Collagen synthesis is required for ascorbic acid-enhanced differentiation of mouse embryonic stem cells into cardiomyocytes

Ascorbic acid has been reported to promote the differentiation of embryonic stem (ES) cells into cardiomyocytes; however, the specific functions of ascorbic acid have not been defined. A stable form of ascorbic acid, namely, l-ascorbic acid 2-phosphate (A2-P), significantly enhanced cardiac differentiation; this was assessed by spontaneous beating of cardiomyocytes and expression of cardiac-specific markers obtained from mouse ES cells. This effect of ascorbic acid was observed only when A2-P was present during the early phase of differentiation. Treatment with two types of collagen synthesis inhibitors, l-2-azetidine carboxylic acid and cis-4-hydroxy-d-proline, significantly inhibited the A2-P-enhanced cardiac differentiation, whereas treatment with the antioxidant N-acetyl cysteine showed no effect. These findings demonstrated that ascorbic acid enhances differentiation of ES cells into cardiomyocytes through collagen synthesis and suggest its potential in the modification of cardiac differentiation of ES cells.     

L-ascorbic acid 2-phosphate stimulates collagen accumulation, cell proliferation, and formation of a three-dimensional tissuelike substance by skin fibroblasts

Proliferation of human skin fibroblasts was stimulated significantly by the presence of L-ascorbic acid 2-phosphate (Asc 2-P). The presence of Asc 2-P (0.1-1.0 mM) in the culture medium for 3 weeks enhanced the relative rate of collagen synthesis to total protein synthesis 2-fold as well as cell growth 4-fold. Coexistence of L-azetidine 2-carboxylic acid (AzC), an inhibitor of collagen synthesis, attenuated both effects of Asc 2-P in a dose-dependent manner. Supplementation of the medium with Asc 2-P also accelerated procollagen processing to collagen and deposition of collagen in the cell layer. Among the acidic glycosaminoglycans (GAG), another major component of extracellular matrix (ECM), deposition of sulfated forms was increased by the additive. Electron microscopic observations showed multilayered, rough endoplasmic reticulum-rich cells surrounded by dense ECM. These results indicate that Asc 2-P is useful in culture systems as a long-acting vitamin C derivative and also that it promotes reorganization of a three-dimensional tissuelike substance from skin fibroblasts in culture by stimulating collagen accumulation in the fibroblasts.     

Ascorbic acid stimulates barrier function of cultured endothelial cell monolayer

The macromolecular permeability of cultured bovine aortic, bovine venous, and human umbilical vein endothelial cell monolayers was decreased significantly in culture medium containing L-ascorbic acid (Asc Acid; 0.01–0.1 mM) and L-ascorbic acid 2-phosphate (Asc 2-P). Dithiothreitol, which shows reducing activity equivalent to that of Asc Acid, did not affect endothelial permeability. Asc Acid induced a sixfold increase in collagen synthesis by the endothelial cells. The coexistence of L-azetidine 2-carboxylic acid, an inhibitor of collagen synthesis, attenuated the effect of Asc 2-P in a dose-dependent manner. Another collagen synthesis inhibitor, ethyl-3,4-dihydroxybenzoate, also inhibited collagen synthesis and increased endothelial permeability. The decrease in permeability of the endothelial monolayer was dependent on a reduction of the permeability coefficient of the endothelial monolayer. These findings indicate that endothelial barrier function is stimulated by Asc Acid via an increase in collagen synthesis. © 1995 Wiley-Liss, Inc.     

Optimization of ascorbic acid-2-phosphate production from ascorbic acid using resting cell of Brevundimonas diminuta

With the aim to produce ascorbic acid-2-phosphate (AsA-2-P) from L-ascorbic acid (AsA, Vitamin C), nine bacteria conferring the ability to transform AsA to AsA-2-P were isolated from soil samples alongside known strains from culture collections. Most isolates were classified to the genus Brevundimonas by 16S phylogenetic analysis. Among them, Brevundimonas diminuta KACC 10306 was selected as the experimental strain because of its the highest productivity of AsA-2-P. The optimum set of conditions for the AsA-2-P production from AsA using resting cells as the source of the enzyme was also investigated. The optimum cultivation time was 16 h and the cell concentration was 120 g/l (wet weight). The optimum concentrations of AsA and pyrophosphate were 550 mM and 450 mM, respectively. The most effective buffer was 50 mM sodium formate. The optimum pH was 4.5 and temperature was 40 degrees C. Under the above conditions, 27.5 g/l of AsA-2-P was produced from AsA after 36 h of incubation, which corresponded to a 19.7% conversion efficiency based on the initial concentration of AsA.     

外源施加AsA和MeJA对乙烯利诱导水稻叶片衰老的影响

以野生型水稻(Oryza sativa)株系中花11(ZH-11)及其抗坏血酸合成关键酶基因GLDH下调株系(GI-2)为材料,研究了外源抗坏血酸(AsA)与茉莉酸甲酯(MeJA)对乙烯利诱导下水稻叶片早衰现象的影响。结果表明,外源AsA提高了水稻GI-2中的抗坏血酸含量、Rubisco含量及叶绿素的含量,减缓了其光合特性参数的下降速率,但对水稻ZH-11没有显著影响。外源MeJA降低了两株系的抗坏血酸、Rubisco及叶绿素含量,加快了叶内光合特性参数的下降速率,且对ZH-11的影响大于GI-2。因此,外源AsA处理能有效缓解乙烯利诱导的水稻叶片早衰现象,使叶片的衰老进程得以延缓,而外源MeJA作用相反。     

Distribution and metabolism of ascorbic acid in apple fruits (Malus domestica Borkh cv. Gala)

The objective of this study was to determine ascorbic acid (AsA) distribution, biosynthesis and recycling in different tissues of young and mature fruit of cv. Gala apple (Malus domestica Borkh). Our results showed that the peel of ‘Gala’ apple had the highest AsA levels among all the tissue types, which resulted from a combination of, lower ascorbate peroxidase (APX, EC 1.11.1.11) activity consuming AsA, and higher dehydroascorbate reductase (DHAR, EC 1.8.5.1) and monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) activities used to recycle AsA. Exogenous feeding of AsA synthesis precursors demonstrated that the peel was capable of de nono AsA biosynthesis via l-galactose and d-galacturonic acid pathways whereas the flesh and seed were only able to synthesize AsA via l-galactose pathway. The young fruit had higher AsA concentration and stronger capability of AsA biosynthesis and recycling. The sun-exposed peel had higher AsA concentration and stronger capability of recycling AsA than the shaded peel, while there was no difference in the flesh between the sun-exposed side and the shaded side. Abundant AsA was found in fruit vascular tissue, which suggests that AsA can be transported to vascular tissues of fruit or vascular tissues could synthesize AsA itself in ‘Gala’ apple.     

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 μM), and dihydrocytochalasin B (3, 10, 20 μM 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 μM). 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. J. Cell. Biochem. 76:84–98, 1999. © 1999 Wiley‐Liss, Inc.     

In vitro effect of ascorbic acid on neutrophil–endothelial cell interaction

The effect of different concentrations (0.06, 0.6 and 6.0 mmol/L) of ascorbic acid on neutrophil–endothelial interaction was studied using an in vitro model of human umbilical cord vein endothelial cells and human neutrophils. The aim of the study was to determine changes in chemiluminescence response of neutrophils during adherence to endothelial cells. Because adherence of neutrophils to endothelial cells is an essential component in inflammatory processes leading to endothelial cell injury, the influence of ascorbic acid on adherence and endothelial cell injury have been investigated. Production of oxygen-derived metabolites, measured by chemiluminescence response of neutrophils, decreased significantly in the presence of 6 mmol/L ascorbic acid during coincubation of neutrophils and endothelial cells (p < 0.025). The adherence of neutrophils to endothelial cells was significantly decreased at a concentration of 6 mmol/L (p < 0.0005). The inhibition of neutrophil adherence to endothelial cells was correlated with a diminished neutrophil-mediated endothelial cell injury during incubation with 6 mmol/L ascorbic acid (p < 0.0005). The present results indicate that ascorbic acid might exert a protective effect on neutrophil-mediated endothelial cell injury by decreasing adherence of neutrophils to endothelial cells and by scavenging reactive oxygen metabolites. Moreover, the current investigation points to probable protective effect of ascorbic acid on oxidant-mediated cell damage in diseases (e.g., Adult Respiratory Distress Syndrome).     

Synthesis, spectroscopic study and anticancer activity of a water-soluble Nb(V) peroxo complex

We synthesized, characterized and studied the anticancer properties of a new water-soluble peroxo niobium complex (K₃[Nb(Asc)(O₂)₃]·4H₂O (Asc = ascorbate anion C₆H₆O₆²⁻))_, as well as that of ascorbic acid, in human leukemic cells. The complex was synthesized and characterized by elemental, IR, Raman, thermogravimetric analysis, detailed NMR and mass spectra analysis. The cytotoxic activity of the complex on HL-60 and K562 human leukemia cell lines has been investigated by assessing vital cellular mechanisms, such as the metabolic activity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTT) and the proliferation capacity (growth curves) of leukemia cells, as well as the structural integrity of cell membrane (trypan blue assay). The complex exerts an increased antiproliferative effect primarily on HL60 human leukemia cells, compared to ascorbic acid alone, as well as an inhibitory effect on necrosis caused by ascorbic acid. Its effect on K562 cells concerns mainly its inhibitory effect upon cell necrosis induced by ascorbic acid alone. Our results support a concentration- and time-dependent enhanced antileukemic effect of the complex, suggesting its significance as a promising tool in the confrontation of leukemia.     

Impaired development of mammary glands in scorbutic rats unable to synthesize ascorbic acid

The effects of ascorbic acid (AsA)-deficiency on the development of mammary glands were investigated using mutant rats (osteogenic disorder syndrome rats; ODS rats) with hereditary inability to synthesize AsA. Female ODS rats of 21 days old were castrated and divided into two groups. One group was given AsA in their drinking water, and the other was not. All the rats received a daily injection of oestradiol-17 beta and progesterone (EP) from day 28 to day 49 of age. After EP treatment, the concentrations of AsA in the mammary glands of rats not given AsA were less than one tenth of those of rats given AsA and the contents of hydroxyproline in the mammary glands of the former rats were about half of those in the latter. Furthermore, the concentration of serum prolactin in rats not given AsA was reduced to about one third of that in rats given AsA. After EP treatment, whole mounts of mammary glands showed that in rats not given AsA the development of ducts was impaired and there was extensive accumulation of endbuds. Consistent with this finding, EP injections did not increase the area of parenchyma in the mammary glands of rats not given AsA, whereas they increased it about 2-fold in rats given AsA. Moreover, after EP treatment the amount of alpha-lactalbumin was significantly less in the mammary parenchyma of rats not given AsA than in that of rats given AsA. On the other hand, AsA deficiency did not impair the response of the mammary cells to insulin or prolactin in terms of DNA synthesis and alpha-lactalbumin production. These findings indicate that AsA deficiency impaired the development of mammary glands. This effect may be partly attributable to a defect in collagen synthesis in the mammary glands and a decrease in the concentration of serum prolactin.     

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.     

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.     

The effect of ascorbic acid on arachidonic acid and prostaglandin E2 metabolism in B16 murine melanoma cells

Ascorbic acid (Asc), arachidonic acid (AA) and prostaglandin E2 (PGE2) are reported to be important in maintaining the stability of the cell matrix. Asc has also been shown to influence fatty acid (FA) and PGE2 synthesis, with the result that effects of Asc on cell growth are suggested to be mediated through the metabolism of these two compounds. This study examined the effect of Asc, supplemented over the concentration range of 0-100 micrograms/ml, on the in vitro cell growth of non-malignant LLCMK (monkey kidney) cells and malignant B16 murine melanoma cells. The effects of Asc supplementation on AA and PGE2 levels in the cell stroma and membrane fractions of the two cell types was also determined. Asc had no significant inhibitory or stimulatory effect on the growth of either the B16 or LLCMK cells. The total percentage AA composition determined in the B16 control cells (combined stroma and membrane fractions), was similar to that determined in the LLCMK control cells. Asc supplementation of the B16 cells, resulted in an inverse relationship between B16 cell growth and total percentage AA composition. PGE2 concentration in the control B16 cells (combined stroma and membrane fractions) was significantly higher than that detected in the control LLCMK cells. No PGE2 was detected in the B16 stroma fraction, with all appearing to be located in the membrane fraction. However, upon the supplementation of the B16 cells with increasing Asc concentrations, PGE2 appeared to be mobilized from the membrane fraction, resulting in increasing PGE2 levels in the stroma fraction relative to the membrane fraction. This was accompanied by a significant decrease in PGE2 concentration, in the membrane fraction. B16 cell growth and total (stroma and membrane fractions) PGE2 concentration in these cells was inversely related, when cultures were supplemented with increasing levels of Asc. Asc supplementation of the LLCMK cells did not appear to have any significant effect on AA or PGE2 metabolism in these cells.     

High-dose ascorbic acid induces carcinostatic effects through hydrogen peroxide and superoxide anion radical generation-induced cell death and growth arrest in human tongue carcinoma cells

High-dose ascorbic acid (AsA) treatment, known as pharmacological AsA, has been shown to exert carcinostatic effects in many types of cancer cells and in vivo tumour models. Although pharmacological AsA has potential as a complementary and alternative medicine for anticancer treatment, its effects on human tongue carcinoma have not yet been elucidated. In this study, we investigated the effect of AsA treatment on human tongue carcinoma HSC-4 cells compared with non-tumourigenic tongue epithelial dysplastic oral keratinocyte (DOK) cells. Our results show that treatment with 1 and 3?mM of AsA for 60?min preferentially inhibits the growth of human tongue carcinoma HSC-4 over DOK cells. Furthermore, AsA-induced effects were accompanied by increased intracellular oxidative stress and were repressed by treatment with a hydrogen peroxide (H₂O₂) scavenger catalase and a superoxide anion radical (O₂^?) scavenger, tempol. Time-lapse observation and thymidine analog EdU incorporation revealed that AsA treatment induces not only cell death but also suppression of DNA synthesis and cell growth. Moreover, the growth arrest was accompanied by abnormal cellular morphologies whereby cells extended dendrite-like pseudopodia. Taken together, our results demonstrate that AsA treatment can induce carcinostatic effects through induction of cell death, growth arrest, and morphological changes mediated by H₂O₂ and O₂^? generation. These findings suggest that high-dose AsA treatment represents an effective treatment for tongue cancer as well as for other types of cancer 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 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.     

Development of a cell line from skin of goldfish, Carassius auratus, and effects of ascorbic acid on collagen deposition

Growth characteristics and collagen expression were investigated in GFSk-S1, a cell line derived from the skin of an adult goldfish (Carassius auratus). These cells are anchorage dependent, grow well in Leibovitz-15 medium with 10% fetal bovine serum, and have been subcultured routinely for 5 years. Cells at various passages have been successfully cryopreserved and thawed. GFSk-S1 cells show mainly a fibroblastic morphology at low density, but at confluence islands of epithelial-shaped cells appear among the fibroblastic cells. The cells require little maintenance, and cultures have been kept viable for more than 3 months without medium changes. Although best growth was observed at room temperature, cell proliferation still occurred at 28°C, and a subline was maintained and passaged for over a year at 25°C. Cells were exposed to various concentrations of ascorbic acid, and its effects on collagen secretion were monitored by light and electron microscopy. Under phase-contrast microscopy, confluent GFSk-S1 cells exposed to ascorbic acid at 50 μg/ml showed distinct development of fibres as early as 3 days after treatment. Histochemical staining for collagen demonstrated a thick network of fibres under a monolayer of ascorbic acid- treated GFSk-S1 cells, and observation by transmission electron microscopy showed collagen fibres with typical banding pattern. This cell line appears to show a stable genotype, as collagen expression was induced at all passages. GFSk-S1 could be useful for studies not only of regulation of protein synthesis, but also of cell differentiation and wound healing     

Influence of some environmental variables on the ascorbic acid status of mullet, Mugil cephalus L., tissues

The seasonal fluctuations in the ascorbic acid (AsA) and ascorbic acid 2-sulphate (AsA 2-sulphate) content of mullet, Mugil cephalus , tissues were examined. Ascorbic acid concentrations in brain, gill and hepatic tissues showed seasonal changes, but the pattern of AsA fluctuations in each tissue differed. The AsA content of mullet brains decreased during the summer, whereas hepatic AsA concentrations increased during this period and were maximal by the end of June. Hepatic AsA reserves declined after environmental water temperatures dropped below 18°C and reached a minimum (20 μg g⁻¹) by the end of January. Greatest fluctuations in AsA content occurred in gill tissues, which had a four-fold range of tissue concentrations. There were also seasonal changes in the AsA 2-sulphate content of brain and hepatic tissues. These differences among mullet tissues in the seasonal patterns of AsA content may be due to diverse effects of environmental variables on tissue AsA reserves. The ability of hepatic and renal tissues of mullet and several other teleost species to synthesize AsA was also investigated. L-gulonolactone oxidase activity was detected in all the species examined, but in all cases the biosynthetic capacity was less than a seventh that in goldfish, Carassius auratus , livers. Mullet appear to have only a limited capacity to synthesize AsA.