Pressure alters endothelial effects upon vascular smooth muscle cells by decreasing smooth muscle cell proliferation and increasing smooth muscle cell apoptosis

Abstract

Background: Although de-endothelialization after vascular intervention is associated with intimal hyperplasia, endothelial cells (ECs) increase smooth muscle cell (SMC) numbers in conventional cocultures. In previously published work, SMCs cocultured with ECs in a chronic high-pressure environment exhibited significantly decreased cell counts compared to monocultured SMCs in the same high pressure. This finding contrasted with SMCs cocultured with ECs in ambient pressure, which exhibited significantly higher cell counts than the monocultured SMCs in ambient pressure. We now hypothesize that extracellular pressure decreases SMC number during coculture with ECs by decreasing SMC proliferation through nuclear protein regulation and by increasing SMC apoptosis. Furthermore, this effect depends on the EC response to pressure.

Methods: Rat aortic SMCs were cultured independently (SMC/0) or cocultured with EC (SMC/EC) under either atmospheric or increased pressure (130-135 mmHg over ambient, SMC/0-P and SMC/EC-P) for 5 days. We assessed SMC proliferative potential by determining c-myc expression (by protein analysis), apoptosis (by cell counting, staining with acridine orange or TUNEL technique), and topoisomerase IIalpha levels. Parallel studies measured the effects of conditioned media from monocultured EC and SMC exposed for 5 days to control or increased pressure on recipient SMC growing in conventional culture.

Results: In high-pressure conditions, SMC/EC-P exhibited 42% less c-myc expression than SMC/0s (P = .00028). Significantly increased apoptotic activity (22 +/- 1.8%) in SMC/EC-Ps compared to SMC/0s was coupled with significantly lower topoisomerase IIalpha levels. Interestingly, pressure (SMC/0-P) and EC coculture (SMC/EC) each separately raised myocyte apoptotic activity to 15 +/- 1.3% and 17 +/- 2.0%, respectively. Conditioned media from pressurized ECs caused a 20% decrease in cell counts in target SMC compared to conditioned media from ECs in atmospheric pressure. Media from pressurized SMCs did not affect target SMCs.

Conclusions: In a model designed to study SMC/EC interactions in a dynamic environment, EC exposure to pressure alters the growth characteristics and apoptotic activity of SMCs via a secreted factor. Extracellular pressure may alter EC regulation of SMC behavior and regulate intimal hyperplasia.