Synthesis of collagen is dysregulated in cultured fibroblasts derived from skin of subjects with varicose veins as it is in venous smooth muscle cells.
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BACKGROUND: The dilatation and tortuosity observed in varicose veins provide evidence for progressive venous wall remodeling associated with abnormalities of smooth muscle cells and extracellular matrix. The present study was designed to examine if the phenotypic modulations observed in the venous smooth muscle cells of patients with varicose veins were also present in their dermal fibroblasts. METHODS AND RESULTS: Collagen type I (collagen I), type III (collagen III), and type V (collagen V) were compared in dermal fibroblasts derived from the skin of control subjects and patients with varicose veins. The synthesis of collagen I, the release of its metabolites, and the expression of its mRNA were increased in fibroblasts from patients with varicose veins, whereas the synthesis of collagen III was decreased but not correlated with a decrease in mRNA expression and in metabolite release. Matrix metalloproteinases (MMP1, 2, 7, 8, 9, and 13) and their inhibitors (TIMP1 and 2) were quantified in both cell types; only the production of proMMP2 was increased in cells derived from patients with varicose veins. CONCLUSIONS: These findings suggest that the synthesis of collagen I and III is dysregulated in dermal fibroblasts derived from patients with varicose veins. These results are comparable with those observed in smooth muscle cells derived from varicose veins, thus suggesting a systemic alteration of tissue remodeling in subjects with varicose veins. (+info)
Thrombospondin-1 mediates smooth muscle cell proliferation induced by interaction with human platelets.
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OBJECTIVES: Platelet adherence and activation are associated with smooth muscle cell (SMC) proliferation and arterial restenosis. This study examined platelet-SMC interaction on fibrillar type I collagen and analyzed the role of thrombospondin (TSP)-1 in platelet-induced SMC proliferation. METHODS AND RESULTS: When SMCs cultured on fibrillar collagen were treated with human platelets (5 preparations), 7.45+/-2.94% of the cells passed through S phase within 24 hours, as determined by bromodeoxyuridine nuclear labeling. The addition of platelets markedly induced SMC TSP-1 mRNA expression and cell surface protein accumulation, which colocalized with adhered platelets, as determined by alpha(IIb) integrin immunostaining. Direct interaction of platelets with SMCs was necessary for its effect on proliferation and TSP-1 accumulation, as determined in the transwell culture system. The anti-TSP-1 blocking antibody strongly inhibited platelet-induced SMC proliferation by approximately 60%. Analysis of the receptors for TSP-1 accumulation on the SMC surface revealed that beta1 integrins are mainly involved. The anti-beta1 integrin blocking antibody, which potently suppressed TSP-1 accumulation on SMCs, also markedly inhibited platelet-stimulated SMC proliferation. CONCLUSIONS: TSP-1 and beta1 integrin interaction is involved in platelet-stimulated SMC proliferation. This in vitro coculture system could prove useful for examining the molecular mechanism underlying platelet-induced vascular remodeling and for studying the mechanism of a tested drug for restenosis. (+info)
Repeated allergen exposure changes collagen composition in airways of sensitised Brown Norway rats.
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Increased or altered collagen deposition in the airway wall is one of the characteristics of airway remodelling in asthma. The mechanisms underlying this increase, and its functional consequences remain to be established further. Representative in vivo animal models might be useful in this respect. In the present study, collagen deposition after prolonged allergen exposure was characterised in the airway wall of Brown Norway rats. Sensitised rats were repeatedly exposed to ovalbumin (OA) or phosphate-buffered saline during 2 and 12 weeks. The deposition of collagen type I, III, IV, V and VI was not altered in animals exposed to OA for 2 weeks. After 12 weeks of OA exposure, more collagen type I was deposited in the inner and outer airway wall and more type V and VI collagen was observed in the outer airway wall. At 12 weeks the number of vessels, identified via type IV collagen staining was not increased, but the total vessel area was. In conclusion, prolonged allergen exposure in sensitised rats is associated with enhanced deposition of type I, V and VI collagens and increased vascularity. This suggests that some aspects of airway remodelling in asthma could be driven by long-term allergen exposure. (+info)
Fibrillar collagen regulation of plasminogen activator inhibitor-1 is involved in altered smooth muscle cell migration.
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OBJECTIVE: Vascular smooth muscle cells (SMCs) cultured on polymerized type I collagen fibrils are arrested in the G1 phase of the cell cycle, and their phenotypic markers and pattern of expressed genes are markedly altered. In this study, we examined polymerized collagen regulation of plasminogen activator inhibitor (PAI)-1 and its involvement in SMC migration. METHODS AND RESULTS: We demonstrate that secretion and cell surface accumulation of PAI-1 are suppressed in SMCs cultured on polymerized collagen compared with SMCs cultured on monomer collagen. SMCs replated on vitronectin after culture on monomer collagen result in PAI-1 accumulation at focal adhesions and colocalization with alpha(v)beta3 integrins. In contrast, polymerized collagen inhibits PAI-1 accumulation at focal adhesions when the SMCs are replated on vitronectin. Furthermore, for SMCs cultured on polymerized collagen, platelet-derived growth factor-stimulated migration on vitronectin is enhanced by PAI-1, with its function counteracted by urinary plasminogen activator. Finally, exogenous addition of PAI-1 appears to partly restore platelet-derived growth factor-stimulated alpha(v)beta3-dependent SMC migration that is specifically suppressed by polymerized collagen. CONCLUSIONS: Polymerized type I collagen fibrils dynamically regulate PAI-1, which may be involved in altered alpha(v)beta3 integrin-dependent SMC migration. (+info)
Role of Cys41 in the N-terminal domain of lumican in ex vivo collagen fibrillogenesis by cultured corneal stromal cells.
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The keratan sulphate proteoglycan lumican regulates collagen fibrillogenesis to maintain the integrity and function of connective tissues such as cornea. We examined the role of a highly conserved cysteine-containing domain proximal to the N-terminus of lumican in collagen fibrillogenesis using site-specific mutagenesis to prepare plasmid DNA encoding wild-type murine lumican (Cys(37)-Xaa(3)-Cys(41)-Xaa-Cys-Xaa(9)-Cys) and a Cys-->Ser (C/S) mutant (Cys(37)-Xaa(3)-Ser(41)-Xaa-Cys-Xaa(9)-Cys). cDNAs were cloned into the pSecTag2A vector, and cultures of MK/T-1 cells (an immortalized cell line from mouse keratocytes) were transfected with the cDNAs. Stable transformants were selected and cloned in the presence of Zeocin. All stable transformants maintained a dendritic morphology and growth rate similar to those of parental MK/T-1 cells. Western blot analysis with anti-lumican antibody detected a 42 kDa lumican protein secreted into the culture medium of both wild-type and C/S mutant lumican cell lines. Ultrastructural analyses by transmission electron microscopy showed both cell lines to form a multi-layered stroma ex vivo, but the matrix assembled by the two cell lines differed. Compared with the mutant cell line, the wild-type cells assembled a more organized matrix with regions containing orthogonal collagen fibrils. In addition, the fibrils in the extracellular matrix formed by the mutant cell line exhibited alterations in fibril packing and structure. Immunostaining analysed by confocal microscopy showed a further difference in this matrix, with the marked occurrence of lumican and collagen I co-localization in the lumican wild-type cells, but a lack thereof in the lumican C/S mutant cells. The results indicate that the cysteine-rich domain of lumican is important in collagen fibrillogenesis and stromal matrix assembly. (+info)
Mimecan/osteoglycin-deficient mice have collagen fibril abnormalities.
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PURPOSE: To study the role of mimecan, a member of the small leucine-rich proteoglycans (SLRPs) gene family and one of the major components of the cornea and other connective tissues, mice that lack a functional mimecan gene were generated and characterized. METHODS: Mimecan-deficient mice were generated by gene-targeting using standard techniques. Mice were genotyped by Southern blot analysis. The absence of mimecan transcripts was confirmed by Northern blot analysis. Corneal clarity was examined by slit lamp biomicroscopy. The strength of the skin was evaluated using a biomechanical skin fragility test. Collagen morphology in cornea and skin preparations from mimecan-null and control wild-type mice was analyzed by transmission electron microscopy. The diameter of collagen fibrils in these tissues was determined by morphometric analysis. RESULTS: Mice lacking mimecan appear to develop normally, are viable and fertile. In a controlled laboratory environment they do not display an evident pathological phenotype compared to wild type mice. Examination of corneal clarity and measurements of corneal thickness show no significant changes in the cornea. However, a skin fragility test revealed a moderate reduction in the tensile strength of skin from mutant mice. Ultrastructural analyses show, on average, thicker collagen fibrils in both corneal and skin preparations from mimecan-null mice. Collagen fibrils from the cornea of mutant mice show an average diameter of 31.84+/-0.322 nm, versus 22.40+/-0.296 nm in their wild type litter-mates. The most pronounced increase in collagen fibril diameter was found in the skin of mimecan-null mice, who demonstrated an average diameter of 130.33+/-1.769 nm, versus 78.82+/-1.157 nm in the wild type mice. In addition, size variability and altered collagen morphology was detected in dorsal and tail skin preparations from the mutant mice. CONCLUSIONS: The results of the present study demonstrate that mimecan, similar to other members of the SLRP gene family, has a role in regulating collagen fibrillogenesis in vivo. Further studies, such as functional challenges, an evaluation of potential compensation by other proteins (including members of the SLRP family), and generation of double-knockouts will be necessary to fully uncover physiological functions of mimecan in mice. (+info)
Enhanced expression of heat shock protein 47 in rat model of peritoneal fibrosis.
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OBJECTIVE: Peritoneal fibrosis is one of the serious complications of continuous ambulatory peritoneal dialysis therapy and is characterized by collagen accumulation. Heat shock protein 47 (HSP-47) is a collagen-specific molecular chaperon and is closely associated with collagen synthesis; however, the involvement of HSP-47 in the progression of peritoneal fibrosis is not fully understood. DESIGN: To examine the serial pathological alterations caused by peritoneal fibrosis, we made an experimental model of peritoneal fibrosis by daily intraperitoneal injection of chlorhexidine gluconate (CG) in rats for 28 days and examined the expression of HSP-47 together with that of types I and III collagen, alpha-smooth muscle actin (aSMA), and ED-1 (a marker for macrophages) using immunohistochemistry. Rats treated with saline containing 15% ethanol were used as the control group. RESULTS: In the control group, the peritoneal tissue was slightly thickened and HSP-47 was expressed in the peritoneum at day 28. In the CG group, the peritoneal tissue serially became thickened and fibrotic. The expression of HSP-47 was evident in mesothelial cells and submesothelial connective tissue after day 7 of treatment with CG, and increased thereafter. The expression of types I and III collagen and aSMA was proportionally strengthened during our experiments. ED-1-positive cells were present in thickened areas with abundant proliferation of collagen fiber. The number of cells positive for ED-1 increased gradually and reached a maximum at day 21. CONCLUSION: Our results indicate that, in a rat experimental model of peritoneal fibrosis, the expression of HSP-47 is associated with the progression of peritoneal fibrosis. (+info)
Elucidation of the potential roles of matrix metalloproteinases in skeletal biology.
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Irreversible destruction of joint structures is a major feature of osteoarthritis and rheumatoid arthritis. Fibrillar collagens in bone, cartilage and other soft tissues are critical for optimal joint form and function. Several approaches can be used to ascertain the role of collagenases, matrix metalloproteinases, in proteolysis of joint collagens in arthritis. These approaches include identifying spontaneous genetic disorders of the enzymes and substrates in humans and animals, as well as engineering mutations in the genes that encode these proteins in mice. Insights gained from such studies can be used to design new therapies to interrupt these catabolic events. (+info)