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

Loss of Cardioprotective Effects at the ADAMTS7 Locus as a Result of Gene-Smoking Interactions

BACKGROUND: Common diseases such as coronary heart disease (CHD) are complex in etiology. The interaction of genetic susceptibility with lifestyle factors may play a prominent role. However, gene-lifestyle interactions for CHD have been difficult to identify. Here, we investigate interaction of smoking behavior, a potent lifestyle factor, with genotypes that have been shown to associate with CHD risk. METHODS: We analyzed data on 60 919 CHD cases and 80 243 controls from 29 studies for gene-smoking interactions for genetic variants at 45 loci previously reported to be associated with CHD risk. We also studied 5 loci associated with smoking behavior. Study-specific gene-smoking interaction effects were calculated and pooled using fixed-effects meta-analyses. Interaction analyses were declared to be significant at a P value of <1.0x10(-3) (Bonferroni correction for 50 tests). RESULTS: We identified novel gene-smoking interaction for a variant upstream of the ADAMTS7 gene. Every T allele of rs7178051 was associated with lower CHD risk by 12% in never-smokers (P= 1.3x10(-16)) in comparison with 5% in ever-smokers (P= 2.5x10(-4)), translating to a 60% loss of CHD protection conferred by this allelic variation in people who smoked tobacco (interaction P value= 8.7x10(-5)). The protective T allele at rs7178051 was also associated with reduced ADAMTS7 expression in human aortic endothelial cells and lymphoblastoid cell lines. Exposure of human coronary artery smooth muscle cells to cigarette smoke extract led to induction of ADAMTS7. CONCLUSIONS: Allelic variation at rs7178051 that associates with reduced ADAMTS7 expression confers stronger CHD protection in never-smokers than in ever-smokers. Increased vascular ADAMTS7 expression may contribute to the loss of CHD protection in smokers.Peer reviewe

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

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

Coronary Artery Disease Associated Transcription Factor TCF21 Regulates Smooth Muscle Precursor Cells That Contribute to the Fibrous Cap

<div><p>Recent genome wide association studies have identified a number of genes that contribute to the risk for coronary heart disease. One such gene, <i>TCF21</i>, encodes a basic-helix-loop-helix transcription factor believed to serve a critical role in the development of epicardial progenitor cells that give rise to coronary artery smooth muscle cells (SMC) and cardiac fibroblasts. Using reporter gene and immunolocalization studies with mouse and human tissues we have found that vascular <i>TCF21</i> expression in the adult is restricted primarily to adventitial cells associated with coronary arteries and also medial SMC in the proximal aorta of mouse. Genome wide RNA-Seq studies in human coronary artery SMC (HCASMC) with siRNA knockdown found a number of putative <i>TCF21</i> downstream pathways identified by enrichment of terms related to CAD, including “vascular disease,” “disorder of artery,” and “occlusion of artery,” as well as disease-related cellular functions including “cellular movement” and “cellular growth and proliferation.” In vitro studies in HCASMC demonstrated that <i>TCF21</i> expression promotes proliferation and migration and inhibits SMC lineage marker expression. Detailed <i>in situ</i> expression studies with reporter gene and lineage tracing revealed that vascular wall cells expressing <i>Tcf21</i> before disease initiation migrate into vascular lesions of <i>ApoE^-/-</i> and <i>Ldlr^-/-</i> mice. While <i>Tcf21</i> lineage traced cells are distributed throughout the early lesions, in mature lesions they contribute to the formation of a subcapsular layer of cells, and others become associated with the fibrous cap. The lineage traced fibrous cap cells activate expression of SMC markers and growth factor receptor genes. Taken together, these data suggest that <i>TCF21</i> may have a role regulating the differentiation state of SMC precursor cells that migrate into vascular lesions and contribute to the fibrous cap and more broadly, in view of the association of this gene with human CAD, provide evidence that these processes may be a mechanism for CAD risk attributable to the vascular wall.</p></div

<i>Tcf21</i> expressing cells associate with the fibrous cap.

<p>Xgal stained lesions in <i>Tcf21</i>^{<i>lacZ/+</i>}, <i>ApoE</i>^<i>-/-</i> mice fed HFD for 20 weeks were evaluated by immunohistochemistry for expression of fibrous cap markers. <i>Tcf21</i> reporter expressing cells (blue indicator) were identified in the media and adventitia (black arrows, middle panels), and in association with the fibrous cap (blue arrows, right panels). β-galactosidase negative cells in the luminal aspect of the fibrous cap stained positive for Tagln, as well as growth factor receptors Tgfbr2 and Pdgfrb (red indicator). In addition, in the region of the fibrous cap, cells were identified that showed staining for <i>Tcf21</i> expression as well as Tagln and growth factor receptors (purple arrows).</p

Rate of cell division in vascular lesions is related to Tcf21 expression.

<p>A) EdU staining was performed in <i>Tcf21</i>^{<i>iCre/+</i>}, <i>ROSA</i>^<i>tdT/+</i>, <i>Ldlr</i>^<i>-/-</i> mice treated with tamoxifen and EdU and placed on HFD for 9, 12, or 15 weeks prior to tissue collection. Co-localization of <i>Tcf21</i> tdT fluorescence (red) and EdU fluorescent staining (green) allowed identification of the sdividing cells that were expressing <i>Tcf21</i> (yellow arrows, image from 12 week timepoint). B) The ratio of <i>Tcf21</i>+EdU+ cells compared to total <i>Tcf21</i>+ cells was significantly increased at 12 weeks of diet, consistent with a proliferative response in <i>Tcf21</i>+ cells. Comparison of percentage differences between 0 and 9 weeks, and from 9 to 12 weeks approached significance, <i>P</i> = 0.06; there was not a statistical difference between 12 and 15 weeks, <i>P</i> = 0.11. C, D) Rates of cell division in vascular lesions were compared between <i>Tcf21</i>^{<i>lacZ/+</i>}, <i>ApoE</i>^<i>-/-</i> animals and <i>ApoE</i>^<i>-/-</i> animals on HFD for 20 wks by quantifying the relative number of dividing cells identified by EdU fluorescence compared to the total number of cells identified by DAPI fluorescence. EdU fluorescence (green) was merged with red pseudocolored DAPI fluorescence, yellow arrows indicate yellow nuclei that are positive for both EdU and DAPI fluorescence. E) A statistically significant decrease in this percentage in <i>Tcf21</i>^{<i>lacZ/+</i>}, <i>ApoE</i>^<i>-/-</i> animals compared to <i>ApoE</i>^<i>-/-</i> animals suggested a correlation between <i>Tcf21</i> expression and rate of cell division in the vascular lesions. In, intima; M, media; Ad, adventitia; FC, fibrous cap, Lu, lumen.</p

<i>Tcf21</i> expressing cells are found in atherosclerotic lesions.

<p><i>Tcf21</i>^{<i>lacZ/+</i>}, <i>ApoE</i>^<i>-/-</i> mice were fed HFD from 4 weeks of age for 4, 8, 12, or 20 weeks, proximal aortic tissues were harvested, and <i>Tcf21</i> gene expression evaluated by Xgal staining to visualize β-galactosidase activity, and sections were counterstained with nuclear fast red to visualize disease lesion architecture. For each timepoint, boxes in low-power images at left indicate regions examined at high power in panels to the right. At 4 weeks of HFD there was no expression in the lesions although clusters of <i>lacZ</i> expressing cells identified in the media in regions below the disease lesions (arrows). At the 8-week timepoint cells with β-galactosidase activity were seen extending from the media to the luminal surface of the lesion (black arrows) in the vicinity of the forming fibrous cap (purple arrows). By 12 weeks of HFD there was extensive labeling of cells in lesions, with the appearance of β-galactosidase positive cells in the vicinity of the fibrous cap (purple arrows). Also, there was extensive staining of cells in areas of disrupted medial structure (white arrows). After 20 weeks of HFD, <i>Tcf21</i>-expressing cells had decreased in the lesions but formed a narrow band of cells associated with the fibrous cap (purple arrows).</p

<i>TCF21</i> regulates basic cellular functions in vascular smooth muscle cells <i>in vitro</i>.

<p>A) HCASMC transduced with <i>TCF21</i> overexpressing lentivirus (pWPI-<i>TCF21</i>) or empty lentivirus (pWPI empty) were labeled with the thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU), which was visualized with a fluorescent azide, allowing identification and quantification of proliferating cells with immunofluorescence microscopy, reported here as a percent with baseline being all DAPI positive cells. HCASMC showed an increase in the percentage of <i>TCF21</i> overexpressing cells compared to DAPI stained cells (33.1% ± 2.3 control vs. 51% ± 4.1 overexpressing cells, <i>P</i><0.001). B) HCASMC transduced with knockdown lentiviruses showed a decreased percentage of dividing cells (43.4% ± 4.4 control vs. 30.8% ± 2.4 knockdown cells, <i>P</i><0.01). C) s<i>iTCF21</i> produced a decrease in apoptosis in HCASMC as measured by a caspase activity assay (41,824±1872 vs. 18,837±1302, <i>P</i><0.0001). D) <i>TCF21</i> regulation of HCASMC migration was evaluated with a gap closure assay. <i>TCF21</i> overexpressing cells transduced with pWPI-<i>TCF21</i> lentivirus covered a significantly larger surface area after 12 hours of study compared to cells transduced with the empty pWPI vector (27.8 ± 2.7 vs. 15.8 ± 1.16, <i>P</i><0.001). E) HCASMC treated with si<i>TCF21</i> compared to siCTRL showed significantly increased expression of <i>ACTA2</i>, <i>TAGLN</i>, and <i>MYH11</i> SMC marker genes (<i>P</i><0.05/<i>P</i><0.01/<i>P</i><0.05 respectively). F) The <i>ACTA2</i> locus as visualized on the University of California Santa Cruz genome browser. Data provided here reveals evidence for a likely enhancer region in the first intron, as indicated by DNase hypersensitivity measured in human aortic SMC, and histone modification data showing enrichment of H3K27Ac and H3K4me1 at this same site, as well as clustering of a number of transcription factor binding sites. G) Chromatin immunoprecipitation for <i>TCF21</i> binding to the enhancer region of the <i>ACTA2</i> locus by ChIP-qPCR (<i>P</i><0.05). H) Dual luciferase assays in rat aortic smooth muscle cells with a reporter construct containing the human <i>ACTA2</i> promoter and first intron (SMA-luc). A <i>TCF21</i> expression construct was transfected with human <i>TCF3 (E12)</i>, <i>TCF3 (E47)</i>, <i>TCF12</i>, and <i>Twist1</i> murine expression vectors, showing specific suppression of transcription of the SMA-luc reporter (1.0 ± 0.01 vs. 0.1 ± 0.009, <i>P</i><0.01 for <i>TCF21</i> alone). I) Similar dual luciferase assays using a 3 E-box containing minimal promoter construct (E-luc) based on the nucleotide sequence of the first intron (n = 3, 3 replicates), again showing <i>TCF21</i> mediated suppression of transcription (1.0 ± 0.29 vs. 0.21 ± 0.02, <i>P</i><0.01 for <i>TCF21</i> alone).</p