Integrative single-cell analysis of cardiogenesis identifies developmental trajectories and non-coding mutations in congenital heart disease.

To define the multi-cellular epigenomic and transcriptional landscape of cardiac cellular development, we generated single-cell chromatin accessibility maps of human fetal heart tissues. We identified eight major differentiation trajectories involving primary cardiac cell types, each associated with dynamic transcription factor (TF) activity signatures. We contrasted regulatory landscapes of iPSC-derived cardiac cell types and their in vivo counterparts, which enabled optimization of in vitro differentiation of epicardial cells. Further, we interpreted sequence based deep learning models of cell-type-resolved chromatin accessibility profiles to decipher underlying TF motif lexicons. De novo mutations predicted to affect chromatin accessibility in arterial endothelium were enriched in congenital heart disease (CHD) cases vs. controls. In vitro studies in iPSCs validated the functional impact of identified variation on the predicted developmental cell types. This work thus defines the cell-type-resolved cis-regulatory sequence determinants of heart development and identifies disruption of cell type-specific regulatory elements in CHD.

Population-scale tissue transcriptomics maps long non-coding RNAs to complex disease.

Long non-coding RNA (lncRNA) genes have well-established and important impacts on molecular and cellular functions. However, among the thousands of lncRNA genes, it is still a major challenge to identify the subset with disease or trait relevance. To systematically characterize these lncRNA genes, we used Genotype Tissue Expression (GTEx) project v8 genetic and multi-tissue transcriptomic data to profile the expression, genetic regulation, cellular contexts, and trait associations of 14,100 lncRNA genes across 49 tissues for 101 distinct complex genetic traits. Using these approaches, we identified 1,432 lncRNA gene-trait associations, 800 of which were not explained by stronger effects of neighboring protein-coding genes. This included associations between lncRNA quantitative trait loci and inflammatory bowel disease, type 1 and type 2 diabetes, and coronary artery disease, as well as rare variant associations to body mass index.

Discovery of Transacting Long Noncoding RNAs That Regulate Smooth Muscle Cell Phenotype.

BACKGROUND: Smooth muscle cells (SMC), the major cell type in atherosclerotic plaques, are vital in coronary artery diseases (CADs). 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, ZIPPOR (ZEB-interacting suppressor) and TNS1-AS2 (TNS1-antisense 2), 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.

Comprehensive Integration of Multiple Single-Cell Transcriptomic Data Sets Defines Distinct Cell Populations and Their Phenotypic Changes in Murine Atherosclerosis.

BACKGROUND: The application of single-cell transcriptomic (single-cell RNA sequencing) analysis to the study of atherosclerosis has provided unique insights into the molecular and genetic mechanisms that mediate disease risk and pathophysiology. However, nonstandardized methodologies and relatively high costs associated with the technique have limited the size and replication of existing data sets and created disparate or contradictory findings that have fostered misunderstanding and controversy. METHODS: To address these uncertainties, we have performed a conservative integration of multiple published single-cell RNA sequencing data sets into a single meta-analysis, performed extended analysis of native resident vascular cells, and used in situ hybridization to map the disease anatomic location of the identified cluster cells. To investigate the transdifferentiation of smooth muscle cells to macrophage phenotype, we have developed a classifying algorithm based on the quantification of reporter transgene expression. RESULTS: The reporter gene expression tool indicates that within the experimental limits of the examined studies, transdifferentiation of smooth muscle cell to the macrophage lineage is extremely rare. Validated transition smooth muscle cell phenotypes were defined by clustering, and the location of these cells was mapped to lesion anatomy with in situ hybridization. We have also characterized 5 endothelial cell phenotypes and linked these cellular species to different vascular structures and functions. Finally, we have identified a transcriptomically unique cellular phenotype that constitutes the aortic valve. CONCLUSIONS: Taken together, these analyses resolve a number of outstanding issues related to differing results reported with vascular disease single-cell RNA sequencing studies, and significantly extend our understanding of the role of resident vascular cells in anatomy and disease.

Genome-Wide Genetic Associations Prioritize Evaluation of Causal Mechanisms of Atherosclerotic Disease Risk.

OBJECTIVE: The goal of this review is to discuss the implementation of genome-wide association studies to identify causal mechanisms of vascular disease risk. APPROACH AND RESULTS: The history of genome-wide association studies is described, the use of imputation and the creation of consortia to conduct meta-analyses with sufficient power to arrive at consistent associated loci for vascular disease. Genomic methods are described that allow the identification of causal variants and causal genes and how they impact the disease process. The power of single-cell analyses to promote genome-wide association studies of causal gene function is described. CONCLUSIONS: Genome-wide association studies represent a paradigm shift in the study of cardiovascular disease, providing identification of genes, cellular phenotypes, and disease pathways that empower the future of targeted drug development.

An integrated approach to identify environmental modulators of genetic risk factors for complex traits.

Complex traits and diseases can be influenced by both genetics and environment. However, given the large number of environmental stimuli and power challenges for gene-by-environment testing, it remains a critical challenge to identify and prioritize specific disease-relevant environmental exposures. We propose a framework for leveraging signals from transcriptional responses to environmental perturbations to identify disease-relevant perturbations that can modulate genetic risk for complex traits and inform the functions of genetic variants associated with complex traits. We perturbed human skeletal-muscle-, fat-, and liver-relevant cell lines with 21 perturbations affecting insulin resistance, glucose homeostasis, and metabolic regulation in humans and identified thousands of environmentally responsive genes. By combining these data with GWASs from 31 distinct polygenic traits, we show that the heritability of multiple traits is enriched in regions surrounding genes responsive to specific perturbations and, further, that environmentally responsive genes are enriched for associations with specific diseases and phenotypes from the GWAS Catalog. Overall, we demonstrate the advantages of large-scale characterization of transcriptional changes in diversely stimulated and pathologically relevant cells to identify disease-relevant perturbations.

Human Coronary Plaque T Cells Are Clonal and Cross-React to Virus and Self.

BACKGROUND: Coronary artery disease is an incurable, life-threatening disease that was once considered primarily a disorder of lipid deposition. Coronary artery disease is now also characterized by chronic inflammation' notable for the buildup of atherosclerotic plaques containing immune cells in various states of activation and differentiation. Understanding how these immune cells contribute to disease progression may lead to the development of novel therapeutic strategies. METHODS: We used single-cell technology and in vitro assays to interrogate the immune microenvironment of human coronary atherosclerotic plaque at different stages of maturity. RESULTS: In addition to macrophages, we found a high proportion of αß T cells in the coronary plaques. Most of these T cells lack high expression of CCR7 and L-selectin, indicating that they are primarily antigen-experienced memory cells. Notably, nearly one-third of these cells express the HLA-DRA surface marker, signifying activation through their TCRs (T-cell receptors). Consistent with this, TCR repertoire analysis confirmed the presence of activated αß T cells (CD4

A single-cell CRISPRi platform for characterizing candidate genes relevant to metabolic disorders in human adipocytes.

CROP-Seq combines gene silencing using CRISPR interference with single-cell RNA sequencing. Here, we applied CROP-Seq to study adipogenesis and adipocyte biology. Human preadipocyte SGBS cell line expressing KRAB-dCas9 was transduced with a sgRNA library. Following selection, individual cells were captured using microfluidics at different timepoints during adipogenesis. Bioinformatic analysis of transcriptomic data was used to determine the knockdown effects, the dysregulated pathways, and to predict cellular phenotypes. Single-cell transcriptomes recapitulated adipogenesis states. For all targets, over 400 differentially expressed genes were identified at least at one timepoint. As a validation of our approach, the knockdown of PPARG and CEBPB (which encode key proadipogenic transcription factors) resulted in the inhibition of adipogenesis. Gene set enrichment analysis generated hypotheses regarding the molecular function of novel genes. MAFF knockdown led to downregulation of transcriptional response to proinflammatory cytokine TNF-α in preadipocytes and to decreased CXCL-16 and IL-6 secretion. TIPARP knockdown resulted in increased expression of adipogenesis markers. In summary, this powerful, hypothesis-free tool can identify novel regulators of adipogenesis, preadipocyte, and adipocyte function associated with metabolic disease.NEW & NOTEWORTHY Genomics efforts led to the identification of many genomic loci that are associated with metabolic traits, many of which are tied to adipose tissue function. However, determination of the causal genes, and their mechanism of action in metabolism, is a time-consuming process. Here, we use an approach to determine the transcriptional outcome of candidate gene knockdown for multiple genes at the same time in a human cell model of adipogenesis.

ZEB2 Shapes the Epigenetic Landscape of Atherosclerosis.

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 ß 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.

Generation of Vascular Smooth Muscle Cells From Induced Pluripotent Stem Cells: Methods, Applications, and Considerations.

The developmental origin of vascular smooth muscle cells (VSMCs) has been increasingly recognized as a major determinant for regional susceptibility or resistance to vascular diseases. As a human material-based complement to animal models and human primary cultures, patient induced pluripotent stem cell iPSC-derived VSMCs have been leveraged to conduct basic research and develop therapeutic applications in vascular diseases. However, iPSC-VSMCs (induced pluripotent stem cell VSMCs) derived by most existing induction protocols are heterogeneous in developmental origins. In this review, we summarize signaling networks that govern in vivo cell fate decisions and in vitro derivation of distinct VSMC progenitors, as well as key regulators that terminally specify lineage-specific VSMCs. We then highlight the significance of leveraging patient-derived iPSC-VSMCs for vascular disease modeling, drug discovery, and vascular tissue engineering and discuss several obstacles that need to be circumvented to fully unleash the potential of induced pluripotent stem cells for precision vascular medicine.

Embryologic Origin Influences Smooth Muscle Cell Phenotypic Modulation Signatures in Murine Marfan Syndrome Aortic Aneurysm.

BACKGROUND: Aortic root smooth muscle cells (SMC) develop from both the second heart field (SHF) and neural crest. Disparate responses to disease-causing Fbn1 variants by these lineages are proposed to promote focal aortic root aneurysm formation in Marfan syndrome (MFS), but lineage-stratified SMC analysis in vivo is lacking. METHODS: We generated SHF lineage-traced MFS mice and performed integrated multiomic (single-cell RNA and assay for transposase-accessible chromatin sequencing) analysis stratified by embryological origin. SMC subtypes were spatially identified via RNA in situ hybridization. Response to TWIST1 overexpression was determined via lentiviral transduction in human aortic SMCs. RESULTS: Lineage stratification enabled nuanced characterization of aortic root cells. We identified heightened SHF-derived SMC heterogeneity including a subset of Tnnt2 (cardiac troponin T)-expressing cells distinguished by altered proteoglycan expression. MFS aneurysm-associated SMC phenotypic modulation was identified in both SHF-traced and nontraced (neural crest-derived) SMCs; however, transcriptomic responses were distinct between lineages. SHF-derived modulated SMCs overexpressed collagen synthetic genes and small leucine-rich proteoglycans while nontraced SMCs activated chondrogenic genes. These modulated SMCs clustered focally in the aneurysmal aortic root at the region of SHF/neural crest lineage overlap. Integrated RNA-assay for transposase-accessible chromatin analysis identified enriched Twist1 and Smad2/3/4 complex binding motifs in SHF-derived modulated SMCs. TWIST1 overexpression promoted collagen and SLRP gene expression in vitro, suggesting TWIST1 may drive SHF-enriched collagen synthesis in MFS aneurysm. CONCLUSIONS: SMCs derived from both SHF and neural crest lineages undergo phenotypic modulation in MFS aneurysm but are defined by subtly distinct transcriptional responses. Enhanced TWIST1 transcription factor activity may contribute to enriched collagen synthetic pathways SHF-derived SMCs in MFS.

Genomic profiling of human vascular cells identifies TWIST1 as a causal gene for common vascular diseases.

Genome-wide association studies have identified multiple novel genomic loci associated with vascular diseases. Many of these loci are common non-coding variants that affect the expression of disease-relevant genes within coronary vascular cells. To identify such genes on a genome-wide level, we performed deep transcriptomic analysis of genotyped primary human coronary artery smooth muscle cells (HCASMCs) and coronary endothelial cells (HCAECs) from the same subjects, including splicing Quantitative Trait Loci (sQTL), allele-specific expression (ASE), and colocalization analyses. We identified sQTLs for TARS2, YAP1, CFDP1, and STAT6 in HCASMCs and HCAECs, and 233 ASE genes, a subset of which are also GTEx eGenes in arterial tissues. Colocalization of GWAS association signals for coronary artery disease (CAD), migraine, stroke and abdominal aortic aneurysm with GTEx eGenes in aorta, coronary artery and tibial artery discovered novel candidate risk genes for these diseases. At the CAD and stroke locus tagged by rs2107595 we demonstrate colocalization with expression of the proximal gene TWIST1. We show that disrupting the rs2107595 locus alters TWIST1 expression and that the risk allele has increased binding of the NOTCH signaling protein RBPJ. Finally, we provide data that TWIST1 expression influences vascular SMC phenotypes, including proliferation and calcification, as a potential mechanism supporting a role for TWIST1 in CAD.

Single-Cell Transcriptomic Census of Endothelial Changes Induced by Matrix Stiffness and the Association with Atherosclerosis.

Vascular endothelial cell (EC) plasticity plays a critical role in the progression of atherosclerosis by giving rise to mesenchymal phenotypes in the plaque lesion. Despite the evidence for arterial stiffening as a major contributor to atherosclerosis, the complex interplay among atherogenic stimuli in vivo has hindered attempts to determine the effects of extracellular matrix (ECM) stiffness on endothelial-mesenchymal transition (EndMT). To study the regulatory effects of ECM stiffness on EndMT, an in vitro model is developed in which human coronary artery ECs are cultured on physiological or pathological stiffness substrates. Leveraging single-cell RNA sequencing, cell clusters with mesenchymal transcriptional features are identified to be more prevalent on pathological substrates than physiological substrates. Trajectory inference analyses reveal a novel mesenchymal-to-endothelial reverse transition, which is blocked by pathological stiffness substrates, in addition to the expected EndMT trajectory. ECs pushed to a mesenchymal character by pathological stiffness substrates are enriched in transcriptional signatures of atherosclerotic ECs from human and murine plaques. This study characterizes at single-cell resolution the transcriptional programs that underpin EC plasticity in both physiological or pathological milieus, and thus serves as a valuable resource for more precisely defining EndMT and the transcriptional programs contributing to atherosclerosis.

Coronary Disease-Associated Gene TCF21 Inhibits Smooth Muscle Cell Differentiation by Blocking the Myocardin-Serum Response Factor Pathway.

RATIONALE: The gene encoding TCF21 (transcription factor 21) has been linked to coronary artery disease risk by human genome-wide association studies in multiple racial ethnic groups. In murine models, Tcf21 is required for phenotypic modulation of smooth muscle cells (SMCs) in atherosclerotic tissues and promotes a fibroblast phenotype in these cells. In humans, TCF21 expression inhibits risk for coronary artery disease. The molecular mechanism by which TCF21 regulates SMC phenotype is not known. OBJECTIVE: To better understand how TCF21 affects the SMC phenotype, we sought to investigate the possible mechanisms by which it regulates the lineage determining MYOCD (myocardin)-SRF (serum response factor) pathway. METHODS AND RESULTS: Modulation of TCF21 expression in human coronary artery SMC revealed that TCF21 suppresses a broad range of SMC markers, as well as key SMC transcription factors MYOCD and SRF, at the RNA and protein level. We conducted chromatin immunoprecipitation-sequencing to map SRF-binding sites in human coronary artery SMC, showing that binding is colocalized in the genome with TCF21, including at a novel enhancer in the SRF gene, and at the MYOCD gene promoter. In vitro genome editing indicated that the SRF enhancer CArG box regulates transcription of the SRF gene, and mutation of this conserved motif in the orthologous mouse SRF enhancer revealed decreased SRF expression in aorta and heart tissues. Direct TCF21 binding and transcriptional inhibition at colocalized sites were established by reporter gene transfection assays. Chromatin immunoprecipitation and protein coimmunoprecipitation studies provided evidence that TCF21 blocks MYOCD and SRF association by direct TCF21-MYOCD interaction. CONCLUSIONS: These data indicate that TCF21 antagonizes the MYOCD-SRF pathway through multiple mechanisms, further establishing a role for this coronary artery disease-associated gene in fundamental SMC processes and indicating the importance of smooth muscle response to vascular stress and phenotypic modulation of this cell type in coronary artery disease risk.

PCSK6 Is a Key Protease in the Control of Smooth Muscle Cell Function in Vascular Remodeling.

RATIONALE: PCSKs (Proprotein convertase subtilisins/kexins) are a protease family with unknown functions in vasculature. Previously, we demonstrated PCSK6 upregulation in human atherosclerotic plaques associated with smooth muscle cells (SMCs), inflammation, extracellular matrix remodeling, and mitogens. OBJECTIVE: Here, we applied a systems biology approach to gain deeper insights into the PCSK6 role in normal and diseased vessel wall. METHODS AND RESULTS: Genetic analyses revealed association of intronic PCSK6 variant rs1531817 with maximum internal carotid intima-media thickness progression in high-cardiovascular risk subjects. This variant was linked with PCSK6 mRNA expression in healthy aortas and plaques but also with overall plaque SMA+ cell content and pericyte fraction. Increased PCSK6 expression was found in several independent human cohorts comparing atherosclerotic lesions versus healthy arteries, using transcriptomic and proteomic datasets. By immunohistochemistry, PCSK6 was localized to fibrous cap SMA+ cells and neovessels in plaques. In human, rat, and mouse intimal hyperplasia, PCSK6 was expressed by proliferating SMA+ cells and upregulated after 5 days in rat carotid balloon injury model, with positive correlation to PDGFB (platelet-derived growth factor subunit B) and MMP (matrix metalloprotease) 2/MMP14. Here, PCSK6 was shown to colocalize and cointeract with MMP2/MMP14 by in situ proximity ligation assay. Microarrays of carotid arteries from Pcsk6-/- versus control mice revealed suppression of contractile SMC markers, extracellular matrix remodeling enzymes, and cytokines/receptors. Pcsk6-/- mice showed reduced intimal hyperplasia response upon carotid ligation in vivo, accompanied by decreased MMP14 activation and impaired SMC outgrowth from aortic rings ex vivo. PCSK6 silencing in human SMCs in vitro leads to downregulation of contractile markers and increase in MMP2 expression. Conversely, PCSK6 overexpression increased PDGFBB (platelet-derived growth factor BB)-induced cell proliferation and particularly migration. CONCLUSIONS: PCSK6 is a novel protease that induces SMC migration in response to PDGFB, mechanistically via modulation of contractile markers and MMP14 activation. This study establishes PCSK6 as a key regulator of SMC function in vascular remodeling. Visual Overview: An online visual overview is available for this article.

CD47-blocking antibodies restore phagocytosis and prevent atherosclerosis.

Atherosclerosis is the disease process that underlies heart attack and stroke. Advanced lesions at risk of rupture are characterized by the pathological accumulation of diseased vascular cells and apoptotic cellular debris. Why these cells are not cleared remains unknown. Here we show that atherogenesis is associated with upregulation of CD47, a key anti-phagocytic molecule that is known to render malignant cells resistant to programmed cell removal, or 'efferocytosis'. We find that administration of CD47-blocking antibodies reverses this defect in efferocytosis, normalizes the clearance of diseased vascular tissue, and ameliorates atherosclerosis in multiple mouse models. Mechanistic studies implicate the pro-atherosclerotic factor TNF-α as a fundamental driver of impaired programmed cell removal, explaining why this process is compromised in vascular disease. Similar to recent observations in cancer, impaired efferocytosis appears to play a pathogenic role in cardiovascular disease, but is not a fixed defect and may represent a novel therapeutic target.

Multi-omics analysis identifies CpGs near G6PC2 mediating the effects of genetic variants on fasting glucose.

AIMS/HYPOTHESIS: An elevated fasting glucose level in non-diabetic individuals is a key predictor of type 2 diabetes. Genome-wide association studies (GWAS) have identified hundreds of SNPs for fasting glucose but most of their functional roles in influencing the trait are unclear. This study aimed to identify the mediation effects of DNA methylation between SNPs identified as significant from GWAS and fasting glucose using Mendelian randomisation (MR) analyses. METHODS: We first performed GWAS analyses for three cohorts (Taiwan Biobank with 18,122 individuals, the Healthy Aging Longitudinal Study in Taiwan with 1989 individuals and the Stanford Asia-Pacific Program for Hypertension and Insulin Resistance with 416 individuals) with individuals of Han Chinese ancestry in Taiwan, followed by a meta-analysis for combining the three GWAS analysis results to identify significant and independent SNPs for fasting glucose. We determined whether these SNPs were methylation quantitative trait loci (meQTLs) by testing their associations with DNA methylation levels at nearby CpG sites using a subsample of 1775 individuals from the Taiwan Biobank. The MR analysis was performed to identify DNA methylation with causal effects on fasting glucose using meQTLs as instrumental variables based on the 1775 individuals. We also used a two-sample MR strategy to perform replication analysis for CpG sites with significant MR effects based on literature data. RESULTS: Our meta-analysis identified 18 significant (p < 5 × 10-8) and independent SNPs for fasting glucose. Interestingly, all 18 SNPs were meQTLs. The MR analysis identified seven CpGs near the G6PC2 gene that mediated the effects of a significant SNP (rs2232326) in the gene on fasting glucose. The MR effects for two CpGs were replicated using summary data based on the European population, using an exonic SNP rs2232328 in G6PC2 as the instrument. CONCLUSIONS/INTERPRETATION: Our analysis results suggest that rs2232326 and rs2232328 in G6PC2 may affect DNA methylation at CpGs near the gene and that the methylation may have downstream effects on fasting glucose. Therefore, SNPs in G6PC2 and CpGs near G6PC2 may reside along the pathway that influences fasting glucose levels. This is the first study to report CpGs near G6PC2, an important gene for regulating insulin secretion, mediating the effects of GWAS-significant SNPs on fasting glucose.

Environment-Sensing Aryl Hydrocarbon Receptor Inhibits the Chondrogenic Fate of Modulated Smooth Muscle Cells in Atherosclerotic Lesions.

BACKGROUND: Smooth 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 risk. We hypothesized that AHR can affect atherosclerosis by regulating phenotypic modulation of SMC. METHODS: We combined RNA-sequencing, chromatin immunoprecipitation followed by sequencing, assay for transposase-accessible chromatin using sequencing, and in vitro assays in human coronary artery SMCs, with single-cell RNA-sequencing, 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. RESULTS: Genomic studies coupled with functional assays in cultured human coronary artery SMCs revealed that AHR modulates the human coronary artery SMC 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 SMCs in the atherosclerotic lesion cap. Furthermore, single-cell RNA-sequencing studies of the SMC-specific Ahr knockout mice showed a significant increase in the proportion of modulated SMCs expressing chondrocyte markers such as Col2a1 and Alpl, which localized to the lesion neointima. These cells, which we term "chondromyocytes," 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 SMCs in the lesion cap, and increased alkaline phosphatase activity in lesions in the Ahr knockout in comparison with wild-type mice. We propose that AHR is likely protective based on these data and inference from human genetic analyses. CONCLUSIONS: Overall, we conclude that AHR promotes the maintenance of lesion cap integrity and diminishes the disease-related SMC-to-chondromyocyte transition in atherosclerotic tissues.

Genetic Regulatory Mechanisms of Smooth Muscle Cells Map to Coronary Artery Disease Risk Loci.

Coronary artery disease (CAD) is the leading cause of death globally. Genome-wide association studies (GWASs) have identified more than 95 independent loci that influence CAD risk, most of which reside in non-coding regions of the genome. To interpret these loci, we generated transcriptome and whole-genome datasets using human coronary artery smooth muscle cells (HCASMCs) from 52 unrelated donors, as well as epigenomic datasets using ATAC-seq on a subset of 8 donors. Through systematic comparison with publicly available datasets from GTEx and ENCODE projects, we identified transcriptomic, epigenetic, and genetic regulatory mechanisms specific to HCASMCs. We assessed the relevance of HCASMCs to CAD risk using transcriptomic and epigenomic level analyses. By jointly modeling eQTL and GWAS datasets, we identified five genes (SIPA1, TCF21, SMAD3, FES, and PDGFRA) that may modulate CAD risk through HCASMCs, all of which have relevant functional roles in vascular remodeling. Comparison with GTEx data suggests that SIPA1 and PDGFRA influence CAD risk predominantly through HCASMCs, while other annotated genes may have multiple cell and tissue targets. Together, these results provide tissue-specific and mechanistic insights into the regulation of a critical vascular cell type associated with CAD in human populations.

Predictive network modeling in human induced pluripotent stem cells identifies key driver genes for insulin responsiveness.

Insulin resistance (IR) precedes the development of type 2 diabetes (T2D) and increases cardiovascular disease risk. Although genome wide association studies (GWAS) have uncovered new loci associated with T2D, their contribution to explain the mechanisms leading to decreased insulin sensitivity has been very limited. Thus, new approaches are necessary to explore the genetic architecture of insulin resistance. To that end, we generated an iPSC library across the spectrum of insulin sensitivity in humans. RNA-seq based analysis of 310 induced pluripotent stem cell (iPSC) clones derived from 100 individuals allowed us to identify differentially expressed genes between insulin resistant and sensitive iPSC lines. Analysis of the co-expression architecture uncovered several insulin sensitivity-relevant gene sub-networks, and predictive network modeling identified a set of key driver genes that regulate these co-expression modules. Functional validation in human adipocytes and skeletal muscle cells (SKMCs) confirmed the relevance of the key driver candidate genes for insulin responsiveness.