Nox Activator 1
A Potential Target for Modulation of Vascular Reactive Oxygen Species in Atherosclerotic Arteries
Background— Despite a concerted effort by many laboratories, the critical subunits that participate in vascular smooth muscle cell (VSMC) NADPH oxidase function have yet to be elucidated. Given the potential therapeutic importance of cell-specific inhibition of NADPH oxidase, we investigated the role of Nox activator 1 (NoxA1), a homolog of p67phox, in VSMC NADPH oxidase function and atherosclerosis.
Methods and Results— The presence of NoxA1 in mouse aortic VSMCs was confirmed by reverse-transcription polymerase chain reaction and sequencing. NoxA1/p47phox interaction after thrombin treatment was observed by immunoprecipitation/Western analysis of lysates from p47phox−/− VSMCs transfected with adenoviral HA-NoxA1 and Myc-p47phox. Infection with adenoviral NoxA1 significantly enhanced thrombin-induced reactive oxygen species generation in wild-type but not in p47phox−/− and Nox1−/− VSMCs. Thrombin-induced reactive oxygen species production and VSMC proliferation were significantly reduced after downregulation of NoxA1 with shRNA. Infection with NoxA1 shRNA but not scrambled shRNA significantly decreased thrombin-induced activation of the redox-sensitive protein kinases (Janus kinase 2, Akt, and p38 mitogen-activated protein kinase) in VSMCs. Adenovirus-mediated overexpression of NoxA1 in guidewire-injured mouse carotid arteries significantly increased superoxide production in medial VSMCs and enhanced neointimal hyperplasia. NoxA1 expression was significantly increased in aortas and atherosclerotic lesions of ApoE−/− mice compared with age-matched wild-type mice. Furthermore, in contrast to p67phox, immunoreactive NoxA1 is present in intimal and medial SMCs of human early carotid atherosclerotic lesions.
Conclusions— NoxA1 is the functional homolog of p67phox in VSMCs that regulates redox signaling and VSMC phenotype. These findings support the potential for modulation of NoxA1 expression as a viable approach for the treatment of vascular diseases.
Received March 11, 2009; accepted November 27, 2009.
NADPH oxidases are the most important of the superoxide- (O2·−) producing enzymes for vascular pathologies such as hypertension and atherosclerosis.1 Vessel wall NADPH oxidases are similar to the well-characterized neutrophil enzyme active in host defense but vary kinetically and in subunit composition, with differences reported among cell types and vascular beds.2
Clinical Perspective on p 559
Given the large number of potential combinations of subunits, NADPH oxidases are categorized on the basis of the catalytic subunit, the Nox protein, and which of the 5 homologs of Nox is present in a particular complex. At least 4 Nox homologs are present in one or another vessel wall cell type. Rodent aortic vascular smooth muscle cells (VSMCs) contain Nox1 and Nox4,3,4 which directly interact with p22phox, a subunit common to all vascular oxidases,5 to form a functional NADPH oxidase. Nox1 is inactive under basal conditions,6 whereas Nox4 is constitutively active.7,8 Nox2 is expressed at extremely low levels in rodent VSMCs,4 and targeted deletion of the NOX2 gene had no effect on agonist-induced O2·− generation in these cells.9 Recently, it has been reported that 4 isoforms of Nox5 are expressed in human aortic SMCs and that Nox5 shRNA significantly inhibited platelet-derived growth factor–stimulated reactive oxygen species (ROS) production and cell proliferation.10
The Nox1-based NADPH oxidase contains regulatory cytosolic components p47phox, Nox activator 1 (NoxA1), and Rac1, in addition to Nox1 and p22phox, which form the membrane-bound cytochrome b558. Both p22phox and p47phox have been shown to be essential for NADPH oxidase activity in VSMCs. Transfection of VSMCs with antisense p22phox cDNA significantly inhibits agonist-stimulated NADH/NADPH-dependent O2·− production.11 We and others have previously demonstrated that genetic deletion of p47phox inhibits agonist-induced O2·− production in VSMCs.9,12,13Both p22phox and p47phox are widely expressed in nonvascular cells. For this reason, a strategy of targeting either p22phox or p47phox is a nonselective means to inhibit vascular wall NADPH oxidase activity. The Nox2-based NADPH oxidase contains p47phox and Rac1 in addition to Nox2. In this complex, p67phox is a necessary subunit for activation.14
Whether p67phox is a necessary component of the VSMC NADPH oxidase and, if not, what subunit substitutes for p67phox remain controversial. We previously reported that p67phox was not expressed in rat aortic VSMCs15; however, p67phox mRNA has been reported in VSMCs from human resistance arteries.16 The report of novel human and mouse homologs of p47phox (Nox organizer 1 [NoxO1]) and p67phox (NoxA1) raised the question of the subunit composition of NADPH oxidases in vascular cells.17,18 The question of the role of p47phox and p67phox versus NoxO1 and NoxA1 is complicated by reports of their interdependence in some systems. Expression of Nox1 with NoxO1 and NoxA1 produced stimulus-independent generation of O2·− in HEK293 cells, and the constitutive activation of Nox1 was lost when NoxO1 was replaced with p47phox.18 The reason is that NoxO1 lacks the autoinhibitory region present in p47phox and therefore can localize to cell membrane under basal conditions, 19 but this interdependence may not exist in VSMCs. NoxA1 is expressed in the cytoplasm of VSMCs, and treatment of cells with epidermal growth factor induces the translocation of NoxA1 and p47phox to the cell membrane.20 In addition, antisense inhibition of NoxA1 attenuates agonist-induced O2·− production in mouse VSMCs. Rac1 is directly involved in the activation of Nox1, perhaps via its interaction with NoxA1.21,22
The present study was designed to determine whether NoxA1 was an important functional component of the VSMC NADPH oxidase and, if so, whether it may represent an appropriate target for therapeutic intervention. We examined the expression of NoxA1 and its interaction with p47phox in mouse aortic VSMCs. Suppression of NoxA1 expression decreased whereas overexpression increased thrombin-induced O2·− production. Localized overexpression of NoxA1 significantly enhanced neointimal hyperplasia in injured mouse carotid arteries. NoxA1 expression is increased in aortas and atherosclerotic lesions from ApoE−/− mice. Furthermore, NoxA1 expression is correlated with the progression of atherosclerotic lesions, whereas both NoxA1 and p67phox were present in advanced atherosclerotic lesions of human carotid arteries. These data suggest that it may be possible to reduce vascular lesion formation and its complications by targeting NoxA1 with specific therapeutic interventions.
Mouse aortic VSMCs were isolated from 4-month-old male C57BL/6J mice as previously described23 (see the Materials section of the online-only Data Supplement).
Reverse-Transcriptase Polymerase Chain Reaction and Sequencing of NoxA1
The first-strand cDNA synthesis was carried out from total RNA isolated from mouse aortic VSMCs with SuperScript II reverse transcriptase (RT; Invitrogen, Carlsbad, Calif) according to the manufacturer’s instructions. Polymerase chain reaction (PCR) was performed with primers generated from the mouse NoxA1 sequence (5′-TTCCAGCTGCAGAGGTTCCAG-3′ as forward primer and 5′-AAGTCCAAATCCTCCGGTCT-3′ as reverse primer) to amplify a 923-bp fragment (247 to 1169 bp) of the NoxA1 gene. RT-PCR to determine the expression of p67phox in mouse VSMCs was performed with primers generated from the mouse p67phox sequence (5′-CTACCTGGAGCCAGTTGAGC-3′ as forward primer and 5′-GGGCAGCCTCATAACTGAAG-3′ as reverse primer) to amplify a 636-bp fragment (876 to 1411 bp) of the p67phox gene (see the Materials section of the online-only Data Supplement for details).
Retroviral Vector Construction and Infection of Mouse Aortic VSMCs
A double-stranded hairpin oligonucleotide designed to target the mouse NoxA1 cDNA nucleotides 376 to 394 (5′-TACAACATGGCATCAGCAC-3′) of NoxA1 gene was cloned into the BglII/HindIII site of pSUPER.retro.puro vector to generate NoxA1 shRNA (see the Materials section of the online-only Data Supplement for details).
Adenovirus Construction and Infection of Mouse Aortic VSMCs
Adenoviral constructs encoding HA-NoxA1 and Myc-p47phox and infection of VSMCs were performed as described in the Materials section of the online-only Data Supplement.
Measurement of Intracellular ROS
Intracellular hydrogen peroxide levels in VSMCs were measured with 2′,7′-dichlorofluorescein diacetate as described previously.24 Superoxide levels in mouse VSMCs25 and in the aortas9 were measured by staining with dihydroethidium. Superoxide anion generation in thrombin-treated mouse VSMC suspension was measured with the chemiluminescence enhancer L012 [8-amino-5-chloro-7-phenylpyridol[3,4-day]pyridazine-1,4(2H,3H)dione] (see the Materials section of the online-only Data Supplement for details).
Immunoprecipitation and Western Blot Analysis
Immunoprecipitation and Western blotting were preformed as described26 (see the Materials section of the online-only Data Supplement).
[3H]Thymidine Incorporation Assay
[3H] Thymidine incorporation into DNA of VSMCs treated with or without thrombin were performed as described26 (see the Materials section of the online-only Data Supplement).
VSMC Migration Assay
VSMC migration was determined in a scratch wound assay (see the Materials section of the online-only Data Supplement).
Real-Time RT-PCR to Measure NoxA1 Expression
Detailed information regarding RNA extraction and real-time RT-PCR for NoxA1 expression is available in the Materials section of the online-only Data Supplement.
Mice and Diet
Wild-type and ApoE−/− mice were purchased from Jackson Laboratory (Bar Harbor, Me). Mice were maintained at 22°C with a 12-hour light/dark cycle and given free access to food and water. Mice on high-fat diet were maintained on standard rodent chow until 4 weeks of age and then fed a Western-type diet (TD88137, Harlan Teklad, Indianapolis, Ind) for 12 weeks. All procedures involving the experimental animals were performed in accordance with protocols approved by University of North Carolina Institutional Animal Care and Use Committee according to National Institutes of Health guidelines.
Carotid Artery Injury and NoxA1 Overexpression
The right external and common carotid arteries were surgically exposed in 10-week-old anesthetized wild-type mice. An angioplasty guidewire, introduced through an arteriotomy made in the external carotid artery, was used to denude the endothelium in the artery. The denuded artery was flushed with PBS; a 1-cm segment was isolated with ligatures; and 50 μL PBS or PBS containing adenoviral NoxA1 or adenoviral green fluorescent protein (GFP) vector (1×109 plaque-forming units) was injected through a catheter and incubated for 30 minutes. After removal of this solution, the external carotid artery was ligated, and blood flow to the internal carotid artery was restored. The arteriotomy site was ligated by tying the previously placed suture, and the skin incision was closed with 7-0 sutures (see the Materials section of the online-only Data Supplement).
Human Carotid Arteries
Fresh-frozen human carotid arteries, collected during autopsy performed 10 to 12 hours after death, were obtained from National Disease Research Interchange (Philadelphia, Pa). Carotid artery endarterectomy samples were collected at St Thomas’ Hospital, London, under informed consent. The use of human specimens was approved by the University of North Carolina Institutional Review Board. Carotid atherosclerotic lesions were graded as follows: normal, no lesions; type I, very initial lesions; type II, first grossly visible lesions characterized by foam cells and lipid droplets in intimal SMCs and droplets of extracellular lipid; and type III, intermediate lesions according to the American Heart Association atherosclerotic plaque classification.27
Histology and Morphometry
Carotid artery sections were stained with combined Masson trichrome elastic stain and Oil Red O, and morphometric analysis was performed (see the Materials section of the online-only Data Supplement).
NoxA1 staining of carotid artery and aortic sections is described online in the Materials section of the online-only Data Supplement.
Data are presented as mean±SEM. All of the data were analyzed with 1-way ANOVA followed by Newman-Keuls multiple-comparison test. Differences were considered significant at P<0.05.
Confirmation of NoxA1 Expression in Mouse Aortic VSMCs
The molecular composition of vascular wall NADPH oxidases continues to be investigated because of its complexity. Multiple possible subunit combinations and variability between cell types, in the same cell type from different species and even in a single-cell type (VSMCs) from different anatomic locations, have been demonstrated.28 An important remaining question is whether one or both activating subunits (p67phox or NoxA1) are functionally important. We began by investigating expression levels and previously reported that p67phox was not detectable in human and rat aortic VSMCs.15 Ambasta et al20 recently reported the expression of NoxA1, a p67phox homolog, in mouse aortic VSMCs by Western analysis. To determine whether NoxA1 is expressed in mouse VSMCs, we performed RT-PCR amplifying a region from 247 to 1169 bp of the NoxA1 coding sequence. We were able to amplify the expected (923 bp) fragment using NoxA1 cDNA but not cellular RNA as the template, indicating that the PCR product was not amplified from the contaminating genomic DNA present in the RNA preparation (Figure 1A). Sequence analysis of the PCR product confirmed the NoxA1 expression in mouse aortic VSMCs. As with human and rat aortic VSMCs, p67phox protein also was not expressed in mouse aortic VSMCs (data not shown). These data indicate that NoxA1 is the p67phox homolog expressed in mouse aortic VSMCs.
NoxA1 Interacts With p47phox
It was previously demonstrated that the fluorescent fusion proteins of NoxA1 and p47phox, when expressed in mouse VSMCs, are translocated from cytosol to the membrane and cytoskeleton after stimulation with epidermal growth factor.20 This is consistent with the hypothesis that p47phox binds NoxA1 and forms a functional NADPH oxidase at the membrane in a manner that is analogous to the association of the complex in phagocytes. We used immunoprecipitation/Western analysis to address the interaction of NoxA1 with p47phox and to determine whether physiological factors that modulate ROS production such as thrombin also affect the NoxA1-p47phox interaction. Because NoxA1-specific antibodies were not available at this time, we infected p47phox-deficient mouse VSMCs with adenoviruses encoding myc-tagged p47phox and HA-tagged NoxA1 (Figure 1B). Cells were treated with or without thrombin for 10 minutes, and immunoprecipitation was performed with anti-HA antibody. Western blot analysis with anti-myc antibody showed a significant increase in the interaction of NoxA1 with p47phox after thrombin stimulation (Figure 1C). These results confirm that interaction of NoxA1 with p47phox is an essential initial event in the assembly of a functional NADPH oxidase on agonist stimulation in mouse VSMCs.
NoxA1 Is Essential for Thrombin-Induced ROS Production in Mouse VSMCs
To determine whether NoxA1 is required for ROS production, wild-type mouse aortic VSMCs were infected with retroviruses encoding either NoxA1-specific shRNA or scrambled shRNA (Figure 2A). NoxA1-specific shRNA infection resulted in an ≈90% decrease in the expression of a 51-kDa band recognized by the NoxA1 antibodies, confirming the effectiveness of shRNA in regulating NoxA1 expression. In addition, regulation of NoxA1 expression with retroviral NoxA1 shRNA or adenoviral NoxA1 constructs had no effect on Nox1 expression as measured by real-time RT-PCR (Figure IA of the online-only Data Supplement). The manipulation of NoxA1 expression by these methods did not cause cellular toxicity (data not shown) or activation of cellular stress response as measured by HSP70 levels (Figure IB of the online-only Data Supplement). In cells not infected with NoxA1 shRNA, treatment with thrombin for 10 minutes significantly increased (P<0.001) ROS production as measured by the oxidation of H2DCF-DA (Figure 2B) and by L012 chemiluminescence (Figure 2C). Thrombin-induced ROS generation was largely inhibited by pretreatment with diphenyleneiodonium (DPI), a flavoprotein inhibitor. Suppression of NoxA1 by infection of VSMCs with retrovirus-encoding NoxA1 shRNA had no significant effect on basal ROS production; however, thrombin-induced ROS generation in mouse VSMCs expressing NoxA1 shRNA was significantly (P<0.01) but not completely inhibited. Infection of VSMCs with retrovirus-encoding scrambled shRNA had no effect on thrombin-induced ROS generation (Figures 2B and 2C). Consistent with these observations, conversion of dihydroethidium (DHE) to ethidium, a sensitive marker for O2·− generation, was markedly attenuated in thrombin-treated VSMCs expressing NoxA1 shRNA but not scrambled shRNA (Figure 2D). Together, these data indicate that NoxA1 is necessary for agonist-induced increases in ROS production in mouse VSMCs. Furthermore, these data are consistent with the notion that either low levels of NoxA1 (or an undetectable level of p67phox) are sufficient for basal ROS production.
Overexpression of NoxA1 Increases ROS Production in Wild-Type but Not p47phox−/− and Nox1−/− VSMCs
It has been reported that in endothelial dysfunction and atherosclerosis, increased ROS production is associated with enhanced expression of the NoxA1 homolog p67phox.29,30 Similarly, increased p67phox and other NADPH oxidase subunit gene expression was observed in vascular cells in response to pathophysiological agonists16 and is correlated with increased ROS production.31 To determine the effect of increased expression of NoxA1 on thrombin-induced ROS generation, wild-type and p47phox knockout mouse aortic VSMCs infected with control adenovirus or adenovirus expressing HA-tagged NoxA1 were quiesced for 72 hours and then left untreated or treated with thrombin for 10 minutes. Infection with HA-tagged NoxA1 adenovirus caused a marked increase in the expression of NoxA1 in both wild-type and p47phox knockout VSMCs (Figure 3A). As previously reported,25 thrombin significantly increased ROS production in wild-type (P<0.05) but not in p47phox−/− (Figure 3B) and Nox1−/− (Figure 3C) VSMCs. NoxA1 overexpression potentiated thrombin-induced ROS generation in wild-type VSMCs (P<0.001) but had no effect on ROS generation in p47phox−/− (Figure 3B) and Nox1−/− (Figure 3C) cells. These data confirm that interaction of p47phox with NoxA1 is necessary for agonist-induced ROS generation and suggest that modulation of NoxA1 under pathophysiological conditions would increase oxidative stress in the vasculature.
Downregulation of NoxA1 Expression Decreases Thrombin-Induced Proliferation of VSMCs
We have previously shown that thrombin promotes VSMC proliferation in a ROS-dependent manner through the activation of NADPH oxidase.9,15 We next examined the role of NoxA1 in thrombin-induced VSMC proliferation (Figure 3D). Growth-arrested mouse aortic VSMCs infected with retroviruses encoding either NoxA1-specific shRNA or scrambled shRNA were treated with or without thrombin in the presence and absence of DPI. Thrombin induced a 2.8-fold increase in DNA synthesis in VSMCs expressing scrambled shRNA as measured by thymidine uptake, an effect that was significantly inhibited by DPI (P<0.001). Thrombin-induced DNA synthesis increased only 1.8-fold in VSMCs expressing NoxA1 shRNA compared with the respective control but was significantly less than that in cells infected with scrambled shRNA (P<0.001). Importantly, DPI pretreatment reduced thrombin-induced increases in DNA synthesis in cells expressing NoxA1 shRNA. This suggests that the residual increase in ROS levels in these cells contributes to increased thymidine uptake. DPI had no effect on basal thymidine uptake in VSMCs expressing either NoxA1 shRNA or scrambled shRNA. Together, these data indicate that NoxA1 contributes to agonist-induced proliferative response of VSMCs.
Suppression of NoxA1 Expression Decreases Thrombin-Induced Activation of Protein Kinases in Mouse Aortic VSMCs
ROS derived from vascular NADPH oxidases serve as second messengers to activate multiple intracellular proteins that play an important role in the physiology and pathophysiology of vascular cells.32 To determine the role of NoxA1-containing NADPH oxidases in thrombin-induced VSMC proliferation, we examined the activation of redox-sensitive protein kinases such as Janus kinase 2 (JAK2), Akt/protein kinase B, and p38 mitogen-activated protein kinase (p38 MAPK) in NoxA1 shRNA and scrambled shRNA expressing wild-type VSMCs treated with thrombin (Figure 4). Western blot analysis of thrombin-stimulated VSMC lysates with a phosphospecific JAK2 antibody showed a significant increase in JAK2 tyrosine phosphorylation at 10 minutes in scrambled shRNA–expressing cells (2.20±0.55-fold increase; P<0.001), which was abrogated by pretreatment with DPI (P<0.001; Figure 4A). In contrast, no such increase in JAK2 phosphorylation in response to thrombin treatment was observed in VSMCs expressing NoxA1 shRNA. Thrombin treatment significantly stimulated Akt/PKB phosphorylation at 10 minutes in both scrambled shRNA– (3.70±0.30-fold increase; P<0.001) and NoxA1 shRNA– (1.57±0.21-fold increase; P<0.01; Figure 4B) expressing VSMCs. Akt/PKB phosphorylation in NoxA1 shRNA–expressing cells was significantly less than that in scrambled shRNA–expressing cells (P<0.001), however, and was completely abrogated by pretreatment with DPI in both cells.
Phosphorylation of p38 MAPK increased significantly in scrambled shRNA– (2.33±0.20–fold increase; P<0.001) and in NoxA1 shRNA– (1.27±0.11–fold increase; P<0.01) expressing VSMCs treated with thrombin (Figure 4C). Similar to its effect on Akt/PKB phosphorylation, p38 MAPK phosphorylation was significantly decreased in NoxA1 shRNA–expressing VSMCs compared with scrambled shRNA–expressing VSMCs (P<0.001). Thrombin-induced p38 MAPK activation in scrambled shRNA–expressing VSMCs was abrogated by DPI pretreatment. These data indicate that NoxA1-containing NADPH oxidase contributes to VSMC proliferation by the activation of mitogenic intracellular signaling pathways.
Tumor Necrosis Factor-α, Not Thrombin, Increased the Expression of NoxA1, and NoxA1 shRNA Inhibited Tumor Necrosis Factor-α–Induced ROS Production in VSMCs
We next examined the effect of thrombin on NoxA1 expression by RT-PCR. Although NoxA1 is essential for thrombin-induced ROS generation, thrombin (1 U/mL) per se did not induce NoxA1 expression in mouse VSMCs (data not shown); however, treatment with tumor necrosis factor-α (TNFα; 20 ng/mL) for 4 hours significantly increased NoxA1 expression (4.35±1.70–fold increase; P<0.05) in mouse VSMCs (Figure 5A). Angiotensin II (100 nmol/L) was a weak inducer of NoxA1 expression (1.92±0.68–fold increase at 4 hours of treatment; Figure 5A). In contrast to the report of Honjo et al33 in human umbilical vein endothelial cell line, oxidized low-density lipoprotein (30 or 60 μg/mL) had no stimulatory effect on NoxA1 expression in mouse VSMCs for up to 24 hours of treatment (data not shown).
Consistent with real-time RT-PCR data, we observed a significant increase in NoxA1 protein expression in VSMCs treated with TNFα for 8 hours (P<0.05; Figures 5B and 5C). As in the case of thrombin, NoxA1 shRNA expression significantly attenuated TNFα-induced ROS generation in mouse VSMCs (Figure 5D). These data indicate that thrombin regulates ROS generation without affecting NoxA1 expression levels, whereas other agonists may enhance ROS production by modulating NoxA1 expression.
NoxA1 Overexpression Increases Superoxide Production in Injured Carotid Arteries and Enhances Neointimal Hyperplasia
We also examined whether the NoxA1-dependent increased production of ROS in VSMCs observed in vitro occurs in arterial VSMCs in vivo and NoxA1-dependent activation of NADPH oxidase results in enhanced neointimal hyperplasia. Mouse carotid arteries were subjected to guidewire injury and then infected with a recombinant adenovirus encoding NoxA1 and GFP, a control empty adenovirus expressing only GFP or only treated with PBS. Three days after adenovirus infection, histochemical analysis revealed NoxA1 overexpression in AdNoxA1-infected vessels (Figure IIA of the online-only Data Supplement). DHE staining of injured carotid arteries showed a significant increase (P<0.001) in O2·− levels in the medial VSMCs (Figure 6A and Figure IIB of the online-only Data Supplement). Fifteen days after guidewire injury and adenovirus infection, neointimal hyperplasia was assessed morphologically and by morphometric analysis (Figure 6B and Figure IIC of the online-only Data Supplement). Compared with empty adenovirus–infected mice, adenovirus NoxA1–expressing mice had significantly increased (P<0.05) neointimal hyperplasia. Furthermore, immunoreactive NoxA1 expression in neointima 15 days after injury was confined to smooth muscle α-actin–positive cells (Figure 6C). Neointimal hyperplasia is correlated with increased intimal SMC proliferation, as shown by enhanced immunostaining with proliferating cell nuclear antigen antibody (Figure 6D). We have also observed that NoxA1 overexpression in VSMCs results in increased migration, as determined by the in vitro scratch assay (Figure III of the online-only Data Supplement). Intriguingly, injury per se had no effect on NoxA1 levels in the carotid artery; however, NoxA1 expression is increased in aortas and atherosclerotic lesions from ApoE−/− mice (Figure 7). Together, these data suggest that in the mouse carotid artery injury model, localized overexpression of NoxA1 stimulates O2·− production, resulting in increased medial VSMC migration and proliferation and enhanced neointima formation.
NoxA1 Is Expressed in Early Atherosclerotic Lesions of Human Carotid Arteries
To determine the clinical relevance of our NoxA1 data from cell culture experiments and mouse atherosclerosis models, we investigated NoxA1 expression in normal, early, and relatively advanced (intermediate) atherosclerotic lesions in human carotid arteries (Figure 8). We analyzed a total of 29 samples (22 male and 7 female). The subjects ranged from 21 to 89 years of age. NoxA1 expression, which was weak in normal carotid arteries, increased significantly in the intimal and medial SMCs of early (type I and II) atherosclerotic lesions. Expression of p67phox was confined to the endothelial layer in both normal and early atherosclerotic arteries, but the expression of both NoxA1 and p67phox increased significantly in the intimal and medial SMCs of early advanced (type III) atherosclerotic lesions. The presence of NoxA1 and p67phox in VSMCs was confirmed by staining with α-actin (Figure 8). Although our small sample size precludes a definitive statement, NoxA1 and p67phox expression in carotid atherosclerotic lesions does not seem to be influenced by the gender of the subjects. These data suggest that NoxA1 also plays a role in the development of human atherosclerosis.
An understanding of the precise molecular composition of vascular NADPH oxidases is essential to elucidate the contribution of cell-specific oxidases in vascular pathology. In this study, we confirmed that NoxA1 is a critical component of VSMC NADPH oxidase.20 Downregulation of NoxA1 expression inhibited thrombin-induced O2·− production, mitogenic protein activation, and proliferation of VSMCs, whereas its upregulation increased thrombin-induced O2·− production. Localized overexpression by adenovirus-mediated delivery of NoxA1 enhanced neointimal hyperplasia in the mouse carotid artery injury model, indicating that regulation of NoxA1 expression could modulate VSMC NADPH oxidase activity under pathophysiological conditions.
It is known that rodent VSMC NADPH oxidase has a unique composition—the presence of Nox1 and Nox4 and the absence of Nox2 isoform—compared with other vascular cells.4,34–36 Nox1-containing VSMC NADPH oxidase is similar to other vascular cell and leukocyte NADPH oxidases in that its activity is dependent on other subunits of the enzyme, p22phox, p47phox, and Rac.9,11,12,37 Recently, Ambasta et al20 have demonstrated that NoxA1 is the p67phox homolog in functional mouse VSMC NADPH oxidase. In the present study, we confirmed this observation by RT-PCR amplification and sequencing of NoxA1 from mouse VSMCs. Our observation that thrombin activates the interaction of p47phox with NoxA1 is consistent with the data of agonist-induced membrane cotranslocation of NoxA1 and p47phox fusion proteins in VSMCs.20 Further support for p47phox and NoxA1 interaction in activating VSMC NADPH oxidase is obtained from the data in the present study that NoxA1 overexpression enhances thrombin-induced ROS production in wild-type but not in p47phox−/− VSMCs. Activated p47phox binds with NoxA1 and interacts with Nox1 at the membrane to increase O2·− production on agonist treatment. This is corroborated by the coimmunoprecipitation of NoxA1 with Nox1 in HEK293 cells transfected with Nox1, NoxO1, and NoxA1 fusion proteins. 20In addition, the critical role of NoxA1 in O2·− production is evident from negligible ROS generation in cells overexpressing Nox1 and NoxO1 in the absence of NoxA1.
NoxA1 plays a critical role in modulating the phenotype of VSMCs because depletion of this protein using retroviral shRNA not only inhibits thrombin-induced ROS generation but also significantly attenuates thrombin-stimulated cell proliferation. Our data also indicate that NoxA1 functions in tandem with phosphorylated p47phox in agonist-induced stimulation of Nox1-containing NADPH oxidase because increased ROS generation was observed in NoxA1-overexpressing VSMCs treated with thrombin and NoxA1 overexpression per se had no effect on ROS generation.
NoxA1 is not expressed in high abundance in mouse VSMCs,20 and we did not observe any significant increase in its expression after thrombin treatment. A robust increase in NoxA1 expression and NoxA1-dependent NADPH oxidase activation observed in response to TNFα suggests, however, that both the presence of NoxA1 and its expression levels play a regulatory role in VSMC NADPH oxidase activation. In this context, it is worth noting that angiotensin II–induced transcriptional regulation of p67phox enhances NADPH oxidase activity in aortic endothelial cells38 and adventitial fibroblasts.39 Interestingly, p67phox has also been proposed as a putative rate-limiting cofactor in NADPH oxidase activation.40,41
We have previously shown that decreased levels of activated mitogenic proteins and attenuated cell proliferation occur downstream of impaired agonist-induced ROS production in p47phox−/− VSMCs.9,42 Our present data showing that depletion of NoxA1 in VSMCs significantly decreases thrombin-induced JAK2, Akt, and p38 MAPK phosphorylation support the hypothesis that agonist-induced NADPH oxidase activation plays an important role in VSMC proliferation. These data are also consistent with redox-sensitive regulation of JAK2, Akt, and p38 MAPK in VSMCs.4,13,26,43
The increased medial O2·− production and enhanced neointimal hyperplasia observed in the present investigation in AdNoxA1-infected injured carotid arteries are consistent with the notion that vascular NADPH oxidase plays a potentially critical role in vascular disease.4,9,42 Furthermore, consistent with the observation of Ambasta et al,20 NoxA1 expression was abundant in proliferating VSMCs in injured carotid arteries. The fact that NoxA1 expression is not upregulated by injury per se but seems to increase in atherosclerotic lesions suggests that NoxA1 expression is fine-tuned to different pathophysiological stimuli. In this context, it is worth mentioning a recent report by Kim et al44 that elucidates a novel mechanism for NoxA1-regulated Nox1 activation in colon cell lines. Protein kinase A–dependent phosphorylation of NoxA1 at Ser172 and Ser461 results in its interaction with 14-3-3ζ and consequent inhibition of Nox1 activity. It would be interesting to see whether a similar mechanism regulates NoxA1-dependent Nox1 activity in vascular cells.
Interestingly, immunoreactive NoxA1 was present in the intimal and medial SMCs of early atherosclerotic lesions (types I and II), whereas NoxA1 and p67phox were present in the intimal and medial SMCs of early advanced (type III) atherosclerotic lesions of human carotid arteries. In this context, it is noteworthy that Clempus et al34 have correlated modulation of rat VSMC phenotypes with relative expression levels of Nox1 and Nox4. It would be interesting to see whether a similar phenomenon occurs with regard to NoxA1- and p67phox-dependent NADPH oxidase activity on cellular phenotype in human aortic VSMCs.
Our results demonstrate that NoxA1 expression regulates NADPH oxidase activity in mouse VSMCs. Furthermore, expression of NoxA1 regulates thrombin-induced activation of redox-sensitive mitogenic protein kinases. Increased neointimal hyperplasia in injured mouse carotid arteries following localized overexpression of NoxA1 and the presence of NoxA1 in mouse and human atherosclerotic lesions suggest that NoxA1 is a potential target for therapeutic intervention. These findings support further investigation of NoxA1 expression in vascular diseases.
We gratefully acknowledge Dr Kathy Griendling for providing Nox1−/− VSMCs.
Sources of Funding
This work was supported in part by National Institutes of Health grant HL-57352 (Dr Runge) and National Institute for Health Research Biomedical Research Center at Guy’s and St Thomas’ National Health Service Foundation Trust (Dr Smith).
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After decades of investigation, there remains a need for preventive strategies that can reduce atherosclerosis and atherothrombosis, which are the most common causes of death and disability in the United States. Despite many important advances in the treatment of cardiovascular diseases, elucidation of specific therapies that target vascular wall cells has been elusive. An important limitation of targeting intracellular signaling pathways in dysfunctional vascular cells is that most of them are present ubiquitously and are necessary for normal cellular function. Many investigators have studied the hypothesis that regulated production of reactive oxygen species and oxidative stress in vascular cells are important in atherogenesis and that it may be possible to specifically inhibit upregulation of reactive oxygen species production in vascular cells, and in doing so, limit atherogenesis and atherothrombosis. The pathways that produce reactive oxygen species—NADPH oxidase, xanthine oxidase, cyclooxygenase, lipogenase, and others—are necessary, however, for normal cellular function. The experiments described here were designed to test the strategy of targeting a specific component of NADPH oxidase, arguably the most important regulated system for reactive oxygen species production in vascular cells. To do so, we examined modulation of NoxA1, an NADPH oxidase component necessary for upregulation of superoxide production in vascular smooth muscle cells. Our studies indicate that NoxA1 is critically important in the NADPH oxidase–mediated overexpression of reactive oxygen species characteristic of vascular diseases. Although specific inhibitors of NoxA1 are not known, this work suggests that the strategy of cell-specific modulation of NADPH oxidase function is a therapeutic approach worthy of further investigation.
↵*Drs Niu, Madamanchi, and Vendrov contributed equally to this work.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.109.908319/DC1.