Optimization of a murine and human tissue model to recapitulate dermal and pulmonary features of systemic sclerosis

PubMed Central

Watanabe, Tomoya; Mlakar, Logan; Heywood, Jonathan; Malaab, Maya; Hoffman, Stanley

2017-01-01

The murine bleomycin (BLM)-induced fibrosis model is the most widely used in systemic sclerosis (SSc) studies. It has been reported that systemic delivery of BLM via continuous diffusion from subcutaneously implanted osmotic minipumps can cause fibrosis of the skin, lungs, and other internal organs. However, the mouse strain, dosage of BLM, administration period, and additional important features differ from one report to the next. In this study, by employing the pump model in C57BL/6J mice, we show a dose-dependent increase in lung fibrosis by day 28 and a transient increase in dermal thickness. Dermal thickness and the level of collagen in skin treated with high-dose BLM was significantly higher than in skin treated with low dose BLM or vehicle. A reduction in the thickness of the adipose layer was noted in both high and low dose groups at earlier time points suggesting that the loss of the fat layer precedes the onset of fibrosis. High-dose BLM also induced dermal fibrosis and increased expression of fibrosis-associated genes ex vivo in human skin, thus confirming and extending the in vivo findings, and demonstrating that a human organ culture model can be used to assess the effect of BLM on skin. In summary, our findings suggest that the BLM pump model is an attractive model to analyze the underlying mechanisms of fibrosis and test the efficacy of potential therapies. However, the choice of mouse strain, duration of BLM administration and dose must be carefully considered when using this model. PMID:28651005

Optimization of a murine and human tissue model to recapitulate dermal and pulmonary features of systemic sclerosis.

PubMed

Watanabe, Tomoya; Nishimoto, Tetsuya; Mlakar, Logan; Heywood, Jonathan; Malaab, Maya; Hoffman, Stanley; Feghali-Bostwick, Carol

2017-01-01

The murine bleomycin (BLM)-induced fibrosis model is the most widely used in systemic sclerosis (SSc) studies. It has been reported that systemic delivery of BLM via continuous diffusion from subcutaneously implanted osmotic minipumps can cause fibrosis of the skin, lungs, and other internal organs. However, the mouse strain, dosage of BLM, administration period, and additional important features differ from one report to the next. In this study, by employing the pump model in C57BL/6J mice, we show a dose-dependent increase in lung fibrosis by day 28 and a transient increase in dermal thickness. Dermal thickness and the level of collagen in skin treated with high-dose BLM was significantly higher than in skin treated with low dose BLM or vehicle. A reduction in the thickness of the adipose layer was noted in both high and low dose groups at earlier time points suggesting that the loss of the fat layer precedes the onset of fibrosis. High-dose BLM also induced dermal fibrosis and increased expression of fibrosis-associated genes ex vivo in human skin, thus confirming and extending the in vivo findings, and demonstrating that a human organ culture model can be used to assess the effect of BLM on skin. In summary, our findings suggest that the BLM pump model is an attractive model to analyze the underlying mechanisms of fibrosis and test the efficacy of potential therapies. However, the choice of mouse strain, duration of BLM administration and dose must be carefully considered when using this model.

Intestinal fibrosis is reduced by early elimination of inflammation in a mouse model of IBD: impact of a "Top-Down" approach to intestinal fibrosis in mice.

PubMed

Johnson, Laura A; Luke, Amy; Sauder, Kay; Moons, David S; Horowitz, Jeffrey C; Higgins, Peter D R

2012-03-01

The natural history of Crohn's disease follows a path of progression from an inflammatory to a fibrostenosing disease, with most patients requiring surgical resection of fibrotic strictures. Potent antiinflammatory therapies reduce inflammation but do not appear to alter the natural history of intestinal fibrosis. The aim of this study was to determine the relationship between intestinal inflammation and fibrogenesis and the impact of a very early "top-down" interventional approach on fibrosis in vivo. In this study we removed the inflammatory stimulus from the Salmonella typhimurium mouse model of intestinal fibrosis by eradicating the S. typhimurium infection with levofloxacin at sequential timepoints during the infection. We evaluated the effect of this elimination of the inflammatory stimulus on the natural history of inflammation and fibrosis as determined by gross pathology, histopathology, mRNA expression, and protein expression. Fibrogenesis is preceded by inflammation. Delayed eradication of the inflammatory stimulus by antibiotic treatment represses inflammation without preventing fibrosis. Early intervention significantly ameliorates but does not completely prevent subsequent fibrosis. This study demonstrates that intestinal fibrosis develops despite removal of an inflammatory stimulus and elimination of inflammation. Early intervention ameliorates but does not abolish subsequent fibrosis, suggesting that fibrosis, once initiated, is self-propagating, suggesting that a very early top-down interventional approach may have the most impact on fibrostenosing disease. Copyright © 2011 Crohn's & Colitis Foundation of America, Inc.

RNA Sequencing Identifies Novel Translational Biomarkers of Kidney Fibrosis

PubMed Central

Craciun, Florin L.; Bijol, Vanesa; Ajay, Amrendra K.; Rao, Poornima; Kumar, Ramya K.; Hutchinson, John; Hofmann, Oliver; Joshi, Nikita; Luyendyk, James P.; Kusebauch, Ulrike; Moss, Christopher L.; Srivastava, Anand; Himmelfarb, Jonathan; Waikar, Sushrut S.; Moritz, Robert L.

2016-01-01

CKD is the gradual, asymptomatic loss of kidney function, but current tests only identify CKD when significant loss has already happened. Several potential biomarkers of CKD have been reported, but none have been approved for preclinical or clinical use. Using RNA sequencing in a mouse model of folic acid-induced nephropathy, we identified ten genes that track kidney fibrosis development, the common pathologic finding in patients with CKD. The gene expression of all ten candidates was confirmed to be significantly higher (approximately ten- to 150-fold) in three well established, mechanistically distinct mouse models of kidney fibrosis than in models of nonfibrotic AKI. Protein expression of these genes was also high in the folic acid model and in patients with biopsy-proven kidney fibrosis. mRNA expression of the ten genes increased with increasing severity of kidney fibrosis, decreased in response to therapeutic intervention, and increased only modestly (approximately two- to five-fold) with liver fibrosis in mice and humans, demonstrating specificity for kidney fibrosis. Using targeted selected reaction monitoring mass spectrometry, we detected three of the ten candidates in human urine: cadherin 11 (CDH11), macrophage mannose receptor C1 (MRC1), and phospholipid transfer protein (PLTP). Furthermore, urinary levels of each of these three proteins distinguished patients with CKD (n=53) from healthy individuals (n=53; P<0.05). In summary, we report the identification of urinary CDH11, MRC1, and PLTP as novel noninvasive biomarkers of CKD. PMID:26449608

Overexpression of SerpinE2/protease nexin-1 Contribute to Pathological Cardiac Fibrosis via increasing Collagen Deposition

PubMed Central

Li, Xuelian; Zhao, Dandan; Guo, Zhenfeng; Li, Tianshi; Qili, Muge; Xu, Bozhi; Qian, Ming; Liang, Haihai; E, Xiaoqiang; Chege Gitau, Samuel; Wang, Lu; Huangfu, Longtao; Wu, Qiuxia; Xu, Chaoqian; Shan, Hongli

2016-01-01

Although increases in cardiovascular load (pressure overload) are known to elicit ventricular remodeling including cardiomyocyte hypertrophy and interstitial fibrosis, the molecular mechanisms of pressure overload or AngII -induced cardiac interstitial fibrosis remain elusive. In this study, serpinE2/protease nexin-1 was over-expressed in a cardiac fibrosis model induced by pressure-overloaded via transverse aortic constriction (TAC) in mouse. Knockdown of serpinE2 attenuates cardiac fibrosis in a mouse model of TAC. At meantime, the results showed that serpinE2 significantly were increased with collagen accumulations induced by AngII or TGF-β stimulation in vitro. Intriguingly, extracellular collagen in myocardial fibroblast was reduced by knockdown of serpinE2 compared with the control in vitro. In stark contrast, the addition of exogenous PN-1 up-regulated the content of collagen in myocardial fibroblast. The MEK1/2- ERK1/2 signaling probably promoted the expression of serpinE2 via transcription factors Elk1 in myocardial fibroblast. In conclusion, stress-induced the ERK1/2 signaling pathway activation up-regulated serpinE2 expression, consequently led accumulation of collagen protein, and contributed to cardiac fibrosis. PMID:27876880

Establishment of a mouse model for pulmonary inflammation and fibrosis by intratracheal instillation of polyhexamethyleneguanidine phosphate

PubMed Central

Lee, Sang Jin; Park, Jong-Hwan; Lee, Jun-Young; Jeong, Yu-Jin; Song, Jeong Ah; Lee, Kyuhong; Kim, Dong-Jae

2016-01-01

Although several animal models have been developed to study human pulmonary fibrosis, lack of a perfect model has raised the need for various animal models of pulmonary fibrosis. In this study, we evaluated the pulmonary effect of polyhexamethyleneguanidine phosphate instillation into the lungs of mice to determine the potential of these mice as a murine model of pulmonary fibrosis. Intratracheal instillation of polyhexamethyleneguanidine phosphate induced severe lung inflammation manifested by the infiltration of mononuclear cells and neutrophils and increased production of IL-6, TNF-α, CCL2 and CXCL1. The lung inflammation gradually increased until 28 days after polyhexamethyleneguanidine phosphate exposure, and increases of collagen deposition and TGF-β production, which are indicators of pulmonary fibrosis, were seen. Our study showed that intratracheal instillation of polyhexamethyleneguanidine phosphate induces pulmonary inflammation and fibrosis in mice. PMID:27182113

Obesity-Induced Diabetes and Lower Urinary Tract Fibrosis Promote Urinary Voiding Dysfunction in a Mouse Model

PubMed Central

Gharaee-Kermani, Mehrnaz; Rodriguez-Nieves, Jose A.; Mehra, Rohit; Vezina, Chad A.; Sarma, Aruna V.; Macoska, Jill A.

2017-01-01

BACKGROUND Progressive aging- and inflammation-associated fibrosis effectively remodels the extracellular matrix (ECM) to increase prostate tissue stiffness and reduce urethral flexibility, resulting in urinary flow obstruction and lower urinary tract symptoms (LUTS). In the current study, we sought to test whether senescence-accelerated mouse prone (SAMP)6 mice, which were reported to develop prostatic fibrosis, would also develop LUTS, and whether these symptoms would be exacerbated by diet-induced obesity and concurrent Type 2 Diabetes Mellitus (T2DM). METHODS To accomplish this, SAMP6 and AKR/J background strain mice were fed regular mouse chow, low fat diet chow, or high fat diet chow for 8 months, then subjected to glucose tolerance tests, assessed for plasma insulin levels, evaluated for urinary voiding function, and assessed for lower urinary tract fibrosis. RESULTS The results of these studies show that SAMP6 mice and AKR/J background strain mice develop diet-induced obesity and T2DM concurrent with urinary voiding dysfunction. Moreover, urinary voiding dysfunction was more severe in SAMP6 than AKR/J mice and was associated with pronounced prostatic and urethral tissue fibrosis. CONCLUSIONS Taken together, these studies suggest that obesity, T2DM, lower urinary tract fibrosis, and urinary voiding dysfunction are inextricably and biologically linked. Prostate. PMID:23532836

New animal models of cystic fibrosis: what are they teaching us?

PubMed Central

Keiser, Nicholas W.; Engelhardt, John F.

2013-01-01

Purpose of review Cystic fibrosis is the first human genetic disease to benefit from the directed engineering of three different species of animal models (mice, pigs, and ferrets). Recent studies on the cystic fibrosis pig and ferret models are providing new information about the pathophysiology of cystic fibrosis in various organ systems. Additionally, new conditional cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice are teaching unexpected lessons about CFTR function in surprising cellular locations. Comparisons between these animal models and the human condition are key to dissecting the complexities of disease pathophysiology in cystic fibrosis. Recent findings Cystic fibrosis pigs and ferrets have provided new models to study the spontaneous development of disease in the lung and pancreas, two organs that are largely spared overt spontaneous disease in cystic fibrosis mice. New cystic fibrosis mouse models are now interrogating CFTR functions involved in growth and inflammation at an organ-based level using conditional knockout technology. Together, these models are providing new insights on the human condition. Summary Basic and clinical cystic fibrosis research will benefit greatly from the comparative pathophysiology of cystic fibrosis mice, pigs, and ferrets. Both similarities and differences between these three cystic fibrosis models will inform pathophysiologically important mechanisms of CFTR function in humans and aid in the development of both organ-specific and general therapies for cystic fibrosis. PMID:21857224

Expression and function of Anoctamin 1/TMEM16A calcium-activated chloride channels in airways of in vivo mouse models for cystic fibrosis research.

PubMed

Hahn, Anne; Salomon, Johanna J; Leitz, Dominik; Feigenbutz, Dennis; Korsch, Lisa; Lisewski, Ina; Schrimpf, Katrin; Millar-Büchner, Pamela; Mall, Marcus A; Frings, Stephan; Möhrlen, Frank

2018-06-02

Physiological processes of vital importance are often safeguarded by compensatory systems that substitute for primary processes in case these are damaged by gene mutation. Ca 2+ -dependent Cl - secretion in airway epithelial cells may provide such a compensatory mechanism for impaired Cl - secretion via cystic fibrosis transmembrane conductance regulator (CFTR) channels in cystic fibrosis (CF). Anoctamin 1 (ANO1) Ca 2+ -gated Cl - channels are known to contribute to calcium-dependent Cl - secretion in tracheal and bronchial epithelia. In the present study, two mouse models of CF were examined to assess a potential protective function of Ca 2+ -dependent Cl - secretion, a CFTR deletion model (cftr -/- ), and a CF pathology model that overexpresses the epithelial Na + channel β-subunit (βENaC), which is encoded by the Scnn1b gene, specifically in airway epithelia (Scnn1b-Tg). The expression levels of ANO1 were examined by mRNA and protein content, and the channel protein distribution between ciliated and non-ciliated epithelial cells was analyzed. Moreover, Ussing chamber experiments were conducted to compare Ca 2+ -dependent Cl - secretion between wild-type animals and the two mouse models. Our results demonstrate that CFTR and ANO1 channels were co-expressed with ENaC in non-ciliated cells of mouse tracheal and bronchial epithelia. Ciliated cells did not express these proteins. Despite co-localization of CFTR and ANO1 in the same cell type, cells in cftr -/- mice displayed no altered expression of ANO1. Similarly, ANO1 expression was unaffected by βENaC overexpression in the Scnn1b-Tg line. These results suggest that the CF-related environment in the two mouse models did not induce ANO1 overexpression as a compensatory system.

Progress toward generating a ferret model of cystic fibrosis by somatic cell nuclear transfer

PubMed Central

Li, Ziyi; Engelhardt, John F

2003-01-01

Mammalian cloning by nuclear transfer from somatic cells has created new opportunities to generate animal models of genetic diseases in species other than mice. Although genetic mouse models play a critical role in basic and applied research for numerous diseases, often mouse models do not adequately reproduce the human disease phenotype. Cystic fibrosis (CF) is one such disease. Targeted ablation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in mice does not adequately replicate spontaneous bacterial infections observed in the human CF lung. Hence, several laboratories are pursuing alternative animal models of CF in larger species such as the pig, sheep, rabbits, and ferrets. Our laboratory has focused on developing the ferret as a CF animal model. Over the past few years, we have investigated several experimental parameters required for gene targeting and nuclear transfer (NT) cloning in the ferret using somatic cells. In this review, we will discuss our progress and the hurdles to NT cloning and gene-targeting that accompany efforts to generate animal models of genetic diseases in species such as the ferret. PMID:14613541

ω-3 PUFAs ameliorate liver fibrosis and inhibit hepatic stellate cells proliferation and activation by promoting YAP/TAZ degradation.

PubMed

Zhang, Kun; Chang, Yanan; Shi, Zhemin; Han, Xiaohui; Han, Yawei; Yao, Qingbin; Hu, Zhimei; Cui, Hongmei; Zheng, Lina; Han, Tao; Hong, Wei

2016-07-20

Elevated levels of the transcriptional regulators Yes-associated protein (YAP) and transcriptional coactivators with PDZ-binding motif (TAZ), key effectors of the Hippo pathway, have been shown to play essential roles in controlling liver cell fate and the activation of hepatic stellate cells (HSCs). The dietary intake of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) has been positively associated with a number of health benefits including prevention and reduction of cardiovascular diseases, inflammation and cancers. However, little is known about the impact of ω-3 PUFAs on liver fibrosis. In this study, we used CCl4-induced liver fibrosis mouse model and found that YAP/TAZ is over-expressed in the fibrotic liver and activated HSCs. Fish oil administration to the model mouse attenuates CCl4-induced liver fibrosis. Further study revealed that ω-3 PUFAs down-regulate the expression of pro-fibrogenic genes in activated HSCs and fibrotic liver, and the down-regulation is mediated via YAP, thus identifying YAP as a target of ω-3 PUFAs. Moreover, ω-3 PUFAs promote YAP/TAZ degradation in a proteasome-dependent manner. Our data have identified a mechanism of ω-3 PUFAs in ameliorating liver fibrosis.

Activation and overexpression of Sirt1 attenuates lung fibrosis via P300.

PubMed

Zeng, Zhilin; Cheng, Sheng; Chen, Huilong; Li, Qinghai; Hu, Yinan; Wang, Qi; Zhu, Xianying; Wang, Jun

2017-05-13

Persistent fibroblast activation is a predominant feature of idiopathic pulmonary fibrosis (IPF), but the transcriptional and epigenetic mechanisms controlling this process are not well understood. Silent information regulator type-1 (Sirt1) is a member of class Ⅲ histone deacetylase with important regulatory roles in a variety of pathophysiologic processes, but its role in fibrotic lung diseases is not clearly elucidated. Sirt1 expression in lung tissues of IPF patients and in a mouse model of bleomycin (BLM)-induced lung fibrosis were evaluated by immunofluorescence. The function of Sirt1 in BLM-induced lung fibrosis in the mouse model or transforming growth factor β1 (TGF-β1)-mediated lung fibroblast cellular model was investigated by Sirt1 activation, overexpression and knockdown of Sirt1. Finally, the involvement of p300 signaling pathways was assessed. In this study, we found up-regulation of Sirt1 in BLM-induced lung fibrosis, as well as in the lungs of IPF patients, including in the aggregated pulmonary fibroblasts of fibrotic foci. Activation or overexpression of Sirt1 attenuated TGF-β1-mediated lung fibroblast differentiation and activation and diminished the severity of experimental lung fibrosis in mice. Whereas knockdown of Sirt1 promoted the pro-fibrogenic activity of TGF-β1 in lung fibroblasts. A potential mechanism for the role of Sirt1 in lung fibrosis was through regulating the expression of p300. Thus, we characterized Sirt1 as an important regulator of lung fibrosis and provides a proof of principle for activation or overexpression of Sirt1 as a potential novel therapeutic strategy for IPF. Copyright © 2017 Elsevier Inc. All rights reserved.

Loss of Matrix Metalloproteinase-13 Attenuates Murine Radiation-Induced Pulmonary Fibrosis

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

Flechsig, Paul; Hartenstein, Bettina; Teurich, Sybille

2010-06-01

Purpose: Pulmonary fibrosis is a disorder of the lungs with limited treatment options. Matrix metalloproteinases (MMPs) constitute a family of proteases that degrade extracellular matrix with roles in fibrosis. Here we studied the role of MMP13 in a radiation-induced lung fibrosis model using a MMP13 knockout mouse. Methods and Materials: We investigated the role of MMP13 in lung fibrosis by investigating the effects of MMP13 deficiency in C57Bl/6 mice after 20-Gy thoracic irradiation (6-MV Linac). The morphologic results in histology were correlated with qualitative and quantitative results of volume computed tomography (VCT), magnetic resonance imaging (MRI), and clinical outcome. Results:more » We found that MMP13 deficient mice developed less pulmonary fibrosis than their wildtype counterparts, showed attenuated acute pulmonary inflammation (days after irradiation), and a reduction of inflammation during the later fibrogenic phase (5-6 months after irradiation). The reduced fibrosis in MMP13 deficient mice was evident in histology with reduced thickening of alveolar septi and reduced remodeling of the lung architecture in good correlation with reduced features of lung fibrosis in qualitative and quantitative VCT and MRI studies. The partial resistance of MMP13-deficient mice to fibrosis was associated with a tendency towards a prolonged mouse survival. Conclusions: Our data indicate that MMP13 has a role in the development of radiation-induced pulmonary fibrosis. Further, our findings suggest that MMP13 constitutes a potential drug target to attenuate radiation-induced lung fibrosis.« less

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

Loss of TRPV4 Function Suppresses Inflammatory Fibrosis Induced by Alkali-Burning Mouse Corneas

PubMed Central

Okada, Yuka; Shirai, Kumi; Miyajima, Masayasu; Reinach, Peter S.; Yamanaka, Osamu; Sumioka, Takayoshi; Kokado, Masahide; Tomoyose, Katsuo; Saika, Shizuya

2016-01-01

In humans suffering from pulmonary disease and a mouse model, transient receptor potential vanilloid 4 (TRPV4) channel activation contributes to fibrosis. As a corneal alkali burn induces the same response, we determined if such an effect is also attributable to TRPV4 activation in mice. Accordingly, we determined if the alkali burn wound healing responses in wild-type (WT) mice are different than those in their TRPV4-null (KO) counterpart. Stromal opacification due to fibrosis in KO (n = 128) mice was markedly reduced after 20 days relative to that in WT (n = 157) mice. Immunohistochemistry revealed that increases in polymorphonuclear leukocytes and macrophage infiltration declined in KO mice. Semi-quantitative real time RT-PCR of ocular KO fibroblast cultures identified increases in proinflammatory and monocyte chemoattractant protein-1 chemoattractant gene expression after injury. Biomarker gene expression of fibrosis, collagen1a1 and α-smooth muscle actin were attenuated along with macrophage release of interleukin-6 whereas transforming growth factor β, release was unchanged. Tail vein reciprocal bone marrow transplantation between WT and KO chimera mouse models mice showed that reduced scarring and inflammation in KO mice are due to loss of TRPV4 expression on both corneal resident immune cells, fibroblasts and infiltrating polymorphonuclear leukocytes and macrophages. Intraperitoneal TRPV4 receptor antagonist injection of HC-067047 (10 mg/kg, daily) into WT mice reproduced the KO-phenotype. Taken together, alkali-induced TRPV4 activation contributes to inducing fibrosis and inflammation since corneal transparency recovery was markedly improved in KO mice. PMID:28030558

Enhancing Autophagy with Drugs or Lung-directed Gene Therapy Reverses the Pathological Effects of Respiratory Epithelial Cell Proteinopathy.

PubMed

Hidvegi, Tunda; Stolz, Donna B; Alcorn, John F; Yousem, Samuel A; Wang, Jieru; Leme, Adriana S; Houghton, A McGarry; Hale, Pamela; Ewing, Michael; Cai, Houming; Garchar, Evelyn Akpadock; Pastore, Nunzia; Annunziata, Patrizia; Kaminski, Naftali; Pilewski, Joseph; Shapiro, Steven D; Pak, Stephen C; Silverman, Gary A; Brunetti-Pierri, Nicola; Perlmutter, David H

2015-12-11

Recent studies have shown that autophagy mitigates the pathological effects of proteinopathies in the liver, heart, and skeletal muscle but this has not been investigated for proteinopathies that affect the lung. This may be due at least in part to the lack of an animal model robust enough for spontaneous pathological effects from proteinopathies even though several rare proteinopathies, surfactant protein A and C deficiencies, cause severe pulmonary fibrosis. In this report we show that the PiZ mouse, transgenic for the common misfolded variant α1-antitrypsin Z, is a model of respiratory epithelial cell proteinopathy with spontaneous pulmonary fibrosis. Intracellular accumulation of misfolded α1-antitrypsin Z in respiratory epithelial cells of the PiZ model resulted in activation of autophagy, leukocyte infiltration, and spontaneous pulmonary fibrosis severe enough to elicit functional restrictive deficits. Treatment with autophagy enhancer drugs or lung-directed gene transfer of TFEB, a master transcriptional activator of the autophagolysosomal system, reversed these proteotoxic consequences. We conclude that this mouse is an excellent model of respiratory epithelial proteinopathy with spontaneous pulmonary fibrosis and that autophagy is an important endogenous proteostasis mechanism and an attractive target for therapy. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Radiation-induced lung fibrosis in a tumor-bearing mouse model is associated with enhanced Type-2 immunity.

PubMed

Chen, Jing; Wang, Yacheng; Mei, Zijie; Zhang, Shimin; Yang, Jie; Li, Xin; Yao, Ye; Xie, Conghua

2016-03-01

Lung fibrosis may be associated with Type-2 polarized inflammation. Herein, we aim to investigate whether radiation can initiate a Type-2 immune response and contribute to the progression of pulmonary fibrosis in tumor-bearing animals. We developed a tumor-bearing mouse model with Lewis lung cancer to receive either radiation therapy alone or radiation combined with Th1 immunomodulator unmethylated cytosine-phosphorothioate-guanine containing oligodeoxynucleotide (CpG-ODN). The Type-2 immune phenotype in tumors and the histological grade of lung fibrosis were evaluated in mice sacrificed three weeks after irradiation. Mouse lung tissues were analyzed for hydroxyproline and the expression of Type-1/Type-2 key transcription factors (T-bet/GATA-3). The concentration of Type-1/Type-2 cytokines in serum was measured by cytometric bead array. Lung fibrosis was observed to be more serious in tumor-bearing mice than in normal mice post-irradiation. The fibrosis score in irradiated tumor-bearing mice on Day 21 was 4.33 ± 0.82, which was higher than that of normal mice (2.00 ± 0.63; P < 0.05). Hydroxyproline and GATA-3 expression were increased in the lung tissues of tumor-bearing mice following irradiation. CpG-ODN attenuated fibrosis by markedly decreasing GATA-3 expression. Serum IL-13 and IL-5 were elevated, whereas INF-γ and IL-12 expression were decreased in irradiated tumor-bearing mice. These changes were reversed after CpG-ODN treatment. Thus, Type-2 immunity in tumors appeared to affect the outcome of radiation damage and might be of interest for future studies on developing approaches in which Type-1-related immunotherapy and radiotherapy are used in combination. © The Author 2015. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

Inactivated Orf virus (Parapoxvirus ovis) elicits antifibrotic activity in models of liver fibrosis.

PubMed

Nowatzky, Janina; Knorr, Andreas; Hirth-Dietrich, Claudia; Siegling, Angela; Volk, Hans-Dieter; Limmer, Andreas; Knolle, Percy; Weber, Olaf

2013-05-01

Inactivated Orf virus (ORFV, Parapoxvirus ovis) demonstrates strong antiviral activity in animal models including a human hepatitis B virus (HBV)-transgenic mouse. In addition, expression of interferon (IFN)-γ and interleukin-10 (IL-10) was induced after administration of inactivated ORFV in these mice. IFN-γ and IL-10 are known to elicit antifibrotic activity. We therefore aimed to study antifibrotic activity of inactivated ORFV in models of liver fibrosis. We characterized ORFV-induced hepatic cytokine expression in rats. We then studied ORFV in two models of liver fibrosis in rats, pig serum-induced liver fibrosis and carbon tetrachloride (CCL4 )-induced liver fibrosis. ORFV induced hepatic expression of IFN-γ and IL-10 in rats. ORFV mediated antifibrotic activity when administrated concomitantly with the fibrosis-inducing agents in both models of liver fibrosis. Importantly, when CCL4 -induced liver fibrosis was already established, ORFV application still showed significant antifibrotic activity. In addition, we were able to demonstrate a direct antifibrotic effect of ORFV on stellate cells. These results establish a potential novel antifibrotic therapeutic approach that not only prevents but also resolves established liver fibrosis. Further studies are required to unravel the details of the mechanisms involved. © 2012 The Japan Society of Hepatology.

Mouse and Rat Models of Induction of Hepatic Fibrosis and Assessment of Portal Hypertension.

PubMed

Klein, Sabine; Schierwagen, Robert; Uschner, Frank Erhard; Trebicka, Jonel

2017-01-01

Portal hypertension either develops due to progressive liver fibrosis or is the consequence of vascular liver diseases such as portal vein thrombosis or non-cirrhotic portal hypertension. This chapter focuses on different rodent models of liver fibrosis with portal hypertension and also in few non-cirrhotic portal hypertension models. Importantly, after the development of portal hypertension, the proper assessment of drug effects in the portal and systemic circulation should be discussed. The last part of the chapter is dedicated in these techniques to assess the in vivo hemodynamics and the ex vivo techniques of the isolated liver perfusion and vascular contractility.

Novel and optimized strategies for inducing fibrosis in vivo: focus on Duchenne Muscular Dystrophy

PubMed Central

2014-01-01

Background Fibrosis, an excessive collagen accumulation, results in scar formation, impairing function of vital organs and tissues. Fibrosis is a hallmark of muscular dystrophies, including the lethal Duchenne muscular dystrophy (DMD), which remains incurable. Substitution of muscle by fibrotic tissue also complicates gene/cell therapies for DMD. Yet, no optimal models to study muscle fibrosis are available. In the widely used mdx mouse model for DMD, extensive fibrosis develops in the diaphragm only at advanced adulthood, and at about two years of age in the ‘easy-to-access’ limb muscles, thus precluding fibrosis research and the testing of novel therapies. Methods We developed distinct experimental strategies, ranging from chronic exercise to increasing muscle damage on limb muscles of young mdx mice, by myotoxin injection, surgically induced trauma (laceration or denervation) or intramuscular delivery of profibrotic growth factors (such as TGFβ). We also extended these approaches to muscle of normal non-dystrophic mice. Results These strategies resulted in advanced and enhanced muscle fibrosis in young mdx mice, which persisted over time, and correlated with reduced muscle force, thus mimicking the severe DMD phenotype. Furthermore, increased fibrosis was also obtained by combining these procedures in muscles of normal mice, mirroring aberrant repair after severe trauma. Conclusions We have developed new and improved experimental strategies to accelerate and enhance muscle fibrosis in vivo. These strategies will allow rapidly assessing fibrosis in the easily accessible limb muscles of young mdx mice, without necessarily having to use old animals. The extension of these fibrogenic regimes to the muscle of non-dystrophic wild-type mice will allow fibrosis assessment in a wide array of pre-existing transgenic mouse lines, which in turn will facilitate understanding the mechanisms of fibrogenesis. These strategies should improve our ability to combat fibrosis-driven dystrophy progression and aberrant regeneration. PMID:25157321

Airway disease phenotypes in animal models of cystic fibrosis.

PubMed

McCarron, Alexandra; Donnelley, Martin; Parsons, David

2018-04-02

In humans, cystic fibrosis (CF) lung disease is characterised by chronic infection, inflammation, airway remodelling, and mucus obstruction. A lack of pulmonary manifestations in CF mouse models has hindered investigations of airway disease pathogenesis, as well as the development and testing of potential therapeutics. However, recently generated CF animal models including rat, ferret and pig models demonstrate a range of well characterised lung disease phenotypes with varying degrees of severity. This review discusses the airway phenotypes of currently available CF animal models and presents potential applications of each model in airway-related CF research.

Grainyhead-like 2 (GRHL2) distribution reveals novel pathophysiological differences between human idiopathic pulmonary fibrosis and mouse models of pulmonary fibrosis

PubMed Central

Mahavadi, Poornima; Sasikumar, Satish; Cushing, Leah; Hyland, Tessa; Rosser, Ann E.; Riccardi, Daniela; Lu, Jining; Kalin, Tanya V.; Kalinichenko, Vladimir V.; Guenther, Andreas; Ramirez, Maria I.; Pardo, Annie; Selman, Moisés; Warburton, David

2013-01-01

Chronic injury of alveolar lung epithelium leads to epithelial disintegrity in idiopathic pulmonary fibrosis (IPF). We had reported earlier that Grhl2, a transcriptional factor, maintains alveolar epithelial cell integrity by directly regulating components of adherens and tight junctions and thus hypothesized an important role of GRHL2 in pathogenesis of IPF. Comparison of GRHL2 distribution at different stages of human lung development showed its abundance in developing lung epithelium and in adult lung epithelium. However, GRHL2 is detected in normal human lung mesenchyme only at early fetal stage (week 9). Similar mesenchymal reexpression of GRHL2 was also observed in IPF. Immunofluorescence analysis in serial sections from three IPF patients revealed at least two subsets of alveolar epithelial cells (AEC), based on differential GRHL2 expression and the converse fluorescence intensities for epithelial vs. mesenchymal markers. Grhl2 was not detected in mesenchyme in intraperitoneal bleomycin-induced injury as well as in spontaneously occurring fibrosis in double-mutant HPS1 and HPS2 mice, whereas in contrast in a radiation-induced fibrosis model, with forced Forkhead box M1 (Foxm1) expression, an overlap of Grhl2 with a mesenchymal marker was observed in fibrotic regions. Grhl2's role in alveolar epithelial cell plasticity was confirmed by altered Grhl2 gene expression analysis in IPF and further validated by in vitro manipulation of its expression in alveolar epithelial cell lines. Our findings reveal important pathophysiological differences between human IPF and specific mouse models of fibrosis and support a crucial role of GRHL2 in epithelial activation in lung fibrosis and perhaps also in epithelial plasticity. PMID:24375798

Growth hormone resistance exacerbates cholestasis-induced murine liver fibrosis

PubMed Central

Stiedl, Patricia; McMahon, Robert; Blaas, Leander; Stanek, Victoria; Svinka, Jasmin; Grabner, Beatrice; Zollner, Gernot; Kessler, Sonja M.; Claudel, Thierry; Müller, Mathias; Mikulits, Wolfgang; Bilban, Martin; Esterbauer, Harald; Eferl, Robert; Haybaeck, Johannes; Trauner, Michael; Casanova, Emilio

2016-01-01

Growth hormone (GH) resistance has been associated with liver cirrhosis in humans but its contribution to the disease remains controversial. In order to elucidate whether GH resistance plays a causal role in the establishment and development of liver fibrosis, or rather represents a major consequence thereof, we challenged mice lacking the Growth hormone receptor gene (Ghr-/-, a model for GH resistance) by crossing them with Mdr2 knockout mice (Mdr2-/-), a mouse model of inflammatory cholestasis and liver fibrosis. Ghr-/-;Mdr2-/- mice showed elevated serum markers associated with liver damage and cholestasis, extensive bile duct proliferation and increased collagen deposition relative to Mdr2 -/- mice, thus suggesting a more severe liver fibrosis phenotype. Additionally, Ghr-/-;Mdr2-/- mice had a pronounced down-regulation of hepato-protective genes Hnf6, Egfr and Igf-1, and significantly increased levels of ROS and apoptosis in hepatocytes, compared to control mice. Moreover, single knockout mice (Ghr-/-) fed with a diet containing 1% cholic acid displayed an increase in hepatocyte ROS production, hepatocyte apoptosis and bile infarcts compared to their wildtype littermates, indicating that loss of Ghr renders hepatocytes more susceptible to toxic bile acid accumulation. Surprisingly, and despite their severe fibrotic phenotype, Ghr-/-;Mdr2-/- mice displayed a significant decrease in tumour incidence compared to Mdr2-/- mice, indicating that loss of Ghr signaling may slow the progression from fibrosis/cirrhosis to cancer in the liver. Conclusion Our findings suggest that GH resistance dramatically exacerbates liver fibrosis in a mouse model of inflammatory cholestasis, therefore suggesting that GH resistance plays a causal role in the disease and provides a novel target for the development of liver fibrosis treatments. PMID:25179284

Muc1 deficiency exacerbates pulmonary fibrosis in a mouse model of silicosis.

PubMed

Kato, Kosuke; Zemskova, Marina A; Hanss, Alec D; Kim, Marianne M; Summer, Ross; Kim, Kwang Chul

2017-11-25

MUC1 (MUC in human and Muc in animals) is a membrane-tethered mucin expressed on the apical surface of lung epithelial cells. However, in the lungs of patients with interstitial lung disease, MUC1 is aberrantly expressed in hyperplastic alveolar type II epithelial (ATII) cells and alveolar macrophages (AM), and elevated levels of extracellular MUC1 are found in bronchoalveolar lavage (BAL) fluid and the serum of these patients. While pro-fibrotic effects of extracellular MUC1 have recently been described in cultured fibroblasts, the contribution of MUC1 to the pathobiology of pulmonary fibrosis is unknown. In this study, we hypothesized that MUC1 deficiency would reduce susceptibility to pulmonary fibrosis in a mouse model of silicosis. We employed human MUC1 transgenic mice, Muc1 deficient mice and wild-type mice on C57BL/6 background in these studies. Some mice received a one-time dose of crystalline silica instilled into their oropharynx in order to induce pulmonary fibrosis and assess the effects of Muc1 deficiency on fibrotic and inflammatory responses in the lung. As previously described in other mouse models of pulmonary fibrosis, we found that extracellular MUC1 levels were markedly increased in whole lung tissues, BALF and serum of human MUC1 transgenic mice after silica. We also detected an increase in total MUC1 levels in the lungs of these mice, indicating that production as well as release contributed to elevated levels after lung injury. Immunohistochemical staining revealed that increased MUC1 expression was mostly confined to ATII cells and AMs in areas of fibrotic remodeling, illustrating a pattern similar to the expression of MUC1 in human fibrotic lung tissues. However, contrary to our hypothesis, we found that Muc1 deficiency resulted in a worsening of fibrotic remodeling in the mouse lung as judged by an increase in number of silicotic nodules, an increase in lung collagen deposition and an increase in the severity of pulmonary inflammation. Altogether, our results indicate that Muc1 has anti-fibrotic properties in the mouse lung and suggest that elevated levels of MUC1 in patients with interstitial lung disease may serve a protective role, which aims to limit the severity of tissue remodeling in the lung. Copyright © 2017. Published by Elsevier Inc.

Dipeptidyl peptidase IV (DPP-IV) inhibition prevents fibrosis in adipose tissue of obese mice.

PubMed

Marques, Ana Patrícia; Cunha-Santos, Janete; Leal, Helena; Sousa-Ferreira, Lígia; Pereira de Almeida, Luís; Cavadas, Cláudia; Rosmaninho-Salgado, Joana

2018-03-01

During the development of obesity the expansion of white adipose tissue (WAT) leads to a dysregulation and an excessive remodeling of extracellular matrix (ECM), leading to fibrosis formation. These ECM changes have high impact on WAT physiology and may change obesity progression. Blocking WAT fibrosis may have beneficial effects on the efficacy of diet regimen or therapeutical approaches in obesity. Since dipeptidyl peptidase IV (DPP-IV) inhibitors prevent fibrosis in tissues, such as heart, liver and kidney, the objective of this study was to assess whether vildagliptin, a DPP-IV inhibitor, prevents fibrosis in WAT in a mouse model of obesity, and to investigate the mechanisms underlying this effect. We evaluated the inhibitory effect of vildagliptin on fibrosis markers on WAT of high-fat diet (HFD)-induced obese mice and on 3T3-L1 cell line of mouse adipocytes treated with a fibrosis inducer, transforming growth factor beta 1 (TGFβ1). Vildagliptin prevents the increase of fibrosis markers in WAT of HFD-fed mice and reduces blood glucose, serum triglycerides, total cholesterol and leptin levels. In the in vitro study, the inhibition of DPP-IV with vildagliptin, neuropeptide Y (NPY) treatment and NPY Y 1 receptor activation prevents ECM deposition and fibrosis markers increase induced by TGFβ1 treatment. Vildagliptin prevents fibrosis formation in adipose tissue in obese mice, at least partially through NPY and NPY Y 1 receptor activation. This study highlights the importance of vildagliptin in the treatment of fibrosis that occur in obesity. Copyright © 2017 Elsevier B.V. All rights reserved.

Vitamin A-coupled liposome system targeting free cholesterol accumulation in hepatic stellate cells offers a beneficial therapeutic strategy for liver fibrosis.

PubMed

Furuhashi, Hirotaka; Tomita, Kengo; Teratani, Toshiaki; Shimizu, Motonori; Nishikawa, Makoto; Higashiyama, Masaaki; Takajo, Takeshi; Shirakabe, Kazuhiko; Maruta, Koji; Okada, Yoshikiyo; Kurihara, Chie; Watanabe, Chikako; Komoto, Shunsuke; Aosasa, Suefumi; Nagao, Shigeaki; Yamamoto, Junji; Miura, Soichiro; Hokari, Ryota

2018-04-01

Liver fibrosis is a life-threatening disorder for which no approved therapy is available. Recently, we reported that mouse hepatic stellate cell (HSC) activation increased free cholesterol (FC) accumulation, partly by enhancing signaling through sterol regulatory element-binding protein 2 (SREBP2) and microRNA-33a (miR-33a), which resulted in HSC sensitization to transforming growth factor-β (TGFβ)-induced activation in a "vicious cycle" of liver fibrosis. Human HSCs were isolated from surgical liver specimens from control patients and patients with liver fibrosis. C57BL/6 mice were treated with carbon tetrachloride for 4 weeks and concurrently given SREBP2-siRNA- or anti-miR-33a-bearing vitamin A-coupled liposomes. In human activated HSCs obtained from patients with liver fibrosis, FC accumulation was enhanced independently of serum cholesterol levels through increased signaling by both SREBP2 and miR-33a. This increased FC accumulation enhanced Toll-like receptor 4 (TLR4) protein levels and lowered the TGFβ-pseudoreceptor Bambi (bone morphogenetic protein and activin membrane-bound inhibitor) mRNA levels in HSCs. Notably, in a mouse liver fibrosis model, reduction of FC accumulation, specifically in activated HSCs by suppression of SREBP2 or miR-33a expression using SREBP2-siRNA- or anti-miR-33a-bearing vitamin A-coupled liposomes, downregulated TLR4 signaling, increased Bambi expression, and consequently ameliorated liver fibrosis. Our results suggest that FC accumulation in HSCs, as an intracellular mediator promoting HSC activation, contributes to a vicious cycle of HSC activation in human and mouse liver fibrosis independent of serum cholesterol levels. Targeting FC accumulation-related molecules in HSCs through a vitamin A-coupled liposomal system represents a favorable therapeutic strategy for liver fibrosis. © 2017 The Japan Society of Hepatology.

Heterogenic transplantation of bone marrow-derived rhesus macaque mesenchymal stem cells ameliorates liver fibrosis induced by carbon tetrachloride in mouse

PubMed Central

Yan, Yaping; Wang, Junfeng; Duan, Yanchao; Li, Shanshan; Yan, Li; Wang, Hong; Chen, Bingbing; Sang, Xiongbo; Ji, Weizhi

2018-01-01

Liver fibrosis is a disease that causes high morbidity and has become a major health problem. Liver fibrosis can lead to the end stage of liver diseases (livercirrhosisand hepatocellularcarcinoma). Currently, liver transplantation is the only effective treatment for end-stage liver disease. However, the shortage of organ donors, high cost of medical surgery, immunological rejection and transplantation complications severely hamper liver transplantation therapy. Mesenchymal stem cells (MSCs) have been regarded as promising cells for clinical applications in stem cell therapy in the treatment of liver diseases due to their unique multipotent differentiation capacity, immunoregulation and paracrine effects. Although liver fibrosis improvements by MSC transplantation in preclinical experiments as well as clinical trials have been reported, the in vivo fate of MSCs after transportation and their therapeutic mechanisms remain unclear. In this present study, we isolated MSCs from the bone marrow of rhesus macaques. The cells exhibited typical MSC markers and could differentiate into chondrocytes, osteocytes, and adipocytes, which were not affected by labeling with enhanced green fluorescent protein (EGFP). The harvested MSCs respond to interferon-γ stimulation and have the ability to inhibit lymphocyte proliferation in vitro. EGFP-labeled MSCs (1 × 106 cells) were transplanted into mice with carbon tetrachloride-induced liver fibrosis via tail vein injection. The ability of the heterogenic MSC infusion to ameliorate liver fibrosis in mice was evaluated by a blood plasma chemistry index, pathological examination and liver fibrosis-associated gene expression. Additionally, a small number of MSCs that homed and engrafted in the mouse liver tissues were evaluated by immunofluorescence analysis. Our results showed that the transplantation of heterogenic MSCs derived from monkey bone marrow can be used to treat liver fibrosis in the mouse model and that the paracrine effects of MSCs may play an important role in the improvement of liver fibrosis. PMID:29456886

S-adenosylmethionine reduces airway inflammation and fibrosis in a murine model of chronic severe asthma via suppression of oxidative stress.

PubMed

Yoon, Sun-Young; Hong, Gyong Hwa; Kwon, Hyouk-Soo; Park, Sunjoo; Park, So Young; Shin, Bomi; Kim, Tae-Bum; Moon, Hee-Bom; Cho, You Sook

2016-06-03

Increased oxidative stress has an important role in asthmatic airway inflammation and remodeling. A potent methyl donor, S-adenosylmethionine (SAMe), is known to protect against tissue injury and fibrosis through modulation of oxidative stress. The aim of this study was to evaluate the effect of SAMe on airway inflammation and remodeling in a murine model of chronic asthma. A mouse model was generated by repeated intranasal challenge with ovalbumin and Aspergillus fungal protease twice a week for 8 weeks. SAMe was orally administered every 24 h for 8 weeks. We performed bronchoalveolar lavage (BAL) fluid analysis and histopathological examination. The levels of various cytokines and 4-hydroxy-2-nonenal (HNE) were measured in the lung tissue. Cultured macrophages and fibroblasts were employed to evaluate the underlying anti-inflammatory and antifibrotic mechanisms of SAMe. The magnitude of airway inflammation and fibrosis, as well as the total BAL cell counts, were significantly suppressed in the SAMe-treated groups. A reduction in T helper type 2 pro-inflammatory cytokines and HNE levels was observed in mouse lung tissue after SAMe administration. Macrophages cultured with SAMe also showed reduced cellular oxidative stress and pro-inflammatory cytokine production. Moreover, SAMe treatment attenuated transforming growth factor-β (TGF-β)-induced fibronectin expression in cultured fibroblasts. SAMe had a suppressive effect on airway inflammation and fibrosis in a mouse model of chronic asthma, at least partially through the attenuation of oxidative stress and TGF-β-induced fibronectin expression. The results of this study suggest a potential role for SAMe as a novel therapeutic agent in chronic asthma.

Radiation Fibrosis of the Vocal Fold: From Man to Mouse

PubMed Central

Johns, Michael M.; Kolachala, Vasantha; Berg, Eric; Muller, Susan; Creighton, Frances X.; Branski, Ryan C.

2013-01-01

Objectives To characterize fundamental late tissue effects in the human vocal fold following radiation therapy. To develop a murine model of radiation fibrosis to ultimately develop both treatment and prevention paradigms. Design Translational study using archived human and fresh murine irradiated vocal fold tissue. Methods 1) Irradiated vocal fold tissue from patients undergoing laryngectomy for loss of function from radiation fibrosis were identified from pathology archives. Histomorphometry, immunohistochemistry, and whole-genome microarray as well as real-time transcriptional analyses was performed. 2) Focused radiation to the head and neck was delivered to mice in a survival fashion. One month following radiation, vocal fold tissue was analyzed with histomorphometry, immunohistochemistry, and real-time PCR transcriptional analysis for selected markers of fibrosis. Results Human irradiated vocal folds demonstrated increased collagen transcription with increased deposition and disorganization of collagen in both the thyroarytenoid muscle and the superficial lamina propria. Fibronectin were increased in the superficial lamina propria. Laminin decreased in the thyroarytenoid muscle. Whole genome microarray analysis demonstrated increased transcription of markers for fibrosis, oxidative stress, inflammation, glycosaminoglycan production and apoptosis. Irradiated murine vocal folds demonstrated increases in collagen and fibronectin transcription and deposition in the lamina propria. Transforming growth factor (TGF)-β increased in the lamina propria. Conclusion Human irradiated vocal folds demonstrate molecular changes leading to fibrosis that underlie loss of vocal fold pliability that occurs in patients following laryngeal irradiation. Irradiated murine tissue demonstrates similar findings, and this mouse model may have utility in creating prevention and treatment strategies for vocal fold radiation fibrosis. PMID:23242839

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.

CD147 promotes liver fibrosis progression via VEGF-A/VEGFR2 signalling-mediated cross-talk between hepatocytes and sinusoidal endothelial cells.

PubMed

Yan, Zhaoyong; Qu, Kai; Zhang, Jing; Huang, Qichao; Qu, Ping; Xu, Xinsen; Yuan, Peng; Huang, Xiaojun; Shao, Yongping; Liu, Chang; Zhang, Hongxin; Xing, Jinliang

2015-10-01

Although previous evidence indicates close involvement of CD147 in the pathogenesis of liver fibrosis, the underlying molecular mechanisms and its therapeutic value remain largely unknown. In the present study, we investigated the biological roles of CD147 in liver fibrosis and assessed its therapeutic value as a target molecule in the CCl4-induced liver fibrosis mouse model. We found that CD147 was highly expressed in both hepatocytes and SECs (sinusoidal endothelial cells) in fibrotic liver tissues. Additionally, it was significantly associated with the fibrosis stage. TGF-β1 (transforming growth factor β1) was found to be mainly responsible for the up-regulation of CD147. Bioinformatic and experimental data suggest a functional link between CD147 expression and VEGF-A (vascular endothelial growth factor A)/VEGR-2 (VEGF receptor 2) signalling-mediated angiogenesis in fibrotic liver tissues. Furthermore, we observed that the CD147-induced activation of the PI3K (phosphoinositide 3-kinase)/Akt signalling pathway promotes the production of VEGF-A in hepatocytes and expression of VEGFR-2 in SECs, which was found to enhance the angiogenic capability of SECs. Finally, our data indicate that blocking of CD147 using an mAb (monoclonal antibody) attenuated liver fibrosis progression via inhibition of VEGF-A/VEGFR-2 signalling and subsequent amelioration of microvascular abnormality in the CCl4-induced mouse model. Our findings suggest a novel functional mechanism that CD147 may promote liver fibrosis progression via inducing the VEGF-A/VEGFR-2 signalling pathway-mediated cross-talk between hepatocytes and SECs. New strategies based on the intervention of CD147 can be expected for prevention of liver fibrosis. © 2015 Authors; published by Portland Press Limited.

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.

Updates on Dietary Models of Nonalcoholic Fatty Liver Disease: Current Studies and Insights

PubMed Central

Stephenson, Kristen; Kennedy, Lindsey; Hargrove, Laura; Demieville, Jennifer; Thomson, Joanne; Alpini, Gianfranco; Francis, Heather

2018-01-01

Nonalcoholic fatty liver disease (NAFLD) is a disease of increasing interest, as its prevalence is on the rise. NAFLD has been linked to metabolic syndrome, which is becoming more common due to the Western diet. Because NAFLD can lead to cirrhosis and related complications including hepatocellular carcinoma, the increasing prevalence is concerning, and medical therapy aimed at treating NAFLD is of great interest. Researchers studying the effects of medical therapy on NAFLD use dietary mouse models. The two main types of mouse model diets are the methionine- and choline-deficient (MCD) diet and the Western-like diet (WD). Although both induce NAFLD, the mechanisms are very different. We reviewed several studies conducted within the last 5 years that used MCD diet or WD mouse models in order to mimic this disease in a way most similar to humans. The MCD diet inconsistently induces NAFLD and fibrosis and does not completely induce metabolic syndrome. Thus, the clinical significance of the MCD diet is questionable. In contrast, WD mouse models consisting of high fat, cholesterol, and a combination of high-fructose corn syrup, sucrose, fructose, or glucose not only lead to metabolic syndrome but also induce NAFLD with fibrosis, making these choices most suitable for research. PMID:29096730

Blockade of CCN4 attenuates CCl4-induced liver fibrosis.

PubMed

Li, Xiaofei; Chen, Yongxin; Ye, Weiwei; Tao, Xingfei; Zhu, Jinhong; Wu, Shuang; Lou, Lianqing

2015-06-19

CCN4, also termed WNT-inducible signaling pathway protein-1 (WISP-1), has important roles in inflammation and tissue injury. This study aimed to investigate the effect of CCN4 inhibition using monoclonal anti-CCN4 antibody (CCN4mAb) on the liver injury and fibrosis in a mouse model of liver fibrosis. The mouse liver fibrosis model was induced by carbon tetrachloride (CCl4). Mice received vehicle (saline/olive oil) by subcutaneous injection, CCl4 by subcutaneous injection or CCl4 (subcutaneous) plus CCN4mAb by subcutaneous injection. The pro-inflammatory and pro-fibrotic factors were determined by Western blot. The biochemistry and histopathology, collagen deposition and nuclear factor (NF)-κB activity were also assessed. Chronic CCl4 treatment caused liver injury and collagen accumulation. The expression levels of CCN4, pro-inflammatory and pro-fibrotic mediators as well as the activity of NF-κB were markedly increased. Treatment with CCN4mAb significantly inhibited CCl4-induced CCN4 expression, leading to attenuated CCl4-induced liver injury and the inflammatory response. CCN4 blockade also significantly reduced the formation of collagen in the liver and the expression of α-smooth muscle actin and transforming growth factor β1. CCN4 inhibition by CCN4mAb in vivo significantly attenuated the CCl4-induced liver injury and the progression of liver fibrosis. CCN4 may represent a novel therapeutic target for liver injury and fibrosis.

Overexpression of Heparin-Binding Epidermal Growth Factor-Like Growth Factor Mediates Liver Fibrosis in Transgenic Mice.

PubMed

Guo, Yongze; Ding, Qian; Chen, Lei; Ji, Chenguang; Hao, Huiyao; Wang, Jia; Qi, Wei; Xie, Xiaoli; Ma, Junji; Li, Aidi; Jiang, Xiaoyu; Li, Xiaotian; Jiang, Huiqing

2017-08-01

The role of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in liver fibrosis is not clear and is sometimes even contradictory. To clarify this role, a HB-EGF transgenic (Tg) mouse model was, for the first time, used to evaluate the functions of HB-EGF in liver fibrosis. For the in vivo study, carbon tetrachloride injection and bile duct ligation treatment were used to induce liver fibrosis in HB-EGF Tg mice and wild-type (WT) mice, respectively. Primary hepatic satellite cells (HSCs) were isolated from HB-EGF Tg and WT mice for the in vitro study. Compared with the WT mice, HB-EGF Tg mice were shown to develop more severe liver fibrosis when treated with carbon tetrachloride or bile duct ligation, with increased matrix metalloproteinases 13 activity and enhanced expression of fibrogenic genes including α-smooth muscle actin and collagen I. HB-EGF gene transfer led to an increase in proliferation and a decrease in apoptosis in primary HSCs. The ERK signaling pathway was more highly activated in primary HSCs from HB-EGF Tg mice than in those from WT mice. Our investigation confirmed the profibrotic effect of HB-EGF on the liver using a Tg mouse model. This result may contribute to the elucidation of HB-EGF as a therapeutic target in liver fibrosis. Copyright © 2017 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.

A mouse model for the cystic fibrosis delta F508 mutation.

PubMed Central

van Doorninck, J H; French, P J; Verbeek, E; Peters, R H; Morreau, H; Bijman, J; Scholte, B J

1995-01-01

Most cystic fibrosis (CF) patients produce a mutant form (delta F508) of the cystic fibrosis transmembrane conductance regulator (CFTR), which is not properly processed in normal cells but is active as a chloride channel in several experimental systems. We used a double homologous recombination ('Hit and Run') procedure to generate a mouse model for the delta F508 mutation. Targeted embryonic stem (ES) cells (Hit clones) were found; of these either 80 or 20% of the clones had lost the delta F508 mutation, depending on the distance between the linearization site in the targeting construct and the delta F508 mutation. Correctly targeted clones underwent a second selection step resulting in ES cell clones (Run clones) heterozygous for the delta F508 mutation with an efficiency of 2-7%. Chimeric mice were generated and offspring homozygous for the delta F508 mutation showed electrophysiological abnormalities in nasal epithelium, gallbladder and in the intestine, and histological abnormalities in the intestine, typical of CF. Our data suggest that the delta F508 mice have residual delta F508 CFTR activity which would explain the mild pathology of the delta F508 mice. The delta F508 mouse may provide a useful model for the study of the processing defect of delta F508 CFTR and for the development of novel therapeutic approaches based on circumvention of the processing block. Images PMID:7556083

Heterozygous disruption of activin receptor-like kinase 1 is associated with increased renal fibrosis in a mouse model of obstructive nephropathy.

PubMed

Muñoz-Félix, José M; López-Novoa, José M; Martínez-Salgado, Carlos

2014-02-01

Tubulointerstitial fibrosis is characterized by an accumulation of extracellular matrix in the renal interstitium, myofibroblast activation, cell infiltration, and tubular cell apoptosis, leading to chronic renal failure. Activin receptor-like kinase 1 (ALK1) is a transforming growth factor-β1 type I receptor with a pivotal role in endothelial proliferation and migration, but its role in the development of renal fibrosis is unknown. To assess this we used the unilateral ureteral obstruction model of tubulointerstitial fibrosis in ALK1 haploinsufficient (ALK1(+/-)) and wild-type mice. After 15 days, there was an increase in extracellular matrix protein expression in the obstructed kidneys from both ALK1(+/+) and ALK1(+/-) mice, but obstructed kidneys from ALK1(+/-) mice showed significantly higher expression of type I collagen than those from wild-type mice. Ureteral obstruction increased kidney myofibroblasts markers (α-smooth muscle actin and S100A4), without differences between mouse genotypes. ALK1 expression was increased after ureteral obstruction, and this increased expression was located in myofibroblasts. Moreover, cultured renal fibroblasts from ALK1(+/-) mice expressed more collagen type I and fibronectin than fibroblasts derived from wild-type mice. Thus, ALK1 modulates obstruction-induced renal fibrosis by increased extracellular matrix synthesis in myofibroblasts, but without differences in myofibroblast number.

Smad4 Loss Synergizes with TGFα Overexpression in Promoting Pancreatic Metaplasia, PanIN Development, and Fibrosis

PubMed Central

Garcia-Carracedo, Dario; Yu, Chih-Chieh; Akhavan, Nathan; Fine, Stuart A.; Schönleben, Frank; Maehara, Naoki; Karg, Dillon C.; Xie, Chuangao; Qiu, Wanglong; Fine, Robert L.; Remotti, Helen E.; Su, Gloria H.

2015-01-01

Aims While overexpression of TGFα has been reported in human pancreatic ductal adenocarcinoma (PDAC), mice with overexpressed TGFα develop premalignant pancreatic acinar-to-ductal metaplasia (ADM) but not PDAC. TGF-β signaling pathway is pivotal to the development of PDAC and tissue fibrosis. Here we sought to investigate the interplay between TGFα and TGF-β signaling in pancreatic tumorigenesis and fibrosis, namely via Smad4 inactivation. Methods The MT-TGFα mouse was crossed with a new Smad4 conditional knock-out mouse (Smad4flox/flox;p48-Cre or S4) to generate Smad4flox/flox;MT-TGFα;p48-Cre (STP). After TGFα overexpression was induced with zinc sulfate water for eight months, the pancreata of the STP, MT-TGFα, and S4 mice were examined for tumor development and fibrotic responses. PanIN lesions and number of ducts were counted, and proliferation was measured by Ki67 immunohistochemistry (IHC). Qualitative analysis of fibrosis was analyzed by Trichrome Masson and Sirius Red staining, while vimentin was used for quantification. Expression analyses of fibrosis, pancreatitis, or desmoplasia associated markers (α-SMA, Shh, COX-2, Muc6, Col1a1, and Ctgf) were performed by IHC and/or qRT-PCR. Results Our STP mice exhibited advanced ADM, increased fibrosis, increased numbers of PanIN lesions, overexpression of chronic pancreatitis-related marker Muc6, and elevated expression of desmoplasia-associated marker Col1A1, compared to the MT-TGFα mice. The inactivation of Smad4 in the exocrine compartment was responsible for both the enhanced PanIN formation and fibrosis in the pancreas. The phenotype of the STP mice represents a transient state from ADMs to PanINs, closely mimicking the interface area seen in human chronic pancreatitis associated with PDAC. Conclusion We have documented a novel mouse model, the STP mice, which displayed histologic presentations reminiscent to those of human chronic pancreatitis with signs of early tumorigenesis. The STP mice could be a suitable animal model for interrogating the transition of chronic pancreatitis to pancreatic cancer. PMID:25803032

Differential Expression of VEGF-Axxx Isoforms Is Critical for Development of Pulmonary Fibrosis.

PubMed

Barratt, Shaney L; Blythe, Thomas; Jarrett, Caroline; Ourradi, Khadija; Shelley-Fraser, Golda; Day, Michael J; Qiu, Yan; Harper, Steve; Maher, Toby M; Oltean, Sebastian; Hames, Thomas J; Scotton, Chris J; Welsh, Gavin I; Bates, David O; Millar, Ann B

2017-08-15

Fibrosis after lung injury is related to poor outcome, and idiopathic pulmonary fibrosis (IPF) can be regarded as an exemplar. Vascular endothelial growth factor (VEGF)-A has been implicated in this context, but there are conflicting reports as to whether it is a contributory or protective factor. Differential splicing of the VEGF-A gene produces multiple functional isoforms including VEGF-A 165 a and VEGF-A 165 b, a member of the inhibitory family. To date there is no clear information on the role of VEGF-A in IPF. To establish VEGF-A isoform expression and functional effects in IPF. We used tissue sections, plasma, and lung fibroblasts from patients with IPF and control subjects. In a bleomycin-induced lung fibrosis model we used wild-type MMTV mice and a triple transgenic mouse SPC-rtTA +/- TetoCre +/- LoxP-VEGF-A +/+ to conditionally induce VEGF-A isoform deletion specifically in the alveolar type II (ATII) cells of adult mice. IPF and normal lung fibroblasts differentially expressed and responded to VEGF-A 165 a and VEGF-A 165 b in terms of proliferation and matrix expression. Increased VEGF-A 165 b was detected in plasma of progressing patients with IPF. In a mouse model of pulmonary fibrosis, ATII-specific deficiency of VEGF-A or constitutive overexpression of VEGF-A 165 b inhibited the development of pulmonary fibrosis, as did treatment with intraperitoneal delivery of VEGF-A 165 b to wild-type mice. These results indicate that changes in the bioavailability of VEGF-A sourced from ATII cells, namely the ratio of VEGF-A xxx a to VEGF-A xxx b, are critical in development of pulmonary fibrosis and may be a paradigm for the regulation of tissue repair.

Comparative biology of cystic fibrosis animal models.

PubMed

Fisher, John T; Zhang, Yulong; Engelhardt, John F

2011-01-01

Animal models of human diseases are critical for dissecting mechanisms of pathophysiology and developing therapies. In the context of cystic fibrosis (CF), mouse models have been the dominant species by which to study CF disease processes in vivo for the past two decades. Although much has been learned through these CF mouse models, limitations in the ability of this species to recapitulate spontaneous lung disease and several other organ abnormalities seen in CF humans have created a need for additional species on which to study CF. To this end, pig and ferret CF models have been generated by somatic cell nuclear transfer and are currently being characterized. These new larger animal models have phenotypes that appear to closely resemble human CF disease seen in newborns, and efforts to characterize their adult phenotypes are ongoing. This chapter will review current knowledge about comparative lung cell biology and cystic fibrosis transmembrane conductance regulator (CFTR) biology among mice, pigs, and ferrets that has implications for CF disease modeling in these species. We will focus on methods used to compare the biology and function of CFTR between these species and their relevance to phenotypes seen in the animal models. These cross-species comparisons and the development of both the pig and the ferret CF models may help elucidate pathophysiologic mechanisms of CF lung disease and lead to new therapeutic approaches.

Fibrosis in connective tissue disease: the role of the myofibroblast and fibroblast-epithelial cell interactions

PubMed Central

Krieg, Thomas; Abraham, David; Lafyatis, Robert

2007-01-01

Fibrosis, characterized by excessive extracellular matrix accumulation, is a common feature of many connective tissue diseases, notably scleroderma (systemic sclerosis). Experimental studies suggest that a complex network of intercellular interactions involving endothelial cells, epithelial cells, fibroblasts and immune cells, using an array of molecular mediators, drives the pathogenic events that lead to fibrosis. Transforming growth factor-β and endothelin-1, which are part of a cytokine hierarchy with connective tissue growth factor, are key mediators of fibrogenesis and are primarily responsible for the differentiation of fibroblasts toward a myofibroblast phenotype. The tight skin mouse (Tsk-1) model of cutaneous fibrosis suggests that numerous other genes may also be important. PMID:17767742

PET/CT with 18F-FDG- and 18F-FBEM-labeled leukocytes for metabolic activity and leukocyte recruitment monitoring in a mouse model of pulmonary fibrosis.

PubMed

Bondue, Benjamin; Sherer, Félicie; Van Simaeys, Gaetan; Doumont, Gilles; Egrise, Dominique; Yakoub, Yousof; Huaux, François; Parmentier, Marc; Rorive, Sandrine; Sauvage, Sébastien; Lacroix, Simon; Vosters, Olivier; De Vuyst, Paul; Goldman, Serge

2015-01-01

Idiopathic pulmonary fibrosis is characterized by a progressive and irreversible respiratory failure. Validated noninvasive methods able to assess disease activity are essential for prognostic purposes as well as for the evaluation of emerging antifibrotic treatments. C57BL/6 mice were used in a murine model of pulmonary fibrosis induced by an intratracheal instillation of bleomycin (control mice were instilled with a saline solution). At different times after instillation, PET/CT with (18)F-FDG- or (18)F-4-fluorobenzamido-N-ethylamino-maleimide ((18)F-FBEM)-labeled leukocytes was performed to assess metabolic activity and leukocyte recruitment, respectively. In bleomycin-treated mice, a higher metabolic activity was measured on (18)F-FDG PET/CT scans from day 7 to day 24 after instillation, with a peak of activity measured at day 14. Of note, lung mean standardized uptake values correlated with bleomycin doses, histologic score of fibrosis, lung hydroxyproline content, and weight loss. Moreover, during the inflammatory phase of the model (day 7), but not the fibrotic phase (day 23), bleomycin-treated mice presented with an enhanced leukocyte recruitment as assessed by (18)F-FBEM-labeled leukocyte PET/CT. Autoradiographic analysis of lung sections and CD45 immunostaining confirm the higher and early recruitment of leukocytes in bleomycin-treated mice, compared with control mice. (18)F-FDG- and (18)F-FBEM-labeled leukocyte PET/CT enable monitoring of metabolic activity and leukocyte recruitment in a mouse model of pulmonary fibrosis. Implications for preclinical evaluation of antifibrotic therapy are expected. © 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Melanocortin 1 receptor and skin pathophysiology: beyond colour, much more than meets the eye.

PubMed

García-Borrón, José Carlos; Olivares, Concepción

2014-06-01

The melanocortin 1 receptor (MC1R), a G protein-coupled receptor preferentially expressed in melanocytes, mediates the pigmentary effects of α melanocyte-stimulating hormone (αMSH). MC1R is also expressed in other cutaneous cell types, particularly keratinocytes and dermal fibroblasts, suggesting non-pigmentary actions of the αMSH/MC1R system. Böhm and Stegemann now report a dramatic effect of mouse Mc1r functional status on susceptibility to skin fibrosis and collagen types I and III metabolism, in a study combining the powerful mouse model provided by the natural Mc1r(e/e) knockout and an established model of skin fibrosis. The study underscores the antifibrotic role for the skin αMSH/MC1R system. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

αvβ6 Integrin Regulates Renal Fibrosis and Inflammation in Alport Mouse

PubMed Central

Hahm, Kyungmin; Lukashev, Matvey E.; Luo, Yi; Yang, William J.; Dolinski, Brian M.; Weinreb, Paul H.; Simon, Kenneth J.; Chun Wang, Li; Leone, Diane R.; Lobb, Roy R.; McCrann, Donald J.; Allaire, Normand E.; Horan, Gerald S.; Fogo, Agnes; Kalluri, Raghu; Shield, Charles F.; Sheppard, Dean; Gardner, Humphrey A.; Violette, Shelia M.

2007-01-01

The transforming growth factor (TGF)-β-inducible integrin αvβ6 is preferentially expressed at sites of epithelial remodeling and has been shown to bind and activate latent precursor TGF-β. Herein, we show that αvβ6 is overexpressed in human kidney epithelium in membranous glomerulonephritis, diabetes mellitus, IgA nephropathy, Goodpasture’s syndrome, and Alport syndrome renal epithelium. To assess the potential regulatory role of αvβ6 in renal disease, we studied the effects of function-blocking αvβ6 monoclonal antibodies (mAbs) and genetic ablation of the β6 subunit on kidney fibrosis in Col4A3−/− mice, a mouse model of Alport syndrome. Expression of αvβ6 in Alport mouse kidneys was observed primarily in cortical tubular epithelial cells and in correlation with the progression of fibrosis. Treatment with αvβ6-blocking mAbs inhibited accumulation of activated fibroblasts and deposition of interstitial collagen matrix. Similar inhibition of renal fibrosis was observed in β6-deficient Alport mice. Transcript profiling of kidney tissues showed that αvβ6-blocking mAbs significantly inhibited disease-associated changes in expression of fibrotic and inflammatory mediators. Similar patterns of transcript modulation were produced with recombinant soluble TGF-β RII treatment, suggesting shared regulatory functions of αvβ6 and TGF-β. These findings demonstrate that αvβ6 can contribute to the regulation of renal fibrosis and suggest this integrin as a potential therapeutic target. PMID:17200187

Characterizing Fibrosis in Mouse Kidney using Label Free Fluorescence Lifetime and Second Harmonic Generation Imaging Microscopy in Unilateral Ureteral Obstruction Model

PubMed Central

Ranjit, Suman; Dobrinskikh, Evgenia; Montford, John; Dvornikov, Alexander; Lehman, Allison; Orlicky, David J.; Nemenoff, Raphael; Gratton, Enrico; Levi, Moshe; Furgeson, Seth

2017-01-01

All forms of progressive renal diseases develop a final pathway of tubulointerstitial fibrosis and glomerulosclerosis. Renal fibrosis is usually quantified using histological staining, a process that is time-consuming and pathologist dependent. The work described here shows the development of a fast and operator-independent method to measure fibrosis. To study renal fibrosis, the unilateral ureteral obstruction (UUO) model was chosen. Mice develop a time-dependent increase in obstructed kidneys; contralateral kidneys are used as controls. After UUO, kidneys were analyzed at three time points: 7 days, 14 days, and 21 days. Fibrosis was investigated using FLIM (Fluorescence Lifetime Imaging) and SHG (Second Harmonic Generation) in the deep tissue imaging microscope called DIVER (Deep Imaging via Enhanced photon Recovery). This microscope was developed for deep tissue and SHG and THG (Third Harmonic Generation) imaging and has extraordinary sensitivity towards harmonic generation. SHG data suggests the presence of more fibrillar collagen in the diseased kidneys. The combinations of short wavelength FLIM and SHG analysis results in a robust analysis procedure independent of observer interpretation and let us create a criterion to quantify the extent of fibrosis directly from the image. The progression of fibrosis in UUO model has been studied using this new FLIM-SHG technique and it shows remarkable improvement in quantification of fibrosis compared to standard histological techniques. PMID:27555119

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

Curcumin alleviates ischemia reperfusion-induced late kidney fibrosis through the APPL1/Akt signaling pathway.

PubMed

Hongtao, Chen; Youling, Fan; Fang, Huang; Huihua, Peng; Jiying, Zhong; Jun, Zhou

2018-05-09

As a major cause of renal failure, transient renal ischemia and reperfusion induce both acute kidney injury and late fibrosis, which are the common pathological manifestations of end-stage renal disease. Curcumin is a biologically active polyphenolic compound found in turmeric. Increasing evidence has demonstrated that curcumin has a protective action against renal fibrosis, whereas mechanisms underlying the anti-fibrosis role of curcumin remain poorly defined. Here, we found that APPL1, an important intracellular binding partner for AdipoR, was involved in the pathogenesis of acute injury or fibrosis and was significantly upregulated by curcumin in a mouse model of ischemia reperfusion-induced late kidney fibrosis. Moreover, Akt signaling was the specific signaling pathway identified downstream of APPL1 in the pathogenesis of fibrosis. Our in vitro experiment demonstrated that curcumin alleviates ischemia reperfusion-induced late kidney fibrosis via the APPL1/Akt pathway. These data are helpful for understanding the anti-fibrosis mechanism of curcumin in the pathogenesis of AKI-induced late fibrosis. © 2018 Wiley Periodicals, Inc.

Absence of microRNA-21 does not reduce muscular dystrophy in mouse models of LAMA2-CMD.

PubMed

Moreira Soares Oliveira, Bernardo; Durbeej, Madeleine; Holmberg, Johan

2017-01-01

MicroRNAs (miRNAs) are short non-coding RNAs that modulate gene expression post-transcriptionally. Current evidence suggests that miR-21 plays a significant role in the progression of fibrosis in muscle diseases. Laminin-deficient congenital muscular dystrophy (LAMA2-CMD) is a severe form of congenital muscular dystrophy caused by mutations in the gene encoding laminin α2 chain. Mouse models dy3K/dy3K and dy2J/dy2J, respectively, adequately mirror severe and milder forms of LAMA2-CMD. Both human and mouse LAMA2-CMD muscles are characterized by extensive fibrosis and considering that fibrosis is the final step that destroys muscle during the disease course, anti-fibrotic therapies may be effective strategies for prevention of LAMA2-CMD. We have previously demonstrated a significant up-regulation of the pro-fibrotic miR-21 in dy3K/dy3K and dy2J/dy2J skeletal muscle. Hence, the objective of this study was to explore if absence of miR-21 reduces fibrogenesis and improves the phenotype of LAMA2-CMD mice. Thus, we generated dy3K/dy3K and dy2J/dy2J mice devoid of miR-21 (dy3K/miR-21 and dy2J/miR-21 mice, respectively). However, the muscular dystrophy phenotype of dy3K/miR-21 and dy2J/miR-21 double knock-out mice was not improved compared to dy3K/dy3K or dy2J/dy2J mice, respectively. Mice displayed the same body weight, dystrophic muscles (with fibrosis) and impaired muscle function. These data indicate that miR-21 may not be involved in the development of fibrosis in LAMA2-CMD.

A novel telomerase activator suppresses lung damage in a murine model of idiopathic pulmonary fibrosis.

PubMed

Le Saux, Claude Jourdan; Davy, Philip; Brampton, Christopher; Ahuja, Seema S; Fauce, Steven; Shivshankar, Pooja; Nguyen, Hieu; Ramaseshan, Mahesh; Tressler, Robert; Pirot, Zhu; Harley, Calvin B; Allsopp, Richard

2013-01-01

The emergence of diseases associated with telomere dysfunction, including AIDS, aplastic anemia and pulmonary fibrosis, has bolstered interest in telomerase activators. We report identification of a new small molecule activator, GRN510, with activity ex vivo and in vivo. Using a novel mouse model, we tested the potential of GRN510 to limit fibrosis induced by bleomycin in mTERT heterozygous mice. Treatment with GRN510 at 10 mg/kg/day activated telomerase 2-4 fold both in hematopoietic progenitors ex vivo and in bone marrow and lung tissue in vivo, respectively. Telomerase activation was countered by co-treatment with Imetelstat (GRN163L), a potent telomerase inhibitor. In this model of bleomycin-induced fibrosis, treatment with GRN510 suppressed the development of fibrosis and accumulation of senescent cells in the lung via a mechanism dependent upon telomerase activation. Treatment of small airway epithelial cells (SAEC) or lung fibroblasts ex vivo with GRN510 revealed telomerase activating and replicative lifespan promoting effects only in the SAEC, suggesting that the mechanism accounting for the protective effects of GRN510 against induced lung fibrosis involves specific types of lung cells. Together, these results support the use of small molecule activators of telomerase in therapies to treat idiopathic pulmonary fibrosis.

Gallic acid attenuates pulmonary fibrosis in a mouse model of transverse aortic contraction-induced heart failure.

PubMed

Jin, Li; Piao, Zhe Hao; Sun, Simei; Liu, Bin; Ryu, Yuhee; Choi, Sin Young; Kim, Gwi Ran; Kim, Hyung-Seok; Kee, Hae Jin; Jeong, Myung Ho

2017-12-01

Gallic acid, a trihydroxybenzoic acid found in tea and other plants, attenuates cardiac hypertrophy, fibrosis, and hypertension in animal models. However, the role of gallic acid in heart failure remains unknown. In this study, we show that gallic acid administration prevents heart failure-induced pulmonary fibrosis. Heart failure induced in mice, 8weeks after transverse aortic constriction (TAC) surgery, was confirmed by echocardiography. Treatment for 2weeks with gallic acid but not furosemide prevented cardiac dysfunction in mice. Gallic acid significantly inhibited TAC-induced pathological changes in the lungs, such as increased lung mass, pulmonary fibrosis, and damaged alveolar morphology. It also decreased the expression of fibrosis-related genes, including collagen types I and III, fibronectin, connective tissue growth factor (CTGF), and phosphorylated Smad3. Further, it inhibited the expression of epithelial-mesenchymal transition (EMT)-related genes, such as N-cadherin, vimentin, E-cadherin, SNAI1, and TWIST1. We suggest that gallic acid has therapeutic potential for the treatment of heart failure-induced pulmonary fibrosis. Copyright © 2017 Elsevier Inc. All rights reserved.

Histochemical and cellular changes accompanying the appearance of lung fibrosis in an experimental mouse model for Hermansky Pudlak syndrome

PubMed Central

Lyerla, Timothy

2010-01-01

Hermansky Pudlak syndrome (HPS) is a heterogeneous recessive genetic disease with a tendency to develop lung fibrosis with aging. A mouse strain with two mutant HPS genes affecting separate vesicle trafficking pathways, C57BL/6-Hps1ep-Ap3b1pe, exhibits severe lung abnormalities at young ages, including enlarged alveolar type II (ATII) cells with giant lamellar bodies and foamy alveolar macrophages (AMs), which are readily identified histologically. In this study, the appearance of lung fibrosis in older animals was studied using classical histological and biochemical methods. The HPS double mutant mice, but not Chediak Higashi syndrome (C57BL/6-Lystbg-J-J, CHS) or C57BL/6J black control (WT) mice, were found to develop lung fibrosis at about 17 months of age using Masson trichrome staining, which was confirmed by hydroxyproline analysis. TGF β1 levels were elevated in bronchial alveolar lavage samples at all ages tested in the double mutant, but not WT or CHS mice, indicative of a prefibrotic condition in this experimental strain; and AMs were highly positive for this cytokine using immunohistochemistry staining. Prosurfactant protein C staining for ATII cells showed redistribution and dysmorphism of these cells with aging, but there was no evidence for epithelial-mesenchymal transition of ATII cells by dual staining for prosurfactant C protein and α-smooth muscle actin. This investigation showed that the HPS double mutant mouse strain develops interstitial pneumonia (HPSIP) past 1 year of age, which may be initiated by abnormal ATII cells and exacerbated by AM activation. With prominent prefibrotic abnormalities, this double mutant may serve as a model for interventive therapy in HPS. PMID:20603711

N-acetylcysteine attenuates the development of cardiac fibrosis and remodeling in a mouse model of heart failure.

PubMed

Giam, Beverly; Chu, Po-Yin; Kuruppu, Sanjaya; Smith, A Ian; Horlock, Duncan; Kiriazis, Helen; Du, Xiao-Jun; Kaye, David M; Rajapakse, Niwanthi W

2016-04-01

Oxidative stress plays a central role in the pathogenesis of heart failure. We aimed to determine whether the antioxidantN-acetylcysteine can attenuate cardiac fibrosis and remodeling in a mouse model of heart failure. Minipumps were implanted subcutaneously in wild-type mice (n = 20) and mice with cardiomyopathy secondary to cardiac specific overexpression of mammalian sterile 20-like kinase 1 (MST-1;n = 18) to administerN-acetylcysteine (40 mg/kg per day) or saline for a period of 8 weeks. At the end of this period, cardiac remodeling and function was assessed via echocardiography. Fibrosis, oxidative stress, and expression of collagen types I andIIIwere quantified in heart tissues. Cardiac perivascular and interstitial fibrosis were greater by 114% and 209%, respectively, inMST-1 compared to wild type (P ≤ 0.001). InMST-1 mice administeredN-acetylcysteine, perivascular and interstitial fibrosis were 40% and 57% less, respectively, compared to those treated with saline (P ≤ 0. 03). Cardiac oxidative stress was 119% greater inMST-1 than in wild type (P < 0.001) andN-acetylcysteine attenuated oxidative stress inMST-1 by 42% (P = 0.005). These data indicate thatN-acetylcysteine can blunt cardiac fibrosis and related remodeling in the setting of heart failure potentially by reducing oxidative stress. This study provides the basis to investigate the role ofN-acetylcysteine in chronic heart failure. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

Surfactant protein D attenuates sub-epithelial fibrosis in allergic airways disease through TGF-β.

PubMed

Ogawa, Hirohisa; Ledford, Julie G; Mukherjee, Sambuddho; Aono, Yoshinori; Nishioka, Yasuhiko; Lee, James J; Izumi, Keisuke; Hollingsworth, John W

2014-11-29

Surfactant protein D (SP-D) can regulate both innate and adaptive immunity. Recently, SP-D has been shown to contribute to the pathogenesis of airway allergic inflammation and bleomycin-induced pulmonary fibrosis. However, in allergic airways disease, the role of SP-D in airway remodeling remains unknown. The objective of this study was to determine the contribution of functional SP-D in regulating sub-epithelial fibrosis in a mouse chronic house dust mite model of allergic airways disease. C57BL/6 wild-type (WT) and SP-D-/- mice (C57BL/6 background) were chronically challenged with house dust mite antigen (Dermatophagoides pteronyssinus, Dp). Studies with SP-D rescue and neutralization of TGF-β were conducted. Lung histopathology and the concentrations of collagen, growth factors, and cytokines present in the airspace and lung tissue were determined. Cultured eosinophils were stimulated by Dp in presence or absence of SP-D. Dp-challenged SP-D-/- mice demonstrate increased sub-epithelial fibrosis, collagen production, eosinophil infiltration, TGF-β1, and IL-13 production, when compared to Dp-challenged WT mice. By immunohistology, we detected an increase in TGF-β1 and IL-13 positive eosinophils in SP-D-/- mice. Purified eosinophils stimulated with Dp produced TGF-β1 and IL-13, which was prevented by co-incubation with SP-D. Additionally, treatment of Dp challenged SP-D-/- mice with exogenous SP-D was able to rescue the phenotypes observed in SP-D-/- mice and neutralization of TGF-β1 reduced sub-epithelial fibrosis in Dp-challenged SP-D-/- mice. These data support a protective role for SP-D in the pathogenesis of sub-epithelial fibrosis in a mouse model of allergic inflammation through regulation of eosinophil-derived TGF-β.

The bleomycin animal model: a useful tool to investigate treatment options for idiopathic pulmonary fibrosis?

PubMed Central

Moeller, Antje; Ask, Kjetil; Warburton, David; Gauldie, Jack; Kolb, Martin

2008-01-01

Different animal models of pulmonary fibrosis have been developed to investigate potential therapies for idiopathic pulmonary fibrosis (IPF). The most common is the bleomycin model in rodents (mouse, rat and hamster). Over the years, numerous agents have been shown to inhibit fibrosis in this model. However, to date none of these compounds are used in the clinical management of IPF and none has shown a comparable antifibrotic effect in humans. We performed a systematic review of publications on drug efficacy studies in the bleomycin model to evaluate the value of this model regarding transferability to clinical use. Between 1980 and 2006 we identified 246 experimental studies describing beneficial antifibrotic compounds in the bleomycin model. In 221 of the studies we found enough details about the timing of drug application to allow inter-study comparison. 211 of those used a preventive regimen (drug given ≤ day 7 after last bleomycin application), only 10 were therapeutic trials (> 7 days after last bleomycin application). It is critical to distinguish between drugs interfering with the inflammatory and early fibrogenic response from those preventing progression of fibrosis, the latter likely much more meaningful for clinical application. All potential antifibrotic compounds should be evaluated in the phase of established fibrosis rather than in the early period of bleomycin-induced inflammation for assessment of its antifibrotic properties. Further care should be taken in extrapolation of drugs successfully tested in the bleomycin model due to partial reversibility of bleomycin induced fibrosis over time. The use of alternative and more robust animal models, which better reflect human IPF, is warranted. PMID:17936056

Inhibiting aerobic glycolysis suppresses renal interstitial fibroblast activation and renal fibrosis.

PubMed

Ding, Hao; Jiang, Lei; Xu, Jing; Bai, Feng; Zhou, Yang; Yuan, Qi; Luo, Jing; Zen, Ke; Yang, Junwei

2017-09-01

Chronic kidney diseases generally lead to renal fibrosis. Despite great progress having been made in identifying molecular mediators of fibrosis, the mechanism that governs renal fibrosis remains unclear, and so far no effective therapeutic antifibrosis strategy is available. Here we demonstrated that a switch of metabolism from oxidative phosphorylation to aerobic glycolysis (Warburg effect) in renal fibroblasts was the primary feature of fibroblast activation during renal fibrosis and that suppressing renal fibroblast aerobic glycolysis could significantly reduce renal fibrosis. Both gene and protein assay showed that the expression of glycolysis enzymes was upregulated in mouse kidneys with unilateral ureter obstruction (UUO) surgery or in transforming growth factor-β1 (TGF-β1)-treated renal interstitial fibroblasts. Aerobic glycolysis flux, indicated by glucose uptake and lactate production, was increased in mouse kidney with UUO nephropathy or TGF-β1-treated renal interstitial fibroblasts and positively correlated with fibrosis process. In line with this, we found that increasing aerobic glycolysis can remarkably induce myofibroblast activation while aerobic glycolysis inhibitors shikonin and 2-deoxyglucose attenuate UUO-induced mouse renal fibrosis and TGF-β1-stimulated myofibroblast activation. Furthermore, mechanistic study indicated that shikonin inhibits renal aerobic glycolysis via reducing phosphorylation of pyruvate kinase type M2, a rate-limiting glycolytic enzyme associated with cell reliance on aerobic glycolysis. In conclusion, our findings demonstrate the critical role of aerobic glycolysis in renal fibrosis and support treatment with aerobic glycolysis inhibitors as a potential antifibrotic strategy. Copyright © 2017 the American Physiological Society.

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.

Therapeutic effects of telomerase in mice with pulmonary fibrosis induced by damage to the lungs and short telomeres.

PubMed

Povedano, Juan Manuel; Martinez, Paula; Serrano, Rosa; Tejera, Águeda; Gómez-López, Gonzalo; Bobadilla, Maria; Flores, Juana Maria; Bosch, Fátima; Blasco, Maria A

2018-01-30

Pulmonary fibrosis is a fatal lung disease characterized by fibrotic foci and inflammatory infiltrates. Short telomeres can impair tissue regeneration and are found both in hereditary and sporadic cases. We show here that telomerase expression using AAV9 vectors shows therapeutic effects in a mouse model of pulmonary fibrosis owing to a low-dose bleomycin insult and short telomeres. AAV9 preferentially targets regenerative alveolar type II cells (ATII). AAV9- Tert -treated mice show improved lung function and lower inflammation and fibrosis at 1-3 weeks after viral treatment, and improvement or disappearance of the fibrosis at 8 weeks after treatment. AAV9- Tert treatment leads to longer telomeres and increased proliferation of ATII cells, as well as lower DNA damage, apoptosis, and senescence. Transcriptome analysis of ATII cells confirms downregulation of fibrosis and inflammation pathways. We provide a proof-of-principle that telomerase activation may represent an effective treatment for pulmonary fibrosis provoked or associated with short telomeres. © 2018, Povedano et al.

Therapeutic effects of telomerase in mice with pulmonary fibrosis induced by damage to the lungs and short telomeres

PubMed Central

Serrano, Rosa; Tejera, Águeda; Gómez-López, Gonzalo; Bobadilla, Maria; Flores, Juana Maria; Bosch, Fátima

2018-01-01

Pulmonary fibrosis is a fatal lung disease characterized by fibrotic foci and inflammatory infiltrates. Short telomeres can impair tissue regeneration and are found both in hereditary and sporadic cases. We show here that telomerase expression using AAV9 vectors shows therapeutic effects in a mouse model of pulmonary fibrosis owing to a low-dose bleomycin insult and short telomeres. AAV9 preferentially targets regenerative alveolar type II cells (ATII). AAV9-Tert-treated mice show improved lung function and lower inflammation and fibrosis at 1–3 weeks after viral treatment, and improvement or disappearance of the fibrosis at 8 weeks after treatment. AAV9-Tert treatment leads to longer telomeres and increased proliferation of ATII cells, as well as lower DNA damage, apoptosis, and senescence. Transcriptome analysis of ATII cells confirms downregulation of fibrosis and inflammation pathways. We provide a proof-of-principle that telomerase activation may represent an effective treatment for pulmonary fibrosis provoked or associated with short telomeres. PMID:29378675

Cardiac fibroblast GSK-3β regulates ventricular remodeling and dysfunction in ischemic heart

PubMed Central

Lal, Hind; Ahmad, Firdos; Zhou, Jibin; Yu, Justine E.; Vagnozzi, Ronald J.; Guo, Yuanjun; Yu, Daohai; Tsai, Emily J.; Woodgett, James; Gao, Erhe; Force, Thomas

2014-01-01

Background Myocardial infarction-induced remodeling includes chamber dilatation, contractile dysfunction, and fibrosis. Of these, fibrosis is the least understood. Following MI, activated cardiac fibroblasts (CFs) deposit extracellular matrix. Current therapies to prevent fibrosis are inadequate and new molecular targets are needed. Methods and Results Herein we report that GSK-3β is phosphorylated (inhibited) in fibrotic tissues from ischemic human and mouse heart. Using two fibroblast-specific GSK-3β knockout mouse models, we show that deletion of GSK-3β in CFs leads to fibrogenesis, left ventricular dysfunction and excessive scarring in the ischemic heart. Deletion of GSK-3β induces a pro-fibrotic myofibroblast phenotype in isolated CFs, in post-MI hearts, and in MEFs deleted for GSK-3β. Mechanistically, GSK-3β inhibits pro-fibrotic TGF-β1-SMAD-3 signaling via interactions with SMAD-3. Moreover, deletion of GSK-3β resulted in the suppression of SMAD-3 transcriptional activity. This pathway is central to the pathology since a small molecule inhibitor of SMAD-3 largely prevented fibrosis and limited LV remodeling. Conclusion These studies support targeting GSK-3β in myocardial fibrotic disorders and establish critical roles of CFs in remodeling and ventricular dysfunction. PMID:24899689

Stable expression of HIF-1α in tubular epithelial cells promotes interstitial fibrosis

PubMed Central

Kimura, Kuniko; Iwano, Masayuki; Higgins, Debra F.; Yamaguchi, Yukinari; Nakatani, Kimihiko; Harada, Koji; Kubo, Atsushi; Akai, Yasuhiro; Rankin, Erinn B.; Neilson, Eric G.; Haase, Volker H.; Saito, Yoshihiko

2008-01-01

Chronic hypoxia accelerates renal fibrosis. The chief mediator of the hypoxic response is hypoxia-inducible factor 1 (HIF-1) and its oxygen-sensitive component HIF-1α. HIF-1 regulates a wide variety of genes, some of which are closely associated with tissue fibrosis. To determine the specific role of HIF-1 in renal fibrosis, we generated a knockout mouse in which tubular epithelial expression of von Hippel-Lindau tumor suppressor (VHL), which acts as a ubiquitin ligase to promote proteolysis of HIF-1α, was targeted. We investigated the effect of VHL deletion (i.e., stable expression of HIF-1α) histologically and used the anti-HIF-1α agent [3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole] (YC-1) to test whether inhibition of HIF-1α could represent a novel approach to treating renal fibrosis. The area of renal fibrosis was significantly increased in a 5/6 renal ablation model of VHL−/− mice and in all VHL−/− mice at least 60 wk of age. Injection of YC-1 inhibited the progression of renal fibrosis in unilateral ureteral obstruction model mice. In conclusion, HIF-1α appears to be a critical contributor to the progression of renal fibrosis and could be a useful target for its treatment. PMID:18667485

Role of Mutant CFTR in Hypersusceptibility of Cystic Fibrosis Patients to Lung Infections

NASA Astrophysics Data System (ADS)

Pier, Gerald B.; Grout, Martha; Zaidi, Tanweer S.; Olsen, John C.; Johnson, Larry G.; Yankaskas, James R.; Goldberg, Joanna B.

1996-01-01

Cystic fibrosis (CF) patients are hypersusceptible to chronic Pseudomonas aeruginosa lung infections. Cultured human airway epithelial cells expressing the ΔF508 allele of the cystic fibrosis transmembrane conductance regulator (CFTR) were defective in uptake of P. aeruginosa compared with cells expressing the wild-type allele. Pseudomonas aeruginosa lipopolysaccharide (LPS)-core oligosaccharide was identified as the bacterial ligand for epithelial cell ingestion; exogenous oligosaccharide inhibited bacterial ingestion in a neonatal mouse model, resulting in increased amounts of bacteria in the lungs. CFTR may contribute to a host-defense mechanism that is important for clearance of P. aeruginosa from the respiratory tract.

Comparative study of two models of combined pulmonary fibrosis and emphysema in mice.

PubMed

Zhang, Wan-Guang; Wu, Si-Si; He, Li; Yang, Qun; Feng, Yi-Kuan; Chen, Yue-Tao; Zhen, Guo-Hua; Xu, Yong-Jian; Zhang, Zhen-Xiang; Zhao, Jian-Ping; Zhang, Hui-Lan

2017-04-01

Combined pulmonary fibrosis and emphysema (CPFE) is an "umbrella term" encompassing emphysema and pulmonary fibrosis, but its pathogenesis is not known. We established two models of CPFE in mice using tracheal instillation with bleomycin (BLM) or murine gammaherpesvirus 68 (MHV-68). Experimental mice were divided randomly into four groups: A (normal control, n=6), B (emphysema, n=6), C (emphysema+MHV-68, n=24), D (emphysema+BLM, n=6). Group C was subdivided into four groups: C1 (sacrificed on day 367, 7 days after tracheal instillation of MHV-68); C2 (day 374; 14days); C3 (day 381; 21days); C4 (day 388; 28days). Conspicuous emphysema and interstitial fibrosis were observed in BLM and MHV-68 CPFE mouse models. However, BLM induced diffuse pulmonary interstitial fibrosis with severely diffuse pulmonary inflammation; MHV-68 induced relatively modest inflammation and fibrosis, and the inflammation and fibrosis were not diffuse, but instead around bronchioles. Inflammation and fibrosis were detectable in the day-7 subgroup and reached a peak in the day-28 subgroup in the emphysema + MHV-68 group. Levels of macrophage chemoattractant protein-1, macrophage inflammatory protein-1α, interleukin-13, and transforming growth factor-β1 in bronchoalveolar lavage fluid were increased significantly in both models. Percentage of apoptotic type-2 lung epithelial cells was significantly higher; however, all four types of cytokine and number of macrophages were significantly lower in the emphysema+MHV-68 group compared with the emphysema +BLM group. The different changes in pathology between BLM and MHV-68 mice models demonstrated different pathology subtypes of CPFE: macrophage infiltration and apoptosis of type-II lung epithelial cells increased with increasing pathology score for pulmonary fibrosis. Copyright © 2017 Elsevier GmbH. All rights reserved.

Progenitor-like cells derived from mouse kidney protect against renal fibrosis in a remnant kidney model via decreased endothelial mesenchymal transition.

PubMed

Chen, C L; Chou, K J; Fang, H C; Hsu, C Y; Huang, W C; Huang, C W; Huang, C K; Chen, H Y; Lee, P T

2015-12-02

Pathophysiological changes associated with chronic kidney disease impair angiogenic processes and increase renal fibrosis. Progenitor-like cells derived from adult kidney have been previously used to promote regeneration in acute kidney injury, even though it remained unclear whether the cells could be beneficial in chronic kidney disease (CKD). In this study, we established a CKD model by five-sixths nephrectomy and mouse kidney progenitor-like cells (MKPCs) were intravenously administered weekly for 5 weeks after establishing CKD. We examined the impact of MKPCs on the progression of renal fibrosis and the potential of MKPCs to preserve the angiogenic process and prevent endothelial mesenchymal transition in vivo and in vitro. Our results demonstrate that the MKPCs delayed interstitial fibrosis and the progression of glomerular sclerosis and ameliorated the decline of kidney function. At 17 weeks, the treated mice exhibited lower blood pressures, higher hematocrit levels, and larger kidney sizes than the control mice. In addition, the MKPC treatment prolonged the survival of the mice with chronic kidney injuries. We observed a decreased recruitment of macrophages and myofibroblasts in the interstitium and the increased tubular proliferation. Notably, MKPC both decreased the level of vascular rarefaction and prevented endothelial mesenchymal transition (EndoMT) in the remnant kidneys. Moreover, the conditioned medium from the MKPCs ameliorated endothelial cell death under hypoxic culture conditions and prevented TGF-β-induced EndoMT through downregulation of phosphorylated Smad 3 in vitro. MKPCs may be a beneficial treatment for kidney diseases characterized by progressive renal fibrosis. The enhanced preservation of angiogenic processes following MKPC injections may be associated with decreased fibrosis in the remnant kidney. These findings provide further understanding of the mechanisms involved in these processes and will help develop new cell-based therapeutic strategies for regenerative medicine in renal fibrosis.

Hepatocellular carcinoma in a mouse model fed a choline-deficient, L-amino acid-defined, high-fat diet.

PubMed

Ikawa-Yoshida, Ayae; Matsuo, Saori; Kato, Atsuhiko; Ohmori, Yusuke; Higashida, Atsuko; Kaneko, Eiji; Matsumoto, Masahiko

2017-08-01

Hepatocellular carcinoma (HCC) is a common cancer worldwide and represents the outcome of the natural history of chronic liver disease. The growing rates of HCC may be partially attributable to increased numbers of people with non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). However, details of the liver-specific molecular mechanisms responsible for the NAFLD-NASH-HCC progression remain unclear, and mouse models that can be used to explore the exact factors that influence the progression of NAFLD/NASH to the more chronic stages of liver disease and subsequent HCC are not yet fully established. We have previously reported a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) as a dietary NASH model with rapidly progressive liver fibrosis in mice. The current study in C57BL/6J mice fed CDAHFD provided evidence for the chronic persistence of advanced hepatic fibrosis in NASH and disease progression towards HCC in a period of 36 weeks. When mice fed CDAHFD were switched back to a standard diet, hepatic steatosis was normalized and NAFLD activity score improved, but HCC incidence increased and the phenotype of fibrosis-associated HCC development was observed. Moreover, when mice continued to be fed CDAHFD for 60 weeks, HCC further developed without severe body weight loss or carcinogenesis in other organs. The autochthonous tumours showed a variety of histological features and architectural patterns including trabecular, pseudoglandular and solid growth. The CDAHFD mouse model might be a useful tool for studying the development of HCC from NAFLD/NASH, and potentially useful for better understanding pathological changes during hepatocarcinogenesis. © 2017 The Authors. International Journal of Experimental Pathology published by John Wiley & Sons Ltd on behalf of Company of the International Journal of Experimental Pathology (CIJEP).

miR-199a-5p Is Upregulated during Fibrogenic Response to Tissue Injury and Mediates TGFbeta-Induced Lung Fibroblast Activation by Targeting Caveolin-1

PubMed Central

Courcot, Elisabeth; Roderburg, Christoph; Cauffiez, Christelle; Aubert, Sébastien; Copin, Marie-Christine; Wallaert, Benoit; Glowacki, François; Dewaeles, Edmone; Milosevic, Jadranka; Maurizio, Julien; Tedrow, John; Marcet, Brice; Lo-Guidice, Jean-Marc; Kaminski, Naftali; Barbry, Pascal; Luedde, Tom; Perrais, Michael

2013-01-01

As miRNAs are associated with normal cellular processes, deregulation of miRNAs is thought to play a causative role in many complex diseases. Nevertheless, the precise contribution of miRNAs in fibrotic lung diseases, especially the idiopathic form (IPF), remains poorly understood. Given the poor response rate of IPF patients to current therapy, new insights into the pathogenic mechanisms controlling lung fibroblasts activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies for this devastating disease. To identify miRNAs with potential roles in lung fibrogenesis, we performed a genome-wide assessment of miRNA expression in lungs from two different mouse strains known for their distinct susceptibility to develop lung fibrosis after bleomycin exposure. This led to the identification of miR-199a-5p as the best miRNA candidate associated with bleomycin response. Importantly, miR-199a-5p pulmonary expression was also significantly increased in IPF patients (94 IPF versus 83 controls). In particular, levels of miR-199a-5p were selectively increased in myofibroblasts from injured mouse lungs and fibroblastic foci, a histologic feature associated with IPF. Therefore, miR-199a-5p profibrotic effects were further investigated in cultured lung fibroblasts: miR-199a-5p expression was induced upon TGFβ exposure, and ectopic expression of miR-199a-5p was sufficient to promote the pathogenic activation of pulmonary fibroblasts including proliferation, migration, invasion, and differentiation into myofibroblasts. In addition, we demonstrated that miR-199a-5p is a key effector of TGFβ signaling in lung fibroblasts by regulating CAV1, a critical mediator of pulmonary fibrosis. Remarkably, aberrant expression of miR-199a-5p was also found in unilateral ureteral obstruction mouse model of kidney fibrosis, as well as in both bile duct ligation and CCl4-induced mouse models of liver fibrosis, suggesting that dysregulation of miR-199a-5p represents a general mechanism contributing to the fibrotic process. MiR-199a-5p thus behaves as a major regulator of tissue fibrosis with therapeutic potency to treat fibroproliferative diseases. PMID:23459460

Glutamine inhibits CCl4 induced liver fibrosis in mice and TGF-β1 mediated epithelial-mesenchymal transition in mouse hepatocytes.

PubMed

Shrestha, Nirajan; Chand, Lokendra; Han, Myung Kwan; Lee, Seung Ok; Kim, Chan Young; Jeong, Yeon Jun

2016-07-01

Glutamine, traditionally a non-essential amino acid, now has been considered as essential in serious illness and injury. It is a major precursor for glutathione synthesis. However, the anti-fibrotic effect of glutamine and its molecular mechanism in experimental liver fibrosis have not been explored. In the present study we aimed to examine the potential role of glutamine in carbon tetrachloride (CCl4) induced liver fibrosis and TGF-β1 mediated epithelial mesenchymal transition (EMT) and apoptosis in mouse hepatocytes. Liver fibrosis was induced by intraperitoneal injection of CCl4 three times a week for 10 weeks. Glutamine treatment effectively attenuated liver injury and oxidative stress. Collagen content was significantly decreased in liver sections of glutamine treated mice compared to CCl4 model mice. Furthermore, glutamine decreased expression level of α-SMA and TGF-β in liver tissue. Our in vitro study showed that TGF-β1 treatment in hepatocytes resulted in loss of E-cadherin and increased expression of mesenchymal markers and EMT related transcription factor. In addition, TGF-β1 increased the expression of apoptotic markers. However, glutamine interestingly suppressed TGF-β1 mediated EMT and apoptosis. In conclusion, our results suggest that glutamine ameliorates CCl4 induced liver fibrosis and suppresses TGF-β1 induced EMT progression and apoptosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Pathogenesis pathways of idiopathic pulmonary fibrosis in bleomycin-induced lung injury model in mice.

PubMed

Shi, Keyun; Jiang, Jianzhong; Ma, Tieliang; Xie, Jing; Duan, Lirong; Chen, Ruhua; Song, Ping; Yu, Zhixin; Liu, Chao; Zhu, Qin; Zheng, Jinxu

2014-01-01

Our objective was to investigate the pathogenesis pathways of idiopathic pulmonary fibrosis (IPF). Bleomycin (BLM) induced animal models of experimental lung fibrosis were used. CHIP assay was executed to find the link between Smad3 and IL-31, and the expressions of TGF-β1, Smad3, IL-31 and STAT1 were detected to find whether they were similar with each other. We found that in the early injury or inflammation of the animal model, BLM promoted the development of inflammation, leading to severe pulmonary fibrosis. Then the expression of TGF-β1 and Smad3 increased. Activated Smad3 bound to the IL-31 promoter region, followed by the activation of JAK-STAT pathways. The inhibitor of TGF-β1 receptor decreased the IL-31 expression and knocking-down of IL-31 also decreased the STAT1 expression. We conclude that there is a pathway of pathogenesis in BLM-induced mouse model that involves the TGF-β, IL-31 and JAKs/STATs pathway. Copyright © 2013 Elsevier B.V. All rights reserved.

Novel form of miR-29b suppresses bleomycin-induced pulmonary fibrosis

PubMed Central

Yamada, Yuko; Takanashi, Masakatsu; Sudo, Katsuko; Ueda, Shinobu; Ohno, Shin-ichiro; Kuroda, Masahiko

2017-01-01

MicroRNA 29b (miR-29b) replacement therapy is effective for suppressing fibrosis in a mouse model. However, to develop clinical applications for miRNA mimics, the side effects of nucleic acid drugs have to be addressed. In this study, we focused on miRNA mimics in order to develop therapies for idiopathic pulmonary fibrosis. We developed a single-stranded RNA, termed “miR-29b Psh-match,” that has a unique structure to avoid problems associated with the therapeutic uses of miRNAs. A comparison of miR-29b Psh-match and double-stranded one, termed “miR-29b mimic” indicated that the single-stranded form was significantly effective towards fibrosis according to both in vivo and in vitro experiments. This novel form of miR-29b may become the foundation for developing an effective therapeutic drug for pulmonary fibrosis. PMID:28234907

Origin and function of myofibroblasts in kidney fibrosis.

PubMed

LeBleu, Valerie S; Taduri, Gangadhar; O'Connell, Joyce; Teng, Yingqi; Cooke, Vesselina G; Woda, Craig; Sugimoto, Hikaru; Kalluri, Raghu

2013-08-01

Myofibroblasts are associated with organ fibrosis, but their precise origin and functional role remain unknown. We used multiple genetically engineered mice to track, fate map and ablate cells to determine the source and function of myofibroblasts in kidney fibrosis. Through this comprehensive analysis, we identified that the total pool of myofibroblasts is split, with 50% arising from local resident fibroblasts through proliferation. The nonproliferating myofibroblasts derive through differentiation from bone marrow (35%), the endothelial-to-mesenchymal transition program (10%) and the epithelial-to-mesenchymal transition program (5%). Specific deletion of Tgfbr2 in α-smooth muscle actin (αSMA)(+) cells revealed the importance of this pathway in the recruitment of myofibroblasts through differentiation. Using genetic mouse models and a fate-mapping strategy, we determined that vascular pericytes probably do not contribute to the emergence of myofibroblasts or fibrosis. Our data suggest that targeting diverse pathways is required to substantially inhibit the composite accumulation of myofibroblasts in kidney fibrosis.

Origin and Function of Myofibroblasts in Kidney Fibrosis

PubMed Central

LeBleu, Valerie S.; Taduri, Gangadhar; O’Connell, Joyce; Teng, Yingqi; Cooke, Vesselina G.; Woda, Craig; Sugimoto, Hikaru; Kalluri, Raghu

2014-01-01

Myofibroblasts are associated with organ fibrosis but their precise origin and functional role remain unknown. We employed multiple genetically engineered mice to track, fate-map and ablate cells to determine the source and function of myofibroblasts in kidney fibrosis. Such comprehensive analysis identified that the total pool of myofibroblasts is split, with 50% arising from local resident fibroblasts via proliferation. The non-proliferating myofibroblasts derive via differentiation from bone marrow (35%), endothelial to mesenchymal transition (EndMT) program (10%) and epithelial to mesenchymal transition (EMT) program (5%). Specific deletion of Tgfbr2 in αSMA+ cells revealed the importance of this pathway in recruitment of myofibroblasts via differentiation. Using genetic mouse models and fate-mapping strategy we determined that vascular pericytes likely do not contribute to the emergence of myofibroblasts or fibrosis. This study suggests that targeting diverse pathways is required to significantly inhibit composite accumulation of myofibroblasts in kidney fibrosis. PMID:23817022

Inhibition of the CXCL12/CXCR4-Axis as Preventive Therapy for Radiation-Induced Pulmonary Fibrosis

PubMed Central

Shu, Hui-Kuo G.; Yoon, Younghyoun; Hong, Samuel; Xu, Kaiming; Gao, Huiying; Hao, Chunhai; Torres-Gonzalez, Edilson; Nayra, Cardenes; Rojas, Mauricio; Shim, Hyunsuk

2013-01-01

Background A devastating late injury caused by radiation is pulmonary fibrosis. This risk may limit the volume of irradiation and compromise potentially curative therapy. Therefore, development of a therapy to prevent this toxicity can be of great benefit for this patient population. Activation of the chemokine receptor CXCR4 by its ligand stromal cell-derived factor 1 (SDF-1/CXCL12) may be important in the development of radiation-induced pulmonary fibrosis. Here, we tested whether MSX-122, a novel small molecule and partial CXCR4 antagonist, can block development of this fibrotic process. Methodology/Principal Findings The radiation-induced lung fibrosis model used was C57BL/6 mice irradiated to the entire thorax or right hemithorax to 20 Gy. Our parabiotic model involved joining a transgenic C57BL/6 mouse expressing GFP with a wild-type mouse that was subsequently irradiated to assess for migration of GFP+ bone marrow-derived progenitor cells to the irradiated lung. CXCL12 levels in the bronchoalveolar lavage fluid (BALF) and serum after irradiation were determined by ELISA. CXCR4 and CXCL12 mRNA in the irradiated lung was determined by RNase protection assay. Irradiated mice were treated daily with AMD3100, an established CXCR4 antagonist; MSX-122; and their corresponding vehicles to determine impact of drug treatment on fibrosis development. Fibrosis was assessed by serial CTs and histology. After irradiation, CXCL12 levels increased in BALF and serum with a corresponding rise in CXCR4 mRNA within irradiated lungs consistent with recruitment of a CXCR4+ cell population. Using our parabiotic model, we demonstrated recruitment of CXCR4+ bone marrow-derived mesenchymal stem cells, identified based on marker expression, to irradiated lungs. Finally, irradiated mice that received MSX-122 had significant reductions in development of pulmonary fibrosis while AMD3100 did not significantly suppress this fibrotic process. Conclusions/Significance CXCR4 inhibition by drugs such as MSX-122 may alleviate potential radiation-induced lung injury, presenting future therapeutic opportunities for patients requiring chest irradiation. PMID:24244561

Inhibition of the CXCL12/CXCR4-axis as preventive therapy for radiation-induced pulmonary fibrosis.

PubMed

Shu, Hui-Kuo G; Yoon, Younghyoun; Hong, Samuel; Xu, Kaiming; Gao, Huiying; Hao, Chunhai; Torres-Gonzalez, Edilson; Nayra, Cardenes; Rojas, Mauricio; Shim, Hyunsuk

2013-01-01

A devastating late injury caused by radiation is pulmonary fibrosis. This risk may limit the volume of irradiation and compromise potentially curative therapy. Therefore, development of a therapy to prevent this toxicity can be of great benefit for this patient population. Activation of the chemokine receptor CXCR4 by its ligand stromal cell-derived factor 1 (SDF-1/CXCL12) may be important in the development of radiation-induced pulmonary fibrosis. Here, we tested whether MSX-122, a novel small molecule and partial CXCR4 antagonist, can block development of this fibrotic process. The radiation-induced lung fibrosis model used was C57BL/6 mice irradiated to the entire thorax or right hemithorax to 20 Gy. Our parabiotic model involved joining a transgenic C57BL/6 mouse expressing GFP with a wild-type mouse that was subsequently irradiated to assess for migration of GFP+ bone marrow-derived progenitor cells to the irradiated lung. CXCL12 levels in the bronchoalveolar lavage fluid (BALF) and serum after irradiation were determined by ELISA. CXCR4 and CXCL12 mRNA in the irradiated lung was determined by RNase protection assay. Irradiated mice were treated daily with AMD3100, an established CXCR4 antagonist; MSX-122; and their corresponding vehicles to determine impact of drug treatment on fibrosis development. Fibrosis was assessed by serial CTs and histology. After irradiation, CXCL12 levels increased in BALF and serum with a corresponding rise in CXCR4 mRNA within irradiated lungs consistent with recruitment of a CXCR4+ cell population. Using our parabiotic model, we demonstrated recruitment of CXCR4+ bone marrow-derived mesenchymal stem cells, identified based on marker expression, to irradiated lungs. Finally, irradiated mice that received MSX-122 had significant reductions in development of pulmonary fibrosis while AMD3100 did not significantly suppress this fibrotic process. CXCR4 inhibition by drugs such as MSX-122 may alleviate potential radiation-induced lung injury, presenting future therapeutic opportunities for patients requiring chest irradiation.

The sexually dimorphic role of adipose and adipocyte estrogen receptors in modulating adipose tissue expansion, inflammation, and fibrosis

PubMed Central

Davis, Kathryn E.; D. Neinast, Michael; Sun, Kai; M. Skiles, William; D. Bills, Jessica; A. Zehr, Jordan; Zeve, Daniel; D. Hahner, Lisa; W. Cox, Derek; M. Gent, Lana; Xu, Yong; V. Wang, Zhao; A. Khan, Sohaib; Clegg, Deborah J.

2013-01-01

Our data demonstrate that estrogens, estrogen receptor-α (ERα), and estrogen receptor-β (ERβ) regulate adipose tissue distribution, inflammation, fibrosis, and glucose homeostasis, by determining that αERKO mice have increased adipose tissue inflammation and fibrosis prior to obesity onset. Selective deletion of adipose tissue ERα in adult mice using a novel viral vector technology recapitulated the findings in the total body ERα null mice. Generation of a novel mouse model, lacking ERα specifically from adipocytes (AdipoERα), demonstrated increased markers of fibrosis and inflammation, especially in the males. Additionally, we found that the beneficial effects of estrogens on adipose tissue require adipocyte ERα. Lastly, we determined the role of ERβ in regulating inflammation and fibrosis, by breeding the AdipoERα into the βERKO background and found that in the absence of adipocyte ERα, ERβ has a protective role. These data suggest that adipose tissue and adipocyte ERα protects against adiposity, inflammation, and fibrosis in both males and females. PMID:24049737

Classical dendritic cells mediate fibrosis directly via the retinoic acid pathway in severe eye allergy

PubMed Central

Ahadome, Sarah D.; Mathew, Rose; Reyes, Nancy J.; Mettu, Priyatham S.; Cousins, Scott W.; Calder, Virginia L.; Saban, Daniel R.

2016-01-01

Fibrosis is a shared end-stage pathway to lung, liver, and heart failure. In the ocular mucosa (conjunctiva), fibrosis leads to blindness in trachoma, pemphigoid, and allergy. The indirect fibrogenic role of DCs via T cell activation and inflammatory cell recruitment is well documented. However, here we demonstrate that DCs can directly induce fibrosis. In the mouse model of allergic eye disease (AED), classical CD11b+ DCs in the ocular mucosa showed increased activity of aldehyde dehydrogenase (ALDH), the enzyme required for retinoic acid synthesis. In vitro, CD11b+ DC–derived ALDH was associated with 9-cis-retinoic acid ligation to retinoid x receptor (RXR), which induced conjunctival fibroblast activation. In vivo, stimulating RXR led to rapid onset of ocular mucosal fibrosis, whereas inhibiting ALDH activity in DCs or selectively depleting DCs markedly reduced fibrosis. Collectively, these data reveal a profibrotic ALDH-dependent pathway by DCs and uncover a role for DC retinoid metabolism. PMID:27595139

The Novel Extracellular Cyclophilin A (CyPA) - Inhibitor MM284 Reduces Myocardial Inflammation and Remodeling in a Mouse Model of Troponin I -Induced Myocarditis.

PubMed

Heinzmann, David; Bangert, Anna; Müller, Anna-Maria; von Ungern-Sternberg, Saskia N I; Emschermann, Frederic; Schönberger, Tanja; Chatterjee, Madhumita; Mack, Andreas F; Klingel, Karin; Kandolf, Reinhard; Malesevic, Miroslav; Borst, Oliver; Gawaz, Meinrad; Langer, Harald F; Katus, Hugo; Fischer, Gunter; May, Andreas E; Kaya, Ziya; Seizer, Peter

2015-01-01

Cyclophilins are a group of highly conserved cytosolic enzymes that have a peptidylprolyl cis/trans isomerase activity. Cyclophilin A (CyPA) can be secreted in the extracellular space by inflammatory cells and upon cell death. The presence of CyPA in patients with non-ischemic cardiomyopathy is associated with poor clinical prognosis. Here, we investigated the inhibition of extracellular CyPA in a mouse model of troponin I-induced autoimmune myocarditis using the strictly extracellular CyPA-inhibitor MM284. Since A/J mice develop severe inflammation and fibrosis after immunization with murine cardiac troponin I (mcTn I), we used this model to analyze the effects of an extracellular CyPA inhibition. As extracellular CyPA-inhibitor we used the recently described CsA-derivate MM284. In vitro studies confirmed that MM284 inhibits CyPA-induced monocytic migration and adhesion. A/J mice immunized with mcTnI were treated with MM284 or vehicle every second day. After 28 days, we found a considerable reduction of myocardial injury and fibrosis. Further analysis revealed a reduced myocardial presence of T-cells and macrophages compared to control treated animals. Whereas MMP-9 expression was reduced significantly by MM284, we observed no significant reduction of inflammatory cytokines such as IL-6 or TNFα. Extracellular CyPA plays an important role in autoimmune myocarditis for myocardial damage and fibrosis. Our data suggest a new pharmacological approach for the treatment of myocardial inflammation and reduction of cardiac fibrosis by inhibition of extracellular CyPA.

Th17 cells and IL-17 promote the skin and lung inflammation and fibrosis process in a bleomycin-induced murine model of systemic sclerosis.

PubMed

Lei, Ling; Zhao, Cheng; Qin, Fang; He, Zhi-Yi; Wang, Xu; Zhong, Xiao-Ning

2016-01-01

Systemic sclerosis (SSc) is characterised by fibrosis of the skin and internal organs, such as the lungs. Enhanced Th17 responses are associated with skin fibrosis in patients with SSc, however, whether they are associated with lung fibrosis has not been clarified. This study aimed to investigate the potential association of Th17 responses with the skin and pulmonary fibrosis as well as the potential mechanisms in a mouse bleomycin (BLM) model of SSc. BALB/c mice were injected subcutaneously with phosphate buffered saline (PBS) (control) or BLM for 28 days and the skin and pulmonary inflammation and fibrosis were characterized by histology. The percentages of circulating, skin and pulmonary infiltrating Th17 cells and the contents of collagen in mice were analysed. The levels of RORγt, IL-17A, IL-6 and TGF-β1 mRNA transcripts in the skin and lungs were determined by quantitative RTPCR and the levels of serum IL-17A, IL-6 and TGF-β1 were determined by ELISA. Furthermore, the effect of rIL-17A on the proliferation of pulmonary fibroblasts and their cytokine expression was analysed. The potential association of Th17 responses with the severity of skin and lung fibrosis was analysed. In comparison with the control mice, significantly increased skin and pulmonary inflammation and fibrosis and higher levels of hydroxyproline were detected in the BLM mice. Significantly higher frequency of circulating, skin and lung infiltrating Th17 cells and higher levels of serum, skin and lung IL-17A, TGF-β1, IL-6 and RORγt were detected in the BLM mice. The concentrations of serum IL-17A were correlated positively with the percentages of Th17 cells and the contents of skin hydroxyproline in the BLM mice. The levels of IL-17A expression were positively correlated with the skin and lung inflammatory scores as well as the skin fibrosis in the BLM mice. In addition, IL-17A significantly enhanced pulmonary fibroblast proliferation and their type I collagen, TGF-β and IL-6 expression in vitro, which were attenuated by treatment with anti-IL-17A. Our results indicate that Th17 cells participate in the pathogenesis of skin and lung fibrosis by enhancing fibroblast proliferation and cytokine production in a mouse BLM model of SSc.

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

Takemura, Takayo; Yoshida, Yuichi; Kiso, Shinichi, E-mail: kiso@gh.med.osaka-u.ac.jp

Highlights: •HB-EGF expression was increased during the development of liver fibrosis. •Conditional HB-EGF knockout mouse showed enhanced experimental liver fibrosis. •HB-EGF antagonized TGF-β-induced activation of hepatic stellate cells. •We report a possible protective role of HB-EGF in cholestatic liver fibrosis. -- Abstract: Our aims were to evaluate the involvement of heparin-binding EGF-like growth factor (HB-EGF) in liver fibrogenesis of humans and mice and to elucidate the effect of HB-EGF deficiency on cholestatic liver fibrosis using conditional HB-EGF knockout (KO) mice. We first demonstrated that gene expression of HB-EGF had a positive significant correlation with that of collagen in human fibroticmore » livers, and was increased in bile duct ligation (BDL)-induced fibrotic livers in mouse. We then generated conditional HB-EGF knockout (KO) mice using the interferon inducible Mx-1 promoter driven Cre recombinase transgene and wild type (WT) and KO mice were subjected to BDL. After BDL, KO mice exhibited enhanced liver fibrosis with increased expression of collagen, compared with WT mice. Finally, we used mouse hepatic stellate cells (HSCs) to examine the role of HB-EGF in the activation of these cells and showed that HB-EGF antagonized TGF-β-induced gene expression of collagen in mouse primary HSCs. Interestingly, HB-EGF did not prevent the TGF-β-induced nuclear accumulation of Smad3, but did lead to stabilization of the Smad transcriptional co-repressor TG-interacting factor. In conclusion, our data suggest a possible protective role of HB-EGF in cholestatic liver fibrosis.« less

Exploring Animal Models That Resemble Idiopathic Pulmonary Fibrosis

PubMed Central

Tashiro, Jun; Rubio, Gustavo A.; Limper, Andrew H.; Williams, Kurt; Elliot, Sharon J.; Ninou, Ioanna; Aidinis, Vassilis; Tzouvelekis, Argyrios; Glassberg, Marilyn K.

2017-01-01

Large multicenter clinical trials have led to two recently approved drugs for patients with idiopathic pulmonary fibrosis (IPF); yet, both of these therapies only slow disease progression and do not provide a definitive cure. Traditionally, preclinical trials have utilized mouse models of bleomycin (BLM)-induced pulmonary fibrosis—though several limitations prevent direct translation to human IPF. Spontaneous pulmonary fibrosis occurs in other animal species, including dogs, horses, donkeys, and cats. While the fibrotic lungs of these animals share many characteristics with lungs of patients with IPF, current veterinary classifications of fibrotic lung disease are not entirely equivalent. Additional studies that profile these examples of spontaneous fibroses in animals for similarities to human IPF should prove useful for both human and animal investigators. In the meantime, studies of BLM-induced fibrosis in aged male mice remain the most clinically relevant model for preclinical study for human IPF. Addressing issues such as time course of treatment, animal size and characteristics, clinically irrelevant treatment endpoints, and reproducibility of therapeutic outcomes will improve the current status of preclinical studies. Elucidating the mechanisms responsible for the development of fibrosis and disrepair associated with aging through a collaborative approach between researchers will promote the development of models that more accurately represent the realm of interstitial lung diseases in humans. PMID:28804709

Amelioration of tissue fibrosis by toll-like receptor 4 knockout in murine models of systemic sclerosis.

PubMed

Takahashi, Takehiro; Asano, Yoshihide; Ichimura, Yohei; Toyama, Tetsuo; Taniguchi, Takashi; Noda, Shinji; Akamata, Kaname; Tada, Yayoi; Sugaya, Makoto; Kadono, Takafumi; Sato, Shinichi

2015-01-01

Bleomycin-induced fibrosis and the tight skin (TSK/+) mouse are well-established experimental murine models of human systemic sclerosis (SSc). Growing evidence has demonstrated the pivotal role of Toll-like receptors (TLRs) in several autoimmune inflammatory diseases, including SSc. This study was undertaken to determine the role of TLR-4 in the fibrotic processes in these murine models. We generated a murine model of bleomycin-induced SSc using TLR-4(-/-) mice and TLR-4(-/-) ;TSK/+ mice. The mechanisms by which TLR-4 contributes to pathologic tissue fibrosis were investigated in these 2 models by histologic examination, hydroxyproline assay, enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and flow cytometry. Dermal and lung fibrosis was attenuated in bleomycin-treated TLR-4(-/-) mice compared with their wild-type counterparts. Inflammatory cell infiltration, expression of various inflammatory cytokines, and pathologic angiogenesis induced by bleomycin treatment were suppressed with TLR-4 deletion. Furthermore, the increased expression of interleukin-6 (IL-6) in fibroblasts, endothelial cells, and immune cells in response to bleomycin in vivo and to lipopolysaccharide in vitro was notably abrogated in the absence of TLR-4. Moreover, TLR-4 deletion was associated with alleviated B cell activation and skew toward a Th2/Th17 response against bleomycin treatment. Importantly, in TSK/+ mice, another SSc murine model, TLR-4 abrogation attenuated hypodermal fibrosis. These results indicate the pivotal contribution of TLR-4 to the pathologic tissue fibrosis of SSc murine models. Our results indicate the critical role of TLR-4 signaling in the development of tissue fibrosis, suggesting that biomolecular TLR-4 targeting might be a potential therapeutic approach to SSc. Copyright © 2015 by the American College of Rheumatology.

Effects of Geniposide from Gardenia Fruit Pomace on Skeletal-Muscle Fibrosis.

PubMed

Pan, Haiou; Li, Yan; Qian, Haifeng; Qi, Xiguang; Wu, Gangcheng; Zhang, Hui; Xu, Meijuan; Rao, Zhiming; Li, Jin-Long; Wang, Li; Ying, Hao

2018-05-30

Geniposide is the main bioactive constituent of gardenia fruit. Skeletal-muscle fibrosis is a common and irreversibly damaging process. Numerous studies have shown that geniposide could improve many chronic diseases, including metabolic syndrome and tumors. However, the effects of geniposide on skeletal-muscle fibrosis are still poorly understood. Here, we found that crude extracts of gardenia fruit pomace could significantly decrease the expression of profibrotic genes in vitro. Moreover, geniposide could also reverse profibrotic-gene expression induced by TGF-β and Smad4, a regulator of skeletal-muscle fibrosis. In addition, geniposide treatment could significantly downregulate profibrotic-gene expression and improve skeletal-muscle injuries in a mouse model of contusion. These results together suggest that geniposide has an antifibrotic effect on skeletal muscle through the suppression of the TGF-β-Smad4 signaling pathway.

Skeletal, cardiac, and respiratory muscle function and histopathology in the P448Lneo- mouse model of FKRP-deficient muscular dystrophy.

PubMed

Yu, Qing; Morales, Melissa; Li, Ning; Fritz, Alexander G; Ruobing, Ren; Blaeser, Anthony; Francois, Ershia; Lu, Qi-Long; Nagaraju, Kanneboyina; Spurney, Christopher F

2018-04-06

Fukutin-related protein (FKRP) mutations are the most common cause of dystroglycanopathies known to cause both limb girdle and congenital muscular dystrophy. The P448Lneo- mouse model has a knock-in mutation in the FKRP gene and develops skeletal, respiratory, and cardiac muscle disease. We studied the natural history of the P448Lneo- mouse model over 9 months and the effects of twice weekly treadmill running. Forelimb and hindlimb grip strength (Columbus Instruments) and overall activity (Omnitech Electronics) assessed skeletal muscle function. Echocardiography was performed using VisualSonics Vevo 770 (FujiFilm VisualSonics). Plethysmography was performed using whole body system (ADInstruments). Histological evaluations included quantification of inflammation, fibrosis, central nucleation, and fiber size variation. P448Lneo- mice had significantly increased normalized tissue weights compared to controls at 9 months of age for the heart, gastrocnemius, soleus, tibialis anterior, quadriceps, and triceps. There were no significant differences seen in forelimb or hindlimb grip strength or activity monitoring in P448Lneo- mice with or without exercise compared to controls. Skeletal muscles demonstrated increased inflammation, fibrosis, central nucleation, and variation in fiber size compared to controls (p < 0.05) and worsened with exercise. Plethysmography showed significant differences in respiratory rates and decreased tidal and minute volumes in P448Lneo- mice (p < 0.01). There was increased fibrosis in the diaphragm compared to controls (p < 0.01). Echocardiography demonstrated decreased systolic function in 9-month-old mutant mice (p < 0.01). There was increased myocardial wall thickness and mass (p < 0.001) with increased fibrosis in 9-month-old P448Lneo- mice compared to controls (p < 0.05). mRNA expression for natriuretic peptide type A (Nppa) was significantly increased in P448Lneo- mice compared to controls at 6 months (p < 0.05) and for natriuretic peptide type B (Nppb) at 6 and 9 months of age (p < 0.05). FKRP-deficient P448Lneo- mice demonstrate significant deficits in cardiac and respiratory functions compared to control mice, and this is associated with increased inflammation and fibrosis. This study provides new functional outcome measures for preclinical trials of FKRP-related muscular dystrophies.

Non-alcoholic fatty liver disease (NAFLD) potentiates autoimmune hepatitis in the CYP2D6 mouse model.

PubMed

Müller, Peter; Messmer, Marie; Bayer, Monika; Pfeilschifter, Josef M; Hintermann, Edith; Christen, Urs

2016-05-01

Non-alcoholic fatty liver disease (NAFLD) and its more severe development non-alcoholic steatohepatitis (NASH) are increasing worldwide. In particular NASH, which is characterized by an active hepatic inflammation, has often severe consequences including progressive fibrosis, cirrhosis, and eventually hepatocellular carcinoma (HCC). Here we investigated how metabolic liver injury is influencing the pathogenesis of autoimmune hepatitis (AIH). We used the CYP2D6 mouse model in which wild type C57BL/6 mice are infected with an Adenovirus expressing the major liver autoantigen cytochrome P450 2D6 (CYP2D6). Such mice display several features of human AIH, including interface hepatitis, formation of LKM-1 antibodies and CYP2D6-specific T cells, as well as hepatic fibrosis. NAFLD was induced with a high-fat diet (HFD). We found that pre-existing NAFLD potentiates the severity of AIH. Mice fed for 12 weeks with a HFD displayed increased cellular infiltration of the liver, enhanced hepatic fibrosis and elevated numbers of liver autoantigen-specific T cells. Our data suggest that a pre-existing metabolic liver injury constitutes an additional risk for the severity of an autoimmune condition of the liver, such as AIH. Copyright © 2016 Elsevier Ltd. All rights reserved.

Investigating Mechanisms of Chronic Kidney Disease in Mouse Models

PubMed Central

Eddy, Allison A.; Okamura, Daryl M.; Yamaguchi, Ikuyo; López-Guisa, Jesús M.

2011-01-01

Animal models of chronic kidney disease (CKD) are important experimental tools that are used to investigate novel mechanistic pathways and to validate potential new therapeutic interventions prior to pre-clinical testing in humans. Over the past several years, mouse CKD models have been extensively used for these purposes. Despite significant limitations, the model of unilateral ureteral obstruction (UUO) has essentially become the high throughput in vivo model, as it recapitulates the fundamental pathogenetic mechanisms that typify all forms of CKD in a relatively short time span. In addition, several alternative mouse models are available that can be used to validate new mechanistic paradigms and/or novel therapies. Several models are reviewed – both genetic and experimentally induced – that provide investigators with an opportunity to include renal functional study end-points together with quantitative measures of fibrosis severity, something that is not possible with the UUO model. PMID:21695449

Serelaxin treatment reverses vascular dysfunction and left ventricular hypertrophy in a mouse model of Type 1 diabetes

PubMed Central

Ng, Hooi Hooi; Leo, Chen Huei; Prakoso, Darnel; Qin, Chengxue; Ritchie, Rebecca H.; Parry, Laura J.

2017-01-01

Serelaxin prevents endothelial dysfunction in the mouse aorta ex vivo and inhibits apoptosis in cardiomyocytes under acute hyperglycaemia. Less is known about the effects of serelaxin in an in vivo mouse model of diabetes. Therefore, we tested the hypothesis in streptozotocin (STZ)-treated mice that serelaxin is able to reverse diabetes-induced vascular dysfunction and cardiac remodelling. Mice were divided into citrate buffer + placebo, STZ + placebo and STZ + serelaxin (0.5 mg/kg/d, 2 weeks) groups. After 12 weeks of diabetes, sensitivity to the endothelium-dependent agonist acetylcholine (ACh) was reduced in the mesenteric artery. This was accompanied by an enhanced vasoconstrictor prostanoid contribution and a decrease in endothelium-derived hyperpolarisation (EDH)-mediated relaxation. Serelaxin restored endothelial function by increasing nitric oxide (NO)-mediated relaxation but not EDH. It also normalised the contribution of vasoconstrictor prostanoids to endothelial dysfunction and suppressed diabetes-induced hyper-responsiveness of the mesenteric artery to angiotensin II. Similarly, diabetes reduced ACh-evoked NO-mediated relaxation in the aorta which was reversed by serelaxin. In the left ventricle, diabetes promoted apoptosis, hypertrophy and fibrosis; serelaxin treatment reversed this ventricular apoptosis and hypertrophy, but had no effect on fibrosis. In summary, serelaxin reversed diabetes-induced endothelial dysfunction by enhancing NO-mediated relaxation in the mouse vasculature and attenuating left ventricular hypertrophy and apoptosis. PMID:28067255

Caveolin-1 regulates chemokine receptor 5-mediated contribution of bone marrow-derived cells to dermal fibrosis

PubMed Central

Lee, Rebecca; Perry, Beth; Heywood, Jonathan; Reese, Charles; Bonner, Michael; Hatfield, Corey M.; Silver, Richard M.; Visconti, Richard P.; Hoffman, Stanley; Tourkina, Elena

2014-01-01

In fibrotic diseases caveolin-1 underexpression in fibroblasts results in collagen overexpression and in monocytes leads to hypermigration. These profibrotic behaviors are blocked by the caveolin-1 scaffolding domain peptide (CSD) which compensates for caveolin-1 deficiency. Monocytes and fibroblasts are related in that monocytes are the progenitors of fibrocytes (CD45+/Collagen I+ cells) that, in turn, are the progenitors of many fibroblasts in fibrotic tissues. In an additional anti-fibrotic activity, CSD blocks monocyte differentiation into fibrocytes. We studied a mouse fibrosis model (Pump Model) involving systemic bleomycin delivery that closely models scleroderma (SSc) in several ways, the most important of which for this study is that fibrosis is observed in the lungs, skin, and internal organs. We show here that dermal thickness is increased 2-fold in the Pump Model and that this effect is almost completely blocked by CSD (p < 0.001). Concomitantly, the subcutaneous fat layer becomes >80% thinner. This effect is also blocked by CSD (p < 0.001). Even in mice receiving vehicle instead of bleomycin, CSD increases the thickness of the fat layer. To study the mechanisms of action of bleomycin and CSD, we examined the accumulation of the chemokine receptor CCR5 and its ligands MIP1α and MIP1β in fibrotic tissue and their roles in monocyte migration. Fibrocytes and other leukocytes expressing CCR5 and its ligands were present at high levels in the fibrotic dermis of SSc patients and Pump Model mice while CSD blocked their accumulation in mouse dermis. Migration toward CCR5 ligands of SSc monocytes and Pump Model bone marrow cells was 3-fold greater than cells from control subjects. This enhanced migration was almost completely blocked by CSD. These results suggest that low monocyte caveolin-1 promotes fibrosis by enhancing the recruitment of fibrocytes and their progenitors into affected tissue. PMID:24966836

A G542X cystic fibrosis mouse model for examining nonsense mutation directed therapies.

PubMed

McHugh, Daniel R; Steele, Miarasa S; Valerio, Dana M; Miron, Alexander; Mann, Rachel J; LePage, David F; Conlon, Ronald A; Cotton, Calvin U; Drumm, Mitchell L; Hodges, Craig A

2018-01-01

Nonsense mutations are present in 10% of patients with CF, produce a premature termination codon in CFTR mRNA causing early termination of translation, and lead to lack of CFTR function. There are no currently available animal models which contain a nonsense mutation in the endogenous Cftr locus that can be utilized to test nonsense mutation therapies. In this study, we create a CF mouse model carrying the G542X nonsense mutation in Cftr using CRISPR/Cas9 gene editing. The G542X mouse model has reduced Cftr mRNA levels, demonstrates absence of CFTR function, and displays characteristic manifestations of CF mice such as reduced growth and intestinal obstruction. Importantly, CFTR restoration is observed in G542X intestinal organoids treated with G418, an aminoglycoside with translational readthrough capabilities. The G542X mouse model provides an invaluable resource for the identification of potential therapies of CF nonsense mutations as well as the assessment of in vivo effectiveness of these potential therapies targeting nonsense mutations.

Genetic disruption of NRF2 promotes the development of necroinflammation and liver fibrosis in a mouse model of HFE-hereditary hemochromatosis.

PubMed

Duarte, Tiago L; Caldas, Carolina; Santos, Ana G; Silva-Gomes, Sandro; Santos-Gonçalves, Andreia; Martins, Maria João; Porto, Graça; Lopes, José Manuel

2017-04-01

In hereditary hemochromatosis, iron deposition in the liver parenchyma may lead to fibrosis, cirrhosis and hepatocellular carcinoma. Most cases are ascribed to a common mutation in the HFE gene, but the extent of clinical expression is greatly influenced by the combined action of yet unidentified genetic and/or environmental modifying factors. In mice, transcription factor NRF2 is a critical determinant of hepatocyte viability during exposure to acute dietary iron overload. We evaluated if the genetic disruption of Nrf2 would prompt the development of liver damage in Hfe -/- mice (an established model of human HFE-hemochromatosis). Wild-type, Nrf2 -/- , Hfe -/- and double knockout (Hfe/Nrf2 -/- ) female mice on C57BL/6 genetic background were sacrificed at the age of 6 (young), 12-18 (middle-aged) or 24 months (old) for evaluation of liver pathology. Despite the parenchymal iron accumulation, Hfe -/- mice presented no liver injury. The combination of iron overload (Hfe -/- ) and defective antioxidant defences (Nrf2 -/- ) increased the number of iron-related necroinflammatory lesions (sideronecrosis), possibly due to the accumulation of toxic oxidation products such as 4-hydroxy-2-nonenal-protein adducts. The engulfment of dead hepatocytes led to a gradual accumulation of iron within macrophages, featuring large aggregates. Myofibroblasts recruited towards the injury areas produced substantial amounts of collagen fibers involving the liver parenchyma of double-knockout animals with increased hepatic fibrosis in an age-dependent manner. The genetic disruption of Nrf2 promotes the transition from iron accumulation (siderosis) to liver injury in Hfe -/- mice, representing the first demonstration of spontaneous hepatic fibrosis in the long term in a mouse model of hereditary hemochromatosis displaying mildly elevated liver iron. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Cardiovascular Benefits of Moderate Exercise Training in Marfan Syndrome: Insights From an Animal Model.

PubMed

Mas-Stachurska, Aleksandra; Siegert, Anna-Maria; Batlle, Monsterrat; Gorbenko Del Blanco, Darya; Meirelles, Thayna; Rubies, Cira; Bonorino, Fabio; Serra-Peinado, Carla; Bijnens, Bart; Baudin, Julio; Sitges, Marta; Mont, Lluís; Guasch, Eduard; Egea, Gustavo

2017-09-25

Marfan syndrome (MF) leads to aortic root dilatation and a predisposition to aortic dissection, mitral valve prolapse, and primary and secondary cardiomyopathy. Overall, regular physical exercise is recommended for a healthy lifestyle, but dynamic sports are strongly discouraged in MF patients. Nonetheless, evidence supporting this recommendation is lacking. Therefore, we studied the role of long-term dynamic exercise of moderate intensity on the MF cardiovascular phenotype. In a transgenic mouse model of MF ( Fbn1 C1039G/+ ), 4-month-old wild-type and MF mice were subjected to training on a treadmill for 5 months; sedentary littermates served as controls for each group. Aortic and cardiac remodeling was assessed by echocardiography and histology. The 4-month-old MF mice showed aortic root dilatation, elastic lamina rupture, and tunica media fibrosis, as well as cardiac hypertrophy, left ventricular fibrosis, and intramyocardial vessel remodeling. Over the 5-month experimental period, aortic root dilation rate was significantly greater in the sedentary MF group, compared with the wild-type group (∆mm, 0.27±0.07 versus 0.13±0.02, respectively). Exercise significantly blunted the aortic root dilation rate in MF mice compared with sedentary MF littermates (∆mm, 0.10±0.04 versus 0.27±0.07, respectively). However, these 2 groups were indistinguishable by aortic root stiffness, tunica media fibrosis, and elastic lamina ruptures. In MF mice, exercise also produced cardiac hypertrophy regression without changes in left ventricular fibrosis. Our results in a transgenic mouse model of MF indicate that moderate dynamic exercise mitigates the progression of the MF cardiovascular phenotype. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

[Effects of fasudil on bleomycin-induced pulmonary fibrosis in mice and on the biological behaviors in NIH3T3 mouse fibroblast cell line].

PubMed

Jiang, Chunguo; Huang, Hui; Liu, Jia; Wang, Yanxun; Zhao, Yuyue; Xu, Zuojun

2014-09-01

To determine the beneficial effects and mechanisms of fasudil, a selective ROCK inhibitor, on bleomycin-induced pulmonary fibrosis in mice and to determine the effects and mechanisms of fasudil on the biological behaviors in NIH3T3 mouse fibroblast cell line. The BPF model was induced by a single dosage of 2.5 mg/kg bleomycin intratracheal injection in mice and fasudil intraperitoneal injection was given to the mice. The fibrosis degree was determined pathologically by using the Ashcroft scoring method and biochemically by hydroxyproline assay in lung tissue. NIH3T3 mouse fibroblast cell line was cultured in vitro and fasudil was given to the cell. The proliferation activity in NIH3T3 cells were detected by MTT assay and flat colony forming experiment. The migration activity in NIH3T3 cells were detected by scratch test and transwell chamber experiment. The expression of CyclinD1, MMP2 and TIMP1 mRNA in NIH3T3 cells was detected by RT-PCR. The expression of CyclinD1, MMP2 and TIMP1 protein and the level of MYPT1 phosphorylation in NIH3T3 cells was detected by Western blot. Compare to the mice administrated by bleomycin, the Ashcroft score and hydroxyproline content were significantly decreased in the mice administered fasudil. Administration of fasudil can reduce the ability of proliferation and migration in a dose-dependent manner in NIH3T3 cells. The effect of fasudil was possibly related to increase the production of TIMP1 and decrease the production of CyclinD1 and MMP2. Administration of fasudil can attenuate pulmonary fibrosis both in vivo and in vitro. These findings suggest that fasudil may be a potential therapeutic candidate for the treatment of pulmonary fibrosis.

Protective Effect of Astaxanthin on Liver Fibrosis through Modulation of TGF-β1 Expression and Autophagy

PubMed Central

Shen, Miao; Chen, Kan; Lu, Jie; Cheng, Ping; Xu, Ling; Dai, Weiqi; Wang, Fan; He, Lei; Zhang, Yan; Chengfen, Wang; Li, Jingjing; Yang, Jing; Zhu, Rong; Zhang, Huawei; Zheng, Yuanyuan; Zhou, Yingqun; Guo, Chuanyong

2014-01-01

Liver fibrosis is a common pathway leading to cirrhosis and a worldwide clinical issue. Astaxanthin is a red carotenoid pigment with antioxidant, anticancer, and anti-inflammatory properties. The aim of this study was to investigate the effect of astaxanthin on liver fibrosis and its potential protective mechanisms. Liver fibrosis was induced in a mouse model using CCL4 (intraperitoneal injection, three times a week for 8 weeks), and astaxanthin was administered everyday at three doses (20, 40, and 80 mg/kg). Pathological results indicated that astaxanthin significantly improved the pathological lesions of liver fibrosis. The levels of alanine aminotransferase aspartate aminotransferase and hydroxyproline were also significantly decreased by astaxanthin. The same results were confirmed in bile duct liagtion, (BDL) model. In addition, astaxanthin inhibited hepatic stellate cells (HSCs) activation and formation of extracellular matrix (ECM) by decreasing the expression of NF-κB and TGF-β1 and maintaining the balance between MMP2 and TIMP1. In addition, astaxanthin reduced energy production in HSCs by downregulating the level of autophagy. These results were simultaneously confirmed in vivo and in vitro. In conclusion, our study showed that 80 mg/kg astaxanthin had a significant protective effect on liver fibrosis by suppressing multiple profibrogenic factors. PMID:24860243

Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular-fibrosis and tumor progression

PubMed Central

Laklai, Hanane; Miroshnikova, Yekaterina A.; Pickup, Michael W.; Collisson, Eric A.; Kim, Grace E.; Barrett, Alex S.; Hill, Ryan C.; Lakins, Johnathon N.; Schlaepfer, David D.; Mouw, Janna K.; LeBleu, Valerie S.; Roy, Nilotpal; Novitskiy, Sergey V.; Johansen, Julia S.; Poli, Valeria; Kalluri, Raghu; Iacobuzio-Donahue, Christine A.; Wood, Laura D.; Hebrok, Matthias; Hansen, Kirk; Moses, Harold L.; Weaver, Valerie M.

2016-01-01

Fibrosis compromises pancreatic ductal carcinoma (PDAC) treatment and contributes to patient mortality yet anti-stromal therapies are controversial. We found that human PDACs with impaired epithelial transforming growth factor β (TGF-β) signaling have elevated epithelial Stat3 activity and develop a stiffer, matricellular-enriched fibrosis associated with high epithelial tension and shorter patient survival. In several Kras-driven mouse models, both the loss of TGF-β signaling and elevated β1-integrin mechanosignaling engaged a positive feedback loop whereby Stat3 signaling promotes tumor progression by increasing matricellular fibrosis and tissue tension. In contrast, epithelial Stat3 ablation attenuated tumor progression by reducing the stromal stiffening and epithelial contractility induced by loss of TGF-β signaling. In PDAC patient biopsies, higher matricellular protein and activated Stat3 associated with SMAD4 mutation and shorter survival. The findings implicate epithelial tension and matricellular fibrosis in the aggressiveness of SMAD4 mutant pancreatic tumors, and highlight Stat3 and mechanics as key drivers of this phenotype. PMID:27089513

Protective effect of α-lipoic acid against radiation-induced fibrosis in mice

PubMed Central

Ryu, Seung-Hee; Park, Eun-Young; Kwak, Sungmin; Heo, Seung-Ho; Ryu, Je-Won; Park, Jin-hong

2016-01-01

Radiation-induced fibrosis (RIF) is one of the most common late complications of radiation therapy. We found that α-lipoic acid (α-LA) effectively prevents RIF. In RIF a mouse model, leg contracture assay was used to test the in vivo efficacy of α-LA. α-LA suppressed the expression of pro-fibrotic genes after irradiation, both in vivo and in vitro, and inhibited the up-regulation of TGF-β1-mediated p300/CBP activity. Thus, α-LA prevents radiation-induced fibrosis (RIF) by inhibiting the transcriptional activity of NF-κB through inhibition of histone acetyltransferase activity. α-LA is a new therapeutic methods that can be used in the prevention-treatment of RIF. PMID:26799284

MicroRNA-327 regulates cardiac hypertrophy and fibrosis induced by pressure overload.

PubMed

Ji, Yue; Qiu, Ming; Shen, Yejiao; Gao, Li; Wang, Yaqing; Sun, Wei; Li, Xinli; Lu, Yan; Kong, Xiangqing

2018-04-01

MicroRNA (miRNA/miR) dysregulation has been reported to be fundamental in the development and progression of cardiac hypertrophy and fibrosis. In the present study, miR-327 levels in fibroblasts were increased in response to cardiac hypertrophy induced by transverse aortic constriction with prominent cardiac fibrosis, particularly when compared with the levels in unstressed cardiomyocytes. In neonatal rat cardiac fibroblasts, induced expression of miR-327 upregulated fibrosis-associated gene expression and activated angiotensin II-induced differentiation into myofibroblasts, as assessed via α-smooth muscle actin staining. By contrast, miR-327 knockdown mitigated angiotensin II-induced differentiation. Cardiac fibroblast proliferation was not affected under either condition. In a mouse model subjected to transverse aortic constriction, miR-327 knockdown through tail-vein injection reduced the development of cardiac fibrosis and ventricular dysfunction. miR-327 was demonstrated to target integrin β3 and diminish the activation of cardiac fibroblasts. Thus, the present study supports the use of miR-327 as a therapeutic target in the reduction of cardiac fibrosis.

Inhibition of pulmonary fibrosis by the chemokine IP-10/CXCL10.

PubMed

Tager, Andrew M; Kradin, Richard L; LaCamera, Peter; Bercury, Scott D; Campanella, Gabriele S V; Leary, Carol P; Polosukhin, Vasiliy; Zhao, Long-Hai; Sakamoto, Hideo; Blackwell, Timothy S; Luster, Andrew D

2004-10-01

Pulmonary fibrosis is an enigmatic and devastating disease with few treatment options, now thought to result from abnormal wound healing in the lung in response to injury. We have previously noted a role for the chemokine interferon gamma-inducible protein of 10 kD (IP-10)/CXC chemokine ligand 10 in the regulation of cutaneous wound healing, and consequently investigated whether IP-10 regulates pulmonary fibrosis. We found that IP-10 is highly expressed in a mouse model of pulmonary fibrosis induced by bleomycin. IP-10-deficient mice exhibited increased pulmonary fibrosis after administration of bleomycin, suggesting that IP-10 limits the development of fibrosis in this model. Substantial fibroblast chemoattractant and proliferative activities were generated in the lung after bleomycin exposure. IP-10 significantly inhibited fibroblast responses to the chemotactic, but not the proliferative activity generated, suggesting that IP-10 may attenuate fibroblast accumulation in bleomycin-induced pulmonary fibrosis by limiting fibroblast migration. Consistent with this inhibitory activity of IP-10 on fibroblast migration, fibroblast accumulation in the lung after bleomycin exposure was dramatically increased in IP-10-deficient mice compared with wild-type mice. Conversely, transgenic mice overexpressing IP-10 were protected from mortality after bleomycin exposure, and demonstrated decreased fibroblast accumulation in the lung after challenge compared with wild-type mice. Our findings suggest that interruption of fibroblast recruitment may represent a novel therapeutic strategy for pulmonary fibrosis, which could have applicability to a wide range of fibrotic illnesses.

Grape seed extract ameliorates bleomycin-induced mouse pulmonary fibrosis.

PubMed

Liu, Qi; Jiang, Jun-Xia; Liu, Ya-Nan; Ge, Ling-Tian; Guan, Yan; Zhao, Wei; Jia, Yong-Liang; Dong, Xin-Wei; Sun, Yun; Xie, Qiang-Min

2017-05-05

Pulmonary fibrosis is common in a variety of inflammatory lung diseases, such as interstitial pneumonia, chronic obstructive pulmonary disease, and silicosis. There is currently no effective clinical drug treatment. It has been reported that grape seed extracts (GSE) has extensive pharmacological effects with minimal toxicity. Although it has been found that GSE can improve the lung collagen deposition and fibrosis pathology induced by bleomycin in rat, its effects on pulmonary function, inflammation, growth factors, matrix metalloproteinases and epithelial-mesenchymal transition remain to be researched. In the present study, we studied whether GSE provided protection against bleomycin (BLM)-induced mouse pulmonary fibrosis. ICR strain mice were treated with BLM in order to establish pulmonary fibrosis models. GSE was given daily via intragastric administration for three weeks starting at one day after intratracheal instillation. GSE at 50 or 100mg/kg significantly reduced BLM-induced inflammatory cells infiltration, proinflammatory factor protein expression, and hydroxyproline in lung tissues, and improved pulmonary function in mice. Additionally, treatment with GSE also significantly impaired BLM-induced increases in lung fibrotic marker expression (collagen type I alpha 1 and fibronectin 1) and decreases in an anti-fibrotic marker (E-cadherin). Further investigation indicated that the possible molecular targets of GSE are matrix metalloproteinases-9 (MMP-9) and TGF-β1, given that treatment with GSE significantly prevented BLM-induced increases in MMP-9 and TGF-β1 expression in the lungs. Together, these results suggest that supplementation with GSE may improve the quality of life of lung fibrosis patients by inhibiting MMP-9 and TGF-β1 expression in the lungs. Copyright © 2017 Elsevier B.V. All rights reserved.

Antifibrotic effects of ambrisentan, an endothelin-A receptor antagonist, in a non-alcoholic steatohepatitis mouse model

PubMed Central

Okamoto, Toshiaki; Koda, Masahiko; Miyoshi, Kennichi; Onoyama, Takumi; Kishina, Manabu; Matono, Tomomitsu; Sugihara, Takaaki; Hosho, Keiko; Okano, Junichi; Isomoto, Hajime; Murawaki, Yoshikazu

2016-01-01

AIM To examine the effects of the endothelin type A receptor antagonist ambrisentan on hepatic steatosis and fibrosis in a steatohepatitis mouse model. METHODS Fatty liver shionogi (FLS) FLS-ob/ob mice (male, 12 wk old) received ambrisentan (2.5 mg/kg orally per day; n = 8) or water as a control (n = 5) for 4 wk. Factors were compared between the two groups, including steatosis, fibrosis, inflammation, and endothelin-related gene expression in the liver. RESULTS In the ambrisentan group, hepatic hydroxyproline content was significantly lower than in the control group (18.0 μg/g ± 6.1 μg/g vs 33.9 μg/g ± 13.5 μg/g liver, respectively, P = 0.014). Hepatic fibrosis estimated by Sirius red staining and areas positive for α-smooth muscle actin, indicative of activated hepatic stellate cells, were also significantly lower in the ambrisentan group (0.46% ± 0.18% vs 1.11% ± 0.28%, respectively, P = 0.0003; and 0.12% ± 0.08% vs 0.25% ± 0.11%, respectively, P = 0.047). Moreover, hepatic RNA expression levels of procollagen-1 and tissue inhibitor of metalloproteinase-1 (TIMP-1) were significantly lower by 60% and 45%, respectively, in the ambrisentan group. Inflammation, steatosis, and endothelin-related mRNA expression in the liver were not significantly different between the groups. CONCLUSION Ambrisentan attenuated the progression of hepatic fibrosis by inhibiting hepatic stellate cell activation and reducing procollagen-1 and TIMP-1 gene expression. Ambrisentan did not affect inflammation or steatosis. PMID:27574547

Antifibrotic effects of ambrisentan, an endothelin-A receptor antagonist, in a non-alcoholic steatohepatitis mouse model.

PubMed

Okamoto, Toshiaki; Koda, Masahiko; Miyoshi, Kennichi; Onoyama, Takumi; Kishina, Manabu; Matono, Tomomitsu; Sugihara, Takaaki; Hosho, Keiko; Okano, Junichi; Isomoto, Hajime; Murawaki, Yoshikazu

2016-08-08

To examine the effects of the endothelin type A receptor antagonist ambrisentan on hepatic steatosis and fibrosis in a steatohepatitis mouse model. Fatty liver shionogi (FLS) FLS-ob/ob mice (male, 12 wk old) received ambrisentan (2.5 mg/kg orally per day; n = 8) or water as a control (n = 5) for 4 wk. Factors were compared between the two groups, including steatosis, fibrosis, inflammation, and endothelin-related gene expression in the liver. In the ambrisentan group, hepatic hydroxyproline content was significantly lower than in the control group (18.0 μg/g ± 6.1 μg/g vs 33.9 μg/g ± 13.5 μg/g liver, respectively, P = 0.014). Hepatic fibrosis estimated by Sirius red staining and areas positive for α-smooth muscle actin, indicative of activated hepatic stellate cells, were also significantly lower in the ambrisentan group (0.46% ± 0.18% vs 1.11% ± 0.28%, respectively, P = 0.0003; and 0.12% ± 0.08% vs 0.25% ± 0.11%, respectively, P = 0.047). Moreover, hepatic RNA expression levels of procollagen-1 and tissue inhibitor of metalloproteinase-1 (TIMP-1) were significantly lower by 60% and 45%, respectively, in the ambrisentan group. Inflammation, steatosis, and endothelin-related mRNA expression in the liver were not significantly different between the groups. Ambrisentan attenuated the progression of hepatic fibrosis by inhibiting hepatic stellate cell activation and reducing procollagen-1 and TIMP-1 gene expression. Ambrisentan did not affect inflammation or steatosis.

Pirfenidone ameliorates murine chronic GVHD through inhibition of macrophage infiltration and TGF-β production

PubMed Central

Du, Jing; Paz, Katelyn; Flynn, Ryan; Vulic, Ante; Robinson, Tara M.; Lineburg, Katie E.; Alexander, Kylie A.; Meng, Jingjing; Roy, Sabita; Panoskaltsis-Mortari, Angela; Loschi, Michael; Hill, Geoffrey R.; Serody, Jonathan S.; Maillard, Ivan; Miklos, David; Koreth, John; Cutler, Corey S.; Antin, Joseph H.; Ritz, Jerome; MacDonald, Kelli P.; Schacker, Timothy W.; Luznik, Leo

2017-01-01

Allogeneic hematopoietic stem cell transplantation is hampered by chronic graft-versus-host disease (cGVHD), resulting in multiorgan fibrosis and diminished function. Fibrosis in lung and skin leads to progressive bronchiolitis obliterans (BO) and scleroderma, respectively, for which new treatments are needed. We evaluated pirfenidone, a Food and Drug Administration (FDA)-approved drug for idiopathic pulmonary fibrosis, for its therapeutic effect in cGVHD mouse models with distinct pathophysiology. In a full major histocompatibility complex (MHC)-mismatched, multiorgan system model with BO, donor T-cell responses that support pathogenic antibody production are required for cGVHD development. Pirfenidone treatment beginning one month post-transplant restored pulmonary function and reversed lung fibrosis, which was associated with reduced macrophage infiltration and transforming growth factor-β production. Pirfenidone dampened splenic germinal center B-cell and T-follicular helper cell frequencies that collaborate to produce antibody. In both a minor histocompatibility antigen–mismatched as well as a MHC-haploidentical model of sclerodermatous cGVHD, pirfenidone significantly reduced macrophages in the skin, although clinical improvement of scleroderma was only seen in one model. In vitro chemotaxis assays demonstrated that pirfenidone impaired macrophage migration to monocyte chemoattractant protein-1 (MCP-1) as well as IL-17A, which has been linked to cGVHD generation. Taken together, our data suggest that pirfenidone is a potential therapeutic agent to ameliorate fibrosis in cGVHD. PMID:28254742

Characterization of nasal potential difference in cftr knockout and F508del-CFTR mice.

PubMed

Saussereau, Emilie Lyne; Roussel, Delphine; Diallo, Siradiou; Debarbieux, Laurent; Edelman, Aleksander; Sermet-Gaudelus, Isabelle

2013-01-01

Treatments designed to correct cystic fibrosis transmembrane conductance regulator (CFTR) defects must first be evaluated in preclinical experiments in the mouse model of cystic fibrosis (CF). Mice nasal mucosa mimics the bioelectric defect seen in humans. The use of nasal potential difference (V(TE)) to assess ionic transport is a powerful test evaluating the restoration of CFTR function. Nasal V(TE) in CF mice must be well characterized for correct interpretation. We performed V(TE) measurements in large-scale studies of two mouse models of CF--B6;129 cftr knockout and FVB F508del-CFTR--and their respective wild-type (WT) littermates. We assessed the repeatability of the test for cftr knockout mice and defined cutoff points distinguishing between WT and F508del-CFTR mice. We determined the typical V(TE) values for CF and WT mice and demonstrated the existence of residual CFTR activity in F508del-CFTR mice. We characterized intra-animal variability in B6;129 mice and defined the cutoff points for F508del-CFTR chloride secretion rescue. Hyperpolarization of more than -2.15 mV after perfusion with a low-concentration Cl(-) solution was considered to indicate a normal response. These data will make it possible to interpret changes in nasal V(TE) in mouse models of CF, in future preclinical studies.

Role of histone deacetylases(HDACs) in progression and reversal of liver fibrosis

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

Li, Xing; Wu, Xiao-Qin; Xu, Tao

Liver fibrosis refers to a reversible wound healing process response to chronic liver injuries. Activation of hepatic stellate cells (HSCs) is closely correlated with the development of liver fibrosis. Histone deacetylases(HDACs) determine the acetylation levels of core histones to modulate expression of genes. To demonstrate the link between HDACs and liver fibrosis, CCl4-induced mouse liver fibrosis model and its spontaneous reversal model were established. Results of the current study demonstrated that deregulation of liver HDACs may involved in the development of liver fibrosis. Among 11 HDACs tested in our study (Class I, II, and IV HDACs), expression of HDAC2 wasmore » maximally increased in CCl4-induced fibrotic livers but decreased after spontaneous recovery. Moreover, expression of HDAC2 was elevated in human liver fibrotic tissues. In this regard, the potential role of HDAC2 in liver fibrosis was further evaluated. Our results showed that administration of HSC-T6 cells with transforming growth factor-beta1 (TGF-β1) resulted in an increase of HDAC2 protein expression in dose- and time-dependent manners. Moreover, HDAC2 deficiency inhibited HSC-T6 cell proliferation and activation induced by TGF-β1. More importantly, the present study showed HDAC2 may regulate HSCs activation by suppressing expression of Smad7, which is a negative modulator in HSCs activation and liver fibrosis. Collectively, these observations revealed that HDAC2 may play a pivotal role in HSCs activation and liver fibrosis while deregulation of HDACs may serve as a novel mechanism underlying liver fibrosis. - Highlights: • This is the first report to systematically examine expressions of HDACs during liver fibrosis and fibrosis reversal. • Aberrant expression of HDAC2 contributes to the development of liver fibrosis. • Provided important foundation for further liver fibrosis conversion studies.« less

A mouse model for Costello syndrome reveals an Ang II–mediated hypertensive condition

PubMed Central

Schuhmacher, Alberto J.; Guerra, Carmen; Sauzeau, Vincent; Cañamero, Marta; Bustelo, Xosé R.; Barbacid, Mariano

2008-01-01

Germline activation of H-RAS oncogenes is the primary cause of Costello syndrome (CS), a neuro-cardio-facio-cutaneous developmental syndrome. Here we describe the generation of a mouse model of CS by introduction of an oncogenic Gly12Val mutation in the mouse H-Ras locus using homologous recombination in ES cells. Germline expression of the endogenous H-RasG12V oncogene, even in homozygosis, resulted in hyperplasia of the mammary gland. However, development of tumors in these mice was rare. H-RasG12V mutant mice closely phenocopied some of the abnormalities observed in patients with CS, including facial dysmorphia and cardiomyopathies. These mice also displayed alterations in the homeostasis of the cardiovascular system, including development of systemic hypertension, extensive vascular remodeling, and fibrosis in both the heart and the kidneys. This phenotype was age dependent and was a consequence of the abnormal upregulation of the renin–Ang II system. Treatment with captopril, an inhibitor of Ang II biosynthesis, prevented development of the hypertension condition, vascular remodeling, and heart and kidney fibrosis. In addition, it partially alleviated the observed cardiomyopathies. These mice should help in elucidating the etiology of CS symptoms, identifying additional defects, and evaluating potential therapeutic strategies. PMID:18483625

Application of small RNA sequencing to identify microRNAs in acute kidney injury and fibrosis

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

Pellegrini, Kathryn L.

Establishing a microRNA (miRNA) expression profile in affected tissues provides an important foundation for the discovery of miRNAs involved in the development or progression of pathologic conditions. We conducted small RNA sequencing to generate a temporal profile of miRNA expression in the kidneys using a mouse model of folic acid-induced (250 mg/kg i.p.) kidney injury and fibrosis. From the 103 miRNAs that were differentially expressed over the time course (> 2-fold, p < 0.05), we chose to further investigate miR-18a-5p, which is expressed during the acute stage of the injury; miR-132-3p, which is upregulated during transition between acute and fibroticmore » injury; and miR-146b-5p, which is highly expressed at the peak of fibrosis. Using qRT-PCR, we confirmed the increased expression of these candidate miRNAs in the folic acid model as well as in other established mouse models of acute injury (ischemia/reperfusion injury) and fibrosis (unilateral ureteral obstruction). In situ hybridization confirmed high expression of miR-18a-5p, miR-132-3p and miR-146b-5p throughout the kidney cortex in mice and humans with severe kidney injury or fibrosis. When primary human proximal tubular epithelial cells were treated with model nephrotoxicants such as cadmium chloride (CdCl{sub 2}), arsenic trioxide, aristolochic acid (AA), potassium dichromate (K{sub 2}Cr{sub 2}O{sub 7}) and cisplatin, miRNA-132-3p was upregulated 4.3-fold after AA treatment and 1.5-fold after K{sub 2}Cr{sub 2}O{sub 7} and CdCl{sub 2} treatment. These results demonstrate the application of temporal small RNA sequencing to identify miR-18a, miR-132 and miR-146b as differentially expressed miRNAs during distinct phases of kidney injury and fibrosis progression. - Highlights: • We used small RNA sequencing to identify differentially expressed miRNAs in kidney. • Distinct patterns were found for acute injury and fibrotic stages in the kidney. • Upregulation of miR-18a, -132 and -146b was confirmed in mice and human kidneys.« less

Quercetin ameliorates pulmonary fibrosis by inhibiting SphK1/S1P signaling.

PubMed

Zhang, Xingcai; Cai, Yuli; Zhang, Wei; Chen, Xianhai

2018-06-25

Idiopathic pulmonary fibrosis (IPF) is an agnogenic chronic disorder with high morbidity and low survival rate. Quercetin is a flavonoid found in a variety of herbs with anti-fibrosis function. In this study, bleomycin was employed to induce a pulmonary fibrosis mouse model. The quercetin administration ameliorated bleomycin-induced pulmonary fibrosis, evidenced by the expression level changes of hydroxyproline, fibronectin, α-smooth muscle actin, Collagen I and Collagen III. The similar results were observed in transforming growth factor (TGF)-β-treated human embryonic lung fibroblast (HELF). The bleomycin or TGF-β administration caused the increase of sphingosine-1-phosphate (S1P) level in pulmonary tissue and HELF cells, as well as its activation-required kinase, sphingosine kinase 1 (SphK1), and its degradation enzyme, sphinogosine-1-phosphate lyase (S1PL). However, the increase of S1P, SphK1 and S1PL was attenuated by application of quercetin. In addition, the effect of quercetin on fibrosis was abolished by the ectopic expression of SphK1. The colocalization of SphK1/S1PL and fibroblast specific protein 1 (FSP1) suggested the roles of fibroblasts in pulmonary fibrosis. In summary, we demonstrated that quercetin ameliorated pulmonary fibrosis in vivo and in vitro by inhibiting SphK1/S1P signaling.

Cystathionine γ-Lyase Deficiency Exacerbates CCl4-Induced Acute Hepatitis and Fibrosis in the Mouse Liver.

PubMed

Ci, Lei; Yang, Xingyu; Gu, Xiaowen; Li, Qing; Guo, Yang; Zhou, Ziping; Zhang, Mengjie; Shi, Jiahao; Yang, Hua; Wang, Zhugang; Fei, Jian

2017-07-20

The present study examined the role of cystathionine γ-lyase (CSE) in carbon tetrachloride (CCl 4 )-induced liver damage. A CSE gene knock-out and luciferase gene knock-in (KI) mouse model was constructed to study the function of CSE and to trace its expression in living status. CCl 4 or lipopolysaccharide markedly downregulated CSE expression in the liver of mice. CSE-deficient mice showed increased serum alanine aminotransferase and aspartate aminotransferase levels, and liver damage after CCl 4 challenge, whereas albumin and endogenous hydrogen sulfide (H 2 S) levels decreased significantly. CSE knockout mice showed increased serum homocysteine levels, upregulation of inflammatory cytokines, and increased autophagy and IκB-α degradation in the liver in response to CCl 4 treatment. The increase in pro-inflammatory cytokines, including tumor necrosis factor-alpha in CSE-deficient mice after CCl 4 challenge, was accompanied by a significant increase in liver tissue hydroxyproline and α-smooth muscle actin and histopathologic changes in the liver. However, H 2 S donor pretreatment effectively attenuated most of these imbalances. Here, a CSE knock-out and luciferase KI mouse model was established for the first time to study the transcriptional regulation of CSE expression in real time in a non-invasive manner, providing information on the effects and potential mechanisms of CSE on CCl 4 -induced liver injury. CSE deficiency increases pro-inflammatory cytokines in the liver and exacerbates acute hepatitis and liver fibrosis by reducing H 2 S production from L-cysteine in the liver. The present data suggest the potential of an H 2 S donor for the treatment of liver diseases such as toxic hepatitis and fibrosis. Antioxid. Redox Signal. 27, 133-149.

Naringin in Ganshuang Granule suppresses activation of hepatic stellate cells for anti-fibrosis effect by inhibition of mammalian target of rapamycin.

PubMed

Shi, Hongbo; Shi, Honglin; Ren, Feng; Chen, Dexi; Chen, Yu; Duan, Zhongping

2017-03-01

A previous study has demonstrated that Ganshuang granule (GSG) plays an anti-fibrotic role partially by deactivation of hepatic stellate cells (HSCs). In HSCs activation, mammalian target of rapamycin (mTOR)-autophagy plays an important role. We attempted to investigate the role of mTOR-autophagy in anti-fibrotic effect of GSG. The cirrhotic mouse model was prepared to demonstrate the anti-fibrosis effect of GSG. High performance liquid chromatography (HPLC) analyses were used to identify the active component of GSG. The primary mouse HSCs were isolated and naringin was added into activated HSCs to observe its anti-fibrotic effect. 3-methyladenine (3-MA) and Insulin-like growth factor-1 (IGF-1) was added, respectively, into fully activated HSCs to explore the role of autophagy and mTOR. GSG played an anti-fibrotic role through deactivation of HSCs in cirrhotic mouse model. The concentration of naringin was highest in GSG by HPLC analyses and naringin markedly suppressed HSCs activation in vitro, which suggested that naringin was the main active component of GSG. The deactivation of HSCs caused by naringin was not because of the autophagic activation but mTOR inhibition, which was supported by the following evidence: first, naringin induced autophagic activation, but when autophagy was blocked by 3-MA, deactivation of HSCs was not attenuated or reversed. Second, naringin inhibited mTOR pathway, meanwhile when mTOR was activated by IGF-1, deactivation of HSCs was reversed. In conclusion, we have demonstrated naringin in GSG suppressed activation of HSCs for anti-fibrosis effect by inhibition of mTOR, indicating a potential therapeutic application for liver cirrhosis. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Asthma progression to airway remodeling and bone marrow eosinophil responses in genetically distinct strains of mice.

PubMed

Hogan, Mary Beth; Piktel, Debra; Hubbs, Ann F; McPherson, Leslie E; Landreth, Kenneth S

2008-12-01

Patient factors that cause long-term airway remodeling are largely unidentified. This suggests that genetic differences may determine which asthmatic patients develop airway remodeling. A murine model with repeated allergen exposure leading to peribronchial fibrosis in complement factor 5 (C5)-deficient A/J mice has been used to study asthma progression. No studies have addressed the systemic effects of allergen sensitization or chronic allergen exposure on bone marrow eosinophilopoiesis in this mouse strain. To investigate bone marrow eosinophil responses during acute sensitization and chronic allergen exposure using genetically distinct mouse strains differing in persistent airway reactivity and remodeling. The C5-sufficient BALB/c and C5-deficient A/J mice were repetitively exposed to intranasal ovalbumin for 12 weeks. Subsequently, the mice were evaluated for airway eosinophilia, mucus-containing goblet cells, and peribronchial fibrosis. Both strains of mice were also acutely sensitized to ovalbumin. Bone marrow eosinophil progenitor cells and mature eosinophils were enumerated. BALB/c and A/J mice have similar bone marrow responses after acute allergen exposure, with elevations in bone marrow eosinophil progenitor cell and eosinophil numbers. After chronic allergen exposure, only C5-deficient A/J mice that developed peribronchial fibrosis exhibited bone marrow eosinophilia. BALB/c mice lacked peribronchial fibrosis and extinguished accelerated eosinophil production after long-term allergen challenge. Chronic airway remodeling after repeated allergen exposure in genetically different mice correlated with differences in long-term bone marrow eosinophilopoiesis. Preventing asthma from progressing to chronic airway remodeling with fibrosis may involve identifying genetically determined influences on bone marrow responses to chronic allergen exposure.

Aryl Hydrocarbon Receptor Ligands in Cigarette Smoke Induce Production of Interleukin-22 to Promote Pancreatic Fibrosis in Models of Chronic Pancreatitis.

PubMed

Xue, Jing; Zhao, Qinglan; Sharma, Vishal; Nguyen, Linh P; Lee, Yvonne N; Pham, Kim L; Edderkaoui, Mouad; Pandol, Stephen J; Park, Walter; Habtezion, Aida

2016-12-01

Cigarette smoke has been identified as an independent risk factor for chronic pancreatitis (CP). Little is known about the mechanisms by which smoking promotes development of CP. We assessed the effects of aryl hydrocarbon receptor (AhR) ligands found in cigarette smoke on immune cell activation in humans and pancreatic fibrosis in animal models of CP. We obtained serum samples from patients with CP treated at Stanford University hospital and healthy individuals (controls) and isolated CD4 + T cells. Levels of interleukin-22 (IL22) were measured by enzyme-linked immunosorbent assay and smoking histories were collected. T cells from healthy nonsmokers and smokers were stimulated and incubated with AhR agonists (2,3,7,8-tetrachlorodibenzo-p-dioxin or benzo[a]pyrene) or antagonists and analyzed by flow cytometry. Mice were given intraperitoneal injections of caerulein or saline, with or without lipopolysaccharide, to induce CP. Some mice were given intraperitoneal injections of AhR agonists at the start of caerulein injection, with or without an antibody against IL22 (anti-IL22) starting 2 weeks after the first caerulein injection, or recombinant mouse IL22 or vehicle (control) intraperitoneally 4 weeks after the first caerulein injection. Mice were exposed to normal air or cigarette smoke for 6 h/d for 7 weeks and expression of AhR gene targets was measured. Pancreata were collected from all mice and analyzed by histology and quantitative reverse transcription polymerase chain reaction. Pancreatic stellate cells and T cells were isolated and studied using immunoblot, immunofluorescence, flow cytometry, and enzyme-linked immunosorbent analyses. Mice given AhR agonists developed more severe pancreatic fibrosis (based on decreased pancreas size, histology, and increased expression of fibrosis-associated genes) than mice not given agonists after caerulein injection. In mice given saline instead of caerulein, AhR ligands did not induce fibrosis. Pancreatic T cells from mice given AhR agonists and caerulein were activated and expressed IL22, but not IL17 or interferon gamma. Human T cells exposed to AhR agonists up-regulated expression of IL22. In mice given anti-IL22, pancreatic fibrosis did not progress, whereas mice given recombinant IL22 had a smaller pancreas and increased fibrosis. Pancreatic stellate cells isolated from mouse and human pancreata expressed the IL22 receptor IL22RA1. Incubation of the pancreatic stellate cells with IL22 induced their expression of the extracellular matrix genes fibronectin 1 and collagen type I α1 chain, but not α2 smooth muscle actin or transforming growth factor-β. Serum samples from smokers had significantly higher levels of IL22 than those from nonsmokers. AhR ligands found in cigarette smoke increase the severity of pancreatic fibrosis in mouse models of pancreatitis via up-regulation of IL22. This pathway might be targeted for treatment of CP and serve as a biomarker of disease. Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.

Characterization of Mouse Models of Early Pancreatic Lesions Induced by Alcohol and Chronic Pancreatitis.

PubMed

Xu, Shiping; Chheda, Chintan; Ouhaddi, Yassine; Benhaddou, Hajar; Bourhim, Mouloud; Grippo, Paul J; Principe, Daniel R; Mascariñas, Emman; DeCant, Brian; Tsukamoto, Hidekazu; Pandol, Stephen J; Edderkaoui, Mouad

2015-08-01

We describe the first mouse model of pancreatic intraepithelial neoplasia (PanIN) lesions induced by alcohol in the presence and absence of chronic pancreatitis. Pdx1-Cre;LSL-K-ras mice were exposed to Lieber-DeCarli alcohol diet for 6 weeks with cerulein injections. The PanIN lesions and markers of fibrosis, inflammation, histone deacetylation, epithelial-to-mesenchymal transition (EMT), and cancer stemness were measured by immunohistochemistry and Western. Exposure of Pdx1-Cre;LSL-K-ras mice to an alcohol diet significantly stimulated fibrosis and slightly but not significantly increased the level of PanIN lesions associated with an increase in tumor-promoting M2 macrophages. Importantly, the alcohol diet did not increase activation of stellate cells. Alcohol diet and cerulein injections resulted in synergistic and additive effects on PanIN lesion and M2 macrophage phenotype induction, respectively. Cerulein pancreatitis caused stellate cell activation, EMT, and cancer stemness in the pancreas. Pancreatitis caused histone deacetylation, which was promoted by the alcohol diet. Pancreatitis increased EMT and cancer stemness markers, which were not further affected by the alcohol diet. The results suggest that alcohol has independent effects on promotion of PDAC associated with fibrosis formed through a stellate cell-independent mechanism and that it further promotes early PDAC and M2 macrophage induction in the context of chronic pancreatitis.

Hepatocyte nuclear receptor SHP suppresses inflammation and fibrosis in a mouse model of nonalcoholic steatohepatitis.

PubMed

Zou, An; Magee, Nancy; Deng, Fengyan; Lehn, Sarah; Zhong, Cuncong; Zhang, Yuxia

2018-06-01

Nonalcoholic fatty liver disease (NAFLD) is a burgeoning health problem worldwide, ranging from nonalcoholic fatty liver (NAFL, steatosis without hepatocellular injury) to the more aggressive nonalcoholic steatohepatitis (NASH, steatosis with ballooning, inflammation, or fibrosis). Although many studies have greatly contributed to the elucidation of NAFLD pathogenesis, the disease progression from NAFL to NASH remains incompletely understood. Nuclear receptor small heterodimer partner (Nr0b2, SHP ) is a transcriptional regulator critical for the regulation of bile acid, glucose, and lipid metabolism. Here, we show that SHP levels are decreased in the livers of patients with NASH and in diet-induced mouse NASH. Exposing primary mouse hepatocytes to palmitic acid and lipopolysaccharide in vitro , we demonstrated that the suppression of Shp expression in hepatocytes is due to c-Jun N-terminal kinase (JNK) activation, which stimulates c-Jun-mediated transcriptional repression of Shp Interestingly, in vivo induction of hepatocyte-specific SHP in steatotic mouse liver ameliorated NASH progression by attenuating liver inflammation and fibrosis, but not steatosis. Moreover, a key mechanism linking the anti-inflammatory role of hepatocyte-specific SHP expression to inflammation involved SHP-induced suppression of NF-κB p65-mediated induction of chemokine (C-C motif) ligand 2 (CCL2), which activates macrophage proinflammatory polarization and migration. In summary, our results indicate that a JNK/SHP/NF-κB/CCL2 regulatory network controls communications between hepatocytes and macrophages and contributes to the disease progression from NAFL to NASH. Our findings may benefit the development of new management or prevention strategies for NASH. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Ivabradine improved left ventricular function and pressure overload-induced cardiomyocyte apoptosis in a transverse aortic constriction mouse model.

PubMed

Yu, Yihui; Hu, Zuoying; Li, Bing; Wang, Zhimei; Chen, Shaoliang

2018-05-22

This study aimed to investigate the effects and molecular mechanisms of ivabradine in preventing cardiac hypertrophy in an established transverse aortic constriction (TAC) mouse model. A total of 56 male C57BL/6 mice were randomly assigned into the following seven groups (8 mice per group): sham, TAC model, Iva-10 (10 mg/kg/day ivabradine), Iva-20 (20 mg/kg/day ivabradine), Iva-40 (40 mg/kg/day ivabradine), Iva-80 (80 mg/kg/day ivabradine), and Rap (rapamycin, a positive control). Echocardiography and left ventricular hemodynamics were performed. Hematoxylin-eosin (H&E), Masson's trichome staining, and TUNEL assays were conducted to evaluate cardiac hypertrophy, fibrosis, and apoptosis, respectively. Western blotting was performed to detect the expression of proteins related to the PI3K/Akt/mTOR/p70S6K pathway. Ivabradine could effectively improve left ventricular dysfunction and hypertrophy induced by TAC in a dose-independent manner. Moreover, no obvious change in heart rate (HR) was observed in the TAC and Rap groups, whereas a significant decrease in HR was found after ivabradine treatment (P < 0.05). Cardiac hypertrophy, fibrosis, and apoptosis induced by TAC were notably suppressed after either rapamycin or ivabradine treatment (P < 0.05). Ivabradine and rapamycin also decreased the expression of PI3K/Akt and mTOR induced by TAC. Ivabradine improved cardiac hypertrophy and fibrosis as well as reduced cardiomyocyte apoptosis via the PI3K/Akt/mTOR/p70S6K pathway in TAC model mice.

Redox nanoparticles as a novel treatment approach for inflammation and fibrosis associated with nonalcoholic steatohepatitis

PubMed Central

Eguchi, Akiko; Yoshitomi, Toru; Lazic, Milos; Johnson, Casey D; Vong, Long Binh; Wree, Alexander; Povero, Davide; Papouchado, Bettina G; Nagasaki, Yukio; Feldstein, Ariel E

2015-01-01

Aim: Oxidative stress (OS) is largely thought to be a central mechanism responsible for liver damage, inflammation and fibrosis in nonalcoholic steatohepatitis (NASH). Our aim was to investigate whether suppression of OS in the liver via redox nanoparticles (RNPs) reduces liver damage in a mouse model of NASH. Materials & methods: RNPs were prepared by self-assembly of redox polymers possessing antioxidant nitroxide radicals and were orally administered by daily gavage for 4 weeks. Results: The redox polymer was delivered to the liver after disintegration of nanoparticle in the stomach. RNP treatment in NASH mice via gavage led to a reduction of liver OS, improvement of fibrosis, and significant reduction of inflammation. Conclusion: These findings uncover RNP as a novel potential NASH therapy. PMID:26020857

Gremlin-1 Overexpression in Mouse Lung Reduces Silica-Induced Lymphocyte Recruitment – A Link to Idiopathic Pulmonary Fibrosis through Negative Correlation with CXCL10 Chemokine

PubMed Central

Koli, Katri; Sutinen, Eva; Rönty, Mikko; Rantakari, Pia; Fortino, Vittorio; Pulkkinen, Ville; Greco, Dario; Sipilä, Petra; Myllärniemi, Marjukka

2016-01-01

Idiopathic pulmonary fibrosis (IPF) is characterized by activation and injury of epithelial cells, the accumulation of connective tissue and changes in the inflammatory microenvironment. The bone morphogenetic protein (BMP) inhibitor protein gremlin-1 is associated with the progression of fibrosis both in human and mouse lung. We generated a transgenic mouse model expressing gremlin-1 in type II lung epithelial cells using the surfactant protein C (SPC) promoter and the Cre-LoxP system. Gremlin-1 protein expression was detected specifically in the lung after birth and did not result in any signs of respiratory insufficiency. Exposure to silicon dioxide resulted in reduced amounts of lymphocyte aggregates in transgenic lungs while no alteration in the fibrotic response was observed. Microarray gene expression profiling and analyses of bronchoalveolar lavage fluid cytokines indicated a reduced lymphocytic response and a downregulation of interferon-induced gene program. Consistent with reduced Th1 response, there was a downregulation of the mRNA and protein expression of the anti-fibrotic chemokine CXCL10, which has been linked to IPF. In human IPF patient samples we also established a strong negative correlation in the mRNA expression levels of gremlin-1 and CXCL10. Our results suggest that in addition to regulation of epithelial-mesenchymal crosstalk during tissue injury, gremlin-1 modulates inflammatory cell recruitment and anti-fibrotic chemokine production in the lung. PMID:27428020

Gremlin-1 Overexpression in Mouse Lung Reduces Silica-Induced Lymphocyte Recruitment - A Link to Idiopathic Pulmonary Fibrosis through Negative Correlation with CXCL10 Chemokine.

PubMed

Koli, Katri; Sutinen, Eva; Rönty, Mikko; Rantakari, Pia; Fortino, Vittorio; Pulkkinen, Ville; Greco, Dario; Sipilä, Petra; Myllärniemi, Marjukka

2016-01-01

Idiopathic pulmonary fibrosis (IPF) is characterized by activation and injury of epithelial cells, the accumulation of connective tissue and changes in the inflammatory microenvironment. The bone morphogenetic protein (BMP) inhibitor protein gremlin-1 is associated with the progression of fibrosis both in human and mouse lung. We generated a transgenic mouse model expressing gremlin-1 in type II lung epithelial cells using the surfactant protein C (SPC) promoter and the Cre-LoxP system. Gremlin-1 protein expression was detected specifically in the lung after birth and did not result in any signs of respiratory insufficiency. Exposure to silicon dioxide resulted in reduced amounts of lymphocyte aggregates in transgenic lungs while no alteration in the fibrotic response was observed. Microarray gene expression profiling and analyses of bronchoalveolar lavage fluid cytokines indicated a reduced lymphocytic response and a downregulation of interferon-induced gene program. Consistent with reduced Th1 response, there was a downregulation of the mRNA and protein expression of the anti-fibrotic chemokine CXCL10, which has been linked to IPF. In human IPF patient samples we also established a strong negative correlation in the mRNA expression levels of gremlin-1 and CXCL10. Our results suggest that in addition to regulation of epithelial-mesenchymal crosstalk during tissue injury, gremlin-1 modulates inflammatory cell recruitment and anti-fibrotic chemokine production in the lung.

OX40L blockade protects against inflammation-driven fibrosis

PubMed Central

Elhai, Muriel; Avouac, Jérôme; Hoffmann-Vold, Anna Maria; Ruzehaji, Nadira; Amiar, Olivia; Ruiz, Barbara; Brahiti, Hassina; Ponsoye, Matthieu; Fréchet, Maxime; Burgevin, Anne; Pezet, Sonia; Sadoine, Jérémy; Guilbert, Thomas; Nicco, Carole; Akiba, Hisaya; Heissmeyer, Vigo; Subramaniam, Arun; Resnick, Robert; Molberg, Øyvind; Kahan, André; Chiocchia, Gilles; Allanore, Yannick

2016-01-01

Treatment for fibrosis represents a critical unmet need, because fibrosis is the leading cause of death in industrialized countries, and there is no effective therapy to counteract the fibrotic process. The development of fibrosis relates to the interplay between vessel injury, immune cell activation, and fibroblast stimulation, which can occur in various tissues. Immunotherapies have provided a breakthrough in the treatment of immune diseases. The glycoprotein OX40–OX40 ligand (OX40L) axis offers the advantage of a targeted approach to costimulatory signals with limited impact on the whole immune response. Using systemic sclerosis (SSc) as a prototypic disease, we report compelling evidence that blockade of OX40L is a promising strategy for the treatment of inflammation-driven fibrosis. OX40L is overexpressed in the fibrotic skin and serum of patients with SSc, particularly in patients with diffuse cutaneous forms. Soluble OX40L was identified as a promising serum biomarker to predict the worsening of lung and skin fibrosis, highlighting the role of this pathway in fibrosis. In vivo, OX40L blockade prevents inflammation-driven skin, lung, and vessel fibrosis and induces the regression of established dermal fibrosis in different complementary mouse models. OX40L exerts potent profibrotic effects by promoting the infiltration of inflammatory cells into lesional tissues and therefore the release of proinflammatory mediators, thereafter leading to fibroblast activation. PMID:27298374

EGF receptor tyrosine kinase inhibitors diminish transforming growth factor-alpha-induced pulmonary fibrosis.

PubMed

Hardie, William D; Davidson, Cynthia; Ikegami, Machiko; Leikauf, George D; Le Cras, Timothy D; Prestridge, Adrienne; Whitsett, Jeffrey A; Korfhagen, Thomas R

2008-06-01

Transforming growth factor-alpha (TGF-alpha) is a ligand for the EGF receptor (EGFR). EGFR activation is associated with fibroproliferative processes in human lung disease and animal models of pulmonary fibrosis. We determined the effects of EGFR tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) on the development and progression of TGF-alpha-induced pulmonary fibrosis. Using a doxycycline-regulatable transgenic mouse model of lung-specific TGF-alpha expression, we determined effects of treatment with gefitinib and erlotinib on changes in lung histology, total lung collagen, pulmonary mechanics, pulmonary hypertension, and expression of genes associated with synthesis of ECM and vascular remodeling. Induction in the lung of TGF-alpha caused progressive pulmonary fibrosis over an 8-wk period. Daily administration of gefitinib or erlotinib prevented development of fibrosis, reduced accumulation of total lung collagen, prevented weight loss, and prevented changes in pulmonary mechanics. Treatment of mice with gefitinib 4 wk after the induction of TGF-alpha prevented further increases in and partially reversed total collagen levels and changes in pulmonary mechanics and pulmonary hypertension. Increases in expression of genes associated with synthesis of ECM as well as decreases of genes associated with vascular remodeling were also prevented or partially reversed. Administration of gefitinib or erlotinib did not cause interstitial fibrosis or increases in lavage cell counts. Administration of small molecule EGFR tyrosine kinase inhibitors prevented further increases in and partially reversed pulmonary fibrosis induced directly by EGFR activation without inducing inflammatory cell influx or additional lung injury.

DC-SIGN activation mediates the differential effects of SAP and CRP on the innate immune system and inhibits fibrosis in mice.

PubMed

Cox, Nehemiah; Pilling, Darrell; Gomer, Richard H

2015-07-07

Fibrosis is caused by scar tissue formation in internal organs and is associated with 45% of deaths in the United States. Two closely related human serum proteins, serum amyloid P (SAP) and C-reactive protein (CRP), strongly affect fibrosis. In multiple animal models, and in Phase 1 and Phase 2 clinical trials, SAP affects several aspects of the innate immune system to reduce fibrosis, whereas CRP appears to potentiate fibrosis. However, SAP and CRP bind the same Fcγ receptors (FcγR) with similar affinities, and why SAP and CRP have opposing effects is unknown. Here, we report that SAP but not CRP binds the receptor DC-SIGN (SIGN-R1) to affect the innate immune system, and that FcγR are not necessary for SAP function. A polycyclic aminothiazole DC-SIGN ligand and anti-DC-SIGN antibodies mimic SAP effects in vitro. In mice, the aminothiazole reduces neutrophil accumulation in a model of acute lung inflammation and, at 0.001 mg/kg, alleviates pulmonary fibrosis by increasing levels of the immunosuppressant IL-10. DC-SIGN (SIGN-R1) is present on mouse lung epithelial cells, and SAP and the aminothiazole potentiate IL-10 production from these cells. Our data suggest that SAP activates DC-SIGN to regulate the innate immune system differently from CRP, and that DC-SIGN is a target for antifibrotics.

Neutrophils alleviate fibrosis in the CCl4-induced mouse chronic liver injury model.

PubMed

Saijou, Eiko; Enomoto, Yutaka; Matsuda, Michitaka; Yuet-Yin Kok, Cindy; Akira, Shizuo; Tanaka, Minoru; Miyajima, Atsushi

2018-06-01

Tribbles pseudokinase 1 ( Trib1 ) is a negative regulator of CCAAT/enhancer binding protein α (C/EBPα) and is known to induce granulopoiesis while suppressing monocyte differentiation. Loss of Trib1 was previously shown to increase the neutrophil population in the spleen but lead to M2-like macrophage reduction. Because M2 macrophages are anti-inflammatory and promote tissue repair by producing fibrogenic factors, we investigated liver fibrosis in Trib1 -deficient mice. Interestingly, loss of Trib1 suppressed fibrosis in the CCl 4 -induced chronic liver injury model. Trib1 knockout increased neutrophils but had a minimal effect on the macrophage population in the liver. Hepatic expressions of neutrophil matrix metalloproteinases ( Mmp ) 8 and Mmp9 were increased, but the production of fibrogenic factors, including transforming growth factor β1, was not affected by loss of Trib1 . These results suggest that neutrophils are responsible for the suppression of fibrosis in Trib1 -deficient liver. Consistently, transplantation of Trib1 -deficient bone marrow cells into wild-type mice alleviated CCl 4 -induced fibrosis. Furthermore, expression of chemokine (C-X-C motif) ligand 1 ( Cxcl1 ) by adeno-associated viral vector in the normal liver recruited neutrophils and suppressed CCl 4 -induced fibrosis; infusion of wild-type neutrophils in CCl 4 -treated mice also ameliorated fibrosis. Using recombinant adeno-associated virus-mediated expression of Mmp8 and Mmp9 alleviated liver fibrosis. Finally, neutrophil depletion by infusion of Ly6G antibody significantly enhanced CCl 4 -induced fibrosis. Conclusion : While neutrophils are well known to exacerbate acute liver injury, our results demonstrate a beneficial role of neutrophils in chronic liver injury by promoting fibrolysis. ( Hepatology Communications 2018;2:703-717).

Neutrophils alleviate fibrosis in the CCl4‐induced mouse chronic liver injury model

PubMed Central

Saijou, Eiko; Enomoto, Yutaka; Matsuda, Michitaka; Yuet‐Yin Kok, Cindy; Akira, Shizuo; Tanaka, Minoru

2018-01-01

Tribbles pseudokinase 1 (Trib1) is a negative regulator of CCAAT/enhancer binding protein α (C/EBPα) and is known to induce granulopoiesis while suppressing monocyte differentiation. Loss of Trib1 was previously shown to increase the neutrophil population in the spleen but lead to M2‐like macrophage reduction. Because M2 macrophages are anti‐inflammatory and promote tissue repair by producing fibrogenic factors, we investigated liver fibrosis in Trib1‐deficient mice. Interestingly, loss of Trib1 suppressed fibrosis in the CCl4‐induced chronic liver injury model. Trib1 knockout increased neutrophils but had a minimal effect on the macrophage population in the liver. Hepatic expressions of neutrophil matrix metalloproteinases (Mmp)8 and Mmp9 were increased, but the production of fibrogenic factors, including transforming growth factor β1, was not affected by loss of Trib1. These results suggest that neutrophils are responsible for the suppression of fibrosis in Trib1‐deficient liver. Consistently, transplantation of Trib1‐deficient bone marrow cells into wild‐type mice alleviated CCl4‐induced fibrosis. Furthermore, expression of chemokine (C‐X‐C motif) ligand 1 (Cxcl1) by adeno‐associated viral vector in the normal liver recruited neutrophils and suppressed CCl4‐induced fibrosis; infusion of wild‐type neutrophils in CCl4‐treated mice also ameliorated fibrosis. Using recombinant adeno‐associated virus‐mediated expression of Mmp8 and Mmp9 alleviated liver fibrosis. Finally, neutrophil depletion by infusion of Ly6G antibody significantly enhanced CCl4‐induced fibrosis. Conclusion: While neutrophils are well known to exacerbate acute liver injury, our results demonstrate a beneficial role of neutrophils in chronic liver injury by promoting fibrolysis. (Hepatology Communications 2018;2:703‐717) PMID:29881822

Mouse genome-wide association study identifies polymorphisms on chromosomes 4, 11, and 15 for age-related cardiac fibrosis.

PubMed

Li, Qiaoli; Berndt, Annerose; Sundberg, Beth A; Silva, Kathleen A; Kennedy, Victoria E; Cario, Clinton L; Richardson, Matthew A; Chase, Thomas H; Schofield, Paul N; Uitto, Jouni; Sundberg, John P

2016-06-01

Dystrophic cardiac calcinosis (DCC), also called epicardial and myocardial fibrosis and mineralization, has been detected in mice of a number of laboratory inbred strains, most commonly C3H/HeJ and DBA/2J. In previous mouse breeding studies between these DCC susceptible and the DCC-resistant strain C57BL/6J, 4 genetic loci harboring genes involved in DCC inheritance were identified and subsequently termed Dyscalc loci 1 through 4. Here, we report susceptibility to cardiac fibrosis, a sub-phenotype of DCC, at 12 and 20 months of age and close to natural death in a survey of 28 inbred mouse strains. Eight strains showed cardiac fibrosis with highest frequency and severity in the moribund mice. Using genotype and phenotype information of the 28 investigated strains, we performed genome-wide association studies (GWAS) and identified the most significant associations on chromosome (Chr) 15 at 72 million base pairs (Mb) (P < 10(-13)) and Chr 4 at 122 Mb (P < 10(-11)) and 134 Mb (P < 10(-7)). At the Chr 15 locus, Col22a1 and Kcnk9 were identified. Both have been reported to be morphologically and functionally important in the heart muscle. The strongest Chr 4 associations were located approximately 6 Mb away from the Dyscalc 2 quantitative trait locus peak within the boundaries of the Extl1 gene and in close proximity to the Trim63 and Cap1 genes. In addition, a single-nucleotide polymorphism association was found on chromosome 11. This study provides evidence for more than the previously reported 4 genetic loci determining cardiac fibrosis and DCC. The study also highlights the power of GWAS in the mouse for dissecting complex genetic traits.

Mouse genome-wide association study identifies polymorphisms on chromosomes 4, 11 and 15 for age-related cardiac fibrosis

PubMed Central

Li, Qiaoli; Berndt, Annerose; Sundberg, Beth A.; Silva, Kathleen A.; Kennedy, Victoria E.; Cario, Clinton L; Richardson, Matthew A.; Chase, Thomas H.; Schofield, Paul N.; Uitto, Jouni; Sundberg, John P.

2017-01-01

Dystrophic cardiac calcinosis (DCC), also called epicardial and myocardial fibrosis and mineralization, has been detected in mice of a number of laboratory inbred strains, most commonly C3H/HeJ and DBA/2J. In previous mouse breeding studies between these DCC susceptible and the DCC resistant strain C57BL/6J, 4 genetic loci harboring genes involved in DCC inheritance were identified and subsequently termed Dyscal loci 1 through 4. Here we report susceptibility to cardiac fibrosis, a sub-phenotype of DCC, at 12 and 20 months of age and close to natural death in a survey of 28 inbred mouse strains. Eight strains showed cardiac fibrosis with highest frequency and severity in the moribund mice. Using genotype and phenotype information of the 28 investigated strains we performed genome-wide association studies (GWAS) and identified the most significant associations on chromosome (Chr) 15 at 72 million base pairs (Mb) (P < 10−13) and Chr 4 at 122 Mb (P < 10−11) and 134 Mb (P < 10−7). At the Chr 15 locus Col22a1 and Kcnk9 were identified. Both have been reported to be morphologically and functionally important in the heart muscle. The strongest Chr 4 associations were located approximate 6 Mb away from the Dyscal 2 quantitative trait locus peak within the boundaries of the Extl1 gene and in close proximity to the Trim63 and Cap1 genes. In addition, a single nucleotide polymorphism association was found on chromosome 11. This study provides evidence for more than the previously reported 4 genetic loci determining cardiac fibrosis and DCC. The study also highlights the power of GWAS in the mouse for dissecting complex genetic traits. PMID:27126641

Mechanotransduction-modulated fibrotic microniches reveal the contribution of angiogenesis in liver fibrosis

NASA Astrophysics Data System (ADS)

Liu, Longwei; You, Zhifeng; Yu, Hongsheng; Zhou, Lyu; Zhao, Hui; Yan, Xiaojun; Li, Dulei; Wang, Bingjie; Zhu, Lu; Xu, Yuzhou; Xia, Tie; Shi, Yan; Huang, Chenyu; Hou, Wei; Du, Yanan

2017-12-01

The role of pathological angiogenesis on liver fibrogenesis is still unknown. Here, we developed fibrotic microniches (FμNs) that recapitulate the interaction of liver sinusoid endothelial cells (LSECs) and hepatic stellate cells (HSCs). We investigated how the mechanical properties of their substrates affect the formation of capillary-like structures and how they relate to the progression of angiogenesis during liver fibrosis. Differences in cell response in the FμNs were synonymous of the early and late stages of liver fibrosis. The stiffness of the early-stage FμNs was significantly elevated due to condensation of collagen fibrils induced by angiogenesis, and led to activation of HSCs by LSECs. We utilized these FμNs to understand the response to anti-angiogenic drugs, and it was evident that these drugs were effective only for early-stage liver fibrosis in vitro and in an in vivo mouse model of liver fibrosis. Late-stage liver fibrosis was not reversed following treatment with anti-angiogenic drugs but rather with inhibitors of collagen condensation. Our work reveals stage-specific angiogenesis-induced liver fibrogenesis via a previously unrevealed mechanotransduction mechanism which may offer precise intervention strategies targeting stage-specific disease progression.

HDL activation of endothelial sphingosine-1-phosphate receptor-1 (S1P1) promotes regeneration and suppresses fibrosis in the liver

PubMed Central

Sun, Yue; Chen, Yutian; Swendeman, Steven L.; Jung, Bongnam; Chavez, Deebly; Cao, Zhongwei; Christoffersen, Christina; Nielsen, Lars Bo; Schwab, Susan R.; Rafii, Shahin; Hla, Timothy

2016-01-01

Regeneration of hepatic sinusoidal vasculature is essential for non-fibrotic liver regrowth and restoration of its metabolic capacity. However, little is known about how this specialized vascular niche is regenerated. Here we show that activation of endothelial sphingosine-1-phosphate receptor-1 (S1P1) by its natural ligand bound to HDL (HDL-S1P) induces liver regeneration and curtails fibrosis. In mice lacking HDL-S1P, liver regeneration after partial hepatectomy was impeded and associated with aberrant vascular remodeling, thrombosis and peri-sinusoidal fibrosis. Notably, this “maladaptive repair” phenotype was recapitulated in mice that lack S1P1 in the endothelium. Reciprocally, enhanced plasma levels of HDL-S1P or administration of SEW2871, a pharmacological agonist specific for S1P1 enhanced regeneration of metabolically functional vasculature and alleviated fibrosis in mouse chronic injury and cholestasis models. This study shows that natural and pharmacological ligands modulate endothelial S1P1 to stimulate liver regeneration and inhibit fibrosis, suggesting that activation of this pathway may be a novel therapeutic strategy for liver fibrosis. PMID:28018969

HDL activation of endothelial sphingosine-1-phosphate receptor-1 (S1P1) promotes regeneration and suppresses fibrosis in the liver.

PubMed

Ding, Bi-Sen; Liu, Catherine H; Sun, Yue; Chen, Yutian; Swendeman, Steven L; Jung, Bongnam; Chavez, Deebly; Cao, Zhongwei; Christoffersen, Christina; Nielsen, Lars Bo; Schwab, Susan R; Rafii, Shahin; Hla, Timothy

2016-12-22

Regeneration of hepatic sinusoidal vasculature is essential for non-fibrotic liver regrowth and restoration of its metabolic capacity. However, little is known about how this specialized vascular niche is regenerated. Here we show that activation of endothelial sphingosine-1-phosphate receptor-1 (S1P 1 ) by its natural ligand bound to HDL (HDL-S1P) induces liver regeneration and curtails fibrosis. In mice lacking HDL-S1P, liver regeneration after partial hepatectomy was impeded and associated with aberrant vascular remodeling, thrombosis and peri-sinusoidal fibrosis. Notably, this "maladaptive repair" phenotype was recapitulated in mice that lack S1P 1 in the endothelium. Reciprocally, enhanced plasma levels of HDL-S1P or administration of SEW2871, a pharmacological agonist specific for S1P 1 enhanced regeneration of metabolically functional vasculature and alleviated fibrosis in mouse chronic injury and cholestasis models. This study shows that natural and pharmacological ligands modulate endothelial S1P 1 to stimulate liver regeneration and inhibit fibrosis, suggesting that activation of this pathway may be a novel therapeutic strategy for liver fibrosis.

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.

A2B Adenosine Receptor–Mediated Induction of IL-6 Promotes CKD

PubMed Central

Dai, Yingbo; Zhang, Weiru; Wen, Jiaming; Zhang, Yujin; Kellems, Rodney E.

2011-01-01

Chronic elevation of adenosine, which occurs in the setting of repeated or prolonged tissue injury, can exacerbate cellular dysfunction, suggesting that it may contribute to the pathogenesis of CKD. Here, mice with chronically elevated levels of adenosine, resulting from a deficiency in adenosine deaminase (ADA), developed renal dysfunction and fibrosis. Both the administration of polyethylene glycol–modified ADA to reduce adenosine levels and the inhibition of the A2B adenosine receptor (A2BR) attenuated renal fibrosis and dysfunction. Furthermore, activation of A2BR promoted renal fibrosis in both mice infused with angiotensin II (Ang II) and mice subjected to unilateral ureteral obstruction (UUO). These three mouse models shared a similar profile of profibrotic gene expression in kidney tissue, suggesting that they share similar signaling pathways that lead to renal fibrosis. Finally, both genetic and pharmacologic approaches showed that the inflammatory cytokine IL-6 mediates adenosine-induced renal fibrosis downstream of A2BR. Taken together, these data suggest that A2BR-mediated induction of IL-6 contributes to renal fibrogenesis and shows potential therapeutic targets for CKD. PMID:21511827

The orphan nuclear receptor ROR alpha and group 3 innate lymphoid cells drive fibrosis in a mouse model of Crohn's disease.

PubMed

Lo, Bernard C; Gold, Matthew J; Hughes, Michael R; Antignano, Frann; Valdez, Yanet; Zaph, Colby; Harder, Kenneth W; McNagny, Kelly M

2016-09-02

Fibrosis is the result of dysregulated tissue regeneration and is characterized by excessive accumulation of matrix proteins that become detrimental to tissue function. In Crohn's disease, this manifests itself as recurrent gastrointestinal strictures for which there is no effective therapy beyond surgical intervention. Using a model of infection-induced chronic gut inflammation, we show that Rora -deficient mice are protected from fibrosis; infected intestinal tissues display diminished pathology, attenuated collagen deposition and reduced fibroblast accumulation. Although Rora is best known for its role in ILC2 development, we find that Salmonella -induced fibrosis is independent of eosinophils, STAT6 signaling and Th2 cytokine production arguing that this process is largely ILC2-independent. Instead, we observe reduced levels of ILC3- and T cell-derived IL-17A and IL-22 in infected gut tissues. Furthermore, using Rora sg/sg / Rag1 -/- bone marrow chimeric mice, we show that restoring ILC function is sufficient to re-establish IL-17A and IL-22 production and a profibrotic phenotype. Our results show that RORα-dependent ILC3 functions are pivotal in mediating gut fibrosis and they offer an avenue for therapeutic intervention in Crohn's-like diseases.

Pirfenidone ameliorates murine chronic GVHD through inhibition of macrophage infiltration and TGF-β production.

PubMed

Du, Jing; Paz, Katelyn; Flynn, Ryan; Vulic, Ante; Robinson, Tara M; Lineburg, Katie E; Alexander, Kylie A; Meng, Jingjing; Roy, Sabita; Panoskaltsis-Mortari, Angela; Loschi, Michael; Hill, Geoffrey R; Serody, Jonathan S; Maillard, Ivan; Miklos, David; Koreth, John; Cutler, Corey S; Antin, Joseph H; Ritz, Jerome; MacDonald, Kelli P; Schacker, Timothy W; Luznik, Leo; Blazar, Bruce R

2017-05-04

Allogeneic hematopoietic stem cell transplantation is hampered by chronic graft-versus-host disease (cGVHD), resulting in multiorgan fibrosis and diminished function. Fibrosis in lung and skin leads to progressive bronchiolitis obliterans (BO) and scleroderma, respectively, for which new treatments are needed. We evaluated pirfenidone, a Food and Drug Administration (FDA)-approved drug for idiopathic pulmonary fibrosis, for its therapeutic effect in cGVHD mouse models with distinct pathophysiology. In a full major histocompatibility complex (MHC)-mismatched, multiorgan system model with BO, donor T-cell responses that support pathogenic antibody production are required for cGVHD development. Pirfenidone treatment beginning one month post-transplant restored pulmonary function and reversed lung fibrosis, which was associated with reduced macrophage infiltration and transforming growth factor-β production. Pirfenidone dampened splenic germinal center B-cell and T-follicular helper cell frequencies that collaborate to produce antibody. In both a minor histocompatibility antigen-mismatched as well as a MHC-haploidentical model of sclerodermatous cGVHD, pirfenidone significantly reduced macrophages in the skin, although clinical improvement of scleroderma was only seen in one model. In vitro chemotaxis assays demonstrated that pirfenidone impaired macrophage migration to monocyte chemoattractant protein-1 (MCP-1) as well as IL-17A, which has been linked to cGVHD generation. Taken together, our data suggest that pirfenidone is a potential therapeutic agent to ameliorate fibrosis in cGVHD. © 2017 by The American Society of Hematology.

Aging enhances liver fibrotic response in mice through hampering extracellular matrix remodeling.

PubMed

Delire, Bénédicte; Lebrun, Valérie; Selvais, Charlotte; Henriet, Patrick; Bertrand, Amélie; Horsmans, Yves; Leclercq, Isabelle A

2016-12-09

Clinical data identify age as a factor for severe liver fibrosis. We evaluate whether and how aging modulates the fibrotic response in a mouse model. Liver fibrosis was induced by CCl 4 injections (thrice weekly for 2 weeks) in 7 weeks- and 15 months-old mice (young and old, respectively). Livers were analyzed for fibrosis, inflammation and remodeling 48 and 96 hours after the last injection. Old mice developed more severe fibrosis compared to young ones as evaluated by sirius red morphometry. Expression of pro-fibrogenic genes was equally induced in the two age-groups but enhanced fibrolysis in young mice was demonstrated by a significantly higher Mmp13 induction and collagenase activity. While fibrosis resolution occurred in young mice within 96 hours, no significant fibrosis attenuation was observed in old mice. Although recruitment of monocytes-derived macrophages was similar in young and old livers, young macrophages had globally a remodeling phenotype while old ones, a pro-fibrogenic phenotype. Moreover, we observed a higher proportion of thick fibers and enhanced expression of enzymes involved in collagen maturation in old mice. Impaired fibrolysis of a matrix less prone to remodeling associated with a pro-inflammatory phenotype of infiltrated macrophages contribute to a more severe fibrosis in old mice.

Gpr110 deficiency decelerates carcinogen-induced hepatocarcinogenesis via activation of the IL-6/STAT3 pathway

PubMed Central

Ma, Benting; Zhu, Junjie; Tan, Juan; Mao, Yulei; Tang, Lingyun; Shen, Chunling; Zhang, Hongxing; Kuang, Ying; Fei, Jian; Yang, Xiao; Wang, Zhugang

2017-01-01

Hepatocarcinogenesis is a complex process that includes pronounced necroinflammation, unregulated hepatocyte damage, subsequent extensive fibrosis, and carcinogenesis. GPR110 was an adhesion G protein-coupled receptor. Analysis of the expression pattern of Gpr110 in mice displayed that Gpr110 was expressed highly in liver, implicating the tissue compartments where Gpr110 could execute its functions, the role of Gpr110 in the physiological and pathological state of liver remains unclear. Based on a Gpr110 knockout mouse model, we evaluated the role of Gpr110 in hepatocarcinogenesis by using a carbon tetrachloride (CCl4)-induced liver injury and fibrosis model, as well as diethylnitrosamine (DEN) plus CCl4-induced liver cancer model. In this study, we found subdued chronic liver injury, reduced compensatory proliferation, lower liver fibrosis, but enhanced inflammation occurred in Gpr110-/- mice during CCl4 challenge. In addition, Gpr110-/- mice were resistant to liver tumorigenesis induced by DEN plus CCl4 injection. Molecular mechanisms underlying these differences correlated with augmented activation of the IL-6/STAT3 pathway, which exerted hepatoprotective effects during liver damage, fibrosis, and oncogenesis in Gpr110-/- mice. Furthermore, pharmacological inhibition of the activation of the IL-6/STAT3 pathway enhanced hepatic fibrosis and promoted DEN plus CCl4-induced carcinogenesis in Gpr110-/- mice. In summary, absence of Gpr110 decelerates liver fibrosis/cirrhosis progressing into tumorigenesis, due to strengthening activation of the IL-6/STAT3 pathway, leading to a weaker liver injury and fibrosis microenvironment. It is indicated that targeting Gpr110 and activating the IL-6/STAT3 pathway may be considered to be preventive methods for some cirrhosis transition. PMID:28401002

Obesity increases inflammation and impairs lymphatic function in a mouse model of lymphedema.

PubMed

Savetsky, Ira L; Torrisi, Jeremy S; Cuzzone, Daniel A; Ghanta, Swapna; Albano, Nicholas J; Gardenier, Jason C; Joseph, Walter J; Mehrara, Babak J

2014-07-15

Although obesity is a major clinical risk factor for lymphedema, the mechanisms that regulate this effect remain unknown. Recent reports have demonstrated that obesity is associated with acquired lymphatic dysfunction. The purpose of this study was to determine how obesity-induced lymphatic dysfunction modulates the pathological effects of lymphatic injury in a mouse model. We used a diet-induced model of obesity in adult male C57BL/6J mice in which experimental animals were fed a high-fat diet and control animals were fed a normal chow diet for 8-10 wk. We then surgically ablated the superficial and deep lymphatics of the midportion of the tail. Six weeks postoperatively, we analyzed changes in lymphatic function, adipose deposition, inflammation, and fibrosis. We also compared responses to acute inflammatory stimuli in obese and lean mice. Compared with lean control mice, obese mice had baseline decreased lymphatic function. Lymphedema in obese mice further impaired lymphatic function and resulted in increased subcutaneous adipose deposition, increased CD45(+) and CD4(+) cell inflammation (P < 0.01), and increased fibrosis, but caused no change in the number of lymphatic vessels. Interestingly, obese mice had a significantly increased acute inflammatory reaction to croton oil application. In conclusion, obese mice have impaired lymphatic function at baseline that is amplified by lymphatic injury. This effect is associated with increased chronic inflammation, fibrosis, and adipose deposition. These findings suggest that obese patients are at higher risk for lymphedema due to impaired baseline lymphatic clearance and an increased propensity for inflammation in response to injury. Copyright © 2014 the American Physiological Society.

Monitoring of Cardiac Remodeling in a Mouse Model of Pressure-Overload Left Ventricular Hypertrophy with [18F]FDG MicroPET.

PubMed

Todica, Andrei; Beetz, Nick L; Günther, Lisa; Zacherl, Mathias J; Grabmaier, Ulrich; Huber, Bruno; Bartenstein, Peter; Brunner, Stefan; Lehner, Sebastian

2018-04-01

This study aims to analyze the left ventricular function parameters, scar load, and hypertrophy in a mouse model of pressure-overload left ventricular (LV) hypertrophy over the course of 8 weeks using 2-deoxy-2-[ 18 F]fluoro-D-glucose ([ 18 F]FDG) micro-positron emission tomography (microPET) imaging. LV hypertrophy was induced in C57BL/6 mice by transverse aortic constriction (TAC). Myocardial hypertrophy developed after 2-4 weeks. ECG-gated microPET scans with [ 18 F]FDG were performed 4 and 8 weeks after surgery. The extent of fibrosis was measured by histopathologic analysis. LV function parameters and scar load were calculated using QGS®/QPS®. LV metabolic volume (LVMV) and percentage injected dose per gram were estimated by threshold-based analysis. The fibrotic tissue volume increased significantly from 4 to 8 weeks after TAC (​1.67 vs. 3.91  mm 3 ; P = 0.044). There was a significant increase of the EDV (4 weeks: 54 ± 15 μl, 8 weeks: 79 ± 32 μl, P < 0.01) and LVMV (4 weeks: 222 ± 24 μl, 8 weeks: 276 ± 52 μl, P < 0.01) as well as a significant decrease of the LVEF (4 weeks: 56 ± 17 %, 8 weeks: 44 ± 20 %, P < 0.01). The increase of LVMV had a high predictive value regarding the amount of ex vivo measured fibrotic tissue (R = 0.905, P < 0.001). The myocardial metabolic defects increased within 4 weeks (P = 0.055) but only moderately correlated with the fibrosis volume (R = 0.502, P = 0.021). The increase in end-diastolic volume showed a positive correlation with the fibrosis at 8 weeks (R = 0.763, P = 0.017). [ 18 F]FDG-PET is applicable for serial in vivo monitoring of the TAC mouse model. Myocardial hypertrophy, the dilation of the left ventricle, and the decrease in LVEF could be reliably quantified over time, as well as the developing localized scar. The increase in volume over time is predictive of a high fibrosis load.

Gain-of-function mutant of angiotensin II receptor, type 1A, causes hypertension and cardiovascular fibrosis in mice

PubMed Central

Billet, Sandrine; Bardin, Sabine; Verp, Sonia; Baudrie, Véronique; Michaud, Annie; Conchon, Sophie; Muffat-Joly, Martine; Escoubet, Brigitte; Souil, Evelyne; Hamard, Ghislaine; Bernstein, Kenneth E.; Gasc, Jean Marie; Elghozi, Jean-Luc; Corvol, Pierre; Clauser, Eric

2007-01-01

The role of the renin-angiotensin system has been investigated by overexpression or inactivation of its different genes in animals. However, there is no data concerning the effect of the constitutive activation of any component of the system. A knockin mouse model has been constructed with a gain-of-function mutant of the Ang II receptor, type 1A (AT1A), associating a constitutively activating mutation (N111S) with a C-terminal deletion, which impairs receptor internalization and desensitization. In vivo consequences of this mutant receptor expression in homozygous mice recapitulate its in vitro characteristics: the pressor response is more sensitive to Ang II and longer lasting. These mice present with a moderate (~20 mmHg) and stable increase in BP. They also develop early and progressive renal fibrosis and cardiac fibrosis and diastolic dysfunction. However, there was no overt cardiac hypertrophy. The hormonal parameters (low-renin and inappropriately normal aldosterone productions) mimic those of low-renin human hypertension. This new model reveals that a constitutive activation of AT1A leads to cardiac and renal fibrosis in spite of a modest effect on BP and will be useful for investigating the role of Ang II in target organs in a model similar to some forms of human hypertension. PMID:17607364

Instability of the insertional mutation in CftrTgH(neoim)Hgu cystic fibrosis mouse model

PubMed Central

Charizopoulou, Nikoletta; Jansen, Silke; Dorsch, Martina; Stanke, Frauke; Dorin, Julia R; Hedrich, Hans-Jürgen; Tümmler, Burkhard

2004-01-01

Background A major boost to the cystic fibrosis disease research was given by the generation of various mouse models using gene targeting in embryonal stem cells. Moreover, the introduction of the same mutation on different inbred strains generating congenic strains facilitated the search for modifier genes. From the original CftrTgH(neoim)Hgu CF mouse model we have generated using strict brother × sister mating two inbred CftrTgH(neoim)Hgu mouse lines (CF/1 and CF/3). Thereafter, the insertional mutation was introgressed from CF/3 into three inbred backgrounds (C57BL/6, BALB/c, DBA/2J) generating congenic animals. In every backcross cycle germline transmission of the insertional mutation was monitored by direct probing the insertion via Southern RFLP. In order to bypass this time consuming procedure we devised an alternative PCR based protocol whereby mouse strains are differentiated at the Cftr locus by Cftr intragenic microsatellite genotypes that are tightly linked to the disrupted locus. Results Using this method we were able to identify animals carrying the insertional mutation based upon the differential haplotypic backgrounds of the three inbred strains and the mutant CftrTgH(neoim)Hgu at the Cftr locus. Moreover, this method facilitated the identification of the precise vector excision from the disrupted Cftr locus in two out of 57 typed animals. This reversion to wild type status took place without any loss of sequence revealing the instability of insertional mutations during the production of congenic animals. Conclusions We present intragenic microsatellite markers as a tool for fast and efficient identification of the introgressed locus of interest in the recipient strain during congenic animal breeding. Moreover, the same genotyping method allowed the identification of a vector excision event, posing questions on the stability of insertional mutations in mice. PMID:15102331

Aldehyde dehydrogenase inhibition blocks mucosal fibrosis in human and mouse ocular scarring

PubMed Central

Ahadome, Sarah D.; Abraham, David J.; Rayapureddi, Suryanarayana; Saw, Valerie P.; Saban, Daniel R.; Calder, Virginia L.; Norman, Jill T.; Ponticos, Markella; Daniels, Julie T.; Dart, John K.

2016-01-01

Mucous membrane pemphigoid (MMP) is a systemic mucosal scarring disease, commonly causing blindness, for which there is no antifibrotic therapy. Aldehyde dehydrogenase family 1 (ALDH1) is upregulated in both ocular MMP (OMMP) conjunctiva and cultured fibroblasts. Application of the ALDH metabolite, retinoic acid (RA), to normal human conjunctival fibroblasts in vitro induced a diseased phenotype. Conversely, application of ALDH inhibitors, including disulfiram, to OMMP fibroblasts in vitro restored their functionality to that of normal controls. ALDH1 is also upregulated in the mucosa of the mouse model of scarring allergic eye disease (AED), used here as a surrogate for OMMP, in which topical application of disulfiram decreased fibrosis in vivo. These data suggest that progressive scarring in OMMP results from ALDH/RA fibroblast autoregulation, that the ALDH1 subfamily has a central role in immune-mediated ocular mucosal scarring, and that ALDH inhibition with disulfiram is a potential and readily translatable antifibrotic therapy. PMID:27699226

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.

Modulation of the Unfolded Protein Response by Tauroursodeoxycholic Acid Counteracts Apoptotic Cell Death and Fibrosis in a Mouse Model for Secondary Biliary Liver Fibrosis

PubMed Central

Paridaens, Annelies; Raevens, Sarah; Devisscher, Lindsey; Bogaerts, Eliene; Verhelst, Xavier; Hoorens, Anne; van Vlierberghe, Hans; Van Grunsven, Leo A.; Geerts, Anja; Colle, Isabelle

2017-01-01

The role of endoplasmic reticulum stress and the unfolded protein response (UPR) in cholestatic liver disease and fibrosis is not fully unraveled. Tauroursodeoxycholic acid (TUDCA), a hydrophilic bile acid, has been shown to reduce endoplasmic reticulum (ER) stress and counteract apoptosis in different pathologies. We aimed to investigate the therapeutic potential of TUDCA in experimental secondary biliary liver fibrosis in mice, induced by common bile duct ligation. The kinetics of the hepatic UPR and apoptosis during the development of biliary fibrosis was studied by measuring markers at six different timepoints post-surgery by qPCR and Western blot. Next, we investigated the therapeutic potential of TUDCA, 10 mg/kg/day in drinking water, on liver damage (AST/ALT levels) and fibrosis (Sirius red-staining), in both a preventive and therapeutic setting. Common bile duct ligation resulted in the increased protein expression of CCAAT/enhancer-binding protein homologous protein (CHOP) at all timepoints, along with upregulation of pro-apoptotic caspase 3 and 12, tumor necrosis factor receptor superfamily, member 1A (TNFRsf1a) and Fas-Associated protein with Death Domain (FADD) expression. Treatment with TUDCA led to a significant reduction of liver fibrosis, accompanied by a slight reduction of liver damage, decreased hepatic protein expression of CHOP and reduced gene and protein expression of pro-apoptotic markers. These data indicate that TUDCA exerts a beneficial effect on liver fibrosis in a model of cholestatic liver disease, and suggest that this effect might, at least in part, be attributed to decreased hepatic UPR signaling and apoptotic cell death. PMID:28117681

Comparison of trichostatin A and valproic acid treatment regimens in a mouse model of kidney fibrosis

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

Van Beneden, Katrien, E-mail: kvbenede@vub.ac.be; Geers, Caroline; Pauwels, Marina

Histone deacetylase (HDAC) inhibitors are promising new compounds for the therapy of fibrotic diseases. In this study we compared the effect of two HDAC inhibitors, trichostatin A and valproic acid, in an experimental model of kidney fibrosis. In mice, doxorubicin (adriamycin) can cause nephropathy characterized by chronic proteinuria, glomerular damage and interstitial inflammation and fibrosis, as seen in human focal segmental glomerulosclerosis. Two treatment regimens were applied, treatment was either started prior to the doxorubicin insult or delayed until a significant degree of proteinuria and fibrosis was present. Pre-treatment of trichostatin A significantly hampered glomerulosclerosis and tubulointerstitial fibrosis, as didmore » the pre-treatment with valproic acid. In contrast, the development of proteinuria was only completely inhibited in the pre-treated valproic acid group, and not in the pre-treated trichostatin A animals. In the postponed treatment with valproic acid, a complete resolution of established doxorubicin-induced proteinuria was achieved within three days, whereas trichostatin A could not correct proteinuria in such a treatment regimen. However, both postponed regimens have comparable efficacy in maintaining the kidney fibrosis to the level reached at the start of the treatments. Moreover, not only the process of fibrosis, but also renal inflammation was attenuated by both HDAC inhibitors. Our data confirm a role for HDACs in renal fibrogenesis and point towards a therapeutic potential for HDAC inhibitors. The effect on renal disease progression and manifestation can however be different for individual HDAC inhibitors. - Highlights: • Valproic acid is a potent antiproteinuric drug, whereas trichostatin A is not. • Trichostatin A and valproic acid reduce kidney fibrosis in doxorubicin nephropathy. • Both valproic acid and trichostatin A attenuate renal inflammation.« less

The increase of microRNA-21 during lung fibrosis and its contribution to epithelial-mesenchymal transition in pulmonary epithelial cells.

PubMed

Yamada, Mitsuhiro; Kubo, Hiroshi; Ota, Chiharu; Takahashi, Toru; Tando, Yukiko; Suzuki, Takaya; Fujino, Naoya; Makiguchi, Tomonori; Takagi, Kiyoshi; Suzuki, Takashi; Ichinose, Masakazu

2013-09-24

The excess and persistent accumulation of fibroblasts due to aberrant tissue repair results in fibrotic diseases such as idiopathic pulmonary fibrosis. Recent reports have revealed significant changes in microRNAs during idiopathic pulmonary fibrosis and evidence in support of a role for microRNAs in myofibroblast differentiation and the epithelial-mesenchymal transition in the context of fibrosis. It has been reported that microRNA-21 is up-regulated in myofibroblasts during fibrosis and promotes transforming growth factor-beta signaling by inhibiting Smad7. However, expression changes in microRNA-21 and the role of microRNA-21 in epithelial-mesenchymal transition during lung fibrosis have not yet been defined. Lungs from saline- or bleomycin-treated C57BL/6 J mice and lung specimens from patients with idiopathic pulmonary fibrosis were analyzed. Enzymatic digestions were performed to isolate single lung cells. Lung epithelial cells were isolated by flow cytometric cell sorting. The expression of microRNA-21 was analyzed using both quantitative PCR and in situ hybridization. To induce epithelial-mesenchymal transition in culture, isolated mouse lung alveolar type II cells were cultured on fibronectin-coated chamber slides in the presence of transforming growth factor-β, thus generating conditions that enhance epithelial-mesenchymal transition. To investigate the role of microRNA-21 in epithelial-mesenchymal transition, we transfected cells with a microRNA-21 inhibitor. Total RNA was isolated from the freshly isolated and cultured cells. MicroRNA-21, as well as mRNAs of genes that are markers of alveolar epithelial or mesenchymal cell differentiation, were quantified using quantitative PCR. The lung epithelial cells isolated from the bleomycin-induced lung fibrosis model system had decreased expression of epithelial marker genes, whereas the expression of mesenchymal marker genes was increased. MicroRNA-21 was significantly upregulated in isolated lung epithelial cells during bleomycin-induced lung fibrosis and human idiopathic pulmonary fibrosis. MicroRNA-21 was also upregulated in the cultured alveolar epithelial cells under the conditions that enhance epithelial-mesenchymal transition. Exogenous administration of a microRNA-21 inhibitor prevented the increased expression of vimentin and alpha-smooth muscle actin in cultured primary mouse alveolar type II cells under culture conditions that induce epithelial-mesenchymal transition. Our experiments demonstrate that microRNA-21 is increased in lung epithelial cells during lung fibrosis and that it promotes epithelial-mesenchymal transition.

Leptospira Interrogans Induces Fibrosis in the Mouse Kidney through Inos-Dependent, TLR- and NLR-Independent Signaling Pathways

PubMed Central

Fanton d'Andon, Martine; Quellard, Nathalie; Fernandez, Béatrice; Ratet, Gwenn; Lacroix-Lamandé, Sonia; Vandewalle, Alain; Boneca, Ivo G.; Goujon, Jean-Michel; Werts, Catherine

2014-01-01

Background Leptospira (L.) interrogans are bacteria responsible for a worldwide reemerging zoonosis. Rodents carry L. interrogans asymptomatically in their kidneys and excrete bacteria in the urine, contaminating the environment. Humans get infected through skin contact and develop a mild or severe leptospirosis that may lead to renal failure and fibrosis. L. interrogans provoke an interstitial nephritis, but the induction of fibrosis caused by L. interrogans has not been studied in murine models. Innate immune receptors from the TLR and NLR families have recently been shown to play a role in the development and progression of tissue fibrosis in the lung, liver and kidneys under different pathophysiological situations. We recently showed that TLR2, TLR4, and NLRP3 receptors were crucial in the defense against leptospirosis. Moreover, infection of a human cell line with L. interrogans was shown to induce TLR2-dependent production of fibronectin, a component of the extracellular matrix. Therefore, we thought to assess the presence of renal fibrosis in L. interrogans infected mice and to analyze the contribution of some innate immune pathways in this process. Methodology/principal findings Here, we characterized by immunohistochemical studies and quantitative real-time PCR, a model of Leptospira-infected C57BL/6J mice, with chronic carriage of L. interrogans inducing mild renal fibrosis. Using various strains of transgenic mice, we determined that the renal infiltrates of T cells and, unexpectedly, TLR and NLR receptors, are not required to generate Leptospira-induced renal fibrosis. We also show that the iNOS enzyme, known to play a role in Leptospira-induced interstitial nephritis, also plays a role in the induction of renal fibrosis. Conclusion/significance To our knowledge, this work provides the first experimental murine model of sustained renal fibrosis induced by a chronic bacterial infection that may be peculiar, since it does not rely on TLR or NLR receptors. This model may prove useful to test future therapeutic strategies to combat Leptospira-induced renal lesions. PMID:24498450

The D prostanoid receptor agonist BW245C [(4S)-(3-[(3R,S)-3-cyclohexyl-3-hydroxypropyl]-2,5-dioxo)-4-imidazolidineheptanoic acid] inhibits fibroblast proliferation and bleomycin-induced lung fibrosis in mice.

PubMed

van den Brule, Sybille; Wallemme, Laurent; Uwambayinema, Francine; Huaux, François; Lison, Dominique

2010-11-01

Prostaglandin (PG) D(2) exerts contrasting activities in the inflamed lung via two receptors, the D prostanoid receptor (DP) and the chemoattractant receptor-homologous molecule expressed on T helper 2 lymphocytes. DP activation is known mainly to inhibit proinflammatory cell functions. We tested the effect of a DP-specific agonist, (4S)-(3-[(3R,S)-3-cyclohexyl-3-hydroxypropyl]-2,5-dioxo)-4-imidazolidineheptanoic acid (BW245C), on pulmonary fibroblast functions in vitro and in a mouse model of lung fibrosis induced by bleomycin. DP mRNA expression was detected in cultured mouse lung primary fibroblasts and human fetal lung fibroblasts and found to be up- and down-regulated by interleukin-13 and transforming growth factor (TGF)-β, respectively. Although micromolar concentrations of BW245C and PGD(2) did not affect mouse fibroblast collagen synthesis or differentiation in myofibroblasts, they both inhibited fibroblast basal and TGF-β-induced proliferation in vitro. The repeated administration of BW245C (500 nmol/kg body weight instilled transorally in the lungs 2 days before and three times per week for 3 weeks) in bleomycin-treated mice significantly decreased both inflammatory cell recruitment and collagen accumulation in the lung (21 days). Our results indicate that BW245C can reduce lung fibrosis in part via its activity on fibroblast proliferation and suggest that DP activation should be considered as a new therapeutic target in fibroproliferative lung diseases.

The pathway to muscle fibrosis depends on myostatin stimulating the differentiation of fibro/adipogenic progenitor cells in chronic kidney disease

PubMed Central

Dong, Jiangling; Dong, Yanjun; Chen, Zihong; Mitch, William E.; Zhang, Liping

2016-01-01

Fibrosis in skeletal muscle develops after injury or in response to chronic kidney disease (CKD) but the origin of cells becoming fibrous tissue and the initiating and sustaining mechanisms causing muscle fibrosis are unclear. We have identified muscle fibro/adipogenic progenitor cells (FAPs) that potentially differentiate into adipose tissues or fibrosis. We also demonstrated that CKD stimulates myostatin production in muscle. Therefore, we tested whether CKD induces myostatin which stimulates fibrotic differentiation of FAPs leading to fibrosis in skeletal muscles. We isolated FAPs from mouse muscles and found that myostatin stimulates their proliferation and conversion into fibrocytes. In vivo, FAPs isolated from EGFP-transgenic mice (FAPs-EGFP) were transplanted into muscles of mice with CKD or into mouse muscles that were treated with myostatin. CKD or myostatin stimulated FAPs-EGFP proliferation in muscle and increased α-smooth muscle actin expression in FAP-EGFP cells. When myostatin was inhibited with a neutralizing peptibody (a chimeric peptide-Fc fusion protein), the FAP proliferation and muscle fibrosis induced by CKD were both suppressed. Knocking down Smad3 in cultured FAPs interrupted their conversion into fibrocytes indicating that myostatin directly converts FAPs into fibrocytes. Thus, counteracting myostatin may be a strategy for preventing the development of fibrosis in skeletal muscles of patients with CKD. PMID:27653838

The pathway to muscle fibrosis depends on myostatin stimulating the differentiation of fibro/adipogenic progenitor cells in chronic kidney disease.

PubMed

Dong, Jiangling; Dong, Yanjun; Chen, Zihong; Mitch, William E; Zhang, Liping

2017-01-01

Fibrosis in skeletal muscle develops after injury or in response to chronic kidney disease (CKD), but the origin of cells becoming fibrous tissue and the initiating and sustaining mechanisms causing muscle fibrosis are unclear. We identified muscle fibro/adipogenic progenitor cells (FAPs) that potentially differentiate into adipose tissues or fibrosis. We also demonstrated that CKD stimulates myostatin production in muscle. Therefore, we tested whether CKD induces myostatin, which stimulates fibrotic differentiation of FAPs leading to fibrosis in skeletal muscles. We isolated FAPs from mouse muscles and found that myostatin stimulates their proliferation and conversion into fibrocytes. In vivo, FAPs isolated from EGFP-transgenic mice (FAPs-EGFP) were transplanted into muscles of mice with CKD or into mouse muscles that were treated with myostatin. CKD or myostatin stimulated FAPs-EGFP proliferation in muscle and increased α-smooth muscle actin expression in FAP-EGFP cells. When myostatin was inhibited with a neutralizing peptibody (a chimeric peptide-Fc fusion protein), the FAP proliferation and muscle fibrosis induced by CKD were both suppressed. Knocking down Smad3 in cultured FAPs interrupted their conversion into fibrocytes, indicating that myostatin directly converts FAPs into fibrocytes. Thus, counteracting myostatin may be a strategy for preventing the development of fibrosis in skeletal muscles of patients with CKD. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

DC-SIGN activation mediates the differential effects of SAP and CRP on the innate immune system and inhibits fibrosis in mice

PubMed Central

Cox, Nehemiah; Pilling, Darrell; Gomer, Richard H.

2015-01-01

Fibrosis is caused by scar tissue formation in internal organs and is associated with 45% of deaths in the United States. Two closely related human serum proteins, serum amyloid P (SAP) and C-reactive protein (CRP), strongly affect fibrosis. In multiple animal models, and in Phase 1 and Phase 2 clinical trials, SAP affects several aspects of the innate immune system to reduce fibrosis, whereas CRP appears to potentiate fibrosis. However, SAP and CRP bind the same Fcγ receptors (FcγR) with similar affinities, and why SAP and CRP have opposing effects is unknown. Here, we report that SAP but not CRP binds the receptor DC-SIGN (SIGN-R1) to affect the innate immune system, and that FcγR are not necessary for SAP function. A polycyclic aminothiazole DC-SIGN ligand and anti–DC-SIGN antibodies mimic SAP effects in vitro. In mice, the aminothiazole reduces neutrophil accumulation in a model of acute lung inflammation and, at 0.001 mg/kg, alleviates pulmonary fibrosis by increasing levels of the immunosuppressant IL-10. DC-SIGN (SIGN-R1) is present on mouse lung epithelial cells, and SAP and the aminothiazole potentiate IL-10 production from these cells. Our data suggest that SAP activates DC-SIGN to regulate the innate immune system differently from CRP, and that DC-SIGN is a target for antifibrotics. PMID:26106150

Translational Profiles of Medullary Myofibroblasts during Kidney Fibrosis

PubMed Central

Grgic, Ivica; Krautzberger, A. Michaela; Hofmeister, Andreas; Lalli, Matthew; DiRocco, Derek P.; Fleig, Susanne V.; Liu, Jing; Duffield, Jeremy S.; McMahon, Andrew P.; Aronow, Bruce

2014-01-01

Myofibroblasts secrete matrix during chronic injury, and their ablation ameliorates fibrosis. Development of new biomarkers and therapies for CKD will be aided by a detailed analysis of myofibroblast gene expression during the early stages of fibrosis. However, dissociating myofibroblasts from fibrotic kidney is challenging. We therefore adapted translational ribosome affinity purification (TRAP) to isolate and profile mRNA from myofibroblasts and their precursors during kidney fibrosis. We generated and characterized a transgenic mouse expressing an enhanced green fluorescent protein (eGFP)–tagged L10a ribosomal subunit protein under control of the collagen1α1 promoter. We developed a one-step procedure for isolation of polysomal RNA from collagen1α1-eGFPL10a mice subject to unilateral ureteral obstruction and analyzed and validated the resulting transcriptional profiles. Pathway analysis revealed strong gene signatures for cell proliferation, migration, and shape change. Numerous novel genes and candidate biomarkers were upregulated during fibrosis, specifically in myofibroblasts, and we validated these results by quantitative PCR, in situ, and Western blot analysis. This study provides a comprehensive analysis of early myofibroblast gene expression during kidney fibrosis and introduces a new technique for cell-specific polysomal mRNA isolation in kidney injury models that is suited for RNA-sequencing technologies. PMID:24652793

Simultaneously Targeting Myofibroblast Contractility and Extracellular Matrix Cross-Linking as a Therapeutic Concept in Airway Fibrosis

PubMed Central

Lin, Yu-chun; Sung, Yon K.; Jiang, Xinguo; Peters-Golden, Marc; Nicolls, Mark R.

2016-01-01

Fibrosis after solid organ transplantation is considered an irreversible process and remains the major cause of graft dysfunction and death with limited therapies. This remodeling is characterized by aberrant accumulation of contractile myofibroblasts that deposit excessive extracellular matrix (ECM) and increase tissue stiffness. However, studies demonstrate that a stiff ECM, itself, promotes fibroblast-to-myofibroblast differentiation, stimulating further ECM production. This creates a positive feedback loop that perpetuates fibrosis. We hypothesized that simultaneously targeting myofibroblast contractility with relaxin and ECM stiffness with lysyl oxidase inhibitors could break the feedback loop, thereby, reversing established fibrosis. To test this, we used the orthotopic tracheal transplanted (OTT) mouse model, which develops robust fibrotic airway remodeling. Mice with established fibrosis were treated with saline, mono-, or combination therapies. While monotherapies had no effect, combining these agents decreased collagen deposition and promoted re-epithelialization of remodeled airways. Relaxin inhibited myofibroblast differentiation and contraction, in a matrix-stiffness-dependent manner through prostaglandin E2 (PGE2). Furthermore, the effect of combination therapy was lost in PGE2 receptor knockout and PGE2 inhibited OTT mice. This study reveals the important synergistic roles of cellular contractility and tissue stiffness in the maintenance of fibrotic tissue and suggests a new therapeutic principle for fibrosis. PMID:27804215

Dual-Functional Nanoparticles Targeting CXCR4 and Delivering Antiangiogenic siRNA Ameliorate Liver Fibrosis.

PubMed

Liu, Chun-Hung; Chan, Kun-Ming; Chiang, Tsaiyu; Liu, Jia-Yu; Chern, Guann-Gen; Hsu, Fu-Fei; Wu, Yu-Hsuan; Liu, Ya-Chi; Chen, Yunching

2016-07-05

The progression of liver fibrosis, an intrinsic response to chronic liver injury, is associated with hepatic hypoxia, angiogenesis, abnormal inflammation, and significant matrix deposition, leading to the development of cirrhosis and hepatocellular carcinoma (HCC). Due to the complex pathogenesis of liver fibrosis, antifibrotic drug development has faced the challenge of efficiently and specifically targeting multiple pathogenic mechanisms. Therefore, CXCR4-targeted nanoparticles (NPs) were formulated to deliver siRNAs against vascular endothelial growth factor (VEGF) into fibrotic livers to block angiogenesis during the progression of liver fibrosis. AMD3100, a CXCR4 antagonist that was incorporated into the NPs, served dual functions: it acted as a targeting moiety and suppressed the progression of fibrosis by inhibiting the proliferation and activation of hepatic stellate cells (HSCs). We demonstrated that CXCR4-targeted NPs could deliver VEGF siRNAs to fibrotic livers, decrease VEGF expression, suppress angiogenesis and normalize the distorted vessels in the fibrotic livers in the carbon tetrachloride (CCl4) induced mouse model. Moreover, blocking SDF-1α/CXCR4 by CXCR4-targeted NPs in combination with VEGF siRNA significantly prevented the progression of liver fibrosis in CCl4-treated mice. In conclusion, the multifunctional CXCR4-targeted NPs delivering VEGF siRNAs provide an effective antifibrotic therapeutic strategy.

The orphan nuclear receptor RORα and group 3 innate lymphoid cells drive fibrosis in a mouse model of Crohn's disease.

PubMed

Lo, Bernard C; Gold, Matthew J; Hughes, Michael R; Antignano, Frann; Valdez, Yanet; Zaph, Colby; Harder, Kenneth W; McNagny, Kelly M

2016-09-02

Fibrosis is the result of dysregulated tissue regeneration and is characterized by excessive accumulation of matrix proteins that become detrimental to tissue function. In Crohn's disease, this manifests itself as recurrent gastrointestinal strictures for which there is no effective therapy beyond surgical intervention. Using a model of infection-induced chronic gut inflammation, we show that Rora -deficient mice are protected from fibrosis; infected intestinal tissues display diminished pathology, attenuated collagen deposition, and reduced fibroblast accumulation. Although Rora is best known for its role in group 2 innate lymphoid cell (ILC2) development, we find that Salmonella -induced fibrosis is independent of eosinophils, signal transducer and activator of transcription 6 signaling, and T helper 2 cytokine production, arguing that this process is largely ILC2-independent. Instead, we observe reduced levels of ILC3- and T cell-derived interleukin-17A (IL-17A) and IL-22 in infected gut tissues. Furthermore, using Rora sg/sg / Rag1 -/- bone marrow chimeric mice, we show that restoring ILC function is sufficient to reestablish IL-17A and IL-22 production and a profibrotic phenotype. Our results show that RORα (retinoic acid receptor-related orphan receptor α)-dependent ILC3 functions are pivotal in mediating gut fibrosis, and they offer an avenue for therapeutic intervention in Crohn's-like diseases. Copyright © 2016, American Association for the Advancement of Science.

X-ray dark-field radiography facilitates the diagnosis of pulmonary fibrosis in a mouse model.

PubMed

Hellbach, Katharina; Yaroshenko, Andre; Willer, Konstantin; Conlon, Thomas M; Braunagel, Margarita B; Auweter, Sigrid; Yildirim, Ali Ö; Eickelberg, Oliver; Pfeiffer, Franz; Reiser, Maximilian F; Meinel, Felix G

2017-03-23

The aim of this study was to evaluate whether diagnosing pulmonary fibrosis with projection radiography can be improved by using X-ray dark-field radiograms. Pulmonary X-ray transmission and dark-field images of C57Bl/6N mice, either treated with bleomycin to induce pulmonary fibrosis or PBS to serve as controls, were acquired with a prototype grating-based small-animal scanner. Two blinded readers, both experienced radiologists and familiar with dark-field imaging, had to assess dark-field and transmission images for the absence or presence of fibrosis. Furthermore readers were asked to grade their stage of diagnostic confidence. Histological evaluation of the lungs served as the standard of reference in this study. Both readers showed a notably higher diagnostic confidence when analyzing the dark-field radiographs (p < 0.001). Diagnostic accuracy improved significantly when evaluating the lungs in dark-field images alone (p = 0.02) or in combination with transmission images (p = 0.01) compared to sole analysis of absorption images. Interreader agreement improved from good when assessing only transmission images to excellent when analyzing dark-field images alone or in combination with transmission images. Adding dark-field images to conventional transmission images in a murine model of pulmonary fibrosis leads to an improved diagnosis of this disease on chest radiographs.

Ferret and pig models of cystic fibrosis: prospects and promise for gene therapy.

PubMed

Yan, Ziying; Stewart, Zoe A; Sinn, Patrick L; Olsen, John C; Hu, Jim; McCray, Paul B; Engelhardt, John F

2015-03-01

Large animal models of genetic diseases are rapidly becoming integral to biomedical research as technologies to manipulate the mammalian genome improve. The creation of cystic fibrosis (CF) ferrets and pigs is an example of such progress in animal modeling, with the disease phenotypes in the ferret and pig models more reflective of human CF disease than mouse models. The ferret and pig CF models also provide unique opportunities to develop and assess the effectiveness of gene and cell therapies to treat affected organs. In this review, we examine the organ disease phenotypes in these new CF models and the opportunities to test gene therapies at various stages of disease progression in affected organs. We then discuss the progress in developing recombinant replication-defective adenoviral, adeno-associated viral, and lentiviral vectors to target genes to the lung and pancreas in ferrets and pigs, the two most affected organs in CF. Through this review, we hope to convey the potential of these new animal models for developing CF gene and cell therapies.

Heat shock transcription factor 1 protects against pressure overload-induced cardiac fibrosis via Smad3.

PubMed

Zhou, Ning; Ye, Yong; Wang, Xingxu; Ma, Ben; Wu, Jian; Li, Lei; Wang, Lin; Wang, Dao Wen; Zou, Yunzeng

2017-04-01

Fibrotic cardiac muscle exhibits high stiffness and low compliance which are major risk factors of heart failure. Although heat shock transcription factor 1 (HSF1) was identified as an intrinsic cardioprotective factor, the role that HSF1 plays in cardiac fibrosis remains unclear. Our study aims to investigate the role of HSF1 in pressure overload-induced cardiac fibrosis and the underlying mechanism. HSF1 phosphorylation was significantly downregulated in transverse aortic constriction (TAC)-treated mouse hearts and mechanically stretched cardiac fibroblasts (cFBs). HSF1 transgenic (TG) mice, HSF1 deficient heterozygote (KO) mice, and their wild-type littermates were subjected to sham or TAC surgery for 4 weeks. HSF1 overexpression significantly attenuated pressure overload-induced cardiac fibrosis and dysfunction. Conversely, HSF1 KO mice showed deteriorated fibrotic response and cardiac dysfunction upon TAC. Moreover, we uncovered that overexpression of HSF1 protected against fibrotic response of cFBs to pressure overload. Mechanistically, we observed that the phosphorylation and the nuclear distribution of the Smad family member 3 (Smad3) were significantly decreased in HSF1-overexpressing mouse hearts, while being greatly increased in HSF1 KO mouse hearts upon TAC, compared to the control hearts, respectively. Similar alteration of Smad3 phosphorylation and nuclear distribution were found in isolated mouse cardiac fibroblasts and mechanically stretched cFBs. Constitutively active Smad3 blocked the anti-fibrotic effect of HSF1 in cFBs. Furthermore, we found a direct binding of phosphorylated HSF1 and Smad3, which can be suppressed by mechanical stress. In conclusion, the present study demonstrated for the first time that HSF1 acts as a novel negative regulator of cardiac fibrosis by blocking Smad3 activation. HSF1 activity is decreased in fibrotic hearts. HSF1 overexpression attenuates pressure overload-induced cardiac fibrosis and dysfunction. Deficiency of HSF1 deteriorates fibrotic response and cardiac dysfunction upon TAC. HSF1 inhibits phosphorylation and nuclear distribution of Smad3 via direct binding to Smad3. Active Smad3 blocks the anti-fibrotic effect of HSF1.

Streptococcus pneumoniae triggers progression of pulmonary fibrosis through pneumolysin.

PubMed

Knippenberg, Sarah; Ueberberg, Bianca; Maus, Regina; Bohling, Jennifer; Ding, Nadine; Tort Tarres, Meritxell; Hoymann, Heinz-Gerd; Jonigk, Danny; Izykowski, Nicole; Paton, James C; Ogunniyi, Abiodun D; Lindig, Sandro; Bauer, Michael; Welte, Tobias; Seeger, Werner; Guenther, Andreas; Sisson, Thomas H; Gauldie, Jack; Kolb, Martin; Maus, Ulrich A

2015-07-01

Respiratory tract infections are common in patients suffering from pulmonary fibrosis. The interplay between bacterial infection and fibrosis is characterised poorly. To assess the effect of Gram-positive bacterial infection on fibrosis exacerbation in mice. Fibrosis progression in response to Streptococcus pneumoniae was examined in two different mouse models of pulmonary fibrosis. We demonstrate that wild-type mice exposed to adenoviral vector delivery of active transforming growth factor-β1 (TGFß1) or diphteria toxin (DT) treatment of transgenic mice expressing the DT receptor (DTR) under control of the surfactant protein C (SPC) promoter (SPC-DTR) to induce pulmonary fibrosis developed progressive fibrosis following infection with Spn, without exhibiting impaired lung protective immunity against Spn. Antibiotic treatment abolished infection-induced fibrosis progression. The cytotoxin pneumolysin (Ply) of Spn caused this phenomenon in a TLR4-independent manner, as Spn lacking Ply (SpnΔply) failed to trigger progressive fibrogenesis, whereas purified recombinant Ply did. Progressive fibrogenesis was also observed in AdTGFβ1-exposed Ply-challenged TLR4 KO mice. Increased apoptotic cell death of alveolar epithelial cells along with an attenuated intrapulmonary release of antifibrogenic prostaglandin E2 was found to underlie progressive fibrogenesis in Ply-challenged AdTGFβ1-exposed mice. Importantly, vaccination of mice with the non-cytotoxic Ply derivative B (PdB) substantially attenuated Ply-induced progression of lung fibrosis in AdTGFβ1-exposed mice. Our data unravel a novel mechanism by which infection with Spn through Ply release induces progression of established lung fibrosis, which can be attenuated by protein-based vaccination of mice. 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.

Uncovering a Predictive Molecular Signature for the Onset of NASH-Related Fibrosis in a Translational NASH Mouse Model.

PubMed

van Koppen, Arianne; Verschuren, Lars; van den Hoek, Anita M; Verheij, Joanne; Morrison, Martine C; Li, Kelvin; Nagabukuro, Hiroshi; Costessi, Adalberto; Caspers, Martien P M; van den Broek, Tim J; Sagartz, John; Kluft, Cornelis; Beysen, Carine; Emson, Claire; van Gool, Alain J; Goldschmeding, Roel; Stoop, Reinout; Bobeldijk-Pastorova, Ivana; Turner, Scott M; Hanauer, Guido; Hanemaaijer, Roeland

2018-01-01

The incidence of nonalcoholic steatohepatitis (NASH) is increasing. The pathophysiological mechanisms of NASH and the sequence of events leading to hepatic fibrosis are incompletely understood. The aim of this study was to gain insight into the dynamics of key molecular processes involved in NASH and to rank early markers for hepatic fibrosis. A time-course study in low-density lipoprotein-receptor knockout. Leiden mice on a high-fat diet was performed to identify the temporal dynamics of key processes contributing to NASH and fibrosis. An integrative systems biology approach was used to elucidate candidate markers linked to the active fibrosis process by combining transcriptomics, dynamic proteomics, and histopathology. The translational value of these findings were confirmed using human NASH data sets. High-fat-diet feeding resulted in obesity, hyperlipidemia, insulin resistance, and NASH with fibrosis in a time-dependent manner. Temporal dynamics of key molecular processes involved in the development of NASH were identified, including lipid metabolism, inflammation, oxidative stress, and fibrosis. A data-integrative approach enabled identification of the active fibrotic process preceding histopathologic detection using a novel molecular fibrosis signature. Human studies were used to identify overlap of genes and processes and to perform a network biology-based prioritization to rank top candidate markers representing the early manifestation of fibrosis. An early predictive molecular signature was identified that marked the active profibrotic process before histopathologic fibrosis becomes manifest. Early detection of the onset of NASH and fibrosis enables identification of novel blood-based biomarkers to stratify patients at risk, development of new therapeutics, and help shorten (pre)clinical experimental time frames.

Oxidative Stress Induced Inflammation Initiates Functional Decline of Tear Production

PubMed Central

Uchino, Yuichi; Kawakita, Tetsuya; Miyazawa, Masaki; Ishii, Takamasa; Onouchi, Hiromi; Yasuda, Kayo; Ogawa, Yoko; Shimmura, Shigeto; Ishii, Naoaki; Tsubota, Kazuo

2012-01-01

Oxidative damage and inflammation are proposed to be involved in an age-related functional decline of exocrine glands. However, the molecular mechanism of how oxidative stress affects the secretory function of exocrine glands is unclear. We developed a novel mev-1 conditional transgenic mouse model (Tet-mev-1) using a modified tetracycline system (Tet-On/Off system). This mouse model demonstrated decreased tear production with morphological changes including leukocytic infiltration and fibrosis. We found that the mev-1 gene encodes Cyt-1, which is the cytochrome b560 large subunit of succinate-ubiquinone oxidoreductase in complex II of mitochondria (homologous to succinate dehydrogenase C subunit (SDHC) in humans). The mev-1 gene induced excessive oxidative stress associated with ocular surface epithelial damage and a decrease in protein and aqueous secretory function. This new model provides evidence that mitochondrial oxidative damage in the lacrimal gland induces lacrimal dysfunction resulting in dry eye disease. Tear volume in Tet-mev-1 mice was lower than in wild type mice and histopathological analyses showed the hallmarks of lacrimal gland inflammation by intense mononuclear leukocytic infiltration and fibrosis in the lacrimal gland of Tet-mev-1 mice. These findings strongly suggest that oxidative stress can be a causative factor for the development of dry eye disease. PMID:23071526

Soluble Dietary Fiber Ameliorates Radiation-Induced Intestinal Epithelial-to-Mesenchymal Transition and Fibrosis.

PubMed

Yang, Jianbo; Ding, Chao; Dai, Xujie; Lv, Tengfei; Xie, Tingbing; Zhang, Tenghui; Gao, Wen; Gong, Jianfeng; Zhu, Weiming; Li, Ning; Li, Jieshou

2017-11-01

Intestinal fibrosis is a late complication of pelvic radiotherapy. Epithelial-to-mesenchymal transition (EMT) plays an important role in tissue fibrosis. The aim of this study was to examine the effect of soluble dietary fiber on radiation-induced intestinal EMT and fibrosis in a mouse model. Apple pectin (4% wt/wt in drinking water) was administered to wild-type and pVillin-Cre-EGFP transgenic mice with intestinal fibrosis induced by a single dose of abdominal irradiation of 10 Gy. The effects of pectin on intestinal EMT and fibrosis, gut microbiota, and short-chain fatty acid (SCFA) concentration were evaluated. Intestinal fibrosis in late radiation enteropathy showed increased submucosal thickness and subepithelial collagen deposition. Enhanced green fluorescent protein (EGFP) + /vimentin + and EGFP + /α-smooth muscle actin (SMA) + coexpressing cells were most clearly observed at 2 weeks after irradiation and gradually decreased at 4 and 12 weeks. Pectin significantly attenuated the thickness of submucosa and collagen deposition at 12 weeks (24.3 vs 27.6 µm in the pectin + radiation-treated group compared with radiation-alone group, respectively, P < .05; 69.0% vs 57.1%, P < .001) and ameliorated EMT at 2 and 4 weeks. Pectin also modulated the intestinal microbiota composition and increased the luminal SCFA concentration. The soluble dietary fiber pectin protected the terminal ileum against radiation-induced fibrosis. This effect might be mediated by altered SCFA concentration in the intestinal lumen and reduced EMT in the ileal epithelium.

Chronic losartan administration reduces mortality and preserves cardiac but not skeletal muscle function in dystrophic mice.

PubMed

Bish, Lawrence T; Yarchoan, Mark; Sleeper, Meg M; Gazzara, Jeffrey A; Morine, Kevin J; Acosta, Pedro; Barton, Elisabeth R; Sweeney, H Lee

2011-01-01

Duchenne muscular dystrophy (DMD) is a degenerative disorder affecting skeletal and cardiac muscle for which there is no effective therapy. Angiotension receptor blockade (ARB) has excellent therapeutic potential in DMD based on recent data demonstrating attenuation of skeletal muscle disease progression during 6-9 months of therapy in the mdx mouse model of DMD. Since cardiac-related death is major cause of mortality in DMD, it is important to evaluate the effect of any novel treatment on the heart. Therefore, we evaluated the long-term impact of ARB on both the skeletal muscle and cardiac phenotype of the mdx mouse. Mdx mice received either losartan (0.6 g/L) (n = 8) or standard drinking water (n = 9) for two years, after which echocardiography was performed to assess cardiac function. Skeletal muscle weight, morphology, and function were assessed. Fibrosis was evaluated in the diaphragm and heart by Trichrome stain and by determination of tissue hydroxyproline content. By the study endpoint, 88% of treated mice were alive compared to only 44% of untreated (p = 0.05). No difference in skeletal muscle morphology, function, or fibrosis was noted in losartan-treated animals. Cardiac function was significantly preserved with losartan treatment, with a trend towards reduction in cardiac fibrosis. We saw no impact on the skeletal muscle disease progression, suggesting that other pathways that trigger fibrosis dominate over angiotensin II in skeletal muscle long term, unlike the situation in the heart. Our study suggests that ARB may be an important prophylactic treatment for DMD-associated cardiomyopathy, but will not impact skeletal muscle disease.

Phosphodiesterase 4 inhibitor and phosphodiesterase 5 inhibitor combination therapy has antifibrotic and anti-inflammatory effects in mdx mice with Duchenne muscular dystrophy.

PubMed

Nio, Yasunori; Tanaka, Masayuki; Hirozane, Yoshihiko; Muraki, Yo; Okawara, Mitsugi; Hazama, Masatoshi; Matsuo, Takanori

2017-12-01

Duchenne muscular dystrophy (DMD) is the most common inherited muscular dystrophy. Patients experience DMD in their 20s from cardiac or respiratory failure related to progressive muscle wasting. Currently, the only treatments for the symptoms of DMD are available. Muscle fibrosis, a DMD feature, leads to reduced muscle function and muscle mass, and hampers pharmaceutical therapeutic efficacy. Although antifibrotic agents may be useful, none is currently approved. Phosphodiesterase 4 (PDE4) inhibitors have exhibited antifibrotic effects in human and animal models. In this study, we showed beneficial effects of the PDE4 inhibitor piclamilast in the DMD mdx mouse. Piclamilast reduced the mRNA level of profibrotic genes, including collagen 1A1, in the gastrocnemius and diaphragm, in the mdx mouse, and significantly reduced the Sirius red staining area. The PDE5 inhibitors sildenafil and tadalafil ameliorated functional muscle ischemia in boys with DMD, and sildenafil reversed cardiac dysfunction in the mdx mouse. Single-treatment piclamilast or sildenafil showed similar antifibrotic effects on the gastrocnemius; combination therapy showed a potent antifibrotic effect, and piclamilast and combination therapy increased peroxisome proliferator-activated receptor γ coactivator-1α mRNA in mouse gastrocnemius. In summary, we confirmed that piclamilast has significant antifibrotic effects in mdx mouse muscle and is a potential treatment for muscle fibrosis in DMD.-Nio, Y., Tanaka, M., Hirozane, Y., Muraki, Y., Okawara, M., Hazama, M., Matsuo, T. Phosphodiesterase 4 inhibitor and phosphodiesterase 5 inhibitor combination therapy has antifibrotic and anti-inflammatory effects in mdx mice with Duchenne muscular dystrophy. © FASEB.

Quantification of Pulmonary Fibrosis in a Bleomycin Mouse Model Using Automated Histological Image Analysis.

PubMed

Gilhodes, Jean-Claude; Julé, Yvon; Kreuz, Sebastian; Stierstorfer, Birgit; Stiller, Detlef; Wollin, Lutz

2017-01-01

Current literature on pulmonary fibrosis induced in animal models highlights the need of an accurate, reliable and reproducible histological quantitative analysis. One of the major limits of histological scoring concerns the fact that it is observer-dependent and consequently subject to variability, which may preclude comparative studies between different laboratories. To achieve a reliable and observer-independent quantification of lung fibrosis we developed an automated software histological image analysis performed from digital image of entire lung sections. This automated analysis was compared to standard evaluation methods with regard to its validation as an end-point measure of fibrosis. Lung fibrosis was induced in mice by intratracheal administration of bleomycin (BLM) at 0.25, 0.5, 0.75 and 1 mg/kg. A detailed characterization of BLM-induced fibrosis was performed 14 days after BLM administration using lung function testing, micro-computed tomography and Ashcroft scoring analysis. Quantification of fibrosis by automated analysis was assessed based on pulmonary tissue density measured from thousands of micro-tiles processed from digital images of entire lung sections. Prior to analysis, large bronchi and vessels were manually excluded from the original images. Measurement of fibrosis has been expressed by two indexes: the mean pulmonary tissue density and the high pulmonary tissue density frequency. We showed that tissue density indexes gave access to a very accurate and reliable quantification of morphological changes induced by BLM even for the lowest concentration used (0.25 mg/kg). A reconstructed 2D-image of the entire lung section at high resolution (3.6 μm/pixel) has been performed from tissue density values allowing the visualization of their distribution throughout fibrotic and non-fibrotic regions. A significant correlation (p<0.0001) was found between automated analysis and the above standard evaluation methods. This correlation establishes automated analysis as a novel end-point measure of BLM-induced lung fibrosis in mice, which will be very valuable for future preclinical drug explorations.

Quantification of Pulmonary Fibrosis in a Bleomycin Mouse Model Using Automated Histological Image Analysis

PubMed Central

Gilhodes, Jean-Claude; Kreuz, Sebastian; Stierstorfer, Birgit; Stiller, Detlef; Wollin, Lutz

2017-01-01

Current literature on pulmonary fibrosis induced in animal models highlights the need of an accurate, reliable and reproducible histological quantitative analysis. One of the major limits of histological scoring concerns the fact that it is observer-dependent and consequently subject to variability, which may preclude comparative studies between different laboratories. To achieve a reliable and observer-independent quantification of lung fibrosis we developed an automated software histological image analysis performed from digital image of entire lung sections. This automated analysis was compared to standard evaluation methods with regard to its validation as an end-point measure of fibrosis. Lung fibrosis was induced in mice by intratracheal administration of bleomycin (BLM) at 0.25, 0.5, 0.75 and 1 mg/kg. A detailed characterization of BLM-induced fibrosis was performed 14 days after BLM administration using lung function testing, micro-computed tomography and Ashcroft scoring analysis. Quantification of fibrosis by automated analysis was assessed based on pulmonary tissue density measured from thousands of micro-tiles processed from digital images of entire lung sections. Prior to analysis, large bronchi and vessels were manually excluded from the original images. Measurement of fibrosis has been expressed by two indexes: the mean pulmonary tissue density and the high pulmonary tissue density frequency. We showed that tissue density indexes gave access to a very accurate and reliable quantification of morphological changes induced by BLM even for the lowest concentration used (0.25 mg/kg). A reconstructed 2D-image of the entire lung section at high resolution (3.6 μm/pixel) has been performed from tissue density values allowing the visualization of their distribution throughout fibrotic and non-fibrotic regions. A significant correlation (p<0.0001) was found between automated analysis and the above standard evaluation methods. This correlation establishes automated analysis as a novel end-point measure of BLM-induced lung fibrosis in mice, which will be very valuable for future preclinical drug explorations. PMID:28107543

Quantitative Assessment of Ultrastructure and Light Scatter in Mouse Corneal Debridement Wounds

PubMed Central

Boote, Craig; Du, Yiqin; Morgan, Sian; Harris, Jonathan; Kamma-Lorger, Christina S.; Hayes, Sally; Lathrop, Kira L.; Roh, Danny S.; Burrow, Michael K.; Hiller, Jennifer; Terrill, Nicholas J.; Funderburgh, James L.; Meek, Keith M.

2012-01-01

Purpose. The mouse has become an important wound healing model with which to study corneal fibrosis, a frequent complication of refractive surgery. The aim of the current study was to quantify changes in stromal ultrastructure and light scatter that characterize fibrosis in mouse corneal debridement wounds. Methods. Epithelial debridement wounds, with and without removal of basement membrane, were produced in C57BL/6 mice. Corneal opacity was measured using optical coherence tomography, and collagen diameter and matrix order were quantified by x-ray scattering. Electron microscopy was used to visualize proteoglycans. Quantitative PCR (Q-PCR) measured mRNA transcript levels for several quiescent and fibrotic markers. Results. Epithelial debridement without basement membrane disruption produced a significant increase in matrix disorder at 8 weeks, but minimal corneal opacity. In contrast, basement membrane penetration led to increases in light scatter, matrix disorder, and collagen diameter, accompanied by the appearance of abnormally large proteoglycans in the subepithelial stroma. This group also demonstrated upregulation of several quiescent and fibrotic markers 2 to 4 weeks after wounding. Conclusions. Fibrotic corneal wound healing in mice involves extensive changes to collagen and proteoglycan ultrastructure, consistent with deposition of opaque scar tissue. Epithelial basement membrane penetration is a deciding factor determining the degree of ultrastructural changes and resulting opacity. PMID:22467580

Longitudinal in vivo microcomputed tomography of mouse lungs: No evidence for radiotoxicity

PubMed Central

Vande Velde, Greetje; De Langhe, Ellen; Poelmans, Jennifer; Bruyndonckx, Peter; d'Agostino, Emiliano; Verbeken, Erik; Bogaerts, Ria; Himmelreich, Uwe

2015-01-01

Before microcomputed tomography (micro-CT) can be exploited to its full potential for longitudinal monitoring of transgenic and experimental mouse models of lung diseases, radiotoxic side effects such as inflammation or fibrosis must be considered. We evaluated dose and potential radiotoxicity to the lungs for long-term respiratory-gated high-resolution micro-CT protocols. Free-breathing C57Bl/6 mice underwent four different retrospectively respiratory gated micro-CT imaging schedules of repeated scans during 5 or 12 wk, followed by ex vivo micro-CT and detailed histological and biochemical assessment of lung damage. Radiation exposure, dose, and absorbed dose were determined by ionization chamber, thermoluminescent dosimeter measurements and Monte Carlo calculations. Despite the relatively large radiation dose delivered per micro-CT acquisition, mice did not show any signs of radiation-induced lung damage or fibrosis when scanned weekly during 5 and up to 12 wk. Doubling the scanning frequency and once tripling the radiation dose as to mimic the instant repetition of a failed scan also stayed without detectable toxicity after 5 wk of scanning. Histological analyses confirmed the absence of radiotoxic damage to the lungs, thereby demonstrating that long-term monitoring of mouse lungs using high-resolution micro-CT is safe. This opens perspectives for longitudinal monitoring of (transgenic) mouse models of lung diseases and therapeutic response on an individual basis with high spatial and temporal resolution, without concerns for radiation toxicity that could potentially influence the readout of micro-CT-derived lung biomarkers. This work further supports the introduction of micro-CT for routine use in the preclinical pulmonary research field where postmortem histological approaches are still the gold standard. PMID:26024893

Mechanical characterization of the mouse diaphragm with optical coherence elastography reveals fibrosis-related change of direction-dependent muscle tissue stiffness

NASA Astrophysics Data System (ADS)

Wang, Shang; Loehr, James A.; Larina, Irina V.; Rodney, George G.; Larin, Kirill V.

2016-03-01

The diaphragm, composed of skeletal muscle, plays an important role in respiration through its dynamic contraction. Genetic and molecular studies of the biomechanics of mouse diaphragm can provide great insights into an improved understanding and potential treatment of the disorders that lead to diaphragm dysfunction (i.e. muscular dystrophy). However, due to the small tissue size, mechanical assessment of mouse diaphragm tissue under its proper physiological conditions has been challenging. Here, we present the application of noncontact optical coherence elastography (OCE) for quantitative elastic characterization of ex vivo mouse diaphragm. Phase-sensitive optical coherence tomography was combined with a focused air-puff system to capture and measure the elastic wave propagation from tissue surface. Experiments were performed on wildtype and dystrophic mouse diaphragm tissues containing different levels of fibrosis. The OCE measurements of elastic wave propagation were conducted along both the longitudinal and transverse axis of the muscle fibers. Cross-correlation of the temporal displacement profiles from different spatial locations was utilized to obtain the propagation time delay, which was used to calculate the wave group velocity and to further quantify the tissue Young's modulus. Prior to and after OCE assessment, peak tetanic force was measured to monitor viability of the tissue during the elasticity measurements. Our experimental results indicate a positive correlation between fibrosis level and tissue stiffness, suggesting this elastic-wave-based OCE method could be a useful tool to monitor mechanical properties of skeletal muscle under physiological and pathological conditions.

Hepatic fibrosis and carcinogenesis in α1-antitrypsin deficiency: a prototype for chronic tissue damage in gain-of-function disorders.

PubMed

Perlmutter, David H; Silverman, Gary A

2011-03-01

In α1-antitrypsin (AT) deficiency, a point mutation renders a hepatic secretory glycoprotein prone to misfolding and polymerization. The mutant protein accumulates in the endoplasmic reticulum of liver cells and causes hepatic fibrosis and hepatocellular carcinoma by a gain-of-function mechanism. Genetic and/or environmental modifiers determine whether an affected homozygote is susceptible to hepatic fibrosis/carcinoma. Two types of proteostasis mechanisms for such modifiers have been postulated: variation in the function of intracellular degradative mechanisms and/or variation in the signal transduction pathways that are activated to protect the cell from protein mislocalization and/or aggregation. In recent studies we found that carbamazepine, a drug that has been used safely as an anticonvulsant and mood stabilizer, reduces the hepatic load of mutant AT and hepatic fibrosis in a mouse model by enhancing autophagic disposal of this mutant protein. These results provide evidence that pharmacological manipulation of endogenous proteostasis mechanisms is an appealing strategy for chemoprophylaxis in disorders involving gain-of-function mechanisms.

Understanding deregulated cellular and molecular dynamics in the haematopoietic stem cell niche to develop novel therapeutics for bone marrow fibrosis.

PubMed

Gleitz, Hélène Fe; Kramann, Rafael; Schneider, Rebekka K

2018-06-01

Bone marrow fibrosis is the continuous replacement of blood-forming cells in the bone marrow with excessive scar tissue, leading to failure of the body to produce blood cells and ultimately to death. Myofibroblasts are fibrosis-driving cells and are well characterized in solid organ fibrosis, but their role and cellular origin in bone marrow fibrosis have remained obscure. Recent work has demonstrated that Gli1 + and leptin receptor + mesenchymal stromal cells are progenitors of fibrosis-causing myofibroblasts in the bone marrow. Genetic ablation or pharmacological inhibition of Gli1 + mesenchymal stromal cells ameliorated fibrosis in mouse models of myelofibrosis. Conditional deletion of the platelet-derived growth factor (PDGF) receptor-α (PDGFRA) gene (Pdgfra) and inhibition of PDGFRA by imatinib in leptin receptor + stromal cells suppressed their expansion and ameliorated bone marrow fibrosis. Understanding the cellular and molecular mechanisms in the haematopoietic stem cell niche that govern the mesenchymal stromal cell-to-myofibroblast transition and myofibroblast expansion will be critical to understand the pathogenesis of bone marrow fibrosis in both malignant and non-malignant conditions, and will guide the development of novel therapeutics. In this review, we summarize recent discoveries of mesenchymal stromal cells as part of the haematopoietic niche and as myofibroblast precursors, and discuss potential therapeutic strategies in the specific targeting of fibrotic transformation in bone marrow fibrosis. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Regulation of renal fibrosis by Smad3 Thr388 phosphorylation.

PubMed

Qu, Xinli; Li, Xueling; Zheng, Yaowu; Ren, Yi; Puelles, Victor G; Caruana, Georgina; Nikolic-Paterson, David J; Li, Jinhua

2014-04-01

Transforming growth factor-β (TGF-β) promotes tissue fibrosis via receptor-mediated phosphorylation of the receptor-activated Smad2/3, together with Smad4. Of these, Smad3 plays a major profibrotic role in mouse models of tissue fibrosis. Transcriptional activity of the Smad3 protein is regulated by phosphorylation of residues in the C-terminal domain and the linker region. Herein, we examined the role of a novel phosphorylation site within the MH2 domain (T388) in the regulation of Smad3 activity. Confocal microscopy using an Smad3 phosphorylated T388-specific antibody identified phosphorylation of Smad3 T388 in myofibroblasts and tubular epithelial cells in human focal and segmental glomerulosclerosis and mouse models of unilateral ureteric obstruction and diabetic nephropathy, whereas phosphorylated T388 was largely absent in normal kidney. In vitro, TGF-β1 induced phosphorylation of Smad3 T388 in a biphasic pattern. A point mutation of T388/V in an Smad3 construct demonstrated that phosphorylation of T388 promotes Smad3 binding to Smad4 and CDK8, but was not necessary for nuclear translocation. Furthermore, T388 phosphorylation was required for TGF-β-induced collagen I gene promoter activity and extracellular matrix production in cultured fibroblasts. In conclusion, our study identifies phosphorylation of T388 in the Smad3 MH2 domain as an important mechanism that regulates the profibrotic TGF-β/Smad3 signaling pathway, which has direct relevance to human and experimental fibrotic kidney disease. Copyright © 2014. Published by Elsevier Inc.

(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.

BRD4 mediates NF-κB-dependent epithelial-mesenchymal transition and pulmonary fibrosis via transcriptional elongation

PubMed Central

Zhao, Yingxin; Sun, Hong; Zhang, Yueqing; Yang, Jun; Brasier, Allan R.

2016-01-01

Chronic epithelial injury triggers a TGF-β-mediated cellular transition from normal epithelium into a mesenchymal-like state that produces subepithelial fibrosis and airway remodeling. Here we examined how TGF-β induces the mesenchymal cell state and determined its mechanism. We observed that TGF-β stimulation activates an inflammatory gene program controlled by the NF-κB/RelA signaling pathway. In the mesenchymal state, NF-κB-dependent immediate-early genes accumulate euchromatin marks and processive RNA polymerase. This program of immediate-early genes is activated by enhanced expression, nuclear translocation, and activating phosphorylation of the NF-κB/RelA transcription factor on Ser276, mediated by a paracrine signal. Phospho-Ser276 RelA binds to the BRD4/CDK9 transcriptional elongation complex, activating the paused RNA Pol II by phosphorylation on Ser2 in its carboxy-terminal domain. RelA-initiated transcriptional elongation is required for expression of the core epithelial-mesenchymal transition transcriptional regulators SNAI1, TWIST1, and ZEB1 and mesenchymal genes. Finally, we observed that pharmacological inhibition of BRD4 can attenuate experimental lung fibrosis induced by repetitive TGF-β challenge in a mouse model. These data provide a detailed mechanism for how activated NF-κB and BRD4 control epithelial-mesenchymal transition initiation and transcriptional elongation in model airway epithelial cells in vitro and in a murine pulmonary fibrosis model in vivo. Our data validate BRD4 as an in vivo target for the treatment of pulmonary fibrosis associated with inflammation-coupled remodeling in chronic lung diseases. PMID:27793799

A pneumocyte-macrophage paracrine lipid axis drives the lung toward fibrosis.

PubMed

Romero, Freddy; Shah, Dilip; Duong, Michelle; Penn, Raymond B; Fessler, Michael B; Madenspacher, Jennifer; Stafstrom, William; Kavuru, Mani; Lu, Bo; Kallen, Caleb B; Walsh, Kenneth; Summer, Ross

2015-07-01

Lipid-laden macrophages, or "foam cells," are observed in the lungs of patients with fibrotic lung disease, but their contribution to disease pathogenesis remains unexplored. Here, we demonstrate that fibrosis induced by bleomycin, silica dust, or thoracic radiation promotes early and sustained accumulation of foam cells in the lung. In the bleomycin model, we show that foam cells arise from neighboring alveolar epithelial type II cells, which respond to injury by dumping lipids into the distal airspaces of the lungs. We demonstrate that oxidized phospholipids accumulate within alveolar macrophages (AMs) after bleomycin injury and that murine and human AMs treated with oxidized phosphatidylcholine (oxPc) become polarized along an M2 phenotype and display enhanced production of transforming growth factor-β1. The direct instillation of oxPc into the mouse lung induces foam cell formation and triggers a severe fibrotic reaction. Further, we show that reducing pulmonary lipid clearance by targeted deletion of the lipid efflux transporter ATP-binding cassette subfamily G member 1 increases foam cell formation and worsens lung fibrosis after bleomycin. Conversely, we found that treatment with granulocyte-macrophage colony-stimulating factor attenuates fibrotic responses, at least in part through its ability to decrease AM lipid accumulation. In summary, this work describes a novel mechanism leading to foam cell formation in the mouse lung and suggests that strategies aimed at blocking foam cell formation might be effective for treating fibrotic lung disorders.

No overt structural or functional changes associated with PEG-coated gold nanoparticles accumulation with acute exposure in the mouse heart.

PubMed

Yang, Chengzhi; Yang, Hui; Wu, Jimin; Meng, Zenghui; Xing, Rui; Tian, Aiju; Tian, Xin; Guo, Lijun; Zhang, Youyi; Nie, Guangjun; Li, Zijian

2013-10-24

In this study, we investigated the cardiac biodistribution of polyethylene glycol (PEG)-coated AuNPs and their effects on cardiac function, structure and inflammation in both normal and cardiac remodeling mice. The model of cardiac remodeling was induced by subcutaneously injection of isoproterenol (ISO), a non-selective beta-adrenergic agonist, for 7 days. After AuNPs were injected intravenously in mice for 7 consecutive days, Au content in different organs was determined quantitatively by inductively coupled plasma mass spectrometry (ICP-MS), cardiac function and structure were measured by echocardiography, cardiac fibrosis was examined with picrosirius red staining, the morphology of cardiomyocytes was observed with hematoxylin and eosin (H & E) staining. The accumulation of AuNPs in hearts did not affect cardiac function or induce cardiac hypertrophy, cardiac fibrosis and cardiac inflammation under normal physiological condition. Cardiac AuNPs content was 6-fold higher in the cardiac remodeling mouse than normal mice. However, the increased accumulation of AuNPs in the heart did not aggravate ISO-induced cardiac hypertrophy, cardiac fibrosis or cardiac inflammation. These observations suggest that PEG-coated AuNPs possess excellent biocompatibility under both physiological and pathological conditions. Thus, AuNPs may be safe for cardiac patients and hold great promise for further development for various biomedical applications. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Standard operating procedures in experimental liver research: thioacetamide model in mice and rats.

PubMed

Wallace, M C; Hamesch, K; Lunova, M; Kim, Y; Weiskirchen, R; Strnad, P; Friedman, S L

2015-04-01

In addition to carbon tetrachloride (CCl4), thioacetamide (TAA) represents a second widely used model for the induction of experimental liver fibrosis, but can also be employed for the development of acute liver failure and liver tumours. While TAA itself is not hepatotoxic, its reactive metabolites covalently bind to proteins and lipids thereby causing oxidative stress and centrilobular necrosis. Compared with CCl4, TAA leads to more periportal infiltrates and more pronounced ductal proliferation. While TAA has been shown to induce liver fibrosis development in several different mouse strains, wide variations in the administration routes, doses and treatment durations have been reported. Therefore, an adoption of a universal standard operating procedure for the administration of TAA is urgently needed. For that purpose, we are presenting here two TAA models (intraperitoneal administration of 150 mg/kg of TAA three times per week for 11 weeks in rats, and TAA administration in drinking water at 300 mg/L for 2-4 months in mice) with which we have had success in reliably and reproducibly developing chronic liver injury and fibrosis. © The Author(s) 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Plasma Gelsolin Induced Glomerular Fibrosis via the TGF-β1/Smads Signal Transduction Pathway in IgA Nephropathy

PubMed Central

Zhang, Lei; Han, Changsong; Ye, Fei; He, Yan; Jin, Yinji; Wang, Tianzhen; Wu, Yiqi; Jiang, Yang; Zhang, Fengmin; Jin, Xiaoming

2017-01-01

Glomerular fibrosis has been shown to be closely related to the progression and prognosis of IgA nephropathy (IgAN). However, mechanism underlying IgAN glomerular fibrosis remains unclear. Recently, our study showed that plasma gelsolin (pGSN) was decreased in the serum of an IgAN mouse model and that pGSN deposition was found in the glomeruli. Another cytokine, TGF-β1, which is closely related to glomerular fibrosis, was also found to be highly expressed in the glomeruli. In the present study, we report that pGSN induces glomerular fibrosis through the TGF-β1/Smads signal transduction pathway. This is supported by the following findings: human mesangial cells (HMCs) show remarkable morphological changes and proliferation in response to co-stimulation with pGSN and polymeric IgA1 (pIgA1) from IgAN patients compared to other controls. Moreover, ELISA assays showed that more TGF-β1 secretion was found in HMCs supernatants in the co-stimulation group. Further experiments showed increased TGF-β1, Smad3, p-Smad2/3, Smad4, and collagen 1 and decreased Smad7 expression in the co-stimulation group. Our present study implied that the synergistic effect of pGSN and pIgA induced glomerular fibrosis via the TGF-β1/Smads signal transduction pathway. This might be a potential mechanism for the glomerular fibrosis observed in IgAN patients. PMID:28208683

Immune-Regulatory Molecule CD69 Controls Peritoneal Fibrosis

PubMed Central

Liappas, Georgios; González-Mateo, Guadalupe Tirma; Sánchez-Díaz, Raquel; Lazcano, Juan José; Lasarte, Sandra; Matesanz-Marín, Adela; Zur, Rafal; Ferrantelli, Evelina; Ramírez, Laura García; Aguilera, Abelardo; Fernández-Ruiz, Elena; Beelen, Robert H.J.; Selgas, Rafael; Sánchez-Madrid, Francisco

2016-01-01

Patients with ESRD undergoing peritoneal dialysis develop progressive peritoneal fibrosis, which may lead to technique failure. Recent data point to Th17-mediated inflammation as a key contributor in peritoneal damage. The leukocyte antigen CD69 modulates the setting and progression of autoimmune and inflammatory diseases by controlling the balance between Th17 and regulatory T cells (Tregs). However, the relevance of CD69 in tissue fibrosis remains largely unknown. Thus, we explored the role of CD69 in fibroproliferative responses using a mouse model of peritoneal fibrosis induced by dialysis fluid exposure under either normal or uremic status. We found that cd69−/− mice compared with wild-type (WT) mice showed enhanced fibrosis, mesothelial to mesenchymal transition, IL-17 production, and Th17 cell infiltration in response to dialysis fluid treatment. Uremia contributed partially to peritoneal inflammatory and fibrotic responses. Additionally, antibody–mediated CD69 blockade in WT mice mimicked the fibrotic response of cd69−/− mice. Finally, IL-17 blockade in cd69−/− mice decreased peritoneal fibrosis to the WT levels, and mixed bone marrow from cd69−/− and Rag2−/−γc−/− mice transplanted into WT mice reproduced the severity of the response to dialysis fluid observed in cd69−/− mice, showing that CD69 exerts its regulatory function within the lymphocyte compartment. Overall, our results indicate that CD69 controls tissue fibrosis by regulating Th17-mediated inflammation. PMID:27151919

Amelioration of radiation-induced pulmonary fibrosis by a water-soluble bifunctional sulfoxide radiation mitigator (MMS350).

PubMed

Kalash, Ronny; Epperly, Michael W; Goff, Julie; Dixon, Tracy; Sprachman, Melissa M; Zhang, Xichen; Shields, Donna; Cao, Shaonan; Franicola, Darcy; Wipf, Peter; Berhane, Hebist; Wang, Hong; Au, Jeremiah; Greenberger, Joel S

2013-11-01

A water-soluble ionizing radiation mitigator would have considerable advantages for the management of acute and chronic effects of ionizing radiation. We report that a novel oxetanyl sulfoxide (MMS350) is effective both as a protector and a mitigator of clonal mouse bone marrow stromal cell lines in vitro, and is an effective in vivo mitigator when administered 24 h after 9.5 Gy (LD100/30) total-body irradiation of C57BL/6NHsd mice, significantly improving survival (P = 0.0097). Furthermore, MMS350 (400 μM) added weekly to drinking water after 20 Gy thoracic irradiation significantly decreased: expression of pulmonary inflammatory and profibrotic gene transcripts and proteins; migration into the lungs of bone marrow origin luciferase+/GFP+ (luc+/GFP+) fibroblast progenitors (in both luc+ marrow chimeric and luc+ stromal cell line injected mouse models) and decreased radiation-induced pulmonary fibrosis (P < 0.0001). This nontoxic and orally administered small molecule may be an effective therapeutic in clinical radiotherapy and as a counter measure against the acute and chronic effects of ionizing radiation.

Amelioration of Radiation-Induced Pulmonary Fibrosis by a Water-Soluble Bifunctional Sulfoxide Radiation Mitigator (MMS350)

PubMed Central

Kalash, Ronny; Epperly, Michael W.; Goff, Julie; Dixon, Tracy; Sprachman, Melissa M.; Zhang, Xichen; Shields, Donna; Cao, Shaonan; Franicola, Darcy; Wipf, Peter; Berhane, Hebist; Wang, Hong; Au, Jeremiah; Greenberger, Joel S.

2014-01-01

A water-soluble ionizing radiation mitigator would have considerable advantages for the management of acute and chronic effects of ionizing radiation. We report that a novel oxetanyl sulfoxide (MMS350) is effective both as a protector and a mitigator of clonal mouse bone marrow stromal cell lines in vitro, and is an effective in vivo mitigator when administered 24 h after 9.5 Gy (LD100/30) total-body irradiation of C57BL/6NHsd mice, significantly improving survival (P =0.0097). Furthermore, MMS350 (400 μM) added weekly to drinking water after 20 Gy thoracic irradiation significantly decreased: expression of pulmonary inflammatory and profibrotic gene transcripts and proteins; migration into the lungs of bone marrow origin luciferase+/GFP+ (luc+/GFP+) fibroblast progenitors (in both luc+ marrow chimeric and luc+ stromal cell line injected mouse models) and decreased radiation-induced pulmonary fibrosis (P < 0.0001). This nontoxic and orally administered small molecule may be an effective therapeutic in clinical radiotherapy and as a counter measure against the acute and chronic effects of ionizing radiation. PMID:24125487

TGF-β and TGF-β/Smad signaling in the interactions between Echinococcus multilocularis and its hosts.

PubMed

Wang, Junhua; Zhang, Chuanshan; Wei, Xufa; Blagosklonov, Oleg; Lv, Guodong; Lu, Xiaomei; Mantion, Georges; Vuitton, Dominique A; Wen, Hao; Lin, Renyong

2013-01-01

Alveolar echinococcosis (AE) is characterized by the development of irreversible fibrosis and of immune tolerance towards Echinococcus multilocularis (E. multilocularis). Very little is known on the presence of transforming growth factor-β (TGF-β) and other components of TGF-β/Smad pathway in the liver, and on their possible influence on fibrosis, over the various stages of infection. Using Western Blot, qRT-PCR and immunohistochemistry, we measured the levels of TGF-β1, TGF-β receptors, and down-stream Smads activation, as well as fibrosis marker expression in both a murine AE model from day 2 to 360 post-infection (p.i.) and in AE patients. TGF-β1, its receptors, and down-stream Smads were markedly expressed in the periparasitic infiltrate and also in the hepatocytes, close to and distant from AE lesions. Fibrosis was significant at 180 days p.i. in the periparasitic infiltrate and was also present in the liver parenchyma, even distant from the lesions. Over the time course after infection TGF-β1 expression was correlated with CD4/CD8 T-cell ratio long described as a hallmark of AE severity. The time course of the various actors of the TGF-β/Smad system in the in vivo mouse model as well as down-regulation of Smad7 in liver areas close to the lesions in human cases highly suggest that TGF-β plays an important role in AE both in immune tolerance against the parasite and in liver fibrosis.

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.

Elevated Incidence of Dental Caries in a Mouse Model of Cystic Fibrosis

PubMed Central

Catalán, Marcelo A.; Scott-Anne, Kathleen; Klein, Marlise I.; Koo, Hyun; Bowen, William H.; Melvin, James E.

2011-01-01

Background Dental caries is the single most prevalent and costly infectious disease worldwide, affecting more than 90% of the population in the U.S. The development of dental cavities requires the colonization of the tooth surface by acid-producing bacteria, such as Streptococcus mutans. Saliva bicarbonate constitutes the main buffering system which neutralizes the pH fall generated by the plaque bacteria during sugar metabolism. We found that the saliva pH is severely decreased in a mouse model of cystic fibrosis disease (CF). Given the close relationship between pH and caries development, we hypothesized that caries incidence might be elevated in the mouse CF model. Methodology/Principal Findings We induced carious lesions in CF and wildtype mice by infecting their oral cavity with S. mutans, a well-studied cariogenic bacterium. After infection, the mice were fed a high-sucrose diet for 5 weeks (diet 2000). The mice were then euthanized and their jaws removed for caries scoring and bacterial counting. A dramatic increase in caries and severity of lesions scores were apparent in CF mice compared to their wildtype littermates. The elevated incidence of carious lesions correlated with a striking increase in the S. mutans viable population in dental plaque (20-fold increase in CF vs. wildtype mice; p value<0.003; t test). We also found that the pilocarpine-stimulated saliva bicarbonate concentration was significantly reduced in CF mice (16±2 mM vs. 31±2 mM, CF and wildtype mice, respectively; p value<0.01; t test). Conclusions/Significance Considering that bicarbonate is the most important pH buffering system in saliva, and the adherence and survival of aciduric bacteria such as S. mutans are enhanced at low pH values, we speculate that the decrease in the bicarbonate content and pH buffering of the saliva is at least partially responsible for the increased severity of lesions observed in the CF mouse. PMID:21304986

Heparanase and macrophage interplay in the onset of liver fibrosis.

PubMed

Secchi, Maria Francesca; Crescenzi, Marika; Masola, Valentina; Russo, Francesco Paolo; Floreani, Annarosa; Onisto, Maurizio

2017-11-02

The heparan sulfate endoglycosidase heparanase (HPSE) is involved in tumor growth, chronic inflammation and fibrosis. Since a role for HPSE in chronic liver disease has not been demonstrated to date, the current study was aimed at investigating the involvement of HPSE in the pathogenesis of chronic liver injury. Herein, we revealed that HPSE expression increased in mouse livers after carbon tetrachloride (CCl 4 )-mediated chronic induction of fibrosis, but with a trend to decline during progression of the disease. In mouse fibrotic liver tissues HPSE immunostaining was restricted in necro-inflammatory areas, co-localizing with F4/80 macrophage marker and TNF-α. TNF-α treatment induced HPSE expression as well as HPSE secretion in U937 macrophages. Moreover, macrophage-secreted HPSE regulated the expression of α-SMA and fibronectin in hepatic stellate LX-2 cells. Finally, HPSE activity increased in the plasma of patients with liver fibrosis but it inversely correlated with liver stiffness. Our results suggest the involvement of HPSE in early phases of reaction to liver damage and inflammatory macrophages as an important source of HPSE. HPSE seems to play a key role in the macrophage-mediated activation of hepatic stellate cells (HSCs), thus suggesting that HPSE targeting could be a new therapeutic option in the treatment of liver fibrosis.

Inducible nitric oxide synthase expression is reduced in cystic fibrosis murine and human airway epithelial cells.

PubMed Central

Kelley, T J; Drumm, M L

1998-01-01

It has been reported that exhaled nitric oxide levels are reduced in cystic fibrosis (CF) patients. We have examined the inducible isoform of nitric oxide synthase (iNOS) in the airways by immunostaining and found that iNOS is constitutively expressed in the airway epithelia of non-CF mouse and human tissues but essentially absent in the epithelium of CF airways. We explored potential consequences of lost iNOS expression and found that iNOS inhibition significantly increases mouse nasal trans-epithelial potential difference, and hindered the ability of excised mouse lungs to prevent growth of Pseudomonas aeruginosa. The absence of continuous nitric oxide production in epithelial cells of CF airways may play a role in two CF-associated characteristics: hyperabsorption of sodium and susceptibility to bacterial infections. PMID:9739054

Single high-dose irradiation aggravates eosinophil-mediated fibrosis through IL-33 secreted from impaired vessels in the skin compared to fractionated irradiation

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

Lee, Eun-Jung, E-mail: forejs2@yuhs.ac; Kim, Jun Won, E-mail: JUNWON@yuhs.ac; Yoo, Hyun, E-mail: gochunghee@yuhs.ac

We have revealed in a porcine skin injury model that eosinophil recruitment was dose-dependently enhanced by a single high-dose irradiation. In this study, we investigated the underlying mechanism of eosinophil-associated skin fibrosis and the effect of high-dose-per-fraction radiation. The dorsal skin of a mini-pig was divided into two sections containing 4-cm{sup 2} fields that were irradiated with 30 Gy in a single fraction or 5 fractions and biopsied regularly over 14 weeks. Eosinophil-related Th2 cytokines such as interleukin (IL)-4, IL-5, and C–C motif chemokine-11 (CCL11/eotaxin) were evaluated by quantitative real-time PCR. RNA-sequencing using 30 Gy-irradiated mouse skin and functional assays in amore » co-culture system of THP-1 and irradiated-human umbilical vein endothelial cells (HUVECs) were performed to investigate the mechanism of eosinophil-mediated radiation fibrosis. Single high-dose-per-fraction irradiation caused pronounced eosinophil accumulation, increased profibrotic factors collagen and transforming growth factor-β, enhanced production of eosinophil-related cytokines including IL-4, IL-5, CCL11, IL-13, and IL-33, and reduced vessels compared with 5-fraction irradiation. IL-33 notably increased in pig and mouse skin vessels after single high-dose irradiation of 30 Gy, as well as in irradiated HUVECs following 12 Gy. Blocking IL-33 suppressed the migration ability of THP-1 cells and cytokine secretion in a co-culture system of THP-1 cells and irradiated HUVECs. Hence, high-dose-per-fraction irradiation appears to enhance eosinophil-mediated fibrotic responses, and IL-33 may be a key molecule operating in eosinophil-mediated fibrosis in high-dose-per fraction irradiated skin. - Highlights: • Single high-dose irradiation aggravates eosinophil-mediated fibrosis through IL-33. • Vascular endothelial cells damaged by high-dose radiation secrete IL-33. • Blocking IL-33 suppressed migration of inflammatory cells and cytokine secretion. • IL-33 is a key in eosinophil-mediated fibrosis in high-dose-per-fraction radiation.« less

Upregulation of distinct collagen transcripts in post-surgery scar tissue: a study of conjunctival fibrosis.

PubMed

Seet, Li-Fong; Toh, Li Zhen; Chu, Stephanie W L; Finger, Sharon N; Chua, Jocelyn L L; Wong, Tina T

2017-06-01

Excessive accumulation of collagen is often used to assess the development of fibrosis. This study aims to identify collagen genes that define fibrosis in the conjunctiva following glaucoma filtration surgery (GFS). Using the mouse model of GFS, we have identified collagen transcripts that were upregulated in the fibrotic phase of wound healing via RNA-seq. The collagen transcripts that were increased the most were encoded by Col8a1 , Col11a1 and Col8a2 Further analysis of the Col8a1 , Col11a1 and Col8a2 transcripts revealed their increase by 67-, 54- and 18-fold, respectively, in the fibrotic phase, compared with 12-fold for Col1a1 , the most commonly evaluated collagen gene for fibrosis. However, only type I collagen was significantly upregulated at the protein level in the fibrotic phase. Type VIII and type I collagens colocalized in fibrous structures and in ACTA2-positive pericytes, and appeared to compensate for each other in expression levels. Type XI collagen showed low colocalization with both type VIII and type I collagens but can be found in association with macrophages. Furthermore, we show that both mouse and human conjunctival fibroblasts expressed elevated levels of the most highly expressed collagen genes in response to TGFβ2 treatment. Importantly, conjunctival tissues from individuals whose GF surgeries have failed due to scarring showed 3.60- and 2.78-fold increases in type VIII and I collagen transcripts, respectively, compared with those from individuals with no prior surgeries. These data demonstrate that distinct collagen transcripts are expressed at high levels in the conjunctiva after surgery and their unique expression profiles may imply differential influences on the fibrotic outcome. © 2017. Published by The Company of Biologists Ltd.

Corona-directed nucleic acid delivery into hepatic stellate cells for liver fibrosis therapy.

PubMed

Zhang, Zhengping; Wang, Chunming; Zha, Yinhe; Hu, Wei; Gao, Zhongfei; Zang, Yuhui; Chen, Jiangning; Zhang, Junfeng; Dong, Lei

2015-03-24

Strategies to modify nanoparticles with biological ligands for targeted drug delivery in vivo have been widely studied but met with limited clinical success. A possible reason is that, in the blood circulation, serum proteins could rapidly form a layer of protein "corona" on the vehicle surface, which might block the modified ligands and hamper their targeting functions. We speculate that strategies for drug delivery can be designed based upon elegant control of the corona formation on the vehicle surfaces. In this study, we demonstrate a retinol-conjugated polyetherimine (RcP) nanoparticle system that selectively recruited the retinol binding protein 4 (RBP) in its corona components. RBP was found to bind retinol, and direct the antisense oligonucleotide (ASO)-laden RcP carrier to hepatic stellate cells (HSC), which play essential roles in the progression of hepatic fibrosis. In both mouse fibrosis models, induced by carbon tetrachloride (CCl4) and bile duct ligation (BDL), respectively, the ASO-laden RcP particles effectively suppressed the expression of type I collagen (collagen I), and consequently ameliorated hepatic fibrosis. Such findings suggest that this delivery system, designed to exploit the power of corona proteins, can serve as a promising tool for targeted delivery of therapeutic agents for the treatment of hepatic fibrosis.

Hepatic stellate cell-specific deletion of SIRT1 exacerbates liver fibrosis in mice.

PubMed

Li, Min; Hong, Wenxuan; Hao, Chenzhi; Li, Luyang; Xu, Huihui; Li, Ping; Xu, Yong

2017-12-01

Liver fibrosis is widely perceived as a host defense mechanism that aids tissue repair following liver injury. Excessive fibrogenesis, however, serves to disrupt normal liver structure and precedes such irrevocable human pathologies as cirrhosis and hepatocellular carcinoma. Activation of hepatic stellate cells (HSCs) is a hallmark event during liver fibrosis. In the present study we investigated the mechanism by which the lysine deacetylase SIRT1 regulates HSC activation. We report here that SIRT1 levels were decreased in the liver in different mouse models and in cultured HSCs undergoing activation. SIRT1 down-regulation paralleled HDAC4 up-regulation. HDAC4 was recruited to the SIRT1 promoter during HSC activation and removed acetylated histones H3 and H4 from the SIRT1 promoter leading to SIRT1 trans-repression. HDAC4 silencing restored SIRT1 expression and attenuated HSC activation in SIRT1-dependent manner. More important, selective deletion of SIRT1 in HSCs exacerbated CCl 4 -induced liver fibrosis in mice. Mechanistically, SIRT1 deacetylated PPARγ to block HSC activation. Together, our data reveal an HDAC4-SIRT1-PPARγ axis that contributes to the regulation of HSC activation and liver fibrosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Life or death by NFκB, Losartan promotes survival in dy2J/dy2J mouse of MDC1A

PubMed Central

Elbaz, M; Yanay, N; Laban, S; Rabie, M; Mitrani-Rosenbaum, S; Nevo, Y

2015-01-01

Inflammation and fibrosis are well-defined mechanisms involved in the pathogenesis of the incurable Laminin α2-deficient congenital muscular dystrophy (MDC1A), while apoptosis mechanism is barely discussed. Our previous study showed treatment with Losartan, an angiotensin II type I receptor antagonist, improved muscle strength and reduced fibrosis through transforming growth factor beta (TGF-β) and mitogen-activated protein kinases (MAPK) signaling inhibition in the dy2J/dy2J mouse model of MDC1A. Here we show for the first time that Losartan treatment up-regulates and shifts the nuclear factor kappa B (NFκB) signaling pathway to favor survival versus apoptosis/damage in this animal model. Losartan treatment was associated with significantly increased serum tumor necrosis factor alpha (TNF-α) level, p65 nuclei accumulation, and decreased muscle IκB-β protein level, indicating NFκB activation. Moreover, NFκB anti-apoptotic target genes TNF receptor-associated factor 1 (TRAF1), TNF receptor-associated factor 2 (TRAF2), cellular inhibitor of apoptosis (cIAP2), and Ferritin heavy chain (FTH1) were increased following Losartan treatment. Losartan induced protein expression toward a pro-survival profile as BCL-2 expression levels were increased and Caspase-3 expression levels were decreased. Muscle apoptosis reduction was further confirmed using terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay. Thus, along with TGF-β and MAPK signaling, NFκB serves as an important regulatory pathway which following Losartan treatment promotes survival in the dy2J/dy2J mouse model of MDC1A. PMID:25766329

Ferret and Pig Models of Cystic Fibrosis: Prospects and Promise for Gene Therapy

PubMed Central

Yan, Ziying; Stewart, Zoe A.; Sinn, Patrick L.; Olsen, John C.; Hu, Jim; McCray, Paul B.

2015-01-01

Abstract Large animal models of genetic diseases are rapidly becoming integral to biomedical research as technologies to manipulate the mammalian genome improve. The creation of cystic fibrosis (CF) ferrets and pigs is an example of such progress in animal modeling, with the disease phenotypes in the ferret and pig models more reflective of human CF disease than mouse models. The ferret and pig CF models also provide unique opportunities to develop and assess the effectiveness of gene and cell therapies to treat affected organs. In this review, we examine the organ disease phenotypes in these new CF models and the opportunities to test gene therapies at various stages of disease progression in affected organs. We then discuss the progress in developing recombinant replication-defective adenoviral, adeno-associated viral, and lentiviral vectors to target genes to the lung and pancreas in ferrets and pigs, the two most affected organs in CF. Through this review, we hope to convey the potential of these new animal models for developing CF gene and cell therapies. PMID:25675143

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.

Systems biology combining human- and animal-data miRNA and mRNA data identifies new targets in ureteropelvic junction obstruction.

PubMed

Papadopoulos, Theofilos; Casemayou, Audrey; Neau, Eric; Breuil, Benjamin; Caubet, Cécile; Calise, Denis; Thornhill, Barbara A; Bachvarova, Magdalena; Belliere, Julie; Chevalier, Robert L; Moulos, Panagiotis; Bachvarov, Dimcho; Buffin-Meyer, Benedicte; Decramer, Stéphane; Auriol, Françoise Conte; Bascands, Jean-Loup; Schanstra, Joost P; Klein, Julie

2017-03-01

Although renal fibrosis and inflammation have shown to be involved in the pathophysiology of obstructive nephropathies, molecular mechanisms underlying evolution of these processes remain undetermined. In an attempt towards improved understanding of obstructive nephropathy and improved translatability of the results to clinical practice we have developed a systems biology approach combining omics data of both human and mouse obstructive nephropathy. We have studied in parallel the urinary miRNome of infants with ureteropelvic junction obstruction and the kidney tissue miRNome and transcriptome of the corresponding neonatal partial unilateral ureteral obstruction (UUO) mouse model. Several hundreds of miRNAs and mRNAs displayed changed abundance during disease. Combination of miRNAs in both species and associated mRNAs let to the prioritization of five miRNAs and 35 mRNAs associated to disease. In vitro and in vivo validation identified consistent dysregulation of let-7a-5p and miR-29-3p and new potential targets, E3 ubiquitin-protein ligase (DTX4) and neuron navigator 1 (NAV1), potentially involved in fibrotic processes, in obstructive nephropathy in both human and mice that would not be identified otherwise. Our study is the first to correlate a mouse model of neonatal partial UUO with human UPJ obstruction in a comprehensive systems biology analysis. Our data revealed let-7a and miR-29b as molecules potentially involved in the development of fibrosis in UPJ obstruction via the control of DTX4 in both man and mice that would not be identified otherwise.

Preventing tissue fibrosis by local biomaterials interfacing of specific cryptic extracellular matrix information

PubMed Central

Horejs, Christine-Maria; St-Pierre, Jean-Philippe; Ojala, Juha R. M.; Steele, Joseph A. M.; da Silva, Patricia Barros; Rynne-Vidal, Angela; Maynard, Stephanie A.; Hansel, Catherine S.; Rodríguez-Fernández, Clara; Mazo, Manuel M.; You, Amanda Y. F.; Wang, Alex J.; von Erlach, Thomas; Tryggvason, Karl; López-Cabrera, Manuel; Stevens, Molly M.

2017-01-01

Matrix metalloproteinases (MMPs) contribute to the breakdown of tissue structures such as the basement membrane, promoting tissue fibrosis. Here we developed an electrospun membrane biofunctionalized with a fragment of the laminin β1-chain to modulate the expression of MMP2 in this context. We demonstrate that interfacing of the β1-fragment with the mesothelium of the peritoneal membrane via a biomaterial abrogates the release of active MMP2 in response to transforming growth factor β1 and rescues tissue integrity ex vivo and in vivo in a mouse model of peritoneal fibrosis. Importantly, our data demonstrate that the membrane inhibits MMP2 expression. Changes in the expression of epithelial-to-mesenchymal transition (EMT)-related molecules further point towards a contribution of the modulation of EMT. Biomaterial-based presentation of regulatory basement membrane signals directly addresses limitations of current therapeutic approaches by enabling a localized and specific method to counteract MMP2 release applicable to a broad range of therapeutic targets. PMID:28593951

Identification of periplakin as a major regulator of lung injury and repair in mice

PubMed Central

Besnard, Valérie; Dagher, Rania; Madjer, Tania; Joannes, Audrey; Jaillet, Madeleine; Kolb, Martin; Bonniaud, Philippe; Murray, Lynne A.; Sleeman, Matthew A.

2018-01-01

Periplakin is a component of the desmosomes that acts as a cytolinker between intermediate filament scaffolding and the desmosomal plaque. Periplakin is strongly expressed by epithelial cells in the lung and is a target antigen for autoimmunity in idiopathic pulmonary fibrosis. The aim of this study was to determine the role of periplakin during lung injury and remodeling in a mouse model of lung fibrosis induced by bleomycin. We found that periplakin expression was downregulated in the whole lung and in alveolar epithelial cells following bleomycin-induced injury. Deletion of the Ppl gene in mice improved survival and reduced lung fibrosis development after bleomycin-induced injury. Notably, Ppl deletion promoted an antiinflammatory alveolar environment linked to profound changes in type 2 alveolar epithelial cells, including overexpression of antiinflammatory cytokines, decreased expression of profibrotic mediators, and altered cell signaling with a reduced response to TGF-β1. These results identify periplakin as a previously unidentified regulator of the response to injury in the lung. PMID:29515024

Preventing tissue fibrosis by local biomaterials interfacing of specific cryptic extracellular matrix information

NASA Astrophysics Data System (ADS)

Horejs, Christine-Maria; St-Pierre, Jean-Philippe; Ojala, Juha R. M.; Steele, Joseph A. M.; da Silva, Patricia Barros; Rynne-Vidal, Angela; Maynard, Stephanie A.; Hansel, Catherine S.; Rodríguez-Fernández, Clara; Mazo, Manuel M.; You, Amanda Y. F.; Wang, Alex J.; von Erlach, Thomas; Tryggvason, Karl; López-Cabrera, Manuel; Stevens, Molly M.

2017-06-01

Matrix metalloproteinases (MMPs) contribute to the breakdown of tissue structures such as the basement membrane, promoting tissue fibrosis. Here we developed an electrospun membrane biofunctionalized with a fragment of the laminin β1-chain to modulate the expression of MMP2 in this context. We demonstrate that interfacing of the β1-fragment with the mesothelium of the peritoneal membrane via a biomaterial abrogates the release of active MMP2 in response to transforming growth factor β1 and rescues tissue integrity ex vivo and in vivo in a mouse model of peritoneal fibrosis. Importantly, our data demonstrate that the membrane inhibits MMP2 expression. Changes in the expression of epithelial-to-mesenchymal transition (EMT)-related molecules further point towards a contribution of the modulation of EMT. Biomaterial-based presentation of regulatory basement membrane signals directly addresses limitations of current therapeutic approaches by enabling a localized and specific method to counteract MMP2 release applicable to a broad range of therapeutic targets.

Overexpression of heterogeneous nuclear ribonucleoprotein F stimulates renal Ace-2 gene expression and prevents TGF-β1-induced kidney injury in a mouse model of diabetes.

PubMed

Lo, Chao-Sheng; Shi, Yixuan; Chang, Shiao-Ying; Abdo, Shaaban; Chenier, Isabelle; Filep, Janos G; Ingelfinger, Julie R; Zhang, Shao-Ling; Chan, John S D

2015-10-01

We investigated whether heterogeneous nuclear ribonucleoprotein F (hnRNP F) stimulates renal ACE-2 expression and prevents TGF-β1 signalling, TGF-β1 inhibition of Ace-2 gene expression and induction of tubulo-fibrosis in an Akita mouse model of type 1 diabetes. Adult male Akita transgenic (Tg) mice overexpressing specifically hnRNP F in their renal proximal tubular cells (RPTCs) were studied. Non-Akita littermates and Akita mice served as controls. Immortalised rat RPTCs stably transfected with plasmid containing either rat Hnrnpf cDNA or rat Ace-2 gene promoter were also studied. Overexpression of hnRNP F attenuated systemic hypertension, glomerular filtration rate, albumin/creatinine ratio, urinary angiotensinogen (AGT) and angiotensin (Ang) II levels, renal fibrosis and profibrotic gene (Agt, Tgf-β1, TGF-β receptor II [Tgf-βrII]) expression, stimulated anti-profibrotic gene (Ace-2 and Ang 1-7 receptor [MasR]) expression, and normalised urinary Ang 1-7 level in Akita Hnrnpf-Tg mice as compared with Akita mice. In vitro, hnRNP F overexpression stimulated Ace-2 gene promoter activity, mRNA and protein expression, and attenuated Agt, Tgf-β1 and Tgf-βrII gene expression. Furthermore, hnRNP F overexpression prevented TGF-β1 signalling and TGF-β1 inhibition of Ace-2 gene expression. These data demonstrate that hnRNP F stimulates Ace-2 gene transcription, prevents TGF-β1 inhibition of Ace-2 gene transcription and induction of kidney injury in diabetes. HnRNP F may be a potential target for treating hypertension and renal fibrosis in diabetes.

Mechanisms and kinetics of proliferation and fibrosis development in a mouse model of thyrocyte hyperplasia.

PubMed

Ciornei, Radu Tudor; Hong, So-Hee; Fang, Yujiang; Zhu, Ziwen; Braley-Mullen, Helen

2016-01-01

IFN-γ(-/-) NOD.H-2h4 mice develop autoimmune disease with extensive hyperplasia and proliferation of thyroid epithelial cells (TEC H/P) and fibrosis. Splenic T cells from donors with severe TEC H/P transfer TEC H/P to SCID recipients. The goal of this study was to determine what factors control TEC H/P development/progression by examining T cells, markers of apoptosis, senescence and proliferation in thyroids of SCID recipients over time. At 28days, T cell infiltration was maximal, thyrocytes were proliferating, and fibrosis was moderate. At days 60 and 90, thyroids were larger with more fibrosis. T cells, cytokines and thyrocyte proliferation decreased, and cell cycle inhibitor proteins, and anti-apoptotic molecules increased. T cells and thyrocytes had foci of phosphorylated histone protein H2A.X, indicative of cellular senescence, when TEC H/P progressed and thyrocyte proliferation declined. Some thyrocytes were regenerating at day 90, with irregularly shaped empty follicles and ciliated epithelium. Proliferating thyrocytes were thyroid transcription factor (TTF1)-positive, suggesting they derived from epithelial cells and not brachial cleft remnants. Copyright © 2016 Elsevier Inc. All rights reserved.

The Smad3/Smad4/CDK9 complex promotes renal fibrosis in mice with unilateral ureteral obstruction.

PubMed

Qu, Xinli; Jiang, Mengjie; Sun, Yu Bo Yang; Jiang, Xiaoyun; Fu, Ping; Ren, Yi; Wang, Die; Dai, Lie; Caruana, Georgina; Bertram, John F; Nikolic-Paterson, David J; Li, Jinhua

2015-12-01

Transforming growth factor-β1 (TGF-β1)/Smad signaling has a central role in the pathogenesis of renal fibrosis. Smad3 and Smad4 are pro-fibrotic, while Smad2 is anti-fibrotic. However, these Smads form heterogeneous complexes, the functions of which are poorly understood. Here we studied Smad complex function in renal fibrosis using the mouse model of unilateral ureteric obstruction. Mice heterozygous for Smad3/4 (Smad3/4 +/- ) exhibited substantial protection from renal fibrosis through day 7 of obstruction, whereas Smad2/3 +/- and Smad2/4 +/- mice showed only modest protection. Formation of Smad3/Smad4/CDK9 complexes was an early event following obstruction in wild-type mice, which involved nuclear phosphorylation of the linker regions of Smad3. Significantly, Smad3 or Smad4 deficiency decreased the formation of Smad4/CDK9 or Smad3/CDK9 complex, Smad3 linker phosphorylation, and fibrosis but at different degrees. In vitro, TGF-β1 stimulation of collagen I promoter activity involved formation of Smad3/Smad4/CDK9 complexes, and overexpression of each component gave additive increases in collagen promoter activity. Co-administration of a CDK9 inhibitor and Smad3-specific inhibition achieved better protection from TGF-β1-induced fibrotic response in vitro and renal interstitial fibrosis in vivo. Thus formation of Smad3/Smad4/CDK9 complex drives renal fibrosis during ureteral obstruction. Formation of this complex represents a novel target for antifibrotic therapies.

A Pneumocyte–Macrophage Paracrine Lipid Axis Drives the Lung toward Fibrosis

PubMed Central

Romero, Freddy; Shah, Dilip; Duong, Michelle; Penn, Raymond B.; Fessler, Michael B.; Madenspacher, Jennifer; Stafstrom, William; Kavuru, Mani; Lu, Bo; Kallen, Caleb B.; Walsh, Kenneth

2015-01-01

Lipid-laden macrophages, or “foam cells,” are observed in the lungs of patients with fibrotic lung disease, but their contribution to disease pathogenesis remains unexplored. Here, we demonstrate that fibrosis induced by bleomycin, silica dust, or thoracic radiation promotes early and sustained accumulation of foam cells in the lung. In the bleomycin model, we show that foam cells arise from neighboring alveolar epithelial type II cells, which respond to injury by dumping lipids into the distal airspaces of the lungs. We demonstrate that oxidized phospholipids accumulate within alveolar macrophages (AMs) after bleomycin injury and that murine and human AMs treated with oxidized phosphatidylcholine (oxPc) become polarized along an M2 phenotype and display enhanced production of transforming growth factor-β1. The direct instillation of oxPc into the mouse lung induces foam cell formation and triggers a severe fibrotic reaction. Further, we show that reducing pulmonary lipid clearance by targeted deletion of the lipid efflux transporter ATP-binding cassette subfamily G member 1 increases foam cell formation and worsens lung fibrosis after bleomycin. Conversely, we found that treatment with granulocyte-macrophage colony-stimulating factor attenuates fibrotic responses, at least in part through its ability to decrease AM lipid accumulation. In summary, this work describes a novel mechanism leading to foam cell formation in the mouse lung and suggests that strategies aimed at blocking foam cell formation might be effective for treating fibrotic lung disorders. PMID:25409201

System-based identification of toxicity pathways associated with multi-walled carbon nanotube-induced pathological responses

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

Snyder-Talkington, Brandi N.; Dymacek, Julian; Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, WV 26506-9300

2013-10-15

The fibrous shape and biopersistence of multi-walled carbon nanotubes (MWCNT) have raised concern over their potential toxicity after pulmonary exposure. As in vivo exposure to MWCNT produced a transient inflammatory and progressive fibrotic response, this study sought to identify significant biological processes associated with lung inflammation and fibrosis pathology data, based upon whole genome mRNA expression, bronchoaveolar lavage scores, and morphometric analysis from C57BL/6J mice exposed by pharyngeal aspiration to 0, 10, 20, 40, or 80 μg MWCNT at 1, 7, 28, or 56 days post-exposure. Using a novel computational model employing non-negative matrix factorization and Monte Carlo Markov Chainmore » simulation, significant biological processes with expression similar to MWCNT-induced lung inflammation and fibrosis pathology data in mice were identified. A subset of genes in these processes was determined to be functionally related to either fibrosis or inflammation by Ingenuity Pathway Analysis and was used to determine potential significant signaling cascades. Two genes determined to be functionally related to inflammation and fibrosis, vascular endothelial growth factor A (vegfa) and C-C motif chemokine 2 (ccl2), were confirmed by in vitro studies of mRNA and protein expression in small airway epithelial cells exposed to MWCNT as concordant with in vivo expression. This study identified that the novel computational model was sufficient to determine biological processes strongly associated with the pathology of lung inflammation and fibrosis and could identify potential toxicity signaling pathways and mechanisms of MWCNT exposure which could be used for future animal studies to support human risk assessment and intervention efforts. - Highlights: • A novel computational model identified toxicity pathways matching in vivo pathology. • Systematic identification of MWCNT-induced biological processes in mouse lungs • MWCNT-induced functional networks of lung inflammation and fibrosis were revealed. • Two functional, representative genes, ccl2 and vegfa, were validated in vitro.« less

Effects of pirfenidone in acute and sub-chronic liver fibrosis, and an initiation-promotion cancer model in the mouse.

PubMed

Seniutkin, Oleksii; Furuya, Shinji; Luo, Yu-Syuan; Cichocki, Joseph A; Fukushima, Hisataka; Kato, Yuki; Sugimoto, Hiromi; Matsumoto, Tomoko; Uehara, Takeki; Rusyn, Ivan

2018-01-15

Liver fibrosis results from chronic tissue damage and excessive regeneration with accumulation of extracellular matrix proteins; it is a precursor of liver cirrhosis and hepatocellular carcinoma. Liver fibrosis treatments are primarily directed at inflammation, with few options to combat fibrogenesis. Pirfenidone is a drug approved for idiopathic pulmonary fibrosis and this study was focused on anti-fibrotic and anti-cancer potential of pirfenidone in the liver of male B6C3F1/J mice. In a dose-finding study, mice were treated with CCl 4 (0.2ml/kg ip, 2×wk for 4weeks) while on a pirfenidone-containing (0-600mg/kg) diet. Pirfenidone at doses of 300 and 600mg/kg had significant anti-fibrotic (collagen) and anti-inflammatory (serum transaminases and "ballooning" hepatocyte) effects. In a sub-chronic study (14weeks), mice received CCl 4 while on pirfenidone (300mg/kg) diet. Pirfenidone significantly reduced collagen deposition, but had little effect of inflammation and injury. In an initiation-promotion cancer study with N-nitrosodiethylamine and CCl 4 , pirfenidone (300mg/kg) did not affect incidence, size, or multiplicity of liver tumors. Overall, we conclude that while pirfenidone exhibits strong anti-fibrotic effects in early stage liver fibrosis, it is less effective in advanced liver fibrosis and was not protective in an initiation-promotion liver cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

L-Endoglin Overexpression Increases Renal Fibrosis after Unilateral Ureteral Obstruction

PubMed Central

Arévalo, Miguel; Núñez-Gómez, Elena; Pérez-Roque, Lucía; Pericacho, Miguel; González-Núñez, María; Langa, Carmen; Martínez-Salgado, Carlos; Perez-Barriocanal, Fernando; Bernabeu, Carmelo; Lopez-Novoa, José M.

2014-01-01

Transforming growth factor-β (TGF-β) plays a pivotal role in renal fibrosis. Endoglin, a 180 KDa membrane glycoprotein, is a TGF-β co-receptor overexpressed in several models of chronic kidney disease, but its function in renal fibrosis remains uncertain. Two membrane isoforms generated by alternative splicing have been described, L-Endoglin (long) and S-Endoglin (short) that differ from each other in their cytoplasmic tails, being L-Endoglin the most abundant isoform. The aim of this study was to assess the effect of L-Endoglin overexpression in renal tubulo-interstitial fibrosis. For this purpose, a transgenic mouse which ubiquitously overexpresses human L-Endoglin (L-ENG+) was generated and unilateral ureteral obstruction (UUO) was performed in L-ENG+ mice and their wild type (WT) littermates. Obstructed kidneys from L-ENG+ mice showed higher amounts of type I collagen and fibronectin but similar levels of α-smooth muscle actin (α-SMA) than obstructed kidneys from WT mice. Smad1 and Smad3 phosphorylation were significantly higher in obstructed kidneys from L-ENG+ than in WT mice. Our results suggest that the higher increase of renal fibrosis observed in L-ENG+ mice is not due to a major abundance of myofibroblasts, as similar levels of α-SMA were observed in both L-ENG+ and WT mice, but to the higher collagen and fibronectin synthesis by these fibroblasts. Furthermore, in vivo L-Endoglin overexpression potentiates Smad1 and Smad3 pathways and this effect is associated with higher renal fibrosis development. PMID:25313562

Calcium-binding protein S100A4 confers mesenchymal progenitor cell fibrogenicity in idiopathic pulmonary fibrosis

PubMed Central

Xia, Hong; Gilbertsen, Adam; Herrera, Jeremy; Racila, Emilian; Peterson, Mark; Griffin, Timothy; Benyumov, Alexey; Yang, Libang; Bitterman, Peter B.; Henke, Craig A.

2017-01-01

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with a prevalence of 1 million persons worldwide. The fibrosis spreads from affected alveoli into contiguous alveoli and leads to death by asphyxiation. We previously discovered that the IPF lung harbors fibrogenic mesenchymal progenitor cells (MPCs) that serve as a cell of origin for disease-mediating myofibroblasts. In a prior genomewide transcriptional analysis, we found that IPF MPCs displayed increased expression of S100 calcium-binding A4 (S100A4), a protein linked to cancer cell proliferation and invasiveness. Here, we have examined whether S100A4 mediates MPC fibrogenicity. Ex vivo analysis revealed that IPF MPCs had increased levels of nuclear S100A4, which interacts with L-isoaspartyl methyltransferase to promote p53 degradation and MPC self-renewal. In vivo, injection of human IPF MPCs converted a self-limited bleomycin-induced mouse model of lung fibrosis to a model of persistent fibrosis in an S100A4-dependent manner. S100A4 gain of function was sufficient to confer fibrotic properties to non-IPF MPCs. In IPF tissue, fibroblastic foci contained cells expressing Ki67 and the MPC markers SSEA4 and S100A4. The expression colocalized in an interface region between myofibroblasts in the focus core and normal alveolar structures, defining this region as an active fibrotic front. Our findings indicate that IPF MPCs are intrinsically fibrogenic and that S100A4 confers MPCs with fibrogenicity. PMID:28530639

Treatment with the matricellular protein CCN3 blocks and/or reverses fibrosis development in obesity with diabetic nephropathy.

PubMed

Riser, Bruce L; Najmabadi, Feridoon; Garchow, Kendra; Barnes, Jeffrey L; Peterson, Darryl R; Sukowski, Ernest J

2014-11-01

Fibrosis is at the core of the high morbidity and mortality rates associated with the complications of diabetes and obesity, including diabetic nephropathy (DN), without any US Food and Drug Administration-approved drugs with this specific target. We recently provided the first evidence that the matricellular protein CCN3 (official symbol NOV) functions in a reciprocal manner, acting on the profibrotic family member CCN2 to inhibit fibrosis in a mesangial cell model of DN. Herein, we used the BT/BR ob/ob mouse as a best model of human obesity and DN progression to determine whether recombinant human CCN3 could be used therapeutically, and the mechanisms involved. Eight weeks of thrice-weekly i.p. injections (0.604 and 6.04 μg/kg of recombinant human CCN3) beginning in early-stage DN completely blocked and/or reversed the up-regulation of mRNA expression of kidney cortex fibrosis genes (CCN2, Col1a2, TGF-β1, and PAI-1) seen in placebo-treated diabetic mice. The treatment completely blocked glomerular fibrosis, as determined by altered mesangial expansion and deposition of laminin. Furthermore, it protected against, or reversed, podocyte loss and kidney function reduction (rise in plasma creatinine concentration); albuminuria was also greatly reduced. This study demonstrates the potential efficacy of recombinant human CCN3 treatment in DN and points to mechanisms operating at multiple levels or pathways, upstream (eg, protecting against cell injury) and downstream (eg, regulating CCN2 activity and extracellular matrix metabolism).

Involvement of Alveolar Epithelial Cell Necroptosis in IPF Pathogenesis.

PubMed

Lee, Ji-Min; Yoshida, Masahiro; Kim, Mi-So; Lee, June-Hyuk; Baek, Ae-Rin; Jang, An Soo; Kim, Do Jin; Minagawa, Shunsuke; Chin, Su Sie; Park, Choon-Sik; Araya, Jun; Kuwano, Kazuyoshi; Park, Sung Woo

2018-02-14

Alveolar epithelial cell (AEC) injury leading to cell death is involved in the process of fibrosis development during idiopathic pulmonary fibrosis (IPF). Among regulated/programmed cell death, the excessive apoptosis of AECs has been widely implicated in IPF pathogenesis. Necroptosis is a type of regulated/programmed necrosis. A multiprotein complex composed of receptor-interacting protein kinase-1 and -3 (RIPK1/3) plays a key regulatory role in initiating necroptosis. Although necroptosis participates in disease pathogeneses through the release of damage-associated molecular patterns (DAMPs), its association with IPF progression remains elusive. In this study, we attempted to illuminate the involvement of RIPK3-regulated necroptosis in IPF pathogenesis. IPF lung tissues were used to detect necroptosis, and the role of RIPK3 was determined using cell culturing models of AECs. Lung fibrosis models of bleomycin (BLM) treatment were also used. RIPK3 expression levels were increased in IPF lungs and both apoptosis and necroptosis were detected mainly in AECs. Necrostatin-1 and RIPK3 knockdown experiments in AECs revealed the participation of necroptosis in BLM and hydrogen peroxide-induced cell death. BLM treatment induced RIPK3 expression in AECs and increased High Mobility Group Box 1 (HMGB1) and interleukin 1β (IL-1β) levels in mouse lungs. The efficient attenuation of BLM-induced lung inflammation and fibrosis was determined in RIPK3 knockout mice and by necrostatin-1 with a concomitant reduction in HMGB1 and IL-1β. RIPK3-regulated necroptosis in AECs is involved in the mechanism of lung fibrosis development through the release of DAMPs as part of the pathogenic sequence of IPF.

Development of a Model of Chronic Kidney Disease in the C57BL/6 Mouse with Properties of Progressive Human CKD

PubMed Central

Cruz, Gaile L.; Lu, Chao; Carlisle, Rachel E.; Werner, Kaitlyn E.; Ask, Kjetil; Dickhout, Jeffrey G.

2015-01-01

Chronic kidney disease (CKD) is a major healthcare problem with increasing prevalence in the population. CKD leads to end stage renal disease and increases the risk of cardiovascular disease. As such, it is important to study the mechanisms underlying CKD progression. To this end, an animal model was developed to allow the testing of new treatment strategies or molecular targets for CKD prevention. Many underlying risk factors result in CKD but the disease itself has common features, including renal interstitial fibrosis, tubular epithelial cell loss through apoptosis, glomerular damage, and renal inflammation. Further, CKD shows differences in prevalence between the genders with premenopausal women being relatively resistant to CKD. We sought to develop and characterize an animal model with these common features of human CKD in the C57BL/6 mouse. Mice of this genetic background have been used to produce transgenic strains that are commercially available. Thus, a CKD model in this strain would allow the testing of the effects of numerous genes on the severity or progression of CKD with minimal cost. This paper describes such a mouse model of CKD utilizing angiotensin II and deoxycorticosterone acetate as inducers. PMID:26064882

Genetic Regulation of Fibroblast Activation and Proliferation in Cardiac Fibrosis.

PubMed

Park, Shuin; Ranjbarvazirj, Sara; Lay, Fides D; Zhao, Peng; Miller, Mark J; Dhaliwal, Jasmeet S; Huertas-Vazquez, Adriana; Wu, Xiuju; Qiao, Rong; Soffer, Justin M; Mikkola, Hanna K A; Lusis, Aldons J; Ardehali, Reza

2018-06-27

Background -Genetic diversity and the heterogeneous nature of cardiac fibroblasts (CFbs) have hindered characterization of the molecular mechanisms that regulate cardiac fibrosis. The Hybrid Mouse Diversity Panel (HMDP) offers a valuable tool to examine genetically diverse cardiac fibroblasts and their role in fibrosis. Methods -Three strains of mice (C57BL/6J, C3H/HeJ, and KK/HlJ) were selected from the HMDP and treated with either isoproterenol (ISO) or saline by an intraperitoneally implanted osmotic pump. After 21 days, cardiac function and levels of fibrosis were measured by echocardiography and trichrome staining, respectively. Activation and proliferation of CFbs were measured by in vitro and in vivo assays under normal and injury conditions. RNA-sequencing was done on isolated CFbs from each strain and results were analyzed by Ingenuity Pathway Analysis (IPA) and validated by reverse transcription-qPCR, immunohistochemistry, and ELISA. Results -ISO treatment in C57BL/6J, C3H/HeJ, and KK/HlJ mice resulted in minimal, moderate, and extensive levels of fibrosis, respectively (n = 7-8 hearts/condition). Isolated CFbs treated with ISO exhibited strain-specific increases in the levels of activation but showed comparable levels of proliferation. Similar results were found in vivo , with fibroblast activation, and not proliferation, correlating with the differential levels of cardiac fibrosis after ISO treatment. RNA-sequencing revealed that CFbs from each strain exhibit unique gene expression changes in response to ISO. We identified Ltbp2 as a commonly upregulated gene after ISO treatment. Expression of LTBP2 was elevated and specifically localized in the fibrotic regions of the myocardium after injury in mice and in human heart failure patients. Conclusions -This study highlights the importance of genetic variation in cardiac fibrosis by using multiple inbred mouse strains to characterize CFbs and their response to ISO treatment. Our data suggest that, while fibroblast activation is a response that parallels the extent of scar formation, proliferation may not necessarily correlate with levels of fibrosis. Additionally, by comparing CFbs from multiple strains, we identified pathways as potential therapeutic targets and LTBP2 as a marker for fibrosis, with relevance to patients with underlying myocardial fibrosis.

Identification of a Cell-of-Origin for Fibroblasts Comprising the Fibrotic Reticulum in Idiopathic Pulmonary Fibrosis

PubMed Central

Xia, Hong; Bodempudi, Vidya; Benyumov, Alexey; Hergert, Polla; Tank, Damien; Herrera, Jeremy; Braziunas, Jeff; Larsson, Ola; Parker, Matthew; Rossi, Daniel; Smith, Karen; Peterson, Mark; Limper, Andrew; Jessurun, Jose; Connett, John; Ingbar, David; Phan, Sem; Bitterman, Peter B.; Henke, Craig A.

2015-01-01

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of the middle aged and elderly with a prevalence of one million persons worldwide. The fibrosis spreads from affected alveoli into contiguous alveoli, creating a reticular network that leads to death by asphyxiation. Lung fibroblasts from patients with IPF have phenotypic hallmarks, distinguishing them from their normal counterparts: pathologically activated Akt signaling axis, increased collagen and α-smooth muscle actin expression, distinct gene expression profile, and ability to form fibrotic lesions in model organisms. Despite the centrality of these fibroblasts in disease pathogenesis, their origin remains uncertain. Here, we report the identification of cells in the lungs of patients with IPF with the properties of mesenchymal progenitors. In contrast to progenitors isolated from nonfibrotic lungs, IPF mesenchymal progenitor cells produce daughter cells manifesting the full spectrum of IPF hallmarks, including the ability to form fibrotic lesions in zebrafish embryos and mouse lungs, and a transcriptional profile reflecting these properties. Morphological analysis of IPF lung tissue revealed that mesenchymal progenitor cells and cells with the characteristics of their progeny comprised the fibrotic reticulum. These data establish that the lungs of patients with IPF contain pathological mesenchymal progenitor cells that are cells of origin for fibrosis-mediating fibroblasts. These fibrogenic mesenchymal progenitors and their progeny represent an unexplored target for novel therapies to interdict fibrosis. PMID:24631025

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

Selective inhibitor of Wnt/β-catenin/CBP signaling ameliorates hepatitis C virus-induced liver fibrosis in mouse model.

PubMed

Tokunaga, Yuko; Osawa, Yosuke; Ohtsuki, Takahiro; Hayashi, Yukiko; Yamaji, Kenzaburo; Yamane, Daisuke; Hara, Mitsuko; Munekata, Keisuke; Tsukiyama-Kohara, Kyoko; Hishima, Tsunekazu; Kojima, Soichi; Kimura, Kiminori; Kohara, Michinori

2017-03-23

Chronic hepatitis C virus (HCV) infection is one of the major causes of serious liver diseases, including liver cirrhosis. There are no anti-fibrotic drugs with efficacy against liver cirrhosis. Wnt/β-catenin signaling has been implicated in the pathogenesis of a variety of tissue fibrosis. In the present study, we investigated the effects of a β-catenin/CBP (cyclic AMP response element binding protein) inhibitor on liver fibrosis. The anti-fibrotic activity of PRI-724, a selective inhibitor of β-catenin/CBP, was assessed in HCV GT1b transgenic mice at 18 months after HCV genome expression. PRI-724 was injected intraperitoneally or subcutaneously in these mice for 6 weeks. PRI-724 reduced liver fibrosis, which was indicated by silver stain, Sirius Red staining, and hepatic hydroxyproline levels, in HCV mice while attenuating αSMA induction. PRI-724 led to increased levels of matrix metalloproteinase (MMP)-8 mRNA in the liver, along with elevated levels of intrahepatic neutrophils and macrophages/monocytes. The induced intrahepatic neutrophils and macrophages/monocytes were identified as the source of MMP-8. In conclusion, PRI-724 ameliorated HCV-induced liver fibrosis in mice. We hypothesize that inhibition of hepatic stellate cells activation and induction of fibrolytic cells expressing MMP-8 contribute to the anti-fibrotic effects of PRI-724. PRI-724 is a drug candidate which possesses anti-fibrotic effect.

SIRT1 antagonizes liver fibrosis by blocking hepatic stellate cell activation in mice.

PubMed

Li, Min; Hong, Wenxuan; Hao, Chenzhi; Li, Luyang; Wu, Dongmei; Shen, Aiguo; Lu, Jun; Zheng, Yuanlin; Li, Ping; Xu, Yong

2018-01-01

Hepatic stellate cells (HSCs) are a major source of fibrogenesis in the liver, contributing to cirrhosis. When activated, HSCs transdifferentiate into myofibroblasts and undergo profound functional alterations paralleling an overhaul of the transcriptome, the mechanism of which remains largely undefined. We investigated the involvement of the class III deacetylase sirtuin [silent information regulator 1 (SIRT1)] in HSC activation and liver fibrosis. SIRT1 levels were down-regulated in the livers in mouse models of liver fibrosis, in patients with cirrhosis, and in activated HSCs as opposed to quiescent HSCs. SIRT1 activation halted, whereas SIRT1 inhibition promoted, HSC transdifferentiation into myofibroblasts. Liver fibrosis was exacerbated in mice with HSC-specific deletion of SIRT1 [conditional knockout (cKO)], receiving CCl 4 (1 mg/kg) injection or subjected to bile duct ligation, compared to wild-type littermates. SIRT1 regulated peroxisome proliferator activated receptor γ (PPARγ) transcription by deacetylating enhancer of zeste homolog 2 (EZH2) in quiescent HSCs. Finally, EZH2 inhibition or PPARγ activation ameliorated fibrogenesis in cKO mice. In summary, our data suggest that SIRT1 plays an essential role guiding the transition of HSC phenotypes.-Li, M., Hong, W., Hao, C., Li, L., Wu, D., Shen, A., Lu, J., Zheng, Y., Li, P., Xu, Y. SIRT1 antagonizes liver fibrosis by blocking hepatic stellate cell activation in mice. © FASEB.

Characterization of the Mouse Beta Defensin 1, Defb1, Mutant Mouse Model

PubMed Central

Morrison, Gillian; Kilanowski, Fiona; Davidson, Donald; Dorin, Julia

2002-01-01

Beta defensins are small cationic antimicrobial peptides present in the respiratory system which have been proposed to be dysfunctional in the environment of the cystic fibrosis lung. Defb1, a murine homologue to the human beta defensins, has also been found to be expressed in the respiratory system and, in order to examine the function of beta defensins in vivo, gene targeting was used to generate Defb1-deficient (Defb1tm1Hgu/Defb1tm1Hgu [Defb1−/−]) mice. The Defb1 synthetic peptide was shown to have a salt-sensitive antimicrobial activity that was stronger against Staphylococcus aureus than against Escherichia coli or Pseudomonas aeruginosa. Defb1−/− mice were found, however, to be effective in the clearance of the cystic fibrosis relevant pathogen S. aureus from the airways after nebulization. Although no overt deleterious phenotype was evident in the Defb1−/− mice, the number of mutant mice found to harbor bacteria of the Staphylococcus species in the bladder was significantly higher (P = 0.008) than that of controls, suggesting a role for these peptides in resistance to urinary tract infection. PMID:12010997

Monocyte-derived alveolar macrophages drive lung fibrosis and persist in the lung over the life span

PubMed Central

Morales-Nebreda, Luisa; Cuda, Carla M.; Walter, James M.; Chen, Ching-I; Anekalla, Kishore R.; Joshi, Nikita; Williams, Kinola J.N.; Abdala-Valencia, Hiam; Yacoub, Tyrone J.; Chi, Monica; Gates, Khalilah; Homan, Philip J.; Soberanes, Saul; Dominguez, Salina; Saber, Rana; Hinchcliff, Monique; Marshall, Stacy A.; Bharat, Ankit; Berdnikovs, Sergejs; Bhorade, Sangeeta M.; Balch, William E.; Chandel, Navdeep S.; Jain, Manu; Ridge, Karen M.; Bagheri, Neda; Shilatifard, Ali

2017-01-01

Little is known about the relative importance of monocyte and tissue-resident macrophages in the development of lung fibrosis. We show that specific genetic deletion of monocyte-derived alveolar macrophages after their recruitment to the lung ameliorated lung fibrosis, whereas tissue-resident alveolar macrophages did not contribute to fibrosis. Using transcriptomic profiling of flow-sorted cells, we found that monocyte to alveolar macrophage differentiation unfolds continuously over the course of fibrosis and its resolution. During the fibrotic phase, monocyte-derived alveolar macrophages differ significantly from tissue-resident alveolar macrophages in their expression of profibrotic genes. A population of monocyte-derived alveolar macrophages persisted in the lung for one year after the resolution of fibrosis, where they became increasingly similar to tissue-resident alveolar macrophages. Human homologues of profibrotic genes expressed by mouse monocyte-derived alveolar macrophages during fibrosis were up-regulated in human alveolar macrophages from fibrotic compared with normal lungs. Our findings suggest that selectively targeting alveolar macrophage differentiation within the lung may ameliorate fibrosis without the adverse consequences associated with global monocyte or tissue-resident alveolar macrophage depletion. PMID:28694385

Progression and Regression of Hepatic Lesions in a Mouse Model of NASH Induced by Dietary Intervention and Its Implications in Pharmacotherapy.

PubMed

Ding, Zhi-Ming; Xiao, Yue; Wu, Xikun; Zou, Haixia; Yang, Shurong; Shen, Yiyun; Xu, Juehua; Workman, Heather C; Usborne, Amy L; Hua, Haiqing

2018-01-01

Understanding of the temporal changes of hepatic lesions in the progression and regression of non-alcoholic steatohepatitis (NASH) is vital to elucidation of the pathogenesis of NASH, and critical to the development of a strategy for NASH pharmacotherapy. There are challenges in studying hepatic lesion progression and regression in NASH patients due to the slow development of NASH in humans, one being the requirement for multiple biopsies during the longitudinal follow-up. Here we studied lesion progression and regression in the diet-induced animal model of NASH by application or removal of the pathogenic diet for multiple time periods. Male C57BL/6 mice fed Western diet developed progressive hepatic steatosis/macrovesicular vacuolation, inflammation, and hepatocyte degeneration, as well as perisinusoidal fibrosis and occasionally portal fibrosis as early as 2 months after initiation of the Western diet. In the same period, the mice exhibited elevated ALT (alanine aminotransferase) and AST (aspartate aminotransferase) enzyme activities, CK18 (cytokeratin-18), PIIINP (N-terminal propeptide of type III collagen), and TIMP-1 (tissue inhibitor of metalloproteinase-1). Hepatic steatosis diminished rapidly when the Western diet was replaced by normal rodent chow diet and hepatic inflammation and hepatocyte degeneration were also reduced. Interestingly, perisinusoidal fibrosis and portal fibrosis regressed 8 months after chow diet replacement. To understand pharmacotherapy for NASH, mice with established NASH hepatic lesions were treated with either FXR agonist obeticholic acid (Ocaliva), or CCR2/5 antagonist Cenicriviroc. Similar to the diet replacement, metabolic modulator Ocaliva markedly reduced steatosis/macrovesicular vacuolation, hepatic inflammation, and hepatocyte degeneration effectively, but exhibited no significant effect on liver fibrosis. Anti-inflammation drug Cenicriviroc, on the other hand, markedly decreased inflammation and hepatocyte degeneration, and mildly decreased liver fibrosis, but exhibited no effect on hepatic steatosis/macrovesicular vacuolation. In conclusion, we found the progression of NASH hepatic steatosis/macrovesicular vacuolation, and inflammation eventually lead to hepatocyte death and fibrosis. Life style change and current pharmacotherapies in development may be effective in treating NASH, but their effects on NASH-induced fibrosis may be mild. Since fibrosis is known to be an independent risk for decompensated cirrhosis, cardiovascular events, and mortality, our study suggests that effective anti-fibrosis therapy should be an essential component of the combined pharmacotherapy for advanced NASH.

Pilot study of the antifibrotic effects of the multikinase inhibitor pacritinib in a mouse model of liver fibrosis.

PubMed

Al-Fayoumi, Suliman; Hashiguchi, Taishi; Shirakata, Yuka; Mascarenhas, John; Singer, Jack W

2018-01-01

Fibrotic diseases result from an exuberant response to chronic inflammation. Myelofibrosis is the end result of inflammation in bone, caused by an inflammatory process triggered by production of abnormal myeloid cells driven by mutations affecting the JAK-STAT pathway. Inflammatory cytokine overproduction leads to increased mesenchymal cell proliferation, culminating in fibrosis. Although JAK2 inhibitors, such as the JAK1/2 inhibitor ruxolitinib and the JAK2/FLT3/CSF1R/IRAK1 inhibitor pacritinib suppress abnormal clone expansion in myelofibrosis, ruxolitinib does not appear to prevent or reverse bone-marrow fibrosis in most patients. In two Phase III clinical trials, pacritinib, however, demonstrated improvements in platelet counts and hemoglobin and reductions in transfusion burden in some patients with baseline cytopenias, suggesting it may improve bone-marrow function. Unlike ruxolitinib, pacritinib suppresses signaling through IRAK1, a key control point for inflammatory and fibrotic signaling. To investigate potential antifibrotic effects of pacritinib in an animal model of liver fibrosis relevant to the observed course of human disease. Pacritinib, negative control (vehicle), and positive control (the angiotensin 2-receptor antagonist and PPARγ partial agonist telmisartan) were assessed in the murine Stelic animal model, which mimics the clinically observed progression from hepatic steatosis to nonalcoholic steatohepatitis, liver fibrosis, and hepatocellular carcinoma. Histopathological analysis used hematoxylin and eosin staining. Body and liver weight changes, nonalcoholic fatty-liver disease activity scores, and plasma cytokeratin 18 fragment levels (a biomarker of hepatic necrosis) were measured. Pacritinib-treated mice had significantly ( P <0.01) reduced fibrotic areas in liver compared to vehicle control and significantly ( P <0.05) lower levels of CK18. The antifibrotic effect of pacritinib was comparable to that of telmisartan, but without significant effects on fat accumulation. These results, the first to demonstrate hepatic antifibrotic effects for pacritinib in an animal model of liver disease, provide preliminary support for potential clinical applications of pacritinib in fibrotic diseases other than myelofibrosis.

Connective tissue growth factor/CCN2-null mouse embryonic fibroblasts retain intact transforming growth factor-{beta} responsiveness

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

Mori, Yasuji; Hinchcliff, Monique; Wu, Minghua

2008-03-10

Background: The matricellular protein connective tissue growth factor (CCN2) has been implicated in pathological fibrosis, but its physiologic role remains elusive. In vitro, transforming growth factor-{beta} (TGF-{beta}) induces CCN2 expression in mesenchymal cells. Because CCN2 can enhance profibrotic responses elicited by TGF-{beta}, it has been proposed that CCN2 functions as an essential downstream signaling mediator for TGF-{beta}. To explore this notion, we characterized TGF-{beta}-induced activation of fibroblasts from CCN2-null (CCN2{sup -/-}) mouse embryos. Methods: The regulation of CCN2 expression was examined in vivo in a model of fibrosis induced by bleomycin. Cellular TGF-{beta} signal transduction and regulation of collagen genemore » expression were examined in CCN2{sup -/-} MEFs by immunohistochemistry, Northern, Western and RT-PCR analysis, immunocytochemistry and transient transfection assays. Results: Bleomycin-induced skin fibrosis in the mouse was associated with substantial CCN2 up-regulation in lesional fibroblasts. Whereas in vitro proliferation rate of CCN2{sup -/-} MEFs was markedly reduced compared to wild type MEFs, TGF-{beta}-induced activation of the Smad pathways, including Smad2 phosphorylation, Smad2/3 and Smad4 nuclear accumulation and Smad-dependent transcriptional responses, were unaffected by loss of CCN2. The stimulation of COL1A2 and fibronectin mRNA expression and promoter activity, and of corresponding protein levels, showed comparable time and dose-response in wild type and CCN2{sup -/-} MEFs, whereas stimulation of alpha smooth muscle actin and myofibroblast transdifferentiation showed subtle impairment in MEFs lacking CCN2. Conclusion: Whereas endogenous CCN2 plays a role in regulation of proliferation and TGF-{beta}-induced myofibroblast transdifferentiation, it appears to be dispensable for Smad-dependent stimulation of collagen and extracellular matrix synthesis in murine embryonic fibroblasts.« less

Endothelial Dysfunction Exacerbates Renal Interstitial Fibrosis through Enhancing Fibroblast Smad3 Linker Phosphorylation in the Mouse Obstructed Kidney

PubMed Central

Sun, Yu Bo Yang; Qu, Xinli; Li, Xueling; Nikolic-Paterson, David J.; Li, Jinhua

2013-01-01

Endothelial dysfunction and enhanced transforming growth factor-β (TGF-β)/Smad3 signalling are common features of progressive renal fibrosis. This study investigated a potential link between these mechanisms. In unilateral ureteric obstruction (UUO) we observed an acute (6 hr) down-regulation of nitric oxide synthase 3 (NOS3/eNOS) levels and increased phosphorylation of the linker region of Smad3 at T179 and S208 in Smad3/JNK complexes. These events preceded Smad3 C-terminal domain phosphorylation and the induction of myofibroblast proliferation at 48 hrs. Mice deficient in NOS3 showed enhanced myofibroblast proliferation and collagen accumulation compared to wild type mice in a 7 day UUO model. This was associated with enhanced phosphorylation of Smad3 T179 and S208 by 92% and 88%, respectively, whereas Smad3-C-terminal phosphorylation was not affected. Resolvin D1 (RvD1) can suppress renal fibrosis in the UUO model, and further analysis herein showed that RvD1 protected against endothelial dysfunction and suppressed Smad3/JNK complex formation with a consequent reduction in phosphorylation of Smad3 T179 and S208 by 78% and 65%, respectively, while Smad3 C-terminal phosphorylation was unaltered. In vitro, conditioned media from mouse microvascular endothelial cells (MMEC) treated with a general inhibitor of nitric oxide synthase (L-NAME) augmented the proliferation and collagen production of renal fibroblasts (NRK49F cells) compared to control MMEC media and this was associated with increased phosphorylation of JNK and Smad3 T179 and S208, whereas Smad3-C-terminal domain phosphorylation was unaffected. The addition of RvD1 to L-NAME treated MMEC abrogated these effects of the conditioned media on renal fibroblasts. Finally, Smad3 T179/V and S208/A mutations significantly inhibit TGF-β1 induced up-regulation collagen I promoter. In conclusion, these data suggest that endothelial dysfunction can exacerbate renal interstitial fibrosis through increased fibroblast proliferation and collagen production via enhanced Smad3 linker phosphorylation. PMID:24391884

Endothelial dysfunction exacerbates renal interstitial fibrosis through enhancing fibroblast Smad3 linker phosphorylation in the mouse obstructed kidney.

PubMed

Sun, Yu Bo Yang; Qu, Xinli; Li, Xueling; Nikolic-Paterson, David J; Li, Jinhua

2013-01-01

Endothelial dysfunction and enhanced transforming growth factor-β (TGF-β)/Smad3 signalling are common features of progressive renal fibrosis. This study investigated a potential link between these mechanisms. In unilateral ureteric obstruction (UUO) we observed an acute (6 hr) down-regulation of nitric oxide synthase 3 (NOS3/eNOS) levels and increased phosphorylation of the linker region of Smad3 at T179 and S208 in Smad3/JNK complexes. These events preceded Smad3 C-terminal domain phosphorylation and the induction of myofibroblast proliferation at 48 hrs. Mice deficient in NOS3 showed enhanced myofibroblast proliferation and collagen accumulation compared to wild type mice in a 7 day UUO model. This was associated with enhanced phosphorylation of Smad3 T179 and S208 by 92% and 88%, respectively, whereas Smad3-C-terminal phosphorylation was not affected. Resolvin D1 (RvD1) can suppress renal fibrosis in the UUO model, and further analysis herein showed that RvD1 protected against endothelial dysfunction and suppressed Smad3/JNK complex formation with a consequent reduction in phosphorylation of Smad3 T179 and S208 by 78% and 65%, respectively, while Smad3 C-terminal phosphorylation was unaltered. In vitro, conditioned media from mouse microvascular endothelial cells (MMEC) treated with a general inhibitor of nitric oxide synthase (L-NAME) augmented the proliferation and collagen production of renal fibroblasts (NRK49F cells) compared to control MMEC media and this was associated with increased phosphorylation of JNK and Smad3 T179 and S208, whereas Smad3-C-terminal domain phosphorylation was unaffected. The addition of RvD1 to L-NAME treated MMEC abrogated these effects of the conditioned media on renal fibroblasts. Finally, Smad3 T179/V and S208/A mutations significantly inhibit TGF-β1 induced up-regulation collagen I promoter. In conclusion, these data suggest that endothelial dysfunction can exacerbate renal interstitial fibrosis through increased fibroblast proliferation and collagen production via enhanced Smad3 linker phosphorylation.

The anticancer drug tamoxifen counteracts the pathology in a mouse model of duchenne muscular dystrophy.

PubMed

Dorchies, Olivier M; Reutenauer-Patte, Julie; Dahmane, Elyes; Ismail, Heham M; Petermann, Olivier; Patthey- Vuadens, Ophélie; Comyn, Sophie A; Gayi, Elinam; Piacenza, Tony; Handa, Robert J; Décosterd, Laurent A; Ruegg, Urs T

2013-02-01

Duchenne muscular dystrophy (DMD) is a severe disorder characterized by progressive muscle wasting,respiratory and cardiac impairments, and premature death. No treatment exists so far, and the identification of active substances to fight DMD is urgently needed. We found that tamoxifen, a drug used to treat estrogen-dependent breast cancer, caused remarkable improvements of muscle force and of diaphragm and cardiac structure in the mdx(5Cv) mouse model of DMD. Oral tamoxifen treatment from 3 weeks of age for 15 months at a dose of 10 mg/kg/day stabilized myofiber membranes, normalized whole body force, and increased force production and resistance to repeated contractions of the triceps muscle above normal values. Tamoxifen improved the structure of leg muscles and diminished cardiac fibrosis by~ 50%. Tamoxifen also reduced fibrosis in the diaphragm, while increasing its thickness,myofiber count, and myofiber diameter, thereby augmenting by 72% the amount of contractile tissue available for respiratory function. Tamoxifen conferred a markedly slower phenotype to the muscles.Tamoxifen and its metabolites were present in nanomolar concentrations in plasma and muscles,suggesting signaling through high-affinity targets. Interestingly, the estrogen receptors ERa and ERb were several times more abundant in dystrophic than in normal muscles, and tamoxifen normalized the relative abundance of ERb isoforms. Our findings suggest that tamoxifen might be a useful therapy for DMD.

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

Chen, Ying; Li, Cuiying; Weng, Dong

Silica exposure can cause lung inflammation and fibrosis, known as silicosis. Interleukin-17A (IL-17A) and Th17 cells play a pivotal role in controlling inflammatory diseases. However, the roles of IL-17A and Th17 cells in the progress of silica-induced inflammation and fibrosis are poorly understood. This study explored the effects of IL-17A on silica-induced inflammation and fibrosis. We used an anti-mouse IL-17A antibody to establish an IL-17A-neutralized mice model, and mice were exposed to silica to establish an experimental silicosis model. We showed that IL-17A neutralization delayed neutrophil accumulation and progression of silica-induced lung inflammation and fibrosis. IL-17A neutralization reduced the percentagemore » of Th17 in CD4 + T cells, decreased IL-6 and IL-1β expression, and increased Tregs at an early phase of silica-induced inflammation. Neutralization of IL-17A delayed silica-induced Th1/Th2 immune and autoimmune responses. These results suggest that IL-17A neutralization alleviates early stage silica-induced lung inflammation and delays progression of silica-induced lung inflammation and fibrosis. Neutralization of IL-17A suppressed Th17 cell development by decreasing IL-6 and/or IL-1β and increased Tregs at an early phase of silica-induced inflammation. Neutralization of IL-17A also delayed the Th1/Th2 immune response during silica-induced lung inflammation and fibrosis. IL-17A may play a pivotal role in the early phase of silica-induced inflammation and may mediate the Th immune response to influence silica-induced lung inflammation and fibrosis in mice. - Highlights: • Neutralization of IL-17A alleviated silica-induced lung inflammation of early stage. • Neutralization of IL-17A decreased Th17 cells and increased Tregs. • IL-17A mediated the reciprocal relationship of Th17/Tregs by IL-6 and/or IL-1β. • Neutralization of IL-17A delayed silica-induced Th1/Th2 immune response. • Neutralization of IL-17A delayed silica-induced lung inflammation and fibrosis.« less

Ultramicronized palmitoylethanolamide (PEA-um(®)) in the treatment of idiopathic pulmonary fibrosis.

PubMed

Di Paola, Rosanna; Impellizzeri, Daniela; Fusco, Roberta; Cordaro, Marika; Siracusa, Rosalba; Crupi, Rosalia; Esposito, Emanuela; Cuzzocrea, Salvatore

2016-09-01

Pulmonary fibrosis is a chronic condition characterized by progressive scarring of lung parenchyma. The aim of this study was to examine the effects of an ultramicronized preparation of palmitoylethanolamide (PEA-um(®)), an endogenous fatty acid amide, in mice subjected to idiopathic pulmonary fibrosis. Idiopathic pulmonary fibrosis was induced in male mice by a single intratracheal administration of saline with bleomycin sulphate (1mg/kg body weight) in a volume of 100μL. PEA-um(®) was injected intraperitoneally at 1, 3 or 10mg/kg 1h after bleomycin instillation and daily thereafter. Animals were sacrificed after 7 and 21days by pentobarbitone overdose. One cohort of mice was sacrificed after seven days of bleomycin administration, followed by bronchoalveloar lavage and determination of myeloperoxidase activity, lung edema and histopathology features. In the 21-day cohort, mortality was assessed daily, and surviving mice were sacrificed followed by the above analyses together with immunohistochemical localization of CD8, tumor necrosis factor-α, CD4, interleukin-1β, transforming growth factor-β, inducible nitric oxide synthase and basic fibroblast growth factor. Compared to bleomycin-treated mice, animals that received also PEA-um(®) (3 or 10mg/kg) had significantly decreased weight loss, mortality, inflammation, lung damage at the histological level, and lung fibrosis at 7 and 21days. PEA-um(®) (1mg/kg) did not significantly inhibit the inflammation response and lung fibrosis. This study demonstrates that PEA-um(®) (3 and 10mg/kg) reduces the extent of lung inflammation in a mouse model of idiopathic pulmonary fibrosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Anti-fibrotic effects of a novel small compound on the regulation of cytokine production in a mouse model of colorectal fibrosis

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

Imai, Jin; Department of Gastroenterology, Tokai University School of Medicine, Kanagawa; Hozumi, Katsuto, E-mail: hozumi@is.icc.u-tokai.ac.jp

Intestinal fibrotic stricture is a major complication of inflammatory bowel disease. Despite its clinical importance, anti-fibrotic therapy has not been implemented. Transforming growth factor-β (TGF-β) is considered to be a major factor contributing to tissue fibrosis. We have previously shown that the administration of a small compound, HSc025, which promotes the nuclear translocation of YB-1 as a downstream effector of IFN-γ and antagonizes TGF-β/Smad signaling, improves fibrosis in several murine tissues. In this study, we evaluated the anti-fibrotic effect of HSc025 on colorectal fibrosis in TNBS-induced murine chronic colitis. Daily oral administration of HSc025 (3, 15 and 75 mg/kg) suppressed collagenmore » production and decreased the severity of colorectal fibrosis in a dose-dependent manner. In addition, the local production of TGF-β was decreased after HSc025 treatment, whereas that of IL-13 and TNF-α was not affected. HSc025 administration maintained the level of IFN-γ production, even at a late stage when IFN-γ production was lost without the drug treatment. These results demonstrate that HSc025 could be a therapeutic candidate for intestinal fibrosis in inflammatory bowel disease that acts by altering the local production of cytokines, as well as by directly suppressing collagen production. - Highlights: • Colorectal fibrosis of TNBS-induced colitis was attenuated by HSc025 administration. • Local production of TGF-b was suppressed by the modulation of TGF-b/IFN-g signaling. • Derepression of IFN-g production was induced by the drug treatment.« less

Ablation of biglycan attenuates cardiac hypertrophy and fibrosis after left ventricular pressure overload.

PubMed

Beetz, Nadine; Rommel, Carolin; Schnick, Tilman; Neumann, Elena; Lother, Achim; Monroy-Ordonez, Elsa Beatriz; Zeeb, Martin; Preissl, Sebastian; Gilsbach, Ralf; Melchior-Becker, Ariane; Rylski, Bartosz; Stoll, Monika; Schaefer, Liliana; Beyersdorf, Friedhelm; Stiller, Brigitte; Hein, Lutz

2016-12-01

Biglycan, a small leucine-rich proteoglycan, has been shown to play an important role in stabilizing fibrotic scars after experimental myocardial infarction. However, the role of biglycan in the development and regression of cardiomyocyte hypertrophy and fibrosis during cardiac pressure overload and unloading remains elusive. Thus, the aim of the present study was to assess the effect of biglycan on cardiac remodeling in a mouse model of left ventricular pressure overload and unloading. Left ventricular pressure overload induced by transverse aortic constriction (TAC) in mice resulted in left ventricular dysfunction, fibrosis and increased biglycan expression. Fluorescence- and magnetic-assisted sorting of cardiac cell types revealed upregulation of biglycan in the fibroblast population, but not in cardiomyocytes, endothelial cells or leukocytes after TAC. Removal of the aortic constriction (rTAC) after short-term pressure overload (3weeks) improved cardiac contractility and reversed ventricular hypertrophy but not fibrosis in wild-type (WT) mice. Biglycan ablation (KO) enhanced functional recovery but did not resolve cardiac fibrosis. After long-term TAC for 9weeks, ablation of biglycan attenuated the development of cardiac hypertrophy and fibrosis. In vitro, biglycan induced hypertrophy of neonatal rat cardiomyocytes and led to activation of a hypertrophic gene program. Putative downstream mediators of biglycan signaling include Rcan1, Abra and Tnfrsf12a. These genes were concordantly induced by TAC in WT but not in biglycan KO mice. Left ventricular pressure overload induces biglycan expression in cardiac fibroblasts. Ablation of biglycan improves cardiac function and attenuates left ventricular hypertrophy and fibrosis after long-term pressure overload. In vitro biglycan induces hypertrophy of cardiomyocytes, suggesting that biglycan may act as a signaling molecule between cell types to modulate cardiac remodeling. Copyright © 2016 Elsevier Ltd. All rights reserved.

Azithromycin attenuates myofibroblast differentiation and lung fibrosis development through proteasomal degradation of NOX4.

PubMed

Tsubouchi, Kazuya; Araya, Jun; Minagawa, Shunsuke; Hara, Hiromichi; Ichikawa, Akihiro; Saito, Nayuta; Kadota, Tsukasa; Sato, Nahoko; Yoshida, Masahiro; Kurita, Yusuke; Kobayashi, Kenji; Ito, Saburo; Fujita, Yu; Utsumi, Hirofumi; Yanagisawa, Haruhiko; Hashimoto, Mitsuo; Wakui, Hiroshi; Yoshii, Yutaka; Ishikawa, Takeo; Numata, Takanori; Kaneko, Yumi; Asano, Hisatoshi; Yamashita, Makoto; Odaka, Makoto; Morikawa, Toshiaki; Nakayama, Katsutoshi; Nakanishi, Yoichi; Kuwano, Kazuyoshi

2017-08-03

Accumulation of profibrotic myofibroblasts is involved in the process of fibrosis development during idiopathic pulmonary fibrosis (IPF) pathogenesis. TGFB (transforming growth factor β) is one of the major profibrotic cytokines for myofibroblast differentiation and NOX4 (NADPH oxidase 4) has an essential role in TGFB-mediated cell signaling. Azithromycin (AZM), a second-generation antibacterial macrolide, has a pleiotropic effect on cellular processes including proteostasis. Hence, we hypothesized that AZM may regulate NOX4 levels by modulating proteostasis machineries, resulting in inhibition of TGFB-associated lung fibrosis development. Human lung fibroblasts (LF) were used to evaluate TGFB-induced myofibroblast differentiation. With respect to NOX4 regulation via proteostasis, assays for macroautophagy/autophagy, the unfolded protein response (UPR), and proteasome activity were performed. The potential anti-fibrotic property of AZM was examined by using bleomycin (BLM)-induced lung fibrosis mouse models. TGFB-induced NOX4 and myofibroblast differentiation were clearly inhibited by AZM treatment in LF. AZM-mediated NOX4 reduction was restored by treatment with MG132, a proteasome inhibitor. AZM inhibited autophagy and enhanced the UPR. Autophagy inhibition by AZM was linked to ubiquitination of NOX4 via increased protein levels of STUB1 (STIP1 homology and U-box containing protein 1), an E3 ubiquitin ligase. An increased UPR by AZM was associated with enhanced proteasome activity. AZM suppressed lung fibrosis development induced by BLM with concomitantly reduced NOX4 protein levels and enhanced proteasome activation. These results suggest that AZM suppresses NOX4 by promoting proteasomal degradation, resulting in inhibition of TGFB-induced myofibroblast differentiation and lung fibrosis development. AZM may be a candidate for the treatment of the fibrotic lung disease IPF.

A small population of liver endothelial cells undergoes endothelial-to-mesenchymal transition in response to chronic liver injury.

PubMed

Ribera, Jordi; Pauta, Montse; Melgar-Lesmes, Pedro; Córdoba, Bernat; Bosch, Anna; Calvo, Maria; Rodrigo-Torres, Daniel; Sancho-Bru, Pau; Mira, Aurea; Jiménez, Wladimiro; Morales-Ruiz, Manuel

2017-11-01

Rising evidence points to endothelial-to-mesenchymal transition (EndMT) as a significant source of the mesenchymal cell population in fibrotic diseases. In this context, we hypothesized that liver endothelial cells undergo EndMT during fibrosis progression. Cirrhosis in mice was induced by CCl 4 A transgenic mouse expressing a red fluorescent protein reporter under the control of Tie2 promoter (Tie2-tdTomato) was used to trace the acquisition of EndMT. Sinusoidal vascular connectivity was evaluated by intravital microscopy and high-resolution three-dimensional confocal microscopy. A modest but significant fraction of liver endothelial cells from both cirrhotic patients and CCl 4 -treated Tie2-tdTomato mice acquired an EndMT phenotype characterized by the coexpression of CD31 and α-smooth muscle actin, compared with noncirrhotic livers. Bone morphogenetic protein-7 (BMP-7) inhibited the acquisition of EndMT induced by transforming growth factor-β1 (TGF-β1) treatment in cultured primary mouse liver endothelial cells from control mice. EndMT was also reduced significantly in vivo in cirrhotic Tie2-tdTomato mice treated intraperitoneally with BMP-7 compared with untreated mice (1.9 ± 0.2 vs. 3.8 ± 0.3%, respectively; P < 0.05). The decrease of EndMT in cirrhotic livers correlated with a significant decrease in liver fibrosis ( P < 0.05) and an improvement in the vascular disorganization rate ( P < 0.05). We demonstrated the acquisition of the EndMT phenotype by a subpopulation of endothelial cells from cirrhotic livers in both animal models and patients. BMP-7 treatment decreases the occurrence of the EndMT phenotype and has a positive impact on the severity of disease by reducing fibrosis and sinusoidal vascular disorganization. NEW & NOTEWORTHY A subpopulation of liver endothelial cells from cirrhotic patients and mice with liver fibrosis undergoes endothelial-to-mesenchymal transition. Liver endothelial cells from healthy mice could transition into a mesenchymal phenotype in culture in response to TGF-β1 treatment. Fibrotic livers treated chronically with BMP-7 showed lower EndMT acquisition, reduced fibrosis, and improved vascular organization. Copyright © 2017 the American Physiological Society.

Vinpocetine Attenuates Pathological Cardiac Remodeling by Inhibiting Cardiac Hypertrophy and Fibrosis

PubMed Central

Wu, Mei-ping; Zhang, Yi-shuai; Xu, Xiangbin; Zhou, Qian

2017-01-01

Purpose Pathological cardiac remodeling, characterized by cardiac hypertrophy and fibrosis, is a pathological feature of many cardiac disorders that leads to heart failure and cardiac arrest. Vinpocetine, a derivative of the alkaloid vincamine, has been used for enhancing cerebral blood flow to treat cognitive impairment. However, its role in pathological cardiac remodeling remains unknown. The aim of this study is to examine the effect of vinpocetine on pathological cardiac remodeling induced by chronic stimulation with angiotensin II (Ang II). Methods Mice received Ang II infusion via osmotic pumps in the presence of vehicle or vinpocetine. Cardiac hypertrophy and fibrosis were assessed by morphological, histological, and biochemical analyses. Mechanistic studies were carried out in vitro with isolated mouse adult cardiac myocytes and fibroblasts. Results We showed that chronic Ang II infusion caused cardiac hypertrophy and fibrosis, which were all significantly attenuated by systemic administration of vinpocetine. In isolated adult mouse cardiomyocytes, vinpocetine suppressed Ang II-stimulated myocyte hypertrophic growth. In cultured cardiac fibroblasts, vinpocetine suppressed TGFβ-induced fibroblast activation and matrix gene expression, consistent with its effect in attenuating cardiac fibrosis. The effects of vinpocetine on cardiac myocyte hypertrophy and fibroblast activation are likely mediated by targeting cyclic nucleotide phosphodiesterase 1 (PDE1). Conclusions Our results reveal a novel protective effect of vinpocetine in attenuating pathological cardiac remodeling through suppressing cardiac myocyte hypertrophic growth and fibroblast activation and fibrotic gene expression. These studies may also shed light on developing novel therapeutic agents for antagonizing pathological cardiac remodeling. PMID:28321644

Vinpocetine Attenuates Pathological Cardiac Remodeling by Inhibiting Cardiac Hypertrophy and Fibrosis.

PubMed

Wu, Mei-Ping; Zhang, Yi-Shuai; Xu, Xiangbin; Zhou, Qian; Li, Jian-Dong; Yan, Chen

2017-04-01

Pathological cardiac remodeling, characterized by cardiac hypertrophy and fibrosis, is a pathological feature of many cardiac disorders that leads to heart failure and cardiac arrest. Vinpocetine, a derivative of the alkaloid vincamine, has been used for enhancing cerebral blood flow to treat cognitive impairment. However, its role in pathological cardiac remodeling remains unknown. The aim of this study is to examine the effect of vinpocetine on pathological cardiac remodeling induced by chronic stimulation with angiotensin II (Ang II). Mice received Ang II infusion via osmotic pumps in the presence of vehicle or vinpocetine. Cardiac hypertrophy and fibrosis were assessed by morphological, histological, and biochemical analyses. Mechanistic studies were carried out in vitro with isolated mouse adult cardiac myocytes and fibroblasts. We showed that chronic Ang II infusion caused cardiac hypertrophy and fibrosis, which were all significantly attenuated by systemic administration of vinpocetine. In isolated adult mouse cardiomyocytes, vinpocetine suppressed Ang II-stimulated myocyte hypertrophic growth. In cultured cardiac fibroblasts, vinpocetine suppressed TGFβ-induced fibroblast activation and matrix gene expression, consistent with its effect in attenuating cardiac fibrosis. The effects of vinpocetine on cardiac myocyte hypertrophy and fibroblast activation are likely mediated by targeting cyclic nucleotide phosphodiesterase 1 (PDE1). Our results reveal a novel protective effect of vinpocetine in attenuating pathological cardiac remodeling through suppressing cardiac myocyte hypertrophic growth and fibroblast activation and fibrotic gene expression. These studies may also shed light on developing novel therapeutic agents for antagonizing pathological cardiac remodeling.

Childhood-Adolescent Obesity in the Cardiorenal Syndrome: Lessons from Animal Models

PubMed Central

Hayden, Melvin R.; Sowers, James R.

2011-01-01

Background/Aims Childhood-adolescent overweight and obesity have grown to pandemic proportions during the past decade. The onset of obesity in younger adults will likely be manifested as earlier onset of myocardial and renal end-organ disease in younger adults. For the first time, it is estimated that the current generation may not live to be as old as their parents. Thus, it is important to develop animal models of childhood obesity to understand fundamental pathological organ changes. Methods In this regard, we utilize transmission electron microscopy evaluation to evaluate early remodeling changes of two adolescent rodent obesity models: the Zucker obese (fa/fa) rat and the db/db mouse models of obesity. We have concentrated on the initial ultrastructural remodeling (obese adipose tissue, skeletal muscle, and islet remodeling) and the associated changes in target end organs (including the myocardium and kidney) in young rodent models of obesity and insulin resistance, collectively manifesting as the cardiorenal metabolic syndrome (CRS). Results Briefly, tissues revealed the following ultrastructural remodeling abnormalities: inflammation, hypertrophy, and early fibrosis in adipose tissue; loss of mitochondria in skeletal muscles, hyperplasia, fibrosis, and depletion of insulin-secretory granules in pancreatic islets; increased intramyocardial lipid accumulation, fibrosis, and mitochondrial deposition in the myocardium, and obesity-related glomerulopathy and tubulopathy in the kidney. Conclusion Based on the current knowledge and ultrastructural observations of organ pathology, we propose mechanisms whereby obesity appears to be the driving force behind the development of the CRS. PMID:22294984

Loss of Cystic Fibrosis Transmembrane Regulator Impairs Intestinal Oxalate Secretion.

PubMed

Knauf, Felix; Thomson, Robert B; Heneghan, John F; Jiang, Zhirong; Adebamiro, Adedotun; Thomson, Claire L; Barone, Christina; Asplin, John R; Egan, Marie E; Alper, Seth L; Aronson, Peter S

2017-01-01

Patients with cystic fibrosis have an increased incidence of hyperoxaluria and calcium oxalate nephrolithiasis. Net intestinal absorption of dietary oxalate results from passive paracellular oxalate absorption as modified by oxalate back secretion mediated by the SLC26A6 oxalate transporter. We used mice deficient in the cystic fibrosis transmembrane conductance regulator gene (Cftr) to test the hypothesis that SLC26A6-mediated oxalate secretion is defective in cystic fibrosis. We mounted isolated intestinal tissue from C57BL/6 (wild-type) and Cftr -/- mice in Ussing chambers and measured transcellular secretion of [ 14 C]oxalate. Intestinal tissue isolated from Cftr -/- mice exhibited significantly less transcellular oxalate secretion than intestinal tissue of wild-type mice. However, glucose absorption, another representative intestinal transport process, did not differ in Cftr -/- tissue. Compared with wild-type mice, Cftr -/- mice showed reduced expression of SLC26A6 in duodenum by immunofluorescence and Western blot analysis. Furthermore, coexpression of CFTR stimulated SLC26A6-mediated Cl - -oxalate exchange in Xenopus oocytes. In association with the profound defect in intestinal oxalate secretion, Cftr -/- mice had serum and urine oxalate levels 2.5-fold greater than those of wild-type mice. We conclude that defective intestinal oxalate secretion mediated by SLC26A6 may contribute to the hyperoxaluria observed in this mouse model of cystic fibrosis. Future studies are needed to address whether similar mechanisms contribute to the increased risk for calcium oxalate stone formation observed in patients with cystic fibrosis. Copyright © 2016 by the American Society of Nephrology.

The antifibrinolytic drug tranexamic acid reduces liver injury and fibrosis in a mouse model of chronic bile duct injury.

PubMed

Joshi, Nikita; Kopec, Anna K; Towery, Keara; Williams, Kurt J; Luyendyk, James P

2014-06-01

Hepatic fibrin deposition has been shown to inhibit hepatocellular injury in mice exposed to the bile duct toxicant α-naphthylisothiocyanate (ANIT). Degradation of fibrin clots by fibrinolysis controls the duration and extent of tissue fibrin deposition. Thus, we sought to determine the effect of treatment with the antifibrinolytic drug tranexamic acid (TA) and plasminogen activator inhibitor-1 (PAI-1) deficiency on ANIT-induced liver injury and fibrosis in mice. Plasmin-dependent lysis of fibrin clots was impaired in plasma from mice treated with TA (1200 mg/kg i.p., administered twice daily). Prophylactic TA administration reduced hepatic inflammation and hepatocellular necrosis in mice fed a diet containing 0.025% ANIT for 2 weeks. Hepatic type 1 collagen mRNA expression and deposition increased markedly in livers of mice fed ANIT diet for 4 weeks. To determine whether TA treatment could inhibit this progression of liver fibrosis, mice were fed ANIT diet for 4 weeks and treated with TA for the last 2 weeks. Interestingly, TA treatment largely prevented increased deposition of type 1 collagen in livers of mice fed ANIT diet for 4 weeks. In contrast, biliary hyperplasia/inflammation and liver fibrosis were significantly increased in PAI-1(-/-) mice fed ANIT diet for 4 weeks. Overall, the results indicate that fibrinolytic activity contributes to ANIT diet-induced liver injury and fibrosis in mice. In addition, these proof-of-principle studies suggest the possibility that therapeutic intervention with an antifibrinolytic drug could form a novel strategy to prevent or reduce liver injury and fibrosis in patients with liver disease.

Myeloid differentiation primary response gene (MyD) 88 signalling is not essential for intestinal fibrosis development.

PubMed

Lutz, C; Weder, B; Hünerwadel, A; Fagagnini, S; Lang, B; Beerenwinkel, N; Rossel, J B; Rogler, G; Misselwitz, B; Hausmann, M

2017-12-15

Dysregulation of the immune response to microbiota is associated with inflammatory bowel disease (IBD), which can trigger intestinal fibrosis. MyD88 is a key component of microbiota signalling but its influence on intestinal fibrosis has not been clarified. Small bowel resections from donor-mice were transplanted subcutaneously into the neck of recipients C57BL/6 B6-MyD88tm1 Aki (MyD88 -/- ) and C57BL/6-Tg(UBC-green fluorescence protein (GFP))30Scha/J (GFP-Tg). Grafts were explanted up to 21 days after transplantation. Collagen layer thickness was determined using Sirius Red stained slides. In the mouse model of fibrosis collagen deposition and transforming growth factor-beta 1 (TGF-β1) expression was equal in MyD88 +/+ and MyD88 -/- , indicating that MyD88 was not essential for fibrogenesis. Matrix metalloproteinase (Mmp)9 expression was significantly decreased in grafts transplanted into MyD88 -/- recipients compared to MyD88 +/+ recipients (0.2 ± 0.1 vs. 153.0 ± 23.1, respectively, p < 0.05), similarly recruitment of neutrophils was significantly reduced (16.3 ± 4.5 vs. 25.4 ± 3.1, respectively, p < 0.05). Development of intestinal fibrosis appears to be independent of MyD88 signalling indicating a minor role of bacterial wall compounds in the process which is in contrast to published concepts and theories. Development of fibrosis appears to be uncoupled from acute inflammation.

Differential susceptibility of inbred mouse strains to chlorine-induced airway fibrosis

PubMed Central

Mo, Yiqun; Chen, Jing; Schlueter, Connie F.

2013-01-01

Chlorine is a reactive gas that is considered a chemical threat agent. Humans who develop acute lung injury from chlorine inhalation typically recover normal lung function; however, a subset can experience chronic airway disease. To examine pathological changes following chlorine-induced lung injury, mice were exposed to a single high dose of chlorine, and repair of the lung was analyzed at multiple times after exposure. In FVB/NJ mice, chlorine inhalation caused pronounced fibrosis of larger airways that developed by day 7 after exposure and was associated with airway hyperreactivity. In contrast, A/J mice had little or no airway fibrosis and had normal lung function at day 7. Unexposed FVB/NJ mice had less keratin 5 staining (basal cell marker) than A/J mice in large intrapulmonary airways where epithelial repair was poor and fibrosis developed after chlorine exposure. FVB/NJ mice had large areas devoid of epithelium on day 1 after exposure leading to fibroproliferative lesions on days 4 and 7. A/J mice had airways covered by squamous keratin 5-stained cells on day 1 that transitioned to a highly proliferative reparative epithelium by day 4 followed by the reappearance of ciliated and Clara cells by day 7. The data suggest that lack of basal cells in the large intrapulmonary airways and failure to effect epithelial repair at these sites are factors contributing to the development of airway fibrosis in FVB/NJ mice. The observed differences in susceptibility to chlorine-induced airway disease provide a model in which mechanisms and treatment of airway fibrosis can be investigated. PMID:23171502

Differential susceptibility of inbred mouse strains to chlorine-induced airway fibrosis.

PubMed

Mo, Yiqun; Chen, Jing; Schlueter, Connie F; Hoyle, Gary W

2013-01-15

Chlorine is a reactive gas that is considered a chemical threat agent. Humans who develop acute lung injury from chlorine inhalation typically recover normal lung function; however, a subset can experience chronic airway disease. To examine pathological changes following chlorine-induced lung injury, mice were exposed to a single high dose of chlorine, and repair of the lung was analyzed at multiple times after exposure. In FVB/NJ mice, chlorine inhalation caused pronounced fibrosis of larger airways that developed by day 7 after exposure and was associated with airway hyperreactivity. In contrast, A/J mice had little or no airway fibrosis and had normal lung function at day 7. Unexposed FVB/NJ mice had less keratin 5 staining (basal cell marker) than A/J mice in large intrapulmonary airways where epithelial repair was poor and fibrosis developed after chlorine exposure. FVB/NJ mice had large areas devoid of epithelium on day 1 after exposure leading to fibroproliferative lesions on days 4 and 7. A/J mice had airways covered by squamous keratin 5-stained cells on day 1 that transitioned to a highly proliferative reparative epithelium by day 4 followed by the reappearance of ciliated and Clara cells by day 7. The data suggest that lack of basal cells in the large intrapulmonary airways and failure to effect epithelial repair at these sites are factors contributing to the development of airway fibrosis in FVB/NJ mice. The observed differences in susceptibility to chlorine-induced airway disease provide a model in which mechanisms and treatment of airway fibrosis can be investigated.

Fibroblast Activation Protein (FAP) Accelerates Collagen Degradation and Clearance from Lungs in Mice*

PubMed Central

Fan, Ming-Hui; Zhu, Qiang; Li, Hui-Hua; Ra, Hyun-Jeong; Majumdar, Sonali; Gulick, Dexter L.; Jerome, Jacob A.; Madsen, Daniel H.; Christofidou-Solomidou, Melpo; Speicher, David W.; Bachovchin, William W.; Feghali-Bostwick, Carol; Puré, Ellen

2016-01-01

Idiopathic pulmonary fibrosis is a disease characterized by progressive, unrelenting lung scarring, with death from respiratory failure within 2–4 years unless lung transplantation is performed. New effective therapies are clearly needed. Fibroblast activation protein (FAP) is a cell surface-associated serine protease up-regulated in the lungs of patients with idiopathic pulmonary fibrosis as well as in wound healing and cancer. We postulate that FAP is not only a marker of disease but influences the development of pulmonary fibrosis after lung injury. In two different models of pulmonary fibrosis, intratracheal bleomycin instillation and thoracic irradiation, we find increased mortality and increased lung fibrosis in FAP-deficient mice compared with wild-type mice. Lung extracellular matrix analysis reveals accumulation of intermediate-sized collagen fragments in FAP-deficient mouse lungs, consistent with in vitro studies showing that FAP mediates ordered proteolytic processing of matrix metalloproteinase (MMP)-derived collagen cleavage products. FAP-mediated collagen processing leads to increased collagen internalization without altering expression of the endocytic collagen receptor, Endo180. Pharmacologic FAP inhibition decreases collagen internalization as expected. Conversely, restoration of FAP expression in the lungs of FAP-deficient mice decreases lung hydroxyproline content after intratracheal bleomycin to levels comparable with that of wild-type controls. Our findings indicate that FAP participates directly, in concert with MMPs, in collagen catabolism and clearance and is an important factor in resolving scar after injury and restoring lung homeostasis. Our study identifies FAP as a novel endogenous regulator of fibrosis and is the first to show FAP's protective effects in the lung. PMID:26663085

A synthetic PPAR-γ agonist triterpenoid ameliorates experimental fibrosis: PPAR-γ-independent suppression of fibrotic responses.

PubMed

Wei, Jun; Zhu, Hongyan; Komura, Kazuhiro; Lord, Gabriel; Tomcik, Michal; Wang, Wenxia; Doniparthi, Sruthi; Tamaki, Zenshiro; Hinchcliff, Monique; Distler, Joerg H W; Varga, John

2014-02-01

Persistent fibroblast activation initiated by transforming growth factor β (TGF-β) is a fundamental event in the pathogenesis of systemic sclerosis, and its pharmacological inhibition represents a potential therapeutic strategy. The nuclear receptor, peroxisome proliferator-activated receptor γ (PPAR-γ), exerts potent fibrotic activity. The synthetic oleanane triterpenoid, 2-cyano-3,12-dioxo-olean-1,9-dien-28-oic acid (CDDO), is a PPAR-γ agonist with potential effects on TGF-β signalling and dermal fibrosis. To examine the modulation of fibrogenesis by CDDO in explanted fibroblasts, skin organ cultures and murine models of scleroderma. The effects of CDDO on experimental fibrosis induced by bleomycin injection or by overexpression of constitutively active type I TGF-β receptor (TgfbR1ca) were evaluated. Modulation of fibrotic gene expression was examined in human skin organ cultures. To delineate the mechanisms underlying the antifibrotic effects of CDDO, explanted skin fibroblasts cultured in two-dimensional monolayers or in three-dimensional full-thickness human skin equivalents were studied. CDDO significantly ameliorated dermal fibrosis in two complementary mouse models of scleroderma, as well as in human skin organ cultures and in three-dimensional human skin equivalents. In two-dimensional monolayer cultures of explanted normal fibroblasts, CDDO abrogated fibrogenic responses induced by TGF-β. These CDDO effects occurred via disruption of Smad-dependent transcription and were associated with inhibition of Akt activation. In scleroderma fibroblasts, CDDO attenuated the elevated synthesis of collagen. Remarkably, the in vitro antifibrotic effects of CDDO were independent of PPAR-γ. The PPAR-γ agonist triterpenoid CDDO attenuates fibrogenesis by antagonistically targeting canonical TGF-β/Smad and Akt signalling in a PPAR-γ-independent manner. These findings identify this synthetic triterpenoid as a potential new therapy for the control of fibrosis.

Cannabidiol attenuates cardiac dysfunction, oxidative stress, fibrosis, inflammatory and cell death signaling pathways in diabetic cardiomyopathy

PubMed Central

Rajesh, Mohanraj; Mukhopadhyay, Partha; Bátkai, Sándor; Patel, Vivek; Saito, Keita; Matsumoto, Shingo; Kashiwaya, Yoshihiro; Horváth, Béla; Mukhopadhyay, Bani; Becker, Lauren; Haskó, György; Liaudet, Lucas; Wink, David A; Veves, Aristidis; Mechoulam, Raphael; Pacher, Pál

2010-01-01

Objectives In this study, we have investigated the effects of cannabidiol (CBD) on myocardial dysfunction, inflammation, oxidative/nitrosative stress, cell death and interrelated signaling pathways, using a mouse model of type I diabetic cardiomyopathy and primary human cardiomyocytes exposed to high glucose. Background CBD, the most abundant nonpsychoactive constituent of Cannabis sativa (marijuana) plant, exerts antiinflammatory effects in various disease models and alleviates pain and spasticity associated with multiple sclerosis in humans. Methods Left ventricular function was measured by pressure-volume system. Oxidative stress, cell death and fibrosis markers were evaluated by molecular biology/biochemical techniques, electron spin resonance spectroscopy and flow cytometry. Results Diabetic cardiomyopathy was characterized by declined diastolic and systolic myocardial performance associated with increased oxidative-nitrosative stress, NF-κB and MAPK (JNK and p-38, p38α) activation, enhanced expression of adhesion molecules (ICAM-1, VCAM-1), TNF-α, markers of fibrosis (TGF-β, CTGF, fibronectin, collagen-1, MMP-2 and MMP-9), enhanced cell death (caspase 3/7 and PARP activity, chromatin fragmentation and TUNEL) and diminished Akt phosphorylation. Remarkably, CBD attenuated myocardial dysfunction, cardiac fibrosis, oxidative/nitrosative stress, inflammation, cell death, and interrelated signaling pathways. Furthermore, CBD also attenuated the high glucose-induced increased reactive oxygen species generation, NF-κB activation and cell death in primary human cardiomyocytes. Conclusions Collectively, these results coupled with the excellent safety and tolerability profile of cannabidiol in humans, strongly suggest that it may have great therapeutic potential in the treatment of diabetic complications, and perhaps other cardiovascular disorders, by attenuating oxidative/nitrosative stress, inflammation, cell death and fibrosis. PMID:21144973

CCN3/CCN2 regulation and the fibrosis of diabetic renal disease.

PubMed

Riser, Bruce L; Najmabadi, Feridoon; Perbal, Bernard; Rambow, Jo Ann; Riser, Melisa L; Sukowski, Ernest; Yeger, Herman; Riser, Sarah C; Peterson, Darryl R

2010-03-01

Prior work in the CCN field, including our own, suggested to us that there might be co-regulatory activity and function as part of the actions of this family of cysteine rich cytokines. CCN2 is now regarded as a major pro-fibrotic molecule acting both down-stream and independent of TGF-beta1, and appears causal in the disease afflicting multiple organs. Since diabetic renal fibrosis is a common complication of diabetes, and a major cause of end stage renal disease (ESRD), we examined the possibility that CCN3 (NOV), might act as an endogenous negative regulator of CCN2 with the capacity to limit the overproduction of extracellular matrix (ECM), and thus prevent, or ameliorate fibrosis. We demonstrate, using an in vitro model of diabetic renal fibrosis, that both exogenous treatment with CCN3 and transfection with the over-expression of the CCN3 gene in mesangial cells markedly down-regulates CCN2 activity and blocks ECM over-accumulation stimulated by TGF-beta1. Conversely, TGF-beta1 treatment reduces endogenous CCN3 expression and increases CCN2 activity and matrix accumulation, indicating an important, novel yin/yang effect. Using the db/db mouse model of diabetic nephropathy, we confirm the expression of CCN3 in the kidney, with temporal localization that supports these in vitro findings. In summary, the results corroborate our hypothesis that one function of CCN3 is to regulate CCN2 activity and at the concentrations and conditions used down-regulates the effects of TGF-beta1, acting to limit ECM turnover and fibrosis in vivo. The findings suggest opportunities for novel endogenous-based therapy either by the administration, or the upregulation of CCN3.

Inhibition of Reticulon-1A-Mediated Endoplasmic Reticulum Stress in Early AKI Attenuates Renal Fibrosis Development.

PubMed

Fan, Ying; Xiao, Wenzhen; Lee, Kyung; Salem, Fadi; Wen, Jiejun; He, Li; Zhang, Jing; Fei, Yang; Cheng, Dongsheng; Bao, Hongda; Liu, Yumei; Lin, Fujun; Jiang, Gengru; Guo, Zhiyong; Wang, Niansong; He, John Cijiang

2017-07-01

Several animal studies have shown an important role for endoplasmic reticulum (ER) stress in AKI, whereas human studies are lacking. We recently reported that Reticulon-1A (RTN1A) is a key mediator of ER stress and kidney cell injury. Here, we investigated whether modulation of RTN1A expression during AKI contributes to the progression to CKD. In a retrospective study of 51 patients with AKI, increased expression of RTN1A and other ER stress markers were associated with the severity of kidney injury and with progression to CKD. In an inducible tubular cell-specific RTN1A-knockdown mouse model subjected to folic acid nephropathy (FAN) or aristolochic acid nephropathy, reduction of RTN1A expression during the initial stage of AKI attenuated ER stress and kidney cell injury in early stages and renal fibrosis development in later stages. Treatment of wild-type mice with tauroursodeoxycholic acid, an inhibitor of ER stress, after the induction of kidney injury with FA facilitated renoprotection similar to that observed in RTN1A-knockdown mice. Conversely, in transgenic mice with inducible tubular cell-specific overexpression of RTN1A subjected to FAN, induction of RTN1A overexpression aggravated ER stress and renal injury at the early stage and renal fibrosis at the late stage of FAN. Together, our human and mouse data suggest that the RTN1A-mediated ER stress response may be an important determinant in the severity of AKI and maladaptive repair that may promote progression to CKD. Copyright © 2017 by the American Society of Nephrology.

The ameliorative effect of silibinin against radiation-induced lung injury: protection of normal tissue without decreasing therapeutic efficacy in lung cancer.

PubMed

Son, Yeonghoon; Lee, Hae June; Rho, Jin Kyung; Chung, Soo Young; Lee, Chang Geun; Yang, Kwangmo; Kim, Sung Ho; Lee, Minyoung; Shin, In Sik; Kim, Joong Sun

2015-07-05

Silibinin has been known for its role in anti-cancer and radio-protective effect. Radiation therapy for treating lung cancer might lead to late-phase pulmonary inflammation and fibrosis. Thus, this study aimed to investigate the effects of silibinin in radiation-induced lung injury with a mouse model. In this study, we examined the ability of silibinin to mitigate lung injury in, and improve survival of, C57BL/6 mice given 13 Gy thoracic irradiation and silibinin treatments orally at 100 mg/kg/day for seven days after irradiation. In addition, Lewis lung cancer (LLC) cells were injected intravenously in C57BL/6 mice to generate lung tumor nodules. Lung tumor-bearing mice were treated with lung radiation therapy at 13 Gy and with silibinin at a dose of 100 mg/day for seven days after irradiation. Silibinin was shown to increase mouse survival, to ameliorate radiation-induced hemorrhage, inflammation and fibrosis in lung tissue, to reduce the number of inflammatory cells in the bronchoalveolar lavage fluid (BALF) and to reduce inflammatory cell infiltration in the respiratory tract. In LLC tumor injected mice, lung tissue from mice treated with both radiation and silibinin showed no differences compared to lung tissue from mice treated with radiation alone. Silibinin treatment mitigated the radiation-induced lung injury possibly by reducing inflammation and fibrosis, which might be related with the improved survival rate. Silibinin might be a useful agent for lung cancer patients as a non-toxic complementary approach to alleviate the side effects by thorax irradiation.

TGF-β mediates early angiogenesis and latent fibrosis in an Emilin1-deficient mouse model of aortic valve disease

PubMed Central

Munjal, Charu; Opoka, Amy M.; Osinska, Hanna; James, Jeanne F.; Bressan, Giorgio M.; Hinton, Robert B.

2014-01-01

Aortic valve disease (AVD) is characterized by elastic fiber fragmentation (EFF), fibrosis and aberrant angiogenesis. Emilin1 is an elastin-binding glycoprotein that regulates elastogenesis and inhibits TGF-β signaling, but the role of Emilin1 in valve tissue is unknown. We tested the hypothesis that Emilin1 deficiency results in AVD, mediated by non-canonical (MAPK/phosphorylated Erk1 and Erk2) TGF-β dysregulation. Using histology, immunohistochemistry, electron microscopy, quantitative gene expression analysis, immunoblotting and echocardiography, we examined the effects of Emilin1 deficiency (Emilin1−/−) in mouse aortic valve tissue. Emilin1 deficiency results in early postnatal cell-matrix defects in aortic valve tissue, including EFF, that progress to latent AVD and premature death. The Emilin1−/− aortic valve displays early aberrant provisional angiogenesis and late neovascularization. In addition, Emilin1−/− aortic valves are characterized by early valve interstitial cell activation and proliferation and late myofibroblast-like cell activation and fibrosis. Interestingly, canonical TGF-β signaling (phosphorylated Smad2 and Smad3) is upregulated constitutively from birth to senescence, whereas non-canonical TGF-β signaling (phosphorylated Erk1 and Erk2) progressively increases over time. Emilin1 deficiency recapitulates human fibrotic AVD, and advanced disease is mediated by non-canonical (MAPK/phosphorylated Erk1 and Erk2) TGF-β activation. The early manifestation of EFF and aberrant angiogenesis suggests that these processes are crucial intermediate factors involved in disease progression and therefore might provide new therapeutic targets for human AVD. PMID:25056700

Accumulation of BDCA1⁺ dendritic cells in interstitial fibrotic lung diseases and Th2-high asthma.

PubMed

Greer, Alexandra M; Matthay, Michael A; Kukreja, Jasleen; Bhakta, Nirav R; Nguyen, Christine P; Wolters, Paul J; Woodruff, Prescott G; Fahy, John V; Shin, Jeoung-Sook

2014-01-01

Dendritic cells (DCs) significantly contribute to the pathology of several mouse lung disease models. However, little is known of the contribution of DCs to human lung diseases. In this study, we examined infiltration with BDCA1⁺ DCs of human lungs in patients with interstitial lung diseases or asthma. Using flow cytometry, we found that these DCs increased by 5∼6 fold in the lungs of patients with idiopathic pulmonary fibrosis or hypersensitivity pneumonitis, which are both characterized by extensive fibrosis in parenchyma. The same DC subset also significantly increased in the lung parenchyma of patients with chronic obstructive pulmonary disease, although the degree of increase was relatively modest. By employing immunofluorescence microscopy using FcεRI and MHCII as the specific markers for BDCA1⁺ DCs, we found that the numbers of BDCA1⁺ DCs also significantly increased in the airway epithelium of Th2 inflammation-associated asthma. These findings suggest a potential contribution of BDCA1⁺ DCs in human lung diseases associated with interstitial fibrosis or Th2 airway inflammation.

Muscle Weakness and Fibrosis Due to Cell Autonomous and Non-cell Autonomous Events in Collagen VI Deficient Congenital Muscular Dystrophy.

PubMed

Noguchi, Satoru; Ogawa, Megumu; Malicdan, May Christine; Nonaka, Ikuya; Nishino, Ichizo

2017-02-01

Congenital muscular dystrophies with collagen VI deficiency are inherited muscle disorders with a broad spectrum of clinical presentation and are caused by mutations in one of COL6A1-3 genes. Muscle pathology is characterized by fiber size variation and increased interstitial fibrosis and adipogenesis. In this study, we define critical events that contribute to muscle weakness and fibrosis in a mouse model with collagen VI deficiency. The Col6a1 GT/GT mice develop non-progressive weakness from younger age, accompanied by stunted muscle growth due to reduced IGF-1 signaling activity. In addition, the Col6a1 GT/GT mice have high numbers of interstitial skeletal muscle mesenchymal progenitor cells, which dramatically increase with repeated myofiber necrosis/regeneration. Our results suggest that impaired neonatal muscle growth and the activation of the mesenchymal cells in skeletal muscles contribute to the pathology of collagen VI deficient muscular dystrophy, and more importantly, provide the insights on the therapeutic strategies for collagen VI deficiency. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Inhaled ENaC antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice.

PubMed

Crosby, Jeff R; Zhao, Chenguang; Jiang, Chong; Bai, Dong; Katz, Melanie; Greenlee, Sarah; Kawabe, Hiroshi; McCaleb, Michael; Rotin, Daniela; Guo, Shuling; Monia, Brett P

2017-11-01

Epithelial sodium channel (ENaC, Scnn1) hyperactivity in the lung leads to airway surface dehydration and mucus accumulation in cystic fibrosis (CF) patients and in mice with CF-like lung disease. We identified several potent ENaC specific antisense oligonucleotides (ASOs) and tested them by inhalation in mouse models of CF-like lung disease. The inhaled ASOs distributed into lung airway epithelial cells and decreased ENaC expression by inducing RNase H1-dependent degradation of the targeted Scnn1a mRNA. Aerosol delivered ENaC ASO down-regulated mucus marker expression and ameliorated goblet cell metaplasia, inflammation, and airway hyper-responsiveness. Lack of systemic activity of ASOs delivered via the aerosol route ensures the safety of this approach. Our results demonstrate that antisense inhibition of ENaC in airway epithelial cells could be an effective and safe approach for the prevention and reversal of lung symptoms in CF and potentially other inflammatory diseases of the lung. Copyright © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Low-dose cadmium exposure exacerbates polyhexamethylene guanidine-induced lung fibrosis in mice.

PubMed

Kim, Min-Seok; Kim, Sung-Hwan; Jeon, Doin; Kim, Hyeon-Young; Han, Jin-Young; Kim, Bumseok; Lee, Kyuhong

2018-01-01

Cadmium (Cd) is a toxic metal present in tobacco smoke, air, food, and water. Inhalation is an important route of Cd exposure, and lungs are one of the main target organs for metal-induced toxicity. Cd inhalation is associated with an increased risk of pulmonary diseases. The present study aimed to assess the effects of repeated exposure to low-dose Cd in a mouse model of polyhexamethylene guanidine (PHMG)-induced lung fibrosis. Mice were grouped into the following groups: vehicle control (VC), PHMG, cadmium chloride (CdCl 2 ), and PHMG + CdCl 2 . Animals in the PHMG group exhibited increased numbers of total cells and inflammatory cells in the bronchoalveolar lavage fluid (BALF) accompanied by inflammation and fibrosis in lung tissues. These parameters were exacerbated in mice in the PHMG + CdCl 2 group. In contrast, mice in the CdCl 2 group alone displayed only minimal inflammation in pulmonary tissue. Expression of inflammatory cytokines and fibrogenic mediators was significantly elevated in lungs of mice in the PHMG group compared with that VC. Further, expression of these cytokines and mediators was enhanced in pulmonary tissue in mice administered PHMG + CdCl 2 . Data demonstrate that repeated exposure to low-dose Cd may enhance the development of PHMG-induced pulmonary fibrosis.

Asbestos and erionite prime and activate the NLRP3 inflammasome that stimulates autocrine cytokine release in human mesothelial cells

PubMed Central

2013-01-01

Background Pleural fibrosis and malignant mesotheliomas (MM) occur after exposures to pathogenic fibers, yet the mechanisms initiating these diseases are unclear. Results We document priming and activation of the NLRP3 inflammasome in human mesothelial cells by asbestos and erionite that is causally related to release of IL-1β, IL-6, IL-8, and Vascular Endothelial Growth Factor (VEGF). Transcription and release of these proteins are inhibited in vitro using Anakinra, an IL-1 receptor antagonist that reduces these cytokines in a human peritoneal MM mouse xenograft model. Conclusions These novel data show that asbestos-induced priming and activation of the NLRP3 inflammasome triggers an autocrine feedback loop modulated via the IL-1 receptor in mesothelial cell type targeted in pleural infection, fibrosis, and carcinogenesis. PMID:23937860

Anti-fibrotic potential of human umbilical cord mononuclear cells and mouse bone marrow cells in CCl4- induced liver fibrosis in mice.

PubMed

Elmahdy, Nageh Ahmed; Sokar, Samia Salem; Salem, Mohamed Labib; Sarhan, Naglaa Ibrahim; Abou-Elela, Sherin Hamed

2017-05-01

Liver fibrosis is the consequence of hepatocyte injury that leads to the activation of hepatic stellate cells (HSC). The treatment of choice is Liver transplantation; however, it has many problems such as surgery-related complications, immunological rejection and high costs associated with the procedure. Stem cell-based therapy would be a potential alternative, so the aim of this study is to investigate the therapeutic potential of human umbilical cord mononuclear cells (MNC) and mouse bone marrow cells (BMC) against carbon tetrachloride (CCl 4 ) induced liver fibrosis in mice and compare it with that of silymarin. In the present study, male albino mice (N=60) were divided into six groups (10 mice each), the first group served as the normal control group while the remaining five groups were rendered fibrotic by intraperitoneal injections of CCl 4 and being left for 6 weeks to develop hepatic fibrosis. Thereafter, the mice were divided into CCl 4 group, CCl 4 group receiving MNC or BMC or silymarin or MNC and silymarin combination. After the specified treatment period, animals were then euthanized, blood and tissue samples were collected for measurement of alanine aminotransferase(ALT), aspartate aminotransferase(AST), malondialdehyde(MDA), reduced glutathione(GSH), collagen, Laminin, transforming growth factor β1(TGFβ1), tumor necrosis factor alpha(TNFα). MNC, BMC, and the combination therapy showed a significant decrease in ALT, AST, MDA, collagen, Laminin, TGFβ1, and TNFα and a significant increase in GSH. The data displayed a similar regression of fibrosis with the histological and immunohistological parameters. In conclusion, MNC, BMC and the combination therapy showed a potential therapeutic effect against liver fibrosis via reducing oxidative stress, inflammatory mediators, and fibrogenic markers. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Secreted Klotho Attenuates Inflammation-Associated Aortic Valve Fibrosis in Senescence-Accelerated Mice P1.

PubMed

Chen, Jianglei; Fan, Jun; Wang, Shirley; Sun, Zhongjie

2018-05-01

Senescence-accelerated mice P1 (SAMP1) is an aging model characterized by shortened lifespan and early signs of senescence. Klotho is an aging-suppressor gene. The purpose of this study is to investigate whether in vivo expression of secreted klotho ( Skl ) gene attenuates aortic valve fibrosis in SAMP1 mice. SAMP1 mice and age-matched (AKR/J) control mice were used. SAMP1 mice developed obvious fibrosis in aortic valves, namely fibrotic aortic valve disease. Serum level of Skl was decreased drastically in SAMP1 mice. Expression of MCP-1 (monocyte chemoattractant protein 1), ICAM-1 (intercellular adhesion molecule 1), F4/80, and CD68 was increased in aortic valves of SAMP1 mice, indicating inflammation. An increase in expression of α-smooth muscle actin (myofibroblast marker), transforming growth factorβ-1, and scleraxis (a transcription factor of collagen synthesis) was also found in aortic valves of SAMP1 mice, suggesting that accelerated aging is associated with myofibroblast transition and collagen gene activation. We constructed adeno-associated virus 2 carrying mouse Skl cDNA for in vivo expression of Skl. Skl gene delivery effectively increased serum Skl of SAMP1 mice to the control level. Skl gene delivery inhibited inflammation and myofibroblastic transition in aortic valves and attenuated fibrotic aortic valve disease in SAMP1 mice. It is concluded that senescence-related fibrotic aortic valve disease in SAMP1 mice is associated with a decrease in serum klotho leading to inflammation, including macrophage infiltration and transforming growth factorβ-1/scleraxis-driven myofibroblast differentiation in aortic valves. Restoration of serum Skl levels by adeno-associated virus 2 carrying mouse Skl cDNA effectively suppresses inflammation and myofibroblastic transition and attenuates aortic valve fibrosis. Skl may be a potential therapeutic target for fibrotic aortic valve disease. © 2018 American Heart Association, Inc.

Longitudinal assessment of bleomycin-induced lung fibrosis by Micro-CT correlates with histological evaluation in mice.

PubMed

Ruscitti, Francesca; Ravanetti, Francesca; Essers, Jeroen; Ridwan, Yanto; Belenkov, Sasha; Vos, Wim; Ferreira, Francisca; KleinJan, Alex; van Heijningen, Paula; Van Holsbeke, Cedric; Cacchioli, Antonio; Villetti, Gino; Stellari, Franco Fabio

2017-01-01

The intratracheal instillation of bleomycin in mice induces early damage to alveolar epithelial cells and development of inflammation followed by fibrotic tissue changes and represents the most widely used model of pulmonary fibrosis to investigate human IPF. Histopathology is the gold standard for assessing lung fibrosis in rodents, however it precludes repeated and longitudinal measurements of disease progression and does not provide information on spatial and temporal distribution of tissue damage. Here we investigated the use of the Micro-CT technique to allow the evaluation of disease onset and progression at different time-points in the mouse bleomycin model of lung fibrosis. Micro-CT was throughout coupled with histological analysis for the validation of the imaging results. In bleomycin-instilled and control mice, airways and lung morphology changes were assessed and reconstructed at baseline, 7, 14 and 21 days post-treatment based on Micro-CT images. Ashcroft score, percentage of collagen content and percentage of alveolar air area were detected on lung slides processed by histology and subsequently compared with Micro-CT parameters. Extent (%) of fibrosis measured by Micro-CT correlated with Ashcroft score, the percentage of collagen content and the percentage of alveolar air area ( r 2  = 0.91; 0.77; 0.94, respectively). Distal airway radius also correlated with the Ashcroft score, the collagen content and alveolar air area percentage ( r 2  = 0.89; 0.78; 0.98, respectively). Micro-CT data were in good agreement with histological read-outs as micro-CT was able to quantify effectively and non-invasively disease progression longitudinally and to reduce the variability and number of animals used to assess the damage. This suggests that this technique is a powerful tool for understanding experimental pulmonary fibrosis and that its use could translate into a more efficient drug discovery process, also helping to fill the gap between preclinical setting and clinical practice.

Obstructive Sleep Apnea and Non-Alcoholic Fatty Liver Disease: Is the Liver Another Target?

PubMed Central

Mirrakhimov, Aibek E.; Polotsky, Vsevolod Y.

2012-01-01

Obstructive sleep apnea (OSA) is recurrent obstruction of the upper airway during sleep leading to intermittent hypoxia (IH). OSA has been associated with all components of the metabolic syndrome as well as with non-alcoholic fatty liver disease (NAFLD). NAFLD is a common condition ranging in severity from uncomplicated hepatic steatosis to steatohepatitis (NASH), liver fibrosis, and cirrhosis. The gold standard for the diagnosis and staging of NAFLD is liver biopsy. Obesity and insulin resistance lead to liver steatosis, but the causes of the progression to NASH are not known. Emerging evidence suggests that OSA may play a role in the progression of hepatic steatosis and the development of NASH. Several cross-sectional studies showed that the severity of IH in patients with OSA predicted the severity of NAFLD on liver biopsy. However, neither prospective nor interventional studies with continuous positive airway pressure treatment have been performed. Studies in a mouse model showed that IH causes triglyceride accumulation in the liver and liver injury as well as hepatic inflammation. The mouse model provided insight in the pathogenesis of liver injury showing that (1) IH accelerates the progression of hepatic steatosis by inducing adipose tissue lipolysis and increasing free fatty acids (FFA) flux into the liver; (2) IH up-regulates lipid biosynthetic pathways in the liver; (3) IH induces oxidative stress in the liver; (4) IH up-regulates hypoxia inducible factor 1 alpha and possibly HIF-2 alpha, which may increase hepatic steatosis and induce liver inflammation and fibrosis. However, the role of FFA and different transcription factors in the pathogenesis of IH-induced NAFLD is yet to be established. Thus, multiple lines of evidence suggest that IH of OSA may contribute to the progression of NAFLD but definitive clinical studies and experiments in the mouse model have yet to be done. PMID:23087670

Sildenafil reduces respiratory muscle weakness and fibrosis in the mdx mouse model of Duchenne muscular dystrophy.

PubMed

Percival, Justin M; Whitehead, Nicholas P; Adams, Marvin E; Adamo, Candace M; Beavo, Joseph A; Froehner, Stanley C

2012-09-01

Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy caused by mutations in the dystrophin gene. Loss of dystrophin initiates a progressive decline in skeletal muscle integrity and contractile capacity which weakens respiratory muscles including the diaphragm, culminating in respiratory failure, the leading cause of morbidity and mortality in DMD patients. At present, corticosteroid treatment is the primary pharmacological intervention in DMD, but has limited efficacy and adverse side effects. Thus, there is an urgent need for new safe, cost-effective, and rapidly implementable treatments that slow disease progression. One promising new approach is the amplification of nitric oxide-cyclic guanosine monophosphate (NO-cGMP) signalling pathways with phosphodiesterase 5 (PDE5) inhibitors. PDE5 inhibitors serve to amplify NO signalling that is attenuated in many neuromuscular diseases including DMD. We report here that a 14-week treatment of the mdx mouse model of DMD with the PDE5 inhibitor sildenafil (Viagra(®), Revatio(®)) significantly reduced mdx diaphragm muscle weakness without impacting fatigue resistance. In addition to enhancing respiratory muscle contractility, sildenafil also promoted normal extracellular matrix organization. PDE5 inhibition slowed the establishment of mdx diaphragm fibrosis and reduced matrix metalloproteinase-13 (MMP-13) expression. Sildenafil also normalized the expression of the pro-fibrotic (and pro-inflammatory) cytokine tumour necrosis factor α (TNFα). Sildenafil-treated mdx diaphragms accumulated significantly less Evans Blue tracer dye than untreated controls, which is also indicative of improved diaphragm muscle health. We conclude that sildenafil-mediated PDE5 inhibition significantly reduces diaphragm respiratory muscle dysfunction and pathology in the mdx mouse model of Duchenne muscular dystrophy. This study provides new insights into the therapeutic utility of targeting defects in NO-cGMP signalling with PDE5 inhibitors in dystrophin-deficient muscle. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Calcium channel blockers ameliorate iron overload-associated hepatic fibrosis by altering iron transport and stellate cell apoptosis

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

Zhang, Ying

Liver fibrosis is the principal cause of morbidity and mortality in patients with iron overload. Calcium channel blockers (CCBs) can antagonize divalent cation entry into renal and myocardial cells and inhibit fibrogenic gene expression. We investigated the potential of CCBs to resolve iron overload-associated hepatic fibrosis. Kunming mice were assigned to nine groups (n = 8 per group): control, iron overload, deferoxamine, high and low dose verapamil, high and low dose nimodipine, and high and low dose diltiazem. Iron deposition and hepatic fibrosis were measured in mouse livers. Expression levels of molecules associated with transmembrane iron transport were determined bymore » molecular biology approaches. In vitro HSC-T6 cells were randomized into nine groups (the same groups as the mice). Changes in proliferation, apoptosis, and metalloproteinase expression in cells were detected to assess the anti-fibrotic effects of CCBs during iron overload conditions. We found that CCBs reduced hepatic iron content, intracellular iron deposition, the number of hepatic fibrotic areas, collagen expression levels, and hydroxyproline content. CCBs rescued abnormal expression of α1C protein in L-type voltage-dependent calcium channel (LVDCC) and down-regulated divalent metal transporter-1 (DMT-1) expression in mouse livers. In iron-overloaded HSC-T6 cells, CCBs reduced iron deposition, inhibited proliferation, induced apoptosis, and elevated expression of matrix metalloproteinase-13 (MMP-13) and tissue inhibitor of metalloproteinase-1 (TIMP-1). CCBs are potential therapeutic agents that can be used to address hepatic fibrosis during iron overload. They resolve hepatic fibrosis probably correlated with regulating transmembrane iron transport and inhibiting HSC growth. - Highlights: • Calcium channel blockers (CCBs) reduced hepatic iron content. • CCBs decreased hepatic fibrotic areas and collagen expression levels. • CCBs resolve fibrosis by regulating iron transport and inhibiting HSC growth.« less

Sinomenine attenuates renal fibrosis through Nrf2-mediated inhibition of oxidative stress and TGFβ signaling

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

Qin, Tian

Renal fibrosis is the common feature of chronic kidney disease and mainly mediated by TGFβ-associated pro-fibrogenic signaling, which causes excessive extracellular matrix accumulation and successive loss of kidney functions. Sinomenine (SIN), an alkaloid derived from medicinal herb extensively used in treatment of rheumatoid arthritis and various inflammatory disorders, displays renal protective properties in experimental animals; however its pharmacological potency against renal fibrosis is not explored. In this study we report that SIN possesses strong anti-renal fibrosis functions in kidney cell and in mouse fibrotic kidney. SIN beneficially modulated the pro-fibrogenic protein expression in TGFβ-treated kidney cells and attenuated the renalmore » fibrotic pathogenesis incurred by unilateral ureteral obstruction (UUO), which correlated with its activation of Nrf2 signaling - the key defender against oxidative stress with anti-fibrotic potentials. Further investigation on its regulation of Nrf2 downstream events revealed that SIN significantly balanced oxidative stress via improving the expression and activity of anti-oxidant and detoxifying enzymes, and interrupted the pro-fibrogenic signaling of TGFβ/Smad and Wnt/β-catenin. Even more impressively SIN achieved its anti-fibrotic activities in an Nrf2-dependent manner, suggesting that SIN regulation of Nrf2-associated anti-fibrotic activities constitutes a critical component of SIN's renoprotective functions. Collectively our studies have demonstrated a novel anti-fibrotic property of SIN and its upstream events and provided a molecular basis for SIN's potential applications in treatment of renal fibrosis-associated kidney disorders. - Highlights: • Sinomenine has strong potency of inhibiting renal fibrosis in UUO mouse kidney. • Sinomenine attenuates the expression of profibrogenic proteins. • Sinomenine balances renal fibrosis-associated oxidative stress. • Sinomenine mitigates profibrogenic signaling of TGBβ/Smad and Wnt/β-catenin. • Sinomenine exerts anti-renal fibrotic functions mainly via activating Nrf2 pathways.« less

Identification of the Gene for Scleroderma in the Tsk/2 Mouse Strain: Implications for Human Scleroderma Pathogenesis and Subset Distinctions

DTIC Science & Technology

2012-07-01

Philadelphia, PA 19104 1 Jul 2011 - 30 Jun 2012Annual01-07-2012 This project is focused on an animal model of the human disease, systemic sclerosis ...earliest indicator of tight-skin in the tissue Animal model, systemic sclerosis , scleroderma, Tsk2/+, fibrosis, gene, genetics, TGFβ 35 eblanken...the  multiple  clinical parameters of fibrotic disease from birth  onward.   BODY  Milestones were assigned to this proposal, with tasks to be

Liquiritigenin attenuates cardiac injury induced by high fructose-feeding through fibrosis and inflammation suppression.

PubMed

Xie, Xiong-Wei

2017-02-01

Diabetes combined with cardiomyopathy is considered as an essential complication, showing diastolic persistently and causing cardiac injury, which is linked to fibrosis progression and inflammation response. Fibrosis and inflammation response are two markers for cardiomyopathy. Liquiritigenin is a flavanone, isolated from Radix glycyrrhiza, which exhibits various biological properties, including anti-cancer and anti-inflammatory activities. Here, in our study, the protective effects and anti-inflammatory activity of liquiritigenin were explored in mice and cardiac muscle cells treated by fructose to reveal the possible mechanism by which liquiritigenin attenuates cardiac injury. The mice were separated into five groups. The diabetic model of mouse was established with 30% high fructose feeding. Liquiritigenin dramatically reduced the lipid accumulation induced by high fructose diet. Compared to mice only treated with high fructose, mice in the presence of liquiritigenin after fructose feeding developed less cardiac fibrosis with lower levels of alpha smooth muscle-actin (α-SMA), Collagen type I, Collagen type II, TGF-β1 and Procol1a1. Additionally, liquiritigenin markedly down-regulated inflammatory cytokines secretion and phosphorylated NF-κB via inhibiting IKKα/IκBα signaling pathway. Our results indicate that liquiritigenin has a protective role in high fructose feeding-triggered cardiac injury through fibrosis and inflammation response suppression by inactivating NF-κB signaling pathway. Thus, liquiritigenin may be a potential candidate for diabetes-associated cardiac injury. Copyright © 2016. Published by Elsevier Masson SAS.

Reducing endoglin activity limits calcineurin and TRPC-6 expression and improves survival in a mouse model of right ventricular pressure overload.

PubMed

Kapur, Navin K; Qiao, Xiaoying; Paruchuri, Vikram; Mackey, Emily E; Daly, Gerard H; Ughreja, Kishan; Ughreja, Keshan; Morine, Kevin J; Levine, Jonathan; Aronovitz, Mark J; Hill, Nicholas S; Jaffe, Iris Z; Letarte, Michelle; Karas, Richard H

2014-07-11

Right ventricular (RV) failure is a major cause of mortality worldwide and is often a consequence of RV pressure overload (RVPO). Endoglin is a coreceptor for the profibrogenic cytokine, transforming growth factor beta 1 (TGF-β1). TGF-β1 signaling by the canonical transient receptor protein channel 6 (TRPC-6) was recently reported to stimulate calcineurin-mediated myofibroblast transformation, a critical component of cardiac fibrosis. We hypothesized that reduced activity of the TGF-β1 coreceptor, endoglin, limits RV calcineurin expression and improves survival in RVPO. We first demonstrate that endoglin is required for TGF-β1-mediated calcineurin/TRPC-6 expression and up-regulation of alpha-smooth muscle antigen (α-SMA), a marker of myofibroblast transformation, in human RV fibroblasts. Using endoglin haploinsufficient mice (Eng(+/-)) we show that reduced endoglin activity preserves RV function, limits RV fibrosis, and attenuates activation of the calcineurin/TRPC-6/α-SMA pathway in a model of angio-obliterative pulmonary hypertension. Next, using Eng(+/-) mice or a neutralizing antibody (Ab) against endoglin (N-Eng) in wild-type mice, we show that reduced endoglin activity improves survival and attenuates RV fibrosis in models of RVPO induced by pulmonary artery constriction. To explore the utility of targeting endoglin, we observed a reversal of RV fibrosis and calcineurin levels in wild-type mice treated with a N-Eng Ab, compared to an immunoglobulin G control. These data establish endoglin as a regulator of TGF-β1 signaling by calcineurin and TRPC-6 in the RV and identify it as a potential therapeutic target to limit RV fibrosis and improve survival in RVPO, a common cause of death in cardiac and pulmonary disease. © 2014 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

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

Harris, Todd R.; Bettaieb, Ahmed; Kodani, Sean

Liver fibrosis is a pathological condition in which chronic inflammation and changes to the extracellular matrix lead to alterations in hepatic tissue architecture and functional degradation of the liver. Inhibitors of the enzyme soluble epoxide hydrolase (sEH) reduce fibrosis in the heart, pancreas and kidney in several disease models. In this study, we assess the effect of sEH inhibition on the development of fibrosis in a carbon tetrachloride (CCl{sub 4})-induced mouse model by monitoring changes in the inflammatory response, matrix remolding and endoplasmic reticulum stress. The sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) was administered in drinking water. Collagen deposition in themore » liver was increased five-fold in the CCl{sub 4}-treated group, and this was returned to control levels by TPPU treatment. Hepatic expression of Col1a2 and 3a1 mRNA was increased over fifteen-fold in the CCl{sub 4}-treated group relative to the Control group, and this increase was reduced by 50% by TPPU treatment. Endoplasmic reticulum (ER) stress observed in the livers of CCl{sub 4}-treated animals was attenuated by TPPU treatment. In order to support the hypothesis that TPPU is acting to reduce the hepatic fibrosis and ER stress through its action as a sEH inhibitor we used a second sEH inhibitor, trans-4-(4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy)-benzoic acid (t-TUCB), and sEH null mice. Taken together, these data indicate that the sEH may play an important role in the development of hepatic fibrosis induced by CCl{sub 4}, presumably by reducing endogenous fatty acid epoxide chemical mediators acting to reduce ER stress. - Highlights: • We administer an inhibitor of sEH in a CCl4 murine model. • sEH inhibition reduces liver collagen deposition and pro-fibrotic gene expression. • sEH inhibition induces MMP-1a activity.« less

Integrative Analysis of PRKAG2 Cardiomyopathy iPS and Microtissue Models Identifies AMPK as a Regulator of Metabolism, Survival, and Fibrosis.

PubMed

Hinson, J Travis; Chopra, Anant; Lowe, Andre; Sheng, Calvin C; Gupta, Rajat M; Kuppusamy, Rajarajan; O'Sullivan, John; Rowe, Glenn; Wakimoto, Hiroko; Gorham, Joshua; Burke, Michael A; Zhang, Kehan; Musunuru, Kiran; Gerszten, Robert E; Wu, Sean M; Chen, Christopher S; Seidman, Jonathan G; Seidman, Christine E

2016-12-20

AMP-activated protein kinase (AMPK) is a metabolic enzyme that can be activated by nutrient stress or genetic mutations. Missense mutations in the regulatory subunit, PRKAG2, activate AMPK and cause left ventricular hypertrophy, glycogen accumulation, and ventricular pre-excitation. Using human iPS cell models combined with three-dimensional cardiac microtissues, we show that activating PRKAG2 mutations increase microtissue twitch force by enhancing myocyte survival. Integrating RNA sequencing with metabolomics, PRKAG2 mutations that activate AMPK remodeled global metabolism by regulating RNA transcripts to favor glycogen storage and oxidative metabolism instead of glycolysis. As in patients with PRKAG2 cardiomyopathy, iPS cell and mouse models are protected from cardiac fibrosis, and we define a crosstalk between AMPK and post-transcriptional regulation of TGFβ isoform signaling that has implications in fibrotic forms of cardiomyopathy. Our results establish critical connections among metabolic sensing, myocyte survival, and TGFβ signaling. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

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

Shen, Wen-jun; Dong, Rui; Chen, Gong, E-mail: chengongzlp@hotmail.com

Highlights: • The RRV infected group showed cholestasis, retardation and extrahepatic biliary atresia. • miR-222 was highly expressed, and PPP2R2A was inhibited in the murine biliary atresia model. • miR-222 profoundly modulated the process of fibrosis in the murine biliary atresia model. • miR-222 might represent a potential target for improving biliary atresia prognosis. - Abstract: microRNA-222 (miR-222) has been shown to initiate the activation of hepatic stellate cells, which plays an important role in the pathogenesis of liver fibrosis. The aim of our study was to evaluate the role of miR-22 in a mouse model of biliary atresia (BA)more » induced by Rhesus Rotavirus (RRV) infection. New-born Balb/c mice were randomized into control and RRV infected groups. The extrahepatic bile ducts were evaluated. The experimental group was divided into BA group and negative group based on histology. The expression of miR-222, protein phosphatase 2 regulatory subunit B alpha (PPP2R2A), proliferating cell nuclear antigen (PCNA) and phospho-Akt were detected. We found that the experimental group showed signs of cholestasis, retardation and extrahepatic biliary atresia. No abnormalities were found in the control group. In the BA group, miR-222, PCNA and Akt were highly expressed, and PPP2R2A expression was significantly inhibited. Our findings suggest that miR-222 profoundly modulated the process of fibrosis in the murine BA model, which might represent a potential target for improving BA prognosis.« less

Saturated fat and cholesterol are critical to inducing murine metabolic syndrome with robust nonalcoholic steatohepatitis.

PubMed

Mells, Jamie E; Fu, Ping P; Kumar, Pradeep; Smith, Tekla; Karpen, Saul J; Anania, Frank A

2015-03-01

Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome (MetS). Up to a third of NAFLD subjects are at risk for developing nonalcoholic steatohepatitis (NASH). Many rodent models fail to replicate both MetS and NASH. The purpose of this study was to develop a reliable mouse model of NASH and MetS using a diet containing cholesterol, saturated fat and carbohydrate that is reflective of Western diets of North Americans. We used adult male C57BL/6 J 4- to 5-week-old mice and administered a solid diet containing 0.2% cholesterol, 45% of its calories from fat, with 30% of the fat in the form of partially hydrogenated vegetable oil. We also provided carbohydrate largely as high-fructose corn syrup equivalent in water. In a separate cohort, we gave the identical diet in the absence of cholesterol. Glucose and insulin tolerance testing was conducted throughout the feeding period. The feeding was conducted for 16 weeks, and the mice were sacrificed for histological analysis, markers of MetS, liver inflammation, circulating lipids, as well as liver staining for fibrosis and alpha smooth muscle actin (α-SMA). We found that cholesterol significantly increased serum leptin, interleukin-6, liver weight and liver weight/body weight ratio, fibrosis and liver α-SMA. Mice administered a diet accurately reflecting patterns associated with humans afflicted with MetS can reliably replicate features of MetS, NASH and significant liver fibrosis. The model we describe significantly reduces the time by several months for development of stage 3 hepatic fibrosis. Copyright © 2015 Elsevier Inc. All rights reserved.

Genetic susceptibility to interstitial pulmonary fibrosis in mice induced by vanadium pentoxide (V2O5)

PubMed Central

Walters, Dianne M.; White, Kevin M.; Patel, Ushma; Davis, Martin J.; Veluci-Marlow, Roberta M.; Bhupanapadu Sunkesula, Solomon Raju; Bonner, James C.; Martin, Jessica R.; Gladwell, Wes; Kleeberger, Steven R.

2014-01-01

Interstitial lung diseases (ILDs) are characterized by injury, inflammation, and scarring of alveoli, leading to impaired function. The etiology of idiopathic forms of ILD is not understood, making them particularly difficult to study due to the lack of appropriate animal models. Consequently, few effective therapies have emerged. We developed an inbred mouse model of ILD using vanadium pentoxide (V2O5), the most common form of a transition metal found in cigarette smoke, fuel ash, mineral ores, and steel alloys. Pulmonary responses to V2O5, including dose-dependent increases in lung permeability, inflammation, collagen content, and dysfunction, were significantly greater in DBA/2J mice compared to C57BL/6J mice. Inflammatory and fibrotic responses persisted for 4 mo in DBA/2J mice, while limited responses in C57BL/6J mice resolved. We investigated the genetic basis for differential responses through genetic mapping of V2O5-induced lung collagen content in BXD recombinant inbred (RI) strains and identified significant linkage on chromosome 4 with candidate genes that associate with V2O5-induced collagen content across the RI strains. Results suggest that V2O5 may induce pulmonary fibrosis through mechanisms distinct from those in other models of pulmonary fibrosis. These findings should further advance our understanding of mechanisms involved in ILD and thereby aid in identification of new therapeutic targets.—Walters, D. M., White, K. M., Patel, U., Davis, M. J., Veluci-Marlow, R. M., Bhupanapadu Sunkesula, S. R., Bonner, J. C., Martin, J. R., Gladwell, W., Kleeberger, S. R. Genetic susceptibility to interstitial pulmonary fibrosis in mice induced by vanadium pentoxide (V2O5). PMID:24285090

Targeting the vascular and perivascular niches as a regenerative therapy for lung and liver fibrosis.

PubMed

Cao, Zhongwei; Ye, Tinghong; Sun, Yue; Ji, Gaili; Shido, Koji; Chen, Yutian; Luo, Lin; Na, Feifei; Li, Xiaoyan; Huang, Zhen; Ko, Jane L; Mittal, Vivek; Qiao, Lina; Chen, Chong; Martinez, Fernando J; Rafii, Shahin; Ding, Bi-Sen

2017-08-30

The regenerative capacity of lung and liver is sometimes impaired by chronic or overwhelming injury. Orthotopic transplantation of parenchymal stem cells to damaged organs might reinstate their self-repair ability. However, parenchymal cell engraftment is frequently hampered by the microenvironment in diseased recipient organs. We show that targeting both the vascular niche and perivascular fibroblasts establishes "hospitable soil" to foster the incorporation of "seed," in this case, the engraftment of parenchymal cells in injured organs. Specifically, ectopic induction of endothelial cell (EC)-expressed paracrine/angiocrine hepatocyte growth factor (HGF) and inhibition of perivascular NOX4 [NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase 4] synergistically enabled reconstitution of mouse and human parenchymal cells in damaged organs. Reciprocally, genetic knockout of Hgf in mouse ECs ( Hgf iΔEC/iΔEC ) aberrantly up-regulated perivascular NOX4 during liver and lung regeneration. Dysregulated HGF and NOX4 pathways subverted the function of vascular and perivascular cells from an epithelially inductive niche to a microenvironment that inhibited parenchymal reconstitution. Perivascular NOX4 induction in Hgf iΔEC/iΔEC mice recapitulated the phenotype of human and mouse liver and lung fibrosis. Consequently, EC-directed HGF and NOX4 inhibitor GKT137831 stimulated regenerative integration of mouse and human parenchymal cells in chronically injured lung and liver. Our data suggest that targeting dysfunctional perivascular and vascular cells in diseased organs can bypass fibrosis and enable reparative cell engraftment to reinstate lung and liver regeneration. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Astragaloside IV suppresses transforming growth factor-β1 induced fibrosis of cultured mouse renal fibroblasts via inhibition of the MAPK and NF-κB signaling pathways

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

Che, Xiajing; Wang, Qin; Xie, Yuanyuan

Renal fibrosis, a progressive process characterized by the accumulation of extracellular matrix (ECM) leading to organ dysfunction, is a characteristic of chronic kidney diseases. Among fibrogenic factors known to regulate the renal fibrotic process, transforming growth factor-β (TGF-β) plays a central role. In the present study, we examined the effect of Astragaloside IV (AS-IV), a component of the traditional Chinese medicinal plant Astragalus membranaceus, on the processes associated with renal fibrosis in cultured mouse renal fibroblasts treated with TGF-β1. RT-PCR, western blotting, immunofluorescence staining and collagen assays showed that AS-IV suppressed TGF-β1 induced fibroblast proliferation, transdifferentiation, and ECM production inmore » a dose-dependent manner. Examination of the underlying mechanisms showed that the effect of AS-IV on the inhibition of fibroblast differentiation and ECM formation were mediated by its modulation of the activity of the MAPK and NF-κB signaling pathways. Taken together, our results indicate that AS-IV alleviates renal interstitial fibrosis via a mechanism involving the MAPK and NF-κB signaling pathways and demonstrate the therapeutic potential of AS-IV for the treatment of chronic kidney diseases. - Highlights: • AS-IV suppressed TGF-β1 induced renal fibroblast proliferation. • AS-IV suppressed TGF-β1 induced renal fibroblast transdifferentiation. • AS-IV suppressed TGF-β1 induced ECM production. • AS-IV alleviates renal fibrosis via the MAPK and NF-κB signaling pathways.« less

Genetic and epigenetic changes in fibrosis-associated hepatocarcinogenesis in mice

PubMed Central

Chappell, Grace; Kutanzi, Kristy; Uehara, Takeki; Tryndyak, Volodymyr; Hong, Hue-Hua; Hoenerhoff, Mark; Beland, Frederick A.; Rusyn, Ivan; Pogribny, Igor P.

2014-01-01

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers and is rising in incidence worldwide. The molecular mechanisms leading to the development of HCC are complex and include both genetic and epigenetic events. To determine the relative contribution of these alterations in liver tumorigenesis, we evaluated epigenetic modifications at both global and gene specific levels, as well as the mutational profile of genes commonly altered in liver tumors. A mouse model of fibrosis-associated liver cancer that was designed to emulate cirrhotic liver, a prevailing disease state observed in most humans with HCC, was used. Tumor and non-tumor liver samples from B6C3F1 mice treated with N-nitrosodiethylamine (DEN; a single ip injection of 1 mg/kg at 14 days of age) and carbon tetrachloride (CCl4; 0.2 ml/kg, 2 times/week ip starting at 8 weeks of age for 14 weeks), as well as corresponding vehicle control animals, were analyzed for genetic and epigenetic alterations. H-ras, Ctnnb1, and Hnf1α genes were not mutated in tumors in mice treated with DEN+CCl4. In contrast, the increased tumor incidence in mice treated with DEN+CCl4 was associated with marked epigenetic changes in liver tumors and non-tumor liver tissue, including demethylation of genomic DNA and repetitive elements, a decrease in histone 3 lysine 9 trimethylation (H3K9me3), and promoter hypermethylation and functional down-regulation of Riz1, a histone lysine methyltransferase tumor suppressor gene. Additionally, the reduction in H3K9me3 was accompanied by increased expression of long interspersed nucleotide elements (LINE) 1 and short interspersed nucleotide elements (SINE) B2, which is an indication of genomic instability. In summary, our results suggest that epigenetic events, rather than mutations in known cancer-related genes, play a prominent role in increased incidence of liver tumors in this mouse model of fibrosis-associated liver cancer. PMID:24242335

Cholecystokinin receptor antagonist halts progression of pancreatic cancer precursor lesions and fibrosis in mice.

PubMed

Smith, Jill P; Cooper, Timothy K; McGovern, Christopher O; Gilius, Evan L; Zhong, Qing; Liao, Jiangang; Molinolo, Alfredo A; Gutkind, J Silvio; Matters, Gail L

2014-10-01

Exogenous administration of cholecystokinin (CCK) induces hypertrophy and hyperplasia of the pancreas with an increase in DNA content. We hypothesized that endogenous CCK is involved in the malignant progression of pancreatic intraepithelial neoplasia (PanIN) lesions and the fibrosis associated with pancreatic cancer. The presence of CCK receptors in early PanIN lesions was examined by immunohistochemistry in mouse and human pancreas. Pdx1-Cre/LSL-Kras transgenic mice were randomized to receive either untreated drinking water or water supplemented with a CCK receptor antagonist (proglumide, 0.1 mg/mL). Pancreas from the mice were removed and examined histologically for number and grade of PanINs after 1, 2, or 4 months of antagonist therapy. Both CCK-A and CCK-B receptors were identified in early stage PanINs from mouse and human pancreas. The grade of PanIN lesions was reversed, and progression to advanced lesions arrested in mice treated with proglumide compared with the controls (P = 0.004). Furthermore, pancreatic fibrosis was significantly reduced in antagonist-treated animals compared with vehicle (P < 0.001). These findings demonstrate that endogenous CCK is in part responsible for the development and progression of pancreatic cancer. The use of CCK receptor antagonists may have a role in cancer prophylaxis in high-risk subjects and may reduce fibrosis in the microenvironment.

CHOLECYSTOKININ RECEPTOR ANTAGONIST HALTS PROGRESSION OF PANCREATIC CANCER PRECURSOR LESIONS AND FIBROSIS IN MICE

PubMed Central

Smith, Jill P.; Cooper, Timothy K.; McGovern, Christopher O.; Gilius, Evan L.; Zhong, Qing; Liao, Jiangang; Molinolo, Alfredo A.; Gutkind, J. Silvio; Matters, Gail L.

2014-01-01

Objectives Exogenous administration of cholecystokinin (CCK) induces hypertrophy and hyperplasia of the pancreas with an increase in DNA content. We hypothesized that endogenous CCK is involved with the malignant progression of pancreatic intraepithelial neoplasia (PanIN) lesions and the fibrosis associated with pancreatic cancer. Methods The presence of CCK receptors in early PanIN lesions was examined by immunohistochemistry in mouse and human pancreas. Pdx1-Cre/LSL-KrasG12D transgenic mice were randomized to receive either untreated drinking water or water supplemented with a CCK-receptor antagonist (proglumide, 0.1mg/ml). Pancreas from mice were removed and examined histologically for number and grade of PanINs after 1, 2 or 4 months of antagonist therapy. Results Both CCK-A and CCK-B receptors were identified in early stage PanINs from mouse and human pancreas. The grade of PanIN lesions was reversed and progression to advanced lesions arrested in mice treated with proglumide compared to controls (p=0.004). Furthermore, pancreatic fibrosis was significantly reduced in antagonist-treated animals compared to vehicle (pitalic>0.001). Conclusions These findings demonstrate that endogenous CCK is in part responsible for the development and progression of pancreatic cancer. Use of CCK-receptor antagonists may have a role in cancer prophylaxis in high risk subjects, and may reduce fibrosis in the microenvironment. PMID:25058882

Curcumin inhibits hepatic stellate cell activation via suppression of succinate-associated HIF-1α induction.

PubMed

She, Linlin; Xu, Dan; Wang, Zixia; Zhang, Yirui; Wei, Qingli; Aa, Jiye; Wang, Guangji; Liu, Baolin; Xie, Yuan

2018-05-07

Aberrant succinate accumulation emerges as a unifying mechanism for inflammation and oxidative stress. This study aims to investigate whether curcumin ameliorates hepatic fibrosis via blocking succinate signaling. We investigated the effects of curcumin on hepatic succinate accumulation and liver fibrosis in mice fed a high-fat diet (HFD). Meanwhile, we stimulated mouse primary hepatic stellate cells (HSCs) with succinate and observed the inhibitory effects of curcumin on succinate signaling. Oral administration of curcumin and metformin combated mitochondrial fatty acid oxidation and reduced hepatic succinate accumulation due to the inhibition of succinate dehydrogenase (SDH) activity and demonstrated inhibitory effect on hepatic fibrosis. In mouse primary HSCs, curcumin prevented succinate- and CoCl 2 -induced hypoxia-inducible transcription factor-1α (HIF-1α) induction via suppression of ROS production and effectively reduced gene expressions of Col1α, Col3α, fibronectin and TGF-β1 with inflammation inhibition. Knockdown of HIF-1α with small interfering RNA blocked the action of succinate to induce HSCs activation, indicative of the essential role of HIF-1α in succinate signaling. Hepatic succinate accumulation served as a metabolic signal to promote liver fibrosis through HIF-1α induction. Curcumin reduced succinate accumulation by combating fatty acid oxidation and prevented HSCs activation by blocking succinate/HIF-1α signaling pathway. Copyright © 2018. Published by Elsevier B.V.

Pkd1 transgenic mice: adult model of polycystic kidney disease with extrarenal and renal phenotypes

PubMed Central

Kurbegovic, Almira; Côté, Olivier; Couillard, Martin; Ward, Christopher J.; Harris, Peter C.; Trudel, Marie

2010-01-01

While high levels of Pkd1 expression are detected in tissues of patients with autosomal dominant polycystic kidney disease (ADPKD), it is unclear whether enhanced expression could be a pathogenetic mechanism for this systemic disorder. Three transgenic mouse lines were generated from a Pkd1-BAC modified by introducing a silent tag via homologous recombination to target a sustained wild-type genomic Pkd1 expression within the native tissue and temporal regulation. These mice specifically overexpressed the Pkd1 transgene in extrarenal and renal tissues from ∼2- to 15-fold over Pkd1 endogenous levels in a copy-dependent manner. All transgenic mice reproducibly developed tubular and glomerular cysts leading to renal insufficiency. Interestingly, Pkd1TAG mice also exhibited renal fibrosis and calcium deposits in papilla reminiscent of nephrolithiasis as frequently observed in ADPKD. Similar to human ADPKD, these mice consistently displayed hepatic fibrosis and ∼15% intrahepatic cysts of the bile ducts affecting females preferentially. Moreover, a significant proportion of mice developed cardiac anomalies with severe left-ventricular hypertrophy, marked aortic arch distention and/or valvular stenosis and calcification that had profound functional impact. Of significance, Pkd1TAG mice displayed occasional cerebral lesions with evidence of ruptured and unruptured cerebral aneurysms. This Pkd1TAG mouse model demonstrates that overexpression of wild-type Pkd1 can trigger the typical adult renal and extrarenal phenotypes resembling human ADPKD. PMID:20053665

Reduction of isoprenaline-induced myocardial TGF-{beta}1 expression and fibrosis in osthole-treated mice

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

Chen Rong; The First Hospital Affiliated to Soochow University, Suzhou 215006, Jiangsu Province; Xue Jie

Peroxisome proliferator-activated receptor (PPAR) {alpha} and PPAR{gamma} ligands can attenuate myocardial fibrosis. Osthole, an active constituent isolated from the fruit of Cnidium monnieri (L.) Cusson, may be a dual PPAR{alpha}/{gamma} agonist, but there has been no report on its effect on myocardial fibrosis. In the present study, we investigated the inhibitory effect of osthole on myocardial fibrotic formation in mice and its possible mechanisms. A mouse model with myocardial fibrosis was induced by hypodermic injection of isoprenaline while the mice were simultaneously treated with 40 and 80 mg/kg osthole for 40 days. After the addition of osthole, the cardiac weightmore » index and hydroxyproline content in the myocardial tissues were decreased, the degree of collagen accumulation in the heart was improved, and the downregulation of myocardial PPAR{alpha}/{gamma} mRNA expression induced by isoprenaline was reversed. Moreover, the mRNA expression of transforming growth factor (TGF)-{beta}1 and the protein levels of nuclear factor (NF)-{kappa}B and TGF-{beta}1 in the myocardial tissues were decreased. These findings suggest that osthole can prevent isoprenaline-induced myocardial fibrosis in mice, and its mechanisms may be related to the reduction of TGF-{beta}1 expression via the activation of PPAR{alpha}/{gamma} and subsequent inhibition of NF-{kappa}B in myocardial tissues. - Highlights: > Osthole could inhibit the myocardial fibrosis induced by isoprenaline in mice. > The mechanism was related to reduction of TGF-{beta}1 expression in myocardial tissue. > The result of osthole was from the activation of PPAR{alpha}/{gamma} and inhibition of NF-{kappa}B.« less

Docosahexaenoic Acid Attenuates Hepatic Inflammation, Oxidative Stress, and Fibrosis without Decreasing Hepatosteatosis in a Ldlr−/− Mouse Model of Western Diet-Induced Nonalcoholic Steatohepatitis123

PubMed Central

Depner, Christopher M.; Philbrick, Kenneth A.; Jump, Donald B.

2013-01-01

The incidence of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) has increased in parallel with the incidence of obesity. While both NAFLD and NASH are characterized by hepatosteatosis, NASH is characterized by hepatic damage, inflammation, oxidative stress, and fibrosis. We previously reported that feeding Ldlr−/− mice a high-fat, high-cholesterol diet containing menhaden oil attenuated several markers of NASH, including hepatosteatosis, inflammation, and fibrosis. Herein, we test the hypothesis that DHA [22:6 (n-3)] is more effective than EPA [20:5 (n-3)] at preventing Western diet (WD)-induced NASH in Ldlr−/− mice. Mice were fed the WD supplemented with either olive oil (OO), EPA, DHA, or EPA + DHA for 16 wk. WD + OO feeding induced a severe NASH phenotype, characterized by robust hepatosteatosis, inflammation, oxidative stress, and fibrosis. Whereas none of the C20–22 (n-3) fatty acid treatments prevented WD-induced hepatosteatosis, all 3 (n-3) PUFA-containing diets significantly attenuated WD-induced inflammation, fibrosis, and hepatic damage. The capacity of dietary DHA to suppress hepatic markers of inflammation (Clec4F, F4/80, Trl4, Trl9, CD14, Myd88), fibrosis (Procol1α1, Tgfβ1), and oxidative stress (NADPH oxidase subunits Nox2, p22phox, p40phox, p47phox, p67phox) was significantly greater than dietary EPA. The effects of DHA on these markers paralleled DHA-mediated suppression of hepatic Fads1 mRNA abundance and hepatic arachidonic acid content. Because DHA suppression of NASH markers does not require a reduction in hepatosteatosis, dietary DHA may be useful in combating NASH in obese humans. PMID:23303872

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

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

Chen, Wei-Yu, E-mail: wychen624@cgmh.org.tw; Chang, Ya-Jen; Su, Chia-Hao

Interstitial fibrosis and loss of parenchymal tubular cells are the common outcomes of progressive renal diseases. Pro-inflammatory cytokines have been known contributing to the damage of tubular cells and fibrosis responses after renal injury. Interleukin (IL)-33 is a tissue-derived nucleus alarmin that drives inflammatory responses. The regulation and function of IL-33 in renal injury, however, is not well understood. To investigate the involvement of cytokines in the pathogenesis of renal injury and fibrosis, we performed the mouse renal injury model induced by unilateral urinary obstruction (UUO) and analyze the differentially upregulated genes between the obstructed and the contralateral unobstructed kidneysmore » using RNA sequencing (RNAseq). Our RNAseq data identified IL33 and its receptor ST2 were upregulated in the UUO kidney. Quantitative analysis confirmed that transcripts of IL33 and ST2 were upregulated in the obstructed kidneys. Immunofluorescent staining revealed that IL-33 was upregulated in Vimentin- and alpha-SMA-positive interstitial cells. By using genetically knockout mice, deletion of IL33 reduced UUO-induced renal fibrosis. Moreover, in combination with BrdU labeling technique, we observed that the numbers of proliferating tubular epithelial cells were increased in the UUO kidneys from IL33-or ST2-deficient mice compared to wild type mice. Collectively, our study demonstrated the upregulation of IL-33/ST2 signaling in the obstructed kidney may promote tubular cell injury and interstitial fibrosis. IL-33 may serve as a biomarker to detect renal injury and that IL-33/ST2 signaling may represent a novel target for treating renal diseases. -- Highlights: •Interleukin (IL)-33 was upregulated in obstructed kidneys. •Interstitial myofibroblasts expressed IL-33 after UUO-induced renal injury. •Deficiency of IL33 reduced interstitial fibrosis and promoted tubular cell proliferation.« less

Autotaxin activity increases locally following lung injury, but is not required for pulmonary lysophosphatidic acid production or fibrosis.

PubMed

Black, Katharine E; Berdyshev, Evgeny; Bain, Gretchen; Castelino, Flavia V; Shea, Barry S; Probst, Clemens K; Fontaine, Benjamin A; Bronova, Irina; Goulet, Lance; Lagares, David; Ahluwalia, Neil; Knipe, Rachel S; Natarajan, Viswanathan; Tager, Andrew M

2016-06-01

Lysophosphatidic acid (LPA) is an important mediator of pulmonary fibrosis. In blood and multiple tumor types, autotaxin produces LPA from lysophosphatidylcholine (LPC) via lysophospholipase D activity, but alternative enzymatic pathways also exist for LPA production. We examined the role of autotaxin (ATX) in pulmonary LPA production during fibrogenesis in a bleomycin mouse model. We found that bleomycin injury increases the bronchoalveolar lavage (BAL) fluid levels of ATX protein 17-fold. However, the LPA and LPC species that increase in BAL of bleomycin-injured mice were discordant, inconsistent with a substrate-product relationship between LPC and LPA in pulmonary fibrosis. LPA species with longer chain polyunsaturated acyl groups predominated in BAL fluid after bleomycin injury, with 22:5 and 22:6 species accounting for 55 and 16% of the total, whereas the predominant BAL LPC species contained shorter chain, saturated acyl groups, with 16:0 and 18:0 species accounting for 56 and 14% of the total. Further, administration of the potent ATX inhibitor PAT-048 to bleomycin-challenged mice markedly decreased ATX activity systemically and in the lung, without effect on pulmonary LPA or fibrosis. Therefore, alternative ATX-independent pathways are likely responsible for local generation of LPA in the injured lung. These pathways will require identification to therapeutically target LPA production in pulmonary fibrosis.-Black, K. E., Berdyshev, E., Bain, G., Castelino, F. V., Shea, B. S., Probst, C. K., Fontaine, B. A., Bronova, I., Goulet, L., Lagares, D., Ahluwalia, N., Knipe, R. S., Natarajan, V., Tager, A. M. Autotaxin activity increases locally following lung injury, but is not required for pulmonary lysophosphatidic acid production or fibrosis. © FASEB.

Fibroblast Activation Protein (FAP) Accelerates Collagen Degradation and Clearance from Lungs in Mice.

PubMed

Fan, Ming-Hui; Zhu, Qiang; Li, Hui-Hua; Ra, Hyun-Jeong; Majumdar, Sonali; Gulick, Dexter L; Jerome, Jacob A; Madsen, Daniel H; Christofidou-Solomidou, Melpo; Speicher, David W; Bachovchin, William W; Feghali-Bostwick, Carol; Puré, Ellen

2016-04-08

Idiopathic pulmonary fibrosis is a disease characterized by progressive, unrelenting lung scarring, with death from respiratory failure within 2-4 years unless lung transplantation is performed. New effective therapies are clearly needed. Fibroblast activation protein (FAP) is a cell surface-associated serine protease up-regulated in the lungs of patients with idiopathic pulmonary fibrosis as well as in wound healing and cancer. We postulate that FAP is not only a marker of disease but influences the development of pulmonary fibrosis after lung injury. In two different models of pulmonary fibrosis, intratracheal bleomycin instillation and thoracic irradiation, we find increased mortality and increased lung fibrosis in FAP-deficient mice compared with wild-type mice. Lung extracellular matrix analysis reveals accumulation of intermediate-sized collagen fragments in FAP-deficient mouse lungs, consistent within vitrostudies showing that FAP mediates ordered proteolytic processing of matrix metalloproteinase (MMP)-derived collagen cleavage products. FAP-mediated collagen processing leads to increased collagen internalization without altering expression of the endocytic collagen receptor, Endo180. Pharmacologic FAP inhibition decreases collagen internalization as expected. Conversely, restoration of FAP expression in the lungs of FAP-deficient mice decreases lung hydroxyproline content after intratracheal bleomycin to levels comparable with that of wild-type controls. Our findings indicate that FAP participates directly, in concert with MMPs, in collagen catabolism and clearance and is an important factor in resolving scar after injury and restoring lung homeostasis. Our study identifies FAP as a novel endogenous regulator of fibrosis and is the first to show FAP's protective effects in the lung. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology

NASA Astrophysics Data System (ADS)

Mederacke, Ingmar; Hsu, Christine C.; Troeger, Juliane S.; Huebener, Peter; Mu, Xueru; Dapito, Dianne H.; Pradere, Jean-Philippe; Schwabe, Robert F.

2013-11-01

Although organ fibrosis causes significant morbidity and mortality in chronic diseases, the lack of detailed knowledge about specific cellular contributors mediating fibrogenesis hampers the design of effective antifibrotic therapies. Different cellular sources, including tissue-resident and bone marrow-derived fibroblasts, pericytes and epithelial cells, have been suggested to give rise to myofibroblasts, but their relative contributions remain controversial, with profound differences between organs and different diseases. Here we employ a novel Cre-transgenic mouse that marks 99% of hepatic stellate cells (HSCs), a liver-specific pericyte population, to demonstrate that HSCs give rise to 82-96% of myofibroblasts in models of toxic, cholestatic and fatty liver disease. Moreover, we exclude that HSCs function as facultative epithelial progenitor cells in the injured liver. On the basis these findings, HSCs should be considered the primary cellular target for antifibrotic therapies across all types of liver disease.

Inactivated ORF virus shows antifibrotic activity and inhibits human hepatitis B virus (HBV) and hepatitis C virus (HCV) replication in preclinical models.

PubMed

Paulsen, Daniela; Urban, Andreas; Knorr, Andreas; Hirth-Dietrich, Claudia; Siegling, Angela; Volk, Hans-Dieter; Mercer, Andrew A; Limmer, Andreas; Schumak, Beatrix; Knolle, Percy; Ruebsamen-Schaeff, Helga; Weber, Olaf

2013-01-01

Inactivated orf virus (iORFV), strain D1701, is a potent immune modulator in various animal species. We recently demonstrated that iORFV induces strong antiviral activity in animal models of acute and chronic viral infections. In addition, we found D1701-mediated antifibrotic effects in different rat models of liver fibrosis. In the present study, we compare iORFV derived from two different strains of ORFV, D1701 and NZ2, respectively, with respect to their antifibrotic potential as well as their potential to induce an antiviral response controlling infections with the hepatotropic pathogens hepatitis C virus (HCV) and hepatitis B virus (HBV). Both strains of ORFV showed anti-viral activity against HCV in vitro and against HBV in a transgenic mouse model without signs of necro-inflammation in vivo. Our experiments suggest that the absence of liver damage is potentially mediated by iORFV-induced downregulation of antigen cross-presentation in liver sinus endothelial cells. Furthermore, both strains showed significant anti-fibrotic activity in rat models of liver fibrosis. iORFV strain NZ2 appeared more potent compared to strain D1701 with respect to both its antiviral and antifibrotic activity on the basis of dosages estimated by titration of active virus. These results show a potential therapeutic approach against two important human liver pathogens HBV and HCV that independently addresses concomitant liver fibrosis. Further studies are required to characterize the details of the mechanisms involved in this novel therapeutic principle.

Imaging Fibrosis and Separating Collagens using Second Harmonic Generation and Phasor Approach to Fluorescence Lifetime Imaging

PubMed Central

Ranjit, Suman; Dvornikov, Alexander; Stakic, Milka; Hong, Suk-Hyun; Levi, Moshe; Evans, Ronald M.; Gratton, Enrico

2015-01-01

In this paper we have used second harmonic generation (SHG) and phasor approach to auto fluorescence lifetime imaging (FLIM) to obtain fingerprints of different collagens and then used these fingerprints to observe bone marrow fibrosis in the mouse femur. This is a label free approach towards fast automatable detection of fibrosis in tissue samples. FLIM has previously been used as a method of contrast in different tissues and in this paper phasor approach to FLIM is used to separate collagen I from collagen III, the markers of fibrosis, the largest groups of disorders that are often without any effective therapy. Often characterized by an increase in collagen content of the corresponding tissue, the samples are usually visualized by histochemical staining, which is pathologist dependent and cannot be automated. PMID:26293987

Apoptosis-Resistant Cardiac Progenitor Cells Modified With Apurinic/Apyrimidinic Endonuclease/Redox Factor 1 Gene Overexpression Regulate Cardiac Repair After Myocardial Infarction.

PubMed

Aonuma, Tatsuya; Takehara, Naofumi; Maruyama, Keisuke; Kabara, Maki; Matsuki, Motoki; Yamauchi, Atsushi; Kawabe, Jun-Ichi; Hasebe, Naoyuki

2016-08-01

: Overcoming the insufficient survival of cell grafts is an essential objective in cell-based therapy. Apurinic/apyrimidinic endonuclease/redox factor 1 (APE1) promotes cell survival and may enhance the therapeutic effect of engrafted cells. The aim of this study is to determine whether APE1 overexpression in cardiac progenitor cells (CPCs) could ameliorate the efficiency of cell-based therapy. CPCs isolated from 8- to 10-week-old C57BL/6 mouse hearts were infected with retrovirus harboring APE1-DsRed (APE1-CPC) or a DsRed control (control-CPC). Oxidative stress-induced apoptosis was then assessed in APE1-CPCs, control-CPCs, and neonatal rat ventricular myocytes (NRVMs) cocultured with these CPCs. This analysis revealed that APE1 overexpression inhibited CPC apoptosis with activation of transforming growth factor β-activated kinase 1 (TAK1) and nuclear factor (NF)-κB. In the coculture model, NRVM apoptosis was inhibited to a greater extent in the presence of APE1-CPCs compared with control-CPCs. Moreover, the number of surviving DsRed-positive CPC grafts was significantly higher 7 days after the transplant of APE1-CPCs into a mouse myocardial infarction model, and the left ventricular ejection fraction showed greater improvement with attenuation of fibrosis 28 days after the transplant of APE1-CPCs compared with control-CPCs. Additionally, fewer inflammatory macrophages and a higher percentage of cardiac α-sarcomeric actinin-positive CPC-grafts were observed in mice injected with APE1-CPCs compared with control-CPCs after 7 days. In conclusion, antiapoptotic APE1-CPC graft, which increased TAK1-NF-κB pathway activation, survived effectively in the ischemic heart, restored cardiac function, and reduced cardiac inflammation and fibrosis. APE1 overexpression in CPCs may serve as a novel strategy to improve cardiac cell therapy. Improving the survival of cell grafts is essential to maximize the efficacy of cell therapy. The authors investigated the role of APE1 in CPCs under ischemic conditions and evaluated the therapeutic efficacy of transplanted APE1-overexpressing CPCs in a mouse model of myocardial infarction. APE1 hindered apoptosis in CPC grafts subjected to oxidative stress caused in part by increased TAK1-NF-κB pathway activation. Furthermore, APE1-CPC grafts that effectively survived in the ischemic heart restored cardiac function and attenuated fibrosis through pleiotropic mechanisms that remain to be characterized. These findings suggest that APE1 overexpression in CPCs may be a novel strategy to reinforce cardiac cell therapy. ©AlphaMed Press.

The downregulation of microRNA let-7a contributes to the excessive expression of type I collagen in systemic and localized scleroderma.

PubMed

Makino, Katsunari; Jinnin, Masatoshi; Hirano, Ayaka; Yamane, Keitaro; Eto, Mitsuhiko; Kusano, Takamitsu; Honda, Noritoshi; Kajihara, Ikko; Makino, Takamitsu; Sakai, Keisuke; Masuguchi, Shinichi; Fukushima, Satoshi; Ihn, Hironobu

2013-04-15

Systemic and localized scleroderma (SSc and LSc) is characterized by excessive deposition of collagen and tissue fibrosis in the skin. Although they have fundamental common characteristics including autoimmunity, little is known about the exact mechanism that mediates the excessive collagen expression in these disorders. In the current study, we tried to evaluate the possibility that microRNAs (miRNAs) play some roles in the pathogenesis of fibrosis seen in these diseases. miRNA expression patterns were evaluated by miRNA array analysis, real-time PCR, and in situ hybridization. The function of miRNAs in dermal fibroblasts was assessed using miRNA inhibitors, precursors, or protectors. In the mouse model of bleomycin-induced dermal sclerosis, the overexpression of miRNAs was performed by i.p. miRNA injection. We demonstrated let-7a expression was downregulated in SSc and LSc skin both in vivo and in vitro, compared with normal or keloid skin. The inhibition or overexpression of let-7a in human or mouse skin fibroblasts affected the protein expression of type I collagen or luciferase activity of collagen 3'-untranslated region. Also, we found let-7a was detectable and quantitative in the serum and investigated serum let-7a levels in patients with SSc or LSc. let-7a concentration was significantly decreased in these patients, especially in LSc patients. Moreover, we revealed that the intermittent overexpression of let-7a in the skin by i.p. miRNA injection improved the skin fibrosis induced by bleomycin in mice. Investigation of more detailed mechanisms of miRNA-mediated regulation of collagen expression may lead to new therapeutic approaches against SSc and LSc.

22-Oxacalcitriol Prevents Progression of Peritoneal Fibrosis in a Mouse Model

PubMed Central

Hirose, Misaki; Nishino, Tomoya; Obata, Yoko; Nakazawa, Masayuki; Nakazawa, Yuka; Furusu, Akira; Abe, Katsushige; Miyazaki, Masanobu; Koji, Takehiko; Kohno, Shigeru

2013-01-01

♦ Objective: Vitamin D plays an important role in calcium homeostasis and is used to treat secondary hyperparathyroidism among dialysis patients. The biologic activity of vitamin D and its analogs is mediated by vitamin D receptor (VDR), which is distributed widely throughout the body. Recent papers have revealed that low vitamin D levels are correlated with severe fibrosis in chronic diseases, including cystic fibrosis and hepatitis. The aim of the present study was to evaluate the protective effects of vitamin D against the progression of peritoneal fibrosis. ♦ Methods: Peritoneal fibrosis was induced by injection of chlorhexidine gluconate (CG) into the peritoneal cavity of mice every other day for 3 weeks. An analog of vitamin D, 22-oxacalcitriol (OCT), was administered subcutaneously daily from initiation of the CG injections. The peritoneal tissue was excised at 3 weeks. Changes in morphology were assessed by hematoxylin and eosin staining. Expression of VDR, alpha smooth muscle actin (as a marker of myofibroblasts), type III collagen, transforming growth factor β(TGF-β), phosphorylated Smad2/3, F4/80 (as a marker of macrophages), and monocyte chemoattractant protein-1 (MCP-1) was examined by immunohistochemistry. Southwestern histochemistry was used to detect activated nuclear factor κB (NF-κB). ♦ Results: In the CG-injected mice, immunohistochemical analysis revealed expression of VDR in mesothelial cells, myofibroblasts, and macrophages in the thickened submesothelial zone. Treatment with OCT significantly prevented peritoneal fibrosis and reduced the accumulation of type III collagen in CG-treated mice. Among the markers of fibrosis, the numbers of myofibroblasts, cells positive for TGF-β, and cells positive for phosphorylated Smad2/3 were significantly decreased in the OCT-treated group compared with the vehicle-treated group. Furthermore, OCT suppressed inflammatory mediators of fibrosis, as shown by the reduced numbers of activated NF-κB cells, macrophages, and MCP-1-expressing cells. ♦ Conclusions: Our results indicate that OCT attenuates peritoneal fibrosis, an effect accompanied by reduced numbers of myofibroblasts, infiltrating macrophages, and TGF-β-positive cells, suggesting that vitamin D has potential as a novel therapeutic agent for preventing peritoneal sclerosis. PMID:23032084

Immunologic aspects of fibrosis in mouse mammary carcinomas.

PubMed

Vaage, J

1992-01-02

The nature of the fibrosis associated with mammary carcinomas MC2 and MC3 was investigated in syngeneic C3H mice. Accelerated and enhanced peri-tumor cellular and fibrotic responses and retarded tumor growth were observed in actively immunized and in adoptively immunized mice, and in mice treated with IL-2. T lymphocytes and, particularly, macrophages were closely associated with collagen deposition at the tumors. The collagen deposition frequently resulted in the encapsulation and regression of the less invasive tumor MC2. A cellular fibrous response was not observed at tumors implanted into athymic C3Hnu/nu mice. The results suggest that tumor fibrosis may in some circumstances be promoted by an immune response.

Macrophage and epithelial cell H-ferritin expression regulates renal inflammation

PubMed Central

Bolisetty, Subhashini; Zarjou, Abolfazl; Hull, Travis D.; Traylor, Amie; Perianayagam, Anjana; Joseph, Reny; Kamal, Ahmed I; Arosio, Paolo; Soares, Miguel P; Jeney, Viktoria; Balla, Jozsef; George, James F.; Agarwal, Anupam

2015-01-01

Inflammation culminating in fibrosis contributes to progressive kidney disease. Crosstalk between the tubular epithelium and interstitial cells regulates inflammation by a coordinated release of cytokines and chemokines. Here we studied the role of heme oxygenase-1 (HO-1) and the heavy subunit of ferritin (FtH) in macrophage polarization and renal inflammation. Deficiency in HO-1 was associated with increased FtH expression, accumulation of macrophages with a dysregulated polarization profile, and increased fibrosis following unilateral ureteral obstruction in mice; a model of renal inflammation and fibrosis. Macrophage polarization in vitro was predominantly dependent on FtH expression in isolated bone marrow-derived mouse monocytes. Utilizing transgenic mice with conditional deletion of FtH in the proximal tubules (FtHPT−/−) or myeloid cells (FtHLysM−/−), we found that myeloid FtH deficiency did not affect polarization or accumulation of macrophages in the injured kidney compared to wild-type (FtH+/+) controls. However, tubular FtH deletion led to a marked increase in pro-inflammatory macrophages. Furthermore, injured kidneys from FtHPT−/− mice expressed significantly higher levels of inflammatory chemokines and fibrosis compared to kidneys from FtH+/+ and FtHLysM−/− mice. Thus, there are differential effects of FtH in macrophages and epithelial cells, which underscores the critical role of FtH in tubular-macrophage crosstalk during kidney injury. PMID:25874599

Deficiency of iNOS-derived NO accelerates lipid accumulation-independent liver fibrosis in non-alcoholic steatohepatitis mouse model.

PubMed

Nozaki, Yuichi; Fujita, Koji; Wada, Koichiro; Yoneda, Masato; Kessoku, Takaomi; Shinohara, Yoshiyasu; Imajo, Kento; Ogawa, Yuji; Nakamuta, Makoto; Saito, Satoru; Masaki, Naohiko; Nagashima, Yoji; Terauchi, Yasuo; Nakajima, Atsushi

2015-04-01

Although many of the factors and molecules closely associated with non-alcoholic steatohepatitis (NASH) have been reported, the role of inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) on the progression of NASH remains unclear. We therefore investigated the role of iNOS-derived NO in NASH pathogenesis with a long-term follow-up study using systemic iNOS-knockout mice under high-fat diet (HFD) conditions. iNOS-knockout and wild-type mice were fed a basal or HFD for 10 or 48 weeks. Lipid accumulation, fibrosis, and inflammation were evaluated, and various factors and molecules closely associated with NASH were analyzed. Marked fibrosis and inflammation (indicators of NASH) were observed in the livers of iNOS-knockout mice compared to wild-type mice after 48 weeks of a HFD; however, lipid accumulation in iNOS-knockout mice livers was less than in the wild-type. Increased expressions of various cytokines that are transcriptionally controlled by NF-kB in iNOS-deficient mice livers were observed during HFD conditions. iNOS-derived NO may play a protective role against the progression to NASH during an HFD by preventing fibrosis and inflammation, which are mediated by NF-kB activation in Kupffer cells. A lack of iNOS-derived NO accelerates progression to NASH without excessive lipid accumulation.

The chemokine receptor CXCR6 contributes to recruitment of bone marrow-derived fibroblast precursors in renal fibrosis

PubMed Central

Xia, Yunfeng; Yan, Jingyin; Jin, Xiaogao; Entman, Mark L.; Wang, Yanlin

2014-01-01

Bone marrow-derived fibroblasts in circulation are of hematopoietic origin, proliferate, differentiate into myofibroblasts, and express the chemokine receptor CXCR6. Since chemokines mediate the trafficking of circulating cells to sites of injury, we studied the role of CXCR6 in mouse models of renal injury. Significantly fewer bone marrow-derived fibroblasts accumulated in the kidney of CXCR6 knockout mice in response to injury, expressed less profibrotic chemokines and cytokines, displayed fewer myofibroblasts, and expressed less α-smooth muscle actin in the obstructed kidneys compared with wild-type mice. CXCR6 deficiency inhibited total collagen deposition and suppressed expression of collagen I and fibronectin in the obstructed kidneys. Furthermore, wild type mice engrafted with CXCR6−/− bone marrow cells displayed fewer bone marrow-derived fibroblasts in the kidneys with obstructive injury and showed less severe renal fibrosis compared with wild-type mice engrafted with CXCR6+/+ bone marrow cells. Transplant of wild type bone marrow into CXCR6−/− recipients restored recruitment of myeloid fibroblasts and susceptibility to fibrosis. Hematopoietic fibroblasts migrate into injured kidney and proliferate and differentiate into myofibroblasts. Thus, CXCR6, together with other chemokines and their receptors, may play important roles in the recruitment of bone marrow-derived fibroblast precursors into the kidney and contribute to the pathogenesis of renal fibrosis. PMID:24646857

The chemokine receptor CXCR6 contributes to recruitment of bone marrow-derived fibroblast precursors in renal fibrosis.

PubMed

Xia, Yunfeng; Yan, Jingyin; Jin, Xiaogao; Entman, Mark L; Wang, Yanlin

2014-08-01

Bone marrow-derived fibroblasts in circulation are of hematopoietic origin, and they proliferate, differentiate into myofibroblasts, and express the chemokine receptor CXCR6. As chemokines mediate the trafficking of circulating cells to sites of injury, we studied the role of CXCR6 in mouse models of renal injury. Significantly, the kidney of CXCR6 knockout mice accumulated fewer bone marrow-derived fibroblasts in response to injury, expressed less profibrotic chemokines and cytokines, displayed fewer myofibroblasts, and expressed less α-smooth muscle actin in the obstructed kidneys compared with wild-type (WT) mice. CXCR6 deficiency inhibited total collagen deposition and suppressed the expression of collagen I and fibronectin in the obstructed kidneys. Furthermore, WT mice engrafted with CXCR6(-/-) bone marrow cells displayed fewer bone marrow-derived fibroblasts in the kidneys with obstructive injury and showed less severe renal fibrosis compared with WT mice engrafted with CXCR6(+/+) bone marrow cells. Transplant of WT bone marrow into CXCR6(-/-) recipients restored recruitment of myeloid fibroblasts and susceptibility to fibrosis. Hematopoietic fibroblasts migrate into injured kidney and proliferate and differentiate into myofibroblasts. Thus, CXCR6, together with other chemokines and their receptors, may have important roles in the recruitment of bone marrow-derived fibroblast precursors into the kidney and contribute to the pathogenesis of renal fibrosis.

FOXO1 content is reduced in cystic fibrosis and increases with IGF-I treatment.

PubMed

Smerieri, Arianna; Montanini, Luisa; Maiuri, Luigi; Bernasconi, Sergio; Street, Maria E

2014-10-08

Cystic fibrosis-related diabetes is to date the most frequent complication in cystic fibrosis (CF). The mechanisms underlying this condition are not well understood, and a possible role of insulin resistance is debated. We investigated insulin signal transduction in CF. Total insulin receptor, IRS1, p85 PI3K, and AKT contents were substantially normal in CF cells (CFBE41o-), whereas winged helix forkhead (FOX)O1 contents were reduced both in baseline conditions and after insulin stimulation. In addition, CF cells showed increased ERK1/2, and reduced β2 arrestin contents. No significant change in SOCS2 was observed. By using a CFTR inhibitor and siRNA, changes in FOXO1 were related to CFTR loss of function. In a CF-affected mouse model, FOXO1 content was reduced in the muscle while no significant difference was observed in liver and adipose tissue compared with wild-type. Insulin-like growth factor 1 (IGF-I) increased FOXO1 content in vitro and in vivo in muscle and adipose tissue. In conclusion; we present the first description of reduced FOXO1 content in CF, which is compatible with reduced gluconeogenesis and increased adipogenesis, both features of insulin insensitivity. IGF-I treatment was effective in increasing FOXO1, thereby suggesting that it could be considered as a potential treatment in CF patients possibly to prevent and treat cystic fibrosis-related diabetes.

Organ- and species-specific biological activity of rosmarinic acid.

PubMed

Iswandana, R; Pham, B T; van Haaften, W T; Luangmonkong, T; Oosterhuis, D; Mutsaers, H A M; Olinga, P

2016-04-01

Rosmarinic acid (RA), a compound found in several plant species, has beneficial properties, including anti-inflammatory and antibacterial effects. We investigated the toxicity, anti-inflammatory, and antifibrotic effects of RA using precision-cut liver slices (PCLS) and precision-cut intestinal slices (PCIS) prepared from human, mouse, and rat tissue. PCLS and PCIS were cultured up to 48 h in the absence or presence of RA. Gene expression of the inflammatory markers: IL-6, IL-8/CXCL1/KC, and IL-1β, as well as the fibrosis markers: pro-collagen 1a1, heat shock protein 47, α-smooth muscle actin, fibronectin (Fn2) and plasminogen activator inhibitor-1 (PAI-1) were evaluated by qPCR. RA was only toxic in murine PCIS. RA failed to mitigate the inflammatory response in most models, while it clearly reduced IL-6 and CXCL1/KC gene expression in murine PCIS at non-toxic concentrations. With regard to fibrosis, RA decreased the gene levels of Fn2 and PAI-1 in murine PCLS, and Fn2 in murine PCIS. Yet, no effect was observed on the gene expression of fibrosis markers in human and rat PCIS. In conclusion, we observed clear organ- and species-specific effects of RA. RA had little influence on inflammation. However, our study further establishes RA as a potential candidate for the treatment of liver fibrosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Repair after nephron ablation reveals limitations of neonatal neonephrogenesis

PubMed Central

Tögel, Florian; Freedman, Benjamin S.; Iatrino, Rossella; Grinstein, Mor; Bonventre, Joseph V.

2017-01-01

The neonatal mouse kidney retains nephron progenitor cells in a nephrogenic zone for 3 days after birth. We evaluated whether de novo nephrogenesis can be induced postnatally beyond 3 days. Given the long-term implications of nephron number for kidney health, it would be useful to enhance nephrogenesis in the neonate. We induced nephron reduction by cryoinjury with or without contralateral nephrectomy during the neonatal period or after 1 week of age. There was no detectable compensatory de novo nephrogenesis, as determined by glomerular counting and lineage tracing. Contralateral nephrectomy resulted in additional adaptive healing, with little or no fibrosis, but did not also stimulate de novo nephrogenesis. In contrast, injury initiated at 1 week of age led to healing with fibrosis. Thus, despite the presence of progenitor cells and ongoing nephron maturation in the newborn mouse kidney, de novo nephrogenesis is not inducible by acute nephron reduction. This indicates that additional nephron progenitors cannot be recruited after birth despite partial renal ablation providing a reparative stimulus and suggests that nephron number in the mouse is predetermined at birth. PMID:28138555

Pilot study of the antifibrotic effects of the multikinase inhibitor pacritinib in a mouse model of liver fibrosis

PubMed Central

Al-Fayoumi, Suliman; Hashiguchi, Taishi; Shirakata, Yuka; Mascarenhas, John; Singer, Jack W

2018-01-01

Background Fibrotic diseases result from an exuberant response to chronic inflammation. Myelofibrosis is the end result of inflammation in bone, caused by an inflammatory process triggered by production of abnormal myeloid cells driven by mutations affecting the JAK–STAT pathway. Inflammatory cytokine overproduction leads to increased mesenchymal cell proliferation, culminating in fibrosis. Although JAK2 inhibitors, such as the JAK1/2 inhibitor ruxolitinib and the JAK2/FLT3/CSF1R/IRAK1 inhibitor pacritinib suppress abnormal clone expansion in myelofibrosis, ruxolitinib does not appear to prevent or reverse bone-marrow fibrosis in most patients. In two Phase III clinical trials, pacritinib, however, demonstrated improvements in platelet counts and hemoglobin and reductions in transfusion burden in some patients with baseline cytopenias, suggesting it may improve bone-marrow function. Unlike ruxolitinib, pacritinib suppresses signaling through IRAK1, a key control point for inflammatory and fibrotic signaling. Purpose To investigate potential antifibrotic effects of pacritinib in an animal model of liver fibrosis relevant to the observed course of human disease. Methods Pacritinib, negative control (vehicle), and positive control (the angiotensin 2-receptor antagonist and PPARγ partial agonist telmisartan) were assessed in the murine Stelic animal model, which mimics the clinically observed progression from hepatic steatosis to nonalcoholic steatohepatitis, liver fibrosis, and hepatocellular carcinoma. Histopathological analysis used hematoxylin and eosin staining. Body and liver weight changes, nonalcoholic fatty-liver disease activity scores, and plasma cytokeratin 18 fragment levels (a biomarker of hepatic necrosis) were measured. Results Pacritinib-treated mice had significantly (P<0.01) reduced fibrotic areas in liver compared to vehicle control and significantly (P<0.05) lower levels of CK18. The antifibrotic effect of pacritinib was comparable to that of telmisartan, but without significant effects on fat accumulation. Conclusion These results, the first to demonstrate hepatic antifibrotic effects for pacritinib in an animal model of liver disease, provide preliminary support for potential clinical applications of pacritinib in fibrotic diseases other than myelofibrosis. PMID:29785143

Naproxcinod shows significant advantages over naproxen in the mdx model of Duchenne Muscular Dystrophy.

PubMed

Miglietta, Daniela; De Palma, Clara; Sciorati, Clara; Vergani, Barbara; Pisa, Viviana; Villa, Antonello; Ongini, Ennio; Clementi, Emilio

2015-08-22

In dystrophin-deficient muscles of Duchenne Muscular Dystrophy (DMD) patients and the mdx mouse model, nitric oxide (NO) signalling is impaired. Previous studies have shown that NO-donating drugs are beneficial in dystrophic mouse models. Recently, a long-term treatment (9 months) of mdx mice with naproxcinod, an NO-donating naproxen, has shown a significant improvement of the dystrophic phenotype with beneficial effects present throughout the disease progression. It remains however to be clearly dissected out which specific effects are due to the NO component compared with the anti-inflammatory activity associated with naproxen. Understanding the contribution of NO vs the anti-inflammatory effect is important, in view of the potential therapeutic perspective, and this is the final aim of this study. Five-week-old mdx mice received either naproxcinod (30 mg/kg) or the equimolar dose of naproxen (20 mg/kg) in the diet for 6 months. Control mdx mice were used as reference. Treatments (or vehicle for control groups) were administered daily in the diet. For the first 3 months the study was performed in sedentary animals, then all mice were subjected to exercise until the sixth month. Skeletal muscle force was assessed by measuring whole body tension in sedentary animals as well as in exercised mice and resistance to fatigue was measured after 3 months of running exercise. At the end of 6 months of treatment, animals were sacrificed for histological analysis and measurement of naproxen levels in blood and skeletal muscle. Naproxcinod significantly ameliorated skeletal muscle force and resistance to fatigue in sedentary as well as in exercised mice, reduced inflammatory infiltrates and fibrosis deposition in both cardiac and diaphragm muscles. Conversely, the equimolar dose of naproxen showed no effects on fibrosis and improved muscle function only in sedentary mice, while the beneficial effects in exercised mice were lost demonstrating a limited and short-term effect. In conclusion, this study shows that NO donation may have an important role, in addition to anti-inflammatory activity, in slowing down the progression of the disease in the mdx mouse model therefore positioning naproxcinod as a promising candidate for treatment of DMD.

Dermal delivery of HSP47 siRNA with NOX4-modulating mesoporous silica-based nanoparticles for treating fibrosis

PubMed Central

Morry, Jingga; Ngamcherdtrakul, Worapol; Gu, Shenda; Goodyear, Shaun M.; Castro, David J.; Reda, Moataz M.; Sangvanich, Thanapon; Yantasee, Wassana

2015-01-01

Fibrotic diseases such as scleroderma have been linked to increased oxidative stress and upregulation of pro-fibrotic genes. Recent work suggests a role of NADPH oxidase 4 (NOX4) and heat shock protein 47 (HSP47) in inducing excessive collagen synthesis, leading to fibrotic diseases. Herein, we elucidate the relationship between NOX4 and HSP47 in fibrogenesis and propose to modulate them altogether as a new strategy to treat fibrosis. We developed a nanoparticle platform consisting of polyethylenimine (PEI) and polyethylene glycol (PEG) coating on a 50-nm mesoporous silica nanoparticle (MSNP) core. The nanoparticles effectively delivered small interfering RNA (siRNA) targeting HSP47 (siHSP47) in an in vitro model of fibrosis based on TGF-β stimulated fibroblasts. The MSNP core also imparted an antioxidant property by scavenging reactive oxygen species (ROS) and subsequently reducing NOX4 levels in the in vitro fibrogenesis model. The nanoparticle was far superior to n-acetyl cysteine (NAC) at modulating pro-fibrotic markers. In vivo evaluation was performed in a bleomycin-induced scleroderma mouse model, which shares many similarities to human scleroderma disease. Intradermal administration of siHSP47-nanoparticles effectively reduced HSP47 protein expression in skin to normal level. In addition, the antioxidant MSNP also played a prominent role in reducing the pro-fibrotic markers, NOX4, alpha smooth muscle actin (α-SMA), and collagen type I (COL I), as well as skin thickness of the mice. PMID:26196532

5'-adenosine monophosphate mediated cooling treatment enhances ΔF508-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) stability in vivo.

PubMed

Zhang, Yueqiang; O'Brien, William G; Zhao, Zhaoyang; Lee, Cheng Chi

2015-09-04

Gene mutations that produce misprocessed proteins are linked to many human disorders. Interestingly, some misprocessed proteins retained their biological function when stabilized by low temperature treatment of cultured cells in vitro. Here we investigate whether low temperature treatment in vivo can rescue misfolded proteins by applying 5'-AMP mediated whole body cooling to a Cystic Fibrosis (CF) mouse model carrying a mutant cystic fibrosis transmembrane conductance regulator (CFTR) with a deletion of the phenylalanine residue in position 508 (ΔF508-CFTR). Low temperature treatment of cultured cells was previously shown to be able to alleviate the processing defect of ΔF508-CFTR, enhancing its plasma membrane localization and its function in mediating chloride ion transport. Here, we report that whole body cooling enhanced the retention of ΔF508-CFTR in intestinal epithelial cells. Functional analysis based on β-adrenergic dependent salivary secretion and post-natal mortality rate revealed a moderate but significant improvement in treated compared with untreated CF mice. Our findings demonstrate that temperature sensitive processing of mutant proteins can be responsive to low temperature treatment in vivo.

Passion fruit peel extract attenuates bleomycin-induced pulmonary fibrosis in mice.

PubMed

Chilakapati, Shanmuga Reddy; Serasanambati, Mamatha; Manikonda, Pavan Kumar; Chilakapati, Damodar Reddy; Watson, Ronald Ross

2014-08-01

Idiopathic pulmonary fibrosis is a progressive fatal lung disease characterized by excessive collagen deposition, with no effective treatments. We investigated the efficacy of natural products with high anti-inflammatory activity, such as passion fruit peel extract (PFPE), in a mouse model of bleomycin-induced pulmonary fibrosis (PF). C57BL/6J mice were subjected to a single intratracheal instillation of bleomycin to induce PF. Daily PFPE treatment significantly reduced loss of body mass and mortality rate in mice compared with those treated with bleomycin. While bleomycin-induced PF resulted in elevated total numbers of inflammatory cells, macrophages, lymphocytes, and neutrophils in bronchoalveolar lavage fluid on both days 7 and 21, PFPE administration significantly attenuated these phenomena compared with bleomycin group. On day 7, the decreased superoxide dismutase and myeloperoxidase activities observed in the bleomycin group were significantly restored with PFPE treatment. On day 21, enhanced hydroxyproline deposition in the bleomycin group was also suppressed by PFPE administration. PFPE treatment significantly attenuated extensive inflammatory cell infiltration and accumulation of collagen in lung tissue sections of bleomycin-induced mice on days 7 and 21, respectively. Our results indicate that administration of PFPE decreased bleomycin-induced PF because of anti-inflammatory and antioxidant activities.

Specifically Formed Corona on Silica Nanoparticles Enhances Transforming Growth Factor β1 Activity in Triggering Lung Fibrosis.

PubMed

Wang, Zhenzhen; Wang, Chunming; Liu, Shang; He, Wei; Wang, Lintao; Gan, JingJing; Huang, Zhen; Wang, Zhenheng; Wei, Haoyang; Zhang, Junfeng; Dong, Lei

2017-02-28

A corona is a layer of macromolecules formed on a nanoparticle surface in vivo. It can substantially change the biological identity of nanomaterials and possibly trigger adverse responses from the body tissues. Dissecting the role of the corona in the development of a particular disease may provide profound insights for understanding toxicity of nanomaterials in general. In our present study, we explored the capability of different silica nanoparticles (SiNPs) to induce silicosis in the mouse lung and analyzed the composition of coronas formed on these particles. We found that SiNPs of certain size and surface chemistry could specifically recruit transforming growth factor β1 (TGF-β1) into their corona, which subsequently induces the development of lung fibrosis. Once embedded into the corona on SiNPs, TGF-β1 was remarkably more stable than in its free form, and its fibrosis-triggering activity was significantly prolonged. Our study meaningfully demonstrates that a specific corona component on a certain nanoparticle could initiate a particular pathogenic process in a clinically relevant disease model. Our findings may shed light on the understanding of molecular mechanisms of human health risks correlated with exposure to small-scale substances.

A role for MCP-1/CCR2 in interstitial lung disease in children

PubMed Central

Hartl, Dominik; Griese, Matthias; Nicolai, Thomas; Zissel, Gernot; Prell, Christine; Reinhardt, Dietrich; Schendel, Dolores J; Krauss-Etschmann, Susanne

2005-01-01

Background Interstitial lung diseases (ILD) are chronic inflammatory disorders leading to pulmonary fibrosis. Monocyte chemotactic protein 1 (MCP-1) promotes collagen synthesis and deletion of the MCP-1 receptor CCR2 protects from pulmonary fibrosis in ILD mouse models. We hypothesized that pulmonary MCP-1 and CCR2+ T cells accumulate in pediatric ILD and are related to disease severity. Methods Bronchoalveolar lavage fluid was obtained from 25 children with ILD and 10 healthy children. Levels of pulmonary MCP-1 and Th1/Th2-associated cytokines were quantified at the protein and the mRNA levels. Pulmonary CCR2+, CCR4+, CCR3+, CCR5+ and CXCR3+ T cells were quantified by flow-cytometry. Results CCR2+ T cells and MCP-1 levels were significantly elevated in children with ILD and correlated with forced vital capacity, total lung capacity and ILD disease severity scores. Children with lung fibrosis had significantly higher MCP-1 levels and CCR2+ T cells in bronchoalveolar lavage fluid compared to non-fibrotic children. Conclusion The results indicate that pulmonary CCR2+ T cells and MCP-1 contribute to the pathogenesis of pediatric ILD and might provide a novel target for therapeutic strategies. PMID:16095529

Multifaceted Therapeutic Benefits of Factors Derived From Dental Pulp Stem Cells for Mouse Liver Fibrosis.

PubMed

Hirata, Marina; Ishigami, Masatoshi; Matsushita, Yoshihiro; Ito, Takanori; Hattori, Hisashi; Hibi, Hideharu; Goto, Hidemi; Ueda, Minoru; Yamamoto, Akihito

2016-10-01

: Chronic liver injury from various causes often results in liver fibrosis (LF). Although the liver possesses endogenous tissue-repairing activities, these can be overcome by sustained inflammation and excessive fibrotic scar formation. Advanced LF leads to irreversible cirrhosis and subsequent liver failure and/or hepatic cancer. Here, using the mouse carbon tetrachloride (CCl 4 )-induced LF model, we showed that a single intravenous administration of stem cells derived from human exfoliated deciduous teeth (SHEDs) or of SHED-derived serum-free conditioned medium (SHED-CM) resulted in fibrotic scar resolution. SHED-CM suppressed the gene expression of proinflammatory mediators, such as TNF-α, IL-1β, and iNOS, and eliminated activated hepatic stellate cells by inducing their apoptosis, but protected parenchymal hepatocytes from undergoing apoptosis. In addition, SHED-CM induced tissue-repairing macrophages that expressed high levels of the profibrinolytic factor, matrix metalloproteinase 13. Furthermore, SHED-CM suppressed the CCl 4 -induced apoptosis of primary cultured hepatocytes. SHED-CM contained a high level of hepatocyte growth factor (HGF). Notably, HGF-depleted SHED-CM (dHGF-CM) did not suppress the proinflammatory response or resolve fibrotic scarring. Furthermore, SHED-CM, but not dHGF-CM, inhibited CCl 4 -induced hepatocyte apoptosis. These results suggest that HGF plays a central role in the SHED-CM-mediated resolution of LF. Taken together, our findings suggest that SHED-CM provides multifaceted therapeutic benefits for the treatment of LF. This study demonstrated that a single intravenous administration of stem cells from human exfoliated deciduous teeth (SHEDs) or of the serum-free conditioned medium (CM) derived from SHEDs markedly improved mouse liver fibrosis (LF). SHED-CM suppressed chronic inflammation, eliminated activated hepatic stellate cells by inducing their apoptosis, protected hepatocytes from undergoing apoptosis, and induced differentiation of tissue-repairing macrophages expressing high levels of the profibrinolytic factor matrix metalloproteinase 13. Furthermore, hepatocyte growth factor played a central role in the SHED-CM-mediated resolution of LF. This is the first report demonstrating the multifaceted therapeutic benefits of secreted factors derived from SHEDs for LF. ©AlphaMed Press.

NADPH Oxidase Signaling Pathway Mediates Mesenchymal Stem Cell-Induced Inhibition of Hepatic Stellate Cell Activation.

PubMed

Qiao, Haowen; Zhou, Yu; Qin, Xingping; Cheng, Jing; He, Yun; Jiang, Yugang

2018-01-01

Bone marrow-derived mesenchymal stem cells (BMSCs) have blossomed into an effective approach with great potential for the treatment of liver fibrosis. The aim of this study was to investigate the underlying antifibrosis mechanisms by which the BMSC inhibit activated hepatic stellate cells (HSCs) in vivo and in vitro. To study the effect of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) on activated HSCs, we used HSCs and the coculture systems to evaluate the inhibition of activated HSCs from the aspects of the apoptosis of activated HSCs. In addition, activation of NADPH oxidase pathway and the changes in liver histopathology were tested by using the carbon tetrachloride- (CCl 4 -) induced liver fibrosis in mice. Introduction of hBM-MSCs significantly inhibited the proliferation of activated HSCs by inducing the apoptosis process of activated HSCs. The effect of hBM-MSCs reduced the signaling pathway of NADPH oxidase in activated HSCs. Besides, the signaling pathway of NADPH oxidase mediated hBM-MSC upregulation of the expression of the peroxisome proliferator-activated receptor γ and downregulation of the expression of α 1(I) collagen and alpha-smooth muscle actin ( α -SMA) in activated HSCs. Moreover, the hBM-MSC-induced decrease in the signaling pathway of NADPH oxidase was accompanied by the decrease of the activated HSC number and liver fibrosis in a mouse model of CCl 4 -induced liver fibrosis. The hBM-MSCs act as a promising drug source against liver fibrosis development with respect to hepatopathy as a therapeutic target.

A Pathogenetic Role for Endothelin-1 in Peritoneal Dialysis-Associated Fibrosis

PubMed Central

Busnadiego, Oscar; Loureiro-Álvarez, Jesús; Sandoval, Pilar; Lagares, David; Dotor, Javier; Pérez-Lozano, María Luisa; López-Armada, María J.; Lamas, Santiago; López-Cabrera, Manuel

2015-01-01

In patients undergoing peritoneal dialysis (PD), chronic exposure to nonphysiologic PD fluids elicits low-grade peritoneal inflammation, leading to fibrosis and angiogenesis. Phenotype conversion of mesothelial cells into myofibroblasts, the so-called mesothelial-to-mesenchymal transition (MMT), significantly contributes to the peritoneal dysfunction related to PD. A number of factors have been described to induce MMT in vitro and in vivo, of which TGF-β1 is probably the most important. The vasoconstrictor peptide endothelin-1 (ET-1) is a transcriptional target of TGF-β1 and mediates excessive scarring and fibrosis in several tissues. This work studied the contribution of ET-1 to the development of peritoneal damage and failure in a mouse model of PD. ET-1 and its receptors were expressed in the peritoneal membrane and upregulated on PD fluid exposure. Administration of an ET receptor antagonist, either bosentan or macitentan, markedly attenuated PD-induced MMT, fibrosis, angiogenesis, and peritoneal functional decline. Adenovirus-mediated overexpression of ET-1 induced MMT in human mesothelial cells in vitro and promoted the early cellular events associated with peritoneal dysfunction in vivo. Notably, TGF-β1–blocking peptides prevented these actions of ET-1. Furthermore, a positive reciprocal relationship was observed between ET-1 expression and TGF-β1 expression in human mesothelial cells. These results strongly support a role for an ET-1/TGF-β1 axis as an inducer of MMT and subsequent peritoneal damage and fibrosis, and they highlight ET-1 as a potential therapeutic target in the treatment of PD-associated dysfunction. PMID:25012164

Docosahexaenoic acid attenuates Western diet-induced hepatic fibrosis in Ldlr−/− mice by targeting the TGFβ-Smad3 pathway[S

PubMed Central

Lytle, Kelli A.; Depner, Christopher M.; Wong, Carmen P.; Jump, Donald B.

2015-01-01

DHA (22:6,ω3), but not EPA (20:5,ω3), attenuates Western diet (WD)-induced hepatic fibrosis in a Ldlr−/− mouse model of nonalcoholic steatohepatitis. We examined the molecular basis for the differential effect of dietary EPA and DHA on WD-induced hepatic fibrosis. DHA was more effective than EPA at preventing WD-induced effects on hepatic transcripts linked to fibrosis, including collagen 1A1 (Col1A1), transforming growth factor-β (TGFβ) signaling and proteins involved in remodeling the extracellular matrix, including metalloproteases, tissue inhibitors of metalloproteases, and lysyl oxidase subtypes. Examination of the TGFβ pathway showed that mice fed the WD supplemented with either olive oil or EPA had a significant (≥2.5-fold) increase in hepatic nuclear abundance of phospho-mothers against decapentaplegic homolog (Smad)3 when compared with mice fed the reference diet (RD); Smad3 is a key regulator of Col1A1 expression in stellate cells. In contrast, mice fed the WD supplemented with DHA had no increase in phospho-Smad3 when compared with mice fed the RD. Changes in hepatic phospho-Smad3 nuclear content correlated with proCol1A1 mRNA and protein abundance. Pretreatment of human LX2 stellate cells with DHA, but not other unsaturated fatty acids, blocked TGFβ1-mediated induction of Col1A1. In conclusion, DHA attenuates WD-induced fibrosis by targeting the TGFβ-Smad3-Col1A1 pathway in stellate cells. PMID:26315048

GDF11 induces kidney fibrosis, renal cell epithelial-to-mesenchymal transition, and kidney dysfunction and failure.

PubMed

Pons, Marianne; Koniaris, Leonidas G; Moe, Sharon M; Gutierrez, Juan C; Esquela-Kerscher, Aurora; Zimmers, Teresa A

2018-05-03

GDF11 modulates embryonic patterning and kidney organogenesis. Herein, we sought to define GDF11 function in the adult kidney and in renal diseases. In vitro renal cell lines, genetic, and murine in vivo renal injury models were examined. Among tissues tested, Gdf11 was highest in normal adult mouse kidney. Expression was increased acutely after 5/6 nephrectomy, ischemia-reperfusion injury, kanamycin toxicity, or unilateral ureteric obstruction. Systemic, high-dose GDF11 administration in adult mice led to renal failure, with accompanying kidney atrophy, interstitial fibrosis, epithelial-to-mesenchymal transition of renal tubular cells, and eventually death. These effects were associated with phosphorylation of SMAD2 and could be blocked by follistatin. In contrast, Gdf11 heterozygous mice showed reduced renal Gdf11 expression, renal fibrosis, and expression of fibrosis-associated genes both at baseline and after unilateral ureteric obstruction compared with wild-type littermates. The kidney-specific consequences of GDF11 dose modulation are direct effects on kidney cells. GDF11 induced proliferation and activation of NRK49f renal fibroblasts and also promoted epithelial-to-mesenchymal transition of IMCD-3 tubular epithelial cells in a SMAD3-dependent manner. Taken together, these data suggest that GDF11 and its downstream signals are critical in vivo mediators of renal injury. These effects are through direct actions of GDF11 on renal tubular cells and fibroblasts. Thus, regulation of GDF11 presents a therapeutic target for diseases involving renal fibrosis and impaired tubular function. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Expression of mutant Sftpcin murine alveolar epithelia drives spontaneous lung fibrosis.

PubMed

Nureki, Shin-Ichi; Tomer, Yaniv; Venosa, Alessandro; Katzen, Jeremy; Russo, Scott J; Jamil, Sarita; Barrett, Matthew; Nguyen, Vivian; Kopp, Meghan; Mulugeta, Surafel; Beers, Michael F

2018-06-19

Epithelial cell dysfunction is postulated as an important component in the pathogenesis of Idiopathic Pulmonary Fibrosis (IPF). Mutations in the Surfactant Protein C [SP-C] gene [SFTPC], an alveolar type 2 (AT2) cell restricted protein, have been found in sporadic and familial IPF. To causally link these events, we developed a knock-in mouse model capable of regulated expression of an IPF-associated Isoleucine to Threonine substitution at codon 73 [I73T] in Sftpc (SP-CI73T). Tamoxifen treated SP-CI73T cohorts developed rapid increases in SftpcI73T mRNA and misprocessed proSP-CI73T protein accompanied by increased early mortality (days 7-14). This acute phase was marked by diffuse parenchymal lung injury, tissue infiltration by monocytes, polycellular alveolitis, and elevations in bronchoalveolar lavage and AT2 mRNA contents of select inflammatory cytokines. Resolution of alveolitis (2-4 weeks), commensurate with a rise in TGFB1, was followed by aberrant remodeling marked by collagen deposition, AT2 cell hyperplasia, a-SMA positive cells, and restrictive lung physiology. The translational relevance of the model was supported by detection of multiple IPF biomarkers previously reported in human cohorts. These data provide proof of principle that mutant SP-C expression in vivo causes spontaneous lung fibrosis strengthening the role of AT2 dysfunction as a key upstream driver of IPF pathogenesis.

Dietary flaxseed administered post thoracic radiation treatment improves survival and mitigates radiation-induced pneumonopathy in mice

PubMed Central

2011-01-01

Background Flaxseed (FS) is a dietary supplement known for its antioxidant and anti-inflammatory properties. Radiation exposure of lung tissues occurs either when given therapeutically to treat intrathoracic malignancies or incidentally, such as in the case of exposure from inhaled radioisotopes released after the detonation of a radiological dispersion devise (RDD). Such exposure is associated with pulmonary inflammation, oxidative tissue damage and irreversible lung fibrosis. We previously reported that dietary FS prevents pneumonopathy in a rodent model of thoracic X-ray radiation therapy (XRT). However, flaxseed's therapeutic usefulness in mitigating radiation effects post-exposure has never been evaluated. Methods We evaluated the effects of a 10%FS or isocaloric control diet given to mice (C57/BL6) in 2 separate experiments (n = 15-25 mice/group) on 0, 2, 4, 6 weeks post a single dose 13.5 Gy thoracic XRT and compared it to an established radiation-protective diet given preventively, starting at 3 weeks prior to XRT. Lungs were evaluated four months post-XRT for blood oxygenation levels, inflammation and fibrosis. Results Irradiated mice fed a 0%FS diet had a 4-month survival rate of 40% as compared to 70-88% survival in irradiated FS-fed mouse groups. Additionally, all irradiated FS-fed mice had decreased fibrosis compared to those fed 0%FS. Lung OH-Proline content ranged from 96.5 ± 7.1 to 110.2 ± 7.7 μg/ml (Mean ± SEM) in all irradiated FS-fed mouse groups, as compared to 138 ± 10.8 μg/ml for mice on 0%FS. Concomitantly, bronchoalveolar lavage (BAL) protein and weight loss associated with radiation cachexia was significantly decreased in all FS-fed groups. Inflammatory cell influx to lungs also decreased significantly except when FS diet was delayed by 4 and 6 weeks post XRT. All FS-fed mice (irradiated or not), maintained a higher blood oxygenation level as compared to mice on 0%FS. Similarly, multiplex cytokine analysis in the BAL fluid revealed a significant decrease of specific inflammatory cytokines in FS-fed mice. Conclusions Dietary FS given post-XRT mitigates radiation effects by decreasing pulmonary fibrosis, inflammation, cytokine secretion and lung damage while enhancing mouse survival. Dietary supplementation of FS may be a useful adjuvant treatment mitigating adverse effects of radiation in individuals exposed to inhaled radioisotopes or incidental radiation. PMID:21702963

Combination effects of alogliptin and pioglitazone on steatosis and hepatic fibrosis formation in a mouse model of non-alcoholic steatohepatitis.

PubMed

Amano, Yuichiro; Tsuchiya, Shuntarou; Imai, Mayumi; Tohyama, Kimio; Matsukawa, Jun; Isono, Osamu; Yasuno, Hironobu; Enya, Kazuaki; Koumura, Emiko; Nagabukuro, Hiroshi

2018-02-26

This study aimed to evaluate the effects of combination therapy with a dipeptidyl peptidase-4 inhibitor, alogliptin, and a peroxisome proliferator-activated receptor-γ agonist, pioglitazone, in a preclinical model of nonalcoholic steatohepatitis using low-density lipoprotein receptor-knockout mice fed a modified choline-deficient l-amino acid-defined diet. Monotherapy with either alogliptin (10-200 mg/kg) or pioglitazone (6-20 mg/kg) significantly decreased hepatic triglyceride content and fibrosis. The concomitant treatment of alogliptin (30 mg/kg), pioglitazone (20 mg/kg) also decreased hepatic triglyceride and hepatic collagen-I mRNA at greater extent compared to monotherapy. Hepatic expression of CD11b mRNA and monocyte chemoattractant protein-1 were also reduced by the concomitant treatment. These results suggest that via an anti-inflammatory potential in addition to anti-metabolic effects, the combination therapy of alogliptin and pioglitazone may provide therapeutic benefits to type 2 diabetes patients with nonalcoholic steatohepatitis, which will be proven in controlled clinical trials. Copyright © 2018 Elsevier Inc. All rights reserved.

Anatomy and bronchoscopy of the porcine lung. A model for translational respiratory medicine.

PubMed

Judge, Eoin P; Hughes, J M Lynne; Egan, Jim J; Maguire, Michael; Molloy, Emer L; O'Dea, Shirley

2014-09-01

The porcine model has contributed significantly to biomedical research over many decades. The similar size and anatomy of pig and human organs make this model particularly beneficial for translational research in areas such as medical device development, therapeutics and xenotransplantation. In recent years, a major limitation with the porcine model was overcome with the successful generation of gene-targeted pigs and the publication of the pig genome. As a result, the role of this model is likely to become even more important. For the respiratory medicine field, the similarities between pig and human lungs give the porcine model particular potential for advancing translational medicine. An increasing number of lung conditions are being studied and modeled in the pig. Genetically modified porcine models of cystic fibrosis have been generated that, unlike mouse models, develop lung disease similar to human cystic fibrosis. However, the scientific literature relating specifically to porcine lung anatomy and airway histology is limited and is largely restricted to veterinary literature and textbooks. Furthermore, methods for in vivo lung procedures in the pig are rarely described. The aims of this review are to collate the disparate literature on porcine lung anatomy, histology, and microbiology; to provide a comparison with the human lung; and to describe appropriate bronchoscopy procedures for the pig lungs to aid clinical researchers working in the area of translational respiratory medicine using the porcine model.

Selective pharmacological inhibition of DDR1 prevents experimentally-induced glomerulonephritis in prevention and therapeutic regime.

PubMed

Moll, Solange; Yasui, Yukari; Abed, Ahmed; Murata, Takeshi; Shimada, Hideaki; Maeda, Akira; Fukushima, Naoshi; Kanamori, Masakazu; Uhles, Sabine; Badi, Laura; Cagarelli, Thomas; Formentini, Ivan; Drawnel, Faye; Georges, Guy; Bergauer, Tobias; Gasser, Rodolfo; Bonfil, R Daniel; Fridman, Rafael; Richter, Hans; Funk, Juergen; Moeller, Marcus J; Chatziantoniou, Christos; Prunotto, Marco

2018-06-01

Discoidin domain receptor 1 (DDR1) is a collagen-activated receptor tyrosine kinase extensively implicated in diseases such as cancer, atherosclerosis and fibrosis. Multiple preclinical studies, performed using either a gene deletion or a gene silencing approaches, have shown this receptor being a major driver target of fibrosis and glomerulosclerosis. The present study investigated the role and relevance of DDR1 in human crescentic glomerulonephritis (GN). Detailed DDR1 expression was first characterized in detail in human GN biopsies using a novel selective anti-DDR1 antibody using immunohistochemistry. Subsequently the protective role of DDR1 was investigated using a highly selective, novel, small molecule inhibitor in a nephrotoxic serum (NTS) GN model in a prophylactic regime and in the NEP25 GN mouse model using a therapeutic intervention regime. DDR1 expression was shown to be mainly limited to renal epithelium. In humans, DDR1 is highly induced in injured podocytes, in bridging cells expressing both parietal epithelial cell (PEC) and podocyte markers and in a subset of PECs forming the cellular crescents in human GN. Pharmacological inhibition of DDR1 in NTS improved both renal function and histological parameters. These results, obtained using a prophylactic regime, were confirmed in the NEP25 GN mouse model using a therapeutic intervention regime. Gene expression analysis of NTS showed that pharmacological blockade of DDR1 specifically reverted fibrotic and inflammatory gene networks and modulated expression of the glomerular cell gene signature, further validating DDR1 as a major mediator of cell fate in podocytes and PECs. Together, these results suggest that DDR1 inhibition might be an attractive and promising pharmacological intervention for the treatment of GN, predominantly by targeting the renal epithelium.

Wnt6 regulates epithelial cell differentiation and is dysregulated in renal fibrosis.

PubMed

Beaton, Hayley; Andrews, Darrell; Parsons, Martin; Murphy, Mary; Gaffney, Andrew; Kavanagh, David; McKay, Gareth J; Maxwell, Alexander P; Taylor, Cormac T; Cummins, Eoin P; Godson, Catherine; Higgins, Debra F; Murphy, Paula; Crean, John

2016-07-01

Diabetic nephropathy is the most common microvascular complication of diabetes mellitus, manifesting as mesangial expansion, glomerular basement membrane thickening, glomerular sclerosis, and progressive tubulointerstitial fibrosis leading to end-stage renal disease. Here we describe the functional characterization of Wnt6, whose expression is progressively lost in diabetic nephropathy and animal models of acute tubular injury and renal fibrosis. We have shown prominent Wnt6 and frizzled 7 (FzD7) expression in the mesonephros of the developing mouse kidney, suggesting a role for Wnt6 in epithelialization. Importantly, TCF/Lef reporter activity is also prominent in the mesonephros. Analysis of Wnt family members in human renal biopsies identified differential expression of Wnt6, correlating with severity of the disease. In animal models of tubular injury and fibrosis, loss of Wnt6 was evident. Wnt6 signals through the canonical pathway in renal epithelial cells as evidenced by increased phosphorylation of GSK3β (Ser9), nuclear accumulation of β-catenin and increased TCF/Lef transcriptional activity. FzD7 was identified as a putative receptor of Wnt6. In vitro Wnt6 expression leads to de novo tubulogenesis in renal epithelial cells grown in three-dimensional culture. Importantly, Wnt6 rescued epithelial cell dedifferentiation in response to transforming growth factor-β (TGF-β); Wnt6 reversed TGF-β-mediated increases in vimentin and loss of epithelial phenotype. Wnt6 inhibited TGF-β-mediated p65-NF-κB nuclear translocation, highlighting cross talk between the two pathways. The critical role of NF-κB in the regulation of vimentin expression was confirmed in both p65(-/-) and IKKα/β(-/-) embryonic fibroblasts. We propose that Wnt6 is involved in epithelialization and loss of Wnt6 expression contributes to the pathogenesis of renal fibrosis. Copyright © 2016 the American Physiological Society.

Maintenance of airway epithelium in acutely rejected orthotopic vascularized mouse lung transplants.

PubMed

Okazaki, Mikio; Gelman, Andrew E; Tietjens, Jeremy R; Ibricevic, Aida; Kornfeld, Christopher G; Huang, Howard J; Richardson, Steven B; Lai, Jiaming; Garbow, Joel R; Patterson, G Alexander; Krupnick, Alexander S; Brody, Steven L; Kreisel, Daniel

2007-12-01

Lung transplantation remains the only therapeutic option for many patients suffering from end-stage pulmonary disease. Long-term success after lung transplantation is severely limited by the development of bronchiolitis obliterans. The murine heterotopic tracheal transplantation model has been widely used for studies investigating pathogenesis of obliterative airway disease and immunosuppressive strategies to prevent its development. Despite its utility, this model employs proximal airway that lacks airflow and is not vascularized. We have developed a novel model of orthotopic vascularized lung transplantation in the mouse, which leads to severe vascular rejection in allogeneic strain combinations. Here we characterize differences in the fate of airway epithelial cells in nonimmunosuppressed heterotopic tracheal and vascularized lung allograft models over 28 days. Up-regulation of growth factors that are thought to be critical for the development of airway fibrosis and interstitial collagen deposition were similar in both models. However, while loss of airway epithelial cells occurred in the tracheal model, airway epithelium remained intact and fully differentiated in lung allografts, despite profound vascular rejection. Moreover, we demonstrate expression of the anti-apoptotic protein Bcl-2 in airway epithelial cells of acutely rejected lung allografts. These findings suggest that in addition to alloimmune responses, other stimuli may be required for the destruction of airway epithelial cells. Thus, the model of vascularized mouse lung transplantation may provide a new and more physiologic experimental tool to study the interaction between immune and nonimmune mechanisms affecting airway pathology in lung allografts.

CCN5 overexpression inhibits profibrotic phenotypes via the PI3K/Akt signaling pathway in lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis and in an in vivo model of lung fibrosis.

PubMed

Zhang, Lin; Li, Yingna; Liang, Chunlian; Yang, Weilin

2014-02-01

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease with unknown etiology and undefined treatment modality. Fibroblasts are regarded as the major cell type that mediates the onset and progression of lung fibrosis by secreting large amounts of extracellular matrix (ECM) proteins, such as connective tissue growth factor (CTGF/CCN2). Current knowledge confers a crucial role of CCN2 in lung fibrosis. CCN5, another member of the CCN family, has been suggested to play an inhibitory role in some fibrotic diseases, such as cardiac fibrosis. However, the role of CCN5 in the process of IPF remains unknown. In the present study, using western blot analysis, we demonstrate that CCN2 is highly expressed in fibroblasts derived from IPF tissue, but is only slightly expressed in normal human lung fibroblasts. However, CCN5 was weakly expressed in all the above cells. qRT-PCR revealed that transforming growth factor (TGF)-β1 stimulation increased CCN2 expression in the IPF-derived cultures of primary human lung fibroblasts (PIFs) in a time- and concentration-dependent manner, but only slightly affected the expression of CCN5. The overexpression of CCN5 induced by the transfection of PIFs with recombinant plasmid did not affect cell viability, proliferation and apoptosis; however, it significantly suppressed the expression of CCN2, α-smooth muscle actin (α-SMA) and collagen type I. The TGF-β1-induced upregulation of the phosphorylation of Akt was reversed by CCN5 overexpression. Our results also demonstrated that adenovirus-mediated CCN5 overexpression in a mouse model of bleomycin-induced IPF significantly decreased the hydroxyproline content in the lungs, as well as TGF-β1 expression in bronchoalveolar lavage fluid. Taken together, our data demonstrate that CCN5 exerts an inhibitory effect on the fibrotic phenotypes of pulmonary fibroblasts in vitro and in vivo, and as such may be a promising target for the treatment of IPF.

Branched-chain amino acids prevent hepatic fibrosis and development of hepatocellular carcinoma in a non-alcoholic steatohepatitis mouse model

PubMed Central

Takegoshi, Kai; Honda, Masao; Okada, Hikari; Takabatake, Riuta; Matsuzawa-Nagata, Naoto; Campbell, Jean S.; Nishikawa, Masashi; Shimakami, Tetsuro; Shirasaki, Takayoshi; Sakai, Yoshio; Yamashita, Taro; Takamura, Toshinari; Tanaka, Takuji; Kaneko, Shuichi

2017-01-01

Oral supplementation with branched-chain amino acids (BCAA; leucine, isoleucine, and valine) in patients with liver cirrhosis potentially suppresses the incidence of hepatocellular carcinoma (HCC) and improves event-free survival. However, the detailed mechanisms of BCAA action have not been fully elucidated. BCAA were administered to atherogenic and high-fat (Ath+HF) diet-induced nonalcoholic steatohepatitis (NASH) model mice. Liver histology, tumor incidence, and gene expression profiles were evaluated. Ath+HF diet mice developed hepatic tumors at a high frequency at 68 weeks. BCAA supplementation significantly improved hepatic steatosis, inflammation, fibrosis, and tumors in Ath+HF mice at 68 weeks. GeneChip analysis demonstrated the significant resolution of pro-fibrotic gene expression by BCAA supplementation. The anti-fibrotic effect of BCAA was confirmed further using platelet-derived growth factor C transgenic mice, which develop hepatic fibrosis and tumors. In vitro, BCAA restored the transforming growth factor (TGF)-β1-stimulated expression of pro-fibrotic genes in hepatic stellate cells (HSC). In hepatocytes, BCAA restored TGF-β1-induced apoptosis, lipogenesis, and Wnt/β-Catenin signaling, and inhibited the transformation of WB-F344 rat liver epithelial stem-like cells. BCAA repressed the promoter activity of TGFβ1R1 by inhibiting the expression of the transcription factor NFY and histone acetyltransferase p300. Interestingly, the inhibitory effect of BCAA on TGF-β1 signaling was mTORC1 activity-dependent, suggesting the presence of negative feedback regulation from mTORC1 to TGF-β1 signaling. Thus, BCAA induce an anti-fibrotic effect in HSC, prevent apoptosis in hepatocytes, and decrease the incidence of HCC; therefore, BCAA supplementation would be beneficial for patients with advanced liver fibrosis with a high risk of HCC. PMID:28212548

Fasting inhibits hepatic stellate cells activation and potentiates anti-cancer activity of Sorafenib in hepatocellular cancer cells.

PubMed

Lo Re, Oriana; Panebianco, Concetta; Porto, Stefania; Cervi, Carlo; Rappa, Francesca; Di Biase, Stefano; Caraglia, Michele; Pazienza, Valerio; Vinciguerra, Manlio

2018-02-01

Hepatocellular carcinoma (HCC) has a poor outcome. Most HCCs develop in the context of liver fibrosis and cirrhosis caused by chronic inflammation. Short-term fasting approaches enhance the activity of chemotherapy in preclinical cancer models, other than HCC. Multi-tyrosine kinase inhibitor Sorafenib is the mainstay of treatment in HCC. However, its benefit is frequently short-lived. Whether fasting can alleviate liver fibrosis and whether combining fasting with Sorafenib is beneficial remains unknown. A 24 hr fasting (2% serum, 0.1% glucose)-induced changes on human hepatic stellate cells (HSC) LX-2 proliferation/viability/cell cycle were assessed by MTT and flow cytometry. Expression of lypolysaccharide (LPS)-induced activation markers (vimentin, αSMA) was evaluated by qPCR and immunoblotting. Liver fibrosis and inflammation were evaluated in a mouse model of steatohepatitis exposed to cycles of fasting, by histological and biochemical analyses. A 24 hr fasting-induced changes were also analyzed on the proliferation/viability/glucose uptake of human HCC cells exposed to Sorafenib. An expression panel of genes involved in survival, inflammation, and metabolism was examined by qPCR in HCC cells exposed to fasting and/or Sorafenib. Fasting decreased the proliferation and the activation of HSC. Repeated cycles of short term starvation were safe in mice but did not improve fibrosis. Fasting synergized with Sorafenib in hampering HCC cell growth and glucose uptake. Finally, fasting normalized the expression levels of genes which are commonly altered by Sorafenib in HCC cells. Fasting or fasting-mimicking diet diets should be evaluated in preclinical studies as a mean to potentiate the activity of Sorafenib in clinical use. © 2017 Wiley Periodicals, Inc.

Branched-chain amino acids prevent hepatic fibrosis and development of hepatocellular carcinoma in a non-alcoholic steatohepatitis mouse model.

PubMed

Takegoshi, Kai; Honda, Masao; Okada, Hikari; Takabatake, Riuta; Matsuzawa-Nagata, Naoto; Campbell, Jean S; Nishikawa, Masashi; Shimakami, Tetsuro; Shirasaki, Takayoshi; Sakai, Yoshio; Yamashita, Taro; Takamura, Toshinari; Tanaka, Takuji; Kaneko, Shuichi

2017-03-14

Oral supplementation with branched-chain amino acids (BCAA; leucine, isoleucine, and valine) in patients with liver cirrhosis potentially suppresses the incidence of hepatocellular carcinoma (HCC) and improves event-free survival. However, the detailed mechanisms of BCAA action have not been fully elucidated. BCAA were administered to atherogenic and high-fat (Ath+HF) diet-induced nonalcoholic steatohepatitis (NASH) model mice. Liver histology, tumor incidence, and gene expression profiles were evaluated. Ath+HF diet mice developed hepatic tumors at a high frequency at 68 weeks. BCAA supplementation significantly improved hepatic steatosis, inflammation, fibrosis, and tumors in Ath+HF mice at 68 weeks. GeneChip analysis demonstrated the significant resolution of pro-fibrotic gene expression by BCAA supplementation. The anti-fibrotic effect of BCAA was confirmed further using platelet-derived growth factor C transgenic mice, which develop hepatic fibrosis and tumors. In vitro, BCAA restored the transforming growth factor (TGF)-β1-stimulated expression of pro-fibrotic genes in hepatic stellate cells (HSC). In hepatocytes, BCAA restored TGF-β1-induced apoptosis, lipogenesis, and Wnt/β-Catenin signaling, and inhibited the transformation of WB-F344 rat liver epithelial stem-like cells. BCAA repressed the promoter activity of TGFβ1R1 by inhibiting the expression of the transcription factor NFY and histone acetyltransferase p300. Interestingly, the inhibitory effect of BCAA on TGF-β1 signaling was mTORC1 activity-dependent, suggesting the presence of negative feedback regulation from mTORC1 to TGF-β1 signaling. Thus, BCAA induce an anti-fibrotic effect in HSC, prevent apoptosis in hepatocytes, and decrease the incidence of HCC; therefore, BCAA supplementation would be beneficial for patients with advanced liver fibrosis with a high risk of HCC.

Fibrocytes Regulate Wilms’ Tumor 1-Positive Cell Accumulation in Severe Fibrotic Lung Disease

PubMed Central

Sontake, Vishwaraj; Shanmukhappa, Shiva K.; DiPasquale, Betsy A.; Reddy, Geereddy B.; Medvedovic, Mario; Hardie, William D.; White, Eric S.; Madala, Satish K.

2015-01-01

Collagen-producing myofibroblast transdifferentiation is considered a crucial determinant in the formation of scar tissue in the lungs of patients with idiopathic pulmonary fibrosis (IPF). Multiple resident pulmonary cell types and bone marrow-derived fibrocytes have been implicated as contributors to fibrotic lesions due to the transdifferentiation potential of these cells into myofibroblasts. In this study, we assessed the expression of Wilms’ tumor 1 (WT1), a known marker of mesothelial cells, in various cell types in normal and fibrotic lungs. We demonstrate that WT1 is expressed by both mesothelial and mesenchymal cells in IPF lungs, but has limited or no expression in normal human lungs. We also demonstrate that WT1-positive cells accumulate in fibrotic lung lesions, using two different mouse models of pulmonary fibrosis and WT1 promoter-driven fluorescent reporter mice. Reconstitution of bone-marrow cells into a transforming growth factor-α transgenic-mouse model demonstrated that fibrocytes do not transform into WT1-positive mesenchymal cells, but do augment accumulation of WT1-positive cells in severe fibrotic lung disease. Importantly, the number of WT1-positive cells in fibrotic lesions were correlated with severity of lung disease as assessed by changes in lung function, histology, and hydroxyproline levels in mice. Finally, inhibition of WT1 expression was sufficient to attenuate collagen and other extracellular-matrix gene production by mesenchymal cells from both murine and human fibrotic lungs. Thus, the results of this study demonstrate a novel association between fibrocyte-driven WT1-positive cell accumulation and severe fibrotic lung disease. PMID:26371248

Long-term treatment with EGFR inhibitor erlotinib attenuates renal inflammatory cytokines but not nephropathy in Alport syndrome mouse model.

PubMed

Omachi, Kohei; Miyakita, Rui; Fukuda, Ryosuke; Kai, Yukari; Suico, Mary Ann; Yokota, Tsubasa; Kamura, Misato; Shuto, Tsuyoshi; Kai, Hirofumi

2017-12-01

Alport syndrome (AS) is a hereditary kidney disease caused by mutation of type IV collagen. Loss of collagen network induces collapse of glomerular basement membrane (GBM) structure. The previous studies showed that upregulation of some tyrosine kinase receptors signaling accompanied GBM disorder in AS mouse model. EGFR signaling is one of the well-known receptor kinase signaling that is involved in glomerular diseases. However, whether EGFR signaling is relevant to AS progression is still uninvestigated. Here, we determined the involvement of EGFR in AS and the effect of suppressing EGFR signaling by erlotinib treatment on AS progression. Phosphorylated EGFR expression was investigated by Western blotting analysis and immunostaining of kidney tissues of Col4a5 mutant mice (a mouse model of X-linked AS). To check the effect of blocking EGFR signaling in AS, we administered erlotinib to AS mice once a day (10 mg/kg/day) orally for 18 weeks. Renal function parameters (proteinuria, serum creatinine, and BUN) and renal histology were assessed, and the gene expressions of inflammatory cytokines were analyzed in renal tissues. Phosphorylated EGFR expression was upregulated in AS mice kidney tissues. Erlotinib slightly reduced the urinary protein and suppressed the expression of renal injury markers (Lcn2, Lysozyme) and inflammatory cytokines (Il-6, Il-1β and KC). Erlotinib did not improve renal pathology, such as glomerular sclerosis and fibrosis. These findings suggest that EGFR signaling is upregulated in kidney, but although inhibiting this signaling pathway suppressed renal inflammatory cytokines, it did not ameliorate renal dysfunction in AS mouse model.

Protective role for miR-9-5p in the fibrogenic transformation of human dermal fibroblasts.

PubMed

Miguel, Verónica; Busnadiego, Oscar; Fierro-Fernández, Marta; Lamas, Santiago

2016-01-01

Excessive accumulation of extracellular matrix (ECM) proteins is the hallmark of fibrotic diseases, including skin fibrosis. This response relies on the activation of dermal fibroblasts that evolve into a pro-fibrogenic phenotype. One of the major players in this process is the cytokine transforming growth factor-β (TGF-β). MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression affecting a wide range of pathophysiological events including fibrogenesis. MicroRNA-9-5p (miR-9-5p) has been shown to exert a protective role in lung and peritoneal fibrosis. This study aimed to evaluate the role of miR-9-5p in skin fibrosis. miR-9-5p is up-regulated in TGF-β1-treated human dermal fibroblasts (HDFs). In silico identification of miR-9-5p targets spotted the type II TGF-β receptor (TGFBR2) as a potential TGF-β signaling-related effector for this miRNA. Consistently, over-expression of miR-9-5p in HDFs down-regulated TGFBR2 at both the mRNA and protein levels and reduced the phosphorylation of Smad2 and the translocation of Smad2/3 to the nucleus. In keeping, over-expression of miR-9-5p significantly delayed TGF-β1-dependent transformation of dermal fibroblasts, decreasing the expression of ECM protein collagen, type I, alpha 1 (Col1α1), and fibronectin (FN), the amount of secreted collagen proteins, and the expression of the archetypal myofibroblast marker alpha-smooth muscle actin (α-SMA). By contrast, specific inhibition of miR-9-5p resulted in enhanced presence of fibrosis markers. The expression of miR-9-5p was also detected in the skin and plasma in the mouse model of bleomycin-induced dermal fibrosis. Using lentiviral constructs, we demonstrated that miR-9-5p over-expression was also capable of deterring fibrogenesis in this same model. miR-9-5p significantly prevents fibrogenesis in skin fibrosis. This is mediated by an abrogation of TGF-β-mediated signaling through the down-regulation of TGFBR2 expression in HDFs. These results may pave the way for future diagnostic or therapeutic developments for skin fibrosis based on miR-9-5p.

α1A-Subtype adrenergic agonist therapy for the failing right ventricle.

PubMed

Cowley, Patrick M; Wang, Guanying; Joshi, Sunil; Swigart, Philip M; Lovett, David H; Simpson, Paul C; Baker, Anthony J

2017-12-01

Failure of the right ventricle (RV) is a serious disease with a poor prognosis and limited treatment options. Signaling by α 1 -adrenergic receptors (α 1 -ARs), in particular the α 1A -subtype, mediate cardioprotective effects in multiple heart failure models. Recent studies have shown that chronic treatment with the α 1A -subtype agonist A61603 improves function and survival in a model of left ventricular failure. The goal of the present study was to determine if chronic A61603 treatment is beneficial in a RV failure model. We used tracheal instillation of the fibrogenic antibiotic bleomycin in mice to induce pulmonary fibrosis, pulmonary hypertension, and RV failure within 2 wk. Some mice were chronically treated with a low dose of A61603 (10 ng·kg -1 ·day -1 ). In the bleomycin model of RV failure, chronic A61603 treatment was associated with improved RV fractional shortening and greater in vitro force development by RV muscle preparations. Cell injury markers were reduced with A61603 treatment (serum cardiac troponin I, RV fibrosis, and expression of matrix metalloproteinase-2). RV oxidative stress was reduced (using the probes dihydroethidium and 4-hydroxynonenal). Consistent with lowered RV oxidative stress, A61603 was associated with an increased level of the cellular antioxidant superoxide dismutase 1 and a lower level of the prooxidant NAD(P)H oxidase isoform NOX4. In summary, in the bleomycin model of RV failure, chronic A61603 treatment reduced RV oxidative stress, RV myocyte necrosis, and RV fibrosis and increased both RV function and in vitro force development. These findings suggest that in the context of pulmonary fibrosis, the α 1A -subtype is a potential therapeutic target to treat the failing RV. NEW & NOTEWORTHY Right ventricular (RV) failure is a serious disease with a poor prognosis and no effective treatments. In the mouse bleomycin model of RV failure, we tested the efficacy of a treatment using the α 1A -adrenergic receptor subtype agonist A61603. Chronic A61603 treatment improved RV contraction and reduced multiple indexes of RV injury, suggesting that the α 1A -subtype is a therapeutic target to treat RV failure.

Collagen VII deficient mice show morphologic and histologic corneal changes that phenotypically mimic human dystrophic epidermolysis bullosa of the eye.

PubMed

Chen, Vicki M; Shelke, Rajani; Nyström, Alexander; Laver, Nora; Sampson, James F; Zhiyi, Cao; Bhat, Najma; Panjwani, Noorjahan

2018-06-16

Absence of collagen VII causes blistering of the skin, eyes and many other tissues. This disease is termed dystrophic epidermolysis bullosa (DEB). Corneal fibrosis occurs in up to 41% and vision loss in up to 64% of patients. Standard treatments are supportive and there is no cure. The immune-histologic and morphologic changes in the corneas of the mouse model for this disease have not been described in the literature. Our purpose is to characterize the eyes of these mice to determine if this is an appropriate model for study of human therapeutics. Western blot analysis (WB) and immunohistochemistry (IHC) were performed to assess the relative collagen VII protein levels and its location within the cornea. Additional IHC for inflammatory and fibrotic biomarkers alpha-smooth muscle actin (α-SMA), transforming growth factor-beta (TGF-β), connective tissue growth factor (CTGF), proteinase 3, tenascin C and collagen III were performed. Clinical photographs documenting opacification of the corneas of animals of differing ages were assessed and scored independently by 2 examiners. Histology was then used to investigate morphologic changes. IHC and WB confirmed that these mice are deficient in collagen VII production at the level of the basement membrane when compared with wild-types. IHC showed anomalous deposition of collagen III throughout the stroma. Of the 5 biomarkers tested, TGF-β showed the strongest and most consistently staining. Photographs documented corneal opacities only in mice older than 10 weeks, opacities were not seen in younger animals. Histology showed multiple abnormalities, including epithelial hyperplasia, ulceration, fibrosis, edema, dysplasia, neovascularization and bullae formation. The collagen VII hypomorphic mouse shows reduced collagen VII production at the level of the corneal basement membrane. Corneal changes are similar to pathology seen in humans with this disease. The presence of anomalous stromal collagen III and TGF-β appear to be the most consistent and strongest staining biomarkers in diseased mice. This mouse appears to mimic human corneal disease. It is an appropriate model for testing of therapeutics to treat EB ocular disease. Copyright © 2018. Published by Elsevier Ltd.

A murine experimental anthracycline extravasation model: pathology and study of the involvement of topoisomerase II alpha and iron in the mechanism of tissue damage.

PubMed

Thougaard, Annemette V; Langer, Seppo W; Hainau, Bo; Grauslund, Morten; Juhl, Birgitte Ravn; Jensen, Peter Buhl; Sehested, Maxwell

2010-02-28

The bisdioxopiperazine topoisomerase II catalytic inhibitor dexrazoxane has successfully been introduced into the clinic as an antidote to accidental anthracycline extravasation based on our preclinical mouse studies. The histology of this mouse extravasation model was investigated and found to be similar to findings in humans: massive necrosis in the subcutis, dermis and epidermis followed by sequestration and healing with granulation tissue, and a graft-versus-host-like reaction with hyperkeratotic and acanthotic keratinocytes, occasional apoptoses, epidermal invasion by lymphocytes and healing with dense dermal connective tissue. The extension of this fibrosis was quantified, and dexrazoxane intervention resulted in a statistically significant decrease in fibrosis extension, as also observed in the clinic. Several mechanisms have been proposed in anthracycline extravasation cytotoxicity, and we tested two major hypotheses: (1) interaction with topoisomerase II alpha and (2) the formation of tissue damaging reactive oxygen species following redox cycling of an anthracycline Fe(2+) complex. Dexrazoxane could minimise skin damage via both mechanisms, as it stops the catalytic activity of topoisomerase II alpha and thereby prevents access of anthracycline to the enzyme and thus cytotoxicity, and also acts as a strong iron chelator following opening of its two bisdioxopiperazine rings. Using the model of extravasation in a dexrazoxane-resistant transgenic mouse with a heterozygous mutation in the topoisomerase II alpha gene (Top2a(Y165S/+)), we found that dexrazoxane provided a protection against anthracycline-induced skin wounds that was indistinguishable from that found in wildtype mice. Thus, interaction with topoisomerase II alpha is not central in the pathogenesis of anthracycline-induced skin damage. In contrast to dexrazoxane, the iron-chelating bisdioxopiperazine ICRF-161 do not inhibit the catalytic cycle of topoisomerase II alpha. This compound was used to isolate and test the importance of iron in the wound pathogenesis. ICRF-161 was found ineffective in the treatment of anthracycline-induced skin damage, suggesting that iron does not play a dominant role in the genesis of wounds. (c) 2010 Elsevier Ireland Ltd. All rights reserved.

Impaired diversity of the lung microbiome predicts progression of idiopathic pulmonary fibrosis.

PubMed

Takahashi, Youhei; Saito, Atsushi; Chiba, Hirofumi; Kuronuma, Koji; Ikeda, Kimiyuki; Kobayashi, Tomofumi; Ariki, Shigeru; Takahashi, Motoko; Sasaki, Yasushi; Takahashi, Hiroki

2018-02-27

Idiopathic pulmonary fibrosis (IPF) is the most frequent and severe form of idiopathic interstitial pneumonias. Although IPF has not been thought to be associated with bacterial communities, recent papers reported the possible role of microbiome composition in IPF. The roles of microbiomes in respiratory functions and as clinical biomarkers for IPF remain unknown. In this study, we aim to identify the relationship between the microbial environment in the lung and clinical findings. Thirty-four subjects diagnosed with IPF were included in this analysis. The 16S rDNA was purified from bronchoalveolar lavage fluid obtained at the time of diagnosis and analyzed using next-generation sequencing techniques to characterize the bacterial communities. Furthermore, microbiomes from mice with bleomycin-induced lung fibrosis were analyzed. The most prevalent lung phyla were Firmicutes, Proteobacteria and Bacteroidetes. Decreased microbial diversity was found in patients with low forced vital capacity (FVC) and early mortality. Additionally, the diversity and relative abundance of Firmicutes, Streptococcaceae, and Veillonellaceae were significantly associated with FVC, 6-min walk distance, and serum surfactant protein D. Bleomycin-induced lung fibrosis resulted in decrease of diversity and alteration of microbiota in PCoA analysis. These results support the observations in human specimens. This study identified relationships between specific taxa in BALF and clinical findings, which were also supported by experiments in a mouse model. Our data suggest the possibility that loss of microbial diversity is associated with disease activities of IPF.

Folic acid prevents cardiac dysfunction and reduces myocardial fibrosis in a mouse model of high-fat diet-induced obesity.

PubMed

Li, Wei; Tang, Renqiao; Ouyang, Shengrong; Ma, Feifei; Liu, Zhuo; Wu, Jianxin

2017-01-01

Folic acid (FA) is an antioxidant that can reduce reactive oxygen species generation and can blunt cardiac dysfunction during ischemia. We hypothesized that FA supplementation prevents cardiac fibrosis and cardiac dysfunction induced by obesity. Six-week-old C57BL6/J mice were fed a high-fat diet (HFD), normal diet (ND), or an HFD supplemented with folic acid (FAD) for 14 weeks. Cardiac function was measured using a transthoracic echocardiographic exam. Phenotypic analysis included measurements of body and heart weight, blood glucose and tissue homocysteine (Hcy) content, and heart oxidative stress status. HFD consumption elevated fasting blood glucose levels and caused obesity and heart enlargement. FA supplementation in HFD-fed mice resulted in reduced fasting blood glucose, heart weight, and heart tissue Hcy content. We also observed a significant cardiac systolic dysfunction when mice were subjected to HFD feeding as indicated by a reduction in the left ventricular ejection fraction and fractional shortening. However, FAD treatment improved cardiac function. FA supplementation protected against cardiac fibrosis induced by HFD. In addition, HFD increased malondialdehyde concentration of the heart tissue and reduced the levels of antioxidant enzyme, glutathione, and catalase. HFD consumption induced myocardial oxidant stress with amelioration by FA treatment. FA supplementation significantly lowers blood glucose levels and heart tissue Hcy content and reverses cardiac dysfunction induced by HFD in mice. These functional improvements of the heart may be mediated by the alleviation of oxidative stress and myocardial fibrosis.

Cardiac remodeling in the mouse model of Marfan syndrome develops into two distinctive phenotypes

PubMed Central

Tae, Hyun-Jin; Marshall, Shannon; Krawczyk, Melissa; Talan, Mark

2015-01-01

Marfan syndrome (MFS) is a systemic disorder of connective tissue caused by mutations in fibrillin-1. Cardiac dysfunction in MFS has not been characterized halting the development of therapies of cardiac complication in MFS. We aimed to study the age-dependent cardiac remodeling in the mouse model of MFS FbnC1039G+/− mouse [Marfan heterozygous (HT) mouse] and its association with valvular regurgitation. Marfan HT mice of 2–4 mo demonstrated a mild hypertrophic cardiac remodeling with predominant decline of diastolic function and increased transforming growth factor-β canonical (p-SMAD2/3) and noncanonical (p-ERK1/2 and p-p38 MAPK) signaling and upregulation of hypertrophic markers natriuretic peptides atrium natriuretic peptide and brain natriuretic peptide. Among older HT mice (6–14 mo), cardiac remodeling was associated with two distinct phenotypes, manifesting either dilated or constricted left ventricular chamber. Dilatation of left ventricular chamber was accompanied by biochemical evidence of greater mechanical stress, including elevated ERK1/2 and p38 MAPK phosphorylation and higher brain natriuretic peptide expression. The aortic valve regurgitation was registered in 20% of the constricted group and 60% of the dilated group, whereas mitral insufficiency was observed in 40% of the constricted group and 100% of the dilated group. Cardiac dysfunction was not associated with the increase of interstitial fibrosis and nonmyocyte proliferation. In the mouse model fibrillin-1, haploinsufficiency results in the early onset of nonfibrotic hypertrophic cardiac remodeling and dysfunction, independently from valvular abnormalities. MFS heart is vulnerable to stress-induced cardiac dilatation in the face of valvular regurgitation, and stress-activated MAPK signals represent a potential target for cardiac management in MFS. PMID:26566724

Cardiac remodeling in the mouse model of Marfan syndrome develops into two distinctive phenotypes.

PubMed

Tae, Hyun-Jin; Petrashevskaya, Natalia; Marshall, Shannon; Krawczyk, Melissa; Talan, Mark

2016-01-15

Marfan syndrome (MFS) is a systemic disorder of connective tissue caused by mutations in fibrillin-1. Cardiac dysfunction in MFS has not been characterized halting the development of therapies of cardiac complication in MFS. We aimed to study the age-dependent cardiac remodeling in the mouse model of MFS FbnC1039G+/- mouse [Marfan heterozygous (HT) mouse] and its association with valvular regurgitation. Marfan HT mice of 2-4 mo demonstrated a mild hypertrophic cardiac remodeling with predominant decline of diastolic function and increased transforming growth factor-β canonical (p-SMAD2/3) and noncanonical (p-ERK1/2 and p-p38 MAPK) signaling and upregulation of hypertrophic markers natriuretic peptides atrium natriuretic peptide and brain natriuretic peptide. Among older HT mice (6-14 mo), cardiac remodeling was associated with two distinct phenotypes, manifesting either dilated or constricted left ventricular chamber. Dilatation of left ventricular chamber was accompanied by biochemical evidence of greater mechanical stress, including elevated ERK1/2 and p38 MAPK phosphorylation and higher brain natriuretic peptide expression. The aortic valve regurgitation was registered in 20% of the constricted group and 60% of the dilated group, whereas mitral insufficiency was observed in 40% of the constricted group and 100% of the dilated group. Cardiac dysfunction was not associated with the increase of interstitial fibrosis and nonmyocyte proliferation. In the mouse model fibrillin-1, haploinsufficiency results in the early onset of nonfibrotic hypertrophic cardiac remodeling and dysfunction, independently from valvular abnormalities. MFS heart is vulnerable to stress-induced cardiac dilatation in the face of valvular regurgitation, and stress-activated MAPK signals represent a potential target for cardiac management in MFS.

Transgenic expression of human neutrophil peptide-1 enhances hepatic fibrosis in mice fed a choline-deficient, L-amino acid-defined diet.

PubMed

Ibusuki, Rie; Uto, Hirofumi; Arima, Shiho; Mawatari, Seiichi; Setoguchi, Yoshiko; Iwashita, Yuji; Hashimoto, Shinichi; Maeda, Takuro; Tanoue, Shiro; Kanmura, Shuji; Oketani, Makoto; Ido, Akio; Tsubouchi, Hirohito

2013-11-01

Neutrophils infiltrate the livers of patients with nonalcoholic steatohepatitis (NASH). Human neutrophil peptides (HNPs) induce cytokine and chemokine production under inflammatory conditions, which may contribute to the progression of NASH. In this study, we focused on the effects of HNP-1 on hepatic steatosis and fibrosis in a mouse model of NASH induced by a choline-deficient, L-amino acid-defined (CDAA) diet. We generated transgenic mice expressing HNP-1 under the control of a β-actin-based promoter. HNP-1 transgenic and wild-type C57BL/6N mice were fed a CDAA diet for 16 weeks to induce hepatic steatosis and fibrosis. Serological and histological features were examined, and the effects of HNP-1 on hepatic stellate cell lines were assessed. HNP-1 transgenic and wild-type mice fed the CDAA diet showed no significant differences in serum alanine aminotransferase levels or the degree of hepatic steatosis based on Oil red O staining and hepatic triglyceride content. In contrast, Sirius Red and Azan staining showed significantly more severe hepatic fibrosis in HNP-1 transgenic mice compared with wild-type mice. In addition, significantly more α-smooth muscle actin-positive hepatic stellate cells were observed in the transgenic mice than in the wild-type mice. Finally, the proliferation of the LI90 hepatic stellate cell line increased in response to HNP-1. Our data indicate that HNP-1 enhances hepatic fibrosis in fatty liver by inducing hepatic stellate cell proliferation. Thus, neutrophil-derived HNP-1 may contribute to the progression of NASH. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Tenascin-C promotes chronic pressure overload-induced cardiac dysfunction, hypertrophy and myocardial fibrosis.

PubMed

Podesser, Bruno K; Kreibich, Maximilian; Dzilic, Elda; Santer, David; Förster, Lorenz; Trojanek, Sandra; Abraham, Dietmar; Krššák, Martin; Klein, Klaus U; Tretter, Eva V; Kaun, Christoph; Wojta, Johann; Kapeller, Barbara; Gonçalves, Inês Fonseca; Trescher, Karola; Kiss, Attila

2018-04-01

Left ventricular (LV) hypertrophy is characterized by cardiomyocyte hypertrophy and interstitial fibrosis ultimately leading to increased myocardial stiffness and reduced contractility. There is substantial evidence that the altered expression of matrix metalloproteinases (MMP) and Tenascin-C (TN-C) are associated with the progression of adverse LV remodeling. However, the role of TN-C in the development of LV hypertrophy because of chronic pressure overload as well as the regulatory role of TN-C on MMPs remains unknown. In a knockout mouse model of TN-C, we investigated the effect of 10 weeks of pressure overload using transverse aortic constriction (TAC). Cardiac function was determined by magnetic resonance imaging. The expression of MMP-2 and MMP-9, CD147 as well as myocardial fibrosis were assessed by immunohistochemistry. The expression of TN-C was assessed by RT-qPCR and ELISA. TN-C knockout mice showed marked reduction in fibrosis (P < 0.001) and individual cardiomyocytes size (P < 0.01), in expression of MMP-2 (P < 0.05) and MMP-9 (P < 0.001) as well as preserved cardiac function (P < 0.01) in comparison with wild-type mice after 10 weeks of TAC. In addition, CD147 expression was markedly increased under pressure overload (P < 0.01), irrespectively of genotype. TN-C significantly increased the expression of the markers of hypertrophy such as ANP and BNP as well as MMP-2 in H9c2 cells (P < 0.05, respectively). Our results are pointed toward a novel signaling mechanism that contributes to LV remodeling via MMPs upregulation, cardiomyocyte hypertrophy as well as myocardial fibrosis by TN-C under chronic pressure overload.

Pirfenidone reduces subchondral bone loss and fibrosis after murine knee cartilage injury.

PubMed

Chan, Deva D; Li, Jun; Luo, Wei; Predescu, Dan N; Cole, Brian J; Plaas, Anna

2018-01-01

Pirfenidone is an anti-inflammatory and anti-fibrotic drug that has shown efficacy in lung and kidney fibrosis. Because inflammation and fibrosis have been linked to the progression of osteoarthritis, we investigated the effects of oral Pirfenidone in a mouse model of cartilage injury, which results in chronic inflammation and joint-wide fibrosis in mice that lack hyaluronan synthase 1 (Has1 -/- ) in comparison to wild-type. Femoral cartilage was surgically injured in wild-type and Has1 -/- mice, and Pirfenidone was administered in food starting after 3 days. At 4 weeks, Pirfenidone reduced the appearance, on micro-computed tomography, of pitting in subchondral bone at, and cortical bone surrounding, the site of cartilage injury. This corresponded with a reduction in fibrotic tissue deposits as observed with gross joint surface photography. Pirfenidone resulted in significant recovery of trabecular bone parameters affected by joint injury in Has1 -/- mice, although the effect in wild-type was less pronounced. Pirfenidone also increased Safranin-O staining of growth plate cartilage after cartilage injury and sham operation in both genotypes. Taken together with the expression of selected extracellular matrix, inflammation, and fibrosis genes, these results indicate that Pirfenidone may confer chondrogenic and bone-protective effects, although the well-known anti-fibrotic effects of Pirfenidone may occur earlier in the wound-healing response than the time point examined in this study. Further investigations to identify the specific cell populations in the joint and signaling pathways that are responsive to Pirfenidone are warranted, as Pirfenidone and other anti-fibrotic drugs may encourage tissue repair and prevent progression of post-traumatic osteoarthritis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:365-376, 2018. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

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

Development of a novel mouse model of hepatocellular carcinoma with nonalcoholic steatohepatitis using a high-fat, choline-deficient diet and intraperitoneal injection of diethylnitrosamine.

PubMed

Kishida, Norihiro; Matsuda, Sachiko; Itano, Osamu; Shinoda, Masahiro; Kitago, Minoru; Yagi, Hiroshi; Abe, Yuta; Hibi, Taizo; Masugi, Yohei; Aiura, Koichi; Sakamoto, Michiie; Kitagawa, Yuko

2016-06-13

The incidence of hepatocellular carcinoma with nonalcoholic steatohepatitis is increasing, and its clinicopathological features are well established. Several animal models of nonalcoholic steatohepatitis have been developed to facilitate its study; however, few fully recapitulate all its clinical features, which include insulin resistance, inflammation, fibrosis, and carcinogenesis. Moreover, these models require a relatively long time to produce hepatocellular carcinoma reliably. The aim of this study was to develop a mouse model of hepatocellular carcinoma with nonalcoholic steatohepatitis that develops quickly and reflects all clinically relevant features. Three-week-old C57BL/6J male mice were fed either a standard diet (MF) or a choline-deficient, high-fat diet (HFCD). The mice in the MF + diethylnitrosamine (DEN) and HFCD + DEN groups received a one-time intraperitoneal injection of DEN at the start of the respective feeding protocols. The mice in the HFCD and HFCD + DEN groups developed obesity early in the experiment and insulin resistance after 12 weeks. Triglyceride levels peaked at 8 weeks for all four groups and decreased thereafter. Alanine aminotransferase levels increased every 4 weeks, with the HFCD and HFCD + DEN groups showing remarkably high levels; the HFCD + DEN group presented the highest incidence of nonalcoholic steatohepatitis. The levels of fibrosis and steatosis varied, but they tended to increase every 4 weeks in the HFCD and HFCD + DEN groups. Computed tomography scans indicated that all the HFCD + DEN mice developed hepatic tumors from 20 weeks, some of which were glutamine synthetase-positive. The nonalcoholic steatohepatitis-hepatocellular carcinoma model we describe here is simple to establish, results in rapid tumor formation, and recapitulates most of the key features of nonalcoholic steatohepatitis. It could therefore facilitate further studies of the development, oncogenic potential, diagnosis, and treatment of this condition.

Long-Term Overexpression of Hsp70 Does Not Protect against Cardiac Dysfunction and Adverse Remodeling in a MURC Transgenic Mouse Model with Chronic Heart Failure and Atrial Fibrillation

PubMed Central

Bernardo, Bianca C.; Sapra, Geeta; Patterson, Natalie L.; Cemerlang, Nelly; Kiriazis, Helen; Ueyama, Tomomi; Febbraio, Mark A.; McMullen, Julie R.

2015-01-01

Previous animal studies had shown that increasing heat shock protein 70 (Hsp70) using a transgenic, gene therapy or pharmacological approach provided cardiac protection in models of acute cardiac stress. Furthermore, clinical studies had reported associations between Hsp70 levels and protection against atrial fibrillation (AF). AF is the most common cardiac arrhythmia presenting in cardiology clinics and is associated with increased rates of heart failure and stroke. Improved therapies for AF and heart failure are urgently required. Despite promising observations in animal studies which targeted Hsp70, we recently reported that increasing Hsp70 was unable to attenuate cardiac dysfunction and pathology in a mouse model which develops heart failure and intermittent AF. Given our somewhat unexpected finding and the extensive literature suggesting Hsp70 provides cardiac protection, it was considered important to assess whether Hsp70 could provide protection in another mouse model of heart failure and AF. The aim of the current study was to determine whether increasing Hsp70 could attenuate adverse cardiac remodeling, cardiac dysfunction and episodes of arrhythmia in a mouse model of heart failure and AF due to overexpression of Muscle-Restricted Coiled-Coil (MURC). Cardiac function and pathology were assessed in mice at approximately 12 months of age. We report here, that chronic overexpression of Hsp70 was unable to provide protection against cardiac dysfunction, conduction abnormalities, fibrosis or characteristic molecular markers of the failing heart. In summary, elevated Hsp70 may provide protection in acute cardiac stress settings, but appears insufficient to protect the heart under chronic cardiac disease conditions. PMID:26660322

Long-Term Overexpression of Hsp70 Does Not Protect against Cardiac Dysfunction and Adverse Remodeling in a MURC Transgenic Mouse Model with Chronic Heart Failure and Atrial Fibrillation.

PubMed

Bernardo, Bianca C; Sapra, Geeta; Patterson, Natalie L; Cemerlang, Nelly; Kiriazis, Helen; Ueyama, Tomomi; Febbraio, Mark A; McMullen, Julie R

2015-01-01

Previous animal studies had shown that increasing heat shock protein 70 (Hsp70) using a transgenic, gene therapy or pharmacological approach provided cardiac protection in models of acute cardiac stress. Furthermore, clinical studies had reported associations between Hsp70 levels and protection against atrial fibrillation (AF). AF is the most common cardiac arrhythmia presenting in cardiology clinics and is associated with increased rates of heart failure and stroke. Improved therapies for AF and heart failure are urgently required. Despite promising observations in animal studies which targeted Hsp70, we recently reported that increasing Hsp70 was unable to attenuate cardiac dysfunction and pathology in a mouse model which develops heart failure and intermittent AF. Given our somewhat unexpected finding and the extensive literature suggesting Hsp70 provides cardiac protection, it was considered important to assess whether Hsp70 could provide protection in another mouse model of heart failure and AF. The aim of the current study was to determine whether increasing Hsp70 could attenuate adverse cardiac remodeling, cardiac dysfunction and episodes of arrhythmia in a mouse model of heart failure and AF due to overexpression of Muscle-Restricted Coiled-Coil (MURC). Cardiac function and pathology were assessed in mice at approximately 12 months of age. We report here, that chronic overexpression of Hsp70 was unable to provide protection against cardiac dysfunction, conduction abnormalities, fibrosis or characteristic molecular markers of the failing heart. In summary, elevated Hsp70 may provide protection in acute cardiac stress settings, but appears insufficient to protect the heart under chronic cardiac disease conditions.

The GSK-3 family as therapeutic target for myocardial diseases

PubMed Central

Lal, Hind; Ahmad, Firdos; Woodgett, James; Force, Thomas

2014-01-01

GSK-3 is one of the very few signaling molecules that regulate a truly astonishing number of critical intracellular signaling pathways. It has been implicated in a number of diseases including heart failure, bipolar disorder, diabetes, Alzheimer’s disease, aging, inflammation and cancer. Furthermore, a recent clinical trial has validated the feasibility of targeting GSK-3 with small molecule inhibitors for human diseases. In the current review we will focus on its expanding role in the heart, concentrating primarily on recent studies that have employed cardiomyocyte- and fibroblast-specific conditional gene deletion in mouse models. We will highlight the role of the GSK-3 isoforms in various pathological conditions including myocardial aging, ischemic injury, myocardial fibrosis and cardiomyocyte proliferation. We will discuss our recent findings that deletion of GSK-3α specifically in cardiomyocytes attenuates ventricular remodeling and cardiac dysfunction post-MI by limiting scar expansion and promoting cardiomyocyte proliferation. The recent emergence of GSK-3β as a regulator of myocardial fibrosis will also be discussed. We will review our very recent findings that specific deletion of GSK-3β in cardiac fibroblasts leads to fibrogenesis, left ventricular dysfunction and excessive scarring in the ischemic heart. Finally, we will examine the underlying mechanisms that drive the aberrant myocardial fibrosis in the models in which GSK-3β is specifically deleted in cardiac fibroblasts. We will summarize these recent results and offer explanations, whenever possible, and hypotheses when not. For these studies we will rely heavily on our models and those of others to reconcile some of the apparent inconsistencies in the literature. PMID:25552693

Paclitaxel modulates TGFbeta signaling in scleroderma skin grafts in immunodeficient mice.

PubMed

Liu, Xialin; Zhu, Shoukang; Wang, Tao; Hummers, Laura; Wigley, Fredrick M; Goldschmidt-Clermont, Pascal J; Dong, Chunming

2005-12-01

Systemic sclerosis (SSc) is characterized by excessive fibrosis and obliterative vascular lesions. Abnormal TGFbeta activation is implicated in the pathogenesis of SSc. Aberrant TGFbeta/Smad signaling can be controlled by stabilization of microtubules with paclitaxel. SSc and healthy human skin biopsies were incubated in the presence or absence of paclitaxel followed by transplantation into severe combined immunodeficient mice. TGFbeta signaling, fibrosis, and neovessel formation were evaluated by quantitative RT-PCR and immunohistochemical staining. Paclitaxel markedly suppressed Smad2 and Smad3 phosphorylation and collagen deposition in SSc grafts. As a result, the autonomous maintenance/reconstitution of the SSc phenotype was prevented. Remarkably, SSc grafts showed a 2-fold increase in neovessel formation relative to normal grafts, regardless of paclitaxel treatment. Angiogenesis in SSc grafts was associated with a substantial increase in mouse PECAM-1 expression, indicating the mouse origin of the neovascular cells. Low-dose paclitaxel can significantly suppress TGFbeta/Smad activity and lessen fibrosis in SCID mice. Transplantation of SSc skin into SCID mice elicits a strong angiogenesis-an effect not affected by paclitaxel. Although prolonged chemotherapy with paclitaxel at higher doses is associated with pro-fibrotic and anti-angiogenic changes, the findings described here indicate that low-dose paclitaxel may have therapeutic benefits for SSc via modulating TGFbeta signaling.

Sulforaphane prevents bleomycin‑induced pulmonary fibrosis in mice by inhibiting oxidative stress via nuclear factor erythroid 2‑related factor‑2 activation.

PubMed

Yan, Bingdi; Ma, Zhongsen; Shi, Shaomin; Hu, Yuxin; Ma, Tiangang; Rong, Gao; Yang, Junling

2017-06-01

Lung fibrosis is associated with inflammation, apoptosis and oxidative damage. The transcription factor nuclear factor erythroid 2‑related factor‑2 (Nrf2) prevents damage to cells from oxidative stress by regulating the expression of antioxidant proteins. Sulforaphane (SFN), an Nrf2 activator, additionally regulates excessive oxidative stress by promoting the expression of endogenous antioxidants. The present study investigated if SFN protects against lung injury induced by bleomycin (BLM). The secondary aim of the present study was to assess if this protection mechanism involves upregulation of Nrf2 and its downstream antioxidants. Pulmonary fibrosis was induced in C57/BL6 mice by intratracheal instillation of BLM. BLM and age‑matched control mice were treated with or without a daily dose of 0.5 mg/kg SFN until sacrifice. On days 7 and 28, mice were assessed for induction of apoptosis, inflammation, fibrosis, oxidative damage and Nrf2 expression in the lungs. The lungs were investigated with histological techniques including haematoxylin and eosin staining, Masson's trichrome staining and terminal deoxynucleotidyl transferase UTP nick end labeling. Inflammatory, fibrotic and apoptotic processes were confirmed by western blot analysis for interleukin‑1β, tumor necrosis factor‑α, transforming growth factor‑β and caspase‑3 protein expressions. Furthermore, protein levels of 3‑nitro‑tyrosine, 4‑hydroxynonenal, superoxide dismutase 1 and catalase were investigated by western blot analysis. It was demonstrated that pulmonary fibrosis induced by BLM significantly increased apoptosis, inflammation, fibrosis and oxidative stress in the lungs at days 7 and 28. Notably, SFN treatment significantly attenuated the infiltration of the inflammatory cells, collagen accumulation, epithelial cell apoptosis and oxidative stress in the lungs. In addition, SFN treatment increased expression of the Nrf2 gene and its downstream targets. In conclusion, these results suggested that SFN treatment of pulmonary fibrosis mouse models may attenuate alveolitis, fibrosis, apoptosis and lung oxidative stress by increasing the expression of antioxidant enzymes, including NAPDH quinone oxidoreductase, heme oxygenase‑1, superoxide dismutase and catalase, via upregulation of Nrf2 gene expression. Thus, the results from the present study may facilitate the development of therapies for BLM‑toxicity and pulmonary fibrosis.

Role of glycogen synthase kinase-3β and PPAR-γ on epithelial-to-mesenchymal transition in DSS-induced colorectal fibrosis.

PubMed

Di Gregorio, Jacopo; Sferra, Roberta; Speca, Silvia; Vetuschi, Antonella; Dubuquoy, Caroline; Desreumaux, Pierre; Pompili, Simona; Cristiano, Loredana; Gaudio, Eugenio; Flati, Vincenzo; Latella, Giovanni

2017-01-01

Intestinal fibrosis is characterized by abnormal production and deposition of extracellular matrix (ECM) proteins by activated myofibroblasts. The main progenitor cells of activated myofibroblasts are the fibroblasts and the epithelial cells, the latter through the epithelial-mesenchymal transition (EMT). To evaluate the action of the new PPAR-γ modulator, GED-0507-34 Levo (GED) on the expression of EMT associated and regulatory proteins such as TGF-β, Smad3, E-cadherin, Snail, ZEB1, β-catenin, and GSK-3β, in a mouse model of DSS-induced intestinal fibrosis. Chronic colitis and fibrosis were induced by oral administration of 2.5% DSS (w/v) for 6 weeks. GW9662 (GW), a selective PPAR-γ inhibitor, was also administered by intraperitoneal injection at the dose of 1 mg/kg/day combined with GED treatment. All drugs were administered at the beginning of the second cycle of DSS (day 12). 65 mice were randomly divided into five groups (H2O as controls n = 10, H2O+GED n = 10, DSS n = 15, DSS+GED n = 15, DSS+GED+GW n = 15). The colon was excised for macroscopic examination and histological and morphometric analyses. The level of expression of molecules involved in EMT and fibrosis, like TGF-β, Smad3, E-cadherin, Snail, ZEB1, β-catenin, GSK-3β and PPAR-γ, was assessed by immunohistochemistry, immunofluorescence, western blot and Real Time PCR. GED improved the DSS-induced chronic colitis and fibrosis. GED was able to reduce the expression of the main fibrosis markers (α-SMA, collagen I-III and fibronectin) as well as the pivotal pro-fibrotic molecules IL-13, TGF-β and Smad3, while it increased the anti-fibrotic PPAR-γ. All these GED effects were nullified by co-administration of GW with GED. Furthermore, GED was able to normalize the expression levels of E-cadherin and β-catenin and upregulated GSK-3β, that are all known to be involved both in EMT and fibrosis. The DSS-induced intestinal fibrosis was improved by the new PPAR-γ modulator GED-0507-34 Levo through the modulation of EMT mediators and pro-fibrotic molecules and through GSK-3β induction.

Free DNA in Cystic Fibrosis Airway Fluids Correlates with Airflow Obstruction

PubMed Central

Marcos, Veronica; Zhou-Suckow, Zhe; Önder Yildirim, Ali; Bohla, Alexander; Hector, Andreas; Vitkov, Ljubomir; Krautgartner, Wolf Dietrich; Stoiber, Walter; Griese, Matthias; Eickelberg, Oliver; Mall, Marcus A.; Hartl, Dominik

2015-01-01

Chronic obstructive lung disease determines morbidity and mortality of patients with cystic fibrosis (CF). CF airways are characterized by a nonresolving neutrophilic inflammation. After pathogen contact or prolonged activation, neutrophils release DNA fibres decorated with antimicrobial proteins, forming neutrophil extracellular traps (NETs). NETs have been described to act in a beneficial way for innate host defense by bactericidal, fungicidal, and virucidal actions. On the other hand, excessive NET formation has been linked to the pathogenesis of autoinflammatory and autoimmune disease conditions. We quantified free DNA structures characteristic of NETs in airway fluids of CF patients and a mouse model with CF-like lung disease. Free DNA levels correlated with airflow obstruction, fungal colonization, and CXC chemokine levels in CF patients and CF-like mice. When viewed in combination, our results demonstrate that neutrophilic inflammation in CF airways is associated with abundant free DNA characteristic for NETosis, and suggest that free DNA may be implicated in lung function decline in patients with CF. PMID:25918476

Endocannabinoids in Liver Disease

PubMed Central

Tam, Joseph; Liu, Jie; Mukhopadhyay, Bani; Cinar, Resat; Godlewski, Grzegorz; Kunos, George

2010-01-01

Endocannabinoids are lipid mediators of the same cannabinoid (CB) receptors that mediate the effects of marijuana. The endocannabinoid system (ECS) consists of CB receptors, endocannabinoids, and the enzymes involved in their biosynthesis and degradation, and is present both in brain and peripheral tissues, including the liver. The hepatic ECS is activated in various liver diseases, which contributes to the underlying pathologies. In cirrhosis of various etiologies, activation of vascular and cardiac CB1 receptors by macrophage- and platelet-derived endocannabinoids contribute to the vasodilated state and cardiomyopathy, which can be reversed by CB1 blockade. In mouse models of liver fibrosis, activation of CB1 receptors on hepatic stellate cells is fibrogenic, and CB1 blockade slows the progression of fibrosis. Fatty liver induced by high-fat diets or chronic alcohol feeding depend on activation of peripheral, including hepatic CB1 receptors, which also contribute to insulin resistance and dyslipidemias. Although the documented therapeutic potential of CB1 blockade is limited by neuropsychiatric side effects, these may be mitigated by using novel, peripherally restricted CB1 antagonists. PMID:21254182

Telomere-driven diseases and telomere-targeting therapies

PubMed Central

2017-01-01

Telomeres, the protective ends of linear chromosomes, shorten throughout an individual’s lifetime. Telomere shortening is proposed to be a primary molecular cause of aging. Short telomeres block the proliferative capacity of stem cells, affecting their potential to regenerate tissues, and trigger the development of age-associated diseases. Mutations in telomere maintenance genes are associated with pathologies referred to as telomere syndromes, including Hoyeraal-Hreidarsson syndrome, dyskeratosis congenita, pulmonary fibrosis, aplastic anemia, and liver fibrosis. Telomere shortening induces chromosomal instability that, in the absence of functional tumor suppressor genes, can contribute to tumorigenesis. In addition, mutations in telomere length maintenance genes and in shelterin components, the protein complex that protects telomeres, have been found to be associated with different types of cancer. These observations have encouraged the development of therapeutic strategies to treat and prevent telomere-associated diseases, namely aging-related diseases, including cancer. Here we review the molecular mechanisms underlying telomere-driven diseases and highlight recent advances in the preclinical development of telomere-targeted therapies using mouse models. PMID:28254828

MicroRNA-155 Deficiency Attenuates Liver Steatosis and Fibrosis without Reducing Inflammation in a Mouse Model of Steatohepatitis

PubMed Central

Lippai, Dora; Kodys, Karen; Catalano, Donna; Iracheta-Vellve, Arvin; Szabo, Gyongyi

2015-01-01

Background & Aim MicroRNAs (miRs) regulate hepatic steatosis, inflammation and fibrosis. Fibrosis is the consequence of chronic tissue damage and inflammation. We hypothesized that deficiency of miR-155, a master regulator of inflammation, attenuates steatohepatitis and fibrosis. Methods Wild type (WT) and miR-155-deficient (KO) mice were fed methionine-choline-deficient (MCD) or -supplemented (MCS) control diet for 5 weeks. Liver injury, inflammation, steatosis and fibrosis were assessed. Results MCD diet resulted in steatohepatitis and increased miR-155 expression in total liver, hepatocytes and Kupffer cells. Steatosis and expression of genes involved in fatty acid metabolism were attenuated in miR-155 KO mice after MCD feeding. In contrast, miR-155 deficiency failed to attenuate inflammatory cell infiltration, nuclear factor κ beta (NF-κB) activation and enhanced the expression of the pro-inflammatory cytokines tumor necrosis factor alpha (TNFα) and monocyte chemoattractant protein-1 (MCP1) in MCD diet-fed mice. We found a significant attenuation of apoptosis (cleaved caspase-3) and reduction in collagen and α smooth muscle actin (αSMA) levels in miR-155 KO mice compared to WTs on MCD diet. In addition, we found attenuation of platelet derived growth factor (PDGF), a pro-fibrotic cytokine; SMAD family member 3 (Smad3), a protein involved in transforming growth factor-β (TGFβ) signal transduction and vimentin, a mesenchymal marker and indirect indicator of epithelial-to-mesenchymal transition (EMT) in miR-155 KO mice. Nuclear binding of CCAAT enhancer binding protein β (C/EBPβ) a miR-155 target involved in EMT was significantly increased in miR-155 KO compared to WT mice. Conclusions Our novel data demonstrate that miR-155 deficiency can reduce steatosis and fibrosis without decreasing inflammation in steatohepatitis. PMID:26042593

LatY136F knock-in mouse model for human IgG4-related disease.

PubMed

Yamada, Kazunori; Zuka, Masahiko; Ito, Kiyoaki; Mizuguchi, Keishi; Kakuchi, Yasushi; Onoe, Tamehito; Suzuki, Yasunori; Yamagishi, Masakazu; Izui, Shozo; Malissen, Marie; Malissen, Bernard; Kawano, Mitsuhiro

2018-01-01

The adaptor protein Linker for activation of T cell (LAT) is a key signaling hub used by the T cell antigen receptor. Mutant mice expressing loss-of-function mutations affecting LAT and including a mutation in which tyrosine 136 is replaced by a phenylalanine (LatY136F) develop lymphoproliferative disorder involving T helper type 2 effector cells capable of triggering a massive polyclonal B cell activation that leads to hypergammaglobulinemia G1 and E and to non-resolving inflammation and autoimmunity. The purpose of this study was to evaluate whether the phenotypes of LatY136F knock-in mice resemble the immunohistopathological features of immunoglobulin G4-related disease (IgG4-RD). LatY136F knock-in mice were sacrificed at 4-20 weeks of age, and pancreas, kidney, salivary gland and lung were obtained. All organs were stained with hematoxylin-eosin and with Azan for estimation of collagen in fibrosis, and the severity scores of inflammation and fibrosis were evaluated. Immunostainings were performed to analyze the types of infiltrating cells. In addition, the effects of corticosteroid treatment on the development of tissue lesions and serum levels of IgG1 were assessed. Tissue lesions characterized by inflammatory mononuclear cell infiltration and fibrosis were detected in pancreas, kidney, and salivary gland starting from 6 weeks of age. Immunostainings showed pronounced infiltration of plasma cells, CD4-positive T cells, and macrophages. Infiltrating plasma cells predominantly expressed IgG1. The extent of inflammation in pancreas and salivary glands was markedly reduced by corticosteroid treatment. LatY136F knock-in mice displayed increased production of Th2-type IgG1 (a homologue of human IgG4) and developed multiple organ tissue lesions reminiscent of those seen in patients with IgG4-RD. Moreover, the development of these tissue lesions was highly sensitive to corticosteroid treatment like in IgG4-RD. For these reasons we consider the LatY136F knock-in mouse strain to represent a promising model for human IgG4-RD.

Glucose Transporter 1–Dependent Glycolysis Is Increased during Aging-Related Lung Fibrosis, and Phloretin Inhibits Lung Fibrosis

PubMed Central

Cho, Soo Jung; Moon, Jong-Seok; Lee, Chang-Min; Choi, Augustine M. K.

2017-01-01

Aging is associated with metabolic diseases such as type 2 diabetes mellitus, cardiovascular disease, cancer, and neurodegeneration. Aging contributes to common processes including metabolic dysfunction, DNA damage, and reactive oxygen species generation. Although glycolysis has been linked to cell growth and proliferation, the mechanisms by which the activation of glycolysis by aging regulates fibrogenesis in the lung remain unclear. The objective of this study was to determine if glucose transporter 1 (GLUT1)–induced glycolysis regulates age-dependent fibrogenesis of the lung. Mouse and human lung tissues were analyzed for GLUT1 and glycolytic markers using immunoblotting. Glycolytic function was measured using a Seahorse apparatus. To study the effect of GLUT1, genetic inhibition of GLUT1 was performed by short hairpin RNA transduction, and phloretin was used for pharmacologic inhibition of GLUT1. GLUT1-dependent glycolysis is activated in aged lung. Genetic and pharmacologic inhibition of GLUT1 suppressed the protein expression of α-smooth muscle actin, a key cytoskeletal component of activated fibroblasts, in mouse primary lung fibroblast cells. Moreover, we demonstrated that the activation of AMP-activated protein kinase, which is regulated by GLUT1-dependent glycolysis, represents a critical metabolic pathway for fibroblast activation. Furthermore, we demonstrated that phloretin, a potent inhibitor of GLUT1, significantly inhibited bleomycin-induced lung fibrosis in vivo. These results suggest that GLUT1-dependent glycolysis regulates fibrogenesis in aged lung and that inhibition of GLUT1 provides a potential target of therapy of age-related lung fibrosis. PMID:27997810

Glucose Transporter 1-Dependent Glycolysis Is Increased during Aging-Related Lung Fibrosis, and Phloretin Inhibits Lung Fibrosis.

PubMed

Cho, Soo Jung; Moon, Jong-Seok; Lee, Chang-Min; Choi, Augustine M K; Stout-Delgado, Heather W

2017-04-01

Aging is associated with metabolic diseases such as type 2 diabetes mellitus, cardiovascular disease, cancer, and neurodegeneration. Aging contributes to common processes including metabolic dysfunction, DNA damage, and reactive oxygen species generation. Although glycolysis has been linked to cell growth and proliferation, the mechanisms by which the activation of glycolysis by aging regulates fibrogenesis in the lung remain unclear. The objective of this study was to determine if glucose transporter 1 (GLUT1)-induced glycolysis regulates age-dependent fibrogenesis of the lung. Mouse and human lung tissues were analyzed for GLUT1 and glycolytic markers using immunoblotting. Glycolytic function was measured using a Seahorse apparatus. To study the effect of GLUT1, genetic inhibition of GLUT1 was performed by short hairpin RNA transduction, and phloretin was used for pharmacologic inhibition of GLUT1. GLUT1-dependent glycolysis is activated in aged lung. Genetic and pharmacologic inhibition of GLUT1 suppressed the protein expression of α-smooth muscle actin, a key cytoskeletal component of activated fibroblasts, in mouse primary lung fibroblast cells. Moreover, we demonstrated that the activation of AMP-activated protein kinase, which is regulated by GLUT1-dependent glycolysis, represents a critical metabolic pathway for fibroblast activation. Furthermore, we demonstrated that phloretin, a potent inhibitor of GLUT1, significantly inhibited bleomycin-induced lung fibrosis in vivo. These results suggest that GLUT1-dependent glycolysis regulates fibrogenesis in aged lung and that inhibition of GLUT1 provides a potential target of therapy of age-related lung fibrosis.

Reducing CTGF/CCN2 slows down mdx muscle dystrophy and improves cell therapy.

PubMed

Morales, Maria Gabriela; Gutierrez, Jaime; Cabello-Verrugio, Claudio; Cabrera, Daniel; Lipson, Kenneth E; Goldschmeding, Roel; Brandan, Enrique

2013-12-15

In Duchenne muscular dystrophy (DMD) and the mdx mouse model, the absence of the cytoskeletal protein dystrophin causes defective anchoring of myofibres to the basal lamina. The resultant myofibre degeneration and necrosis lead to a progressive loss of muscle mass, increased fibrosis and ultimately fatal weakness. Connective tissue growth factor (CTGF/CCN-2) is critically involved in several chronic fibro-degenerative diseases. In DMD, the role of CTGF might extend well beyond replacement fibrosis secondary to loss of muscle fibres, since its overexpression in skeletal muscle could by itself induce a dystrophic phenotype. Using two independent approaches, we here show that mdx mice with reduced CTGF availability do indeed have less severe muscular dystrophy. Mdx mice with hemizygous CTGF deletion (mdx-Ctgf+/-), and mdx mice treated with a neutralizing anti-CTGF monoclonal antibody (FG-3019), performed better in an exercise endurance test, had better muscle strength in isolated muscles and reduced skeletal muscle impairment, apoptotic damage and fibrosis. Transforming growth factor type-β (TGF-β), pERK1/2 and p38 signalling remained unaffected during CTGF suppression. Moreover, both mdx-Ctgf+/- and FG-3019 treated mdx mice had improved grafting upon intramuscular injection of dystrophin-positive satellite cells. These findings reveal the potential of targeting CTGF to reduce disease progression and to improve cell therapy in DMD.

Toward an animal model of cystic fibrosis: Targeted interruption of exon 10 of the cystic fibrosis transmembrane regulator gene in embryonic stem cells

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

Koller, B.H.; Hyungsuk Kim; Latour, A.M.

1991-12-01

A gene-targeting construct was made containing 7.8 kilobases of DNA spanning exon 10 of the mouse cystic fibrosis transmembrane regulator (CFTR) gene in which part of the exon has been replaced by two neomycin-resistance (Neo) genes driven by different promoters. (This replacement introduces a chain-termination codon at amino acid position 489 in the CFTR sequence). A herpes simplex thymidine kinase gene was on each end of the construct, which was electroporated into embryonic stem (ES) cells. Colonies resistant to G418, or to G418 plus ganciclovir, were selected and screened by Southern blotting or by PCR amplification. Five pools of G418-resistantmore » cells gave PCR products diagnostic of targeting. Four independent clones of ES cells with a disrupted CFTR gene have been isolated from these pools. The frequency of targeting was 1/2500 G418-resistant colonies. This low frequency is not the consequence of marginal expression of the Neo genes in the targeted cells. The CFTR targeting events were clustered among our experiments in a manner suggesting that some unidentified factor(s), possible passage number, influences the recovery of CFTR-targeted cells.« less

A Novel Platelet-Activating Factor Receptor Antagonist Inhibits Choroidal Neovascularization and Subretinal Fibrosis

PubMed Central

Zhang, Han; Yang, Yang; Takeda, Atsunobu; Yoshimura, Takeru; Oshima, Yuji; Sonoda, Koh-Hei; Ishibashi, Tatsuro

2013-01-01

Choroidal neovascularization (CNV) is a critical pathogenesis in age-related macular degeneration (AMD), the most common cause of blindness in developed countries. To date, the precise molecular and cellular mechanisms underlying CNV have not been elucidated. Platelet-activating factor (PAF) has been previously implicated in angiogenesis; however, the roles of PAF and its receptor (PAF-R) in CNV have not been addressed. The present study reveals several important findings concerning the relationship of the PAF-R signaling with CNV. PAF-R was detected in a mouse model of laser-induced CNV and was upregulated during CNV development. Experimental CNV was suppressed by administering WEB2086, a novel PAF-R antagonist. WEB2086-dependent suppression of CNV occurred via the inhibition of macrophage infiltration and the expression of proangiogenic (vascular endothelial growth factor) and proinflammatory molecules (monocyte chemotactic protein-1 and IL-6) in the retinal pigment epithelium–choroid complex. Additionally, WEB2086-induced PAF-R blockage suppresses experimentally induced subretinal fibrosis, which resembles the fibrotic subretinal scarring observed in neovascular AMD. As optimal treatment modalities for neovascular AMD would target the multiple mechanisms of AMD-associated vision loss, including neovascularization, inflammation and fibrosis, our results suggest PAF-R as an attractive molecular target in the treatment of AMD. PMID:23826375

PAI1 mediates fibroblast-mast cell interactions in skin fibrosis.

PubMed

Pincha, Neha; Hajam, Edries Yousaf; Badarinath, Krithika; Batta, Surya Prakash Rao; Masudi, Tafheem; Dey, Rakesh; Andreasen, Peter; Kawakami, Toshiaki; Samuel, Rekha; George, Renu; Danda, Debashish; Jacob, Paul Mazhuvanchary; Jamora, Colin

2018-05-01

Fibrosis is a prevalent pathological condition arising from the chronic activation of fibroblasts. This activation results from the extensive intercellular crosstalk mediated by both soluble factors and direct cell-cell connections. Prominent among these are the interactions of fibroblasts with immune cells, in which the fibroblast-mast cell connection, although acknowledged, is relatively unexplored. We have used a Tg mouse model of skin fibrosis, based on expression of the transcription factor Snail in the epidermis, to probe the mechanisms regulating mast cell activity and the contribution of these cells to this pathology. We have discovered that Snail-expressing keratinocytes secrete plasminogen activator inhibitor type 1 (PAI1), which functions as a chemotactic factor to increase mast cell infiltration into the skin. Moreover, we have determined that PAI1 upregulates intercellular adhesion molecule type 1 (ICAM1) expression on dermal fibroblasts, rendering them competent to bind to mast cells. This heterotypic cell-cell adhesion, also observed in the skin fibrotic disorder scleroderma, culminates in the reciprocal activation of both mast cells and fibroblasts, leading to the cascade of events that promote fibrogenesis. Thus, we have identified roles for PAI1 in the multifactorial program of fibrogenesis that expand its functional repertoire beyond its canonical role in plasmin-dependent processes.

Comparison of mathematical models of fibrosis. Comment on "Towards a unified approach in the modeling of fibrosis: A review with research perspectives" by M. Ben Amar and C. Bianca

NASA Astrophysics Data System (ADS)

Kachapova, Farida

2016-07-01

Mathematical and computational models in biology and medicine help to improve diagnostics and medical treatments. Modeling of pathological fibrosis is reviewed by M. Ben Amar and C. Bianca in [4]. Pathological fibrosis is the process when excessive fibrous tissue is deposited on an organ or tissue during a wound healing and can obliterate their normal function. In [4] the phenomena of fibrosis are briefly explained including the causes, mechanism and management; research models of pathological fibrosis are described, compared and critically analyzed. Different models are suitable at different levels: molecular, cellular and tissue. The main goal of mathematical modeling of fibrosis is to predict long term behavior of the system depending on bifurcation parameters; there are two main trends: inhibition of fibrosis due to an active immune system and swelling of fibrosis because of a weak immune system.

Modeling the mechanical properties of liver fibrosis in rats.

PubMed

Zhu, Ying; Chen, Xin; Zhang, Xinyu; Chen, Siping; Shen, Yuanyuan; Song, Liang

2016-06-14

The progression of liver fibrosis changes the biomechanical properties of liver tissue. This study characterized and compared different liver fibrosis stages in rats in terms of viscoelasticity. Three viscoelastic models, the Voigt, Maxwell, and Zener models, were applied to experimental data from rheometer tests and then the elasticity and viscosity were estimated for each fibrosis stage. The study found that both elasticity and viscosity are correlated with the various stages of liver fibrosis. The study revealed that the Zener model is the optimal model for describing the mechanical properties of each fibrosis stage, but there is no significant difference between the Zener and Voigt models in their performance on liver fibrosis staging. Therefore the Voigt model can still be effectively used for liver fibrosis grading. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mesenchymal stem cell-derived inflammatory fibroblasts mediate interstitial fibrosis in the aging heart.

PubMed

Trial, JoAnn; Entman, Mark L; Cieslik, Katarzyna A

2016-02-01

Pathologic fibrosis in the aging mouse heart is associated with dysregulated resident mesenchymal stem cells (MSC) arising from reduced stemness and aberrant differentiation into dysfunctional inflammatory fibroblasts. Fibroblasts derived from aging MSC secrete higher levels of 1) collagen type 1 (Col1) that directly contributes to fibrosis, 2) monocyte chemoattractant protein-1 (MCP-1) that attracts leukocytes from the blood and 3) interleukin-6 (IL-6) that facilitates transition of monocytes into myeloid fibroblasts. The transcriptional activation of these proteins is controlled via the farnesyltransferase (FTase)-Ras-Erk pathway. The intrinsic change in the MSC phenotype acquired by advanced age is specific for the heart since MSC originating from bone wall (BW-MSC) or fibroblasts derived from them were free of these defects. The potential therapeutic interventions other than clinically approved strategies based on findings presented in this review are discussed as well. This article is a part of a Special Issue entitled "Fibrosis and Myocardial Remodeling". Copyright © 2015 Elsevier Ltd. All rights reserved.

Lessons from Hepatocyte-Specific Cyp51 Knockout Mice: Impaired Cholesterol Synthesis Leads to Oval Cell-Driven Liver Injury

NASA Astrophysics Data System (ADS)

Lorbek, Gregor; Perše, Martina; Jeruc, Jera; Juvan, Peter; Gutierrez-Mariscal, Francisco M.; Lewinska, Monika; Gebhardt, Rolf; Keber, Rok; Horvat, Simon; Björkhem, Ingemar; Rozman, Damjana

2015-03-01

We demonstrate unequivocally that defective cholesterol synthesis is an independent determinant of liver inflammation and fibrosis. We prepared a mouse hepatocyte-specific knockout (LKO) of lanosterol 14α-demethylase (CYP51) from the part of cholesterol synthesis that is already committed to cholesterol. LKO mice developed hepatomegaly with oval cell proliferation, fibrosis and inflammation, but without steatosis. The key trigger was reduced cholesterol esters that provoked cell cycle arrest, senescence-associated secretory phenotype and ultimately the oval cell response, while elevated CYP51 substrates promoted the integrated stress response. In spite of the oval cell-driven fibrosis being histologically similar in both sexes, data indicates a female-biased down-regulation of primary metabolism pathways and a stronger immune response in males. Liver injury was ameliorated by dietary fats predominantly in females, whereas dietary cholesterol rectified fibrosis in both sexes. Our data place defective cholesterol synthesis as a focus of sex-dependent liver pathologies.

Reduced Collagen Deposition in Infarcted Myocardium Facilitates Induced Pluripotent Stem Cell Engraftment and Angiomyogenesis for Improvement of Left Ventricular Function

PubMed Central

Dai, Bo; Huang, Wei; Xu, Meifeng; Millard, Ronald W.; Gao, Mei Hua; Hammond, H. Kirk; Menick, Donald R.; Ashraf, Muhammad; Wang, Yigang

2012-01-01

Objectives The purpose of this study was to assess the effect of scar tissue composition on engraftment of progenitor cells into infarcted myocardium. Background Scar tissue formation after myocardial infarction creates a barrier that severely compromises tissue regeneration, limiting potential functional recovery. Methods In vitro: A tricell patch (Tri-P) was created from peritoneum seeded and cultured with induced pluripotent stem cell–derived cardiomyocytes, endothelial cells, and mouse embryonic fibroblasts. The expression of fibrosis-related molecules from mouse embryonic fibroblasts and infarcted heart was measured by Western blot and quantitative reverse transcriptase polymerase chain reaction. In vivo: A Tri-P was affixed over the entire infarcted area 7 days after myocardial infarction in mice overexpressing adenylyl cyclase 6 (AC6). Engraftment efficiency of progenitor cells in hearts of AC6 mice was compared with that of control wild-type (WT) mice using a combination of in vivo bioluminescence imaging, post-mortem ex vivo tissue analysis, and the number of green fluorescent protein–positive cells. Echocardiography of left ventricular (LV) function was performed weekly. Hearts were harvested for analysis 4 weeks after Tri-P application. Mouse embryonic fibroblasts were stimulated with forskolin before an anoxia/reoxygenation protocol. Fibrosis-related molecules were analyzed. Results In AC6 mice, infarcted hearts treated with Tri-P showed significantly higher bioluminescence imaging intensity and numbers of green fluorescent protein–positive cells than in WT mice. LV function improved progressively in AC6 mice from weeks 2 to 4 and was associated with reduced LV fibrosis. Conclusions Application of a Tri-P in AC6 mice resulted in significantly higher induced pluripotent stem cell engraftment accompanied by angiomyogenesis in the infarcted area and improvement in LV function. PMID:22051336

Expression of profibrotic growth factors and their receptors by mouse lung macrophages and fibroblasts under conditions of acute viral inflammation in influenza A/H5N1 virus.

PubMed

Anikina, A G; Shkurupii, V A; Potapova, O V; Kovner, A V; Shestopalov, A M

2014-04-01

Morphological signs of early interstitial fibrosis, developing under conditions of acute viral inflammation (postinfection days 1-14), were observed in C57Bl/6 mice infected with influenza A/H5N1 A/goose/Krasnoozerskoye/627/05 virus. The development of fibrosis was confirmed by an increase in the number of lung cells expressing TNF-α. These changes were recorded in the presence of a many-fold increase in the counts of macrophages and fibroblasts expressing FGF, EGF, and their receptors.

Lysyl Oxidase as a Serum Biomarker of Liver Fibrosis in Patients with Severe Obesity and Obstructive Sleep Apnea.

PubMed

Mesarwi, Omar A; Shin, Mi-Kyung; Drager, Luciano F; Bevans-Fonti, Shannon; Jun, Jonathan C; Putcha, Nirupama; Torbenson, Michael S; Pedrosa, Rodrigo P; Lorenzi-Filho, Geraldo; Steele, Kimberley E; Schweitzer, Michael A; Magnuson, Thomas H; Lidor, Anne O; Schwartz, Alan R; Polotsky, Vsevolod Y

2015-10-01

Obstructive sleep apnea (OSA) is associated with the progression of nonalcoholic fatty liver disease (NAFLD). We hypothesized that the hypoxia of OSA increases hepatic production of lysyl oxidase (LOX), an enzyme that cross-links collagen, and that LOX may serve as a biomarker of hepatic fibrosis. Thirty-five patients with severe obesity underwent liver biopsy, polysomnography, and serum LOX testing. A separate group with severe OSA had serum LOX measured before and after 3 mo of CPAP or no therapy, as did age-matched controls. LOX expression and secretion were measured in mouse hepatocytes following exposure to hypoxia. The Johns Hopkins Bayview Sleep Disorders Center, and the Hypertension Unit of the Heart Institute at the University of São Paulo Medical School. In the bariatric cohort, the apnea-hypopnea index was higher in patients with hepatic fibrosis than in those without fibrosis (42.7 ± 30.2 events/h, versus 16.2 ± 15.5 events/h; P = 0.002), as was serum LOX (84.64 ± 29.71 ng/mL, versus 45.46 ± 17.16 ng/mL; P < 0.001). In the sleep clinic sample, patients with severe OSA had higher baseline LOX than healthy controls (70.75 ng/mL versus 52.36 ng/mL, P = 0.046), and serum LOX decreased in patients with OSA on CPAP (mean decrease 20.49 ng/mL) but not in untreated patients (mean decrease 0.19 ng/mL). Hypoxic mouse hepatocytes demonstrated 5.9-fold increased LOX transcription (P = 0.046), and enhanced LOX protein secretion. The hypoxic stress of obstructive sleep apnea may increase circulating lysyl oxidase (LOX) levels. LOX may serve as a biomarker of liver fibrosis in patients with severe obesity and nonalcoholic fatty liver disease. © 2015 Associated Professional Sleep Societies, LLC.

Renal proximal tubule function is preserved in Cftrtm2camΔF508 cystic fibrosis mice

PubMed Central

Kibble, J D; Balloch, K J D; Neal, A M; Hill, C; White, S; Robson, L; Green, R; Taylor, C J

2001-01-01

Changes in proximal tubule function have been reported in cystic fibrosis patients. The aim of this study was to investigate proximal tubule function in the Cftrtm2camΔF508 cystic fibrosis (CF) mouse model. A range of techniques were used including renal clearance studies, in situ microperfusion, RT-PCR and whole-cell patch clamping. Renal Na+ clearance was similar in wild-type (1.4 ± 0.3 μl min−1, number of animals, N= 12) and CF mice (1.6 ± 0.4 μl min−1, N= 7) under control conditions. Acute extracellular volume expansion resulted in significant natriuresis in wild-type (7.0 ± 0.8 μl min−1, N= 8) and CF mice (9.3 ± 1.4 μl min−1, N= 9); no difference between genotypes was observed. In situ microperfusion revealed that fluid absorptive rate (Jv) was similar under control conditions between wild-type (2.2 ± 0.4 nl mm−1 min−1, n= 10) and CF mice (1.9 ± 0.3 nl mm−1 min−1, n= 11). Addition of a forskolin-dibutyryl cAMP (db-cAMP) cocktail to the perfusate caused no significant change in Jv in either wild-type (2.6 ± 0.7 nl mm−1 min−1, n= 10) or Cftrtm2camΔF508 mice (2.0 ± 0.5 nl mm−1 min−1, n= 10). CFTR expression was confirmed in samples of outer cortex using RT-PCR. However, no evidence for functional CFTR was obtained when outer cortical cells were stimulated with protein kinase A or forskolin-db-cAMP using whole-cell patch clamping. In conclusion, no functional deficit in proximal tubule function was found in Cftrtm2camΔF508 mice. This may be a consequence of a lack of whole-cell cAMP-dependent Cl− conductance in mouse proximal tubule cells. PMID:11306663

Bromide supplementation exacerbated the renal dysfunction, injury and fibrosis in a mouse model of Alport syndrome.

PubMed

Yokota, Tsubasa; Omachi, Kohei; Suico, Mary Ann; Kojima, Haruka; Kamura, Misato; Teramoto, Keisuke; Kaseda, Shota; Kuwazuru, Jun; Shuto, Tsuyoshi; Kai, Hirofumi

2017-01-01

A seminal study recently demonstrated that bromide (Br-) has a critical function in the assembly of type IV collagen in basement membrane (BM), and suggested that Br- supplementation has therapeutic potential for BM diseases. Because salts of bromide (KBr and NaBr) have been used as antiepileptic drugs for several decades, repositioning of Br- for BM diseases is probable. However, the effects of Br- on glomerular basement membrane (GBM) disease such as Alport syndrome (AS) and its impact on the kidney are still unknown. In this study, we administered daily for 16 weeks 75 mg/kg or 250 mg/kg (within clinical dosage) NaBr or NaCl (control) via drinking water to 6-week-old AS mice (mouse model of X-linked AS). Treatment with 75 mg/kg NaBr had no effect on AS progression. Surprisingly, compared with 250 mg/kg NaCl, 250 mg/kg NaBr exacerbated the progressive proteinuria and increased the serum creatinine and blood urea nitrogen in AS mice. Histological analysis revealed that glomerular injury, renal inflammation and fibrosis were exacerbated in mice treated with 250 mg/kg NaBr compared with NaCl. The expressions of renal injury markers (Lcn2, Lysozyme), matrix metalloproteinase (Mmp-12), pro-inflammatory cytokines (Il-6, Il-8, Tnf-α, Il-1β) and pro-fibrotic genes (Tgf-β, Col1a1, α-Sma) were also exacerbated by 250 mg/kg NaBr treatment. Notably, the exacerbating effects of Br- were not observed in wild-type mice. These findings suggest that Br- supplementation needs to be carefully evaluated for real positive health benefits and for the absence of adverse side effects especially in GBM diseases such as AS.

Induction of Scleroderma Fibrosis in Skin-Humanized Mice by Administration of Anti-Platelet-Derived Growth Factor Receptor Agonistic Autoantibodies.

PubMed

Luchetti, Michele M; Moroncini, Gianluca; Jose Escamez, Maria; Svegliati Baroni, Silvia; Spadoni, Tatiana; Grieco, Antonella; Paolini, Chiara; Funaro, Ada; Avvedimento, Enrico V; Larcher, Fernando; Del Rio, Marcela; Gabrielli, Armando

2016-09-01

To describe a skin-SCID mouse chimeric model of systemic sclerosis (SSc; scleroderma) fibrosis based on engraftment of ex vivo-bioengineered skin using skin cells derived either from scleroderma patients or from healthy donors. Three-dimensional bioengineered skin containing human keratinocytes and fibroblasts isolated from skin biopsy specimens from healthy donors or SSc patients was generated ex vivo and then grafted onto the backs of SCID mice. The features of the skin grafts were analyzed by immunohistochemistry, and the functional profile of the graft fibroblasts was defined before and after treatment with IgG from healthy controls or SSc patients. Two procedures were used to investigate the involvement of platelet-derived growth factor receptor (PDGFR): 1) nilotinib, a tyrosine kinase inhibitor, was administered to mice before injection of IgG from SSc patient sera (SSc IgG) into the grafts, and 2) human anti-PDGFR monoclonal antibodies were injected into the grafts. Depending on the type of bioengineered skin grafted, the regenerated human skin exhibited either the typical scleroderma phenotype or the healthy human skin architecture. Treatment of animals carrying healthy donor skin grafts with SSc IgG resulted in the appearance of a bona fide scleroderma phenotype, as confirmed by increased collagen deposition and fibroblast activation markers. Results of the experiments involving administration of nilotinib or monoclonal antibodies confirmed the involvement of PDGFR. Our results provide the first in vivo demonstration of the fibrotic properties of anti-PDGFR agonistic antibodies. This bioengineered skin-humanized mouse model can be used to test in vivo the progression of the disease and to monitor response to antifibrotic drugs. © 2016, American College of Rheumatology.

Abnormal sodium current properties contribute to cardiac electrical and contractile dysfunction in a mouse model of myotonic dystrophy type 1.

PubMed

Algalarrondo, Vincent; Wahbi, Karim; Sebag, Frédéric; Gourdon, Geneviève; Beldjord, Chérif; Azibi, Kamel; Balse, Elise; Coulombe, Alain; Fischmeister, Rodolphe; Eymard, Bruno; Duboc, Denis; Hatem, Stéphane N

2015-04-01

Myotonic dystrophy type 1 (DM1) is the most common neuromuscular disorder and is associated with cardiac conduction defects. However, the mechanisms of cardiac arrhythmias in DM1 are unknown. We tested the hypothesis that abnormalities in the cardiac sodium current (INa) are involved, and used a transgenic mouse model reproducing the expression of triplet expansion observed in DM1 (DMSXL mouse). The injection of the class-I antiarrhythmic agent flecainide induced prominent conduction abnormalities and significantly lowered the radial tissular velocities and strain rate in DMSXL mice compared to WT. These abnormalities were more pronounced in 8-month-old mice than in 3-month-old mice. Ventricular action potentials recorded by standard glass microelectrode technique exhibited a lower maximum upstroke velocity [dV/dt](max) in DMSXL. This decreased [dV/dt](max) was associated with a 1.7 fold faster inactivation of INa in DMSXL myocytes measured by the whole-cell patch-clamp technique. Finally in the DMSXL mouse, no mutation in the Scn5a gene was detected and neither cardiac fibrosis nor abnormalities of expression of the sodium channel protein were observed. Therefore, alterations in the sodium current markedly contributed to electrical conduction block in DM1. This result should guide pharmaceutical and clinical research toward better therapy for the cardiac arrhythmias associated with DM1. Copyright © 2014 Elsevier B.V. All rights reserved.

Phenylquinoxalinone CFTR activator as potential prosecretory therapy for constipation

PubMed Central

CIL, ONUR; PHUAN, PUAY-WAH; SON, JUNG-HO; ZHU, JIE S.; KU, COLTON K.; TABIB, NILOUFAR AKHAVAN; TEUTHORN, ANDREW P.; FERRERA, LORETTA; ZACHOS, NICHOLAS C.; LIN, RUXIAN; GALIETTA, LUIS J. V.; DONOWITZ, MARK; KURTH, MARK J.; VERKMAN, ALAN S.

2017-01-01

Constipation is a common condition for which current treatments can have limited efficacy. By high-throughput screening, we recently identified a phenylquinoxalinone activator of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel that stimulated intestinal fluid secretion and normalized stool output in a mouse model of opioid-induced constipation. Here, we report phenylquinoxalinone structure-activity analysis, mechanism of action, animal efficacy data in acute and chronic models of constipation, and functional data in ex vivo primary cultured human enterocytes. Structure-activity analysis was done on 175 phenylquinoxalinone analogs, including 15 synthesized compounds. The most potent compound, CFTRact-J027, activated CFTR with EC50 ~ 200 nM, with patch-clamp analysis showing a linear CFTR current-voltage relationship with direct CFTR activation. CFTRact-J027 corrected reduced stool output and hydration in a mouse model of acute constipation produced by scopolamine and in a chronically constipated mouse strain (C3H/HeJ). Direct comparison with the approved prosecretory drugs lubiprostone and linaclotide showed substantially greater intestinal fluid secretion with CFTRact-J027, as well as greater efficacy in a constipation model. As evidence to support efficacy in human constipation, CFTRact-J027 increased transepithelial fluid transport in enteroids generated from normal human small intestine. Also, CFTRact-J027 was rapidly metabolized in vitro in human hepatic microsomes, suggesting minimal systemic exposure upon oral administration. These data establish structure-activity and mechanistic data for phenylquinoxalinone CFTR activators, and support their potential efficacy in human constipation. PMID:27815136

Prostaglandin D2 Attenuates Bleomycin-Induced Lung Inflammation and Pulmonary Fibrosis.

PubMed

Kida, Taiki; Ayabe, Shinya; Omori, Keisuke; Nakamura, Tatsuro; Maehara, Toko; Aritake, Kosuke; Urade, Yoshihiro; Murata, Takahisa

2016-01-01

Pulmonary fibrosis is a progressive and fatal lung disease with limited therapeutic options. Although it is well known that lipid mediator prostaglandins are involved in the development of pulmonary fibrosis, the role of prostaglandin D2 (PGD2) remains unknown. Here, we investigated whether genetic disruption of hematopoietic PGD synthase (H-PGDS) affects the bleomycin-induced lung inflammation and pulmonary fibrosis in mouse. Compared with H-PGDS naïve (WT) mice, H-PGDS-deficient mice (H-PGDS-/-) represented increased collagen deposition in lungs 14 days after the bleomycin injection. The enhanced fibrotic response was accompanied by an increased mRNA expression of inflammatory mediators, including tumor necrosis factor-α, monocyte chemoattractant protein-1, and cyclooxygenase-2 on day 3. H-PGDS deficiency also increased vascular permeability on day 3 and infiltration of neutrophils and macrophages in lungs on day 3 and 7. Immunostaining showed that the neutrophils and macrophages expressed H-PGDS, and its mRNA expression was increased on day 3and 7 in WT lungs. These observations suggest that H-PGDS-derived PGD2 plays a protective role in bleomycin-induced lung inflammation and pulmonary fibrosis.

Targeting the vascular and perivascular niches as a regenerative therapy for lung and liver fibrosis

PubMed Central

Cao, Zhongwei; Ye, Tinghong; Sun, Yue; Ji, Gaili; Shido, Koji; Chen, Yutian; Luo, Lin; Na, Feifei; Li, Xiaoyan; Huang, Zhen; Ko, Jane L.; Mittal, Vivek; Qiao, Lina; Chen, Chong; Martinez, Fernando J.; Rafii, Shahin; Ding, Bi-Sen

2017-01-01

The regenerative capacity of lung and liver is sometimes impaired by chronic or overwhelming injury. Orthotopic transplantation of parenchymal stem cells to damaged organs might reinstate their self-repair ability. However, parenchymal cell engraftment is frequently hampered by the microenvironment in diseased recipient organs. Here, we show that targeting both the vascular niche and perivascular fibroblasts establishes “hospitable soil” to foster incorporation of “seed”, in this case the engraftment of parenchymal cells in injured organs. Specifically, ectopic induction of endothelial cell (EC)-expressed paracrine/angiocrine hepatocyte growth factor (HGF) and inhibition of perivascular NADPH Oxidase 4 (NOX4) synergistically enabled reconstitution of mouse and human parenchymal cells in damaged organs. Reciprocally, genetic knockout of Hgf in mouse ECs (HgfiΔEC/iΔEC) aberrantly upregulated perivascular NOX4 during liver and lung regeneration. Dysregulated HGF and NOX4 pathways subverted the function of vascular and perivascular cells from an epithelially-inductive niche to a microenvironment that inhibited parenchymal reconstitution. Perivascular NOX4 induction in HgfiΔEC/iΔEC mice recapitulated the phenotype of human and mouse fibrotic livers and lungs. Consequently, EC-directed HGF and NOX4 inhibitor GKT137831 stimulated regenerative integration of mouse and human parenchymal cells in chronically injured lung and liver. Our data suggest that targeting dysfunctional perivascular and vascular cells in diseased organs can bypass fibrosis and enable reparative cell engraftment to reinstate lung and liver regeneration. PMID:28855398

Electrophysiological Evidence for the Presence of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in Mouse Sperm

PubMed Central

Dulce, Figueiras Fierro; José, Acevedo Juan; Pablo, Martínez; Escoffier, Jessica; Sepúlveda, Francisco V.; Enrique, Balderas; Gerardo, Orta; Pablo, Visconti; Alberto, Darszon

2014-01-01

Mammalian sperm must undergo a maturational process, named capacitation, in the female reproductive tract to fertilize the egg. Sperm capacitation is regulated by a cAMP/PKA pathway and involves increases in intracellular Ca2+, pH, Cl−, protein tyrosine phosphorylation, and in mouse and some other mammals a membrane potential hyperpolarization. The cystic fibrosis transmembrane conductance regulator (CFTR), a Cl− channel modulated by cAMP/PKA and ATP, was detected in mammalian sperm and proposed to modulate capacitation. Our whole-cell patch-clamp recordings from testicular mouse sperm now reveal a Cl− selective component to membrane current that is ATP-dependent, stimulated by cAMP, cGMP and genistein (a CFTR agonist, at low concentrations), and inhibited by DPC and CFTRinh-172, two well-known CFTR antagonists. Furthermore, the Cl− current component activated by cAMP and inhibited by CFTRinh-172 is absent in recordings on testicular sperm from mice possessing the CFTR ΔF508 loss-of-function mutation, indicating that CFTR is responsible for this component. A Cl− selective like current component displaying CFTR characteristics was also found in wild type epididymal sperm bearing the cytoplasmatic droplet. Capacitated sperm treated with CFTRinh-172 undergo a shape change, suggesting that CFTR is involved in cell volume regulation. These findings indicate that functional CFTR channels are present in mouse sperm and their biophysical properties are consistent with their proposed participation in capacitation. PMID:22833409

Three distinct cell populations express extracellular matrix proteins and increase in number during skeletal muscle fibrosis.

PubMed

Chapman, Mark A; Mukund, Kavitha; Subramaniam, Shankar; Brenner, David; Lieber, Richard L

2017-02-01

Tissue extracellular matrix (ECM) provides structural support and creates unique environments for resident cells (Bateman JF, Boot-Handford RP, Lamandé SR. Nat Rev Genet 10: 173-183, 2009; Kjaer M. Physiol Rev 84: 649-98, 2004). However, the identities of cells responsible for creating specific ECM components have not been determined. In striated muscle, the identity of these cells becomes important in disease when ECM changes result in fibrosis and subsequent increased tissue stiffness and dysfunction. Here we describe a novel approach to isolate and identify cells that maintain the ECM in both healthy and fibrotic muscle. Using a collagen I reporter mouse, we show that there are three distinct cell populations that express collagen I in both healthy and fibrotic skeletal muscle. Interestingly, the number of collagen I-expressing cells in all three cell populations increases proportionally in fibrotic muscle, indicating that all cell types participate in the fibrosis process. Furthermore, while some profibrotic ECM and ECM-associated genes are significantly upregulated in fibrotic muscle, the fibrillar collagen gene expression profile is not qualitatively altered. This suggests that muscle fibrosis in this model results from an increased number of collagen I-expressing cells and not the initiation of a specific fibrotic collagen gene expression program. Finally, in fibrotic muscle, we show that these collagen I-expressing cell populations differentially express distinct ECM proteins-fibroblasts express the fibrillar components of ECM, fibro/adipogenic progenitors cells differentially express basal laminar proteins, and skeletal muscle progenitor cells differentially express genes important for the satellite cell. Copyright © 2017 the American Physiological Society.

Collagen-binding vascular endothelial growth factor attenuates CCl4-induced liver fibrosis in mice

PubMed Central

Wu, Kangkang; Huang, Rui; Wu, Hongyan; Liu, Yong; Yang, Chenchen; Cao, Shufeng; Hou, Xianglin; Chen, Bing; Dai, Jianwu; Wu, Chao

2016-01-01

Vascular endothelial growth factor (VEGF) serves an important role in promoting angiogenesis and tissue regeneration. However, the lack of an effective delivery system that can target this growth factor to the injured site reduces its therapeutic efficacy. Therefore, in the current study, collagen-binding VEGF was constructed by fusing a collagen-binding domain (CBD) to the N-terminal of native VEGF. The CBD-VEGF can specifically bind to collagen which is the major component of the extracellular matrix in fibrotic liver. The anti-fibrotic effects of this novel material were investigated by the carbon tetrachloride (CCl4)-induced liver fibrotic mouse model. Mice were injected with CCl4 intraperitoneally to induce liver fibrosis. CBD-VEGF was injected directly into the liver tissue of mice. The liver tissues were stained with hematoxylin and eosin for general observation or with Masson's trichrome staining for detection of collagen deposition. The hepatic stellate cell activation, blood vessel formation and hepatocyte proliferation were measured by immunohistochemical staining for α-smooth muscle actin, CD31 and Ki67 in the liver tissue. The fluorescent TUNEL assay was performed to evaluate the hepatocyte apoptosis. The present study identified that the CBD-VEGF injection could significantly promote vascularization of the liver tissue of fibrotic mice and attenuate liver fibrosis. Furthermore, hepatocyte apoptosis and hepatic stellate cell activation were attenuated by CBD-VEGF treatment. CBD-VEGF treatment could additionally promote hepatocyte regeneration in the liver tissue of fibrotic mice. Thus, it was suggested that CBD-VEGF may be used as a novel therapeutic intervention for liver fibrosis. PMID:27748931

Targeting Hypoxia-Inducible Factor-1α/Pyruvate Dehydrogenase Kinase 1 Axis by Dichloroacetate Suppresses Bleomycin-induced Pulmonary Fibrosis.

PubMed

Goodwin, Justin; Choi, Hyunsung; Hsieh, Meng-Hsiung; Neugent, Michael L; Ahn, Jung-Mo; Hayenga, Heather N; Singh, Pankaj K; Shackelford, David B; Lee, In-Kyu; Shulaev, Vladimir; Dhar, Shanta; Takeda, Norihiko; Kim, Jung-Whan

2018-02-01

Hypoxia has long been implicated in the pathogenesis of fibrotic diseases. Aberrantly activated myofibroblasts are the primary pathological driver of fibrotic progression, yet how various microenvironmental influences, such as hypoxia, contribute to their sustained activation and differentiation is poorly understood. As a defining feature of hypoxia is its impact on cellular metabolism, we sought to investigate how hypoxia-induced metabolic reprogramming affects myofibroblast differentiation and fibrotic progression, and to test the preclinical efficacy of targeting glycolytic metabolism for the treatment of pulmonary fibrosis. Bleomycin-induced pulmonary fibrotic progression was evaluated in two independent, fibroblast-specific, promoter-driven, hypoxia-inducible factor (Hif) 1A knockout mouse models and in glycolytic inhibitor, dichloroacetate-treated mice. Genetic and pharmacological approaches were used to explicate the role of metabolic reprogramming in myofibroblast differentiation. Hypoxia significantly enhanced transforming growth factor-β-induced myofibroblast differentiation through HIF-1α, whereas overexpression of the critical HIF-1α-mediated glycolytic switch, pyruvate dehydrogenase kinase 1 (PDK1) was sufficient to activate glycolysis and potentiate myofibroblast differentiation, even in the absence of HIF-1α. Inhibition of the HIF-1α/PDK1 axis by genomic deletion of Hif1A or pharmacological inhibition of PDK1 significantly attenuated bleomycin-induced pulmonary fibrosis. Our findings suggest that HIF-1α/PDK1-mediated glycolytic reprogramming is a critical metabolic alteration that acts to promote myofibroblast differentiation and fibrotic progression, and demonstrate that targeting glycolytic metabolism may prove to be a potential therapeutic strategy for the treatment of pulmonary fibrosis.

Three distinct cell populations express extracellular matrix proteins and increase in number during skeletal muscle fibrosis

PubMed Central

Chapman, Mark A.; Mukund, Kavitha; Subramaniam, Shankar; Brenner, David

2017-01-01

Tissue extracellular matrix (ECM) provides structural support and creates unique environments for resident cells (Bateman JF, Boot-Handford RP, Lamandé SR. Nat Rev Genet 10: 173–183, 2009; Kjaer M. Physiol Rev 84: 649–98, 2004). However, the identities of cells responsible for creating specific ECM components have not been determined. In striated muscle, the identity of these cells becomes important in disease when ECM changes result in fibrosis and subsequent increased tissue stiffness and dysfunction. Here we describe a novel approach to isolate and identify cells that maintain the ECM in both healthy and fibrotic muscle. Using a collagen I reporter mouse, we show that there are three distinct cell populations that express collagen I in both healthy and fibrotic skeletal muscle. Interestingly, the number of collagen I-expressing cells in all three cell populations increases proportionally in fibrotic muscle, indicating that all cell types participate in the fibrosis process. Furthermore, while some profibrotic ECM and ECM-associated genes are significantly upregulated in fibrotic muscle, the fibrillar collagen gene expression profile is not qualitatively altered. This suggests that muscle fibrosis in this model results from an increased number of collagen I-expressing cells and not the initiation of a specific fibrotic collagen gene expression program. Finally, in fibrotic muscle, we show that these collagen I-expressing cell populations differentially express distinct ECM proteins—fibroblasts express the fibrillar components of ECM, fibro/adipogenic progenitors cells differentially express basal laminar proteins, and skeletal muscle progenitor cells differentially express genes important for the satellite cell. PMID:27881411

C-type natriuretic peptide ameliorates pulmonary fibrosis by acting on lung fibroblasts in mice.

PubMed

Kimura, Toru; Nojiri, Takashi; Hino, Jun; Hosoda, Hiroshi; Miura, Koichi; Shintani, Yasushi; Inoue, Masayoshi; Zenitani, Masahiro; Takabatake, Hiroyuki; Miyazato, Mikiya; Okumura, Meinoshin; Kangawa, Kenji

2016-02-19

Pulmonary fibrosis has high rates of mortality and morbidity; however, no effective pharmacological therapy has been established. C-type natriuretic peptide (CNP), a member of the natriuretic peptide family, selectively binds to the transmembrane guanylyl cyclase (GC)-B receptor and exerts anti-inflammatory and anti-fibrotic effects in various organs through vascular endothelial cells and fibroblasts that have a cell-surface GC-B receptor. Given the pathophysiological importance of fibroblast activation in pulmonary fibrosis, we hypothesized that the anti-fibrotic and anti-inflammatory effects of exogenous CNP against bleomycin (BLM)-induced pulmonary fibrosis were exerted in part by the effect of CNP on pulmonary fibroblasts. C57BL/6 mice were divided into two groups, CNP-treated (2.5 μg/kg/min) and vehicle, to evaluate BLM-induced (1 mg/kg) pulmonary fibrosis and inflammation. A periostin-CNP transgenic mouse model exhibiting CNP overexpression in fibroblasts was generated and examined for the anti-inflammatory and anti-fibrotic effects of CNP via fibroblasts in vivo. Additionally, we assessed CNP attenuation of TGF-β-induced differentiation into myofibroblasts by using immortalized human lung fibroblasts stably expressing GC-B receptors. Furthermore, to investigate whether CNP acts on human lung fibroblasts in a clinical setting, we obtained primary-cultured fibroblasts from surgically resected lungs of patients with lung cancer and analyzed levels of GC-B mRNA transcription. CNP reduced mRNA levels of the profibrotic cytokines interleukin (IL)-1β and IL-6, as well as collagen deposition and the fibrotic area in lungs of mice with bleomycin-induced pulmonary fibrosis. Furthermore, similar CNP effects were observed in transgenic mice exhibiting fibroblast-specific CNP overexpression. In cultured-lung fibroblasts, CNP treatment attenuated TGF-β-induced phosphorylation of Smad2 and increased mRNA and protein expression of α-smooth muscle actin and SM22α, indicating that CNP suppresses fibroblast differentiation into myofibroblasts. Furthermore, human lung fibroblasts from patients with or without interstitial lung disease substantially expressed GC-B receptor mRNA. These data suggest that CNP ameliorates bleomycin-induced pulmonary fibrosis by suppressing TGF-β signaling and myofibroblastic differentiation in lung fibroblasts. Therefore, we propose consideration of CNP for clinical application to pulmonary fibrosis treatment.

The OTT-MAL fusion oncogene activates RBPJ-mediated transcription and induces acute megakaryoblastic leukemia in a knockin mouse model

PubMed Central

Mercher, Thomas; Raffel, Glen D.; Moore, Sandra A.; Cornejo, Melanie G.; Baudry-Bluteau, Dominique; Cagnard, Nicolas; Jesneck, Jonathan L.; Pikman, Yana; Cullen, Dana; Williams, Ifor R.; Akashi, Koichi; Shigematsu, Hirokazu; Bourquin, Jean-Pierre; Giovannini, Marco; Vainchenker, William; Levine, Ross L.; Lee, Benjamin H.; Bernard, Olivier A.; Gilliland, D. Gary

2009-01-01

Acute megakaryoblastic leukemia (AMKL) is a form of acute myeloid leukemia (AML) associated with a poor prognosis. The genetics and pathophysiology of AMKL are not well understood. We generated a knockin mouse model of the one twenty-two–megakaryocytic acute leukemia (OTT-MAL) fusion oncogene that results from the t(1;22)(p13;q13) translocation specifically associated with a subtype of pediatric AMKL. We report here that OTT-MAL expression deregulated transcriptional activity of the canonical Notch signaling pathway transcription factor recombination signal binding protein for immunoglobulin κ J region (RBPJ) and caused abnormal fetal megakaryopoiesis. Furthermore, cooperation between OTT-MAL and an activating mutation of the thrombopoietin receptor myeloproliferative leukemia virus oncogene (MPL) efficiently induced a short-latency AMKL that recapitulated all the features of human AMKL, including megakaryoblast hyperproliferation and maturation block, thrombocytopenia, organomegaly, and extensive fibrosis. Our results establish that concomitant activation of RBPJ (Notch signaling) and MPL (cytokine signaling) transforms cells of the megakaryocytic lineage and suggest that specific targeting of these pathways could be of therapeutic value for human AMKL. PMID:19287095

Mitochondrial biogenesis in the pulmonary vasculature during inhalation lung injury and fibrosis

EPA Science Inventory

Cell survival and injury repair is facilitated by mitochondrial biogenesis; however, the role of this process in lung repair is unknown. We evaluated mitochondrial biogenesis in the mouse lung in two injuries that cause acute inflammation and in two that cause chronic inflammatio...

Impaired anti-fibrotic effect of bone marrow-derived mesenchymal stem cell in a mouse model of pulmonary paracoccidioidomycosis

PubMed Central

Arango, Julián Camilo; Puerta-Arias, Juan David; Pino-Tamayo, Paula Andrea; Salazar-Peláez, Lina María; Rojas, Mauricio

2017-01-01

Bone marrow-derived mesenchymal stem cells (BMMSCs) have been consider as a promising therapy in fibrotic diseases. Experimental models suggest that BMMSCs may be used as an alternative therapy to treat chemical- or physical-induced pulmonary fibrosis. We investigated the anti-fibrotic potential of BMMSCs in an experimental model of lung fibrosis by infection with Paracoccidioides brasiliensis. BMMSCs were isolated and purified from BALB/c mice using standardized methods. BALB/c male mice were inoculated by intranasal infection of 1.5x106 P. brasiliensis yeasts. Then, 1x106 BMMSCs were administered intra venous at 8th week post-infection (p.i.). An additional group of mice was treated with itraconazole (ITC) two weeks before BMMSCs administration. Animals were sacrificed at 12th week p.i. Histopathological examination, fibrocytes counts, soluble collagen and fibrosis-related genes expression in lungs were evaluated. Additionally, human fibroblasts were treated with homogenized lung supernatants (HLS) to determine induction of collagen expression. Histological analysis showed an increase of granulomatous inflammatory areas in BMMSCs-treated mice. A significant increase of fibrocytes count, soluble collagen and collagen-3α1, TGF-β3, MMP-8 and MMP-15 genes expression were also observed in those mice. Interestingly, when combined therapy BMMSCs/ITC was used there is a decrease of TIMP-1 and MMP-13 gene expression in infected mice. Finally, human fibroblasts stimulated with HLS from infected and BMMSCs-transplanted mice showed a higher expression of collagen I. In conclusion, our findings indicate that late infusion of BMMSCs into mice infected with P. brasiliensis does not have any anti-fibrotic effect; possibly because their interaction with the fungus promotes collagen expression and tissue remodeling. PMID:29040281

Curcumin Modulates the Inflammatory Response and Inhibits Subsequent Fibrosis in a Mouse Model of Viral-induced Acute Respiratory Distress Syndrome

PubMed Central

Liu, Guangliang; Wang, Ruixue; London, Steven D.; London, Lucille

2013-01-01

Acute Respiratory Distress Syndrome (ARDS) is a clinical syndrome characterized by diffuse alveolar damage usually secondary to an intense host inflammatory response of the lung to a pulmonary or extrapulmonary infectious or non-infectious insult often leading to the development of intra-alveolar and interstitial fibrosis. Curcumin, the principal curcumoid of the popular Indian spice turmeric, has been demonstrated as an anti-oxidant and anti-inflammatory agent in a broad spectrum of diseases. Using our well-established model of reovirus 1/L-induced acute viral pneumonia, which displays many of the characteristics of the human ALI/ARDS, we evaluated the anti-inflammatory and anti-fibrotic effects of curcumin. Female CBA/J mice were treated with curcumin (50 mg/kg) 5 days prior to intranasal inoculation with 107 pfu reovirus 1/L and daily, thereafter. Mice were evaluated for key features associated with ALI/ARDS. Administration of curcumin significantly modulated inflammation and fibrosis, as revealed by histological and biochemical analysis. The expression of IL-6, IL-10, IFNγ, and MCP-1, key chemokines/cytokines implicated in the development of ALI/ARDS, from both the inflammatory infiltrate and whole lung tissue were modulated by curcumin potentially through a reduction in the phosphorylated form of NFκB p65. While the expression of TGFß1 was not modulated by curcumin, TGFß Receptor II, which is required for TGFß signaling, was significantly reduced. In addition, curcumin also significantly inhibited the expression of α-smooth muscle actin and Tenascin-C, key markers of myofibroblast activation. This data strongly supports a role for curcumin in modulating the pathogenesis of viral-induced ALI/ARDS in a pre-clinical model potentially manifested through the alteration of inflammation and myofibroblast differentiation. PMID:23437361

Therapy with mesenchymal stromal cells or conditioned medium reverse cardiac alterations in a high-fat diet-induced obesity model.

PubMed

Daltro, P S; Barreto, B C; Silva, P G; Neto, P Chenaud; Sousa Filho, P H F; Santana Neta, D; Carvalho, G B; Silva, D N; Paredes, B D; de Alcantara, A C; Freitas, L A R; Couto, R D; Santos, R R; Souza, B S F; Soares, M B P; Macambira, S G

2017-10-01

Obesity is associated with numerous cardiac complications, including arrhythmias, cardiac fibrosis, remodeling and heart failure. Here we evaluated the therapeutic potential of mesenchymal stromal cells (MSCs) and their conditioned medium (CM) to treat cardiac complications in a mouse model of high-fat diet (HFD)-induced obesity. After obesity induction and HFD withdrawal, obese mice were treated with MSCs, CM or vehicle. Cardiac function was assessed using electrocardiography, echocardiography and treadmill test. Body weight and biochemical parameters were evaluated. Cardiac tissue was used for real time (RT)-polymerase chain reaction (PCR) and histopathologic analysis. Characterization of CM by protein array showed the presence of different cytokines and growth factors, including chemokines, osteopontin, cystatin C, Serpin E1 and Gas 6. HFD-fed mice presented cardiac arrhythmias, altered cardiac gene expression and fibrosis reflected in physical exercise incapacity associated with obesity and diabetes. Administration of MSCs or CM improved arrhythmias and exercise capacity. This functional improvement correlated with normalization of GATA4 gene expression in the hearts of MSC- or CM-treated mice. The gene expression of connexin 43, troponin I, adiponectin, transforming growth factor (TGF) β, peroxisome proliferator activated receptor gamma (PPARγ), insulin-like growth factor 1 (IGF-1), matrix metalloproteinase-9 (MMP9) and tissue inhibitor of metalloproteinases 1 (TIMP1) were significantly reduced in MSCs, but not in CM-treated mice. Moreover, MSC or CM administration reduced the intensity of cardiac fibrosis. Our results suggest that MSCs and CM have a recovery effect on cardiac disturbances due to obesity and corroborate to the paracrine action of MSCs in heart disease models. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Aqueous Extract of Gumiganghwal-tang, a Traditional Herbal Medicine, Reduces Pulmonary Fibrosis by Transforming Growth Factor-β1/Smad Signaling Pathway in Murine Model of Chronic Asthma.

PubMed

Jeon, Woo-Young; Shin, In-Sik; Shin, Hyeun-Kyoo; Jin, Seong Eun; Lee, Mee-Young

2016-01-01

Gumiganghwal-tang is a traditional herbal prescription that is used widely for the treatment of the common cold and inflammatory diseases in Korea and other Asian countries. In this study, we investigated the protective effects of a Gumiganghwal-tang aqueous extract (GGTA) against airway inflammation and pulmonary fibrosis using a mouse model of chronic asthma. Chronic asthma was modeled in BALB/c mice via sensitization/challenge with an intraperitoneal injection of 1% ovalbumin (OVA) and inhalation of nebulized 1% OVA for 4 weeks. GGTA (100 mg/kg or 200 mg/kg) was also administered by oral gavage once a day for 4 weeks. We investigated the number of inflammatory cells, production of T-helper type 2 (Th2) cytokines, chemokine and the total transforming growth factor-β1 (TGF-β1) in bronchoalveolar lavage fluid (BALF); the levels of immunoglobulin E (IgE) in the plasma; the infiltration of inflammatory cells in lung tissue; and the expression of TGF-β1, Smad-3, and collagen in lung tissue. Our results revealed that GGTA lowered the recruitment of inflammatory cells (particularly, lymphocyte); and decreased the production of Th2 cytokines, chemokine and total TGF-β1; and attenuated the levels of total and OVA-specific IgE; and decreased the infiltration of inflammatory cells. Moreover, GGTA significantly reduced the expression of TGF-β1 and Smad-3, and lowered collagen deposition. These results indicate that GGTA reduces airway inflammation and pulmonary fibrosis by regulating Th2 cytokines production and the TGF-β1/Smad-3 pathway, thus providing a potential treatment for chronic asthma.

Mechanism of tacrolimus-induced chronic renal fibrosis following transplantation is regulated by ox-LDL and its receptor, LOX-1

PubMed Central

Deng, Shi; Jin, Tao; Zhang, Li; Bu, Hong; Zhang, Peng

2016-01-01

Chronic renal allograft dysfunction (CRAD) is the most common cause of graft failure following renal transplantation. However, the underlying mechanisms remain to be fully elucidated. Immunosuppressants and hyperlipidemia are associated with renal fibrosis following long-term use. The present study aimed to determine the effects of tacrolimus (FK506) and lipid metabolism disorder on CRAD. In vitro and in vivo models were used for this investigation. Cells of the mouse proximal renal tubular epithelial cell strain, NRK-52E, were cultured either with oxidized low-density lipoprotein (ox-LDL), FK506, ox-LDL combined with FK506, or vehicle, respectively. Changes in cell morphology and changes in the levels of lectin-like ox-LDL receptor-1 (LOX-1), reactive oxygen species (ROS), hydrogen peroxide and fibrosis-associated genes were evaluated at 24, 48 and 72 h. In separate experiment, total of 60 Sprague-Dawley rats were divided randomly into four groups, which included a high-fat group, FK506 group, high-fat combined with FK506 group, and control group. After 2, 4 and 8 weeks, the serum lipid levels, the levels of ox-LDL, ROS, and the expression levels of transforming growth factor (TGF)-β1 and connective tissue growth factor were determined. The in vitro and in vivo models revealed that lipid metabolism disorder and FK506 caused oxidative stress and a fibrogenic response. In addition, decreased levels of LOX-1 markedly reduced the levels of TGF-β1 in the in vitro model. Taken together, FK506 and dyslipidemia were found to be associated with CRAD following transplantation. PMID:27633115

Curcumin modulates the inflammatory response and inhibits subsequent fibrosis in a mouse model of viral-induced acute respiratory distress syndrome.

PubMed

Avasarala, Sreedevi; Zhang, Fangfang; Liu, Guangliang; Wang, Ruixue; London, Steven D; London, Lucille

2013-01-01

Acute Respiratory Distress Syndrome (ARDS) is a clinical syndrome characterized by diffuse alveolar damage usually secondary to an intense host inflammatory response of the lung to a pulmonary or extrapulmonary infectious or non-infectious insult often leading to the development of intra-alveolar and interstitial fibrosis. Curcumin, the principal curcumoid of the popular Indian spice turmeric, has been demonstrated as an anti-oxidant and anti-inflammatory agent in a broad spectrum of diseases. Using our well-established model of reovirus 1/L-induced acute viral pneumonia, which displays many of the characteristics of the human ALI/ARDS, we evaluated the anti-inflammatory and anti-fibrotic effects of curcumin. Female CBA/J mice were treated with curcumin (50 mg/kg) 5 days prior to intranasal inoculation with 10(7)pfu reovirus 1/L and daily, thereafter. Mice were evaluated for key features associated with ALI/ARDS. Administration of curcumin significantly modulated inflammation and fibrosis, as revealed by histological and biochemical analysis. The expression of IL-6, IL-10, IFNγ, and MCP-1, key chemokines/cytokines implicated in the development of ALI/ARDS, from both the inflammatory infiltrate and whole lung tissue were modulated by curcumin potentially through a reduction in the phosphorylated form of NFκB p65. While the expression of TGFß1 was not modulated by curcumin, TGFß Receptor II, which is required for TGFß signaling, was significantly reduced. In addition, curcumin also significantly inhibited the expression of α-smooth muscle actin and Tenascin-C, key markers of myofibroblast activation. This data strongly supports a role for curcumin in modulating the pathogenesis of viral-induced ALI/ARDS in a pre-clinical model potentially manifested through the alteration of inflammation and myofibroblast differentiation.

Loss of Nrf2 promotes alveolar type 2 cell loss in irradiated, fibrotic lung.

PubMed

Traver, Geri; Mont, Stacey; Gius, David; Lawson, William E; Ding, George X; Sekhar, Konjeti R; Freeman, Michael L

2017-11-01

The development of radiation-induced pulmonary fibrosis represents a critical clinical issue limiting delivery of therapeutic doses of radiation to non-small cell lung cancer. Identification of the cell types whose injury initiates a fibrotic response and the underlying biological factors that govern that response are needed for developing strategies that prevent or mitigate fibrosis. C57BL/6 mice (wild type, Nrf2 null, Nrf2 flox/flox , and Nrf2 Δ/Δ ; SPC-Cre) were administered a thoracic dose of 12Gy and allowed to recover for 250 days. Whole slide digital and confocal microscopy imaging of H&E, Masson's trichrome and immunostaining were used to assess tissue remodeling, collagen deposition and cell renewal/mobilization during the regenerative process. Histological assessment of irradiated, fibrotic wild type lung revealed significant loss of alveolar type 2 cells 250 days after irradiation. Type 2 cell loss and the corresponding development of fibrosis were enhanced in the Nrf2 null mouse. Yet, conditional deletion of Nrf2 in alveolar type 2 cells in irradiated lung did not impair type 2 cell survival nor yield an increased fibrotic phenotype. Instead, radiation-induced ΔNp63 stem/progenitor cell mobilization was inhibited in the Nrf2 null mouse while the propensity for radiation-induced myofibroblasts derived from alveolar type 2 cells was magnified. In summary, these results indicate that Nrf2 is an important regulator of irradiated lung's capacity to maintain alveolar type 2 cells, whose injury can initiate a fibrotic phenotype. Loss of Nrf2 inhibits ΔNp63 stem/progenitor mobilization, a key event for reconstitution of injured lung, while promoting a myofibroblast phenotype that is central for fibrosis. Copyright © 2017 Elsevier Inc. All rights reserved.

Downregulation of S100A4 Alleviates Cardiac Fibrosis via Wnt/β -Catenin Pathway in Mice.

PubMed

Qian, LiJun; Hong, Jian; Zhang, YanMei; Zhu, MengLin; Wang, XinChun; Zhang, YanJuan; Chu, Ming; Yao, Jing; Xu, Di

2018-05-07

Cardiac fibrosis is a pathological change leading to cardiac remodeling during the progression of myocardial ischemic diseases, and its therapeutic strategy remains to be explored. S100A4, a calcium-binding protein, participates in fibrotic diseases with an unclear mechanism. This study aimed to investigate the role of S100A4 in cardiac fibrosis. Cardiac fibroblasts from neonatal C57BL/6 mouse hearts were isolated and cultured. Myocardial infarction was induced by ligating the left anterior descending coronary artery (LAD). The ligation was not performed in the sham group. A volume of 5×105pfu/g adenovirus or 5 µM/g ICG-001 was intramyocardially injected into five parts bordering the infarction zone or normal region. We used Western blotting, quantitative RT-PCR, immunofluorescence, immunohistochemistry and Masson's trichrome staining to explore the function of S100A4. We found significant increases of S100A4 level and cardiac fibrosis markers, and β-catenin signaling activation in vitro and in vivo. In addition, knockdown of S100A4 significantly reduced cardiac fibrosis and β-catenin levels. Moreover, the expression of S100A4 decreased after ICG-001 inhibited β-catenin signal pathway. Downregulation of S100A4 alleviates cardiac fibrosis via Wnt/β -catenin pathway in mice. S100A4 may be a therapeutic target of cardiac fibrosis. © 2018 The Author(s). Published by S. Karger AG, Basel.

Nano-risk Science: application of toxicogenomics in an adverse outcome pathway framework for risk assessment of multi-walled carbon nanotubes.

PubMed

Labib, Sarah; Williams, Andrew; Yauk, Carole L; Nikota, Jake K; Wallin, Håkan; Vogel, Ulla; Halappanavar, Sabina

2016-03-15

A diverse class of engineered nanomaterials (ENMs) exhibiting a wide array of physical-chemical properties that are associated with toxicological effects in experimental animals is in commercial use. However, an integrated framework for human health risk assessment (HHRA) of ENMs has yet to be established. Rodent 2-year cancer bioassays, clinical chemistry, and histopathological endpoints are still considered the 'gold standard' for detecting substance-induced toxicity in animal models. However, the use of data derived from alternative toxicological tools, such as genome-wide expression profiling and in vitro high-throughput assays, are gaining acceptance by the regulatory community for hazard identification and for understanding the underlying mode-of-action. Here, we conducted a case study to evaluate the application of global gene expression data in deriving pathway-based points of departure (PODs) for multi-walled carbon nanotube (MWCNT)-induced lung fibrosis, a non-cancer endpoint of regulatory importance. Gene expression profiles from the lungs of mice exposed to three individual MWCNTs with different physical-chemical properties were used within the framework of an adverse outcome pathway (AOP) for lung fibrosis to identify key biological events linking MWCNT exposure to lung fibrosis. Significantly perturbed pathways were categorized along the key events described in the AOP. Benchmark doses (BMDs) were calculated for each perturbed pathway and were used to derive transcriptional BMDs for each MWCNT. Similar biological pathways were perturbed by the different MWCNT types across the doses and post-exposure time points studied. The pathway BMD values showed a time-dependent trend, with lower BMDs for pathways perturbed at the earlier post-exposure time points (24 h, 3d). The transcriptional BMDs were compared to the apical BMDs derived by the National Institute for Occupational Safety and Health (NIOSH) using alveolar septal thickness and fibrotic lesions endpoints. We found that regardless of the type of MWCNT, the BMD values for pathways associated with fibrosis were 14.0-30.4 μg/mouse, which are comparable to the BMDs derived by NIOSH for MWCNT-induced lung fibrotic lesions (21.0-27.1 μg/mouse). The results demonstrate that transcriptomic data can be used to as an effective mechanism-based method to derive acceptable levels of exposure to nanomaterials in product development when epidemiological data are unavailable.

Pulmonary Bacteriophage Therapy on Pseudomonas aeruginosa Cystic Fibrosis Strains: First Steps Towards Treatment and Prevention

PubMed Central

Morello, Eric; Saussereau, Emilie; Maura, Damien; Huerre, Michel; Touqui, Lhousseine; Debarbieux, Laurent

2011-01-01

Multidrug-resistant bacteria are the cause of an increasing number of deadly pulmonary infections. Because there is currently a paucity of novel antibiotics, phage therapy—the use of specific viruses that infect bacteria—is now more frequently being considered as a potential treatment for bacterial infections. Using a mouse lung-infection model caused by a multidrug resistant Pseudomonas aeruginosa mucoid strain isolated from a cystic fibrosis patient, we evaluated bacteriophage treatments. New bacteriophages were isolated from environmental samples and characterized. Bacteria and bacteriophages were applied intranasally to the immunocompetent mice. Survival was monitored and bronchoalveolar fluids were analysed. Quantification of bacteria, bacteriophages, pro-inflammatory and cytotoxicity markers, as well as histology and immunohistochemistry analyses were performed. A curative treatment (one single dose) administrated 2 h after the onset of the infection allowed over 95% survival. A four-day preventive treatment (one single dose) resulted in a 100% survival. All of the parameters measured correlated with the efficacy of both curative and preventive bacteriophage treatments. We also showed that in vitro optimization of a bacteriophage towards a clinical strain improved both its efficacy on in vivo treatments and its host range on a panel of 20 P. aeruginosa cystic fibrosis strains. This work provides an incentive to develop clinical studies on pulmonary bacteriophage therapy to combat multidrug-resistant lung infections. PMID:21347240

SDF-1 in Mammary Fibroblasts of Bovine with Mastitis Induces EMT and Inflammatory Response of Epithelial Cells.

PubMed

He, Guiliang; Ma, Mengru; Yang, Wei; Wang, Hao; Zhang, Yong; Gao, Ming-Qing

2017-01-01

Fibroblasts constitute the majority of the stromal cells within bovine mammary gland, yet the functional contributions of these cells to mastitis and fibrosis and the mechanism are poorly understood. In this study, we demonstrate that inflammation-associated fibroblasts (INFs) extracted from bovine mammary glands with clinical mastitis had different expression pattern regarding to several extracellular matrix (ECM) proteins, chemokines and cytokines compared to normal fibroblasts (NFs) from dairy cows during lactation. The INFs induced epithelial-mesenchymal transition (EMT) and inflammatory responses of mammary epithelial cells in a vitro co-culture model. These functional contributions of INFs to normal epithelial cells were mediated through their ability to secrete stromal cell-derived factor 1 (SDF-1). SDF-1 was highly secreted/expressed by INFs, lipopolysaccharide (LPS) -treated NFs, lipoteichoic acid (LTA) -treated NFs, as well as mastitic tissue compared to their counterparts. Exogenous SDF-1 promoted EMT on epithelial cells through activating NF-κB pathway, induced inflammation response and inhibited proliferation of epithelial cells. In addition, SDF-1 was able to induce mastitis and slight fibrosis of mouse mammary gland, which was attenuated by a specific inhibitor of the receptor of SDF-1. Our findings indicate that stromal fibroblasts within mammary glands with mastitis contribute to EMT and inflammatory responses of epithelial cells through the secretion of SDF-1, which could result in the inflammation spread and fibrosis within mammary gland.

Image Analysis Algorithms for Immunohistochemical Assessment of Cell Death Events and Fibrosis in Tissue Sections

PubMed Central

Krajewska, Maryla; Smith, Layton H.; Rong, Juan; Huang, Xianshu; Hyer, Marc L.; Zeps, Nikolajs; Iacopetta, Barry; Linke, Steven P.; Olson, Allen H.; Reed, John C.; Krajewski, Stan

2009-01-01

Cell death is of broad physiological and pathological importance, making quantification of biochemical events associated with cell demise a high priority for experimental pathology. Fibrosis is a common consequence of tissue injury involving necrotic cell death. Using tissue specimens from experimental mouse models of traumatic brain injury, cardiac fibrosis, and cancer, as well as human tumor specimens assembled in tissue microarray (TMA) format, we undertook computer-assisted quantification of specific immunohistochemical and histological parameters that characterize processes associated with cell death. In this study, we demonstrated the utility of image analysis algorithms for color deconvolution, colocalization, and nuclear morphometry to characterize cell death events in tissue specimens: (a) subjected to immunostaining for detecting cleaved caspase-3, cleaved poly(ADP-ribose)-polymerase, cleaved lamin-A, phosphorylated histone H2AX, and Bcl-2; (b) analyzed by terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling assay to detect DNA fragmentation; and (c) evaluated with Masson's trichrome staining. We developed novel algorithm-based scoring methods and validated them using TMAs as a high-throughput format. The proposed computer-assisted scoring methods for digital images by brightfield microscopy permit linear quantification of immunohistochemical and histochemical stainings. Examples are provided of digital image analysis performed in automated or semiautomated fashion for successful quantification of molecular events associated with cell death in tissue sections. (J Histochem Cytochem 57:649–663, 2009) PMID:19289554

Extracellular signal-regulated kinase (ERK) activation preserves cardiac function in pressure overload induced hypertrophy.

PubMed

Mutlak, Michael; Schlesinger-Laufer, Michal; Haas, Tali; Shofti, Rona; Ballan, Nimer; Lewis, Yair E; Zuler, Mor; Zohar, Yaniv; Caspi, Lilac H; Kehat, Izhak

2018-05-24

Chronic pressure overload and a variety of mediators induce concentric cardiac hypertrophy. When prolonged, cardiac hypertrophy culminates in decreased myocardial function and heart failure. Activation of the extracellular signal-regulated kinase (ERK) is consistently observed in animal models of hypertrophy and in human patients, but its role in the process is controversial. We generated transgenic mouse lines with cardiomyocyte restricted overexpression of intrinsically active ERK1, which similar to the observations in hypertrophy is phosphorylated on both the TEY and the Thr207 motifs and is overexpressed at pathophysiological levels. The activated ERK1 transgenic mice developed a modest adaptive hypertrophy with increased contractile function and without fibrosis. Following induction of pressure-overload, where multiple pathways are stimulated, this activation did not further increase the degree of hypertrophy but protected the heart through a decrease in the degree of fibrosis and maintenance of ventricular contractile function. The ERK pathway acts to promote a compensated hypertrophic response, with enhanced contractile function and reduced fibrosis. The activation of this pathway may be a therapeutic strategy to preserve contractile function when the pressure overload cannot be easily alleviated. The inhibition of this pathway, which is increasingly being used for cancer therapy on the other hand, should be used with caution in the presence of pressure-overload. Copyright © 2017. Published by Elsevier B.V.

A Mouse Model of Cardiomyopathy Induced by Mutations in the Hemochromatosis HFE Gene.

PubMed

Djemai, Haidar; Thomasson, Rémi; Trzaskus, Yvan; Mougenot, Nathalie; Meziani, Amira; Toussaint, Jean-François; Noirez, Philippe; Vitiello, Damien

2017-07-01

The heart is 1 of the organs most affected by hereditary hemochromatosis (HH). The clinical impact of cardiomyopathy in patients with HH requires a particular diagnosis and less invasive treatments. We developed a model of cardiomyopathy in knockout (KO) mice for the high-Fe (HFE) gene and assessed left ventricular (LV) function and structure from 7-20 months. Male wild-type (WT) heterozygous and KO SV129 mice for the HFE gene were used in this study. Twenty-four mice were used to assess LV function and structure by echocardiography at 7, 14, 18, and 20 months. Evaluations of LV function and structure and myocardial fibrosis were performed at 7 and 20 months. The percent decrease of LV thickness-to-radius ratio between 7 and 20 months was higher in KO mice compared with WT mice (-30.2% ± 5.3% vs -10.5% ± 4.9%; P < 0.01). The LV diameters were higher in old mice compared with young mice (+13% at end-diastole; +33% at end-systole; P < 0.001). The LV ejection fraction values were lower in KO mice compared with WT mice between 7 and 20 months. The highest difference was found at 14 months (60.0% ± 7.6% vs 78.1% ± 3.5%; P < 0.001). Myocardial fibrosis was higher in old KO mice compared with old WT mice (+55%; P < 0.001), and myocardial iron deposition was slightly increased in old KO mice compared with old WT mice (1.31% ± 0.33% vs 0.84% ± 0.22%; P = 0.056). The present mouse model has the potential to allow the determination of underlying mechanisms involved in the cardiomyopathy induced by HFE-related hemochromatosis. Copyright © 2017 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.

Performance of non-invasive models of fibrosis in predicting mild to moderate fibrosis in patients with non-alcoholic fatty liver disease.

PubMed

Siddiqui, Mohammad S; Patidar, Kavish R; Boyett, Sherry; Luketic, Velimir A; Puri, Puneet; Sanyal, Arun J

2016-04-01

In non-alcoholic fatty liver disease, presence of fibrosis is predictive of long-term liver-related complications. Currently, there are no reliable and non-invasive means of quantifying fibrosis in those with non-alcoholic fatty liver disease. Therefore, we aimed to evaluate the performance of a panel of non-invasive models in predicting fibrosis in non-alcoholic fatty liver disease. The accuracy of FibroMeter non-alcoholic fatty liver disease, fibrosis 4 and four other non-invasive models in predicting fibrosis in those with biopsy proven non-alcoholic fatty liver disease was compared. These models were constructed post hoc in patients who had necessary clinical information collected within 2 months of a liver biopsy. The areas under receiver operating characteristics curves were compared for each model using Delong analysis. Optimum cut-off for each model and fibrosis stage were calculated using the Youden index. The area under receiver operating characteristics curves for F ≥ 1 fibrosis for fibrosis 4 and FibroMeter non-alcoholic fatty liver disease was 0.821 and 0.801 respectively. For F ≥ 3, the area under receiver operating characteristics curves was 0.866 for fibrosis 4 and 0.862 for FibroMeter non-alcoholic fatty liver disease. Delong analysis showed the area under receiver operating characteristics curves was statistically different for fibrosis 4 and FibroMeter non-alcoholic fatty liver disease compared with BARD, BAAT and aspartate aminotransferase:alanine aminotransferase ratio for F ≥ 1 and F ≥ 3. Area under receiver operating characteristics curves were significantly different for fibrosis 4 and FibroMeter non-alcoholic fatty liver disease for F ≥ 3 compared with non-alcoholic fatty liver disease fibrosis score. At a fixed sensitivity of 90%, FibroMeter non-alcoholic fatty liver disease had the highest specificity for F ≥ 1 (52.4%) and F ≥ 3 (63.8%). In contrast, at a fixed specificity of 90%, fibrosis 4 outperformed other models with a sensitivity of 60.2% for F ≥ 1 and 70.6% for F ≥ 3 fibrosis. These non-invasive models of fibrosis can predict varying degrees of fibrosis from routinely collected clinical information in non-alcoholic fatty liver disease. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Experimental Mouse Model of Lumbar Ligamentum Flavum Hypertrophy.

PubMed

Saito, Takeyuki; Yokota, Kazuya; Kobayakawa, Kazu; Hara, Masamitsu; Kubota, Kensuke; Harimaya, Katsumi; Kawaguchi, Kenichi; Hayashida, Mitsumasa; Matsumoto, Yoshihiro; Doi, Toshio; Shiba, Keiichiro; Nakashima, Yasuharu; Okada, Seiji

2017-01-01

Lumbar spinal canal stenosis (LSCS) is one of the most common spinal disorders in elderly people, with the number of LSCS patients increasing due to the aging of the population. The ligamentum flavum (LF) is a spinal ligament located in the interior of the vertebral canal, and hypertrophy of the LF, which causes the direct compression of the nerve roots and/or cauda equine, is a major cause of LSCS. Although there have been previous studies on LF hypertrophy, its pathomechanism remains unclear. The purpose of this study is to establish a relevant mouse model of LF hypertrophy and to examine disease-related factors. First, we focused on mechanical stress and developed a loading device for applying consecutive mechanical flexion-extension stress to the mouse LF. After 12 weeks of mechanical stress loading, we found that the LF thickness in the stress group was significantly increased in comparison to the control group. In addition, there were significant increases in the area of collagen fibers, the number of LF cells, and the gene expression of several fibrosis-related factors. However, in this mecnanical stress model, there was no macrophage infiltration, angiogenesis, or increase in the expression of transforming growth factor-β1 (TGF-β1), which are characteristic features of LF hypertrophy in LSCS patients. We therefore examined the influence of infiltrating macrophages on LF hypertrophy. After inducing macrophage infiltration by micro-injury to the mouse LF, we found excessive collagen synthesis in the injured site with the increased TGF-β1 expression at 2 weeks after injury, and further confirmed LF hypertrophy at 6 weeks after injury. Our findings demonstrate that mechanical stress is a causative factor for LF hypertrophy and strongly suggest the importance of macrophage infiltration in the progression of LF hypertrophy via the stimulation of collagen production.

HS-173, a Novel PI3K Inhibitor, Attenuates the Activation of Hepatic Stellate Cells in Liver Fibrosis

PubMed Central

Son, Mi Kwon; Ryu, Ye-Lim; Jung, Kyung Hee; Lee, Hyunseung; Lee, Hee Seung; Yan, Hong Hua; Park, Heon Joo; Ryu, Ji-Kan; Suh, Jun–Kyu; Hong, Sungwoo; Hong, Soon-Sun

2013-01-01

Hepatic stellate cells (HSCs) are the primary source of matrix components in liver disease such as fibrosis. Phosphatidylinositol 3-kinase (PI3K) signaling in HSCs has been shown to induce fibrogenesis. In this study, we evaluated the anti-fibrotic activity of a novel imidazopyridine analogue (HS-173) in human HSCs as well as mouse liver fibrosis. HS-173 strongly suppressed the growth and proliferation of HSCs and induced the arrest at the G2/M phase and apoptosis in HSCs. Furthermore, it reduced the expression of extracellular matrix components such as collagen type I, which was confirmed by an in vivo study. We also observed that HS-173 blocked the PI3K/Akt signaling pathway in vitro and in vivo. Taken together, HS-173 suppressed fibrotic responses such as cell proliferation and collagen synthesis by blocking PI3K/Akt signaling. Therefore, we suggest that this compound may be an effective therapeutic agent for ameliorating liver fibrosis through the inhibition of PI3K signaling. PMID:24326778

The diagnostic value of a globulin/platelet model for evaluating liver fibrosis in chronic hepatitis B patients.

PubMed

Coskun, Banu Demet; Altinkaya, Engin; Sevinc, Eylem; Ozen, Mustafa; Karaman, Hatice; Karaman, Ahmet; Poyrazoglu, Orhan

2015-12-01

Liver biopsy, which is considered the best method for evaluating hepatic fibrosis, has important adverse events. Therefore, non-invasive tests have been developed to determine the degree of hepatic fibrosis in patients with chronic hepatitis B. To verify the usefulness of a new fibrosis index the globulin/platelet model in patients with chronic hepatitis B and to compare it with other noninvasive tests for predicting significant fibrosis. This study was the second to evaluate the globulin/platelet model in HBV patients. We retrospectively investigated 228 patients with chronic hepatitis B who performed liver biopsy from 2013 to 2014. The globulin/platelet model, APGA [AST/Platelet/Gamma-glutamyl transpeptidase/Alfa-fetoprotein], FIB4, fibrosis index, cirrhosis discriminate score, and Fibro-quotient were calculated, and the diagnostic accuracies of all of the fibrosis indices were compared between the F0-2 (no-mild fibrosis) and F3-6 (significant fibrosis) groups. All of the noninvasive markers were significantly correlated with the stage of liver fibrosis (p < 0,001). To predict significant fibrosis (F ≥ 3), the area under the curve (95% CI) was found to be greatest for APGA (0.83 [0.74-0.86]), followed by FIB-4 (0.75[0.69-0.80]), the globulin/platelet model (0.74 [0.68-0.79]), fibrosis index (0.72 [0.6-0.78], cirrhosis discriminate score (0.71 [0.64-0.76]) and Fibro-quotient (0.62 [0.55-0.7]). The area under the receiver operating characteristic curves of APGA was significantly higher than that of the other noninvasive fibrosis markers (p < 0.05). While the APGA index was found to be the most valuable test for the prediction significant fibrosis in patients with chronic hepatitis B, GP model was the thirth valuable test. Therefore, we recommended that APGA could be used instead of the GP model for prediction liver fibrosis.

Thermoneutral housing exacerbates non-alcoholic fatty liver disease in mice and allows for sex-independent disease modeling

PubMed Central

Giles, Daniel A; Moreno-Fernandez, Maria E; Stankiewicz, Traci E; Graspeuntner, Simon; Cappelletti, Monica; Wu, David; Mukherjee, Rajib; Chan, Calvin C; Lawson, Matthew J; Klarquist, Jared; Sünderhauf, Annika; Softic, Samir; Kahn, C Ronald; Stemmer, Kerstin; Iwakura, Yoichiro; Aronow, Bruce J; Karns, Rebekah; Steinbrecher, Kris A; Karp, Christopher L; Sheridan, Rachel; Shanmukhappa, Shiva K; Reynaud, Damien; Haslam, David B; Sina, Christian; Rupp, Jan; Hogan, Simon P; Divanovic, Senad

2017-01-01

Non-alcoholic fatty liver disease (NAFLD), a common prelude to cirrhosis and hepatocellular carcinoma, is the most common chronic liver disease worldwide. Defining the molecular mechanisms underlying the pathogenesis of NAFLD has been hampered by a lack of animal models that closely recapitulate the severe end of the human disease spectrum, including bridging hepatic fibrosis. Here, we demonstrate that a novel experimental model employing thermoneutral housing, as opposed to standard housing, resulted in lower stress-driven production of corticosterone, augmented mouse proinflammatory immune responses and markedly exacerbated high fat diet (HFD)-induced NAFLD pathogenesis. Disease exacerbation at thermoneutrality was conserved across multiple mouse strains and was associated with augmented intestinal permeability, an altered microbiome and activation of inflammatory pathways associated with human disease. Depletion of Gram-negative microbiota, hematopoietic cell deletion of Toll-like receptor 4 (TLR4) and inactivation of the interleukin-17 (IL-17) axis resulted in altered immune responsiveness and protection from thermoneutral housing-driven NAFLD amplification. Finally, female mice, typically resistant to HFD-induced obesity and NAFLD, develop full-blown disease at thermoneutrality. Thus, thermoneutral housing provides a sex-independent model of exacerbated NAFLD in mice and represents a novel approach for interrogation of the cellular and molecular mechanisms underlying disease pathogenesis. PMID:28604704

Taxifolin protects against cardiac hypertrophy and fibrosis during biomechanical stress of pressure overload

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

Guo, Haipeng; Zhang, Xin; Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Qilu Hospital of Shandong University, Jinan

Cardiac hypertrophy is a key pathophysiological component to biomechanical stress, which has been considered to be an independent and predictive risk factor for adverse cardiovascular events. Taxifolin (TAX) is a typical plant flavonoid, which has long been used clinically for treatment of cardiovascular and cerebrovascular diseases. However, very little is known about whether TAX can influence the development of cardiac hypertrophy. In vitro studies, we found that TAX concentration-dependently inhibited angiotensin II (Ang II) induced hypertrophy and protein synthesis in cardiac myocytes. Then we established a mouse model by transverse aortic constriction (TAC) to further confirm our findings. It wasmore » demonstrated that TAX prevented pressure overload induced cardiac hypertrophy in mice, as assessed by ventricular mass/body weight, echocardiographic parameters, myocyte cross-sectional area, and the expression of ANP, BNP and β-MHC. The excess production of reactive oxygen species (ROS) played critical role in the development of cardiac hypertrophy. TAX arrested oxidative stress and decreased the expression of 4-HNE induced by pressure overload. Moreover, TAX negatively modulated TAC-induced phosphorylation of ERK1/2 and JNK1/2. Further studies showed that TAX significantly attenuated left ventricular fibrosis and collagen synthesis through abrogating the phosphorylation of Smad2 and Smad2/3 nuclear translocation. These results demonstrated that TAX could inhibit cardiac hypertrophy and attenuate ventricular fibrosis after pressure overload. These beneficial effects were at least through the inhibition of the excess production of ROS, ERK1/2, JNK1/2 and Smad signaling pathways. Therefore, TAX might be a potential candidate for the treatment of cardiac hypertrophy and fibrosis. - Highlights: • We focus on the protective effect of taxifolin on cardiac remodeling. • Taxifolin inhibited cardiac hypertrophy and attenuated ventricular fibrosis. • Taxifolin suppressed oxidative stress and the excess production of ROS. • Taxifolin blocked ERK1/2, JNK1/2 and Smad signaling pathways. • We reported that taxifolin had the potential to be a candidate for cardiac hypertrophy treatment.« less

Reduced Cx43 expression triggers increased fibrosis due to enhanced fibroblast activity.

PubMed

Jansen, John A; van Veen, Toon A B; de Jong, Sanne; van der Nagel, Roel; van Stuijvenberg, Leonie; Driessen, Helen; Labzowski, Ronald; Oefner, Carolin M; Bosch, Astrid A; Nguyen, Tri Q; Goldschmeding, Roel; Vos, Marc A; de Bakker, Jacques M T; van Rijen, Harold V M

2012-04-01

Arrhythmogenic ventricular remodeling is hallmarked by both reduced gap junction expression and increased collagen deposition. We hypothesized that reduced connexin43 (Cx43) expression is responsible for enhanced fibrosis in the remodeled heart, resulting in an arrhythmogenic substrate. Therefore, we investigated the effect of normal or reduced Cx43 expression on the formation of fibrosis in a physiological (aging) and pathophysiological (transverse aortic constriction [TAC]) mouse model. The Cx43(fl/fl) and Cx43(CreER(T)/fl) mice were aged 18 to 21 months or, at the age of 3 months, either TAC or sham operated and euthanized after 16 weeks. Epicardial activation mapping of the right and left ventricles was performed on Langendorff perfused hearts. Sustained ventricular arrhythmias were induced in 0 of 11 aged Cx43(fl/fl) and 10 of 15 Cx43(Cre-ER(T)/fl) mice (P<0.01). Cx43 expression was reduced by half in aged Cx43(CreER(T)/fl) compared with aged Cx43(fl/fl) mice, whereas collagen deposition was significantly increased from 1.1±0.2% to 7.4±1.3%. Aged Cx43(CreER(T)/fl) mice with arrhythmias had significantly higher levels of fibrosis and conduction heterogeneity than aged Cx43(CreER(T)/fl) mice without arrhythmias. The TAC operation significantly increased fibrosis in control compared with sham (4.0±1.2% versus 0.4±0.06%), but this increase was significantly higher in Cx43(CreER(T)/fl) mice (10.8±1.4%). Discoidin domain receptor 2 expression was unchanged, but procollagen peptide I and III expression and collagen type 1α2 mRNA levels were higher in TAC-operated Cx43HZ mice. Reduced cellular coupling results in more excessive collagen deposition during aging or pressure overload in mice due to enhanced fibroblast activity, leading to increased conduction in homogeneity and proarrhythmia.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) increases necroinflammation and hepatic stellate cell activation but does not exacerbate experimental liver fibrosis in mice

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

Lamb, Cheri L.; Cholico, Giovan N.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant and high-affinity ligand for the aryl hydrocarbon receptor (AhR). Increasing evidence indicates that AhR signaling contributes to wound healing, which involves the coordinated deposition and remodeling of the extracellular matrix. In the liver, wound healing is attributed to the activation of hepatic stellate cells (HSCs), which mediate fibrogenesis through the production of soluble mediators and collagen type I. We recently reported that TCDD treatment increases the activation of human HSCs in vitro. The goal of this study was to determine how TCDD impacts HSC activation in vivo using a mouse model of experimentalmore » liver fibrosis. To elicit fibrosis, C57BL6/male mice were treated twice weekly for 8 weeks with 0.5 ml/kg carbon tetrachloride (CCl{sub 4}). TCDD (20 μg/kg) or peanut oil (vehicle) was administered once a week during the last 2 weeks. Results indicate that TCDD increased liver-body-weight ratios, serum alanine aminotransferase activity, and hepatic necroinflammation in CCl{sub 4}-treated mice. Likewise, TCDD treatment increased mRNA expression of HSC activation and fibrogenesis genes, namely α-smooth muscle actin, desmin, delta-like homolog-1, TGF-β1, and collagen type I. However, TCDD treatment did not exacerbate fibrosis, nor did it increase the collagen content of the liver. Instead, TCDD increased hepatic collagenase activity and increased expression of matrix metalloproteinase (MMP)-13 and the matrix regulatory proteins, TIMP-1 and PAI-1. These results support the conclusion that TCDD increases CCl{sub 4}-induced liver damage and exacerbates HSC activation, yet collagen deposition and the development of fibrosis may be limited by TCDD-mediated changes in extracellular matrix remodeling. - Highlights: • TCDD increased liver damage and inflammation in mice treated with CCl{sub 4}. • TCDD treatment enhanced markers of hepatic stellate cell activation and fibrogenesis. • TCDD did not increase the deposition of collagen type I or the severity of liver fibrosis. • TCDD increased hepatic collagenase activity and expression of matrix metalloproteinase-13.« less

Pleiotrophin triggers inflammation and increased peritoneal permeability leading to peritoneal fibrosis.

PubMed

Yokoi, Hideki; Kasahara, Masato; Mori, Kiyoshi; Ogawa, Yoshihisa; Kuwabara, Takashige; Imamaki, Hirotaka; Kawanishi, Tomoko; Koga, Kenichi; Ishii, Akira; Kato, Yukiko; Mori, Keita P; Toda, Naohiro; Ohno, Shoko; Muramatsu, Hisako; Muramatsu, Takashi; Sugawara, Akira; Mukoyama, Masashi; Nakao, Kazuwa

2012-01-01

Long-term peritoneal dialysis induces peritoneal fibrosis with submesothelial fibrotic tissue. Although angiogenesis and inflammatory mediators are involved in peritoneal fibrosis, precise molecular mechanisms are undefined. To study this, we used microarray analysis and compared gene expression profiles of the peritoneum in control and chlorhexidine gluconate (CG)-induced peritoneal fibrosis mice. One of the 43 highly upregulated genes was pleiotrophin, a midkine family member, the expression of which was also upregulated by the solution used to treat mice by peritoneal dialysis. This growth factor was found in fibroblasts and mesothelial cells within the underlying submesothelial compact zones of mice, and in human peritoneal biopsy samples and peritoneal dialysate effluent. Recombinant pleiotrophin stimulated mitogenesis and migration of mouse mesothelial cells in culture. We found that in wild-type mice, CG treatment increased peritoneal permeability (measured by equilibration), increased mRNA expression of TGF-β1, connective tissue growth factor and fibronectin, TNF-α and IL-1β expression, and resulted in infiltration of CD3-positive T cells, and caused a high number of Ki-67-positive proliferating cells. All of these parameters were decreased in peritoneal tissues of CG-treated pleiotrophin-knockout mice. Thus, an upregulation of pleiotrophin appears to play a role in fibrosis and inflammation during peritoneal injury.

ADAM10-mediated ephrin-B2 shedding promotes myofibroblast activation and organ fibrosis

PubMed Central

Lagares, David; Ghassemi-Kakroodi, Parisa; Tremblay, Caroline; Santos, Alba; Probst, Clemens K.; Franklin, Alicia; Santos, Daniela M.; Grasberger, Paula; Ahluwalia, Neil; Montesi, Sydney B.; Shea, Barry S.; Black, Katharine E.; Knipe, Rachel; Blati, Meryem; Baron, Murray; Wu, Brian; Fahmi, Hassan; Gandhi, Rajiv; Pardo, Annie; Selman, Moisés; Wu, Jiangping; Pelletier, Jean-Pierre; Martel-Pelletier, Johanne; Tager, Andrew M.; Kapoor, Mohit

2017-01-01

Maladaptive wound healing responses to chronic tissue injury result in organ fibrosis. Fibrosis, which entails excessive extracellular matrix (ECM) deposition and tissue remodelling by activated myofibroblasts, leads to loss of proper tissue architecture and organ function; however the molecular mediators of myofibroblast activation remain to be fully identified. Here we identify soluble ephrin-B2 as a novel pro-fibrotic mediator in lung and skin fibrosis. We provide molecular, functional and translational evidence that the ectodomain of membrane-bound ephrin-B2 is shed from fibroblasts into the alveolar airspace after lung injury. Shedding of soluble ephrin-B2 (sEphrin-B2) promotes fibroblast chemotaxis and activation via EphB3/EphB4 receptor signaling. We found that mice lacking ephrin-B2 in fibroblasts are protected from skin and lung fibrosis and that a distintegrin and metalloproteinase 10 (ADAM10) is the major ephrin-B2 sheddase in fibroblasts. ADAM10 is induced by transforming growth factor-β1 (TGF-β1), and ADAM10-mediated sEphrin-B2 generation is required for TGF-β1–induced myofibroblast activation. Pharmacological inhibition of ADAM10 reduces sEphrin-B2 levels in bronchoalveolar lavage and prevents lung fibrosis in mice. Consistent with the mouse data, ADAM10/sEphrin-B2 signaling is upregulated in fibroblasts from human subjects with idiopathic pulmonary fibrosis. These results uncover a new molecular mechanism of tissue fibrogenesis and identify sEphrin-B2, its receptors Eph3/Eph4, and ADAM10 as potential therapeutic targets in the treatment of fibrotic diseases. PMID:29058717

The role of microRNA-155/liver X receptor pathway in experimental and idiopathic pulmonary fibrosis.

PubMed

Kurowska-Stolarska, Mariola; Hasoo, Manhl K; Welsh, David J; Stewart, Lynn; McIntyre, Donna; Morton, Brian E; Johnstone, Steven; Miller, Ashley M; Asquith, Darren L; Millar, Neal L; Millar, Ann B; Feghali-Bostwick, Carol A; Hirani, Nikhil; Crick, Peter J; Wang, Yuqin; Griffiths, William J; McInnes, Iain B; McSharry, Charles

2017-06-01

Idiopathic pulmonary fibrosis (IPF) is progressive and rapidly fatal. Improved understanding of pathogenesis is required to prosper novel therapeutics. Epigenetic changes contribute to IPF; therefore, microRNAs may reveal novel pathogenic pathways. We sought to determine the regulatory role of microRNA (miR)-155 in the profibrotic function of murine lung macrophages and fibroblasts, IPF lung fibroblasts, and its contribution to experimental pulmonary fibrosis. Bleomycin-induced lung fibrosis in wild-type and miR-155 -/- mice was analyzed by histology, collagen, and profibrotic gene expression. Mechanisms were identified by in silico and molecular approaches and validated in mouse lung fibroblasts and macrophages, and in IPF lung fibroblasts, using loss-and-gain of function assays, and in vivo using specific inhibitors. miR-155 -/- mice developed exacerbated lung fibrosis, increased collagen deposition, collagen 1 and 3 mRNA expression, TGF-β production, and activation of alternatively activated macrophages, contributed by deregulation of the miR-155 target gene the liver X receptor (LXR)α in lung fibroblasts and macrophages. Inhibition of LXRα in experimental lung fibrosis and in IPF lung fibroblasts reduced the exacerbated fibrotic response. Similarly, enforced expression of miR-155 reduced the profibrotic phenotype of IPF and miR-155 -/- fibroblasts. We describe herein a molecular pathway comprising miR-155 and its epigenetic LXRα target that when deregulated enables pathogenic pulmonary fibrosis. Manipulation of the miR-155/LXR pathway may have therapeutic potential for IPF. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Pharmacologic inhibition of the enzymatic effects of tissue transglutaminase reduces cardiac fibrosis and attenuates cardiomyocyte hypertrophy following pressure overload.

PubMed

Shinde, Arti V; Su, Ya; Palanski, Brad A; Fujikura, Kana; Garcia, Mario J; Frangogiannis, Nikolaos G

2018-04-01

Tissue transglutaminase (tTG) is a multifunctional protein with a wide range of enzymatic and non-enzymatic functions. We have recently demonstrated that tTG expression is upregulated in the pressure-overloaded myocardium and exerts fibrogenic actions promoting diastolic dysfunction, while preventing chamber dilation. Our current investigation dissects the in vivo and in vitro roles of the enzymatic effects of tTG on fibrotic remodeling in pressure-overloaded myocardium. Using a mouse model of transverse aortic constriction, we demonstrated perivascular and interstitial tTG activation in the remodeling pressure-overloaded heart. tTG inhibition through administration of the selective small molecule tTG inhibitor ERW1041E attenuated left ventricular diastolic dysfunction and reduced cardiomyocyte hypertrophy and interstitial fibrosis in the pressure-overloaded heart, without affecting chamber dimensions and ejection fraction. In vivo, tTG inhibition markedly reduced myocardial collagen mRNA and protein levels and attenuated transcription of fibrosis-associated genes. In contrast, addition of exogenous recombinant tTG to fibroblast-populated collagen pads had no significant effects on collagen transcription, and instead increased synthesis of matrix metalloproteinase (MMP)3 and tissue inhibitor of metalloproteinases (TIMP)1 through transamidase-independent actions. However, enzymatic effects of matrix-bound tTG increased the thickness of pericellular collagen in fibroblast-populated pads. tTG exerts distinct enzymatic and non-enzymatic functions in the remodeling pressure-overloaded heart. The enzymatic effects of tTG are fibrogenic and promote diastolic dysfunction, but do not directly modulate the pro-fibrotic transcriptional program of fibroblasts. Targeting transamidase-dependent actions of tTG may be a promising therapeutic strategy in patients with heart failure and fibrosis-associated diastolic dysfunction. Copyright © 2018 Elsevier Ltd. All rights reserved.

N-Acetyl-Seryl-Aspartyl-Lysyl-Proline: mechanisms of renal protection in mouse model of systemic lupus erythematosus

PubMed Central

Liao, Tang-Dong; Nakagawa, Pablo; Janic, Branislava; D'Ambrosio, Martin; Worou, Morel E.; Peterson, Edward L.; Rhaleb, Nour-Eddine; Yang, Xiao-Ping

2015-01-01

Systemic lupus erythematosus is an autoimmune disease characterized by the development of auto antibodies against a variety of self-antigens and deposition of immune complexes that lead to inflammation, fibrosis, and end-organ damage. Up to 60% of lupus patients develop nephritis and renal dysfunction leading to kidney failure. N-acetyl-seryl-aspartyl-lysyl-proline, i.e., Ac-SDKP, is a natural tetrapeptide that in hypertension prevents inflammation and fibrosis in heart, kidney, and vasculature. In experimental autoimmune myocarditis, Ac-SDKP prevents cardiac dysfunction by decreasing innate and adaptive immunity. It has also been reported that Ac-SDKP ameliorates lupus nephritis in mice. We hypothesize that Ac-SDKP prevents lupus nephritis in mice by decreasing complement C5-9, proinflammatory cytokines, and immune cell infiltration. Lupus mice treated with Ac-SDKP for 20 wk had significantly lower renal levels of macrophage and T cell infiltration and proinflammatory chemokine/cytokines. In addition, our data demonstrate for the first time that in lupus mouse Ac-SDKP prevented the increase in complement C5-9, RANTES, MCP-5, and ICAM-1 kidney expression and it prevented the decline of glomerular filtration rate. Ac-SDKP-treated lupus mice had a significant improvement in renal function and lower levels of glomerular damage. Ac-SDKP had no effect on the production of autoantibodies. The protective Ac-SDKP effect is most likely achieved by targeting the expression of proinflammatory chemokines/cytokines, ICAM-1, and immune cell infiltration in the kidney, either directly or via C5-9 proinflammatory arm of complement system. PMID:25740596

Relative contribution of Prevotella intermedia and Pseudomonas aeruginosa to lung pathology in airways of patients with cystic fibrosis.

PubMed

Ulrich, Martina; Beer, Isabelle; Braitmaier, Peter; Dierkes, Michaela; Kummer, Florian; Krismer, Bernhard; Schumacher, Ulrike; Gräpler-Mainka, Ute; Riethmüller, Joachim; Jensen, Peter Ø; Bjarnsholt, Thomas; Høiby, Niels; Bellon, Gabriel; Döring, Gerd

2010-11-01

Patients with cystic fibrosis (CF) with Pseudomonas aeruginosa lung infections produce endobronchial mucus plugs allowing growth of obligate anaerobes including Prevotella spp. Whether obligate anaerobes contribute to the pathophysiology of CF lung disease is unknown. The virulence of Prevotella intermedia and Ps aeruginosa was investigated in vitro and in mice, antibodies against P intermedia in CF sera were assessed and a culture-independent detection method for P intermedia/P nigrescens in CF sputum was tested. P intermedia reached cell numbers of >10(5)->10(7) colony-forming units (CFU)/ml sputum. The majority of patients with CF (16/17; 94.1%) produced antibodies against two immunoreactive antigens of P intermedia. Culture supernatant fluids, collected from 10(9) P intermedia cells, were more cytotoxic to respiratory epithelial cells in vitro and inflammatory in mouse lungs than respective fluids from anaerobically grown Ps aeruginosa, while fluids from aerobically grown Ps aeruginosa had the highest cytotoxicity and inflammation. Both pathological effects were largely reduced when culture supernatant fluids from 10(7) cells of either species were used. P intermedia cells (∼10(6)CFU/lung) did not induce mortality in the agar beads lung infection mouse model, while Ps aeruginosa cells caused death in 30% of mice due to rapid multiplication. A P intermedia/P nigrescens-specific PNA probe was significantly more sensitive than culture-dependent diagnostic assays to detect these strict anaerobes. Ps aeruginosa and P intermedia become significantly virulent in vitro and in vivo when cell numbers exceed 10(8) CFU/lung.

Comparative biology of rAAV transduction in ferret, pig and human airway epithelia.

PubMed

Liu, X; Luo, M; Guo, C; Yan, Z; Wang, Y; Engelhardt, J F

2007-11-01

Differences between rodent and human airway cell biology have made it difficult to translate recombinant adeno-associated virus (rAAV)-mediated gene therapies to the lung for cystic fibrosis (CF). As new ferret and pig models for CF become available, knowledge about host cell/vector interactions in these species will become increasingly important for testing potential gene therapies. To this end, we have compared the transduction biology of three rAAV serotypes (AAV1, 2 and 5) in human, ferret, pig and mouse-polarized airway epithelia. Our results indicate that apical transduction of ferret and pig airway epithelia with these rAAV serotypes closely mirrors that observed in human epithelia (rAAV1>rAAV2 congruent withrAAV5), while transduction of mouse epithelia was significantly different (rAAV1>rAAV5>rAAV2). Similarly, ferret, pig and human epithelia also shared serotype-specific differences in the polarity (apical vs basolateral) and proteasome dependence of rAAV transduction. Despite these parallels, N-linked sialic acid receptors were required for rAAV1 and rAAV5 transduction of human and mouse airway epithelia, but not ferret or pig airway epithelia. Hence, although the airway tropisms of rAAV serotypes 1, 2 and 5 are conserved better among ferret, pig and human as compared to mouse, viral receptors/co-receptors appear to maintain considerable species diversity.

Augmented sphingosine 1 phosphate receptor-1 signaling in cardiac fibroblasts induces cardiac hypertrophy and fibrosis through angiotensin II and interleukin-6

PubMed Central

Ohkura, Sei-ichiro; Takashima, Shin-ichiro; Yoshioka, Kazuaki; Okamoto, Yasuo; Inagaki, Yutaka; Sugimoto, Naotoshi; Kitano, Teppei; Takamura, Masayuki; Wada, Takashi; Kaneko, Shuichi; Takuwa, Yoh

2017-01-01

Background: Cardiac fibroblasts, together with cardiomyocytes, occupy the majority of cells in the myocardium and are involved in myocardial remodeling. The lysophospholipid mediator sphigosine-1-phosphate (S1P) regulates functions of cardiovascular cells through multiple receptors including S1PR1–S1PR3. S1PR1 but not other S1P receptors was upregulated in angiotensin II-induced hypertrophic hearts. Therefore, we investigated a role of S1PR1 in fibroblasts for cardiac remodeling by employing transgenic mice that overexpressed S1PR1 under the control of α-smooth muscle actin promoter. In S1PR1-transgenic mouse heart, fibroblasts and/or myofibroblasts were hyperplastic, and those cells as well as vascular smooth muscle cells overexpressed S1PR1. Transgenic mice developed bi-ventricular hypertrophy by 12-week-old and diffuse interstitial fibrosis by 24-week-old without hemodynamic stress. Cardiac remodeling in transgenic mice was associated with greater ERK phosphorylation, upregulation of fetal genes, and systolic dysfunction. Transgenic mouse heart showed increased mRNA expression of angiotensin-converting enzyme and interleukin-6 (IL-6). Isolated fibroblasts from transgenic mice exhibited enhanced generation of angiotensin II, which in turn stimulated IL-6 release. Either an AT1 blocker or angiotensin-converting enzyme inhibitor prevented development of cardiac hypertrophy and fibrosis, systolic dysfunction and increased IL-6 expression in transgenic mice. Finally, administration of anti-IL-6 antibody abolished an increase in tyrosine phosphorylation of STAT3, a major signaling molecule downstream of IL-6, in the transgenic mouse heart and prevented development of cardiac hypertrophy in transgenic mice. These results demonstrate a promoting role of S1PR1 in cardiac fibroblasts for cardiac remodeling, in which angiotensin II—AT1 and IL-6 are involved. PMID:28771545

Effective silencing of ENaC by siRNA delivered with epithelial-targeted nanocomplexes in human cystic fibrosis cells and in mouse lung.

PubMed

Tagalakis, Aristides D; Munye, Mustafa M; Ivanova, Rositsa; Chen, Hanpeng; Smith, Claire M; Aldossary, Ahmad M; Rosa, Luca Z; Moulding, Dale; Barnes, Josephine L; Kafetzis, Konstantinos N; Jones, Stuart A; Baines, Deborah L; Moss, Guy W J; O'Callaghan, Christopher; McAnulty, Robin J; Hart, Stephen L

2018-05-10

Loss of the cystic fibrosis transmembrane conductance regulator in cystic fibrosis (CF) leads to hyperabsorption of sodium and fluid from the airway due to upregulation of the epithelial sodium channel (ENaC). Thickened mucus and depleted airway surface liquid (ASL) then lead to impaired mucociliary clearance. ENaC regulation is thus a promising target for CF therapy. Our aim was to develop siRNA nanocomplexes that mediate effective silencing of airway epithelial ENaC in vitro and in vivo with functional correction of epithelial ion and fluid transport. We investigated translocation of nanocomplexes through mucus and their transfection efficiency in primary CF epithelial cells grown at air-liquid interface (ALI).Short interfering RNA (SiRNA)-mediated silencing was examined by quantitative RT-PCR and western analysis of ENaC. Transepithelial potential (V t ), short circuit current (I sc ), ASL depth and ciliary beat frequency (CBF) were measured for functional analysis. Inflammation was analysed by histological analysis of normal mouse lung tissue sections. Nanocomplexes translocated more rapidly than siRNA alone through mucus. Transfections of primary CF epithelial cells with nanocomplexes targeting αENaC siRNA, reduced αENaC and βENaC mRNA by 30%. Transfections reduced V t , the amiloride-sensitive I sc and mucus protein concentration while increasing ASL depth and CBF to normal levels. A single dose of siRNA in mouse lung silenced ENaC by approximately 30%, which persisted for at least 7 days. Three doses of siRNA increased silencing to approximately 50%. Nanoparticle-mediated delivery of ENaCsiRNA to ALI cultures corrected aspects of the mucociliary defect in human CF cells and offers effective delivery and silencing in vivo. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

miR-34a Inhibits Lung Fibrosis by Inducing Lung Fibroblast Senescence.

PubMed

Cui, Huachun; Ge, Jing; Xie, Na; Banerjee, Sami; Zhou, Yong; Antony, Veena B; Thannickal, Victor J; Liu, Gang

2017-02-01

Cellular senescence has been implicated in diverse pathologies. However, there is conflicting evidence regarding the role of this process in tissue fibrosis. Although dysregulation of microRNAs is a key mechanism in the pathogenesis of lung fibrosis, it is unclear whether microRNAs function by regulating cellular senescence in the disease. In this study, we found that miR-34a demonstrated greater expression in the lungs of patients with idiopathic pulmonary fibrosis and in mice with experimental pulmonary fibrosis, with its primary localization in lung fibroblasts. More importantly, miR-34a was up-regulated significantly in both human and mouse lung myofibroblasts. We found that mice with miR-34a ablation developed more severe pulmonary fibrosis than did wild-type animals after fibrotic lung injury. Mechanistically, we found that miR-34a induced a senescent phenotype in lung fibroblasts because this microRNA increased senescence-associated β-galactosidase activity, enhanced expression of senescence markers, and decreased cell proliferative capacities. Consistently, we found that primary lung fibroblasts from fibrotic lungs of miR-34a-deficient mice had a diminished senescent phenotype and enhanced resistance to apoptosis as compared with those from wild-type animals. We also identified multiple miR-34a targets that likely mediated its activities in inducing senescence in lung fibroblasts. In conclusion, our data suggest that miR-34a functions through a negative feedback mechanism to restrain fibrotic response in the lungs by promoting senescence of pulmonary fibroblasts.

Identification of the Gene for Scleroderma in the Tsk/2 Mouse Strain: Implications for Human Scleroderma Pathogenesis and Subset Distinctions

DTIC Science & Technology

2015-09-01

skin)and)used)as) a)marker)of)fibrosis)[3,)4]),)we)assessed)both) protein )and)mRNA)levels)in)fibroblasts)that)received)DNA)from)a) plasmid...containing)a)single)allele)of)a)single)Col3a1%gene.)In)three)independent)experiments,)COL1A1) protein ) was)significantly)elevated)after)48)hours)of)transfection...Mouse& Col3a1eKO& fibroblasts& transfected& with& a& plasmid& bearing&the&mutant&Col3a1Tsk2&express&34%&more&COL1A1& protein &than& Col3a1WT

Renal Impairment with Sublethal Tubular Cell Injury in a Chronic Liver Disease Mouse Model

PubMed Central

Ishida, Tokiko; Kotani, Hirokazu; Miyao, Masashi; Kawai, Chihiro; Jemail, Leila; Abiru, Hitoshi; Tamaki, Keiji

2016-01-01

The pathogenesis of renal impairment in chronic liver diseases (CLDs) has been primarily studied in the advanced stages of hepatic injury. Meanwhile, the pathology of renal impairment in the early phase of CLDs is poorly understood, and animal models to elucidate its mechanisms are needed. Thus, we investigated whether an existing mouse model of CLD induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) shows renal impairment in the early phase. Renal injury markers, renal histology (including immunohistochemistry for tubular injury markers and transmission electron microscopy), autophagy, and oxidative stress were studied longitudinally in DDC- and standard diet–fed BALB/c mice. Slight but significant renal dysfunction was evident in DDC-fed mice from the early phase. Meanwhile, histological examinations of the kidneys with routine light microscopy did not show definitive morphological findings, and electron microscopic analyses were required to detect limited injuries such as loss of brush border microvilli and mitochondrial deformities. Limited injuries have been recently designated as sublethal tubular cell injury. As humans with renal impairment, either with or without CLD, often show almost normal tubules, sublethal injury has been of particular interest. In this study, the injuries were associated with mitochondrial aberrations and oxidative stress, a possible mechanism for sublethal injury. Intriguingly, two defense mechanisms were associated with this injury that prevent it from progressing to apparent cell death: autophagy and single-cell extrusion with regeneration. Furthermore, the renal impairment of this model progressed to chronic kidney disease with interstitial fibrosis after long-term DDC feeding. These findings indicated that DDC induces renal impairment with sublethal tubular cell injury from the early phase, leading to chronic kidney disease. Importantly, this CLD mouse model could be useful for studying the pathophysiological mechanisms of sublethal tubular cell injury. PMID:26752420

High-fat, high-calorie diet promotes early pancreatic neoplasia in the conditional KrasG12D mouse model.

PubMed

Dawson, David W; Hertzer, Kathleen; Moro, Aune; Donald, Graham; Chang, Hui-Hua; Go, Vay Liang; Pandol, Steven J; Lugea, Aurelia; Gukovskaya, Anna S; Li, Gang; Hines, Oscar J; Rozengurt, Enrique; Eibl, Guido

2013-10-01

There is epidemiologic evidence that obesity increases the risk of cancers. Several underlying mechanisms, including inflammation and insulin resistance, are proposed. However, the driving mechanisms in pancreatic cancer are poorly understood. The goal of the present study was to develop a model of diet-induced obesity and pancreatic cancer development in a state-of-the-art mouse model, which resembles important clinical features of human obesity, for example, weight gain and metabolic disturbances. Offspring of Pdx-1-Cre and LSL-KrasG12D mice were allocated to either a high-fat, high-calorie diet (HFCD; ∼4,535 kcal/kg; 40% of calories from fats) or control diet (∼3,725 kcal/kg; 12% of calories from fats) for 3 months. Compared with control animals, mice fed with the HFCD significantly gained more weight and developed hyperinsulinemia, hyperglycemia, hyperleptinemia, and elevated levels of insulin-like growth factor I (IGF-I). The pancreas of HFCD-fed animals showed robust signs of inflammation with increased numbers of infiltrating inflammatory cells (macrophages and T cells), elevated levels of several cytokines and chemokines, increased stromal fibrosis, and more advanced PanIN lesions. Our results show that a diet high in fats and calories leads to obesity and metabolic disturbances similar to humans and accelerates early pancreatic neoplasia in the conditional KrasG12D mouse model. This model and findings will provide the basis for more robust studies attempting to unravel the mechanisms underlying the cancer-promoting properties of obesity, as well as to evaluate dietary- and chemopreventive strategies targeting obesity-associated pancreatic cancer development.

SHEAR WAVE DISPERSION MEASURES LIVER STEATOSIS

PubMed Central

Barry, Christopher T.; Mills, Bradley; Hah, Zaegyoo; Mooney, Robert A.; Ryan, Charlotte K.; Rubens, Deborah J.; Parker, Kevin J.

2012-01-01

Crawling waves, which are interfering shear wave patterns, can be generated in liver tissue over a range of frequencies. Some important biomechanical properties of the liver can be determined by imaging the crawling waves using Doppler techniques and analyzing the patterns. We report that the dispersion of shear wave velocity and attenuation, that is, the frequency dependence of these parameters, are strongly correlated with the degree of steatosis in a mouse liver model, ex vivo. The results demonstrate the possibility of assessing liver steatosis using noninvasive imaging methods that are compatible with color Doppler scanners and, furthermore, suggest that liver steatosis can be separated from fibrosis by assessing the dispersion or frequency dependence of shear wave propagations. PMID:22178165

Limited fibrosis accompanies triple-negative breast cancer metastasis in multiple model systems and is not a preventive target.

PubMed

Brooks, Danielle; Zimmer, Alexandra; Wakefield, Lalage; Lyle, L Tiffany; Difilippantonio, Simone; Tucci, Fabio C; Illiano, Stephane; Annunziata, Christina M; Steeg, Patricia S

2018-05-04

The lysophosphatidic acid receptor 1 (LPAR1) is mechanistically implicated in both tumor metastasis and tissue fibrosis. Previously, metastasis was increased when fulminant fibrosis was first induced in mice, suggesting a direct connection between these processes. The current report examined the extent of metastasis-induced fibrosis in breast cancer model systems, and tested the metastasis preventive efficacy and fibrosis attenuation of antagonists for LPAR1 and Idiopathic Pulmonary Fibrosis (IPF) in breast and ovarian cancer models. Staining analysis demonstrated only focal, low-moderate levels of fibrosis in lungs from eleven metastasis model systems. Two orally available LPAR1 antagonists, SAR100842 and EPGN9878, significantly inhibited breast cancer motility to LPA in vitro . Both compounds were negative for metastasis prevention and failed to reduce fibrosis in the experimental MDA-MB-231T and spontaneous murine 4T1 in vivo breast cancer metastasis models. SAR100842 demonstrated only occasional reductions in invasive metastases in the SKOV3 and OVCAR5 ovarian cancer experimental metastasis models. Two approved drugs for IPF, nintedanib and pirfenidone, were investigated. Both were ineffective at preventing MDA-MB-231T metastasis, with no attenuation of fibrosis. In summary, metastasis-induced fibrosis is only a minor component of metastasis in untreated progressive breast cancer. LPAR1 antagonists, despite in vitro evidence of specificity and efficacy, were ineffective in vivo as oral agents, as were approved IPF drugs. The data argue against LPAR1 and fibrosis as monotherapy targets for metastasis prevention in triple-negative breast cancer and ovarian cancer.

A new model of diabetic nephropathy in C57BL/6 mice challenged with advanced oxidation protein products.

PubMed

Bai, Xiaoyan; Li, Xiao; Tian, Jianwei; Xu, Liting; Wan, Jiao; Liu, Youhua

2018-04-01

There remains a lack of robust mouse models with key features of advanced human diabetic nephropathy (DN). Few options of murine models of DN require mutations to be superimposed to obtain desired phenotypic characteristics. Most genetically modified mice are on the C57BL/6 background; however, they are notorious for resistance to develop DN. To overcome these conundrums, this study reports a novel DN model by challenging with advanced oxidation protein products (AOPPs) in streptozotocin-induced diabetic C57BL/6 mice. AOPPs-challenged diabetic C57BL/6 mice were more sensitive to develop progressive proteinuria, causing a 5.59-fold increase in urine albumin to creatinine ratio as compared to diabetic controls by 24 weeks. Typical lesions were present as demonstrated by significant diffuse mesangial expansion, diffuse podocyte foot process effacement, increased glomerular basement membrane thickness, focal arteriolar hyalinosis, mesangiolysis, and mild interstitial fibrosis. These changes were alleviated by losartan treatment. Collectively, these results suggest that AOPPs can accelerate the progression of DN in the resistant C57BL/6 mouse strain. Our studies offer a novel model for studying the pathogenesis of DN that resembles human diabetic kidney disease. It also makes it possible to interrogate the role of specific genetic modifications and to evaluate novel therapeutics to treat DN in preclinical setting. Copyright © 2018. Published by Elsevier Inc.

Cardiac-Specific IGF-1 Receptor Transgenic Expression Protects Against Cardiac Fibrosis and Diastolic Dysfunction in a Mouse Model of Diabetic Cardiomyopathy

PubMed Central

Huynh, Karina; McMullen, Julie R.; Julius, Tracey L.; Tan, Joon Win; Love, Jane E.; Cemerlang, Nelly; Kiriazis, Helen; Du, Xiao-Jun; Ritchie, Rebecca H.

2010-01-01

OBJECTIVE Compelling epidemiological and clinical evidence has identified a specific cardiomyopathy in diabetes, characterized by early diastolic dysfunction and adverse structural remodeling. Activation of the insulin-like growth factor 1 (IGF-1) receptor (IGF-1R) promotes physiological cardiac growth and enhances contractile function. The aim of the present study was to examine whether cardiac-specific overexpression of IGF-1R prevents diabetes-induced myocardial remodeling and dysfunction associated with a murine model of diabetes. RESEARCH DESIGN AND METHODS Type 1 diabetes was induced in 7-week-old male IGF-1R transgenic mice using streptozotocin and followed for 8 weeks. Diastolic and systolic function was assessed using Doppler and M-mode echocardiography, respectively, in addition to cardiac catheterization. Cardiac fibrosis and cardiomyocyte width, heart weight index, gene expression, Akt activity, and IGF-1R protein content were also assessed. RESULTS Nontransgenic (Ntg) diabetic mice had reduced initial (E)-to-second (A) blood flow velocity ratio (E:A ratio) and prolonged deceleration times on Doppler echocardiography compared with nondiabetic counterparts, indicative markers of diastolic dysfunction. Diabetes also increased cardiomyocyte width, collagen deposition, and prohypertrophic and profibrotic gene expression compared with Ntg nondiabetic littermates. Overexpression of the IGF-1R transgene markedly reduced collagen deposition, accompanied by a reduction in the incidence of diastolic dysfunction. Akt phosphorylation was elevated ∼15-fold in IGF-1R nondiabetic mice compared with Ntg, and this was maintained in a setting of diabetes. CONCLUSIONS The current study suggests that cardiac overexpression of IGF-1R prevented diabetes-induced cardiac fibrosis and diastolic dysfunction. Targeting IGF-1R–Akt signaling may represent a therapeutic target for the treatment of diabetic cardiac disease. PMID:20215428

Functional and molecular effects of arginine butyrate and prednisone on muscle and heart in the mdx mouse model of Duchenne Muscular Dystrophy.

PubMed

Guerron, Alfredo D; Rawat, Rashmi; Sali, Arpana; Spurney, Christopher F; Pistilli, Emidio; Cha, Hee-Jae; Pandey, Gouri S; Gernapudi, Ramkishore; Francia, Dwight; Farajian, Viken; Escolar, Diana M; Bossi, Laura; Becker, Magali; Zerr, Patricia; de la Porte, Sabine; Gordish-Dressman, Heather; Partridge, Terence; Hoffman, Eric P; Nagaraju, Kanneboyina

2010-06-21

The number of promising therapeutic interventions for Duchenne Muscular Dystrophy (DMD) is increasing rapidly. One of the proposed strategies is to use drugs that are known to act by multiple different mechanisms including inducing of homologous fetal form of adult genes, for example utrophin in place of dystrophin. In this study, we have treated mdx mice with arginine butyrate, prednisone, or a combination of arginine butyrate and prednisone for 6 months, beginning at 3 months of age, and have comprehensively evaluated the functional, biochemical, histological, and molecular effects of the treatments in this DMD model. Arginine butyrate treatment improved grip strength and decreased fibrosis in the gastrocnemius muscle, but did not produce significant improvement in muscle and cardiac histology, heart function, behavioral measurements, or serum creatine kinase levels. In contrast, 6 months of chronic continuous prednisone treatment resulted in deterioration in functional, histological, and biochemical measures. Arginine butyrate-treated mice gene expression profiling experiments revealed that several genes that control cell proliferation, growth and differentiation are differentially expressed consistent with its histone deacetylase inhibitory activity when compared to control (saline-treated) mdx mice. Prednisone and combination treated groups showed alterations in the expression of genes that control fibrosis, inflammation, myogenesis and atrophy. These data indicate that 6 months treatment with arginine butyrate can produce modest beneficial effects on dystrophic pathology in mdx mice by reducing fibrosis and promoting muscle function while chronic continuous treatment with prednisone showed deleterious effects to skeletal and cardiac muscle. Our results clearly indicate the usefulness of multiple assays systems to monitor both beneficial and toxic effects of drugs with broad range of in vivo activity.

Complement Component 5 Mediates Development of Fibrosis, via Activation of Stellate Cells, in 2 Mouse Models of Chronic Pancreatitis

PubMed Central

Sendler, Matthias; Beyer, Georg; Mahajan, Ujjwal M.; Kauschke, Vivien; Maertin, Sandrina; Schurmann, Claudia; Homuth, Georg; Völker, Uwe; Völzke, Henry; Halangk, Walter; Wartmann, Thomas; Weiss, Frank-Ulrich; Hegyi, Peter; Lerch, Markus M.; Mayerle, Julia

2015-01-01

Background & Aims Little is known about the pathogenic mechanisms of chronic pancreatitis. We investigated the roles of complement component 5 (C5) in pancreatic fibrogenesis in mice and patients. Methods Chronic pancreatitis was induced by ligation of the midpancreatic duct, followed by a single supramaximal intraperitoneal injection of cerulein, in C57Bl6 (control) and C5-deficient mice. Some mice were given injections of 2 different antagonists of the receptor for C5a over 21 days. In a separate model, mice were given injections of cerulein for 10 weeks to induce chronic pancreatitis. Direct effects of C5 were studied in cultured primary cells. We performed genotype analysis for the single-nucleotide polymorphisms rs 17611 and rs 2300929 in C5 in patients with pancreatitis and healthy individuals (controls). Blood cells from 976 subjects were analyzed by transcriptional profiling. Results During the initial phase of pancreatitis, levels of pancreatic damage were similar between C5-deficient and control mice. During later stages of pancreatitis, C5-deficient mice and mice given injections of C5a-receptor antagonists developed significantly less pancreatic fibrosis than control mice. Primary pancreatic stellate cells were activated in vitro by C5a. There were no differences in the rs 2300929 SNP between subjects with or without pancreatitis, but the minor allele rs17611 was associated with a significant increase in levels of C5 in whole blood. Conclusions In mice, loss of C5 or injection of a C5a-receptor antagonist significantly reduced the level of fibrosis of chronic pancreatitis, but this was not a consequence of milder disease in early stages of pancreatitis. C5 might be a therapeutic target for chronic pancreatitis. PMID:26001927

Functional and Molecular Effects of Arginine Butyrate and Prednisone on Muscle and Heart in the mdx Mouse Model of Duchenne Muscular Dystrophy

PubMed Central

Guerron, Alfredo D.; Rawat, Rashmi; Sali, Arpana; Spurney, Christopher F.; Pistilli, Emidio; Cha, Hee-Jae; Pandey, Gouri S.; Gernapudi, Ramkishore; Francia, Dwight; Farajian, Viken; Escolar, Diana M.; Bossi, Laura; Becker, Magali; Zerr, Patricia; de la Porte, Sabine; Gordish-Dressman, Heather; Partridge, Terence; Hoffman, Eric P.; Nagaraju, Kanneboyina

2010-01-01

Background The number of promising therapeutic interventions for Duchenne Muscular Dystrophy (DMD) is increasing rapidly. One of the proposed strategies is to use drugs that are known to act by multiple different mechanisms including inducing of homologous fetal form of adult genes, for example utrophin in place of dystrophin. Methodology/Principal Findings In this study, we have treated mdx mice with arginine butyrate, prednisone, or a combination of arginine butyrate and prednisone for 6 months, beginning at 3 months of age, and have comprehensively evaluated the functional, biochemical, histological, and molecular effects of the treatments in this DMD model. Arginine butyrate treatment improved grip strength and decreased fibrosis in the gastrocnemius muscle, but did not produce significant improvement in muscle and cardiac histology, heart function, behavioral measurements, or serum creatine kinase levels. In contrast, 6 months of chronic continuous prednisone treatment resulted in deterioration in functional, histological, and biochemical measures. Arginine butyrate-treated mice gene expression profiling experiments revealed that several genes that control cell proliferation, growth and differentiation are differentially expressed consistent with its histone deacetylase inhibitory activity when compared to control (saline-treated) mdx mice. Prednisone and combination treated groups showed alterations in the expression of genes that control fibrosis, inflammation, myogenesis and atrophy. Conclusions/Significance These data indicate that 6 months treatment with arginine butyrate can produce modest beneficial effects on dystrophic pathology in mdx mice by reducing fibrosis and promoting muscle function while chronic continuous treatment with prednisone showed deleterious effects to skeletal and cardiac muscle. Our results clearly indicate the usefulness of multiple assays systems to monitor both beneficial and toxic effects of drugs with broad range of in vivo activity. PMID:20574530

Animal models of intestinal fibrosis: new tools for the understanding of pathogenesis and therapy of human disease

PubMed Central

Rieder, Florian; Kessler, Sean; Sans, Miquel

2012-01-01

Fibrosis is a serious condition complicating chronic inflammatory processes affecting the intestinal tract. Advances in this field that rely on human studies have been slow and seriously restricted by practical and logistic reasons. As a consequence, well-characterized animal models of intestinal fibrosis have emerged as logical and essential systems to better define and understand the pathophysiology of fibrosis. In point of fact, animal models allow the execution of mechanistic studies as well as the implementation of clinical trials with novel, pathophysiology-based therapeutic approaches. This review provides an overview of the currently available animal models of intestinal fibrosis, taking into consideration the methods of induction, key characteristics of each model, and underlying mechanisms. Currently available models will be classified into seven categories: spontaneous, gene-targeted, chemical-, immune-, bacteria-, and radiation-induced as well as postoperative fibrosis. Each model will be discussed in regard to its potential to create research opportunities to gain insights into the mechanisms of intestinal fibrosis and stricture formation and assist in the development of effective and specific antifibrotic therapies. PMID:22878121

Mouse Models of Diet-Induced Nonalcoholic Steatohepatitis Reproduce the Heterogeneity of the Human Disease

PubMed Central

Machado, Mariana Verdelho; Michelotti, Gregory Alexander; Xie, Guanhua; de Almeida, Thiago Pereira; Boursier, Jerome; Bohnic, Brittany; Guy, Cynthia D.; Diehl, Anna Mae

2015-01-01

Background and aims Non-alcoholic steatohepatitis (NASH), the potentially progressive form of nonalcoholic fatty liver disease (NAFLD), is the pandemic liver disease of our time. Although there are several animal models of NASH, consensus regarding the optimal model is lacking. We aimed to compare features of NASH in the two most widely-used mouse models: methionine-choline deficient (MCD) diet and Western diet. Methods Mice were fed standard chow, MCD diet for 8 weeks, or Western diet (45% energy from fat, predominantly saturated fat, with 0.2% cholesterol, plus drinking water supplemented with fructose and glucose) for 16 weeks. Liver pathology and metabolic profile were compared. Results The metabolic profile associated with human NASH was better mimicked by Western diet. Although hepatic steatosis (i.e., triglyceride accumulation) was also more severe, liver non-esterified fatty acid content was lower than in the MCD diet group. NASH was also less severe and less reproducible in the Western diet model, as evidenced by less liver cell death/apoptosis, inflammation, ductular reaction, and fibrosis. Various mechanisms implicated in human NASH pathogenesis/progression were also less robust in the Western diet model, including oxidative stress, ER stress, autophagy deregulation, and hedgehog pathway activation. Conclusion Feeding mice a Western diet models metabolic perturbations that are common in humans with mild NASH, whereas administration of a MCD diet better models the pathobiological mechanisms that cause human NAFLD to progress to advanced NASH. PMID:26017539

Thalidomide Prevents the Progression of Peritoneal Fibrosis in Mice

PubMed Central

Arai, Hideyuki; Furusu, Akira; Nishino, Tomoya; Obata, Yoko; Nakazawa, Yuka; Nakazawa, Masayuki; Hirose, Misaki; Abe, Katsushige; Koji, Takehiko; Kohno, Shigeru

2011-01-01

Thalidomide is clinically recognized as a therapeutic agent for multiple myeloma and has been known to exert anti-angiogenic actions. Recent studies have suggested the involvement of angiogenesis in the progression of peritoneal fibrosis. The present study investigated the effects of thalidomide on the development of peritoneal fibrosis induced by injection of chlorhexidine gluconate (CG) into the mouse peritoneal cavity every other day for 3 weeks. Thalidomide was given orally every day. Peritoneal tissues were dissected out 21 days after CG injection. Expression of CD31 (as a marker of endothelial cells), proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF), α-smooth muscle actin (as a marker of myofibroblasts), type III collagen and transforming growth factor (TGF)-β was examined using immunohistochemistry. CG group showed thickening of the submesothelial zone and increased numbers of vessels and myofibroblasts. Large numbers of VEGF-, PCNA-, and TGF-β-positive cells were observed in the submesothelial area. Thalidomide treatment significantly ameliorated submesothelial thickening and angiogenesis, and decreased numbers of PCNA- and VEGF-expressing cells, myofibroblasts, and TGF-β-positive cells. Moreover, thalidomide attenuated peritoneal permeability for creatinine, compared to the CG group. Our results indicate the potential utility of thalidomide for preventing peritoneal fibrosis. PMID:21614166

Oxidative Lung Damage Resulting from Repeated Exposure to Radiation and Hyperoxia Associated with Space Exploration.

PubMed

Pietrofesa, Ralph A; Turowski, Jason B; Arguiri, Evguenia; Milovanova, Tatyana N; Solomides, Charalambos C; Thom, Stephen R; Christofidou-Solomidou, Melpo

2013-09-30

Spaceflight missions may require crewmembers to conduct Extravehicular Activities (EVA) for repair, maintenance or scientific purposes. Pre-breathe protocols in preparation for an EVA entail 100% hyperoxia exposure that may last for a few hours (5-8 hours), and may be repeated 2-3 times weekly. Each EVA is associated with additional challenges such as low levels of total body cosmic/galactic radiation exposure that may present a threat to crewmember health and therefore, pose a threat to the success of the mission. We have developed a murine model of combined, hyperoxia and radiation exposure (double-hit) in the context of evaluating countermeasures to oxidative lung damage associated with space flight. In the current study, our objective was to characterize the early and chronic effects of repeated single and double-hit challenge on lung tissue using a novel murine model of repeated exposure to low-level total body radiation and hyperoxia. This is the first study of its kind evaluating lung damage relevant to space exploration in a rodent model. Mouse cohorts (n=5-15/group) were exposed to repeated: a) normoxia; b) >95% O 2 (O 2 ); c) 0.25Gy single fraction gamma radiation (IR); or d) a combination of O 2 and IR (O 2 +IR) given 3 times per week for 4 weeks. Lungs were evaluated for oxidative damage, active TGFβ1 levels, cell apoptosis, inflammation, injury, and fibrosis at 1, 2, 4, 8, 12, 16, and 20 weeks post-initiation of exposure. Mouse cohorts exposed to all challenge conditions displayed decreased bodyweight compared to untreated controls at 4 and 8 weeks post-challenge initiation. Chronic oxidative lung damage to lipids (malondialdehyde levels), DNA (TUNEL, cleaved Caspase 3, cleaved PARP positivity) leading to apoptotic cell death and to proteins (nitrotyrosine levels) was elevated all treatment groups. Importantly, significant systemic oxidative stress was also noted at the late phase in mouse plasma, BAL fluid, and urine. Importantly, however, late oxidative damage across all parameters that we measured was significantly higher than controls in all cohorts but was exacerbated by the combined exposure to O 2 and IR. Additionally, impaired levels of arterial blood oxygenation were noted in all exposure cohorts. Significant but transient elevation of lung tissue fibrosis ( p <0.05), determined by lung hydroxyproline content, was detected as early as 2 week in mice exposed to challenge conditions and persisted for 4-8 weeks only. Interestingly, active TGFβ1 levels in +BAL fluid was also transiently elevated during the exposure time only (1-4 weeks). Inflammation and lung edema/lung injury was also significantly elevated in all groups at both early and late time points, especially the double-hit group. We have characterized significant, early and chronic lung changes consistent with oxidative tissue damage in our murine model of repeated radiation and hyperoxia exposure relevant to space travel. Lung tissue changes, detectable several months after the original exposure, include significant oxidative lung damage (lipid peroxidation, DNA damage and protein nitrosative stress) and increased pulmonary fibrosis. These findings, along with increased oxidative stress in diverse body fluids and the observed decreases in blood oxygenation levels in all challenge conditions (whether single or in combination), lead us to conclude that in our model of repeated exposure to oxidative stressors, chronic tissue changes are detected that persist even months after the exposure to the stressor has ended. This data will provide useful information in the design of countermeasures to tissue oxidative damage associated with space exploration.

Oxidative Lung Damage Resulting from Repeated Exposure to Radiation and Hyperoxia Associated with Space Exploration

PubMed Central

Pietrofesa, Ralph A; Turowski, Jason B; Arguiri, Evguenia; Milovanova, Tatyana N; Solomides, Charalambos C; Thom, Stephen R; Christofidou-Solomidou, Melpo

2013-01-01

Background Spaceflight missions may require crewmembers to conduct Extravehicular Activities (EVA) for repair, maintenance or scientific purposes. Pre-breathe protocols in preparation for an EVA entail 100% hyperoxia exposure that may last for a few hours (5-8 hours), and may be repeated 2-3 times weekly. Each EVA is associated with additional challenges such as low levels of total body cosmic/galactic radiation exposure that may present a threat to crewmember health and therefore, pose a threat to the success of the mission. We have developed a murine model of combined, hyperoxia and radiation exposure (double-hit) in the context of evaluating countermeasures to oxidative lung damage associated with space flight. In the current study, our objective was to characterize the early and chronic effects of repeated single and double-hit challenge on lung tissue using a novel murine model of repeated exposure to low-level total body radiation and hyperoxia. This is the first study of its kind evaluating lung damage relevant to space exploration in a rodent model. Methods Mouse cohorts (n=5-15/group) were exposed to repeated: a) normoxia; b) >95% O2 (O2); c) 0.25Gy single fraction gamma radiation (IR); or d) a combination of O2 and IR (O2+IR) given 3 times per week for 4 weeks. Lungs were evaluated for oxidative damage, active TGFβ1 levels, cell apoptosis, inflammation, injury, and fibrosis at 1, 2, 4, 8, 12, 16, and 20 weeks post-initiation of exposure. Results Mouse cohorts exposed to all challenge conditions displayed decreased bodyweight compared to untreated controls at 4 and 8 weeks post-challenge initiation. Chronic oxidative lung damage to lipids (malondialdehyde levels), DNA (TUNEL, cleaved Caspase 3, cleaved PARP positivity) leading to apoptotic cell death and to proteins (nitrotyrosine levels) was elevated all treatment groups. Importantly, significant systemic oxidative stress was also noted at the late phase in mouse plasma, BAL fluid, and urine. Importantly, however, late oxidative damage across all parameters that we measured was significantly higher than controls in all cohorts but was exacerbated by the combined exposure to O2 and IR. Additionally, impaired levels of arterial blood oxygenation were noted in all exposure cohorts. Significant but transient elevation of lung tissue fibrosis (p<0.05), determined by lung hydroxyproline content, was detected as early as 2 week in mice exposed to challenge conditions and persisted for 4-8 weeks only. Interestingly, active TGFβ1 levels in +BAL fluid was also transiently elevated during the exposure time only (1-4 weeks). Inflammation and lung edema/lung injury was also significantly elevated in all groups at both early and late time points, especially the double-hit group. Conclusion We have characterized significant, early and chronic lung changes consistent with oxidative tissue damage in our murine model of repeated radiation and hyperoxia exposure relevant to space travel. Lung tissue changes, detectable several months after the original exposure, include significant oxidative lung damage (lipid peroxidation, DNA damage and protein nitrosative stress) and increased pulmonary fibrosis. These findings, along with increased oxidative stress in diverse body fluids and the observed decreases in blood oxygenation levels in all challenge conditions (whether single or in combination), lead us to conclude that in our model of repeated exposure to oxidative stressors, chronic tissue changes are detected that persist even months after the exposure to the stressor has ended. This data will provide useful information in the design of countermeasures to tissue oxidative damage associated with space exploration. PMID:24358450

Human Umbilical Cord Mesenchymal Stem Cells Reduce Fibrosis of Bleomycin-Induced Lung Injury

PubMed Central

Moodley, Yuben; Atienza, Daniel; Manuelpillai, Ursula; Samuel, Chrishan S.; Tchongue, Jorge; Ilancheran, Sivakami; Boyd, Richard; Trounson, Alan

2009-01-01

Acute respiratory distress syndrome is characterized by loss of lung tissue as a result of inflammation and fibrosis. Augmenting tissue repair by the use of mesenchymal stem cells may be an important advance in treating this condition. We evaluated the role of term human umbilical cord cells derived from Wharton’s jelly with a phenotype consistent with mesenchymal stem cells (uMSCs) in the treatment of a bleomycin-induced mouse model of lung injury. uMSCs were administered systemically, and lungs were harvested at 7, 14, and 28 days post-bleomycin. Injected uMSCs were located in the lung 2 weeks later only in areas of inflammation and fibrosis but not in healthy lung tissue. The administration of uMSCs reduced inflammation and inhibited the expression of transforming growth factor-β, interferon-γ, and the proinflammatory cytokines macrophage migratory inhibitory factor and tumor necrosis factor-α. Collagen concentration in the lung was significantly reduced by uMSC treatment, which may have been a consequence of the simultaneous reduction in Smad2 phosphorylation (transforming growth factor-β activity). uMSCs also increased matrix metalloproteinase-2 levels and reduced their endogenous inhibitors, tissue inhibitors of matrix metalloproteinases, favoring a pro-degradative milieu following collagen deposition. Notably, injected human lung fibroblasts did not influence either collagen or matrix metalloproteinase levels in the lung. The results of this study suggest that uMSCs have antifibrotic properties and may augment lung repair if used to treat acute respiratory distress syndrome. PMID:19497992

Integrin αvβ6 critically regulates hepatic progenitor cell function and promotes ductular reaction, fibrosis, and tumorigenesis.

PubMed

Peng, Zhen-Wei; Ikenaga, Naoki; Liu, Susan B; Sverdlov, Deanna Y; Vaid, Kahini A; Dixit, Richa; Weinreb, Paul H; Violette, Shelia; Sheppard, Dean; Schuppan, Detlef; Popov, Yury

2016-01-01

Integrin αvβ6 is rapidly up-regulated on cells of epithelial lineage during tissue injury, where one of its primary functions is activation of latent transforming growth factor beta 1 (TGFβ1). In human liver cirrhosis, αvβ6 is overexpressed by cells comprising the ductular reaction, and its inhibition suppresses experimental biliary fibrosis in rodents. Here, we show that αvβ6 is expressed on the actively proliferating subset of hepatic progenitor cells and is required for their progenitor function in vivo and in vitro through integrin αvβ6-dependent TGFβ1 activation. Freshly isolated αvβ6(+) liver cells demonstrate clonogenic potential and differentiate into cholangiocytes and functional hepatocytes in vitro, whereas colony formation by epithelial cell adhesion molecule-positive progenitor cells is blocked by αvβ6-neutralizing antibody and in integrin beta 6-deficient cells. Inhibition of progenitors by anti-αvβ6 antibody is recapitulated by TGFβ1 neutralization and rescued by addition of bioactive TGFβ1. Genetic disruption or selective targeting of αvβ6 with 3G9 antibody potently inhibits progenitor cell responses in mouse models of chronic biliary injury and protects from liver fibrosis and tumorigenesis, two conditions clinically associated with exacerbated ductular reaction. These results suggest that αvβ6 is a promising target for chronic fibrotic liver diseases and associated cancers. © 2015 by the American Association for the Study of Liver Diseases.

Adipose Stem Cell Therapy Mitigates Chronic Pancreatitis via Differentiation into Acinar-like Cells in Mice.

PubMed

Sun, Zhen; Gou, Wenyu; Kim, Do-Sung; Dong, Xiao; Strange, Charlie; Tan, Yu; Adams, David B; Wang, Hongjun

2017-11-01

The objective of this study was to assess the capacity of adipose-derived mesenchymal stem cells (ASCs) to mitigate disease progression in an experimental chronic pancreatitis mouse model. Chronic pancreatitis (CP) was induced in C57BL/6 mice by repeated ethanol and cerulein injection, and mice were then infused with 4 × 10 5 or 1 × 10 6 GFP + ASCs. Pancreas morphology, fibrosis, inflammation, and presence of GFP + ASCs in pancreases were assessed 2 weeks after treatment. We found that ASC infusion attenuated pancreatic damage, preserved pancreas morphology, and reduced pancreatic fibrosis and cell death. GFP + ASCs migrated to pancreas and differentiated into amylase + cells. In further confirmation of the plasticity of ASCs, ASCs co-cultured with acinar cells in a Transwell system differentiated into amylase + cells with increased expression of acinar cell-specific genes including amylase and chymoB1. Furthermore, culture of acinar or pancreatic stellate cell lines in ASC-conditioned medium attenuated ethanol and cerulein-induced pro-inflammatory cytokine production in vitro. Our data show that a single intravenous injection of ASCs ameliorated CP progression, likely by directly differentiating into acinar-like cells and by suppressing inflammation, fibrosis, and pancreatic tissue damage. These results suggest that ASC cell therapy has the potential to be a valuable treatment for patients with pancreatitis. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Cystic fibrosis transmembrane conductance regulator regulates epithelial cell response to Aspergillus and resultant pulmonary inflammation.

PubMed

Chaudhary, Neelkamal; Datta, Kausik; Askin, Frederic B; Staab, Janet F; Marr, Kieren A

2012-02-01

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) alter epithelial cell (EC) interactions with multiple microbes, such that dysregulated inflammation and injury occur with airway colonization in people with cystic fibrosis (CF). Aspergillus fumigatus frequently colonizes CF airways, but it has been assumed to be an innocent saprophyte; its potential role as a cause of lung disease is controversial. To study the interactions between Aspergillus and EC, and the role of the fungus in evoking inflammatory responses. A. fumigatus expressing green fluorescent protein was developed for in vitro and in vivo models, which used cell lines and mouse tracheal EC. Fungal spores (conidia) are rapidly ingested by ECs derived from bronchial cell lines and murine tracheas, supporting a role for EC in early airway clearance. Bronchial ECs harboring CFTR mutations (ΔF508) or deletion demonstrate impaired uptake and killing of conidia, and ECs with CFTR mutation undergo more conidial-induced apoptosis. Germinated (hyphal) forms of the fungus evoke secretion of inflammatory mediators, with CFTR mutation resulting in increased airway levels of macrophage inflammatory protein 2 and KC, and higher lung monocyte chemotactic protein-1. After A. fumigatus inhalation, CFTR(-/-) mice develop exaggerated lymphocytic inflammation, mucin accumulation, and lung injury. Data demonstrate a critical role for CFTR in mediating EC responses to A. fumigatus. Results suggest that the fungus elicits aberrant pulmonary inflammation in the setting of CFTR mutation, supporting the potential role of antifungals to halt progressive CF lung disease.

miR-9-5p suppresses pro-fibrogenic transformation of fibroblasts and prevents organ fibrosis by targeting NOX4 and TGFBR2.

PubMed

Fierro-Fernández, Marta; Busnadiego, Óscar; Sandoval, Pilar; Espinosa-Díez, Cristina; Blanco-Ruiz, Eva; Rodríguez, Macarena; Pian, Héctor; Ramos, Ricardo; López-Cabrera, Manuel; García-Bermejo, Maria Laura; Lamas, Santiago

2015-10-01

Uncontrolled extracellular matrix (ECM) production by fibroblasts in response to injury contributes to fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). Reactive oxygen species (ROS) generation is involved in the pathogenesis of IPF. Transforming growth factor-β1 (TGF-β1) stimulates the production of NADPH oxidase 4 (NOX4)-dependent ROS, promoting lung fibrosis (LF). Dysregulation of microRNAs (miRNAs) has been shown to contribute to LF. To identify miRNAs involved in redox regulation relevant for IPF, we performed arrays in human lung fibroblasts exposed to ROS. miR-9-5p was selected as the best candidate and we demonstrate its inhibitory effect on TGF-β receptor type II (TGFBR2) and NOX4 expression. Increased expression of miR-9-5p abrogates TGF-β1-dependent myofibroblast phenotypic transformation. In the mouse model of bleomycin-induced LF, miR-9-5p dramatically reduces fibrogenesis and inhibition of miR-9-5p and prevents its anti-fibrotic effect both in vitro and in vivo. In lung specimens from patients with IPF, high levels of miR-9-5p are found. In omentum-derived mesothelial cells (MCs) from patients subjected to peritoneal dialysis (PD), miR-9-5p also inhibits mesothelial to myofibroblast transformation. We propose that TGF-β1 induces miR-9-5p expression as a self-limiting homeostatic response. © 2015 The Authors.

Anti-fibrotic efficacy of nintedanib in pulmonary fibrosis via the inhibition of fibrocyte activity.

PubMed

Sato, Seidai; Shinohara, Shintaro; Hayashi, Shinya; Morizumi, Shun; Abe, Shuichi; Okazaki, Hiroyasu; Chen, Yanjuan; Goto, Hisatsugu; Aono, Yoshinori; Ogawa, Hirohisa; Koyama, Kazuya; Nishimura, Haruka; Kawano, Hiroshi; Toyoda, Yuko; Uehara, Hisanori; Nishioka, Yasuhiko

2017-09-15

Nintedanib, a tyrosine kinase inhibitor that is specific for platelet-derived growth factor receptors (PDGFR), fibroblast growth factor receptors (FGFR), and vascular endothelial growth factor receptors (VEGFR), has recently been approved for idiopathic pulmonary fibrosis. Fibrocytes are bone marrow-derived progenitor cells that produce growth factors and contribute to fibrogenesis in the lungs. However, the effects of nintedanib on the functions of fibrocytes remain unclear. Human monocytes were isolated from the peripheral blood of healthy volunteers. The expression of growth factors and their receptors in fibrocytes was analyzed using ELISA and Western blotting. The effects of nintedanib on the ability of fibrocytes to stimulate lung fibroblasts were examined in terms of their proliferation. The direct effects of nintedanib on the differentiation and migration of fibrocytes were also assessed. We investigated whether nintedanib affected the accumulation of fibrocytes in mouse lungs treated with bleomycin. Human fibrocytes produced PDGF, FGF2, and VEGF-A. Nintedanib and specific inhibitors for each growth factor receptor significantly inhibited the proliferation of lung fibroblasts stimulated by the supernatant of fibrocytes. Nintedanib inhibited the migration and differentiation of fibrocytes induced by growth factors in vitro. The number of fibrocytes in the bleomycin-induced lung fibrosis model was reduced by the administration of nintedanib, and this was associated with anti-fibrotic effects. These results support the role of fibrocytes as producers of and responders to growth factors, and suggest that the anti-fibrotic effects of nintedanib are at least partly mediated by suppression of fibrocyte function.

Prevention of diabetic nephropathy in Ins2(+/)⁻(AkitaJ) mice by the mitochondria-targeted therapy MitoQ.

PubMed

Chacko, Balu K; Reily, Colin; Srivastava, Anup; Johnson, Michelle S; Ye, Yaozu; Ulasova, Elena; Agarwal, Anupam; Zinn, Kurt R; Murphy, Michael P; Kalyanaraman, Balaraman; Darley-Usmar, Victor

2010-11-15

Mitochondrial production of ROS (reactive oxygen species) is thought to be associated with the cellular damage resulting from chronic exposure to high glucose in long-term diabetic patients. We hypothesized that a mitochondria-targeted antioxidant would prevent kidney damage in the Ins2(+/)⁻(AkitaJ) mouse model (Akita mice) of Type 1 diabetes. To test this we orally administered a mitochondria-targeted ubiquinone (MitoQ) over a 12-week period and assessed tubular and glomerular function. Fibrosis and pro-fibrotic signalling pathways were determined by immunohistochemical analysis, and mitochondria were isolated from the kidney for functional assessment. MitoQ treatment improved tubular and glomerular function in the Ins2(+/)⁻(AkitaJ) mice. MitoQ did not have a significant effect on plasma creatinine levels, but decreased urinary albumin levels to the same level as non-diabetic controls. Consistent with previous studies, renal mitochondrial function showed no significant change between any of the diabetic or wild-type groups. Importantly, interstitial fibrosis and glomerular damage were significantly reduced in the treated animals. The pro-fibrotic transcription factors phospho-Smad2/3 and β-catenin showed a nuclear accumulation in the Ins2(+/)⁻(AkitaJ) mice, which was prevented by MitoQ treatment. These results support the hypothesis that mitochondrially targeted therapies may be beneficial in the treatment of diabetic nephropathy. They also highlight a relatively unexplored aspect of mitochondrial ROS signalling in the control of fibrosis.

Prevention of diabetic nephropathy in Ins2+/−AkitaJ mice by the mitochondria-targeted therapy MitoQ

PubMed Central

Chacko, Balu K.; Reily, Colin; Srivastava, Anup; Johnson, Michelle S.; Ye, Yaozu; Ulasova, Elena; Agarwal, Anupam; Zinn, Kurt R.; Murphy, Michael P.; Kalyanaraman, Balaraman; Darley-Usmar, Victor

2010-01-01

Mitochondrial production of ROS (reactive oxygen species) is thought to be associated with the cellular damage resulting from chronic exposure to high glucose in long-term diabetic patients. We hypothesized that a mitochondria-targeted antioxidant would prevent kidney damage in the Ins2+/−AkitaJ mouse model (Akita mice) of Type 1 diabetes. To test this we orally administered a mitochondria-targeted ubiquinone (MitoQ) over a 12-week period and assessed tubular and glomerular function. Fibrosis and pro-fibrotic signalling pathways were determined by immunohistochemical analysis, and mitochondria were isolated from the kidney for functional assessment. MitoQ treatment improved tubular and glomerular function in the Ins2+/−AkitaJ mice. MitoQ did not have a significant effect on plasma creatinine levels, but decreased urinary albumin levels to the same level as non-diabetic controls. Consistent with previous studies, renal mitochondrial function showed no significant change between any of the diabetic or wild-type groups. Importantly, interstitial fibrosis and glomerular damage were significantly reduced in the treated animals. The pro-fibrotic transcription factors phospho-Smad2/3 and β-catenin showed a nuclear accumulation in the Ins2+/−AkitaJ mice, which was prevented by MitoQ treatment. These results support the hypothesis that mitochondrially targeted therapies may be beneficial in the treatment of diabetic nephropathy. They also highlight a relatively unexplored aspect of mitochondrial ROS signalling in the control of fibrosis. PMID:20825366

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.

Inflammation and Fibrosis in Perirenal Adipose Tissue of Patients With Aldosterone-Producing Adenoma.

PubMed

Wu, Chunyan; Zhang, Huijian; Zhang, Jiajun; Xie, Cuihua; Fan, Cunxia; Zhang, Hongbin; Wu, Peng; Wei, Qiang; Tan, Wanlong; Xu, Lingling; Wang, Ling; Xue, Yaoming; Guan, Meiping

2018-01-01

The prevalence of primary aldosteronism is much higher than previously thought. Recent studies have shown that primary aldosteronism is related to a higher risk of cardiovascular events. However, the underlying mechanism is not yet clear. Here we investigate the characteristics, including inflammation, fibrosis, and adipokine expression, of adipose tissues from different deposits in patients with aldosterone-producing adenoma (APA). Inflammation and fibrosis changes were evaluated in perirenal and subcutaneous adipose tissues obtained from patients with APA (n = 16), normotension (NT; n = 10), and essential hypertension (EH; n = 5) undergoing laparoscopic surgery. We also evaluated the effect of aldosterone in isolated human perirenal adipose tissue stromal vascular fraction (SVF) cells and investigated the effect of aldosterone in mouse 3T3-L1 and brown preadipocytes. Compared with the EH group, significantly higher levels of interleukin-6 (IL-6) and tumor necrosis factor-α messenger RNA (mRNA) and protein were observed in perirenal adipose tissue of patients with APA. Expression of genes related to fibrosis and adipogenesis in perirenal adipose tissue was notably higher in patients with APA than in patients with NT and EH. Aldosterone significantly induced IL-6 and fibrosis gene mRNA expression in differentiated SVF cells. Aldosterone treatment enhanced mRNA expression of genes associated with inflammation and fibrosis and stimulated differentiation of 3T3-L1 and brown preadipocytes. In conclusion, these data indicate that high aldosterone in patients with APA may induce perirenal adipose tissue dysfunction and lead to inflammation and fibrosis, which may be involved in the high risk of cardiovascular events observed in patients with primary aldosteronism. Copyright © 2018 Endocrine Society.

The role of PTEN in regulation of hepatic macrophages activation and function in progression and reversal of liver fibrosis

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

Cheng, Yahui; Tian, Yuanyao; Xia, Jialu

Activation of Kupffer cells (KCs) plays a pivotal role in the pathogenesis of liver fibrosis. The progression and reversal of CCl{sub 4}-induced mouse liver fibrosis showed a mixed induction of hepatic classical (M1) and alternative (M2) macrophage markers. Although the role of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in modulating myeloid cell activation has recently been identified, its function in macrophage activation during hepatic fibrosis remains to be fully appreciated. In our study, PTEN expression of KCs was remarkably decreased in CCl{sub 4}-induced mice but increased to a near-normal level in reversed mice. Moreover, PTEN was significantlymore » decreased in IL4-induced RAW 264.7 cells in vitro and lower expression of PTEN was observed in M2 macrophages in vivo. In addition, loss- and gain-of-function studies suggested that PTEN regulates M2 macrophages polarization via activation of PI3K/Akt/STAT6 signaling, but had a limited effect on M1 macrophages polarization in vitro. Additionally, Ly294002, a chemical inhibitor of PI3K/Akt, could dramatically down-regulate the hallmarks of M2 macrophages. In conclusion, PTEN mediates macrophages activation by PI3K/Akt/STAT6 signaling pathway, which provides novel compelling evidences on the potential of PTEN in liver injury and opens new cellular target for the pharmacological therapy of liver fibrosis. - Highlights: • CCl{sub 4} treatment triggered a mixed M1/M2 macrophage phenotype in fibrosis. • Lower expression of PTEN in murine M2 macrophages in vivo and vitro. • PTEN modulates M2 macrophages activation via PI3K/Akt/STAT6 signaling. • Provide a new cellular target modulate macrophage mediated hepatic fibrosis.« less

The transport of drug in fibrosis. Comment on "Towards a unified approach in the modeling of fibrosis: A review with research perspectives" by Martine Ben Amar and Carlo Bianca

NASA Astrophysics Data System (ADS)

Ivancevic, Vladimir

2016-07-01

The topic of the review article [1] is the derivation of a multiscale paradigm for the modeling of fibrosis. Firstly, the biological process of the physiological and pathological fibrosis including therapeutical actions is reviewed. Fibrosis can be a consequence of tissue damage, infections and autoimmune diseases, foreign material, tumors. Some questions regarding the pathogenesis, progression and possible regression of fibrosis are lacking. At each scale of observation, different theoretical tools coming from computational, mathematical and physical biology have been proposed. However a complete framework that takes into account the different mechanisms occurring at different scales is still missing. Therefore with the main aim to define a multiscale approach for the modeling of fibrosis, the authors of [1] have presented different top-down and bottom-up approaches that have been developed in the literature. Specifically, their description refers to models for fibrosis diseases based on ordinary and partial differential equation, agents [2], thermostatted kinetic theory [3-5], coarse-grained structures [6-8] and constitutive laws for fibrous collagen networks [9]. A critical analysis has been addressed for all frameworks discussed in the paper. Open problems and future research directions referring to both biological and modeling insight of fibrosis are presented. The paper concludes with the ambitious aim of a multiscale model.

Inflammation and Fibrosis in Polycystic Kidney Disease.

PubMed

Song, Cheng Jack; Zimmerman, Kurt A; Henke, Scott J; Yoder, Bradley K

Polycystic kidney disease (PKD) is a commonly inherited disorder characterized by cyst formation and fibrosis (Wilson, N Engl J Med 350:151-164, 2004) and is caused by mutations in cilia or cilia-related proteins, such as polycystin 1 or 2 (Oh and Katsanis, Development 139:443-448, 2012; Kotsis et al., Nephrol Dial Transplant 28:518-526, 2013). A major pathological feature of PKD is the development of interstitial inflammation and fibrosis with an associated accumulation of inflammatory cells (Grantham, N Engl J Med 359:1477-1485, 2008; Zeier et al., Kidney Int 42:1259-1265, 1992; Ibrahim, Sci World J 7:1757-1767, 2007). It is unclear whether inflammation is a driving force for cyst formation or a consequence of the pathology (Ta et al., Nephrology 18:317-330, 2013) as in some murine models cysts are present prior to the increase in inflammatory cells (Phillips et al., Kidney Blood Press Res 30:129-144, 2007; Takahashi et al., J Am Soc Nephrol JASN 1:980-989, 1991), while in other models the increase in inflammatory cells is present prior to or coincident with cyst initiation (Cowley et al., Kidney Int 43:522-534, 1993, Kidney Int 60:2087-2096, 2001). Additional support for inflammation as an important contributor to cystic kidney disease is the increased expression of many pro-inflammatory cytokines in murine models and human patients with cystic kidney disease (Karihaloo et al., J Am Soc Nephrol JASN 22:1809-1814, 2011; Swenson-Fields et al., Kidney Int, 2013; Li et al., Nat Med 14:863-868, 2008a). Based on these data, an emerging model in the field is that disruption of primary cilia on tubule epithelial cells leads to abnormal cytokine cross talk between the epithelium and the inflammatory cells contributing to cyst growth and fibrosis (Ta et al., Nephrology 18:317-330, 2013). These cytokines are produced by interstitial fibroblasts, inflammatory cells, and tubule epithelial cells and activate multiple pathways including the JAK-STAT and NF-κB signaling (Qin et al., J Am Soc Nephrol JASN 23:1309-1318, 2012; Park et al., Am J Nephrol 32:169-178, 2010; Bhunia et al., Cell 109:157-168, 2002). Indeed, inflammatory cells are responsible for producing several of the pro-fibrotic growth factors observed in PKD patients with fibrosis (Nakamura et al., Am J Nephrol 20:32-36, 2000; Wilson et al., J Cell Physiol 150:360-369, 1992; Song et al., Hum Mol Genet 18:2328-2343, 2009; Schieren et al., Nephrol Dial Transplant 21:1816-1824, 2006). These growth factors trigger epithelial cell proliferation and myofibroblast activation that stimulate the production of extracellular matrix (ECM) genes including collagen types 1 and 3 and fibronectin, leading to reduced glomerular function with approximately 50% of ADPKD patients progressing to end-stage renal disease (ESRD). Therefore, treatments designed to reduce inflammation and slow the rate of fibrosis are becoming important targets that hold promise to improve patient life span and quality of life. In fact, recent studies in several PKD mouse models indicate that depletion of macrophages reduces cyst severity. In this chapter, we review the potential mechanisms of interstitial inflammation in PKD with a focus on ADPKD and discuss the role of interstitial inflammation in progression to fibrosis and ESRD.

A structure-based extracellular matrix expansion mechanism of fibrous tissue growth.

PubMed

Kalson, Nicholas S; Lu, Yinhui; Taylor, Susan H; Starborg, Tobias; Holmes, David F; Kadler, Karl E

2015-05-20

Embryonic growth occurs predominately by an increase in cell number; little is known about growth mechanisms later in development when fibrous tissues account for the bulk of adult vertebrate mass. We present a model for fibrous tissue growth based on 3D-electron microscopy of mouse tendon. We show that the number of collagen fibrils increases during embryonic development and then remains constant during postnatal growth. Embryonic growth was explained predominately by increases in fibril number and length. Postnatal growth arose predominately from increases in fibril length and diameter. A helical crimp structure was established in embryogenesis, and persisted postnatally. The data support a model where the shape and size of tendon is determined by the number and position of embryonic fibroblasts. The collagen fibrils that these cells synthesise provide a template for postnatal growth by structure-based matrix expansion. The model has important implications for growth of other fibrous tissues and fibrosis.

PD-1/PD-L1 Pathway Mediates the Alleviation of Pulmonary Fibrosis by Human Mesenchymal Stem Cells in Humanized Mice.

PubMed

Ni, Ke; Liu, Ming; Zheng, Jian; Wen, Liyan; Chen, Qingyun; Xiang, Zheng; Lam, Kowk-Tai; Liu, Yinping; Chan, Godfrey Chi-Fung; Lau, Yu-Lung; Tu, Wenwei

2018-06-01

Pulmonary fibrosis is a chronic progressive lung disease with few treatments. Human mesenchymal stem cells (MSCs) have been shown to be beneficial in pulmonary fibrosis because they have immunomodulatory capacity. However, there is no reliable model to test the therapeutic effect of human MSCs in vivo. To mimic pulmonary fibrosis in humans, we established a novel bleomycin-induced pulmonary fibrosis model in humanized mice. With this model, the benefit of human MSCs in pulmonary fibrosis and the underlying mechanisms were investigated. In addition, the relevant parameters in patients with pulmonary fibrosis were examined. We demonstrate that human CD8 + T cells were critical for the induction of pulmonary fibrosis in humanized mice. Human MSCs could alleviate pulmonary fibrosis and improve lung function by suppressing bleomycin-induced human T-cell infiltration and proinflammatory cytokine production in the lungs of humanized mice. Importantly, alleviation of pulmonary fibrosis by human MSCs was mediated by the PD-1/programmed death-ligand 1 pathway. Moreover, abnormal PD-1 expression was found in circulating T cells and lung tissues of patients with pulmonary fibrosis. Our study supports the potential benefit of targeting the PD-1/programmed death-ligand 1 pathway in the treatment of pulmonary fibrosis.

Rheb/mTORC1 Signaling Promotes Kidney Fibroblast Activation and Fibrosis

PubMed Central

Jiang, Lei; Xu, Lingling; Mao, Junhua; Li, Jianzhong; Fang, Li; Zhou, Yang; Liu, Wei; He, Weichun; Zhao, Allan Zijian

2013-01-01

Ras homolog enriched in brain (Rheb) is a small GTPase that regulates cell growth, differentiation, and survival by upregulating mammalian target of rapamycin complex 1 (mTORC1) signaling. The role of Rheb/mTORC1 signaling in the activation of kidney fibroblasts and the development of kidney fibrosis remains largely unknown. In this study, we found that Rheb/mTORC1 signaling was activated in interstitial myofibroblasts from fibrotic kidneys. Treatment of rat kidney interstitial fibroblasts (NRK-49F cell line) with TGFβ1 also activated Rheb/mTORC1 signaling. Blocking Rheb/mTORC1 signaling with rapamycin or Rheb small interfering RNA abolished TGFβ1-induced fibroblast activation. In a transgenic mouse, ectopic expression of Rheb activated kidney fibroblasts. These Rheb transgenic mice exhibited increased activation of mTORC1 signaling in both kidney tubular and interstitial cells as well as progressive interstitial renal fibrosis; rapamycin inhibited these effects. Similarly, mice with fibroblast-specific deletion of Tsc1, a negative regulator of Rheb, exhibited activated mTORC1 signaling in kidney interstitial fibroblasts and increased renal fibrosis, both of which rapamycin abolished. Taken together, these results suggest that Rheb/mTORC1 signaling promotes the activation of kidney fibroblasts and contributes to the development of interstitial fibrosis, possibly providing a therapeutic target for progressive renal disease. PMID:23661807

Preoperatively staging liver fibrosis using noninvasive method in Hepatitis B virus-infected hepatocellular carcinoma patients

PubMed Central

Gao, Hengyi; Zhu, Feng; Wang, Min; Zhang, Hang; Ye, Dawei; Yang, Jiayin; Jiang, Li; Liu, Chang; Qin, Renyi; Yan, Lunan; Xiao, Guangqin

2017-01-01

Background Advanced liver fibrosis can result in serious complications (even patient’s death) after partial hepatectomy. Preoperatively percutaneous liver biopsy is an invasive and expensive method to assess liver fibrosis. We aim to establish a noninvasive model, on the basis of preoperative biomarkers, to predict liver fibrosis in hepatocellular carcinoma (HCC) patients with hepatitis B virus (HBV) infection. Methods The HBV-infected liver cancer patients who had received hepatectomy were retrospectively and prospectively enrolled in this study. Univariate analysis was used to compare the variables of the patients with mild to moderate liver fibrosis and with severe liver fibrosis. The significant factors were selected into binary logistic regression analysis. Factors determined to be significant were used to establish a noninvasive model. Then the diagnostic accuracy of this novel model was examined based on sensitivity, specificity and area under the receiver-operating characteristic curve (AUC). Results This study included 2,176 HBV-infected HCC patients who had undergone partial hepatectomy (1,682 retrospective subjects and 494 prospective subjects). Regression analysis indicated that total bilirubin and prothrombin time had positive correlation with liver fibrosis. It also demonstrated that blood platelet count and fibrinogen had negative correlation with liver fibrosis. The AUC values of the model based on these four factors for predicting significant fibrosis, advanced fibrosis and cirrhosis were 0.79-0.83, 0.83-0.85 and 0.85-0.88, respectively. Conclusion The results showed that this novel preoperative model was an excellent noninvasive method for assessing liver fibrosis in HBV-infected HCC patients. PMID:28008144

C-X-C Chemokine Receptor Type 4 Plays a Crucial Role in Mediating Oxidative Stress-Induced Podocyte Injury.

PubMed

Mo, Hongyan; Wu, Qinyu; Miao, Jinhua; Luo, Congwei; Hong, Xue; Wang, Yongping; Tang, Lan; Hou, Fan Fan; Liu, Youhua; Zhou, Lili

2017-08-20

Oxidative stress plays a role in mediating podocyte injury and proteinuria. However, the underlying mechanism remains poorly understood. In this study, we investigated the potential role of C-X-C chemokine receptor type 4 (CXCR4), the receptor for stromal cell-derived factor 1α (SDF-1α), in mediating oxidative stress-induced podocyte injury. In mouse model of adriamycin nephropathy (ADR), CXCR4 expression was significantly induced in podocytes as early as 3 days. This was accompanied by an increased upregulation of oxidative stress in podocyte, as demonstrated by malondialdehyde assay, nitrotyrosine staining and secretion of 8-hydroxy-2'-deoxyguanosine in urine, and induction of NOX2 and NOX4, major subunits of NADPH oxidase. CXCR4 was also induced in human kidney biopsies with proteinuric kidney diseases and colocalized with advanced oxidation protein products (AOPPs), an established oxidative stress trigger. Using cultured podocytes and mouse model, we found that AOPPs induced significant loss of podocyte marker Wilms tumor 1 (WT1), nephrin, and podocalyxin, accompanied by upregulation of desmin both in vitro and in vivo. Furthermore, AOPPs worsened proteinuria and aggravated glomerulosclerosis in ADR. These effects were associated with marked activation of SDF-1α/CXCR4 axis in podocytes. Administration of AMD3100, a specific inhibitor of CXCR4, reduced proteinuria and ameliorated podocyte dysfunction and renal fibrosis triggered by AOPPs in mice. In glomerular miniorgan culture, AOPPs also induced CXCR4 expression and downregulated nephrin and WT1. Innovation and Conclusion: These results suggest that chemokine receptor CXCR4 plays a crucial role in mediating oxidative stress-induced podocyte injury, proteinuria, and renal fibrosis. CXCR4 could be a new target for mitigating podocyte injury, proteinuria, and glomerular sclerosis in proteinuric chronic kidney disease. Antioxid. Redox Signal. 27, 345-362.

STAT3 inhibition attenuates the progressive phenotypes of Alport syndrome mouse model.

PubMed

Yokota, Tsubasa; Omachi, Kohei; Suico, Mary Ann; Kamura, Misato; Kojima, Haruka; Fukuda, Ryosuke; Motomura, Keishi; Teramoto, Keisuke; Kaseda, Shota; Kuwazuru, Jun; Takeo, Toru; Nakagata, Naomi; Shuto, Tsuyoshi; Kai, Hirofumi

2018-02-01

Alport syndrome (AS) is a hereditary, progressive nephritis caused by mutation of type IV collagen. Previous studies have shown that activation of signal transducer and activator of transcription 3 (STAT3) exacerbates other renal diseases, but whether STAT3 activation exacerbates AS pathology is still unknown. Here we aim to investigate the involvement of STAT3 in the progression of AS. Phosphorylated STAT3 expression was assessed by immunoblotting analysis of kidneys and glomeruli of an AS mouse model (Col4a5 G5X mutant). To determine the effect of blocking STAT3 signaling, we treated AS mice with the STAT3 inhibitor stattic (10 mg/kg i.p., three times per week for 10 weeks; n = 10). We assessed the renal function [proteinuria, blood urea nitrogen (BUN), serum creatinine] and analyzed the glomerular injury score, fibrosis and inflammatory cell invasion by histological staining. Moreover, we analyzed the gene expression of nephritis-associated molecules. Phosphorylated STAT3 was upregulated in AS kidneys and glomeruli. Treatment with stattic ameliorated the progressive renal dysfunction, such as increased levels of proteinuria, BUN and serum creatinine. Stattic also significantly suppressed the gene expression levels of renal injury markers (Lcn2, Kim-1), pro-inflammatory cytokines (Il-6, KC), pro-fibrotic genes (Tgf-β, Col1a1, α-Sma) and Mmp9. Stattic treatment decreased the renal fibrosis congruently with the decrease of transforming growth factor beta (TGF-β) protein and increase of antifibrosis-associated markers p-Smad1, 5 and 8, which are negative regulators of TGF-β signaling. STAT3 inhibition significantly ameliorated the renal dysfunction in AS mice. Our finding identifies STAT3 as an important regulator in AS progression and provides a promising therapeutic target for AS. © The Author 2017. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

Adiponectin attenuates kidney injury and fibrosis in deoxycorticosterone acetate-salt and angiotensin II induced CKD mice.

PubMed

Tian, Mi; Tang, Li; Wu, Yuanyuan; Beddhu, Srinivasan; Huang, Yufeng

2018-06-06

Adiponectin (ApN) is a multifunctional adipokine. However high, rather than low, concentrations of ApN are unexpectedly found in patients with chronic kidney disease (CKD) via an as yet unknown mechanism and the role of ApN in CKD is unclear. We, herein, investigated the effect of ApN overexpression on the progressive renal injury resulted from deoxycorticosterone acetate-salt (DOCA) and angiotensin II (Ang-II) infusion using a transgenic, inducible ApN-overexpressing mouse model. Three groups of mice (wild type receiving no infusion (WT), WT and cyp1a1 ApN transgenic mice (ApN-Tg) receiving DOCA+Ang-II infusion (WT/DOCA+Ang-II and ApN-Tg/DOCA+Ang-II)) were assigned to receive a normal food containing 0.15% of the transgene inducer indol-3-carbinol (I3C) for 3 weeks. The I3C-induced ApN-Tg/DOCA+Ang-II mice, not the WT or WT/DOCA+Ang-II mice, overexpressing ApN in liver resulted in 3.15-fold increases in circulating ApN than non-transgenic controls. Of note, these transgenic mice receiving DOCA+Ang-II infusion were still hypertensive but had much less albuminuria and glomerular and tubulointerstitial fibrosis, which were associated with ameliorated podocyte injury determined by ameliorated podocyte loss and foot process effacement; and alleviated tubular injury determined by ameliorated mRNA overexpression of KIM-1 and NGAL and mRNA decreases of cubilin and megalin in tubular cells, compared with WT/DOCA+Ang-II mice. In addition, renal production of NF-kB-p65, NAPDH oxidase-2 and p47phox, and MAPK-related cellular proliferation, which were induced in WT/DOCA+Ang-II mice, were markedly reduced in ApN-Tg/DOCA+Ang-II mice. These results indicate that elevated ApN in CKD mouse model is renal protective. Enhancing adiponectin production or signaling may have therapeutic potential for CKD.

Cytokine and Chemokine Expression in Kidneys during Chronic Leptospirosis in Reservoir and Susceptible Animal Models

PubMed Central

Matsui, Mariko; Roche, Louise; Geroult, Sophie; Soupé-Gilbert, Marie-Estelle; Monchy, Didier; Huerre, Michel; Goarant, Cyrille

2016-01-01

Leptospirosis is caused by pathogenic spirochetes of the genus Leptospira. Humans can be infected after exposure to contaminated urine of reservoir animals, usually rodents, regarded as typical asymptomatic carriers of leptospires. In contrast, accidental hosts may present an acute form of leptospirosis with a range of clinical symptoms including the development of Acute Kidney Injury (AKI). Chronic Kidney Disease (CKD) is considered as a possible AKI-residual sequela but little is known about the renal pathophysiology consequent to leptospirosis infection. Herein, we studied the renal morphological alterations in relation with the regulation of inflammatory cytokines and chemokines, comparing two experimental models of chronic leptospirosis, the golden Syrian hamster that survived the infection, becoming carrier of virulent leptospires, and the OF1 mouse, a usual reservoir of the bacteria. Animals were monitored until 28 days after injection with a virulent L. borgpetersenii serogroup Ballum to assess chronic infection. Hamsters developed morphological alterations in the kidneys with tubulointerstitial nephritis and fibrosis. Grading of lesions revealed higher scores in hamsters compared to the slight alterations observed in the mouse kidneys, irrespective of the bacterial load. Interestingly, pro-fibrotic TGF-β was downregulated in mouse kidneys. Moreover, cytokines IL-1β and IL-10, and chemokines MIP-1α/CCL3 and IP-10/CXCL-10 were significantly upregulated in hamster kidneys compared to mice. These results suggest a possible maintenance of inflammatory processes in the hamster kidneys with the infiltration of inflammatory cells in response to bacterial carriage, resulting in alterations of renal tissues. In contrast, lower expression levels in mouse kidneys indicated a better regulation of the inflammatory response and possible resolution processes likely related to resistance mechanisms. PMID:27219334

Longitudinal assessment of mouse renal injury using high-resolution anatomic and magnetization transfer MR imaging.

PubMed

Wang, Feng; Jiang, Rosie; Takahashi, Keiko; Gore, John; Harris, Raymond C; Takahashi, Takamune; Quarles, C Chad

2014-11-01

The purpose of this study is to evaluate the utility of high-resolution non-invasive endogenous high-field MRI methods for the longitudinal structural and quantitative assessments of mouse kidney disease using the model of unilateral ureter obstruction (UUO). T1-weighted, T2-weighted and magnetization transfer (MT) imaging protocols were optimized to improve the regional contrast in mouse kidney. Conventional T1 and T2 weighted images were collected in UUO mice on day 0 (~3h), day 1, day 3 and day 6 after injury, on a 7 T small animal MRI system. Cortical and medullary thickness, corticomedullary contrast and Magnetization Transfer Ratio (MTR) were assessed longitudinally. Masson trichrome staining was used to histologically assess changes in tissue microstructure. Over the course of UUO progression there were significant (p<0.05) changes in thickness of cortex and outer medulla, and regional changes in T2 signal intensity and MTR values. Histological changes included tubular cell death, tubular dilation, urine retention, and interstitial fibrosis, assessed by histology. The MRI measures of renal cortical and medullary atrophy, cortical-medullary differentiation and MTR changes provide an endogenous, non-invasive and quantitative evaluation of renal morphology and tissue composition during UUO progression. Copyright © 2014 Elsevier Inc. All rights reserved.

Chronic CCl4 intoxication causes liver and bone damage similar to the human pathology of hepatic osteodystrophy: a mouse model to analyse the liver-bone axis.

PubMed

Nussler, Andreas K; Wildemann, Britt; Freude, Thomas; Litzka, Christian; Soldo, Petra; Friess, Helmut; Hammad, Seddik; Hengstler, Jan G; Braun, Karl F; Trak-Smayra, Viviane; Godoy, Patricio; Ehnert, Sabrina

2014-04-01

Patients with chronic liver diseases frequently exhibit decreased bone mineral densities (BMD), which is defined as hepatic osteodystrophy (HOD). HOD is a multifactorial disease whose regulatory mechanisms are barely understood. Thus, an early diagnosis and therapy is hardly possible. Therefore, the aim of our study consisted in characterizing a mouse model reflecting the human pathomechanism. Serum samples were collected from patients with chronic liver diseases and 12-week old C57Bl6/N mice after 6-week treatment with carbon tetrachloride (CCl4). Repetitive injections of CCl4 induced liver damage in mice, resembling liver fibrosis in patients, as assessed by serum analysis and histological staining. Although CCl4 did not affect primary osteoblast cultures, μCT analysis revealed significantly decreased BMD, bone volume, trabecular number and thickness in CCl4-treated mice. In both HOD patients and CCl4-treated mice, an altered vitamin D metabolism with decreased CYP27A1, CYP2R1, vitamin D-binding protein GC and increased 7-dehydrocholesterol reductase hepatic gene expression, results in decreased 25-OH vitamin D serum levels. Moreover, both groups exhibit excessively high active transforming growth factor-beta (TGF-β) serum levels, inhibiting osteoblast function in vitro. Summarizing, our mouse model presents possible mediators of HOD, e.g. altered vitamin D metabolism and increased active TGF-β. Liver damage and significant changes in bone structure and mineralization are already visible by μCT analysis after 6 weeks of CCl4 treatment. This fast response and easy transferability makes it an ideal model to investigate specific gene functions in HOD.

Epithelial neoplasia coincides with exacerbated injury and fibrotic response in the lungs of Gprc5a-knockout mice following silica exposure

PubMed Central

Zhong, Shuangshuang; Song, Hongyong; Sun, Beibei; Zhou, Binhua P.; Deng, Jiong; Han, Baohui

2015-01-01

Exposure to crystalline silica is suggested to increase the risk for a variety of lung diseases, including fibrosis and lung cancer. However, epidemiological evidences for the exposure-risk relationship are ambiguous and conflicting, and experimental study from a reliable animal model to explore the relationship is lacking. We reasoned that a mouse model that is sensitive to both lung injury and tumorigenesis would be appropriate to evaluate the exposure-risk relationship. Previously, we showed that, Gprc5a−/− mice are susceptible to both lung tumorigenesis and endotoxin-induced acute lung injury. In this study, we investigated the biological consequences in Gprc5a−/− mouse model following silica exposure. Intra-tracheal administration of fine silica particles in Gprc5a−/− mice resulted in more severe lung injury and pulmonary inflammation than in wild-type mice. Moreover, an enhanced fibrogenic response, including EMT-like characteristics, was induced in the lungs of Gprc5a−/− mice compared to those from wild-type ones. Importantly, increased hyperplasia or neoplasia coincided with silica-induced tissue injury and fibrogenic response in lungs from Gprc5a−/− mice. Consistently, expression of MMP9, TGFβ1 and EGFR was significantly increased in lungs from silica-treated Gprc5a−/− mice compared to those untreated or wild-type ones. These results suggest that, the process of tissue repair coincides with tissue damages; whereas persistent tissue damages leads to abnormal repair or neoplasia. Thus, silica-induced pulmonary inflammation and injury contribute to increased neoplasia development in lungs from Gprc5a−/− mouse model. PMID:26447616

A time for multi-scale modeling of anti-fibrotic therapies. Comment on "Towards a unified approach in the modeling of fibrosis: A review with research perspectives" by Martine Ben Amar and Carlo Bianca

NASA Astrophysics Data System (ADS)

Wu, Min

2016-07-01

The development of anti-fibrotic therapies in diversities of diseases becomes more and more urgent recently, such as in pulmonary, renal and liver fibrosis [1,2], as well as in malignant tumor growths [3]. As reviewed by Ben Amar and Bianca [4], various theoretical, experimental and in-silico models have been developed to understand the fibrosis process, where the implication on therapeutic strategies has also been frequently demonstrated (e.g., [5-7]). In [4], these models are analyzed and sorted according to their approaches, and in the end of [4], a unified multi-scale approach was proposed to understand fibrosis. While one of the major purposes of extensive modeling of fibrosis is to shed light on therapeutic strategies, the theoretical, experimental and in-silico studies of anti-fibrosis therapies should be conducted more intensively.

Regulation of STATs by polycystin-1 and their role in polycystic kidney disease.

PubMed

Weimbs, Thomas; Olsan, Erin E; Talbot, Jeffrey J

2013-04-01

Autosomal-dominant polycystic kidney disease (ADPKD) is a common genetic disease caused by mutations in the gene coding for polycystin-1 (PC1). PC1 can regulate STAT transcription factors by a novel, dual mechanism. STAT3 and STAT6 are aberrantly activated in renal cysts. Genetic and pharmacological approaches to inhibit STAT3 or STAT6 have led to promising results in ADPKD mouse models. Here, we review current findings that lead to a model of PC1 as a key regulator of STAT signaling in renal tubule cells. We discuss how PC1 may orchestrate appropriate epithelial responses to renal injury, and how this system may lead to aberrant STAT activation in ADPKD thereby causing inappropriate activation of tissue repair programs that culminate in renal cyst growth and fibrosis.

Alcoholic Liver Disease: A Mouse Model Reveals Protection by Lactobacillus fermentum

PubMed Central

Barone, Rosario; Rappa, Francesca; Macaluso, Filippo; Caruso Bavisotto, Celeste; Sangiorgi, Claudia; Di Paola, Gaia; Tomasello, Giovanni; Di Felice, Valentina; Marcianò, Vito; Farina, Felicia; Zummo, Giovanni; Conway de Macario, Everly; J.L. Macario, Alberto; Cocchi, Massimo; Cappello, MD, Francesco; Marino Gammazza, Antonella

2016-01-01

Objectives: Alcoholism is one of the most devastating diseases with high incidence, but knowledge of its pathology and treatment is still plagued with gaps mostly because of the inherent limitations of research with patients. We developed an animal model for studying liver histopathology, Hsp (heat-shock protein)-chaperones involvement, and response to treatment. Methods: The system was standardized using mice to which ethanol was orally administered alone or in combination with Lactobacillus fermentum following a precise schedule over time and applying, at predetermined intervals, a battery of techniques (histology, immunohistochemistry, western blotting, real-time PCR, immunoprecipitation, 3-nitrotyrosine labeling) to assess liver pathology (e.g., steatosis, fibrosis), and Hsp60 and iNOS (inducible form of nitric oxide synthase) gene expression and protein levels, and post-translational modifications. Results: Typical ethanol-induced liver pathology occurred and the effect of the probiotic could be reliably monitored. Steatosis score, iNOS levels, and nitrated proteins (e.g., Hsp60) decreased after probiotic intake. Conclusions: We describe a mouse model useful for studying liver disease induced by chronic ethanol intake and for testing pertinent therapeutic agents, e.g., probiotics. We tested L. fermentum, which reduced considerably ethanol-induced tissue damage and deleterious post-translational modifications of the chaperone Hsp60. The model is available to test other agents and probiotics with therapeutic potential in alcoholic liver disease. PMID:26795070

Human dental pulp pluripotent-like stem cells promote wound healing and muscle regeneration.

PubMed

Martínez-Sarrà, Ester; Montori, Sheyla; Gil-Recio, Carlos; Núñez-Toldrà, Raquel; Costamagna, Domiziana; Rotini, Alessio; Atari, Maher; Luttun, Aernout; Sampaolesi, Maurilio

2017-07-27

Dental pulp represents an easily accessible autologous source of adult stem cells. A subset of these cells, named dental pulp pluripotent-like stem cells (DPPSC), shows high plasticity and can undergo multiple population doublings, making DPPSC an appealing tool for tissue repair or maintenance. DPPSC were harvested from the dental pulp of third molars extracted from young patients. Growth factors released by DPPSC were analysed using antibody arrays. Cells were cultured in specific differentiation media and their endothelial, smooth and skeletal muscle differentiation potential was evaluated. The therapeutic potential of DPPSC was tested in a wound healing mouse model and in two genetic mouse models of muscular dystrophy (Scid/mdx and Sgcb-null Rag2-null γc-null). DPPSC secreted several growth factors involved in angiogenesis and extracellular matrix deposition and improved vascularisation in all three murine models. Moreover, DPPSC stimulated re-epithelialisation and ameliorated collagen deposition and organisation in healing wounds. In dystrophic mice, DPPSC engrafted in the skeletal muscle of both dystrophic murine models and showed integration in muscular fibres and vessels. In addition, DPPSC treatment resulted in reduced fibrosis and collagen content, larger cross-sectional area of type II fast-glycolytic fibres and infiltration of higher numbers of proangiogenic CD206 + macrophages. Overall, DPPSC represent a potential source of stem cells to enhance the wound healing process and slow down dystrophic muscle degeneration.

ASK1 Inhibition Halts Disease Progression in Preclinical Models of Pulmonary Arterial Hypertension.

PubMed

Budas, Grant R; Boehm, Mario; Kojonazarov, Baktybek; Viswanathan, Gayathri; Tian, Xia; Veeroju, Swathi; Novoyatleva, Tatyana; Grimminger, Friedrich; Hinojosa-Kirschenbaum, Ford; Ghofrani, Hossein A; Weissmann, Norbert; Seeger, Werner; Liles, John T; Schermuly, Ralph T

2018-02-01

Progression of pulmonary arterial hypertension (PAH) is associated with pathological remodeling of the pulmonary vasculature and the right ventricle (RV). Oxidative stress drives the remodeling process through activation of MAPKs (mitogen-activated protein kinases), which stimulate apoptosis, inflammation, and fibrosis. We investigated whether pharmacological inhibition of the redox-sensitive apical MAPK, ASK1 (apoptosis signal-regulating kinase 1), can halt the progression of pulmonary vascular and RV remodeling. A selective, orally available ASK1 inhibitor, GS-444217, was administered to two preclinical rat models of PAH (monocrotaline and Sugen/hypoxia), a murine model of RV pressure overload induced by pulmonary artery banding, and cellular models. Oral administration of GS-444217 dose dependently reduced pulmonary arterial pressure and reduced RV hypertrophy in PAH models. The therapeutic efficacy of GS-444217 was associated with reduced ASK1 phosphorylation, reduced muscularization of the pulmonary arteries, and reduced fibrotic gene expression in the RV. Importantly, efficacy was observed when GS-444217 was administered to animals with established disease and also directly reduced cardiac fibrosis and improved cardiac function in a model of isolated RV pressure overload. In cellular models, GS-444217 reduced phosphorylation of p38 and JNK (c-Jun N-terminal kinase) induced by adenoviral overexpression of ASK1 in rat cardiomyocytes and reduced activation/migration of primary mouse cardiac fibroblasts and human pulmonary adventitial fibroblasts derived from patients with PAH. ASK1 inhibition reduced pathological remodeling of the pulmonary vasculature and the right ventricle and halted progression of pulmonary hypertension in rodent models. These preclinical data inform the first description of a causal role of ASK1 in PAH disease pathogenesis.

Assessment of Hepatic Fibrosis with the Stiffness of Liver and the Dynamic of Blood in Liver

NASA Astrophysics Data System (ADS)

Chen, Hao; Ye, Lihong; Li, Zhenyan; Jiang, Yi

Cirrhosis affects liver functions, and is a significant public health problem. Early stages of liver fibrosis are difficult to diagnose. The mechanism of fibrosis changing the mechanical properties of the liver tissue and altering the dynamic of blood flow is still unclear. In collaboration with clinicians specialized in hepatic fibrosis, we have developed a mechanical model to integrate our empirical understanding of fibrosis development and connect the fibrosis stage to mechanical properties of tissue and the consequential blood flow pattern changes. We modeled toxin distribution in the liver that leads to tissue damage and collagen deposition. We showed that the excessive collagen forms polygonal patterns, resembling those found in pathology images. Treating the collagen bundles as elastic spring networks, we also showed a nonlinear relationship between liver stiffness and fibrosis stage, which is consistent with experimental observations. We further modeled the stiffness affecting the mechanical properties of the portal veins, resulting in altered blood flow pattern. These results are supported by ultrasound Doppler measurements from hepatic fibrosis patients. These results promise a new noninvasive diagnostic tool for early fibrosis.

Regulatory T Cells Promote β-Catenin–Mediated Epithelium-to-Mesenchyme Transition During Radiation-Induced Pulmonary Fibrosis

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

Xiong, Shanshan; Pan, Xiujie; Xu, Long

Purpose: Radiation-induced pulmonary fibrosis results from thoracic radiation therapy and severely limits radiation therapy approaches. CD4{sup +}CD25{sup +}FoxP3{sup +} regulatory T cells (Tregs) as well as epithelium-to-mesenchyme transition (EMT) cells are involved in pulmonary fibrosis induced by multiple factors. However, the mechanisms of Tregs and EMT cells in irradiation-induced pulmonary fibrosis remain unclear. In the present study, we investigated the influence of Tregs on EMT in radiation-induced pulmonary fibrosis. Methods and Materials: Mice thoraxes were irradiated (20 Gy), and Tregs were depleted by intraperitoneal injection of a monoclonal anti-CD25 antibody 2 hours after irradiation and every 7 days thereafter. Mice were treated onmore » days 3, 7, and 14 and 1, 3, and 6 months post irradiation. The effectiveness of Treg depletion was assayed via flow cytometry. EMT and β-catenin in lung tissues were detected by immunohistochemistry. Tregs isolated from murine spleens were cultured with mouse lung epithelial (MLE) 12 cells, and short interfering RNA (siRNA) knockdown of β-catenin in MLE 12 cells was used to explore the effects of Tregs on EMT and β-catenin via flow cytometry and Western blotting. Results: Anti-CD25 antibody treatment depleted Tregs efficiently, attenuated the process of radiation-induced pulmonary fibrosis, hindered EMT, and reduced β-catenin accumulation in lung epithelial cells in vivo. The coculture of Tregs with irradiated MLE 12 cells showed that Tregs could promote EMT in MLE 12 cells and that the effect of Tregs on EMT was partially abrogated by β-catenin knockdown in vitro. Conclusions: Tregs can promote EMT in accelerating radiation-induced pulmonary fibrosis. This process is partially mediated through β-catenin. Our study suggests a new mechanism for EMT, promoted by Tregs, that accelerates radiation-induced pulmonary fibrosis.« less

Cellular Plasticity in the Diabetic Myocardium

DTIC Science & Technology

2017-09-01

pathogenesis of cardiac dysfunction associated with metabolic disease . 8 Fig. 1: The db/db mouse recapitulates features of human Heart failure with ...patients, hypertensive heart disease is associated with development of interstitial and periarteriolar fibrosis even in the absence of significant cor...increased secretion of structural matrix proteins. The cardiac ECM in metabolic disease Diabetics exhibit a high incidence of heart failure with

[Value of non-invasive models of liver fibrosis in judgment of treatment timing in chronic hepatitis B patients with ALT < 2×upper limit of normal].

PubMed

Zhou, Q Q; Hu, Y B; Zhou, K; Zhang, W W; Li, M H; Dong, P; Di, J G; Hong, L; Du, Q W; Xie, Y; Sun, Q F

2016-09-20

Objective: To investigate the value of non-invasive liver fibrosis models, FIB-4, S index, aspartate aminotransferase to platelet ratio index(APRI), globulin-platelet(GP)model, aspartate aminotransferase/platelet/gamma-glutamyl transpeptidase/alpha-fetoprotein(APGA), and platelet/age/phosphatase/alpha-fetoprotein/aspartate aminotransferase(PAPAS), in the diagnosis of marked liver fibrosis in chronic hepatitis B(CHB)patients with ALT < 2×upper limit of normal(ULN), as well as treatment timing for this population. Methods: A total of 389 CHB patients with ALT < 2×ULN who were admitted to Beijing Ditan Hospital and whose treatment timing was difficult to judge were enrolled. Transdermal liver biopsy was performed to obtain pathological results, and routine serological tests were performed, including routine blood test, serum biochemical parameters, hepatitis B virus(HBV)markers, and HBV DNA. According to liver pathology, the patients were divided into non-marked liver fibrosis group(S < 2)with 324 patients and marked liver fibrosis group(S≥2)with 65 patients. The non-invasive models for predicting liver fibrosis was established with reference to original articles. SPSS 19.0 software was used for statistical analysis, and the receiver operating characteristic(ROC)curve was used to compare the value of different non-invasive models in predicting marked liver fibrosis in this population. Results: All the non-invasive models had a certain diagnostic value for liver fibrosis degree in these patients, and the areas under the ROC curve for APRI, FIB-4, APGA, S index, PAPAS, and GP model were 0.718, 0.691, 0.758, 0.729, 0.673, and 0.691, respectively. APGA had the largest area under the ROC curve(0.758, 95% CI 0.673-0.844), and gamma-glutamyl transpeptidase was significantly positively correlated with liver fibrosis degree. Conclusion: The non-invasive models of liver fibrosis can identify marked liver fibrosis in CHB patients with ALT < 2×ULN in whom it is difficult to judge treatment timing and help to determine treatment timing for them. APGA model has the highest value and can reduce the need for liver biopsy to the certain degree.

A biomarker panel for non-alcoholic steatohepatitis (NASH) and NASH-related fibrosis.

PubMed

Younossi, Zobair M; Page, Sandra; Rafiq, Nila; Birerdinc, Aybike; Stepanova, Maria; Hossain, Noreen; Afendy, Arian; Younoszai, Zahra; Goodman, Zachary; Baranova, Ancha

2011-04-01

Patients with biopsy-proven NASH and especially those with fibrosis are at risk for progressive liver disease, emphasizing the clinical importance of developing non-invasive biomarkers for NASH and NASH-related fibrosis. This study examines the performance of a new biomarker panel for NASH and NASH-related fibrosis with a combination of clinical and laboratory variables. Enrolled patients had biopsy-proven NAFLD. Clinical data, laboratory data, and serum samples were collected at the time of biopsy. Fasting serum was assayed for adiponectin, resistin, glucose, M30, M65, Tissue inhibitor of metalloproteinases-1 (Timp-1), ProCollagen 3 N-terminal peptide (PIIINP), and hyaluronic acid (HA). Regression models predictive of NASH, NASH-related fibrosis, and NASH-related advanced fibrosis were designed and cross-validated. Of the 79 enrolled NAFLD patients, 40 had biopsy-proven NASH and 39 had non-NASH NAFLD. Clinical and laboratory data were from this cohort were used to develop a NAFLD Diagnostic Panel that includes three models (models for NASH, NASH-related fibrosis, and NASH-related advanced fibrosis). The model for predicting NASH includes diabetes, gender, BMI, triglycerides, M30 (apoptosis), and M65-M30 (necrosis) [AUC: 0.81, 95% CI, 0.70-0.89, 300 p value <9E 301 (-06)]. The NASH-related fibrosis prediction model includes the same predictors [AUC: 0.80, 95% CI 0.68-0.88, 307 p value <0.00014]. Finally, the NASH-related advanced fibrosis model includes type 2 diabetes, serum triglycerides, Timp-1, and AST [AUC: 0.81, 95% CI, 0.70-0.89; p value, 0.000062]. This NAFLD Diagnostic Panel based on a clinical and laboratory data has good performance characteristics and is easy to use. This biomarker panel could become useful in the management of patients with NAFLD.

Development of Hepatocellular Carcinoma in a Murine Model of Nonalcoholic Steatohepatitis Induced by Use of a High-Fat/Fructose Diet and Sedentary Lifestyle

PubMed Central

Dowman, Joanna K.; Hopkins, Laurence J.; Reynolds, Gary M.; Nikolaou, Nikolaos; Armstrong, Matthew J.; Shaw, Jean C.; Houlihan, Diarmaid D.; Lalor, Patricia F.; Tomlinson, Jeremy W.; Hübscher, Stefan G.; Newsome, Philip N.

2014-01-01

Obesity is increasingly prevalent, strongly associated with nonalcoholic liver disease, and a risk factor for numerous cancers. Here, we describe the liver-related consequences of long-term diet-induced obesity. Mice were exposed to an extended obesity model comprising a diet high in trans-fats and fructose corn syrup concurrent with a sedentary lifestyle. Livers were assessed histologically using the nonalcoholic fatty liver disease (NAFLD) activity score (Kleiner system). Mice in the American Lifestyle-Induced Obesity Syndrome (ALIOS) model developed features of early nonalcoholic steatohepatitis at 6 months (mean NAFLD activity score = 2.4) and features of more advanced nonalcoholic steatohepatitis at 12 months, including liver inflammation and bridging fibrosis (mean NAFLD activity score = 5.0). Hepatic expression of lipid metabolism and insulin signaling genes were increased in ALIOS mice compared with normal chow-fed mice. Progressive activation of the mouse hepatic stem cell niche in response to ALIOS correlated with steatosis, fibrosis, and inflammation. Hepatocellular neoplasms were observed in 6 of 10 ALIOS mice after 12 months. Tumors displayed cytological atypia, absence of biliary epithelia, loss of reticulin, alteration of normal perivenular glutamine synthetase staining (absent or diffuse), and variable α-fetoprotein expression. Notably, perivascular tumor cells expressed hepatic stem cell markers. These studies indicate an adipogenic lifestyle alone is sufficient for the development of nonalcoholic steatohepatitis, hepatic stem cell activation, and hepatocarcinogenesis in wild-type mice. PMID:24650559

Protease Activated Receptor-2 Contributes to Heart Failure

PubMed Central

Antoniak, Silvio; Sparkenbaugh, Erica M.; Tencati, Michael; Rojas, Mauricio; Mackman, Nigel; Pawlinski, Rafal

2013-01-01

Heart failure is a major clinical problem worldwide. Previous studies have demonstrated an important role for G protein-coupled receptors, including protease-activated receptors (PARs), in the pathology of heart hypertrophy and failure. Activation of PAR-2 on cardiomyocytes has been shown to induce hypertrophic growth in vitro. PAR-2 also contributes to myocardial infarction and heart remodeling after ischemia/reperfusion injury. In this study, we found that PAR-2 induced hypertrophic growth of cultured rat neonatal cardiomyocytes in a MEK1/2 and p38 dependent manner. In addition, PAR-2 activation on mouse cardiomyocytes increased expression of the pro-fibrotic chemokine MCP-1. Furthermore, cardiomyocyte-specific overexpression of PAR-2 in mice induced heart hypertrophy, cardiac fibrosis, inflammation and heart failure. Finally, in a mouse model of myocardial infarction induced by permanent ligation of the left anterior descending coronary artery, PAR-2 deficiency attenuated heart remodeling and improved heart function independently of its contribution to the size of the initial infarct. Taken together, our data indicate that PAR-2 signaling contributes to the pathogenesis of hypertrophy and heart failure. PMID:24312345

Role of glutathione biosynthesis in endothelial dysfunction and fibrosis.

PubMed

Espinosa-Díez, Cristina; Miguel, Verónica; Vallejo, Susana; Sánchez, Francisco J; Sandoval, Elena; Blanco, Eva; Cannata, Pablo; Peiró, Concepción; Sánchez-Ferrer, Carlos F; Lamas, Santiago

2018-04-01

Glutathione (GSH) biosynthesis is essential for cellular redox homeostasis and antioxidant defense. The rate-limiting step requires glutamate-cysteine ligase (GCL), which is composed of the catalytic (GCLc) and the modulatory (GCLm) subunits. To evaluate the contribution of GCLc to endothelial function we generated an endothelial-specific Gclc haplo-insufficient mouse model (Gclc e/+ mice). In murine lung endothelial cells (MLEC) derived from these mice we observed a 50% reduction in GCLc levels compared to lung fibroblasts from the same mice. MLEC obtained from haplo-insufficient mice showed significant reduction in GSH levels as well as increased basal and stimulated ROS levels, reduced phosphorylation of eNOS (Ser 1177) and increased eNOS S-glutathionylation, compared to MLEC from wild type (WT) mice. Studies in mesenteric arteries demonstrated impaired endothelium-dependent vasodilation in Gclc(e/+) male mice, which was corrected by pre-incubation with GSH-ethyl-ester and BH 4 . To study the contribution of endothelial GSH synthesis to renal fibrosis we employed the unilateral ureteral obstruction model in WT and Gclc(e/+) mice. We observed that obstructed kidneys from Gclc(e/+) mice exhibited increased deposition of fibrotic markers and reduced Nrf2 levels. We conclude that the preservation of endothelial GSH biosynthesis is not only critical for endothelial function but also in anti-fibrotic responses. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Lung pathology in response to repeated exposure to Staphylococcus aureus in congenic residual function cystic fibrosis mice does not increase in response to decreased CFTR levels or increased bacterial load.

PubMed

Davidson, Donald J; Webb, Sheila; Teague, Peter; Govan, John R W; Dorin, Julia R

2004-01-01

To establish the role of defects in murine Cftr in the susceptibility to Staphylococcus aureus lung disease using mouse models of cystic fibrosis (CF), congenic or inbred strains. We describe the histopathological analyses of CF mice repeatedly exposed by aerosolisation to a CF isolate of S. aureus, using residual function Cftr mice and compound heterozygotes generated by intercrossing these with Cftr 'null' mice, all congenic on the C57Bl6/N background. We demonstrate that mice congenic on the C57Bl/6 background develop significantly more severe lung pathology than non-CF littermates in response to repeated exposure to the most frequent early CF lung pathogen S. aureus. Furthermore, reducing the level of Cftr by half in compound heterozygote mice does not impact upon disease severity, even in response to an increased bacterial dose. These results are consistent with an airway clearance defect, or abnormal inflammatory response secondary to Cftr mutation. These studies confirm the primary role for Cftr mutation in the development of this lung phenotype. In addition, these results demonstrate that a further 50% decrease in residual wild-type Cftr mRNA levels in this model does not impact the severity of the histopathological response to S. aureus, suggesting a critical threshold level for functional CFTR. Copyright 2004 S. Karger AG, Basel

Simpler score of routine laboratory tests predicts liver fibrosis in patients with chronic hepatitis B.

PubMed

Zhou, Kun; Gao, Chun-Fang; Zhao, Yun-Peng; Liu, Hai-Lin; Zheng, Rui-Dan; Xian, Jian-Chun; Xu, Hong-Tao; Mao, Yi-Min; Zeng, Min-De; Lu, Lun-Gen

2010-09-01

In recent years, a great interest has been dedicated to the development of noninvasive predictive models to substitute liver biopsy for fibrosis assessment and follow-up. Our aim was to provide a simpler model consisting of routine laboratory markers for predicting liver fibrosis in patients chronically infected with hepatitis B virus (HBV) in order to optimize their clinical management. Liver fibrosis was staged in 386 chronic HBV carriers who underwent liver biopsy and routine laboratory testing. Correlations between routine laboratory markers and fibrosis stage were statistically assessed. After logistic regression analysis, a novel predictive model was constructed. This S index was validated in an independent cohort of 146 chronic HBV carriers in comparison to the SLFG model, Fibrometer, Hepascore, Hui model, Forns score and APRI using receiver operating characteristic (ROC) curves. The diagnostic values of each marker panels were better than single routine laboratory markers. The S index consisting of gamma-glutamyltransferase (GGT), platelets (PLT) and albumin (ALB) (S-index: 1000 x GGT/(PLT x ALB(2))) had a higher diagnostic accuracy in predicting degree of fibrosis than any other mathematical model tested. The areas under the ROC curves (AUROC) were 0.812 and 0.890 for predicting significant fibrosis and cirrhosis in the validation cohort, respectively. The S index, a simpler mathematical model consisting of routine laboratory markers predicts significant fibrosis and cirrhosis in patients with chronic HBV infection with a high degree of accuracy, potentially decreasing the need for liver biopsy.

Cystic Fibrosis Associated with Worse Survival After Liver Transplantation.

PubMed

Black, Sylvester M; Woodley, Frederick W; Tumin, Dmitry; Mumtaz, Khalid; Whitson, Bryan A; Tobias, Joseph D; Hayes, Don

2016-04-01

Survival in cystic fibrosis patients after liver transplantation and liver-lung transplantation is not well studied. To discern survival rates after liver transplantation and liver-lung transplantation in patients with and without cystic fibrosis. The United Network for Organ Sharing database was queried from 1987 to 2013. Univariate Cox proportional hazards, multivariate Cox models, and propensity score matching were performed. Liver transplant and liver-lung transplant were performed in 212 and 53 patients with cystic fibrosis, respectively. Univariate Cox proportional hazards regression identified lower survival in cystic fibrosis after liver transplant compared to a reference non-cystic fibrosis liver transplant cohort (HR 1.248; 95 % CI 1.012, 1.541; p = 0.039). Supplementary analysis found graft survival was similar across the 3 recipient categories (log-rank test: χ(2) 2.68; p = 0.262). Multivariate Cox models identified increased mortality hazard among cystic fibrosis patients undergoing liver transplantation (HR 2.439; 95 % CI 1.709, 3.482; p < 0.001) and liver-lung transplantation (HR 2.753; 95 % CI 1.560, 4.861; p < 0.001). Propensity score matching of cystic fibrosis patients undergoing liver transplantation to non-cystic fibrosis controls identified a greater mortality hazard in the cystic fibrosis cohort using a Cox proportional hazards model stratified on matched pairs (HR 3.167; 95 % CI 1.265, 7.929, p = 0.014). Liver transplantation in cystic fibrosis is associated with poorer long-term patient survival compared to non-cystic fibrosis patients, although the difference is not due to graft survival.

Novel non-invasive biological predictive index for liver fibrosis in hepatitis C virus genotype 4 patients

PubMed Central

Khattab, Mahmoud; Sakr, Mohamed Amin; Fattah, Mohamed Abdel; Mousa, Youssef; Soliman, Elwy; Breedy, Ashraf; Fathi, Mona; Gaber, Salwa; Altaweil, Ahmed; Osman, Ashraf; Hassouna, Ahmed; Motawea, Ibrahim

2016-01-01

AIM To investigate the diagnostic ability of a non-invasive biological marker to predict liver fibrosis in hepatitis C genotype 4 patients with high accuracy. METHODS A cohort of 332 patients infected with hepatitis C genotype 4 was included in this cross-sectional study. Fasting plasma glucose, insulin, C-peptide, and angiotensin-converting enzyme serum levels were measured. Insulin resistance was mathematically calculated using the homeostasis model of insulin resistance (HOMA-IR). RESULTS Fibrosis stages were distributed based on Metavir score as follows: F0 = 43, F1 = 136, F2 = 64, F3 = 45 and F4 = 44. Statistical analysis relied upon reclassification of fibrosis stages into mild fibrosis (F0-F) = 179, moderate fibrosis (F2) = 64, and advanced fibrosis (F3-F4) = 89. Univariate analysis indicated that age, log aspartate amino transaminase, log HOMA-IR and log platelet count were independent predictors of liver fibrosis stage (P < 0.0001). A stepwise multivariate discriminant functional analysis was used to drive a discriminative model for liver fibrosis. Our index used cut-off values of ≥ 0.86 and ≤ -0.31 to diagnose advanced and mild fibrosis, respectively, with receiving operating characteristics of 0.91 and 0.88, respectively. The sensitivity, specificity, positive predictive value, negative predictive value and positive likelihood ratio were: 73%, 91%, 75%, 90% and 8.0 respectively for advanced fibrosis, and 67%, 88%, 84%, 70% and 4.9, respectively, for mild fibrosis. CONCLUSION Our predictive model is easily available and reproducible, and predicted liver fibrosis with acceptable accuracy. PMID:27917265

Novel non-invasive biological predictive index for liver fibrosis in hepatitis C virus genotype 4 patients.

PubMed

Khattab, Mahmoud; Sakr, Mohamed Amin; Fattah, Mohamed Abdel; Mousa, Youssef; Soliman, Elwy; Breedy, Ashraf; Fathi, Mona; Gaber, Salwa; Altaweil, Ahmed; Osman, Ashraf; Hassouna, Ahmed; Motawea, Ibrahim

2016-11-18

To investigate the diagnostic ability of a non-invasive biological marker to predict liver fibrosis in hepatitis C genotype 4 patients with high accuracy. A cohort of 332 patients infected with hepatitis C genotype 4 was included in this cross-sectional study. Fasting plasma glucose, insulin, C-peptide, and angiotensin-converting enzyme serum levels were measured. Insulin resistance was mathematically calculated using the homeostasis model of insulin resistance (HOMA-IR). Fibrosis stages were distributed based on Metavir score as follows: F0 = 43, F1 = 136, F2 = 64, F3 = 45 and F4 = 44. Statistical analysis relied upon reclassification of fibrosis stages into mild fibrosis (F0-F) = 179, moderate fibrosis (F2) = 64, and advanced fibrosis (F3-F4) = 89. Univariate analysis indicated that age, log aspartate amino transaminase, log HOMA-IR and log platelet count were independent predictors of liver fibrosis stage ( P < 0.0001). A stepwise multivariate discriminant functional analysis was used to drive a discriminative model for liver fibrosis. Our index used cut-off values of ≥ 0.86 and ≤ -0.31 to diagnose advanced and mild fibrosis, respectively, with receiving operating characteristics of 0.91 and 0.88, respectively. The sensitivity, specificity, positive predictive value, negative predictive value and positive likelihood ratio were: 73%, 91%, 75%, 90% and 8.0 respectively for advanced fibrosis, and 67%, 88%, 84%, 70% and 4.9, respectively, for mild fibrosis. Our predictive model is easily available and reproducible, and predicted liver fibrosis with acceptable accuracy.

Nitrated fatty acids reverse pulmonary fibrosis by dedifferentiating myofibroblasts and promoting collagen uptake by alveolar macrophages

PubMed Central

Reddy, Aravind T.; Lakshmi, Sowmya P.; Zhang, Yingze; Reddy, Raju C.

2014-01-01

Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal disease, thought to be largely transforming growth factor β (TGFβ) driven, for which there is no effective therapy. We assessed the potential benefits in IPF of nitrated fatty acids (NFAs), which are unique endogenous agonists of peroxisome proliferator-activated receptor γ (PPARγ), a nuclear hormone receptor that exhibits wound-healing and antifibrotic properties potentially useful for IPF therapy. We found that pulmonary PPARγ is down-regulated in patients with IPF. In vitro, knockdown or knockout of PPARγ expression in isolated human and mouse lung fibroblasts induced a profibrotic phenotype, whereas treating human fibroblasts with NFAs up-regulated PPARγ and blocked TGFβ signaling and actions. NFAs also converted TGFβ to inactive monomers in cell-free solution, suggesting an additional mechanism through which they may inhibit TGFβ. In vivo, treating mice bearing experimental pulmonary fibrosis with NFAs reduced disease severity. Also, NFAs up-regulated the collagen-targeting factor milk fat globule-EGF factor 8 (MFG-E8), stimulated collagen uptake and degradation by alveolar macrophages, and promoted myofibroblast dedifferentiation. Moreover, treating mice with established pulmonary fibrosis using NFAs reversed their existing myofibroblast differentiation and collagen deposition. These findings raise the prospect of treating IPF with NFAs to halt and perhaps even reverse the progress of IPF.—Reddy, A. T., Lakshmi, S. P., Zhang, Y., Reddy, R. C. Nitrated fatty acids reverse pulmonary fibrosis by dedifferentiating myofibroblasts and promoting collagen uptake by alveolar macrophages. PMID:25252739

Tubule-Derived Wnts Are Required for Fibroblast Activation and Kidney Fibrosis.

PubMed

Zhou, Dong; Fu, Haiyan; Zhang, Lu; Zhang, Ke; Min, Yali; Xiao, Liangxiang; Lin, Lin; Bastacky, Sheldon I; Liu, Youhua

2017-08-01

Cell-cell communication via Wnt ligands is necessary in regulating embryonic development and has been implicated in CKD. Because Wnt ligands are ubiquitously expressed, the exact cellular source of the Wnts involved in CKD remains undefined. To address this issue, we generated two conditional knockout mouse lines in which Wntless (Wls), a dedicated cargo receptor that is obligatory for Wnt secretion, was selectively ablated in tubular epithelial cells or interstitial fibroblasts. Blockade of Wnt secretion by genetic deletion of Wls in renal tubules markedly inhibited myofibroblast activation and reduced renal fibrosis after unilateral ureteral obstruction. This effect associated with decreased activation of β -catenin and downstream gene expression and preserved tubular epithelial integrity. In contrast, fibroblast-specific deletion of Wls exhibited little effect on the severity of renal fibrosis after obstructive or ischemia-reperfusion injury. In vitro , incubation of normal rat kidney fibroblasts with tubule-derived Wnts promoted fibroblast proliferation and activation. Furthermore, compared with kidney specimens from patients without CKD, biopsy specimens from patients with CKD also displayed increased expression of multiple Wnt proteins, predominantly in renal tubular epithelium. These results illustrate that tubule-derived Wnts have an essential role in promoting fibroblast activation and kidney fibrosis via epithelial-mesenchymal communication. Copyright © 2017 by the American Society of Nephrology.

Human germline hedgehog pathway mutations predispose to fatty liver.

PubMed

Guillen-Sacoto, Maria J; Martinez, Ariel F; Abe, Yu; Kruszka, Paul; Weiss, Karin; Everson, Joshua L; Bataller, Ramon; Kleiner, David E; Ward, Jerrold M; Sulik, Kathleen K; Lipinski, Robert J; Solomon, Benjamin D; Muenke, Maximilian

2017-10-01

Non-alcoholic fatty liver disease (NAFLD) is the most common form of liver disease. Activation of hedgehog (Hh) signaling has been implicated in the progression of NAFLD and proposed as a therapeutic target; however, the effects of Hh signaling inhibition have not been studied in humans with germline mutations that affect this pathway. Patients with holoprosencephaly (HPE), a disorder associated with germline mutations disrupting Sonic hedgehog (SHH) signaling, were clinically evaluated for NAFLD. A combined mouse model of Hh signaling attenuation (Gli2 heterozygous null: Gli2 +/- ) and diet-induced NAFLD was used to examine aspects of NAFLD and hepatic gene expression profiles, including molecular markers of hepatic fibrosis and inflammation. Patients with HPE had a higher prevalence of liver steatosis compared to the general population, independent of obesity. Exposure of Gli2 +/- mice to fatty liver-inducing diets resulted in increased liver steatosis compared to wild-type mice. Similar to humans, this effect was independent of obesity in the mutant mice and was associated with decreased expression of pro-fibrotic and pro-inflammatory genes, and increased expression of PPARγ, a potent anti-fibrogenic and anti-inflammatory regulator. Interestingly, tumor suppressors p53 and p16INK4 were found to be downregulated in the Gli2 +/- mice exposed to a high-fat diet. Our results indicate that germline mutations disrupting Hh signaling promotes liver steatosis, independent of obesity, with reduced fibrosis. While Hh signaling inhibition has been associated with a better NAFLD prognosis, further studies are required to evaluate the long-term effects of mutations affecting this pathway. Lay summary: Non-alcoholic fatty liver disease (NAFLD) is characterized by excess fat deposition in the liver predominantly due to high calorie intake and a sedentary lifestyle. NAFLD progression is usually accompanied by activation of the Sonic hedgehog (SHH) pathway leading to fibrous buildup (scar tissue) and inflammation of the liver tissue. For the first time patients with holoprosencephaly, a disease caused by SHH signaling mutations, are shown to have increased liver steatosis independent of obesity. This observation was recapitulated in a mouse model of attenuated SHH signaling that also showed increased liver steatosis but with decreased fibrosis and inflammation. While SHH inhibition is associated with a good NAFLD prognosis, this increase in liver fat accumulation in the context of SHH signaling inhibition must be studied prospectively to evaluate its long-term effects, especially in individuals with Western-type dietary habits. Published by Elsevier B.V.

Radiotherapy induced dermatitis is a strong predictor for late fibrosis in head and neck cancer. The development of a predictive model for late fibrosis.

PubMed

Nevens, Daan; Duprez, Fréderic; Daisne, Jean Francois; Laenen, Annouschka; De Neve, Wilfried; Nuyts, Sandra

2017-02-01

To determine if the severity of radiodermatitis at the end of radio(chemo)therapy (R(C)T) for head and neck cancer (HNC) is a predictive factor for late fibrosis of the neck and to find a model to predict neck fibrosis grade⩾2 (fibrosis RTOG 2-4 ) at 6months following R(C)T for HNC. 161 patients were prospectively included. We correlated radiodermatitis at the end of RCT, age, sex, T/N stage, tumor site, concomitant chemotherapy, upfront neck dissection, neo-adjuvant chemotherapy, accelerated RT, smoking, alcohol consumption, HPV status and the dose prescribed to the elective neck with fibrosis RTOG 2-4 6months after the end of treatment. Radiodermatitis at the end of R(C)T ⩾grade 3 proved to be associated with the incidence of fibrosis RTOG 2-4 at 6months (p<0.01). Furthermore, upfront neck dissection (p<0.01), increasing N stage (p<0.01) and tumor site (p=0.02) are significantly associated in univariate analysis with fibrosis RTOG 2-4 at 6months of follow-up. Upfront neck dissection and radiodermatitis grade⩾3 at the end of R(C)T were identified by our multivariate model. Additionally, increasing N stage was selected as an independent predictor variable. The AUC for this model was 0.92. A model for the prediction of fibrosis RTOG 2-4 following R(C)T for head and neck cancer is presented with an AUC of 0.92. Interestingly, radiodermatitis grade⩾3 at the end of R(C)T is associated with RTOG 2-4 fibrosis at 6months. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Comparison of serological assessments in the diagnosis of liver fibrosis in bile duct ligation mice.

PubMed

Xie, Chengxia; Ma, Bo; Wang, Ning; Wan, Lin

2017-08-01

Liver fibrosis assessment is essential to make a prognosis and to determine the appropriate anti-fibrosis treatment. Non-invasive serum markers are widely studied in patients to assess liver fibrosis due to the limitations of liver biopsy. When using animal models to study the mechanism and intervention of hepatic fibrosis, serum markers might be useful for the continuous assessment of liver fibrosis in individual animals, which could avoid the influence of biological differences between individuals. However, it is unclear whether serum markers can assess hepatic fibrosis in the animal model. In the present study, we evaluated and compared the ability of four serum markers to assess liver fibrosis in bile duct ligation mice. According to the stages of liver fibrosis assessed by pathological changes, mice in this study were divided into five groups (F0, F1, F2, F3, and F4). Subsequently, four serum markers, aspartate aminotransferase-to-alanine aminotransferase ratio (AAR), aspartate aminotransferase-to-platelet ratio index (APRI), fibrosis index based on the 4 factors (FIB-4), and Forns Index, were calculated for each group. Furthermore, the correlations between serum markers and pathological stages and the ability of serological markers to evaluate liver fibrosis were analyzed. AAR, APRI, FIB-4, and Forns Index could significantly distinguish F0-2 from F3-4 mice. APRI, FIB-4, and Forns Index could detect F0-3 from F4 mice. Among these four markers, FIB-4 was the best able to distinguish ≥F2 and ≥F3, with area under the curve values of 0.882 and 0.92, respectively. Forns Index was best for diagnosing F4 with area under the curve value of 0.879. These results demonstrated that serum markers could be used for assessing liver fibrosis in bile duct ligation mice, and therefore, these markers might lead to more accurate diagnostic and therapeutic studies through continuous monitoring in individual animals. Impact statement The assessment of liver fibrosis is essential for making a prognosis and determining the appropriate anti-fibrosis treatment. In studies focusing on the mechanism and treatment of liver fibrosis using animal models, it would be more accurate to continuously evaluate liver fibrosis in a single animal to avoid individual biological differences. Unfortunately, it is difficult to perform continuous assessment through liver biopsy in the most commonly used rodent models. It is unclear whether serum markers, which have been used in hepatic fibrosis patients, could be used in animal models. Our results demonstrate that serum markers could be used for assessing liver fibrosis in bile duct ligation mice. This study might contribute to more accurate diagnostic and therapeutic studies through continuous monitoring in individual animals.

Tauroursodeoxycholic acid (TUDCA) attenuates pressure overload-induced cardiac remodeling by reducing endoplasmic reticulum stress

PubMed Central

Rani, Shilpa; Sreenivasaiah, Pradeep Kumar; Kim, Jin Ock; Lee, Mi Young; Kang, Wan Seok; Kim, Yong Sook; Ahn, Youngkeun; Park, Woo Jin; Cho, Chunghee

2017-01-01

Pressure overload in the heart induces pathological hypertrophy and is associated with cardiac dysfunction. Apoptosis and fibrosis signaling initiated by the endoplasmic reticulum stress (ERS) is known to contribute to these maladaptive effects. The aim of this study was to investigate whether reduction of ERS by a known chemical chaperone, tauroursodeoxycholic acid (TUDCA) can attenuate pressure overload-induced cardiac remodeling in a mouse model of transverse aortic constriction (TAC). Oral administration of TUDCA at a dose of 300 mg/kg body weight (BW) in the TUDCA-TAC group reduced ERS markers (GRP78, p-PERK, and p-eIf2α), compared to the Vehicle (Veh)-TAC group. TUDCA administration, for 4 weeks after TAC significantly reduced cardiac hypertrophy as shown by the reduced heart weight (HW) to BW ratio, and expression of hypertrophic marker genes (ANF, BNP, and α-SKA). Masson's trichrome staining showed that myocardial fibrosis and collagen deposition were also significantly reduced in the TUDCA-TAC group. We also found that TUDCA significantly decreased expression of TGF-β signaling proteins and collagen isoforms. TUDCA administration also reduced cardiac apoptosis and the related proteins in the TUDCA-TAC group. Microarray analysis followed by gene ontology (GO) and pathway analysis demonstrated that extracellular matrix genes responsible for hypertrophy and fibrosis, and mitochondrial genes responsible for apoptosis and fatty acid metabolism were significantly altered in the Veh-TAC group, but the alterations were normalized in the TUDCA-TAC group, suggesting potential of TUDCA in treatment of heart diseases related to pressure-overload. PMID:28426781

Cannabidiol limits Tcell-mediated chronic autoimmune myocarditis: implications to autoimmune disorders and organ transplantation.

PubMed

Lee, Wen-Shin; Erdelyi, Katalin; Matyas, Csaba; Mukhopadhyay, Partha; Varga, Zoltan V; Liaudet, Lucas; Haskó, György; Čiháková, Daniela; Mechoulam, Raphael; Pacher, Pal

2016-01-08

Myocarditis is a major cause of heart failure and sudden cardiac death in young adults and adolescents. Many cases of myocarditis are associated with autoimmune processes in which cardiac myosin is a major autoantigen. Conventional immunosuppressive therapies often provide unsatisfactory results and are associated with adverse toxicities during the treatment of autoimmune myocarditis. Cannabidiol (CBD) is a non-psychoactive constituent of Marijuana which exerts antiinflammatory effects independent from classical cannabinoid receptors. Recently 80 clinical trials have been reported investigating the effects of CBD in various diseases from inflammatory bowel disease to graft-versus-host disease. CBD-based formulations are used for the management of multiple sclerosis in numerous countries, and CBD also received FDA approval for the treatment of refractory childhood epilepsy and glioblastoma multiforme. Herein, using a well-established mouse model of experimental autoimmune myocarditis (EAM) induced by immunization with cardiac myosin emmulsified in adjuvant resulting in T cell-mediated inflammation, cardiomyocyte cell death, fibrosis and myocardial dysfunction, we studied the potential beneficial effects of CBD. EAM was characterized by marked myocardial T cell-infiltration, profound inflammatory response, fibrosis (measured by qRT-PCR, histology and immunohistochemistry analyses) accompanied by marked attenuation of both systolic and diastolic cardiac functions measured with pressure-volume conductance catheter technique. Chronic treatment with CBD largely attenuated the CD3+ and CD4+ mediated inflammatory response and injury, myocardial fibrosis and cardiac dysfunction in mice. CBD may represent a promising novel treatment for management of autoimmune myocarditis and possibly other autoimmune disorders, and organ transplantation.

Cannabidiol Limits T Cell–Mediated Chronic Autoimmune Myocarditis: Implications to Autoimmune Disorders and Organ Transplantation

PubMed Central

Lee, Wen-Shin; Erdelyi, Katalin; Matyas, Csaba; Mukhopadhyay, Partha; Varga, Zoltan V; Liaudet, Lucas; Hask’, György; ’iháková, Daniela; Mechoulam, Raphael; Pacher, Pal

2016-01-01

Myocarditis is a major cause of heart failure and sudden cardiac death in young adults and adolescents. Many cases of myocarditis are associated with autoimmune processes in which cardiac myosin is a major autoantigen. Conventional immunosuppressive therapies often provide unsatisfactory results and are associated with adverse toxicities during the treatment of autoimmune myocarditis. Cannabidiol (CBD) is a nonpsychoactive constituent of marijuana that exerts antiinflammatory effects independent of classical cannabinoid receptors. Recently, 80 clinical trials have investigated the effects of CBD in various diseases from inflammatory bowel disease to graft versus host disease. CBD-based formulations are used for the management of multiple sclerosis in numerous countries, and CBD also received U.S. Food and Drug Administration approval for the treatment of refractory childhood epilepsy and glioblastoma multiforme. Herein, using a well-established mouse model of experimental autoimmune myocarditis (EAM) induced by immunization with cardiac myosin emmulsified in adjuvant resulting in T cell–mediated inflammation, cardiomyocyte cell death, fibrosis and myocardial dysfunction, we studied the potential beneficial effects of CBD. EAM was characterized by marked myocardial T-cell infiltration, profound inflammatory response and fibrosis (measured by quantitative real-time polymerase chain reaction, histology and immunohistochemistry analyses) accompanied by marked attenuation of both systolic and diastolic cardiac functions measured with a pressure-volume conductance catheter technique. Chronic treatment with CBD largely attenuated the CD3+ and CD4+ T cell–mediated inflammatory response and injury, myocardial fibrosis and cardiac dysfunction in mice. In conclusion, CBD may represent a promising novel treatment for managing autoimmune myocarditis and possibly other autoimmune disorders and organ transplantation. PMID:26772776

Cystic Fibrosis Transmembrane Conductance Regulator Regulates Epithelial Cell Response to Aspergillus and Resultant Pulmonary Inflammation

PubMed Central

Chaudhary, Neelkamal; Datta, Kausik; Askin, Frederic B.; Staab, Janet F.

2012-01-01

Rationale: Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) alter epithelial cell (EC) interactions with multiple microbes, such that dysregulated inflammation and injury occur with airway colonization in people with cystic fibrosis (CF). Aspergillus fumigatus frequently colonizes CF airways, but it has been assumed to be an innocent saprophyte; its potential role as a cause of lung disease is controversial. Objectives: To study the interactions between Aspergillus and EC, and the role of the fungus in evoking inflammatory responses. Methods: A. fumigatus expressing green fluorescent protein was developed for in vitro and in vivo models, which used cell lines and mouse tracheal EC. Measurements and Main Results: Fungal spores (conidia) are rapidly ingested by ECs derived from bronchial cell lines and murine tracheas, supporting a role for EC in early airway clearance. Bronchial ECs harboring CFTR mutations (ΔF508) or deletion demonstrate impaired uptake and killing of conidia, and ECs with CFTR mutation undergo more conidial-induced apoptosis. Germinated (hyphal) forms of the fungus evoke secretion of inflammatory mediators, with CFTR mutation resulting in increased airway levels of macrophage inflammatory protein 2 and KC, and higher lung monocyte chemotactic protein-1. After A. fumigatus inhalation, CFTR−/− mice develop exaggerated lymphocytic inflammation, mucin accumulation, and lung injury. Conclusions: Data demonstrate a critical role for CFTR in mediating EC responses to A. fumigatus. Results suggest that the fungus elicits aberrant pulmonary inflammation in the setting of CFTR mutation, supporting the potential role of antifungals to halt progressive CF lung disease. PMID:22135344

Vitamin B5 and N-acetylcysteine in nonalcoholic steatohepatitis: a pre-clinical study in a dietary mouse model

PubMed Central

Machado, Mariana Verdelho; Kruger, Leandi; Jewell, Mark L.; Michelotti, Gregory Alexander; de Almeida Pereira, Thiago; Xie, Guanhua; Moylan, Cynthia A.; Diehl, Anna Mae

2015-01-01

Background Nonalcoholic fatty liver disease (NAFLD) is the number one cause of chronic liver disease and second indication for liver transplantation in the Western world. Effective therapy is still not available. Previously we showed a critical role for caspase-2 in the pathogenesis of nonalcoholic steatohepatitis (NASH), the potentially progressive form of NAFLD. An imbalance between free Coenzyme A (CoA) and acyl-CoA ratio is known to induce caspase-2 activation. Objectives We aimed to evaluate CoA metabolism and the effects of supplementation with CoA precursors, pantothenate and cysteine, in mouse models of NASH. Methods CoA metabolism was evaluated in methionine-choline deficient (MCD) and Western diet mouse models of NASH. MCD-diet fed mice were treated with pantothenate and N-acetylcysteine or placebo to determine effects on NASH. Results Liver free CoA content was reduced, pantothenate kinase (PANK), the rate-limiting enzyme in the CoA biosynthesis pathway, was down-regulated, and CoA degrading enzymes were increased in mice with NASH. Decreased hepatic free CoA content was associated with increased caspase-2 activity, and correlated with worse liver cell apoptosis, inflammation and fibrosis. Treatment with pantothenate and N-acetylcysteine did not inhibit caspase-2 activation, improve NASH, normalize PANK expression, or restore free CoA levels in MCD diet-fed mice. Conclusion In mice with NASH, hepatic CoA metabolism is impaired, leading to decreased free CoA content, activation of caspase-2, and increased liver cell apoptosis. Dietary supplementation with CoA precursors did not restore CoA levels or improve NASH, suggesting that alternative approaches are necessary to normalize free CoA during NASH. PMID:26403427

Inflammation-Dependent IL18 Signaling Restricts Hepatocellular Carcinoma Growth by Enhancing the Accumulation and Activity of Tumor-Infiltrating Lymphocytes.

PubMed

Markowitz, Geoffrey J; Yang, Pengyuan; Fu, Jing; Michelotti, Gregory A; Chen, Rui; Sui, Jianhua; Yang, Bin; Qin, Wen-Hao; Zhang, Zheng; Wang, Fu-Sheng; Diehl, Anna Mae; Li, Qi-Jing; Wang, Hongyang; Wang, Xiao-Fan

2016-04-15

Chronic inflammation in liver tissue is an underlying cause of hepatocellular carcinoma. High levels of inflammatory cytokine IL18 in the circulation of patients with hepatocellular carcinoma correlates with poor prognosis. However, conflicting results have been reported for IL18 in hepatocellular carcinoma development and progression. In this study, we used tissue specimens from hepatocellular carcinoma patients and clinically relevant mouse models of hepatocellular carcinoma to evaluate IL18 expression and function. In a mouse model of liver fibrosis that recapitulates a tumor-promoting microenvironment, global deletion of the IL18 receptor IL18R1 enhanced tumor growth and burden. Similarly, in a carcinogen-induced model of liver tumorigenesis, IL18R1 deletion increased tumor burden. Mechanistically, we found that IL18 exerted inflammation-dependent tumor-suppressive effects largely by promoting the differentiation, activity, and survival of tumor-infiltrating T cells. Finally, differences in the expression of IL18 in tumor tissue versus nontumor tissue were more predictive of patient outcome than overall tissue expression. Taken together, our findings resolve a long-standing contradiction regarding a tumor-suppressive role for IL18 in established hepatocellular carcinoma and provide a mechanistic explanation for the complex relationship between its expression pattern and hepatocellular carcinoma prognosis. Cancer Res; 76(8); 2394-405. ©2016 AACR. ©2016 American Association for Cancer Research.

Periostin promotes fibrosis and predicts progression in patients with idiopathic pulmonary fibrosis

PubMed Central

Naik, Payal K.; Bozyk, Paul D.; Bentley, J. Kelley; Popova, Antonia P.; Birch, Carolyn M.; Wilke, Carol A.; Fry, Christopher D.; White, Eric S.; Sisson, Thomas H.; Tayob, Nabihah; Carnemolla, Barbara; Orecchia, Paola; Flaherty, Kevin R.; Hershenson, Marc B.; Murray, Susan; Martinez, Fernando J.

2012-01-01

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease without effective therapeutics. Periostin has been reported to be elevated in IPF patients relative to controls, but its sources and mechanisms of action remain unclear. We confirm excess periostin in lungs of IPF patients and show that IPF fibroblasts produce periostin. Blood was obtained from 54 IPF patients (all but 1 with 48 wk of follow-up). We show that periostin levels predict clinical progression at 48 wk (hazard ratio = 1.47, 95% confidence interval = 1.03–2.10, P < 0.05). Monocytes and fibrocytes are sources of periostin in circulation in IPF patients. Previous studies suggest that periostin may regulate the inflammatory phase of bleomycin-induced lung injury, but periostin effects during the fibroproliferative phase of the disease are unknown. Wild-type and periostin-deficient (periostin−/−) mice were anesthetized and challenged with bleomycin. Wild-type mice were injected with bleomycin and then treated with OC-20 Ab (which blocks periostin and integrin interactions) or control Ab during the fibroproliferative phase of disease, and fibrosis and survival were assessed. Periostin expression was upregulated quickly after treatment with bleomycin and remained elevated. Periostin−/− mice were protected from bleomycin-induced fibrosis. Instillation of OC-20 during the fibroproliferative phase improved survival and limited collagen deposition. Chimeric mouse studies suggest that hematopoietic and structural sources of periostin contribute to lung fibrogenesis. Periostin was upregulated by transforming growth factor-β in lung mesenchymal cells, and periostin promoted extracellular matrix deposition, mesenchymal cell proliferation, and wound closure. Thus periostin plays a vital role in late stages of pulmonary fibrosis and is a potential biomarker for disease progression and a target for therapeutic intervention. PMID:23043074

Plexin C1 deficiency permits synaptotagmin 7–mediated macrophage migration and enhances mammalian lung fibrosis

PubMed Central

Peng, Xueyan; Moore, Meagan; Mathur, Aditi; Zhou, Yang; Sun, Huanxing; Gan, Ye; Herazo-Maya, Jose D.; Kaminski, Naftali; Hu, Xinyuan; Pan, Hongyi; Ryu, Changwan; Osafo-Addo, Awo; Homer, Robert J.; Feghali-Bostwick, Carol; Fares, Wassim H.; Gulati, Mridu; Hu, Buqu; Lee, Chun-Geun; Elias, Jack A.; Herzog, Erica L.

2016-01-01

Pulmonary fibrosis is a progressive and often fatal condition that is believed to be partially orchestrated by macrophages. Mechanisms that control migration of these cells into and within the lung remain undefined. We evaluated the contributions of the semaphorin receptor, plexin C1 (PLXNC1), and the exocytic calcium sensor, synaptotagmin 7 (Syt7), in these processes. We evaluated the role of PLXNC1 in macrophage migration by using Boyden chambers and scratch tests, characterized its contribution to experimentally induced lung fibrosis in mice, and defined the mechanism for our observations. Our findings reveal that relative to control participants, patients with idiopathic pulmonary fibrosis demonstrate excessive monocyte migration and underexpression of PLXNC1 in the lungs and circulation, a finding that is recapitulated in the setting of scleroderma-related interstitial lung disease. Relative to wild type, PLXNC1−/− mouse macrophages are excessively migratory, and PLXNC1−/− mice show exacerbated collagen accumulation in response to either inhaled bleomycin or inducible lung targeted TGF-β1 overexpression. These findings are ameliorated by replacement of PLXNC1 on bone marrow–derived cells or by genetic deletion of Syt7. These data demonstrate the previously unrecognized observation that PLXNC1 deficiency permits Syt7-mediated macrophage migration and enhances mammalian lung fibrosis.—Peng, X., Moore, M., Mathur, A., Zhou, Y., Sun, H., Gan, Y., Herazo-Maya, J. D., Kaminski, N., Hu, X., Pan, H., Ryu, C., Osafo-Addo, A., Homer, R. J., Feghali-Bostwick, C., Fares, W. H., Gulati, M., Hu, B., Lee, C.-G., Elias, J. A., Herzog, E. L. Plexin C1 deficiency permits synaptotagmin 7–mediated macrophage migration and enhances mammalian lung fibrosis. PMID:27609773

Lentiviral gene transfer regenerates hematopoietic stem cells in a mouse model for Mpl-deficient aplastic anemia.

PubMed

Heckl, Dirk; Wicke, Daniel C; Brugman, Martijn H; Meyer, Johann; Schambach, Axel; Büsche, Guntram; Ballmaier, Matthias; Baum, Christopher; Modlich, Ute

2011-04-07

Thpo/Mpl signaling plays an important role in the maintenance of hematopoietic stem cells (HSCs) in addition to its role in megakaryopoiesis. Patients with inactivating mutations in Mpl develop thrombocytopenia and aplastic anemia because of progressive loss of HSCs. Yet, it is unknown whether this loss of HSCs is an irreversible process. In this study, we used the Mpl knockout (Mpl(-/-)) mouse model and expressed Mpl from newly developed lentiviral vectors specifically in the physiologic Mpl target populations, namely, HSCs and megakaryocytes. After validating lineage-specific expression in vivo using lentiviral eGFP reporter vectors, we performed bone marrow transplantation of transduced Mpl(-/-) bone marrow cells into Mpl(-/-) mice. We show that restoration of Mpl expression from transcriptionally targeted vectors prevents lethal adverse reactions of ectopic Mpl expression, replenishes the HSC pool, restores stem cell properties, and corrects platelet production. In some mice, megakaryocyte counts were atypically high, accompanied by bone neo-formation and marrow fibrosis. Gene-corrected Mpl(-/-) cells had increased long-term repopulating potential, with a marked increase in lineage(-)Sca1(+)cKit(+) cells and early progenitor populations in reconstituted mice. Transcriptome analysis of lineage(-)Sca1(+)cKit(+) cells in Mpl-corrected mice showed functional adjustment of genes involved in HSC self-renewal.

Modeling the Chagas’ disease after stem cell transplantation

NASA Astrophysics Data System (ADS)

Galvão, Viviane; Miranda, José Garcia Vivas

2009-04-01

A recent model for Chagas’ disease after stem cell transplantation is extended for a three-dimensional multi-agent-based model. The computational model includes six different types of autonomous agents: inflammatory cell, fibrosis, cardiomyocyte, proinflammatory cytokine tumor necrosis factor- α, Trypanosoma cruzi, and bone marrow stem cell. Only fibrosis is fixed and the other types of agents can move randomly through the empty spaces using the three-dimensional Moore neighborhood. Bone marrow stem cells can promote apoptosis in inflammatory cells, fibrosis regression and can differentiate in cardiomyocyte. T. cruzi can increase the number of inflammatory cells. Inflammatory cells and tumor necrosis factor- α can increase the quantity of fibrosis. Our results were compared with experimental data giving a fairly fit and they suggest that the inflammatory cells are important for the development of fibrosis.

Induced Autologous Stem Cell Transplantation for Treatment of Rabbit Renal Interstitial Fibrosis

PubMed Central

Ruan, Guang-Ping; Xu, Fan; Li, Zi-An; Zhu, Guang-Xu; Pang, Rong-Qing; Wang, Jin-Xiang; Cai, Xue-Min; He, Jie; Yao, Xiang; Ruan, Guang-Hong; Xu, Xin-Ming; Pan, Xing-Hua

2013-01-01

Introduction Renal interstitial fibrosis (RIF) is a significant cause of end-stage renal failure. The goal of this study was to characterize the distribution of transplanted induced autologous stem cells in a rabbit model of renal interstitial fibrosis and evaluate its therapeutic efficacy for treatment of renal interstitial fibrosis. Methods A rabbit model of renal interstitial fibrosis was established. Autologous fibroblasts were cultured, induced and labeled with green fluorescent protein (GFP). These labeled stem cells were transplanted into the renal artery of model animals at 8 weeks. Results Eight weeks following transplantation of induced autologous stem cells, significant reductions (P < 0.05) were observed in serum creatinine (SCr) (14.8 ± 1.9 mmol/L to 10.1 ± 2.1 mmol/L) and blood urea nitrogen (BUN) (119 ± 22 µmol/L to 97 ± 13 µmol/L), indicating improvement in renal function. Conclusions We successfully established a rabbit model of renal interstitial fibrosis and demonstrated that transplantation of induced autologous stem cells can repair kidney damage within 8 weeks. The repair occurred by both inhibition of further development of renal interstitial fibrosis and partial reversal of pre-existing renal interstitial fibrosis. These beneficial effects lead to the development of normal tissue structure and improved renal function. PMID:24367598

Development of experimental fibrotic liver diseases animal model by Carbon Tetracholoride.

PubMed

Gitiara, Atoosa; Tokhanbigli, Samaneh; Mazhari, Sogol; Baghaei, Kaveh; Hatami, Behzad; Hashemi, Seyed Mahmoud; Asadi Rad, Ali; Moradi, Afshin; Nasiri, Meyam; Zarrabi Ahrabi, Nakisa; Zali, Mohammad Reza

2017-01-01

This study is presenting an effective method of inducing liver fibrosis by CCL4 as a toxin in two different breeds of rat models. Liver fibrosis is a result of inflammation and liver injury caused by wound healing responses which ultimately lead to liver failure. Consequently, after liver fibrosis, the progression will be continued to liver cirrhosis and at the end stage hepatocellular carcinoma (HCC). Many studies have demonstrated that one of the most important causes of liver fibrosis is Non-alcoholic steatohepatitis (NASH). Fibrotic Liver is affected by an excessive accumulation of extracellular matrix (ECM) proteins like collagen and α-SMA. In two different experiments, male Vistar, and Sprague Dawley Rat models ranging from 200±60, corresponding to an age of approximately 10 weeks were utilized in order to induce CCL4 treated liver fibrosis. After 6 weeks of CCL4 injection, different tests have been carried out to verify the liver fibrosis including serum markers such as Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT), molecular tests containing, laminin and α-SMA and also pathological observation by Hematoxylin and eosin staining in both fibrosis and control group. The results of Pathology and Real-time PCR showed that fibrosis was induced much more effectively in Sprague Dawley rat model compared with Wistar rats.

Genetic deletion of keratin 8 corrects the altered bone formation and osteopenia in a mouse model of cystic fibrosis.

PubMed

Le Henaff, Carole; Faria Da Cunha, Mélanie; Hatton, Aurélie; Tondelier, Danielle; Marty, Caroline; Collet, Corinne; Zarka, Mylène; Geoffroy, Valérie; Zatloukal, Kurt; Laplantine, Emmanuel; Edelman, Aleksander; Sermet-Gaudelus, Isabelle; Marie, Pierre J

2016-04-01

Patients with cystic fibrosis (CF) display low bone mass and alterations in bone formation. Mice carrying the F508del genetic mutation in the cystic fibrosis conductance regulator (Cftr) gene display reduced bone formation and decreased bone mass. However, the underlying molecular mechanisms leading to these skeletal defects are unknown, which precludes the development of an efficient anti-osteoporotic therapeutic strategy. Here we report a key role for the intermediate filament protein keratin 8 (Krt8), in the osteoblast dysfunctions in F508del-Cftr mice. We found that murine and human osteoblasts express Cftr and Krt8 at low levels. Genetic studies showed that Krt8 deletion (Krt8(-/-)) in F508del-Cftr mice increased the levels of circulating markers of bone formation, corrected the expression of osteoblast phenotypic genes, promoted trabecular bone formation and improved bone mass and microarchitecture. Mechanistically, Krt8 deletion in F508del-Cftr mice corrected overactive NF-κB signaling and decreased Wnt-β-catenin signaling induced by the F508del-Cftr mutation in osteoblasts. In vitro, treatment with compound 407, which specifically disrupts the Krt8-F508del-Cftr interaction in epithelial cells, corrected the abnormal NF-κB and Wnt-β-catenin signaling and the altered phenotypic gene expression in F508del-Cftr osteoblasts. In vivo, short-term treatment with 407 corrected the altered Wnt-β-catenin signaling and bone formation in F508del-Cftr mice. Collectively, the results show that genetic or pharmacologic targeting of Krt8 leads to correction of osteoblast dysfunctions, altered bone formation and osteopenia in F508del-Cftr mice, providing a therapeutic strategy targeting the Krt8-F508del-CFTR interaction to correct the abnormal bone formation and bone loss in cystic fibrosis. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Lipopolysaccharide promotes pulmonary fibrosis in acute respiratory distress syndrome (ARDS) via lincRNA-p21 induced inhibition of Thy-1 expression.

PubMed

Zhou, Wen-Qin; Wang, Peng; Shao, Qiu-Ping; Wang, Jian

2016-08-01

Acute respiratory distress syndrome (ARDS) is a common clinical disorder characterized by pulmonary edema leading to acute lung damage and arterial hypoxemia. Pulmonary fibrosis is a progressive, fibrotic lung disorder, whose pathogenesis in ARDS remains speculative. LincRNA-p21 was a novel regulator of cell proliferation, apoptosis and DNA damage response. This study aims to investigate the effects and mechanism of lincRNA-p21 on pulmonary fibrosis in ARDS. Purified 10 mg/kg LPS was dropped into airways of C57BL/6 mice. Expression levels of lincRNA-p21 and Thy-1 were measured by real-time PCR or western blotting. Proliferation of lung fibroblasts was analyzed by BrdU incorporation assay. Lung and BAL collagen contents were estimated using colorimetric Sircol assay. LincRNA-p21 expression was time-dependently increased and Thy-1 expression was time-dependently reduced in a mouse model of ARDS and in LPS-treated lung fibroblasts. Meanwhile, lung fibroblast proliferation was also time-dependently elevated in LPS-treated lung fibroblasts. In addition, lung fibroblast proliferation could be promoted by lincRNA-p21 overexpression and LPS treatment, however, the elevated lung fibroblast proliferation was further abrogated by Thy-1 overexpression or lincRNA-p21 interference. And Thy-1 interference could elevate cell viability of lung fibroblasts and rescue the reduction of lung fibroblast proliferation induced by lincRNA-p21 interference. Moreover, lincRNA-p21 overexpression dramatically inhibited acetylation of H3 and H4 at the Thy-1 promoter and Thy-1 expression levels in HLF1 cells. Finally, lincRNA-p21 interference rescued LPS-induced increase of lung and BAL collagen contents. LincRNA-p21 could lead to pulmonary fibrosis in ARDS by inhibition of the expression of Thy-1.

Genetically manipulated mouse models of lung disease: potential and pitfalls

PubMed Central

Choi, Alexander J. S.; Owen, Caroline A.; Choi, Augustine M. K.

2012-01-01

Gene targeting in mice (transgenic and knockout) has provided investigators with an unparalleled armamentarium in recent decades to dissect the cellular and molecular basis of critical pathophysiological states. Fruitful information has been derived from studies using these genetically engineered mice with significant impact on our understanding, not only of specific biological processes spanning cell proliferation to cell death, but also of critical molecular events involved in the pathogenesis of human disease. This review will focus on the use of gene-targeted mice to study various models of lung disease including airways diseases such as asthma and chronic obstructive pulmonary disease, and parenchymal lung diseases including idiopathic pulmonary fibrosis, pulmonary hypertension, pneumonia, and acute lung injury. We will attempt to review the current technological approaches of generating gene-targeted mice and the enormous dataset derived from these studies, providing a template for lung investigators. PMID:22198907

Utility of texture analysis for quantifying hepatic fibrosis on proton density MRI.

PubMed

Yu, HeiShun; Buch, Karen; Li, Baojun; O'Brien, Michael; Soto, Jorge; Jara, Hernan; Anderson, Stephan W

2015-11-01

To evaluate the potential utility of texture analysis of proton density maps for quantifying hepatic fibrosis in a murine model of hepatic fibrosis. Following Institutional Animal Care and Use Committee (IACUC) approval, a dietary model of hepatic fibrosis was used and 15 ex vivo murine liver tissues were examined. All images were acquired using a 30 mm bore 11.7T magnetic resonance imaging (MRI) scanner with a multiecho spin-echo sequence. A texture analysis was employed extracting multiple texture features including histogram-based, gray-level co-occurrence matrix-based (GLCM), gray-level run-length-based features (GLRL), gray level gradient matrix (GLGM), and Laws' features. Texture features were correlated with histopathologic and digital image analysis of hepatic fibrosis. Histogram features demonstrated very weak to moderate correlations (r = -0.29 to 0.51) with hepatic fibrosis. GLCM features correlation and contrast demonstrated moderate-to-strong correlations (r = -0.71 and 0.59, respectively) with hepatic fibrosis. Moderate correlations were seen between hepatic fibrosis and the GLRL feature short run low gray-level emphasis (SRLGE) (r = -0. 51). GLGM features demonstrate very weak to weak correlations with hepatic fibrosis (r = -0.27 to 0.09). Moderate correlations were seen between hepatic fibrosis and Laws' features L6 and L7 (r = 0.58). This study demonstrates the utility of texture analysis applied to proton density MRI in a murine liver fibrosis model and validates the potential utility of texture-based features for the noninvasive, quantitative assessment of hepatic fibrosis. © 2015 Wiley Periodicals, Inc.

Phosphoproteomic biomarkers predicting histologic nonalcoholic steatohepatitis and fibrosis.

PubMed

Younossi, Zobair M; Baranova, Ancha; Stepanova, Maria; Page, Sandra; Calvert, Valerie S; Afendy, Arian; Goodman, Zachary; Chandhoke, Vikas; Liotta, Lance; Petricoin, Emanuel

2010-06-04

The progression of nonalcoholic fatty liver disease (NAFLD) has been linked to deregulated exchange of the endocrine signaling between adipose and liver tissue. Proteomic assays for the phosphorylation events that characterize the activated or deactivated state of the kinase-driven signaling cascades in visceral adipose tissue (VAT) could shed light on the pathogenesis of nonalcoholic steatohepatitis (NASH) and related fibrosis. Reverse-phase protein microarrays (RPMA) were used to develop biomarkers for NASH and fibrosis using VAT collected from 167 NAFLD patients (training cohort, N = 117; testing cohort, N = 50). Three types of models were developed for NASH and advanced fibrosis: clinical models, proteomics models, and combination models. NASH was predicted by a model that included measurements of two components of the insulin signaling pathway: AKT kinase and insulin receptor substrate 1 (IRS1). The models for fibrosis were less reliable when predictions were based on phosphoproteomic, clinical, or the combination data. The best performing model relied on levels of the phosphorylation of GSK3 as well as on two subunits of cyclic AMP regulated protein kinase A (PKA). Phosphoproteomics technology could potentially be used to provide pathogenic information about NASH and NASH-related fibrosis. This information can lead to a clinically relevant diagnostic/prognostic biomarker for NASH.

Diagnostic accuracy of APRI and FIB-4 for predicting hepatitis B virus-related liver fibrosis accompanied with hepatocellular carcinoma.

PubMed

Xiao, Guangqin; Zhu, Feng; Wang, Min; Zhang, Hang; Ye, Dawei; Yang, Jiayin; Jiang, Li; Liu, Chang; Yan, Lunan; Qin, Renyi

2016-10-01

Aspartate aminotransferase to platelet ratio index (APRI) and the fibrosis index based on four factors (FIB-4) are the two most focused non-invasive models to assess liver fibrosis. We aimed to examine the validity of these two models for predicting hepatitis B virus (HBV)-related liver fibrosis accompanied with hepatocellular carcinoma (HCC). We enrolled HBV-infected patients with liver cancer who had received hepatectomy. The accuracy of APRI and FIB-4 for diagnosing liver fibrosis was assessed based on their sensitivity, specificity, diagnostic efficiency, positive predictive value (PPV), negative predictive value (NPV), kappa (κ) value and area under the receiver-operating characteristic curve (AUC). Finally 2176 patients were included, with 1682 retrospective subjects and 494 prospective subjects. APRI (rs=0.310) and FIB-4 (rs=0.278) were positively correlated with liver fibrosis. And χ(2) analysis demonstrated that APRI and FIB-4 values correlated with different levels of liver fibrosis with all P values less than 0.01. The AUC values for APRI and FIB-4 were 0.685 and 0.626 (P=0.73) for predicting significant fibrosis, 0.681 and 0.648 (P=0.81) for differentiation of advanced fibrosis and 0.676 and 0.652 (P=0.77) for diagnosing cirrhosis. APRI and FIB-4 correlate with liver fibrosis. However these two models have low accuracy for predicting HBV-related liver fibrosis in HCC patients. Copyright © 2016. Published by Elsevier Ltd.

Vascular endothelial growth factor receptor-3 is a novel target to improve net ultrafiltration in methylglyoxal-induced peritoneal injury.

PubMed

Terabayashi, Takeshi; Ito, Yasuhiko; Mizuno, Masashi; Suzuki, Yasuhiro; Kinashi, Hiroshi; Sakata, Fumiko; Tomita, Takako; Iguchi, Daiki; Tawada, Mitsuhiro; Nishio, Ryosuke; Maruyama, Shoichi; Imai, Enyu; Matsuo, Seiichi; Takei, Yoshifumi

2015-09-01

Appropriate fluid balance is important for good clinical outcomes and survival in patients on peritoneal dialysis. We recently reported that lymphangiogenesis associated with fibrosis developed in the peritoneal cavity via the transforming growth factor-β1-vascular endothelial growth factor-C (VEGF-C) pathway. We investigated whether VEGF receptor-3 (VEGFR-3), the receptor for VEGF-C and -D, might be a new target to improve net ultrafiltration by using adenovirus-expressing soluble VEGFR-3 (Adeno-sVEGFR-3) in rodent models of peritoneal injury induced by methylglyoxal (MGO). We demonstrated that lymphangiogenesis developed in these MGO models, especially in the diaphragm, indicating that lymphangiogenesis is a common feature in the peritoneal cavity with inflammation and fibrosis. In MGO models, VEGF-D was significantly increased in the diaphragm; however, VEGF-C was not significantly upregulated. Adeno-sVEGFR-3, which was detected on day 50 after administration via tail vein injections, successfully suppressed lymphangiogenesis in the diaphragm and parietal peritoneum in mouse MGO models without significant effects on fibrosis, inflammation, or neoangiogenesis. Drained volume in the peritoneal equilibration test using a 7.5% icodextrin peritoneal dialysis solution (the 7.5% icodextrin peritoneal equilibration test) was improved by Adeno-sVEGFR-3 on day 22 (P<0.05) and day 50 after reduction of inflammation (P<0.01), indicating that the 7.5% icodextrin peritoneal equilibration test identifies changes in lymphangiogenesis. The solute transport rate was not affected by suppression of lymphangiogenesis. In human peritoneal dialysis patients, the dialysate to plasma ratio of creatinine positively correlated with the dialysate VEGF-D concentration (P<0.001). VEGF-D mRNA was significantly higher in the peritoneal membranes of patients with ultrafiltration failure, indicating that VEGF-D is involved in the development of lymphangiogenesis in peritoneal dialysis patients. These results indicate that VEGFR-3 is a new target to improve net ultrafiltration by suppressing lymphatic absorption and that the 7.5% icodextrin peritoneal equilibration test is useful for estimation of lymphatic absorption.

Experimental models of liver fibrosis.

PubMed

Yanguas, Sara Crespo; Cogliati, Bruno; Willebrords, Joost; Maes, Michaël; Colle, Isabelle; van den Bossche, Bert; de Oliveira, Claudia Pinto Marques Souza; Andraus, Wellington; Alves, Venâncio Avancini Ferreira; Leclercq, Isabelle; Vinken, Mathieu

2016-05-01

Hepatic fibrosis is a wound healing response to insults and as such affects the entire world population. In industrialized countries, the main causes of liver fibrosis include alcohol abuse, chronic hepatitis virus infection and non-alcoholic steatohepatitis. A central event in liver fibrosis is the activation of hepatic stellate cells, which is triggered by a plethora of signaling pathways. Liver fibrosis can progress into more severe stages, known as cirrhosis, when liver acini are substituted by nodules, and further to hepatocellular carcinoma. Considerable efforts are currently devoted to liver fibrosis research, not only with the goal of further elucidating the molecular mechanisms that drive this disease, but equally in view of establishing effective diagnostic and therapeutic strategies. The present paper provides a state-of-the-art overview of in vivo and in vitro models used in the field of experimental liver fibrosis research.

Oxidative Stress in Dilated Cardiomyopathy Caused by MYBPC3 Mutation

PubMed Central

Lynch, Thomas L.; Sivaguru, Mayandi; Velayutham, Murugesan; Cardounel, Arturo J.; Michels, Michelle; Barefield, David; Govindan, Suresh; dos Remedios, Cristobal; van der Velden, Jolanda; Sadayappan, Sakthivel

2015-01-01

Cardiomyopathies can result from mutations in genes encoding sarcomere proteins including MYBPC3, which encodes cardiac myosin binding protein-C (cMyBP-C). However, whether oxidative stress is augmented due to contractile dysfunction and cardiomyocyte damage in MYBPC3-mutated cardiomyopathies has not been elucidated. To determine whether oxidative stress markers were elevated in MYBPC3-mutated cardiomyopathies, a previously characterized 3-month-old mouse model of dilated cardiomyopathy (DCM) expressing a homozygous MYBPC3 mutation (cMyBP-C(t/t)) was used, compared to wild-type (WT) mice. Echocardiography confirmed decreased percentage of fractional shortening in DCM versus WT hearts. Histopathological analysis indicated a significant increase in myocardial disarray and fibrosis while the second harmonic generation imaging revealed disorganized sarcomeric structure and myocyte damage in DCM hearts when compared to WT hearts. Intriguingly, DCM mouse heart homogenates had decreased glutathione (GSH/GSSG) ratio and increased protein carbonyl and lipid malondialdehyde content compared to WT heart homogenates, consistent with elevated oxidative stress. Importantly, a similar result was observed in human cardiomyopathy heart homogenate samples. These results were further supported by reduced signals for mitochondrial semiquinone radicals and Fe-S clusters in DCM mouse hearts measured using electron paramagnetic resonance spectroscopy. In conclusion, we demonstrate elevated oxidative stress in MYPBC3-mutated DCM mice, which may exacerbate the development of heart failure. PMID:26508994

Strain Analysis in the Assessment of a Mouse Model of Cardiotoxicity due to Chemotherapy: Sample for Preclinical Research.

PubMed

Rea, Domenica; Coppola, Carmela; Barbieri, Antonio; Monti, Maria Gaia; Misso, Gabriella; Palma, Giuseppe; Bimonte, Sabrina; Zarone, Mayra Rachele; Luciano, Antonio; Liccardo, Davide; Maiolino, Piera; Cittadini, Antonio; Ciliberto, Gennaro; Arra, Claudio; Maurea, Nicola

2016-01-01

In recent years, the development of more effective anticancer drugs has provided great benefits in patients' quality of life by improving both prognosis and disease-free survival. Nevertheless, the frequency and severity of side-effects, with particular reference to cardiac toxicity, have gained particular attention. The purpose of this study was to create a precise and sensitive preclinical model, able to identify early contractile dysfunction in mice treated with chemotherapy, through use of speckle-tracking echocardiography. We generated a mouse model of cardiotoxicity induced by doxorubicin. C57BL 6 mice were divided into two groups, treated for 7 days by intraperitoneal injections of placebo (vehicle) or doxorubicin (2.17 mg/kg), in order to characterize the cardiac phenotype in vivo. We demonstrated that doxorubicin caused ealy remodeling of the left ventricle: after two days of therapy, the radial, circumferential and strain rates were reduced respectively by 35%, 34%, and 39% (p-value ≤0.001). Moreover, histological analysis revealed that doxorubicin treatment increased fibrosis, cardiomyocyte diameter and apoptosis. In a murine model of doxorubicin-induced cardiac injury, we detected left ventricular dysfunction followed by alterations in conventional echocardiographic indices. Our study suggests that a change in strain could be an effective early marker of myocardial dysfunction for new anticancer treatments and, in preclinical studies, it might also be a valuable indicator for the assessment of activity of cardioprotective agents. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Local CD34-positive capillaries decrease in mouse models of kidney disease associating with the severity of glomerular and tubulointerstitial lesions.

PubMed

Masum, Md Abdul; Ichii, Osamu; Elewa, Yaser Hosny Ali; Nakamura, Teppei; Kon, Yasuhiro

2017-09-04

The renal vasculature plays important roles in both homeostasis and pathology. In this study, we examined pathological changes in the renal microvascular in mouse models of kidney diseases. Glomerular lesions (GLs) in autoimmune disease-prone male BXSB/MpJ-Yaa (Yaa) mice and tubulointerstitial lesions (TILs) in male C57BL/6 mice subjected to unilateral ureteral obstruction (UUO) for 7 days were studied. Collected kidneys were examined using histopathological techniques. A nonparametric Mann-Whitney U test (P < 0.05) was performed to compare healthy controls and the experimental mice. The Kruskal-Wallis test was used to compare three or more groups, and multiple comparisons were performed using Scheffe's method when significant differences were observed (P < 0.05). Yaa mice developed severe autoimmune glomerulonephritis, and the number of CD34 + glomerular capillaries decreased significantly in GLs compared to that in control mice. However, UUO-treated mice showed severe TILs only, and CD34 + tubulointerstitial capillaries were decreased significantly in TILs with the progression of tubulointerstitial fibrosis compared to those in untreated control kidneys. Infiltrations of B-cells, T-cells, and macrophages increased significantly in the respective lesions of both disease models (P < 0.05). In observations of vascular corrosion casts by scanning electron microscopy and of microfil rubber-perfused thick kidney sections by fluorescence microscopy, segmental absences of capillaries were observed in the GLs and TILs of Yaa and UUO-treated mice, respectively. Further, transmission electron microscopy revealed capillary endothelial injury in the respective lesions of both models. The numbers of CD34 + glomerular and tubulointerstitial capillaries were negatively correlated with all examined parameters in GLs (P < 0.05) and TILs (P < 0.01), respectively. From the analysis of mouse models, we identified inverse pathological correlations between the number of local capillaries in GLs and TILs and the severity of kidney diseases.

Effect of Rifaximin on Hepatic Fibrosis in Bile Duct-ligated Rat Model.

PubMed

Shin, Seung Kak; Kwon, Oh Sang; Lee, Jong Joon; Park, Yeon Ho; Choi, Cheol Soo; Jeong, Sung Hwan; Choi, Duck Joo; Kim, Yun Soo; Kim, Ju Hyun

2017-11-25

The translocation of bacteria and their lipopolysaccharides from the gut can promote fibrosis in cirrhotic patients. The aim of this study was to investigate the effects of rifaximin on hepatic fibrosis in a bile duct-ligated rat model. The bile duct ligation (BDL) was carried out for eight days (acute injury model: sham-operated rats [G1], BDL rats [G2], and BDL rats treated with rifaximin [G3]) or 22 days (chronic injury model: sham-operated rats [G4], BDL rats [G5], and BDL rats treated with rifaximin [G6]). Rifaximin (50 mg/kg/day) was administered daily via gavage after BDL. Liver function, serum tumor necrosis factor-alpha (TNF-α), and hepatic hydroxyproline levels were measured. Moreover, a histological analysis of fibrosis contents was performed using sirius red stain. In the acute injury model, the liver function and TNF-α level were not improved after the rifaximin treatment. The hydroxyproline levels (µg/g liver tissue) in G1, G2, and G3 were 236.4±103.1, 444.8±114.4, and 312.5±131.6, respectively; and fibrosis contents (%) were 0.22±0.04, 1.64±0.53, and 1.66±0.44, respectively. The rifaximin treatment did not ameliorate acute BDL-induced fibrosis. In the chronic injury model, the hydroxyproline levels in G4, G5, and G6 were 311.5±72.9, 1,110.3±357.9, and 944.3±209.3, respectively; and fibrosis contents (%) were 0.19±0.03, 5.04±0.18, and 4.42±0.68, respectively (G5 vs. G6, p=0.059). The rifaximin treatment marginally ameliorated chronic BDL-induced fibrosis. Rifaximin did not reduce inflammation and fibrosis in bile duct-ligated rat model.

Adeno-associated virus–targeted disruption of the CFTR gene in cloned ferrets

PubMed Central

Sun, Xingshen; Yan, Ziying; Yi, Yaling; Li, Ziyi; Lei, Diana; Rogers, Christopher S.; Chen, Juan; Zhang, Yulong; Welsh, Michael J.; Leno, Gregory H.; Engelhardt, John F.

2008-01-01

Somatic cell gene targeting combined with nuclear transfer cloning presents tremendous potential for the creation of new, large-animal models of human diseases. Mouse disease models often fail to reproduce human phenotypes, underscoring the need for the generation and study of alternative disease models. Mice deficient for CFTR have been poor models for cystic fibrosis (CF), lacking many aspects of human CF lung disease. In this study, we describe the production of a CFTR gene–deficient model in the domestic ferret using recombinant adeno-associated virus–mediated gene targeting in fibroblasts, followed by nuclear transfer cloning. As part of this approach, we developed a somatic cell rejuvenation protocol using serial nuclear transfer to produce live CFTR-deficient clones from senescent gene-targeted fibroblasts. We transferred 472 reconstructed embryos into 11 recipient jills and obtained 8 healthy male ferret clones heterozygous for a disruption in exon 10 of the CFTR gene. To our knowledge, this study represents the first description of genetically engineered ferrets and describes an approach that may be of substantial utility in modeling not only CF, but also other genetic diseases. PMID:18324338

Gut Microbiome-Based Metagenomic Signature for Non-invasive Detection of Advanced Fibrosis in Human Nonalcoholic Fatty Liver Disease.

PubMed

Loomba, Rohit; Seguritan, Victor; Li, Weizhong; Long, Tao; Klitgord, Niels; Bhatt, Archana; Dulai, Parambir Singh; Caussy, Cyrielle; Bettencourt, Richele; Highlander, Sarah K; Jones, Marcus B; Sirlin, Claude B; Schnabl, Bernd; Brinkac, Lauren; Schork, Nicholas; Chen, Chi-Hua; Brenner, David A; Biggs, William; Yooseph, Shibu; Venter, J Craig; Nelson, Karen E

2017-05-02

The presence of advanced fibrosis in nonalcoholic fatty liver disease (NAFLD) is the most important predictor of liver mortality. There are limited data on the diagnostic accuracy of gut microbiota-derived signature for predicting the presence of advanced fibrosis. In this prospective study, we characterized the gut microbiome compositions using whole-genome shotgun sequencing of DNA extracted from stool samples. This study included 86 uniquely well-characterized patients with biopsy-proven NAFLD, of which 72 had mild/moderate (stage 0-2 fibrosis) NAFLD, and 14 had advanced fibrosis (stage 3 or 4 fibrosis). We identified a set of 40 features (p < 0.006), which included 37 bacterial species that were used to construct a Random Forest classifier model to distinguish mild/moderate NAFLD from advanced fibrosis. The model had a robust diagnostic accuracy (AUC 0.936) for detecting advanced fibrosis. This study provides preliminary evidence for a fecal-microbiome-derived metagenomic signature to detect advanced fibrosis in NAFLD. Copyright © 2017 Elsevier Inc. All rights reserved.

Development of experimental fibrotic liver diseases animal model by Carbon Tetracholoride

PubMed Central

Gitiara, Atoosa; Tokhanbigli, Samaneh; Mazhari, Sogol; Baghaei, Kaveh; Hatami, Behzad; Hashemi, Seyed Mahmoud; Asadi Rad, Ali; Moradi, Afshin; Nasiri, Meyam; Zarrabi Ahrabi, Nakisa; Zali, Mohammad Reza

2017-01-01

Aim: This study is presenting an effective method of inducing liver fibrosis by CCL4 as a toxin in two different breeds of rat models. Background: Liver fibrosis is a result of inflammation and liver injury caused by wound healing responses which ultimately lead to liver failure. Consequently, after liver fibrosis, the progression will be continued to liver cirrhosis and at the end stage hepatocellular carcinoma (HCC). Many studies have demonstrated that one of the most important causes of liver fibrosis is Non-alcoholic steatohepatitis (NASH). Fibrotic Liver is affected by an excessive accumulation of extracellular matrix (ECM) proteins like collagen and α-SMA. Methods: In two different experiments, male Vistar, and Sprague Dawley Rat models ranging from 200±60, corresponding to an age of approximately 10 weeks were utilized in order to induce CCL4 treated liver fibrosis. Results: After 6 weeks of CCL4 injection, different tests have been carried out to verify the liver fibrosis including serum markers such as Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT), molecular tests containing, laminin and α-SMA and also pathological observation by Hematoxylin and eosin staining in both fibrosis and control group. Conclusion: The results of Pathology and Real-time PCR showed that fibrosis was induced much more effectively in Sprague Dawley rat model compared with Wistar rats. PMID:29511482

The Association of Inbreeding With Lung Fibrosis Incidence in Beagle Dogs That Inhaled 238PuO2 or 239PuO2.

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

Wilson, Dulaney A.; Brigantic, Andrea M.; Morgan, William F.

Studies of health effects in animals after exposure to internally deposited radionuclides were intended to supplement observational studies in humans. Both nuclear workers and Beagle dogs have exhibited plutonium associated lung fibrosis; however, the dogs smaller gene pool may limit the applicability of findings to humans. Data on Beagles that inhaled either plutonium-238 dioxide (238PuO2) or plutonium-239 dioxide (239PuO2) were analyzed. Wright's Coefficient of Inbreeding was used to measure genetic or familial susceptibility and was assessed as an explanatory variable when modeling the association between lung fibrosis incidence and plutonium exposure. Lung fibrosis was diagnosed in approximately 80% of themore » exposed dogs compared with 23.7% of the control dogs. The maximum degree of inbreeding was 9.4%. Regardless of isotope, the addition of inbreeding significantly improved the model in female dogs but not in males. In female dogs an increased inbreeding coefficient predicted decreased hazard of a lung fibrosis diagnosis. Lung fibrosis was common in these dogs with inbreeding affecting models of lung fibrosis incidence in females but not in males. The apparent protective effect in females predicted by these models of lung fibrosis incidence is likely to be minimal given the small degree of inbreeding in these groups.« less