Leptospira santorosai Serovar Shermani detergent extract induces an increase in fibronectin production through a Toll-like receptor 2-mediated pathway.

PubMed

Tian, Ya-Chung; Hung, Cheng-Chieh; Li, Yi-Jung; Chen, Yung-Chang; Chang, Ming-Yang; Yen, Tzung-Hai; Hsu, Hsiang-Hao; Wu, Mai-Szu; Phillips, Aled; Yang, Chih-Wei

2011-03-01

Leptospirosis can activate inflammatory responses through Toll-like receptors (TLRs) and may cause renal tubulointerstitial fibrosis characterized by the accumulation of extracellular matrix (ECM). We have previously demonstrated that Leptospira santorosai serovar Shermani detergent extract stimulates ECM accumulation in vitro. The aim of this study was to examine the mechanistic basis of these previous observations and, in particular, to examine the potential involvement of TLRs. The addition of serovar Shermani detergent extract led to an increase in fibronectin gene expression and production. Inhibition of TLR2 but not TLR4 expression abrogated serovar Shermani detergent extract-mediated increases in fibronectin production. This response was also blocked by the knockdown of the gene expression of the TLR2 downstream transducers myeloid differentiation factor 88 (MyD88) and tumor necrosis factor receptor-associated factor 6 (TRAF6). Serovar Shermani detergent extract also activated nuclear factor-κB, and its inhibition by curcumin-attenuated serovar Shermani detergent extract induced increases in fibronectin production. These effects were also mimicked by the specific TLR2 agonist, Pam(3)CsK(4), a response that was also abrogated by the knockdown of MyD88 and TRAF6. Similarly, the administration of live leptospires to cells also induced fibronectin production that was blocked by inhibition of TLR2 and MyD88 expression. In conclusion, serovar Shermani detergent extract can induce fibronectin production through the TLR2-associated cascade, providing evidence of an association between TLRs and leptospirosis-mediated ECM deposition.

A clinicopathological study of the expression of extracellular matrix components in urothelial carcinoma.

PubMed

Ioachim, Elli; Michael, Michalis; Stavropoulos, Nicolaos E; Kitsiou, Evangelia; Salmas, Marios; Malamou-Mitsi, Vasiliki

2005-03-01

To measure the immunohistochemical expression of the extracellular matrix (ECM) components tenascin, fibronectin, collagen type IV and laminin in urothelial carcinomas, and to correlate their expression with clinicopathological features to clarify the prognostic value of these molecules and their role in tumour progression. Tumour specimens obtained during transurethral resection of bladder tumour (TURBT) from 103 patients (82 men and 2 1 women, mean age 66.7 years, range 27-89) were studied retrospectively. The expression of tenascin, fibronectin, collagen type IV and laminin was correlated with clinicopathological features (tumour grade and stage, multiplicity, simultaneous in situ component, the proliferative activity as estimated by the two proliferation associated indices, Ki-67 and proliferating cell nuclear antigen, the recurrence rate, and the progression of invading tumour). Specimens investigated for tenascin expression from patients with superficial bladder cancers were categorized into 28 treated by TURBT only and 53 who had TURBT followed by intravesical instillations of interferon. Cytoplasmic tenascin expression was detected in tumour cells in 20% of specimens. Tenascin was expressed in the tumour stroma in 76% of specimens, and was positively correlated with tumour grade and stage. Stromal tenascin expression was positively correlated with proliferative activity, and with the expression of fibronectin and collagen type IV. Fibronectin was expressed in the tumour stroma in 89% of specimens and was positively correlated with tumour stage, proliferative activity, and expression of collagen type IV and laminin. Collagen type IV was expressed in 93% of specimens, and was positively correlated with tumour grade and stage. Laminin was expressed in 78% of specimens and had no significant correlation with the clinicopathological features. Patients treated with TURBT alone and who had low levels of tenascin had a longer tumour-free interval than those with high levels of tenascin. Levels of tenascin might be valuable for predicting the risk of early recurrence. The expression of tenascin, fibronectin and collagen type IV seems to be correlated with more aggressive tumour behaviour. Furthermore, their interrelationships could indicate that they are involved in the remodelling of bladder cancer tissue, probably influencing tumour progression.

Upregulation of adhesion complex proteins and fibronectin by human keratinocytes treated with an aqueous extract from the leaves of Chromolaena odorata (Eupolin).

PubMed

Phan, T T; Allen, J; Hughes, M A; Cherry, G; Wojnarowska, F

2000-01-01

The fresh leaves and extract of the plant Chromolaena odorata are a traditional herbal treatment in developing countries for burns, soft tissue wounds and skin infections. We have previously shown that the extract had an effect on the growth and proliferation of keratinocytes and fibroblasts in culture. This study has demonstrated that Eupolin extract increased expression of several components of the adhesion complex and fibronectin by human keratinocytes. Using indirect immunofluorescence we found increased expression (dose-dependent) of laminin 5, laminin 1, collagen IV, and fibronectin. The expression of the b1 and b4 integrins was upregulated by the extract at low concentrations (0.1 and 1 microg/ml), but the expression was decreased at higher doses of Eupolin (10 microg-150 microg/ml). A number of clinical studies carried out by Vietnamese and international medical investigators have demonstrated the efficacy of this extract on the wound healing process. In this study we have shown that Eupolin stimulated the expression of many proteins of the adhesion complex and fibronectin by human keratinocytes. The adhesion complex proteins are essential to stabilise epithelium and this effect could contribute to the clinical efficacy of Eupolin in healing.

Biosynthesis of fibronectin by rabbit aorta.

PubMed

Takasaki, I; Chobanian, A V; Brecher, P

1991-09-15

The in vitro interactions between vascular cells and fibronectin have been shown to influence phenotypic expression of both cultured endothelial and smooth muscle cells. To more effectively assess the potential functional role of fibronectin in vivo in modulating vascular phenotypes, we have established methodology for studying fibronectin biosynthesis in the rabbit aorta using aortic rings that are morphologically and functionally intact and metabolically active. Aortic rings were incubated with 35S-labeled methionine in a supplemented physiological salt solution. The tissue was fractionated, and quantitative immunoprecipitation was performed using a polyclonal antibody directed against human plasma fibronectin. Newly synthesized fibronectin was most abundant in the fraction solubilized using 4% sodium dodecyl sulfate and in the incubation medium. In all fractions studied, fibronectin was present predominantly as a dimer with no detectable aggregates of fibronectin. Pulse-chase experiments showed that a substantial amount of newly synthesized fibronectin was found in the 4% sodium dodecyl sulfate extract after only 1 h, suggesting that fibronectin was rapidly incorporated into the extracellular matrix. The more soluble forms of newly synthesized fibronectin appeared to be the precursors for secreted fibronectin, and no precursor-product relationship between soluble and insoluble fibronectin was found. Dissection of aortic rings following incubation with labeled methionine showed that newly synthesized fibronectin was uniformally distributed in both intima-media and media-adventitia segments. Endothelial cell denudation caused only a 20% decrease of fibronectin biosynthesis concomitant with similar changes in total protein biosynthesis, consistent with the medial smooth muscle cell as the major source of newly synthesized fibronectin. Biosynthesis of fibronectin was increased following a 24-h preincubation of the aortic rings, and concomitant increases in steady state mRNA for fibronectin were found. These in vitro studies documented the utility of aortic rings for the general purpose of studying protein synthesis in vascular cells and provide new information on the characteristics of fibronectin biosynthesis by aortic tissue.

A novel mechanism of UV-A and riboflavin-mediated corneal cross-linking through induction of tissue transglutaminases.

PubMed

Kopsachilis, Nikolaos; Tsaousis, Konstantinos T; Tsinopoulos, Ioannis T; Kruse, Friedrich E; Welge-Luessen, Ulrich

2013-07-01

Collagen cross-linking using UV-A irradiation combined with the photosensitizer riboflavin is a new technique for treating progressive keratoconus. The purposes of this study were to examine whether primary human corneal keratocytes (HCKs) are capable of expressing and secreting fibronectin and tissue transglutaminase (tTgase), an enzyme cross-linking extracellular matrix protein, and to examine whether fibronectin and tTgase are increased after the treatment of HCK cells with UV-A irradiation combined with riboflavin (RFUV-A), thus providing another possible physiological mechanism of the cross-linking pathway. Cell cultures established from HCKs were treated with 0.025% riboflavin solution and UV-A (370 nm) irradiance 3 mW/cm2 for 30 minutes. Induction of fibronectin and tTgase was investigated by immunohistochemistry, real-time polymerase chain reaction, and Western blot analysis. Cell viability was quantified by a microscopic live-dead assay. External tTgase activity was measured by the ability to form polymerized fibronectin and the incorporation of biotinylated cadaverine into fibronectin. Treatment of cultured HCK cells with RFUV-A increased the fibronectin and tTgase messenger RNA and protein levels. This effect was not observed in cells treated with riboflavin or UV-A radiation alone. Incorporation of biotinylated cadaverine was significantly increased when HCK cells were treated with RFUV-A. The enzymes tTgase and fibronectin are expressed by RFUV-A treatment in cultured HCK cells. This mechanism provides more information about the physiology of corneal cross-linking.

Fibronectin on extracellular vesicles from microvascular endothelial cells is involved in the vesicle uptake into oligodendrocyte precursor cells

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

Osawa, Sho; Department of Molecular and Cellular Neurobiology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511; Kurachi, Masashi

We previously reported transplantation of brain microvascular endothelial cells (MVECs) into cerebral white matter infarction model improved the animal's behavioral outcome by increasing the number of oligodendrocyte precursor cells (OPCs). We also revealed extracellular vesicles (EVs) derived from MVECs promoted survival and proliferation of OPCs in vitro. In this study, we investigated the mechanism how EVs derived from MVECs contribute to OPC survival and proliferation. Protein mass spectrometry and enzyme-linked immunosorbent assay revealed fibronectin was abundant on the surface of EVs from MVECs. As fibronectin has been reported to promote OPC survival and proliferation via integrin signaling pathway, we blocked themore » binding between fibronectin and integrins using RGD sequence mimics. Blocking the binding, however, did not attenuate the survival and proliferation promoting effect of EVs on OPCs. Flow cytometric and imaging analyses revealed fibronectin on EVs mediates their internalization into OPCs by its binding to heparan sulfate proteoglycan on OPCs. OPC survival and proliferation promoted by EVs were attenuated by blocking the internalization of EVs into OPCs. These lines of evidence suggest that fibronectin on EVs mediates their internalization into OPCs, and the cargo of EVs promotes survival and proliferation of OPCs, independent of integrin signaling pathway. - Highlights: • Fibronectin exists on the surface of extracellular vesicles from endothelial cells. • Integrin signaling is not involved in effects of extracellular vesicles on OPCs. • Fibronectin on the surface of extracellular vesicles mediates their uptake into OPCs.« less

Fibronectin Deposition Participates in Extracellular Matrix Assembly and Vascular Morphogenesis

PubMed Central

Hielscher, Abigail; Ellis, Kim; Qiu, Connie; Porterfield, Josh; Gerecht, Sharon

2016-01-01

The extracellular matrix (ECM) has been demonstrated to facilitate angiogenesis. In particular, fibronectin has been documented to activate endothelial cells, resulting in their transition from a quiescent state to an active state in which the cells exhibit enhanced migration and proliferation. The goal of this study is to examine the role of polymerized fibronectin during vascular tubulogenesis using a 3 dimensional (3D) cell-derived de-cellularized matrix. A fibronectin-rich 3D de-cellularized ECM was used as a scaffold to study vascular morphogenesis of endothelial cells (ECs). Confocal analyses of several matrix proteins reveal high intra- and extra-cellular deposition of fibronectin in formed vascular structures. Using a small peptide inhibitor of fibronectin polymerization, we demonstrate that inhibition of fibronectin fibrillogenesis in ECs cultured atop de-cellularized ECM resulted in decreased vascular morphogenesis. Further, immunofluorescence and ultrastructural analyses reveal decreased expression of stromal matrix proteins in the absence of polymerized fibronectin with high co-localization of matrix proteins found in association with polymerized fibronectin. Evaluating vascular kinetics, live cell imaging showed that migration, migration velocity, and mean square displacement, are disrupted in structures grown in the absence of polymerized fibronectin. Additionally, vascular organization failed to occur in the absence of a polymerized fibronectin matrix. Consistent with these observations, we tested vascular morphogenesis following the disruption of EC adhesion to polymerized fibronectin, demonstrating that block of integrins α5β1 and αvβ3, abrogated vascular morphogenesis. Overall, fibronectin deposition in a 3D cell-derived de-cellularized ECM appears to be imperative for matrix assembly and vascular morphogenesis. PMID:26811931

Expression of tumour necrosis factor alpha and accumulation of fibronectin in coronary artery restenotic lesions retrieved by atherectomy.

PubMed Central

Clausell, N.; de Lima, V. C.; Molossi, S.; Liu, P.; Turley, E.; Gotlieb, A. I.; Adelman, A. G.; Rabinovitch, M.

1995-01-01

BACKGROUND--The formation of coronary artery neointima experimentally induced in piglets after cardiac transplantation is related to an immune-inflammatory reaction associated with increased expression of T cells and inflammatory mediators (tumour necrosis factor alpha and interleukin 1 beta) and upregulation of fibronectin. In vivo blockade of tumour necrosis factor alpha in rabbits after cardiac transplantation results in reduced neointimal formation. The objective of this study was to investigate the hypothesis that coronary restenosis after atherectomy or percutaneous balloon angioplasty is associated with a similar inflammatory cascade initiated by mechanical injury. METHODS--Specimens taken at coronary atherectomy were analysed from 16 patients. Nine had had the procedure performed twice, firstly, to remove a primary lesion, and secondly, to remove a restenotic lesion. Seven had percutaneous balloon angioplasty after removal of restenotic tissue. Coronary atherectomy specimens were analysed by immunohistochemistry for the presence of T cells, macrophages, major histocompatibility complex II, interleukin 1 beta, tumour necrosis factor alpha, fibronectin, and the receptor for hyaluronan mediated motility. RESULTS--The groups were clinically and angiographically similar with equivalent lumens before and after atherectomy. Restenotic lesions had increased expression of tumour necrosis factor alpha and fibronectin compared with the primary lesions (P < 0.05 for both). There was also a trend towards a greater number of T cells and increased expression of interleukin 1 beta. CONCLUSIONS--Restenosis is associated with increased expression of tumour necrosis factor alpha and fibronectin, suggesting that an immune-inflammatory reaction probably contributes to neointimal formation and may represent a form of wound healing and repair secondary to mechanical injury. Images PMID:7626352

Elucidation of the therapeutic role of mitochondrial biogenesis transducers NRF-1 in the regulation of renal fibrosis

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

Hsieh, Pei-Fang; Graduate Institute of Medical Laboratory Science and Biotechnology, Chung Hwa University of Medical Technology, Tainan, Taiwan; Liu, Shu-Fen

Background: Mitochondrial dysfunction is a newly established risk factor for the development of renal fibrosis. Cell survival and injury repair is facilitated by mitochondrial biogenesis. Nuclear respiratory factor 1 (NRF-1) is a transcriptional regulation factor that plays a central role in the regulation of mitochondrial biogenesis. However, the transcription factor of this process in renal fibrosis is unknown. Thus, we hereby discussed the correlations of NRF-1 and renal interstitial fibrosis. Materials and methods: In vitro fibrosis model was established by treatment with transforming growth factor-β1 (TGF-β1) in NRK-49F (Normal Rat kidney fibroblast). We investigated the ROS production, mitochondrial biogenesis andmore » fibrogenic marker (e.q. fibronectin) during the progression of renal fibrosis by kit and Western blotting assay. Here, we used that two distinct mechanisms regulate NRF-1 activation and degradation of NRF-1. NRF-1 was transfect by pcDNA-NRF-1 overexpression gene to evaluate the NRF-1 activity of the therapeutic effect in renal fibrosis. In addition, NRF-1 was silenced by shRNA-NRF-1 to evaluate the significance of NRF-1. ELISA was used to evaluate the secreted fibronectin. Immunofluorescence staining was used to assay the in situ expression of proteins (e.g. fibronectin, NRF-1). Results: Under renal fibrosis conditions, TGF-β1 (5 ng/ml) increased ROS. Simultaneously, TGF-β1-induced extracellular fibronectin by ELISA assay. In addition, TGF-β1 decreased expression of mitochondrial biogenesis. This is the first time to demonstrate that expression of NRF-1 is significantly decreased in renal fibrosis. However, NRK49F was a transfection with pcDNA-NRF-1 (2 μg/ml) expression vector dramatically reverse TGF-β1-induced cellular fibrosis concomitantly with the suppression of fibronectin (both intracellular and extracellular fibronectin). More importantly, transfection with shRNA-NRF-1 (2 μg/ml) significantly increased the expression of fibronectin of both intercellular and extracellular origins in NRK-49F cells. Discussion: These finding suggest that NRF-1 plays a pivotal role on renal cellular fibrosis. Moreover, NRF-1 might act as a novel renal fibrosis antagonist by down-regulating fibrosis signaling in renal fibroblast cells.« less

Elucidation of the therapeutic role of mitochondrial biogenesis transducers NRF-1 in the regulation of renal fibrosis.

PubMed

Hsieh, Pei-Fang; Liu, Shu-Fen; Hung, Tsung-Jen; Hung, Chien-Ya; Liu, Guo-Zheng; Chuang, Lea-Yea; Chen, Mei-Fen; Wang, Jue-Long; Shi, Ming-Der; Hsu, Chen Hung; Shiue, Yow-Ling; Yang, Yu-Lin

2016-11-15

Mitochondrial dysfunction is a newly established risk factor for the development of renal fibrosis. Cell survival and injury repair is facilitated by mitochondrial biogenesis. Nuclear respiratory factor 1 (NRF-1) is a transcriptional regulation factor that plays a central role in the regulation of mitochondrial biogenesis. However, the transcription factor of this process in renal fibrosis is unknown. Thus, we hereby discussed the correlations of NRF-1 and renal interstitial fibrosis. In vitro fibrosis model was established by treatment with transforming growth factor-β1 (TGF-β1) in NRK-49F (Normal Rat kidney fibroblast). We investigated the ROS production, mitochondrial biogenesis and fibrogenic marker (e.q. fibronectin) during the progression of renal fibrosis by kit and Western blotting assay. Here, we used that two distinct mechanisms regulate NRF-1 activation and degradation of NRF-1. NRF-1 was transfect by pcDNA-NRF-1 overexpression gene to evaluate the NRF-1 activity of the therapeutic effect in renal fibrosis. In addition, NRF-1 was silenced by shRNA-NRF-1 to evaluate the significance of NRF-1. ELISA was used to evaluate the secreted fibronectin. Immunofluorescence staining was used to assay the in situ expression of proteins (e.g. fibronectin, NRF-1). Under renal fibrosis conditions, TGF-β1 (5ng/ml) increased ROS. Simultaneously, TGF-β1-induced extracellular fibronectin by ELISA assay. In addition, TGF-β1 decreased expression of mitochondrial biogenesis. This is the first time to demonstrate that expression of NRF-1 is significantly decreased in renal fibrosis. However, NRK49F was a transfection with pcDNA-NRF-1 (2μg/ml) expression vector dramatically reverse TGF-β1-induced cellular fibrosis concomitantly with the suppression of fibronectin (both intracellular and extracellular fibronectin). More importantly, transfection with shRNA-NRF-1 (2μg/ml) significantly increased the expression of fibronectin of both intercellular and extracellular origins in NRK-49F cells. These finding suggest that NRF-1 plays a pivotal role on renal cellular fibrosis. Moreover, NRF-1 might act as a novel renal fibrosis antagonist by down-regulating fibrosis signaling in renal fibroblast cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Propolis Modulates Fibronectin Expression in the Matrix of Thermal Injury

PubMed Central

Komosinska-Vassev, Katarzyna; Wisowski, Grzegorz; Mencner, Lukasz; Stojko, Jerzy; Kozma, Ewa M.

2014-01-01

The aim of the study was to assess the propolis effect on fibronectin metabolism in the course of burn wounds healing process. A model of burn wound healing of pig skin was applied. The amount of the released glycoprotein was assessed by a surface plasmon resonance. The profile of extracted fibronectin components was also assessed by an electrophoresis in polyacrylamide gel, with a subsequent immunodetection by Western Blotting. Propolis burn treatment decreased the release of fibronectin components from healing wounds in relation to damages treated with silver sulfadiazine. The main reason of decreased extraction of fibronectin components from wounds treated with propolis was a substantial decrease of degradation product release of the mentioned glycoprotein, which was observed particularly from the 3rd to 5th day of the repair. Wounds treatment with propolis demonstrated, especially in relation to damages treated with silver sulfadiazine, the decreased release of synthesized fibronectin molecules. The obtained results suggest that propolis modifies fibronectin metabolism in the course of wound healing process. The influence of propolis is reflected in prevention of fibronectin biosynthesis as well as its degradation in the wound area. The above-mentioned metabolic changes may decrease the risk of complications in the repair wounds process. PMID:24738072

Immunohistochemical study of integrin α₅β₁, fibronectin, and Bcl-2 in normal oral mucosa, inflammatory fibroepithelial hyperplasia, oral epithelial dysplasia, and oral squamous cell carcinoma.

PubMed

Núñez, Manuel Antonio Gordón; de Matos, Felipe Rodrigues; Freitas, Roseana de Almeida; Galvão, Hébel Cavalcanti

2013-07-01

The objective of this study was to compare the immunoexpression of integrin α₅β₁, fibronectin, and the Bcl-2 protein in normal oral mucosa (NOM), inflammatory fibroepithelial hyperplasia (IFH), oral epithelial dysplasia (OED), and oral squamous cell carcinoma (OSCC). Eleven cases of NOM, 16 IFH, 20 OED, and 27 OSCC were selected for analysis of the immunoexpression of integrin α₅β₁, fibronectin, and bcl-2 protein. There was an association between the intensity and location of the integrin α₅β₁ expression, especially in the OSCC, that 48.1% of cases showed weak immunoreactivity and 40.7% in the suprabasal layer (P < 0.05). There was an association between the pattern and distribution of fibronectin expression in basement membrane, where 90% of NOM showed a pattern of linear continuous and 80% of OED exhibited focal distribution (P < 0.05). The fibronectin expression in connective tissue was predominantly intense with an association of staining pattern among the different specimens, where 37% of OSCC showed a reticular pattern (P < 0.05). There was an association of bcl-2 protein among the types of specimens, especially in IFH and OSCC, where 100% of the cases exhibited scores 1 of staining (P < 0.05). Within this context, the interaction of integrin α₅β₁ with its main ligand in the extracellular matrix, fibronectin, is suggested to influence the survival of tumor cells and to favor their proliferation by modulating apoptosis through the upregulation of antiapoptotic proteins or the suppression of apoptotic mediators.

Distinct Effects of RGD-glycoproteins on Integrin-Mediated Adhesion and Osteogenic Differentiation of Human Mesenchymal Stem Cells

PubMed Central

Schwab, Elisabeth H.; Halbig, Maria; Glenske, Kristina; Wagner, Alena-Svenja; Wenisch, Sabine; Cavalcanti-Adam, Elisabetta A.

2013-01-01

The detailed interactions of mesenchymal stem cells (MSCs) with their extracellular matrix (ECM) and the resulting effects on MSC differentiation are still largely unknown. Integrins are the main mediators of cell-ECM interaction. In this study, we investigated the adhesion of human MSCs to fibronectin, vitronectin and osteopontin, three ECM glycoproteins which contain an integrin-binding sequence, the RGD motif. We then assayed MSCs for their osteogenic commitment in the presence of the different ECM proteins. As early as 2 hours after seeding, human MSCs displayed increased adhesion when plated on fibronectin, whereas no significant difference was observed when adhering either to vitronectin or osteopontin. Over a 10-day observation period, cell proliferation was increased when cells were cultured on fibronectin and osteopontin, albeit after 5 days in culture. The adhesive role of fibronectin was further confirmed by measurements of cell area, which was significantly increased on this type of substrate. However, integrin-mediated clusters, namely focal adhesions, were larger and more mature in MSCs adhering to vitronectin and osteopontin. Adhesion to fibronectin induced elevated expression of α5-integrin, which was further upregulated under osteogenic conditions also for vitronectin and osteopontin. In contrast, during osteogenic differentiation the expression level of β3-integrin was decreased in MSCs adhering to the different ECM proteins. When MSCs were cultured under osteogenic conditions, their commitment to the osteoblast lineage and their ability to form a mineralized matrix in vitro was increased in presence of fibronectin and osteopontin. Taken together these results indicate a distinct role of ECM proteins in regulating cell adhesion, lineage commitment and phenotype of MSCs, which is due to the modulation of the expression of specific integrin subunits during growth or osteogenic differentiation. PMID:24324361

Distinct effects of RGD-glycoproteins on Integrin-mediated adhesion and osteogenic differentiation of human mesenchymal stem cells.

PubMed

Schwab, Elisabeth H; Halbig, Maria; Glenske, Kristina; Wagner, Alena-Svenja; Wenisch, Sabine; Cavalcanti-Adam, Elisabetta A

2013-01-01

The detailed interactions of mesenchymal stem cells (MSCs) with their extracellular matrix (ECM) and the resulting effects on MSC differentiation are still largely unknown. Integrins are the main mediators of cell-ECM interaction. In this study, we investigated the adhesion of human MSCs to fibronectin, vitronectin and osteopontin, three ECM glycoproteins which contain an integrin-binding sequence, the RGD motif. We then assayed MSCs for their osteogenic commitment in the presence of the different ECM proteins. As early as 2 hours after seeding, human MSCs displayed increased adhesion when plated on fibronectin, whereas no significant difference was observed when adhering either to vitronectin or osteopontin. Over a 10-day observation period, cell proliferation was increased when cells were cultured on fibronectin and osteopontin, albeit after 5 days in culture. The adhesive role of fibronectin was further confirmed by measurements of cell area, which was significantly increased on this type of substrate. However, integrin-mediated clusters, namely focal adhesions, were larger and more mature in MSCs adhering to vitronectin and osteopontin. Adhesion to fibronectin induced elevated expression of α₅-integrin, which was further upregulated under osteogenic conditions also for vitronectin and osteopontin. In contrast, during osteogenic differentiation the expression level of β₃-integrin was decreased in MSCs adhering to the different ECM proteins. When MSCs were cultured under osteogenic conditions, their commitment to the osteoblast lineage and their ability to form a mineralized matrix in vitro was increased in presence of fibronectin and osteopontin. Taken together these results indicate a distinct role of ECM proteins in regulating cell adhesion, lineage commitment and phenotype of MSCs, which is due to the modulation of the expression of specific integrin subunits during growth or osteogenic differentiation.

Tissue distribution of very late activation antigens-1/6 and very late activation antigen ligands in the normal thymus and in thymoma.

PubMed Central

Ruco, L. P.; Paradiso, P.; Pittiglio, M.; Diodoro, M. G.; Gearing, A. J.; Mainiero, F.; Gismondi, A.; Santoni, A.; Baroni, C. D.

1993-01-01

The expression of very late activation antigens (VLAs)-1/6 was correlated with that of the VLA ligands fibronectin, laminin, collagen, and vascular cell adhesion molecule-1 in sections of normal thymus, in thymocyte suspensions, and in 10 cases of thymoma. Capsular epithelial cells are VLA-2+, VLA-3+, and VLA-6+ and face the thymic basement membrane, which is rich in fibronectin, laminin, and collagen type IV. Cortical epithelial cells are VLA-2+ and are embedded in a reticular meshwork of nonorganized extracellular matrix (ECM) that is rich in fibronectin. Cortical thymocytes, identified as CD3dim cells by using immunofluorescence in suspension, are highly positive for VLA-4, a fibronectin ligand. Most cortical macrophages are positive for vascular cell adhesion molecule-1, a molecule recognized by VLA-4. Medullary epithelial cells are VLA-2+/VLA-3+ and are codistributed with fibrous strands of organized ECM that are positive for fibronectin, collagen, and laminin. Medullary thymocytes, identified as CD3bright cells, are positive for VLA-4 and VLA-6, a ligand for laminin. Our findings suggest that intrathymic thymocyte maturation is associated with changes in expression of VLA molecules, which are apparently correlated with the presence of VLA ligands in the tissue microenvironment. Thymomas were classified as cortical (three), common (five), or medullary (two) type. Expression of VLA molecules and distribution of ECM in the three histological subtypes were reminiscent of those observed in the respective regions of the normal thymus. All cases of thymoma were characterized by overexpression of VLA molecules on neoplastic cells, which was associated with increased deposition of organized ECM rich in fibronectin, laminin, and collagen. Images Figure 1 Figure 3 PMID:8456937

Matrix stiffness-upregulated LOXL2 promotes fibronectin production, MMP9 and CXCL12 expression and BMDCs recruitment to assist pre-metastatic niche formation.

PubMed

Wu, Sifan; Zheng, Qiongdan; Xing, Xiaoxia; Dong, Yinying; Wang, Yaohui; You, Yang; Chen, Rongxin; Hu, Chao; Chen, Jie; Gao, Dongmei; Zhao, Yan; Wang, Zhiming; Xue, Tongchun; Ren, Zhenggang; Cui, Jiefeng

2018-05-04

Higher matrix stiffness affects biological behavior of tumor cells, regulates tumor-associated gene/miRNA expression and stemness characteristic, and contributes to tumor invasion and metastasis. However, the linkage between higher matrix stiffness and pre-metastatic niche in hepatocellular carcinoma (HCC) is still largely unknown. We comparatively analyzed the expressions of LOX family members in HCC cells grown on different stiffness substrates, and speculated that the secreted LOXL2 may mediate the linkage between higher matrix stiffness and pre-metastatic niche. Subsequently, we investigated the underlying molecular mechanism by which matrix stiffness induced LOXL2 expression in HCC cells, and explored the effects of LOXL2 on pre-metastatic niche formation, such as BMCs recruitment, fibronectin production, MMPs and CXCL12 expression, cell adhesion, etc. RESULTS: Higher matrix stiffness significantly upregulated LOXL2 expression in HCC cells, and activated JNK/c-JUN signaling pathway. Knockdown of integrin β1 and α5 suppressed LOXL2 expression and reversed the activation of above signaling pathway. Additionally, JNK inhibitor attenuated the expressions of p-JNK, p-c-JUN, c-JUN and LOXL2, and shRNA-c-JUN also decreased LOXL2 expression. CM-LV-LOXL2-OE and rhLOXL2 upregulated MMP9 expression and fibronectin production obviously in lung fibroblasts. Moreover, activation of Akt pathway contributed to LOXL2-induced fibronectin upregulation. LOXL2 in CM as chemoattractant increased motility and invasion of BMCs, implicating a significant role of LOXL2 in BMCs recruitment. Except that, CM-LV-LOXL2-OE as chemoattractant also increased the number of migrated HCC cells, and improved chemokine CXCL12 expression in lung fibroblasts. The number of HCC cells adhered to surface of lung fibroblasts treated with CM-LV-LOXL2-OE was remarkably higher than that of the control cells. These results indicated that the secreted LOXL2 facilitated the motility of HCC cells and strengthened CTCs settlement on the remodeled matrix "soil". Integrin β1/α5/JNK/c-JUN signaling pathway participates in higher matrix stiffness-induced LOXL2 upregulation in HCC cells. The secreted LOXL2 promotes fibronectin production, MMP9 and CXCL12 expression and BMDCs recruitment to assist pre-metastatic niche formation.

Interactions between integrin receptors and fibronectin are required for calvarial osteoblast differentiation in vitro

NASA Technical Reports Server (NTRS)

Moursi, A. M.; Globus, R. K.; Damsky, C. H.

1997-01-01

We previously showed that anti-fibronectin antibodies or soluble fibronectin fragments containing the central cell-binding domain inhibit formation of mineralized nodules by fetal calvarial osteoblasts in vitro. These findings suggest a critical role for fibronectin in osteoblast differentiation and morphogenesis. In this study we tested the hypothesis that fibronectin's effects on osteogenesis are mediated via direct interactions with integrin receptors for fibronectin on osteoblasts. Immunocytochemical analysis identified the integrin fibronectin receptor alpha5ss1 in fetal rat calvarial tissue and in cultured osteoblasts at all stages of differentiation. Three other integrins, alpha3ss1, alpha8ss1 and alphavss3, which can bind fibronectin, as well as other matrix components, were also identified in tissue and at all stages of cell culture. Immunoprecipitation data showed that alpha5ss1 levels are constant throughout osteoblast differentiation whereas levels of alpha3ss1 and alpha8ss1 decline in mature mineralized cultures. To determine whether integrin fibronectin receptors are required for osteoblast formation of mineralized nodules, we examined the extent of nodule formation in the presence and absence of function-perturbing anti-integrin antibodies. The antibodies were present continuously in cultures beginning at confluence (day 3), and nodule formation was measured at days 10 and 20. An anti-alpha5 integrin subunit antibody reduced nodule formation to less than 5% of control values at both time points. Inhibition of nodule formation was reversible and did not affect cell attachment and viability. Function-perturbing antibodies against alpha3ss1 and alpha8ss1 also reduced nodule formation, to less than 20% of control values. In contrast, function-perturbing antibodies to alphavss3 and alphavss5 did not affect nodule formation, indicating that the inhibitions noted were indeed specific. To determine the effect of antibody treatment on gene expression, steady-state mRNA expression was examined and found to be suppressed for osteoblast markers alkaline phosphatase and osteocalcin. Together, these results indicate that direct osteoblast interactions with the extracellular matrix are mediated by a select group of integrin receptors that includes alpha5ss1, alpha3ss1 and alpha8ss1. We further conclude that the specific alpha5ss1 fibronectin receptor mediates critical interactions between osteoblasts and fibronectin required for both bone morphogenesis and osteoblast differentiation.

CD44-mediated activation of α5β1-integrin, cortactin and paxillin signaling underpins adhesion of basal-like breast cancer cells to endothelium and Fibronectin-enriched matrices

PubMed Central

McFarlane, Suzanne; McFarlane, Cheryl; Montgomery, Nicola; Hill, Ashleigh; Waugh, David J.J.

2015-01-01

CD44 expression is elevated in basal-like breast cancer (BLBC) tissue, and correlates with increased efficiency of distant metastasis in patients and experimental models. We sought to characterize mechanisms underpinning CD44-promoted adhesion of BLBC cells to vascular endothelial monolayers and extracellular matrix (ECM) substrates. Stimulation with hyaluronan (HA), the native ligand for CD44, increased expression and activation of β1-integrin receptors, and increased α5-integrin subunit expression. Adhesion assays confirmed that CD44-signalling potentiated BLBC cell adhesion to endothelium and Fibronectin in an α5B1-integrin-dependent mechanism. Co-immunoprecipitation experiments confirmed HA-promoted association of CD44 with talin and the β1-integrin chain in BLBC cells. Knockdown of talin inhibited CD44 complexing with β1-integrin and repressed HA-induced, CD44-mediated activation of β1-integrin receptors. Immunoblotting confirmed that HA induced rapid phosphorylation of cortactin and paxillin, through a CD44-dependent and β1-integrin-dependent mechanism. Knockdown of CD44, cortactin or paxillin independently attenuated the adhesion of BL-BCa cells to endothelial monolayers and Fibronectin. Accordingly, we conclude that CD44 induced, integrin-mediated signaling not only underpins efficient adhesion of BLBC cells to BMECs to facilitate extravasation but initiates their adhesion to Fibronectin, enabling penetrant cancer cells to adhere more efficiently to underlying Fibronectin-enriched matrix present within the metastatic niche. PMID:26447611

Cellular origin of fibronectin in interspecies hybrid kidneys

PubMed Central

1984-01-01

The cellular origin of fibronectin in the kidney was studied in three experimental models. Immunohistochemical techniques that use cross- reacting or species-specific antibodies against mouse or chicken fibronectin were employed. In the first model studied, initially avascular mouse kidneys cultured on avian chorioallantoic membranes differentiate into epithelial kidney tubules and become vascularized by chorioallantoic vessels. Subsequently, hybrid glomeruli composed of mouse podocytes and avian endothelial-mesangial cells form. In immunohistochemical studies, cross-reacting antibodies to fibronectin stained vascular walls, tubular basement membranes, interstitium, and glomeruli of mouse kidney grafts. The species-specific antibodies reacting only with mouse fibronectin stained interstitial areas and tubular basement membranes, but showed no reaction with hybrid glomeruli and avian vascular walls. In contrast, species-specific antibodies against chicken fibronectin stained both the interstitial areas and the vascular walls as well as the endothelial-mesangial areas of the hybrid glomeruli, but did not stain the mouse-derived epithelial structures of the kidneys. In the second model, embryonic kidneys cultured under avascular conditions in vitro develop glomerular tufts, which are devoid of endothelial cells. These explants showed fluorescence staining for fibronectin only in tubular basement membranes and in interstitium. The avascular, purely epithelial glomerular bodies remained unstained. Finally, in outgrowths of separated embryonic glomeruli, the cross-reacting fibronectin antibodies revealed two populations of cells: one devoid of fibronectin and another expressing fibronectin in strong fibrillar and granular patterns. These results favor the idea that the main endogenous cellular sources for fibronectin in the embryonic kidney are the interstitial and vascular cells. All experiments presented here suggest that fibronectin is not synthesized by glomerular epithelial cells in vivo. PMID:6389571

Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells

PubMed Central

Hunt, Geoffrey C.; Singh, Purva; Schwarzbauer, Jean E.

2012-01-01

Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomitantly inducing the loss of Nanog and Oct4 self-renewal markers. Conversely, reducing fibronectin production by mES cells growing on a feeder-free gelatin substrate caused loss of cell adhesion, decreased integrin signaling, and decreased expression of self-renewal markers. These effects were reversed by providing the cells with exogenous fibronectin, thereby restoring adhesion to the gelatin substrate. Interestingly, mES cells do not adhere directly to the gelatin substrate, but rather adhere indirectly through gelatin-bound fibronectin, which facilitates self-renewal via its effects on cell adhesion. These results provide new insights into the mechanism of regulation of self-renewal by growth on a gelatin-coated surface. The effects of increasing or decreasing fibronectin levels show that self-renewal depends on an intermediate level of cell-fibronectin interactions. By providing cell adhesive signals that can act with other self-renewal factors to maintain mES cell pluripotency, fibronectin is therefore a necessary component of the self-renewal signaling pathway in culture. PMID:22710062

Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells.

PubMed

Hunt, Geoffrey C; Singh, Purva; Schwarzbauer, Jean E

2012-09-10

Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomitantly inducing the loss of Nanog and Oct4 self-renewal markers. Conversely, reducing fibronectin production by mES cells growing on a feeder-free gelatin substrate caused loss of cell adhesion, decreased integrin signaling, and decreased expression of self-renewal markers. These effects were reversed by providing the cells with exogenous fibronectin, thereby restoring adhesion to the gelatin substrate. Interestingly, mES cells do not adhere directly to the gelatin substrate, but rather adhere indirectly through gelatin-bound fibronectin, which facilitates self-renewal via its effects on cell adhesion. These results provide new insights into the mechanism of regulation of self-renewal by growth on a gelatin-coated surface. The effects of increasing or decreasing fibronectin levels show that self-renewal depends on an intermediate level of cell-fibronectin interactions. By providing cell adhesive signals that can act with other self-renewal factors to maintain mES cell pluripotency, fibronectin is therefore a necessary component of the self-renewal signaling pathway in culture. Copyright © 2012 Elsevier Inc. All rights reserved.

Platelet-Derived Growth Factor-BB Stimulates Fibronectin Gene Expression in Fibroblasts Isolated from Rat Thoracic Aorta

DTIC Science & Technology

1994-06-13

MARYLAND 20814-4799 TEACHING HOSPITALS WALTER REED ARMY MEDtCA L CENTER APPROVAL SHEET NAVAL HOSPITAL. BETHESDA MALCOLM GROW AIR FORCE MEDICAL ...CENTER WILFORD HALL "IR FORCE MEDICAL CENTER Title of Dissertation: "Platelet-derived growth factor-BB stimulates fibronectin gene expression in...fascinating world of basic medical science. His dedication and pursuit of excellence in all facets of his work are standards by which I will guide my own

Silibinin inhibits fibronectin induced motility, invasiveness and survival in human prostate carcinoma PC3 cells via targeting integrin signaling

PubMed Central

Deep, Gagan; Kumar, Rahul; Jain, Anil K; Agarwal, Chapla; Agarwal, Rajesh

2014-01-01

Prostate cancer (PCA) is the 2nd leading cause of cancer-related deaths among men in the United States. Preventing or inhibiting metastasis-related events through non-toxic agents could be a useful approach for lowering high mortality among PCA patients. We have earlier reported that natural flavonoid silibinin possesses strong anti-metastatic efficacy against PCA however, mechanism/s of its action still remains largely unknown. One of the major events during metastasis is the replacement of cell-cell interaction with integrins-based cell-matrix interaction that controls motility, invasiveness and survival of cancer cells. Accordingly, here we examined silibinin effect on advanced human PCA PC3 cells' interaction with extracellular matrix component fibronectin. Silibinin (50-200 μM) treatment significantly decreased the fibronectin (5 μg/ml)-induced motile morphology via targeting actin cytoskeleton organization in PC3 cells. Silibinin also decreased the fibronectin-induced cell proliferation and motility but significantly increased cell death in PC3 cells. Silibinin also inhibited the PC3 cells invasiveness in Transwell invasion assays with fibronectin or cancer associated fibroblasts (CAFs) serving as chemoattractant. Importantly, PC3-luc cells cultured on fibronectin showed rapid dissemination and localized in lungs following tail vein injection in athymic male nude mice; however, in silibinin-treated PC3-luc cells, dissemination and lung localization was largely compromised. Molecular analyses revealed that silibinin treatment modulated the fibronectin-induced expression of integrins (α5, αV, β1 and β3), actin-remodeling (FAK, Src, GTPases, ARP2 and cortactin), apoptosis (cPARP and cleaved caspase 3), EMT (E-cadherin and β-catenin), and cell survival (survivin and Akt) related signaling molecules in PC3 cells. Furthermore, PC3-xenograft tissue analyses confirmed the inhibitory effect of silibinin on fibronectin and integrins expression. Together, these results showed that silibinin targets PCA cells' interaction with fibronectin and inhibits their motility, invasiveness and survival; thus further supporting silibinin use in PCA intervention including its metastatic progression. PMID:25285031

Silibinin inhibits fibronectin induced motility, invasiveness and survival in human prostate carcinoma PC3 cells via targeting integrin signaling.

PubMed

Deep, Gagan; Kumar, Rahul; Jain, Anil K; Agarwal, Chapla; Agarwal, Rajesh

2014-10-01

Prostate cancer (PCA) is the 2nd leading cause of cancer-related deaths among men in the United States. Preventing or inhibiting metastasis-related events through non-toxic agents could be a useful approach for lowering high mortality among PCA patients. We have earlier reported that natural flavonoid silibinin possesses strong anti-metastatic efficacy against PCA however, mechanism/s of its action still remains largely unknown. One of the major events during metastasis is the replacement of cell-cell interaction with integrins-based cell-matrix interaction that controls motility, invasiveness and survival of cancer cells. Accordingly, here we examined silibinin effect on advanced human PCA PC3 cells' interaction with extracellular matrix component fibronectin. Silibinin (50-200 μM) treatment significantly decreased the fibronectin (5 μg/ml)-induced motile morphology via targeting actin cytoskeleton organization in PC3 cells. Silibinin also decreased the fibronectin-induced cell proliferation and motility but significantly increased cell death in PC3 cells. Silibinin also inhibited the PC3 cells invasiveness in Transwell invasion assays with fibronectin or cancer associated fibroblasts (CAFs) serving as chemoattractant. Importantly, PC3-luc cells cultured on fibronectin showed rapid dissemination and localized in lungs following tail vein injection in athymic male nude mice; however, in silibinin-treated PC3-luc cells, dissemination and lung localization was largely compromised. Molecular analyses revealed that silibinin treatment modulated the fibronectin-induced expression of integrins (α5, αV, β1 and β3), actin-remodeling (FAK, Src, GTPases, ARP2 and cortactin), apoptosis (cPARP and cleaved caspase 3), EMT (E-cadherin and β-catenin), and cell survival (survivin and Akt) related signaling molecules in PC3 cells. Furthermore, PC3-xenograft tissue analyses confirmed the inhibitory effect of silibinin on fibronectin and integrins expression. Together, these results showed that silibinin targets PCA cells' interaction with fibronectin and inhibits their motility, invasiveness and survival; thus further supporting silibinin use in PCA intervention including its metastatic progression.

Fibronectin matrix assembly suppresses dispersal of glioblastoma cells.

PubMed

Sabari, Joshua; Lax, Daniel; Connors, Daniel; Brotman, Ian; Mindrebo, Eric; Butler, Christine; Entersz, Ildiko; Jia, Dongxuan; Foty, Ramsey A

2011-01-01

Glioblastoma (GBM), the most aggressive and most common form of primary brain tumor, has a median survival of 12-15 months. Surgical excision, radiation and chemotherapy are rarely curative since tumor cells broadly disperse within the brain. Preventing dispersal could be of therapeutic benefit. Previous studies have reported that increased cell-cell cohesion can markedly reduce invasion by discouraging cell detachment from the tumor mass. We have previously reported that α5β1 integrin-fibronectin interaction is a powerful mediator of indirect cell-cell cohesion and that the process of fibronectin matrix assembly (FNMA) is crucial to establishing strong bonds between cells in 3D tumor-like spheroids. Here, we explore a potential role for FNMA in preventing dispersal of GBM cells from a tumor-like mass. Using a series of GBM-derived cell lines we developed an in vitro assay to measure the dispersal velocity of aggregates on a solid substrate. Despite their similar pathologic grade, aggregates from these lines spread at markedly different rates. Spreading velocity is inversely proportional to capacity for FNMA and restoring FNMA in GBM cells markedly reduces spreading velocity by keeping cells more connected. Blocking FNMA using the 70 KDa fibronectin fragment in FNMA-restored cells rescues spreading velocity, establishing a functional role for FNMA in mediating dispersal. Collectively, the data support a functional causation between restoration of FNMA and decreased dispersal velocity. This is a first demonstration that FNMA can play a suppressive role in GBM dispersal.

The Agr quorum-sensing system regulates fibronectin binding but not hemolysis in the absence of a functional electron transport chain.

PubMed

Pader, Vera; James, Ellen H; Painter, Kimberley L; Wigneshweraraj, Sivaramesh; Edwards, Andrew M

2014-10-01

Staphylococcus aureus is responsible for numerous chronic and recurrent infections, which are frequently associated with the emergence of small-colony variants (SCVs) that lack a functional electron transport chain. SCVs exhibit enhanced expression of fibronectin-binding protein (FnBP) and greatly reduced hemolysin production, although the basis for this is unclear. One hypothesis is that these phenotypes are a consequence of the reduced Agr activity of SCVs, while an alternative is that the lack of a functional electron transport chain and the resulting reduction in ATP production are responsible. Disruption of the electron transport chain of S. aureus genetically (hemB and menD) or chemically, using 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), inhibited both growth and Agr activity and conferred an SCV phenotype. Supplementation of the culture medium with synthetic autoinducing peptide (sAIP) significantly increased Agr expression in both hemB mutant strains and S. aureus grown with HQNO and significantly reduced staphylococcal adhesion to fibronectin. However, sAIP did not promote hemolysin expression in hemB mutant strains or S. aureus grown with HQNO. Therefore, while Agr regulates fibronectin binding in SCVs, it cannot promote hemolysin production in the absence of a functional electron transport chain. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

The Agr Quorum-Sensing System Regulates Fibronectin Binding but Not Hemolysis in the Absence of a Functional Electron Transport Chain

PubMed Central

Pader, Vera; James, Ellen H.; Painter, Kimberley L.; Wigneshweraraj, Sivaramesh

2014-01-01

Staphylococcus aureus is responsible for numerous chronic and recurrent infections, which are frequently associated with the emergence of small-colony variants (SCVs) that lack a functional electron transport chain. SCVs exhibit enhanced expression of fibronectin-binding protein (FnBP) and greatly reduced hemolysin production, although the basis for this is unclear. One hypothesis is that these phenotypes are a consequence of the reduced Agr activity of SCVs, while an alternative is that the lack of a functional electron transport chain and the resulting reduction in ATP production are responsible. Disruption of the electron transport chain of S. aureus genetically (hemB and menD) or chemically, using 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), inhibited both growth and Agr activity and conferred an SCV phenotype. Supplementation of the culture medium with synthetic autoinducing peptide (sAIP) significantly increased Agr expression in both hemB mutant strains and S. aureus grown with HQNO and significantly reduced staphylococcal adhesion to fibronectin. However, sAIP did not promote hemolysin expression in hemB mutant strains or S. aureus grown with HQNO. Therefore, while Agr regulates fibronectin binding in SCVs, it cannot promote hemolysin production in the absence of a functional electron transport chain. PMID:25092909

Tlx acts as a proangiogenic switch by regulating extracellular assembly of fibronectin matrices in retinal astrocytes.

PubMed

Uemura, Akiyoshi; Kusuhara, Sentaro; Wiegand, Stanley J; Yu, Ruth T; Nishikawa, Shin-ichi

2006-02-01

In response to hypoxia, hypoxia-inducible factors act as the primary proangiogenic triggers by regulating transcription levels of target genes, including VEGF. However, little is known about the specific factors that control other components of the angiogenic process, particularly formation of matrix scaffolds that promote adhesion and migration of endothelial cells. We show that in the postnatal mouse retina, the orphan nuclear receptor tailless (Tlx) is strongly expressed in the proangiogenic astrocytes, which secrete VEGF and fibronectin. Tlx expression by retinal astrocytes is controlled by oxygen concentration and rapidly downregulated upon contact with blood vessels. In mice null for Tlx, retinal astrocytes maintain VEGF expression; however, the extracellular assembly of fibronectin matrices by astrocytes is severely impaired, leading to defective scaffold formation and a complete failure of normal retinal vascular development. This work identifies Tlx as an essential component of the molecular network involved in the hypoxia-inducible proangiogenic switch in retinal astrocytes.

Tlx acts as a proangiogenic switch by regulating extracellular assembly of fibronectin matrices in retinal astrocytes

PubMed Central

Uemura, Akiyoshi; Kusuhara, Sentaro; Wiegand, Stanley J.; Yu, Ruth T.; Nishikawa, Shin-Ichi

2006-01-01

In response to hypoxia, hypoxia-inducible factors act as the primary proangiogenic triggers by regulating transcription levels of target genes, including VEGF. However, little is known about the specific factors that control other components of the angiogenic process, particularly formation of matrix scaffolds that promote adhesion and migration of endothelial cells. We show that in the postnatal mouse retina, the orphan nuclear receptor tailless (Tlx) is strongly expressed in the proangiogenic astrocytes, which secrete VEGF and fibronectin. Tlx expression by retinal astrocytes is controlled by oxygen concentration and rapidly downregulated upon contact with blood vessels. In mice null for Tlx, retinal astrocytes maintain VEGF expression; however, the extracellular assembly of fibronectin matrices by astrocytes is severely impaired, leading to defective scaffold formation and a complete failure of normal retinal vascular development. This work identifies Tlx as an essential component of the molecular network involved in the hypoxia-inducible proangiogenic switch in retinal astrocytes. PMID:16424942

Living biointerfaces based on non-pathogenic bacteria support stem cell differentiation

NASA Astrophysics Data System (ADS)

Hay, Jake J.; Rodrigo-Navarro, Aleixandre; Hassi, Karoliina; Moulisova, Vladimira; Dalby, Matthew J.; Salmeron-Sanchez, Manuel

2016-02-01

Lactococcus lactis, a non-pathogenic bacteria, has been genetically engineered to express the III7-10 fragment of human fibronectin as a membrane protein. The engineered L. lactis is able to develop biofilms on different surfaces (such as glass and synthetic polymers) and serves as a long-term substrate for mammalian cell culture, specifically human mesenchymal stem cells (hMSC). This system constitutes a living interface between biomaterials and stem cells. The engineered biofilms remain stable and viable for up to 28 days while the expressed fibronectin fragment induces hMSC adhesion. We have optimised conditions to allow long-term mammalian cell culture, and found that the biofilm is functionally equivalent to a fibronectin-coated surface in terms of osteoblastic differentiation using bone morphogenetic protein 2 (BMP-2) added to the medium. This living bacteria interface holds promise as a dynamic substrate for stem cell differentiation that can be further engineered to express other biochemical cues to control hMSC differentiation.

A new method to determine wound age in early vital skin injuries: a probability scoring system using expression levels of Fibronectin, CD62p and Factor VIII in wound hemorrhage.

PubMed

van de Goot, Franklin R W; Korkmaz, H Ibrahim; Fronczek, Judith; Witte, Birgit I; Visser, Rob; Ulrich, Magda M W; Begieneman, Mark P V; Rozendaal, Lawrence; Krijnen, Paul A J; Niessen, Hans W M

2014-11-01

In forensic autopsies it is important to determine the age of early vital skin wounds as accurate as possible. In addition to inflammation, coagulation is also induced in vital wounds. Analysis of blood coagulation markers in wound hemorrhage could therefore be an important additional discriminating factor in wound age determination. The aim of this study was to develop a wound age probability scoring system, based on the immunohistochemical expression levels of Fibronectin, CD62p and Factor VIII in wound hemorrhage. Tissue samples of (A) non injured control skin (n=383), and samples of mechanically induced skin injuries of known wound age, (B) injuries inflicted shortly before death (up to a few minutes old) (n=382), and (C) injuries inflicted 15-30 min before death (n=42) were obtained at autopsy in order to validate wound age estimation. Tissue slides were stained for Fibronectin, CD62p and Factor VIII and were subsequently scored for staining intensity (IHC score) in wound hemorrhage (1=minor, 2=moderate, 3=strong positive). Finally, probability scores of these markers were calculated. In at most 14% of the non-injured control samples, hemorrhage was found, with mean±standard deviation IHC scores of 0.1±0.4, 0.2±0.4 and 0.2±0.5 for Fibronectin, CD62p, and Factor VIII, respectively. Expression of these markers significantly increased to mean IHC scores 1.4±0.8 (Fibronectin), 1.2±0.6 (CD62p), and 1.6±0.8 (Factor VIII) in wounds inflicted shortly before death (few minutes old) and to 2.6±0.5 (Fibronectin), 2.5±0.6 (CD62p), and 2.8±0.4 (Factor VIII) in 15-30 min old wounds. The probabilities that a wound was non vital in case of an IH score 0 were 87%, 88% and 90% for Fibronectin, CD62p, and Factor VIII, respectively. In case of an IHC score 1 or 2, the probabilities that a wound was a few minutes old were 82/90%, 82/83% and 72/93%. Finally, in case of an IHC score 3, the probabilities that a wound was 15-30 min old were 65%, 76% and 55%. Based on the expression of Fibronectin, CD62p and Factor VIII in wound hemorrhage, we developed a probability scoring system that can be used in forensic autopsies to improve wound age estimation in early skin injuries. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Influenza viral neuraminidase primes bacterial coinfection through TGF-β-mediated expression of host cell receptors.

PubMed

Li, Ning; Ren, Aihui; Wang, Xiaoshuang; Fan, Xin; Zhao, Yong; Gao, George F; Cleary, Patrick; Wang, Beinan

2015-01-06

Influenza infection predisposes the host to secondary bacterial pneumonia, which is a major cause of mortality during influenza epidemics. The molecular mechanisms underlying the bacterial coinfection remain elusive. Neuraminidase (NA) of influenza A virus (IAV) enhances bacterial adherence and also activates TGF-β. Because TGF-β can up-regulate host adhesion molecules such as fibronectin and integrins for bacterial binding, we hypothesized that activated TGF-β during IAV infection contributes to secondary bacterial infection by up-regulating these host adhesion molecules. Flow cytometric analyses of a human lung epithelial cell line indicated that the expression of fibronectin and α5 integrin was up-regulated after IAV infection or treatment with recombinant NA and was reversed through the inhibition of TGF-β signaling. IAV-promoted adherence of group A Streptococcus (GAS) and other coinfective pathogens that require fibronectin for binding was prevented significantly by the inhibition of TGF-β. However, IAV did not promote the adherence of Lactococcus lactis unless this bacterium expressed the fibronectin-binding protein of GAS. Mouse experiments showed that IAV infection enhanced GAS colonization in the lungs of wild-type animals but not in the lungs of mice deficient in TGF-β signaling. Taken together, these results reveal a previously unrecognized mechanism: IAV NA enhances the expression of cellular adhesins through the activation of TGF-β, leading to increased bacterial loading in the lungs. Our results suggest that TGF-β and cellular adhesins may be potential pharmaceutical targets for the prevention of coinfection.

Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions.

PubMed

Purushothaman, Anurag; Bandari, Shyam Kumar; Liu, Jian; Mobley, James A; Brown, Elizabeth E; Sanderson, Ralph D

2016-01-22

Exosomes regulate cell behavior by binding to and delivering their cargo to target cells; however, the mechanisms mediating exosome-cell interactions are poorly understood. Heparan sulfates on target cell surfaces can act as receptors for exosome uptake, but the ligand for heparan sulfate on exosomes has not been identified. Using exosomes isolated from myeloma cell lines and from myeloma patients, we identify exosomal fibronectin as a key heparan sulfate-binding ligand and mediator of exosome-cell interactions. We discovered that heparan sulfate plays a dual role in exosome-cell interaction; heparan sulfate on exosomes captures fibronectin, and on target cells it acts as a receptor for fibronectin. Removal of heparan sulfate from the exosome surface releases fibronectin and dramatically inhibits exosome-target cell interaction. Antibody specific for the Hep-II heparin-binding domain of fibronectin blocks exosome interaction with tumor cells or with marrow stromal cells. Regarding exosome function, fibronectin-mediated binding of exosomes to myeloma cells activated p38 and pERK signaling and expression of downstream target genes DKK1 and MMP-9, two molecules that promote myeloma progression. Antibody against fibronectin inhibited the ability of myeloma-derived exosomes to stimulate endothelial cell invasion. Heparin or heparin mimetics including Roneparstat, a modified heparin in phase I trials in myeloma patients, significantly inhibited exosome-cell interactions. These studies provide the first evidence that fibronectin binding to heparan sulfate mediates exosome-cell interactions, revealing a fundamental mechanism important for exosome-mediated cross-talk within tumor microenvironments. Moreover, these results imply that therapeutic disruption of fibronectin-heparan sulfate interactions will negatively impact myeloma tumor growth and progression. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions*

PubMed Central

Purushothaman, Anurag; Bandari, Shyam Kumar; Liu, Jian; Mobley, James A.; Brown, Elizabeth E.; Sanderson, Ralph D.

2016-01-01

Exosomes regulate cell behavior by binding to and delivering their cargo to target cells; however, the mechanisms mediating exosome-cell interactions are poorly understood. Heparan sulfates on target cell surfaces can act as receptors for exosome uptake, but the ligand for heparan sulfate on exosomes has not been identified. Using exosomes isolated from myeloma cell lines and from myeloma patients, we identify exosomal fibronectin as a key heparan sulfate-binding ligand and mediator of exosome-cell interactions. We discovered that heparan sulfate plays a dual role in exosome-cell interaction; heparan sulfate on exosomes captures fibronectin, and on target cells it acts as a receptor for fibronectin. Removal of heparan sulfate from the exosome surface releases fibronectin and dramatically inhibits exosome-target cell interaction. Antibody specific for the Hep-II heparin-binding domain of fibronectin blocks exosome interaction with tumor cells or with marrow stromal cells. Regarding exosome function, fibronectin-mediated binding of exosomes to myeloma cells activated p38 and pERK signaling and expression of downstream target genes DKK1 and MMP-9, two molecules that promote myeloma progression. Antibody against fibronectin inhibited the ability of myeloma-derived exosomes to stimulate endothelial cell invasion. Heparin or heparin mimetics including Roneparstat, a modified heparin in phase I trials in myeloma patients, significantly inhibited exosome-cell interactions. These studies provide the first evidence that fibronectin binding to heparan sulfate mediates exosome-cell interactions, revealing a fundamental mechanism important for exosome-mediated cross-talk within tumor microenvironments. Moreover, these results imply that therapeutic disruption of fibronectin-heparan sulfate interactions will negatively impact myeloma tumor growth and progression. PMID:26601950

Fibronectin matrix assembly is essential for cell condensation during chondrogenesis

PubMed Central

Singh, Purva; Schwarzbauer, Jean E.

2014-01-01

ABSTRACT Mesenchymal cell condensation is the initiating event in endochondral bone formation. Cell condensation is followed by differentiation into chondrocytes, which is accompanied by induction of chondrogenic gene expression. Gene mutations involved in chondrogenesis cause chondrodysplasias and other skeletal defects. Using mesenchymal stem cells (MSCs) in an in vitro chondrogenesis assay, we found that knockdown of the diastrophic dysplasia (DTD) sulfate transporter (DTDST, also known as SLC26A2), which is required for normal cartilage development, blocked cell condensation and caused a significant reduction in fibronectin matrix. Knockdown of fibronectin with small interfering RNAs (siRNAs) also blocked condensation. Fibrillar fibronectin matrix was detected prior to cell condensation, and its levels increased during and after condensation. Inhibition of fibronectin matrix assembly by use of the functional upstream domain (FUD) of adhesin F1 from Streptococcus pyogenes prevented cell condensation by MSCs and also by the chondrogenic cell line ATDC5. Our data show that cell condensation and induction of chondrogenesis depend on fibronectin matrix assembly and DTDST, and indicate that this transporter is required earlier in chondrogenesis than previously appreciated. They also raise the possibility that certain of the skeletal defects in DTD patients might derive from the link between DTDST, fibronectin matrix and condensation. PMID:25146392

Fibronectin matrix assembly is essential for cell condensation during chondrogenesis.

PubMed

Singh, Purva; Schwarzbauer, Jean E

2014-10-15

Mesenchymal cell condensation is the initiating event in endochondral bone formation. Cell condensation is followed by differentiation into chondrocytes, which is accompanied by induction of chondrogenic gene expression. Gene mutations involved in chondrogenesis cause chondrodysplasias and other skeletal defects. Using mesenchymal stem cells (MSCs) in an in vitro chondrogenesis assay, we found that knockdown of the diastrophic dysplasia (DTD) sulfate transporter (DTDST, also known as SLC26A2), which is required for normal cartilage development, blocked cell condensation and caused a significant reduction in fibronectin matrix. Knockdown of fibronectin with small interfering RNAs (siRNAs) also blocked condensation. Fibrillar fibronectin matrix was detected prior to cell condensation, and its levels increased during and after condensation. Inhibition of fibronectin matrix assembly by use of the functional upstream domain (FUD) of adhesin F1 from Streptococcus pyogenes prevented cell condensation by MSCs and also by the chondrogenic cell line ATDC5. Our data show that cell condensation and induction of chondrogenesis depend on fibronectin matrix assembly and DTDST, and indicate that this transporter is required earlier in chondrogenesis than previously appreciated. They also raise the possibility that certain of the skeletal defects in DTD patients might derive from the link between DTDST, fibronectin matrix and condensation. © 2014. Published by The Company of Biologists Ltd.

Women’s perspectives of the fetal fibronectin testing process: a qualitative descriptive study

PubMed Central

2014-01-01

Background In 2009 the Ontario Ministry of Health and Long Term Care funded the implementation of province-wide fetal fibronectin testing in Ontario hospitals. This paper reports results from the provincial evaluation that sought to describe the experience of fetal fibronectin testing from the perspective of women with symptoms of preterm labour. Methods A descriptive qualitative design was used, employing semi-structured telephone and face-to-face interviews with women who had fetal fibronectin testing. Results Five hospitals participated in recruiting women for the study and 17 women were interviewed. Women described their experiences of fetal fibronectin testing as an emotional process that moves from expecting, to feeling, to hoping for reassurance; and then to re-defining what is required to feel reassured. Women described feeling anxious while waiting for fetal fibronectin results. When test results were negative, women described feeling a sense of relief that their symptoms would not likely lead to an imminent preterm birth. Women with positive results expressed feeling reassured by the care decisions and quick action taken by the health care team. Conclusion Fetal fibronectin testing was acceptable and beneficial to these women with symptoms of preterm labour. Implications for practice and future research are suggested. PMID:24894630

Effects of Coating a Titanium Alloy with Fibronectin on the Expression of Osteoblast Gene Markers in the MC3T3 Osteoprogenitor Cell Line

PubMed Central

Rapuano, Bruce E.; Hackshaw, Kyle M.; Schniepp, Hannes C.; MacDonald, Daniel E.

2013-01-01

Purpose A number of environmental and patient-related factors contribute to implant failure. A significant fraction of these failures can be attributed to limited osseointegration resulting from poor bone healing responses. The overall goal of this study was to determine whether surface treatment of a titanium-aluminum-vanadium alloy (Ti-6Al-4V) implant material with a biomimetic protein coating could promote the differentiation of attached osteoblastic cells. The specific aims of the study were to investigate whether osteoprogenitor cells cultured on a rigorously cleaned implant specimen showed a normal pattern of differentiation and whether preadsorbed fibronectin accelerated or enhanced osteoblast differentiation. Materials and Methods Ti-6Al-4V disks were rigorously cleaned, passivated in nitric acid, and dry heat–sterilized; some of the disks were then coated with 1 nmol/L fibronectin. MC3T3 osteoprogenitor cells were then cultured on the pretreated disks for several weeks. Quantitative real-time polymerase chain reaction was performed to measure changes over time in the mRNA levels of osteoblast genes. Results Fibronectin increased the peak expression of all analyzed osteoblast gene markers. “Early” genes that normally mark the proliferative phase (0 to 10 days) of osteoblastic development showed peak expression within the first 10 days after cell attachment to the titanium alloy. In contrast, “late” genes that normally mark the differentiation (10 to 20 days) and mineralization (20 to 36 days) phases of osteoblastogenesis achieved peak expression only after approximately 3 to 4 weeks of culture. Conclusions Osteoprogenitors cultured on a rigorously cleaned Ti-6Al-4V alloy were found to demonstrate a normal pattern of osteoblast differentiation. Preadsorbed fibronectin was observed to stimulate osteoblast differentiation during the mineralization phase of osteoblastogenesis. PMID:23057020

Antisense GLUT-1 protects mesangial cells from glucose induction of GLUT-1 and fibronectin expression.

PubMed

Heilig, C W; Kreisberg, J I; Freytag, S; Murakami, T; Ebina, Y; Guo, L; Heilig, K; Loberg, R; Qu, X; Jin, Y; Henry, D; Brosius, F C

2001-04-01

A stable clone of rat mesangial cells expressing antisense GLUT-1 (i.e., MCGT1AS cells) was developed to protect them from high glucose exposure. GLUT-1 protein was reduced 50%, and the 2-deoxy-[(3)H]glucose uptake rate was reduced 33% in MCGT1AS. MCLacZ control cells and MCGT1 GLUT-1-overexpressing cells were used for comparisons. In MCLacZ, 20 mM D-glucose increased GLUT-1 transcription 90% vs. no increase in MCGT1AS. Glucose (8 mM) and 12 mM xylitol [a hexose monophosphate (HMP) shunt substrate] did not stimulate GLUT-1 transcription. An 87% replacement of the standard 8 mM D-glucose with 3-O-methylglucose reduced GLUT-1 transcription 80%. D-Glucose (20 mM) increased fibronectin mRNA and protein by 47 and 100%, respectively, in MCLacZ vs. no increases in MCGT1AS. Fibronectin synthesis was elevated 48% in MCGT1 and reduced 44% in MCGT1AS. We conclude that 1) transcription of GLUT-1 in response to D-glucose depends on glucose metabolism, although not through the HMP shunt, and 2) antisense GLUT-1 treatment of mesangial cells blocks D-glucose-induced GLUT-1 and fibronectin expression, thereby demonstrating a protective effect that could be beneficial in the setting of diabetes.

HGF potentiates extracellular matrix-driven migration of human myoblasts: involvement of matrix metalloproteinases and MAPK/ERK pathway.

PubMed

González, Mariela Natacha; de Mello, Wallace; Butler-Browne, Gillian S; Silva-Barbosa, Suse Dayse; Mouly, Vincent; Savino, Wilson; Riederer, Ingo

2017-10-10

The hepatocyte growth factor (HGF) is required for the activation of muscle progenitor cells called satellite cells (SC), plays a role in the migration of proliferating SC (myoblasts), and is present as a soluble factor during muscle regeneration, along with extracellular matrix (ECM) molecules. In this study, we aimed at determining whether HGF is able to interact with ECM proteins, particularly laminin 111 and fibronectin, and to modulate human myoblast migration. We evaluated the expression of the HGF-receptor c-Met, laminin, and fibronectin receptors by immunoblotting, flow cytometry, or immunofluorescence and used Transwell assays to analyze myoblast migration on laminin 111 and fibronectin in the absence or presence of HGF. Zymography was used to check whether HGF could modulate the production of matrix metalloproteinases by human myoblasts, and the activation of MAPK/ERK pathways was evaluated by immunoblotting. We demonstrated that human myoblasts express c-Met, together with laminin and fibronectin receptors. We observed that human laminin 111 and fibronectin have a chemotactic effect on myoblast migration, and this was synergistically increased when low doses of HGF were added. We detected an increase in MMP-2 activity in myoblasts treated with HGF. Conversely, MMP-2 inhibition decreased the HGF-associated stimulation of cell migration triggered by laminin or fibronectin. HGF treatment also induced in human myoblasts activation of MAPK/ERK pathways, whose specific inhibition decreased the HGF-associated stimulus of cell migration triggered by laminin 111 or fibronectin. We demonstrate that HGF induces ERK phosphorylation and MMP production, thus stimulating human myoblast migration on ECM molecules. Conceptually, these data state that the mechanisms involved in the migration of human myoblasts comprise both soluble and insoluble moieties. This should be taken into account to optimize the design of therapeutic cell transplantation strategies by improving the migration of donor cells within the host tissue, a main issue regarding this approach.

The Cellular Form of Human Fibronectin as an Adhesion Target for the S Fimbriae of Meningitis-Associated Escherichia coli

PubMed Central

Sarén, Anne; Virkola, Ritva; Hacker, Jörg; Korhonen, Timo K.

1999-01-01

The adhesion of the S fimbriae of meningitis-associated Escherichia coli O18ac:K1:H7 to the cellular and the plasma forms of human fibronectin was studied. E. coli HB101(pAZZ50) expressing the complete S-fimbria II gene cluster of E. coli O18 adhered to cellular fibronectin (cFn) on glass but not to plasma fibronectin (pFn). Adhesion to cFn was specifically inhibited by neuraminidase treatment of cFn as well as by incubation of the bacteria with sialyl-α2-3-lactose, a receptor analog of the S fimbriae. No significant adhesion to cFn or pFn was detected with E. coli HB101(pAZZ50-67) expressing S fimbriae lacking the SfaS lectin subunit. Strain HB101(pAZZ50) also adhered to a human fibroblast cell culture known to be rich in cFn, and the adhesion was specifically inhibited in the presence of polyclonal antibodies to cFn. The results show that the SfaS lectin of the S fimbriae mediates the adherence of meningitis-associated E. coli to sialyl oligosaccharide chains of cFn. PMID:10225941

The FasX Small Regulatory RNA Negatively Regulates the Expression of Two Fibronectin-Binding Proteins in Group A Streptococcus.

PubMed

Danger, Jessica L; Makthal, Nishanth; Kumaraswami, Muthiah; Sumby, Paul

2015-12-01

The group A Streptococcus (GAS; Streptococcus pyogenes) causes more than 700 million human infections each year. The success of this pathogen can be traced in part to the extensive arsenal of virulence factors that are available for expression in temporally and spatially specific manners. To modify the expression of these virulence factors, GAS use both protein- and RNA-based regulators, with the best-characterized RNA-based regulator being the small regulatory RNA (sRNA) FasX. FasX is a 205-nucleotide sRNA that contributes to GAS virulence by enhancing the expression of the thrombolytic secreted virulence factor streptokinase and by repressing the expression of the collagen-binding cell surface pili. Here, we have expanded the FasX regulon, showing that this sRNA also negatively regulates the expression of the adhesion- and internalization-promoting, fibronectin-binding proteins PrtF1 and PrtF2. FasX posttranscriptionally regulates the expression of PrtF1/2 through a mechanism that involves base pairing to the prtF1 and prtF2 mRNAs within their 5' untranslated regions, overlapping the mRNA ribosome-binding sites. Thus, duplex formation between FasX and the prtF1 and prtF2 mRNAs blocks ribosome access, leading to an inhibition of mRNA translation. Given that FasX positively regulates the expression of the spreading factor streptokinase and negatively regulates the expression of the collagen-binding pili and of the fibronectin-binding PrtF1/2, our data are consistent with FasX functioning as a molecular switch that governs the transition of GAS between the colonization and dissemination stages of infection. More than half a million deaths each year are a consequence of infections caused by GAS. Insights into how this pathogen regulates the production of proteins during infection may facilitate the development of novel therapeutic or preventative regimens aimed at inhibiting this activity. Here, we have expanded insight into the regulatory activity of the GAS small RNA FasX. In addition to identifying that FasX reduces the abundance of the cell surface-located fibronectin-binding proteins PrtF1/2, fibronectin is present in high abundance in human tissues, and we have determined the mechanism behind this regulation. Importantly, as FasX is the only mechanistically characterized regulatory RNA in GAS, it serves as a model RNA in this and related pathogens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Age-specific function of α5β1 integrin in microglial migration during early colonization of the developing mouse cortex.

PubMed

Smolders, Sophie Marie-Thérèse; Swinnen, Nina; Kessels, Sofie; Arnauts, Kaline; Smolders, Silke; Le Bras, Barbara; Rigo, Jean-Michel; Legendre, Pascal; Brône, Bert

2017-07-01

Microglia, the immune cells of the central nervous system, take part in brain development and homeostasis. They derive from primitive myeloid progenitors that originate in the yolk sac and colonize the brain mainly through intensive migration. During development, microglial migration speed declines which suggests that their interaction with the microenvironment changes. However, the matrix-cell interactions allowing dispersion within the parenchyma are unknown. Therefore, we aimed to better characterize the migration behavior and to assess the role of matrix-integrin interactions during microglial migration in the embryonic brain ex vivo. We focused on microglia-fibronectin interactions mediated through the fibronectin receptor α5β1 integrin because in vitro work indirectly suggested a role for this ligand-receptor pair. Using 2-photon time-lapse microscopy on acute ex vivo embryonic brain slices, we found that migration occurs in a saltatory pattern and is developmentally regulated. Most importantly, there is an age-specific function of the α5β1 integrin during microglial cortex colonization. At embryonic day (E) 13.5, α5β1 facilitates migration while from E15.5, it inhibits migration. These results indicate a developmentally regulated function of α5β1 integrin in microglial migration during colonization of the embryonic brain. © 2017 Wiley Periodicals, Inc.

Epithelial-mesenchymal status influences how cells deposit fibrillin microfibrils.

PubMed

Baldwin, Andrew K; Cain, Stuart A; Lennon, Rachel; Godwin, Alan; Merry, Catherine L R; Kielty, Cay M

2014-01-01

Here, we show that epithelial-mesenchymal status influences how cells deposit extracellular matrix. Retinal pigmented epithelial (RPE) cells that expressed high levels of E-cadherin and had cell-cell junctions rich in zona occludens (ZO)-1, β-catenin and heparan sulfate, required syndecan-4 but not fibronectin or protein kinase C α (PKCα) to assemble extracellular matrix (fibrillin microfibrils and perlecan). In contrast, RPE cells that strongly expressed mesenchymal smooth muscle α-actin but little ZO-1 or E-cadherin, required fibronectin (like fibroblasts) and PKCα, but not syndecan-4. Integrins α5β1 and/or α8β1 and actomyosin tension were common requirements for microfibril deposition, as was heparan sulfate biosynthesis. TGFβ, which stimulates epithelial-mesenchymal transition, altered gene expression and overcame the dependency on syndecan-4 for microfibril deposition in epithelial RPE cells, whereas blocking cadherin interactions disrupted microfibril deposition. Renal podocytes had a transitional phenotype with pericellular β-catenin but little ZO-1; they required syndecan-4 and fibronectin for efficient microfibril deposition. Thus, epithelial-mesenchymal status modulates microfibril deposition.

Mifepristone inhibits extracellular matrix formation in uterine leiomyoma.

PubMed

Patel, Amrita; Malik, Minnie; Britten, Joy; Cox, Jeris; Catherino, William H

2016-04-01

To characterize the efficacy of mifepristone treatment on extracellular matrix (ECM) production in leiomyomas. Laboratory study. University research laboratory. None. Treatment of human immortalized two-dimensional (2D) and three-dimensional (3D) leiomyoma and myometrial cells with mifepristone and the progestin promegestone (R5020). Expression of COL1A1, fibronectin, versican variant V0, and dermatopontin in treated leiomyoma cells by Western blot analysis and confirmatory immunohistochemistry staining of treated 3D cultures. Treatment with progestin stimulated production of COL1A1, fibronectin, versican, and dermatopontin. Mifepristone treatment inhibited protein production of these genes, most notably with versican expression. Combination treatment with both the agonist and antagonist further inhibited protein expression of these genes. Immunohistochemistry performed on 3D cultures demonstrated generalized inhibition of ECM protein concentration. Our study demonstrated that the progesterone agonist R5020 directly stimulated extracellular matrix components COL1A1, fibronectin, versican, and dermatopontin production in human leiomyoma cells. Progesterone antagonist mifepristone decreased protein production of these genes to levels comparable with untreated leiomyoma cells. Published by Elsevier Inc.

The E1 beta-subunit of pyruvate dehydrogenase is surface-expressed in Lactobacillus plantarum and binds fibronectin.

PubMed

Vastano, Valeria; Salzillo, Marzia; Siciliano, Rosa A; Muscariello, Lidia; Sacco, Margherita; Marasco, Rosangela

2014-01-01

Lactobacillus plantarum is among the species with a probiotic activity. Adhesion of probiotic bacteria to host tissues is an important principle for strain selection, because it represents a crucial step in the colonization process of either pathogens or commensals. Most bacterial adhesins are proteins, and a major target for them is fibronectin, an extracellular matrix glycoprotein. In this study we demonstrate that PDHB, a component of the pyruvate dehydrogenase complex, is a factor contributing to fibronectin-binding in L. plantarum LM3. By means of fibronectin overlay immunoblotting assay, we identified a L. plantarum LM3 surface protein with apparent molecular mass of 35 kDa. Mass spectrometric analysis shows that this protein is the pyruvate dehydrogenase E1 beta-subunit (PDHB). The corresponding pdhB gene is located in a 4-gene cluster encoding pyruvate dehydrogenase. In LM3-B1, carrying a null mutation in pdhB, the 35 kDa adhesin was not anymore detectable by immunoblotting assay. Nevertheless, the pdhB null mutation did not abolish pdhA, pdhC, and pdhD transcription in LM3-B1. By adhesion assays, we show that LM3-B1 cells bind to immobilized fibronectin less efficiently than wild type cells. Moreover, we show that pdhB expression is negatively regulated by the CcpA protein and is induced by bile. Copyright © 2013. Published by Elsevier GmbH.

The influence of rat mesenchymal stem cell CD44 surface markers on cell growth, fibronectin expression, and cardiomyogenic differentiation on silk fibroin - Hyaluronic acid cardiac patches.

PubMed

Yang, Ming-Chia; Chi, Nai-Hsin; Chou, Nai-Kuan; Huang, Yi-You; Chung, Tze-Wen; Chang, Yu-Lin; Liu, Hwa-Chang; Shieh, Ming-Jium; Wang, Shoei-Shen

2010-02-01

Since MSCs contain an abundant of CD44 surface markers, it is of interesting to investigate whether CD44 on rat MSC (rMSCs) influenced cell growth, fibronectin expression and cardiomyogenic differentiation on new SF/HA cardiac patches. For this investigation, we examined the influences of rMSCs with or without a CD44-blockage treatment on the aforementioned issues after they were cultivated, and further induced by 5-aza on SF and SF/HA patches. The results showed that the relative growth rates of rMSCs cultured on cultural wells, SF/HA patches without or with a CD44-blockage treatment were 100%, 208.9+/-7.1 (%) or 48.4+/-6.0 (%) (n=3, for all), respectively, after five days of cultivations. Moreover, rMSCs cultivated on SF/HA patches highly promoted fibronectin expressions (e.g., 1.8x10(5)/cell, in fluorescent intensity) while cells with a CD44-blockage treatment markedly diminished the expressions (e.g., 1.1x10(4)/cell, in fluorescent intensity) on same patches. For investigating possible influences of CD44 surface markers of rMSCs on their cardiomyogenic differentiation, the expressions of specific cardiac genes of cells were examined by using real-time PCR analysis. The results indicated that 5-aza inducing rMSCs significantly promoted the expressions of Gata4, Nkx2.5, Tnnt2 and Actc1 genes (all, P<0.01 or better, n=3) on SF/HA patches compared with those expressions on SF patches and for cells with a CD44-blockage treatment on SF/HA patches. Furthermore, the intensity of the expressions of cardiotin and connexin 43 of 5-aza inducing rMSCs were markedly higher than those of cells with a CD44-blockage treatment after they were cultured on SF/HA patches. Through this study, we reported that CD44 surface markers of rMSCs highly influenced the proliferations, fibronectin expressions and cardiomyogenic differentiation of rMSCs cultivated on cardiac SF/HA patches.

Role of a bacillus Calmette-Guérin fibronectin attachment protein in BCG-induced antitumor activity.

PubMed

Zhao, W; Schorey, J S; Bong-Mastek, M; Ritchey, J; Brown, E J; Ratliff, T L

2000-04-01

Intravesical Mycobacterium bovis bacillus Calmette-Gu*erin (BCG) is the treatment of choice for superficial bladder cancer. Previous studies showed that attachment of BCG to fibronectin within the bladder was necessary for mediation of the antitumor response. Further studies identified a bacterial receptor, fibronectin attachment protein (FAP), as an important mediator of BCG attachment to fibronectin. In vitro studies showed that a stable BCG/fibronectin interaction was dependent on FAP binding to fibronectin; however, no role for FAP in the attachment of BCG in vivo has been characterized. We now report the cloning of the M. bovis BCG FAP (FAP-B) and demonstrate an important role for FAP in the in vivo attachment of BCG to the bladder wall and in the induction of BCG-mediated antitumor activity. The predicted amino acid sequence for FAP-B shows 61% and 71% homology, respectively, with Mycobacterium avium FAP (FAP-A) and Mycobacterium leprae FAP (FAP-L). Rabbit polyclonal antibodies against Mycobacterium vaccae FAP (FAP-V) reacted with all 3 recombinant FAP proteins on Western blots. Functional studies show FAP-B to bind fibronectin via the highly conserved attachment regions previously identified for FAP-A and FAP-L and also to competitively inhibit attachment of BCG to matrix fibronectin. In vivo studies show FAP to be a necessary protein for the stable attachment of BCG to the bladder wall. Moreover, stable binding of BCG via FAP was shown to be necessary for the expression of BCG-induced antitumor activity. Our results demonstrate a biological role for FAP in the mediation of BCG-induced antitumor activity.

The transcription factor ETS-1 regulates angiotensin II-stimulated fibronectin production in mesangial cells.

PubMed

Hua, Ping; Feng, Wenguang; Rezonzew, Gabriel; Chumley, Phillip; Jaimes, Edgar A

2012-06-01

Angiotensin II (ANG II) produced as result of activation of the renin-angiotensin system (RAS) plays a critical role in the pathogenesis of chronic kidney disease via its hemodynamic effects on the renal microcirculation as well as by its nonhemodynamic actions including the production of extracellular matrix proteins such as fibronectin, a multifunctional extracellular matrix protein that plays a major role in cell adhesion and migration as well as in the development of glomerulosclerosis. ETS-1 is an important transcription factor essential for normal kidney development and glomerular integrity. We previously showed that ANG II increases ETS-1 expression and is required for fibronectin production in mesangial cells. In these studies, we determined that ANG II induces phosphorylation of ETS-1 via activation of the type 1 ANG II receptor and that Erk1/2 and Akt/PKB phosphorylation are required for these effects. In addition, we characterized the role of ETS-1 on the transcriptional activation of fibronectin production in mesangial cells. We determined that ETS-1 directly activates the fibronectin promoter and by utilizing gel shift assays and chromatin immunoprecipitation assays identified two different ETS-1 binding sites that promote the transcriptional activation of fibronectin in response to ANG II. In addition, we identified the essential role of CREB and its coactivator p300 on the transcriptional activation of fibronectin by ETS-1. These studies unveil novel mechanisms involved in RAS-induced production of the extracellular matrix protein fibronectin in mesangial cells and establish the role of the transcription factor ETS-1 as a direct mediator of these effects.

Disruption of fibronectin matrix affects type IV collagen, fibrillin and laminin deposition into extracellular matrix of human trabecular meshwork (HTM) cells.

PubMed

Filla, Mark S; Dimeo, Kaylee D; Tong, Tiegang; Peters, Donna M

2017-12-01

Fibronectin fibrils are a major component of the extracellular matrix (ECM) of the trabecular meshwork (TM). They are a key mediator of the formation of the ECM which controls aqueous humor outflow and contributes to the pathogenesis of glaucoma. The purpose of this work was to determine if a fibronectin-binding peptide called FUD, derived from the Streptococcus pyogenes Functional Upstream Domain of the F1 adhesin protein, could be used to control fibronectin fibrillogenesis and hence ECM formation under conditions where its expression was induced by treatment with the glucocorticoid dexamethasone. FUD was very effective at preventing fibronectin fibrillogenesis in the presence or absence of steroid treatment as well as the removal of existing fibronectin fibrils. Disruption of fibronectin fibrillogenesis by FUD also disrupted the incorporation of type IV collagen, laminin and fibrillin into the ECM. The effect of FUD on these other protein matrices, however, was found to be dependent upon the maturity of the ECM when FUD was added. FUD effectively disrupted the incorporation of these other proteins into matrices when added to newly confluent cells that were forming a nascent ECM. In contrast, FUD had no effect on these other protein matrices if the cell cultures already possessed a pre-formed, mature ECM. Our studies indicate that FUD can be used to control fibronectin fibrillogenesis and that these fibrils play a role in regulating the assembly of other ECM protein into matrices involving type IV collagen, laminin, and fibrillin within the TM. This suggests that under in vivo conditions, FUD would selectively disrupt fibronectin fibrils and de novo assembly of other proteins into the ECM. Finally, our studies suggest that targeting fibronectin fibril assembly may be a viable treatment for POAG as well as other glaucomas involving excessive or abnormal matrix deposition of the ECM. Copyright © 2017 Elsevier Ltd. All rights reserved.

Platelet-rich plasma and chronic wounds: remaining fibronectin may influence matrix remodeling and regeneration success.

PubMed

Moroz, Andrei; Deffune, Elenice

2013-11-01

Platelet-rich plasma has been largely used as a therapeutic option for the treatment of chronic wounds of different etiologies. The enhanced regeneration observed after the use of platelet-rich plasma has been systematically attributed to the growth factors that are present inside platelets' granules. We hypothesize that the remaining plasma and platelet-bound fibronectin may act as a further bioactive protein in platelet-rich plasma preparations. Recent reports were analyzed and presented as direct evidences of this hypotheses. Fibronectin may directly influence the extracellular matrix remodeling during wound repair. This effect is probably through matrix metalloproteinase expression, thus exerting an extra effect on chronic wound regeneration. Physicians should be well aware of the possible fibronectin-induced effects in their future endeavors with PRP in chronic wound treatment. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Novel mechanism of transcriptional regulation of cell matrix protein through CREB

PubMed Central

Habib, Samy L; Mohan, Sumathy; Liang, Sitai; Li, Baojie; Yadav, Mukesh

2015-01-01

The transcription mechanism(s) of renal cell matrix accumulation in diabetes does not explored. Phosphorylation of the transcription factor cAMP-responsive element binding protein (CREB) significantly increased in cells treated with high glucose (HG) compared to cell grown in normal glucose (NG). Cells pretreated with rapamycin before exposure to HG showed significant decrease phosphorylation of CREB, increase in AMPK activity and decrease protein/mRNA and promoter activity of fibronectin. In addition, cells transfected with siRNA against CREB showed significant increase in AMPK activity, decrease in protein/mRNA and promoter activity of fibronectin. Cells treated with HG showed nuclear localization of p-CREB while pretreated cells with rapamycin reversed HG effect. Moreover, gel shift analysis shows increase binding of CREB to fibronectin promoter in cells treated with HG while cells pretreated with rapamycin reversed the effect of HG. Furthermore, db/db mice treated with rapamycin showed significant increase in AMPK activity, decrease in expression of p-CREB and protein/mRNA of fibronectin. Strong staining of fibronectin and p-CREB was detected in kidney cortex of db/db mice while treated mice with rapamycin reversed hyperglycemia effect. In summary, our data provide a novel mechanism of transcriptional regulation of fibronectin through CREB that may be used as therapeutic approach to prevent diabetes complications. PMID:26115221

Novel Potential Interacting Partners of Fibronectin in Spontaneous Animal Model of Interstitial Cystitis

PubMed Central

Treutlein, Gudrun; Dorsch, Roswitha; Euler, Kerstin N.; Hauck, Stefanie M.; Amann, Barbara; Hartmann, Katrin; Deeg, Cornelia A.

2012-01-01

Feline idiopathic cystitis (FIC) is the only spontaneous animal model for human interstitial cystitis (IC), as both possess a distinctive chronical and relapsing character. Underlying pathomechanisms of both diseases are not clearly established yet. We recently detected increased urine fibronectin levels in FIC cases. The purpose of this study was to gain further insight into the pathogenesis by assessing interacting partners of fibronectin in urine of FIC affected cats. Several candidate proteins were identified via immunoprecipitation and mass spectrometry. Considerable changes in FIC conditions compared to physiological expression of co-purified proteins were detected by Western blot and immunohistochemistry. Compared to controls, complement C4a and thioredoxin were present in higher levels in urine of FIC patients whereas loss of signal intensity was detected in FIC affected tissue. Galectin-7 was exclusively detected in urine of FIC cats, pointing to an important role of this molecule in FIC pathogenesis. Moderate physiological signal intensity of galectin-7 in transitional epithelium shifted to distinct expression in transitional epithelium under pathophysiological conditions. I-FABP expression was reduced in urine and urinary bladder tissue of FIC cats. Additionally, transduction molecules of thioredoxin, NF-κB p65 and p38 MAPK, were examined. In FIC affected tissue, colocalization of thioredoxin and NF-κB p65 could be demonstrated compared to absent coexpression of thioredoxin and p38 MAPK. These considerable changes in expression level and pattern point to an important role for co-purified proteins of fibronectin and thioredoxin-regulated signal transduction pathways in FIC pathogenesis. These results could provide a promising starting point for novel therapeutic approaches in the future. PMID:23236492

Minireview: Fibronectin in retinal disease.

PubMed

Miller, Charles G; Budoff, Greg; Prenner, Jonathan L; Schwarzbauer, Jean E

2017-01-01

Retinal fibrosis, characterized by dysregulation of extracellular matrix (ECM) protein deposition by retinal endothelial cells, pigment epithelial cells, and other resident cell-types, is a unifying feature of several common retinal diseases. Fibronectin is an early constituent of newly deposited ECM and serves as a template for assembly of other ECM proteins, including collagens. Under physiologic conditions, fibronectin is found in all layers of Bruch's membrane. Proliferative vitreoretinopathy (PVR), a complication of retinal surgery, is characterized by ECM accumulation. Among the earliest histologic manifestations of diabetic retinopathy (DR) is capillary basement membrane thickening, which occurs due to perturbations in ECM homeostasis. Neovascularization, the hallmark of late stage DR as well as exudative age-related macular degeneration (AMD), involves ECM assembly as a scaffold for the aberrant new vessel architecture. Rodent models of retinal injury demonstrate a key role for fibronectin in complications characteristic of PVR, including retinal detachment. In mouse models of DR, reducing fibronectin gene expression has been shown to arrest the accumulation of ECM in the capillary basement membrane. Alterations in matrix metalloproteinase activity thought to be important in the pathogenesis of AMD impact the turnover of fibronectin matrix as well as collagens. Growth factors involved in PVR, AMD, and DR, such as PDGF and TGFβ, are known to stimulate fibronectin matrix assembly. A deeper understanding of how pathologic ECM deposition contributes to disease progression may help to identify novel targets for therapeutic intervention. © 2016 by the Society for Experimental Biology and Medicine.

The Circular RNA Interacts with STAT3, Increasing Its Nuclear Translocation and Wound Repair by Modulating Dnmt3a and miR-17 Function.

PubMed

Yang, Zhen-Guo; Awan, Faryal Mehwish; Du, William W; Zeng, Yan; Lyu, Juanjuan; Wu, De; Gupta, Shaan; Yang, Weining; Yang, Burton B

2017-09-06

Delayed or impaired wound healing is a major health issue worldwide, especially in patients with diabetes and atherosclerosis. Here we show that expression of the circular RNA circ-Amotl1 accelerated healing process in a mouse excisional wound model. Further studies showed that ectopic circ-Amotl1 increased protein levels of Stat3 and Dnmt3a. The increased Dnmt3a then methylated the promoter of microRNA miR-17, decreasing miR-17-5p levels but increasing fibronectin expression. We found that Stat3, similar to Dnmt3a and fibronectin, was a target of miR-17-5p. Decreased miR-17-5p levels would increase expression of fibronectin, Dnmt3a, and Stat3. All of these led to increased cell adhesion, migration, proliferation, survival, and wound repair. Furthermore, we found that circ-Amotl1 not only increased Stat3 expression but also facilitated Stat3 nuclear translocation. Thus, the ectopic expressed circ-Amotl1 and Stat3 were mainly translocated to nucleus. In the presence of circ-Amotl1, Stat3 interacted with Dnmt3a promoter with increased affinity, facilitating Dnmt3a transcription. Ectopic application of circ-Amotl1 accelerating wound repair may shed light on skin wound healing clinically. Copyright © 2017. Published by Elsevier Inc.

Lactococcus lactis expressing either Staphylococcus aureus fibronectin-binding protein A or Listeria monocytogenes internalin A can efficiently internalize and deliver DNA in human epithelial cells.

PubMed

Innocentin, Silvia; Guimarães, Valeria; Miyoshi, Anderson; Azevedo, Vasco; Langella, Philippe; Chatel, Jean-Marc; Lefèvre, François

2009-07-01

Lactococci are noninvasive bacteria frequently used as protein delivery vectors and, more recently, as in vitro and in vivo DNA delivery vehicles. We previously showed that a functional eukaryotic enhanced green fluorescent protein (eGFP) expression plasmid vector was delivered in epithelial cells by Lactococcus lactis producing Listeria monocytogenes internalin A (L. lactis InlA(+)), but this strategy is limited in vivo to transgenic mice and guinea pigs. In this study, we compare the internalization ability of L. lactis InlA(+) and L. lactis producing either the fibronectin-binding protein A of Staphylococcus aureus (L. lactis FnBPA(+)) or its fibronectin binding domains C and D (L. lactis CD(+)). L. lactis FnBPA(+) and L. lactis InlA(+) showed comparable internalization rates in Caco-2 cells, while the internalization rate observed with L. lactis CD(+) was lower. As visualized by conventional and confocal fluorescence microscopy, large clusters of L. lactis FnBPA(+), L. lactis CD(+), and L. lactis InlA(+) were present in the cytoplasm of Caco-2 cells after internalization. Moreover, the internalization rates of Lactobacillus acidophilus NCFM and of an NCFM mutant strain with the gene coding for the fibronectin-binding protein (fbpA) inactivated were also evaluated in Caco-2 cells. Similar low internalization rates were observed for both wild-type L. acidophilus NCFM and the fbpA mutant, suggesting that commensal fibronectin binding proteins have a role in adhesion but not in invasion. L. lactis FnBPA(+), L. lactis CD(+), and L. lactis InlA(+) were then used to deliver a eukaryotic eGFP expression plasmid in Caco-2 cells: flow cytometry analysis showed that the highest percentage of green fluorescent Caco-2 cells was observed after coculture with either L. lactis FnBPA(+) or L. lactis InlA(+). Analysis of the in vivo efficiency of these invasive recombinant strains is currently in progress to validate their potential as DNA vaccine delivery vehicles.

Liarozole inhibits transforming growth factor-β3–mediated extracellular matrix formation in human three-dimensional leiomyoma cultures

PubMed Central

Levy, Gary; Malik, Minnie; Britten, Joy; Gilden, Melissa; Segars, James; Catherino, William H.

2014-01-01

Objective To investigate the impact of liarozole on transforming growth factor-β3 (TGF-β3) expression, TGF-β3 controlled profibrotic cytokines, and extracellular matrix formation in a three-dimensional (3D) leiomyoma model system. Design Molecular and immunohistochemical analysis in a cell line evaluated in a three-dimensional culture. Setting Laboratory study. Patient(s) None. Intervention(s) Treatment of leiomyoma and myometrial cells with liarozole and TGF-β3 in a three-dimensional culture system. Main Outcome Measure(s) Quantitative real-time reverse-transcriptase polymerase chain reaction and Western blotting to assess fold gene and protein expression of TGF-β3 and TGF-β3 regulated fibrotic cytokines: collagen 1A1 (COL1A1), fibronectin, and versican before and after treatment with liarozole, and confirmatory immunohistochemical stains of treated three-dimensional cultures. Result(s) Both TGF-β3 gene and protein expression were elevated in leiomyoma cells compared with myometrium in two-dimensional and 3D cultures. Treatment with liarozole decreased TGF-β3 gene and protein expression. Extracellular matrix components versican, COL1A1, and fibronectin were also decreased by liarozole treatment in 3D cultures. Treatment of 3D cultures with TGF-β3 increased gene expression and protein production of COL1A1, fibronectin, and versican. Conclusion(s) Liarozole decreased TGF-β3 and TGF-β3–mediated extracellular matrix expression in a 3D uterine leiomyoma culture system. PMID:24825427

RAC1 GTP-ase signals Wnt-beta-catenin pathway mediated integrin-directed metastasis-associated tumor cell phenotypes in triple negative breast cancers.

PubMed

De, Pradip; Carlson, Jennifer H; Jepperson, Tyler; Willis, Scooter; Leyland-Jones, Brian; Dey, Nandini

2017-01-10

The acquisition of integrin-directed metastasis-associated (ID-MA) phenotypes by Triple-Negative Breast Cancer (TNBC) cells is caused by an upregulation of the Wnt-beta-catenin pathway (WP). We reported that WP is one of the salient genetic features of TNBC. RAC-GTPases, small G-proteins which transduce signals from cell surface proteins including integrins, have been implicated in tumorigenesis and metastasis by their role in essential cellular functions like motility. The collective percentage of alteration(s) in RAC1 in ER+ve BC was lower as compared to ER-ve BC (35% vs 57%) (brca/tcga/pub2015). High expression of RAC1 was associated with poor outcome for RFS with HR=1.48 [CI: 1.15-1.9] p=0.0019 in the Hungarian ER-veBC cohort. Here we examined how WP signals are transduced via RAC1 in the context of ID-MA phenotypes in TNBC. Using pharmacological agents (sulindac sulfide), genetic tools (beta-catenin siRNA), WP modulators (Wnt-C59, XAV939), RAC1 inhibitors (NSC23766, W56) and WP stimulations (LWnt3ACM, Wnt3A recombinant) in a panel of 6-7 TNBC cell lines, we studied fibronectin-directed (1) migration, (2) matrigel invasion, (3) RAC1 and Cdc42 activation, (4) actin dynamics (confocal microscopy) and (5) podia-parameters. An attenuation of WP, which (a) decreased cellular levels of beta-catenin, as well as its nuclear active-form, (b) decreased fibronectin-induced migration, (c) decreased invasion, (d) altered actin dynamics and (e) decreased podia-parameters was successful in blocking fibronectin-mediated RAC1/Cdc42 activity. Both Wnt-antagonists and RAC1 inhibitors blocked fibronectin-induced RAC1 activation and inhibited the fibronectin-induced ID-MA phenotypes following specific WP stimulation by LWnt3ACM as well as Wnt3A recombinant protein. To test a direct involvement of RAC1-activation in WP-mediated ID-MA phenotypes, we stimulated brain-metastasis specific MDA-MB231BR cells with LWnt3ACM. LWnt3ACM-stimulated fibronectin-directed migration was blocked by RAC1 inhibition in MDA-MB231BR cells. In the light of our previous report that WP upregulation causes ID-MA phenotypes in TNBC tumor cells, here we provide the first mechanism based evidence to demonstrate that WP upregulation signals ID-MA tumor cell phenotypes in a RAC1-GTPase dependent manner involving exchange-factors like TIAM1 and VAV2. Our study demonstrates for the first time that beta-catenin-RAC1 cascade signals integrin-directed metastasis-associated tumor cell phenotypes in TNBC.

Dexamethasone-Mediated Activation of Fibronectin Matrix Assembly Reduces Dispersal of Primary Human Glioblastoma Cells

PubMed Central

Shannon, Stephen; Vaca, Connan; Jia, Dongxuan; Entersz, Ildiko; Schaer, Andrew; Carcione, Jonathan; Weaver, Michael; Avidar, Yoav; Pettit, Ryan; Nair, Mohan; Khan, Atif; Foty, Ramsey A.

2015-01-01

Despite resection and adjuvant therapy, the 5-year survival for patients with Glioblastoma multiforme (GBM) is less than 10%. This poor outcome is largely attributed to rapid tumor growth and early dispersal of cells, factors that contribute to a high recurrence rate and poor prognosis. An understanding of the cellular and molecular machinery that drive growth and dispersal is essential if we are to impact long-term survival. Our previous studies utilizing a series of immortalized GBM cell lines established a functional causation between activation of fibronectin matrix assembly (FNMA), increased tumor cohesion, and decreased dispersal. Activation of FNMA was accomplished by treatment with Dexamethasone (Dex), a drug routinely used to treat brain tumor related edema. Here, we utilize a broad range of qualitative and quantitative assays and the use of a human GBM tissue microarray and freshly-isolated primary human GBM cells grown both as conventional 2D cultures and as 3D spheroids to explore the role of Dex and FNMA in modulating various parameters that can significantly influence tumor cell dispersal. We show that the expression and processing of fibronectin in a human GBM tissue-microarray is variable, with 90% of tumors displaying some abnormality or lack in capacity to secrete fibronectin or assemble it into a matrix. We also show that low-passage primary GBM cells vary in their capacity for FNMA and that Dex treatment reactivates this process. Activation of FNMA effectively “glues” cells together and prevents cells from detaching from the primary mass. Dex treatment also significantly increases the strength of cell-ECM adhesion and decreases motility. The combination of increased cohesion and decreased motility discourages in vitro and ex vivo dispersal. By increasing cell-cell cohesion, Dex also decreases growth rate of 3D spheroids. These effects could all be reversed by an inhibitor of FNMA and by the glucocorticoid receptor antagonist, RU-486. Our results describe a new role for Dex as a suppressor of GBM dispersal and growth. PMID:26284619

Migratory capabilities of human umbilical cord blood-derived neural stem cells (HUCB-NSC) in vitro.

PubMed

Janowski, Miroslaw; Lukomska, Barbara; Domanska-Janik, Krystyna

2011-01-01

Many types of neural progenitors from various sources have been evaluated for therapy of CNS disorders. Prerequisite for success in cell therapy is the ability for transplanted cells to reach appropriate target such as stroke lesion. We have established neural stem cell line from human umbilical cord blood neural stem (HUCB-NSC). In the present study we evaluated migratory capabilities of cells (HUCB-NSC) and the presence of various migration-related receptors. Immunocytochemical analysis revealed abundant expression of CXCR4, PDGFR-alpha, PDGFR-beta, c-Met, VEGFR, IGF-1R and PSA-NCAM receptors in non-adherent population of HUCB-NSC cultured in serum free (SF) conditions (SF cells). Biological activity of selected receptors was confirmed by HUCB-NSC in vitro migration towards SDF-1 and IGF-1 ligands. Additionally, rat brain-derived homogenates have been assessed for their chemoattractive activity of HUCB-NSC. Our experiments unveiled that brain tissue was more attracted for HUCB-NSC than single ligands with higher potency of injured than intact brain. Moreover, adherent HUCB-NSC cultured in low serum (LS) conditions (LS cells) were employed to investigate an impact of different extracellular matrix (ECM) proteins on cell motility. It turned out that laminin provided most permissive microenvironment for cell migration, followed by fibronectin and gelatin. Unexpected nuclear localization of CXCR4 in SF cells prompted us to characterize intracellular pattern of this expression in relation to developmental stage of cells cultured in different conditions. Continuous culture of LS cells revealed cytoplasmatic pattern of CXCR4 expression while HUCB-NSC cultured in high serum conditions (HS cells) resulted in gradual translocation of CXCR4 from nucleus to cytoplasm and then to arising processes. Terminal differentiation of HUCB-NSC was followed by CXCR4 expression decline.

αV-class integrins exert dual roles on α5β1 integrins to strengthen adhesion to fibronectin

PubMed Central

Bharadwaj, Mitasha; Strohmeyer, Nico; Colo, Georgina P.; Helenius, Jonne; Beerenwinkel, Niko; Schiller, Herbert B.; Fässler, Reinhard; Müller, Daniel J.

2017-01-01

Upon binding to the extracellular matrix protein, fibronectin, αV-class and α5β1 integrins trigger the recruitment of large protein assemblies and strengthen cell adhesion. Both integrin classes have been functionally specified, however their specific roles in immediate phases of cell attachment remain uncharacterized. Here, we quantify the adhesion of αV-class and/or α5β1 integrins expressing fibroblasts initiating attachment to fibronectin (≤120 s) by single-cell force spectroscopy. Our data reveals that αV-class integrins outcompete α5β1 integrins. Once engaged, αV-class integrins signal to α5β1 integrins to establish additional adhesion sites to fibronectin, away from those formed by αV-class integrins. This crosstalk, which strengthens cell adhesion, induces α5β1 integrin clustering by RhoA/ROCK/myosin-II and Arp2/3-mediated signalling, whereas overall cell adhesion depends on formins. The dual role of both fibronectin-binding integrin classes commencing with an initial competition followed by a cooperative crosstalk appears to be a basic cellular mechanism in assembling focal adhesions to the extracellular matrix. PMID:28128308

Counterbalancing anti-adhesive effects of Tenascin-C through fibronectin expression in endothelial cells.

PubMed

Radwanska, Agata; Grall, Dominique; Schaub, Sébastien; Divonne, Stéphanie Beghelli-de la Forest; Ciais, Delphine; Rekima, Samah; Rupp, Tristan; Sudaka, Anne; Orend, Gertraud; Van Obberghen-Schilling, Ellen

2017-10-06

Cellular fibronectin (FN) and tenascin-C (TNC) are prominent development- and disease-associated matrix components with pro- and anti-adhesive activity, respectively. Whereas both are present in the tumour vasculature, their functional interplay on vascular endothelial cells remains unclear. We have previously shown that basally-oriented deposition of a FN matrix restricts motility and promotes junctional stability in cultured endothelial cells and that this effect is tightly coupled to expression of FN. Here we report that TNC induces FN expression in endothelial cells. This effect counteracts the potent anti-adhesive activity of TNC and leads to the assembly of a dense highly-branched subendothelial matrix that enhances tubulogenic activity. These findings suggest that pro-angiogenic remodelling of the perivascular matrix may involve TNC-induced upregulation of FN in endothelial cells.

Small interfering RNA mediated knockdown of irisin suppresses food intake and modulates appetite regulatory peptides in zebrafish.

PubMed

Sundarrajan, Lakshminarasimhan; Unniappan, Suraj

2017-10-01

Irisin is a myokine encoded in fibronectin type III domain containing 5 (FNDC5). FNDC5 forms an integral part of the muscle post-exercise, and causes an increase in energy expenditure in mammals. Irisin is abundantly expressed in cardiac and skeletal muscles and is secreted upon activation of peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1 alpha). Irisin regulates feeding behaviour and cardiovascular function in mammals. More recently, irisin has gained importance as a potential biomarker for myocardial infarction due to its abundance in cardiac muscle. The goal of this research was to determine whether irisin influences feeding, and regulates appetite regulatory peptides in zebrafish. Intraperitoneal injection of irisin [0.1, 1, 10 and 100ng/g body weight (BW)] did not affect feeding, but its knockdown using siRNA (10ng/g BW) caused a significant reduction in food intake. Knockdown of irisin reduced ghrelin and orexin-A mRNA expression, and increased cocaine and amphetamine regulated transcript mRNA expression in zebrafish brain and gut. siRNA mediated knockdown of irisin also downregulated brain derived neurotrophic factor mRNA in zebrafish. The role of endogenous irisin on food intake is likely mediated by its actions on other metabolic peptides. Collectively, these results indicate that unaltered endogenous irisin is required to maintain food intake in zebrafish. Copyright © 2017 Elsevier Inc. All rights reserved.

klf2a couples mechanotransduction and zebrafish valve morphogenesis through fibronectin synthesis

PubMed Central

Steed, Emily; Faggianelli, Nathalie; Roth, Stéphane; Ramspacher, Caroline; Concordet, Jean-Paul; Vermot, Julien

2016-01-01

The heartbeat and blood flow signal to endocardial cell progenitors through mechanosensitive proteins that modulate the genetic program controlling heart valve morphogenesis. To date, the mechanism by which mechanical forces coordinate tissue morphogenesis is poorly understood. Here we use high-resolution imaging to uncover the coordinated cell behaviours leading to heart valve formation. We find that heart valves originate from progenitors located in the ventricle and atrium that generate the valve leaflets through a coordinated set of endocardial tissue movements. Gene profiling analyses and live imaging reveal that this reorganization is dependent on extracellular matrix proteins, in particular on the expression of fibronectin1b. We show that blood flow and klf2a, a major endocardial flow-responsive gene, control these cell behaviours and fibronectin1b synthesis. Our results uncover a unique multicellular layering process leading to leaflet formation and demonstrate that endocardial mechanotransduction and valve morphogenesis are coupled via cellular rearrangements mediated by fibronectin synthesis. PMID:27221222

Osteoblast mineralization requires β1 integrin/ICAP-1–dependent fibronectin deposition

PubMed Central

Brunner, Molly; Millon-Frémillon, Angélique; Chevalier, Genevieve; Nakchbandi, Inaam A.; Mosher, Deane; Block, Marc R.

2011-01-01

The morphogenetic and differentiation events required for bone formation are orchestrated by diffusible and insoluble factors that are localized within the extracellular matrix. In mice, the deletion of ICAP-1, a modulator of β1 integrin activation, leads to severe defects in osteoblast proliferation, differentiation, and mineralization and to a delay in bone formation. Deposition of fibronectin and maturation of fibrillar adhesions, adhesive structures that accompany fibronectin deposition, are impaired upon ICAP-1 loss, as are type I collagen deposition and mineralization. Expression of β1 integrin with a mutated binding site for ICAP-1 recapitulates the ICAP-1–null phenotype. Follow-up experiments demonstrated that ICAP-1 negatively regulates kindlin-2 recruitment onto the β1 integrin cytoplasmic domain, whereas an excess of kindlin-2 binding has a deleterious effect on fibrillar adhesion formation. These results suggest that ICAP-1 works in concert with kindlin-2 to control the dynamics of β1 integrin–containing fibrillar adhesions and, thereby, regulates fibronectin deposition and osteoblast mineralization. PMID:21768292

FbpA, a novel multifunctional Listeria monocytogenes virulence factor.

PubMed

Dramsi, S; Bourdichon, F; Cabanes, D; Lecuit, M; Fsihi, H; Cossart, P

2004-07-01

Listeria monocytogenes is a Gram-positive intracellular bacterium responsible for severe opportunistic infections in humans and animals. Signature-tagged mutagenesis (STM) was used to identify a gene named fbpA, required for efficient liver colonization of mice inoculated intravenously. FbpA was also shown to be required for intestinal and liver colonization after oral infection of transgenic mice expressing human E-cadherin. fbpA encodes a 570-amino-acid polypeptide that has strong homologies to atypical fibronectin-binding proteins. FbpA binds to immobilized human fibronectin in a dose-dependent and saturable manner and increases adherence of wild-type L. monocytogenes to HEp-2 cells in the presence of exogenous fibronectin. Despite the lack of conventional secretion/anchoring signals, FbpA is detected using an antibody generated against the recombinant FbpA protein on the bacterial surface by immunofluorescence, and in the membrane compartment by Western blot analysis of cell extracts. Strikingly, FbpA expression affects the protein levels of two virulence factors, listeriolysin O (LLO) and InlB, but not that of InlA or ActA. FbpA co-immunoprecipitates with LLO and InlB, but not with InlA or ActA. Thus, FbpA, in addition to being a fibronectin-binding protein, behaves as a chaperone or an escort protein for two important virulence factors and appears as a novel multifunctional virulence factor of L. monocytogenes.

Inflammatory and fibrotic proteins proteomically identified as key protein constituents in urine and stone matrix of patients with kidney calculi.

PubMed

Boonla, Chanchai; Tosukhowong, Piyaratana; Spittau, Björn; Schlosser, Andreas; Pimratana, Chaowat; Krieglstein, Kerstin

2014-02-15

To uncover whether urinary proteins are incorporated into stones, the proteomic profiles of kidney stones and urine collected from the same patients have to be explored. We employed 1D-PAGE and nanoHPLC-ESI-MS/MS to analyze the proteomes of kidney stone matrix (n=16), nephrolithiatic urine (n=14) and healthy urine (n=3). We identified 62, 66 and 22 proteins in stone matrix, nephrolithiatic urine and healthy urine, respectively. Inflammation- and fibrosis-associated proteins were frequently detected in the stone matrix and nephrolithiatic urine. Eighteen proteins were exclusively found in the stone matrix and nephrolithiatic urine, considered as candidate biomarkers for kidney stone formation. S100A8 and fibronectin, representatives of inflammation and fibrosis, respectively, were up-regulated in nephrolithiasis renal tissues. S100A8 was strongly expressed in infiltrated leukocytes. Fibronectin was over-expressed in renal tubular cells. S100A8 and fibronectin were immunologically confirmed to exist in nephrolithiatic urine and stone matrix, but in healthy urine they were undetectable. Conclusion, both kidney stones and urine obtained from the same patients greatly contained inflammatory and fibrotic proteins. S100A8 and fibronectin were up-regulated in stone-baring kidneys and nephrolithiatic urine. Therefore, inflammation and fibrosis are suggested to be involved in the formation of kidney calculi. Copyright © 2013 Elsevier B.V. All rights reserved.

Fibronectin Extra Domain A Promotes Liver Sinusoid Repair following Hepatectomy.

PubMed

Sackey-Aboagye, Bridget; Olsen, Abby L; Mukherjee, Sarmistha M; Ventriglia, Alexander; Yokosaki, Yasuyuki; Greenbaum, Linda E; Lee, Gi Yun; Naga, Hani; Wells, Rebecca G

2016-01-01

Liver sinusoidal endothelial cells (LSECs) are the main endothelial cells in the liver and are important for maintaining liver homeostasis as well as responding to injury. LSECs express cellular fibronectin containing the alternatively spliced extra domain A (EIIIA-cFN) and increase expression of this isoform after liver injury, although its function is not well understood. Here, we examined the role of EIIIA-cFN in liver regeneration following partial hepatectomy. We carried out two-thirds partial hepatectomies in mice lacking EIIIA-cFN and in their wild type littermates, studied liver endothelial cell adhesion on decellularized, EIIIA-cFN-containing matrices and investigated the role of cellular fibronectins in liver endothelial cell tubulogenesis. We found that liver weight recovery following hepatectomy was significantly delayed and that sinusoidal repair was impaired in EIIIA-cFN null mice, especially females, as was the lipid accumulation typical of the post-hepatectomy liver. In vitro, we found that liver endothelial cells were more adhesive to cell-deposited matrices containing the EIIIA domain and that cellular fibronectin enhanced tubulogenesis and vascular cord formation. The integrin α9β1, which specifically binds EIIIA-cFN, promoted tubulogenesis and adhesion of liver endothelial cells to EIIIA-cFN. Our findings identify a role for EIIIA-cFN in liver regeneration and tubulogenesis. We suggest that sinusoidal repair is enhanced by increased LSEC adhesion, which is mediated by EIIIA-cFN.

Fibronectin Extra Domain A Promotes Liver Sinusoid Repair following Hepatectomy

PubMed Central

Sackey-Aboagye, Bridget; Olsen, Abby L.; Mukherjee, Sarmistha M.; Ventriglia, Alexander; Yokosaki, Yasuyuki; Greenbaum, Linda E.; Lee, Gi Yun; Naga, Hani

2016-01-01

Liver sinusoidal endothelial cells (LSECs) are the main endothelial cells in the liver and are important for maintaining liver homeostasis as well as responding to injury. LSECs express cellular fibronectin containing the alternatively spliced extra domain A (EIIIA-cFN) and increase expression of this isoform after liver injury, although its function is not well understood. Here, we examined the role of EIIIA-cFN in liver regeneration following partial hepatectomy. We carried out two-thirds partial hepatectomies in mice lacking EIIIA-cFN and in their wild type littermates, studied liver endothelial cell adhesion on decellularized, EIIIA-cFN-containing matrices and investigated the role of cellular fibronectins in liver endothelial cell tubulogenesis. We found that liver weight recovery following hepatectomy was significantly delayed and that sinusoidal repair was impaired in EIIIA-cFN null mice, especially females, as was the lipid accumulation typical of the post-hepatectomy liver. In vitro, we found that liver endothelial cells were more adhesive to cell-deposited matrices containing the EIIIA domain and that cellular fibronectin enhanced tubulogenesis and vascular cord formation. The integrin α9β1, which specifically binds EIIIA-cFN, promoted tubulogenesis and adhesion of liver endothelial cells to EIIIA-cFN. Our findings identify a role for EIIIA-cFN in liver regeneration and tubulogenesis. We suggest that sinusoidal repair is enhanced by increased LSEC adhesion, which is mediated by EIIIA-cFN. PMID:27741254

Complementary roles of KCa3.1 channels and β1-integrin during alveolar epithelial repair.

PubMed

Girault, Alban; Chebli, Jasmine; Privé, Anik; Trinh, Nguyen Thu Ngan; Maillé, Emilie; Grygorczyk, Ryszard; Brochiero, Emmanuelle

2015-09-04

Extensive alveolar epithelial injury and remodelling is a common feature of acute lung injury and acute respiratory distress syndrome (ARDS) and it has been established that epithelial regeneration, and secondary lung oedema resorption, is crucial for ARDS resolution. Much evidence indicates that K(+) channels are regulating epithelial repair processes; however, involvement of the KCa3.1 channels in alveolar repair has never been investigated before. Wound-healing assays demonstrated that the repair rates were increased in primary rat alveolar cell monolayers grown on a fibronectin matrix compared to non-coated supports, whereas an anti-β1-integrin antibody reduced it. KCa3.1 inhibition/silencing impaired the fibronectin-stimulated wound-healing rates, as well as cell migration and proliferation, but had no effect in the absence of coating. We then evaluated a putative relationship between KCa3.1 channel and the migratory machinery protein β1-integrin, which is activated by fibronectin. Co-immunoprecipitation and immunofluorescence experiments indicated a link between the two proteins and revealed their cellular co-distribution. In addition, we demonstrated that KCa3.1 channel and β1-integrin membrane expressions were increased on a fibronectin matrix. We also showed increased intracellular calcium concentrations as well as enhanced expression of TRPC4, a voltage-independent calcium channel belonging to the large TRP channel family, on a fibronectin matrix. Finally, wound-healing assays showed additive effects of KCa3.1 and TRPC4 inhibitors on alveolar epithelial repair. Taken together, our data demonstrate for the first time complementary roles of KCa3.1 and TRPC4 channels with extracellular matrix and β1-integrin in the regulation of alveolar repair processes.

Progesterone and gravidity differentially regulate expression of extracellular matrix components in the pregnant rat myometrium.

PubMed

Shynlova, Oksana; Mitchell, Jennifer A; Tsampalieros, Anne; Langille, B Lowell; Lye, Stephen J

2004-04-01

Myometrial growth and remodeling during pregnancy depends on increased synthesis of interstitial matrix proteins. We hypothesize that the presence of mechanical tension in a specific hormonal environment regulates the expression of extracellular matrix (ECM) components in the uterus. Myometrial tissue was collected from pregnant rats on Gestational Days 0, 12, 15, 17, 19, 21, 22, 23 (labor), and 1 day postpartum and ECM expression was analyzed by Northern blotting. Expression of fibronectin, laminin beta2, and collagen IV mRNA was low during early gestation but increased dramatically on Day 23 during labor. Expression of fibrillar collagens (type I and III) peaked Day 19 and decreased near term. In contrast, elastin mRNA remained elevated from midgestation onward. Injection of progesterone (P4) on Days 20-23 (to maintain elevated plasma P4 levels) delayed the onset of labor, caused dramatic reductions in the levels of fibronectin and laminin mRNA, and prevented the fall of collagen III mRNA levels on Day 23. Treatment of pregnant rats with the progesterone receptor antagonist RU486 on Day 19 induced preterm labor on Day 20 and a premature increase in mRNA levels of collagen IV, fibronectin, and laminin. Analysis of the uterine tissue from unilaterally pregnant rats revealed that most of the changes in ECM gene expression occurred specifically in the gravid horn. Our results show a decrease in expression of fibrillar collagens and a coordinated temporal increase in expression of components of the basement membrane near term associated with decreased P4 and increased mechanical tension. These ECM changes contribute to myometrial growth and remodeling during late pregnancy and the preparation for the synchronized contractions of labor.

Disruption of β-catenin/CBP signaling inhibits human airway epithelial-mesenchymal transition and repair.

PubMed

Moheimani, Fatemeh; Roth, Hollis M; Cross, Jennifer; Reid, Andrew T; Shaheen, Furquan; Warner, Stephanie M; Hirota, Jeremy A; Kicic, Anthony; Hallstrand, Teal S; Kahn, Michael; Stick, Stephen M; Hansbro, Philip M; Hackett, Tillie-Louise; Knight, Darryl A

2015-11-01

The epithelium of asthmatics is characterized by reduced expression of E-cadherin and increased expression of the basal cell markers ck-5 and p63 that is indicative of a relatively undifferentiated repairing epithelium. This phenotype correlates with increased proliferation, compromised wound healing and an enhanced capacity to undergo epithelial-mesenchymal transition (EMT). The transcription factor β-catenin plays a vital role in epithelial cell differentiation and regeneration, depending on the co-factor recruited. Transcriptional programs driven by the β-catenin/CBP axis are critical for maintaining an undifferentiated and proliferative state, whereas the β-catenin/p300 axis is associated with cell differentiation. We hypothesized that disrupting the β-catenin/CBP signaling axis would promote epithelial differentiation and inhibit EMT. We treated monolayer cultures of human airway epithelial cells with TGFβ1 in the presence or absence of the selective small molecule ICG-001 to inhibit β-catenin/CBP signaling. We used western blots to assess expression of an EMT signature, CBP, p300, β-catenin, fibronectin and ITGβ1 and scratch wound assays to assess epithelial cell migration. Snai-1 and -2 expressions were determined using q-PCR. Exposure to TGFβ1 induced EMT, characterized by reduced E-cadherin expression with increased expression of α-smooth muscle actin and EDA-fibronectin. Either co-treatment or therapeutic administration of ICG-001 completely inhibited TGFβ1-induced EMT. ICG-001 also reduced the expression of ck-5 and -19 independent of TGFβ1. Exposure to ICG-001 significantly inhibited epithelial cell proliferation and migration, coincident with a down regulation of ITGβ1 and fibronectin expression. These data support our hypothesis that modulating the β-catenin/CBP signaling axis plays a key role in epithelial plasticity and function. Copyright © 2015 Elsevier Ltd. All rights reserved.

An anthocyanin rich strawberry extract induces apoptosis and ROS while decreases glycolysis and fibrosis in human uterine leiomyoma cells

PubMed Central

Janjusevic, Milijana; Gasparrini, Massimiliano; Forbes-Hernandez, Tamara Y.; Mazzoni, Luca; Greco, Stefania; Giannubilo, Stefano Raffaele; Ciavattini, Andrea; Mezzetti, Bruno; Capocasa, Franco; Castellucci, Mario; Battino, Maurizio; Ciarmela, Pasquapina

2017-01-01

Uterine leiomyomas are highly prevalent benign tumors in reproductive aged women. Unfortunately, medical treatments are still limited and no preventive therapies have been developed. In the present study, we investigated the therapeutic effects of strawberry extract on uterine leiomyoma cells. Leiomyoma and myometrial cells were treated with strawberry (cultivar Alba) extract (250 μg/ml) for 48 h to measure apoptosis, reactive oxygen species (ROS), oxidative phosphorylation (OCR, oxygen consumption rate) and glycolysis (ECAR, extracellular acidification rate) as well as fibrosis associated gene and/or protein expression. In leiomyoma cells, strawberry increased the percentage of apoptotic and dead cells. Strawberry significantly increased ROS concentration in leiomyoma cells, while decreased it in myometrial cells. After strawberry treatment, leiomyoma cells showed a significant decreased rate of ECAR, while OCR was unchanged in both myometrial and leiomyoma cells. Strawberry significantly decreased collagen1A1, fibronectin and versican mRNA expression in leiomyoma cells. The reduced protein expression of fibronectin was observed by strawberry extract in leiomyoma cells as well. Furthermore, strawberry was able to reduce activin A induced fibronectin, collagen1A1, and versican as well as activin A and PAI-1 mRNA expression in leiomyoma cells. This study suggests that strawberry can be developed as therapeutic and/or preventive agent for uterine leiomyomas. PMID:28212568

An anthocyanin rich strawberry extract induces apoptosis and ROS while decreases glycolysis and fibrosis in human uterine leiomyoma cells.

PubMed

Islam, Md Soriful; Giampieri, Francesca; Janjusevic, Milijana; Gasparrini, Massimiliano; Forbes-Hernandez, Tamara Y; Mazzoni, Luca; Greco, Stefania; Giannubilo, Stefano Raffaele; Ciavattini, Andrea; Mezzetti, Bruno; Capocasa, Franco; Castellucci, Mario; Battino, Maurizio; Ciarmela, Pasquapina

2017-04-04

Uterine leiomyomas are highly prevalent benign tumors in reproductive aged women. Unfortunately, medical treatments are still limited and no preventive therapies have been developed. In the present study, we investigated the therapeutic effects of strawberry extract on uterine leiomyoma cells. Leiomyoma and myometrial cells were treated with strawberry (cultivar Alba) extract (250 μg/ml) for 48 h to measure apoptosis, reactive oxygen species (ROS), oxidative phosphorylation (OCR, oxygen consumption rate) and glycolysis (ECAR, extracellular acidification rate) as well as fibrosis associated gene and/or protein expression. In leiomyoma cells, strawberry increased the percentage of apoptotic and dead cells. Strawberry significantly increased ROS concentration in leiomyoma cells, while decreased it in myometrial cells. After strawberry treatment, leiomyoma cells showed a significant decreased rate of ECAR, while OCR was unchanged in both myometrial and leiomyoma cells. Strawberry significantly decreased collagen1A1, fibronectin and versican mRNA expression in leiomyoma cells. The reduced protein expression of fibronectin was observed by strawberry extract in leiomyoma cells as well. Furthermore, strawberry was able to reduce activin A induced fibronectin, collagen1A1, and versican as well as activin A and PAI-1 mRNA expression in leiomyoma cells. This study suggests that strawberry can be developed as therapeutic and/or preventive agent for uterine leiomyomas.

Eosinophils enhance WNT-5a and TGF-β1 genes expression in airway smooth muscle cells and promote their proliferation by increased extracellular matrix proteins production in asthma.

PubMed

Januskevicius, Andrius; Vaitkiene, Simona; Gosens, Reinoud; Janulaityte, Ieva; Hoppenot, Deimante; Sakalauskas, Raimundas; Malakauskas, Kestutis

2016-06-13

Recent studies have suggested that eosinophils may have a direct effect on airway smooth muscle cells (ASMC), causing their proliferation in patients with asthma, but the precise mechanism of the interaction between these cells remains unknown. We propose that changes in Wnt signaling activity and extracellular matrix (ECM) production may help explain these findings. Therefore, the aim of this study was to investigate the effect of eosinophils from asthmatic and non-asthmatic subjects on Wnt-5a, transforming growth factor β1 (TGF-β1), and ECM protein (fibronectin and collagen) gene expression and ASMC proliferation. A total of 18 subjects were involved in the study: 8 steroid-free asthma patients and 10 healthy subjects. Peripheral blood eosinophils were isolated using centrifugation and magnetic separation. An individual co-culture of eosinophils with human ASMC was prepared for each study subject. Adhesion of eosinophils to ASMC (evaluated by assaying eosinophil peroxidase activity) was determined following various incubation periods (30, 45, 60, 120, and 240 min). The expression of Wnt-5a, TGF-β1, and ECM protein genes in ASMC was measured using quantitative real-time polymerase chain reaction (PCR) after 24 h of co-culture. Proliferation of ASMC was measured using the Alamar blue method after 48 h and 72 h of co-culture with eosinophils. Eosinophils from asthmatic subjects demonstrated increased adhesion to ASMC compared with eosinophils from healthy subjects (p < 0.05) in vitro. The expression of Wnt-5a, TGF-β1, collagen, and fibronectin genes in ASMC was significantly higher after 24 h of co-culture with eosinophils from asthmatic subjects, while co-culture of ASMC with eosinophils from healthy subjects increased only TGF-β1 and fibronectin gene expression. ASMC proliferation was augmented after co-culture with eosinophils from asthma patients compared with co-culture with eosinophils from healthy subjects (p < 0.05). Eosinophils enhance Wnt-5a, TGF-β1, fibronectin, and collagen gene expression in ASMC and promote proliferation of these cells in asthma. ClinicalTrials.gov Identifier: NCT02648074 .

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

PubMed Central

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

2017-01-01

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

Recombinant probes for visualizing endogenous synaptic proteins in living neurons

PubMed Central

Gross, Garrett G.; Junge, Jason A.; Mora, Rudy J.; Kwon, Hyung-Bae; Olson, C. Anders; Takahashi, Terry T.; Liman, Emily R.; Ellis-Davies, Graham C.R.; McGee, Aaron W.; Sabatini, Bernardo L.; Roberts, Richard W.; Arnold, Don B.

2013-01-01

Summary The ability to visualize endogenous proteins in living neurons provides a powerful means to interrogate neuronal structure and function. Here we generate recombinant antibody-like proteins, termed FingRs (Fibronectin intrabodies generated with mRNA display), that bind endogenous neuronal proteins PSD-95 and Gephyrin with high affinity and which, when fused to GFP, allow excitatory and inhibitory synapses to be visualized in living neurons. Design of the FingR incorporates a novel transcriptional regulation system that ties FingR expression to the level of the target and reduces background fluorescence. In dissociated neurons and brain slices FingRs generated against PSD-95 and Gephyrin did not affect the expression patterns of their endogenous target proteins or the number or strength of synapses. Together, our data indicate that PSD-95 and Gephyrin FingRs can report the localization and amount of endogenous synaptic proteins in living neurons and thus may be used to study changes in synaptic strength in vivo. PMID:23791193

Evidence for the in vivo polymerization of ependymin: a brain extracellular glycoprotein.

PubMed

Shashoua, V E; Hesse, G W; Milinazzo, B

1990-07-09

Ependymin, a glycoprotein of the brain extracellular fluid, has been implicated in synaptic changes associated with the consolidation process of long-term memory formation and the activity-dependent sharpening of connections of regenerating optic nerve. In vitro experiments have demonstrated that ependymin has the capacity to form fibrous insoluble polymers (FIP) when the solvent Ca2+ concentration is reduced by the addition of EGTA. Such products, once formed, do not dissolve in 2% sodium dodecyl sulfate (SDS) in 5 M urea. This property was used to develop a method for isolating brain FIP. A reproducible quantity of FIP was found in goldfish and mouse brain. This was highly concentrated in the synaptosomal fraction and had identical immunoreactivity properties to FIP obtained by the polymerization of pure ependymin in vitro as well as a cross-reactivity to other protein components of the extracellular matrix such as fibronectin and laminin. Labeling studies with [35S]methionine showed that labeled FIP aggregates are synthesized in vivo and become associated with the synaptosomal fraction. A comparison of the amino acid sequence of ependymin with those for proteins of the extracellular matrix indicated that common sequences 5-6 amino acids long exist in the molecules. These homologies may explain why antibodies to fibronectin, laminin and tubulin can recognize the FIP prepared from pure ependymin. These results suggest that ependymin can polymerize in vivo to form FIP aggregates which have similar immunoreactivity properties to major components of the brain extracellular matrix.

Fibronectin Matrix Remodeling in the Regulation of the Inflammatory Response within the Lung: An Early Step in Lung Cancer Progression

DTIC Science & Technology

2011-09-01

such as that which occurs in chronic obstructive pulmonary disease (COPD) and emphysema , is associated with increased risk of lung cancer. These...effect of the fibronectin III-1c peptide on the expression of inflammatory genes by human lung fibroblasts, lung cancer cells, and pulmonary ...CXCR2 in bleomycin-induced pulmonary inflammation and fibrosis. Am J Respir Cell Mol Biol. 2009; 40:410-21. 7. Barnes PJ. New therapies for chronic

Cerebrovascular Remodeling and Neuroinflammation is a Late Effect of Radiation-Induced Brain Injury in Non-Human Primates.

PubMed

Andrews, Rachel N; Metheny-Barlow, Linda J; Peiffer, Ann M; Hanbury, David B; Tooze, Janet A; Bourland, J Daniel; Hampson, Robert E; Deadwyler, Samuel A; Cline, J Mark

2017-05-01

Fractionated whole-brain irradiation (fWBI) is a mainstay of treatment for patients with intracranial neoplasia; however late-delayed radiation-induced normal tissue injury remains a major adverse consequence of treatment, with deleterious effects on quality of life for affected patients. We hypothesize that cerebrovascular injury and remodeling after fWBI results in ischemic injury to dependent white matter, which contributes to the observed cognitive dysfunction. To evaluate molecular effectors of radiation-induced brain injury (RIBI), real-time quantitative polymerase chain reaction (RT-qPCR) was performed on the dorsolateral prefrontal cortex (DLPFC, Brodmann area 46), hippocampus and temporal white matter of 4 male Rhesus macaques (age 6-11 years), which had received 40 Gray (Gy) fWBI (8 fractions of 5 Gy each, twice per week), and 3 control comparators. All fWBI animals developed neurologic impairment; humane euthanasia was elected at a median of 6 months. Radiation-induced brain injury was confirmed histopathologically in all animals, characterized by white matter degeneration and necrosis, and multifocal cerebrovascular injury consisting of perivascular edema, abnormal angiogenesis and perivascular extracellular matrix deposition. Herein we demonstrate that RIBI is associated with white matter-specific up-regulation of hypoxia-associated lactate dehydrogenase A (LDHA) and that increased gene expression of fibronectin 1 (FN1), SERPINE1 and matrix metalloprotease 2 (MMP2) may contribute to cerebrovascular remodeling in late-delayed RIBI. Additionally, vascular stability and maturation associated tumor necrosis super family member 15 (TNFSF15) and vascular endothelial growth factor beta (VEGFB) mRNAs were increased within temporal white matter. We also demonstrate that radiation-induced brain injury is associated with decreases in white matter-specific expression of neurotransmitter receptors SYP, GRIN2A and GRIA4. We additionally provide evidence that macrophage/microglial mediated neuroinflammation may contribute to RIBI through increased gene expression of the macrophage chemoattractant CCL2 and macrophage/microglia associated CD68. Global patterns in cerebral gene expression varied significantly between regions examined (P < 0.0001, Friedman's test), with effects most prominent within cerebral white matter.

Functional integrins from normal and glycosylation-deficient baby hamster kidney cells. Terminal processing of asparagine-linked oligosaccharides is not correlated with fibronectin-binding activity.

PubMed

Koyama, T; Hughes, R C

1992-12-25

We have examined the properties of the alpha 5 beta 1 integrin of baby hamster kidney (BHK) cells, a ricin-resistant variant Ric14 lacking N-acetylglucosaminyl transferase I, and hence unable to complete assembly of hybrid- or complex-type N-glycans, and BHK cells treated with 1-deoxymannojirimycin (dMM), an inhibitor of Golgi mannosidases involved in the initial processing of N-glycan precursors. Comparable amounts of alpha 5 beta 1 integrin were isolated from these cells by chromatography of detergent extracts on a fibronectin cell-binding fragment affinity column and elution with EDTA. The alpha 5 beta 1 integrin obtained from normal BHK cells by fibronectin affinity chromatography contained mainly endoglycosidase H-resistant oligosaccharides, whereas in RicR14 cells or dMM-treated BHK cells these were entirely endoglycosidase H-sensitive. Analysis of lactoperoxidase labeled or long term biosynthetically 35S-labeled proteins from cultures of normal or glycosylation deficient cells showed similar steady state levels of alpha 5 beta 1 integrin and expression at the cell surface. Pulse-chase experiments in normal BHK cells showed rapid conversion of the alpha 5 subunit into a mature form containing oligosaccharides resistant to endoglycosidase H and slower maturation of a precursor beta 1 subunit, as in other cell types. In Ric14 cells the precursor beta 1 subunit was found to carry glycans larger than the fully processed Man5GlcNAc2 glycan of the mature subunit, indicating that the bulk precursor pool had not been translocated into the cis-Golgi compartment containing mannosidase I. We conclude that in BHK cells terminal oligosaccharide processing of alpha 5 beta 1 integrin subunits is not required for dimer formation, surface expression, and fibronectin binding, and that expression of the glycosylation defect of Ric14 cells on the alpha 5 beta 1 integrin does not account for the reduced adhesiveness of these cells on fibronectin compared with normal and dMM-treated BHK cells.

Molecular cloning of NILE glycoprotein and evidence for its continued expression in mature rat CNS.

PubMed

Prince, J T; Alberti, L; Healy, P A; Nauman, S J; Stallcup, W B

1991-11-01

The NILE glycoprotein is a rat neuronal cell adhesion molecule which has been reported to be very similar in structure, function, and distribution to the mouse L1 glycoprotein. Here we report the complete nucleotide sequence of the NILE message (5,208 nucleotides) and the deduced amino acid sequence of the NILE polypeptide (1,257 amino acids). The predicted NILE protein is 96% identical to L1 at the amino acid level, confirming that the two molecules are homologues. The sequence information shows that NILE is a transmembrane molecule with an extensive ectodomain and a much smaller cytoplasmic domain. The extracellular portion of the molecule contains six immunoglobulin C-2 type domains followed by five fibronectin type III repeats. These two structural motifs are characteristic of several other cell adhesion molecules. The cytoplasmic tails of NILE and L1 are identical to each other and distinct from the cytoplasmic regions of all other cell adhesion molecules except Ng-CAM and neuroglian. Several possible sites for phosphorylation are present in the cytoplasmic tail of NILE. Antisera were produced against two NILE-beta-galactosidase fusion proteins containing distinct segments of the NILE polypeptide: the cytoplasmic domain and the segment containing fibronectin type III repeats. Immunoblots with these antisera and Northern blots with a NILE cDNA probe indicate that NILE continues to be expressed in most areas of the mature rat brain. This contradicts previous immunofluorescence data, which suggested that NILE was substantially down-regulated in maturing nerve fiber tracts. This raises the possibility that NILE could be masked in situ by interactions with other cell surface molecules.

Leishmania infection modulates beta-1 integrin activation and alters the kinetics of monocyte spreading over fibronectin.

PubMed

Figueira, Cláudio Pereira; Carvalhal, Djalma Gomes Ferrão; Almeida, Rafaela Andrade; Hermida, Micely d' El-Rei; Touchard, Dominique; Robert, Phillipe; Pierres, Anne; Bongrand, Pierre; dos-Santos, Washington L C

2015-08-07

Contact with Leishmania leads to a decreases in mononuclear phagocyte adherence to connective tissue. In this work, we studied the early stages of bond formation between VLA4 and fibronectin, measured the kinetics of membrane alignment and the monocyte cytoplasm spreading area over a fibronectin-coated surface, and studied the expression of high affinity integrin epitope in uninfected and Leishmania-infected human monocytes. Our results show that the initial VLA4-mediated interaction of Leishmania-infected monocyte with a fibronectin-coated surface is preserved, however, the later stage, leukocyte spreading over the substrate is abrogated in Leishmania-infected cells. The median of spreading area was 72 [55-89] μm(2) for uninfected and 41 [34-51] μm(2) for Leishmania-infected monocyte. This cytoplasm spread was inhibited using an anti-VLA4 blocking antibody. After the initial contact with the fibronectrin-coated surface, uninfected monocyte quickly spread the cytoplasm at a 15 μm(2) s(-1) ratio whilst Leishmania-infected monocytes only made small contacts at a 5.5 μm(2) s(-1) ratio. The expression of high affinity epitope by VLA4 (from 39 ± 21% to 14 ± 3%); and LFA1 (from 37 ± 32% to 18 ± 16%) molecules was reduced in Leishmania-infected monocytes. These changes in phagocyte function may be important for parasite dissemination and distribution of lesions in leishmaniasis.

Mechanism of mast cell adhesion to human tenocytes in vitro.

PubMed

Behzad, Hayedeh; Tsai, Shu-Huei; Nassab, Paulina; Mousavizadeh, Rouhollah; McCormack, Robert G; Scott, Alex

2015-01-01

Mast cells and fibroblasts are two key players involved in many fibrotic and degenerative disorders. In the present study we examined the nature of binding interactions between human mast cells and tendon fibroblasts (tenocytes). In the mast cell-fibroblast co-culture model, mast cells were shown to spontaneously bind to tenocytes, in a process that was partially mediated by α5β1 integrin receptors. The same receptors on mast cells significantly mediated binding of these cells to tissue culture plates in the presence of tenocyte-conditioned media; the tenocyte-derived fibronectin in the media was shown to also play a major role in these binding activities. Upon binding to tenocytes or tissue culture plates, mast cells acquired an elongated phenotype, which was dependent on α5β1 integrin and tenocyte fibronectin. Additionally, tenocyte-derived fibronectin significantly enhanced mRNA expression of the adhesion molecule, THY1, by mast cells. Our data suggests that α5β1 integrin mediates binding of mast cells to human tenocyte and to tenocyte-derived ECM proteins, in particular fibronectin. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Neurite outgrowth on a fibronectin isoform expressed during peripheral nerve regeneration is mediated by the interaction of paxillin with α4β1 integrins

PubMed Central

Vogelezang, Mariette; Forster, Ulrike B; Han, Jaewon; Ginsberg, Mark H; ffrench-Constant, Charles

2007-01-01

Background The regeneration of peripheral nerve is associated with a change in the alternative splicing of the fibronectin primary gene transcript to re-express embryonic isoforms containing a binding site for α4β1 integrins that promote neurite outgrowth. Here we use PC12 cells to examine the role of the interaction between paxillin and the α4 integrin cytoplasmic domain in neurite outgrowth. Results Expression of α4 with mutations in the paxillin-binding domain reduced neurite outgrowth on recombinant embryonic fibronectin fragments relative to wild type α4. Over-expression of paxillin promoted neurite outgrowth while a mutant isoform lacking the LD4 domain implicated in the regulation of ARF and Rac GTPases was less effective. Optimal α4-mediated migration in leucocytes requires spatial regulation of α4 phosphorylation at Ser988, a post-translational modification that blocks paxillin binding to the integrin cytoplasmic domain. In keeping with this α4(S988D), which mimics phosphorylated α4, did not promote neurite outgrowth. However, α4 was not phosphorylated in the PC12 cells, and a non-phosphorylatable α4(S988A) mutant promoted neurite outgrowth indistinguishably from the wild type integrin. Conclusion We establish the importance of the α4 integrin-paxillin interaction in a model of axonal regeneration and highlight differing dependence on phosphorylation of α4 for extension of neuronal growth cones and migration of non-neural cells. PMID:17603879

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

PubMed

Matsui, Futoshi; Babitz, Stephen A; Rhee, Audrey; Hile, Karen L; Zhang, Hongji; Meldrum, Kirstan K

2017-01-01

STAT3 is a transcription factor implicated in renal fibrotic injury, but the role of STAT3 in mesenchymal stem cell (MSC)-induced renoprotection during renal fibrosis remains unknown. We hypothesized that MSCs protect against obstruction-induced renal fibrosis by downregulating STAT3 activation and STAT3-induced matrix metalloproteinase-9 (MMP-9) expression. Male Sprague-Dawley rats underwent renal arterial injection of vehicle or MSCs (1 × 10 6 /rat) immediately before sham operation or induction of unilateral ureteral obstruction (UUO). The kidneys were harvested after 4 wk and analyzed for collagen I and III gene expression, collagen deposition (Masson's trichrome), fibronectin, α-smooth muscle actin, active STAT3 (p-STAT3), MMP-9, and tissue inhibitor of matrix metalloproteinases 1 (TIMP-1) expression. In a separate arm, the STAT3 inhibitor S3I-201 (10 mg/kg) vs. vehicle was administered to rats intraperitoneally just after induction of UUO and daily for 14 days thereafter. The kidneys were harvested after 2 wk and analyzed for p-STAT3 and MMP-9 expression, and collagen and fibronectin deposition. Renal obstruction induced a significant increase in collagen, fibronectin, α-SMA, p-STAT3, MMP-9, and TIMP-1 expression while exogenously administered MSCs significantly reduced these indicators of obstruction-induced renal fibrosis. STAT3 inhibition with S3I-201 significantly reduced obstruction-induced MMP-9 expression and tubulointerstitial fibrosis. These results demonstrate that MSCs protect against obstruction-induced renal fibrosis, in part, by decreasing STAT3 activation and STAT3-dependent MMP-9 production. Copyright © 2017 the American Physiological Society.

Immunohistochemical expression of basement membrane proteins of verrucous carcinoma of the oral mucosa.

PubMed

Arduino, Paolo G; Carrozzo, Marco; Pagano, Marco; Broccoletti, Roberto; Scully, Crispian; Gandolfo, Sergio

2010-06-01

Squamous cell carcinoma (SCC) of the oral cavity is an extremely invasive tumour of stratified squamous epithelium that spreads throughout degradation of the basement membrane (BM) and extra-cellular matrix. Oral verrucous carcinoma (VC) is a rare low-grade variant of oral SCC that penetrates into the subepithelial connective tissue. It also has a different clinical behaviour from classical oral SCC. We investigated the immunohistochemical expression of laminin, laminin-5, collagen IV and fibronectin in VC, severe epithelial dysplasia (SED) and SCC in order to analyse if the pattern of these molecules expression contributes to the differences in the biological behaviour of these diseases. The staining pattern of laminin was less intensive in SCC compared with SED and VC, and collagen IV expression was increased in VC compared with SED. Discontinuities of laminin, collagen IV and fibronectin were more evident in SED than in VC. This study indicates that VC has a biological behaviour different from SED or SCC, observable by immunohistochemistry in the BM zone.

rse, a novel receptor-type tyrosine kinase with homology to Axl/Ufo, is expressed at high levels in the brain.

PubMed

Mark, M R; Scadden, D T; Wang, Z; Gu, Q; Goddard, A; Godowski, P J

1994-04-08

We have isolated cDNA clones that encode the human and murine forms of a novel receptor-type tyrosine kinase termed Rse. Sequence analysis indicates that human Rse contains 890 amino acids, with an extracellular region composed of two immunoglobulin-like domains followed by two fibronectin type III domains. Murine Rse contains 880 amino acids and shares 90% amino acid identity with its human counterpart. Rse is structurally similar to the receptor-type tyrosine kinase Axl/Ufo, and the two proteins have 35 and 63% sequence identity in their extracellular and intracellular domains, respectively. To study the synthesis and activation of this putative receptor-type tyrosine kinase, we constructed a version of Rse (termed gD-Rse, where gD represents glycoprotein D) that contains an NH2-terminal epitope tag. NIH3T3 cells were engineered to express gD-Rse, which could be detected at the cell surface by fluorescence-activated cell sorting. Moreover, gD-Rse was rapidly phosphorylated on tyrosine residues upon incubation of the cells with an antibody directed against the epitope tag, suggesting that rse encodes an active tyrosine kinase. In the human tissues we examined, the highest level of expression of rse mRNA was observed in the brain; rse mRNA was also detected in the premegakaryocytopoietic cell lines CMK11-5 and Dami. The gene for rse was localized to human chromosome 15.

Female receptivity phenotype of icebox mutants caused by a mutation in the L1-type cell adhesion molecule neuroglian.

PubMed

Carhan, A; Allen, F; Armstrong, J D; Hortsch, M; Goodwin, S F; O'Dell, K M C

2005-11-01

Relatively little is known about the genes and brain structures that enable virgin female Drosophila to make the decision to mate or not. Classical genetic approaches have identified several mutant females that have a reluctance-to-mate phenotype, but most of these have additional behavioral defects. However, the icebox (ibx) mutation was previously reported to lower the sexual receptivity of females, without apparently affecting any other aspect of female behavior. We have shown that the ibx mutation maps to the 7F region of the Drosophila X chromosome to form a complex complementation group with both lethal and viable alleles of neuroglian (nrg). The L1-type cell adhesion molecule encoded by nrg consists of six immunoglobulin-like domains, five fibronectin-like domains, one transmembrane domain and one alternatively spliced intracellular domain. The ibx strain has a missense mutation causing a glycine-to-arginine change at amino acid 92 in the first immunoglobulin domain of nrg. Defects in the central brain of ibx mutants are similar to those observed in another nrg mutant, central brain deranged(1) (ceb(1)). However, both ceb(1) homozygous and ceb(1)/ibx heterozygous females are receptive. The expression of a transgene containing the non-neural isoform of nrg rescues both the receptivity and the brain structure phenotypes of ibx females.

Expression, immunogenicity and variation of iron-regulated surface protein A from bovine isolates of Staphylococcus aureus.

PubMed

Misra, Neha; Wines, Tyler F; Knopp, Colton L; McGuire, Mark A; Tinker, Juliette K

2017-05-01

Staphylococcus aureus iron-regulated surface protein A (IsdA) is a fibrinogen and fibronectin adhesin that also contributes to iron sequestration and resistance to innate immunity. IsdA is conserved in human isolates and has been investigated as a human vaccine candidate. Here we report the expression of isdA, the efficacy of anti-IsdA responses and the existence of IsdA sequence variants from bovine Staphylococcus. Clinical staphylococci were obtained from US dairy farms and assayed by PCR for the presence and expression of isdA. isdA-positive species from bovines included S. aureus, S. haemolyticus and S. chromogenes. Immunoassays on bovine milk and serum confirmed the induction and opsonophagocytic activity of anti-IsdA humoral responses. The variable region of isdA was sequenced and protein alignments predicted the presence of two main variants consistent with those from human S. aureus. Mouse antibodies against one IsdA variant reduced staphylococcal binding to fibronectin in vitro in an isotype-dependent manner. Purified IsdA variants bound distinctly to fibronectin and fibrinogen. Our findings demonstrate that variability within the C-terminus of this adhesin affects immune reactivity and binding specificity, but are consistent with the significance of IsdA in bovine disease and relevant for vaccine development. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Manganese inhibits the ability of astrocytes to promote neuronal differentiation

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

Giordano, Gennaro; Pizzurro, Daniella; VanDeMark, Kathryn

Manganese (Mn) is a known neurotoxicant and developmental neurotoxicant. As Mn has been shown to accumulate in astrocytes, we sought to investigate whether Mn would alter astrocyte-neuronal interactions, specifically the ability of astrocytes to promote differentiation of neurons. We found that exposure of rat cortical astrocytes to Mn (50-500 {mu}M) impaired their ability to promote axonal and neurite outgrowth in hippocampal neurons. This effect of Mn appeared to be mediated by oxidative stress, as it was reversed by antioxidants (melatonin and PBN) and by increasing glutathione levels, while it was potentiated by glutathione depletion in astrocytes. As the extracellular matrixmore » protein fibronectin plays an important role in astrocyte-mediated neuronal neurite outgrowth, we also investigated the effect of Mn on fibronectin. Mn caused a concentration-dependent decrease of fibronectin protein and mRNA in astrocytes lysate and of fibronectin protein in astrocyte medium; these effects were also antagonized by antioxidants. Exposure of astrocytes to two oxidants, H{sub 2}O{sub 2} and DMNQ, similarly impaired their neuritogenic action, and led to a decreased expression of fibronectin. Mn had no inhibitory effect on neurite outgrowth when applied directly onto hippocampal neurons, where it actually caused a small increase in neuritogenesis. These results indicate that Mn, by targeting astrocytes, affects their ability to promote neuronal differentiation by a mechanism which is likely to involve oxidative stress.« less

Characterization of the Modular Design of the Autolysin/Adhesin Aaa from Staphylococcus Aureus

PubMed Central

Hirschhausen, Nina; Schlesier, Tim; Peters, Georg; Heilmann, Christine

2012-01-01

Background Staphylococcus aureus is a frequent cause of serious and life-threatening infections, such as endocarditis, osteomyelitis, pneumonia, and sepsis. Its adherence to various host structures is crucial for the establishment of diseases. Adherence may be mediated by a variety of adhesins, among them the autolysin/adhesins Atl and Aaa. Aaa is composed of three N-terminal repeated sequences homologous to a lysin motif (LysM) that can confer cell wall attachment and a C-terminally located cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain having bacteriolytic activity in many proteins. Methodology/Principal Findings Here, we show by surface plasmon resonance that the LysM domain binds to fibrinogen, fibronectin, and vitronectin respresenting a novel adhesive function for this domain. Moreover, we demonstrated that the CHAP domain not only mediates the bacteriolytic activity, but also adherence to fibrinogen, fibronectin, and vitronectin, thus demonstrating for the first time an adhesive function for this domain. Adherence of an S. aureus aaa mutant and the complemented aaa mutant is slightly decreased and increased, respectively, to vitronectin, but not to fibrinogen and fibronectin, which might at least in part result from an increased expression of atl in the aaa mutant. Furthermore, an S. aureus atl mutant that showed enhanced adherence to fibrinogen, fibronectin, and endothelial cells also demonstrated increased aaa expression and production of Aaa. Thus, the redundant functions of Aaa and Atl might at least in part be interchangeable. Lastly, RT-PCR and zymographic analysis revealed that aaa is negatively regulated by the global virulence gene regulators agr and SarA. Conclusions/Significance We identified novel functions for two widely distributed protein domains, LysM and CHAP, i.e. the adherence to the extracellular matrix proteins fibrinogen, fibronectin, and vitronectin. The adhesive properties of Aaa might promote S. aureus colonization of host extracellular matrix and tissue, suggesting a role for Aaa in the pathogenesis of S. aureus infections. PMID:22768285

Characterization of the modular design of the autolysin/adhesin Aaa from Staphylococcus aureus.

PubMed

Hirschhausen, Nina; Schlesier, Tim; Peters, Georg; Heilmann, Christine

2012-01-01

Staphylococcus aureus is a frequent cause of serious and life-threatening infections, such as endocarditis, osteomyelitis, pneumonia, and sepsis. Its adherence to various host structures is crucial for the establishment of diseases. Adherence may be mediated by a variety of adhesins, among them the autolysin/adhesins Atl and Aaa. Aaa is composed of three N-terminal repeated sequences homologous to a lysin motif (LysM) that can confer cell wall attachment and a C-terminally located cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain having bacteriolytic activity in many proteins. Here, we show by surface plasmon resonance that the LysM domain binds to fibrinogen, fibronectin, and vitronectin respresenting a novel adhesive function for this domain. Moreover, we demonstrated that the CHAP domain not only mediates the bacteriolytic activity, but also adherence to fibrinogen, fibronectin, and vitronectin, thus demonstrating for the first time an adhesive function for this domain. Adherence of an S. aureus aaa mutant and the complemented aaa mutant is slightly decreased and increased, respectively, to vitronectin, but not to fibrinogen and fibronectin, which might at least in part result from an increased expression of atl in the aaa mutant. Furthermore, an S. aureus atl mutant that showed enhanced adherence to fibrinogen, fibronectin, and endothelial cells also demonstrated increased aaa expression and production of Aaa. Thus, the redundant functions of Aaa and Atl might at least in part be interchangeable. Lastly, RT-PCR and zymographic analysis revealed that aaa is negatively regulated by the global virulence gene regulators agr and SarA. We identified novel functions for two widely distributed protein domains, LysM and CHAP, i.e. the adherence to the extracellular matrix proteins fibrinogen, fibronectin, and vitronectin. The adhesive properties of Aaa might promote S. aureus colonization of host extracellular matrix and tissue, suggesting a role for Aaa in the pathogenesis of S. aureus infections.

High Glucose Forces a Positive Feedback Loop Connecting Akt Kinase and FoxO1 Transcription Factor to Activate mTORC1 Kinase for Mesangial Cell Hypertrophy and Matrix Protein Expression*

PubMed Central

Das, Falguni; Ghosh-Choudhury, Nandini; Dey, Nirmalya; Bera, Amit; Mariappan, Meenalakshmi M.; Kasinath, Balakuntalam S.; Ghosh Choudhury, Goutam

2014-01-01

High glucose-induced Akt acts as a signaling hub for mesangial cell hypertrophy and matrix expansion, which are recognized as cardinal signatures for the development of diabetic nephropathy. How mesangial cells sustain the activated state of Akt is not clearly understood. Here we show Akt-dependent phosphorylation of the transcription factor FoxO1 by high glucose. Phosphorylation-deficient, constitutively active FoxO1 inhibited the high glucose-induced phosphorylation of Akt to suppress the phosphorylation/inactivation of PRAS40 and mTORC1 activity. In contrast, dominant negative FoxO1 increased the phosphorylation of Akt, resulting in increased mTORC1 activity similar to high glucose treatment. Notably, FoxO1 regulates high glucose-induced protein synthesis, hypertrophy, and expression of fibronectin and PAI-1. High glucose paves the way for complications of diabetic nephropathy through the production of reactive oxygen species (ROS). We considered whether the FoxO1 target antioxidant enzyme catalase contributes to sustained activation of Akt. High glucose-inactivated FoxO1 decreases the expression of catalase to increase the production of ROS. Moreover, we show that catalase blocks high glucose-stimulated Akt phosphorylation to attenuate the inactivation of FoxO1 and PRAS40, resulting in the inhibition of mTORC1 and mesangial cell hypertrophy and fibronectin and PAI-1 expression. Finally, using kidney cortices from type 1 diabetic OVE26 mice, we show that increased FoxO1 phosphorylation is associated with decreased catalase expression and increased fibronectin and PAI-1 expression. Together, our results provide the first evidence for the presence of a positive feedback loop for the sustained activation of Akt involving inactivated FoxO1 and a decrease in catalase expression, leading to increased ROS and mesangial cell hypertrophy and matrix protein expression. PMID:25288788

Altered expression of extracellular matrix molecules and their receptors in chronic pancreatitis and pancreatic adenocarcinoma in comparison with normal pancreas.

PubMed

Shimoyama, S; Gansauge, F; Gansauge, S; Oohara, T; Beger, H G

1995-12-01

The aim of this study was to elucidate the expression and distribution patterns of both integrins and extracellular matrix (ECM) molecules in chronic pancreatitis (CP) and pancreatic adenocarcinoma (PC) compared with normal pancreas (NP). Expression of nine alpha-subunits (alpha 2-alpha 6, alpha V, alpha L, alpha M, and alpha X), four beta-subunits (beta 1, beta 3-beta 5), and four ECM molecules (type IV collagen, laminin, fibronectin, and vitronectin) was investigated immunohistochemically. In CP, all integrins except alpha V showed nearly the same staining patterns compared with NP. Some acinar cells in CP expressed alpha V. Whereas alpha 2, alpha 3, and alpha 6 expression was stronger and diffuse, no alpha 5 expression was seen in PC. Basement membrane (BM) showed continuous staining in CP, whereas it showed discontinuous/absent staining in PC with antitype IV collagen, laminin, and vitronectin antibodies. Some carcinoma cells showed reverse correlation between alpha 2, alpha 3, and alpha 6 expression and type IV collagen and laminin expression. Fibronectin showed diffuse stromal expression in CP and PC. Some acinar cells or duct cells in CP carcinoma cells in PC showed intracellular VN expression. These results suggest that these integrins and ECM molecules are involved in inflammatory and malignant processes in pancreas.

TGFβ1-mediated expression and alternative splicing of Fibronectin Extra Domain A in human podocyte culture.

PubMed

Madne, Tarunkumar Hemraj; Dockrell, Mark Edward Carl

2018-02-28

Alternative splicing is a fundamental phenomenon to build protein diversity in health and diseases. Extra Domain A+ Fibronectin (EDA+Fn) is an alternatively spliced form of fibronectin protein present in the extra cellular matrix (ECM) in renal fibrosis. Podocytes are spectacular cell type and play a key role in filtration and synthesise ECM proteins in renal physiology and pathology. TGFβ1 is a strong stimulator of ECM proteins in renal injury. In this study, we have investigated alternative splicing of EDA+ Fn in human podocytes in response to TGFβ1. We have performed western blotting and immunofluorescence to characterise the expression of the EDA+Fn protein, real-time PCR for RNA expression and RT-PCR to look for alternative splicing of EDA+Fn in conditionally immortalised human podocytes culture.We used TGFβ1 as a stimulator and SB431542 and SRPIN340 for inhibitory studies. In this work, for the first time we have demonstrated in human podocytes culture EDA+Fn is expressed in the basal condition and TGFβ1 2.5ng/ml induced the Fn mRNA and EDA+Fn protein expression demonstrated by real-time PCR, western blotting and immunofluorescence. TGFβ1 2.5ng/ml induced the alternative splicing of EDA+Fn shown by conventional RT-PCR. Studies with ALK5 inhibitor SB431542 and SRPIN340 show that TGFβ1 induced alternative splicing of EDA+Fn was by the ALK5 receptor and the SR proteins.  In human podocytes culture, alternative splicing of EDA+Fn occurs at basal conditions and TGFβ1 further induced the alternative splicing of EDA+Fn via ALK5 receptor activation and SR proteins. This is the first evidence of basal and TGFβ1 mediated alternative splicing of EDA+Fn in human podocytes culture.

Impaired Integrin-mediated Adhesion and Signaling in Fibroblasts Expressing a Dominant-negative Mutant PTP1B

PubMed Central

Arregui, Carlos O.; Balsamo, Janne; Lilien, Jack

1998-01-01

To investigate the role of nonreceptor protein tyrosine phosphatase 1B (PTP1B) in β1-integrin– mediated adhesion and signaling, we transfected mouse L cells with normal and catalytically inactive forms of the phosphatase. Parental cells and cells expressing the wild-type or mutant PTP1B were assayed for (a) adhesion, (b) spreading, (c) presence of focal adhesions and stress fibers, and (d) tyrosine phosphorylation. Parental cells and cells expressing wild-type PTP1B show similar morphology, are able to attach and spread on fibronectin, and form focal adhesions and stress fibers. In contrast, cells expressing the inactive PTP1B have a spindle-shaped morphology, reduced adhesion and spreading on fibronectin, and almost a complete absence of focal adhesions and stress fibers. Attachment to fibronectin induces tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin in parental cells and cells transfected with the wild-type PTP1B, while in cells transfected with the mutant PTP1B, such induction is not observed. Additionally, in cells expressing the mutant PTP1B, tyrosine phosphorylation of Src is enhanced and activity is reduced. Lysophosphatidic acid temporarily reverses the effects of the mutant PTP1B, suggesting the existence of a signaling pathway triggering focal adhesion assembly that bypasses the need for active PTP1B. PTP1B coimmunoprecipitates with β1-integrin from nonionic detergent extracts and colocalizes with vinculin and the ends of actin stress fibers in focal adhesions. Our data suggest that PTP1B is a critical regulatory component of integrin signaling pathways, which is essential for adhesion, spreading, and formation of focal adhesions. PMID:9813103

Leishmania infection modulates beta-1 integrin activation and alters the kinetics of monocyte spreading over fibronectin

PubMed Central

Figueira, Cláudio Pereira; Carvalhal, Djalma Gomes Ferrão; Almeida, Rafaela Andrade; Hermida, Micely d’ El-Rei; Touchard, Dominique; Robert, Phillipe; Pierres, Anne; Bongrand, Pierre; dos-Santos, Washington LC

2015-01-01

Contact with Leishmania leads to a decreases in mononuclear phagocyte adherence to connective tissue. In this work, we studied the early stages of bond formation between VLA4 and fibronectin, measured the kinetics of membrane alignment and the monocyte cytoplasm spreading area over a fibronectin-coated surface, and studied the expression of high affinity integrin epitope in uninfected and Leishmania-infected human monocytes. Our results show that the initial VLA4-mediated interaction of Leishmania-infected monocyte with a fibronectin-coated surface is preserved, however, the later stage, leukocyte spreading over the substrate is abrogated in Leishmania-infected cells. The median of spreading area was 72 [55–89] μm2 for uninfected and 41 [34–51] μm2 for Leishmania-infected monocyte. This cytoplasm spread was inhibited using an anti-VLA4 blocking antibody. After the initial contact with the fibronectrin-coated surface, uninfected monocyte quickly spread the cytoplasm at a 15 μm2 s−1 ratio whilst Leishmania-infected monocytes only made small contacts at a 5.5 μm2 s−1 ratio. The expression of high affinity epitope by VLA4 (from 39 ± 21% to 14 ± 3%); and LFA1 (from 37 ± 32% to 18 ± 16%) molecules was reduced in Leishmania-infected monocytes. These changes in phagocyte function may be important for parasite dissemination and distribution of lesions in leishmaniasis. PMID:26249106

[The influence of immobilized fibronectin on karyotypic variability of two rat kangaroo kidney cell lines].

PubMed

Polianskaia, G G; Goriachaia, T S; Pinaev, G P

2007-01-01

The numerical and structural karyotypic variability has been investigated in "markerless" Rat kangaroo kidney cell lines NBL-3-17 and NBL-3-11 when cultivating on a fibronectin-coated surface. In cell line NBL-3-17, cultivated on the fibronectin-coated surface for 1, 2, 4 and 8 days, the character of cell distribution for the chromosome number has changed. These changes involve a significant decrease in frequency of cells with modal number of chromosomes, and an increase in frequency of cells with lower chromosomal number. Many new additional structural variants of the karyotype (SVK) appear. The observed alterations seem to be due preference adhesion of cells with lower chromosome number, disturbances of mitotic apparatus and selection of SVK, which are more adopted to changes in culture conditions. Detachment of cells from the fibronectin-coated surface, followed by 5 days cultivation on a hydrophilic surface restored control distribution. In cell line NBL-3-11, cultivated on the fibronectin-coated surface for 1, 2, 4 and 8 days, the character of numerical karyotypic variability did not change compared to control variants. In cell line NBL-3-17 the frequency of chromosomal aberrations under cultivation on the fibronectin-coated surface for 1, 2, 4 and 8 days did not change relative to control variants. In cell line NBL-3-11 the frequency of chromosomal aberrations under the same conditions significantly increases, mainly at the expence of chromosomal, chromatid breaks and dicentrics (telomeric association) relative to control variants. We discuss possible reasons of differences in the character of numerical and structural karyotypic variability between cell lines NBL-3-17 (hypotriploid) and NBL-3-11 (hypodiploid) under cultivation on fibronectin. The reasons of the observed interline karyotypic differences possibly consist in peculiarity of karyotypic structure of cell line NBL-3-11 and in the change of gene expression, namely in a dose of certain functioning genes in the hypotryploid cell line NBL-3-17.

Electrically evoked local muscle contractions cause an increase in hippocampal BDNF.

PubMed

Maekawa, Takahiro; Ogasawara, Riki; Tsutaki, Arata; Lee, Kihyuk; Nakada, Satoshi; Nakazato, Koichi; Ishii, Naokata

2018-05-01

High-intensity exercise has recently been shown to cause an increase in brain-derived neurotropic factor (BDNF) in the hippocampus. Some studies have suggested that myokines secreted from contracting skeletal muscle, such as irisin (one of the truncated form of fibronectin type III domain-containing protein 5 (FNDC5)), play important roles in this process. Thus, we hypothesized that locally evoked muscle contractions may cause an increase of BDNF in the hippocampus through some afferent mechanisms. Under anesthesia, Sprague-Dawley rats were fixed on a custom-made dynamometer and their triceps surae muscles were made to maximally contract via delivery of electric stimulations of the sciatic nerve (100 Hz with 1-ms pulse and 3-s duration). Following 50 repeated maximal isometric contractions, the protein expressions of BDNF and activation of its receptor in the hippocampus significantly increased compared with the sham-operated control rats. However, the expression of both BDNF and FNDC5 within stimulated muscles did not significantly increase, nor did their serum concentrations change. These results indicate that local muscular contractions under unconsciousness can induce BDNF expression in the hippocampus. This effect may be mediated by peripheral reception of muscle contraction, but not by systemic factors.

Alpha lipoic acid selectively inhibits proliferation and adhesion to fibronectin of v-H-ras-transformed 3Y1 cells.

PubMed

Yamasaki, Masao; Iwase, Masahiro; Kawano, Kazuo; Sakakibara, Yoichi; Suiko, Masahito; Nishiyama, Kazuo

2012-05-01

Here, we focused on the effects of racemic α-lipoic acid on proliferation and adhesion properties of 3Y1 rat fibroblasts and the v-H-ras-transformed derivative, HR-3Y1-2 cells. Racemic α-lipoic acid inhibited proliferation of HR-3Y1-2 but not 3Y1 cells at 0.3 and 1.0 mM. R-(+)-α-lipoic acid also inhibited proliferation of HR-3Y1-2 cells equivalent to that of racemic α-lipoic acid. In addition, racemic α-lipoic acid decreased intracellular reactive oxygen species levels in HR-3Y1 cells but not 3Y1 cells. Next, we evaluated the effects of racemic α-lipoic acid on cell adhesion to fibronectin. The results indicated that racemic α-lipoic acid decreased adhesive ability of HR-3Y1-2 cells to fibronectin-coated plates. As blocking antibody experiment revealed that β1-integrin plays a key role in cell adhesion in this experimental system, the effects of racemic α-lipoic acid on the expression of β1-integrin were examined. The results indicated that racemic α-lipoic acid selectively downregulated the expression of cell surface β1-integrin expression in HR-3Y1-2 cells. Intriguingly, exogenous hydrogen peroxide upregulated cell surface β1-integrin expression in 3Y1 cells. Taken together, these data suggest that reduction of intracellular reactive oxygen species levels by α-lipoic acid could be an effective means of ameliorating abnormal growth and adhesive properties in v-H-ras transformed cells.

Fish Rhabdovirus Cell Entry Is Mediated by Fibronectin

PubMed Central

Bearzotti, Monique; Delmas, Bernard; Lamoureux, Annie; Loustau, Anne-Marie; Chilmonczyk, Stefan; Bremont, Michel

1999-01-01

Three monoclonal antibodies (MAbs) generated against rainbow trout gonad cells (RTG-2) have been selected for their ability to protect cells from the viral hemorrhagic septicemia virus (VHSV) infection, a salmonid rhabdovirus. Protection from infection was restricted to the salmonid-derived cell lines indicating species specificity of the blocking MAbs. Surprisingly, the blocking activity of these MAbs was also effective against other nonantigenically related fish rhabdoviruses. Indirect immunofluorescence and immunoelectron microscopy observations demonstrated that the three MAbs were all directed against an abundant cell plasma membrane component, and immunoprecipitation studies indicated that the target consisted of a heterodimeric complex with molecular masses of 200 and 44 kDa. Biochemical data provided the following evidence that fibronectin is part of this complex and that it could represent the main receptor for fish rhabdoviruses. (i) An antiserum generated against the 200-kDa protein reacted against the recombinant rainbow trout fibronectin expressed in Escherichia coli. (ii) The purified rainbow trout fibronectin was able to bind specifically to VHSV. To our knowledge, this is the first identification of a cellular component acting as a primary receptor for a virus replicating in lower vertebrates and, more interestingly, for viruses belonging to the Rhabdoviridae family. PMID:10438860

Attachment, invasion, chemotaxis, and proteinase expression of B16-BL6 melanoma cells exhibiting a low metastatic phenotype after exposure to dietary restriction of tyrosine and phenylalanine.

PubMed

Uhlenkott, C E; Huijzer, J C; Cardeiro, D J; Elstad, C A; Meadows, G G

1996-03-01

We previously reported that low levels of tyrosine (Tyr) and phenylalanine (Phe) alter the metastatic phenotype of B16-BL6 (BL6) murine melanoma and select for tumor cell populations with decreased lung colonizing ability. To more specifically characterize the effects of Tyr and Phe restriction on the malignant phenotype of BL6, we investigated in vitro attachment, invasion, proteinase expression, and chemotaxis of high and low metastatic BL6 variants. High metastatic variant cells were isolated from subcutaneous tumors of mice fed a nutritionally complete diet (ND cells) and low metastatic variant cells were isolated from mice fed a diet restricted in Tyr and Phe (LTP cells). Results indicate that attachment to reconstituted basement membrane (Matrigel) was significantly reduced in LTP cells as compared to ND cells. Attachment to collagen IV, laminin, and fibronectin were similar between the two variants. Invasion through Matrigel and growth factor-reduced Matrigel were significantly decreased in LTP cells as compared to ND cells. Zymography revealed the presence of M(r) 92,000 and M(r) 72,000 progelatinases, tissue plasminogen activator, and urokinase plasminogen activator in the conditioned medium of both variants; however, there were no differences in activity of these secreted proteinases between the two variants. Growth of the variants on growth factor-reduced Matrigel similarly induced expression of the M(r) 92,000 progelatinase. The variants exhibited similar chemotactic responses toward laminin. However, the chemotactic response toward fibronectin by LTP cells was significantly increased. MFR5, a monoclonal antibody which selectively blocks function of the alpha 5 chain of the alpha 5 beta 1 integrin, VLA-5, decreased the chemotactic response toward fibronectin of ND cells by 37%; the chemotactic response by LTP cells was reduced by 49%. This effect was specific for fibronectin-mediated chemotaxis since the chemotaxis toward laminin and invasion through Matrigel were not altered by the presence of MFR5. The surface expression of VLA-5 was significantly increased in LTP cells as compared to ND cells by flow cytometric analysis. These observations suggest that limitation of Tyr and Phe either directly modifies BL6 or selects for subpopulations with altered in vitro invasion, chemotaxis, and integrin expression.

Reinke's edema: investigations on the role of MIB-1 and hepatocyte growth factor.

PubMed

Artico, M; Bronzetti, E; Ionta, B; Bruno, M; Greco, A; Ruoppolo, G; De Virgilio, A; Longo, L; De Vincentiis, M

2010-07-08

Reinke's edema is a benign disease of the human vocal fold, which mainly affects the sub-epithelial layer of the vocal fold. Microscopic observations show a strongly oedematous epithelium with loosened intercellular junctions, a disruption of the extracellular connections between mucosal epithelium and connective tissue, closely adherent to the thyroarytenoid muscle. Thickening of the basal layer of epithelium, known as Reinke's space, high deposition of fibronectin and chronic inflammatory infiltration it is also visible. We analyzed, together with the hepatocyte growth factor (HGF), the expression level of MIB-1 in samples harvested from patients affected by Reinke's edema, in order to define its biological role and consider it as a possible prognostic factor in the follow-up after surgical treatment. We observed a moderate expression of HGF in the lamina propria of the human vocal fold and in the basal membrane of the mucosal epithelium. Our finding suggests that this growth factor acts as an antifibrotic agent in Reinke's space and affects the fibronectin deposition in the lamina propria. MIB-1, on the contrary, showed a weak expression in the basement membrane of the mucosal epithelium and a total absence in the lamina propria deep layer, thus suggesting that only the superficial layer is actively involved in the reparatory process with a high regenerative capacity, together with a high deposition of fibronectin. The latter is necessary for the cellular connections reconstruction, after the inflammatory infiltration.

Reinke's Edema: investigations on the role of MIB-1 and hepatocyte growth factor

PubMed Central

Artico, M.; Bronzetti, E.; Ionta, B.; Bruno, M.; Greco, A.; Ruoppolo, G.; De Virgilio, A.; Longo, L.; De Vincentiis, M.

2010-01-01

Reinke's edema is a benign disease of the human vocal fold, which mainly affects the sub-epithelial layer of the vocal fold. Microscopic observations show a strongly oedematous epithelium with loosened intercellular junctions, a disruption of the extracellular connections between mucosal epithelium and connective tissue, closely adherent to the thyroarytenoid muscle. Thickening of the basal layer of epithelium, known as Reinke's space, high deposition of fibronectin and chronic inflammatory infiltration it is also visible. We analyzed, together with the hepatocyte growth factor (HGF), the expression level of MIB-1 in samples harvested from patients affected by Reinke's edema, in order to define its biological role and consider it as a possible prognostic factor in the follow-up after surgical treatment. We observed a moderate expression of HGF in the lamina propria of the human vocal fold and in the basal membrane of the mucosal epithelium. Our finding suggests that this growth factor acts as an anti - fibrotic agent in Reinke's space and affects the fibronectin deposition in the lamina propria. MIB-1, on the contrary, showed a weak expression in the basement membrane of the mucosal epithelium and a total absence in the lamina propria deep layer, thus suggesting that only the superficial layer is actively involved in the reparatory process with a high regenerative capacity, together with a high deposition of fibronectin. The latter is necessary for the cellular connections reconstruction, after the inflammatory infiltration. PMID:20819770

Biological Signatures of Brain Damage Associated with High Serum Ferritin Levels in Patients with Acute Ischemic Stroke and Thrombolytic Treatment

PubMed Central

Millán, Mónica; Sobrino, Tomás; Arenillas, Juan Francisco; Rodríguez-Yáñez, Manuel; García, María; Nombela, Florentino; Castellanos, Mar; de la Ossa, Natalia Pérez; Cuadras, Patricia; Serena, Joaquín; Castillo, José; Dávalos, Antoni

2008-01-01

Background and purpose: Increased body iron stores have been related to greater oxidative stress and brain injury in clinical and experimental cerebral ischemia and reperfusion. We aimed to investigate the biological signatures of excitotoxicity, inflammation and blood brain barrier disruption potentially associated with high serum ferritin levels-related damage in acute stroke patients treated with i.v. t-PA. Methods: Serum levels of ferritin (as index of increased cellular iron stores), glutamate, interleukin-6, matrix metalloproteinase-9 and cellular fibronectin were determined in 134 patients treated with i.v. t-PA within 3 hours from stroke onset in blood samples obtained before t-PA treatment, at 24 and 72 hours. Results: Serum ferritin levels before t-PA infusion correlated to glutamate (r = 0.59, p < 0.001) and interleukin-6 (r = 0.55, p <0.001) levels at baseline, and with glutamate (r = 0.57,p <0.001), interleukin-6 (r = 0.49,p <0.001), metalloproteinase-9 (r = 0.23, p = 0.007) and cellular fibronectin (r = 0.27, p = 0.002) levels measured at 24 hours and glutamate (r = 0.415, p < 0.001), interleukin-6 (r = 0.359, p < 0.001) and metalloproteinase-9 (r = 0.261, p = 0.004) at 72 hours. The association between ferritin and glutamate levels remained after adjustment for confounding factors in generalized linear models. Conclusions: Brain damage associated with increased iron stores in acute ischemic stroke patients treated with iv. tPA may be mediated by mechanisms linked to excitotoxic damage. The role of inflammation, blood brain barrier disruption and oxidative stress in this condition needs further research. PMID:19096131

Fibronectin regulates Wnt7a signaling and satellite cell expansion

PubMed Central

Bentzinger, C. Florian; Wang, Yu Xin; von Maltzahn, Julia; Soleimani, Vahab D.; Yin, Hang; Rudnicki, Michael A.

2012-01-01

SUMMARY The influence of the extracellular matrix (ECM) within the stem cell niche remains poorly understood. We found that Syndecan-4 (Sdc4) and Frizzled-7 (Fzd7) form a co-receptor complex in satellite cells and that binding of the ECM glycoprotein Fibronectin (FN) to Sdc4 stimulates the ability of Wnt7a to induce the symmetric expansion of satellite stem cells. Newly activated satellite cells dynamically remodel their niche by transient high-level expression of FN. Knockdown of FN in prospectively isolated satellite cells severely impaired their ability to repopulate the satellite cell niche. Conversely, in vivo over-expression of FN with Wnt7a dramatically stimulated the expansion of satellite stem cells in regenerating muscle. Therefore, activating satellite cells remodel their niche through autologous expression of FN that provides feedback to stimulate Wnt7a signaling through the Fzd7/Sdc4 co-receptor complex. Thus, FN and Wnt7a together regulate the homeostatic levels of satellite stem cells and satellite myogenic cells during regenerative myogenesis. PMID:23290138

Inhalation of Carbon Black Nanoparticles Aggravates Pulmonary Inflammation in Mice

PubMed Central

Saputra, Devina; Yoon, Jin-ha; Park, Hyunju; Heo, Yongju; Yang, Hyoseon; Lee, Eun Ji; Lee, Sangjin; Song, Chang-Woo; Lee, Kyuhong

2014-01-01

An increasing number of recent studies have focused on the impact of particulate matter on human health. As a model for atmospheric particulate inhalation, we investigated the effects of inhaled carbon black nanoparticles (CBNP) on mice with bleomycin-induced pulmonary fibrosis. The CNBPs were generated by a novel aerosolization process, and the mice were exposed to the aerosol for 4 hours. We found that CBNP inhalation exacerbated lung inflammation, as evidenced by histopathology analysis and by the expression levels of interleukin-6 protein, fibronectin, and interferon-γ mRNAs in lung tissues. Notably, fibronectin mRNA expression showed a statistically significant increase in expression after CBNP exposure. These data suggest that the concentration of CBNPs delivered (calculated to be 12.5 μg/m3) can aggravate lung inflammation in mice. Our results also suggest that the inhalation of ultrafine particles like PM 2.5 is an impactful environmental risk factor for humans, particularly in susceptible populations with predisposing lung conditions. PMID:25071917

Role of extracellular matrix in regulation of staurosporine-induced apoptosis in breast cancer cells.

PubMed

Vasaturo, F; Malacrino, C; Sallusti, E; Coppotelli, G; Birarelli, P; Giuffrida, A; Albonici, L; Simonelli, L; Modesti, A; Modesti, M; Scarpa, S

2005-04-01

Autocrine and paracrine mechanisms modulate the synthesis and secretion of extracellular matrix (ECM); moreover, each component of the ECM is capable of modulating the synthesis and release of other ECM molecules. Therefore, the synthesis of ECM glycoprotein fibronectin and laminin was studied in the human breast cancer cell lines MCF7 and MDA MB 23, plated on different ECM. Our results showed that the cells plated on a fibronectin substrate increased laminin synthesis: this event correlated with an increase in alpha2 and alpha3 integrin subunits. Staurosporine-induced apoptosis was then analyzed in the cell lines plated on different ECM. Staurosporine treatment determined the apoptosis of 35 and 33% respectively of MDA MB 231 and MCF7; these values increased to 60 and 64% in cells plated on laminin, to 48 and 63% in cells plated on fibronectin and to 64 and 69% in cells plated on matrigel. Moreover, staurosporine treatment decreased bcl-2 expression in the cells plated on fibronectin and laminin. Yet, staurosporine treatment determined PARP cleavage and PARP partial disappearance when the cells were plated on matrigel. Finally, a partial loss of function mutant Ras protein that activated only Raf pathway, was expressed in MCF7, in order to identify whether the increase of apoptosis induced by extracellular matrix involved the Raf/MAP kinase pathway. The increase of apoptosis of the cells plated on matrigel suggested that the activation of the Raf pathway is probably involved in the decrease of survival on matrigel. These data demonstrate that the modification of ECM modulates the apoptotic process of breast cancer cells and suggest that it is worthwhile to dissect the role of ECM in the control of apoptotic process.

MT1-MMP regulates the turnover and endocytosis of extracellular matrix fibronectin

PubMed Central

Shi, Feng; Sottile, Jane

2011-01-01

The extracellular matrix (ECM) is dynamically remodeled by cells during development, normal tissue homeostasis and in a variety of disease processes. We previously showed that fibronectin is an important regulator of ECM remodeling. The deposition and/or polymerization of fibronectin into the ECM controls the deposition and stability of other ECM molecules. In addition, agents that inhibit fibronectin polymerization promote the turnover of fibronectin fibrils and enhance ECM fibronectin endocytosis and intracellular degradation. Endocytosis of ECM fibronectin is regulated by β1 integrins, including α5β1 integrin. We have examined the role of extracellular proteases in regulating ECM fibronectin turnover. Our data show that membrane type matrix metalloproteinase 1 (MT1-MMP; also known as MMP14) is a crucial regulator of fibronectin turnover. Cells lacking MT1-MMP show reduced turnover and endocytosis of ECM fibronectin. MT1-MMP regulates ECM fibronectin remodeling by promoting extracellular cleavage of fibronectin and by regulating α5β1-integrin endocytosis. Our data also show that fibronectin polymerization stabilizes fibronectin fibrils and inhibits ECM fibronectin endocytosis by inhibiting α5β1-integrin endocytosis. These data are the first to show that an ECM protein and its modifying enzyme can regulate integrin endocytosis. These data also show that integrin trafficking plays a major role in modulating ECM fibronectin remodeling. The dual dependence of ECM fibronectin turnover on extracellular proteolysis and endocytosis highlights the complex regulatory mechanisms that control ECM remodeling to ensure maintenance of proper tissue function. PMID:22159414

Adhesion mechanisms in embryogenesis and in cancer invasion and metastasis.

PubMed

Thiery, J P; Boyer, B; Tucker, G; Gavrilovic, J; Valles, A M

1988-01-01

Cell-substratum and cell-cell adhesion mechanisms contribute to the development of animal form. The adhesive status of embryonic cells has been analysed during epithelial-mesenchymal cell interconversion and in cell migrations. Clear-cut examples of the modulation of cell adhesion molecules (CAMs) have been described at critical periods of morphogenesis. In chick embryos the three primary CAMs (N-CAM. L-CAM and N-cadherin) present early in embryogenesis are expressed later in a defined pattern during morphogenesis and histogenesis. The axial mesoderm derived from gastrulating cells expresses increasing amounts of N-cadherin and N-CAM. During metamerization these two adhesion molecules become abundant at somitic cell surfaces. Both CAMs are functional in an in vitro aggregation assay; however, the calcium-dependent adhesion molecule N-cadherin is more sensitive to perturbation by specific antibodies. Neural crest cells which separate from the neural epithelium lose their primary CAMs in a defined time-sequence. Adhesion to fibronectins via specific surface receptors becomes a predominant interaction during the migratory process, while some primary and secondary CAMs are expressed de novo during the ontogeny of the peripheral nervous system. In vitro, different fibronectin functional domains have been identified in the attachment, spreading and migration of neural crest cells. The fibronectin receptors which transduce the adhesive signals play a key role in the control of cell movement. All these results have prompted us to examine whether similar mechanisms operate in carcinoma cell invasion and metastasis. In vitro, rat bladder transitional carcinoma cells convert reversibly into invasive mesenchymal cells. A rapid modulation of adhesive properties is found during the epithelial-mesenchymal carcinoma cell interconversion. The different model systems analysed demonstrate that a limited repertoire of adhesion molecules, expressed in a well-defined spatiotemporal pattern, is involved in tissue formation and in key processes of tumour spread.

Dietary docosahexaenoic acid ameliorates, but rapeseed oil and safflower oil accelerate renal injury in stroke-prone spontaneously hypertensive rats as compared with soybean oil, which is associated with expression for renal transforming growth factor-beta, fibronectin and renin.

PubMed

Miyazaki, M; Takemura, N; Watanabe, S; Hata, N; Misawa, Y; Okuyama, H

2000-01-03

We have noted that n-3 fatty acid-rich oils, such as fish oil, perilla oil and flaxseed oil as well as ethyl docosahexaenoate (DHA) prolonged the survival time of stroke-prone spontaneously hypertensive rats (SHRSP) rats by approximately 10% as compared with linoleate (n-6)-rich safflower oil. Rapeseed oil with a relatively low n-6/n-3 ratio unusually shortened the survival time by approximately 40%, suggesting the presence of minor components unfavorable to SHRSP rats. This study examined the effects of dietary oils and DHA on renal injury and gene expression related to renal injury in SHRSP rats. Rats fed rapeseed oil- and safflower oil-supplemented diets developed more severe proteinuria than those fed soybean oil-supplemented diet used as a control, but there were no significant differences in blood pressure. In contrast, the DHA-supplemented diet inhibited the development of proteinuria and suppressed hypertension. The mRNA levels for renal TGF-beta, fibronectin and renin were higher in the rapeseed oil and safflower oil groups after 9 weeks of feeding of the experimental diet than in the soybean oil and DHA groups. The fatty acid composition of kidney phospholipids was markedly affected by these diets. These results indicate that the renal injury observed in the groups fed safflower oil with a high n-6/n-3 ratio and rapeseed oil with presumed minor components is accompanied by increased expression of the TGF-beta, renin and fibronectin genes, and that dietary DHA suppresses renal injury and gene expression as compared with soybean oil.

The eighth fibronectin type III domain of protein tyrosine phosphatase receptor J influences the formation of protein complexes and cell localization.

PubMed

Iuliano, Rodolfo; Raso, Cinzia; Quintiero, Alfina; Pera, Ilaria Le; Pichiorri, Flavia; Palumbo, Tiziana; Palmieri, Dario; Pattarozzi, Alessandra; Florio, Tullio; Viglietto, Giuseppe; Trapasso, Francesco; Croce, Carlo Maria; Fusco, Alfredo

2009-03-01

Regulation of receptor-type phosphatases can involve the formation of higher-order structures, but the exact role played in this process by protein domains is not well understood. In this study we show the formation of different higher-order structures of the receptor-type phosphatase PTPRJ, detected in HEK293A cells transfected with different PTPRJ expression constructs. In the plasma membrane PTPRJ forms dimers detectable by treatment with the cross-linking reagent BS(3) (bis[sulfosuccinimidyl]suberate). However, other PTPRJ complexes, dependent on the formation of disulfide bonds, are detected by treatment with the oxidant agent H(2)O(2) or by a mutation Asp872Cys, located in the eighth fibronectin type III domain of PTPRJ. A deletion in the eighth fibronectin domain of PTPRJ impairs its dimerization in the plasma membrane and increases the formation of PTPRJ complexes dependent on disulfide bonds that remain trapped in the cytoplasm. The deletion mutant maintains the catalytic activity but is unable to carry out inhibition of proliferation on HeLa cells, achieved by the wild type form, since it does not reach the plasma membrane. Therefore, the intact structure of the eighth fibronectin domain of PTPRJ is critical for its localization in plasma membrane and biological function.

Cerebrovascular Remodeling and Neuroinflammation is a Late Effect of Radiation-Induced Brain Injury in Non-Human Primates

PubMed Central

Andrews, Rachel N.; Metheny-Barlow, Linda J.; Peiffer, Ann M.; Hanbury, David B.; Tooze, Janet A.; Bourland, J. Daniel; Hampson, Robert E.; Deadwyler, Samuel A.; Cline, J. Mark

2017-01-01

Andrews, R. N., Metheny-Barlow, L. J., Peiffer, A. M., Hanbury, D. B., Tooze, J. A., Bourland, J. D., Hampson, R. E., Deadwyler, S. A. and Cline, J. M. Cerebrovascular Remodeling and Neuroinflammation is a Late Effect of Radiation-Induced Brain Injury in Non-Human Primates. Radiat. Res. 187, 599–611 (2017). Fractionated whole-brain irradiation (fWBI) is a mainstay of treatment for patients with intracranial neoplasia; however late-delayed radiation-induced normal tissue injury remains a major adverse consequence of treatment, with deleterious effects on quality of life for affected patients. We hypothesize that cerebrovascular injury and remodeling after fWBI results in ischemic injury to dependent white matter, which contributes to the observed cognitive dysfunction. To evaluate molecular effectors of radiation-induced brain injury (RIBI), real-time quantitative polymerase chain reaction (RT-qPCR) was performed on the dorsolateral prefrontal cortex (DLPFC, Brodmann area 46), hippocampus and temporal white matter of 4 male Rhesus macaques (age 6–11 years), which had received 40 Gray (Gy) fWBI (8 fractions of 5 Gy each, twice per week), and 3 control comparators. All fWBI animals developed neurologic impairment; humane euthanasia was elected at a median of 6 months. Radiation-induced brain injury was confirmed histopathologically in all animals, characterized by white matter degeneration and necrosis, and multifocal cerebrovascular injury consisting of perivascular edema, abnormal angiogenesis and perivascular extracellular matrix deposition. Herein we demonstrate that RIBI is associated with white matter-specific up-regulation of hypoxia-associated lactate dehydrogenase A (LDHA) and that increased gene expression of fibronectin 1 (FN1), SERPINE1 and matrix metalloprotease 2 (MMP2) may contribute to cerebrovascular remodeling in late-delayed RIBI. Additionally, vascular stability and maturation associated tumor necrosis super family member 15 (TNFSF15) and vascular endothelial growth factor beta (VEGFB) mRNAs were increased within temporal white matter. We also demonstrate that radiation-induced brain injury is associated with decreases in white matter-specific expression of neurotransmitter receptors SYP, GRIN2A and GRIA4. We additionally provide evidence that macrophage/microglial mediated neuroinflammation may contribute to RIBI through increased gene expression of the macrophage chemoattractant CCL2 and macrophage/ microglia associated CD68. Global patterns in cerebral gene expression varied significantly between regions examined (P < 0.0001, Friedman’s test), with effects most prominent within cerebral white matter. PMID:28398880

Brain-Derived Neurotrophic Factor Val66Met Human Polymorphism Impairs the Beneficial Exercise-Induced Neurobiological Changes in Mice.

PubMed

Ieraci, Alessandro; Madaio, Alessandro I; Mallei, Alessandra; Lee, Francis S; Popoli, Maurizio

2016-12-01

Several studies have shown that exercise improves cognitive functions and emotional behaviors. Positive effects of exercise have been associated with enhanced brain plasticity, adult hippocampal neurogenesis, and increased levels of brain-derived neurotrophic factor (BDNF). However, a substantial variability of individual response to exercise has been described, which may be accounted for by individual genetic variants. Here, we have assessed whether and how the common human BDNF Val66Met polymorphism influences the neurobiological effects modulated by exercise in BDNF Val66Met knock-in male mice. Wild-type (BDNF Val/Val ) and homozygous BDNF Val66Met (BDNF Met/Met ) male mice were housed in cages equipped with or without running wheels for 4 weeks. Changes in behavioral phenotype, hippocampal adult neurogenesis, and gene expression were evaluated in exercised and sedentary control mice. We found that exercise reduced the latency to feed in the novelty suppressed feeding and the immobility time in the forced swimming test in BDNF Val/Val but not in BDNF Met/Met mice. Hippocampal neurogenesis was reduced in BDNF Met/Met mice compared with BDNF Val/Val mice. BDNF Met/Met mice had lower basal BDNF protein levels in the hippocampus, which was not recovered following exercise. Moreover, exercise-induced expression of total BDNF, BDNF splice variants 1, 2, 4, 6 and fibronectin type III domain-containing protein 5 (FNDC5) mRNA levels were absent or reduced in the dentate gyrus of BDNF Met/Met mice. Exercise failed to enhance PGC-1α and FNDC5 mRNA levels in the BDNF Met/Met muscle. Overall these results indicate that, in adult male mice, the BDNF Val66Met polymorphism impairs the beneficial behavioral and neuroplasticity effects induced by physical exercise.

Brain-Derived Neurotrophic Factor Val66Met Human Polymorphism Impairs the Beneficial Exercise-Induced Neurobiological Changes in Mice

PubMed Central

Ieraci, Alessandro; Madaio, Alessandro I; Mallei, Alessandra; Lee, Francis S; Popoli, Maurizio

2016-01-01

Several studies have shown that exercise improves cognitive functions and emotional behaviors. Positive effects of exercise have been associated with enhanced brain plasticity, adult hippocampal neurogenesis, and increased levels of brain-derived neurotrophic factor (BDNF). However, a substantial variability of individual response to exercise has been described, which may be accounted for by individual genetic variants. Here, we have assessed whether and how the common human BDNF Val66Met polymorphism influences the neurobiological effects modulated by exercise in BDNF Val66Met knock-in male mice. Wild-type (BDNFVal/Val) and homozygous BDNF Val66Met (BDNFMet/Met) male mice were housed in cages equipped with or without running wheels for 4 weeks. Changes in behavioral phenotype, hippocampal adult neurogenesis, and gene expression were evaluated in exercised and sedentary control mice. We found that exercise reduced the latency to feed in the novelty suppressed feeding and the immobility time in the forced swimming test in BDNFVal/Val but not in BDNFMet/Met mice. Hippocampal neurogenesis was reduced in BDNFMet/Met mice compared with BDNFVal/Val mice. BDNFMet/Met mice had lower basal BDNF protein levels in the hippocampus, which was not recovered following exercise. Moreover, exercise-induced expression of total BDNF, BDNF splice variants 1, 2, 4, 6 and fibronectin type III domain-containing protein 5 (FNDC5) mRNA levels were absent or reduced in the dentate gyrus of BDNFMet/Met mice. Exercise failed to enhance PGC-1α and FNDC5 mRNA levels in the BDNFMet/Met muscle. Overall these results indicate that, in adult male mice, the BDNF Val66Met polymorphism impairs the beneficial behavioral and neuroplasticity effects induced by physical exercise. PMID:27388329

Fibronectin is a survival factor for differentiated osteoblasts

NASA Technical Reports Server (NTRS)

Globus, R. K.; Doty, S. B.; Lull, J. C.; Holmuhamedov, E.; Humphries, M. J.; Damsky, C. H.

1998-01-01

The skeletal extracellular matrix produced by osteoblasts contains the glycoprotein fibronectin, which regulates the adhesion, differentiation and function of various adherent cells. Interactions with fibronectin are required for osteoblast differentiation in vitro, since fibronectin antagonists added to cultures of immature fetal calvarial osteoblasts inhibit their progressive differentiation. To determine if fibronectin plays a unique role in fully differentiated osteoblasts, cultures that had already formed mineralized nodules in vitro were treated with fibronectin antagonists. Fibronectin antibodies caused >95% of the cells in the mature cultures to display characteristic features of apoptosis (nuclear condensation, apoptotic body formation, DNA laddering) within 24 hours. Cells appeared to acquire sensitivity to fibronectin antibody-induced apoptosis as a consequence of differentiation, since antibodies failed to kill immature cells and the first cells killed were those associated with mature nodules. Intact plasma fibronectin, as well as fragments corresponding to the amino-terminal, cell-binding, and carboxy-terminal domains of fibronectin, independently induced apoptosis of mature (day-13), but not immature (day-4), osteoblasts. Finally, transforming growth factor-beta1 partially protected cells from the apoptotic effects of fibronectin antagonists. Thus, in the course of maturation cultured osteoblasts switch from depending on fibronectin for differentiation to depending on fibronectin for survival. These data suggest that fibronectin, together with transforming growth factor-beta1, may affect bone formation, in part by regulating the survival of osteoblasts.

Binding of the fibronectin-mimetic peptide, PR_b, to α5β1 on pig islet cells increases fibronectin production and facilitates internalization of PR_b functionalized liposomes

PubMed Central

Atchison, Nicole A.; Fan, Wei; Papas, Klearchos K.; Hering, Bernhard J.; Tsapatsis, Michael; Kokkoli, Efrosini

2010-01-01

Islet transplantation is a promising treatment for type 1 diabetes. Recent studies have demonstrated that human islet allografts can restore insulin independence to patients with this disease. As islet isolation and immunotherapeutic techniques improve, the demand for this cell-based therapy will dictate the need for other sources of islets. Pig islets could provide an unlimited supply for xenotransplantation and have shown promise as an alternative to human islet allografts. However, stresses imposed during islet isolation and transplantation decrease islet viability, leading to loss of graft function. In this study, we investigated the ability of a fibronectin-mimetic peptide, PR_b, which specifically binds to the α5β1 integrin, to reestablish lost extracellular matrix (ECM) around isolated pig islets and increase internalization of liposomes. Confocal microscopy and western blotting were used to show the presence of the integrin α5β1 on the pig islets on day 0 (day of isolation), as well as different days of islet culture. Islets cultured in medium supplemented with free PR_b for 48 hours were found to have increased levels of ECM fibronectin secretion compared to islets in normal culture conditions. Using confocal microscopy and flow cytometry we found that PR_b peptide-amphiphile functionalized liposomes delivered to the pig islets internalized into the cells in a PR_b concentration dependent manner, and non-functionalized liposomes showed minimal internalization. These studies proved that the fibronectin-mimetic peptide, PR_b, is an appropriate peptide bullet for applications involving α5β1 expressing pig islet cells. Fibronectin production stimulated through α5β1 PR_b binding may decrease apoptosis and therefore increase islet viability in culture. In addition, PR_b peptide-amphiphile functionalized liposomes may be used for targeted delivery of different agents to pig islet cells. PMID:20704278

Cellular Interactions and Immune Response of Spherical Nucleic Acid (SNA) Nanoconjugates

NASA Astrophysics Data System (ADS)

Massich, Matthew David

Spherical nucleic acid (SNA) nanoconjugates consist of a densely packed monolayer shell of highly-oriented oligonucleotides covalently bound to a gold nanoparticle core. The nanoconjugates exhibit several important qualities, which make them useful for various biological applications, such as antisense gene regulation strategies and the intracellular detection of biomolecules. The focus of this thesis was to characterize the nanoconjugates interaction with cultured cells and specifically the immune response to their intracellular presence. The immune response of macrophage cells to internalized nanoconjugates was studied, and due to the dense functionalization of oligonucleotides on the surface of the nanoparticle and the resulting high localized salt concentration the innate immune response to the nanoconjugates is ˜25-fold less when compared to a lipoplex carrying the same sequence. Additionally, genome-wide expression profiling was used to study the biological response of cultured cells to the nanoconjugates. The biological response of HeLa cells to gold nanoparticles stabilized by weakly bound ligands was significant, yet when these same nanoparticles were stably functionalized with covalently attached oligonucleotides the cells showed no measurable response. In human keratinocytes, the oligonucleotide sequences caused 427 genes to be differentially expressed when complexed with Dharmafect, but when the oligonucleotides were conjugated to nanoparticles only 7 genes were differentially expressed. Beyond characterizing the cellular interactions and immune response of the nanoconjugates, the optimal length of siRNA (from 19--34 base pairs) that induces the most gene knockdown while maintaining limited immune activation was determined to be 24 base pairs. Further, the SNAs were shown to be useful as a potential antiviral gene therapy by demonstrating approximately 50% knockdown of the Ebola VP35 gene. Lastly, a scanning probe-enabled method was used to rapidly create nanoscale fibronectin patterns over large areas with a range of feature sizes, thereby opening the field of nanocombinatorics. This allowed the investigation of the relationship between fibronectin feature size and stem cell fate. MSCs cultured on nanoscale fibronectin features directed differentiation toward osteogenesis to a greater extent than cells grown on both microscale features and cells grown on non-patterned fibronectin substrates with osteogenic inducing media, demonstrating a new method for controlling stem cell fate.

Promotion of osteoblast differentiation in 3D biomaterial micro-chip arrays comprising fibronectin-coated poly(methyl methacrylate) polycarbonate.

PubMed

Altmann, Brigitte; Steinberg, Thorsten; Giselbrecht, Stefan; Gottwald, Eric; Tomakidi, Pascal; Bächle-Haas, Maria; Kohal, Ralf-Joachim

2011-12-01

Due to the architecture of solid body tissues including bone, three-dimensional (3D) in vitro microenvironments appear favorable, since herein cell growth proceeds under more physiological conditions compared to conventional 2D systems. In the present study we show that a 3D microenvironment comprising a fibronectin-coated PMMA/PC-based micro-chip promotes differentiation of primary human osteoblasts as reflected by the densely-packed 3D bone cell aggregates and expression of biomarkers indicating osteoblast differentiation. Morphogenesis and fluorescence dye-based live/dead staining revealed homogenous cell coverage of the microcavities of the chip array, whereat cells showed high viability up to 14 days. Moreover, Azur II staining proved formation of uniform sized multilayered aggregates, exhibiting progressive intracellular deposition of extracellular bone matrix constituents comprising fibronectin, osteocalcin and osteonectin from day 7 on. Compared to 2D monolayers, osteoblasts grown in the 3D chip environment displayed differential mostly higher gene expression for osteocalcin, osteonectin, and alkaline phosphatase, while collagen type I remained fairly constant in both culture environments. Our results indicate that the 3D microenvironment, based on the PMMA biomaterial chip array promotes osteoblast differentiation, and hereby renders a promising tool for tissue-specific in vitro preconditioning of osteoblasts designated for clinically-oriented bone augmentation or regeneration. Copyright © 2011 Elsevier Ltd. All rights reserved.

Evidence for the evolution of tenascin and fibronectin early in the chordate lineage.

PubMed

Tucker, Richard P; Chiquet-Ehrismann, Ruth

2009-02-01

Fibronectin and tenascin are extracellular matrix glycoproteins that play important roles in cell adhesion and motility. In a previous study we provided evidence that tenascin first appeared early in the chordate lineage. As tenascin has been proposed to act, in part, through modulation of cell-fibronectin interactions, we sought here to identify fibronectin genes in non-vertebrate chordates and other invertebrates to determine if tenascin and fibronectin evolved separately or together, and to identify phylogenetically conserved features of both proteins. We found that the genome of the urochordate Ciona savignyi contains both a tenascin gene and a gene encoding a fibronectin-like protein with fibronectin type 1, 2 and 3 repeats. The genome of the cephalochordate Branchiostoma floridae (amphioxus) also has a tenascin gene. However, we could not identify a fibronectin-like gene in B. floridae, nor could we identify fibronectin or tenascin genes in echinoderms, protostomes or cnidarians. If urochordates are more closely related to vertebrates, tenascin may have evolved before fibronectin in an ancestor common to tunicates and amphioxus. Alternatively, tenascin and fibronectin may have evolved in an ancestor common to B. floridae and C. savignyi and the fibronectin gene was subsequently lost in the cephalochordate lineage. The fibronectin-like gene from C. savignyi does not encode the RGD motif for integrin binding found in all vertebrate fibronectins, and it lacks most of the fibronectin type 1 domains believed to be critical for fibrillogenesis. In contrast, the tenascin gene in B. floridae encodes multiple RGD motifs, suggesting that integrin binding is fundamental to tenascin function.

Mechanism of acute depletion of plasma fibronectin following thermal injury in rats. Appearance of a gelatinlike ligand in plasma.

PubMed Central

Deno, D C; McCafferty, M H; Saba, T M; Blumenstock, F A

1984-01-01

Plasma fibronectin was depleted within 15 min following sublethal burn, followed by partial recovery at 8 h and complete restoration by 24 h in anesthetized rats. Radiolabeled 75Se-plasma fibronectin, injected intravenously before burn, was rapidly sequestered in burn skin as well as the liver. Fibronectin levels at 2 h postburn as detected by immunoassay vs. 75Se-plasma fibronectin indicated that more fibronectin was in the plasma than detected by electroimmunoassay. Crossed immunoelectrophoretic analysis of fibronectin in early postburn plasma demonstrated a reduced electrophoretic mobility of the fibronectin antigen. Addition of heparin or fibrin, both of which have affinity for fibronectin, to normal plasma was unable to reproduce this altered fibronectin electrophoretic pattern. In contrast, addition of gelatin or native collagen to normal plasma reproduced the abnormal electrophoretic pattern of fibronectin seen in burn plasma. Extracts of burned skin, but not extracts of normal skin, when added to normal plasma, elicited a similar altered electrophoretic pattern for fibronectin. By gel filtration, fibronectin in burn plasma had an apparent molecular weight approximately 40% greater than that observed in normal plasma. These data suggest the release into the blood of a gelatinlike ligand from burned skin, which complexes with plasma fibronectin. Thus, fibronectin deficiency acutely postburn appears mediated by (a) its accumulation at the site of burn injury; (b) its removal from the circulation by the liver; and (c) its presence in the plasma in a form that is less detectable by immunoassay. Images PMID:6690478

Nitric oxide donors rescue diabetic nephropathy through oxidative-stress-and nitrosative-stress-mediated Wnt signaling pathways

PubMed Central

Hsu, Yung-Chien; Lee, Pei-Hsien; Lei, Chen-Chou; Ho, Cheng; Shih, Ya-Hsueh; Lin, Chun-Liang

2015-01-01

Aims/Introduction The role of the renal nitric oxide (NO) system in the pathophysiology of diabetic nephropathy constitutes a very challenging and fertile field for future investigation. The purpose of the present study was to investigate whether NO donors can attenuate diabetic renal fibrosis and apoptosis through modulating oxidative-and nitrosative-stress, and Wnt signaling using in vivo diabetic models. Materials and Methods Diabetic rat was induced by a single intraperitoneal injection of streptozotocin. Rats in each group were intraperitoneally given 2,2′-(hydroxynitrosohydrazino)bis-ethanamine (1 U/kg/day) and vehicle for 28 and 56 consecutive days. Expression of the oxidative-and nitrosative-stress, and Wnt signaling components were examined in kidneys from diabetic animals by quantitative reverse transcription polymerase chain reaction, western blot analysis and immunohistochemical staining. Results NO donor treatment significantly reduced the ratio of kidney weight to bodyweight and proteinuria. This treatment also significantly restored the suppressive effect of diabetes on urinary NO2 + NO3 levels. Immunohistochemistry showed that NO donor treatment significantly reduced transforming growth factor (TGF)-β1, fibronectin, cleaved caspase-3 and triphosphate-biotin nick end-labeling expression in the glomeruli of diabetic rats. We found that diabetes promoted 8-hydroxy-2′-deoxyguanosine, and peroxynitrite expression coincided with reduced endothelial NO synthase expression in glomeruli. Interestingly, NO donor treatment completely removed oxidative stress and nitrosative stress, and restored endothelial NO synthase expression in diabetic renal glomeruli. Immunohistomorphometry results showed that NO donor treatment significantly restored suppressed Wnt5a expression and β-catenin immunoreactivities in glomeruli. Based on laser-captured microdissection for quantitative reverse transcription polymerase chain reaction, diabetes significantly increased TGF-β1, and fibronectin expression coincided with depressed Wnt5a expression. NO donor treatment reduced TGF-β1, fibronectin activation, and the suppressing effect of diabetes on Wnt5a and β-catenin expression in renal glomeruli. Conclusions NO donor treatment alleviates extracellular matrix accumulation and apoptosis in diabetic nephropathy in vivo by not only preventing the diabetes-mediated oxidative and nitrostative stress, but also restoring downregulation of endothelial NO synthase expression and Wnt/β-catenin signaling. These findings suggest that modulation of NO is a viable alternative strategy for rescuing diabetic renal injury. PMID:25621130

The role of Mycobacterium avium complex fibronectin attachment protein in adherence to the human respiratory mucosa.

PubMed

Middleton, A M; Chadwick, M V; Nicholson, A G; Dewar, A; Groger, R K; Brown, E J; Wilson, R

2000-10-01

Mycobacterium avium complex (MAC) are opportunistic respiratory pathogens that infect non-immunocompromised patients with established lung disease, although they can also cause primary infections. The ability to bind fibronectin is conserved among many mycobacterial species. We have investigated the adherence of a sputum isolate of MAC to the mucosa of organ cultures constructed with human tissue and the contribution of M. avium fibronectin attachment protein (FAP) to the process. MAC adhered to fibrous, but not globular mucus, and to extracellular matrix (ECM) in areas of epithelial damage, but not to intact extruded cells and collagen fibres. Bacteria occasionally adhered to healthy unciliated epithelium and to cells that had degenerated exposing their contents, but never to ciliated cells. The results obtained with different respiratory tissues were similar. Two ATCC strains of MAC gave similar results. There was a significant reduction (P < 0.05) in the number of bacteria adhering to ECM after preincubation of bacteria with fibronectin and after preincubation of the tissue with M. avium FAP in a concentration-dependant manner. The number of bacteria adhering to fibrous mucus was unchanged. Immunogold labelling demonstrated fibronectin in ECM as well as in other areas of epithelial damage, but only ECM bound FAP. A Mycobacterium smegmatis strain had the same pattern of adherence to the mucosa as MAC. When the FAP gene was deleted, the strain demonstrated reduced adherence to ECM, and adherence was restored when the strain was transfected with an M. avium FAP expression construct. We conclude that MAC adheres to ECM in areas of epithelial damage via FAP and to mucus with a fibrous appearance via another adhesin. Epithelial damage exposing ECM and poor mucus clearance will predispose to MAC airway infection.

Fndc5 knockdown induced suppression of mitochondrial integrity and significantly decreased cardiac differentiation of mouse embryonic stem cells.

PubMed

Nazem, Shima; Rabiee, Farzaneh; Ghaedi, Kamran; Babashah, Sadegh; Sadeghizadeh, Majid; Nasr-Esfahani, Mohammad Hossein

2018-06-01

Fibronectin type III domain-containing 5 protein (Fndc5) is a glycosylated protein with elevated expression in high energy demanded tissues as heart, brain, and muscle. It has been shown that upregulation of Fndc5 is regulated by peroxisome proliferator-activated receptor-γ coactivator-1 alpha (PGC-1α), which is known as a master regulator of mitochondrial function and biogenesis. Also, our group indicated that Fndc5 expression increases gradually during cardiac differentiation of mouse embryonic stem cells (mESCs). In this paper, to clarify the importance of Fndc5 in cardiac differentiation, we south to knock down Fndc5 expression by generation a stably transduced mESC line that derives the expression of a short hairpin RNA (shRNA) against Fndc5 gene following doxycycline (Dox) induction. Knock-down of Fndc5 demonstrated a considerable decrease in expression of cardiac progenitor and cardiomyocyte markers. Considering the fact that mitochondria play a crucial role in cardiac differentiation of ESCs, we investigated the role of Fndc5, as a downstream target of PGC1-α, on mitochondrial indices. Results showed that expression of nuclear encoded mitochondrial genes including PGC1-α, Atp5b, Ndufb5, and SOD2 significantly decreased. Moreover, mitochondrial membrane potential (ΔΨm) and relative ATP content of cardiomyocytes decreased markedly with relative ROS level increase. Together, our results suggest that Fndc5 attenuates process of cardiac differentiation of mESCs which is associated with modulation of mitochondrial function and gene expression. © 2017 Wiley Periodicals, Inc.

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

Siddiqui, Shabeena; Ahsan, Haseeb; Khan, Mohammad Rashid

Dyslipidemia is common in patients with diabetes mellitus (DM) and is considered a risk factor for the progression of diabetic nephropathy (DN). Hyperlipidemia and hyperglycemia act synergistically to induce renal injury. The present study was designed to investigate the protective effects of tocotrienols as tocotrienol-rich fraction (TRF) extracted from palm (PO) and rice bran oils (RBO) against lipid induced nephropathy in type-2 diabetic rats and its probable molecular mechanism. Male Wistar rats (175–200 g) were divided into four groups. The first group served as diabetic control, while the second and third groups received PO-TRF and RBO-TRF, respectively by gavage overmore » a period of sixteen weeks post-induction of diabetes. The fourth group comprised of age-matched rats that served as normal control. The effects of TRF on serum lipid profile, oxidative stress markers, expression of TGF-β, fibronectin and collagen type IV were analyzed in the kidney of diabetic rats. Treatment with PO-TRF and RBO-TRF significantly improved glycemic status, serum lipid profile and renal function in type-2 diabetic rats. In addition, TRF supplementation down-regulated the expression of TGF-β, fibronectin and collagen type IV in the kidney of diabetic rats. Transforming growth factor-β (TGF-β) plays a critical role in progression of DN, but its modulation by tocotrienols in DN remains unexplored. TRF ameliorated lipid induced nephropathy in type-2 diabetes by its hypoglycemic, hypolipidemic and antioxidant activities as well as by modulation of TGF-β to prevent increased expression of collagen type IV and fibrinogen. We finally propose a mechanism for the expression of molecular markers that are significant in the events leading to diabetic nephropathy and its modulation by tocotrienols/TRF. - Highlights: • The nephroprotective effect of TRF in type-2 diabetic rats was investigated. • Treatment with TRF improved glycemic status, lipid profile and renal functions in rats. • TRF down-regulated the expression of TGF-β, fibronectin and collagen in rats' kidney. • TRF ameliorated nephropathy by hypoglycemic, hypolipidemic and antioxidant activity. • Tocotrienols modulate the expression of TGF-β in DN in type-2 diabetic rats.« less

Disentangling the multifactorial contributions of fibronectin, collagen and cyclic strain on MMP expression and extracellular matrix remodeling by fibroblasts.

PubMed

Zhang, Yang; Lin, Zhe; Foolen, Jasper; Schoen, Ingmar; Santoro, Alberto; Zenobi-Wong, Marcy; Vogel, Viola

2014-11-01

Early wound healing is associated with fibroblasts assembling a provisional fibronectin-rich extracellular matrix (ECM), which is subsequently remodeled and interlaced by type I collagen. This exposes fibroblasts to time-variant sets of matrices during different stages of wound healing. Our goal was thus to gain insight into the ECM-driven functional regulation of human foreskin fibroblasts (HFFs) being either anchored to a fibronectin (Fn) or to a collagen-decorated matrix, in the absence or presence of cyclic mechanical strain. While the cells reoriented in response to the onset of uniaxial cyclic strain, cells assembled exogenously added Fn with a preferential Fn-fiber alignment along their new orientation. Exposure of HFFs to exogenous Fn resulted in an increase in matrix metalloproteinase (MMP) expression levels, i.e. MMP-15 (RT-qPCR), and MMP-9 activity (zymography), while subsequent exposure to collagen slightly reduced MMP-15 expression and MMP-9 activity compared to Fn-exposure alone. Cyclic strain upregulated Fn fibrillogenesis and actin stress fiber formation, but had comparatively little effect on MMP activity. We thus propose that the appearance of collagen might start to steer HFFs towards homeostasis, as it decreased both MMP secretion and the tension of Fn matrix fibrils as assessed by Fluorescence Resonance Energy Transfer. These results suggest that HFFs might have a high ECM remodeling or repair capacity in contact with Fn alone (early event), which is reduced in the presence of Col1 (later event), thereby down-tuning HFF activity, a processes which would be required in a tissue repair process to finally reach tissue homeostasis. Copyright © 2014. Published by Elsevier B.V.

Gonadotropin-releasing hormone analogues inhibit leiomyoma extracellular matrix despite presence of gonadal hormones.

PubMed

Malik, Minnie; Britten, Joy; Cox, Jeris; Patel, Amrita; Catherino, William H

2016-01-01

To determine the effect of GnRH analogues (GnRH-a) leuprolide acetate (LA) and cetrorelix acetate on gonadal hormone-regulated expression of extracellular matrix in uterine leiomyoma three-dimensional (3D) cultures. Laboratory study. University research laboratory. Women undergoing hysterectomy for symptomatic leiomyomas. The 3D cell cultures, protein analysis, Western blot, immunohistochemistry. Expression of extracellular matrix proteins, collagen 1, fibronectin, and versican in leiomyoma cells 3D cultures exposed to E2, P, LA, cetrorelix acetate, and combinations for 24- and 72-hour time points. The 3D leiomyoma cultures exposed to E2 for 24 hours demonstrated an increased expression of collagen-1 and fibronectin, which was maintained for up to 72 hours, a time point at which versican was up-regulated significantly. Although P up-regulated collagen-1 protein (1.29 ± 0.04) within 24 hours of exposure, significant increase in all extracellular matrix (ECM) proteins was observed when the gonadal hormones were used concomitantly. Significant decrease in the amount of ECM proteins was observed on use of GnRH-a, LA and cetrorelix, with 24-hour exposure. Both the compounds also significantly decreased ECM protein concentration despite the presence of E2 or both gonadal hormones. This study demonstrates that GnRH-a directly affect the gonadal hormone-regulated collagen-1, fibronectin, and versican production in their presence. These findings suggest that localized therapy with GnRH-a may inhibit leiomyoma growth even in the presence of endogenous gonadal hormone exposure, thereby providing a mechanism to eliminate the hypoestrogenic side effects associated with GnRH-a therapy. Published by Elsevier Inc.

In vivo investigation on connective tissue healing to polished surfaces with different surface wettability.

PubMed

Kloss, Frank R; Steinmüller-Nethl, Doris; Stigler, Robert G; Ennemoser, Thomas; Rasse, Michael; Hächl, Oliver

2011-07-01

Connective tissue in contact to transgingival/-dermal implants presents itself as tight scar formation. Although rough surfaces support the attachment they increase bacterial colonisation as well. In contrast to surface roughness, little is known about the influence of surface wettability on soft-tissue healing in vivo. We therefore investigated the influence of different surface wettabilities on connective tissue healing at polished implant surfaces in vivo. Three polished experimental groups (titanium, titanium coated with hydrophobic nano-crystalline diamond (H-NCD) and titanium coated with hydrophilic nano-crystalline diamond (O-NCD) were inserted into the subcutaneous connective tissue of the abdominal wall of 24 rats. Animals were sacrificed after 1 and 4 weeks resulting in eight specimen per group per time point. Specimen were subjected to histological evaluation (van Giesson's staining) and immunohistochemistry staining for proliferating cell nuclear antigen (PCNA), fibronectin and tumour necrosis factor-alpha (TNF-α). Histological evaluation revealed dense scar formation at the titanium and H-NCD surfaces. In contrast, the connective tissue was loose at the O-NCD surface with a significantly higher number of cells after 4 weeks. O-NCD demonstrated a strong expression of PCNA and fibronectin but a weak expression of TNF-α. In contrast, the PCNA and fibronectin expression was low at the titanium and H-NCD, with a strong signal of TNF-α at the H-NCD surface. Hydrophilicity influences the connective tissue healing at polished implant surfaces in vivo positively. The attachment of connective tissue and the number of cells in contact to the surface were increased. Moreover, the inflammatory response is decreased at the hydrophilic surface. © 2010 John Wiley & Sons A/S.

Leptospiral outer membrane protein microarray, a novel approach to identification of host ligand-binding proteins.

PubMed

Pinne, Marija; Matsunaga, James; Haake, David A

2012-11-01

Leptospirosis is a zoonosis with worldwide distribution caused by pathogenic spirochetes belonging to the genus Leptospira. The leptospiral life cycle involves transmission via freshwater and colonization of the renal tubules of their reservoir hosts. Infection requires adherence to cell surfaces and extracellular matrix components of host tissues. These host-pathogen interactions involve outer membrane proteins (OMPs) expressed on the bacterial surface. In this study, we developed an Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 OMP microarray containing all predicted lipoproteins and transmembrane OMPs. A total of 401 leptospiral genes or their fragments were transcribed and translated in vitro and printed on nitrocellulose-coated glass slides. We investigated the potential of this protein microarray to screen for interactions between leptospiral OMPs and fibronectin (Fn). This approach resulted in the identification of the recently described fibronectin-binding protein, LIC10258 (MFn8, Lsa66), and 14 novel Fn-binding proteins, denoted Microarray Fn-binding proteins (MFns). We confirmed Fn binding of purified recombinant LIC11612 (MFn1), LIC10714 (MFn2), LIC11051 (MFn6), LIC11436 (MFn7), LIC10258 (MFn8, Lsa66), and LIC10537 (MFn9) by far-Western blot assays. Moreover, we obtained specific antibodies to MFn1, MFn7, MFn8 (Lsa66), and MFn9 and demonstrated that MFn1, MFn7, and MFn9 are expressed and surface exposed under in vitro growth conditions. Further, we demonstrated that MFn1, MFn4 (LIC12631, Sph2), and MFn7 enable leptospires to bind fibronectin when expressed in the saprophyte, Leptospira biflexa. Protein microarrays are valuable tools for high-throughput identification of novel host ligand-binding proteins that have the potential to play key roles in the virulence mechanisms of pathogens.

Production of Fibronectin by the Human Alveolar Macrophage: Mechanism for the Recruitment of Fibroblasts to Sites of Tissue Injury in Interstitial Lung Diseases

NASA Astrophysics Data System (ADS)

Rennard, Stephen I.; Hunninghake, Gary W.; Bitterman, Peter B.; Crystal, Ronald G.

1981-11-01

Because cells of the mononuclear phagocyte system are known to produce fibronectin and because alveolar macrophages are activated in many interstitial lung diseases, the present study was designed to evaluate a role for the alveolar macrophage as a source of the increased levels of fibronectin found in the lower respiratory tract in interstitial lung diseases and to determine if such fibronectin might contribute to the development of the fibrosis found in these disorders by being a chemoattractant for human lung fibroblasts. Production of fibronectin by human alveolar macrophages obtained by bronchoalveolar lavage and maintained in short-term culture in serum-free conditions was demonstrated; de novo synthesis was confirmed by the incorporation of [14C]proline. This fibronectin had a monomer molecular weight of 220,000 and was antigenically similar to plasma fibronectin. Macrophages from patients with idiopathic pulmonary fibrosis produced fibronectin at a rate 20 times higher than did normal macrophages; macrophages from patients with pulmonary sarcoidosis produced fibronectin at 10 times the normal rate. Macrophages from 6 of 10 patients with various other interstitial disorders produced fibronectin at rates greater than the rate of highest normal control. Human alveolar macrophage fibronectin was chemotactic for human lung fibroblasts, suggesting a functional role for this fibronectin in the derangement of the alveolar structures that is characteristic of these disorders.

Stretching Fibroblasts Remodels Fibronectin and Alters Cancer Cell Migration

NASA Astrophysics Data System (ADS)

Ao, Mingfang; Brewer, Bryson M.; Yang, Lijie; Franco Coronel, Omar E.; Hayward, Simon W.; Webb, Donna J.; Li, Deyu

2015-02-01

Most investigations of cancer-stroma interactions have focused on biochemical signaling effects, with much less attention being paid to biophysical factors. In this study, we investigated the role of mechanical stimuli on human prostatic fibroblasts using a microfluidic platform that was adapted for our experiments and further developed for both repeatable performance among multiple assays and for compatibility with high-resolution confocal microscopy. Results show that mechanical stretching of normal tissue-associated fibroblasts (NAFs) alters the structure of secreted fibronectin. Specifically, unstretched NAFs deposit and assemble fibronectin in a random, mesh-like arrangement, while stretched NAFs produce matrix with a more organized, linearly aligned structure. Moreover, the stretched NAFs exhibited an enhanced capability for directing co-cultured cancer cell migration in a persistent manner. Furthermore, we show that stretching NAFs triggers complex biochemical signaling events through the observation of increased expression of platelet derived growth factor receptor α (PDGFRα). A comparison of these behaviors with those of cancer-associated fibroblasts (CAFs) indicates that the observed phenotypes of stretched NAFs are similar to those associated with CAFs, suggesting that mechanical stress is a critical factor in NAF activation and CAF genesis.

Extracellular matrix components expression in human pluripotent stem cell-derived retinal organoids recapitulates retinogenesis in vivo and reveals an important role for IMPG1 and CD44 in the development of photoreceptors and interphotoreceptor matrix.

PubMed

Felemban, Majed; Dorgau, Birthe; Hunt, Nicola Claire; Hallam, Dean; Zerti, Darin; Bauer, Roman; Ding, Yuchun; Collin, Joseph; Steel, David; Krasnogor, Natalio; Al-Aama, Jumana; Lindsay, Susan; Mellough, Carla; Lako, Majlinda

2018-05-17

The extracellular matrix (ECM) plays an important role in numerous processes including cellular proliferation, differentiation, migration, maturation, adhesion guidance and axonal growth. To date, there has been no detailed analysis of the ECM distribution during retinal ontogenesis in humans and the functional importance of many ECM components is poorly understood. In this study, the expression of key ECM components in adult mouse and monkey retina, developing and adult human retina and retinal organoids derived from human pluripotent stem cells was studied. Our data indicate that basement membrane ECMs (Fibronectin and Collagen IV) were expressed in Bruch's membrane and the inner limiting membrane of the developing human retina, whilst the hyalectins (Versican and Brevican), cluster of differentiation 44 (CD44), photoreceptor-specific ECMs Interphotoreceptor Matrix Proteoglycan 1 (IMPG1) and Interphotoreceptor Matrix Proteoglycan 2 (IMPG2) were detected in the developing interphotoreceptor matrix (IPM). The expression of IMPG1, Versican and Brevican in the developing IPM was conserved between human developing retina and human pluripotent stem cell-derived retinal organoids. Blocking the action of CD44 and IMPG1 in pluripotent stem cell derived retinal organoids affected the development of photoreceptors, their inner/outer segments and connecting cilia and disrupted IPM formation, with IMPG1 having an earlier and more significant impact. Together, our data suggest an important role for IMPG1 and CD44 in the development of photoreceptors and IPM formation during human retinogenesis. The expression and the role of many extracellular matrix (ECM) components during human retinal development is not fully understood. In this study, expression of key ECM components (Collagen IV, Fibronectin, Brevican, Versican, IMPG1 and IMPG2) was investigated during human retinal ontogenesis. Collagen IV and Fibronectin were expressed in Bruch's membrane; whereas Brevican, Versican, IMPG1 & IMPG2 in the developing interphotoreceptor matrix (IPM). Retinal organoids were successfully generated from pluripotent stem cells. The expression of ECM components was examined in the retinal organoids and found to recapitulate human retinal development in vivo. Using functional blocking experiments, we were able to highlight an important role for IMPG1 and CD44 in the development of photoreceptors and IPM formation. Copyright © 2018 Acta Materialia Inc. All rights reserved.

[Morphological features of the myometrium in connective tissue dysplasia in women with uterine inertia].

PubMed

Konovalov, P V; Mitrofanova, L B; Gorshkov, A N; Ovsyannikov, F A

2015-01-01

to reveal the morphological features of the lower uterine segment myometrium in connective tissue dysplasia (CTD) in women with uterine inertia. Histological, immunohistochemical (with antibodies against collagen types I and III, matrix metalloproteinases 1 and 9 (MMR-1, MMP-9), tissue inhibitor of metalloproteinase 1 (TIMP-1), fibronectin; fibulin-5, connexin-43), electron microscopic, and electron immunocytochemical studies with morphometry of myometrial fragments from 15 parturient women with CTD and uterine inertia (a study group) and those from 10 women without CTD (a control group). The myometrium in CTD exhibited the decreased expression of connextin-43, fibulin-5, TIMP-1, collagens types I and III with collagen type III predominance and the unchanged levels of fibronectin and MMP-1 and MMP-9. Electron microscopy and immunocytochemistry showed fewer intercellular contacts and the dramatically lower expression of connexin-43 than in the control. A set of found myometrial changes in women with uterine inertia is a manifestation of CTD.

The role played by the group A streptococcal negative regulator Nra on bacterial interactions with epithelial cells.

PubMed

Molinari, G; Rohde, M; Talay, S R; Chhatwal, G S; Beckert, S; Podbielski, A

2001-04-01

Group A streptococci (GAS) specifically attach to and internalize into human epithelial host cells. In some GAS isolates, fibronectin-binding proteins were identified as being responsible for these virulence traits. In the present study, the previously identified global negative regulator Nra was shown to control the binding of soluble fibronectin probably via regulation of protein F2 and/or SfbII expression in the serotype M49 strain 591. According to results from a conventional invasion assay based on the recovery of viable intracellular bacteria, the increased fibronectin binding did not affect bacterial adherence to HEp-2 epithelial cells, but was associated with a reduction in the internalization rates. However, when examined by confocal and electron microscopy techniques, the nra-mutant bacteria were shown to exhibit higher adherence and internalization rates than the corresponding wild type. The mutant bacteria escaped from the phagocytic vacuoles much faster, promoting consistent morphological changes which resulted in severe host cell damage. The apoptotic and lytic processes observed in nra-mutant infected host cells were correlated with an increased expression of the genes encoding superantigen SpeA, the cysteine protease SpeB, and streptolysin S in the nra-mutant bacteria. Adherence and internalization rates of a nra/speB-double mutant at wild-type levels indicated that the altered speB expression in the nra mutant contributed to the observed changes in both processes. The Nra-dependent effects on bacterial virulence were confined to infections carried out with stationary growth phase bacteria. In conclusion, the obtained results demonstrated that the global GAS regulator Nra modulates virulence genes, which are involved in host cell damage. Thus, by helping to achieve a critical balance of virulence factor expression that avoids the injury of target cells, Nra may facilitate GAS persistence in a safe intracellular niche.

Release of Matrix Metalloproteinases-2 and 9 by S-Nitrosylated Caveolin-1 Contributes to Degradation of Extracellular Matrix in tPA-Treated Hypoxic Endothelial Cells

PubMed Central

Bi, Gang; Zhu, Yihui; Jun, Wei; Ma, Wenlin; Wu, Huimin

2016-01-01

Intracranial hemorrhage remains the most feared complication in tissue plasminogen activator (tPA) thrombolysis for ischemic stroke. However, the underlying molecular mechanisms are still poorly elucidated. In this study, we reported an important role of caveolin-1 (Cav-1) s-nitrosylation in matrix metalloproteinase (MMP)-2 and 9 secretion from tPA-treated ischemic endothelial cells. Brain vascular endothelial cells (bEND3) were exposed to oxygen-glucose deprivation (OGD) for 2 h before adding recombinant human tPA for 6 h. This treatment induced a significant increase of MMP2 and 9 in the media of bEND3 cells and a simultaneous degradation of fibronectin and laminin β-1, the two main components of extracellular matrix (ECM). Inhibition of MMP2 and 9 with SB-3CT completely blocked the degradation of fibronectin and laminin β-1. ODG+tPA treatment led to Cav-1 shedding from bEND3 cells into the media. Notably, OGD triggered nitric oxide (NO) production and S-nitrosylationof Cav-1 (SNCav-1). Meanwhile tPA induced activation of ERK signal pathway and stimulates the secretion of SNCav-1. Pretreatment of bEND3 cells with C-PTIO (a NO scavenger) or U0126 (a specific ERK inhibitor) significantly reduced OGD-induced S-nitrosylation of Cav-1 in cells and blocked the secretion of Cav-1 and MMP2 and 9 into the media as well as the degradation of fibronectin and laminin β-1 in OGD and tPA-treated cells. These data indicate that OGD-triggered Cav-1 S-nitrosylation interacts with tPA-induced ERK activation to augment MMP2 and 9 secretion and subsequent ECM degradation, which may account for the exacerbation of ischemic blood brain barrier damage following tPA thrombolysis for ischemic stroke. PMID:26881424

Release of Matrix Metalloproteinases-2 and 9 by S-Nitrosylated Caveolin-1 Contributes to Degradation of Extracellular Matrix in tPA-Treated Hypoxic Endothelial Cells.

PubMed

Song, Haoming; Cheng, Youjun; Bi, Gang; Zhu, Yihui; Jun, Wei; Ma, Wenlin; Wu, Huimin

2016-01-01

Intracranial hemorrhage remains the most feared complication in tissue plasminogen activator (tPA) thrombolysis for ischemic stroke. However, the underlying molecular mechanisms are still poorly elucidated. In this study, we reported an important role of caveolin-1 (Cav-1) s-nitrosylation in matrix metalloproteinase (MMP)-2 and 9 secretion from tPA-treated ischemic endothelial cells. Brain vascular endothelial cells (bEND3) were exposed to oxygen-glucose deprivation (OGD) for 2 h before adding recombinant human tPA for 6 h. This treatment induced a significant increase of MMP2 and 9 in the media of bEND3 cells and a simultaneous degradation of fibronectin and laminin β-1, the two main components of extracellular matrix (ECM). Inhibition of MMP2 and 9 with SB-3CT completely blocked the degradation of fibronectin and laminin β-1. ODG+tPA treatment led to Cav-1 shedding from bEND3 cells into the media. Notably, OGD triggered nitric oxide (NO) production and S-nitrosylationof Cav-1 (SNCav-1). Meanwhile tPA induced activation of ERK signal pathway and stimulates the secretion of SNCav-1. Pretreatment of bEND3 cells with C-PTIO (a NO scavenger) or U0126 (a specific ERK inhibitor) significantly reduced OGD-induced S-nitrosylation of Cav-1 in cells and blocked the secretion of Cav-1 and MMP2 and 9 into the media as well as the degradation of fibronectin and laminin β-1 in OGD and tPA-treated cells. These data indicate that OGD-triggered Cav-1 S-nitrosylation interacts with tPA-induced ERK activation to augment MMP2 and 9 secretion and subsequent ECM degradation, which may account for the exacerbation of ischemic blood brain barrier damage following tPA thrombolysis for ischemic stroke.

Extracellular matrix components direct porcine muscle stem cell behavior

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

Wilschut, Karlijn J.; Haagsman, Henk P.; Roelen, Bernard A.J., E-mail: b.a.j.roelen@uu.nl

2010-02-01

In muscle tissue, extracellular matrix proteins, together with the vasculature system, muscle-residence cells and muscle fibers, create the niche for muscle stem cells. The niche is important in controlling proliferation and directing differentiation of muscle stem cells to sustain muscle tissue. Mimicking the extracellular muscle environment improves tools exploring the behavior of primary muscle cells. Optimizing cell culture conditions to maintain muscle commitment is important in stem cell-based studies concerning toxicology screening, ex vivo skeletal muscle tissue engineering and in the enhancement of clinical efficiency. We used the muscle extracellular matrix proteins collagen type I, fibronectin, laminin, and also gelatinmore » and Matrigel as surface coatings of tissue culture plastic to resemble the muscle extracellular matrix. Several important factors that determine myogenic commitment of the primary muscle cells were characterized by quantitative real-time RT-PCR and immunofluorescence. Adhesion of high PAX7 expressing satellite cells was improved if the cells were cultured on fibronectin or laminin coatings. Cells cultured on Matrigel and laminin coatings showed dominant integrin expression levels and exhibited an activated Wnt pathway. Under these conditions both stem cell proliferation and myogenic differentiation capacity were superior if compared to cells cultured on collagen type I, fibronectin and gelatin. In conclusion, Matrigel and laminin are the preferred coatings to sustain the proliferation and myogenic differentiation capacity of the primary porcine muscle stem cells, when cells are removed from their natural environment for in vitro culture.« less

Myofibroblasts in interstitial lung diseases show diverse electron microscopic and invasive features.

PubMed

Karvonen, Henna M; Lehtonen, Siri T; Sormunen, Raija T; Harju, Terttu H; Lappi-Blanco, Elisa; Bloigu, Risto S; Kaarteenaho, Riitta L

2012-09-01

The characteristic features of myofibroblasts in various lung disorders are poorly understood. We have evaluated the ultrastructure and invasive capacities of myofibroblasts cultured from small volumes of diagnostic bronchoalveolar lavage (BAL) fluid samples from patients with different types of lung diseases. Cells were cultured from samples of BAL fluid collected from 51 patients that had undergone bronchoscopy and BAL for diagnostic purposes. The cells were visualized by transmission electron microscopy and immunoelectron microscopy to achieve ultrastructural localization of alpha-smooth muscle actin (α-SMA) and fibronectin. The levels of α-SMA protein and mRNA and fibronectin mRNA were measured by western blot and quantitative real-time reverse transcriptase polymerase chain reaction. The invasive capacities of the cells were evaluated. The cultured cells were either fibroblasts or myofibroblasts. The structure of the fibronexus, and the amounts of intracellular actin, extracellular fibronectin and cell junctions of myofibroblasts varied in different diseases. In electron and immunoelectron microscopy, cells cultured from interstitial lung diseases (ILDs) expressed more actin filaments and α-SMA than normal lung. The invasive capacity of the cells obtained from patients with idiopathic pulmonary fibrosis was higher than that from patients with other type of ILDs. Cells expressing more actin filaments had a higher invasion capacity. It is concluded that electron and immunoelectron microscopic studies of myofibroblasts can reveal differential features in various diseases. An analysis of myofibroblasts cultured from diagnostic BAL fluid samples may represent a new kind of tool for diagnostics and research into lung diseases.

Recognition of fibronectin by Penicillium marneffei conidia via a sialic acid-dependent process and its relationship to the interaction between conidia and laminin.

PubMed

Hamilton, A J; Jeavons, L; Youngchim, S; Vanittanakom, N

1999-10-01

Adhesion of Penicillium marneffei conidia to the extracellular matrix protein laminin via a sialic acid-dependent process has previously been demonstrated. This study describes the interaction of P. marneffei conidia with fibronectin and examines the relationship of this process to the recognition of laminin via conidia. Immunofluorescence microscopy demonstrated that fibronectin bound to the surface of conidia and to phialides, but not to hyphae, in a pattern similar to that reported for laminin. Conidia were able to bind to fibronectin immobilized on microtiter plates in a concentration-dependent manner. However, binding to fibronectin (at any given concentration of protein and conidia) was less than that to laminin under equivalent conditions. Soluble fibronectin and antifibronectin antibody inhibited adherence of conidia to fibronectin in the plate adherence assay; soluble laminin also caused pronounced inhibition. Various monosaccharides and several peptides had no effect on adherence to fibronectin. However, N-acetylneuraminic acid abolished adherence to fibronectin, indicating that the interaction was mediated through a sialic acid-dependent process; the latter parallels observations of laminin binding by conidia. Fibronectin binding (and binding of laminin) was considerably reduced by prolonged preincubation of conidia with chymotrypsin, suggesting the protein nature of the binding site. Conidia from older cultures were more adherent to both immobilized fibronectin and laminin than conidia from younger cultures. Ligand affinity binding demonstrated the presence of a 20-kDa protein with the ability to bind both fibronectin and laminin. There would therefore appear to be a common receptor for the binding of fibronectin and laminin on the surface of P. marneffei, and the interaction described here maybe important in mediating attachment of the fungus to host tissue.

Aliskiren Regulates Neonatal Fc Receptor and IgG Metabolism with Attenuation of Anti-GBM Glomerulonephritis in Mice.

PubMed

Kang, Ju Hyung; Baik, Haing Woon; Yoo, Seung-Min; Kim, Joo Heon; Cheong, Hae Il; Park, Chung-Gyu; Kang, Hee Gyung; Ha, Il-Soo

2016-01-01

Renin, in addition to its activation of the renin-angiotensin system, binds to the (pro)renin receptor (PRR) and triggers inflammatory and fibrogenic signaling in tissue. In addition, aliskiren, a direct renin inhibitor, has been shown to affect IgG metabolism by altering PRR and neonatal Fc receptors (FcRns). We investigated the effect of aliskiren on proteinuria, glomerular extracellular matrix, expressions of fibronectin, transforming growth factor β1 (TGF-β1), PRR, FcRn and renal metabolism of IgG in a mice model of anti-glomerular basement membrane glomerulonephritis (anti-GBM GN). IgG deposition and expressions of FcRn and PRR were enhanced at glomeruli and urinary IgG levels increased in anti-GBM GN. Aliskiren attenuated anti-GBM GN with reduction of proteinuria and cortical expressions of fibronectin and TGF-β1. In addition, aliskiren suppressed the renal cortical expressions of FcRn and PRR. Aliskiren also reduced the glomerular IgG depositions and the urinary IgG levels albeit with increased circulating serum IgG levels. These results suggest that suppression of FcRn and PRR and regulation of IgG metabolism may be related to the attenuation of anti-GBM GN by aliskiren. © 2016 S. Karger AG, Basel.

Phenotypic changes in neutrophils related to anti-inflammatory therapy.

PubMed

Barton, A E; Bayley, D L; Mikami, M; Llewellyn-Jones, C G; Stockley, R A

2000-01-03

Previous work from the group has shown that non-steroidal anti-inflammatory agents given to volunteers and patients inhibit PMN function possibly by affecting the developing neutrophil during the differentiation process. In this study indomethacin treatment in vivo reduced neutrophil chemotaxis and proteolytic degradation of fibronectin, with a maximal effect after 14 days. Stimulated neutrophil adherence to fibronectin was also reduced but this was not due to quantitative changes in beta(2) integrin expression or function. L-Selectin expression on resting and stimulated neutrophils was increased after 14 days and there was a small decrease in plasma levels of soluble L-selectin. These effects, however, could not be reproduced by treatment of neutrophils with indomethacin in vitro, suggesting they are due to effects on differentiating/maturing PMNs. In an attempt to interpret these changes, studies were performed with dexamethasone, which is known to alter neutrophil function and kinetics. Dexamethasone treatment reduced chemotaxis and increased superoxide generation after 1 day and was associated with increased expression of activated beta(2) integrins and reduced L-selectin expression on resting neutrophils. This suggests the appearance of mainly 'activated' cells as a result of demargination and indicates that the effects of indomethacin are distinctive and not related to changes in compartmentalisation.

Effects of mechanical strain on human mesenchymal stem cells and ligament fibroblasts in a textured poly(L-lactide) scaffold for ligament tissue engineering.

PubMed

Kreja, Ludwika; Liedert, Astrid; Schlenker, Heiter; Brenner, Rolf E; Fiedler, Jörg; Friemert, Benedikt; Dürselen, Lutz; Ignatius, Anita

2012-10-01

The purpose of this study was to prove the effect of cyclic uniaxial intermittent strain on the mRNA expression of ligament-specific marker genes in human mesenchymal stem cells (MSC) and anterior cruciate ligament-derived fibroblasts (ACL-fibroblasts) seeded onto a novel textured poly(L-lactide) scaffold (PLA scaffold). Cell-seeded scaffolds were mechanically stimulated by cyclic uniaxial stretching. The expression of ligament matrix gene markers: collagen types I and III, fibronectin, tenascin C and decorin, as well as the proteolytic enzymes matrix metalloproteinase MMP-1 and MMP-2 and their tissue specific inhibitors TIMP-1 and TIMP-2 was investigated by analysing the mRNA expression using reverse transcriptase polymerase chain reaction and related to the static control. In ACL-fibroblasts seeded on PLA, mechanical load induced up-regulation of collagen types I and III, fibronectin and tenascin C. No effect of mechanical stimulation on the expression of ligament marker genes was found in undifferentiated MSC seeded on PLA. The results indicated that the new textured PLA scaffold could transfer the mechanical load to the ACL-fibroblasts and improved their ligament phenotype. This scaffold might be suitable as a cell-carrying component of ACL prostheses.

Factor XIII as a modulator of plasma fibronectin alterations during experimental bacteremia.

PubMed

Kiener, J L; Cho, E; Saba, T M

1986-11-01

Fibronectin is found in plasma as well as in association with connective tissue and cell surfaces. Depletion of plasma fibronectin is often observed in septic trauma and burned patients, while experimental rats often manifest hyperfibronectinemia with sepsis. Since Factor XIII may influence the rate of clearance and deposition of plasma fibronectin into tissues, we evaluated the temporal changes in plasma fibronectin and plasma Factor XIII following bacteremia and RE blockade in rats in an attempt to understand the mechanism leading to elevation of fibronectin levels in bacteremic rats, which is distinct from that observed with RE blockade. Clearance of exogenously administered fibronectin after bacteremia was also determined. Rats received either saline, Pseudomonas aeruginosa (1 X 10(9) organisms), gelatinized RE test lipid emulsion (50 mg/100 gm B.W.), or emulsion followed by Pseudomonas. Plasma fibronectin and Factor XIII were determined at 0, 2, 24, and 48 hours post-blockade or bacteremia. At 24 and 48 hr following bacteremia alone or bacteremia after RE blockade, there was a significant elevation (p less than 0.05) of plasma fibronectin and a concomitant decrease (p less than 0.05) of plasma factor XIII activity. Extractable tissue fibronectin from liver and spleen was also increased at 24 and 48 hours following R.E. blockade plus bacteremia. In addition, the plasma clearance of human fibronectin was significantly prolonged (p less than 0.05) following bacterial challenge. Infusion of activated Factor XIII (20 units/rat) during a period of hyperfibronectinemia (908.0 +/- 55.1 micrograms/ml) resulted in a significant (p less than 0.05) decrease in plasma fibronectin (548.5 +/- 49.9 micrograms/ml) within 30 min. Thus Factor XIII deficiency in rats with bacteremia may contribute to the elevation in plasma fibronectin by altering kinetics associated with the clearance of fibronectin from the blood.

Intravesical Bacillus Calmette-Guérin therapy for murine bladder tumors: initiation of the response by fibronectin-mediated attachment of Bacillus Calmette-Guérin.

PubMed

Ratliff, T L; Palmer, J O; McGarr, J A; Brown, E J

1987-04-01

Intravesical Bacillus Calmette-Guérin (BCG) is considered to be one of the most effective treatments for superficial bladder cancer. Although the mechanisms by which BCG inhibits tumor growth are not known, previous studies have shown that systemic immunization to BCG and the local expression of the immune response in the bladder are associated with a favorable response to BCG therapy. We have investigated the conditions required for the initiation of an immunological response after the intravesical instillation of BCG. Initial histological studies showed that BCG attached to the bladder wall only in areas where the urothelium was damaged by electrocautery and suggested that attachment was associated with the fibrin clot. Quantitative studies verified the histological observations. Minimal BCG attachment (mean less than 10(2) colony forming units) was observed in normal bladders in contrast with a mean of 1.42 X 10(4) colony forming units/bladder in bladders damaged by electrocautery (10 separate experiments). BCG attachment to the bladder wall was durable since organisms were observed in bladders 48 h after instillation. To investigate the proteins to which BCG attached, we tested the binding of BCG to extracellular matrix and inflammatory proteins which comprise a significant portion of the fibrin clot. BCG bound in vitro to coverslips coated in vivo with extracellular matrix proteins but did not bind to control albumin-coated coverslips. BCG also bound to coverslips coated with purified plasma fibronectin but not to coverslips coated with other purified extracellular matrix proteins including laminin, fibrinogen, and type IV collagen. BCG attachment to coverslips coated with either extracellular matrix proteins or purified fibronectin was inhibited by antibodies specific for fibronectin. Moreover, BCG attachment to cauterized bladders in vivo was inhibited by antifibronectin antibodies. These results demonstrate that fibronectin mediates the attachment of BCG to surfaces and suggest that it is the primary component mediating attachment within the bladder. Moreover, the data suggest that the BCG-fibronectin interaction may be a requisite first step for the initiation of the antitumor activity in intravesical BCG for bladder cancer.

Plasma fibronectin: three steps to purification and stability.

PubMed

Poulouin, L; Gallet, O; Rouahi, M; Imhoff, J M

1999-10-01

Large amounts of soluble fibronectin were easily purified from cryoprecipitated or fresh citrated human blood plasma by a three-step combination of gelatin and heparin-cellufine affinity chromatography. The elution conditions were optimized to obtain a homogeneous fraction on SDS-PAGE and Western blot under reducing condition. No proteolytic activities were detected by zymography at acidic or neutral pH. Furthermore, the fibronectin preparation was stable over time up to 456 h at 37 degrees C in the presence of calcium, zinc, or mercury. This preparation of very stable fibronectin, called highly purified fibronectin (hpFN), gave a yield of 7.00 +/- 0.77 mg of fibronectin per gram of cryoprecipitated plasma and 0.16 mg of fibronectin per milliliter of fresh citrated, giving a yield of 32 to 53% (from presumed fibronectin concentration). This preparation may be useful for cellular tests and interaction analysis. Copyright 1999 Academic Press.

A novel role for the integrin-binding III-10 module in fibronectin matrix assembly.

PubMed

Hocking, D C; Smith, R K; McKeown-Longo, P J

1996-04-01

Fibronectin matrix assembly is a cell-dependent process which is upregulated in tissues at various times during development and wound repair to support the functions of cell adhesion, migration, and differentiation. Previous studies have demonstrated that the alpha 5 beta 1 integrin and fibronectin's amino terminus and III-1 module are important in fibronectin polymerization. We have recently shown that fibronectin's III-1 module contains a conformationally sensitive binding site for fibronectin's amino terminus (Hocking, D.C., J. Sottile, and P.J. McKeown-Longo. 1994. J. Biol. Chem. 269: 19183-19191). The present study was undertaken to define the relationship between the alpha 5 beta 1 integrin and fibronectin polymerization. Solid phase binding assays using recombinant III-10 and III-1 modules of human plasma fibronectin indicated that the III-10 module contains a conformation-dependent binding site for the III-1 module of fibronectin. Unfolded III-10 could support the formation of a ternary complex containing both III-1 and the amino-terminal 70-kD fragment, suggesting that the III-1 module can support the simultaneous binding of III-10 and 70 kD. Both unfolded III-10 and unfolded III-1 could support fibronectin binding, but only III-10 could promote the formation of disulfide-bonded multimers of fibronectin in the absence of cells. III-10-dependent multimer formation was inhibited by both the anti-III-1 monoclonal antibody, 9D2, and amino-terminal fragments of fibronectin. A fragment of III-10, termed III-10/A, was able to block matrix assembly in fibroblast monolayers. Similar results were obtained using the III-10A/RGE fragment, in which the RGD site had been mutated to RGE, indicating that III-I0/A was blocking matrix assembly by a mechanism distinct from disruption of integrin binding. Texas red-conjugated recombinant III-1,2 localized to beta 1-containing sites of focal adhesions on cells plated on fibronectin or the III-9,10 modules of fibronectin. Monoclonal antibodies against the III-1 or the III-9,10 modules of fibronectin blocked binding of III-1,2 to cells without disrupting focal adhesions. These data suggest that a role of the alpha 5 beta 1 integrin in matrix assembly is to regulate a series of sequential self-interactions which result in the polymerization of fibronectin.

[Changes in the thrombophilic status in patients with pre-eclampsia].

PubMed

Baptista-González, H A; Rosenfeld-Mann, F; Saavedra-Trejo, M R; Castro-López, J L; Peñuela-Olaya, M A

1999-04-01

The object of this study was to evaluate the changes in fibrinolysis and clotting inhibitors in patients with preeclampsia and to describe the connection between preeclampsia and blood pressure values. Two groups of pregnant women were prospectively studied at delivery: group 1 women without preeclampsia and group 2 patients with preeclampsia. The variables that were registered are: diastolic blood pressure (DBP), systolic blood pressure (SBP), mean blood pressure (MBP), hemoglobin (Hb), platelet count (Plt), lupus like inhibitor, anticardiolipin antibodies (ACA), antinuclear antibodies (ANA), fibronectina, D dimer, protein S (PS), protein C (PC) and vo Willebrand factor (vWF). 62 pregnant women were included. The patients of group 2 presented high values of Hb (p 0.01), fibronectin (p 0.0001), D-dimer (p 0.01) and lower PC (p 0.04). We found an association between fibronectin and higher values of SBP, DBP, MBP and Hb (p 0.0007) versus lower values of VFW and PC (p 0.002). The low values of total PS were associated with high D-dimer and SBP results (p 0.04 and 0.002 respectively). All patients were ACA/ANA negative. In preclampsia there is a increased hemoconcentration and drop in clotting inhibitors (PC), without fibrinolytic compensatory response (lower D-dimer) and remarked vasopressive effect (hig fibronectin). This changes depend on the stratification of blood pressure. Th SBP and MBP values depend on the haemodynamic changes (Hb, fibronectin), while the increase in DBP expresses a non compensated thrombophilic state.

Hibiscus syriacus Extract from an Established Cell Culture Stimulates Skin Wound Healing.

PubMed

di Martino, O; Tito, A; De Lucia, A; Cimmino, A; Cicotti, F; Apone, F; Colucci, G; Calabrò, V

2017-01-01

Higher plants are the source of a wide array of bioactive compounds that support skin integrity and health. Hibiscus syriacus , family Malvaceae, is a plant of Chinese origin known for its antipyretic, anthelmintic, and antifungal properties. The aim of this study was to assess the healing and hydration properties of H. syriacus ethanolic extract (HSEE). We established a cell culture from Hibiscus syriacus leaves and obtained an ethanol soluble extract from cultured cells. The properties of the extract were tested by gene expression and functional analyses on human fibroblast, keratinocytes, and skin explants. HSEE treatment increased the healing potential of fibroblasts and keratinocytes. Specifically, HSEE significantly stimulated fibronectin and collagen synthesis by 16 and 60%, respectively, while fibroblasts contractility was enhanced by 30%. These results were confirmed on skin explants, where HSEE accelerated the wound healing activity in terms of epithelium formation and fibronectin production. Moreover, HSEE increased the expression of genes involved in skin hydration and homeostasis. Specifically, aquaporin 3 and filaggrin genes were enhanced by 20 and 58%, respectively. Our data show that HSEE contains compounds capable of stimulating expression of biomarkers relevant to skin regeneration and hydration thereby counteracting molecular pathways leading to skin damage and aging.

Hibiscus syriacus Extract from an Established Cell Culture Stimulates Skin Wound Healing

PubMed Central

di Martino, O.; Tito, A.; De Lucia, A.; Cimmino, A.; Cicotti, F.; Apone, F.; Colucci, G.

2017-01-01

Higher plants are the source of a wide array of bioactive compounds that support skin integrity and health. Hibiscus syriacus, family Malvaceae, is a plant of Chinese origin known for its antipyretic, anthelmintic, and antifungal properties. The aim of this study was to assess the healing and hydration properties of H. syriacus ethanolic extract (HSEE). We established a cell culture from Hibiscus syriacus leaves and obtained an ethanol soluble extract from cultured cells. The properties of the extract were tested by gene expression and functional analyses on human fibroblast, keratinocytes, and skin explants. HSEE treatment increased the healing potential of fibroblasts and keratinocytes. Specifically, HSEE significantly stimulated fibronectin and collagen synthesis by 16 and 60%, respectively, while fibroblasts contractility was enhanced by 30%. These results were confirmed on skin explants, where HSEE accelerated the wound healing activity in terms of epithelium formation and fibronectin production. Moreover, HSEE increased the expression of genes involved in skin hydration and homeostasis. Specifically, aquaporin 3 and filaggrin genes were enhanced by 20 and 58%, respectively. Our data show that HSEE contains compounds capable of stimulating expression of biomarkers relevant to skin regeneration and hydration thereby counteracting molecular pathways leading to skin damage and aging. PMID:29333453

Fibronectin potentiates topical erythropoietin-induced wound repair in diabetic mice.

PubMed

Hamed, Saher; Ullmann, Yehuda; Egozi, Dana; Daod, Essam; Hellou, Elias; Ashkar, Manal; Gilhar, Amos; Teot, Luc

2011-06-01

Diabetes mellitus disrupts all phases of the wound repair cascade and leads to development of chronic wounds. We previously showed that topical erythropoietin (EPO) can promote wound repair in diabetic rats. Fibronectin (FN) has a critical role throughout the process of wound healing, yet it is deficient in wound tissues of diabetic patients. Therefore, we investigated the effect of topical treatment of both EPO and FN (EPO/FN) on wound repair in diabetic mice. Full-thickness excisional skin wounds in diabetic and nondiabetic mice were treated with a cream containing vehicle, EPO, FN, or EPO/FN. We assessed the rate of wound closure, angiogenesis, apoptosis, and expression of inflammatory cytokines, endothelial nitric oxide synthase (eNOS) and β1-integrin, in the wound tissues. We also investigated the effect of EPO, FN, and EPO/FN on human dermal microvascular endothelial cells and fibroblasts cultured on fibrin-coated plates, or in high glucose concentrations. EPO/FN treatment significantly increased the rate of wound closure and this effect was associated with increased angiogenesis, increased eNOS and β1-integrin expression, and reduced expression of inflammatory cytokines and apoptosis. Our findings show that EPO and FN have an additive effect on wound repair in diabetic mice.

The predictive value of quantitative fibronectin testing in combination with cervical length measurement in symptomatic women.

PubMed

Bruijn, Merel M C; Kamphuis, Esme I; Hoesli, Irene M; Martinez de Tejada, Begoña; Loccufier, Anne R; Kühnert, Maritta; Helmer, Hanns; Franz, Marie; Porath, Martina M; Oudijk, Martijn A; Jacquemyn, Yves; Schulzke, Sven M; Vetter, Grit; Hoste, Griet; Vis, Jolande Y; Kok, Marjolein; Mol, Ben W J; van Baaren, Gert-Jan

2016-12-01

The combination of the qualitative fetal fibronectin test and cervical length measurement has a high negative predictive value for preterm birth within 7 days; however, positive prediction is poor. A new bedside quantitative fetal fibronectin test showed potential additional value over the conventional qualitative test, but there is limited evidence on the combination with cervical length measurement. The purpose of this study was to compare quantitative fetal fibronectin and qualitative fetal fibronectin testing in the prediction of spontaneous preterm birth within 7 days in symptomatic women who undergo cervical length measurement. We performed a European multicenter cohort study in 10 perinatal centers in 5 countries. Women between 24 and 34 weeks of gestation with signs of active labor and intact membranes underwent quantitative fibronectin testing and cervical length measurement. We assessed the risk of preterm birth within 7 days in predefined strata based on fibronectin concentration and cervical length. Of 455 women who were included in the study, 48 women (11%) delivered within 7 days. A combination of cervical length and qualitative fibronectin resulted in the identification of 246 women who were at low risk: 164 women with a cervix between 15 and 30 mm and a negative fibronectin test (<50 ng/mL; preterm birth rate, 2%) and 82 women with a cervix at >30 mm (preterm birth rate, 2%). Use of quantitative fibronectin alone resulted in a predicted risk of preterm birth within 7 days that ranged from 2% in the group with the lowest fibronectin level (<10 ng/mL) to 38% in the group with the highest fibronectin level (>500 ng/mL), with similar accuracy as that of the combination of cervical length and qualitative fibronectin. Combining cervical length and quantitative fibronectin resulted in the identification of an additional 19 women at low risk (preterm birth rate, 5%), using a threshold of 10 ng/mL in women with a cervix at <15 mm, and 6 women at high risk (preterm birth rate, 33%) using a threshold of >500 ng/mL in women with a cervix at >30 mm. In women with threatened preterm birth, quantitative fibronectin testing alone performs equal to the combination of cervical length and qualitative fibronectin. Possibly, the combination of quantitative fibronectin testing and cervical length increases this predictive capacity. Cost-effectiveness analysis and the availability of these tests in a local setting should determine the final choice. Copyright © 2016 Elsevier Inc. All rights reserved.

Fibronectin alters the rate of formation and structure of the fibrin matrix.

PubMed

Ramanathan, Anand; Karuri, Nancy

2014-01-10

Plasma fibronectin is a vital component of the fibrin clot; however its role on clot structure is not clearly understood. The goal of this study was to examine the influence of fibronectin on the kinetics of formation, structural characteristics and composition of reconstituted fibrin clots or fibrin matrices. Fibrin matrices were formed by adding thrombin to 1, 2 or 4 mg/ml fibrinogen supplemented with 0-0.4 mg/ml fibronectin. The rate of fibrin matrix formation was then monitored by measuring light absorbance properties at different time points. Confocal microscopy of fluorescein conjugated fibrinogen was used to visualize the structural characteristics of fibrin matrices. The amount of fibronectin in fibrin matrices was determined through electrophoresis and immunoblotting of solubilized matrices. Fibronectin concentration positively correlated with the initial rate of fibrin matrix formation and with steady state light absorbance values of fibrin matrices. An increase in fibronectin concentration resulted in thinner and denser fibers in the fibrin matrices. Electrophoresis and immunoblotting showed that fibronectin was covalently and non-covalently bound to fibrin matrices and in the form of high molecular weight multimers. The formation of fibronectin multimers was attributed to cross-linking of fibronectin by trace amounts Factor XIIIa. These findings are novel because they link results from light absorbance studies to microcopy analyses and demonstrate an influence of fibronectin on fibrin matrix structural characteristics. This data is important in developing therapies that destabilize fibrin clots. Copyright © 2014. Published by Elsevier Inc.

Heterologous expression of pathogen-specific genes ligA and ligB in the saprophyte Leptospira biflexa confers enhanced adhesion to cultured cells and fibronectin.

PubMed

Figueira, Cláudio Pereira; Croda, Julio; Choy, Henry A; Haake, David A; Reis, Mitermayer G; Ko, Albert I; Picardeau, Mathieu

2011-06-09

In comparison to other bacterial pathogens, our knowledge of the molecular basis of the pathogenesis of leptospirosis is extremely limited. An improved understanding of leptospiral pathogenetic mechanisms requires reliable tools for functional genetic analysis. Leptospiral immunoglobulin-like (Lig) proteins are surface proteins found in pathogenic Leptospira, but not in saprophytes. Here, we describe a system for heterologous expression of the Leptospira interrogans genes ligA and ligB in the saprophyte Leptospira biflexa serovar Patoc. The genes encoding LigA and LigB under the control of a constitutive spirochaetal promoter were inserted into the L. biflexa replicative plasmid. We were able to demonstrate expression and surface localization of LigA and LigB in L. biflexa. We found that the expression of the lig genes significantly enhanced the ability of transformed L. biflexa to adhere in vitro to extracellular matrix components and cultured cells, suggesting the involvement of Lig proteins in cell adhesion. This work reports a complete description of the system we have developed for heterologous expression of pathogen-specific proteins in the saprophytic L. biflexa. We show that expression of LigA and LigB proteins from the pathogen confers a virulence-associated phenotype on L. biflexa, namely adhesion to eukaryotic cells and fibronectin in vitro. This study indicates that L. biflexa can serve as a surrogate host to characterize the role of key virulence factors of the causative agent of leptospirosis.

The amino-terminal matrix assembly domain of fibronectin stabilizes cell shape and prevents cell cycle progression.

PubMed

Christopher, R A; Judge, S R; Vincent, P A; Higgins, P J; McKeown-Longo, P J

1999-10-01

Adhesion to the extracellular matrix modulates the cellular response to growth factors and is critical for cell cycle progression. The present study was designed to address the relationship between fibronectin matrix assembly and cell shape or shape dependent cellular processes. The binding of fibronectin's amino-terminal matrix assembly domain to adherent cells represents the initial step in the assembly of exogenous fibronectin into the extracellular matrix. When added to monolayers of pulmonary artery endothelial cells, the 70 kDa fragment of fibronectin (which contains the matrix assembly domain) stabilized both the extracellular fibronectin matrix as well as the actin cytoskeleton against cytochalasin D-mediated structural reorganization. This activity appeared to require specific fibronectin sequences as fibronectin fragments containing the cell adhesion domain as well as purified vitronectin were ineffective inhibitors of cytochalasin D-induced cytoarchitectural restructuring. Such pronounced morphologic consequences associated with exposure to the 70 kDa fragment suggested that this region of the fibronectin molecule may affect specific growth traits known to be influenced by cell shape. To assess this possibility, the 70 kDa fragment was added to scrape-wounded monolayers of bovine microvessel endothelium and the effects on two shape-dependent processes (i.e. migration and proliferation) were measured as a function of time after injury and location from the wound. The addition of amino-terminal fragments of fibronectin to the monolayer significantly inhibited (by >50%) wound closure. Staining of wounded monolayers with BrdU, moreover, indicated that either the 70 kDa or 25 kDa amino-terminal fragments of fibronectin, but not the 40 kDa collagen binding fragment, also inhibited cell cycle progression. These results suggest that the binding of fibronectin's amino-terminal region to endothelial cell layers inhibits cell cycle progression by stabilizing cell shape.

Sorption of fibronectin to human root surfaces in vitro

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

Mendieta, C.; Caravana, C.; Fine, D.H.

1990-05-01

The purpose of this study was to determine the conditions that favor the sorption and retention of human plasma fibronectin to cementum. Rectangular root segments prepared from teeth extracted for orthodontic reasons were mounted on a capillary pipette and immersed in solutions of {sup 125}I fibronectin for assay of cementum sorption under various conditions. Kinetic studies showed sorption to be rapid, with 77% of the maximum fibronectin sorption occurring within 1 minute. Fibronectin sorption was reduced when added in conjunction with serum and was inhibited by monovalent ions (such as sodium), but enhanced in the presence of divalent cations (suchmore » as calcium). Exposure of cementum to serum partially blocked subsequent sorption of fibronectin, while cementum bound fibronectin was eluted by subsequent exposure to serum. Treatment of cementum with citric acid pH 1.1 (4 minutes) followed by 5% sodium hypochlorite (5 minutes) caused a significant increase in fibronectin sorption with maximum retention upon subsequent exposure to serum (P less than 0.05). Fibronectin sorption to cementum was: rapid, electrostatic in nature, competitive, reversible, Ca+(+)-facilitated, and maximized by prior treatment of the root with citric acid and sodium hypochlorite. It is concluded that sorption of fibronectin to cementum can be achieved for clinical gain; however, conditions of application can significantly influence both accumulation and subsequent release of root sorbed material.« less

EDB Fibronectin Specific Peptide for Prostate Cancer Targeting.

PubMed

Han, Zheng; Zhou, Zhuxian; Shi, Xiaoyue; Wang, Junpeng; Wu, Xiaohui; Sun, Da; Chen, Yinghua; Zhu, Hui; Magi-Galluzzi, Cristina; Lu, Zheng-Rong

2015-05-20

Extradomain-B fibronectin (EDB-FN), one of the oncofetal fibronectin (onfFN) isoforms, is a high-molecular-weight glycoprotein that mediates cell adhesion and migration. The expression of EDB-FN is associated with a number of cancer-related biological processes such as tumorigenesis, angiogenesis, and epithelial-to-mesenchymal transition (EMT). Here, we report the development of a small peptide specific to EDB-FN for targeting prostate cancer. A cyclic nonapeptide, CTVRTSADC (ZD2), was identified using peptide phage display. A ZD2-Cy5 conjugate was synthesized to accomplish molecular imaging of prostate cancer in vitro and in vivo. ZD2-Cy5 demonstrated effective binding to up-regulated EDB-FN secreted by TGF-β-induced PC3 cancer cells following EMT. Following intravenous injections, the targeted fluorescent probe specifically bound to and delineated PC3-GFP prostate tumors in nude mice bearing the tumor xenografts. ZD2-Cy5 also showed stronger binding to human prostate tumor specimens with a higher Gleason score (GS9) compared to those with a lower score (GS 7), with no binding in benign prostatic hyperplasia (BPH). Thus, the ZD2 peptide is a promising strategy for molecular imaging and targeted therapy of prostate cancer.

The solid state environment orchestrates embryonic development and tissue remodeling

NASA Technical Reports Server (NTRS)

Damsky, C. H.; Moursi, A.; Zhou, Y.; Fisher, S. J.; Globus, R. K.

1997-01-01

Cell interactions with extracellular matrix and with other cells play critical roles in morphogenesis during development and in tissue homeostasis and remodeling throughout life. Extracellular matrix is information-rich, not only because it is comprised of multifunctional structural ligands for cell surface adhesion receptors, but also because it contains peptide signaling factors, and proteinases and their inhibitors. The functions of these groups of molecules are extensively interrelated. In this review, three primary cell culture models are described that focus on adhesion receptors and their roles in complex aspects of morphogenesis and remodeling: the regulation of proteinase expression by fibronectin and integrins in synovial fibroblasts; the regulation of osteoblast differentiation and survival by fibronectin, and the regulation of trophoblast differentiation and invasion by integrins, cadherins and immunoglobulin family adhesion receptors.

Effects of exercise training on brain-derived neurotrophic factor in skeletal muscle and heart of rats post myocardial infarction.

PubMed

Lee, Heow Won; Ahmad, Monir; Wang, Hong-Wei; Leenen, Frans H H

2017-03-01

What is the central question of this study? Exercise training increases brain-derived neurotrophic factor (BDNF) in the hippocampus, which depends on a myokine, fibronectin type III domain-containing protein 5 (FNDC5). Whether exercise training after myocardial infarction induces parallel increases in FNDC5 and BDNF expression in skeletal muscle and the heart has not yet been studied. What is the main finding and its importance? Exercise training after myocardial infarction increases BDNF protein in skeletal muscle and the non-infarct area of the LV without changes in FNDC5 protein, suggesting that BDNF is not regulated by FNDC5 in skeletal muscle and heart. An increase in cardiac BDNF may contribute to the improvement of cardiac function by exercise training. Exercise training after myocardial infarction (MI) attenuates progressive left ventricular (LV) remodelling and dysfunction, but the peripheral stimuli induced by exercise that trigger these beneficial effects are still unclear. We investigated as possible mediators fibronectin type III domain-containing protein 5 (FNDC5) and brain-derived neurotrophic factor (BDNF) in the skeletal muscle and heart. Male Wistar rats underwent either sham surgery or ligation of the left descending coronary artery, and surviving MI rats were allocated to either a sedentary (Sed-MI) or an exercise group (ExT-MI). Exercise training was done for 4 weeks on a motor-driven treadmill. At the end, LV function was evaluated, and FNDC5 and BDNF mRNA and protein were assessed in soleus muscle, quadriceps and non-, peri- and infarct areas of the LV. At 5 weeks post MI, FNDC5 mRNA was decreased in soleus muscle and all areas of the LV, but FNDC5 protein was increased in the soleus muscle and the infarct area. Mature BDNF (mBDNF) protein was decreased in the infarct area without a change in mRNA. Exercise training attenuated the decrease in ejection fraction and the increase in LV end-diastolic pressure post MI. Exercise training had no effect on FNDC5 mRNA and protein, but increased mBDNF protein in soleus muscle, quadriceps and the non-infarct area of the LV. The mBDNF protein in the non-infarct area correlated positively with ejection fraction and inversely with LV end-diastolic pressure. In conclusion, mBDNF is induced by exercise training in skeletal muscle and the non-infarct area of the LV, which may contribute to improvement of muscle dysfunction and cardiac function post MI. © 2017 The Authors. Experimental Physiology © 2017 The Physiological Society.

BIGH3 modulates adhesion and migration of hematopoietic stem and progenitor cells

PubMed Central

Klamer, Sofieke E; Kuijk, Carlijn GM; Hordijk, Peter L; van der Schoot, C Ellen; von Lindern, Marieke; van Hennik, Paula B; Voermans, Carlijn

2013-01-01

Cell adhesion and migration are important determinants of homing and development of hematopoietic stem and progenitor cells (HSPCs) in bone marrow (BM) niches. The extracellular matrix protein transforming growth factor-β (TGF-β) inducible gene H3 (BIGH3) is involved in adhesion and migration, although the effect of BIGH3 is highly cell type-dependent. BIGH3 is abundantly expressed by mesenchymal stromal cells, while its expression in HSPCs is relatively low unless induced by certain BM stressors. Here, we set out to determine how BIGH3 modulates HSPC adhesion and migration. We show that primary HSPCs adhere to BIGH3-coated substrates, which is, in part, integrin-dependent. Overexpression of BIGH3 in HSPCs and HL60 cells reduced the adhesion to the substrate fibronectin in adhesion assays, which was even more profound in electrical cell-substrate impedance sensing (ECIS) assays. Accordingly, the CXCL12 induced migration over fibronectin-coated surface was reduced in BIGH3-expressing HSPCs. The integrin expression profile of HSPCs was not altered upon BIGH3 expression. Although expression of BIGH3 did not alter actin polymerization in response to CXCL12, it inhibited the PMA-induced activation of the small GTPase RAC1 as well as the phosphorylation and activation of extracellular-regulated kinases (ERKs). Reduced activation of ERK and RAC1 may be responsible for the inhibition of cell adhesion and migration by BIGH3 in HSPCs. Induced BIGH3 expression upon BM stress may contribute to the regulation of BM homeostasis. PMID:24152593

Effects of extracellular matrix proteins on macrophage differentiation, growth, and function: comparison of liquid and agar culture systems

NASA Technical Reports Server (NTRS)

Armstrong, J. W.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

1994-01-01

Both spaceflight and skeletal unloading suppress the haematopoietic differentiation of macrophages (Sonnenfeld et al., Aviat. Space Environ. Med., 61:648-653, 1990; Armstrong et al., J. Appl. Physiol., 75:2734-2739, 1993). The mechanism behind this reduction in haematopoiesis has yet to be elucidated. However, changes in bone marrow extracellular matrix (ECM) may be involved. To further understand the role of ECM products in macrophage differentiation, we have performed experiments evaluating the effects of fibronectin, laminin, collagen type I, and collagen type IV on macrophage development and function. Bone marrow-derived macrophages cultured on four different ECM substrates in liquid culture medium showed less growth than those cultured on plastic. Significant morphological differences were seen on each of the substrates used. Phenotypically and functionally, as measured by class II major histocompatibility molecule (MHCII) expression, MAC-2 expression, and the secretion of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), these macrophages were similar. In contrast, bone marrow-derived macrophages cultured in suspension, using agar, showed no difference in growth when exposed to ECM proteins. However, IL-6 and TNF-alpha secretion was affected by fibronectin, laminin, collagen type I, and collagen type IV in a concentration-dependent manner. We conclude that the ECM products fibronectin, laminin, collagen type I, and collagen type IV have profound effects on macrophage development and function. Additionally, we suggest that an ECM-supplemented agar culture system provides an environment more analogous to in vivo bone marrow than does a traditional liquid culture system.

Fibronectin matrix-mediated cohesion suppresses invasion of prostate cancer cells.

PubMed

Jia, Dongxuan; Entersz, Ildiko; Butler, Christine; Foty, Ramsey A

2012-03-20

Invasion is an important early step in the metastatic cascade and is the primary cause of death of prostate cancer patients. In order to invade, cells must detach from the primary tumor. Cell-cell and cell-ECM interactions are important regulators of cohesion--a property previously demonstrated to mediate cell detachment and invasion. The studies reported here propose a novel role for α5β1 integrin--the principle mediator of fibronectin matrix assembly (FNMA)--as an invasion suppressor of prostate cancer cells. Using a combination of biophysical and cell biological methods, and well-characterized prostate cancer cell lines of varying invasiveness, we explore the relationship between cohesion, invasiveness, and FNMA. We show that cohesion is inversely proportional to invasive capacity. We also show that more invasive cells express lower levels of α5β1 integrin and lack the capacity for FNMA. Cells were generated to over-express either wild-type α5 integrin or an integrin in which the cytoplasmic domain of α5 was replaced with that of α2. The α2 construct does not promote FNMA. We show that only wild-type α5 integrin promotes aggregate compaction, increases cohesion, and reduces invasion of the more aggressive cells, and that these effects can be blocked by the 70-kDa fibronectin fragment. We propose that restoring capacity for FNMA in deficient cells can increase tumor intercellular cohesion to a point that significantly reduces cell detachment and subsequent invasion. In prostate cancer, this could be of therapeutic benefit by blocking an early key step in the metastatic cascade.

Suppression of the epidermal growth factor receptor inhibits epithelial-mesenchymal transition in human pancreatic cancer PANC-1 cells.

PubMed

Chang, Zhi-Gang; Wei, Jun-Min; Qin, Chang-Fu; Hao, Kun; Tian, Xiao-Dong; Xie, Kun; Xie, Xue-Hai; Yang, Yin-Mo

2012-05-01

Aberrant expression of epidermal growth factor receptor (EGFR) has been detected in pancreatic cancer; however, the mechanisms of EGFR in inducing pancreatic cancer development have not been adequately elucidated. The objective of this study was to determine the role of EGFR in mediating epithelial-mesenchymal transition (EMT) in pancreatic cancer cells. Pancreatic cancer cell line PANC-1 was transfected with small interfering RNA of EGFR by use of a lentiviral expression vector to establish an EGFR-knockdown cell line (si-PANC-1). PANC-1 cells transfected with lentiviral vector expressing negative control sequence were used as negative control (NC-PANC-1). Scratch assay and transwell study were used to analyze cell migration and invasion. Real-time PCR and Western blotting were used to detect the expression of EMT markers E-cadherin, N-cadherin, vimentin, and fibronectin and transcription factors snail, slug, twist1, and sip1 in PANC-1, NC-PANC-1, and si-PANC-1 cells. Immunofluorescent staining with these antibodies and confocal microscopy were used to observe their cellular location and morphologic changes. After RNA interference of EGFR, the migration and invasion ability of si-PANC-1 cells decreased significantly. The expression of epithelial phenotype marker E-cadherin increased and the expression of mesenchymal phenotype markers N-cadherin, vimentin, and fibronectin decreased, indicating reversion of EMT. We also observed intracellular translocation of E-cadherin. Expression of transcription factors snail and slug in si-PANC-1 cells decreased significantly. Suppression of EGFR expression can significantly inhibit EMT of pancreatic cancer PANC-1 cells. The mechanism may be related with the down-regulation of the expression of transcription factors snail and slug.

Cellular Fibronectin Expression in Human Trabecular Meshwork and Induction by Transforming Growth Factor-β2

PubMed Central

Medina-Ortiz, Wanda E.; Belmares, Ricardo; Neubauer, Sandra; Wordinger, Robert J.; Clark, Abbot F.

2013-01-01

Purpose. Levels of TGF-β2 are higher in POAG aqueous humor, causing deposition of extracellular matrix (ECM) proteins, including fibronectin (FN), in the glaucomatous human trabecular meshwork (HTM) that may be responsible for elevated IOP. The purpose of this study was to identify the expression of cellular FN (cFN) isoforms (EDA and EDB) in HTM cells and tissues, and to determine whether TGF-β2 can induce cFN expression and fibril formation in cultured HTM cells. Methods. Expression of cFN mRNA isoforms and induction by recombinant TGF-β2 (5 ng/mL) were determined by quantitative RT-PCR. The TGF-β2 induction of EDA isoform protein expression and FN fibril formation were analyzed using Western immunoblots and immunocytochemistry (ICC), respectively. Immunohistochemistry (IHC) analysis was used to examine total FN and EDA isoform expression in normal (NTM) and glaucomatous (GTM) trabecular meshwork (TM) tissues. Results. Both cFN mRNA isoforms were expressed in cultured HTM cells and were induced by TGF-β2 after 2, 4, and 7 days (P < 0.05). Similarly, EDA isoform protein and fibril formation were increased after 4 and 7 days of TGF-β2 treatment. Finally, GTM tissues had significantly greater EDA isoform protein levels (1.7-fold, P < 0.05) compared to NTM tissues. Conclusions. This study demonstrated that cFN isoforms are expressed and induced in HTM cells by TGF-β2. Also, increased EDA isoform protein levels were seen in GTM tissues. Our findings suggest that induction of cFN isoform expression in the TM ECM may be a novel pathologic mechanism involved in the TM changes associated with glaucoma. PMID:24030464

Pirfenidone inhibits TGF-β1-induced over-expression of collagen type I and heat shock protein 47 in A549 cells

PubMed Central

2012-01-01

Background Pirfenidone is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis (IPF). We previously showed that pirfenidone inhibits the over-expression of collagen type I and of heat shock protein (HSP) 47, a collagen-specific molecular chaperone, in human lung fibroblasts stimulated with transforming growth factor (TGF)-β1 in vitro. The increased numbers of HSP47-positive type II pneumocytes as well as fibroblasts were also diminished by pirfenidone in an animal model of pulmonary fibrosis induced by bleomycin. The present study evaluates the effects of pirfenidone on collagen type I and HSP47 expression in the human alveolar epithelial cell line, A549 cells in vitro. Methods The expression of collagen type I, HSP47 and E-cadherin mRNAs in A549 cells stimulated with TGF-β1 was evaluated by Northern blotting or real-time PCR. The expression of collagen type I, HSP47 and fibronectin proteins was assessed by immunocytochemical staining. Results TGF-β1 stimulated collagen type I and HSP47 mRNA and protein expression in A549 cells, and pirfenidone significantly inhibited this process. Pirfenidone also inhibited over-expression of the fibroblast phenotypic marker fibronectin in A549 cells induced by TGF-β1. Conclusion We concluded that the anti-fibrotic effects of pirfenidone might be mediated not only through the direct inhibition of collagen type I expression but also through the inhibition of HSP47 expression in alveolar epithelial cells, which results in reduced collagen synthesis in lung fibrosis. Furthermore, pirfenidone might partially inhibit the epithelial-mesenchymal transition. PMID:22694981

Fibronectin-mediation cell adhesion is required for induction of 92-kDa type IV collagenase/gelatinase (MMP-9) gene expression during macrophage differentiation : the signaling role of protein kinase C-{beta}.

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

Xie, B.; Laouar, A.; Huberman, E.

1998-05-08

Induction of the 92-kDa gelatinase (MMP-9) gene expression is associated with macrophage differentiation. In this study, we explored the regulatory mechanisms underlying this differentiation-associated MMP-9 gene expression in human HL-60 myeloid leukemia cells and human peripheral blood monocytes. Phorbol 12-myristate 13-acetate (PMA) markedly induced MMP-9 gene expression in HL-60 cells; the induction closely paralleled the timing and extent of PMA-induced cell adhesion and spreading, a hallmark of macrophage differentiation. Similarly, treatment with PMA or macrophage-colony stimulating factor stimulated adherence and spreading of blood monocytes with a concurrent 7- or 5-fold increase in MMP-9 production, respectively. In protein kinase C (PKC)-betamore » -deficient HL-60 variant cells (HL-525), PMA failed to induce cell adhesion and MMP-9 gene expression. Transfecting HL-525 cells with a PKC-beta expression plasmid restored PKC-beta levels and PMA inducibility of cell adhesion and spreading as well as MMP-9 gene expression. Induction of cell adhesion and MMP-9 gene expression in HL-60 cells and blood monocytes was strongly inhibited by neutralizing monoclonal antibodies to fibronectin (FN) and its receptor {alpha}5{beta}1 integrin. HL-525 cells, which constitutively display high levels of surface {alpha}5{beta}1 integrin, adhered and spread on immobilized FN with concomitant induction of MMP-9 gene expression. Cytochalasins B and D were each a potent inhibitor of MMP-9 production. Our results suggest that {alpha}5{beta}1 integrin-mediated interaction of immature hematopoietic cells with FN plays a critical role in modulating matrix-degrading activities during macrophage differentiation.« less

Optimizing a multifunctional microsphere scaffold to improve neural precursor cell transplantation for traumatic brain injury repair.

PubMed

Skop, Nolan B; Calderon, Frances; Cho, Cheul H; Gandhi, Chirag D; Levison, Steven W

2016-10-01

Tissue engineering using stem cells is widely used to repair damaged tissues in diverse biological systems; however, this approach has met with less success in regenerating the central nervous system (CNS). In this study we optimized and characterized the surface chemistry of chitosan-based scaffolds for CNS repair. To maintain radial glial cell (RGC) character of primitive neural precursors, fibronectin was adsorbed to chitosan. The chitosan was further modified by covalently linking heparin using genipin, which then served as a linker to immobilize fibroblast growth factor-2 (FGF-2), creating a multifunctional film. Fetal rat neural precursors plated onto this multifunctional film proliferated and remained multipotent for at least 3 days without providing soluble FGF-2. Moreover, they remained less mature and more highly proliferative than cells maintained on fibronectin-coated substrates in culture medium supplemented with soluble FGF-2. To create a vehicle for cell transplantation, a 3% chitosan solution was electrosprayed into a coagulation bath to generate microspheres (range 30-100 µm, mean 64 µm) that were subsequently modified. Radial glial cells seeded onto these multifunctional microspheres proliferated for at least 7 days in culture and the microspheres containing cells were small enough to be injected, using 23 Gauge Hamilton syringes, into the brains of adult rats that had previously sustained cortical contusion injuries. When analysed 3 days later, the transplanted RGCs were positive for the stem cell/progenitor marker Nestin. These results demonstrate that this multifunctional scaffold can be used as a cellular and growth factor delivery vehicle for the use in developing cell transplantation therapies for traumatic brain injuries. Copyright © 2013 John Wiley & Sons, Ltd. Copyright © 2013 John Wiley & Sons, Ltd.

A fibronectin receptor on Candida albicans mediates adherence of the fungus to extracellular matrix

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

Klotz, S.A.; Smith, R.L.

1991-03-01

Binding of fibronectin, an extracellular matrix (ECM) protein, to Candida albicans was measured, and adherence of the fungus to immobilized ECM proteins, fibronectin, laminin, types I and IV collagen, and subendothelial ECM was studied. 125I-labeled fibronectin was inhibited from binding to the fungus by unlabeled human plasma fibronectin and by Arg-Gly-Asp (RGD), Gly-Arg-Gly-Glu-Ser-Pro (GRGESP), and Gly-Arg-Gly-Asp-Thr-Pro (GRGDTP), but binding was not inhibited by Gly-Arg-Gly-Asp-Ser-Pro. Soluble fibronectin, RGD, GRGESP, and GRGDTP also inhibited fungal adherence to the individual immobilized ECM proteins in a complex pattern, but only soluble fibronectin (10(-7) M) inhibited fungal adherence to subendothelial ECM. Thus, C. albicans possessesmore » at least one type of cell surface receptor for binding soluble fibronectin that can be inhibited with peptides. This receptor apparently is used to bind the fungus to immobilized ECM proteins and to subendothelial ECM and may play a role in the initiation of disseminated disease by bloodborne fungi by providing for adherence of the microorganisms to ECM proteins.« less

Protein F, a fibronectin-binding protein, is an adhesin of the group A streptococcus Streptococcus pyogenes.

PubMed

Hanski, E; Caparon, M

1992-07-01

Binding to fibronectin has been suggested to play an important role in adherence of the group A streptococcus Streptococcus pyrogenes to host epithelial cells; however, the identity of the streptococcal fibronectin receptor has been elusive. Here we demonstrate that the fibronectin-binding property of S. pyogenes is mediated by protein F, a bacterial surface protein that binds fibronectin at high affinity. The gene encoding protein F (prtF) produced a functional fibronectin-binding protein in Escherichia coli. Insertional mutagenesis of the cloned gene generated a mutation that resulted in the loss of fibronectin-binding activity. When this mutation was introduced into the S. pyrogenes chromosome by homologous recombination with the wild-type allele, the resulting strains no longer produced protein F and lost their ability to bind fibronectin. The mutation could be complemented by prtF introduced on a plasmid. Mutants lacking protein F had a much lower capacity to adhere to respiratory epithelial cells. These results demonstrate that protein F is an important adhesin of S. pyogenes.

Synergistic Effects of a Calcium Phosphate/Fibronectin Coating on the Adhesion of Periodontal Ligament Stem Cells Onto Decellularized Dental Root Surfaces.

PubMed

Lee, Jung-Seok; Kim, Hyun-Suk; Park, So-Yon; Kim, Tae-Wan; Jung, Jae-Suk; Lee, Jong-Bin; Kim, Chang-Sung

2015-01-01

This study aimed to enhance the attachment of periodontal ligament stem cells (PDLSCs) onto the decellularized dental root surface using surface coating with fibronectin and/or calcium phosphate (CaP) and to evaluate the activity of PDLSCs attached to a coated dental root surface following tooth replantation. PDLSCs were isolated from five dogs, and the other dental roots were used as a scaffold for carrying PDLSCs and then assigned to one of four groups according to whether their surface was coated with CaP, fibronectin, CaP/fibronectin, or left uncoated (control). Fibronectin increased the adhesion of PDLSCs onto dental root surfaces compared to both the control and CaP-coated groups, and simultaneous surface coating with CaP and fibronectin significantly accelerated and increased PDLSC adhesion compared to the fibronectin-only group. On in vivo tooth replantation, functionally oriented periodontal new attachment was observed on the CaP/fibronectin-coated dental roots to which autologous PDLSCs had adhered, while in the control condition, dental root replantation was associated only with root resorption and ankylosis along the entire root length. CaP and fibronectin synergistically enhanced the attachment of PDLSCs onto dental root surfaces, and autologous PDLSCs could produce de novo periodontal new attachment in an experimental in vivo model.

The signaling pathway of Campylobacter jejuni-induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2

PubMed Central

2011-01-01

Background Host cell invasion by the foodborne pathogen Campylobacter jejuni is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin-/-, integrin-beta1-/-, focal adhesion kinase (FAK)-/- and Src/Yes/Fyn-/- deficient mice, and wild-type control cells, to investigate C. jejuni-induced mechanisms leading to Cdc42 activation and bacterial uptake. Results Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR) during the course of infection, correlating with accumulating Cdc42-GTP levels and C. jejuni invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA) and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1/2-/- knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker molecule between Cdc42 and activated EGFR/PDGFR/PI3-kinase. Using C. jejuni mutant strains we further demonstrated that the fibronectin-binding protein CadF and intact flagella are involved in Cdc42-GTP induction, indicating that the bacteria may directly target the fibronectin/integrin complex for inducing signaling leading to its host cell entry. Conclusion Collectively, our findings led us propose that C. jejuni infection triggers a novel fibronectin→integrin-beta1→FAK/Src→EGFR/PDGFR→PI3-kinase→Vav2 signaling cascade, which plays a crucial role for Cdc42 GTPase activity associated with filopodia formation and enhances bacterial invasion. PMID:22204307

Selective reversible inhibition of autophagy in hypoxic breast cancer cells promotes pulmonary metastasis

PubMed Central

Dower, Christopher M.; Bhat, Neema; Wang, Edward W.; Wang, Hong-Gang

2016-01-01

Autophagy influences how cancer cells respond to nutrient deprivation and hypoxic stress, two hallmarks of the tumor microenvironment (TME). In this study, we explored the impact of autophagy on the pathophysiology of breast cancer cells, using a novel hypoxia-dependent, reversible dominant negative strategy to regulate autophagy at the cellular level within the TME. Suppression of autophagy via hypoxia-induced expression of the kinase-dead unc-51 like autophagy activating kinase (ULK1) mutant K46N increased lung metastases in MDA-MB-231 xenograft mouse models. Consistent with this effect, expressing a dominant-negative mutant of ULK1 or ATG4b or a ULK1-targeting shRNA facilitated cell migration in vitro. Functional proteomic and transcriptome analysis revealed that loss of hypoxia-regulated autophagy promotes metastasis via induction of the fibronectin integrin signaling axis. Indeed, loss of ULK1 function increased fibronectin deposition in the hypoxic TME. Together, our results indicated that hypoxia-regulated autophagy suppresses metastasis in breast cancer by preventing tumor fibrosis. These results also suggest cautions in the development of autophagy-based strategies for cancer treatment. PMID:28115361

Lack of Fibronectin Extra Domain A Alternative Splicing Exacerbates Endothelial Dysfunction in Diabetes

PubMed Central

Gortan Cappellari, Gianluca; Barazzoni, Rocco; Cattin, Luigi; Muro, Andrés F.; Zanetti, Michela

2016-01-01

Glucose-induced changes of artery anatomy and function account for diabetic vascular complications, which heavily impact disease morbidity and mortality. Since fibronectin containing extra domain A (EDA + FN) is increased in diabetic vessels and participates to vascular remodeling, we wanted to elucidate whether and how EDA + FN is implicated in diabetes-induced endothelial dysfunction using isometric-tension recording in a murine model of diabetes. In thoracic aortas of EDA−/−, EDA+/+ (constitutively lacking and expressing EDA + FN respectively), and of wild-type mice (EDAwt/wt), streptozotocin (STZ)-induced diabetes impaired endothelial vasodilation to acetylcholine, irrespective of genotype. However STZ + EDA−/− mice exhibited increased endothelial dysfunction compared with STZ + EDA+/+ and with STZ + EDAwt/wt. Analysis of the underlying mechanisms revealed that STZ + EDA−/− mice show increased oxidative stress as demonstrated by enhanced aortic superoxide anion, nitrotyrosine levels and expression of NADPH oxidase NOX4 and TGF-β1, the last two being reverted by treatment with the antioxidant n-acetylcysteine. In contrast, NOX1 expression and antioxidant potential were similar in aortas from the three genotypes. Interestingly, reduced eNOS expression in STZ + EDA+/+ vessels is counteracted by increased eNOS coupling and function. Although EDA + FN participates to vascular remodelling, these findings show that it plays a crucial role in limiting diabetic endothelial dysfunction by preventing vascular oxidative stress. PMID:27897258

Advanced Glycation End-Products Induce Connective Tissue Growth Factor-Mediated Renal Fibrosis Predominantly through Transforming Growth Factor β-Independent Pathway

PubMed Central

Zhou, Guihua; Li, Cai; Cai, Lu

2004-01-01

Advanced glycation end-products (AGEs) play a critical role in diabetic nephropathy by stimulating extracellular matrix (ECM) synthesis. Connective tissue growth factor (CTGF) is a potent inducer of ECM synthesis and increases in the diabetic kidneys. To determine the critical role of CTGF in AGE-induced ECM accumulation leading to diabetic nephropathy, rats were given AGEs by intravenous injection for 6 weeks. AGE treatment induced a significant renal ECM accumulation, as shown by increases in periodic acid-Schiff-positive materials, fibronectin, and type IV collagen (Col IV) accumulation in glomeruli, and a mild renal dysfunction, as shown by increases in urinary volume and protein content. AGE treatment also caused significant increases in renal CTGF and transforming growth factor (TGF)-β1 mRNA and protein expression. Direct exposure of rat mesangial cells to AGEs in vitro significantly induced increases in fibronectin and Col IV production, which could be completely prevented by pretreatment with anti-CTGF antibody. AGE treatment also significantly increased both TGF-β1 and CTGF mRNA expression; however, inhibition of TGF-β1 mRNA expression by shRNA or neutralization of TGF-β1 protein by anti-TGF-β1 antibody did not significantly prevent AGE-increased expression of CTGF mRNA and protein. These results suggest that AGE-induced CTGF expression, predominantly through a TGF-β1-independent pathway, plays a critical role in renal ECM accumulation leading to diabetic nephropathy. PMID:15579446

Expression of fibronectin ED-A+ and ED-B+ isoforms by human and experimental colorectal cancer. Contribution of cancer cells and tumor-associated myofibroblasts.

PubMed Central

Pujuguet, P.; Hammann, A.; Moutet, M.; Samuel, J. L.; Martin, F.; Martin, M.

1996-01-01

Alternative splicing of primary fibronectin (FN) mRNA results in the synthesis of different isoforms. ED-A+ and ED-B+ FN isoforms are absent from plasma FN and are representative of cellular FN. Their expression was studied in human and rat normal colon, in human colorectal carcinomas, and in transplanted tumors derived from a chemically-induced rat colon cancer. In normal colon, only the ED-A+ FN isoform was expressed as a thin deposit between crypt colonocytes and pericryptal myofibroblasts. Conversely, heavy ED-A+ FN deposits and lighter ED-B+ FN expression were found in the stroma of colorectal tumors in association with myofibroblasts surrounding tumor glands. Some colonic cancer cells also contained intracellular FN isoform granules and expressed FN mRNA. Tumor-associated myofibroblasts and some cancer cell lines were able to synthesize and deposit extracellular ED-A+ and ED-B+ FN in vitro. FN isoform deposition by tumor-associated myofibroblasts was not modulated by colon cancer cell-conditioned medium, but was strongly enhanced when myofibroblasts were cultured on colon cancer cell extracellular matrix or on laminin. These results show that the ED-A+ and ED-B+ FN isoforms were overexpressed in colorectal cancer. Cancer cells can deposit these FN isoforms directly and also stimulate their deposition by tumor-associated myofibroblasts. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 7 PMID:8579120

The extracellular matrix protein laminin-10 promotes blood-brain barrier repair after hypoxia and inflammation in vitro.

PubMed

Kangwantas, Korakoch; Pinteaux, Emmanuel; Penny, Jeffrey

2016-02-01

The blood-brain barrier (BBB) of the central nervous system (CNS) is essential for normal brain function. However, the loss of BBB integrity that occurs after ischaemic injury is associated with extracellular matrix (ECM) remodelling and inflammation, and contributes to poor outcome. ECM remodelling also contributes to BBB repair after injury, but the precise mechanisms and contribution of specific ECM molecules involved are unknown. Here, we investigated the mechanisms by which hypoxia and inflammation trigger loss of BBB integrity and tested the hypothesis ECM changes could contribute to BBB repair in vitro. We used an in vitro model of the BBB, composed of primary rat brain endothelial cells grown on collagen (Col) I-, Col IV-, fibronectin (FN)-, laminin (LM) 8-, or LM10-coated tissue culture plates, either as a single monolayer culture or on Transwell® inserts above mixed glial cell cultures. Cultures were exposed to oxygen-glucose deprivation (OGD) and/or reoxygenation, in the absence or the presence of recombinant interleukin-1β (IL-1β). Cell adhesion to ECM molecules was assessed by cell attachment and cell spreading assays. BBB dysfunction was assessed by immunocytochemistry for tight junction proteins occludin and zona occludens-1 (ZO-1) and measurement of trans-endothelial electrical resistance (TEER). Change in endothelial expression of ECM molecules was assessed by semi-quantitative RT-PCR. OGD and/or IL-1 induce dramatic changes associated with loss of BBB integrity, including cytoplasmic relocalisation of membrane-associated tight junction proteins occludin and ZO-1, cell swelling, and decreased TEER. OGD and IL-1 also induced gene expression of key ECM molecules associated with the BBB, including FN, Col IV, LM 8, and LM10. Importantly, we found that LM10, but not FN, Col IV, nor LM8, plays a key role in maintenance of BBB integrity and reversed most of the key hallmarks of BBB dysfunction induced by IL-1. Our data unravel new mechanisms of BBB dysfunction induced by hypoxia and inflammation and identify LM10 as a key ECM molecule involved in BBB repair after hypoxic injury and inflammation.

Chondrocyte heterogeneity: immunohistologically defined variation of integrin expression at different sites in human fetal knees.

PubMed

Salter, D M; Godolphin, J L; Gourlay, M S

1995-04-01

During development and at maturity different forms of cartilage vary in morphology and macromolecular content. This reflects heterogeneity of chondrocyte activity, in part involving differential interactions with the adjacent extracellular matrix via specialized cell surface receptors such as integrins. We undertook an immunohistological study on a series of human fetal knee joints to assess variation in the expression of integrins by chondrocytes and potential matrix ligands in articular, epiphyseal, growth plate, and meniscal cartilage. The results show that articular chondrocytes (beta 1+, beta 5 alpha V+, alpha 1+, alpha 2+/-, alpha 5+, weakly alpha 6+, alpha V+) differed from epiphyseal (beta 1+, beta 5 alpha V+, alpha 1+/-, alpha 2+/-, alpha 5+, alpha 6+, alpha V+) growth plate (beta 1+, beta 5 alpha V+, alpha 1-, alpha 2-, alpha 5+, alpha 6+, alpha V+), and meniscal cells (beta 1+, beta 5 alpha V+, alpha 1+, strongly alpha 2+, alpha 5+, alpha 6+, alpha V+ in expression of integrin subunits. There was no expression of beta 3, beta 4, beta 6, or alpha 3 by chondrocytes. These results differ from previous reports on the expression of integrins by adult articular cartilage, where alpha 2 and alpha 6 are not seen. Variation in distribution of matrix ligands was also seen. Fibronectin, laminin and Type VI collagen were expressed in all cartilages but there was restricted expression of tenascin, ED-A and ED-B fibronectin isoforms (articular cartilage and meniscus), and vitronectin (absent from growth plate cartilage). Regulated expression of integrins by chondrocytes, associated with changes in the pericellular matrix composition, is of potential importance in control of cartilage differentiation and function in health and disease.

Improved penetration of wild ginseng extracts into the skin using low-temperature atmospheric pressure plasma

NASA Astrophysics Data System (ADS)

Nam, Seoul Hee; Hae Choi, Jeong; Song, Yeon Suk; Lee, Hae-June; Hong, Jin-Woo; Kim, Gyoo Cheon

2018-04-01

Wild ginseng (WG) is a well-known traditional medicinal plant that grows in natural environments in deep mountains. WG has been thought to exert potent physiological and medicinal effects, and, recently, its use in skin care has attracted much interest. This study investigated the efficient penetration of WG extracts into the skin by means of low-temperature atmospheric pressure plasma (LTAPP), and its effects on the skin at the cellular and tissue levels. NIH3T3 mouse embryonic fibroblasts and HRM-2 hairless mice were used to confirm the improved absorption of WG extracts into the skin using LTAPP. The gene expression levels in NIH3T3 cells and morphological changes in skin tissues after WG treatment were monitored using both in vitro and in vivo experiments. Although WG extracts did not show any significant effects on proliferative activity and cytotoxicity, at a concentration of 1:800, it significantly increased the expression of fibronectin and vascular endothelial growth factor. In the in vivo study, the combinational treatment of LTAPP and WG markedly induced the expression of fibronectin and integrin α6, and it thickened. Our results showed that LTAPP treatment safely and effectively accelerated the penetration of the WG extracts into the skin, thereby increasing the effects of WG on the skin.

Tissue-associated self-antigens containing exosomes: Role in allograft rejection.

PubMed

Sharma, Monal; Ravichandran, Ranjithkumar; Bansal, Sandhya; Bremner, Ross M; Smith, Michael A; Mohanakumar, T

2018-06-15

Exosomes are extracellular vesicles that express self-antigens (SAgs) and donor human leukocyte antigens. Tissue-specific exosomes can be detected in the circulation following lung, heart, kidney and islet cell transplantations. We collected serum samples from patients who had undergone lung (n = 30), heart (n = 8), or kidney (n = 15) transplantations to isolate circulating exosomes. Exosome purity was analyzed by Western blot, using CD9 exosome-specific markers. Tissue-associated lung SAgs, collagen V (Col-V) and K-alpha 1 tubulin (Kα1T), heart SAgs, myosin and vimentin, and kidney SAgs, fibronectin and collagen IV (Col-IV), were identified using western blot. Lung transplant recipients diagnosed with bronchiolitis obliterans syndrome had exosomes with higher expression of Col-V (4.2-fold) and Kα1T (37.1-fold) than stable. Exosomes isolated from heart transplant recipients diagnosed with coronary artery vasculopathy had a 3.9-fold increase in myosin and a 4.7-fold increase in vimentin compared with stable. Further, Kidney transplant recipients diagnosed with transplant glomerulopathy had circulating exosomes with a 2-fold increased expression of fibronectin and 2.5-fold increase in Col-IV compared with stable. We conclude that circulating exosomes with tissue associated SAgs have the potential to be a noninvasive biomarker for allograft rejection. Copyright © 2018. Published by Elsevier Inc.

Erhuang Formula ameliorates renal damage in adenine-induced chronic renal failure rats via inhibiting inflammatory and fibrotic responses.

PubMed

Zhang, Chun-Yan; Zhu, Jian-Yong; Ye, Ying; Zhang, Miao; Zhang, Li-Jun; Wang, Su-Juan; Song, Ya-Nan; Zhang, Hong

2017-11-01

The present study aimed to evaluate the protective effects of Erhuang Formula (EHF) and explore its pharmacological mechanisms on adenine-induced chronic renal failure (CRF). The compounds in EHF were analyzed by HPLC/MS. Adenine-induced CRF rats were administrated by EHF. The effects were evaluated by renal function examination and histology staining. Immunostaining of some proteins related cell adhesion was performedin renal tissues, including E-cadherin, β-catenin, fibronectin and laminin. The qRT-PCR was carried out determination of gene expression related inflammation and fibrosis including NF-κB, TNF-α, TGF-β1, α-SMA and osteopontin (OPN). Ten compounds in EHF were identified including liquiritigenin, farnesene, vaccarin, pachymic acid, cycloastragenol, astilbin, 3,5,6,7,8,3',4'-heptemthoxyflavone, physcion, emodin and curzerene. Abnormal renal function and histology had significant improvements by EHF treatment. The protein expression of β-catenin, fibronectin and laminin were significantly increased and the protein expression of E-cadherin significantly decreased in CRF groups. However, these protein expressions were restored to normal levels in EHF group. Furthermore, low expression of PPARγ and high expression of NF-κB, TNF-α, TGF-β1, α-SMA and OPN were substantially restored by EHF treatment in a dose-dependent manner. EHF ameliorated renal damage in adenine-induced CRF rats, and the mechanisms might involve in the inhibition of inflammatory and fibrotic responses and the regulation of PPARγ, NF-κB and TGF-β signaling pathways. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Heterologous expression of pathogen-specific genes ligA and ligB in the saprophyte Leptospira biflexa confers enhanced adhesion to cultured cells and fibronectin

PubMed Central

2011-01-01

Background In comparison to other bacterial pathogens, our knowledge of the molecular basis of the pathogenesis of leptospirosis is extremely limited. An improved understanding of leptospiral pathogenetic mechanisms requires reliable tools for functional genetic analysis. Leptospiral immunoglobulin-like (Lig) proteins are surface proteins found in pathogenic Leptospira, but not in saprophytes. Here, we describe a system for heterologous expression of the Leptospira interrogans genes ligA and ligB in the saprophyte Leptospira biflexa serovar Patoc. Results The genes encoding LigA and LigB under the control of a constitutive spirochaetal promoter were inserted into the L. biflexa replicative plasmid. We were able to demonstrate expression and surface localization of LigA and LigB in L. biflexa. We found that the expression of the lig genes significantly enhanced the ability of transformed L. biflexa to adhere in vitro to extracellular matrix components and cultured cells, suggesting the involvement of Lig proteins in cell adhesion. Conclusions This work reports a complete description of the system we have developed for heterologous expression of pathogen-specific proteins in the saprophytic L. biflexa. We show that expression of LigA and LigB proteins from the pathogen confers a virulence-associated phenotype on L. biflexa, namely adhesion to eukaryotic cells and fibronectin in vitro. This study indicates that L. biflexa can serve as a surrogate host to characterize the role of key virulence factors of the causative agent of leptospirosis. PMID:21658265

A 3'-untranslated region (3'UTR) induces organ adhesion by regulating miR-199a* functions.

PubMed

Lee, Daniel Y; Shatseva, Tatiana; Jeyapalan, Zina; Du, William W; Deng, Zhaoqun; Yang, Burton B

2009-01-01

Mature microRNAs (miRNAs) are single-stranded RNAs of 18-24 nucleotides that repress post-transcriptional gene expression. However, it is unknown whether the functions of mature miRNAs can be regulated. Here we report that expression of versican 3'UTR induces organ adhesion in transgenic mice by modulating miR-199a* activities. The study was initiated by the hypothesis that the non-coding 3'UTR plays a role in the regulation of miRNA function. Transgenic mice expressing a construct harboring the 3'UTR of versican exhibits the adhesion of organs. Computational analysis indicated that a large number of microRNAs could bind to this fragment potentially including miR-199a*. Expression of versican and fibronectin, two targets of miR-199a*, are up-regulated in transgenic mice, suggesting that the 3'UTR binds and modulates miR-199a* activities, freeing mRNAs of versican and fibronectin from being repressed by miR-199a*. Confirmation of the binding was performed by PCR using mature miR-199a* as a primer and the targeting was performed by luciferase assays. Enhanced adhesion by expression of the 3'UTR was confirmed by in vitro assays. Our results demonstrated that upon arrival in cytoplasm, miRNA activities can be modulated locally by the 3'UTR. Our assay may be developed as sophisticated approaches for studying the mutual regulation of miRNAs and mRNAs in vitro and in vivo. We anticipate that expression of the 3'UTR may be an approach in the development of gene therapy.

Lectins as probes for assessing the accessibility of N-linked glycans in relation to the conformational changes of fibronectin.

PubMed

Agniel, Rémy; Vendrely, Charlotte; Poulouin, Laurent; Bascetin, Rümeyza; Benachour, Hamanou; Gallet, Olivier; Leroy-Dudal, Johanne

2015-12-01

Fibronectin, a ≈ 450-kDa protein with 4-9% (w/w) glycosylation, is a key component of extracellular matrices and has a high conformational lability regarding its functions. However, the accessibility and the role of glycosylated moieties associated with the conformational changes of fibronectin are poorly understood. Using lectins as probes, we developed an approach comprising dynamic light scattering, turbidimetry measurements, and isothermal titration calorimetry to assess the accessibility of glycosylated moieties of fibronectin undergoing thermal-induced conformational changes. Among a set of 14 lectins, fibronectin mainly reacted with mannose-binding lectins, specifically concanavalin A. When temperature was raised from 25 to 50 °C, fibronectin underwent progressive unfolding, but the conformation of concanavalin A was unaffected. Dynamic light scattering, turbidimetry measurements, and isothermal titration calorimetry showed increased concanavalin A binding to fibronectin during progressive thermal-induced unfolding of the protein core. Such data suggest that mannosylated residues are progressively exposed as fibronectin unfolds. Because oligosaccharide moieties can be differently exposed to cells, and the cell's responses could be modified physiologically or pathologically, modulation of fibronectin sugar chains could be relevant to its biological functions. Thus, lectins might be useful tools to probe the glycosylation accessibility accompanying changes in protein core folding, for which a better understanding would be of value for biological and biomedical research. Copyright © 2015 John Wiley & Sons, Ltd.

Fibronectin in cell adhesion and migration via N-glycosylation

PubMed Central

Hsiao, Cheng-Te; Cheng, Hung-Wei; Huang, Chi-Ming; Li, Hao-Ru; Ou, Meng-Hsin; Huang, Jie-Rong; Khoo, Kay-Hooi; Yu, Helen Wenshin; Chen, Yin-Quan; Wang, Yang-Kao; Chiou, Arthur; Kuo, Jean-Cheng

2017-01-01

Directed cell migration is an important step in effective wound healing and requires the dynamic control of the formation of cell-extracellular matrix interactions. Plasma fibronectin is an extracellular matrix glycoprotein present in blood plasma that plays crucial roles in modulating cellular adhesion and migration and thereby helping to mediate all steps of wound healing. In order to seek safe sources of plasma fibronectin for its practical use in wound dressing, we isolated fibronectin from human (homo) and porcine plasma and demonstrated that both have a similar ability as a suitable substrate for the stimulation of cell adhesion and for directing cell migration. In addition, we also defined the N-glycosylation sites and N-glycans present on homo and porcine plasma fibronectin. These N-glycosylation modifications of the plasma fibronectin synergistically support the integrin-mediated signals to bring about mediating cellular adhesion and directed cell migration. This study not only determines the important function of N-glycans in both homo and porcine plasma fibronectin-mediated cell adhesion and directed cell migration, but also reveals the potential applications of porcine plasma fibronectin if it was applied as a material for clinical wound healing and tissue repair. PMID:29050309

Involvement of Receptor-like Protein Tyrosine Phosphatase ζ/RPTPβ and Its Ligand Pleiotrophin/Heparin-binding Growth-associated Molecule (HB-GAM) in Neuronal Migration

PubMed Central

Maeda, Nobuaki; Noda, Masaharu

1998-01-01

Pleiotrophin/heparin-binding growth-associated molecule (HB-GAM) is a specific ligand of protein tyrosine phosphatase ζ (PTPζ)/receptor-like protein tyrosine phosphatase β (RPTPβ) expressed in the brain as a chondroitin sulfate proteoglycan. Pleiotrophin and PTPζ isoforms are localized along the radial glial fibers, a scaffold for neuronal migration, suggesting that these molecules are involved in migratory processes of neurons during brain development. In this study, we examined the roles of pleiotrophin-PTPζ interaction in the neuronal migration using cell migration assay systems with glass fibers and Boyden chambers. Pleiotrophin and poly-l-lysine coated on the substratums stimulated cell migration of cortical neurons, while laminin, fibronectin, and tenascin exerted almost no effect. Pleiotrophin-induced and poly-l-lysine–induced neuronal migrations showed significant differences in sensitivity to various molecules and reagents. Polyclonal antibodies against the extracellular domain of PTPζ, PTPζ-S, an extracellular secreted form of PTPζ, and sodium vanadate, a protein tyrosine phosphatase inhibitor, added into the culture medium strongly suppressed specifically the pleiotrophin-induced neuronal migration. Furthermore, chondroitin sulfate C but not chondroitin sulfate A inhibited pleiotrophin-induced neuronal migration, in good accordance with our previous findings that chondroitin sulfate constitutes a part of the pleiotrophin-binding site of PTPζ, and PTPζ-pleiotrophin binding is inhibited by chondroitin sulfate C but not by chondroitin sulfate A. Immunocytochemical analysis indicated that the transmembrane forms of PTPζ are expressed on the migrating neurons especially at the lamellipodia along the leading processes. These results suggest that PTPζ is involved in the neuronal migration as a neuronal receptor of pleiotrophin distributed along radial glial fibers. PMID:9660874

Spray-painted human fibronectin coating as an effective strategy to enhance graft ligamentization of a polyethylene terephthalate artificial ligament.

PubMed

Li, Hong; Chen, Chen; Ge, Yunsheng; Chen, Shiyi

2014-05-01

To enhance graft ligamentization after anterior cruciate ligament (ACL) reconstruction, human fibronectin (FN) was coated on polyethylene terephthalate (PET) ligaments by spray painting. The FN-coated PET ligaments were investigated in vitro using rat mesenchymal stromal cells (MSCs). MSCs cultured on FN-coated grafts resulted in similar cell densities and amounts of proliferating cells with control grafts without coating. The FN-coated group not only gave rise to MSC-derived collagen-like tissues but also enhanced the expression of collagen-I gene. Furthermore, rat ACL reconstruction models were used to evaluate the effect of the FN coating in vivo. The FN coating significantly promoted new ligament tissue regeneration into the graft fibers. In conclusion, sprayed FN coating had a positive effect to enhance graft ligamentization of PET artificial ligament.

Pili of oral Streptococcus sanguinis bind to fibronectin and contribute to cell adhesion.

PubMed

Okahashi, Nobuo; Nakata, Masanobu; Sakurai, Atsuo; Terao, Yutaka; Hoshino, Tomonori; Yamaguchi, Masaya; Isoda, Ryutaro; Sumitomo, Tomoko; Nakano, Kazuhiko; Kawabata, Shigetada; Ooshima, Takashi

2010-01-08

Streptococcus sanguinis is a predominant bacterium in the human oral cavity and occasionally causes infective endocarditis. We identified a unique cell surface polymeric structure named pili in this species and investigated its functions in regard to its potential virulence. Pili of S. sanguinis strain SK36 were shown to be composed of three distinctive pilus proteins (PilA, PilB, and PilC), and a pili-deficient mutant demonstrated reduced bacterial adherence to HeLa and human oral epithelial cells. PilC showed a binding ability to fibronectin, suggesting that pili are involved in colonization by this species. In addition, ATCC10556, a standard S. sanguinis strain, was unable to produce pili due to defective pilus genes, which indicates a diversity of pilus expression among various S. sanguinis strains. Copyright 2009 Elsevier Inc. All rights reserved.

A role for exon sequences in alternative splicing of the human fibronectin gene.

PubMed Central

Mardon, H J; Sebastio, G; Baralle, F E

1987-01-01

Exon EDIIIA of the fibronectin (Fn) gene is alternatively spliced via pathways which either skip or include the whole exon in the messenger RNA (mRNA). We have investigated the role of EDIIIA exon sequences in the human Fn gene in determining alternative splicing of this exon during transient expression of alpha globin/Fn minigene hybrids in HeLa cells. We demonstrate that a DNA sequence of 81bp within the central region of exon EDIIIA is required for alternative splicing during processing of the primary transcript to generate both EDIIIA+ and EDIIIA- mRNA's. Furthermore, alternative splicing of EDIIIA only occurs when this sequence is present in the correct orientation since when it is in antisense orientation splicing always occurs via exon-skipping generating EDIIIA- mRNA. Images PMID:3671064

Role of Altered Sialylation of the I-Like Domain of β1 Integrin in the Binding of Fibronectin to β1 Integrin: Thermodynamics and Conformational Analyses

PubMed Central

Pan, Di; Song, Yuhua

2010-01-01

Abstract N-glycosylation of the I-like domain of β1 integrin plays an essential role in integrin structure and function, and the altered sialylation of β1 integrin regulates β1 integrin binding to fibronectin. However, the structural basis underlying the effect of altered sialylation of the β1 I-like domain on β1 integrin binding to fibronectin remains largely unknown. In this study, we used a combination of molecular dynamics simulations and binding free energy analyses to investigate changes in binding thermodynamics and in conformation of the glycosylated β1 I-like domain-FN-III9-10 complex caused by altered sialylation of the β1 I-like domain. Binding free energy analyses showed that desialylation of β1 I-like domain increased β1 integrin binding to fibronectin, consistent with experimental results. Interaction analyses showed that altered sialylation of the β1 I-like domain resulted in significant changes in the interaction of the N-glycans of the I-like domain with both the I-like domain and fibronectin, and these changes could directly affect the allosteric regulation of the interaction between the I-like domain and fibronectin. Altered sialylation of the β1 I-like domain caused significant conformational changes in key functional sites of both the β1 I-like domain and fibronectin. In addition, altered sialylation of the β1 I-like domain resulted in changes in the degree of correlated motions between residues in the I-like domain and residues in fibronectin, and in the degree of motion changes in fibronectin, which could affect β1 integrin binding to fibronectin. We believe results from this study provide thermodynamic and structural evidence for a role of altered sialylation of β1 integrin in regulating β1 integrin binding to fibronectin and it's induced cellular activities. PMID:20655849

Cell-Matrix Interactions in Breast Carcinoma Invasion.

DTIC Science & Technology

1998-01-01

concentrated in hemidesmosomes, adhesive junctions which connect the basement membrane to the intracellular keratin cytoskeleton. In virtually all...fibronectin receptor contribute to the adhesive abnormalities of transformed fibroblasts by overexpressing this integrin in Chinese hamster ovary (CHO) cells...normal breast epithelium , the integrins expressed in breast carcinoma cells are diffusely distributed over the cell surface (Zutter et al., 1990

Persistent Transmissible Gastroenteritis Virus Infection Enhances Enterotoxigenic Escherichia coli K88 Adhesion by Promoting Epithelial-Mesenchymal Transition in Intestinal Epithelial Cells.

PubMed

Xia, Lu; Dai, Lei; Yu, Qinghua; Yang, Qian

2017-11-01

Transmissible gastroenteritis virus (TGEV) is a coronavirus characterized by diarrhea and high morbidity rates, and the mortality rate is 100% in piglets less than 2 weeks old. Pigs infected with TGEV often suffer secondary infection by other pathogens, which aggravates the severity of diarrhea, but the mechanisms remain unknown. Here, we hypothesized that persistent TGEV infection stimulates the epithelial-mesenchymal transition (EMT), and thus enterotoxigenic Escherichia coli (ETEC) can more easily adhere to generating cells. Intestinal epithelial cells are the primary targets of TGEV and ETEC infections. We found that TGEV can persistently infect porcine intestinal columnar epithelial cells (IPEC-J2) and cause EMT, consistent with multiple changes in key cell characteristics. Infected cells display fibroblast-like shapes; exhibit increases in levels of mesenchymal markers with a corresponding loss of epithelial markers; have enhanced expression levels of interleukin-1β (IL-1β), IL-6, IL-8, transforming growth factor β (TGF-β), and tumor necrosis factor alpha (TNF-α) mRNAs; and demonstrate increases in migratory and invasive behaviors. Additional experiments showed that the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) signaling pathways via TGF-β is critical for the TGEV-mediated EMT process. Cellular uptake is also modified in cells that have undergone EMT. TGEV-infected cells have higher levels of integrin α5 and fibronectin and exhibit enhanced ETEC K88 adhesion. Reversal of EMT reduces ETEC K88 adhesion and inhibits the expression of integrin α5 and fibronectin. Overall, these results suggest that TGEV infection induces EMT in IPEC-J2 cells, increasing the adhesion of ETEC K88 in the intestine and facilitating dual infection. IMPORTANCE Transmissible gastroenteritis virus (TGEV) causes pig diarrhea and is often followed by secondary infection by other pathogens. In this study, we showed that persistent TGEV infection induces an EMT in porcine intestinal columnar epithelial cells (IPEC-J2) and enhances the adhesion of the secondary pathogen ETEC K88. Additional experiments suggest that integrin α5 and fibronectin play an important role in TGEV-enhanced ETEC K88 adhesion. Reversal of EMT reduces the expression of integrin α5 and fibronectin and also reduces ETEC K88 adhesion. We conclude that TGEV infection triggers EMT and facilitates dual infection. Our results provide new insights into secondary infection and suggest that targeted anti-EMT therapy may have implications for the prevention and treatment of secondary infection. Copyright © 2017 American Society for Microbiology.

Cyclic Mechanical Stretch Up-regulates Hepatoma-Derived Growth Factor Expression in Cultured Rat Aortic Smooth Muscle Cells.

PubMed

Kao, Ying-Hsien; Chen, Po-Han; Sun, Cheuk-Kwan; Chang, Yo-Chen; Lin, Yu-Chun; Tsai, Ming-Shian; Lee, Po-Huang; Cheng, Cheng-I

2018-02-21

Hepatoma-derived growth factor (HDGF) is a potent mitogen for vascular smooth muscle cells (SMCs) during embryogenesis and injury repair of vessel walls. Whether mechanical stimuli modulate HDGF expression remains unknown. This study aimed at investigating whether cyclic mechanical stretch plays a regulatory role in HDGF expression and regenerative cytokine production in aortic SMCs. A SMC cell line was grown on a silicone-based elastomer chamber with extracellular matrix coatings (either type I collagen or fibronectin) and received cyclic and uni-axial mechanical stretches with 10% deformation at frequency 1 Hz. Morphological observation showed that fibronectin coating provided better cell adhesion and spreading and that consecutive 6 hours of cyclic mechanical stretch remarkably induced reorientation and realignment of SMCs. Western blotting detection demonstrated that continuous mechanical stimuli elicited up-regulation of HDGF and PCNA, a cell proliferative marker. Signal kinetic profiling study indicated that cyclic mechanical stretch induced signaling activity in RhoA/ROCK and PI3K/Akt cascades. Kinase inhibition study further showed that blockade of PI3K activity suppressed the stretch-induced TNF-a, whereas RhoA/ROCK inhibition significantly blunted the IL-6 production and HDGF over-expression. Moreover, siRNA-mediated HDGF gene silencing significantly suppressed constitutive expression of IL-6, but not TNF-α, in SMCs. These findings support the role of HDGF in maintaining vascular expression of IL-6, which has been regarded a crucial regenerative factor for acute vascular injury. In conclusion, cyclic mechanical stretch may maintain constitutive expression of HDGF in vascular walls and be regarded an important biophysical regulator in vascular regeneration. ©2018 The Author(s).

NF-κB Mediates the Stimulation of Cytokine and Chemokine Expression by Human Articular Chondrocytes in Response to Fibronectin Fragments1

PubMed Central

Pulai, Judit I.; Chen, Hong; Im, Hee-Jeong; Kumar, Sanjay; Hanning, Charles; Hegde, Priti S.; Loeser, Richard F.

2010-01-01

Fibronectin fragments (FN-f) that bind to the α5β1 integrin stimulate chondrocyte-mediated cartilage destruction and could play an important role in the progression of arthritis. The objective of this study was to identify potential cytokine mediators of cartilage inflammation and destruction induced by FN-f and to investigate the mechanism of their stimulation. Human articular chondrocytes, isolated from normal ankle cartilage obtained from tissue donors, were treated with a 110-kDa FN-f in serum-free culture, and expression of various cytokine genes was analyzed by cDNA microarray and by a cytokine protein array. Compared with untreated control cultures, stimulation by FN-f resulted in a >2-fold increase in IL-6, IL-8, MCP-1, and growth-related oncogene β (GRO-β). Constitutive and FN-f-inducible expression of GRO-α and GRO-γ were also noted by RT-PCR and confirmed by immunoblotting. Previous reports of IL-1β expression induced by FN-f were also confirmed, while TNF expression was found to be very low. Inhibitor studies revealed that FN-f-induced stimulation of chondrocyte chemokine expression was dependent on NF-κB activity, but independent of IL-1 autocrine signaling. The ability of FN-f to stimulate chondrocyte expression of multiple proinflammatory cytokines and chemokines suggests that damage to the cartilage matrix is capable of inducing a proinflammatory state responsible for further progressive matrix destruction, which also includes the chemoattraction of inflammatory cells. Targeting the signaling pathways activated by FN-f may be an effective means of inhibiting production of multiple mediators of cartilage destruction. PMID:15843581

Pirfenidone may revert the epithelial-to-mesenchymal transition in human lung adenocarcinoma.

PubMed

Kurimoto, Ryota; Ebata, Takahiro; Iwasawa, Shunichiro; Ishiwata, Tsukasa; Tada, Yuji; Tatsumi, Koichiro; Takiguchi, Yuichi

2017-07-01

The epithelial-to-mesenchymal transition (EMT) in cancer is associated with invasion, metastasis and chemoresistance. Recent studies have revealed the increased expression of programmed death-ligand 1 (PD-L1) in cells undergoing EMT. The underlying mechanism of EMT involves transforming growth factor-β (TGF-β) and fibroblast growth factor-2 (FGF-2). Pirfenidone and the known EMT-suppressor nintedanib suppress pulmonary fibrosis partially through suppression of TGF-β. The present study aimed to determine whether pirfenidone has the potential to induce EMT-reversion, using nintedanib as a reference. The human lung adenocarcinoma cell lines A-549, HCC-827, and PC-9 were treated with TGF-β and FGF-2 to induce EMT. The EMT-induced cells were further treated with pirfenidone or nintedanib. Phenotypic alterations associated with EMT were assessed by examining the following: i) The expression levels of E-cadherin, vimentin, fibronectin and slug, using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and fluorescent immunohistochemistry; ii) cell motility via wound-healing assays; and iii) the expression of PD-L1 using RT-qPCR. The combination of TGF-β and FGF-2 successfully induced EMT in all three cell lines, characterized by a significant reduction in E-cadherin expression in the A-549 and HCC-827 cells, increased expression levels of vimentin, fibronectin, slug and PD-L1, and increased cell motility in all three cell lines. Pirfenidone and nintedanib reverted all of these phenotypes, with the exception of unaltered E-cadherin expression in all three cell lines, and inconsistent expression of vimentin in the HCC-827 and PC-9 cells. Thus, pirfenidone and nintedanib have the ability to induce EMT-reversion in human lung adenocarcinoma.

Syndecan-2 is upregulated in colorectal cancer cells through interactions with extracellular matrix produced by stromal fibroblasts.

PubMed

Vicente, Carolina Meloni; Ricci, Ritchelli; Nader, Helena Bonciani; Toma, Leny

2013-05-25

The extracellular matrix (ECM) influences the structure, viability and functions of cells and tissues. Recent evidence indicates that tumor cells and stromal cells interact through direct cell-cell contact, the production of ECM components and the secretion of growth factors. Syndecans are a family of transmembrane heparan sulfate proteoglycans that are involved in cell adhesion, motility, proliferation and differentiation. Syndecan-2 has been found to be highly expressed in colorectal cancer cell lines and appears to be critical for cancer cell behavior. We have examined the effect of stromal fibroblast-produced ECM on the production of proteoglycans by colorectal cancer cell lines. Our results showed that in a highly metastatic colorectal cancer cell line, HCT-116, syndecan-2 expression is enhanced by fibroblast ECM, while the expression of other syndecans decreased. Of the various components of the stromal ECM, fibronectin was the most important in stimulating the increase in syndecan-2 expression. The co-localization of syndecan-2 and fibronectin suggests that these two molecules are involved in the adhesion of HCT-116 cells to the ECM. Additionally, we demonstrated an increase in the expression of integrins alpha-2 and beta-1, in addition to an increase in the expression of phospho-FAK in the presence of fibroblast ECM. Furthermore, blocking syndecan-2 with a specific antibody resulted in a decrease in cell adhesion, migration, and organization of actin filaments. Overall, these results show that interactions between cancer cells and stromal ECM proteins induce significant changes in the behavior of cancer cells. In particular, a shift from the expression of anti-tumorigenic syndecans to the tumorigenic syndecan-2 may have implications in the migratory behavior of highly metastatic tumor cells.

Decreased Fibronectin Production Significantly Contributes to Dysregulated Repair of Asthmatic Epithelium

PubMed Central

Kicic, Anthony; Hallstrand, Teal S.; Sutanto, Erika N.; Stevens, Paul T.; Kobor, Michael S.; Taplin, Christopher; Paré, Peter D.; Beyer, Richard P.; Stick, Stephen M.; Knight, Darryl A.

2010-01-01

Rationale: Damage to airway epithelium is followed by deposition of extracellular matrix (ECM) and migration of adjacent epithelial cells. We have shown that epithelial cells from children with asthma fail to heal a wound in vitro. Objectives: To determine whether dysregulated ECM production by the epithelium plays a role in aberrant repair in asthma. Methods: Airway epithelial cells (AEC) from children with asthma (n = 36), healthy atopic control subjects (n = 23), and healthy nonatopic control subjects (n = 53) were investigated by microarray, gene expression and silencing, transcript regulation analysis, and ability to close mechanical wounds. Measurements and Main Results: Time to repair a mechanical wound in vitro by AEC from healthy and atopic children was not significantly different and both were faster than AEC from children with asthma. Microarray analysis revealed differential expression of multiple gene sets associated with repair and remodeling in asthmatic AEC. Fibronectin (FN) was the only ECM component whose expression was significantly lower in asthmatic AEC. Expression differences were verified by quantitative polymerase chain reaction and ELISA, and reduced FN expression persisted in asthmatic cells over passage. Silencing of FN expression in nonasthmatic AEC inhibited wound repair, whereas addition of FN to asthmatic AEC restored reparative capacity. Asthmatic AEC failed to synthesize FN in response to wounding or cytokine/growth factor stimulation. Exposure to 5′, 2′deoxyazacytidine had no effect on FN expression and subsequent analysis of the FN promoter did not show evidence of DNA methylation. Conclusions: These data show that the reduced capacity of asthmatic epithelial cells to secrete FN is an important contributor to the dysregulated AEC repair observed in these cells. PMID:20110557

The effect of statin on epithelial-mesenchymal transition in peritoneal mesothelial cells.

PubMed

Chang, Tae Ik; Kang, Hye-Young; Kim, Kyung Sik; Lee, Sun Ha; Nam, Bo Young; Paeng, Jisun; Kim, Seonghun; Park, Jung Tak; Yoo, Tae-Hyun; Kang, Shin-Wook; Han, Seung Hyeok

2014-01-01

Statins have recently been highlighted for their pleiotropic actions distinct from cholesterol-lowering effects. Despite this interest, it is currently unknown whether statin therapy inhibits peritoneal dialysis (PD)-related epithelial-mesenchymal transition (EMT). In vitro, human peritoneal mesothelial cells (HPMCs) were exposed to 5.6 mM glucose (NG) or 100 mM glucose (HG) with or without simvastatin (1 µM). In vivo, PD catheters were inserted into 32 Sprague-Dawley rats, and saline (C, n = 16) or 4.25% peritoneal dialysis fluid (PDF) (PD, n = 16) was infused for 4 weeks. Eight rats from each group were treated with 5 mg/kg/day of simvastatin intraperitoneally. Changes in the protein expression of EMT markers such as E-cadherin, α-SMA, Snail, and fibronectin in HPMCs and the peritoneum were evaluated by Western blot analysis and immunofluorescence or immunohistochemical staining. We also explored whether activation of the mevalonate pathway and its downstream small GTPases were involved in dialysis-related peritoneal EMT and could be inhibited by statin treatment. Compared to NG cells, E-cadherin expression was significantly decreased, while α-SMA, Snail, and fibronectin expression were significantly increased in HPMCs exposed to HG, and these changes were abrogated by simvastatin (p<0.05). In addition, the cobblestone-like appearance of normal HPMCs was converted into a fibroblast-like morphology after HG treatment, which was reversed by simvastatin. These EMT-like changes were also observed in HPMCs treated with geranyl-geranyl pyrophosphate (5 µM). HG significantly increased the protein expression of RhoA and Rac1 in the membrane fractions, and these increases were ameliorated by simvastatin (p<0.05). In PD rats, E-cadherin in the peritoneum was significantly decreased, whereas α-SMA, Snail, and fibronectin expression were significantly increased (p<0.05) compared to C rats. The thickness of the mesothelial layer in the peritoneum were also significantly greater in PD rats than in C rats (p<0.05). These changes of the peritoneum in PD rats were significantly attenuated by simvastatin. This study demonstrated that PD-related EMT was mediated via the mevalonate pathway, and statin treatment inhibited the EMT changes in HG-treated HPMCs and PDF-stimulated PD rats. These findings suggest that statins may be a promising therapeutic strategy for preservation of peritoneal membrane integrity in long-term PD patients.

Inhibition of Pseudomonas aeruginosa adhesion to fibronectin by PA-IL and monosaccharides: involvement of a lectin-like process.

PubMed

Rebiere-Huët, Julie; Di Martino, Patrick; Hulen, Christian

2004-05-01

Pseudomonas aeruginosa adherence to fibronectin has been shown to be important to bacterial colonization and infection. To better understand the mechanisms involved in this interaction, the role of the carbohydrate moiety of the fibronectin molecule in P. aeruginosa adhesion was studied. Strain NK 125 502 adhered to immobilized fibronectin with an adherence index of 4.8 x 10(5) CFU/ micro g. Periodic oxidation of fibronectin markedly reduced the adhesion of P. aeruginosa, while a neuraminidase treatment increased bacteria adhesion. N-Acetylgalactosamine, N-acetylglucosamine, sialic acid, and also lectin PA-IL worked as efficient inhibitors in adhesion assays: 59%, 70.7%, 100%, and 60% of inhibition, respectively. We have demonstrated here the involvement of a lectin-like process in the interaction of P. aeruginosa NK 125 502 with immobilized fibronectin.

SPR imaging biosensor for the quantitation of fibronectin concentration in blood samples.

PubMed

Sankiewicz, Anna; Romanowicz, Lech; Pyc, Marlena; Hermanowicz, Adam; Gorodkiewicz, Ewa

2018-02-20

The purpose of this study was presentation of a new biosensor capable of determination of fibronectin. This biosensor was based on the specific interaction of anti-fibronectin antibody produced in rabbit with fibronectin. The surface plasmon resonance imaging (SPRI) technique was used as a detecting method. Optimization and characterization properties of the biosensor were studied. The determination of fibronectin concentration in natural samples was done. The results were compared with a reference method (Enzyme-Linked Immunosorbent Assay-ELISA). The analytically useful dynamic response range of biosensor is between 5 and 400ngmL -1 . The detection limit is 1.5ngmL -1 and limit quantification is 5ngmL -1 . The proposed SPRI biosensor showed good selectivity for potential interferences. It was applied to determine fibronectin concentrations in plasma of healthy donors and of patients after thermal injury. Good correlations between results obtained using the SPRI biosensor and ELISA test (correlation coefficients for healthy donors 0.996, for patients 0.984) were obtained. The average fibronectin concentration of healthy donors was 140.5±24.6μgmL -1 and the average fibronectin concentration of patients was 601.5±72.1μgmL -1 , which was in agreement with results obtained by other investigators. The obtained results indicate that the developed biosensor may be a candidate for monitoring fibronectin concentration in blood samples. Copyright © 2017 Elsevier B.V. All rights reserved.

TMPRSS4 regulates levels of integrin α5 in NSCLC through miR-205 activity to promote metastasis.

PubMed

Larzabal, L; de Aberasturi, A L; Redrado, M; Rueda, P; Rodriguez, M J; Bodegas, M E; Montuenga, L M; Calvo, A

2014-02-04

TMPRSS4 is a membrane-anchored protease involved in cell migration and invasion in different cancer types including lung cancer. TMPRSS4 expression is increased in NSCLC and its inhibition through shRNA reduces lung metastasis. However, molecular mechanisms leading to the protumorigenic regulation of TMPRSS4 in lung cancer are unknown. miR-205 was identified as an overexpressed gene upon TMPRSS4 downregulation through microarray analysis. Cell migration and invasion assays and in vivo lung primary tumour and metastasis models were used for functional analysis of miR-205 overexpression in H2170 and H441 cell lines. Luciferase assays were used to identify a new miR-205 direct target in NSCLC. miR-205 overexpression promoted an epithelial phenotype with increased E-cadherin and reduced fibronectin. Furthermore, miR-205 expression caused a G0/G1 cell cycle arrest and inhibition of cell growth, migration, attachment to fibronectin, primary tumour growth and metastasis formation in vivo. Integrin α5 (a proinvasive protein) was identified as a new miR-205 direct target in NSCLC. Integrin α5 downregulation in lung cancer cells resulted in complete abrogation of cell migration, a decreased capacity to adhere to fibronectin and reduced in vivo tumour growth, compared with control cells. TMPRSS4 silencing resulted in a concomitant reduction of integrin α5 levels. We have demonstrated for the first time a new molecular pathway that connects TMPRSS4 and integrin α5 through miR-205 to regulate cancer cell invasion and metastasis. Our results will help designing new therapeutic strategies to inhibit this novel pathway in NSCLC.

Identification of a Novel Virulence Determinant with Serum Opacification Activity in Streptococcus suis

PubMed Central

Baums, Christoph G.; Kaim, Ute; Fulde, Marcus; Ramachandran, Girish; Goethe, Ralph; Valentin-Weigand, Peter

2006-01-01

Streptococcus suis serotype 2 is a porcine and human pathogen with adhesive and invasive properties. In other streptococci, large surface-associated proteins (>100 kDa) of the MSCRAMM family (microbial surface components recognizing adhesive matrix molecules) are key players in interactions with host tissue. In this study, we identified a novel opacity factor of S. suis (OFS) with structural homology to members of the MSCRAMM family. The N-terminal region of OFS is homologous to the respective regions of fibronectin-binding protein A (FnBA) of Streptococcus dysgalactiae and the serum opacity factor (SOF) of Streptococcus pyogenes. Similar to these two proteins, the N-terminal domain of OFS opacified horse serum. Serum opacification activity was detectable in sodium dodecyl sulfate extracts of wild-type S. suis but not in extracts of isogenic ofs knockout mutants. Heterologous expression of OFS in Lactococcus lactis demonstrated that a high level of expression of OFS is sufficient to provide surface-associated serum opacification activity. Furthermore, serum opacification could be inhibited by an antiserum against recombinant OFS. The C-terminal repetitive sequence elements of OFS differed significantly from the respective repeat regions of FnBA and SOF as well as from the consensus sequence of the fibronectin-binding repeats of MSCRAMMs. Accordingly, fibronectin binding was not detectable in recombinant OFS. To investigate the putative function of OFS in the pathogenesis of invasive S. suis diseases, piglets were experimentally infected with an isogenic mutant strain in which the ofs gene had been knocked out by an in-frame deletion. The mutant was severely attenuated in virulence but not in colonization, demonstrating that OFS represents a novel virulence determinant of S. suis. PMID:17057090

CCAAT/Enhancer Binding Protein–α Regulates the Protease/Antiprotease Balance Required for Bronchiolar Epithelium Regeneration

PubMed Central

Sato, Atsuyasu; Xu, Yan; Whitsett, Jeffrey A.

2012-01-01

Many transcription factors that regulate lung morphogenesis during development are reactivated to mediate repairs of the injured adult lung. We hypothesized that CCAAT/enhancer binding protein–α (C/EBPα), a transcription factor critical for perinatal lung maturation, regulates genes required for the normal repair of the bronchiolar epithelium after injury. Transgenic CebpαΔ/Δ mice, in which Cebpa was conditionally deleted from Clara cells and Type II cells after birth, were used in this study. Airway injury was induced in mice by the intraperitoneal administration of naphthalene to ablate bronchiolar epithelial cells. Although the deletion of C/EBPα did not influence lung structure and function under unstressed conditions, C/EBPα was required for the normal repair of terminal bronchiolar epithelium after naphthalene injury. To identify cellular processes that are influenced by C/EBPα during repair, mRNA microarray was performed on terminal bronchiolar epithelial cells isolated by laser-capture microdissection. Normal repair of the terminal bronchiolar epithelium was highly associated with the mRNAs regulating antiprotease activities, and their induction required C/EBPα. The defective deposition of fibronectin in CebpαΔ/Δ mice was associated with increased protease activity and delayed differentiation of FoxJ1-expressing ciliated cells. The fibronectin and ciliated cells were restored by the intratracheal treatment of CebpαΔ/Δ mice with the serine protease inhibitor. In conclusion, C/EBPα regulates the expression of serine protease inhibitors that are required for the normal increase of fibronectin and the restoration of ciliated cells after injury. Treatment with serine protease inhibitor may aid in the recovery of injured bronchiolar epithelial cells, and prevent common chronic lung diseases. PMID:22652201

A conserved gene family encodes transmembrane proteins with fibronectin, immunoglobulin and leucine-rich repeat domains (FIGLER)

PubMed Central

Munfus, Delicia L; Haga, Christopher L; Burrows, Peter D; Cooper, Max D

2007-01-01

Background In mouse the cytokine interleukin-7 (IL-7) is required for generation of B lymphocytes, but human IL-7 does not appear to have this function. A bioinformatics approach was therefore used to identify IL-7 receptor related genes in the hope of identifying the elusive human cytokine. Results Our database search identified a family of nine gene candidates, which we have provisionally named fibronectin immunoglobulin leucine-rich repeat (FIGLER). The FIGLER 1–9 genes are predicted to encode type I transmembrane glycoproteins with 6–12 leucine-rich repeats (LRR), a C2 type Ig domain, a fibronectin type III domain, a hydrophobic transmembrane domain, and a cytoplasmic domain containing one to four tyrosine residues. Members of this multichromosomal gene family possess 20–47% overall amino acid identity and are differentially expressed in cell lines and primary hematopoietic lineage cells. Genes for FIGLER homologs were identified in macaque, orangutan, chimpanzee, mouse, rat, dog, chicken, toad, and puffer fish databases. The non-human FIGLER homologs share 38–99% overall amino acid identity with their human counterpart. Conclusion The extracellular domain structure and absence of recognizable cytoplasmic signaling motifs in members of the highly conserved FIGLER gene family suggest a trophic or cell adhesion function for these molecules. PMID:17854505

A Biosynthetic Nerve Guide Conduit Based on Silk/SWNT/Fibronectin Nanocomposite for Peripheral Nerve Regeneration

PubMed Central

Mottaghitalab, Fatemeh; Farokhi, Mehdi; Zaminy, Arash; Kokabi, Mehrdad; Soleimani, Masoud; Mirahmadi, Fereshteh

2013-01-01

As a contribution to the functionality of nerve guide conduits (NGCs) in nerve tissue engineering, here we report a conduit processing technique through introduction and evaluation of topographical, physical and chemical cues. Porous structure of NGCs based on freeze-dried silk/single walled carbon nanotubes (SF/SWNTs) has shown a uniform chemical and physical structure with suitable electrical conductivity. Moreover, fibronectin (FN) containing nanofibers within the structure of SF/SWNT conduits produced through electrospinning process have shown aligned fashion with appropriate porosity and diameter. Moreover, fibronectin remained its bioactivity and influenced the adhesion and growth of U373 cell lines. The conduits were then implanted to 10 mm left sciatic nerve defects in rats. The histological assessment has shown that nerve regeneration has taken places in proximal region of implanted nerve after 5 weeks following surgery. Furthermore, nerve conduction velocities (NCV) and more myelinated axons were observed in SF/SWNT and SF/SWNT/FN groups after 5 weeks post implantation, indicating a functional recovery for the injured nerves. With immunohistochemistry, the higher S-100 expression of Schwann cells in SF/SWNT/FN conduits in comparison to other groups was confirmed. In conclusion, an oriented conduit of biocompatible SF/SWNT/FN has been fabricated with acceptable structure that is particularly applicable in nerve grafts. PMID:24098649

Cellular Interaction of Integrin α3β1 with Laminin 5 Promotes Gap Junctional Communication

PubMed Central

Lampe, Paul D.; Nguyen, Beth P.; Gil, Susana; Usui, Marcia; Olerud, John; Takada, Yoshikazu; Carter, William G.

1998-01-01

Wounding of skin activates epidermal cell migration over exposed dermal collagen and fibronectin and over laminin 5 secreted into the provisional basement membrane. Gap junctional intercellular communication (GJIC) has been proposed to integrate the individual motile cells into a synchronized colony. We found that outgrowths of human keratinocytes in wounds or epibole cultures display parallel changes in the expression of laminin 5, integrin α3β1, E-cadherin, and the gap junctional protein connexin 43. Adhesion of keratinocytes on laminin 5, collagen, and fibronectin was found to differentially regulate GJIC. When keratinocytes were adhered on laminin 5, both structural (assembly of connexin 43 in gap junctions) and functional (dye transfer) assays showed a two- to threefold increase compared with collagen and five- to eightfold over fibronectin. Based on studies with immobilized integrin antibody and integrin-transfected Chinese hamster ovary cells, the interaction of integrin α3β1 with laminin 5 was sufficient to promote GJIC. Mapping of intermediate steps in the pathway linking α3β1–laminin 5 interactions to GJIC indicated that protein trafficking and Rho signaling were both required. We suggest that adhesion of epithelial cells to laminin 5 in the basement membrane via α3β1 promotes GJIC that integrates individual cells into synchronized epiboles. PMID:9852164

Laminin and Fibronectin in Cell Adhesion: Enhanced Adhesion of Cells from Regenerating Liver to Laminin

NASA Astrophysics Data System (ADS)

Carlsson, Roland; Engvall, Eva; Freeman, Aaron; Ruoslahti, Erkki

1981-04-01

Laminin, a basement membrane glycoprotein isolated from cultures of mouse endodermal cells and rat yolk sac carcinoma cells, promoted the attachment of liver cells obtained from regenerating mouse liver. Cells from normal mouse liver attached readily to dishes coated with fibronectin but attached poorly to surfaces coated with laminin. Both proteins efficiently promoted the attachment of cells from livers undergoing regeneration. After regeneration, the attachment to laminin returned to the low levels found in animals not subjected to partial hepatectomy but attachment to fibronectin remained high. Immunofluorescent staining of sections of normal liver with antilaminin revealed the presence of laminin in or adjacent to the walls of the bile ducts and blood vessels. After induction of regeneration by partial hepatectomy, increased amounts of laminin appeared in the sinusoidal areas. After carbon tetrachloride poisoning, staining for laminin was especially pronounced in the necrotic and postnecrotic areas around the central veins. This additional expression of laminin was transient. It reached a maximum around 5-6 days after the injury and then gradually disappeared. These findings show that laminin is an adhesive protein. The increase of laminin in regenerating liver and the adhesiveness of cells from such livers to laminin suggest a role for laminin in the maintenance of a proper tissue organization during liver regeneration.

[The role of fibronectin in adhesion of corynebacteria to vaginal epitheliocytes].

PubMed

Gladysheva, I V; Cherkasov, S V

2014-01-01

Determination of the role of fibronectin in adhesion of corynebacteria to vaginal epitheliocytes. Corynebacterium genus microorganisms and primary epitheliocytes isolated from the lower reproductive tract of women were used. Adhesive ability of corynebacteria was studied in polystyrene plates against fixed fibronectin and on the model of vaginal epitheliocytes. Changes in adhesion of corynebacteria to vaginal epitheliocytes was evaluated after treatment of the latter with fibronectin. All the studied strains had the adhesion ability to fibronectin and vaginal epitheliocytes. The same strains were attributed to groups of high, moderate or low adhesive using both plate method and method utilizing vaginal epitheliocytes model, that tells of their comparability. During the addition of fibronectin to epitheliocytes, an enhancement of adhesion of all the studied corynebacteria strains took place. Adhesion index in strains isolated from healthy women increased by an average of 46.6%, adhesion index by 10.5 bact. cells/epith. In strains isolated from women with micro-ecologic disruption, adhesion increase was by 15.3% and 4.9 bact. cells/epith., respectively. Fibronectin is a factor that determines adhesion of corynebacteria to vaginal epitheliocytes and thus is important for formation of associative symbiosis of reproductive tract of women. The data obtained open perspective of use of fibronectin with the aim of colonizing ability increase of probiotics.

MC3T3-E1 Cells on Titanium Surfaces with Nanometer Smoothness and Fibronectin Immobilization

PubMed Central

Hayakawa, Tohru; Yoshida, Eiji; Yoshimura, Yoshitaka; Uo, Motohiro; Yoshinari, Masao

2012-01-01

The present study was aimed to evaluate the viability and total protein contents of osteoblast-like cells on the titanium surface with different surface mechanical treatment, namely, nanometer smoothing (Ra: approximately 2.0 nm) and sandblasting (Ra: approximately 1.0 μm), and biochemical treatment, namely, with or without fibronectin immobilization. Fibronectin could be easily immobilized by tresyl chloride-activation technique. MC3T3-E1 cells were seeded on the different titanium surfaces. Cell viability was determined by MTT assay. At 1 day of cell culture, there were no significant differences in cell viability among four different titanium surfaces. At 11 days, sandblasted titanium surface with fibronectin immobilization showed the significantly highest cell viability than other titanium surface. No significant differences existed for total protein contents among four different titanium surfaces at 11 days of cell culture. Scanning electron microscopy observation revealed that smoothness of titanium surface produced more spread cell morphologies, but that fibronectin immobilization did not cause any changes of the morphologies of attached cells. Fibronectin immobilization provided greater amount of the number of attached cells and better arrangement of attached cells. In conclusion, the combination of sandblasting and fibronectin immobilization enhanced the cell viability and fibronectin immobilization providing better arrangements of attached cells. PMID:22675359

Adhesive interactions of human multiple myeloma cell lines with different extracellular matrix molecules.

PubMed

Kibler, C; Schermutzki, F; Waller, H D; Timpl, R; Müller, C A; Klein, G

1998-06-01

Multiple myeloma represents a human B cell malignancy which is characterized by a predominant localization of the malignant cell clone within the bone marrow. With the exception of the terminal stage of the disease the myeloma tumor cells do not circulate in the peripheral blood. The bone marrow microenvironment is believed to play an important role in homing, proliferation and terminal differentiation of myeloma cells. Here we have studied the expression of several extracellular matrix (ECM) molecules in the bone marrow of multiple myeloma patients and analyzed their adhesive capacities with four different human myeloma-derived cell lines. All ECM molecules analyzed (tenascin, laminin, fibronectin, collagen types I, III, V and VI) could be detected in bone marrow cryostat sections of multiple myeloma patients. Adhesion assays showed that only laminin, the microfibrillar collagen type VI and fibronectin were strong adhesive components for the myeloma cell lines U266, IM-9, OPM-2 and NCI-H929. Tenascin and collagen type I were only weak adhesive substrates for these myeloma cells. Adhesion to laminin and fibronectin was beta 1-integrin-mediated since addition of anti-beta 1-integrin antibodies could inhibit the binding of the four different cell types to both matrix molecules. In contrast, integrins do not seem to be involved in binding of the myeloma cells to collagen type VI. Instead, inhibition of binding by heparin suggested that membrane-bound heparan sulfate proteoglycans are responsible ligands for binding to collagen type VI. Adhesion assays with several B-cell lines resembling earlier differentiation stages revealed only weak interactions with tenascin and no interactions with collagen type VI, laminin or fibronectin. In summary, the interactions of human myeloma cells with the extracellular matrix may explain the specific retention of the plasma cells within the bone marrow.

Enhanced expression of FNDC5 in human embryonic stem cell-derived neural cells along with relevant embryonic neural tissues.

PubMed

Ghahrizjani, Fatemeh Ahmadi; Ghaedi, Kamran; Salamian, Ahmad; Tanhaei, Somayeh; Nejati, Alireza Shoaraye; Salehi, Hossein; Nabiuni, Mohammad; Baharvand, Hossein; Nasr-Esfahani, Mohammad Hossein

2015-02-25

Availability of human embryonic stem cells (hESCs) has enhanced the capability of basic and clinical research in the context of human neural differentiation. Derivation of neural progenitor (NP) cells from hESCs facilitates the process of human embryonic development through the generation of neuronal subtypes. We have recently indicated that fibronectin type III domain containing 5 protein (FNDC5) expression is required for appropriate neural differentiation of mouse embryonic stem cells (mESCs). Bioinformatics analyses have shown the presence of three isoforms for human FNDC5 mRNA. To differentiate which isoform of FNDC5 is involved in the process of human neural differentiation, we have used hESCs as an in vitro model for neural differentiation by retinoic acid (RA) induction. The hESC line, Royan H5, was differentiated into a neural lineage in defined adherent culture treated by RA and basic fibroblast growth factor (bFGF). We collected all cell types that included hESCs, rosette structures, and neural cells in an attempt to assess the expression of FNDC5 isoforms. There was a contiguous increase in all three FNDC5 isoforms during the neural differentiation process. Furthermore, the highest level of expression of the isoforms was significantly observed in neural cells compared to hESCs and the rosette structures known as neural precursor cells (NPCs). High expression levels of FNDC5 in human fetal brain and spinal cord tissues have suggested the involvement of this gene in neural tube development. Additional research is necessary to determine the major function of FDNC5 in this process. Copyright © 2014 Elsevier B.V. All rights reserved.

Proteomic Analysis of Gingival Tissue and Alveolar Bone during Alveolar Bone Healing*

PubMed Central

Yang, Hee-Young; Kwon, Joseph; Kook, Min-Suk; Kang, Seong Soo; Kim, Se Eun; Sohn, Sungoh; Jung, Seunggon; Kwon, Sang-Oh; Kim, Hyung-Seok; Lee, Jae Hyuk; Lee, Tae-Hoon

2013-01-01

Bone tissue regeneration is orchestrated by the surrounding supporting tissues and involves the build-up of osteogenic cells, which orchestrate remodeling/healing through the expression of numerous mediators and signaling molecules. Periodontal regeneration models have proven useful for studying the interaction and communication between alveolar bone and supporting soft tissue. We applied a quantitative proteomic approach to analyze and compare proteins with altered expression in gingival soft tissue and alveolar bone following tooth extraction. For target identification and validation, hard and soft tissue were extracted from mini-pigs at the indicated times after tooth extraction. From triplicate experiments, 56 proteins in soft tissue and 27 proteins in alveolar bone were found to be differentially expressed before and after tooth extraction. The expression of 21 of those proteins was altered in both soft tissue and bone. Comparison of the activated networks in soft tissue and alveolar bone highlighted their distinct responsibilities in bone and tissue healing. Moreover, we found that there is crosstalk between identified proteins in soft tissue and alveolar bone with respect to cellular assembly, organization, and communication. Among these proteins, we examined in detail the expression patterns and associated networks of ATP5B and fibronectin 1. ATP5B is involved in nucleic acid metabolism, small molecule biochemistry, and neurological disease, and fibronectin 1 is involved in cellular assembly, organization, and maintenance. Collectively, our findings indicate that bone regeneration is accompanied by a profound interaction among networks regulating cellular resources, and they provide novel insight into the molecular mechanisms involved in the healing of periodontal tissue after tooth extraction. PMID:23824910

Role of dextran in maintaining adhesive and stiffness properties of prestripped DMEK lenticules.

PubMed

Parekh, Mohit; Ruzza, Alessandro; Di Mundo, Rosa; Ferrari, Stefano; Recchia, Giuseppina; Elbadawy, Hossein; Carbone, Giuseppe; Ponzin, Diego

2017-05-11

To investigate the adhesive and stiffness properties of prestripped Descemet membrane endothelial keratoplasty (DMEK) lenticules in different preservation conditions (with and without dextran). The study included 3 conditions: (C1) tissues collected from tissue culture media (TCM), stripped and preserved in TCM; (C2) tissues collected from transport media (TM) (TCM supplemented with 6% dextran T-500), stripped and preserved in TM; and (C3) tissues collected from TCM, stripped and preserved in TM. Using a hinge, 9.5-mm stripped DMEK lenticules were restored back on the stroma and preserved for 4 days at room temperature (RT) in different conditions as above. Nine tissues, 3 from each condition, were used to check the adhesive (fibronectin, laminin, and vitronectin) and elastic properties (fibrillin, elastin, and collagen VI) using different antibodies. Six tissues, 2 from each condition, were used to check the stiffness properties after preservation using atomic force microscopy (AFM) nanoindentation method. On the Descemet membrane, fibronectin was strongly expressed in C2 and C3, whereas laminin was intense in C2 postpreservation. Vitronectin was expressed in all the conditions. Elastic proteins were not expressed in either of the conditions apart from collagen VI, which was expressed on the posterior stroma. Atomic force microscopy showed higher stiffness in C3 and an insignificant but lower rigidity in C2 as compared to C1. The tissues from C2 showed expression of adherent proteins and lower stiffness. Dextran may be suitable in preservation of DMEK grafts before and after preparation. Less stiff tissues may help reduce manipulations required in the recipient eye during DMEK surgery.

Baicalin ameliorates renal fibrosis via inhibition of transforming growth factor β1 production and downstream signal transduction

PubMed Central

Zheng, Long; Zhang, Chao; Li, Long; Hu, Chao; Hu, Mushuang; Sidikejiang, Niyazi; Wang, Xuanchuan; Lin, Miao; Rong, Ruiming

2017-01-01

Previous studies have demonstrated the potential antifibrotic effects of baicalin in vitro, via examination of 21 compounds isolated from plants. However, its biological activity and underlying mechanisms of action in vivo remain to be elucidated. The present study aimed to evaluate the effect of baicalin on renal fibrosis in vivo, and the potential signaling pathways involved. A unilateral ureteral obstruction (UUO)-induced renal fibrosis model was established using Sprague-Dawley rats. Baicalin was administrated intraperitoneally every 2 days for 10 days. The degree of renal damage and fibrosis was investigated by histological assessment, and detection of fibronectin and collagen I mRNA expression levels. Epithelial-mesenchymal transition (EMT) markers, transforming growth factor-β1 (TGF-β1) levels and downstream phosphorylation of mothers against decapentaplegic 2/3 (Smad2/3) were examined in vivo and in an NRK-52E rat renal tubular cell line in vitro. Baicalin was demonstrated to markedly ameliorate renal fibrosis and suppress EMT, as evidenced by reduced interstitial collagen accumulation, decreased fibronectin and collagen I mRNA expression levels, upregulation of N- and E-cadherin expression levels, and downregulation of α-smooth muscle actin and vimentin expression. Furthermore, baicalin decreased TGF-β1 expression levels in serum and kidney tissue following UUO, and suppressed Smad2/3 phosphorylation in rat kidney tissue. In vitro studies identified that baicalin may inhibit the phosphorylation of Smad2/3 under the same TGF-β1 concentration. In conclusion, baicalin may protect against renal fibrosis, potentially via inhibition of TGF-β1 production and its downstream signal transduction. PMID:28260014

Acellular dermal matrix scaffolds coated with connective tissue growth factor accelerate diabetic wound healing by increasing fibronectin through PKC signalling pathway.

PubMed

Yan, Wenxia; Liu, Hanping; Deng, Xiaoyuan; Jin, Ying; Wang, Ning; Chu, Jing

2018-03-01

The regional injection of connective tissue growth factor (CTGF) for diabetic wound healing requires multiple components and results in a substantial loss of its biological activity. Acellular dermal matrix (ADM) scaffolds are optimal candidates for delivering these factors to local ischaemic environments. In this study, we explored whether CTGF loaded on ADM scaffolds can enhance fibronectin (FN) expression to accelerate diabetic wound healing via the protein kinase C (PKC) signalling pathway. The performance of CTGF and CTGF + PKC inhibitor, which were loaded on ADM scaffolds to treat dorsal skin wounds in streptozotocin-induced diabetic mice, was evaluated with naked ADM as a control. Wound closure showed that ADM scaffolds loaded with CTGF induced greater diabetic wound healing in the early stage of the wound in diabetic mice. Moreover, ADM scaffolds loaded with CTGF obviously increased the expression of FN both at the mRNA and protein levels, whereas the expression of FN was significantly reduced in the inhibitor group. Furthermore, the ADM + CTGF group, which produce FN, obviously promoted alpha-smooth muscle actin and transforming growth factor-beta expression and enhanced neovasculature and collagen synthesis at the wound sites. ADM scaffolds loaded with CTGF + PKC inhibitor delayed diabetic wound healing, indicating that FN expression was mediated by the PKC signalling pathway. Our findings offer new perspectives for the treatment of diabetic wound healing and suggest a rationale for the clinical evaluation of CTGF use in diabetic wound healing. Copyright © 2017 John Wiley & Sons, Ltd.

Effect of Negative Pressure Wound Therapy on Cellular Fibronectin and Transforming Growth Factor-β1 Expression in Diabetic Foot Wounds.

PubMed

Yang, Shao Ling; Zhu, Lv Yun; Han, Rui; Sun, Lei Lei; Dou, Jing Tao

2017-08-01

Chronic diabetic foot wounds are a leading cause of amputation, morbidity, and hospitalization for patients with diabetes. Negative-pressure wound therapy (NPWT) can putatively facilitate wound healing, but the underlying mechanisms remain unclear. Cellular fibronectin (cFN) and transforming growth factor-β1 (TGF-β1) play an important role in wound healing. This prospective randomized controlled trial evaluated the effects of NPWT on the production of cFN and the expression of TGF-β1 in diabetic foot wounds of patients. From January 2012 to January 2015, 40 patients with diabetic foot wounds were randomly and equally apportioned to receive either NPWT or advanced moist wound therapy (control) for 7 days. Granulation tissue was harvested before and after treatment. Immunohistochemistry and Western blot were performed to evaluate protein levels of cFN and TGF-β1, and real-time polymerase chain reaction (PCR) to measure corresponding mRNA expressions. NPWT facilitated the expression of cFN and TGF-β1 in diabetic foot wounds. Immunohistochemical analysis revealed higher levels of cFN and TGF-β1 in the NPWT group than in the control group. Western blot and real-time PCR analysis further showed that protein and mRNA levels of cFN or TGF-β1 were higher in the NPWT group than that in the control group ( P < .01, both). Our results showed that NPWT facilitated the production of cFN and the expression of TGF-β1 in granulation tissue in diabetic foot ulcers. Level I, randomized controlled study.

Thy-1 Expression Regulates the Ability of Rat Lung Fibroblasts to Activate Transforming Growth Factor-β in Response to Fibrogenic Stimuli

PubMed Central

Zhou, Yong; Hagood, James S.; Murphy-Ullrich, Joanne E.

2004-01-01

Distinct subpopulations of fibroblasts contribute to lung fibrosis, although the mechanisms underlying fibrogenesis in these subpopulations are not clear. Differential expression of the glycophosphatidylinositol-linked protein Thy-1 affects proliferation and myofibroblast differentiation. Lung fibroblast populations selected on the basis of Thy-1 expression by cell sorting were examined for responses to fibrogenic stimuli. Thy-1 (−) and Thy-1 (+) fibroblast populations were treated with platelet-derived growth factor-BB, interleukin-1β, interleukin-4, or bleomycin and assessed for activation of transforming growth factor (TGF)-β, Smad3 phosphorylation, and α-smooth muscle actin and fibronectin expression. Thy-1 (−) fibroblasts responded to these stimuli with increased TGF-β activity, Smad3 phosphorylation, and expression of α-smooth muscle actin and fibronectin, whereas Thy-1 (+) fibroblasts resisted stimulation. The unresponsiveness of Thy-1 (+) cells is not because of defective TGF-β signaling because both subsets respond to exogenous active TGF-β. Rather, Thy-1 (−) fibroblasts activate latent TGF-β in response to fibrogenic stimuli, whereas Thy-1 (+) cells fail to do so. Defective activation is common to multiple mechanisms of TGF-β activation, including thrombospondin 1, matrix metalloproteinase, or plasmin. Thy-1 (−) lung fibroblasts transfected with Thy-1 also become resistant to fibrogenic stimulation, indicating that Thy-1 is a critical biological response modifier that protects against fibrotic progression by controlling TGF-β activation. These studies provide a molecular basis for understanding the differential roles of fibroblast subpopulations in fibrotic lung disease through control of latent TGF-β activation. PMID:15277239

Expression of the serine/threonine kinase hSGK1 in chronic viral hepatitis.

PubMed

Fillon, Sophie; Klingel, Karin; Wärntges, Simone; Sauter, Martina; Gabrysch, Sabine; Pestel, Sabine; Tanneur, Valerie; Waldegger, Siegfried; Zipfel, Annette; Viebahn, Richard; Häussinger, Dieter; Bröer, Stefan; Kandolf, Reinhard; Lang, Florian

2002-01-01

The human serine/threonine kinase hSGK1 is expressed ubiquitously with highest transcript levels in pancreas and liver. This study has been performed to determine the hSGK1 distribution in normal liver and its putative role in fibrosing liver disease. HSGK1-localization was determined by in situ hybridization, regulation of hSGK1-transcription by Northern blotting, fibronectin synthesis and hSGK1 phosphorylation by Western blotting. In normal liver hSGK1 was mainly transcribed by Kupffer cells. In liver tissue from patients with chronic viral hepatitis, hSGK1 transcript levels were excessively high in numerous activated Kupffer cells and inflammatory cells localized within fibrous septum formations. HSGK1 transcripts were also detected in activated hepatic stellate cells. Accordingly, Western blotting revealed that tissue from fibrotic liver expresses excessive hSGK1 protein as compared to normal liver. TGF-beta1 (2 ng/ml) increases hSGK1 transcription in both human U937 macro-phages and HepG2 hepatoma cells. H(2)O(2) (0.3 mM) activated hSGK1 and increased fibronectin formation in HepG2 cells overexpressing hSGK1 but not in HepG2 cells expressing the inactive mutant hSGK1(K127R). In conclusion hSGK1 is upregulated by TGF-beta1 during hepatitis and may contribute to enhanced matrix formation during fibrosing liver disease. Copyright 2002 S. Karger AG, Basel

Use of the Uteroglobin Platform for the Expression of a Bivalent Antibody against Oncofetal Fibronectin in Escherichia coli

PubMed Central

Ventura, Elisa; Riondato, Mattia; Sambuceti, Gianmario; Salis, Annalisa; Damonte, Gianluca; Cordazzo, Cinzia; Besir, Hüseyin; Pistoia, Vito; Zardi, Luciano

2013-01-01

Escherichia coli is a robust, economic and rapid expression system for the production of recombinant therapeutic proteins. However, the expression in bacterial systems of complex molecules such as antibodies and fusion proteins is still affected by several drawbacks. We have previously described a procedure based on uteroglobin (UG) for the engineering of very soluble and stable polyvalent and polyspecific fusion proteins in mammalian cells (Ventura et al. 2009. J. Biol. Chem. 284∶26646–26654.) Here, we applied the UG platform to achieve the expression in E. coli of a bivalent human recombinant antibody (L19) toward the oncofetal fibronectin (B-FN), a pan-tumor target. Purified bacterial L19-UG was highly soluble, stable, and, in all molecules, the L19 moiety maintained its immunoreactivity. About 50–70% of the molecules were covalent homodimer, however after refolding with the redox couple reduced-glutathione/oxidized-glutathione (GSH/GSSG), 100% of molecules were covalent dimers. Mass spectrometry studies showed that the proteins produced by E. coli and mammalian cells have an identical molecular mass and that both proteins are not glycosylated. L19-UG from bacteria can be freeze-dried without any loss of protein and immunoreactivity. In vivo, in tumor-bearing mice, radio-iodinated L19-UG selectively accumulated in neoplastic tissues showing the same performance of L19-UG from mammalian cells. The UG-platform may represent a general procedure for production of various biological therapeutics in E. coli. PMID:24367567

Planar cell polarity proteins differentially regulate extracellular matrix organization and assembly during zebrafish gastrulation.

PubMed

Dohn, Michael R; Mundell, Nathan A; Sawyer, Leah M; Dunlap, Julie A; Jessen, Jason R

2013-11-01

Zebrafish gastrulation cell movements occur in the context of dynamic changes in extracellular matrix (ECM) organization and require the concerted action of planar cell polarity (PCP) proteins that regulate cell elongation and mediolateral alignment. Data obtained using Xenopus laevis gastrulae have shown that integrin-fibronectin interactions underlie the formation of polarized cell protrusions necessary for PCP and have implicated PCP proteins themselves as regulators of ECM. By contrast, the relationship between establishment of PCP and ECM assembly/remodeling during zebrafish gastrulation is unclear. We previously showed that zebrafish embryos carrying a null mutation in the four-pass transmembrane PCP protein vang-like 2 (vangl2) exhibit increased matrix metalloproteinase activity and decreased immunolabeling of fibronectin. These data implicated for the first time a core PCP protein in the regulation of pericellular proteolysis of ECM substrates and raised the question of whether other zebrafish PCP proteins also impact ECM organization. In Drosophila melanogaster, the cytoplasmic PCP protein Prickle binds Van Gogh and regulates its function. Here we report that similar to vangl2, loss of zebrafish prickle1a decreases fibronectin protein levels in gastrula embryos. We further show that Prickle1a physically binds Vangl2 and regulates both the subcellular distribution and total protein level of Vangl2. These data suggest that the ability of Prickle1a to impact fibronectin organization is at least partly due to effects on Vangl2. In contrast to loss of either Vangl2 or Prickle1a function, we find that glypican4 (a Wnt co-receptor) and frizzled7 mutant gastrula embryos with disrupted non-canonical Wnt signaling exhibit the opposite phenotype, namely increased fibronectin assembly. Our data show that glypican4 mutants do not have decreased proteolysis of ECM substrates, but instead have increased cell surface cadherin protein expression and increased intercellular adhesion. These data indicate that Wnt/Glypican4/Frizzled signaling regulates ECM assembly through effects on cadherin-mediated cell cohesion. Together, our results demonstrate that zebrafish Vangl2/Prickle1a and non-canonical Wnt/Frizzled signaling have opposing effects on ECM organization underlying PCP and gastrulation cell movements. © 2013 Elsevier Inc. All rights reserved.

Avian pathogenic Escherichia coli bind fibronectin and laminin.

PubMed

Ramírez, Rosa María; Almanza, Yolanda; González, Rafael; García, Santos; Heredia, Norma

2009-04-01

Avian colisepticemia frequently occurs after respiratory tract damage, the primary site for infection allows bacteria to encounter an exposed basement membrane, where laminin and fibronectin are important components. We investigated the ability of an isolate of avian pathogenic Escherichia coli to bind fibronectin and laminin. Using Far-western dot blot analysis, we demonstrated the ability of this microorganism to bind basement membrane proteins fibronectin and laminin. Results from an ELISA-based approach indicate that the binding to these membrane proteins was bacterial-dose dependent. Furthermore, two specific E. coli polypeptides, of 32 kDa and 130 kDa, reacted with laminin and fibronectin, respectively. Further evaluation of these potential bacterial adhesins may provide insights into the pathogenesis of colibacillosis.

Porins of Pseudomonas fluorescens MFO as fibronectin-binding proteins.

PubMed

Rebière-Huët, J; Guérillon, J; Pimenta, A L; Di Martino, P; Orange, N; Hulen, C

2002-09-24

Bacterial adherence is a complex phenomenon involving specific interactions between receptors, including matricial fibronectin, and bacterial ligands. We show here that fibronectin and outer membrane proteins of Pseudomonas fluorescens were able to inhibit adherence of P. fluorescens to fibronectin-coated wells. We identified at least six fibronectin-binding proteins with molecular masses of 70, 55, 44, 37, 32 and 28 kDa. The presence of native (32 kDa) and heat-modified forms (37 kDa) of OprF was revealed by immuno-analysis and the 44-kDa band was composed of three proteins, their N-terminal sequences showing homologies with Pseudomonas aeruginosa porins (OprD, OprE1 and OprE3).

Quantification of fibronectin as a method to assess ex vivo extracellular matrix remodeling

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

Bager, C.L., E-mail: cba@nordicbioscience.com; Technical University of Denmark; Gudmann, N.

Altered architecture, composition and quality of the extracellular matrix (ECM) are pathological hallmarks of several inflammatory and fibro-proliferative pathological processes such as osteoarthritis (OA), rheumatoid arthritis (RA), fibrosis and cancer. One of the most important components of the ECM is fibronectin. Fibronectin serves as an adhesion molecule anchoring cells to the underlying basement membrane through direct interaction with integrin receptors. Fibronectin hereby modulates the properties of the ECM and affects cellular processes. Quantification of fibronectin remodeling could therefore be used to assess the changes in the ECM that occur during progression of fibro-proliferative pathologies. Ex vivo models are becoming state-of-the-art toolsmore » to study ECM remodeling as the cellular composition and the organization of the ECM are preserved. Ex vivo models may therefore be a valuable tool to study the ECM remodeling that occurs during progression of fibro-proliferative pathologies. The aim of this study was to quantify fibronectin remodeling in ex vivo models of cartilage and cancer. A competitive The enzyme-linked immunosorbent assay (ELISA) against the C-terminus of fibronectin was developed (FBN-C). The assay was evaluated in relation to specificity, technical performance and as a marker for quantification of fibronectin in cartilage and cancer ex vivo models. The ELISA was specific and technically stable. Cleavage of tumor tissue with MMP-2 released significantly higher levels of FBN-C compared to tissue with buffer only and western blot analysis revealed that FBN-C recognizes both full length and degraded fibronectin. When ex vivo cartilage cultures were stimulated with the anabolic factor TGFβ and catabolic factors TNF-α and OSM, significantly higher levels of FBN-C were found in the conditioned media. Lastly, FBN-C was released from a cancer ex vivo model. In conclusion, we were able to quantify fibronectin remodeling in ex vivo models of cartilage and cancer. Quantification of fibronectin remodeling could be a valuable tool to understand ECM remodeling in ex vivo models of fibro-proliferative pathologies.« less

Corrosion resistance and biocompatibility of magnesium alloy modified by alkali heating treatment followed by the immobilization of poly (ethylene glycol), fibronectin and heparin.

PubMed

Pan, Changjiang; Hu, Youdong; Hou, Yu; Liu, Tao; Lin, Yuebin; Ye, Wei; Hou, Yanhua; Gong, Tao

2017-01-01

In recent years, magnesium alloys are attracting more and more attention as a kind of biodegradable metallic biomaterials, however, their uncontrollable biodegradation speed in vivo and the limited surface biocompatibility hinder their clinical applications. In the present study, with the aim of improving the corrosion resistance and biocompatibility, the magnesium alloy (AZ31B) surface was modified by alkali heating treatment followed by the self-assembly of 3-aminopropyltrimethoxysilane (APTMS). Subsequently, poly (ethylene glycol) (PEG) and fibronectin or fibronectin/heparin complex were sequentially immobilized on the modified surface. The results of attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS) confirmed that the above molecules were successfully immobilized on the magnesium alloy surface. An excellent hydrophilic surface was obtained after the alkali heating treatment while the hydrophilicity decreased to some degree after the self-assembly of APTMS, the surface hydrophilicity was gradually improved again after the immobilization of PEG, fibronectin or fibronectin/heparin complex. The corrosion resistance of the control magnesium alloy was significantly improved by the alkali heating treatment. The self-assembly of APTMS and the following immobilization of PEG further enhanced the corrosion resistance of the substrates, however, the grafting of fibronectin or fibronectin/heparin complex slightly lowered the corrosion resistance. As compared to the pristine magnesium alloy, the samples modified by the immobilization of PEG and fibronectin/heparin complex presented better blood compatibility according to the results of hemolysis assay and platelet adhesion as well as the activated partial thromboplastin time (APTT). In addition, the modified substrates had better cytocompatibility to endothelial cells due to the improved anticorrosion and the introduction of fibronectin. The substrates modified by fibronectin or fibronectin/heparin complex can significantly promote endothelial cell adhesion and proliferation. Taking all these results into consideration, the method of the present study can be used for the surface modification of the magnesium alloy to simultaneously impart it better corrosion resistance, favorable blood compatibility and good cytocompatibility to endothelial cells. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed Central

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

2018-01-01

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

The effects of pleiotrophin in proliferative vitreoretinopathy.

PubMed

Ding, Xue; Bai, Yujing; Zhu, Xuemei; Li, Tianqi; Jin, Enzhong; Huang, Lvzhen; Yu, Wenzhen; Zhao, Mingwei

2017-05-01

The purpose of our study was to investigate the effects of pleiotrophin (PTN) in proliferative vitreoretinopathy (PVR) both in vitro and in vivo. Immunofluorescence was used to observe the PTN expression in periretinal membrane samples from patients with PVR and controls. ARPE-19 cells were exposed to TGF-β1. The epithelial-to-mesenchymal transition (EMT) of the ARPE-19 cells was confirmed by observed morphological changes and the increased expression of α-SMA and fibronectin at both the mRNA and protein levels. We used specific small interfering (si)RNA to knock down the expression of PTN. The subsequent effects of PTN inhibition were assessed with regard to the EMT, migration, proliferation, cytoskeletal arrangement, TGF-β signaling, PTN signaling, integral tight junction protein expression (e.g., claudin-1 and occludin), and p38 MAPK and p-p38 MAPK levels. Additionally, a PVR rat model was established by the intravitreal injection of ARPE-19 cells transfected with PTN-siRNA and was evaluated accordingly. PTN was highly expressed in PVR membranes compared to controls. PTN knockdown attenuated the TGF-β1-induced migration, proliferation, cytoskeletal rearrangement, and expression of EMT markers such as α-SMA and fibronectin in the ARPE-19 cells, and these effects may have been mediated through p38 MAPK signaling pathway activation. PTN silencing inhibited the up-regulation of claudin-1 and occludin stimulated by TGF-β1, and PTN knockdown inhibited the proliferative aspects of severe PVR in vivo. PTN is involved in the process of EMT induced by TGF-β1 in human ARPE-19 cells in vitro, and PTN knockdown attenuated the progression of experimental PVR in vivo. These findings provide new insights into the pathogenesis of PVR.

A 3′-Untranslated Region (3′UTR) Induces Organ Adhesion by Regulating miR-199a* Functions

PubMed Central

Lee, Daniel Y.; Shatseva, Tatiana; Jeyapalan, Zina; Du, William W.; Deng, Zhaoqun; Yang, Burton B.

2009-01-01

Mature microRNAs (miRNAs) are single-stranded RNAs of 18–24 nucleotides that repress post-transcriptional gene expression. However, it is unknown whether the functions of mature miRNAs can be regulated. Here we report that expression of versican 3′UTR induces organ adhesion in transgenic mice by modulating miR-199a* activities. The study was initiated by the hypothesis that the non-coding 3′UTR plays a role in the regulation of miRNA function. Transgenic mice expressing a construct harboring the 3′UTR of versican exhibits the adhesion of organs. Computational analysis indicated that a large number of microRNAs could bind to this fragment potentially including miR-199a*. Expression of versican and fibronectin, two targets of miR-199a*, are up-regulated in transgenic mice, suggesting that the 3′UTR binds and modulates miR-199a* activities, freeing mRNAs of versican and fibronectin from being repressed by miR-199a*. Confirmation of the binding was performed by PCR using mature miR-199a* as a primer and the targeting was performed by luciferase assays. Enhanced adhesion by expression of the 3′UTR was confirmed by in vitro assays. Our results demonstrated that upon arrival in cytoplasm, miRNA activities can be modulated locally by the 3′UTR. Our assay may be developed as sophisticated approaches for studying the mutual regulation of miRNAs and mRNAs in vitro and in vivo. We anticipate that expression of the 3′UTR may be an approach in the development of gene therapy. PMID:19223980

EDA-Fibronectin Originating from Osteoblasts Inhibits the Immune Response against Cancer

PubMed Central

Rossnagl, Stephanie; Altrock, Eva; Sens, Carla; Kraft, Sabrina; Rau, Katrin; Giese, Thomas; Samstag, Yvonne; Nakchbandi, Inaam A.

2016-01-01

Osteoblasts lining the inner surface of bone support hematopoietic stem cell differentiation by virtue of proximity to the bone marrow. The osteoblasts also modify their own differentiation by producing various isoforms of fibronectin (FN). Despite evidence for immune regulation by osteoblasts, there is limited knowledge of how osteoblasts modulate cells of the immune system. Here, we show that extra domain A (EDA)-FN produced by osteoblasts increases arginase production in myeloid-derived cells, and we identify α5β1 as the mediating receptor. In different mouse models of cancer, osteoblasts or EDA-FN was found to up-regulate arginase-1 expression in myeloid-derived cells, resulting in increased cancer growth. This harmful effect can be reduced by interfering with the integrin α5β1 receptor or inhibiting arginase. Conversely, in tissue injury, the expression of arginase-1 is normally beneficial as it dampens the immune response to allow wound healing. We show that EDA-FN protects against excessive fibrotic tissue formation in a liver fibrosis model. Our results establish an immune regulatory function for EDA-FN originating from the osteoblasts and identify new avenues for enhancing the immune reaction against cancer. PMID:27653627

Transforming Growth Factor Beta 1 Drives a Switch in Connexin Mediated Cell-to-Cell Communication in Tubular Cells of the Diabetic Kidney.

PubMed

Hills, Claire; Price, Gareth William; Wall, Mark John; Kaufmann, Timothy John; Chi-Wai Tang, Sidney; Yiu, Wai Han; Squires, Paul Edward

2018-01-01

Changes in cell-to-cell communication have been linked to several secondary complications of diabetes, but the mechanism by which connexins affect disease progression in the kidney is poorly understood. This study examines a role for glucose-evoked changes in the beta1 isoform of transforming growth factor (TGFβ1), on connexin expression, gap-junction mediated intercellular communication (GJIC) and hemi-channel ATP release from tubular epithelial cells of the proximal renal nephron. Biopsy material from patients with and without diabetic nephropathy was stained for connexin-26 (CX26) and connexin-43 (CX43). Changes in expression were corroborated by immunoblot analysis in human primary proximal tubule epithelial cells (hPTECs) and model epithelial cells from human renal proximal tubules (HK2) cultured in either low glucose (5mmol/L) ± TGFβ1 (2-10ng/ml) or high glucose (25mmol/L) for 48h or 7days. Secretion of the cytokine was determined by ELISA. Paired whole cell patch clamp recordings were used to measure junctional conductance in control versus TGFβ1 treated (10ng/ml) HK2 cells, with carboxyfluorescein uptake and ATP-biosensing assessing hemi-channel function. A downstream role for ATP in mediating the effects of TGF-β1 on connexin mediated cell communication was assessed by incubating cells with ATPγS (1-100µM) or TGF-β1 +/- apyrase (5 Units/ml). Implications of ATP release were measured through immunoblot analysis of interleukin 6 (IL-6) and fibronectin expression. Biopsy material from patients with diabetic nephropathy exhibited increased tubular expression of CX26 and CX43 (P<0.01, n=10), data corroborated in HK2 and hPTEC cells cultured in TGFβ1 (10ng/ml) for 7days (P<0.001, n=3). High glucose significantly increased TGFβ1 secretion from tubular epithelial cells (P<0.001, n=3). The cytokine (10ng/ml) reduced junctional conductance between HK2 cells from 4.5±1.3nS in control to 1.15±0.9nS following 48h TGFβ1 and to 0.42±0.2nS after 7days TGFβ1 incubation (P<0.05, n=5). Acute (48h) and chronic (7day) challenge with TGFβ1 produced a carbenoxolone (200µM)-sensitive increase in carboxyfluorescein loading, matched by an increase in ATP release from 0.29±0.06μM in control to 1.99±0.47μM after 48hr incubation with TGFβ1 (10ng/ml; P<0.05, n=3). TGF-β1 (2-10ng/ml) and ATPγs (1-100µM) increased expression of IL-6 (P<0.001 n=3) and fibronectin (P<0.01 n=3). The effect of TGF-β1 on IL-6 and fibronectin expression was partially blunted when preincubated with apyrase (n=3). These data suggest that chronic exposure to glucose-evoked TGFβ1 induce an increase in CX26 and CX43 expression, consistent with changes observed in tubular epithelia from patients with diabetic nephropathy. Despite increased connexin expression, direct GJIC communication decreases, whilst hemichannel expression/function and paracrine release of ATP increases, changes that trigger increased levels of expression of interleukin 6 and fibronectin. Linked to inflammation and fibrosis, local increases in purinergic signals may exacerbate disease progression and highlight connexin mediated cell communication as a future therapeutic target for diabetic nephropathy. © 2018 The Author(s). Published by S. Karger AG, Basel.

Fibronectin type III domain containing 5 attenuates NLRP3 inflammasome activation and phenotypic transformation of adventitial fibroblasts in spontaneously hypertensive rats.

PubMed

Ling, Li; Chen, Dan; Tong, Ying; Zang, Ying-Hao; Ren, Xing-Sheng; Zhou, Hong; Qi, Xiao-Hong; Chen, Qi; Li, Yue-Hua; Kang, Yu-Ming; Zhu, Guo-Qing

2018-05-01

Phenotypic transformation of adventitial fibroblasts is important in the pathogenesis of hypertension. This study was designed to determine whether fibronectin type III domain containing 5 (FNDC5) alleviates the phenotypic transformation of adventitial fibroblasts in hypertension and the underlying mechanisms. Experiments were carried out in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) and primary aortic adventitial fibroblasts. FNDC5 was downregulated and NLRP3 inflammasome was activated in aortic adventitia of SHR. FNDC5 overexpression attenuated adventitial fibroblasts phenotypic transformation, excessive synthesis and secretion of matrix components, NLRP3 inflammasome activation and inflammation in adventitial fibroblasts from SHR. Moreover, FNDC5 overexpression reduced NADPH oxidase 2 (NOX2) expression and reactive oxygen species (ROS) production in adventitial fibroblasts from SHR. Similarly, exogenous FNDC5 inhibited adventitial fibroblasts phenotypic transformation, expression of matrix components, NLRP3 inflammasome activation and NOX2 expression in adventitial fibroblasts from SHR. FNDC5 overexpression in rats attenuated phenotypic transformation, inflammation and reactive oxygen species (ROS) production in the aortic adventitia of SHR. Furthermore, FNDC5 overexpression reduced blood pressure and alleviated vascular remodeling in SHR. FNDC5 reduces NOX2-derived ROS production, NLRP3 inflammasome activation and phenotypic transformation in adventitial fibroblasts of SHR. FNDC5 plays a beneficial role in attenuating vascular inflammation, vascular remodeling and hypertension in SHR.

Effect of the Direct Renin Inhibitor Aliskiren on Urinary Albumin Excretion in Spontaneous Type 2 Diabetic KK-A y Mouse

PubMed Central

Furukawa, Masako; Horikoshi, Satoshi; Funabiki, Kazuhiko; Tomino, Yasuhiko

2013-01-01

Objective. Although angiotensin II-mediated inflammation and extracellular matrix accumulation are considered to be associated with the progression of diabetic nephropathy, these processes have not yet been sufficiently clarified. The objective of this study was to determine whether the correction of the abnormal renal expression of MMPs and its inhibitors (MMPs/TIMPs) and cytokines following the administration of aliskiren to KK-A y mice results in a renoprotective effect. Methods. KK-A y mice were divided into two groups, that is, untreated (saline) and treated (aliskiren) groups. Systolic BP, HbA1c levels, and the albumin-creatinine ratio (ACR) were measured. The renal expression of MMPs/TIMPs, fibronectin, type IV collagen, MCP-1, and (pro)renin receptor ((P)RR) was examined using real-time PCR and/or immunohistochemical staining. Renal MAPK and NF-κB activity were also examined by Western blot analyses and ELISA, respectively. Results. Significant decreases in systolic BP and ACR levels were observed in treated KK-A y mice compared with the findings in untreated KK-A y mice. Furthermore, increases in MMPs/TIMPs, fibronectin, type IV collagen, MCP-1, and (P)RR expression, in addition to MAPK and NF-κB activity, were significantly attenuated by aliskiren administration. Conclusions. It appears that aliskiren improves albuminuria and renal fibrosis by regulating inflammation and the alteration of collagen synthesis and degradation. PMID:23819050

Emodin attenuates high glucose-induced TGF-β1 and fibronectin expression in mesangial cells through inhibition of NF-κB pathway

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

Yang, Jie; Zeng, Zhi; Wu, Teng

The activation of nuclear factor-κB (NF-κB) and the subsequent overexpression of its downstream targets transforming growth factor-β1 (TGF-β1) and fibronectin (FN) are among the hallmarks for the progressive diabetic nephropathy. Our previous studies demonstrated that emodin ameliorated renal injury and inhibited extracellular matrix accumulation in kidney and mesangial cells under diabetic condition. However, the molecular mechanism has not been fully elucidated. Here, we showed that emodin significantly attenuated high glucose-induced NF-κB nuclear translocation in mesangial cells. Interestingly, emodin also inhibited the DNA-binding activity and transcriptional activity of NF-κB. Furthermore, NF-κB-mediated TGF-β1 and FN expression was significantly decreased by emodin. Thesemore » results demonstrated that emodin suppressed TGF-β1 and FN overexpression through inhibition of NF-κB activation, suggesting that emodin-mediated inhibition of the NF-κB pathway could protect against diabetic nephropathy. - Highlights: • Emodin decreased high glucose-induced p65 phosphorylation in MCs. • Emodin decreased high glucose-induced IκB-α degradation in MCs. • Emodin decreased high glucose-induced p65 translocation in MCs. • Emodin blocked high glucose-induced NF-κB activity. • Emodin blocked high glucose-induced the expression of TGF-β1 and FN.« less

Biosynthetic processing of the oligosaccharide chains of cellular fibronectin.

PubMed

Olden, K; Hunter, V A; Yamada, K M

1980-10-15

We have examined the maturation or processing of the oligosaccharides of cellular fibronectin in cultured chick embryo fibroblasts. Fibronectin was pulse-labeled with [2-3H]mannose of [35S]methionine, and the turnover rates of carbohydrate and polypeptide portions of immunoprecipitated fibronectin were compared. The oligosaccharides on fibronectin were analyzed by gel electrophoresis for alterations in sensitivity to the enzyme endo-beta-N-acetylgluosaminidase H, which specifically cleaves the 'high-mannose' class of asparagine-linked oligosaccharide. Incorporated mannose was removed only at early time points, suggesting that the structure of fibronectin oligosaccharides was altered due to processing. This possibility was confirmed by the analysis of glycopeptides generated by exhaustive pronase digestion. Two major glycopeptide structures were detected; their properties correspond to a 'high-mannose' oligosaccharide precursor and a 'complex' carbohydrate product. The precursor-product relationship of these two forms of oligosaccharide chains was demonstrated by pulse-chase labeling experiments. The precursor glycopeptide had an apparent size (Mr 2100) comparable to (Man)9GlcNAc (Mr 2080), and was sensitive to endo-beta-N-acetylglucosaminidase H; nearly all of the labeled mannose incorporated in a 10 min pulse was released from fibronectin glycopeptides by this enzyme. During a 90 min chase period, the glycopeptides became larger and increasingly resistant to endo-beta-N-acetylglucosaminidase H cleavage. The final 'complex' or processed oligosaccharide structure contained approximately two-thirds less [3H]mannose, was insensitive to endo-beta-N-acetylglucosaminidase H and had an apparent Mr of 2300 as estimated by gel filtration. We conclude that the carbohydrate portion of fibronectin is synthesized as a 'high-mannose' intermediate and is subsequently processed to give the characteristic 'complex' oligosaccharide chains of fibronectin.

Surface Oxide Net Charge of a Titanium Alloy ; Modulation of Fibronectin-Activated Attachment and Spreading of Osteogenic Cells

PubMed Central

Rapuano, Bruce E.; MacDonald, Daniel E.

2010-01-01

In the current study, we have altered the surface oxide properties of a Ti6Al4V alloy using heat treatment or radiofrequency glow discharge (RFGD) in order to evaluate the relationship between the physico-chemical and biological properties of the alloy's surface oxide. The effects of surface pretreatments on the attachment of cells from two osteogenic cell lines (MG63 and MC3T3) and a mesenchymal stem cell line (C3H10T1/2) to fibronectin adsorbed to the alloy were measured. Both heat and RFGD pretreatments produced a several-fold increase in the number of cells that attached to fibronectin adsorbed to the alloy (0.001 and 10 nM FN) for each cell line tested. An antibody (HFN7.1) directed against the central integrin binding domain of fibronectin produced a 65-70% inhibition of cell attachment to fibronectin-coated disks, incdicating that cell attachment to the metal discs was dependent on fibronectin binding to cell integrin receptors. Both treatments also accelerated the cell spreading response manifested by extensive flattening and an increase in mean cellular area. The treatment-induced increases in the cell attachment activity of adsorbed fibronectin were correlated with previously demonstrated increases in Ti6Al4V oxide negative net surface charge at physiological pH produced by both heat and RFGD pretreatments. Since neither treatment increased the adsorption mass of fibronectin, these findings suggest that negatively charged surface oxide functional groups in Ti6Al4V can modulate fibronectin's integrin receptor activity by altering the adsorbed protein's conformation. Our results further suggest that negatively charged functional groups in the surface oxide can play a prominent role in the osseointegration of metallic implant materials. PMID:20884181

Solution and surface effects on plasma fibronectin structure

PubMed Central

1983-01-01

As assessed by electron microscopy, the reported shape of the plasma fibronectin molecule ranges from that of a compact particle to an elongated, rod-like structure. In this study, we evaluated the effects of solution and surface conditions on fibronectin shape. Freeze-dried, unstained human plasma fibronectin molecules deposited at pH 7.0-7.4 onto carbon films and examined by scanning transmission electron microscopy appeared relatively compact and pleiomorphic, with approximate average dimensions of 24 nm X 16 nm. Negatively stained molecules also had a similar shape but revealed greater detail in that we observed irregular, yarn-like structures. Glutaraldehyde-induced intramolecular cross-linking did not alter the appearance of plasma fibronectin. Molecules deposited at pH 2.8, pH 9.3, or after succinylation were less compact than those deposited at neutral pH. In contrast, fibronectin molecules sprayed onto mica surfaces at pH 7, rotary shadowed, and examined by transmission electron microscopy were elongated and nodular with a contour length of 120-130 nm. Sedimentation velocity experiments and electron microscopic observations indicate that fibronectin unfolds when it is succinylated, when the ionic strength is raised at pH 7, or when the pH is adjusted to 9.3 or 2.8. Greater unfolding is observed at pH 2.8 at low ionic strength (less than 0.01) compared with material at that pH in 0.15 M NaCl solution. We conclude that (a) the shape assumed by the fibronectin molecule can be strongly affected by solution conditions and by deposition onto certain surfaces; and that (b) the images of fibronectin seen by scanning transmission electron microscopy at neutral pH on carbon film are representative of molecules in physiologic solution. PMID:6417145

Epigenetic Regulation of Galectin-3 Expression by β1 Integrins Promotes Cell Adhesion and Migration*

PubMed Central

Margadant, Coert; van den Bout, Iman; van Boxtel, Antonius L.; Thijssen, Victor L.; Sonnenberg, Arnoud

2012-01-01

Introduction of the integrin β1- but not the β3-subunit in GE11 cells induces an epithelial-to-mesenchymal-transition (EMT)-like phenomenon that is characterized by the loss of cell-cell contacts, cell scattering, increased cell migration and RhoA activity, and fibronectin fibrillogenesis. Because galactose-binding lectins (galectins) have been implicated in these phenomena, we investigated whether galectins are involved in the β1-induced phenotype. We examined 9 galectins and, intriguingly, found that the expression of galectin-3 (Gal-3) is specifically induced by β1 but not by β3. Using β1-β3 chimeric integrins, we show that the induction of Gal-3 expression requires the hypervariable region in the extracellular domain of β1, but not its cytoplasmic tail. Furthermore, Gal-3 expression does not depend on RhoA signaling, serum factors, or any of the major signal transduction pathways involving protein kinase C (PKC), p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase-1/-2 (ERK-1/2), phosphatidylinositol-3-OH kinase (PI3-K), or Src kinases. Instead, Gal-3 expression is controlled in an epigenetic manner. Whereas DNA methylation of the Lgals3 promoter maintains Gal-3 silencing in GE11 cells, expression of β1 causes its demethylation, leading to transcriptional activation of the Lgals3 gene. In turn, Gal-3 expression enhances β1 integrin-mediated cell adhesion to fibronectin (FN) and laminin (LN), as well as cell migration. Gal-3 also promotes β1-mediated cell adhesion to LN and Collagen-1 (Col)-1 in cells that endogenously express Gal-3 and β1 integrins. In conclusion, we identify a functional feedback-loop between β1 integrins and Gal-3 that involves the epigenetic induction of Gal-3 expression during integrin-induced EMT and cell scattering. PMID:23118221

Role of ID Proteins in BMP4 Inhibition of Profibrotic Effects of TGF-β2 in Human TM Cells.

PubMed

Mody, Avani A; Wordinger, Robert J; Clark, Abbot F

2017-02-01

Increased expression of TGF-β2 in primary open-angle glaucoma (POAG) aqueous humor (AH) and trabecular meshwork (TM) causes deposition of extracellular matrix (ECM) in the TM and elevated IOP. Bone morphogenetic proteins (BMPs) regulate TGF-β2-induced ECM production. The underlying mechanism for BMP4 inhibition of TGF-β2-induced fibrosis remains undetermined. Bone morphogenic protein 4 induces inhibitor of DNA binding proteins (ID1, ID3), which suppress transcription factor activities to regulate gene expression. Our study will determine whether ID1and ID3 proteins are downstream targets of BMP4, which attenuates TGF-β2 induction of ECM proteins in TM cells. Primary human TM cells were treated with BMP4, and ID1 and ID3 mRNA, and protein expression was determined by quantitative PCR (Q-PCR) and Western immunoblotting. Intracellular ID1 and ID3 protein localization was studied by immunocytochemistry. Transformed human TM cells (GTM3 cells) were transfected with ID1 or ID3 expression vectors to determine their potential inhibitory effects on TGF-β2-induced fibronectin and plasminogen activator inhibitor-I (PAI-1) protein expression. Basal expression of ID1-3 was detected in primary human TM cells. Bone morphogenic protein 4 significantly induced early expression of ID1 and ID3 mRNA (P < 0.05) and protein in primary TM cells, and a BMP receptor inhibitor blocked this induction. Overexpression of ID1 and ID3 significantly inhibited TGF-β2-induced expression of fibronectin and PAI-1 in TM cells (P < 0.01). Bone morphogenic protein 4 induced ID1 and ID3 expression suppresses TGF-β2 profibrotic activity in human TM cells. In the future, targeting specific regulators may control the TGF-β2 profibrotic effects on the TM, leading to disease modifying IOP lowering therapies.

The glycation of fibronectin by glycolaldehyde and methylglyoxal as a model for aging in Bruch's membrane.

PubMed

Thao, Mai T; Gaillard, Elizabeth R

2016-07-01

The purpose of the study is to identify the sites of modification when fibronectin reacts with glycolaldehyde or methylglyoxal as a model system for aging of Bruch's membrane. A synthetic peptide consisting of the α5β1 integrin binding region of fibronectin was incubated with glycolaldehyde for 12 h or with methylglyoxal for 1 h at 37 °C. After tryptic digestion, the samples were analyzed with liquid chromatography-mass spectrometry (LC/MS). Tandem MS was used to determine the sites of modification. The adducts, aldoamine and N (ε)-carboxymethyl-lysine, attached preferably at lysine residues when the fibronectin peptide reacted with glycolaldehyde. When the fibronectin peptide reacted with methylglyoxal, modifications occurred at lysine and arginine residues. At lysine residues, N (ε)-carboxyethyl-lysine adducts were present. At arginine residues, hydroimidazolone and tetrapyrimidine adducts were present. Several advanced glycation endproducts were generated when fibronectin was glycated via glycolaldehyde and methylglyoxal. These results can help explain the structural changes Bruch's membrane undergoes during aging.

The Fibronectin-Binding Protein Fnm Contributes to Adherence to Extracellular Matrix Components and Virulence of Enterococcus faecium

PubMed Central

Somarajan, Sudha R.; La Rosa, Sabina Leanti; Singh, Kavindra V.; Roh, Jung H.; Höök, Magnus

2015-01-01

The interaction between bacteria and fibronectin is believed to play an important role in the pathogenicity of clinically important Gram-positive cocci. In the present study, we identified a gene encoding a predicted fibronectin-binding protein of Enterococcus faecium (fnm), a homologue of Streptococcus pneumoniae pavA, in the genomes of E. faecium strain TX82 and all other sequenced E. faecium isolates. Full-length recombinant Fnm from strain TX82 bound to immobilized fibronectin in a concentration-dependent manner and also appeared to bind collagen type V and laminin, but not other proteins, such as transferrin, heparin, bovine serum albumin, mucin, or collagen IV. We demonstrated that the N-terminal fragment of Fnm is required for full fibronectin binding, since truncation of this region caused a 2.4-fold decrease (P < 0.05) in the adhesion of E. faecium TX82 to fibronectin. Deletion of fnm resulted in a significant reduction (P < 0.001) in the ability of the mutant, TX6128, to bind fibronectin relative to that of the wild-type strain; in situ reconstitution of fnm in the deletion mutant strain restored adherence. In addition, the Δfnm mutant was highly attenuated relative to TX82 (P ≤ 0.0001) in a mixed-inoculum rat endocarditis model. Taken together, these results demonstrate that Fnm affects the adherence of E. faecium to fibronectin and is important in the pathogenesis of experimental endocarditis. PMID:26371130

Osteograft, plastic material for regenerative medicine

NASA Astrophysics Data System (ADS)

Zaidman, A. M.; Korel, A. V.; Shevchenko, A. I.; Shchelkunova, E. I.; Sherman, K. M.; Predein, Yu. A.; Kosareva, O. S.

2016-08-01

Creating tissue-engineering constructs based on the mechanism of cartilage-bone evolution is promising for traumatology and orthopedics. Such a graft was obtained from a chondrograft by transdifferentiation. The hondrograft placed in osteogenic medium is undergoing osteogenic differentiation for 14-30 days. Tissue specificity of the osteograft was studied by morphology, immunohistochemistry, electron microscopy, and the expression of the corresponding genes was estimated. The expression of osteonectin, fibronectin, collagen of type I, izolektin and CD 44 is determined. Alkaline phosphatase and matrix vesicles are determined in osteoblasts. Calcificates are observed in the matrix. Chondrogenic proteins expression is absent. These findings evidence the tissue specificity of the developed osteograft.

Amyloid β-Protein as a Substrate Interacts with Extracellular Matrix to Promote Neurite Outgrowth

NASA Astrophysics Data System (ADS)

Koo, Edward H.; Park, Lisa; Selkoe, Dennis J.

1993-05-01

Progressive deposition of amyloid β-protein (Aβ) in brain parenchyma and blood vessels is a characteristic feature of Alzheimer disease. Recent evidence suggests that addition of solubilized synthetic Aβ to medium may produce toxic or trophic effects on cultured hippocampal neurons. Because soluble Aβ may not accumulate in significant quantities in brain, we asked whether immobilized Aβ peptide as a substrate alters neurite outgrowth from cultured rat peripheral sensory neurons. This paradigm may closely mimic the conditions in Alzheimer disease brain tissue, in which neurites contact insoluble, extracellular aggregates of β-amyloid. We detected no detrimental effects of Aβ substrate on neurite outgrowth. Rather, Aβ in combination with low doses of laminin or fibronectin enhanced neurite out-growth from these neuronal explants. Our results suggest that insoluble Aβ in the cerebral neuropil may serve as a neurite-promoting matrix, perhaps explaining the apparent regenerative response of neurites observed around amyloid plaques in Alzheimer disease. Moreover, in concert with the recent discovery of Aβ production by cultured neurons, our data suggest that Aβ plays a normal physiological role in brain by complexing with the extracellular matrix.

Pregestational type 2 diabetes mellitus induces cardiac hypertrophy in the murine embryo through cardiac remodeling and fibrosis.

PubMed

Lin, Xue; Yang, Penghua; Reece, E Albert; Yang, Peixin

2017-08-01

Cardiac hypertrophy is highly prevalent in patients with type 2 diabetes mellitus. Experimental evidence has implied that pregnant women with type 2 diabetes mellitus and their children are at an increased risk of cardiovascular diseases. Our previous mouse model study revealed that maternal type 2 diabetes mellitus induces structural heart defects in their offspring. This study aims to determine whether maternal type 2 diabetes mellitus induces embryonic heart hypertrophy in a murine model of diabetic embryopathy. The type 2 diabetes mellitus embryopathy model was established by feeding 4-week-old female C57BL/6J mice with a high-fat diet for 15 weeks. Cardiac hypertrophy in embryos at embryonic day 17.5 was characterized by measuring heart size and thickness of the right and left ventricle walls and the interventricular septum, as well as the expression of β-myosin heavy chain, atrial natriuretic peptide, insulin-like growth factor-1, desmin, and adrenomedullin. Cardiac remodeling was determined by collagen synthesis and fibronectin synthesis. Fibrosis was evaluated by Masson staining and determining the expression of connective tissue growth factor, osteopontin, and galectin-3 genes. Cell apoptosis also was measured in the developing heart. The thicknesses of the left ventricle walls and the interventricular septum of embryonic hearts exposed to maternal diabetes were significantly thicker than those in the nondiabetic group. Maternal diabetes significantly increased β-myosin heavy chain, atrial natriuretic peptide, insulin-like growth factor-1, and desmin expression, but decreased expression of adrenomedullin. Moreover, collagen synthesis was significantly elevated, whereas fibronectin synthesis was suppressed, in embryonic hearts from diabetic dams, suggesting that cardiac remodeling is a contributing factor to cardiac hypertrophy. The cardiac fibrosis marker, galectin-3, was induced by maternal diabetes. Furthermore, maternal type 2 diabetes mellitus activated the proapoptotic c-Jun-N-terminal kinase 1/2 stress signaling and triggered cell apoptosis by increasing the number of terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeling-positive cells (10.4 ± 2.2% of the type 2 diabetes mellitus group vs 3.8 ± 0.7% of the nondiabetic group, P < .05). Maternal type 2 diabetes mellitus induces cardiac hypertrophy in embryonic hearts. Adverse cardiac remodeling, including elevated collagen synthesis, suppressed fibronectin synthesis, profibrosis, and apoptosis, is implicated as the etiology of cardiac hypertrophy. Copyright © 2017 Elsevier Inc. All rights reserved.

The TLR4 agonist fibronectin extra domain A is cryptic, exposed by elastase-2; use in a fibrin matrix cancer vaccine

DOE PAGES

Julier, Ziad; Martino, Mikaël M.; de Titta, Alexandre; ...

2015-02-24

Fibronectin (FN) is an extracellular matrix (ECM) protein including numerous fibronectin type III (FNIII) repeats with different functions. The alternatively spliced FN variant containing the extra domain A (FNIII EDA), located between FNIII 11 and FNIII 12, is expressed in sites of injury, chronic inflammation, and solid tumors. Although its function is not well understood, FNIII EDA is known to agonize Toll-like receptor 4 (TLR4). Here, by producing various FN fragments containing FNIII EDA, we found that FNIII EDA's immunological activity depends upon its local intramolecular context within the FN chain. N-terminal extension of the isolated FNIII EDA with itsmore » neighboring FNIII repeats (FNIII 9-10-11) enhanced its activity in agonizing TLR4, while C-terminal extension with the native FNIII 12-13-14 heparin-binding domain abrogated it. We reveal that an elastase 2 cleavage site is present between FNIII EDA and FNIII 12. Activity of the C-terminally extended FNIII EDA could be restored after cleavage of the FNIII 12-13-14 domain by elastase 2. FN being naturally bound to the ECM, we immobilized FNIII EDA-containing FN fragments within a fibrin matrix model along with antigenic peptides. Such matrices were shown to stimulate cytotoxic CD8 + T cell responses in two murine cancer models.« less

In Vitro Analysis of Fibronectin-Modified Titanium Surfaces

PubMed Central

Chang, Yu-Chi; Lee, Wei-Fang; Feng, Sheng-Wei; Huang, Haw-Ming; Lin, Che-Tong; Teng, Nai-Chia; Chang, Wei Jen

2016-01-01

Background Glow discharge plasma (GDP) procedure is an effective method for grafting various proteins, including albumin, type I collagen, and fibronectin, onto a titanium surface. However, the behavior and impact of titanium (Ti) surface modification is yet to be unraveled. Purpose The purpose of this study is to evaluate and analyze the biological properties of fibronectin-grafted Ti surfaces treated by GDP. Materials and Methods Grade II Ti discs were initially cleaned and autoclaved to obtain original specimens. Subsequently, the specimens were GDP treated and grafted with fibronectin to form Ar-GDP (Argon GDP treatment only) and GDP-fib (fibronectin coating following GDP treatment) groups. Blood coagulation test and MG-63 cell culture were performed to evaluate the biological effects on the specimen. Results There was no significant difference between Ar-GDP and GDP-fib groups in blood compatibility analysis. While in the MTT test, cellular proliferation was benefited from the presence of fibronectin coating. The numbers of cells on Ar-GDP and GDP-fib specimens were greater than those in the original specimens after 24 h of culturing. Conclusions GDP treatment combined with fibronectin grafting favored MG-63 cell adhesion, migration, and proliferation on titanium surfaces, which could be attributed to the improved surface properties. PMID:26731536

Structural determinants of the interaction between the Haemophilus influenzae Hap autotransporter and fibronectin.

PubMed

Spahich, Nicole A; Kenjale, Roma; McCann, Jessica; Meng, Guoyu; Ohashi, Tomoo; Erickson, Harold P; St Geme, Joseph W

2014-06-01

Haemophilus influenzae is a Gram-negative cocco-bacillus that initiates infection by colonizing the upper respiratory tract. Hap is an H. influenzae serine protease autotransporter protein that mediates adherence, invasion and microcolony formation in assays with human epithelial cells and is presumed to facilitate the process of colonization. Additionally, Hap mediates adherence to fibronectin, laminin and collagen IV, extracellular matrix (ECM) proteins that are present in the respiratory tract and are probably important targets for H. influenzae colonization. The region of Hap responsible for adherence to ECM proteins has been localized to the C-terminal 511 aa of the Hap passenger domain (HapS). In this study, we characterized the structural determinants of the interaction between HapS and fibronectin. Using defined fibronectin fragments, we established that Hap interacts with the fibronectin repeat fragment called FNIII(1-2). Using site-directed mutagenesis, we found a series of motifs in the C-terminal region of HapS that contribute to the interaction with fibronectin. Most of these motifs are located on the F1 and F3 faces of the HapS structure, suggesting that the F1 and F3 faces may be responsible for the HapS-fibronectin interaction. © 2014 The Authors.

Structural determinants of the interaction between the Haemophilus influenzae Hap autotransporter and fibronectin

PubMed Central

Spahich, Nicole A.; Kenjale, Roma; McCann, Jessica; Meng, Guoyu; Ohashi, Tomoo; Erickson, Harold P.

2014-01-01

Haemophilus influenzae is a Gram-negative cocco-bacillus that initiates infection by colonizing the upper respiratory tract. Hap is an H. influenzae serine protease autotransporter protein that mediates adherence, invasion and microcolony formation in assays with human epithelial cells and is presumed to facilitate the process of colonization. Additionally, Hap mediates adherence to fibronectin, laminin and collagen IV, extracellular matrix (ECM) proteins that are present in the respiratory tract and are probably important targets for H. influenzae colonization. The region of Hap responsible for adherence to ECM proteins has been localized to the C-terminal 511 aa of the Hap passenger domain (HapS). In this study, we characterized the structural determinants of the interaction between HapS and fibronectin. Using defined fibronectin fragments, we established that Hap interacts with the fibronectin repeat fragment called FNIII(1–2). Using site-directed mutagenesis, we found a series of motifs in the C-terminal region of HapS that contribute to the interaction with fibronectin. Most of these motifs are located on the F1 and F3 faces of the HapS structure, suggesting that the F1 and F3 faces may be responsible for the HapS–fibronectin interaction. PMID:24687948

The effect of Centella asiatica, vitamins, glycolic acid and their mixtures preparations in stimulating collagen and fibronectin synthesis in cultured human skin fibroblast.

PubMed

Hashim, Puziah

2014-03-01

Centella asiatica (Linn.) Urban is well known in promoting wound healing and provides significant benefits in skin care and therapeutic products formulation. Glycolic acid and vitamins also play a role in the enhancement of collagen and fibronectin synthesis. Here, we evaluate the specific effect of Centella asiatica (CA), vitamins, glycolic acid and their mixture preparations to stimulate collagen and fibronectin synthesis in cultured human fibroblast cells. The fibroblast cells are incubated with CA, glycolic acid, vitamins and their mixture preparations for 48 h. The cell lysates were analyzed for protein content and collagen synthesis by direct binding enzyme immunoassay. The fibronectin of the cultured supernatant was measured by sandwich enzyme immunoassay. The results showed that CA, glycolic acid, vitamins A, E and C significantly stimulate collagen and fibronectin synthesis in the fibroblast. Addition of glycolic acid and vitamins to CA further increased the levels of collagen and fibronectin synthesis to 8.55 and 23.75 μg/100 μg, respectively. CA, glycolic acid, vitamins A, E, and C, and their mixtures demonstrated stimulatory effect on both extra-cellular matrix synthesis of collagen and fibronectin in in vitro studies on human foreskin fibroblasts, which is beneficial to skin care and therapeutic products formulation.

Development of siRNA Technology to Prevent Scar Formation in Tendon Repair

DTIC Science & Technology

2013-12-01

Anti-sense RNA technologies: Under normal conditions cells produce small interfering (si) RNAs that inhibit protein synthesis and stimulate...stimulation of fibroblast proliferation and migration, collagen and fibronectin synthesis , and altered tissue remodeling through regulation of MMPs...expression by an antisense oligonucleotide protects mice from fulminant hepatitis. Nat Biotechnol 2000;18:862-7. 7. Guha M, Xu ZG, Tung D, Lanting L

pH-Sensitive PEGylated liposomes functionalized with a fibronectin-mimetic peptide show enhanced intracellular delivery to colon cancer cell.

PubMed

Garg, Ashish; Kokkoli, Efrosini

2011-08-01

pH-sensitive liposomes undergo rapid destabilization under mildly acidic conditions such as those found in endocytotic vesicles. Though this makes them promising drug carriers, their application is limited due to their rapid clearance from circulation by the reticulo-endothelial system. Researchers have therefore used pH-sensitive liposomes that are sterically stabilized by polyethylene glycol (PEG) molecules (stealth liposomes) on the liposome surface. The goal of this study is to bring bio-functionality to pH-sensitive PEGylated liposomes in order to facilitate their potential use as a targeted drug delivery agent. To improve the selectivity of these nanoparticles, we included a targeting moiety, PR_b which specifically recognizes and binds to integrin α(5)β(1) expressing cells. PR_b (KSSPHSRN(SG)(5)RGDSP) is a novel fibronectin-mimetic peptide sequence that mimics the cell adhesion domain of fibronectin. Integrin α(5)β(1) is expressed on several types of cancer cells, including colon cancer, and plays an important role in tumor growth and metastasis. We have thoroughly studied the release of calcein from pH-sensitive PEGylated liposomes by varying the lipid composition of the liposomes in the absence and presence of the targeting peptide, PR_b, and accounting for the first time for the effect of both pH and time (photo-bleaching effect) on the fluorescence signal of calcein. We have demonstrated that we can design PR_b-targeted pH-sensitive PEGylated liposomes, which can undergo destabilization under mildly acidic conditions and have shown that incorporating the PR_b peptide does not significantly affect the pH-sensitivity of the liposomes. PR_b-targeted pH-sensitive PEGylated liposomes bind to CT26.WT colon carcinoma cells that express integrin α(5)β(1), undergo cellular internalization, and release their load intracellularly in a short period of time as compared to other formulations. Our studies demonstrate that PR_b-functionalized pH-sensitive targeted delivery systems have the potential to deliver a payload directly to cancer cells in an efficient and specific manner.

Live Imaging of Type I Collagen Assembly Dynamics in Osteoblasts Stably Expressing GFP and mCherry-Tagged Collagen Constructs.

PubMed

Lu, Yongbo; Kamel-El Sayed, Suzan A; Wang, Kun; Tiede-Lewis, LeAnn M; Grillo, Michael A; Veno, Patricia A; Dusevich, Vladimir; Phillips, Charlotte L; Bonewald, Lynda F; Dallas, Sarah L

2018-06-01

Type I collagen is the most abundant extracellular matrix protein in bone and other connective tissues and plays key roles in normal and pathological bone formation as well as in connective tissue disorders and fibrosis. Although much is known about the collagen biosynthetic pathway and its regulatory steps, the mechanisms by which it is assembled extracellularly are less clear. We have generated GFPtpz and mCherry-tagged collagen fusion constructs for live imaging of type I collagen assembly by replacing the α2(I)-procollagen N-terminal propeptide with GFPtpz or mCherry. These novel imaging probes were stably transfected into MLO-A5 osteoblast-like cells and fibronectin-null mouse embryonic fibroblasts (FN-null-MEFs) and used for imaging type I collagen assembly dynamics and its dependence on fibronectin. Both fusion proteins co-precipitated with α1(I)-collagen and remained intracellular without ascorbate but were assembled into α1(I) collagen-containing extracellular fibrils in the presence of ascorbate. Immunogold-EM confirmed their ultrastuctural localization in banded collagen fibrils. Live cell imaging in stably transfected MLO-A5 cells revealed the highly dynamic nature of collagen assembly and showed that during assembly the fibril networks are continually stretched and contracted due to the underlying cell motion. We also observed that cell-generated forces can physically reshape the collagen fibrils. Using co-cultures of mCherry- and GFPtpz-collagen expressing cells, we show that multiple cells contribute collagen to form collagen fiber bundles. Immuno-EM further showed that individual collagen fibrils can receive contributions of collagen from more than one cell. Live cell imaging in FN-null-MEFs expressing GFPtpz-collagen showed that collagen assembly was both dependent upon and dynamically integrated with fibronectin assembly. These GFP-collagen fusion constructs provide a powerful tool for imaging collagen in living cells and have revealed novel and fundamental insights into the dynamic mechanisms for the extracellular assembly of collagen. © 2018 American Society for Bone and Mineral Research. © 2018 American Society for Bone and Mineral Research.

Type XVII collagen (BP180) can function as a cell-matrix adhesion molecule via binding to laminin 332

PubMed Central

Van den Bergh, F.; Eliason, S.L.; Giudice, G.J.

2010-01-01

Collagen XVII (COL17) is a transmembrane glycoprotein that is expressed on the basal surface of basal epidermal keratinocytes. Previous observations have led to the hypothesis that an interaction between COL17 and laminin 332, an extracellular matrix protein, contributes to the attachment of the basal keratinocyte to the basement membrane. In order to isolate and manipulate COL17 interactions with ECM components, we induced COL17 expression in two cells lines, SK-MEL1 and K562, that exhibit little or no capacity to attach to our test substrates, including laminin 332, types I and IV collagen, and fibronectin. Cells expressing high levels of COL17 preferentially adhered to a laminin 332 matrix, and, to a lesser extent, type IV collagen, while showing little or no binding to type I collagen or fibronectin. A quantitative analysis of cell adhesive forces revealed that, compared with COL17-negative cells, COL17-positive cells required over 7-fold greater force to achieve 50% detachment from a laminin 332 substrate. When a cell preparation (either K562 or SK-MEL1) with heterogeneous COL17 expression levels was allowed to attach to a laminin 332 matrix, the COL17-positive and COL17-negative cells differentially sorted to the bound and unbound cell fractions, respectively. COL17-dependent attachment to laminin 332 could be reduced or abolished by siRNA-mediated knockdown of COL17 expression or by adding to the assay wells specific antibodies against COL17 or laminin 332. These findings provide strong support for the hypothesis that cell surface COL17 can interact with laminin 332 and, together, participate in the adherence of a cell to the extracellular matrix. PMID:21034821

Streptococcal modulation of cellular invasion via TGF-beta1 signaling.

PubMed

Wang, Beinan; Li, Shaoying; Southern, Peter J; Cleary, Patrick P

2006-02-14

Group A Streptococcus (GAS) and other bacterial pathogens are known to interact with integrins as an initial step in a complex pathway of bacterial ingestion by host cells. Efficient GAS invasion depends on the interaction of bound fibronectin (Fn) with integrins and activation of integrin signaling. TGF-beta1 regulates expression of integrins, Fn, and other extracellular matrix proteins, and positively controls the integrin signaling pathway. Therefore, we postulated that TGF-beta1 levels could influence streptococcal invasion of mammalian cells. Pretreatment of HEp-2 cells with TGF-beta1 increased their capacity to ingest GAS when the bacteria expressed fibronectin-binding proteins (M1 or PrtF1). Western blots revealed significant induction of alpha5 integrin and Fn expression by HEp-2 cells in response to TGF-beta1. Increased ingestion of streptococci by these cells was blocked by a specific inhibitor of the TGF-beta1 receptor I and antibodies directed against alpha5 integrin and Fn, indicating that increased invasion depends on TGF-beta1 up-regulation of both the alpha5 integrin and Fn. The capacity of TGF-beta1 to up-regulate integrin expression and intracellular invasion by GAS was reproduced in primary human tonsil fibroblasts, which could be a source of TGF-beta1 in chronically infected tonsils. The relationship between TGF-beta1 and GAS invasion was strengthened by the observation that TGF-beta1 production was stimulated in GAS-infected primary human tonsil fibroblasts. These findings suggest a mechanism by which GAS induce a cascade of changes in mammalian tissue leading to elevated expression of the alpha5beta1 receptor, enhanced invasion, and increased opportunity for survival and persistence in their human host.

Protective effects of tocotrienols against lipid-induced nephropathy in experimental type-2 diabetic rats by modulation in TGF-β expression.

PubMed

Siddiqui, Shabeena; Ahsan, Haseeb; Khan, Mohammad Rashid; Siddiqui, Waseem A

2013-12-01

Dyslipidemia is common in patients with diabetes mellitus (DM) and is considered a risk factor for the progression of diabetic nephropathy (DN). Hyperlipidemia and hyperglycemia act synergistically to induce renal injury. The present study was designed to investigate the protective effects of tocotrienols as tocotrienol-rich fraction (TRF) extracted from palm (PO) and rice bran oils (RBO) against lipid induced nephropathy in type-2 diabetic rats and its probable molecular mechanism. Male Wistar rats (175-200 g) were divided into four groups. The first group served as diabetic control, while the second and third groups received PO-TRF and RBO-TRF, respectively by gavage over a period of sixteen weeks post-induction of diabetes. The fourth group comprised of age-matched rats that served as normal control. The effects of TRF on serum lipid profile, oxidative stress markers, expression of TGF-β, fibronectin and collagen type IV were analyzed in the kidney of diabetic rats. Treatment with PO-TRF and RBO-TRF significantly improved glycemic status, serum lipid profile and renal function in type-2 diabetic rats. In addition, TRF supplementation down-regulated the expression of TGF-β, fibronectin and collagen type IV in the kidney of diabetic rats. Transforming growth factor-β (TGF-β) plays a critical role in progression of DN, but its modulation by tocotrienols in DN remains unexplored. TRF ameliorated lipid induced nephropathy in type-2 diabetes by its hypoglycemic, hypolipidemic and antioxidant activities as well as by modulation of TGF-β to prevent increased expression of collagen type IV and fibrinogen. We finally propose a mechanism for the expression of molecular markers that are significant in the events leading to diabetic nephropathy and its modulation by tocotrienols/TRF. © 2013.

Potential role of recombinant secretory leucoprotease inhibitor in the prevention of neutrophil mediated matrix degradation.

PubMed

Llewellyn-Jones, C G; Lomas, D A; Stockley, R A

1994-06-01

Neutrophil elastase is able to degrade connective tissue matrices and is thought to be involved in the pathogenesis of destructive lung diseases. The ability of recombinant secretory leucoprotease inhibitor (rSLPI) to inhibit neutrophil mediated degradation of fibronectin in vitro is demonstrated and its efficacy compared with native alpha-1-proteinase inhibitor (n alpha 1-PI), recombinant alpha-1-proteinase inhibitor (r alpha 1-PI), and the chemical elastase inhibitor ICI 200,355. When preincubated with neutrophils both rSLPI and r alpha 1-PI were effective inhibitors of fibronectin degradation although n alpha 1-PI and ICI 200,355 were less effective. Recombinant SLPI was the most effective inhibitor when the cells were allowed to adhere to fibronectin before the addition of the inhibitors. Preincubation of rSLPI (0.1 mumol/l) with the fibronectin plate resulted in almost total inhibition of fibronectin degradation (reduced to 3.3 (SE 0.9)% of control). Pretreating the fibronectin plate with 1 mumol/l rSLPI, r alpha 1-PI and ICI 200,355 followed by thorough washing before the addition of cells resulted in no inhibition of fibronectin degradation with r alpha 1-PI and the ICI inhibitor, but rSLPI retained its inhibitory effect. This effect could be reduced by adding rSLPI in high pH buffer or 2 mol/1 NaCl. It is postulated that rSLPI binds to fibronectin to form a protective layer which prevents its degradation by neutrophil elastase. It may prove to be the most useful therapeutic agent in the prevention of neutrophil mediated lung damage.

Characterization of a highly conserved human homolog to the chicken neural cell surface protein Bravo/Nr-CAM that maps to chromosome band 7q31

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

Lane, R.P.; Vielmetter, J.; Dreyer, W.J.

1996-08-01

The neuronal cell adhesion molecule Bravo/Nr-CAM is a cell surface protein of the immunoglobulin (Ig) superfamily and is closely related to the L1/NgCAM and neurofascin molecules, all of which contain six immunoglobulin domains, five fibronectin repeats, a transmembrane region, and an intracellular domain. Chicken Bravo/Nr-CAM has been shown to interact with other cell surface molecules of the Ig superfamily and has been implicated in specific pathfinding roles of axonal growth cones in the developing nervous system. We now report the characterization of cDNA clones encoding the human Bravo/Nr-CAM protein, which, like its chicken homolog, is composed of six V-like Igmore » domains and five fibronectin type III repeats. The human Bravo/Nr-CAM homolog also contains a transmembrane and intracellular domain, both of which are 100% conserved at the amino acid level compared to its chicken homolog. Overall, the human Bravo/Nr-CAM homolog is 82% identical to the chicken Bravo/Nr-CAM amino acid sequence. Independent cDNAs encoding four different isoforms were also identified, all of which contain alternatively spliced variants around the fifth fibronectin type III repeat, including one isoform that had been previously identified for chicken Bravo/Nr-CAM. Northern blot analysis reveals one mRNA species of approximately 7.0 kb in adult human brain tissue. Fluorescence in situ hybridization maps the gene for human Bravo/Nr-CAM to human chromosome 7q31.1-q31.2. This chromosomal locus has been previously identified as containing a tumore suppressor candidate gene commonly deleted in certain human cancer tissues. 38 refs., 5 figs.« less

Accumulation of Extracellular Matrix in Advanced Lesions of Canine Distemper Demyelinating Encephalitis

PubMed Central

Seehusen, Frauke; Al-Azreg, Seham A.; Raddatz, Barbara B.; Haist, Verena; Puff, Christina; Spitzbarth, Ingo; Ulrich, Reiner; Baumgärtner, Wolfgang

2016-01-01

In demyelinating diseases, changes in the quality and quantity of the extracellular matrix (ECM) may contribute to demyelination and failure of myelin repair and axonal sprouting, especially in chronic lesions. To characterize changes in the ECM in canine distemper demyelinating leukoencephalitis (DL), histochemical and immunohistochemical investigations of formalin-fixed paraffin-embedded cerebella using azan, picrosirius red and Gomori`s silver stain as well as antibodies directed against aggrecan, type I and IV collagen, fibronectin, laminin and phosphacan showed alterations of the ECM in CDV-infected dogs. A significantly increased amount of aggrecan was detected in early and late white matter lesions. In addition, the positive signal for collagens I and IV as well as fibronectin was significantly increased in late lesions. Conversely, the expression of phosphacan was significantly decreased in early and more pronounced in late lesions compared to controls. Furthermore, a set of genes involved in ECM was extracted from a publically available microarray data set and was analyzed for differential gene expression. Gene expression of ECM molecules, their biosynthesis pathways, and pro-fibrotic factors was mildly up-regulated whereas expression of matrix remodeling enzymes was up-regulated to a relatively higher extent. Summarized, the observed findings indicate that changes in the quality and content of ECM molecules represent important, mainly post-transcriptional features in advanced canine distemper lesions. Considering the insufficiency of morphological regeneration in chronic distemper lesions, the accumulated ECM seems to play a crucial role upon regenerative processes and may explain the relatively small regenerative potential in late stages of this disease. PMID:27441688

Accumulation of Extracellular Matrix in Advanced Lesions of Canine Distemper Demyelinating Encephalitis.

PubMed

Seehusen, Frauke; Al-Azreg, Seham A; Raddatz, Barbara B; Haist, Verena; Puff, Christina; Spitzbarth, Ingo; Ulrich, Reiner; Baumgärtner, Wolfgang

2016-01-01

In demyelinating diseases, changes in the quality and quantity of the extracellular matrix (ECM) may contribute to demyelination and failure of myelin repair and axonal sprouting, especially in chronic lesions. To characterize changes in the ECM in canine distemper demyelinating leukoencephalitis (DL), histochemical and immunohistochemical investigations of formalin-fixed paraffin-embedded cerebella using azan, picrosirius red and Gomori`s silver stain as well as antibodies directed against aggrecan, type I and IV collagen, fibronectin, laminin and phosphacan showed alterations of the ECM in CDV-infected dogs. A significantly increased amount of aggrecan was detected in early and late white matter lesions. In addition, the positive signal for collagens I and IV as well as fibronectin was significantly increased in late lesions. Conversely, the expression of phosphacan was significantly decreased in early and more pronounced in late lesions compared to controls. Furthermore, a set of genes involved in ECM was extracted from a publically available microarray data set and was analyzed for differential gene expression. Gene expression of ECM molecules, their biosynthesis pathways, and pro-fibrotic factors was mildly up-regulated whereas expression of matrix remodeling enzymes was up-regulated to a relatively higher extent. Summarized, the observed findings indicate that changes in the quality and content of ECM molecules represent important, mainly post-transcriptional features in advanced canine distemper lesions. Considering the insufficiency of morphological regeneration in chronic distemper lesions, the accumulated ECM seems to play a crucial role upon regenerative processes and may explain the relatively small regenerative potential in late stages of this disease.

Heme oxygenase-1 upregulation modulates tone and fibroelastic properties of internal anal sphincter

PubMed Central

Krishna, Chadalavada Vijay; Singh, Jagmohan; Kumar, Sumit

2014-01-01

A compromise in the internal anal sphincter (IAS) tone and fibroelastic properties (FEP) plays an important role in rectoanal incontinence. Herein, we examined the effects of heme oxygenase (HO)-1 upregulation on these IAS characteristics in young rats. We determined the effect of HO-1 upregulator hemin on HO-1 mRNA and protein expressions and on basal IAS tone and its FEP before and after HO-1 inhibitor tin protoporphyrin IX. For FEP, we determined the kinetics of the IAS smooth muscle responses, by the velocities of relaxation, and recovery of the IAS tone following 0 Ca2+ and electrical field stimulation. To characterize the underlying signal transduction for these changes, we determined the effects of hemin on RhoA-associated kinase (RhoA)/Rho kinase (ROCK) II, myosin-binding subunit of myosin light chain phosphatase 1, fibronectin, and elastin expression levels. Hemin increased HO-1 mRNA and protein similar to the increases in the basal tone, and in the FEP of the IAS. Underlying mechanisms in the IAS characteristics are associated with increases in the genetic and translational expressions of RhoA/ROCKII, and elastin. Fibronectin expression levels on the other hand were found to be decreased following HO-1 upregulation. The results of our study show that the hemin/HO-1 system regulates the tone and FEP of IAS. The hemin/HO-1 system thus provides a potential target for the development of new interventions aimed at treatment of gastrointestinal motility disorders, specifically the age-related IAS dysfunction. PMID:25035109

Fibronectin non-amyloid glomerulopathy.

PubMed

Yong, Jim L; Killingsworth, Murray C; Spicer, S Timothy; Wu, Xiao-Juan

2009-11-20

A 41-year-old Burmese man presented with nephrotic syndrome, a creatinine level of 150 micromol/L and limited clinical history. His renal biopsy demonstrated glomerulopathy with large eosinophilic deposits in the mesangium and capillary loops that were negative for Congo red, slightly positive for periodic acid-Schiff and blue with Masson trichrome stain. Immunofluorescence microscopy with a routine antibody panel was unhelpful. Electron microscopy demonstrated extensive, moderately electron-dense deposits in the subendothelial space, subepithelial space and mesangium that could be differentiated from adjacent basement membrane by their increased electron density. The deposits contained finely granular material and occasional filaments with variable diameter ranging from 9-16 nm. Fibronectin glomerulopathy was suspected from anti-fibronectin immunohistochemistry that showed positive staining of thickened capillary loops. This staining was subsequently confirmed by immunoelectron microscopy demonstrating the presence of cellular fibronectin (cFN) in deposits. Significantly, deposition of fibronectin appeared to have occurred in the absence of thickening or folding of the adjacent basement membrane. The prominent mesangial location of deposits containing a cFN isotype may indicate that retention of local fibronectin produced in the mesangium has contributed to this pathology.

Fibronectin non-amyloid glomerulopathy

PubMed Central

Yong, Jim L; Killingsworth, Murray C; Spicer, S Timothy; Wu, Xiao-Juan

2010-01-01

A 41-year-old Burmese man presented with nephrotic syndrome, a creatinine level of 150 µmol/L and limited clinical history. His renal biopsy demonstrated glomerulopathy with large eosinophilic deposits in the mesangium and capillary loops that were negative for Congo red, slightly positive for periodic acid-Schiff and blue with Masson trichrome stain. Immunofluorescence microscopy with a routine antibody panel was unhelpful. Electron microscopy demonstrated extensive, moderately electron-dense deposits in the subendothelial space, subepithelial space and mesangium that could be differentiated from adjacent basement membrane by their increased electron density. The deposits contained finely granular material and occasional filaments with variable diameter ranging from 9-16 nm. Fibronectin glomerulopathy was suspected from anti-fibronectin immunohistochemistry that showed positive staining of thickened capillary loops. This staining was subsequently confirmed by immunoelectron microscopy demonstrating the presence of cellular fibronectin (cFN) in deposits. Significantly, deposition of fibronectin appeared to have occurred in the absence of thickening or folding of the adjacent basement membrane. The prominent mesangial location of deposits containing a cFN isotype may indicate that retention of local fibronectin produced in the mesangium has contributed to this pathology. PMID:20126589

Interaction between integrin α5 and PDE4D regulates endothelial inflammatory signalling

PubMed Central

Yun, Sanguk; Budatha, Madhusudhan; Dahlman, James E.; Coon, Brian G.; Cameron, Ryan T.; Langer, Robert; Anderson, Daniel G.; Baillie, George; Schwartz, Martin A.

2016-01-01

Atherosclerosis is primarily a disease of lipid metabolism and inflammation; however, it is also closely associated with endothelial extracellular matrix (ECM) remodelling, with fibronectin accumulating in the laminin–collagen basement membrane. To investigate how fibronectin modulates inflammation in arteries, we replaced the cytoplasmic tail of the fibronectin receptor integrin α5 with that of the collagen/laminin receptor integrin α2. This chimaera suppressed inflammatory signalling in endothelial cells on fibronectin and in knock-in mice. Fibronectin promoted inflammation by suppressing anti-inflammatory cAMP. cAMP was activated through endothelial prostacyclin secretion; however, this was ECM-independent. Instead, cells on fibronectin suppressed cAMP via enhanced phosphodiesterase (PDE) activity, through direct binding of integrin α5 to phosphodiesterase-4D5 (PDE4D5), which induced PP2A-dependent dephosphorylation of PDE4D5 on the inhibitory site Ser651. In vivo knockdown of PDE4D5 inhibited inflammation at athero-prone sites. These data elucidate a molecular mechanism linking ECM remodelling and inflammation, thereby identifying a new class of therapeutic targets. PMID:27595237

Crystal structure of tandem type III fibronectin domains from Drosophila neuroglian at 2.0 A.

PubMed

Huber, A H; Wang, Y M; Bieber, A J; Bjorkman, P J

1994-04-01

We report the crystal structure of two adjacent fibronectin type III repeats from the Drosophila neural cell adhesion molecule neuroglian. Each domain consists of two antiparallel beta sheets and is folded topologically identically to single fibronectin type III domains from the extracellular matrix proteins tenascin and fibronectin. beta bulges and left-handed polyproline II helices disrupt the regular beta sheet structure of both neuroglian domains. The hydrophobic interdomain interface includes a metal-binding site, presumably involved in stabilizing the relative orientation between domains and predicted by sequence comparision to be present in the vertebrate homolog molecule L1. The neuroglian domains are related by a near perfect 2-fold screw axis along the longest molecular dimension. Using this relationship, a model for arrays of tandem fibronectin type III repeats in neuroglian and other molecules is proposed.

Inductions of granulosa cell luteinization and cumulus expansion are dependent on the fibronectin-integrin pathway during ovulation process in mice.

PubMed

Kitasaka, Hiroya; Kawai, Tomoko; Hoque, S A Masudul; Umehara, Takashi; Fujita, Youko; Shimada, Masayuki

2018-01-01

It has been known that EGF-like factor secreted from LH-stimulated granuloma cells acts on granulosa cells and cumulus cells to induce ovulation process. Granulosa cells are changed the morphology with differentiating cell functions to produce progesterone. Cumulus cells are detached to make a space between the cells to accumulate hyaluronan rich matrix. LH also changes extracellular matrix (ECM) components including fibronectin in the follicular walls and granulosa cell layers. EGF like factor and fibronectin synergistically play important roles in numerous cell functions, especially cancer cell migration, estimating that fibronectin would impact on granulosa cells and cumulus cells. To clear this hypothesis, the localizations of fibronectin and its receptor integrin were observed by immunofluorescence technique. The functions were monitored by the detection of downstream signaling pathway, focal adhesion kinase (FAK). The pharmacological approach in both in vivo and in vitro were used for analyzing the physiological roles of FAK during ovulation process. The immunofluorescence staining revealed that fibronectin and integrin were observed in granulosa cells, cumulus cells and the space between cumulus cells and oocyte at 4 and 8 h after hCG injection. Concomitantly with the changes of fibronectin-integrin localization, FAK was phosphorylated in periovulatory follicles. The injection of FAK inhibitor suppressed not only ovulation but also luteinization of granulosa cells and cumulus expansion. In cultured-granulosa cells, fibronectin-integrin synergistically activated FAK with amphiregulin (AREG). Such cooperative stimulations induced a morphological change in granulosa cells, which resulted in the maximum level of progesterone production via the induction of Hsd3b. When cumulus-oocyte complexes (COCs) were cultured with AREG in the presence of serum, the maximum level of cumulus expansion was observed. The AREG-induced cumulus expansion was also suppressed by FAK inhibitor. Thus, it is concluded that fibronectin and AREG synergistically activate FAK not only in granulosa cells and cumulus cells to induce successful ovulation process.

PBP-2 Negative Methicillin Resistant Staphylococcus schleiferi Bacteremia from a Prostate Abscess: An Unusual Occurrence

PubMed Central

Merchant, Chandni; Villanueva, Daphne-Dominique; Lalani, Ishan; Eng, Margaret; Kang, Yong

2016-01-01

Staphylococcus schleiferi subsp. schleiferi is a coagulase-negative Staphylococcus which has been described as a pathogen responsible for various nosocomial infections including bacteremia, brain abscess, and infection of intravenous pacemakers. Recently, such bacteria have been described to be found typically on skin and mucosal surfaces. It is also believed to be a part of the preaxillary human flora and more frequently found in men. It is very similar in its pathogenicity with Staphylococcus aureus group and expresses a fibronectin binding protein. Literature on this pathogen reveals that it commonly causes otitis among dogs because of its location in the auditory meatus of canines. Also, it has strong association with pyoderma in dogs. The prime concern with this organism is the antibiotic resistance and relapse even after appropriate treatment. Very rarely, if any, cases have been reported about prostatic abscess (PA) with this microbe. Our patient had a history of recurrent UTIs and subsequent PA resulting in S. schleiferi bacteremia in contrast to gram negative bacteremia commonly associated with UTI. This organism was found to be resistant to methicillin, in spite of being negative for PBP2, which is a rare phenomenon and needs further studies. PMID:27092283

Suppression of the Epidermal Growth Factor-like Domain 7 and Inhibition of Migration and Epithelial-Mesenchymal Transition in Human Pancreatic Cancer PANC-1 Cells.

PubMed

Wang, Yun-Liang; Dong, Feng-Lin; Yang, Jian; Li, Zhi; Zhi, Qiao-Ming; Zhao, Xin; Yang, Yong; Li, De-Chun; Shen, Xiao-Chun; Zhou, Jin

2015-01-01

Epidermal growth factor-like domain multiple 7 (EGFL7), a secreted protein specifically expressed by endothelial cells during embryogenesis, recently was identified as a critical gene in tumor metastasis. Epithelial-mesenchymal transition (EMT) was found to be closely related with tumor progression. Accordingly, it is important to investigate the migration and EMT change after knock-down of EGFL7 gene expression in human pancreatic cancer cells. EGFL7 expression was firstly testified in 4 pancreatic cancer cell lines by real-time polymerase chain reaction (Real-time PCR) and western blot, and the highest expression of EGFL7 was found in PANC-1 cell line. Then, PANC-1 cells transfected with small interference RNA (siRNA) of EGFL7 using plasmid vector were named si-PANC-1, while transfected with negative control plasmid vector were called NC-PANC-1. Transwell assay was used to analyze the migration of PANC-1 cells. Real-time PCR and western blotting were used to detect the expression change of EGFL7 gene, EMT markers like E-Cadherin, N-Cadherin, Vimentin, Fibronectin and transcription factors like snail, slug in PANC-1, NC- PANC-1, and si-PANC-1 cells, respectively. After successful plasmid transfection, EGFL7 gene were dramatically knock-down by RNA interference in si-PANC-1 group. Meanwhile, migration ability decreased significantly, compared with PANC-1 and NC-PANC-1 group. Meanwhile, the expression of epithelial phenotype marker E-Cadherin increased and that of mesenchymal phenotype markers N-Cadherin, Vimentin, Fibronectin dramatically decreased in si-PANC-1 group, indicating a reversion of EMT. Also, transcription factors snail and slug decreased significantly after RNA interference. Current study suggested that highly-expressed EGFL7 promotes migration of PANC-1 cells and acts through transcription factors snail and slug to induce EMT, and further study is needed to confirm this issue.

Hepatocyte growth factor upregulation promotes carcinogenesis and epithelial-mesenchymal transition in hepatocellular carcinoma via Akt and COX-2 pathways

PubMed Central

Ogunwobi, Olorunseun O.

2013-01-01

Advanced hepatocellular carcinoma (HCC) is an important cause of cancer mortality. Epithelial-mesenchymal transition (EMT) has been shown to be an important biological process in cancer progression and metastasis. We have focused on elucidating factors that induce EMT to promote carcinogenesis and subsequent metastasis in HCC using the BNL CL.2 (BNL) and BNL 1ME A. 7R.1 (1MEA) cell lines. BNL cells are normal hepatocytes whereas the 1MEA cells are HCC cells derived from chemical transformation of the BNL cells. Their morphological characteristics were examined. Expression levels of hepatocyte growth factor (HGF), markers of EMT and mediators of HGF signaling were determined and functional characteristics were compared. BNL cells were treated with HGF and effects on EMT-marker and mediators of HGF signaling were analyzed. BNL cells display characteristic epithelial morphology whereas 1MEA cells display mesenchymal characteristics. 1MEA cells express and secrete more HGF than BNL cells. There was significantly decreased expression of E-cadherin, albumin, AAT and increased expression of fibronectin, collagen-1, vimentin, snail and slug in 1MEA cells. There was also increased expression of cyclooxygenase-2 (COX-2), Akt and phosphorylated Akt (pAkt) in 1MEA cells. Moreover, 1MEA cells had increased migratory capacity inhibited by inhibition of COX-2 and Akt but not extracellular signal regulated kinase (ERK). Molecular mesenchymal characteristics of 1MEA cells were reversed by inhibition of COX-2, Akt and ERK. Treatment of BNL cells with HGF led to decreased expression of E-cadherin and increased expression of fibronectin, vimentin, snail, slug, COX-2, Akt, pAkt and increased migration, invasiveness and clonogenicity. We conclude that development of HCC is associated with upregulation of HGF which promotes EMT and carcinogenesis via upregulation of COX-2 and Akt. Consequently, HGF signaling may be targeted for therapy in advanced and metastatic HCC. PMID:21744257

Hepatocyte growth factor upregulation promotes carcinogenesis and epithelial-mesenchymal transition in hepatocellular carcinoma via Akt and COX-2 pathways.

PubMed

Ogunwobi, Olorunseun O; Liu, Chen

2011-12-01

Advanced hepatocellular carcinoma (HCC) is an important cause of cancer mortality. Epithelial-mesenchymal transition (EMT) has been shown to be an important biological process in cancer progression and metastasis. We have focused on elucidating factors that induce EMT to promote carcinogenesis and subsequent metastasis in HCC using the BNL CL.2 (BNL) and BNL 1ME A. 7R.1 (1MEA) cell lines. BNL cells are normal hepatocytes whereas the 1MEA cells are HCC cells derived from chemical transformation of the BNL cells. Their morphological characteristics were examined. Expression levels of hepatocyte growth factor (HGF), markers of EMT and mediators of HGF signaling were determined and functional characteristics were compared. BNL cells were treated with HGF and effects on EMT-marker and mediators of HGF signaling were analyzed. BNL cells display characteristic epithelial morphology whereas 1MEA cells display mesenchymal characteristics. 1MEA cells express and secrete more HGF than BNL cells. There was significantly decreased expression of E-cadherin, albumin, AAT and increased expression of fibronectin, collagen-1, vimentin, snail and slug in 1MEA cells. There was also increased expression of cyclooxygenase-2 (COX-2), Akt and phosphorylated Akt (pAkt) in 1MEA cells. Moreover, 1MEA cells had increased migratory capacity inhibited by inhibition of COX-2 and Akt but not extracellular signal regulated kinase (ERK). Molecular mesenchymal characteristics of 1MEA cells were reversed by inhibition of COX-2, Akt and ERK. Treatment of BNL cells with HGF led to decreased expression of E-cadherin and increased expression of fibronectin, vimentin, snail, slug, COX-2, Akt, pAkt and increased migration, invasiveness and clonogenicity. We conclude that development of HCC is associated with upregulation of HGF which promotes EMT and carcinogenesis via upregulation of COX-2 and Akt. Consequently, HGF signaling may be targeted for therapy in advanced and metastatic HCC.

Suppression of TGF-β pathway by pirfenidone decreases extracellular matrix deposition in ocular fibroblasts in vitro.

PubMed

Stahnke, Thomas; Kowtharapu, Bhavani S; Stachs, Oliver; Schmitz, Klaus-Peter; Wurm, Johannes; Wree, Andreas; Guthoff, Rudolf Friedrich; Hovakimyan, Marina

2017-01-01

In glaucoma surgery, fibrotic processes occur, leading to impairment of liquid outflow. Activated fibroblasts are responsible for postoperative scarring. The transforming growth factor-β (TGF-β) pathway plays a key role in fibroblast function, differentiation and proliferation. The aim of this study was the characterization of the fibrotic potential of two subtypes of primary human ocular fibroblasts and the attempt to inhibit fibrotic processes specifically, without impairing cell viability. For fibrosis inhibition we focused on the small molecule pirfenidone, which has been shown to prevent pulmonary fibrosis by the decrease of the expression of TGF-β1, TGF-β2 and TGF-β3 cytokines. For in vitro examinations, isolated human primary fibroblasts from Tenon capsule and human intraconal orbital fat tissues were used. These fibroblast subpopulations were analyzed in terms of the expression of matrix components responsible for postoperative scarring. We concentrated on the expression of collagen I, III, VI and fibronectin. Additionally, we analyzed the expression of α-smooth muscle actin, which serves as a marker for fibrosis and indicates transformation of fibroblasts into myofibroblasts. Gene expression was analyzed by rtPCR and synthesized proteins were examined by immunofluorescence and Western blot methods. Proliferation of fibroblasts under different culture conditions was assessed using BrdU assay. TGF-β1 induced a significant increase of cell proliferation in both cell types. Also the expression of some fibrotic markers was elevated. In contrast, pirfenidone decreased cell proliferation and matrix synthesis in both fibroblast subpopulations. Pirfenidone slightly attenuated TGF-β1 induced expression of fibronectin and α-smooth muscle actin in fibroblast cultures, without impairing cell viability. To summarize, manipulation of the TGF-β signaling pathway by pirfenidone represents a specific antifibrotic approach with no toxic side effects in two human orbital fibroblast subtypes. We presume that pirfenidone is a promising candidate for the treatment of fibrosis following glaucoma surgery.

Potential role of recombinant secretory leucoprotease inhibitor in the prevention of neutrophil mediated matrix degradation.

PubMed Central

Llewellyn-Jones, C. G.; Lomas, D. A.; Stockley, R. A.

1994-01-01

BACKGROUND--Neutrophil elastase is able to degrade connective tissue matrices and is thought to be involved in the pathogenesis of destructive lung diseases. METHODS--The ability of recombinant secretory leucoprotease inhibitor (rSLPI) to inhibit neutrophil mediated degradation of fibronectin in vitro is demonstrated and its efficacy compared with native alpha-1-proteinase inhibitor (n alpha 1-PI), recombinant alpha-1-proteinase inhibitor (r alpha 1-PI), and the chemical elastase inhibitor ICI 200,355. RESULTS--When preincubated with neutrophils both rSLPI and r alpha 1-PI were effective inhibitors of fibronectin degradation although n alpha 1-PI and ICI 200,355 were less effective. Recombinant SLPI was the most effective inhibitor when the cells were allowed to adhere to fibronectin before the addition of the inhibitors. Preincubation of rSLPI (0.1 mumol/l) with the fibronectin plate resulted in almost total inhibition of fibronectin degradation (reduced to 3.3 (SE 0.9)% of control). Pretreating the fibronectin plate with 1 mumol/l rSLPI, r alpha 1-PI and ICI 200,355 followed by thorough washing before the addition of cells resulted in no inhibition of fibronectin degradation with r alpha 1-PI and the ICI inhibitor, but rSLPI retained its inhibitory effect. This effect could be reduced by adding rSLPI in high pH buffer or 2 mol/1 NaCl. CONCLUSIONS--It is postulated that rSLPI binds to fibronectin to form a protective layer which prevents its degradation by neutrophil elastase. It may prove to be the most useful therapeutic agent in the prevention of neutrophil mediated lung damage. Images PMID:7912452

Novel insights into a retinoic-acid-induced cleft palate based on Rac1 regulation of the fibronectin arrangement.

PubMed

Tang, Qinghuang; Li, Liwen; Lee, Min-Jung; Ge, Qing; Lee, Jong-Min; Jung, Han-Sung

2016-03-01

Retinoic acid (RA)-induced cleft palate results from both extrinsic obstructions by the tongue and internal factors within the palatal shelves. Our previous study showed that the spatiotemporal expression of Rac1 regulates the fibronectin (FN) arrangement through cell density alterations that play an important role in palate development. In this study, we investigate the involvement of the Rac1 regulation of the FN arrangement in RA-induced cleft palate. Our results demonstrate that RA-induced intrinsic alterations in palatal shelves, including a delayed progress of cell condensation, delay palate development, even after the removal of the tongue. Further analysis shows that RA treatment diminishes the region-distinctive expression of Rac1 within the palatal shelves, which reversely alters the fibrillar arrangement of FN. Furthermore, RA treatment disrupts the formation of lamellipodia, which are indicative structures of cell migration that are regulated by Rac1. These results suggest that the Rac1 regulation of the FN arrangement is involved in RA-induced cleft palate through the regulation of cell migration, which delays the progress of cell condensation and subsequently influences the FN arrangement, inducing a delay in palate development. Our study provides new insights into the RA-induced impairment of palatal shelf elevation based on cell migration dynamics.

Comparison of nephron-protective effects of enalapril and GLP analogues (exenatide) in diabetic nephropathy.

PubMed

Çavusoglu, T; Erbas, O; Karadeniz, T; Akdemir, O; Acikgoz, E; Karadeniz, M; Tuglu, M I; Ates, U

2014-06-01

One of the major concerns is a nephropathy in diabetes, which applies many different kinds of medicines. However, required level of the treatment of renal disease has not been achieved. To investigate and compare the effect of the enalapril and the exenatide on diabetic nephropathy in rats developed diabetes by streptozosin. 32 male Sprague Dawley rats were divided into 4 groups: (1) Control, (2) Diabetic (DM), (3) DM+ Enalapril, and (4) DM+ exenatide groups. Then, the animals were euthanized and their blood samples were collected by cardiac puncture for blood glucose; blood urea nitrogen (BUN), creatinin, and nephrectomy were performed for histopathologic examination, and urine samples were taken on stick for proteinuria. Administration of the enalapril or the exenatide in diabetic rats resulted in a significant reduction both fibronectin, induced nitric oxide synthase (i-NOS) expression in glomerular area and urine protein levels. It was shown that both of enalapril and exenatide protected the renal glomerulus more than diabetic group in the nephropathy histopathologically. The beneficial effects of enalapril and exenatide which reduces fibronectin, i-NOS expression and urine protein levels or increases recovery of glomerules, might be used for preventing the harmful effects of diabetic nephropathy. © Georg Thieme Verlag KG Stuttgart · New York.

Expression and putative function of fibronectin and its receptor (integrin alpha(5)beta(1)) in male and female gametes during bovine fertilization in vitro.

PubMed

Thys, Mirjan; Nauwynck, Hans; Maes, Dominiek; Hoogewijs, Maarten; Vercauteren, Dries; Rijsselaere, Tom; Favoreel, Herman; Van Soom, Ann

2009-09-01

Fibronectin (Fn) is a 440 kDa glycoprotein assumed to participate in sperm-egg interaction in human. Recently, it has been demonstrated that Fn--when present during bovine IVF--strongly inhibits sperm penetration. The present study was conducted firstly to evaluate the expression of Fn and its integrin receptor (alpha(5)beta(1)) on male and female bovine gametes using indirect immunofluorescence and secondly, to determine the function of Fn during bovine IVF. Endogenous Fn was detected underneath the zona pellucida (ZP) and integrin alpha(5) on the oolemma of cumulus-denuded oocytes. Bovine spermatozoa displayed integrin alpha(5) at their equatorial segment after acrosome reaction. We established that the main inhibitory effect of exogenously supplemented Fn was located at the sperm-oolemma binding, with a (concurrent) effect on fusion, and this can probably be attributed to the binding of Fn to spermatozoa at the equatorial segment, as shown by means of Alexa Fluor 488-conjugated Fn. Combining these results, the inhibitory effect of exogenously supplemented Fn seemed to be exerted on the male gamete by binding to the exposed integrin alpha(5)beta(1) receptor after acrosome reaction. The presence of endogenous Fn underneath the ZP together with integrin alpha(5) expression on oolemma and acrosome-reacted (AR) sperm cell surface suggests a 'velcro' interaction between the endogenous Fn ligand and corresponding receptors on both (AR) sperm cell and oolemma, initiating sperm-egg binding.

The N-terminal domain of the thermo-regulated surface protein PrpA of Enterococcus faecium binds to fibrinogen, fibronectin and platelets

PubMed Central

Guzmán Prieto, Ana M.; Urbanus, Rolf T.; Zhang, Xinglin; Bierschenk, Damien; Koekman, C. Arnold; van Luit-Asbroek, Miranda; Ouwerkerk, Janneke P.; Pape, Marieke; Paganelli, Fernanda L.; Wobser, Dominique; Huebner, Johannes; Hendrickx, Antoni P. A.; Bonten, Marc J. M.; Willems, Rob J. L.; van Schaik, Willem

2015-01-01

Enterococcus faecium is a commensal of the mammalian gastrointestinal tract, but is also found in non-enteric environments where it can grow between 10 °C and 45 °C. E. faecium has recently emerged as a multi-drug resistant nosocomial pathogen. We hypothesized that genes involved in the colonization and infection of mammals exhibit temperature-regulated expression control and we therefore performed a transcriptome analysis of the clinical isolate E. faecium E1162, during mid-exponential growth at 25 °C and 37 °C. One of the genes that exhibited differential expression between 25 °C and 37 °C, was predicted to encode a peptidoglycan-anchored surface protein. The N-terminal domain of this protein is unique to E. faecium and closely related enterococci, while the C-terminal domain is homologous to the Streptococcus agalactiae surface protein BibA. This region of the protein contains proline-rich repeats, leading us to name the protein PrpA for proline-rich protein A. We found that PrpA is a surface-exposed protein which is most abundant during exponential growth at 37 °C in E. faecium E1162. The heterologously expressed and purified N-terminal domain of PrpA was able to bind to the extracellular matrix proteins fibrinogen and fibronectin. In addition, the N-terminal domain of PrpA interacted with both non-activated and activated platelets. PMID:26675410

The N-terminal domain of the thermo-regulated surface protein PrpA of Enterococcus faecium binds to fibrinogen, fibronectin and platelets.

PubMed

Guzmán Prieto, Ana M; Urbanus, Rolf T; Zhang, Xinglin; Bierschenk, Damien; Koekman, C Arnold; van Luit-Asbroek, Miranda; Ouwerkerk, Janneke P; Pape, Marieke; Paganelli, Fernanda L; Wobser, Dominique; Huebner, Johannes; Hendrickx, Antoni P A; Bonten, Marc J M; Willems, Rob J L; van Schaik, Willem

2015-12-17

Enterococcus faecium is a commensal of the mammalian gastrointestinal tract, but is also found in non-enteric environments where it can grow between 10 °C and 45 °C. E. faecium has recently emerged as a multi-drug resistant nosocomial pathogen. We hypothesized that genes involved in the colonization and infection of mammals exhibit temperature-regulated expression control and we therefore performed a transcriptome analysis of the clinical isolate E. faecium E1162, during mid-exponential growth at 25 °C and 37 °C. One of the genes that exhibited differential expression between 25 °C and 37 °C, was predicted to encode a peptidoglycan-anchored surface protein. The N-terminal domain of this protein is unique to E. faecium and closely related enterococci, while the C-terminal domain is homologous to the Streptococcus agalactiae surface protein BibA. This region of the protein contains proline-rich repeats, leading us to name the protein PrpA for proline-rich protein A. We found that PrpA is a surface-exposed protein which is most abundant during exponential growth at 37 °C in E. faecium E1162. The heterologously expressed and purified N-terminal domain of PrpA was able to bind to the extracellular matrix proteins fibrinogen and fibronectin. In addition, the N-terminal domain of PrpA interacted with both non-activated and activated platelets.

Chemical Chaperone of Endoplasmic Reticulum Stress Inhibits Epithelial-Mesenchymal Transition Induced by TGF-β1 in Airway Epithelium via the c-Src Pathway

PubMed Central

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

2017-01-01

Epithelial-mesenchymal transition (EMT) is a biological process that allows epithelial cells to assume a mesenchymal cell phenotype. EMT is considered as a therapeutic target for several persistent inflammatory airway diseases related to tissue remodeling. Herein, we investigated the role of endoplasmic reticulum (ER) stress and c-Src in TGF-β1-induced EMT. A549 cells, primary nasal epithelial cells (PNECs), and inferior nasal turbinate organ cultures were exposed to 4-phenylbutylic acid (4PBA) or PP2 and then stimulated with TGF-β1. We found that E-cadherin, vimentin, fibronectin, and α-SMA expression was increased in nasal polyps compared to inferior turbinates. TGF-β1 increased the expression of EMT markers such as E-cadherin, fibronectin, vimentin, and α-SMA and ER stress markers (XBP-1s and GRP78), an effect that was blocked by PBA or PP2 treatment. 4-PBA and PP2 also blocked the effect of TGF-β1 on migration of A549 cells and suppressed TGF-β1-induced expression of EMT markers in PNECs and organ cultures of inferior turbinate. In conclusion, we demonstrated that 4PBA inhibits TGF-β1-induced EMT via the c-Src pathway in A549 cells, PNECs, and inferior turbinate organ cultures. These results suggest an important role for ER stress and a diverse role for TGF-β1 in upper airway chronic inflammatory disease such as CRS. PMID:28804222

Chemical Chaperone of Endoplasmic Reticulum Stress Inhibits Epithelial-Mesenchymal Transition Induced by TGF-β1 in Airway Epithelium via the c-Src Pathway.

PubMed

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

2017-01-01

Epithelial-mesenchymal transition (EMT) is a biological process that allows epithelial cells to assume a mesenchymal cell phenotype. EMT is considered as a therapeutic target for several persistent inflammatory airway diseases related to tissue remodeling. Herein, we investigated the role of endoplasmic reticulum (ER) stress and c-Src in TGF- β 1-induced EMT. A549 cells, primary nasal epithelial cells (PNECs), and inferior nasal turbinate organ cultures were exposed to 4-phenylbutylic acid (4PBA) or PP2 and then stimulated with TGF- β 1. We found that E-cadherin, vimentin, fibronectin, and α -SMA expression was increased in nasal polyps compared to inferior turbinates. TGF- β 1 increased the expression of EMT markers such as E-cadherin, fibronectin, vimentin, and α -SMA and ER stress markers (XBP-1s and GRP78), an effect that was blocked by PBA or PP2 treatment. 4-PBA and PP2 also blocked the effect of TGF- β 1 on migration of A549 cells and suppressed TGF- β 1-induced expression of EMT markers in PNECs and organ cultures of inferior turbinate. In conclusion, we demonstrated that 4PBA inhibits TGF- β 1-induced EMT via the c-Src pathway in A549 cells, PNECs, and inferior turbinate organ cultures. These results suggest an important role for ER stress and a diverse role for TGF- β 1 in upper airway chronic inflammatory disease such as CRS.

Granzyme B mediates both direct and indirect cleavage of extracellular matrix in skin after chronic low-dose ultraviolet light irradiation

PubMed Central

Parkinson, Leigh G; Toro, Ana; Zhao, Hongyan; Brown, Keddie; Tebbutt, Scott J; Granville, David J

2015-01-01

Extracellular matrix (ECM) degradation is a hallmark of many chronic inflammatory diseases that can lead to a loss of function, aging, and disease progression. Ultraviolet light (UV) irradiation from the sun is widely considered as the major cause of visible human skin aging, causing increased inflammation and enhanced ECM degradation. Granzyme B (GzmB), a serine protease that is expressed by a variety of cells, accumulates in the extracellular milieu during chronic inflammation and cleaves a number of ECM proteins. We hypothesized that GzmB contributes to ECM degradation in the skin after UV irradiation through both direct cleavage of ECM proteins and indirectly through the induction of other proteinases. Wild-type and GzmB-knockout mice were repeatedly exposed to minimal erythemal doses of solar-simulated UV irradiation for 20 weeks. GzmB expression was significantly increased in wild-type treated skin compared to nonirradiated controls, colocalizing to keratinocytes and to an increased mast cell population. GzmB deficiency significantly protected against the formation of wrinkles and the loss of dermal collagen density, which was related to the cleavage of decorin, an abundant proteoglycan involved in collagen fibrillogenesis and integrity. GzmB also cleaved fibronectin, and GzmB-mediated fibronectin fragments increased the expression of collagen-degrading matrix metalloproteinase-1 (MMP-1) in fibroblasts. Collectively, these findings indicate a significant role for GzmB in ECM degradation that may have implications in many age-related chronic inflammatory diseases. PMID:25495009

Progression of renal fibrosis in congenital CKD model rats with reduced number of nephrons.

PubMed

Yasuda, Hidenori; Tochigi, Yuki; Katayama, Kentaro; Suzuki, Hiroetsu

2017-06-14

A congenital reduction in the number of nephrons is a critical risk factor for both onset of chronic kidney disease (CKD) and its progression to end-stage kidney disease (ESKD). Hypoplastic kidney (HPK) rats have only about 20% of the normal number of nephrons and show progressive CKD. This study used an immunohistological method to assess glomerular and interstitial pathogenesis in male HPK rats aged 35-210days. CD68 positive-macrophages were found to infiltrate into glomeruli in HPK rats aged 35 and 70days and to infiltrate into interstitial tissue in rats aged 140 and 210days. HPK rats aged 35 and 70days showed glomerular hypertrophy, loss of normal linear immunostaining of podocine, and increased expression of PDGFr-β, TGF-β, collagens, and fibronectin, with all of these alterations gradually deteriorating with age. α-SMA-positive myofibroblasts were rarely detected in glomerular tufts, whereas α-SMA-positive glomerular parietal epithelium (GPE) cells were frequently observed along Bowman's capsular walls. The numbers of PDGFr-β-positive fibroblasts in interstitial tissue were increased in rats aged 35days and older, whereas interstitial fibrosis, characterized by the increased expression of tubular PDGF-BB, the appearance of myofibroblasts doubly positive for PDGFr-β and α-SMA, and increased expression of collagens and fibronectin, were observed in rats aged 70 and older. These results clearly indicate that congenital CKD with only 20% of nephrons cause renal fibrosis in rats. Copyright © 2017 Elsevier GmbH. All rights reserved.

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.

Fibronectins containing extradomain A or B enhance osteoblast differentiation via distinct integrins

PubMed Central

Sens, Carla; Huck, Katrin; Pettera, Stefan; Uebel, Stephan; Wabnitz, Guido; Moser, Markus; Nakchbandi, Inaam A.

2017-01-01

Fibronectin is a multidomain protein secreted by various cell types. It forms a network of fibers within the extracellular matrix and impacts intracellular processes by binding to various molecules, primarily integrin receptors on the cells. Both the presence of several isoforms and the ability of the various domains and isoforms to bind to a variety of integrins result in a wide range of effects. In vivo findings suggest that fibronectin isoforms produced by the osteoblasts enhance their differentiation. Here we report that the isoform characterized by the presence of extradomain A activates α4β1 integrin and augments osteoblast differentiation. In addition, the isoform containing extradomain B enhances the binding of fibronectin through the RGD sequence to β3-containing integrin, resulting in increased mineralization by and differentiation of osteoblasts. Our study thus reveals novel functions for two fibronectin isoforms and the mediating receptors in osteoblast differentiation. PMID:28325836

The EphA8 Receptor Regulates Integrin Activity through p110γ Phosphatidylinositol-3 Kinase in a Tyrosine Kinase Activity-Independent Manner

PubMed Central

Gu, Changkyu; Park, Soochul

2001-01-01

Recent genetic studies suggest that ephrins may function in a kinase-independent Eph receptor pathway. Here we report that expression of EphA8 in either NIH 3T3 or HEK293 cells enhanced cell adhesion to fibronectin via α5β1- or β3 integrins. Interestingly, a kinase-inactive EphA8 mutant also markedly promoted cell attachment to fibronectin in these cell lines. Using a panel of EphA8 point mutants, we have demonstrated that EphA8 kinase activity does not correlate with its ability to promote cell attachment to fibronectin. Analysis using EphA8 extracellular and intracellular domain mutants has revealed that enhanced cell adhesion is dependent on ephrin A binding to the extracellular domain and the juxtamembrane segment of the cytoplasmic domain of the receptor. EphA8-promoted adhesion was efficiently inhibited by wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor. Additionally, we found that EphA8 had associated PI 3-kinase activity and that the p110γ isoform of PI 3-kinase is associated with EphA8. In vitro binding experiments revealed that the EphA8 juxtamembrane segment was sufficient for the formation of a stable complex with p110γ. Similar results were obtained in assay using cells stripped of endogenous ephrin A ligands by treatment with preclustered ephrin A5-Fc proteins. In addition, a membrane-targeted lipid kinase-inactive p110γ mutant was demonstrated to stably associate with EphA8 and suppress EphA8-promoted cell adhesion to fibronectin. Taken together, these results suggest the presence of a novel mechanism by which the EphA8 receptor localizes p110γ PI 3-kinase to the plasma membrane in a tyrosine kinase-independent fashion, thereby allowing access to lipid substrates to enable the signals required for integrin-mediated cell adhesion. PMID:11416136

Gene editing of the extra domain A positive fibronectin in various tumors, amplified the effects of CRISPR/Cas system on the inhibition of tumor progression.

PubMed

Lv, Wan-Qi; Wang, Hai-Cheng; Peng, Jing; Wang, Yi-Xiang; Jiang, Jiu-Hui; Li, Cui-Ying

2017-12-01

The low efficiency of clustered, regularly interspaced, palindromic repeats-associated Cas (CRISPR/Cas) system editing genes in vivo limits the application. A components of the extracellular matrix (ECM), the extra domain A positive fibronectin (EDA+FN), may be a target for CRISPR/Cas system for the pro-oncogenic effects. The exclusion of EDA exon would alter the microenvironment and inhibit tumor progression, even the frequency of gene editing is still limited. The pro-oncogenic effects were confirmed by the exclusion of EDA exon from the fibronectin gene, as illustrated by the down-regulated proliferation, migration and invasion of CNE-2Z or SW480 cells (P<0.05). Furthermore, although the efficacy of EDA exon knockout through CRISPR/Cas system was shown to be low in vivo , the EDA+FN protein levels decrease obviously, inhibiting the tumor growth rate significantly (P<0.05), which was accompanied by a decrease in Ki-67 expression and microvessel numbers, and increased E-cadherin or decreased Vimentin expression (P<0.05). Human nasopharyngeal carcinoma cell line CNE-2Z, and the colorectal carcinoma cell line SW480 were transfected with CRISPR/Cas9 plasmids targeting EDA exon. The effects of the exclusion of EDA on the cell proliferation, motility and epithelial-mesenchymal transition (EMT) were investigated, and the western blot and real-time PCR were performed to analyze the underlying mechanisms. Furthermore, CRISPR/Cas9 plasmids were injected into xenograft tumors to knockout EDA exon in vivo , and tumor growth, cell proliferation, EMT rate, or vascularization were investigated using western blot, PCR and immunohistochemistry. CRISPR/Cas system targeting ECM components was shown to be an effective method for the inhibition of tumor progression, as these paracrine or autocrine molecules are necessary for various tumor cells. This may represent a novel strategy for overcoming the drug evasion or resistance, in addition, circumventing the low efficiency of CRISPR/Cas system in vivo .

Human Fibronectin Extra-Domain B (EDB)-Specific Aptide (APTEDB) Radiolabelling with Technetium-99m as a Potent Targeted Tumour-Imaging Agent.

PubMed

Mohammadgholi, Mohsen; Sadeghzadeh, Nourollah; Erfani, Mostafa; Abediankenari, Saeid; Abedi, Seyed Mohammad; Emrarian, Iman; Jafari, Narjes; Behzadi, Ramezan

2018-01-01

Human fibronectin extra-domain B (EDB) is particularly expressed during angiogenesis progression. It is, thus, a promising marker of tumour growth. Aptides are a novel class of peptides with high-affinity binding to specific protein targets. APTEDB is an antagonist-like ligand that especially interacts with human fibronectin EDB. This study was the first attempt in which the hydrazinonicotinamide (HYNIC)-conjugated APTEDB was labelled with technetium-99m (99mTc) as an appropriate radiotracer and tricine/EDDA exchange labeling. Radiochemical purity, normal saline, and serum stability were evaluated by HPLC and radio-isotope TLC scanner. Other examinations, such as protein-binding calculation, dissociation radioligand binding assay, and partition coefficient constant determination, were also carried out. The cellular-specific binding of 99mTc- HYNIC-conjugated APTEDB was assessed in two EDB-positive (U87MG) and EDB-negative (U373MG) cell lines. Bio-distribution was investigated in normal mice as well as in U87MG and U373MG tumour-bearing mice. Eventually, the radiolabelled APTEDB was used for tumour imaging using planar SPECT. Radiolabelling was achieved with high purity (up to 97%) and accompanied by high solution (over 90% after overnight) and serum (80% after 2 hours) stability. The obtained cellular-specific binding ratio was greater than nine-fold. In-vivo experiments showed rapid blood clearance with mainly renal excretion and tumour uptake specificity (0.48±0.03% ID/g after 1h). The results of the imaging also confirmed considerable tumour uptake for EDB-positive cell line compared with the EDB-negative one. Aptides are considered to be a potent candidate for biopharmaceutical applications. They can be modified with imaging or therapeutic agents. This report shows the capability of 99mTc-HYNIC-APTEDB for human EDB-expressing tumours detection. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

The multifunctional LigB adhesin binds homeostatic proteins with potential roles in cutaneous infection by pathogenic Leptospira interrogans.

PubMed

Choy, Henry A; Kelley, Melissa M; Croda, Julio; Matsunaga, James; Babbitt, Jane T; Ko, Albert I; Picardeau, Mathieu; Haake, David A

2011-02-09

Leptospirosis is a potentially fatal zoonotic disease in humans and animals caused by pathogenic spirochetes, such as Leptospira interrogans. The mode of transmission is commonly limited to the exposure of mucous membrane or damaged skin to water contaminated by leptospires shed in the urine of carriers, such as rats. Infection occurs during seasonal flooding of impoverished tropical urban habitats with large rat populations, but also during recreational activity in open water, suggesting it is very efficient. LigA and LigB are surface localized proteins in pathogenic Leptospira strains with properties that could facilitate the infection of damaged skin. Their expression is rapidly induced by the increase in osmolarity encountered by leptospires upon transition from water to host. In addition, the immunoglobulin-like repeats of the Lig proteins bind proteins that mediate attachment to host tissue, such as fibronectin, fibrinogen, collagens, laminin, and elastin, some of which are important in cutaneous wound healing and repair. Hemostasis is critical in a fresh injury, where fibrinogen from damaged vasculature mediates coagulation. We show that fibrinogen binding by recombinant LigB inhibits fibrin formation, which could aid leptospiral entry into the circulation, dissemination, and further infection by impairing healing. LigB also binds fibroblast fibronectin and type III collagen, two proteins prevalent in wound repair, thus potentially enhancing leptospiral adhesion to skin openings. LigA or LigB expression by transformation of a nonpathogenic saprophyte, L. biflexa, enhances bacterial adhesion to fibrinogen. Our results suggest that by binding homeostatic proteins found in cutaneous wounds, LigB could facilitate leptospirosis transmission. Both fibronectin and fibrinogen binding have been mapped to an overlapping domain in LigB comprising repeats 9-11, with repeat 11 possibly enhancing binding by a conformational effect. Leptospirosis patient antibodies react with the LigB domain, suggesting applications in diagnosis and vaccines that are currently limited by the strain-specific leptospiral lipopolysaccharide coats.

The Multifunctional LigB Adhesin Binds Homeostatic Proteins with Potential Roles in Cutaneous Infection by Pathogenic Leptospira interrogans

PubMed Central

Choy, Henry A.; Kelley, Melissa M.; Croda, Julio; Matsunaga, James; Babbitt, Jane T.; Ko, Albert I.; Picardeau, Mathieu; Haake, David A.

2011-01-01

Leptospirosis is a potentially fatal zoonotic disease in humans and animals caused by pathogenic spirochetes, such as Leptospira interrogans. The mode of transmission is commonly limited to the exposure of mucous membrane or damaged skin to water contaminated by leptospires shed in the urine of carriers, such as rats. Infection occurs during seasonal flooding of impoverished tropical urban habitats with large rat populations, but also during recreational activity in open water, suggesting it is very efficient. LigA and LigB are surface localized proteins in pathogenic Leptospira strains with properties that could facilitate the infection of damaged skin. Their expression is rapidly induced by the increase in osmolarity encountered by leptospires upon transition from water to host. In addition, the immunoglobulin-like repeats of the Lig proteins bind proteins that mediate attachment to host tissue, such as fibronectin, fibrinogen, collagens, laminin, and elastin, some of which are important in cutaneous wound healing and repair. Hemostasis is critical in a fresh injury, where fibrinogen from damaged vasculature mediates coagulation. We show that fibrinogen binding by recombinant LigB inhibits fibrin formation, which could aid leptospiral entry into the circulation, dissemination, and further infection by impairing healing. LigB also binds fibroblast fibronectin and type III collagen, two proteins prevalent in wound repair, thus potentially enhancing leptospiral adhesion to skin openings. LigA or LigB expression by transformation of a nonpathogenic saprophyte, L. biflexa, enhances bacterial adhesion to fibrinogen. Our results suggest that by binding homeostatic proteins found in cutaneous wounds, LigB could facilitate leptospirosis transmission. Both fibronectin and fibrinogen binding have been mapped to an overlapping domain in LigB comprising repeats 9–11, with repeat 11 possibly enhancing binding by a conformational effect. Leptospirosis patient antibodies react with the LigB domain, suggesting applications in diagnosis and vaccines that are currently limited by the strain-specific leptospiral lipopolysaccharide coats. PMID:21347378

Osteograft, plastic material for regenerative medicine

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

Zaidman, A. M., E-mail: AZaydman@niito.ru; Korel, A. V., E-mail: AKorel@niito.ru; Shchelkunova, E. I., E-mail: EShelkunova@niito.ru

Creating tissue-engineering constructs based on the mechanism of cartilage-bone evolution is promising for traumatology and orthopedics. Such a graft was obtained from a chondrograft by transdifferentiation. The hondrograft placed in osteogenic medium is undergoing osteogenic differentiation for 14–30 days. Tissue specificity of the osteograft was studied by morphology, immunohistochemistry, electron microscopy, and the expression of the corresponding genes was estimated. The expression of osteonectin, fibronectin, collagen of type I, izolektin and CD 44 is determined. Alkaline phosphatase and matrix vesicles are determined in osteoblasts. Calcificates are observed in the matrix. Chondrogenic proteins expression is absent. These findings evidence the tissuemore » specificity of the developed osteograft.« less

Fibronectin gene polymorphism in patients with lung cancer.

PubMed

Siemianowicz, K; Gminski, J; Francuz, T; Syzdol, M; Polanska, D; Machalski, M; Brulinski, K; Magiera-Molendowska, H

2001-01-01

Fibronectin (FN) is a glycoprotein component of connective tissue. It is involved in cancer progression. FN plays a role in non-neoplasmatic lung pathology in which fibronectin gene polymorphisms (RFLPs) have been studied. The aim of our work was to evaluate the frequency of two of fibronectin RFLPs: genotypes AB, AA, BB (HaeIII) and CD, CC, DD (MspI) in patients with lung cancer. The studied group consisted of 63 patients with squamous cell lung cancer and 53 controls without any malignant or proliferative disease. There were no statistically significant differences in the distribution of studied genotypes between lung cancer patients and controls.

GD1a Overcomes Inhibition of Myelination by Fibronectin via Activation of Protein Kinase A: Implications for Multiple Sclerosis.

PubMed

Qin, Jing; Sikkema, Arend H; van der Bij, Kristine; de Jonge, Jenny C; Klappe, Karin; Nies, Vera; Jonker, Johan W; Kok, Jan Willem; Hoekstra, Dick; Baron, Wia

2017-10-11

Remyelination failure by oligodendrocytes contributes to the functional impairment that characterizes the demyelinating disease multiple sclerosis (MS). Since incomplete remyelination will irreversibly damage axonal connections, treatments effectively promoting remyelination are pivotal in halting disease progression. Our previous findings suggest that fibronectin aggregates, as an environmental factor, contribute to remyelination failure by perturbing oligodendrocyte progenitor cell (OPC) maturation. Here, we aim at elucidating whether exogenously added gangliosides (i.e., cell surface lipids with a potential to modulate signaling pathways) could counteract fibronectin-mediated inhibition of OPC maturation. Exclusive exposure of rat oligodendrocytes to GD1a, but not other gangliosides, overcomes aggregated fibronectin-induced inhibition of myelin membrane formation, in vitro , and OPC differentiation in fibronectin aggregate containing cuprizone-induced demyelinated lesions in male mice. GD1a exerts its effect on OPCs by inducing their proliferation and, at a late stage, by modulating OPC maturation. Kinase activity profiling revealed that GD1a activated a protein kinase A (PKA)-dependent signaling pathway and increased phosphorylation of the transcription factor cAMP response element-binding protein. Consistently, the effect of GD1a in restoring myelin membrane formation in the presence of fibronectin aggregates was abolished by the PKA inhibitor H89, whereas the effect of GD1a was mimicked by the PKA activator dibutyryl-cAMP. Together, GD1a overcomes the inhibiting effect of aggregated fibronectin on OPC maturation by activating a PKA-dependent signaling pathway. Given the persistent presence of fibronectin aggregates in MS lesions, ganglioside GD1a might act as a potential novel therapeutic tool to selectively modulate the detrimental signaling environment that precludes remyelination. SIGNIFICANCE STATEMENT As an environmental factor, aggregates of the extracellular matrix protein fibronectin perturb the maturation of oligodendrocyte progenitor cells (OPCs), thereby impeding remyelination, in the demyelinating disease multiple sclerosis (MS). Here we demonstrate that exogenous addition of ganglioside GD1a overcomes the inhibiting effect of aggregated fibronectin on OPC maturation, both in vitro and in vivo , by activating a PKA-dependent signaling pathway. We propose that targeted delivery of GD1a to MS lesions may act as a potential novel molecular tool to boost maturation of resident OPCs to overcome remyelination failure and halt disease progression. Copyright © 2017 the authors 0270-6474/17/379925-14$15.00/0.

Sphingosine kinase 1 mediates AGEs-induced fibronectin upregulation in diabetic nephropathy.

PubMed

Chen, Cheng; Gong, Wenyan; Li, Changzheng; Xiong, Fengxiao; Wang, Shaogui; Huang, Junying; Wang, Yu; Chen, Zhiquan; Chen, Qiuhong; Liu, Peiqing; Lan, Tian; Huang, Heqing

2017-10-03

Activation of sphingosine kinase 1 (SphK1) signaling pathway mediates fibronectin (FN) upregulation in glomerular mesangial cells (GMCs) under high glucose (HG) condition. However, the roles of SphK1 in advanced glycation end products (AGEs)-induced DN have not been elucidated. Here we show that AGEs upregulated FN and SphK1 and SphK1 activity. Inhibition of SphK1 signaling attenuated AGEs-induced FN synthesis in GMCs. Inhibition of AGE receptor (RAGE) signaling reduced the upregulation of FN and SphK1 and SphK1 activity in GMCs induced by AGEs. Treatment of aminoguanidine ameliorates the renal injury and fibrosis in STZ-induced diabetic mice and attenuated SphK1 expression and activity in diabetic mouse kidneys. The renal injury and fibrosis in diabetic SphK1 -/- mice was significantly attenuated than WT mice. Furthermore, AGEs upregulated SphK1 by reducing its degradation and prolonging its half-life. SphK1 mediates AGEs-induced FN synthesis in GMCs and diabetic mice under hyperglycemic condition .

Cranial neural crest recycle surface integrins in a substratum-dependent manner to promote rapid motility.

PubMed

Strachan, Lauren R; Condic, Maureen L

2004-11-08

Cell migration is essential for proper development of numerous structures derived from embryonic neural crest cells (NCCs). Although the migratory pathways of NCCs have been determined, the molecular mechanisms regulating NCC motility remain unclear. NCC migration is integrin dependent, and recent work has shown that surface expression levels of particular integrin alpha subunits are important determinants of NCC motility in vitro. Here, we provide evidence that rapid cranial NCC motility on laminin requires integrin recycling. NCCs showed both ligand- and receptor-specific integrin regulation in vitro. On laminin, NCCs accumulated internalized laminin but not fibronectin receptors over 20 min, whereas on fibronectin neither type of receptor accumulated internally beyond 2 min. Internalized laminin receptors colocalized with receptor recycling vesicles and were subsequently recycled back to the cell surface. Blocking receptor recycling with bafilomycin A inhibited NCC motility on laminin, indicating that substratum-dependent integrin recycling is essential for rapid cranial neural crest migration.

Predictive value of cervical length measurement and fibronectin testing in threatened preterm labor.

PubMed

van Baaren, Gert-Jan; Vis, Jolande Y; Wilms, Femke F; Oudijk, Martijn A; Kwee, Anneke; Porath, Martina M; Oei, Guid; Scheepers, Hubertina C J; Spaanderman, Marc E A; Bloemenkamp, Kitty W M; Haak, Monique C; Bolte, Antoinette C; Bax, Caroline J; Cornette, Jérôme M J; Duvekot, Johannes J; Nij Bijvanck, Bas W A; van Eyck, Jim; Franssen, Maureen T M; Sollie, Krystyna M; Vandenbussche, Frank P H A; Woiski, Mallory; Grobman, William A; van der Post, Joris A M; Bossuyt, Patrick M M; Opmeer, Brent C; Mol, Ben W J

2014-06-01

To estimate the performance of combining cervical length measurement with fetal fibronectin testing in predicting delivery in women with symptoms of preterm labor. We conducted a prospective nationwide cohort study in all 10 perinatal centers in The Netherlands. Women with symptoms of preterm labor between 24 and 34 weeks of gestation with intact membranes were included. In all women, qualitative fibronectin testing (0.050-microgram/mL cutoff) and cervical length measurement were performed. Logistic regression was used to predict spontaneous preterm delivery within 7 days after testing. A risk less than 5%, corresponding to the risk for women with a cervical length of at least 25 mm, was considered as low risk. Between December 2009 and August 2012, 714 women were enrolled. Fibronectin results and cervical length were available for 665 women, of whom 80 (12%) delivered within 7 days. Women with a cervical length of at least 30 mm or with a cervical length between 15 and 30 mm with a negative fibronectin result were at low risk (less than 5%) of spontaneous delivery within 7 days. Fibronectin testing in case of a cervical length between 15 and 30 mm additionally classified 103 women (15% of the cohort) as low risk and 36 women (5% of the cohort) as high risk. Cervical length measurement, combined with fetal fibronectin testing in case of a cervical length between 15 and 30 mm, improves identification of women with a low risk to deliver spontaneously within 7 days. II.

Sequestration of latent TGF-β binding protein 1 into CADASIL-related Notch3-ECD deposits.

PubMed

Kast, Jessica; Hanecker, Patrizia; Beaufort, Nathalie; Giese, Armin; Joutel, Anne; Dichgans, Martin; Opherk, Christian; Haffner, Christof

2014-08-13

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) represents the most common hereditary form of cerebral small vessel disease characterized by early-onset stroke and premature dementia. It is caused by mutations in the transmembrane receptor Notch3, which promote the aggregation and accumulation of the Notch3 extracellular domain (Notch3-ECD) within blood vessel walls. This process is believed to mediate the abnormal recruitment and dysregulation of additional factors including extracellular matrix (ECM) proteins resulting in brain vessel dysfunction. Based on recent evidence indicating a role for the transforming growth factor-β (TGF-β) pathway in sporadic and familial small vessel disease we studied fibronectin, fibrillin-1 and latent TGF-β binding protein 1 (LTBP-1), three ECM constituents involved in the regulation of TGF-β bioavailability, in post-mortem brain tissue from CADASIL patients and control subjects. Fibronectin and fibrillin-1 were found to be enriched in CADASIL vessels without co-localizing with Notch3-ECD deposits, likely as a result of fibrotic processes secondary to aggregate formation. In contrast, LTBP-1 showed both an accumulation and a striking co-localization with Notch3-ECD deposits suggesting specific recruitment into aggregates. We also detected increased levels of the TGF-β prodomain (also known as latency-associated peptide, LAP) indicating dysregulation of the TGF-β pathway in CADASIL development. In vitro analyses revealed a direct interaction between LTBP-1 and Notch3-ECD and demonstrated a specific co-aggregation of LTBP-1 with mutant Notch3. We propose LTBP-1 as a novel component of Notch3-ECD deposits and suggest its involvement in pathological processes triggered by Notch3-ECD aggregation.

Neuropeptides, via specific receptors, regulate T cell adhesion to fibronectin.

PubMed

Levite, M; Cahalon, L; Hershkoviz, R; Steinman, L; Lider, O

1998-01-15

The ability of T cells to adhere to and interact with components of the blood vessel walls and the extracellular matrix is essential for their extravasation and migration into inflamed sites. We have found that the beta1 integrin-mediated adhesion of resting human T cells to fibronectin, a major glycoprotein component of the extracellular matrix, is induced by physiologic concentrations of three neuropeptides: calcitonin gene-related protein (CGRP), neuropeptide Y, and somatostatin; each acts via its own specific receptor on the T cell membrane. In contrast, substance P (SP), which coexists with CGRP in the majority of peripheral endings of sensory nerves, including those innervating the lymphoid organs, blocks T cell adhesion to fibronectin when induced by CGRP, neuropeptide Y, somatostatin, macrophage inflammatory protein-1beta, and PMA. Inhibition of T cell adhesion was obtained both by the intact SP peptide and by its 1-4 N-terminal and its 4-11, 5-11, and 6-11 C-terminal fragments, used at similar nanomolar concentrations. The inhibitory effects of the parent SP peptide and its fragments were abrogated by an SP NK-1 receptor antagonist, suggesting they all act through the same SP NK-1 receptor. These findings suggest that neuropeptides, by activating their specific T cell-expressed receptors, can provide the T cells with both positive (proadhesive) and negative (antiadhesive) signals and thereby regulate their function. Thus, neuropeptides may influence diverse physiologic processes involving integrins, including leukocyte-mediated migration and inflammation.

Bcr-Abl induces abnormal cytoskeleton remodeling, beta1 integrin clustering and increased cell adhesion to fibronectin through the Abl interactor 1 pathway.

PubMed

Li, Yingzhu; Clough, Nancy; Sun, Xiaolin; Yu, Weidong; Abbott, Brian L; Hogan, Christopher J; Dai, Zonghan

2007-04-15

Hematopoietic cells isolated from patients with Bcr-Abl-positive leukemia exhibit multiple abnormalities of cytoskeletal and integrin function. These abnormalities are thought to play a role in the pathogenesis of leukemia; however, the molecular events leading to these abnormalities are not fully understood. We show here that the Abi1 pathway is required for Bcr-Abl to stimulate actin cytoskeleton remodeling, integrin clustering and cell adhesion. Expression of Bcr-Abl induces tyrosine phosphorylation of Abi1. This is accompanied by a subcellular translocation of Abi1/WAVE2 to a site adjacent to membrane, where an F-actin-enriched structure containing the adhesion molecules such as beta1-integrin, paxillin and vinculin is assembled. Bcr-Abl-induced membrane translocation of Abi1/WAVE2 requires direct interaction between Abi1 and Bcr-Abl, but is independent of the phosphoinositide 3-kinase pathway. Formation of the F-actin-rich complex correlates with an increased cell adhesion to fibronectin. More importantly, disruption of the interaction between Bcr-Abl and Abi1 by mutations either in Bcr-Abl or Abi1 not only abolished tyrosine phosphorylation of Abi1 and membrane translocation of Abi1/WAVE2, but also inhibited Bcr-Abl-stimulated actin cytoskeleton remodeling, integrin clustering and cell adhesion to fibronectin. Together, these data define Abi1/WAVE2 as a downstream pathway that contributes to Bcr-Abl-induced abnormalities of cytoskeletal and integrin function.

Placental Alpha Microglobulin-1 Compared With Fetal Fibronectin to Predict Preterm Delivery in Symptomatic Women.

PubMed

Wing, Deborah A; Haeri, Sina; Silber, Angela C; Roth, Cheryl K; Weiner, Carl P; Echebiri, Nelson C; Franco, Albert; Pappas, Lanissa M; Yeast, John D; Brebnor, Angelle A; Quirk, J Gerald; Murphy, Aisling M; Laurent, Louise C; Field, Nancy T; Norton, Mary E

2017-12-01

To compare the rapid bedside test for placental α microglobulin-1 with the instrumented fetal fibronectin test for prediction of imminent spontaneous preterm delivery among women with symptoms of preterm labor. We conducted a prospective observational study on pregnant women with signs or symptoms suggestive of preterm labor between 24 and 35 weeks of gestation with intact membranes and cervical dilatation less than 3 cm. Participants were prospectively enrolled at 15 U.S. academic and community centers. Placental α microglobulin-1 samples did not require a speculum examination. Health care providers were blinded to placental α microglobulin-1 results. Fetal fibronectin samples were collected through speculum examination per manufacturer requirements and sent to a certified laboratory for testing using a cutoff of 50 ng/mL. The coprimary endpoints were positive predictive value (PPV) superiority and negative predictive value (NPV) noninferiority of placental α microglobulin-1 compared with fetal fibronectin for the prediction of spontaneous preterm birth within 7 days and within 14 days. Of 796 women included in the study cohort, 711 (89.3%) had both placental α microglobulin-1 and fetal fibronectin results and valid delivery outcomes available for analysis. The overall rate of preterm birth was 2.4% (17/711) within 7 days of testing and 4.2% (30/711) within 14 days of testing with respective rates of spontaneous preterm birth of 1.3% (9/703) and 2.9% (20/701). Fetal fibronectin was detected in 15.5% (110/711), and placental α microglobulin-1 was detected in 2.4% (17/711). The PPVs for spontaneous preterm delivery within 7 days or less among singleton gestations (n=13) for placental α microglobulin-1 and fetal fibronectin were 23.1% (3/13) and 4.3% (4/94), respectively (P<.025 for superiority). The NPVs were 99.5% (619/622) and 99.6% (539/541) for placental α microglobulin-1 and fetal fibronectin, respectively (P<.001 for noninferiority). Although placental α microglobulin-1 performed the same as fetal fibronectin in ruling out spontaneous preterm delivery among symptomatic women, it demonstrated statistical superiority in predicting it.

Cellular viability and genetic expression of human gingival fibroblasts to zirconia with enamel matrix derivative (Emdogain®)

PubMed Central

Kwon, Yong-Dae; Choi, Hyun-jung; Lee, Heesu; Lee, Jung-Woo; Weber, Hans-Peter

2014-01-01

PURPOSE The objective of this study was to investigate the biologic effects of enamel matrix derivative (EMD) with different concentrations on cell viability and the genetic expression of human gingival fibroblasts (HGF) to zirconia surfaces. MATERIALS AND METHODS Immortalized human gingival fibroblasts (HGF) were cultured (1) without EMD, (2) with EMD 25 µg/mL, and (3) with EMD 100 µg/mL on zirconia discs. MTT assay was performed to evaluate the cell proliferation activity and SEM was carried out to examine the cellular morphology and attachment. The mRNA expression of collagen type I, osteopontin, fibronectin, and TGF-β1 was evaluated with the real-time polymerase chain reaction (RT-PCR). RESULTS From MTT assay, HGF showed more proliferation in EMD 25 µg/mL group than control and EMD 100 µg/mL group (P<.05). HGFs showed more flattened cellular morphology on the experimental groups than on the control group after 4h culture and more cellular attachments were observed on EMD 25 µg/mL group and EMD 100 µg/mL group after 24h culture. After 48h of culture, cellular attachment was similar in all groups. The mRNA expression of type I collagen increased in a concentration dependent manner. The genetic expression of osteopontin, fibronectin, and TGF-β1 was increased at EMD 100 µg/mL. However, the mRNA expression of proteins associated with cellular attachment was decreased at EMD 25 µg/mL. CONCLUSION Through this short term culture of HGF on zirconium discs, we conclude that EMD affects the proliferation, attachment, and cell morphology of HGF cells. Also, EMD stimulates production of extracellular matrix collagen, osteopontin, and TGF-β1 in high concentration levels. CLINICAL RELEVANCE With the use of EMD, protective barrier between attached gingiva and transmucosal zirconia abutment may be enhanced leading to final esthetic results with implants. PMID:25352963

αMβ2-integrin-intercellular adhesion molecule-1 interactions drive the flow-dependent trafficking of Guillain-Barré syndrome patient derived mononuclear leukocytes at the blood-nerve barrier in vitro

PubMed Central

Yosef, Nejla; Ubogu, Eroboghene E.

2012-01-01

The mechanisms of hematogenous leukocyte trafficking at the human blood-nerve barrier (BNB) are largely unknown. Intercellular adhesion molecule-1 (ICAM-1) has been implicated in the pathogenesis of Guillain-Barré syndrome (GBS). We developed a cytokine-activated human in vitro BNB model using primary endoneurial endothelial cells. Endothelial treatment with 10 U/mL tissue necrosis factor-α and 20 U/mL interferon-γ resulted in de novo expression of proinflammatory chemokines CCL2, CXCL9, CXCL11 and CCL20, with increased expression of CXCL2-3, CXCL8 and CXCL10 relative to basal levels. Cytokine treatment induced/ enhanced ICAM-1, E- and P-selectin, vascular cell adhesion molecule-1 and the alternatively spliced pro-adhesive fibronectin variant, fibronectin connecting segment-1 expression in a time-dependent manner, without alterations in junctional adhesion molecule-A expression. Lymphocytes and monocytes from untreated GBS patients express ICAM-1 counterligands, αM- and αL-integrin, with differential regulation of αM-integrin expression compared to healthy controls. Under flow conditions that mimic capillary hemodynamics in vivo, there was a >3-fold increase in total GBS patient and healthy control mononuclear leukocyte adhesion/ migration at the BNB following cytokine treatment relative to the untreated state. Function neutralizing monoclonal antibodies against human αM-integrin (CD11b) and ICAM-1 reduced untreated GBS patient mononuclear leukocyte trafficking at the BNB by 59% and 64.2% respectively. Monoclonal antibodies against αL-integrin (CD11a) and human intravenous immunoglobulin reduced total leukocyte adhesion/migration by 22.8% and 17.6% respectively. This study demonstrates differential regulation of αM-integrin on circulating mononuclear cells in GBS, as well as an important role for αM-integrin-ICAM-1 interactions in pathogenic GBS patient leukocyte trafficking at the human BNB in vitro. PMID:22552879

Epidermal growth factor and tumor necrosis factor α cooperatively promote the motility of hepatocellular carcinoma cell lines via synergistic induction of fibronectin by NF-κB/p65.

PubMed

Liu, Zong-Cai; Ning, Fen; Wang, Hai-Fang; Chen, Dan-Yang; Cai, Yan-Na; Sheng, Hui-Ying; Lash, Gendie E; Liu, Li; Du, Jun

2017-11-01

The interaction between hepatocellular carcinoma (HCC) cells and their microenvironment plays a fundamental role in tumor metastasis. The HCC microenvironment is rich in epidermal growth factor (EGF) and tumor necrosis factor α (TNFα), which may cooperatively, rather than individually, interact with tumor cells to influence their biological behavior. Immunohistochemistry was performed to study the expression of EGF and TNFα in HCCs. Western blotting, immunofluorescence, qRT-PCR, wound healing scratch and invasion assay, and chromatin immunoprecipitation assays were used to study the combined roles of EGF and TNFα in the motility of HCC cells in vitro. We demonstrated that both EGF and TNFα were highly expressed in HCCs, and HCCs with higher expression of both EGF and TNFα were more frequently rated as high-grade tumors. In vitro, EGF and TNFα cooperatively promoted the motility of HCC cells mainly via synergistic induction of an extracellular matrix glycoprotein fibronectin (FN). Mechanistically, EGF and TNFα jointly increased the nuclear translocation and PKC mediated phosphorylation of NF-κB/p65 which could bind to the -356bp to -259bp fragment of the FN promoter, leading to a markedly increased activity of the FN promoter in HCC cells. HCCs with higher expression of both EGF and TNFα were more frequently rated as high-grade tumors. EGF and TNFα cooperatively promoted the motility of HCC cells mainly through NF-κB/p65 mediated synergistic induction of FN in vitro. These findings highlight the crosstalk between EGF and TNFα in promoting HCC, and provide potential targets for HCC prevention and treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

Cells involved in extracellular matrix remodeling after acute myocardial infarction

PubMed Central

Garcia, Larissa Ferraz; Mataveli, Fábio D’Aguiar; Mader, Ana Maria Amaral Antônio; Theodoro, Thérèse Rachell; Justo, Giselle Zenker; Pinhal, Maria Aparecida da Silva

2015-01-01

Objective Evaluate the effects of VEGF165 gene transfer in the process of remodeling of the extracellular matrix after an acute myocardial infarct. Methods Wistar rats were submitted to myocardial infarction, after the ligation of the left descending artery, and the left ventricle ejection fraction was used to classify the infarcts into large and small. The animals were divided into groups of ten, according to the size of infarcted area (large or small), and received or not VEGF165 treatment. Evaluation of different markers was performed using immunohistochemistry and digital quantification. The primary antibodies used in the analysis were anti-fibronectin, anti-vimentin, anti-CD44, anti-E-cadherin, anti-CD24, anti-alpha-1-actin, and anti-PCNA. The results were expressed as mean and standard error, and analyzed by ANOVA, considering statistically significant if p≤0.05. Results There was a significant increase in the expression of undifferentiated cell markers, such as fibronectin (protein present in the extracellular matrix) and CD44 (glycoprotein present in the endothelial cells). However, there was decreased expression of vimentin and PCNA, indicating a possible decrease in the process of cell proliferation after treatment with VEGF165. Markers of differentiated cells, E-cadherin (adhesion protein between myocardial cells), CD24 (protein present in the blood vessels), and alpha-1-actin (specific myocyte marker), showed higher expression in the groups submitted to gene therapy, compared to non-treated group. The value obtained by the relation between alpha-1-actin and vimentin was approximately three times higher in the groups treated with VEGF165, suggesting greater tissue differentiation. Conclusion The results demonstrated the important role of myocytes in the process of tissue remodeling, confirming that VEGF165 seems to provide a protective effect in the treatment of acute myocardial infarct. PMID:25993074

Unique Footprint in the scl1.3 Locus Affects Adhesion and Biofilm Formation of the Invasive M3-Type Group A Streptococcus.

PubMed

Bachert, Beth A; Choi, Soo J; LaSala, Paul R; Harper, Tiffany I; McNitt, Dudley H; Boehm, Dylan T; Caswell, Clayton C; Ciborowski, Pawel; Keene, Douglas R; Flores, Anthony R; Musser, James M; Squeglia, Flavia; Marasco, Daniela; Berisio, Rita; Lukomski, Slawomir

2016-01-01

The streptococcal collagen-like proteins 1 and 2 (Scl1 and Scl2) are major surface adhesins that are ubiquitous among group A Streptococcus (GAS). Invasive M3-type strains, however, have evolved two unique conserved features in the scl1 locus: (i) an IS1548 element insertion in the scl1 promoter region and (ii) a nonsense mutation within the scl1 coding sequence. The scl1 transcript is drastically reduced in M3-type GAS, contrasting with a high transcription level of scl1 allele in invasive M1-type GAS. This leads to a lack of Scl1 expression in M3 strains. In contrast, while scl2 transcription and Scl2 production are elevated in M3 strains, M1 GAS lack Scl2 surface expression. M3-type strains were shown to have reduced biofilm formation on inanimate surfaces coated with cellular fibronectin and laminin, and in human skin equivalents. Repair of the nonsense mutation and restoration of Scl1 expression on M3-GAS cells, restores biofilm formation on cellular fibronectin and laminin coatings. Inactivation of scl1 in biofilm-capable M28 and M41 strains results in larger skin lesions in a mouse model, indicating that lack of Scl1 adhesin promotes bacterial spread over localized infection. These studies suggest the uniquely evolved scl1 locus in the M3-type strains, which prevents surface expression of the major Scl1 adhesin, contributed to the emergence of the invasive M3-type strains. Furthermore these studies provide insight into the molecular mechanisms mediating colonization, biofilm formation, and pathogenesis of group A streptococci.

Extracellular matrix remodeling and matrix metalloproteinases (ajMMP-2 like and ajMMP-16 like) characterization during intestine regeneration of sea cucumber Apostichopus japonicus.

PubMed

Miao, Ting; Wan, Zixuan; Sun, Lina; Li, Xiaoni; Xing, Lili; Bai, Yucen; Wang, Fang; Yang, Hongsheng

2017-10-01

Remodeling of extracellular matrix (ECM) regulated by matrix metalloproteinases (MMPs) is essential for tissue regeneration. In the present study, we used immunohistochemistry (IHC) techniques against ECM components to reveal changes of ECM during intestine regeneration of Apostichopus japonicus. The expression of collagen I and laminin reduced apparently from the eviscerated intestine, while fibronectin exhibited continuous expression in all regeneration stages observed. Meanwhile, we cloned two MMP genes from A. japonicus by RACE PCR. The full-length cDNA of ajMMP-2 like is 2733bp and contains a predicted open reading frame (ORF) of 1716bp encoding 572 amino acids. The full-length cDNA of ajMMP-16 like is 2705bp and contains an ORF of 1452bp encoding 484 amino acids. The predicted protein sequences of each MMP contain two conserved domains, ZnMc_MMP and HX. Homology and phylogenetic analysis revealed that ajMMP-2 like and ajMMP-16 like share high sequence similarity with MMP-2 and MMP-16 from Strongylocentrotus purpuratus, respectively. Then we investigated spatio-temporal expression of ajMMP-2 like and ajMMP-16 like during different regeneration stages by qRT-PCR and IHC. The expression pattern of them showed a roughly opposite trend from that of ECM components. According to our results, a fibronectin-dominate temporary matrix is created in intestine regeneration, and it might provide structural integrity for matrix and promote cell movement. We also hypothesize that ajMMP-2 like and ajMMP-16 like could accelerate cell migration and regulate interaction between ECM components and growth factors. This work provides new evidence of ECM and MMPs involvement in sea cucumber regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

Beneficial effects of previous exercise training on renal changes in streptozotocin-induced diabetic female rats

PubMed Central

Amaral, Liliany S de Brito; Silva, Fernanda A; Correia, Vicente B; Andrade, Clara EF; Dutra, Bárbara A; Oliveira, Márcio V; de Magalhães, Amélia CM; Volpini, Rildo A; Seguro, Antonio C; Coimbra, Terezila M

2016-01-01

This study evaluated the effects of aerobic exercise performed both previously and after the induction of diabetes mellitus on changes of renal function and structure in streptozotocin-induced diabetic rats. Female wistar rats were divided into five groups: sedentary control (C + Se); trained control (C + Ex); sedentary diabetic (D + Se); trained diabetic (D + Ex) and previously trained diabetic (D + PEx). The previous exercise consisted of treadmill running for four weeks before the induction of diabetes mellitus. After induction of diabetes mellitus with streptozotocin, the D + PEx, D + Ex and C + Ex groups were submitted to eight weeks of aerobic exercise. At the end of the training protocol, we evaluate the serum glucose, insulin and 17β-estradiol levels, renal function and structure, proteinuria, and fibronectin, collagen IV and transforming growth factor beta 1 (TGF-β1) renal expressions. Induction of diabetes mellitus reduced the insulin and did not alter 17β-estradiol levels, and exercise did not affect any of these parameters. Previous exercise training attenuated the loss of body weight, the blood glucose, the increase of glomerular filtration rate and prevented the proteinuria in the D + PEx group compared to D + Se group. Previous exercise also reduced glomerular hypertrophy, tubular and glomerular injury, as well as the expressions of fibronectin and collagen IV. These expressions were associated with reduced expression of TGF-β1. In conclusion, our study shows that regular aerobic exercise especially performed previously to induction of diabetes mellitus improved metabolic control and has renoprotective action on the diabetic kidney. PMID:26490345

Beneficial effects of previous exercise training on renal changes in streptozotocin-induced diabetic female rats.

PubMed

Amaral, Liliany S de Brito; Silva, Fernanda A; Correia, Vicente B; Andrade, Clara E F; Dutra, Bárbara A; Oliveira, Márcio V; de Magalhães, Amélia C M; Volpini, Rildo A; Seguro, Antonio C; Coimbra, Terezila M; Soares, Telma de J

2016-02-01

This study evaluated the effects of aerobic exercise performed both previously and after the induction of diabetes mellitus on changes of renal function and structure in streptozotocin-induced diabetic rats. Female wistar rats were divided into five groups: sedentary control (C + Se); trained control (C + Ex); sedentary diabetic (D + Se); trained diabetic (D + Ex) and previously trained diabetic (D + PEx). The previous exercise consisted of treadmill running for four weeks before the induction of diabetes mellitus. After induction of diabetes mellitus with streptozotocin, the D + PEx, D + Ex and C + Ex groups were submitted to eight weeks of aerobic exercise. At the end of the training protocol, we evaluate the serum glucose, insulin and 17β-estradiol levels, renal function and structure, proteinuria, and fibronectin, collagen IV and transforming growth factor beta 1 (TGF-β1) renal expressions. Induction of diabetes mellitus reduced the insulin and did not alter 17β-estradiol levels, and exercise did not affect any of these parameters. Previous exercise training attenuated the loss of body weight, the blood glucose, the increase of glomerular filtration rate and prevented the proteinuria in the D + PEx group compared to D + Se group. Previous exercise also reduced glomerular hypertrophy, tubular and glomerular injury, as well as the expressions of fibronectin and collagen IV. These expressions were associated with reduced expression of TGF-β1. In conclusion, our study shows that regular aerobic exercise especially performed previously to induction of diabetes mellitus improved metabolic control and has renoprotective action on the diabetic kidney. © 2016 by the Society for Experimental Biology and Medicine.

Qualitative research of alternatively splice variants of fibronectin in different development stage of mice heart.

PubMed

Lu, Feng; Ma, Fang-Fang; Zhang, Wei; Li, Ying; Wei, Fei-Yu; Zhou, Lei

2015-12-01

Fibronectin (FN) plays vital roles in cell adhesion, differentiation, proliferation and migration. It is involved in the process of embryonic development and is highly conserved during evolution. The EIIIA and EIIIB of FN show a very high degree of homology among vertebrates. Embryos deleting both EIIIA and EIIIB displayed multiple embryonic cardiovascular defects, implying their crucial role during embryogenesis. The correlation of spliced EIIIB, EIIIA, and IIICS of FN to heart development was studied by observing their chronological expression in mice heart. C57 mice embryos at E11.5, E12.5, E13.5, E14.5, E15.5, E16.5, E17.5, E18.5, E19.5 days, postnatal day 1 (P1d), and adult male mice (3 months) were used. For each alternatively spliced FN1 domain (EIIIB, EIIIA and IIICS), primer pairs were designed for specific amplification. Total RNA was extracted from the heart tissue, reverse transcripted to cDNA, followed by RT-PCR with specific primers. The PCR amplification was verified by agarose gel electrophoresis, showing specific fragments of the expected sizes. In adult mice heart, only alternatively splice variants of EIIIA-, EIIIB-, IIICS+ were expressed. While in embryonic mice, spliced variant of EIIIA+/-, EIIIB+/-, IIICS+ were observed. The expression of EIIIA and EIIIB changed during heart development. FN is crucial for the normal development of the embryonic heart by modulating cardiac neural crest (CNC) proliferation and survival, and maintenance of CNC cells. FN1 gene seems to play a significant role by expression of highly conserved EIIIA and EIIIB in embryonic heart development.

Vitronectin and fibronectin function as glucan binding proteins augmenting macrophage responses to Pneumocystis carinii.

PubMed

Vassallo, R; Kottom, T J; Standing, J E; Limper, A H

2001-08-01

beta-glucans represent major structural components of fungal cell walls. We recently reported that Pneumocystis carinii beta-glucans stimulate alveolar macrophages to release proinflammatory cytokines. Macrophage activation by beta-glucan is augmented by serum, implying the presence of circulating factors that interact with beta-glucans and enhance their ability to stimulate macrophages. Using beta-glucan-enriched cell wall fractions from P. carinii and Saccharomyces cerevisiae, two prominent proteins were precipitated from serum and demonstrated to be vitronectin (VN) and fibronectin (FN) by immune analysis. Preincubation of beta-glucan with VN or FN enhanced macrophage activation in response to this cell wall component. Because VN and FN accumulate in the lungs during P. carinii pneumonia, we further investigated hepatic and pulmonary expression of VN and FN messenger RNA during infection. P. carinii pneumonia in rodents is associated with increased hepatic expression of VN and FN as well as increased local expression of FN in the lung. Because interleukin (IL)-6 represents the major regulator of VN and FN expression during inflammatory conditions, we measured macrophage IL-6 release in response to stimulation with P. carinii beta-glucan. Stimulation of macrophages with P. carinii beta-glucan induced significant release of IL-6. Elevated concentrations of IL-6 were noted in the blood of infected animals compared with uninfected control animals. These studies indicate that VN and FN bind to beta-glucan components of P. carinii and augment macrophage inflammatory responses. P. carinii cell wall beta-glucan stimulates secretion of IL-6 by macrophages, thereby enhancing hepatic synthesis of both VN and FN, and lung synthesis of FN during pneumonia.

Prenatal Exposure to Nicotine and Childhood Asthma: Role of Nicotine Acetylcholine Receptors, Neuropeptides and Fibronectin Expression in Lung

DTIC Science & Technology

2008-12-01

of apoptosis , how it might be affected by nicotine, and how it might be involved in lung development. This idea was based on observations generated...Furthermore, we recently found that agents capable of inhibiting apoptosis (zinc and autocarboxylic acid) inhibit lung branching morphogenesis...cell apoptosis in lung development. We discovered that apoptosis is most prominent in pseudoglandular-stage lungs coinciding with the time period of

Fabrication of functional fibronectin patterns by nanosecond excimer laser direct write for tissue engineering applications.

PubMed

Grigorescu, S; Hindié, M; Axente, E; Carreiras, F; Anselme, K; Werckmann, J; Mihailescu, I N; Gallet, O

2013-07-01

Laser direct write techniques represent a prospective alternative for engineering a new generation of hybrid biomaterials via the creation of patterns consisting of biological proteins onto practically any type of substrate. In this paper we report on the characterization of fibronectin features obtained onto titanium substrates by UV nanosecond laser transfer. Fourier-transform infrared spectroscopy measurements evidenced no modification in the secondary structure of the post-transferred protein. The molecular weight of the transferred protein was identical to the initial fibronectin, no fragment bands being found in the transferred protein's Western blot migration profile. The presence of the cell-binding domain sequence and the mannose groups within the transferred molecules was revealed by anti-fibronectin monoclonal antibody immunolabelling and FITC-Concanavalin-A staining, respectively. The in vitro tests performed with MC3T3-E1 osteoblast-like cells and Swiss-3T3 fibroblasts showed that the cells' morphology and spreading were strongly influenced by the presence of the fibronectin spots.

Fibronectin regulates the activation of THP-1 cells by TGF-beta1.

PubMed

Wang, A C; Fu, L

2001-03-01

To determine how fibronectin regulates the immunomodulatory effects of transforming growth factor (TGF)-beta on THP-1 cells. THP-1 monocytic cell line. THP-1 cells were primed for 48 h in the presence or absence of 250 pM TGF-beta1. Assays or assessments carried out, together with statistical test applied. We found that adherence to fibronectin dramatically modulates the effects of TGF-beta1 on the human monocytic cell line THP-1. TGF-beta did not significantly affect constitutive interleukin (IL)-8 secretion or IL-1beta-induced IL-8 secretion from suspended cells. In contrast, TGF-beta stimulated IL-8 secretion as well as augmented IL-1beta-induced IL-8 secretion from adherent cells. The differential effects of TGF-beta1 on IL-8 secretion from suspended and adherent cells could not be explained by differences in IL-1 receptor antagonist production. The effects of fibronectin on TGF-beta1 induced IL-8 secretion from THP-1 cells were mimicked by adhesion to immobilized anti-a4beta1 integrin antibody and to a fibronectin fragment containing the CS-1 domain. These results indicate that alpha4beta1-mediated adhesion to fibronectin may play a key role during inflammation by profoundly influencing the effects of TGF-beta1 on monocytes.

Osseoconductivity of a Specific Streptavidin-Biotin-Fibronectin Surface Coating of Biotinylated Titanium Implants - A Rabbit Animal Study.

PubMed

Kämmerer, Peer W; Lehnert, Michael; Al-Nawas, Bilal; Kumar, Vinay V; Hagmann, Sebastien; Alshihri, Abdulmonem; Frerich, Bernhard; Veith, Michael

2015-10-01

Biofunctionalized implant surfaces may accelerate bony integration and increase long-term stability. The aim of the study was to evaluate the osseous reaction toward biomimetic titanium implants surfaces coated with quasicovalent immobilized fibronectin in an in vivo animal model. A total of 84 implants (uncoated [control 1, n = 36], streptavidin-biotin coated [test 1, n = 24], streptavidin-biotin-fibronectin coated [test 2, n = 24]) were inserted 1 mm supracortically in the proximal tibia of 12 rabbits. The samples were examined after 3 and 6 weeks. Total bone-implant contact (tBIC; %), bone-implant contact in the cortical (cBIC; %) and in the spongious bone (sBIC; %) as well as the percentage of linear bone fill (PLF; %) were evaluated. After 3 weeks, streptavidin-biotin-fibronectin implants had a significant higher sBIC (p = .043) and PLF (p = .007) compared with the uncoated samples. After 6 weeks, this difference was significant for tBIC (p = .016) and cBIC (p < .001). Additionally, uncoated screws showed a significant higher sBIC when compared with the fibronectin coating (p < .001). Streptavidin-biotin-coated implants showed less bone growth at both time points of all examined parameters when compared with their counterparts (all p < .001). Quasicovalent immobilization of biotinylated fibronectin with the streptavidin-biotin-fibronectin system on smooth surface titanium shows a beneficial faster osseous healing in vivo. Besides, an antifouling effect of the streptavidin-biotin coating was proven. © 2015 Wiley Periodicals, Inc.

Connection between integrins and cell activation in rat adrenal glomerulosa cells: a role for Arg-Gly-Asp peptide in the activation of the p42/p44(mapk) pathway and intracellular calcium.

PubMed

Campbell, Shirley; Otis, Melissa; Côté, Mylène; Gallo-Payet, Nicole; Payet, Marcel Daniel

2003-04-01

Integrins are responsible for adhesion and activation of several intracellular cascades. The present study was aimed at determining whether the interaction between fibronectin and integrins could generate pathways involved in physiological functions of rat adrenal glomerulosa cells. Immunofluorescence studies and adhesion assays showed that fibronectin was the best matrix in promoting the formation of focal adhesion. Binding of glomerulosa cells to fibronectin, but not to collagen I or poly-L-lysine, involved the integrin-binding sequence Arg-Gly-Asp (RGD). Activation of glomerulosa cells with Arg-Gly-Asp-Ser (RGDS) induced an increase in [Ca(2+)](i), whereas fibronectin triggered a release of Ca(2+) from InsP(3)-sensitive Ca(2+) stores. Aldosterone secretion induced by ACTH, angiotensin II, and RGDS and proliferation were improved on fibronectin, compared with poly-L-lysine. The RGDS peptide induced a transient increase in the activity of the p42/p44(mapk), independent of phosphatidylinositol-3 kinase and protein kinase C. Integrins alpha(5) and alpha(V) as well as their fibronectin receptor partners beta(1) and beta(3), were identified. These results suggest that in rat adrenal glomerulosa cells, binding of the alpha(5)beta(1), alpha(v)beta(1), or alpha(v)beta(3) integrins to fibronectin is involved in the generation of two important signaling events, increase in intracellular calcium, and activation of the p42/p44(mapk) cascade, leading to cell proliferation and aldosterone secretion.

Spatial Anisotropies and Temporal Fluctuations in Extracellular Matrix Network Texture during Early Embryogenesis

PubMed Central

Loganathan, Rajprasad; Potetz, Brian R.; Rongish, Brenda J.; Little, Charles D.

2012-01-01

Early stages of vertebrate embryogenesis are characterized by a remarkable series of shape changes. The resulting morphological complexity is driven by molecular, cellular, and tissue-scale biophysical alterations. Operating at the cellular level, extracellular matrix (ECM) networks facilitate cell motility. At the tissue level, ECM networks provide material properties required to accommodate the large-scale deformations and forces that shape amniote embryos. In other words, the primordial biomaterial from which reptilian, avian, and mammalian embryos are molded is a dynamic composite comprised of cells and ECM. Despite its central importance during early morphogenesis we know little about the intrinsic micrometer-scale surface properties of primordial ECM networks. Here we computed, using avian embryos, five textural properties of fluorescently tagged ECM networks — (a) inertia, (b) correlation, (c) uniformity, (d) homogeneity, and (e) entropy. We analyzed fibronectin and fibrillin-2 as examples of fibrous ECM constituents. Our quantitative data demonstrated differences in the surface texture between the fibronectin and fibrillin-2 network in Day 1 (gastrulating) embryos, with the fibronectin network being relatively coarse compared to the fibrillin-2 network. Stage-specific regional anisotropy in fibronectin texture was also discovered. Relatively smooth fibronectin texture was exhibited in medial regions adjoining the primitive streak (PS) compared with the fibronectin network investing the lateral plate mesoderm (LPM), at embryonic stage 5. However, the texture differences had changed by embryonic stage 6, with the LPM fibronectin network exhibiting a relatively smooth texture compared with the medial PS-oriented network. Our data identify, and partially characterize, stage-specific regional anisotropy of fibronectin texture within tissues of a warm-blooded embryo. The data suggest that changes in ECM textural properties reflect orderly time-dependent rearrangements of a primordial biomaterial. We conclude that the ECM microenvironment changes markedly in time and space during the most important period of amniote morphogenesis—as determined by fluctuating textural properties. PMID:22693609

[Dracorhodin perchlorate inhibit high glucose induce serum and glucocorticoid induced protein kinase 1 and fibronectin expression in human mesangial cells].

PubMed

Xie, Yifeng; Wang, Quansheng; Liu, Jianguo; Xie, Jiwen; Xue, Kaming; Tang, Qing

2010-08-01

To investigate the effect of dracorhodin perchlorate (DP) on inhibiting high glucose-induced serum and glucocorticoid induced protein kinase 1 (SGK1) and fibronectin (FN) expression in human mesangial cells (HMC), and its mechanism of prevention and treatment on renal fibrosis in diabetic nephropathy (DN) . The HMC were divided into normal glucose group (NG group, 5.5 mmol x L(-1) D-glucose), normal glucose +low DP group (NG + LDP group, 5.5 mmol x L(-1) D-glucose +7.5 micromol x L(-1) DP), normal glucose +high DP group (NG + HDP group, 5.5 mmol x L(-1) D-glucose + 15 micromol x L(-1) DP), high glucose group (HG group,25 mmol x L(-1) D-glucose), high glucose +low DP group (HG + LDP group, 25 mmol x L(-1) D-glucose + 7.5 micromol x L(-1) DP)and high glucose +high DP group (HG +HDP group, 25 mmol x L(-1) D-glucose + 15 micromol x L(-1) DP). Each group was examined at 24 hours. The levels of SGK1 and FN mRNA was detected by real-time fluorescence quantitative PCR,and the expression of SGK1 and FN protein was detected by Western blot or indirect immunofluorescence. A basal level of SGK1 and FN in HMC were detected in NG group, and the level of SGK1 and FN mRNA and protein were not evidently different compared to that of NG group adding 7.5 micromol x L(-1) DP for 24 hours. On the other hand, the levels of SGK1 and FN mRNA and protein were obviously decreased by adding 15 micromol x L(-1) DP for 24 hours. Compared to NG group, the levels of SGK1 and FN mRNA and protein were increased in HG group after stimulating for 24 hours (P < 0.01). Compared to HG group, the level of SGK1 and FN mRNA and protein were evidently reduced in HG + LDP and HG + HDP groups by adding 7.5 micromol x L(-1) DP and 15 micromol x L(-1) DP for 24 hours (P < 0.01). Dracorhodin perchlorate can inhibit high glucose-induced serum and glucocorticoid induced protein kinase 1 (SGK1) and fibronectin(FN) expression in human mesangial cells, and this may be part of the mechanism of preventing and treating renal fibrosis of DN.

Transgenic FingRs for Live Mapping of Synaptic Dynamics in Genetically-Defined Neurons

PubMed Central

Son, Jong-Hyun; Keefe, Matthew D.; Stevenson, Tamara J.; Barrios, Joshua P.; Anjewierden, Scott; Newton, James B.; Douglass, Adam D.; Bonkowsky, Joshua L.

2016-01-01

Tools for genetically-determined visualization of synaptic circuits and interactions are necessary to build connectomics of the vertebrate brain and to screen synaptic properties in neurological disease models. Here we develop a transgenic FingR (fibronectin intrabodies generated by mRNA display) technology for monitoring synapses in live zebrafish. We demonstrate FingR labeling of defined excitatory and inhibitory synapses, and show FingR applicability for dissecting synapse dynamics in normal and disease states. Using our system we show that chronic hypoxia, associated with neurological defects in preterm birth, affects dopaminergic neuron synapse number depending on the developmental timing of hypoxia. PMID:26728131

The extracellular Leucine-Rich Repeat superfamily; a comparative survey and analysis of evolutionary relationships and expression patterns

PubMed Central

Dolan, Jackie; Walshe, Karen; Alsbury, Samantha; Hokamp, Karsten; O'Keeffe, Sean; Okafuji, Tatsuya; Miller, Suzanne FC; Tear, Guy; Mitchell, Kevin J

2007-01-01

Background Leucine-rich repeats (LRRs) are highly versatile and evolvable protein-ligand interaction motifs found in a large number of proteins with diverse functions, including innate immunity and nervous system development. Here we catalogue all of the extracellular LRR (eLRR) proteins in worms, flies, mice and humans. We use convergent evidence from several transmembrane-prediction and motif-detection programs, including a customised algorithm, LRRscan, to identify eLRR proteins, and a hierarchical clustering method based on TribeMCL to establish their evolutionary relationships. Results This yields a total of 369 proteins (29 in worm, 66 in fly, 135 in mouse and 139 in human), many of them of unknown function. We group eLRR proteins into several classes: those with only LRRs, those that cluster with Toll-like receptors (Tlrs), those with immunoglobulin or fibronectin-type 3 (FN3) domains and those with some other domain. These groups show differential patterns of expansion and diversification across species. Our analyses reveal several clusters of novel genes, including two Elfn genes, encoding transmembrane proteins with eLRRs and an FN3 domain, and six genes encoding transmembrane proteins with eLRRs only (the Elron cluster). Many of these are expressed in discrete patterns in the developing mouse brain, notably in the thalamus and cortex. We have also identified a number of novel fly eLRR proteins with discrete expression in the embryonic nervous system. Conclusion This study provides the necessary foundation for a systematic analysis of the functions of this class of genes, which are likely to include prominently innate immunity, inflammation and neural development, especially the specification of neuronal connectivity. PMID:17868438

Acquisition of epithelial-mesenchymal transition phenotype in the tamoxifen-resistant breast cancer cell: a new role for G protein-coupled estrogen receptor in mediating tamoxifen resistance through cancer-associated fibroblast-derived fibronectin and β1-integrin signaling pathway in tumor cells.

PubMed

Yuan, Jie; Liu, Manran; Yang, Li; Tu, Gang; Zhu, Qing; Chen, Maoshan; Cheng, Hong; Luo, Haojun; Fu, Weijie; Li, Zhenhua; Yang, Guanglun

2015-05-21

Acquired tamoxifen resistance remains the major obstacle to breast cancer endocrine therapy. β1-integrin was identified as one of the target genes of G protein-coupled estrogen receptor (GPER), a novel estrogen receptor recognized as an initiator of tamoxifen resistance. Here, we investigated the role of β1-integrin in GPER-mediated tamoxifen resistance in breast cancer. The expression of β1-integrin and biomarkers of epithelial-mesenchymal transition were evaluated immunohistochemically in 53 specimens of metastases and paired primary tumors. The function of β1-integrin was investigated in tamoxifen-resistant (MCF-7R) subclones, derived from parental MCF-7 cells, and MCF-7R β1-integrin-silenced subclones in MTT and Transwell assays. Involved signaling pathways were identified using specific inhibitors and Western blotting analysis. GPER, β1-integrin and mesenchymal biomarkers (vimentin and fibronectin) expression in metastases increased compared to the corresponding primary tumors; a close expression pattern of β1-integrin and GPER were in metastases. Increased β1-integrin expression was also confirmed in MCF-7R cells compared with MCF-7 cells. This upregulation of β1-integrin was induced by agonists of GPER and blocked by both antagonist and knockdown of it in MCF-7R cells. Moreover, the epidermal growth factor receptor/extracellular regulated protein kinase (EGFR/ERK) signaling pathway was involved in this transcriptional regulation since specific inhibitors of these kinases also reduced the GPER-induced upregulation of β1-integrin. Interestingly, silencing of β1-integrin partially rescued the sensitivity of MCF-7R cells to tamoxifen and the α5β1-integrin subunit is probably responsible for this phenomenon. Importantly, the cell migration and epithelial-mesenchymal transition induced by cancer-associated fibroblasts, or the product of cancer-associated fibroblasts, fibronectin, were reduced by knockdown of β1-integrin in MCF-7R cells. In addition, the downstream kinases of β1-integrin including focal adhesion kinase, Src and AKT were activated in MCF-7R cells and may be involved in the interaction between cancer cells and cancer-associated fibroblasts. GPER/EGFR/ERK signaling upregulates β1-integrin expression and activates downstream kinases, which contributes to cancer-associated fibroblast-induced cell migration and epithelial-mesenchymal transition, in MCF-7R cells. GPER probably contributes to tamoxifen resistance via interaction with the tumor microenvironment in a β1-integrin-dependent pattern. Thus, β1-integrin may be a potential target to improve anti-hormone therapy responses in breast cancer patients.

Molecular characterization and in situ mRNA localization of the neural recognition molecule J1-160/180: a modular structure similar to tenascin

PubMed Central

1993-01-01

The oligodendrocyte-derived extracellular matrix glycoprotein J1- 160/180 is a recognition molecule expressed exclusively in the central nervous system. J1-160/180 has been shown to be adhesive for astrocytes and repellent towards neurons and growth cones. We report here the complete nucleotide sequence of J1-160/180 in the rat. The predicted amino acid sequence showed a structural architecture very similar to tenascin: a cysteine-rich amino terminal region is followed by 4.5 epidermal growth factor-like repeats, 9 fibronectin type III homologous repeats and a domain homologous to fibrinogen. Sequence comparison analysis revealed highest homology of rat J1-160/180 to mouse tenascin and chicken restrictin with a similarity of 66% and 85%, respectively. The J1-160/180-coding mRNA is derived from a single copy gene. Using the polymerase chain reaction we could show that two J1-160/180 isoforms are generated by alternative splicing of the sixth fibronectin type III homologous repeat. Localization of J1-160/180 mRNA by in situ hybridization in the cerebellum, hippocampus and olfactory bulb confirmed the expression of J1-160/180 by oligodendrocytes with a peak of transcription at 7-14 d after birth, indicating a functional role during myelination. In addition, J1-160/180-specific RNA was found in a small subset of neurons in all three structures of the CNS analyzed. These neurons continue to express J1-160/180 in the adult. PMID:7679676

Effect of doxycycline on epithelial-mesenchymal transition via the p38/Smad pathway in respiratory epithelial cells.

PubMed

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

2017-03-01

Doxycycline has antibacterial and anti-inflammatory effects, and it also suppresses collagen biosynthesis. This study aimed to confirm the effects and mechanism of doxycycline on transforming growth factor (TGF) beta 1 induced epithelial-mesenchymal transition and cell migration in A549 and primary nasal epithelial cells. A 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay and phalloidin-fluorescein isothiocyanate staining were used to evaluate cytotoxicity and cellular morphologic changes. Western blot and immunofluorescence staining were used to determine the expression levels of E-cadherin, vimentin, alpha-smooth muscle actin, fibronectin, phosphorylated Smad2/3, and mitogen-activated protein kinases. Scratch and transwell migration assays were used to assess cellular migration ability. Doxycycline (0-10 μg/mL) had no significant cytotoxic effects in A549 and primary nasal epithelial cells. Increased expression of mesenchymal markers, including vimentin, alpha-smooth muscle actin, and fibronectin in TGF beta 1 induced A549 cells were downregulated by doxycycline treatment. In contrast, E-cadherin expression was upregulated in TGF beta 1 induced A549 cells. An in vitro cell migration assay showed that doxycycline also inhibited the ability of TGF beta 1 induced migration. Doxycycline treatment suppressed the activation of Smad2/3 and p38, whereas its inhibitory effects were similar to each element-specific inhibitor in A549 and primary nasal epithelial cells. Doxycycline inhibited TGF beta 1 induced epithelial-to-mesenchymal transition and migration by targeting Smad2/3 and p38 signal pathways in respiratory epithelial cells.

Granzyme B mediates both direct and indirect cleavage of extracellular matrix in skin after chronic low-dose ultraviolet light irradiation.

PubMed

Parkinson, Leigh G; Toro, Ana; Zhao, Hongyan; Brown, Keddie; Tebbutt, Scott J; Granville, David J

2015-02-01

Extracellular matrix (ECM) degradation is a hallmark of many chronic inflammatory diseases that can lead to a loss of function, aging, and disease progression. Ultraviolet light (UV) irradiation from the sun is widely considered as the major cause of visible human skin aging, causing increased inflammation and enhanced ECM degradation. Granzyme B (GzmB), a serine protease that is expressed by a variety of cells, accumulates in the extracellular milieu during chronic inflammation and cleaves a number of ECM proteins. We hypothesized that GzmB contributes to ECM degradation in the skin after UV irradiation through both direct cleavage of ECM proteins and indirectly through the induction of other proteinases. Wild-type and GzmB-knockout mice were repeatedly exposed to minimal erythemal doses of solar-simulated UV irradiation for 20 weeks. GzmB expression was significantly increased in wild-type treated skin compared to nonirradiated controls, colocalizing to keratinocytes and to an increased mast cell population. GzmB deficiency significantly protected against the formation of wrinkles and the loss of dermal collagen density, which was related to the cleavage of decorin, an abundant proteoglycan involved in collagen fibrillogenesis and integrity. GzmB also cleaved fibronectin, and GzmB-mediated fibronectin fragments increased the expression of collagen-degrading matrix metalloproteinase-1 (MMP-1) in fibroblasts. Collectively, these findings indicate a significant role for GzmB in ECM degradation that may have implications in many age-related chronic inflammatory diseases. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Matrix-specific protein kinase A signaling regulates p21 activated kinase activation by flow in endothelial cells

PubMed Central

Funk, Steven Daniel; Yurdagul, Arif; Green, Jonette M.; Jhaveri, Krishna A.; Schwartz, Martin Alexander; Orr, A. Wayne

2010-01-01

Rationale Atherosclerosis is initiated by blood flow patterns that activate inflammatory pathways in endothelial cells. Activation of inflammatory signaling by fluid shear stress is highly dependent on the composition of the subendothelial extracellular matrix. The basement membrane proteins laminin and collagen found in normal vessels suppress flow-induced p21 activated kinase (PAK) and NF-κB activation. By contrast, the provisional matrix proteins fibronectin and fibrinogen found in wounded or inflamed vessels support flow-induced PAK and NF-κB activation. PAK mediates both flow-induced permeability and matrix-specific activation of NF-κB. Objective To elucidate the mechanisms regulating matrix-specific PAK activation. Methods and Results We now show that matrix composition does not affect the upstream pathway by which flow activates PAK (integrin activation, Rac). Instead basement membrane proteins enhance flow-induced protein kinase A (PKA) activation, which suppresses PAK. Inhibiting PKA restored flow-induced PAK and NF-κB activation in cells on basement membrane proteins, whereas stimulating PKA inhibited flow-induced activation of inflammatory signaling in cells on fibronectin. PKA suppressed inflammatory signaling through PAK inhibition. Activating PKA by injection of the PGI2 analog iloprost reduced PAK activation and inflammatory gene expression at sites of disturbed flow in vivo, whereas inhibiting PKA by PKI injection enhanced PAK activation and inflammatory gene expression. Inhibiting PAK prevented the enhancement of inflammatory gene expression by PKI. Conclusions Basement membrane proteins inhibit inflammatory signaling in endothelial cells via PKA-dependent inhibition of PAK. PMID:20224042

Epilobium angustifolium extract demonstrates multiple effects on dermal fibroblasts in vitro and skin photo-protection in vivo.

PubMed

Ruszová, Ema; Cheel, José; Pávek, Stanislav; Moravcová, Martina; Hermannová, Martina; Matějková, Ilona; Spilková, Jiřina; Velebný, Vladimír; Kubala, Lukáš

2013-09-01

Stress-induced fibroblast senescence is thought to contribute to skin aging. Ultraviolet light (UV) radiation is the most potent environmental risk factor in these processes. An Epilobium angustifolium (EA) extract was evaluated for its capacity to reverse the senescent response of normal human dermal fibroblasts (NHDF) in vitro and to exhibit skin photo-protection in vivo. The HPLC-UV-MS analysis of the EA preparation identified three major polyphenol groups: tannins (oenothein B), phenolic acids (gallic and chlorogenic acids) and flavonoids. EA extract increased the cell viability of senescent NHDF induced by serum deprivation. It diminished connective tissue growth factor and fibronectin gene expressions in senescent NHDF. Down-regulation of the UV-induced release of both matrix metalloproteinase-1 and -3 and the tissue inhibitor of matrix metalloproteinases-1 and -2, and also down-regulation of the gene expression of hyaluronidase 2 were observed in repeatedly UV-irradiated NHDF after EA extract treatment. Interestingly, EA extract diminished the down-regulation of sirtuin 1 dampened by UV-irradiation. The application of EA extract using a sub-irritating dose protected skin against UV-induced erythema formation in vivo. In summary, EA extract diminished stress-induced effects on NHDF, particularly on connective tissue growth factor, fibronectin and matrix metalloproteinases. These results collectively suggest that EA extract may possess anti-aging properties and that the EA polyphenols might account for these benefits.

Tenascin-C, proliferation and subendothelial fibronectin in progressive pulmonary vascular disease.

PubMed Central

Jones, P. L.; Cowan, K. N.; Rabinovitch, M.

1997-01-01

Progressive pulmonary hypertension is characterized by smooth muscle cell proliferation and migration leading to occlusive arterial lesions. Previously, using cultured smooth muscle cells, we demonstrated that epidermal growth factor (EGF)-dependent proliferation and migration are dependent on tenascin-C (Tn) and cellular fibronectin (Fn), respectively. In this study we applied immunohistochemistry to lung biopsy tissue from patients with congenital heart defects and pulmonary hypertension to determine how the distribution and intensity of Tn, EGF, proliferating cell nuclear antigen (PCNA), and Fn expression related to arterial abnormalities. With mildly increased wall thickness, minimal Tn, PCNA, and EGF was evident. With progressive hypertrophy, moderately intense foci of Tn were apparent in the adventitia, periendothelium, and occasionally the media but not consistently co-distributing with EGF and PCNA. With obstructive lesions, intense neointimal Tn expression co-localized with EGF and PCNA. Fn accumulation in the periendothelium increased with medial hypertrophy and became more widespread in a diffuse pattern with neointimal formation. The neointima was predominantly composed of alpha-smooth-muscle-actin-positive cells, occasional inflammatory cells with no evidence of apoptosis. These studies are consistent with Tn modulating EGF-dependent neointimal smooth muscle cell proliferation and Fn providing a gradient for smooth muscle cell migration from media to neointima. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:9094991

ER stress-mediated cell damage contributes to the release of EDA+ fibronectin from hepatocytes in nonalcoholic fatty liver disease.

PubMed

He, Lei; Yuan, Fa-Hu; Chen, Ting; Huang, Qiang; Wang, Yu; Liu, Zhi-Guo

2017-04-01

Fibronectin containing extra domain A (EDA + FN), a functional glycoprotein participating in several cellular processes, correlates with chronic liver disease. Herein, we aim to investigate the expression and secretion of EDA + FN from hepatocytes in nonalcoholic fatty liver disease (NAFLD) and the underlying mechanisms. Circulating levels of EDA + FN were determined by ELISA in clinical samples. Western blotting and flow cytometry were performed on L02 and HepG2 cell lines to analyze whether the levels of EDA + FN were associated with endoplasmic reticulum (ER) stress-related cell death. Circulating levels of EDA + FN in NAFLD patients were significantly higher than those in control subjects, and positively related with severity of ultrasonographic steatosis score. In cultured hepatocytes, palmitate up-regulated the expression of EDA + FN in a dose-dependent manner. Conversely, when the cells were pretreated with 4-phenylbutyrate, a specific inhibitor of ER stress, up-regulation of EDA + FN could be abrogated. Moreover, silencing CHOP by shRNA enhanced the release of EDA + FN from hepatocytes following palmitate treatment, which was involved in ER stress-related cell damage. These findings suggest that the up-regulated level of EDA + FN is associated with liver damage in NAFLD, and ER stress-mediated cell damage contributes to the release of EDA + FN from hepatocytes.

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

PubMed Central

Feng, Xiaowen; Zhang, Feng; Xie, Haiyang

2017-01-01

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

Anti-fibrotic effects of pirfenidone and rapamycin in primary IPF fibroblasts and human alveolar epithelial cells.

PubMed

Molina-Molina, M; Machahua-Huamani, C; Vicens-Zygmunt, V; Llatjós, R; Escobar, I; Sala-Llinas, E; Luburich-Hernaiz, P; Dorca, J; Montes-Worboys, A

2018-04-27

Pirfenidone, a pleiotropic anti-fibrotic treatment, has been shown to slow down disease progression of idiopathic pulmonary fibrosis (IPF), a fatal and devastating lung disease. Rapamycin, an inhibitor of fibroblast proliferation could be a potential anti-fibrotic drug to improve the effects of pirfenidone. Primary lung fibroblasts from IPF patients and human alveolar epithelial cells (A549) were treated in vitro with pirfenidone and rapamycin in the presence or absence of transforming growth factor β1 (TGF-β). Extracellular matrix protein and gene expression of markers involved in lung fibrosis (tenascin-c, fibronectin, collagen I [COL1A1], collagen III [COL3A1] and α-smooth muscle actin [α-SMA]) were analyzed. A cell migration assay in pirfenidone, rapamycin and TGF-β-containing media was performed. Gene and protein expression of tenascin-c and fibronectin of fibrotic fibroblasts were reduced by pirfenidone or rapamycin treatment. Pirfenidone-rapamycin treatment did not revert the epithelial to mesenchymal transition pathway activated by TGF-β. However, the drug combination significantly abrogated fibroblast to myofibroblast transition. The inhibitory effect of pirfenidone on fibroblast migration in the scratch-wound assay was potentiated by rapamycin combination. These findings indicate that the combination of pirfenidone and rapamycin widen the inhibition range of fibrogenic markers and prevents fibroblast migration. These results would open a new line of research for an anti-fibrotic combination therapeutic approach.

The integrin expression profile modulates orientation and dynamics of force transmission at cell-matrix adhesions.

PubMed

Balcioglu, Hayri E; van Hoorn, Hedde; Donato, Dominique M; Schmidt, Thomas; Danen, Erik H J

2015-04-01

Integrin adhesion receptors connect the extracellular matrix (ECM) to the cytoskeleton and serve as bidirectional mechanotransducers. During development, angiogenesis, wound healing and cancer progression, the relative abundance of fibronectin receptors, including integrins α5β1 and αvβ3, changes, thus altering the integrin composition of cell-matrix adhesions. Here, we show that enhanced αvβ3 expression can fully compensate for loss of α5β1 and other β1 integrins to support outside-in and inside-out force transmission. α5β1 and αvβ3 each mediate actin cytoskeletal remodeling in response to stiffening or cyclic stretching of the ECM. Likewise, α5β1 and αvβ3 support cellular traction forces of comparable magnitudes and similarly increase these forces in response to ECM stiffening. However, cells using αvβ3 respond to lower stiffness ranges, reorganize their actin cytoskeleton more substantially in response to stretch, and show more randomly oriented traction forces. Centripetal traction force orientation requires long stress fibers that are formed through the action of Rho kinase (ROCK) and myosin II, and that are supported by α5β1. Thus, altering the relative abundance of fibronectin-binding integrins in cell-matrix adhesions affects the spatiotemporal organization of force transmission. © 2015. Published by The Company of Biologists Ltd.

Impedimetric Analysis of the Effect of Decellularized Porcine Heart Scaffold on Human Fibrosarcoma, Endothelial, and Cardiomyocyte Cell Lines

PubMed Central

Bäcker, Henrik; Polgár, Livia; Soós, Pal; Lajkó, Eszter; Láng, Orsolya; Merkely, Bela; Szabó, Gabor; Dohmen, Pascal M.; Weymann, Alexander; Kőhidai, Laszlo

2017-01-01

Background Experiments on porcine heart scaffold represent significant assays in development of immunoneutral materials for cardiac surgery. Characterization of cell-cell and cell-scaffold interactions is essential to understand the homing process of cardiac cells into the scaffolds. Material/Methods In the present study, the highly sensitive and real-time impedimetric technique of xCELLigence SP was used to monitor cell adhesion, which is the key process of recellularization in heart scaffolds. Our objectives were: (i) to characterize the effect of decellularized porcine heart scaffold on cell adhesion of human cardiovascular cells potentially used in the recellularization process; and (ii) to investigate cell-extracellular matrix element interactions for building artificial multi-layer systems, applied as cellular models of recellularization experiments. Human fibrosarcoma, endothelial, and cardiomyocyte cells were investigated and the effect of decellularized porcine heart scaffold (HS) and fibronectin on cell adhesion was examined. Adhesion was quantified as slope of curves. Results Heart scaffold had neutral effect on cardiomyocytes as well as on endothelial cells. Adhesion of cardiomyocytes was increased by fibronectin (1.480±0.021) compared to control (0.745±0.029). The combination of fibronectin and HS induced stronger adhesion of cardiomyocytes (2.407±0.634) than fibronectin alone. Endothelial and fibrosarcoma cells showed similarly strong adhesion profiles with marked enhancer effect by fibronectin. Conclusions Decellularized porcine HS does not inhibit adhesion of human cardiovascular cells at the cell biological level, while fibronectin has strong cell adhesion-inducer effect, as well as an enhancer effect on activity of HS. Consequently, decellularized porcine hearts could be used as scaffolds for recellularization with cardiomyocytes and endothelial cells with fibronectin acting as a regulator, leading to construction of working bioartificial hearts. PMID:28493851

Diagnostic value of fibronectin discriminant score for predicting liver fibrosis stages in chronic hepatitis C virus patients.

PubMed

Attallah, Abdelfattah M; Abdallah, Sanaa O; Attallah, Ahmed A; Omran, Mohamed M; Farid, Khaled; Nasif, Wesam A; Shiha, Gamal E; Abdel-Aziz, Abdel-Aziz F; Rasafy, Nancy; Shaker, Yehia M

2013-01-01

Several noninvasive predictive models were developed to substitute liver biopsy for fibrosis assessment. To evaluate the diagnostic value of fibronectin which reflect extracellular matrix metabolism and standard liver functions tests which reflect alterations in hepatic functions. Chronic hepatitis C (CHC) patients (n = 145) were evaluated using ROC curves and stepwise multivariate discriminant analysis (MDA) and was validated in 180 additional patients. Liver biochemical profile including transaminases, bilirubin, alkaline phosphatase, albumin, complete blood count were estimated. Fibronectin concentration was determined using monoclonal antibody and ELISA. A novel index named fibronectin discriminant score (FDS) based on fibronectin, APRI and albumin was developed. FDS produced areas under ROC curves (AUC) of 0.91 for significant fibrosis and 0.81 for advanced fibrosis. The FDS correctly classified 79% of the significant liver fibrosis patients (F2-F4) with 87% sensitivity and 75% specificity. The relative risk [odds ratio (OR)] of having significant liver fibrosis using the cut-off values determined by ROC curve analyses were 6.1 for fibronectin, 4.9 for APRI, and 4.2 for albumin. FDS predicted liver fibrosis with an OR of 16.8 for significant fibrosis and 8.6 for advanced fibrosis. The FDS had similar AUC and OR in the validation group to the estimation group without statistically significant difference. FDS predicted liver fibrosis with high degree of accuracy, potentially decreasing the number of liver biopsy required.

Endothelin and sex hormones modulate the fibronectin synthesis by cultured human skin scleroderma fibroblasts

PubMed Central

Soldano, S; Montagna, P; Villaggio, B; Parodi, A; Gianotti, G; Sulli, A; Seriolo, B; Secchi, M E; Cutolo, M

2009-01-01

Objective: To evaluate the influence of endothelin-1 (ET-1) and sex hormones on cell proliferation and extracellular matrix (ECM) synthesis (ie, fibronectin, laminin) by cultured normal and scleroderma (SSc) human skin fibroblasts (FBs). Methods: Primary cultures of FBs were treated with ET-1 and sex hormones (17β-oestradiol or testosterone) for 24 h. Cell growth was analysed by methiltetrazolium salt test, ECM synthesis was evaluated by immunocytochemistry and western blot, both at 24 h. Results: In normal FBs, ET-1 and 17β-oestradiol, as well as their combination, increased cell growth (p<0.001, p<0.001, p<0.01 vs untreated cells (control), respectively) and fibronectin synthesis (p<0.05, p<0.05, p<0.01 vs control, respectively). By contrast, testosterone either alone or in combination with ET-1 did not influence cell proliferation, but decreased fibronectin synthesis (p<0.05, testosterone vs control). In SSc FBs, ET-1 and 17β-oestradiol alone or their combination induced an increased fibronectin synthesis (p<0.05, p<0.05, p<0.01 vs control, respectively). Unexpectedly, testosterone induced an increase of fibronectin synthesis (p<0.05 vs control). Conclusions: ET-1 and 17β-oestradiol seem to exert a profibrotic effect in normal and SSc culture FBs and might suggest their synergistic effect in the pathogenesis of the fibrotic process in SSc. PMID:18952637

Chemically grafted fibronectin for use in QCM-D cell studies

PubMed Central

Sobolewski, Peter; Tomczyk, Nancy; Composto, Russell J.; Eckmann, David M.

2014-01-01

Traditionally, fibronectin has been used as a physisorbed surface coating (physFN) in cell culture experiments due to its critical role in cell adhesion. However, because the resulting layer is thick, unstable, and of unpredictable uniformity, this method of fibronectin deposition is unsuitable for some types of research, including quartz crystal microbalance (QCM) experiments involving cells. Here, we present a new method for chemical immobilization of fibronectin onto silicon oxide surfaces, including QCM crystals pre-coated with silicon oxide. We characterize these chemically coated fibronectin surfaces (chemFN) as well as physFN ones using surface ellipsometry (SE), Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), and contact angle measurements. A cell culture model demonstrates that cells on chemFN and physFN surfaces exhibit similar viability, structure, adhesion and metabolism. Finally, we perform QCM experiments using cells on both surfaces which demonstrate the superior suitability of chemFN coatings for QCM research, and provide real-time QCM-D data from cells subjected to an actin depolymerizing agent. Overall, our method of chemical immobilization of fibronectin yields great potential for furthering cellular experiments in which thin, stable and uniform coatings are desirable. As QCM research with cells has been rather limited in success thus far, we anticipate that this new technique will particularly benefit this experimental system by availing it to the much broader field of cell mechanics. PMID:24657645

The Haemophilus influenzae Hap Autotransporter Binds to Fibronectin, Laminin, and Collagen IV

PubMed Central

Fink, Doran L.; Green, Bruce A.; St. Geme III, Joseph W.

2002-01-01

Nontypeable Haemophilus influenzae (NTHI) initiates infection by colonizing the upper respiratory tract mucosa. NTHI disease frequently occurs in the context of respiratory tract inflammation, where organisms encounter damaged epithelium and exposed basement membrane. In this study, we examined interactions between the H. influenzae Hap adhesin and selected extracellular matrix proteins. Hap is an autotransporter protein that undergoes autoproteolytic cleavage, with release of the adhesive passenger domain, Haps, from the bacterial cell surface. We found that Hap promotes bacterial adherence to purified fibronectin, laminin, and collagen IV and that Hap-mediated adherence is enhanced by inhibition of autoproteolysis. Adherence is inhibited by pretreatment of bacteria with a polyclonal antiserum recognizing Haps. Purified Haps binds with high affinity to fibronectin, laminin, and collagen IV but not to collagen II. Binding of Haps to fibronectin involves interaction with the 45-kDa gelatin-binding domain but not the 30-kDa heparin-binding domain of fibronectin. Taken together, these observations suggest that interactions between Hap and extracellular matrix proteins may play an important role in NTHI colonization of the respiratory tract. PMID:12183535

Glycogen synthase kinase-3 (GSK-3) regulates TGF-β1-induced differentiation of pulmonary fibroblasts

PubMed Central

Baarsma, Hoeke A; Engelbertink, Lilian HJM; van Hees, Lonneke J; Menzen, Mark H; Meurs, Herman; Timens, Wim; Postma, Dirkje S; Kerstjens, Huib AM; Gosens, Reinoud

2013-01-01

Background Chronic lung diseases such as asthma, COPD and pulmonary fibrosis are characterized by abnormal extracellular matrix (ECM) turnover. TGF-β is a key mediator stimulating ECM production by recruiting and activating lung fibroblasts and initiating their differentiation process into more active myofibroblasts. Glycogen synthase kinase-3 (GSK-3) regulates various intracellular signalling pathways; its role in TGF-β1-induced myofibroblast differentiation is currently largely unknown. Purpose To determine the contribution of GSK-3 signalling in TGF-β1-induced myofibroblast differentiation. Experimental Approach We used MRC5 human lung fibroblasts and primary pulmonary fibroblasts of individuals with and without COPD. Protein and mRNA expression were determined by immunoblotting and RT-PCR analysis respectively. Results Stimulation of MRC5 and primary human lung fibroblasts with TGF-β1 resulted in time- and dose-dependent increases of α-sm-actin and fibronectin expression, indicative of myofibroblast differentiation. Pharmacological inhibition of GSK-3 by SB216763 dose-dependently attenuated TGF-β1-induced expression of these myofibroblasts markers. Moreover, silencing of GSK-3 by siRNA or pharmacological inhibition by CT/CHIR99021 fully inhibited the TGF-β1-induced expression of α-sm-actin and fibronectin. The effect of GSK-3 inhibition on α-sm-actin expression was similar in fibroblasts from individuals with and without COPD. Neither smad, NF-κB nor ERK1/2 were involved in the inhibitory actions of GSK-3 inhibition by SB126763 on myofibroblast differentiation. Rather, SB216763 increased the phosphorylation of CREB, which in its phosphorylated form acts as a functional antagonist of TGF-β/smad signalling. Conclusion and Implication We demonstrate that GSK-3 signalling regulates TGF-β1-induced myofibroblast differentiation by regulating CREB phosphorylation. GSK-3 may constitute a useful target for treatment of chronic lung diseases. PMID:23297769

Effects of protein-coated nanofibers on conformation of gingival fibroblast spheroids: potential utility for connective tissue regeneration.

PubMed

Kaufman, Gili; Whitescarver, Ryan A; Nunes, Laiz; Palmer, Xavier-Lewis; Skrtic, Drago; Tutak, Wojtek

2018-01-24

Deep wounds in the gingiva caused by trauma or surgery require a rapid and robust healing of connective tissues. We propose utilizing gas-brushed nanofibers coated with collagen and fibrin for that purpose. Our hypotheses are that protein-coated nanofibers will: (i) attract and mobilize cells in various spatial orientations, and (ii) regulate the expression levels of specific extracellular matrix (ECM)-associated proteins, determining the initial conformational nature of dense and soft connective tissues. Gingival fibroblast monolayers and 3D spheroids were cultured on ECM substrate and covered with gas-blown poly-(DL-lactide-co-glycolide) (PLGA) nanofibers (uncoated/coated with collagen and fibrin). Cell attraction and rearrangement was followed by F-actin staining and confocal microscopy. Thicknesses of the cell layers, developed within the nanofibers, were quantified by ImageJ software. The expression of collagen1α1 chain (Col1α1), fibronectin, and metalloproteinase 2 (MMP2) encoding genes was determined by quantitative reverse transcription analysis. Collagen- and fibrin- coated nanofibers induced cell migration toward fibers and supported cellular growth within the scaffolds. Both proteins affected the spatial rearrangement of fibroblasts by favoring packed cell clusters or intermittent cell spreading. These cell arrangements resembled the structural characteristic of dense and soft connective tissues, respectively. Within three days of incubation, fibroblast spheroids interacted with the fibers, and grew robustly by increasing their thickness compared to monolayers. While the ECM key components, such as fibronectin and MMP2 encoding genes, were expressed in both protein groups, Col1α1 was predominantly expressed in bundled fibroblasts grown on collagen fibers. This enhanced expression of collagen1 is typical for dense connective tissue. Based on results of this study, our gas-blown, collagen- and fibrin-coated PLGA nanofibers are viable candidates for engineering soft and dense connective tissues with the required structural characteristics and functions needed for wound healing applications. Rapid regeneration of these layers should enhance healing of open wounds in a harsh oral environment.

Vascular dysfunction and fibrosis in stroke-prone spontaneously hypertensive rats: The aldosterone-mineralocorticoid receptor-Nox1 axis.

PubMed

Harvey, Adam P; Montezano, Augusto C; Hood, Katie Y; Lopes, Rheure A; Rios, Francisco; Ceravolo, Graziela; Graham, Delyth; Touyz, Rhian M

2017-06-15

We questioned whether aldosterone and oxidative stress play a role in vascular damage in severe hypertension and investigated the role of Nox1 in this process. We studied mesenteric arteries, aortas and vascular smooth muscle cells (VSMC) from WKY and SHRSP rats. Vascular effects of eplerenone or canrenoic acid (CA) (mineralocorticoid receptor (MR) blockers), ML171 (Nox1 inhibitor) and EHT1864 (Rac1/2 inhibitor) were assessed. Nox1-knockout mice were also studied. Vessels and VSMCs were probed for Noxs, reactive oxygen species (ROS) and pro-fibrotic/inflammatory signaling. Blood pressure and plasma levels of aldosterone and galectin-3 were increased in SHRSP versus WKY. Acetylcholine-induced vasorelaxation was decreased (61% vs 115%) and phenylephrine-induced contraction increased in SHRSP versus WKY (E max 132.8% vs 96.9%, p<0.05). Eplerenone, ML171 and EHT1864 attenuated hypercontractility in SHRSP. Vascular expression of collagen, fibronectin, TGFβ, MCP-1, RANTES, MMP2, MMP9 and p66Shc was increased in SHRSP versus WKY. These changes were associated with increased ROS generation, 3-nitrotyrosine expression and Nox1 upregulation. Activation of vascular p66Shc and increased expression of Nox1 and collagen I were prevented by CA in SHRSP. Nox1 expression was increased in aldosterone-stimulated WKY VSMCs, an effect that was amplified in SHRSP VSMCs (5.2vs9.9 fold-increase). ML171 prevented aldosterone-induced VSMC Nox1-ROS production. Aldosterone increased vascular expression of fibronectin and PAI-1 in wild-type mice but not in Nox1-knockout mice. Our findings suggest that aldosterone, which is increased in SHRSP, induces vascular damage through MR-Nox1-p66Shc-mediated processes that modulate pro-fibrotic and pro-inflammatory signaling pathways. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

FOXC1 Regulates Expression of Prostaglandin Receptors Leading to an Attenuated Response to Latanoprost.

PubMed

Doucette, Lance P; Footz, Tim; Walter, Michael A

2018-05-01

This study examines the effect of FOXC1 on the prostaglandin pathway in order to explore FOXC1's role in the prostaglandin-resistant glaucoma phenotype commonly seen in Axenfeld-Rieger syndrome. Binding and transcriptional activity of FOXC1 to the gene coding for the EP3 prostaglandin receptor (PTGER3) were evaluated through ChIP-qPCR and luciferase-based assays. Immortalized trabecular meshwork cells (TM1) and HeLa cells had FOXC1 mRNA reduced via siRNA interference. qPCR and Western blot experiments were conducted to examine the changes in prostaglandin receptor expression brought about by lowered FOXC1. TM1 cells were then treated with 10 μM latanoprost acid and/or an siRNA for FOXC1. The expression of fibronectin and matrix metalloproteinase 9 were evaluated via qPCR in each treatment condition. ChIP-qPCR and luciferase experiments confirmed that FOXC1 binds to and activates transcription of the EP3 gene prostaglandin receptor. qPCR and Western experiments in HeLa and TM1 cells showed that FOXC1 siRNA knockdown results in significantly lowered EP3 levels (protein and RNA). In addition, RNA levels of the other prostaglandin receptor genes EP1 (PTGER1), EP2 (PTGER2), EP4 (PTGER4), and FP (PTGFR) were altered when FOXC1 was knocked down in TM1 and HeLa cells. Analysis of fibronectin expression in TM1 cells after treatment with 10 μM latanoprost acid showed a statistically significant increase in expression; this increase was abrogated by cotreatment with a siRNA for FOXC1. We show the abrogation of latanoprost signalling when FOXC1 is knocked down via siRNA in a trabecular meshwork cell line. We propose that the lower levels of active FOXC1 in Axenfeld-Rieger syndrome patients with glaucoma account for the lack of response to prostaglandin-based medications.

Shortening of the Lactobacillus paracasei subsp. paracasei BGNJ1-64 AggLb Protein Switches Its Activity from Auto-aggregation to Biofilm Formation.

PubMed

Miljkovic, Marija; Bertani, Iris; Fira, Djordje; Jovcic, Branko; Novovic, Katarina; Venturi, Vittorio; Kojic, Milan

2016-01-01

AggLb is the largest (318.6 kDa) aggregation-promoting protein of Lactobacillus paracasei subsp. paracasei BGNJ1-64 responsible for forming large cell aggregates, which causes auto-aggregation, collagen binding and pathogen exclusion in vitro. It contains an N-terminus leader peptide, followed by six successive collagen binding domains, 20 successive repeats (CnaB-like domains) and an LPXTG sorting signal at the C-terminus for cell wall anchoring. Experimental information about the roles of the domains of AggLb is currently unknown. To define the domain that confers cell aggregation and the key domains for interactions of specific affinity between AggLb and components of the extracellular matrix, we constructed a series of variants of the aggLb gene and expressed them in Lactococcus lactis subsp. lactis BGKP1-20 using a lactococcal promoter. All of the variants contained a leader peptide, an inter collagen binding-CnaB domain region (used to raise an anti-AggLb antibody), an anchor domain and a different number of collagen binding and CnaB-like domains. The role of the collagen binding repeats of the N-terminus in auto-aggregation and binding to collagen and fibronectin was confirmed. Deletion of the collagen binding repeats II, III, and IV resulted in a loss of the strong auto-aggregation, collagen and fibronectin binding abilities whereas the biofilm formation capability was increased. The strong auto-aggregation, collagen and fibronectin binding abilities of AggLb were negatively correlated to biofilm formation.

Tensile forces drive a reversible fibroblast-to-myofibroblast transition during tissue growth in engineered clefts

PubMed Central

Kollmannsberger, Philip; Bidan, Cécile M.; Dunlop, John W. C.; Fratzl, Peter; Vogel, Viola

2018-01-01

Myofibroblasts orchestrate wound healing processes, and if they remain activated, they drive disease progression such as fibrosis and cancer. Besides growth factor signaling, the local extracellular matrix (ECM) and its mechanical properties are central regulators of these processes. It remains unknown whether transforming growth factor–β (TGF-β) and tensile forces work synergistically in up-regulating the transition of fibroblasts into myofibroblasts and whether myofibroblasts undergo apoptosis or become deactivated by other means once tissue homeostasis is reached. We used three-dimensional microtissues grown in vitro from fibroblasts in macroscopically engineered clefts for several weeks and found that fibroblasts transitioned into myofibroblasts at the highly tensed growth front as the microtissue progressively closed the cleft, in analogy to closing a wound site. Proliferation was up-regulated at the growth front, and new highly stretched fibronectin fibers were deposited, as revealed by fibronectin fluorescence resonance energy transfer probes. As the tissue was growing, the ECM underneath matured into a collagen-rich tissue containing mostly fibroblasts instead of myofibroblasts, and the fibronectin fibers were under reduced tension. This correlated with a progressive rounding of cells from the growth front inward, with decreased α–smooth muscle actin expression, YAP nuclear translocation, and cell proliferation. Together, this suggests that the myofibroblast phenotype is stabilized at the growth front by tensile forces, even in the absence of endogenously supplemented TGF-β, and reverts into a quiescent fibroblast phenotype already 10 μm behind the growth front, thus giving rise to a myofibroblast-to-fibroblast transition. This is the hallmark of reaching prohealing homeostasis. PMID:29349300

Alteration of medial-edge epithelium cell adhesion in two Tgf-β3 null mouse strains

PubMed Central

Martínez-Sanz, Elena; Del Río, Aurora; Barrio, Carmen; Murillo, Jorge; Maldonado, Estela; Garcillán, Beatriz; Amorós, María; Fuerte, Tamara; Fernández, Álvaro; Trinidad, Eva; Rabadán, M Ángeles; López, Yamila; Martínez, M Luisa; Martínez-Álvarez, Concepción

2008-01-01

Although palatal shelf adhesion is a crucial event during palate development, little work has been carried out to determine which molecules are responsible for this process. Furthermore, whether altered palatal shelf adhesion causes the cleft palate presented by Tgf-β3 null mutant mice has not yet been clarified. Here, we study the presence/distribution of some extracellular matrix and cell adhesion molecules at the time of the contact of palatal shelves in both wild-type and Tgf-β3 null mutant palates of two strains of mice (C57/BL/6J (C57), and MF1) that develop cleft palates of different severity. We have performed immunohistochemistry with antibodies against collagens IV and IX, laminin, fibronectin, the α5- and β1-integrins, and ICAM-1; in situ hybridization with a Nectin-1 riboprobe; and palatal shelf cultures treated or untreated with TGF-β3 or neutralizing antibodies against fibronectin or the α5-integrin. Our results show the location of these molecules in the wild-type mouse medial edge epithelium (MEE) of both strains at the time of the contact of palatal shelves; the heavier (C57) and milder (MF1) alteration of their presence in the Tgf-β3 null mutants; the importance of TGF-β3 to restore their normal pattern of expression; and the crucial role of fibronectin and the α5-integrin in palatal shelf adhesion. We thus provide insight into the molecular bases of this important process and the cleft palate presented by Tgf-β3 null mutant mice. PMID:18431835

G protein-coupled receptor kinase 2 positively regulates epithelial cell migration

PubMed Central

Penela, Petronila; Ribas, Catalina; Aymerich, Ivette; Eijkelkamp, Niels; Barreiro, Olga; Heijnen, Cobi J; Kavelaars, Annemieke; Sánchez-Madrid, Francisco; Mayor, Federico

2008-01-01

Cell migration requires integration of signals arising from both the extracellular matrix and messengers acting through G protein-coupled receptors (GPCRs). We find that increased levels of G protein-coupled receptor kinase 2 (GRK2), a key player in GPCR regulation, potentiate migration of epithelial cells towards fibronectin, whereas such process is decreased in embryonic fibroblasts from hemizygous GRK2 mice or upon knockdown of GRK2 expression. Interestingly, the GRK2 effect on fibronectin-mediated cell migration involves the paracrine/autocrine activation of a sphingosine-1-phosphate (S1P) Gi-coupled GPCR. GRK2 positively modulates the activity of the Rac/PAK/MEK/ERK pathway in response to adhesion and S1P by a mechanism involving the phosphorylation-dependent, dynamic interaction of GRK2 with GIT1, a key scaffolding protein in cell migration processes. Furthermore, decreased GRK2 levels in hemizygous mice result in delayed wound healing rate in vivo, consistent with a physiological role of GRK2 as a regulator of coordinated integrin and GPCR-directed epithelial cell migration. PMID:18369319

Fetal Fibronectin Signaling Induces Matrix Metalloproteases and Cyclooxygenase-2 (COX-2) in Amnion Cells and Preterm Birth in Mice*

PubMed Central

Mogami, Haruta; Kishore, Annavarapu Hari; Shi, Haolin; Keller, Patrick W.; Akgul, Yucel; Word, R. Ann

2013-01-01

Fetal fibronectin (fFN) in cervical and vaginal secretions has been used as a predictor of preterm delivery. Here, we clarified the pathological function of fFN on cell type-specific matrix metalloproteinases (MMPs) and prostaglandin synthesis in fetal membranes. Treatment of amnion mesenchymal cells with fFN resulted in dramatic increases in MMP-1 and MMP-9 mRNA and enzymatic activity as well as COX-2 mRNA and prostaglandin E2 synthesis, activating both NFκB and ERK1/2 signaling. Fetal FN-induced increases in MMPs and COX-2 were mediated through its extra domain A and Toll-like receptor 4 expressed in mesenchymal cells. Lipopolysaccharide and TNF-α increased the release of free FN in medium of amnion epithelial cells in culture. Finally, injection of fFN in pregnant mice resulted in preterm birth. Collectively, these results indicate that fFN is not only a marker of preterm delivery but also plays a significant role in the pathogenesis of preterm labor and premature rupture of fetal membranes. PMID:23184961

Sphingosine kinase 1 mediates AGEs-induced fibronectin upregulation in diabetic nephropathy

PubMed Central

Chen, Cheng; Gong, Wenyan; Li, Changzheng; Xiong, Fengxiao; Wang, Shaogui; Huang, Junying; Wang, Yu; Chen, Zhiquan; Chen, Qiuhong; Liu, Peiqing; Lan, Tian; Huang, Heqing

2017-01-01

Activation of sphingosine kinase 1 (SphK1) signaling pathway mediates fibronectin (FN) upregulation in glomerular mesangial cells (GMCs) under high glucose (HG) condition. However, the roles of SphK1 in advanced glycation end products (AGEs)-induced DN have not been elucidated. Here we show that AGEs upregulated FN and SphK1 and SphK1 activity. Inhibition of SphK1 signaling attenuated AGEs-induced FN synthesis in GMCs. Inhibition of AGE receptor (RAGE) signaling reduced the upregulation of FN and SphK1 and SphK1 activity in GMCs induced by AGEs. Treatment of aminoguanidine ameliorates the renal injury and fibrosis in STZ-induced diabetic mice and attenuated SphK1 expression and activity in diabetic mouse kidneys. The renal injury and fibrosis in diabetic SphK1-/- mice was significantly attenuated than WT mice. Furthermore, AGEs upregulated SphK1 by reducing its degradation and prolonging its half-life. Conclusion: SphK1 mediates AGEs-induced FN synthesis in GMCs and diabetic mice under hyperglycemic condition. PMID:29108256

Streptococcus mutans autolysin AtlA is a fibronectin-binding protein and contributes to bacterial survival in the bloodstream and virulence for infective endocarditis.

PubMed

Jung, Chiau-Jing; Zheng, Quan-Hau; Shieh, Ya-Hsiung; Lin, Chi-Shuan; Chia, Jean-San

2009-11-01

Streptococcus mutans, a commensal of the human oral cavity, can survive in the bloodstream and cause infective endocarditis (IE). However, the virulence factors associated with this manifestation of disease are not known. Here, we demonstrate that AtlA, an autolysin of S. mutans is a newly identified fibronectin (Fn) binding protein and contributes to bacterial resistance to phagocytosis and survival in the bloodstream. Interestingly, prior exposure to plasma at low concentrations was sufficient to enhance bacterial survival in the circulation. Calcium ions at physiological plasma concentrations induced maturation of AtlA from the 104-90 kDa isoform resulting in increased Fn binding and resistance to phagocytosis. An isogenic mutant strain defective in AtlA expression exhibited reduced survival and virulence when tested in a rat model of IE compared with the wild-type and complemented strains. The data presented suggest that plasma components utilized by S. mutans enhanced survival in the circulation and AtlA is a virulence factor associated with infective endocarditis.

Cell-micropatterning by micromolding in capillary technique based on UV polymerization

NASA Astrophysics Data System (ADS)

Park, Min J.; Choi, Won M.; Park, O. O.

2006-01-01

Although optical lithography or photolithography is one of the most well-established techniques for micro, nano-fabrication, its usage with proteins and cells is restricted by steps that must be carried out in harsh organic solvents. Here, we present simple methods for cell-micropatterning using poly(dimethylsiloxane) (PDMS) as a mold. Cell non-adhesive surface or nonfouling surface providing a physico-chemical barrier to cell attachment was introduced for biomaterial pattering, where cells fail to interact with the surface over desired periods of time determined by each application. Poly(ethylene glycol) (PEG) was selected as nonfouling material to inhibit protein adsorption from biological media. The fouling resistance of PEG polymer is often explained by a steric repulsion interaction, resulting from the compression of PEG chains as proteins approach the surface. We also chose fibronectin to direct cell attachment because it is an extracellular matrix protein that is involved in the adhesion and spreading of anchorage-dependent cells. In our experiment, we propose two methods by application of micromolding in capillary (MIMIC) method based on UV polymerization to obtain a surface of alternating PEG and fibronectin. First to fabricate PEG microstructure via MIMIC method, a pre-patterned PDMS mold is placed on a desired substrate, and then the relief structure in the mold forms a network of empty channels. A drop of ethylene glycol monomer solution containing initiator for UV polymerization is placed at the open ends of the network of channels, which is then polymerized by exposure to UV light at room temperature. Once PEG microstructure is fabricated, incubation of the patterned surface in a fibronectin-containing solution allows back-filling of only the bare regions with fibronectin via adsorption. In the alternative method, a substrate is first incubated in a fibronectin-containing solution, leading to the adsorption of fibronectin over the entire surface, and the fibronectin-adsorbed substrate is then micropatterned with the PEG by MIMIC based on UV polymerization. Both methods create reproducible alternating PEG and fibronectin patterns applicable to cell-surface interactions on the microscale.

Carbon Ion Irradiation Inhibits Glioma Cell Migration Through Downregulation of Integrin Expression

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

Rieken, Stefan, E-mail: Stefan.Rieken@med.uni-heidelberg.de; Habermehl, Daniel; Wuerth, Lena

2012-05-01

Purpose: To investigate the effect of carbon ion irradiation on glioma cell migration. Methods and Materials: U87 and Ln229 glioma cells were irradiated with photons and carbon ions. Migration was analyzed 24 h after irradiation. Fluorescence-activated cell sorting analysis was performed in order to quantify surface expression of integrins. Results: Single photon doses of 2 Gy and 10 Gy enhanced {alpha}{sub {nu}}{beta}{sub 3} and {alpha}{sub {nu}}{beta}{sub 5} integrin expression and caused tumor cell hypermigration on both vitronectin (Vn) and fibronectin (Fn). Compared to integrin expression in unirradiated cells, carbon ion irradiation caused decreased integrin expression and inhibited cell migration onmore » both Vn and Fn. Conclusion: Photon radiotherapy (RT) enhances the risk of tumor cell migration and subsequently promotes locoregional spread via photon induction of integrin expression. In contrast to photon RT, carbon ion RT causes decreased integrin expression and suppresses glioma cell migration on both Vn and Fn, thus promising improved local control.« less

Insulin-Like Growth Factor-I Stimulates Fibronectin Gene Expression in Rat Vascular Smooth Muscle Cells and Glomerular Mesangial Cells

DTIC Science & Technology

1993-09-21

negative feedback on GH secretion. The GH/IGF- I hormonal axis is further strengthened by clinical presentations of acromegaly and Laron dwarfism... Acromegaly patients afflicted with high levels of GH have elevated levels of IGF-I (Clemmons et al., 1980) . The opposite is true for Laron dwarfs who...Kjellberg, R.N.; Van Wyk, J.J. Estradiol Treatment of Acromegaly . Reduction of Immunoreactive Somatomedin-C and Improvement of Metabolic Status

The mouse neuronal cell surface protein F3: a phosphatidylinositol- anchored member of the immunoglobulin superfamily related to chicken contactin

PubMed Central

1989-01-01

Several members of the Ig superfamily are expressed on neural cells where they participate in surface interactions between cell bodies and processes. Their Ig domains are more closely related to each other than to Ig variable and constant domains and have been grouped into the C2 set. Here, we report the cloning and characterization of another member of this group, the mouse neuronal cell surface antigen F3. The F3 cDNA sequence contains an open reading frame that could encode a 1,020-amino acid protein consisting of a signal sequence, six Ig-like domains of the C2 type, a long premembrane region containing two segments that exhibit sequence similarity to fibronectin type III repeats and a moderately hydrophobic COOH-terminal sequence. The protein does not contain a typical transmembrane segment but appears to be attached to the membrane by a phosphatidylinositol anchor. Antibodies against the F3 protein recognize a prominent 135-kD protein in mouse brain. In fetal brain cultures, they stain the neuronal cell surface and, in cultures maintained in chemically defined medium, most prominently neurites and neurite bundles. The mouse f3 gene maps to band F of chromosome 15. The gene transcripts detected in the brain by F3 cDNA probes are developmentally regulated, the highest amounts being expressed between 1 and 2 wk after birth. The F3 nucleotide and deduced amino acid sequence show striking similarity to the recently published sequence of the chicken neuronal cell surface protein contactin. However, there are important differences between the two molecules. In contrast to F3, contactin has a transmembrane and a cytoplasmic domain. Whereas contactin is insoluble in nonionic detergent and is tightly associated with the cytoskeleton, about equal amounts of F3 distribute between buffer-soluble, nonionic detergent-soluble, and detergent- insoluble fractions. Among other neural cell surface proteins, F3 most resembles the neuronal cell adhesion protein L1, with 25% amino acid identity between their extracellular domains. Based on its structural similarity with known cell adhesion proteins of nervous tissue and with L1 in particular, we propose that F3 mediates cell surface interactions during nervous system development. PMID:2474555

Molecular cloning of a novel receptor tyrosine kinase, tif, highly expressed in human ovary and testis.

PubMed

Dai, W; Pan, H; Hassanain, H; Gupta, S L; Murphy, M J

1994-03-01

Using a combination of polymerase chain reaction and conventional cDNA library screening approaches, we have cloned and characterized a putative receptor tyrosine kinase termed tif. The extracellular domain of tif has an immunoglobulin-like loop and a fibronectin type III structure. The intracellular domain contains a tyrosine kinase domain. Compared with ryk, a ubiquitously expressed receptor tyrosine kinase, tif expression is tissue-specific with human ovary and testis containing the highest amount of tif mRNA. Many other tested human tissues such as heart, liver, pancreas and thymus do not contain detectable levels of tif mRNA. The molecular cloning and characterization of tif cDNA will facilitate the identification of a potential ligand(s) for the putative receptor and the study of its biological role.

Flagellin and GroEL mediates in vitro binding of an atypical enteropathogenic Escherichia coli to cellular fibronectin.

PubMed

Moraes, Claudia T P; Polatto, Juliana M; Rossato, Sarita S; Izquierdo, Mariana; Munhoz, Danielle D; Martins, Fernando H; Pimenta, Daniel C; Farfan, Mauricio J; Elias, Waldir P; Barbosa, Ângela S; Piazza, Roxane M F

2015-12-18

Enteropathogenic Escherichia coli (EPEC) is distinguished mainly by the presence of EPEC adherence factor plasmid (pEAF) in typical EPEC (tEPEC) and its absence in atypical EPEC (aEPEC). The initial adherence to the intestinal mucosa is complex and mediated by adhesins other than bundle-forming pilus, which is not produced by aEPEC. Extracellular matrix (ECM) proteins of eukaryotic cells are commonly recognized by bacterial adhesins. Therefore, binding to ECM proteins may facilitate colonization, invasion and/or signaling by intestinal pathogens. Previous studies from our group demonstrated that aEPEC O26:H11 (strain BA2103) showed high binding activity to fibronectin, not shared by its counterpart, aEPEC O26:HNM. In the present study, using mass spectrometry after fibronectin-associated immunoprecipitation, two proteins, flagellin (50 kDa) and GroEL (52 kDa), were identified and BA2103 binding ability to fibronectin was inhibited in the presence of anti-H11 and anti-GroEL sera, but not by either naïve rabbit or other unrelated sera. It was also observed that the presence of purified flagellin inhibits adhesion of BA2103 to cellular fibronectin in a dose-dependent manner. Additionally, BA2103 GroEL is similar to the same protein of uropathogenic E. coli. Our results suggest that flagellin may play a role in the in vitro interaction of BA2103 with cellular fibronectin, and GroEL can be an accessory protein in this process.

Polymorphisms in Fibronectin Binding Protein A of Staphylococcus Aureus are Associated with Infection of Cardiovascular Devices

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

Lower, Steven; Lamlertthon, Supaporn; Casillas-Ituarte, Nadia

Medical implants, like cardiovascular devices, improve the quality of life for countless individuals but may become infected with bacteria like Staphylococcus aureus. Such infections take the form of a bio-film, a structured community of bacterial cells adherent to the surface of a solid substrate. Every bio-film begins with an attractive force or bond between bacterium and substratum. We used atomic force microscopy to probe experimentally forces between a fibronectin-coated surface (i.e., proxy for an implanted cardiac device) and fibronectin-binding receptors on the surface of individual living bacteria from each of 80 clinical isolates of S. aureus. These isolates originated frommore » humans with infected cardiac devices (CDI; n = 26), uninfected cardiac devices (n = 20), and the anterior nares of asymptomatic subjects (n = 34). CDI isolates exhibited a distinct bindingforce signature and had speci!c single amino acid polymorphisms in fibronectin-binding protein A corresponding to E652D, H782Q, and K786N. In silico molecular dynamics simulations demonstrate that residues D652, Q782, and N786 in fibronectin-binding protein A form extra hydrogen bonds with fibronectin, complementing the higher binding force and energy measured by atomic force microscopy for the CDI isolates. This study is significant, because it links pathogenic bacteria biofilms from the length scale of bonds acting across a nanometer-scale space to the clinical presentation of disease at the human dimension.« less

Fibronectin connecting segment-1 peptide inhibits pathogenic leukocyte trafficking and inflammatory demyelination in experimental models of chronic inflammatory demyelinating polyradiculoneuropathy.

PubMed

Dong, Chaoling; Greathouse, Kelsey M; Beacham, Rebecca L; Palladino, Steven P; Helton, E Scott; Ubogu, Eroboghene E

2017-06-01

The molecular determinants of pathogenic leukocyte migration across the blood-nerve barrier (BNB) in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) are unknown. Specific disease modifying therapies for CIDP are also lacking. Fibronectin connecting segment-1 (FNCS1), an alternatively spliced fibronectin variant expressed by microvascular endothelial cells at sites of inflammation in vitro and in situ, is a counterligand for leukocyte α 4 integrin (also known as CD49d) implicated in pathogenic leukocyte trafficking in multiple sclerosis and inflammatory bowel disease. We sought to determine the role of FNCS1 in CIDP patient leukocyte trafficking across the BNB in vitro and in severe chronic demyelinating neuritis in vivo using a representative spontaneous murine CIDP model. Peripheral blood mononuclear leukocytes from 7 untreated CIDP patients were independently infused into a cytokine-treated, flow-dependent in vitro BNB model system. Time-lapse digital video microscopy was performed to visualize and quantify leukocyte trafficking, comparing FNCS1 peptide blockade to relevant controls. Fifty 24-week old female B7-2 deficient non-obese diabetic mice with spontaneous autoimmune peripheral polyneuropathy (SAPP) were treated daily with 2mg/kg FNCS1 peptide for 5days via intraperitoneal injection with appropriate controls. Neurobehavioral measures of disease severity, motor nerve electrophysiology assessments and histopathological quantification of inflammation and morphometric assessment of demyelination were performed to determine in vivo efficacy. The biological relevance of FNCS1 and CD49d in CIDP was evaluated by immunohistochemical detection in affected patient sural nerve biopsies. 25μM FNCS1 peptide maximally inhibited CIDP leukocyte trafficking at the human BNB in vitro. FNCS1 peptide treatment resulted in significant improvements in disease severity, motor electrophysiological parameters of demyelination and histological measures of inflammatory demyelination. Microvessels demonstrating FNCS1 expression and CD49d+ leukocytes were seen within the endoneurium of patient nerve biopsies. Taken together, these results imply a role for FNCS1 in pathogenic leukocyte trafficking in CIDP, providing a potential target for therapeutic modulation. Copyright © 2017 Elsevier Inc. All rights reserved.

A low-molecular-weight galactofucan from the seaweed, Spatoglossum schröederi, binds fibronectin and inhibits capillary-like tube formation in vitro.

PubMed

Menezes, Maira Maria; Nobre, Leonardo Thiago Duarte Barreto; Rossi, Gustavo Rodrigues; Almeida-Lima, Jailma; Melo-Silveira, Raniere Fagundes; Franco, Celia Regina Cavichiolo; Trindade, Edvaldo Silva; Nader, Helena Bonciani; Rocha, Hugo Alexandre Oliveira

2018-05-01

A low-molecular-weight (LMW) heterofucan (designated fucan B) was obtained from the brown seaweed, Spatoglossum schröederi, and its activity as an inhibitor of capillary-like tube formation by endothelial cells (ECs) was analyzed. Chemical, infrared and electrophoretic analyses confirmed the identity of fucan B. In contrast to other LMW fucans, fucan B (0.012-0.1 mg/mL) inhibited ECs capillary-like tube formation in a concentration-dependent manner. In addition, fucan B (0.01-0.05 mg/mL) did not affect ECs proliferation. Fucan B also inhibited ECs migration on a fibronectin-coated surface, but not on laminin- or collagen-coated surfaces. Biotinylated fucan B was used as a probe to identify its localization. Confocal microscopy experiments revealed that biotinylated fucan did not bind to the cell surface, but rather only to fibronectin. Our findings suggest that fucan B inhibits ECs capillary-like tube formation and migration by binding directly to fibronectin and blocking fibronectin sites recognized by cell surface ligands. However, further studies are needed to evaluate the in vivo effects of fucan B. Copyright © 2018 Elsevier B.V. All rights reserved.

Functional Analysis of an S-Layer-Associated Fibronectin-Binding Protein in Lactobacillus acidophilus NCFM

PubMed Central

Hymes, Jeffrey P.; Johnson, Brant R.; Barrangou, Rodolphe

2016-01-01

Bacterial surface layers (S-layers) are crystalline arrays of self-assembling proteinaceous subunits called S-layer proteins (Slps) that comprise the outermost layer of the cell envelope. Many additional proteins that are associated with or embedded within the S-layer have been identified in Lactobacillus acidophilus NCFM, an S-layer-forming bacterium that is widely used in fermented dairy products and probiotic supplements. One putative S-layer-associated protein (SLAP), LBA0191, was predicted to mediate adhesion to fibronectin based on the in silico detection of a fibronectin-binding domain. Fibronectin is a major component of the extracellular matrix (ECM) of intestinal epithelial cells. Adhesion to intestinal epithelial cells is considered an important trait for probiotic microorganisms during transit and potential association with the intestinal mucosa. To investigate the functional role of LBA0191 (designated FbpB) in L. acidophilus NCFM, an fbpB-deficient strain was constructed. The L. acidophilus mutant with a deletion of fbpB lost the ability to adhere to mucin and fibronectin in vitro. Homologues of fbpB were identified in five additional putative S-layer-forming species, but no homologues were detected in species outside the L. acidophilus homology group. PMID:26921419

Scaffold composition affects cytoskeleton organization, cell-matrix interaction and the cellular fate of human mesenchymal stem cells upon chondrogenic differentiation.

PubMed

Li, Yuk Yin; Choy, Tze Hang; Ho, Fu Chak; Chan, Pui Barbara

2015-06-01

The stem cell niche, or microenvironment, consists of soluble, matrix, cell and mechanical factors that together determine the cellular fates and/or differentiation patterns of stem cells. Collagen and glycosaminoglycans (GAGs) are important scaffolding materials that can mimic the natural matrix niche. Here, we hypothesize that imposing changes in the scaffold composition or, more specifically, incorporating GAGs into the collagen meshwork, will affect the morphology, cytoskeletal organization and integrin expression profiles, and hence the fate of human mesenchymal stem cells (MSCs) upon the induction of differentiation. Using chondrogenesis as an example, we microencapsulated MSCs in three scaffold systems that had varying matrix compositions: collagen alone (C), aminated collagen (AC) and aminated collagen with GAGs (ACG). We then induced the MSCs to differentiate toward a chondrogenic lineage, after which, we characterized the cell viability and morphology, as well as the level of cytoskeletal organization and the integrin expression profile. We also studied the fate of the MSCs by evaluating the major chondrogenic markers at both the gene and protein level. In C, MSC chondrogenesis was successfully induced and MSCs that spread in the scaffolds had a clear actin cytoskeleton; they expressed integrin α2β1, α5 and αv; promoted sox9 nuclear localization transcription activation; and upregulated the expression of chondrogenic matrix markers. In AC, MSC chondrogenesis was completely inhibited but the scaffold still supported cell survival. The MSCs did not spread and they had no actin cytoskeleton; did not express integrin α2 or αv; they failed to differentiate into chondrogenic lineage cells even on chemical induction; and there was little colocalization or functional interaction between integrin α5 and fibronectin. In ACG, although the MSCs did not express integrin α2, they did express integrin αv and there was strong co-localization and hence functional binding between αv and fibronectin. In addition, vimentin was the dominant cytoskeletal protein in these cells, and the chondrogenic marker genes were expressed but at a much lower level than in the MSCs encapsulated in C alone. This work suggests the importance of controlling the matrix composition as a strategy to manipulate cell-matrix interactions (through changes in the integrin expression profile and cytoskeleton organization), and hence stem cell fates. Copyright © 2015 Elsevier Ltd. All rights reserved.

Biochemical basis of vocal fold mobilization after microflap surgery in a rabbit model.

PubMed

Mitchell, Joshua R; Kojima, Tsuyoshi; Wu, Hongmei; Garrett, C Gaelyn; Rousseau, Bernard

2014-02-01

To investigate phonation-related extracellular matrix (ECM) changes in the vocal fold lamina propria after microflap surgery using an in vivo rabbit phonation model. Prospective animal study. Twenty-four New Zealand White rabbits were used in this study. Quantitative polymerase chain reaction and immunohistochemistry were used to investigate alterations in vocal fold ECM proinflammatory and profibrotic gene, and protein expression from a control group of animals receiving a microflap without phonation and a separate group of animals receiving experimentally induced phonation on postmicroflap days 0, 3, and 7. IHC demonstrated the highest concentration of CD45 in vocal folds on postoperative day 0. Staining for CD45 was absent by postoperative day 7, with no differences in CD45 staining between groups. Fibronectin gene expression increased significantly on postoperative day 3 in the control and experimentally induced phonation groups, with maximal staining of fibronectin around the microflap incision on postoperative day 7. No alterations in cyclooxygenase-2, interleukin-1β, and transforming growth factor-β1 gene expression were observed between groups. Results of the present study revealed an acute inflammatory response in the vocal fold at the time of microflap (day 0) and up to 3 days post-microflap. By post-operative day 3, staining of CD45 positive cells decreased, with essentially no evidence of inflammation by post-operative day 7. With the end of the acute inflammatory response occurring around day 3, these data may provide support for mobilizing tissue after inflammation has subsided and the process of active tissue remodeling has ensued (days 3-7). N/A. © 2013 The American Laryngological, Rhinological and Otological Society, Inc.

Morinda citrifolia Linn. Reduces Parasite Load and Modulates Cytokines and Extracellular Matrix Proteins in C57BL/6 Mice Infected with Leishmania (Leishmania) amazonensis.

PubMed

Almeida-Souza, Fernando; Cardoso, Flávia de Oliveira; Souza, Bruno Vinicius da Conceição; do Valle, Tânia Zaverucha; de Sá, Joicy Cortez; Oliveira, Iara Dos Santos da Silva; de Souza, Celeste da Silva Freitas; Moragas Tellis, Carla Junqueira; Chagas, Maria do Socorro Dos Santos; Behrens, Maria Dutra; Abreu-Silva, Ana Lúcia; Calabrese, Kátia da Silva

2016-08-01

The absence of an effective vaccine and the debilitating chemotherapy for Leishmaniasis demonstrate the need for developing alternative treatments. Several studies conducted with Morinda citrifolia have shown various biological activities, including antileishmanial activity, however its mechanisms of action are unknown. This study aimed to analyze the in vivo activity of M. citrifolia fruit juice (Noni) against Leishmania (Leishmania) amazonensis in C57BL/6 mice. M. citrifolia fruit juice from the Brazilian Amazon has shown the same constitution of other juices produced around the world and liquid chromatography-mass spectrometry analysis identified five compounds: deacetylasperulosidic acid, asperulosidic acid, rutin, nonioside B and nonioside C. Daily intragastric treatment with Noni was carried out after 55 days of L. (L.) amazonensis infection in C57BL/6 mice. Parasitic loads, cytokine and extracellular protein matrix expressions of the lesion site were analyzed by qPCR. Histopathology of the lesion site, lymph nodes and liver were performed to evaluate the inflammatory processes. Cytokines and biochemical parameters of toxicity from sera were also evaluated. The Noni treatment at 500 mg.kg-1.day-1 for 60 days decreased the lesion size and parasitic load in the footpad infected with L. (L.) amazonensis. The site of infection also showed decreased inflammatory infiltrates and decreased cytokine expressions for IL-12, TNF-α, TGF-β and IL-10. On the other hand, Noni treatment enhanced the extracellular matrix protein expressions of collagen IV, fibronectin and laminin in the infected footpad as well collagen I and II, fibronectin and laminin in the mock-infected footpads. No toxicity was observed at the end of treatment. These data show the efficacy of Noni treatment.

Differential Effects of Tissue Culture Coating Substrates on Prostate Cancer Cell Adherence, Morphology and Behavior

PubMed Central

Liberio, Michelle S.; Sadowski, Martin C.; Soekmadji, Carolina; Davis, Rohan A.; Nelson, Colleen C.

2014-01-01

Weak cell-surface adhesion of cell lines to tissue culture surfaces is a common problem and presents technical limitations to the design of experiments. To overcome this problem, various surface coating protocols have been developed. However, a comparative and precise real-time measurement of their impact on cell behavior has not been conducted. The prostate cancer cell line LNCaP, derived from a patient lymph node metastasis, is a commonly used model system in prostate cancer research. However, the cells’ characteristically weak attachment to the surface of tissue culture vessels and cover slips has impeded their manipulation and analysis and use in high throughput screening. To improve the adherence of LNCaP cells to the culture surface, we compared different coating reagents (poly-l-lysine, poly-l-ornithine, collagen type IV, fibronectin, and laminin) and culturing conditions and analyzed their impact on cell proliferation, adhesion, morphology, mobility and gene expression using real-time technologies. The results showed that fibronectin, poly-l-lysine and poly-l-ornithine improved LNCaP cells adherence and provoked cell morphology alterations, such as increase of nuclear and cellular area. These coating reagents also induced a higher expression of F-actin and reduced cell mobility. In contrast, laminin and collagen type IV did not improve adherence but promoted cell aggregation and affected cell morphology. Cells cultured in the presence of laminin displayed higher mobility than control cells. All the coating conditions significantly affected cell viability; however, they did not affect the expression of androgen receptor-regulated genes. Our comparative findings provide important insight for the selection of the ideal coating reagent and culture conditions for the cancer cell lines with respect to their effect on proliferation rate, attachment, morphology, migration, transcriptional response and cellular cytoskeleton arrangement. PMID:25375165

Morinda citrifolia Linn. Reduces Parasite Load and Modulates Cytokines and Extracellular Matrix Proteins in C57BL/6 Mice Infected with Leishmania (Leishmania) amazonensis

PubMed Central

Almeida-Souza, Fernando; Cardoso, Flávia de Oliveira; Souza, Bruno Vinicius da Conceição; do Valle, Tânia Zaverucha; de Sá, Joicy Cortez; Oliveira, Iara dos Santos da Silva; de Souza, Celeste da Silva Freitas; Moragas Tellis, Carla Junqueira; Chagas, Maria do Socorro dos Santos; Behrens, Maria Dutra

2016-01-01

The absence of an effective vaccine and the debilitating chemotherapy for Leishmaniasis demonstrate the need for developing alternative treatments. Several studies conducted with Morinda citrifolia have shown various biological activities, including antileishmanial activity, however its mechanisms of action are unknown. This study aimed to analyze the in vivo activity of M. citrifolia fruit juice (Noni) against Leishmania (Leishmania) amazonensis in C57BL/6 mice. M. citrifolia fruit juice from the Brazilian Amazon has shown the same constitution of other juices produced around the world and liquid chromatography–mass spectrometry analysis identified five compounds: deacetylasperulosidic acid, asperulosidic acid, rutin, nonioside B and nonioside C. Daily intragastric treatment with Noni was carried out after 55 days of L. (L.) amazonensis infection in C57BL/6 mice. Parasitic loads, cytokine and extracellular protein matrix expressions of the lesion site were analyzed by qPCR. Histopathology of the lesion site, lymph nodes and liver were performed to evaluate the inflammatory processes. Cytokines and biochemical parameters of toxicity from sera were also evaluated. The Noni treatment at 500 mg.kg-1.day-1 for 60 days decreased the lesion size and parasitic load in the footpad infected with L. (L.) amazonensis. The site of infection also showed decreased inflammatory infiltrates and decreased cytokine expressions for IL-12, TNF-α, TGF-β and IL-10. On the other hand, Noni treatment enhanced the extracellular matrix protein expressions of collagen IV, fibronectin and laminin in the infected footpad as well collagen I and II, fibronectin and laminin in the mock-infected footpads. No toxicity was observed at the end of treatment. These data show the efficacy of Noni treatment. PMID:27579922

A Novel Antifibrotic Mechanism of Nintedanib and Pirfenidone. Inhibition of Collagen Fibril Assembly.

PubMed

Knüppel, Larissa; Ishikawa, Yoshihiro; Aichler, Michaela; Heinzelmann, Katharina; Hatz, Rudolf; Behr, Jürgen; Walch, Axel; Bächinger, Hans Peter; Eickelberg, Oliver; Staab-Weijnitz, Claudia A

2017-07-01

Idiopathic pulmonary fibrosis (IPF) is characterized by excessive deposition of extracellular matrix, in particular, collagens. Two IPF therapeutics, nintedanib and pirfenidone, decelerate lung function decline, but their underlying mechanisms of action are poorly understood. In this study, we sought to analyze their effects on collagen synthesis and maturation at important regulatory levels. Primary human fibroblasts from patients with IPF and healthy donors were treated with nintedanib (0.01-1.0 μM) or pirfenidone (100-1,000 μM) in the absence or presence of transforming growth factor-β1. Effects on collagen, fibronectin, FKBP10, and HSP47 expression, and collagen I and III secretion, were analyzed by quantitative polymerase chain reaction and Western blot. The appearance of collagen fibrils was monitored by scanning electron microscopy, and the kinetics of collagen fibril assembly was assessed using a light-scattering approach. In IPF fibroblasts, nintedanib reduced the expression of collagen I and V, fibronectin, and FKBP10 and attenuated the secretion of collagen I and III. Pirfenidone also down-regulated collagen V but otherwise showed fewer and less pronounced effects. By and large, the effects were similar in donor fibroblasts. For both drugs, electron microscopy of IPF fibroblast cultures revealed fewer and thinner collagen fibrils compared with untreated controls. Finally, both drugs dose-dependently delayed fibril formation of purified collagen I. In summary, both drugs act on important regulatory levels in collagen synthesis and processing. Nintedanib was more effective in down-regulating profibrotic gene expression and collagen secretion. Importantly, both drugs inhibited collagen I fibril formation and caused a reduction in and an altered appearance of collagen fibril bundles, representing a completely novel mechanism of action for both drugs.

Differentiation of Human Dental Pulp Stem Cells into Dopaminergic Neuron-like Cells in Vitro.

PubMed

Chun, So Young; Soker, Shay; Jang, Yu-Jin; Kwon, Tae Gyun; Yoo, Eun Sang

2016-02-01

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.

Role for transforming growth factor-beta1 in alport renal disease progression.

PubMed

Sayers, R; Kalluri, R; Rodgers, K D; Shield, C F; Meehan, D T; Cosgrove, D

1999-11-01

Alport syndrome results from mutations in either the alpha3(IV), alpha4(IV), or alpha5(IV) collagen genes. The disease is characterized by a progressive glomerulonephritis usually associated with a high-frequency sensorineural hearing loss. A mouse model for an autosomal form of Alport syndrome [collagen alpha3(IV) knockout] was produced and characterized. In this study, the model was exploited to demonstrate a potential role for transforming growth factor-beta1 (TGF-beta1) in Alport renal disease pathogenesis. Kidneys from normal and Alport mice, taken at different stages during the course of renal disease progression, were analyzed by Northern blot, in situ hybridization, and immunohistology for expression of TGF-beta1 and components of the extracellular matrix. Normal and Alport human kidney was examined for TGF-beta1 expression using RNase protection. The mRNAs encoding TGF-beta1 (in both mouse and human), entactin, fibronectin, and the collagen alpha1(IV) and alpha2(IV) chains were significantly induced in total kidney as a function of Alport renal disease progression. The induction of these specific mRNAs was observed in the glomerular podocytes of animals with advanced disease. Type IV collagen, laminin-1, and fibronectin were markedly elevated in the tubulointerstitium at 10 weeks, but not at 6 weeks, suggesting that elevated expression of specific mRNAs on Northern blots reflects events associated with tubulointerstitial fibrosis. The concomitant accumulation of mRNAs encoding TGF-beta1 and extracellular matrix components in the podocytes of diseased kidneys may reflect key events in Alport renal disease progression. These data suggest a role for TGF-beta1 in both glomerular and tubulointerstitial damage associated with Alport syndrome.

Cellular fibronectin response to supervised moderate aerobic training in patients with type 2 diabetes

PubMed Central

Alghadir, Ahmad H.; Gabr, Sami A.; Al-Eisa, Einas

2016-01-01

[Purpose] Physical activity is one of the most pivotal targets for the prevention and management of vascular complications, especially endothelial dysfunctions. Cellular fibronectin is an endothelium-derived protein involved in subendothelial matrix assembly. Its plasma levels reflect matrix alterations and vessel wall destruction in patients with type II diabetes. This study investigated the influence of 12 weeks of supervised aerobic training on cellular fibronectin and its relationship with insulin resistance and body weight in type II diabetic subjects. [Subjects and Methods] This study included 50 men with type II diabetes who had a mean age of 48.8 ± 14.6 years and were randomly divided into two groups: an aerobic exercise group (12 weeks, three 50 minutes sessions per week) and control group. To examine changes in cellular fibronectin, glycosylated hemoglobin, insulin resistance, fasting insulin, fasting blood sugar, and lipid profile, 5 ml of blood was taken from the brachial vein of patients before and 48 hours after completion of the exercise period and after 12 hours of fasting at rest. Data analysis was performed using the SPSS-16 software with the independent and paired t-tests. [Results] A significant decrease was observed in body mass index and body fat percentage in the experimental group. Compared with the control group, the aerobic exercise group showed a significant decrease in cellular fibronectin, glycosylated hemoglobin, insulin resistance, fasting insulin, fasting blood sugar, and lipid profile after 12 weeks of aerobic exercise. The change in cellular fibronectin showed positive significant correlation with body mass index, diabetic biomarkers, and physical activity level. [Conclusion] The results showed that supervised aerobic exercise as a stimulus can change the levels of cellular fibronectin as matrix metalloproteinase protein a long with improvement of insulin sensitivity and glycosylated hemoglobin in order to prevent cardiovascular diseases in men with diabetes PMID:27190433

PDGF-A suppresses contact inhibition during directional collective cell migration.

PubMed

Nagel, Martina; Winklbauer, Rudolf

2018-06-08

The leading edge mesendoderm (LEM) of the Xenopus gastrula moves as an aggregate by collective migration. However, LEM cells on fibronectin in vitro show contact inhibition of locomotion by quickly retracting lamellipodia upon mutual contact. We found that a fibronectin-integrin-syndecan module acts between p21-activated kinase-1 upstream and ephrinB1 downstream to promote the contact-induced collapse of lamellipodia. To function in this module, fibronectin has to be present as puncta on the surface of LEM cells. To overcome contact inhibition in LEM cell aggregates, PDGF-A deposited in the endogenous substratum of LEM migration blocks the fibronectin-integrin-syndecan module at the integrin level. This stabilizes lamellipodia preferentially in the direction of normal LEM movement and supports cell orientation and the directional migration of the coherent LEM cell mass. © 2018. Published by The Company of Biologists Ltd.

Single-molecule dynamic force spectroscopy of the fibronectin-heparin interaction

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

Mitchell, Gabriel; Lamontagne, Charles-Antoine; Lebel, Rejean

2007-12-21

The integrity of cohesive tissues strongly depends on the presence of the extracellular matrix, which provides support and anchorage for cells. The fibronectin protein and the heparin-like glycosaminoglycans are key components of this dynamic structural network. In this report, atomic force spectroscopy was used to gain insight into the compliance and the resistance of the fibronectin-heparin interaction. We found that this interaction can be described by an energetic barrier width of 3.1 {+-} 0.2 A and an off-rate of 0.2 {+-} 0.1 s{sup -1}. These dissociation parameters are similar to those of other carbohydrate-protein interactions and to off-rate values reportedmore » for more complex interactions between cells and extracellular matrix components. Our results indicate that the function of the fibronectin-heparin interaction is supported by its capacity to sustain significant deformations and considerable external mechanical forces.« less

Nickel-induced Epithelial-Mesenchymal Transition by Reactive Oxygen Species Generation and E-cadherin Promoter Hypermethylation*

PubMed Central

Wu, Chih-Hsien; Tang, Sheau-Chung; Wang, Po-Hui; Lee, Huei; Ko, Jiunn-Liang

2012-01-01

Epithelial-mesenchymal transition (EMT) is considered a critical event in the pathogenesis of lung fibrosis and tumor metastasis. During EMT, the expression of differentiation markers switches from cell-cell junction proteins such as E-cadherin to mesenchymal markers such as fibronectin. Although nickel-containing compounds have been shown to be associated with lung carcinogenesis, the role of nickel in the EMT process in bronchial epithelial cells is not clear. The aim of this study was to examine whether nickel contributes to EMT in human bronchial epithelial cells. We also attempted to clarify the mechanisms involved in NiCl2-induced EMT. Our results showed that NiCl2 induced EMT phenotype marker alterations such as up-regulation of fibronectin and down-regulation of E-cadherin. In addition, the potent antioxidant N-acetylcysteine blocked EMT and expression of HIF-1α induced by NiCl2, whereas the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine restored the down-regulation of E-cadherin induced by NiCl2. Promoter hypermethylation of E-cadherin, determined by quantitative real time methyl-specific PCR and bisulfate sequencing, was also induced by NiCl2. These results shed new light on the contribution of NiCl2 to carcinogenesis. Specifically, NiCl2 induces down-regulation of E-cadherin by reactive oxygen species generation and promoter hypermethylation. This study demonstrates for the first time that nickel induces EMT in bronchial epithelial cells. PMID:22648416

Selective Modulation of Integrin-mediated Cell Migration by Distinct ADAM Family MembersV⃞

PubMed Central

Huang, Jing; Bridges, Lance C.; White, Judith M.

2005-01-01

A disintegrin and a metalloprotease (ADAM) family members have been implicated in many biological processes. Although it is recognized that recombinant ADAM disintegrin domains can interact with integrins, little is known about ADAM-integrin interactions in cellular context. Here, we tested whether ADAMs can selectively regulate integrin-mediated cell migration. ADAMs were expressed in Chinese hamster ovary cells that express defined integrins (α4β1, α5β1, or both), and cell migration on full-length fibronectin or on its α4β1 or α5β1 binding fragments was studied. We found that ADAMs inhibit integrin-mediated cell migration in patterns dictated by the integrin binding profiles of their isolated disintegrin domains. ADAM12 inhibited cell migration mediated by the α4β1 but not the α5β1 integrin. ADAM17 had the reciprocal effect; it inhibited α5β1- but not α4β1-mediated cell migration. ADAM19 and ADAM33 inhibited migration mediated by both α4β1 and α5β1 integrins. A point mutation in the ADAM12 disintegrin loop partially reduced the inhibitory effect of ADAM12 on cell migration on the α4β1 binding fragment of fibronectin, whereas mutations that block metalloprotease activity had no effect. Our results indicate that distinct ADAMs can modulate cell migration mediated by specific integrins in a pattern dictated, at least in part, by their disintegrin domains. PMID:16079176

Sperm-binding fibronectin type II-module proteins are genetically linked and functionally related.

PubMed

Ekhlasi-Hundrieser, Mahnaz; Schäfer, Bettina; Philipp, Ute; Kuiper, Heidi; Leeb, Tosso; Mehta, Meenal; Kirchhoff, Christiane; Töpfer-Petersen, Edda

2007-05-01

Fibronectin type II (Fn2) module-containing proteins in the male genital tract are characterized by different numbers of Fn2 modules. Predominantly two classes exist which are distinct by having either two or four Fn2 modules. Minor variants with three Fn2 modules were also found in the human and the porcine epididymis. To reveal their relationship, mRNAs and proteins of representatives of these classes were studied in human, in Sus scrofa, and in rodents. Adult boars expressed members of both classes, i.e. ELSPBP1 and pB1, in subsequent regions of the epididymis, and both were under androgenic control. Human and rodent epididymides, on the other hand, alternatively contained only representatives of one of these two classes, i.e. ELSPBP1 in the human and two different pB1-related counterparts in rodents. ELSPBP1 and pB1-related genomic sequences were closely linked in chromosomal regions HSA 19q and SSC 6 q11-q21; conserved synteny between these regions is well established. On the other hand, in a syntenic region on mouse chromosome 7, ELSPBP1-related sequences were lacking. Tight binding to the sperm membrane via a choline-mediated mechanism was a common feature of the two classes of Fn2-module proteins, suggesting related function(s). However, differences in their regionalized expression patterns along the male genital tract as well as in association sites on the sperm surface suggested a species-specific sequential order in sperm binding.

Heparin-induced conformational changes of fibronectin within the extracellular matrix promote hMSC osteogenic differentiation.

PubMed

Li, Bojun; Lin, Zhe; Mitsi, Maria; Zhang, Yang; Vogel, Viola

2015-01-01

An increasing body of evidence suggests important roles of extracellular matrix (ECM) in regulating stem cell fate. This knowledge can be exploited in tissue engineering applications for the design of ECM scaffolds appropriate to direct stem cell differentiation. By probing the conformation of fibronectin (Fn) using fluorescence resonance energy transfer (FRET), we show here that heparin treatment of the fibroblast-derived ECM scaffolds resulted in more extended conformations of fibrillar Fn in ECM. Since heparin is a highly negatively charged molecule while fibronectin contains segments of positively charged modules, including FnIII13, electrostatic interactions between Fn and heparin might interfere with residual quaternary structure in relaxed fibronectin fibers thereby opening up buried sites. The conformation of modules FnIII12-14 in particular, which contain one of the heparin binding sites as well as binding sites for many growth factors, may be activated by heparin, resulting in alterations in growth factor binding to Fn. Indeed, upregulated osteogenic differentiation was observed when hMSCs were seeded on ECM scaffolds that had been treated with heparin and were subsequently chemically fixed. In contrast, either rigidifying relaxed fibers by fixation alone, or heparin treatment without fixation had no effect. We hypothesize that fibronectin's conformations within the ECM are activated by heparin such as to coordinate with other factors to upregulate hMSC osteogenic differentiation. Thus, the conformational changes of fibronectin within the ECM could serve as a 'converter' to tune hMSC differentiation in extracellular matrices. This knowledge could also be exploited to promote osteogenic stem cell differentiation on biomedical surfaces.

Silk-based biomaterials functionalized with fibronectin type II promotes cell adhesion.

PubMed

Pereira, Ana Margarida; Machado, Raul; da Costa, André; Ribeiro, Artur; Collins, Tony; Gomes, Andreia C; Leonor, Isabel B; Kaplan, David L; Reis, Rui L; Casal, Margarida

2017-01-01

The objective of this work was to exploit the fibronectin type II (FNII) module from human matrix metalloproteinase-2 as a functional domain for the development of silk-based biopolymer blends that display enhanced cell adhesion properties. The DNA sequence of spider dragline silk protein (6mer) was genetically fused with the FNII coding sequence and expressed in Escherichia coli. The chimeric protein 6mer+FNII was purified by non-chromatographic methods. Films prepared from 6mer+FNII by solvent casting promoted only limited cell adhesion of human skin fibroblasts. However, the performance of the material in terms of cell adhesion was significantly improved when 6mer+FNII was combined with a silk-elastin-like protein in a concentration-dependent behavior. With this work we describe a novel class of biopolymer that promote cell adhesion and potentially useful as biomaterials for tissue engineering and regenerative medicine. This work reports the development of biocompatible silk-based composites with enhanced cell adhesion properties suitable for biomedical applications in regenerative medicine. The biocomposites were produced by combining a genetically engineered silk-elastin-like protein with a genetically engineered spider-silk-based polypeptide carrying the three domains of the fibronectin type II module from human metalloproteinase-2. These composites were processed into free-standing films by solvent casting and characterized for their biological behavior. To our knowledge this is the first report of the exploitation of all three FNII domains as a functional domain for the development of bioinspired materials with improved biological performance. The present study highlights the potential of using genetically engineered protein-based composites as a platform for the development of new bioinspired biomaterials. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Origin-Specific Adhesive Interactions of Mesenchymal Stem Cells with Platelets Influence Their Behavior After Infusion.

PubMed

Sheriff, Lozan; Alanazi, Asma; Ward, Lewis S C; Ward, Carl; Munir, Hafsa; Rayes, Julie; Alassiri, Mohammed; Watson, Steve P; Newsome, Phil N; Rainger, G E; Kalia, Neena; Frampton, Jon; McGettrick, Helen M; Nash, Gerard B

2018-02-28

We investigated the adhesive behavior of mesenchymal stem cells (MSC) in blood, which might influence their fate when infused as therapy. Isolated human bone marrow MSC (BMMSC) or umbilical cord MSC (UCMSC) adhered efficiently from flow to the matrix proteins, collagen, or fibronectin, but did not adhere to endothelial selectins. However, when suspended in blood, BMMSC no longer adhered to collagen, while UCMSC adhered along with many aggregated platelets. Neither MSC adhered to fibronectin from flowing blood, although the fibronectin surface did become coated with a platelet monolayer. UCMSC induced platelet aggregation in platelet rich plasma, and caused a marked drop in platelet count when mixed with whole human or mouse blood in vitro, or when infused into mice. In contrast, BMMSC did not activate platelets or induce changes in platelet count. Interestingly, isolated UCMSC and BMMSC both adhered to predeposited platelets. The differences in behavior in blood were attributable to expression of podoplanin (an activating ligand for the platelet receptor CLEC-2), which was detected on UCMSC, but not BMMSC. Thus, platelets were activated when bound to UCMSC, but not BMMSC. Platelet aggregation by UCMSC was inhibited by recombinant soluble CLEC-2, and UCMSC did not cause a reduction in platelet count when mixed with blood from mice deficient in CLEC-2. We predict that both MSC would carry platelets in the blood, but their interaction with vascular endothelium would depend on podoplanin-induced activation of the bound platelets. Such interactions with platelets might target MSC to damaged tissue, but could also be thrombotic. Stem Cells 2018. © 2018 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Developmental Competence of Buffalo (Bubalus bubalis) Pluripotent Embryonic Stem Cells Over Different Homologous Feeder Layers and the Comparative Evaluation with Various Extracellular Matrices.

PubMed

Sharma, Manjinder; Dubey, Pawan K; Kumar, Rajesh; Nath, Amar; Kumar, G Sai; Sharma, G Taru

2013-05-01

Use of somatic cells as a feeder layer to maintain the embryonic stem cells (ESCs) in undifferentiated state limits the stem cell research design, since experimental data may result from a combined ESCs and feeder cell response to various stimuli. Therefore, present study was designed to evaluate the developmental competence of the buffalo ESCs over different homogenous feeders and compare with various extracellular matrices using different concentrations of LIF. Inner cell masses (ICMs) of in vitro hatched blastocysts were cultured onto homologous feeders viz. fetal fibroblast, granulosa and oviductal cell feeder layers and synthetic matrices viz. fibronectin, collagen type I and matrigel in culture medium. Developmental efficiency was found higher for ESCs cultured on fetal fibroblast and granulosa layers (83.33%) followed by fibronectin (77.78%) at 30 ng LIF. Oviductal feeder was found to be the least efficient feeder showing only 11.11% undifferentiated primary ESC colonies at 30 ng LIF. However, neither feeder layer nor synthetic matrix could support the development of primary colonies at 10 ng LIF. Expression of SSEA- 4, TRA-1-60 and Oct-4 were found positive in ESC colonies from all the feeders and synthetic matrices with 20 ng and 30 ng LIF. Fetal fibroblast and granulosa cell while, amongst synthetic matrices, fibronectin were found to be equally efficient to support the growth and maintenance of ESCs pluripotency with 30 ng LIF. This well-defined culture conditions may provide an animal model for culturing human embryonic stem cells in the xeno-free or feeder-free conditions for future clinical applications.

Quantitative fetal fibronectin and cervical length to predict preterm birth in asymptomatic women with previous cervical surgery.

PubMed

Vandermolen, Brooke I; Hezelgrave, Natasha L; Smout, Elizabeth M; Abbott, Danielle S; Seed, Paul T; Shennan, Andrew H

2016-10-01

Quantitative fetal fibronectin testing has demonstrated accuracy for prediction of spontaneous preterm birth in asymptomatic women with a history of preterm birth. Predictive accuracy in women with previous cervical surgery (a potentially different risk mechanism) is not known. We sought to compare the predictive accuracy of cervicovaginal fluid quantitative fetal fibronectin and cervical length testing in asymptomatic women with previous cervical surgery to that in women with 1 previous preterm birth. We conducted a prospective blinded secondary analysis of a larger observational study of cervicovaginal fluid quantitative fetal fibronectin concentration in asymptomatic women measured with a Hologic 10Q system (Hologic, Marlborough, MA). Prediction of spontaneous preterm birth (<30, <34, and <37 weeks) with cervicovaginal fluid quantitative fetal fibronectin concentration in primiparous women who had undergone at least 1 invasive cervical procedure (n = 473) was compared with prediction in women who had previous spontaneous preterm birth, preterm prelabor rupture of membranes, or late miscarriage (n = 821). Relationship with cervical length was explored. The rate of spontaneous preterm birth <34 weeks in the cervical surgery group was 3% compared with 9% in previous spontaneous preterm birth group. Receiver operating characteristic curves comparing quantitative fetal fibronectin for prediction at all 3 gestational end points were comparable between the cervical surgery and previous spontaneous preterm birth groups (34 weeks: area under the curve, 0.78 [95% confidence interval 0.64-0.93] vs 0.71 [95% confidence interval 0.64-0.78]; P = .39). Prediction of spontaneous preterm birth using cervical length compared with quantitative fetal fibronectin for prediction of preterm birth <34 weeks of gestation offered similar prediction (area under the curve, 0.88 [95% confidence interval 0.79-0.96] vs 0.77 [95% confidence interval 0.62-0.92], P = .12 in the cervical surgery group; and 0.77 [95% confidence interval 0.70-0.84] vs 0.74 [95% confidence interval 0.67-0.81], P = .32 in the previous spontaneous preterm birth group). Prediction of spontaneous preterm birth using cervicovaginal fluid quantitative fetal fibronectin in asymptomatic women with cervical surgery is valid, and has comparative accuracy to that in women with a history of spontaneous preterm birth. Copyright © 2016 Elsevier Inc. All rights reserved.

Uric acid promotes vascular stiffness, maladaptive inflammatory responses and proteinuria in western diet fed mice.

PubMed

Aroor, Annayya R; Jia, Guanghong; Habibi, Javad; Sun, Zhe; Ramirez-Perez, Francisco I; Brady, Barron; Chen, Dongqing; Martinez-Lemus, Luis A; Manrique, Camila; Nistala, Ravi; Whaley-Connell, Adam T; Demarco, Vincent G; Meininger, Gerald A; Sowers, James R

2017-09-01

Aortic vascular stiffness has been implicated in the development of cardiovascular disease (CVD) and chronic kidney disease (CKD) in obese individuals. However, the mechanism promoting these adverse effects are unclear. In this context, promotion of obesity through consumption of a western diet (WD) high in fat and fructose leads to excess circulating uric acid. There is accumulating data implicating elevated uric acid in the promotion of CVD and CKD. Accordingly, we hypothesized that xanthine oxidase(XO) inhibition with allopurinol would prevent a rise in vascular stiffness and proteinuria in a translationally relevant model of WD-induced obesity. Four-week-old C57BL6/J male mice were fed a WD with excess fat (46%) and fructose (17.5%) with or without allopurinol (125mg/L in drinking water) for 16weeks. Aortic endothelial and extracellular matrix/vascular smooth muscle stiffness was evaluated by atomic force microscopy. Aortic XO activity, 3-nitrotyrosine (3-NT) and aortic endothelial sodium channel (EnNaC) expression were evaluated along with aortic expression of inflammatory markers. In the kidney, expression of toll like receptor 4 (TLR4) and fibronectin were assessed along with evaluation of proteinuria. XO inhibition significantly attenuated WD-induced increases in plasma uric acid, vascular XO activity and oxidative stress, in concert with reductions in proteinuria. Further, XO inhibition prevented WD-induced increases in aortic EnNaC expression and associated endothelial and subendothelial stiffness. XO inhibition also reduced vascular pro-inflammatory and maladaptive immune responses induced by consumption of a WD. XO inhibition also decreased WD-induced increases in renal TLR4 and fibronectin that associated proteinuria. Consumption of a WD leads to elevations in plasma uric acid, increased vascular XO activity, oxidative stress, vascular stiffness, and proteinuria all of which are attenuated with allopurinol administration. Copyright © 2017 Elsevier Inc. All rights reserved.

TGFβ1-mediated PI3K/Akt and p38 MAP kinase dependent alternative splicing of fibronectin extra domain A in human podocyte culture.

PubMed

Madne, Tarunkumar Hemraj; Dockrell, Mark Edward Carl

2018-04-30

Alternative splicing is an important gene regulation process to distribute proteins in health and diseases. Extra Domain A+ Fibronectin (EDA+Fn) is an alternatively spliced form of fibronectin (Fn) protein, present in the extra cellular matrix (ECM) and a recognised marker of various pathologies. TGFβ1 has been shown to induce alternative splicing of EDA+Fn in many cell types. Podocytes are spectacular cell type and play a key role in filtration and synthesise ECM proteins in renal physiology and pathology. In our previous study we have demonstrated expression and alternative splicing of EDA+Fn in basal condition in human podocytes culture. TGFβ1 further induced the basal expression and alternative splicing of EDA+Fn through Alk5 receptor and SR proteins. In this study, we have investigated TGFβ1 mediated signalling involved in alternative splicing of EDA+Fn in human podocytes. We have performed western blotting to characterise the expression of the EDA+Fn protein and other signalling proteins and RT-PCR to look for signalling pathways involved in regulation of alternative splicing of EDA+Fn in conditionally immortalised human podocytes culture.We have used TGFβ1 as a stimulator and SB431542, SB202190 and LY294002 for inhibitory studies. In this work, we have demonstrated in human podocytes culture TGFβ1 2.5ng/ml induced phosphorylation of Smad1/5/8, Smad2 and Smad3 via the ALK5 receptor. TGFβ1 significantly induced the PI3K/Akt pathway and the PI3K/Akt pathway inhibitor LY294002 significantly downregulated basal as well as TGFβ1 induced alternative splicing of EDA+Fn in human podocytes. In addition to this, TGFβ1 significantly induced the p38 MAP kinase signalling pathway and p38 MAP kinase signalling pathway inhibitor SB202190 downregulated the TGFβ1-mediated alternative splicing of EDA+Fn in human podocytes. The results with PI3K and p38 MAP kinase signalling pathway suggest that inhibiting PI3K signalling pathway downregulated the basal alternative splicing of EDA+Fn in human podocytes and its the inhibition of p38 Map Kinase signalling pathway which had specifically downregulated the TGFβ1 mediated alternative splicing of EDA+Fn in human podocytes culture. Activation of TGFβ1-mediated Smad1/5/8 via Alk5 receptor suggests that TGFβ1 signalling pathway involved Alk5/Alk1 receptor axis signalling in human podocytes.

Effect of obesity and exercise on the expression of the novel myokines, Myonectin and Fibronectin type III domain containing 5

PubMed Central

Mart, Ryan; Bond, Cherie E.

2014-01-01

Metabolic dysfunction in skeletal muscle is a major contributor to the development of type 2 diabetes. Endurance exercise training has long been established as an effective means to directly restore skeletal muscle glucose and lipid uptake and metabolism. However, in addition to the direct effects of skeletal muscle on glucose and lipids, there is renewed interest in the ability of skeletal muscle to coordinate metabolic activity of other tissues, such as adipose tissue and liver. The purpose of this study was to examine the effects of endurance exercise on the expression level of two novel muscle-derived secreted factors, or myokines, Myonectin and Fibronectin type III domain containing 5 (FNDC5), the precursor for Irisin. Methods. We performed immunoblot analysis and quantitative real-time PCR analysis of Myonectin and FNDC5 in the diaphragm muscles of obese Zucker rat (OZR) and lean Zucker rat (LZR) with 9 weeks of aerobic training on a motorized treadmill. Results. We show that myonectin gene expression is increased in the OZR model of obesity and decreases with exercise in both lean and obese Zucker rats. Conversely, myonectin protein concentration was elevated with exercise. Similarly, FNDC5 mRNA levels are significantly higher in the OZR, however exercise training had no effect on the expression level of FNDC5 in either the LZR or OZR. We did not observe any difference in muscle protein content of Irisin with obesity or exercise. Conclusion. Our data shows that exercise training does not increase either FNDC5 or myonectin gene expression, indicating that increased transcriptional regulation of these myokines is not induced by exercise. However, our data also indicates a yet to be explored disconnect between myonectin gene expression and protein content. Further, this report highlights the importance of verifying reference genes when completing gene expression analysis. We found that many commonly used reference genes varied significantly by obesity and/or exercise and would have skewed the results of this study if used to normalize gene expression data. The unstable reference genes include: beta-Actin, beta-2-microglobulin, Non-POU domain containing, octamer-binding, Peptidylprolyl isomerase H, 18S ribosomal RNA, TATA box binding protein and Transferrin receptor. PMID:25289190

Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase.

PubMed

Schlaepfer, D D; Hanks, S K; Hunter, T; van der Geer, P

The cytoplasmic focal adhesion protein-tyrosine kinase (FAK) localizes with surface integrin receptors at sites where cells attach to the extracellular matrix. Increased FAK tyrosine phosphorylation occurs upon integrin engagement with fibronectin. Here we show that adhesion of murine NIH3T3 fibroblasts to fibronectin promotes SH2-domain-mediated association of the GRB2 adaptor protein and the c-Src protein-tyrosine kinase (PTK) with FAK in vivo, and also results in activation of mitogen-activated protein kinase (MAPK). In v-Src-transformed NIH3T3, the association of v-Src, GRB2 and Sos with FAK is independent of cell adhesion to fibronectin. The GRB2 SH2 domain binds directly to tyrosine-phosphorylated FAK. Mutation of tyrosine residue 925 of FAK (YENV motif) to phenylalanine blocks GRB2 SH2-domain binding to FAK in vitro. Our results show that fibronectin binding to integrins on NIH3T3 fibroblasts promotes c-Src and FAK association and formation of an integrin-activated signalling complex. Phosphorylation of FAK at Tyr 925 upon fibronectin stimulation creates an SH2-binding site for GRB2 which may link integrin engagement to the activation of the Ras/MAPK signal transduction pathway.

Hyaluronic acid (with fibronectin) as a bioimplant for the vocal fold mucosa.

PubMed

Chan, R W; Titze, I R

1999-07-01

To measure the viscoelastic shear properties of hyaluronic acid, with and without fibronectin, and to compare them with those of the human vocal fold mucosa and other phonosurgical biomaterials. Viscoelastic shear properties of various implantable biomaterials (Teflon, gelatin, collagen, fat, hyaluronic acid, and hyaluronic acid with fibronectin) were measured with a parallel-plate rotational rheometer. Elastic and viscous shear properties were quantified as a function of oscillation frequency (0.01-15 Hz) at 37 degrees C. The shear properties of hyaluronic acid were relatively close to those of human vocal fold mucosal tissues reported previously. Hyaluronic acid at specific concentrations (0.5%-1%), with or without fibronectin, was found to exhibit viscous shear properties (viscous shear modulus and dynamic viscosity) similar to those of the average male and female vocal fold mucosa. According to a theory that establishes the effects of tissue shear properties on vocal fold oscillation, phonation threshold pressure (a measure of the ease of phonation) is directly related to the viscous shear modulus of the vibrating vocal fold mucosa. Therefore, our findings suggest that hyaluronic acid, either by itself or mixed with fibronectin, may be a potentially optimal bioimplant for the surgical management of vocal fold mucosal defects and lamina propria deficiencies (e.g., scarring) from a biomechanical standpoint.

Complementary effects of multi-protein components on biomineralization in vitro

PubMed Central

Ba, Xiaolan; Rafailovich, Miriam; Meng, Yizhi; Pernodet, Nadine; Wirick, Sue; Füredi-Milhofer, Helga; Qin, Yi-Xian; DiMasi, Elaine

2016-01-01

The extracellular matrix (ECM) is composed of mixed protein fibers whose precise composition affects biomineralization. New methods are needed to probe the interactions of these proteins with calcium phosphate mineral and with each other. Here we follow calcium phosphate mineralization on protein fibers self-assembled in vitro from solutions of fibronectin, elastin and their mixture. We probe the surface morphology and mechanical properties of the protein fibers during the early stages. The development of mineral crystals on the protein matrices is also investigated. In physiological mineralization solution, the elastic modulus of the fibers in the fibronectin-elastin mixture increases to a greater extent than that of the fibers from either pure protein. In the presence of fibronectin, longer exposure in the mineral solution leads to the formation of amorphous calcium phosphate particles templated along the self-assembled fibers, while elastin fibers only collect calcium without any mineral observed during early stage. TEM images confirm that small needle-shape crystals are confined inside elastin fibers which suppress the release of mineral outside the fibers during late stage, while hydroxyapatite crystals form when fibronectin is present. These results demonstrate complementary actions of the two ECM proteins fibronectin and elastin to collect cations and template mineral, respectively. PMID:20035875

Functional Analysis of an S-Layer-Associated Fibronectin-Binding Protein in Lactobacillus acidophilus NCFM.

PubMed

Hymes, Jeffrey P; Johnson, Brant R; Barrangou, Rodolphe; Klaenhammer, Todd R

2016-05-01

Bacterial surface layers (S-layers) are crystalline arrays of self-assembling proteinaceous subunits called S-layer proteins (Slps) that comprise the outermost layer of the cell envelope. Many additional proteins that are associated with or embedded within the S-layer have been identified in Lactobacillus acidophilus NCFM, an S-layer-forming bacterium that is widely used in fermented dairy products and probiotic supplements. One putative S-layer-associated protein (SLAP), LBA0191, was predicted to mediate adhesion to fibronectin based on the in silico detection of a fibronectin-binding domain. Fibronectin is a major component of the extracellular matrix (ECM) of intestinal epithelial cells. Adhesion to intestinal epithelial cells is considered an important trait for probiotic microorganisms during transit and potential association with the intestinal mucosa. To investigate the functional role of LBA0191 (designated FbpB) in L. acidophilus NCFM, an fbpB-deficient strain was constructed. The L. acidophilus mutant with a deletion off bpB lost the ability to adhere to mucin and fibronectin in vitro Homologues off bpB were identified in five additional putative S-layer-forming species, but no homologues were detected in species outside theL. acidophilus homology group. Copyright © 2016 Hymes et al.

Adhesion of a monolayer of fibroblast cells to fibronectin under sonic vibrations in a bioreactor.

PubMed

Titze, Ingo R; Klemuk, Sarah A; Lu, Xiaoying

2012-06-01

We examined cell adhesion to a surface under vibrational forces approximating those of phonation. A monolayer of human fibroblast cells was seeded on a fibronectin-coated glass coverslip, which was attached to either the rotating part or the stationary part of a rheometer-bioreactor. The temperature, humidity, carbon dioxide level, nutrients, and cell seeding density were controlled. The cell density was on the order of 1,000 to 5,000 cells per square millimeter. Target stresses above 1 kPa at an oscillatory frequency of 100 Hz were chosen to reflect conditions of vocal fold tissue vibration. Fibronectin coating provided enough adhesion to support at least 2 kPa of oscillating stress, but only about 0.1 kPa of steady rotational shear. For stresses exceeding those limits, the cells were not able to adhere to the thin film of fibronectin. Cells will adhere to a planar surface under stresses typical of phonation, which provide a more stringent test than adherence in a 3-dimensional matrix. The density of cell seeding on the coverslip played a role in cell-extracellular matrix adhesion, in that the cells adhered to each other more than to the fibronectin coating when the cells were nearly confluent.

BMP2 and BMP7 play antagonistic roles in feather induction

PubMed Central

Michon, Frédéric; Forest, Loïc; Collomb, Elodie; Demongeot, Jacques; Dhouailly, Danielle

2008-01-01

Summary During embryonic development, feathers first appear as primordia consisting of an epidermal placode associated with a dermal condensation. In most previous studies, the BMPs have been proposed to function as inhibitors of the formation of cutaneous appendages. We showed that the function of BMPs is quite nuanced: BMP-2 and BMP-7, which are expressed in both skin components, act antagonistically and yet are both involved in the dermal condensations formation. BMP-7, the first to be expressed, is implicated in chemotaxis which leads to cell recruitment to the condensation, whereas BMP-2, which is expressed later, leads to an arrest of cell migration, likely via its modulation of EIIIA Fibronectin domain and α4-Integrin expression. We also propose a mathematical model, a reaction-diffusion system, based on cell proliferation, chemotaxis and the timing of BMP-2 and BMP-7 expression, which simulates the endogenous situation and reproduces the negative effects of excess BMP-2 or BMP-7 on feather patterning. PMID:18635609

Neisseria meningitidis Opc invasin binds to the sulphated tyrosines of activated vitronectin to attach to and invade human brain endothelial cells.

PubMed

Sa E Cunha, Claudia; Griffiths, Natalie J; Virji, Mumtaz

2010-05-20

The host vasculature is believed to constitute the principal route of dissemination of Neisseria meningitidis (Nm) throughout the body, resulting in septicaemia and meningitis in susceptible humans. In vitro, the Nm outer membrane protein Opc can enhance cellular entry and exit, utilising serum factors to anchor to endothelial integrins; but the mechanisms of binding to serum factors are poorly characterised. This study demonstrates that Nm Opc expressed in acapsulate as well as capsulate bacteria can increase human brain endothelial cell line (HBMEC) adhesion and entry by first binding to serum vitronectin and, to a lesser extent, fibronectin. This study also demonstrates that Opc binds preferentially to the activated form of human vitronectin, but not to native vitronectin unless the latter is treated to relax its closed conformation. The direct binding of vitronectin occurs at its Connecting Region (CR) requiring sulphated tyrosines Y(56) and Y(59). Accordingly, Opc/vitronectin interaction could be inhibited with a conformation-dependent monoclonal antibody 8E6 that targets the sulphotyrosines, and with synthetic sulphated (but not phosphorylated or unmodified) peptides spanning the vitronectin residues 43-68. Most importantly, the 26-mer sulphated peptide bearing the cell-binding domain (45)RGD(47) was sufficient for efficient meningococcal invasion of HBMECs. To our knowledge, this is the first study describing the binding of a bacterial adhesin to sulphated tyrosines of the host receptor. Our data also show that a single region of Opc is likely to interact with the sulphated regions of both vitronectin and of heparin. As such, in the absence of heparin, Opc-expressing Nm interact directly at the CR but when precoated with heparin, they bind via heparin to the heparin-binding domain of the activated vitronectin, although with a lower affinity than at the CR. Such redundancy suggests the importance of Opc/vitronectin interaction in meningococcal pathogenesis and may enable the bacterium to harness the benefits of the physiological processes in which the host effector molecule participates.

Chondroitin sulphates A, B and C, collagen types I-IV and fibronectin in venous sinus of the red pulp in human spleen.

PubMed

Rovenská, E; Michalka, P; Papincák, J; Durdík, S; Jakubovský, J

2005-01-01

The morphological relationship of chondroitin sulphates A, B, and C, collagen types I-IV and fibronectin in the wall of venous sinuses of the red pulp in human spleen has not been a focus of interest among morphologists. Regarding the hypothesis that the structure of the spleen lends it the function of a blood filter the substances described in our study might play a significant role in the functional morphology. Of 146 human spleen surgical specimens, groups of 12 specimens each were examined under a light microscope using the method of antibodies against fibronectin, against collagen types I-IV and against chondroitin sulphates A, B, and C. The sections of the red pulp of human spleen stained with hematoxylin and eosin provided limited information about the wall of the sinuses. Chondroitin sulphates A and B were observed on the surface of sinus-lining cells (SLC), and fibronectin was detected on the surface of the annular fibers. Collagen type 11 was observed almost in the same places as chondroitin sulphates A and B. Collagen type IV was present in annular fibers of the wall of the sinus and in the basement membrane, like fibronectin. Chondroitin sulphate was not present in the walls of sinuses. Binding of antibodies against chondroitin sulphate A and against chondroitin sulphate B indicates the presence of chondroitin sulfates on the surface of SLC, where they probably play a role in helping the human organism to recognize alien and self substances. The presence of chondroitin,sulphates A and B probably affects inhibition of binding of cells with collagen type I, but not with fibronectin.

Type of MRI contrast, tissue gadolinium, and fibrosis.

PubMed

Do, Catherine; Barnes, Jeffrey L; Tan, Chunyan; Wagner, Brent

2014-10-01

It has been presupposed that the thermodynamic stability constant (K(therm)) of gadolinium-based MRI chelates relate to the risk of precipitating nephrogenic systemic fibrosis. The present study compared low-K(therm) gadodiamide with high-K(therm) gadoteridol in cultured fibroblasts and rats with uninephrectomies. Gadolinium content was assessed using scanning electron microscopy equipped with energy-dispersive X-ray spectroscopy in paraffin-embedded tissues. In vitro, fibroblasts demonstrated dose-dependent fibronectin generation, transforming growth factor-β production, and expression of activated myofibroblast stress fiber protein α-smooth muscle actin. There were negligible differences with respect to toxicity or proliferation between the two contrast agents. In the rodent model, gadodiamide treatment led to greater skin fibrosis and dermal cellularity than gadoteridol. In the kidney, both contrast agents led to proximal tubule vacuolization and increased fibronectin accumulation. Despite large detectable gadolinium signals in the spleen, skin, muscle, and liver from the gadodiamide-treated group, contrast-induced fibrosis appeared to be limited to the skin and kidney. These findings support the hypothesis that low-K(therm) chelates have a greater propensity to elicit nephrogenic systemic fibrosis and demonstrate that certain tissues are resistant to these effects.

Morphological responses of dissociated sponge cells to different organic substrata.

PubMed

Gaino, E; Magnino, G; Burlando, B; Sara', M

1993-06-01

To study interactions between sponge cells and components of the extracellular matrix (ECM), cells of the calcareous sponge Clathrina cerebrum were investigated in vitro by scanning electron microscopy. Cells were settled on glass coverslips, used as controls, and on coverslips coated with various ECM components (laminin, collagens and fibronectin), and with an adhesive substance (polylysine). Cells tended to conserve a rounded shape, producing thin, stiff processes (scleropodia) and lamellipodia, whose shape and extension varied according to the substrata. Spreading was observed only on polylysine, inducing cells to assume a fibroblast-like aspect. On laminin, cell adhesion was assured only by scleropodia. On fibronectin, scleropodia and lamellipodia were present, but reduced in size and length. On collagens, laminar processes occurred among prevailing scleropodia. Measurements of cell area and perimeter allowed statistical comparison of substrata, on the basis of their induction of cell flattening and protuberance formation. In summary, sponge cells were found to modulate their morphology in response to the external environment, expressing features for dynamic activities most fully in the presence of substances close to their natural ECM constituents. These results are discussed in the context of tissue rearrangement as a basic adaptation occurring throughout the life span of these organisms.

Large Gradient High Magnetic Fields Affect Osteoblast Ultrastructure and Function by Disrupting Collagen I or Fibronectin/αβ1 Integrin

PubMed Central

Qian, Ai-Rong; Gao, Xiang; Zhang, Wei; Li, Jing-Bao; Wang, Yang; Di, Sheng-Meng; Hu, Li-Fang; Shang, Peng

2013-01-01

The superconducting magnet generates a field and field gradient product that can levitate diamagnetic materials. In this study a specially designed superconducting magnet with a large gradient high magnetic field (LG-HMF), which can provide three apparent gravity levels (μ-g, 1-g, and 2-g), was used to simulate a space-like gravity environment. The effects of LG-HMF on the ultrastructure and function of osteoblast-like cells (MG-63 and MC3T3-E1) and the underlying mechanism were investigated by transmission electromicroscopy (TEM), MTT, and cell western (ICW) assays. Under LG-HMF significant morphologic changes in osteoblast-like cells occurred, including expansion of endoplasmic reticulum and mitochondria, an increased number of lysosomes, distorted microvilli, and aggregates of actin filaments. Compared to controls, cell viability and alkaline phosphatase (ALP) secretion were significantly increased, and collagen I (col I), fibronectin (FN), vinculin, integrin α3, αv, and β1 expression were changed under LG-HMF conditions. In conclusion, LG-HMF affects osteoblast ultrastructure, cell viability, and ALP secretion, and the changes caused by LG-HMF may be related to disrupting col I or FN/αβ1 integrin. PMID:23382804

Hepatocyte growth factor: a regulator of extracellular matrix genes in mouse mesangial cells.

PubMed

Laping, N J; Olson, B A; Ho, T; Ziyadeh, F N; Albrightson, C R

2000-04-01

The potential role of hepatocyte growth factor (HGF) in regulating extracellular matrix in mouse mesangial cells (MMC) was evaluated. Functional HGF receptors were deed in MMC by HGF-induced extracellular acidification, a response that was inhibited by the HGF inhibitor HGF/NK2, a splice variant expressing the N-terminal domain through the second kringle domain HGF also increased fibronectin and collagen alpha1 (IV) mRNA levels in these cells; the increases were associated with a concentration-dependent increase in transcriptional activity of the collagen alpha1 (IV) gene. HGF also stimulated fibronectin and collagen alpha1 (IV) mRNA levels in primary rabbit proximal tubule epithelial cells To evaluate the potential consequences of chronic elevation of HGF on renal fuction, HGF was administered continuously for 18 days to normal and diabetic C57BLKS/J lepr(db) mice. In the diabetic mice, HGF reduced creatinine clearance and increased microalbuminuria, indicating that chronic exposure to HGF impairs renal function. Thus, chronically elevated HGF may contribute to the progression of chronic renal disease in diabetes by decreasing the glomerular filtration rate and possibly promoting the accumulation of extracellular matrix.

EDA-containing fibronectin increases proliferation of embryonic stem cells.

PubMed

Losino, Noelia; Waisman, Ariel; Solari, Claudia; Luzzani, Carlos; Espinosa, Darío Fernández; Sassone, Alina; Muro, Andrés F; Miriuka, Santiago; Sevlever, Gustavo; Barañao, Lino; Guberman, Alejandra

2013-01-01

Embryonic stem cells (ESC) need a set of specific factors to be propagated. They can also grow in conditioned medium (CM) derived from a bovine granulosa cell line BGC (BGC-CM), a medium that not only preserves their main features but also increases ESC´s proliferation rate. The mitogenic properties of this medium were previously reported, ascribing this effect to an alternative spliced generated fibronectin isoform that contains the extra domain A (FN EDA(+)). Here, we investigated if the FN EDA(+) isoform increased proliferation of mouse and human ES cells. We analyzed cell proliferation using conditioned media produced by different mouse embryonic fibroblast (MEF) lines genetically engineered to express FN constitutively including or excluding the EDA domain (FN EDA(-)), and in media supplemented with recombinant peptides containing or not the EDA. We found that the presence of EDA in the medium increased mouse and human ESC's proliferation rate. Here we showed for the first time that this FN isoform enhances ESC's proliferation. These findings suggest a possible conserved behavior for regulation of ES cells proliferation by this FN isoform and could contribute to improve their culturing conditions both for research and cell therapy.

EDA-Containing Fibronectin Increases Proliferation of Embryonic Stem Cells

PubMed Central

Losino, Noelia; Waisman, Ariel; Solari, Claudia; Luzzani, Carlos; Espinosa, Darío Fernández; Sassone, Alina; Muro, Andrés F.; Miriuka, Santiago; Sevlever, Gustavo; Barañao, Lino; Guberman, Alejandra

2013-01-01

Embryonic stem cells (ESC) need a set of specific factors to be propagated. They can also grow in conditioned medium (CM) derived from a bovine granulosa cell line BGC (BGC-CM), a medium that not only preserves their main features but also increases ESC´s proliferation rate. The mitogenic properties of this medium were previously reported, ascribing this effect to an alternative spliced generated fibronectin isoform that contains the extra domain A (FN EDA+). Here, we investigated if the FN EDA+ isoform increased proliferation of mouse and human ES cells. We analyzed cell proliferation using conditioned media produced by different mouse embryonic fibroblast (MEF) lines genetically engineered to express FN constitutively including or excluding the EDA domain (FN EDA-), and in media supplemented with recombinant peptides containing or not the EDA. We found that the presence of EDA in the medium increased mouse and human ESC’s proliferation rate. Here we showed for the first time that this FN isoform enhances ESC’s proliferation. These findings suggest a possible conserved behavior for regulation of ES cells proliferation by this FN isoform and could contribute to improve their culturing conditions both for research and cell therapy. PMID:24244705

The role of integrin α8β1 in fetal lung morphogenesis and injury

PubMed Central

Benjamin, John T.; Gaston, David C.; Halloran, Brian A.; Schnapp, Lynn M.; Zent, Roy; Prince, Lawrence S.

2009-01-01

Prenatal inflammation prevents normal lung morphogenesis and leads to bronchopulmonary dysplasia (BPD), a common complication of preterm birth. We previously demonstrated in a bacterial endotoxin mouse model of BPD that disrupting fibronectin localization in the fetal lung mesenchyme causes arrested saccular airway branching. In this study we show that expression of the fibronectin receptor, integrin α8β1, is decreased in the lung mesenchyme in the same inflammation model suggesting it is required for normal lung development. We verified a role for integrin α8β1 in lung development using integrin α8-null mice, which develop fusion of the medial and caudal lobes as well as abnormalities in airway division. We further show in vivo and vitro that α8-null fetal lung mesenchymal cells fail to form stable adhesions and have increased migration. Thus we propose that integrin α8β1 plays a critical role in lung morphogenesis by regulating mesenchymal cell adhesion and migration. Furthermore, our data suggests that disruption of the interactions between extracellular matrix and integrin α8β1 may contribute to the pathogenesis of BPD. PMID:19769957

Selenium nanoparticles involve HSP-70 and SIRT1 in preventing the progression of type 1 diabetic nephropathy.

PubMed

Kumar, Goru Santosh; Kulkarni, Apoorva; Khurana, Amit; Kaur, Jasmine; Tikoo, Kulbhushan

2014-11-05

The present study was undertaken to examine the protective effect of selenium nanoparticles (SeNPs) in the progression of diabetic nephropathy (DN). Diabetes was induced in male Sprague Dawley (SD) rats by injecting streptozotocin (STZ) (55mg/kg, i.p). DN was then assessed by measuring blood urea nitrogen (BUN), creatinine, albumin, fibronectin and collagen. Changes in the expression of cytoprotective and apoptotic proteins in the kidney of rats were also examined. Herein we show that SeNPs effectively lowered the levels of BUN, creatinine, fibronectin and collagen and elevated the levels of albumin in diabetic rats. Histological observation corroborated with the above protective effects of SeNPs. Interestingly, SeNPs elevated the levels of heat shock protein (HSP-70), longevity protein SIRT 1 and also modulated apoptotic proteins Bax and Bcl-2 in diabetic kidney. Our data represents a paradigm shift in our understanding about the therapeutic potential of SeNPs in preventing DN by not only quenching oxidative stress but also by activating cyto-protective protein HSP70 and longevity protein SIRT1. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Large gradient high magnetic fields affect osteoblast ultrastructure and function by disrupting collagen I or fibronectin/αβ1 integrin.

PubMed

Qian, Ai-Rong; Gao, Xiang; Zhang, Wei; Li, Jing-Bao; Wang, Yang; Di, Sheng-Meng; Hu, Li-Fang; Shang, Peng

2013-01-01

The superconducting magnet generates a field and field gradient product that can levitate diamagnetic materials. In this study a specially designed superconducting magnet with a large gradient high magnetic field (LG-HMF), which can provide three apparent gravity levels (μ-g, 1-g, and 2-g), was used to simulate a space-like gravity environment. The effects of LG-HMF on the ultrastructure and function of osteoblast-like cells (MG-63 and MC3T3-E1) and the underlying mechanism were investigated by transmission electromicroscopy (TEM), MTT, and cell western (ICW) assays. Under LG-HMF significant morphologic changes in osteoblast-like cells occurred, including expansion of endoplasmic reticulum and mitochondria, an increased number of lysosomes, distorted microvilli, and aggregates of actin filaments. Compared to controls, cell viability and alkaline phosphatase (ALP) secretion were significantly increased, and collagen I (col I), fibronectin (FN), vinculin, integrin α3, αv, and β1 expression were changed under LG-HMF conditions. In conclusion, LG-HMF affects osteoblast ultrastructure, cell viability, and ALP secretion, and the changes caused by LG-HMF may be related to disrupting col I or FN/αβ1 integrin.

Fibronectin is an acute phase reactant in mice.

PubMed

Dyck, R F; Rogers, S L

1985-01-01

Tissue injury and inflammation are potent stimuli for the immediate increased synthesis of several plasma proteins collectively known as acute phase phase reactants. This dramatic phenomenon is thought to play an important role in inflammation and tissue repair. Plasma fibronectin is a normal plasma glycoprotein and a major non-specific opsonin apparently involved in maintaining the integrity of the mononuclear phagocytic system. Because of its ability to mediate clearance of intravascular particulate matter, increased production following tissue injury could be of benefit to the organism. We now report that plasma fibronectin is a significant acute phase reactant in mice with levels increasing from a baseline mean value of 257 ug/ml to 595 ug/ml by 24 hours (p less than 0.01) after a subcutaneous injection of silver nitrate. Similar findings were observed when subcutaneous casein was used as the acute phase stimulus. This data provides further circumstantial evidence that plasma fibronectin is involved in host defence and tissue repair.

Effects of protein-coated nanofibers on conformation of gingival fibroblast spheroids: potential utility for connective tissues regeneration.

PubMed

Kaufman, Gili; Whitescarver, Ryan; Nunes, Laiz; Palmer, Xavier-Lewis; Skrtic, Drago; Tutak, Wojtek

2017-10-09

Deep wounds in the gingiva caused by trauma or surgery require a rapid and robust healing of connective tissues. We propose utilizing gas-brushed nanofibers coated with collagen and fibrin for that purpose. Our hypotheses are that protein-coated nanofibers will: (i) attract and mobilize cells in various spatial orientations, and (ii) regulate the expression levels of specific extracellular matrix (ECM)-associated proteins, determining the initial conformational nature of dense and soft connective tissues. Gingival fibroblast monolayers and 3D spheroids were cultured on ECM substrate and covered with gas-blown poly-(DL-lactide-co-glycolide) (PLGA) nanofibers (uncoated/coated with collagen and fibrin). Cell attraction and rearrangement was followed by F-actin staining and confocal microscopy. Thicknesses of the cell layers, developed within the nanofibers, were quantified by imageJ software. The expression of collagen1α1 chain (Col1α1), fibronectin, and metalloproteinase 2 (MMP2) encoding genes was determined by quantitative reverse transcription analysis. Collagen- and fibrin- coated nanofibers induced cell migration toward fibers and supported cellular growth within the scaffolds. Both proteins affected the spatial rearrangement of fibroblasts by favoring packed cell clusters or intermittent cell spreading. These cell arrangements resembled the structural characteristic of dense and soft connective tissues, respectively. Within 3 days of incubation, fibroblast spheroids interacted with the fibers and grew robustly by increasing their thickness compared to monolayers. While the ECM key components, such as fibronectin and MMP2 encoding genes, were expressed in both protein groups, Col1α1 was predominantly expressed in bundled fibroblasts grown on collagen fibers. This enhanced expression of collagen1 is typical for dense connective tissue. Based on results of this study, our gas-blown, collagen- and fibrin-coated PLGA nanofibers are viable candidates for engineering soft and dense connective tissues with the required structural characteristics and functions needed for wound healing applications. Rapid regeneration of these layers should enhance healing of open wounds in a harsh oral environment. © 2017 IOP Publishing Ltd.

The Structures of Fibronectin Adsorbed on Polyelectrolyte Thin Films

NASA Astrophysics Data System (ADS)

Shin, Kwanwoo; Satija, Sushil; Fang, Xiao-Hua; Li, Bin-Quan; Nadine, Pernodet; Miriam, Rafailovich; Sokolov, Jonathan; Arach, Goldar; Roser, Steve

2002-03-01

We have shown that it is possible to form a fibrilar network of fibronectin on a polyelectrolyte polymer film whose dimensions are similar to those reported on the extra cellular matrix. The fibronectin network was observed to form only when the charge density of the polymer was in excess of the natural charge density of the cell wall. Furthermore, the self-organized fibronectin layer was much thicker than the polymer film, indicating that long ranged interaction may play a key role in the assembly process. It is therefore important to understand the structure of the polymer layer/protein interface. Here we report on a neutron reflectivity study where we explore the structure of the polyelectrolyte layer, in this case sulfonated polystyrene (PSS_x.), with varying degree of sulfonation (x<30%), as a function of sulfur content and counter ion concentration. These results are then correlated with systemic study of the adsorption and the multilayer formation of fibronectin as a function of incubation time for various sulfonation levels of PSS_x. Furthermore, the surface charge on the substrates can be strongly influenced by the presence of salt ions, it is important to understand changes due to electrostatic interactions occurring in the various salt conditions. Complementary X-ray reflection was used to determine the salt density profile associating with the internal ionic polymer matrix. This work was funded in part of the NSF-MRSEC program.

Interaction of intraocular lenses with fibronectin and human lens epithelial cells: Effect of chemical composition and aging.

PubMed

Tortolano, Lionel; Serrano, Carole; Jubeli, Emile; Saunier, Johanna; Yagoubi, Najet

2015-12-01

The aim of this study is to investigate in vitro interactions between hydrophobic acrylate intraocular lenses (IOLs) and their biological environment. The influence of lens chemical composition and aging on fibronectin (FN) adsorption and on IOLs cytotoxicity on human lens epithelial cells was examined. Cytotoxicity of acrylate monomers used in IOLs manufacture was also investigated. Four different IOLs were included in the study: Acrysof(®), Tecnis(®), EnVista(®), and iSert(®). Implants were artificially aged in a xenon arc chamber to simulate 2 years of light exposure. Fibronectin adsorption on IOL surface was quantified using ELISA and correlated to surface roughness determined with AFM. Direct contact cytotoxicity was determined with the MTT assay and cell morphology was observed with light microscopy. Results showed that fibronectin adsorption did not differ significantly among IOLs, whatever their chemical composition. Moreover, aging conditions did not impact fibronectin adsorption. All IOLs were biocompatible even after applying 2-year aging conditions, with cell viability higher than 70%. Five acrylate monomers appeared to be toxic in the range of concentrations tested, but no monomer release from the IOLs could be detected during accelerated 2-year incubation with saline solution. This study did not reveal an influence of chemical composition and aging on protein adsorption and on biocompatibility. © 2015 Wiley Periodicals, Inc.

Fibronectin Modulates Cell Adhesion and Signaling to Promote Single Cell Migration of Highly Invasive Oral Squamous Cell Carcinoma

PubMed Central

Ramos, Grasieli de Oliveira; Bernardi, Lisiane; Lauxen, Isabel; Sant’Ana Filho, Manoel; Horwitz, Alan Rick; Lamers, Marcelo Lazzaron

2016-01-01

Cell migration is regulated by adhesion to the extracellular matrix (ECM) through integrins and activation of small RhoGTPases, such as RhoA and Rac1, resulting in changes to actomyosin organization. During invasion, epithelial-derived tumor cells switch from laminin-enriched basal membrane to collagen and fibronectin-enriched connective tissue. How this switch affects the tumor migration is still unclear. We tested the hypothesis that ECM dictates the invasiveness of Oral Squamous Cell Carcinoma (OSCC). We analyzed the migratory properties of two OSCC lines, a low invasive cell line with high e-cadherin levels (Linv/HE-cad) or a highly invasive cell line with low e-cadherin levels (Hinv/LE-cad), plated on different ECM components. Compared to laminin, fibronectin induced non-directional collective migration and decreased RhoA activity in Linv/HE-cad OSCC. For Hinv/LE-cad OSCC, fibronectin increased Rac1 activity and induced smaller adhesions, resulting in a fast single cell migration in both 2D and 3D environments. Consistent with these observations, human OSCC biopsies exhibited similar changes in cell-ECM adhesion distribution at the invasive front of the tumor, where cells encounter fibronectin. Our results indicate that ECM composition might induce a switch from collective to single cell migration according to tumor invasiveness due to changes in cell-ECM adhesion and the resulting signaling pathways that alter actomyosin organization. PMID:26978651

Fibronectin EDA forms the chronic fibrotic scar after contusive spinal cord injury.

PubMed

Cooper, John G; Jeong, Su Ji; McGuire, Tammy L; Sharma, Sripadh; Wang, Wenxia; Bhattacharyya, Swati; Varga, John; Kessler, John A

2018-04-27

Gliosis and fibrosis after spinal cord injury (SCI) lead to formation of a scar that is an impediment to axonal regeneration. Fibrotic scarring is characterized by the accumulation of fibronectin, collagen, and fibroblasts at the lesion site. The mechanisms regulating fibrotic scarring after SCI and its effects on axonal elongation and functional recovery are not well understood. In this study, we examined the effects of eliminating an isoform of fibronectin containing the Extra Domain A domain (FnEDA) on both fibrosis and on functional recovery after contusion SCI using male and female FnEDA-null mice. Eliminating FnEDA did not reduce the acute fibrotic response but markedly diminished chronic fibrotic scarring after SCI. Glial scarring was unchanged after SCI in FnEDA-null mice. We found that FnEDA was important for the long-term stability of the assembled fibronectin matrix during both the subacute and chronic phases of SCI. Motor functional recovery was significantly improved, and there were increased numbers of axons in the lesion site compared to wildtype mice, suggesting that the chronic fibrotic response is detrimental to recovery. Our data provide insight into the mechanisms of fibrosis after SCI and suggest that disruption of fibronectin matrix stability by targeting FnEDA represents a potential therapeutic strategy for promoting recovery after SCI. Copyright © 2018 Elsevier Inc. All rights reserved.

Substrate engagement of integrins α5β1 and αvβ3 is necessary, but not sufficient, for high directional persistence in migration on fibronectin

PubMed Central

Missirlis, Dimitris; Haraszti, Tamás; Scheele, Catharina v. C.; Wiegand, Tina; Diaz, Carolina; Neubauer, Stefanie; Rechenmacher, Florian; Kessler, Horst; Spatz, Joachim P.

2016-01-01

The interplay between specific integrin-mediated matrix adhesion and directional persistence in cell migration is not well understood. Here, we characterized fibroblast adhesion and migration on the extracellular matrix glycoproteins fibronectin and vitronectin, focusing on the role of α5β1 and αvβ3 integrins. Fibroblasts manifested high directional persistence in migration on fibronectin-, but not vitronectin-coated substrates, in a ligand density-dependent manner. Fibronectin stimulated α5β1-dependent organization of the actin cytoskeleton into oriented, ventral stress fibers, and assembly of dynamic, polarized protrusions, characterized as regions free of stress fibers and rich in nascent adhesions at their edge. Such protrusions correlated with persistent, local leading edge advancement, but were not sufficient, nor necessary for directional migration over longer times. Selective blocking of αvβ3 or α5β1 integrins using small molecule integrin antagonists reduced directional persistence on fibronectin, indicating integrin cooperativity in maintaining directionality. On the other hand, patterned substrates, designed to selectively engage either integrin, or their combination, were not sufficient to establish directional migration. Overall, our study demonstrates adhesive coating-dependent regulation of directional persistence in fibroblast migration and challenges the generality of the previously suggested role of β1 and β3 integrins in directional migration. PMID:26987342

Splicing factors PTBP1 and PTBP2 promote proliferation and migration of glioma cell lines

PubMed Central

Cheung, Hannah C.; Hai, Tao; Zhu, Wen; Baggerly, Keith A.; Tsavachidis, Spiridon; Krahe, Ralf

2009-01-01

Polypyrimidine tract-binding protein 1 (PTBP1) is a multi-functional RNA-binding protein that is aberrantly overexpressed in glioma. PTBP1 and its brain-specific homologue polypyrimidine tract-binding protein 2 (PTBP2) regulate neural precursor cell differentiation. However, the overlapping and non-overlapping target transcripts involved in this process are still unclear. To determine why PTBP1 and not PTBP2 would promote glial cell-derived tumours, both PTBP1 and PTBP2 were knocked down in the human glioma cell lines U251 and LN229 to determine the role of these proteins in cell proliferation, migration, and adhesion. Surprisingly, removal of both PTBP1 and PTBP2 slowed cell proliferation, with the double knockdown having no additive effects. Decreased expression of both proteins individually and in combination inhibited cell migration and increased adhesion of cells to fibronectin and vitronectin. A global survey of differential exon expression was performed following PTBP1 knockdown in U251 cells using the Affymetrix Exon Array to identify PTBP1-specific splicing targets that enhance gliomagenesis. In the PTBP1 knockdown, previously determined targets were unaltered in their splicing patterns. A single gene, RTN4 (Nogo) had significantly enhanced inclusion of exon 3 when PTBP1 was removed. Overexpression of the splice isoform containing exon 3 decreased cell proliferation to a similar degree as the removal of PTBP1. These results provide the first evidence that RNA-binding proteins affect the invasive and rapid growth characteristics of glioma cell lines. Its actions on proliferation appear to be mediated, in part, through alternative splicing of RTN4. PMID:19506066

EFFECT OF GROWTH FACTOR-FIBRONECTIN MATRIX INTERACTION ON RAT TYPE II CELL ADHESION AND DNA SYTHESIS

EPA Science Inventory

ABSTRACT

Type II cells attach, migrate and proliferate on a provisional fibronectin-rich matrix during alveolar wall repair after lung injury. The combination of cell-substratum interactions via integrin receptors and exposure to local growth factors are likely to initiat...

Changes in p53 expression in mouse fibroblasts can modify motility and extracellular matrix organization.

PubMed

Alexandrova, A; Ivanov, A; Chumakov, P; Kopnin, B; Vasiliev, J

2000-11-23

Effects of p53 expression on cell morphology and motility were studied using the derivatives of p53-null 10(1) mouse fibroblasts with tetracycline-regulated expression of exogenous human p53. Induction of p53 expression was accompanied by significant decrease in extracellular matrix (fibronectin) and reduction of matrix fibrils, diminution of the number and size of focal contacts, decrease of cell areas, establishment of more elongated cell shape and alterations of actin cytoskeleton (actin bundles became thinner, their number and size decreased). Expression of His175 and Gln22/ Ser23 p53 mutants caused no such effects. To study the influence of p53 expression on cell motility we used wound technique and videomicroscopy observation of single living cells. It was found that induction of p53 expression led to increase of lamellar activity of cell edge. However, in spite of enhanced lamellar activity p53-expressing cells migrated to shorter distance and filled the narrow wound in longer time as compared with their p53-null counterparts. Possible mechanisms of the influence of p53 expression on cell morphology and motility are discussed.

Vesicle-associated membrane protein 2 mediates trafficking of {alpha}5{beta}1 integrin to the plasma membrane

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

Hasan, Nazarul; Hu, Chuan, E-mail: chuan.hu@louisville.edu

2010-01-01

Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of {alpha}5{beta}1 integrin. VAMP2 was present on vesicles containing endocytosed {beta}1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cellmore » surface {alpha}5{beta}1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of {alpha}5{beta}1, without altering cell surface expression of {alpha}2{beta}1 integrin or {alpha}3{beta}1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of {alpha}5{beta}1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.« less

Vinculin contributes to Cell Invasion by Regulating Contractile Activation

NASA Astrophysics Data System (ADS)

Mierke, Claudia Tanja

2008-07-01

Vinculin is a component of the focal adhesion complex and is described as a mechano-coupling protein connecting the integrin receptor and the actin cytoskeleton. Vinculin knock-out (k.o.) cells (vin-/-) displayed increased migration on a 2-D collagen- or fibronectin-coated substrate compared to wildtype cells, but the role of vinculin in cell migration through a 3-D connective tissue is unknown. We determined the invasiveness of established tumor cell lines using a 3-D collagen invasion assay. Gene expression analysis of 4 invasive and 4 non-invasive tumor cell lines revealed that vinculin expression was significantly increased in invasive tumor cell lines. To analyze the mechanisms by which vinculin increased cell invasion in a 3-D gel, we studied mouse embryonic fibroblasts wildtype and vin-/- cells. Wildtype cells were 3-fold more invasive compared vin-/- cells. We hypothesized that the ability to generate sufficient traction forces is a prerequisite for tumor cell migration in a 3-D connective tissue matrix. Using traction microscopy, we found that wildtype exerted 3-fold higher tractions on fibronectin-coated polyacrylamide gels compared to vin-/- cells. These results show that vinculin controls two fundamental functions that lead to opposite effects on cell migration in a 2-D vs. a 3-D environment: On the one hand, vinculin stabilizes the focal adhesions (mechano-coupling function) and thereby reduces motility in 2-D. On the other hand, vinculin is also a potent activator of traction generation (mechano-regulating function) that is important for cell invasion in a 3-D environment.

Lsa30, a novel adhesin of Leptospira interrogans binds human plasminogen and the complement regulator C4bp.

PubMed

Souza, Natalie M; Vieira, Monica L; Alves, Ivy J; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

2012-09-01

Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Surface proteins have the potential to promote several activities, including adhesion. This work aimed to study the leptospiral coding sequence (CDS) LIC11087, genome annotated as hypothetical outer membrane protein. The LIC11087 gene was cloned and expressed in Escherichia coli BL21 (DE3) strain by using the expression vector pAE. The recombinant protein tagged with N-terminal 6XHis was purified by metal-charged chromatography and characterized by circular dichroism (CD) spectroscopy. The recombinant protein has the ability to mediate attachment to the extracellular matrix (ECM) components, laminin and plasma fibronectin, and was named Lsa30 (Leptospiral surface adhesin of 30 kDa). Lsa30 binds to laminin and to plasma fibronectin in a dose-dependent and saturable manner, with dissociation equilibrium constants (K(D)) of 292 ± 24 nm and 157 ± 35 nm, respectively. Moreover, the Lsa30 is a plasminogen (PLG) receptor, capable of generating plasmin, in the presence of activator. This protein may interfere with the complement cascade by interacting with C4bp regulator. The Lsa30 is probably a new surface protein of Leptospira as revealed by immunofluorescence assays with living organisms and the reactivity with antibodies present in serum samples of experimentally infected hamsters. Thus, Lsa30 is a novel versatile protein that may play a role in mediating adhesion and may help pathogenic Leptospira to overcome tissue barriers and to escape the immune system. Copyright © 2012 Elsevier Ltd. All rights reserved.

Fibronectin gene expression, synthesis and accumulation during in vitro differentiation of chicken osteoblasts

NASA Technical Reports Server (NTRS)

Winnard, R. G.; Gerstenfeld, L. C.; Toma, C. D.; Franceschi, R. T.; Landis, W. J. (Principal Investigator)

1995-01-01

A well-defined chicken osteoblast culture system(18) has been used to examine fibronectin (FN) mRNA levels, synthesis, and accumulation during in vitro differentiation and matrix mineralization. Immunofluorescent staining of cells after 6 or 18 days in culture revealed that FN was initially associated with the cell surface and in partial coalignment with cytoskeletal elements while at the latter time most FN was associated with the extracellular matrix as a ubiquitous fibrillar network. Western blot analysis of total cell-associated proteins also detected FN at all culture times. However, when results were normalized to cellular DNA, FN levels increased until 12-16 and remained relatively constant thereafter. Similarly, FN synthesis as measured by [35S]-methionine labeling, and immunoprecipitation was greatest in early cultures (culture day 3) and then declined such that synthesis decreased 60% at day 18 and 94% after 24-31 days. FN mRNA levels as measured by Northern blot analysis were well correlated with FN synthesis. These results clearly show that FN is made by primary osteoblasts during their in vitro maturation. In contrast to other osteoblast markers such as alkaline phosphatase, osteocalcin, and osteopontin, whose expression increases as cells differentiate, FN accumulates in the matrix during periods of early cell growth and attachment and then remains proportional to cell number. Results with FN differ from those obtained with collagen which continues to accumulate in the extracellular matrix during osteoblast maturation. These results are consistent with FN being important for the initial attachment of early osteoblasts or osteoblast precursors to the pericellular matrix.

Podocyte-derived microparticles promote proximal tubule fibrotic signaling via p38 MAPK and CD36

PubMed Central

Munkonda, Mercedes N.; Akbari, Shareef; Landry, Chloe; Sun, Suzy; Xiao, Fengxia; Turner, Maddison; Holterman, Chet E.; Nasrallah, Rania; Hébert, Richard L.; Kennedy, Christopher R. J.; Burger, Dylan

2018-01-01

ABSTRACT Tubulointerstitial fibrosis is a hallmark of advanced diabetic kidney disease that is linked to a decline in renal function, however the pathogenic mechanisms are poorly understood. Microparticles (MPs) are 100–1000 nm vesicles shed from injured cells that are implicated in intercellular signalling. Our lab recently observed the formation of MPs from podocytes and their release into urine of animal models of type 1 and 2 diabetes and in humans with type 1 diabetes. The purpose of the present study was to examine the role of podocyte MPs in tubular epithelial cell fibrotic responses. MPs were isolated from the media of differentiated, untreated human podocytes (hPODs) and administered to cultured human proximal tubule epithelial cells (PTECs). Treatment with podocyte MPs increased p38 and Smad3 phosphorylation and expression of the extracellular matrix (ECM) proteins fibronectin and collagen type IV. MP-induced responses were attenuated by co-treatment with the p38 inhibitor SB202190. A transforming growth factor beta (TGF-β) receptor inhibitor (LY2109761) blocked MP-induced Smad3 phosphorylation and ECM protein expression but not p38 phosphorylation suggesting that these responses occurred downstream of p38. Finally, blockade of the class B scavenger receptor CD36 completely abrogated MP-mediated p38 phosphorylation, downstream Smad3 activation and fibronectin/collagen type IV induction. Taken together our results suggest that podocyte MPs interact with proximal tubule cells and induce pro-fibrotic responses. Such interactions may contribute to the development of tubular fibrosis in glomerular disease. PMID:29435202

TrmFO, a Fibronectin-Binding Adhesin of Mycoplasma bovis.

PubMed

Guo, Yongpeng; Zhu, Hongmei; Wang, Jiayao; Huang, Jing; Khan, Farhan Anwar; Zhang, Jingjing; Guo, Aizhen; Chen, Xi

2017-08-09

Mycoplasma bovis is an important pathogenic mycoplasma, causing the cattle industry serious economic losses. Adhesion is a crucial step in the mycoplasmas' infection and colonization process; fibronectin (Fn), an extracellular matrix glycoprotein, is a molecular bridge between the bacterial adhesins and host cell receptors. The present study was designed to characterize the Fn-binding ability of methylenetetrahydrofolate-tRNA-(uracil-5-)-methyltransferase (TrmFO) and its role in M. bovis cytoadherence. The trmFO ( MBOV_RS00785 ) gene was cloned and expressed in E. coli BL21, and polyclonal antibodies against the recombinant TrmFO (rTrmFO) were raised in rabbits. Immunoblotting demonstrated that TrmFO was an immunogenic component, and the TrmFO expression was conserved in different M. bovis isolates. The mycoplasmacidal assay further showed that in the presence of complement, rabbit anti-recombinant TrmFO serum exhibited remarkable mycoplasmacidal efficacy. TrmFO was detected in both the M. bovis membrane and cytoplasm. By ligand dot blot and enzyme-linked immunosorbent assay (ELISA) binding assay, we found that rTrmFO bound Fn in a dose-dependent manner. Immunostaining visualized by confocal laser scanning microscopy showed that rTrmFO had capacity to adhere to the embryonic bovine lung (EBL) cells. In addition, the adhesion of M. bovis and rTrmFO to EBL cells could be inhibited by anti-rTrmFO antibodies. To the best of our knowledge, this is the first report to characterize the Fn-binding ability of TrmFO and its role in the bacterial adhesion to host cells.

TrmFO, a Fibronectin-Binding Adhesin of Mycoplasma bovis

PubMed Central

Guo, Yongpeng; Zhu, Hongmei; Wang, Jiayao; Huang, Jing; Khan, Farhan Anwar; Zhang, Jingjing; Guo, Aizhen; Chen, Xi

2017-01-01

Mycoplasma bovis is an important pathogenic mycoplasma, causing the cattle industry serious economic losses. Adhesion is a crucial step in the mycoplasmas’ infection and colonization process; fibronectin (Fn), an extracellular matrix glycoprotein, is a molecular bridge between the bacterial adhesins and host cell receptors. The present study was designed to characterize the Fn-binding ability of methylenetetrahydrofolate-tRNA-(uracil-5-)-methyltransferase (TrmFO) and its role in M. bovis cytoadherence. The trmFO (MBOV_RS00785) gene was cloned and expressed in E. coli BL21, and polyclonal antibodies against the recombinant TrmFO (rTrmFO) were raised in rabbits. Immunoblotting demonstrated that TrmFO was an immunogenic component, and the TrmFO expression was conserved in different M. bovis isolates. The mycoplasmacidal assay further showed that in the presence of complement, rabbit anti-recombinant TrmFO serum exhibited remarkable mycoplasmacidal efficacy. TrmFO was detected in both the M. bovis membrane and cytoplasm. By ligand dot blot and enzyme-linked immunosorbent assay (ELISA) binding assay, we found that rTrmFO bound Fn in a dose-dependent manner. Immunostaining visualized by confocal laser scanning microscopy showed that rTrmFO had capacity to adhere to the embryonic bovine lung (EBL) cells. In addition, the adhesion of M. bovis and rTrmFO to EBL cells could be inhibited by anti-rTrmFO antibodies. To the best of our knowledge, this is the first report to characterize the Fn-binding ability of TrmFO and its role in the bacterial adhesion to host cells. PMID:28792486

Repurposed drug screen identifies cardiac glycosides as inhibitors of TGF-β-induced cancer-associated fibroblast differentiation.

PubMed

Coleman, David T; Gray, Alana L; Stephens, Charles A; Scott, Matthew L; Cardelli, James A

2016-05-31

The tumor microenvironment, primarily composed of myofibroblasts, directly influences the progression of solid tumors. Through secretion of growth factors, extracellular matrix deposition, and contractile mechanotransduction, myofibroblasts, or cancer-associated fibroblasts (CAFs), support angiogenesis and cancer cell invasion and metastasis. The differentiation of fibroblasts to CAFs is primarily induced by TGF-β from cancer cells. To discover agents capable of blocking CAF differentiation, we developed a high content immunofluorescence-based assay to screen repurposed chemical libraries utilizing fibronectin expression as an initial CAF marker. Screening of the Prestwick chemical library and NIH Clinical Collection repurposed drug library, totaling over 1700 compounds, identified cardiac glycosides as particularly potent CAF blocking agents. Cardiac glycosides are traditionally used to regulate intracellular calcium by inhibiting the Na+/K+ ATPase to control cardiac contractility. Herein, we report that multiple cardiac glycoside compounds, including digoxin, are able to inhibit TGF-β-induced fibronectin expression at low nanomolar concentrations without undesirable cell toxicity. We found this inhibition to hold true for multiple fibroblast cell lines. Using real-time qPCR, we determined that digoxin prevented induction of multiple CAF markers. Furthermore, we report that digoxin is able to prevent TGF-β-induced fibroblast contraction of extracellular matrix, a major phenotypic consequence of CAF differentiation. Assessing the mechanism of inhibition, we found digoxin reduced SMAD promoter activity downstream of TGF-β, and we provide data that the effect is through inhibition of its known target, the Na+/K+ ATPase. These findings support a critical role for calcium signaling during CAF differentiation and highlight a novel, repurposable modality for cancer therapy.

α5β1 Integrin-Fibronectin Interactions Specify Liquid to Solid Phase Transition of 3D Cellular Aggregates

PubMed Central

Caicedo-Carvajal, Carlos E.; Shinbrot, Troy; Foty, Ramsey A.

2010-01-01

Background Tissue organization during embryonic development and wound healing depends on the ability of cells on the one hand to exchange adhesive bonds during active rearrangement and on the other to become fixed in place as tissue homeostasis is reached. Cells achieve these contradictory tasks by regulating either cell-cell adhesive bonds, mediated by cadherins, or cell-extracellular matrix (ECM) connections, regulated by integrins. Integrin α5β1 and soluble fibronectin (sFN) are key players in cell-ECM force generation and in ECM polymerization. Here, we explore the interplay between integrin α5β1 and sFN and its influence on tissue mechanical properties and cell sorting behavior. Methodology/Principal Findings We generated a series of cell lines varying in α5β1 receptor density. We then systematically explored the effects of different sFN concentrations on aggregate biomechanical properties using tissue surface tensiometry. We found previously unreported complex behaviors including the observation that interactions between fibronectin and integrin α5β1 generates biphasic tissue cohesion profiles. Specifically, we show that at constant sFn concentration, aggregate cohesion increases linearly as α5β1 receptor density is increased from low to moderate levels, producing a transition from viscoelastic-liquid to pseudo viscoelastic-solid behavior. However, further increase in receptor density causes an abrupt drop in tissue cohesion and a transition back to viscoelastic-liquid properties. We propose that this may be due to depletion of sFn below a critical value in the aggregate microenvironment at high α5β1 levels. We also show that differential expression of α5β1 integrin can promote phase-separation between cells. Conclusions/Significance The interplay between α5-integrin and sFn contributes significantly to tissue cohesion and, depending on their level of expression, can mediate a shift from liquid to elastic behavior. This interplay represents a tunable level of control between integrins and the ECM that can influence tissue cohesion and other mechanical properties, which may translate to the specification of tissue structure and function. These studies provide insights into important biological processes such as embryonic development, wound healing, and for tissue engineering applications. PMID:20686611

Preferential Enhancement of Sensory and Motor Axon Regeneration by Combining Extracellular Matrix Components with Neurotrophic Factors

PubMed Central

Santos, Daniel; González-Pérez, Francisco; Giudetti, Guido; Micera, Silvestro; Udina, Esther; Del Valle, Jaume; Navarro, Xavier

2016-01-01

After peripheral nerve injury, motor and sensory axons are able to regenerate but inaccuracy of target reinnervation leads to poor functional recovery. Extracellular matrix (ECM) components and neurotrophic factors (NTFs) exert their effect on different neuronal populations creating a suitable environment to promote axonal growth. Here, we assessed in vitro and in vivo the selective effects of combining different ECM components with NTFs on motor and sensory axons regeneration and target reinnervation. Organotypic cultures with collagen, laminin and nerve growth factor (NGF)/neurotrophin-3 (NT3) or collagen, fibronectin and brain-derived neurotrophic factor (BDNF) selectively enhanced sensory neurite outgrowth of DRG neurons and motor neurite outgrowth from spinal cord slices respectively. For in vivo studies, the rat sciatic nerve was transected and repaired with a silicone tube filled with a collagen and laminin matrix with NGF/NT3 encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres (MP) (LM + MP.NGF/NT3), or a collagen and fibronectin matrix with BDNF in PLGA MPs (FN + MP.BDNF). Retrograde labeling and functional tests showed that LM + MP.NGF/NT3 increased the number of regenerated sensory neurons and improved sensory functional recovery, whereas FN + MP.BDNF preferentially increased regenerated motoneurons and enhanced motor functional recovery. Therefore, combination of ECM molecules with NTFs may be a good approach to selectively enhance motor and sensory axons regeneration and promote appropriate target reinnervation. PMID:28036084

Recombinant anti-tenascin antibody constructs

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

ZALUTSKY, MICHAEL R

2006-08-29

The general objective of this research is to combine genetically derived molecular constructs reactive with tenascin, with appropriate radionuclides and labeling methods in order to generate more effective diagnostic and therapeutic reagents for oncologic nuclear medicine. Tenascin, a polymorphic extracellular matrix glycoprotein, is of interest because of its high expression on glioma, melanoma, as well as prostate and breast carcinoma. Recently, we have also documented high levels of tenascin in lymphomas, particularly those of higher grade, making the potential clinical impact of tenascin-specific radiodiagnostics and therapeutics even greater. An essential feature of our work plan is the ability to exploitmore » our extensive clinical experience in order to design second-generation constructs with properties which could improve clinical efficacy. To date, we have treated over 150 brain tumor patients with 131I-labeled murine 81C6, an antibody which binds specifically to the alternatively spliced fibronectin type III repeats CD of the tenascin molecule. During the current grant period, we have made several observations which form the basis for our proposed specific aims. First, tissue distribution and catabolism experiments in animal models have demonstrated enhanced stability for a chimeric construct composed of murine variable regions and human IgG2 constant domains. Furthermore, pharmacokinetic studies in patients with 131I-labeled chimeric 81C6 have shown significantly longer retention in glioma tumor resection cavities compared with its murine parent. Second, we have initiated the first clinical trial of an endoradiotherapeutic labeled with the 7.2-hr -particle emitter 211At. Twelve glioma patients have received 211At-labeled chimeric 81C6 directly into their brain tumor resection cavity, and very encouraging results have been obtained. Now that the feasibility of human studies with 211At, has been demonstrated, the development and evaluation of anti-tenascin constructs with optimized properties for use in tandem with short half life radionuclides such as 211At ( as well as 1.8-hr 18F for PET imaging) is warranted. Our specific aims are: 1) to construct a bivalent, anti-tenascin molecule containing murine 81C6 variable regions and the human IgG2 hinge region. Both the CH2 domain deletion construct (CH2) and F(ab’)2 will be investigated; 2) to construct a single-chain Fv dimer or multimer with adequate stability, affinity and immunoreactivity for use in tandem with 211At for therapy and 18F for imaging; 3) to generate higher affinity scFv constructs reactive with the alternatively spliced fibronectin type III repeats CD of the tenascin molecule via phage display technology and site-directed mutagenesis; 4) to label promising anti-tenascin constructs with radioiodine, 211At, and 18F and evaluate their potential as radiodiagnostic and radiotherapeutic agents. The proposed studies include: characterization of affinity and immunoreactivity after labeling; evaluation of tissue distribution and projected dosimetry in normal mice, and athymic rodents with subcutaneous, intracranial and neoplastic meningitis xenografts; investigation of the nature of low and high molecular weight labeled catabolites generated in mice; and assessment of cytotoxicity in vitro and in vivo models of human glioma, and possibly, other tenascin expressing tumors; and 5) to investigate strategies for labeling scFv monomers and dimers which will minimize retention of the radiohalogen in the kidneys through the use of negatively charged templates.« less

Lack of Adipocyte-Fndc5/Irisin Expression and Secretion Reduces Thermogenesis and Enhances Adipogenesis.

PubMed

Pérez-Sotelo, D; Roca-Rivada, A; Baamonde, I; Baltar, J; Castro, A I; Domínguez, E; Collado, M; Casanueva, F F; Pardo, M

2017-11-24

Irisin is a browning-stimulating molecule secreted from the fibronectin type III domain containing 5 precursor (FNDC5) by muscle tissue upon exercise stimulation. Despite its beneficial role, there is an unmet and clamorous need to discern many essential aspects of this protein and its mechanism of action not only as a myokine but also as an adipokine. Here we contribute to address this topic by revealing the nature and role of FNDC5/irisin in adipose tissue. First, we show that FNDC5/irisin expression and secretion are induced by adipocyte differentiation and confirm its over-secretion by human obese visceral (VAT) and subcutaneous (SAT) adipose tissues. Second, we show how secreted factors from human obese VAT and SAT decrease PGC1α, FNDC5 and UCP1 gene expression on differentiating adipocytes; this effect over UCP1 is blunted by blocking irisin in obese secretomes. Finally, by stable gene silencing FNDC5 we reveal that FNDC5-KO adipocytes show reduced UCP1 expression and enhanced adipogenesis.

Novel leukocyte protein, Trojan, differentially expressed during thymocyte development.

PubMed

Petrov, Petar; Motobu, Maki; Salmi, Jussi; Uchida, Tatsuya; Vainio, Olli

2010-04-01

"Trojan" is a novel cell surface protein, discovered from chicken embryonic thymocytes on the purpose to identify molecules involved in T cell differentiation. The molecule is predicted as a type I transmembrane protein having a Sushi and two fibronectin type III domains and a pair of intracellular phosphorylation sites. Its transcript expression is specific for lymphoid tissues and the presence of the protein on the surface of recirculating lymphocytes and macrophages was confirmed by immunofluorescence analysis. In thymus, about half of the double negative (CD4(-) CD8(-)) and CD8 single positive and the majority of CD4 single positive cells express Trojan with a relatively high intensity. However, only a minority of the double positive (CD4(+) CD8(+)) cells are positive for Trojan. This expression pattern, similar to that of some proteins with anti-apoptotic and function, like IL-7Ralpha, makes Trojan an attractive candidate of having an anti-apoptotic role. Copyright 2010 Elsevier Ltd. All rights reserved.

Separation of integrin-dependent adhesion from morphological changes based on differential PLC specificities.

PubMed

Wooten, D K; Teague, T K; McIntyre, B W

1999-01-01

In normal lymphocytes an inside-out signal up-regulating integrin adhesion is followed by a ligand-mediated outside-in cell spreading signal. Protein kinase C (PKC) inhibition blocks lymphocyte adherence to and spreading on fibronectin. In contrast, putative PLC inhibitors yield distinct differences with respect to adhesion and morphology. The phosphatidylinositol-specific phospholipase C (PLC) inhibitor neomycin blocked spreading of CD3/CD28-activated T cells on fibronectin by disrupting adhesion. Furthermore, when an additional inside-out signal for fibronectin adhesion is unnecessary such as with HPB-ALL T leukemic or phorbol-myristate-acetate-treated normal T cells, neomycin treatment does not alter adhesion or morphology. However, the phosphatidylcholine-specific PLC inhibitor D609 abrogates cell spreading without affecting adhesion to fibronectin in these cells as well as the CD3/CD28-activated T cells. These results strongly suggest that inside-out signaling for the integrin alpha4beta1 in lymphocytes proceeds through phosphatidylinositol-specific PLC and PKC, whereas the outside-in signal utilizes phosphatidylcholine-specific PLC and PKC.

Structural and functional analysis of an anchorless fibronectin-binding protein FBPS from Gram-positive bacterium Streptococcus suis.

PubMed

Musyoki, Abednego Moki; Shi, Zhongyu; Xuan, Chunling; Lu, Guangwen; Qi, Jianxun; Gao, Feng; Zheng, Beiwen; Zhang, Qiangmin; Li, Yan; Haywood, Joel; Liu, Cuihua; Yan, Jinghua; Shi, Yi; Gao, George F

2016-11-29

The anchorless fibronectin-binding proteins (FnBPs) are a group of important virulence factors for which the structures are not available and the functions are not well defined. In this study we performed comprehensive studies on a prototypic member of this group: the fibronectin-/fibrinogen-binding protein from Streptococcus suis (FBPS). The structures of the N- and C-terminal halves (FBPS-N and FBPS-C), which together cover the full-length protein in sequence, were solved at a resolution of 2.1 and 2.6 Å, respectively, and each was found to be composed of two domains with unique folds. Furthermore, we have elucidated the organization of these domains by small-angle X-ray scattering. We further showed that the fibronectin-binding site is located in FBPS-C and that FBPS promotes the adherence of S suis to host cells by attaching the bacteria via FBPS-N. Finally, we demonstrated that FBPS functions both as an adhesin, promoting S suis attachment to host cells, and as a bacterial factor, activating signaling pathways via β1 integrin receptors to induce chemokine production.

The α5β1 Integrin Mediates Elimination of Amyloid-β Peptide and Protects Against Apoptosis

PubMed Central

Matter, Michelle L.; Zhang, Zhuohua; Nordstedt, Christer; Ruoslahti, Erkki

1998-01-01

The amyloid-β peptide (Aβ) can mediate cell attachment by binding to β1 integrins through an arg-his-asp sequence. We show here that the α5β1 integrin, a fibronectin receptor, is an efficient binder of Aβ, and mediates cell attachment to nonfibrillar Aβ. Cells engineered to express α5β1 internalized and degraded more added Aβ1-40 than did α5β1-negative control cells. Deposition of an insoluble Aβ1-40 matrix around the α5β1-expressing cells was reduced, and the cells showed less apoptosis than the control cells. Thus, the α5β1 integrin may protect against Aβ deposition and toxicity, which is a course of Alzheimer's disease lesions. PMID:9585419

Characteristics of allelic gene expression in human brain cells from single-cell RNA-seq data analysis.

PubMed

Zhao, Dejian; Lin, Mingyan; Pedrosa, Erika; Lachman, Herbert M; Zheng, Deyou

2017-11-10

Monoallelic expression of autosomal genes has been implicated in human psychiatric disorders. However, there is a paucity of allelic expression studies in human brain cells at the single cell and genome wide levels. In this report, we reanalyzed a previously published single-cell RNA-seq dataset from several postmortem human brains and observed pervasive monoallelic expression in individual cells, largely in a random manner. Examining single nucleotide variants with a predicted functional disruption, we found that the "damaged" alleles were overall expressed in fewer brain cells than their counterparts, and at a lower level in cells where their expression was detected. We also identified many brain cell type-specific monoallelically expressed genes. Interestingly, many of these cell type-specific monoallelically expressed genes were enriched for functions important for those brain cell types. In addition, function analysis showed that genes displaying monoallelic expression and correlated expression across neuronal cells from different individual brains were implicated in the regulation of synaptic function. Our findings suggest that monoallelic gene expression is prevalent in human brain cells, which may play a role in generating cellular identity and neuronal diversity and thus increasing the complexity and diversity of brain cell functions.

[Expression of c-jun protein after experimental rat brain concussion].

PubMed

Wang, Feng; Li, Yong-hong

2010-02-01

To observe e-jun protein expression after rat brain concussion and explore the forensic pathologic markers following brain concussion. Fifty-five rats were randomly divided into brain concussion group and control group. The expression of c-jun protein was observed by immunohistochemistry. There were weak positive expression of c-jun protein in control group. In brain concussion group, however, some neutrons showed positive expression of c-jun protein at 15 min after brain concussion, and reach to the peak at 3 h after brain concussion. The research results suggest that detection of c-jun protein could be a marker to determine brain concussion and estimate injury time after brain concussion.

Ethanol-induced hyponatremia augments brain edema after traumatic brain injury.

PubMed

Katada, Ryuichi; Watanabe, Satoshi; Ishizaka, Atsushi; Mizuo, Keisuke; Okazaki, Shunichiro; Matsumoto, Hiroshi

2012-04-01

Alcohol consumption augments brain edema by expression of brain aquaporin-4 after traumatic brain injury. However, how ethanol induces brain aquaporin-4 expression remains unclear. Aquaporin-4 can operate with some of ion channels and transporters. Therefore, we hypothesized that ethanol may affect electrolytes through regulating ion channels, leading to express aquaporin-4. To clarify the hypothesis, we examined role of AQP4 expression in ethanol-induced brain edema and changes of electrolyte levels after traumatic brain injury in the rat. In the rat traumatic brain injury model, ethanol administration reduced sodium ion concentration in blood significantly 24 hr after injury. An aquaporin-4 inhibitor recovered sodium ion concentration in blood to normal. We observed low sodium ion concentration in blood and the increase of brain aquaporin-4 in cadaver with traumatic brain injury. Therefore, ethanol increases brain edema by the increase of aquaporin-4 expression with hyponatremia after traumatic brain injury.

Effect of a nutrient mixture on the localization of extracellular matrix proteins in HeLa human cervical cancer xenografts in female nude mice.

PubMed

Roomi, M Waheed; Cha, John; Kalinovsky, Tatiana; Roomi, Nusrath; Niedzwiecki, Aleksandra; Rath, Matthias

2015-09-01

Cervical cancer is one of the most commonly diagnosed cancers and a significant cause of mortality in women worldwide. Although cervical cancer is fully treatable in the early stages, once it has metastasized, patient outcome is poor. The objective of the present study was to investigate the effect of dietary supplementation with a nutrient mixture (NM) containing lysine, ascorbic acid, proline, green tea extract and other micronutrients on the expression of extracellular matrix (ECM) proteins in HeLa cell xenografts in nude female mice. After housing for 1 week, female athymic nude mice between 5 and 6 weeks of age (n=12) were inoculated subcutaneously with 3×10 6 HeLa cells in phosphate-buffered saline and Matrigel and randomly divided into two groups. These were the control group, in which the mice were fed with regular mouse chow, and the NM group, in which the mice were fed with the regular diet supplemented with 0.5% NM (w/w). After 4 weeks, the tumors were excised and processed for histology. Tumor growth was evaluated and the tumors were stained for the ECM proteins collagen I, collagen IV, fibronectin, laminin, periodic acid-Schiff (PAS) and elastin. NM strongly inhibited (by 59%, P=0.001) the growth of HeLa xenografts in nude mice. Tumors from control mice exhibited little to no collagen I expression either internally or in the fibrous capsule, while tumors from the NM group expressed collagen I in the fibrous capsule and within the tumor. Tumors from the control group showed diffuse cytoplasmic and capsular collagen IV with abundant nucleated cells. NM treatment substantially increased collagen IV production and induced a dense fibrous network of collagen IV with chambers that surrounded live nucleated cells and large amounts of necrotic cell debris. Tumors from the mice fed with the NM exhibited a well-defined border of fibronectin in the capsule and intense areas of staining internally whereas control group tumors showed less overall fibronectin with sporadic internal staining and little in the fibrous capsule. Although laminin appeared abundantly in control and NM-treated tumors, the NM group tumors exhibited a chamber-like network of laminin internally. Tumors from the control group exhibited internal areas of intense PAS staining, whereas tumors from the NM-treated group exhibited a more uniform diffuse pattern of PAS staining. In conclusion, NM supplementation of HeLa xenograft-bearing female nude mice demonstrated a potent inhibition of tumor growth and enhancement of ECM proteins, suggesting the therapeutic value of this specific nutrient complex in the treatment of cervical cancer.

Microparticles Engineered to Highly Express Peroxisome Proliferator-Activated Receptor-γ Decreased Inflammatory Mediator Production and Increased Adhesion of Recipient Monocytes

PubMed Central

Sahler, Julie; Woeller, Collynn F.; Phipps, Richard P.

2014-01-01

Circulating blood microparticles are submicron vesicles released primarily by megakaryocytes and platelets that act as transcellular communicators. Inflammatory conditions exhibit elevated blood microparticle numbers compared to healthy conditions. Direct functional consequences of microparticle composition, especially internal composition, on recipient cells are poorly understood. Our objective was to evaluate if microparticle composition could impact the function of recipient cells, particularly during inflammatory provocation. We therefore engineered the composition of megakaryocyte culture-derived microparticles to generate distinct microparticle populations that were given to human monocytes to assay for influences recipient cell function. Herein, we tested the responses of monocytes exposed to either control microparticles or microparticles that contain the anti-inflammatory transcription factor, peroxisome proliferator-activated receptor-γ (PPARγ). In order to normalize relative microparticle abundance from two microparticle populations, we implemented a novel approach that utilizes a Nanodrop Spectrophotometer to assay for microparticle density rather than concentration. We found that when given to peripheral blood mononuclear cells, microparticles were preferentially internalized by CD11b+ cells, and furthermore, microparticle composition had a profound functional impact on recipient monocytes. Specifically, microparticles containing PPARγ reduced activated monocyte production of the proinflammatory cytokines interleukin-8 and monocyte chemotactic protein-1 compared to activated monocytes exposed to control microparticles. Additionally, treatment with PPARγ microparticles greatly increased monocyte cell adherence. This change in morphology occurred simultaneously with increased production of the key extracellular matrix protein, fibronectin and increased expression of the fibronectin-binding integrin, ITGA5. PPARγ microparticles also changed monocyte mRNA levels of several genes including those under PPARγ control. Overall, the delivery of PPARγ from microparticles to human monocytes influenced gene expression, decreased inflammatory mediator production and increased monocyte adherence. These results support the concept that the composition of blood microparticles has a profound impact on the function of cells with which they interact, and likely plays a role in vascular inflammation. PMID:25426628

Transglutaminases factor XIII-A and TG2 regulate resorption, adipogenesis and plasma fibronectin homeostasis in bone and bone marrow

PubMed Central

Mousa, Aisha; Cui, Cui; Song, Aimei; Myneni, Vamsee D; Sun, Huifang; Li, Jin Jin; Murshed, Monzur; Melino, Gerry; Kaartinen, Mari T

2017-01-01

Appropriate bone mass is maintained by bone-forming osteoblast and bone-resorbing osteoclasts. Mesenchymal stem cell (MSC) lineage cells control osteoclastogenesis via expression of RANKL and OPG (receptor activator of nuclear factor κB ligand and osteoprotegerin), which promote and inhibit bone resorption, respectively. Protein crosslinking enzymes transglutaminase 2 (TG2) and Factor XIII-A (FXIII-A) have been linked to activity of myeloid and MSC lineage cells; however, in vivo evidence has been lacking to support their function. In this study, we show in mice that TG2 and FXIII-A control monocyte-macrophage cell differentiation into osteoclasts as well as RANKL production in MSCs and in adipocytes. Long bones of mice lacking TG2 and FXIII-A transglutaminases, show compromised biomechanical properties and trabecular bone loss in axial and appendicular skeleton. This was caused by increased osteoclastogenesis, a cellular phenotype that persists in vitro. The increased potential of TG2 and FXIII-A deficient monocytes to form osteoclasts was reversed by chemical inhibition of TG activity, which revealed the presence of TG1 in osteoclasts and assigned different roles for the TGs as regulators of osteoclastogenesis. TG2- and FXIII-A-deficient mice had normal osteoblast activity, but increased bone marrow adipogenesis, MSCs lacking TG2 and FXIII-A showed high adipogenic potential and significantly increased RANKL expression as well as upregulated TG1 expression. Chemical inhibition of TG activity in the null cells further increased adipogenic potential and RANKL production. Altered differentiation of TG2 and FXIII-A null MSCs was associated with plasma fibronectin (FN) assembly defect in cultures and FN retention in serum and marrow in vivo instead of assembly into bone. Our findings provide new functions for TG2, FXIII-A and TG1 in bone cells and identify them as novel regulators of bone mass, plasma FN homeostasis, RANKL production and myeloid and MSC cell differentiation. PMID:28387755

Microparticles engineered to highly express peroxisome proliferator-activated receptor-γ decreased inflammatory mediator production and increased adhesion of recipient monocytes.

PubMed

Sahler, Julie; Woeller, Collynn F; Phipps, Richard P

2014-01-01

Circulating blood microparticles are submicron vesicles released primarily by megakaryocytes and platelets that act as transcellular communicators. Inflammatory conditions exhibit elevated blood microparticle numbers compared to healthy conditions. Direct functional consequences of microparticle composition, especially internal composition, on recipient cells are poorly understood. Our objective was to evaluate if microparticle composition could impact the function of recipient cells, particularly during inflammatory provocation. We therefore engineered the composition of megakaryocyte culture-derived microparticles to generate distinct microparticle populations that were given to human monocytes to assay for influences recipient cell function. Herein, we tested the responses of monocytes exposed to either control microparticles or microparticles that contain the anti-inflammatory transcription factor, peroxisome proliferator-activated receptor-γ (PPARγ). In order to normalize relative microparticle abundance from two microparticle populations, we implemented a novel approach that utilizes a Nanodrop Spectrophotometer to assay for microparticle density rather than concentration. We found that when given to peripheral blood mononuclear cells, microparticles were preferentially internalized by CD11b+ cells, and furthermore, microparticle composition had a profound functional impact on recipient monocytes. Specifically, microparticles containing PPARγ reduced activated monocyte production of the proinflammatory cytokines interleukin-8 and monocyte chemotactic protein-1 compared to activated monocytes exposed to control microparticles. Additionally, treatment with PPARγ microparticles greatly increased monocyte cell adherence. This change in morphology occurred simultaneously with increased production of the key extracellular matrix protein, fibronectin and increased expression of the fibronectin-binding integrin, ITGA5. PPARγ microparticles also changed monocyte mRNA levels of several genes including those under PPARγ control. Overall, the delivery of PPARγ from microparticles to human monocytes influenced gene expression, decreased inflammatory mediator production and increased monocyte adherence. These results support the concept that the composition of blood microparticles has a profound impact on the function of cells with which they interact, and likely plays a role in vascular inflammation.

Irisin inhibition of growth hormone secretion in cultured tilapia pituitary cells.

PubMed

Lian, Anji; Li, Xin; Jiang, Quan

2017-01-05

Irisin, the product of fibronectin type III domain-containing protein 5 (FNDC5) gene, is well-documented to be a regulator of energy metabolism. At present, not much is known about its biological function in non-mammalian species. In this study, a full-length tilapia FDNC5 was cloned and its tissue expression pattern has been confirmed. Based on the sequence obtained, we produced and purified recombinant irisin which could induce uncoupling protein 1 (UCP1) gene expression in tilapia hepatocytes. Further, the rabbit polyclonal irisin antiserum was produced and its specificity was confirmed by antiserum preabsorption. In tilapia pituitary cells, irisin inhibited growth hormone (GH) gene expression and secretion and triggered rapid phosphorylation of Akt, Erk1/2, and p38 MAPK. Furthermore, irisin-inhibited GH mRNA expression could be prevented by inhibiting PI3K/Akt, MEK1/2, and p38 MAPK, respectively. Apparently, fish irisin can act directly at the pituitary level to inhibit GH transcript expression via multiple signaling pathways. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

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

PubMed

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

2017-08-01

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

Dysregulated fibronectin trafficking by Hsp90 inhibition restricts prostate cancer cell invasion.

PubMed

Armstrong, Heather K; Gillis, Joanna L; Johnson, Ian R D; Nassar, Zeyad D; Moldovan, Max; Levrier, Claire; Sadowski, Martin C; Chin, Mei Yieng; Tomlinson Guns, Emma S; Tarulli, Gerard; Lynn, David J; Brooks, Douglas A; Selth, Luke A; Centenera, Margaret M; Butler, Lisa M

2018-02-01

The molecular chaperone Hsp90 is overexpressed in prostate cancer (PCa) and is responsible for the folding, stabilization and maturation of multiple oncoproteins, which are implicated in PCa progression. Compared to first-in-class Hsp90 inhibitors such as 17-allylamino-demethoxygeldanamycin (17-AAG) that were clinically ineffective, second generation inhibitor AUY922 has greater solubility and efficacy. Here, transcriptomic and proteomic analyses of patient-derived PCa explants identified cytoskeletal organization as highly enriched with AUY922 treatment. Validation in PCa cell lines revealed that AUY922 caused marked alterations to cell morphology, and suppressed cell motility and invasion compared to vehicle or 17-AAG, concomitant with dysregulation of key extracellular matrix proteins such as fibronectin (FN1). Interestingly, while the expression of FN1 was increased by AUY922, FN1 secretion was significantly decreased. This resulted in cytosolic accumulation of FN1 protein within late endosomes, suggesting that AUY922 disrupts vesicular secretory trafficking pathways. Depletion of FN1 by siRNA knockdown markedly reduced the invasive capacity of PCa cells, phenocopying AUY922. These results highlight a novel mechanism of action for AUY922 beyond its established effects on cellular mitosis and survival and, furthermore, identifies extracellular matrix cargo delivery as a potential therapeutic target for the treatment of aggressive PCa.

Adipose progenitor cells increase fibronectin matrix strain and unfolding in breast tumors

NASA Astrophysics Data System (ADS)

Chandler, E. M.; Saunders, M. P.; Yoon, C. J.; Gourdon, D.; Fischbach, C.

2011-02-01

Increased stiffness represents a hallmark of breast cancer that has been attributed to the altered physicochemical properties of the extracellular matrix (ECM). However, the role of fibronectin (Fn) in modulating the composition and mechanical properties of the tumor-associated ECM remains unclear. We have utilized a combination of biochemical and physical science tools to evaluate whether paracrine signaling between breast cancer cells and adipose progenitor cells regulates Fn matrix assembly and stiffness enhancement in the tumor stroma. In particular, we utilized fluorescence resonance energy transfer imaging to map the molecular conformation and stiffness of Fn that has been assembled by 3T3-L1 preadipocytes in response to conditioned media from MDA-MB231 breast cancer cells. Our results reveal that soluble factors secreted by tumor cells promote Fn expression, unfolding, and stiffening by adipose progenitor cells and that transforming growth factor-β serves as a soluble cue underlying these changes. In vivo experiments using orthotopic co-transplantation of primary human adipose-derived stem cells and MDA-MB231 into SCID mice support the pathological relevance of our results. Insights gained by these studies advance our understanding of the role of Fn in mammary tumorigenesis and may ultimately lead to improved anti-cancer therapies.

Coding SNP in tenascin-C Fn-III-D domain associates with adult asthma.

PubMed

Matsuda, Akira; Hirota, Tomomitsu; Akahoshi, Mitsuteru; Shimizu, Makiko; Tamari, Mayumi; Miyatake, Akihiko; Takahashi, Atsushi; Nakashima, Kazuko; Takahashi, Naomi; Obara, Kazuhiko; Yuyama, Noriko; Doi, Satoru; Kamogawa, Yumiko; Enomoto, Tadao; Ohshima, Koichi; Tsunoda, Tatsuhiko; Miyatake, Shoichiro; Fujita, Kimie; Kusakabe, Moriaki; Izuhara, Kenji; Nakamura, Yusuke; Hopkin, Julian; Shirakawa, Taro

2005-10-01

The extracellular matrix glycoprotein tenascin-C (TNC) has been accepted as a valuable histopathological subepithelial marker for evaluating the severity of asthmatic disease and the therapeutic response to drugs. We found an association between an adult asthma and an SNP encoding TNC fibronectin type III-D (Fn-III-D) domain in a case-control study between a Japanese population including 446 adult asthmatic patients and 658 normal healthy controls. The SNP (44513A/T in exon 17) strongly associates with adult bronchial asthma (chi2 test, P=0.00019, Odds ratio=1.76, 95% confidence interval=1.31-2.36). This coding SNP induces an amino acid substitution (Leu1677Ile) within the Fn-III-D domain of the alternative splicing region. Computer-assisted protein structure modeling suggests that the substituted amino acid locates at the outer edge of the beta-sheet in Fn-III-D domain and causes instability of this beta-sheet. As the TNC fibronectin-III domain has molecular elasticity, the structural change may affect the integrity and stiffness of asthmatic airways. In addition, TNC expression in lung fibroblasts increases with Th2 immune cytokine stimulation. Thus, Leu1677Ile may be valuable marker for evaluating the risk for developing asthma and plays a role in its pathogenesis.

Articular Chondrocytes Express the Receptor for Advanced Glycation End Products

PubMed Central

Loeser, Richard F.; Yammani, Raghunatha R.; Carlson, Cathy S.; Chen, Hong; Cole, Ada; Im, Hee-Jeong; Bursch, Laura S.; Yan, Shi Du

2006-01-01

Objective The receptor for advanced glycation end products (RAGE) binds multiple ligands, including S100 proteins, high mobility group box chromosomal protein 1 (HMGB-1), and AGEs, all of which are present in articular cartilage. Stimulation of RAGE signaling can lead to MAP kinase activation and increased NF-κB activity. The objective of the present study was to determine if chondrocytes express functional RAGE. Methods The presence of chondrocyte RAGE was analyzed by immunohistochemistry using normal and osteoarthritic (OA) cartilage from young and old monkeys and humans, immunoblotting of chondrocyte lysates and human cartilage extracts, and reverse transcription–polymerase chain reaction (RT-PCR) analysis of RNA from chondrocytes treated with interleukin-1 (IL-1) and fibronectin fragments. RAGE signaling was evaluated by stimulating chondrocytes with S100B and HMGB-1 and analyzing for activation of the ERK MAP kinase and NF-κB. The ability of S100B and HMGB-1 to stimulate matrix metalloproteinase 13 (MMP-13) production was also assessed. A pull-down assay using biotin-labeled S100B was used to demonstrate binding to RAGE. Results RAGE was detected in sections of monkey knee cartilage and human knee and ankle cartilage. Increased immunostaining for RAGE was noted in cartilage from older adult monkeys and humans and was further increased in OA tissue. RAGE was also detected by immunoblotting and by RT-PCR, where IL-1β and fibronectin fragments were found to stimulate RAGE expression. Stimulation of chondrocytes with S100B or HMGB-1 increased phosphorylation of the ERK MAP kinase and the p65 subunit of NF-κB and increased the production of MMP-13. This signaling was inhibited in cells pretreated with soluble RAGE, and S100B was shown to bind to chondrocyte RAGE. Conclusion Articular chondrocytes express functional RAGE. The increase in RAGE noted in OA cartilage and the ability of RAGE ligands to stimulate chondrocyte MAP kinase and NF-κB activity and to stimulate MMP-13 production suggests that chondrocyte RAGE signaling could play a role in OA. PMID:16052547

Interaction with extracellular matrix proteins influences Lsh/Ity/Bcg (candidate Nramp) gene regulation of macrophage priming/activation for tumour necrosis factor-alpha and nitrite release.

PubMed

Formica, S; Roach, T I; Blackwell, J M

1994-05-01

The murine resistance gene Lsh/Ity/Bcg regulates activation of macrophages for tumour necrosis factor-alpha (TNF-alpha)-dependent production of nitric oxide mediating antimicrobial activity against Leishmania, Salmonella and Mycobacterium. As Lsh is differentially expressed in macrophages from different tissue sites, experiments were performed to determine whether interaction with extracellular matrix (ECM) proteins would influence the macrophage TNF-alpha response. Plating of bone marrow-derived macrophages onto purified fibrinogen or fibronectin-rich L929 cell-derived matrices, but not onto mannan, was itself sufficient to stimulate TNF-alpha release, with significantly higher levels released from congenic B10.L-Lshr compared to C57BL/10ScSn (Lshs) macrophages. Only macrophages plated onto fibrinogen also released measurable levels of nitrites, again higher in Lshr compared to Lshs macrophages. Addition of interferon-gamma (IFN-gamma), but not bacterial lipopolysaccharide or mycobacterial lipoarabinomannan, as a second signal enhanced the TNF-alpha and nitrite responses of macrophages plated onto fibrinogen, particularly in the Lshr macrophages. Interaction with fibrinogen and fibronectin also primed macrophages for an enhanced TNF-alpha response to leishmanial parasites, but this was only translated into enhanced nitrite responses in the presence of IFN-gamma. In these experiments, Lshr macrophages remained superior in their TNF-alpha responses throughout, but to a degree which reflected the magnitude of the difference observed on ECM alone. Hence, the specificity for the enhanced TNF-alpha responses of Lshr macrophages lay in their interaction with fibrinogen and fibronectin ECM, while a differential nitrite response was only observed with fibrinogen and/or IFN-gamma. The results are discussed in relation to the possible function of the recently cloned candidate gene Nramp, which has structural identity to eukaryote transporters and an N-terminal cytoplasmic proline/serine-rich putative SH3 binding domain.

Binding of extracellular matrix proteins to Aspergillus fumigatus conidia.

PubMed Central

Gil, M L; Peñalver, M C; Lopez-Ribot, J L; O'Connor, J E; Martinez, J P

1996-01-01

As detected by confocal immunofluorescence microscopy, binding of fibronectin and laminin appeared to be associated with the protrusions present on the outer cell wall layer of resting Aspergillus fumigatus conidia. Flow cytometry confirmed that binding of laminin to conidia was dose dependent and saturable. Laminin binding was virtually eliminated in trypsin-treated organisms, thus suggesting the protein nature of the binding site. Conidia were also able to specifically adhere to laminin immobilized on microtiter plates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) with laminin and antilaminin antibody of whole conidial homogenates allowed identification, among the complex array of protein and glycoprotein species, of one polypeptide with an apparent molecular mass of 37 kDa which specifically interacts with laminin. The fact that binding of conidia to soluble or immobilized laminin or fibronectin was inhibited by fibronectin or laminin, respectively, suggests the existence of common binding sites for both ligands on the surface of conidia. Intact conidia were also able to adhere to type I and IV collagen immobilized on microtiter plates; adhesion was found to be dose dependent and saturable. Adhesion to immobilized type I and IV collagen was markedly inhibited by laminin and weakly inhibited by fibronectin. Coincubation of conidia with Arg-Gly-Asp (RGD) peptides caused a dose-dependent decrease in binding of cells to immobilized or soluble fibronectin, yet interaction of cells with soluble or immobilized laminin and type I and IV collagen remained unaffected. Interactions described here could be important in mediating attachment of the fungus to host tissues, thus playing a role in the establishment of the disease. PMID:8945572

Enhanced expression by the brain matrix of P-glycoprotein in brain capillary endothelial cells.

PubMed

Tatsuta, T; Naito, M; Mikami, K; Tsuruo, T

1994-10-01

P-glycoprotein (PGP), an active efflux pump of antitumor agents in multidrug-resistant tumor cells, exists in brain capillary endothelium and could be functionally involved in the blood-brain barrier. To study the regulatory mechanism of PGP expression in brain capillary endothelium, various mouse tissue matrices were tested for their abilities to enhance the expression of PGP in mouse brain capillary endothelial cells (MBEC), which express relatively small amounts of PGP. Of the four tissue matrices we examined, PGP expression in MBEC cultured on the brain matrix increased 2.0-fold. The PGP-inducing activity was similarly detected in bovine brain matrix, and the activity was enriched in the fraction of pl 9.0 by isoelectric focusing. The fraction, named PIC-fraction (PGP-inducing component), increased the PGP expression in MBEC 3.5-fold. By Northern blot analysis, a 3.3-fold enhancement of mdr gene expression was observed in MBEC cultured on the PIC-fraction. The PGP-inducing activity of the PIC-fraction was reduced by the treatment with trypsin but not with collagenase, suggesting that a proteinaceous factor distinct from type I collagen might be responsible for the PGP-inducing activity of PIC-fraction. Although the PIC-fraction increased the PGP expression in other mouse brain capillary endothelial cells, the PIC-fraction did not increase PGP expression in mouse aortic endothelial cells and KB carcinoma cell lines expressing various amounts of PGP. These observations suggest that PGP expression in brain capillary endothelium is specifically regulated by a tissue-specific factor in the brain matrix.

Accelerated recruitment of new brain development genes into the human genome.

PubMed

Zhang, Yong E; Landback, Patrick; Vibranovski, Maria D; Long, Manyuan

2011-10-01

How the human brain evolved has attracted tremendous interests for decades. Motivated by case studies of primate-specific genes implicated in brain function, we examined whether or not the young genes, those emerging genome-wide in the lineages specific to the primates or rodents, showed distinct spatial and temporal patterns of transcription compared to old genes, which had existed before primate and rodent split. We found consistent patterns across different sources of expression data: there is a significantly larger proportion of young genes expressed in the fetal or infant brain of humans than in mouse, and more young genes in humans have expression biased toward early developing brains than old genes. Most of these young genes are expressed in the evolutionarily newest part of human brain, the neocortex. Remarkably, we also identified a number of human-specific genes which are expressed in the prefrontal cortex, which is implicated in complex cognitive behaviors. The young genes upregulated in the early developing human brain play diverse functional roles, with a significant enrichment of transcription factors. Genes originating from different mechanisms show a similar expression bias in the developing brain. Moreover, we found that the young genes upregulated in early brain development showed rapid protein evolution compared to old genes also expressed in the fetal brain. Strikingly, genes expressed in the neocortex arose soon after its morphological origin. These four lines of evidence suggest that positive selection for brain function may have contributed to the origination of young genes expressed in the developing brain. These data demonstrate a striking recruitment of new genes into the early development of the human brain.

Behavior of sea urchin primary mesenchyme cells in artificial extracellular matrices.

PubMed

Katow, H

1986-02-01

The primary mesenchyme cells (PMCs) were separated from the mesenchyme blastulae of Pseudocentrotus depressus using differential adhesiveness of these cells to plastic Petri dishes. These cells were incubated in various artificial extracellular matrices (ECMs) including horse serum plasma fibronectin, mouse EHS sarcoma laminin, mouse EHS sarcoma type IV collagen, and porcine skin dermatan sulfate. The cell behavior was monitored by a time-lapse videomicrograph and analysed with a microcomputer. The ultrastructure of the artificial ECM was examined by transmission electron microscopy (TEM), while the ultrastructure of the PMCs was examined by scanning electron microscopy (SEM). The PMCs did not migrate in type IV collagen gel, laminin or dermatan sulfate matrix either with or without collagen gel, whereas PMCs in the matrix which was composed of fibronectin and collagen gel migrated considerably. However, the most active and extensive PMC migration was seen in the matrix which contained dermatan sulfate in addition to fibronectin and collagen gel. This PMC migration involved an increase not only of migration speed but also of proportion of migration-promoted cells. These results support the hypothesis that the mechanism of PMC migration involves fibronectin, collagen and sulfated proteoglycans which contain dermatan sulfate.

Controlled Assembly of Fibronectin Nanofibrils Triggered by Random Copolymer Chemistry.

PubMed

Mnatsakanyan, Hayk; Rico, Patricia; Grigoriou, Eleni; Candelas, Aarón Maturana; Rodrigo-Navarro, Aleixandre; Salmeron-Sanchez, Manuel; Sabater i Serra, Roser

2015-08-19

Fibronectin fibrillogenesis is the physiological process by which cells elaborate a fibrous FN matrix. Poly(ethyl acrylate), PEA, has been described to induce a similar process upon simple adsorption of fibronectin (FN) from a protein solution-in the absence of cells-leading to the so-called material-driven fibronectin fibrillogenesis. Poly(methyl acrylate), PMA, is a polymer with very similar chemistry to PEA, on which FN is adsorbed, keeping the globular conformation of the protein in solution. We have used radical polymerization to synthesize copolymers with controlled EA/MA ratio, seeking to modulate the degree of FN fibrillogenesis. The physicochemical properties of the system were studied using dynamic-mechanical analysis, differential scanning calorimetry, and water contact angle. Both the degree of FN fibrillogenesis and the availability of the integrin binding region of FN directly depend on the percentage of EA in the copolymer, whereas the same total amount of FN was adsorbed regardless the EA/MA ratio. Cell morphology adhesion and differentiation of murine C2C12 were shown to depend on the degree of FN fibrillogenesis previously attained on the material surface. Myogenic differentiation was enhanced on the copolymers with higher EA content, i.e. more interconnected FN fibrils.

Protein nanocoatings on synthetic polymeric nanofibrous membranes designed as carriers for skin cells.

PubMed

Bacakova, Marketa; Pajorova, Julia; Stranska, Denisa; Hadraba, Daniel; Lopot, Frantisek; Riedel, Tomas; Brynda, Eduard; Zaloudkova, Margit; Bacakova, Lucie

2017-01-01

Protein-coated resorbable synthetic polymeric nanofibrous membranes are promising for the fabrication of advanced skin substitutes. We fabricated electrospun polylactic acid and poly(lactide- co -glycolic acid) nanofibrous membranes and coated them with fibrin or collagen I. Fibronectin was attached to a fibrin or collagen nanocoating, in order further to enhance the cell adhesion and spreading. Fibrin regularly formed a coating around individual nanofibers in the membranes, and also formed a thin noncontinuous nanofibrous mesh on top of the membranes. Collagen also coated most of the fibers of the membrane and randomly created a soft gel on the membrane surface. Fibronectin predominantly adsorbed onto a thin fibrin mesh or a collagen gel, and formed a thin nanofibrous structure. Fibrin nanocoating greatly improved the attachment, spreading, and proliferation of human dermal fibroblasts, whereas collagen nanocoating had a positive influence on the behavior of human HaCaT keratinocytes. In addition, fibrin stimulated the fibroblasts to synthesize fibronectin and to deposit it as an extracellular matrix. Fibrin coating also showed a tendency to improve the ultimate tensile strength of the nanofibrous membranes. Fibronectin attached to fibrin or to a collagen coating further enhanced the adhesion, spreading, and proliferation of both cell types.

Protein nanocoatings on synthetic polymeric nanofibrous membranes designed as carriers for skin cells

PubMed Central

Bacakova, Marketa; Pajorova, Julia; Stranska, Denisa; Hadraba, Daniel; Lopot, Frantisek; Riedel, Tomas; Brynda, Eduard; Zaloudkova, Margit; Bacakova, Lucie

2017-01-01

Protein-coated resorbable synthetic polymeric nanofibrous membranes are promising for the fabrication of advanced skin substitutes. We fabricated electrospun polylactic acid and poly(lactide-co-glycolic acid) nanofibrous membranes and coated them with fibrin or collagen I. Fibronectin was attached to a fibrin or collagen nanocoating, in order further to enhance the cell adhesion and spreading. Fibrin regularly formed a coating around individual nanofibers in the membranes, and also formed a thin noncontinuous nanofibrous mesh on top of the membranes. Collagen also coated most of the fibers of the membrane and randomly created a soft gel on the membrane surface. Fibronectin predominantly adsorbed onto a thin fibrin mesh or a collagen gel, and formed a thin nanofibrous structure. Fibrin nanocoating greatly improved the attachment, spreading, and proliferation of human dermal fibroblasts, whereas collagen nanocoating had a positive influence on the behavior of human HaCaT keratinocytes. In addition, fibrin stimulated the fibroblasts to synthesize fibronectin and to deposit it as an extracellular matrix. Fibrin coating also showed a tendency to improve the ultimate tensile strength of the nanofibrous membranes. Fibronectin attached to fibrin or to a collagen coating further enhanced the adhesion, spreading, and proliferation of both cell types. PMID:28223803

Rapid and Localized Mechanical Stimulation and Adhesion Assay: TRPM7 Involvement in Calcium Signaling and Cell Adhesion

PubMed Central

Nishitani, Wagner Shin; Alencar, Adriano Mesquita; Wang, Yingxiao

2015-01-01

A cell mechanical stimulation equipment, based on cell substrate deformation, and a more sensitive method for measuring adhesion of cells were developed. A probe, precisely positioned close to the cell, was capable of a vertical localized mechanical stimulation with a temporal frequency of 207 Hz, and strain magnitude of 50%. This setup was characterized and used to probe the response of Human Umbilical Endothelial Vein Cells (HUVECs) in terms of calcium signaling. The intracellular calcium ion concentration was measured by the genetically encoded Cameleon biosensor, with the Transient Receptor Potential cation channel, subfamily M, member 7 (TRPM7) expression inhibited. As TRPM7 expression also regulates adhesion, a relatively simple method for measuring adhesion of cells was also developed, tested and used to study the effect of adhesion alone. Three adhesion conditions of HUVECs on polyacrylamide gel dishes were compared. In the first condition, the substrate is fully treated with Sulfo-SANPAH crosslinking and fibronectin. The other two conditions had increasingly reduced adhesion: partially treated (only coated with fibronectin, with no use of Sulfo-SANPAH, at 5% of the normal amount) and non-treated polyacrylamide gels. The cells showed adhesion and calcium response to the mechanical stimulation correlated to the degree of gel treatment: highest for fully treated gels and lowest for non-treated ones. TRPM7 inhibition by siRNA on HUVECs caused an increase in adhesion relative to control (no siRNA treatment) and non-targeting siRNA, but a decrease to 80% of calcium response relative to non-targeting siRNA which confirms the important role of TRPM7 in mechanotransduction despite the increase in adhesion. PMID:25946314

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

PubMed

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

2016-03-01

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

Apigenin ameliorates streptozotocin-induced diabetic nephropathy in rats via MAPK-NF-κB-TNF-α and TGF-β1-MAPK-fibronectin pathways.

PubMed

Malik, Salma; Suchal, Kapil; Khan, Sana Irfan; Bhatia, Jagriti; Kishore, Kamal; Dinda, Amit Kumar; Arya, Dharamvir Singh

2017-08-01

Diabetic nephropathy (DN), a microvascular complication of diabetes, has emerged as an important health problem worldwide. There is strong evidence to suggest that oxidative stress, inflammation, and fibrosis play a pivotal role in the progression of DN. Apigenin has been shown to possess antioxidant, anti-inflammatory, antiapoptotic, antifibrotic, as well as antidiabetic properties. Hence, we evaluated whether apigenin halts the development and progression of DN in streptozotocin (STZ)-induced diabetic rats. Male albino Wistar rats were divided into control, diabetic control, and apigenin treatment groups (5-20 mg/kg po, respectively), apigenin per se (20 mg/kg po), and ramipril treatment group (2 mg/kg po). A single injection of STZ (55 mg/kg ip) was administered to all of the groups except control and per se groups to induce type 1 diabetes mellitus. Rats with fasting blood glucose >250 mg/dl were included in the study and randomized to different groups. Thereafter, the protocol was continued for 8 mo in all of the groups. Apigenin (20 mg/kg) treatment attenuated renal dysfunction, oxidative stress, and fibrosis (decreased transforming growth factor-β1, fibronectin, and type IV collagen) in the diabetic rats. It also significantly prevented MAPK activation, which inhibited inflammation (reduced TNF-α, IL-6, and NF-κB expression) and apoptosis (increased expression of Bcl-2 and decreased Bax and caspase-3). Furthermore, histopathological examination demonstrated reduced inflammation, collagen deposition, and glomerulosclerosis in the renal tissue. In addition, all of these changes were comparable with those produced by ramipril. Hence, apigenin ameliorated renal damage due to DN by suppressing oxidative stress and fibrosis and by inhibiting MAPK pathway. Copyright © 2017 the American Physiological Society.

The Glycolytic Enzyme Triosephosphate Isomerase of Trichomonas vaginalis Is a Surface-Associated Protein Induced by Glucose That Functions as a Laminin- and Fibronectin-Binding Protein

PubMed Central

Miranda-Ozuna, Jesús F. T.; Hernández-García, Mar S.; Brieba, Luis G.; Benítez-Cardoza, Claudia G.; Ortega-López, Jaime; González-Robles, Arturo

2016-01-01

Triosephosphate isomerase of Trichomonas vaginalis (TvTIM) is a 27-kDa cytoplasmic protein encoded by two genes, tvtim1 and tvtim2, that participates in glucose metabolism. TvTIM is also localized to the parasite surface. Thus, the goal of this study was to identify the novel functions of the surface-associated TvTIM in T. vaginalis and to assess the effect of glucose as an environmental factor that regulates its expression and localization. Reverse transcription-PCR (RT-PCR) showed that the tvtim genes were differentially expressed in response to glucose concentration. tvtim1 was overexpressed under glucose-restricted (GR) conditions, whereas tvtim2 was overexpressed under glucose-rich, or high-glucose (HG), conditions. Western blot and indirect immunofluorescence assays also showed that glucose positively affected the amount and surface localization of TvTIM in T. vaginalis. Affinity ligand assays demonstrated that the recombinant TvTIM1 and TvTIM2 proteins bound to laminin (Lm) and fibronectin (Fn) but not to plasminogen. Moreover, higher levels of adherence to Lm and Fn were detected in parasites grown under HG conditions than in those grown under GR conditions. Furthermore, pretreatment of trichomonads with an anti-TvTIMr polyclonal antibody or pretreatment of Lm- or Fn-coated wells with both recombinant proteins (TvTIM1r and TvTIM2r) specifically reduced the binding of live parasites to Lm and Fn in a concentration-dependent manner. Moreover, T. vaginalis was exposed to different glucose concentrations during vaginal infection of women with trichomoniasis. Our data indicate that TvTIM is a surface-associated protein under HG conditions that mediates specific binding to Lm and Fn as a novel virulence factor of T. vaginalis. PMID:27481251

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

PubMed Central

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

2016-01-01

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

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

PubMed

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

2015-01-01

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

Delta-like 1/Fetal Antigen-1 (Dlk1/FA1) Is a Novel Regulator of Chondrogenic Cell Differentiation via Inhibition of the Akt Kinase-dependent Pathway*

PubMed Central

Chen, Li; Qanie, Diyako; Jafari, Abbas; Taipaleenmaki, Hanna; Jensen, Charlotte H.; Säämänen, Anna-Marja; Sanz, Maria Luisa Nueda; Laborda, Jorge; Abdallah, Basem M.; Kassem, Moustapha

2011-01-01

Delta-like 1 (Dlk1, also known as fetal antigen-1, FA1) is a member of Notch/Delta family that inhibits adipocyte and osteoblast differentiation; however, its role in chondrogenesis is still not clear. Thus, we overexpressed Dlk1/FA1 in mouse embryonic ATDC5 cells and tested its effects on chondrogenic differentiation. Dlk1/FA1 inhibited insulin-induced chondrogenic differentiation as evidenced by reduction of cartilage nodule formation and gene expression of aggrecan, collagen Type II and X. Similar effects were obtained either by using Dlk1/FA1-conditioned medium or by addition of a purified, secreted, form of Dlk1 (FA1) directly to the induction medium. The inhibitory effects of Dlk1/FA1 were dose-dependent and occurred irrespective of the chondrogenic differentiation stage: proliferation, differentiation, maturation, or hypertrophic conversion. Overexpression or addition of the Dlk1/FA1 protein to the medium strongly inhibited the activation of Akt, but not the ERK1/2, or p38 MAPK pathways, and the inhibition of Akt by Dlk1/FA1 was mediated through PI3K activation. Interestingly, inhibition of fibronectin expression by siRNA rescued the Dlk1/FA1-mediated inhibition of Akt, suggesting interaction of Dlk1/FA1 and fibronectin in chondrogenic cells. Our results identify Dlk1/FA1 as a novel regulator of chondrogenesis and suggest Dlk1/FA1 acts as an inhibitor of the PI3K/Akt pathways that leads to its inhibitory effects on chondrogenesis. PMID:21724852

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

PubMed

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

2011-08-31

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

Aliskiren ameliorates chlorhexidine digluconate-induced peritoneal fibrosis in rats.

PubMed

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

2010-04-01

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

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.

Maintaining Elastogenicity of Mesenchymal Stem Cell-Derived Smooth Muscle Cells in Two-Dimensional Culture.

PubMed

Dahal, Shataakshi; Broekelman, Thomas; Mecham, Robert P; Ramamurthi, Anand

2018-06-01

Abdominal aortic aneurysms (AAAs) are localized expansions of the abdominal aorta that grow slowly to rupture. AAA growth is driven by irreversible elastic matrix breakdown in the aorta wall by chronically upregulated matrix metalloproteases (MMPs). Since adult vascular smooth muscle cells (SMCs) poorly regenerate elastic matrix, we previously explored utility of bone marrow mesenchymal stem cells and SMCs derived therefrom (BM-SMCs) for this purpose. One specific differentiated phenotype (cBM-SMCs) generated on a fibronectin substrate in presence of exogenous transforming growth factor-β and platelet-derived growth factor exhibited superior elastogenicity versus other phenotypes, and usefully provided proelastogenic and antiproteolytic stimuli to aneurysmal SMCs. Since in vivo cell therapy demands large cell inoculates, these derived SMCs must be propagated in vitro while maintaining their superior elastogenic, proelastogenic, and antiproteolytic characteristics. In this work, we thus investigated the culture conditions that must be provided to this propagation phase, which ensure that the differentiated SMCs maintain their phenotype and matrix regenerative benefits. Our results indicate that our BM-SMCs retain their phenotype in long-term culture even in the absence of differentiation growth factors and fibronectin substrate, but these conditions must be continued to be provided during postdifferentiation propagation if they are to maintain their superior elastic matrix deposition, crosslinking, and fiber formation properties. Our study, however, showed that cells propagated under these conditions exhibit higher expression of MMP-2, but favorably, no expression of elastolytic MMP-9. Hence, the study outcomes provide crucial guidelines to maintain phenotypic stability of cBM-SMCs during their propagation in two-dimensional culture before their delivery to the AAA wall for therapy.

The molecular mechanism of mediation of adsorbed serum proteins to endothelial cells adhesion and growth on biomaterials.

PubMed

Yang, Dayun; Lü, Xiaoying; Hong, Ying; Xi, Tingfei; Zhang, Deyuan

2013-07-01

To explore molecular mechanism of mediation of adsorbed proteins to cell adhesion and growth on biomaterials, this study examined endothelial cell adhesion, morphology and viability on bare and titanium nitride (TiN) coated nickel titanium (NiTi) alloys and chitosan film firstly, and then identified the type and amount of serum proteins adsorbed on the three surfaces by proteomic technology. Subsequently, the mediation role of the identified proteins to cell adhesion and growth was investigated with bioinformatics analyses, and further confirmed by a series of cellular and molecular biological experiments. Results showed that the type and amount of adsorbed serum proteins associated with cell adhesion and growth was obviously higher on the alloys than on the chitosan film, and these proteins mediated endothelial cell adhesion and growth on the alloys via four ways. First, proteins such as adiponectin in the adsorbed protein layer bound with cell surface receptors to generate signal transduction, which activated cell surface integrins through increasing intracellular calcium level. Another way, thrombospondin 1 in the adsorbed protein layer promoted TGF-β signaling pathway activation and enhanced integrins expression. The third, RGD sequence containing proteins such as fibronectin 1, vitronectin and thrombospondin 1 in the adsorbed protein layer bound with activated integrins to activate focal adhesion pathway, increased focal adhesion formation and actin cytoskeleton organization and mediated cell adhesion and spreading. In addition, the activated focal adhesion pathway promoted the expression of cell growth related genes and resulted in cell proliferation. The fourth route, coagulation factor II (F2) and fibronectin 1 in the adsorbed protein layer bound with cell surface F2 receptor and integrin, activated regulation of actin cytoskeleton pathway and regulated actin cytoskeleton organization. Copyright © 2013 Elsevier Ltd. All rights reserved.

Establishment of substratum polarity in the blastocoel roof of the Xenopus embryo.

PubMed

Nagel, M; Winklbauer, R

1999-05-01

The fibronectin fibril matrix on the blastocoel roof of the Xenopus gastrula contains guidance cues that determine the direction of mesoderm cell migration. The underlying guidance-related polarity of the blastocoel roof is established in the late blastula under the influence of an instructive signal from the vegetal half of the embryo, in particular from the mesoderm. Formation of an oriented substratum depends on functional activin and FGF signaling pathways in the blastocoel roof. Besides being involved in tissue polarization, activin and FGF also affect fibronectin matrix assembly. Activin treatment of the blastocoel roof inhibits fibril formation, whereas FGF modulates the structure of the fibril network. The presence of intact fibronectin fibrils is permissive for directional mesoderm migration on the blastocoel roof extracellular matrix.

Global differential expression of genes located in the Down Syndrome Critical Region in normal human brain

PubMed Central

Montoya, Julio Cesar; Fajardo, Dianora; Peña, Angela; Sánchez, Adalberto; Domínguez, Martha C; Satizábal, José María

2014-01-01

Background: The information of gene expression obtained from databases, have made possible the extraction and analysis of data related with several molecular processes involving not only in brain homeostasis but its disruption in some neuropathologies; principally in Down syndrome and the Alzheimer disease. Objective: To correlate the levels of transcription of 19 genes located in the Down Syndrome Critical Region (DSCR) with their expression in several substructures of normal human brain. Methods: There were obtained expression profiles of 19 DSCR genes in 42 brain substructures, from gene expression values available at the database of the human brain of the Brain Atlas of the Allen Institute for Brain Sciences", (http://human.brain-map.org/). The co-expression patterns of DSCR genes in brain were calculated by using multivariate statistical methods. Results: Highest levels of gene expression were registered at caudate nucleus, nucleus accumbens and putamen among central areas of cerebral cortex. Increased expression levels of RCAN1 that encode by a protein involved in signal transduction process of the CNS were recorded for PCP4 that participates in the binding to calmodulin and TTC3; a protein that is associated with differentiation of neurons. That previously identified brain structures play a crucial role in the learning process, in different class of memory and in motor skills. Conclusion: The precise regulation of DSCR gene expression is crucial to maintain the brain homeostasis, especially in those areas with high levels of gene expression associated with a remarkable process of learning and cognition. PMID:25767303

Neuron-Enriched Gene Expression Patterns are Regionally Anti-Correlated with Oligodendrocyte-Enriched Patterns in the Adult Mouse and Human Brain

PubMed Central

Tan, Powell Patrick Cheng; French, Leon; Pavlidis, Paul

2013-01-01

An important goal in neuroscience is to understand gene expression patterns in the brain. The recent availability of comprehensive and detailed expression atlases for mouse and human creates opportunities to discover global patterns and perform cross-species comparisons. Recently we reported that the major source of variation in gene transcript expression in the adult normal mouse brain can be parsimoniously explained as reflecting regional variation in glia to neuron ratios, and is correlated with degree of connectivity and location in the brain along the anterior-posterior axis. Here we extend this investigation to two gene expression assays of adult normal human brains that consisted of over 300 brain region samples, and perform comparative analyses of brain-wide expression patterns to the mouse. We performed principal components analysis (PCA) on the regional gene expression of the adult human brain to identify the expression pattern that has the largest variance. As in the mouse, we observed that the first principal component is composed of two anti-correlated patterns enriched in oligodendrocyte and neuron markers respectively. However, we also observed interesting discordant patterns between the two species. For example, a few mouse neuron markers show expression patterns that are more correlated with the human oligodendrocyte-enriched pattern and vice-versa. In conclusion, our work provides insights into human brain function and evolution by probing global relationships between regional cell type marker expression patterns in the human and mouse brain. PMID:23440889

Neuron-Enriched Gene Expression Patterns are Regionally Anti-Correlated with Oligodendrocyte-Enriched Patterns in the Adult Mouse and Human Brain.

PubMed

Tan, Powell Patrick Cheng; French, Leon; Pavlidis, Paul

2013-01-01

An important goal in neuroscience is to understand gene expression patterns in the brain. The recent availability of comprehensive and detailed expression atlases for mouse and human creates opportunities to discover global patterns and perform cross-species comparisons. Recently we reported that the major source of variation in gene transcript expression in the adult normal mouse brain can be parsimoniously explained as reflecting regional variation in glia to neuron ratios, and is correlated with degree of connectivity and location in the brain along the anterior-posterior axis. Here we extend this investigation to two gene expression assays of adult normal human brains that consisted of over 300 brain region samples, and perform comparative analyses of brain-wide expression patterns to the mouse. We performed principal components analysis (PCA) on the regional gene expression of the adult human brain to identify the expression pattern that has the largest variance. As in the mouse, we observed that the first principal component is composed of two anti-correlated patterns enriched in oligodendrocyte and neuron markers respectively. However, we also observed interesting discordant patterns between the two species. For example, a few mouse neuron markers show expression patterns that are more correlated with the human oligodendrocyte-enriched pattern and vice-versa. In conclusion, our work provides insights into human brain function and evolution by probing global relationships between regional cell type marker expression patterns in the human and mouse brain.

The Effect of Gravity Fields on Cellular Gene Expression

NASA Technical Reports Server (NTRS)

Hughes-Fulford, Millie

1999-01-01

Early theoretical analysis predicted that microgravity effects on the isolated cell would be minuscule at the subcellular level; however, these speculations have not proven true in the real world. Astronauts experience a significant bone and muscle loss in as little as 2 weeks of spaceflight and changes are seen at the cellular level soon after exposure to microgravity. Changes in biological systems may be primarily due to the lack of gravity and the resulting loss of mechanical stress on tissues and cells. Recent ground and flight studies examining the effects of gravity or mechanical stress on cells demonstrate marked changes in gene expression when relatively small changes in mechanical forces or gravity fields were made. Several immediate early genes (IEG) like c-fos and c-myc are induced by mechanical stimulation within minutes. In contrast, several investigators report that the absence of mechanical forces during space flight result in decreased sera response element (SRE) activity and attenuation of expression of IEGs such as c-fos, c-jun and cox-2 mRNAs. Clearly, these early changes in gene expression may have long term consequences on mechanically sensitive cells. In our early studies on STS-56, we reported four major changes in the osteoblast; 1) prostaglandin synthesis in flight, 2) changes in cellular morphology, 3) altered actin cytoskeleton and 4) reduced osteoblast growth after four days exposure to microgravity. Initially, it was believed that changes in fibronectin (FN) RNA, FN protein synthesis or subsequent FN matrix formation might account for the changes in cytoskeleton and/ or reduction of growth. However our recent studies on Biorack (STS-76, STS-81 and STS-84), using ground and in-flight 1-G controls, demonstrated that fibronectin synthesis and matrix formation were normal in microgravity. In addition, in our most recent Biorack paper, our laboratory has documented that relative protein synthesis and mRNA synthesis are not changed after 24 hours exposure to microgravity. We did, however, find significant changes in osteoblast gene expression of IEGs, c-fos and cox-2 in microgravity exposure as compared to ground and in-flight 1-G controls. Subsequent ground studies suggest that the molecular mechanism underlying these changes may involve prostaglandin c-AMP receptors (EPs) and/or subsequent alteration of intracellular signaling in the absence of gravity.

The HCM-linked W792R mutation in cardiac myosin-binding protein C reduces C6 FnIII domain stability.

PubMed

Smelter, Dan F; de Lange, Willem J; Cai, Wenxuan; Ge, Ying; Ralphe, J Carter

2018-06-01

Cardiac myosin-binding protein C (cMyBP-C) is a functional sarcomeric protein that regulates contractility in response to contractile demand, and many mutations in cMyBP-C lead to hypertrophic cardiomyopathy (HCM). To gain insight into the effects of disease-causing cMyBP-C missense mutations on contractile function, we expressed the pathogenic W792R mutation (substitution of a highly conserved tryptophan residue by an arginine residue at position 792) in mouse cardiomyocytes lacking endogenous cMyBP-C and studied the functional effects using three-dimensional engineered cardiac tissue constructs (mECTs). Based on complete conservation of tryptophan at this location in fibronectin type II (FnIII) domains, we hypothesized that the W792R mutation affects folding of the C6 FnIII domain, destabilizing the mutant protein. Adenoviral transduction of wild-type (WT) and W792R cDNA achieved equivalent mRNA transcript abundance, but not equivalent protein levels, with W792R compared with WT controls. mECTs expressing W792R demonstrated abnormal contractile kinetics compared with WT mECTs that were nearly identical to cMyBP-C-deficient mECTs. We studied whether common pathways of protein degradation were responsible for the rapid degradation of W792R cMyBP-C. Inhibition of both ubiquitin-proteasome and lysosomal degradation pathways failed to increase full-length mutant protein abundance to WT equivalence, suggesting rapid cytosolic degradation. Bacterial expression of WT and W792R protein fragments demonstrated decreased mutant stability with altered thermal denaturation and increased susceptibility to trypsin digestion. These data suggest that the W792R mutation destabilizes the C6 FnIII domain of cMyBP-C, resulting in decreased full-length protein expression. This study highlights the vulnerability of FnIII-like domains to mutations that alter domain stability and further indicates that missense mutations in cMyBP-C can cause disease through a mechanism of haploinsufficiency. NEW & NOTEWORTHY This study is one of the first to describe a disease mechanism for a missense mutation in cardiac myosin-binding protein C linked to hypertrophic cardiomyopathy. The mutation decreases stability of the fibronectin type III domain and results in substantially reduced mutant protein expression dissonant to transcript abundance.

Ligand-induced Epitope Masking: DISSOCIATION OF INTEGRIN α5β1-FIBRONECTIN COMPLEXES ONLY BY MONOCLONAL ANTIBODIES WITH AN ALLOSTERIC MODE OF ACTION.

PubMed

Mould, A Paul; Askari, Janet A; Byron, Adam; Takada, Yoshikazu; Jowitt, Thomas A; Humphries, Martin J

2016-09-30

We previously demonstrated that Arg-Gly-Asp (RGD)-containing ligand-mimetic inhibitors of integrins are unable to dissociate pre-formed integrin-fibronectin complexes (IFCs). These observations suggested that amino acid residues involved in integrin-fibronectin binding become obscured in the ligand-occupied state. Because the epitopes of some function-blocking anti-integrin monoclonal antibodies (mAbs) lie near the ligand-binding pocket, it follows that the epitopes of these mAbs may become shielded in the ligand-occupied state. Here, we tested whether function-blocking mAbs directed against α5β1 can interact with the integrin after it forms a complex with an RGD-containing fragment of fibronectin. We showed that the anti-α5 subunit mAbs JBS5, SNAKA52, 16, and P1D6 failed to disrupt IFCs and hence appeared unable to bind to the ligand-occupied state. In contrast, the allosteric anti-β1 subunit mAbs 13, 4B4, and AIIB2 could dissociate IFCs and therefore were able to interact with the ligand-bound state. However, another class of function-blocking anti-β1 mAbs, exemplified by Lia1/2, could not disrupt IFCs. This second class of mAbs was also distinguished from 13, 4B4, and AIIB2 by their ability to induce homotypic cell aggregation. Although the epitope of Lia1/2 was closely overlapping with those of 13, 4B4, and AIIB2, it appeared to lie closer to the ligand-binding pocket. A new model of the α5β1-fibronectin complex supports our hypothesis that the epitopes of mAbs that fail to bind to the ligand-occupied state lie within, or very close to, the integrin-fibronectin interface. Importantly, our findings imply that the efficacy of some therapeutic anti-integrin mAbs could be limited by epitope masking. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Increased expression of Interleukin-13 and connective tissue growth factor, and their potential roles during foreign body encapsulation of subcutaneous implants.

PubMed

Ward, W Kenneth; Li, Allen G; Siddiqui, Yasmin; Federiuk, Isaac F; Wang, Xiao-Jing

2008-01-01

The purpose of this study was to better understand whether interleukin-13 (IL-13) and connective tissue growth factor (CTGF) are highly expressed during foreign body encapsulation of subcutaneous devices. Mock biosensors were implanted into rats for three lengths of time (7-, 21- and 48-55 days) to address different stages of the foreign body response. Using quantitative real-time PCR and immunofluorescence, the expression of IL13, CTGF, collagen 1, decorin and fibronectin were measured in this tissue. IL-13, a product of Th2 cells, was highly expressed at all time points, with greatest expression at day 21. The IL-13 expression was paralleled by increased presence of T-cells at all time points. CTGF was also found to be more highly expressed in foreign body tissue than in controls. Collagen and decorin were highly expressed at the middle and later stages. Given the increased expression of IL-13 and CTGF in foreign body tissue, and their roles in other fibrotic disorders, these cytokines may well contribute to the formation of the foreign body capsule. Since the peak gene expression of IL-13 occurred later than the previously-reported TGFbeta expression peak, IL-13 is probably not the major stimulus to TGFbeta expression during foreign body encapsulation and may contribute to fibrosis independently.

The murine ufo receptor: molecular cloning, chromosomal localization and in situ expression analysis.

PubMed

Faust, M; Ebensperger, C; Schulz, A S; Schleithoff, L; Hameister, H; Bartram, C R; Janssen, J W

1992-07-01

We have cloned the mouse homologue of the ufo oncogene. It encodes a novel tyrosine kinase receptor characterized by a unique extracellular domain containing two immunoglobulin-like and two fibronectin type III repeats. Comparison of the predicted ufo amino acid sequences of mouse and man revealed an overall identity of 87.6%. The ufo locus maps to mouse chromosome 7A3-B1 and thereby extends the known conserved linkage group between mouse chromosome 7 and human chromosome 19. RNA in situ hybridization analysis established the onset of specific ufo expression in the late embryogenesis at day 12.5 post coitum (p.c.) and localized ufo transcription to distinct substructures of a broad spectrum of developing tissues (e.g. subepidermal cells of the skin, mesenchymal cells of the periosteum). In adult animals ufo is expressed in cells forming organ capsules as well as in connective tissue structures. ufo may function as a signal transducer between specific cell types of mesodermal origin.

Drosophila neuroglian: a member of the immunoglobulin superfamily with extensive homology to the vertebrate neural adhesion molecule L1.

PubMed

Bieber, A J; Snow, P M; Hortsch, M; Patel, N H; Jacobs, J R; Traquina, Z R; Schilling, J; Goodman, C S

1989-11-03

Drosophila neuroglian is an integral membrane glycoprotein that is expressed on a variety of cell types in the Drosophila embryo, including expression on a large subset of glial and neuronal cell bodies in the central and peripheral nervous systems and on the fasciculating axons that extend along them. Neuroglian cDNA clones were isolated by expression cloning. cDNA sequence analysis reveals that neuroglian is a member of the immunoglobulin superfamily. The extracellular portion of the protein consists of six immunoglobulin C2-type domains followed by five fibronectin type III domains. Neuroglian is closely related to the immunoglobulin-like vertebrate neural adhesion molecules and, among them, shows most extensive homology to mouse L1. Its homology to L1 and its embryonic localization suggest that neuroglian may play a role in neural and glial cell adhesion in the developing Drosophila embryo. We report here on the identification of a lethal mutation in the neuroglian gene.

Chymase inhibition prevents fibronectin and myofibrillar loss and improves cardiomyocyte function and LV torsion angle in dogs with isolated mitral regurgitation.

PubMed

Pat, Betty; Chen, Yuanwen; Killingsworth, Cheryl; Gladden, James D; Shi, Ke; Zheng, Junying; Powell, Pamela C; Walcott, Greg; Ahmed, Mustafa I; Gupta, Himanshu; Desai, Ravi; Wei, Chih-Chang; Hase, Naoki; Kobayashi, Tsunefumi; Sabri, Abdelkarim; Granzier, Henk; Denney, Thomas; Tillson, Michael; Dillon, A Ray; Husain, Ahsan; Dell'italia, Louis J

2010-10-12

The left ventricular (LV) dilatation of isolated mitral regurgitation (MR) is associated with an increase in chymase and a decrease in interstitial collagen and extracellular matrix. In addition to profibrotic effects, chymase has significant antifibrotic actions because it activates matrix metalloproteinases and kallikrein and degrades fibronectin. Thus, we hypothesize that chymase inhibitor (CI) will attenuate extracellular matrix loss and LV remodeling in MR. We studied dogs with 4 months of untreated MR (MR; n=9) or MR treated with CI (MR+CI; n=8). Cine MRI demonstrated a >40% increase in LV end-diastolic volume in both groups, consistent with a failure of CI to improve a 25% decrease in interstitial collagen in MR. However, LV cardiomyocyte fractional shortening was decreased in MR versus normal dogs (3.71±0.24% versus 4.81±0.31%; P<0.05) and normalized in MR+CI dogs (4.85±0.44%). MRI with tissue tagging demonstrated an increase in LV torsion angle in MR+CI versus MR dogs. CI normalized the significant decrease in fibronectin and FAK phosphorylation and prevented cardiomyocyte myofibrillar degeneration in MR dogs. In addition, total titin and its stiffer isoform were increased in the LV epicardium and paralleled the changes in fibronectin and FAK phosphorylation in MR+CI dogs. These results suggest that chymase disrupts cell surface-fibronectin connections and FAK phosphorylation that can adversely affect cardiomyocyte myofibrillar structure and function. The greater effect of CI on epicardial versus endocardial titin and noncollagen cell surface proteins may be responsible for the increase in torsion angle in chronic MR.

Expression of the ADHD candidate gene Diras2 in the brain.

PubMed

Grünewald, Lena; Becker, Nils; Camphausen, Annika; O'Leary, Aet; Lesch, Klaus-Peter; Freudenberg, Florian; Reif, Andreas

2018-06-01

The distinct subgroup of the Ras family member 2 (DIRAS2) gene has been found to be associated with attention-deficit/hyperactivity disorder (ADHD) in one of our previous studies. This gene is coding for a small Ras GTPase with unknown function. DIRAS2 is highly expressed in the brain. However, the exact neural expression pattern of this gene was unknown so far. Therefore, we investigated the expressional profile of DIRAS2 in the human and murine brain. In the present study, qPCR analyses in the human and in the developing mouse brain, immunocytological double staining on murine hippocampal primary cells and RNA in situ hybridization (ISH) on brain sections of C57BL/6J wild-type mice, have been used to reveal the expression pattern of DIRAS2 in the brain. We could show that DIRAS2 expression in the human brain is the highest in the hippocampus and the cerebral cortex, which is in line with the ISH results in the mouse brain. During mouse brain development, Diras2 levels strongly increase from prenatal to late postnatal stages. Co-expression studies indicate Diras2 expression in glutamatergic and catecholaminergic neurons. Our findings support the idea of DIRAS2 as a candidate gene for ADHD as the timeline of its expression as well as the brain regions and cell types that show Diras2 expression correspond to those assumed to underlie the pathomechanisms of the disease.

Brain Region–Specific Alterations in the Gene Expression of Cytokines, Immune Cell Markers and Cholinergic System Components during Peripheral Endotoxin–Induced Inflammation

PubMed Central

Silverman, Harold A; Dancho, Meghan; Regnier-Golanov, Angelique; Nasim, Mansoor; Ochani, Mahendar; Olofsson, Peder S; Ahmed, Mohamed; Miller, Edmund J; Chavan, Sangeeta S; Golanov, Eugene; Metz, Christine N; Tracey, Kevin J; Pavlov, Valentin A

2014-01-01

Inflammatory conditions characterized by excessive peripheral immune responses are associated with diverse alterations in brain function, and brain-derived neural pathways regulate peripheral inflammation. Important aspects of this bidirectional peripheral immune–brain communication, including the impact of peripheral inflammation on brain region–specific cytokine responses, and brain cholinergic signaling (which plays a role in controlling peripheral cytokine levels), remain unclear. To provide insight, we studied gene expression of cytokines, immune cell markers and brain cholinergic system components in the cortex, cerebellum, brainstem, hippocampus, hypothalamus, striatum and thalamus in mice after an intraperitoneal lipopolysaccharide injection. Endotoxemia was accompanied by elevated serum levels of interleukin (IL)-1β, IL-6 and other cytokines and brain region–specific increases in Il1b (the highest increase, relative to basal level, was in cortex; the lowest increase was in cerebellum) and Il6 (highest increase in cerebellum; lowest increase in striatum) mRNA expression. Gene expression of brain Gfap (astrocyte marker) was also differentially increased. However, Iba1 (microglia marker) mRNA expression was decreased in the cortex, hippocampus and other brain regions in parallel with morphological changes, indicating microglia activation. Brain choline acetyltransferase (Chat ) mRNA expression was decreased in the striatum, acetylcholinesterase (Ache) mRNA expression was decreased in the cortex and increased in the hippocampus, and M1 muscarinic acetylcholine receptor (Chrm1) mRNA expression was decreased in the cortex and the brainstem. These results reveal a previously unrecognized regional specificity in brain immunoregulatory and cholinergic system gene expression in the context of peripheral inflammation and are of interest for designing future antiinflammatory approaches. PMID:25299421

Expression analysis of extracellular matrix components in brush biopsies of oral lesions.

PubMed

Driemel, Oliver; Kosmehl, Hartwig; Rosenhahn, Julia; Berndt, Alexander; Reichert, Torsten E; Zardi, Luciano; Dahse, Regine

2007-01-01

Oral brush biopsies have proved to be a promising new non-invasive methodology in the diagnosis of oral lesions. The extracellular matrix (ECM) molecules gamma2 chain of laminin-5 (L5gamma2), tenascin-c (Tn-C) and the fibronectin isoform containing EDB (EDB-fn) are involved in matrix remodeling during malignant transformation in oral carcinoma. Expression of L5gamma2, Tn-C and EDB-fn was analysed in brush biopsy-obtained cells of benign inflammatory or hyperproliferative lesions and primary oral squamous cell carcinoma (OSCC) using the Roche LightCycler 2.0 System. Oral carcinoma are detectable with mRNA resynthesis of the ECM molecules L5gamma2 and Tn-C in oral brush biopsies. EDB-fn mRNA was not detected--the stroma myofibroblasts are apparently a preferential source of EDB-fn and sampling by oral brush biopsy harvests epithelial cells and does not reach the cells which do express EDB-fn. The performance of gene expression analysis in brush biopsies is limited by a high RNase activity in the oral cavity.

Neural cell adhesion molecule-deficient beta-cell tumorigenesis results in diminished extracellular matrix molecule expression and tumour cell-matrix adhesion.

PubMed

Håkansson, Joakim; Xian, Xiaojie; He, Liqun; Ståhlberg, Anders; Nelander, Sven; Samuelsson, Tore; Kubista, Mikael; Semb, Henrik

2005-01-01

To understand by which mechanism neural cell adhesion molecule (N-CAM) limits beta tumour cell disaggregation and dissemination, we searched for potential downstream genes of N-CAM during beta tumour cell progression by gene expression profiling. Here, we show that N-CAM-deficient beta-cell tumorigenesis is associated with changes in the expression of genes involved in cell-matrix adhesion and cytoskeletal dynamics, biological processes known to affect the invasive and metastatic behaviour of tumour cells. The extracellular matrix (ECM) molecules emerged as the primary target, i.e. N-CAM deficiency resulted in down-regulated mRNA expression of a broad range of ECM molecules. Consistent with this result, deficient deposition of major ECM stromal components, such as fibronectin, laminin 1 and collagen IV, was observed. Moreover, N-CAM-deficient tumour cells displayed defective matrix adhesion. These results offer a potential mechanism for tumour cell disaggregation during N-CAM-deficient beta tumour cell progression. Prospective consequences of these findings for the role of N-CAM in beta tumour cell dissemination are discussed.

Correlation Between Extravasation and Alterations of Cerebrovascular Laminin and β-Dystroglycan Immunoreactivity Following Cryogenic Lesions in Rats.

PubMed

Kálmán, Mihály; Tóth, László; Szöllosi, Dávid; Oszwald, Erzsébet; Mahalek, Judit; Sadeghian, Sam

2017-11-01

The blood-brain barrier becomes "leaky" following lesions. Former studies revealed that following lesions the immunoreactivity of cerebrovascular laminin becomes detectable whereas that of β-dystroglycan disappears. These alterations may be indicators of glio-vascular decoupling that may result in the impairment of the blood-brain-barrier. This study investigates correlation between the post-lesion extravasation and the above-mentioned immunohistochemical alterations. Following cryogenic lesions, the survival periods lasted 5, 10, 30 minutes, 1 or 12 hours, or 1 day. Some brains were fixed immediately post-lesion. Immunofluorescent reactions were performed in floating sections. The extravasation was detected with immunostaining for plasma fibronectin and rat immunoglobulins. When the survival period was 30 minutes or longer, the area of extravasation corresponded to the area of altered laminin and β-dystroglycan immunoreactivities. Following immediate fixation some laminin immunoreactivity was already detected. The extravasation seemed to precede this early appearance of laminin immunoreactivity. The β-dystroglycan immunoreactivity disappeared later. When the extravasation spread into the corpus callosum, vascular laminin immunoreactivity appeared but the β-dystroglycan immunoreactivity persisted. It seems that extravasation separates the glial and vascular basal laminae, which results in the appearance of laminin immunoreactivity. The disappearance of β-dystroglycan immunoreactivity is neither a condition nor an inevitable consequence of the 2 other phenomena. © 2017 American Association of Neuropathologists, Inc. All rights reserved.

Curli mediate bacterial adhesion to fibronectin via tensile multiple bonds

NASA Astrophysics Data System (ADS)

Oh, Yoo Jin; Hubauer-Brenner, Michael; Gruber, Hermann J.; Cui, Yidan; Traxler, Lukas; Siligan, Christine; Park, Sungsu; Hinterdorfer, Peter

2016-09-01

Many enteric bacteria including pathogenic Escherichia coli and Salmonella strains produce curli fibers that bind to host surfaces, leading to bacterial internalization into host cells. By using a nanomechanical force-sensing approach, we obtained real-time information about the distribution of molecular bonds involved in the adhesion of curliated bacteria to fibronectin. We found that curliated E. coli and fibronectin formed dense quantized and multiple specific bonds with high tensile strength, resulting in tight bacterial binding. Nanomechanical recognition measurements revealed that approximately 10 bonds were disrupted either sequentially or simultaneously under force load. Thus the curli formation of bacterial surfaces leads to multi-bond structural components of fibrous nature, which may explain the strong mechanical binding of curliated bacteria to host cells and unveil the functions of these proteins in bacterial internalization and invasion.

An Examination of Dynamic Gene Expression Changes in the Mouse Brain During Pregnancy and the Postpartum Period.

PubMed

Ray, Surjyendu; Tzeng, Ruei-Ying; DiCarlo, Lisa M; Bundy, Joseph L; Vied, Cynthia; Tyson, Gary; Nowakowski, Richard; Arbeitman, Michelle N

2015-11-23

The developmental transition to motherhood requires gene expression changes that alter the brain to drive the female to perform maternal behaviors. We broadly examined the global transcriptional response in the mouse maternal brain, by examining four brain regions: hypothalamus, hippocampus, neocortex, and cerebellum, in virgin females, two pregnancy time points, and three postpartum time points. We find that overall there are hundreds of differentially expressed genes, but each brain region and time point shows a unique molecular signature, with only 49 genes differentially expressed in all four regions. Interestingly, a set of "early-response genes" is repressed in all brain regions during pregnancy and postpartum stages. Several genes previously implicated in underlying postpartum depression change expression. This study serves as an atlas of gene expression changes in the maternal brain, with the results demonstrating that pregnancy, parturition, and postpartum maternal experience substantially impact diverse brain regions. Copyright © 2016 Ray et al.

A regulatory toolbox of MiniPromoters to drive selective expression in the brain.

PubMed

Portales-Casamar, Elodie; Swanson, Douglas J; Liu, Li; de Leeuw, Charles N; Banks, Kathleen G; Ho Sui, Shannan J; Fulton, Debra L; Ali, Johar; Amirabbasi, Mahsa; Arenillas, David J; Babyak, Nazar; Black, Sonia F; Bonaguro, Russell J; Brauer, Erich; Candido, Tara R; Castellarin, Mauro; Chen, Jing; Chen, Ying; Cheng, Jason C Y; Chopra, Vik; Docking, T Roderick; Dreolini, Lisa; D'Souza, Cletus A; Flynn, Erin K; Glenn, Randy; Hatakka, Kristi; Hearty, Taryn G; Imanian, Behzad; Jiang, Steven; Khorasan-zadeh, Shadi; Komljenovic, Ivana; Laprise, Stéphanie; Liao, Nancy Y; Lim, Jonathan S; Lithwick, Stuart; Liu, Flora; Liu, Jun; Lu, Meifen; McConechy, Melissa; McLeod, Andrea J; Milisavljevic, Marko; Mis, Jacek; O'Connor, Katie; Palma, Betty; Palmquist, Diana L; Schmouth, Jean-François; Swanson, Magdalena I; Tam, Bonny; Ticoll, Amy; Turner, Jenna L; Varhol, Richard; Vermeulen, Jenny; Watkins, Russell F; Wilson, Gary; Wong, Bibiana K Y; Wong, Siaw H; Wong, Tony Y T; Yang, George S; Ypsilanti, Athena R; Jones, Steven J M; Holt, Robert A; Goldowitz, Daniel; Wasserman, Wyeth W; Simpson, Elizabeth M

2010-09-21

The Pleiades Promoter Project integrates genomewide bioinformatics with large-scale knockin mouse production and histological examination of expression patterns to develop MiniPromoters and related tools designed to study and treat the brain by directed gene expression. Genes with brain expression patterns of interest are subjected to bioinformatic analysis to delineate candidate regulatory regions, which are then incorporated into a panel of compact human MiniPromoters to drive expression to brain regions and cell types of interest. Using single-copy, homologous-recombination "knockins" in embryonic stem cells, each MiniPromoter reporter is integrated immediately 5' of the Hprt locus in the mouse genome. MiniPromoter expression profiles are characterized in differentiation assays of the transgenic cells or in mouse brains following transgenic mouse production. Histological examination of adult brains, eyes, and spinal cords for reporter gene activity is coupled to costaining with cell-type-specific markers to define expression. The publicly available Pleiades MiniPromoter Project is a key resource to facilitate research on brain development and therapies.

EPA or DHA enhanced oxidative stress and aging protein expression in brain of d-galactose treated mice.

PubMed

Hsu, Yuan-Man; Yin, Mei-Chin

2016-06-01

Effects of eicosapentaenoic acid (EPA, 20:5) and docosahexaenoic acid (DHA, 22:6) upon fatty acid composition, oxidative and inflammatory factors and aging proteins in brain of d-galactose (DG) treated aging mice were examined. Each fatty acid at 7 mg/kg BW/week was supplied for 8 weeks. Brain aging was induced by DG treatment (100 mg/kg body weight) via daily subcutaneous injection for 8 weeks. DG, EPA and DHA treatments changed brain fatty acid composition. DG down-regulated brain Bcl-2 expression and up-regulated Bax expression. Compared with DG groups, EPA and DHA further enhanced Bax expression. DG decreased glutathione content, increased reactive oxygen species (ROS) and oxidized glutathione (GSSG) production, the intake of EPA or DHA caused greater ROS and GSSG formation. DG treatments up-regulated the protein expression of p47(phox) and gp91(phox), and the intake of EPA or DHA led to greater p47(phox) and gp91(phox) expression. DG increased brain prostaglandin E2 (PGE2) levels, and cyclooxygenase (COX)-2 expression and activity, the intake of EPA or DHA reduced brain COX-2 activity and PGE2 formation. DG enhanced brain p53, p16 and p21 expression. EPA and DHA intake led to greater p21 expression, and EPA only caused greater p53 and p16 expression. These findings suggest that these two PUFAs have toxic effects toward aging brain.

Modulation of Human Plasma Fibronectin Levels Following Exercise,

DTIC Science & Technology

1988-01-01

forms of this large molecular weight (440 kilodaltons) glycoprotein,(17. While the tissue type is cell-associated and important to cell adhesion and...increased under conditions of pathology, such as in obesity (6). cancer (3). proteinuria (4). diabetic retinopathy (5). and preeclampsia (27). in the absence...Res. 1977: 22:709-716. 27. Stubbs. T.M.. Lazarchick. J.. and Horger. E.O. Plasma fibronectin levels in preeclampsia : A possible biochemical marker

Interaction of Mycobacterium tuberculosis with human respiratory mucosa.

PubMed

Middleton, A M; Chadwick, M V; Nicholson, A G; Dewar, A; Groger, R K; Brown, E J; Ratliff, T L; Wilson, R

2002-01-01

Endobronchial infection is associated with pulmonary tuberculosis in the majority of cases. We have investigated the adherence of Mycobacterium tuberculosis to the human respiratory mucosa. Organ cultures constructed with human tissue were infected with M. tuberculosis in the presence or absence of mycobacterial fibronectin attachment cell surface proteins and examined by scanning electron microscopy. M. tuberculosis adhered mainly to extracellular matrix (ECM) in areas of mucosal damage, but not to ciliated mucosa, intact extruded cells, basement membrane or collagen fibres. Bacteria also adhered to fibrous but not globular mucus and occasionally to healthy unciliated mucosa, open tight junctions and to extruded cells that had degenerated, exposing their contents. There was a significant reduction (p<0.05) in the number of bacteria adhering to ECM after pre-incubation of bacteria with fibronectin and after pre-incubation of the tissue with M. avium fibronectin attachment protein (FAP) and M. bovis antigen 85B protein, in a concentration dependent manner. The combined effect of FAP and antigen 85B protein was significantly greater than either protein alone. Bacterial adherence to fibrous mucus was not influenced by fibronectin. We conclude that M. tuberculosis adheres to ECM in areas of mucosal damage at least in part via FAP and antigen 85B protein.

Development of a microfabricated artificial limbus with micropockets for cell delivery to the cornea.

PubMed

Ortega, Ílida; Deshpande, Pallavi; Gill, Andrew A; MacNeil, Sheila; Claeyssens, Frederik

2013-06-01

The aim of this study was to develop a synthetic alternative to the human corneal limbus for use initially as an ex vivo model in which to study corneal stem cell function within a niche environment and ultimately to develop an implantable limbus for future clinical use. Microstereolithography was used for the fabrication of polyethylene glycol diacrylate (PEGDA) based rings on a macroscopic (1.2 cm) scale containing unique microfeatures (pockets) which were then modified with fibronectin to promote cell adhesion. These rings were designed to mimic the limbal area of the eye containing structures of the approximate size and shape of the stem cell microenvironments found in the palisades of Vogt. The attachment of rabbit limbal fibroblasts and rabbit limbal epithelial cells to the PEGDA rings was increased by pretreating the microfabricated structures with biotinylated fibronectin. Cell outgrowth from fibronectin coated microfabricated structures was 50% greater than from rings without structures or fibronectin coating. The cell loaded rings were then placed on an ex vivo wounded cornea model and the outgrowth of cells to form a multilayered epithelium was observed. We suggest this is a new approach to investigating limbal stem cells niches and the first steps towards a new approach for corneal regeneration.

Is vitronectin the velcro that binds the gametes together?

PubMed

Fusi, F M; Bernocchi, N; Ferrari, A; Bronson, R A

1996-11-01

Evidence has been presented that the adhesion of human spermatozoa to the oolemma is mediated by integrins recognizing the Arg-Gly-Asp sequence (RGD). Fibronectin and vitronectin, glycoproteins that contain functional RGD sequences, are both present on human spermatozoa, and integrins that recognize these ligands have been detected on spermatozoa and eggs. In this work, we studied the effects of oligopeptides specifically designed to block fibronectin or vitronectin receptors on the interaction of human spermatozoa with zona-free hamster oocytes. GRGDdSP, a peptide blocking cell attachment to fibronectin, was without effect, while GdRGDSP, which blocks both fibronectin and vitronectin receptors, significantly inhibited the binding of human spermatozoa to the oolemma of zona-free hamster eggs, in a concentration-dependent manner, over a range 1-100 microM. As these experiments suggested that a vitronectin receptor plays a role in sperm-oolemmal adhesion, we performed a series of experiments studying the effects of exogenous vitronectin, when added to spermatozoa and oocytes, on gamete interactions. Sperm-oolemmal adherence, as well as sperm aggregation, was promoted by vitronectin, over range of 2.2 nM to 1 microM, but only in the presence of calcium ions. We propose that vitronectin released during the sperm acrosome reaction is recognized by both gametes and plays a role in their adhesion.

The relationship between resolution of asymptomatic bacterial vaginosis and spontaneous preterm birth in fetal fibronectin-positive women.

PubMed

Hendler, Israel; Andrews, William W; Carey, Christopher J; Klebanoff, Mark A; Noble, William D; Sibai, Baha M; Hillier, Sharon L; Dudley, Donald; Ernest, Joseph M; Leveno, Kenneth J; Wapner, Ronald; Iams, Jay D; Varner, Michael; Moawad, Atef; Miodovnik, Menachem; O'Sullivan, Mary J; Van Dorsten, Peter J

2007-11-01

The purpose of this study was to determine the impact of persistent bacterial vaginosis (BV) on the occurrence of spontaneous preterm birth (SPB) in women who test positive for fetal fibronectin. This is a secondary analysis of a subset of pregnant women who tested positive for BV and fetal fibronectin between 16(0/7) and 25(6/7) weeks of gestation and who participated in randomized placebo controlled trials of antibiotic therapy. Nugent's criteria were used for the diagnosis of BV. Patients were reassessed for the presence of BV after treatment. The rate of SPB at <34 weeks of gestation was analyzed on the basis of treatment mode and BV status at the follow-up visit. The primary studies included a total of 3285 women. A subset of 215 women met the criteria for this analysis. Seventy-seven of 100 patients (77%) in the antibiotics group vs 33 of the 115 patients (28.7%) in the placebo group became BV negative (P < .0001). The rate of SPB at <34 weeks of gestation was lower for BV resolution compared with persistent BV (0 vs 5.7%, respectively; P = .01). In women who tested positive for fetal fibronectin and BV, resolution of BV is associated with less SPB before 34 weeks of gestation.

A simple in vitro model for investigating epithelial/mesenchymal interactions: keratinocyte inhibition of fibroblast proliferation and fibronectin synthesis.

PubMed

Harrison, Caroline A; Dalley, Andrew J; Mac Neil, Sheila

2005-01-01

Hypertrophic scarring and graft contracture are major causes of morbidity after burn injuries. It is well established that application of a split-thickness skin graft reduces scarring and contraction, and cultured epithelial autografts have a similar effect. To investigate the influence of keratinocytes on fibroblast proliferation and fibronectin synthesis, we used an in vitro separated co-culture model in which epithelial sheets were cultured above fibroblast monolayers without physical contact. We also investigated the response of fibroblasts to keratinocyte-conditioned medium (KCM) obtained from confluent and subconfluent keratinocyte monolayers. Both cultured epithelial sheets, composed of adherent fully confluent keratinocytes, and their conditioned medium, reduced fibroblast proliferation. However, KCM from subconfluent keratinocytes stimulated fibroblast proliferation at low concentrations while inhibiting it at higher concentrations, indicating that keratinocytes can produce both mitogenic and growth-inhibiting factors for fibroblasts. KCM, but not epithelial sheet co-culture, also inhibited fibroblast fibronectin synthesis. This indicates regulation of fibroblast phenotype by soluble factors released by the keratinocyte and also suggests that there is a dialogue between keratinocytes and fibroblasts with respect to fibronectin production. We conclude that this separated co-culture model is a simple way to study epithelial/mesenchymal communication particularly with respect to the role of the fibroblast in wound healing.

Vitronectin as a Micromanager of Cell Response in Material-Driven Fibronectin Nanonetworks.

PubMed

Cantini, Marco; Gomide, Karina; Moulisova, Vladimira; González-García, Cristina; Salmerón-Sánchez, Manuel

2017-09-01

Surface functionalization strategies of synthetic materials for regenerative medicine applications comprise the development of microenvironments that recapitulate the physical and biochemical cues of physiological extracellular matrices. In this context, material-driven fibronectin (FN) nanonetworks obtained from the adsorption of the protein on poly(ethyl acrylate) provide a robust system to control cell behavior, particularly to enhance differentiation. This study aims at augmenting the complexity of these fibrillar matrices by introducing vitronectin, a lower-molecular-weight multifunctional glycoprotein and main adhesive component of serum. A cooperative effect during co-adsorption of the proteins is observed, as the addition of vitronectin leads to increased fibronectin adsorption, improved fibril formation, and enhanced vitronectin exposure. The mobility of the protein at the material interface increases, and this, in turn, facilitates the reorganization of the adsorbed FN by cells. Furthermore, the interplay between interface mobility and engagement of vitronectin receptors controls the level of cell fusion and the degree of cell differentiation. Ultimately, this work reveals that substrate-induced protein interfaces resulting from the cooperative adsorption of fibronectin and vitronectin fine-tune cell behavior, as vitronectin micromanages the local properties of the microenvironment and consequently short-term cell response to the protein interface and higher order cellular functions such as differentiation.

Induction of indolamine 2,3-dioxygenase and kynurenine 3-monooxygenase in rat brain following a systemic inflammatory challenge: a role for IFN-gamma?

PubMed

Connor, Thomas J; Starr, Neasa; O'Sullivan, Joan B; Harkin, Andrew

2008-08-15

Inflammation-mediated dysregulation of the kynurenine pathway has been implicated as a contributor to a number of major brain disorders. Consequently, we examined the impact of a systemic inflammatory challenge on kynurenine pathway enzyme expression in rat brain. Indoleamine 2,3-dioxygenase (IDO) expression was induced in cortex and hippocampus following systemic lipopolysaccharide (LPS) administration. Whilst IDO expression was paralleled by increased circulating interferon (IFN)-gamma concentrations, IFN-gamma expression in the brain was only modestly altered following LPS administration. In contrast, induction of IDO was associated with increased central tumour necrosis factor (TNF)-alpha and interleukin (IL)-6 expression. Similarly, in cultured glial cells LPS-induced IDO expression was accompanied by increased TNF-alpha and IL-6 expression, whereas IFN-gamma was not detectable. These findings indicate that IFN-gamma is not required for LPS-induced IDO expression in brain. A robust increase in kynurenine-3-monooxygenase (KMO) expression was observed in rat brain 24h post LPS, without any change in kynurenine aminotransferase II (KAT II) expression. In addition, we report that constitutive expression of KAT II is approximately 8-fold higher than KMO in cortex and 20-fold higher in hippocampus. Similarly, in glial cells constitutive expression of KAT II was approximately 16-fold higher than KMO, and expression of KMO but not KAT II was induced by LPS. These data are the first to demonstrate that a systemic inflammatory challenge stimulates KMO expression in brain; a situation that is likely to favour kynurenine metabolism in a neurotoxic direction. However, our observation that expression of KAT II is much higher than KMO in rat brain is likely to counteract potential neurotoxicity that could arise from KMO induction following an acute inflammation.

Snail1 transcription factor controls telomere transcription and integrity

PubMed Central

Mazzolini, Rocco; Gonzàlez, Núria; Garcia-Garijo, Andrea; Millanes-Romero, Alba; Peiró, Sandra; Smith, Susan

2018-01-01

Abstract Besides controlling epithelial-to-mesenchymal transition (EMT) and cell invasion, the Snail1 transcriptional factor also provides cells with cancer stem cell features. Since telomere maintenance is essential for stemness, we have examined the control of telomere integrity by Snail1. Fluorescence in situ hybridization (FISH) analysis indicates that Snail1-depleted mouse mesenchymal stem cells (MSC) have both a dramatic increase of telomere alterations and shorter telomeres. Remarkably, Snail1-deficient MSC present higher levels of both telomerase activity and the long non-coding RNA called telomeric repeat-containing RNA (TERRA), an RNA that controls telomere integrity. Accordingly, Snail1 expression downregulates expression of the telomerase gene (TERT) as well as of TERRA 2q, 11q and 18q. TERRA and TERT are transiently downregulated during TGFβ-induced EMT in NMuMG cells, correlating with Snail1 expression. Global transcriptome analysis indicates that ectopic expression of TERRA affects the transcription of some genes induced during EMT, such as fibronectin, whereas that of TERT does not modify those genes. We propose that Snail1 repression of TERRA is required not only for telomere maintenance but also for the expression of a subset of mesenchymal genes. PMID:29059385

GDNF family receptor α-1 in the catfish: Possible implication to brain dopaminergic activity.

PubMed

Mamta, Sajwan-Khatri; Senthilkumaran, Balasubramanian

2018-05-31

Glial cell line-derived neurotrophic factor (GDNF)is a potent trophic factor that preferentially binds to GDNF family receptor α-1 (GFRα-1)by regulating dopaminergic (DA-ergic) neuronsin brain. Present study aimed to evaluate the significance of GFRα-1 expression during early brain development in catfish. Initially, the full-length cDNA of GFRα-1 was cloned from adult brain which showed high homology with other vertebrate counterparts. Quantitative PCR analysis of tissue distribution revealed ubiquitous expression of GFRα-1 in the tissues analyzed with high levels in female brain and ovary. Significant high expression was evident in brain at 75 and 100 days post hatch females than the respective age-match males. Expression of GFRα-1 was high in brain during the spawning phase when compared to other reproductive phases. Localization of GFRα-1 revealed its presence in preoptic area-hypothalamus which correlated well with the expression profile in discrete areas of brain in adult catfish. Transient silencing of GFRα-1through siRNA lowered expression levels of GFRα-1, which further down regulated the expression of certain brain-specific genes. Expression of GFRα-1 in brain declined significantly upon treatment with the 1-methyl-1,2,3,6-tetrahydropyridinecausing neurodegeneration which further correlated with catecholamines (CA), L-3,4-dihydroxyphenylalanine, DA and norepinephrine levels. Taken together, GFRα-1 plausibly entrains gonadotropin-releasing hormone and gonadotropin axiseither directly or indirectly, at least by partially targeting CA-ergic activity. Copyright © 2018 Elsevier Inc. All rights reserved.

Mucosal adjuvanticity of fibronectin-binding peptide (FBP) fused with Echinococcus multilocularis tetraspanin 3: systemic and local antibody responses.

PubMed

Dang, Zhisheng; Feng, Jinchao; Yagi, Kinpei; Sugimoto, Chihiro; Li, Wei; Oku, Yuzaburo

2012-01-01

Studies have shown that a bacterial fibronectin attachment protein (FAP) is able to stimulate strong systemic and mucosal antibody responses when it is used alone or co-administrated with other antigens (Ags). Thus, it has been suggested to be a promising adjuvant candidate for the development of efficient vaccines. However, the co-administered Ags and FAP were cloned, expressed and purified individually to date. In a recent study, we first evaluated the adjuvanticity of a fibronectin-binding peptide (FBP, 24 amino acids) of Mycobacterium avium FAP fused with Echinococcus multilocularis tetraspanin 3 (Em-TSP3) by detecting systemic and local antibody responses in intranasally (i.n.) immunized BALB/c mice. Em-TSP3 and FBP fragments were linked with a GSGGSG linker and expressed as a single fusion protein (Em-TSP3-FBP) using the pBAD/Thio-TOPO expression vector. BALB/c mice were immunized i.n. with recombinant Em-TSP3-FBP (rEm-TSP3-FBP) and rEm-TSP3+CpG and the systemic and local antibody responses were detected by ELISA. The results showed that both rEm-TSP3-FBP and rEm-TSP3+CpG evoked strong serum IgG (p<0.001) and IgG1 responses (p<0.001), whereas only the latter induced a high level IgG2α production (p<0.001), compared to that of rEm-TSP3 alone without any adjuvant. There were no significant differences in IgG and IgG1 production between the groups. Low level of serum IgA and IgM were detected in both groups. The tendency of Th1 and Th2 cell immune responses were assessed via detecting the IgG1/IgG2α ratio after the second and third immunizations. The results indicated that i.n. immunization with rEm-TSP3-FBP resulted in an increased IgG1/IgG2α ratio (a Th2 tendency), while rEm-TSP3+CpG caused a rapid Th1 response that later shifted to a Th2 response. Immunization with rEm-TSP3-FBP provoked significantly stronger IgA antibody responses in intestine (p<0.05), lung (p<0.001) and spleen (p<0.001) compared to those by rEm-TSP3+CpG. Significantly high level IgA antibodies were detected in nasal cavity (p<0.05) and liver (p<0.05) samples from both groups when compared to rEm-TSP3 alone without any adjuvant, with no significant difference between them. I.n. administration of rEm-TSP3-FBP can induce strong systemic and mucosal antibody responses in immunized BALB/c mice, suggesting that fusion of Em-TSP3 with FBP is a novel, prospective strategy for developing safe and efficient human mucosal vaccines against alveolar echinococcosis (AE).

Gender-Specific Gene Expression in Post-Mortem Human Brain: Localization to Sex Chromosomes

PubMed Central

Vawter, Marquis P; Evans, Simon; Choudary, Prabhakara; Tomita, Hiroaki; Meador-Woodruff, Jim; Molnar, Margherita; Li, Jun; Lopez, Juan F; Myers, Rick; Cox, David; Watson, Stanley J; Akil, Huda; Jones, Edward G; Bunney, William E

2011-01-01

Gender differences in brain development and in the prevalence of neuropsychiatric disorders such as depression have been reported. Gender differences in human brain might be related to patterns of gene expression. Microarray technology is one useful method for investigation of gene expression in brain. We investigated gene expression, cell types, and regional expression patterns of differentially expressed sex chromosome genes in brain. We profiled gene expression in male and female dorsolateral prefrontal cortex, anterior cingulate cortex, and cerebellum using the Affymetrix oligonucleotide microarray platform. Differentially expressed genes between males and females on the Y chromosome (DBY, SMCY, UTY, RPS4Y, and USP9Y) and X chromosome (XIST) were confirmed using real-time PCR measurements. In situ hybridization confirmed the differential expression of gender-specific genes and neuronal expression of XIST, RPS4Y, SMCY, and UTY in three brain regions examined. The XIST gene, which silences gene expression on regions of the X chromosome, is expressed in a subset of neurons. Since a subset of neurons express gender-specific genes, neural subpopulations may exhibit a subtle sexual dimorphism at the level of differences in gene regulation and function. The distinctive pattern of neuronal expression of XIST, RPS4Y, SMCY, and UTY and other sex chromosome genes in neuronal subpopulations may possibly contribute to gender differences in prevalence noted for some neuropsychiatric disorders. Studies of the protein expression of these sex- chromosome-linked genes in brain tissue are required to address the functional consequences of the observed gene expression differences. PMID:14583743