PROJECT SUMMARY/ABSTRACT Coronary artery disease (CAD), the leading cause of death in the U.S., is caused by a narrowing of coronary arteries, the result of which is reduced cardiac perfusion and potentially myocardial infarction and/or heart failure. One promising approach to treating CAD is to regenerate arteries and restore blood flow to ischemic heart tissue. In order to make arterial regeneration a reality for CAD treatment, however, we need a more detailed understanding of how arteries are formed. Previous studies indicate that exposure of endothelial cells (ECs) to blood flow is critical for coronary artery development. Coronary artery formation is a stepwise process involving 1) specification of ECs to an arterial phenotype and 2) migration of capillary ECs into developing arteries. The mechanisms by which blood flow stimulates each of these morphogenic processes are unclear. Previous studies have shown that exposing cultured ECs to shear stress leads to upregulation of artery-specific genes, one key step in arterial EC specification. Furthermore, findings from our laboratory and others have shown that expression of the chemokine Cxcl12 is enriched in the arterial endothelium, a high shear stress environment. We also found that Cxcl12-Cxcr4 (Cxcl12 receptor) signaling promotes the migration of ECs against the direction of flow in vitro. These observations have led me to hypothesize that shear stress both fully arterializes progenitor ECs and stimulates them to release chemokines which attract nearby ECs to the developing artery. I will test this hypothesis by addressing the following Specific Aims. In Specific Aim 1, I will determine the effects of shear stress on arterial specification of ECs. To accomplish this goal, I will utilize a novel in vitro system of human arterial differentiation in which pure populations of arterial ECs can be generated from pluripotent stem cells treated with arterializing biochemical signals. Measuring arterial EC specification in response to different combinations of shear stress and arterializing biochemical signals will reveal molecular mechanisms by which shear stress drives ECs towards an arterial fate. In Specific Aim 2, I will determine the role of chemokines in orchestrating flow-induced EC migration in vivo. Namely, I will perturb EC Cxcl12 – Cxcr4 signaling by using mice in which either Cxcl12 is deleted from arterial ECs or Cxcr4 is deleted from capillary ECs. Assessing coronary artery formation in these mice will allow me to determine whether arterial Cxcl12 – capillary Cxcr4 signaling directs the migration of ECs from environments of low (capillary) to high (artery) shear stress. Results from these studies will generate substantial insight into the mechanisms by which shear stress promotes arterial specification and coronary artery remodeling. Findings from this work may be leveraged therapeutically to develop strategies for regenerating arteries in vivo or generating tissue-engineered arteries ...