PROJECT SUMMARY Coronary microvascular dysfunction (CMD) is linked to coronary artery disease (CAD), diabetic cardiomyopathy, and HFpEF (heart failure with preserved ejection fraction). In healthy adults, nitric oxide (NO) mediates endothelium-dependent dilation, whereas hydrogen peroxide (H2O2) is the principal endothelial dilator in patients with CAD. Such a transition might be an early pathogenic step in the progression of CAD, but little is known about the mechanisms underlying this switch, and whether under chronic conditions the switch can be reversed. This proposal addresses the mechanisms underlying the NO to H2O2 switch by using preclinical models that recapitulate the clinical CAD observations by the Gutterman group. We hypothesize that “normal” coronary microvascular function (restoring endothelial-dependent dilation) can be restored by modulating microRNA-21 (miR-21) expression and that miR-21 regulates endothelial (EC) and endothelial progenitor cell (EPC) function in diabetes. To test this hypothesis, we proposed three specific aims that utilize genetic miR-21 deletion strategies to address the impact of the miR-21 deficiency in a HFHS model of diabetes. Aim 1 addresses key aspects of EPC function and signaling pathways via single cell RNA sequencing. Aim 2 utilizes intramyocardial transplantation EPCs and myocardial blood flow measurements to determine if EPCs can reverse the NO to H2O2 switch. Aim 3 investigates the role of miR-21 specifically in ECs to determine the impact on preventing / reversing the NO to H2O2 switch and assesses vasomotor function and myocardial blood flow in diabetes. Overall, the current proposal merges vascular physiological concepts and methodologies with state- of-the genetic tools and instrumentation to gain novel insight into the mechanisms of CMD in diabetes by studying the EPC-EC-microvascular function axis and the physiological and pathological consequences surrounding miR- 21. To ensure scientific rigor, quality of research, and career development, the training plan laid out for this fellowship entails a combination of technical skill acquisition and regular scientific engagement among peers, colleagues, and professors to facilitate critical thinking and scientific communication skills. The research environment at Northeast Ohio Medical University can be described as highly collaborative, ethical, and proficient.