Cyclin-dependent kinase inhibitor 2B regulates efferocytosis and atherosclerosis
Yoko Kojima, … , Tom Quertermous, Nicholas J. Leeper
Yoko Kojima, … , Tom Quertermous, Nicholas J. Leeper
Published February 17, 2014
Citation Information: J Clin Invest. ;124(3):1083-1097. https://doi.org/10.1172/JCI70391.
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Research Article

Cyclin-dependent kinase inhibitor 2B regulates efferocytosis and atherosclerosis

Abstract

Genetic variation at the chromosome 9p21 risk locus promotes cardiovascular disease; however, it is unclear how or which proteins encoded at this locus contribute to disease. We have previously demonstrated that loss of one candidate gene at this locus, cyclin-dependent kinase inhibitor 2B (Cdkn2b), in mice promotes vascular SMC apoptosis and aneurysm progression. Here, we investigated the role of Cdnk2b in atherogenesis and found that in a mouse model of atherosclerosis, deletion of Cdnk2b promoted advanced development of atherosclerotic plaques composed of large necrotic cores. Furthermore, human carriers of the 9p21 risk allele had reduced expression of CDKN2B in atherosclerotic plaques, which was associated with impaired expression of calreticulin, a ligand required for activation of engulfment receptors on phagocytic cells. As a result of decreased calreticulin, CDKN2B-deficient apoptotic bodies were resistant to efferocytosis and not efficiently cleared by neighboring macrophages. These uncleared SMCs elicited a series of proatherogenic juxtacrine responses associated with increased foam cell formation and inflammatory cytokine elaboration. The addition of exogenous calreticulin reversed defects associated with loss of Cdkn2b and normalized engulfment of Cdkn2b-deficient cells. Together, these data suggest that loss of CDKN2B promotes atherosclerosis by increasing the size and complexity of the lipid-laden necrotic core through impaired efferocytosis.

Authors

Yoko Kojima, Kelly Downing, Ramendra Kundu, Clint Miller, Frederick Dewey, Hope Lancero, Uwe Raaz, Ljubica Perisic, Ulf Hedin, Eric Schadt, Lars Maegdefessel, Tom Quertermous, Nicholas J. Leeper

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Figure 4

Loss of CDKN2B renders apoptotic SMCs “inedible.” (A) Flow cytometry-based phagocytosis assays revealed that apoptotic CDKN2B-deficient SMCs were significantly less likely to be cleared by primary macrophages than control ABs.

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Loss of CDKN2B renders apoptotic SMCs “inedible.” (A) Flow cytometry-bas...
(B) Apoptotic CDKN2B-deficient HCASMCs were also resistant to efferocytic clearance by neighboring SMCs, which are known to function as nonprofessional phagocytes in conditions such as atherosclerosis. (C) Efferocytosis competition assays revealed that control-transfected ABs were more likely to be phagocytosed than CDKN2B-deficient ABs when cocultured with phagocytes in equal numbers. Original magnification, ×10. (D) Qualitative electron microscopy images revealed evidence of SMC necrosis in plaques from Cdkn2b–/–,ApoE–/– mice, as indicated by cells with condensed chromatin and disrupted plasma membranes (black arrow) and extracellular lysosomes, suggestive of cellular rupture (black arrowhead). Conversely, plaques from Cdkn2b+/+,ApoE–/– mice harbored little necrotic debris and were populated by phagocytes that had each engulfed numerous ABs, suggesting more robust efferocytic clearance (white arrow). Original magnification, ×1,200. Additional representative images are provided in Supplemental Figure 4. #P < 0.01; †P < 0.03.