Program Director/Principal Investigator (Last, First, Middle): Naik, Jay, S Impaired endothelial cell (EC) function is one of the first indicators of systemic cardiovascular disease. EC dysfunction impairs local organ blood flow regulation, a primary cause of end-organ damage in most cardiovascular diseases. The discovery that ECs also synthesize carbon monoxide (CO) and hydrogen sulfide (H2S), in addition to nitric oxide (NO), which elicits vasodilation has opened a new chapter in endothelial gasotransmitter biology. While increasing evidence supports a key role for H2S in cardiovascular homeostasis, disparate findings in previous studies leaves a significant gap in knowledge on the regulation and significance of H2S signaling in the vasculature. Recently, we identified a novel regulator of H2S signaling that leads to profound differences in vasodilatory sensitivity between primary and tertiary segments of the circulation. Intriguingly, our preliminary data show that H2S dilates small (resistance) arteries in an endothelium-dependent manner at concentrations that have no effect in large arteries. However, depleting EC membrane cholesterol in large arteries unmasks H2S-mediated vasodilation, suggesting membrane lipid content and domains regulate H2S signaling. Moreover, our preliminary data show that native EC cholesterol content is greater in large arteries than resistance arteries. The concept that innate regional differences in EC membrane cholesterol content mediate functional differences in EC dilation is wholly novel, and our preliminary data (Fig. 6) demonstrate this may be caused by increased cholesterol efflux via ATP-binding cassette family a1 (Abca1) and phospholipid transfer protein (Pltp). Importantly, we have previously shown that changes in EC membrane cholesterol appear to contribute to dysfunction in disease. Therefore, EC membrane cholesterol is an important but uninvestigated variable in vascular function. The overall goal of this project is two-fold. First, to elucidate mechanisms leading to the functionally significant differences we have observed in EC membrane cholesterol content between large and small arteries. The second is to define ways in vitro and in vivo that these differences in EC membrane cholesterol control EC function, specifically H2S-induced dilation. Thus, we hypothesize that augmented cholesterol efflux in EC of resistance arteries enhances downstream H2S signaling Aim 1: Determine the mechanism(s) leading to heterogeneous membrane cholesterol content between large and small arteries. Aim 2: Determine the mechanism(s) by which membrane cholesterol regulates H2S signaling in EC. Completing the proposed studies will fill an existing knowledge gap by elucidating the regulation of H2S dilation to identify differences in EC function between small and large arteries. Conceptually, the project is very innovative in its focus on the novel ability of membrane cholesterol trafficking to act as a regulator of vasodil...