Increased age, presence of other cardiovascular risk factors or previous treatment with anti-cancer therapy are among the leading risk factors for development of coronary artery disease (CAD). While CAD is traditionally viewed as a large vessel disease substantial recent data indicate that impaired microvascular function contributes substantially to pathophysiology and outcomes in cardiovascular disease. Subjects with a clinical diagnosis of CAD exhibit loss of NO-mediated microvascular flow-mediated dilation (FMD) concurrent with upregulation of mitochondrial hydrogen peroxide (H2O2), promoting local inflammation and cellular proliferation. Understanding the contributing mechanisms that regulate the switch from NO to H2O2 may help to reduce the risk of tissue injury from vascular paracrine redox toxicity. We have identified several components of the signaling pathway that changes the mediator of FMD from NO to H2O2. The common feature of each of these pathways is excess endothelial mitochondrial ROS generation. Mitochondrial fission and fusion, known regulators of ROS production, are tightly regulated by a group of pro- fission and pro-fusion proteins suggesting the possibility that these factors determine the mediator of FMD in the human microvasculature, an unexplored question. The goal of this study is to test the hypothesis that mitochondrial fission/fusion is critically linked to the mediator of FMD in the human microcirculation. Based on preliminary data we expect that regulators of fission/fusion are fundamental mediators of mitochondrial ROS production and determinants of whether shear elicits release of endothelial NO or H2O2. Mitochondria and ROS are also involved in hypoxic preconditioning (HPC), a stimulus that improves tissue tolerance to stressors and protects against disease. Very little is known about HPC and vascular protection with no studies in the microcirculation. Our preliminary data support a role for mitochondrial fission and fusion in mediating HPC. This potential mechanism for HPC induced vascular protection will be explored. We will study fresh human coronary and adipose arterioles and primary human microvascular endothelial cells in vitro using pharmacological and genetic tools to manipulate fission and fusion mediators and determine how these changes contribute to alterations in mechanisms of FMD observed in CAD or after acute stress (elevated glucose, intraluminal pressure). We will test the overreaching hypothesis that mitochondrial fission is associated with H2O2 while mitochondrial fusion promotes physiological NO mediated dilation to flow. Aim 1: Changes in fission/fusion or its regulators are necessary and sufficient to explain the transition in the mediator of FMD from NO to H2O2 during CAD or vascular stress (IILP or HG) Aim 2: Investigate whether the mechanism by which hypoxic vascular preconditioning improves microvascular function after acute stress (IILP, HG) or in subjects with CAD involves an increase in mitochondrial fusion.

In response to shear, microvascular dilation occurs via endothelial release of NO in healthy individuals and mitochondrial derived H2O2 in stress or in patients with coronary disease. We hypothesize that mitochondrial fission/fusion is responsible for this change in mediator of dilation. Resulting information could have profound impact on regional blood flow and on oxidative-stress induced cardiovascular disease.