silymarin, administered in the early stages of fibrotic changes is capable of inhibiting the fibrogenetic mechanism and the progression of initial liver fibrosis.

Hydrogen Rich Water Attenuates Renal Injury and Fibrosis by Regulation Transforming Growth Factor-β Induced Sirt1.

PubMed

Xing, Zhaoyu; Pan, Wanma; Zhang, Jing; Xu, Xianlin; Zhang, Xuemei; He, Xiaozhou; Fan, Min

2017-01-01

The current research was designed to study the role of hydrogen in renal fibrosis and the renal epithelial to mesenchymal transition (EMT) induced by transforming growth factor-β1 (TGF-β1). Hydrogen rich water (HW) was used to treat animal and cell models. Unilateral ureteral obstruction (UUO) was performed on Balb/c mice to create a model of renal fibrosis. Human kidney proximal tubular epithelial cells (HK-2 cells) were treated with TGF-β1 for 36 h to induce EMT. Serum creatinine (Scr) and blood urea nitrogen (BUN) were measured to test renal function, in addition, kidney histology and immunohistochemical staining of alpha-smooth muscle actin (α-SMA) positive cells was performed to examine the morphological changes. The treatment with UUO induced a robust fibrosis of renal interstitium, shrink of glomerulus and partial fracture of basement membrane. Renal function was also impaired in the experimental group with UUO, with an increase of Scr and BUN in serum. After that, Western-blot was performed to examine the expression of α-SMA, fibronectin, E-cadherin, Smad2 and Sirtuin-1 (Sirt1). The treatment with HW attenuated the development of fibrosis and deterioration of renal function in UUO model. In HK-2 cells, the pretreatment of HW abolished EMT induced by TGF-β1. The down-regulation the expression of Sirt1 induced by TGF-β1 which was dampened by the treatment with HW. Sirtinol, a Sirt1 inhibitor, reversed the effect of HW on EMT induced by TGF-β1. HW can inhibit the development of fibrosis in kidney and prevents HK-2 cells from undergoing EMT which is mediated through Sirt1, a downstream molecule of TGF-β1.

Apelin attenuates TGF-β1-induced epithelial to mesenchymal transition via activation of PKC-ε in human renal tubular epithelial cells.

PubMed

Wang, Li-Yan; Diao, Zong-Li; Zheng, Jun-Fang; Wu, Yi-Ru; Zhang, Qi-Dong; Liu, Wen-Hu

2017-10-01

Epithelial to mesenchymal transition (EMT), a process whereby fully differentiated epithelial cells transition to a mesenchymal phenotype, has been implicated in the pathogenesis of renal fibrosis. Apelin, a bioactive peptide, has recently been recognized to protect against renal profibrotic activity, but the underlying mechanism has not yet been elucidated. In this study, we investigated the regulation of EMT in the presence of apelin-13 in vitro. Expression of the mesenchymal marker alpha-smooth muscle actin (α-SMA) and the epithelial marker E-cadherin was examined by immunofluorescence and western blotting in transforming growth factor beta 1 (TGF-β1)-stimulated human proximal tubular epithelial cells. Expression of extracellular matrix, fibronectin and collagen-I was examined by quantitative real-time PCR and ELISA. F13A, an antagonist of the apelin receptor APJ, and small interfering RNA targeting protein kinase C epsilon (PKC-ε) were used to explore the relevant signaling pathways. Apelin attenuated TGF-β1-induced EMT, and inhibited the EMT-associated increase in α-SMA, loss of E-cadherin, and secretion of extracellular matrix. Moreover, apelin activated PKC-ε in tubular epithelial cells, which in turn decreased phospho-Smad2/3 levels and increased Smad-7 levels. APJ inhibition or PKC-ε deletion diminished apelin-induced modulation of Smad signaling and suppression of tubular EMT. Our findings identify a novel PKC-ε-dependent mechanism in which apelin suppresses TGF-β1-mediated activation of Smad signaling pathways and thereby inhibits tubular EMT. These results suggest that apelin may be a new agent that can suppress renal fibrosis and retard chronic kidney disease progression. Copyright © 2017 Elsevier Inc. All rights reserved.

Long-term pathological and immunohistochemical features in the liver after intraoperative whole-liver irradiation in rats.

PubMed

Imaeda, Masumi; Ishikawa, Hitoshi; Yoshida, Yukari; Takahashi, Takeo; Ohkubo, Yu; Musha, Atsushi; Komachi, Mayumi; Nakazato, Yoichi; Nakano, Takashi

2014-07-01

Radiation therapy (RT) has become particularly important recently for treatment of liver tumors, but there are few experimental investigations pertaining to radiation-induced liver injuries over long-term follow-up periods. Thus, the present study examined pathological liver features over a 10-month period using an intraoperative whole-liver irradiation model. Liver function tests were performed in blood samples, whereas cell death, cell proliferation, and fibrotic changes were evaluated pathologically in liver tissues, which were collected from irradiated rats 24 h, 1, 2, 4 and 40 weeks following administration of single irradiation doses of 0 (control), 15 or 30 Gy. The impaired liver function, increased hepatocyte number, and decreased apoptotic cell proportion observed in the 15 Gy group, but not the 30 Gy group, returned to control group levels after 40 weeks; however, the Ki-67 indexes in the 15 Gy group were still higher than those in the control group after 40 weeks. Azan staining showed a fibrotic pattern in the irradiated liver in the 30 Gy group only, but the expression levels of alpha smooth muscle actin (α-SMA) and transforming growth factor-beta 1 (TGF-β1) in both the 15 and 30 Gy groups were significantly higher than those in the control group (P < 0.05). There were differences in the pathological features of the irradiated livers between the 15 Gy and 30 Gy groups, but TGF-β1 and α-SMA expression patterns supported the gradual progression of radiation-induced liver fibrosis in both groups. These findings will be useful in the future development of protective drugs for radiation-induced liver injury. © The Author 2014. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

Pericellular Versican Regulates the Fibroblast-Myofibroblast Transition

PubMed Central

Hattori, Noriko; Carrino, David A.; Lauer, Mark E.; Vasanji, Amit; Wylie, James D.; Nelson, Courtney M.; Apte, Suneel S.

2011-01-01

The cell and its glycosaminoglycan-rich pericellular matrix (PCM) comprise a functional unit. Because modification of PCM influences cell behavior, we investigated molecular mechanisms that regulate PCM volume and composition. In fibroblasts and other cells, aggregates of hyaluronan and versican are found in the PCM. Dermal fibroblasts from Adamts5−/− mice, which lack a versican-degrading protease, ADAMTS5, had reduced versican proteolysis, increased PCM, altered cell shape, enhanced α-smooth muscle actin (SMA) expression and increased contractility within three-dimensional collagen gels. The myofibroblast-like phenotype was associated with activation of TGFβ signaling. We tested the hypothesis that fibroblast-myofibroblast transition in Adamts5−/− cells resulted from versican accumulation in PCM. First, we noted that versican overexpression in human dermal fibroblasts led to increased SMA expression, enhanced contractility, and increased Smad2 phosphorylation. In contrast, dermal fibroblasts from Vcan haploinsufficient (Vcanhdf/+) mice had reduced contractility relative to wild type fibroblasts. Using a genetic approach to directly test if myofibroblast transition in Adamts5−/− cells resulted from increased PCM versican content, we generated Adamts5−/−;Vcanhdf/+ mice and isolated their dermal fibroblasts for comparison with dermal fibroblasts from Adamts5−/− mice. In Adamts5−/− fibroblasts, Vcan haploinsufficiency or exogenous ADAMTS5 restored normal fibroblast contractility. These findings demonstrate that altering PCM versican content through proteolytic activity of ADAMTS5 profoundly influenced the dermal fibroblast phenotype and may regulate a phenotypic continuum between the fibroblast and its alter ego, the myofibroblast. We propose that a physiological function of ADAMTS5 in dermal fibroblasts is to maintain optimal versican content and PCM volume by continually trimming versican in hyaluronan-versican aggregates. PMID:21828051

Doxycycline inhibits TGF-β1-induced extracellular matrix production in nasal polyp-derived fibroblasts.

PubMed

Shin, Jae-Min; Park, Joo-Hoo; Park, Il-Ho; Lee, Heung-Man

2016-03-01

Doxycycline has been shown to have antibacterial and anti-inflammatory effects and suppresses collagen biosynthesis. The purpose of this study was to evaluate the effects of doxycycline on transforming growth factor (TGF)-β1-induced myofibroblast differentiation and extracellular matrix production in nasal polyp-derived fibroblasts (NPDFs). We also determined the molecular mechanisms of action for doxycycline. NPDFs were isolated from nasal polyps from 8 patients. Doxycycline was used to pretreat TGF-β1-induced NPDFs. Cytotoxicity was evaluated using a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay. Expression levels of α-smooth muscle actin (SMA) and fibronectin were measured using Western blot, reverse-transcription polymerase chain reaction, and immunofluorescence staining. Total collagen production was analyzed with the Sircol collagen assay, while mitogen-activated protein kinase (MAPK) and NF-κB activation were determined using Western blot analysis. Luciferase assay was used to evaluate the transcriptional activity of NF-κB. Although doxycycline (0 to 40 μg/mL) had no significant cytotoxic effects in TGF-β1-induced NPDFs, it significantly reduced the expression levels of α-SMA, fibronectin, and collagen in TGF-β1-induced NPDFs in a dose-dependent manner. Doxycycline also inhibited the TGF-β1-induced activation of p38, c-Jun NH2 -terminal kinase (JNK), and NF-κB, and its inhibitory effects were similar to those of the specific inhibitors for each. Doxycycline has an inhibitory effect on TGF-β1-induced myofibroblast differentiation and extracellular matrix production via the p38 and JNK/NF-κB signal pathways in NPDFs. © 2015 ARS-AAOA, LLC.

Radical-Containing Ultrafine Particulate Matter Initiates Epithelial-to-Mesenchymal Transitions in Airway Epithelial Cells

PubMed Central

Thevenot, Paul T.; Saravia, Jordy; Jin, Nili; Giaimo, Joseph D.; Chustz, Regina E.; Mahne, Sarah; Kelley, Matthew A.; Hebert, Valeria Y.; Dellinger, Barry; Dugas, Tammy R.; DeMayo, Francesco J.

2013-01-01

Environmentally persistent free radicals (EPFRs) in combustion-generated particulate matter (PM) are capable of inducing pulmonary pathologies and contributing to the development of environmental asthma. In vivo exposure of infant rats to EPFRs demonstrates their ability to induce airway hyperresponsiveness to methacholine, a hallmark of asthma. However, the mechanisms by which combustion-derived EPFRs elicit in vivo responses remain elusive. In this study, we used a chemically defined EPFR consisting of approximately 0.2 μm amorphrous silica containing 3% cupric oxide with the organic pollutant 1,2-dichlorobenzene (DCB-230). DCB-230 possesses similar radical content to urban-collected EPFRs but offers several advantages, including lack of contaminants and chemical uniformity. DCB-230 was readily taken up by BEAS-2B and at high doses (200 μg/cm2) caused substantial necrosis. At low doses (20 μg/cm2), DCB-230 particles caused lysosomal membrane permeabilization, oxidative stress, and lipid peroxidation within 24 hours of exposure. During this period, BEAS-2B underwent epithelial-to-mesenchymal transition (EMT), including loss of epithelial cell morphology, decreased E-cadherin expression, and increased α–smooth muscle actin (α-SMA) and collagen I production. Similar results were observed in neonatal air–liquid interface culture (i.e., disruption of epithelial integrity and EMT). Acute exposure of infant mice to DCB-230 resulted in EMT, as confirmed by lineage tracing studies and evidenced by coexpression of epithelial E-cadherin and mesenchymal α-SMA proteins in airway cells and increased SNAI1 expression in the lungs. EMT in neonatal mouse lungs after EPFR exposure may provide an explanation for epidemiological evidence supporting PM exposure and increased risk of asthma. PMID:23087054

Baicalin Down-Regulates IL-1β-Stimulated Extracellular Matrix Production in Nasal Fibroblasts

PubMed Central

Shin, Jae-Min; Kang, Ju-Hyung; Lee, Seoung-Ae; Park, Il-Ho; Lee, Heung-Man

2016-01-01

Purpose Baicalin, a Chinese herbal medicine, has anti-fibrotic and anti-inflammatory effects. The aims of present study were to investigate the effects of baicalin on the myofibroblast differentiation, extracellular matrix production, migration, and collagen contraction of interleukin (IL)-1β-stimulated nasal fibroblasts and to determine the molecular mechanism of baicalin in nasal fibroblasts. Methods Nasal fibroblasts were isolated from the inferior turbinate of patients. Baicalin was used to treat IL-1β-stimulated nasal fibroblasts. To evaluate cytotoxicity, a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay was used. The expression levels of α-smooth muscle actin (SMA), fibronectin, phospho-mitogen-activated protein kinase (p-MAPK), p-Akt, p-p50, p-p65, and p-IκBα were measured by western blotting, reverse transcription-polymerase chain reaction (RT—PCR),or immunofluorescence staining. Fibroblast migration was analyzed with scratch assays and transwell migration assays. Total collagen was evaluated with the Sircol collagen assay. Contractile activity was measured with a collagen gel contraction assay. Results Baicalin (0–50 μM) had no significant cytotoxic effects in nasal fibroblasts. The expression of α–SMA and fibronectin were significantly down-regulated in baicalin-treated nasal fibroblasts. Migration, collagen production, and contraction of IL-1β-stimulated nasal fibroblasts were significantly inhibited by baicalin treatment. Baicalin also significantly down-regulated p-MAPK, p-Akt, p-p50, p-p65, and p-IκBα in IL-1β-stimulated nasal fibroblasts. Conclusions We showed that baicalin down-regulated myofibroblast differentiation, extracellular matrix production, migration, and collagen contraction via the MAPK and Akt/ NF-κB pathways in IL-1β-stimulated nasal fibroblasts. PMID:28002421

Beneficial effects of enalapril on chlorhexidine digluconate-induced liver peritoneal fibrosis in rats.

PubMed

Lee, Chung-Jen; Subeq, Yi-Maun; Lee, Ru-Ping; Ke, Chen-Yen; Lin, Nien-Tsung; Hsu, Bang-Gee

2011-08-31

Peritoneal fibrosis (PF) is a recognized complication of long-term peritoneal dialysis (PD) and can lead to ultrafiltration failure. The present study was designed to investigate the protective effects of enalapril on chlorhexidine digluconate-induced liver PF by decreasing transforming growth factor-β1 (TGF-β1) production in rats. PF was induced in Sprague-Dawley rats by daily administration of 0.5 ml 0.1% chlorhexidine digluconate in normal saline via PD tube for one week. Rats received daily intravenous injections of low dose enalapril (1 mg/kg), or high dose enalapril (2.5 mg/kg), for one week. After 7 days, conventional 4.25% Dianeal (30 ml) was administered via a PD catheter with a dwell time of 4 h and assessment of peritoneal function. At the end of dialysis, the rats were sacrificed and liver peritoneum was harvested for microscopic examination and immunohistochemistry. There was no significant difference in mean arterial pressure and heart rate between groups. After 4 h of PD, the D₄/P₄(urea) level was reduced, the D₄/D₀ glucose level, serum and the dialysate TGF-β1 level was increased, the liver peritoneum was markedly thicker, and the expression of TGF-β1, alpha-smooth muscle actin (α-SMA), fibronectin, collagen and vascular endothelial growth factor (VEGF) were elevated in the PF group compared with the vehicle group. High dose of enalapril decreased the serum and dialysate TGF-β1 levels, decreased the thickness of the liver peritoneum, and decreased the expression of TGF-β1, α-SMA, fibronectin, collagen and VEGF-positive cells in the liver peritoneum. Low dose of enalapril did not protect against chlorhexidine digluconate-induced PF in the rat. Enalapril protected against chlorhexidine digluconate-induced PF in rats by decreasing TGF-β1 production.

Aliskiren ameliorates chlorhexidine digluconate-induced peritoneal fibrosis in rats.

PubMed

Ke, Chun-Yen; Lee, Chia-Chi; Lee, Chung-Jen; Subeq, Yi-Maun; Lee, Ru-Ping; Hsu, Bang-Gee

2010-04-01

Peritoneal fibrosis (PF) is a recognized complication of long-term peritoneal dialysis (PD) and can lead to ultrafiltration failure. The present study was designed to investigate the protective effects of aliskiren on chlorhexidine digluconate-induced PF in rats. The PF was induced in Sprague-Dawley rats by daily administration of 0.5 mL 0.1% chlorhexidine digluconate in normal saline via PD tube for 1 week. Rats received daily intravenous injections of low-dose aliskiren (1 mg kg(-1)) or high-dose aliskiren (10 mg kg(-1)) for 1 week. After 7 days, conventional 4.25% Dianeal (30 mL) was administered via a PD catheter with a dwell time of 4 h and assessed of peritoneal function. At the end of dialysis, rats were sacrificed and the liver peritoneum was harvested for microscopically and immunohistochemistry. There was no significant difference in mean arterial pressure and heart rate between groups. After 4 h of PD, the D(4)/P(4) urea level was reduced, the D(4)/D(0) glucose level, serum and dialysate transforming growth factor-beta1 (TGF-beta1) level was increased, the liver peritoneum was markedly thicker, and the expression of TGF-beta1, alpha-smooth muscle actin (alpha-SMA), fibronectin, collagen, and vascular endothelial growth factor (VEGF) were elevated in the PS group compared with the vehicle group. Aliskiren decreased the serum and dialysate TGF-beta1 level, decreased the thickness of the liver peritoneum, and decreased the expression of TGF-beta1, alpha-SMA, fibronectin, collagen, and VEGF-positive cells in liver peritoneum. Moreover, high-dose aliskiren had better protective effects against PF than low dose in rats. Aliskiren protected against chlorhexidine digluconate-induced PF in rats by decreasing TGF-beta1 production.

Topical naltrexone accelerates full-thickness wound closure in type 1 diabetic rats by stimulating angiogenesis.

PubMed

McLaughlin, Patricia J; Immonen, Jessica A; Zagon, Ian S

2013-07-01

Delays in wound healing often result in infection, chronic ulceration, and possible amputation of extremities. Impaired wound healing is a major complication of the 23 million people in the USA with diabetes, and financial and medical burdens are demanding new treatments for wound healing. Previous studies have demonstrated that topical application of the opioid antagonist naltrexone (NTX) dissolved in moisturizing cream reverses delays in wound closure in rats with streptozotocin-induced type 1 diabetes. A target of NTX's action is DNA synthesis and cell proliferation. In this study, granulation tissue was evaluated to ascertain the specific cellular targets that were impaired in diabetic wounds, as well as those that were enhanced following NTX application. Mast cell number as well as the number of new blood vessels immunoreactive to fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), and alpha smooth muscle actin (α-SMA) antibodies were recorded at 3, 5, 8, 10, 15, and 20 days following creation of full-thickness dorsal cutaneous wounds in normal and type 1 diabetic rats. Diabetic rats displayed delays in wound closure as well as a reduction in the number of mast cells responding to the injury, and delays in the spatial and temporal expression of FGF-2, VEGF, and α-SMA in capillaries. Topical NTX accelerated the rate of wound closure and stimulated expression of angiogenic factors within granulation tissue of diabetic rats relative to control animals receiving saline in moisturizing cream. These data support observations that a novel biological pathway is impaired under diabetic conditions and can be modulated by topical NTX to enhance proliferative events in wound healing.

Combination of three angiogenic growth factors has synergistic effects on sprouting of endothelial cell/mesenchymal stem cell-based spheroids in a 3D matrix.

PubMed

Kim, Sook Kyoung; Lee, Jaeyeon; Song, Myeongjin; Kim, Mirim; Hwang, Soon Jung; Jang, Hwanseok; Park, Yongdoo

2016-11-01

Combinations of angiogenic growth factors have been shown to have synergistic effects on angiogenesis and natural wound healing in various animal models. Each growth factor has unique roles during angiogenesis; vascular endothelial growth factor (VEGF) plays a key role during the initial step of angiogenesis, whereas PDGF functions in the maturation of blood vessels. We used a combination of three angiogenic growth factors to increase angiogenesis in vitro and in vivo. We chose VEGF as a basic factor and added platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) to induce angiogenesis in three in vitro and in vivo models: 3D angiogenesis assay, 3D co-culture, and matrigel plug implantation assay. Cell proliferation was significantly higher in co-cultured cells treated with PDGF + VEGF + FGF than in the control, single, or dual combination groups. mRNA expression of α-smooth muscle actin (α-SMA), von Willebrand factor (vWF), and CD105 was higher in the triple group (PDGF + VEGF + FGF) than in control, single, or dual combination groups. In the PDGF + VEGF + FGF group, the length and number of branches of spheroids was also significantly higher than in the control, single, or dual combination groups. Furthermore, in a nude mouse model, α-SMA expression was significantly higher in the PDGF + VEGF + FGF group than in other groups. In conclusion, the addition of PDGF and FGF to VEGF showed synergistic effects on angiogenesis in vitro and in vivo. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1535-1543, 2016. © 2015 Wiley Periodicals, Inc.

Progesterone attenuates airway remodeling and glucocorticoid resistance in a murine model of exposing to ozone.

PubMed

Zhang, Xue; Bao, Wuping; Fei, Xia; Zhang, Yingying; Zhang, Guoqing; Zhou, Xin; Zhang, Min

2018-04-01

Airway remodeling is a vital component of chronic obstructive pulmonary disease (COPD). Despite the broad anti-inflammation effects of glucocorticoids, they exhibit relatively little therapeutic benefit in COPD, indicating the accelerating demands of new agents for COPD. We aim to explore the effect of progesterone on airway remodeling in a murine modeling of exposing to ozone and to further examine the potential effect of progesterone on glucocorticoid insensitivity. C57/BL6 mice were exposed to ozone for 12 times over 6 weeks, and were administered with progesterone alone or combined with budesonide (BUD) after each exposure until the 10th week. The peribronchial collagen deposition was measured. The protein levels of MMP8 and MMP9 in bronchoalveolar lavage fluid (BALF) and lungs were assessed. Western blot analysis was used to detect the levels of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), a-smooth muscle actin (α-SMA), glycogen synthase kinase-3β (GSK-3β). The expression of VEGF and histone deacetylase 2 (HDAC2) in the lung were determined by Immunohistochemical analyses. We observe that progesterone attenuates the peribronchial collagen deposition, as well as the expression of MMP8, MMP9, HIF-1α, VEGF, α-SMA, and GSK-3β in BALF or lung tissues. Progesterone or BUD monotherapy has no effect on HDAC2 production. Progesterone combines with BUD induce dramatically enhanced effects. Thus, these results demonstrate novel roles of progesterone for the pathogenesis and airway remodeling in COPD. Progesterone plus BUD administration exerts more significant inhibition on airway remodeling with dose-independent. Additionally, progesterone may, to some extent, improve the glucocorticoid insensitivity. Copyright © 2018. Published by Elsevier Ltd.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jin Lim, Min; Ahn, Jiyeon; Youn Yi, Jae

Fibrosis is one of the most serious side effects in cancer patients undergoing radio-/ chemo-therapy, especially of the lung, pancreas or kidney. Based on our previous finding that galectin-1 (Gal-1) was significantly increased during radiation-induced lung fibrosis in areas of pulmonary fibrosis, we herein clarified the roles and action mechanisms of Gal-1 during fibrosis. Our results revealed that treatment with TGF-β1 induced the differentiation of fibroblast cell lines (NIH3T3 and IMR-90) to myofibroblasts, as evidenced by increased expression of the fibrotic markers smooth muscle actin-alpha (α-SMA), fibronectin, and collagen (Col-1). We also observed marked and time-dependent increases in the expressionmore » level and nuclear accumulation of Gal-1. The TGF-β1-induced increases in Gal-1, α-SMA and Col-1 were decreased by inhibitors of PI3-kinase and p38 MAPK, but not ERK. Gal-1 knockdown using shRNA decreased the phosphorylation and nuclear retention of Smad2, preventing the differentiation of fibroblasts. Gal-1 interacted with Smad2 and phosphorylated Smad2, which may accelerate fibrotic processes. In addition, up-regulation of Gal-1 expression was demonstrated in a bleomycin (BLM)-induced mouse model of lung fibrosis in vivo. Together, our results indicate that Gal-1 may promote the TGF-β1-induced differentiation of fibroblasts by sustaining nuclear localization of Smad2, and could be a potential target for the treatment of pulmonary fibrotic diseases. - Highlights: • Galectin-1 (Gal-1) promotes TGF-β-induced fibroblast differentiation via activation of PI3-kinase and p38 MAPK. • Gal-1 binds to Smad2 and phosphorylated Smad2. • GAl-1 may be a new therapeutic target for attenuating lung fibrotic process.« less

Vascular Permeability and Remodelling Coincide with Inflammatory and Reparative Processes after Joint Bleeding in Factor VIII-Deficient Mice.

PubMed

Cooke, Esther J; Zhou, Jenny Y; Wyseure, Tine; Joshi, Shweta; Bhat, Vikas; Durden, Donald L; Mosnier, Laurent O; Drygalski, Annette von

2018-06-01

Vascular remodelling is a prominent feature of haemophilic arthropathy (HA) that may underlie re-bleeding, yet the nature of vascular changes and underlying mechanisms remain largely unknown. Here, we aimed to characterize synovial vascular remodelling and vessel integrity after haemarthrosis, as well as temporal changes in inflammatory and tissue-reparative pathways. Thirty acutely painful joints in patients with haemophilia (PWH) were imaged by musculoskeletal ultrasound with Power Doppler (MSKUS/PD) to detect vascular abnormalities and bloody effusions. Nineteen out of 30 painful joint episodes in PWH were associated with haemarthrosis, and abnormal vascular perfusion was unique to bleeding joints. A model of induced haemarthrosis in factor VIII (FVIII)-deficient mice was used for histological assessment of vascular remodelling (α-smooth muscle actin [αSMA] expression), and monitoring of in vivo vascular perfusion and permeability by MSKUS/PD and albumin extravasation, respectively. Inflammatory (M1) and reparative (M2) macrophage markers were quantified in murine synovium over a 10-week time course by real-time polymerase chain reaction. The abnormal vascular perfusion observed in PWH was recapitulated in FVIII-deficient mice after induced haemarthrosis. Neovascularization and increased vessel permeability were apparent 2 weeks post-bleed in FVIII-deficient mice, after a transient elevation of inflammatory macrophage M1 markers. These vascular changes subsided by week 4, while vascular remodelling, evidenced by architectural changes and pronounced αSMA expression, persisted alongside a reparative macrophage M2 response. In conclusion, haemarthrosis leads to transient inflammation coupled with neovascularization and associated vascular permeability, while subsequent tissue repair mechanisms coincide with vascular remodelling. Together, these vascular changes may promote re-bleeding and HA progression. Schattauer GmbH Stuttgart.

Inhibition of the K+ channel K(Ca)3.1 reduces TGF-β1-induced premature senescence, myofibroblast phenotype transition and proliferation of mesangial cells.

PubMed

Fu, Rong-Guo; Zhang, Tao; Wang, Li; Du, Yan; Jia, Li-Ning; Hou, Jing-Jing; Yao, Gang-Lian; Liu, Xiao-Dan; Zhang, Lei; Chen, Ling; Gui, Bao-Song; Xue, Rong-Liang

2014-01-01

K(Ca)3.1 channel participates in many important cellular functions. This study planned to investigate the potential involvement of K(Ca)3.1 channel in premature senescence, myofibroblast phenotype transition and proliferation of mesangial cells. Rat mesangial cells were cultured together with TGF-β1 (2 ng/ml) and TGF-β1 (2 ng/ml) + TRAM-34 (16 nM) separately for specified times from 0 min to 60 min. The cells without treatment served as controls. The location of K(Ca)3.1 channels in mesangial cells was determined with Confocal laser microscope, the cell cycle of mesangial cells was assessed with flow cytometry, the protein and mRNA expression of K(Ca)3.1, α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP-1) were detected with Western blot and RT-PCR. One-way analysis of variance (ANOVA) and Student-Newman-Keuls-q test (SNK-q) were used to do statistical analysis. Statistical significance was considered at P<0.05. Kca3.1 channels were located in the cell membranes and/or in the cytoplasm of mesangial cells. The percentage of cells in G0-G1 phase and the expression of K(ca)3.1, α-SMA and FSP-1 were elevated under the induction of TGF-β1 when compared to the control and decreased under the induction of TGF-β1+TRAM-34 when compared to the TGF-β1 induced (P<0.05 or P<0.01). Targeted disruption of K(Ca)3.1 inhibits TGF-β1-induced premature aging, myofibroblast-like phenotype transdifferentiation and proliferation of mesangial cells.

Effect of [6]-gingerol on myofibroblast differentiation in transforming growth factor beta 1-induced nasal polyp-derived fibroblasts.

PubMed

Park, Sook A; Park, Il-Ho; Cho, Jung-Sun; Moon, You-Mi; Lee, Seung Hoon; Kim, Tae Hoon; Lee, Sang Hag; Lee, Heung-Man

2012-01-01

[6]-Gingerol is one of the major pungent principles of ginger and has diverse effects, including anti-inflammatory, and antioxidative effects. Reactive oxygen species (ROS) are released during the phenotypic transformation of fibroblasts to myofibroblasts, a process that is involved in the growth of nasal polyps by inducing extracellular matrix (ECM) accumulation. The purpose of this study was to determine the effect of [6]-gingerol on myofibroblast differentiation and collagen production of nasal polyp-derived fibroblasts (NPDFs) and to determine if the effect of [6]-gingerol is linked to an antioxidant effect. NPDFs were incubated and treated with transforming growth factor (TGF) beta 1. The ROS generated by NPDFs were determined using 2″,7″-dichlorfluorescein-diacetate. The fluorescence was captured by a fluorescent microscope and measured using a fluorometer. The expression of alpha-smooth muscle actin (SMA) and collagen type IV mRNA was determined by a reverse transcription-polymerase chain reaction, and the expression of α-SMA protein and pSmad2/3 was determined by immunofluorescence microscopy and or Western blotting. The amount of total soluble collagen production was analyzed by the SirCol collagen dye-binding assay. TGF-beta 1 stimulation increased ROS production by NPDFs. [6]-Gingerol decreased the production of ROS in TGF-beta 1-induced NPDFs. Myofibroblast differentiation, collagen production, and phosphorylation of Smad2/3 were prevented by [6]-gingerol and inhibition of ROS generation with antioxidant such as diphenyliodonium, N-acetylcysteine, and ebselen. These results suggest the possibility that [6]-gingerol may play an important role in inhibiting the production of the ECM in the development of nasal polyps through an antioxidant effect.

Non-Aggregating Tau Phosphorylation by Cyclin-Dependent Kinase 5 Contributes to Motor Neuron Degeneration in Spinal Muscular Atrophy

PubMed Central

Miller, Nimrod; Feng, Zhihua; Edens, Brittany M.; Yang, Ben; Shi, Han; Sze, Christie C.; Hong, Benjamin Taige; Su, Susan C.; Cantu, Jorge A.; Topczewski, Jacek; Crawford, Thomas O.; Ko, Chien-Ping; Sumner, Charlotte J.; Ma, Long

2015-01-01

Mechanisms underlying motor neuron degeneration in spinal muscular atrophy (SMA), the leading inherited cause of infant mortality, remain largely unknown. Many studies have established the importance of hyperphosphorylation of the microtubule-associated protein tau in various neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. However, tau phosphorylation in SMA pathogenesis has yet to be investigated. Here we show that tau phosphorylation on serine 202 (S202) and threonine 205 (T205) is increased significantly in SMA motor neurons using two SMA mouse models and human SMA patient spinal cord samples. Interestingly, phosphorylated tau does not form aggregates in motor neurons or neuromuscular junctions (NMJs), even at late stages of SMA disease, distinguishing it from other tauopathies. Hyperphosphorylation of tau on S202 and T205 is mediated by cyclin-dependent kinase 5 (Cdk5) in SMA disease condition, because tau phosphorylation at these sites is significantly reduced in Cdk5 knock-out mice; genetic knock-out of Cdk5 activating subunit p35 in an SMA mouse model also leads to reduced tau phosphorylation on S202 and T205 in the SMA;p35−/− compound mutant mice. In addition, expression of the phosphorylation-deficient tauS202A,T205A mutant alleviates motor neuron defects in a zebrafish SMA model in vivo and mouse motor neuron degeneration in culture, whereas expression of phosphorylation-mimetic tauS202E,T205E promotes motor neuron defects. More importantly, genetic knock-out of tau in SMA mice rescues synapse stripping on motor neurons, NMJ denervation, and motor neuron degeneration in vivo. Altogether, our findings suggest a novel mechanism for SMA pathogenesis in which hyperphosphorylation of non-aggregating tau by Cdk5 contributes to motor neuron degeneration. PMID:25878277

A Murine Hypertrophic Cardiomyopathy Model: The DBA/2J Strain.

PubMed

Zhao, Wenyuan; Zhao, Tieqiang; Chen, Yuanjian; Zhao, Fengbo; Gu, Qingqing; Williams, Robert W; Bhattacharya, Syamal K; Lu, Lu; Sun, Yao

2015-01-01

Familial hypertrophic cardiomyopathy (HCM) is attributed to mutations in genes that encode for the sarcomere proteins, especially Mybpc3 and Myh7. Genotype-phenotype correlation studies show significant variability in HCM phenotypes among affected individuals with identical causal mutations. Morphological changes and clinical expression of HCM are the result of interactions with modifier genes. With the exceptions of angiotensin converting enzyme, these modifiers have not been identified. Although mouse models have been used to investigate the genetics of many complex diseases, natural murine models for HCM are still lacking. In this study we show that the DBA/2J (D2) strain of mouse has sequence variants in Mybpc3 and Myh7, relative to widely used C57BL/6J (B6) reference strain and the key features of human HCM. Four-month-old of male D2 mice exhibit hallmarks of HCM including increased heart weight and cardiomyocyte size relative to B6 mice, as well as elevated markers for cardiac hypertrophy including β-myosin heavy chain (MHC), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and skeletal muscle alpha actin (α1-actin). Furthermore, cardiac interstitial fibrosis, another feature of HCM, is also evident in the D2 strain, and is accompanied by up-regulation of type I collagen and α-smooth muscle actin (SMA)-markers of fibrosis. Of great interest, blood pressure and cardiac function are within the normal range in the D2 strain, demonstrating that cardiac hypertrophy and fibrosis are not secondary to hypertension, myocardial infarction, or heart failure. Because D2 and B6 strains have been used to generate a large family of recombinant inbred strains, the BXD cohort, the D2 model can be effectively exploited for in-depth genetic analysis of HCM susceptibility and modifier screens.

Searching Novel Therapeutic Targets for Scleroderma: P2X7-Receptor Is Up-regulated and Promotes a Fibrogenic Phenotype in Systemic Sclerosis Fibroblasts

PubMed Central

Gentile, Daniela; Lazzerini, Pietro E.; Gamberucci, Alessandra; Natale, Mariarita; Selvi, Enrico; Vanni, Francesca; Alì, Alessandra; Taddeucci, Paolo; Del-Ry, Silvia; Cabiati, Manuela; Della-Latta, Veronica; Abraham, David J.; Morales, Maria A.; Fulceri, Rosella; Laghi-Pasini, Franco; Capecchi, Pier L.

2017-01-01

Objectives: Systemic sclerosis (SSc) is a connective tissue disorder presenting fibrosis of the skin and internal organs, for which no effective treatments are currently available. Increasing evidence indicates that the P2X7 receptor (P2X7R), a nucleotide-gated ionotropic channel primarily involved in the inflammatory response, may also have a key role in the development of tissue fibrosis in different body districts. This study was aimed at investigating P2X7R expression and function in promoting a fibrogenic phenotype in dermal fibroblasts from SSc patients, also analyzing putative underlying mechanistic pathways. Methods: Fibroblasts were isolated by skin biopsy from 9 SSc patients and 8 healthy controls. P2X7R expression, and function (cytosolic free Ca2+ fluxes, α-smooth muscle actin [α-SMA] expression, cell migration, and collagen release) were studied. Moreover, the role of cytokine (interleukin-1β, interleukin-6) and connective tissue growth factor (CTGF) production, and extracellular signal-regulated kinases (ERK) activation in mediating P2X7R-dependent pro-fibrotic effects in SSc fibroblasts was evaluated. Results: P2X7R expression and Ca2+ permeability induced by the selective P2X7R agonist 2′-3′-O-(4-benzoylbenzoyl)ATP (BzATP) were markedly higher in SSc than control fibroblasts. Moreover, increased αSMA expression, cell migration, CTGF, and collagen release were observed in lipopolysaccharides-primed SSc fibroblasts after BzATP stimulation. While P2X7-induced cytokine changes did not affect collagen production, it was completely abrogated by inhibition of the ERK pathway. Conclusion: In SSc fibroblasts, P2X7R is overexpressed and its stimulation induces Ca2+-signaling activation and a fibrogenic phenotype characterized by increased migration and collagen production. These data point to the P2X7R as a potential, novel therapeutic target for controlling exaggerated collagen deposition and tissue fibrosis in patients with SSc. PMID:28955239

Curcumin accelerates cutaneous wound healing via multiple biological actions: The involvement of TNF-α, MMP-9, α-SMA, and collagen.

PubMed

Yen, Yu-Hsiu; Pu, Chi-Ming; Liu, Chen-Wei; Chen, Ya-Chun; Chen, Yu-Chen; Liang, Chan-Jung; Hsieh, Jung-Hsien; Huang, Hui-Fu; Chen, Yuh-Lien

2018-04-16

Curcumin, a constituent of the turmeric plant, has antitumor, anti-inflammatory, and antioxidative effects, but its effects on wound healing are unclear. We created back wounds in 72 mice and treated them with or without topical curcumin (0.2 mg/mL) in Pluronic F127 gel (20%) daily for 3, 5, 7, 9, and 12 days. Healing in wounds was evaluated from gross appearance, microscopically by haematoxylin and eosin staining, by immunohistochemistry for tumour necrosis factor alpha and alpha smooth muscle actin, and by polymerase chain reaction amplification of mRNA expression levels. Treatment caused fast wound closure with well-formed granulation tissue dominated by collagen deposition and regenerating epithelium. Curcumin increased the levels of tumour necrosis factor alpha mRNA and protein in the early phase of healing, which then decreased significantly. However, these levels remained high in controls. Levels of collagen were significantly higher in curcumin-treated wounds. Immunohistochemical staining for alpha smooth muscle actin was increased in curcumin-treated mice on days 7 and 12. Curcumin treatment significantly suppressed matrix metallopeptidase-9 and stimulated alpha smooth muscle levels in tumour necrosis factor alpha-treated fibroblasts via nuclear factor kappa B signalling. Thus, topical curcumin accelerated wound healing in mice by regulating the levels of various cytokines. © 2018 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

Pirfenidone inhibits post-traumatic proliferative vitreoretinopathy.

PubMed

Khanum, B N M K; Guha, R; Sur, V P; Nandi, S; Basak, S K; Konar, A; Hazra, S

2017-09-01

PurposeThe purpose of the study was to evaluate the efficacy and safety of intravitreal pirfenidone for inhibition of proliferative vitreoretinopathy (PVR) in a model of penetrating ocular injury.Patients and methodsPenetrating trauma was induced on the retina of rabbit and treated either with 0.1 ml of phosphate-buffered saline (PBS) or 0.1 ml of 0.5% pirfenidone, and development of PVR was evaluated clinically and graded after 1 month. Histopathology and immunohistochemistry with transforming growth factor beta (TGFβ), alpha smooth muscle actin (αSMA), and collagen-1 were performed to assess the fibrotic changes. Expression of cytokines in the vitro-retinal tissues at different time points following pirfenidone and PBS injection was examined by RT-PCR. Availability of pirfenidone in the vitreous of rabbit at various time points was determined by high-performance liquid chromatography following injection of 0.1 ml of 0.5% pirfenidone. In normal rabbit eye, 0.1 ml of 0.5% pirfenidone was injected to evaluate any toxic effect.ResultsClinical assessment and grading revealed prevention of PVR formation in pirfenidone-treated animals, gross histology, and histopathology confirmed the observation. Immunohistochemistry showed prevention in the expression of collagen-I, αSMA, and TGFβ in the pirfenidone-treated eyes compared to the PBS-treated eyes. Pirfenidone inhibited increased gene expression of cytokines observed in control eyes. Pirfenidone could be detected up to 48 h in the vitreous of rabbit eye following single intravitreal injection. Pirfenidone did not show any adverse effect following intravitreal injection; eyes were devoid of any abnormal clinical sign, intraocular pressure, and electroretinography did not show any significant change and histology of retina remained unchanged.ConclusionThis animal study shows that pirfenidone might be a potential therapy for PVR. Further clinical study will be useful to evaluate the clinical application of pirfenidone.

Cathepsin K expression in a wide spectrum of perivascular epithelioid cell neoplasms (PEComas): a clinicopathological study emphasizing extrarenal PEComas.

PubMed

Rao, Qiu; Cheng, Liang; Xia, Qiu-yuan; Liu, Biao; Li, Li; Shi, Qun-li; Shi, Shan-shan; Yu, Bo; Zhang, Ru-song; Ma, Heng-hui; Lu, Zhen-feng; Tu, Pin; Zhou, Xiao-jun

2013-03-01

Recent studies have demonstrated that cathepsin K seems to be a powerful marker in identifying renal perivascular epithelioid cell neoplasms (PEComas). However, the expression in extrarenal PEComas has not been well characterized due to their rare incidence. Our aim was to investigate the expression of cathepsin K in a wide spectrum of extrarenal PEComas and evaluate its potential diagnostic usefulness in comparison with other commonly used markers. Twenty-three cases of PEComa (liver, n = 9; lung, n = 1; broad ligament of uterus, n = 1; vertex subcutaneous soft tissue, n = 1; abdominal wall, n = 1; and kidney, n = 10) were selected for study. All displayed a high percentage of cells with moderately to strongly positive reactions for cathepsin K (mean 91%; range 80-100%). HMB45, Melan-A and smooth muscle actin (SMA) were expressed in 78, 87 and 87% of cases, respectively, with various percentages of positive cells (mean, 34, 40 and 38%; range 0-80, 0-90 and 0-90%). Transcription factor E3 (TFE3) was expressed strongly in only three cases; none exhibited evidence of TFE3 gene fusion or amplification. Cathepsin K appears to be more powerful than other commonly used markers in diagnosing a wide spectrum of PEComas and distinguishing them from the majority of human cancers. © 2012 Blackwell Publishing Ltd.

ERα inhibited myocardin-induced differentiation in uterine fibroids.

PubMed

Liao, Xing-Hua; Li, Jun-Yan; Dong, Xiu-Mei; Wang, Xiuhong; Xiang, Yuan; Li, Hui; Yu, Cheng-Xi; Li, Jia-Peng; Yuan, Bai-Yin; Zhou, Jun; Zhang, Tong-Cun

2017-01-01

Uterine fibroids, also known as uterine leiomyomas, are a benign tumor of the human uterus and the commonest estrogen-dependent benign tumor found in women. Myocardin is an important transcriptional regulator in smooth and cardiac muscle development. The role of myocardin and its relationship with ERα in uterine fibroids have barely been addressed. We noticed that the expression of myocardin was markedly reduced in human uterine fibroid tissue compared with corresponding normal or adjacent myometrium tissue. Here we reported that myocardin induced the transcription and expression of differentiation markers SM22α and alpha smooth muscle actin (α-SMA) in rat primary uterine smooth muscle cells (USMCs) and this effect was inhibited by ERα. Notably, we showed that, ERα induced expression of proliferation markers PCNA and ki-67 in rat primary USMCs. We also found ERα interacted with myocardin and formed complex to bind to CArG box and inhibit the SM22α promoter activity. Furthermore, ERα inhibited the transcription and expression of myocardin, and reduced the levels of transcription and expression of downstream target SM22α, a SMC differentiation marker. Our data thus provided important and novel insights into how ERα and myocardin interact to control the cell differentiation and proliferation of USMCs. Thus, it may provide potential therapeutic target for uterine fibroids. Copyright © 2016 Elsevier Inc. All rights reserved.

Actin cytoskeletal rearrangement and dysfunction due to activation of the receptor for advanced glycation end products is inhibited by thymosin beta 4

PubMed Central

Kim, Sokho; Kwon, Jungkee

2015-01-01

The receptor of advanced glycation end products (RAGE) is a cell-surface receptor that is a key factor in the pathogenesis of diabetic complications, including vascular disorders. Dysfunction of the actin cytoskeleton contributes to disruption of cell membrane repair in response to various type of endothelial cell damage. However, mechanism underlying RAGE remodelling of the actin cytoskeleton, by which globular actin (G-actin) forms to filamentous actin (F-actin), remains unclear. In this study we examined the role of thymosin beta 4 (Tβ4) – which binds to actin, blocks actin polymerization, and maintains the dynamic equilibrium between G-actin and F-actin in human umbilical vein endothelial cells (HUVECs) – in the response to RAGE. Tβ4 increased cell viability and decreased levels of reactive oxygen species in HUVECs incubated with AGEs. Tβ4 reduced the expression of RAGE, consistent with a down-regulation of the F-actin to G-actin ratio. The effect of remodelling of the actin cytoskeleton on RAGE expression was clarified by adding Phalloidin, which stabilizes F-actin. Moreover, small interfering RNA was used to determine whether intrinsic Tβ4 regulates RAGE expression in the actin cytoskeleton. The absence of intrinsic Tβ4 in HUVECs evoked actin cytoskeleton disorder and increased RAGE expression. These findings suggest that regulation of the actin cytoskeleton by Tβ4 plays a pivotal role in the RAGE response to AGEs. PMID:25640761

The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis

PubMed Central

Spracklen, Andrew J.; Fagan, Tiffany N.; Lovander, Kaylee E.; Tootle, Tina L.

2015-01-01

Dynamic remodeling of the actin cytoskeleton is required for both development and tissue homeostasis. While fixed image analysis has provided significant insight into such events, a complete understanding of cytoskeletal dynamics requires live imaging. Numerous tools for the live imaging of actin have been generated by fusing the actin-binding domain from an actin-interacting protein to a fluorescent protein. Here we comparatively assess the utility of three such tools – Utrophin, Lifeact, and F-tractin – for characterizing the actin remodeling events occurring within the germline-derived nurse cells during Drosophila mid-oogenesis or follicle development. Specifically, we used the UAS/GAL4 system to express these tools at different levels and in different cells, and analyzed these tools for effects on fertility, alterations in the actin cytoskeleton, and ability to label filamentous actin (F-actin) structures by both fixed and live imaging. While both Utrophin and Lifeact robustly label F-actin structures within the Drosophila germline, when strongly expressed they cause sterility and severe actin defects including cortical actin breakdown resulting in multi-nucleate nurse cells, early F-actin filament and aggregate formation during stage 9 (S9), and disorganized parallel actin filament bundles during stage 10B (S10B). However, by using a weaker germline GAL4 driver in combination with a higher temperature, Utrophin can label F-actin with minimal defects. Additionally, strong Utrophin expression within the germline causes F-actin formation in the nurse cell nuclei and germinal vesicle during mid-oogenesis. Similarly, Lifeact expression results in nuclear F-actin only within the germinal vesicle. F-tractin expresses at a lower level than the other two labeling tools, but labels cytoplasmic F-actin structures well without causing sterility or striking actin defects. Together these studies reveal how critical it is to evaluate the utility of each actin labeling tool within the tissue and cell type of interest in order to identify the tool that represents the best compromise between acceptable labeling and minimal disruption of the phenomenon being observed. In this case, we find that F-tractin, and perhaps Utrophin, when Utrophin expression levels are optimized to label efficiently without causing actin defects, can be used to study F-actin dynamics within the Drosophila nurse cells. PMID:24995797

Disturbed P53-MDM2 Feedback Loop Contributes to Thoracic Aortic Dissection Formation and May be a Result of TRIM25 Overexpression.

PubMed

Gong, Bin; Wang, Zhiwei; Zhang, Min; Hu, Zhipeng; Ren, Zongli; Tang, Zheng; Jiang, Wanli; Cheng, Lianghao; Huang, Jun; Ren, Wei; Wang, Qingtao

2017-04-01

The development of thoracic aortic dissection (TAD) is attributed to a broad range of degenerative, genetic, structural, oxidative, apoptotic, and acquired disease states. In this study, we examined the role of the disturbed p53-MDM2 (murine double minute 2) feedback loop in the formation of TAD, and one of a potential feedback loop regulator, TRIM25 (tripartite motif protein-25). Surgical specimens of the aorta from TAD patients (n = 10) and controls (n = 10) were tested for α-smooth muscle actin (α-SMA), p53, MDM2, and TRIM25 by western blot, immunohistochemical staining, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), respectively. When compared with controls, western blot shows that the protein levels of p53, MDM2, and TRIM25 were increased significantly in the aortic media of TAD patients. qRT-PCR further verified that the mRNA expression of MDM2 and TRIM25 was also increased 6- and 4-folds, respectively, in the TAD media of the aortic wall. Immunohistochemistry results showed significantly decreased staining of α-SMA, smooth muscle cells, and more collagen deposition in the media of the aortic wall from patients with TAD. This study provided a new insight into the disturbed p53-MDM2 feedback loop in the pathogenesis of TAD, and this may be because of the TRIM25 overexpression. Copyright © 2016 Elsevier Inc. All rights reserved.

Adenovirus-mediated E2-EPF UCP Gene Transfer Prevents Autoamputation in a Mouse Model of Hindlimb Ischemia

PubMed Central

Lim, Jung Hwa; Shin, Hyo Jung; Park, Kyeong-Su; Lee, Chan Hee; Jung, Cho-Rok; Im, Dong-Soo

2012-01-01

E2-EPF ubiquitin carrier protein (UCP) stabilizes hypoxia-inducible factor-1α (HIF-1α) inducing ischemic vascular responses. Here, we investigated the effect of UCP gene transfer on therapeutic angiogenesis. Adenovirus-encoded UCP (Ad-F-UCP) increased the expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) in cells and mice. Conditioned media from UCP-overexpressing cells promoted proliferation, tubule formation, and invasion of human umbilical-vascular-endothelial cells (HUVECs), and vascularization in chorioallantoic membrane (CAM) assay. Ad-F-UCP increased the vessel density in the Martigel plug assay, and generated copious vessel-like structures in the explanted muscle. The UCP effect on angiogenesis was dependent on VEGF and FGF-2. In mouse hindlimb ischemia model (N = 30/group), autoamputation (limb loss) occurred in 87% and 68% of the mice with saline and Ad encoding β-galactosidase (Ad-LacZ), respectively, whereas only 23% of the mice injected with Ad-F-UCP showed autoamputation after 21 days of treatment. Ad-F-UCP increased protein levels of HIF-1α, platelet-endothelial cell adhesion molecule-1 (PECAM-1), smooth muscle cell actin (SMA) in the ischemic muscle, and augmented blood vessels doubly positive for PECAM-1 and SMA. Consequently, UCP gene transfer prevented muscle degeneration and autoamputation of ischemic limb. The results suggest that E2-EPF UCP may be a target for therapeutic angiogenesis. PMID:22294149

Adenovirus-mediated E2-EPF UCP gene transfer prevents autoamputation in a mouse model of hindlimb ischemia.

PubMed

Lim, Jung Hwa; Shin, Hyo Jung; Park, Kyeong-Su; Lee, Chan Hee; Jung, Cho-Rok; Im, Dong-Soo

2012-04-01

E2-EPF ubiquitin carrier protein (UCP) stabilizes hypoxia-inducible factor-1α (HIF-1α) inducing ischemic vascular responses. Here, we investigated the effect of UCP gene transfer on therapeutic angiogenesis. Adenovirus-encoded UCP (Ad-F-UCP) increased the expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) in cells and mice. Conditioned media from UCP-overexpressing cells promoted proliferation, tubule formation, and invasion of human umbilical-vascular-endothelial cells (HUVECs), and vascularization in chorioallantoic membrane (CAM) assay. Ad-F-UCP increased the vessel density in the Martigel plug assay, and generated copious vessel-like structures in the explanted muscle. The UCP effect on angiogenesis was dependent on VEGF and FGF-2. In mouse hindlimb ischemia model (N = 30/group), autoamputation (limb loss) occurred in 87% and 68% of the mice with saline and Ad encoding β-galactosidase (Ad-LacZ), respectively, whereas only 23% of the mice injected with Ad-F-UCP showed autoamputation after 21 days of treatment. Ad-F-UCP increased protein levels of HIF-1α, platelet-endothelial cell adhesion molecule-1 (PECAM-1), smooth muscle cell actin (SMA) in the ischemic muscle, and augmented blood vessels doubly positive for PECAM-1 and SMA. Consequently, UCP gene transfer prevented muscle degeneration and autoamputation of ischemic limb. The results suggest that E2-EPF UCP may be a target for therapeutic angiogenesis.

An in-vitro scaffold-free epithelial-fibroblast coculture model for the larynx

PubMed Central

Walimbe, Tanaya; Panitch, Alyssa; Sivasankar, M. Preeti

2017-01-01

Objective Physiologically relevant, well-characterized in vitro vocal fold coculture models are needed to test the effects of various challenges and therapeutics on vocal fold physiology. We characterize a healthy state coculture model, created by using bronchial/tracheal epithelial cells and immortalized vocal fold fibroblasts. We also demonstrate that this model can be induced into a fibroplastic state to overexpress stress fibers using TGFβ1. Method Cell metabolic activity of immortalized human vocal fold fibroblasts incubated in different media combinations were confirmed with MTT assay. Fibroblasts were grown to confluence and primary bronchial/tracheal epithelial cells suspended in coculture media were seeded directly over the base layer of the fibroblasts. Cells were treated with TGFβ1 to induce myofibroblast formation. Cell shape and position was confirmed by live cell tracking, fibrosis was confirmed by probing for α smooth muscle actin (α-SMA) and phenotype was confirmed by immunostaining for vimentin and E-cadherin. Results Fibroblasts retain metabolic activity in coculture epithelial media. Live cell imaging revealed a layer of epithelial cells atop fibroblasts. α-SMA expression was enhanced in TGFβ1 treated cells, confirming that both cell types maintained a healthy phenotype in coculture, and can be induced into overexpressing stress fibers. Vimentin and E-cadherin immunostaining show that cells retain phenotype in coculture. Conclusion These data lay effective groundwork for a functional coculture model that retains the reproducibility necessary to serve as a viable diagnostic and therapeutic screening platform. Level of Evidence NA PMID:27859361

Twelve actin-encoding cDNAs from the American lobster, Homarus americanus: cloning and tissue expression of eight skeletal muscle, one heart, and three cytoplasmic isoforms.

PubMed

Kim, Bo Kwang; Kim, Kyoung Sun; Oh, Chul-Woong; Mykles, Donald L; Lee, Sung Gu; Kim, Hak Jun; Kim, Hyun-Woo

2009-06-01

Lobster muscles express a diverse array of myofibrillar protein isoforms. Three fiber types (fast, slow-twitch or S1, and slow-tonic or S2) differ qualitatively and quantitatively in myosin heavy and light chains, troponin-T, -I, and -C, paramyosin, and tropomyosin variants. However, little is known about the diversity of actin isoforms present in crustacean tissues. In this report we characterized cDNAs that encode twelve actin isoforms in the American lobster, Homarus americanus: eight from skeletal muscle (Ha-ActinSK1-8), one from heart (Ha-ActinHT1), and three cytoplasmic type actins from hepatopancreas (Ha-ActinCT1-3). All twelve cDNAs were products of distinct genes, as indicated by differences in the 3'-untranslated regions (UTRs). The open reading frames specified polypeptides 376 or 377 amino acids in length. Although key amino residues are conserved in the lobster actins, variations in nearby sequences may affect actin polymerization and/or interactions with other myofibrillar proteins. Quantitative reverse transcription-polymerase chain reaction showed muscle fiber type- and tissue-specific expression patterns. Ha-Actin-HT1 was expressed exclusively in heart (87% of the total; 12% of the total was Ha-ActinCT1). Ha-ActinCT1 was expressed in all tissues, while CT2 and CT3 were expressed only in hepatopancreas, with Ha-ActinCT2 as the major isoform (93% of the total). Ha-ActinSK1 and SK2 were the major isoforms (88% and 12% of the total, respectively) in the S1 fibers of crusher claw closer muscle. Fast fibers in the cutter claw closer and deep abdominal muscles differed in SK isoforms. Ha-ActinSK3, SK4, and SK5 were the major isoforms in cutter claw closer muscle (12%, 48%, and 37% of the total, respectively). Ha-ActinSK5 and SK8 were the major isoforms in deep abdominal flexor (31% and 65% of the total, respectively) and extensor (46% and 53% of the total, respectively) muscles, with SK6 and SK7 expressed at low levels. These data indicate that fast fibers in cutter claw and abdominal muscles show a phenotypic plasticity with respect to the expression of actin isoforms and may constitute discrete subtypes that differ in contractile properties.

The role of shear stress and altered tissue properties on endothelial to mesenchymal transformation and tumor-endothelial cell interaction.

PubMed

Mina, Sara G; Huang, Peter; Murray, Bruce T; Mahler, Gretchen J

2017-07-01

Tumor development is influenced by stromal cells in aspects including invasion, growth, angiogenesis, and metastasis. Activated fibroblasts are one group of stromal cells involved in cancer metastasis, and one source of activated fibroblasts is endothelial to mesenchymal transformation (EndMT). EndMT begins when the endothelial cells delaminate from the cell monolayer, lose cell-cell contacts, lose endothelial markers such as vascular endothelial-cadherin (VE-cadherin), gain mesenchymal markers like alpha-smooth muscle actin (α-SMA), and acquire mesenchymal cell-like properties. A three-dimensional (3D) culture microfluidic device was developed for investigating the role of steady low shear stress (1 dyne/cm 2 ) and altered extracellular matrix (ECM) composition and stiffness on EndMT. Shear stresses resulting from fluid flow within tumor tissue are relevant to both cancer metastasis and treatment effectiveness. Low and oscillatory shear stress rates have been shown to enhance the invasion of metastatic cancer cells through specific changes in actin and tubulin remodeling. The 3D ECM within the device was composed of type I collagen and glycosaminoglycans (GAGs), hyaluronic acid and chondroitin sulfate. An increase in collagen and GAGs has been observed in the solid tumor microenvironment and has been correlated with poor prognosis in many different cancer types. In this study, it was found that ECM composition and low shear stress upregulated EndMT, including upregulation of mesenchymal-like markers (α-SMA and Snail) and downregulated endothelial marker protein and gene expression (VE-cadherin). Furthermore, this novel model was utilized to investigate the role of EndMT in breast cancer cell proliferation and migration. Cancer cell spheroids were embedded within the 3D ECM of the microfluidic device. The results using this device show for the first time that the breast cancer spheroid size is dependent on shear stress and that the cancer cell migration rate, distance, and proliferation are induced by EndMT-derived activated fibroblasts. This model can be used to explore new therapeutics in a tumor microenvironment.

Tumor Microenvironment Gene Signature as a Prognostic Classifier and Therapeutic Target

DTIC Science & Technology

2016-06-01

smooth muscle actin (αSMA, encoded by gene ACTA2), fibroblast activation protein ( FAP ), podoplanin (PDPN), palladin (PALLD), tenascin-C (TNC...0.73 0.75 0.752 4 COL3A1 0.85 0.7 0.88 0.64 0.85 0.68 0.63 0.83 0.86 0.85 0.54 0.66 0.79 0.751 5 FAP 0.77 0.72 0.83 0.55 0.69 0.75 0.63 0.77 0.77 0.85

Comparative immunohistochemical characterization of interstitial cells in the urinary bladder of human, guinea pig and pig.

PubMed

Steiner, Clara; Gevaert, Thomas; Ganzer, Roman; De Ridder, Dirk; Neuhaus, Jochen

2018-05-01

Interstitial cells (ICs) are thought to play a functional role in urinary bladder. Animal models are commonly used to elucidate bladder physiology and pathophysiology. However, inter-species comparative studies on ICs are rare. We therefore analyzed ICs and their distribution in the upper lamina propria (ULP), the deeper lamina propria (DLP) and the detrusor muscular layer (DET) of human, guinea pig (GP) and pig. Paraffin slices were examined by immunohistochemistry and 3D confocal immunofluorescence of the mesenchymal intermediate filament vimentin (VIM), alpha-smooth muscle actin (αSMA), platelet-derived growth factor receptor alpha (PDGFRα) and transient receptor potential cation channel A1 (TRPA1). Image stacks were processed for analysis using Huygens software; quantitative analysis was performed with Fiji macros. ICs were identified by immunoreactivity for VIM (excluding blood vessels). In all species ≥ 75% of ULP ICs were VIM + /PDGFRα + and ≥ 90% were VIM + /TRPA1 + . In human and pig ≥ 74% of ULP ICs were VIM + /αSMA + , while in GP the percentage differed significantly with only 37% VIM + /αSMA + ICs. Additionally, over 90% of αSMA + ICs were also TRPA1 + and PDGFRα + in human, GP and pig. In all three species, TRPA1 + and PDGFRα + ICs point to an active role for these cells in bladder physiology, regarding afferent signaling processes and signal modification. We hypothesize that decline in αSMA-positivity in GP reflects adaptation of bladder histology to smaller bladder size. In our experiments, pig bladder proved to be highly comparable to human urinary bladder and seems to provide safer interpretation of experimental findings than GP.

Actin genes and their expression in pacific white shrimp, Litopenaeus vannamei.

PubMed

Zhang, Xiaoxi; Zhang, Xiaojun; Yuan, Jianbo; Du, Jiangli; Li, Fuhua; Xiang, Jianhai

2018-04-01

Actin is a multi-functional gene family that can be divided into muscle-type actins and non-muscle-type actins. In this study, 37 unigenes encoding actins were identified from RNA-Seq data of Pacific white shrimp, Litopenaeus vannamei. According to phylogenetic analysis, four and three cDNAs belong to cytoplasmic- and heart-type actins and were named LvActinCT and LvActinHT, respectively. 10 cDNAs belong to the slow-type skeletal muscle actins, and 18 belong to the fast-type skeletal muscle actins; they were designated LvActinSSK and LvActinFSK, respectively. Some muscle actin genes formed gene clusters in the genome. Multiple alternative transcription starts sites (ATSSs) were found for LvActinCT1. Based on the early developmental expression profile, almost all LvActins were highly expressed between the early limb bud and post-larval stages. Using LvActinSSK5 as probes, slow-type muscle was localized in pleopod muscle and superficial ventral muscle. We also found three actin genes that were down-regulated in the hemocytes of white spot syndrome virus (WSSV)- and Vibrio parahaemolyticus-infected L. vannamei. This study provides valuable information on the actin gene structure of shrimp, furthers our understanding of the shrimp muscle system and helps us develop strategies for disease control and sustainable shrimp farming.

Mutant soluble ectodomain of fibroblast growth factor receptor-2 IIIc attenuates bleomycin-induced pulmonary fibrosis in mice.

PubMed

Yu, Zhi-hong; Wang, Ding-ding; Zhou, Zhi-you; He, Shui-lian; Chen, An-an; Wang, Ju

2012-01-01

We have developed a strong inhibitor (S252W mutant soluble ectodomain of fibroblast growth factor recptor-2 IIIc, msFGFR2) that binds FGFs strongly and blocks the activation of FGFRs. In vitro, msFGFR2 could inhibit the promoting effect of transforming growth factor (TGF)-β1 on the proliferation of primary lung fibroblasts. In vivo, msFGFR2 alleviated lung fibrosis through inhibiting the expression of α-smooth muscle actin (SMA) and collagen deposit. In Western blotting of the right lung tissues and immunohistochemical assay, we found the level of p-FGFRs, p-mitogen activated protein kinase (MAPK) and p-Smad3 in the mice of bleomycin (BLM) group treated with msFGFR2 was down dramatically compared with the mice of BLM group, which suggested the activations of FGF and TGF-β signals were blocked meanwhile. In summary, msFGFR2 attenuated BLM-induced fibrosis and is an attractive therapeutic candidate for human pulmonary fibrosis.

Intrinsic healing response of the human anterior cruciate ligament: an histological study of reattached ACL remnants.

PubMed

Nguyen, Duy Tan; Ramwadhdoebe, Tamara H; van der Hart, Cor P; Blankevoort, Leendert; Tak, Paul Peter; van Dijk, Cornelis Niek

2014-02-01

A reattachment of the tibial remnant of the torn anterior cruciate ligament (ACL) to the posterior cruciate ligament is sometimes observed during surgery and apparently implies that the human ACL does have a healing response. The aim of this study was to investigate whether this reattachment tissue has similar histological characteristics of a healing response as the medial collateral ligament (MCL), which can heal spontaneously. Standard histology and immunostaining of α-smooth muscle actin and collagen type 3 was performed. The results shows that the reattached tissue has typical characteristics of a healing response: there attached ACL remnant could not be released by forceful traction; microscopy showed that the collagen fibers of the reattached tissue were disorganized with no preferred direction; increased neovascularization; the presence of lipid vacuoles; the mean number of cells within the biopsy tissue was 631±269 cells per mm2; and 68±20% was expressing α-SMA; semi-quantitative analysis of collagen type 3 expression showed that collagen type 3 had an high expression with an average score of 3. In conclusion, this study shows that the human proximal 1/3 ACL has an intrinsic healing response with typical histological characteristics similar to the MCL. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Protection effect of piper betel leaf extract against carbon tetrachloride-induced liver fibrosis in rats.

PubMed

Young, Shun-Chieh; Wang, Chau-Jong; Lin, Jing-Jing; Peng, Pei-Ling; Hsu, Jui-Ling; Chou, Fen-Pi

2007-01-01

Piper betel leaves (PBL) are used in Chinese folk medicine for the treatment of various disorders. PBL has the biological capabilities of detoxication, antioxidation, and antimutation. In this study, we evaluated the antihepatotoxic effect of PBL extract on the carbon tetrachloride (CCl(4))-induced liver injury in a rat model. Fibrosis and hepatic damage, as reveled by histology and the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were induced in rats by an administration of CCl(4) (8%, 1 ml/kg body weight) thrice a week for 4 weeks. PBL extract significantly inhibited the elevated AST and ALT activities caused by CCl(4) intoxication. It also attenuated total glutathione S-transferase (GST) activity and GST alpha isoform activity, and on the other hand, enhanced superoxide dismutase (SOD) and catalase (CAT) activities. The histological examination showed the PBL extract protected liver from the damage induced by CCl(4) by decreasing alpha-smooth muscle actin (alpha-sma) expression, inducing active matrix metalloproteinase-2 (MMP2) expression though Ras/Erk pathway, and inhibiting TIMP2 level that consequently attenuated the fibrosis of liver. The data of this study support a chemopreventive potential of PBL against liver fibrosis.

Targeting the Myofibroblast Genetic Switch: Inhibitors of Myocardin-Related Transcription Factor/Serum Response Factor–Regulated Gene Transcription Prevent Fibrosis in a Murine Model of Skin Injury

PubMed Central

Haak, Andrew J.; Tsou, Pei-Suen; Amin, Mohammad A.; Ruth, Jeffrey H.; Campbell, Phillip; Fox, David A.; Khanna, Dinesh; Larsen, Scott D.

2014-01-01

Systemic sclerosis (SSc), or scleroderma, similar to many fibrotic disorders, lacks effective therapies. Current trials focus on anti-inflammatory drugs or targeted approaches aimed at one of the many receptor mechanisms initiating fibrosis. In light of evidence that a myocardin-related transcription factor (MRTF)–and serum response factor (SRF)–regulated gene transcriptional program induced by Rho GTPases is essential for myofibroblast activation, we explored the hypothesis that inhibitors of this pathway may represent novel antifibrotics. MRTF/SRF-regulated genes show spontaneously increased expression in primary dermal fibroblasts from patients with diffuse cutaneous SSc. A novel small-molecule inhibitor of MRTF/SRF-regulated transcription (CCG-203971) inhibits expression of connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), and collagen 1 (COL1A2) in both SSc fibroblasts and in lysophosphatidic acid (LPA)–and transforming growth factor β (TGFβ)–stimulated fibroblasts. In vivo treatment with CCG-203971 also prevented bleomycin-induced skin thickening and collagen deposition. Thus, targeting the MRTF/SRF gene transcription pathway could provide an efficacious new approach to therapy for SSc and other fibrotic disorders. PMID:24706986

Increased IGF-1 in muscle modulates the phenotype of severe SMA mice

PubMed Central

Bosch-Marcé, Marta; Wee, Claribel D.; Martinez, Tara L.; Lipkes, Celeste E.; Choe, Dong W.; Kong, Lingling; Van Meerbeke, James P.; Musarò, Antonio; Sumner, Charlotte J.

2011-01-01

Spinal muscular atrophy (SMA) is an inherited motor neuron disease caused by the mutation of the survival motor neuron 1 (SMN1) gene and deficiency of the SMN protein. Severe SMA mice have abnormal motor function and small, immature myofibers early in development suggesting that SMN protein deficiency results in retarded muscle growth. Insulin-like growth factor 1 (IGF-1) stimulates myoblast proliferation, induces myogenic differentiation and generates myocyte hypertrophy in vitro and in vivo. We hypothesized that increased expression of IGF-1 specifically in skeletal muscle would attenuate disease features of SMAΔ7 mice. SMAΔ7 mice overexpressing a local isoform of IGF-1 (mIGF-1) in muscle showed enlarged myofibers and a 40% increase in median survival compared with mIGF-1-negative SMA littermates (median survival = 14 versus 10 days, respectively, log-rank P = 0.025). Surprisingly, this was not associated with a significant improvement in motor behavior. Treatment of both mIGF-1NEG and mIGF-1POS SMA mice with the histone deacetylase inhibitor, trichostatin A (TSA), resulted in a further extension of survival and improved motor behavior, but the combination of mIGF-1 and TSA treatment was not synergistic. These results show that increased mIGF-1 expression restricted to muscle can modulate the phenotype of SMA mice indicating that therapeutics targeted to muscle alone should not be discounted as potential disease-modifying therapies in SMA. IGF-1 may warrant further investigation in mild SMA animal models and perhaps SMA patients. PMID:21325354

Enhancement of SMN protein levels in a mouse model of spinal muscular atrophy using novel drug-like compounds

PubMed Central

Cherry, Jonathan J; Osman, Erkan Y; Evans, Matthew C; Choi, Sungwoon; Xing, Xuechao; Cuny, Gregory D; Glicksman, Marcie A; Lorson, Christian L; Androphy, Elliot J

2013-01-01

Spinal muscular atrophy (SMA) is a neurodegenerative disease that causes progressive muscle weakness, which primarily targets proximal muscles. About 95% of SMA cases are caused by the loss of both copies of the SMN1 gene. SMN2 is a nearly identical copy of SMN1, which expresses much less functional SMN protein. SMN2 is unable to fully compensate for the loss of SMN1 in motor neurons but does provide an excellent target for therapeutic intervention. Increased expression of functional full-length SMN protein from the endogenous SMN2 gene should lessen disease severity. We have developed and implemented a new high-throughput screening assay to identify small molecules that increase the expression of full-length SMN from a SMN2 reporter gene. Here, we characterize two novel compounds that increased SMN protein levels in both reporter cells and SMA fibroblasts and show that one increases lifespan, motor function, and SMN protein levels in a severe mouse model of SMA. PMID:23740718

Adrenomedullin and angiopoietin-1 additively restore erectile function in diabetic rats: comparison with the combination therapy of vascular endothelial growth factor and angiopoietin-1.

PubMed

Nishimatsu, Hiroaki; Suzuki, Etsu; Nomiya, Akira; Niimi, Aya; Suzuki, Motofumi; Fujimura, Tetsuya; Fukuhara, Hiroshi; Homma, Yukio

2013-07-01

Erectile dysfunction (ED) is a major health problem. We have shown that adrenomedullin (AM) restores erectile function in diabetic rats. The aim of this study is to explore a better treatment for ED, we examined whether combination of AM and angiopoietin-1 (Ang-1) was more effective to treat ED than treatment with AM alone or Ang-1 alone. We also compared the effect of the combination therapy with that of treatment with vascular endothelial growth factor-A (VEGF-A). Male Wistar rats were injected with streptozotocin (STZ) to induce diabetes. Adenoviruses expressing AM (AdAM), Ang-1 (AdAng-1), and VEGF-A (AdVEGF-A) were injected into the penis 6 weeks after STZ administration. Erectile function, penile histology, and protein expression were analyzed 4 weeks after the injection of the adenoviruses. Intracavernous pressure and mean arterial pressure were measured to evaluate erectile function. The morphology of the penis was analyzed by Elastica van Gieson stain and immunohistochemistry. The expression of α-smooth muscle actin (SMA), VE-cadherin and type I collagen was assessed by Western blot analysis. Infection with AdAM plus AdAng-1 more effectively restored erectile function than infection with AdAM alone or AdAng-1 alone. This combination therapy restored erectile function to a level similar to that observed in the age-matched Wistar rats. Expression of SMA and VE-cadherin increased more significantly in the AdAM plus AdAng-1-treated group than in the AdAM- or AdAng-1-treated group. Although AdVEGF-A infection restored erectile function significantly, it also caused enlargement of the trabeculae of the cavernous body, aberrant angiogenesis, and overproduction of type I collagen. These results suggested that combination therapy with AM and Ang-1 potently restored erectile function and normal morphology of the cavernous body compared with VEGF-A administration. This combination therapy will be useful to treat ED patients with a severely damaged cavernous body. © 2013 International Society for Sexual Medicine.

Expression of JAKs/STATs pathway molecules in rat model of rapid focal segmental glomerulosclerosis.

PubMed

Liang, Yaojun; Jin, Yu; Li, Yuning

2009-09-01

The objective of this study was to investigate the role of the Janus kinase-signal transducers and activators of transcription (JAKs/STATs) pathway in focal segmental glomerulosclerosis. Sixty specific pathogen-free male Wistar rats were randomly divided into two groups: a model group (MG) and a control group (CG). In the MG group, nephropathy was induced by unilateral nephrectomy and a single tail vein injection of adriamycin (5 mg/kg). Ten rats were sacrificed every 2 weeks in each group. The expressions of smooth muscle alpha actin (alpha-SMA), collagen (COL)-IV, STAT1, and STAT3 were examined using histochemical techniques, and Western blotting was used to examine the protein levels of STAT1, STAT3, phosphorylated (P)-STAT1, P-STAT3, and transforming growth factor beta1 (TGFbeta(1)). The expressions of JAK1, JAK2, STAT1, STAT3, suppressors of cytokine signaling (SOCS)1, SOCS3, protein inhibitors of activated STAT (PIAS)1, and PIAS3 were also measured by real-time quantitative reverse transcriptase-PCR. A steady and significant increase in the expressions of alpha-SMA, COL-IV and TGFbeta(1) were observed in MG rats over the whole experimental course. Increased STAT1 and P-STAT1 levels in MG rats were observed by week 6, whereas increased levels of STAT3 and P-STAT3 were noted by week 2. At the mRNA levels, JAK1, STAT1, and PIAS1 were significantly increased in MG rats in week 2, whereas JAK2 mRNA showed a significant decrease by weeks 2 and 4, followed by an significant increase in week 6. Significantly increased STAT3 levels were noted in week 2, followed by a steady and significant decrease in weeks 4 and 6. Significantly reduced levels of SOCS1, SOCS3, and PIAS3 mRNA were noted at all time points. We conclude that the JAKs/STATs signaling pathway may play an important role in the pathological process of rapid focal segmental glomerulosclerosis in the rat model.

Starvation is more efficient than the washing technique for purification of rat Sertoli cells.

PubMed

Ghasemzadeh-Hasankolaei, Mohammad; Eslaminejad, Mohamadreza Baghaban; Sedighi-Gilani, Mohammadali; Mokarizadeh, Aram

2014-09-01

Sertoli cells (SCs), one of the most important components of seminiferous tubules, are vital for normal spermatogenesis and male fertility. In recent years, numerous in vitro studies have shown the potential and actual activities of SCs. However, pure SCs are necessary for various in vitro studies. In this study, we have evaluated the efficiency of the starvation method for SC purification as compared with the washing method. Seminiferous tubule-derived cells (STDCs) of rats' testes underwent two different techniques for SC purification. In the first group (washing group), the medium was changed every 3-4 d, and cells were washed twice with phosphate-buffered saline that lacked CaC12 and MgSO4 (PBS(-)) before the addition of fresh medium. In the second group (starvation), the medium was changed every 7-8 d. Primary culture (P0), passage 1 (P1), and passage 2 (P2) cells were analyzed for the expression of SC-specific genes, vimentin, Wilm's tumor 1 (WT1), germ cell gene (vasa), Leydig cell marker, 17beta-hydroxysteroid dehydrogenase type 3 (Hsd17b3), and a marker of peritubular myoid cells, alpha smooth muscle actin (αSma), by reverse transcriptase polymerase chain reaction (RT-PCR) and real-time RT-PCR. Gene expression analysis showed that P0 cells expressed all tested genes except Hsd17b3. The starvation method caused significant downregulation of vasa and αSma expression in P0, P1, and P2 cells, whereas vimentin and WT1 were upregulated. In contrast, the washing method was less effective than the starvation method for the removal of germ and pretubular myoid cells (p < 0.001). Totally, the results have revealed that although washing is the only common technique for elimination of contaminant cells in SC cultures, starvation has a stronger effect and is a suitable, affordable technique for SC purification. We propose that starvation is an efficient, inexpensive method that can be used for purification of SCs in animal species.

GREM1 is expressed in the cancer-associated myofibroblasts of basal cell carcinomas.

PubMed

Kim, Hye Sung; Shin, Myung Soo; Cheon, Min Seok; Kim, Jae Wang; Lee, Cheol; Kim, Woo Ho; Kim, Young Sill; Jang, Bo Gun

2017-01-01

Cancer-associated fibroblasts (CAFs) play important roles in cancer progression through their complex interactions with cancer cells. The secreted bone morphogenetic protein antagonist, gremlin1 (GREM1) is expressed by the CAFs of basal cell carcinomas (BCCs), and promotes the growth of cancer cells. In this study, we investigated the expression of GREM1 mRNAs in various benign and malignant skin tumors, including various BCC subtypes. Analysis by RNA in situ hybridization (ISH) revealed that fibroblasts in the scar tissue expressed GREM1 and α-smooth muscle actin (α-SMA), whereas resident fibroblasts in the dermis of the normal skin did not express GREM1. Real-time polymerase chain reaction analysis showed significantly higher GREM1 expression in skin cancers and pilomatricomas (PMCs) than in other benign skin tumors. Tissue microarrays analyzed by RNA ISH for GREM1 expression also demonstrated that 23% of BCCs, 42% of squamous cell carcinomas, 20% of melanomas, and 90% of PMCs were positive for GREM1 expression, whereas trichoepitheliomas, eccrine poromas, hidradenomas, and spiradenomas were negative for GREM1 expression. Most BCCs that were GREM1 expression positive were of desmoplastic or mixed subtypes, and GREM1 expression was localized to activated myofibroblasts at the tumoral-stromal interface. Interestingly, most PMCs harbored GREM1-expressing fibroblasts, probably because of the inflammatory responses caused by foreign body reactions to keratin. Additionally, in BCCs, stromal GREM1 expression had a strong correlation with CD10 expression. In conclusion, GREM1 is frequently expressed by myofibroblasts in scars or in the stroma of basal cell carcinomas, suggesting that GREM1 expression can be a marker for activated myofibroblasts in the cancer stroma or in scar tissue.

Induced Expression of Rnd3 Is Associated with Transformation of Polarized Epithelial Cells by the Raf–MEK–Extracellular Signal-Regulated Kinase Pathway†

PubMed Central

Hansen, Steen H.; Zegers, Mirjam M. P.; Woodrow, Melissa; Rodriguez-Viciana, Pablo; Chardin, Pierre; Mostov, Keith E.; McMahon, Martin

2000-01-01

Madin-Darby canine kidney (MDCK) epithelial cells transformed by oncogenic Ras and Raf exhibit cell multilayering and alterations in the actin cytoskeleton. The changes in the actin cytoskeleton comprise a loss of actin stress fibers and enhanced cortical actin. Using MDCK cells expressing a conditionally active form of Raf, we have explored the molecular mechanisms that underlie these observations. Raf activation elicited a robust increase in Rac1 activity consistent with the observed increase in cortical actin. Loss of actin stress fibers is indicative of attenuated Rho function, but no change in Rho-GTP levels was detected following Raf activation. However, the loss of actin stress fibers in Raf-transformed cells was preceded by the induced expression of Rnd3, an endogenous inhibitor of Rho protein function. Expression of Rnd3 alone at levels equivalent to those observed following Raf transformation led to a substantial loss of actin stress fibers. Moreover, cells expressing activated RhoA failed to multilayer in response to Raf. Pharmacological inhibition of MEK activation prevented all of the biological and biochemical changes described above. Consequently, the data are consistent with a role for induced Rnd3 expression downstream of the Raf–MEK–extracellular signal-regulated kinase pathway in epithelial oncogenesis. PMID:11094087

[The role of RNA splicing in the pathogenesis of spinal muscular atrophy and development of its therapeutics].

PubMed

Sahashi, Kentaro; Sobue, Gen

2014-12-01

Loss-of-function mutations in SMN1 cause spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. Degeneration of alpha-motor neurons that results in progressive paralysis is a pathological hallmark of SMA. Recently, peripheral-tissue involvement has also been reported in SMA. Patients have low levels of functional SMN which is attributed to alternative splicing in SMN2, a gene closely-related to SMN1. This decrease in the expression of SMN, a ubiquitously expressed protein involved in promoting snRNP assembly required for splicing, is responsible for SMA. However, the mechanism through which decrease in SMN levels causes SMA remains unclear. Currently, no curative treatment is available for SMA, but SMN restoration is thought to be necessary and sufficient for cure. Antisense oligonucleotides (ASOs) can be designed to specifically alter splicing patterns of target pre-mRNAs. We identified an ASO that redirects SMN2 splicing and is currently in clinical trials for use as RNA-targeting therapeutics. Further, we have also reported a novel application of splicing-modulating ASOs--creation of animal phenocopy models of diseases by inducing mis-splicing. Exploring the relationship between the spatial and temporal effects of therapeutic and pathogenic ASOs yields relevant insights into the roles of SMN in SMA pathogenesis and into its normal physiological functions. This knowledge, in turn, contributes to the ongoing development of targeted therapeutics.

In vivo responses of macrophages and perisinusoidal cells to cholestatic liver injury.

PubMed Central

Hines, J. E.; Johnson, S. J.; Burt, A. D.

1993-01-01

We investigated the response of macrophages and perisinusoidal (Ito) cells (PSCs) during the development of secondary biliary cirrhosis after ligation and division of the common bile duct. Liver tissue was obtained from three groups of male Wistar rats: 1) untreated controls (n = 3); 2) common bile duct-ligated (CBDL) animals (n = 15); and 3) sham-operated controls (n = 15). Material from animal groups 2 and 3 was obtained on days 3, 7, 14, 21, and 28 after operation; in all animals 5-bromo-2-deoxyuridine was administered intraperitoneally before death. Monocytes and macrophages were detected using the monoclonal antibody ED1 and tissue macrophages using the antibody ED2. Cell proliferation within the macrophage population was demonstrated by double labeling for ED2 and incorporated 5-bromo-2-deoxyuridine. PSCs were demonstrated in tissue sections by immunolocalization of desmin; proliferating PSCs were identified by double labeling for desmin and incorporated 5-bromo-2-deoxyuridine. Evidence of phenotypic modulation of PSCs was sought using anti-alpha-smooth muscle actin (alpha-SMA) antibody. Increased numbers of ED1- and ED2-positive cells were seen in CBDL animals at all time points. Local proliferation of macrophages could be identified and reached a peak at day 3, thereafter falling toward control values. Compared with those of controls, livers of CBDL animals showed increased numbers of desmin-positive PSCs in periportal zones from day 3 on, reaching a peak at day 14 (127.8 +/- 10.99 cells/0.635 mm2) and followed by a plateau. PSC proliferation peaked at days 3 and 7 (labeling indices 11.2% and 11.2%, respectively) and thereafter fell toward control values; no expansion of the PSC population was seen in sham-operated rats. Increased alpha-SMA-positive cells were also noted from day 3, with a peak at day 21 (231.1 +/- 11.52 cells/0.635 mm2) and followed by a plateau. En face labeling experiments in days 14, 21, and 28 CBDL animals showed cells co-expressing alpha-SMA and desmin and cells expressing alpha-SMA alone. These results indicate that in response to chronic cholestatic liver injury, PSCs proliferate and undergo phenotypic modulation toward "myofibroblast-like" cells. The kinetics of the response are similar to those of the ED2-positive cell population in keeping with a hypothesis that PSC proliferation and activation may be mediated by factors released by macrophages in response to various forms of liver injury. We conclude that the responses of macrophages and PSCs to cholestatic injury are similar to those after toxin-induced hepatocyte necrosis. Images Figure 2 Figure 4 Figure 5 Figure 6 PMID:8434646

Cells Lacking β-Actin are Genetically Reprogrammed and Maintain Conditional Migratory Capacity*

PubMed Central

Tondeleir, Davina; Lambrechts, Anja; Müller, Matthias; Jonckheere, Veronique; Doll, Thierry; Vandamme, Drieke; Bakkali, Karima; Waterschoot, Davy; Lemaistre, Marianne; Debeir, Olivier; Decaestecker, Christine; Hinz, Boris; Staes, An; Timmerman, Evy; Colaert, Niklaas; Gevaert, Kris; Vandekerckhove, Joël; Ampe, Christophe

2012-01-01

Vertebrate nonmuscle cells express two actin isoforms: cytoplasmic β- and γ-actin. Because of the presence and localized translation of β-actin at the leading edge, this isoform is generally accepted to specifically generate protrusive forces for cell migration. Recent evidence also implicates β-actin in gene regulation. Cell migration without β-actin has remained unstudied until recently and it is unclear whether other actin isoforms can compensate for this cytoplasmic function and/or for its nuclear role. Primary mouse embryonic fibroblasts lacking β-actin display compensatory expression of other actin isoforms. Consistent with this preservation of polymerization capacity, β-actin knockout cells have unchanged lamellipodial protrusion rates despite a severe migration defect. To solve this paradox we applied quantitative proteomics revealing a broad genetic reprogramming of β-actin knockout cells. This also explains why reintroducing β-actin in knockout cells does not restore the affected cell migration. Pathway analysis suggested increased Rho-ROCK signaling, consistent with observed phenotypic changes. We therefore developed and tested a model explaining the phenotypes in β-actin knockout cells based on increased Rho-ROCK signaling and increased TGFβ production resulting in increased adhesion and contractility in the knockout cells. Inhibiting ROCK or myosin restores migration of β-actin knockout cells indicating that other actins compensate for β-actin in this process. Consequently, isoactins act redundantly in providing propulsive forces for cell migration, but β-actin has a unique nuclear function, regulating expression on transcriptional and post-translational levels, thereby preventing myogenic differentiation. PMID:22448045

Pollen specific expression of maize genes encoding actin depolymerizing factor-like proteins.

PubMed Central

Lopez, I; Anthony, R G; Maciver, S K; Jiang, C J; Khan, S; Weeds, A G; Hussey, P J

1996-01-01

In pollen development, a dramatic reorganization of the actin cytoskeleton takes place during the passage of the pollen grain into dormancy and on activation of pollen tube growth. A role for actin-binding proteins is implicated and we report here the identification of a small gene family in maize that encodes actin depolymerizing factor (ADF)-like proteins. The ADF group of proteins are believed to control actin polymerization and depolymerization in response to both intracellular and extracellular signals. Two of the maize genes ZmABP1 and ZmABP2 are expressed specifically in pollen and germinating pollen suggesting that the protein products may be involved in pollen actin reorganization. A third gene, ZmABP3, encodes a protein only 56% and 58% identical to ZmABP1 and ZmABP2, respectively, and its expression is suppressed in pollen and germinated pollen. The fundamental biochemical characteristics of the ZmABP proteins has been elucidated using bacterially expressed ZmABP3 protein. This has the ability to bind monomeric actin (G-actin) and filamentous actin (F-actin). Moreover, it decreases the viscosity of polymerized actin solutions consistent with an ability to depolymerize filaments. These biochemical characteristics, taken together with the sequence comparisons, support the inclusion of the ZmABP proteins in the ADF group. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8693008

Protective effects of butyrate-based compounds on a mouse model for spinal muscular atrophy.

PubMed

Butchbach, Matthew E R; Lumpkin, Casey J; Harris, Ashlee W; Saieva, Luciano; Edwards, Jonathan D; Workman, Eileen; Simard, Louise R; Pellizzoni, Livio; Burghes, Arthur H M

2016-05-01

Proximal spinal muscular atrophy (SMA) is a childhood-onset degenerative disease resulting from the selective loss of motor neurons in the spinal cord. SMA is caused by the loss of SMN1 (survival motor neuron 1) but retention of SMN2. The number of copies of SMN2 modifies disease severity in SMA patients as well as in mouse models, making SMN2 a target for therapeutics development. Sodium butyrate (BA) and its analog (4PBA) have been shown to increase SMN2 expression in SMA cultured cells. In this study, we examined the effects of BA, 4PBA as well as two BA prodrugs-glyceryl tributyrate (BA3G) and VX563-on the phenotype of SMNΔ7 SMA mice. Treatment with 4PBA, BA3G and VX563 but not BA beginning at PND04 significantly improved the lifespan and delayed disease end stage, with administration of VX563 also improving the growth rate of these mice. 4PBA and VX563 improved the motor phenotype of SMNΔ7 SMA mice and prevented spinal motor neuron loss. Interestingly, neither 4PBA nor VX563 had an effect on SMN expression in the spinal cords of treated SMNΔ7 SMA mice; however, they inhibited histone deacetylase (HDAC) activity and restored the normal phosphorylation states of Akt and glycogen synthase kinase 3β, both of which are altered by SMN deficiency in vivo. These observations show that BA-based compounds with favorable pharmacokinetics ameliorate SMA pathology possibly by modulating HDAC and Akt signaling. Copyright © 2016 Elsevier Inc. All rights reserved.

Protective Effects of Butyrate-based Compounds on a Mouse Model for Spinal Muscular Atrophy

PubMed Central

Butchbach, Matthew E. R.; Lumpkin, Casey J.; Harris, Ashlee W.; Saieva, Luciano; Edwards, Jonathan D.; Workman, Eileen; Simard, Louise R.; Pellizzoni, Livio; Burghes, Arthur H. M.

2016-01-01

Proximal spinal muscular atrophy (SMA) is a childhood-onset degenerative disease resulting from the selective loss of motor neurons in the spinal cord. SMA is caused by the loss of SMN1 (survival motor neuron 1) but retention of SMN2. The number of copies of SMN2 modifies disease severity in SMA patients as well as in mouse models, making SMN2 a target for therapeutics development. Sodium butyrate (BA) and its analogue (4PBA) have been shown to increase SMN2 expression in SMA cultured cells. In this study, we examined the effects of BA, 4PBA as well as two BA prodrugs—glyceryl tributyrate (BA3G) and VX563—on the phenotype of SMNΔ7 SMA mice. Treatment with 4PBA, BA3G and VX563 but not BA beginning at PND04 significantly improved the lifespan and delayed disease end stage, with administration of VX563 also improving the growth rate of these mice. 4PBA and VX563 improved the motor phenotype of SMNΔ7 SMA mice and prevented spinal motor neuron loss. Interestingly, neither 4PBA nor VX563 had an effect on SMN expression in the spinal cords of treated SMNΔ7 SMA mice; however, they inhibited histone deacetylase (HDAC) activity and restored the normal phosphorylation states of Akt and glycogen synthase kinase 3β, both of which are altered by SMN deficiency in vivo. These observations show that BA-based compounds with favourable pharmacokinetics ameliorate SMA pathology possibly by modulating HDAC and Akt signaling. PMID:26892876

Molecular Mechanisms of Neurodegeneration in Spinal Muscular Atrophy.

PubMed

Ahmad, Saif; Bhatia, Kanchan; Kannan, Annapoorna; Gangwani, Laxman

2016-01-01

Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease with a high incidence and is the most common genetic cause of infant mortality. SMA is primarily characterized by degeneration of the spinal motor neurons that leads to skeletal muscle atrophy followed by symmetric limb paralysis, respiratory failure, and death. In humans, mutation of the Survival Motor Neuron 1 (SMN1) gene shifts the load of expression of SMN protein to the SMN2 gene that produces low levels of full-length SMN protein because of alternative splicing, which are sufficient for embryonic development and survival but result in SMA. The molecular mechanisms of the (a) regulation of SMN gene expression and (b) degeneration of motor neurons caused by low levels of SMN are unclear. However, some progress has been made in recent years that have provided new insights into understanding of the cellular and molecular basis of SMA pathogenesis. In this review, we have briefly summarized recent advances toward understanding of the molecular mechanisms of regulation of SMN levels and signaling mechanisms that mediate neurodegeneration in SMA.

Metoprolol Inhibits Cardiac Apoptosis and Fibrosis in a Canine Model of Chronic Obstructive Sleep Apnea.

PubMed

Li, Wenpeng; Yan, Sen; Zhao, Jing; Ding, Xue; Zhang, Song; Wang, Dingyu; Liu, Lei; Peng, Wenpeng; Li, Hui; Wang, Dongyang; Liu, Zhaorui; Li, Yue

2015-01-01

Emerging evidence suggested that obstructive sleep apnea (OSA) was independently associated with the development of heart failure. In this study, we explored the influence of chronic OSA on left ventricular structural remodeling in canines, and the potential therapeutical role of metoprolol. Chronic OSA model was established by stopping the ventilator and closing the airway for 4 h/day apnea-ventilation cycles every other day for 12 weeks while metoprolol (5 mg· kg(-1)· day(-1)) were administered continuously. Norepinephrine concentration was measured by Enzyme Linked Immunosorbent Assay. Transmission electron microscopy, Hematoxylin and eosin, TUNEL and Masson trichrome staining were employed to detect the morphology, apoptosis and fibrosis of cardiomyocytes. Protein expression of apoptosis and fibrosis-related factors including apoptosis-inducing factor (AIF), caspase 3, Bcl-2, Bax, α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), and p38 mitogen-activated protein kinase (MAPK) were examined by Western blotting. Norepinephrine concentration was markedly increased in chronic OSA dogs and reduced by metoprolol. Both the apoptotic ratio and collagen volume fraction were significantly increased in left ventricular myocytes of chronic OSA dogs, and was reversed by metoprolol. Moreover, chronic OSA-induced upregulation of AIF, cleaved caspase 3, Bax, α-SMA, and TGF-β1 as well as downregulation of Bcl-2 was markedly recovered by metoprolol, which was mediated by p38 MAPK. Metoprolol protects against chronic OSA-induced cardiac apoptosis and fibrosis in left ventricular myocytes of canines, which may provide new potential strategy for drug therapy of OSA. © 2015 S. Karger AG, Basel.

Polymer-Based Reconstruction of the Inferior Vena Cava in Rat: Stem Cells or RGD Peptide?

PubMed Central

Pontailler, Margaux; Illangakoon, Eranka; Williams, Gareth R.; Marijon, Camille; Bellamy, Valérie; Balvay, Daniel; Autret, Gwenhael; Vanneaux, Valérie; Larghero, Jérôme; Planat-Benard, Valérie; Perier, Marie-Cécile; Bruneval, Patrick; Menasché, Philippe

2015-01-01

As part of a program targeted at developing a resorbable valved tube for replacement of the right ventricular outflow tract, we compared three biopolymers (polyurethane [PU], polyhydroxyalkanoate (the poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxyvalerate) [PHBVV]), and polydioxanone [PDO]) and two biofunctionalization techniques (using adipose-derived stem cells [ADSCs] or the arginine-glycine-aspartate [RGD] peptide) in a rat model of partial inferior vena cava (IVC) replacement. Fifty-three Wistar rats first underwent partial replacement of the IVC with an acellular electrospun PDO, PU, or PHBVV patch, and 31 nude rats subsequently underwent the same procedure using a PDO patch biofunctionalized either by ADSC or RGD. Results were assessed both in vitro (proliferation and survival of ADSC seeded onto the different materials) and in vivo by magnetic resonance imaging (MRI), histology, immunohistochemistry [against markers of vascular cells (von Willebrand factor [vWF], smooth muscle actin [SMA]), and macrophages ([ED1 and ED2] immunostaining)], and enzyme-linked immunosorbent assay (ELISA; for the expression of various cytokines and inducible NO synthase). PDO showed the best in vitro properties. Six weeks after implantation, MRI did not detect significant luminal changes in any group. All biopolymers were evenly lined by vWF-positive cells, but only PDO and PHBVV showed a continuous layer of SMA-positive cells at 3 months. PU patches resulted in a marked granulomatous inflammatory reaction. The ADSC and RGD biofunctionalization yielded similar outcomes. These data confirm the good biocompatibility of PDO and support the concept that appropriately peptide-functionalized polymers may be successfully substituted for cell-loaded materials. PMID:25611092

Response of human bone marrow-derived MSCs on triphasic Ca-P substrate with various HA/TCP ratio.

PubMed

Bajpai, Indu; Kim, Duk Yeon; Kyong-Jin, Jung; Song, In-Hwan; Kim, Sukyoung

2017-01-01

Calcium phosphates (Ca-P) are used commonly as artificial bone substitutes to control the biodegradation rate of an implant in the body fluid. This study examined the in vitro proliferation of human bone marrow-derived mesenchymal stem cells (hBMSCs) on triphasic Ca-P samples. For this aspect, hydroxyapatite (HA), dicalcium phosphate dehydrate (DCPD), and calcium hydroxide (Ca(OH) 2 ) were mixed at various ratios, cold compacted, and sintered at 1250°C in air. X-ray diffraction showed that the β-tricalcium phosphate (TCP) to α-TCP phase transformation increased with increasing DCPD/HA ratio. The micro-hardness deceased with increasing TCP content, whereas the mean grain size and porosity increased with increasing TCP concentration. To evaluate the in vitro degree of adhesion and proliferation on the HA/TCP samples, human BMSCs were incubated on the HA/TCP samples and analyzed by a cells proliferation assay, expression of the extracellular matrix (ECM) genes, such as α-smooth muscle actin (α-SMA) and fibronectin (FN), and FITC-phalloidin fluorescent staining. In terms of the interactions of human BMSCs with the triphasic Ca-P samples, H50T50 (Ca/P = 1.59) markedly enhanced cell spreading, proliferation, FN, and α-SMA compared with H100T0 (Ca/P = 1.67). Interestingly, these results show that among the five HA/TCP samples, H50T50 is the optimal Ca-P composition for in vitro cell proliferation. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 72-80, 2017. © 2015 Wiley Periodicals, Inc.

Bioenergetic status modulates motor neuron vulnerability and pathogenesis in a zebrafish model of spinal muscular atrophy

PubMed Central

Boyd, Penelope J.; Shorrock, Hannah K.; Carter, Roderick N.; Powis, Rachael A.; Thomson, Sophie R.; Thomson, Derek; Graham, Laura C.; Motyl, Anna A. L.; Highley, J. Robin; Becker, Thomas; Becker, Catherina G.; Heath, Paul R.

2017-01-01

Degeneration and loss of lower motor neurons is the major pathological hallmark of spinal muscular atrophy (SMA), resulting from low levels of ubiquitously-expressed survival motor neuron (SMN) protein. One remarkable, yet unresolved, feature of SMA is that not all motor neurons are equally affected, with some populations displaying a robust resistance to the disease. Here, we demonstrate that selective vulnerability of distinct motor neuron pools arises from fundamental modifications to their basal molecular profiles. Comparative gene expression profiling of motor neurons innervating the extensor digitorum longus (disease-resistant), gastrocnemius (intermediate vulnerability), and tibialis anterior (vulnerable) muscles in mice revealed that disease susceptibility correlates strongly with a modified bioenergetic profile. Targeting of identified bioenergetic pathways by enhancing mitochondrial biogenesis rescued motor axon defects in SMA zebrafish. Moreover, targeting of a single bioenergetic protein, phosphoglycerate kinase 1 (Pgk1), was found to modulate motor neuron vulnerability in vivo. Knockdown of pgk1 alone was sufficient to partially mimic the SMA phenotype in wild-type zebrafish. Conversely, Pgk1 overexpression, or treatment with terazosin (an FDA-approved small molecule that binds and activates Pgk1), rescued motor axon phenotypes in SMA zebrafish. We conclude that global bioenergetics pathways can be therapeutically manipulated to ameliorate SMA motor neuron phenotypes in vivo. PMID:28426667

The DcpS inhibitor RG3039 improves motor function in SMA mice

PubMed Central

Van Meerbeke, James P.; Gibbs, Rebecca M.; Plasterer, Heather L.; Miao, Wenyan; Feng, Zhihua; Lin, Ming-Yi; Rucki, Agnieszka A.; Wee, Claribel D.; Xia, Bing; Sharma, Shefali; Jacques, Vincent; Li, Darrick K.; Pellizzoni, Livio; Rusche, James R.; Ko, Chien-Ping; Sumner, Charlotte J.

2013-01-01

Spinal muscular atrophy (SMA) is caused by mutations of the survival motor neuron 1 (SMN1) gene, retention of the survival motor neuron 2 (SMN2) gene and insufficient expression of full-length survival motor neuron (SMN) protein. Quinazolines increase SMN2 promoter activity and inhibit the ribonucleic acid scavenger enzyme DcpS. The quinazoline derivative RG3039 has advanced to early phase clinical trials. In preparation for efficacy studies in SMA patients, we investigated the effects of RG3039 in severe SMA mice. Here, we show that RG3039 distributed to central nervous system tissues where it robustly inhibited DcpS enzyme activity, but minimally activated SMN expression or the assembly of small nuclear ribonucleoproteins. Nonetheless, treated SMA mice showed a dose-dependent increase in survival, weight and motor function. This was associated with improved motor neuron somal and neuromuscular junction synaptic innervation and function and increased muscle size. RG3039 also enhanced survival of conditional SMA mice in which SMN had been genetically restored to motor neurons. As this systemically delivered drug may have therapeutic benefits that extend beyond motor neurons, it could act additively with SMN-restoring therapies delivered directly to the central nervous system such as antisense oligonucleotides or gene therapy. PMID:23727836

Stiffening hydrogels for investigating the dynamics of hepatic stellate cell mechanotransduction during myofibroblast activation

NASA Astrophysics Data System (ADS)

Caliari, Steven R.; Perepelyuk, Maryna; Cosgrove, Brian D.; Tsai, Shannon J.; Lee, Gi Yun; Mauck, Robert L.; Wells, Rebecca G.; Burdick, Jason A.

2016-02-01

Tissue fibrosis contributes to nearly half of all deaths in the developed world and is characterized by progressive matrix stiffening. Despite this, nearly all in vitro disease models are mechanically static. Here, we used visible light-mediated stiffening hydrogels to investigate cell mechanotransduction in a disease-relevant system. Primary hepatic stellate cell-seeded hydrogels stiffened in situ at later time points (following a recovery phase post-isolation) displayed accelerated signaling kinetics of both early (Yes-associated protein/Transcriptional coactivator with PDZ-binding motif, YAP/TAZ) and late (alpha-smooth muscle actin, α-SMA) markers of myofibroblast differentiation, resulting in a time course similar to observed in vivo activation dynamics. We further validated this system by showing that α-SMA inhibition following substrate stiffening resulted in attenuated stellate cell activation, with reduced YAP/TAZ nuclear shuttling and traction force generation. Together, these data suggest that stiffening hydrogels may be more faithful models for studying myofibroblast activation than static substrates and could inform the development of disease therapeutics.

Beta-Actin Is Required for Proper Mouse Neural Crest Ontogeny

PubMed Central

Tondeleir, Davina; Noelanders, Rivka; Bakkali, Karima; Ampe, Christophe

2014-01-01

The mouse genome consists of six functional actin genes of which the expression patterns are temporally and spatially regulated during development and in the adult organism. Deletion of beta-actin in mouse is lethal during embryonic development, although there is compensatory expression of other actin isoforms. This suggests different isoform specific functions and, more in particular, an important function for beta-actin during early mammalian development. We here report a role for beta-actin during neural crest ontogeny. Although beta-actin null neural crest cells show expression of neural crest markers, less cells delaminate and their migration arrests shortly after. These phenotypes were associated with elevated apoptosis levels in neural crest cells, whereas proliferation levels were unchanged. Specifically the pre-migratory neural crest cells displayed higher levels of apoptosis, suggesting increased apoptosis in the neural tube accounts for the decreased amount of migrating neural crest cells seen in the beta-actin null embryos. These cells additionally displayed a lack of membrane bound N-cadherin and dramatic decrease in cadherin-11 expression which was more pronounced in the pre-migratory neural crest population, potentially indicating linkage between the cadherin-11 expression and apoptosis. By inhibiting ROCK ex vivo, the knockout neural crest cells regained migratory capacity and cadherin-11 expression was upregulated. We conclude that the presence of beta-actin is vital for survival, specifically of pre-migratory neural crest cells, their proper emigration from the neural tube and their subsequent migration. Furthermore, the absence of beta-actin affects cadherin-11 and N-cadherin function, which could partly be alleviated by ROCK inhibition, situating the Rho-ROCK signaling in a feedback loop with cadherin-11. PMID:24409333

SENP1 attenuates the liver fibrosis through down-regulating the expression of SMAD2.

PubMed

Wu, Linshi; Qiu, Weiqing; Sun, Jianhua; Wang, Jian

2018-01-01

To investigate whether SENP1 could play a regulating role in the liver fibrosis process, the Sprague-Dawley (SD) rats were used to establish the liver fibrosis rat models by intraperitoneally injecting with 1 ml/kg of 10% CCl 4 , while the control normal rats were injected with olive oil. Then confirmation experiments to verify the successful establishment of these models were conducted by detecting the cellular and lobular architecture, and liver function indexes using hematoxylin-eosin staining, Masson's trichrome staining and microplate method, respectively. In addition, the expression levels of fibrosis markers including collagen I, collagen III, α-SMA and TGF-β1 were inspected using quantitative real-time PCR (qRT-PCR), as well as SMAD2. Subsequently, the relative mRNA and protein level of SENP1 was also determined via qRT-PCR and western blot analysis. Next, the HSC-T6 cells of SENP1 knock-down were constructed and used to test the relative protein expression levels of α-SMA and SMAD2 in these cells. The results of hematoxylin-eosin staining, Masson's trichrome staining and microplate method turned out that the rat liver fibrosis models were constructed successfully, which was further confirmed by the increased expression of collagen I, collagen III, α-SMA and TGF-β1 in mRNA and protein level, as well as SMAD2. Then the expression of SENP1 was overexpressed in the rat liver fibrosis models induced by CCl 4 and the TGF-β1 treatment could increase the protein expression level of collagen I, collagen III and α-SMA. Lastly, the SENP1 knockdown HSC-T6 cells were successfully constructed, while the silence of SENP1 down-regulated the protein expression of α-SMA and SMAD2. In conclusion, this study provided a new regulation mechanism about the liver fibrosis process. Copyright © 2017 Elsevier Inc. All rights reserved.

[Effect of multi-glycoside of Tripterygium wilfordii Hook. f. in intervening TGF-beta1/Smad signaling pathway of adriamycin-induced nephropathy model rat].

PubMed

Wan, Yi-gang; Sun, Wei; Dou, Chen-hui

2011-04-01

To explore the potential molecular mechanisms of multi-glycoside of Tripterygium wilfordii Hook. f. (GTW) for ameliorating glomerulosclerosis (GS) by observing its intervention effect on transforming growth factor (TGF)-beta1/Smad signaling pathway in adriamycin-induced nephropathy (ADRN) model rat. Fifteen female Sprague-Dawley (SD) rats were randomly divided into three groups, the sham-operation group (A), the untreated model group (B), and the GTW treated model group (C). Rats in Group B and C were made into ADRN model by right nephrectomy and intravenous injection of adriamycin (ADR, 0. 4 mL and 0. 2 mL respectively in 4 weeks). After the model was successfully established, rats in Group C were orally given GTW (50 mg/kg per day), while rats in Group B were intervened with distilled water. The intervention for two groups was 6 weeks. Rats' body weight were weighed and 24 h urinary protein excretion (Upro) detected by the end of the 2nd, 4th, 8th and 10th week. All rats were sacrificed at the end of 10th week after operation to withdraw blood and kidney tissue to examine serum biochemical parameters, glomerular morphological changes, alpha-smooth muscle actin (alpha-SMA), and collagen type I expression. Besides, the mRNA expressions of TGF-beta1, Smad3 and Smad7, as well as protein expressions of TGF-beta1, and phosphorylated Smad2/3 (p-Smad2/3) in glomeruli were detected by RT-PCR or Western blotting. As compared with Group B, in Group C, Upro and serum albumin were improved significantly, but no difference between groups was found in levels of blood urea nitrogen(BUN), serum creatinine(SCr), or hepatic cell injury. Mesangial cell proliferation, extracellular matrix (ECM) and collagen deposition were suppressed by GTW. Expressions of alpha-SMA and collagen type I decreased, and the characteristic changes of GS were attenuated. The mRNA expressions of TGF-P,31, Smad3 and protein expression of TGF-beta1, p-Smad2/3 in renal tissues were down-regulated, while the protein expression of Smad7 mRNA was up-regulated. GTW showed effect in ameliorating GS in vivo. It could reduce the ECM deposition and improve GS by way of intervening TGF-beta1/Smad signaling pathway in the kidney through regulating the mRNA or protein expressions of key signal molecules, such as Smad3 and p-Smad2/3.

Kidney protection against ischemia/reperfusion injury by myofibrillogenesis regulator-1.

PubMed

Wang, Xiaoreng; Tao, Tianqi; Ding, Rui; Song, Dandan; Liu, Mi; Xie, Yuansheng; Liu, Xiuhua

2014-01-01

Ischemia/reperfusion (I/R) injury is characterized by cytoskeletal reorganization and loss of polarity in proximal tubule epithelial cells. Previously, we showed that myofibrillogenesis regulator (MR)-1 promoted actin organization in cardiomyocytes. MR-1 is also expressed in the kidney. In this study, we investigated MR-1 expression in acute renal failure induced by I/R in Sprague-Dawley rats. We determined the MR-1 expression and the ratio of fibrous actin (F-actin) to globular actin (G-actin). HK-2 cells were treated with or without hypoxia/reoxygenation (H/R), and MR-1 levels were increased by adenoviral overexpression or silenced by RNA interference. I/R and H/R resulted in cellular injury and decreases of MR-1, the F-/G-actin ratio, and myosin light chain (MLC)-2. MR-1 overexpression attenuated H/R-induced cell injury and loss of surface membrane polarity of actin. MR-1 overexpression also increased the expression and phosphorylation of MLC-2 and MLC kinase, which were decreased in MR-1-silenced and H/R-treated cells. Together, these data show that MR-1 promoted actin polarity on the membrane surface and protected HK-2 cells from H/R injury. The mechanism might involve the rapid organization of F-actin through the upregulation and phosphorylation of MLC-2.

Effects of selexipag and its active metabolite in contrasting the profibrotic myofibroblast activity in cultured scleroderma skin fibroblasts.

PubMed

Cutolo, Maurizio; Ruaro, Barbara; Montagna, Paola; Brizzolara, Renata; Stratta, Emanuela; Trombetta, Amelia Chiara; Scabini, Stefano; Tavilla, Pier Paolo; Parodi, Aurora; Corallo, Claudio; Giordano, Nicola; Paolino, Sabrina; Pizzorni, Carmen; Sulli, Alberto; Smith, Vanessa; Soldano, Stefano

2018-05-02

Myofibroblasts contribute to fibrosis through the overproduction of extracellular matrix (ECM) proteins, primarily type I collagen (COL-1) and fibronectin (FN), a process which is mediated in systemic sclerosis (SSc) by the activation of fibrogenic intracellular signaling transduction molecules, including extracellular signal-regulated kinases 1 and 2 (Erk1/2) and protein kinase B (Akt). Selexipag is a prostacyclin receptor agonist synthesized for the treatment of pulmonary arterial hypertension. The study investigated the possibility for selexipag and its active metabolite (ACT-333679) to downregulate the profibrotic activity in primary cultures of SSc fibroblasts/myofibroblasts and the fibrogenic signaling molecules involved. Fibroblasts from skin biopsies obtained with Ethics Committee (EC) approval from patients with SSc, after giving signed informed consent, were cultured until the 3 rd culture passage and then either maintained in normal growth medium (untreated cells) or independently treated with different concentrations of selexipag (from 30 μM to 0.3 μM) or ACT-333679 (from 10 μM to 0.1 μM) for 48 h. Protein and gene expressions of α-smooth muscle actin (α-SMA), fibroblast specific protein-1 (S100A4), COL-1, and FN were investigated by western blotting and quantitative real-time PCR. Erk1/2 and Akt phosphorylation was investigated in untreated and ACT-333679-treated cells by western botting. Selexipag and ACT-333679 significantly reduced protein synthesis and gene expression of α-SMA, S100A4, and COL-1 in cultured SSc fibroblasts/myofibroblasts compared to untreated cells, whereas FN was significantly downregulated at the protein level. Interestingly, ACT-333679 significantly reduced the phosphorylation of Erk1/2 and Akt in cultured SSc fibroblasts/myofibroblasts. Selexipag and mainly its active metabolite ACT-333679 were found for the first time to potentially interfere with the profibrotic activity of cultured SSc fibroblasts/myofibroblasts at least in vitro, possibly through the downregulation of fibrogenic Erk1/2 and Akt signaling molecules.

Smooth muscle cells differentiated from mesenchymal stem cells are regulated by microRNAs and suitable for vascular tissue grafts.

PubMed

Gu, Wenduo; Hong, Xuechong; Le Bras, Alexandra; Nowak, Witold N; Issa Bhaloo, Shirin; Deng, Jiacheng; Xie, Yao; Hu, Yanhua; Ruan, Xiong Z; Xu, Qingbo

2018-05-25

Tissue-engineered vascular grafts with long-term patency are greatly needed in the clinical settings, and smooth muscle cells (SMCs) are a critical graft component. Human mesenchymal stem cells (MSCs) are used for generating SMCs, and understanding the underlying regulatory mechanisms of the MSC-to-SMC differentiation process could improve SMC generation in the clinic. Here, we found that in response to stimulation of transforming growth factor-β1 (TGFβ1), human umbilical cord-derived MSCs abundantly express the SMC markers α-smooth muscle actin (αSMA), smooth muscle protein 22 (SM22), calponin, and smooth muscle myosin heavy chain (SMMHC) at both gene and protein levels. Functionally, MSC-derived SMCs displayed contracting capacity in vitro and supported vascular structure formation in the Matrigel plug assay in vivo More importantly, SMCs differentiated from human MSCs could migrate into decellularized mouse aorta and give rise to the smooth muscle layer of vascular grafts, indicating the potential of utilizing human MSC-derived SMCs to generate vascular grafts. Of note, microRNA (miR) array analysis and TaqMan microRNA assays identified miR-503 and miR-222-5p as potential regulators of MSC differentiation into SMCs at early time points. Mechanistically, miR-503 promoted SMC differentiation by directly targeting SMAD7, a suppressor of SMAD-related, TGFβ1-mediated signaling pathways. Moreover, miR-503 expression was SMAD4-dependent. SMAD4 was enriched at the miR-503 promoter. Furthermore, miR-222-5p inhibited SMC differentiation by targeting and down-regulating ROCK2 and αSMA. In conclusion, MSC differentiation into SMCs is regulated by miR-503 and miR-222-5p and yields functional SMCs for use in vascular grafts. © 2018 Gu et al.

Pirfenidone inhibits the proliferation of fibroblasts from patients with active Crohn's disease.

PubMed

Kadir, Sara-Irini; Wenzel Kragstrup, Tue; Dige, Anders; Kok Jensen, Simon; Dahlerup, Jens Frederik; Kelsen, Jens

2016-11-01

One-third of Crohn's disease (CD) patients develop intestinal strictures that require repeated surgical intervention. Current anti-inflammatory therapies have limited effect on stricture development, which necessitates the exploration of new pharmacological approaches. Pirfenidone (PFD), a novel anti-fibrotic agent, was recently approved in Europe for the treatment of idiopathic pulmonary fibrosis (IPF). We hypothesized that observations in IPF could be transferable to intestinal fibrosis and that PFD inhibits the proliferation and extracellular matrix (ECM) turnover of gut-derived fibroblasts from CD patients. Fibroblasts were isolated from biopsies of inflamed (n = 8) and non-inflamed (n = 5) colonic mucosa. Expression of CD90 and alpha-smooth muscle actin (αSMA) expression was determined by flow cytometry. The fibroblasts were cultured with PFD (0.5, 1.0 and 2.0 mg/ml). Proliferation was evaluated with CellTiter 96(®) AQueous One Solution Cell Proliferation Assay. Production of matrix metalloproteinase-3 (MMP-3), tissue inhibitor of metalloproteinases-1 (TIMP-1) and collagen were assessed using ELISA and calorimetric assays, respectively. The majority of the fibroblasts were αSMA-positive myofibroblasts. PFD inhibited fibroblast proliferation [0.94 (PFD 0.5 mg/ml); 0.76 (1.0 mg/ml); 0.58 (2.0 mg/ml)] and production of MMP-3 [0.85 (0.5 mg/ml); 0.74 (1.0 mg/ml); 0.63 (2.0 mg/ml)] dose-dependently (both p = 0.0001). The anti-proliferative effect of PFD was reversible (p = 0.0001), indicating that PFD does not act by an irreversible cytotoxic mechanism. PFD did not influence neither TIMP-1 nor collagen production. PFD inhibited the proliferation and the production of MMP-3 dose-dependently in gut-derived fibroblast from CD patients. Our observations support further studies on PFD in stricturing CD.

Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells

PubMed Central

Ando, Seijitsu; Otani, Hitomi; Yagi, Yasuhiro; Kawai, Kenzo; Araki, Hiromasa; Fukuhara, Shirou; Inagaki, Chiyoko

2007-01-01

Background Proteinase-activated receptors (PARs; PAR1–4) that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR4, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR4 stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT) which contributes to the increase in myofibroblast population. Methods EMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, α-smooth muscle actin (α-SMA) for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells). Results Stimulation of PAR with thrombin (1 U/ml) or a synthetic PAR4 agonist peptide (AYPGKF-NH2, 100 μM) for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR4 stimulation decreased E-cadherin-like immunoreactivity and increased α-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-β (TGF-β). Western blot analyses of PAR4-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR4-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR) kinase and Src. PAR4-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR4 stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor. Conclusion PAR4 stimulation of alveolar epithelial cells induced epithelial-mesenchymal transition (EMT) as monitored by cell shapes, and epithelial or myofibroblast marker at least partly through EGFR transactivation via receptor-linked Src activation. PMID:17433115

Ultraviolet B Radiation Stimulates the Interaction between Nuclear Factor of Activated T Cells 5 (NFAT5) and Nuclear Factor-Kappa B (NF-κB) in Human Lens Epithelial Cells.

PubMed

Chung, Inyoung; Hah, Young-Sool; Ju, SunMi; Kim, Ji-Hye; Yoo, Woong-Sun; Cho, Hee-Young; Yoo, Ji-Myong; Seo, Seong-Wook; Choi, Wan-Sung; Kim, Seong-Jae

2017-07-01

Nuclear factor-kappa B (NF-κB) has been proposed as a therapeutic target for the treatment of cataracts. The authors investigated the relationship between nuclear factor of activated T cells 5 (NFAT5) and NF-κB in ultraviolet B (UVB)-irradiated human lens epithelial (HLE) cells. Human lens epithelial B-3 (HLE-B3) cells were exposed to UVB light at a dose of 10 mJ/cm 2 and then incubated for 24 h. Cell viability was assessed by using the Cell Counting Kit-8 (CCK-8) assay. Gene expression level of NFAT5 was determined using real-time quantitative polymerase chain reaction (qPCR). Protein expression levels of NFAT5, NF-κB p65, and α-smooth muscle actin (α-SMA) and the association of NFAT5 with the NF-κB p65 subunit were measured by Western blot analysis and a co-immunoprecipitation assay, respectively. The cellular distribution of NFAT5 and NF-κB p65 was examined by triple immunofluorescence staining. At 24 h after UVB exposure, cell viability significantly decreased in a dose-dependent manner, and UVB light (15 and 20 mJ/cm 2 ) significantly increased the ROS generation. UVB irradiation increased NFAT5 mRNA and protein levels and increased phosphorylation of NF-κB in HLE-B3 cells. α-SMA protein levels were increased in the irradiated cells. In addition, NFAT5 and NF-κB translocated from the cytoplasm to the nucleus, and binding between the p65 subunit and NFAT5 was increased. Exposure to UVB radiation induces nuclear translocation and stimulates binding between NFAT5 and NF-κB proteins in HLE-B3 cells. These interactions may form part of the biochemical mechanism of cataractogenesis in UVB-irradiated HLECs.

Valsartan decreases TGF-β1 production and protects against chlorhexidine digluconate-induced liver peritoneal fibrosis in rats.

PubMed

Subeq, Yi-Maun; Ke, Chen-Yen; Lin, Nien-Tsung; Lee, Chung-Jen; Chiu, Yi-Han; Hsu, Bang-Gee

2011-02-01

Peritoneal fibrosis (PF) is a recognized complication of long-term peritoneal dialysis (PD) and can lead to ultrafiltration failure. The present study was designed to investigate the protective effects of valsartan on chlorhexidine digluconate-induced PF by decreasing TGF-β1 production in rats. PF was induced in Sprague-Dawley rats by daily administration of 0.5 ml 0.1% chlorhexidine digluconate in normal saline via peritoneal dialysis (PD) tube for 1 week. Rats received daily intravenous injections of low dose valsartan (1 mg/kg) or high dose valsartan (3 mg/kg) for 1 week. After 7 days, conventional 4.25% Dianeal (30 ml) was administered via a PD catheter with a dwell time of 4 h and assessed of peritoneal function. At the end of dialysis, rats were sacrificed and the liver peritoneum was harvested for microscopically and immunohistochemistry. There was no significant difference in mean arterial pressure and heart rate between groups. After 4 h of PD, the D₄/P(4Urea) level was reduced, the D₄/D₀ glucose level, serum and dialysate transforming growth factor-β1 (TGF-β1) level was increased, the liver peritoneum was markedly thicker, and the expression of TGF-β1, alpha-smooth muscle actin (α-SMA), fibronectin, collagen, and vascular endothelial growth factor (VEGF) were elevated in the PF group compared with the vehicle group. High dose of valsartan decreased the serum and dialysate TGF-β1 level, decreased the thickness of the liver peritoneum, and decreased the expression of TGF-β1, α-SMA, fibronectin, collagen, and VEGF-positive cells in liver peritoneum. The low dose of valsartan did not protect against chlorhexidine digluconate-induced PF in rat. Valsartan protected against chlorhexidine digluconate-induced PF in rats by decreasing TGF-β1 production. Copyright © 2010 Elsevier Ltd. All rights reserved.

Combination of glycopyrronium and indacaterol inhibits carbachol-induced ERK5 signal in fibrotic processes.

PubMed

Namba, Yukiko; Togo, Shinsaku; Tulafu, Miniwan; Kadoya, Kotaro; Nagahama, Kumi Yoneda; Taka, Hikari; Kaga, Naoko; Orimo, Akira; Liu, Xiangde; Takahashi, Kazuhisa

2017-03-11

Airway fibrosis is one of the pathological features of chronic obstructive pulmonary disease (COPD), and recent studies revealed that acetylcholine plays an important role in the development of airway remodeling by stimulating proliferation and collagen synthesis of lung fibroblasts. This study was designed to examine the effects of a long-acting muscarinic receptor antagonist (LAMA) glycopyrronium and a long-acting β2 adrenergic receptor agonist (LABA) indacaterol on acetylcholine-mediated fibrotic responses in lung fibroblasts. After carbachol (CCh) or transforming growth factor-β1 (TGF-β1) exposure, the response to glycopyrronium and indacaterol was determined in vitro in fibroblasts isolated from mild-to-moderate COPD lung tissue. The ability of fibroblasts to mediate the contraction of collagen gels was assessed. The expression of α-smooth muscle actin (α-SMA) and the phosphorylation of extracellular-signal-regulated kinase 5 (ERK5) were determined by immunoblot. TGF-β1 was quantified by ELISA and acetylcholine was quantified by liquid chromatography tandem-mass spectrometry. CCh stimulated fibroblast-mediated collagen gel contraction and α-SMA expression and TGF-β1 release by fibroblasts. Blockade of autocrine TGF-β1 attenuated CCh-mediated fibrotic responses, while TGF-β1 did not stimulate acetylcholine release. Glycopyrronium plus indacaterol significantly attenuated CCh- and TGF-β1-mediated fibrotic responses through inhibition of ERK5 phosphorylation. Notably, the magnitudes of CCh- and TGF-β1-stimulated gel contraction, CCh-induced TGF-β1 release, and ERK5 phosphorylation were greater in fibroblasts isolated from COPD subjects than in those from non-smokers. CCh induced TGF-β1 self-sustaining signaling loops by potentiating ERK5 signaling and promoted myofibroblast activity. This autocrine signaling mechanism may be an attractive therapeutic target to block the fibrotic response, which was modulated by the combination of glycopyrronium and indacaterol.

Limbal Fibroblasts Maintain Normal Phenotype in 3D RAFT Tissue Equivalents Suggesting Potential for Safe Clinical Use in Treatment of Ocular Surface Failure.

PubMed

Massie, Isobel; Dale, Sarah B; Daniels, Julie T

2015-06-01

Limbal epithelial stem cell deficiency can cause blindness, but transplantation of these cells on a carrier such as human amniotic membrane can restore vision. Unfortunately, clinical graft manufacture using amnion can be inconsistent. Therefore, we have developed an alternative substrate, Real Architecture for 3D Tissue (RAFT), which supports human limbal epithelial cells (hLE) expansion. Epithelial organization is improved when human limbal fibroblasts (hLF) are incorporated into RAFT tissue equivalent (TE). However, hLF have the potential to transdifferentiate into a pro-scarring cell type, which would be incompatible with therapeutic transplantation. The aim of this work was to assess the scarring phenotype of hLF in RAFT TEs in hLE+ and hLE- RAFT TEs and in nonairlifted and airlifted RAFT TEs. Diseased fibroblasts (dFib) isolated from the fibrotic conjunctivae of ocular mucous membrane pemphigoid (Oc-MMP) patients were used as a pro-scarring positive control against which hLF were compared using surrogate scarring parameters: matrix metalloproteinase (MMP) activity, de novo collagen synthesis, α-smooth muscle actin (α-SMA) expression, and transforming growth factor-β (TGF-β) secretion. Normal hLF and dFib maintained different phenotypes in RAFT TE. MMP-2 and -9 activity, de novo collagen synthesis, and α-SMA expression were all increased in dFib cf. normal hLF RAFT TEs, although TGF-β1 secretion did not differ between normal hLF and dFib RAFT TEs. Normal hLF do not progress toward a scarring-like phenotype during culture in RAFT TEs and, therefore, may be safe to include in therapeutic RAFT TE, where they can support hLE, although in vivo work is required to confirm this. dFib RAFT TEs (used in this study as a positive control) may be useful toward the development of an ex vivo disease model of Oc-MMP.

Limbal Fibroblasts Maintain Normal Phenotype in 3D RAFT Tissue Equivalents Suggesting Potential for Safe Clinical Use in Treatment of Ocular Surface Failure

PubMed Central

Dale, Sarah B.; Daniels, Julie T.

2015-01-01

Limbal epithelial stem cell deficiency can cause blindness, but transplantation of these cells on a carrier such as human amniotic membrane can restore vision. Unfortunately, clinical graft manufacture using amnion can be inconsistent. Therefore, we have developed an alternative substrate, Real Architecture for 3D Tissue (RAFT), which supports human limbal epithelial cells (hLE) expansion. Epithelial organization is improved when human limbal fibroblasts (hLF) are incorporated into RAFT tissue equivalent (TE). However, hLF have the potential to transdifferentiate into a pro-scarring cell type, which would be incompatible with therapeutic transplantation. The aim of this work was to assess the scarring phenotype of hLF in RAFT TEs in hLE+ and hLE− RAFT TEs and in nonairlifted and airlifted RAFT TEs. Diseased fibroblasts (dFib) isolated from the fibrotic conjunctivae of ocular mucous membrane pemphigoid (Oc-MMP) patients were used as a pro-scarring positive control against which hLF were compared using surrogate scarring parameters: matrix metalloproteinase (MMP) activity, de novo collagen synthesis, α-smooth muscle actin (α-SMA) expression, and transforming growth factor-β (TGF-β) secretion. Normal hLF and dFib maintained different phenotypes in RAFT TE. MMP-2 and -9 activity, de novo collagen synthesis, and α-SMA expression were all increased in dFib cf. normal hLF RAFT TEs, although TGF-β1 secretion did not differ between normal hLF and dFib RAFT TEs. Normal hLF do not progress toward a scarring-like phenotype during culture in RAFT TEs and, therefore, may be safe to include in therapeutic RAFT TE, where they can support hLE, although in vivo work is required to confirm this. dFib RAFT TEs (used in this study as a positive control) may be useful toward the development of an ex vivo disease model of Oc-MMP. PMID:25380529

Prophylactic and therapeutic treatment with a synthetic analogue of a parasitic worm product prevents experimental arthritis and inhibits IL-1β production via NRF2-mediated counter-regulation of the inflammasome.

PubMed

Rzepecka, Justyna; Pineda, Miguel A; Al-Riyami, Lamyaa; Rodgers, David T; Huggan, Judith K; Lumb, Felicity E; Khalaf, Abedawn I; Meakin, Paul J; Corbet, Marlene; Ashford, Michael L; Suckling, Colin J; Harnett, Margaret M; Harnett, William

2015-06-01

Rheumatoid arthritis (RA) remains a debilitating autoimmune condition as many patients are refractory to existing conventional and biologic therapies, and hence successful development of novel treatments remains a critical requirement. Towards this, we now describe a synthetic drug-like small molecule analogue, SMA-12b, of an immunomodulatory parasitic worm product, ES-62, which acts both prophylactically and therapeutically against collagen-induced arthritis (CIA) in mice. Mechanistic analysis revealed that SMA-12b modifies the expression of a number of inflammatory response genes, particularly those associated with the inflammasome in mouse bone marrow-derived macrophages and indeed IL-1β was the most down-regulated gene. Consistent with this, IL-1β was significantly reduced in the joints of mice with CIA treated with SMA-12b. SMA-12b also increased the expression of a number of genes associated with anti-oxidant responses that are controlled by the transcription factor NRF2 and critically, was unable to inhibit expression of IL-1β by macrophages derived from the bone marrow of NRF2(-/-) mice. Collectively, these data suggest that SMA-12b could provide the basis of an entirely novel approach to fulfilling the urgent need for new treatments for RA. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

A Perturbed MicroRNA Expression Pattern Characterizes Embryonic Neural Stem Cells Derived from a Severe Mouse Model of Spinal Muscular Atrophy (SMA)

PubMed Central

Luchetti, Andrea; Ciafrè, Silvia Anna; Murdocca, Michela; Malgieri, Arianna; Masotti, Andrea; Sanchez, Massimo; Farace, Maria Giulia; Novelli, Giuseppe; Sangiuolo, Federica

2015-01-01

Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder and the leading genetic cause of death in infants. Despite the disease-causing gene, survival motor neuron (SMN1), encodes a ubiquitous protein, SMN1 deficiency preferentially affects spinal motor neurons (MNs), leaving the basis of this selective cell damage still unexplained. As neural stem cells (NSCs) are multipotent self-renewing cells that can differentiate into neurons, they represent an in vitro model for elucidating the pathogenetic mechanism of neurodegenerative diseases such as SMA. Here we characterize for the first time neural stem cells (NSCs) derived from embryonic spinal cords of a severe SMNΔ7 SMA mouse model. SMNΔ7 NSCs behave as their wild type (WT) counterparts, when we consider neurosphere formation ability and the expression levels of specific regional and self-renewal markers. However, they show a perturbed cell cycle phase distribution and an increased proliferation rate compared to wild type cells. Moreover, SMNΔ7 NSCs are characterized by the differential expression of a limited number of miRNAs, among which miR-335-5p and miR-100-5p, reduced in SMNΔ7 NSCs compared to WT cells. We suggest that such miRNAs may be related to the proliferation differences characterizing SMNΔ7 NSCs, and may be potentially involved in the molecular mechanisms of SMA. PMID:26258776

A Perturbed MicroRNA Expression Pattern Characterizes Embryonic Neural Stem Cells Derived from a Severe Mouse Model of Spinal Muscular Atrophy (SMA).

PubMed

Luchetti, Andrea; Ciafrè, Silvia Anna; Murdocca, Michela; Malgieri, Arianna; Masotti, Andrea; Sanchez, Massimo; Farace, Maria Giulia; Novelli, Giuseppe; Sangiuolo, Federica

2015-08-06

Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder and the leading genetic cause of death in infants. Despite the disease-causing gene, survival motor neuron (SMN1), encodes a ubiquitous protein, SMN1 deficiency preferentially affects spinal motor neurons (MNs), leaving the basis of this selective cell damage still unexplained. As neural stem cells (NSCs) are multipotent self-renewing cells that can differentiate into neurons, they represent an in vitro model for elucidating the pathogenetic mechanism of neurodegenerative diseases such as SMA. Here we characterize for the first time neural stem cells (NSCs) derived from embryonic spinal cords of a severe SMNΔ7 SMA mouse model. SMNΔ7 NSCs behave as their wild type (WT) counterparts, when we consider neurosphere formation ability and the expression levels of specific regional and self-renewal markers. However, they show a perturbed cell cycle phase distribution and an increased proliferation rate compared to wild type cells. Moreover, SMNΔ7 NSCs are characterized by the differential expression of a limited number of miRNAs, among which miR-335-5p and miR-100-5p, reduced in SMNΔ7 NSCs compared to WT cells. We suggest that such miRNAs may be related to the proliferation differences characterizing SMNΔ7 NSCs, and may be potentially involved in the molecular mechanisms of SMA.

Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells.

PubMed

Genz, Berit; Thomas, Maria; Pützer, Brigitte M; Siatkowski, Marcin; Fuellen, Georg; Vollmar, Brigitte; Abshagen, Kerstin

2014-11-01

Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluated an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC. Copyright © 2014 Elsevier Inc. All rights reserved.

Endothelial-monocyte activating polypeptide II disrupts alveolar epithelial type II to type I cell transdifferentiation

PubMed Central

2012-01-01

Background Distal alveolar morphogenesis is marked by differentiation of alveolar type (AT)-II to AT-I cells that give rise to the primary site of gas exchange, the alveolar/vascular interface. Endothelial-Monocyte Activating Polypeptide (EMAP) II, an endogenous protein with anti-angiogenic properties, profoundly disrupts distal lung neovascularization and alveolar formation during lung morphogenesis, and is robustly expressed in the dysplastic alveolar regions of infants with Bronchopulmonary dysplasia. Determination as to whether EMAP II has a direct or indirect affect on ATII→ATI trans-differentiation has not been explored. Method In a controlled nonvascular environment, an in vitro model of ATII→ATI cell trans-differentiation was utilized to demonstrate the contribution that one vascular mediator has on distal epithelial cell differentiation. Results Here, we show that EMAP II significantly blocked ATII→ATI cell transdifferentiation by increasing cellular apoptosis and inhibiting expression of ATI markers. Moreover, EMAP II-treated ATII cells displayed myofibroblast characteristics, including elevated cellular proliferation, increased actin cytoskeleton stress fibers and Rho-GTPase activity, and increased nuclear:cytoplasmic volume. However, EMAP II-treated cells did not express the myofibroblast markers desmin or αSMA. Conclusion Our findings demonstrate that EMAP II interferes with ATII → ATI transdifferentiation resulting in a proliferating non-myofibroblast cell. These data identify the transdifferentiating alveolar cell as a possible target for EMAP II's induction of alveolar dysplasia. PMID:22214516

Total Glucosides of Danggui Buxue Tang Attenuate BLM-Induced Pulmonary Fibrosis via Regulating Oxidative Stress by Inhibiting NOX4

PubMed Central

Zhao, Ping; Zhou, Wen-Cheng; Li, De-Lin; Mo, Xiao-Ting; Xu, Liang; Li, Liu-Cheng; Cui, Wen-Hui; Gao, Jian

2015-01-01

Pulmonary fibrosis (PF) is a serious chronic lung disease with unknown pathogenesis. Researches have confirmed that oxidative stress which is regulated by NADPH oxidase-4 (NOX4), a main source of reactive oxygen species (ROS), is an important molecular mechanism underlying PF. Previous studies showed that total glucosides of Danggui Buxue Tang (DBTG), an extract from a classical traditional Chinese herbal formula, Danggui Buxue Tang (DBT), attenuated bleomycin-induced PF in rats. However, the mechanisms of DBTG are still not clear. We hypothesize that DBTG attenuates PF through regulating the level of oxidative stress by inhibiting NOX4. And we found that fibrosis indexes hydroxyproline (HYP) and type I collagen (Col-I) were lower in DBTG groups compared with the model group. In addition, the expression of transforming growth factor-β1 (TGF-β1) and expression of alpha smooth muscle actin (α-SMA) were also much more decreased than the model group. For oxidative stress indicators, DBTG blunted the decrease of superoxide dismutase (SOD) activity, total antioxidant capacity (T-AOC), and the increase in malondialdehyde (MDA), 8-iso-prostaglandin in lung homogenates. Treatment with DBTG restrained the expression of NOX4 compared to the model group. Present study confirms that DBTG inhibits BLM-induced PF by modulating the level of oxidative stress via suppressing NOX4. PMID:26347805

Trichostatin A increases SMN expression and survival in a mouse model of spinal muscular atrophy

PubMed Central

Avila, Amy M.; Burnett, Barrington G.; Taye, Addis A.; Gabanella, Francesca; Knight, Melanie A.; Hartenstein, Parvana; Cizman, Ziga; Di Prospero, Nicholas A.; Pellizzoni, Livio; Fischbeck, Kenneth H.; Sumner, Charlotte J.

2007-01-01

The inherited motor neuron disease spinal muscular atrophy (SMA) is caused by mutation of the telomeric survival motor neuron 1 (SMN1) gene with retention of the centromeric SMN2 gene. We sought to establish whether the potent and specific hydroxamic acid class of histone deacetylase (HDAC) inhibitors activates SMN2 gene expression in vivo and modulates the SMA disease phenotype when delivered after disease onset. Single intraperitoneal doses of 10 mg/kg trichostatin A (TSA) in nontransgenic and SMA model mice resulted in increased levels of acetylated H3 and H4 histones and modest increases in SMN gene expression. Repeated daily doses of TSA caused increases in both SMN2-derived transcript and SMN protein levels in neural tissues and muscle, which were associated with an improvement in small nuclear ribonucleoprotein (snRNP) assembly. When TSA was delivered daily beginning on P5, after the onset of weight loss and motor deficit, there was improved survival, attenuated weight loss, and enhanced motor behavior. Pathological analysis showed increased myofiber size and number and increased anterior horn cell size. These results indicate that the hydroxamic acid class of HDAC inhibitors activates SMN2 gene expression in vivo and has an ameliorating effect on the SMA disease phenotype when administered after disease onset. PMID:17318264

Actin expression in trypanosomatids (Euglenozoa: Kinetoplastea)

PubMed Central

Souza, Ligia Cristina Kalb; Pinho, Rosana Elisa Gonçalves Gonçalves; Lima, Carla Vanessa de Paula; Fragoso, Stênio Perdigão; Soares, Maurilio José

2013-01-01

Heteroxenic and monoxenic trypanosomatids were screened for the presence of actin using a mouse polyclonal antibody produced against the entire sequence of the Trypanosoma cruzi actin gene, encoding a 41.9 kDa protein. Western blot analysis showed that this antibody reacted with a polypeptide of approximately 42 kDa in the whole-cell lysates of parasites targeting mammals (T. cruzi, Trypanosoma brucei and Leishmania major), insects (Angomonas deanei, Crithidia fasciculata, Herpetomonas samuelpessoai and Strigomonas culicis) and plants (Phytomonas serpens). A single polypeptide of approximately 42 kDa was detected in the whole-cell lysates of T. cruzi cultured epimastigotes, metacyclic trypomastigotes and amastigotes at similar protein expression levels. Confocal microscopy showed that actin was expressed throughout the cytoplasm of all the tested trypanosomatids. These data demonstrate that actin expression is widespread in trypanosomatids. PMID:23903980

(Pro)renin Receptor Is an Amplifier of Wnt/β-Catenin Signaling in Kidney Injury and Fibrosis.

PubMed

Li, Zhen; Zhou, Lili; Wang, Yongping; Miao, Jinhua; Hong, Xue; Hou, Fan Fan; Liu, Youhua

2017-08-01

The (pro)renin receptor (PRR) is a transmembrane protein with multiple functions. However, its regulation and role in the pathogenesis of CKD remain poorly defined. Here, we report that PRR is a downstream target and an essential component of Wnt/ β -catenin signaling. In mouse models, induction of CKD by ischemia-reperfusion injury (IRI), adriamycin, or angiotensin II infusion upregulated PRR expression in kidney tubular epithelium. Immunohistochemical staining of kidney biopsy specimens also revealed induction of renal PRR in human CKD. Overexpression of either Wnt1 or β -catenin induced PRR mRNA and protein expression in vitro Notably, forced expression of PRR potentiated Wnt1-mediated β -catenin activation and augmented the expression of downstream targets such as fibronectin, plasminogen activator inhibitor 1, and α -smooth muscle actin ( α -SMA). Conversely, knockdown of PRR by siRNA abolished β -catenin activation. PRR potentiation of Wnt/ β -catenin signaling did not require renin, but required vacuolar H + ATPase activity. In the mouse model of IRI, transfection with PRR or Wnt1 expression vectors promoted β -catenin activation, aggravated kidney dysfunction, and worsened renal inflammation and fibrotic lesions. Coexpression of PRR and Wnt1 had a synergistic effect. In contrast, knockdown of PRR expression ameliorated kidney injury and fibrosis after IRI. These results indicate that PRR is both a downstream target and a crucial element in Wnt signal transmission. We conclude that PRR can promote kidney injury and fibrosis by amplifying Wnt/ β -catenin signaling. Copyright © 2017 by the American Society of Nephrology.

Mesenchymal stem cells protect against obstruction-induced renal fibrosis by decreasing STAT3 activation and STAT3-dependent MMP-9 production

PubMed Central

Matsui, Futoshi; Babitz, Stephen A.; Rhee, Audrey; Hile, Karen L.; Zhang, Hongji

2017-01-01

STAT3 is a transcription factor implicated in renal fibrotic injury, but the role of STAT3 in mesenchymal stem cell (MSC)-induced renoprotection during renal fibrosis remains unknown. We hypothesized that MSCs protect against obstruction-induced renal fibrosis by downregulating STAT3 activation and STAT3-induced matrix metalloproteinase-9 (MMP-9) expression. Male Sprague-Dawley rats underwent renal arterial injection of vehicle or MSCs (1 × 106/rat) immediately before sham operation or induction of unilateral ureteral obstruction (UUO). The kidneys were harvested after 4 wk and analyzed for collagen I and III gene expression, collagen deposition (Masson’s trichrome), fibronectin, α-smooth muscle actin, active STAT3 (p-STAT3), MMP-9, and tissue inhibitor of matrix metalloproteinases 1 (TIMP-1) expression. In a separate arm, the STAT3 inhibitor S3I-201 (10 mg/kg) vs. vehicle was administered to rats intraperitoneally just after induction of UUO and daily for 14 days thereafter. The kidneys were harvested after 2 wk and analyzed for p-STAT3 and MMP-9 expression, and collagen and fibronectin deposition. Renal obstruction induced a significant increase in collagen, fibronectin, α-SMA, p-STAT3, MMP-9, and TIMP-1 expression while exogenously administered MSCs significantly reduced these indicators of obstruction-induced renal fibrosis. STAT3 inhibition with S3I-201 significantly reduced obstruction-induced MMP-9 expression and tubulointerstitial fibrosis. These results demonstrate that MSCs protect against obstruction-induced renal fibrosis, in part, by decreasing STAT3 activation and STAT3-dependent MMP-9 production. PMID:27760767

Alteration of human umbilical vein endothelial cell gene expression in different biomechanical environments.

PubMed

Shoajei, Shahrokh; Tafazzoli-Shahdpour, Mohammad; Shokrgozar, Mohammad Ali; Haghighipour, Nooshin

2014-05-01

Biomechanical environments affect the function of cells. In this study we analysed the effects of five mechanical stimuli on the gene expression of human umbilical vein endothelial cells (HUVECs) in mRNA level using real-time PCR. The following loading regimes were applied on HUVECs for 48 h: intermittent (0-5 dyn/cm(2) , 1 Hz) and uniform (5 dyn/cm(2) ) shear stresses concomitant by 10% intermittent equiaxial stretch (1 Hz), uniform shear stress alone (5 dyn/cm(2) ), and intermittent uniaxial and equiaxial stretches (10%, 1 Hz). A new bioreactor was made to apply uniform/cyclic shear and tensile loadings. Three endothelial suggestive specific genes (vascular endothelial growth factor receptor-2 (VEGFR-2, also known as FLK-1), von Willebrand Factor (vWF) and vascular endothelial-cadherin (VE-cadherin)), and two smooth muscle genes (α-smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain (SMMHC)) were chosen for assessment of alteration in gene expression of endothelial cells and transdifferentiation toward smooth cells following load applications. Shear stress alone enhanced the endothelial gene expression significantly, while stretching alone was identified as a transdifferentiating factor. Cyclic equiaxial stretch contributed less to elevation of smooth muscle genes compared to uniaxial stretch. Cyclic shear stress in comparison to uniform shear stress concurrent with cyclic stretch was more influential on promotion of endothelial genes expression. Influence of different mechanical stimuli on gene expression may open a wider horizon to regulate functions of cell for tissue engineering purposes. © 2013 International Federation for Cell Biology.

[Expression of integrin alpha5 and actin in the cells of intervertebral disc under cyclic hydrostatic pressure in vitro].

PubMed

Yu, Sheng-ji; Qiu, Gui-xing; Burton, Yang; Sandra, Roth; Cari, Whyne; Albert, Yee

2005-12-15

To investigate the expression of integrin alpha5 and actin in the cells of intervertebral disc under cyclic hydrostatic pressure in vitro. The porcine lumbar intervertebral disc cells were isolated and cultured in vitro, and the cells underwent cyclic hydrostatic loading. After that, the expression of integrin alpha5 and actin in intervertebral disc cells were studied by means of morphology observing, Western blot and immunohistochemistry staining. The morphology of intervertebral disc cells were changed into smaller and flatten shape, and the expression of integrin alpha5 and actin were decreased after loading. The expression of integrin alpha5 decreases under cyclic hydrostatic pressure, and the actin is affected at the same time when signals are transferred into the cells by integrin alpha5. That may be one of the important mechanisms of the mechanotransduction in the cells of intervertebral disc.

Pathological Roles of Interleukin-22 in the Development of Recurrent Hepatitis C after Liver Transplantation

PubMed Central

Li, Jin; Cheung, Eddie; Li, Hanwei; Zhao, Jingmin; Liu, Hongling; Liu, Zhenwen; Zhang, Min

2016-01-01

Objective The aim of this study was to longitudinally evaluate and analyze the role of interleukin-22-producing CD4 positive cells (IL-22) in the pathogenesis of Hepatitis C Virus recurrence after Orthotopic Liver Transplantation (HCV-OLT). Methods 15 HCV-OLT, 15 age- and gender- matched non-HCV post-OLT (OLT) and 15 hepatitis C virus infected (HCV) patients were enrolled into our study from the liver transplantation and research center at Beijing 302 Hospital. We determined the frequencies of IL-22 using flow cytometry and expression of IL-22 mRNA using PCR in peripheral blood and liver tissue. We also divided HCV-OLT patients into rapid fibrosis progression (RFP) and slow fibrosis progression (SFP), examined IL-22 cells and analyzed the correlations between IL-22 frequencies and liver injury, fibrosis and clinical parameters. Moreover, we investigated the role of IL-22 in Human Hepatic Stellate Cells (HSCs). Results The levels of serum IL-22, frequencies of IL-22 producing cells in peripheral blood mononuclear cells, and expression of IL-22 mRNA and protein in the liver in the HCV-OLT group were significantly higher than that in the HCV and OLT groups. Furthermore, eight (53.3%) patients developed RFP after two years; another three patients were diagnosed liver cirrhosis. The frequencies of IL-22 were much higher in RFP compared with SFP, while no significant difference existed between OLT and SFP. Intrahepatic IL-22 positive cells were located in fibrotic areas and significantly correlated with α-smooth muscle actin (α-SMA) and fibrosis staging scores, not with grading scores and HCRVNA. In vitro, IL-22 administration prevented HSCs apoptosis, promoted HSCs proliferation and activation, up-regulated the expression of HSC-sourced growth factors including α-SMA, TGF-β and TIMP-1, and increased the production of liver fibrosis markers including laminin, hyaluronic acid and collagen type IV. Conclusion Peripheral and intrahepatic IL-22 is up-regulated and plays a pathological role in exacerbating liver fibrosis by activating HSCs in HCV-OLT patients, which may predict RFP and serve as an attractive target for anti-fibrotic therapy. PMID:27123854

San-Cao Granule () ameliorates hepatic fibrosis through high mobility group box-1 protein/smad signaling pathway.

PubMed

Wei, Shi-Zhang; Luo, Sheng-Qiang; Wang, Jian; Wang, Jia-Bo; Li, Rui-Sheng; Zhang, Xiao-Mei; Guo, Yan-Lei; Chen, Chang; Ma, Xiao; Chen, Zhe; Liu, Hong-Hong; Yang, Zhi-Rui; Li, Jian-Yu; Wang, Rui-Lin; Zhang, Ya-Ming; Yang, Hui-Yin; Xiao, Xiao-He; Zhao, Yan-Ling

2015-12-19

To investigate the possible mechanism of San-Cao Granule (SCG, ) mediating antiliver fifibrosis. A total of 60 male Sprague-Dawley rats were randomly divided into the normal control group, porcine serum-treated group, ursodesoxycholic acid (UDCA, 60 mg/kg), SCG (3.6 g/kg) group, SCG (1.8 g/kg) group and SCG (0.9 g/kg) group, with 10 rats in each group. Liver fifibrosis was induced with porcine serum by intraperitoneal injection for 8 weeks, except for the normal control group. Then, the rats in the three SCG-treated groups and UDCA group were administered SCG and UDCA respectively for 4 weeks. The serum levels of alanine transaminase (ALT), aspartate transaminase (AST), albumin (ALB), total bilirubin (TBIL), hyaluronic acid (HA), laminin (LN), and type IVcollagen (IVC) were examined using commercial kits and hepatic histopathology was examined with hematoxylin and eosin and Masson staining. Moreover, the protein expression levels of high mobility group box-1 protein (HMGB1), transforming growth factor β1 (TGF-β1), phosphorylated mothers against decapentaplegic homolog 3 (p-Smad3), Smad7, toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear factor-kappa B (NF-κB) and α-smooth muscle actin (α-SMA) were determined by western blot, immunohistochemistry and real time quantitativereverse transcription polymerase. Both SCG (3.6 and 1.8 g/kg) and UDCA signifificantly ameliorated the liver fifibrosis induced by porcine serum as indicated by retarding the serum levels increasing of ALT, AST, TBIL, HA, LN and IVC and preventing the serum level reducing of ALB compared with the model group (all P<0.01). Meanwhile, the collagen deposition was attenuated by SCG and UDCA treatment. Furthermore, SCG markedly reduced the expressions of HMGB1, TGF-β1, p-Smad3, TLR4, MyD88, NF-κB and α-SMA, and enhanced the expression of the Smad7 compared with the model group (all P<0.01). SCG ameliorates hepatic fifibrosis possibly through inhibiting HMGB1, TLR4/NF-κB and TGF-β1/Smad signaling pathway.

Protective effects of seed melon extract on CCl4-induced hepatic fibrosis in mice.

PubMed

Zhan, Yuan-Yuan; Wang, Jin-Hui; Tian, Xing; Feng, Shi-Xiu; Xue, Lin; Tian, Li-Ping

2016-12-04

Citrullus lanatus ssp. vulgaris var. megalaspermus Lin et Chao, was also known as watermelon belongs to family Cucurbitaceae, variously used as healthy food and in the treatment of liver and lungs problems. Currently, Citrullus lanatus has become a major economic crop of medicinal and edible effects with regional characteristics. This study was designed to evaluate the hepatoprotective and antioxidant activity of the seed melon (Citrullus lanatus ssp. vulgaris var. megalaspermus Lin et Chao) extract (SME) against carbon tetrachloride (CCl 4 ) induced hepatic fibrosis in mice. In this study, mice were randomly divided into 7 groups, including normal control, model, silymarin tablets as the positive control, SME 100, 200, 400, and 800mg/kg. After 8 weeks, activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), hyaluronic acid (HA) and laminin (LN) were checked. The levels of antioxidant enzymes such as superoxide dismutase (SOD), glutataion (GSH) and glutathione peroxidase (GSH-Px) were determined after SME administration. The hydroxyproline (HYP) levels, malondialdehyde (MDA) levels and histopathologic examinations of hepatocyte fibrosis were also determined. Additionally, effects of SME on alpha-smooth muscle actin (α-SMA) and transforming growth factor beta-1(TGF-β1) protein expressions were determined. We found that SME could significantly lower the serum levels of hepatic enzyme markers AST, ALT, HA and LN (P<0.01). Compared with the CCl 4 -only treatment group, levels of hepatic SOD and GSH-Px were significantly increased, and the MDA levels were remarkably decreased in mice treated by SME at medium dose (400mg/kg) and high dose (800mg/kg) (P<0.01). A histological examination of the liver showed that lesions, including necrosis, lymphocyte infiltration and fatty degeneration, were partially healed by treatment with SME. The results of protein expressions studies displayed that SME could inhibit α-SMA and TGF-β1 protein expression (P<0.01). The present results suggested that protective effect of SME against CCl 4 -induced hepatic fibrosis may rely on its effect on reducing oxidative stress and improving drug metabolizing enzyme activity in liver. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Vitamin D inhibits development of liver fibrosis in an animal model but cannot ameliorate established cirrhosis.

PubMed

Abramovitch, Shirley; Sharvit, Efrat; Weisman, Yosef; Bentov, Amir; Brazowski, Eli; Cohen, Gili; Volovelsky, Oded; Reif, Shimon

2015-01-15

1,25(OH)2D3, the active form of vitamin D, has an antiproliferative and antifibrotic effect on hepatic stellate cells. Our aim was to investigate the potential of 1,25(OH)2D3 to inhibit the development of liver fibrosis and to ameliorate established fibrosis in vivo. The antifibrotic effect of 1,25(OH)2D3 was investigated in a thioacetamide (TAA) model (as a preventive treatment and as a remedial treatment) and in a bile duct ligation model. In the preventive model, rats received simultaneously intraperitoneum injection of TAA and/or 1,25(OH)2D3 for 10 wk. In the remedial model, rats were treated with TAA for 10 wk and then received 1,25(OH)2D3 or saline for 8 wk. Fibrotic score was determined by Masson staining. Collagen I, α-smooth muscle actin (α-SMA), tissue inhibitor of metalloproteinase-1 (TIMP1), platelet-derived growth factor (PDGF), and transforming growth factor-β (TGF-β) expression were measured by Western blot analysis and real-time PCR. Hypercalemia was detected by chemistry measurements. Preventive treatment of 1,25(OH)2D3 significantly suppressed liver fibrosis both macroscopically and microscopically and significantly lowered the fibrotic score of the TAA + 1,25(OH)2D3 group compared with the TAA group. 1,25(OH)2D3 significantly inhibited expression of PDGF and TGF-β by ∼50% and suppressed the expression of collagen Iα1, TIMP1, and α-SMA by approximately three-, two-, and threefold, respectively. In contrast, 1,25(OH)2D3 was inefficient in amelioration of established liver fibrosis. Administration of 1,25(OH)2D3 to bile duct ligation rats led to a high mortality rate probably caused by hypercalcemia. We conclude that 1,25(OH)2D3 may be considered as a potential preventive treatment in an in vivo model but failed to ameliorate established cirrhosis. Copyright © 2015 the American Physiological Society.

Adipose tissue fibrosis in human cancer cachexia: the role of TGFβ pathway.

PubMed

Alves, Michele Joana; Figuerêdo, Raquel Galvão; Azevedo, Flavia Figueiredo; Cavallaro, Diego Alexandre; Neto, Nelson Inácio Pinto; Lima, Joanna Darck Carola; Matos-Neto, Emidio; Radloff, Katrin; Riccardi, Daniela Mendes; Camargo, Rodolfo Gonzalez; De Alcântara, Paulo Sérgio Martins; Otoch, José Pinhata; Junior, Miguel Luiz Batista; Seelaender, Marília

2017-03-14

Cancer cachexia is a multifactorial syndrome that dramatically decreases survival. Loss of white adipose tissue (WAT) is one of the key characteristics of cachexia. WAT wasting is paralleled by microarchitectural remodeling in cachectic cancer patients. Fibrosis results from uncontrolled ECM synthesis, a process in which, transforming growth factor-beta (TGFβ) plays a pivotal role. So far, the mechanisms involved in adipose tissue (AT) re-arrangement, and the role of TGFβ in inducing AT remodeling in weight-losing cancer patients are poorly understood. This study examined the modulation of ECM components mediated by TGFβ pathway in fibrotic AT obtained from cachectic gastrointestinal cancer patients. After signing the informed consent form, patients were enrolled into the following groups: cancer cachexia (CC, n = 21), weight-stable cancer (WSC, n = 17), and control (n = 21). The total amount of collagen and elastic fibers in the subcutaneous AT was assessed by histological analysis and by immunohistochemistry. TGFβ isoforms expression was analyzed by Multiplex assay and by immunohistochemistry. Alpha-smooth muscle actin (αSMA), fibroblast-specific protein (FSP1), Smad3 and 4 were quantified by qPCR and/or by immunohistochemistry. Interleukin (IL) 2, IL5, IL8, IL13 and IL17 content, cytokines known to be associated with fibrosis, was measured by Multiplex assay. There was an accumulation of collagen and elastic fibers in the AT of CC, as compared with WSC and controls. Collagens type I, III, VI, and fibronectin expression was enhanced in the tissue of CC, compared with both WSC and control. The pronounced expression of αSMA in the surrounding of adipocytes, and the increased mRNA content for FSP1 (20-fold) indicate the presence of activated myofibroblasts; particularly in CC. TGFβ1 and TGFβ3 levels were up-regulated by cachexia in AT, as well in the isolated adipocytes. Smad3 and Smad4 labeling was found to be more evident in the fibrotic areas of CC adipose tissue. Cancer cachexia promotes the development of AT fibrosis, in association with altered TGFβ signaling, compromising AT organization and function.

Low-intensity treadmill exercise promotes rat dorsal wound healing.

PubMed

Zhou, Wu; Liu, Guo-hui; Yang, Shu-hua; Mi, Bo-bin; Ye, Shu-nan

2016-02-01

In order to investigate the promoting effect of low-intensity treadmill exercise on rat dorsal wound healing and the mechanism, 20 Sprague-Dawley rats were randomly divided into two groups: exercise group (Ex) and non-exercise group (non-ex). The rats in Ex group were given treadmill exercise for one month, and those in non-ex group raised on the same conditions without treadmill exercise. Both groups received dorsal wound operation with free access to food and water. By two-week continuous observation and recording of the wound area, the healing rate was analyzed. The blood sample was collected at day 14 post-operation via cardiac puncture for determination of the number of endothelial progenitor cells (EPCs) by flow cytometry, and the concentrations of relevant cytokines such as basic fibroblast growth factor (bFGF), endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) were measured by ELISA. The skin tissue around the wound was dissected to observe the vascular density under the microscope after HE staining, to detect the mRNA level of VEGFR2 and angiopoietin-1 (Ang-1) receptor using RT-qPCR, and protein expression of a-smooth muscle actin (αSMA) and type III collagen (ColIII) using Western blotting. It was found that the wound area in Ex group was smaller at the same time point than in non-ex group. The number of circulating EPCs was greater and the concentrations of vasoactive factors such as VEGF, eNOS and bFGF were higher in Ex group than in non-ex group. HE staining displayed a higher vessel density in Ex group than in non-ex group. Moreover, the mRNA expression of VEGFR2 and Ang-1 detected in the wound tissue in Ex group was higher than in non-ex group. Meanwhile, the protein expression of αSMA and ColIII was more abundant in Ex group than in non-ex group. Conclusively, the above results demonstrate Ex rats had a higher wound healing rate, suggesting low-intensity treadmill exercise accelerates wound healing. The present work may provide some hint for future study of treating refractory wound.

Protective effect of Ac-SDKP on alveolar epithelial cells through inhibition of EMT via TGF-β1/ROCK1 pathway in silicosis in rat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng, Haijing; Xu, Hong; Zhang, Xianghong

The epithelial–mesenchymal transition (EMT) is a critical stage during the development of silicosis fibrosis. In the current study, we hypothesized that the anti-fibrotic tetrapeptide, N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) may exert its anti-fibrotic effects via activation of the TGF-β1/ROCK1 pathway, leading to inhibition of EMT. To address this hypothesis, we first examined the effect of Ac-SDKP upon EMT using an in vivo rat silicosis model, as well as in an in vitro model of TGF-β1-induced EMT. Confocal laser scanning microscopy was used to examine colocalization of surfactant protein A (SP-A), fibroblast specific protein-1 (FSP-1) and α-smooth muscle actin (α-SMA) in vivo. Western blotmore » analysis was used to examine for changes in the protein levels of E-cadherin (E-cad) and SP-A (epithelial cell markers), vimentin (mesenchymal cell marker), α-SMA (active myofibroblast marker), and collagen I and III in both in vivo and in vitro experiments. Secondly, we utilized Western blot analysis and confocal laser scanning microscopy to examine the protein expression of TGF-β1 and ROCK1 in in vivo and in vitro studies. The results revealed that Ac-SDKP treatment prevented increases in the expression of mesenchymal markers as well as TGF-β1, ROCK1, collagen I and III. Furthermore, Ac-SDKP treatment prevented decreases in the expression of epithelial cell markers in both in vivo and in vitro experiments. Based on the results, we conclude that Ac-SDKP inhibits the transition of epithelial cell-myofibroblast in silicosis via activation of the TGF-β1/ROCK1 signaling pathway, which may serve as a novel mechanism by which it exerts its anti-fibrosis properties. - Highlights: • EMT is a critical stage during the development of silicosis fibrosis. • Ac-SDKP inhibits the EMT process in silicosis both in vivo and in vitro. • Ac-SDKP inhibits the EMT process in silicosis via TGF-β1/ROCK1 pathway.« less

Chemical chaperon 4-phenylbutyrate protects against the endoplasmic reticulum stress-mediated renal fibrosis in vivo and in vitro.

PubMed

Liu, Shing-Hwa; Yang, Ching-Chin; Chan, Ding-Cheng; Wu, Cheng-Tien; Chen, Li-Ping; Huang, Jenq-Wen; Hung, Kuan-Yu; Chiang, Chih-Kang

2016-04-19

Renal tubulointerstitial fibrosis is the common and final pathologic change of kidney in end-stage renal disease. Interesting, endoplasmic reticulum (ER) stress is known to contribute to the pathophysiological mechanisms during the development of renal fibrosis. Here, we investigated the effects of chemical chaperon sodium 4-phenylbutyrate (4-PBA) on renal fibrosis in vivo and in vitro. In a rat unilateral ureteral obstruction (UUO) model, 4-PBA mimicked endogenous ER chaperon in the kidneys and significantly reduced glucose regulated protein 78 (GRP78), CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP), activating transcription factor 4 (ATF4), and phosphorylated JNK protein expressions as well as restored spliced X-box-binding protein 1 (XBP1) expressions in the kidneys of UUO rats. 4-PBA also attenuated the increases of α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF) protein expressions, tubulointerstitial fibrosis, and apoptosis in the kidneys of UUO rats. Moreover, transforming growth factor (TGF)-β markedly increased ER stress-associated molecules, profibrotic factors, and apoptotic markers in the renal tubular cells (NRK-52E), all of which could be significantly counteracted by 4-PBA treatment. 4-PBA also diminished TGF-β-increased CTGF promoter activity and CTGF mRNA expression in NRK-52E cells. Taken together, our results indicated that 4-PBA acts as an ER chaperone to ameliorate ER stress-induced renal tubular cell apoptosis and renal fibrosis.

Actin stress in cell reprogramming

PubMed Central

Guo, Jun; Wang, Yuexiu; Sachs, Frederick; Meng, Fanjie

2014-01-01

Cell mechanics plays a role in stem cell reprogramming and differentiation. To understand this process better, we created a genetically encoded optical probe, named actin–cpstFRET–actin (AcpA), to report forces in actin in living cells in real time. We showed that stemness was associated with increased force in actin. We reprogrammed HEK-293 cells into stem-like cells using no transcription factors but simply by softening the substrate. However, Madin-Darby canine kidney (MDCK) cell reprogramming required, in addition to a soft substrate, Harvey rat sarcoma viral oncogene homolog expression. Replating the stem-like cells on glass led to redifferentiation and reduced force in actin. The actin force probe was a FRET sensor, called cpstFRET (circularly permuted stretch sensitive FRET), flanked by g-actin subunits. The labeled actin expressed efficiently in HEK, MDCK, 3T3, and bovine aortic endothelial cells and in multiple stable cell lines created from those cells. The viability of the cell lines demonstrated that labeled actin did not significantly affect cell physiology. The labeled actin distribution was similar to that observed with GFP-tagged actin. We also examined the stress in the actin cross-linker actinin. Actinin force was not always correlated with actin force, emphasizing the need for addressing protein specificity when discussing forces. Because actin is a primary structural protein in animal cells, understanding its force distribution is central to understanding animal cell physiology and the many linked reactions such as stress-induced gene expression. This new probe permits measuring actin forces in a wide range of experiments on preparations ranging from isolated proteins to transgenic animals. PMID:25422450

SMN blood levels in a Porcine Model of Spinal Muscular Atrophy

PubMed Central

Iyer, Chitra; Wang, Xueqian; Renusch, Samantha R.; Duque, Sandra I.; Wehr, Allison M.; Mo, Xiaokui-Molly; McGovern, Vicki L.; Arnold, W. David; Burghes, Arthur H.M.; Kolb, Stephen J.

2017-01-01

Spinal Muscular Atrophy (SMA) is an autosomal recessive motor neuron disease that results in loss of spinal motor neurons, muscular weakness and, in severe cases, respiratory failure and death. SMA is caused by a deletion or mutation of the SMN1 gene and retention of the SMN2 gene that leads to low SMN expression levels. The measurement of SMN mRNA levels in peripheral blood samples has been used in SMA clinical studies as a pharmacodynamic biomarker for response to therapies designed to increase SMN levels. We recently developed a postnatal porcine model of SMA by the viral delivery of a short-hairpin RNA (shRNA) targeting porcine pSMN. scAAV9-mediated knockdown of pSMN mRNA at postnatal day 5 reliably resulted in denervation, weakness and motor neuron and ventral root axon loss that began 3–4 weeks after viral delivery, and this phenotype could be ameliorated by subsequent viral delivery of human SMN (hSMN). To determine if the effect of modulating SMN levels using gene therapy can be measured in blood, we measured expression of pSMN mRNA and hSMN mRNA by quantitative droplet digital PCR (ddPCR). We found that the endogenous expression of pSMN mRNA in blood increases in the first month of life. However, there were no significant differences in blood levels of pSMN mRNA after knock-down or of human SMN mRNA after gene therapy. Our results, obtained in a large animal model of SMA that is similar in size and anatomy to human infants, suggest that measurement of SMN mRNA levels in blood may not be informative in SMA clinical trials involving intrathecal delivery of SMN-modulating therapies. PMID:28269795

Electrically Stimulated Adipose Stem Cells on Polypyrrole-Coated Scaffolds for Smooth Muscle Tissue Engineering.

PubMed

Björninen, Miina; Gilmore, Kerry; Pelto, Jani; Seppänen-Kaijansinkko, Riitta; Kellomäki, Minna; Miettinen, Susanna; Wallace, Gordon; Grijpma, Dirk; Haimi, Suvi

2017-04-01

We investigated the use of polypyrrole (PPy)-coated polymer scaffolds and electrical stimulation (ES) to differentiate adipose stem cells (ASCs) towards smooth muscle cells (SMCs). Since tissue engineering lacks robust and reusable 3D ES devices we developed a device that can deliver ES in a reliable, repeatable, and cost-efficient way in a 3D environment. Long pulse (1 ms) or short pulse (0.25 ms) biphasic electric current at a frequency of 10 Hz was applied to ASCs to study the effects of ES on ASC viability and differentiation towards SMCs on the PPy-coated scaffolds. PPy-coated scaffolds promoted proliferation and induced stronger calponin, myosin heavy chain (MHC) and smooth muscle actin (SMA) expression in ASCs compared to uncoated scaffolds. ES with 1 ms pulse width increased the number of viable cells by day 7 compared to controls and remained at similar levels to controls by day 14, whereas shorter pulses significantly decreased viability compared to the other groups. Both ES protocols supported smooth muscle expression markers. Our results indicate that electrical stimulation on PPy-coated scaffolds applied through the novel 3D ES device is a valid approach for vascular smooth muscle tissue engineering.

Triazole RGD antagonist reverts TGFβ1-induced endothelial-to-mesenchymal transition in endothelial precursor cells.

PubMed

Bianchini, Francesca; Peppicelli, Silvia; Fabbrizzi, Pierangelo; Biagioni, Alessio; Mazzanti, Benedetta; Menchi, Gloria; Calorini, Lido; Pupi, Alberto; Trabocchi, Andrea

2017-01-01

Fibrosis is the dramatic consequence of a dysregulated reparative process in which activated fibroblasts (myofibroblasts) and Transforming Growth Factor β1 (TGFβ1) play a central role. When exposed to TGFβ1, fibroblast and epithelial cells differentiate in myofibroblasts; in addition, endothelial cells may undergo endothelial-to-mesenchymal transition (EndoMT) and actively participate to the progression of fibrosis. Recently, the role of αv integrins, which recognize the Arg-Gly-Asp (RGD) tripeptide, in the release and signal transduction activation of TGFβ1 became evident. In this study, we present a class of triazole-derived RGD antagonists that interact with αvβ3 integrin. Above different compounds, the RGD-2 specifically interferes with integrin-dependent TGFβ1 EndoMT in Endothelial Colony-Forming Cells (ECPCs) derived from circulating Endothelial Precursor Cells (ECPCs). The RGD-2 decreases the amount of membrane-associated TGFβ1, and reduces both ALK5/TGFβ1 type I receptor expression and Smad2 phosphorylation in ECPCs. We found that RGD-2 antagonist reverts EndoMT, reducing α-smooth muscle actin (α-SMA) and vimentin expression in differentiated ECPCs. Our results outline the critical role of integrin in fibrosis progression and account for the opportunity of using integrins as target for anti-fibrotic therapeutic treatment.

Corneal Wound Healing Requires IKB kinase β Signaling in Keratocytes

PubMed Central

Chen, Liang; Mongan, Maureen; Meng, Qinghang; Wang, Qin; Kao, Winston; Xia, Ying

2016-01-01

IkB kinase β (IKKβ) is a key signaling kinase for inflammatory responses, but it also plays diverse cell type-specific roles that are not yet fully understood. Here we investigated the role of IKKβ in the cornea using IkkβΔCS mice in which the Ikkβ gene was specifically deleted in the corneal stromal keratocytes. The IkkβΔCS corneas had normal morphology, transparency and thickness; however, they did not heal well from mild alkali burn injury. In contrast to the IkkβF/F corneas that restored transparency in 2 weeks after injury, over 50% of the IkkβΔCS corneas failed to fully recover. They instead developed recurrent haze with increased stromal thickness, severe inflammation and apoptosis. This pathogenesis correlated with sustained myofibroblast transformation with increased α smooth muscle actin (α-SMA) expression, higher levels of senescence β-Gal activity and scar tissue formation at the late stage of wound healing. In addition, the IkkβΔCS corneas displayed elevated expression of hemo-oxygenase-1 (HO-1), a marker of oxidative stress, and activation of stress signaling pathways with increased JNK, c-Jun and SMAD2/3 phosphorylation. These data suggest that IKKβ in keratocytes is required to repress oxidative stress and attenuate fibrogenesis and senescence in corneal wound healing. PMID:26987064

Human MSC gene expression under simulated microgravity (RPM)

NASA Astrophysics Data System (ADS)

Buravkova, Ludmila; Gershovich, Pavel; Grigoriev, Anatoly

It is generally supposed that microgravity cell response is mediated by some structures of actin cytoskeleton that can be implicated in cell mechanosensitivity. Cytoskeletal reorganization in the microgravity environment can affect gene expression, which results in alterations of cell function. However the direct impact of microgravity on expression of some cytoskeletal genes and encoded proteins remains unknown. Multipotential adult mesechymal stromal cells (MSCs) are the early precursors of bone marrow that can be induced to differentiate into bone-like cells as well as to the other mesenchymal tissues. In our previous experiments we revealed cytoskele-ton alterations and reduced human MSCs growth and osteogenesis in simulated microgravity by Random Positioning Machine. The purpose of this study was to determine the impact of low gravity on F-actin organization and gene expression level of α-, β-, γ-actin, vinculin, cofilin, small GTPase RhoA, Rho kinase (ROCK) and protein expression of some adhesion molecules in cultured hMSCs. Fluorescent microscopy have shown that even 30 min of SMG results in rearrangement of F-actin and the lack of stress fibers in cultured hMSCs. Cell number with abnormal F-actin organization was increased after 6 h, 24 h and 48 h of SMG. On the other hand, after 120 hours of SMG cells displayed partial restoration of F-actin fibers in comparison with 24 h and 48 h. Similarly, near the same restoration was seen in F-actin after readaptation for 24 h in 1g environment after 24 h of SMG. However, the observed alterations in F-actin dimensional organization were accompanied by changes in related proteins gene expression. Real-time PCR revealed slight up-regulation of α-actin expression that became more signifi-cant after 48 h of SMG. Down-regulation of γ-actin was observed after 48 hours of exposure in RPM. Moreover the up-regulation of β-tubulin, cofilin and small GTPase RhoA gene expres-sion was also detected after 48 h of SMG. On the contrary, there was no significant difference between SMG and 1g control group after 120 h of exposure, except up regulation of β-tubulin and, firstly appeared down regulation of vinculin. The same results were obtained when hMSCs were exposed to 24 h readaptation after 24 h of SMG, there were no changes in expression level of all genes of interest. Thus our study has demonstrated that prolonged exposure (more than 120 h) to SMG leads to restoration of hMSC actin cytoskeleton organization. The transient changes in expression level of some genes associated with actin cytoskeleton are supposed to be one of the possible mechanisms which can contribute to first stage of precursor's cellular adaptation to microgravity.

From Cytoskeleton to Gene Expression: Actin in the Nucleus.

PubMed

Viita, Tiina; Vartiainen, Maria K

2017-01-01

Although most people still associate actin mainly with the cytoskeleton, several lines of evidence, with the earliest studies dating back to decades ago, have emphasized the importance of actin also inside the cell nucleus. Actin has been linked to many gene expression processes from gene activation to chromatin remodeling, but also to maintenance of genomic integrity and intranuclear movement of chromosomes and chromosomal loci. Recent advances in visualizing different forms and dynamic properties of nuclear actin have clearly advanced our understanding of the basic concepts by which actin operates in the nucleus. In this chapter we address the different breakthroughs in nuclear actin studies, as well as discuss the regulation nuclear actin and the importance of nuclear actin dynamics in relation to its different nuclear functions. Our aim is to highlight the fact that actin should be considered as an essential component of the cell nucleus, and its nuclear actions should be taken into account also in experiments on cytoplasmic actin networks.

Isolation, culture and biological characteristics of multipotent porcine tendon-derived stem cells.

PubMed

Yang, Jinjuan; Zhao, Qianjun; Wang, Kunfu; Ma, Caiyun; Liu, Hao; Liu, Yingjie; Guan, Weijun

2018-06-01

Tendon-derived stem cells (TDSCs), a postulated multi-potential stem cell population, play significant role in the postnatal replenishment of tendon injuries. However, the majority of experimental materials were obtained from horse, rat, human and rabbit, but rarely from pig. In this research, 1‑day‑old pig was chosen as experimental sample source to isolate and culture TDSCs in vitro. Specific markers of TDSCs were then characterized by immunofluorescence and reverse transcription polymerase chain reaction (RT‑PCR) assays. The results showed that TDSCs could be expanded for 11 passages in vitro. The expression of specific markers, such as collagen Ⅰ, collagen Ⅲ, α‑smooth muscle actin (α‑SMA), CD105 and CD90 were observed by immunofluorescence and RT‑PCR. TDSCs were induced to differentiate into adipocytes, osteoblasts and chondrocytes, respectively. These results suggest that TDSCs isolated from porcine tendon exhibit the characteristics of multipotent stem cells. TDSCs, therefore, may be potential candidates for cellular transplantation therapy and tissue engineering in tendon injuries.

Rare paratesticular aggressive angiomyxoma with negative oestrogen and progesterone receptors in a male patient.

PubMed

Neyaz, Azfar; Husain, Nuzhat; Anand, Nidhi; Srivastava, Pallavi

2018-06-04

Aggressive angiomyxoma (AAM) is a rare mesenchymal myxoid tumour localised to the pelvis and/or perineum in adult females in reproductive age group. AAM is very rare in males, with <50 cases described in literature, and involves scrotum, spermatic cord and perineum. It is slow growing, with a marked tendency for local recurrence after excision, but without metastatic potential. We present a rare case of a paratesticular AAM in a man aged 53 years. Tumour cells were immunoreactive for desmin, smooth muscle actin (SMA), vimentin, CD34 and were negative for S100. Unlike AAMs in females which express oestrogen receptor (ER) and/or progesterone receptor (PR) in >90% cases, the tumour cells in our case were negative for ER and PR, suggesting that the hypothesis that these markers play a role in tumour development and pathogenesis, does not apply in males. Androgen receptor positivity was noted in 2%-5% tumour cells. © BMJ Publishing Group Ltd (unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Changes in beta-actin mRNA expression in remodeling canine myocardium.

PubMed

Carlyle, W C; Toher, C A; Vandervelde, J R; McDonald, K M; Homans, D C; Cohn, J N

1996-01-01

Beta-actin, a cytoskeletal protein important in the maintenance of cytoarchitecture, has long been thought to be expressed constitutively in myocardial tissue. As such, beta-actin mRNA has been used as a control gene in a wide range of experiments. However, we have uncovered consistent changes in beta-actin mRNA expression in canine myocardium remodeling as a result of insult to the left ventricle. The experimental canine models used were either DC shock damage to the left ventricle or volume overload resulting from severe mitral regurgitation. The remodeling process in both canine models is characterized by an increase in left ventricular mass. PCR amplification using primers designed to selectively amplify the 3' end and a portion of the 3' untranslated region of beta-actin mRNA resulted in the generation of a 297 base pair product predominant only in normal canine myocardium and a 472 base pair product that became increasingly prominent from 1 to 30 days after DC shock damage to the left ventricle and from 10 to 90 days after creation of mitral regurgitation. Northern analysis showed a three-fold increase in beta-actin mRNA after either DC shock or creation of mitral regurgitation. Western analysis revealed an early increase in beta-actin protein followed by an apparent decrease to below baseline levels. These observations suggest that changes in beta-actin mRNA expression accompany the structural alterations that occur in response to myocardial damage. Whether or not the changes in beta-actin mRNA expression play a role in mediating these structural alterations remains to be determined.

Astrocytes influence the severity of spinal muscular atrophy

PubMed Central

Rindt, Hansjörg; Feng, Zhihua; Mazzasette, Chiara; Glascock, Jacqueline J.; Valdivia, David; Pyles, Noah; Crawford, Thomas O.; Swoboda, Kathryn J.; Patitucci, Teresa N.; Ebert, Allison D.; Sumner, Charlotte J.; Ko, Chien-Ping; Lorson, Christian L.

2015-01-01

Systemically low levels of survival motor neuron-1 (SMN1) protein cause spinal muscular atrophy (SMA). α-Motor neurons of the spinal cord are considered particularly vulnerable in this genetic disorder and their dysfunction and loss cause progressive muscle weakness, paralysis and eventually premature death of afflicted individuals. Historically, SMA was therefore considered a motor neuron-autonomous disease. However, depletion of SMN in motor neurons of normal mice elicited only a very mild phenotype. Conversely, restoration of SMN to motor neurons in an SMA mouse model had only modest effects on the SMA phenotype and survival. Collectively, these results suggested that additional cell types contribute to the pathogenesis of SMA, and understanding the non-autonomous requirements is crucial for developing effective therapies. Astrocytes are critical for regulating synapse formation and function as well as metabolic support for neurons. We hypothesized that astrocyte functions are disrupted in SMA, exacerbating disease progression. Using viral-based restoration of SMN specifically to astrocytes, survival in severe and intermediate SMA mice was observed. In addition, neuromuscular circuitry was improved. Astrogliosis was prominent in end-stage SMA mice and in post-mortem patient spinal cords. Increased expression of proinflammatory cytokines was partially normalized in treated mice, suggesting that astrocytes contribute to the pathogenesis of SMA. PMID:25911676

Heat Shock Protein 27 Plays a Pivotal Role in Myofibroblast Differentiation and in the Development of Bleomycin-Induced Pulmonary Fibrosis

PubMed Central

Park, Ah-Mee; Kanai, Kyosuke; Itoh, Tatsuki; Sato, Takao; Tsukui, Tatsuya; Inagaki, Yutaka; Selman, Moises; Matsushima, Kouji; Yoshie, Osamu

2016-01-01

Heat shock protein 27 (HSP27) is a member of the small molecular weight HSP family. Upon treatment with transforming growth factor β1 (TGF-β1), we observed upregulation of HSP27 along with that of α-smooth muscle actin (α-SMA), a marker of myofibroblast differentiation, in cultured human and mouse lung fibroblasts. Furthermore, by using siRNA knockdown, we demonstrated that HSP27 was involved in cell survival and upregulation of fibronectin, osteopontin (OPN) and type 1 collagen, all functional markers of myofibroblast differentiation, in TGF-β1-treated MRC-5 cells. In lung tissues of bleomycin-treated mice, HSP27 was strongly upregulated and substantially co-localized with α-SMA, OPN and type I collagen but not with proSP-C (a marker of type II alveolar epithelial cells), E-cadherin (a marker of epithelial cells) or F4/80 (a marker of macrophages). A similar co-localization of HSP27 and α-SMA was observed in lung tissues of patients with idiopathic pulmonary fibrosis. Furthermore, airway delivery of HSP27 siRNA effectively suppressed bleomycin-induced pulmonary fibrosis in mice. Collectively, our findings indicate that HSP27 is critically involved in myofibroblast differentiation of lung fibroblasts and may be a promising therapeutic target for lung fibrotic diseases. PMID:26859835

Loss of cytokeratin 10 indicates malignant transformation in actinic cheilitis.

PubMed

Garcia, Natália Galvão; Oliveira, Denise Tostes; Lauris, José Roberto Pereira; Domingues, Maria Aparecida Custódio; Minicucci, Eliana Maria; Soares, Cléverson Teixeira

2016-05-01

The aim of this study was to investigate the relationship the expression of cytokeratins (CK10 and CK13) and the cell proliferation index determined by Ki-67 of lip squamous cell carcinoma and actinic cheilitis with different degrees of dysplasia. Forty-five paraffin-embedded actinic cheilitis with and without dysplasia and 20 lip squamous cell carcinoma were analyzed by immunohistochemistry using anti-human anti-CK10, anti-CK13, and anti-Ki-67 antibodies. The majority of actinic cheilitis showed immunopositivity for CK10 and CK13 with decrease or loss of expression in dysplastic areas. In lip squamous cell carcinoma of the lip, heterogeneous expression of CK13 and immunonegativity for CK10 were observed. There was a statistically significant difference between CK10 expression in lip squamous cell carcinoma and in actinic cheilitis with or without dysplasia (p < 0.001). The cell proliferation index was higher in actinic cheilitis with dysplasia and lip squamous cell carcinoma than in actinic cheilitis without epithelial dysplasia. A significant correlation was found between the intensity of the epithelial dysplasia and the cell proliferation index (p < 0.001). These results provide evidence that there is a downregulation of CK10 expression in dysplastic areas of patients with actinic cheilitis and in those with lip squamous cell carcinoma (LSCC) and that the index of cell proliferation, determined by Ki-67, is directly correlated with the intensity of the epithelial dysplasia. Altogether, these results suggest that CK10 expression and the epithelial cell proliferation index can help to identify malignant transformation in the lip region.

Emerging treatment options for spinal muscular atrophy.

PubMed

Burnett, Barrington G; Crawford, Thomas O; Sumner, Charlotte J

2009-03-01

The motor neuron disease spinal muscular atrophy (SMA) is one of the leading genetic killers of infants worldwide. SMA is caused by mutation of the survival motor neuron 1 (SMN1) gene and deficiency of the survival motor neuron (SMN) protein. All patients retain one or more copies of the SMN2 gene, which (by producing a small amount of the SMN protein) rescues embryonic lethality and modifies disease severity. Rapid progress continues in dissecting the cellular functions of the SMN protein, but the mechanisms linking SMN deficiency with dysfunction and loss of functioning motor units remain poorly defined. Clinically, SMA should to be distinguished from other neuromuscular disorders, and the diagnosis can be readily confirmed with genetic testing. Quality of life and survival of SMA patients are improved with aggressive supportive care including optimized respiratory and nutritional care and management of scoliosis and contractures. Because SMA is caused by inadequate amounts of SMN protein, one aim of current SMA therapeutics development is to increase SMN protein levels in SMA patients by activating SMN2 gene expression and/or increasing levels of full-length SMN2 transcripts. Several potential therapeutic compounds are currently being studied in clinical trials in SMA patients.

SYNERGISTIC EFFECTS of CYCLIC TENSION and TRANSFORMING GROWTH FACTOR-β1 on the AORTIC VALVE MYOFIBROBLAST

PubMed Central

Merryman, W. David; Lukoff, Howard D.; Long, Rebecca A.; Engelmayr, George C.; Hopkins, Richard A.; Sacks, Michael S.

2007-01-01

Background Phenotypically, the aortic valve interstitial cell (AVIC) is a dynamic myofibroblast, appearing contractile and activated in times of development, disease, and remodeling. The precise mechanism of phenotypic modulation is unclear, but it is speculated that both biomechanical and biochemical factors are influential. Therefore, we hypothesized that isolated and combined treatments of cyclic tension and TGF-β1 would alter the phenotype and subsequent collagen biosynthesis of AVICs in situ. Methods and Results Porcine aortic valve leaflets received 7 and 14 day treatments of 15% cyclic stretch (Tension), 0.5 ng/ml TGF-β1 (TGF), 15% cyclic stretch and 0.5 ng/ml TGF-β1 (Tension+TGF), or neither mechanical nor cytokine stimuli (Null). Tissues were homogenized and assayed for AVIC phenotype (smooth muscle α-actin (SMA)) and collagen biosynthesis (via heat shock protein 47 (Hsp47) which was further verified with type I collagen C-terminal propeptide (CICP)). At both 7 and 14 days, SMA, Hsp47, and CICP quantities were significantly greater (p<0.001) in the Tension+TGF group compared to all other groups. Additionally, Tension alone appeared to maintain SMA and Hsp47 levels that were measured at day 0, while TGF alone elicited an increase in SMA and Hsp47 compared to day 0 levels. Null treatment revealed diminished proteins at both time points. Conclusions Elevated TGF-β1 levels, in the presence of cyclic mechanical tension, resulted in synergistic increases in the contractile and biosynthetic proteins in AVICs. Since cyclic mechanical stimuli can never be relieved in vivo, the presence of TGF-β1 (possibly from infiltrating macrophages) may result in overly biosynthetic AVICs, leading to altered ECM architecture, compromised valve function, and ultimately degenerative valvular disease. PMID:17868877

Ocular myxoid leiomyosarcoma in a cat.

PubMed

Labelle, Philippe; Holmberg, Bradford J

2010-01-01

A case of myxoid leiomyosarcoma likely of iris dilator muscle origin in the enucleated eye of a 6-year-old domestic short haired cat is reported. The poorly demarcated mass expanded the iris, partially filled the globe and extended into the optic nerve. The mass was composed of spindle cells separated by abundant matrix positive for mucopolysaccharides with alcian blue. The neoplastic cells were immunoreactive for smooth muscle actin (SMA), S100 and vimentin, and negative for cytokeratin, Melan-A, glial fibrillary protein (GFAP) and desmin. There was no evidence of recurrence or metastasis 6 months after enucleation.

Triptolide disrupts the actin-based Sertoli-germ cells adherens junctions by inhibiting Rho GTPases expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Xiang; Zhao, Fang

Triptolide (TP), derived from the medicinal plant Triterygium wilfordii Hook. f. (TWHF), is a diterpene triepoxide with variety biological and pharmacological activities. However, TP has been restricted in clinical application due to its narrow therapeutic window especially in reproductive system. During spermatogenesis, Sertoli cell cytoskeleton plays an essential role in facilitating germ cell movement and cell-cell actin-based adherens junctions (AJ). At Sertoli cell-spermatid interface, the anchoring device is a kind of AJ, known as ectoplasmic specializations (ES). In this study, we demonstrate that β-actin, an important component of cytoskeleton, has been significantly down-regulated after TP treatment. TP can inhibit themore » expression of Rho GTPase such as, RhoA, RhoB, Cdc42 and Rac1. Downstream of Rho GTPase, Rho-associated protein kinase (ROCKs) gene expressions were also suppressed by TP. F-actin immunofluorescence proved that TP disrupts Sertoli cells cytoskeleton network. As a result of β-actin down-regulation, TP treatment increased expression of testin, which indicating ES has been disassembled. In summary, this report illustrates that TP induces cytoskeleton dysfunction and disrupts cell-cell adherens junctions via inhibition of Rho GTPases. - Highlights: • Triptolide induced the disruption of Sertoli-germ cell adherens junction. • Rho GTPases expression and actin dynamics have been suppressed by triptolide. • Actin-based adherens junction is a potential antifertility target of triptolide. • Rho-Rock is involved in the regulation of actin dynamics.« less

Unique ζ-chain motifs mediate a direct TCR-actin linkage critical for immunological synapse formation and T-cell activation.

PubMed

Klieger, Yair; Almogi-Hazan, Osnat; Ish-Shalom, Eliran; Pato, Aviad; Pauker, Maor H; Barda-Saad, Mira; Wang, Lynn; Baniyash, Michal

2014-01-01

TCR-mediated activation induces receptor microclusters that evolve to a defined immune synapse (IS). Many studies showed that actin polymerization and remodeling, which create a scaffold critical to IS formation and stabilization, are TCR mediated. However, the mechanisms controlling simultaneous TCR and actin dynamic rearrangement in the IS are yet not fully understood. Herein, we identify two novel TCR ζ-chain motifs, mediating the TCR's direct interaction with actin and inducing actin bundling. While T cells expressing the ζ-chain mutated in these motifs lack cytoskeleton (actin) associated (cska)-TCRs, they express normal levels of non-cska and surface TCRs as cells expressing wild-type ζ-chain. However, such mutant cells are unable to display activation-dependent TCR clustering, IS formation, expression of CD25/CD69 activation markers, or produce/secrete cytokine, effects also seen in the corresponding APCs. We are the first to show a direct TCR-actin linkage, providing the missing gap linking between TCR-mediated Ag recognition, specific cytoskeleton orientation toward the T-cell-APC interacting pole and long-lived IS maintenance. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Neuroprotective Effect of Non-viral Gene Therapy Treatment Based on Tetanus Toxin C-fragment in a Severe Mouse Model of Spinal Muscular Atrophy.

PubMed

Oliván, Sara; Calvo, Ana C; Rando, Amaya; Herrando-Grabulosa, Mireia; Manzano, Raquel; Zaragoza, Pilar; Tizzano, Eduardo F; Aquilera, Jose; Osta, Rosario

2016-01-01

Spinal muscular atrophy (SMA) is a hereditary childhood disease that causes paralysis and progressive degeneration of skeletal muscles and spinal motor neurons. SMA is associated with reduced levels of full-length Survival of Motor Neuron (SMN) protein, due to mutations in the Survival of Motor Neuron 1 gene. Nowadays there are no effective therapies available to treat patients with SMA, so our aim was to test whether the non-toxic carboxy-terminal fragment of tetanus toxin heavy chain (TTC), which exhibits neurotrophic properties, might have a therapeutic role or benefit in SMA. In this manuscript, we have demonstrated that TTC enhance the SMN expression in motor neurons "in vitro" and evaluated the effect of intramuscular injection of TTC-encoding plasmid in the spinal cord and the skeletal muscle of SMNdelta7 mice. For this purpose, we studied the weight and the survival time, as well as, the survival and cell death pathways and muscular atrophy. Our results showed that TTC treatment reduced the expression of autophagy markers (Becn1, Atg5, Lc3, and p62) and pro-apoptotic genes such as Bax and Casp3 in spinal cord. In skeletal muscle, TTC was able to downregulate the expression of the main marker of autophagy, Lc3, to wild-type levels and the expression of the apoptosis effector protein, Casp3. Regarding the genes related to muscular atrophy (Ankrd1, Calm1, Col19a1, Fbox32, Mt2, Myod1, NogoA, Pax7, Rrad, and Sln), TTC suggest a compensatory effect for muscle damage response, diminished oxidative stress and modulated calcium homeostasis. These preliminary findings suggest the need for further experiments to depth study the effect of TTC in SMA disease.

The enteric serotonergic system is altered in patients with diverticular disease.

PubMed

Böttner, Martina; Barrenschee, Martina; Hellwig, Ines; Harde, Jonas; Egberts, Jan-Hendrik; Becker, Thomas; Zorenkov, Dimitri; Wedel, Thilo

2013-12-01

Disturbances of the enteric serotonergic system have been implicated in several intestinal motility disorders. Patients with diverticular disease (DD) have been reported to exhibit abnormal intestinal motility and innervation patterns. Gene expression profiles of the serotonergic system and distribution of the serotonin type 4 receptor (5HT-4R) were thus studied in patients with DD. Colonic specimens from patients with DD and controls were subjected to quantitative PCR for serotonin receptors 2B, 3A, 4, serotonin transporter and synthesising enzyme tryptophan hydroxylase. Localisation of 5HT-4R was determined by dual-label immunocytochemistry using smooth muscle actin (α-SMA) and pan-neuronal markers (PGP 9.5) and quantitative analysis was carried out. Site-specific gene expression analysis of 5HT-4R was assessed within myenteric ganglia and muscle layers. Correlation of 5HT-4R with muscarinic receptors 2 and 3 (M2R, M3R) messenger RNA expression was determined. 5HT-4R mRNA expression was downregulated in the tunica muscularis and upregulated in the mucosa of patients with DD, whereas the other components of the serotonergic system remained unchanged. 5HT-4R was detected in ganglia and muscle layers, but was decreased in the circular muscle layer and myenteric ganglia of patients with DD. 5HT-4R mRNA expression correlated with M2R/M3R mRNA expression in controls, but not in patients with DD. The serotonergic system is compromised in DD. Altered expression of 5HT-4R at mRNA and protein levels may contribute to intestinal motor disturbances reported in patients with DD. The findings support the hypothesis that DD is associated and possibly promoted by an enteric neuromuscular pathology.

Immunohistochemical characterization of hemangiopericytomas and other spindle cell tumors in the dog.

PubMed

Pérez, J; Bautista, M J; Rollón, E; de Lara, F C; Carrasco, L; Martin de las Mulas, J

1996-07-01

The immunohistochemical expression of muscle actin has been studied in 45 canine hemangiopericytomas (CHP) using a monoclonal antibody (HHF35) and formalin-fixed, paraffin-embedded specimens. The distribution of vimentin, desmin, cytokeratins, lysozyme, factor VIII-related antigen, S-100 protein, and glial fibrillary acidic protein was studied both in CHP and in some canine soft-tissue neoplasms (seven fibrosarcomas, seven benign schwannomas, seven benign fibrous histiocytomas, and six leiomyosarcomas) used as controls for differential diagnosis. All CHP and control tumors expressed vimentin. Twenty-three CHP expressed muscle actin, whereas all control tumors analyzed were muscle actin-negative, with the exception of leiomyosarcomas. Among muscle actin- and vimentin-positive CHP, one case could be reclassified as leiomyosarcoma because it was desmin-positive, two cases expressed lysozyme, and nine cases expressed S-100 protein. Among muscle actin-negative and vimentin-positive CHP, seven expressed S-100 protein. In addition, S-100 protein was detected in five schwannomas. All CHP and control tumors analyzed were negative for cytokeratins, factor VIII-related antigen, and glial fibrillary acidic protein. Our results support the hypothesis of a pericytic origin of CHP, and suggest that muscle actin, desmin, vimentin, and lysozyme could be useful for the differential diagnosis of canine spindle cell tumors, but not all these neoplasms can be identified with these tumor tissue markers.

Insights into deregulated TNF and IL-10 production in malaria: implications for understanding severe malarial anaemia.

PubMed

Boeuf, Philippe S; Loizon, Séverine; Awandare, Gordon A; Tetteh, John K A; Addae, Michael M; Adjei, George O; Goka, Bamenla; Kurtzhals, Jørgen A L; Puijalon, Odile; Hviid, Lars; Akanmori, Bartholomew D; Behr, Charlotte

2012-08-01

Severe malarial anaemia (SMA) is a major life-threatening complication of paediatric malaria. Protracted production of pro-inflammatory cytokines promoting erythrophagocytosis and depressing erythropoiesis is thought to play an important role in SMA, which is characterized by a high TNF/IL-10 ratio. Whether this TNF/IL-10 imbalance results from an intrinsic incapacity of SMA patients to produce IL-10 or from an IL-10 unresponsiveness to infection is unknown. Monocytes and T cells are recognized as the main sources of TNF and IL-10 in vivo, but little is known about the activation status of those cells in SMA patients. The IL-10 and TNF production capacity and the activation phenotype of monocytes and T cells were compared in samples collected from 332 Ghanaian children with non-overlapping SMA (n = 108), cerebral malaria (CM) (n = 144) or uncomplicated malaria (UM) (n = 80) syndromes. Activation status of monocytes and T cells was ascertained by measuring HLA-DR+ and/or CD69+ surface expression by flow cytometry. The TNF and IL-10 production was assessed in a whole-blood assay after or not stimulation with lipopolysaccharide (LPS) or phytohaemaglutinin (PHA) used as surrogate of unspecific monocyte and T cell stimulant. The number of circulating pigmented monocytes was also determined. Monocytes and T cells from SMA and CM patients showed similar activation profiles with a comparable decreased HLA-DR expression on monocytes and increased frequency of CD69+ and HLA-DR+ T cells. In contrast, the acute-phase IL-10 production was markedly decreased in SMA compared to CM (P = .003) and UM (P = .004). Although in SMA the IL-10 response to LPS-stimulation was larger in amplitude than in CM (P = .0082), the absolute levels of IL-10 reached were lower (P = .013). Both the amplitude and levels of TNF produced in response to LPS-stimulation were larger in SMA than CM (P = .019). In response to PHA-stimulation, absolute levels of IL-10 produced in SMA were lower than in CM (P = .005) contrasting with TNF levels, which were higher (P = .001). These data reveal that SMA patients have the potential to mount efficient IL-10 responses and that the TNF/IL-10 imbalance may reflect a specific monocyte and T cell programming/polarization pattern in response to infection.

Effects of nicotine on corneal wound healing following acute alkali burn.

PubMed

Kim, Jong Won; Lim, Chae Woong; Kim, Bumseok

2017-01-01

Epidemiological studies have indicated that smoking is a pivotal risk factor for the progression of several chronic diseases. Nicotine, the addictive component of cigarettes, has powerful pathophysiological properties in the body. Although the effects of cigarette smoking on corneal re-epithelialization have been studied, the effects of nicotine on corneal wound healing-related neovascularization and fibrosis have not been fully demonstrated. The aim of this study was to evaluate the effects of chronic administration of nicotine on corneal wound healing following acute insult induced by an alkali burn. BALB/C female mice randomly received either vehicle (2% saccharin) or nicotine (100 or 200 μg/ml in 2% saccharin) in drinking water ad libitum. After 1 week, animals were re-randomized and the experimental group was subjected to a corneal alkali burn, and then nicotine was administered until day 14 after the alkali burn. A corneal alkali burn model was generated by placing a piece of 2 mm-diameter filter paper soaked in 1N NaOH on the right eye. Histopathological analysis and the expression level of the pro-angiogenic genes vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP9) revealed that chronic nicotine administration enhanced alkali burn-induced corneal neovascularization. Furthermore, the mRNA expression of the pro-fibrogenic factors α-smooth muscle actin (αSMA), transforming growth factor-β (TGF-β), and collagen α1 (Col1) was enhanced in the high-concentration nicotine-treated group compared with the vehicle group after corneal injury. Immunohistochemical analysis also showed that the αSMA-positive area was increased in chronic nicotine-treated mice after corneal alkali burn. An in vitro assay found that expression of the α3, α7, and β1 nicotinic acetylcholine receptor (nAChR) subunits was significantly increased by chemical injury in human corneal fibroblast cells. Moreover, alkali-induced fibrogenic gene expression and proliferation of fibroblast cells were further increased by treatment with nicotine and cotinine. The proliferation of such cells induced by treatment of nicotine and cotinine was reduced by inhibition of the PI3K and PKC pathways using specific inhibitors. In conclusion, chronic administration of nicotine accelerated the angiogenic and fibrogenic healing processes in alkali-burned corneal tissue.

Proteomic profiling of bone marrow mesenchymal stem cells upon TGF-beta stimulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Daojing; Park, Jennifer S.; Chu, Julia S.F.

Bone marrow mesenchymal stem cells (MSCs) can differentiate into different types of cells, and have tremendous potential for cell therapy and tissue engineering. Transforming growth factor {beta}1 (TGF-{beta}) plays an important role in cell differentiation and vascular remodeling. We showed that TGF-{beta} induced cell morphology change and an increase in actin fibers in MSCs. To determine the global effects of TGF-{beta} on MSCs, we employed a proteomic strategy to analyze the effect of TGF-{beta} on the human MSC proteome. By using two-dimensional gel electrophoresis and electrospray ionization coupled to Quadrupole/time-of-flight tandem mass spectrometers, we have generated a proteome reference mapmore » of MSCs, and identified {approx}30 proteins with an increase or decrease in expression or phosphorylation in response to TGF-{beta}. The proteins regulated by TGF-{beta} included cytoskeletal proteins, matrix synthesis proteins, membrane proteins, metabolic enzymes, etc. TGF-{beta} increased the expression of smooth muscle (SM) {alpha}-actin and decreased the expression of gelsolin. Over-expression of gelsolin inhibited TGF-{beta}-induced assembly of SM {alpha}-actin; on the other hand, knocking down gelsolin expression enhanced the assembly of {alpha}-actin and actin filaments without significantly affecting {alpha}-actin expression. These results suggest that TGF-{beta} coordinates the increase of {alpha}-actin and the decrease of gelsolin to promote MSC differentiation. This study demonstrates that proteomic tools are valuable in studying stem cell differentiation and elucidating the underlying molecular mechanisms.« less

A plasma-based biomatrix mixed with endothelial progenitor cells and keratinocytes promotes matrix formation, angiogenesis, and reepithelialization in full-thickness wounds.

PubMed

Vermeulen, Pieter; Dickens, Stijn; Degezelle, Karlien; Van den Berge, Stefaan; Hendrickx, Benoit; Vranckx, Jan Jeroen

2009-07-01

In search of an autologous vascularized skin substitute, we treated full-thickness wounds (FTWs) with autologous platelet-rich plasma gel (APG) in which we embedded endothelial progenitor cells (EPCs) and basal cell keratinocytes (KCs). We cultivated autologous KCs in low-serum conditions and expanded autologous EPCs from venous blood. FTWs (n = 55) were created on the backs of four pigs, covered with wound chambers, and randomly assigned to the following treatments: (1) APG, (2) APG + KCs, (3) APG + EPCs, (4) APG + KCs + EPCs, and (5) saline. All wounds were biopsied to measure neovascularization (lectin Bandeiraea Simplicifolia-1 (BS-1), alpha smooth muscle actin [alphaSMA], and membrane type 1 matrix metalloproteinase (MT1-MMP)), matrix deposition (fibronectin, collagen type I/III, and alphavbeta3), and reepithelialization. Wound fluids were analyzed for protein expression. All APG-treated wounds showed more vascular structures (p < 0.001), and the addition of EPCs further improved neovascularization, as confirmed by higher lectin, alphaSMA, and MT1-MMP. APG groups had higher collagen I/III (p < 0.05), alphavbeta3, and fibronectin content (p < 0.001), and they exhibited higher concentrations of platelet-derived growth factor subunit bb, basic fibroblast growth factor, hepatocyte growth factor, insulin growth factor-1, transforming growth factor-beta1 and -beta3, matrix metalloproteinase-1 and -z9, and tissue-inhibiting matrix metalloproteinase-1 and -2. Applying APG + KCs resulted in the highest reepithelialization rates (p < 0.001). No differences were found for wound contraction by planimetry. In this porcine FTW model, APG acts as a supportive biomatrix that, along with the embedded cells, improves extracellular matrix organization, promotes angiogenesis, and accelerates reepithelialization.

Secreted factors from metastatic prostate cancer cells stimulate mesenchymal stem cell transition to a pro-tumourigenic 'activated' state that enhances prostate cancer cell migration.

PubMed

Ridge, Sarah M; Bhattacharyya, Dibyangana; Dervan, Eoin; Naicker, Serika D; Burke, Amy J; Murphy, J M; O'leary, Karen; Greene, John; Ryan, Aideen E; Sullivan, Francis J; Glynn, Sharon A

2018-05-15

Mesenchymal stem cells (MSCs) are a heterogeneous population of multipotent cells that are capable of differentiating into osteocytes, chondrocytes and adipocytes. Recently, MSCs have been found to home to the tumour site and engraft in the tumour stroma. However, it is not yet known whether they have a tumour promoting or suppressive function. We investigated the interaction between prostate cancer cell lines 22Rv1, DU145 and PC3, and bone marrow-derived MSCs. MSCs were 'educated' for extended periods in prostate cancer cell conditioned media and PC3-educated MSCs were found to be the most responsive with a secretory profile rich in pro-inflammatory cytokines. PC3-educated MSCs secreted increased osteopontin (OPN), interleukin-8 (IL-8) and fibroblast growth factor-2 (FGF-2) and decreased soluble fms-like tyrosine kinase-1 (sFlt-1) compared to untreated MSCs. PC3-educated MSCs showed a reduced migration and proliferation capacity that was dependent on exposure to PC3-conditioned medium. Vimentin and α-smooth muscle actin (αSMA) expression was decreased in PC3-educated MSCs compared to untreated MSCs. PC3 and DU145 education of healthy donor and prostate cancer patient-derived MSCs led to a reduced proportion of FAP+ αSMA+ cells contrary to characteristics commonly associated with cancer associated fibroblasts (CAFs). The migration of PC3 cells was increased toward both PC3-educated and DU145-educated MSCs compared to untreated MSCs, while DU145 migration was only enhanced toward patient-derived MSCs. In summary, MSCs developed an altered phenotype in response to prostate cancer conditioned medium which resulted in increased secretion of pro-inflammatory cytokines, modified functional activity and the chemoattraction of prostate cancer cells. © 2017 UICC.

Targeted Injection of a Biocomposite Material Alters Macrophage and Fibroblast Phenotype and Function following Myocardial Infarction: Relation to Left Ventricular Remodeling

PubMed Central

McGarvey, Jeremy R.; Pettaway, Sara; Shuman, James A.; Novack, Craig P.; Zellars, Kia N.; Freels, Parker D.; Echols, Randall L.; Burdick, Jason A.; Gorman, Joseph H.; Gorman, Robert C.

2014-01-01

A treatment target for progressive left ventricular (LV) remodeling prevention following myocardial infarction (MI) is to affect structural changes directly within the MI region. One approach is through targeted injection of biocomposite materials, such as calcium hydroxyapatite microspheres (CHAM), into the MI region. In this study, the effects of CHAM injections upon key cell types responsible for the MI remodeling process, the macrophage and fibroblast, were examined. MI was induced in adult pigs before randomization to CHAM injections (20 targeted 0.1-ml injections within MI region) or saline. At 7 or 21 days post-MI (n = 6/time point per group), cardiac magnetic resonance imaging was performed, followed by macrophage and fibroblast isolation. Isolated macrophage profiles for monocyte chemotactic macrophage inflammatory protein-1 as measured by real-time polymerase chain reaction increased at 7 days post-MI in the CHAM group compared with MI only (16.3 ± 6.6 versus 1.7 ± 0.6 cycle times values, P < 0.05), and were similar by 21 days post-MI. Temporal changes in fibroblast function and smooth muscle actin (SMA) expression relative to referent control (n = 5) occurred with MI. CHAM induced increases in fibroblast proliferation, migration, and SMA expression—indicative of fibroblast transformation. By 21 days, CHAM reduced LV dilation (diastolic volume: 75 ± 2 versus 97 ± 4 ml) and increased function (ejection fraction: 48 ± 2% versus 38 ± 2%) compared with MI only (both P < 0.05). This study identified that effects on macrophage and fibroblast differentiation occurred with injection of biocomposite material within the MI, which translated into reduced adverse LV remodeling. These unique findings demonstrate that biomaterial injections impart biologic effects upon the MI remodeling process over any biophysical effects. PMID:25022514

Decursin attenuates hepatic fibrogenesis through interrupting TGF-beta-mediated NAD(P)H oxidase activation and Smad signaling in vivo and in vitro.

PubMed

Choi, Young Ji; Kim, Da Hye; Kim, Sang Jun; Kim, Ju; Jeong, Seung-Il; Chung, Chang Ho; Yu, Kang-Yeol; Kim, Seon-Young

2014-07-17

We studied that a potent antifibrotic effect of decursin on in vivo liver damage model and the mechanism in inhibiting which transforming growth factor (TGF)-β1-induced human hepatic stellate cells (HSCs) activation. Liver injury was induced in vivo by intraperitoneal injection of carbon tetrachloride (CCl4) with or without decursin for 4weeks in mice. Human hepatic stellate cell line, an immortalized human HSC line, was used in in vitro assay system. The effects of decursin on HSC activation were measured by analyzing the expression of α-smooth muscle actin (α-SMA) and collagen I in liver tissue and human HSCs. Decursin treatment significantly reduced the ratio of liver/body weight, α-SMA activation, and type I collagen overexpression in CCl4 treated mice liver. The elevated serum levels, including ALT, AST, and ALP, were also decreased by decursin treatment. Treatment of decursin markedly proved the generation of reactive oxygen species, NAD(P)H oxidase (NOX) protein (1, 2, and 4) upregulation, NOX activity, and superoxide anion production in HSCs by TGF-β1. It also significantly reduced TGF-β1-induced Smad 2/3 phosphorylation, nuclear translocation of Smad 4, and association of Smad 2/3-Smad 4 complex. Consistent with in vitro results, decursin treatment effectively blocked the levels of NOX protein, and Smad 2/3 phosphorylation in injured mice liver. Decursin blocked CCl4-induced liver fibrosis and inhibited TGF-β1-mediated HSC activation in vitro. These data demonstrated that decursin exhibited hepatoprotective effects on experimental fibrosis, potentially by inhibiting the TGF-β1 induced NOX activation and Smad signaling. Copyright © 2014 Elsevier Inc. All rights reserved.

Pulmonary Arterial Lesions in New World Camelids in Association With Dicrocoelium dendriticum and Fasciola hepatica Infection.

PubMed

Hilbe, M; Robert, N; Pospischil, A; Gerspach, C

2015-11-01

In Switzerland, dicrocoeliasis is regarded as the most significant parasitic infection of llamas and alpacas. Fasciola hepatica infestation is also a problem but less common. The aim of the present retrospective study was to evaluate the lungs of New World camelids (NWCs) for evidence of arterial hypertension in association with liver changes due to liver fluke infestation. The lungs of 20 llamas and 20 alpacas with liver fluke infestation were histologically evaluated. The hematoxylin and eosin and van Gieson (VG)-elastica stains as well as immunohistology for the expression of α-smooth muscle actin (α-SMA) were used to visualize the structures of arterial walls. Parasitology of fecal matter (11 llamas and 17 alpacas) confirmed that most of these animals were infested with both Dicrocoelium dendriticum and other gastrointestinal parasites. In most cases (10/12 llamas, 4/6 alpacas), liver enzyme activity in serum was elevated. Histologically, arteries in the lungs of 9 of 20 llamas (45%) and 3 of 20 alpacas (15%) showed severe intimal and adventitial and slight to moderate medial thickening, which was confirmed with α-SMA and VG-elastica staining. All animals exhibited typical liver changes, such as fibrosis and biliary hyperplasia, in association with the presence of liver flukes. This study shows that liver flukes can induce proliferative changes in lung arteries in NWCs that resemble those seen with pulmonary arterial hypertension due to liver parasites in humans. However, the degree of liver fluke infestation was not correlated with the extent of liver damage, or with the amount of thoracic or abdominal effusion or pulmonary arterial changes. © The Author(s) 2015.

Importance of Pulmonary Vein Preferential Fibrosis for Atrial Fibrillation Promotion in Hypertensive Rat Hearts.

PubMed

Iwasaki, Yu-Ki; Yamashita, Takeshi; Sekiguchi, Akiko; Hayami, Noriyuki; Shimizu, Wataru

2016-06-01

Hypertension is one of the independent risk factors for atrial fibrillation (AF). Pulmonary veins (PVs) play an important role as the substrate for AF and triggers of AF. The purpose of this study was to determine the structural remodelling of the PVs and its effect on promoting AF in hypertensive (HT) rat hearts. Eighteen-week-old Dahl salt-sensitive HT rats and their controls were used for histological and immunohistological analyses, and electrophysiological studies were performed in Langendorff perfused hearts. Masson-trichrome staining revealed that hypertension significantly increased the fibrosis in the PVs, particularly in subendocardial and perivascular areas, compared with that in control rats, however, at this early stage of hypertension, left atrial fibrosis was not prominent. In the HT rat hearts with PVs, electrical stimulation significantly increased the number of repetitive atrial firing and atrial tachycardia inducibility, which significantly diminished after the excision of the PVs. An immunofluorescent analysis revealed that HT rats had PV specific endocardial smooth muscle actin (αSMA)-positive cells with remarkable proliferation of platelet-derived growth factor (PDGF)-C and vascular endothelial growth factor (VEGF), which was lacking in the left atrial structures of the control and the HT rats. Pretreatment with imatinib, a PDGF receptor activity blocker, in HT rats reduced the αSMA-positive cell proliferation and fibrosis in the PVs and also induced a significant reduction in VEGF expression. Also, the drug pretreatment effectively prevented repetitive atrial firing promotion without affecting the blood pressure. PV preferential fibrosis might play an important role in the arrhythmogenic substrate of AF in HT rat hearts. Copyright © 2016 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.

Daam1 regulates fascin for actin assembly in mouse oocyte meiosis.

PubMed

Lu, Yujie; Zhang, Yu; Pan, Meng-Hao; Kim, Nam-Hyung; Sun, Shao-Chen; Cui, Xiang-Shun

2017-07-18

As a formin protein, Daam1 (Dishevelled-associated activator of morphogenesis 1) is reported to regulate series of cell processes like endocytosis, cell morphology and migration via its effects on actin assembly in mitosis. However, whether Daam1 plays roles in female meiosis remains uncertain. In this study, we investigated the expression and functions of Daam1 during mouse oocyte meiosis. Our results indicated that Daam1 localized at the cortex of oocytes, which was similar with actin filaments. After Daam1 morpholino (MO) microinjection, the expression of Daam1 significantly decreased, which resulted in the failure of oocyte polar body extrusion. These results might be due to the defects of actin assembly, since the decreased fluorescence intensity of actin filaments in oocyte cortex and cytoplasm were observed. However, Daam1 knockdown seemed not to affect the meiotic spindle movement. In addition, we found that fascin might be the down effector of Daam1, since the protein expression of fascin decreased after Daam1 knockdown. Thus, our data suggested that Daam1 affected actin assembly during oocyte meiotic division via the regulation of fascin expression.

Essential Role of Cofilin-1 in Regulating Thrombin-induced RelA/p65 Nuclear Translocation and Intercellular Adhesion Molecule 1 (ICAM-1) Expression in Endothelial Cells*

PubMed Central

Fazal, Fabeha; Bijli, Kaiser M.; Minhajuddin, Mohd; Rein, Theo; Finkelstein, Jacob N.; Rahman, Arshad

2009-01-01

Activation of RhoA/Rho-associated kinase (ROCK) pathway and the associated changes in actin cytoskeleton induced by thrombin are crucial for activation of NF-κB and expression of its target gene ICAM-1 in endothelial cells. However, the events acting downstream of RhoA/ROCK to mediate these responses remain unclear. Here, we show a central role of cofilin-1, an actin-binding protein that promotes actin depolymerization, in linking RhoA/ROCK pathway to dynamic alterations in actin cytoskeleton that are necessary for activation of NF-κB and thereby expression of ICAM-1 in these cells. Stimulation of human umbilical vein endothelial cells with thrombin resulted in Ser3 phosphorylation/inactivation of cofilin and formation of actin stress fibers in a ROCK-dependent manner. RNA interference knockdown of cofilin-1 stabilized the actin filaments and inhibited thrombin- and RhoA-induced NF-κB activity. Similarly, constitutively inactive mutant of cofilin-1 (Cof1-S3D), known to stabilize the actin cytoskeleton, inhibited NF-κB activity by thrombin. Overexpression of wild type cofilin-1 or constitutively active cofilin-1 mutant (Cof1-S3A), known to destabilize the actin cytoskeleton, also impaired thrombin-induced NF-κB activity. Additionally, depletion of cofilin-1 was associated with a marked reduction in ICAM-1 expression induced by thrombin. The effect of cofilin-1 depletion on NF-κB activity and ICAM-1 expression occurred downstream of IκBα degradation and was a result of impaired RelA/p65 nuclear translocation and consequently, RelA/p65 binding to DNA. Together, these data show that cofilin-1 occupies a central position in RhoA-actin pathway mediating nuclear translocation of RelA/p65 and expression of ICAM-1 in endothelial cells. PMID:19483084

Essential role of cofilin-1 in regulating thrombin-induced RelA/p65 nuclear translocation and intercellular adhesion molecule 1 (ICAM-1) expression in endothelial cells.

PubMed

Fazal, Fabeha; Bijli, Kaiser M; Minhajuddin, Mohd; Rein, Theo; Finkelstein, Jacob N; Rahman, Arshad

2009-07-31

Activation of RhoA/Rho-associated kinase (ROCK) pathway and the associated changes in actin cytoskeleton induced by thrombin are crucial for activation of NF-kappaB and expression of its target gene ICAM-1 in endothelial cells. However, the events acting downstream of RhoA/ROCK to mediate these responses remain unclear. Here, we show a central role of cofilin-1, an actin-binding protein that promotes actin depolymerization, in linking RhoA/ROCK pathway to dynamic alterations in actin cytoskeleton that are necessary for activation of NF-kappaB and thereby expression of ICAM-1 in these cells. Stimulation of human umbilical vein endothelial cells with thrombin resulted in Ser(3) phosphorylation/inactivation of cofilin and formation of actin stress fibers in a ROCK-dependent manner. RNA interference knockdown of cofilin-1 stabilized the actin filaments and inhibited thrombin- and RhoA-induced NF-kappaB activity. Similarly, constitutively inactive mutant of cofilin-1 (Cof1-S3D), known to stabilize the actin cytoskeleton, inhibited NF-kappaB activity by thrombin. Overexpression of wild type cofilin-1 or constitutively active cofilin-1 mutant (Cof1-S3A), known to destabilize the actin cytoskeleton, also impaired thrombin-induced NF-kappaB activity. Additionally, depletion of cofilin-1 was associated with a marked reduction in ICAM-1 expression induced by thrombin. The effect of cofilin-1 depletion on NF-kappaB activity and ICAM-1 expression occurred downstream of IkappaBalpha degradation and was a result of impaired RelA/p65 nuclear translocation and consequently, RelA/p65 binding to DNA. Together, these data show that cofilin-1 occupies a central position in RhoA-actin pathway mediating nuclear translocation of RelA/p65 and expression of ICAM-1 in endothelial cells.

Wiskott-Aldrich syndrome protein is required for NK cell cytotoxicity and colocalizes with actin to NK cell-activating immunologic synapses

NASA Astrophysics Data System (ADS)

Orange, Jordan S.; Ramesh, Narayanaswamy; Remold-O'Donnell, Eileen; Sasahara, Yoji; Koopman, Louise; Byrne, Michael; Bonilla, Francisco A.; Rosen, Fred S.; Geha, Raif S.; Strominger, Jack L.

2002-08-01

The Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency disorder caused by a mutation in WAS protein (WASp) that results in defective actin polymerization. Although the function of many hematopoietic cells requires WASp, the specific expression and function of this molecule in natural killer (NK) cells is unknown. Here, we report that WAS patients have increased percentages of peripheral blood NK cells and that fresh enriched NK cells from two patients with a WASp mutation have defective cytolytic function. In normal NK cells, WASp was expressed and localized to the activating immunologic synapse (IS) with filamentous actin (F-actin). Perforin also localized to the NK cell-activating IS but at a lesser frequency than F-actin and WASp. The accumulation of F-actin and WASp at the activating IS was decreased significantly in NK cells that had been treated with the inhibitor of actin polymerization, cytochalasin D. NK cells from WAS patients lacked expression of WASp and accumulated F-actin at the activating IS infrequently. Thus, WASp has an important function in NK cells. In patients with WASp mutations, the resulting NK cell defects are likely to contribute to their disease.

Edaravone is a candidate agent for spinal muscular atrophy: In vitro analysis using a human induced pluripotent stem cells-derived disease model.

PubMed

Ando, Shiori; Funato, Michinori; Ohuchi, Kazuki; Kameyama, Tsubasa; Inagaki, Satoshi; Seki, Junko; Kawase, Chizuru; Tsuruma, Kazuhiro; Shimazawa, Masamitsu; Kaneko, Hideo; Hara, Hideaki

2017-11-05

Spinal muscular atrophy (SMA) is an intractable disease characterized by a progressive loss of spinal motor neurons, which leads to skeletal muscle weakness and atrophy. Currently, there are no curative agents for SMA, although it is understood to be caused by reduced levels of survival motor neuron (SMN) protein. Additionally, why reduced SMN protein level results in selective apoptosis in spinal motor neurons is still not understood. Our purpose in this study was to evaluate the therapeutic potential of edaravone, a free radical scavenger, by using induced pluripotent stem cells from an SMA patient (SMA-iPSCs) and to address oxidative stress-induced apoptosis in spinal motor neurons. We first found that edaravone could improve impaired neural development of SMA-iPSCs-derived spinal motor neurons with limited effect on nuclear SMN protein expression. Furthermore, edaravone inhibited the generation of reactive oxygen species and mitochondrial reactive oxygen species upregulated in SMA-iPSCs-derived spinal motor neurons, and reversed oxidative-stress induced apoptosis. In this study, we suggest that oxidative stress might be partly the reason for selective apoptosis in spinal motor neurons in SMA pathology, and that oxidative stress-induced apoptosis might be the therapeutic target of SMA. Copyright © 2017 Elsevier B.V. All rights reserved.

Cordyceps sinensis attenuates renal fibrosis and suppresses BAG3 induction in obstructed rat kidney.

PubMed

Du, Feng; Li, Si; Wang, Tian; Zhang, Hai-Yan; Zong, Zhi-Hong; Du, Zhen-Xian; Li, De-Tian; Wang, Hua-Qin; Liu, Bo; Miao, Jia-Ning; Bian, Xiao-Hui

2015-01-01

BAG3 regulates a number of cellular processes, including cell proliferation, apoptosis, adhesion and migration, and epithelial-mesenchymal transition (EMT). However, the role of BAG3 in renal tubular EMT and renal interstitial fibrosis remains elusive. This study aimed to examine the dynamic expression of BAG3 during renal fibrosis, and to investigate the efficacy of Cordyceps sinensis (C. sinensis) on renal fibrosis. A rat model of unilateral ureteral obstruction (UUO) was established, and the expression of BAG3 and α-SMA, and the efficacy of C. sinensis on renal fibrosis induced by UUO were examined. The results showed that UUO led to collagen accumulation, which was significantly suppressed by C. sinensis. UUO increased the expression of BAG3 and α-SMA, a mesenchymal marker, while UUO induced BAG3 and α-SMA expression was significantly inhibited by C. sinensis. In addition, immunohistochemical staining demonstrated that BAG3 immunoreactivity was restricted to tubular epithelium. In conclusion, BAG3 is a potential target for the prevention and/or treatment of renal fibrosis, and C. Sinensis is a promising agent for renal fibrosis.

NOTCH3 regulates stem-to-mural cell differentiation in infantile hemangioma.

PubMed

Edwards, Andrew K; Glithero, Kyle; Grzesik, Peter; Kitajewski, Alison A; Munabi, Naikhoba Co; Hardy, Krista; Tan, Qian Kun; Schonning, Michael; Kangsamaksin, Thaned; Kitajewski, Jan K; Shawber, Carrie J; Wu, June K

2017-11-02

Infantile hemangioma (IH) is a vascular tumor that begins with rapid vascular proliferation shortly after birth, followed by vascular involution in early childhood. We have found that NOTCH3, a critical regulator of mural cell differentiation and maturation, is expressed in hemangioma stem cells (HemSCs), suggesting that NOTCH3 may function in HemSC-to-mural cell differentiation and pathological vessel stabilization. Here, we demonstrate that NOTCH3 is expressed in NG2+PDGFRβ+ perivascular HemSCs and CD31+GLUT1+ hemangioma endothelial cells (HemECs) in proliferating IHs and becomes mostly restricted to the αSMA+NG2loPDGFRβlo mural cells in involuting IHs. NOTCH3 knockdown in HemSCs inhibited in vitro mural cell differentiation and perturbed αSMA expression. In a mouse model of IH, NOTCH3 knockdown or systemic expression of the NOTCH3 inhibitor, NOTCH3 Decoy, significantly decreased IH blood flow, vessel caliber, and αSMA+ perivascular cell coverage. Thus, NOTCH3 is necessary for HemSC-to-mural cell differentiation, and adequate perivascular cell coverage of IH vessels is required for IH vessel stability.

Mesenchymal stem cells protect against obstruction-induced renal fibrosis by decreasing STAT3 activation and STAT3-dependent MMP-9 production.

PubMed

Matsui, Futoshi; Babitz, Stephen A; Rhee, Audrey; Hile, Karen L; Zhang, Hongji; Meldrum, Kirstan K

2017-01-01

STAT3 is a transcription factor implicated in renal fibrotic injury, but the role of STAT3 in mesenchymal stem cell (MSC)-induced renoprotection during renal fibrosis remains unknown. We hypothesized that MSCs protect against obstruction-induced renal fibrosis by downregulating STAT3 activation and STAT3-induced matrix metalloproteinase-9 (MMP-9) expression. Male Sprague-Dawley rats underwent renal arterial injection of vehicle or MSCs (1 × 10 6 /rat) immediately before sham operation or induction of unilateral ureteral obstruction (UUO). The kidneys were harvested after 4 wk and analyzed for collagen I and III gene expression, collagen deposition (Masson's trichrome), fibronectin, α-smooth muscle actin, active STAT3 (p-STAT3), MMP-9, and tissue inhibitor of matrix metalloproteinases 1 (TIMP-1) expression. In a separate arm, the STAT3 inhibitor S3I-201 (10 mg/kg) vs. vehicle was administered to rats intraperitoneally just after induction of UUO and daily for 14 days thereafter. The kidneys were harvested after 2 wk and analyzed for p-STAT3 and MMP-9 expression, and collagen and fibronectin deposition. Renal obstruction induced a significant increase in collagen, fibronectin, α-SMA, p-STAT3, MMP-9, and TIMP-1 expression while exogenously administered MSCs significantly reduced these indicators of obstruction-induced renal fibrosis. STAT3 inhibition with S3I-201 significantly reduced obstruction-induced MMP-9 expression and tubulointerstitial fibrosis. These results demonstrate that MSCs protect against obstruction-induced renal fibrosis, in part, by decreasing STAT3 activation and STAT3-dependent MMP-9 production. Copyright © 2017 the American Physiological Society.

Fluid shear-induced mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton-integrin interactions

NASA Technical Reports Server (NTRS)

Pavalko, F. M.; Chen, N. X.; Turner, C. H.; Burr, D. B.; Atkinson, S.; Hsieh, Y. F.; Qiu, J.; Duncan, R. L.

1998-01-01

Mechanical stimulation of bone induces new bone formation in vivo and increases the metabolic activity and gene expression of osteoblasts in culture. We investigated the role of the actin cytoskeleton and actin-membrane interactions in the transmission of mechanical signals leading to altered gene expression in cultured MC3T3-E1 osteoblasts. Application of fluid shear to osteoblasts caused reorganization of actin filaments into contractile stress fibers and involved recruitment of beta1-integrins and alpha-actinin to focal adhesions. Fluid shear also increased expression of two proteins linked to mechanotransduction in vivo, cyclooxygenase-2 (COX-2) and the early response gene product c-fos. Inhibition of actin stress fiber development by treatment of cells with cytochalasin D, by expression of a dominant negative form of the small GTPase Rho, or by microinjection into cells of a proteolytic fragment of alpha-actinin that inhibits alpha-actinin-mediated anchoring of actin filaments to integrins at the plasma membrane each blocked fluid-shear-induced gene expression in osteoblasts. We conclude that fluid shear-induced mechanical signaling in osteoblasts leads to increased expression of COX-2 and c-Fos through a mechanism that involves reorganization of the actin cytoskeleton. Thus Rho-mediated stress fiber formation and the alpha-actinin-dependent anchorage of stress fibers to integrins in focal adhesions may promote fluid shear-induced metabolic changes in bone cells.

Growth of arterioles in chronically stimulated adult rat skeletal muscle.

PubMed

Hansen-Smith, F; Egginton, S; Hudlicka, O

1998-01-01

The purpose of this study was to test the hypothesis that capillary growth induced by chronic electrical stimulation of skeletal muscle is accompanied by the growth of small arterioles. Lower limb flexor muscles of Sprague-Dawley rats were stimulated by electrodes implanted in the vicinity of the peroneal nerve at 10 Hz for 8 h/d for 2 and 7 days. Cryostat sections from the proximal, middle, and distal regions of the extensor digitorum longus muscle (EDL) were fluorescently immunolabeled with alpha-smooth muscle actin (alpha SMA) and myosin heavy chain (MHC) to identify mature (alpha SMA and MHC-positive) and immature (alpha SMA-positive, MHC-negative) arterioles. The fluorescent derivative of the lectin Griffonia simplicifolia I (GSI) was used to identify all microvessels, including arterioles, capillaries, and venules. The number of vessels positive for GSI or alpha SMA surrounding muscle fibers was similar in all three muscle regions (proximal, middle, distal). The mean values +/- SEM for GSI-positive vessels from all regions were similar in control (4.3 +/- 0.07) and 2-day stimulated (4.7 +/- 0.08) but higher in 7-day stimulated muscles (6.7 +/- 0.1, p < 0.05), thus confirming the previous findings on capillary growth. A similar increase was found in the number of alpha SMA positive vessels < or = 10 microns outer diameter (1.3 +/- 0.09 versus 0.4 +/- 0.03 around muscle fibers in controls). The density of terminal arterioles (< or = 10 microns) was slightly but not significantly higher after 2 days of stimulation (19.5 +/- 4 versus 15.6 +/- 2 profiles/mm2 in control muscles) and significantly higher after 7 days (33 +/- 7). While a similar increase was observed in the density of preterminal arterioles > 10 microns (17 +/- 3 control, 22 +/- 3 at 2 days and 40 +/- 5 at 7 days), the density of MHC-positive vessels muscles stimulated for 7 days was unchanged. Seven-day stimulated muscle also had a fivefold higher density of microvessel profiles < or = 10 microns that were only partially surrounded by alpha SMA. This considerably exceeds the relative increase in the number of capillaries and thus supports the concept of arteriolar growth by transformation from capillaries. Chronic electrical stimulation results in an early increase in the number of immature (MHG-negative), but not mature (MHC-positive) arterioles, a process that accompanies the increase in capillarization. The great increase in the number of microvessels only partially covered by alpha SMA suggests arteriolization of capillaries as a contributing mechanism in this growth.

SMN regulates axonal local translation via miR-183/mTOR pathway

PubMed Central

Kye, Min Jeong; Niederst, Emily D.; Wertz, Mary H.; Gonçalves, Inês do Carmo G.; Akten, Bikem; Dover, Katarzyna Z.; Peters, Miriam; Riessland, Markus; Neveu, Pierre; Wirth, Brunhilde; Kosik, Kenneth S.; Sardi, S. Pablo; Monani, Umrao R.; Passini, Marco A.; Sahin, Mustafa

2014-01-01

Reduced expression of SMN protein causes spinal muscular atrophy (SMA), a neurodegenerative disorder leading to motor neuron dysfunction and loss. However, the molecular mechanisms by which SMN regulates neuronal dysfunction are not fully understood. Here, we report that reduced SMN protein level alters miRNA expression and distribution in neurons. In particular, miR-183 levels are increased in neurites of SMN-deficient neurons. We demonstrate that miR-183 regulates translation of mTor via direct binding to its 3′ UTR. Interestingly, local axonal translation of mTor is reduced in SMN-deficient neurons, and this can be recovered by miR-183 inhibition. Finally, inhibition of miR-183 expression in the spinal cord of an SMA mouse model prolongs survival and improves motor function of Smn-mutant mice. Together, these observations suggest that axonal miRNAs and the mTOR pathway are previously unidentified molecular mechanisms contributing to SMA pathology. PMID:25055867

Development of siRNA expression vector utilizing rock bream beta-actin promoter: a potential therapeutic tool against viral infection in fish.

PubMed

Zenke, Kosuke; Nam, Yoon Kwon; Kim, Ki Hong

2010-01-01

In the present study, we have developed short interfering RNA (siRNA) expression vector utilizing rock bream beta-actin promoter and examined the possible use for the inhibition of highly pathogenic fish virus, rock bream iridovirus (RBIV), replication in vitro. Initially, in order to express siRNA effectively, we added several modifications to wild-type rock bream beta-actin promoter. Next, we succeeded in knocking down the expression of enhanced green fluorescent protein reporter gene expression in fish cells using newly developed vector more effectively than the fugu U6 promoter-driven vector we described previously. Finally, we could observe that cells transfected with modified rock bream beta-actin promoter-driven siRNA expression vector targeting major capsid protein (MCP) gene of RBIV exhibited more resistance to RBIV challenge than other control cells. Our results indicate that this novel siRNA expression vector can be used as a new tool for therapeutics in virus infection in fish species.

Heterogeneity of keratin expression and actin distribution in benign and malignant mammary diseases.

PubMed

Wada, T; Yasutomi, M; Yamada, K; Hashimura, K; Kunikata, M; Tanaka, T; Huang, J W; Mori, M

1991-01-01

Immunoreactivity of monoclonal anti-cytokeratin KL1, PKK1, K8.12 and anti-actin antibodies in 101 cases of diseased human breast lesions showed irregular keratin distribution in luminal cells of terminal ductal-lobular unit and basal layer cells of the interlobular and main duct. Actin staining was confined to myoepithelial cells. Benign lesions showed great heterogeneity in luminal cells of the terminal ductal-lobular units. Breast carcinoma showed a reduced staining for keratins, heterogeneity of keratin expression was found in solid tubular carcinoma, and actin was usually absent: however, papillo-ductal or comedo type had actin positive myoepithelial cells around carcinoma foci.

Members of the Cyr61/CTGF/NOV Protein Family: Emerging Players in Hepatic Progenitor Cell Activation and Intrahepatic Cholangiocarcinoma

PubMed Central

Jorgensen, Marda; Song, Joanna; Zhou, Junmei; Liu, Chen

2016-01-01

Hepatic stem/progenitor cells (HPC) reside quiescently in normal biliary trees and are activated in the form of ductular reactions during severe liver damage when the replicative ability of hepatocytes is inhibited. HPC niches are full of profibrotic stimuli favoring scarring and hepatocarcinogenesis. The Cyr61/CTGF/NOV (CCN) protein family consists of six members, CCN1/CYR61, CCN2/CTGF, CCN3/NOV, CCN4/WISP1, CCN5/WISP2, and CCN6/WISP3, which function as extracellular signaling modulators to mediate cell-matrix interaction during angiogenesis, wound healing, fibrosis, and tumorigenesis. This study investigated expression patterns of CCN proteins in HPC and cholangiocarcinoma (CCA). Mouse HPC were induced by the biliary toxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Differential expression patterns of CCN proteins were found in HPC from DDC damaged mice and in human CCA tumors. In addition, we utilized reporter mice that carried Ccn2/Ctgf promoter driven GFP and detected strong Ccn2/Ctgf expression in epithelial cell adhesion molecule (EpCAM)+ HPC under normal conditions and in DDC-induced liver damage. Abundant CCN2/CTGF protein was also found in cytokeratin 19 (CK19)+ human HPC that were surrounded by α-smooth muscle actin (α-SMA)+ myofibroblast cells in intrahepatic CCA tumors. These results suggest that CCN proteins, particularly CCN2/CTGF, function in HPC activation and CCA development. PMID:27829832

TM4SF5-Mediated Roles in the Development of Fibrotic Phenotypes

PubMed Central

Ryu, Jihye

2017-01-01

Transmembrane 4 L six family member 5 (TM4SF5) can form tetraspanin-enriched microdomains (TERMs) on the cell's surface. TERMs contain protein-protein complexes comprised of tetraspanins, growth factor receptors, and integrins. These complexes regulate communication between extracellular and intracellular spaces to control diverse cellular functions. TM4SF5 influences the epithelial-mesenchymal transition (EMT), aberrant multilayer cellular growth, drug resistance, enhanced migration and invasion, circulation through the bloodstream, tumor-initiation property, metastasis, and muscle development in zebrafish. Here, current data on TM4SF5's roles in the development of fibrotic phenotypes are reviewed. TM4SF5 is induced by transforming growth factor β1 (TGFβ1) signaling via a collaboration with epidermal growth factor receptor (EGFR) activation. TM4SF5, by itself or in concert with other receptors, transduces signals intracellularly. In hepatocytes, TM4SF5 expression regulates cell cycle progression, migration, and expression of extracellular matrix components. In CCl4-treated mice, TM4SF5, α-smooth muscle actin (α-SMA), and collagen I expression are observed together along the fibrotic septa regions of the liver. These fibrotic phenotypes are diminished by anti-TM4SF5 reagents, such as a specific small compound [TSAHC, 4′-(p-toluenesulfonylamido)-4-hydroxychalcone] or a chimeric antibody. This review discusses the antifibrotic strategies that target TM4SF5 and its associated protein networks that regulate the intracellular signaling necessary for fibrotic functions of hepatocytes. PMID:28458469

STVNa attenuates right ventricle hypertrophy and pulmonary artery remodeling in rats induced by transverse aortic constriction.

PubMed

Liu, Qing; Hu, Hui; Hu, Tingting; Han, Ting; Wang, Ahui; Huang, Lijie; Tan, Qiwen; Tan, Wen

2018-05-01

Right heart failure and pulmonary artery remodeling resulting from increased left heart pressure are prevalent in a clinical setting, and the specific pathological feature exhibits cancer-like cell proliferation in lung. STVNa has been previously demonstrated its anti-proliferation property. In this study, we want to verify the therapeutic effect of STVNa against right ventricle hypertrophy and pulmonary artery remodeling in rats induced by transverse aortic constriction (TAC). The results show that TAC surgery increased mean right ventricle pressure (mRVP) less in the STVNa group than that in the vehicle group (11.81 vs 22.71 mmHg/ml, p < 0.01). STVNa treatment reduced the right ventricle cardiomyocyte area (p < 0.05) and the proliferation of pathological smooth muscle cells proving by PCNA immunohistochemical staining. Gene expression of brain natriuretic peptide (BNP), smooth muscle actin (SMA) and CD31 assessed by real-time polymerase chain reaction were confirmed the above results. Also, STVNa treatment decreased the lung fibrosis content and alleviated the inflammation infiltration. The expression of ET-1 and the phosphorylation of signal-regulated kinase (ERK) were lower in STVNa group compared to vehicle group (p < 0.05). In summary, STVNa could relieve right ventricle hypertrophy and pulmonary artery remodeling formation in rats after 9 weeks of TAC surgery by reducing ET-1 expression and suppressing ERK phosphorylation signal and subsequently inhibiting cell proliferation. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Connective tissue growth factor stimulates the proliferation, migration and differentiation of lung fibroblasts during paraquat-induced pulmonary fibrosis.

PubMed

Yang, Zhizhou; Sun, Zhaorui; Liu, Hongmei; Ren, Yi; Shao, Danbing; Zhang, Wei; Lin, Jinfeng; Wolfram, Joy; Wang, Feng; Nie, Shinan

2015-07-01

It is well established that paraquat (PQ) poisoning can cause severe lung injury during the early stages of exposure, finally leading to irreversible pulmonary fibrosis. Connective tissue growth factor (CTGF) is an essential growth factor that is involved in tissue repair and pulmonary fibrogenesis. In the present study, the role of CTGF was examined in a rat model of pulmonary fibrosis induced by PQ poisoning. Histological examination revealed interstitial edema and extensive cellular thickening of interalveolar septa at the early stages of poisoning. At 2 weeks after PQ administration, lung tissue sections exhibited a marked thickening of the alveolar walls with an accumulation of interstitial cells with a fibroblastic appearance. Masson's trichrome staining revealed a patchy distribution of collagen deposition, indicating pulmonary fibrogenesis. Western blot analysis and immunohistochemical staining of tissue samples demonstrated that CTGF expression was significantly upregulated in the PQ-treated group. Similarly, PQ treatment of MRC-5 human lung fibroblast cells caused an increase in CTGF in a dose-dependent manner. Furthermore, the addition of CTGF to MRC-5 cells triggered cellular proliferation and migration. In addition, CTGF induced the differentiation of fibroblasts to myofibroblasts, as was evident from increased expression of α-smooth muscle actin (α-SMA) and collagen. These findings demonstrate that PQ causes increased CTGF expression, which triggers proliferation, migration and differentiation of lung fibroblasts. Therefore, CTGF may be important in PQ-induced pulmonary fibrogenesis, rendering this growth factor a potential pharmacological target for reducing lung injury.

Bee venom inhibits hepatic fibrosis through suppression of pro-fibrogenic cytokine expression.

PubMed

Kim, Soo-Jung; Park, Ji-Hyun; Kim, Kyung-Hyun; Lee, Woo-Ram; Chang, Young-Chae; Park, Kwan-Kyu; Lee, Kwang-Gill; Han, Sang-Mi; Yeo, Joo-Hong; Pak, Sok Cheon

2010-01-01

Bee venom (BV) has a long tradition of use for the control of pain and inflammation in various chronic diseases. Carbon tetrachloride (CCl4) is known to induce hepatotoxicity after being metabolized to the highly reactive trichloromethyl free radical and its peroxy radical. The purpose of the current study was to examine whether BV regulates the pro-inflammation and fibrosis related genes against a mouse model of hepatic fibrosis induced by CCl4 and ethanol-treated hepatocytes (ETH). Test mice were administered with CCl4 (2 ml/mg) and hepatocytes were treated with 25 mM ethanol. BV was added to the final concentration of 0.05-0.5 mg/kg and 1-100 ng/ml for in vivo and in vitro testing, respectively. Fibrotic livers and ETH were used for the measurement of hepatocyte necrosis, pro-inflammatory cytokines and fibrogenic genes. BV suppressed CCl4-induced hepatocyte necrosis markers of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT). It also inhibited the secretion of interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha. Moreover, BV inhibited CCl4-induced expression of transforming growth factor (TGF)-beta1, alpha-smooth muscle actin (SMA) and fibronectin. Similarly, ETH exhibited significant suppression of IL-1beta, TNF-alpha, TGF-beta1 and fibronectin when cultured with BV. These results suggest that BV possesses anti-fibrogenic properties that are mediated by the suppression of pro-inflammatory cytokines and fibrogenic gene expression. BV has substantial therapeutic potential for the treatment of fibrotic diseases.

Survival motor neuron protein in motor neurons determines synaptic integrity in spinal muscular atrophy.

PubMed

Martinez, Tara L; Kong, Lingling; Wang, Xueyong; Osborne, Melissa A; Crowder, Melissa E; Van Meerbeke, James P; Xu, Xixi; Davis, Crystal; Wooley, Joe; Goldhamer, David J; Lutz, Cathleen M; Rich, Mark M; Sumner, Charlotte J

2012-06-20

The inherited motor neuron disease spinal muscular atrophy (SMA) is caused by deficient expression of survival motor neuron (SMN) protein and results in severe muscle weakness. In SMA mice, synaptic dysfunction of both neuromuscular junctions (NMJs) and central sensorimotor synapses precedes motor neuron cell death. To address whether this synaptic dysfunction is due to SMN deficiency in motor neurons, muscle, or both, we generated three lines of conditional SMA mice with tissue-specific increases in SMN expression. All three lines of mice showed increased survival, weights, and improved motor behavior. While increased SMN expression in motor neurons prevented synaptic dysfunction at the NMJ and restored motor neuron somal synapses, increased SMN expression in muscle did not affect synaptic function although it did improve myofiber size. Together these data indicate that both peripheral and central synaptic integrity are dependent on motor neurons in SMA, but SMN may have variable roles in the maintenance of these different synapses. At the NMJ, it functions at the presynaptic terminal in a cell-autonomous fashion, but may be necessary for retrograde trophic signaling to presynaptic inputs onto motor neurons. Importantly, SMN also appears to function in muscle growth and/or maintenance independent of motor neurons. Our data suggest that SMN plays distinct roles in muscle, NMJs, and motor neuron somal synapses and that restored function of SMN at all three sites will be necessary for full recovery of muscle power.

Relationship between ultrasound elastography and myofibroblast distribution in breast cancer and its clinical significance

PubMed Central

Hao, Yi; Guo, Xia; Ma, Binlin; Zhu, Lin; Liu, Lisha

2016-01-01

The study investigated the relationship between ultrasound elastography (USE) scoring and myofibroblast distribution with expression features of α-SMA + /CD34− in patients of Uyghur and Han ethnicities with breast masses in Xinjiang, China. The data was used to evaluate its clinical significance in the early diagnosis of breast cancer. A total of 300 patients with breast masses were included in the study, which involved conventional sonography and USE, with histopathologic diagnosis as the reference standard. Myofibroblast distribution was investigated by detecting the expression levels of α-SMA and CD34 in lesions using immunohistochemistry and real-time PCR. Out of 300 lesions, 185 were histologically malignant and 115 benign. The mean elasticity score for malignant lesions was significantly higher than for benign lesions. The expression level of α-SMA was elevated while the expression level of CD34 was lower in malignancies, compared with benign lesions. The expression of α-SMA was positively associated with the USE scores, while a negative relationship was observed between CD34 expression and USE scoring. The combination of USE and molecular diagnosis provides a promising modality for the early diagnosis and evaluation of the risks in particular types of breast cancer. PMID:26846996

Protective effect of a coffee preparation (Nescafe pure) against carbon tetrachloride-induced liver fibrosis in rats.

PubMed

Shi, Hongyang; Dong, Lei; Zhang, Yong; Bai, Yanhua; Zhao, Juhui; Zhang, Li

2010-06-01

We examined the effects of a coffee preparation on liver fibrosis induced by carbon tetrachloride (CCl(4)) and explored the possible mechanisms. Rats were divided randomly into four groups: control, CCl(4), and two coffee preparation groups. Except for the control group, liver fibrosis was induced in male Sprague-Dawley (SD) rats by subcutaneous injection with 40% CCl(4) twice a week for 8 weeks. At the same time, a coffee preparation (300 mg/kg and 150 mg/kg) was administered to the two coffee preparation groups intragastrically once daily. Upon pathological examination, a coffee preparation treatment significantly reduced liver damage and symptoms of liver fibrosis. The mRNA expression of collagen I, collagen III, bcl-2, vascular endothelial growth factor (VEGF) and transforming growth factor-beta1 (TGF-beta1) were markedly increased by CCl(4) treatment but suppressed by a coffee preparation treatment. Whereas compared with the CCl(4) group, the mRNA expression of Bax was increased in the coffee preparation group. The protein expression of Bax and bcl-2 were confirmed by western blot. Intragastric administration of a coffee preparation reduced the protein expression of alpha-smooth muscle actin (alpha-SMA) and the glucose-regulated proteins (GRP) 78 and 94 in rats increased by CCl(4). Our data indicate that a coffee preparation can efficiently inhibit CCl(4)-induced liver fibrosis in rats. The coffee preparation may therefore be a potential functional food for preventing liver fibrosis. Copyright 2009 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Sirtuin 3 (SIRT3) Regulates α-Smooth Muscle Actin (α-SMA) Production through the Succinate Dehydrogenase-G Protein-coupled Receptor 91 (GPR91) Pathway in Hepatic Stellate Cells*

PubMed Central

Li, Ying Hui; Choi, Dae Hee; Lee, Eun Hye; Seo, Su Ryeon; Lee, Seungkoo

2016-01-01

Sirtuin 3 (SIRT3) is an NAD+-dependent protein deacetylase. Recent studies have shown that SIRT3 expression is decreased in nonalcoholic fatty liver disease (NAFLD). Moreover, SIRT3 is a key regulator of succinate dehydrogenase (SDH), which catalyzes the oxidation of succinate to fumarate. Increased succinate concentrations and the specific G protein-coupled receptor 91 (GPR91) are involved in the activation of hepatic stellate cells (HSCs). In this study, we aimed to establish whether SIRT3 regulated the SDH activity, succinate, and GPR91 expression in HSCs and an animal model of NAFLD. Our goal was also to determine whether succinate released from hepatocytes regulated HSC activation. Inhibiting SIRT3 using SIRT3 siRNA exacerbated HSC activation via the SDH-succinate-GPR91 pathway, and SIRT3 overexpression or honokiol treatment attenuated HSC activation in vitro. In isolated liver and HSCs from methionine- and choline-deficient (MCD) diet-induced NAFLD, the expression of SIRT3 and SDH activity was decreased, and the succinate concentrations and GPR91 expression were increased. Moreover, we found that GPR91 knockdown or resveratrol treatment improved the steatosis in MCD diet-fed mice. This investigation revealed a novel mechanism of the SIRT3-SDH-GPR91 cascade in MCD diet-induced HSC activation in NAFLD. These findings highlight the biological significance of novel strategies aimed at targeting SIRT3 and GPR91 in HSCs with the goal of improving NAFLD treatment. PMID:26912655

Sirtuin 3 (SIRT3) Regulates α-Smooth Muscle Actin (α-SMA) Production through the Succinate Dehydrogenase-G Protein-coupled Receptor 91 (GPR91) Pathway in Hepatic Stellate Cells.

PubMed

Li, Ying Hui; Choi, Dae Hee; Lee, Eun Hye; Seo, Su Ryeon; Lee, Seungkoo; Cho, Eun-Hee

2016-05-06

Sirtuin 3 (SIRT3) is an NAD(+)-dependent protein deacetylase. Recent studies have shown that SIRT3 expression is decreased in nonalcoholic fatty liver disease (NAFLD). Moreover, SIRT3 is a key regulator of succinate dehydrogenase (SDH), which catalyzes the oxidation of succinate to fumarate. Increased succinate concentrations and the specific G protein-coupled receptor 91 (GPR91) are involved in the activation of hepatic stellate cells (HSCs). In this study, we aimed to establish whether SIRT3 regulated the SDH activity, succinate, and GPR91 expression in HSCs and an animal model of NAFLD. Our goal was also to determine whether succinate released from hepatocytes regulated HSC activation. Inhibiting SIRT3 using SIRT3 siRNA exacerbated HSC activation via the SDH-succinate-GPR91 pathway, and SIRT3 overexpression or honokiol treatment attenuated HSC activation in vitro In isolated liver and HSCs from methionine- and choline-deficient (MCD) diet-induced NAFLD, the expression of SIRT3 and SDH activity was decreased, and the succinate concentrations and GPR91 expression were increased. Moreover, we found that GPR91 knockdown or resveratrol treatment improved the steatosis in MCD diet-fed mice. This investigation revealed a novel mechanism of the SIRT3-SDH-GPR91 cascade in MCD diet-induced HSC activation in NAFLD. These findings highlight the biological significance of novel strategies aimed at targeting SIRT3 and GPR91 in HSCs with the goal of improving NAFLD treatment. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The Progression of Haze Formation in Rabbit Corneas Following Phototherapeutic Keratectomy

PubMed Central

Gibson, Daniel J.; Tuli, Sonal S.; Schultz, Gregory S.

2013-01-01

Purpose. To determine the topographical location and time course of development of corneal haze in a phototherapeutic keratectomy model using slit lamp examination, macrophotography, quantitative image analysis, and immunofluorescence staining of corneal sections. Methods. Rabbit corneas were ablated with an excimer laser and were observed and graded for haze via slit lamp, imaged, and graded by macrophotography. Corneal sections were stained for α-smooth muscle actin (α-SMA) and tenascin-C (TNC). The distribution of haze imaged in the macrophotographs and density of α-SMA and TNC staining were compared. A daily image time course of haze formation was generated using macrophotography. Results. The first signs of corneal haze were apparent shortly after reepithelialization. The haze was distributed as a ring at the wound margin in all cases, while nearly all corneas also had some central islands of haze initiation. With time, the haze spread within the ablated zone and intensified. The pattern of immunofluorescent staining for α-SMA and TNC at the wound margin mirrored the haze distribution, spread, and intensification with time. Conclusions. The initiation and spread of subepithelial haze begins shortly after reepithelialization. The haze then spreads from the loci of initiation and becomes more dense with time, maturing as early as 14 days after wounding. The improved temporal and spatial resolution provided by these data improve the current model of light-scattering haze formation in wounded corneas, which will improve the design of studies aimed at maintaining corneal clarity following acute injury or surgery. PMID:23800768

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Xuejiao, E-mail: liuxuejiao0615@163.com; Institute for Liver Diseases, Anhui Medical University, Hefei 230032; Wu, Yuting

Background: NLRC5, as the largest member of NLRs family, has recently been identified as a critical regulator of immune responses through negatively regulating NF-κB which is associated with the development of hepatic fibrosis. However, the expression and potential roles of NLRC5 in hepatic fibrosis and its reversal are still to be defined. Methods: C57BL/6 mice were treatment with carbon tetrachloride (CCl{sub 4}) induce hepatic fibrosis and its reversal. In vitro, models of hepatic fibrosis and its reversal are established by the treatment with TGF-β and MDI. The expression of NLRC5 was determined by RT-PCR, Western blot and immunohistochemistry. Consequently, NLRC5more » was overexpressed or knockdown by transfecting PEGFP-C2-NLRC5 or NLRC5-siRNA respectively in the reversal of hepatic fibrosis, and the expression of fibrogenic genes such as α-SMA and Col1α1 was quantified. The NF-κB activity was detected as well. Results: Immunohistochemistry, RT-PCR and Western blot analysis with liver tissues and primary HSCs showed that NLRC5 was highly expressed in hepatic fibrosis and correspondingly decreased in the reversal stage. The differential expression of NLRC5 was confirmed in vitro. Enforced NLRC5 expression increased the expression of α-SMA and Col1α1, and blockade of NLRC5 reduced the fibrotic response. While the opposite expression of phosphorylated NF-kB p65 and phospho-IκBα was found. Conclusion: NLRC5 is differentially expressed in hepatic tissues and hepatic stellate cells during hepatic fibrosis and its reversal. All the data indicated that NLRC5 may play a crucial role in regulating the reversal of hepatic fibrosis through NF-κB signaling pathway. - Highlights: • The activated HSCs can be reverted to quiescent HSCs by MDI treatment. • NLRC5 expressed differentially during different stages of hepatic fibrosis and its reversal. • Enforced NLRC5 in reverted LX-c cells boosted the expression of α-SMA and Col1α1. • Blockade of NLRC5 diminished α-SMA and Col1α1 expression. • NLRC5 might provide a novel therapeutic approach for liver fibrosis.« less

Actin expression in some Platyhelminthe species.

PubMed

Fagotti, A; Panara, F; Di Rosa, I; Simoncelli, F; Gabbiani, G; Pascolini, R

1994-10-01

Actin expression in some Platyhelminthe species was demonstrated by western-blotting and immunocytochemical analysis using two distinct anti-actin antibodies: the anti-total actin that reacts against all actin isoforms of higher vertebrates and the anti-alpha SM-1 that recognizes the alpha-smooth muscle (alpha SM) isotype of endothermic vertebrates (Skalli et al., 1986). Western-blotting experiments showed that all species tested, including some free-living Platyhelminthes (Tricladida and Rhabdocoela) and the parasitic Fasciola hepatica, were stained by anti-total actin antibody while only Dugesidae and Dendrocoelidae showed a positive immunoreactivity against anti-alpha SM-1. These results were confirmed by cytochemical immunolocalization using both avidin biotin conjugated peroxidase reaction on paraffin sections, and immunogold staining on Lowicryl 4KM embedded specimens. Our findings may contribute to the understanding of Platyhelminthes phylogeny.

More than a bystander: the contributions of intrinsic skeletal muscle defects in motor neuron diseases

PubMed Central

Boyer, Justin G.; Ferrier, Andrew; Kothary, Rashmi

2013-01-01

Spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), and spinal-bulbar muscular atrophy (SBMA) are devastating diseases characterized by the degeneration of motor neurons. Although the molecular causes underlying these diseases differ, recent findings have highlighted the contribution of intrinsic skeletal muscle defects in motor neuron diseases. The use of cell culture and animal models has led to the important finding that muscle defects occur prior to and independently of motor neuron degeneration in motor neuron diseases. In SMA for instance, the muscle specific requirements of the SMA disease-causing gene have been demonstrated by a series of genetic rescue experiments in SMA models. Conditional ALS mouse models expressing a muscle specific mutant SOD1 gene develop atrophy and muscle degeneration in the absence of motor neuron pathology. Treating SBMA mice by over-expressing IGF-1 in a skeletal muscle-specific manner attenuates disease severity and improves motor neuron pathology. In the present review, we provide an in depth description of muscle intrinsic defects, and discuss how they impact muscle function in these diseases. Furthermore, we discuss muscle-specific therapeutic strategies used to treat animal models of SMA, ALS, and SBMA. The study of intrinsic skeletal muscle defects is crucial for the understanding of the pathophysiology of these diseases and will open new therapeutic options for the treatment of motor neuron diseases. PMID:24391590

Changes in the microarchitecture of the pancreatic cancer stroma are linked to neutrophil-dependent reprogramming of stellate cells and reflected by diffusion-weighted magnetic resonance imaging

PubMed Central

Mayer, Philipp; Dinkic, Christine; Jesenofsky, Ralf; Klauss, Miriam; Schirmacher, Peter; Dapunt, Ulrike; Hackert, Thilo; Uhle, Florian; Hänsch, G. Maria; Gaida, Matthias M.

2018-01-01

In pancreatic cancer (PDAC) intratumor infiltration of polymorphonuclear neutrophils (PMN) is associated with histologically apparent alterations of the tumor growth pattern. The aim of this study was to examine possible associations between PMN infiltration, tumor microarchitecture, and water diffusivity in diffusion-weighted magnetic resonance imaging (DW-MRI), and to further asses the underlying mechanisms. Methods: DW-MRI was performed in 33 PDAC patients prior to surgery. In parallel, tissue specimen were examined histologically for growth pattern, azurocidin-positive PMN infiltrates, and the presence of alpha-smooth muscle actin (α-SMA) and metalloproteinase 9 (MMP9)-positive myofibroblastic cells. For confirmation of the histological findings, a tissue microarray of a second cohort of patients (n=109) was prepared and examined similarly. For in vitro studies, the pancreatic stellate cell line RLT was co-cultivated either with isolated PMN, PMN-lysates, or recombinant azurocidin and characterized by Western blot, flow cytometry, and proteome profiler arrays. Results: Tumors with high PMN density showed restricted water diffusion in DW-MRI and histologic apparent alterations of the tumor microarchitecture (microglandular, micropapillary, or overall poorly differentiated growth pattern) as opposed to tumors with scattered PMN. Areas with altered growth pattern lacked α-SMA-positive myofibroblastic cells. Tissue microarrays confirmed a close association of high PMN density with alterations of the tumor microarchitecture and revealed a significant association of high PMN density with poor histologic grade of differentiation (G3). In vitro experiments provided evidence for direct effects of PMN on stellate cells, where a change to a spindle shaped cell morphology in response to PMN and to PMN-derived azurocidin was seen. Azurocidin incorporated into stellate cells, where it associated with F-actin. Down-regulation of α-SMA was seen within hours, as was activation of the p38-cofilin axis, up-regulation of MMP9, and acquisition of intracellular lipid droplets, which together indicate a phenotype switch of the stellate cells. Conclusion: In PDAC, PMN infiltrates are associated with alterations of the tumor microarchitecture. As a causal relationship, we propose a reprogramming of stellate cells by PMN-derived azurocidin towards a phenotype, which affects the microarchitecture of the tumor. PMID:29290790

Galectin-1 mediates TGF-β-induced transformation from normal fibroblasts into carcinoma-associated fibroblasts and promotes tumor progression in gastric cancer

PubMed Central

Zheng, Lingyan; Xu, Cong; Guan, Zhonghai; Su, Xingyun; Xu, Zhenzhen; Cao, Jiang; Teng, Lisong

2016-01-01

Rcinoma-associated fibroblasts (CAFs) are a major constituent of the tumor microenvironment. Cancer cells can induce the transformation from normal fibroblasts (NFs) into CAFs, reciprocally, CAFs promote tumor invasion and proliferation. TGF-β has been the mostly accepted factor to fuel NFs transformation into CAFs. Galectin-1 (Gal1) is highly upregulated in CAFs of multiple human cancers, and overexpression of Gal1 in CAFs promotes tumor progression. The effect of Gal1 on TGF-β-induced CAFs activation has not yet been established in gastric cancer (GC). In this study, we show that Gal1 expression in stroma is positively related to TGF-β in epithelial cells by retrospective analysis of GC patient samples. Meanwhile, conditioned media (CMs) from gastric cancer cells induce expression of both Gal1 and the CAFs marker alpha smooth muscle actin (α-SMA) in NFs via TGF-β secretion. Knockdown of Gal1 prevents TGF-β-induced the conversion of NFs to CAFs. CMs from fibroblasts overexpressing Gal1 inhibits cancer cells apoptosis, promotes migration and invasion in vitro. Thus, Gal1 is significantly involved in the development of tumor-promoting microenvironment by enhancing TGF-β signaling in a positive feedback loop. Targeting Gal1 in tumor stroma should be considered as a potential therapeutic target for GC. PMID:27186290

Anti-fibrotic effects of pirfenidone and rapamycin in primary IPF fibroblasts and human alveolar epithelial cells.

PubMed

Molina-Molina, M; Machahua-Huamani, C; Vicens-Zygmunt, V; Llatjós, R; Escobar, I; Sala-Llinas, E; Luburich-Hernaiz, P; Dorca, J; Montes-Worboys, A

2018-04-27

Pirfenidone, a pleiotropic anti-fibrotic treatment, has been shown to slow down disease progression of idiopathic pulmonary fibrosis (IPF), a fatal and devastating lung disease. Rapamycin, an inhibitor of fibroblast proliferation could be a potential anti-fibrotic drug to improve the effects of pirfenidone. Primary lung fibroblasts from IPF patients and human alveolar epithelial cells (A549) were treated in vitro with pirfenidone and rapamycin in the presence or absence of transforming growth factor β1 (TGF-β). Extracellular matrix protein and gene expression of markers involved in lung fibrosis (tenascin-c, fibronectin, collagen I [COL1A1], collagen III [COL3A1] and α-smooth muscle actin [α-SMA]) were analyzed. A cell migration assay in pirfenidone, rapamycin and TGF-β-containing media was performed. Gene and protein expression of tenascin-c and fibronectin of fibrotic fibroblasts were reduced by pirfenidone or rapamycin treatment. Pirfenidone-rapamycin treatment did not revert the epithelial to mesenchymal transition pathway activated by TGF-β. However, the drug combination significantly abrogated fibroblast to myofibroblast transition. The inhibitory effect of pirfenidone on fibroblast migration in the scratch-wound assay was potentiated by rapamycin combination. These findings indicate that the combination of pirfenidone and rapamycin widen the inhibition range of fibrogenic markers and prevents fibroblast migration. These results would open a new line of research for an anti-fibrotic combination therapeutic approach.

Shifts in renin-angiotensin system components, angiogenesis, and oxidative stress-related protein expression in the lamina cribrosa region of streptozotocin-induced diabetic mice.

PubMed

Qian, Xiaobing; Lin, Leilei; Zong, Yao; Yuan, Yongguang; Dong, Yanmin; Fu, Yue; Shao, Wanwen; Li, Yujie; Gao, Qianying

2018-03-01

This study aimed to analyse shifts in renin-angiotensin system (RAS) components, angiogenesis, and oxidative stress-related protein expression in the lamina cribrosa (LC) region in streptozotocin (STZ)-induced diabetic mice. Six months after diabetes induction, the retinal vessels of male C57BL/6 J mice were observed by colour photography, fundus fluorescein angiography (FFA), and immunofluorescent staining following incubation with CD31. Immunofluorescence for glial fibrillary acidic protein (GFAP), alpha-smooth muscle actin (α-SMA),and NG2 was also performed. Angiotensin-converting enzyme 1 (ACE1), angiotensin II type I receptor (AT1R), renin, hypoxia-inducible factor 1-alpha (HIF-1α), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR2), and haeme oxygenase 1 (HO-1) expression levels were confirmed by immunohistochemical and western blotting analyses. Compared with control mice, diabetic mice had significantly higher blood glucose concentrations (p < 0.001) and significantly lower body weights (p < 0.001). Colour photography and FFA did not reveal any vessel abnormalities in the diabetic mice; however, immunostaining of whole-mount retinas revealed an increased number of retinal vessels. Furthermore, histopathological staining showed significant reduction in the whole retinal thickness. GFAP expression was slightly higher, whereas fewer NG2 + pericytes were observed in diabetic mice than in control mice. ACE1, AT1R, renin, HIF-1α, VEGF, VEGFR2, and HO-1 expression were up-regulated in the LC of the STZ-induced diabetic mice. Collectively, ACE 1, AT1R, HIF-1α, VEGF, VEGFR2, and HO-1 activation in the LC region in diabetic mice may be involved in diabetes via the RAS and induction of angiogenesis and oxidative stress.

MiR-277/4989 regulate transcriptional landscape during juvenile to adult transition in the parasitic helminth Schistosoma mansoni

PubMed Central

van Dongen, Stijn; Collins, Julie; Quintais, Leonor; Ribeiro, Diogo M.; Sessler, Florian; Hunt, Martin; Rinaldi, Gabriel; Collins, James J.; Enright, Anton J.; Berriman, Matthew

2017-01-01

Schistosomes are parasitic helminths that cause schistosomiasis, a disease affecting circa 200 million people, primarily in underprivileged regions of the world. Schistosoma mansoni is the most experimentally tractable schistosome species due to its ease of propagation in the laboratory and the high quality of its genome assembly and annotation. Although there is growing interest in microRNAs (miRNAs) in trematodes, little is known about the role these molecules play in the context of developmental processes. We use the completely unaware “miRNA-blind” bioinformatics tool Sylamer to analyse the 3’-UTRs of transcripts differentially expressed between the juvenile and adult stages. We show that the miR-277/4989 family target sequence is the only one significantly enriched in the transition from juvenile to adult worms. Further, we describe a novel miRNA, sma-miR-4989 showing that its proximal genomic location to sma-miR-277 suggests that they form a miRNA cluster, and we propose hairpin folds for both miRNAs compatible with the miRNA pathway. In addition, we found that expression of sma-miR-277/4989 miRNAs are up-regulated in adults while their predicted targets are characterised by significant down-regulation in paired adult worms but remain largely undisturbed in immature “virgin” females. Finally, we show that sma-miR-4989 is expressed in tegumental cells located proximal to the oesophagus gland and also distributed throughout the male worms’ body. Our results indicate that sma-miR-277/4989 might play a dominant role in post-transcriptional regulation during development of juvenile worms and suggest an important role in the sexual development of female schistosomes. PMID:28542189

Hypertrophic Stimulation Increases β-actin Dynamics in Adult Feline Cardiomyocytes

PubMed Central

Balasubramanian, Sundaravadivel; Mani, Santhosh K.; Kasiganesan, Harinath; Baicu, Catalin C.; Kuppuswamy, Dhandapani

2010-01-01

The myocardium responds to hemodynamic stress through cellular growth and organ hypertrophy. The impact of cytoskeletal elements on this process, however, is not fully understood. While α-actin in cardiomyocytes governs muscle contraction in combination with the myosin motor, the exact role of β-actin has not been established. We hypothesized that in adult cardiomyocytes, as in non-myocytes, β-actin can facilitate cytoskeletal rearrangement within cytoskeletal structures such as Z-discs. Using a feline right ventricular pressure overload (RVPO) model, we measured the level and distribution of β-actin in normal and pressure overloaded myocardium. Resulting data demonstrated enriched levels of β-actin and enhanced translocation to the Triton-insoluble cytoskeletal and membrane skeletal complexes. In addition, RVPO in vivo and in vitro hypertrophic stimulation with endothelin (ET) or insulin in isolated adult cardiomyocytes enhanced the content of polymerized fraction (F-actin) of β-actin. To determine the localization and dynamics of β-actin, we adenovirally expressed GFP-tagged β-actin in isolated adult cardiomyocytes. The ectopically expressed β-actin-GFP localized to the Z-discs, costameres, and cell termini. Fluorescence recovery after photobleaching (FRAP) measurements of β-actin dynamics revealed that β-actin at the Z-discs is constantly being exchanged with β-actin from cytoplasmic pools and that this exchange is faster upon hypertrophic stimulation with ET or insulin. In addition, in electrically stimulated isolated adult cardiomyocytes, while β-actin overexpression improved cardiomyocyte contractility, immunoneutralization of β-actin resulted in a reduced contractility suggesting that β-actin could be important for the contractile function of adult cardiomyocytes. These studies demonstrate the presence and dynamics of β-actin in the adult cardiomyocyte and reinforce its usefulness in measuring cardiac cytoskeletal rearrangement during hypertrophic stimulation. PMID:20635003

Hypertrophic stimulation increases beta-actin dynamics in adult feline cardiomyocytes.

PubMed

Balasubramanian, Sundaravadivel; Mani, Santhosh K; Kasiganesan, Harinath; Baicu, Catalin C; Kuppuswamy, Dhandapani

2010-07-12

The myocardium responds to hemodynamic stress through cellular growth and organ hypertrophy. The impact of cytoskeletal elements on this process, however, is not fully understood. While alpha-actin in cardiomyocytes governs muscle contraction in combination with the myosin motor, the exact role of beta-actin has not been established. We hypothesized that in adult cardiomyocytes, as in non-myocytes, beta-actin can facilitate cytoskeletal rearrangement within cytoskeletal structures such as Z-discs. Using a feline right ventricular pressure overload (RVPO) model, we measured the level and distribution of beta-actin in normal and pressure overloaded myocardium. Resulting data demonstrated enriched levels of beta-actin and enhanced translocation to the Triton-insoluble cytoskeletal and membrane skeletal complexes. In addition, RVPO in vivo and in vitro hypertrophic stimulation with endothelin (ET) or insulin in isolated adult cardiomyocytes enhanced the content of polymerized fraction (F-actin) of beta-actin. To determine the localization and dynamics of beta-actin, we adenovirally expressed GFP-tagged beta-actin in isolated adult cardiomyocytes. The ectopically expressed beta-actin-GFP localized to the Z-discs, costameres, and cell termini. Fluorescence recovery after photobleaching (FRAP) measurements of beta-actin dynamics revealed that beta-actin at the Z-discs is constantly being exchanged with beta-actin from cytoplasmic pools and that this exchange is faster upon hypertrophic stimulation with ET or insulin. In addition, in electrically stimulated isolated adult cardiomyocytes, while beta-actin overexpression improved cardiomyocyte contractility, immunoneutralization of beta-actin resulted in a reduced contractility suggesting that beta-actin could be important for the contractile function of adult cardiomyocytes. These studies demonstrate the presence and dynamics of beta-actin in the adult cardiomyocyte and reinforce its usefulness in measuring cardiac cytoskeletal rearrangement during hypertrophic stimulation.

Long-term exercise-specific neuroprotection in spinal muscular atrophy-like mice.

PubMed

Chali, Farah; Desseille, Céline; Houdebine, Léo; Benoit, Evelyne; Rouquet, Thaïs; Bariohay, Bruno; Lopes, Philippe; Branchu, Julien; Della Gaspera, Bruno; Pariset, Claude; Chanoine, Christophe; Charbonnier, Frédéric; Biondi, Olivier

2016-04-01

The real impact of physical exercise parameters, i.e. intensity, type of contraction and solicited energetic metabolism, on neuroprotection in the specific context of neurodegeneration remains poorly explored. In this study behavioural, biochemical and cellular analyses were conducted to compare the effects of two different long-term exercise protocols, high intensity swimming and low intensity running, on motor units of a type 3 spinal muscular atrophy (SMA)-like mouse model. Our data revealed a preferential SMA-induced death of intermediate and fast motor neurons which was limited by the swimming protocol only, suggesting a close relationship between neuron-specific protection and their activation levels by specific exercise. The exercise-induced neuroprotection was independent of SMN protein expression and associated with specific metabolic and behavioural adaptations with notably a swimming-induced reduction of muscle fatigability. Our results provide new insight into the motor units' adaptations to different physical exercise parameters and will contribute to the design of new active physiotherapy protocols for patient care. Spinal muscular atrophy (SMA) is a group of autosomal recessive neurodegenerative diseases differing in their clinical outcome, characterized by the specific loss of spinal motor neurons, caused by insufficient level of expression of the protein survival of motor neuron (SMN). No cure is at present available for SMA. While physical exercise might represent a promising approach for alleviating SMA symptoms, the lack of data dealing with the effects of different exercise types on diseased motor units still precludes the use of active physiotherapy in SMA patients. In the present study, we have evaluated the efficiency of two long-term physical exercise paradigms, based on either high intensity swimming or low intensity running, in alleviating SMA symptoms in a mild type 3 SMA-like mouse model. We found that 10 months' physical training induced significant benefits in terms of resistance to muscle damage, energetic metabolism, muscle fatigue and motor behaviour. Both exercise types significantly enhanced motor neuron survival, independently of SMN expression, leading to the maintenance of neuromuscular junctions and skeletal muscle phenotypes, particularly in the soleus, plantaris and tibialis of trained mice. Most importantly, both exercises significantly improved neuromuscular excitability properties. Further, all these training-induced benefits were quantitatively and qualitatively related to the specific characteristics of each exercise, suggesting that the related neuroprotection is strongly dependent on the specific activation of some motor neuron subpopulations. Taken together, the present data show significant long-term exercise benefits in type 3 SMA-like mice providing important clues for designing rehabilitation programmes in patients. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Differential effects of LifeAct-GFP and actin-GFP on cell mechanics assessed using micropipette aspiration.

PubMed

Sliogeryte, Kristina; Thorpe, Stephen D; Wang, Zhao; Thompson, Clare L; Gavara, Nuria; Knight, Martin M

2016-01-25

The actin cytoskeleton forms a dynamic structure involved in many fundamental cellular processes including the control of cell morphology, migration and biomechanics. Recently LifeAct-GFP (green fluorescent protein) has been proposed for visualising actin structure and dynamics in live cells as an alternative to actin-GFP which has been shown to affect cell mechanics. Here we compare the two approaches in terms of their effect on cellular mechanical behaviour. Human mesenchymal stem cells (hMSCs) were analysed using micropipette aspiration and the effective cellular equilibrium and instantaneous moduli calculated using the standard linear solid model. We show that LifeAct-GFP provides clearer visualisation of F-actin organisation and dynamics. Furthermore, LifeAct-GFP does not alter effective cellular mechanical properties whereas actin-GFP expression causes an increase in the cell modulus. Interestingly, LifeAct-GFP expression did produce a small (~10%) increase in the percentage of cells exhibiting aspiration-induced membrane bleb formation, whilst actin-GFP expression reduced blebbing. Further studies examined the influence of LifeAct-GFP in other cell types, namely chondrogenically differentiated hMSCs and murine chondrocytes. LifeAct-GFP also had no effect on the moduli of these non-blebbing cells for which mechanical properties are largely dependent on the actin cortex. In conclusion we show that LifeAct-GFP enables clearer visualisation of actin organisation and dynamics without disruption of the biomechanical properties of either the whole cell or the actin cortex. Thus the study provides new evidence supporting the use of LifeAct-GFP rather than actin-GFP for live cell microscopy and the study of cellular mechanobiology. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Tobacco Arp3 is localized to actin-nucleating sites in vivo

PubMed Central

Maisch, Jan; Fišerová, Jindřiška; Fischer, Lukáš; Nick, Peter

2009-01-01

The polarity of actin is a central determinant of intracellular transport in plant cells. To visualize actin polarity in living plant cells, the tobacco homologue of the actin-related protein 3 (ARP3) was cloned and a fusion with the red fluorescent protein (RFP) was generated. Upon transient expression of these fusions in the tobacco cell line BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2), punctate structures were observed near the nuclear envelope and in the cortical plasma. These dots could be shown to decorate actin filaments by expressing RFP–ARP3 in a marker line, where actin was tagged by GFP (green fluorescent protein)–FABD (fimbrin actin-binding domain 2). When actin filaments were disrupted by latrunculin B or by prolonged cold treatment, and subsequently allowed to recover, the actin filaments reformed from the RFP–ARP3 structures, that therefore represented actin nucleation sites. The intracellular distribution of these sites was followed during the formation of pluricellular files, and it was observed that the density of RFP–ARP3 increased in the apex of the polarized, terminal cells of a file, whereas it was equally distributed in the central cells of a file. These findings are interpreted in terms of position-dependent differences of actin organization. PMID:19129161

Rice actin-binding protein RMD is a key link in the auxin-actin regulatory loop that controls cell growth.

PubMed

Li, Gang; Liang, Wanqi; Zhang, Xiaoqing; Ren, Haiyun; Hu, Jianping; Bennett, Malcolm J; Zhang, Dabing

2014-07-15

The plant hormone auxin plays a central role in plant growth and development. Auxin transport and signaling depend on actin organization. Despite its functional importance, the mechanistic link between actin filaments (F-actin) and auxin intracellular signaling remains unclear. Here, we report that the actin-organizing protein Rice Morphology Determinant (RMD), a type II formin from rice (Oryza sativa), provides a key link. Mutants lacking RMD display abnormal cell growth and altered configuration of F-actin array direction. The rmd mutants also exhibit an inhibition of auxin-mediated cell elongation, decreased polar auxin transport, altered auxin distribution gradients in root tips, and suppression of plasma membrane localization of auxin transporters O. sativa PIN-FORMED 1b (OsPIN1b) and OsPIN2 in root cells. We demonstrate that RMD is required for endocytosis, exocytosis, and auxin-mediated OsPIN2 recycling to the plasma membrane. Moreover, RMD expression is directly regulated by heterodimerized O. sativa auxin response factor 23 (OsARF23) and OsARF24, providing evidence that auxin modulates the orientation of F-actin arrays through RMD. In support of this regulatory loop, osarf23 and lines with reduced expression of both OsARF23 and OsARF24 display reduced RMD expression, disrupted F-actin organization and cell growth, less sensitivity to auxin response, and altered auxin distribution and OsPIN localization. Our findings establish RMD as a crucial component of the auxin-actin self-organizing regulatory loop from the nucleus to cytoplasm that controls rice cell growth and morphogenesis.

MicroRNA-31 is a positive modulator of endothelial-mesenchymal transition and associated secretory phenotype induced by TGF-β.

PubMed

Katsura, Akihiro; Suzuki, Hiroshi I; Ueno, Toshihide; Mihira, Hajime; Yamazaki, Tomoko; Yasuda, Takahiko; Watabe, Tetsuro; Mano, Hiroyuki; Yamada, Yoshitsugu; Miyazono, Kohei

2016-01-01

Transforming growth factor-β (TGF-β) plays central roles in endothelial-mesenchymal transition (EndMT) involved in development and pathogenesis. Although EndMT and epithelial-mesenchymal transition are similar processes, roles of microRNAs in EndMT are largely unknown. Here, we report that constitutively active microRNA-31 (miR-31) is a positive regulator of TGF-β-induced EndMT. Although the expression is not induced by TGF-β, miR-31 is required for induction of mesenchymal genes including α-SMA, actin reorganization and MRTF-A activation during EndMT. We identified VAV3, a regulator of actin remodeling and MRTF-A activity, as a miR-31 target. Global transcriptome analysis further showed that miR-31 positively regulates EndMT-associated unique secretory phenotype (EndMT-SP) characterized by induction of multiple inflammatory chemokines and cytokines including CCL17, CX3CL1, CXCL16, IL-6 and Angptl2. As a mechanism for this phenomenon, TGF-β and miR-31 suppress Stk40, a negative regulator of NF-κB pathway. Interestingly, TGF-β induces alternative polyadenylation (APA)-coupled miR-31-dependent Stk40 suppression without concomitant miR-31 induction, and APA-mediated exclusion of internal poly(A) sequence in Stk40 3'UTR enhances target efficiency of Stk40. Finally, miR-31 functions as a molecular hub to integrate TGF-β and TNF-α signaling to enhance EndMT. These data confirm that constitutively active microRNAs, as well as inducible microRNAs, serve as phenotypic modifiers interconnected with transcriptome dynamics during EndMT. © 2015 The Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

Evidence for actin cytoskeleton-dependent and -independent pathways for RelA/p65 nuclear translocation in endothelial cells.

PubMed

Fazal, Fabeha; Minhajuddin, Mohd; Bijli, Kaiser M; McGrath, James L; Rahman, Arshad

2007-02-09

Activation of the transcription factor NF-kappaB involves its release from the inhibitory protein IkappaBalpha in the cytoplasm and subsequently, its translocation to the nucleus. Whereas the events responsible for its release have been elucidated, mechanisms regulating the nuclear transport of NF-kappaB remain elusive. We now provide evidence for actin cytoskeleton-dependent and -independent mechanisms of RelA/p65 nuclear transport using the proinflammatory mediators, thrombin and tumor necrosis factor alpha, respectively. We demonstrate that thrombin alters the actin cytoskeleton in endothelial cells and interfering with these alterations, whether by stabilizing or destabilizing the actin filaments, prevents thrombin-induced NF-kappaB activation and consequently, expression of its target gene, ICAM-1. The blockade of NF-kappaB activation occurs downstream of IkappaBalpha degradation and is associated with impaired RelA/p65 nuclear translocation. Importantly, thrombin induces association of RelA/p65 with actin and this interaction is sensitive to stabilization/destabilization of the actin filaments. In parallel studies, stabilizing or destabilizing the actin filaments fails to inhibit RelA/p65 nuclear accumulation and ICAM-1 expression by tumor necrosis factor alpha, consistent with its inability to induce actin filament formation comparable with thrombin. Thus, these studies reveal the existence of actin cytoskeleton-dependent and -independent pathways that may be engaged in a stimulus-specific manner to facilitate RelA/p65 nuclear import and thereby ICAM-1 expression in endothelial cells.

Neurocalcin Delta Suppression Protects against Spinal Muscular Atrophy in Humans and across Species by Restoring Impaired Endocytosis.

PubMed

Riessland, Markus; Kaczmarek, Anna; Schneider, Svenja; Swoboda, Kathryn J; Löhr, Heiko; Bradler, Cathleen; Grysko, Vanessa; Dimitriadi, Maria; Hosseinibarkooie, Seyyedmohsen; Torres-Benito, Laura; Peters, Miriam; Upadhyay, Aaradhita; Biglari, Nasim; Kröber, Sandra; Hölker, Irmgard; Garbes, Lutz; Gilissen, Christian; Hoischen, Alexander; Nürnberg, Gudrun; Nürnberg, Peter; Walter, Michael; Rigo, Frank; Bennett, C Frank; Kye, Min Jeong; Hart, Anne C; Hammerschmidt, Matthias; Kloppenburg, Peter; Wirth, Brunhilde

2017-02-02

Homozygous SMN1 loss causes spinal muscular atrophy (SMA), the most common lethal genetic childhood motor neuron disease. SMN1 encodes SMN, a ubiquitous housekeeping protein, which makes the primarily motor neuron-specific phenotype rather unexpected. SMA-affected individuals harbor low SMN expression from one to six SMN2 copies, which is insufficient to functionally compensate for SMN1 loss. However, rarely individuals with homozygous absence of SMN1 and only three to four SMN2 copies are fully asymptomatic, suggesting protection through genetic modifier(s). Previously, we identified plastin 3 (PLS3) overexpression as an SMA protective modifier in humans and showed that SMN deficit impairs endocytosis, which is rescued by elevated PLS3 levels. Here, we identify reduction of the neuronal calcium sensor Neurocalcin delta (NCALD) as a protective SMA modifier in five asymptomatic SMN1-deleted individuals carrying only four SMN2 copies. We demonstrate that NCALD is a Ca 2+ -dependent negative regulator of endocytosis, as NCALD knockdown improves endocytosis in SMA models and ameliorates pharmacologically induced endocytosis defects in zebrafish. Importantly, NCALD knockdown effectively ameliorates SMA-associated pathological defects across species, including worm, zebrafish, and mouse. In conclusion, our study identifies a previously unknown protective SMA modifier in humans, demonstrates modifier impact in three different SMA animal models, and suggests a potential combinatorial therapeutic strategy to efficiently treat SMA. Since both protective modifiers restore endocytosis, our results confirm that endocytosis is a major cellular mechanism perturbed in SMA and emphasize the power of protective modifiers for understanding disease mechanism and developing therapies. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

A SMN-Dependent U12 Splicing Event Essential for Motor Circuit Function

PubMed Central

Lotti, Francesco; Imlach, Wendy L.; Saieva, Luciano; Beck, Erin S.; Hao, Le T.; Li, Darrick K.; Jiao, Wei; Mentis, George Z.; Beattie, Christine E.; McCabe, Brian D.; Pellizzoni, Livio

2012-01-01

SUMMARY Spinal muscular atrophy (SMA) is a motor neuron disease caused by deficiency of the ubiquitous survival motor neuron (SMN) protein. To define the mechanisms of selective neuronal dysfunction in SMA, we investigated the role of SMN-dependent U12 splicing events in the regulation of motor circuit activity. We show that SMN deficiency perturbs splicing and decreases the expression of a subset of U12 intron-containing genes in mammalian cells and Drosophila larvae. Analysis of these SMN target genes identifies Stasimon as a novel protein required for motor circuit function. Restoration of Stasimon expression in the motor circuit corrects defects in neuromuscular junction transmission and muscle growth in Drosophila SMN mutants and aberrant motor neuron development in SMN-deficient zebrafish. These findings directly link defective splicing of critical neuronal genes induced by SMN deficiency to motor circuit dysfunction, establishing a molecular framework for the selective pathology of SMA. PMID:23063131

Increasing expression and decreasing degradation of SMN ameliorate the spinal muscular atrophy phenotype in mice

PubMed Central

Kwon, Deborah Y.; Motley, William W.; Fischbeck, Kenneth H.; Burnett, Barrington G.

2011-01-01

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by reduced levels of the survival motor neuron (SMN) protein. Here we show that the proteasome inhibitor, bortezomib, increases SMN in cultured cells and in peripheral tissues of SMA model mice. Bortezomib-treated animals had improved motor function, which was associated with reduced spinal cord and muscle pathology and improved neuromuscular junction size, but no change in survival. Combining bortezomib with the histone deacetylase inhibitor trichostatin A (TSA) resulted in a synergistic increase in SMN protein levels in mouse tissue and extended survival of SMA mice more than TSA alone. Our results demonstrate that a combined regimen of drugs that decrease SMN protein degradation and increase SMN gene transcription synergistically increases SMN levels and improves the lifespan of SMA model mice. Moreover, this study indicates that while increasing SMN levels in the central nervous system may help extend survival, peripheral tissues can also be targeted to improve the SMA disease phenotype. PMID:21693563

VPAC2 receptor agonist BAY 55-9837 increases SMN protein levels and moderates disease phenotype in severe spinal muscular atrophy mouse models.

PubMed

Hadwen, Jeremiah; MacKenzie, Duncan; Shamim, Fahad; Mongeon, Kevin; Holcik, Martin; MacKenzie, Alex; Farooq, Faraz

2014-01-09

Spinal Muscular Atrophy (SMA) is one of the most common inherited causes of infant death and is caused by the loss of functional survival motor neuron (SMN) protein due to mutations or deletion in the SMN1 gene. One of the treatment strategies for SMA is to induce the expression of the protein from the homologous SMN2 gene, a rescuing paralog for SMA. Here we demonstrate the promise of pharmacological modulation of SMN2 gene by BAY 55-9837, an agonist of the vasoactive intestinal peptide receptor 2 (VPAC2), a member of G protein coupled receptor family. Treatment with BAY 55-9837 lead to induction of SMN protein levels via activation of MAPK14 or p38 pathway in vitro. Importantly, BAY 55-9837 also ameliorated disease phenotype in severe SMA mouse models. Our findings suggest the VPAC2 pathway is a potential SMA therapeutic target.

Inhibitory effect of ginsenoside Rg1 on extracellular matrix production via extracellular signal-regulated protein kinase/activator protein 1 pathway in nasal polyp-derived fibroblasts.

PubMed

Cho, Jung-Sun; Moon, You-Mi; Um, Ji-Young; Moon, Jun-Hyeok; Park, Il-Ho; Lee, Heung-Man

2012-06-01

Nasal polyps are associated with chronic inflammation of the sinonasal mucosa and are involved in myofibroblast differentiation and extracellular matrix (ECM) accumulation. Ginsenoside Rg1, a compound derived from Panax ginseng, shows antifibrotic and anticancer effects. However, the molecular effects of Rg1 on myofibroblast differentiation and ECM production remain unknown. The aims of this study were to investigate the effect of Rg1 on transforming growth factor (TGF)-β1-induced myofibroblast differentiation and ECM production and to determine the molecular mechanism of Rg1 in nasal polyp-derived fibroblasts (NPDFs). NPDFs were isolated from nasal polyps of seven patients who had chronic rhinosinusitis with nasal polyp. NPDFs were exposed to TGF-β1 with or without Rg1. Expression levels of α-smooth muscle actin (SMA), fibronectin and collagen type Iα1 were determined by reverse transcription polymerase chain reaction, Western blot and immunofluorescent staining. TGF-β1 signaling molecules, including Smad2/3, extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 were analyzed by Western blotting. Transcription factors involved with TGF-β1 signaling, nuclear factor (NF)-κB and activator protein 1 (AP-1) were also assessed by Western blot. The cytotoxic effect of Rg1 was measured by an established viability assay. The mRNA and protein expression levels of α-SMA, fibronectin and collagen type Iα1 were increased in TGF-β1-induced NPDFs. Rg1 inhibited these effects. The inhibitory molecular mechanism of Rg1 was involved in the ERK pathway. Rg1 inhibited the transcription factor activation of AP-1. Rg1 itself was not cytotoxic. The ginsenoside Rg1 has inhibitory effects on myofibroblast differentiation and ECM production. The inhibitory mechanism of Rg1 is involved with the ERK and AP-1 signaling pathways. Rg1 may be useful as an inhibitor of ECM deposition, and has potential to be used as a novel treatment option for nasal polyps.

Histopathological and Immunohistochemical Evaluation of Pannus Tissue in Patients with Prosthetic Valve Dysfunction.

PubMed

Karakoyun, Süleyman; Ozan Gürsoy, Mustafa; Yesin, Mahmut; Kalçık, Macit; Astarcıoğlu, Mehmet Ali; Gündüz, Sabahattin; Emrah Oğuz, Ali; Çoban Kökten, Şermin; Nimet Karadayı, Ayşe; Tuncer, Altuğ; Köksal, Cengiz; Gökdeniz, Tayyar; Özkan, Mehmet

2016-01-01

Prosthetic valve dysfunction due to pannus formation is a rare but serious complication. Currently, limited data are available concerning the pathogenesis and immunohistochemical properties of pannus. The study aim was to investigate the morphological, histopathological and immunohistochemical characteristics of pannus formation in patients with prosthetic valve dysfunction. A total of 35 patients (10 males, 25 females; mean age 44 ± 16 years) who had undergone re-do valve surgery due to prosthetic valve obstruction was enrolled in the study. Immunohistochemical studies were aimed at evaluating the expression of alphasmooth muscle actin (α-SMA) and desmin in myofibroblasts and smooth muscle cells; epithelial membrane antigen (EMA) in epithelial cells; and CD34, Factor VIII and vascular endothelial growth factor (VEGF) in endothelial cells. Matrix metalloproteinases (MMPs) -2 and -9, and transforming growth factor-beta (TGF-β) were used to demonstrate cytokine release from macrophages, leukocytes, fibroblasts and myofibroblasts. Pannus appeared as a tough and thick tissue hyperplasia which began from outside the suture ring in the periannular region and extended to the inflow and outflow surfaces of the prosthetic valves. Histopathological analysis showed the pannus tissue to consist of chronic inflammatory cells (lymphocytes, plasma cells, macrophages and foreign body giant cells), spindle cells such as myofibroblasts, capillary blood vessels and endothelial cells laying down the lumens. Calcification was present in the pannus tissue of 19 explanted prostheses. Immunohistochemical studies revealed positive α-SMA expression in all patients, whereas 60.5% of patients were positive for desmin, 50% for EMA, 42.1% for VEGF, 39.5% for TBF-β, 42.1% for MMP-2, 86.8% for CD34, and 97.4% for Factor VIII. MMP-9 was negative in all patients. Pannus tissue appears to be formed as the result of a neointimal response in periannular regions of prosthetic valves that consist of periannular tissue migration, myofibroblast and extracellular matrix proliferation with vascular components. It is a chronic active process in which mediators such as TGF-β, VEGF and MMP-2 play roles in both matrix formation and degradation.

Hydroxyapatite and Calcified Elastin Induce Osteoblast-like Differentiation in Rat Aortic Smooth Muscle Cells

PubMed Central

Lei, Yang; Sinha, Aditi; Nosoudi, Nasim; Grover, Ankit; Vyavahare, Naren

2014-01-01

Vascular calcification can be categorized into two different types. Intimal calcification related to atherosclerosis and elastin-specific medial arterial calcification (MAC). Osteoblast-like differentiation of vascular smooth muscle cells (VSMCs) has been shown in both types; however, how this relates to initiation of vascular calcification is unclear. We hypothesize that the initial deposition of hydroxyapatite-like mineral in MAC occurs on degraded elastin first and that causes osteogenic transformation of VSMCs. To test this, rat aortic smooth muscle cells (RASMCs) were cultured on hydroxyapatite crystals and calcified aortic elastin. Using RT-PCR and specific protein assays, we demonstrate that RASMCs lose their smooth muscle lineage markers like alpha smooth muscle actin (SMA) and myosin heavy chain (MHC) and undergo chondrogenic/osteogenic transformation. This is indicated by an increase in the expression of typical chondrogenic proteins such as aggrecan, collagen type II alpha 1(Col2a1) and bone proteins such as runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN). Furthermore, when calcified conditions are removed, cells return to their original phenotype. Our data supports the hypothesis that elastin degradation and calcification precedes VSMCs' osteoblast-like differentiation. PMID:24447384

Oral administration of Saccharomyces boulardii ameliorates carbon tetrachloride-induced liver fibrosis in rats via reducing intestinal permeability and modulating gut microbial composition.

PubMed

Li, Ming; Zhu, Lin; Xie, Ao; Yuan, Jieli

2015-02-01

To investigate the effects of orally administrated Saccharomyces boulardii (S. boulardii) on the progress of carbon tetrachloride (CCl4)-induced liver fibrosis, 34 male Wistar rats were randomly divided into four experimental groups including the control group (n = 8), the cirrhotic group (n = 10), the preventive group (n = 8), and the treatment group (n = 8). Results showed that the liver expression levels of collagen, type I, alpha 1 (Col1A1), alpha smooth muscle actin (αSMA), transforming growth factor beta (TGF-β) and the serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and malondialdehyde (MDA) increased significantly in cirrhotic rats compared with control and decreased by S. boulardii administration. Treatment of S. boulardii also attenuated the increased endotoxin levels and pro-inflammatory cytokines in CCl4-treated rats. And, these were associated with the changes of intestinal permeability and fecal microbial composition. Our study suggested that oral administration of S. boulardii can promote the liver function of CCl4-treated rats, and the preventive treatment of this probiotic yeast may decelerate the progress of liver fibrosis.

Competitive inhibition of leptin signaling results in amelioration of liver fibrosis through modulation of stellate cell function.

PubMed

Elinav, Eran; Ali, Mohammad; Bruck, Rafi; Brazowski, Eli; Phillips, Adam; Shapira, Yami; Katz, Meirav; Solomon, Gila; Halpern, Zamir; Gertler, Arieh

2009-01-01

Leptin signaling is involved in T-cell polarization and is required for profibrotic function of hepatic stellate cells (HSCs). Leptin-deficient ob/ob mice do not develop liver fibrosis despite the presence of severe long-standing steatohepatitis. Here, we blocked leptin signaling with our recently generated mouse leptin antagonist (MLA), and examined the effects on chronic liver fibrosis in vivo using the chronic thioacetamide (TAA) fibrosis model, and in vitro using freshly-isolated primary HSCs. In the chronic TAA fibrosis model, leptin administration was associated with significantly enhanced liver disease and a 100% 5-week to 8-week mortality rate, while administration or coadministration of MLA markedly improved survival, attenuated liver fibrosis, and reduced interferon gamma (IFN-gamma) levels. No significant changes in weight, serum cholesterol, or triglycerides were noted. In vitro administration of rat leptin antagonist (RLA), either alone or with leptin, to rat primary HSCs reduced leptin-stimulated effects such as increased expression of alpha-smooth muscle actin (alpha-SMA), and activation of alpha1 procollagen promoter. Inhibition of leptin-enhanced hepatic fibrosis may hold promise as a future antifibrotic therapeutic modality.

Cytoplasmic YY1 Is Associated with Increased Smooth Muscle-Specific Gene Expression

PubMed Central

Favot, Laure; Hall, Susan M.; Haworth, Sheila G.; Kemp, Paul R.

2005-01-01

Immediately after birth the adluminal vascular SMCs of the pulmonary elastic arteries undergo transient actin cytoskeletal remodeling as well as cellular de-differentiation and proliferation. Vascular smooth muscle phenotype is regulated by serum response factor, which is itself regulated in part by the negative regulator YY1. We therefore studied the subcellular localization of YY1 in arteries of normal newborn piglets and piglets affected by neonatal pulmonary hypertension. We found that YY1 localization changed during development and that expression of γ-smooth muscle actin correlated with expression of cytoplasmic rather than nuclear YY1. Analysis of the regulation of YY1 localization in vitro demonstrated that polymerized γ-actin sequestered EGFP-YY1 in the cytoplasm and that YY1 activation of c-myc promoter activity was inhibited by LIM kinase, which increases actin polymerization. Consistent with these data siRNA-mediated down-regulation of YY1 in C2C12 cells increased SM22-α expression and inhibited cell proliferation. Thus, actin polymerization controls subcellular YY1 localization, which contributes to vascular SMC proliferation and differentiation in normal pulmonary artery development. In the absence of actin depolymerization, YY1 does not relocate to the nucleus, and this lack of relocation may contribute to the pathobiology of pulmonary hypertension. PMID:16314465

β-actin as a loading control for plasma-based Western blot analysis of major depressive disorder patients.

PubMed

Zhang, Rufang; Yang, Deyu; Zhou, Chanjuan; Cheng, Ke; Liu, Zhao; Chen, Liang; Fang, Liang; Xie, Peng

2012-08-15

Western blot analysis is a commonly used technique for determining specific protein levels in clinical samples. For normalization of protein levels in Western blot, a suitable loading control is required. On account of its relatively high and constant expression, β-actin has been widely employed in Western blot of cell cultures and tissue extracts. However, β-actin's presence in human plasma and this protein's putative role as a plasma-based loading control for Western blot analysis remain unknown. In this study, an enzyme-linked immunosorbent assay was used to determine the concentration of β-actin in human plasma, which is 6.29±0.54 ng/ml. In addition, the linearity of β-actin immunostaining and loaded protein amount was evaluated by Western blot, and a fine linearity (R²=0.974±0.012) was observed. Furthermore, the expression of plasma β-actin in major depressive disorder subjects and healthy controls was compared. The data revealed no statistically significant difference between these two groups. Moreover, the total coefficient of variation for β-actin expression in the two groups was 9.2±1.2%. These findings demonstrate that β-actin is present in human plasma and may possibly be used as a suitable loading control for plasma-based Western blot analysis in major depressive disorder. Copyright © 2012 Elsevier Inc. All rights reserved.

F-actin clustering and cell dysmotility induced by the pathological W148R missense mutation of filamin B at the actin-binding domain.

PubMed

Zhao, Yongtong; Shapiro, Sandor S; Eto, Masumi

2016-01-01

Filamin B (FLNB) is a dimeric actin-binding protein that orchestrates the reorganization of the actin cytoskeleton. Congenital mutations of FLNB at the actin-binding domain (ABD) are known to cause abnormalities of skeletal development, such as atelosteogenesis types I and III and Larsen's syndrome, although the underlying mechanisms are poorly understood. Here, using fluorescence microscopy, we characterized the reorganization of the actin cytoskeleton in cells expressing each of six pathological FLNB mutants that have been linked to skeletal abnormalities. The subfractionation assay showed a greater accumulation of the FLNB ABD mutants W148R and E227K than the wild-type protein to the cytoskeleton. Ectopic expression of FLNB-W148R and, to a lesser extent, FLNB-E227K induced prominent F-actin accumulations and the consequent rearrangement of focal adhesions, myosin II, and septin filaments and results in a delayed directional migration of the cells. The W148R protein-induced cytoskeletal rearrangement was partially attenuated by the inhibition of myosin II, p21-activated protein kinase, or Rho-associated protein kinase. The expression of a single-head ABD fragment with the mutations partially mimicked the rearrangement induced by the dimer. The F-actin clustering through the interaction with the mutant FLNB ABD may limit the cytoskeletal reorganization, preventing normal skeletal development. Copyright © 2016 the American Physiological Society.

Inhibition of arsenic induced-rat liver injury by grape seed exact through suppression of NADPH oxidase and TGF-{beta}/Smad activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pan Xinjuan; Dai Yujie; Li Xing

2011-08-01

Chronic arsenic exposure induces oxidative damage to liver leading to liver fibrosis. We aimed to define the effect of grape seed extract (GSE), an antioxidant dietary supplement, on arsenic-induced liver injury. First, Male Sprague-Dawley rats were exposed to a low level of arsenic in drinking water (30 ppm) with or without GSE (100 mg/kg, every other day by oral gavage) for 12 months and the effect of GSE on arsenic-induced hepatotoxicity was examined. The results from this study revealed that GSE co-treatment significantly attenuated arsenic-induced low antioxidant defense, oxidative damage, proinflammatory cytokines and fibrogenic genes. Moreover, GSE reduced arsenic-stimulated Smad2/3more » phosphorylation and protein levels of NADPH oxidase subunits (Nox2, Nox4 and p47phox). Next, we explored the molecular mechanisms underlying GSE inhibition of arsenic toxicity using cultured rat hepatic stellate cells (HSCs). From the in vitro study, we found that GSE dose-dependently reduced arsenic-stimulated ROS production and NADPH oxidase activities. Both NADPH oxidases flavoprotein inhibitor DPI and Nox4 siRNA blocked arsenic-induced ROS production, whereas Nox4 overexpression suppressed the inhibitory effects of GSE on arsenic-induced ROS production and NADPH oxidase activities, as well as expression of TGF-{beta}1, type I procollagen (Coll-I) and {alpha}-smooth muscle actin ({alpha}-SMA) mRNA. We also observed that GSE dose-dependently inhibited TGF-{beta}1-induced transactivation of the TGF-{beta}-induced smad response element p3TP-Lux, and that forced expression of Smad3 attenuated the inhibitory effects of GSE on TGF-{beta}1-induced mRNA expression of Coll-I and {alpha}-SMA. Collectively, GSE could be a potential dietary therapeutic agent for arsenic-induced liver injury through suppression of NADPH oxidase and TGF-{beta}/Smad activation. - Research Highlights: > GSE attenuated arsenic-induced low antioxidant defense, oxidative damage, proinflammatory cytokines and fibrogenic genes. > GSE reduced arsenic-mediated Smad2/3 phosphorylation and NADPH oxidase subunits (Nox2, Nox4 and p47phox). > Beneficial effects of GSE on As-induced liver injury was via inhibition of NADPH oxidase and TGF-{beta}/Smad activation.« less

TGF-β1-Mediated Differentiation of Fibroblasts Is Associated with Increased Mitochondrial Content and Cellular Respiration

PubMed Central

Negmadjanov, Ulugbek; Godic, Zarko; Rizvi, Farhan; Emelyanova, Larisa; Ross, Gracious; Richards, John; Holmuhamedov, Ekhson L.; Jahangir, Arshad

2015-01-01

Objectivs Cytokine-dependent activation of fibroblasts to myofibroblasts, a key event in fibrosis, is accompanied by phenotypic changes with increased secretory and contractile properties dependent on increased energy utilization, yet changes in the energetic profile of these cells are not fully described. We hypothesize that the TGF-β1-mediated transformation of myofibroblasts is associated with an increase in mitochondrial content and function when compared to naive fibroblasts. Methods Cultured NIH/3T3 mouse fibroblasts treated with TGF-β1, a profibrotic cytokine, or vehicle were assessed for transformation to myofibroblasts (appearance of α-smooth muscle actin [α-SMA] stress fibers) and associated changes in mitochondrial content and functions using laser confocal microscopy, Seahorse respirometry, multi-well plate reader and biochemical protocols. Expression of mitochondrial-specific proteins was determined using western blotting, and the mitochondrial DNA quantified using Mitochondrial DNA isolation kit. Results Treatment with TGF-β1 (5 ng/mL) induced transformation of naive fibroblasts into myofibroblasts with a threefold increase in the expression of α-SMA (6.85 ± 0.27 RU) compared to cells not treated with TGF-β1 (2.52 ± 0.11 RU). TGF-β1 exposure increased the number of mitochondria in the cells, as monitored by membrane potential sensitive dye tetramethylrhodamine, and expression of mitochondria-specific proteins; voltage-dependent anion channels (0.54 ± 0.05 vs. 0.23 ± 0.05 RU) and adenine nucleotide transporter (0.61 ± 0.11 vs. 0.22 ± 0.05 RU), as well as mitochondrial DNA content (530 ± 12 μg DNA/106 cells vs. 307 ± 9 μg DNA/106 cells in control). TGF-β1 treatment was associated with an increase in mitochondrial function with a twofold increase in baseline oxygen consumption rate (2.25 ± 0.03 vs. 1.13 ± 0.1 nmol O2/min/106 cells) and FCCP-induced mitochondrial respiration (2.87 ± 0.03 vs. 1.46 ± 0.15 nmol O2/min/106 cells). Conclusions TGF-β1 induced differentiation of fibroblasts is accompanied by energetic remodeling of myofibroblasts with an increase in mitochondrial respiration and mitochondrial content. PMID:25849590

Histopathologic and immunohistochemical features of capsular tissue around failed Ahmed glaucoma valves.

PubMed

Mahale, Alka; Fikri, Fatma; Al Hati, Khitam; Al Shahwan, Sami; Al Jadaan, Ibrahim; Al Katan, Hind; Khandekar, Rajiv; Maktabi, Azza; Edward, Deepak P

2017-01-01

Impervious encapsulation around Ahmed glaucoma valve (AGV) results in surgical failure raising intraocular pressure (IOP). Dysregulation of extracellular matrix (ECM) molecules and cellular factors might contribute to increased hydraulic resistance to aqueous drainage. Therefore, we examined these molecules in failed AGV capsular tissue. Immunostaining for ECM molecules (collagen I, collagen III, decorin, lumican, chondroitin sulfate, aggrecan and keratan sulfate) and cellular factors (αSMA and TGFβ) was performed on excised capsules from failed AGVs and control tenon's tissue. Staining intensity of ECM molecules was assessed using Image J. Cellular factors were assessed based on positive cell counts. Histopathologically two distinct layers were visible in capsules. The inner layer (proximal to the AGV) showed significant decrease in most ECM molecules compared to outer layer. Furthermore, collagen III (p = 0.004), decorin (p = 0.02), lumican (p = 0.01) and chondroitin sulfate (p = 0.02) was significantly less in inner layer compared to tenon's tissue. Outer layer labelling however was similar to control tenon's for most ECM molecules. Significantly increased cellular expression of αSMA (p = 0.02) and TGFβ (p = 0.008) was detected within capsular tissue compared to controls. Our results suggest profibrotic activity indicated by increased αSMA and TGFβ expression and decreased expression of proteoglycan (decorin and lumican) and glycosaminoglycans (chondroitin sulfate). Additionally, we observed decreased collagen III which might reflect increased myofibroblast contractility when coupled with increased TGFβ and αSMA expression. Together these events lead to tissue dysfunction potentially resulting in hydraulic resistance that may affect aqueous flow through the capsular wall.

The atypical Rho GTPase RhoD is a regulator of actin cytoskeleton dynamics and directed cell migration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blom, Magdalena; Reis, Katarina; Heldin, Johan

RhoD belongs to the Rho GTPases, a protein family responsible for the regulation and organization of the actin cytoskeleton, and, consequently, many cellular processes like cell migration, cell division and vesicle trafficking. Here, we demonstrate that the actin cytoskeleton is dynamically regulated by increased or decreased protein levels of RhoD. Ectopic expression of RhoD has previously been shown to give an intertwined weave of actin filaments. We show that this RhoD-dependent effect is detected in several cell types and results in a less dynamic actin filament system. In contrast, RhoD depletion leads to increased actin filament-containing structures, such as corticalmore » actin, stress fibers and edge ruffles. Moreover, vital cellular functions such as cell migration and proliferation are defective when RhoD is silenced. Taken together, we present data suggesting that RhoD is an important component in the control of actin dynamics and directed cell migration. - Highlights: • Increased RhoD expression leads to loss of actin structures, e.g. stress fibers and gives rise to decreased actin dynamics. • RhoD knockdown induces various actin-containing structures such as edge ruffles, stress fibers and cortical actin, in a cell-type specific manner. • RhoD induces specific actin rearrangements depending on its subcellular localization. • RhoD knockdown has effects on cellular processes, such as directed cell migration and proliferation.« less

Dendrite architecture organized by transcriptional control of the F-actin nucleator Spire.

PubMed

Ferreira, Tiago; Ou, Yimiao; Li, Sally; Giniger, Edward; van Meyel, Donald J

2014-02-01

The architectures of dendritic trees are crucial for the wiring and function of neuronal circuits because they determine coverage of receptive territories, as well as the nature and strength of sensory or synaptic inputs. Here, we describe a cell-intrinsic pathway sculpting dendritic arborization (da) neurons in Drosophila that requires Longitudinals Lacking (Lola), a BTB/POZ transcription factor, and its control of the F-actin cytoskeleton through Spire (Spir), an actin nucleation protein. Loss of Lola from da neurons reduced the overall length of dendritic arbors, increased the expression of Spir, and produced inappropriate F-actin-rich dendrites at positions too near the cell soma. Selective removal of Lola from only class IV da neurons decreased the evasive responses of larvae to nociception. The increased Spir expression contributed to the abnormal F-actin-rich dendrites and the decreased nocifensive responses because both were suppressed by reduced dose of Spir. Thus, an important role of Lola is to limit expression of Spir to appropriate levels within da neurons. We found Spir to be expressed in dendritic arbors and to be important for their development. Removal of Spir from class IV da neurons reduced F-actin levels and total branch number, shifted the position of greatest branch density away from the cell soma, and compromised nocifensive behavior. We conclude that the Lola-Spir pathway is crucial for the spatial arrangement of branches within dendritic trees and for neural circuit function because it provides balanced control of the F-actin cytoskeleton.

Sinonasal haemangiopericytoma: histomorphology and differential diagnoses.

PubMed

Leow, Wei Qiang; Sng, Ivy

2015-04-01

A 39-year-old female presented with a fleshy nasal polyp occluding the left nasal cavity, associated with haemopurulent discharge. Computed Tomography (CT) and Magnetic Resonance Imaging (MRI) scans of the paranasal cavities revealed a large polypoid tumour arising from the left middle turbinate and obstructing the left maxillary sinus ostium. However, no bony or intracranial involvement was identified. A biopsy revealed a tumour with small blue round cell morphology. The tumour cells showed diffuse strong membranous CD99 positivity and patchy CD34 positivity. Ancillary cytogenetic tests for the EWSR1 and SS18/SYT gene translocations were negative. In view of the non-invasive nature of the tumour and the low cell proliferative index (Ki-67) of 5%, a medial maxillectomy resection was performed. The resection revealed additional areas with spindle-cell morphology and focal haemangiopericytic vasculature. The tumour continued to show immunoreactivity to CD99 and CD34, as well as Smooth Muscle Actin (SMA) and Muscle Specific Actin (MSA). The overall findings are in keeping with a sinonasal haemangiopericytoma. With clear surgical resection margins, the patient is on routine follow-up and is currently disease-free.

Rab8 Regulates the Actin-based Movement of MelanosomesV⃞

PubMed Central

Chabrillat, Marion L.; Wilhelm, Claire; Wasmeier, Christina; Sviderskaya, Elena V.; Louvard, Daniel; Coudrier, Evelyne

2005-01-01

Rab GTPases have been implicated in the regulation of specific microtubule- and actin-based motor proteins. We devised an in vitro motility assay reconstituting the movement of melanosomes on actin bundles in the presence of ATP to investigate the role of Rab proteins in the actin-dependent movement of melanosomes. Using this assay, we confirmed that Rab27 is required for the actin-dependent movement of melanosomes, and we showed that a second Rab protein, Rab8, also regulates this movement. Rab8 was partially associated with mature melanosomes. Expression of Rab8Q67L perturbed the cellular distribution and increased the frequency of microtubule-independent movement of melanosomes in vivo. Furthermore, anti-Rab8 antibodies decreased the number of melanosomes moving in vitro on actin bundles, whereas melanosomes isolated from cells expressing Rab8Q67L exhibited 70% more movements than wild-type melanosomes. Together, our observations suggest that Rab8 is involved in regulating the actin-dependent movement of melanosomes. PMID:15673612

HIV compromises integrity of the podocyte actin cytoskeleton through downregulation of the vitamin D receptor

PubMed Central

Chandel, Nirupama; Sharma, Bipin; Husain, Mohammad; Salhan, Divya; Singh, Tejinder; Rai, Partab; Mathieson, Peter W.; Saleem, Moin A.; Malhotra, Ashwani

2013-01-01

Alterations in the podocyte actin cytoskeleton have been implicated in the development of proteinuric kidney diseases. In the present study, we evaluated the effect of HIV on the podocyte actin cytoskeleton and the mechanism involved. We hypothesized that HIV may be compromising the actin cytoskeleton via downregulation of the vitamin D receptor (VDR) of conditionally immortalized differentiated human podocytes (CIDHPs). HIV-transduced podocytes (HIV/CIDHPs) not only displayed downregulation of VDR but also showed activation of the renin-angiotensin system (RAS) in the form of enhanced expression of renin and increased production of ANG II. Moreover, CIDHPs lacking VDR displayed enhanced ANG II production, and treatment of HIV/CIDHPs with EB1089 (vitamin D3; VD) attenuated ANG II production. HIV/CIDHPs as well as ANG II-treated CIDHPs exhibited enhanced expression of cathepsin (CTS) L. Additionally, losartan (an ANG II type I receptor blocker) inhibited both HIV- and ANG II-induced podocyte cathepsin L expression. Furthermore, VD downregulated HIV-induced podocyte CTSL expression. Both losartan and free radical scavengers attenuated HIV- and ANG II-induced podocyte reactive oxygen species (ROS) generation. HIV also led to cytosolic CTSL accumulation through enhancement of podocyte lysosomal membrane permeabilization; on the other hand, VD, losartan, and superoxide dismutase (SOD) attenuated HIV-induced enhanced podocyte cytosolic CTSL accumulation. Morphological evaluation of HIV/CIDHPs revealed sparse actin filaments and attenuated expression of dynamin. Interestingly, podocytes lacking CTSL displayed enhanced dynamin expression, and HIV/CIDHPs expressing CTSL exhibited downregulation of dynamin. These findings indicate that HIV-induced downregulation of podocyte VDR and associated RAS activation and cytosolic CTSL accumulation compromised the actin cytoskeleton. PMID:23467424

[Intervention effects of Dan-fang capsule in rats with hepatic fibrosis].

PubMed

Hu, X H; Wu, J; Lu, S

2017-09-19

Objective: To investigate the interventional effect of Dan-fang capsule on liver fibrosis in rats. Methods: Sixty one-week aged male healthy SD rats [weight (180±20) g] were randomly divided into normal control group (group A), hepatic fibrosis model group (group B), Fu-Fang-Bie-Jia-Ruan-Gan tablet group (group C), Dan-fang capsule groups at high, middle and low dose group (group D, E, F, respectively). Except for the normal control group, hepatic fibrosis was induced in other groups by intraperitoneal injection of porcine serum.Simultaneously, rats in Dan-fang capsule groups were administered by gavage with Dan-fang capsule at doses of 4.32, 2.16, 0.54 g/kg, respectively.Rats in Fu-Fang-Bie-Jia-Ruan-Gan tablet group were orally administered by gavage with Fu-Fang-Bie-Jia-Ruan-Gan tablet (0.54 g/kg) every day and the normal control group received saline alone.All rats were killed at the end of the 12th week. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and PⅢnp clia (PⅢNP) were measured in the groups.Pathology changes of hepatic tissue were evaluated by hematoxylin-eosin (HE) and Masson staining.The proteinic expressions of alpha-smooth muscle actin (α-SMA), collagen-Ⅰ (COL-Ⅰ) and collagen-Ⅲ (COL-Ⅲ) were observed with the method of immunohistochemistry.Analysis of variance was applied when data were compared among groups. Results: Compared with those in the group A, the levels of ALT, AST and PⅢNP in serum and the expressions of α-SMA, COL-Ⅰ and COL-Ⅲ in liver tissues were significantly higher in group B [(68.3±3.4) vs (51.5±6.3) U/L, (205±52) vs (135±24) U/L, (3.1±1.4) vs (1.6±0.6) μg/L and 0.35±0.02 vs 0.13±0.02, 0.37±0.02 vs 0.13±0.02, 0.43±0.13 vs 0.13±0.01, t =17.020, 71.053, 1.552, 0.214, 0.241, 0.292, all P <0.01], and the degree of liver fibrosis significantly increased in group B than that in group A. Compared with those in group B, the levels of ALT, AST, PⅢNP and the expressions of α-SMA, COL-Ⅰ, COL-Ⅲ were all significantly lower in group D, E and F ( t =-58.232--0.104, all P <0.01). The degree of liver fibrosis significantly reduced in group D, E and F than that in group B ( Z =3.82, 3.76, 3.90, all P <0.05). Conclusion: Dan-fang capsule has certain preventive effect on liver fibrosis that caused by porcine serum in rats.

Glutathione depletion triggers actin cytoskeleton changes via actin-binding proteins.

PubMed

Zepeta-Flores, Nahum; Valverde, Mahara; Lopez-Saavedra, Alejandro; Rojas, Emilio

2018-06-04

The importance of glutathione (GSH) in alternative cellular roles to the canonically proposed, were analyzed in a model unable to synthesize GSH. Gene expression analysis shows that the regulation of the actin cytoskeleton pathway is strongly impacted by the absence of GSH. To test this hypothesis, we evaluate the effect of GSH depletion via buthionine sulfoximine (5 and 12.5 mM) in human neuroblastoma MSN cells. In the present study, 70% of GSH reduction did not induce reactive oxygen species, lipoperoxidation, or cytotoxicity, which enabled us to evaluate the effect of glutathione in the absence of oxidative stress. The cells with decreasing GSH levels acquired morphology changes that depended on the actin cytoskeleton and not on tubulin. We evaluated the expression of three actin-binding proteins: thymosin β4, profilin and gelsolin, showing a reduced expression, both at gene and protein levels at 24 hours of treatment; however, this suppression disappears after 48 hours of treatment. These changes were sufficient to trigger the co-localization of the three proteins towards cytoplasmic projections. Our data confirm that a decrease in GSH in the absence of oxidative stress can transiently inhibit the actin binding proteins and that this stimulus is sufficient to induce changes in cellular morphology via the actin cytoskeleton.

Plant vegetative and animal cytoplasmic actins share functional competence for spatial development with protists.

PubMed

Kandasamy, Muthugapatti K; McKinney, Elizabeth C; Roy, Eileen; Meagher, Richard B

2012-05-01

Actin is an essential multifunctional protein encoded by two distinct ancient classes of genes in animals (cytoplasmic and muscle) and plants (vegetative and reproductive). The prevailing view is that each class of actin variants is functionally distinct. However, we propose that the vegetative plant and cytoplasmic animal variants have conserved functional competence for spatial development inherited from an ancestral protist actin sequence. To test this idea, we ectopically expressed animal and protist actins in Arabidopsis thaliana double vegetative actin mutants that are dramatically altered in cell and organ morphologies. We found that expression of cytoplasmic actins from humans and even a highly divergent invertebrate Ciona intestinalis qualitatively and quantitatively suppressed the root cell polarity and organ defects of act8 act7 mutants and moderately suppressed the root-hairless phenotype of act2 act8 mutants. By contrast, human muscle actins were unable to support prominently any aspect of plant development. Furthermore, actins from three protists representing Choanozoa, Archamoeba, and green algae efficiently suppressed all the phenotypes of both the plant mutants. Remarkably, these data imply that actin's competence to carry out a complex suite of processes essential for multicellular development was already fully developed in single-celled protists and evolved nonprogressively from protists to plants and animals.

Enigma interacts with adaptor protein with PH and SH2 domains to control insulin-induced actin cytoskeleton remodeling and glucose transporter 4 translocation.

PubMed

Barrès, Romain; Grémeaux, Thierry; Gual, Philippe; Gonzalez, Teresa; Gugenheim, Jean; Tran, Albert; Le Marchand-Brustel, Yannick; Tanti, Jean-François

2006-11-01

APS (adaptor protein with PH and SH2 domains) initiates a phosphatidylinositol 3-kinase-independent pathway involved in insulin-stimulated glucose transport. We recently identified Enigma, a PDZ and LIM domain-containing protein, as a partner of APS and showed that APS-Enigma complex plays a critical role in actin cytoskeleton organization in fibroblastic cells. Because actin rearrangement is important for insulin-induced glucose transporter 4 (Glut 4) translocation, we studied the potential involvement of Enigma in insulin-induced glucose transport in 3T3-L1 adipocytes. Enigma mRNA was expressed in differentiated adipocytes and APS and Enigma were colocalized with cortical actin. Expression of an APS mutant unable to bind Enigma increased the insulin-induced Glut 4 translocation to the plasma membrane. By contrast, overexpression of Enigma inhibited insulin-stimulated glucose transport and Glut 4 translocation without alterations in proximal insulin signaling. This inhibitory effect was prevented with the deletion of the LIM domains of Enigma. Using time-lapse fluorescent microscopy of green fluorescent protein-actin, we demonstrated that the overexpression of Enigma altered insulin-induced actin rearrangements, whereas the expression of Enigma without its LIM domains was without effect. A physiological link between increased expression of Enigma and an alteration in insulin-induced glucose uptake was suggested by the increase in Enigma mRNA expression in adipose tissue of diabetic obese patients. Taken together, these data strongly suggest that the interaction between APS and Enigma is involved in insulin-induced Glut 4 translocation by regulating cortical actin remodeling and raise the possibility that modification of APS/Enigma ratio could participate in the alteration of insulin-induced glucose uptake in adipose tissue.

{beta}-Catenin regulates airway smooth muscle contraction.

PubMed

Jansen, Sepp R; Van Ziel, Anna M; Baarsma, Hoeke A; Gosens, Reinoud

2010-08-01

beta-Catenin is an 88-kDa member of the armadillo family of proteins that is associated with the cadherin-catenin complex in the plasma membrane. This complex interacts dynamically with the actin cytoskeleton to stabilize adherens junctions, which play a central role in force transmission by smooth muscle cells. Therefore, in the present study, we hypothesized a role for beta-catenin in the regulation of smooth muscle force production. beta-Catenin colocalized with smooth muscle alpha-actin (sm-alpha-actin) and N-cadherin in plasma membrane fractions and coimmunoprecipitated with sm-alpha-actin and N-cadherin in lysates of bovine tracheal smooth muscle (BTSM) strips. Moreover, immunocytochemistry of cultured BTSM cells revealed clear and specific colocalization of sm-alpha-actin and beta-catenin at the sites of cell-cell contact. Treatment of BTSM strips with the pharmacological beta-catenin/T cell factor-4 (TCF4) inhibitor PKF115-584 (100 nM) reduced beta-catenin expression in BTSM whole tissue lysates and in plasma membrane fractions and reduced maximal KCl- and methacholine-induced force production. These changes in force production were not accompanied by changes in the expression of sm-alpha-actin or sm-myosin heavy chain (MHC). Likewise, small interfering RNA (siRNA) knockdown of beta-catenin in BTSM strips reduced beta-catenin expression and attenuated maximal KCl- and methacholine-induced contractions without affecting sm-alpha-actin or sm-MHC expression. Conversely, pharmacological (SB-216763, LiCl) or insulin-induced inhibition of glycogen synthase kinase-3 (GSK-3) enhanced the expression of beta-catenin and augmented maximal KCl- and methacholine-induced contractions. We conclude that beta-catenin is a plasma membrane-associated protein in airway smooth muscle that regulates active tension development, presumably by stabilizing cell-cell contacts and thereby supporting force transmission between neighboring cells.

G-actin sequestering protein thymosin-β4 regulates the activity of myocardin-related transcription factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morita, Tsuyoshi, E-mail: tsuyo@nbiochem.med.osaka-u.ac.jp; Hayashi, Ken’ichiro

2013-08-02

Highlights: •Tβ4 competed with MRTF-A for G-actin binding. •Tβ4 activated the MRTF–SRF signaling pathway. •Tβ4 increased the endogenous expression of SRF-dependent genes. -- Abstract: Myocardin-related transcription factors (MRTFs) are robust coactivators of serum response factor (SRF). MRTFs contain three copies of the RPEL motif at their N-terminus, and they bind to monomeric globular actin (G-actin). Previous studies illustrate that G-actin binding inhibits MRTF activity by preventing the MRTFs nuclear accumulation. In the living cells, the majority of G-actin is sequestered by G-actin binding proteins that prevent spontaneous actin polymerization. Here, we demonstrate that the most abundant G-actin sequestering protein thymosin-β4more » (Tβ4) was involved in the regulation of subcellular localization and activity of MRTF-A. Tβ4 competed with MRTF-A for G-actin binding; thus, interfering with G-actin–MRTF-A complex formation. Tβ4 overexpression induced the MRTF-A nuclear accumulation and activation of MRTF–SRF signaling. The activation rate of MRTF-A by the Tβ4 mutant L17A, whose affinity for G-actin is very low, was lower than that by wild-type Tβ4. In contrast, the β-actin mutant 3DA, which has a lower affinity for Tβ4, more effectively suppressed MRTF-A activity than wild-type β-actin. Furthermore, ectopic Tβ4 increased the endogenous expression of SRF-dependent actin cytoskeletal genes. Thus, Tβ4 is an important MRTF regulator that controls the G-actin–MRTFs interaction.« less

Urinary podocalyxin, the novel biomarker for detecting early renal change in obesity.

PubMed

Suwanpen, Chayanut; Nouanthong, Phonethipsavanh; Jaruvongvanich, Veeravich; Pongpirul, Krit; Pongpirul, Wannarat Amornnimit; Leelahavanichkul, Asada; Kanjanabuch, Talerngsak

2016-02-01

The prevalence of obesity is increasing during the past decade along with obesity-related glomerulopathy (ORG), glomeruli injury due to the obesity. The major pathogenesis of ORG is the shedding of podocytes from the glomerular cell barrier into urine. Podocalyxin (PCX), a main surface antigen of podocyte, correlates well with glomerulosclerosis progression and glomerular injury severity, and might be a potential biomarker for early renal alteration in obesity. In addition, vascular endothelial growth factor (VEGF) and alpha-smooth muscle actin (α-SMA) also play a role in promoting glomerulosclerosis. The aim of this study was to explore whether obese subjects without other diseases excrete more PCX-positive (PCX+) cells than non-obese individuals, in comparison with urine protein-creatinine ratio (UPCR) and glomerular filtration rate (GFR) as traditional renal markers. Moreover, the effect of body mass index (BMI) on urinary VEGF, PCX or α-SMA positive cells was also investigated. Forty-eight obese and 13 non-obese adults were included. Exfoliated cells from fresh first void morning urine were harvested, stained with PCX, VEGF, and α-SMA antibody, and quantified by flow cytometry. Correlation between interested urinary biomarkers (cells positive for PCX, VEGF plus PCX and α-SMA), UPCR and GFR with BMI and metabolic risk factors were analyzed. Obese patients had significantly higher PCX+ cells than non-obese [0.62 (0.00-13.13) vs. 0.15 (0.00-0.72) cells/ml × mg cr, p < 0.05]. There was no significant difference in GFR and UPCR between the groups. Of interest, BMI demonstrated a correlation with PCX+ cells (r = 0.343, p = 0.008) and cells positive for PCX plus VEGF (r = 0.374, p = 0.004). Obese subjects without other diseases and with normal UPCR and GFR showed evidence of renal alteration through the detection of a higher number of PCX+ cells. Increasing BMI also resulted in higher number of PCX+ cells.

Molecular Characterization of abLIM, a Novel Actin-binding and Double Zinc Finger Protein

PubMed Central

Roof, Dorothy J.; Hayes, Annmarie; Adamian, Michael; Chishti, Athar H.; Li, Tiansen

1997-01-01

Molecules that couple the actin-based cytoskeleton to intracellular signaling pathways are central to the processes of cellular morphogenesis and differentiation. We have characterized a novel protein, the actin-binding LIM (abLIM) protein, which could mediate such interactions between actin filaments and cytoplasmic targets. abLIM protein consists of a COOH-terminal cytoskeletal domain that is fused to an NH2-terminal domain consisting of four double zinc finger motifs. The cytoskeletal domain is ∼50% identical to erythrocyte dematin, an actin-bundling protein of the red cell membrane skeleton, while the zinc finger domains conform to the LIM motif consensus sequence. In vitro expression studies demonstrate that abLIM protein can bind to F-actin through the dematin-like domain. Transcripts corresponding to three distinct isoforms have a widespread tissue distribution. However, a polypeptide corresponding to the full-length isoform is found exclusively in the retina and is enriched in biochemical extracts of retinal rod inner segments. abLIM protein also undergoes extensive phosphorylation in light-adapted retinas in vivo, and its developmental expression in the retina coincides with the elaboration of photoreceptor inner and outer segments. Based on the composite primary structure of abLIM protein, actin-binding capacity, potential regulation via phosphorylation, and isoform expression pattern, we speculate that abLIM may play a general role in bridging the actin-based cytoskeleton with an array of potential LIM protein-binding partners. The developmental time course of abLIM expression in the retina suggests that the retina-specific isoform may have a specialized role in the development or elaboration of photoreceptor inner and outer segments. PMID:9245787

Assays for the Identification and Prioritization of Drug Candidates for Spinal Muscular Atrophy

PubMed Central

Cherry, Jonathan J.; Kobayashi, Dione T.; Lynes, Maureen M.; Naryshkin, Nikolai N.; Tiziano, Francesco Danilo; Zaworski, Phillip G.; Rubin, Lee L.

2014-01-01

Abstract Spinal muscular atrophy (SMA) is an autosomal recessive genetic disorder resulting in degeneration of α-motor neurons of the anterior horn and proximal muscle weakness. It is the leading cause of genetic mortality in children younger than 2 years. It affects ∼1 in 11,000 live births. In 95% of cases, SMA is caused by homozygous deletion of the SMN1 gene. In addition, all patients possess at least one copy of an almost identical gene called SMN2. A single point mutation in exon 7 of the SMN2 gene results in the production of low levels of full-length survival of motor neuron (SMN) protein at amounts insufficient to compensate for the loss of the SMN1 gene. Although no drug treatments are available for SMA, a number of drug discovery and development programs are ongoing, with several currently in clinical trials. This review describes the assays used to identify candidate drugs for SMA that modulate SMN2 gene expression by various means. Specifically, it discusses the use of high-throughput screening to identify candidate molecules from primary screens, as well as the technical aspects of a number of widely used secondary assays to assess SMN messenger ribonucleic acid (mRNA) and protein expression, localization, and function. Finally, it describes the process of iterative drug optimization utilized during preclinical SMA drug development to identify clinical candidates for testing in human clinical trials. PMID:25147906

Paratesticular dedifferentiated liposarcoma with leiomyosarcomatous differentiation: a case report with a review of literature.

PubMed

Hatanaka, Kazuhito; Yoshioka, Takako; Tasaki, Takashi; Tanimoto, Akihide

2013-08-23

Paratesticular liposarcoma is a rare neoplasm, described in single case studies or components of larger studies, as histologically well-differentiated liposarcoma (WDL) and dedifferentiated liposarcoma (DL). However, leiomyosarcomatous differentiation is an extremely rare occurrence in WDL and DL. We report a case of leiomyosarcomatous differentiation in a 77-year-old man. The patient presented with a painless right scrotal mass. Magnetic resonance imaging showed a large mass along the right spermatic cord. The resected mass, measuring 17.5 × 12 × 5 cm, was composed of a high-grade pleomorphic undifferentiated sarcomatous component with necrosis. Atypical smooth muscle differentiation was also detected. Additional tumor sampling revealed the presence of a WDL component. Immunohistochemical analysis of the pleomorphic sarcomatous component showed positive staining for MDM2 and CDK4, and negative staining for alpha smooth muscle actin (αSMA) and desmin. The smooth muscle component was positive for αSMA and desmin, and negative for MDM2 and CDK4. Extension from primary retroperitoneal sarcoma was not proved. We diagnosed of DL with leiomyosarcomatous differentiation. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1484291498104021.

Paratesticular dedifferentiated liposarcoma with leiomyosarcomatous differentiation: a case report with a review of literature

PubMed Central

2013-01-01

Abstract Paratesticular liposarcoma is a rare neoplasm, described in single case studies or components of larger studies, as histologically well-differentiated liposarcoma (WDL) and dedifferentiated liposarcoma (DL). However, leiomyosarcomatous differentiation is an extremely rare occurrence in WDL and DL. We report a case of leiomyosarcomatous differentiation in a 77-year-old man. The patient presented with a painless right scrotal mass. Magnetic resonance imaging showed a large mass along the right spermatic cord. The resected mass, measuring 17.5 × 12 × 5 cm, was composed of a high-grade pleomorphic undifferentiated sarcomatous component with necrosis. Atypical smooth muscle differentiation was also detected. Additional tumor sampling revealed the presence of a WDL component. Immunohistochemical analysis of the pleomorphic sarcomatous component showed positive staining for MDM2 and CDK4, and negative staining for alpha smooth muscle actin (αSMA) and desmin. The smooth muscle component was positive for αSMA and desmin, and negative for MDM2 and CDK4. Extension from primary retroperitoneal sarcoma was not proved. We diagnosed of DL with leiomyosarcomatous differentiation. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1484291498104021. PMID:23971887

Coordination of Actin- and Microtubule-Based Cytoskeletons Supports Transport of Spermatids and Residual Bodies/Phagosomes During Spermatogenesis in the Rat Testis

PubMed Central

Tang, Elizabeth I.; Lee, Will M.

2016-01-01

Germ cell transport across the seminiferous epithelium during spermatogenesis requires the intricate coordination of cell junctions, signaling proteins, and both actin- and microtubule (MT)-based cytoskeletons. Although the involvement of cytoskeletons in germ cell transport has been suggested, the precise mechanism(s) remains elusive. Based on growing evidence that actin and MT interactions underlie fundamental cellular processes, such as cell motility, it is unlikely that actin- and MT-based cytoskeletons work independently to regulate germ cell transport in the testis. Using rats treated with adjudin, a potential male contraceptive that disrupts spermatid adhesion and transport in the testis, as a study model, we show herein that actin- and MT-based cytoskeletons are both necessary for transport of spermatids and residual bodies/phagosomes across the seminiferous epithelium in adult rat testes. Analysis of intratubular expression of F-actin and tubulin revealed disruption of both actin and MT networks, concomitant with misdirected spermatids and phagosomes in rats treated with adjudin. Actin regulatory proteins, epidermal growth factor receptor pathway substrate 8 and actin-related protein 3, were mislocalized and down-regulated at the actin-rich anchoring junction between germ and Sertoli cells (apical ectoplasmic specialization) after adjudin treatment. Nonreceptor tyrosine kinase p-FAK-Tyr407, known to regulate F-actin nucleation via actin-related protein 3, was also mislocalized and down-regulated at the apical ectoplasmic specialization, corroborating the observation of actin cytoskeleton disruption. Additionally, spatiotemporal expression of MT regulatory protein end-binding protein 1, shown to be involved in MT-actin cross talk herein, was also disrupted after adjudin treatment. In summary, spermatid/phagosome transport across the epithelium during spermatogenesis requires the coordination between actin- and MT-based cytoskeletons. PMID:26894662

Myosin II Motor Activity in the Lateral Amygdala Is Required for Fear Memory Consolidation

ERIC Educational Resources Information Center

Gavin, Cristin F.; Rubio, Maria D.; Young, Erica; Miller, Courtney; Rumbaugh, Gavin

2012-01-01

Learning induces dynamic changes to the actin cytoskeleton that are required to support memory formation. However, the molecular mechanisms that mediate filamentous actin (F-actin) dynamics during learning and memory are poorly understood. Myosin II motors are highly expressed in actin-rich growth structures including dendritic spines, and we have…

Identification of specific gravity sensitive signal transduction pathways in human A431 carcinoma cells

NASA Astrophysics Data System (ADS)

Rijken, P. J.; de Groot, R. P.; Kruijer, W.; de Laat, S. W.; Verkleij, A. J.; Boonstra, J.

Epidermal growth factor (EGF) activates a well characterized signal transduction cascade in human A431 epidermoid carcinoma cells. The influence of gravity on EGF-induced EGF-receptor clustering and early gene expression as well as on actin polymerization and actin organization have been investigated. Different signalling pathways induced by the agents TPA, forskolin and A23187 that activate gene expression were tested for sensitivity to gravity. EGF-induced c-fos and c-jun expression were decreased in microgravity. However, constitutive β-2 microglobulin expression remained unaltered. Under simulated weightlessness conditions EGF- and TPA-induced c-fos expression was decreased, while forskolin- and A23187-induced c-fos expression was independent of the gravity conditions. These results suggest that gravity affects specific signalling pathways. Preliminary results indicate that EGF-induced EGF-receptor clustering remained unaltered irrespective of the gravity conditions. Furthermore, the relative filamentous actin content of steady state A431 cells was enhanced under microgravity conditions and actin filament organization was altered. Under simulated weightlessness actin filament organization in steady state cells as well as in EGF-treated cells was altered as compared to the 1 G reference experiment. Interestingly the microtubule and keratin organization in untreated cells showed no difference with the normal gravity samples. This indicates that gravity may affect specific components of the signal transduction circuitry.

p53 AND MDM2 PROTEIN EXPRESSION IN ACTINIC CHEILITIS

PubMed Central

de Freitas, Maria da Conceição Andrade; Ramalho, Luciana Maria Pedreira; Xavier, Flávia Caló Aquino; Moreira, André Luis Gomes; Reis, Sílvia Regina Almeida

2008-01-01

Actinic cheilitis is a potentially malignant lip lesion caused by excessive and prolonged exposure to ultraviolet radiation, which can lead to histomorphological alterations indicative of abnormal cell differentiation. In this pathology, varying degrees of epithelial dysplasia may be found. There are few published studies regarding the p53 and MDM2 proteins in actinic cheilitis. Fifty-eight cases diagnosed with actinic cheilitis were histologically evaluated using Banóczy and Csiba (1976) parameters, and were subjected to immunohistochemical analysis using the streptavidin-biotin method in order to assess p53 and MDM2 protein expression. All studied cases expressed p53 proteins in basal and suprabasal layers. In the basal layer, the nuclei testing positive for p53 were stained intensely, while in the suprabasal layer, cells with slightly stained nuclei were predominant. All cases also tested positive for the MDM2 protein, but with varying degrees of nuclear expression and a predominance of slightly stained cells. A statistically significant correlation between the percentage of p53 and MDM2-positive cells was established, regardless of the degree of epithelial dysplasia. The expression of p53 and MDM2 proteins in actinic cheilitis can be an important indicator in lip carcinogenesis, regardless of the degree of epithelial dysplasia. PMID:19082401

p53 and MDM2 protein expression in actinic cheilitis.

PubMed

de Freitas, Maria da Conceição Andrade; Ramalho, Luciana Maria Pedreira; Xavier, Flávia Caló Aquino; Moreira, André Luis Gomes; Reis, Sílvia Regina Almeida

2008-01-01

Actinic cheilitis is a potentially malignant lip lesion caused by excessive and prolonged exposure to ultraviolet radiation, which can lead to histomorphological alterations indicative of abnormal cell differentiation. In this pathology, varying degrees of epithelial dysplasia may be found. There are few published studies regarding the p53 and MDM2 proteins in actinic cheilitis. Fifty-eight cases diagnosed with actinic cheilitis were histologically evaluated using Banóczy and Csiba (1976) parameters, and were subjected to immunohistochemical analysis using the streptavidin-biotin method in order to assess p53 and MDM2 protein expression. All studied cases expressed p53 proteins in basal and suprabasal layers. In the basal layer, the nuclei testing positive for p53 were stained intensely, while in the suprabasal layer, cells with slightly stained nuclei were predominant. All cases also tested positive for the MDM2 protein, but with varying degrees of nuclear expression and a predominance of slightly stained cells. A statistically significant correlation between the percentage of p53 and MDM2-positive cells was established, regardless of the degree of epithelial dysplasia. The expression of p53 and MDM2 proteins in actinic cheilitis can be an important indicator in lip carcinogenesis, regardless of the degree of epithelial dysplasia.

A baculovirus (Bombyx mori nuclear polyhedrosis virus) repeat element functions as a powerful constitutive enhancer in transfected insect cells.

PubMed

Lu, M; Farrell, P J; Johnson, R; Iatrou, K

1997-12-05

It has been previously reported that baculovirus homologous regions, the regions of baculovirus genomes that contain the origins of DNA replication, can augment the expression of a small number of baculovirus genes in vitro. We are now reporting that a region of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) containing the homologous region 3 (HR3) acts as an enhancer for the promoter of a nonviral gene, the cytoplasmic actin gene of the silkmoth B. mori. Incorporation of the HR3 sequences of BmNPV into an actin promoter-based expression cassette results in an augmentation of transgene expression in transfected cells by two orders of magnitude relative to the control recombinant expression cassette. This increase is due to a corresponding increase in the rate of transcription from the actin promoter and not to replication of the expression cassette and occurs only when the HR3 element is linked to the expression cassette in cis. A comparable degree of enhancement in the activity of the silkworm actin promoter occurs also in heterologous lepidopteran cells. Concomitant supplementation of transfected cells with the BmIE1 trans-activator, which was previously shown to be capable of functioning in vitro as a transcriptional co-activator of the cytoplasmic actin gene promoter, results in more than a 1,000-fold increase in the level of expression of recombinant proteins placed under the control of the actin gene promoter. These findings provide the foundation for the development of a nonlytic insect cell expression system for continuous high-level expression of recombinant proteins. Such a system should provide levels of expression of recombinant proteins comparable to those obtained from baculovirus expression systems and should also have the additional advantage of continuous production in a cellular environment that, in contrast to that generated by a baculovirus infection, supports continuously proper posttranslational modifications of recombinant proteins and the capability of expression of proteins from genomic as well as cDNA sequences.

Lenticular cytoprotection, part 2: link between glycogen synthase kinase-3β, epithelial to mesenchymal transition, and mitochondrial depolarization.

PubMed

Neelam, Sudha; Brooks, Morgan M; Cammarata, Patrick R

2014-01-01

The inhibition of GSK-3β blocks mitochondrial membrane permeability transition (mMPT) for HLE-B3 cells in atmospheric oxygen. GSK-3β, as part of a multifactorial complex, also regulates nuclear levels of β-catenin, a known coordinator of cell survival and adhesion. The purpose of these studies was to demonstrate a novel, but likely disadvantageous, link between β-catenin's influence on the expression of the pro-survival protein, vascular endothelial growth factor (VEGF), resulting in enhanced lens epithelial cell mitochondrial protection against depolarization and nuclear β-catenin as an inducer of epithelial to mesenchymal transition (EMT). Virally transformed human lens epithelial cells (HLE-B3) were treated with SB216763, a specific inhibitor of GSK-3β catalytic activity and XAV939, a specific β-catenin inhibitor that bars the translocation of β-catenin from cytoplasm to the nucleus. Western blot analysis was employed to detect the levels of cytoplasmic and nuclear β-catenin and phospho-β-catenin, pBcl-2 and the EMT proteins, α-smooth muscle actin (α-SMA), and fibronectin. ELISA was used to measure the levels of VEGF in cell culture supernatants. JC-1 analysis was performed to analyze the influence of either SB216763 or XAV939 on mitochondrial depolarization. Cultured lens epithelial cells maintained in hypoxia (1% oxygen) and subsequently reintroduced into atmospheric oxygen and treated with the GSK-3β inhibitor SB216763 illustrated a marked inhibition of phosphorylation of glycogen synthase (downstream substrate of GSK-3β) and significant increase in nuclear translocation of β-catenin. The augmented nuclear β-catenin levels positively correlated with increased expression of α-SMA and fibronectin, both marker proteins indicative of EMT. The enhanced nuclear β-catenin activity also elicited increased VEGF and pBcl-2 expression, resulting in increased resistance to mitochondrial depolarization. Treatment of the cells with the β-catenin inhibitor XAV939 resulted in decreased expression of nuclear β-catenin, VEGF levels, pBcl-2, and EMT proteins, as well as increased mitochondrial depolarization. The data support a model whereby the onset of epithelial to mesenchymal transition may circuitously benefit from the enhanced synthesis of VEGF by setting up a potentially harmful situation whereby the resulting mesenchymal cell population may be more resistant to mitochondrial depolarization than the lens epithelial cell population from which it originated. These findings support the potential therapeutic relevance of developing strategies to undermine the progression of normal cells to mesenchymal transition without subverting cell viability.

CD34(+) fibrocytes in normal cervical stroma, cervical intraepithelial neoplasia III, and invasive squamous cell carcinoma of the cervix uteri.

PubMed

Barth, Peter J; Ramaswamy, Annette; Moll, Roland

2002-12-01

CD34(+) fibrocytes are widely distributed in normal connective tissues but have been reported to be absent within the stroma associated with invasive carcinomas. In the present study we investigated the presence and distribution of CD34(+) fibrocytes and alpha-smooth muscle actin (alpha-SMA) positive myofibroblasts in cervical intraepithelial neoplasia III (CIN III; n=8), invasive carcinoma of the cervix ( n=18) and adjacent normal cervical stroma. Normal cervical stroma and the stroma adjacent to CIN III disclosed a dense network of CD34(+) fibrocytes, whereas the stroma of invasive carcinoma was virtually free of this cell population. Early stromal invasion by squamous carcinoma was characterized by a focal loss of CD34(+) fibrocytes. alpha-SMA-positive myofibroblasts were not seen in the normal cervical stroma but occurred in six of eight cases of CIN III adjacent to the atypical epithelium. The stroma of invasive carcinoma was made up of large amounts of haphazardly arranged alpha-SMA-positive myofibroblasts. In the setting of the present study, a loss of CD34(+) fibrocytes was specific for stromal alterations associated with invasive carcinoma and proved to be a sensitive tool in detecting small foci of stromal invasion. Therefore, detection of a loss of CD34(+) fibrocytes may constitute an adjunctive tool in detecting (1) early stromal invasion and (2) invasive carcinoma in small biopsy specimens. Moreover, the present study shows that CD34(+) fibrocytes and myofibroblasts play an important role in stromal remodeling associated with invasive squamous cell carcinoma of the cervix.

Nucleation promoting factors regulate the expression and localization of Arp2/3 complex during meiosis of mouse oocytes.

PubMed

Liu, Jun; Wang, Qiao-Chu; Wang, Fei; Duan, Xing; Dai, Xiao-Xin; Wang, Teng; Liu, Hong-Lin; Cui, Xiang-Shun; Kim, Nam-Hyung; Sun, Shao-Chen

2012-01-01

The actin nucleation factor Arp2/3 complex is a main regulator of actin assembly and is involved in multiple processes like cell migration and adhesion, endocytosis, and the establishment of cell polarity in mitosis. Our previous work showed that the Arp2/3 complex was involved in the actin-mediated mammalian oocyte asymmetric division. However, the regulatory mechanisms and signaling pathway of Arp2/3 complex in meiosis is still unclear. In the present work, we identified that the nucleation promoting factors (NPFs) JMY and WAVE2 were necessary for the expression and localization of Arp2/3 complex in mouse oocytes. RNAi of both caused the degradation of actin cap intensity, indicating the roles of NPFs in the formation of actin cap. Moreover, JMY and WAVE2 RNAi decreased the expression of ARP2, a key component of Arp2/3 complex. However, knock down of Arp2/3 complex by Arpc2 and Arpc3 siRNA microinjection did not affect the expression and localization of JMY and WAVE2. Our results indicate that the NPFs, JMY and WAVE2, are upstream regulators of Arp2/3 complex in mammalian oocyte asymmetric division.

Connective tissue growth factor stimulates the proliferation, migration and differentiation of lung fibroblasts during paraquat-induced pulmonary fibrosis

PubMed Central

YANG, ZHIZHOU; SUN, ZHAORUI; LIU, HONGMEI; REN, YI; SHAO, DANBING; ZHANG, WEI; LIN, JINFENG; WOLFRAM, JOY; WANG, FENG; NIE, SHINAN

2015-01-01

It is well established that paraquat (PQ) poisoning can cause severe lung injury during the early stages of exposure, finally leading to irreversible pulmonary fibrosis. Connective tissue growth factor (CTGF) is an essential growth factor that is involved in tissue repair and pulmonary fibrogenesis. In the present study, the role of CTGF was examined in a rat model of pulmonary fibrosis induced by PQ poisoning. Histological examination revealed interstitial edema and extensive cellular thickening of interalveolar septa at the early stages of poisoning. At 2 weeks after PQ administration, lung tissue sections exhibited a marked thickening of the alveolar walls with an accumulation of interstitial cells with a fibroblastic appearance. Masson’s trichrome staining revealed a patchy distribution of collagen deposition, indicating pulmonary fibrogenesis. Western blot analysis and immunohistochemical staining of tissue samples demonstrated that CTGF expression was significantly upregulated in the PQ-treated group. Similarly, PQ treatment of MRC-5 human lung fibroblast cells caused an increase in CTGF in a dose-dependent manner. Furthermore, the addition of CTGF to MRC-5 cells triggered cellular proliferation and migration. In addition, CTGF induced the differentiation of fibroblasts to myofibroblasts, as was evident from increased expression of α-smooth muscle actin (α-SMA) and collagen. These findings demonstrate that PQ causes increased CTGF expression, which triggers proliferation, migration and differentiation of lung fibroblasts. Therefore, CTGF may be important in PQ-induced pulmonary fibrogenesis, rendering this growth factor a potential pharmacological target for reducing lung injury. PMID:25815693

Identification of Histological Patterns in Clinically Affected and Unaffected Palm Regions in Dupuytren's Disease

PubMed Central

Alfonso-Rodríguez, Camilo-Andrés; Garzón, Ingrid; Garrido-Gómez, Juan; Oliveira, Ana-Celeste-Ximenes; Martín-Piedra, Miguel-Ángel; Scionti, Giuseppe; Carriel, Víctor; Hernández-Cortés, Pedro; Campos, Antonio; Alaminos, Miguel

2014-01-01

Dupuytren's disease is a fibro-proliferative disease characterized by a disorder of the extracellular matrix (ECM) and high myofibroblast proliferation. However, studies failed to determine if the whole palm fascia is affected by the disease. The objective of this study was to analyze several components of the extracellular matrix of three types of tissues—Dupuytren's diseased contracture cords (DDC), palmar fascia clinically unaffected by Dupuytren's disease contracture (NPF), and normal forehand fascia (NFF). Histological analysis, quantification of cells recultured from each type of tissue, mRNA microarrays and immunohistochemistry for smooth muscle actin (SMA), fibrillar ECM components and non-fibrillar ECM components were carried out. The results showed that DDC samples had abundant fibrosis with reticular fibers and few elastic fibers, high cell proliferation and myofibroblasts, laminin and glycoproteins, whereas NFF did not show any of these findings. Interestingly, NPF tissues had more cells showing myofibroblasts differentiation and more collagen and reticular fibers, laminin and glycoproteins than NFF, although at lower level than DDC, with similar elastic fibers than DDC. Immunohistochemical expression of decorin was high in DDC, whereas versican was highly expressed NFF, with no differences for aggrecan. Cluster analysis revealed that the global expression profile of NPF was very similar to DDC, and reculturing methods showed that cells corresponding to DDC tissues proliferated more actively than NPF, and NPF more actively than NFF. All these results suggest that NPF tissues may be affected, and that a modification of the therapeutic approach used for the treatment of Dupuytren's disease should be considered. PMID:25379672

Identification of histological patterns in clinically affected and unaffected palm regions in dupuytren's disease.

PubMed

Alfonso-Rodríguez, Camilo-Andrés; Garzón, Ingrid; Garrido-Gómez, Juan; Oliveira, Ana-Celeste-Ximenes; Martín-Piedra, Miguel-Ángel; Scionti, Giuseppe; Carriel, Víctor; Hernández-Cortés, Pedro; Campos, Antonio; Alaminos, Miguel

2014-01-01

Dupuytren's disease is a fibro-proliferative disease characterized by a disorder of the extracellular matrix (ECM) and high myofibroblast proliferation. However, studies failed to determine if the whole palm fascia is affected by the disease. The objective of this study was to analyze several components of the extracellular matrix of three types of tissues-Dupuytren's diseased contracture cords (DDC), palmar fascia clinically unaffected by Dupuytren's disease contracture (NPF), and normal forehand fascia (NFF). Histological analysis, quantification of cells recultured from each type of tissue, mRNA microarrays and immunohistochemistry for smooth muscle actin (SMA), fibrillar ECM components and non-fibrillar ECM components were carried out. The results showed that DDC samples had abundant fibrosis with reticular fibers and few elastic fibers, high cell proliferation and myofibroblasts, laminin and glycoproteins, whereas NFF did not show any of these findings. Interestingly, NPF tissues had more cells showing myofibroblasts differentiation and more collagen and reticular fibers, laminin and glycoproteins than NFF, although at lower level than DDC, with similar elastic fibers than DDC. Immunohistochemical expression of decorin was high in DDC, whereas versican was highly expressed NFF, with no differences for aggrecan. Cluster analysis revealed that the global expression profile of NPF was very similar to DDC, and reculturing methods showed that cells corresponding to DDC tissues proliferated more actively than NPF, and NPF more actively than NFF. All these results suggest that NPF tissues may be affected, and that a modification of the therapeutic approach used for the treatment of Dupuytren's disease should be considered.

CCN5 attenuates profibrotic phenotypes of fibroblasts through the Smad6-CCN2 pathway: Potential role in epidural fibrosis.

PubMed

Xu, Honghai; Liu, Cong; Sun, Zhengming; Guo, Xiong; Zhang, Yuelin; Liu, Mengting; Li, Peng

2015-07-01

Epidural fibrosis is characterized by the development of dense and thick scar tissue adjacent to the dural mater and ranked as the major contributor for post-operative pain recurrence after laminectomy or discectomy. Recently, CCN5 exhibited an inhibitory effect on connective tissue growth factor (CTGF)/CCN2 (a critical regulator for fibrotic disease)‑mediated fibrogenesis. However, its function in epidural fibrosis and the underlying mechanisms involved remain to be determined. In this study, an obvious downregulation of CCN5 was observed in scar tissues from laminectomized rats, concomitant with a marked upregulation of CCN2, suggesting a potential negative regulatory role of CCN5 in fibrogenesis. Furthermore, CCN5 overexpression notably mitigated transforming growth factor‑β1-enhanced fibroblast viability and proliferation. Of note, CCN5 upregulation inhibited the switch of fibroblasts into myofibroblasts as its overexpression abrogated the expression of the myofibroblast marker, α-smooth muscle actin (α-SMA). CCN5 upregulation also reduced an increase in collagen type I, α1 (COL1A1) and total collagen concentrations. Additionally, CCN5 over-expression decreased CCN2 expression and increased Smad6 phosphorylation. Mechanism analysis revealed that blocking Smad6 signaling significantly ameliorated the inhibitory effect of CCN5 on the CCN2 levels, accompanied by the reduction in cell proliferation and collagen production. These results confirm that CCN5 exerts an anti-fibrotic function by regulating the Smad6-CCN2 pathway, thereby indicating a potential approach for ameliorating epidural fibrosis after laminectomy.

Stimulation of Transforming Growth Factor-β1-Induced Endothelial-To-Mesenchymal Transition and Tissue Fibrosis by Endothelin-1 (ET-1): A Novel Profibrotic Effect of ET-1.

PubMed

Wermuth, Peter J; Li, Zhaodong; Mendoza, Fabian A; Jimenez, Sergio A

2016-01-01

TGF-β-induced endothelial-to-mesenchymal transition (EndoMT) is a newly recognized source of profibrotic activated myofibroblasts and has been suggested to play a role in the pathogenesis of various fibrotic processes. Endothelin-1 (ET-1) has been implicated in the development of tissue fibrosis but its participation in TGF-β-induced EndoMT has not been studied. Here we evaluated the role of ET-1 on TGF-β1-induced EndoMT in immunopurified CD31+/CD102+ murine lung microvascular endothelial cells. The expression levels of α-smooth muscle actin (α-SMA), of relevant profibrotic genes, and of various transcription factors involved in the EndoMT process were assessed employing quantitative RT-PCR, immunofluorescence histology and Western blot analysis. TGF-β1 caused potent induction of EndoMT whereas ET-1 alone had a minimal effect. However, ET-1 potentiated TGF-β1-induced EndoMT and TGF-β1-stimulated expression of mesenchymal cell specific and profibrotic genes and proteins. ET-1 also induced expression of the TGF-β receptor 1 and 2 genes, suggesting a plausible autocrine mechanism to potentiate TGF-β-mediated EndoMT and fibrosis. Stimulation of TGF-β1-induced skin and lung fibrosis by ET-1 was confirmed in vivo in an animal model of TGF-β1-induced tissue fibrosis. These results suggest a novel role for ET-1 in the establishment and progression of tissue fibrosis.

Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Genz, Berit; Thomas, Maria; Pützer, Brigitte M.

2014-11-01

Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluatedmore » an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC. - Highlights: • We performed adenoviral overexpression of Lhx2 in primary hepatic stellate cells. • Hepatic stellate cells expressed stem cell markers during cultivation. • Cell migration and contractility was slightly hampered upon Lhx2 overexpression. • Lhx2 overexpression did not affect stem cell character of hepatic stellate cells.« less

Standardized Salvia miltiorrhiza extract suppresses hepatic stellate cell activation and attenuates steatohepatitis induced by a methionine-choline deficient diet in mice.

PubMed

Lee, Hak Sung; Son, Woo-Chan; Ryu, Jae-Eun; Koo, Bon Am; Kim, Yeong Shik

2014-06-17

The aim of this study was to examine the effect of standardized extract of Salvia miltiorrhiza (SME) on gene and protein expression of non-alcoholic steatohepatitis (NASH)-related factors in activated human hepatic stellate cells (HSC), and in mice with steatohepatitis induced by a methionine-choline deficient (MCD) diet. Male C57BL/6J mice were placed on an MCD or control diet for 8 weeks and SME (0, 0.1, 0.5 and 1 mg/kg body weight) was administered orally every other day for 4 or 6 weeks. HSCs from the LX-2 cell line were treated with transforming growth factor β-1 (TGF-β1) or TGF-β1 plus SME (0.1-10 μg/mL). To investigate the effect of SME on reactive oxygen species (ROS)-induced condition, LX-2 cells were treated with hydrogen peroxide (H2O2) or H2O2 plus SME (0.1-100 μg/mL). MCD administration for 12 weeks increased mRNA expression of tumor necrosis factor (TNF-α), TGF-β1, interleukin-1β (IL-1β), C-reactive protein (CRP), α-smooth muscle actin (α-SMA), type I collagen, matrix metalloproteinase-2 (MMP-2) and MMP-9. TGF-β1-induced LX-2 cells exhibited similar gene expression patterns. SME treatment significantly reduced the mRNA and protein expression of NASH-related factors in the mouse model and HSCs. Histopathological liver analysis showed improved non-alcoholic fatty liver disease (NAFLD) activity and fibrosis score in SME-treated mice. The in vivo studies showed that SME had a significant effect at low doses. These results suggest that SME might be a potential therapeutic candidate for NAFLD treatment.

Thymidylate synthase (TS) gene expression in primary lung cancer patients: a large-scale study in Japanese population.

PubMed

Tanaka, F; Wada, H; Fukui, Y; Fukushima, M

2011-08-01

Previous small-sized studies showed lower thymidylate synthase (TS) expression in adenocarcinoma of the lung, which may explain higher antitumor activity of TS-inhibiting agents such as pemetrexed. To quantitatively measure TS gene expression in a large-scale Japanese population (n = 2621) with primary lung cancer, laser-captured microdissected sections were cut from primary tumors, surrounding normal lung tissues and involved nodes. TS gene expression level in primary tumor was significantly higher than that in normal lung tissue (mean TS/β-actin, 3.4 and 1.0, respectively; P < 0.01), and TS gene expression level was further higher in involved node (mean TS/β-actin, 7.7; P < 0.01). Analyses of TS gene expression levels in primary tumor according to histologic cell type revealed that small-cell carcinoma showed highest TS expression (mean TS/β-actin, 13.8) and that squamous cell carcinoma showed higher TS expression as compared with adenocarcinoma (mean TS/β-actin, 4.3 and 2.3, respectively; P < 0.01); TS gene expression was significantly increased along with a decrease in the grade of tumor cell differentiation. There was no significant difference in TS gene expression according to any other patient characteristics including tumor progression. Lower TS expression in adenocarcinoma of the lung was confirmed in a large-scale study.

Plant Vegetative and Animal Cytoplasmic Actins Share Functional Competence for Spatial Development with Protists[W][OA

PubMed Central

Kandasamy, Muthugapatti K.; McKinney, Elizabeth C.; Roy, Eileen; Meagher, Richard B.

2012-01-01

Actin is an essential multifunctional protein encoded by two distinct ancient classes of genes in animals (cytoplasmic and muscle) and plants (vegetative and reproductive). The prevailing view is that each class of actin variants is functionally distinct. However, we propose that the vegetative plant and cytoplasmic animal variants have conserved functional competence for spatial development inherited from an ancestral protist actin sequence. To test this idea, we ectopically expressed animal and protist actins in Arabidopsis thaliana double vegetative actin mutants that are dramatically altered in cell and organ morphologies. We found that expression of cytoplasmic actins from humans and even a highly divergent invertebrate Ciona intestinalis qualitatively and quantitatively suppressed the root cell polarity and organ defects of act8 act7 mutants and moderately suppressed the root-hairless phenotype of act2 act8 mutants. By contrast, human muscle actins were unable to support prominently any aspect of plant development. Furthermore, actins from three protists representing Choanozoa, Archamoeba, and green algae efficiently suppressed all the phenotypes of both the plant mutants. Remarkably, these data imply that actin’s competence to carry out a complex suite of processes essential for multicellular development was already fully developed in single-celled protists and evolved nonprogressively from protists to plants and animals. PMID:22589468

NHERF1 regulates actin cytoskeleton organization through modulation of α-actinin-4 stability.

PubMed

Sun, Licui; Zheng, Junfang; Wang, Qiqi; Song, Ran; Liu, Hua; Meng, Ran; Tao, Tao; Si, Yang; Jiang, Wenguo; He, Junqi

2016-02-01

The actin cytoskeleton is composed of a highly dynamic network of filamentous proteins, yet the molecular mechanism that regulates its organization and remodeling remains elusive. In this study, Na(+)/H(+) exchanger regulatory factor (NHERF)-1 loss-of-function and gain-of-function experiments reveal that polymerized actin cytoskeleton (F-actin) in HeLa cells is disorganized by NHERF1, whereas actin protein expression levels exhibit no detectable change. To elucidate the molecular mechanism underlying actin cytoskeleton disorganization by NHERF1, a combined 2-dimensional electrophoresis-matrix-assisted laser desorption/ionization-time of flight mass spectrometry approach was used to screen for proteins regulated by NHERF1 in HeLa cells. α-Actinin-4, an actin cross-linking protein, was identified. Glutathione S-transferase pull-down and coimmunoprecipitation studies showed the α-actinin-4 carboxyl-terminal region specifically interacted with the NHERF1 postsynaptic density 95/disc-large/zona occludens-1 domain. The NHERF1/α-actinin-4 interaction increased α-actinin-4 ubiquitination and decreased its expression levels, resulting in actin cytoskeleton disassembly. Our study identified α-actinin-4 as a novel NHERF1 interaction partner and provided new insights into the regulatory mechanism of the actin cytoskeleton by NHERF1. © FASEB.

Intrauterine human chorionic gonadotropin infusion in oocyte donors promotes endometrial synchrony and induction of early decidual markers for stromal survival: a randomized clinical trial.

PubMed

Strug, Michael R; Su, Renwei; Young, James E; Dodds, William G; Shavell, Valerie I; Díaz-Gimeno, Patricia; Ruíz-Alonso, Maria; Simón, Carlos; Lessey, Bruce A; Leach, Richard E; Fazleabas, Asgerally T

2016-07-01

Does a single intrauterine infusion of human chorionic gonadotropin (hCG) at the time corresponding to a Day 3 embryo transfer in oocyte donors induce favorable molecular changes in the endometrium for embryo implantation? Intrauterine hCG was associated with endometrial synchronization between endometrial glands and stroma following ovarian stimulation and the induction of early decidual markers associated with stromal cell survival. The clinical potential for increasing IVF success rates using an intrauterine hCG infusion prior to embryo transfer remains unclear based on previously reported positive and non-significant findings. However, infusion of CG in the non-human primate increases the expression of pro-survival early decidual markers important for endometrial receptivity, including α-smooth muscle actin (α-SMA) and NOTCH1. Oocyte donors (n=15) were randomly assigned to receive an intrauterine infusion of 500 IU hCG (n=7) or embryo culture media vehicle (n=8) 3 days following oocyte retrieval during their donor stimulation cycle. Endometrial biopsies were performed 2 days later, followed by either RNA isolation or tissue fixation in formalin and paraffin embedding. Reverse transcription of total RNA from endometrial biopsies generated cDNA, which was used for analysis in the endometrial receptivity array (ERA; n = 5/group) or quantitative RT-PCR to determine relative expression of ESR1, PGR, C3 and NOTCH1. Tissue sections were stained with hematoxylin and eosin followed by blinded staging analysis for dating of endometrial glands and stroma. Immunostaining for ESR1, PGR, α-SMA, C3 and NOTCH1 was performed to determine their tissue localization. Intrauterine hCG infusion was associated with endometrial synchrony and reprograming of stromal development following ovarian stimulation. ESR1 and PGR were significantly elevated in the endometrium of hCG-treated patients, consistent with earlier staging. The ERA did not predict an overall positive impact of intrauterine hCG on endometrial receptivity. However, ACTA2, encoding α-SMA was significantly increased in response to intrauterine hCG. Similar to the hCG-treated non-human primate, sub-epithelial and peri-vascular α-SMA expression was induced in women following hCG infusion. Other known targets of hCG in the baboon were also found to be increased, including C3 and NOTCH1, which have known roles in endometrial receptivity. This study differs from our previous work in the hCG-treated non-human primate along with clinical studies in infertile patients. Specifically, we performed a single intrauterine infusion in oocyte donors instead of either continuous hCG via an osmotic mini-pump in the baboon or infusion followed by blastocyst-derived hCG in infertile women undergoing embryo transfer. Therefore, the full impact of intrauterine hCG in promoting endometrial receptivity may not have been evident. Our findings suggest a potential clinical benefit for intrauterine hCG prior to embryo transfer on Day 3 in counteracting endometrial dyssynchrony from ovarian stimulation and promoting expression of markers important for stromal survival. Finally, there were no obvious negative effects of intrauterine hCG treatment. Funding for this work was provided by NICHD R01 HD042280 (A.T.F.) and NICHD F30 HD082951 (M.R.S.). C.S. and P.D.-G are co-inventors of the patented ERA, which is owned by IGENOMIX SL and was used in this study, and C.S. is a shareholder in IGENOMIX SL. M.R.-A. is employed by IGENOMIX SL. No other authors have any conflicts of interest to report. This study was registered with ClinicalTrials.gov (NCT01786252). 5 February 2013. 10 May 2013. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Dimerization and actin-bundling properties of villin and its role in the assembly of epithelial cell brush borders.

PubMed

George, Sudeep P; Wang, Yaohong; Mathew, Sijo; Srinivasan, Kamalakkannan; Khurana, Seema

2007-09-07

Villin is a major actin-bundling protein in the brush border of epithelial cells. In this study we demonstrate for the first time that villin can bundle actin filaments using a single F-actin binding site, because it has the ability to self-associate. Using fluorescence resonance energy transfer, we demonstrate villin self-association in living cells in microvilli and in growth factor-stimulated cells in membrane ruffles and lamellipodia. Using sucrose density gradient, size-exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight, the majority of villin was identified as a monomer or dimer. Villin dimers were also identified in Caco-2 cells, which endogenously express villin and Madin-Darby canine kidney cells that ectopically express villin. Using truncation mutants of villin, site-directed mutagenesis, and fluorescence resonance energy transfer, an amino-terminal dimerization site was identified that regulated villin self-association in parallel conformation as well as actin bundling by villin. This detailed analysis describes for the first time microvillus assembly by villin, redefines the actin-bundling function of villin, and provides a molecular mechanism for actin bundling by villin, which could have wider implications for other actin cross-linking proteins that share a villin-like headpiece domain. Our study also provides a molecular basis to separate the morphologically distinct actin-severing and actin-bundling properties of villin.

Conserved pattern of embryonic actin gene expression in several sea urchins and a sand dollar.

PubMed

Bushman, F D; Crain, W R

1983-08-01

An examination of the size and relative abundance of actin-coding RNA in embryos of four sea urchins (Strongylocentrotus purpuratus, Strongylocentrotus droebachiensis, Arbacia punctulata, Lytechinus variegatus) and one sand dollar (Echinarachnius parma) reveals a generally conserved program of expression. In each species the relative abundance of these sequences is low in early embryos and begins to rise during late cleavage or blastula stages. In the four sea urchins, actin-coding RNAs increase between approximately 9- and 35-fold by pluteus or an earlier stage, and in the sand dollar about 5.5-fold by blastula. A major actin-coding RNA class of 2.0-2.2 kilobases (kb) is found in each species. A smaller actin-coding RNA class, which accumulates during embryogenesis, is also present in S. purpuratus (1.8 kb), S. droebachiensis (1.9 kb), and A. punctulata (1.6 kb), but apparently absent in L. variegatus and E. parma. In S. droebachiensis, actin-coding RNA is relatively abundant in unfertilized eggs and drops sharply by the 16-cell stage. This is in contrast to the other sea urchins where the actin message content is relatively low in eggs and does not change substantially in the embryos throughout early cleavage. The observations in this study suggest that the pattern of embryonic expression of at least some members of this gene family is ancient and conserved.

The clinical landscape for SMA in a new therapeutic era

PubMed Central

Talbot, K; Tizzano, E F

2017-01-01

Despite significant advances in basic research, the treatment of degenerative diseases of the nervous system remains one of the greatest challenges for translational medicine. The childhood onset motor neuron disorder spinal muscular atrophy (SMA) has been viewed as one of the more tractable targets for molecular therapy due to a detailed understanding of the molecular genetic basis of the disease. In SMA, inactivating mutations in the SMN1 gene can be partially compensated for by limited expression of SMN protein from a variable number of copies of the SMN2 gene, which provides both a molecular explanation for phenotypic severity and a target for therapy. The advent of the first tailored molecular therapy for SMA, based on modulating the splicing behaviour of the SMN2 gene provides, for the first time, a treatment which alters the natural history of motor neuron degeneration. Here we consider how this will change the landscape for diagnosis, clinical management and future therapeutic trials in SMA, as well as the implications for the molecular therapy of other neurological diseases. PMID:28644430

Low-intensity infrared lasers alter actin gene expression in skin and muscle tissue

NASA Astrophysics Data System (ADS)

Fonseca, A. S.; Mencalha, A. L.; Campos, V. M. A.; Ferreira-Machado, S. C.; Peregrino, A. A. F.; Magalhães, L. A. G.; Geller, M.; Paoli, F.

2013-02-01

The biostimulative effect of low-intensity lasers is the basis for treatment of diseases in soft tissues. However, data about the influence of biostimulative lasers on gene expression are still scarce. The aim of this work was to evaluate the effects of low-intensity infrared lasers on the expression of actin mRNA in skin and muscle tissue. Skin and muscle tissue of Wistar rats was exposed to low-intensity infrared laser radiation at different fluences and frequencies. One and 24 hours after laser exposure, tissue samples were withdrawn for total RNA extraction, cDNA synthesis and evaluation of actin gene expression by quantitative polymerase chain reaction. The data obtained show that laser radiation alters the expression of actin mRNA differently in skin and muscle tissue of Wistar rats depending of the fluence, frequency and time after exposure. The results could be useful for laser dosimetry, as well as to justify the therapeutic protocols for treatment of diseases of skin and muscle tissues based on low-intensity infrared laser radiation.

Notch1 stimulation induces a vascularization switch with pericyte-like cell differentiation of glioblastoma stem cells.

PubMed

Guichet, Pierre-Olivier; Guelfi, Sophie; Teigell, Marisa; Hoppe, Liesa; Bakalara, Norbert; Bauchet, Luc; Duffau, Hugues; Lamszus, Katrin; Rothhut, Bernard; Hugnot, Jean-Philippe

2015-01-01

Glioblastoma multiforms (GBMs) are highly vascularized brain tumors containing a subpopulation of multipotent cancer stem cells. These cells closely interact with endothelial cells in neurovascular niches. In this study, we have uncovered a close link between the Notch1 pathway and the tumoral vascularization process of GBM stem cells. We observed that although the Notch1 receptor was activated, the typical target proteins (HES5, HEY1, and HEY2) were not or barely expressed in two explored GBM stem cell cultures. Notch1 signaling activation by expression of the intracellular form (NICD) in these cells was found to reduce their growth rate and migration, which was accompanied by the sharp reduction in neural stem cell transcription factor expression (ASCL1, OLIG2, and SOX2), while HEY1/2, KLF9, and SNAI2 transcription factors were upregulated. Expression of OLIG2 and growth were restored after termination of Notch1 stimulation. Remarkably, NICD expression induced the expression of pericyte cell markers (NG2, PDGFRβ, and α-smooth muscle actin [αSMA]) in GBM stem cells. This was paralleled with the induction of several angiogenesis-related factors most notably cytokines (heparin binding epidermal growth factor [HB-EGF], IL8, and PLGF), matrix metalloproteinases (MMP9), and adhesion proteins (vascular cell adhesion molecule 1 [VCAM1], intercellular adhesion molecule 1 [ICAM1], and integrin alpha 9 [ITGA9]). In xenotransplantation experiments, contrasting with the infiltrative and poorly vascularized tumors obtained with control GBM stem cells, Notch1 stimulation resulted in poorly disseminating but highly vascularized grafts containing large vessels with lumen. Notch1-stimulated GBM cells expressed pericyte cell markers and closely associated with endothelial cells. These results reveal an important role for the Notch1 pathway in regulating GBM stem cell plasticity and angiogenic properties. © 2014 AlphaMed Press.

Notch Signaling Pathway Is Activated in Motoneurons of Spinal Muscular Atrophy

PubMed Central

Caraballo-Miralles, Víctor; Cardona-Rossinyol, Andrea; Garcera, Ana; Torres-Benito, Laura; Soler, Rosa M.; Tabares, Lucía; Lladó, Jerònia; Olmos, Gabriel

2013-01-01

Spinal muscular atrophy (SMA) is a neurodegenerative disease produced by low levels of Survival Motor Neuron (SMN) protein that affects alpha motoneurons in the spinal cord. Notch signaling is a cell-cell communication system well known as a master regulator of neural development, but also with important roles in the adult central nervous system. Aberrant Notch function is associated with several developmental neurological disorders; however, the potential implication of the Notch pathway in SMA pathogenesis has not been studied yet. We report here that SMN deficiency, induced in the astroglioma cell line U87MG after lentiviral transduction with a shSMN construct, was associated with an increase in the expression of the main components of Notch signaling pathway, namely its ligands, Jagged1 and Delta1, the Notch receptor and its active intracellular form (NICD). In the SMNΔ7 mouse model of SMA we also found increased astrocyte processes positive for Jagged1 and Delta1 in intimate contact with lumbar spinal cord motoneurons. In these motoneurons an increased Notch signaling was found, as denoted by increased NICD levels and reduced expression of the proneural gene neurogenin 3, whose transcription is negatively regulated by Notch. Together, these findings may be relevant to understand some pathologic attributes of SMA motoneurons. PMID:23759991

A Role for SMN Exon 7 Splicing in the Selective Vulnerability of Motor Neurons in Spinal Muscular Atrophy

PubMed Central

Ruggiu, Matteo; McGovern, Vicki L.; Lotti, Francesco; Saieva, Luciano; Li, Darrick K.; Kariya, Shingo; Monani, Umrao R.; Burghes, Arthur H. M.

2012-01-01

Spinal muscular atrophy (SMA) is an inherited motor neuron disease caused by homozygous loss of the Survival Motor Neuron 1 (SMN1) gene. In the absence of SMN1, inefficient inclusion of exon 7 in transcripts from the nearly identical SMN2 gene results in ubiquitous SMN decrease but selective motor neuron degeneration. Here we investigated whether cell type-specific differences in the efficiency of exon 7 splicing contribute to the vulnerability of SMA motor neurons. We show that normal motor neurons express markedly lower levels of full-length SMN mRNA from SMN2 than do other cells in the spinal cord. This is due to inefficient exon 7 splicing that is intrinsic to motor neurons under normal conditions. We also find that SMN depletion in mammalian cells decreases exon 7 inclusion through a negative feedback loop affecting the splicing of its own mRNA. This mechanism is active in vivo and further decreases the efficiency of exon 7 inclusion specifically in motor neurons of severe-SMA mice. Consistent with expression of lower levels of full-length SMN, we find that SMN-dependent downstream molecular defects are exacerbated in SMA motor neurons. These findings suggest a mechanism to explain the selective vulnerability of motor neurons to loss of SMN1. PMID:22037760

Uric acid causes kidney injury through inducing fibroblast expansion, Endothelin-1 expression, and inflammation.

PubMed

Romi, Muhammad Mansyur; Arfian, Nur; Tranggono, Untung; Setyaningsih, Wiwit Ananda Wahyu; Sari, Dwi Cahyani Ratna

2017-10-31

Uric acid (UA) plays important roles in inducing renal inflammation, intra-renal vasoconstriction and renal damage. Endothelin-1 (ET-1) is a well-known profibrotic factor in the kidney and is associated with fibroblast expansion. We examined the role of hyperuricemia conditions in causing elevation of ET-1 expression and kidney injury. Hyperuricemia was induced in mice using daily intraperitoneal injection of uric acid 125 mg/Kg body weight. An NaCl injection was used in control mice. Mice were euthanized on days-7 (UA7) and 14 (UA14). We also added allopurinol groups (UAL7 and UAL14) with supplementation of allopurinol 50 mg/Kg body weight orally. Uric acid and creatinine serum were measured from blood serum. Periodic Acid Schiff (PAS) and Sirius Red staining were done for glomerulosclerosis, tubular injury and fibrosis quantification. mRNA expression examination was performed for nephrin, podocin, preproEndothelin-1 (ppET-1), MCP-1 and ICAM-1. PDGFRβ immunostaining was done for quantification of fibroblast, while α-SMA immunostaining was done for localizing myofibroblast. Western blot analysis was conducted to quantify TGF-β1, α-SMA and Endothelin A Receptor (ETAR) protein expression. Uric acid and creatinine levels were elevated after 7 and 14 days and followed by significant increase of glomerulosclerosis and tubular injury score in the uric acid group (p < 0.05 vs. control). Both UA7 and UA14 groups had higher fibrosis, tubular injury and glomerulosclerosis with significant increase of fibroblast cell number compared with control. RT-PCR revealed down-regulation of nephrin and podocin expression (p < 0.05 vs. control), and up-regulation of MCP-1, ET-1 and ICAM-1 expression (p < 0.05 vs. control). Western blot revealed higher expression of TGF-β1 and α-SMA protein expression. Determination of allopurinol attenuated kidney injury was based on reduction of fibroblast cell number, inflammation mediators and ppET-1 expression with reduction of TGF-β1 and α-SMA protein expression. UA induced glomerulosclerosis, tubular injury and renal fibrosis with reduction of podocyte function and inflammatory mediator elevation. ET-1 and fibroblast expansion might modulate hyperuricemia induced renal fibrosis.

Strong fascin expression promotes metastasis independent of its F-actin bundling activity.

PubMed

Heinz, Lisa S; Muhs, Stefanie; Schiewek, Johanna; Grüb, Saskia; Nalaskowski, Marcus; Lin, Yuan-Na; Wikman, Harriet; Oliveira-Ferrer, Leticia; Lange, Tobias; Wellbrock, Jasmin; Konietzny, Anja; Mikhaylova, Marina; Windhorst, Sabine

2017-12-15

High expression of the actin bundling protein Fascin increases the malignancy of tumor cells. Here we show that fascin expression is up-regulated in more malignant sub-cell lines of MDA-MB-231 cells as compared to parental cells. Since also parental MDA-MB-231 cells exhibit high fascin levels, increased fascin expression was termed as "hyperexpression". To examine the effect of fascin hyperexpression, fascin was hyperexpressed in parental MDA-MB-231 cells and metastasis was analyzed in NOD scid gamma (NSG) mice. In addition, the effect of fascin mutants with inactive or constitutively active actin bundling activity was examined. Unexpectedly, we found that hyperexpression of both, wildtype (wt) and mutant fascin strongly increased metastasis in vivo , showing that the effect of fascin hyperexpression did not depend on its actin bundling activity. Cellular assays revealed that hyperexpression of wt and mutant fascin increased adhesion of MDA-MB-231 cells while transmigration and proliferation were not affected. Since it has been shown that fascin controls adhesion by directly interacting with microtubules ( MTs), we analyzed if fascin hyperexpression affects MT dynamics. We found that at high concentrations fascin significantly increased MT dynamics in cells and in cell-free approaches. In summary our data show that strong expression of fascin in breast cancer cells increases metastasis independent of its actin bundling activity. Thus, it seems that the mechanism of fascin-stimulated metastasis depends on its concentration.

PHD3-mediated prolyl hydroxylation of nonmuscle actin impairs polymerization and cell motility

PubMed Central

Luo, Weibo; Lin, Benjamin; Wang, Yingfei; Zhong, Jun; O'Meally, Robert; Cole, Robert N.; Pandey, Akhilesh; Levchenko, Andre; Semenza, Gregg L.

2014-01-01

Actin filaments play an essential role in cell movement, and many posttranslational modifications regulate actin filament assembly. Here we report that prolyl hydroxylase 3 (PHD3) interacts with nonmuscle actin in human cells and catalyzes hydroxylation of actin at proline residues 307 and 322. Blocking PHD3 expression or catalytic activity by short hairpin RNA knockdown or pharmacological inhibition, respectively, decreased actin prolyl hydroxylation. PHD3 knockdown increased filamentous F-actin assembly, which was reversed by PHD3 overexpression. PHD3 knockdown increased cell velocity and migration distance. Inhibition of PHD3 prolyl hydroxylase activity by dimethyloxalylglycine also increased actin polymerization and cell migration. These data reveal a novel role for PHD3 as a negative regulator of cell motility through posttranslational modification of nonmuscle actins. PMID:25079693

Migration and differentiation potential of stem cells in the cnidarian Hydractinia analysed in eGFP-transgenic animals and chimeras.

PubMed

Künzel, Timo; Heiermann, Reinhard; Frank, Uri; Müller, Werner; Tilmann, Wido; Bause, Markus; Nonn, Anja; Helling, Matthias; Schwarz, Ryan S; Plickert, Günter

2010-12-01

To analyse cell migration and the differentiation potential of migratory stem cells in Hydractinia, we generated animals with an eGFP reporter gene stably expressed and transmitted via the germline. The transgene was placed under the control of two different actin promoters and the promoter of elongation factor-1α. One actin promoter (Act-II) and the EF-1α promoter enabled expression of the transgene in all cells, the other actin promoter (Act-I) in epithelial and gametogenic cells, but not in the pluripotent migratory stem cells. We produced chimeric animals consisting of histocompatible wild type and transgenic parts. When the transgene was under the control of the epithelial cell specific actin-I promoter, non-fluorescent transgenic stem cells immigrated into wild type tissue, stopped migration and differentiated into epithelial cells which then commenced eGFP-expression. Migratory stem cells are therefore pluripotent and can give rise not only to germ cells, nematocytes and nerve cells, but also to epithelial cells. While in somatic cells expression of the act-I promoter was restricted to epithelial cells it became also active in gametogenesis. The act-I gene is expressed in spermatogonia, oogonia and oocytes. In males the expression pattern showed that migratory stem cells are the precursors of both the spermatogonia and their somatic envelopes. Comparative expression studies using the promoters of the actin-II gene and the elongation factor-1α gene revealed the potential of transgenic techniques to trace the development of the nervous system. Copyright © 2010 Elsevier Inc. All rights reserved.

Cardiomyogenic Differentiation of Human Dental Follicle-derived Stem Cells by Suberoylanilide Hydroxamic Acid and Their In Vivo Homing Property.

PubMed

Sung, Iel-Yong; Son, Han-Na; Ullah, Imran; Bharti, Dinesh; Park, Ju-Mi; Cho, Yeong-Cheol; Byun, June-Ho; Kang, Young-Hoon; Sung, Su-Jin; Kim, Jong-Woo; Rho, Gyu-Jin; Park, Bong-Wook

2016-01-01

The purpose of the present study was to investigate the in vitro cardiomyogenic differentiation potential of human dental follicle-derived stem cells (DFCs) under the influence of suberoylanilide hydroxamic acid (SAHA), a member of the histone deacetylase inhibitor family, and analyze the in vivo homing capacity of induced cardiomyocytes (iCMs) when transplanted systemically. DFCs from extracted wisdom teeth showed mesenchymal stem cell (MSC) characteristics such as plate adherent growing, expression of MSC markers (CD44, CD90, and CD105), and mesenchymal lineage-specific differentiation potential. Adding SAHA to the culture medium induced the successful in vitro differentiation of DFCs into cardiomyocytes. These iCMs expressed cardiomyogenic markers, including alpha-smooth muscle actin (α-SMA), cardiac muscle troponin T (TNNT2), Desmin, and cardiac muscle alpha actin (ACTC1) , at both the mRNA and protein level. For the assessment of homing capacity, PKH26 labeled iCMs were intraperitoneally injected (1×10 6 cells in 100 µL of PBS) into the experimental mice, and the ratios of PKH26 positive cells to the total number of injected cells, in multiple organs were determined. The calculated homing ratios, 14 days after systemic cell transplantation, were 5.6 ± 1.0%, 3.6 ± 1.1%, and 11.6 ± 2.7% in heart, liver, and kidney respectively. There was no difference in the serum levels of interleukin-2 and interleukin-10 at 14 days after transplantation, between the experimental (iCM injected) and control (no injection or PBS injection) groups. These results demonstrate that DFCs can be an excellent source for cardiomyocyte differentiation and regeneration. Moreover, the iCMs can be delivered into heart muscle via systemic administration without eliciting inflammatory or immune response. This can serve as the pilot study for further investigations into the in vitro cardiomyogenic differentiation potential of DFCs under the influence of SAHA and the in vivo homing capacity of the iCMs into the heart muscle, when injected systemically.

SKAP2 regulates Arp2/3 complex for actin-mediated asymmetric cytokinesis by interacting with WAVE2 in mouse oocytes.

PubMed

He, Shu-Wen; Xu, Bai-Hui; Liu, Yu; Wang, Ya-Long; Chen, Ming-Huang; Xu, Lin; Liao, Bao-Qiong; Lui, Rui; Li, Fei-Ping; Lin, Yan-Hong; Fu, Xian-Pei; Fu, Bin-Bin; Hong, Zi-Wei; Liu, Yu-Xin; Qi, Zhong-Quan; Wang, Hai-Long

2017-01-01

SKAP2 (Src kinase-associated phosphoprotein 2), a substrate of Src family kinases, has been suggested to be involved in actin-mediated cellular processes. However, little is known about its role in mouse oocyte maturation. In this study, we thus investigated the expression, localization, and functions of SKAP2 during mouse oocyte asymmetric division. SKAP2 protein expression was detected at all developmental stages in mouse oocytes. Immunofluorescent staining showed that SKAP2 was mainly distributed at the cortex of the oocytes during maturation. Treatment with cytochalasin B in oocytes confirmed that SKAP2 was co-localized with actin. Depletion of SKAP2 by injection with specific short interfering RNA caused failure of spindle migration, polar body extrusion, and cytokinesis defects. Meanwhile, the staining of actin filaments at the oocyte membrane and in the cytoplasm was significantly reduced after these treatments. SKAP2 depletion also disrupted actin cap and cortical granule-free domain formation, and arrested a large proportion of oocytes at the telophase stage. Moreover, Arp2/3 complex and WAVE2 expression was decreased after the depletion of SKAP2 activity. Our results indicate that SKAP2 regulates the Arp2/3 complex and is essential for actin-mediated asymmetric cytokinesis by interacting with WAVE2 in mouse oocytes.

Reversal of nicotine-induced alveolar lipofibroblast-to-myofibroblast transdifferentiation by stimulants of parathyroid hormone-related protein signaling.

PubMed

Rehan, Virender K; Sakurai, Reiko; Wang, Ying; Santos, Jamie; Huynh, Kyle; Torday, John S

2007-01-01

Nicotine exposure disrupts the parathyroid hormone-related protein (PTHrP)-driven alveolar epithelial-mesenchymal paracrine-signaling pathway, resulting in the transdifferentiation of pulmonary lipofibroblasts (LIFs) to myofibroblasts (MYFs), which seems to be central to altered pulmonary development and function in infants born to mothers who smoke during pregnancy. Modulation of PTHrP-driven signaling can almost completely prevent nicotine-induced LIF-to-MYF transdifferentiation. However, once this process has occurred, whether it can be reversed is not known. Our objective was to determine if nicotine-induced LIF-to-MYF transdifferentiation could be reversed by specifically targeting the PTHrP-mediated alveolar epithelial-mesenchymal paracrine signaling. WI38 cells, a human embryonic pulmonary fibroblast cell line, were initially treated with nicotine for 7 days and LIF-to-MYF transdifferentiation was confirmed by determining the downregulation of the key lipogenic marker, peroxisome proliferator-activated receptor gamma (PPARgamma) and upregulation of the key myogenic marker, alpha-smooth muscle actin (alphaSMA). Because downregulation of the PPARgamma signaling pathway is the key determinant of LIF-to-MYF transdifferentiation, cells were treated with three agonists of this pathway, PTHrP, dibutryl cAMP (DBcAMP), or rosiglitazone (RGZ) for 7 days, and the expression of the PTHrP receptor, PPARgamma, alphaSMA, and calponin was determined by Western analysis and immunohistochemistry. Simultaneously, fibroblast function was characterized by measuring their capacity to take up triglycerides. Nicotine-induced LIF-to-MYF transdifferentiation was almost completely reversed by treatment with RGZ, PTHrP, or DBcAMP, as determined by protein and functional assays. Using a specific molecular approach and targeting specific molecular intermediates in the PTHrP signaling pathway, to our knowledge, this for the first time, demonstrates the reversibility of nicotine-induced LIF-to-MYF transdifferentiation, suggesting not only the possibility of prevention but also the potential for reversal of nicotine-induced lung injury.

Pirfenidone and nintedanib modulate properties of fibroblasts and myofibroblasts in idiopathic pulmonary fibrosis.

PubMed

Lehtonen, Siri T; Veijola, Anniina; Karvonen, Henna; Lappi-Blanco, Elisa; Sormunen, Raija; Korpela, Saara; Zagai, Ulrika; Sköld, Magnus C; Kaarteenaho, Riitta

2016-02-04

Idiopathic pulmonary fibrosis (IPF) is an incurable lung disease with a poor prognosis. Fibroblasts and myofibroblasts are the key cells in the fibrotic process. Recently two drugs, pirfenidone and nintedanib, were approved for clinical use as they are able to slow down the disease progression. The mechanisms by which these two drugs act in in vitro cell systems are not known. The aim of this study was therefore to examine the effects of pirfenidone and nintedanib on fibroblasts and myofibroblasts structure and function established from patients with or without IPF. Stromal cells were collected and cultured from control lung (n = 4) or IPF (n = 7). The cells were treated with pirfenidone and/or nintedanib and the effect of treatment was evaluated by measuring cell proliferation, alpha smooth muscle actin (α-SMA) and fibronectin expression by Western analysis and/or immunoelectron microscopy, ultrastructural properties by transmission electron microscopy and functional properties by collagen gel contraction and invasion assays. Both pirfenidone and nintedanib reduced in vitro proliferation of fibroblastic cells in a dose dependent manner. The number of cells from control lung was reduced to 47 % (p = 0.04) and of IPF cells to 42 % (p = 0.04) by 1 mM pirfenidone and correspondingly to 67 % (p = 0.04) and 68 % (p = 0.04), by 1 μM nintedanib. If both drugs were used together, a further reduced proliferation was observed. Both pirfenidone and nintedanib were able to reduce the amount of α-SMA and the myofibroblastic appearance although the level of reduction was cell line dependent. In functional assays, the effect of both drugs was also variable. We conclude that the ultrastructure and function of fibroblasts and myofibroblasts are affected by pirfenidone and nintedanib. Combination of the drugs reduced cell proliferation more than either of them individually. Human lung derived cell culture systems represent a potential platform for screening and testing drugs for fibrotic diseases.

Effectiveness of Losartan-Loaded Hyaluronic Acid (HA) Micelles for the Reduction of Advanced Hepatic Fibrosis in C3H/HeN Mice Model

PubMed Central

Thomas, Reju George; Moon, Myeong Ju; Kim, Jo Heon; Lee, Jae Hyuk; Jeong, Yong Yeon

2015-01-01

Advanced hepatic fibrosis therapy using drug-delivering nanoparticles is a relatively unexplored area. Angiotensin type 1 (AT1) receptor blockers such as losartan can be delivered to hepatic stellate cells (HSC), blocking their activation and thereby reducing fibrosis progression in the liver. In our study, we analyzed the possibility of utilizing drug-loaded vehicles such as hyaluronic acid (HA) micelles carrying losartan to attenuate HSC activation. Losartan, which exhibits inherent lipophilicity, was loaded into the hydrophobic core of HA micelles with a 19.5% drug loading efficiency. An advanced liver fibrosis model was developed using C3H/HeN mice subjected to 20 weeks of prolonged TAA/ethanol weight-adapted treatment. The cytocompatibility and cell uptake profile of losartan-HA micelles were studied in murine fibroblast cells (NIH3T3), human hepatic stellate cells (hHSC) and FL83B cells (hepatocyte cell line). The ability of these nanoparticles to attenuate HSC activation was studied in activated HSC cells based on alpha smooth muscle actin (α-sma) expression. Mice treated with oral losartan or losartan-HA micelles were analyzed for serum enzyme levels (ALT/AST, CK and LDH) and collagen deposition (hydroxyproline levels) in the liver. The accumulation of HA micelles was observed in fibrotic livers, which suggests increased delivery of losartan compared to normal livers and specific uptake by HSC. Active reduction of α-sma was observed in hHSC and the liver sections of losartan-HA micelle-treated mice. The serum enzyme levels and collagen deposition of losartan-HA micelle-treated mice was reduced significantly compared to the oral losartan group. Losartan-HA micelles demonstrated significant attenuation of hepatic fibrosis via an HSC-targeting mechanism in our in vitro and in vivo studies. These nanoparticles can be considered as an alternative therapy for liver fibrosis. PMID:26714035

Effectiveness of Losartan-Loaded Hyaluronic Acid (HA) Micelles for the Reduction of Advanced Hepatic Fibrosis in C3H/HeN Mice Model.

PubMed

Thomas, Reju George; Moon, Myeong Ju; Kim, Jo Heon; Lee, Jae Hyuk; Jeong, Yong Yeon

2015-01-01

Advanced hepatic fibrosis therapy using drug-delivering nanoparticles is a relatively unexplored area. Angiotensin type 1 (AT1) receptor blockers such as losartan can be delivered to hepatic stellate cells (HSC), blocking their activation and thereby reducing fibrosis progression in the liver. In our study, we analyzed the possibility of utilizing drug-loaded vehicles such as hyaluronic acid (HA) micelles carrying losartan to attenuate HSC activation. Losartan, which exhibits inherent lipophilicity, was loaded into the hydrophobic core of HA micelles with a 19.5% drug loading efficiency. An advanced liver fibrosis model was developed using C3H/HeN mice subjected to 20 weeks of prolonged TAA/ethanol weight-adapted treatment. The cytocompatibility and cell uptake profile of losartan-HA micelles were studied in murine fibroblast cells (NIH3T3), human hepatic stellate cells (hHSC) and FL83B cells (hepatocyte cell line). The ability of these nanoparticles to attenuate HSC activation was studied in activated HSC cells based on alpha smooth muscle actin (α-sma) expression. Mice treated with oral losartan or losartan-HA micelles were analyzed for serum enzyme levels (ALT/AST, CK and LDH) and collagen deposition (hydroxyproline levels) in the liver. The accumulation of HA micelles was observed in fibrotic livers, which suggests increased delivery of losartan compared to normal livers and specific uptake by HSC. Active reduction of α-sma was observed in hHSC and the liver sections of losartan-HA micelle-treated mice. The serum enzyme levels and collagen deposition of losartan-HA micelle-treated mice was reduced significantly compared to the oral losartan group. Losartan-HA micelles demonstrated significant attenuation of hepatic fibrosis via an HSC-targeting mechanism in our in vitro and in vivo studies. These nanoparticles can be considered as an alternative therapy for liver fibrosis.

The effect of sulindac, a non-steroidal anti-inflammatory drug, attenuates inflammation and fibrosis in a mouse model of chronic pancreatitis

PubMed Central

2012-01-01

Background Chronic pancreatitis is characterized by progressive fibrosis, pain and loss of exocrine and endocrine functions. The long-standing chronic pancreatitis and its associated pancreatic fibrosis are the most common pathogenic events involved in human pancreatic carcinogenesis, but the therapeutic strategies to chronic pancreatitis and the chemoprevention of pancreatic carcinogenesis are very limited. Methods We investigated the effect of sulindac, a non-steroidal anti-inflammatory drug (NSAID), on inhibition of chronic pancreatitis in a caerulein induced chronic pancreatitis mouse model. Results Sulindac significantly reduced the severity of chronic pancreatitis including the extent of acini loss, inflammatory cell infiltration and stromal fibrosis. The protein expression of phosphorylation of MEK/ERK was inhibited in the chronic pancreatic tissues by sulindac treatment as measured by Western blot assay. The levels of inflammatory cytokines including TNF-α and MCP-1 were also significantly decreased with sulindac treatment, as well as the expression of TGF-β, PDGF-β, SHH and Gli in the chronic pancreatic tissue detected by qPCR assay and confirmed by western blot assay. The activation of pancreatic satellet cells was also inhibited by sulindac as measured by the activity of α-smooth muscle actin (α-SMA) in the pancreatic tissue of chronic pancreatitis. Conclusions Sulindac is a promising reagent for the treatment of chronic pancreatitis via inhibition of inflammatory cell infiltration and stromal fibrosis, the inhibitory effect of sulindac on chronic pancreatitis may through targeting the activation ERK/MAPK signaling pathway. PMID:22920325

Inhibition of soluble epoxide hydrolase lowers portal hypertension in cirrhotic rats by ameliorating endothelial dysfunction and liver fibrosis.

PubMed

Deng, Wensheng; Zhu, Yiming; Lin, Jiayun; Zheng, Lei; Zhang, Chihao; Luo, Meng

2017-07-01

Epoxyeicostrienoic acids (EETs) are arachidonic acid derived meditators which are catalyzed by soluble epoxide hydrolase (sEH) to less active dihydroeicostrienoics acids (DHETS). The aim of our study is to investigate the effects of sEH inhibition on hepatic and systemic hemodynamics, hepatic endothelial dysfunction, and hepatic fibrosis in CCl4 cirrhotic rats. The sEH inhibitor,trans-4-{4-[3-(4-trifluoromethoxyphenyl)-ureido]cyclohexyloxy}benzoic acid (t-TUCB) was administered to stabilize hepatic EETs by gavage at a dose of 1mg/kg/d. Our results showed that hepatic sEH expression was markedly increased in portal hypertension, and led to a lower ratio of EETs/DHETs which was effectively reversed by t-TUCB administration. t-TUCB significantly decreased portal pressure without significant changes in systemic hemodynamics, which was associated with the attenuation of intrahepatic vascular resistance (IHVR) and liver fibrosis. t-TUCB ameliorated endothelial dysfunction, increased hepatic endothelial nitric oxide synthase (eNOS) phosphorylation and nitric oxide (NO) production. In addition, t-TUCB significantly reduced alpha-Smooth Muscle Actin (α-SMA) expression and liver fibrosis, which was associated with a decrease in NF-κB signaling. Taken together, inhibition of sEH reduces portal pressure, liver fibrosis and attenuates hepatic endothelial dysfunction in cirrhotic rats. Our results indicate that sEH inhbitors may be useful in the treatment of portal hypertension in patients with cirrhosis. Copyright © 2017 Elsevier Inc. All rights reserved.

Design and evaluation of Actichip, a thematic microarray for the study of the actin cytoskeleton

PubMed Central

Muller, Jean; Mehlen, André; Vetter, Guillaume; Yatskou, Mikalai; Muller, Arnaud; Chalmel, Frédéric; Poch, Olivier; Friederich, Evelyne; Vallar, Laurent

2007-01-01

Background The actin cytoskeleton plays a crucial role in supporting and regulating numerous cellular processes. Mutations or alterations in the expression levels affecting the actin cytoskeleton system or related regulatory mechanisms are often associated with complex diseases such as cancer. Understanding how qualitative or quantitative changes in expression of the set of actin cytoskeleton genes are integrated to control actin dynamics and organisation is currently a challenge and should provide insights in identifying potential targets for drug discovery. Here we report the development of a dedicated microarray, the Actichip, containing 60-mer oligonucleotide probes for 327 genes selected for transcriptome analysis of the human actin cytoskeleton. Results Genomic data and sequence analysis features were retrieved from GenBank and stored in an integrative database called Actinome. From these data, probes were designed using a home-made program (CADO4MI) allowing sequence refinement and improved probe specificity by combining the complementary information recovered from the UniGene and RefSeq databases. Actichip performance was analysed by hybridisation with RNAs extracted from epithelial MCF-7 cells and human skeletal muscle. Using thoroughly standardised procedures, we obtained microarray images with excellent quality resulting in high data reproducibility. Actichip displayed a large dynamic range extending over three logs with a limit of sensitivity between one and ten copies of transcript per cell. The array allowed accurate detection of small changes in gene expression and reliable classification of samples based on the expression profiles of tissue-specific genes. When compared to two other oligonucleotide microarray platforms, Actichip showed similar sensitivity and concordant expression ratios. Moreover, Actichip was able to discriminate the highly similar actin isoforms whereas the two other platforms did not. Conclusion Our data demonstrate that Actichip is a powerful alternative to commercial high density microarrays for cytoskeleton gene profiling in normal or pathological samples. Actichip is available upon request. PMID:17727702

Nucleation Promoting Factors Regulate the Expression and Localization of Arp2/3 Complex during Meiosis of Mouse Oocytes

PubMed Central

Liu, Jun; Wang, Qiao-Chu; Wang, Fei; Duan, Xing; Dai, Xiao-Xin; Wang, Teng; Liu, Hong-Lin; Cui, Xiang-Shun; Kim, Nam-Hyung; Sun, Shao-Chen

2012-01-01

The actin nucleation factor Arp2/3 complex is a main regulator of actin assembly and is involved in multiple processes like cell migration and adhesion, endocytosis, and the establishment of cell polarity in mitosis. Our previous work showed that the Arp2/3 complex was involved in the actin-mediated mammalian oocyte asymmetric division. However, the regulatory mechanisms and signaling pathway of Arp2/3 complex in meiosis is still unclear. In the present work, we identified that the nucleation promoting factors (NPFs) JMY and WAVE2 were necessary for the expression and localization of Arp2/3 complex in mouse oocytes. RNAi of both caused the degradation of actin cap intensity, indicating the roles of NPFs in the formation of actin cap. Moreover, JMY and WAVE2 RNAi decreased the expression of ARP2, a key component of Arp2/3 complex. However, knock down of Arp2/3 complex by Arpc2 and Arpc3 siRNA microinjection did not affect the expression and localization of JMY and WAVE2. Our results indicate that the NPFs, JMY and WAVE2, are upstream regulators of Arp2/3 complex in mammalian oocyte asymmetric division. PMID:23272233

Lifeact-mEGFP Reveals a Dynamic Apical F-Actin Network in Tip Growing Plant Cells

PubMed Central

Hepler, Peter K.; Bezanilla, Magdalena

2009-01-01

Background Actin is essential for tip growth in plants. However, imaging actin in live plant cells has heretofore presented challenges. In previous studies, fluorescent probes derived from actin-binding proteins often alter growth, cause actin bundling and fail to resolve actin microfilaments. Methodology/Principal Findings In this report we use Lifeact-mEGFP, an actin probe that does not affect the dynamics of actin, to visualize actin in the moss Physcomitrella patens and pollen tubes from Lilium formosanum and Nicotiana tobaccum. Lifeact-mEGFP robustly labels actin microfilaments, particularly in the apex, in both moss protonemata and pollen tubes. Lifeact-mEGFP also labels filamentous actin structures in other moss cell types, including cells of the gametophore. Conclusions/Significance Lifeact-mEGFP, when expressed at optimal levels does not alter moss protonemal or pollen tube growth. We suggest that Lifeact-mEGFP represents an exciting new versatile probe for further studies of actin's role in tip growing plant cells. PMID:19478943

[Effect of shenluotong decoction on renal interstitial fibrosis in rats with obstructive nephropathy].

PubMed

Wang, Zheng; Liang, Li-juan; Wang, Cong-hui; Wang, Rui; Jiang, Guo-wang; Zhang, Xiao-man; An, Ya-juan; Xu, Qing-you

2014-10-01

To observe the effect of Shenluotong Decoction (SD) on serum levels of aldosterone, monocyte chemoattractant protein-1 (MCP-1), α-smooth muscle protein (α-SMA), and nuclear factor-KB (NF-κB) in obstructive nephropathy rats, and to explore the initial mechanism of SD for inhibiting renal interstitial fibrosis. Totally 48 healthy Wistar rats were randomly divided into the sham-operation group (n =12) and the model group (n =36). Renal interstitial fibrosis rat model was established by unilateral ureteral obstruction (UUO). After successful modeling, 36 rats were randomly divided into the model group, the Chinese medicine group, and the Western medicine group, 12 in each group. Eplerenone was added in the forage at the daily dose of 100 mg/kg for rats in the Western medicine group. Chinese medicine was added in the forage at the daily dose of 26 g/kg for rats in the Chinese medicine group. Equal volume of normal saline was administered to rats in the sham-operation group and the model group. All medication was performed once daily. The obstructive kidneys were extracted ten days after medication. The pathomorphological changes were observed. The contents of serum aldosterone and MCP-1, and the protein or mRNA expression of MCP-1, α-SMA, and NF-KB were detected. Compared with the sham-operation group, infiltration of a large amount of inflammatory cells and collagen deposition significantly increased, serum contents of aldosterone and MCP-1 obviously increased (P < 0.01), the expression of MCP-1 mRNA and protein were significantly up-regulated (P <0.01), the protein expression of α-SMA and NF-KB were significantly enhanced in the model group (P <0.01). Com- pared with the model group, infiltration of inflammatory cells and renal collagen deposition were attenua- ted in the Chinese medicine group and the Western medicine group, the serum MCP-1 level were reduced, and the mRNA and protein expression of MCP-1 were significantly down-regulated (P <0.01), the protein expression of α-SMA and NF-KB were obviously inhibited (P <0. 01). At the same time, serum aldosterone level was reduced in the Chinese medicine group (P <0.01). inflammatory lesions of the renal tissue could promote the progress of interstitial fibrosis in rats with obstructive nephropathy. SD could attenuate interstitial fibrosis through reducing serum contents of aldosterone and MCP-1, down-regulating MCP-1/ NF-KB, and inhibiting the expression of α-SMA.

Characterization of the Biochemical Properties and Biological Function of the Formin Homology Domains of Drosophila DAAM*

PubMed Central

Barkó, Szilvia; Bugyi, Beáta; Carlier, Marie-France; Gombos, Rita; Matusek, Tamás; Mihály, József; Nyitrai, Miklós

2010-01-01

We characterized the properties of Drosophila melanogaster DAAM-FH2 and DAAM-FH1-FH2 fragments and their interactions with actin and profilin by using various biophysical methods and in vivo experiments. The results show that although the DAAM-FH2 fragment does not have any conspicuous effect on actin assembly in vivo, in cells expressing the DAAM-FH1-FH2 fragment, a profilin-dependent increase in the formation of actin structures is observed. The trachea-specific expression of DAAM-FH1-FH2 also induces phenotypic effects, leading to the collapse of the tracheal tube and lethality in the larval stages. In vitro, both DAAM fragments catalyze actin nucleation but severely decrease both the elongation and depolymerization rate of the filaments. Profilin acts as a molecular switch in DAAM function. DAAM-FH1-FH2, remaining bound to barbed ends, drives processive assembly of profilin-actin, whereas DAAM-FH2 forms an abortive complex with barbed ends that does not support profilin-actin assembly. Both DAAM fragments also bind to the sides of the actin filaments and induce actin bundling. These observations show that the D. melanogaster DAAM formin represents an extreme class of barbed end regulators gated by profilin. PMID:20177055

ROCK inhibition as a therapy for spinal muscular atrophy: understanding the repercussions on multiple cellular targets

PubMed Central

Coque, Emmanuelle; Raoul, Cédric; Bowerman, Mélissa

2014-01-01

Spinal muscular atrophy (SMA) is the most common genetic disease causing infant death, due to an extended loss of motoneurons. This neuromuscular disorder results from deletions and/or mutations within the Survival Motor Neuron 1 (SMN1) gene, leading to a pathological decreased expression of functional full-length SMN protein. Emerging studies suggest that the small GTPase RhoA and its major downstream effector Rho kinase (ROCK), which both play an instrumental role in cytoskeleton organization, contribute to the pathology of motoneuron diseases. Indeed, an enhanced activation of RhoA and ROCK has been reported in the spinal cord of an SMA mouse model. Moreover, the treatment of SMA mice with ROCK inhibitors leads to an increased lifespan as well as improved skeletal muscle and neuromuscular junction pathology, without preventing motoneuron degeneration. Although motoneurons are the primary target in SMA, an increasing number of reports show that other cell types inside and outside the central nervous system contribute to SMA pathogenesis. As administration of ROCK inhibitors to SMA mice was systemic, the improvement in survival and phenotype could therefore be attributed to specific effects on motoneurons and/or on other non-neuronal cell types. In the present review, we will present the various roles of the RhoA/ROCK pathway in several SMA cellular targets including neurons, myoblasts, glial cells, cardiomyocytes and pancreatic cells as well as discuss how ROCK inhibition may ameliorate their health and function. It is most likely a concerted influence of ROCK modulation on all these cell types that ultimately lead to the observed benefits of pharmacological ROCK inhibition in SMA mice. PMID:25221469

Expression of matrix metalloproteinases 9 and 12 in actinic cheilitis

PubMed Central

Poulopoulos, Athanasios K; Andreadis, Dimitrios; Markopoulos, Anastasios K

2013-01-01

AIM: To investigate the role of matrix-degrading metalloproteinases 9, 12 (MMPs), as mediators of functional connective tissue damage in actinic cheilitis. METHODS: Thirty five formalin-fixed, paraffin embedded specimens of actinic cheilitis, and twelve specimens of normal lower lip vermillion, which were obtained by the archives of the Department of Oral Medicine and Maxillofacial Pathology, were examined. From each block, 5 μm thick sections were cut and routinely stained with Hematoxylin and Eosin. Immunohistochemical studies were performed on 4-μm thick sections of formalin-fixed paraffin embedded actinic cheilitis lesions and of normal lower lip vermillion, for MMP-9 and MMP-12 in serial sections of our specimens. Appropriate positive and negative controls were performed to confirm the specificity of the staining reaction. MMP immunohistochemistry was evaluated using a semiquantitative immunoreactive score. RESULTS: Haematoxylin and eosin staining revealed in actinic cheilitis lesions atrophic stratified squamous cell epithelium, or focally and irregularly hyperplastic of variable thickness, in some areas was observed marked keratin production. Varying degrees of epithelial dysplasia were noticed with a wide spectrum of change within the same specimen. Characteristic was the appearance of chronic inflammatory infiltration, and a band of amorphous acellular, basophilic change like solar elastosis (elastin replacement of collagen). In normal lower lip specimens weak and scanty positive expression of MMP-9 and MMP-12 was observed. Anti-MMP-9 antibody showed a weak reaction, in actinic cheilitis lesions, focal in the elastotic material, in chronic inflammatory cells and mostly in macrophages and neutrophils. Strong and in some cases diffused immunohistochemical expression of MMP-12 was detected in actinic cheilitis lesions in the areas of the fragmented, distorted and thickened elastic fibers. MMP-12 was also expressed in chronic inflammatory cells and mostly macrophages. MMP-12 was significantly higher in actinic cheilitis specimens compared with the normal lower lip specimens (P = 0.0029). CONCLUSION: Our results suggest that especially MMP-12 may play an important role in remodeling events occurring in the connective tissue during long-term exposure to sunlight in the actinic cheilitis lesions. PMID:24520545

Roles of Asp179 and Glu270 in ADP-Ribosylation of Actin by Clostridium perfringens Iota Toxin

PubMed Central

Belyy, Alexander; Tabakova, Irina; Lang, Alexander E.; Jank, Thomas; Belyi, Yury; Aktories, Klaus

2015-01-01

Clostridium perfringens iota toxin is a binary toxin composed of the enzymatically active component Ia and receptor binding component Ib. Ia is an ADP-ribosyltransferase, which modifies Arg177 of actin. The previously determined crystal structure of the actin-Ia complex suggested involvement of Asp179 of actin in the ADP-ribosylation reaction. To gain more insights into the structural requirements of actin to serve as a substrate for toxin-catalyzed ADP-ribosylation, we engineered Saccharomyces cerevisiae strains, in which wild type actin was replaced by actin variants with substitutions in residues located on the Ia-actin interface. Expression of the actin mutant Arg177Lys resulted in complete resistance towards Ia. Actin mutation of Asp179 did not change Ia-induced ADP-ribosylation and growth inhibition of S. cerevisiae. By contrast, substitution of Glu270 of actin inhibited the toxic action of Ia and the ADP-ribosylation of actin. In vitro transcribed/translated human β-actin confirmed the crucial role of Glu270 in ADP-ribosylation of actin by Ia. PMID:26713879

Increased expression of enhancer of Zeste Homolog 2 (EZH2) differentiates squamous cell carcinoma from normal skin and actinic keratosis.

PubMed

Xie, Qiang; Wang, Hongbei; Heilman, Edward R; Walsh, Michael G; Haseeb, M A; Gupta, Raavi

2014-01-01

Enhancer of Zeste Homolog 2 (EZH2) is a polycomb group protein that has been shown to be involved in the progression of multiple human cancers including melanoma. The expression of EZH2 in normal skin and in pre-malignant and malignant cutaneous squamous cell carcinoma (SCC) has not been studied. We examined the expression of EZH2 in normal skin, actinic keratosis (AK), SCC in situ, well-differentiated (SCC-WD), moderately-differentiated (SCC-MD) and poorly-differentiated SCC (SCC-PD) to ascertain whether EZH2 expression differentiates these conditions. Immunohistochemical staining for EZH2 was performed on formalin-fixed paraffin-embedded biopsies and a tissue microarray containing normal skin, AK, SCC in situ, and SCC of different grades. In comparison to the normal skin, EZH2 expression in actinic keratosis was increased (p=0.03). Similarly, EZH2 expression in all of the neoplastic conditions studied (SCC in situ, SCC-WD, SCC-MD and SCC-PD) was greatly increased in comparison to both the normal skin and actinic keratosis (p≤0.001). EZH2 expression increases incrementally from normal skin to AK and further to SCC, suggesting a role for EZH2 in the progression and differentiation of SCC. EZH2 expression may be used as a diagnostic marker for differentiating SCC from AK or normal skin.

Defining adipose tissue-derived stem cells in tissue and in culture.

PubMed

Lin, Ching-Shwun; Xin, Zhong-Cheng; Deng, Chun-Hua; Ning, Hongxiu; Lin, Guiting; Lue, Tom F

2010-06-01

Adipose tissue-derived stem cells (ADSC) are routinely isolated from the stromal vascular fraction (SVF) of homogenized adipose tissue. Similar to other types of mesenchymal stem cells (MSC), ADSC remain difficult to define due to the lack of definitive cellular markers. Still, many types of MSC, including ADSC, have been shown to reside in a perivascular location, and increasing evidence shows that both MSC and ADSC may in fact be vascular stem cells (VSC). Locally, these cells differentiate into smooth muscle and endothelial cells that are assembled into newly formed blood vessels during angiogenesis and neovasculogenesis. Additionally, MSC or ADSC can also differentiate into tissue cells such as adipocytes in the adipose tissue. Systematically, MSC or ADSC are recruited to injury sites where they participate in the repair/regeneration of the injured tissue. Due to the vasculature's dynamic capacity for growth and multipotential nature for diversification, VSC in tissue are individually at various stages and on different paths of differentiation. Therefore, when isolated and put in culture, these cells are expected to be heterogeneous in marker expression, renewal capacity, and differentiation potential. Although this heterogeneity of VSC does impose difficulties and cause confusions in basic science studies, its impact on the development of VSC as a therapeutic cell source has not been as apparent, as many preclinical and clinical trials have reported favorable outcomes. With this understanding, ADSC are generally defined as CD34+CD31- although loss of CD34 expression in culture is well documented. In adipose tissue, CD34 is localized to the intima and adventitia of blood vessels but not the media where cells expressing alpha-smooth muscle actin (SMA) exist. By excluding the intima, which contains the CD34+CD31+ endothelial cells, and the media, which contains the CD34-CD31- smooth muscle cells, it leaves the adventitia as the only possible location for the CD34+ ADSC. In the capillary, CD34 and CD140b (a pericyte marker) are mutually exclusively expressed, thus suggesting that pericytes are not the CD34+ ADSC. Many other cellular markers for vascular cells, stem cells, and stem cell niche have also been investigated as possible ADSC markers. Particularly the best-known MSC marker STRO-1 has been found either expressed or not expressed in cultured ADSC. In the adipose tissue, STRO-1 appears to be expressed exclusively in the endothelium of certain but not all blood vessels, and thus not associated with the CD34+ ADSC. In conclusion, we believe that ADSC exist as CD34+CD31-CD104b-SMA- cells in the capillary and in the adventitia of larger vessels. In the capillary these cells coexist with pericytes and endothelial cells, both of which are possibly progenies of ADSC (or more precisely VSC). In the larger vessels, these ADSC or VSC exist as specialized fibroblasts (having stem cell properties) in the adventitia.

Pattern of galectins expression in actinic cheilitis with different risks of malignant transformation.

PubMed

Lopes, Maria Luiza Diniz de Sousa; Nonaka, Cassiano Francisco Weege; Queiroz, Lélia Maria Guedes; de Souza, Lélia Batista; Miguel, Márcia Cristina da Costa; da Silveira, Éricka Janine Dantas

2016-09-01

Actinic cheilitis (AC) is a chronic inflammatory lesion that in some situations can turn into squamous cell carcinoma of the lip. The molecular mechanisms involved in this process are not yet completely understood. This study aimed to investigate the expression pattern of galectins in actinic cheilitis according to the histopathological grading. Immunoexpression of galectin-1, galectin-3, galectin-7, and galectin-9 was semiquantitatively analyzed in 65 cases of actinic cheilitis graded as low risk (n = 40) or high risk (n = 25) of malignant transformation. Association between the location of the galectins in the cellular compartments and histopathological grading was analyzed. Galectin-1 was mainly observed in the cell cytoplasm, and was elevated (score 3) in 60% of cases, regardless of the histopathological grade (P > 0.05). Galectin-3 expression was higher in high-risk group than in the low-risk group (P < 0.05), with a predominant expression in the cytoplasm and nucleus of low-risk (67.5%), and only in the cytoplasm of high-risk cases (60%) (P < 0.05). Galectin-7 expression did not show significant differences between low-risk and high-risk groups (P > 0.05). With respect to galectin-9, 89.2% of cases were positive, showing decrease in median of scores as there was an increase in histological grade (P < 0.001), with predominant expression in the nucleus and cytoplasm. This study is the first indication of galectins involvement in the pathogenesis and morphologic progression of actinic cheilitis, particularly galectin-3 and galectin-9. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A WAVE2-Abi1 complex mediates CSF-1-induced F-actin-rich membrane protrusions and migration in macrophages.

PubMed

Kheir, Wassim Abou; Gevrey, Jean-Claude; Yamaguchi, Hideki; Isaac, Beth; Cox, Dianne

2005-11-15

Colony-stimulating factor 1 (CSF-1) is an important physiological chemoattractant for macrophages. The mechanisms by which CSF-1 elicits the formation of filamentous actin (F-actin)-rich membrane protrusions and induces macrophage migration are not fully understood. In particular, very little is known regarding the contribution of the different members of the Wiskott-Aldrich Syndrome protein (WASP) family of actin regulators in response to CSF-1. Although a role for WASP itself in macrophage chemotaxis has been previously identified, no data was available regarding the function of WASP family verprolin-homologous (WAVE) proteins in this cell type. We found that WAVE2 was the predominant isoform to be expressed in primary macrophages and in cells derived from the murine monocyte/macrophage RAW264.7 cell line (RAW/LR5). CSF-1 treatment of macrophages resulted in WAVE2 accumulation in F-actin-rich protrusions induced by CSF-1. Inhibition of WAVE2 function by expressing a dominant-negative mutant or introducing anti-WAVE2 antibodies in RAW/LR5 cells, as well as reduction of endogenous WAVE2 expression by RNA-mediated interference (RNAi), resulted in a significant reduction of CSF-1-elicited F-actin protrusions. WAVE2 was found in a protein complex together with Abelson kinase interactor 1 (Abi1) in resting or stimulated cells. Both WAVE2 and Abi1 were recruited to and necessary for the formation of F-actin protrusions in response to CSF-1. Reducing the levels of WAVE2, directly or by targeting Abi1, resulted in an impaired cell migration to CSF-1. Altogether these data identify a WAVE2-Abi1 complex crucial for the normal actin cytoskeleton reorganization and migration of macrophages in response to CSF-1.

Aneurysmal and haemangiopericytoma-like fibrous histiocytoma.

PubMed Central

Zelger, B W; Zelger, B G; Steiner, H; Ofner, D

1996-01-01

AIM: To describe the clinicopathological features of 33 aneurysmal fibrous histiocytomas (AFH), including five cases with a haemangiopericytoma-like pattern. METHODS: Thirty three cases of AFH were studied by using routine histology and immunohistochemistry for factor XIIIa, the "cell activity marker" E9 (anti-metallothionein), NK1C3 (CD57), smooth muscle actin (SMA), factor VIII, ulex europaeus agglutinin, JC70A (CD31), and QBEND10 (CD34). The time dependent variation in histopathological features was evaluated by statistical methods (Pearson chi 2, likelihood ratio chi 2). RESULTS: Of the AFHs, 29 of 33 occurred on the extremities of adults (age range 30 to 50 years), six of which were associated with rapid growth, probably caused by trauma, and pain. Twenty one lesions were thought to be vascular and/or melanocytic lesions, including two melanomas, because of a bluish-black and/or cystic appearance. Histologically, large areas of haemorrhage, up to 50% of the tumour bulk, lacking an endothelial lining were seen in otherwise typical fibrous histiocytomas. Five cases resembled nodular stages of Kaposi's sarcoma. Variable haemosiderin deposition in histiocytes (18/33) and giant cells (11/33) was suggestive of haemosiderotic histiocytoma. A haemangiopericytoma-like pattern was seen in five otherwise indistinguishable cases. On immunohistochemistry, variable reactivity was seen for factor XIIIa (18/30), with E9 (18/30), NK1C3 (19/30), and for SMA (14/30), but labelling for vascular markers was not detected. Early lesions without iron deposition were factor XIIIa positive; late lesions with iron deposition were factor XIIIa negative. Labelling for SMA correlated with prominent sclerosis. CONCLUSION: AFHs, including a haemangiopericytoma-like variant, have a characteristic time dependent histological and immunophenotypic profile, clearly different from nodular type Kaposi's sarcoma. Images PMID:8655708

Immunohistochemical evaluation of myofibroblasts in odontogenic cysts and tumors: A comparative study.

PubMed

Syamala, Deepa; Suresh, Rakesh; Janardhanan, Mahija; Savithri, Vindhya; Anand, Prem P; Jose, Amrutha

2016-01-01

Myofibroblasts are fibroblasts with smooth muscle-like features characterized by the presence of a contractile apparatus and found in the connective tissue stroma of normal tissues such as blood vessels and lymph nodes. They are now thought to play a role in the synthesis and reorganization of extracellular matrix, which could contribute to the aggressive biologic behavior of the lesions. To compare the mean number of stromal myofibroblasts in dentigerous cysts (DCs), keratocystic odontogenic tumor (KCOT) and ameloblastoma; and to derive a correlation between the stromal myofibroblasts and the known biologic behavior of the lesions. A cross-sectional immunohistochemical analysis of cases of DC, KCOT and ameloblastoma. Twenty paraffin-embedded tissue blocks each of DC, KCOT and multicystic ameloblastoma were selected for the study and diagnosis confirmed through hematoxylin and eosin staining. Tissue sections were analyzed for the number of myofibroblasts using alpha smooth muscle actin (α-SMA) immunostaining. Differences in the mean number of α-SMA positive cells in each group were analyzed using one-way ANOVA test. Intergroup comparisons of mean values of α-SMA positive cells were performed using Mann-Whitney U-test. Ameloblastoma showed the highest number of myofibroblasts, whereas DC showed the lowest. Among the groups, there were significant differences between the myofibroblast counts among DC and KCOT and between DC and ameloblastoma, whereas the difference in counts was not statistically significant between KCOT and ameloblastoma. A positive correlation was observed between the myofibroblast count and the known biologic behavior of the lesions. Myofibroblasts may act in close association with the epithelial cells to bring about changes in stromal microenvironment, favorable to the growth and progression of the lesion. They may be of great value in predicting the biologic behavior and growth potential of such lesions.

The Effect of Microporous Polysaccharide Hemospheres on Wound Healing and Scarring in Wild-Type and db/db Mice.

PubMed

Miller, Kyle J; Cao, Wei; Ibrahim, Mohamed M; Levinson, Howard

2017-04-01

Hemostasis, the initial phase of wound healing, sets the stage for tissue repair. Microporous polysaccharide hemosphere powder (MPH) is an FDA-approved hemostatic agent that may impact the wound-healing process. This study examined the role of MPH in murine wild-type and diabetic (db/db) wound-healing models and a foreign body response scarring model. The powder was topically applied to excisional wounds in wild-type C57BL/6 mice and db/db mice. The effect of MPH on scarring was evaluated by applying it to the expanded polytetrafluoroethylene tube implantation model. In wild-type mice, topically applied MPH increased epithelial thickness. Levels of α-smooth muscle actin (α-SMA) were decreased in MPH-treated wild-type wounds, whereas Rho-associated protein kinase 2 (ROCK2) and transforming growth factor β levels were increased. In db/db mice, topical wound MPH application decreased epithelial thickness and delayed wound closure. The db/db wounds displayed an increased collagen index. The ROCK2 was increased in a similar manner to wild-type mice, whereas α-SMA and transforming growth factor β levels were decreased. The MPH-treated expanded polytetrafluoroethylene tube mice showed increased α-SMA levels and depressed ROCK2 levels. There were no changes in histologic parameters of the foreign body response. The results suggest that MPH does not adversely impact wound healing in wild-type mice, both topically and around implants, but prolongs time to closure and diminishes thickness in db/db wounds. The MPH application alters contractile proteins in all wound models. These changes could have downstream effects on the wound healing process, and further investigation into the use of MPH in altered or impaired states of wound healing is warranted.

Bone-repair properties of biodegradable hydroxyapatite nano-rod superstructures

NASA Astrophysics Data System (ADS)

D'Elía, Noelia L.; Mathieu, Colleen; Hoemann, Caroline D.; Laiuppa, Juan A.; Santillán, Graciela E.; Messina, Paula V.

2015-11-01

Nano-hydroxyapatite (nano-HAp) materials show an analogous chemical composition to the biogenic mineral components of calcified tissues and depending on their topography they may mimic the specific arrangement of the crystals in bone. In this work, we have evaluated the potential of four synthesized nano-HAp superstructures for the in vitro conditions of bone-repair. Experiments are underway to investigate the effects of the material microstructure, surface roughness and hydrophilicity on their osseo-integration, osteo-conduction and osteo-induction abilities. Materials were tested in the presence of both, rat primary osteoblasts and rabbit mesenchymal stem cells. The following aspects are discussed: (i) cytotoxicity and material degradation; (ii) rat osteoblast spreading, proliferation and differentiation; and (iii) rabbit mesenchymal stem cell adhesion on nano-HAp and nano-HAp/collagen type I coatings. We effectively prepared a material based on biomimetic HAp nano-rods displaying the appropriate surface topography, hydrophilicity and degradation properties to induce the in vitro desired cellular responses for bone bonding and healing. Cells seeded on the selected material readily attached, proliferated and differentiated, as confirmed by cell viability, mitochondrial metabolic activity, alkaline phosphatase (ALP) activity and cytoskeletal integrity analysis by immunofluorescence localization of alpha-smooth muscle actin (α-SMA) protein. These results highlight the influence of material's surface characteristics to determine their tissue regeneration potential and their future use in engineering osteogenic scaffolds for orthopedic implants.Nano-hydroxyapatite (nano-HAp) materials show an analogous chemical composition to the biogenic mineral components of calcified tissues and depending on their topography they may mimic the specific arrangement of the crystals in bone. In this work, we have evaluated the potential of four synthesized nano-HAp superstructures for the in vitro conditions of bone-repair. Experiments are underway to investigate the effects of the material microstructure, surface roughness and hydrophilicity on their osseo-integration, osteo-conduction and osteo-induction abilities. Materials were tested in the presence of both, rat primary osteoblasts and rabbit mesenchymal stem cells. The following aspects are discussed: (i) cytotoxicity and material degradation; (ii) rat osteoblast spreading, proliferation and differentiation; and (iii) rabbit mesenchymal stem cell adhesion on nano-HAp and nano-HAp/collagen type I coatings. We effectively prepared a material based on biomimetic HAp nano-rods displaying the appropriate surface topography, hydrophilicity and degradation properties to induce the in vitro desired cellular responses for bone bonding and healing. Cells seeded on the selected material readily attached, proliferated and differentiated, as confirmed by cell viability, mitochondrial metabolic activity, alkaline phosphatase (ALP) activity and cytoskeletal integrity analysis by immunofluorescence localization of alpha-smooth muscle actin (α-SMA) protein. These results highlight the influence of material's surface characteristics to determine their tissue regeneration potential and their future use in engineering osteogenic scaffolds for orthopedic implants. Electronic supplementary information (ESI) available: Calculation of roughness parameters Rz, Rz,max, and Rz, prom. Nano-HAp powder degradation after immersion in phosphate buffer (pH = 7.4). Optical phase contrast microphotographs of MSC adhesion on nano-HAp and nano-HAp/Co I coatings at different concentrations. Laser scanning confocal microphotographs of MSCs' α-SMA expression spreading on large amounts of nano-HAp (MI) coatings. Immunofluorescence microphotograph analysis by image software. See DOI: 10.1039/c5nr04850h

[Salidroside inhibits hypoxia-induced phenotypic modulation of corpus cavernosum smooth muscle cells in vitro].

PubMed

Chen, Gang; Huang, Xiao-Jun; Lü, Bo-Dong; Chen, Shi-Tao; Zhang, Shi-Geng; Yang, Ke-Bing

2013-08-01

To explore the effects of salidroside on the phenotypic modulation of corpus cavernosum smooth muscle cells (CCSMC) in hypoxic SD rats. CCSMCs were cultured in vitro and identified by immunohistochemistry. The cells were divided into six groups: normal control (21% O2), hypoxia (1% O2), hypoxia + salidroside 1 mg/L, hypoxia + salidroside 3 mg/L, hypoxia + salidroside 5 mg/L and hypoxia + PGE1 0.4 microg/L, and then cultured for 48 hours. The relative expressions of alpha-actin and osteopontin (OPN) in each group were determined by RT-PCR. The in vitro cultured CCSMCs grew well, with anti-alpha-smooth muscle actin monoclonal antibodies immunohistochemically positive. The relative expression of alpha-actin was markedly decreased while that of OPN remarkably increased in the hypoxia group as compared with the normal control group (P < 0.01). The hypoxia + salidroside 5 mg/L group showed a significantly higher expression of alpha-actin and lower expression of OPN than the hypoxia group (P < 0.01), but exhibited no significant differences from the hypoxia + PGE group (P > 0.05). Hypoxia can reduce the relative expression level of alpha-actin and increase that of OPN in the CCSMCs of SD rats, namely, induce their phenotypic modulation from the contraction to the non-contraction type. Salidroside can restrain hypoxia-induced phenotypic modulation of CCSMCs, and its inhibitory effect at 5 mg/L is similar to that of PGE1.

Homologous peptide of connective tissue growth factor ameliorates epithelial to mesenchymal transition of tubular epithelial cells.

PubMed

Shi, Yujun; Tu, Zhidan; Wang, Wei; Li, Qing; Ye, Feng; Wang, Jinjing; Qiu, Jing; Zhang, Li; Bu, Hong; Li, Youping

2006-10-01

The hallmark of failing renal transplants is tubular atrophy and interstitial fibrosis. The cytokine connective tissue growth factor (CTGF or CCN2) plays an important role in epithelial-mesenchymal transition (EMT) of tubular epithelial cells (TECs). A unique domain within CTGF (IRTPKISKPIKFELSG) which binds to its potential receptor integrin alpha v beta3 has been identified. This study was carried out to further characterize a synthetic hexadeca-peptide (P2) homologous to this domain and to determine its effect on CTGF-mediated solid phase cell adhesion, EMT induction and fibrogenesis in rat renal NRK-52E cells. Results showed that both P2 and recombinant CTGF bound to NRK-52E cells. Unlike CTGF, P2 had little effect on EMT induction including cytoskeleton remodeling and expression of alpha-smooth muscle actin (alpha-SMA) and E-cadherin, nor did it have effect on fibrogenic induction including alternation of extracellular matrix (ECM) proteins, collagen type I and IV at gene and protein levels. All data showed that P2 bound preferably on the surface of NRK-52E cells and inhibited the effect of CTGF on EMT induction and cell fibrogenesis, probably by occupying the binding sites of CTGF within its potential receptors. Therefore, P2 may be used as a potential anti-fibrotic agent.

Expression of cytoskeletal proteins, cross-reacting with anti-CYP1A, in Mytilus sp. exposed to organic contaminants.

PubMed

Jonsson, Henrik; Schiedek, Doris; Goksøyr, Anders; Grøsvik, Bjørn Einar

2006-06-01

The possible use of cytoskeletal components as biomarkers of organic pollution in mussels has been investigated. Responses of non-muscular actin and tropomyosin (TM), two bivalve proteins that were recently demonstrated to cross-react with anti-fish-CYP1A, were analysed in digestive tissue of blue mussels (Mytilus sp.) exposed to a wide range of organic contaminants. The results were evaluated with ELISA and Western blot assays, utilising commercial monoclonal antibodies, and compared with expression of Hsp70, a marker of chemical stress. Furthermore, mussels were sampled from the Baltic Sea at sites with different degrees of pollution to assess the expression of these proteins, and to monitor seasonal changes in relation to energy reserves and water temperature. The results demonstrated that expression of microsomal actin was significantly higher (p<0.02) in mussels exposed to a brominated flame retardant (BDE-47), and lower, however not significantly, in specimens exposed to crude oil, alone and spiked with alkylphenols and PAHs. Hsp70 was strongly induced in all exposure groups, which also included bisphenol A and diallylphthalate. Furthermore, microsomal actin exhibited seasonal variations, and expression was negatively correlated with water temperature. No correlation was seen between actin and the microfilament-binding protein TM, indicating that regulation of these two cytoskeletal components are not coupled. Furthermore, parallel and significant (p<0.05) up-regulations of TM and Hsp70 were seen in individuals sampled from a strongly polluted field site, whereas the seasonal analysis showed that TM expression was positively correlated with energy reserves (total glycogen content) in mussels, suggesting the use of TM as a marker of growth. In conclusion, this study has demonstrated the cytoskeleton to be a target of contaminants in mussels, calling for further attention. Exposure-induced increase of microsomal actin can be interpreted either as stimulated actin synthesis, or re-arrangements of the dynamic microfilaments.

Systemic peptide-mediated oligonucleotide therapy improves long-term survival in spinal muscular atrophy

PubMed Central

Hazell, Gareth; Shabanpoor, Fazel; Saleh, Amer F.; Bowerman, Melissa; Meijboom, Katharina E.; Zhou, Haiyan; Muntoni, Francesco; Talbot, Kevin; Gait, Michael J.; Wood, Matthew J. A.

2016-01-01

The development of antisense oligonucleotide therapy is an important advance in the identification of corrective therapy for neuromuscular diseases, such as spinal muscular atrophy (SMA). Because of difficulties of delivering single-stranded oligonucleotides to the CNS, current approaches have been restricted to using invasive intrathecal single-stranded oligonucleotide delivery. Here, we report an advanced peptide-oligonucleotide, Pip6a-morpholino phosphorodiamidate oligomer (PMO), which demonstrates potent efficacy in both the CNS and peripheral tissues in severe SMA mice following systemic administration. SMA results from reduced levels of the ubiquitously expressed survival motor neuron (SMN) protein because of loss-of-function mutations in the SMN1 gene. Therapeutic splice-switching oligonucleotides (SSOs) modulate exon 7 splicing of the nearly identical SMN2 gene to generate functional SMN protein. Pip6a-PMO yields SMN expression at high efficiency in peripheral and CNS tissues, resulting in profound phenotypic correction at doses an order-of-magnitude lower than required by standard naked SSOs. Survival is dramatically extended from 12 d to a mean of 456 d, with improvement in neuromuscular junction morphology, down-regulation of transcripts related to programmed cell death in the spinal cord, and normalization of circulating insulin-like growth factor 1. The potent systemic efficacy of Pip6a-PMO, targeting both peripheral as well as CNS tissues, demonstrates the high clinical potential of peptide-PMO therapy for SMA. PMID:27621445

Importance of Interaction between Integrin and Actin Cytoskeleton in Suspension Adaptation of CHO cells.

PubMed

Walther, Christa G; Whitfield, Robert; James, David C

2016-04-01

The biopharmaceutical production process relies upon mammalian cell technology where single cells proliferate in suspension in a chemically defined synthetic environment. This environment lacks exogenous growth factors, usually contributing to proliferation of fibroblastic cell types such as Chinese hamster ovary (CHO) cells. Use of CHO cells for production hence requires a lengthy 'adaptation' process to select clones capable of proliferation as single cells in suspension. The underlying molecular changes permitting proliferation in suspension are not known. Comparison of the non-suspension-adapted clone CHO-AD and a suspension-adapted propriety cell line CHO-SA by flow cytometric analysis revealed a highly variable bi-modal expression pattern for cell-to-cell contact proteins in contrast to the expression pattern seen for integrins. Those have a uni-modal expression on suspension and adherent cells. Integrins showed a conformation distinguished by regularly distributed clusters forming a sphere on the cell membrane of suspension-adapted cells. Actin cytoskeleton analysis revealed reorganisation from the typical fibrillar morphology found in adherent cells to an enforced spherical subcortical actin sheath in suspension cells. The uni-modal expression and specific clustering of integrins could be confirmed for CHO-S, another suspension cell line. Cytochalasin D treatment resulted in breakdown of the actin sheath and the sphere-like integrin conformation demonstrating the link between integrins and actin in suspension-adapted CHO cells. The data demonstrates the importance of signalling changes, leading to an integrin rearrangement on the cell surface, and the necessity of the reinforcement of the actin cytoskeleton for proliferation in suspension conditions.

Effects of selective bile duct ligation on liver parenchyma in young animals: histologic and molecular evaluations.

PubMed

Tannuri, Ana Cristina A; Coelho, Maria Cecília M; de Oliveira Gonçalves, Josiane; Santos, Maria Mercês; Ferraz da Silva, Luiz Fernando; Bendit, Israel; Tannuri, Uenis

2012-03-01

The mechanisms of increased collagen production and liver parenchyma fibrosis are poorly understood. These phenomena are observed mainly in children with biliary obstruction (BO), and in a great number of patients, the evolution to biliary cirrhosis and hepatic failure leads to the need for liver transplantation before adolescence. However, pediatric liver transplantation presents with biliary complications in 20% to 30% of cases in the postoperative period. Intra- or extrahepatic stenosis of bile ducts is frequent and may lead to secondary biliary cirrhosis and the need for retransplantation. It is unknown whether biliary stenosis involving isolated segments or lobes may affect the adjacent nonobstructed lobes by paracrine or endocrine means, leading to fibrosis in this parenchyma. Therefore, the present study aimed to create an experimental model of selective biliary duct ligation in young animals with a subsequent evaluation of the histologic and molecular alterations in liver parenchyma of the obstructed and nonobstructed lobes. After a pilot study to standardize the surgical procedures, weaning rats underwent ligation of the bile ducts of the median, left lateral, and caudate liver lobes. The bile duct of the right lateral lobe was kept intact. To avoid intrahepatic biliary duct collaterals neoformation, the parenchymal connection between the right lateral and median lobes was clamped. The animals were divided into groups according to the time of death: 1, 2, 3, 4, and 8 weeks after surgical procedure. After death, the median and left lateral lobes (with BO) and the right lateral lobe (without BO [NBO]) were harvested separately. A group of 8 healthy nonoperated on animals served as controls. Liver tissues were subjected to histologic evaluation and quantification of the ductular proliferation and of the portal fibrosis. The expressions of smooth muscle α-actin (α-SMA), desmin, and transforming growth factor β1 genes were studied by molecular analyses (semiquantitative reverse transcriptase-polymerase chain reaction and real-time polymerase chain reaction, a quantitative method). Histologic analyses revealed the occurrence of ductular proliferation and collagen formation in the portal spaces of both BO and NBO lobes. These phenomena were observed later in NBO than BO. Bile duct density significantly increased 1 week after duct ligation; it decreased after 2 and 3 weeks and then increased again after 4 and 8 weeks in both BO and NBO lobes. The portal space collagen area increased after 2 weeks in both BO and NBO lobes. After 3 weeks, collagen deposition in BO was even higher, and in NBO, the collagen area started decreasing after 2 weeks. Molecular analyses revealed increased expression of the α-SMA gene in both BO and NBO lobes. The semiquantitative and quantitative methods showed concordant results. The ligation of a duct responsible for biliary drainage of the liver lobe promoted alterations in the parenchyma and in the adjacent nonobstructed parenchyma by paracrine and/or endocrine means. This was supported by histologic findings and increased expression of α-SMA, a protein related to hepatic fibrogenesis. Copyright © 2012 Elsevier Inc. All rights reserved.

SMA-Causing Missense Mutations in Survival motor neuron (Smn) Display a Wide Range of Phenotypes When Modeled in Drosophila

PubMed Central

Praveen, Kavita; Wen, Ying; Gray, Kelsey M.; Noto, John J.; Patlolla, Akash R.; Van Duyne, Gregory D.; Matera, A. Gregory

2014-01-01

Mutations in the human survival motor neuron 1 (SMN) gene are the primary cause of spinal muscular atrophy (SMA), a devastating neuromuscular disorder. SMN protein has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. Additional tissue-specific and global functions have been ascribed to SMN; however, their relevance to SMA pathology is poorly understood and controversial. Using Drosophila as a model system, we created an allelic series of twelve Smn missense mutations, originally identified in human SMA patients. We show that animals expressing these SMA-causing mutations display a broad range of phenotypic severities, similar to the human disease. Furthermore, specific interactions with other proteins known to be important for SMN's role in RNP assembly are conserved. Intragenic complementation analyses revealed that the three most severe mutations, all of which map to the YG box self-oligomerization domain of SMN, display a stronger phenotype than the null allele and behave in a dominant fashion. In support of this finding, the severe YG box mutants are defective in self-interaction assays, yet maintain their ability to heterodimerize with wild-type SMN. When expressed at high levels, wild-type SMN is able to suppress the activity of the mutant protein. These results suggest that certain SMN mutants can sequester the wild-type protein into inactive complexes. Molecular modeling of the SMN YG box dimer provides a structural basis for this dominant phenotype. These data demonstrate that important structural and functional features of the SMN YG box are conserved between vertebrates and invertebrates, emphasizing the importance of self-interaction to the proper functioning of SMN. PMID:25144193

SMA-causing missense mutations in survival motor neuron (Smn) display a wide range of phenotypes when modeled in Drosophila.

PubMed

Praveen, Kavita; Wen, Ying; Gray, Kelsey M; Noto, John J; Patlolla, Akash R; Van Duyne, Gregory D; Matera, A Gregory

2014-08-01

Mutations in the human survival motor neuron 1 (SMN) gene are the primary cause of spinal muscular atrophy (SMA), a devastating neuromuscular disorder. SMN protein has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. Additional tissue-specific and global functions have been ascribed to SMN; however, their relevance to SMA pathology is poorly understood and controversial. Using Drosophila as a model system, we created an allelic series of twelve Smn missense mutations, originally identified in human SMA patients. We show that animals expressing these SMA-causing mutations display a broad range of phenotypic severities, similar to the human disease. Furthermore, specific interactions with other proteins known to be important for SMN's role in RNP assembly are conserved. Intragenic complementation analyses revealed that the three most severe mutations, all of which map to the YG box self-oligomerization domain of SMN, display a stronger phenotype than the null allele and behave in a dominant fashion. In support of this finding, the severe YG box mutants are defective in self-interaction assays, yet maintain their ability to heterodimerize with wild-type SMN. When expressed at high levels, wild-type SMN is able to suppress the activity of the mutant protein. These results suggest that certain SMN mutants can sequester the wild-type protein into inactive complexes. Molecular modeling of the SMN YG box dimer provides a structural basis for this dominant phenotype. These data demonstrate that important structural and functional features of the SMN YG box are conserved between vertebrates and invertebrates, emphasizing the importance of self-interaction to the proper functioning of SMN.

Folic acid-conjugated amphiphilic alternating copolymer as a new active tumor targeting drug delivery platform.

PubMed

Li, Xia; Szewczuk, Myron R; Malardier-Jugroot, Cecile

2016-01-01

Targeted drug delivery using polymeric nanostructures is an emerging cancer research area, engineered for safer, more efficient, and effective use of chemotherapeutic drugs. A pH-responsive, active targeting delivery system was designed using folic acid functionalized amphiphilic alternating copolymer poly(styrene-alt-maleic anhydride) (FA-DABA-SMA) via a biodegradable linker 2,4-diaminobutyric acid (DABA). The polymeric template is pH responsive, forming amphiphilic nanostructures at pH 7, allowing the encapsulation of hydrophobic drugs on its interior. Moreover, the structure is stable only at neutral pH and collapses in the acidic tumor microenvironment, releasing drugs on-site from its core. The delivery vehicle is investigated using human pancreatic PANC-1 cancer cells and RAW-Blue™ mouse macrophage reporter cell line, both of which have overly expression of folic acid receptors. To trace the cellular uptake by both cell lines, curcumin was selected as a dye and drug mimic owing to its fluorescence nature and hydrophobic properties. Fluorescent microscopy of FA-DABA-SMA loaded with curcumin revealed a significant internalization of the dye by human pancreatic PANC-1 cancer cells compared to those with unfunctionalized polymers (SMA). Moreover, the FA-DABA-SMA polymers exhibit rodlike association specific to the cells. Both empty SMA and FA-DABA-SMA show little toxicity to PANC-1 cells as characterized by WST-1 cell proliferation assay. These results clearly indicate that FA-DABA-SMA polymers show potential as an active tumor targeting drug delivery system with the ability to internalize hydrophobic chemotherapeutics after they specifically attach to cancer cells.

Morphological Changes of Myoepithelial Cells in the Rat Submandibular Gland Following the Application of Surgical Stimuli.

PubMed

Kawabe, Yoshihiro; Mizobe, Kenich; Bando, Yasuhiko; Sakiyama, Koji; Taira, Fuyoko; Tomomura, Akito; Araki, Hisao; Amano, Osamu

2016-12-28

Myoepithelial cells (MECs) exist on the basal surface of acini in major exocrine glands, include myofilaments and various constructive proteins, and share characteristics with smooth muscle and epithelial cells. MECs project several ramified processes to invest acini, and possibly contract to compress acini to support the secretion by the glandular cells. However, the functional roles of MECs in salivary secretion are still unclear. We investigated morphological changes in immunostained MECs using the anti-α-smooth muscle actin (αSMA) antibody in operated or non-operated contralateral (NC) submandibular glands after partial or total resection. Furthermore, we investigated and discuss other salivary glands of rats. MECs in the parotid, sublingual and submandibular gland of adult rats exhibited different shapes and localizations. After surgery, in both operated and NC glands, the number of MECs and αSMA-immunopositive areas increased significantly. Three-dimensional analysis using a confocal laser-scanning microscope revealed that substantial and significant enhancement became evident in the number, length, and thickness of MEC-processes covering acini of the operated and NC submandibular glands. The preset findings indicate that MECs alter the morphology of their processes in operated and NC glands after surgery of the partial or total resection. It is suggested that MECs promote salivary secretion using elongated, thickened, and more ramified processes.

Contractile actin cables induced by Bacillus anthracis lethal toxin depend on the histone acetylation machinery.

PubMed

Rolando, Monica; Stefani, Caroline; Doye, Anne; Acosta, Maria I; Visvikis, Orane; Yevick, Hannah G; Buchrieser, Carmen; Mettouchi, Amel; Bassereau, Patricia; Lemichez, Emmanuel

2015-10-01

It remains a challenge to decode the molecular basis of the long-term actin cytoskeleton rearrangements that are governed by the reprogramming of gene expression. Bacillus anthracis lethal toxin (LT) inhibits mitogen-activated protein kinase (MAPK) signaling, thereby modulating gene expression, with major consequences for actin cytoskeleton organization and the loss of endothelial barrier function. Using a laser ablation approach, we characterized the contractile and tensile mechanical properties of LT-induced stress fibers. These actin cables resist pulling forces that are transmitted at cell-matrix interfaces and at cell-cell discontinuous adherens junctions. We report that treating the cells with trichostatin A (TSA), a broad range inhibitor of histone deacetylases (HDACs), or with MS-275, which targets HDAC1, 2 and 3, induces stress fibers. LT decreased the cellular levels of HDAC1, 2 and 3 and reduced the global HDAC activity in the nucleus. Both the LT and TSA treatments induced Rnd3 expression, which is required for the LT-mediated induction of actin stress fibers. Furthermore, we reveal that treating the LT-intoxicated cells with garcinol, an inhibitor of histone acetyl-transferases (HATs), disrupts the stress fibers and limits the monolayer barrier dysfunctions. These data demonstrate the importance of modulating the flux of protein acetylation in order to control actin cytoskeleton organization and the endothelial cell monolayer barrier. © 2015 Wiley Periodicals, Inc.

SKAP2 regulates Arp2/3 complex for actin-mediated asymmetric cytokinesis by interacting with WAVE2 in mouse oocytes

PubMed Central

Xu, Bai-Hui; Liu, Yu; Wang, Ya-Long; Chen, Ming-Huang; Xu, Lin; Liao, Bao-Qiong; Lui, Rui; Li, Fei-Ping; Lin, Yan-Hong; Fu, Xian-Pei; Fu, Bin-Bin; Hong, Zi-Wei; Qi, Zhong-Quan

2017-01-01

ABSTRACT SKAP2 (Src kinase-associated phosphoprotein 2), a substrate of Src family kinases, has been suggested to be involved in actin-mediated cellular processes. However, little is known about its role in mouse oocyte maturation. In this study, we thus investigated the expression, localization, and functions of SKAP2 during mouse oocyte asymmetric division. SKAP2 protein expression was detected at all developmental stages in mouse oocytes. Immunofluorescent staining showed that SKAP2 was mainly distributed at the cortex of the oocytes during maturation. Treatment with cytochalasin B in oocytes confirmed that SKAP2 was co-localized with actin. Depletion of SKAP2 by injection with specific short interfering RNA caused failure of spindle migration, polar body extrusion, and cytokinesis defects. Meanwhile, the staining of actin filaments at the oocyte membrane and in the cytoplasm was significantly reduced after these treatments. SKAP2 depletion also disrupted actin cap and cortical granule-free domain formation, and arrested a large proportion of oocytes at the telophase stage. Moreover, Arp2/3 complex and WAVE2 expression was decreased after the depletion of SKAP2 activity. Our results indicate that SKAP2 regulates the Arp2/3 complex and is essential for actin-mediated asymmetric cytokinesis by interacting with WAVE2 in mouse oocytes. PMID:28933599

Microcompartmentation of cytosolic aldolase by interaction with the actin cytoskeleton in Arabidopsis.

PubMed

Garagounis, Constantine; Kostaki, Kalliopi-Ioanna; Hawkins, Tim J; Cummins, Ian; Fricker, Mark D; Hussey, Patrick J; Hetherington, Alistair M; Sweetlove, Lee J

2017-02-01

Evidence is accumulating for molecular microcompartments formed when proteins interact in localized domains with the cytoskeleton, organelle surfaces, and intracellular membranes. To understand the potential functional significance of protein microcompartmentation in plants, we studied the interaction of the glycolytic enzyme fructose bisphosphate aldolase with actin in Arabidopsis thaliana. Homology modelling of a major cytosolic isozyme of aldolase, FBA8, suggested that the tetrameric holoenzyme has two actin binding sites and could therefore act as an actin-bundling protein, as was reported for animal aldolases. This was confirmed by in vitro measurements of an increase in viscosity of F-actin polymerized in the presence of recombinant FBA8. Simultaneously, interaction with F-actin caused non-competitive inhibition of aldolase activity. We did not detect co-localization of an FBA8-RFP fusion protein, expressed in an fba8-knockout background, with the actin cytoskeleton using confocal laser-scanning microscopy. However, we did find evidence for a low level of interaction using FRET-FLIM analysis of FBA8-RFP co-expressed with the actin-binding protein GFP-Lifeact. Furthermore, knockout of FBA8 caused minor alterations of guard cell actin cytoskeleton morphology and resulted in a reduced rate of stomatal closure in response to decreased humidity. We conclude that cytosolic aldolase can be microcompartmented in vivo by interaction with the actin cytoskeleton and may subtly modulate guard cell behaviour as a result. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Vasodilator-stimulated Phosphoprotein (VASP) Regulates Actin Polymerization and Contraction in Airway Smooth Muscle by a Vinculin-dependent Mechanism*

PubMed Central

Wu, Yidi; Gunst, Susan J.

2015-01-01

Vasodilator-stimulated phosphoprotein (VASP) can catalyze actin polymerization by elongating actin filaments. The elongation mechanism involves VASP oligomerization and its binding to profilin, a G-actin chaperone. Actin polymerization is required for tension generation during the contraction of airway smooth muscle (ASM); however, the role of VASP in regulating actin dynamics in ASM is not known. We stimulated ASM cells and tissues with the contractile agonist acetylcholine (ACh) or the adenylyl cyclase activator, forskolin (FSK), a dilatory agent. ACh and FSK stimulated VASP Ser157 phosphorylation by different kinases. Inhibition of VASP Ser157 phosphorylation by expression of the mutant VASP S157A in ASM tissues suppressed VASP phosphorylation and membrane localization in response to ACh, and also inhibited contraction and actin polymerization. ACh but not FSK triggered the formation of VASP-VASP complexes as well as VASP-vinculin and VASP-profilin complexes at membrane sites. VASP-VASP complex formation and the interaction of VASP with vinculin and profilin were inhibited by expression of the inactive vinculin mutant, vinculin Y1065F, but VASP phosphorylation and membrane localization were unaffected. We conclude that VASP phosphorylation at Ser157 mediates its localization at the membrane, but that VASP Ser157 phosphorylation and membrane localization are not sufficient to activate its actin catalytic activity. The interaction of VASP with activated vinculin at membrane adhesion sites is a necessary prerequisite for VASP-mediated molecular processes necessary for actin polymerization. Our results show that VASP is a critical regulator of actin dynamics and tension generation during the contractile activation of ASM. PMID:25759389

A Legionella Effector Disrupts Host Cytoskeletal Structure by Cleaving Actin

DOE PAGES

Liu, Yao; Zhu, Wenhan; Tan, Yunhao; ...

2017-01-27

Legionella pneumophila, the etiological agent of Legionnaires' disease, replicates intracellularly in protozoan and human hosts. Successful colonization and replication of this pathogen in host cells requires the Dot/Icm type IVB secretion system, which translocates approximately 300 effector proteins into the host cell to modulate various cellular processes. In this study, we identified RavK as a Dot/Icm substrate that targets the host cytoskeleton and reduces actin filament abundance in mammalian cells upon ectopic expression. RavK harbors an H 95E XXH 99 motif associated with diverse metalloproteases, which is essential for the inhibition of yeast growth and for the induction of cellmore » rounding in HEK293T cells. We demonstrate that the actin protein itself is the cellular target of RavK and that this effector cleaves actin at a site between residues Thr351 and Phe352. Importantly, RavK-mediated actin cleavage also occurs during L. pneumophila infection. Cleavage by RavK abolishes the ability of actin to form polymers. Furthermore, an F352A mutation renders actin resistant to RavK-mediated cleavage; expression of the mutant in mammalian cells suppresses the cell rounding phenotype caused by RavK, further establishing that actin is the physiological substrate of RavK. Furthermore, L. pneumophila exploits components of the host cytoskeleton by multiple effectors with distinct mechanisms, highlighting the importance of modulating cellular processes governed by the actin cytoskeleton in the intracellular life cycle of this pathogen.« less

Comparative proteomic analysis of fibrotic liver of rats fed high fat diet contained lard versus corn oil.

PubMed

Wang, Hualin; Sit, Wat-Hung; Tipoe, George Lim; Liu, Zhiguo; Wan, Jennifer Man-Fan

2017-02-01

The influences of dietary fatty acids on the progress of chronic liver diseases have attracted lots of attentions, but the mechanisms of the effects of lipids rich in saturated fatty acids or PUFAs on hepatic fibrogenesis remain unclear. Female Fischer 344 rats were fed normal chow or chow plus 20% (w/w) of corn oil or lard, respectively, and injected CCl 4 twice a week for 4 weeks to induce liver fibrosis. Masson's staining was adopted to illustrate the fibrosis level. The mRNA expression level of α-SMA and the DNA methylation level of its promoter region were analyzed. A 2-DE gel based proteomic approach was constructed to investigate the differential expression level of hepatic proteome between three diet groups. Histological evaluations and α-SMA expression analysis illustrated the high corn oil intake has no effects on hepatic fibrogenesis, but lard intake aggravated liver fibrosis, partly attributed to DNA demethylation of α-SMA promoter region. 2-DE Gel based proteomic study demonstrated excessive lard consumption elevated the expression of fibrosis related alpha-1-antitrypsin precursor, and endoplasmic reticulum stress related proteins such as heat shock cognate 71 kDa, eukaryotic translation initiation factor 4A1 and protein disulfide isomerase associated 3. Moreover, unlike corn oil rich in PUFAs, lard had no effects to elevate the expression of glutathione S-transferases, but decreased the expression of iron store related proteins heme binding protein 1 and ferritin. Lard intake aggravates CCl 4 induced liver fibrosis via enhancing the expression of fibrogenesis and ER stress related proteins, and disturbing the hepatic transmethylation reaction. Copyright © 2015 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Toll like receptor 4: A novel signaling pathway during renal fibrogenesis

PubMed Central

Campbell, Matthew T.; Hile, Karen L; Zhang, Hongji; Asanuma, Hiroshi; Vanderbrink, Brian A.; Rink, Richard R.; Meldrum, Kirstan K.

2010-01-01

Background The toll like receptor (TLR) family serves an important regulatory role in the innate immune system, and recent evidence has implicated TLR signaling in the pro-inflammatory response of a variety of endogenous and exogenous stimuli within the kidney. The role of TLR signaling in fibrotic renal injury; however, remains unknown. Materials and Methods C3H/HeJ TLR4 hyporesponsive mice (TLR4Lps-d) or WT controls (C3H/Heou/J) underwent either sham operation or 1 week of unilateral ureteral obstruction (UUO). The kidneys were harvested and tissues were analyzed for TLR4 expression (Western Blot; RTPCR), E-cadherin and α-SMA expression (Western Blot), fibroblast accumulation (fibroblast specific protein (FSP-1+) staining), renal fibrosis (collagen I RTPCR, total collagen assay, Masson's trichrome staining), cytokine gene expression (tumor necrosis factor-α (TNF-α) and transforming growth factor-beta1 (TGF-β1) RTPCR), and pSMAD2 and integrin α1 expression (Western Blot). Results Mice with intact TLR4 signaling demonstrate a significant increase in TLR4 expression, α-SMA expression, fibroblast accumulation, collagen deposition, and interstitial fibrosis, and a significant decrease in E-cadherin expression in response to UUO. TLR4 deficient mice; however, exhibit a significant reduction in obstruction-induced α-SMA expression, fibroblast accumulation, and renal fibrosis, with preservation of E-cadherin expression. TLR4's influence on fibroblast accumulation and renal fibrosis occurred independent of any alterations in TNF-α,TGF-β1, or pSMAD2 expression, but did involve alterations integrin α1 expression. Conclusion TLR4 appears to be a significant mediator of fibrotic renal injury. While TLR4 signaling is recognized as a critical component of the innate immune response, this is the first study to demonstrate a novel role for TLR4 in renal fibroblast accumulation and tubulointerstitial fibrosis. PMID:20089260

Antimicrobial peptide KSL-W promotes gingival fibroblast healing properties in vitro.

PubMed

Park, Hyun-Jin; Salem, Mabrouka; Semlali, Abdelhabib; Leung, Kai P; Rouabhia, Mahmoud

2017-07-01

We investigated the effect of synthetic antimicrobial decapeptide KSL-W (KKVVFWVKFK) on normal human gingival fibroblast growth, migration, collagen gel contraction, and α-smooth muscle actin protein expression. Results show that in addition to promoting fibroblast adhesion by increasing F-actin production, peptide KSL-W promoted cell growth by increasing the S and G2/M cell cycle phases, and enhanced the secretion of metalloproteinase (MMP)-1 and MMP-2 by upregulating MMP inhibitors, such as tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 in fibroblasts. An in vitro wound healing assay confirmed that peptide KSL-W promoted fibroblast migration and contraction of a collagen gel matrix. We also demonstrated a high expression of α-smooth muscle actin by gingival fibroblasts being exposed to KSL-W. This work shows that peptide KSL-W enhances gingival fibroblast growth, migration, and metalloproteinase secretion, and the expression of α-smooth muscle actin, thus promoting wound healing. Copyright © 2017 Elsevier Inc. All rights reserved.

Design of a muscle cell-specific expression vector utilising human vascular smooth muscle alpha-actin regulatory elements.

PubMed

Keogh, M C; Chen, D; Schmitt, J F; Dennehy, U; Kakkar, V V; Lemoine, N R

1999-04-01

The facility to direct tissue-specific expression of therapeutic gene constructs is desirable for many gene therapy applications. We describe the creation of a muscle-selective expression vector which supports transcription in vascular smooth muscle, cardiac muscle and skeletal muscle, while it is essentially silent in other cell types such as endothelial cells, hepatocytes and fibroblasts. Specific transcriptional regulatory elements have been identified in the human vascular smooth muscle cell (VSMC) alpha-actin gene, and used to create an expression vector which directs the expression of genes in cis to muscle cells. The vector contains an enhancer element we have identified in the 5' flanking region of the human VSMC alpha-actin gene involved in mediating VSMC expression. Heterologous pairing experiments have shown that the enhancer does not interact with the basal transcription complex recruited at the minimal SV40 early promoter. Such a vector has direct application in the modulation of VSMC proliferation associated with intimal hyperplasia/restenosis.

Ezrin is down-regulated in diabetic kidney glomeruli and regulates actin reorganization and glucose uptake via GLUT1 in cultured podocytes.

PubMed

Wasik, Anita A; Koskelainen, Susanna; Hyvönen, Mervi E; Musante, Luca; Lehtonen, Eero; Koskenniemi, Kerttu; Tienari, Jukka; Vaheri, Antti; Kerjaschki, Dontscho; Szalay, Csaba; Révész, Csaba; Varmanen, Pekka; Nyman, Tuula A; Hamar, Peter; Holthöfer, Harry; Lehtonen, Sanna

2014-06-01

Diabetic nephropathy is a complication of diabetes and a major cause of end-stage renal disease. To characterize the early pathophysiological mechanisms leading to glomerular podocyte injury in diabetic nephropathy, we performed quantitative proteomic profiling of glomeruli isolated from rats with streptozotocin-induced diabetes and controls. Fluorescence-based two-dimensional difference gel electrophoresis, coupled with mass spectrometry, identified 29 differentially expressed spots, including actin-binding protein ezrin and its interaction partner, NHERF2, which were down-regulated in the streptozotocin group. Knockdown of ezrin by siRNA in cultured podocytes increased glucose uptake compared with control siRNA-transfected cells, apparently by increasing translocation of glucose transporter GLUT1 to the plasma membrane. Knockdown of ezrin also induced actin remodeling under basal conditions, but reduced insulin-stimulated actin reorganization. Ezrin-dependent actin remodeling involved cofilin-1 that is essential for the turnover and reorganization of actin filaments. Phosphorylated, inactive cofilin-1 was up-regulated in diabetic glomeruli, suggesting altered actin dynamics. Furthermore, IHC analysis revealed reduced expression of ezrin in the podocytes of patients with diabetes. Our findings suggest that ezrin may play a role in the development of the renal complication in diabetes by regulating transport of glucose and organization of the actin cytoskeleton in podocytes. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

MAP18 regulates the direction of pollen tube growth in Arabidopsis by modulating F-actin organization.

PubMed

Zhu, Lei; Zhang, Yan; Kang, Erfang; Xu, Qiangyi; Wang, Miaoying; Rui, Yue; Liu, Baoquan; Yuan, Ming; Fu, Ying

2013-03-01

For fertilization to occur in plants, the pollen tube must be guided to enter the ovule via the micropyle. Previous reports have implicated actin filaments, actin binding proteins, and the tip-focused calcium gradient as key contributors to polar growth of pollen tubes; however, the regulation of directional pollen tube growth is largely unknown. We reported previously that Arabidopsis thaliana MICROTUBULE-ASSOCIATED PROTEIN18 (MAP18) contributes to directional cell growth and cortical microtubule organization. The preferential expression of MAP18 in pollen and in pollen tubes suggests that MAP18 also may function in pollen tube growth. In this study, we demonstrate that MAP18 functions in pollen tubes by influencing actin organization, rather than microtubule assembly. In vitro biochemical results indicate that MAP18 exhibits Ca(2+)-dependent filamentous (F)-actin-severing activity. Abnormal expression of MAP18 in map18 and MAP18 OX plants was associated with disorganization of the actin cytoskeleton in the tube apex, resulting in aberrant pollen tube growth patterns and morphologies, inaccurate micropyle targeting, and fewer fertilization events. Experiments with MAP18 mutants created by site-directed mutagenesis suggest that F-actin-severing activity is essential to the effects of MAP18 on pollen tube growth direction. Our study demonstrates that in Arabidopsis, MAP18 guides the direction of pollen tube growth by modulating actin filaments.

GSK-3β inhibitor attenuates urinary albumin excretion in type 2 diabetic db/db mice, and delays epithelial-to-mesenchymal transition in mouse kidneys and podocytes.

PubMed

Wan, Jia; Li, Peng; Liu, Dong-Wei; Chen, Ying; Mo, Hai-Zhen; Liu, Ben-Guo; Chen, Wen-Jie; Lu, Xiao-Qing; Guo, Jia; Zhang, Qian; Qiao, Ying-Jin; Liu, Zhang-Suo; Wan, Guang-Rui

2016-08-01

The mechanism underlying epithelial‑to‑mesenchymal transition (EMT) caused by high glucose (HG) stimulation in diabetic nephropathy (DN) remains to be fully elucidated. The present study investigated the effects of HG on EMT and the activity of glycogen synthase kinase 3β (GSK‑3β) in podocytes and the kidneys of db/db mice, and assessed the effects of (2'Z, 3'E)‑6‑bromoindirubin‑3'‑oxime (BIO), an inhibitor of GSK‑3β, on EMT and glomerular injury. The resulting data showed that the activity of GSK‑3β was upregulated by HG and downregulated by BIO in the podocytes and the renal cortex. The expression levels of epithelial markers, including nephrin, podocin and synaptopodin, were decreased by HG and increased by BIO, whereas the reverse were true for mesenchymal markers, including α‑smooth muscle actin (α‑SMA) and fibronectin. The expression levels of β‑catenin and Snail, in contrast to current understanding of the Wnt signaling pathway, were increased by HG and decreased by BIO. In addition, expression of the vitamin D receptor (VDR) was decreased by HG and increased by BIO. In conclusion, the present study revealed that the mechanism by which BIO inhibited HG‑mediated EMT in podocytes and the renal cortex was primarily due to the VDR. Treatment with BIO protected renal function by maintaining the integrity of the filtration membrane and decreasing UAE, but not by regulating blood glucose. Therefore, GSK‑3β may be used as a sensitive biomarker of DN, and its inhibition by BIO may be effective in the treatment of DN.

MicroRNA-29b regulates TGF-β1-mediated epithelial–mesenchymal transition of retinal pigment epithelial cells by targeting AKT2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Min; Li, Hui; Liu, Xiaoqiang

2016-07-15

The role of microRNA (miRNA) in proliferative vitreoretinopathy (PVR) progression has not been studied extensively, especially in retinal pigment epithelial–mesenchymal transition (EMT) which is the main reason for formation of PVR. In this study, we first investigated the miRNA expression profile in transforming growth factor beta 1 (TGF-β1) mediated EMT of ARPE-19 cells. Among the five changed miRNAs, miR-29b showed the most significant downregulation. Enhanced expression of miR-29b could reverse TGF-β1 induced EMT through targeting Akt2. Akt2 downregulation could inhibit TGF-β1-induced EMT. Furthermore, inhibition of miR-29b in ARPE-19 cells directly triggered EMT process, which characterized by the phenotypic transition andmore » the upregulation of α-smooth muscle actin (α-SMA) and downregulation of E-cadherin and zona occludin-1 (ZO-1) with increased cell migration. Akt2-shRNA also inhibited miR-29 inhibitor-induced EMT process. These data indicate that miR-29b plays an important role in TGF-β1-mediated EMT in ARPE-19 cells by targeting Akt2. - Highlights: • MiR-29b expression is decreased in TGF-β1-induced EMT of ARPE-19 cells. • MiR-29b inhibits TGF-β1-induced EMT in ARPE-19 cells. • MiR-29b inhibitor induces EMT in ARPE-19 cells. • Akt2 is the target for miR-29b. • Downregulation of Akt2 prevents TGF-β1-induced EMT of ARPE-19 cells.« less

Sildenafil protects against bile duct ligation induced hepatic fibrosis in rats: Potential role for silent information regulator 1 (SIRT1).

PubMed

Abd El Motteleb, Dalia M; Ibrahim, Islam A A E-H; Elshazly, Shimaa M

2017-11-15

Hepatic fibrosis is a potential health problem that may end with life-threatening cirrhosis and primary liver cancer. Recent studies point out to the protective effects of silent information regulator1 (SIRT1), against different models of organs fibrosis. This work aimed to investigate the possible protective effect of sildenafil (SIRT1 activator) against hepatic fibrosis induced by bile duct ligation (BDL). Firstly, three different doses of sildenafil (5, 10, 20mg/kg/day) were investigated; to detect the most protective one against BDL induced liver dysfunction and hepatic fibrosis. The most protective dose is then used; to study its effect on BDL induced SIRT1 downregulation, imbalance of oxidant/antioxidant status, increased inflammatory cytokines and fibrosis. Sildenafil (20mg/kg/day) was the most protective one, it caused upregulation of SIRT1, reduction of hepatic malondialdehyde (MDA) content, increase in expression of nuclear factor erythroid 2-related factor 2 (Nrf2), hemeoxygenease (HO)-1, reduced glutathione (GSH) content and superoxide dismutase (SOD) activity. Hepatic content of tumor necrosis factor-α (TNF-α) and nuclear factor κB (NFκB) expression & content displayed significant reductions with sildenafil treatment, Furthermore, sildenafil caused marked reductions of transforming growth factor (TGF)-β content, expression of plasminogen activator inhibitor-1 (PAI-1), matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), α-smooth muscle actin (α-SMA), fibronectin, collagen I (α1) and hydroxyproline content. However, sildenafil protective effects were significantly reduced by co-administration of EX527 (SIRT1 inhibitor). Our work showed, for the first time that, sildenafil has promising protective effects against BDL induced liver dysfunction and hepatic fibrosis. These effects may be, in part, mediated by up regulation of SIRT1. Copyright © 2017. Published by Elsevier Inc.

Hepatoprotective effect of Forsythiae Fructus water extract against carbon tetrachloride-induced liver fibrosis in mice.

PubMed

Zhang, Yi; Miao, Hui; Yan, Hongyu; Sheng, Yuchen; Ji, Lili

2018-05-23

The fruit of Forsythia suspensa (Thunb.) Vahl, named Forsythiae Fructus (Lian-Qiao), is a well-known traditional Chinese medicine (TCM) used for clearing away heat and toxic material, eliminating the mass and relieving swelling. This study aims to observe the attenuation of the water extract of Forsythiae Fructus (FSE) on carbon tetrachloride (CCl 4 )-induced hepatic fibrosis in male C57BL/6 mice. Hepatic fibrosis was induced in male C57BL/6 mice by intraperitoneal injection with 2 ml/kg CCl 4 (mixed 1: 3 in olive oil) twice a week for 4 weeks. At the same time, the mice were orally given with FSE (1, 2 g/kg) every day for 4 weeks. Serum biochemical parameters, gene and protein expression related to liver fibrosis were analyzed. The contents of forsythiaside A and forsythin in FSE were measured by high-performance liquid chromatography (HPLC). Results of serum alanine/aspartate aminotransferase (ALT/AST) activity and liver histological evaluation both showed the protection of FSE against CCl 4 -induced liver injury. Further, the anti-fibrotic effects of FSE was evidenced by the results of Masson's trichrome and Sirius red staining, liver hydroxyproline content, and serum amounts of hyaluronic acid, laminin, collagen Ⅳ and type III procollagen (PCIII). FSE also reduced the expression of α-smooth muscle actin (α-SMA) in livers from CCl 4 -injured mice. Additionally, FSE decreased the increased hepatic expression of fibroblast-specific protein 1 (FSP1) and vimentin induced by CCl 4 in mice. FSE attenuates CCl 4 -induced liver fibrosis in mice by inhibiting hepatic stellate cells (HSCs) activation, reducing hepatic extracellular matrix (ECM) disposition and reversing epithelial-mesenchymal transition (EMT). Copyright © 2018 Elsevier B.V. All rights reserved.

Overexpression of Snail in retinal pigment epithelial triggered epithelial–mesenchymal transition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Hui; Li, Min; Xu, Ding

2014-03-28

Highlights: • First reported overexpression of Snail in RPE cells could directly trigger EMT. • Further confirmed the regulator role of Snail in RPE cells EMT in vitro. • Snail may be a potential therapeutic target to prevent the fibrosis of PVR. - Abstract: Snail transcription factor has been implicated as an important regulator in epithelial–mesenchymal transition (EMT) during tumourigenesis and fibrogenesis. Our previous work showed that Snail transcription factor was activated in transforming growth factor β1 (TGF-β1) induced EMT in retinal pigment epithelial (RPE) cells and may contribute to the development of retinal fibrotic disease such as proliferative vitreoretinopathymore » (PVR). However, whether Snail alone has a direct role on retinal pigment epithelial–mesenchymal transition has not been investigated. Here, we analyzed the capacity of Snail to drive EMT in human RPE cells. A vector encoding Snail gene or an empty vector were transfected into human RPE cell lines ARPE-19 respectively. Snail overexpression in ARPE-19 cells resulted in EMT, which was characterized by the expected phenotypic transition from a typical epithelial morphology to mesenchymal spindle-shaped. The expression of epithelial markers E-cadherin and Zona occludin-1 (ZO-1) were down-regulated, whereas mesenchymal markers a-smooth muscle actin (a-SMA) and fibronectin were up-regulated in Snail expression vector transfected cells. In addition, ectopic expression of Snail significantly enhanced ARPE-19 cell motility and migration. The present data suggest that overexpression of Snail in ARPE-19 cells could directly trigger EMT. These results may provide novel insight into understanding the regulator role of Snail in the development of retinal pigment epithelial–mesenchymal transition.« less

How do culture media influence in vitro perivascular cell behavior?

PubMed

Huber, Birgit; Volz, Ann-Cathrin; Kluger, Petra Juliane

2015-12-01

Perivascular cells are multilineage cells located around the vessel wall and important for wall stabilization. In this study, we evaluated a stem cell media and a perivascular cell-specific media for the culture of primary perivascular cells regarding their cell morphology, doubling time, stem cell properties, and expression of cell type-specific markers. When the two cell culture media were compared to each other, perivascular cells cultured in the stem cell medium had a more elongated morphology and a faster doubling rate and cells cultured in the pericyte medium had a more typical morphology, with several filopodia, and a slower doubling rate. To evaluate stem cell properties, perivascular cells, CD146(-) cells, and mesenchymal stem cells (MSCs) were differentiated into the adipogenic, osteogenic, and chondrogenic lineages. It was seen that perivascular cells, as well as CD146(-) cells and MSCs, cultured in stem cell medium showed greater differentiation than cells cultured in pericyte-specific medium. The expression of pericyte-specific markers CD146, neural/glial antigen 2 (NG2), platelet-derived growth factor receptor-β (PDGFR-β), myosin, and α-smooth muscle actin (α-SMA) could be found in both pericyte cultures, as well as to varying amounts in CD146(-) cells, MSCs, and endothelial cells. The here presented work shows that perivascular cells can adapt to their in vitro environment and cell culture conditions influence cell functionality, such as doubling rate or differentiation behavior. Pericyte-specific markers were shown to be expressed also from cells other than perivascular cells. We can further conclude that CD146(+) perivascular cells are inhomogeneous cell population probably containing stem cell subpopulations, which are located perivascular around capillaries. © 2015 International Federation for Cell Biology.

A retinoic acid receptor β2 agonist reduces hepatic stellate cell activation in nonalcoholic fatty liver disease.

PubMed

Trasino, Steven E; Tang, Xiao-Han; Jessurun, Jose; Gudas, Lorraine J

2016-10-01

Hepatic stellate cells (HSCs) are an important cellular target for the development of novel pharmacological therapies to prevent and treat nonalcoholic fatty liver diseases (NAFLD). Using a high fat diet (HFD) model of NAFLD, we sought to determine if synthetic selective agonists for retinoic acid receptor β2 (RARβ2) and RARγ can mitigate HSC activation and HSC relevant signaling pathways during early stages of NAFLD, before the onset of liver injury. We demonstrate that the highly selective RARβ2 agonist, AC261066, can reduce the activation of HSCs, marked by decreased HSC expression of α-smooth muscle actin (α-SMA), in mice with HFD-induced NAFLD. Livers of HFD-fed mice treated with AC261066 exhibited reduced steatosis, oxidative stress, and expression of pro-inflammatory mediators, such as tumor necrosis factor-alpha (TNFα), interleukin 1β (IL-1β), and monocyte chemotactic protein-1 (MCP-1). Kupffer cell (macrophage) expression of transforming growth factor-β1 (TGF-β1), which plays a critical role in early HSC activation, was markedly reduced in AC261066-treated, HFD-fed mice. In contrast, HFD-fed mice treated with an RARγ agonist (CD1530) showed no decreases in steatosis, HSC activation, or Kupffer cell TGF-β1 levels. In conclusion, our data demonstrate that RARβ2 is an attractive target for development of NAFLD therapies. • Hepatic stellate cells (HSCs) are an important pharmacological target for the prevention of nonalcoholic fatty liver diseases (NAFLD). • Retinoids and retinoic acid receptors (RARs) possess favorable metabolic modulating properties. • We show that an agonist for retinoic acid receptor-β2 (RARβ2), but not RARγ, mitigates HSC activation and NAFLD.

Carnosic Acid Alleviates BDL-Induced Liver Fibrosis through miR-29b-3p-Mediated Inhibition of the High-Mobility Group Box 1/Toll-Like Receptor 4 Signaling Pathway in Rats

PubMed Central

Zhang, Shuai; Wang, Zhecheng; Zhu, Jie; Xu, Ting; Zhao, Yan; Zhao, Huanyu; Tang, Fan; Li, Zhenlu; Zhou, Junjun; Gao, Dongyan; Tian, Xiaofeng; Yao, Jihong

2018-01-01

Fibrosis reflects a progression to liver cancer or cirrhosis of the liver. Recent studies have shown that high-mobility group box-1 (HMGB1) plays a major role in hepatic injury and fibrosis. Carnosic acid (CA), a compound extracted from rosemary, has been reported to alleviate alcoholic and non-alcoholic fatty liver injury. CA can also alleviate renal fibrosis. We hypothesized that CA might exert anti-liver fibrosis properties through an HMGB1-related pathway, and the results of the present study showed that CA treatment significantly protected against hepatic fibrosis in a bile duct ligation (BDL) rat model. CA reduced the liver expression of α-smooth muscle actin (α-SMA) and collagen 1 (Col-1). Importantly, we found that CA ameliorated the increase in HMGB1 and Toll-like receptor 4 (TLR4) caused by BDL, and inhibited NF-κB p65 nuclear translocation in fibrotic livers. In vitro, CA inhibited LX2 cell activation by inhibiting HMGB1/TLR4 signaling pathway. Furthermore, miR-29b-3p decreased HMGB1 expression, and a dual-luciferase assay validated these results. Moreover, CA down-regulated HMGB1 and inhibited LX2 cell activation, and these effects were significantly counteracted by antago-miR-29b-3p, indicating that the CA-mediated inhibition of HMGB1 expression might be miR-29b-3p dependent. Collectively, the results demonstrate that a miR-29b-3p/HMGB1/TLR4/NF-κB signaling pathway, which can be modulated by CA, is important in liver fibrosis, and indicate that CA might be a prospective therapeutic drug for liver fibrosis. PMID:29403377

Combinatorial screening of 3D biomaterial properties that promote myofibrogenesis for mesenchymal stromal cell-based heart valve tissue engineering.

PubMed

Usprech, Jenna; Romero, David A; Amon, Cristina H; Simmons, Craig A

2017-08-01

The physical and chemical properties of a biomaterial integrate with soluble cues in the cell microenvironment to direct cell fate and function. Predictable biomaterial-based control of integrated cell responses has been investigated with two-dimensional (2D) screening platforms, but integrated responses in 3D have largely not been explored systematically. To address this need, we developed a screening platform using polyethylene glycol norbornene (PEG-NB) as a model biomaterial with which the polymer wt% (to control elastic modulus) and adhesion peptide types (RGD, DGEA, YIGSR) and densities could be controlled independently and combinatorially in arrays of 3D hydrogels. We applied this platform and regression modeling to identify combinations of biomaterial and soluble biochemical (TGF-β1) factors that best promoted myofibrogenesis of human mesenchymal stromal cells (hMSCs) in order to inform our understanding of regenerative processes for heart valve tissue engineering. In contrast to 2D culture, our screens revealed that soft hydrogels (low PEG-NB wt%) best promoted spread myofibroblastic cells that expressed high levels of α-smooth muscle actin (α-SMA) and collagen type I. High concentrations of RGD enhanced α-SMA expression in the presence of TGF-β1 and cell spreading regardless of whether TGF-β1 was in the culture medium. Strikingly, combinations of peptides that maximized collagen expression depended on the presence or absence of TGF-β1, indicating that biomaterial properties can modulate MSC response to soluble signals. This combination of a 3D biomaterial array screening platform with statistical modeling is broadly applicable to systematically identify combinations of biomaterial and microenvironmental conditions that optimally guide cell responses. We present a novel screening platform and methodology to model and identify how combinations of biomaterial and microenvironmental conditions guide cell phenotypes in 3D. Our approach to systematically identify complex relationships between microenvironmental cues and cell responses enables greater predictive power over cell fate in conditions with interacting material design factors. We demonstrate that this approach not only predicts that mesenchymal stromal cell (MSC) myofibrogenesis is promoted by soft, porous 3D biomaterials, but also generated new insights which demonstrate how biomaterial properties can differentially modulate MSC response to soluble signals. An additional benefit of the process includes utilizing both parametric and non parametric analyses which can demonstrate dominant significant trends as well as subtle interactions between biochemical and biomaterial cues. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Atorvastatin prevents advanced glycation end products (AGEs)-induced cardiac fibrosis via activating peroxisome proliferator-activated receptor gamma (PPAR-γ).

PubMed

Chen, Miao; Li, Hongwei; Wang, Guoxing; Shen, Xuhua; Zhao, Shumei; Su, Wen

2016-04-01

Previous studies have shown that the activation of advanced glycation end products (AGEs) contributed to the cardiac fibrosis in diabetic patients. Although it had been reported that statins have beneficial effects on cardiac fibrosis in hypertension and myocardial ischemia models, their effects on AGEs models have not been studied. We aimed to investigate the effects of atorvastatin (Ator) on the AGEs-induced cardiac fibrosis both in vitro and vivo. Male Sprague-Dawley rats were randomly divided into four groups: Control, AGEs, Ator or AGEs+Ator. The cardiac function was evaluated with the echocardiography at the second and the third month. Fibrosis area, α-SMA and RAGE expression in cardiac tissue were measured. For in vitro study, rat cardiac fibroblasts were treated with PD98059 (ERK inhibitor), Ator or Ator+GW9662 (PPAR-γ antagonist), and then were stimulated with AGEs. Fibroblasts proliferation, ERK1/2, phosphorylated ERK1/2, α-SMA, and RAGE expression were studied. Compared with the control group, in vivo treatment with Ator significantly retarded the AGEs-induced diastolic function and attenuated cardiac fibrosis, α-SMA, and RAGE over expression induced by AGEs. Consistently, Ator prominently downregulated RAGE and α-SMA, while inhibited phosphorylation of ERK1/2 and fibroblast proliferation induced by AGEs in vitro. The GW9662 neutralized these effects of Ator on cardiac fibroblasts stimulated by AGEs. In this study, we demonstrated that AGEs-induced fibroblast proliferation and differentiation were dependent on AGEs-RAGE-ERK1/2 pathway and that atorvastatin could block this pathway via activating PPAR-γ. Copyright © 2016 Elsevier Inc. All rights reserved.

Baculovirus AC102 Is a Nucleocapsid Protein That Is Crucial for Nuclear Actin Polymerization and Nucleocapsid Morphogenesis.

PubMed

Hepp, Susan E; Borgo, Gina M; Ticau, Simina; Ohkawa, Taro; Welch, Matthew D

2018-06-01

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the type species of alphabaculoviruses, is an enveloped DNA virus that infects lepidopteran insects and is commonly known as a vector for protein expression and cell transduction. AcMNPV belongs to a diverse group of viral and bacterial pathogens that target the host cell actin cytoskeleton during infection. AcMNPV is unusual, however, in that it absolutely requires actin translocation into the nucleus early in infection and actin polymerization within the nucleus late in infection coincident with viral replication. Of the six viral factors that are sufficient, when coexpressed, to induce the nuclear localization of actin, only AC102 is essential for viral replication and the nuclear accumulation of actin. We therefore sought to better understand the role of AC102 in actin mobilization in the nucleus early and late in infection. Although AC102 was proposed to function early in infection, we found that AC102 is predominantly expressed as a late protein. In addition, we observed that AC102 is required for F-actin assembly in the nucleus during late infection, as well as for proper formation of viral replication structures and nucleocapsid morphogenesis. Finally, we found that AC102 is a nucleocapsid protein and a newly recognized member of a complex consisting of the viral proteins EC27, C42, and the actin polymerization protein P78/83. Taken together, our findings suggest that AC102 is necessary for nucleocapsid morphogenesis and actin assembly during late infection through its role as a component of the P78/83-C42-EC27-AC102 protein complex. IMPORTANCE The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is an important biotechnological tool for protein expression and cell transduction, and related nucleopolyhedroviruses are also used as environmentally benign insecticides. One impact of our work is to better understand the fundamental mechanisms through which AcMNPV exploits the cellular machinery of the host for replication, which may aid in the development of improved baculovirus-based research and industrial tools. Moreover, AcMNPV's ability to mobilize the host actin cytoskeleton within the cell's nucleus during infection makes it a powerful cell biological tool. It is becoming increasingly clear that actin plays important roles in the cell's nucleus, and yet the regulation and function of nuclear actin is poorly understood. Our work to better understand how AcMNPV relocalizes and polymerizes actin within the nucleus may reveal fundamental mechanisms that govern nuclear actin regulation and function, even in the absence of viral infection. Copyright © 2018 American Society for Microbiology.