Project Summary Coronary microvascular dysfunction or disease (CMD) may lead to angina pectoris or acute coronary syndrome (ACS) without obstructive coronary artery disease (CAD), which is considered as one of major mechanisms of ischemia heart disease (IHD). However, the pathogenesis of CMD remains unclear. Based on literature and our previous studies, we wondered whether exosomes or other microvesicles-mediated signaling from endothelial cells (ECs) serve as a crucial mechanism mediating CMD because much thinner endothelium- smooth muscle (EC-SM) space in small coronary arteries (CAs) or arterioles than large arteries may limit the development of typical atherogenic pathology, but the exosome-carrying molecules may result in functional abnormality in these small coronary arteries. Our preliminary studies indeed demonstrated that exosomes were increasingly secreted into subendothelial space in small coronary arteries in mice fed the Western diet (WD) and that deletion of lysosomal acid ceramidase (AC) in coronary ECs from Asah1fl/fl/ECcre mice, a strain of EC- specific AC gene knockout mice (Asah1 is mouse gene code of AC) resulted in accumulation or trapping of exosomes in the EC-SM space, leading to CMD-mediated myocardial ischemia without atherosclerosis and obstructive CAD. The present grant proposal will test a central hypothesis that endothelium-derived exosomes regulated by lysosomal AC-mediated sphingolipid metabolism plays a crucial role in the control of small CA function and that subendothelial exosome accumulation is critically implicated in the pathogenesis of CMD. To test this hypothesis, three Specific Aims are proposed. Specific Aim 1 will determine whether exosome secretion from coronary arterial ECs is fine controlled by lysosomal AC activity and whether the deficiency of this AC regulation causes subendothelial accumulation of exosomes in small CAs of Asah1fl/fl/ECcre mice, leading to CMD and myocardial ischemia without atherosclerosis and obstructive CAD. Specific Aim 2 attempts to test whether lysosomal AC-mediated sphingolipid signaling regulates lysosome trafficking to and fusion with multivesicular bodies (MVBs) to limit exosome secretion from ECs and whether pathologically released exosomes from ECs with gene deletion from Asah1fl/fl/ECcre mice induce dysfunction of coronary arterial SMCs. In Specific Aim 3, we will address whether the AC regulation of lysosome trafficking and exosome secretion from coronary ECs are attributed to its action on lysosomal TRPML1 channel activity and associated Ca2+ release and whether the deficiency of this regulatory mechanism leads to subendothelial exosome accumulation and CMD. To our knowledge, these proposed studies will represent the first effort to investigate the pathogenic role of subendothelial exosomes in CMD and consequent myocardial ischemia and the findings will provide new insights into the pathogenesis of CMD and IHD without atherosclerosis or obstructive CAD.