Ascorbic acid modulates collagen type I gene expression by cells from an eye tissue--trabecular meshwork.

The trabecular meshwork, a specialized tissue in the anterior chamber of the eye, plays a major role in the regulation of aqueous humor outflow. We studied the effects of ascorbic acid, a significant component in the aqueous humor, on gene expression of type I collagen in cultures of bovine trabecular meshwork cells. These cells were plated for 6 days, exposed to ascorbic acid in concentrations of 100, 250 and 500 micrograms/ml for 3 days and labeled with (3H)proline for the last 24 hrs. Cultures that did not receive ascorbic acid served as controls. Bacterial collagenase assays showed enhanced incorporation of (3H)proline into collagenous proteins in cultures treated with 100 and 250 micrograms/ml of ascorbic acid. Gel electrophoresis and fluorography revealed that ascorbic acid caused a 2.6- to 4.9-fold increase in production of alpha 1 (I) and alpha 2(I) collagen chains by trabecular meshwork cells. Such an increase was found, using a cDNA probe specific for pro alpha 1(I) chains, to be accompanied by an increase in steady-state mRNA levels. Similar findings were also yielded from in situ hybridization experiments. These results, coupled with previously demonstrated ascorbate-induced effects on glycosaminoglycan, fibronectin and laminin synthesis, suggest that ascorbic acid is a key mediator of the extracellular matrix production by trabecular meshwork cells. Fluctuations in its concentration may lead to alterations in the makeup and assembly of matrices underlying the cells.     

Collagen biosynthesis by human skin fibroblasts. III. The effects of ascorbic acid on procollagen production and prolyl hydroxylase activity

Human skin fibroblasts were cultured under conditions optimized for collagen synthesis, and the effects of ascorbic acid on procollagen production, proline hydroxylation and the activity of prolyl hydroxylase were examined in cultures. the results indicated that addition of ascorbic acid to confluent monolayer cultures of adult human skin fibroblasts markedly increased the amount of [3H]hydroxyproline synthesized. Ascorbic acid, however, did not increase the synthesis of 3H-labeled collagenous polypeptides assayed independently of hydroxylation of proline residues, nor did it affect the amount of prolyl hydroxylase detectable by an in vitro enzyme assay. Also long-term cultures of the cells or initiation of fibroblast cultures in the presence of ascorbic acid did not lead to an apparent selection of a cell population which might be abnormally responsive to ascorbic acid. Thus, ascorbic acid appears to have one primary action on the synthesis of procollagen by cultured human skin fibroblasts: it is necessary for synthesis of hydroxyproline, and consequently for proper triple helix formation and secretion of procollagen.     

Collagen biosynthesis by human skin fibroblasts III. The effects of ascorbic acid on procollagen production and prolyl hydroxylase activity

Human skin fibroblasts were cultured under conditions optimized for collagen synthesis, and the effects of ascorbic acid on procollagen production, proline hydroxylation and the activity of prolyl hydroxylase were examined in cultures. The results indicated that addition of ascorbic acid to confluent monolayer cultures of adult human skin fibroblasts markedly increased tha amount of [³H]hydroxyproline syntehsized. Ascorbic acid, however, did not increase the synthesis of ³H-labeled collagenous polypeptides assayed independently of hydroxylation of proline residues, nor did it affect the amount of prolyl hydroxylase detectable by an in vitro enzyme assay. Also long-term cultures of the cells or initiation of fibroblast cultures in the presence of ascorbic acid did not lead to an apparent selection of a cell population which might be abnormally responsive to ascorbic acid. Thus, ascorbic acid appears to have one primary action on the synthesis of procollagen by cultured human skin fibroblasts: it is necessary for synthesis of hydroxyproline, and consequently for proper triple helix formation and selection of procollagen.     

Evaluation of the effect of ascorbic acid treatment on wound healing in mice exposed to different doses of fractionated gamma radiation

Alteration of the radiation-induced changes in wound contraction, collagen synthesis and wound histology by ascorbic acid was studied in mice exposed to 10, 16 and 20 Gy of fractionated (2 Gy/fraction) gamma radiation. The animals were given double-distilled water or ascorbic acid daily before exposure to 2 Gy/day of fractionated irradiation. A full-thickness skin wound was created on the dorsum of the irradiated mice, and the progression of wound contraction and collagen synthesis were examined and histological evaluations were carried out at various times after wounding. Irradiation caused a dose-dependent delay in wound contraction, and pretreatment with ascorbic acid resulted in a significant increase in wound contraction. The greatest increase in wound contraction was observed 6 and 9 days after wounding in both groups. Pretreatment with ascorbic acid augmented the synthesis of collagen significantly as revealed by an increase in hydroxyproline content. The collagen deposition and fibroblast and vasculature densities declined in a dose-dependent manner in groups receiving radiation alone as indicated by histological evaluation. Pretreatment with ascorbic acid ameliorated the observed effect significantly. These studies demonstrate that pretreatment with ascorbic acid resulted in a significant reduction of radiation-induced delay in wound healing as shown by earlier wound closure and increased collagen content and fibroblast and vascular densities.     

Opposing effects of ascorbate on collagen and elastin deposition in the neonatal rat aorta

Ascorbic acid plays an important role in connective tissue metabolism, where, among other effects, it acts as a reducing factor in the reactions catalyzed by prolyl and lysyl hydroxylases. In vitro, ascorbic acid has been shown to have a positive influence on collagen synthesis at pre- and/or post-translational levels and a negative effect on elastin production. In the present work, the effects of vitamin C on extracellular matrix deposition have been studied in vivo. Stereological analysis on electron micrographs showed, compared to age-matched controls, a 50 to 60% increase of collagen deposition in the media and in the adventitia of the aorta of rats treated for 30 days from the 18th day of life with 10% ascorbate in their drinking water. By contrast, elastin volume density was significantly reduced by the treatment at all ages examined. These morphological data were supported by in situ hybridization observations showing enhanced collagen type I mRNA and reduced elastin mRNA expression upon treatment. Although vitamin C did not inhibit lysyl oxidase activity in vivo, being only slightly higher than in controls, enzyme activity was significantly reduced, when high doses of ascorbate were added in vitro. Lysyl oxidase activity may be a function of enhanced collagen metabolism rather than a direct effect of the vitamin on the enzyme activity. These data indicate that ascorbate exerts opposite effects on the deposition of two major components of the extracellular matrix in vivo, at least during periods of rapid growth.     

Regulation of Collagen Synthesis in Human Dermal Fibroblasts in Contracted Collagen Gels by Ascorbic Acid, Growth Factors, and Inhibitors of Lipid Peroxidation

Ascorbic acid has been shown to stimulate collagen synthesis in monolayer cultures of human dermal fibroblasts. In the present studies, we examined whether the presence of a collagen matrix influences this response of dermal fibroblasts to ascorbic acid. Fibroblasts and collagen were mixed and allowed to gel and contract for 6 days to form a matrix prior to determining the concentration and time dependence for ascorbic acid to affect collagen synthesis by fibroblasts within the matrix. Collagen synthesis was stimulated at levels at or above 10 μM ascorbic acid and was maximal after 2 days of treatment. This concentration and time dependence is similar to that of cells grown in monolayer cultures. The effects of transforming growth factor-β (TGF-β) and fibroblast growth factor (FGF) were also examined in this model. TGF-β increased and FGF inhibited collagen synthesis in the gels, as has been shown for cells in monolayer cultures. The effects of potential inhibitors of lipid peroxidation induced by ascorbic acid were also examined in these matrices and compared to previous results obtained in monolayer cultures. Propyl gallate, cobalt chloride, α,α-dipyridyl, and α-tocopherol inhibited the ascorbic acid-mediated stimulation of collagen synthesis while mannitol had no effect. Natural retinoids inhibited total protein synthesis without the specific effect on collagen synthesis that was seen in monolayer cultures. These results indicate that ascorbic acid stimulates collagen synthesis in fibroblasts grown in a collagen matrix in a manner similar to that found in monolayer cultures. In contracting collagen gels, however, the magnitude of the effect is less and retinoids do not specifically inhibit collagen synthesis.     

Factors that promote progressive development of the osteoblast phenotype in cultured fetal rat calvaria cells

Rat calvaria osteoblasts derived from 21-day-old fetal rat pups undergo a temporal expression of markers of the osteoblast phenotype during a 5 week culture period. Alkaline phosphatase and osteocalcin are sequentially expressed in relation to collagen accumulation and mineralization. This pattern of expression of these osteoblast parameters in cultured rat osteoblasts (ROB) is analogous to that seen in vivo in developing fetal rat calvaria tissue (Yoon et. al: Biochem. Biophis. Res. Commun. 148:1129, 1987) and is similar to that observed in cultures of subcultivated 16-day-old embryonic chick calvaria-derived osteoblasts (COB) (Gerstenfeld, et.al: Dev. Biol. 122:46, 1987). While the cellular organization of subcultivated COB and primary ROB cultures are somewhat different, the temporal expression of the parameters remains. Both the rat and chick culture systems support formation of matrix mineralization even in the absence of beta-glycerol-phosphate. A systematic examination of factors which constitute conditions supporting complete expression of the osteoblast phenotype in ROB cultures indicate requirements for specific serum lots, ascorbic acid and the ordered deposition of mineral in the extracellular matrix. The present studies suggest that formation of a collagenous matrix, dependent on ascorbic acid, is requisite for expression of the osteoblast phenotype. In ROB cultures, expression of osteocalcin synthesis occurs subsequent to initiation of alkaline phosphatase activity and accompanies the formation of mineralized nodules. Thus, extracellular matrix mineralization (deposition of hydroxyapatite) is required for complete development of the osteoblast phenotype, as reflected by a 200-fold increase in osteocalcin synthesis. These data show the temporal expression of the various osteoblast parameters during the formation and mineralization of an extracellular matrix can provide markers reflective of various stages of osteoblast differentiation/maturation in vitro.     

Regulation of angiogenesis in vitro by collagen metabolism

Summary The role of collagen in microvascular growth was investigated using the aortic ring model of angiogenesis. Collagen production by vasoformative outgrowths in plasma clot culture of rat aorta was either stimulated with ascorbic acid or inhibited with the proline analogue cis-hydroxyproline. Microvessels proliferating in the absence of ascorbic acid supplements became ectatic and developed large lumina. In contrast, newly formed microvessels in the presence of ascorbic acid remained small and maintained thin lumina throughout the angiogenic process. Biochemical studies demonstrated enhanced collagen production and deposition in cultures treated with ascorbic acid. Ultrastructural studies of these cultures showed a marked increase in newly formed interstitial collagen in the perivascular matrix and in regions of the plasma clot containing nonendothelial mesenchymal cells. Small microvessels with thin lumina similar to the ones observed in ascorbic acid-treated plasma clot cultures were obtained by growing aortic explants in gels of interstitial collagen in the absence of ascorbic acid. Inhibition of collagen production with the proline analogue cis-hydroxyproline had a marked anti-angiogenic effect in both plasma clot and collagen gel cultures. The anti-angiogenic effect of cis-hydroxyproline was abolished by addingl-proline to the culture medium, thereby restoring normal metabolism. These results support the hypothesis that angiogenesis is regulated by collagen production and suggest that the size of newly formed microvessels is influenced by the degree of collagenization of the extracellular matrix.     

Inhibitory effects of 1,25(OH)2 vitamin D3 on collagen type I,osteopontin, and osteocalcin gene expression in chicken osteoblasts

Seventeen day chicken embryonic osteoblasts treated over a 30-day period with 1,25(OH)₂ D₃ showed a 2–10-fold decrease in collagen, osteopontin and osteocalcin protein accumulation, alkaline phosphatase enzyme activity, and mineral deposition. Comparable inhibition in the steady state mRNA levels for α₁(I) and α₂(I) collagen, osteocalcin, and osteopontin were observed, and the inhibitory action of the hormone was shown to be specific for only the late release populations of cells from sequential enzyme digestions of the chick calvaria. In order to determine whether the continuous hormone treatment blocked osteoblast differentiation, the cells were acutely treated for 24 h with 1,25(OH)₂ D₃ at culture periods when the cells proliferate (day 5), a culture period when the cells cease further cell division and are increasing in the expression of their differentiated functions (day 17), and a culture period when the cells are encapsulated within a mineralized extracellular matrix (day 30). Inhibition of the expression of collagen, osteocalcin, and osteopontin were observed at days 17 and 30, while no effect could be detected for the 5-day cultures. To further define whether the inhibitory effect was specific for cells expressing their differentiated phenotype, 1,25(OH)₂ D₃ treatment was initiated at day 17 and continued to day 30 after the cells have established their collagenous matrix. In these experiments further collagenous matrix deposition, mineral deposition, alkaline phosphatase activity, and osteocalcin synthesis were also inhibited after the hormone treatment was initiated. These results, in summary, show that 1,25(OH)₂ D₃ in primary avian osteoblast cultures derived from 17-day embryonic calvaria inhibits the expression of several genes associated with differentiated osteoblast function and inhibit extracellular matrix mineral deposition.     

Procollagen mRNA metabolism during the fibroblast cell cycle and its synthesis in transformed cells.

Procollagen mRNA was isolated from mouse embryos and used for the synthesis of a highly labelled cDNA probe complementary to collagen mRNA. This probe was used for the investigation of procollagen mRNA metabolism during the cell cycle of 3T6 mouse embryo fibroblasts in culture. Titration hybridization experiments revealed that procollagen mRNA was present throughout the cell cycle following stumulation of confluent monolayers. Procollagen mRNA levels of sparse cultures appeared similar to those of unstimulated monolayers. The fluctuating levels of collagen synthesis during the cell cycle can be ascribed to changes in the amount of collagen mRNA present. In mouse sarcoma virus transformed 3T3 cells only 20--30% of the amount of procollagen mRNA in 3T3 cells is present indicating that the decline in collagen synthesis is due to mRNA availability.     

Mechanical Load Enhances Procollagen Processing in Dermal Fibroblasts by Regulating Levels of Procollagen C-Proteinase

Mechanical forces are emerging as key regulators of cell function. We hypothesize that mechanical load may influence dermal fibroblast activity. We assessed the direct effects of mechanical load on human dermal fibroblast procollagen synthesis and processing in vitro. Cells were loaded in a biaxial loading system (Flexercell 3000). Hydroxyproline levels were measured in the medium and cell layer as an estimate of procollagen synthesis and processing to insoluble collagen. Mechanical load (in the presence of serum or TGF-beta) enhanced procollagen synthesis by 45 +/- 3% (P < 0.001), and 38 +/- 4% (P < 0.001), respectively, over unloaded growth factor controls after 48 h. Insoluble collagen deposition was enhanced in the same cultures by 115 +/- 8% (P < 0.01) and 72% +/- 9% (P < 0.01), respectively. This effect was inhibited using l-arginine suggesting that procollagen C-proteinase, the enzyme which directly cleaves the C-terminal propeptide of procollagen to form insoluble collagen, is required for the fiber formation observed. Procollagen mRNA levels in loaded samples increased by more than two-fold in both serum and TGF-beta-treated cultures at 48 h. Procollagen C-proteinase mRNA levels were also enhanced by a similar magnitude, although the increase was observed at 24 h. Procollagen C-proteinase protein levels were also increased at this time. Protein and mRNA levels of the procollagen C-proteinase enhancer protein, which binds the C-terminal propeptide of procollagen to enhance the rate of peptide cleavage, were unaffected by mechanical load. This study demonstrates that mechanical load promotes procollagen synthesis in dermal fibroblasts by enhancing gene expression and posttranslational processing of procollagen.     

Collagen processing, crosslinking, and fibril bundle assembly in matrix produced by fibroblasts in long-term cultures supplemented with ascorbic acid

Human foreskin fibroblasts were cultured for up to 6 weeks in medium supplemented with ascorbic acid. During this time, the cells produced an extensive new connective tissue matrix in which the accumulated collagen (mostly type I) amounted to about 0.25 mg/10(6) cells. The matrix was highly differentiated as shown by complete processing of procollagen to collagen alpha-chains and covalent crosslinking of the collagen. Alignment of collagen fibrils occurred as the fibrils were deposited between cells, and binding of adjacent fibrils to the cell surface appeared to hold the fibrils in register. Groups of aligned fibrils were subdivided into bundles by cell-surface folds. If beta-aminopropionitrile was added to the medium, collagen crosslinking was inhibited, but not collagen synthesis or fibril bundle organization. If ascorbic acid was omitted from the culture medium, the extensive new connective tissue matrix was not produced. Our results indicate that fibroblasts in long-term cultures supplemented with ascorbic acid produce a connective tissue matrix with many in vivo-like properties including supermolecular organization of collagen.     

Regulation of collagenase-3 and osteocalcin gene expression by collagen and osteopontin in differentiating MC3T3-E1 cells

Both collagenase-3 and osteocalcin mRNAs are expressed maximally during the later stages of osteoblast differentiation. Here, we demonstrate that collagenase-3 mRNA expression in differentiating MC3T3-E1 cells is dependent upon the presence of ascorbic acid, is inhibited in the presence of the collagen synthesis inhibitor, 3,4-dehydroproline, and is stimulated by growth on collagen in the absence of ascorbic acid. Transient transfection studies show that collagenase-3 promoter activity increases during cell differentiation and requires the presence of ascorbic acid. Additionally, we show that, in differentiating MC3T3-E1 cells, collagenase-3 gene expression increases in the presence of an anti-osteopontin monoclonal antibody that binds near the RGD motif of this protein, whereas osteocalcin expression is inhibited. Furthermore, an RGD peptidomimetic compound, designed to block interaction of ligands to the alpha(v) integrin subunit, increases osteocalcin expression and inhibits collagenase-3 expression, suggesting that the RGD peptidomimetic initiates certain alpha(v) integrin signaling in osteoblastic cells. Overall, these studies demonstrate that stimulation of collagenase-3 expression during osteoblast differentiation requires synthesis of a collagenous matrix and that osteopontin and alpha(v) integrins exert divergent regulation of collagenase-3 and osteocalcin expression during osteoblast differentiation.     

Characterization and localization of collagens synthesized by cultured muscle cells stimulated with collagen-inducing factor from embryonic brain extracts

A low molecular weight fraction (Mr = 500-1500) was isolated by membrane and gel filtration from rat embryonic brain extract. This fraction was shown to stimulate collagen production in muscle cultures. Procollagen, intermediate collagenous proteins, and collagens of types I, III, and V were identified by analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and DEAE- and Cm-cellulose chromatography and by selective degradation by pepsin, bacterial collagenase, and cyanogen bromide. Immunofluorescent staining with antisera to collagen types I, III, IV, and V of muscle cultures treated with the collagen-inducing factor revealed a large increase in staining compared to control cultures and a distinct pattern of staining on the surface of the treated myotubes.     

Rapid chondrocyte maturation by serum-free culture with BMP-2 and ascorbic acid

In serum-containing medium, ascorbic acid induces maturation of prehypertrophic chick embryo sternal chondrocytes. Recently, cultured chondrocytes have also been reported to undergo maturation in the presence of bone morphogenetic proteins or in serum-free medium supplemented with thyroxine. In the present study, we have examined the combined effect of ascorbic acid, BMP-2, and serum-free conditions on the induction of alkaline phosphatase and type X collagen in chick sternal chondrocytes. Addition of either ascorbate or rhBMP-2 to nonconfluent cephalic sternal chondrocytes produced elevated alkaline phosphatase levels within 24–72 h, and simultaneous exposure to both ascorbate and BMP yielded enzyme levels at least threefold those of either inducer alone. The effects of ascorbate and BMP were markedly potentiated by culture in serum-free medium, and alkaline phosphatase levels of preconfluent serum-free cultures treated for 48 h with BMP + ascorbate were equivalent to those reached in serum-containing medium only after confluence. While ascorbate addition was required for maximal alkaline phosphatase activity, it did not induce a rapid increase in type X collagen mRNA. In contrast, BMP added to serum-free medium induced a three- to fourfold increase in type X collagen mRNA within 24 h even in the presence of cyclohexamide, indicating that new protein synthesis was not required. Addition of thyroid hormone to serum-free medium was required for maximal ascorbate effects but not for BMP stimulation. Neither ascorbate nor BMP induced alkaline phosphatase activity in caudal sternal chondrocytes, which do not undergo hypertrophy during embryonic development. These results indicate that ascorbate + BMP in serum-free culture induces rapid chondrocyte maturation of prehypertrophic chondrocytes. The mechanisms for ascorbate and BMP action appear to be distinct, while BMP and thyroid hormone may share a similar mechanism for induction. J. Cell. Biochem. 66:394–403, 1997. © 1997 Wiley-Liss, Inc.     

Collagen synthesis in human fibroblasts: Effects of ascorbic acid and regulation by hydrocortisone

The effects of hydrocortisone and ascorbic acid on collagen and noncollagen protein synthesis, and on growth were examined in fibroblasts derived from normal human dermis. When the medium was supplemented with 0.28 mM ascorbic acid, the apparent rate of collagen production increased 2--3 fold over the culture cycle. Ascorbic acid also caused a small increase in the apparent rate of synthesis of noncollagen protein and an elevation in growth rate and maximum cell density. Growth was not required for the increase in collagen production since addition of ascorbate to confluent cultures induced a similar increase. Hydrocortisone (1.5 μM) blocked the ascorbate-related increase in collagen production during growth and in confluent cultures. The hormone simultaneously increased the apparent rate of noncollagen protein production and maximum cell density, suggesting that the effect on collagen synthesis was specific. Inhibition of collagen production by hydrocortisone was observed only in the presence of ascorbate, while the increase in growth and noncollagen protein production occurred in the presence and absence of the vitamin.     

Regulation of growth, protein synthesis, and maturation of fetal bovine epiphyseal chondrocytes grown in high-density culture in the presence of ascorbic acid, retinoic acid, and dihydrocytochalasin B

Phenotypic expression of chondrocytes can be modulated in vitro by changing the culture technique and by agents such vitamins and growth factors. We studied the effects of ascorbic acid, retinoic acid (0.5 and 10 microM), and dihydrocytochalasin B (3, 10, 20 microM DHCB), separately or in combination (ascorbic acid + retinoic acid or ascorbic acid + DHCB), on the induction of maturation of fetal bovine epiphyseal chondrocytes grown for up to 4 weeks at high density in medium containing 10% fetal calf serum and the various agents. In the absence of any agent or with retinoic acid or DHCB alone, the metabolic activity of the cells remained very low after day 6, with no induction of type I or X collagen synthesis nor increase in alkaline phosphatase activity. Chondrocytes treated with fresh ascorbic acid showed active protein synthesis associated with expression of types I and X after 6 and 13 days, respectively. This maturation was not accompanied by obvious hypertrophy of the cells or high alkaline phosphatase activity. Addition of retinoic acid to the ascorbic acid-treated cultures decreased the level of type II collagen synthesis and delayed the induction of types I and X collagen, which were present only after 30 days. A striking increase in alkaline phosphatase activity (15-20-fold) was observed in the presence of both ascorbic acid and the highest dose of retinoic acid (10 microM). DHCB was also a potent inhibitor of the maturation induced by treatment with ascorbic acid, as the chondrocytes maintained their rounded shape and synthesized type II collagen without induction of type I or X collagen. The pattern of protein secretion was compared under all culture conditions by two-dimensional gel electrophoresis. The different regulations of chondrocyte differentiation by ascorbic acid, retinoic acid, and DHCB were confirmed by the important qualitative and quantitative changes in the pattern of secreted proteins observed by two-dimensional gel electrophoresis along the study.