Effect of the reducing environment on the accumulation of elastin and collagen in cultured smooth-muscle cells.

We show here that cultured neonatal-rabbit aortic smooth-muscle cells produce and accumulate significant amounts of insoluble elastin. When grown in the presence of ascorbic acid, the amount of insoluble elastin in these cultures decreases, whereas the accumulation of collagen increases. These changes have been attributed to increased hydroxylation of proline in elastin. The function of ascorbic acid in proline hydroxylation is thought to be that of a reductive cofactor that maintains the proper oxidation state of molecular iron in the enzyme complex. This study shows that both ascorbic and isoascorbic acids act similarly to modify the accumulation of elastin and collagen in culture. On the other hand, cultures grown in the presence of dithiothreitol, a reducing agent previously shown to act as a cofactor for prolyl hydroxylase, do not demonstrate altered elastin accumulation. These studies are consistent with the suggestion that there is a specific role for ascorbic acid in this cellular system that cannot be replaced by other reducing cofactors.     

Studies in vivo on the biosynthesis of collagen and elastin in ascorbic acid-deficient guinea pigs

1. After the administration of labelled proline to guinea pigs deprived of ascorbic acid for 15 days, the dorsal skin was examined 5 days later in an attempt to detect the presence of hydroxyproline-deficient collagen (protocollagen). The extent of incorporation of proline into skin collagens indicated a severe impairment of collagen synthesis. 2. A comparison of proline and hydroxyproline specific radioactivities in diffusible peptides obtained by treatment with collagenase of either purified skin collagens or direct hot-trichloroacetic acid extracts of skin failed to indicate the presence of protocollagen. Possible reasons for this are discussed. 3. The incorporation results did not indicate an inability of normal collagen, i.e. collagen hydroxylated to the normal degree, to cross-link in scurvy. 4. Incorporation of labelled proline into aortic elastin isolated from the same animals did not indicate a decrease in elastin biosynthesis in ascorbic acid deficiency, beyond that attributable to the inanition accompanying the vitamin deficiency. The proline/hydroxyproline specific-radioactivity ratio in elastin from scorbutic guinea pigs was about 6:1 in contrast with the 1:1 ratio in control groups. It is concluded that the formation of elastin hydroxyproline was ascorbate-dependent and that a hydroxyproline-deficient elastin is formed and retained in scurvy. The formation of desmosines was unimpaired in scorbutic animals. 5. Studies with chick embryos confirmed the formation of elastin hydroxyproline from free proline. Incorporation of free hydroxyproline into elastin hydroxyproline was negligible. 6. Digestion of solubilized samples with collagenase indicated that the hydroxyproline in guinea-pig aortic elastin preparations was not derived from contamination by collagen. It is suggested that most if not all of the hydroxyproline in the guinea pig elastin preparations investigated can be considered an integral part of the elastin molecule.     

Isolation of alpha-amino adipic acid from mature dermal collagen and elastin. Evidence for an oxidative pathway in the maturation of collagen and elastin.

Human serum alpha-2-macroglobulin has been found to be a major cadmium-binding protein $\text{in vitro}$. Serum and alpha-2-macroglobulin equilibrated with cadmium at the 0.20 ppm level were chromatographed over Sephadex and agarose gels to separate and estimate the molecular weights of the proteins. Alpha-2-macroglobulin was found to fragment into reproducible fragments when chromatographed on agarose gels showing different metal-binding fractions for cadmium and endogenous zinc. The distribution of cadmium on serum protein chromatograms was correlated with alpha-2-macroglobulin chromatograms. Cadmium was bound to fractions with molecular weights as high as 800,000 daltons with an affinity greater than that observed for serum albumin.     

The effect of ascorbic acid on collagen polypeptide synthesis and proline hydroxylation during the growth of cultured fibroblasts

The rate of collagen synthesis relative to the rate of synthesis of noncollagen protein was determined in several lines of cultured fibroblasts using an assay which measures [¹⁴C]proline incorporation into the polypeptide chains of collagen. In this assay procedure, collagen is degraded by protease-free collagenase regardless of whether proline and lysine residues are hydroxylated, thus separating the process of polypeptide synthesis from hydroxylation. It was found that the relative rate of collagen synthesis in L-929 cells was approximately 0.8–1% at all stages of growth. There was no significant increase in the relative rate of collagen synthesis in stationary phase compared to log phase cells in the lines Balb 3T3, 3T6, 3T12, and Swiss mouse 3T6. In all cases, the absolute incorporation of [¹⁴C]proline into both collagen and noncollagen proteins expressed as radioactivity incorporated per milligram of cellular protein, was 2–10 times higher in log phase cells, depending on the line examined.     

Heterotrophic production of ascorbic acid by microalgae

An aerobic fermentation process has been developed for the production of L-ascorbic acid (vitamin C). After an extensive screening program for microorganisms capable of heterotrophically synthesizing L-ascorbic acid, a unicellular green microalga,Chlorella pyrenoidosa, was selected. This organism has a number of characteristics that recommend it as an industrial organism: (1) it can double every 3.5 h when growing aerobically in the dark on a glucose-minimal salts medium; (2) its small size and tough cell wall make it very insensitive to shear, allowing very high impeller velocities; (3) it can be grown to 100 g L^–1 cell dry weight; (4) it is readily mutable by classical mutagenesis techniques; and (5) it has efficient growth kinetics with respect to yield of cell mass on glucose and oxygen. Fermentation process development and classical strain improvement have resulted in a greater than 70-fold increase in intracellular ascorbic acid concentration compared to the parent strainC. pyrenoidosa UTEX 1663. The process is compatible with existing industrial fermentation technology and equipment and is described in U.S. Patent 5,001,059. Patents have been submitted for a process in which the ascorbic acid accumulates extracellularly.     

The oxidation of endogenous ascorbic acid during histamine secretion by rat peritoneal mast cells

Endogenous ascorbic acid is oxidized to the free radical species by rat mast cells during histamine secretion. This antioxidant may function as a radical scavenger of Superoxide and other membrane-damaging radicals known to be liberated by this process. The high levels of ascorbic acid in mast cells may, therefore, function to protect the cell membrane from oxidative damage and thereby promote cell survival after an extensive secretory response.     

Protective effect of ascorbic acid in high altitude hypoxia in the rat.

Control (physiological saline treated) and ascorbic acid (AA) treated (1 mg.g-1 b.w. one hour before exposure) 18-day-old rats were exposed for 1 hour to high altitude in a hypobaric chamber and the mean lethal altitudes were calculated. AA displayed a protective effect, so that in two identical experiments the mean lethal altitude was 10,900 and 10,150 m in controls, while it was 11,500 and 11,450 m in AA treated animals.     

The effect of step-wise increased stretching on rat calvarial osteoblast collagen production

Mechanical forces regulate the function of bone cells. In this paper, the effects of cyclic stretching on osteoblasts derived from rat calvaria were studied at a magnitude occurring in physiological loaded bone tissue. A four-point bending apparatus was used to apply cyclic stretching on osteoblasts. Stretching at 500 microepsilon for 2-24 h resulted in an increase in matrix synthesis(P<0.01). In contrast, the cyclic stretching at 1000 and 1500 microepsilon for 2-24 h inhibited osteoblast collagen production (P<0.01). We also described our new loading method to increase strain magnitude step-by-step. The strain magnitude increased by 500 microepsilon increments from 500 to 1500 microepsilon every 2 or 12 h, respectively. Results showed that osteoblasts could absorb large amount of proline for collagen synthesis when stretched at 500 microepsilon. However, not all the absorbed proline was used to synthesize collagen. Some of it was stored in cells. When the suitable signal (500 microepsilon) was changed to an inhibiting signal (1000 microepsilon), cells responded to it accordingly and released proline to medium. These results demonstrate that the response of osteoblasts is dependent on the magnitude of the strain applied and cells can adjust their bio-chemical response to adapt to the changing environmental stimulation.     

The effect of ascorbic acid on Helicobacter pylori induced cyclooxygenase 2 expression and prostaglandin E2 production by gastric epithelial cells in vitro

BACKGROUND: Cyclooxygenase 2 (COX-2) is induced by the presence of Helicobacter pylori (H. pylori) on the gastric mucosa as part of the inflammatory response; this results in the synthesis of prostaglandins that amplify the local inflammatory response. The presence of H. pylori inhibits the secretion of ascorbate into the gastric lumen. Interestingly, ascorbate inhibits the growth of H. pylori and low dietary levels are associated with an increased risk of gastric adenocarcinoma. We therefore investigated the effect of ascorbate on H. pylori mediated COX-2 induction and prostaglandin production in vitro. METHODS: H. pylori was cocultured with gastric epithelial cells in the presence of ascorbate at physiological concentrations. The expression of COX-2 was assessed by Western blotting and prostaglandin E(2) (PGE(2)) was assessed by ELISA. RESULTS: Ascorbate inhibited gastric cell PGE(2) synthesis but not in COX-2 expression in response to H. pylori. In the absence of the organism, ascorbate also reduced PGE(2) expression in cells that constitutively express COX-2, again with no reduction of COX-2 protein expression. CONCLUSIONS: Physiological concentrations of ascorbate inhibit PGE(2) but not COX-2 expression in response to H. pylori in gastric epithelial cells.     

Synergistic effect of ascorbic acid and collagen addition on the increase in type 2 collagen accumulation in cartilage-like MSC sheet

Aiming to increase the content of type 2 collagen in scaffold-free cartilage-like cell sheets prepared using human bone marrow mesenchymal stem cells, the effect of several kinds of additives in a chondrogenic medium was investigated. Addition of ascorbic acid 2 phosphate (VCP) at a high concentration (250 µg/ml) and type 1 atelocollagen (5 µg/ml) increased the accumulation of type 2 collagen by fourfold and twofold, respectively. On the other hand, an antioxidant, glutathione showed no such effect. The synergistic effect of VCP and type 1 atelocollagen resulted in an eightfold increase in the accumulation level of type 2 collagen. Furthermore, the gene expression level of type 2 collagen increased and that of matrix metalloproteinase-13 (MMP-13) decreased to approximately one-third of the control. The increase in type 2 collagen accumulation in the scaffold-free cartilage-like cell sheet might be due to not only the enhancement of the synthesis but also the suppression of the degradation of type 2 collagen by MMP-13.