Natriuretic peptide receptor-C perturbs mitochondrial respiration in white adipose tissue.

Natriuretic peptide receptor-C (NPR-C) is highly expressed in adipose tissues and regulates obesity-related diseases; however, the detailed mechanism remains unknown. In this research, we aimed to explore the potential role of NPR-C in cold exposure and high-fat/high-sugar (HF/HS) diet-induced metabolic changes, especially in regulating white adipose tissue (WAT) mitochondrial function. Our findings showed that NPR-C expression, especially in epididymal WAT (eWAT), was reduced after cold exposure. Global Npr3 (gene encoding NPR-C protein) deficiency led to reduced body weight, increased WAT browning, thermogenesis, and enhanced expression of genes related to mitochondrial biogenesis. RNA-sequencing of eWAT showed that Npr3 deficiency enhanced the expression of mitochondrial respiratory chain complex genes and promoted mitochondrial oxidative phosphorylation in response to cold exposure. In addition, Npr3 KO mice were able to resist obesity induced by HF/HS diet. Npr3 knockdown in stromal vascular fraction (SVF)-induced white adipocytes promoted the expression of proliferator-activated receptor gamma coactivator 1α (PGC1α), uncoupling protein one (UCP1), and mitochondrial respiratory chain complexes. Mechanistically, NPR-C inhibited cGMP and calcium signaling in an NPR-B-dependent manner but suppressed cAMP signaling in an NPR-B-independent manner. Moreover, Npr3 knockdown induced browning via AKT and p38 pathway activation, which were attenuated by Npr2 knockdown. Importantly, treatment with the NPR-C-specific antagonist, AP-811, decreased WAT mass and increased PGC-1α, UCP1, and mitochondrial complex expression. Our findings reveal that NPR-C deficiency enhances mitochondrial function and energy expenditure in white adipose tissue, contributing to improved metabolic health and resistance to obesity.

Brown Adipocyte ADRB3 Mediates Cardioprotection via Suppressing Exosomal iNOS.

BACKGROUND: The ADRB3 (ß3-adrenergic receptors), which is predominantly expressed in brown adipose tissue (BAT), can activate BAT and improve metabolic health. Previous studies indicate that the endocrine function of BAT is associated with cardiac homeostasis and diseases. Here, we investigate the role of ADRB3 activation-mediated BAT function in cardiac remodeling. METHODS: BKO (brown adipocyte-specific ADRB3 knockout) and littermate control mice were subjected to Ang II (angiotensin II) for 28 days. Exosomes from ADRB3 antagonist SR59230A (SR-exo) or agonist mirabegron (MR-exo) treated brown adipocytes were intravenously injected to Ang II-infused mice. RESULTS: BKO markedly accelerated cardiac hypertrophy and fibrosis compared with control mice after Ang II infusion. In vitro, ADRB3 KO rather than control brown adipocytes aggravated expression of fibrotic genes in cardiac fibroblasts, and this difference was not detected after exosome inhibitor treatment. Consistently, BKO brown adipocyte-derived exosomes accelerated Ang II-induced cardiac fibroblast dysfunction compared with control exosomes. Furthermore, SR-exo significantly aggravated Ang II-induced cardiac remodeling, whereas MR-exo attenuated cardiac dysfunction. Mechanistically, ADRB3 KO or SR59230A treatment in brown adipocytes resulted an increase of iNOS (inducible nitric oxide synthase) in exosomes. Knockdown of iNOS in brown adipocytes reversed SR-exo-aggravated cardiac remodeling. CONCLUSIONS: Our data illustrated a new endocrine pattern of BAT in regulating cardiac remodeling, suggesting that activation of ADRB3 in brown adipocytes offers cardiac protection through suppressing exosomal iNOS.

C-atrial natriuretic peptide (ANP)4-23 attenuates renal fibrosis in deoxycorticosterone-acetate-salt hypertensive mice.

Epithelial-mesenchymal transition (EMT) plays a critical role in hypertension-induced renal fibrosis, a final pathway that leads to end-stage renal failure. C-Atrial natriuretic peptide (ANP)4-23, a specific agonist of natriuretic peptide receptor-C (NPR-C), has been reported to have protective effects against hypertension. However, the role of C-ANP4-23 in hypertension-associated renal fibrosis has not yet been elucidated. In this study, mice were randomly divided into SHAM group, DOCA-salt group and DOCA-salt + C-ANP4-23 group. Renal morphology changes, renal function and fibrosis were detected. Human proximal tubular epithelial cells (HK2) stimulated by aldosterone were used for cell function and mechanism study. The DOCA-salt treated mice exhibited hypertension, kidney fibrosis and renal dysfunction, which were attenuated by C-ANP4-23. Moreover, C-ANP4-23 inhibited DOCA-salt treatment-induced renal EMT as evidenced by decrease of the mesenchymal marker alpha-smooth muscle actin (ACTA2) and vimentin and increase of epithelial cell marker E-cadherin. In HK2 cells, aldosterone induced EMT response, which was also suppressed by C-ANP4-23. The key transcription factors (twist, snail, slug and ZEB1) involved in EMT were increased in the kidney of DOCA-salt-treated mice, which were also suppressed by C-ANP4-23. Mechanistically, C-ANP4-23 inhibited the aldosterone-induced translocation of MR from cytosol to nucleus without change of MR expression. Furthermore, C-ANP4-23 rescued the enhanced expression of NADPH oxidase (NOX) 4 and oxidative stress after aldosterone stimulation. Aldosterone-induced Akt and Erk1/2 activation was also suppressed by C-ANP4-23. Our data suggest that C-ANP4-23 attenuates renal fibrosis, likely through inhibition of MR activation, enhanced oxidative stress and Akt and Erk1/2 signaling pathway.

Mitogen-Activated Protein Kinases Mediate Adventitial Fibroblast Activation and Neointima Formation via GATA4/Cyclin D1 Axis.

PURPOSE: Activation of mitogen-activated protein kinases (MAPKs) by pathological stimuli participates in cardiovascular diseases. Dysfunction of adventitial fibroblast has emerged as a critical regulator in vascular remodeling, while the potential mechanism remains unclear. In this study, we sought to determine the effect of different activation of MAPKs in adventitial fibroblast contributing to neointima formation. METHODS: Balloon injury procedure was performed in male 12-week-old Sprague-Dawley rats. After injury, MAPK inhibitors were applied to the adventitia of injured arteries to suppress MAPK activation. Adventitial fibroblasts were stimulated by platelet-derived growth factor-BB (PDGF-BB) with or without MAPK inhibitors. RNA sequencing was performed to investigate the change of pathway and cell function. Wound healing, transwell assay, and flow cytometry were used to analyze adventitial fibroblast function. RESULTS: Phosphorylation of p38, c-Jun N-terminal kinase (JNK), and extracellular regulated kinases 1/2 (ERK1/2) was increased in injured arteries after balloon injury. In primary culture of adventitial fibroblasts, PDGF-BB increased phosphorylation of p38, JNK, ERK1/2, and extracellular regulated kinase 5 (ERK5) in a short time, which was normalized by their inhibitors respectively. Compared with the injury group, perivascular administration of four MAPK inhibitors significantly attenuated neointima formation by quantitative analysis of neointimal area, intima to media (I/M) ratio, and lumen area. RNA sequencing of adventitial fibroblasts treated with PDGF-BB with or without four inhibitors demonstrated differentially expressed genes involved in multiple biological processes, including cell adhesion, proliferation, migration, and inflammatory response. Wound healing and transwell assays showed that four inhibitors suppressed PDGF-BB-induced adventitial fibroblast migration. Cell cycle analysis by flow cytometry demonstrated that JNK, ERK1/2, and ERK5 but not p38 inhibitor blocked PDGF-BB-induced G1 phase release associated with decrease expression of cell cycle protein Cyclin D1 and transcription factor GATA4. Moreover, four inhibitors decreased macrophage infiltration into adventitia and monocyte chemoattractant protein-1 (MCP-1) expression. CONCLUSION: These results suggest that MAPKs differentially regulate activation of adventitial fibroblast through GATA4/Cyclin D1 axis that participates in neointima formation.

Adenosine A2A receptor activation prevents DOCA-salt induced hypertensive cardiac remodeling via iBAT.

Hypertensive cardiac remodeling is a constellation of abnormalities that includes cardiomyocyte hypertrophy and death and tissue fibrosis. Adenosine is a long-known vasodilator, through interacting with its four cell surface receptor subtypes in cardiovascular system. However, it is unclear that whether adenosine A2A receptor (A2AR) activation is involved in the cardiac remodeling in hypertension. WT mice were utilized to induce DOCA-salt sensitive hypertension and received A2AR agonist CGS21680 or antagonist KW6002 treatment. Cardiac functional phenotyping measurement by echocardiography showed that CGS21680 improved cardiac dysfunction in DOCA-salt mice. Moreover, CGS21680 reduced cardiomyocyte hypertrophy, cardiac inflammation and fibrosis. However, iBAT depletion surgery induces dramatic cardiac remodeling in DOCA-salt mice, and the protective function of CGS21680 was blocked without intact iBAT. Mechanistically, A2AR agonist CGS21680 increased iBAT-derived fibroblast growth factor 21 (FGF21). Our data suggest that activation of A2AR could be a potential therapeutic strategy in preventing heart damage in hypertension.

Transactivation domain of Krüppel-like factor 15 negatively regulates angiotensin II-induced adventitial inflammation and fibrosis.

Krüppel-like factor (KLF) 15 has emerged as a critical regulator of fibrosis in cardiovascular diseases. However, the precise role that KLF15 and its functional domain played in adventitial inflammation and fibrosis remains unclear. This study aims to investigate the role of the transactivation domain (TAD) of KLF15 in angiotensin II (Ang II)-induced adventitial pathologic changes. KLF15 expression was decreased in the vascular adventitia of Ang II-infused mice (1000 ng/kg/min, 14 d) and in adventitial fibroblasts (AFs) stimulated by Ang II (10-7 M). Adenovirus-mediated KLF15 overexpression normalized Ang II-induced vascular hypertrophy, increased collagen deposition, macrophage infiltration, and CCL2 and VCAM-1 expression. Interestingly, KLF15-ΔTAD (KLF15 with deletion of TAD at amino acids 132-152) overexpression showed no effect on the above pathologic changes. Similarly, perivascularly overexpression of KLF15 but not KLF15-ΔTAD in carotid arteries also attenuated Ang II-induced vascular inflammation and fibrosis. Furthermore, KLF15 overexpression after Ang II infusion rescued Ang II-induced vascular remodeling. CCL2 or VCAM-1-mediated monocyte and macrophage migration or adhesion to AFs in response to Ang II was negatively regulated by KLF15 through TAD. Ang II-enhanced Smad2/3 activation and adventitial migration, proliferation, and differentiation of AFs were suppressed by KLF15 but not KLF15-ΔTAD overexpression. Conversely, small interfering RNA knockdown of KLF15 aggravated Ang II-induced Smad2/3 activation and dysfunction of AFs. Luciferase, coimmunoprecipitation, and chromatin immunoprecipitation assay were used to demonstrate that interaction of KLF15 with Smad2/3 suppressed CCL2 expression through TAD. Mechanistically, activation of Ang II type 1 receptor/phospholipase Cγ 1/ERK1/2 signaling resulted in a decrease of KLF15 expression. In conclusion, these results demonstrate that KLF15 negatively regulates activation of AFs through TAD, which plays an important role in Ang II-induced adventitial inflammation and fibrosis.-Lu, Y.-Y., Li, X.-D., Zhou, H.-D., Shao, S., He, S., Hong, M.-N., Liu, J.-C., Xu, Y.-L., Wu, Y.-J., Zhu, D.-L., Wang, J.-G., Gao, P.-J. Transactivation domain of Krüppel-like factor 15 negatively regulates angiotensin II-induced adventitial inflammation and fibrosis.

Deficiency of Complement C3a and C5a Receptors Prevents Angiotensin II-Induced Hypertension via Regulatory T Cells.

RATIONALE: Inflammation and immunity play crucial roles in the development of hypertension. Complement activation-mediated innate immune response is involved in the regulation of hypertension and target-organ damage. However, whether complement-mediated T-cell functions could regulate blood pressure elevation in hypertension is still unclear. OBJECTIVE: We aim to determine whether C3aR (complement component 3a receptor) and C5aR (complement component 5a receptor) could regulate blood pressure via modulating regulatory T cells (Tregs). METHODS AND RESULTS: We showed that angiotensin II (Ang II)-induced hypertension resulted in an elevated expression of C3aR and C5aR in Foxp3 (forkhead box P3)+ Tregs. By using C3aR and C5aR DKO (double knockout) mice, we showed that C3aR and C5aR deficiency together strikingly decreased both systolic and diastolic blood pressure in response to Ang II compared with WT (wild type), single C3aR-deficient (C3aR-/-), or C5aR-deficient (C5aR-/-) mice. Flow cytometric analysis showed that Ang II-induced Treg reduction in the kidney and blood was also blocked in DKO mice. Histological analysis indicated that renal and vascular structure remodeling and damage after Ang II treatment were attenuated in DKO mice compared with WT mice. In vitro, Ang II was able to stimulate C3aR and C5aR expression in cultured CD4+CD25+ natural Tregs. CD3 and CD28 antibody stimuli downregulated Foxp3 expression in WT but not DKO Tregs. More important, depletion of Tregs with CD25 antibody abolished the protective effects against Ang II-induced hypertension and target-organ damage in DKO mice. Adoptive transfer of DKO Tregs showed much more profound protective effects against Ang II-induced hypertension than WT Treg transfer. Furthermore, we demonstrated that C5aR expression in Foxp3+ Tregs was higher in hypertensive patients compared with normotensive individuals. CONCLUSIONS: C3aR and C5aR DKO-mediated Treg function prevents Ang II-induced hypertension and target-organ damage. Targeting C3aR and C5aR in Tregs specifically may be an alternative novel approach for hypertension treatment.

Membrane raft redox signalling contributes to endothelial dysfunction and vascular remodelling of thoracic aorta in angiotensin II-infused rats.

NEW FINDINGS: What is the central question of this study? Is the membrane raft redox signalling pathway involved in blood pressure increase, endothelial dysfunction and vascular remodelling in an angiotensin II-induced hypertensive animal model? What is the main finding and its importance? The membrane raft redox signalling pathway was involved in endothelial dysfunction and medial remodelling in angiotensin II-induced hypertension. ABSTRACT: The membrane raft (MR) redox pathway is characterized by NADPH oxidase activation via the clustering of its subunits through lysosome fusion and the activation of acid sphingomyelinase (ASMase). Our previous study shows that the MR redox signalling pathway is associated with angiontensin II (AngII)-induced production of reactive oxygen species (ROS) and endothelial dysfunction in rat mesenteric arteries. In the present study, we hypothesized that this signalling pathway is involved in blood pressure increase, endothelial dysfunction and vascular remodelling in an AngII-induced hypertensive animal model. Sixteen-week-old male Sprague-Dawley rats were subjected to AngII infusion for 2 weeks with or without treatment with the lysosome fusion inhibitor bafilomycin A1 and ASMase inhibitor amitriptyline. After treatments, aortas were harvested for further study. The results showed that the MR redox signalling pathway was activated as indicated by the increase of MR formation, ASMase activity and ROS production in aorta from AngII-infused rats compared with that from control rats. MR formation and ROS production were significantly inhibited in thoracic aorta from AngII-induced rats treated with bafilomycin A1 and amitriptyline. Both treatments significantly attenuated blood pressure increase, endothelial dysfunction and vascular remodelling including medial hypertrophy, and increased collagen and fibronectin deposition in thoracic aortas from AngII-infused rats. Finally, both treatments significantly prevented the increase of inflammatory factors including monocyte chemotactic protein 1, intercellular adhesion molecule 1 and tumour necrosis factor α in thoracic aorta from AngII-infused rats. In conclusion, the present study demonstrates that the MR redox signalling pathway was involved in endothelial dysfunction and medial remodelling in AngII-induced hypertension.

Complement 5a-mediated trophoblasts dysfunction is involved in the development of pre-eclampsia.

Pre-eclampsia (PE) is a life-threatening multisystem disorder leading to maternal and neonatal mortality and morbidity. Emerging evidence showed that activation of the complement system is implicated in the pathological processes of PE. However, little is known about the detailed cellular and molecular mechanism of complement activation in the development of PE. In this study, we reported that complement 5a (C5a) plays a pivotal role in aberrant placentation, which is essential for the onset of PE. We detected an elevated C5a deposition in macrophages and C5a receptor (C5aR) expression in trophoblasts of pre-eclamptic placentas. Further study showed that C5a stimulated trophoblasts towards an anti-angiogenic phenotype by mediating the imbalance of angiogenic factors such as soluble fms-like tyrosine kinase 1 (sFlt1) and placental growth factor (PIGF). Additionally, C5a inhibited the migration and tube formation of trophoblasts, while, C5aR knockdown with siRNA rescued migration and tube formation abilities. We also found that maternal C5a serum level was increased in women with PE and was positively correlated with maternal blood pressure and arterial stiffness. These results demonstrated that the placental C5a/C5aR pathway contributed to the development of PE by regulating placental trophoblasts dysfunctions, suggesting that C5a may be a novel therapeutic possibility for the disease.

Membrane rafts-redox signalling pathway contributes to renal fibrosis via modulation of the renal tubular epithelial-mesenchymal transition.

KEY POINTS: Membrane rafts (MRs)-redox signalling pathway is activated in response to transforming growth factor-ß1 (TGF-ß1) stimulation in renal tubular cells. This pathway contributes to TGF-1ß-induced epithelial-mesenchymal transition (EMT) in renal tubular cells. The the MRs-redox signalling pathway is activated in renal tubular cells isolated from angiotensin II (AngII)-induced hypertensive rats. Inhibition of this pathway attenuated renal inflammation and fibrosis in AngII-induced hypertension. ABSTRACT: The membrane rafts (MRs)-redox pathway is characterized by NADPH oxidase subunit clustering and activation through lysosome fusion, V-type proton ATPase subunit E2 (encoded by the Atp6v1e2 gene) translocation and sphingomyelin phosphodiesterase 1 (SMPD1, encoded by the SMPD1 gene) activation. In the present study, we hypothesized that the MRs-redox-derived reactive oxygen species (ROS) are involved in renal inflammation and fibrosis by promoting renal tubular epithelial-mesenchymal transition (EMT). Results show that transforming growth factor-ß1 (TGF-ß1) acutely induced MR formation and ROS production in NRK-52E cells, a rat renal tubular cell line. In addition, transfection of Atp6v1e2 small hairpin RNAs (shRNA) and SMPD1 shRNA attenuated TGF-ß1-induced changes in EMT markers, including E-cadherin, α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP-1) in NRK-52E cells. Moreover, Erk1/2 activation may be a downstream regulator of the MRs-redox-derived ROS, because both shRNAs significantly inhibited TGF-ß1-induced Erk1/2 phosphorylation. Further in vivo study shows that the renal tubular the MRs-redox signalling pathway was activated in angiotensin II (AngII)-induced hypertension, as indicated by the increased NADPH oxidase subunit Nox4 fraction in the MR domain, SMPD1 activation and increased ROS content in isolated renal tubular cells. Finally, renal transfection of Atp6v1e2 shRNA and SMPD1 shRNA significantly prevented renal fibrosis and inflammation, as indicated by the decrease of α-SMA, fibronectin, collagen I, monocyte chemoattractant protein-1 (MCP-1), intercellular cell adhesion molecule-1 (ICAM-1) and tumour necrosis factor-α (TNF-α) in kidneys from AngII-infused rats. It was concluded that the the MRs-redox signalling pathway is involved in TGF-ß1-induced renal tubular EMT and renal inflammation/fibrosis in AngII-induced hypertension.

Complement-mediated inhibition of adiponectin regulates perivascular inflammation and vascular injury in hypertension.

Perivascular adipose tissue (PVAT)-derived adiponectin (APN) is a secreted adipokine that protects against hypertension-related cardiovascular injury. However, the regulation of APN expression in hypertension remains to be explored. In this study, we demonstrated that down-regulation of APN was associated with complement activation in the PVAT of desoxycorticosterone acetate (DOCA)-salt hypertensive mice. Complement 3-deficient hypertensive mice were protected from ANP decrease in the PVAT. APN deficiency blockaded the protective effects of complement inhibition against hypertensive vascular injury. Mechanistically, complement 5a (C5a)-induced TNF-α secretion from macrophages is required for inhibiting APN expression in adipocytes. Macrophage depletion reversed C5a agonist peptide-induced TNF-α up-regulation and APN down-regulation in the PVAT of DOCA mice. Moreover, we detected increased macrophage infiltration and C5a expression associated with decreased APN expression in adipose tissue from patients with aldosterone-producing adenoma. These results identify a novel interaction between macrophages and adipocytes in the PVAT, where complement-mediated inhibition of APN acts as a potential risk factor for hypertensive vascular inflammation.-Ruan, C.-C., Ma, Y., Ge, Q., Li, Y., Zhu, L.-M., Zhang, Y., Kong, L.-R., Wu, Q-H., Li, F., Cheng, L., Zhao, A. Z., Zhu, D.-L., Gao, P.-J. Complement-mediated inhibition of adiponectin regulates perivascular inflammation and vascular injury in hypertension.

Loss of miR-146b-3p Inhibits Perivascular Adipocyte Browning with Cold Exposure During Aging.

PURPOSE: Pathological changes of the perivascular adipose tissue (PVAT) are directly associated with increased risk of age-related vascular diseases. MicroRNAs regulate adipocyte biological functions including adipogenic differentiation and white adipocyte browning. The present study aims to determine whether miR-146b-3p is involved in the regulation of perivascular adipocyte browning during aging. METHODS: We utilized a cold-induced animal model to investigate the effect of aging on perivascular adipocyte browning. We also detected the miR-146b-3p expression in the PVAT of young or old mice after cold stimulus. We further investigated the role of miR-146b-3p in regulating perivascular adipocyte browning in vitro and in vivo via administrating miRNA mimics or inhibitors. RESULTS: Old mice showed decrease of perivascular adipocyte browning and downregulation of miR-146b-3p expression in the PVAT after cold stimulus. Oil red O staining and qPCR indicated that aging perturbed preadipocyte to brown adipocyte differentiation, and expression of miR-146b-3p gradually increased during differentiation. MiR-146b-3p inhibitors blocked brown adipocyte differentiation in young preadipocytes, whereas miR-146b-3p mimics rescued the differentiation of the old preadipocytes. Finally, miR-146b-3p knocks down inhibited perivascular adipocyte browning in young mice after cold stimulus. CONCLUSION: Aging inhibits perivascular adipocyte browning, and loss of miR-146b-3p is a potential regulator for this process.

Activating transcription factor 3 SUMOylation is involved in angiotensin II-induced endothelial cell inflammation and dysfunction.

Activating transcription factor 3 (ATF3) is an adaptive-response protein induced by various environmental stresses and is implicated in the pathogenesis of many disease states. However, the role of ATF3 SUMOylation in hypertension-induced vascular injury remains poorly understood. Here we investigated the function of ATF3 SUMOylation in vascular endothelial cells (ECs). The expression of ATF3 and small ubiquitin-like modifier 1 (SUMO1) was increased in angiotensin II (Ang II)-induced human umbilical vein endothelial cells (HUVECs). Microscopic analyses further revealed that the expression of ATF3 and SUMO1 is upregulated and colocalized in the endothelium of thoracic aortas from Ang II-induced hypertensive mice. However, Ang II-induced upregulation of ATF3 and SUMO1 in vitro and in vivo was blocked by Ang II type I receptor antagonist olmesartan. Moreover, Ang II induced ATF3 SUMOylation at lysine 42, which is SUMO1 dependent. ATF3 SUMOylation attenuated ATF3 ubiquitination and in turn promoted ATF3 protein stability. ATF3 or SUMO1 knockdown inhibited Ang II-induced expression of inflammatory molecules such as tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8. Wild type ATF3 but not ATF3-K42R (SUMOylation defective mutant) reduced the production of nitric oxide (NO), a key indicator of EC function. Consistently, ginkgolic acid, an inhibitor of SUMOylation, increased NO production in HUVECs and significantly improved vasodilatation of aorta from Ang II-induced hypertensive mice. Our findings demonstrated that ATF3 SUMOylation is involved in Ang II-induced EC inflammation and dysfunction in vitro and in vivo through inhibiting ATF3 ubiquitination and increasing ATF3 protein stability.

Protease-activated receptor 1 and 2 contribute to angiotensin II-induced activation of adventitial fibroblasts from rat aorta.

Adventitial fibroblasts (AFs) can be activated by angiotensin II (Ang II) and exert pro-fibrotic and pro-inflammatory effects in vascular remodeling. Protease-activated receptor (PAR) 1 and 2 play a significant role in fibrogenic and inflammatory diseases. The present study hypothesized that PAR1 and PAR2 are involved in Ang II-induced AF activation and contribute to adventitial remodeling. We found that direct activation of PAR1 and PAR2 with PAR1-AP and PAR2-AP led to AF activation, including proliferation and differentiation of AFs, extracellular matrix synthesis, as well as production of pro-fibrotic cytokine TGF-ß and pro-inflammatory cytokines IL-6 and MCP-1. Furthermore, PAR1 and PAR2 mediated Ang II-induced AF activation, since both PAR1 and PAR2 antagonists inhibited Ang II-induced proliferation, migration, differentiation, extracellular matrix synthesis and production of pro-fibrotic and pro-inflammatory cytokines in AFs. Finally, mechanistic study showed that Ang II, via Ang II type I receptor (AT1R), upregulated both PAR1 and PAR2 expression, and transactivated PAR1 and PAR2, as denoted by internalization of both proteins. In conclusion, our results suggest that PAR1 and PAR2 play a critical role in Ang II-induced AF activation, and this may contribute to adventitia-related pathological changes.

Adenovirus-Mediated Overexpression of Septin 2 Attenuates α-Smooth Muscle Actin Expression and Adventitial Myofibroblast Migration Induced by Angiotensin II.

Phenotypic transformation from adventitial fibroblasts (AFs) to myofibroblasts (MFs) is critical for vascular remodeling. Septin 2 was found to be downregulated during the differentiation of AFs to MFs induced by angiotensin II (Ang II); however, the role of septin 2 in this process is still unknown. In this study, we investigate whether septin 2 contributes to the adventitial MF phenotypic modulation caused by Ang II. The decreased level of septin 2 and the increased expression of α-smooth muscle actin (α-SMA), a marker of MFs, were readily observed in Ang II-stimulated MF differentiation. After gene transfer of septin 2, the expression of α-SMA was markedly decreased and the MF migration response to Ang II was inhibited. Furthermore, the inhibition of RhoA, another molecule involved in MF phenotypic modulation, decreased the motility of MFs and the expression of septin 2 triggered in Ang II. Finally, transfection of septin 2 rescued the level of acetyl-α-tubulin in MFs. These findings demonstrate that, as a downstream molecule of RhoA, septin 2 blunted the responses of AFs to Ang II by protecting α-tubulin acetylation, which suggests that septin 2 may serve as a potential therapeutic target for vascular injury.

Downregulation of dynamin-related protein 1 contributes to impaired autophagic flux and angiogenic function in senescent endothelial cells.

OBJECTIVE: Recent studies have shown that altered mitochondrial dynamics impairs the function in senescent endothelial cells (ECs). However, the underlying molecular mechanism remains to be elucidated. Herein, we investigated the role and underlying mechanism of mitochondrial fission protein dynamin-related protein 1 (DRP1) in vascular aging. APPROACH AND RESULTS: We found that DRP1 expression is decreased in senescent ECs, accompanied with long interconnected mitochondria and impaired angiogenic function. In addition, there was marked increase of autophagosomes but not of autolysosomes (assessed as punctate dual fluorescent mCherry-GFP (green fluorescent protein) tandem-tagged light chain 3 expression) in senescent ECs, indicating impaired autophagic flux. DRP1 knockdown or pharmacological inhibition in young ECs resulted in elongated mitochondria, suppressed autophagic flux, premature senescence, and impaired angiogenic function. In contrast, adenoviral-mediated overexpression of DRP1 in senescent ECs restored autophagic flux and improved angiogenic function. EC senescence was associated with the increase of mitochondrial reactive oxygen species and antioxidant N-acetyl-cysteine restored autophagosome clearance and improved angiogenic function. Consistently, en face staining of old rat thoracic aorta revealed a decrease of DRP1 expression and increase of autophagosomes accumulation. Furthermore, in vivo knockdown of Drp1 in common carotid arteries significantly impaired the autophagosome clearance. Importantly, downregulation of Drp1 directly abrogated microvessels outgrowth from ex vivo aortic rings. CONCLUSIONS: These results suggest that loss of DRP1 during senescence exacerbates ECs dysfunction by increasing mitochondrial reactive oxygen species and subsequently inhibiting autophagic flux.

Complement-mediated macrophage polarization in perivascular adipose tissue contributes to vascular injury in deoxycorticosterone acetate-salt mice.

OBJECTIVE: We have previously shown an increased expression of complement 3 (C3) in the perivascular adipose tissue (PVAT) in the deoxycorticosterone acetate (DOCA)-salt hypertensive model. This study aims to examine the role and underlying mechanism of C3 in PVAT for understanding the pathogenesis of hypertensive vascular remodeling further. APPROACH AND RESULTS: The role of C3 in macrophage polarization was investigated using peritoneal macrophages from wild-type and C3-deficient (C3KO) mice because we found that C3 was primarily expressed in macrophages in PVAT of blood vessels from DOCA-salt mice, and results showed a decreased expression of M1 phenotypic marker in contrast to an increased level of M2 marker in the C3KO macrophages. Bone marrow transplantation studies further showed in vivo that DOCA-salt recipient mice had fewer M1 but more M2 macrophages in PVAT when the donor bone marrows were from C3KO compared with those from wild-type mice. Of note, this macrophage polarization shift was accompanied with an ameliorated vascular injury. Furthermore, we identified the complement 5a (C5a) as the major C3 activation product that was involved in macrophage polarization and DOCA-salt-induced vascular injury. Consistently, in vivo depletion of macrophages prevented the induction of C3 and C5a in PVAT, and ameliorated hypertensive vascular injury as well. CONCLUSIONS: The presence and activation of bone marrow-derived macrophages in PVAT are crucial for complement activation in hypertensive vascular inflammation, and C5a plays a critical role in DOCA-salt-induced vascular injury by stimulating macrophage polarization toward a proinflammatory M1 phenotype in PVAT.

Mitochondrial division inhibitor Mdivi-1 ameliorates angiotensin II-induced endothelial dysfunction.

Mitochondrial fission can occur via activation of dynamin-related protein 1 (Drp1), which participates in the mitochondrial membrane scission process. The present study was designed to investigate the effect of angiotensin II (AngII) on mitochondrial fission and fusion in human umbilical vascular endothelial cells (HUVECs). And we further inquire into whether Mdivi-1, a newly identified pharmacological inhibitor of Drp1, can prevent endothelial dysfunction induced by AngII. The HUVECs were treated with AngII alone or in combination with Mdivi-1. Western blot was used to detect protein expressions of Drp1, endothelial nitric oxide synthase (eNOS) and apoptosis-related enzymes. MitoTracker Red and JC-1 dye were used to detect mitochondrial morphology and membrane potential, respectively. DCFH-DA probe was used to access intracellular reactive oxygen species (ROS) generation. Transwell assay was used to evaluate cell migration. Annexin V/PI staining was used to assess cellular apoptosis. The results showed that, in cultured HUVECs, AngII (1 × 10-7 mol/L, 12 h) treatment significantly upregulated the expression of Drp1 followed by increased apoptosis and decreased eNOS expression. The treatment of AngII resulted in a change in mitochondrial morphology from elongated to uniformly punctate organelles, which was accompanied by decreased mitochondrial membrane potential. Furthermore, Mdivi-1 significantly protected against AngII-induced endothelial dysfunction, as shown by increased mitochondrial membrane potential and eNOS expression, reduced ROS level, decreased apoptosis and migration ability. Taking together, our data suggest that inhibition of Drp1 with Mdivi-1 can restore AngII-induced endothelial dysfunction.

Deletion of angiotensin-converting enzyme 2 exacerbates renal inflammation and injury in apolipoprotein E-deficient mice through modulation of the nephrin and TNF-alpha-TNFRSF1A signaling.

BACKGROUND: The renin-angiotensin system (RAS) has been implicated in atherosclerotic lesions and progression to chronic kidney diseases. We examined regulatory roles of angiotensin-converting enzyme 2 (ACE2) in the apolipoprotein E (ApoE) knockout (KO) kidneys. METHODS: The 3-month-old wild-type, ApoEKO, ACE2KO and ApoE/ACE2 double-KO (DKO) mice in a C57BL/6 background were used. The ApoEKO mice were randomized to daily deliver either Ang II (1.5 mg/kg) and/or human recombinant ACE2 (rhACE2; 2 mg/kg) for 2 weeks. We examined changes in pro-inflammatory cytokines, renal ultrastructure, and pathological signaling in mouse kidneys. RESULTS: Downregulation of ACE2 and nephrin levels was observed in ApoEKO kidneys. Genetic ACE2 deletion resulted in modest elevations in systolic blood pressure levels and Ang II type 1 receptor expression and reduced nephrin expression in kidneys of the ApoE/ACE2 DKO mice with a decrease in renal Ang-(1-7) levels. These changes were linked with marked increases in renal superoxide generation, NADPH oxidase (NOX) 4 and proinflammatory factors levels, including interleukin (IL)-1beta, IL-6, IL-17A, RANTES, ICAM-1, Tumor necrosis factor-alpha (TNF-alpha) and TNFRSF1A. Renal dysfunction and ultrastructure injury were aggravated in the ApoE/ACE2 DKO mice and Ang II-infused ApoEKO mice with increased plasma levels of creatinine, blood urea nitrogen and enhanced levels of Ang II in plasma and kidneys. The Ang II-mediated reductions of renal ACE2 and nephrin levels in ApoEKO mice were remarkably rescued by rhACE2 supplementation, along with augmentation of renal Ang-(1-7) levels. More importantly, rhACE2 treatment significantly reversed Ang II-induced renal inflammation, superoxide generation, kidney dysfunction and adverse renal injury in ApoEKO mice with suppression of the NOX4 and TNF-alpha-TNFRSF1A signaling. However, rhACE2 had no effect on renal NOX2 and TNFRSF1B expression and circulating lipid levels. CONCLUSIONS: ACE2 deficiency exacerbates kidney inflammation, oxidative stress and adverse renal injury in the ApoE-mutant mice through modulation of the nephrin, NOX4 and TNF-alpha-TNFRSF1A signaling. While rhACE2 supplementation alleviates inflammation, renal dysfunction and glomerulus injury in the ApoE-mutant mice associated with upregulations of Ang-(1-7) levels and nephrin expression and suppression of the TNF-alpha-TNFRSF1A signaling. Strategies aimed at enhancing the ACE2/Ang-(1-7) actions may have important therapeutic potential for atherosclerotic renal injury and kidney diseases.

[Angiotensin II induces expression of inflammatory mediators in vascular adventitial fibroblasts].

Vascular adventitial fibroblasts (AF) may play an important role in vascular inflammation. This study was aimed to investigate the expression pattern of inflammatory mediators in AF induced by angiotensin II (AngII) and to explore the effects of AF-derived inflammatory mediators on the adhesion and migration of macrophages both in vitro and in vivo. We used real-time RT-PCR to detect the mRNA expression of inflammatory mediators in cultured AF. The results showed that AngII (1 × 10(-7) mol/L) up-regulated mRNA expression of 4 inflammatory mediators, including P-selectin, ICAM-1, IL-6 and MCP-1, in cultured AF. Western blot analysis or ELISA revealed that AngII up-regulated P-selectin and ICAM-1 protein expression and IL-6 secretion in cultured AF, but did not alter MCP-1 secretion. We further detected the effects of AF-derived inflammatory mediators on the adhesion and chemotaxis of RAW264.7, a macrophage cell line. We found that AF stimulated with AngII could enhance the adhesion of RAW264.7 and the conditioned medium from AngII-stimulated AF could enhance the migration of RAW264.7. Immunofluorescence study showed an enhanced accumulation of CD68 positive cells and the up-regulation of P-selectin, ICAM-1, IL-6 and MCP-1 in aortic adventitia of AngII-infused (200 ng/kg per min for 2 weeks) rats. We concluded that AF may contribute to vascular inflammation via expression of certain inflammatory mediators and the subsequent adhesion and chemotaxis of macrophages.