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p47phox is 1 of the 5 subunits that makes up the multisubunit complex of the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox2), a major source of cellular reactive oxygen species (ROS).1 The prototype Nox2 comprises 2 transmembrane subunits (gp91phox [Nox2] and p22phox) and 3 cytosolic subunits (p47phox, p67phox, and p40phox).2,3 Nox2 is characteristically found in phagocytic cells, where it has a critical antimicrobial function through the production of superoxide anion.2 Nox2, as well as the other Nox homologues, including Nox1, Nox4, and Nox5, is expressed in many cell types, including those of the cardiovascular system.3 Although the function of phagocytic Nox2 is clearly defined, the exact pathophysiological significance of cardiovascular Nox2 is still unclear. Article, see p 1542 p47phox is a major regulator of Nox2.1 In the resting state, it localizes in the cytoplasm where its activity is inhibited through an autoinhibitory phosphorylation. However, on cell stimulation, p47phox interacts with cytosolic subunits p67phox and p40phox and the complex translocates to the cell membrane to assemble the active oxidase, which generates superoxide anion.2 This process is triggered by phosphorylation of p47phox, which is the key for activation of Nox and consequent ROS production. Upregulation of Nox and associated increased ROS generation (oxidative stress) have been implicated in many cardiovascular pathologies, processes that are p47phox dependent because deletion of this subunit reduces oxidative stress and ameliorates pathological cardiac and vascular inflammation and remodeling.4–7 On the basis of this paradigm, in this issue of Circulation Research , Patel et al8 questioned the putative protective effect of p47phox deletion/downregulation in a mouse model of transverse aortic constriction–induced pressure overload and heart failure. Intriguingly and …

Maintaining intracellular redox balance through tightly controlled systems that regulate production (pro-oxidants) and scavenging (antioxidants) of reactive oxygen species (ROS) is critical for normal cell function. In the heart, ROS are produced primarily by mitochondria as a byproduct of electron transport and also by extramitochondrial enzymes, such as nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. Multiple antioxidants including superoxide dismutase, catalase, glutathione peroxidase, and thioredoxin/thioredoxin-reductase systems counterbalance ROS generation, preserving redox equilibrium. Disruption in this equilibrium leads to redox stress, of which 2 main types have been defined: oxidative stress and reductive stress. Oxidative stress, characterized by a shift in the oxidative:reductive potential to a more oxidative state because of increased ROS production by pro-oxidant enzymes and reduced antioxidant defense mechanisms to scavenge excessive ROS, plays a physiological role in aging and wound healing, and a pathophysiological role in atherosclerosis, hypertension, heart failure, and ischemia–reperfusion injury.1 Reductive stress, however, is characterized by an aberrant increase in reducing equivalents, such as reduced glutathione and reduced NADPH, increased activation of antioxidant enzymes and reduced pro-oxidant capacity, leading to a shift in the redox balance from an oxidative to a reduced state (Figure).2 Intuitively, one would consider activation of antioxidant systems as being redox-protective; however, growing evidence indicates that reductive stress may be even more injurious than oxidative stress through processes, whereby intrinsic cytoprotective defences negatively target the exact systems they should protect.3 In the heart, reductive stress has been linked to mitochondrial dysfunction, heart failure, myocardial ischemia–reperfusion injury, cardiac hypertrophy, and cardiomyopathy. These are the exact pathological conditions associated with oxidative stress. Then, how can both an increase in pro-oxidants and an increase in antioxidants or reductants lead to the same outcome? Figure. Schematic demonstration of the interplay between processes that cause oxidative and reductive stress in cardiomyocytes. Cardiac stressors, …

High doses of Ang II receptor (AT1R) blockers (ARBs) are renoprotective in diabetes. Underlying mechanisms remain unclear. We evaluated whether high/ultra-high doses of candesartan (ARB) up-regulate angiotensin-converting enzyme 2 (ACE2)/Ang II type 2 receptor (AT2R)/Mas receptor [protective axis of the of the renin–angiotensin system (RAS)] in diabetic mice. Systolic blood pressure (SBP), albuminuria and expression/activity of RAS components were assessed in diabetic db/db and control db/+ mice treated with increasing candesartan doses (intermediate, 1 mg/kg/d; high, 5 mg/kg/d; ultra-high, 25 and 75 mg/kg/d; 4 weeks). Lower doses candesartan did not influence SBP, but ultra-high doses reduced SBP in both groups. Plasma glucose and albuminuria were increased in db/db compared with db/+ mice. In diabetic mice treated with intermediate dose candesartan, renal tubular damage and albuminuria were ameliorated and expression of ACE2, AT2R and Mas and activity of ACE2 were increased, effects associated with reduced ERK1/2 phosphorylation, decreased fibrosis and renal protection. Ultra-high doses did not influence the ACE2/AT2R/Mas axis and promoted renal injury with increased renal ERK1/2 activation and exaggerated fibronectin expression in db/db mice. Our study demonstrates dose-related effects of candesartan in diabetic nephropathy: intermediate–high dose candesartan is renoprotective, whereas ultra-high dose candesartan induces renal damage. Molecular processes associated with these effects involve differential modulation of the ACE2/AT2R/Mas axis: intermediate–high dose candesartan up-regulating RAS protective components and attenuating pro-fibrotic processes, and ultra-high doses having opposite effects. These findings suggest novel mechanisms through the protective RAS axis, whereby candesartan may ameliorate diabetic nephropathy. Our findings also highlight potential injurious renal effects of ultra-high dose candesartan in diabetes.

Hypertension (HT) and CADASIL are clinical conditions of small vessel disease. Vascular dementia is a major feature in CADASIL, and a serious consequence of HT. CADASIL is a monogenic condition due to mutations in NOTCH3 , which is expressed almost exclusively in VSMCs. We hypothesised that altered NOTCH3 signalling in CADASIL and HT are associated with small vessel disease. Small arteries from gluteal biopsies from CADASIL patients (n=14), HT patients (n=3) and healthy controls (n=10) were investigated. Vascular function was assessed by myography. Cultured VSMCs were used to assess signaling through NOTCH3, NO, ER stress (gene array) and Rho kinase (ELISA). CADASIL and HT patients exhibited endothelial dysfunction (Max response: CADASIL 41.7±3%, HT 54.1±2% vs Control 98.2±4%). Pre-incubation with N-acetyl-cysteine ameliorated vasorelaxation. Only CADASIL displayed impaired endothelium-independent relaxation (Max response: CADASIL 53±1.9% vs Control 93±8.9%) and contraction (Max response: CADASIL 78±1.3% vs control 102±5%) (p1 R mRNA expression (HT: 5.1; CADASIL: 3.8; fold vs control; p

TRPM7 is a cationic ion channel and with a serine/threonine kinase important for cellular Mg 2+ homeostasis. We recently showed that TRPM7-kinase plays a role in aldosterone-mediated vascular effects and inflammation. Here we explored the role of TRPM7-kinase in cardiac fibrosis and vascular function in aldosterone-induced hypertension in mice. Wild-type (WT) or heterozygote TRPM7-kinase domain (TRPM7+/-) were treated with infused aldosterone (600 μg/Kg/day) and NaCl 1% in drinking water (aldo/salt) for 4 weeks. Blood pressure (BP) was evaluated by tail-cuff. Vessel function was investigated in mesenteric arteries by wire and pressure myography. Protein expression was assessed in cardiac tissue by western-blot and histology. Aldo/salt increased BP in TRPM7+/- and WT to similar levels (137mmHg vs control 118mmHg). Mesenteric arteries from untreated TRPM7+/- mice were more sensitive to relaxation induced by acetylcholine (LogEC50: 7.6±0.1 vs 7.1±0.2, TRPM7+/- and WT, respectively), effects that were reduced by Aldo/salt treatment (LogEC50: 7.2±0.1). Phenylephrine-contraction and sodium nitroprusside-relaxation curves were similar among groups. Pressure myography showed that in WT, aldo/salt increase the diameter (26%) and cross-sectional area (40%), resulting in hypertrophic outward remodelling, whereas in TRPM7+/-, the treatment decreased the diameter (16%) and increase the wall/lumen ration (82%), resulting in eutrophic inward remodelling. Hearts from TRPM7+/- presented decreased expression of annexin-1, which is a target protein of TRPM7-kinase, that was further decreased by aldo-salt. Hearts from untreated TRPM7+/- mice had increased fibrotic markers: plasma galectin-3 (2.5ng/mL) vs WT (1.4ng/mL) and protein expression for fibronectin (2.4-fold) and TGFβ (2-fold), and the aging marker p-P66Sch (47%) which were similar to WT-aldo/salt. Aldo/salt induced higher collagen expression in TRPM7+/- than in WT animals (15%), as observed by picrosirius red staining. Our findings provide some insights into aldosterone signalling through TRPM7-kinase and suggest that this chanzyme may have protective actions, which when downregulated, promotes vascular remodelling and cardiac fibrosis in aldosterone-induced hypertension.

Osteoprotegerin (OPG) levels are increased in metabolic diseases, and are a biomarker of vascular dysfunction and cardiovascular risk. Mechanisms related to OPG-induced vascular dysfunction and its role in hypertension are not fully understood, but we previously demonstrated that OPG induces vascular dysfunction through ROS-dependent mechanisms. Here we assessed the molecular mechanisms whereby OPG regulates ROS and vascular function, with a focus on syndecan-1. VSMCs from normotensive (WKY) and hypertensive (SHRSP) rats were stimulated with OPG (50 ng/mL). ROS production was measured by lucigenin, amplex red and ELISA. In VSMCs from WKY rats, OPG increased ROS generation (158±15% vs veh, p2 O 2 (2 fold) and ONOO - (1.5 fold) levels in VSMCs (p2 O 2 further stimulates ROS levels and redox signalling through activation of TRPM2, a redox-sensitive Ca 2+ channel. TRPM2 inhibitors, 8-bromo-ADPR (8Br) and N-(p-amylcinnamoyl)anthranilic acid (ACA), did not block OPG-induced ROS generation in VSMCs from WKY rats. Syndecan-1 activation leads to FAK and c-Src activation, which are redox-sensitive signalling proteins. FAK, but not c-Src, activation (117±2%, p

Nox5 is a unique Ca 2+ -sensitive Nox isoform that is expressed in human vascular smooth muscle cells (VSMC). Although Nox5 has been implicated in diabetic nephropathy, its role in vascular function and development of hypertension remain unclear. Nox5 is not expressed in rodents, and accordingly we generated humanised Nox5 mice with Nox5 expressed in a VSMC-specific manner (Nox5SM22). Control (wild-type) and Nox5SM22 mice were infused with Ang II (600 ng/Kg/day). Blood pressure (BP) was assessed by tail-cuff. Vascular function and structure of resistance arteries were measured by myography. Ang II increased BP in WT (182.5±10 mmHg) and Nox5SM22 mice (173.1±5 mmHg) with no significant differences. Arteries from Nox5SM22 mice exhibited reduced endothelium-dependent relaxation versus WT controls (%ACh relaxation: 55.1±4 vs ctl: 81.6±7%). Fasudil (Rho kinase inhibitor)-induced relaxation was reduced in Nox5SM22 mice versus controls (%Fas: 111.3±11 vs ctl: 166.6±8%) (p

TRPM7 is a Mg2 + channel linked to a kinase domain important in cell proliferation and survival. We demonstrated that cells deficient in TRPM7-kinase domain are prone to aldosterone (aldo)-induced oxidative stress and inflammation. Here, we investigated whether TRPM7-kinase plays a role in inflammatory responses in aldo-induced hypertension. Wild-type (WT) or heterozygote TRPM7-kinase domain (TRPM7+/-) mice were infused with aldo (600μg/Kg/day - osmotic minipumps) and 1% NaCl in the drinking water (aldo-salt) for 4 weeks. Inflammatory responses were evaluated by examining T cells (CD4+ and CD8+) and macrophages (M1- and M2-phenotype) infiltration in kidneys and spleens, using flow cytometry. Gene and protein expression was assessed by real-time PCR and immunoblot respectively. ROS was evaluated by lucigenin chemiluminescence. Aldo-salt increased kidney mass and urinary levels of albumin, Ca 2+ , Mg 2+ and K + , similarly in WT and TRPM7+/- (p6 vs WT 0.22x10 6 cells/g), TCD4+ cells (27% vs WT 19%), and macrophages (47% vs WT 31%) (p8 vs WT 2.0x10 8 cells/mg). Splenic macrophages were higher in untreated TRPM7+/- (8.8% vs WT 5.9%) and presented an increase in CD206 M2-marker (16% vs WT 7%), higher TCD4+ (68% vs WT 50%) and TCD8+ (26% vs WT 17%), values that were similar to WT aldo-salt. Our data provide insights into the importance of the TRPM7-kinase domain in the immune system activation, which when down regulated provokes an increase in inflammatory cell infiltration in kidneys and spleens in aldo-salt-induced hypertension.

In hypertension (HTN) mechanisms whereby protein oxidation regulates vascular function remain unclear. We hypothesise that increased ROS promote a shift of oxidative post-translational protein modifications from reversible to irreversible forms, leading to aberrant redox signalling and vascular injury. VSMC from mesenteric arteries of normotensive (WKY) and hypertensive (SHRSP) rats were stimulated with Ang II (10 -7 M) in the presence/absence of PEG-catalase (1000U/ml) or tempol (10 -5 M). Protein carbonylation was assessed by oxyblot and protein sulfenylation by DCP-Rho1 fluorescent probe. Protein tyrosine phosphatase (PTP)-oxidation, peroxiredoxin hyperoxidation (PRXSO3), γH2AX, Bcl2 levels were assessed by immunoblotting. DiGE and CyDye labelling screened for reversibly oxidised thiol proteome. Irreversible carbonylation and PRXSO3 were increased in SHRSP (fold change (FC)=1.29 and 2.77, p2, detected 1777 spots with 21% being differentially oxidised between WKY and SHRSP. Candidate proteins differentially oxidized between WKY and SHRSP include annexin A1 (-2.29) and galectin-1 (2.83). These results demonstrate altered redox status in HTN characterised by increased protein hyperoxidation and decreased reversible oxidation, in combination with decreased anti-oxidant capacity. Moreover, our findings identify novel candidate oxidized proteins implicated in VSMC motility, proliferation and signalling which may contribute to oxidative vascular injury in HTN.

Fetuin-A (FetA) is an endogenous agonist to toll-like receptor 4 (TLR4) and regulates insulin resistance and inflammation. FetA has been associated with endothelial dysfunction during metabolic diseases. Exact mechanisms whereby FetA influences vascular function in pathological conditions remain unknown, but we demonstrated that FetA regulates vascular function by Nox1 and TLR4 activation. Here we hypothesized that FetA, through changes in cell metabolism and activation of TLR4-Nox1 axis induces ROS formation and inflammation in hypertension. Normotensive (WKY) and hypertensive (SHRSP) vascular cells, as well as human microvascular endothelial cells, were stimulated with FetA (50 ng/mL). ROS production was measured by lucigenin and Amplex red, while gene expression was assessed by qPCR. FetA increased ROS production (131±49.2%), decreased H 2 O 2 intracellular levels (63±14%) and increased gene levels of IL6 (2 fold), IL1β (1 fold), RANTES (1 fold) and MMP2/9 (2 fold) in rat endothelial cells ( vs. veh, p

Supplemental Digital Content is available in the text., Estrogen and oxidative stress have been implicated in pulmonary arterial hypertension (PAH). Mechanisms linking these systems are elusive. We hypothesized that estrogen metabolite, 16α-hydroxyestrone (16αOHE1), stimulates nicotinamide adenine dinucleotide phosphate oxidase (Nox)–induced reactive oxygen species (ROS) generation and proliferative responses in human pulmonary artery smooth muscle cells (hPASMCs) and that in PAH aberrant growth signaling promotes vascular remodeling. The pathophysiological significance of estrogen–Nox–dependent processes was studied in female Nox1−/− and Nox4−/− mice with PAH. PASMCs from control subjects (control hPASMCs) and PAH patients (PAH-hPASMCs) were exposed to estrogen and 16αOHE1 in the presence/absence of inhibitors of Nox, cytochrome P450 1B1, and estrogen receptors. Estrogen, through estrogen receptor-α, increased Nox-derived ROS and redox-sensitive growth in hPASMCs, with greater effects in PAH-hPASMCs versus control hPASMCs. Estrogen effects were inhibited by cytochrome P450 1B1 blockade. 16αOHE1 stimulated transient ROS production in hPASMCs, with sustained responses in PAH-hPASMCs. Basal expression of Nox1/Nox4 was potentiated in PAH-hPASMCs. In hPASMCs, 16αOHE1 increased Nox1 expression, stimulated irreversible oxidation of protein tyrosine phosphatases, decreased nuclear factor erythroid–related factor 2 activity and expression of nuclear factor erythroid–related factor 2–regulated antioxidant genes, and promoted proliferation. This was further amplified in PAH-hPASMCs. Nox1−/− but not Nox4−/− mice were protected against PAH and vascular remodeling. Our findings demonstrate that in PAH-hPASMCs, 16αOHE1 stimulates redox-sensitive cell growth primarily through Nox1. Supporting this, in vivo studies exhibited protection against pulmonary hypertension and remodeling in Nox1−/− mice. This study provides new insights through Nox1/ROS and nuclear factor erythroid–related factor 2 whereby 16αOHE1 influences hPASMC function, which when upregulated may contribute to vascular injury in PAH, particularly important in women.

Hypertension is a major cardiovascular risk factor. Of the many processes involved in the pathophysiology of hypertension, cardiac, vascular and renal damage due to oxidative stress (excess bioavailability of reactive oxygen species (ROS)) is important. Physiologically, ROS regulate cell function through redox-sensitive pathways. In hypertension, oxidative stress promotes endothelial dysfunction, vascular remodeling and inflammation, leading to vascular damage. While experimental evidence indicates a causative role for oxidative stress in hypertension, human data are less convincing. This may relate to sub-optimal approaches to accurately measure ROS in humans. Various methods have been developed to assess the extent and nature of oxidative stress, including markers of protein oxidation, lipid oxidation, and anti-oxidant status. These approaches are, in general, indirect and measure indices of redox state. While large clinical studies to establish whether biomarkers of oxidative stress accurately predict disease risk are still needed, oxidative biomarkers have provided important mechanistic insights regarding redox-sensitive processes of hypertension. Here we briefly describe the importance of ROS in redox signaling and hypertension and discuss biomarkers of oxidative stress in human hypertension.

Most diabetes-related complications and causes of death arise from cardiovascular disease and end-stage renal disease. Amongst the major complications of diabetes mellitus are retinopathy, neuropathy, nephropathy and accelerated atherosclerosis. Increased bioavailability of reactive oxygen species (ROS) (termed oxidative stress), derived in large part from the NADPH oxidase (Nox) family of free radical producing enzymes, has been demonstrated in experimental and clinical diabetes and has been implicated in the cardiovascular and renal complications of diabetes. The present review focuses on the role of Noxs and oxidative stress in some major complications of diabetes, including nephropathy, retinopathy and atherosclerosis. We also discuss Nox isoforms as potential targets for therapy.

Reactive oxygen species (ROS) are signaling molecules that influence many physiological processes. Increased ROS bioavailability and altered redox signaling (oxidative stress) have been implicated in chronic diseases including hypertension. Although oxidative stress may not be the sole cause of hypertension, it amplifies blood pressure elevation in the presence of other prohypertensive factors (salt, renin-angiotensin system, sympathetic hyperactivity). A major source for cardiovascular ROS is a family of non-phagocytic NADPH oxidases (Nox1, Nox2, Nox4, Nox5). Other sources of ROS involve mitochondrial electron transport enzymes, xanthine oxidase, and uncoupled nitric oxide synthase. Although evidence from experimental and animal studies supports a role for oxidative stress in the pathogenesis of hypertension, there is still no convincing proof that oxidative stress is a cause of human hypertension. However, what is clear is that oxidative stress is important in the molecular mechanisms associated with cardiovascular and renal injury in hypertension and that hypertension itself can contribute to oxidative stress. The present review addresses the putative function of ROS in the pathogenesis of hypertension and focuses on the role of Noxs in ROS generation in vessels and the kidney. Implications of oxidative stress in human hypertension are discussed, and clinical uncertainties are highlighted.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Canadian Institutes of Health Research Testosterone has been implicated in vascular remodeling associated with hypertension. Molecular mechanisms underlying this are elusive, but oxidative stress may be important. We hypothesized that testosterone stimulates generation of reactive oxygen species (ROS) and migration of vascular smooth muscle cells (VSMCs), with enhanced effects in cells from spontaneously hypertensive rats (SHRs). the mechanisms (genomic and nongenomic) whereby testosterone induces ROS generation and the role of c-Src, a regulator of redox-sensitive migration, were determined. VSMCs from male Wistar-Kyoto rats and SHRs were stimulated with testosterone (10(-7) mol/L, 0-120 minutes). Testosterone increased ROS generation, assessed by dihydroethidium fluorescence and lucigenin-enhanced chemiluminescence (30 minutes [SHR] and 60 minutes [both strains]). Flutamide (androgen receptor antagonist) and actinomycin D (gene transcription inhibitor) diminished ROS production (60 minutes). Testosterone increased Nox1 and Nox4 mRNA levels and p47phox protein expression, determined by real-time PCR and immunoblotting, respectively. Flutamide, actinomycin D, and cycloheximide (protein synthesis inhibitor) diminished testosterone effects on p47phox. c-Src phosphorylation was observed at 30 minutes (SHR) and 120 minutes (Wistar-Kyoto rat). Testosterone-induced ROS generation was repressed by 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-day]pyrimidin-4-amine (c-Src inhibitor) in SHRs and reduced by apocynin (antioxidant/NADPH oxidase inhibitor) in both strains. Testosterone stimulated VSMCs migration, assessed by the wound healing technique, with greater effects in SHRs. Flutamide, apocynin, and 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-day] pyrimidin-4-amine blocked testosterone-induced VSMCs migration in both strains. Our study demonstrates that testosterone induces VSMCs migration via NADPH oxidase-derived ROS and c-Src-dependent pathways by genomic and nongenomic mechanisms, which are differentially regulated in VSMCs from Wistar-Kyoto rats and SHRs. (Hypertension. 2012; 59: 1263-1271.). Online Data Supplement Univ São Paulo, Med Sch Ribeirao Preto, Dept Pharmacol, BR-14049900 Ribeirao Preto, SP, Brazil Univ São Paulo, Inst Biomed Sci, São Paulo, Brazil Univ São Paulo, Sch Med, Inst Heart, São Paulo, Brazil Univ Ottawa, Ottawa Hosp Res Inst, Kidney Res Ctr, Ottawa, ON, Canada Universidade Federal de São Paulo, Dept Pharmacol, Sect Expt Endocrinol, São Paulo, Brazil Universidade Federal de São Paulo, Dept Pharmacol, Sect Expt Endocrinol, São Paulo, Brazil Web of Science

Many factors have been implicated in the pathophysiology of hypertension such as upregulation of the renin-angiotensin-aldosterone system, activation of the sympathetic nervous system, perturbed G protein-coupled receptor signalling, inflammation, and altered T-cell function. Common to these processes is increased bioavailability of reactive oxygen species (ROS) (termed oxidative stress) due to excess ROS generation, decreased nitric oxide (NO) levels, and reduced antioxidant capacity in the cardiovascular, renal, and nervous systems. Although oxidative stress may not be the sole etiology of hypertension, it amplifies blood pressure elevation in the presence of other prohypertensive factors. In the cardiovascular system ROS play a physiological role in controlling endothelial function, vascular tone, and cardiac function, and a pathophysiological role in inflammation, hypertrophy, proliferation, apoptosis, migration, fibrosis, angiogenesis, and rarefaction, all of which are important processes contributing to endothelial dysfunction and cardiovascular remodelling in hypertension. A major source for cardiovascular ROS is a family of nonphagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Nox1, Nox2, Nox4, and Nox5). Other sources include mitochondrial enzymes, xanthine oxidase, and uncoupled NO synthase (NOS). Although convincing data from animal studies support a causative role for oxidative stress in the pathogenesis of hypertension, there is still no solid evidence that oxidative stress causes hypertension in humans. However, biomarkers of excess ROS are increased in patients with hypertension and oxidative damage is important in the molecular mechanisms associated with cardiovascular and renal injury in hypertension. Although clinical trials failed to show beneficial antihypertensive effects of antioxidants, strategies that combat oxidative stress by targeting Noxs in an isoform-specific manner may have therapeutic potential.

Little is known about vascular MAPK regulation in response to mechanical strain. Whether mechanically-sensitive pathways are altered in hypertension is unclear. We examined effects of stretch and Ang II on activation of p38MAPK in vascular smooth muscle cells (VSMC) from WKY and SHR. The role of c-Src and redox-sensitive pathways in stretch-induced effects were examined. VSMC from mesenteric arteries were plated onto flexible silastic plates and exposed to acute or chronic cyclic stretch (10%, 1 Hz) with or without Ang II (0.1 uM). Acute stretch stimulated p38MAPK activation in WKY and SHR, independently of c-Src and reactive oxygen species (ROS), since PP2 (c-Src inhibitor) and apocynin (NADPH oxidase inhibitor), failed to alter stretch-mediated p38MAPK. Chronic stretch blunted p38MAPK phosphorylation in WKY and increased phosphorylation in SHR. Stretch, in the presence of Ang II, induced an increase in procollagen-1 expression. This was blocked by SB203580 (p38MAPK inhibitor). Accordingly, vascular p38MAPK is a mechano-sensitive MAPK, differentially regulated by acute and chronic stretch in WKY and SHR. Functionally, stretch and Ang II, amplify profibrotic responses in a p38MAPK-dependent manner, responses that are perturbed in SHR. Such molecular process may influence vascular fibrosis in hypertension and appear to be independent of c-Src and ROS.

The interplay between reactive oxygen species (ROS) and Ca 2?+ plays a major role in the regulation of vascular function. However, mechanisms underlying ROS-induced Ca 2+ -influx and signalling are not fully established. The transient receptor potential melastatin 2 cation channel (TRPM2) is a redox-sensitive cation channel that promotes influx of Ca 2+ after activation by H 2 O 2 through PARP-ADPR-dependent mechanisms in inflammatory cells. TRPM2 also regulates Na + influx and by increasing intracellular Na + content, it could interfere with the function of the Na + -Ca 2+ exchanger (NCX), which may confer a novel mechanism whereby ROS influences Ca 2+ influx and signalling. Here, we postulated that redox-sensitive Ca 2+ regulation involves TRPM2 and NCX; a process exacerbated in hypertension leading to vascular dysfunction. We also interrogated the role of Nox4 in these processes. Mesenteric arteries from wild-type (WT), LinA3 (chronic Ang II-induced mouse model of hypertension), Nox4 -/- , and LinA3/Nox4 -/- and VSMCs cultures from human arteries were used. Vascular function, assessed by wire myography, demonstrated that mesenteric arteries from LinA3 mice present increased Phe-induced vasoconstriction (Emax – LinA3 vs WT: 9.37±0.51 vs 6.79±0.29); an effect ameliorated by olaparib (PARP inhibitor) and 2-APB (TRPM2 blocker). The mRNA expression of NOX4 (fold change: 3.05±0.30), TRPM2 (fold change: 1.38±0.24), and NCX exchanger (fold change: 1.97±0.34) were increased in LinA3 mice; an effect not observed in LinA3/Nox4 -/- mice (a model with reduced H2O2 levels). Ang II stimulation increased Ca 2+ influx in human VSMC from normotensive (AUC-Ex490/Em535: 15400±917.5) and hypertensive subjects (AUC-Ex490/Em535: 22460±2388). TRPM2 activation inhibitors, such as 2-APB, olaparib and 8-Br, as well as, NCX inhibitors benzamil, KB-R7943 and YM244769, ameliorated Ang II-induced Ca 2+ influx in human VSMC. In conclusion, TRPM2/NCX-induced increase in intracellular levels of calcium may be involved in hypertension-associated vascular dysfunction. Our data also suggests that oxidative stress regulates Ca 2+ homeostasis through TRPM2-dependent mechanisms.

The healthy endothelium prevents platelet aggregation and leucocyte adhesion, controls permeability to plasma components and maintains vascular integrity. Damage to the endothelium promotes endothelial dysfunction characterized by: altered endothelium-mediated vasodilation, increased vascular reactivity, platelet aggregation, thrombus formation, increased permeability, leucocyte adhesion and monocyte migration. Molecular processes contributing to these phenomena include increased expression of adhesion molecules, synthesis of pro-inflammatory and pro-thrombotic factors and increased endothelin-1 secretion. Decreased nitric oxide bioavailability and increased generation of reactive oxygen species (ROS) are among the major molecular changes associated with endothelial dysfunction. A critical source of endothelial ROS is a family of non-phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, including the prototypic Nox2-based NADPH oxidases, Nox1, Nox4 and Nox5. Other possible sources include mitochondrial electron transport enzymes, xanthine oxidase, cyclooxygenase, lipoxygenase and uncoupled nitric oxide synthase (NOS). Cross-talk between ROS-generating enzymes, such as mitochondrial oxidases and Noxs, is increasingly implicated in cellular ROS production. The present review discusses the importance of endothelial ROS in health and disease and focuses on the major ROS-generating systems in the endothelium, namely uncoupled endothelial nitric oxide synthase and NADPH oxidases.

Adipose tissue influences vascular function through adipocyte-derived factors, including components of the renin-angiotensin-aldosterone system. Molecular mechanisms underlying these phenomena are elusive. We investigated the role of adipocyte-derived factors on mitogen-activated protein kinases (MAPKs), proinflammatory status, apoptosis, and mitogenic signaling in vascular smooth muscle cells (VSMCs) and questioned whether these effects involve mineralocorticoid receptor (MR), glucocorticoid receptor (GR), and angiotensin II type 1 receptor (AT 1 R). Cultured mouse VSMCs were exposed to adipocyte-conditioned medium (ACM) from differentiated 3T3-L1 adipocytes. ACM induced phosphorylation of stress-activated protein kinase/c-Jun N-terminal kinase, p38MAPK, and extracellular signal–regulated kinase 1/2 and increased expression of proinflammatory and proliferative markers in VSMCs. Eplerenone (MR antagonist), mifepristone (GR antagonist), and candesartan (AT 1 R antagonist) inhibited ACM-induced effects on extracellular signal–regulated kinase 1/2, p38MAPK, and proliferating cell nuclear antigen, without influencing apoptosis (Bax, Bcl, and caspase 3). Stress-activated protein kinase/c-Jun N-terminal kinase phosphorylation was inhibited by mifepristone and candesartan but not by eplerenone. ACM-induced increase of fibronectin, vascular cell adhesion molecule 1, and plasminogen activator inhibitor 1 expression was blocked by MR and AT 1 R antagonism but not by GR inhibition. ACM has no effect on GR, MR, and AT 1 R expression. Our data show that adipocyte-derived factors influence MAPK signaling, leading to VSMC proinflammatory and profibrotic responses through distinct pathways. Although ACM stimulates p38MAPK and extracellular signal–regulated kinase 1/2 phosphorylation through MR, GR, and AT 1 R, activation of stress-activated protein kinase/c-Jun N-terminal kinase involves GR and AT 1 R. These findings suggest that adipocyte-derived factors regulate VSMC function through specific MAPKs linked to MR, GR, and AT 1 R, a posttranslational phenomenon, because ACM did not influence receptor expression. Such cross-talk between adipocytes and VSMCs may provide a potential molecular mechanism linking renin-angiotensin-aldosterone system, adipocytes, and vascular function.

Objectives:ROS regulates Ca2+ homeostasis by mechanisms still not fully understood. TRPM2, a redox-sensitive channel permeable to Ca2+ and Na+, is activated by H2O2 via oxidation or ADPR production. We tested the hypothesis that TRPM2 is a molecular target of ROS leading to regulation of Ca2+ levels

Rationale : Although Nox5 (Nox2 homolog) has been identified in the vasculature, its regulation and functional significance remain unclear. Objectives : We sought to test whether vasoactive agents regulate Nox5 through Ca 2+ /calmodulin-dependent processes and whether Ca 2+ -sensitive Nox5, associated with Rac-1, generates superoxide (O 2 ·− ) and activates growth and inflammatory responses via mitogen-activated protein kinases in human endothelial cells (ECs). Methods and Results : Cultured ECs, exposed to angiotensin II (Ang II) and endothelin (ET)-1 in the absence and presence of diltiazem (Ca 2+ channel blocker), calmidazolium (calmodulin inhibitor), and EHT1864 (Rac-1 inhibitor), were studied. Nox5 was downregulated with small interfering RNA. Ang II and ET-1 increased Nox5 expression (mRNA and protein). Effects were inhibited by actinomycin D and cycloheximide and blunted by diltiazem, calmidazolium and low extracellular Ca 2+ ([Ca 2+ ] e ). Ang II and ET-1 activated NADPH oxidase, an effect blocked by low [Ca 2+ ] e , but not by EHT1864. Nox5 knockdown abrogated agonist-stimulated O 2 ·− production and inhibited phosphorylation of extracellular signal-regulated kinase (ERK)1/2, but not p38 MAPK (mitogen-activated protein kinase) or SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase). Nox5 small interfering RNA blunted Ang II-induced, but not ET-1-induced, upregulation of proliferating-cell nuclear antigen and vascular cell adhesion molecule-1, important in growth and inflammation. Conclusions : Human ECs possess functionally active Nox5, regulated by Ang II and ET-1 through Ca 2+ /calmodulin-dependent, Rac-1-independent mechanisms. Nox5 activation by Ang II and ET-1 induces ROS generation and ERK1/2 phosphorylation. Nox5 is involved in ERK1/2-regulated growth and inflammatory signaling by Ang II but not by ET-1. We elucidate novel mechanisms whereby vasoactive peptides regulate Nox5 in human ECs and demonstrate differential Nox5-mediated functional responses by Ang II and ET-1. Such phenomena link Ca 2+ /calmodulin to Nox5 signaling, potentially important in the regulation of endothelial function by Ang II and ET-1.

Background and purpose: There are interactions between endothelin-1 (ET-1) and endothelial vascular injury in hyperhomocysteinemia (HHcy), but the underlying mechanisms are poorly understood. Here we evaluated the effects of HHcy on the endothelin system in rat carotid arteries. Experimental approach: Vascular reactivity to ET-1 and ETA and ETB receptor antagonists was assessed in rings of carotid arteries from normal rats and those with HHcy. ETA and ETB receptor expression was assessed by mRNA (RT-PCR), immunohistochemistry and binding of [125I]-ET-1. Key results: HHcy enhanced ET-1-induced contractions of carotid rings with intact endothelium. Selective antagonism of ETA or ETB receptors produced concentration-dependent rightward displacements of ET-1 concentration response curves. Antagonism of ETA but not of ETB receptors abolished enhancement in HHcy tissues. ETA and ETB receptor gene expressions were not up-regulated. ETA receptor expression in the arterial media was higher in HHcy arteries. Contractions to big ET-1 served as indicators of endothelin-converting enzyme activity, which was decreased by HHcy, without reduction of ET-1 levels. ET-1-induced Rho-kinase activity, calcium release and influx were increased by HHcy. Pre-treatment with indomethacin reversed enhanced responses to ET-1 in HHcy tissues, which were reduced also by a thromboxane A2 receptor antagonist. Induced relaxation was reduced by BQ788, absent in endothelium-denuded arteries and was decreased in HHcy due to reduced bioavailability of NO. Conclusions and implications: Increased ETA receptor density plays a fundamental role in endothelial injury induced by HHcy. ET-1 activation of ETA receptors in HHcy changed the balance between endothelium-derived relaxing and contracting factors, favouring enhanced contractility. British Journal of Pharmacology (2009) 157, 568–580; doi:10.1111/j.1476-5381.2009.00165.x; published online 9 April 2009 This article is part of a themed section on Endothelium in Pharmacology. For a list of all articles in this section see the end of this paper, or visit: http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009

Transient receptor potential melastatin 7 (TRPM7) channels have been identified in the vasculature. However, their regulation and function remain unclear.Here, we tested the hypothesis that bradykinin and its second messenger cAMP upregulate TRPM7, which stimulates activation of annexin-1 (TRPM7 substrate) and increases transmembrane Mg2+ transport and vascular smooth muscle cell (VSMC) migration. Human and rat VSMCs were studied. TRPM7 phosphorylation was assessed by immunoprecipitation:immunoblotting using antiphospho-serine/threonine and anti-TRPM7 antibodies. [Mg2+]i was measured by mag-fura-2. TRPM7 was downregulated by small interfering RNA and 2-aminoethoxydiphenyl borate. Annexin-1 activity was assessed by cytosol-to-membrane translocation. Cell migration and invasion, functional responses to bradykinin, were assessed in transwell chambers.Bradykinin increased expression of TRPM7 and annexin-1. TRPM7 was rapidly (5 min) phosphorylated on serine/threonine but not on tyrosine residues by bradykinin. [Mg2+]i was increased in bradykinin-stimulated cells (0.53 versus 0.68 mmol/l, basal versus bradykinin, P0.01). Annexin-1 activation was increased by bradykinin and inhibited by 2-aminoethoxydiphenyl borate. Although Hoe 140 (B2 receptor antagonist), U-73122 (phospholipase C inhibitor), 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (c-Src inhibitor) and chelerythrine (protein kinase C inhibitor) blocked bradykinin actions, dibutyryl-c-AMP was without effect. In small interfering RNA-transfected and in 2-aminoethoxydiphenyl borate-treated cells, bradykinin-induced Mg2+ influx and VSMC migration were reduced.Our results demonstrate that bradykinin regulates TRPM7 and its downstream target annexin-1 through phospholipase C-dependent, protein kinase C-dependent and c-Src-dependent and cAMP-independent pathways; effects are mediated through bradykinin type 2 receptor; and bradykinin regulates VSMC [Mg2+]i and migration through TRPM7. These data identify TRPM7/annexin-1/Mg2+ as a novel pathway in bradykinin signaling.

Objective— Synergistic interactions between aldosterone (Aldo) and angiotensin II (Ang II) have been implicated in vascular inflammation, fibrosis, and remodeling. Molecular mechanisms underlying this are unclear. We tested the hypothesis that c-Src activation, through receptor tyrosine kinase transactivation, is critically involved in synergistic interactions between Aldo and Ang II and that it is upstream of promigratory signaling pathways in vascular smooth muscle cells (VSMCs). Methods and Results— VSMCs from WKY rats were studied. At low concentrations (10 −10 mol/L) Aldo and Ang II alone did not influence c-Src activation, whereas in combination they rapidly increased phosphorylation ( P P Conclusions— Aldo/Ang II synergistically activate c-Src, an immediate signaling response, through EGFR and PDGFR, but not IGFR transactivation. This is associated with activation of redox-regulated RhoA/Rho kinase, which controls VSMC migration. Although Aldo and Ang II interact to stimulate ERK1/2, such effects are c-Src-independent. These findings indicate differential signaling in Aldo-Ang II crosstalk and highlight the importance of c-Src in redox-sensitive RhoA, but not ERK1/2 signaling. Blockade of Aldo/Ang II may be therapeutically useful in vascular remodeling associated with abnormal VSMC migration.

We demonstrated previously that, in mice with chronic angiotensin II–dependent hypertension, gp91phox-containing NADPH oxidase is not involved in the development of high blood pressure, despite being important in redox signaling. Here we sought to determine whether a gp91phox homologue, Nox1, may be important in blood pressure elevation and activation of redox-sensitive pathways in a model in which the renin-angiotensin system is chronically upregulated. Nox1-deficient mice and transgenic mice expressing human renin (TTRhRen) were crossed, and 4 genotypes were generated: control, TTRhRen, Nox1-deficient, and TTRhRen Nox1-deficient. Blood pressure and oxidative stress (systemic and renal) were increased in TTRhRen mice ( P P

The ETs (endothelins) comprise a family of three 21-amino-acid peptides (ET-1, ET-2 and ET-3) and 31-amino-acid ETs (ET-11–31, ET-21–31 and ET-31–31). ET-1 is synthesized from a biologically inactive precursor, big ET-1, by ECEs (ET-converting enzymes). The actions of ET-1 are mediated through activation of the G-protein-coupled ETA and ETB receptors, which are found in a variety of cells in the cardiovascular and renal systems. ET-1 has potent vasoconstrictor, mitogenic, pro-inflammatory and antinatriuretic properties, which have been implicated in the pathophysiology of a number of cardiovascular diseases. Overexpression of ET-1 has been consistently described in salt-sensitive models of hypertension and in models of renal failure, and has been associated with disease progression. Sex differences are observed in many aspects of mammalian cardiovascular function and pathology. Hypertension, as well as other cardiovascular diseases, is more common in men than in women of similar age. In experimental models of hypertension, males develop an earlier and more severe form of hypertension than do females. Although the reasons for these differences are not well established, the effects of gonadal hormones on arterial, neural and renal mechanisms that control blood pressure are considered contributing factors. Sex differences in the ET-1 pathway, with males displaying higher ET-1 levels, greater ET-1-mediated vasoconstrictor and enhanced pressor responses in comparison with females, are addressed in the present review. Sex-associated differences in the number and function of ETB receptors appear to be particularly important in the specific characteristics of hypertension between females and males. Although the gonadal hormones modulate some of the differences in the ET pathway in the cardiovascular system, a better understanding of the exact mechanisms involved in sex-related differences in this peptidergic system is needed. With further insights into these differences, we may learn that men and women could require different antihypertensive regimens.

The existence of blood pressure quantitative trait loci exaggerated by salt on rat chromosome 2 has been confirmed previously using congenic strains derived from stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto (WKY) rats. This study aimed to dissect the implicated chromosome 2 region and to identify candidate genes based on microarray expression profiling and real-time PCR. A marker-assisted breeding strategy generated congenic strains SP.WKYGla2a (D2Rat13-D2Rat157), SP.WKYGla2c* (D2Wox9-D2Mgh12), and SP.WKYGla2k (D2Mit21-D2Rat157) using SHRSP as the recipient and WKY as the donor strain. The SP.WKYGla2k strain contains a 10-cM congenic interval, which is encompassed within the larger (64-cM) SP.WKYGla2a congenic region. Salt-loaded systolic blood pressure, measured by radiotelemetry, was significantly lower in the SP.WKYGla2a and SP.WKYGla2k strains compared with SHRSP. Salt sensitivity in SP.WKYGla2c* was not significantly different from SHRSP. Exclusion mapping identified a 6-Mbp region harboring genes responsible for salt-sensitive blood pressure regulation. Microarray expression profiling was carried out in whole homogenized kidneys from parental and SP.WKYGla2a strains. Examination of expression data within the minimal congenic interval identified the positional candidates Edg1 and Vcam1 , demonstrating significantly elevated renal RNA expression levels in the SHRSP compared with WKY and SP.WKYGla2a congenic strains. These results were confirmed by quantitative real-time PCR. DNA sequencing identified SNPs in both Edg1 and Vcam1 between SHRSP and WKY rats. In conclusion, we have identified a suggestive minimal interval encompassing a 6-Mbp region on rat chromosome 2. This region contains several physiological candidate genes for salt-sensitive hypertension in the SHRSP, including Edg1 and Vcam1 , which are differentially expressed and lie on common and functionally important pathways.

Diabetes and hypertension increasingly are recognized as pro-inflammatory conditions. We tested the hypothesis that in patients with hypertension and type 2 diabetes, blood pressure (BP) reduction with an angiotensin receptor blocker (ARB), valsartan, or with a beta blocker, atenolol, is associated with a decreased inflammatory response. Normotensive subjects and hypertensive patients with type 2 diabetes (40 to 70 years of age) participated in the study. Patients (n = 28) were randomized to double-blind treatment for 1 year with valsartan (80–160 mg) or atenolol (50–100 mg) daily, added to previous therapy. Age-matched controls (n = 12) were also studied. Serum levels of cytokines (IL-6, IL-18), chemokines (MCP-1), and adhesion molecules (sICAM, sE-selectin) were measured by enzyme-linked immunosorbent assay (ELISA) as indices of systemic and vascular inflammation, before and 1 year after treatment. BP was similarly reduced by valsartan and atenolol. Glycemic control and lipid profiles were comparable in the two groups and did not change significantly with antihypertensive therapy. Serum levels of all inflammatory markers were increased in patients before treatment (by two- to four-fold vs. controls, P < .05). IL-6, IL-18, sICAM, and MCP-1 levels were reduced by valsartan (three-fold, P < .05). Only IL-18 was reduced by atenolol compared with pretreatment levels (P < .05). These data indicate that proinflammatory mediators are significantly increased in hypertensive type 2 diabetic patients and that despite similar BP lowering by valsartan and atenolol and similar glucose levels in both treated groups, global inflammatory status was improved only in the valsartan group. Our findings suggest that antihypertensive treatment, particularly with an ARB, ameliorates inflammatory processes in diabetic hypertensive patients. Such effects, which are independent of BP and glycemic control, may contribute to cardiovascular protection.

The present study evaluated the effects of endothelin (ET)-1 and the peroxisome proliferator activated receptor gamma (PPAR-gamma) agonist, rosiglitazone, on inflammatory markers in vascular smooth muscle cells (VSMCs) from normotensive (WKY) and hypertensive (SHRSP) rats. Rat VSMC-derived mesenteric arteries from WKY and SHRSP were treated with ET-1 (100 mmol/L) and rosiglitazone (1mumol/L) or ET type A (ET(A)) or type B (ET(B)) receptor antagonists. Nuclear factor kappa-B (NFkappaB) binding activity was assessed by electrophoretic mobility shift assay and phospho-inhibitory kappaB (IkappaB); vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, and cyclooxygenase (COX)-2 expression was determined using Western blotting. ET-1 significantly increased NFkappaB binding, and VCAM-1, ICAM, and COX-2 expression to a greater degree in SHRSP than in WKY VSMC. These changes were associated with increased phosphorylation of IkappaB, thus resulting in decreased NFkappaB inhibition. Co-incubation with PPAR-gamma activator rosiglitazone, or ET(A) or ET(B) receptor antagonism prevented ET-1-stimulated vascular proinflammatory effects in both WKY and SHRSP VSMC. Proinflammatory effects of ET-1 in VSMCs are mediated via both ET(A) and ET(B) receptor subtypes. These effects may be abrogated by the PPAR-gamma activator rosiglitazone. PPAR-gamma activators may thus prevent deleterious ET-1-dependent proinflammatory vascular effects in VSMC in hypertension.

Purpose of review: Lipid rafts are emerging as key players in the integration of cellular responses. Alterations in these highly regulated signaling cascades are important in structural, mechanical and functional abnormalities that underlie vascular pathological processes. The present review focuses on recent advances in signal transduction through caveolae/lipid rafts, implicated in hypertensive processes. Recent findings: Caveolae/lipid rafts function as sites of dynamic regulatory events in receptor-induced signal transduction. Mediators of vascular function, including G-protein coupled receptors, Src family tyrosine kinases, receptor tyrosine kinases, protein phosphatases and nitric oxide synthase, are concentrated within these microdomains. The assembly of functionally active nicotinamide adenine dinucleotide phosphate oxidase and subsequent reactive oxygen species production are also dependent on interactions within the caveolae/lipid rafts. Recent findings have also demonstrated the importance of actin-cytoskeleton and focal adhesion sites for protein interactions with caveolae/lipid raft. Summary: Many vascular signaling processes are altered in hypertension. Whether these events involve lipid rafts/caveolae remains unclear. A better understanding of how signaling molecules compartmentalize in lipid rafts/caveolae will provide further insights into molecular mechanisms underlying vascular damage in cardiovascular disease.

Elevated blood pressure was an unexpected outcome in some cholesteryl ester transfer protein (CETP) inhibitor trials, possibly due to vascular effects of these drugs. We investigated whether CETP inhibitors (torcetrapib, dalcetrapib, anacetrapib) influence vascular function and explored the putative underlying molecular mechanisms. Resistance arteries and vascular smooth muscle cells (VSMC) from rats, which lack the CETP gene, were studied. CETP inhibitors increased phenylephrine-stimulated vascular contraction (logEC50 (:) 6.6 ± 0.1; 6.4 ± 0.06, and 6.2 ± 0.09 for torcetrapib, dalcetrapib, and anacetrapib, respectively, versus control 5.9 ± 0.05). Only torcetrapib reduced endothelium-dependent vasorelaxation. The CETP inhibitor effects were ameliorated by N-acetylcysteine (NAC), a reactive oxygen species (ROS) scavenger, and by S3I-201 [2-hydroxy-4-[[2-(4-methylphenyl)sulfonyloxyacetyl]amino]benzoic acid], a signal transducer and activator of transcription 3 (STAT3) inhibitor. CETP inhibitors increased the phosphorylation (2- to 3-fold) of vascular myosin light chain (MLC) and myosin phosphatase target subunit 1 (MYPT1) (procontractile proteins) and stimulated ROS production. CETP inhibitors increased the phosphorylation of STAT3 (by 3- to 4-fold), a transcription factor important in cell activation. Activation of MLC was reduced by NAC, GKT137831 [2-(2-chlorophenyl)-4-[3-(dimethylamino)phenyl]-5-methyl-1H-pyrazolo[4,3-c]pyridine-3,6-dione] (Nox1/4 inhibitor), and S3I-201. The phosphorylation of STAT3 was unaffected by NAC and GKT137831. CETP inhibitors did not influence activation of mitogen-activated proteins kinases (MAPK) or c-Src. Our data demonstrate that CETP inhibitors influence vascular function and contraction through redox-sensitive, STAT3-dependent, and MAPK-independent processes. These phenomena do not involve CETP because the CETP gene is absent in rodents. Findings from our study indicate that CETP inhibitors have vasoactive properties, which may contribute to the adverse cardiovascular effects of these drugs such as hypertension.

Oxidative stress has been implicated in the pathophysiology of hypertension (HTN) through redox-sensitive processes that cause vascular damage. Despite recent advances in the field of vascular redox signalling in HTN, it still remains unclear exactly how ROS cause vascular injury. We hypothesise that regulation of redox-sensitive protein tyrosine phosphatases (PTP) through post-translational oxidative modification, is impaired in HTN where ROS levels are increased. Vascular smooth muscle cells (VSMC) from small arteries of normotensive (NT) and hypertensive (HT) individuals were stimulated with AngII (10-7 M) and ET-1 (10-7 M). Irreversible oxidation of proteins and PTPs was assessed by oxyblot and immunoblotting, respectively. Differential gel electrophoresis (DiGE) and CyDye thiol labelling were employed for screening of reversibly oxidised proteome. Irreversible protein oxidation was not affected by AngII or ET-1 in VSMCs from NT and HT subjects. PTP hyperoxidation tended to increase in VSMCs from NT upon stimulation with AngII (FC=2.12 at 60min) and ET-1 (FC=1.60 at 60min), whereas a similar trend was observed only after AngII treatment (FC=1.38 at 60min) in HTN (p>0.05). Proteomic data, filtered for FC>2, detected 2051 spots with 1899 (92.5%) being equally oxidised between NT and HT. In addition, oxidation of 57 (2.9%) spots was increased, while 95 (4.6%) were decreased in HT. Candidate proteins exhibiting consistent changes across three experimental replicates included β-actin (FC=-2.86), annexin A1 (-2.23), galectin-1 (-1.67), FK506 binding protein (-2.35) and polymerase I and transcript release factor (PTRF, -1.92). Stimulation with AngII altered the redox status in 2-3% of proteins, both in HT and NT. However, vimentin was the only target changing consistently across the replicates (FC=2.48). Our findings indicate that pro-hypertensive agents may not impact significantly on irreversible protein and PTP oxidation in health and disease, but may have effects on reversible oxidation. Our proteomic data, in agreement with our previous rat studies, support decreased reversible thiol oxidation in HTN. Moreover, these novel findings identify differentially oxidised proteins which may contribute to oxidative vascular injury in HTN.

ACE2 and Ang 1-7 have been shown to protect against pulmonary hypertension (PH). Mechanisms remain unclear. Considering the important role of ET-1 in PH pathophysiology and endothelial dysfunction, we asked whether Ang 1-7 influences ET-1 signaling in endothelial cells and whether Ang 1-7 treatment influences the ET-1 system in PH. Human microvascular endothelial cells (HMEC) were stimulated with ET-1 in absence/presence of Ang 1-7 and showed that Ang 1-7 increased preproET-1 mRNA (250%), ET-1 release (125%) and ETBR protein (50%), p To investigate pathophysiological significance of our findings, we investigated whether Ang 1-7 treatment ameliorates PH and whether this is associated with altered ET-1 status. Hypoxia was used to induce PH in mice: normoxic controls (NC), hypoxic PH (HP), normoxic (NA) and hypoxic PH (HA) treated with Ang 1-7 30μg/kg/day. In HP mice, RVSP (18.7 NC vs. 47.6mmHg HP, p These findings indicate that vasoprotective effects of Ang 1-7 may be mediated through MAS:ETBR dimerization. In vivo studies support a relationship between Ang 1-7/MAS and ET-1 systems. In conclusion we have identified a novel link between Ang 1-7 and ET-1 through physical interactions between MAS and ETBR.

Adipose tissue releases many adipokines, including chemerin, which produces its effects through activation of chemokine-like receptor 1 (ChemR23). Chemerin influences major aspects of the metabolic syndrome, including adipogenesis and insulin sensitivity in adipocytes and skeletal muscle cells. Considering that chemerin impairs vascular reactivity and that amongst its many actions, insulin also influences vascular function, we postulate that chemerin decreases insulin-induced vasodilatation, by reducing PI3K/Akt signaling, and impairs glucose uptake by vascular smooth muscle cells (VSMC). Mesenteric arteries, endothelial cells (EC) or VSMC from C57Bl6 mice were incubated with chemerin (0.5 ng/mL, 1 h) or vehicle (veh). Chemerin decreased insulin-induced relaxation (0.1 - 3000 ng/mL, pD2: chemerin: 94.5±0.1 vs. veh: 23.6±0,1), which was prevented by the ChemR23 antagonist CCX 832 (pD2: veh= 16.9±0.1; chem= 174.1±0.1, CCX+chem= 37.4±0.1) and a PI3K activator (YS-49) (pD2: veh= 23.0±0.1; chem= 108.5±0.1, YS-49+chem= 28.5±0.1). Chemerin also decreased PI3K (0.8±0.1 vs. veh 1.1±0.1), Akt (0.8±0.1 vs. veh 1.2±0.1) and AMPK [0.7±0.2 vs. veh 1.0 ±0.1] phosphorylation in VSMC. IRS1 and IRS2 gene expression was decreased in VSMC stimulated with chemerin. In addition, chemerin decreased insulin-stimulated NO production in EC through activation of ChemR23 and PI3K and inhibited insulin-stimulated glucose uptake by VSMC (counts per minute: 5668±729.0 vs. veh 9923±662.7), which was mediated by ChemR23, AMPK and PI3K. Importantly, ChemR23 antagonism in diabetic (db/db) mice partially reversed decreased insulin-induced vasodilatation (mesenteric resistance arteries; pD2: db/m: 6.1±1.0; db/db: 7.3±0.2; db/db+CCX832: 6.9±0.3). In conclusion, chemerin decreases vascular insulin responses by reducing PI3K/Akt and AMPK signaling. Moreover, chemerin/ChemR23 system plays a crucial role in impaired vascular insulin signaling in diabetes, suggesting its involvement in the pathogenesis of vascular insulin resistance. Our study may contribute to a better understanding of the role of chemerin in vascular insulin dysfunction in diabetes- and obesity-associated diseases. Financial Support: FAPESP, Brazil.

Fetuin-A (FetA) regulates calcium and phosphate homeostasis. It is also an agonist to toll-like receptor 4 (TLR4) and is related to insulin resistance and inflammation. FetA has also been associated with endothelial dysfunction, which is regulated by oxidative stress. Mechanisms whereby FetA influences vascular function are unknown. We hypothesized that FetA through TLR4 and ROS production induces vascular dysfunction. Mesenteric arteries and vascular cells from WKY rats were studied. Vascular function was analysed by wire myography in the presence or absence of FetA (50 ng/mL) and/or CLI095 (CLI - 10-6M - TLR4 inhibitor). Levels of reactive oxygen species (ROS) were measured by chemiluminescence, Amplex Red (H2O2) and ELISA (nitrotyrosine) Protein oxidation and levels were measured by immunoblotting. WKY vessels exposed to FetA were less sensitive to acetylcholine (Ach)-induced and sodium nitroprusside (SNP)-induced relaxation, while sensitivity to phenylephrine was increased by FetA; an effect blocked by N-acetylcysteine (antioxidant) and ML171 (Nox1 inhibitor). Inhibition of TLR4 blocked FetA effects on endothelial-dependent relaxation and contraction, but not on endothelial-independent relaxation. FetA increased ROS production (131±49.2%), but decreased H2O2 intracellular levels (63±14%) in endothelial cells (EC) (vs. veh, p

Oxidative stress and Noxs have been implicated in the pathogenesis of experimental and human pulmonary arterial hypertension (PH). Gender differences in PH may be, in part, due to increased formation of NADPH oxidase (Nox) derived reactive oxygen species (ROS). A large body of evidence implicates E2 in the pathogenesis of PAH and an interaction between E2 and Noxs has been suggested. We hypothesised that i) Estrogen (E2) leads to Nox-induced oxidative stress, which promotes PASMC damage, ii) E2-induced Nox activation may account for gender differences in PH. Cultured human PASMC were stimulated with E2 (1nM). ROS production was assessed by chemiluminescence (O2-) and proliferation by BrdU assay. E2 increased superoxide (219%; p In vivo studies were conducted to assess the role of Nox4 in hypoxia-induced pulmonary hypertension in male and female mice. Hypobaric hypoxia was used to induce PH in mice, which were divided into 8 groups: normoxic WT (NWT), hypoxic WT (HWT), normoxic Nox4-/- (NKO) and hypoxic Nox4-/- (HKO) for both male and female mice. In male HWT mice, RVSP (20.5 NWT vs. 45.2mmHg HWT, p Hypoxia-induced endothelial dysfunction in both male and female WT pulmonary arteries was improved in male HKO, however, endothelial dysfunction remained in HKO females. In female Nox4-/- mice, increased spleen and uterine weight (which has been associated with altered ovarian hormone biogenesis) suggests a role for inflammatory and fibrotic processes. In conclusion, genetic ablation of Nox4 in males, but not females, protects against the development of hypoxic PH. The effects of E2 on oxidative stress is present in PASMCs where Nox4-derived ROS may be an important regulator of, and impact on, molecular processes contributing to vascular remodelling in PAH.

Oxidative stress plays an important role in vascular dysfunction in diabetes, an important risk factor for erectile dysfunction (ED). Functional and structural changes in internal pudendal arteries (IPAs), which provide blood supply to the corpus cavernosum, can lead to ED. We hypothesized that downregulation of Nrf2-regulated enzymes, consequent to increased NOX1-derived ROS, impairs IPAs function in diabetic mice. IPAs and cultured vascular smooth muscle cells (VSMC) from C57BL/6 (control) and NOX1 knockout (KO) mice were used; diabetes (DM) was induced by streptozotocin in C57BL/6 mice. Vascular function assessment, by wire myography, demonstrated that IPAs from diabetic mice displayed increased contractility to phenylephrine (Phe, Emax: control 138.5±9.5 vs. DM 191.8±15.5) and decreased endothelial-dependent relaxation to acetylcholine (ACh, Emax: control 98.3±2.65 vs. DM 76.1±4.5). These responses were corrected by incubation of IPAs with tiron (ROS scavenger), bardoxolone (Nrf2 activator), ML171 (NOX1 inhibitor) and Y27632 (Rho Kinase inhibitor). IPAs from diabetic mice exhibited decreased Y276732-induced vasodilatation (LogEC50: control 5.8±0.1 vs. DM 5.3±0.1). Cultured VSMC from IPAs, maintained in high glucose (HG) medium, displayed increased levels of superoxide anion (59.6%±10.43) and nitrotyrosine (40.8%±9.8), as well as decreased NO production (37.7%±10.42) and nuclear accumulation of Nrf2 (20.42%±3.6), effects not observed in VSMC from IPAs isolated from NOX1 KO mice. The expression of Nrf2-regulated genes was also decreased in VSMC from IPAs maintained in HG [catalase (25.6%±0.05), HO-1 (21%±0.1) and NQO-1 (22%±0.1)], an effect prevented by the incubation with ML171. HG decreased H2O2 levels in control VSMC (29%±5.0), but increased H2O2 in NOX1 KO VSMC (45.8%±7.1), effects not observed when cells were pre-incubated with GKT 137831 (NOX1/4 inhibitor). In conclusion, diabetes-associated IPAs dysfunction involves increased NOX-1 derived ROS, decreased expression of Nrf2-regulated enzymes and activation of the Rho kinase pathway. These data suggest that Nrf2 may be vasoprotective in diabetes-associated ED. Financial Support: FAPESP, Brazil.

The vascular phenotype in hypertension is characterised by features typically observed in the ageing vasculature. Pathophysiological processes underlying premature vascular aging in hypertension remains unclear but aldosterone (aldo) and oxidative stress may be important. We postulated that physiological aging is amplified in hypertension due to increased aldo-induced Nox activation and redox signalling. We used arteries from adult WKY (18 weeks), aged WKY (52 weeks) and adult stroke-prone spontaneously hypertensive (SHRSP) rats. Blood pressure was measured by tail-cuff. Vascular function/structure was analysed by myography. Gene level was assessed by qPCR and protein by immunoblotting. BP was increased in SHRSP (180.7±2.5 vs. 127±2.7 mmHg, p

VEGF/VEGFR inhibitors, used as anti-angiogenic drugs to treat cancer, induce severe hypertension. Molecular mechanisms whereby VEGF inhibitors cause hypertension are unclear, but nitric oxide (NO) and oxidative stress may be involved. We questioned whether reactive oxygen species (ROS) and Ang II, important regulators of vascular function in hypertension, also play a role in VEGF inhibitor-induced vascular dysfunction. Human microvascular endothelial cells (HMECs) were stimulated with vatalanib (VAT-VEGFR inhibitor) and gefitinib (GEF-EGFR inhibitor) in the absence/presence of Ang II. Activation of eNOS and MAPKs were assessed by immunoblotting. Antioxidant enzyme mRNA was analysed by qPCR. Microparticle levels were measured by flow cytometry. Endothelial microparticles, biomarkers of endothelial damage, tend to increase in subjects treated with VEGFR inhibitors. Phosphorylation of eNOS activation site (Ser1177) (28.3% ± 7.1) was decreased by VAT, while no changes were observed after exposure of HMECs to GEF (p

Osteoprotegerin (OPG), an inhibitor of vascular calcification, has pleiotropic vascular effects independently of its actions on calcification. OPG has been associated with vascular inflammation and remodelling and may be important in cardiovascular disease where OPG levels may be elevated. Molecular mechanisms and functional consequences of OPG stimulation in the vasculature are unclear. We propose that syndecan-1, a membrane glycoprotein, may be important and that reactive oxygen species (ROS) play a role in OPG signalling. Vascular reactivity of resistance arteries from WKY rats was studied by wire myography in the presence or absence of OPG (50 ng/mL) and/or synstatin (SSNT - 10-6M - syndecan-1 inhibitor). Rat endothelial cells (EC) and vascular smooth muscle cells (VSMC) were studied. Levels of ROS were measured by chemiluminescence, Amplex Red (H2O2) and ELISA (nitrotyrosine; peroxynitrite - ONOO-). Protein oxidation and levels were measured by immunoblotting. Exposure of resistance arteries to OPG induced endothelial (decreased relaxation to acetylcholine) and VSMC (decreased relaxation to sodium nitroprusside - SNP) dysfunction, as well as, increased contraction to phenylephrine. All responses were blocked by SSNT, N-acetylcisteine (antioxidant) and ML171 (Nox inhibitor). In EC, OPG-induced ROS production (240±46.1% increase vs. veh, p

Oxidative stress is implicated in vascular dysfunction in hypertension. Although mechanisms regulating vascular pro-oxidants are emerging, there is a paucity of information on antioxidant systems, particularly nuclear factor erythroid 2–related factor (Nrf2), a master regulator of antioxidants enzymes. We evaluated the vascular regulatory role of Nrf2 in hypertension and examined molecular mechanisms, whereby Nrf2 influences redox signaling in small arteries and vascular smooth muscle cells from Wistar Kyoto (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP). Cells were stimulated with angiotensin II in the absence/presence of Nrf2 activators (bardoxolone/L-sulforaphane). Increased vascular reactive oxygen species production (chemiluminescence and amplex red) was associated with reduced Nrf2 activity in arteries (18%) and vascular smooth muscle cells (48%) in SHRSP ( P P E max WKY 113.4±5.6 versus SHRSP 159.0±8.3) and decreased endothelial-dependent relaxation ( E max WKY 88.6±3.1 versus SHRSP 74.6±3.2, P

Disturbed magnesium (Mg2+) homoeostasis and increased levels of OS (oxidative stress) are associated with poor clinical outcomes in patients suffering from neurodegenerative, cardiovascular and metabolic diseases. Data from clinical and animal studies suggest that MD (Mg2+ deficiency) is correlated with increased production of ROS (reactive oxygen species) in cells, but a straightforward causal relationship (including molecular mechanisms) between the two conditions is lacking. The multifactorial protein PARK7/DJ-1 is a major antioxidant protein, playing a key role in cellular redox homoeostasis, and is a positive regulator of AR (androgen receptor)-dependent transcription. SLC41A1 (solute carrier family 41 member 1), the gene encoding a ubiquitous cellular Mg2+E (Mg2+efflux) system, has been shown to be regulated by activated AR. We hypothesize that overexpression/up-regulation of PARK7/DJ-1, attributable to OS and related activation of AR, is an important event regulating the expression of SLC41A1 and consequently, modulating the Mg2+E capacity. This would involve changes in the transcriptional activity of PARK7/DJ-1, AR and SLC41A1, which may serve as biomarkers of intracellular MD and may have clinical relevance. Imipramine, in use as an antidepressant, has been shown to reduce the Mg2+E activity of SLC41A1 and OS. We therefore hypothesize further that administration of imipramine or related drugs will be beneficial in MD- and OS-associated diseases, especially when combined with Mg2+ supplementation. If proved true, the OS-responsive functional axis, PARK7/DJ-1–AR–SLC41A1, may be a putative mechanism underlying intracellular MD secondary to OS caused by pro-oxidative stimuli, including extracellular MD. Furthermore, it will advance our understanding of the link between OS and MD.