MagC, Magnesium Ascorbyl Phosphate, Ascorbic Acid Derivatives and Collagen

Ono M; Aratani Y; Kitagawa I; Kitagawa Y. (1990) Ascorbic acid phosphate stimulates type IV collagen synthesis and accelerates adipose conversion of 3T3-L1 cells., Exp Cell Res; 187(2):309-14

Abstract:

We showed that the synthesis and secretion of type IV collagen, entactin, and laminin were enhanced when adipose conversion of 3T3-L1 cells at confluence was stimulated by hormones (Y. Aratani and Y. Kitagawa (1988) J. Biol. Chem. 263, 16163-16169). Ascorbic acid phosphate (Asc-P) stimulated the synthesis and secretion of type IV collagen and other collagens from both 3T3-L1 preadipocytes and adipocytes. The synthesis and secretion of laminin and entactin were not affected by Asc-P. The continuous addition of Asc-P stimulated cell growth and increased cell density at confluence 1.3-fold. Concomitantly, Asc-P remarkably accelerated the emergence of lipoprotein lipase, glycerophosphate dehydrogenase, and Oil Red O-stainable lipid droplets. These findings suggest an important role for type IV collagen in adipocyte differentiation.

Saika S; Kanagawa R; Uenoyama K; Hiroi K; Hiraoka J. (1991) L-ascorbic acid 2-phosphate, a phosphate derivative of L-ascorbic acid, enhances the growth of cultured rabbit keratocytes., Graefes Arch Clin Exp Ophthalmol; 229(1):79-83

Abstract:

We examined the effect of L-ascorbic acid 2-phosphate (P-Asc) on the proliferation of cultured rabbit keratocytes. P-Asc is a phosphate derivative of L-ascorbic acid and has more prolonged vitamin C activity in solution than does L-ascorbic acid. The proliferation of cultured keratocytes was promoted by the presence of P-Asc in culture medium. Transmission electron microscopic observations revealed that cells were more multi-layered after culture in the presence of P-Asc (0.1 mM) for 30 days than were those cultured in the absence of P-Asc. The effect of P-Asc was abrogated by L-azetidine 2-carboxylic acid, which is an analogue of proline that inhibits the production and secretion of collagen. Our observations support a therapeutic role for P-Asc in the repair of corneal stromal damage such as that caused by a corneal chemical burn.

Saika S; Uenoyama K; Hiroi K; Ooshima A. (1992) L-ascorbic acid 2-phosphate enhances the production of type I and type III collagen peptides in cultured rabbit keratocytes., Ophthalmic Res; 24(2):68-72

Abstract:

We studied the effect of L-ascorbic acid 2-phosphate (P-Asc), a long-acting phosphate derivative of L-ascorbic acid, on the production and secretion of type I and type III collagen peptides in cultured rabbit keratocytes using immunohistochemistry and enzyme immunoassay. P-Asc enhanced production and secretion of these collagen peptides. Our observations support a therapeutic role for P-Asc in the repair of corneal stromal damage such as caused by corneal chemical burn.

Yamamoto, I., Muto, N., Murakami, K. and Akiyama, JI. (1992) Collagen Synthesis in human skin fibroblasts is stimulated by a stable form of ascorbate, 2-0-Ol-D-Glucopyranosyl-L-Ascorbic Acid, American Institute of Nutrition; 871-76.

Kurata, SI., Sendoo, H, and Hata, RI. (1993) Transcriptional activation of type I collagen genes by ascorbic acid 2-phosphate in human skin fibroblasts and its failure in cells from a patient with (1)-chain-defective Ehlers-Danios syndrome, Experimental Cell Research; 206: 63-71.

Hata R; Senoo H. (1989) L-ascorbic acid 2-phosphate stimulates collagen accumulation, cell proliferation, and formation of a three-dimensional tissuelike substance by skin fibroblasts., J Cell Physiol; 138(1):8-16

Abstract:

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.

Kurata, SI., and Hata, RI. (1991) Epidermal growth factor inhibits transcription of type I collagen genes and production of type I collagen in cultured human skin fibroblasts in the presence and absence of L-ascorbic acid 2-phosphate, a long-acting vitamin C derivative, The Journal of Biological Chemistry; 266: No. 15, 9997-10002.

Geesin JC; Gordon JS; Berg RA.(1993) Regulation of collagen synthesis in human dermal fibroblasts by the sodium and magnesium salts of ascorbyl-2-phosphate, Skin Pharmacol; 6(1):65-71

Abstract:

Ascorbic acid has been shown to stimulate collagen synthesis in dermal fibroblasts by increasing the rate of transcription of collagen genes. Experiments involving the use of ascorbic acid require daily supplementation due to the instability of the molecule in aqueous solutions. In order to provide a more stable alternative to ascorbic acid, two salts of ascorbyl-2-phosphate, having a greater chemical stability than ascorbic acid, were tested for their ability to stimulate collagen synthesis in monolayer fibroblast cultures. The concentration and time dependence of their activities were compared with ascorbic acid. The magnesium salt of ascorbyl-2-phosphate was found to be equivalent to ascorbic acid in stimulating collagen synthesis in these assays, while the sodium salt required at least a tenfold greater concentration to produce the same effect as ascorbic acid. Solutions of either ascorbic acid or the ascorbyl-2-phosphate analogs (at 10 mM) in phosphate-buffered saline (PBS) were relatively stable as shown by their decay rates and their ability to stimulate collagen synthesis even after nine days in solution prior to testing their effects on cultured cells. Ascorbic acid was unstable at neutral pH compared to solutions of either sodium or magnesium ascorbyl-2-phosphate. These data support the use of magnesium ascorbyl-2-phosphate in experiments where stability of ascorbic acid is a concern, e.g. in long-term cultures or in in vivo studies.

Ishikawa O; Yokoyama Y; Miyachi Y. (1994) Disaccharide analysis of dermal fibroblast-derived glycosaminoglycans in the three-dimensional culture., J Dermatol Sci; 8(3):203-7

Abstract:

We performed the quantitative and qualitative analysis on the main disaccharide units of glycosaminoglycans produced by human dermal fibroblasts in the 3-dimensional culture supplemented with L-ascorbic acid 2-phosphate (Asc 2-p) comparing with the monolayer culture system. The addition of Asc 2-p rendered fibroblasts to the organization of the dermis-like 3-dimensional structure in vitro without any pre-treatments with the plastic dish. Main disaccharide units were analyzed using HPLC after 1-phenyl-3-methyl-5-pyrasolone (PMP) labeling. The addition of Asc 2-p significantly increased the total amount of main disaccharide units and, furthermore, the composition revealed it to be more similar to that of the dermis. This 3-dimensional culture may offer a simple and useful system to investigate the glycosaminoglycan metabolism of human dermal fibroblasts in vitro.

Senoo H; Hata R . (1994) Extracellular matrix regulates cell morphology, proliferation, and tissue formation. Kaibogaku Zasshi; 69(6):719-33

Abstract:

The roles of extracellular matrix (ECM) components such as collagen, elastin, proteoglycan, and adhesive glycoprotein in the regulation of cell morphology, proliferation, and tissue formation were investigated. On a basement membrane gel, the perisinusoidal stellate cells (lipocytes, fat-storing cells, Ito cells) formed a mesh-like structure, proliferated slowly, and synthesized only a small amount of collagen. On polystyrene or type I collagen-coated culture dishes, the stellate cells spread well and extended cellular processes. The stellate cells proliferated better and synthesized more collagen on type I collagen-coated dishes than on polystyrene dishes. Co-cultures of hepatic parenchymal cells and fibroblasts formed a three-dimensional hepatic cord-like architecture in the medium supplemented with a long-acting vitamin C derivative, L-ascorbic acid 2-phosphate (Asc 2-P). Skin fibroblasts formed a three-dimensional dermis-like structure in the medium supplemented with Asc 2-P. Asc 2-P stimulated collagen synthesis of these cells. The stimulative effects of Asc 2-P on tissue formation were suppressed when collagen synthesis in these cells was inhibited. These data indicate that ECM can regulate cell morphology, proliferation and tissue formation. Regulation of cellular functions in other tissues such as mammary gland, thymus and prostate by ECM was also reviewed, and the molecular mechanisms of the regulation are discussed.

Cinatl J; Cinatl J; Weber B; Rabenau H; Gumbel HO; Chenot JF; Scholz M; Encke A; Doerr HW. (1995) In vitro inhibition of human cytomegalovirus replication in human foreskin fibroblasts and endothelial cells by ascorbic acid 2-phosphate., Antiviral Res; 27(4):405-18

Abstract:

Antiviral activity of L-ascorbic acid-2-phosphate (ASC-2P), a long-acting derivative of L-ascorbic acid, against several human cytomegalovirus (CMV) strains was examined in cultures of human foreskin fibroblasts (HFF) and endothelial cells (EC). ASC-2P at concentrations ranging from 0.2 to 2 mM had no effect on the number of cells expressing 72 kDa CMV immediate early antigen (IEA) while it inhibited expression of 68 kDa late antigen (LA) in infected cultures of both cell types (30% and 55% reduction for EC and HFF, respectively). In HFF cells, virus yield was reduced up to 4-fold, when ASC-2P was added after CMV infection. Antiviral effects were significantly increased in cultures pretreated with ASC-2P. In HFF and EC pretreated for three subcultures (18 days) with 0.2 mM ASC-2P, a significant reduction of cells expressing IEA (75% and 80% reduction in EC and HFF, respectively) and LA (92% and 90% reduction for EC and HFF, respectively) was observed. Pretreatment for three subcultures with ASC-2P inhibited virus yield 50- to 100-fold in EC and 100- to 1000-fold in HFF. The continuous presence of ASC-2P was not required for its antiviral activity. A significantly higher reduction of virus replication with ganciclovir and foscarnet was obtained in ASC-2P pretreated cells than in untreated controls. The results showed that ASC-2P provides L-ascorbic acid with long-lasting antiviral activity against CMV. ASC-2P may be of benefit for the adjunctive treatment of CMV infection.