Enhanced apoptosis in prolonged cultures of senescent porcine pulmonary artery endothelial cells

Abstract

Senescent or aged endothelial cells in culture remain metabolically active after cessation of division, and are generally believed to eventually die. However, mechanisms underlying the terminal aging of cultured cells, i.e. from senescence to death, are poorly understood. Here, we report that culturing of replicative senescent endothelial cells for a prolonged period of time without passaging leads to enhanced programmed cell death or apoptosis. Senescent (passage 45) and young (passage 3) porcine pulmonary artery endothelial cells (PAEC) were cultured for 0-42 days post confluence. The cells attached to culture dishes and floating in medium were collected at 0, 7, 14, 21, 28, 35 and 42 days post confluence and were assessed for markers of apoptosis. Morphology studies showed that ratios between senescent and young cells attached to dishes declined to 45% after 42 days postconfluence. Apoptotic cells in prolonged cultures of senescent PAEC increased from 5 to 35% as determined by protein mass, DNA breakage, and caspase-3 activation. Steady state levels of Bcl-2, an anti-apoptotic protein, in senescent prolonged cultures decreased to less than 20% for all time points compared with young cells. Relative levels of Bad, a pro-apoptotic protein, in senescent cells were elevated from 60 to 130% during prolonged culturing. These results indicate that terminal cellular aging enhances apoptosis and the levels of Bcl-2/Bad may be associated with the apoptotic process in porcine lung endothelial cells.