Background: Smooth muscle cells (SMC) transition into a number of different phenotypes during atherosclerosis, including those that resemble fibroblasts and chondrocytes, and make up the majority of cells in the atherosclerotic plaque. To better understand the epigenetic and transcriptional mechanisms that mediate these cell state changes, and how they relate to risk for coronary artery disease (CAD), we have investigated the causality and function of transcription factors (TFs) at genome wide associated loci. Methods: We employed CRISPR-Cas 9 genome and epigenome editing to identify the causal gene and cell(s) for a complex CAD GWAS signal at 2q22.3. Subsequently, single-cell epigenetic and transcriptomic profiling in murine models and human coronary artery smooth muscle cells were employed to understand the cellular and molecular mechanism by which this CAD risk gene exerts its function. Results: CRISPR-Cas 9 genome and epigenome editing showed that the complex CAD genetic signals within a genomic region at 2q22.3 lie within smooth muscle long-distance enhancers for ZEB2 , a TF extensively studied in the context of epithelial mesenchymal transition (EMT) in development and cancer. ZEB2 regulates SMC phenotypic transition through chromatin remodeling that obviates accessibility and disrupts both Notch and TGFβ signaling, thus altering the epigenetic trajectory of SMC transitions. SMC specific loss of ZEB2 resulted in an inability of transitioning SMCs to turn off contractile programing and take on a fibroblast-like phenotype, but accelerated the formation of chondromyocytes, mirroring features of high-risk atherosclerotic plaques in human coronary arteries. Conclusions: These studies identify ZEB2 as a new CAD GWAS gene that affects features of plaque vulnerability through direct effects on the epigenome, providing a new thereapeutic approach to target vascular disease.

Genome wide association studies for coronary artery disease (CAD) have identified a risk locus at 11q22.3. Here, we verify with mechanistic studies that rs2019090 and PDGFD represent the functional variant and gene at this locus. Further, FOXC1/C2 transcription factor binding at rs2019090 is shown to promote PDGFD transcription through the CAD promoting allele. With single cell transcriptomic and histology studies with Pdgfd knockdown in an SMC lineage tracing male atherosclerosis mouse model we find that Pdgfd promotes expansion, migration, and transition of SMC lineage cells to the chondromyocyte phenotype. Pdgfd also increases adventitial fibroblast and pericyte expression of chemokines and leukocyte adhesion molecules, which is linked to plaque macrophage recruitment. Despite these changes there is no effect of Pdgfd deletion on overall plaque burden. These findings suggest that PDGFD mediates CAD risk by promoting deleterious phenotypic changes in SMC, along with an inflammatory response that is primarily focused in the adventitia.

BACKGROUND: Smooth muscle cells (SMCs), the major cell type in atherosclerotic plaques, are vital in coronary artery diseases (CADs). Smooth muscle cell (SMC) phenotypic transition, which leads to the formation of various cell types in atherosclerotic plaques, is regulated by a network of genetic and epigenetic mechanisms and governs the risk of disease. The involvement of long noncoding RNAs (lncRNAs) has been increasingly identified in cardiovascular disease. However, SMC lncRNAs have not been comprehensively characterized, and their regulatory role in SMC state transition remains unknown. METHODS: A discovery pipeline was constructed and applied to deeply strand-specific RNA sequencing from perturbed human coronary artery SMC with different disease-related stimuli, to allow for the detection of novel lncRNAs. The functional relevance of a select few novel lncRNAs were verified in vitro. RESULTS: We identified 4579 known and 13 655 de novo lncRNAs in human coronary artery SMC. Consistent with previous long noncoding RNA studies, these lncRNAs overall have fewer exons, are shorter in length than protein-coding genes (pcGenes), and have relatively low expression level. Genomic location of these long noncoding RNA is disproportionately enriched near CAD-related TFs (transcription factors), genetic loci, and gene regulators of SMC identity, suggesting the importance of their function in disease. Two de novo lncRNAs, ZEB-interacting suppressor ( ZIPPOR ) and TNS1 -antisense ( TNS1-AS2 ), were identified by our screen. Combining transcriptional data and in silico modeling along with in vitro validation, we identified CAD gene ZEB2 as a target through which these lncRNAs exert their function in SMC phenotypic transition. CONCLUSIONS: Expression of a large and diverse set of lncRNAs in human coronary artery SMC are highly dynamic in response to CAD-related stimuli. The dynamic changes in expression of these lncRNAs correspond to alterations in transcriptional programs that are relevant to CAD, suggesting a critical role for lncRNAs in SMC phenotypic transition and human atherosclerotic disease.

Platelet derived growth factor (PDGF) signaling has been extensively studied in the context of vascular disease, but the genetics of this pathway remain to be established. Genome wide association studies (GWAS) for coronary artery disease (CAD) have identified a risk locus at 11q22.3, and we have verified with fine mapping approaches that the regulatory variant rs2019090 and PDGFD represent the functional variant and putative functional gene. Further, FOXC1/C2 transcription factor (TF) binding at rs2019090 was found to promote PDGFD transcription through the CAD promoting allele. Employing a constitutive Pdgfd knockout allele along with SMC lineage tracing in a male atherosclerosis mouse model we mapped single cell transcriptomic, cell state, and lesion anatomical changes associated with gene loss. These studies revealed that Pdgfd promotes expansion, migration, and transition of SMC lineage cells to the chondromyocyte phenotype and vascular calcification. This is in contrast to protective CAD genes TCF21, ZEB2, and SMAD3 which we have shown to promote the fibroblast-like cell transition or perturb the pattern or extent of transition to the chondromyocyte phenotype. Further, Pdgfd expressing fibroblasts and pericytes exhibited greater expression of chemokines and leukocyte adhesion molecules, consistent with observed increased macrophage recruitment to the plaque. Despite these changes there was no effect of Pdgfd deletion on SMC contribution to the fibrous cap or overall lesion burden. These findings suggest that PDGFD mediates CAD risk through promoting SMC expansion and migration, in conjunction with deleterious phenotypic changes, and through promoting an inflammatory response that is primarily focused in the adventitia where it contributes to leukocyte trafficking to the diseased vessel wall.