Substratum influence on collagen and fibronectin biosynthesis by arterial smooth muscle cells in vitro

Collagen, fibronectin, and nonfibrous protein biosynthesis were examined in cultures of rabbit arterial smooth muscle cells grown on tissue culture plastic precoated either with rabbit plasma fibronectin or bovine serum albumin. Cells seeded into fibronectin-coated wells appeared to reach confluence more quickly than counterparts grown on albumin-coated surfaces. Measurement 3H-thymidine incorporation into DNA by these cultures suggested that this was probably a consequence of more rapid and efficient cell attachment rather than an increased rate of proliferation of smooth muscle cells grown on fibronectin. In preconfluent cultures, the rates of collagen and fibronectin biosynthesis were reduced to 34 and 57%, respectively, on a per-cell basis in cultures grown on fibronectin-coated surfaces compared with cells grown on albumin-coated plasticware. In preconfluent cultures grown on fibronectin-coated surfaces, a greater percentage of the total fibronectin synthesized was incorporated into the cell layer. The distribution of newly synthesized collagen between culture medium and cell layer, however, was not affected by alteration of substratum composition. There was no difference in the rate of synthesis of noncollagen proteins between the two groups of preconfluent cells. In postconfluent cultures the rates of collagen and fibronectin biosynthesis were equivalent in both albumin- and fibronectin-treated cultureware. In preconfluent cultures, analyses of procollagens showed that the overall amounts of both types I and III procollagens were reduced in fibronectin-treated wells, indicating the reduction in collagen synthesis to be general and not type-specific. Although type V procollagen biosynthesis was not detected in either preconfluent group, it was found in postconfluent cultures. The reduction of fibronectin synthesis in cells grown in fibronectin-coated wells was significant as early as 4 hours after plating. Together, these findings suggest that cultured arterial smooth muscle cells are capable of deriving information from their substratum and regulating the biosynthetic rates of extracellular matrix components in response to the immediate needs of the cell.     

Expression of constitutive and inducible HSP70 and HSP47 is enhanced in cells persistently spread on OPN1 or collagen

Cells persistently spread on OPN or collagen survive heat shock better than cells transiently spread on fibronectin or tissue culture plates. Thus, a central question is whether constitutively or inducible stress proteins are enhanced in cells grown on adhesive proteins that maintain a persistent spread cell shape. Levels of Hsp 72,73, and colligin/Hsp47 were determined by Western blot analyses. The inducible Hsp 72 was prominently expressed following heat shock in cells grown on OPN or collagen, but not in cells plated on fibronectin coated substratum or on tissue culture plates. Colligin/Hsp 47 and Hsp 73 manifested a similar pattern of expression indicating that these adhesive attachment proteins accommodate cell function through organization of cell architecture.     

Role of fibronectin in collagen deposition: Fab' to the gelatin-binding domain of fibronectin inhibits both fibronectin and collagen organization in fibroblast extracellular matrix

We report the effect of Fab' (anti-60k) to a 60,000 mol wt gelatin binding domain of fibronectin (1981, J. Biol. Chem. 256:5583) on diploid fibroblast (IMR-90) extracellular fibronectin and collagen organization. Anti-60k Fab' did not inhibit IMR-90 attachment or proliferation in fibronectin-depleted medium. Fibroblasts cultured with preimmune Fab' deposited a dense extracellular network of fibronectin and collagen detectable by immunofluorescence, while anti-60k Fab' prevented extracellular collagen and fibronectin fibril deposition. Matrix fibronectin and collagen deposition remained decreased in cultures containing anti-60k Fab' until cells became bilayered or more dense, when fibronectin and collagen began to appear in lower cell layers. Anti-60k Fab' added to confluent cultures 24 h before fixation and staining had no effect on matrix fibronectin or collagen, so anti- 60k Fab' did not simply block immunostaining. Confluent cultures grown in anti-60k Fab' and labeled for 24 h with [3H]proline incorporated identical amounts of [3H]proline and [3H]hydroxyproline, but [3H]hydroxyproline deposition in the cell layer was significantly decreased by anti-60k Fab' (P less than 0.01). Extracellular matrix collagen does not appear to form a scaffold for fibronectin deposition, as neither gelatin nor a gelatin-binding fragment of plasma fibronectin inhibited deposition of matrix fibronectin. Our results suggest that interstitial collagens and fibronectin interact to form a fibrillar component of the extracellular matrix, and that fibronectin is required for normal collagen organization and deposition by fibroblasts in vitro. Domain-specific antibodies to fibronectin are powerful tools to study the biological role of fibronectin in extracellular matrix organization and other processes.     

Carbohydrate specific binding of fibronectin to Vibrio cholerae cells

Cells of 10 strains of Vibrio cholerae were grown on Trypticase Soy Broth and were tested for different surface porperties such as expression of surface haemagglutinins, cell-surface hydrophobicity and binding to 3 connective tissue proteins: fibronectin, type II collagen and fibrinogen.All strains bound fibronectin and one selected strain was shown to bind in a time-dependent and saturable manner.The binding of ¹²⁵I-labelled fibronectin could be completely inhibited by unlabelled fibronectin, and also partly by some other glycoproteins. Mannose inhibited binding of fibronectin up to 60%. The data indicate that carbohydrate structures within the 40 kDa (gelatin binding) and 105 kDa (cell binding) fragments of fibronectin are recognized by lectins on V. cholerae. The binding of collagen or fibrinogen was low or negligible.     

Different substrates influence the expression of intermediate filaments and the deposition of basement membrane proteins

Summary A primary culture of serous cystadenocarcinoma of the ovary was used to study the expression of intermediate filament proteins and the deposition of basal lamina proteins. It was found that cells grown on type I and IV collagens or in collagen gels failed to express vimentin, which was readily demonstrable in cultures of the same cells grown on plastic or glass. Furthermore cells grown in collagen gels formed colonies demonstrating a cystic architecture Unlike what is commonly observed on glass or plastic where laminin and fibronectin are deposited as disorganized fibrils in the extracellular space, in or on collagen these proteins appear solely at the interface between the epithelial cells and matrix. The results suggest that the extracellular matrix influences the cytoskeletal organization of the intermediate filaments and determines cell polarity. They confirm that collagen substrates permit epithelial cell cultures to progress toward a more differentiated state. Supported by grants from the Italian Assciation for Cancer Research (AIRC).     

The use of colloidal gold complexes in an ultrastructural study of lectin-binding sites and matrix deposition of normal human primary breast epithelium on collagen gels

Summary Colloidal gold probes were used to study the distribution of peanut agglutinin binding sites and the deposition of extracellular fibronectin and type IV collagen in cultured human breast cells grown on type I collagen gels. Qualitative analysis was performed at the ultrastructural level and appraised in relation to the possible role of peanut agglutinin, fibronectin and type IV collagen as functional markers for distinguishing cell types using this methodology.Peanut agglutinin bound to the surface of cuboidal epithelial cells but not on basal, putative myoepithelial cells in the cell islands, suggesting that it may be a useful functional marker. The binding on the epithelial cells was markedly increased by pre-treatment of the cells with neuraminidase. No correlation was seen between the amount of binding and either the surface topography or cellular ultrastructure.Fibronectin and type IV collagen were demonstrated on the fibrillar network left on the collagen gels after removal of the cell sheet. Any cells still adhering to the gel surface showed no evidence of gold probe binding on their upper surfaces. Examination of the under surfaces of the cell sheet showed gold probe binding equivalent to that found on the gels under the cells. However, it was not proven conclusively which cells produce the fibronectin and type IV collagen.     

INTEGRIN EXPRESSION AND COLLAGEN TYPE II IMPLICATED IN MAINTENANCE OF CHONDROCYTE SHAPE IN MONOLAYER CULTURE: AN IMMUNOMORPHOLOGICAL STUDY

Chondrocytes grown in monolayer culture at low density, with serum added, either dedifferentiate after several days whereby their cell shape changes or they are overgrown by fibroblast-like cells. The aim of this study was to optimize the cultivation of chondrocytes in monolayer culture and to slow down their transformation or their overgrowth by fibroblast-like cells. For this purpose freshly isolated chondrocytes of cartilage anlagen from 17-day-old mouse embryos were grown on plastic or collagen type II-coated substrates. With this model: (a) chondrocytes grown on plastic substrates had almost completely changed to fibroblast-like cells after 5 days in culture. (b) When grown on collagen type II, the chondrocytes maintained their round phenotype for more than 2 weeks in culture. (c) Immunomorphological investigations showed that chondrocytes produce collagen type II and fibronectin and express specific surface receptors (integrins of the β1-group) on the membrane from day 1 until the end of the culture period when grown on collagen type II. (d) Treatment with β1-integrin antibodies clearly reduces chondrocyte adhesion on collagen type II by about 70%. Hence, these data indicate that the most probable influence of collagen type II on cellular behaviour depends on the integrins participating in a chondrocyte—collagen type II interaction, and this model represents a pure chondrocyte culture which allows cell growth for an extended period.     

Involvement of plasma membrane dipeptidyl peptidase IV in fibronectin-mediated adhesion of cells on collagen

Dipeptidyl peptidase IV is an exopeptidase found in the serum and in plasma membranes of most animal tissues. The role of this enzyme in cell-matrix interaction of BHK cells and hepatocytes grown on collagen-coated surfaces was investigated by three different approaches. 1) Glass surfaces were derivatized with bovine serum albumin which resulted in a cell-repulsing substratum. When it was further modified with Gly-Pro-Ala tripeptide, which is a substrate for dipeptidyl peptidase IV, BHK fibroblasts spread on it rapidly. The spreading could be inhibited by addition of free Gly-Pro-Ala or other substrates of the enzyme as well as by an inhibitor peptide Val-Pro-Leu. It was not influenced by tripeptides which were neither substrates nor inhibitors of dipeptidyl peptidase IV. 2) The addition of Gly-Pro-Ala to seeded cells slowed down the initial process of cell spreading on denatured collagen in the presence of fibronectin. The presence of both collagen and fibronectin was a necessary precondition for the spreading of cells in a manner sensitive to Gly-Pro-Ala. 3) Antiserum raised against mouse liver dipeptidyl peptidase IV added to the medium delayed the spreading of rat hepatocytes on denatured collagen in the presence of fibronectin in a manner similar to when Gly-Pro-Ala was added to the medium. These observations lead to the conclusion that plasma membrane dipeptidyl peptidase IV may be involved in the initial phase of fibronectin-mediated cell spreading on collagen.     

Collagen can modulate cell interactions with fibronectin

We have examined the effects of soluble collagen on the function of fibronectin in baby hamster kidney (BHK) cells. Collagen and its purified alpha1(l) chain noncompetitively inhibited cell spreading on substrates precoated with fibronectin or a 75,000-D cell-binding fragment of fibronectin. Neither preincubation of cells with collagen followed by washing nor the addition of collagen to previously spread cells had any inhibitory effect on cell spreading, which indicates a requirement for the concurrent presence of collagen during the process of spreading. Treatment of collagen or alpha1(l) chain with collagenase abolished the inhibitory effect on fibronectin-mediated cell spreading. However, direct attachment of BHK cells to fibronectin-coated or 75,000-D fragment-coated substrates was not inhibited by collagen or by the alpha1(l) chain. Moreover, the binding of [3H]fibronectin or the 3'-75,000-D fragment to cell surfaces was not inhibited by the presence of soluble collagen, whereas soluble fibronectin inhibited binding. Although the binding of [3H]fibronectin-coated beads to BHK cell surfaces was also not inhibited by collagen, the phagocytosis of such beads was inhibited by the presence of collagen. On the other hand, soluble fibronectin partially inhibited the binding of fibronectin-coated beads but did not inhibit phagocytosis of the beads that did bind. The mechanism of the inhibition of fibronectin function by collagen and the possible interactions of two different kinds of receptors on the cell surface are discussed.     

Analysis of heterogeneity of human keratinocyte populations by their adhesion to substrate and keratin 19 to actin ratio

Keratin 19 is considered to be a marker for skin stem cells that assume a particular subpopulation of these cells in the keratinocyte population. It is also known that keratinocytes have a natural affinity for extracellular matrix proteins. The aim of this work was to identify subpopulations of keratinocytes by cultivation on various substrates (type-I collagen, laminin 2/4 and fibronectin) and by measuring keratin 19/actin ratio (G/R). The areas of cell projection on a substrate, perimeter, the spreading coefficient, and G/R have been assayed. Keratinocyte populations were morphologically homogeneous both on fibronectin and laminin 2/4. Large cells comprised 12 and 20% of the populations, respectively. No correlation between morphological parameters of cells and keratin 19/actin ratio was found in keratinocytes cultivated in fibronectin. The cells grown on collagen behaved as a heterogenous population with large cells comprising more than 50% of the population. On average, the size of the cells grown on collagen was twice as large as the cells cultivated on fibronectin. On the other hand, the correlation between cell size and G/R was revealed in cells grown on both collagen and laminin 2/4. The cells with lower G/R values display a larger size and higher spreading. We assume that this correlation is determined by the presence of α1 and α2 integrin subunits in these cells. Keratin 19 assay did not reveal keratinocyte subpopulations. Our results raised doubts regarding the presence of stem cells in cultures of humanskin keratinocytes.     

Transforming growth factor beta type 1 binds to collagen IV of basement membrane matrix: implications for development

The interaction of transforming growth factor beta (TGF beta) with extracellular matrix macromolecules was examined by using radiolabeled TGF beta and various matrix macromolecules immobilized on nitrocellulose. TGF beta bound to collagen IV with greater affinity than to other extracellular matrix macromolecules tested. Neither laminin nor fibronectin, both of which bind type IV collagen, interfered with the binding of TGF beta to type IV collagen. TGF beta 2 competed effectively with TGF beta 1 for binding to type IV collagen. The biological effect of TGF beta was tested by an assay based on inhibition of proliferation of an osteoblast cell line, MC3T3-E1. The results demonstrated that the effect of TGF beta 1 was sustained when cells were grown on type IV collagen compared to cells grown on laminin, collagen type I, and plastic. These results demonstrate that extracellular matrix components may function as an affinity matrix for binding and immobilizing soluble growth and differentiation factors. In view of the demonstrated role of basement membranes in development the present results imply an important function for transforming growth factor beta bound to collagen IV in local regulation of cell proliferation and differentiation.     

Basement Membrane Proteins Influence Brain Capillary Endothelial Barrier Function In Vitro

Abstract: The influence of basement membrane proteins on cellular barrier properties of primary cultures of porcine brain capillary endothelial cells grown on permeable filter inserts has been investigated. Measurements of transcellular electrical resistance (TER) by impedance spectroscopy were performed with cells cultured on type IV collagen, fibronectin, laminin, and one-to-one mixtures of these proteins. Moreover, a one-to-one combination of type IV collagen and SPARC (secreted protein acidic and rich in cysteine) has been studied. Rat tail collagen has been used as a reference substratum. If TERs of cells from a given preparation were low (∼350 Ω× cm²) on the reference substratum, type IV collagen, fibronectin, and laminin as well as one-to-one combinations of these proteins elevated transcellular resistances significantly (2.3- to 2.9-fold) compared with rat tail collagen. TER of cells exhibiting a high reference level (∼1,000 Ω× cm²) could, by contrast, be increased only 1.1- to 1.2-fold. The type IV collagen/SPARC mixture did not elevate TER. Our findings suggest that type IV collagen, fibronectin, and laminin are involved in tight junction formation between cerebral capillary endothelial cells. The differential effects observed for individual preparations probably reflect more or less dedifferentiated states of the endothelium, in which basement membrane proteins can influence cellular differentiation more or less strongly. However, our results indicate that type IV collagen, fibronectin, and laminin enhance the reliability and suitability of primary microvascular endothelial cell cultures as an in vitro model of the blood-brain barrier.     

The human villous cytotrophoblast: Interactions with extracellular matrix proteins, endocrine function, and cytoplasmic differentiation in the absence of syncytium formation

Human syncytiotrophoblasts are derived from villous cytotrophoblasts by cell fusion. Coincident with this morphologic transformation, trophoblasts acquire specific endocrine functions, including elaboration of chorionic gonadotropin (hCG). We wondered if syncytia formation was a prerequisite for biochemical differentiation or simply was one part of the differentiation program. By growing purified human cytotrophoblasts under serum-free conditions and manipulating the culture surface, we were able to disassociate morphologic from biochemical differentiation. We have shown previously (Endocrinology 1986, 118:1567) that human cytotrophoblasts grown in the presence of fetal calf serum flatten out, aggregate, and form functional syncytiotrophoblasts in vitro over 24-96 hr. Here we demonstrate that when grown in the absence of serum, the cells do not undergo these morphologic changes, but remain as individual spherical cells. If the culture surface was precoated with fibronectin or a variety of collagens, but not albumin, the cells regained their ability to flatten, aggregate, and form syncytia. Attachment to and syncytia formation on fibronectin was blocked by the addition of the R-G-D-S-containing peptide, Gly-Arg-Gly-Asp-Ser-Pro. Attachment to and syncytia formation on type I collagen was blocked by anti-human fibronectin F(ab')2 fragments, while association with type IV collagen was not affected by this antibody, suggesting that fibronectin mediates trophoblast association with type I collagen, but not type IV. Although syncytia formation did not occur when cytotrophoblasts were cultured under serum-free conditions in the absence of ECM proteins, biochemical differentiation was not affected. These cells secreted hCG at the same rate under serum-free conditions whether they were plated on plastic only--which prevented syncytia formation--or fibronectin, laminin or, type IV collagen--which allowed syncytia formation to occur. Furthermore, cytoplasmic differentiation in the absence of syncytia formation was confirmed by performing transmission electron microscopy on cytotrophoblasts grown under serum-free conditions in the presence of 8-bromo-cAMP. We conclude that syncytia formation is not a prerequisite for biochemical differentiation, but simply part of the trophoblast differentiation program.     

Nitrate reductase and the membrane composition of pleiotropic chlorate resistant mutants of Escherichia coli K-12.

Attachment of rat hepatocytes to collagen but not to fibronectin substrata was efficiently inhibited by antibodies against the hepatocyte surface. Further analyses of this inhibition suggested that hepatocyte attachment to collagen involves cell surface antigens which are not identical to membrane bound fibronectin or collagen.Rabbit antibodies against rat fibronectin inhibited hepatocyte attachment to rat fibronectin but not to collagen or rabbit fibronectin. After plasmin digestion of fibronectin, peptides were isolated that lacked affinity for collagen but could serve as a substratum for hepatocyte attachment. These results suggested that attachment to fibronectin does not involve membrane bound fibronectin or collagen.     

Substrate adhesion of rat hepatocytes: on the mechanism of attachment to fibronectin

We examined the mechanisms of cell attachment to fibronectin-coated substrates. Inhibition of cell attachment was obtained by species- specific antifibronectin antibodies, which presumably recognize a distinct antigenic structure in the protein located at, or in the immediate vicinity of, the cell-binding site. The inhibiting antibodies could be adsorbed on a column of Sepharose substituted with plasma fibronectin. The initial phase of cell attachment was also inhibited by addition of soluble fibronectin to the incubation medium in a reaction that exhibited specificity and concentration dependence. These data suggest that cell-binding sites are available in an active form on the surface of soluble fibronectin. However, the inhibitory effect of fibronectin was greatly enhanced by adding the protein together with heparin, heparan sulfate, collagen, or a fibronectin-binding collagen peptide (CB-7), which is consistent with an "activation" of fibronectin on binding to these matrix components. A similar activation of fibronectin was obtained by cleaving the protein with trypsin. We discuss these findings in relation to conformational rearrangements in the fibronectin molecule. Data is presented supporting a mechanism of cell attachment to fibronectin involving multiple weak interactions between cellular receptors and substrate molecules, although some steps in the attachment process appear to disobey the requirements for such a mechanism.     

Regulation of cell adhesion signaling by synthetic glycopolymer matrix in primary cultured hepatocyte

Control of cell-matrix interactions is a central principle for the design of biomaterial in tissue engineering. In this study, we evaluated a synthetic glycopolymer, which is recognized by the asialoglycoprotein receptor (ASGPR) expressed on the surface of hepatocytes, as an artificial matrix to regulate integrin-mediated signaling. The phosphorylation of focal adhesion kinase was restricted in hepatocytes cultured on the glycopolymer compared with fibronectin. In addition, there was no reorganization of cytoskeleton-related proteins such as actin filaments, microtubules, and vinculin in hepatocytes cultured on the glycopolymer. DNA synthesis and cyclin D1 expression were suppressed in hepatocytes grown on the glycopolymer as compared with those grown on fibronectin and collagen. The data suggest that the glycopolymer will be a good artificial matrix to regulate integrin-mediated signaling and cell growth through the unique ASGPR-carbohydrate interaction.     

Response of basal epithelial cell surface and Cytoskeleton to solubilized extracellular matrix molecules

Corneal epithelium removed from underlying extracellular matrix (ECM) extends numerous cytoplasmic processes (blebs) from the formerly smooth basal surface. If blebbing epithelia are grown on collagen gels or lens capsules in vitro, the basal surface flattens and takes on the smooth contour typical of epithelium in contact with basal lamina in situ. This study examines the effect of soluble extracellular matrix components on the basal surface. Corneal epithelia from 9- to 11-d-old chick embryos were isolated with trypsin-collagenase or ethylenediamine tetraacetic acid, then placed on Millipore filters (Millipore Corp., Bedford, Mass.), and cultured at the medium-air interface. Media were prepared with no serum, with 10% of calf serum, or with serum from which plasma fibronectin was removed. Epithelia grown on filters in this medium continue to bleb for the duration of the experiments (12-14 h). If soluble collagen, laminin, or fibronectin is added to the medium, however, blebs are withdrawn and by 2-6 h the basal surface is flat. Epithelia grown on filters in the presence of albumin, IgG, or glycosaminoglycans continue to bleb. Epithelia cultured on solid substrata, such as glass, also continue to bleb if ECM is absent from the medium. The basal cell cortex in situ contains a compact cortical mat of filaments that decorate with S-1 myosin subfragments; some, if not all, of these filaments point away from the plasmalemma. The actin filaments disperse into the cytoplasmic processes during blebbing and now many appear to point toward the plasmalemma. In isolated epithelia that flatten in response to soluble collagens, laminin, and fibronectin, the actin filaments reform the basal cortical mat typical or epithelial in situ. Thus, extracellular macromolecules influence and organize not only the basal cell surface but also the actin-rich basal cell cortex of epithelial cells.     

Effect of the triterpenoid fraction of Centella asiatica on macromolecules of the connective matrix in human skin fibroblast cultures

The mechanism of action of the total triterpenoid fraction extracted from Centella Asiatica (TTFCA) was evaluated using human skin fibroblasts cultures as the experimental system. In particular its influence on the biosynthesis of collagen, fibronectin and proteoglycans was considered. The presence of TTFCA (25 micrograms/ml) does not seem to affect cell proliferation, total protein synthesis or the biosynthesis of proteoglycans in a significant way. A statistically important increase was observed in the percentage of collagen and, as revealed by immunofluorescence measurements, in cell layer fibronectin. This effect on collagen and fibronectin may help to explain the action of TTFCA in promoting wound healing, and suggests an interesting working hypothesis for its action on basal endothelia.     

Effects of substrata on the polarization of bovine endometrial epithelial cells in vitro

Summary Epithelial-cell function requires cellular polarity in which apical membrane surfaces have unique characteristics and cellular organelles are stratified. Physiological investigations of endometrial, epithelial cells would be enhanced greatly by the ability of a method to polarize cells in culture. This study investigates the effects of different substrata on polarization of cultured bovine endometrial epithelial cells. Fetal bovine endometrial epithelial-cell lines were developed from explant outgrowth. Epithelial monolayers were subcultured onto amniotic membranes, Millicell-HA membranes, or Millicell-CM membranes coated with rat-tail collagen, Matrigel, laminin, Vitrogen,or fibronectin. Cultures on these substrata were maintained at the air/liquid interface. Cells grown on either collagen-coated or uncoated Milli-cell membranes also were maintained submerged in medium. Excellent polarized morphology was attained in cultures grown at the air/liquid interface on amniotic membranes and rat-tail collagen-coated membranes. Lectin-binding patterns, to apical membranes of polarized epithelial cell cultures paralleled patterns of binding to bovine endometrial surfaces in vivo. Cultures on rat-tail collagen were maintained for several weeks. These methods provide a valuable system for studying the endometrium in vitro.