Collagen synthesis by bone marrow stromal cells: a quantitative study

Collagen-producing, marrow-derived adherent cells (MDAC) provide a microenvironment which supports haemopoietic stem cell (HSC) proliferation in vitro . To investigate the relationship between collagen-producing cells in intact marrow and in MDAC cultures, quantitative studies of collagen synthesis were performed.
Collagen synthesis by fresh murine bone marrow cells was 0.22% of total protein synthesis and collagen types I and III were synthesized in a ratio of approximately 5:1. During MDAC cultivation, the collagen synthesis fraction increased to 2.3%. The relative amounts of type I and type III remained more or less constant. The MDAC monolayers showed characteristic pleomorphism, and were shown to support HSC proliferation in continuous bone marrow culture for periods of at least 5 weeks.
These results suggest that MDAC culture, under the conditions reported, selects a specific type of bone marrow connective tissue cell, probably a reticulum cell, which synthesizes collagen types I and III in a ratio of approximately 5:1.     

Characterization of Marrow-Derived Adherent Cells

Cultured, marrow-derived, adherent cells (MDAC) provide a microenvironment which supports the proliferation of haemopoietic stem cells (HSC) for extended periods in vitro. Morphological characterization suggested that MDAC populations consisted of a variety of cell types, including mononuclear phagocytes, fibroblastoid cells, fat cells and vascular endothelial cells. Recently performed functional characterization studies suggest that they consist largely of collagen-producing, fibroblastic cells. MDAC were not, however, examined systematically for endothelial cell characteristics. Unrecharged cultures of MDAC, shown in parallel studies to support in vitro haemopoiesis, were examined for endothelial cell markers. These included the presence of Weibel-Palade bodies and synthesis of factor VIII related antigen. They were also examined biochemically for synthesis of basement membrane (type IV) collagen. The results of these investigations were negative in all cultures examined. It is thus concluded that vascular endothelial cells are not present as a significant component of the unrecharged MDAC population and do not, therefore, contribute to the functional haemopoietic microenvironment in vitro or in vivo.     

Characterization of marrow-derived adherent cells. Evidence against an endothelial subpopulation

Cultured, marrow-derived, adherent cells (MDAC) provide a microenvironment which supports the proliferation of haemopoietic stem cells (HSC) for extended periods in vitro. Morphological characterization suggested that MDAC populations consisted of a variety of cell types, including mononuclear phagocytes, fibroblastoid cells, fat cells and vascular endothelial cells. Recently performed functional characterization studies suggest that they consist largely of collagen-producing, fibroblastic cells. MDAC were not, however, examined systematically for endothelial cell characteristics. Unrecharged cultures of MDAC, shown in parallel studies to support in vitro haemopoiesis, were examined for endothelial cell markers. These included the presence of Weibel-Palade bodies and synthesis of factor VII related antigen. They were also examined biochemically for synthesis of basement membrane (type IV) collagen. The results of these investigations were negative in all cultures examined. It is thus concluded that vascular endothelial cells are not present as a significant component of the unrecharged MDAC population and do not, therefore, contribute to the functional haemopoietic microenvironment in vitro or in vitro.     

Codistribution of pericellular matrix proteins in cultured fibroblasts and loss in transformation: fibronectin and procollagen.

Antibodies to fibronectin and to distinct types of procollagens and collagens were used in immunofluorescent staining to localize these proteins in cell cultures. Normal human skin or lung fibroblasts produced a fibrillar pericellular matrix in which fibronectin and procollagen (types I and III) showed extensive codistribution. Fibronectin and procollagen were synthesized by the same cells as judged by double-stain immunofluorescence. Pericellular procollagen was specifically digested with collagenase without an effect on the fibrillar distribution of matrix fibronectin. Brief treatment with trypsin removed both matrix proteins. The human tumor cell lines HT-1080 (fibrosarcoma) and RD (rhabdomyosarcoma) produced little or no matrix fibronectin or procollagen. At sites of cell contact, simian virus 40-transformed lung fibroblasts (VA13) produced small amounts of pericellular fibrillar matrix fibronectin that codistributed with procollagen type I. Intracellular fibronectin and procollagen were visualized in all of these human sarcoma cell lines. When chicken embryo fibroblasts infected with a T class mutant (NY68) of Rous sarcoma virus temperature-sensitive for transformation were maintained at the nonpermissive temperature (41 degrees ) the cells had normal phenotype and a fibrillar matrix containing fibronectin and procollagen was present. At the permissive temperature (35 degrees ), the cells showed transformed phenotype and the matrix was lost. The failure to produce a pericellular fibronectin/collagen matrix may account for several phenotypic characteristics of transformed cultured fibroblasts.     

Acetaldehyde and lactate stimulate collagen synthesis of cultured baboon liver myofibroblasts

Cells with electron-microscopic characteristics of myofibroblasts were isolated from baboon liver biopsy specimens by collagenase digestion and Percoll density gradient centrifugation and then cultured. The cultures consisted of only one cell type. By immunofluorescence, these cells synthesized collagen types I, III, and IV and laminin. Typical features of myofibroblasts were maintained throughout many passages in the culture. To study the effects of ethanol (and its oxidation product acetaldehyde and associated metabolite lactate) on myofibroblast collagen synthesis, the cell cultures were incubated for 24 h in a medium containing either 50 mM ethanol, 200 microM acetaldehyde, or 5 mM lactate. The cells did not contain significant alcohol dehydrogenase activity. Acetaldehyde stimulated significantly (p less than 0.05) myofibroblast collagen synthesis without changing noncollagen protein synthesis or proline pools. Lactate caused a significant (p less than 0.02) increase in intracellular proline pool and collagen synthesis. Ethanol itself did not have any effect on collagen synthesis of myofibroblasts. The stimulation of collagen synthesis of hepatic myofibroblasts by acetaldehyde and lactate may contribute to the development of alcoholic liver fibrosis, as alcohol intake is known to elevate acetaldehyde and lactate in tissues and blood.     

Laminin inhibits estrogen action in human breast cancer cells

Breast tumors that lack estrogen responsiveness have a poor prognosis. Despite the critical importance to breast cancer treatment, little is known about the loss of estrogen responsiveness and the development of antiestrogen resistance. We have examined the regulation of estrogen-induced proliferation, estrogen regulation of progesterone receptor (PR) expression, and estrogen signaling pathways in estrogen receptor (ER) positive (MCF-7 and T47D) breast cancer cell lines by specific extracellular matrix proteins (ECM) under serum-free conditions. Estrogen, supplemented with submaximal concentrations of insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF), stimulated DNA synthesis of MCF-7 cells 7- to 10-fold and T47D cells 2-fold on collagen I or fibronectin. However, estrogen-induced proliferation was greatly reduced on laminin. In contrast, IGF-I or EGF, alone, stimulated proliferation of MCF-7 and T47D cells on all ECM. Thus, ER+ breast cancer cells were not refractory to mitogens when cultured on laminin. Similarly, estrogen induction of PR occurred on fibronectin or collagen I, but not on laminin. While ER content was similar on all ECM, estrogen stimulation of estrogen response element (ERE)-luciferase activity was significantly lower in MCF-7 cells cultured on laminin. Therefore, changes in ECM composition that occur in breast cancer may alter estrogen-responsiveness and the effectiveness of antiestrogen therapies in ER+ breast cancer cells.     

Extracellular matrix production by the adherent cells of long-term murine bone marrow cultures

We have studied the deposition of extracellular matrix proteins in the adherent stroma of long-term murine bone marrow cultures. Stable hematopoiesis was maintained for greater than 12 wk. At selected intervals, culture dishes were sacrificed by removing all nonadherent cells and air drying the dishes. The adherent stromal layer was analyzed for the presence of intracellular and extracellular collagen, fibronectin, and laminin using double immunofluorescent staining with specific antisera against these matrix components. In cultures examined during the first 2 wk, large numbers of stromal cells contained collagen, fibronectin, and laminin. Over the next 2 wk, an extensive extracellular network of fibronectin, laminin, and collagen was deposited on the dishes, which persisted throughout the life of the cultures. In contrast to a previous report, we detected substantial numbers of endothelial cells by means of immunofluorescent staining of stromal cells with antisera to type IV collagen, laminin, and factor VIII antigen. Although deposition of these extracellular matrix proteins coincides with onset of active hematopoietic cell production, the relative roles of the stromal cells and the extracellular matrix in supporting hematopoiesis in murine bone marrow cell cultures remain to be determined.     

Factor XIII of blood coagulation modulates collagen biosynthesis by fibroblasts in vitro

The effect of activated factor XIII (FXIIIa), the transglutaminase of blood coagulation, on some cellular functions was studied in skin and lung fibroblasts in vitro. FXIIIa repressed the overall protein synthesis and mainly collagen synthesis in a concentration-dependent manner and induced modifications in the proportion of the different types of newly synthesized collagen. The repression of collagen synthesis occurred in cells cultured on plastic (-40%), on coated fibronectin (-53%), on coated collagens (-38%) and within a collagen lattice (-16%). Preincubation of the cells with FXIIIa and labelling in its absence also resulted in such an inhibition. However, when embedded into a fibrin lattice cross-linked by FXIIIa, fibroblasts displayed a higher biosynthetic activity than in untreated fibrin gel. These results suggest that FXIIIa acts through a modulation of the cell-matrix interactions.     

Integrin receptors on aortic smooth muscle cells mediate adhesion to fibronectin, laminin, and collagen.

Extracellular matrix receptors on vascular smooth muscle cells help in anchoring the cells during contraction and in promoting cellular migration after vessel injury. We found that rat aortic smooth muscle cells attach to surfaces coated with fibronectin, laminin, and collagen types I and IV. Cell attachment to these substrates appears to be mediated by members of the beta 1 integrin family of extracellular matrix receptors. Antibodies to the beta 1 subunit not only demonstrated the presence of integrin complexes in focal adhesion plaques but also blocked cell adhesion to the different substrates. Ligand-affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis isolated a series of receptor complexes that were recognized by antisera to beta 1 integrin receptors. Each of the receptors appeared to be a heterodimer in which one of several alpha subunits shared a common 120-kDa (nonreduced) beta 1 subunit protein. The rat aortic smooth muscle cells had one alpha subunit (150 kDa nonreduced, 140 kDa reduced) that bound exclusively to fibronectin. There was a second alpha subunit (150 kDa nonreduced, 160 kDa reduced) that bound exclusively to collagen type I. In addition, there was a third alpha subunit (185 kDa nonreduced, 200 kDa reduced) that was promiscuous and bound to collagen types I and IV as well as to laminin; the 185-kDa alpha subunit appeared to bind to collagen more efficiently than it did to laminin. Thus, smooth muscle cells express multiple integrin receptors with different ligand specificities that appear to mediate cell interactions with the extracellular matrix.     

Extracellular matrix constituents affect superficial gastric epithelial cell adhesion

Abstract The interfoveolar and upper gastric pit cells become necrotic and slough off after superficial luminal injury to the gastric mucosa. The subsequent rapid epithelial restitution of the wound is dependent on an intact basal lamina upon which viable mucous cells migrate. Several lines of evidence suggest that migrating mucous cells recognize specific moieties in the basal lamina which would then affect restitution and the ability of the gastric mucosa to be repaired. Therefore, this study examined the effect of three individual protein constituents of the extracellular matrix, laminin, fibronectin and type IV collagen as well as a synthetic basal lamina, Matrigel, on adherence of mucous cells isolated from guinea-pig stomach to these substrates in culture. After 3 h, approximately 40% of the cells adhered to Matrigel, 25% to both collagen IV and fibronectin, but only about 10% to laminin and 3% to uncoated plastic substrates. Disruption of protein synthesis by pre-incubation with cyclohexamide significantly reduced adherence to Matrigel and collagen IV but not laminin, fibronectin or plastic substrates. These results suggest that gastric mucous cells have multiple receptors for extracellular matrix proteins (ligands) which influence the adherence and probably the migration of these cells. Furthermore, some of these receptors are synthesized in response to moieties in the substrate itself.     

Cellular interactions promote tissue-specific function, biomatrix deposition and junctional communication of primary cultured hepatocytes

Hepatocytes, prepared from normal adult rat liver, were seeded onto a collagen substratum and cultured alone or in the presence of rat liver endothelial cells. When hepatocytes were cultured alone in a hormonally defined serum-free medium, decreased albumin production and rapid morphological deterioration of bile canaliculi structures and gap junctions occurred within 4 to 5 days. In contrast, hepatocytes cocultured with liver mesenchymal cells remained morphologically intact and biochemically functional for at least 4 weeks. They reorganized into small islands, continued to secrete high levels of albumin, did not express alpha-fetoprotein (a fetal marker), and remained strongly dye coupled. All of the hepatocytes synthesized albumin and retained their gap junctional channels. No junctional communication was observed between hepatocytes and endothelial cells. Long fibers containing fibronectin, Type I collagen and laminin distributed over the hepatocytes were induced in coculture but never appeared in hepatocytes cultured alone. Moreover, supplementation of the hormonally defined medium with phenobarbital and dimethyl sulfoxide, both of which improve the life span and functional activities of cultured hepatocytes, failed to induce reticulin fiber formation in pure culture of hepatocytes. The modulation of albumin secretion, biomatrix deposition and junctional communication observed in hepatocytes cultured with sinusoidal liver cells was also obtained when hepatocytes were in association with various epithelial or mesenchymal cells [rat liver epithelial cells (T51B), mouse embryonic fibroblasts (NIH 3T3), human or rat dermal fibroblasts and bovine aorta endothelial cells (AG 4762)].     

Matrix metalloproteinase-2 activation in human hepatic fibrosis regulation by cell-matrix interactions.

Matrix metalloproteinase-2 (MMP-2) is involved in extracellular matrix remodeling. It is secreted as a proenzyme and activated by membrane type-MMPs (MT-MMP), such as MT1-MMP. In liver fibrosis, MMP-2 is highly expressed in myofibroblasts and may have a profibrogenic role. The mechanisms of its activation in the liver are still unclear. The aim of this work was to show that pro-MMP-2 is efficiently activated in human fibrotic liver and to investigate the role of cell-matrix interactions in this process. Liver specimens obtained from patients with active cirrhosis were compared to normal liver specimens. Human hepatic myofibroblasts were cultured either on plastic, fibronectin, laminin, or on collagen I gels. MMP-2 activity was visualized by gelatin zymography. MMP-2 active form (59 kd) was detected in active cirrhosis but not in normal liver. Myofibroblasts cultured on plastic, fibronectin, or laminin predominantly expressed inactive pro-MMP-2 (66 kd). In contrast, myofibroblasts cultured on collagen I markedly activated the enzyme. Similar results were obtained using membrane fractions from cells previously cultured on collagen or plastic. Activation was inhibited by the tissue inhibitor of metalloproteinases-2 but not by tissue inhibitor of metalloproteinases-1, implicating a MT-MMP-mediated process. Culture on collagen I up-regulated MT1-MMP protein detected by Western blotting, but decreased MT1-MMP mRNA. This study shows that MMP-2 is activated in fibrotic liver. It suggests that interactions between collagen I and myofibroblasts promote this process through a post-translational increase of MT1-MMP expression in these cells.     

Dexamethasone alters messenger RNA levels but not synthesis of collagens,fibronectin, or laminin by cultured rat fat-storing cells

Glucocorticoids have been shown to suppress collagen synthesis and gene expression by fibroblasts. However, little is known about their effects on fat-storing cells, the major matrix-producing cells in liver fibrosis. In this study we investigated the effect of dexamethasone on the extracellular matrix expression by cultured rat fat-storing cells. Fat-storing cells were isolated from male Wistar rats by collagenase/pronase digestion and purified by density gradient centrifugation. Fat-storing cells in early primary culture (3-day-old, representing a relatively quiescent phenotype) and in subculture (one passage, about 2-week-old, representing an activated phenotype) were treated with 10^-6 mol/L dexamethasone for messenger RNA (mRNA) study or with 10^-8 to 10^-6 mol/L dexamethasone for protein study. Expression of collagen type I, III, IV, fibronectin, and laminin was analyzed at the mRNA level by Northern hybridization, and at the protein level by metabolic labeling and immunoprecipitation. Dexamethasone had a variable effect on the expression of collagen alpha1(I) mRNA level. While a tendency for modest suppression was observed (5%-50%) in primary cells, the difference was not statistically significant. Variable response was observed in subcultured cells. Collagen alpha1(III) mRNA level showed a tendency for stimulation. Dexamethasone stimulated the expression of collagen alpha1 (IV), fibronectin, and laminin B1 mRNA levels by 1.4-, 2.4-, and 1.6-fold respectively, in primary fat-storing cells. Subcultured cells showed a similar response, but the magnitude of stimulation was more variable than that of primary cells. Unexpectedly, at the protein level dexamethasone had no effect on the expression of these proteins. Our results indicate that glucocorticoids do not possess a net suppressive effect on extracellular matrix synthesis by fat-storing cells. Beneficial effects of glucocorticoids may be attributable to other mechanisms of action, such as their anti-inflammatory effect. (Hepatology 1996 Jun;23(6):1673-81)     

Hormonal and extracellular matrix regulation of plasminogen activator in a bovine mammary epithelial cell line

The effects of lactogenic hormones on urokinase plasminogen activator (u-PA) produced by bovine mammary epithelial cells (MAC-T) were examined. High levels of u-PA activity were detected in growth arrested cells cultured on plastic. This suggests that high levels of PA activity alone are not sufficient to induce proliferation of bovine mammary epithelial cells. Cells were cultured on various extracellular matrices: plastic, fibronectin, collagen, matrigel, and laminin. Basal levels of u-PA activity in media from MAC-T cells cultured on matrigel were 1.6-, 2-, 2- and 3.5-fold higher than that of cells cultured on plastic, fibronectin, collagen, and laminin, respectively. Insulin increased (P<0.01) mammary epithelial u-PA activity on a per cell basis, an effect observed irrespectively of the type of extracellular matrix onto which cells were cultured. Not unexpectedly, insulin-like growth factor (IGF)-I, Des(1-3) IGF-I and IGF-II increased mammary epithelial u-PA activity on a per cell basis and u-PA mRNA levels, thus, mimicking the effect of insulin. Dexamethasone suppressed (P<0.01) u-PA activity but was unable to suppress the insulin-induced increase in u-PA activity of cells cultured on various extracellular matrices. These data indicate that u-PA activity is modulated by both lactogenic hormones and the type of extracellular matrix.     

Transforming growth factor-beta effects on morphology of immature rat Leydig cells

Transforming growth factor-beta (TGF beta) has been shown to regulate steroid production and DNA synthesis in rat Leydig cells. We have investigated the effects of TGF beta on the secretion of extracellular matrix (ECM) proteins and on the cytoskeleton of immature rat Leydig cells in vitro. TGF beta caused significant morphological changes in Leydig cells, which were accompanied by significant increases in secretion of fibronectin, laminin and collagen IV and rearrangement of actin filaments in TGF beta-treated cells. The cells cultured on plates pre-coated with fibronectin or fibronectin plus laminin and collagen IV, displayed morphological and cytoskeletal changes similar to those induced by TGF beta. Immunofluorescence localization studies revealed significantly higher fibronectin staining in Leydig cells in adult animals and in LH-treated immature animals than those in untreated immature animals. We conclude that TGF beta participates in the morphological differentiation of immature Leydig cells into adult Leydig cells in the rat testis by inducing the expression of ECM proteins.     

Extracellular matrix proteins in cardiac fibroblasts derived from rat hearts with chronic pressure overload: effects of beta-receptor blockade

Left ventricular hypertrophy (LVH) is accompanied by progressive accumulations of extracellular matrix proteins. They are produced predominantly by cardiac fibroblasts that surround the cardiac myocytes. The aim of this study was to emphasize the role of a combined approach using both in vivo and in vitro studies to elucidate the effects of carvedilol on cardiac remodeling. We therefore used an established model of supravalvular aortic banding and cardiac fibroblasts. LVH was induced by banding of the ascending aorta. Male Wistar rats were allocated to four groups: sham-operated, sham+carvedilol, aortic stenosis (AS), and AS+carvedilol. Treatment time was four weeks. Fibroblasts were isolated from the entire left ventricle of sham and AS rats. Carvedilol/metoprolol/prazosin were added (0.1, 1.0 and 10 microM; 24 h). In addition, interferon- gamma was applied for 24 h (10, 100 and 1000 IU). AS rats revealed increased LV weights (+27%) and cardiomyocyte widths as compared to sham-operated rats (1.6-fold, P<0.01). Carvedilol reduced LVH by 20%. This finding was accompanied by a decrease of laminin, fibronectin, collagen I and III in vivo. Collagen I/III and fibronectin were increased in fibroblasts of AS v sham rats (P<0.0001, each). Carvedilol reduced collagen I, III and fibronectin by 40/60/35% (0.1 microM; P<0.001) irrespective of LVH. Carvedilol had no effects on collagen IV and laminin. Carvedilol dose-dependently reduced the proliferation rate by 20% at 0.1 microM(P<0.0001). Metoprolol and prazosin had no effect on the expression of extracellular matrix proteins and on the proliferation of the cells of either origin. Interferon- gamma blunted the proliferation rate of cultured fibroblasts and lead to a significant decrease in extracellular matrix deposits. These results indicate that the effects of carvedilol may be due to the antiproliferative or antioxidative properties of this unselective beta-adrenergic receptor antagonist. These changes of the extracellular matrix represent a new mechanism of carvedilol that may contribute to the observed beneficial effects in congestive heart failure.     

Establishment of an adherent cell feeder layer from human umbilical cord blood for support of long-term hematopoietic progenitor cell growth.

Previous attempts to establish a stromal cell feeder layer from human umbilical cord blood (HUCB) have met with very limited success. It has been suggested that there is an insufficient number of stromal precursor cells in HUCB to form a hematopoietic-supporting feeder layer in primary cultures. The present study shows that HUCB does contain a significant accessory cell population that routinely develops into a confluent, adherent cell layer under defined primary culture conditions. HUCB-derived adherent layers were shown to support long-term hematopoietic activity for an average of 4 months. This was achieved by using a customized coverslip with a modified surface structure as the cell attachment substratum and using a specialized culture feeding regime. We have characterized the various cell types (including fibroblasts, macrophages, and endothelial cells) and extracellular matrix proteins (including fibronectin, collagen III, and laminin) that were present in abundance in the HUCB-derived adherent cell layer. In contrast, oil red O-staining fat cells were rarely detected. ELISA and bioassays showed that stem cell factor and interleukin 6 were produced by the HUCB stromal cell cultures, but interleukin 3 or granulocyte/macrophage colony-stimulating factor was not detected. Application of this hematopoietic culture system to transgenic and gene therapy studies of stem cells is discussed.     

Expression and function of receptors for extracellular matrix molecules in the differentiation of human megakaryocytes in vitro

The role of adhesive interactions with the extracellular matrix components of the bone marrow (BM) stroma has been widely studied in the differentiation of erythroid and myelomonocytic cells, but not in the megakaryocytic lineage. The development of efficient culture techniques for the production of megakaryocytes (Mks) from CD34⁺ purified BM cells, enables the study of the expression and function of adhesion receptors for collagen (VLA-2), fibronectin (VLA-4 and VLA-5) and laminin (VLA-6) during the maturation of Mks. We have shown that a significant percentage of CFU-MK (roughly 20%) adhere to fibronectin but not to collagen and laminin. The expression and adhesion of Mks developing in liquid culture from BM-CD34⁺ cells were tested at days 4, 7 and 10 of incubation. The expression of VLA-2, VLA-5 and VLA-6 on day 10 cultured Mks enabled purification of intermediate and large polyploid Mks by FACS sorting whereas VLA-4 appeared to label only immature Mks and myeloid cells. We observed that only a small proportion of mature Mks was able to adhere to collagen without spreading at day 10 of culture, whereas 30% of Mks adhered to fibronectin as early as day 4 of incubation, 40% of which also attached to laminin. Our data suggest that VLA-4 may be involved in the adhesion of CFU-MK and immature Mks on fibronectin, then replaced by VLA-5 in the final stages of maturation. The expression of VLA-6 and the number of adherent Mks on laminin increased sharply between day 7 and 10 of incubation. A number of mature polyploid Mks found in day 10 of culture exhibited characteristic features of intense spreading on laminin and fibronectin which were not observed on collagen.     

Effect of high-glucose concentrations on the expression of collagens and fibronectin by fibroblasts in culture

Extracellular matrix macromolecules such as collagen and fibronectin are progressively altered during aging and age-related diseases like diabetes. We investigated the effect of high-glucose concentration (mimicking diabetic conditions) and the influence of in vitro cell aging [comparing 4th-passage fibroblasts (P4) to 15th-passage fibroblasts (P15)] on collagen and fibronectin synthesis. Fibroblasts were incubated at postconfluency with radiolabeled precursors, [³H] proline for collagen, [³⁵S] methionine for fibronectin. We report that in control conditions (5 mM glucose) collagen III production increased with in vitro cell aging. High glucose concentrations (10 and 15 mM) increased specifically collagen III synthesis both at the mRNA and protein levels, without alteration of collagen I production in P4 and P15 cells. Fibronectin synthesis was also increased both during in vitro cell aging and in high glucose-treated P4 fibroblasts. Taken together, these data suggest similarities between changes of phenotypic expression of collagen and fibronectin induced by in vitro cell aging and conditions imitating diabetes.