Age-related changes in the glycosaminoglycans and collagen of cultured pig endothelial cells.

Pig aortic endothelial cells were grown from aortic intimal explants. Tissue culture samples were obtained at regular intervals between the 2nd and 21st day (time-course experiments) and between the 2nd and 15th passage (influence of subcultivation). Glycosaminoglycans (GAG-s) were isolated separately from the cells and from the medium. The various GAG-s were identified by electrophoresis on cellulose acetate strips, by differential degradations with HNO2 and by digestion with selective enzymes, and their concentration was measured. Protein and collagen content was also determined. Parallel tissue culture monolayers were investigated by microscopical methods. Total GAG and collagen concentrations showed maximum curves when plotted as a function of the time of incubation, with peak values on the 9th day, both in the cells and in the medium. From among the individual GAG-s the amount of hyaluronic acid and chondroitin-sulfate increased gradually, while heparan-sulfate content changed in the opposite direction. Chondroitin was detectable only in the culture medium; its concentration decreased steeply until the tenth day. Alterations in the dermatansulfate content showed inverse tendency. The production and excretion of collagen and of GAG-s increased during the first six passages, both in the cells and in the media, and then declined during the four following ones. The ratio of the individual GAG-s did not alter until the tenth subculture. Further subcultivation resulted in a gradual increase in the relative amount of chondroitin-sulfate and a decrease in heparan sulfate contents.