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Atherosclerotic plaques consist mostly of smooth muscle cells (SMC), and genes that influence SMC phenotype can modulate coronary artery disease (CAD) risk. Allelic variation at 15q22.33 has been identified by genome-wide association studies to modify the risk of CAD and is associated with the expression of

RationaleVascular beds have distinct susceptibility to atherosclerosis and aneurysm, yet the biological underpinnings of vascular bed specific disease risk are largely unknown. Vascular tissues have different developmental origins which may influence global chromatin accessibility. Understanding chromatin accessibility and gene expression profiles on single cell resolution is crucial to gain insight into vascular bed specific disease risk.ObjectiveWe aim to understand, at single cell resolution, the global chromatin accessibility and gene expression profiles across distinct vascular beds in the healthy adult mouse to provide insight into the potential mechanisms of vascular bed specific disease risk.Methods and ResultsWe performed single cell chromatin accessibility (scATACseq) and gene expression profiling (scRNAseq) of healthy adult mouse vascular tissue from three vascular beds, 1) aortic root and ascending aorta, 2) brachiocephalic and carotid artery, and 3) descending thoracic aorta. By integrating datasets and comparing vascular beds within cell type, we identified thousands of differentially accessible chromatin peaks within smooth muscle cells, fibroblasts, and endothelial cells, demonstrating numerous enhancers to be vascular bed specific. We revealed an epigenetic ‘memory’ of embryonic origin with differential chromatin accessibility of key developmental transcription factors such asTbx20,Hand2,Gata4, andHoxbfamily members. Increased transcription factor motif accessibility in ascending fibroblasts compared to descending further highlights SMAD2/3 functions and suggests a differential susceptibility to TGFβ. By isolating primary adventitial fibroblasts from ascending and descending thoracic aorta from adult mice, we demonstrate ascending fibroblasts to have a distinctly higher transcriptional response to TGFβ compared to descending fibroblasts, highlighting that distinct chromatin accessibility between vascular beds is retained following primaryin vitroculture and influences responsiveness to disease relevant signaling.ConclusionsThis work supports a paradigm that the epigenomic and transcriptional landscapes of vascular cells are cell type and vascular bed specific and that differentially accessible regions are enriched for disease risk genes.

Background: Smooth muscle cells (SMCs) 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 at genome-wide associated loci. Methods: We used CRISPR-Cas 9 genome and epigenome editing to identify the causal gene and cells for a complex CAD genome-wide association study signal at 2q22.3. Single-cell epigenetic and transcriptomic profiling in murine models and human coronary artery smooth muscle cells were used 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 transcription factor extensively studied in the context of epithelial mesenchymal transition in development of cancer. Zeb2 regulates SMC phenotypic transition through chromatin remodeling that obviates accessibility and disrupts both Notch and transforming growth factor β 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 genome-wide association study gene that affects features of plaque vulnerability through direct effects on the epigenome, providing a new therapeutic approach to target vascular disease.

Atherosclerotic plaques consist mostly of smooth muscle cells (SMC), and genes that influence SMC biology can modulate coronary artery disease (CAD) risk. Allelic variation at 15q22.33 has been identified by genome-wide association studies to modify the risk of CAD, and is associated with expression of SMAD3 in SMC, but the mechanism by which this gene modifies CAD risk remains poorly understood. SMC-specific deletion of Smad3 in a murine atherosclerosis model resulted in greater plaque burden, more positive remodeling, and increased vascular calcification. Single-cell transcriptomic analyses revealed that loss of Smad3 altered SMC transition cell state toward two fates: a novel SMC phenotype that governs both vascular remodeling and recruitment of inflammatory cells, as well as a chondromyocyte fate. The remodeling population was marked by uniquely high Mmp3 and Cxcl12 expression, and its appearance correlated with higher risk plaque features such as increased positive remodeling and macrophage content. Further, investigation of transcriptional mechanisms by which Smad3 alters SMC cell fate revealed novel roles for Hox and Sox transcription factors whose direct interaction with Smad3 regulate an extensive transcriptional program balancing remodeling and vascular extracellular matrix with significant implications for atherosclerotic and Mendelian aortic aneurysmal diseases. Together, these data suggest that Smad3 expression in SMC inhibits the emergence of specific SMC phenotypic transition cells that mediate adverse plaque features, including positive remodeling, monocyte recruitment, and vascular calcification.