Effect of subcellular matrix on glycosaminoglycan synthesis by human lung fibroblasts

The influences modulating glycosaminoglycan production by lung cells are not well understood. We examined the effect of three different subcellular matrices, plastic, type I collagen, and reconstituted basement membrane-like material (RBM), on the synthesis of sulfated glycosaminoglycans by cultured IMR-90 human lung fibroblasts. Accumulation of 35SO4-labeled glycosaminoglycans into the cell-matrix layer or medium was measured. Cells on collagen synthesized significantly less total glycosaminoglycans than cells on plastic but had a higher fraction of labeled glycosaminoglycans present in the cell-matrix layer (35 vs. 18%) with the increases being highest for dermatan and chondroitin sulfates. Cells grown on the RBM synthesized significantly more glycosaminoglycans than cells on plastic or collagen and also had 260% more labeled glycosaminoglycans present in the cell-matrix layer than cells on plastic. We conclude that the matrix to which lung fibroblasts are exposed can influence the amount and type of glycosaminoglycans synthesized and the degree of incorporation into the matrix. This may be relevant to fibrotic lungs with increased type I collagen or to severely injured lungs in which intra-alveolar fibroblasts are in contact with denuded basement membranes.     

Cell substratum modulates responses of preosteoblasts to retinoic acid

The aim of this study was to determine the role of ECM components of bone in regulating the differentiation and function of cells of the osteoblast lineage. Rat UMR 201 cells, phenotypically preosteoblast, were plated onto plastic tissue culture dishes or dishes coated with gelled type I collagen or reconstituted basement membrane (matrigel). Acute cell attachment assays showed that cells adhered to substrates in the following order: collagen > matrigel ? plastic. Proliferation rate up to 96 hr were similar on each substrate. However, if cells were treated with 10^?6 M retinoic acid (RA), proliferation rates were reduced compared with control for cells grown on collagen and matrigel but not on plastic. Morphological changes were matrix-specific; in subconfluent cultures, long thin processes were seen with cells grown on collagen and a pattern of interconnecting cell processes formed when cells were plated on matrigel. Striking differences were observed in the constitutive or RA-induced gene expression of cells grown on the different substrates. When cells plated on collagen were treated with RA, induction of mRNA for alkaline phosphatase (ALP) as well as ALP enzyme activity were much less than with cells grown on plastic. In contrast, RA treatment induced osteopontin (OP) mRNA expression more strongly in cells plated on collagen compared with plastic within 24 hr and this was maintained for 72 hr. RA treatment produced a two fold increase of pro-α 1(I) collagen mRNA in cells grown on plastic and matrigel but not in cells grown on collagen. Growth on collagen produced changes in the way UMR 201 cells responded to RA from which they did not fully recover in subsequent 48-hr growth periods on plastic. These results indicate that ECM components regulate the function of and are capable of modulating RA-induced differentiation of preosteoblasts. © 1993 Wiley-Liss, Inc.     

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.     

Regulation of gene expression in adult rat hepatocytes cultured on a basement membrane matrix

Freshly isolated adult rat hepatocytes, when cultured on type I collagen (commercially available as Vitrogen), assume a polygonal shape, form a stable monolayer within 24 hours, but lose the capacity to express some liver-specific functions over time in culture. We incubated hepatocytes in a serum-free medium on a reconstituted basement membrane gel, "matrigel" (prepared from an extract of extracellular matrix of the murine Engelbreth-Holm-Swarm sarcoma), and observed that the cells adhered firmly, remained rounded as single cells or clusters, and maintained liver-specific gene expression for more than 1 week in vitro. Hepatocytes on matrigel secreted substantially higher amounts of albumin, transferrin, haptoglobin, and hemopexin, Northern blot analyses of extracted cellular RNA, expressed increased amounts of mRNA for the liver-specific protein albumin (as compared with cells on vitrogen). In cultures treated with phenobarbital, cytochrome P-450b, and cytochrome P-450e, mRNAs and proteins were barely detectable in cells on Vitrogen but were induced to levels similar to those in the liver in vivo in matrigel cultures. Likewise, the use of matrigel greatly enhanced the induction of mRNA and protein for P-450c by 3-methylcholanthrene and for P-450p by steroidal and nonsteroidal inducers. However, neither substratum permitted induction of P-450d by 3-methylcholanthrene, suggesting that the effects of matrigel are selective even for expression in liver of members of the superfamily of cytochrome P-450 genes. Within 5 days in cultures on Vitrogen, hepatocytes expressed detectable amounts of fetal liver aldolase activity and also mRNA for vimentin and type I collagen, each considered a phenotypic change reflecting hepatocyte "dedifferentiation." None of these was present in cells on matrigel. Responsiveness to mitogenic stimuli, as judged by incorporation of 3H-thymidine into DNA, was also decreased in hepatocytes cultured on matrigel. Finally, there was a remarkable increase in the levels of both matrices during the first 2 days in culture. However, the continuously cytoskeleton mRNA over time in culture than did the rounded cells on matrigel. We conclude that hepatocytes cultured on matrigel, as opposed to the standard collagen, exhibit remarkably enhanced expression of many liver-specific functions.     

The effects of cell density, attachment substratum and dexamethasone on spontaneous apoptosis of rat hepatocytes in primary culture

Summary The rates of spontaneous cell detachment, cell viability, and apoptosis in primary cultures of rat hepatocytes plated at high and low density were compared. Apoptosis was frequent in detached cells, and the rates of cell detachment and apoptosis were greater in high-density than in low-density cultures. Among attached cells, more cells had condensed or fragmented nuclei in high-density than in low-density cultures. Further, ladder-like DNA fragmentation was not seen in low-cell-density cultures but was clearly evident in high-density cultures. Bax was more highly expressed in cells cultured at high density, and on collagen vs. matrigel, whereas changes of Bcl-2 and Fas expression observed in culture appeared unrelated to the rate of apoptosis. The rate of hepatocyte apoptosis appeared to be identical in low-density cultures on collagen 1 and matrigel, but when cells were cultured at high density, matrigel suppressed apoptosis by more than 50% at 36 h. In hepatocytes cultured on collagen 1, dexamethasone (0.1 μM) suppressed apoptosis in both low- and high-density cultures; higher doses had no further effects. In high density cultures, aurintricarboxylic acid (10 μM) suppressed apoptosis and this improved cell attachment at 48 h. It is concluded that cell viability in primary cultures of rat hepatocytes grown on collagen I is dependent on optimal culture density and that the cell population is regulated, at least in part, by apoptosis. Corticosteroids suppress spontaneous apoptosis of cultured hepatocytes in a non-dose-dependent manner, whereas matrigel abolishes apoptosis induced by increasing cell density. Bax may be an important protein in the cell density and cell matrix-dependent regulation of apoptosis in cultured hepatocytes.     

Retinol and extracellular collagen matrices modulate hepatic Ito cell collagen phenotype and cellular retinol binding protein levels

The hepatic vitamin A-storing Ito cell has been implicated as a causative cell in hepatic fibrogenesis. Using a modification of a recent method (Friedman, S. L., Roll, F. J., Boyles, J., and Bissell, D. M. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 8681-8685), rat Ito cells were isolated and passaged in vitro on collagen-coated plastic dishes through cell generation 40-50. The collagen synthetic phenotype for Ito cells grown on various extracellular matrices was demonstrated by immunofluorescence and quantitated by competition enzyme-linked immunosorbent assays. When grown on a type I collagen matrix, Ito cells produced type IV greater than type III greater than type I collagen. When grown on a type IV collagen matrix, the cells produced relatively equal amounts of types I and III collagen. The absolute amounts of type I collagen produced were greater when cells were grown on type IV versus type I matrix. When 10(-5) M retinol was added to cell cultures, there was a uniform increase in type III collagen regardless of matrix type but a decrease in type I collagen when cells were grown on a type IV matrix and a large increase in type I collagen when cells were grown on a type I collagen matrix. The levels of cellular retinol binding protein, a key cytosolic retinol transport protein, were quantitated by high performance liquid chromatography and compared for cells grown on type I versus type IV collagen matrices. It was found that cells on a type I matrix contain 4.96 +/- 2.8 times more cellular retinol binding protein than do cells grown on a type IV matrix. In conclusion, Ito cell collagen synthesis may be altered by underlying extracellular matrix and exogenous retinol. This in vitro culture system should allow the study of regulatory factors and possible therapeutic anti-fibrogenic mediators.     

Influence of collagen gel substratum on response to low-density lipoprotein by cultured human skin fibroblasts

The effect of low-density lipoprotein (LDL) on accumulation of glycosaminoglycans (GAG) was compared in cultures of human skin fibroblasts on a conventional plastic substratum and in a native type I collagen gel. The 24-h incorporation of [3H]glucosamine and Na2(35)SO4 into GAG secreted into the medium or associated with the substratum and cell surface (SCA) was measured in cells at subconfluent densities. When cells were grown on plastic, 13-25% of the labeled GAG was in the SCA pool. Cells cultured within a collagen gel matrix incorporated three times more [3H]glucosamine and up to five times more [35S]sulfate into this pool. The addition of LDL (300 micrograms protein/mL) to the medium increased the level of total GAG incorporation of [3H]glucosamine by 40-50% and of [35S]sulfate by 15-20% on both substrata. For cells on plastic the relative increase in the medium and SCA pool was similar, whereas for cells in collagen gel the response to LDL was twice as great in the SCA pool as in the medium. The distribution of GAG types was unaffected by LDL; hyaluronic acid remained the principal GAG in the media pools of both substrata, heparan sulfate remained the main SCA GAG in cultures on plastic, and dermatan sulfate remained the dominant GAG in the SCA pool of collagen gel cultures. LDL degradation was measured at intervals up to 48 h after the addition of 125I-labeled LDL. The rate of accumulation of degraded LDL products was lower in collagen gel cultures, but the final levels achieved were the same in the two substrata. Concentrations of total cell cholesterol were similar, although the increases in free cholesterol induced by LDL were 26% greater in cells within collagen gel than in those on plastic. We conclude that fibroblasts grown within a collagen gel, as compared with those on a plastic substratum, (i) accumulate more GAG that remain attached to the substratum and cell surface; (ii) respond to LDL with a similar degree of increase in GAG accumulation, but more of the increase is found in the substratum and cell surface compartment; and (iii) accumulate more intracellular free cholesterol in response to LDL.     

Stress-relaxation and contraction of a collagen matrix induces expression of TGF-beta and triggers apoptosis in dermal fibroblasts.

Extracellular matrix serves as a scaffold for cells and can also regulate gene expression and ultimately cell behaviour. In this study, we compared the effects of three forms of type I collagen matrix, which differed only in their mechanical properties, and plastic on the expression of transforming growth factor-beta1 (TGF-beta1), matrix metalloproteinase-1 (collagenase), and type I collagen and on the growth and survival of human dermal fibroblasts. These effects were correlated with alterations in cell morphology and organization of intracellular actin. Cells in detached or stress-relaxed matrices were spherical, lacked stress fibres, and showed increased TGF-beta1 mRNA compared to the cells in anchored collagen matrices or on plastic, which were polygonal or bipolar and formed stress fibres. The levels of TGF-beta measured by bioassay were higher in detached and stress-relaxed collagen matrices, than in anchored collagen matrices. Cells on plastic contained little or no immunoreactive TGF-beta, while most cells in collagen matrices were stained. The levels of collagenase mRNA were significantly higher in all the collagen matrix cultures compared to those on plastic, but there were no statistically significant differences between them. Levels of mRNA for procollagen type I were not significantly affected by culture in the collagen matrices. Apoptotic fibroblasts were detected by the TUNEL assay in detached (5.7%) and to a lesser extent in stress-relaxed (2.2%) matrices, but none were observed in anchored collagen matrices or on plastic. These results show that alterations in the mechanical properties of matrix can induce the expression of TGF-beta and trigger apoptosis in dermal fibroblasts. They further suggest that inability to reorganize this matrix could be responsible for the maintenance of the fibroproliferative phenotype associated with fibroblasts in hypertrophic scarring.     

Functional differentiation of alveolar type II epithelial cells in vitro: effects of cell shape, cell-matrix interactions and cell-cell interactions

Alveolar type II epithelial cells rapidly lose characteristics of differentiated function when cultured on plastic dishes. We have attempted to circumvent this problem by culturing type II cells under conditions that might better reproduce their environment in vivo. Cell-matrix interactions were studied by culturing isolated adult rat type II cells on Engelbreth-Holm-Swarm (EHS) tumor basement membrane. Aggregates of type II cells formed on the surface of the matrix during 4 days in culture. Microscopic examination of these aggregates revealed cuboidal cells that retained more characteristics of differentiated type II cells than did cells cultured on plastic. Type II cells cultured on EHS matrix incorporated a higher percentage of acetate into phosphatidylcholine (PC) than did cells on plastic, and a higher percentage of this PC was saturated. Phosphatidylglycerol (PG) synthesis by these cells was no different from that seen in cells on plastic. The effects of cell-cell interactions and cell shape were evaluated by culturing type II cells on feeder layers that in turn were grown on collagen gels. The feeder layer cells included fetal rat lung fibroblasts, adult rat lung fibroblasts, fetal rat skin fibroblasts, bovine aortic endothelial cells, and rat mammary tumor epithelial cells. One-half of the gels remained attached to the culture dish and one-half of the gels were detached after 24 h and allowed to float free in the medium. Type II cells grown in association with any of the attached feeder layers became flattened and lost their differentiated phenotype. These cells incorporated no greater percentage of acetate into PC than did cells on plastic. Saturated PC synthesis was modestly increased. PG synthesis declined in parallel with that seen in cells cultured on plastic. Type II cells cultured on feeder layers that were detached assumed their native cuboidal shape and also exhibited many morphological characteristics of differentiated function. These cells incorporated a significantly greater percentage of acetate into PC compared to cells on either plastic or attached feeder layers. Saturated PC synthesis also increased markedly. These cells, however, incorporated no greater percentage of acetate into PG than did cells on plastic or attached feeder layers. These data suggest an important role for cell shape and cell-matrix interactions and maintenance of type II cell differentiation. The effects of cell-cell interactions, while beneficial, appear to be non-specific.     

Collagen-induced proMMP-2 activation by MT1-MMP in human dermal fibroblasts and the possible role of alpha2beta1 integrins

Culture of human dermal fibroblasts within a three-dimensional matrix composed of native type I collagen fibrils is widely used to study the cellular responses to the extracellular matrix. Upon contact with native type I collagen fibrils human skin fibroblasts activate latent 72-kDa type IV collagenase/ gelatinase (MMP-2) to its active 59- and 62-kDa forms. This activation did not occur when cells were cultured on plastic dishes coated with monomeric type I collagen or its denatured form, gelatin. Activation could be inhibited by antibodies against MT1-MMP, by the addition of TIMP-2 and by prevention of MT1-MMP processing. MT1-MMP protein was detected at low levels as active protein in fibroblasts cultured as monolayers. In collagen gel cultures, an increase of the active, 60-kDa MT1-MMP and an additional 63-kDa protein corresponding to inactive MT1-MMP was detected. Incubation of medium containing latent MMP-2 with cell membranes isolated from fibroblasts grown in collagen gels caused activation of the enzyme. Furthermore, regulation of MT1-MMP expression in collagen cultures seems to be mediated by alpha2beta1 integrins. These studies suggest that activation of the proMMP-2 is regulated at the cell surface by a mechanism which is sensitive to cell culture in contact with physiologically relevant matrices and which depends on the ratio of proenzyme and the specific inhibitor TIMP-2.     

Influence of the extracellular matrix on the proliferative response of human skin fibroblasts to serum and purified platelet-derived growth factor

The culture of adult human skin fibroblasts on reconstituted bovine type 1 fibrillar collagen gels, ranging in concentration from 2.5-35.0 mg/ml, results in a reduction in proliferation rate by 40%-60% as measured by (3H) thymidine incorporation. The suppressive effect was noted when cells were cultured in both human and bovine serum. Drying the gels into thin films abolishes the suppressive effect of the fibrillar collagen on cell proliferation. Cell attachment studies showed that differences in the proliferation rate of cells on the various substrata were not simply due to differences in initial attachment. Studies with purified platelet-derived growth factor (PDGF) demonstrated that the reduced responsiveness of cells to this factor, when cultured on collagen gels as compared to plastic, was largely responsible for the reduced proliferative activity of the cells when cultured in the presence of serum. The reduced proliferative activity of fibroblasts in response to PDGF, when cultured on collagen gels, was confirmed by total DNA determination. It was shown that the reduced responsiveness of cells to PDGF was not simply because the factor bound to the fibrillar collagen gel or was inaccessible to the cells. The data indicate that the reduced proliferation rate of fibroblasts cultured on collagen gels is a direct result of the influence of the extracellular matrix on the cells' ability to respond to a soluble mitogenic mediator.     

Extracellular matrix regulates Sertoli cell differentiation, testicular cord formation, and germ cell development in vitro

Sertoli cell preparations isolated from 10-day-old rats were cultured on three different substrates: plastic, a matrix deposited by co-culture of Sertoli and peritubular myoid cells, and a reconstituted basement membrane gel from the EHS tumor. When grown on plastic, Sertoli cells formed a squamous monolayer that did not retain contaminating germ cells. Grown on the matrix deposited by Sertoli-myoid cell co-cultures, Sertoli cells were more cuboidal and supported some germ cells but did not allow them to differentiate. After 3 wk however, the Sertoli cells flattened to resemble those grown on plastic. In contrast, the Sertoli cells grown on top of the reconstituted basement membrane formed polarized monolayers virtually identical to Sertoli cells in vivo. They were columnar with an elaborate cytoskeleton. In addition, they had characteristic basally located tight junctions and maintained germ cells for at least 5 wk in the basal aspect of the monolayer. However, germ cells did not differentiate. Total protein, androgen binding protein, transferrin, and type I collagen secretion were markedly greater when Sertoli cells were grown on the extracellular matrices than when they were grown on plastic. When Sertoli cells were cultured within rather than on top of reconstituted basement membrane gels they reorganized into cords. After one week, tight junctional complexes formed between adjacent Sertoli cells, functionally compartmentalizing the cords into central (adluminal) and peripheral (basal) compartments. Germ cells within the cords continued to differentiate. Thus, Sertoli cells cultured on top of extracellular matrix components assume a phenotype and morphology more characteristic of the in vivo, differentiated cells. Growing Sertoli cells within reconstituted basement membrane gels induces a morphogenesis of the cells into cords, which closely resemble the organ from which the cells were dissociated and which provide an environment permissive for germ cell differentiation.     

Regulation of collagen synthesis in fibroblasts within a three-dimensional collagen gel

Fibroblasts cultivated within a three-dimensional collagen gel display an elongated, spindle-like morphology, reduce their proliferation rate, contact the gel to a very dense tissue, and modify their metabolic activity as compared to monolayer cultures. Collagen synthesis measured as protein-bound hydroxyproline is reduced to 5% of the values found in monolayer culture. The reduction involving type I and type III collagen is due to decreased de novo synthesis and not to enhanced degradation. Dot blot hybridization, Northern blot analysis, and in situ hybridization using collagen I- and III-specific cDNA probes demonstrate that reduced biosynthesis rates are reflected by a marked reduction of pro alpha 1 (I), pro alpha 2 (I), and pro alpha 1 (III) collagen mRNA indicating pretranslational regulation. A similar reduction was observed for actin mRNA whereas levels of tubulin mRNA were similar for fibroblasts in monolayer culture or cultivated within the three-dimensional collagen gels. The data suggest a specific reprogramming of various cellular activities in response to contact with the reconstituted extracellular matrix.     

Pulmonary macrophages alter the collagen phenotype of lung fibroblasts

Cultured lung fibroblasts produced and secreted interstitial collagen types I and III. The relative proportion of type III collagen increased as a linear function of cell density, with confluent cultures producing 8.6% type III collagen. When human lung fibroblasts were cultured in the presence of newly harvested lung macrophages, the proportion of type III collagen secreted rose to 15.5%. This high level of type III collagen synthesis was greater than could be induced by withdrawal of serum, a perturbation known to alter the proportion of types I and III collagen synthesized by fibroblasts. This effect on fibroblast phenotype was independent of cell density, as both low and high density cultures of fibroblasts responded similarly when cultured with macrophages. There was no evidence that fibroblasts synthesize new or different collagen types (such as type I trimer) in response to macrophages. Optimal conditions for eliciting an effect on fibroblast connective tissue metabolism required interaction of the two cell types for 5–8 days. These in vitro changes are analogous to the sequence of interactions and changes in connective tissue metabolism seen during recovery from tissue injury.     

A comparison of the effects of different substrata on chondrocyte morphology and the synthesis of collagen types IX and X

Summary Embryonic chick sternal chondrocytes were cultured either within three dimensional gels of type I collagen, type II collagen or agar, or as monolayers on plastic dishes coated with air-dried films of these matrix macromolecules. It was observed that cell shape and cell growth varied markedly between the different culture conditions. Flattened monolayers of cells on plastic or films of type I or type II collagen, proliferated more rapidly and reached a higher final cell density per culture than the more rounded cells found in the cultures on agar films or within three-dimensional gels. Biosynthetic studies demonstrated that in addition to the synthesis of type II collagen, all the cultures were producing collagen types IX and X. Chondrocytes cultured on plastic or films of the different matrix macromolecules all showed a similar expression of types IX and X collagen, independent of whether they displayed a flattened or round cell morphology. In contrast, marked variations in the proportions of the minor collagens, particularly type X collagen, were observed when the cells were cultured within three-dimensional gels. The data suggest that direct interaction of the cell surface with matrix constituents displaying a particular spatial array could be an important aspect in the control of type IX and X collagen expression by chondrocytes. The financial support of the Arthritis & Rheumatism Council and the Medical Research Council is gratefully acknowledged.     

Growth of cells from esophageal squamous cell carcinoma biopsies in a reconstituted basement membrane

Cells from esophageal carcinoma biopsies were cultured on or inside a three-dimensional basement membrane matrix (matrigel). Their growth was compared to cells derived from control esophageal biopsies. Cells from both normal and neoplastic tissue attached poorly to tissue culture plastic. Matrigel coating improved adhesion and growth. When cells were grown inside a matrigel matrix, a striking difference was noticed between carcinoma cells and controls. Carcinoma cells grew invasively in the three-dimensional substrate and digested the matrix after a few weeks; control cells did not grow and only a few necrotic cells were visible with time. Matrigel provided a better growth substrate than plastic for esophageal derived cells and discriminated between carcinoma-derived and control cells when used as a three-dimensional growth substrate. Our studies suggest a possible use of matrigel for the selective growth of tumor cells derived directly from tissue biopsies.     

Specific association of iduronic acid-rich dermatan sulphate with the extracellular matrix of human skin fibroblasts cultured on collagen gels.

Human skin fibroblasts cultured on collagen gels produced two dermatan sulphate species, one, enriched in iduronic acid residues, that bound specifically to the collagenous fibres of the gel, the other, enriched in glucuronic acid, that accumulated in the culture medium. Collagen-binding and collagen-non-binding dermatan sulphates were also produced by cells grown on plastic surfaces, but in these cultures each constituent was released into the growth medium. Net synthesis of dermatan sulphate was 3-fold higher in cells maintained on collagen gels. In contrast, heparan sulphate synthesis was not influenced by the nature of the culture surface. The concentration of heparan sulphate in surface-membrane extracts was similar for cells grown on plastic and on collagen gels, but cells cultured on collagen showed a notable increase in the content of surface-membrane dermatan sulphate. The patterns of synthesis and distribution of sulphated glycosaminoglycans observed in skin fibroblasts maintained on collagen gels may reflect differentiated cellular functions.     

Effect of protein-hydroxyethylmethacrylate hydrogels on cultured endothelial cells

The use of protein hydroxy ethylmethacrylate (HEMA) hydrogels to control cell morphology and growth, as well as the synthesis of extracellular matrix components, is described in this communication. HEMA hydrogels prepared with collagen support growth of embryonic lung fibroblasts (IMR-90), as well as bovine aortic and pulmonary artery endothelial cells at a level comparable to the respective cells grown on tissue culture surfaces. On the other hand, HEMA hydrogels prepared with solubilized elastin inhibit the fibroblast growth and prevent both types of endothelial cell cultures from achieving their normal morphology. These morphologically altered endothelial cells resume a normal cobblestone-like appearance when subcultivated from the elastin-HEMA hydrogels to tissue culture plastic. When pulsed with [14C]proline, the procollagens synthesized by the endothelial cells on the different surfaces vary, as shown by immunoprecipitation and polyacrylamide gel electrophoresis. On the standard tissue culture plastic, the confluent cells produce mainly type III procollagen in the medium, whereas those endothelial cells grown on collagen and elastin-HEMA hydrogels synthesize primarily type I procollagen (much like sprouting cells on tissue culture plastic), regardless of their morphology.     

Maintenance of differentiated phenotype of cultured rat hepatic lipocytes by basement membrane matrix

This study examined the role of extracellular matrix in regulating matrix phenotype of hepatic lipocytes, the major source of matrix in liver. Lipocytes (Ito, stellate, or fat-storing cells) were purified from normal rat liver and established in primary culture on either uncoated plastic, plastic coated with individual matrix proteins, or a "complete" gel matrix, a basement membrane-like matrix derived from the Engelbreth-Holm-Swarm (EHS) murine tumor. The ultrastructure of lipocytes cultured on the gel matrix resembled that of cells in normal liver, whereas lipocytes on plastic had dispersed nuclear chromatin and expanded rough endoplasmic reticulum, consistent with active proliferation and secretion. Lipocytes on the gel matrix exhibited no proliferative activity; cells maintained on plastic proliferated and produced type I collagen predominantly. Total collagen secretion by lipocytes on the gel matrix was 29% of that of cells on plastic, and consisted of type III collagen only. This difference extended to proteoglycan production, which was less than 5% of the amount produced by cells in conventional culture on plastic. The effects of the EHS gel were not reproduced by the individual components of the gel (laminin, type IV collagen, and heparan sulfate proteoglycan) or by a type I collagen gel. They were also reversible upon transfer of the cells to conventional culture. In contrast to lipocytes, collagen synthesis by hepatocytes was similar whether cultured on EHS gel or on plastic. These results show that the extracellular matrix can modulate matrix protein production by lipocytes and imply that, in early hepatic inflammation, changes in the hepatic subendothelial matrix may underlie stimulation of lipocyte matrix production and progression of the fibrotic process.     

Type I collagen synthesis by corneal endothelial cells modulated by polymorphonuclear leukocytes

When primary corneal endothelial cells were grown in polymorphonuclear leukocyte (PMN)-conditioned medium, a minor population of cells acquired fibroblastic morphology. Such modulated endothelial cells supported by PMN-conditioned medium grew much faster than the major nonresponding polygonal endothelial cell. Upon serial passages, the modulated endothelial cells became the dominant cell type and eventually formed a homogeneous fibroblastic culture. At the same time, phenotypic changes of collagen were observed. The primary endothelial cells grown in PMN-conditioned medium, consisting of responding elongated cells and nonresponding polygonal endothelial cells, produced predominantly type IV collagen with type III collagen as a minor component. As cells were subcultured and fibroblastic cells became dominant, type IV collagen synthesis was dramatically decreased and type I collagen synthesis was increased in parallel. When they reached the fully modulated stage, the cultures synthesized types I and III collagen, with type I accounting for 75-85% of the total. Type I collagen synthesized by the fibroblastic endothelial cells shared common characteristics with known type I collagen, such as migration behavior on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, CNBr peptide profiles, and immunologic identity. Thus, PMNs apparently contribute to the modulation of corneal endothelial cells, causing them to acquire characteristics of fibroblasts, cell multilayering, and deposition of interstitial extracellular matrix composed predominantly of interstitial type I collagen.