Therapeutically inhibiting macrophage necroptosis reduces inflammation‐driven atherosclerosis and promotes plaque stability

Abstract

Atherosclerosis is a maladaptive disease driven primarily by macrophages. As the atherosclerotic plaque progresses, inflammatory and macrophage cell death pathways drive the formation of complex, unstable and rupture‐prone lesions with large necrotic core(s). Necroptosis is an emerging cell death pathway involving RIP1, RIP3 and MLKL kinases that, in contrast to apoptosis, induces a pro‐inflammatory state. We hypothesize that inhibiting RIP1 and RIP3 reduces macrophage inflammation and necroptosis to reduce atherosclerosis. Oxidized LDL (oxLDL) induces necroptotic cell death in macrophages, which can be blocked by Nec‐1, a RIP1‐RIP3 inhibitor. Additionally, RIP3−/− macrophages are more resistant to oxLDL‐induced cell death. Treatment with oxLDL increased macrophage expression of necroptotic genes RIP1, RIP3 and MLKL through ROS‐dependent activation of the promoter region. ApoE−/− mice fed a western diet (WD) and simultaneously injected with 100mg/kg RIP1 anti‐sense oligonucleotides (ASO) for 8 weeks had a marked reduction in aortic lesion area compared to both control ASO‐ and PBS‐treated controls (58.8% reduction, p<0.001). In addition, serum inflammatory cytokine profiling suggested a marked reduction in circulating inflammatory cytokines (e.g. IL‐1α, IL‐3 and IL‐17A etc) in RIP1 ASO treated mice compared to control ASO. These results are consistent in vitro, where peritoneal macrophages treated with RIP1 ASO had a reduction in the gene expression of inflammatory genes (e.g. IL‐1α, TNFα) relative to control ASO. Next, in the same experimental model of established atherosclerosis (i.e. Apoe−/− mice fed a western diet for 6 weeks), therapeutic intervention with Nec‐1 these mice reduced lesion size (p<0.05) and increased markers of plaque instability, including reduced necrotic core formation (62.6% reduction, p<0.01). In addition, we developed a novel 123I‐Nec‐1 radiotracer that targets this pathway to specifically localize to and tightly correlate with atherosclerotic lesions. Lastly, in humans with unstable carotid atherosclerosis, expression of the necroptotic genes RIP1, RIP3 and MLKL is elevated and MLKL phosphorylation is detected in advanced atheromas, indicating that the necroptotic pathway is highly activated in vulnerable plaques. In conclusion, our findings offer molecular insight into the inflammatory and cell death mechanisms by which atherogenic ligands drives the formation of the vulnerable plaque and identify innovative therapeutic tools to treat atherosclerosis.