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.

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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