A recent research letter published within the journal Nature Cardiovascular Research describes two latest genetic loci related to coronary artery calcification (CAC).
CAC is a measure of atherosclerosis and predicts coronary artery disease (CAD) events. Coronary calcification is a manifestation of atherosclerotic plaque. It is usually recommended to contribute to plaque rupture when present as microcalcifications, while more extensive calcification sheets are related to stabilizing the plaque.
Vascular calcification is characterised by the contractile-to-osteogenic phenotype switch of vascular smooth muscle cells (VSMCs). The osteogenic phenotype is characterised by the expression of markers, including the master regulator of the switch, Runt-related transcription factor 2 (RUNX2), and others similar to alkaline phosphatase (ALPL) and bone gamma-carboxyglutamate protein (BGLAP).
Genome-wide association studies (GWASs) have identified greater than 200 loci related to CAD events and only 4 for CAC. The 4 CAC loci are also related to CAD events, and their effects are mainly related to the progression of atherosclerosis. Identifying additional loci for CAC will provide insights into the pathogenic mechanism of atherosclerotic heart problems.
Letter: Whole-genome sequencing uncovers two loci for coronary artery calcification and identifies ARSE as a regulator of vascular calcification. Image Credit: sciencepics / Shutterstock
The study and findings
In the current study, researchers performed a GWAS to discover CAC loci. Around 22,400 individuals from 10 studies, with whole-genome sequencing and CAC data, were included and stratified based on race/ethnicity. Participants were a mean age of 58, 53% male and 47% female. Half the population had detectable CAC. In single-variant analyses, over 28.5 million variants with minor allele count ≥ 50 were tested for associations with CAC.
Genetic variants showing a big association with CAC were detected at six loci – 9p21, matrix metallopeptidase 16 (MMP16), apolipoprotein B (APOB), APOE, arylsulfatase E (ARSE), and phosphatase and actin regulator 1/endothelin 1 (PHACTR1/EDN1). Next, rare variants (minor allele frequency < 1%) were tested for associations with CAC using gene-based variant aggregation and filtering. This resulted in a genome-wide significant aggregation unit for CAC mapping to APOB.
Nevertheless, the associations were insignificant when adjusted for the index variant identified at that locus within the single-variant evaluation. Genetic variants in MMP16 and ARSE weren’t previously reported to be related to CAD or CAC at a genome-wide significance level. The inverse association between CAC and the G allele of the ARSE index variant (rs5982944) followed a recessive mode of inheritance.
Further, the index variant at ARSE was related to carotid plaque, low- (LDL-C) and high-density lipoprotein cholesterol (HDL-C), and systolic blood pressure. It was also related to thoracic ascending and descending aorta calcification. Alternatively, the MMP16 index variant (rs13268080) showed nominal associations with CAD events and was not significantly related to every other atherosclerotic phenotypes.
Furthermore, the credible set evaluation suggested that the ARSE index variant was the causal variant with an 85% posterior probability. This variant was also related to ARSE gene expression levels in various cells and tissues, similar to the aorta and cultured fibroblasts. The G allele of the ARSE index variant was related to lower ARSE expression in cultured fibroblasts, suggesting that increased ARSE levels might stimulate arterial calcification.
Likewise, the MMP16 index variant was related to MMP16 gene expression. The G allele of this variant within the aorta was related to reduced MMP16 messenger RNA (mRNA) levels. Further analyses suggested that increased MMP16 expression might inhibit calcification. Next, functional perturbation analyses were performed in coronary artery VSMCs to look at whether ARSE and MMP16 regulate the phenotypic switch and calcification.
MMP16 expression in VSMCs was reduced by 75% when grown in osteogenic media relative to normal media. Silencing MMP16 had no effect on the osteogenic phenotype or calcification of VSMCs. In contrast, ARSE expression increased five-fold (mRNA) and 1.7-fold (protein) when VSMCs were grown in osteogenic media in comparison with normal media.
Silencing ARSE decreased the expression of osteogenic phenotype markers (RUNX2, ALPL, and BGLAP) and increased that of calponin (contractile phenotype marker). It also reduced calcium deposition by cells in osteogenic media by 60% and increased cellular contractility. Increasing ARSE levels in VSMCs in normal media caused a seven-fold increase in RUNX2 and a 70% reduction in calponin levels.
Further, coronary VSMC calcification increased four-fold, and coronary artery contractility declined by 70%. When experiments were performed using aortic VSMCs, results were much like those in coronary artery VSMCs. Next, ARSE expression was examined within the coronary arteries of ischemic CAD patients and controls.
ARSE expression was low in coronary arteries from controls but significantly higher in patients. RUNX2 expression was also higher in calcified ischemic arteries, and it co-localized with ARSE. Additional experiments revealed that the ARSE index variant could influence ARSE gene expression in HEK293 cells and human aortic and coronary artery smooth muscle cells.
Together, six loci related to CAC were identified. The ARSE index variant was restricted to populations with African ancestry. The findings implicate ARSE as a regulator of the phenotype switch and calcification. ARSE silencing increased VSMC contractility but decreased osteogenic phenotype markers and calcification, while its overexpression induced opposite effects. Overall, the outcomes underscore ARSE as a possible drug goal for vascular calcific disease.