The Integrin α2β1 (GPIa/IIa)-I-Domain Inhibits Platelet-Collagen Interaction

1997 ◽  
Vol 77 (05) ◽  
pp. 0981-0985 ◽  
Author(s):  
H Depraetere ◽  
C Wille ◽  
Y Gansemans ◽  
P Stanssens ◽  
M Lauwereys ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Collagen Type ◽  
Stop Codon ◽  
Collagen Type I ◽  
Type I ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
C Terminus ◽  
A Domains ◽  
Domain I

SummaryThe integrin α2β1 is a major cellular receptor for collagen. The α2subunit contains an ± 200 amino acids inserted domain (I-domain) in the N-terminal region. A certain degree of homology exists between the I-domains found in integrins, collagen and the A-domains of vWF.The α2-I-domain encoding region (aa residues D145 to S334) was obtained by RT-PCR from mRNA of non stimulated human PBL’s. The primers were designed to introduce the necessary restriction sites for cloning of the DNA fragment in frame downstream of the malE gene, as well as a stop codon after the last triplet. The resulting construct pMAL-c2-α2-I allows the expression of the I-domain, fused to the C-terminus of maltose binding protein (mal). The α2-I-mal is purified from the bacterial extract by affinity chromatography on an amylose column.The purified α2-I-mal has been characterized by ELISA’s. The animal bound to immobilised collagen type I in a concentration dependent manner and could be blocked by the functional monoclonal anti-α2β1 antibody 6F1.The interaction of α2-I-mal with collagen furthermore is Mg2+- dependent since the binding was inhibited in the presence of 10 mM EDTA or 10 mM Ca2+ but sustained in the presence of 10 mM Mg2+.Finally, α2-I-mal itself was able to inhibit adhesion of washed platelets to collagen immobilised on a microtiterplate in a dose-dependent manner (α2-I-mal IC50:0.7 μ M) as well as platelet aggregation induced by collagen type I (α2-I-mal IC50: 0.7 μM).With these results we could confirm that the α2-I-domain represents the collagen-binding site of α2β1 and we furthermore could indicate that this domain is able to prevent platelet adhesion to collagen and collagen-induced platelet aggregation, pointing to the primordial role of α2-I-mal and hence of α2β1 in platelet-collagen interaction.

scholarly journals A Newly Identified Leptospiral Adhesin Mediates Attachment to Laminin

2006 ◽  
Vol 74 (11) ◽  
pp. 6356-6364 ◽  
Author(s):  
Angela S. Barbosa ◽  
Patricia A. E. Abreu ◽  
Fernanda O. Neves ◽  
Marina V. Atzingen ◽  
Mônica M. Watanabe ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Collagen Type ◽  
Outer Membrane Proteins ◽  
Collagen Type I ◽  
Surface Proteins ◽  
Collagen Type Iv ◽  
Type I ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Sodium Metaperiodate

ABSTRACT Pathogenic leptospires have the ability to survive and disseminate to multiple organs after penetrating the host. Several pathogens, including spirochetes, have been shown to express surface proteins that interact with the extracellular matrix (ECM). This adhesin-mediated binding process seems to be a crucial step in the colonization of host tissues. This study examined the interaction of putative leptospiral outer membrane proteins with laminin, collagen type I, collagen type IV, cellular fibronectin, and plasma fibronectin. Six predicted coding sequences selected from the Leptospira interrogans serovar Copenhageni genome were cloned, and proteins were expressed, purified by metal affinity chromatography, and characterized by circular dichroism spectroscopy. Their capacity to mediate attachment to ECM components was evaluated by binding assays. We have identified a leptospiral protein encoded by LIC12906, named Lsa24 (leptospiral surface adhesin; 24 kDa) that binds strongly to laminin. Attachment of Lsa24 to laminin was specific, dose dependent, and saturable. Laminin oxidation by sodium metaperiodate reduced the protein-laminin interaction in a concentration-dependent manner, indicating that laminin sugar moieties are crucial for this interaction. Triton X-114-solubilized extract of L. interrogans and phase partitioning showed that Lsa24 was exclusively in the detergent phase, indicating that it is a component of the leptospiral membrane. Moreover, Lsa24 partially inhibited leptospiral adherence to immobilized laminin. This newly identified membrane protein may play a role in mediating adhesion of L. interrogans to the host. To our knowledge, this is the first leptospiral adhesin with laminin-binding properties reported to date.

Integrin alpha 2 I-domain is a binding site for collagens

1995 ◽  
Vol 108 (4) ◽  
pp. 1629-1637 ◽  
Author(s):  
D. Tuckwell ◽  
D.A. Calderwood ◽  
L.J. Green ◽  
M.J. Humphries
Keyword(s):  
Type I Collagen ◽  
Type I ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Collagen Binding ◽  
Functional Antibodies ◽  
A Domains ◽  
Domain I ◽  
Von Willebrand ◽  
Willebrand Factor

Integrins alpha 1 beta 1 and alpha 2 beta 1 are major cellular receptors for collagens. The alpha 1 and alpha 2 subunits contain a approximately 200 amino acid inserted domain (I-domain) in their N-terminal region and, because of the homology between the I-domains and the collagen-binding A-domains of von Willebrand factor, it has been suggested that the I-domains might mediate the collagen-binding functions of alpha 1 beta 1 and alpha 2 beta 1. In order to fully investigate this hypothesis, we have generated recombinant human alpha 2 I-domain (r alpha 2I) by reverse transcriptase-polymerase chain reaction/bacterial expression and tested its ability to mediate the collagen-binding functions of alpha 2 beta 1. R alpha 2 I binds specifically to type I collagen in a concentration-dependent manner: binding is cation dependent and, like the complete receptor, is supported by magnesium and manganese ions but not by calcium ions. R alpha 2I is recognised by anti-functional anti-alpha 2 monoclonal antibodies 6F1, 5E8 and P1E6 in capture ELISAs, and anti-functional antibodies inhibited r alpha 2I-collagen binding. In addition, r alpha 2I inhibits cell spreading on collagen. R alpha 2I is therefore a collagen-binding domain and can account for many of the collagen-binding functions of integrin alpha 2 beta 1. We have also determined the collagen specificity of r alpha 2I and found that it binds types I, II and XI collagen.

scholarly journals SAT0298 IS INTERLEUKIN 6 A FACTOR OF FIBROGENESIS IN DERMAL FIBROBLASTS?

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1094.1-1094
Author(s):  
A. S. Siebuhr ◽  
P. Juhl ◽  
M. Karsdal ◽  
A. C. Bay-Jensen
Keyword(s):  
Gene Expression ◽  
Type I Collagen ◽  
Collagen Type ◽  
Collagen Type I ◽  
Dermal Fibroblasts ◽  
Type I ◽  
Dependent Manner ◽  
Full Time ◽  
Collagen Formation ◽  
Type Iii

Background:Interleukin 6 (IL-6) is known to have both pro- and anti-inflammatory properties, depending on the receptor activation. The classical IL-6 signaling via the membrane bound receptor is mainly anti-inflammatory, whereas signaling through the soluble receptor (sIL-6R) is pro-inflammatory/pro-fibrotic. However, the direct fibrotic effect of IL-6 stimulation on dermal fibroblasts is unknown.Objectives:We investigated the fibrotic effect of IL-6 + sIL-6R in a dermal fibroblast model and assessed fibrosis by neo-epitope biomarkers of extracellular matrix proteins.Methods:Primary healthy human dermal fibroblasts were grown for up to 17 days in DMEM medium with 0.4% fetal calf serum, ficoll (to produce a crowded environment) and ascorbic acid. IL-6 [1-90 nM]+sIL-6R [0.1-9 nM] alone or in combination with TGFβ [1 nM] were tested in three different donors. TGFβ [1 nM], PDGF-AB [3 nM] and non-stimulated cells (w/o) were used as controls. Tocilizumab (TCZ) with TGFβ + IL-6 + sIL-6R stimulation was tested in one donor. Collagen type I, III and VI formation (PRO-C1, PRO-C3 and PRO-C6) and fibronectin (FBN-C) were evaluated by validated ELISAs (Nordic Bioscience). Western blot analysis investigated signal cascades. Gene expression of selected ECM proteins was analyzed. Statistical analyses included One-way and 2-way ANOVA and area under the curve analysis.Results:formation by the end of the culture period. The fibronectin and collagen type VI signal were consistent between the three tested donors, whereas the formation of type III collagen was only increased in one donor, but in several trials. Type I collagen formation was unchanged by IL-6 + sIL-6R stimulation. The gene expression of type I collagen was induced by IL-6 + sIL-6R. Western blot analysis validated trans-signaling by the IL-6+sIL-6R stimulation as expected.IL-6 + sIL-6R stimulation in combination with TGFβ decreased fibronectin levels compared to TGFβ alone but did not reach the level of unstimulated fibroblasts. The formation of collagen type IV was generally unchanged with IL-6 + sIL-6R + TGFβ compared to TGFβ alone. Collagen type I and III formation was more scattered in the signals when IL-6 + sIL-6R was in combination with TGFβ, as the biomarker level could be either decreased or increased compared to TGFβ alone. In two studies the type I collagen level was synergistic increased by IL-6 + sIL-6R + TGFβ, whereas another study found the level to be decreased compared to TGFβ alone. The gene expression of fibronectin and type I collagen was increased with TGFβ +IL-6+sIL-6R compared to TGFβ alone.Inhibition of IL-6R by TCZ in combination with IL-6 + sIL-6R did only decrease the fibronectin level with the lowest TCZ concentration (p=0.03). TCZ alone decreased the fibronectin level in a dose-dependent manner (One-way ANOVA p=0.0002).Conclusion:We investigated the fibrotic response of dermal fibroblasts to IL-6 + sIL-6R stimulation. IL-6 modulated the fibronectin level and modulated the collagen type III formation level in a somewhat dose-dependent manner. In combination with TGFβ, IL-6 decreased collagen type I and IV formation and fibronectin. However, in this study inhibition of IL-6R by TCZ did not change the fibrotic response of the dermal fibroblasts. This study indicated that IL-6 did not induce collagen formation in dermal fibroblasts, except type III collagen formation with high IL-6 concentration.Figure:Disclosure of Interests:Anne Sofie Siebuhr Employee of: Nordic Bioscience, Pernille Juhl Employee of: Nordic Bioscience, Morten Karsdal Shareholder of: Nordic Bioscience A/S., Employee of: Full time employee at Nordic Bioscience A/S., Anne-Christine Bay-Jensen Shareholder of: Nordic Bioscience A/S, Employee of: Full time employee at Nordic Bioscience A/S.

scholarly journals Collagen microarchitecture mechanically controls myofibroblast differentiation

2020 ◽  
Vol 117 (21) ◽  
pp. 11387-11398 ◽  
Author(s):  
Bo Ri Seo ◽  
Xingyu Chen ◽  
Lu Ling ◽  
Young Hye Song ◽  
Adrian A. Shimpi ◽  
...  
Keyword(s):  
Collagen Type ◽  
Collagen Type I ◽  
Type I ◽  
Myofibroblast Differentiation ◽  
Dependent Manner ◽  
Fibrillar Collagen ◽  
Collagen Gels ◽  
Gelation Temperature ◽  
Rheological Characterization ◽  
Fiber Thickness

Altered microarchitecture of collagen type I is a hallmark of wound healing and cancer that is commonly attributed to myofibroblasts. However, it remains unknown which effect collagen microarchitecture has on myofibroblast differentiation. Here, we combined experimental and computational approaches to investigate the hypothesis that the microarchitecture of fibrillar collagen networks mechanically regulates myofibroblast differentiation of adipose stromal cells (ASCs) independent of bulk stiffness. Collagen gels with controlled fiber thickness and pore size were microfabricated by adjusting the gelation temperature while keeping their concentration constant. Rheological characterization and simulation data indicated that networks with thicker fibers and larger pores exhibited increased strain-stiffening relative to networks with thinner fibers and smaller pores. Accordingly, ASCs cultured in scaffolds with thicker fibers were more contractile, expressed myofibroblast markers, and deposited more extended fibronectin fibers. Consistent with elevated myofibroblast differentiation, ASCs in scaffolds with thicker fibers exhibited a more proangiogenic phenotype that promoted endothelial sprouting in a contractility-dependent manner. Our findings suggest that changes of collagen microarchitecture regulate myofibroblast differentiation and fibrosis independent of collagen quantity and bulk stiffness by locally modulating cellular mechanosignaling. These findings have implications for regenerative medicine and anticancer treatments.

scholarly journals Toxicity Effects of Methylene Blue on Rat Intervertebral Disc Annulus Fibrosus Cells

2019 ◽  
Vol 2 (22.2) ◽  
pp. 155-164
Author(s):  
Liang Zhang
Keyword(s):  
Methylene Blue ◽  
Cell Apoptosis ◽  
Annulus Fibrosus ◽  
Caspase 3 ◽  
In Vitro Study ◽  
Collagen Type I ◽  
Type I ◽  
Dependent Manner ◽  
Concentration Dependent Manner

Background: There is an increasing local application of methylene blue (MB) in the treatment of discogenic low back pain (LBP) and percutaneous transforaminal endoscopic discectomy (PTED) procedures. MB could generate DNA damage and induce apoptosis in different cell types; however, the effects of MB on intervertebral disc (IVD) annulus fibrosus (AF) cells are not clearly understood. Objective: The objective of this study was to investigate the effects of different concentrations of MB on rat AF cells in vitro. Study Design: This study used an experimental design. Setting: This research was conducted at the Orthopaedic Institute of the Clinical Medical College of Yangzhou University. Methods: AF cells were isolated and cultured with different concentrations of MB (0, 2, 20, and 200 μg/mL) and assessed to determine the possible cytotoxic effects of MB. The cell proliferation was detected by Cell Counting Kit-8 (CCK-8) assay. The inverted phase-contrast microscopy was used to perform morphological observation of apoptotic cells, and flow cytometry was used to measure the incidence of cell apoptosis. The mRNA and protein expression levels of apoptosis-associated genes (caspase-3, Bcl-2, and Bax) and other related genes (collagen type I, transforming growth factor β1 [TGF-β1], fibroblast growth factor [bFGF], and tissue inhibitor of metalloproteinase-1 [TIMP-1]) were analyzed by quantitative real-time PCR (RT-PCR) and Western blotting. Results: Our results indicated that MB reduced cell viability in a concentration- and timedependent manner. MB also induced marked AF cell apoptosis in a concentration-dependent manner observed by inverted phase-contrast microscopy, flow cytometry, and indicated by the increased expression of caspase-3. Both RT-PCR and Western blotting revealed significant upregulation of Bax and caspase-3 expression levels accompanied by decreased expression of Bcl2 in a concentration-dependent manner. Moreover, collagen type I, TGF-β1, bFGF, and TIMP-1 mRNA and protein levels were also found to be decreased by MB in a concentration-dependent manner. Limitations: Limitations of this study were the in vitro study design and lack of in vivo validation of the observed effects of MB on human IVD cells. Conclusions: Our results indicate that a high concentration of MB can not only inhibit proliferation and paracrine function of AF cells, but can also induce cell apoptosis in a concentration-dependent manner, suggesting that it is necessary to choose low concentrations of MB in practical application and limit the use of MB in the treatment of discogenic LBP to research protocols. Key words: Methylene blue, annulus fibrosus cell, proliferation, apoptosis, paracrine

scholarly journals Fibrin coating of polymer vascular graft does not reduce its thromboresistance

2020 ◽  
Vol 5 (2) ◽  
pp. 22-29
Author(s):  
E. A. Velikanova ◽  
T. V. Glushkova ◽  
T. N. Akentyeva ◽  
V. G. Matveeva ◽  
M. Yu. Khanova ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Platelet Aggregation ◽  
Vascular Graft ◽  
Collagen Type ◽  
Platelet Rich Plasma ◽  
Small Diameter ◽  
Collagen Type I ◽  
Type I ◽  
Nuclear Staining ◽  
Human Coronary Artery

Aim. To evaluate biocompatibility along with adhesion and aggregation of platelets on the surface of uncoated and fibrin-coated poly(3-hydroxybutyrate- co-3-hydroxyvalerate)/poly(ε-caprolactone) (PHBV/PCL) small-diameter vascular grafts.Materials and Methods. 4 mm diameter grafts were fabricated by electrospinning from PHBV/ PCL (1:2) blend dissolved in 1,1,1,3,3,3-hexafluoro- 2-propanol. Inner wall of the grafts was produced using co-electrospinning of the polymer blend and collagen type I (5 mg/mL) from two different syringes. Fibrinogen was obtained from the blood of healthy donors by a cryoprecipitation procedure. Sterile polymer scaffolds were impregnated into a fibrinogen solution and immersed in a thrombin/calcium chloride blend for polymerization. To assess the biocompatibility of the grafts, primary human coronary artery endothelial cells were seeded on the luminal surface and counted under a fluorescence microscope after nuclear staining. Hemocompatibility was tested by incubation of the grafts with human platelet-rich plasma. Platelet aggregation was assessed using a platelet aggregation analyser. Surface morphology, platelet adhesion and activation were evaluated by scanning electron microscopy.Results. Fibrin coating promoted cell adhesion and proliferation and improved the graft biocompatibility as evidenced by a higher number of endothelial cells. Fibrin coating did not increase platelet aggregation, adhesion, and activation and therefore did not reduce the thromboresistance of vascular graft.Conclusion. The fibrin modification of polymer grafts from PHBV/PCL blend and collagen type I improves the surface biocompatibility and does not reduce its thromboresistance.

The Development of Small Molecule Inhibitors of Collagen Binding to the Integrin α2β1 as Antithrombotic Drugs.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3677-3677
Author(s):  
Sungwook Choi ◽  
Seth E. Snyder ◽  
David T. Moore ◽  
Gaston Vilaire ◽  
Joel S. Bennett ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Small Molecule ◽  
Platelet Adhesion ◽  
Collagen Type ◽  
Small Molecule Inhibitors ◽  
Collagen Type I ◽  
Mouse Tumor ◽  
Type I ◽  
Immunoglobulin Gene ◽  
Collagen Binding

Abstract Platelets tether to collagen in the subendothelial matrix that is exposed by vascular damage. Collagen is a particularly important matrix component in this context, not only because it is a substrate for platelet adhesion, but because it is an agonist for platelet aggregation and secretion as well. There are two platelet collagen receptors, the immunoglobulin gene superfamily member GPVI and the integrin α2Β1. Both are involved in adhesion to exposed collagen and generate downstream activating signals. α2Β1 is widely expressed and has been implicated in hemostasis and thrombosis, as well as cancer metastasis, wound healing, and angiogenesis. In mice, α2Β1 deficiency results in decreased ex vivo platelet aggregation, but normal bleeding times. In mouse tumor models, α2Β1 blockade reduces both metastasis and angiogenesis. Humans lacking α2Β1 have a mild bleeding diathesis. Given this background, α2Β1 appears to be an appropriate target for the development of small-molecule inhibitors to serve as relatively safe anti-platelet and anti-tumor agents, either acting alone or in synergy with other anti-platelet or anti-tumor agents. We have developed two classes of small-molecule α2Β1 inhibitors. The first class is targeted against the collagen binding site located on the α2 I-domain and was designed using molecular modeling to superimpose a dipeptide scaffold onto the published crystal structure of the I-domain bound to a collagen-mimetic peptide (GFOGER). These molecules block recombinant human I-domain binding to immobilized collagen type I with IC50s as low as 10 μM. Although the molecules inhibit platelet adhesion to collagen only at higher concentrations, they readily inhibit melanoma cell adhesion to collagen mimetics. It is also noteworthy that the molecules induce platelet protein phosphorylation and potentiate platelet aggregation induced by other platelet agonists, both of which can be prevented by pre-incubating platelets with monoclonal antibodies directed against the α2 I-domain, but not against GPVI. The second class of molecules was derived from proline-substituted 2,3-diaminopropionic acids and is directed against the Β1 I-like domain, an allosteric site that regulates ligand binding. These molecules are potent inhibitors of platelet adhesion to immobilized soluble collagen type I with IC50s of 10–50 nM and inhibit the adhesion of melanoma cells to collagen mimetics with IC50s of 250–350 nM. These molecules do not inhibit platelet aggregation, nor do they inhibit I-domain binding to immobilized collagen type I, behavior consistent with binding to the Β1 I-like domain. In a murine model of ferric-chloride induced carotid thrombosis, the molecules synergize with aspirin to prevent arterial thrombosis. In summary, we have developed two classes of small molecule inhibitors that impair the interaction of collagen with the integrin α2Β1. Although both classes of inhibitors bind to α2Β1, their effects on its function are substantially different, indicating that there are multiple potential strategies for inhibiting integrin function pharmacologically. Further development of these inhibitors may lead to agents that will be clinically useful in the treatment of thrombosis and cancer.

scholarly journals P045 Oncostatin M induces strong fibrotic and chemokine responses from primary colonic subepithelial myofibroblasts

2021 ◽  
Vol 15 (Supplement_1) ◽  
pp. S154-S155
Author(s):  
G Kokkotis ◽  
G Tarapatzi ◽  
I Drygiannakis ◽  
E Filidou ◽  
L Kandilogiannakis ◽  
...  
Keyword(s):  
Collagen Type ◽  
Intestinal Inflammation ◽  
Collagen Type I ◽  
Oncostatin M ◽  
Type I ◽  
Dependent Manner ◽  
Chemokine Expression ◽  
Basal Expression ◽  
Tnf Α ◽  
Chronic Intestinal Inflammation

Abstract Background Oncostatin M (OSM) may play an important role in Inflammatory Bowel Disease (IBD) pathogenesis. Specifically, both OSM and its receptor are upregulated in inflamed colonic regions of IBD patients, and high OSM expression has been associated with failure to respond to anti-TNF therapy. Our aim was to investigate the effect of OSM in fibrotic factors and chemokine expression on primary colonic subepithelial myofibroblasts (SEMFs) from healthy individuals (HI). Methods Primary SEMFs were isolated from endoscopically-obtained colonic biopsies from HI. SEMFs were stimulated with 1, 10, or 100ng/ml OSM for 6 hours, with or without pre-stimulation with 5ng/ml IL-1α plus 50ng/ml TNF-α for 24h. Total RNA was collected and mRNA transcripts for collagen type I, type III, fibronectin, and the chemokines CCL2, CXCL9, CXCL10 and CXCL11 were measured by reverse transcription quantitative PCR. Results Unstimulated SEMFs had a basal expression of collagen type I, III, fibronectin, CCL2, CXCL9, CXCL10 and CXCL11. OSM stimulation augmented chemokine mRNA expression in a dose-dependent manner (Table 1) but had no effect on fibrotic factors expression. Pre-stimulation of myofibroblasts with TNF-α and IL-1α resulted in augmented expression of collagens I and III and fibronectin, in addition to further increases in chemokine expression in response to subsequent stimulation by OSM (Table 2). Conclusion Our results show that stimulation with OSM induces fibrotic and chemokine responses by SEMFs. Our findings further support the hypothesis that SEMFs may play a key role in regulating chronic intestinal inflammation and response to biological therapy.

Stretching-Induced Collagen Type I Synthesis in Human Tendon Fibroblasts Is Mediated by TGF-β1

2003 ◽  
Author(s):  
Guoguang Yang ◽  
Richard C. Crawford ◽  
James H.-C. Wang
Keyword(s):  
Protein Synthesis ◽  
Transforming Growth Factor Beta ◽  
Collagen Type ◽  
Transforming Growth Factor ◽  
Collagen Type I ◽  
Type I ◽  
Dependent Manner ◽  
Gene And Protein Expression ◽  
Mechanical Stretching ◽  
Tgf Β1

This study investigated the effect of cyclic mechanical stretching on the collagen gene expression and protein synthesis of human patellar tendon fibroblasts (HPTFs). We hypothesized that cyclic mechanical stretching of HPTFs would increase collagen synthesis via transforming growth factor-beta 1 (TGF-β1). To test the hypothesis, the tendon fibroblasts were cultured on microgrooved surfaces of silicone dishes under serum-free conditions. The cells were subjected to cyclic uniaxial stretching with a constant frequency and duration (0.5Hz, 4hr), and one of three stretching magnitudes (no stretch, 4%, and 8%) followed by 4 hours of rest. It was found that the gene and protein expression of both collagen type I and TGF-β1 were significantly increased in a stretching-magnitude dependent manner, whereas collagen type III gene and protein levels were not significantly changed. The exogenous addition of antibody to TGF-β1 eliminated the stretching-induced increase in collagen type I protein synthesis. The results therefore confirmed our working hypothesis and suggest that mechanical stretching of tendon fibroblasts can lead to matrix remodeling by modulating the collagen production of tendon fibroblasts, a process at least particially mediated by TGF-β1.

scholarly journals High Glucose Stimulates Proliferation and Collagen Type I Synthesis in Renal Cortical Fibroblasts

1999 ◽  
Vol 10 (9) ◽  
pp. 1891-1899 ◽  
Author(s):  
DONG CHEOL HAN ◽  
MOTOHIDE ISONO ◽  
BRENDA B. HOFFMAN ◽  
FUAD N. ZIYADEH
Keyword(s):  
High Glucose ◽  
Type I Collagen ◽  
Collagen Type ◽  
Transforming Growth Factor ◽  
Thymidine Incorporation ◽  
Collagen Type I ◽  
Type I ◽  
Dependent Manner ◽  
Collagen Mrna ◽  
Tgf Β1

Abstract. Renal tubular epithelial cells and interstitial fibroblasts are active participants in tubulointerstitial fibrosis, the best correlate of decreased glomerular filtration in diabetic nephropathy. It was reported previously that high ambient glucose stimulates transforming growth factor-β (TGF-β) mRNA and bioactivity, promotes cellular hypertrophy, and increases collagen synthesis in proximal tubular cells. This study evaluates the effects of high glucose and TGF-β on the behavior of murine renal cortical fibroblasts (TFB) in culture. High glucose (450 mg/dl) significantly increased [3H]-thymidine incorporation (by 60 to 80% after 24 to 72 h) and cell number, without significantly increasing cell death when compared with normal glucose (100 mg/dl). There also was a transient increase in the mRNA of the c-mycandegr-1early-response genes. Exogenous TGF-β1 was promitogenic rather than antiproliferative in contrast to other renal cell types. Northern blot analysis demonstrated constitutive expression of TGF-β1, -β2, and -β3 transcripts. Exposure to high glucose increased all three TGF-β isoforms in a time-dependent manner. High glucose as well as exogenous TGF-β1 also increased [3H]-proline incorporation, α2(I) collagen mRNA, and type I collagen protein (measured by immunoassay). Treatment with a neutralizing pan-selective monoclonal anti-TGF-β antibody markedly attenuated the stimulation by high ambient glucose of thymidine incorporation, TGF-β1 mRNA, and type I collagen mRNA and protein levels. It is concluded that high ambient glucose and exogenous TGF-β1 share similar actions on renal fibroblasts. Moreover, the stimulation of cell proliferation and collagen type I synthesis in these cells by high ambient glucose are mediated by activation of an autocrine TGF-β system.

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