Guoying Yao

Expression of a Functionally Active gp91phox-Containing Neutrophil-Type NAD(P)H Oxidase in Smooth Muscle Cells From Human Resistance Arteries Regulation by Angiotensin II

A major source of vascular smooth muscle cell (VSMC) superoxide is NAD(P)H oxidase. However, the molecular characteristics and regulation of this enzyme are unclear. We investigated whether VSMCs from human resistance arteries (HVSMCs) possess a functionally active, angiotensin II (Ang II)–regulated NAD(P)H oxidase that contains neutrophil oxidase subunits, including p22phox, gp91phox, p40phox, p47phox, and p67phox. mRNA expression of gp91phox homologues, nox1 and nox4, was also assessed in HVSMCs, human aortic smooth muscle cells, and rat VSMCs. HVSMCs were obtained from resistance arteries from gluteal biopsies of healthy subjects. gp91phox and nox4, but not nox1, were detected in HVSMCs. Nox1 and nox4, but not gp91phox, were expressed in human aortic smooth muscle cells and rat VSMCs. All NAD(P)H oxidase subunits were present in HVSMCs as detected by reverse transcriptase–polymerase chain reaction and immunoblotting. Ang II increased NAD(P)H oxidase subunit abundance. These effects were inhibited by cycloheximide. Acute Ang II stimulation (10 to 15 minutes) increased p47phox serine phosphorylation and induced p47phox and p67phox translocation. This was associated with NAD(P)H oxidase activation. In cells transfected with gp91phox antisense oligonucleotides, Ang II–mediated actions were abrogated. NADPH-induced superoxide generation was reduced by gp91ds-tat and apocynin, inhibitors of p47phox-gp91phox interactions. Our results suggest that HVSMCs possess a functionally active gp91phox-containing neutrophil-like NAD(P)H oxidase. Ang II regulates the enzyme by inducing phosphorylation of p47phox, translocation of cytosolic subunits, and de novo protein synthesis. These novel findings provide insight into the molecular regulation of NAD(P)H oxidase by Ang II in HVSMCs. Furthermore, we identify differences in gp91phox homologue expression in VSMCs from rats and human small and large arteries.

c-Src Induces Phosphorylation and Translocation of p47phox: Role in Superoxide Generation by Angiotensin II in Human Vascular Smooth Muscle Cells

Objectives—The aim of this study was to determine molecular mechanisms whereby c-Src regulates angiotensin II (Ang II)-mediated NAD(P)H oxidase-derived ·O2− in human vascular smooth muscle cells (VSMCs). Methods and Results—VSMCs from human small arteries were studied. Ang II increased NAD(P)H oxidase-mediated generation of ·O2− and H2O2 (P <0.01). PP2, c-Src inhibitor, attenuated these effects by 70% to 80%. Immunoprecipitation of p47phox, followed by immunoblotting with antiphosphoserine antibody, demonstrated a rapid increase (1.5- to 2-fold) in p47phox phosphorylation in Ang II-stimulated cells. This was associated with p47phox translocation from cytosol to membrane, as assessed by immunoblotting and immunofluorescence. PP2 abrogated these effects. Long-term Ang II stimulation (6 to 24 hours) increased NAD(P)H oxidase subunit expression. c-Src inhibition decreased abundance of gp91phox, p22phox, and p47phox. Confirmation of c-Src-dependent regulation of NAD(P)H oxidase was tested in VSMCs from c-Src−/− mice. Ang II-induced ·O2− generation was lower in c-Src−/− than c-Src+/+ counterparts. This was associated with decreased p47phox phosphorylation, blunted Ang II-stimulated NAD(P)H oxidase activation, and failure of Ang II to increase subunit expression. Conclusions—c-Src regulates NAD(P)H oxidase-derived ·O2− generation acutely by stimulating p47phox phosphorylation and translocation and chronically by increasing protein content of gp91phox, p22phox, and p47phox in Ang II-stimulated cells. These novel findings identify NAD(P)H oxidase subunits, particularly p47phox, as downstream targets of c-Src.

Angiotensin II and endothelin-1 regulate MAP kinases through different redox-dependent mechanisms in human vascular smooth muscle cells.

Objective The role of reactive oxygen species (ROS) in mitogen-activated protein kinase (MAPK) signaling by angiotensin (Ang) II and endothelin-1 (ET-1) in human vascular smooth muscle cells (VSMC) was investigated. Design VSMCs were derived from resistance arteries from healthy subjects. MAPK activity was assessed using phospho-specific antibodies. ROS generation was measured by CMH2DCFDA fluorescence and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity by lucigenin chemiluminescence. Results Ang II and ET-1 increased MAPK phosphorylation (P < 0.01). Pre-treatment with Tiron and Tempol, •O2− scavengers, attenuated agonist-stimulated phosphorylation of p38MAPK, c-Jun N-terminal kinases (JNK) and ERK5, but not of ERK1/2 (extracellular signal-regulated kinases). Apocynin and diphenylene iodinium (DPI), NAD(P)H oxidase inhibitors, decreased Ang II-induced responses 60–70%. ET-1-mediated MAPK phosphorylation was unaffected by apocynin but was reduced (> 50%) by thenoyltrifluoroacetone (TIFT) and carboxyl cyanide-m-chlorophenylhydrazone (CCCP), mitochondrial inhibitors. Allopurinol and Nω-nitro-l-arginine methyl ester (l-NAME), xanthine oxidase and nitric oxide synthase (NOS) inhibitors, respectively, did not influence MAPK activation. Intracellular ROS generation, was increased by Ang II and ET-1 (P < 0.01). DPI inhibited Ang II- but not ET-1-mediated ROS production. Expression of p22phox and p47phox and activation of NAD(P)H oxidase were increased by Ang II but not by ET-1. CCCP and TIFT significantly attenuated ET-1-mediated ROS formation (P < 0.05), without influencing Ang II effects. Conclusions Ang II activates p38MAPK, JNK and ERK5 primarily through NAD(P)H oxidase-generated ROS. ET-1 stimulates these kinases via redox-sensitive processes that involve mitochondrial-derived ROS. These data suggest that redox-dependent activation of MAPKs by Ang II and ET-1 occur through distinct ROS-generating systems that could contribute to differential signaling by these agonists in VSMCs.

Transient Receptor Potential Melastatin 7 Ion Channels Regulate Magnesium Homeostasis in Vascular Smooth Muscle Cells: Role of Angiotensin II

Magnesium modulates vascular smooth muscle cell (VSMC) function. However, molecular mechanisms regulating VSMC Mg2+ remain unknown. Using biochemical, pharmacological, and genetic tools, the role of transient receptor potential membrane melastatin 7 (TRPM7) cation channel in VSMC Mg2+ homeostasis was evaluated. Rat, mouse, and human VSMCs were studied. Reverse transcriptase polymerase chain reaction and immunoblotting demonstrated TRPM7 presence in VSMCs (membrane and cytosol). Angiotensin II (Ang II) and aldosterone increased TRPM7 expression. Gene silencing using small interfering RNA (siRNA) against TRPM7, downregulated TRPM7 (mRNA and protein). Basal [Mg2+]i, measured by mag fura-2AM, was reduced in siRNA-transfected cells (0.39±0.01 mmol/L) versus controls (0.54±0.01 mmol/L; P<0.01). Extracellular Mg2+ dose-dependently increased [Mg2+]i in control cells (Emax 0.70±0.02 mmol/L) and nonsilencing siRNA-transfected cells (Emax 0.71±0.04 mmol/L), but not in siRNA-transfected cells (Emax 0.5±0.01 mmol/L). The functional significance of TRPM7 was evaluated by assessing [Mg2+]i and growth responses to Ang II in TRPM7 knockdown cells. Acute Ang II stimulation decreased [Mg2+]i in control and TRPM7-deficient cells in a Na+-dependent manner. Chronic stimulation increased [Mg2+]i in control, but not in siRNA-transfected VSMCs. Ang II–induced DNA and protein synthesis, measured by 3[H]-thymidine and 3[H]-leucine incorporation, respectively, were increased in control and nonsilencing cells, but not in TRPM7 knockdown VSMCs. Our data indicate that VSMCs possess membrane-associated, Ang II–, and aldosterone-regulated TRPM7 channels, which play a role in regulating basal [Mg2+]i, transmembrane Mg2+ transport and DNA and protein synthesis. These novel findings identify TRPM7 as a functionally important regulator of Mg2+ homeostasis and growth in VSMCs.

p47phox Associates With the Cytoskeleton Through Cortactin in Human Vascular Smooth Muscle Cells: Role in NAD(P)H Oxidase Regulation by Angiotensin II

Objective—We tested the hypothesis that p47phox associates with the actin cytoskeleton, enabling site-directed activation of NAD(P)H oxidase, and assessed whether these actions influence reactive oxygen species (ROS) generation and signaling by angiotensin II (Ang II) in vascular smooth muscle cells (VSMCs) from human resistance and coronary arteries. Methods and Results—Electroporation of anti-p47phox antibody into VSMCs abrogated Ang II–mediated &OV0151; generation, establishing the requirement for p47phox in this response. Immunfluorescence confocal microscopy demonstrated a cytosolic distribution of p47phox in basal conditions. After Ang II stimulation, p47phox rearranged in a linear fashion, colocalizing with F-actin. Co-immunoprecipitation studies confirmed an association between p47phox and actin and demonstrated an interaction with the actin-binding protein cortactin. Cytoskeletal disruption with cytochalasin prevented p47phox:actin interaction and attenuated ROS formation and p38MAP kinase and Akt phosphorylation by Ang II. Intracellular ROS generation in response to LY83583 (&OV0151; generator) or exogenous H2O2 and Ang II–induced ERK1/2 activation were unaltered by cytochalasin. Conclusions—The p47phox:actin interaction, through cortactin, plays an important role in Ang II–mediated site-directed assembly of functionally active NAD(P)H oxidase, ROS generation, and activation of redox-sensitive p38MAP kinase and Akt, but not ERK1/2. These findings demonstrate the importance of an intact actin–cytoskeleton in NAD(P)H oxidase regulation and redox signaling by Ang II in human VSMCs.

Angiotensin II-Dependent Chronic Hypertension and Cardiac Hypertrophy Are Unaffected by gp91phox-Containing NADPH Oxidase

The gp91phox-containing NADPH oxidase is the major source of reactive oxygen species (ROS) in the cardiovascular system and inactivation of gp91phox has been reported to blunt hypertension and cardiac hypertrophy seen in angiotensin (Ang) II-infused animals. In the current study, we sought to determine the role of gp91phox-derived ROS on cardiovascular outcomes of chronic exposure to Ang II. The gp91phox-deficient mice were crossed with transgenic mice expressing active human renin in the liver (TTRhRen). TTRhRen mice exhibit chronic Ang II–dependent hypertension and frank cardiac hypertrophy by age 10 to 12 weeks. Four genotypes of mice were generated: control, TTRhRen trangenics (TTRhRen), gp91phox-deficient (gp91−), and TTRhRen transgenic gp91phox-deficient (TTRhRen/gp91−). Eight to 10 mice/group were studied. ROS levels were significantly reduced (P <0.05) in the heart and aorta of TTRhRen/gp91− and gp91−mice compared with control counterparts, and this was associated with reduced cardiac, aortic, and renal NADPH oxidase activity (P <0.05). Systolic blood pressure (SBP), cardiac mass, and cardiac fibrosis were increased in TTRhRen versus controls. In contrast to its action on ROS generation, gp91phox inactivation had no effect on development of hypertension or cardiac hypertrophy in TTRhRen mice, although interstitial fibrosis was reduced. Cardiac and renal expression of gp91phox homologues, Nox1 and Nox4, was not different between groups. Thus, although eliminating gp91phox-associated ROS production may be important in cardiovascular consequences in acute insult models, it does not prevent the development of hypertension and cardiac hypertrophy in a model in which the endogenous renin-angiotensin system is chronically upregulated.

Redox-Sensitive Signaling by Angiotensin II Involves Oxidative Inactivation and Blunted Phosphorylation of Protein Tyrosine Phosphatase SHP-2 in Vascular Smooth Muscle Cells From SHR

Angiotensin II (Ang II) signaling in vascular smooth muscle cells (VSMCs) involves reactive oxygen species (ROS) through unknown mechanisms. We propose that Ang II induces phosphorylation of growth signaling kinases by redox-sensitive regulation of protein tyrosine phosphatases (PTP) in VSMCs and that augmented Ang II signaling in spontaneously hypertensive rats (SHRs) involves oxidation/inactivation and blunted phosphorylation of the PTP, SHP-2. PTP oxidation was assessed by the in-gel PTP method. SHP-2 expression and activity were evaluated by immunoblotting and by a PTP activity assay, respectively. SHP-2 and Nox1 were downregulated by siRNA. Ang II induced oxidation of multiple PTPs, including SHP-2. Basal SHP-2 content was lower in SHRs versus WKY. Ang II increased SHP-2 phosphorylation and activity with blunted responses in SHRs. Ang II—induced SHP-2 effects were inhibited by valsartan (AT1R blocker), apocynin (NAD(P)H oxidase inhibitor), and Nox1 siRNA. Ang II stimulation increased activation of ERK1/2, p38MAPK, and AKT, with enhanced effects in SHR. SHP-2 knockdown resulted in increased AKT phosphorylation, without effect on ERK1/2 or p38MAPK. Nox1 downregulation attenuated Ang II–mediated AKT activation in SHRs. Hence, Ang II regulates PTP/SHP-2 in VSMCs through AT1R and Nox1-based NAD(P)H oxidase via two mechanisms, oxidation and phosphorylation. In SHR Ang II–stimulated PTP oxidation/inactivation is enhanced, basal SHP-2 expression is reduced, and Ang II–induced PTP/SHP-2 phosphorylation is blunted. These SHP-2 actions are associated with augmented AKT signaling. We identify a novel redox-sensitive SHP-2–dependent pathway for Ang II in VSMCs. SHP-2 dysregulation by increased Nox1-derived ROS in SHR is associated with altered Ang II–AKT signaling.

Negative regulation of RhoA/Rho kinase by angiotensin II type 2 receptor in vascular smooth muscle cells: role in angiotensin II-induced vasodilation in stroke-prone spontaneously hypertensive rats.

Objective To test whether angiotensin II (Ang II) through the Ang II type 2 receptor (AT2R), downregulates RhoA/Rho kinase, which plays a role in AT1 receptor (AT1R)-mediated function. Methods In vitro studies were performed in A10 vascular smooth muscle cells (VSMC) and in vivo studies in mesenteric arteries from Wistar–Kyoto (WKY) and stroke-prone spontaneously hypertensive (SHRSP) rats. VSMC were stimulated with Ang II (10−7 mol/l), CGP42112A (10−7 mol/l, a selective AT2R agonist) ± valsartan (10−5 mol/l, an AT1R antagonist), or the Rho kinase inhibitor fasudil (10−5 mol/l). AT1R and AT2R expression and myosin light chain (MLC) phosphorylation were determined by immunoblotting. RhoA activity was assessed by measuring membrane translocation. Functional significance between AT2R, RhoA/Rho kinase and vasodilation was assessed in arteries from valsartan-treated (30 mg/kg per day, 14 days) WKY and SHRSP rats. Vasodilatory responses to Ang II (10−9–10−6 mol/l) were performed in norepinephrine pre-contracted vessels ± valsartan(10−6 mol/l), PD123319 (10−6 mol/l, an AT2R antagonist) or fasudil (10−6 mol/l). Results A10 VSMC expressed AT1R and AT2R. In valsartan-treated cells, Ang II-induced RhoA translocation was reduced versus controls (42 ± 6%, P < 0.05). Similar responses were obtained with CGP42112A (45 ± 6%, P < 0.05). This was associated with decreased MLC activation. Fasudil abrogated Ang II- and CGP42112A-mediated effects. Ang II evoked a significant vasodilatory response only in valsartan-treated SHRSP (max dilation 40 ± 7%). PD123319 blocked these effects. Fasudil increased AngII-induced relaxation in SHRSP vessels. AT2R expression was increased by valsartan (two- to three-fold) in SHRSP arteries. RhoA translocation was increased two-fold in untreated SHRSP (P < 0.05) and was reduced by valsartan (P < 0.05). These changes were associated with decreased MLC phosphorylation. Conclusions Ang II/AT2R negatively regulates vascular RhoA/Rho kinase/MLC phosphorylation. These processes may play a role in Ang II-mediated vasodilation in conditions associated with vascular AT2R upregulation, such as in SHRSP chronically treated with AT1R blockers, which may contribute to blood pressure lowering by these antihypertensive agents.

Increased systemic inflammation and oxidative stress in patients with worsening congestive heart failure: improvement after short-term inotropic support.

In the present study, we evaluated circulating pro-inflammatory mediators and markers of oxidative stress in patients with decompensated CHF (congestive heart failure) and assessed whether clinical recompensation by short-term inotropic therapy influences these parameters. Patients with worsening CHF (n=29, aged 61.9+/-2.7 years), NYHA (New York Heart Association) class III-IV, and left ventricular ejection fraction of 23.7+/-1.8% were studied. Controls comprised age-matched healthy volunteers (n=15; 54.1+/-3.2 years). Plasma levels of cytokines [IL (interleukin)-6 and IL-18], chemokines [MCP-1 (monocyte chemotactic protein-1)], adhesion molecules [sICAM (soluble intercellular adhesion molecule), sE-selectin (soluble E-selectin)], systemic markers of oxidation [TBARS (thiobarbituric acid-reactive substances), 8-isoprostaglandin F(2alpha) and nitrotyrosine] and hs-CRP (high-sensitivity C-reactive protein) were measured by ELISA and colorimetric assays at admission and 30 days following 72-h milrinone (n=15) or dobutamine (n=14) infusion. Plasma IL-6, IL-18, sICAM, E-selectin, hs-CRP and oxidative markers were significantly higher in patients on admission before inotropic treatment compared with controls (P<0.05). Short-term inotropic support improved clinical status as assessed by NYHA classification and by the 6-min walk test and significantly decreased plasma levels of IL-6, IL-18, sICAM, hs-CRP and markers of oxidation (P<0.05) at 30 days. The effects of milrinone and dobutamine were similar. In conclusion, our results demonstrate that patients with decompensated CHF have marked systemic inflammation and increased production of oxygen free radicals. Short-term inotropic support improves functional status and reduces indices of inflammation and oxidative stress in patients with decompensated CHF.

Effects of low dietary magnesium intake on development of hypertension in stroke-prone spontaneously hypertensive rats: role of reactive oxygen species.

Objectives To investigate whether low dietary Mg2+ intake influences the development of hypertension in stroke-prone spontaneously hypertensive rats (spSHRs) and whether these effects are associated with vascular functional and structural changes, and to assess the role of reactive oxygen species and the activation of vascular mitogen-activated protein (MAP) kinases in these processes. Methods Six-week-old male spSHRs (n = 18) were divided into three groups: control (normal chow, 0.21% Mg2+), low Mg2+ group (Mg2+-free diet), and high Mg2+ group (Mg2+-rich diet, 0.75%). Systolic blood pressure (SBP) was assessed weekly for 16 weeks. In a second series of experiments, 6-week-old spSHRs (n = 18) were divided into three groups and studied weekly for 7 weeks: control group, low Mg2+ group, and low Mg2+ group receiving the superoxide dismutase mimetic, tempol (1 mmol/l). Results The low Mg2+ diet caused an initial decrease in SBP followed, 5 weeks later, by an exacerbated development of hypertension. This was associated with a transient reduction in the plasma concentrations of substances associated with the thiobarbituric acid reaction (markers of oxidative stress), which increased rapidly 2 weeks later. In the low Mg2+ group, acetylcholine-induced vasodilatation was decreased compared with that in controls (P < 0.05). The media : lumen ratio was greater in rats receiving a low Mg2+ diet than in those fed a high Mg2+ diet (P < 0.05). Mg2+ depletion was associated with increased vascular superoxide anion compared with that in Mg2+-supplemented rats (1.2 ± 0.24 compared with 0.65 ± 0.1 nmol/min per mg). Phosphorylation of MAP kinases was increased two- to threefold in Mg2+-deficient rats. Tempol prevented the progression of hypertension and normalized the vascular changes in rats fed a low Mg2+ diet. Conclusions Chronic Mg2+ deficiency leads to development of severe hypertension, endothelial dysfunction and vascular remodelling. These processes are associated with oxidative stress and upregulation of redox-dependent MAP kinases. Tempol normalized vascular changes and attenuated the development of hypertension. Our findings suggest that reactive oxygen species play an important part in vascular processes that are associated with progression of hypertension in Mg2+-deficient spSHRs.