Extracellular Matrix Contraction Research Articles

Matrix Contraction by Dermal Fibroblasts Requires Transforming Growth Factor-β/Activin-Linked Kinase 5, Heparan Sulfate-Containing Proteoglycans, and MEK/ERK: Insights into Pathological Scarring in Chronic Fibrotic Disease

Scarring is characterized by excessive synthesis and contraction of extracellular matrix. Here, we show that fibroblasts from scarred (lesional) areas of patients with the chronic fibrotic disorder diffuse scleroderma [diffuse systemic sclerosis (dSSc)] show an enhanced ability to adhere to and contract extracellular matrix, relative to fibroblasts from unscarred (nonlesional) areas of dSSc patients and dermal fibroblasts from normal, healthy individuals. The contractile abilities of normal and dSSc dermal fibroblasts were suppressed by blocking heparin sulfate-containing proteoglycan biosynthesis or antagonizing transforming growth factor-beta receptor type I [activin-linked kinase (ALK5)] or ras/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK). Compared with both normal and nonlesional fibroblasts, lesional dSSc fibroblasts overexpressed the heparin sulfate-containing proteoglycan syndecan 4. We also found that the procontractile signals from transforming growth factor (TGF)-beta were integrated through syndecan 4 and MEK/ERK because the ability of TGFbeta to induce contraction of dermal fibroblasts was prevented by MEK antagonism. TGFbeta could not induce a contractile phenotype or phosphorylate ERK in syndecan 4(-/-) dermal fibroblasts. These results suggest that integrating TGFbeta and ERK signals via syndecan 4 is essential for the contractile ability of dermal fibroblasts. We conclude that antagonizing MEK/ERK, TGFbeta1/ALK5, or syndecan 4 may alleviate scarring in chronic fibrotic disease.

Distinct expression profile in fumarate-hydratase-deficient uterine fibroids

Defects in mitochondrial enzymes predispose to severe developmental defects as well as tumorigenesis. Heterozygous germline mutations in the nuclear gene encoding fumarate hydratase (FH), an enzyme catalyzing the hydration of fumarate in the Krebs tricarboxylic acid cycle, cause hereditary leiomyomatosis and renal cell cancer; yet the connection between disruption of mitochondrial metabolic pathways and neoplasia remains to be discovered. We have used an expression microarray approach for studying differences in global gene expression pattern caused by mutations in FH. Seven uterine fibroids carrying FH mutations were compared with 15 fibroids with wild-type FH. The two groups showed markedly different expression profiles, and multiple differentially expressed genes were detected. The most significant increase in FH mutants was seen in the expression of carbohydrate metabolism- and glycolysis-related genes. Other significantly up-regulated gene categories in FH mutants were, for example, iron ion homeostasis and oxidoreduction. Genes with lower expression in FH-mutant fibroids belonged to groups such as extracellular matrix, cell adhesion, muscle development and cell contraction. We show that FH mutations alter significantly the expression profiles of fibroids, most strikingly increasing the expression of genes involved in glycolysis.

Mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) pathways differently regulate retinal pigment epithelial cell-mediated collagen gel contraction

Retinal pigment epithelial (RPE) cell-mediated extracellular matrix contraction is believed to contribute to developing proliferative vitreoretinopathy. It has been shown that platelet-derived growth factor (PDGF) and its intracellular signaling pathway, including mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K), are mainly involved in this process. The aim of this study is to investigate how these downstream signaling pathways are related to RPE-mediated collagen gel contraction. We performed the gel contraction assay to evaluate the effect of PDGF in cultured ARPE-19 cells under the presence or absence of PD98059, MAPK inhibitor or wortmannin, PI3K inhibitor. Experiments treated with neutralizing antibody for various subtypes of integrin were also performed and the effect on PDGF-induced gel contraction was investigated. Expression changes of integrin α1, α2 and β1 after PDGF stimulation was evaluated using quantitative real-time PCR and flow cytometry. The results showed that PDGF up-regulated ARPE-19 cell-mediated gel contractile activity. PDGF-induced collagen gel contraction was attenuated under presence of PD98059, wortmannin, or neutralizing antibody for integrin α1, α2, or β1, all of which are critical subset for binding with type I collagen. The expression of integrin α1 and α2 was increased after PDGF stimulation in both real-time PCR and flow cytometry, however β1 expression was not increased. PD98059 significantly attenuated integrin α1 and α2 expressions. However, wortmannin did not have the same effect. In conclusion, PDGF promotes ARPE-19 cell-mediated gel contraction via both MAPK and PI3K. This was probably due to an increased expression of integrin α1 and α2, which is mediated by MAPK, but not by PI3K. PI3K may regulate collagen gel contraction by another mechanism other than the up-regulation of integrin expression.

Fibronectin fragmentation promotes α4β1 integrin-mediated contraction of a fibrin–fibronectin provisional matrix

In injured tissues, the fibrin–fibronectin (FN) provisional matrix provides a framework for cell adhesion, migration, and repair. Effective repair and remodeling require a proper balance between extracellular matrix (ECM) deposition, contraction, and turnover. We utilized a three-dimensional (3D) fibrin–FN provisional matrix model to determine the contributions of the FN-binding integrin receptors α5β1 and α4β1 to matrix contraction. CHOα5 cells expressing α5β1, a receptor for FN's RGD cell-binding domain, were highly contractile, and cells were well spread on a 3D fibrin–FN matrix. In contrast, CHOα4 cells expressing the α4β1 receptor for FN's alternatively spliced V region attached less efficiently to FN and were deficient in fibrin–FN matrix contraction. Surprisingly, cell adhesion and matrix contraction by CHOα4 cells were dramatically enhanced, to levels equivalent to CHOα5 cells, when proteolyzed FN was used in place of intact FN in the fibrin–FN matrix. Similar enhancement was observed when ligand binding by α4β1 integrins was activated by treatment with Mn ++, but not by stimulation of actin organization with LPA. Therefore, α4β1-dependent cell responses to the provisional matrix are modulated by cleavage of matrix components.

Extracellular matrix changes in stented human coronary arteries.

Restenosis after stenting occurs secondary to the accumulation of smooth muscle cells (SMCs) and extracellular matrix (ECM), with the ECM accounting for >50% of the neointimal volume. The composition of the in-stent ECM has not been well characterized in humans. Postmortem human coronary arteries (n=45) containing stents underwent histological assessment of neointimal proteoglycans, hyaluronan, collagen (types I and III), SMCs, and CD44 (a cell surface receptor for hyaluronan). The mean duration of stent implantation was 18.7 months; stents in place > or =3 to <9 months (n=17) were assigned to group 1, stents > or =9 to <18 months old (n=19) to group 2, and stents > or =18 months old (n=9) to group 3. In groups 1 and 2, neointimal versican and hyaluronan staining was strongly positive, colocalized with alpha-actin-positive SMCs, and was greater in intensity compared with group 3. Conversely, decorin staining was greatest in group 3. The neointima of both group 1 and 2 stents was rich in type III collagen, with reduced staining in group 3. Type I collagen staining was weakest in group 1 stents, with progressively stronger staining in groups 2 and 3. SMC density and stent stenosis were significantly reduced in group 3 stents compared with groups 1 and 2. CD44 staining colocalized with macrophages and was associated with increased neointimal thickness. The ECM within human coronary stents resembles a wound that is not fully healed until 18 months after deployment, followed by neointimal retraction. ECM contraction may be a target for therapies aimed at stent restenosis prevention.

TGF-beta signaling and the fibrotic response.

The cause of fibrotic diseases, pathologies characterized by excessive production, deposition, and contraction of extracellular matrix, is unknown. To understand the molecular basis of fibrotic disease, it is essential to appreciate how matrix deposition is normally controlled and how this process is dysregulated in fibrogenesis. This review discusses the current state of knowledge concerning interactions among the profibrotic proteins transforming growth factor-beta (TGF-beta), connective tissue growth factor (CTGF, CCN2), and ED-A fibronectin (ED-A FN) and the antifibrotic proteins tumor necrosis factor-alpha (TNF-alpha) and gamma-interferon (IFN-gamma).

Modulation of cultured corneal keratocyte phenotype by growth factors/cytokines control in vitro contractility and extracellular matrix contraction

The purpose of this study was to evaluate specific keratocyte phenotypes (keratocyte, fibroblast, myofibroblast) for cell contractility and ability to contract extracellular matrix. Rabbit keratocyte phenotype was modulated by exposure to optimal proliferative doses of IGF-I, IL-1α, FGF2, PDGF-AB, and TGFβ 1. Cells were then evaluated by immunocytochemistry, western blot, collagen gel contraction and LPA stimulation to measure: (1) focal adhesion (FA), fibronectin (FN) and f-actin assembly; (2) expression of α-smooth muscle actin (α-SMA); (3) ability to contract extracellular matrix and (4) determine contractile ability, respectively. Untreated keratocytes showed no ability to contract collagen matrix. IGF-I and IL-1α increased cell proliferation (70·2 and 74·3%, respectively) but did not alter keratocyte phenotype or ability to contract matrix. FGF2 and PDGF induced fibroblast differentiation with FA and FN assembly and significant ( p<0·05) extracellular matrix contraction. TGFβ 1 induced myofibroblast differentiation with prominent FA and FN assembly, expression of α-SMA and significantly greater ( p<0·05) matrix contraction. Addition of LPA induced actin filament assembly in growth factor starved fibroblasts and myofibroblasts but had no effect on the cultured keratocyte phenotype. We report for the first time that the keratocyte phenotype is non-contractile and that cell quiescence is not a defining characteristic. We further establish that changes in environmental conditions modulate the keratocyte phenotype resulting in physiologically functional differences regarding cell contractility and capacity to contract extracellular matrix.

α-Smooth muscle actin is expressed by fibroblastic cells of the ovine anterior cruciate ligament and its free tendon graft during remodeling

Contractile fibroblastic cells expressing the α-smooth muscle actin isoform, so-called myofibroblasts, have been identified to play a possible role during the healing of the medial collateral ligament by means of restoring the tissues in situ strain via extracellular matrix contraction. Recently, these cells have also been identified to be a normal part of the human anterior cruciate ligament. It has been hypothesized that myofibroblasts play a role in the wrinkling of the extracellular matrix. The goal of the present study was to identify myofibroblasts in the intact ovine anterior cruciate ligament and a free autologous tendon graft during remodeling after anterior cruciate ligament reconstruction. In 36 mature merino sheep the anterior cruciate ligament was replaced with an ipsilateral Achilles tendon split graft. Midsubstance tissue samples were immunostained for α-smooth muscle actin at 6, 9, 12, 24, 52, and 104 weeks. Myofibroblasts were identified in the intact ovine anterior cruciate ligament as well as in the Achilles tendon graft prior to implantation. During remodeling the first myofibroblasts were found at six weeks within newly formed fiber bundles. At 24, 52, and 104 weeks myofibroblast distribution and cell density were similar to those of the intact ovine anterior cruciate ligament. These findings indicate that α-smooth muscle actin containing fibroblastic cells are a regular part of the intact as well as the remodeled anterior cruciate ligament. There is evidence that myofibroblasts may be involved in maintaining tissue homeostasis in the mature ligament e.g., by means of crimp formation. The presence of these cells during the early remodeling may further indicate that α-smooth muscle actin containing fibroblastic cells are involved in the earliest stages of fiber bundle formation. The role and function of this special cell type for the anterior cruciate ligament needs to be further clarified.

Regulation of α-smooth muscle actin gene expression in myofibroblast differentiation from rat lung fibroblasts

Myofibroblasts express α-smooth muscle actin and have a phenotype intermediate between fibroblasts and smooth muscle cells. Their emergence can be induced by cytokines such as transforming growth factor β; but the regulatory mechanism for induction of α-smooth muscle actin gene expression in myofibroblast differentiation has not been determined. To examine this mechanism at the level of the α-smooth muscle actin promoter, rat lung fibroblasts were transfected with varying lengths of the α-smooth muscle actin promoter linked to the chloramphenicol acetyl transferase reporter gene and treated with transforming growth factor β1. The results show that the shortest inducible promoter was 150 base pairs long, suggesting the presence in this region of cis-elements of potential importance in transforming growth factor β1 induced myofibroblast differentiation. Transfection of “decoy” oligonucleotides corresponding to sequences for four suspected regulatory factors demonstrated that only the transforming growth factor β control element is involved in the regulation of transforming growth factor β1-induced α-smooth muscle actin expression in myofibroblast differentiation. Consistent with this conclusion is the finding that a mutation in the transforming growth factor β control element caused a significant reduction in promoter activity. These observations taken together show that α-smooth muscle actin promoter regulation during myofibroblast differentiation is uniquely different from that in smooth muscle cells and other cell lines. Since myofibroblasts play a key role in wound contraction and synthesis of extracellular matrix, clarification of this differentiation mechanism should provide new insight into fibrogenesis and suggest future novel strategies for modulation of wound healing and controlling fibrosis.

Effect of vitamin C and its derivatives on collagen synthesis and cross‐linking by normal human fibroblasts

Vitamin C (VitC) plays a critical role in the maintenance of a normal mature collagen network in humans (anti-scurvy properties) by preventing the auto-inactivation of lysyl and prolyl hyroxylase, two key enzymes in collagen biosynthesis. In this study two in vitro models were designed to evaluate the effects of VitC on collagen biosynthesis and cross-linking at cellular and tissue levels. It was shown that VitC induced a dose-dependent increase in collagen type I deposits by normal human fibroblasts (NHF) cultured in monolayer, and enhanced extracellular matrix contraction by NHF in a lattice model, in a non-cytotoxic range of concentrations (103m, 104m, 105m). Exogenous VitC supply could thus contribute to the maintenance of optimal collagenic density in the dermis and locally strengthen the collagen network. Vitamin C-phosphate (VitC-P) and vitamin C-glucoside (VitC-Glu) (two VitC derivatives presenting higher chemical stability in aqueous solution) were also tested in our two models, and showed similar biological properties, but with different potencies. These two compounds can be considered as pro-vitamins for skin, and could thus advantageously substitute for VitC in VitC-based anti-ageing products, as they allow the development of stable, easy-to-use formulations.

Retinal pigment epithelial cells: autocrine and paracrine stimulation of extracellular matrix contraction.

This study was carried out to examine the biological activity of contraction promoters produced by dedifferentiating retinal pigment epithelial cells (RPE) and to evaluate the importance of autocrine and paracrine effects within a semi-closed environment like the vitreal cavity. RPE at different stages of dedifferentiation in culture were examined for their ability (a) to generate tractional forces in vitro, with and without serum stimulation, and (b) to produce and release contraction-stimulating proteins. Autocrine versus paracrine effects of cell-secreted promoters were tested by using RPE or human dermal fibroblasts (HDF) as target cells. The contraction-stimulating activity of the cell-secreted promoters was partially characterized and compared to the activity of defined promoters. Our study confirmed that RPE can synthesize and secrete cell-contraction-promoting factor(s) active in stimulating the development of tractional forces by RPE as well as HDF. The quantity of biological activity secreted per cell decreases with progressive dedifferentiation, yet the responsiveness of the cell to contraction promoters increases. The contraction promoter(s) synthesized by RPE is partially distinct from the promoters in serum, TGF-beta 1 and beta 2, IGF-1, ET-1 and PDGF. The contraction-promoting effects of the RPE product(s) can be completely blocked by staurosporine. De-differentiation of RPE is characterized by increasing capacity to generate tractional forces and decreasing synthetic capacity. RPE within a semi-closed system like the vitreal cavity can, theoretically, act both as promoting and active component of traction-related events (tractional retinal detachment).

Contraction of extracellular matrix by transdifferentiated retinal pigment epithelial cells, inducers and inhibitors

Transdifferentiated retinal pigment epithelial cells (RPE) display enhanced contractile potentials and have been implicated in the development of tractional retinal detachment. This study determines the activity of contraction-promoting factors, examines some involved mechanisms and evaluates inhibitors. Using an in vitro contraction assay, we demonstrated that collagen matrix contraction by transdifferentiated RPE cells is effectively stimulated by serum, platelet-derived growth factor and insulin-like growth factor-1. Endothelin-1 and transforming growth factor-beta 1 and -beta 2 have a more discrete or marginal effect. Tractional forces promoted by these peptides are completely protein synthesis dependent. Contraction stimulated by serum is only partly dependent on de novo protein synthesis, suggesting different active factors and/or pathways. Staurosporine, a broad-spectrum kinase inhibitor, effectively inhibited collagen matrix contraction by transdifferentiated RPE cells regardless of the promoter.

Protein kinase C has two different major roles in lattice compaction enhanced by cerebrospinal fluid from patients with subarachnoid hemorrhage.

Compaction of extracellular matrix (ECM) lattices by cultured fibroblasts was accelerated by cerebrospinal fluid (CSF) from patients with subarachnoid hemorrhage (SAH). The rate of acceleration was significantly related to the clinical grade of vasospasm. However, the mechanism remains unclear. Evidence exists for an important role in cerebral vasospasm for protein kinase C (PKC). The purpose of this study was to help clarify whether PKC has a role in contraction of the ECM. We studied the effects of a myristoylated PKC peptide inhibitor (Myr-Arg-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln-Lys-Asn-Val) (PKC peptide inhibitor), (5-isoquinolinesulfonyl)-homopiperazine (HA-1077) (inhibitor of protein kinase A, myosin light-chain kinase, and protein kinase G), 7-deacetyl-6-(N-ace-tylglycyl)-forskolin (forskolin) (adenyl cyclase activator), and diacylglycerol-lactone (DAG-lactone) (PKC activator) on fibroblast-populated collagen lattice compaction with or without CSF from SAH patients. Four sets of fibroblasts were used: three explanted from skin and one from cerebral artery. Moderate and high concentrations of PKC peptide inhibitor inhibited lattice compaction with or without acceleration by CSF. Low concentration of PKC peptide inhibitor enhanced acceleration by CSF but had no effects without CSF. HA-1077 could not inhibit lattice compaction. Forskolin inhibited compaction. DAG-lactone accelerated compaction in early phases. In the mechanism of acceleration of contraction of ECM under the influence of CSF, PKC seems to have two different roles. Protein kinase A and myosin light-chain kinase apparently play more minor roles than PKC in the mechanism, but no evidence was found of a role for protein kinase G activation in matrix compaction.

In vitro changes in nonfacial human skin following CO2 laser resurfacing: A comparison study

Background and Objective We evaluated the physical changes in human skin following CO2 laser cutaneous resurfacing with either the Sharplan SilkTouch® handpiece or the Coherent UltraPulse® laser. Study Design/Materials and Methods Three-hundred five human tissue samples and matched controls were used. Up to five laser passes were performed per specimen. Parameters evaluated included: lateral skin shrinkage, transient temperature change, isometric tension development, elasticity change, and histologic change. Results Skin shrinkage increased in direct proportion to laser pass number. Isometric tension exponentially increased and elasticity exponentially decreased with successive laser passes. The zone of thermal denaturation for the SilkTouch® handpiece was 115 ± 15 μm, and was independent of laser pass number. The zone of thermal denaturation was patchy for the UltraPulse® laser treatments, regardless of pass number. A greater temperature increase was also measured for SilkTouch® irradiation than with the UltraPulse® laser. Conclusion The observed alterations in tissue length, tension development, and elasticity obtained with SilkTouch® or UltraPulse® treatment may contribute to the changes in clinical appearance associated with laser cutaneous resurfacing. Our findings support a role for extracellular matrix contraction in the mechanism of action for CO2 lasers in cutaneous resurfacing. © 1996 Wiley-Liss, Inc.

Dependence of collagen remodelling on alpha-smooth muscle actin expression by fibroblasts.

To study the relation between expression of the putative myofibroblast marker alpha-smooth muscle actin and the remodelling of extracellular matrix, immunocytochemical, gel electrophoresis, and collagen gel contraction studies were performed on two human fibroblast subtypes. Double immunolabelling for total actins and alpha-smooth muscle (sm) actin as well as affinity labelling of filamentous and monomeric actins in gingival fibroblasts demonstrated that alpha-sm was colocalized in stress fibres and in regions with high levels of monomeric actin throughout the cytoplasm. alpha-sm comprised up to 14% of total cellular actin as assessed by 2D gel electrophoresis. Thirteen different gingival and seven different periodontal ligament fibroblast lines constitutively expressed on alpha-sm actin. These cells exhibited up to 60% inter-line variations of fluorescence due to alpha-sm actin and up to 70% and 45% inter-line variation in the rate of collagen gel contraction. Quantitative, single cell fluorimetry of alpha-sm actin immunoreactivity demonstrated a linear relation between gel contraction and alpha-sm actin (correlation coefficients of 0.71 for gingival and 0.61 for periodontal ligament cells), but there was no detectable relationship between total actin content and gel contraction. In contrast, flow cytometry demonstrated that 99% of the total gated cells from cell lines exhibiting rapid gel contraction showed alpha-sm actin staining above background fluorescence as compared to only 35% of cells with slow rates of gel contraction. Contracting collagen gels stained with FITC-phalloidin showed cells with well-developed stress fibres that were progressively more compact and elongated during the time of maximal gel contraction. To examine the dependence of gel contraction on assembly of monomeric actin into actin filaments, cells were electroporated in the presence of phalloidin or cytochalasin D. Collagen gels exhibited up to 100% inhibition of gel contraction that was dose-dependent. Gel contraction was inhibited 93% by electroinjection of cells with alpha-sm actin antibody prior to incubation, but the antibody did not inhibit actin assembly after attachment and spreading on substrates. These data indicate that gel contraction is dependent on alpha-sm actin expression and that alpha-sm actin is a functional marker for a fibroblast subtype that rapidly remodels the extracellular matrix.

Extracellular matrix contraction by cultured mesangial cells: Modulation by transforming growth factor-β and matrix components

Cultured glomerular mesangial cells (MCs) have the ability to contract the surrounding collagen gel matrix (CGM). To investigate this phenomenon, we examined the effect of growth factors and extracellular matrix (ECM) components. Among some growth factors tested, transforming growth factor-β (TGF-β) and fetal calf serum (FCS) enhanced CGM contraction dose dependently. These factors acted through distinct mechanisms because: (1) when growth-arrested MCs were used, the effect of FCS was inhibited partially but that of TGF-β was not; and (2) anti-TGF-β had no influence on CGM contraction induced by FCS. Among the ECM components such as laminin, fibronectin, type IV collagen, and heparin-like proteoglycans (heparan sulfate and heparin), which were each mixed separately with CGM before gelling, heparin-like proteoglycans and type IV collagen inhibited CGM contraction by MCs. The inhibitory effect of heparin was mediated by the interaction both with CGM and with MCs because: (1) when heparin was added to the culture medium, not into the gel, the inhibitory effect was diminished but still noted; and (2) using growth-arrested MCs, the inhibitory effect of heparin in the medium was reduced but still observed. This culture assay is useful for elucidating the tensional interaction between MCs and surrounding ECM.

Extracellular matrix contraction by fibroblasts: peptide promoters and second messengers.

Cells contracting connective tissue matrices generate tractional forces in tissues. Studies of fibroblast contraction, using collagen gels in an in vitro model, demonstrate that it involves the actin cytoskeleton, specific extracellular matrix receptors and requires stimulation by exogenous promoters. Fibroblast contraction is stimulated by factors released by platelets and potentially secreted within the contracting tissue. Endothelial cells secrete a potent promoter of fibroblast contraction which has been identified as endothelin 1. The pathway through which fibroblast contraction is stimulated appears to require activation of protein kinase C. Tumor cells can also secrete endothelin. These mechanisms may be relevant to tumor progression.

Endothelium and growth factors in vascular remodeling of hypertension.

The pioneering work of Furchgott, Moncada, and Vane has clearly established that the endothelium plays a critical role in the regulation of vascular tone. The endothelium produces several potent vasoactive substances, including vasodilators such as endothelium-derived relaxing factor and prostacyclin as well as vasoconstrictors such as angiotensin II and endothelin. These vasoactive substances not only have short-term effects on vascular tone but also appear to induce long-term effects on vascular structure. This process of vascular remodeling involves cell growth or regression and extracellular matrix expansion or contraction. As the interface between the bloodstream and the vessel wall, the endothelium plays a pivotal role in sensing and transducing the stimulus that induces vascular remodeling as well as producing the mediators that alter cell growth and the composition of the extracellular matrix. The endothelium maintains homeostasis within the vasculature by a balance between vasodilators and vasoconstrictors or growth inhibitors and growth promoters. Endothelial cell dysfunction may alter the delicate balance of mediators necessary to maintain homeostasis. Indeed, endothelial cell dysfunction is a common pathogenetic defect in animal models of hypertension as well as in hypertensive humans. Further understanding of the cellular and molecular mechanisms of vascular remodeling induced by endothelial cell-derived mediators may have important implications in the pathogenesis and treatment of hypertension.

Extracellular matrix contraction by cultured mesangial cells: An assay system for mesangial cell-matrix interaction

To investigate the interaction between glomerular mesangial cells (MCs) and extracellular matrix (ECM), we have developed a quantitative assay using a floating culture of MC-populated collagen gel matrix (MCGM). Since MCs can contract such a matrix consisting of type I collagen, MCGM exhibited marked contraction. The apparent cause was tensional interaction between MCs and collagen fibers, because: (1) MCs in the gel attached tightly to surrounding collagen fibers; (2) collagen fibrils surrounding MCs were ordered in a radial array; (3) collagen fibers aggregated around MCs in the contracted gel, but not in an uncontracted gel; (4) cytochalasin B, an actin polymerization blocker, inhibited gel contraction in a dose-dependent manner. We found that this interaction was modulated by some factors. Serum in the medium stimulated the contraction of MCGM. The degree of MCGM contraction was proportional to the number of MCs embedded above 2.3 × 10 4 cells/ml of gel. In MCGM containing a basement membrane-type gel matrix (BGM) gel contraction was increasingly inhibited as the content of BGM rose. Our method is useful for elucidating physiological and pathological interactions between MCs and ECM.