Ang II-induced Reactive Oxygen Species Production Research Articles

Phospholipase C-β3 is dispensable for vascular constriction but indispensable for vascular hyperplasia

Angiotensin II (AngII) induces the contraction and proliferation of vascular smooth muscle cells (VSMCs). AngII activates phospholipase C-β (PLC-β), thereby inducing Ca2+ mobilization as well as the production of reactive oxygen species (ROS). Since contraction is a unique property of contractile VSMCs, signaling cascades related to the proliferation of VSMCs may differ. However, the specific molecular mechanism that controls the contraction or proliferation of VSMCs remains unclear. AngII-induced ROS production, migration, and proliferation were suppressed by inhibiting PLC-β3, inositol trisphosphate (IP3) receptor, and NOX or by silencing PLC-β3 or NOX1 but not by NOX4. However, pharmacological inhibition or silencing of PLC-β3 or NOX did not affect AngII-induced VSMC contraction. Furthermore, the AngII-dependent constriction of mesenteric arteries isolated from PLC-β3∆SMC, NOX1−/−, NOX4−/− and normal control mice was similar. AngII-induced VSMC contraction and mesenteric artery constriction were blocked by inhibiting the L-type calcium channel Rho-associated kinase 2 (ROCK2) or myosin light chain kinase (MLCK). The activation of ROCK2 and MLCK was significantly induced in PLC-β3∆SMC mice, whereas the depletion of Ca2+ in the extracellular medium suppressed the AngII-induced activation of ROCK2, MLCK, and vasoconstriction. AngII-induced hypertension was significantly induced in NOX1−/− and PLC-β3∆SMC mice, whereas LCCA ligation-induced neointima formation was significantly suppressed in NOX1−/− and PLC-β3∆SMC mice. These results suggest that PLC-β3 is essential for vascular hyperplasia through NOX1-mediated ROS production but is nonessential for vascular constriction or blood pressure regulation.

Pemafibrate Prevents Rupture of Angiotensin II-Induced Abdominal Aortic Aneurysms.

BackgroundAbdominal aortic aneurysm (AAA) is a life-threatening disease that lacks effective preventive therapies. This study aimed to evaluate the effect of pemafibrate, a selective peroxisome proliferator-activated receptor alpha (PPARα) agonist, on AAA formation and rupture.MethodsExperimental AAA was induced by subcutaneous angiotensin II (AngII) infusion in ApoE–/– mice for 4 weeks. Pemafibrate (0.1 mg/kg/day) was administered orally. Dihydroethidium staining was used to evaluate the reactive oxygen species (ROS).ResultsThe size of the AngII-induced AAA did not differ between pemafibrate- and vehicle-treated groups. However, a decreased mortality rate due to AAA rupture was observed in pemafibrate-treated mice. Pemafibrate ameliorated AngII-induced ROS and reduced the mRNA expression of interleukin-6 and tumor necrosis factor-α in the aortic wall. Gelatin zymography analysis demonstrated significant inhibition of matrix metalloproteinase-2 activity by pemafibrate. AngII-induced ROS production in human vascular smooth muscle cells was inhibited by pre-treatment with pemafibrate and was accompanied by an increase in catalase activity. Small interfering RNA-mediated knockdown of catalase or PPARα significantly attenuated the anti-oxidative effect of pemafibrate.ConclusionPemafibrate prevented AAA rupture in a murine model, concomitant with reduced ROS, inflammation, and extracellular matrix degradation in the aortic wall. The protective effect against AAA rupture was partly mediated by the anti-oxidative effect of catalase induced by pemafibrate in the smooth muscle cells.

Sirt3 Protects Against Thoracic Aortic Dissection Formation by Reducing Reactive Oxygen Species, Vascular Inflammation, and Apoptosis of Smooth Muscle Cells.

Sirtuin3 (Sirt3) is a histone deacetylase involved in the regulation of many cellular processes. Sirt3 deficiency is known to increase oxidative stress. Reactive oxygen species (ROS) promote degradation of the extracellular matrix and vascular smooth muscle cell (VSMC) apoptosis. Reducing oxidative stress by Sirt3 overexpression could have therapeutic potential for limiting thoracic aortic dissection (TAD) development. We hypothesized that Sirt3 deficiency could increase the risk for TAD by decreasing ROS elimination and that Sirt3 overexpression (Sirt3OE) could provide an alternative option for TAD treatment. Mice with TAD had significantly lower Sirt3 expression than normal subjects. Sirt3 KO mice exhibit significantly increased TAD incidence rate and increased aortic diameters. Moreover, Sirt3 overexpression reduced Ang II-induced ROS production, NF-kB activation, and apoptosis in human aortic smooth muscle cells (HASMCs). Sirt3 overexpression attenuated aneurysm formation and decreased aortic expansion. In conclusion, our data showed that Sirt3 deficiency increases susceptibility to TAD formation by attenuating anti-ROS effects and increasing VSMC apoptosis and vascular inflammation.

LRRC8A contributes to angiotensin II-induced cardiac hypertrophy by interacting with NADPH oxidases via the C-terminal leucine-rich repeat domain

Cardiac hypertrophy, an important cause of heart failure, is characterized by an increase in heart weight, the ventricular wall, and cardiomyocyte volume. The volume regulatory anion channel (VRAC) is an important regulator of cell volume. However, its role in cardiac hypertrophy remains unclear. The purpose of this study was to investigate the effect of leucine-rich repeat-containing 8A (LRRC8A), an essential component of the VRAC, on angiotensin II (AngII)-induced cardiac hypertrophy. Our results showed that LRRC8A expression, NADPH oxidase activity, and reactive oxygen species (ROS) production were increased in AngII-induced hypertrophic neonatal mouse cardiomyocytes and the myocardium of C57/BL/6 mice. In addition, AngII activated VRAC currents in cardiomyocytes. The delivery of adeno-associated viral (AAV9) bearing siRNA against mouse LRRC8A into the left ventricular wall inhibited AngII-induced cardiac hypertrophy and fibrosis. Accordingly, the knockdown of LRRC8A attenuated AngII-induced cardiomyocyte hypertrophy and VRAC currents in vitro. Furthermore, knockdown of LRRC8A suppressed AngII-induced ROS production, NADPH oxidase activity, the expression of NADPH oxidase membrane-bound subunits Nox2, Nox4, and p22phox, and the translocation of NADPH oxidase cytosolic subunits p47phox and p67phox. Immunofluorescent staining showed that LRRC8A co-localized with NADPH oxidase membrane subunits Nox2, Nox4, and p22phox. Co-immunoprecipitation and analysis of a C-terminal leucine-rich repeat domain (LRRD) mutant showed that LRRC8A physically interacts with Nox2, Nox4, and p22phox via the LRRD. Taken together, the results of this study suggested that LRRC8A might play an important role in promoting AngII-induced cardiac hypertrophy by interacting with NADPH oxidases via the LRRD.

Aldehyde dehydrogenase 2 protects against abdominal aortic aneurysm formation by reducing reactive oxygen species, vascular inflammation, and apoptosis of vascular smooth muscle cells

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is an enzyme that detoxifies aldehydes by converting them to carboxylic acids. ALDH2 deficiency is known to increase oxidative stress. Increased oxidative stress plays a pivotal role in abdominal aortic aneurysm (AAA) pathogenesis. Reactive oxygen species (ROS) promote degradation of the extracellular matrix (ECM) and vascular smooth muscle cell (VSMC) apoptosis. Reducing oxidative stress by an ALDH2 activator could have therapeutic potential for limiting AAA development. We hypothesized that ALDH2 deficiency could increase the risk for AAA by decreasing ROS elimination and that an ALDH2 activator could provide an alternative option for AAA treatment. The National Center for Biotechnology (NCBI) Gene Expression Omnibus (GEO) database was used. Human aortic smooth muscle cells (HASMCs) were used for the in vitro experiments. Gene-targeted ALDH2*2 KI knock-in mice on a C57BL/6J background and apolipoprotein E knockout (ApoE KO) mice were obtained. An animal model of AAA was constructed using osmotic minipumps to deliver 1000ng/kg/min angiotensin II (AngII) for 28days. Patients with AAA had significantly lower ALDH2 expression levels than normal subjects. ALDH2*2 KI mice were susceptible to AngII administration, exhibiting significantly increased AAA incidence rates and increased aortic diameters. Alda-1, an ALDH2 activator, reduced AngII-induced ROS production, NF-kB activation, and apoptosis in HASMCs. Alda-1 attenuated AngII-induced aneurysm formation and decreased aortic expansion in ApoE KO mice. We concluded that ALDH2 deficiency is associated with the development of AAAs in humans and a murine disease model. ALDH2 deficiency increases susceptibility to AngII-induced AAA formation by attenuating anti-ROS effects and increasing VSMC apoptosis and vascular inflammation. Alda-1 was shown to attenuate the progression of experimental AAA in a murine model.

Blockade of intercellular adhesion molecule-1 prevents angiotensin II-induced hypertension and vascular dysfunction

Monocyte and adhesion infiltration into the arterial subendothelium are initial steps in hypertension development. The endothelial intercellular adhesion molecule-1 (ICAM-1) has been implicated in the recruitment and adhesion of leukocytes in several cardiac diseases. However, the role of ICAM-1 in angiotensin II (Ang II)-induced hypertension development remains unknown. Hypertension was induced by administering an infusion of Ang II (1000 ng/kg/min) to wild-type (WT) mice treated with an IgG control or ICAM-1 neutralizing antibody (1 and 2 mg/mouse/day, respectively). Blood pressure was determined using the tail-cuff system. Vascular remodeling was assessed by performing a histological examination. Inflammation and reactive oxygen species (ROS) levels were determined by using immunostaining. Vascular dysfunction was assessed by aortic ring assay. The expression of fibrotic markers, cytokines and NOX was evaluated by quantitative real-time PCR analysis. Our results demonstrate that Ang II infusion markedly increased the ICAM-1 level in the aorta. Blocking ICAM-1 with a neutralizing antibody significantly attenuated Ang II-induced arterial hypertension, vascular hypertrophy, fibrosis, macrophage infiltration, and ROS production and improved vascular relaxation. In conclusion, ICAM-1-mediated monocyte adhesion and migration play a critical role in Ang II-induced arterial hypertension and vascular dysfunction. ICAM-1 inhibitors may represent a new therapeutic strategy for the treatment of this disease.

ClC-3 promotes angiotensin II-induced reactive oxygen species production in endothelial cells by facilitating Nox2 NADPH oxidase complex formation.

Recent evidence suggests that ClC-3, a member of the ClC family of Cl- channels or Cl-/H+ antiporters, plays a critical role in NADPH oxidase-derived reactive oxygen species (ROS) generation. However, the underling mechanisms remain unclear. In this study we investigated the effects and mechanisms of ClC-3 on NADPH oxidase activation and ROS generation in endothelial cells. Treatment with angiotensin II (Ang II, 1 μmol/L) significantly elevated ClC-3 expression in cultured human umbilical vein endothelial cells (HUVECs). Furthermore, Ang II treatment increased ROS production and NADPH oxidase activity, an effect that could be significantly inhibited by knockdown of ClC-3, and further enhanced by overexpression of ClC-3. SA-β-galactosidase staining showed that ClC-3 silencing abolished Ang II-induced HUVEC senescence, whereas ClC-3 overexpression caused the opposite effects. We further showed that Ang II treatment increased the translocation of p47phox and p67phox from the cytosol to membrane, accompanied by elevated Nox2 and p22phox expression, which was significantly attenuated by knockdown of ClC-3 and potentiated by overexpression of ClC-3. Moreover, overexpression of ClC-3 increased Ang II-induced phosphorylation of p47phox and p38 MAPK in HUVECs. Pretreatment with a p38 inhibitor SB203580 abolished ClC-3 overexpression-induced increase in p47phox phosphorylation, as well as NADPH oxidase activity and ROS generation. Our results demonstrate that ClC-3 acts as a positive regulator of Ang II-induced NADPH oxidase activation and ROS production in endothelial cells, possibly via promoting both Nox2/p22phox expression and p38 MAPK-dependent p47phox/p67phox membrane translocation, then increasing Nox2 NADPH oxidase complex formation.

Role of PI3‐kinase in Angiotensin II‐induced Cardiac Hypertrophy: Shift from Class III to Class I

Cardiac hypertrophy is initially an adaptive response to increased hemodynamic workload or defective contractile machinery; however, in the long‐term, it contributes to heart failure and sudden cardiac death. The extracellular regulatory factors and intracellular signaling pathways involved are not fully clear. Here, we studied the chronic effect of Ang II infusion on cardiac hypertrophy and fibrosis, and evaluated the role of class I PI3‐kinase in these actions of Ang II using lentiviral vector‐mediated expression of a dominant negative form of Class I PI3‐kinase subunit p85α in the heart of rats. Subcutaneous 4‐week infusion of Ang II induced severe cardiac hypertrophy and perivascular fibrosis in the heart. Cardiac transduction of Lv‐DNp85 abolished Ang II‐induced phosphorylation of Akt in the heart, as well as Ang II‐induced cardiac hypertrophy and perivascular fibrosis. Next, we examined intracellular mechanisms underlying Ang II‐induced cardiac hypertrophy in cultured cardiomyocytes. It is well known that mitochondrial oxidative stress mediates Ang II‐induced cardiac hypertrophy and the major source of reactive oxygen species (ROS), damaged mitochondria, is cleared by autophagy (also called mitophagy) in cardiac myocytes. Treatment of cardiomyocytes with Ang II (10−9 to 10−5 M) significantly increased autophagy with two phases (increases at low doses, decreases at high doses, peaked at 10−7 M). Ang II (10−6 M)‐induced increases in autophagy were significantly potentiated by PI3‐kinase Class I inhibitor, LY294002 (1 μM) and dramatically attenuated by 3‐Methyladenine (3‐MA, 1 μM), a PI3‐kinase Class III inhibitor. Ang II‐induced mitochondrial ROS production, measured using MitoSOX, was attenuated by LY294002 and potentiated by 3‐MA significantly. PI3‐kinases activity were measured using ELISA in cardiomyocytes, demonstrating that Ang II (10−6 M, i.e. high dose) significantly increased the activity of both Class I and Class III PI3‐kinases; however, Ang II (10−9 M, i.e. low dose) selectively elevated PI3‐kinase Class III activity. Together these data suggest that Ang II could have dual effects in the heart: 1) a protective arm to prevent ROS over‐generation in physiological cardiac hypertrophy, which is mediated by a Class III PI3‐kinase/autophagy‐dependent pathway; and 2) a deleterious arm due to increased mitochondrial ROS production through inhibition of autophagy by a Class I PI3‐kinase pathway in cardiomyocytes.Support or Funding InformationSupported by NIH NS55008.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 032: Microglial TLR4 Mediate Angiotensin II-induced Reactive Oxygen Species Production within the Paraventricular Nucleus in Hypertension

Angiotensin II (AngII) contribution to hypertension involves CNS inflammation that includes cytokine release and reactive oxygen species (ROS) production. The innate immune system, via TLR4 signaling, has been implicated in AngII-mediated inflammatory responses. Yet, whether microglia, the immune cells of the CNS, are key cell targets mediating these effects is still unknown. Thus, we studied here whether TLR4 is a molecular link connecting AngII mediated microglia activation and ROS production within the paraventricular nucleus (PVN) during hypertension. TLR4 and AngII type 1 receptor (AT1) mRNA expression was found in isolated PVN microglia, providing molecular evidence for AT1/TLR4 crosstalk. Isolated microglia TLR4 mRNA expression was 2.56*-fold higher in hypertension. TLR4 and microglia (IBA1) immunoreactivity density were increased in the PVN of SHR vs WKY (TLR4 1.8±0.02 vs 1.3±0.1*; IBA1 23±3.03 vs 13.6±0.6* Arbitrary units - AU). Altered density was attenuated in SHR treated with AT1 blocker Losartan (1.3±0.05 and 14.9±0.9 AU). Higher TLR4/IBA1 co-localization was found in SHR vs WKY (4.6±0.9 vs 1.3±0.2* AU), supporting microglia activation and TLR4 upregulation in hypertension. To study whether AngII-induced microglia activation and ROS production involved TLR4, we used TLR4 deficient (TLR4-D, C3H/HeJ) and sufficient (TLR4-S, C3H/OuJ) mice. Acute hypothalamic slices exposed to AngII (1μM, 60 min) showed increased IBA1 density in the PVN of TLR4-S (3.9±0.1 to 6.5±0.3*%) and WKY (9.8±1.1 to 16.2±1.4*%). This effect was blunted in TLR4-D (4.3±0.1 to 5.2±0.5%). In SHR, AngII failed to further promote microglia activation (17.3±1.8 vs 14.1±1.1 AU). AngII (1 μΜ) increased dihydroethidium (DHE) staining (an indirect measure of ROS) in the PVN of WKY (166±2*) and TLR4-S (129±2*) compared to vehicle (100±3%). This effect was blunted in TLR4-D (102±2%). AngII-induced ROS production was attenuated by the microglia inhibitor minocycline (100 μΜ, 131±2*%) and blunted by Losartan (2 μΜ, 111±6%), suggesting AngII-ROS production involves microglia and AT1. Our results support a major contribution of microglial TLR4 to AngII-mediated ROS and microglia activation within the PVN, actions that are upregulated in hypertension. *P<0.05.

TLR4 is a critical regulator of angiotensin II-induced vascular remodeling: the roles of extracellular SOD and NADPH oxidase.

Toll-like receptor 4 (TLR4) and angiotensin II (AngII) induce vascular remodeling through the production of reactive oxygen species (ROS). AngII has also been shown to increase antioxidant enzyme extracellular superoxide dismutase (ecSOD). However, the roles of TLR4 in Ang II-induced ROS production, vascular remodeling and hypertension remain unknown. Mice lacking TLR4 function showed significant inhibition of vascular remodeling in response to chronic AngII infusion, with no impact on blood pressure. The increases in ROS level and NADPH oxidase activity in response to AngII infusion were markedly blunted in TLR4-deficient mice. Similar effects were observed in wild-type (WT) mice treated with a sub-depressor dose of the AT1 receptor antagonist irbesartan, which had no effects on TLR4-deficient mice. Intriguingly, the AngII infusion-induced increases in ecSOD activity and expression were rather enhanced in TLR4-deficient mice compared with WT mice, whereas the expression of the proinflammatory chemokine MCP-1 was decreased. Importantly, AngII-induced vascular remodeling was positively correlated with NADPH oxidase activity, ROS levels and MCP-1 expression levels. Notably, chronic norepinephrine infusion, which elevates blood pressure without increasing ROS production, did not induce significant vascular remodeling in WT mice. Taken together, these findings suggest that ROS elevation is required for accelerating vascular remodeling but not for hypertensive effects in this model. We demonstrated that TLR4 plays a pivotal role in regulating AngII-induced vascular ROS levels by inhibiting the expression and activity of the antioxidant enzyme ecSOD, as well as by activating NADPH oxidase, which enhances inflammation to facilitate the progression of vascular remodeling.

Qian Yang Yu Yin Granule-containing serum inhibits angiotensin II-induced proliferation, reactive oxygen species production, and inflammation in human mesangial cells via an NADPH oxidase 4-dependent pathway.

BackgroundQian Yang Yu Yin Granule (QYYYG), a traditional Chinese herbal medicine, has been indicated for renal damage in hypertension for decades in China, but little remains known regarding its underlying molecular mechanism. Therefore, we performed the current study in order to investigate the underlying molecular mechanism of QYYYG in the treatment of hypertensive renal damage.MethodsWe hypothesize that QYYYG relieves hypertensive renal injury through an angiotensin II (Ang II)-nicotinamide adenine dinucleotide phosphate (NAPDH)-oxidase (NOX)-reactive oxygen species (ROS) pathway. In this study, we investigated the effects of QYYYG-containing serum (QYGS) in human mesangial cells (HMCs) against Ang II-induced cell proliferation, ROS production, and inflammation through the seropharmacological method.ResultsWe found that QYGS could inhibit cell proliferation in Ang II-treated HMCs. In addition, QYGS considerably suppressed production of ROS, decreased mRNA and protein expression of NAPDH-oxidase 4 (NOX4), p22phox, and activated Ras-related C3 botulinum toxin substrate 1 (GTP-Rac1); as well as counteracted the up-regulation of inflammatory markers including tumor necrosis factor-α (TNF-α), nuclear factor-κB (NF-κB) p65, and interleukin 6 (IL-6). These effects were further confirmed in HMCs transfected with specific small interfering RNA (siRNA) targeting NOX4.ConclusionsTaken together, these results suggest that a NOX4-dependent pathway plays an important role in regulating the inhibitory effect of QYGS. Our findings provide new insights into the molecular mechanisms of QYYYG and their role in the treatment of hypertensive nephropathy.

Endothelial cell-specific reactive oxygen species production increases susceptibility to aortic dissection.

Increased production of reactive oxygen species (ROS) throughout the vascular wall is a feature of cardiovascular disease states, but therapeutic strategies remain limited by our incomplete understanding of the role and contribution of specific vascular cell ROS to disease pathogenesis. To investigate the specific role of endothelial cell (EC) ROS in the development of structural vascular disease, we generated a mouse model of endothelium-specific Nox2 overexpression and tested the susceptibility to aortic dissection after angiotensin II (Ang II) infusion. A specific increase in endothelial ROS production in Nox2 transgenic mice was sufficient to cause Ang II-mediated aortic dissection, which was never observed in wild-type mice. Nox2 transgenic aortas had increased endothelial ROS production, endothelial vascular cell adhesion molecule-1 expression, matrix metalloproteinase activity, and CD45(+) inflammatory cell infiltration. Conditioned media from Nox2 transgenic ECs induced greater Erk1/2 phosphorylation in vascular smooth muscle cells compared with wild-type controls through secreted cyclophilin A (CypA). Nox2 transgenic ECs (but not vascular smooth muscle cells) and aortas had greater secretion of CypA both at baseline and in response to Ang II stimulation. Knockdown of CypA in ECs abolished the increase in vascular smooth muscle cell Erk1/2 phosphorylation conferred by EC conditioned media, and preincubation with CypA augmented Ang II-induced vascular smooth muscle cell ROS production. These findings demonstrate a pivotal role for EC-derived ROS in the determination of the susceptibility of the aortic wall to Ang II-mediated aortic dissection. ROS-dependent CypA secretion by ECs is an important signaling mechanism through which EC ROS regulate susceptibility of structural components of the aortic wall to aortic dissection.

Alpha-Asarone Protects Endothelial Cells from Injury by Angiotensin II.

α-Asarone is the major therapeutical constituent of Acorus tatarinowii Schott. In this study, the potential protective effects of α-asarone against endothelial cell injury induced by angiotensin II were investigated in vitro. The EA.hy926 cell line derived from human umbilical vein endothelial cells was pretreated with α-asarone (10, 50, 100 µmol/L) for 1 h, followed by coincubation with Ang II (0.1 µmol/L) for 24 h. Intracellular nitric oxide (NO) and reactive oxygen species (ROS) were detected by fluorescent dyes, and phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser1177 was determined by Western blotting. α-Asarone dose-dependently mitigated the Ang II-induced intracellular NO reduction (P < 0.01 versus model) and ROS production (P < 0.01 versus model). Furthermore, eNOS phosphorylation (Ser1177) by acetylcholine was significantly inhibited by Ang II, while pretreatment for 1 h with α-asarone partially prevented this effect (P < 0.05 versus model). Additionally, cell viability determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (105~114.5% versus control, P > 0.05) was not affected after 24 h of incubation with α-asarone at 1–100 µmol/L. Therefore, α-asarone protects against Ang II-mediated damage of endothelial cells and may be developed to prevent injury to cardiovascular tissues.

B-type natriuretic peptide inhibits angiotensin II-induced proliferation and migration of pulmonary arterial smooth muscle cells

Pulmonary vascular remodeling, characterized by disordered proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), is a pathognomonic feature of pulmonary arterial hypertension. Thus, pharmacologic strategy targeting on anti-proliferation and anti-migration of PASMCs may have therapeutic implications for PAH. Here we investigated the effects and underlying mechanisms of B-type natriuretic peptide (BNP) on angiotensin II (Ang II)-induced proliferation and migration of PASMCs. Proliferation and migration of PASMCs cultured from Wistar rats were induced by Ang II, with or without BNP treatment. In addition, potential underlying mechanisms including cell cycle progression, Ca(2+) overload, reactive oxygen species (ROS) production, signal transduction of MAPK and Akt, and the cGMP/PKG pathway were examined. We found that BNP inhibited Ang II-induced PASMCs proliferation and migration dose dependently. BNP could also arrest the cell cycle progression in the G0/G1-phase. In addition, BNP attenuated intracellular calcium overload caused by Ang II. Moreover, Ang II-induced ROS production was mitigated by BNP, with associated down-regulation of NAD(P)H oxidase 1 (Nox1) and reduced mitochondrial ROS production. Finally, Ang II-activated MAPKs and Akt were also counteracted by BNP. Of note, all these effects of BNP were abolished by a PKG inhibitor (Rp-8-Br-PET-cGMPS). In conclusion, BNP inhibits Ang II-induced PASMCs proliferation and migration. These effects are potentially mediated by decreased calcium influx, reduced ROS production by Nox1 and mitochondria, and down-regulation of MAPK and Akt signal transduction, through the cGMP/PKG pathway. Therefore, this study implicates that BNP may have a therapeutic role in pulmonary vascular remodeling.

Cyclophilin A Is Required for Angiotensin II–Induced p47phox Translocation to Caveolae in Vascular Smooth Muscle Cells

Angiotensin II (AngII) signal transduction in vascular smooth muscle cells (VSMC) is mediated by reactive oxygen species (ROS). Cyclophilin A (CyPA) is a ubiquitously expressed cytosolic protein that possesses peptidyl-prolyl cis-trans isomerase activity, scaffold function, and significantly enhances AngII-induced ROS production in VSMC. We hypothesized that CyPA regulates AngII-induced ROS generation by promoting translocation of NADPH oxidase cytosolic subunit p47phox to caveolae of the plasma membrane. Overexpression of CyPA in CyPA-deficient VSMC (CyPA(-/-)VSMC) significantly increased AngII-stimulated ROS production. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors (VAS2870 or diphenylene iodonium) significantly attenuated AngII-induced ROS production in CyPA and p47phox-overexpressing CyPA(-/-)VSMC. Cell fractionation and sucrose gradient analyses showed that AngII-induced p47phox plasma membrane translocation, specifically to the caveolae, was reduced in CyPA(-/-)VSMC compared with wild-type-VSMC. Immunofluorescence studies demonstrated that AngII increased p47phox and CyPA colocalization and translocation to the plasma membrane. In addition, immunoprecipitation of CyPA followed by immunoblotting of p47phox and actin showed that AngII increased CyPA and p47phox interaction. AngII-induced p47phox and actin cell cytoskeleton association was attenuated in CyPA(-/-)VSMC. Mechanistically, inhibition of p47phox phosphorylation and phox homology domain deletion attenuated CyPA and p47phox interaction. Finally, cyclosporine A and CyPA-peptidyl-prolyl cis-trans isomerase mutant, R55A, inhibited AngII-stimulated CyPA and p47phox association in VSMC, suggesting that peptidyl-prolyl cis-trans isomerase activity was required for their interaction. These findings provide the mechanism by which CyPA is an important regulator for AngII-induced ROS generation in VSMC through interaction with p47phox and cell cytoskeleton, which enhances the translocation of p47phox to caveolae.

Antihypertensive effect of gomisin A from Schisandra chinensis on angiotensin II-induced hypertension via preservation of nitric oxide bioavailability

Gomisin A (GA) is a small molecular weight lignan present in Schisandra chinensis, and has been demonstrated to have vasodilatory activity. In the present study, we investigated the effect of GA on blood pressure (BP) in angiotensin II (Ang II)-induced hypertensive mice. C57/BL6 mice infused subcutaneously with Ang II (1 and 2 μg kg−1 per min for 2 weeks) showed an increase in BP with a decrease in nitric oxide (NO) metabolites in plasma, and a negative correlation between these two parameters was demonstrated. In the thoracic aorta from Ang II-induced hypertensive mice, a decrease in vascular NO that was accompanied by a diminution of phosphorylated endothelial nitric oxide synthase (eNOS), as well as by increased reactive oxygen species (ROS) production, was demonstrated. These alterations in BP, eNOS phosphorylation and ROS production in the vasculature of Ang II-treated mice were markedly and dose-dependently reversed by simultaneous administration of GA (2 and 10 μg kg−1 per min). In addition, Ang II-induced ROS production in cultured vascular cells such as endothelial cells and vascular smooth muscle cells was markedly attenuated by GA. These results suggested that GA attenuated the increase in BP via preservation of vascular NO bioavailability not only by inhibiting ROS production but also by preventing the impairment of eNOS function in the vasculature of Ang II-induced hypertensive mice.

Angiotensin II type 2 receptor-coupled nitric oxide production modulates free radical availability and voltage-gated Ca2+ currents in NTS neurons.

The medial region of the nucleus tractus solitarius (mNTS) is a key brain stem site controlling cardiovascular function, wherein ANG II modulates neuronal L-type Ca(2+) currents via activation of ANG II type 1 receptors (AT(1)R) and production of reactive oxygen species (ROS). ANG II type 2 receptors (AT(2)R) induce production of nitric oxide (NO), which may interact with ROS and modulate AT(1)R signaling. We sought to determine whether AT(2)R-mediated NO production occurs in mNTS neurons and, if so, to elucidate the NO source and the functional interaction with AT(1)R-induced ROS or Ca(2+) influx. Electron microscopic (EM) immunolabeling showed that AT(2)R and neuronal NO synthase (nNOS) are coexpressed in neuronal somata and dendrites receiving synapses in the mNTS. In the presence of the AT(1)R antagonist losartan, ANG II increased NO production in isolated mNTS neurons, an effect blocked by the AT(2)R antagonist PD123319, but not the angiotensin (1-7) antagonist D-Ala. Studies in mNTS neurons of nNOS-null or endothelial NOS (eNOS)-null mice established nNOS as the source of NO. ANG II-induced ROS production was enhanced by PD123319, the NOS inhibitor N(G)-nitro-l-arginine (LNNA), or in nNOS-null mice. Moreover, in the presence of losartan, ANG II reduced voltage-gated L-type Ca(2+) current, an effect blocked by PD123319 or LNNA. We conclude that AT(2)R are closely associated and functionally coupled with nNOS in mNTS neurons. The resulting NO production antagonizes AT(1)R-mediated ROS and dampens L-type Ca(2+) currents. The ensuing signaling changes in the NTS may counteract the deleterious effects of AT(1)R on cardiovascular function.

Inactivation of Adenosine A2A Receptor Attenuates Basal and Angiotensin II-induced ROS Production by Nox2 in Endothelial Cells

Endothelial cells (ECs) express a Nox2 enzyme, which, by generating reactive oxygen species (ROS), contributes to EC redox signaling and angiotensin II (AngII)-induced endothelial dysfunction. ECs also express abundantly an adenosine A2A receptor (A2AR), but its role in EC ROS production remains unknown. In this study, we investigated the role of A2AR in the regulation of Nox2 activity and signaling in ECs with or without acute AngII stimulation. In cultured ECs (SVEC4–10), AngII (100 nm, 30 min) significantly increased Nox2 membrane translocation and association with A2AR. These were accompanied by p47phox, ERK1/2, p38 MAPK, and Akt phosphorylation and an increased ROS production (169 ± 0.04%). These AngII effects were inhibited back to the control levels by a specific A2AR antagonist (SCH58261), or adenosine deaminase, or by knockdown of A2AR or Nox2 using specific siRNAs. Knockdown of A2AR, as determined by Western blotting, decreased Nox2 and p47phox expression. In wild-type mouse aorta, SCH58261 significantly reduced acute AngII-induced ROS production and preserved endothelium-dependent vessel relaxation to acetylcholine. These results were further confirmed by using aortas from A2AR knock-out mice. In conclusion, A2AR is involved in the regulation of EC ROS production by Nox2. Inhibition or blockade of A2AR protects ECs from acute AngII-induced oxidative stress, MAPK activation, and endothelium dysfunction.

BAS/BSCR28 Blockade of adenosine A2A receptor attenuates angiotensin II-induced reactive oxygen species production and impairment of endothelium-dependent vessel relaxation in mouse aortas

The endothelium expresses abundantly an adenosine A2A receptor (A2AR) which has important roles in the regulation of vascular function. The endothelium expresses also the AT1-type receptor for angiotensin II (AngII)...

EGCG reduces Ang II‐induced NAD(P)H oxidase and NO synthase expression in HUVECs

Atherosclerosis is a chronic systemic disease of the vasculature with an inflammatory component. The impairment of vascular endothelial cell function which is an important determinant of atherosclerosis progression is in part due to increased release of reactive oxygen species (ROS). Angiotensin II (Ang II) causes endothelial dysfunction by increasing NAD(P)H oxidase‐mediated vascular superoxide production. Epigallocatechin‐3‐gallate (EGCG), the major bioactive polyphenol present in green tea and a potent antioxidant, has been reported to lower the risk of cardiovascular diseases such as atherosclerosis and hypertension. In this study, we investigated the effects of EGCG on Ang II‐induced expression of NAD(P)H oxidase and nitric oxide synthase (NOS), which may contribute to oxidative stress. We observed that Ang II stimulated mRNA and protein expression of NAD(P)H oxidase subunit and iNOS in human umbilical vein endothelial cells (HUVECs). EGCG inhibited Ang II‐induced expression of NAD(P)H oxidase subunit and iNOS in a concentration‐dependent manner. However, EGCG did not affect Ang II‐induced ROS production. Surprisingly, EGCG itself produced ROS in HUVECs. These results suggest that anti‐atherosclerotic activity of EGCG is likely not due to decreased ROS formation although there may be some benefit through inhibition of expression of NAD(P)H oxidase and NOS induced by Ang II.