Project Summary Elevated levels of plasma ceramide are an independent risk factor for major adverse cardiac events (MACE) and are associated with cardiovascular diseases including coronary artery disease (CAD) and heart failure with preserved ejection fraction (HFpEF). Endothelial microvascular dysfunction, the loss of nitric oxide (NO)- mediated dilation to flow (flow-induced dilation; FID), precedes the development of CAD and occurs following chronic exposure to exogenous ceramide. During disease, following acute stress (e.g. high pressure), or after chronic ceramide treatment, FID is maintained by utilizing mitochondrial-derived hydrogen peroxide (H2O2). Although effective at eliciting dilation, unlike the anti-inflammatory effects of NO, H2O2 promotes an inflammatory environment within the vasculature and surrounding parenchymal tissue. The mechanism(s) by which ceramide promotes mitochondrial H2O2-mediated FID remains unknown. Interestingly, ceramide has also been implicated as a critical signaling component in the generation of NO. The ceramide metabolite sphingosine-1-phosphate (S1P) exerts opposing effects on the endothelium, promotes the formation of NO, and may explain the positive vascular effects associated with ceramide. A large knowledge gap exists regarding the dual functionality of ceramide within the human microvascular endothelium. We hypothesize that while ceramide formation is a critical mechanistic component in NO-mediated FID, prolonged exposure initiates a signaling cascade that results in the release of mitochondrial H2O2 in response to shear. Our aims are as follows; 1) determine the necessary role of ceramide in maintaining NO-mediated FID within the human microcirculation, and 2) investigate the mechanism(s) by which ceramide formation during stress or disease initiates the transition in FID mediator from NO to mitochondrial-derived H2O2. Using a novel approach, these mechanistic studies will be complemented by the first human in vivo study to examine the effect of elevated plasma ceramide on peripheral microvascular function. The translational studies proposed in this application will enhance our understanding of ceramide signaling during health, disease, and following acute stress. This information will provide new targets for therapeutic intervention in individuals at risk for developing cardiovascular disease including CAD and HFpEF.