Important Role of Nox4 Type NADPH Oxidase in Angiogenic Responses in Human Microvascular Endothelial Cells In Vitro
Objective— Redox signaling mediated by Nox2-containing NADPH oxidase has been implicated in angiogenic responses both in vitro and in vivo. Because Nox4 type NADPH oxidase is also highly expressed in endothelial cells, we studied the role of Nox4 in angiogenic responses in human endothelial cells in culture.
Methods and Results— Inhibition of Nox4 expression by small interfering RNA reduced angiogenic responses as assessed by the tube formation and wound healing assays, in both human microvascular and umbilical vein endothelial cells. Overexpression of wild-type Nox4 enhanced, whereas expression of a dominant negative form of Nox4 suppressed the angiogenic responses in endothelial cells. These effects were mimicked by exogenous H2O2 and the antioxidant compound ebselen, respectively. Overexpression of Nox4 enhanced receptor tyrosine kinase phosphorylation and the activation of extracellular signal-regulated kinase (Erk). Inhibition of the Erk pathway reduced the endothelial angiogenic responses. Nox4 expression also promotes proliferation and migration of endothelial cells, and reduced serum deprivation–induced apoptosis.
Conclusions— Nox4 type NADPH oxidase promotes endothelial angiogenic responses, at least partly, via enhanced activation of receptor tyrosine kinases and the downstream Erk pathway.
Angiogenesis is a fundamental developmental and adult physiological process, requiring the coordinated action of a variety of growth factors and cell-adhesion molecules in endothelial and mural cells. Understanding the molecular and cellular mechanisms of angiogenesis has profound clinical implications. Inhibiting angiogenesis is a promising strategy for treatment of cancer and several other disorders, including age-related macular degeneration, whereas therapeutic angiogenesis (promoting new vessel growth to treat ischemic disorders) is an exciting frontier of cardiovascular medicine.1 In both cases, a thorough understanding of the signaling pathways involved in angiogenic process is needed.
Vascular endothelial growth factor (VEGF) is one of the most important angiogenic stimuli of angiogenic responses in vascular endothelial cells.2 It is shown that VEGF stimulates generation of reactive oxygen species (ROS) in endothelial cells via activation of the reduced β-nicotinamide adenine dinucleotide phosphate (NADPH) oxidase,3 which is involved in modulating the angiogenic activities of endothelial cells.4 Endogenously generated ROS are known to serve as second messengers activating multiple intracellular signaling pathways that have key roles in endothelial cell biology.5 There is evidence that the Nox2 type NADPH oxidase has an important role in mediating angiogenic responses both in vitro6 and in vivo.7 In Nox2-deficient animals, both VEGF- and tissue ischemia-induced new blood vessel formation is blunted,6,7 indicating that Nox2-dependent redox signaling is important in modulating angiogenesis.
We have previously shown that Nox4 is important for ROS generation in human endothelial cells,8 which is consistent with the findings by others.9,10 Moreover, there is evidence that Nox4 is involved in modulating endothelial cell proliferation.11 Together with the finding that the level of Nox4 expression in endothelial cells in vivo is high as compared with other Nox isoforms,12 these data strongly suggest that Nox4 also has a critical role in mediating endothelial redox signaling and is functionally important in modulating endothelial cell physiology. In this study we examined the role of Nox4 in angiogenic responses in human endothelial cells in vitro.
An expanded Methods section has been published online (please see http://atvb.ahajournals.org).
Human microvascular endothelial cells (HMECs) are a gift from Professor Philip Hogg (University of New South Wales, Sydney, Australia). Human umbilical vein endothelial cells (HUVECs) were obtained from ATCC. Cells were cultured in EGM-2 BulletKit (Cambrex Corporation) containing 5% FCS in a CO2/O2 incubator at 37°C.
Transfection was performed as described previously.8 The Silencer Predesigned small interfering RNA (siRNA) sequence targeting the exon 2 of human Nox4 was obtained from Ambion (Catalogue No. AM16706, sequence ID #118807). The Negative Control siRNA #1 (Catalogue No. AM4611, Ambion) was used as control.
Western blot analysis was performed as described previously.8
Cell Migration Assay
Cell migration was examined in a modified Boyden chamber system (Neuro Probe Inc) as described previously.13
Preparation of Adenoviral Vectors
The replication-deficient adenoviral vectors encoding β-galactosidase (Ad-LacZ), human wild-type Nox4 (Ad-Nox4WT), or a truncated form of Nox4 lacking the NADPH binding domain (Ad-Nox4ΔNADPH) were kindly provided by Dr Barry Goldstein (Thomas Jefferson University, Philadelphia, Pa).14 Cell transduction was performed by incubating the cells with the virus preparation for 48 hour in the absence (for Ad-Nox4WT) or presence (for Ad-Nox4ΔNADPH) of 5% serum.
Real-Time Polymerase Chain Reaction
Quantitative real-time polymerase chain reaction (PCR) analysis was performed as described previously.8
Endothelial cell apoptosis was measured by caspase 3/7 activation using the Caspase-Glo 3/7 Luminescent Assay kit (Promega) as per manufacturer’s instruction.
Intracellular ROS generation was measured by 2′,7′-dichlorofluorescin diacetate (DCFH-DA) fluorescence as described previously.15
Data and Statistical Analysis
Data are expressed as mean±SEM. The mean data were analyzed with Student t test or 1-way ANOVA followed by Newman-Keuls t test as appropriate. A value of P<0.05 was regarded as statistically significant.
Nox4 Is Important for ROS Generation in Endothelial Cells
To demonstrate that the Nox4 subunit is important in catalyzing ROS generation in endothelial cells, we used Nox4 siRNA-mediated gene silencing to modulate Nox4 expression. In HMECs, Nox4 siRNA (100 nmol/L) significantly reduced the expression of Nox4, whereas Nox2 was unchanged (Figure 1a and the supplemental Figure Ia), which is consistent with our previous results.8 The Nox4 siRNA significantly reduced the ROS production from HMECs by about 40% (supplemental Figure Ic).
To further explore the role of Nox4 in endothelial ROS generation, we used adenoviral vector-mediated gene transfer. To determine the efficiency of cell transduction, we infected the HMECs with different concentrations of Ad-LacZ for 48 hours, and the transgene expression was examined by X-gal staining. We found that at a concentration of 3×105 pfu/mL, > 95% of the cells were stained positive (blue) (supplemental Figure II). Using the same viral concentration, we then examined the effects of wild-type Nox4 (AdNox4WT) overexpression. We found that AdNox4WT treatment significantly increased the Nox4 protein level, without any effect on Nox2 expression (Figure 1b and supplemental Figure Ib). Wild-type Nox4 overexpression significantly increased endothelial ROS production (supplemental Figure Id). Moreover, expression of a dominant negative form of Nox4 (Ad-Nox4ΔNADPH)14 significantly reduced ROS production in HMECs (supplemental Figure Id). However, the Nox4 antibody used in our study does not react with the truncated form of Nox4, thus it is difficult to directly analyze the expression level of the dominant negative Nox4 protein.
Angiogenic Responses Were Inhibited by Nox4 Gene Silencing
To clarify the role of Nox4 in angiogenic responses of endothelial cells, we examined the effects of Nox4 gene silencing on the tube formation and wound healing responses in HMECs. As shown in Figure 2a and 2b, knocking down of Nox4 expression significantly reduced both of the tube formation and wound healing responses. To further confirm these findings, we repeated the experiments in HUVECs and similar results were observed (Figure 2a and 2b). The quantitative data are shown in supplemental Figure III.
Angiogenic Responses Were Enhanced by Wild-Type, but Inhibited by Dominant Negative, Nox4 Expression
To further examine the role of Nox4 in angiogenic responses in endothelial cells, we expressed the wild-type and dominant negative Nox4 in HMECs. As shown in Figure 3a and 3b, Ad-Nox4WT treatment significantly enhanced the wound healing response, whereas Ad-Nox4ΔNADPH significantly reduced the response. In the tube formation assay, however, we found that transduction with the viral vectors impaired the morphogenetic capacity of the HMECs. In Ad-LacZ–treated cells, no obvious tube structures could be identified up to 24 hours after cell plating. In contrast, a few tubes could be observed between cells in Ad-Nox4WT–transduced cells at 6 hours, although these tubes remained premature till 24 hours and no interconnecting capillary-like networks formed as in normal cells (supplemental Figure IV).
To clarify whether these effects of Ad-Nox4WT and Nox4ΔNADPH can be mimicked by directly modulating the intracellular ROS levels, we examined the effects of exogenous H2O2 and a ROS scavenger ebselen. As shown in supplemental Figure V, both of the wound healing and tube formation responses were enhanced by exogenous H2O2 but attenuated by ebselen. The inhibitory effect on wound healing by higher concentrations of H2O2 may be because of the prolonged incubation time (18 hours) used in this assay, which resulted in oxidative cellular injury.
Nox4 Is Important in Promoting Endothelial Cell Migration and Proliferation
To clarify the effects of Nox4 on endothelial cell migration and proliferation, we tested the effects of Nox4 gene silencing and Nox4 overexpression in HMECs. As shown in Figure 4a, in cells maintained in low-serum medium overnight and without exogenous VEGF, the basal spontaneous migration of the cells was significantly reduced by the Nox4 siRNA treatment. Addition of VEGF (from Sigma) in the lower compartment of the Boyden chamber induced chemotaxis of the cells toward VEGF, which was blunted by Nox4 siRNA treatment. In cells maintained in medium containing 5% serum, the basal migration rate was much elevated, and VEGF failed to induce further chemotaxis under such a condition. In both situations, Nox4 siRNA significantly inhibited cell migration. The motility of HMECs in both low- or 5%-serum medium, or in the presence of a VEGF gradient, was all significantly enhanced in wild-type Nox4-overexpressing cells as compared with Ad-LacZ transduced cells (online Figure VIa).
We also examined the role of Nox4 in cell proliferation in HMECs. Cells treated with Nox4 siRNA exhibited decreased growth rates in the presence of serum, VEGF, or their combination (Figure 4b). Interestingly, Nox4 siRNA treatment also slightly decreased the cell number under quiescent conditions (low serum without growth factor). We propose that this may be attributable to impaired cell survival in Nox4 siRNA-treated cells during serum deprivation. Conversely, the growth rate in HMECs was enhanced in cells overexpressing Nox4 as compared with the LacZ-expressing cells (supplemental Figure VIb). In the experiments of supplemental Figure VIb, the lack of stimulatory effects of VEGF and serum on cell growth was likely because of the prolonged serum starvation (48 hours) during viral vector infection.
Nox4 Modulates Receptor Tyrosine Kinase-Mediated Signaling
There is evidence that, in adipocytes, Nox4-dependent ROS production may modulate intracellular signaling by inactivating protein tyrosine phosphatases (PTPs),14 resulting in enhanced tyrosine phosphorylation events. To elucidate whether this mechanism is also involved in endothelial cells, we firstly examined whether Nox4 expression affects receptor tyrosine kinase activation. We analyzed VEGF receptor-2 (VEGFR-2/KDR) phosphorylation. However, Western blot could not detect VEGFR-2 in HMECs. Unexpectedly, we found that the platelet-derived growth factor (PDGF) receptor-α could be readily detected. Using a phospho-specific antibody (from Sigma), we found that the level of phosphorylation of PDGFR-α was enhanced in Ad-Nox4WT-transduced cells, but decreased in Ad-Nox4ΔNADPH-transduced cells (Figure 5a). Interestingly, these PDGF receptors were phosphorylated without PDGF stimulation under the current experimental conditions, whereas PDGF did not further increase the phosphorylation status of the receptors (data not shown). To further confirm that Nox4 is important in modulating growth factor–induced downstream signaling, we analyzed the effects of Nox4 overexpression on extracellular signal-related kinase (Erk) 1/2 activation. As shown in Figure 5b, VEGF stimulated Erk1/2 phosphorylation, which peaked at 5 to 10 minutes and remained elevated up to 20 minutes. Nox4 overexpression increased the basal level of Erk1/2 phosphorylation, and enhanced that after VEGF stimulation at all time points examined. To clarify whether Nox4 may modulate endothelial angiogenic responses via the Erk pathway, we treated the cells with the MEK (MAPK/ERK kinase) inhibitor U0126. As shown in supplemental Figure VII, U0126 significantly reduced the wound healing and tube formation responses, indicating that the Erk pathway is directly involved in endothelial angiogenic responses. This is consistent with the findings by other groups.16,17 Finally, we examined whether VEGF affects Nox4 expression. Real-time PCR experiments demonstrated that VEGF did not significantly change Nox4 mRNA levels from 2 to 24 hours (supplemental Figure VIII).
The Effects of Nox4 on Angiogenesis Is Independent of Nitric Oxide Functions
It is known that endothelial nitric oxide (NO) has an important role in modulating angiogenesis.18 To clarify whether the effects of Nox4 on angiogenic responses in endothelial cells involve modulation of the endothelial NO function, we examined the endothelial nitric oxide synthase (eNOS) expression and NO availability in HMECs. As compared with primary HUVECs, the expression level of eNOS in the microvascular endothelial cells is low (supplemental Figure IXa). Then we measured the intracellular NO level with the NO-sensitive probe 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM, Invitrogen) and fluorescent microscopy. As shown in supplemental Figure IXb, treatment with the calcium ionophore A23187 failed to induce DAF-FM fluorescence in HMECs, whereas the NO donor S-nitroso-N-acetylpenicillamine (SNAP) increased the intracellular fluorescence above the background. Although we found that in wild-type Nox4-expressing cells, the NO level after loading with SNAP was significantly lower that in Ad-LacZ–treated cells (supplemental Figure IXc), in separate experiments we observed that treatment with the NOS inhibitor NG-nitro-l-arginine methyl ester (L-NAME) did not change the wound healing response (supplemental Figure IXd). Together with the low eNOS expression in HMECs, these results indicate that the endogenous NO is unlikely to have a major role in modulating angiogenesis in these cells. However, we could not exclude the involvement of NO in other endothelial cells that express higher amounts of eNOS.
Nox4 Is Antiapoptotic
Finally, we studied how Nox4 modulates endothelial cell apoptosis in HMECs. Apoptosis was induced by serum deprivation for 48 hours. Overexpression of wild-type Nox4 inhibited serum deprivation-induced activation of Caspase 3/7 by about 40% (supplemental Figure X). The NOS inhibitor L-NAME did not modify the apoptotic response to serum deprivation (data not shown), suggesting that peroxynitrite formation by NO and superoxide is unlikely to be involved.
Previous studies have shown that the Nox4 type NADPH oxidase is abundantly expressed in endothelial cells,10,12 and this subunit is important in mediating NADPH oxidase-dependent superoxide generation in these cells because Nox4 gene silencing induced by either antisense oligonucleotide10 or siRNA8 suppressed endothelial superoxide release. Our previous study has demonstrated that in vascular smooth muscle cells, Nox4 expression is increased after cytokine stimulation and is responsible for the resultant increase in ROS production, indicating that Nox4 may be involved in mediating vascular oxidative stress under inflammatory conditions.19 However, the role of constitutively expressed Nox4 under physiological conditions in vascular endothelial cells, remains to be defined. In this study, we provided evidence that Nox4 has an important role in modulating angiogenic responses of human endothelial cells in culture. To assess the angiogenic activity of endothelial cells in vitro, we carried out a variety of assays, including proliferation, migration, tube formation, and wound healing assays. In all these experiments, we found that the angiogenic responses were consistently inhibited by Nox4 siRNA or overexpression of a dominant negative Nox4. Moreover, overexpression of the wild-type Nox4 enhanced the angiogenic responses in endothelial cells, and these effects were mimicked by exogenous H2O2. The observation that inhibition of Nox4 expression suppressed endothelial cell proliferation is consistent with the results reported recently.11 All these data suggest that Nox4 is an important modulator of the regenerative function of endothelial cells, and Nox4 expression in these cells is required to support an efficient angiogenic process. Our study warrants further investigation of the role of Nox4 in angiogenesis in vivo.
Our results complement the previous finding that the Nox2 NADPH oxidase is essential for mediating angiogenic responses both in vitro and in vivo.6,7 In endothelial cells, both Nox2 and Nox4 are involved in endothelial ROS generation.10,20 Moreover, inhibition of the expression of either Nox2 or Nox4 suppressed endothelial cell proliferation similarly.11 Several groups have studied the intracellular localization of Nox2 and Nox4 in endothelial cells and have unequivocally shown that these Nox subunits have a similar pattern of intracellular distribution, predominantly existing in the cytoplasm in association with the endoplasmic reticulum or cytoskeleton,11,21–23 in contrast to the membrane localization of the neutrophil Nox2. All these results indicate that both Nox2 and Nox4 are equivalently important in modulating endothelial cell functions. Whether they have different roles in endothelial cell biology is unclear, although there is evidence that in endothelial cells, the mRNA transcripts of Nox4 are expressed more highly than that of Nox2.10,23 Given the results by Ushio-Fukai et al,6 our study suggests that Nox2 and Nox4 have nonredundant effects on modulating angiogenic process, as separate inhibition of each isoform similarly decreased the angiogenic activities in ECs. Therefore it would be interesting to study whether simultaneous inhibition of both isoforms has synergistic effects on the endothelial angiogenic response.
The involvement of Nox4-dependent redox mechanisms in regulating intracellular signaling and cellular functions has been studied in nonendothelial cells. For example, in insulin-sensitive adipocytes, expression of the dominant negative Nox4 attenuated insulin-stimulated ROS generation, insulin receptor tyrosine phosphorylation, activation of downstream serine kinases, and glucose uptake, indicating that Nox4 is involved in promoting insulin-induced signaling by intracellular ROS generation.14 One well established target of intracellular ROS-mediated signaling is the thiol-containing PTPs, which terminate growth factor-induced signals by dephosphorylation of the receptor tyrosine kinases. Inactivation of PTPs may prolong the activation of the receptor tyrosine kinases and thereby enhance the downstream signaling cascades.24 Consistent with this notion, we demonstrated that overexpression of wild-type Nox4 enhanced receptor tyrosine kinase phosphorylation and enhanced the activation of Erk. To our knowledge, this is the first evidence that Nox4 is involved in modulating receptor tyrosine kinase activation in endothelial cells. Our results support the idea that Nox4 has primary physiological roles in maintaining normal cell signaling and functions, although exaggerated expression of this subunit may be involved in cellular oxidant stress.
We also found that Nox4 overexpression prevents endothelial cell apoptosis. This observation is consistent with previous reports in pancreatic cancer cells that ROS produced by Nox4 NADPH oxidase inhibited apoptosis, whereas inhibition of Nox4 expression activated apoptosis in these cells.25,26 The mechanisms by which Nox4 produces antiapoptotic effects are not totally understood. In pancreatic cancer cells, it has been shown that Nox4 gene silencing or blockade of NADPH oxidase by diphenyleneiodonium suppressed phosphorylation and activation of the cell survival kinase Akt, suggesting that Nox4-induced cell survival involves the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway. This argument is further supported by a recent study in the mouse heart.27 Whether this pathway is also responsible for the antiapoptotic actions of Nox4 in endothelial cells has not been examined. Nonetheless, our observation that Nox4 expression enhanced VEGF-induced signaling supports this notion, because PI3-kinase/Akt is downstream of receptor tyrosine kinase activation. On the other hand, it is known that high levels of ROS may induce cell death. Therefore the regulatory effects of Nox4 on cell apoptosis and survival may vary under different conditions, for there is evidence that Nox4 is involved in promoting apoptosis of human aortic smooth muscle cells during endoplasmic reticulum stress induced by oxidized lipids.28
In summary, our results suggest that the Nox4 type NADPH oxidase-dependent redox signaling is an important positive modulator of angiogenic responses in cultured endothelial cells, and this effect is, at least partly, mediated by enhanced VEGF-induced intracellular signaling.
The authors thank Nancy Guo for technical assistance with the real-time PCR.
Sources of Funding
This study is supported by project grants from the Australian National Health and Medical Research Council and Grants-in-Aid from the National Heart Foundation. G.J.D. is a NHMRC Principal Research Fellow.
Original received February 12, 2007; final version accepted August 13, 2007.
Griendling KK, Sorescu D, Ushio-Fukai M. NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res. 2000; 86: 494–501.
Kunsch C, Medford RM. Oxidative stress as a regulator of gene expression in the vasculature. Circ Res. 1999; 85: 753–766.
Ushio-Fukai M, Tang Y, Fukai T, Dikalov SI, Ma Y, Fujimoto M, Quinn MT, Pagano PJ, Johnson C, Alexander RW. Novel role of gp91(phox)-containing NAD(P)H oxidase in vascular endothelial growth factor-induced signaling and angiogenesis. Circ Res. 2002; 91: 1160–1167.
Tojo T, Ushio-Fukai M, Yamaoka-Tojo M, Ikeda S, Patrushev N, Alexander RW. Role of gp91phox (Nox2)-containing NAD(P)H oxidase in angiogenesis in response to hindlimb ischemia. Circulation. 2005; 111: 2347–2355.
Jiang F, Roberts SJ, raju Datla S, Dusting GJ. Nitric oxide modulates NADPH oxidase function via heme oxygenase-1 in human endothelial cells. Hypertension. 2006; 48: 950–957.
Thum T, Borlak J. Mechanistic role of cytochrome P450 monooxygenases in oxidized low-density lipoprotein-induced vascular injury: therapy through LOX-1 receptor antagonism? Circ Res. 2004; 94: e1–e13.
Ago T, Kitazono T, Ooboshi H, Iyama T, Han YH, Takada J, Wakisaka M, Ibayashi S, Utsumi H, Iida M. Nox4 as the major catalytic component of an endothelial NAD(P)H oxidase. Circulation. 2004; 109: 227–233.
Sorescu D, Weiss D, Lassegue B, Clempus RE, Szocs K, Sorescu GP, Valppu L, Quinn MT, Lambeth JD, Vega JD, Taylor WR, Griendling KK. Superoxide production and expression of nox family proteins in human atherosclerosis. Circulation. 2002; 105: 1429–1435.
Mahadev K, Motoshima H, Wu X, Ruddy JM, Arnold RS, Cheng G, Lambeth JD, Goldstein BJ. The NAD(P)H oxidase homolog Nox4 modulates insulin-stimulated generation of H2O2 and plays an integral role in insulin signal transduction. Mol Cell Biol. 2004; 24: 1844–1854.
Meadows KN, Bryant P, Pumiglia K. Vascular endothelial growth factor induction of the angiogenic phenotype requires Ras activation. J Biol Chem. 2001; 276: 49289–49298.
Bhagwat SV, Petrovic N, Okamoto Y, Shapiro LH. The angiogenic regulator CD13/APN is a transcriptional target of Ras signaling pathways in endothelial morphogenesis. Blood. 2003; 101: 1818–1826.
Bayraktutan U, Blayney L, Shah AM. Molecular characterization and localization of the NAD(P)H oxidase components gp91-phox and p22-phox in endothelial cells. Arterioscler Thromb Vasc Biol. 2000; 20: 1903–1911.
Li JM, Shah AM. Intracellular localization and preassembly of the NADPH oxidase complex in cultured endothelial cells. J Biol Chem. 2002; 277: 19952–19960.
Mochizuki T, Furuta S, Mitsushita J, Shang WH, Ito M, Yokoo Y, Yamaura M, Ishizone S, Nakayama J, Konagai A, Hirose K, Kiyosawa K, Kamata T. Inhibition of NADPH oxidase 4 activates apoptosis via the AKT/apoptosis signal-regulating kinase 1 pathway in pancreatic cancer PANC-1 cells. Oncogene. 2006; 25: 3699–3707.
Vaquero EC, Edderkaoui M, Pandol SJ, Gukovsky I, Gukovskaya AS. Reactive oxygen species produced by NAD(P)H oxidase inhibit apoptosis in pancreatic cancer cells. J Biol Chem. 2004; 279: 34643–34654.
Chen JX, Zeng H, Tuo QH, Yu H, Meyrick B, Aschner JL. NADPH oxidase modulates myocardial Akt, ERK1/2 activation, and angiogenesis after hypoxia-reoxygenation. Am J Physiol Heart Circ Physiol. 2007; 292: H1664–H1674.
Pedruzzi E, Guichard C, Ollivier V, Driss F, Fay M, Prunet C, Marie JC, Pouzet C, Samadi M, Elbim C, O’Dowd Y, Bens M, Vandewalle A, Gougerot-Pocidalo MA, Lizard G, Ogier-Denis E. NAD(P)H oxidase Nox-4 mediates 7-ketocholesterol-induced endoplasmic reticulum stress and apoptosis in human aortic smooth muscle cells. Mol Cell Biol. 2004; 24: 10703–10717.