Rac and PI3 kinase mediate endothelial cell-reactive oxygen species generation during normoxic lung ischemia

Abrupt reduction of flow (ischemia) leads to endothelial cell membrane depolarization, NADPH oxidase activation, and reactive oxygen species (ROS) generation in isolated rat and mouse lungs and in flow-adapted endothelial cells in vitro. Here we evaluated the role of PI-3-kinase and rac in activation of endothelial NADPH oxidase. Endothelium of isolated perfused mouse lungs labeled with 2',7'-dichlorodihydrofluorescein (H(2)DCF) or hydroethidine (HE) showed increased ROS generation with ischemia; these results were supported by TBARS measurement in whole-lung homogenate and by in vitro studies using flow-adapted mouse pulmonary microvascular endothelial cells. Ischemia-induced ROS generation in intact lung or isolated cells was blocked by pretreatment with Clostridium difficile toxin B, a rac inhibitor, and by wortmannin or LY294002, PI3 kinase inhibitors. In cells, immunofluorescence and immunoblot after subcellular fractionation showed ischemia-induced translocation of rac, p47(phox), and p67(phox) to the plasma membrane. Increased extracellular K(+) also resulted in rac translocation, providing evidence that this pathway is sensitive to alterations of endothelial cell membrane potential. These results indicate that PI-3-kinase and the small G protein rac are involved in the activation of endothelial cell NADPH oxidase that is associated with the acute loss of shear stress.     

TGF-beta-induced p38 activation is mediated by Rac1-regulated generation of reactive oxygen species in cultured human keratinocytes

p38 has been shown to be involved in TGF-beta-induced gene expression, but the upstream of the signaling pathway leading to the activation of p38 is left undefined. We investigated the pathway in cultured human keratinocytes (HaCat cells). Western blot analysis revealed that TGF-beta induced the activation of p38 within 1 h post TGF-beta treatment. H2O2 also strongly induced p38 activation in a time dependent manner. We also observed that TGF-beta-induced p38 activation was inhibited by PDTC, pyrrolidinedithiocarbamate, a known antioxidant, and DPI, diphenylene iodonium chloride, one of the known NADPH oxidase inhibitors. In contrast, TGF-beta-induced Smad2 phosphorylation was not affected. To test whether reactive oxygen species (ROS) is involved in TGF-beta-induced p38 activation, we examined the generation of ROS and activation of NADPH oxidase. FACS analysis showed that TGF-beta induced generation of ROS in time-dependent manner. DPI, an inhibitor of NADPH oxidase, inhibited TGF-beta-induced ROS production. Lucigenin-based NADPH oxidase assay indicated that TGF-beta-induced NADPH oxidase activity started as early as 5 min following treatment and peaked at about 15 min with induction of about 2-folds. The activity remained elevated up to 1 h. Immunofluorescence microscopy study showed that Rac1, one of the subunits of NADPH oxidase, translocated from cytoplasm to the membrane within 5 min. Pretreatment with DPI dramatically reduced TGF-beta-induced NADPH oxidase activity. Collectively, our data suggest that TGF-beta-induced p38 activation is mediated by Rac1-regulated generation of reactive oxygen species in cultured human keratinocytes.     

二烯丙基三硫诱导人髓样白血病HL-60细胞活性氧产生及其细胞毒性作用

目的：探讨二烯丙基三硫(DATS)诱导人白血病HL-60细胞产生活性氧(ROS)及其细胞毒性作用。方法: 用浓度为50、100、150、200 μmol/L DATS处理HL-60细胞1、3、6、12、24 h后，流式细胞计数法检测HL-60细胞内的ROS水平，NBT还原实验分析NADPH氧化酶的活性，分别用NADPH氧化酶特异性阻断剂apocynin与抗氧化剂NAC（N-acetyl-L-cystein）预处理30 min后，观察DATS对HL-60细胞产生活性氧的影响，分光光度法检测细胞质膜氧化产物丙二醛（MDA）与细胞蛋白氧化产物羰基化蛋白（protein carbonyl）。结果: DATS能诱导人白血病HL-60细胞产生ROS，当DATS处理细胞1-3 h 时，HL-60细胞产生ROS随DATS浓度的升高和处理时间的延长而升高，当浓度为150 μmol/L DATS处理细胞3h时，ROS的荧光强度达到最高峰，其后维持在一个较高水平。NADPH氧化酶活性与ROS的产生一致。HL-60细胞的脂质过氧化和羰基化蛋白浓度与DATS诱导ROS产生的趋势基本一致，都在3 h、150 μmol/L处理点达到最高值。应用NADPH氧化酶特异性阻断剂apocynin或抗氧化剂NAC后，能显著降低HL-60细胞ROS的产生和细胞损伤。结论: NADPH氧化酶是DATS诱导 HL-60 细胞产生ROS的主要酶系，ROS能氧化细胞质膜和蛋白质。     

Effects of N-acetylcysteine on nicotinamide dinucleotide phosphate oxidase activation and antioxidant status in heart, lung, liver and kidney in streptozotocin-induced diabetic rats

Purpose

Hyperglycemia increases reactive oxygen species (ROS) and the resulting oxidative stress plays a key role in the pathogenesis of diabetic complications. Nicotinamide dinucleotide phosphate (NADPH) oxidase is one of the major sources of ROS production in diabetes. We, therefore, examined the possibility that NADPH oxidase activation is increased in various tissues, and that the antioxidant N-acetylcysteine (NAC) may have tissue specific effects on NADPH oxidase and tissue antioxidant status in diabetes.

Materials and Methods

Control (C) and streptozotocin-induced diabetic (D) rats were treated either with NAC (1.5 g/kg/day) orally or placebo for 4 weeks. The plasma, heart, lung, liver, kidney were harvested immediately and stored for biochemical or immunoblot analysis.

Results

levels of free 15-F_2t-isoprostane were increased in plasma, heart, lung, liver and kidney tissues in diabetic rats, accompanied with significantly increased membrane translocation of the NADPH oxidase subunit p67phox in all tissues and increased expression of the membrane-bound subunit p22phox in heart, lung and kidney. The tissue antioxidant activity in lung, liver and kidney was decreased in diabetic rats, while it was increased in heart tissue. NAC reduced the expression of p22phox and p67phox, suppressed p67phox membrane translocation, and reduced free 15-F_2t-isoprostane levels in all tissues. NAC increased antioxidant activity in liver and lung, but did not significantly affect antioxidant activity in heart and kidney.

Conclusion

The current study shows that NAC inhibits NADPH oxidase activation in diabetes and attenuates tissue oxidative damage in all organs, even though its effects on antioxidant activity are tissue specific.     

胆固醇通过NADPH氧化酶诱导ROS升高,NF-κB活化进而导致内皮细胞损伤

目的:研究胆固醇对人脐静脉内皮细胞(HUVECs)的损伤是否与细胞内活性氧(ROS)升高有关,明确此种情况下ROS的主要来源。方法:体外培养HUVECs-12,设置正常对照组、溶媒(无水乙醇)组、胆固醇组、N-乙酰半胱氨酸(NAC)作用1 h后再加入胆固醇作用48 h组。以DCFH-DA为荧光探针,流式细胞术检测细胞内ROS水平;免疫细胞化学方法检测细胞内核因子-κB亚单位p65核移位阳性细胞数;分别用比色法、硝酸酶还原法及ELISA检测细胞培养液中乳酸脱氢酶(LDH)活性、一氧化氮(NO)及单核细胞趋化蛋白-1(MCP-1)浓度。并观察加入产生ROS的4种酶抑制剂二联苯碘(DPI)、鱼藤酮(rotenone)、奥苷嘌醇(oxypurinol)、N-硝基-L-精氨酸甲酯(L-NAME)后细胞内ROS水平。结果:(1)与正常对照组比较,50 mg/L胆固醇能升高细胞内ROS(P0.01),并能激活细胞内NF-κB p65的核移位(P0.01),升高细胞培养液中LDH活性、MCP-1浓度,降低NO浓度(P0.01);(2)与胆固醇作用组相比,NADPH氧化酶抑制剂DPI预作用组细胞内ROS明显降低(P0.01),retenone也可以部分抑制ROS的产生(P0.05),oxypurinol和L-NAME预作用则几乎不影响胆固醇所致的ROS生成增加(P0.05)。结论:一定浓度的游离胆固醇能引起内皮细胞内ROS升高,激活细胞内NF-κB,进而导致内皮细胞损伤;此情况下细胞内产生的ROS主要来源于NADPH氧化酶。     

Aldose reductase regulates platelet-derived growth factor-induced proliferation through mediating cell cycle progression in rat mesangial cells

Aldose reductase (AR), the first and the rate-limiting enzyme of the polyol pathway, has been implicated in platelet-derived growth factor (PDGF)-induced proliferation of rat mesangial cells (MsCs). It is well known that AR plays an important role in various chronic diabetic complications, for example, diabetic nephropathy. Moreover, our previous studies have demonstrated that an AR inhibitor (ARI) significantly reduced the proliferation of rat MsCs induced by PDGF, however, the mechanism remains unclear. The aim of the present study was to elucidate the molecular mechanisms through which AR regulates PDGF-induced rat MsC proliferation. It was demonstrated that PDGF-induced MsC proliferation was significantly inhibited by pretreatment with ARI. Cell cycle analysis by flow cytometry revealed that ARI prevented the entry of cells from the G1 into the S?phase. Furthermore, the effect of the PI3K/Akt signaling pathway on the cell cycle was analyzed. The PI3K/Akt pathway was activated with PDGF treatment. However, ARI blocked Akt activation in response to PDGF. Moreover, PDGF increased the levels of p21Cip1 cyclin kinase inhibitor protein in MsC, which was markedly inhibited by pretreatment with ARI. Conversely, PDGF significantly reduced the levels of the p27Kip1 cyclin kinase inhibitor protein, which was also restored by pretreatment with ARI. In conclusion, AR is involved in PDGF-induced rat MsC proliferation, and may serve as a potential target for the inhibition of MsC proliferation in several types of glomerulonephritis.     

Phagocyte NADPH oxidase: a multicomponent enzyme essential for host defenses

Phagocytes such as neutrophils and monocytes play an essential role in host defenses against microbial pathogens. Reactive oxygen species (ROS), such as superoxide anion, hydrogen peroxide, the hydroxyl radical, and hypochlorous acid, together with microbicidal peptides and proteases, constitute their antimicrobial arsenal. The enzyme responsible for superoxide anion production and, consequently, ROS generation, is called NADPH oxidase or respiratory burst oxidase. This multicomponent enzyme system is composed of cytosolic proteins (p47phox, p67phox, p40phox, and rac1/2) and membrane proteins (p22phox and gp91phox, which form cytochrome b558) which assemble at membrane sites upon cell activation. The importance of this enzyme in host defenses is illustrated by a life-threatening genetic disorder called chronic granulomatous disease in which the phagocyte enzyme is dysfunctional, leading to life-threatening bacterial and fungal infections. Also, because ROS can damage surrounding tissues, their production, and thus NADPH oxidase activation, must be tightly regulated. This review describes the structure and activation of the neutrophil NADPH enzyme complex.     

Signalling mechanisms in sphingosine 1-phosphate-promoted mesangial cell proliferation

BACKGROUND: The bioactive sphingolipid sphingosine 1-phosphate (S1P) is formed by the activation of sphingosine kinase (SPHK) in diverse stimuli, such as platelet-derived growth factor (PDGF). S1P acts not only as an extracellular mediator but also as an intracellular second messenger, resulting in the proliferation of various different types of cells. However, the signal transduction mechanism in S1P-induced proliferation of mesangial cells is poorly known. RESULTS: We examined the signalling mechanisms by which S1P and dihydro-S1P (DHS1P), another S1P receptor agonist, induce mesangial cell proliferation. We first observed that exogenous S1P/DHS1P had additive effects on the PDGF-promoted proliferation of mesangial cells. Treatment of mesangial cells with pertussis toxin almost completely inhibited S1P- and DHS1P-induced, and slightly inhibited PDGF-induced cell proliferation. Additionally, the ERK kinase inhibitor PD98059 partially blocked the proliferation of mesangial cells induced by all these ligands. N,N-dimethylsphingosine, a competitive inhibitor of SPHK, reduced PDGF-induced mesangial cell proliferation, whereas over-expression of SPHK promoted it. We also revealed that PDGF induces SPHK mRNA expression and SPHK activity, suggesting that SPHK, which links the PDGF to the S1P signalling cascade, is, at least in part, involved in PDGF-induced mesangial cell proliferation. Moreover, we found that extracellular S1P stimulates two S1P receptors, EDG3 and EDG5, which leads to cell proliferation and survival. CONCLUSIONS: The data show that S1P-induced mesangial cell proliferation is mediated by EDG-dependent and -independent signalling pathways. S1P may cooperate with PDGF to increase the proliferation of mesangial cells during pathophysiological processes.     

Aluminum induces oxidative burst,cell wall NADH peroxidase activity,and DNA damage in root cells of Allium cepa L

Plants under stress incur an oxidative burst that involves a rapid and transient overproduction of reactive oxygen species (ROS: O₂^??, H₂O₂, ^?OH). We hypothesized that aluminum (Al), an established soil pollutant that causes plant stress, would induce an oxidative burst through the activation of cell wall‐NADH peroxidase (NADH‐PX) and/or plasma membrane‐associated NADPH oxidase (NADPH‐OX), leading to DNA damage in the root cells of Allium cepa L. Growing roots of A. cepa were treated with Al³⁺ (800 μM of AlCl₃) for 3 or 6 hr without or with the pretreatment of inhibitors specific to NADH‐PX and NADPH‐OX for 2 hr. At the end of the treatment, the extent of ROS generation, cell death, and DNA damage were determined. The cell wall‐bound protein (CWP) fractions extracted from the untreated control and the Al‐treated roots under the aforementioned experimental conditions were also subjected to in vitro studies, which measured the extent of activation of peroxidase/oxidase, generation of ^?OH, and DNA damage. Overall, the present study demonstrates that the cell wall‐bound NADH‐PX contributes to the Al‐induced oxidative burst through the generation of ROS that lead to cell death and DNA damage in the root cells of A. cepa. Furthermore, the in vitro studies revealed that the CWP fraction by itself caused DNA damage in the presence of NADH, supporting a role for NADH‐PX in the stress response. Altogether, this study underscores the crucial function of the cell wall‐bound NADH‐PX in the oxidative burst‐mediated cell death and DNA damage in plants under Al stress. Environ. Mol. Mutagen., 2012. © 2012 Wiley Periodicals, Inc.     

Th17 development and autoimmune arthritis in the absence of reactive oxygen species

Dendritic cells (DC) express a functional NADPH oxidase and produce reactive oxygen species (ROS) upon interaction with microbes and T cells. Exposure to ROS leads to DC activation and maturation, as evidenced by phenotypic and functional changes. We have evaluated how endogenous ROS production affects the cytokine secretion pattern and T cell-activating capacity of bone marrow-derived murine DC. DC treated with ROS scavengers, as well as DC from mice that lack a functional NADPH oxidase (and thereby inherently deficient in ROS production) produced significantly increased levels of IL-1beta, IL-6, TNF-alpha and TGF-beta in response to microbial activation. DC deficient in ROS production induced high levels of IFN-gamma and IL-17 in responding T cells after Ag-specific or superantigen-induced activation. Finally, we show that ROS deficiency affected the induction of a T cell-dependent inflammatory condition, collagen-induced arthritis (CIA). C57BL/6 mice that lack a functional NADPH oxidase developed a severe and erosive CD4-dependent CIA, whereas the majority of the congenic wild-type animals remained healthy. These data suggest that ROS act as immunomodulators in DC-driven T cell activation and perhaps also in T cell-dependent immunopathology.     

Arachidonic acid metabolites mediate angiotensin II-induced NADH/NADPH oxidase activity and hypertrophy in vascular smooth muscle cells

Previously, we showed that angiotensin II stimulation of the NADH/NADPH oxidase is involved in hypertrophy of cultured vascular smooth muscle cells (VSMC). Here, we examine the pathways leading to oxidase activation, and demonstrate that arachidonic acid metabolites mediate hypertrophy by activating the p22phox-based NADH/NADPH oxidase. Angiotensin II stimulates phospholipase A2, releasing arachidonic acid, which stimulates oxidase activity in vitro. When arachidonic acid metabolism is blocked with 5,8,11,14-eicosatetraynoic acid (ETYA) or nordihydroguaiaretic acid (NDGA), oxidase activity decreases by 80 +/- 10%. In VSMC transfected with antisense p22phox to attenuate NADH/NADPH oxidase expression, arachidonic acid is unable to stimulate NADH/NADPH-dependent superoxide production. In these cells, or in cells in which NADH/NADPH oxidase activity is inhibited by diphenylene iodonium, angiotensin II-induced [3H]leucine incorporation is also inhibited. Attenuation of oxidase activation by inhibiting arachidonic acid metabolism with ETYA, NDGA, baicalein, or SKF-525A also inhibits angiotensin II-stimulated protein synthesis (74 +/- 2% and 34 +/- 1%, respectively). Thus, endogenous noncyclooxygenase arachidonic acid metabolites mediate angiotensin II-stimulated protein synthesis in cultured VSMC by activating the NADH/NADPH oxidase, providing mechanistic evidence for redox control of VSMC hypertrophy.     

胰岛素通过活性氧的产生促进VEGF表达及血管平滑肌细胞迁移和增殖

 目的:探讨活性氧（reactive oxygen species, ROS）在胰岛素促进的血管平滑肌细胞迁移和增殖中的作用及分子机制。方法:采用原代培养的大鼠主动脉血管平滑肌细胞,应用DCF-DA荧光探针检测细胞内ROS的生成;应用实时定量PCR、Western blotting和ELISA法检测mRNA和蛋白的表达;应用转染报告基因的方法检测基因的转录活性;划痕法测定细胞迁移;CCK-8法测定细胞增殖。结果:胰岛素处理后血管平滑肌细胞内ROS产生明显增加。过氧化氢酶和NADPH氧化酶抑制剂二亚苯基碘鎓（DPI）明显抑制胰岛素促进的ROS生成及p-Akt、p-p70S6K1和p-ERK1/2蛋白的表达。过氧化氢酶和DPI明显降低胰岛素促进的血管内皮生长因子（vascular endothelial growth factor, VEGF）的mRNA和蛋白表达及转录激活。抑制ROS产生明显抑制胰岛素刺激的血管平滑肌细胞迁移和增殖。结论: 胰岛素通过NADPH氧化酶途径促进血管平滑肌细胞ROS产生。ROS介导了胰岛素促进的Akt/p70S6K1和ERK信号通路的激活、VEGF表达及血管平滑肌细胞的迁移和增殖。     

二氢青蒿素通过抑制SIRT1的表达而增强5-氟尿嘧啶对胃癌的抗肿瘤活性

目的:探讨二氢青蒿素对5-氟尿嘧啶治疗胃癌的辅助作用并研究其机制。方法:实验分为对照组、二氢青蒿素组、5-氟尿嘧啶组、5-氟尿嘧啶联合二氢青蒿素组和5-氟尿嘧啶+二氢青蒿素+SIRT1质粒组。MTT法检测胃癌细胞系BGC-823在5-氟尿嘧啶联合二氢青蒿素处理下的细胞活力。Western blot实验检测5-氟尿嘧啶联合二氢青蒿素对BGC-823细胞SIRT1和NADPH氧化酶表达水平,caspase-9和caspase-3活化水平及凋亡信号调节激酶1(ASK1)和c-Jun氨基末端激酶(JNK)蛋白磷酸化水平的影响。流式细胞术检测BGC-823细胞在5-氟尿嘧啶和二氢青蒿素联合处理下的活性氧簇(ROS)生成水平和细胞凋亡率。结果:二氢青蒿素处理能显著抑制BGC-823细胞SIRT1的表达并增加NADPH氧化酶的蛋白水平,明显提高BGC-823细胞对5-氟尿嘧啶的敏感性,降低5-氟尿嘧啶的半数抑制浓度;转染SIRT1表达质粒后,二氢青蒿素联合5-氟尿嘧啶对BGC-823细胞的杀伤活性受到显著抑制(P0.05)。二氢青蒿素能明显促进5-氟尿嘧啶对BGC-823细胞生成ROS的诱导效应和ASK1及JNK的磷酸化(P0.05)。用ROS清除剂N-乙酰半胱氨酸(NAC)或JNK特异性抑制剂SP600125处理后,二氢青蒿素联合5-氟尿嘧啶对BGC-823细胞的杀伤活性和caspase-9及caspase-3的活化均受到明显抑制(P0.05)。另外,NAC能显著抑制二氢青蒿素联合5-氟尿嘧啶对JNK磷酸化的促进作用,而SP600125却不能影响BGC-823细胞ROS的产生,表明JNK是ROS的下游分子。结论:二氢青蒿素联合5-氟尿嘧啶通过SIRT1/NADPH氧化酶/ROS/JNK通路诱导胃癌细胞发生caspase依赖的凋亡。     

Lipoxins inhibit Akt/PKB activation and cell cycle progression in human mesangial cells

Lipoxins (LX) are endogenously produced eicosanoids with a spectrum of bioactions that suggest anti-inflammatory, pro-resolution roles for these agents. Mesangial cell (MC) proliferation plays a pivotal role in the pathophysiology of glomerular inflammation and is coupled to sclerosis and tubulointerstitial fibrosis. We have previously reported that LXA4 acts through a specific G-protein-coupled-receptor (GPCR) to modulate MC proliferation in response to the proinflammatory mediators LTD4 and platelet-derived growth factor (PDGF). Further investigations revealed that these effects were mediated by modulation of receptor tyrosine kinase activity. Here we have explored the underlying mechanisms and report inhibition of growth factor (PDGF; epithelial growth factor) activation of Akt/PKB by LXA4. LXA4 (10 nmol/L) modulates PDGF-induced (10 ng/ml, 24 hours) decrements in the levels of cyclin kinase inhibitors p21Cip1 and p27Kip1. PDGF-induced increases in CDK2-cyclin E complex formation are also inhibited by LXA4. The potential of LXA4 as an anti-inflammatory therapeutic is compromised by its degradation; this has been circumvented by synthesis of stable analogs. We report that 15-(R/S)-methyl-LXA4 and 16-phenoxy-LXA4 mimic the native compound with respect to modulation of cell proliferation and PDGF-induced changes in cell cycle proteins. In vivo, MC proliferation in response to PDGF is associated with TGFbeta1 production and the subsequent development of renal fibrosis. Here we demonstrate that prolonged (24 to 48 hours) exposure to PDGF is associated with autocrine TGFbeta1 production, which is significantly reduced by LXA4. In aggregate these data demonstrate that LX inhibit PDGF stimulated proliferation via modulation of the PI-3-kinase pathway preventing mitogen-elicited G1-S phase progression and suggest the therapeutic potential of LX as anti-fibrotic agents.     

Evidence for a NADH/NADPH oxidase in human umbilical vein endothelial cells using electron spin resonance

A growing body of evidence has suggested that a membrane-bound NADH/NADPH oxidase is the predominant source of reactive oxygen species (ROS) in vascular cells. Prior studies have used indirect assessments of superoxide including lucigenin-enhanced chemiluminescence, cytochrome c, and fluorescent dye techniques. The present study was performed to determine if NADH/NADPH oxidase function could be detected human endothelial cells using electron spin resonance. Human umbilical vein endothelial cells (HUVEC) were homogenized and fractionated into cytosolic and membrane components. Cell fractions were incubated in buffer containing either NADH or NADPH (100 microM for each) and the spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO). EPR signals were obtained in a Bruker EMX spectrometer. Cytoplasmic fractions were devoid of activity. In contrast, incubation of membrane fractions with NADH produced a signal with a total peak intensity of 1,038 +/- 64, which was significantly greater than that observed with NADPH (540 +/- 101). The signal was completely inhibited by either manganese superoxide dismutase (MnSOD, 100 U/ml) or the flavoprotein inhibitor diphenylene iodinium (DPI, 100 microM). Rotenone (100 microM) did not significantly alter the signal intensity, (833 +/- 88). These data demonstrate direct evidence for a functional NADH/NADPH oxidase in human endothelial cells and show that electron spin resonance is a useful tool for study of this enzyme system.     

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.     

Pigment epithelium-derived factor (PEDF) blocks angiotensin II-induced T cell proliferation by suppressing autocrine production of interleukin-2

Angiotensin II (Ang II) elicits numerous inflammatory-proliferative responses in vascular cells, thereby being involved in atherosclerosis. We have previously shown that pigment epithelium-derived factor (PEDF) blocks the Ang II-induced endothelial cell activation, thus suggesting that PEDF may play a role in atherosclerosis. However, effects of PEDF on T cell activation, another key steps of atherosclerosis, remain to be elucidated. In this study, we examined whether PEDF could inhibit the Ang II-induced MOLT-3 T cell proliferation in vitro and the way that it might achieve this effect. Ang II significantly stimulated DNA synthesis in MOLT-3 T cells, which was inhibited by PEDF, olmesartan, an Ang II type I receptor blocker, an anti-oxidant N-acetylcysteine (NAC), or antibodies directed against IL-2. PEDF or NAC suppressed gene expression of interleukin-2 (IL-2) in Ang II-exposed MOLT-3 T cells. Furthermore, PEDF blocked the Ang II-induced reactive oxygen species (ROS) generation and NADPH oxidase activity in MOLT-3 T cells. These results demonstrate that PEDF inhibits the Ang II-induced T cell proliferation by blocking autocrine production of IL-2 via suppression of NADPH oxidase-mediated ROS generation. Blockade by PEDF of T cell activation may become a novel therapeutic target for atherosclerosis.     

Regulation of platelet-derived growth factor-induced Ras signaling by poliovirus receptor Necl-5 and negative growth regulator Sprouty2

Necl-5, known as a poliovirus receptor and up-regulated in many cancer cells, enhances platelet-derived growth factor (PDGF)-induced activation of Ras-Raf-MEK-ERK signaling, but not PDGF-induced tyrosine phosphorylation of PDGF receptor, resulting in facilitation of cell proliferation. Here, we showed that Necl-5 interacted with Sprouty2, known to be a negative regulator of growth factor-induced signaling, and reduced the inhibitory effect of Sprouty2 on PDGF-induced Ras signaling. Necl-5 was reported to be down-regulated by its trans-interaction with nectin-3 upon cell-cell contact, initiating cooperative cell-cell adhesion with cadherin. This down-regulation of Necl-5 caused tyrosine phosphorylation of Sprouty2 by c-Src, which was activated by PDGF receptor in response to PDGF, and inhibited PDGF-induced Ras signaling. Thus, Necl-5 and Sprouty2 cooperatively regulate PDGF-induced Ras signaling. The roles of Necl-5 and Sprouty2 in contact inhibition for cell proliferation are also discussed.     

The ROS-NOX connection in cancer and angiogenesis

Initially viewed as dangerous byproducts of aerobic life, reactive oxygen species (ROS) nowadays appear to be essential secondary messengers of many signaling cascades and cellular functions. The establishment of ROS as important signaling molecules has been confirmed by the existence of specialized ROS producing complexes expressed in nonphagocytic cells, the NADPH oxidase complex (NOX). Because of the diversity of their proteic targets (besides lipids and DNA), ROS have multiple and sometimes contradictory functions. In the present review, we focus on several different signaling pathways influenced by ROS and NOX in tumorigenesis, focusing on proliferation and angiogenesis. We review the ROS targets regulating proliferation, including cellular signaling (phosphatases, AP1, and nuclear factor-kappa B [NF-kappaB]) and cell cycle targets (CDC25, cyclin D, and forkhead proteins), and the role of NOX during proliferation. Finally, we review the direct and indirect involvement of ROS and NOX in (tumor) angiogenesis through the regulation of different biologic systems such as vascular endothelial growth factor, angiotensin II, hypoxia-inducible factor, AP1, and inflammation.