We have identified TCF21 as the coronary artery disease (CAD) associated gene mapped by genome-wide association studies at 6q23.2 and employed numerous mechanistic approaches to show that it promotes a smooth muscle cell (SMC) transition to a fibroblast like “fibromyocyte” phenotype, and the contribution of these cells to the protective fibrous cap. Our studies with another CAD associated gene, the aryl hydrocarbon receptor (AHR), have characterized the transition of SMC to a second, chondrogenic “chondromyocyte” phenotype. To extend this work and investigate the mechanisms of epigenetic signaling upstream of TCF21, AHR, and other factors that mediate SMC cell state, we are focusing efforts on the CAD associated platelet derived growth factor D gene (PDGFD). We have shown that PDGFD regulates TCF21 and other validated CAD genes including LMOD1, CXCL12, and SMAD3, and is expressed primarily in disease transition SMC that also express the PDGFRB receptor. Together, these data suggest that PDGFD activates an autocrine signaling pathway that modulates SMC phenotype and CAD risk. The hypothesis directing this research postulates that PDGFD promotes CAD risk through its regulation of TCF21 and other key disease related transcription factors that mediate the SMC phenotypic response to vascular stress. The primary goals of the work proposed here are thus to identify the PDGFD target transcription factors (TFs) that regulate SMC transitions and characterize their transcriptional program in this cell type. Specifically, in Aim 1 we will employ Pdgfd knockout and SMC lineage tracing in the ApoE null mouse atherosclerosis model to characterize the effect of this gene on SMC cell state transitions, and the impact of perturbing these transitions on disease morphology and cellular anatomy. In Aim 2, we will conduct single cell RNA sequencing (scRNAseq) in Pdgfd null and wildtype atherosclerotic mice to characterize the SMC gene expression program downstream of Pdgfd in this cell type. Single cell ATAC sequencing (scATACseq) in the same animals will map enhancers genome- wide that are differentially regulated in SMC phenotypic transitions, and identify specific TFs that bind these enhancers to regulate expression of fibromyocyte and chondromyocyte specific genes. In Aim 3, we will perturb candidate SMC transition promoting TFs that are identified in Aim 2, in vitro in a PDGFD stimulated human coronary artery smooth muscle cell de-differentiation model, and the resulting transcriptomic and cell state effects interpreted in the context of PDGFD function in this model. These studies will link PDGFD to CAD associated genes that we have characterized in the context of SMC phenotypic transition (TCF21, AHR, SMAD3, TWIST1), and to additional high probability CAD genes that regulate SMC phenotype, to expand the disease transcriptional network in this vascular cell type. This work will advance our understanding of atherosclerosis pathophysiology and promote efforts to ta...