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

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 in <i>ApoE</i>^<i>-/-</i> lesions give rise to smooth muscle cells in the fibrous cap.

<p><i>Tcf21</i>^{<i>iCre/+</i>}, <i>ApoE</i>^<i>-/-</i> mice were administered tamoxifen to activate expression of an inducible MerCreMer construct knocked into the <i>Tcf21</i> locus [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005155#pgen.1005155.ref031" target="_blank">31</a>]. Cre mediated recombination of a tomato (tdT) reporter at the constitutively expressed <i>ROSA26</i> locus allowed lineage tracing of <i>Tcf21</i> expressing cells. Animals received tamoxifen at 6–8 weeks of HFD and tissues were harvested at 12 weeks of diet (A-D), or received tamoxifen 4–6 weeks of age prior to HFD and tissues harvested at 16 weeks (E). A) Intramyocardial coronary artery showing <i>Tcf21</i> expression (tdT) in adventitial cells surrounding the Tagln positive medial SMC. B) In an early lesion without a defined fibrous cap, tdT positive cells were seen throughout the lesion and in the media (pink arrow), with Tagln staining (green arrows) being restricted to cells in the media. C) In the Tagln and tdT merged imaging (right panel), lesion Tagln positive cells are identified in the fibrous cap (green arrows) and some of these cells co-stain for tdT fluorescence (yellow arrows), indicating that they previously expressed <i>Tcf21</i>. Tagln expressing cells in the media (green arrows, Tagln only imaging) do not colocalize with tdT staining in this location. D) Tagln positive cells in the fibrous cap were also positive for tdT fluorescence, suggesting that the identified SMC had expressed <i>Tcf21</i>. Also, co-staining of tdT and Tagln is noted in the media (yellow color). E) Animals received tamoxifen at 4 weeks of age prior to HFD and tissues were harvested at 12 weeks of diet. In an early lesion without a defined fibrous cap, tdT positive cells are seen throughout the adventitia, in the media and in the plaque. In, intima; M, media; Ad, adventitia; Lu, lumen.</p

<i>Tcf21</i> lineage traced cells in <i>Ldlr</i>^<i>-/-</i> lesions give rise to smooth muscle cells in the fibrous cap.

<p><i>Tcf21</i>^{<i>iCre/+</i>}, <i>Ldlr</i>^<i>-/-</i> mice were administered tamoxifen to activate expression of an inducible MerCreMer construct knocked into the <i>Tcf21</i> locus to produce recombination and thus expression of a floxed STOP tandem dimer tomato (tdT) reporter gene. Animals received tamoxifen chow at 3–5 weeks of age and were subsequently fed HFD for 16–20 weeks before sacrifice. A) tdT positive cells are seen throughout the lesion and in the adventitia with Tagln staining cells identified in the media and fibrous cap. B) A number of tdT lineage traced cells in the fibrous cap express the SMC marker Acta2 as shown by colocalization of fluorescent markers (yellow arrows). tdT positive cells within the lesion did not express Tagln, as shown by red color (red arrow). C) Some cells in the adventitia stain for periostin expression and colocalize with tdT staining in this location (yellow arrows). Staining for periostin and colocalization with <i>Tcf21</i> lineage cells was also observed in the fibrous cap and subcap (yellow arrows). D) Extensive colocalization was noted for Pdgfra antibody staining and tdT fluorescence in the adventitia and fibrous cap. 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

Functional annotation derived from analysis of differentially expressed genes in si<i>TCF21</i> RNA-Seq experiments.

<p>Functional annotation derived from analysis of differentially expressed genes in si<i>TCF21</i> RNA-Seq experiments.</p

Differential <i>TCF21</i> gene expression in fibrous cap of stable vs. ruptured atherosclerotic plaque.

<p>A) Microdissection laser capture was employed to harvest tissue from endarterectomy samples from vessels with stable plaque or evidence of plaque rupture. An example of a stable plaque and a ruptured plaque with necrotic core (NC) and related luminal thrombus (T) are shown. These representative sections show the types of plaque regions that were harvested for RNA isolation. B) qPCR quantitation of <i>TCF21</i> mRNA levels in harvested tissue. TCF21 expression was significantly lower in fibrous cap tissue from the ruptured plaque.</p