Genetic Regulation of Atherosclerosis-Relevant Phenotypes in Human Vascular Smooth Muscle Cells

Abstract

Rationale: Coronary artery disease (CAD) is a major cause of morbidity and mortality worldwide. Recent genome-wide association studies (GWAS) revealed 163 loci associated with CAD. However, the precise molecular mechanisms by which the majority of these loci increase CAD risk are not known. Vascular smooth muscle cells (VSMCs) are critical in the development of CAD. They can play either beneficial or detrimental roles in lesion pathogenesis, depending on the nature of their phenotypic changes. Objective: To identify genetic variants associated with atherosclerosis-relevant phenotypes in VSMCs Methods and Results: We quantified twelve atherosclerosis-relevant phenotypes related to calcification, proliferation, and migration in VSMCs isolated from 151 multi-ethnic heart transplant donors. After genotyping and imputation, we performed association mapping using 6.3 million genetic variants. We demonstrated significant variations in calcification, proliferation, and migration. These phenotypes were not correlated with each other. We performed GWAS for twelve atherosclerosis-relevant phenotypes and identified four genome-wide significant loci associated with at least one VSMC phenotype. We overlapped the previously identified CAD GWAS loci with our dataset and found nominally significant associations at 79 loci. One of them was the chromosome 1q41 locus, which harbors MIA3. The G allele of the lead risk SNP rs67180937 was associated with lower VSMC MIA3 expression and lower proliferation. Lentivirus-mediated silencing of MIA3 in VSMCs resulted in lower proliferation, consistent with human genetics findings. Further, we observed a significant reduction of MIA3 protein in VSMCs in thin fibrous caps of late-stage atherosclerotic plaques compared to early fibroatheroma with thick and protective fibrous caps in mice and humans. Conclusions: Our data demonstrate that genetic variants have significant influences on VSMC function relevant to the development of atherosclerosis. Further, high MIA3 expression may promote atheroprotective VSMC phenotypic transitions, including increased proliferation, which is essential in the formation or maintenance of a protective fibrous cap.

Deficiency of ZC3HC1 modulates vascular smooth muscle cell phenotype and increases neointima formation

“…In addition, a lower level of ZC3HC1 mRNA significantly enhanced the proliferation of SMCs at 72 h, but not at earlier time points. This finding was in agreement with the results from genotyped SMCs derived from 151 human donors 16 , in which no significant correlation was observed between the T allele and cell proliferation at 24 h. Transcriptional profiling of ZC3HC1 downregulation in human SMCs: To determine the transcriptional profile of ZC3HC1 downregulation, transcriptome analysis using RNA sequencing was performed in aortic SMCs in the presence or absence of 5 ng/mL PDGF-BB.…”

“…Analysis of the publicly available GTEx dataset eQTL (V8) showed that the genetic variant rs11556924-T results in reduced ZC3HC1 gene expression in heart (atrial appendage and left ventricle) and skin samples. Because eQTL effects are often cell type specific, we tested whether an eQTL was present in aortic SMCs from 151 heart transplant donors 16 , finding that SMCs from donors carrying the rs11556924-T allele have lower ZC3HC1 expression and migrate faster than SMCs from donors carrying the rs11556924-C allele. The absence of a significant association between ZC3HC1 expression and rs11556924 in blood samples 9 , monocytes/macrophages 28 and aortic endothelial cells 29 suggests that the regulatory impact of the variant at the ZC3HC1 locus is specific to SMCs.…”

“…Cell migration was monitored over 24 h. Data were analyzed using RTCA software version 1.2 (Acea Biosciences Inc., San Diego, CA) combined with R software. The association between the genotype of rs11556924 and migration was calculated using linear mixed model to account for multiethnic population composition as previously been described 16 .…”

“…Human aortic SMCs isolated from the ascending aortas of 151 heart transplant donors at UCLA or obtained from commercial suppliers (Lonza and PromoCell) as this has been previously described 16 . All SMCs were maintained in Smooth muscle cell Basal Medium qPCR analysis: These analyses were performed as described 21 22 .…”

The Contribution of Endothelial-Mesenchymal Transition to Atherosclerosis

“…Cigarette smoking constitutes a major environmental risk factor for atherosclerosis. Gene-environment interactions have also been demonstrated [99,100]. Indeed, a synergistic effect between cigarette smoking and the carrier state of the ApoE epsilon4 allele increases the risk of atherosclerosis [101].…”