Objective: Inflammatory activation changes the mitochondrial function of macrophages from oxidative phosphorylation to reactive oxygen species production, which may promote necrotic core formation in atherosclerotic lesions. In hypoxic and cancer cells, HIF-1α (hypoxia-inducible factor) promotes oxygen-independent energy production by microRNAs. Therefore, we studied the role of HIF-1α in the regulation of macrophage energy metabolism in the context of atherosclerosis. Approach and Results: Myeloid cell–specific deletion of Hif1a reduced atherosclerosis and necrotic core formation by limiting macrophage necroptosis in apolipoprotein E-deficient mice. In inflammatory bone marrow–derived macrophages, deletion of Hif1a increased oxidative phosphorylation, ATP levels, and the expression of genes encoding mitochondrial proteins and reduced reactive oxygen species production and necroptosis. microRNA expression profiling showed that HIF-1α upregulates miR-210 and downregulates miR-383 levels in lesional macrophages and inflammatory bone marrow–derived macrophages. In contrast to miR-210 , which inhibited oxidative phosphorylation and enhanced mitochondrial reactive oxygen species production, miR-383 increased ATP levels and inhibited necroptosis. The effect of miR-210 was due to targeting 2,4-dienoyl-CoA reductase, which is essential in the β oxidation of unsaturated fatty acids. miR-383 affected the DNA damage repair pathway in bone marrow–derived macrophages by targeting poly(ADP-ribose)-glycohydrolase (Parg), which reduced energy consumption and increased cell survival. Blocking the targeting of Parg by miR-383 prevented the protective effect of Hif1a deletion in macrophages on atherosclerosis and necrotic core formation in mice. Conclusions: Our findings unveil a new mechanism by which activation of HIF-1α in inflammatory macrophages increases necroptosis through microRNA-mediated ATP depletion, thus increasing atherosclerosis by necrotic core formation.

Background: The necrotic core partly formed by ineffective efferocytosis increases the risk of an atherosclerotic plaque rupture. microRNAs contribute to necrotic core formation by regulating efferocytosis and macrophage apoptosis. Atherosclerotic plaque rupture occurs at increased frequency in the early morning, indicating diurnal changes in plaque vulnerability. Although circadian rhythms play a role in atherosclerosis, the molecular clock output pathways that control plaque composition and rupture susceptibility are unclear. Methods: Circadian gene expression, necrotic core size, and apoptosis and efferocytosis in aortic lesions were investigated at different times of the day in Apoe -/- Mir21 +/+ mice and Apoe -/- Mir21 -/- mice after consumption of a high-fat diet for 12 weeks feeding. Genome-wide gene expression and lesion formation were analyzed in bone marrow (BM)-transplanted mice. Diurnal changes in apoptosis and clock gene expression were determined in human atherosclerotic lesions. Results: The expression of molecular clock genes, lesional apoptosis, and necrotic core size were diurnally regulated in Apoe -/- mice. Efferocytosis did not match the diurnal increase in apoptosis at the beginning of the active phase. However, in parallel with apoptosis, expression levels of oscillating Mir21 strands decreased in the mouse atherosclerotic aorta. Mir21 knockout abolished circadian regulation of apoptosis and reduced necrotic core size, but did not affect core clock gene expression. Further, Mir21 knockout upregulated expression of pro-apoptotic XIAP associated factor 1 ( Xaf1 ) in the atherosclerotic aorta, which abolished circadian expression of Xaf1. The anti-apoptotic effect of Mir21 was mediated by non-canonical targeting of Xaf1 through both Mir21 strands. Mir21 knockout in BM cells also reduced atherosclerosis and necrotic core size. Circadian regulation of clock gene expression was confirmed in human atherosclerotic lesions. Apoptosis oscillated diurnally in phase with XAF1 expression, demonstrating an early morning peak anti-phase to that of the Mir21 strands. Conclusions: Our findings suggest that the molecular clock in atherosclerotic lesions induces a diurnal rhythm of apoptosis regulated by circadian Mir21 expression in macrophages that is not matched by efferocytosis, thus increasing the size of the necrotic core.

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