Angiotensin II-induced miR-31-5p upregulation promotes vascular smooth muscle cell proliferation and migration.

Angiotensin II (Ang II) plays a central role in vascular smooth muscle cell (VSMC) proliferation and migration, being key to regulate vascular function and promote vascular remodeling in cardiovascular diseases. We recently showed that miR-31-5p promoted oxidative stress in spontaneously hypertensive rats. In this study, we aim to investigate whether miR-31-5p and fibronectin type III domain-containing 5 (FNDC5) contribute to Ang II-induced VSMC proliferation and migration. Experiments were performed in primary VSMCs of wide-type (WT) and FNDC5-/- mice as well as the rat A7r5 cell line. We found that Ang II increased miR-31-5p level, reduced FNDC5 expression and stimulated VSMC proliferation and migration, which were aggravated by miR-31-5p mimic, and prevented by miR-31-5p inhibitor in VSMCs. The Ang II-induced VSMC proliferation were prevented by exogenous FNDC5 in both WT and FNDC5-/- mice, while the effects were more significant in FNDC5-/- mice. Furthermore, exogenous FNDC5 reversed the effects of miR-31-5p mimic on VSMC proliferation and migration in Ang II-treated VSMCs. Meanwhile, FNDC5 deficiency prevented the effects of miR-31-5p inhibitor on VSMC proliferation and migration in Ang II-treated VSMCs. In conclusion, our findings demonstrate that the miR-31-5p upregulation and the following FNDC5 downregulation contribute to Ang II-induced VSMC proliferation and migration.

Norepinephrine acting on adventitial fibroblasts stimulates vascular smooth muscle cell proliferation via promoting small extracellular vesicle release.

Excessive sympathetic activity and norepinephrine (NE) release play crucial roles in the pathogeneses of hypertension. Sympathetic fibers innervate adventitia rather than media of arteries. However, the roles of NE in adventitial fibroblasts (AFs) are unknown. This study investigated the roles of NE in regulating AFs-derived extracellular vesicles (EVs) release and vascular smooth muscle cells (VSMCs) proliferation in hypertension. Methods: AFs and VSMCs were prepared from aorta of Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). AFs were treated with NE (10 µM) for 24 h (every 6 h, 4 times), and cultured in exosomes-depleted medium for 48 h. EVs were isolated from AFs medium with ultracentrifugation for identification and transfer to VSMCs. Results: NE promoted AFs phenotypic transformation and proliferation, which were prevented by α-receptor antagonist phentolamine rather than ß-receptor antagonist propranolol. NE-treated AFs conditioned medium stimulated VSMCs proliferation, which was inhibited by either exosome inhibitor GW4869 or phentolamine. NE increased small EVs number, diameter and angiotensin converting enzyme (ACE) contents. The NE-induced EVs release was abolished by GW4869. The EVs from NE-treated AFs stimulated VSMCs proliferation, which was prevented by angiotensin II type 1 receptor antagonist losartan. The EVs from the ACE knockdown-treated AFs showed lower ACE contents, and lost their roles in stimulating VSMCs proliferation. Conclusion: NE promotes AFs-derived small EVs release and ACE transfer, and then causes VSMCs proliferation in hypertension. Intervention of AFs-derived EVs release may be potential therapeutics for excessive sympathetic activation-related vascular remodeling in hypertension.

Chronic infusion of ELABELA alleviates vascular remodeling in spontaneously hypertensive rats via anti-inflammatory, anti-oxidative and anti-proliferative effects.

Inflammatory activation and oxidative stress promote the proliferation of vascular smooth muscle cells (VSMCs), which accounts for pathological vascular remodeling in hypertension. ELABELA (ELA) is the second endogenous ligand for angiotensin receptor-like 1 (APJ) receptor that has been discovered thus far. In this study, we investigated whether ELA regulated VSMC proliferation and vascular remodeling in spontaneously hypertensive rats (SHRs). We showed that compared to that in Wistar-Kyoto rats (WKYs), ELA expression was markedly decreased in the VSMCs of SHRs. Exogenous ELA-21 significantly inhibited inflammatory cytokines and NADPH oxidase 1 expression, reactive oxygen species production and VSMC proliferation and increased the nuclear translocation of nuclear factor erythroid 2-related factor (Nrf2) in VSMCs. Osmotic minipump infusion of exogenous ELA-21 in SHRs for 4 weeks significantly decreased diastolic blood pressure, alleviated vascular remodeling and ameliorated vascular inflammation and oxidative stress in SHRs. In VSMCs of WKY, angiotensin II (Ang II)-induced inflammatory activation, oxidative stress and VSMC proliferation were attenuated by pretreatment with exogenous ELA-21 but were exacerbated by ELA knockdown. Moreover, ELA-21 inhibited the expression of matrix metalloproteinase 2 and 9 in both SHR-VSMCs and Ang II-treated WKY-VSMCs. We further revealed that exogenous ELA-21-induced inhibition of proliferation and PI3K/Akt signaling were amplified by the PI3K/Akt inhibitor LY294002, while the APJ receptor antagonist F13A abolished ELA-21-induced PI3K/Akt inhibition and Nrf2 activation in VSMCs. In conclusion, we demonstrate that ELA-21 alleviates vascular remodeling through anti-inflammatory, anti-oxidative and anti-proliferative effects in SHRs, indicating that ELA-21 may be a therapeutic agent for treating hypertension.

RND3 attenuates oxidative stress and vascular remodeling in spontaneously hypertensive rat via inhibiting ROCK1 signaling.

Superoxide and vascular smooth muscle cells (VSMCs) migration and proliferation play crucial roles in the vascular remodeling. Vascular remodeling contributes to the development and complications of hypertension. Rho family GTPase 3 (RND3 or RhoE), an atypical small Rho-GTPase, is known to be involved in cancer development and metastasis. However, the roles of RND3 in superoxide production and cardiovascular remodeling are unknown. Here, we uncovered the critical roles of RND3 in attenuating superoxide production, VSMCs migration and proliferation, and vascular remodeling in hypertension and its underline mechanisms. VSMCs were isolated and prepared from thoracic aorta of Male Wistar-Kyoto rat (WKY) and spontaneously hypertensive rat (SHR). RND3 mRNA and protein expressions in arteries and VSMCs were down-regulated in SHR. RND3 overexpression in VSMCs reduced NAD(P)H oxidase (NOX) activity, NOX1 and NOX2 expressions, mitochondria superoxide generation, and H2O2 production in SHR. Moreover, the RND3 overexpression inhibited VSMCs migration and proliferation in SHR, which were similar to the effects of NOX1 inhibitor ML171 plus NOX2 inhibitor GSK2795039. Rho-associated kinase 1 (ROCK1) and RhoA expressions and myosin phosphatase targeting protein 1 (MYPT1) phosphorylation in VSMCs were increased in SHR, which were prevented by RND3 overexpression. ROCK1 overexpression promoted NOX1 and NOX2 expressions, superoxide and H2O2 production, VSMCs migration and proliferation in both WKY and SHR, which were attenuated by RND3 overexpression. Adenoviral-mediated RND3 overexpression in SHR attenuated hypertension, vascular remodeling and oxidative stress. These results indicate that RND3 attenuates VSMCs migration and proliferation, hypertension and vascular remodeling in SHR via inhibiting ROCK1-NOX1/2 and mitochondria superoxide signaling.

Multiparametric MRI analysis for the evaluation of renal function in patients with hyperuricemia: a preliminary study.

BACKGROUND: To investigate the renal dysfunction in patients with hyperuricemia by employing a multiparametric MRI protocol, consisting of quantitative water molecule diffusion, microstructure, microscopic perfusion, and oxygenation measurements in kidneys. MATERIALS AND METHODS: A total of 48 patients with hyperuricemia (HU) and 22 age-matched healthy control subjects (HC) were enrolled in the study. For each participant, three different functional magnetic resonance imaging (fMRI) sequences were acquired and analyzed, including intravoxel incoherent motion imaging (IVIM), diffusion tensor imaging (DTI), and blood-oxygen-level-dependent MRI (BOLD). Thereafter, an independent two-sample t-test was applied to discover the significant differences of MRI indices between the hyperuricemia (HU) and HC groups, and the specific potential biomarkers between two subgroups of HU group (asymptomatic hyperuricemia group (AH) and gouty arthritis group (GA)). Further, multivariate logistic regression analyses were performed to classify the AH from the GA group using the MRI indices with significant between-group differences. The receiver operating characteristic (ROC) curve was plotted, and the area under the ROC curve (AUC) was calculated to assess the performance of each MR index for differentiation between the AH and GA groups. RESULTS: Ten parametric values of the HU group were significantly lower than those of the HC group among the 14 fMRI parameters (P < 0.05). The cortical D, D*, and f values and medullary D and R2*values had significant differences between the AH and GA groups (P < 0.05). Combining the cortical D and f values and medullary R2* value gave the best diagnostic efficacy, yielding an AUC, sensitivity, and specificity of 0.967 ± 0.022, 91.67%, and 95.83%, respectively. CONCLUSIONS: A multiparametric MR analysis plays an important role in the evaluation of renal dysfunction in hyperuricemia from multiple perspectives. It could be a promising method for noninvasive detection and identification of the early-stage renal damage induced by hyperuricemia.

Extracellular vesicle-mediated miR135a-5p transfer in hypertensive rat contributes to vascular smooth muscle cell proliferation via targeting FNDC5.

Background Extracellular vesicles (EVs) from vascular adventitial fibroblasts (AFs) contribute to the proliferation of vascular smooth muscle cells (VSMCs) and vascular remodeling in spontaneously hypertensive rat (SHR). This study shows the crucial roles of EVs-mediated miR135a-5p transfer in VSMC proliferation and the underlying mechanisms in hypertension. Methods AFs and VSMCs were obtained from the aorta of Wistar-Kyoto rat (WKY) and SHR. EVs were isolated from the culture of AFs with ultracentrifugation method. Results MiR135a-5p level in SHR-EVs was significantly increased. MiR135a-5p inhibitor prevented the SHR-EVs-induced VSMC proliferation. Fibronectin type III domain containing 5 (FNDC5) was a target gene of miR135a-5p. FNDC5 level was lower in VSMCs of SHR. MiR135a-5p inhibitor not only increased FNDC5 expression, but reversed the SHR-EVs-induced FNDC5 downregulation in VSMCs of SHR. MiR135a-5p mimic inhibited FNDC5 expression, but failed to promote the SHR-EVs-induced FNDC5 downregulation in VSMCs of SHR. Exogenous FNDC5 prevented the SHR-EVs-induced VSMC proliferation of both WKY and SHR. Knockdown of miR135a-5p in fibroblasts completely prevented the upregulation of miR135a-5p in the EVs. The SHR-EVs from the miR135a-5p knockdown-treated fibroblasts lost their roles in inhibiting FNDC5 expression and promoting proliferation in VSMCs of both WKY and SHR. Conclusions Increased miR135a-5p in the SHR-EVs promoted VSMC proliferation of WKY and SHR via inhibiting FNDC5 expression. MiR135a-5p and FNDC5 are crucial targets for intervention of VSMC proliferation in hypertension.

Exacerbated pressor and sympathoexcitatory effects of central Elabela in spontaneously hypertensive rats.

Elabela (ELA) is a newly discovered peptide that acts as a novel endogenous ligand of angiotensin receptor-like 1 (APJ) receptor. This study was designed to evaluate the effects of ELA-21 in paraventricular nucleus (PVN) on blood pressure and sympathetic nerve activity in spontaneously hypertensive rats (SHR). Experiments were performed in male Wistar-Kyoto rats (WKY) and SHR. ELA expression was upregulated in PVN of SHR. PVN microinjection of ELA-21 increased renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP), heart rate (HR), plasma norepinephrine, and arginine vasopressin (AVP) levels in SHR. Intravenous injection of ELA-21 significantly decreased MAP and HR in both WKY and SHR, but only induced a slight decrease in RSNA. APJ antagonist F13A in PVN abolished the effects of ELA-21 on RSNA, MAP and HR. Intravenous infusion of both ganglionic blocker hexamethonium and AVP V1a receptor antagonist SR49059 caused significant reduction in the effects of ELA-21 on RSNA, MAP and HR in SHR, while combined administration of hexamethonium and SR49059 abolished the effects of ELA-21. ELA-21 microinjection stimulated Akt and p85α subunit of phosphatidylinositol 3-kinase (PI3K) phosphorylation in PVN, whereas PI3K inhibitor LY294002 or Akt inhibitor MK-2206 almost abolished the effects of ELA-21 on RSNA, MAP, and HR. Chronic PVN infusion of ELA-21 induced sympathetic activation, hypertension, and AVP release accompanied with cardiovascular remodeling in normotensive WKY. In conclusion, ELA-21 in PVN induces exacerbated pressor and sympathoexcitatory effects in hypertensive rats via PI3K-Akt pathway.NEW & NOTEWORTHY We demonstrated that PVN microinjection of ELA-21 increases sympathetic nerve activity and blood pressure, which can be abolished by pretreatment of APJ antagonist. This is the first demonstration that central ELA can induce hypertension. The pressor effects in PVN are mediated by both sympathetic activation and vasopressin release via PI3K-Akt pathway. Our data confirm that ELA is upregulated in the PVN of SHR and so may be involved in the pressor and sympathoexcitatory effects in hypertension.

MiR155-5p in adventitial fibroblasts-derived extracellular vesicles inhibits vascular smooth muscle cell proliferation via suppressing angiotensin-converting enzyme expression.

Proliferation of vascular smooth muscle cells (VSMCs) plays crucial roles in vascular remodelling and stiffening in hypertension. Vascular adventitial fibroblasts are a key regulator of vascular wall function and structure. This study is designed to investigate the roles of adventitial fibroblasts-derived extracellular vesicles (EVs) in VSMC proliferation and vascular remodelling in normotensive Wistar-Kyoto rat (WKY) and spontaneously hypertensive rat (SHR), an animal model of human essential hypertension. EVs were isolated from aortic adventitial fibroblasts of WKY (WKY-EVs) and SHR (SHR-EVs). Compared with WKY-EVs, miR155-5p content was reduced, while angiotensin-converting enzyme (ACE) content was increased in SHR-EVs. WKY-EVs inhibited VSMC proliferation of SHR, which was prevented by miR155-5p inhibitor. SHR-EVs promoted VSMC proliferation of both strains, which was enhanced by miR155-5p inhibitor, but abolished by captopril or losartan. Dual luciferase reporter assay showed that ACE was a target gene of miR155-5p. MiR155-5p mimic or overexpression inhibited VSMC proliferation and ACE upregulation of SHR. WKY-EVs reduced ACE mRNA and protein expressions while SHR-EVs only increased ACE protein level in VSMCs of both strains. However, the SHR-EVs-derived from the ACE knockdown-treated adventitial fibroblasts lost the roles in promoting VSMC proliferation and ACE upregulation. Systemic miR155-5p overexpression reduced vascular ACE, angiotensin II and proliferating cell nuclear antigen levels, and attenuated hypertension and vascular remodelling in SHR. Repetitive intravenous injection of SHR-EVs increased blood pressure and vascular ACE contents, and promoted vascular remodelling in both strains, while WKY-EVs reduced vascular ACE contents and attenuated hypertension and vascular remodelling in SHR. We concluded that WKY-EVs-mediated miR155-5p transfer attenuates VSMC proliferation and vascular remodelling in SHR via suppressing ACE expression, while SHR-EVs-mediated ACE transfer promotes VSMC proliferation and vascular remodelling.

Adipose afferent reflex is enhanced by TNFα in paraventricular nucleus through NADPH oxidase-dependent ROS generation in obesity-related hypertensive rats.

BACKGROUND: The adipose afferent reflex (AAR), a sympatho-excitatory reflex, can promote the elevation of sympathetic nerve activity (SNA) and blood pressure (BP). Inflammation in the paraventricular nucleus (PVN) involves sympathetic abnormality in some cardiovascular diseases such as hypertension. This study was designed to explore the effects of tumor necrosis factor alpha (TNFα) in the PVN on the AAR and SNA in rats with obesity-related hypertension (OH) induced by a high-fat diet for 12 weeks. METHODS: Renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were continuously recorded in anesthetized rats, and their responses to capsaicin (CAP) stimulation of the right inguinal white adipose tissue were used to evaluate the AAR. RESULTS: Compared to the control rats, the systolic blood pressure (SBP), plasma norepinephrine (NE, indicating SNA) and TNFα levels, TNFα mRNA and protein levels, reactive oxygen species (ROS) content and NADPH oxidase activity in the PVN were significantly elevated in rats with OH. TNFα in the PVN markedly enhanced sympathoexcitation and AAR. Moreover, the enhancement of AAR caused by TNFα can be significantly strengthened by the pretreatment of diethyldithiocarbamate (DETC), a superoxide dismutase inhibitor, but attenuated by TNF-α receptor antagonist R-7050, superoxide scavenger PEG-SOD and NADPH oxidase inhibitor apocynin (Apo) in rats with OH. Acute microinjection of TNF-α into the PVN significantly increased the activity of NADPH oxidase and ROS levels in rats with OH, which were effectively blocked by R-7050. Furthermore, our results also showed that the increased levels of ROS, TNFα and NADPH oxidase subunits mRNA and protein in the PVN of rats with OH were significantly reversed by pentoxifylline (PTX, 30 mg/kg daily ip; in 10% ethanol) application, a cytokine blocker, for a period of 5 weeks. PTX administration also significantly decreased SBP, AAR and plasma NE levels in rats with OH. CONCLUSIONS: TNFα in the PVN modulates AAR and contributes to sympathoexcitation in OH possibly through NADPH oxidase-dependent ROS generation. TNFα blockade attenuates AAR and sympathoexcitation that unveils TNFα in the PVN may be a possible therapeutic target for the intervention of OH.

FNDC5 attenuates obesity-induced cardiac hypertrophy by inactivating JAK2/STAT3-associated inflammation and oxidative stress.

BACKGROUND: Chronic low-grade inflammation and oxidative stress play important roles in the development of obesity-induced cardiac hypertrophy. Here, we investigated the role of Fibronectin type III domain containing 5 (FNDC5) in cardiac inflammation and oxidative stress in obesity-induced cardiac hypertrophy. METHODS: Male wild-type and FNDC5-/- mice were fed normal chow or high fat diet (HFD) for 20 weeks to induce obesity, and primary cardiomyocytes and H9c2 cells treated with palmitate (PA) were used as in vitro model. The therapeutic effects of lentiviral vector-mediated FNDC5 overexpression were also examined in HFD-induced cardiac hypertrophy. RESULTS: High fat diet manifested significant increases in body weight and cardiac hypertrophy marker genes expression, while FNDC5 deficiency aggravated cardiac hypertrophy evidenced by increased Nppa, Nppb and Myh7 mRNA level and cardiomyocytes area, in association with enhanced cardiac inflammatory cytokines expression, oxidative stress level and JAK2/STAT3 activation in HFD-fed mice. FNDC5 deficiency in primary cardiomyocytes or FNDC5 knockdown in H9c2 cells enhanced PA-induced inflammatory responses and NOX4 expression. Exogenous FNDC5 pretreatment attenuated PA-induced cardiomyocytes hypertrophy, inflammatory cytokines up-regulation and oxidative stress in primary cardiomyocytes and H9c2 cells. FNDC5 overexpression attenuated cardiac hypertrophy as well as cardiac inflammation and oxidative stress in HFD-fed mice. CONCLUSIONS: FNDC5 attenuates obesity-induced cardiac hypertrophy by inactivating JAK2/STAT3 associated-cardiac inflammation and oxidative stress. The cardio-protective role of FNDC5 shed light on future therapeutic interventions in obesity and related cardiovascular complications.

Exosome-Mediated Transfer of ACE (Angiotensin-Converting Enzyme) From Adventitial Fibroblasts of Spontaneously Hypertensive Rats Promotes Vascular Smooth Muscle Cell Migration.

Migration of vascular smooth muscle cells (VSMCs) is pivotal for vascular remodeling in hypertension. Vascular adventitial fibroblasts (AFs) are important in the homeostasis of vascular structure. This study is designed to investigate the roles of AF exosomes (AFE) in VSMC migration and underling mechanism. Primary VSMCs and AFs were obtained from the aorta of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. VSMC migration was evaluated with Boyden chamber assay and wound healing assay. AFE from WKY rats and SHR were isolated and identified. AFE from SHR promoted but AFE from WKY rats had no significant effect on VSMC migration. The effects of AFE on VSMC migration were prevented by an exosome inhibitor GW4869, an AT1R (Ang II [angiotensin II] type 1 receptor) antagonist losartan, or an inhibitor of ACE (angiotensin-converting enzyme) captopril. ACE contents and activity were much higher in AFE from SHR than those from WKY rats. There were no significant difference in Ang II and AT1R mRNA and protein levels between AFE from SHR and AFE from WKY rats. AFE from SHR increased Ang II and ACE contents and ACE activity in VSMCs of WKY rats and SHR. The changes of Ang II contents and ACE activity were prevented by captopril. ACE knockdown in AFs reduced ACE contents and activity in AFE from SHR and inhibited AFE-induced migration of VSMCs of WKY rats and those of SHR. These results indicate that exosomes from AFs of SHR transfer ACE to VSMCs, which increases Ang II levels and activates AT1R in VSMCs and thereby promotes VSMC migration.

FNDC5 attenuates adipose tissue inflammation and insulin resistance via AMPK-mediated macrophage polarization in obesity.

BACKGROUND: Obesity-induced chronic inflammation is critical in the pathogenesis of insulin resistance, and the recruitment and proinflammatory activation of adipose tissue macrophages (ATMs) is important for the development of this process. Here, we examined the effects of fibronectin type III domain-containing 5 (FNDC5) on inflammation and insulin resistance in high-fat diet-induced obese mice. MATERIALS AND METHODS: Male wild-type (WT) and FNDC5-/- mice were fed with standard chow (Ctrl) or high fat diet (HFD) for 20 weeks to induce obesity and insulin resistance. Firstly, effects of FNDC5 gene deletion on obesity, insulin resistance, macrophage accumulation and polarization and adipose tissue inflammation were determined in mice. Secondly, the macrophage polarity shift was further examined with flow cytometry in isolated stromal vascular fraction (SVF). Thirdly, the effects of exogenous FNDC5 on lipopolysaccharide (LPS)-induced macrophage polarization, inflammation and the underlying signaling mechanism were investigated in RAW264.7 macrophages and primary mouse peritoneal cavity macrophages (PMs). Finally, the therapeutic effects of FNDC5 overexpression were examined in HFD-induced obese WT and FNDC5-/- mice. RESULTS: FNDC5 gene deletion aggravated obesity, insulin resistance, fat accumulation and inflammation accompanied with enhanced AMPK inhibition, macrophages recruitment and M1 polarization in mice fed with HFD. Exogenous FNDC5 inhibited LPS-induced M1 macrophage polarization and inflammatory cytokine production via AMPK phosphorylation in both RAW264.7 macrophages and PMs. FNDC5 overexpression attenuated insulin resistance, AMPK inhibition, M1 macrophage polarization and inflammatory cytokine production in adipose tissue of obese WT and FNDC5-/- mice. CONCLUSIONS: FNDC5 attenuates adipose tissue inflammation and insulin resistance via AMPK-mediated macrophage polarization in HFD-induced obesity. FNDC5 plays several beneficial roles in obesity and may be used as a therapeutic regimen for preventing inflammation and insulin resistance in obesity and diabetes.

NLRP3 inflammasome activation contributes to VSMC phenotypic transformation and proliferation in hypertension.

Inflammation is involved in pathogenesis of hypertension. NLRP3 inflammasome activation is a powerful mediator of inflammatory response via caspase-1 activation. The present study was designed to determine the roles and mechanisms of NLRP3 inflammasome in phenotypic modulation and proliferation of vascular smooth muscle cells (VSMCs) in hypertension. Experiments were conducted in spontaneously hypertensive rats (SHR) and primary aortic VSMCs. NLRP3 inflammasome activation was observed in the media of aorta in SHR and in the VSMCs from SHR. Knockdown of NLRP3 inhibited inflammasome activation, VSMC phenotypic transformation and proliferation in SHR-derived VSMCs. Increased NFκB activation, histone acetylation and histone acetyltransferase expression were observed in SHR-derived VSMCs and in media of aorta in SHR. Chromatin immunoprecipitation analysis revealed the increased histone acetylation, p65-NFκB and Pol II occupancy at the NLRP3 promoter in vivo and in vitro. Inhibition of NFκB with BAY11-7082 or inhibition of histone acetyltransferase with curcumin prevented the NLRP3 inflammasome activation, VSMC phenotype switching and proliferation in VSMCs from SHR. Moreover, curcumin repressed NFκB activation. Silencing of NLRP3 gene ameliorated hypertension, vascular remodeling, NLRP3 inflammasome activation and phenotype switching in the aorta of SHR. These results indicate that NLRP3 inflammasome activation response to histone acetylation and NFκB activation contributes to VSMC phenotype switching and proliferation and vascular remodeling in hypertension.

BCL6 attenuates renal inflammation via negative regulation of NLRP3 transcription.

Renal inflammation contributes to the pathogeneses of hypertension. This study was designed to determine whether B-cell lymphoma 6 (BCL6) attenuates renal NLRP3 inflammasome activation and inflammation and its underlying mechanism. Male spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were used in the present study. Angiotensin (Ang) II or lipopolysaccharides (LPS) was used to induce inflammation in HK-2 cells, a human renal tubular epithelial (RTE) cell line. NLRP3 inflammasome was activated and BCL6 was downregulated in the kidneys of SHR. Either Ang II or LPS suppressed BCL6 expression in HK-2 cells. BCL6 overexpression in HK-2 cells attenuated Ang II-induced NLRP3 upregulation, inflammation and cell injury. The inhibitory effects of BCL6 overexpression on NLRP3 expression and inflammation were also observed in LPS-treated HK-2 cells. BCL6 inhibited the NLRP3 transcription via binding to the NLRP3 promoter. BCL6 knockdown with shRNA increased NLRP3 and mature IL-1ß expression levels in both PBS- or Ang II-treated HK-2 cells but had no significant effects on ASC, pro-caspase-1 and pro-IL-1ß expression levels. BCL6 overexpression caused by recombinant lentivirus expressing BCL6 reduced blood pressure in SHR. BCL6 overexpression prevented the upregulation of NLRP3 and mature IL-1ß expression levels in the renal cortex of SHR. The results indicate that BCL6 attenuates Ang II- or LPS-induced inflammation in HK-2 cells via negative regulation of NLRP3 transcription. BCL6 overexpression in SHR reduced blood pressure, NLRP3 expression and inflammation in the renal cortex of SHR.

Salusin-ß contributes to oxidative stress and inflammation in diabetic cardiomyopathy.

Salusin-ß accelerates inflammatory responses in vascular endothelial cells, and increases oxidative stress in vascular smooth muscle cells. Plasma salusin-ß levels were increased in diabetic patients. This study was designed to determine whether salusin-ß is involved in the pathogenesis of diabetic cardiomyopathy (DCM), and whether knockdown of salusin-ß attenuates cardiac inflammation and oxidative stress in rats with DCM. H9c2 or neonatal rat cardiomyocytes were incubated with 33.3 mM of glucose to mimic the high glucose (HG) in diabetes. Streptozotocin and high-fat diet were used to induce type 2 diabetes in rats. HG induced salusin-ß expression in H9c2 cells. Salusin-ß caused greater responses of oxidative stress, NFκB activation and inflammation in HG-treated H9c2 cells than these in control H9c2 cells. Diphenyleneiodonium (a NAD(P)H oxidase inhibitor) or N-acetylcysteine (an antioxidant) inhibited the salusin-ß-induced NFκB activation and inflammation. Bay11-7082 (a NFκB inhibitor) attenuated salusin-ß-induced inflammation but not oxidative stress. Knockdown of salusin-ß prevented the HG-induced oxidative stress, NFκB activation and inflammation in neonatal rat cardiomyocytes. Silencing salusin-ß with adenoviruse-mediated shRNA had no significant effects on blood glucose and insulin resistance, but attenuated ventricular dysfunction in diabetic rats. Oxidative stress, NFκB activation, inflammation, salusin-ß upregulation in myocardium of diabetic rats were prevented by knockdown of salusin-ß. These results indicate that salusin-ß contributes to inflammation in DCM via NOX2/ROS/NFκB signaling, and that knockdown of salusin-ß attenuates cardiac dysfunction, oxidative stress and inflammation in DCM.

FNDC5 Alleviates Hepatosteatosis by Restoring AMPK/mTOR-Mediated Autophagy, Fatty Acid Oxidation, and Lipogenesis in Mice.

Fibronectin type III domain-containing 5 (FNDC5) protein induces browning of subcutaneous fat and mediates the beneficial effects of exercise on metabolism. However, whether FNDC5 is associated with hepatic steatosis, autophagy, fatty acid oxidation (FAO), and lipogenesis remains unknown. Herein, we show the roles and mechanisms of FNDC5 in hepatic steatosis, autophagy, and lipid metabolism. Fasted FNDC5-/- mice exhibited severe steatosis, reduced autophagy, and FAO, and enhanced lipogenesis in the liver compared with wild-type mice. Energy deprivation-induced autophagy, FAO, and AMPK activity were attenuated in FNDC5-/- hepatocytes, which were restored by activating AMPK with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). Inhibition of mammalian target of rapamycin (mTOR) complex 1 with rapamycin enhanced autophagy and FAO and attenuated lipogenesis and steatosis in FNDC5-/- livers. FNDC5 deficiency exacerbated hyperlipemia, hepatic FAO and autophagy impairment, hepatic lipogenesis, and lipid accumulation in obese mice. Exogenous FNDC5 stimulated autophagy and FAO gene expression in hepatocytes and repaired the attenuated autophagy and palmitate-induced steatosis in FNDC5-/- hepatocytes. FNDC5 overexpression prevented hyperlipemia, hepatic FAO and autophagy impairment, hepatic lipogenesis, and lipid accumulation in obese mice. These results indicate that FNDC5 deficiency impairs autophagy and FAO and enhances lipogenesis via the AMPK/mTOR pathway. FNDC5 deficiency aggravates whereas FNDC5 overexpression prevents the HFD-induced hyperlipemia, hepatic lipid accumulation, and impaired FAO and autophagy in the liver.

ß-aminoisobutyric acid attenuates hepatic endoplasmic reticulum stress and glucose/lipid metabolic disturbance in mice with type 2 diabetes.

ß-aminoisobutyric acid (BAIBA) is a nature thymine catabolite, and contributes to exercise-induced protection from metabolic diseases. Here we show the therapeutical effects of BAIBA on hepatic endoplasmic reticulum (ER) stress and glucose/lipid metabolic disturbance in diabetes. Type 2 diabetes was induced by combined streptozotocin (STZ) and high-fat diet (HFD) in mice. Oral administration of BAIBA for 4 weeks reduced blood glucose and lipids levels, hepatic key enzymes of gluconeogenesis and lipogenesis expressions, attenuated hepatic insulin resistance and lipid accumulation, and improved insulin signaling in type 2 diabetic mice. BAIBA reduced hepatic ER stress and apoptosis in type 2 diabetic mice. Furthermore, BAIBA alleviated ER stress in human hepatocellular carcinoma (HepG2) cells with glucosamine-induced insulin resistance. Hepatic AMPK phosphorylation was reduced in STZ/HFD mice and glucosamine-treated HepG2 cells, which were restored by BAIBA treatment. The suppressive effects of BAIBA on glucosamine-induced ER stress were reversed by knockdown of AMPK with siRNA. In addition, BAIBA prevented thapsigargin- or tunicamycin-induced ER stress, and tunicamycin-induced apoptosis in HepG2 cells. These results indicate that BAIBA attenuates hepatic ER stress, apoptosis and glucose/lipid metabolic disturbance in mice with type 2 diabetes. AMPK signaling is involved to the role of BAIBA in attenuating ER stress.

GABA in Paraventricular Nucleus Regulates Adipose Afferent Reflex in Rats.

BACKGROUND: Chemical stimulation of white adipose tissue (WAT) induces adipose afferent reflex (AAR), and thereby causes a general sympathetic activation. Paraventricular nucleus (PVN) is important in control of sympathetic outflow. This study was designed to investigate the role of γ-aminobutyric acid (GABA) in PVN in regulating the AAR. METHODOLOGY/PRINCIPAL FINDINGS: Experiments were carried out in anesthetized rats. Renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were continuously recorded. AAR was evaluated by the RSNA and MAP responses to electrical stimulation of the right epididymal WAT (eWAT) afferent nerve. Electrical stimulation of eWAT afferent nerve increase RSNA. Bilateral microinjection of the GABAA receptor agonist isoguvacine or the GABAB receptor agonist baclofen attenuated the AAR. The effect of isoguvacine on the AAR was greater than that of baclofen. The GABAA receptor antagonist gabazine enhanced the AAR, while the GABAB receptor antagonist CGP-35348 had no significant effect on the AAR. Bilateral PVN microinjection of vigabatrin, a selective GABA-transaminase inhibitor, to increase endogenous GABA levels in the PVN abolished the AAR. The inhibitory effect of vigabatrin on the AAR was attenuated by the pretreatment with gabazine or CGP-35348. Pretreatment with combined gabazine and CGP-35348 abolished the effects of vigabatrin. CONCLUSIONS: Activation of GABAA or GABAB receptors in the PVN inhibits the AAR. Blockade of GABAA receptors in the PVN enhances the AAR. Endogenous GABA in the PVN plays an important role in regulating the AAR.

Irisin inhibits hepatic gluconeogenesis and increases glycogen synthesis via the PI3K/Akt pathway in type 2 diabetic mice and hepatocytes.

Increased glucose production and reduced hepatic glycogen storage contribute to metabolic abnormalities in diabetes. Irisin, a newly identified myokine, induces the browning of white adipose tissue, but its effects on gluconeogenesis and glycogenesis are unknown. In the present study, we investigated the effects and underlying mechanisms of irisin on gluconeogenesis and glycogenesis in hepatocytes with insulin resistance, and its therapeutic role in type 2 diabetic mice. Insulin resistance was induced by glucosamine (GlcN) or palmitate in human hepatocellular carcinoma (HepG2) cells and mouse primary hepatocytes. Type 2 diabetes was induced by streptozotocin/high-fat diet (STZ/HFD) in mice. In HepG2 cells, irisin ameliorated the GlcN-induced increases in glucose production, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) expression, and glycogen synthase (GS) phosphorylation; it prevented GlcN-induced decreases in glycogen content and the phosphoinositide 3-kinase (PI3K) p110α subunit level, and the phosphorylation of Akt/protein kinase B, forkhead box transcription factor O1 (FOXO1) and glycogen synthase kinase-3 (GSK3). These effects of irisin were abolished by the inhibition of PI3K or Akt. The effects of irisin were confirmed in mouse primary hepatocytes with GlcN-induced insulin resistance and in human HepG2 cells with palmitate-induced insulin resistance. In diabetic mice, persistent subcutaneous perfusion of irisin improved the insulin sensitivity, reduced fasting blood glucose, increased GSK3 and Akt phosphorylation, glycogen content and irisin level, and suppressed GS phosphorylation and PEPCK and G6Pase expression in the liver. Irisin improves glucose homoeostasis by reducing gluconeogenesis via PI3K/Akt/FOXO1-mediated PEPCK and G6Pase down-regulation and increasing glycogenesis via PI3K/Akt/GSK3-mediated GS activation. Irisin may be regarded as a novel therapeutic strategy for insulin resistance and type 2 diabetes.

FNDC5 overexpression and irisin ameliorate glucose/lipid metabolic derangements and enhance lipolysis in obesity.

Irisin is a cleaved and secreted fragment of fibronectin type III domain containing 5 (FNDC5), and contributes to the beneficial effects of exercise on metabolism. Here we report the therapeutical effects of FNDC5/irisin on metabolic derangements and insulin resistance in obesity, and show the lipolysis effect of irisin and its signal molecular mechanism. In obese mice, lentivirus mediated-FNDC5 overexpression enhanced energy expenditure, lipolysis and insulin sensitivity, and reduced hyperlipidemia, hyperglycemia, hyperinsulinism, blood pressure and norepinephrine levels; it increased hormone-sensitive lipase (HSL) expression and phosphorylation, and reduced perilipin level and adipocyte diameter in adipose tissues. Subcutaneous perfusion of irisin reduced hyperlipidemia and hyperglycemia, and improved insulin resistance. Either FNDC5 overexpression or irisin perfusion only induced a tendency toward a slight decrease in body weight in obese mice. In 3T3-L1 adipocytes, irisin enhanced basal lipolysis rather than isoproterenol-induced lipolysis, which were prevented by inhibition of adenylate cyclase or PKA; irisin increased the HSL and perilipin phosphorylation; it increased PKA activity, and cAMP and HSL mRNA levels, but reduced perilipin expression. These results indicate that FNDC5/irisin ameliorates glucose/lipid metabolic derangements and insulin resistance in obese mice, and enhances lipolysis via cAMP-PKA-HSL/perilipin pathway. FNDC5 or irisin can be taken as an effective therapeutic strategy for metabolic disorders.