Melatonin stabilizes rupture‐prone vulnerable plaques via regulating macrophage polarization in a nuclear circadian receptor RORα‐dependent manner

Abstract

Rupture of vulnerable plaques is the main trigger of acute cardio-cerebral vascular events, but mechanisms responsible for transforming a stable atherosclerotic into a vulnerable plaque remain largely unknown. Melatonin, an indoleamine hormone secreted by the pineal gland, plays pleiotropic roles in the cardiovascular system; however, the effect of melatonin on vulnerable plaque rupture and its underlying mechanisms remains unknown. Here, we generated a rupture-prone vulnerable carotid plaque model induced by endogenous renovascular hypertension combined with low shear stress in hypercholesterolemic ApoE −/− mice. Melatonin (10 mg/kg/d by oral administration for 9 weeks) significantly prevented vulnerable plaque rupture, with lower incidence of intraplaque hemorrhage (42.9% vs. 9.5%, P = 0.014) and of spontaneous plaque rupture with intraluminal thrombus formation (38.1% vs. 9.5%, P = 0.029). Mechanistic studies indicated that melatonin ameliorated intraplaque inflammation by suppressing the differentiation of intraplaque macrophages toward the proinflammatory M1 phenotype, and circadian nuclear receptor retinoid acid receptor-related orphan receptor-α (RORα) mediated melatonin-exerted vasoprotection against vulnerable plaque instability and intraplaque macrophage polarization. Further analysis in human monocyte-derived macrophages confirmed the role of melatonin in regulating macrophage polarization by regulating the AMPKα-STATs pathway in a RORα-dependent manner. In summary, our data provided the first evidence that melatonin-RORα axis acts as a novel endogenous protective signaling pathway in the vasculature, regulates intraplaque inflammation, and stabilizes rupture-prone vulnerable plaques. K E Y W O R D S atherosclerosis, macrophage polarization, melatonin, nuclear receptor, RAR-related orphan receptor, vulnerable plaques 2 of 15 | DING et al. 12 of 15 | DING et al. F I G U R E 7 Melatonin regulates macrophage polarization through the signal transducer and activator of transcription (STAT) pathway.A, Flow cytometry analysis of cell surface markers for M1 (CD80 and CD197) and M2 (CD163 and CD206) macrophages during M1 and M2 polarization with vehicle or melatonin/SR3335 treatment. The results showed that co-culture with melatonin significantly reduced IFN-γ and LPSinduced CD80 and CD197 expression, whereas RORα agonism with SR3335 markedly promoted M1 polarization (n = 6 per group). *P < 0.05 or **P < 0.01 versus monocyte; # P < 0.05 versus M0. B and C, Western blotting was conducted on cell lysates with antibodies against P-STAT1, P-STAT3, and P-STAT6; total STAT1, STAT3, and STAT6; and GAPDH. Relative densities of phosphorylated STAT compared with total STAT are shown as histograms (n = 6 per group). *P < 0.05 or **P < 0.01 versus control; ##P < 0.01 versus M1 + siRORα in C. D and E, Western blotting was conducted on cell lysates with antibodies against RORα, P-AMPKα, total AMPKα, and GAPDH. *P < 0.05 versus Mel (0 mmol/L); ##P < 0.01 versus control siRNA

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