NADPH oxidase-derived superoxide anion mediates angiotensin II-induced pressor effect via activation of p38 mitogen-activated protein kinase in the rostral ventrolateral medulla

The rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons are located, is a central site via which angiotensin II (Ang II) elicits its pressor effect. We tested the hypothesis that NADPH oxidase-derived superoxide anion (O2*-) in the RVLM mediates Ang II-induced pressor response via activation of mitogen-activated protein kinase (MAPK) signaling pathways. Bilateral microinjection of Ang II into the RVLM resulted in an angiotensin subtype 1 (AT1) receptor-dependent phosphorylation of p38 MAPK and extracellular signal-regulated protein kinase (ERK)1/2, but not stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK), in the ventrolateral medulla. The Ang II-induced p38 MAPK or ERK1/2 phosphorylation was attenuated by application into the RVLM of a NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI), an antisense oligonucleotide that targets against p22phox or p47phox subunit of NADPH oxidase mRNA, or the superoxide dismutase mimetic tempol. DPI or antisense p22phox or p47phox oligonucleotide treatment also attenuated the AT1 receptor-dependent increase in O2*- production in the ventrolateral medulla elicited by Ang II at the RVLM. Functionally, Ang II-elicited pressor response in the RVLM was attenuated by DPI, tempol, or a p38 MAPK inhibitor, SB203580. The AT1 receptor-mediated enhancement of the frequency of glutamate-sensitive spontaneous excitatory postsynaptic currents induced by Ang II in RVLM neurons was also abolished by SB203580. These results suggest that NADPH oxidase-derived O2*- underlies the activation of p38 MAPK or ERK1/2 by Ang II in the ventrolateral medulla. Furthermore, the p38 MAPK signaling pathway may mediate Ang II-induced pressor response via enhancement of presynaptic release of glutamate to RVLM neurons.     

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 N-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.     

Abrupt reoxygenation of microvascular endothelial cells after hypoxia activates ERK1/2 and JNK1, leading to NADPH oxidase-dependent oxidant production

OBJECTIVE: Mitogen-activated protein kinases (MAPK) in microvascular endothelial cells (EC) may participate in organ pathophysiology following hypoxia/reoxygenation (H/R). The authors aimed to determine the role of MAPK in H/R-induced reactive oxygen species (ROS) generation in mouse microvascular EC. METHODS: Cultured EC derived from skeletal muscle of male wild-type (WT), gp91phox-/- or p47phox-/- mice were subjected to hypoxia (0.1% O2, 1 h) followed by abrupt reoxygenation, H/RA (hypoxic medium quickly replaced by normoxic medium), or slow reoxygenation, H/RS (O2 diffused to cells through hypoxic medium). Cells were analyzed for ERK, JNK, and p38 MAPK phosphorylation, NADPH oxidase activation, and ROS generation. RESULTS: In WT cells, H/RA but not H/RS rapidly phosphorylated ERK1/2 and JNK1 and subsequently increased ROS production. H/RA did not affect p38. MAPK phosphorylation persisted despite inhibition of NADPH oxidase, mitochondrial respiration, protein tyrosine kinase, or PKC. ROS increase during H/RA was prevented by deletion of gp91phox or p47phox, or MAPK inhibition. CONCLUSIONS: Abrupt reoxygenation after hypoxia activates ERK1/2 and JNK1 in mouse microvascular endothelial cells via a tyrosine kinase-, PKC-, and NADPH oxidase-insensitive mechanism, leading to increased NADPH oxidase-dependent ROS production. The results suggest that MAPK activation in the microvascular endothelium is O2-sensitive, contributing critically to tissue pathophysiology after H/R.     

ERK and p38 MAPK, but not NF-kappaB, are critically involved in reactive oxygen species-mediated induction of IL-6 by angiotensin II in cardiac fibroblasts

We recently reported that angiotensin II (Ang II) induced IL-6 mRNA expression in cardiac fibroblasts, which played an important role in Ang II-induced cardiac hypertrophy in paracrine fashion. The present study investigated the regulatory mechanism of Ang II-induced IL-6 gene expression, focusing especially on reactive oxygen species (ROS)-mediated signaling in cardiac fibroblasts. Ang II increased intracellular ROS in cardiac fibroblasts, and the increase was completely inhibited by the AT-1 blocker candesartan and the NADH/NADPH oxidase inhibitor diphenyleneiodonium (DPI). We first confirmed that antioxidant N-acetylcysteine, superoxide scavenger Tiron, and DPI suppressed Ang II-induced IL-6 expression. Because we observed that exogenous H(2)O(2) also increased IL-6 mRNA, the signaling pathways downstream of Ang II and exogenous H(2)O(2) were compared. Ang II, as well as exogenous H(2)O(2), activated ERK, p38 MAPK, and JNK, which were significantly inhibited by N-acetylcysteine and DPI. In contrast with exogenous H(2)O(2), however, Ang II did not influence phosphorylation and degradation of IkappaB-alpha/beta or nuclear translocation of p65, nor did it increase NF-kappaB promoter activity. PD98059 and SB203580 inhibited Ang II-induced IL-6 expression. Truncation and mutational analysis of the IL-6 gene promoter showed that CRE was an important cis-element in Ang II-induced IL-6 gene expression. NF-kappaB-binding site was important for the basal expression of IL-6, but was not activated by Ang II. Ang II phosphorylated CREB through the ERK and p38 MAPK pathway in a ROS-sensitive manner. Collectively, these data indicated that Ang II stimulated ROS production via the AT1 receptor and NADH/NADPH oxidase, and that these ROS mediated activation of MAPKs, which culminated in IL-6 gene expression through a CRE-dependent, but not NF-kappaB-dependent, pathway in cardiac fibroblasts.     

Rac2 GTPase activation by angiotensin II is modulated by Ca2+/calcineurin and mitogen-activated protein kinases in human neutrophils

Angiotensin II (Ang II) highly stimulates superoxide anion production by neutrophils. The G-protein Rac2 modulates the activity of NADPH oxidase in response to various stimuli. Here, we describe that Ang II induced both Rac2 translocation from the cytosol to the plasma membrane and Rac2 GTP-binding activity. Furthermore, Clostridium difficile toxin A, an inhibitor of the Rho-GTPases family Rho, Rac and Cdc42, prevented Ang II-elicited O2-/ROS production, phosphorylation of the mitogen-activated protein kinases (MAPKs) p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase 1/2, and Rac2 activation. Rac2 GTPase inhibition by C. difficile toxin A was accompanied by a robust reduction of the cytosolic Ca(2)(+) elevation induced by Ang II in human neutrophils. Furthermore, SB203580 and PD098059 act as inhibitors of p38MAPK and ERK1/2 respectively, wortmannin, an inhibitor of phosphatidylinositol-3-kinase, and cyclosporin A, a calcineurin inhibitor, hindered both translocation of Rac2 from the cytosol to the plasma membrane and enhancement of Rac2 GTP-binding elicited by Ang II. These results provide evidence that the activation of Rac2 by Ang II is exerted through multiple signalling pathways, involving Ca(2)(+)/calcineurin and protein kinases, the elucidation of which should be insightful in the design of new therapies aimed at reversing the inflammation of vessel walls found in a number of cardiovascular diseases.     

Stimulation of NADPH oxidase by angiotensin II in human neutrophils is mediated by ERK, p38 MAP-kinase and cytosolic phospholipase A2

OBJECTIVE: The present research was designed to study the involvement of ERK and p38 MAP-kinase in cytosolic phospholipase A2 (cPLA2) and NADPH-oxidase activation by angiotensin II (Ang II) in human neutrophils. METHODS: NADPH-oxidase activity was measured by reduction of cytochrome C. cPLA2 activity was measured in cell lysate using sonicated dispersions of 1-stearoyl-2-[C]arachidonyl phosphatidylcholine. Cells were incubated with MEK inhibitor UO126 or with p38 MAP-kinase inhibitor SB202190 prior to stimulation with Ang II. Translocation of p47, p67 and cPLA2 and phosphorylation of ERK and p38 MAP-kinase were measured by immunoblot analysis. RESULTS: Ang II induced a dose-dependent activation of NADPH oxidase in neutrophils and monocytes as well as in differentiated PLB-985 cells towards neutrophil or monocyte lineages, but not in cPLA2-deficient differentiated PLB-985 cells. An immediate activation of both ERK and p38 MAP-kinase and of cPLA2 was induced by Ang II in human neutrophils. In addition, Ang II induced translocation of the cytosolic oxidase components, detected by translocation of p47, which preceded the translocation of cPLA2 induced by this agonist. The p38 MAP-kinase inhibitor SB202190 or the MEK-ERK pathway inhibitor UO126 totally inhibited the activation of both NADPH oxidase and cPLA2 as well as the translocation of cytosolic oxidase components and of cPLA2 to the membrane fractions. CONCLUSIONS: These results suggest that either ERK or p38 MAP-kinase are involved in the activation of both cPLA2 and NADPH oxidase, and that cPLA2 is required for activation of the NADPH oxidase by Ang II in human neutrophils.     

Redox regulation of angiotensin II preconditioning of the myocardium requires MAP kinase signaling

A recent study documented reactive oxygen species (ROS), generated through NADPH oxidase by angiotensin II (Ang II) with the activation of NADPH oxidase subunits, p22phox and gp91phox, to be responsible for the preconditioning effect of Ang II. The present study was designed to determine if similar to ischemic preconditioning (PC), mitogen-activated protein (MAP) kinases are also involved in Ang II PC of the heart. Isolated working rat hearts were perfused for 15 min with KHB (Krebs-Henseleit bicarbonate) buffer containing Ang II in the absence or presence of an Erk (1/2) inhibitor, PD 098059, a p38MAPK inhibitor, SB 202190, a JNK inhibitor, SP 600125 or a ROS scavenger, N-acetyl cysteine (NAC). All hearts were subsequently subjected to 30 min global ischemia followed by 2 h reperfusion with KHB buffer only. Cardioprotection was examined by determining infarct size, cardiomyocyte apoptosis and ventricular recovery. Redox and MAP kinase regulation were studied by determining the survival signaling mediated by Akt and Bcl-2. In consistent with previous results, Ang II preconditioned the heart as evidenced by improved postischemic ventricular recovery and reduced infarct size and decreases cardiomyocyte apoptosis. Ang II phosphorylated both Akt, Bcl-2 and Bad, which was blocked by NAC, PD 098059 or SP 600125, but not by SB 202190. NAC, PD 098059 and SP600125, but not SB202190, also abolished the cardioprotective effect of Ang II preconditioning. The results indicate that Ang II preconditioning is potentiated through MAP kinases that are regulated by redox signaling.     

Angiotensin II-induced over-activation of p47phox in fibroblasts from hypertensives: which role in the enhanced ERK1/2 responsiveness to angiotensin II?

BACKGROUND: Fibroblasts are involved in the remodeling of the heart and of the vasculature associated to arterial hypertension, and an abnormal extracellular signal-regulated kinase 1/2 (ERK1/2) activation by angiotensin II (Ang II) plays a pivotal role in this process. However, the intracellular pathways leading to cell hypertrophy and hyperplasia, as well as to collagen production, are still incompletely known. OBJECTIVE: To investigate the role of superoxide anion (O2) and of nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase in Ang II-stimulated ERK1/2 over-activation in fibroblasts from hypertensive patients. METHODS: O2 production was measured in skin fibroblasts from hypertensives (HT, n = 11) and from normotensive controls (NT, n = 10) by electron spin resonance technique. ERK1/2 phosphorylation and p47phox NAD(P)H oxidase subunit translocation were measured by western blot. RESULTS: Ang II (1 micromol/l) induced a larger p47phox subunit translocation and increased intracellular O2 production to a larger extent in HT in comparison to NT and this effect was blocked by apocynin, an inhibitor of the NAD(P)H oxidase. Ang II increased ERK1/2 phosphorylation more in HT than in NT. The Ang II-induced ERK1/2 phosphorylation was inhibited by apocynin in a dose-dependent manner in NT, but not in HT. CONCLUSIONS: The chain of cellular events leading to increased ERK1/2 responsiveness to Ang II in hypertension include an exaggerated response of p47phox, NAD(P)H oxidase and O2, but it is partially resistant to apocynin. Therefore, NAD(P)H-dependent reactive oxygen species (ROS) production is not the only determinant of the exaggerated ERK1/2 responsiveness in fibroblasts of hypertensives (HT).     

p40phox: the last NADPH oxidase subunit

The phagocytic NADPH-oxidase is a multiprotein system activated during the inflammatory response to produce superoxide anion (O2-), which is the substrate for formation of additional reactive oxygen species (ROS). The importance of this system for innate immunity is established by chronic granulomatous disease (CGD), a primary immunodeficiency caused by defects in the NADPH oxidase. In this review, we present and discuss recent knowledge about p40phox, the last NADPH oxidase component to be identified. Furthermore, its interaction with cellular pathways outside of the NADPH oxidase is discussed. Described in this review is evidence that p40phox participates in NADPH oxidase dynamics within cells, what is known about its role in the oxidase, the possibility that p40phox participates in non-NADPH oxidase processes in phagocytic and non-phagocytic cells and whether p40phox could mediate a similar function in other NADPH oxidases. An improved understanding of p40phox should provide new insights about NADPH oxidase, the physiology of phagocytic cells and the innate immune system.     

Role of NAD(P)H oxidase- and mitochondria-derived reactive oxygen species in cardioprotection of ischemic reperfusion injury by angiotensin II

Reactive oxygen species (ROS) participate in cardioprotection of ischemic reperfusion (I/R) injury via preconditioning mechanisms. Mitochondrial ROS have been shown to play a key role in this process. Angiotensin II (Ang II) exhibits pharmacological preconditioning; however, the involvement of NAD(P)H oxidase, known as an ROS-generating enzyme responsive to Ang II stimuli, in the preconditioning process remains unclear. We compared the effects of 5-hydroxydecanoate (5-HD; an inhibitor of mitochondrial ATP-sensitive potassium channels), apocynin (an NAD(P)H oxidase inhibitor), and 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl (tempol; a membrane permeable radical scavenger) on pharmacological preconditioning by Ang II in rat cardiac I/R injury in vivo. Treatment with a pressor dose of Ang II before a 30-minute coronary occlusion reduced infarct size as determined 24 hours after reperfusion. The protective effects of Ang II were eliminated by pretreatment with 5-HD or apocynin, similar to tempol. Both 5-HD and apocynin suppressed the enhanced cardiac lipid peroxidation and activation of the apoptosis signal-regulating kinase/p38, c-Jun NH2-terminal kinase (JNK) pathways, but not the Raf/MEK/extracellular signal-regulated kinase pathway, elicited by acutely administered Ang II. Apocynin but not 5-HD suppressed Ang II-induced augmentations of the NAD(P)H oxidase complex formation (p47phox, p22phox, and Rac-1) and its activity in the heart. Finally, 5-HD suppressed superoxide production by isolated cardiac mitochondria without any effect on their respiration. These results suggest that the preconditioning effects of Ang II for cardiac I/R injury may be mediated by cardiac mitochondria-derived ROS enhanced through NAD(P)H oxidase via JNK and p38 mitogen-activated protein kinase activation.     

Activation of vascular p38MAPK by mechanical stretch is independent of c-Src and NADPH oxidase: influence of hypertension and angiotensin II

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.     

Homocysteine enhances superoxide anion release and NADPH oxidase assembly by human neutrophils. Effects on MAPK activation and neutrophil migration

Hyperhomocysteinaemia has recently been recognized as a risk factor of cardiovascular disease. However, the action mechanisms of homocysteine (Hcy) are not well understood. Given that Hcy may be involved in the recruitment of monocytes and neutrophils to the vascular wall, we have investigated the role of Hcy in essential functions of human neutrophils. We show that Hcy increased superoxide anion (O2*-) release by neutrophils to the extracellular medium, and that this effect was inhibited by superoxide dismutase and diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase activity. The enzyme from rat peritoneal macrophages displayed a similar response. These effects were accompanied by a time-dependent increased translocation of p47phox and p67phox subunits of NADPH oxidase to the plasma membrane. We also show that Hcy increased intracellular H2O2 production by neutrophils, that Hcy enhanced the activation and phosphorylation of mitogen-activated protein kinases (MAPKs), specifically p38-MAPK and ERK1/2, and that the migration of neutrophils was increased by Hcy. Present results are the first evidence that Hcy enhances the oxidative stress of neutrophils, and underscore the potential role of phagocytic cells in vascular wall injury through O2*- release in hyperhomocysteinaemia conditions.     

Antioxidants inhibit JNK and p38 MAPK activation but not ERK 1/2 activation by angiotensin II in rat aortic smooth muscle cells.

Angiotensin II (Ang II) induces vascular smooth muscle cell (VSMC) hypertrophy, which results in several cardiovascular diseases. Ang II-induced cellular events have been mediated, in part, by reactive oxygen species (ROS) which also involve activation of mitogen-activated protein (MAP) kinases. Although it has been proposed that the therapeutic administration of antioxidants is useful for vascular diseases, the precise mechanisms which regulate ROS-sensitive signaling events have not been well characterized. Thus, we hypothesized that antioxidants may affect ROS-mediated MAP kinases activation induced by Ang II. The present findings showed that Ang II stimulated rapid and significant activation of ERK 1/2, JNK and p38 MAPK in cultured rat aortic smooth muscle cells (RASMC). Ang II-induced ERK 1/2 activation was not affected by all antioxidants examined, whereas JNK was sensitive to all antioxidants. In contrast, p38 MAPK activation was inhibited by DPI and ascorbic acid concentration-dependently, but by NAC only at high concentration. DETC and Trolox C had no effects on p38 MAPK activation by Ang II. We further examined the effects of antioxidants on Ang II-induced increases in oxygen consumption as an index of ROS generation in RASMC. DPI strongly inhibited Ang II-induced increases in oxygen consumption. DETC also inhibited Ang II-induced oxygen consumption, whereas ascorbic acid markedly augmented it. These findings suggest that the inhibitory effects of antioxidants on MAP kinases activation in VSMC are attributable, in part, to their modulating effects on ROS generation by Ang II in VSMC. Thus, inhibition of MAP kinases by antioxidants may imply their usefulness for relief of cardiovascular diseases.     

Involvement of reactive oxygen species in the activation of tyrosine kinase and extracellular signal-regulated kinase by angiotensin II

Reactive oxygen species (ROS) have been proposed to mediate vascular hypertrophy induced by angiotensin II (Ang II). Recently, we and others have shown that growth-promoting signals by Ang II involve protein tyrosine kinase (PTK) and extracellular signal-regulated kinase (ERK). However, whether ROS contribute to the Ang II-induced PTK and/or ERK activation in vascular smooth muscle cells (VSMCs) remains largely unclear. Here, we have investigated the possible involvement of ROS in Ang II-induced PTK and ERK activation. In the presence of a NADH/NADPH oxidase inhibitor, diphenyleneiodonium (DPI) or an antioxidant, alpha-tocopherol, Ang II-induced protein tyrosine phosphorylation of two major proteins (p120, p70) and ERK activation were markedly reduced, whereas ERK activation by epidermal growth factor was unaffected. DPI also inhibited Ang II-induced H2O2 production and PTK activation. In this regard, H2O2 and a membrane permeable thiol-oxidizing agent, diamide, stimulated protein tyrosine phosphorylation of p120 and p70, and ERK activation in VSMCs. H2O2 also enhanced PTK activity. From these data, we conclude that ROS play a critical role in the Ang II-induced PTK and ERK activation in VSMCs, thereby contributing to vascular growth associated with enhanced Ang II activity.     

Angiotensin II impairs neurovascular coupling in neocortex through NADPH oxidase-derived radicals

Angiotensin II (Ang II) exerts detrimental effects on cerebral circulation, the mechanisms of which have not been elucidated. In particular, Ang II impairs the increase in cerebral blood flow (CBF) produced by neural activity, a critical mechanism that matches substrate delivery with energy demands in brain. We investigated whether Ang II exerts its deleterious actions by activating Ang II type 1 (AT1) receptors on cerebral blood vessels and producing reactive oxygen species (ROS) through NADPH oxidase. Somatosensory cortex CBF was monitored in anesthetized mice by laser-Doppler flowmetry. Ang II (0.25 microg/kg per minute IV) attenuated the CBF increase produced by mechanical stimulation of the vibrissae. The effect was blocked by the AT1 antagonist losartan and by ROS scavenger superoxide dismutase or tiron and was not observed in mice lacking the gp91phox subunit of NADPH oxidase or in wild-type mice treated with the NADPH oxidase peptide inhibitor gp91ds-tat. Ang II increased ROS production in cerebral microvessels, an effect blocked by the ROS scavenger Mn(III)tetrakis (4-benzoic acid) porphyrin and by the NADPH oxidase assembly inhibitor apocynin. Ang II did not increase ROS production in gp91-null mice. Double-label immunoelectron microscopy demonstrated that AT1 and gp91phox immunoreactivities were present in endothelium and adventitia of neocortical arterioles. Collectively, these findings suggest that Ang II impairs functional hyperemia by activating AT1 receptors and inducing ROS production via a gp91phox containing NADPH oxidase. The data provide the mechanistic basis for the cerebrovascular dysregulation induced by Ang II and suggest novel therapeutic strategies to counteract the effects of hypertension on the brain.     

Redox signaling in angiogenesis: role of NADPH oxidase

Angiogenesis, a process of new blood vessel formation, is a key process involved in normal development and wound repair as well as in the various pathophysiologies such as ischemic heart and limb diseases and atherosclerosis. Reactive oxygen species (ROS) such as superoxide and H(2)O(2) function as signaling molecules in many aspects of growth factor-mediated responses including angiogenesis. Vascular endothelial growth factor (VEGF) is a key angiogenic growth factor and stimulates proliferation, migration, and tube formation of endothelial cells (ECs) primarily through the VEGF receptor type2 (VEGR2, KDR/Flk1). VEGF binding initiates autophosphorylation of VEGFR2, which results in activation of downstream signaling enzymes including ERK1/2, Akt, and eNOS in ECs, thereby stimulating angiogenesis. The major source of ROS in EC is a NADPH oxidase which consists of Nox1, Nox2 (gp91phox), Nox4, p22phox, p47phox, p67phox and the small G protein Rac1. The endothelial NADPH oxidase is activated by angiogenic factors including VEGF and angiopoietin-1. ROS derived from this enzyme stimulate diverse redox signaling pathways leading to angiogenesis-related gene induction as well as EC migration and proliferation, which may contribute to postnatal angiogenesis in vivo. The aim of this review is to provide an overview of the recent progress on the emerging area of the role of ROS derived from NADPH oxidase and redox signaling in angiogenesis. Understanding these mechanisms may provide insight into the NADPH oxidase and redox signaling components as potential therapeutic targets for treatment of angiogenesis-dependent cardiovascular diseases and for promoting angiogenesis in ischemic limb and heart diseases.     

C-reactive protein stimulates superoxide anion release and tissue factor activity in vivo

C-reactive protein (CRP), the prototypic marker of inflammation, is a cardiovascular risk marker and recent in vitro studies suggest that it may promote atherogenesis. CRP promotes oxidative stress in vitro and induces tissue factor (TF) release. However, there is a paucity of data examining the effects of CRP on oxidative stress and tissue factor procoagulant activity (PCA) in vivo. Thus, we tested the effects of CRP administration on superoxide anion release and tissue factor activity and examined mechanistic pathways using a rat sterile air pouch model. Intraperitoneal administration of CRP (20mg/kg body weight) compared to human serum albumin (HuSA) increased superoxide anion release and tissue factor activity from peritoneal macrophages in vivo (p<0.01). This was confirmed using intrapouch administration of CRP (25mug/mL) compared to HuSA. Pretreatment with reactive oxygen species (ROS) scavengers or protein kinase C (PKC) inhibitor significantly abrogated CRP-induced superoxide anion release and tissue factor activity. Pretreatment with extracellular signal-regulated kinase (ERK) and Jun N-terminal kinase (JNK) inhibitors, but not p38 mitogen-activated protein kinase (p38MAPK) significantly decreased CRP-induced superoxide anion release from macrophages in vivo. CRP-induced tissue factor activity in vivo was abrogated by pretreatment with inhibitors to p38MAPK, JNK and NFkappab (nuclear factor-kappab), but not ERK. Antibodies to Fc gamma receptors, CD32 and CD64 resulted in significant reduction in CRP-induced superoxide and tissue factor activity in vivo. Thus, CRP appears to induce oxidative stress in vivo by stimulating NADPH oxidase via PKC, ERK and JNK phosphorylation, and induces tissue factor PCA in vivo via upregulation of PKC, p38MAPK, JNK, ROS and NFkappab. CRP-induced ROS appears to precede tissue factor release. These effects are abrogated by blocking Fc gamma receptors, CD32 and CD64. This in vivo demonstration provides further evidence for a role for CRP in atherothrombosis.