Differential responsiveness of early and late passage (SENESCENT) endothelial cells to flow

Abstract

Hemodynamic forces such as shear stress are implicated in the development of atherosclerosis and neointimal hyperplasia. To determine whether in vitro studies of flow using early passage endothelial cells (EC) are relevant to elderly patients, the effects of flow on early passage and late passage (senescent) EC were compared. Early (p3–7) and late (p28–32) passage bovine aortic EC were exposed to arterial levels of flow (11 dyn/cm 2) using an orbital shaker; control cells were exposed to static conditions (0 dyn/cm 2). Cell proliferation was counted with a Coulter counter. PCNA was evaluated by immunohistochemistry. Phosphorylated and total Akt were assessed with Western blotting. Conditioned media from early and late passage EC under static and flow conditions (16 h) were evaluated for their chemoattractant activities by assessing smooth muscle cell (SMC) migration (4 h, Boyden chamber). Early passage cells demonstrated increased proliferation with flow compared to static conditions (36% increase, day 5, P = 0.03); late passage cells had no statistically significant increase ( P = 0.42). PCNA staining confirmed lack of proliferation in late passage cells under static or flow conditions. Western blotting demonstrated increased Akt phosphorylation with flow in early, but not late, passage EC ( n = 6, P = 0.01). Senescent cells induced 26% less SMC migration than early passage cells ( n = 3, P = 0.03). Late passage EC do not proliferate in response to turbulent flow as younger EC do. Late passage EC also do not phosphorylate Akt, and attract fewer SMC, than early passage EC, in response to flow. These results suggest that hemodynamic forces may have different effects on senescent compared to younger EC and implies that treatment of vascular disease in elderly patients may require different therapeutic strategies than those based on strategies designed for younger patients.