Circadian rhythm disruption or decrease in levels of circadian hormones such as melatonin increases ischemic heart disease risk. The nuclear melatonin receptors RORs are pivotally involved in circadian rhythm regulation and melatonin effects mediation. However, the functional roles of RORs in the heart have never been investigated and were therefore the subject of this study on myocardial ischemia/reperfusion (MI/R) injury pathogenesis. RORα and RORγ subtypes were detected in the adult mouse heart, and RORα but not RORγ was downregulated after MI/R. To determine the pathological consequence of MI/R-induced reduction of RORα, we subjected RORα-deficient staggerer mice and wild-type (WT) littermates to MI/R injury, resulting in significantly increased myocardial infarct size, myocardial apoptosis and exacerbated contractile dysfunction in the former. Mechanistically, RORα deficiency promoted MI/R-induced endoplasmic reticulum stress, mitochondrial impairments, and autophagy dysfunction. Moreover, RORα deficiency augmented MI/R-induced oxidative/nitrative stress. Given the emerging evidence of RORα as an essential melatonin effects mediator, we further investigated the RORα roles in melatonin-exerted cardioprotection, in particular against MI/R injury, which was significantly attenuated in RORα-deficient mice, but negligibly affected by cardiac-specific silencing of RORγ. Finally, to determine cell type-specific effects of RORα, we generated mice with cardiomyocyte-specific RORα overexpression and they were less vulnerable to MI/R injury. In summary, our study provides the first direct evidence that the nuclear melatonin receptor RORα is a novel endogenous protective receptor against MI/R injury and an important mediator of melatonin-exerted cardioprotection; melatonin-RORα axis signaling thus appears important in protection against ischemic heart injury.

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

Diabetic cardiomyopathy is a major complication that significantly contributes to morbidity and mortality in diabetics with few therapies. Moreover, antidiabetic drugs reported inconsistent or even adverse cardiovascular effects, suggesting that it is important to exploit novel therapeutic targets against diabetic cardiomyopathy. Here, we observed that the nuclear melatonin receptor, the retinoic acid-related orphan receptor-α (RORα), was downregulated in diabetic hearts. By utilizing a mouse line with RORα disruption, we demonstrated that RORα deficiency led to significantly augmented diastolic dysfunction and cardiac remodeling induced by diabetes. Microscopic and molecular analyses further indicated that the detrimental effects of RORα deficiency were associated with aggravated myocardial apoptosis, autophagy dysfunction, and oxidative stress by disrupting antioxidant gene expression. By contrast, restoration of cardiac RORα levels in transgenic mice significantly improved cardiac functional and structural parameters at 8 weeks after diabetes induction. Consistent with genetic manipulation, pharmacological activation of RORα by melatonin and SR1078 (a synthetic agonist) showed beneficial effects against diabetic cardiomyopathy, while the RORα inhibitor SR3335 significantly exacerbated cardiac impairments in diabetic mice. Collectively, these findings suggest that cardiac-targeted manipulation of nuclear melatonin receptor RORα may hold promise for delaying diabetic cardiomyopathy development.

Context:Both polycystic ovary syndrome (PCOS) and obesity are associated with specific reproductive health complications, including lower oocyte quality and clinical pregnancy rates in assisted conception cycles, which may be a result of metabolism-induced changes in the oocyte through the microenvironment of follicular fluid. Free fatty acids (FFAs) are important biomedical indicators of abnormal lipid metabolism and have pronounced effects on cells, leading to changes in metabolism, cell growth, and differentiationObjective:Our objective was to determine the effect of FFA metabolism in plasma and follicular fluid on oocyte quality in the women with PCOS undergoing in vitro fertilization.Design and Setting:Ninety-three women undergoing in vitro fertilization treatment, including 55 with PCOS and 38 age-matched controls, were recruited. PCOS patients were divided into obese and nonobese subgroups on the basis of their body mass index.Main Outcome Measures:Embryo quality was morphologically assessed, and serum sex hormone and insulin levels were measured. FFAs in plasma and follicular fluid were measured using gas chromatography-mass spectrometry.Results:PCOS was found to be associated with significantly higher LH/FSH, total T, free androgen index (FAI), and lower SHBG levels, independent of obesity(P < .05). Obese women with PCOS had a significantly higher total T level, FAI, fasting insulin, insulin resistance index as determined by homeostasis model assessment for insulin resistance, and lower SHBG levels than the nonobese women with PCOS (P < .05). The embryo fragmentation score was significantly positively correlated with the oleic acid concentration in all PCOS patients (r = 0.22, P = .04, for nonobese patients and r = 0.25, P = .03, for obese patients).Conclusions:Our findings clearly demonstrated that PCOS is associated with significantly higher FAI and insulin resistance levels and decreased plasma SHBG levels, independent of body mass index. Obese PCOS patients had higher palmitoleic acid and oleic acid levels in both the plasma and follicular fluid than did the control subject and nonobese PCOS patients. Our results indicated that developmental competence is associated with oleic and stearic acid concentrations, which may contribute to the poor pregnancy outcomes in patients with PCOS.

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Nonalcoholic fatty liver disease (NAFLD), characterized by hepatic steatosis, insulin resistance and inflammation, poses a high risk of cardiometabolic disorders. Ubiquitin specific protease 4 (USP4), a deubiquitinating enzyme, is pivotally involved in regulating multiple inflammatory pathways; however, the role of USP4 in NAFLD is unknown. Here we report that USP4 expression was dramatically downregulated in the livers from NAFLD patients and different NAFLD mouse models induced by a high fat diet (HFD) or a genetic deficiency (ob/ob) as well as in palmitate-treated hepatocytes. Hepatocyte-specific USP4 depletion exacerbated hepatic steatosis, insulin resistance and inflammatory response in HFD-induced NAFLD mice. Conversely, hepatic USP4 overexpression notably alleviated the pathological alterations in two different NAFLD models. Mechanistically, hepatocyte USP4 directly bound to and deubiquitinated transforming growth factor-β activated kinase 1 (TAK1), leading to a suppression of the activation of downstream NF-κB and JNK cascades, which in turn reversed the disruption of the IRS-AKT-GSK3β signaling. In addition, USP4-TAK1 interaction and subsequent TAK1 deubiquitination were required for the amelioration of metabolic dysfunctions. Collectively, the present study provides the first evidence that USP4 functions as a pivotal suppressor in NAFLD and related metabolic disorders. This article is protected by copyright. All rights reserved.

Ischemic stroke, caused by obstruction of blood flow to the brain, would initiate microglia activation which contributes to neuronal damage. Therefore, inhibition of microglia-mediated neuroinflammation could be a therapeutic strategy for ischemic stroke. This study was aimed to elucidate the anti-inflammatory effects of alpha-lipoic acid and etanercept given either singly or in combination in rats subjected to middle cerebral artery occlusion. Both α-lipoic acid and etanercept markedly reduced cerebral infarct, blood-brain barrier disruption, and neurological motor deficits with the former drug being more effective with the dosage used. Furthermore, when used in combination, the reduction was more substantial. Remarkably, a greater diminution in the serum levels of tumor necrosis factor-alpha as well as the brain levels of microglial activation (e.g., microgliosis, amoeboid microglia, and microglial overexpression of tumor necrosis factor-α) was observed with the combined drug treatment as compared to the drugs given separately. We conclude that inhibition of peripheral tumor necrosis factor-alpha as well as downregulation of brain microglial activation by alpha-lipoic acid or etanercept protect rat brain against ischemic stroke. Moreover, when both drugs were used in combination, the stroke recovery was promoted more extensively.

Abstract-Dysregulation of the ubiquitin proteasome system components ubiquitin ligases and proteasome plays an important role in the pathogenesis of cardiac hypertrophy. However, little is known about the role of another ubiquitin proteasome system component, the deubiquitinating enzymes, in cardiac hypertrophy. Here, we revealed a crucial role of ubiquitin specific protease 4 (USP4), a deubiquitinating enzyme prominently expressed in the heart, in attenuating pathological cardiac hypertrophy and dysfunction. USP4 levels were consistently decreased in human failing hearts and in murine hypertrophied hearts. Adenovirus-mediated gain-and loss-of-function approaches indicated that deficiency of endogenous USP4 promoted myocyte hypertrophy induced by angiotensin II in vitro, whereas restoration of USP4 significantly attenuated the prohypertrophic effect of angiotensin II. To corroborate the role of USP4 in vivo, we generated USP4 global knockout mice and mice with cardiac-specific overexpression of USP4. Consistent with the in vitro study, USP4 depletion exacerbated the hypertrophic phenotype and cardiac dysfunction in mice subjected to pressure overload, whereas USP4 transgenic mice presented ameliorated pathological cardiac hypertrophy compared with their control littermates. Molecular analysis revealed that USP4 deficiency augmented the activation of the transforming growth factor β-activated kinase 1 (TAK1)-(JNK1/2)/P38 signaling in response to hypertrophic stress, and blockage of TAK1 activation abolished the pathological effects of USP4 deficiency in vivo. These findings provide the first evidence for the involvement of USP4 in cardiac hypertrophy, and shed light on the therapeutic potential of targeting USP4 in the treatment of cardiac hypertrophy. promotes ionizing radiation-induced cell apoptosis by specifically reducing p53 expression. 13 Of note, USP4 is a putative proto-oncogene, which promotes epithelial to mesenchymal transition and breast cancer cell migration. 18 Interestingly, mounting evidence supports pivotal roles of proto-oncogenes in modulating cardiac hypertrophy, 19,20 indicating a potential involvement of USP4 in cardiac hypertrophy. We therefore posited that USP4, a DUB prominently expressed in the heart, might be a potential signaling regulator implicated in cardiac hypertrophy.In this study, we observed reduced levels of USP4 in hearts from patients with dilated cardiomyopathy and in animal models of cardiac hypertrophy induced by pressure overload. By subjecting USP4 knockout mice and transgenic mice with cardiac-specific USP4 overexpression to aortic banding (AB), we observed that USP4 deficiency aggravated hypertrophic growth and cardiac dysfunction. Conversely, restoration of USP4 level remarkably protected the heart against pathological hypertrophy. Mechanistically, the beneficial effect of USP4 was largely dependent on the blockade of the transforming growth factor β-activated kinase 1 (TAK1)-(JNK1/2)/P38 signaling. Materials and MethodsThe animal protocol was approved by the Instit...