Objective:

The “pleiotropic benefits of statins” may result from their impact on vascular inflammation. The molecular process underlying this phenomenon is not fully elucidated.

Results:

We performed RNA sequencing designed to investigate gene expression patterns following CD47-SIRPα inhibition. Using Qiagen’s Ingenuity Pathway Analysis, we found that a first generation HMG-CoA reductase inhibitor was one of the top activated “upstream regulators” and the only drug in the database. The unanticipated crosstalk prompted us to test whether combined treatment of CD47-SIRPα blockade (by targeting either CD47 or SIRPα’s downstream effector molecule SHP-1) and HMG-CoA reductase inhibition (using atorvastatin) has additive effects on the atherosclerotic plaque activity in vivo. We found that combined treatment not only decreased lesion size but also reduced necrotic core area in high-fat diet-fed Apoe-/- mice. Next, we reasoned that atorvastatin might increase the efferocytosis rate and thus beneficially impact lesion development. In vitro, we observed by flow cytometry a relevant increase of the efferocytic rate of apoptotic cells upon combined treatment compared to single therapies. Furthermore, we confirmed these observations in vivo by investigating the cleaved caspase-3 activity and the number of “free” apoptotic bodies not associated with an intraplaque macrophage, both reliable measures of accumulation of apoptotic bodies and thus efferocytosis in tissue specimens. Having identified the efferocytic rate as a pivotal link for additivity of combined treatment, we sought to elucidate the underlying mechanism. We identified a direct effect of atorvastatin on the CD47 expression on both RNA and protein levels in smooth muscle cells. Dual luciferase reporter assay confirmed that atorvastatin was able to inhibit the tumor necrosis factor-α induced CD47 promoter activity and Western blot analyses revealed that atorvastatin directly reduced the pathological CD47 upregulation in atherosclerosis via inhibition of the pro-inflammatory factor NFκB1 p50, which is a known key transcriptional factor for CD47. Intriguingly, analyses of carotid endarterectomy samples from the Munich Vascular Biobank also confirmed that patients receiving statin treatment had lower CD47 expression than a propensity score matched cohort without such a medication, highlighting the potential translational implications.

Conclusion:
Our findings identify efferocytosis and CD47 as a pivotal mediator of “statin pleiotropy”. In turn, statins amplify the anti-atherosclerotic effects of pro-phagocytic therapies independently of any lipid-lowering effect.