Collagen VI deficiency affects the organization of fibronectin in the extracellular matrix of cultured fibroblasts.

Fibronectin is one of the main components of the extracellular matrix and associates with a variety of other matrix molecules including collagens. We demonstrate that the absence of secreted type VI collagen in cultured primary fibroblasts affects the arrangement of fibronectin in the extracellular matrix. We observed a fine network of collagen VI filaments and fibronectin fibrils in the extracellular matrix of normal murine and human fibroblasts. The two microfibrillar systems did not colocalize, but were interconnected at some discrete sites which could be revealed by immunoelectron microscopy. Direct interaction between collagen VI and fibronectin was also demonstrated by far western assay. When primary fibroblasts from Col6a1 null mutant mice were cultured, collagen VI was not detected in the extracellular matrix and a different pattern of fibronectin organization was observed, with fibrils running parallel to the long axis of the cells. Similarly, an abnormal fibronectin deposition was observed in fibroblasts from a patient affected by Bethlem myopathy, where collagen VI secretion was drastically reduced. The same pattern was also observed in normal fibroblasts after in vivo perturbation of collagen VI-fibronectin interaction with the 3C4 anti-collagen VI monoclonal antibody. Competition experiments with soluble peptides indicated that the organization of fibronectin in the extracellular matrix was impaired by added soluble collagen VI, but not by its triple helical (pepsin-resistant) fragments. These results indicate that collagen VI mediates the three-dimensional organization of fibronectin in the extracellular matrix of cultured fibroblasts.     

Effects of elevated glucose levels on interactions of cardiac fibroblasts with the extracellular matrix

Exposure of fibroblasts to high glucose levels promotes a fibrotic response characterized by increased expression of extracellular matrix components including interstitial collagens. Little is known about the effects of glucose levels on other aspects of fibroblast function. Fibroblasts in the myocardium are surrounded by an extensive extracellular matrix composed predominantly of type I collagen. Interactions between fibroblasts and the myocardial extracellular matrix are thought to affect heart function by altering ventricular diastolic properties. The purpose of the present study was to determine the effects of elevated glucose levels on the interactions between heart fibroblasts and the collagenous extracellular matrix. Studies were performed to determine the effects of relative glucose levels on the ability of fibroblasts to migrate on and contract a three-dimensional collagenous substratum. These experiments illustrated that exposure of cardiac fibroblasts to high glucose levels (25 mM) resulted in decreased migratory activity of fibroblasts on a collagen matrix and decreased fibroblast proliferation. In addition, high glucose stimulated collagen and collagen-binding integrin expression and contraction of three-dimensional collagen gels by cardiac fibroblasts. These studies illustrate that altered glucose levels induce important changes in the interactions of cardiac fibroblasts with the collagenous extracellular matrix. Xiaoyi Zhang and James A. Stewart, Jr. are co-first authors.     

Fibroblast adhesion results in the induction of a matrix remodeling gene expression program

Fibrosis is believed to occur through the failure to terminate the normal tissue remodeling program. Tissue repair intimately involves the ability of fibroblasts to attach to extracellular matrix (ECM), resulting in cell migration and ECM contraction. Elevated, activated adhesive signaling is a key phenotypic hallmark of fibrotic cells. The precise contribution of adhesion to tissue remodeling and repair and fibrotic responses in fibroblasts is unclear, but involves focal adhesion kinase (FAK). FAK signals downstream of integrin-mediates attachment of fibroblasts to extracellular matrix. In this report, we show that FAK is required for the expression of a cohort of mRNAs encoding ECM and matrix remodeling genes including CCN2, alpha-smooth muscle actin (SMA) and type I collagen. Adhesion of fibroblasts to fibronectin, a component of the provisional matrix deposited in the initial phases of tissue repair, also resulted in the induction of CCN2, alpha-SMA and type I collagen mRNAs. Endothelin-1 (ET-1), a key inducer of pro-fibrotic gene expression, was also induced upon fibroblast attachment to ECM, and antagonism of the ET-1 receptors significantly reduced the ability of adhesion to induce expression of CCN2, alpha-SMA and type I collagen mRNAs. These results suggest that adhesion of fibroblasts to matrix during the initial phases of tissue remodeling and repair may actively contribute to the tissue repair program through the induction of pro-fibrotic gene expression.     

Modulation of heart fibroblast migration and collagen gel contraction by IGF-I

Dynamic interactions between cells and the extracellular matrix are essential in the regulation of a number of cellular processes including migration, adhesion, proliferation and differentiation. A variety of factors have been identified which modulate these interactions including transforming growth factor-beta, platelet-derived growth factor and others. Insulin-like growth factors have been shown to regulate collagen production by heart fibroblasts; however, the effects of this growth factor on the interactions of heart fibroblasts with the extracellular matrix have not been examined. The present studies were carried out to determine the effects of IGF-I on the ability of fibroblasts to interact with the extracellular matrix and to begin to determine the mechanisms of this response. These experiments illustrate that IGF-I treatment results in increased migration, collagen reorganization and gel contraction by heart fibroblasts. IGF-I has been shown to activate both the mitogen-activated protein kinase and phophatidylinositol-3 kinase pathways in isolated cells. Experiments with pharmacological antagonists of these pathways indicate that the mitogen-activated protein kinase pathway is essential for IGF-I stimulated collagen gel contraction by fibroblasts. These studies illustrate that IGF-I modulates the ability of fibroblasts to interact with the collagen matrix and that activation of multiple signaling pathways by IGF-I may produce distinct downstream responses in these cells.     

Extracellular collagenases and the endocytic receptor, urokinase plasminogen activator receptor-associated protein/Endo180, cooperate in fibroblast-mediated collagen degradation

The collagens of the extracellular matrix are the most abundant structural proteins in the mammalian body. In tissue remodeling and in the invasive growth of malignant tumors, collagens constitute an important barrier, and consequently, the turnover of collagen is a rate-limiting process in these events. A recently discovered turnover route with importance for tumor growth involves intracellular collagen degradation and is governed by the collagen receptor, urokinase plasminogen activator receptor-associated protein (uPARAP or Endo180). The interplay between this mechanism and extracellular collagenolysis is not known. In this report, we demonstrate the existence of a new, composite collagen breakdown pathway. Thus, fibroblast-mediated collagen degradation proceeds preferentially as a sequential mechanism in which extracellular collagenolysis is followed by uPARAP/Endo180-mediated endocytosis of large collagen fragments. First, we show that collagen that has been pre-cleaved by a mammalian collagenase is taken up much more efficiently than intact, native collagen by uPARAP/Endo180-positive cells. Second, we demonstrate that this preference is governed by the acquisition of a gelatin-like structure by the collagen, occurring upon collagenase-mediated cleavage under native conditions. Third, we demonstrate that the growth of uPARAP/Endo180-deficient fibroblasts on a native collagen matrix leads to substantial extracellular accumulation of well defined collagen fragments, whereas, wild-type fibroblasts possess the ability to direct an organized and complete degradation sequence comprising both the initial cleavage, the endocytic uptake, and the intracellular breakdown of collagen.     

Modulation of Heart Fibroblast Migration and Collagen Gel Contraction by IGF-I

Dynamic interactions between cells and the extracellular matrix are essential in the regulation of a number of cellular processes including migration, adhesion, proliferation and differentiation. A variety of factors have been identified which modulate these interactions including transforming growth factor+, platelet-derived growth factor and others. Insulin-like growth factors have been shown to regulate collagen production by heart fibroblasts; however, the effects of this growth factor on the interactions of heart fibroblasts with the extracellular matrix have not been examined. The present studies were carried out to determine the effects of IGF-I on the ability of fibroblasts to interact with the extracellular matrix and to begin to determine the mechanisms of this response. These experiments illustrate that IGF-I treatment results in increased migration, collagen reorganization and gel contraction by heart fibroblasts. IGF-I has been shown to activate both the mitogen-activated protein kinase and phophatidylinositol-3 kinase pathways in isolated cells. Experiments with pharmacological antagonists of these pathways indicate that the mitogen-activated protein kinase pathway is essential for IGF-I stimulated collagen gel contraction by fibroblasts. These studies illustrate that IGF-I modulates the ability of fibroblasts to interact with the collagen matrix and that activation of multiple signaling pathways by IGF-I may produce distinct downstream responses in these cells.     

Extracellular matrix organization modulates fibroblast growth and growth factor responsiveness

To learn more about the relationship between extracellular matrix organization, cell shape, and cell growth control, we studied DNA synthesis by fibroblasts in collagen gels that were either attached to culture dishes or floating in culture medium during gel contraction. After 4 days of contraction, the collagen density (initially 1.5 mg/ml) reached 22 mg/ml in attached gels and 55 mg/ml in floating gels. After contraction, attached collagen gels were well organized; collagen fibrils were aligned in the plane of cell spreading; and fibroblasts had an elongated, bipolar morphology. Floating collagen gels, however, were unorganized; collagen fibrils were arranged randomly; and fibroblasts had a stellate morphology. DNA synthesis by fibroblasts in contracted collagen gels was suppressed if the gels were floating in medium but not if the gels were attached, and inhibition was independent of the extent of gel contraction. Therefore, growth of fibroblasts in contracted collagen gels could be regulated by differences in extracellular matrix organization and cell shape independently of extracellular matrix density. We also compared the responses of fibroblasts in contracted collagen gels and monolayer culture to peptide growth factors including fibroblast growth factor, platelet-derived growth factor, transforming growth factor-beta, and interleukin 1. Cells in floating collagen gels were generally unresponsive to any of the growth factors. Cells in attached collagen gels and monolayer culture were affected similarly by fibroblast growth factor but not by the others. Our results indicate that extracellular matrix organization influenced not only cell growth, but also fibroblast responsiveness to peptide growth factors.     

Extracellular matrix proteins: A positive feedback loop in lung fibrosis?

Lung fibrosis is characterized by excessive deposition of extracellular matrix. This not only affects tissue architecture and function, but it also influences fibroblast behavior and thus disease progression. Here we describe the expression of elastin, type V collagen and tenascin C during the development of bleomycin-induced lung fibrosis. We further report in vitro experiments clarifying both the effect of myofibroblast differentiation on this expression and the effect of extracellular elastin on myofibroblast differentiation.Lung fibrosis was induced in female C57Bl/6 mice by bleomycin instillation. Animals were sacrificed at zero to five weeks after fibrosis induction. Collagen synthesized during the week prior to sacrifice was labeled with deuterium. After sacrifice, lung tissue was collected for determination of new collagen formation, microarray analysis, and histology. Human lung fibroblasts were grown on tissue culture plastic or BioFlex culture plates coated with type I collagen or elastin, and stimulated to undergo myofibroblast differentiation by 0–10 ng/ml transforming growth factor (TGF)β₁. mRNA expression was analyzed by quantitative real-time PCR.New collagen formation during bleomycin-induced fibrosis was highly correlated to gene expression of elastin, type V collagen and tenascin C. At the protein level, elastin, type V collagen and tenascin C were highly expressed in fibrotic areas as seen in histological sections of the lung. Type V collagen and tenascin C were transiently increased. Human lung fibroblasts stimulated with TGFβ₁ strongly increased gene expression of elastin, type V collagen and tenascin C. The extracellular presence of elastin increased gene expression of the myofibroblastic markers α smooth muscle actin and type I collagen.The extracellular matrix composition changes dramatically during the development of lung fibrosis. The increased levels of elastin, type V collagen and tenascin C are probably the result of increased expression by fibroblastic cells; reversely, elastin influences myofibroblast differentiation. This suggests a reciprocal interaction between fibroblasts and the extracellular matrix composition that could enhance the development of lung fibrosis.     

Contraction of fibroblast-containing collagen gels: Initial collagen concentration regulates the degree of contraction and cell survival

Remodeling of extracellular matrix involves a number of steps including the recruitment, accumulation, and eventual apoptosis of parenchymal cells as well as the production, organization, and rearrangement of extracellular matrix produced by these cells. The culture of fibroblasts in three-dimensional gels made of type I collagen has been used as a model of tissue contraction which characterizes both wound repair and fibrosis. The current study was designed to determine the effect of initial collagen concentration on the ability of fibroblasts to contract collagen gels and on cell survival. Native type I collagen was extracted from rat tail tendons and used to prepare collagen gels with varying collagen concentrations (0.75-2.0 mg/ml). Human lung fibroblasts (HFL-1) were cast into the gels and cultured in Dulbecco modified Eagle medium with 0.1% fetal calf serum for 2 wk. The gel size, collagen content, and deoxyribonucleic acid (DNA) content were determined. Gels prepared with an initial concentration of 0.75 mg/ml contracted more rapidly and to a smaller final size than gels prepared from 2 mg/ml initial collagen concentration (final size 7.1 versus 36.4% of initial size, P < 0.01). There was no significant degradation of the collagen in the gels under either condition. Hence, the dramatically increased contraction of the lower density gels resulted in a higher final density (P < 0.01). Cell density was estimated from DNA content. In low initial density gels, the final DNA content was significantly less than that in higher initial density gels (0.73 versus 1.88 microg/gel, P < 0.05). This was accompanied by an increased percentage of apoptotic cells at day 14 (43.3 versus 34.1%, P < 0.05). If the gels were maintained in the attached state which largely prevents contraction, apoptosis was significantly reduced, suggesting that contraction rather than matrix composition was a requirement for the increased apoptosis. In summary, these findings indicate that the initial matrix composition can lead to differing outcomes during fibroblast-mediated wound contraction.     

Modulation of collagen metabolism by the nucleolar protein fibrillarin.

Metabolic functions of fibroblasts are tightly regulated by the extracellular environment. When cultivated in tridimensional collagen lattices, fibroblasts exhibit a lowered activity of protein synthesis, especially concerning extracellular matrix proteins. We have previously shown that extracellular collagen impaired the processing of ribosomal RNA (rRNA) in nucleoli by generating changes in the expression of nucleolar proteins and a premature degradation of neosynthesized rRNA. In this study, we have investigated whether inhibiting the synthesis of fibrillarin, a major nucleolar protein with decreased expression in collagen lattices, could mimic the effects of extracellular matrix. Monolayer-cultured fibroblasts were transfected with anti-fibrillarin antisense oligodeoxynucleotides, which significantly decreased fibrillarin content. Downregulation of fibrillarin expression inhibited procollagen secretion into the extracellular medium, without altering total collagen production. No changes of pro1(I)collagen mRNA expression or proline hydroxylation were found. A concomitant intracellular retention of collagen and its chaperone protein HSP47 was found, but no effect on the production of other extracellular matrix macromolecules or remodelling enzymes was observed. These data show that collagen processing depends on unknown mechanisms, involving proteins primarily located in the nucleolar compartment with other demonstrated functions, and suggest specific links between nucleolar machinery and extracellular matrix.     

Interactions of Human Skin Fibroblasts with Monomeric or Fibrillar Collagens Induce Different Organization of the Cytoskeleton

Fibrillar collagens represent the most abundant extracellular matrix components surrounding fibroblasts. Although there is a large heterogeneity in the collagen composition and in the physiological functions of different tissues, interactions between cells and native collagens monomers are mediated by only two integrins, the α1β1 and α2β1 integrins. In tissue, fibroblasts are exposed to collagen polymers, supramolecular assemblies which might play a role on the availability of the cell-binding sites at the surface of the fibrils. We have addressed this issue by investigating the patterns of adhesion structures in normal human skin fibroblasts exposed to collagen monomers or polymers. Our results showed that cell morphology, cell adhesion pattern, actin organization, and distribution of integrin subunits, talin, vinculin, and phosphotyrosine-containing proteins are dependent on the supramolecular organization of the collagens. In particular, compared to monomers, collagen polymers induced a looser organization of the actin network and a linear clustering of integrins, talin, vinculin, and phosphotyrosine-containing proteins. These results emphasize the role of the physical state of collagen on cellular interactions and underline the role of the extracellular matrix in the phenotypic modulation of fibroblasts. Furthermore, our studies suggest the existence of a local heterogeneity in the biological activity of collagen fibrils.     

SPARC regulates processing of procollagen I and collagen fibrillogenesis in dermal fibroblasts

A characterization of the factors that control collagen fibril formation is critical for an understanding of tissue organization and the mechanisms that lead to fibrosis. SPARC (secreted protein acidic and rich in cysteine) is a counter-adhesive protein that binds collagens. Herein we show that collagen fibrils in SPARC-null skin from mice 1 month of age were inefficient in fibril aggregation and accumulated in the diameter range of 60-70 nm, a proposed intermediate in collagen fibril growth. In vitro, procollagen I produced by SPARC-null dermal fibroblasts demonstrated an initial preferential association with cell layers, in comparison to that produced by wild-type fibroblasts. However, the collagen I produced by SPARC-null cells was not efficiently incorporated into detergent-insoluble fractions. Coincident with an initial increase in cell association, greater amounts of total collagen I were present as processed forms in SPARC-null versus wild-type cells. Addition of recombinant SPARC reversed collagen I association with cell layers and decreased the processing of procollagen I in SPARC-null cells. Although collagen fibers formed on the surface of SPARC-null fibroblasts earlier than those on wild-type cells, fibers on SPARC-null fibroblasts did not persist. We conclude that SPARC mediates the association of procollagen I with cells, as well as its processing and incorporation into the extracellular matrix.     

Stiffening of human skin fibroblasts with age

Changes in mechanical properties are an essential characteristic of the aging process of human skin. Previous studies attribute these changes predominantly to the altered collagen and elastin organization and density of the extracellular matrix. Here, we show that individual dermal fibroblasts also exhibit a significant increase in stiffness during aging in vivo. With the laser-based optical cell stretcher we examined the viscoelastic biomechanics of dermal fibroblasts isolated from 14 human donors aged 27 to 80. Increasing age was clearly accompanied by a stiffening of the investigated cells. We found that fibroblasts from old donors exhibited an increase in rigidity of ∼60% with respect to cells of the youngest donors. A FACS analysis of the content of the cytoskeletal polymers shows a shift from monomeric G-actin to polymerized, filamentous F-actin, but no significant changes in the vimentin and microtubule content. The rheological analysis of fibroblast-populated collagen gels demonstrates that cell stiffening directly results in altered viscoelastic properties of the collagen matrix. These results identify a new mechanism that may contribute to the age-related impairment of elastic properties in human skin. The altered mechanical behavior might influence cell functions involving the cytoskeleton, such as contractility, motility, and proliferation, which are essential for reorganization of the extracellular matrix.     

Effects of interleukin-18 on cardiac fibroblast function and gene expression

Fibroblasts are the primary cell type responsible for synthesis and remodeling of the extracellular matrix in the heart. A number of factors including growth factors, hormones and mechanical forces have been identified that modulate the production of extracellular matrix by cardiac fibroblasts. Inflammatory mediators including pro-inflammatory cytokines and chemokines also impact fibrosis of the heart. Recent studies have illustrated that interleukin-18 promotes a pro-fibrotic response in cardiac fibroblasts; however the effects of this cytokine on other aspects of fibroblast function have not been examined. While fibroblasts have long been known for their role in production and remodeling of the extracellular matrix, other functions of these cells are only now beginning to be appreciated. We hypothesize that exposure to interleukin-18 will stimulate other aspects of fibroblast behavior important in myocardial remodeling including proliferation, migration and collagen reorganization. Fibroblasts were isolated from adult male rat hearts and bioassays performed to determine the effects of interleukin-18 on fibroblast function. Treatment of fibroblasts with interleukin-18 (1-100ng/ml) resulted in increased production of extracellular matrix components and remodeling or contraction of three-dimensional collagen scaffolds by these cells. Furthermore, exposure to interleukin-18 stimulated fibroblast migration and proliferation. Treatment of heart fibroblasts with interleukin-18 resulted in the rapid activation of the c-Jun N-terminal kinase (JNK) and phosphoinositide 3-kinase (PI3-kinase) pathways. Studies with pharmacological inhibitors illustrated that activation of these pathways is critical to interleukin-18 mediated alterations in fibroblast function. These studies illustrate that interleukin-18 plays a role in modulation of cardiac fibroblast function and may be an important component of the inflammation-fibrosis cascade during pathological myocardial remodeling.     

Decreased number of nucleolar organizing regions in collagen lattice cultured fibroblasts

Dermal fibroblasts cultivated in tridimensional matrices (lattices) of collagen exhibit a very low metabolic activity, and a low protein synthesis in particular. We have previously shown that ribosomal RNA content and half-life were decreased in collagen lattice cultured fibroblasts when compared to monolayer cultured fibroblasts. In this study, we seeded fibroblasts in collagen lattices and investigated the influence of matrix on the number of nucleolar organizing regions. We found that fibroblasts in fully retracted lattices exhibited a significant decrease of 45 % (P < 0.001) in the number of nucleolar organizing regions when compared to monolayer cultured fibroblasts. This decrease was correlated to the decrease in ribosomal RNA content. These data suggest that extracellular matrix induces early alterations of synthesis and/or processing of ribosomal RNAs, explaining, at least partly, the resulting low metabolic activity.     

Coalignment of plasma membrane channels and protrusions (fibripositors) specifies the parallelism of tendon

The functional properties of tendon require an extracellular matrix (ECM) rich in elongated collagen fibrils in parallel register. We sought to understand how embryonic fibroblasts elaborate this exquisite arrangement of fibrils. We show that procollagen processing and collagen fibrillogenesis are initiated in Golgi to plasma membrane carriers (GPCs). These carriers and their cargo of 28-nm-diam fibrils are targeted to previously unidentified plasma membrane (PM) protrusions (here designated "fibripositors") that are parallel to the tendon axis and project into parallel channels between cells. The base of the fibripositor lumen (buried several microns within the cell) is a nucleation site of collagen fibrillogenesis. The tip of the fibripositor is the site of fibril deposition to the ECM. Fibripositors are absent at postnatal stages when fibrils increase in diameter by accretion of extracellular collagen, thereby maintaining parallelism of the tendon. Thus, we show that the parallelism of tendon is determined by the late secretory pathway and interaction of adjacent PMs to form extracellular channels.     

Collagenase gene expression in fibroblasts is regulated by a three-dimensional contact with collagen

Collagenase activity in fibroblasts is regulated by cytokines and the interaction with the extracellular matrix. In this study we demonstrate that fibroblasts cultured within a three-dimensional collagen gel show a strong induction of collagenase gene expression. In addition to increased de novo synthesis most of the secreted enzyme was found to be activated leading to a high collagenolytic activity and complete degradation of collagen matrices after removal of fetal calf serum. Collagen I gene expression was found to be reduced under these conditions. These data suggest a specific modulation of cellular metabolism in response to contact with a three-dimensional collagenous matrix resulting in the divergent regulation of collagen and collagenase.