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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, positive remodeling, and increased vascular calcification. Single-cell transcriptomic analyses revealed that loss of Smad3 altered SMC progeny phenotype toward the previously described chondromyocyte fate, but importantly also promoted transition to a novel cell-state that governs remodeling and recruitment of inflammatory cells. This new 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 ECM with significant implications for human 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.

IntroductionSmooth muscle cells (SMC) play a critical role in atherosclerosis. The Aryl hydrocarbon receptor (AHR) is an environment-sensing transcription factor that contributes to vascular development, and has been implicated in coronary artery disease (CAD) risk. We hypothesized that AHR can affect atherosclerosis by regulating phenotypic modulation of SMC.MethodsWe combined RNA-Seq, ChIP-Seq, ATAC-Seq and in-vitro assays in human coronary artery SMC (HCASMC), with single-cell RNA-Seq (scRNA-Seq), histology, and RNAscope in an SMC-specific lineage-tracing Ahr knockout mouse model of atherosclerosis to better understand the role of AHR in vascular disease.ResultsGenomic studies coupled with functional assays in cultured HCASMC revealed that AHR modulates HCASMC phenotype and suppresses ossification in these cells. Lineage tracing and activity tracing studies in the mouse aortic sinus showed that the Ahr pathway is active in modulated SMC in the atherosclerotic lesion cap. Furthermore, scRNA-Seq studies of the SMC-specific Ahr knockout mice showed a significant increase in the proportion of modulated SMC expressing chondrocyte markers such as Col2a1 and Alpl, which localized to the lesion neointima. These cells, which we term “chondromyocytes” (CMC), were also identified in the neointima of human coronary arteries. In histological analyses, these changes manifested as larger lesion size, increased lineage-traced SMC participation in the lesion, decreased lineage-traced SMC in the lesion cap, and increased alkaline phosphatase activity in lesions in the Ahr knockout compared to wild-type mice. We propose that AHR is likely protective based on these data and inference from human genetic analyses.ConclusionOverall, we conclude that AHR promotes maintenance of lesion cap integrity and diminishes the disease related SMC-to-CMC transition in atherosclerotic tissues.

BackgroundTo investigate the epigenetic and transcriptional mechanisms of coronary artery disease (CAD) risk, as well as the functional regulation of chromatin structure and function, we have created a catalog of genetic variants associated with three stages of transcriptionalcis-regulation in primary human coronary artery vascular smooth muscle cells (HCASMC).ResultsTo this end, we have used a pooling approach with HCASMC lines to map regulatory variation that mediates binding of the CAD associated transcription factor TCF21 with ChIPseq studies (bQTLs), variation that regulates chromatin accessibility with ATACseq studies (caQTLs), and chromosomal looping with HiC methods (clQTLs). We show significant overlap of the QTLs, and their relationship to smooth muscle specific genes and the binding of smooth muscle transcription factors. Further, we use multiple analyses to show that these QTLs are highly associated with CAD GWAS loci and correlated to lead SNPs in these loci where they show allelic effects. We have verified with genome editing that identified functional variants can regulate both chromatin accessibility and chromosomal looping, providing new insights into functional mechanisms regulating chromatin state and chromosomal structure. Finally, we directly link the disease associatedTGFβ1-SMAD3pathway to the CAD associatedFN1gene through a response QTL that modulates both chromatin accessibility and chromosomal looping.ConclusionsTogether, these studies represent the most thorough mapping of multiple QTL types in a highly disease relevant primary cultured cell type, and provide novel insights into their functional overlap and mechanisms that underlie these genomic features and their relationship to disease risk.

Long noncoding RNAs (lncRNA) comprise an underlying regulatory network in the human genome that remains largely unexplored and could represent a molecular mechanism connecting GWAS risk variants with the disease causal genes. Human coronary artery smooth muscle cells (HCASMC) are one of the most important cells in the regulation of atherosclerotic disease progression. In this study, we aimed to discover the HCASMC-specific lncRNA collection and to explore the mechanistic relationship between HCAMSC lncRNAs with increased disease risk for coronary artery disease. We applied 6 different stimuli relevant to athero-condition, including stimulations with TGFbeta, TNFalpha, PDGFD and serum, and two transcription factor knock-downs TCF21 and SMAD3, and performed deep RNA sequencing on 48 HCASMC samples. We designed a lncRNA discovery pipeline to maximize the detection of tissue specific and low expressed lncRNAs. This generated a set of 53076 lncRNAs, that showed increased association with CAD GWAS variants, various HCASMC eQTL data sets and GTEx eQTLs for tissues enriched in smooth muscle. Module analysis revealed lncRNAs highly associated with TGFbeta and general pro-differentiation traits located in the CAD GWAS loci, such as, FES/FURIN, COL4A1/A2, CDKN2A/B, TGFB1, and FN1. Transcription factor motif analysis revealed the presence of HCASMC relevant factors, such as, TCF21, ZEB1, ZEB2, JUN, JUND, and an SRF cofactor ELK4, near the boundaries of HCASMC lncRNA. We defined lncRNA QTLs using the GTEx Coronary Artery dataset, and showed colocalization with other HCASMC QTLs, such as expression QTLs, chromatin looping QTLs, chromatin accessibility QTLs and TCF21 binding QTLs. We show several examples of CAD GWAS loci where lncRNAs show regulatory function, such as FES/FURIN, FN1, COL4A1/A2 and TGFB1. We unraveled a complex network of regulatory interactions at FES/FURIN locus, involving TCF21 and lncRNA 43779.9, and present a model in which TCF21 inhibited lncRNA 43779.9 regulates FES gene and indirectly the pro-differentiation FURIN gene by creating a looping interaction with the intron 1 of FES transcript. We define 5 regulatory variants at the FES/FURIN locus and propose a causal variant, rs35346340, that disrupts TCF21 binding and subsequently influences 43779.9 expression. Finally, we show the presence of lncRNAs in deeply sequenced TCF21 pooled ChIPSeq and discover TCF21 binding lncRNAs, 3938.1 and 3852.1 as ACTA2-regulating transcripts. This study defines lncRNAs as essential regulators of GWAS loci and shows the importance of using deep sequencing approach to further explore the genomic regulatory landscape of lncRNAs in human tissues.