Project Summary Chronic Hypoxia (CH)-induced pulmonary hypertension (PH) is a significant source of morbidity and mortality in patients with chronic obstructive pulmonary diseases. It is widely recognized that vasoconstriction is a critical mediator of PH, although the mechanisms involved are poorly understood. Our previous studies have demonstrated that enhanced vasoconstrictor sensitivity following CH involves a requisite reduction in pulmonary arterial smooth muscle cell (PASMC) membrane cholesterol content. We have also demonstrated that CH augments vasoconstrictor reactivity by a switch in signaling from primarily calcium-dependent mechanisms to a Ca2+ sensitization pathway that involves the epidermal growth factor receptor (EGFR) and reactive oxygen species (ROS). However, the mechanisms by which CH decreases membrane cholesterol and how this unmasks EGFR-dependent vasoconstriction has yet to be assessed. The proposed studies will investigate the central hypothesis that coupling of vasoconstrictor stimuli to EGFR signaling following CH promotes PASMC hypercontractility through a ROS-dependent decrease in membrane cholesterol. To test this hypothesis, protocols will employ both in vivo and in vitro approaches using a variety of experimental preparations from molecular and single cell imaging studies to video-microscopy of pressurized small pulmonary arteries using a rat model of CH-induced PH. We plan to pursue the following specific aims: Specific Aim 1: Determine the mechanism by which CH decreases PASMC membrane cholesterol. Hypothesis: Elevated ROS production during CH diminishes membrane cholesterol. Specific Aim 2: Determine the mechanism by which decreased PASMC membrane cholesterol augments vasoconstrictor sensitivity following CH. Hypothesis: Decreased PASMC membrane cholesterol in response to CH unmasks EGFR-dependent pulmonary vasoconstriction through regulation of NOX2 and Rac1. The applicant will be immersed in a rich training environment in the Vascular Physiology Group at the UNM School of Medicine through a unique, multi-sponsor mentoring team that will facilitate his research training in defining novel mechanisms by which ROS alter the PASMC membrane microenvironment to affect cellular function in CH-induced PH. The proposed training plan will afford the applicant intensive training experiences in a variety of new experimental approaches, refinement of his oral and written communication skills, and professional development training that will aid him in achieving his goal as an independent, academic physician-scientist in pulmonary research.