Background: Vascular diseases are a leading cause of mortality worldwide. Epidemiological data suggest significant sex-based differences in vascular disease risk, but the mechanisms underlying these differences remain unclear. Clinical observations indicate that key sex-specific differences may exist in the vessel wall. Hypothesis: We hypothesize that sex differences in vascular diseases are regulated by sex-specific epigenetic factors/features that alter cellular responses to pathologic triggers in the vessel wall. Methods: Single cell transcriptomic (scRNA-seq) and epigenetic (scATAC-seq) analyses were performed on male (M) and female (F) ApoE mice at different stages of atherosclerosis progression (0, 16 wks) to uncover sex-specific responses to vascular insult. Comparative analyses were performed on M and F human coronary arteries to identify biologically conserved sex-specific regulatory features shared between humans and mice. These features were integrated with human genetic data to identify the biological processes and regulatory regions driving sex-specific responses. Key findings were subsequently validated in vitro using primary human vascular cells. Results: Transcriptomic differences between M and F cells were most profound in smooth muscle cells (SMC) and fibroblasts in human and murine arteries. In mouse, the sex-specific transcriptomic differences drastically increased after high fat diet exposures, with number of differentially expressed genes increasing from 29 to 140 in SMC, and 63 to 181 in fibroblasts. Furthermore, we found F SMC to more readily undergo phenotypic modulation despite lower lipid levels. Gene set enrichment analysis showed increased pathways related to extracellular matrix organization and epigenetic modification, as well as key regulators of PDGF and TGFB signaling in mice and human female SMC. Additionally, scATAC-seq identified thousands of sex-specific differentially accessible regions enriching for human genetic signals of vascular diseases. In particular, X-escapee genes Kdm6a and Kdm5c were identified which may work in concert to alter the epigenetic landscape. Conclusion: This study reveals significant sex-specific, vascular cell-type specific transcriptional and epigenetic mechanisms in vascular disease. These findings provide insights into the genetic and molecular basis of sex differences in vascular diseases and potential therapeutic targets.

Vascular diseases, including atherosclerotic disease, are the leading cause of mortality worldwide. Epidemiological data points to sex-based differences in vascular disease risk, but the pathophysiological mechanisms remain poorly understood. Clinical observations suggest these differences originate within the vessel wall, but the mechanism that governs these sex-specific difference is not known. We hypothesized that sex-specific vascular disease susceptibility arises from interactions between sex chromosome–encoded epigenetic regulators and environmental exposures such as cigarette smoke. To study this, male and female ApoE knockout mice were exposed to cigarette smoke for 7 weeks and fed high-fat diet (HFD) for 8 weeks (Exposed cohort). As a control cohort, the other set were only fed HFD for 8 weeks. Throughout the exposure period, weekly weights and blood pressures were obtained. The aortic roots and descending aortae were harvested and processed for single-cell transcriptomic analysis (scRNA-seq). The data was integrated with human genetic data to identify the biological processes and regulatory regions driving sex-specific responses. A set of the aortic root and descending aorta were also used for immunohistology. At the end of the exposure period, there was gross evidence of early plaque development in the aortic root and descending aorta. Differential gene expression analysis of each segmental cell population reveals increased sex differences in smooth muscle cells (SMCs) and fibroblasts, compared to other cell types present. Increased transcriptomic differences show up in both male and female SMCs and fibroblasts for smoking and high-fat diet exposure relative to HFD alone. Further, GSEA reveals over-enrichment of pathways related to epigenetic modifications in female cells, compared to male cells. Notably, the X-linked histone demethylase Kdm6a and Kdm5c emerged as a top differentially expressed gene in descending SMCs with strong sex-specific significance, while showing modest regulation in root-derived cells. Smoking exposure downregulated Kdm6a and Kdm5c for both the sex, females more than males, as compared to HFD only which further affected matrix remodeling pathway. This study reveals significant sex-specific, vascular cell-type specific transcriptional and epigenetic mechanisms in vascular disease and shows sex chromosome-encoded epigenetic modifiers, particularly Kdm6a and Kdm5c, as pivotal in vascular cell plasticity in atherosclerosis.

Smooth muscle cell (SMC) phenotypic modulation and proliferation are a conserved cellular response to vascular insult in the pathogenesis of atherosclerosis, aneurysm and vascular injury. Pan-SMC lineage tracing previously revealed that few SMCs oligo-clonally expand in these diseases, while the majority remain dormant. However, it remains unclear whether SMC clone selection is stochastic or predetermined. Understanding which SMCs expand, and how, could reveal major new therapeutic targets for vascular disease. Notch3 and Sox9 are critical for arterial SMC specification during development. Using tamoxifen-inducible lineage-tracing and scRNAseq, we characterised rare, distinct populations of medial SMCs retaining Notch3 and Sox9 expression in healthy adult vessels. Despite low baseline abundance, lineage-traced Notch3/Sox9 + SMC progeny combined to contribute over half of SMC-derived lesional cells, suggesting that oligo-clonal SMC expansion arises mainly from pre-determined progenitors rather than stochastically. Further, Notch3/Sox9+ SMCs showed distinct transcriptomic profiles at baseline and differing fates within the final lesion, suggesting differing roles in pathogenesis. At baseline, Sox9+ SMCs displayed downregulation of contractile markers and upregulation of a complement of genes typically only detected in lesional modulated SMCs, suggesting cell priming. Moreover, compared with other SMC-derived plaque cells, progeny of Notch3+ cells expressed a distinct inflammatory profile and were preferentially fated to the fibrous cap while avoiding chondrogenic transformation. Finally, at baseline, Notch3/Sox9+ progenitor abundance varied between arterial regions, with lower frequency of both in the athero-resistant descending aorta and a higher Sox9+:Notch3+ ratio in the brachiocephalic artery, which forms more calcified plaque relative to fibrous cap. Overall, these findings suggest that the distinct, prespecified SMC progenitor populations may play functional roles in governing plaque structure and ultimately disease risk.

Introduction: Coronary artery disease (CAD) is a leading cause of death worldwide. It is now generally accepted that residual risk for CAD resides in genetic variation that regulates cellular processes of smooth muscle cells (SMCs) in the vessel wall. Methods: We applied CRISPRi Perturb-seq using split-pool barcoding (SPLiT-seq) in immortalized human coronary artery SMCs, targeting 971 genome wide association study (GWAS) nominated genes with 3,163 guides across 954,000 cells. In vitro Perturb-seq transcriptomic effects were measured using single-cell RNA-seq. Explanted coronary arteries from cardiac transplant recipients was collected to generate a high quality scMultiome human coronary artery cell atlas (n=9). Together, this provided orthogonal references to map gene programs using consensus non-negative matrix factorization (cNMF) with robust cross-validation of gene programs. In vivo Perturb-seq via SMC-targeted AAV delivery is performed for mechanistic validation and characterization of in vivo effects. Results: cNMF decomposition performed on the Perturb-seq and human scMultiome atlas revealed 50 in vitro, and 40 in vivo gene programs, respectively. In total, 84% of our candidate genes demonstrated statistically robust knockdown. This allowed systematic integration of GWAS variants to gene linking followed by analysis of perturbation-associated differential gene signatures with SMC-specificity and CAD risk gene enrichment that converged across both cNMF references. Together, this enabled quantitative ranking of functional disease genes by their impact on transcriptional circuits. To extend upon this framework, SMC-targeted in vivo Perturb-seq demonstrated robust knockdown across 19 target genes using 123 guides, enabling rapid simultaneous characterization of cellular functions. Integration of these analyses highlighted convergence of CAD-associated perturbations on risk associated vascular regulatory programs such as TGFB signaling and nominated ZEB1 as a high-confidence mediator of CAD genetic risk. Transcriptomic analysis of in vivo effects revealed a shift in SMC cellular state marked by ectopic activation of epithelial markers and upregulation of immune signaling. Conclusion: We combine SPLiT-seq CRISPRi screening with multi-modal gene program references for robust hypothesis generation and in vivo Perturb-seq for rapid functional validation of CAD risk associated loci, identifying ZEB1 as a novel regulator of SMC identity and disease risk.

Cardiac allograft vasculopathy (CAV) is the leading cause of mortality after heart transplantation, yet no targeted therapies exist to prevent or reverse disease progression, and patients with CAV ultimately require a retransplant. CAV is characterized by progressive neointimal hyperplasia in donor coronary arteries, resulting in luminal occlusion and eventual allograft failure. Although immune and stromal cell interactions are thought to drive disease, the key cellular and molecular mechanisms remain poorly defined. Here we integrate single-cell RNA sequencing and spatial transcriptomics of human coronary arteries to characterize the CAV neointimal microenvironment. By comparing arteries with CAV with atherosclerotic coronary artery disease and non-diseased controls, we identify a distinct transcriptional signature of CAV. Our analysis reveals that modulated vascular smooth muscle cells and macrophage subsets dominate the neointima and interact to promote type 1 interferon (IFN)-mediated inflammation. Using a mouse model of CAV, we show that IFN signaling blockade with ruxolitinib significantly reduces CAV incidence and prolongs allograft survival. These findings define key cellular drivers of CAV and highlight IFN signaling as a potential therapeutic target.

Although genetic risk in coronary artery disease (CAD) is linked to changes in gene expression, recent discoveries have revealed a major role for A-to-I RNA editing in CAD. ADAR1 edits immunogenic double-stranded RNA (dsRNA), preventing activation of the dsRNA sensor MDA5 (IFIH1) and downstream interferon-stimulated gene signaling. Using human plaque analysis and human coronary artery smooth muscle cells (SMCs), here, we show that SMCs uniquely require RNA editing and that MDA5 activation regulates SMC phenotype. In a conditional SMC-specific Adar deletion mouse model on an atherosclerosis-prone background, combined with Ifih1 deletion and single-cell RNA sequencing, we demonstrate that ADAR1 preserves vascular integrity and limits atherosclerosis and calcification by suppressing MDA5 activation. Analysis of the Athero-Express carotid endarterectomy cohort further shows that interferon-stimulated gene expression correlates with SMC modulation, plaque instability and calcification. These findings reveal a fundamental mechanism of CAD, where cell type and context-specific RNA editing modulates genetic risk and vascular disease progression. Weldy et al. show that smooth muscle expression of the RNA editing enzyme ADAR1 regulates activation of the double-stranded RNA sensor MDA5 in a novel mechanism of atherosclerosis.

The fibrous cap of atherosclerotic plaques is essential for plaque stability. Rupture of the fibrous cap leads to heart attacks and strokes, and causes tens of millions of deaths globally every year. Identifying and understanding the cellular origins and plasticity of the fibrous cap is critical to developing therapeutic strategies to stabilize the atherosclerotic plaques. The fibrous cap is thought to arise oligoclonal from medial smooth muscle cells (SMCs), but whether all SMCs can give rise to the fibrous cap is unknown. Furthermore, conflicting data exist regarding whether plaque cells deeper in the lesion can give rise to the fibrous cap or vice vera. Murine SMC-lineage traced scRNAseq data revealed a transcriptomically distinct population of Notch3 and Elastin high population of cells that localizes to the fibrous cap. Utilizing a lineage tracing mouse model driven by endogenous Notch3, we demonstrated that fibrous cap cells arise from a predefined population of SMC that expresses Notch3 at baseline. After pulse-labeling the Notch3 CreERT2 ; ROSA lsl-tdTomato ; Apoe -/- mice with tamoxifen before high fat diet and then feeding them with high fat diet for 16 weeks, Notch3 -lineage traced cells stain positive for SMC markers ( Tagln , Cnn1 ) and are nearly exclusively found at the fibrous cap in multiple atherosclerotic prone beds. Furthermore, Notch3 -lineage traced SMCs and chondrogenic SMCs are mutually exclusive in the plaque, as demonstrated by the minimal overlap of tdTomato with chondrogenic SMC markers, including Col2a1 and Sox9. The Notch3 lineage labeled fibrous cap-SMCs display different inflammatory and extracellular matrix program from the non-labeled SMC progenies, as demonstrated by single cell transcriptomic sequencing. Consistently, Notch3 -lineage traced cells are committed to the fibrous cap fate and excluded from the calcified portions of the lesion and acellular core. Altogether, these lineage tracing studies highlight previously unrecognized medial SMC heterogeneity in healthy vessels. Unique Notch3 + populations of SMCs in normal media are fated to form the lesion cap. Once the Notch3 program is turned on, cells are locked into a fibrous cap fate and do not give rise to osteochondrogenic SMCs. Importantly, Notch3 CreERT2 mice can be used as a cap-specific genetic manipulation tool to further elucidate the role of fibrous cap specific genetic programs.

Vascular sites have distinct susceptibility to atherosclerosis and aneurysm, yet the biological underpinning of vascular site-specific disease risk is largely unknown. Vascular tissues have different developmental origins that may influence global chromatin accessibility, and understanding differential chromatin accessibility, gene expression profiles, and gene regulatory networks (GRN) on single cell resolution may give key insight into vascular site-specific disease risk. Here, we performed single cell chromatin accessibility (scATACseq) and gene expression profiling (scRNAseq) of healthy adult mouse vascular tissue from three vascular sites, 1) aortic root and ascending aorta, 2) brachiocephalic and carotid artery, and 3) descending thoracic aorta. Through a comprehensive analysis at single cell resolution, we discovered key regulatory enhancers to not only be cell type, but vascular site specific in vascular smooth muscle (SMC), fibroblasts, and endothelial cells. We identified epigenetic markers of embryonic origin with differential chromatin accessibility of key developmental transcription factors such as Tbx20 , Hand2 , Gata4 , and Hoxb family members and discovered transcription factor motif accessibility to be cell type and vascular site specific. Notably, we found ascending fibroblasts to have distinct epigenomic patterns, highlighting SMAD2/3 function to suggest a differential susceptibility to TGFβ, a finding we confirmed through in vitro culture of primary adventitial fibroblasts. Finally, to understand how vascular site-specific enhancers may regulate human genetic risk for disease, we integrated genome wide association study (GWAS) data for ascending and descending aortic dimension, and through using a distinct base resolution deep learning model to predict variant effect on chromatin accessibility, ChromBPNet, to predict variant effects in SMC, Fibroblasts, and Endothelial cells within ascending aorta, carotid, and descending aorta sites of origin. We reveal that although cell type remains a primary influence on variant effects, vascular site modifies cell type transcription and highlights genomic regions that are enriched for specific TF motif footprints — including MEF2A, SMAD3, and HAND2. This work supports a paradigm that the epigenomic and transcriptomic landscape of vascular cells are cell type and vascular site-specific and that site-specific enhancers govern complex genetic drivers of disease risk.

Vascular sites have distinct susceptibility to atherosclerosis and aneurysm, yet the epigenomic and transcriptomic underpinning of vascular site-specific disease risk is largely unknown. Here, we performed single-cell chromatin accessibility (scATACseq) and gene expression profiling (scRNAseq) of mouse vascular tissue from three vascular sites. Through interrogation of epigenomic enhancers and gene regulatory networks, we discovered key regulatory enhancers to not only be cell type, but vascular site-specific. We identified epigenetic markers of embryonic origin including developmental transcription factors such as Tbx20, Hand2, Gata4, and Hoxb family members and discovered transcription factor motif accessibility to be vascular site-specific for smooth muscle, fibroblasts, and endothelial cells. We further integrated genome-wide association data for aortic dimension, and using a deep learning model to predict variant effect on chromatin accessibility, ChromBPNet, we predicted variant effects across cell type and vascular site of origin, revealing genomic regions enriched for specific TF motif footprints-including MEF2A, SMAD3, and HAND2. This work supports a paradigm that cell type and vascular site-specific enhancers govern complex genetic drivers of disease risk.

To systematically identify causal genetic mechanisms that confer risk for coronary artery disease (CAD) in GWAS loci, we mapped genome-wide variant-to-enhancer-to-gene (V2E2G) links in vascular smooth muscle cells (SMC). Enhancers identified by active chromatin features, and further prioritized by base-resolution deep learning models of chromatin accessibility in 108 CAD loci, were studied with CRISPRi targeting and Direct-Capture Targeted Perturb-seq (DC-TAP-seq) evaluation of 470 genes. Seventy-six V2E2G links were identified for 59 candidate CAD genes representing gene programs including epithelial-mesenchymal transformation, ubiquitination, and protein folding as well as BMP and TGFB signaling. Similar methods employed with an independent focused screen targeting one candidate locus at 9p21.3 identified 10 enhancers regulating expression of multiple genes at this location. Detailed molecular studies revealed that two enhancers mediating transcription factor binding and transcriptional regulation contribute to ancestry-specific and sex-specific risk for CAD and the surrogate biomarker vascular calcification. Together, these studies advance our identification of GWAS CAD V2E2G links across the genome, and specific mechanisms of risk at the complex 9p21.3 locus.

Arterial segments show differing disease propensities, yet mechanisms remain unknown. We compiled a transcriptomic and spatial atlas of healthy human arterial cells across multiple segments to understand these differences. Arteries demonstrated a stereotyped pattern of cell-specific, segmental heterogeneity not captured by common marker genes. Arterial identities are encoded in fibroblast and smooth muscle cell (SMC) transcriptomes. Differentially expressed genes enrich for disease loci. Fibroblast gene expression enriches for a disproportionate number of disease loci, highlighting an underrecognized role for fibroblasts in disease risk. Cells of different segments cluster more by embryonic origin than anatomy. Global analysis of disease regulons in fibroblasts and SMCs identified developmental transcription factors that persist into adulthood, suggesting a functional role of these factors in disease. Lastly, the heterogeneity of non-coding transcriptomes rivals that of protein-coding transcriptomes. Differentially expressed lncRNAs enrich for genetic signals for vascular diseases, suggesting a role for lncRNAs in vascular disease.

The fibrous cap of atherosclerotic plaques is essential for the plaque stability. Rupture of the fibrous cap leads to heart attacks and strokes, and causes tens of millions of deaths globally every year. Identifying and understanding the cellular origins and plasticity of the fibrous cap is critical to developing therapeutic strategies to stabilize the atherosclerotic plaques. Utilizing the lineage tracing mouse model, we demonstrated that fibrous cap cells arise from a predefined population of cells that expresses Notch3 at baseline. After pulse-labeling the Notch3 CreERT2 ; ROSA lsltdTomato ; Apoe -/- mice with tamoxifen before high fat diet and then feeding them with high fat diet for 16 weeks, Notch3 lineage traced cells stain positive for SMC markers (TAGLN, CNN1) and are nearly exclusively found at the fibrous cap in multiple atherosclerotic prone beds. Furthermore, Notch3-lineage traced SMCs and chondrogenic SMCs are mutually exclusive in the plaque, as demonstrated by the minimal overlap of tdTomato with chondrogenic SMC markers, including Col2a1 and Sox9. Not all fibrous caps are marked, which could reflect poor Cre efficiency or new cap cells that form later. Increase tamoxifen treatment frequencing throughout high fat diet (every 10 days) significantly improved labeling efficiency and led to fully labeled lesion cap of the whole aortic root plaque. Consistently, Notch3 + cells are committed to fibrous cap formation and excluded from the calcified portions of the lesion and acellular core. Collectively, these lineage tracing studies highlight previously unrecognized medial SMC heterogeneity in healthy vessels. Unique Notch3 + populations of SMCs in normal media are fated to form the lesion cap. Once the Notch3 program is turned on, cells are locked into a fibrous cap fate and do not give rise to osteochondrogenic SMCs. Importantly, Notch3 CreERT2 mice can be used as a cap-specific genetic manipulation tool.

Background: Vascular smooth muscle cells (SMCs) contribute significantly to heritable coronary artery disease (CAD) risk and undergo phenotypic transitions in the intimal plaque during atherosclerosis. ZEB1 is a master regulator of epithelial-to-mesenchymal transition, that has been associated with CAD through human genetic studies. ZEB1 orchestrates cell state changes through modulation of TGFβ signaling and numerous epigenetic regulators. However, the mechanisms underlying the genetic association between ZEB1 and CAD remain unexplored. Methods: Single-cell RNA and ATAC sequencing on lineage-traced SMCs from the atherosclerotic aortic roots of SMC-specific Zeb1 knockout mice ( Zeb1 -KO; Myh11Cre ERT2 , ROSA tdT/+ , ApoE -/- ) were collected after 16 weeks on high-fat diet to characterize the changes in transcriptomic and epigenetic landscape. siRNA knockdown of ZEB1 in combination with TGFβ and IFNγ stimulations were performed in vitro on human coronary artery SMCs (HCASMCs) to validate in vivo findings. Results: Zeb1 -KO single cell RNA and ATAC sequencing showed the emergence of a novel epithelial-like SMC state characterized by robust ectopic activation of tricellular tight junction and cell polarity genes. Despite this increase in epithelial features, Zeb1 -KO also led to an increase in phenotypically transitioning SMCs contributing to the intimal plaque, accompanied by multi-fold enrichment in interferon-gamma (IFNγ) signaling targets. Motif analysis revealed near complete opening of chromatin accessibility at previously closed ZEB1 binding motifs, implicating ZEB1 mediated epigenetic repression. In vitro co-stimulation with TGFβ and IFNγ showed that si ZEB1 abolished the inhibitory effect of TGFβ on IFNγ signaling and demonstrated enriched for migratory and growth factor response functions by bulk RNAseq. Cell-cell signaling analysis by MultiNicheNet identified ectopic expression of migratory marker Lamc2 and concurrent increases in its interacting integrin partners in Zeb1 -KO SMCs. Furthermore, si ZEB1 HCASMCs also showed elevated expression of LAMC2 , its integrin interacting partners, as well as increased wound healing capacity by scratch assay. Conclusion: We identify ZEB1 as a critical epigenetic repressor required for the maintenance of the SMC cell state, suppressing epithelial marker expression and modifying cellular response to interferon signaling to ultimately augment SMC phenotypic transitions, linking genetic association between ZEB1 and CAD risk.

Introduction:Environmental exposure to dioxin has been linked to increased myocardial infarction. Smooth muscle cells (SMC) in the coronary vasculature play a critical role in atherosclerotic plaque remodeling due to their phenotypic plasticity, however, the detailed mechanism linking dioxin exposure to adverse SMC modulation is not well understood. Methods:Single-cell RNA and ATAC sequencing and histological analyses were performed on the aorta from mouse models of atherosclerosis exposed to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) or control. Primary human coronary artery SMC (HCASMC) treated in culture with TCDD were used to perform RNA-Seq, ATAC-Seq, and functional phenotypic assays. ChIP-Seq was performed with antibodies against Aryl-hydrocarbon receptor (AHR) and TCF21, two of known SMC modulating transcription factors. Results:Modulated SMC were the most transcriptionally responsive cell type to dioxin in the atherosclerotic aorta. Dioxin accelerated disease phenotype by promoting a modulated SMC phenotype early, resulting in increased lesion size, migration of SMC, and macrophage recruitment to the lesion. We found C3 expressing modulated SMCs to be likely contributing to the increased macrophage recruitment and inflammation. Analysis of the RNA-Seq data from HCASMC treated with TCDD showed differential enrichment of biological pathways related to cell migration, localization, and inflammation. Furthermore, ATAC-Seq data showed a significant activation for pathways regulating vascular development, cell migration, inflammation, and apoptosis. With TCDD treatment, there was also enrichment of AHR ChIP-Seq peaks, while the TCF21 enrichment decreased significantly. The SMC-specific Ahr knockout resulted in increased oxidative stress in SMC, increased lesion size and macrophage content, and loss of SMC lineage cells in the lesion cap when exposed to TCDD, consistent with a more vulnerable plaque phenotype. Conclusion:Dioxin adversely remodels atherosclerotic plaque by accelerating the SMC- phenotypic modulation, and increasing inflammation and oxidative stress resulting in increased macrophage recruitment and lesion size. Dioxin may adversely affect the SMC phenotype and disease state by affecting the TCF21 occupancy in the open chromatin regions. Furthermore, we observed that SMC-specific deletion of Ahr in mice resulted in worsening of dioxin mediated SMC modulation and atherosclerosis, suggesting that Ahr in SMC confers protection against dioxin by promoting a stable plaque phenotype and reducing dioxin induced oxidative stress. Summary:Exposure to dioxin, an environmental pollutant present in tobacco smoke and air pollution, accelerates smooth muscle cell modulation, and atherosclerosis.Dioxin exposure leads to inflammatory smooth muscle cell phenotype characterized by complement pathway activation and increased macrophage recruitment to plaqueAryl-hydrocarbon receptor in SMC protects against oxidative stress, and promotes a stable plaque phenotype.

The majority of variants identified by genome-wide association studies (GWAS) that influence coronary artery disease (CAD) risk reside in noncoding regions of the genome, making it challenging to link them with the genes they regulate. The 9p21.3 locus is the most impactful genetic risk locus for CAD. Due to the complexity of this locus, the causal genes and molecular mechanisms are poorly understood. Enhancers are cell type specific, and vascular smooth muscle cells (SMC) are known to have the highest heritable risk for CAD and play a major role in the atherosclerotic plaque formation. Here, we report efforts to systematically map SMC specific enhancers to neighboring genes within the 9p21.3 locus by implementing single cell CRISPRi enhancer screens and validating single causal variants in these enhancers. First, we intersected CAD GWAS loci with human coronary artery SMC (HCASMC) ATAC-seq and H3K27ac ChIP-seq datasets to focus on the disease relevant SNPs. This analysis identified 27 SNPs in 11 enhancers, that we targeted with CRISPRi machinery and analyzed at 5- and 10-days post transduction. As target genes in the locus are lowly expressed, we employed the targeted Perturb-seq (TAP-seq) approach for library generation and sequencing. We identified several enhancer-gene -pairs, including a strong enhancer-gene connection to both CDKN2A and CDKN2B . Additionally, we identified multiple enhancer regions that control MTAP expression, with smaller but significant effects. We followed up with individual validation of enhancer-gene pairs through qPCR. Furthermore, these results are consistent with chromosomal interaction data obtained from our previous HiChIP. Notably, enhancers 5 and 6 were strong regulators of CDNK2B and CDKN2A expression, so we investigated how variants in these enhancers might directly disrupt transcription factor (TF) binding. By using luciferase enhancer assays, CHIPseq and phenotypic in vitro assays we linked this variation with TFs that drive vascular calcification in SMCs. Our results identify new variant to gene links and suggest how the genetic risk in 9p21 is mediated in the vascular wall, providing mechanistic understanding of vascular calcification and genetic risk of CAD and suggesting a novel mechanism of how 9p21.3 mediates disease risk.

Despite decades of progress, coronary artery disease (CAD) remains the top cause of death worldwide. Additionally, trends in outcomes have worsened recently, highlighting the critical need for additional treatments. Human genetics has identified over 300 loci associated with CAD, but understanding the molecular mechanisms leading to disease remains a huge barrier to developing new therapies. These CAD-associated loci are enriched in smooth muscle cells (SMC) of the vascular wall, but no current therapies target these cells. Since insulin resistance is an important risk factor for CAD, understanding the molecular functions of genes associated with both insulin resistance and CAD will prioritize therapeutic candidates, especially if expressed in SMCs. One promising candidate is the gene PRDM16 (PR domain containing 16), which is highly expressed in vascular tissue. PRDM16 regulates cell fate decisions and insulin resistance in adipose tissue, but its role in atherosclerosis and SMC function is unknown. Recent advances in lineage tracing and conditional SMC knockout mouse models in conjunction with single cell technologies have demonstrated the cellular trajectories of SMC into several cellular states, including fibromyocytes (FMC) and chondromyocytes (CMC). Here, we employ this innovative approach to understand the role of Prdm16 on SMC phenotypic modulation in vivo and its role in the development of FMC and CMC. We also demonstrate the impact of Prdm16 in atherosclerosis and other important lesion characteristics relating to disease risk. We validate these effects in vitro and employ epigenetic analysis to identify the gene regulatory mechanisms whereby PRDM16 mediates its effects on SMC phenotypic modulation. Collectively these data demonstrate that PRDM16 is a causal factor that promotes risk of CAD.

We have recently identified rs2019090 and PDGFD as the functional variant and gene mediating CAD risk at the 11q22.3 locus, with our initial analysis using a global knockout (KO) model showing that this gene may promote phenotypic changes in smooth muscle cells (SMCs) in the plaque and contribute to neointimal vascular calcification. Nonetheless, the specific cell-type and phenotypic states through which PDGFD may confer disease risk remains unexplored.To delineate the impact of SMC-derived PDGFD signalling on cell state transitions and plaque progression in atherosclerosis, we have developed a novel SMC-specific lineage tracing and Pdgfd KO mouse ( Pdgfd ΔSMC/ΔSMC , Myh11 CreERT2 , ROSA tdT/+ , ApoE -/- ) allowing us to confidently define the impact of SMC-derived Pdgfd in the vascular SMC lineage as well as in neighbouring populations. Leveraging our lineage tracing KO mutants on a hypercholesterolemic diet, we have employed single cell RNA sequencing (scRNAseq) and histological analyses to characterize the cellular and molecular effect of Pdgfd in vascular disease. SMC-specific Pdgfd deletion resulted in alterations in the distribution of transitioning SMC populations, as well as previously unexplored gene expression differences within these cell types. This was accompanied by a significant reduction in atherosclerotic burden and plaque size across the aorta, including aortic root as well as descending abdominal aorta. Histological analysis revealed decreased monocyte recruitment, show by quantifying CD68+ cells within the plaque. To explore this further we interrogated the scRNAseq dataset to identify major pathways of cell-cell communication and the impact of altered Pdgfd signalling across different cell types. Overall, these data reveal that SMC-derived PDGFD substantially alters lesional SMC cell phenotype transitions, as well as inflammatory cell recruitment, implicating it as a major regulator of atherosclerotic disease progression.