Targeting macrophage necroptosis for therapeutic and diagnostic interventions in atherosclerosis

Atherosclerosis results from maladaptive inflammation driven primarily by macrophages, whose recruitment and proliferation drive plaque progression. In advanced plaques, macrophage death contributes centrally to the formation of plaque necrosis, which underlies the instability that promotes plaque rupture and myocardial infarction. Hence, targeting macrophage cell death pathways may offer promise for the stabilization of vulnerable plaques. Necroptosis is a recently discovered pathway of programmed cell necrosis regulated by RIP3 and MLKL kinases that, in contrast to apoptosis, induces a proinflammatory state. We show herein that necroptotic cell death is activated in human advanced atherosclerotic plaques and can be targeted in experimental atherosclerosis for both therapeutic and diagnostic interventions. In humans with unstable carotid atherosclerosis, expression of RIP3 and MLKL is increased, and MLKL phosphorylation, a key step in the commitment to necroptosis, is detected in advanced atheromas. Investigation of the molecular mechanisms underlying necroptosis showed that atherogenic forms of low-density lipoprotein increase RIP3 and MLKL transcription and phosphorylation—two critical steps in the execution of necroptosis. Using a radiotracer developed with the necroptosis inhibitor necrostatin-1 (Nec-1), we show that ¹²³I-Nec-1 localizes specifically to atherosclerotic plaques in Apoe⁻/⁻ mice, and its uptake is tightly correlated to lesion areas by ex vivo nuclear imaging. Furthermore, treatment of Apoe⁻/⁻ mice with established atherosclerosis with Nec-1 reduced lesion size and markers of plaque instability, including necrotic core formation. Collectively, our findings offer molecular insight into the mechanisms of macrophage cell death that drive necrotic core formation in atherosclerosis and suggest that this pathway can be used as both a diagnostic and therapeutic tool for the treatment of unstable atherosclerosis.