Nox1 Is Involved in Angiotensin II–Mediated Hypertension
A Study in Nox1-Deficient Mice
Background— Increased production of reactive oxygen species (ROSs) by angiotensin II (Ang II) is involved in the initiation and progression of cardiovascular diseases. NADPH oxidase is a major source of superoxide generated in vascular tissues. Although Nox1 has been identified in vascular smooth muscle cells as a new homolog of gp91phox (Nox2), a catalytic subunit of NADPH oxidase, the pathophysiological function of Nox1-derived ROSs has not been fully elucidated. To clarify the role of Nox1 in Ang II–mediated hypertension, we generated Nox1-deficient (−/Y) mice.
Methods and Results— No difference in the baseline blood pressure was observed between Nox1+/Y and Nox1−/Y. Infusion of Ang II induced a significant increase in mean blood pressure, accompanied by augmented expression of Nox1 mRNA and superoxide production in the aorta of Nox1+/Y, whereas the elevation in blood pressure and production of superoxide were significantly blunted in Nox1−/Y. Conversely, the infusion of pressor as well as subpressor doses of Ang II did elicit marked hypertrophy in the thoracic aorta of Nox1−/Y similar to Nox1+/Y. Administration of a nitric oxide synthase inhibitor (L-NAME) to Nox1+/Y did not affect the Ang II–mediated increase in blood pressure, but it abolished the suppressed pressor response to Ang II in Nox1−/Y. Finally, endothelium-dependent relaxation and the level of cGMP in the isolated aorta were preserved in Nox1−/Y infused with Ang II.
Conclusions— A pivotal role for ROSs derived from Nox1/NADPH oxidase was suggested in the pressor response to Ang II by reducing the bioavailability of nitric oxide.
Received July 6, 2005; revision received August 17, 2005; accepted August 18, 2005.
Accumulating evidence indicates that angiotensin (Ang) II, the principal effector peptide of the renin-angiotensin system, plays a major role in the initiation and progression of such vascular diseases as hypertension, vascular hypertrophy, and atherosclerosis.1 These effects of Ang II are mediated by reactive oxygen species (ROSs) generated by membrane-bound nicotinamide adenine dinucleotide phosphate (NADPH) oxidase localized in the vascular wall.2,3 The ROSs originating from the vascular oxidase have been held responsible for endothelial dysfunction and also recognized as important signaling molecules involved in vascular remodeling.4
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Early studies on the source of oxidant generation were typically limited to the prototypical NADPH oxidase of phagocytic cells, which is a multisubunit enzyme comprising a membrane-associated cytochrome b558 (a heterodimer of a catalytic subunit gp91phox and p22phox) and several cytosolic regulatory subunits (p47phox, p40phox, p67phox, and Rac1 or Rac2). In recent years, 4 homologs of gp91phox (Nox2), named Nox1,5 Nox3, Nox4, and Nox5,6 have been identified as components of nonphagocyte-type NADPH oxidase. Among these Nox isoforms expressed primarily in nonphagocyte cells, Nox1 is highly expressed in colon epithelial cells.5 In vessels, Nox1 mRNA has been detected in vascular smooth muscle cells (VSMCs) and endothelial cells but not in adventitial cells. Conversely, the phagocyte-type subunit Nox2 is localized primarily in endothelial and adventitial cells, whereas Nox4 is abundantly expressed in all of the vessel constituents.7–11
Although genetic approaches have been used in recent investigations, there is a relative paucity of information on the role of Nox isoforms in Ang II–mediated vascular disorders.12–14 In knockout mice genetically deficient in Nox2, the basal blood pressure was lower than wild-type counterparts, whereas Ang II–dependent hypertension was unaffected.14,15 In the aortic media of these knockout mice, hypertrophic responses to Ang II infusion were significantly attenuated.14 Conversely, the hypertensive response to Ang II was significantly reduced in knockout mice of p47phox, a regulatory subunit of NADPH oxidase.16 These results suggest that Nox1 may participate in Ang II–induced hypertension, because p47phox can regulate both Nox2 and Nox1.17 To clarify the role of the Nox1 isoform in the pathogenesis of Ang II–induced hypertension, we generated Nox1-deficient mice and administered Ang II by osmotic minipumps. We report here results indicating a pivotal role for Nox1/NADPH oxidase in the pressor response to Ang II.
Generation of Nox1-Deficient Mice
Mouse genomic clones containing the Nox1 locus were isolated from a 129/SvJ mouse genomic library constructed in lambda FixII (Stratagene) using a murine Nox1 partial cDNA fragment as a probe. A 6.5-kb SacI-HindIII fragment and a 1.0-kb HindIII-BamHI fragment were cloned into pBluescript II-KS (+), a plasmid containing a neomycin (neo) expression cassette driven by the murine phosphoglycerate kinase promoter. At the 3′ end of the vector, a diphtheria toxin A fragment was included to serve as a negative selection marker. The Nox1 targeting vector was designed to replace the 1.5 kb of the genomic locus containing exon 3 to 6 with the neo cassette.
R1 embryonic stem (ES) cells were transfected with the linearized targeting vector and selected with G418. Targeted ES clones were identified by polymerase chain reaction (PCR) screening and verified by Southern hybridization using genomic probes located on the 5′ and 3′ sides of the Nox1 gene. Correctly targeted ES cells were used to make chimeric mice by aggregating the cells in E2.5 embryos and transferring the aggregated embryos to pseudopregnant females.18 Male chimeras were crossed with C57BL/6 females to generate heterozygous mice. Heterozygous females were crossed with C57BL/6 males to obtain Nox1-deficient mice (Nox1−/Y). The present study was performed with the approval of the Committee for Animal Research at Kyoto Prefectural University of Medicine.
Nox1-deficient mice and their control littermates (8 to 12 weeks old) were anesthetized with sodium pentobarbital (80 mg/kg IP). The intrascapular region was shaved, and an osmotic minipump (Alzet model 2002; Durect Corp) that contained [Val5] angiotensin II (Sigma) or vehicle (phosphate-buffered saline, PBS) was inserted to permit subcutaneous infusion of Ang II (0.7 mg · kg−1 · d−1). In an additional series of experiments, a subpressor dose of Ang II (0.14 mg · kg−1 · d−1) was administered for 28 days to induce vascular hypertrophy by use of osmotic minipumps (Alzet model 2004). NG-Nitro-l-arginine methyl ester (L-NAME) purchased from Nacalai Tesque was administered in the drinking water for 14 days (1.4 mg/d).
On day 7 of Ang II administration, the thoracic aorta was dissected and snap-frozen in liquid nitrogen after being embedded in Tissue-Tek O.C.T. compound (Sakura Finetechnical Co). Unfixed frozen ring segments were cut into 30-μm-thick sections and placed on a glass slide. Dihydroethidium (DHE, 10 μmol/L, Molecular Probes) was topically applied to each tissue section and coverslipped. Slides were incubated in a light-protected humidified chamber at 37°C for 30 minutes. For the detection of ethidium bromide, a 543-nm He-Ne laser combined with a 560-nm long-pass filter was used.
Detection of Superoxide Production
Superoxide (O2−) production was measured with the L-012 chemiluminescence assay as described previously.19 L-012 (Wako Pure Chemical Industries, Ltd) is a luminol derivative with high sensitivity to superoxide radicals that does not exert redox cycling itself.20 After the infusion of Ang II for 7 days, aortic rings (0.5 cm) were dissected and incubated for 30 minutes in Krebs-HEPES buffer at 37°C. Rings were transferred to scintillation vials containing 100 μmol/L L-012 in Krebs-HEPES buffer and incubated for 5 minutes at 37°C in the dark. After incubation, chemiluminescence was measured by luminometer (Lumat LB 9507, Berthold Technologies Ltd) over a period of 10 minutes at 1-minute intervals. The L-012 chemiluminescence was expressed as relative light units per milligram of dry tissue weight per minute.
Blood Pressure and Heart Rate Measurements
Blood pressure and heart rate in conscious mice were measured by the tail-cuff system using BP98A (Softron Co). Before the osmotic pump was implanted, at least 3 days of training were conducted to accustom mice to the procedure. For each time point, 5 measurements were obtained and averaged for each mouse. Mean blood pressure (MBP) was used for all data analysis.
The thoracic aorta of mice was dissected and snap-frozen in liquid nitrogen. Total RNA was isolated by the acid guanidinium thiocyanate/phenol/chloroform method. Real-time PCR was performed by use of the GeneAmp 5700 Sequence Detection System (Applied Biosystems) with the SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit (Invitrogen Corp). The primer sequences used are shown in the Data Supplement Table (http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.105.573709/DC1). Dissociation curves were monitored to check the aberrant formation of primer dimers. PCR-amplified products were electrophoresed on 2% agarose gels to confirm the presence of a single band. Copy numbers were calculated on the basis of standard curves generated with genuine Nox1, Nox2, and Nox4 cDNA templates. Data were expressed as copies per microgram RNA or levels relative to day 0 (%).
After the infusion of Ang II, mice were anesthetized and perfused transcardially with 10 mL of PBS followed by 10 mL of 4% paraformaldehyde phosphate buffer under pressure (100 mm Hg). The aorta, placed in 4% formalin overnight, was processed and embedded in paraffin. Three sections (6 μm) were obtained from each descending thoracic aorta, 3 mm distal to the left subclavian artery at 500-μm intervals, and stained with elastica van Gieson stain. The medial areas and circumference of the external elastic lamina were measured by use of ImageJ software. The medial thickness was calculated by dividing the medial area by the circumference of the external elastic lamina.
Measurement of cGMP Levels
After the infusion of Ang II for 7 days, the thoracic aorta was dissected and connective tissue was removed. Vessels were immediately snap-frozen in liquid nitrogen and homogenized in ice-cold 5% trichloroacetic acid. The level of cGMP in the supernatant fraction of the homogenate was measured by an enzyme immunoassay (Cayman Chemical Co) and expressed as picomoles per milligram of the dry trichloroacetic acid precipitate.
Isolated Vascular Strip Experiments
Endothelium-dependent and -independent relaxations of the isolated vessels were measured in ex vivo organ chamber baths as described previously.21 Briefly, after the infusion of Ang II for 7 days, mice were anesthetized, and thoracic aorta was dissected free from surrounding connective tissue. The aorta was cut into helical strips (10 mm long and 1 mm wide). The resting tension was adjusted to 0.2 g, which is optimal for induction of a maximal contraction. The strips were equilibrated for 90 minutes in organ baths containing Tyrode’s solution. After equilibration, the strips were precontracted with norepinephrine (30 nmol/L, Sankyo Co). At the maximal constriction level, acetylcholine (ACh, Daiichi Pharmaceutical Co) or sodium pentacyanonitrosylferrate dihydrate (Nacalai Tesque, Kyoto, Japan) was added to evaluate vasodilator function. After a stable relaxation was achieved with ACh, papaverine (100 μmol/L, Sanko Seiyaku Kogyo Co) was added to induce a maximal relaxation. Relaxation was assessed by percent relaxation relative to papaverine-mediated relaxation (100%).
The results are expressed as the mean±SEM. For multiple treatment groups, repeated-measures, 2-way, or Latin-square design ANOVA followed by a Tukey-Kramer test was applied. For expression levels of Nox isoforms, a Kruskal-Wallis test was performed, followed by a Dunnett test.
Generation of Nox1-Deficient Mice
Nox1-deficient mice were generated by replacing exon 3 to 6 containing presumed membrane-spanning regions with the neo cassette (Figure 1a). Two independent ES clones yielded germ-line chimeras and their heterozygous mutant F1 mice. Because the locus of the Nox1 gene is on the X chromosome, heterozygotes obtained at F1 generation were female. The heterozygous females were crossed with C57BL/6 males to obtain Nox1-deficient (−/Y) mice. The ratio of genotypes of the offspring did not deviate significantly from the expected 1:1 distribution of male Nox1+/Y and Nox1−/Y offspring. Southern blot analysis of the PstI-digested genomic DNA obtained from F2 offspring demonstrated a 7.2-kb band for the wild-type allele and a 3.8-kb band for the mutant allele (Figure 1b). Because mouse Nox1 mRNA is most abundantly expressed in the colon,5 we verified the expression of Nox1 mRNA in the colon. Although a 902-base band was clearly detected in Nox1+/Y by reverse transcription–PCR, Nox1 mRNA was absent in Nox1−/Y (Figure 1c). Compared with their control littermates, Nox1−/Y grew with normal weight gain and without obvious abnormalities in their general appearance.
Ang II–Induced Superoxide Production Was Reduced in Nox1-Deficient Mice
The effect of Nox1 gene disruption on vascular superoxide production was first investigated by DHE staining of the frozen sections of the aorta (Figure 2A). A low level of DHE fluorescence was detected in the thoracic aorta of Nox1+/Y or Nox1−/Y after PBS infusion for 7 days. Infusion of Ang II for 7 days markedly increased DHE fluorescence in Nox1+/Y aorta throughout the vessel wall. Conversely, DHE fluorescence in Ang II–treated Nox1−/Y was significantly attenuated in the media.
We next performed the L-012 chemiluminescence assay. No difference in L-012 chemiluminescence was detected in the thoracic aorta of Nox1+/Y or Nox1−/Y after the infusion of PBS for 7 days. In Ang II–infused mice, the chemiluminescence of the aorta was significantly less intense in Nox1−/Y than Nox1+/Y (Figure 2B).
Pressor Response to Ang II Was Suppressed in Nox1-Deficient Mice
Between the age-matched Nox1+/Y and Nox1−/Y mice, no difference in initial body weight was observed. There was no difference in the baseline MBP determined between these experimental groups. In response to continuous infusion of 0.7 mg · kg−1 · d−1 of Ang II, MBP levels were similarly elevated in both Nox1+/Y and Nox1−/Y until day 5 of treatment. On day 7 of the infusion, however, the increase was significantly suppressed, and a lower MBP was demonstrated in Nox1−/Y compared with Nox1+/Y until day 14 of infusion (Figure 3). Conversely, there was no difference in the basal heart rate between Nox1+/Y and Nox1−/Y (+/Y, 605.3±24.8 versus −/Y, 602.0±25.1 bpm, N=8 to 9). No difference in heart rate of Ang II–infused mice was observed between these groups on day 14 (+/Y, 598.2±18.8 versus −/Y, 599.0±22.0, N=8 to 9).
Ang II–Upregulated Expression of Nox Isoforms in the Aorta
The Ang II–induced elevation in MBP was blunted in Nox1−/Y at 7 days of treatment. We therefore investigated expression levels of Nox1, Nox2, and Nox4 mRNA in the thoracic aorta of mice treated with Ang II. As shown in Figure 4a, Nox1 mRNA levels in Nox1+/Y significantly increased on day 7, and increased levels were sustained during the course of Ang II infusion. A concomitant increase in Nox2 mRNA level was demonstrated on day 5, which returned to the basal level on day 14 (Figure 4b). Conversely, increased Nox4 mRNA levels were observed on days 7 and 14 of Ang II infusion (Figure 4c). In Nox1−/Y mice, similar increases in Nox2 and Nox4 mRNAs were demonstrated at 0, 7, and 14 days of treatment (Figure 4d).
Nox1 Was Not Involved in the Ang II–Induced Vascular Hypertrophy
We previously reported the involvement of Nox1 in prostaglandin F2α (PGF2α)–induced hypertrophy of VSMCs in culture.22 Because vascular hypertrophy is closely linked to elevated blood pressure, we investigated the effect of Ang II infusion on the vascular architecture in Nox1−/Y. When medial area and thickness in cross sections of the thoracic aorta were compared, the infusion of Ang II for 14 days was found to have induced significant hypertrophy in both Nox1+/Y and Nox1−/Y (Figure 5a). Contrary to the results of in vitro studies, medial area and thickness of Nox1−/Y were markedly increased, similar to those of Nox1+/Y (Figure 5b). No change in these parameters was observed in control mice infused with vehicle (PBS) for 14 days.
We further investigated the effect of a subpressor dose of Ang II (0.14 mg · kg−1 · d−1) in Nox1-deficient mice. As shown in Figure 5c, no change in MBP was observed in mice infused with a subpressor dose of Ang II for 28 days. Under equivalent blood pressure conditions, significant hypertrophy in the media was observed in both Nox1+/Y and Nox1−/Y. Medial area and thickness of the thoracic aorta increased in Nox1−/Y to an extent similar to that in Nox1+/Y.
L-NAME Abolished the Suppression of Pressor Response to Ang II in Nox1-Deficient Mice
In Ang II–induced hypertension, superoxide derived from vascular NADPH oxidase has been reported to impair endothelium-dependent relaxation by inactivating nitric oxide (NO), an endothelial vasodilator. To investigate the possible interaction between Nox1 deficiency and endogenous NO, we administered 1.4 mg/d of L-NAME, an NO synthase inhibitor, to Nox1+/Y and Nox1−/Y (Figure 6A). Administration of L-NAME for 14 days slightly elevated basal MBP levels in Nox1+/Y and Nox1−/Y, although the effect was statistically insignificant. The pressor response to Ang II in Nox1+/Y was unaffected by L-NAME, whereas in Ang II–infused Nox1−/Y, MBP was significantly elevated by administration of L-NAME. Consequently, no difference in MBP levels was observed between Nox1+/Y and Nox1−/Y treated with Ang II along with L-NAME.
cGMP Level in the Aorta Was Preserved in Nox1-Deficient Mice Infused With Ang II
To evaluate the effect of disruption of Nox1/NADPH oxidase on NO bioactivity, we measured the level of cGMP in the thoracic aorta. As shown in Figure 6B, no difference in cGMP levels was observed between Nox1+/Y and Nox1−/Y infused with vehicle (PBS) for 7 days. In Ang II–infused mice, conversely, cGMP levels were significantly decreased in Nox1+/Y, whereas the levels in Nox1−/Y were retained. No difference in the activity of NO synthase was demonstrated between Nox1+/Y and Nox1−/Y treated with Ang II (data not shown).
Endothelium-Dependent Vasodilatation Was Preserved in Nox1-Deficient Mice Infused With Ang II
Finally, vascular relaxations in aortic strips were studied to determine whether Nox1-derived ROSs are involved in the Ang II–induced alteration in vascular reactivity. In the vehicle-infused Nox1−/Y, the response to ACh was slightly better than that in Nox1+/Y, without statistical significance (Figure 7a). After the infusion of Ang II for 7 days, the response to ACh was significantly attenuated in Nox1+/Y. In contrast, Ang II infusion did not affect the ACh-induced relaxation in Nox1−/Y. As shown in Figure 7b, endothelium-independent relaxations to sodium pentacyanonitrosylferrate dihydrate were similar in aortic strips obtained from all experimental groups. Usage of PGF2α as an alternative contractile substance gave similar results, and no difference in the contractile response to norepinephrine or Ang II was observed between Nox1+/Y and Nox1−/Y infused with vehicle or Ang II (data not shown). These findings suggest that scavenging of NO by Nox1-derived ROSs underlies the development of Ang II–induced hypertension.
In this study, we clarified for the first time the role of Nox1 in the pathogenesis of Ang II–mediated hypertension using Nox1-deficient mice. The principal findings obtained were that ROSs derived from Nox1/NADPH oxidase are crucial to the pressor response to Ang II by reducing the bioavailability of NO.
The present findings suggest that Nox1 is involved in the late phase but not in the early phase of the pressor response to Ang II. In both Nox1+/Y and Nox1−/Y, MBP was increased up to day 5 of Ang II infusion, whereas in Nox1−/Y, the pressor response was blunted after 7 days. Meanwhile, Nox1 mRNA expression was markedly upregulated from day 7 in Nox1+/Y. Vascular superoxide production is known to impair vascular relaxation through the inactivation of endothelial NO, which serves as an important component in the development and maintenance of increased blood pressure.23 It has been reported that administration of tempol, a superoxide dismutase mimetic, abolishes the pressor response to Ang II.24 We also observed that administration of tempol almost completely abolished the pressor response to Ang II in both Nox1+/Y and Nox1−/Y (data not shown). It therefore seems likely that ROSs derived from another source participate in the early phase of the pressor response to Ang II. In the present Ang II–treated mice, a marked increase in Nox2 mRNA in the aorta was demonstrated at day 5 and 7. In a previous study using Nox2-deficient mice, however, infusion of Ang II for 6 days caused an increase in systolic blood pressure similar to that in wild-type mice.14 A recent study also reported that inactivation of Nox2 had no effect on the development of hypertension in a model in which the endogenous renin-angiotensin system is chronically upregulated.15 These findings therefore suggest that Nox2 does not participate in the late phase of the hypertensive response to Ang II in a murine animal model. Meanwhile, a gradual increase in Nox4 mRNA was observed during Ang II infusion, but the level was unchanged at day 3, when the early phase of the pressor response was already depicted. Although the source of ROSs responsible for the early phase of increased MBP in Ang II–infused Nox1−/Y is still unclear, an alternative possibility may be that a rapid increase in superoxide production by activated Nox2 or Nox4/NADPH oxidase takes part in the early phase of the hypertensive response in Nox1−/Y. Ang II–induced production of ROSs by NADPH oxidase is known to take place instantly, which depends on the activation of protein kinase C and a small G protein, Rac.25
Ang II induces vascular hypertrophy, and it is well known that vascular hypertrophy is closely linked to elevation in blood pressure. We previously demonstrated that depletion of Nox1 mRNA by ribozymes significantly reduced the increased protein synthesis induced by PGF2α in a rat VSMC-derived cell line.22 Nox1 in fact mediates Ang II–induced activation of the redox-sensitive signaling molecules, p38 mitogen-activated protein kinase and Akt, both of which are required for hypertrophy of VSMCs.12 However, our study demonstrated that hypertrophic responses to pressor and subpressor doses of Ang II in Nox1−/Y were similar to those in Nox1+/Y. These findings clearly indicate that Nox1 is not associated with the development of vascular hypertrophy induced by Ang II, which is inconsistent with the previous studies in vitro. Why such inconsistent findings have come about is a subject for further investigation. Under conditions in vivo, however, the ROSs responsible for vascular hypertrophy may derive primarily from Nox2 localized in the endothelial and adventitial cells. ROSs derived from Nox1 may have a limited effect because of its low level of activity compared with that of Nox2.4 Because such growth-promoting ROSs as hydrogen peroxide diffuse freely across the cell membrane, VSMCs in Ang II–infused Nox1−/Y may yet be exposed to a high level of ambient ROSs, whereas isolated VSMCs in culture are unaffected by Nox2-derived ROSs. Also to be considered is the fact that VSMCs removed from their tissue of origin and placed in cell culture transform from a contractile to a synthetic phenotype.26 In vascular injury and atherosclerosis, VSMCs also transform from a contractile to a synthetic phenotype.27,28 Although hypertrophy in the aorta of Ang II–infused mice developed in the absence of Nox1, the findings in cell culture suggest that Nox1 may be involved in vascular remodeling under different experimental or pathological conditions.
Administration of L-NAME abolished the effect of Nox1 gene disruption on the pressor response to Ang II, whereas the level of cGMP, a second messenger of the NO signaling cascade, was preserved in Ang II–infused Nox1−/Y vessels. It has been generally accepted that ROSs decrease the bioavailability of NO by scavenging NO and causing endothelial NO synthase uncoupling, thereby contributing to the pathogenesis of hypertension.29 Uncoupling of endothelial NO synthase appears not to be involved in the pressor response to Ang II under our experimental conditions, because pretreatment with L-NAME did not affect superoxide production in aortic rings isolated from Ang II–treated mice (data not shown). Our findings thus suggest that the preservation of the availability of NO through depletion of Nox1-derived ROSs is the underlying mechanism of the suppressed pressor response in Nox1−/Y. The fact that endothelium-dependent vascular relaxation was maintained in Ang II–infused Nox1−/Y aorta further strengthens this view.
It should be noted that Nox1-derived ROSs participated primarily in the pressor response to Ang II, although an Ang II–induced increase in superoxide production could be attributed to augmented expression of Nox2 and Nox4 in the vascular wall as well. In the thoracic aorta, quantitative determination of mRNA levels illustrated dominant expression of Nox2 and Nox4 compared with Nox1. However, superoxide production in isolated aorta was significantly attenuated in Ang II–infused Nox1−/Y. Such a discrepancy in the levels of Nox transcripts expressed in the aorta and their relative contribution to superoxide production may be explained by distribution and interaction of these catalytic subunits with other subunits required for full enzyme activity.23 ROSs generated by NADPH oxidase act as intracellular and intercellular signaling molecules,12 and Nox1-derived ROSs in the kidney may also participate in the regulation of the pressor response to Ang II.30 In this context, multiple sites of action may be involved in the antihypertensive response in Nox1−/Y.
To date, no isoform-specific inhibitors of the Nox family have been developed. Knockout mice are therefore important tools with which to clarify the functional roles of Nox isoforms in vivo. A previous report using Nox2-deficient mice demonstrated the involvement of Nox2 in the regulation of the basal blood pressure and in Ang II–induced vascular hypertrophy but not in the pressor response to Ang II.14,15 Taking these findings into consideration, it is notable that each Nox isoform plays a distinct role in the development of vascular disorders. Isoform-specific inhibitors of the Nox family may therefore become ideal therapeutic agents for the treatment of vascular disorders with diverse pathological backgrounds.
We thank Dr N. Urao of the Department of Cardiovascular Medicine and Drs T. Nishinaka, M. Ibi, and N. Arakawa of the Department of Pharmacology for valuable discussion and advice. We are also grateful to Drs T. Aihara, K. Amagase, and E. Nakamura of the Department of Applied Pharmacology, Kyoto Pharmaceutical University, for valuable advice and assistance. The authors are indebted to Dr J. Takeda of the Department of Social and Environmental Medicine, Osaka University, for initial guidance with the gene targeting study. This work was supported in part by a Grant-in-Aid for Young Scientists (B) 16790310 (to K. Iwata) and 14770036 (to Dr Katsuyama) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
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Involvement of reactive oxygen species (ROSs) generated by membrane-bound NADPH oxidase has been suggested in the pathogenesis of various cardiovascular diseases. NADPH oxidase is a multisubunit enzyme comprising a membrane-associated catalytic subunit (Nox) and several cytosolic regulatory subunits. Among the Nox isoforms recently identified, vascular Nox1 is detected in smooth muscle cells and endothelial cells, whereas Nox2 is localized in endothelial and adventitial cells. A previous report using Nox2-deficient mice demonstrated the involvement of Nox2 in the regulation of the basal blood pressure and in Ang II–induced vascular hypertrophy, but not in the pressor response to Ang II. In this study, Nox1-deficient mice were generated and chronically infused with Ang II. The elevation of blood pressure was significantly blunted compared with wild-type mice, whereas the infusion of pressor as well as subpressor doses of Ang II did elicit marked hypertrophy in the Nox1-deficient aorta. The underlying mechanism of the suppressed pressor response to Ang II was indicated to be the preservation of the availability of nitric oxide through depletion of Nox1-derived ROSs. Nox1/NADPH oxidase, as a downstream unit of Ang II, thus plays a major role in renin-angiotensin system–mediated chronic hypertension. Certainly, roles for NADPH oxidase in ischemic heart disease, cardiac hypertrophy, and arteriosclerosis should be further clarified in Nox-deficient mice. Because each Nox isoform appears to play a distinct role in the development of vascular disorders, isoform-specific inhibitors of the Nox family may become a new generation of therapeutic agents for the treatment of cardiovascular diseases with diverse pathological backgrounds.
The online-only Data Supplement, which contains a table, can be found at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.105.573709/DC1.