Published online before print March 15, 2007, doi: 10.1161/01.RES.0000263381.83835.7b
© 2007 American Heart Association, Inc.
Molecular Medicine |
Endothelial Nox2 Overexpression Potentiates Vascular Oxidative Stress and Hemodynamic Response to Angiotensin II
Studies in Endothelial-Targeted Nox2 Transgenic Mice
From the Department of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital, OX39DU, UK.
Correspondence to Prof Keith M. Channon, Department of Cardiovascular Medicine, John Radcliffe Hospital, Oxford, OX3 9DU, UK. E-mail keith.channon{at}cardiov.ox.ac.uk
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Abstract |
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Vascular disease states are associated with endothelial dysfunction and increased production of reactive oxygen species (ROS) derived from vascular NADPH oxidases in both vascular smooth muscle cells (VSMCs) and endothelial cells. Recent evidence suggests an important role for VSMC NADPH oxidases in vascular ROS production. However, it is unclear whether increased NADPH oxidase activity in endothelial cells alone is sufficient to alter overall vascular ROS production and hemodynamics. We sought to address these questions using transgenic mice with endothelial-targeted overexpression of the catalytic subunit of NADPH oxidase, Nox2. Aortas of Nox2 transgenic (Nox2-Tg) mice had increased total Nox2 mRNA and protein levels compared with wild-type littermates. Both p22phox mRNA and protein levels were also significantly elevated in Nox2-Tg aortas. Aortic superoxide production was significantly increased in Nox2-Tg mice compared with wild-type, but this difference was abolished by endothelial removal. Superoxide dismutase inhibition increased superoxide release and levels of Mn superoxide dismutase protein were significantly elevated in aortas from Nox2-Tg mice compared with wild type. Increased ROS production from endothelial Nox2 overexpression led to increased endothelial nitric oxide synthase protein and extracellular signal-regulated kinase 1/2 phosphorylation in transgenic aortas. Basal blood pressure was similar, however the pressor responses to both acute and chronic angiotensin II administration were significantly increased in Nox2-Tg mice compared with wild type. These results demonstrate that endothelial-targeted Nox2 overexpression is sufficient to increase vascular NADPH oxidase activity, activate downstream signaling pathways, and potentiate the hemodynamic response to angiotensin II, despite compensatory increases in vascular antioxidant enzymes. Endothelial cell Nox2-containing NADPH oxidase plays an important functional role in vascular redox signaling.
Key Words: NADPH oxidase • oxidative stress • endothelium • hemodynamics
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Introduction |
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Many vascular disease states are associated with an increase in the production of reactive oxygen species (ROS) in the vessel wall, which is associated with reduced nitric oxide (NO) bioavailability, leading to endothelial dysfunction.1,2 The production of ROS, in particular superoxide (O2–·), reduces vascular NO bioavailability by scavenging. In addition, ROS are involved in the activation of important intracellular signaling molecules, such as mitogen-activated protein kinases (MAPKs), that regulate numerous signaling pathways involved in cell growth, gene expression and apoptosis.3,4
Recent studies have shown that the phagocyte-type NADPH oxidases are a major source of ROS in the vasculature.5–7 These oxidases are expressed in several different cell types in the vessel wall, including endothelial cells, vascular smooth muscle cells (VSMCs), adventitial fibroblasts, and inflammatory cells. NADPH oxidases are multisubunit flavoprotein complexes that, in phagocytic cells, consist of the membrane-bound cytochrome b558, comprising the catalytic gp91phox subunit and the p22phox subunit, as well as 4 regulatory subunits: p47phox, p67phox, p40phox, and rac1. Recently, novel gp91phox (renamed Nox2) homologs have been identified in nonphagocytic cells, named Nox1 to 5, including Nox1, Nox2, and Nox4 in the vasculature.8,9 NADPH oxidases in endothelial cells use Nox2 and Nox4, whereas VSMCs express only low levels of Nox2 and predominantly use Nox1 and Nox4.10
Compelling evidence implicates NADPH oxidase-derived ROS from VSMCs in various forms of hypertension, including angiotensin II (Ang II)-mediated hypertension.5,11,12 Mice deficient in Nox2 have reduced basal blood pressure compared with wild-types,9,13 whereas Nox1-deficient mice have similar basal blood pressure yet significantly suppressed pressor responses to Ang II.14 Furthermore, mice with vascular smooth muscle-targeted overexpression of either Nox114 or p22phox15 have increased vascular ROS production, vascular smooth muscle hypertrophy, and enhanced pressor responses to Ang II.
Although these studies establish an important role for NADPH oxidase-derived ROS in VSMCs, whether NADPH oxidase(s) in endothelial cells have specific functional importance in hypertension and other vascular disease states remains unclear. This is an important question, because ROS production in endothelial cells may have specific pathophysiologic roles in vascular diseases, for example, through NO scavenging and through oxidation of tetrahydrobiopterin, the essential endothelial nitric oxide synthase (eNOS) cofactor.16 Furthermore, VSMC and endothelial cell NADPH oxidases appear to have distinct intracellular characteristics that may lead to cell- and isoform-specific roles. In VSMCs, Nox1-containing NADPH oxidase is colocalized with caveolin on the cell surface and is involved in cell growth, whereas Nox4-containing NADPH oxidase is localized in focal adhesions and is implicated in cellular senescence.17 Endothelial NADPH oxidase is associated mainly with the cytoskeleton in a perinuclear distribution.18 Finally, it is unclear how vascular antioxidant defenses may respond to a primary increase in endothelial cell ROS production.
Accordingly, we aimed to determine the specific importance of endothelial Nox2-containing NADPH oxidase in regulating vascular oxidative stress and blood pressure by generating transgenic mice with targeted overexpression of Nox2 in endothelial cells.
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Materials and Methods |
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Generation of Nox2 Transgenic Mice
To target Nox2 gene expression to the vascular endothelium, we constructed a human Nox2 transgene incorporating the murine Tie2 promoter and intronic enhancer (Figure 1A).19 The Tie2-Nox2 transgene underwent pronuclear microinjection into fertilized eggs from superovulated C57BL/6xCBA mice. Transgenic founders were then back-crossed on to the C57BL/6J strain. Fluorescent in situ hybridization was performed to ascertain the chromosomal site(s) of transgene integration into the mouse genome. For more details, see the online data supplement, available at http://circres.ahajournals.org. Experiments were performed in accordance with the Guidance on the Operation of Animals (Scientific Procedures) Act, 1986/UK) on mice housed in individually ventilated cages with 12-hour light/dark cycle and controlled temperature (20-22°C) and fed normal chow and water ad libitum.
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Isolation of Murine Endothelial Cells
Primary endothelial cells were isolated from lungs by immunoselection with CD31 antibody–coated magnetic beads. See the online data supplement.
Quantitative Real-Time RT-PCR
Quantitative RT-PCR was performed on RNA extracted from lung, aorta, and spleen and on CD31-positive and -negative cells isolated from lung (endothelial and nonendothelial cells, respectively; n
3 per group) to measure human, murine, or total Nox2 mRNA expression. See the online data supplement.
Western Blot Analysis
Western blot analysis was performed to measure protein levels of Nox2, p22phox, (phospho-)extracellular signal-regulated kinase (ERK)1/2, (phospho-)p38 MAPK, (phospho-)c-Jun N-terminal kinase (JNK), eNOS, Cu/Zn superoxide dismutase (Cu/ZnSOD), extracellular superoxide dismutase (ecSOD), Mn superoxide dismutase (MnSOD), and catalase in aortas from wild-type and Nox2-Tg mice (n4 per group; see the online data supplement).
Lucigenin-Enhanced Chemiluminescence
O2–· production was measured in both left ventricular (LV) homogenate (n=5 to 11 per group) and from intact whole or endothelial-denuded aortas using lucigenin-enhanced (5 to 20 µmol/L) chemiluminescence according to methods previously described.20 See the online data supplement.
Oxidative Fluorescent Microtopography
O2–· production was detected in tissue sections of mouse aorta (n=3 to 4 per group) using the fluorescent probe dihydroethidium (DHE), as previously described.20 See the online data supplement.
Isometric Tension Vasomotor Studies
Aortic vasomotor function was assessed using isometric tension studies in a wire myograph (Multi-Myograph 610M, Danish Myo Technology, Aarhus, Denmark); see the online data supplement.
Measurement of Arterial Blood Pressure
Blood pressure was measured in anesthetized wild-type and Nox2-Tg mice (n=5 to 8 per group) using the Millar catheter system; see the online data supplement.
In Vivo Ang II Infusion and Measurement of Systemic Blood Pressure
Wild-type and Nox2-Tg mice were implanted with osmotic minipumps containing Ang II (infusion rate 0.4 mg/kg per day) as previously described,21 and systolic blood pressure was obtained using the tail-cuff system in conscious animals; see the online data supplement.
Statistical Analysis
One-way ANOVA tests were used to compare data sets, with appropriate post hoc correction for multiple comparisons. P<0.05 was considered significant. Data are expressed as means±SEM.
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Results |
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Generation and Characterization of Endothelial-Targeted Nox2 Transgenic Mice
Generation of Nox2-Tg mice was confirmed by PCR, RT-PCR, and fluorescent in situ hybridization analysis (Figure 1). Two independent Nox2-Tg founders were identified: Nox2-Tg1 and Nox2-Tg2. Both transmitted the transgene to offspring (Figure 1B) and expressed human Nox2 mRNA, as confirmed by RT-PCR using human-specific Nox2 primers (Figure 1C). Fluorescent in situ hybridization revealed a single transgenic integration site for both colonies (Figure 1D): on chromosome 17 for Nox2-Tg1 and on chromosome 12 for Nox2-Tg2 mice. The endogenous signal for Tie2 was observed on both chromosome 4 homologs for both colonies. Compared with littermate wild types, Nox2-Tg1 and Nox2-Tg2 mice were overtly normal.
Neither body weights (BWs) nor heart-weight-to-BW ratios were significantly different between Nox2-Tg mice and wild types (heart-weight-to-BW ratios, 4.61±0.1 versus 4.66±0.1, respectively).
Expression of Nox2 mRNA and Protein Production
We verified the endothelial specificity of human Nox2 transgene expression by measuring human Nox2 mRNA in primary endothelial cells and nonendothelial cells, isolated by immunomagnetic bead selection, from wild-type and Nox2-Tg mice. Importantly, human Nox2 transgene expression was present in only the endothelial cell population from Nox2-Tg mice; none was detected in the nonendothelial cell population nor in either the endothelial or nonendothelial cells from wild types (Figure 2A). As expected, mouse Nox2 mRNA was expressed in both the endothelial and nonendothelial populations from both groups.
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We next quantified the relative levels of transgene expression between the 2 colonies of founder lines using fluorescence quantitative RT-PCR and primers specific for the transgenic human Nox2 mRNA in total RNA extracted from lung. Human Nox2 mRNA, not detected in wild types, was detected in all samples from Nox2-Tg1 and Nox2-Tg2 mice, but expression was significantly greater in Nox2-Tg1 compared with Nox2-Tg2 mice (16.8±1.8 versus 5.9±0.2 arbitrary units, respectively; n=6 per group, P<0.05). Nox2-Tg1 mice were selected for further experiments.
We next determined the levels of transgenic (human), native (mouse), and total (both) Nox2 mRNA expression in organs with different proportions of endothelial cells (lung, aorta, and spleen). Total Nox2 mRNA expression was 
2-fold higher in lung and 3.5-fold higher in aorta from Nox2-Tg mice compared with wild types (P<0.05 for both; Figure 2B). In spleen, there was no significant difference in total Nox2 expression between Nox2-Tg and wild-type mice, reflecting the high contribution of native Nox2 expression in spleen and the proportionately fewer endothelial cells. Native mouse Nox2 expression in spleen was >2-fold higher than in lung and almost 6-fold higher than in aorta, consistent with known high levels of Nox2 in spleen. Native Nox2 expression in lung, aorta, or spleen was similar between Nox2-Tg and wild-type mice, indicating that transgenic Nox2 expression had no effect on expression of the native gene (Figure 2C). Western blot analysis confirmed that Nox2 protein levels were significantly increased in Nox2-Tg aortas compared with wild-type (Figure 2D).
Levels of p22phox Protein and mRNA Expression
We then determined the effects of endothelial Nox2 overexpression on the levels of p22phox, the other NADPH oxidase catalytic subunit required to form the active multimeric enzyme. Western blot analysis revealed that p22phox protein was significantly elevated in lung homogenates from Nox2-Tg mice compared with wild types (P<0.05; Figure 3A and 3B). RT-PCR revealed that p22phox mRNA expression was also significantly increased in Nox2-Tg mice compared with wild types (P<0.05; Figure 3C).
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Superoxide Production
To investigate whether endothelial Nox2 overexpression, and the associated increase in p22phox, would increase overall NADPH oxidase activity, we first measured O2–· production in tissue lysates. NADPH-stimulated O2–· production was significantly increased in unfractionated (total) LV lysates from Nox2-Tg mice compared with wild types (P<0.05; Figure 4A). Subcellular fractionation into particulate (membrane) and soluble (cytosolic) fractions revealed that the majority of the NADPH-stimulated O2–· production was localized to the membrane in both Nox2-Tg and wild-type mice (P<0.001; Figure 4A). O2–· production remained significantly elevated in LV membrane fractions from Nox2-Tg mice (>2-fold; P<0.05; Figure 4A). Importantly, O2–· production was also increased, although to a lesser extent, in Nox2-Tg2 mice compared with counterpart wild types (26.4±8.6 versus 13.8±5.8 relative light units per second per microgram of protein, respectively), correlating with the relative levels of transgene expression between the 2 colonies. The NADPH oxidase flavoprotein inhibitor, diphenylene iodonium, abolished the membrane-derived O2–· signal in both colonies (P<0.001).
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We next measured NADPH-stimulated O2–· production in LV membrane fractions in the presence of the SOD inhibitor, diethyl-dithiocarbamate (DETC), to investigate the effects of potential changes in SOD activity in transgenic animals. SOD inhibition significantly increased the O2–· signal in both groups but to a greater extent in Nox2-Tg mice, thereby further enhancing the difference between Nox2-Tg and wild types. This suggests that endogenous SOD activity was increased in Nox2-Tg animals (Figure 4B). Notably, O2–· production was also significantly elevated in Nox2-Tg2 mice compared with counterpart wild types in the presence of DETC (P<0.05; data not shown).
To further evaluate the effects of Nox2 overexpression on endothelial NADPH oxidase activity, we measured O2–· production in intact aortas using both lucigenin-enhanced chemiluminescence and DHE fluorescence. In intact aortas, NADPH-stimulated O2–· production was significantly increased in Nox2-Tg mice compared with wild types (Figure 4C, P<0.05). This O2–· signal was almost completely abolished by diphenylene iodonium (P<0.001). Endothelial denudation abolished the difference in both basal and NADPH-stimulated O2–· production between wild-type and Nox2-Tg aortas (Figure 4C). Furthermore, oxidative confocal microtopography revealed that endothelial DHE fluorescence in aortic tissue sections was increased 2-fold in Nox2-Tg compared with wild-type mice (Figure 4D). Together with the RT-PCR data demonstrating Nox2 transgene expression exclusively in endothelial cells, these results demonstrate the endothelial specificity of Nox2 overexpression and increased Nox2-derived O2–· production in Nox2-Tg mice.
Antioxidant Enzymes, eNOS, and MAPK Activation
To investigate whether increased vascular ROS production in Nox2-Tg mice altered antioxidant defenses, we measured protein levels of antioxidant enzymes by Western blot. There was no change in protein levels of Cu/ZnSOD, ecSOD, or catalase between Nox2-Tg and wild-type aortas (Figure 5A, 5B, and 5D). However, MnSOD protein was significantly increased in Nox2-Tg aortas (P<0.01; Figure 5C). eNOS protein levels were also significantly elevated in Nox2-Tg aortas compared with wild-type (P<0.05; Figure 6A).
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To determine the effects of Nox2 overexpression on downstream signaling pathways, we examined MAPK phosphorylation. Phospho-ERK1/2, as a proportion of total ERK1/2, was significantly increased in Nox2-Tg animals compared with wild-type (P<0.05; Figure 6B). These data indicate that increased endothelial O2–· production in Nox2-Tg mice is sufficient to activate downstream ROS-sensitive signaling pathways despite increased expression of vascular antioxidant enzymes. However, phospho-JNK and phospho–p38 MAPK levels were not significantly increased in Nox2-Tg mice (Figure 6C and 6D), suggesting that nonendothelial cell types dominate levels of these kinases in the vascular wall.
Endothelial Vasomotor Function
To determine whether increased endothelial O2–· production would alter NO-mediated endothelial function in Nox2-Tg animals, we measured endothelium-dependent vasorelaxation of aortic rings. Contraction responses to phenylephrine were similar between wild-type and Nox2-Tg aortas. Endothelium-dependent and -independent relaxations to acetylcholine and sodium nitroprusside, respectively, were similar between the 2 groups (Figure I in the online data supplement).
Hemodynamic Response to Acute Ang II Infusion
To determine the functional importance of increased endothelial O2–· production in Nox2-Tg mice, we first measured systolic blood pressure using a Millar catheter in the left carotid artery. Heart rates were similar between Nox2-Tg and wild-type animals (496±20 versus 538±32 bpm, respectively). Basal systolic blood pressure was similar between Nox2-Tg and wild-type mice (100.4±2.1 versus 101.1±1.4 mm Hg, respectively). Acute administration of Ang II (10 µg/kg) caused a significantly greater increase in systolic blood pressure in Nox2-Tg mice compared with wild types (P<0.01; Figure 7A and 7B), although heart rates remained no different (482±24 versus 545±35 bpm, respectively).
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) in systolic blood pressure following Ang II administration below (n=5 to 8 per group). C, Systolic blood pressure, measured by tail-cuff, in wild-type (empty triangles) and Nox2-Tg (filled squares) mice during 7 days of Ang II infusion (0.4 mg/kg per day) (n=8 to 11 per group). *P<0.05, **P<0.01 vs wild types.
Hemodynamic Response to Chronic In Vivo Ang II Infusion
We then measured systemic blood pressure in nonanesthetized Ang II–infused (0.4 mg/kg per day) wild-type and Nox2-Tg mice using the tail-cuff method. BW, heart rates, and heart-weight-to-BW ratios were similar between Ang II–infused Nox2-Tg and wild-type mice (heart rates, 736±7 versus 704±11 bpm, respectively; heart-weight-to-BW ratios, 5.65±0.3 versus 5.57±0.3, respectively). We again found no difference in basal systemic blood pressure, however chronic Ang II infusion significantly increased blood pressure in Nox2-Tg mice compared with wild types after just 3 days (P<0.05; Figure 7C).
Superoxide Production in Ang II–Infused Mice
Ang II infusion caused a marked increase in O2–· production, measured by lucigenin chemiluminescence, in both wild-type and Nox2-Tg aortas compared with untreated animals (P<0.05 for both; Figure 8A) but to a similar extent. It is likely that Ang II is activating VSMC NADPH oxidases, as well the endothelial oxidase, potentially masking any difference in endothelial NADPH oxidase-derived O2–· production between Nox2-Tg and wild-type aortas.
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To test this hypothesis, we also measured O2–· production in aortic endothelium from Ang II–infused mice using DHE fluorescence. Importantly, endothelial fluorescence was significantly increased in Ang II–infused Nox2-Tg mice compared with wild types (Figure 8B), whereas total wall fluorescence was similar between the 2 groups.
To determine the effects of chronic Ang II infusion on downstream signaling pathways in Nox2-Tg mice, we measured aortic MAPK phosphorylation. Ang II infusion significantly increased phospho–p38 MAPK and phospho-JNK protein in both Nox2-Tg and wild-type mice compared with counterpart non–Ang II–infused animals (P<0.01 for all; data not shown) although to a similar extent in both groups. Ang II infusion also significantly increased phospho-ERK1/2 protein levels in Ang II–treated wild-type mice compared with untreated counterparts but not in the Nox2-Tg group. However, as reported above, non–Ang II–treated Nox2-Tg mice had significantly increased phospho-ERK1/2 levels compared with corresponding wild types (Figure 6B).
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Discussion |
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In this study, we describe a novel transgenic mouse model with targeted overexpression of Nox2 in the endothelium to investigate the specific role of endothelial Nox2-containing NADPH oxidase in regulating vascular oxidative stress and hemodynamic responses. The major findings of this study are as follows. (1) Endothelial-specific Nox2 overexpression leads to an increase in total Nox2 mRNA and protein in Nox2-Tg mice compared with wild-type littermates. (2) Endothelial Nox2 overexpression is associated with an increase in p22phox mRNA and protein levels, and these are together sufficient to augment endothelial NADPH oxidase activity. (3) This increase in vascular O2–· production leads to a compensatory upregulation of MnSOD and eNOS protein and activates downstream vascular signaling pathways, as evidenced by ERK1/2 phosphorylation. (4) Increased activity of endothelial Nox2-containing NADPH oxidase does not alter basal blood pressure but significantly potentiates the pressor response to both acute and chronic Ang II stimulation.
These findings provide important insights into the role and importance of endothelial Nox2 in vascular ROS production. Previous studies have demonstrated that vascular NADPH oxidase-derived ROS play important roles in vascular diseases such as hypertension and atherosclerosis.2,5,6 However, the recent discovery of multiple Nox homologs, expressed in different vascular cell types, has raised critical questions about the specific roles of these enzymes in vascular function and blood pressure regulation. Because endothelial cells express Nox2 and Nox4, but not Nox1 or Nox3,9,22,23 we generated a novel transgenic mouse model directing human Nox2 transgene expression to the endothelium under the control of the murine Tie2 promotor. This endothelial-specific Nox2-Tg mouse enabled us to assess the importance of endothelial-specific Nox2/NADPH oxidase responses, independently from those of the vascular smooth muscle and adventitia. As predicted, human Nox2 mRNA expression was detected in only endothelial cells, not in nonendothelial cells, from Nox2-Tg animals. Total Nox2 mRNA was increased in only endothelial-rich tissues in these mice (in lung and aorta but not in spleen), and Nox2 protein was elevated in Nox2-Tg aortas compared with wild types. Importantly, native Nox2 mRNA expression was unchanged in Nox2-Tg mice, indicating that transgene expression had no effect on transcriptional regulation of the native gene.
An interesting finding in this study is that overexpression of Nox2 was associated with marked upregulation of p22phox mRNA expression and protein levels. Previous studies in phagocytes have reported that when assembled as the transmembrane heterodimer b558, Nox2 and p22phox are significantly more stable than either uncomplexed protein subunit,24 suggesting that transgenic overexpression of Nox2 may be accompanied by increased p22phox levels through effects on protein stability. Indeed, Laude et al15 recently reported that mice overexpressing vascular smooth muscle p22phox had increased aortic Nox1 protein. We now demonstrate that in endothelial cells Nox2, overexpression is also accompanied by increased p22phox mRNA expression, suggesting that the increase in p22phox protein is likely attributable to effects on gene expression, in addition to the potential increase in protein stability. Although the mechanism for this remains unclear, it is possible that regulation of p22phox mRNA expression is redox-sensitive. In support of this, recent in vitro experiments in endothelial cells have demonstrated that ROS can upregulate p22phox mRNA and protein and that this upregulation can be prevented by NADPH oxidase inhibition.25 These observations suggest the potential for a "positive feedback" mechanism in endothelial cells, where small initial increases in Nox2 may lead to larger changes in overall NADPH oxidase activity through associated changes in p22phox mRNA and protein levels.
We demonstrated that overexpression of the Nox2 subunit, in conjunction with the associated increase in p22phox levels, was sufficient to increase total NADPH oxidase activity, as determined by measuring O2–· production in both LV lysates and in fresh, intact aorta. Importantly, O2–· production was similar in endothelial-denuded aorta from wild-type and Nox2-Tg mice, further supporting the endothelial specificity of the Nox2 overexpression. The observation that the majority of NADPH oxidase activity was detectable in the membrane fraction rather than the cytosol of LV lysates is consistent with previous reports and the known localization of the active NADPH oxidase complex on the membrane.2,18,26 Importantly, 2 separate founder lines expressing different levels of Nox2 mRNA both demonstrated an increase in O2–· production from membrane fractions that correlated with their level of transgene expression. Nox2-Tg2 mice, which had significantly lower human Nox2 mRNA expression compared with Nox2-Tg1 animals, demonstrated only a modest increase in NADPH-dependent O2–· production that was significant only with SOD inhibition. However, a significant 2-fold increase in NADPH-dependent O2–· release was detectable in both LV membrane fractions and intact aorta from Nox2-Tg1 animals, suggesting that a sufficiently large increase in Nox2 expression is required to increase overall NADPH oxidase activity and overcome antioxidant defenses. Indeed, we observed significant activation of ERK1/2 in aortas from Nox2-Tg mice compared with wild types, characteristic of activation of downstream signaling molecules typical of increased NADPH oxidase activity and ROS production.3,4,27 We did not observe increased levels of either JNK or p38 MAPK phosphorylation, suggesting either that these pathways are less important in mediating Nox2 redox effects in endothelial cells or that the levels of JNK and p38 MAPK phosphorylation in other cell types in the vascular wall are sufficient to mask changes within the endothelium.
It is well known that the renin–angiotensin system plays an important role in the control of arterial blood pressure. Numerous studies have shown that Ang II induces hypertension, which is in part mediated by vascular NADPH oxidase–derived ROS.5,11,12,28,29 For example, basal blood pressure is reduced in Nox2-deficient mice.13,21 In the present study, we found no change in either the basal blood pressure or in endothelium-dependent vasorelaxations in Nox2-Tg mice, despite clear evidence of increased vascular ROS production and activation of redox-sensitive targets in the vascular wall. However, increased endothelial O2–· release in Nox2-Tg mice is likely to lead to compensatory mechanisms that tend to balance vascular redox status and preserve normal hemodynamics. Indeed, we observed an increase in the protein levels of MnSOD and eNOS, both of which may contribute to the maintenance of normal basal blood pressure and endothelium-dependent vasorelaxation through enhanced O2–· removal and NO production. Although SOD enzymatic activity was not specifically measured, SOD inhibition led to a greater increase in NADPH-dependent O2–· release in LV membrane fractions from Nox2-Tg compared with wild-type mice, suggesting that overall SOD activity is elevated in transgenic animals. This notion is supported by recent studies reporting the effects of vascular smooth muscle-targeted Nox1 or p22phox overexpression that found no change in basal blood pressure in transgenic animals14,15; MnSOD and eNOS protein levels were also increased in these animals.
We clearly demonstrated that both acute and chronic Ang II stimulation led to a significant pressor response in Nox2-Tg animals but not in wild-type littermates. As expected, Ang II led to a significant increase in vascular O2–· release, as has been previously reported.5,11,12,28,29 However aortic O2–· production was similar between Ang II–treated wild-type and Nox2-Tg animals, likely because Ang II is well known to increase expression of a number of NADPH oxidase subunits in the endothelium, medial VSMCs, and adventitia. This substantial increase in total vascular NADPH oxidase activity is likely to have masked any difference in endothelial-specific O2–· release. However, when we measured in situ O2–· generation specifically in the endothelium, we did observe a significant increase in Nox2-Tg mice compared with wild types. In line with the increased vascular oxidative stress, we detected significant MAPK phosphorylation in both Ang II–infused wild-type and Nox2-Tg mice compared with untreated animals, which was similar between groups. Again, this is likely to reflect the global increase in ROS production throughout the vessel wall in both wild-type and Nox2-Tg animals subjected to Ang II.
Our findings have important implications for understanding how endothelial NADPH oxidases contribute to ROS-dependent signaling in the vascular wall. Whereas vascular NADPH oxidases in general, and VSMC oxidases in particular, are known to play key roles, the importance of endothelial cell NADPH oxidases is less clear. Importantly, endothelial cells express Nox2 and Nox4 rather than Nox1 that predominates in VSMCs,17,18 and these different NADPH oxidases appear to locate in different cellular compartments, suggesting cell- and Nox-specific signaling roles for vascular NADPH oxidase–derived ROS. We now demonstrate that endothelial Nox2-containing NADPH oxidase is sufficient to alter total vascular ROS production and modulate the hemodynamic response to Ang II. Future studies need to address how endothelial and VSMC NADPH oxidases together contribute to vascular ROS production in the pathogenesis of vascular disease states, and how distinct or complementary roles for these different oxidases might provide new targets for novel therapies.
To conclude, our studies using a novel transgenic mouse model overexpressing Nox2 specifically in endothelium provide valuable new insights into the role of this Nox homolog in vascular ROS production. Endothelial-targeted overexpression of Nox2 leads to upregulation of p22phox and increases vascular NADPH oxidase–derived O2–· production. Basal blood pressure in Nox2-Tg mice is likely preserved by compensatory mechanisms, including changes in MnSOD and eNOS. However, these compensatory mechanisms are overcome by Ang II administration, leading to a potentiated pressor response. Together, these data suggest that endothelial Nox2-containing NADPH oxidase plays a specific and critical role in vascular oxidative stress and in mediating the hemodynamic response to Ang II.
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Acknowledgments |
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Sources of Funding
This work was supported by the British Heart Foundation (RG02/007). D.A. is Wellcome Trust Cardiovascular Research Initiative Clinical Training Fellow. J.d.B. is a Bristol Myers Squibb Research Fellow.
Disclosures
None.
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Footnotes |
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Original received February 16, 2006; resubmission received January 31, 2007; revised resubmission received February 21, 2007; accepted March 1, 2007.
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References |
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