Abstract 13551: RhoA Mediates Pressure-Induced Endothelial Hyperpermeability

Abstract

Pulmonary edema is a severe complication of heart failure. It is often caused by the abrupt increase in lung pulmonary capillary pressure that leads to endothelial barrier instability and disruption. The mechanisms whereby high pressure causes pulmonary edema is unresolved. However, eNOS uncoupling and downstream protein nitration may play a role. We have previously shown that eNOS uncoupling, leading to RhoA activation, is key to the development of pulmonary edema in a model of hypertensive heart failure. Herein, we sought to determine if RhoA activation plays a role in pressure-induced pulmonary edema and the molecular mechanisms that govern this process. Human lung microvascular endothelial cells (HLMEC) were exposed to 0cm (control) or 30cm (high pressure) H 2 O pressure for 5 to 60 minutes. Phosphorylation of eNOS at T495 and PKCα activation were assessed by Western blot, RhoA activation and nitration was assessed by ELISA and dot blot assays and endothelial barrier stability was assessed by VE-cadherin staining. The effects of pressure on barrier function were assessed by transendothelial electrical resistance (TEER). High hydrostatic pressure induced PKCα activation (1.42±0.2 vs 0.8±0.05 control, p=0.029; N=5) and the phosphorylation of eNOS at T495 (1.6±0.1 vs 0.62±0.08 control; p=0.0319; N=3), which was used as a correlation to eNOS uncoupling. This was associated with increased RhoA activity (0.009±0.001 vs. 0.005±0.001 control; p=0.0247; N=5), RhoA nitration (2.1±0.4 vs 1.0±0.02 control, p=0.0511; N=4) and VE-cadherin internalization. HLMEC exposed to control and high pressure conditions were transferred to gold electrodes (ECIS assay) to monitor TEER as indices of barrier recovery. High pressure caused a delay in barrier recovery and a decrease in the maximum TEER after 24 hours (0.952±0.16 vs 1.55±.019 control; p=0.0412; N=4). Fasudil, a Rho inhibitor, mitigated the effects of pressure on barrier recovery (1.996±0.79 vs 0.968±0.44 pressure; p<0.0001; N=4). Altogether, these findings suggest that pressure induces endothelial barrier disruption via PKCα-mediated eNOS uncoupling and subsequent activation of RhoA. This may be important in conditions in which pulmonary pressure can increase abruptly, including heart failure.