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

BackgroundGenome-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. MethodsWe 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. ResultsWe 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. ConclusionsOur 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. HighlightsThe 9p21.3 CAD risk locus promotes the transition of iPSC-derived Vascular Smooth Muscle Cells (iPSC-VSMCs) to an osteochondrogenic phenotype, both transcriptionally and functionally. iPSC-VSMCs carrying the risk haplotype at 9p21.3 display a distinct transcriptomic signature, including osteochondrogenic markers SOX9 and COL2A1, along with two novel markers: LIMCH1 and CRABP1. Knockout of the entire haplotype reverts this signature to a non-risk state, demonstrating a clear genotype-to-phenotype causal effect of the risk allele. Short isoform 12 of the lncRNA ANRIL, which partially overlaps the 9p21.3 locus, is sufficient to induce this transcriptional signature. The iPSC-VSMC transcriptional profile strongly resembles that of ex-vivo human arterial VSMCs, providing a human-specific model to study VSMC phenotypic alterations and investigate the effects of large CAD haplotypes on vascular wall cells.