The enigmatic role of VCAM‐1 expressed by macrophages in mitochondrial metabolism and atherosclerosis

Abstract

BackgroundAtherosclerosis is a primary underlying cause of most cardiovascular diseases, including myocardial infarction, heart failure and stroke. It has been shown that vascular cell adhesion protein 1(VCAM‐1) expressed by endothelial cells mediates rolling and adhesion of monocytes to endothelial cells under atherosclerotic conditions. However, the role of VCAM1 expressed by monocyte and macrophage in atherosclerosis pathology has not been explored. Here, we show that VCAM1 in macrophages signals via CMPK2 and STING to modulate mitochondrial biogenesis and promote atherosclerosis.MethodsLyzMcre/+VCAM‐1fl/fl mice were used to characterize the role of macrophage‐specific VCAM1 in the pathogenesis of atherosclerosis, LyzM+/+VCAM‐1fl/flmice were used as controls. Bone marrow cells were transplanted from VCAM‐1‐deficient donor into Ldlr‐/‐fed atherogenic western diet. Additionally, we also generated Apoe‐/‐ LyzMcre/+VCAM‐1fl/fl mice as a second model to understand the function of myeloid VCAM‐1 in atherosclerosis. Flow cytometry was performed to enumerate atheroma immune cell populations and sort plaque macrophages from the aorta. RNA‐seq was performed on isolated aortic macrophages from VCAM‐1 deficient mice (wild‐type controls). Mitochondrial respiration and complex activity were measured by Seahorse Agilent bioanalyzer. Mitochondrial volume and membrane potential were quantified by flow cytometry and immunofluorescence using MitoTracker dyes. Mitophagy was assessed in mtKeima bone marrow derived macrophages (BMDMs) after silencing VCAM1 and treatment with oxLDL. RT‐PCR was performed to quantify expression of inflammatory cytokine, retention factor and mitochondrial biogenesis genes. Gene silencing was performed using siRNA.ResultsAtherosclerotic plaque macrophages expressed high levels of VCAM‐1 in humans and mice. Concomitantly, plaque macrophages had increased mitochondrial volume, mitochondrial DNA synthesis, oxidized mitochondrial DNA and oxidative phosphorylation. We observed that mice lacking Vcam1 in macrophages had reduced atherosclerotic plaque and necrotic core areas. Additionally, Vcam1 silencing in macrophages diminished inflammation, oxidative phosphorylation, mitochondrial biogenesis and the expression of the genes involved in mitochondrial DNA synthesis including Cmpk2. Cmpk2 knock down in macrophages after oxidized LDL treatment reduced inflammatory mediators that aggravate atherosclerosis. RNA sequencing analysis of Vcam‐1‐deficient plaque macrophages and analysis of macrophages lacking Cmpk2 identified Fcor and Lyz1 as the target genes of Vcam1 and Cmpk2. Interestingly, atherosclerotic plaque macrophages deficient of Sting, which mediates inflammatory signaling in response to oxidized mitochondrial DNA, had increased levels of Fcor and Lyz1.ConclusionWe conclude that macrophage‐specific VCAM‐1 augments mitochondrial biogenesis and DNA oxidation via Cmpk2. Oxidized mitochondrial DNA in plaque macrophages increases inflammation via Sting, resulting in exacerbation of atherosclerosis.