Cdkn2a is an atherosclerosis modifier locus that regulates monocyte/macrophage proliferation

Abstract

Objective: Common genetic variants in a 58-kb region of chromosome 9p21, near the CDKN2A/CDKN2B tumor suppressor locus, are strongly associated with coronary artery disease. However, the underlying mechanism of action remains unknown.

Methods and results: We previously reported a congenic mouse model harboring an atherosclerosis susceptibility locus and the region of homology with the human 9p21 locus. Microarray and transcript-specific expression analyses showed markedly decreased Cdkn2a expression, including both p16(INK4a) and p19(ARF), but not Cdkn2b (p15(INK4b)), in macrophages derived from congenic mice compared with controls. Atherosclerosis studies in subcongenic strains revealed genetic complexity and narrowed 1 locus to a small interval including Cdkn2a/b. Bone marrow (BM) transplantation studies implicated myeloid lineage cells as the culprit cell type, rather than resident vascular cells. To directly test the role of BM-derived Cdkn2a transcripts in atherogenesis and inflammatory cell proliferation, we performed a transplantation study using Cdkn2a(-/-) cells in the Ldlr(-/-) mouse model. Cdkn2a-deficient BM recipients exhibited accelerated atherosclerosis, increased Ly6C proinflammatory monocytes, and increased monocyte/macrophage proliferation compared with controls.

Conclusion: These data provide a plausible mechanism for accelerated atherogenesis in susceptible congenic mice, involving decreased expression of Cdkn2a and increased proliferation of monocyte/macrophages, with possible relevance to the 9p21 human locus.

Figure 1. Refined mapping of an atherosclerosis susceptibility locus to a 5.4-9-Mb region including the region of homology with a human ASVD risk interval on 9p21

A, Physical map of mouse chr 4 and MOLF donor intervals (white boxes) carried by congenic strains in the B6-Ldlr^−/− background. Mb, megabase; b/b, homozygosity for B6 alleles; m/m, homozygosity for MOLF alleles. The rectangle indicates the narrowed interval defined by data in (b). B, Mean lesion areas in control (b/b) and congenic strains fed 9-wk WTD. Two-factor ANOVA performed with square root transformation. Horizontal bars indicate group means for males (circles) and females (triangles). NS, not significant. C, Microsatellite markers delineating proximal (D4Mit349/D4Mit27) and distal (D4Mit350/D4Mit154) recombination breakpoints of the refined risk interval. The interval includes the region of homology with a human risk interval on 9p21.

Figure 2. Decreased expression of p16^INK4a and p19^ARF, but not p15^INK4b, cell proliferation inhibitor transcripts in BM-derived cells from chr 4 congenic mice compared to controls

rtPCR results in cells derived from B6-Ldlr^−/− (b/b) and congenic (54- or 17-Mb) mice. A, concanavalin A-elicited peritoneal macrophages, 6-wk WTD, n=10 mice/group; B, resident peritoneal macrophages, 9-wk WTD, n=8–12 mice/group; C, splenic CD11b⁺ monocyte/macrophages, 15-wk WTD. n=7–9 mice/group. ANOVA (A) or t-test (B, C) performed with log transformation. *p≤0.05, **p≤0.005, †p≤0.0005 compared to b/b controls.

Figure 3. BM-specific Cdkn2a deficiency is sufficient to promote atherosclerosis in B6-Ldlr^−/− mice

A–B, Transcript-specific rtPCR results for Cdkn2a (p16^INK4a and p19^ARF) and p18^INK4c (another INK4-class gene encoded by a distant region of mouse chr 4). N=7 mice/group. Unpaired t-test performed with log transformation. C, Mean lesion areas from B6-Ldlr^−/− mice transplanted with B6-Ldr^+/− or B6-Ldr^+/−, Cdkn2a^+/− BM and fed 10-wk WTD. Two-factor ANOVA performed with square root transformation. Horizontal bars indicate group means for males (circles) and females (triangles). *p≤0.05.

Figure 4. Increased inflammatory Ly6C^hi monocytes, mediated by increased cell proliferation, in the circulation of B6-Ldlr^−/− mice transplanted with Ldlr^+/−, Cdkn2a^+/− BM compared to controls

A, Flow cytometry analysis of blood monocytes from Ldlr^+/− or Ldlr^+/−, Cdkn2a^+/− BM recipients fed chow or WTD. Monocytes were gated as CD45⁺CD115⁺ Ly6C^hi or CD45⁺CD115⁺Ly6C^lo. B, Quantification of Ly6C^hi/ Ly6C^lo cells among total CD45⁺CD115⁺ cells. C, Analysis of proliferating CD45⁺CD115+ monocytes at the 10-wk timepoint. Cells were gated as Ly6C^hi BrdU⁺. D, Quantification of BrdU⁺ cells among total CD45⁺CD115⁺ monocytes. N=11 mice/group. *p≤0.05, **p≤0.005, †p≤0.0005.

Figure 5. Increased proliferation of tissue macrophages derived from B6-Ldlr^−/− mice transplanted with Ldlr^+/−, Cdkn2a^+/− BM compared to controls

A, Flow cytometry analysis of concanavalin A-elicited peritoneal macrophages from B6-Ldlr^−/−or B6-Ldlr^−/−, Cdkn2a^+/− mice fed 4–5 wks WTD. Macrophages were gated as CD45⁺CD115⁺F4/80⁺. B, Analysis of proliferating CD45⁺CD115⁺F4/80⁺ macrophages at the 4–5 wk timepoint. Cells were gated as BrdU⁺. C, Quantification of BrdU⁺ cells among total CD45⁺CD115⁺F4/80⁺ macrophages. N=6–7 mice/group. *p≤0.05.