Background Genome-wide association studies (GWAS) have established ADAMTS7 as a locus for coronary artery disease (CAD) in humans. Yet, these studies fail to provide directionality for the association between ADAMTS7 and CAD. Previous reports have implicated ADAMTS7 in the regulation of vascular smooth muscle cell (VSMC) migration, but a role and direction of impact for this gene in atherogenesis has not been shown in relevant model systems. Methods and Results We bred an Adamts7 whole body knockout (KO) mouse onto both the Ldlr and Apoe KO hyperlipidemic mouse models. Adamts7−/−/Ldlr−/− and Adamts7−/−/Apoe−/− mice displayed significant reductions in lesion formation in aortas and aortic roots as compared to controls. Adamts7 KO mice also showed reduced neointimal formation after femoral wire injury. Adamts7 expression was induced in response to injury and hyperlipidemia but was absent at later timepoints, and primary Adamts7 KO VSMCs showed reduced migration in the setting of TNFα stimulation. ADAMTS7 localized to cells positive for SMC markers in human CAD lesions, and sub-cellular localization studies in cultured VSMCs placed ADAMTS7 at the cytoplasm and cell membrane, where it co-localized with markers of podosomes. Conclusions These data represent the first in vivo experimental validation of the association of Adamts7 with atherogenesis, likely through modulation of vascular cell migration and matrix in atherosclerotic lesions. These results demonstrate that Adamts7 is proatherogenic, lending directionality to the original genetic association and supporting the concept that pharmacological inhibition of ADAMTS7 should be atheroprotective in humans, making it an attractive target for novel therapeutic interventions.

Rationale Hemodynamic disturbed flow is associated with susceptibility to atherosclerosis. Endothelial KLF4 is an important anti-inflammatory atheroprotective transcription factor that is suppressed in regions of disturbed flow. Objective The plasticity of epigenomic KLF4 transcriptional regulation by flow-mediated DNA methylation was investigated in vitro and in arterial tissue. Methods and Results To recapitulate dominant flow characteristics of atheroprotected and atherosusceptible arteries, human aortic endothelial cells (HAEC) were subjected to pulsatile undisturbed flow (UF) or oscillatory disturbed flow (DF) containing a flow-reversing phase. Differential CpG site methylation was measured by methylation specific PCR, bisulfite pyrosequencing and restriction enzyme-PCR. The methylation profiles of endothelium from disturbed and undisturbed flow sites of adult swine aortas were also investigated. In vitro, DF increased DNA methylation of CpG islands within the KLF4 promoter that significantly contributed to suppression of KLF4 transcription; the effects were mitigated by DNA methyltransferase (DNMT) inhibitors and knock-down of DNMT3A. Contributory mechanisms included DF-induced increase of DNMT3A protein (1.7 fold), DNMT3A enrichment (11-fold) on the KLF4 promoter, and competitive blocking of a MEF2 binding site in the KLF4 promoter near the TSS. DF also induced DNMT-sensitive pro-pathological expression of downstream KLF4 transcription targets NOS3, thrombomodulin (THBD) and MCP-1. In support of the in vitro findings, swine aortic endothelium isolated from DF regions expressed significantly lower KLF4 and NOS3, and bisulfite sequencing of KLF4 promoter identified a hypermethylated MEF2 binding site. Conclusions Hemodynamics influence endothelial KLF4 expression through DNMT enrichment/MEF2 inhibition mechanisms of KLF4 promoter CpG methylation with regional consequences for atherosusceptibility.

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Type 2 diabetes mellitus (T2DM) is characterized by the inability of the insulin-producing β-cells to overcome insulin resistance. We previously identified an imprinted region on chromosome 14, the DLK1-MEG3 locus, as being downregulated in islets from humans with T2DM. In this study, using targeted epigenetic modifiers, we prove that increased methylation at the promoter of Meg3 in mouse βTC6 β-cells results in decreased transcription of the maternal transcripts associated with this locus. As a result, the sensitivity of β-cells to cytokine-mediated oxidative stress was increased. Additionally, we demonstrate that an evolutionarily conserved intronic region at the MEG3 locus can function as an enhancer in βTC6 β-cells. Using circular chromosome conformation capture followed by high-throughput sequencing, we demonstrate that the promoter of MEG3 physically interacts with this novel enhancer and other putative regulatory elements in this imprinted region in human islets. Remarkably, this enhancer is bound in an allele-specific manner by the transcription factors FOXA2, PDX1, and NKX2.2. Overall, these data suggest that the intronic MEG3 enhancer plays an important role in the regulation of allele-specific expression at the imprinted DLK1-MEG3 locus in human β-cells, which in turn impacts the sensitivity of β-cells to cytokine-mediated oxidative stress.

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-environment 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 associate 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 < 1.0×10−3 (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-value: 1.3×10−16) compared to 5% in ever-smokers (P-value: 2.5×10−4) translating to a 60% loss of CHD protection conferred by this allelic variation in people who smoked tobacco (Interaction P-value: 8.7×10−5). 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” compared to “ever-smokers”. Increased vascular ADAMTS7 expression may contribute to the loss of CHD protection in smokers.