Transforming growth factor-beta1 regulates basement membrane formation by alveolar epithelial cells in vitro

Immortalized alveolar type II epithelial (SV40-T2) cells formed a continuous, thin lamina densa when they were cultured on collagen fibrils with the supplement of 1.0 ng/ml TGF-beta1. Corresponding to lamina densa formation, immunohistochemical analysis of laminin, type IV collagen, perlecan, and entactin (nidogen) indicated integration of these components in a linear array beneath the SV40-T2 cells. Synthesis of these basement membrane constituents was significantly enhanced by TGF-beta1 in a dose-dependent manner. On the other hand, TGF-beta1 did not affect the synthesis of extracellular matrix-regulatory enzymes and their inhibitors, such as type II transglutaminase, matrix metalloproteinase-2, plasminogen activator inhibitor-1, or tissue inhibitor of matrix metalloproteinase-1. These results indicate that basement membrane formation in the presence of 1.0 ng/ml TGF-beta1 is attributable to enhanced synthesis of basement membrane constituents. However, formation of a continuous basement membrane was inhibited at a TGF-beta1 concentration of 5.0 ng/ml. Synthesis of the basement membrane constituents was further enhanced at this concentration and the extracellular matrix-regulatory enzymes remained unchanged. The deposits of cellular fibronectin and type I collagen beneath SV40-T2 cells were significantly augmented. Thus excessive production of interstitial extracellular matrix components appears to obstruct the integration of basement membrane constituents into a continuous architecture. These results indicate that the basement membrane formation by SV40-T2 cells is achieved at the optimal TGF-beta1 concentration.     

Interleukin-1β and tumor necrosis factor-α have opposite effects on fibroblasts and epithelial cells during basement membrane formation

Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) are typical proinflammatory cytokines that influence various cellular functions, including metabolism of the extracellular matrix. We examined the roles of IL-1beta and TNF-alpha in basement membrane formation in an in vitro model of alveolar epithelial tissue composed of alveolar epithelial cells and pulmonary fibroblasts. Formation of the basement membrane by immortalized rat alveolar type II epithelial (SV40-T2) cells, which ordinarily do not form a continuous basement membrane, was dose-dependently upregulated in the presence of 2 ng/ml IL-1beta or 5 ng/ml TNF-alpha. IL-1beta or TNF-alpha alone induced increased secretion of type IV collagen, laminin-1, and nidogen-1/entactin, all of which contributed to this upregulation. In contrast, while SV40-T2 cells cultured with a fibroblasts-embedded type I collagen gel were able to form a continuous basement membrane, they failed to form a continuous basement membrane in the presence of IL-1beta or TNF-alpha. Fibroblasts treated with IL-1beta or TNF-alpha secreted matrix metalloproteinase (MMP)-9 and MMP-2, and these MMPs inhibited basement membrane formation and degraded the basement membrane architecture. Neither IL-1beta- nor TNF-alpha-treated SV40-T2 cells increased the secretion of MMP-9 and MMP-2. These results suggest that IL-1beta participates in basement membrane formation in two ways. One is the induction of MMP-2 and MMP-9 secretion by fibroblasts, which inhibits basement membrane formation, and the other is induction of basement membrane component secretion from alveolar epithelial cells to enhance basement membrane formation.     

Expression of extracellular matrix components is regulated by substratum

Reconstituted basement membranes and extracellular matrices have been demonstrated to affect, positively and dramatically, the production of milk proteins in cultured mammary epithelial cells. Here we show that both the expression and the deposition of extracellular matrix components themselves are regulated by substratum. The steady-state levels of the laminin, type IV collagen, and fibronectin mRNAs in mammary epithelial cells cultured on plastic dishes and on type I collagen gels have been examined, as has the ability of these cells to synthesize, secrete, and deposit laminin and other, extracellular matrix proteins. We demonstrate de novo synthesis of a basement membrane by cells cultured on type I collagen gels which have been floated into the medium. Expression of the mRNA and proteins of basement membranes, however, are quite low in these cultures. In contrast, the levels of laminin, type IV collagen, and fibronectin mRNAs are highest in cells cultured on plastic surfaces, where no basement membrane is deposited. It is suggested that the interaction between epithelial cells and both basement membrane and stromally derived matrices exerts a negative influence on the expression of mRNA for extracellular matrix components. In addition, we show that the capacity for lactational differentiation correlates with conditions that favor the deposition of a continuous basement membrane, and argue that the interaction between specialized epithelial cells and stroma enables them to create their own microenvironment for accurate signal transduction and phenotypic function.     

HGF-Induced Tubulogenesis and Branching of Epithelial Cells Is Modulated by Extracellular Matrix and TGF-β

Beginning with the observation that hepatocyte growth factor (HGF) induces the formation of branching tubular structures in Madin-Darby canine kidney (MDCK) cells cultured in Type I collagen gels but not in basement membrane Matrigel, we examined the individual components within this complex basement membrane extract to determine the effect of these proteins on the morphogenetic changes mediated by HGF. After extraction of several growth factors from Matrigel, HGF was still unable to induce process formation, an early event in tubulogenesis, indicating that one or more of the remaining extracellular matrix (ECM) proteins or growth factors were exerting the inhibitory effect. By individually adding back these components to MDCK cells grown in Type I collagen gels in the presence of HGF, we were able to establish that: (1) certain ECM proteins, such as laminin, entactin, and fibronectln, actually facilitated the formation of branching tubular structures and increased their complexity; (2) other ECM proteins, such as Type IV collagen, heparan sulfate proteoglycan, and vitronectin, caused marked inhibition of HGF-induced morphogenesis; and (3) not only did transforming growth factor-β (TGF-β) inhibit the formation of tubular structures, but those which did form exhibited little branching, thereby suggesting that TGF-β modulates tubulogenesis as well as branching. These results suggest that a tubulogenic morphogen such as HGF and a tubulogenesis-inhibitory morphogen such as TGF-β can, in the context of the dynamic matrix known to exist during epithelial tissue development, modulate the degree of tubule (or ductal) formation, the length of these tubules, and the extent of their arborization. The relevance of these findings to tubulogenesis and branching during kidney development is discussed.     

Immunohistochemical studies on the tissue localization of collagen types I, III, IV, V and VI in schwannomas. Correlation with ultrastructural features of the extracellular matrix

The distinctive tissue localization of collagen types in typical schwannomas with Antoni type A and B areas was demonstrated immunohistochemically using affinity-purified antibodies against types I, III, IV, V and VI collagen and comparative ultrastructural studies were made on the extracellular matrix components. Antoni type A tissue, which was composed of tightly packed spindle cells with long cytoplasmic processes surrounded by a continuous basement membrane and a few fibrillar components of the extracellular matrix, was almost exclusively immunoreactive for type IV collagen, presumably representing the basement membrane. Verocay bodies, which are organoid structures of Antoni type A tissue, had a variety of more abundant extracellular fibrous components, such as banded collagen fibrils, fibrous long-spacing fibrils and microfibrils. These were positive for type I and III, as well as type IV collagen. In Antoni type B areas, where two types to tumor cells designated Schwann cell-like and fibroblast-like were scattered in large amounts of amorphous extracellular matrix containing microfibrils and thick banded collagen fibrils, type VI collagen as well as types I, III and IV collagen were consistently detected. Type V collagen was localized in dense fibrous tissue areas and around blood vessels. These findings indicate that the differently organized cellular patterns of schwannomas, identified as Antoni types A and B, are characterized not only by the ultrastructural features of the extracellular matrix, but also by the distinctive collagen types produced by neoplastic Schwann cells.     

Reconstituted basement membrane enhances neurite outgrowth in PC12 cells induced by nerve growth factor

Rat pheochromocytoma cells (PC12) respond to nerve growth factor (NGF) by elaborating neurites. We have studied the effect of extracellular matrix proteins on responsiveness of PC12 cells to NGF. NGF elicited neurite outgrowth in cells plated on collagen, fibronectin, or laminin within 48-72 h. In contrast, cells entrapped in reconstituted basement membrane prepared from the Engelbreth-Holm-Swarm tumor (EHS-BM) responded within 24 h. Cells plated on collagen or fibronectin required a minimal dose of 50 ng/ml NGF to achieve the same effect as cells entrapped in EHS-BM at 1 ng/ml NGF. Neurites displayed by cells plated in EHS-BM were more extensively branched and remained intact up to 21 days following a single administration of NGF.     

Basic fibroblast growth factor stimulates hepatocyte growth factor/scatter factor secretion by human mesenchymal cells

Basic fibroblast growth factor (bFGF) together with other pleiotropic factors plays an important role in many complex physiological processes such as embryonic development, angiogenesis, and wound repair. Among these factors, hepatocyte growth factor/scatter factor (HGF/SF) which is secreted by cells of mesodermal origin exerts its mito- and motogenic activities on cells of epithelial and endothelial origin. Knowledge of the regulatory mechanisms of HGF/SF may contribute to the understanding of its role in physio-pathological processes. We observed that the secretion of HGF/SF by MRC-5 cells and by other fibroblast-derived cell cultures in conditioned media was enhanced by exposure to bFGF. HGF/SF was measured by the scatter assay, a bioassay for cell motility, and was further characterized by Western blot analysis with anti-HGF/SF antibodies. Exposure of MRC-5 cultures to 10 ng/ml of bFGF resulted already 6 h posttreatment in a threefold higher amount of scatter factor secreted into the medium as compared to untreated cultures. HGF/SF secretion was sustained after bFGF treatment for the following 72 h when increased amounts of HGF/SF were detected both in conditioned media as well as associated to the extracellular matrix. The secretion of HGF/SF in cell supernatants increased dose dependently upon treatment with bFGF starting from basal levels of 6 U/ml and reaching 27 U/ml at 30 ng/ml bFGF, plateauing thereafter. Upregulation of HGF/SF by IL-1, already described by others, was confirmed in this study. Based on our findings an articulated interaction can be speculated for bFGF, HGF/SF, and IL-1, e.g., in tissue regeneration during inflammatory processes or in wound healing. © 1996 Wiley-Liss, Inc.     

Differential Effects of TGF-β1 on Hyaluronan Synthesis by Fetal and Adult Skin Fibroblasts: Implications for Cell Migration and Wound Healing

Fetal wound healing differs from its adult counterpart in that it is regenerative and occurs without scarring. The matrix macromolecule hyaluronan (HA) and various cytokines, including members of the TGF-β family, have been implicated in the control of scarring. We have previously reported that adult and fetal fibroblasts differ with respect to the effect of cell density on HA synthesis when cultured on plastic tissue culture dishes. Data regarding the effects of substratum and TGF-β1 on HA synthesis by these cells are presented in this communication. Our results indicate that HA synthesis by both fetal and adult fibroblasts is (a) up-regulated by culture on a collagen substratum and (b) differentially regulated by TGF-β1 in a manner which is dependent upon both substratum and cell density. TGF-β1 stimulated HA synthesis by confluent fetal fibroblasts growing on a plastic substratum, but inhibited HA synthesis on a collagen substratum; these data underscore the important role of the substratum in determining the precise effect of TGF-β1 on cell behavior. Related studies indicated that the migration of fetal and adult fibroblasts into the collagen substrata was modulated by TGF-β1 in a manner identical to its effect on HA synthesis. These observations are discussed in terms of the contribution of distinct fibroblast subpopulations to wound healing and the manner in which this is regulated by matrix and cytokines.     

Regulation of Human (Caco-2) Intestinal Epithelial Cell Differentiation by Extracellular Matrix Proteins

Extracellular matrix regulation of intestinal epithelial differentiation may affect development, differentiation during migration to villus tips, healing, inflammatory bowel disease, and malignant transformation. Cell culture studies of intestinal epithelial biology may also depend on the matrix substrate used. We evaluated matrix effects on differentiation and proliferation in human intestinal Caco-2 epithelial cells, a model for intestinal epithelial differentiation. Proliferation, brush border enzyme specific activity, and spreading were compared in cells cultured on tissue culture plastic with interstitial collagen I and the basement membrane constituents collagen IV and laminin. Each matrix significantly increased alkaline phosphatase, dipeptidyl peptidase, lactase, sucrase-isomaltase, and cell spreading in comparison to plastic. However, the basement membrane proteins collagen IV and laminin further promoted all four brush border enzymes but inhibited spreading compared to collagen I. Proliferation was most rapid on type I collagen and slowest on laminin and tissue culture plastic. Basement membrane matrix proteins may promote intestinal epithelial differentiation and inhibit proliferation compared with interstitial collagen I.     

Epithelial-mesenchymal interactions in the production of basement membrane components in the gut

The production and deposition of extracellular matrix proteins and the cellular origin of type-IV collagen have been analysed immunocytochemically in cocultured or transplanted intestinal epithelial-mesenchymal cell associations. In the first experimental model, rat intestinal endodermal cells were cultured on top of confluent mono-layers of rat intestinal or skin fibroblastic cells. Under these conditions, interstitial matrix and basement membrane proteins were deposited within the fibroblastic layer over the whole culture period; interactions between the epithelial cells and the fibroblastic cell population, whatever their organ of origin, were required for the production of the basement membrane. In addition, its formation was progressive as assessed by the shift of a spot-like labelling to a continuous linear pattern at the epithelial-mesenchymal interface, and paralleled epithelial cell differentiation. In the second experimental model, chick-rat epithelial-mesenchymal recombinants developed as intracoelomic grafts were used, and the immunocytochemical detection of a basement membrane protein, type-IV collagen, was performed with species-specific antibodies. The major role of the mesenchyme in the deposition of type-IV collagen is supported by the fact that anti-chick but not anti-mammalian antibodies stained this antigen in chick mesenchyme-rat endoderm recombinants. These observations emphasize the role of tissue interactions in the formation of a basement membrane and show that the mesenchymal compartment is the principal endogenous source of type-IV collagen.     

Growth factors and extracellular matrix proteins during wound healing promoted with frozen cultured sheets of human epidermal keratinocytes

In a murine model of full-thickness wounds, healing is stimulated by the application of human frozen cultured epidermal sheets. With immunofluorescence techniques, we studied, during this process, the spatial and temporal pattern of expression of: transforming growth factor-alpha (TGF-alpha); transforming growth factor-beta (TGF-beta) isoforms 1, 2, and 3; platelet-derived growth factor (PDGF); and the extracellular matrix proteins fibronectin, collagen IV, and tenascin. The growth factors, with the exception of PDGF, were found to be located in the frozen cultured sheet of keratinocytes before and after its application to the wound, whereas collagen IV and tenascin were deposited in the connective tissue under the frozen cultures. None of these factors were detected in control wound beds. Monoclonal antibodies against collagen IV and tenascin showed that both were of murine origin. We propose that the frozen cultures of human keratinocytes promote faster reepithelialization through the release of growth factors such as TGF-alpha which directly enhance migration and proliferation of murine keratinocytes, and through the stimulation of murine subepithelial cells, by TGF-beta, to secrete basement membrane proteins such as collagen IV, laminin, and tenascin, which provide a provisional substrate that improves migration of the murine epidermal cells.     

Polarity reversal of inside-out thyroid follicles cultured on the surface of a reconstituted basement membrane matrix.

When cultured in suspension, epithelial thyroid cells organized into inside-out follicles. We studied the behavior of these structures after seeding on polystyrene, type I collagen, and reconstituted basement membrane (RBM) gel. When seeded on plastic, type I collagen or mixed type I collagen-RBM gel, inside-out follicles attached and spread, forming polarized cell monolayers. In contrast, on thick RBM gel, inside-out follicles attached penetrated into the gel, and reorganized into properly oriented follicular structures. Polarity of the cell layer was progressively inverted while, after adhesion, cells penetrated the soft RBM gel. In the process of reorientation, cells with hybrid polarity were observed. The fraction of the apical pole which was not yet in the gel showed an inside-out orientation, while a modified orientation was observed in contact with the RBM gel. Cells which had penetrated completely in the matrix formed a new apical pole and displayed an opposite orientation of their polarity. A continuous basement membrane was observed, lining the basal cell surface when native RBM gel was present in the substratum.     

Chitins and chitosans for the repair of wounded skin,nerve, cartilage and bone

This review provides a balanced integration of the most recent chemical, biochemical and medical information on the unique characteristics of chitins and chitosans in the area of animal/human tissue regeneration. Hemostasis is immediately obtained after application of most of the commercial chitin-based dressings to traumatic and surgical wounds: platelets are activated by chitin with redundant effects and superior performances compared with known hemostatic materials. To promote angiogenesis, necessary to support physiologically ordered tissue formation, the production of the vascular endothelial growth factor is strongly up-regulated in wound healing when macrophages are activated by chitin/chitosan. The inhibition of activation and expression of matrix metalloproteinases in primary human dermal fibroblasts by low MW chitosans prevents or solves problems caused by metalloproteinase-2 such as the hydrolysis of the basement membrane collagen IV. Experimental biocompatible wound dressings derived from chitin are today available in the form of hydrogels, xerogels, powders, composites, films and scaffolds: the latter are easily colonized by human cells in view of the restoration of tissue defects, with the advantage of avoiding retractive scar formation. The growth of nerve tissue has been guided with chitin tubes covalently coated with oligopeptides derived from laminin. The regeneration of cartilage is also feasible because chitosan maintains the correct morphology of chondrocytes and preserves their capacity to synthesize cell-specific extracellular matrix: chitosan scaffolds incorporating growth factors and morphogenetic proteins have been developed. Impressive advances have been made with osteogenic chitosan composites in treating bone defects, particularly with osteoblasts from mesenchymal stem cells in porous hydroxyapatite-chitin matrices. The introduction of azido functions in chitosan has provided photo-sensitive hydrogels that crosslink in a matter of seconds, thus paving the way to cytocompatible hydrogels for surgical use as coatings, scaffolds, drug carriers and implants capable to deliver cells and growth factors. The peculiar biochemical properties of chitins and chitosans remain unmatched by other polysaccharides.     

Hepatocyte growth factor modulates migration and proliferation of human microvascular endothelial cells in culture.

Epidermal growth factor (EGF) induces tubular formation of cultured human omental microvascular endothelial (HOME) cells and EGF also stimulates cell migration as well as expression of tissue type plasminogen activator (t-PA). Here we studied the effects of hepatocyte growth factor (HGF) on cell proliferation, cell migration and expression of t-PA and other related genes. Migration of confluent HOME cells into the denuded space was stimulated by HGF after being wounded with razor blade, but at a reduced rate in comparison with EGF. HOME cells could be proliferated in response to exogenous 100 ng/ml of HGF at rates comparable to that of 20 ng/ml EGF. The chemotactic activity of HOME cells was significantly stimulated by HGF in a dose-dependent manner when assayed by Boyden chamber. HGF did not efficiently enhance expression of both the t-PA gene and a tissue inhibitor of metalloproteinase gene whereas it stimulated expression of plasminogen activator inhibitor-1. Our present study provides a new evidence that some of the biological effects of HGF on HOME cells in culture are similar to those of EGF.     

The Anti-Scar Effects of Basic Fibroblast Growth Factor on the Wound Repair In Vitro and In Vivo

Hypertrophic scars (HTS) and keloids are challenging problems. Their pathogenesis results from an overproduction of fibroblasts and excessive deposition of collagen. Studies suggest a possible anti-scarring effect of basic fibroblast growth factor (bFGF) during wound healing, but the precise mechanisms of bFGF are still unclear. In view of this, we investigated the therapeutic effects of bFGF on HTS animal model as well as human scar fibroblasts (HSF) model. We show that bFGF promoted wound healing and reduced the area of flattened non-pathological scars in rat skin wounds and HTS in the rabbit ear. We provide evidence of a new therapeutic strategy: bFGF administration for the treatment of HTS. The scar elevation index (SEI) and epidermal thickness index (ETI) was also significantly reduced. Histological reveal that bFGF exhibited significant amelioration of the collagen tissue. bFGF regulated extracellular matrix (ECM) synthesis and degradation via interference in the collagen distribution, the α-smooth muscle actin (α-SMA) and transforming growth factor-1 (TGF-β1) expression. In addition, bFGF reduced scarring and promoted wound healing by inhibiting TGFβ1/SMAD-dependent pathway. The levels of fibronectin (FN), tissue inhibitor of metalloproteinase-1 (TIMP-1) collagen I, and collagen III were evidently decreased, and matrix metalloproteinase-1 (MMP-1) and apoptosis cells were markedly increased. These results suggest that bFGF possesses favorable therapeutic effects on hypertrophic scars in vitro and in vivo, which may be an effective cure for human hypertrophic scars.     

Stimulation of chondrogenesis in limb bud mesoderm cells by recombinant human bone morphogenetic protein 2B (BMP-2B) and modulation by transforming growth factor beta 1 and beta 2

Bone morphogenetic protein 2B (BMP-2B) also called BMP-4 is one of a family of cartilage and bone-inductive proteins derived from bone matrix and belongs to the transforming growth factor beta (TGF-beta) superfamily. These bone-inductive proteins isolated from adult bone may be involved in bone repair. However, they may also play a role in cartilage and bone formation during embryonic development. To test whether BMP-2B influences cartilage formation by embryonic cells, recombinant human BMP-2B was applied to cultured limb bud mesoderm plated at three different densities. BMP-2B stimulated cartilage formation as assessed by Alcian blue staining and incorporation of radioactive sulfate into sulfated proteoglycans. Cells cultured at all three densities in the presence of 10 ng/ml BMP-2B formed a nearly continuous sheet of cartilage with abundant extracellular matrix and type II collagen. In addition, when cells were cultured in 0.5% serum in the presence of 10 ng/ml of BMP-2B for 5 days there was an increase in alkaline phosphatase as detected by histochemical and biochemical methods. Transforming growth factor beta isoforms (TGF-beta 1 and TGF-beta 2) inhibited sulfate incorporation into proteoglycans in a dose-dependent manner. This inhibition by TGF beta was overcome by recombinant BMP-2B. This study demonstrates that recombinant BMP-2B stimulates cartilage formation by chick limb bud mesoderm in vitro and is further modulated by TGF-beta isoforms.     

Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures

We have defined a signal responsible for the morphological differentiation of human umbilical vein and human dermal microvascular endothelial cells in vitro. We find that human umbilical vein endothelial cells deprived of growth factors undergo morphological differentiation with tube formation after 6-12 wk, and that human dermal microvascular endothelial cells differentiate after 1 wk of growth factor deprivation. Here, we report that morphological differentiation of both types of endothelial cells is markedly accelerated by culture on a reconstituted gel composed of basement membrane proteins. Under these conditions, tube formation begins in 1-2 h and is complete by 24 h. The tubes are maintained for greater than 2 wk. Little or no proliferation occurs under these conditions, although the cells, when trypsinized and replated on fibronectin-coated tissue culture dishes, resume division. Ultrastructurally, the tubes possess a lumen surrounded by endothelial cells attached to one another by junctional complexes. The cells possess Weibel-Palade bodies and factor VIII-related antigens, and take up acetylated low density lipoproteins. Tubule formation does not occur on tissue culture plastic coated with laminin or collagen IV, either alone or in combination, or on an agarose or a collagen I gel. However, endothelial cells cultured on a collagen I gel supplemented with laminin form tubules, while supplementation with collagen IV induces a lesser degree of tubule formation. Preincubation of endothelial cells with antibodies to laminin prevented tubule formation while antibodies to collagen IV were less inhibitory. Preincubation of endothelial cells with synthetic peptides derived from the laminin B1 chain that bind to the laminin cell surface receptor or incorporation of these peptides into the gel matrix blocked tubule formation, whereas control peptides did not. These observations indicate that endothelial cells can rapidly differentiate on a basement membrane-like matrix and that laminin is the principal factor in inducing this change.