ascorbic-acid and diisopropyl-1-3-dithiol-2-ylidenemalonate

To assess the migratory response of fibroblasts in vitro, normal human dermal fibroblasts (NHDF) were cultured in the presence of L-ascorbic acid 2-phosphate to induce a multilayered structure. Round wounds were made by punching, and the migratory response was evaluated by counting the number of migrating cells in the wounded areas. Collagenase activity in the culture-medium was then measured. When the wound model was treated with bFGF, IL-1 alpha or PDGF, the migratory response was facilitated with increased collagenase secretion. In contrast, treatment with TGF-beta reduced the migratory response and collagenase secretion. Since the multilayered structure is rich in collagenous matrix, degradation of the matrix by secreted collagenase is probably necessary for the cells to migrate into the wounded areas. Furthermore, malotilate, which is now under development as an agent for wound therapy, facilitated the migratory response of NHDF with increased collagenase secretion in this wound model, suggesting that the wound healing effect of malotilate is in part attributable to stimulated migration of fibroblasts to wounded areas subsequent to extracellular matrix-degradation.

Topics: Ascorbic Acid; Cell Movement; Collagenases; Culture Media; Cytokines; Cytological Techniques; Fibroblasts; Humans; Immunoblotting; Malonates; Microscopy, Electron; Skin; Stimulation, Chemical; Wound Healing

Confluent bovine aortic endothelial cells (BAECs) formed a cobblestone-shaped cell monolayer when cultured on a collagen gel in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's-F12 supplemented with 5% newborn calf serum. Within a few days, however, they lost cell-cell attachment and became fibroblastic. When BAECs were cultured in the same culture medium but further supplemented with either 10 micrograms/ml malotilate or 1 mM phosphoascorbic acid, the monolayer organization and the cobblestone-like cell morphology were maintained for more than 2 weeks although many sprout cells were observed underneath the monolayer. In contrast, if both malotilate and phosphoascorbic acid were present in the culture medium, a tight monolayer without underlying sprout cells was maintained for at least 4 months and the cells expressed factor VIII-related antigens and massively internalized acetylated low density lipoprotein. By electron microscopy, we observed well-developed gap and adherence junctions, Golgi apparatuses and vesicles many of which were open to the outside by fusing with either the apical or the basal surface, indicating high metabolic activity of the cells cultured for weeks in the same dish. Although malotilate-treated BAEC monolayers secreted increased levels of prostacyclin (PGI2), the drug did not appear to directly affect the PGI2 production pathway since the similarly increased PGI2 production was noted in tight monolayers formed without the use of malotilate. Our results indicate that malotilate and phosphoascorbic acid together preserve differentiated phenotypes in cultured endothelial cells.

Topics: Animals; Aorta; Ascorbic Acid; Cattle; Culture Media; Endothelium, Vascular; Factor VIII; Lipoproteins, LDL; Malonates; Phenotype