Andreas Schober1,2,3,4,5, Richard M Blay1,2,3,4,5, Saffiyeh Saboor Maleki1,2,3,4,5, Farima Zahedi1,2,3,4,5, Anja E Winklmaier1,2,3,4,5, Mati Y Kakar1,2,3,4,5, Isabelle M Baatsch1,2,3,4,5, Mengyu Zhu1,2,3,4,5, Claudia Geißler1,2,3,4,5, Anja E Fusco1,2,3,4,5, Anna Eberlein1,2,3,4,5, Nan Li1,2,3,4,5, Remco T A Megens1,2,3,4,5, Ramin Banafsche1,2,3,4,5, Jörg Kumbrink1,2,3,4,5, Christian Weber1,2,3,4,5, Maliheh Nazari-Jahantigh1,2,3,4,5. 1. Institute for Cardiovascular Prevention (A.S., R.M.B., S.S.M., F.Z., M.Y.K., I.M.B., M.Z., C.G., A.E.F., A.E., N.L., R.T.A.M., C.W., M.N.-J.), Ludwig-Maximilians-University, Munich, Germany. 2. Department of Vascular Surgery (A.E.W., R.B.), Ludwig-Maximilians-University, Munich, Germany. 3. University Hospital, Institute for Pathology (J.K.), Ludwig-Maximilians-University, Munich, Germany. 4. Deutschen Zentrum für Herz-Kreislauf-Forschung (German Center for Cardiovascular Research), German Centre for Cardiovascular Research, Partner Site Munich Heart Alliance (A.S., C.W., M.N.-J.). 5. Cardiovascular Research Institute Maastricht, Department of Biomedical Engineering, Maastricht University, The Netherlands (R.T.A.M.).
Abstract
BACKGROUND: The necrotic core partly formed by ineffective efferocytosis increases the risk of an atherosclerotic plaque rupture. Microribonucleic acids 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, 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. Genome-wide gene expression and lesion formation were analyzed in bone marrow-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 proapoptotic Xaf1 (XIAP-associated factor 1) in the atherosclerotic aorta, which abolished circadian expression of Xaf1. The antiapoptotic effect of Mir21 was mediated by noncanonical targeting of Xaf1 through both Mir21 strands. Mir21 knockout in bone marrow 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 antiphase 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.
BACKGROUND: The necrotic core partly formed by ineffective efferocytosis increases the risk of an atherosclerotic plaque rupture. Microribonucleic acids 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, 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. Genome-wide gene expression and lesion formation were analyzed in bone marrow-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 proapoptotic Xaf1 (XIAP-associated factor 1) in the atherosclerotic aorta, which abolished circadian expression of Xaf1. The antiapoptotic effect of Mir21 was mediated by noncanonical targeting of Xaf1 through both Mir21 strands. Mir21 knockout in bone marrow 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 antiphase 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.