Deletion of the murine ortholog of human 9p21.3 locus promotes atherosclerosis by increasing macrophage proinflammatory activity

BACKGROUND: Several genome-wide association studies have reported a risk locus for coronary artery disease (CAD) in the 9p21. 3 chromosomal region. This region encodes a lncRNA in the INK4 locus (ANRIL) and its genetic variance has a strong association with CAD, but its mechanisms in atherogenesis remain unclear. OBJECTIVES: This study aimed to investigate the role of the murine ortholog of human 9p21.3 locus in atherogenesis in hypercholesterolemic mice. METHODS: Murine 9p21.3 ortholog knockout mice (Chr4(Δ70kb/Δ70kb)) were crossbred with Ldlr(−/−)ApoB(100/100) mice, and atherosclerotic plaque size and morphology were analyzed on a standard or a high-fat diet (HFD). The hematopoietic cell-specific effect of Chr4(Δ70kb/Δ70kb) on atherosclerotic plaque development was studied via bone marrow (BM) transplantation, where Chr4(Δ70kb/Δ70kb) or wild-type BM was transplanted into Ldlr(−/−)ApoB(100/100) mice. The role of Chr4(Δ70kb/Δ70kb) in macrophage M1/M2 polarization was studied. In addition, single-cell sequencing data from human and mouse atheroma were analyzed to show the expression profiles of ANRIL and its murine equivalent, Ak148321, in the plaques. RESULTS: Both systemic and hematopoietic Chr4(Δ70kb/Δ70kb) increased atherosclerosis in Ldlr(−/−)ApoB(100/100) mice after 12 weeks of HFD. The systemic Chr4(Δ70kb/Δ70kb) also elevated the number of circulating leukocytes. Chr4(Δ70kb/Δ70kb) BMDMs showed enhanced M1 polarization in vitro. Single-cell sequencing data from human and mouse atheroma revealed that ANRIL and Ak148321 were mainly expressed in the immune cells. CONCLUSION: These data demonstrate that both systemic and BM-specific deletion of the murine 9p21.3 risk locus ortholog promotes atherosclerosis and regulates macrophage pro-inflammatory activity, suggesting the inflammation-driven mechanisms of the risk locus on atherogenesis.