Abstract
Impairment of efferocytosis in apoptotic macrophages is a known determinant of the severity of atherosclerosis and the vulnerability of plaques to rupture. The precise mechanisms involved in impaired efferocytosis are unclear. Given the well-recognized role of the inflammatory cytokine cyclophilin A (Cyp A) in modulating several atherogenic mechanisms in high-glucose primed monocytes, we investigated the role of Cyp A in macrophage efferocytosis. The efficiency of efferocytosis in RAW 264.7 macrophages grown in vitro and primed with cyclophilin A was assessed using flow cytometry and confocal assays. Cholesterol content in cells was measured using cell-based cholesterol efflux assay. Proteomic analysis and bioinformatics tools were employed to decipher the link between cyclophilin A and the known ligand receptors involved in efferocytosis. Cyclophilin A was found to impair efferocytosis in apoptotic macrophages by reducing ABCA1-mediated cholesterol efflux in foam cells derived from macrophages. Cyclophilin A-primed macrophages showed an increase in expression of the don’t-eat-me signal CD 47 and a decrease in the expression of the eat-me signal, calreticulin. Phagocytosis was restored upon silencing of cyclophilin A. New Zealand white rabbits were fed a high-fat diet, and lesions in their aortae were analyzed histologically for evidence of atherosclerosis and the expression of Cyp A, CD 47 and calreticulin, the ligand receptor involved in efferocytosis. Gene and protein expressions in aortae and macrophages were analyzed by real-time PCR and Western blotting. Cyclophilin A, via its effects on the expression of CD 47 and calreticulin, impairs efferocytosis in apoptotic macrophages. Together with its impact on cholesterol efflux from macrophages, these effects can amplify other mechanisms of Cyp A in accelerating the progression of atherosclerosis.
Highlights
Cyclophilin A (Cyp A) is a universally distributed protein that exists in both intracellular and extracellular forms [1,2]
Our studies indicate that cyclophilin A (Cyp A) impairs macrophage efferocytosis
A decrease in ABCA1-mediated reverse cholesterol efflux and impaired efferocytosis mediated by enhanced CD 47 expression over calreticulin can lead to progression of atherosclerosis lesions (Figure 5)
Summary
Cyclophilin A (Cyp A) is a universally distributed protein that exists in both intracellular and extracellular forms [1,2] Several cells, such as endothelial cells, vascular smooth muscle cells and platelets, secrete Cyp A in response to increased oxidative stress [3]. Efferocytosis involves several sequential events, including finding of apoptotic cells by phagocytes, recognition of apoptotic cells (ACs) by phagocytic receptors, activation of intracellular signaling cascades with cytoskeletal rearrangements for AC engulfment and post-engulfment processes for phagosome maturation and AC degradation [5]. These mechanisms are regulated by several signal molecules, such as ‘find-me’, ‘eat-me’ and ‘don’t-eat-me’ signals. The necrotic core is the hallmark of the vulnerable atherosclerotic plaque
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