VEGF signaling through NADPH oxidase-derived ROS

Angiogenesis is a key process involved in normal development and wound repair, as well as ischemic heart and limb diseases, and atherosclerosis. Vascular endothelial growth factor (VEGF), a potent angiogenesis factor, stimulates proliferation, migration, and tube formation of endothelial cells (ECs), primarily through the VEGF receptor type2 (VEGFR2). Reactive oxygen species (ROS) function as signaling molecules to mediate biological responses. In ECs, NADPH oxidase is one of the major sources of ROS and consists of catalytic subunits (Nox1, Nox2, and Nox4), p22phox, p47phox, p67phox, and the small GTPase Rac1. VEGF stimulates ROS production via activation of gp91phox (Nox2)-based NADPH oxidase, and ROS are involved in VEGFR2-mediated signaling linked to EC migration and proliferation. Moreover, ROS derived from NADPH oxidase are involved in postnatal angiogenesis. Localizing NADPH oxidase and its regulators at the specific subcellular compartment is an important mechanism for activating specific redox signaling events. This review focuses on a role of NADPH oxidase-derived ROS in angiogenesis and critical regulators involved in generation of spatially and temporally restricted ROS-dependent VEGF signaling at leading edge, focal adhesions/complexes, caveolae/lipid rafts, and cell-cell junctions in ECs. Understanding these mechanisms should facilitate the development of new therapeutic strategies to modulate new blood vessel formation.     

Regulation of NADPH oxidase in vascular endothelium: the role of phospholipases, protein kinases, and cytoskeletal proteins

The generation of reactive oxygen species (ROS) in the vasculature plays a major role in the genesis of endothelial cell (EC) activation and barrier function. Of the several potential sources of ROS in the vasculature, the endothelial NADPH oxidase family of proteins is a major contributor of ROS associated with lung inflammation, ischemia/reperfusion injury, sepsis, hyperoxia, and ventilator-associated lung injury. The NADPH oxidase in lung ECs has most of the components found in phagocytic oxidase, and recent studies show the expression of several homologues of Nox proteins in vascular cells. Activation of NADPH oxidase of nonphagocytic vascular cells is complex and involves assembly of the cytosolic (p47(phox), p67(phox), and Rac1) and membrane-associated components (Noxes and p22(phox)). Signaling pathways leading to NADPH oxidase activation are not completely defined; however, they do appear to involve the cytoskeleton and posttranslation modification of the components regulated by protein kinases, protein phosphatases, and phospholipases. Furthermore, several key components regulating NADPH oxidase recruitment, assembly, and activation are enriched in lipid microdomains to form a functional signaling platform. Future studies on temporal and spatial localization of Nox isoforms will provide new insights into the role of NADPH oxidase-derived ROS in the pathobiology of lung diseases.     

The NADPH oxidase family and its inhibitors

The classical nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was originally detected in neutrophils as a multicomponent enzyme that catalyzes the generation of superoxide from oxygen and the reduced form of NADPH. This enzyme is composed of two membrane-bound subunits (p22phox and gp91phox), three cytosolic subunits (p67phox, p47phox, and p40phox) and a small G-protein Rac (Rac1 and Rac2). Recently, it has been demonstrated that there are several isoforms of nonphagocytic NADPH oxidase. Endothelial cells, vascular smooth muscle cells or adventitial fibroblasts possess multiple isoforms of this enzyme. The new homologs, along with gp91phox are now designated the Nox family of NADPH oxidases and are key sources of reactive oxygen species in the vasculature. Reactive oxygen species play a significant role in regulating endothelial function and vascular tone. However, besides the participation in the processes of physiological cell, these enzymes can also be the perpetrator of oxidative stress that causes endothelial dysfunction. This review summarizes the current state of knowledge of the structure and functions of NADPH oxidase and NADPH oxidase inhibitors in the treatment of disorders with endothelial damage.     

Nox enzymes in immune cells

The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase of phagocytes is a multi-component electron transferase that uses cytoplasmic NADPH to convert molecular oxygen to superoxide anion, consequently delivering reactive oxygen species to the site of invading microorganisms. Together with soluble factors and other phagocyte-derived agents, the resultant toxic species kill and degrade the ingested microbe. Flavocytochrome b (558), a heterodimeric protein composed of gp91 phox and p22 phox, is the membrane component of the NADPH oxidase and was previously thought to be uniquely expressed in phagocytes. Based on structural homology with gp91 phox, recent studies have defined a family of NADPH oxidase proteins (Nox) that is widely distributed throughout the plant and animal kingdoms and in many tissues in multicellular organisms. The goals of this review are to review features of the phagocyte NADPH oxidase that serve as a paradigm for exploiting oxidants for host defense, and to discuss contributions of other Nox proteins to innate immunity.     

Role of NADPH oxidases in the control of vascular gene expression

All vascular cells, including endothelial cells and smooth muscle cells, express components of the leukocyte NADPH oxidase such as p22phox, p47phox, and Rac. Endothelial cells and fibroblasts also express the leukocyte NADPH oxidase subunit gp91phox/nox2, whereas in smooth muscle cells nox1 and nox4 are found. The different vascular NADPH oxidases represent important sources for the basal as well as the agonist-induced superoxide anion (O(2) .-) generation in the vasculature. In vascular smooth muscle cells, activation of the NADPH oxidases and the subsequent formation of O(2) .- has been demonstrated for various agents including angiotensin II, thrombin, lysophosphatidylcholine, and tumor necrosis factor alpha. By influencing the activity of p38 mitogen-activated protein kinase and AKT, NADPH oxidase-derived O(2) .- increases the expression of several pro-arteriosclerotic genes, such as monocyte chemoattractant protein-1, tissue factor, and vascular endothelial growth factor. Thus, the vascular NADPH oxidases play an important role in mediating the signal transduction cascade of pro-arteriosclerotic stimuli.     

阿托伐他汀通过抑制PKC的激活对抗高糖诱导的人脐静脉内皮细胞氧化应激反应

目的:探讨阿托伐他汀对高糖诱导的人脐静脉血管内皮细胞(HUVECs)产生氧化应激的影响及其作用机制。方法:体外培养HUVECs,以25 mmol/L葡萄糖干预,模拟糖尿病患者体内环境,通过流式细胞术和共聚焦显微镜检测细胞内的活性氧(ROS)水平,采用Lucigenin分析方法测定还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶活性,分别应用实时荧光定量PCR和免疫印迹杂交的方法检测 NADPH氧化酶亚基Nox4和Nox2/gp91^phox的表达水平,用免疫印迹杂交方法检测蛋白激酶C(PKC)蛋白的磷酸化水平。结果:(1)在高糖环境(终浓度为25 mmol/L)下,HUVECs内ROS生成显著增加,NADPH氧化酶的活性显著增强,NADPH 氧化酶Nox4和Nox2/gp91^phox亚基的mRNA和蛋白表达水平显著上调;(2)阿托伐他汀可显著抑制高糖诱导的ROS 生成、NADPH氧化酶活性的增强及NADPH 氧化酶Nox4和Nox2/gp91^phox亚基表达水平的增加幅度,且具有浓度依赖性;(3)PKC抑制剂(PKC inhibitor peptide, 20 μmol/L)可显著抑制高糖环境下ROS的生成、NADPH氧化酶活性的增强及NADPH 氧化酶Nox4和Nox2/gp91^phox亚基表达水平的增加幅度;(4)阿托伐他汀可抑制高糖诱导的PKC蛋白的磷酸化。结论:PKC的活化参与了高糖诱导的HUVECs产生的氧化应激反应。阿托伐他汀通过抑制PKC蛋白的活化对抗高糖诱导的内皮细胞产生的氧化应激反应。     

The role of NADPH oxidase in chronic intermittent hypoxia-induced pulmonary hypertension in mice

Obstructive sleep apnea, characterized by intermittent periods of hypoxemia, is an independent risk factor for the development of pulmonary hypertension. However, the exact mechanisms of this disorder remain to be defined. Enhanced NADPH oxidase expression and superoxide (O2(-).) generation in the pulmonary vasculature play a critical role in hypoxia-induced pulmonary hypertension. Therefore, the current study explores the hypothesis that chronic intermittent hypoxia (CIH) causes pulmonary hypertension, in part, by increasing NADPH oxidase-derived reactive oxygen species (ROS) that contribute to pulmonary vascular remodeling and hypertension. To test this hypothesis, male C57Bl/6 mice and gp91phox knockout mice were exposed to CIH for 8 hours per day, 5 days per week for 8 weeks. CIH mice were placed in a chamber where the oxygen concentration was cycled between 21% and 10% O2 45 times per hour. Exposure to CIH for 8 weeks increased right ventricular systolic pressure (RVSP), right ventricle (RV):left ventricle (LV) + septum (S) weight ratio, an index of RV hypertrophy, and thickness of the right ventricular anterior wall as measured by echocardiography. CIH exposure also caused pulmonary vascular remodeling as demonstrated by increased muscularization of the distal pulmonary vasculature. CIH-induced pulmonary hypertension was associated with increased lung levels of the NADPH oxidase subunits, Nox4 and p22phox, as well as increased activity of platelet-derived growth factor receptor beta and its associated downstream effector, Akt kinase. These CIH-induced derangements were attenuated in similarly treated gp91phox knockout mice. These findings demonstrate that NADPH oxidase-derived ROS contribute to the development of pulmonary vascular remodeling and hypertension caused by CIH.     

Mechanisms of expression of NADPH oxidase components in human cultured monocytes: role of cytokines and transcriptional regulators involved

Human blood monocytes lose their capability to produce microbicidal oxidants during culture. We report that this process is associated with decreased gp91phox, p22phox and p47phox expression, release of PU.1 and CP-1 from gp91phox promoter, and PU.1 from p47phox promoter. However, in presence of IFN-gamma or TNF-alpha, the superoxide anion (O(2)(-)) production, the p47phox, gp91phox and p22phox expression, and the binding of PU.1 and CP-1 to DNA are maintained at the high levels observed in blood monocytes. To clarify the role of PU.1 in the expression of NADPH oxidase components, oligonucleotides competing for PU.1-DNA binding were added to cultured monocytes. These oligonucleotides abrogated the maintenance of gp91phox and p22phox expression by IFN-gamma and TNF-alpha, but did not inhibit the effect of these cytokines on p47phox expression and O(2)(-) production. Our results indicate that in monocytes the IFN-gamma- and TNF-alpha-induced expression of gp91phox and p22phox, but not p47phox, requires the binding of PU.1 to gp91phox promoter. However, the preservation of O(2)(-) production by IFN-gamma and TNF-alpha is unrelated to their effect on gp91phox and p22phox expression.     

Assembly of the phagocyte NADPH oxidase complex: chimeric constructs derived from the cytosolic components as tools for exploring structure-function relationships

Phagocytes generate superoxide (O2*-) by an enzyme complex known as reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Its catalytic component, responsible for the NADPH-driven reduction of oxygen to O2*-, is flavocytochrome b559, located in the membrane and consisting of gp91phox and p22phox subunits. NADPH oxidase activation is initiated by the translocation to the membrane of the cytosolic components p47phox, p67phox, and the GTPase Rac. Cytochrome b559 is converted to an active form by the interaction of gp91phox with p67phox, leading to a conformational change in gp91phox and the induction of electron flow. We designed a new family of NADPH oxidase activators, represented by chimeras comprising various segments of p67phox and Rac1. The prototype chimera p67phox (1-212)-Rac1 (1-192) is a potent activator in a cell-free system, also containing membrane p47phox and an anionic amphiphile. Chimeras behave like bona fide GTPases and can be prenylated, and prenylated (p67phox -Rac1) chimeras activate the oxidase in the absence of p47phox and amphiphile. Experiments involving truncations, mutagenesis, and supplementation with Rac1 demonstrated that the presence of intrachimeric bonds between the p67phox and Rac1 moieties is an absolute requirement for the ability to activate the oxidase. The presence or absence of intrachimeric bonds has a major impact on the conformation of the chimeras, as demonstrated by fluorescence resonance energy transfer, small angle X-ray scattering, and gel filtration. Based on this, a "propagated wave" model of NADPH oxidase activation is proposed in which a conformational change initiated in Rac is propagated to p67phox and from p67phox to gp91phox.     

Mechanism and characteristics of stimuli-dependent ROS generation in undifferentiated HL-60 cells

It has been widely believed that undifferentiated human promyelocytic leukemia cells (HL-60) have no ability to generate reactive oxygen species (ROS) responding to stimuli. We report here that undifferentiated HL-60 cells possess NADPH oxidase and that generation of superoxide can be measured using a highly sensitive chemiluminescence dye, L-012. Five subunits of NADPH oxidase, namely, gp91(phox), p22(phox), p67(phox), p47(phox), and Rac 2, were detected in undifferentiated HL-60 cells by immunoblotting analysis. The contents of these NADPH oxidase components in the cells were increased with the differentiation induced by phorbol myristate acetate (PMA), except for p22(phox). Messenger RNAs of these subunits were also detected by the RT-PCR method, and their expressions increased except that of p22(phox) with the differentiation induced by PMA. Kinetic analysis using L-012 revealed that HL-60 cells generated substantial amounts of ROS by various stimulants, including formylmethionyl-leucyl-phenylalanine, PMA, myristic acid, and a Ca2+ ionophore, A23187. Both diphenyleneiodonium (an inhibitor of FAD-dependent oxidase) and apocynin (a specific inhibitor of NADPH oxidase) suppressed this stimuli-dependent ROS generation. Genistein, staurosporine, uric acid, and sodium azide inhibited the ROS generation in undifferentiated HL-60 cells in a similar way to that in undifferentiated neutrophils. These results suggested that the mechanism of ROS generation in undifferentiated HL-60 cells is the same as that in primed neutrophils.     

The C-terminal flavin domain of gp91phox bound to plasma membranes of granulocyte-like X-CGD PLB-985 cells is sufficient to anchor cytosolic oxidase components and support NADPH oxidase-associated diaphorase activity independent of cytosolic phospholipase A2 regulation

We have previously established a model of cytosolic phospholipase A(2) (cPLA(2))-deficient PLB-985 cells and demonstrated that cPLA(2)-generated arachidonic acid (AA) is essential for reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and NADPH-dependent diaphorase activity. The present study focuses on the C-terminal cytoplasmic domain of gp91(phox) (residues 283-570), which contains the NADPH binding and flavin adenine dinucleotide-reducing center, to determine if this portion is regulated by AA. The gp91(phox) C-terminal reductase domain was expressed in X-CGD PLB-985 cells lacking normal gp91(phox) (X-CGD PLB 91CT cells) and was detected in the plasma membrane. It appears to be bound electrostatically to the plasma membrane, as it is eluted by high salt. Permeabilized, granulocyte-like X-CGD PLB 91CT cells lacking cPLA(2) protein and activity, as well as AA release after stimulation, supported NADPH-dependent diaphorase activity after stimulation, similar to granulocyte-like X-CGD PLB 91CT cells. Normal translocation of p47(phox) and p67(phox) to the membrane fractions of both stimulated cell types indicated that the gp91(phox) C-terminal region is sufficient to anchor the cytosolic oxidase components to the membranes. cPLA(2) translocated to membranes and bound the assembled oxidase in granulocyte-like X-CGD PLB 91CT cells after stimulation. Therefore, the assembled membrane-bound oxidase complex encompassing the flavin domain of gp91(phox) provides a docking site for cPLA(2) but is not the site of AA-based regulation of oxidase activity.     

Anaplasma phagocytophilum utilizes multiple host evasion mechanisms to thwart NADPH oxidase-mediated killing during neutrophil infection

Anaplasma phagocytophilum, the etiologic agent of human anaplasmosis, is a bacterial pathogen that specifically colonizes neutrophils. Neutrophils utilize the NADPH oxidase complex to generate superoxide (O(2)(-)) and initiate oxidative killing of microorganisms. A. phagocytophilum's unique tropism for neutrophils, however, indicates that it subverts and/or avoids oxidative killing. We therefore examined the effects of A. phagocytophilum infection on neutrophil NADPH oxidase assembly and reactive oxygen species (ROS) production. Following neutrophil binding, Anaplasma invasion requires at least 240 min. During its prolonged association with the neutrophil plasma membrane, A. phagocytophilum stimulates NADPH oxidase assembly, as indicated by increased cytochrome b(558) mobilization to the membrane, as well as colocalization of Rac and p22(phox). This initial stimulation taxes the host neutrophil's finite oxidase reserves, as demonstrated by time- and bacterial-dose-dependent decreases in secondary activation by N-formyl-methionyl-leucyl-phenylalanine (FMLP) or phorbol myristate acetate (PMA). This stimulation is modest, however, and does not diminish oxidase stores to nearly the extent that Escherichia coli, serum-opsonized zymosan, FMLP, or PMA do. Despite the apparent activation of NADPH oxidase, no change in ROS-dependent chemiluminescence is observed upon the addition of A. phagocytophilum to neutrophils, indicating that the bacterium may scavenge exogenous O(2)(-). Indeed, A. phagocytophilum rapidly detoxifies O(2)(-) in a cell-free system. Once internalized, the bacterium resides within a protective vacuole that excludes p22(phox) and gp91(phox). Thus, A. phagocytophilum employs at least two strategies to protect itself from neutrophil NADPH oxidase-mediated killing.     

Genetic ablation of NADPH oxidase enhances susceptibility to cigarette smoke-induced lung inflammation and emphysema in mice

Cigarette smoke (CS) induces recruitment of inflammatory cells in the lungs leading to the generation of reactive oxygen species (ROS), which are involved in lung inflammation and injury. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a multimeric system that is responsible for ROS production in mammalian cells. We hypothesized that NADPH oxidase-derived ROS play an important role in lung inflammation and injury and that targeted ablation of components of NADPH oxidase (p47(phox) and gp91(phox)) would protect lungs against the detrimental effects of CS. To test this hypothesis, we exposed p47(phox-/-) and gp91(phox-/-) mice to CS and examined inflammatory response and injury in the lung. Surprisingly, although CS-induced ROS production was decreased in the lungs of p47(phox-/-) and gp91(phox-/-) mice compared with wild-type mice, the inflammatory response was significantly increased and was accompanied by development of distal airspace enlargement and alveolar destruction. This pathological abnormality was associated with enhanced activation of the TLR4-nuclear factor-kappaB pathway in response to CS exposure in p47(phox-/-) and gp91(phox-/-) mice. This phenomenon was confirmed by in vitro studies in which treatment of peritoneal macrophages with a nuclear factor-kappaB inhibitor reversed the CS-induced release of proinflammatory mediators. Thus, these data suggest that genetic ablation of components of NADPH oxidase enhances susceptibility to the proinflammatory effects of CS leading to airspace enlargement and alveolar damage.     

Role of Nox4 and Nox2 in hyperoxia-induced reactive oxygen species generation and migration of human lung endothelial cells

In vascular endothelium, the major research focus has been on reactive oxygen species (ROS) derived from Nox2. The role of Nox4 in endothelial signal transduction, ROS production, and cytoskeletal reorganization is not well defined. In this study, we show that human pulmonary artery endothelial cells (HPAECs) and human lung microvascular endothelial cells (HLMVECs) express higher levels of Nox4 and p22(phox) compared to Nox1, Nox2, Nox3, or Nox5. Immunofluorescence microscopy and Western blot analysis revealed that Nox4 and p22(phox), but not Nox2 or p47(phox), are localized in nuclei of HPAECs. Further, knockdown of Nox4 with siRNA decreased Nox4 nuclear expression significantly. Exposure of HPAECs to hyperoxia (3-24 h) enhanced mRNA and protein expression of Nox4, and Nox4 siRNA decreased hyperoxia-induced ROS production. Interestingly, Nox4 or Nox2 knockdown with siRNA upregulated the mRNA and protein expression of the other, suggesting activation of compensatory mechanisms. A similar upregulation of Nox4 mRNA was observed in Nox2 2(-/-) ko mice. Downregulation of Nox4, or pretreatment with N-acetylcysteine, attenuated hyperoxia-induced cell migration and capillary tube formation, suggesting that ROS generated by Nox4 regulate endothelial cell motility. These results indicate that Nox4 and Nox2 play a physiological role in hyperoxia-induced ROS production and migration of ECs.     

Critical role of the NADPH oxidase subunit p47phox on vascular TLR expression and neointimal lesion formation in high-fat diet-induced obesity

Reactive oxygen species (ROS) formation is associated with inflammation and vasculature dysfunction. We investigated the potential role of the NADPH oxidase on vascular Toll-like receptor (TLR) expression and carotid neointimal formation in high-fat (HF) diet-induced obesity (DIO) model. Using mice DIO and common carotid artery flow cessation-induced lesion formation models, we examined vascular TLR2 and TLR4 expression and neointimal formation in NADPH oxidase subunit p47(phox)-deficient (p47(phox-/-)) mice. Feeding C57BL/6J mice an HF diet for 22 weeks resulted in significant increases in p47(phox), TLR2 and TLR4 expression in vascular tissues compared with mice fed a low-fat (LF) diet. Minimal changes in TLR2 and TLR4 expression was detected in p47(phox-/-) DIO mice. Furthermore, flow cessation-induced angiogenic and inflammatory response and neointimal formation were significantly attenuated in p47(phox-/-) DIO mice compared with wild-type DIO mice. In addition, exposure of endothelial cells to leptin led to ROS formation; this was accompanied by upregulation of TLR2, TLR4 expression and its downstream signaling. Leptin also increased endothelial cell migration and proliferation. Pharmacological inhibition of NADPH oxidase or genetic deletion of p47(phox) significantly diminished these alterations. Obesity increases neointimal formation via a mechanism involving p47(phox)-TLRs signaling, suggesting that the NADPH oxidase may represent a potential novel therapeutic target for the treatment of obesity-associated vascular inflammation and dysfunction.     

First evidence for a crosstalk between mitochondrial and NADPH oxidase-derived reactive oxygen species in nitroglycerin-triggered vascular dysfunction

Chronic nitroglycerin treatment results in development of nitrate tolerance associated with endothelial dysfunction (ED). We sought to clarify how mitochondria- and NADPH oxidase (Nox)-derived reactive oxygen species (ROS) contribute to nitrate tolerance and nitroglycerin-induced ED. Nitrate tolerance was induced by nitroglycerin infusion in male Wistar rats (100 microg/h/4 day) and in C57/Bl6, p47(phox/) and gp91(phox/) mice (50 microg/h/4 day). Protein and mRNA expression of Nox subunits were unaltered by chronic nitroglycerin treatment. Oxidative stress was determined in vascular rings and mitochondrial fractions of nitroglycerin-treated animals by L-012 enhanced chemiluminescence, revealing a dominant role of mitochondria for nitrate tolerance development. Isometric tension studies revealed that genetic deletion or inhibition (apocynin, 0.35 mg/h/4 day) of Nox improved ED, whereas nitrate tolerance was unaltered. Vice versa, nitrate tolerance was attenuated by co-treatment with the respiratory chain complex I inhibitor rotenone (100 microg/h/4 day) or the mitochondrial permeability transition pore blocker cyclosporine A (50 microg/h/4 day). Both compounds improved ED, suggesting a link between mitochondrial and Nox-derived ROS. Mitochondrial respiratory chain-derived ROS are critical for the development of nitrate tolerance, whereas Nox-derived ROS mediate nitrate tolerance-associated ED. This suggests a crosstalk between mitochondrial and Nox-derived ROS with distinct mechanistic effects and sites for pharmacological intervention.