Important Role of Nox4 Type NADPH Oxidase in Angiogenic Responses in Human Microvascular Endothelial Cells In Vitro

Cell Culture

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.

siRNA Transfection

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

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-Nox4^WT), or a truncated form of Nox4 lacking the NADPH binding domain (Ad-Nox4^ΔNADPH) 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-Nox4^WT) or presence (for Ad-Nox4^ΔNADPH) of 5% serum.

Real-Time Polymerase Chain Reaction

Quantitative real-time polymerase chain reaction (PCR) analysis was performed as described previously.⁸

Apoptosis

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.

ROS Measurement

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).

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Figure 1. Western blots (n=3 to 4) showing that Nox4 expression in HMECs was modulated by (a) Nox4 siRNA and (b) Ad-Nox4^WT. The level of Nox2 was unchanged by these treatments. The densitometry data are shown in supplemental Figure I.

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 (AdNox4^WT) overexpression. We found that AdNox4^WT 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-Nox4^ΔNADPH)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.

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Figure 2. Effects of Nox4 siRNA (transfected for 48 to 72 hours) on tube formation (a) and wound healing (b) responses in HUVECs and HMECs. n=4 to 5. A high magnification image of the HMEC cells used in Figure 2b (white box) is shown in supplemental Figure IIIc.

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-Nox4^WT treatment significantly enhanced the wound healing response, whereas Ad-Nox4^ΔNADPH 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-Nox4^WT–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).

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Figure 3. Effects of (a) wild-type and (b) dominant negative Nox4 gene transfer on wound healing responses in HMECs. Cells were incubated with adenoviral vectors in medium containing 0.5% (for Nox4^WT) or 5% (for Nox4^ΔNADPH) for 48 hours. P<0.05 vs Ad-LacZ, n=3 to 4.

To clarify whether these effects of Ad-Nox4^WT and Nox4^ΔNADPH 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).

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Figure 4. Effects of Nox4 siRNA on (a) migration and (b) proliferation in HMECs. Cell proliferation was assessed with a CellTiter-96 AQueous One Solution kit (Promega). *P<0.05 vs control siRNA, n=3 to 4.

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-Nox4^WT-transduced cells, but decreased in Ad-Nox4^ΔNADPH-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).

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Figure 5. Effects of wild-type and dominant negative Nox4 on (a) tyrosine phosphorylation of PDGFR-α (*P<0.05 vs Ad-LacZ, n=4), and (b) Erk1/2 phosphorylation under basal conditions and after VEGF (10 ng/mL) stimulation (example from 2 independent experiments).

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 N^G-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.