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.

Intermedin in Paraventricular Nucleus Attenuates Sympathoexcitation and Decreases TLR4-Mediated Sympathetic Activation via Adrenomedullin Receptors in Rats with Obesity-Related Hypertension.

Intermedin/adrenomedullin-2 (IMD/AM2), a member of the calcitonin gene-related peptide/AM family, plays an important role in protecting the cardiovascular system. However, its role in the enhanced sympathoexcitation in obesity-related hypertension is unknown. In this study, we investigated the effects of IMD in the paraventricular nucleus (PVN) of the hypothalamus on sympathetic nerve activity (SNA), and lipopolysaccharide (LPS)-induced sympathetic activation in obesity-related hypertensive (OH) rats induced by a high-fat diet for 12 weeks. Acute experiments were performed under anesthesia. The dynamic alterations of sympathetic outflow were evaluated as changes in renal SNA and mean arterial pressure (MAP) in response to specific drugs. Male rats were fed a control diet (12% kcal as fat) or a high-fat diet (42% kcal as fat) for 12 weeks to induce OH. The results showed that IMD protein in the PVN was downregulated, but Toll-like receptor 4 (TLR4) and plasma norepinephrine (NE, indicating sympathetic hyperactivity) levels, and systolic blood pressure were increased in OH rats. LPS (0.5 µg/50 nL)-induced enhancement of renal SNA and MAP was greater in OH rats than in obese or control rats. Bilateral PVN microinjection of IMD (50 pmol) caused greater decreases in renal SNA and MAP in OH rats than in control rats, and inhibited LPS-induced sympathetic activation, and these were effectively prevented in OH rats by pretreatment with the AM receptor antagonist AM22-52. The mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) inhibitor U0126 in the PVN partially reversed the LPS-induced enhancement of SNA. However, IMD in the PVN decreased the LPS-induced ERK activation, which was also effectively prevented by AM22-52. Chronic IMD administration resulted in significant reductions in the plasma NE level and blood pressure in OH rats. Moreover, IMD lowered the TLR4 protein expression and ERK activation in the PVN, and decreased the LPS-induced sympathetic overactivity. These results indicate that IMD in the PVN attenuates SNA and hypertension, and decreases the ERK activation implicated in the LPS-induced enhancement of SNA in OH rats, and this is mediated by AM receptors.

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.

Fibronectin type III domain containing 5 attenuates NLRP3 inflammasome activation and phenotypic transformation of adventitial fibroblasts in spontaneously hypertensive rats.

OBJECTIVE: Phenotypic transformation of adventitial fibroblasts is important in the pathogenesis of hypertension. This study was designed to determine whether fibronectin type III domain containing 5 (FNDC5) alleviates the phenotypic transformation of adventitial fibroblasts in hypertension and the underlying mechanisms. METHODS AND RESULTS: Experiments were carried out in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) and primary aortic adventitial fibroblasts. FNDC5 was downregulated and NLRP3 inflammasome was activated in aortic adventitia of SHR. FNDC5 overexpression attenuated adventitial fibroblasts phenotypic transformation, excessive synthesis and secretion of matrix components, NLRP3 inflammasome activation and inflammation in adventitial fibroblasts from SHR. Moreover, FNDC5 overexpression reduced NADPH oxidase 2 (NOX2) expression and reactive oxygen species (ROS) production in adventitial fibroblasts from SHR. Similarly, exogenous FNDC5 inhibited adventitial fibroblasts phenotypic transformation, expression of matrix components, NLRP3 inflammasome activation and NOX2 expression in adventitial fibroblasts from SHR. FNDC5 overexpression in rats attenuated phenotypic transformation, inflammation and reactive oxygen species (ROS) production in the aortic adventitia of SHR. Furthermore, FNDC5 overexpression reduced blood pressure and alleviated vascular remodeling in SHR. CONCLUSION: FNDC5 reduces NOX2-derived ROS production, NLRP3 inflammasome activation and phenotypic transformation in adventitial fibroblasts of SHR. FNDC5 plays a beneficial role in attenuating vascular inflammation, vascular remodeling and hypertension in SHR.

NLRP3 Gene Deletion Attenuates Angiotensin II-Induced Phenotypic Transformation of Vascular Smooth Muscle Cells and Vascular Remodeling.

BACKGROUND/AIMS: Angiotensin (Ang) II plays vital roles in vascular inflammation and remodeling in hypertension. Phenotypic transformation of vascular smooth muscle cells (VSMCs) is a major initiating factor for vascular remodeling. The present study was designed to determine the roles of NLRP3 inflammasome activation in Ang II-induced VSMC phenotypic transformation and vascular remodeling in hypertension. METHODS: Primary VSMCs from the aorta of NLRP3 knockout (NLRP3-/-) mice and wild-type (WT) mice were treated with Ang II for 24 h. Subcutaneous infusion of Ang II via osmotic minipump for 2 weeks was used to induce vascular remodeling and hypertension in WT and NLRP3-/- mice. RESULTS: NLRP3 gene deletion attenuates Ang II-induced NLRP3 inflammasome activation, phenotypic transformation from a contractile phenotype to a synthetic phenotype and proliferation in primary mice VSMCs. Ang II-induced hypertension and vascular remodeling in WT mice were attenuated in NLRP3-/- mice. Furthermore, Ang II-induced NLRP3 inflammasome activation, phenotypic transformation and proliferating cell nuclear antigen (PCNA) upregulation were inhibited in the media of aorta of NLRP3-/- mice. CONCLUSIONS: NLRP3 inflammasome activation contributes to Ang II-induced VSMC phenotypic transformation and proliferation as well as vascular remodeling and hypertension.

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.

Silencing salusin-ß attenuates cardiovascular remodeling and hypertension in spontaneously hypertensive rats.

Salusin-ß is a bioactive peptide involved in vascular smooth muscle cell proliferation, vascular fibrosis and hypertension. The present study was designed to determine the effects of silencing salusin-ß on hypertension and cardiovascular remodeling in spontaneously hypertensive rats (SHR). Thirteen-week-old male SHR and normotensive Wistar-Kyoto rats (WKY) were subjected to intravenous injection of PBS, adenoviral vectors encoding salusin-ß shRNA (Ad-Sal-shRNA) or a scramble shRNA. Salusin-ß levels in plasma, myocardium and mesenteric artery were increased in SHR. Silencing salusin-ß had no significant effect on blood pressure in WKY, but reduced blood pressure in SHR. It reduced the ratio of left ventricle weight to body weight, cross-sectional areas of cardiocytes and perivascular fibrosis, and decreased the media thickness and the media/lumen ratio of arteries in SHR. Silencing salusin-ß almost normalized plasma norepinephrine and angiotensin II levels in SHR. It prevented the upregulation of angiotensin II and AT1 receptors, and reduced the NAD(P)H oxidase activity and superoxide anion levels in myocardium and mesenteric artery of SHR. Knockdown of salusin-ß attenuated cell proliferation and fibrosis in vascular smooth muscle cells from SHR. These results indicate that silencing salusin-ß attenuates hypertension and cardiovascular remodeling in SHR.

Reduced lipolysis response to adipose afferent reflex involved in impaired activation of adrenoceptor-cAMP-PKA-hormone sensitive lipase pathway in obesity.

Chemical stimulation of white adipose tissue (WAT) causes adipose afferent reflex (AAR) and sympathetic activation. This study is to investigate the effects of AAR on lipolysis and the mechanisms of attenuated lipolysis response to enhanced AAR in obesity. Obesity was caused by high-fat diet for 12 weeks in rats. AAR was induced by injection of capsaicin into inguinal WAT or electrical stimulation of epididymal WAT afferent nerve. AAR caused sympathetic activation, which was enhanced in obesity rats. AAR increased cAMP levels and PKA activity, promoted hormone sensitive lipase (HSL) and perilipin phosphorylation, and increased lipolysis in WAT, which were attenuated in obesity rats. PKA activity, cAMP, perilipin and ß-adrenoceptor levels were reduced, while HSL was upregulated in adipocytes from obesity rats. In primary adipocytes, isoproterenol increased cAMP levels and PKA activity, promoted HSL and perilipin phosphorylation, and increased lipolysis, which were attenuated in obesity rats. The attenuated effects of isoproterenol in adipocytes from obesity rats were prevented by a cAMP analogue dbcAMP. The results indicate that reduced lipolysis response to enhanced AAR in obesity is attributed to the impaired activation of ß-adrenoceptor-cAMP-PKA-HSL pathway. Increased cAMP level in adipocytes rectifies the attenuated lipolysis in obesity.

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.

Salusin-ß induces foam cell formation and monocyte adhesion in human vascular smooth muscle cells via miR155/NOX2/NFκB pathway.

Vascular smooth muscle cells (VSMCs) are indispensible components in foam cell formation. Salusin-ß is a stimulator in the progression of atherosclerosis. Here, we showed that salusin-ß increased foam cell formation evidenced by accumulation of lipid droplets and intracellular cholesterol content, and promoted monocyte adhesion in human VSMCs. Salusin-ß increased the expressions and activity of acyl coenzyme A:cholesterol acyltransferase-1 (ACAT-1) and vascular cell adhesion molecule-1 (VCAM-1) in VSMCs. Silencing of ACAT-1 abolished the salusin-ß-induced lipid accumulation, and silencing of VCAM-1 prevented the salusin-ß-induced monocyte adhesion in VSMCs. Salusin-ß caused p65-NFκB nuclear translocation and increased p65 occupancy at the ACAT-1 and VCAM-1 promoter. Inhibition of NFκB with Bay 11-7082 prevented the salusin-ß-induced ACAT-1 and VCAM-1 upregulation, foam cell formation and monocyte adhesion in VSMCs. Scavenging ROS, inhibiting NADPH oxidase or knockdown of NOX2 abolished the effects of salusin-ß on ACAT-1 and VCAM-1 expressions, p65-NFκB nuclear translocation, lipid accumulation and monocyte adhesion in VSMCs. Salusin-ß increased miR155 expression, and knockdown of miR155 prevented the effects of salusin-ß on ACAT-1 and VCAM-1 expressions, p65-NFκB nuclear translocation, lipid accumulation, monocyte adhesion and ROS production in VSMCs. These results indicate that salusin-ß induces foam formation and monocyte adhesion via miR155/NOX2/NFκB-mediated ACAT-1 and VCAM-1 expressions in VSMCs.

Salusin-ß Promotes Vascular Smooth Muscle Cell Migration and Intimal Hyperplasia After Vascular Injury via ROS/NFκB/MMP-9 Pathway.

AIMS: Media-to-intima migration of vascular smooth muscle cells (VSMCs) is critical to intimal thickening in atherosclerosis and restenosis after coronary angioplasty. The aim of this study is to determine the effects of salusin-ß on VSMC migration and intimal hyperplasia after vascular injury and the underlying mechanism. RESULTS: In vitro, salusin-ß promoted VSMC migration, which was attenuated by matrix metalloproteinase (MMP)-9 inhibition. Inhibition or knockdown of p65-nuclear factor kappa beta (NFκB) in VSMCs suppressed salusin-ß-induced MMP-9 expression and VSMC migration. Salusin-ß increased NADPH oxidase 2 (NOX2) expression and reactive oxygen species (ROS) production, which were prevented by NOX2-small interfering RNA (siRNA) transfection. Salusin-ß-induced p65-NFκB translocation, MMP-9 expression, and VSMC migration were inhibited by ROS scavenger, NADPH oxidase inhibitor, or NOX2-siRNA. In vivo, carotid artery ligation-induced vascular injury resulted in intimal hyperplasia in injured artery in rats. Salusin-ß was upregulated in the injured carotid arteries of rats, which was attributed to reduced miR-133a-3p expression. Knockdown of salusin-ß with siRNA attenuated the vascular injury-induced intimal thickening, p65-NFκB nuclear translocation, and NOX2 and MMP-9 expressions in rats. INNOVATION: Salusin-ß is a critical modulator in VSMC migration and neointima formation in response to vascular injury. CONCLUSIONS: Salusin-ß promotes VSMC migration and vascular injury-induced intimal hyperplasia via MMP-9 accumulation due to NOX2 activation, followed by ROS production, IκBα phosphorylation and degradation, and p65-NFκB translocation. We propose that salusin-ß may be important in the VSMC migration and neointima of some vascular diseases. Antioxid. Redox Signal. 24, 1045-1057.