Alterations in the protein composition and surface structure of human endothelial cells during growth in primary culture

Abstract

The protein composition and surface structure of human endothelial cells in primary culture at different ages and cell densities have been examined by lactoperoxidase-catalyzed iodination, followed by electrophoresis of the solubilized cells on sodium dodecylsulphate-polyacrylamide gels. Electrophoresis followed by carbohydrate staining with the periodic acid Schiff's reagent of cells grown for at least 5–6 days revealed the presence of 4 glycopolypeptide bands. The corresponding molecular weights were estimated to be 225 000, 190 000, 145 000 and 135 000. In contrast, glycopolypeptide bands were rarely seen when cells from younger cultures were examined. Similarly, the distribution of radioactivity varied according to the age of the culture. In cultures grown for less than 5–6 days the dominant peaks of radioactivity appeared between 90 000 and 50 000 daltons. As the cultures increased in age a peak of radioactivity at 225 000 daltons became successively more prominent. This age-dependent change in the distribution of radioactivity occurred both in sparse cultures that did not reach confluence and in denser cultures. Electrophoresis of solubilized endothelial cells followed by protein staining with Coomassie Brilliant Blue revealed a characteristic pattern of seven major bands ranging in molecular weight from 225 000 to 40 000. These bands were detected regardless of the age or cell density of the cultures. However, with increasing age or cell density of the cultures the relative intensity of the 225 000-dalton band increased compared to the 190 000-dalton band. Our findings demonstrate that alterations in protein composition and surface structure of human endothelial cells take place during growth in primary culture under conditions aimed to mimic the normal physiological situation. The present technique may also provide a means of studying alterations of the endothelial cell-surface proteins that take place when the cells are exposed to atherogenic agents.