Abstract Body (Do not enter title and authors here): Background: The genetic underpinning of coronary artery disease is well established, with 9p21 risk loci among the strongest associated with CAD. However, the mechanistic underpinning of risk is poorly understood. TAM receptors (TYRO3, AXL, MERTK) and their ligand GAS6 are known regulators of vascular smooth muscle cell (VSMC) survival, anti-inflammatory signaling, and matrix remodeling—critical in atherosclerosis.

Hypothesis: We hypothesize that the 9p21 risk haplotype will lead to a downregulation of GAS6 and TAM, resulting in loss of protective anti-inflammatory effects promoting a pro-fibrotic, pre-senescent VSMC phenotype driving risk for CAD.

Methods: Previously generated iPSC from homozygous risk haplotype (RR), non-risk haplotype (NN), and risk haplotype knockout (RRKO) of the 9p21 locus were differentiated towards VSMC fate (iVSMCs). Single-cell RNA sequencing (scRNAseq) was performed for the iVSMCs, and differential gene expression was analyzed. Differential gene expression (DGE) data was assessed by average log2 fold change and p-values, with significant DGE for GAS6 and TAM. CellChat ligand-receptor interactions were analyzed to compare outgoing and incoming signaling specificity in coronary atherosclerosis versus non-atherosclerotic samples.

Results: In RR iVSMCs, both AXL (log2FC= -1.11, p=1.06E-52) and GAS6 (log2FC= -1.99, p=7.36E-102) were significantly downregulated compared to NN iVSMCs. This was accompanied by an upregulation of pro-inflammatory markers in RR, such as CCL2 (log2FC= 2.54, p= 5.82E-18) and CD36 (log2FC= 3.60, p= 0.02). Compared to RR, RRKO iVSMCs re-expressed AXL (log2FC= -1.79, p=1.02E-89) and GAS6 (log2FC= -2.20, p=2.38E-89) returning expression similar to NN iVSMCs, indicating 9p21’s involvement in GAS6/TAM regulation.

Conclusions: The 9p21 risk haplotype downregulates GAS6 and AXL expression and shifts cell-cell signaling in iVSMCs, potentially impairing anti-inflammatory and vascular homeostasis pathways. Reversal of this expression was observed with deletion of the risk loci. These findings suggest that the TAM pathway plays an integral role in pathogenesis conveyed by the 9p21 risk haplotype to modulate CAD progression.