The 9p21.3 coronary artery disease risk locus drives vascular smooth muscle cells to an osteochondrogenic state

Background. Genome-wide association studies have identified common genetic variants at ~300 human genomic loci linked to coronary artery disease (CAD) susceptibility. Among these genomic regions, the most impactful is the 9p21.3 CAD risk locus, which spans a 60 kb gene desert and encompasses ~80 SNPs in high linkage disequilibrium. Despite nearly two decades since its discovery, the role of the 9p21.3 locus in cells of the vasculature remains incompletely resolved. Methods. We differentiated induced pluripotent stem cells (iPSCs) from risk and non-risk donors at 9p21.3 into vascular smooth muscle cells. We performed single-cell transcriptomic profiling, including co embedding and comparison with publicly available human arterial datasets. We conducted functional characterization using migration and calcification assays and confirmed our findings on iPSC-VSMCs derived from additional donors. Finally, we used overexpression of ANRIL followed by gene expression analysis. Results. We demonstrated that iPSC-VSMCs harboring the 9p21.3 risk haplotype preferentially adopt an osteochondrogenic state and show remarkable similarity to fibrochondrocytes from human artery tissue. The transcriptional profile and functional assessment of migration and calcification capacity across iPSC VSMCs lines from multiple donors concordantly resemble an osteochondrogenic state. Importantly, we identified numerous transcription factors driving different VSMC state trajectories. Additionally, we prioritized LIMCH1 and CRABP1 as signature genes critical for defining the risk transcriptional program. Finally, overexpression of a short isoform of ANRIL in non-risk cells was sufficient to induce the osteochondrogenic transcriptional signature. Conclusions. Our study provides new insights into the mechanism of the 9p21.3 risk locus and defines its previously undescribed role in driving a disease-prone transcriptional and functional state in VSMCs concordant with an osteochondrogenic-like state. Our data suggest that the 9p21.3 risk haplotype likely promotes arterial calcification, through altered expression of ANRIL, in a cell-type specific and cell autonomous manner, providing insight into potential risk assessment and treatment for carriers.