Abstract Body: Introduction. GWAS studies have identified approximately 300 genomic loci linked to increased coronary artery disease (CAD) risk, of which the most impactful is the 9p21.3 locus. This ~60 kb region is a gene desert containing ~80 SNPs in high linkage disequilibrium, defining two possible alleles, risk or non-risk. Despite its identification nearly 20 years ago, the function of the 9p21.3 risk locus remains incompletely understood. We hypothesize that the 9p21.3 region contributes to CAD risk by mediating aberrant phenotypic modulation in vascular smooth muscle cells (VSMC).
Method. We used induced pluripotent stem cells (iPSCs) from 9p21.3 risk and non-risk donors to create isogenic lines with a full haplotype deletion, generating four genotypes: risk WT, risk KO, non-risk WT, and non-risk KO. Using two independent iPSC lines per genotype (8 lines total), we conducted a differentiation to VSMC and employed single-cell transcriptomic profiling. We then performed co-embedding and comparison with publicly available human coronary artery scRNAseq datasets, validated the hits from our analysis by immunocytochemistry, and performed functional assays to test the impact of the transcriptional changes. Finally, we used knockdown and overexpression experiments to further examine the impact of key genes identified in our analysis.
Results. Our study shows that iPSC-VSMCs carrying the 9p21.3 risk haplotype adopt an osteochondrogenic state. The altered transcriptional profile, decreased migration capacity, and increased calcification reveal a phenotype closely resembling fibrochondrocytes, an osteochondrogenic state known to contribute to CAD pathogenesis. Additionally, we observe a strong transcriptional overlap between iPSC-VSMCs and native human VSMCs from heart arteries, validating the utility of our model to study human disease. We also identify two novel 9p21.3 risk-biased signature genes, LIMCH1 and CRABP1, which appear to be critical in defining the risk state.
Conclusions. Our study demonstrates that 9p21.3 drives disease-relevant phenotypic modulation in VSMCs consistent with an osteochondrogenic-like state called fibrochondrocytes. We observe that the 9p21.3 risk region exerts its influence in a cell type-specific and cell-autonomous manner, promoting CAD-relevant phenotypes like arterial stiffening and calcification. This work contributes to our knowledge of how 9p21.3 can increase risk of CAD, thus aiding efforts to mitigate genetic risk for the disease.