Collagen type I modulates the platelet-derived growth factor (PDGF) regulation of the growth and expression of beta1 integrins by rat mesangial cells

Mesangial cell (MC) proliferation and the deposition of collagen type I (collagen I) are the major pathological features in many types of glomerulonephritis (GN). Recent work suggested that beta-integrins play a critical role in the cell proliferation and extracellular matrix (ECM) remodeling observed in tissue repair after injury. To examine the involvement of beta-integrins in MC proliferation in association with the interaction of MCs with pathological collagen I, we investigated the effect of a prominent mitogen, platelet-derived growth factor-BB (PDGF-BB) on the growth and expression of beta-integrins by MCs cultured on plastic or in a three-dimensional collagen I gel. Immunoprecipitation using 35S-metabolic labeling, flow cytometry and a 3H-thymidine-uptake analysis demonstrated that PDGF-BB stimulated the cell mitogenicity and the expression of alpha5beta1 integrin (a fibronectin receptor), but not alpha1beta1 integrin (a collagen and laminin receptor) of MCs on plastic, in a dose-dependent manner. In contrast, MCs in the collagen I gels showed no significant changes in mitogenicity or alpha1beta1 and alpha5beta1 integrin expression, but increased alpha1beta1 integrin-mediated gel contraction was observed after PDGF-BB stimulation. Thus, the parallel up-regulation of MC-mitogenicity and alpha5beta1 integrin expression by PDGF-BB suggested that alpha5beta1 integrin is an important ECM receptor involved in the proliferative phenotype of MC. A spatial interaction between MCs and pathological collagen I in GN may influence the PDGF regulation of the MC phenotype regarding the cell growth and the expression of beta1 integrins.     

Neonatal estrogen stimulates proliferation of periductal fibroblasts and alters the extracellular matrix composition in the rat prostate

The purpose of this study was to examine whether changes in extracellular matrix (ECM) molecules are associated with the growth inhibition and differentiation defects of the prostate gland following neonatal exposure to estradiol. Using immunocytochemistry (ICC), laminin and collagen IV were localized to the basement membrane (BM) as well to the basal lamina of the periductal smooth muscle of the control developing prostates. In contrast, fibronectin and collagen III were localized throughout the stromal ECM. Exposure to neonatal estrogen altered the staining profile for specific ECM molecules. In the estrogenized rats, a thick layer of cells negative for laminin and collagen IV was observed adjacent to the BM. Electron microscopy and ICC for alpha-actin, fibronectin, and vimentin identified this multicellular layer of periductal cells as differentiated fibroblasts. Peripheral to these fibroblasts, actin-positive smooth muscle formed a second layer of periductal stromal cells. PCNA labeling showed that estrogen exposure increased the fibroblast proliferation. Because many periductal fibroblasts were positive for estrogen receptor alpha (ER alpha) in estrogenized rats, a direct effect of estradiol on their proliferation is suggested. Gelatinolytic gels revealed that estrogen exposure did not alter the activity of matrix metalloproteinases associated with tissue remodeling during prostate morphogenesis. However, the periductal fibroblast layer in estrogenized prostates was devoid of urokinase- and tissue-plasminogen activator, which may potentially alter the localized proteolysis involved in matrix remodeling. It is proposed that proliferation of a multicellular layer of periductal fibroblasts in estrogenized prostates results in a physical barrier that constrains branching morphogenesis and blocks paracrine communications between smooth muscle and epithelial cells which normally regulate differentiation.     

Studies in vitro on the role of alpha v and beta 1 integrins in the adhesion of human dermal fibroblasts to provisional matrix proteins fibronectin, vitronectin, and fibrinogen

Fibroblasts that migrate into a wound during the early stages of repair use cell surface integrins to interact with extracellular molecules as they move away from the interstitial matrix of normal tissue and into the provisional matrix of the wound. Therefore, to understand a critical phase of wound healing, it is necessary to understand the details of integrin involvement. Normal adult human dermal fibroblasts in culture express many receptors for the provisional matrix proteins fibronectin, vitronectin, and fibrinogen, including the integrins alpha 3 beta 1, alpha 4 beta 1, alpha 5 beta 1, alpha v beta 1, alpha v beta 3, and alpha v beta 5. We used quantitative flow cytometry to estimate the relative numbers of these receptors and immunoprecipitation to confirm the expression of alpha v beta 1. Adult human dermal fibroblasts primarily use beta 1 integrins, alpha 4 beta 1, alpha 5 beta 1, and possibly alpha v beta 1, for attachment to fibronectin. alpha v beta 3 and perhaps other integrins containing the alpha v subunit serve fibroblasts as secondary or auxiliary receptors for fibronectin. In contrast, these cells use alpha v integrins but probably not beta 1 integrins for attachment to vitronectin. alpha v beta 3 and alpha v beta 5 apparently act in concert to mediate attachment to vitronectin, and these two integrins may perform different functions during wound repair. Fibroblast adhesion to certain preparations of fibrinogen occurs, at least partially, through the small amount of fibronectin present in the preparations. Fibroblast attachment to fibrinogen purified free of fibronectin also occurs, and that was demonstrated with a sensitive new assay called electrical cell-substrate impedance sensing. Fibroblast attachment to pure fibrinogen can be inhibited by RGD peptide, suggesting that integrins are involved.     

Discordant growth in twin pregnancy--value of Doppler ultrasound

Fibroblast accumulation in a cutaneous wound requires phenotypic modulation of fibroblasts. In response to injury, resident fibroblasts in the surrounding tissue proliferate for the first 3 days and then at day 4 migrate into the wounded site. Once within the wound, they produce type I procollagen as well as other matrix molecules and deposit these extracellular matrix molecules in the local milieu. By day 7, abundant extracellular matrix has accumulated and fibroblasts switch to a myofibroblast phenotype replete with actin bundles along the cytoplasmic face of the plasma membrane. Wound contraction occurs as these myofibroblast gather in the wound extracellular matrix by extending pseudopodia, attaching to extracellular matrix molecules, such as fibronectin and collagen, then retracting the pseudopodia. Once these processes have been accomplished, the fibroblasts appear to undergo apoptosis. Therefore, during cutaneous wound repair, fibroblasts appear to progress through four phenotypes: first proliferating, second migrating, third synthesizing extracellular matrix molecules, and fourth expressing thick actin bundles as myofibroblasts.     

Boron modulates extracellular matrix and TNF alpha synthesis in human fibroblasts

Boric acid was not mitogenic for human fibroblasts and it did not change cell viability until 0.5% (w/v). Boric acid treatment affected the metabolism of human dermal fibroblasts in culture, decreasing the synthesis of extracellular matrix macromolecules such as proteoglycans, collagen, and total proteins. It also increased the release of these molecules into the culture medium. The principal proteins secreted into the medium after boric acid treatment had molecular masses of 90, 70, 58, 49, and 43 kDa and faint bands were detected by electrophoresis between 14 and 30 kDa. hsp 70 and TNF alpha were detected among the secreted proteins by immunoblotting, and the amount of TNF alpha released was quantified by radioimmunoassay. Total mRNA levels were higher after boric acid treatment and peaked after 6 h of treatment. TNF alpha mRNA was undetectable in unstimulated fibroblasts and two TNF alpha mRNA bands were detected after stimulation: immature mRNA (4.8 kb) and mature TNF alpha mRNA (1.9 kb). Thus, the effects of boric acid observed in wound repair in vivo may be due to TNF alpha synthesis and secretion.     

Control of fibrosis in systemic scleroderma

Scleroderma is characterized by an excessive deposition of collagen in all involved organs. This is due to an overproduction of extracellular matrix (ECM) molecules following induction of gene expression, whereas there is no evidence that the composition of the connective tissue matrix is altered. Several in vivo studies and in vitro experiments suggest that a close interaction between inflammatory cells and fibroblasts is required for the initial activation of fibroblasts. TGF-beta presumably plays an important role, but other cytokines, e.g., PDGF or FGF, may also be involved. Many of the ECM molecules have been shown to interact closely with fibroblasts and provide signals that regulate fibroblast metabolism. The cellular response towards those signals is a further aspect of fibrosis that has attracted attention during recent years. The altered expression of receptor proteins on the cell surface of scleroderma fibroblasts for example might explain in part the lack of down-regulation of collagen synthesis in late phases of the disease. This review summarizes the alterations of connective tissue in scleroderma, and discusses the role of cytokines as well as the ECM for the regulation of fibroblast function and their implication for the development of fibrosis.     

Extracellular matrix in renal cell carcinomas

Extracellular matrix (ECM) may be divided into interstitial matrix and the basement membrane (BM). ECM influences a variety of epithelial cell behaviours, including proliferation, differentiation, and morphogenesis, maybe most widely studied in kidney morphogenesis. In carcinomas, including renal cell carcinomas (RCCs), these properties and interactions of cells with interstitial matrix and BM are disturbed. As a carcinoma with a tendency to spread to distant sites, RCC is an interesting target for the study of epithelial-stromal interactions. Among interstitial collagens, type VI collagen appears to be widely distributed in RCCs. Also EDA-fibronectin (EDA-Fn) as well as tenascin-C (Tn) are important stromal components especially in poorly differentiated carcinomas. BMs of RCC islets and those of tumor blood vessel endothelia may merge in poorly differentiated carcinomas. As a dynamic component of BMs, laminins (Ln) are important in kidney development and RCC progression. Type IV collagen and nidogen, other components of BMs in RCCs, are produced by stromal as well as epithelial cells. ECM proteins may function in RCC progression by binding and regulating the activity of growth factors e.g. transforming growth factor beta 1 and basic fibroblast growth factor. Also the expression of cell surface receptors for ECM is disturbed in RCCs. At least alpha v integrin (Int) and CD44 emerge in renal epithelial cells during malignant transformation. Papillary renal neoplasms differ from RCCs by cell adhesion receptor expression and BM composition as well as by ECM avascularity and capacity to bind growth factors, thus suggesting a distinct property for this renal tumor.     

Regulated production of type I collagen and inflammatory cytokines by peripheral blood fibrocytes

We recently described a novel population of blood-borne cells, termed fibrocytes, that display a distinct cell surface phenotype (collagen+/CD13+/CD34+/CD45+), rapidly enter sites of tissue injury, and contribute to scar formation. To further characterize the role of these cells in vivo, we examined the expression of type I collagen and cytokine mRNAs by cells isolated from wound chambers implanted into mice. Five days after chamber implantation, CD34+ fibrocytes but not CD14+ monocytes or CD90+ T cells expressed mRNA for type I collagen. Fibrocytes purified from wound chambers also were found to express mRNA for IL-1beta, IL-10, TNF-alpha, JE/MCP, MIP-1alpha, MIP-1beta, MIP-2, PDGF-A, TGF-beta1, and M-CSF. The addition of IL-1beta (1-100 ng/ml), a critical mediator in wound healing, to fibrocytes isolated from human peripheral blood induced the secretion of chemokines (MIP-1alpha, MIP-1beta, MCP-1, IL-8, and GRO alpha), hemopoietic growth factors (IL-6, IL-10, and macrophage-CSF), and the fibrogenic cytokine TNF-alpha. By contrast, IL-1beta decreased the constitutive secretion of type I collagen as measured by ELISA. Additional evidence for a role for fibrocytes in collagen production in vivo was obtained in studies of livers obtained from Schistosoma japonicum-infected mice. Mouse fibrocytes localized to areas of granuloma formation and connective matrix deposition. We conclude that fibrocytes are an important source of cytokines and type I collagen during both the inflammatory and the repair phase of the wound healing response. Furthermore, IL-1beta may act on fibrocytes to effect a phenotypic transition between a repair/remodeling and a proinflammatory mode.     

Enhancement of cell interactions with collagen/glycosaminoglycan matrices by RGD derivatization

The interaction of three cell types important to the wound repair process with collagen/glycosaminoglycan (GAG) dermal regeneration matrices covalently modified with an Arg-Gly-Asp (RGD)-containing peptide was characterized. Function-blocking monoclonal antibodies directed against various integrin subunits were used to demonstrate that human fibroblasts attached to the unmodified matrix through the integrin, alpha2beta1. Human endothelial cells and human keratinocytes, however, attached minimally to the unmodified matrix. After modification of the collagen/GAG matrix with RGD-containing peptide, endothelial cells and keratinocytes attached and spread well on the matrix. This attachment was RGD dependent as evidenced by essentially complete inhibition with competing soluble peptide. In terms of overall cell number, fibroblast cell attachment remained unchanged on the RGD peptide-modified matrix compared to the unmodified material. Antibody and peptide inhibition studies demonstrate, however, that attachment to the modified matrix was mediated by both alpha2beta1 and RGD-binding integrins. We have successfully introduced a specific RGD receptor-mediated attachment site on collagen/GAG dermal regeneration matrices, resulting in enhanced cell interaction of important wound healing cell types. This modification could have important implications for the performance of these matrices in promoting dermal regeneration.     

TGF-beta1 influences early gingival wound healing in rats: an immunohistochemical evaluation of stromal remodelling by extracellular matrix molecules and PCNA

The effect of topically applied transforming growth factor beta1 (TGF-beta1) on the rat gingival wound healing process after flap surgery was evaluated by immunohistochemistry for extracellular matrix molecules (ECM), such as tenascin, heparan sulfate proteoglycan (HSPG) and type IV collagen, and for proliferating cell nuclear antigen (PCNA) in fibroblasts. TGF-beta1 solution was applied to the surgical wound experimental sites. Two microg/microl were applied at the time of the operation, and 1 microg/microl at days 1 and 2 after surgery, with contralateral control sites receiving the vehicle alone. Periodontal tissues were histologically examined at 3 and 7 days post-surgery. Tenascin was found to be more strongly stained in the granulation tissue from experimental sites at 3 days post-surgery. At 7 days postsurgery, HSPG-positive areas in granulation tissue had become smaller and there was a prominent proliferation of PCNA-positive fibroblast-like cells and type IV collagen-positive blood vessels. These results suggest that TGF-beta1 applied to surgical wounds influences early proliferation of gingival fibroblast-like cells, the formation of blood vessels, and ECM remodelling. In conclusion, TGF-beta1 application appears to promote granulation tissue formation in periodontal wound healing.     

The reversed-flow medio-distal fasciocutaneous island thigh flap: anatomic basis and clinical applications

To investigate changes in retinal pigment epithelial (RPE) cells during wound healing, we evaluated the deposition of newly synthesized extracellular matrix (ECM) over time during wound healing in rat RPE cultures. We also estimated the effect of growth factors on the healing rate and ECM synthesis. After preparing rat RPE cell sheet cultures, we made round 1-mm defects in the cultures. Fibronectin, laminin, and collagen IV synthesis were evaluated with immunocytochemistry every 12 hours after wounding. S-phase cell distribution was analyzed every 12 hours by 5-bromodeoxyuridine uptake. We added either platelet-derived growth factor (PDGF), epidermal growth factor (EGF), or transforming growth factor- beta2 (TGF-beta2) to cultures at concentrations of 1, 10, and 100 ng/mL and immunocytochemically analyzed the effects on ECM and estimated the rate of wound closure. Although approximately 50% closure was achieved 24 hours after wounding, fibronectin deposits first appeared at that time. Laminin and collagen IV were first detected at 36 hours and fibronectin staining had extended toward the wound center. S-phase cells were distributed in concentric rings that moved centripetally over time and corresponded to the leading edge of the area stained with anti-ECM antibodies. TGF-beta2 enhanced ECM deposition, but EGF and PDGF did not. TGF-beta2 decreased the healing rate in a dose-dependent manner, whereas PDGF promoted wound closure. EGF enhanced closure at the highest concentration only. In summary, wound healing in RPE may be initiated when cells at the wound edge slide or migrate toward the wound center, which is followed by cell proliferation and then ECM synthesis. ECM components may be produced in a specific sequence during healing. TGF-beta2 may promote RPE cell differentiation, and PDGF may enhance proliferation during wound healing of the RPE.     

MIP-1alpha as a critical macrophage chemoattractant in murine wound repair

At sites of injury, macrophages secrete growth factors and proteins that promote tissue repair. While this central role of the macrophage has been well studied, the specific stimuli that recruit macrophages into sites of injury are not well understood. This study examines the role of macrophage inflammatory protein 1alpha (MIP-1alpha), a C-C chemokine with monocyte chemoattractant capability, in excisional wound repair. Both MIP-1alpha mRNA and protein were detectable in murine wounds from 12 h through 5 d after injury. MIP-1alpha protein levels peaked 3 d after injury, coinciding with maximum macrophage infiltration. The contribution of MIP-1alpha to monocyte recruitment into wounds was assessed by treating mice with neutralizing anti-MIP-1alpha antiserum before injury. Wounds of mice treated with anti-MIP-1alpha antiserum had significantly fewer macrophages than control (41% decrease, P < 0. 01). This decrease in wound macrophages was paralleled by decreased angiogenic activity and collagen synthesis. When tested in the corneal micropocket assay, wound homogenates from mice treated with anti-MIP-1alpha contained significantly less angiogenic activity than control wound homogenates (27% positive for angiogenic activity versus 91% positive in the control group, P < 0.01). Collagen production was also significantly reduced in the wounds from anti-MIP-1alpha treated animals (29% decrease, P < 0.05). The results demonstrate that MIP-1alpha plays a critical role in macrophage recruitment into wounds, and suggest that appropriate tissue repair is dependent upon this recruitment.     

Inhibition of glomerular mesangial cell proliferation and extracellular matrix deposition by halofuginone

Mesangial cell proliferation, increased deposition of collagen, and expansion of the mesangial extracellular matrix (ECM) are key features in the development of mesangioproliferative diseases. Halofuginone, a low molecular weight anti-coccidial quinoazolinone derivative, inhibits collagen type alpha 1(I) gene expression and synthesis. We investigated the effect of halofuginone on both normal and SV40 transformed mesangial cell proliferation, collagen synthesis, and ECM deposition. Proliferation of both cell types was almost completely inhibited in the presence of 50 ng/ml halofuginone. The cells were arrested in the late G1 phase of the cell cycle and resumed their normal growth rate following removal of the compound from the culture medium. The antiproliferative effect of halofuginone was associated with inhibition of tyrosine phosphorylation of cellular proteins. Similar results were obtained whether the mesangial cells were seeded on regular tissue culture plastic or in close contact with a naturally produced ECM resembling their local environment in vivo. Halofuginone also inhibited synthesis and deposition of ECM by mesangial cells as indicated by a substantial reduction in 14C-glycine and Na2(35)SO4 incorporation into the ECM, and by the inhibition of collagen type I synthesis and gene expression. It is proposed that by inhibiting collagen type I synthesis and matrix deposition, halofuginone exerts a potent antiproliferative effect that may be applied to inhibit mesangial cell proliferation and matrix expansion in a variety of chronic progressive glomerular diseases.     

Increase in the expression of biglycan mRNA expression Co-localized closely with that of type I collagen mRNA in the infarct zone after experimentally-induced myocardial infarction in rats

Biglycan, a small dermatan sulphate proteoglycan, has been postulated to interact with other components of the extracellular matrix (ECM), specifically collagens. We hypothesized that biglycan messenger ribonucleic acid (mRNA) is increased in the myocardial infarct zone. Biglycan mRNA expression after acute myocardial infarction (AMI) in rats was determined with the use of Northern blotting and in situ hybridization, and its expression pattern was compared to that of type I collagen mRNA [alpha1(I) collagen]. The left coronary artery was ligated in male Sprague-Dawley rats, and the hearts were excised on days 2 and 7. The Northern blot analysis demonstrated that expression of biglycan mRNA in the infarct on days 2 and 7 were 4.0- and 6.8-fold higher, respectively, compared to the sham-operated hearts. The in situ hybridization revealed intense signals for both biglycan and alpha1(I) collagen mRNA on day 2 in the spindle-shaped mesenchymal cells located between the surviving myocytes in the infarct peripheral zone. On day 7, biglycan mRNA signals were observed in the interior of the infarct around the infarct granulation tissue, a distribution that was essentially the same as that of alpha1(I) collagen. These results demonstrated that the increases in the infarct biglycan mRNA expression produced by mesenchymal cells (presumably myofibroblasts and fibroblasts) was closely co-localized with that of type I collagen mRNA, indicating that biglycan contributes to the infarct healing processes.     

Radiation-induced alteration of rat mesangial cell transforming growth factor-beta and expression of the genes associated with the extracellular matrix

We hypothesized that the altered mesangial cell phenotype observed in radiation nephropathy reflects, at least partly, radiation-induced changes in expression of the genes associated with transforming growth factor-beta (TGF-beta) and extracellular matrix (ECM). To test this hypothesis, rat mesangial cells were used between passages 7 and 11 after primary isolation from glomeruli. Cells were placed in serum-free medium 24 h prior to irradiation and irradiated with single doses of 5-20 Gy 137Cs gamma rays; control cells received sham irradiation. After irradiation, the cells were maintained in serum-free medium for up to 48 h postirradiation. Total RNA was isolated, and Northern analysis was performed using cDNA probes for TGF-beta 1, beta 2 and beta 3 and several ECM genes. Irradiation resulted in isoform-specific alterations in TGF-beta mRNA; TGF-beta 1 levels showed a dose-independent increase 24-48 h postirradiation; TGF-beta 3 mRNA levels showed a progressive dose-independent decrease over the same period, decreasing to levels approximately 25% of those seen in controls. These changes were associated with a concomitant increase in levels of mRNA expressed by genes for the components of the ECM; no changes were observed in TGF-beta 2, collagen I, collagen III or decorin. Thus radiation can alter mesangial cell TGF-beta and the expression of the genes involved in ECM, although the nature of this alteration varies for the TGF-beta isoforms and specific ECM genes.     

Integrin alpha 2 beta 1 is a positive regulator of collagenase (MMP-1) and collagen alpha 1(I) gene expression

A classical model for studying the effects of extracellular matrix is to culture cells inside a three-dimensional collagen gel. When surrounded by fibrillar collagen, many cell types decrease the production of type I collagen, and the expression of interstitial collagenase (matrix metalloproteinase-1; MMP-1) is simultaneously induced. To study the role of the collagen-binding integrins alpha 1 beta 1 and alpha 2 beta 1 in this process, we used three different osteogenic cell lines with distinct patterns of putative collagen receptors: HOS cells, which express only alpha 1 beta 1 integrin, MG-63 cells, which express only alpha 2 beta 1 integrin, and KHOS-240 cells, which express both. Inside collagen gels, alpha 1 (I) collagen mRNA levels were decreased in HOS and KHOS-240 cells but not in MG-63 cells. In contrast, MMP-1 expression was induced in KHOS-240 and MG-63 cells but not in HOS cells. Transfection of MG-63 cells with alpha 2 integrin cDNA in an antisense orientation reduced the expression level of alpha 2 integrin. These cell clones showed induction and reduction of mRNA levels for MMP-1, respectively. HOS cells normally lacking alpha 2 beta 1 integrin were forced to express it, and this prevented the down-regulation in the levels of alpha 1 (I) collagen mRNA when cells were grown inside collagen gels. The data indicate that the level of MMP-1 expression is regulated by the collagen receptor alpha 2 beta 1 integrin. The down-regulation of collagen alpha 1 (I) is mediated by another receptor. Integrin alpha 2 beta 1 may compete with it and thus be a positive regulator of collagen synthesis.