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Cardiovascular Research 2005 65(2):495-504; doi:10.1016/j.cardiores.2004.10.026
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Copyright © 2004, European Society of Cardiology

The contribution of Nox4 to NADPH oxidase activity in mouse vascular smooth muscle

Sara H.M. Ellmark, Gregory J. Dusting*, Mark Ng Tang Fui, Nancy Guzzo-Pernell and Grant R. Drummond1

Howard Florey Institute, University of Melbourne, Victoria 3010, Australia

* Corresponding author. Tel.: +61 3 8344 1961; fax: +61 3 9348 1707. Email address: g.dusting{at}hfi.unimelb.edu.au Grant.Drummond{at}med.monash.edu.au

Received 2 March 2004; revised 12 October 2004; accepted 20 October 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: NADPH oxidases are important sources of reactive oxygen species (ROS) in the vasculature. In phagocytic cells, the catalytic subunit of NADPH oxidase is a glycoprotein, gp91phox. However, vascular smooth muscle cells (VSMCs), which show prominent NADPH oxidase activity, lack gp91phox. Hence, we examined the role of Nox4, a gp91phox homologue, in superoxide production in mouse-cultured VSMCs.

Methods and results: Incubation of VSMCs with NADPH increased ROS production whether detected by lucigenin-enhanced chemiluminescence or dichlorofluorescein. Superoxide production was inhibited by the NADPH oxidase inhibitors, diphenyleneiodonium and apocynin, but not by inhibitors of other potential sources of superoxide. In unstimulated VSMCs, phosphorothioate antisense oligonucleotides against Nox4 down-regulated mRNA expression of the subunit by 65% and attenuated superoxide production by 41% without affecting Nox1 expression. Interleukin-1β (IL-β) thrombin and platelet-derived growth factor (PDGF) also reduced Nox4 mRNA expression after 3 h without affecting Nox1 levels. Of these stimuli, only IL-β reduced superoxide, but this effect was more rapid (≥ 30 min) than its actions on Nox4.

Conclusions: Under resting conditions, NADPH oxidase activity in VSMCs is largely dependent upon Nox4 expression. Proinflammatory mediators down-regulated Nox4 but did not affect Nox1 expression, so other factors must compensate to regulate superoxide production.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Reactive oxygen species (ROS) play central roles in vascular smooth muscle cell (VSMC) biology and pathobiology. For example, ROS are important physiological and pathophysiological regulators of vascular tone eliciting effects ranging from direct H2O2-induced K+ channel opening and vasodilatation [1] to indirect vasoconstriction mediated by superoxide-dependent inactivation of endothelium-derived nitric oxide [2–4]. ROS are also involved in VSMC growth and migration; processes essential for blood vessel development, injury repair and remodelling due to vascular disease. The production of ROS in VSMCs is reportedly increased by growth factors such as platelet-derived growth factor (PDGF) [5], angiotensin II [6] and thrombin [7]. Moreover, ROS scavengers such as N-acetylcysteine and catalase not only prevent mitogen-induced stimulation of protein kinases [8,9] and transcription factors [10,11] but also inhibit VSMC proliferation [12] and hypertrophy [6].

The major source of ROS in VSMCs is an NADPH oxidase complex that is similar, although not identical, to the enzyme complex in neutrophils responsible for the respiratory burst. The neutrophil NADPH oxidase complex contains 5 protein subunits: a membrane-bound cytochrome b558 domain comprising gp91phox (Nox2) and p22phox, and three cytosolic regulatory subunits, p47phox, p67phox and p40phox [13]. Whereas both p22phox [14] and p47phox [15] appear to be essential for NADPH oxidase activity in VSMCs, targeted-deletion of the gp91phox gene in mice had no effect on superoxide production in these cells [16]. Given that gp91phox contains the NADPH-binding site and catalytic domain of the neutrophil oxidase, this suggests that there must be a substitute gp91phox homologue(s) in VSMCs.

Two homologues of gp91phox, termed Nox1 and Nox4, have been identified in rat cultured VSMCs [17–20]. A full-length cDNA antisense against Nox1 reduced VSMC superoxide production in response to angiotensin II but did not inhibit basal superoxide production [18]. Thus, the source of VSMC superoxide generated under resting conditions remains to be determined. The role of Nox4 in VSMC superoxide production has not been addressed directly despite the fact that Nox4 appears to be a critical NADPH oxidase subunit in mouse osteoclasts [21], rat mesangial cells [22], human embryonic kidney (HEK293) cells [23] and endothelial cells [24].

We set out to investigate the role of the Nox4 subunit in NADPH oxidase-dependent superoxide production in mouse VSMCs. Specific suppression of Nox4 mRNA expression with phosphorothioate antisense oligonucleotides led to a marked decrease in NADPH-dependent superoxide production. Interestingly, while proatherogenic stimuli interleukin-1β (IL-1β), thrombin and PDGF each caused significant reductions in Nox4 mRNA expression after 3–6 h, only IL-β decreased superoxide production, and this occurred before mRNA was altered.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996).

2.1. Mice
Thirteen-week-old male C57BL6/J mice, purchased from the Animal Resource Centre (Australia) and maintained on a normal chow diet, were used for these studies (Howard Florey Institute Animal Ethics No. 99023). For all experiments, mice were heparinised (250 IU, i.p.) and anaesthetised with Isoflo (Abbot, USA) prior to being killed by decapitation.

2.2. VSMC culture
For each culture, thoracic aortas from two mice were isolated and cleared of fat and connective tissue, before being placed in digestion medium [i.e., Dulbecco's Modified Eagles Medium (DMEM; CSL, Australia) containing 0.5 mg/ml elastase, 1.0 mg/ml collagenase and 1.25 mg/ml trypsin], and incubated at 37 °C for 5 min. The adventitial layers of the blood vessels were then peeled off with forceps, and digestion medium was flushed through the vessel lumen to dislodge endothelial cells. The remaining tube of medial smooth muscle cells was then cut into ring segments (2–3 mm) and transferred to a microcentrifuge tube containing 500 µL of digestion medium. After incubating for 90 min at 37 °C, VSMCs were dispersed with a P1000 pipette tip and plated onto a 60-mm culture dish containing 5 mL DMEM supplemented with 10% heat-inactivated foetal bovine serum (FBS, CSL), 2 mmol/L L-glutamine (CSL), 50 U/ml penicillin and 50 µg/ml streptomycin (CSL). The cells were maintained at 37 °C in a 5% CO2 humidified incubator and passaged weekly in a 1:4 ratio. Cultures were confirmed to consist primarily (>95%) of VSMCs both morphologically, by their classical ‘hill and valley’ appearance, and immunohistochemically, by {alpha}-actin immunoreactivity. Cells between passages 4 and 20 were used for experiments.

2.3. Reactive oxygen species detection
Superoxide production in mouse VSMCs was assessed by lucigenin-enhanced chemiluminescence. To examine the effects of pharmacological agents on superoxide production, VSMCs were plated onto 96-well ViewPlates (Packard, USA) and allowed to grow to confluence. Twenty-four hours prior to assaying for superoxide, the regular cell culture media were exchanged for DMEM containing a reduced FBS concentration (5%) along with L-glutamine and antibiotics. VSMCs were incubated in 5% FBS rather than in serum-free media because in preliminary experiments, we found that overnight serum-deprivation caused a dramatic reduction in cell viability. Therefore, in pilot experiments, we examined the effects of a range of FBS concentrations (0.5–20%) on superoxide production and Nox4 expression and found no effect on either measure up to concentrations of at least 5% serum. In some experiments, the 5% serum-containing media was further supplemented with apocynin (10–1000 µmol/L), IL-1β (10 ng/mL), thrombin (2 U/mL), PDGF (20 ng/mL) or vehicle [dimethyl sulphoxide (DMSO; Merck, Australia) 0.1%]. The following day, the cell culture media was exchanged for a Krebs–HEPES preincubation solution containing diethyldithiocarbamic acid (DETCA; 3 mmol/L, inhibitor of Cu2+/Zn2+-superoxide dismutase, SOD) and one or more of the following compounds: NADPH (3–3000 µmol/L); NADH (100 µmol/L); apocynin (10–1000 µmol/L); diphenyleneiodonium (DPI; 0.03–1000 nmol/L); N{omega}-nitro-L-arginine (L-NAME; 100 µmol/L); indomethacin (3 µmol/L); 17-octadecynoic acid (17-ODYA; 3 µmol/L); allopurinol (100 µmol/L); rotenone (1 µmol/L); SOD (600 U/mL); tiron (10 mmol/L); M40403 (10 µmol/L; Metaphore); thrombin (2 U/mL); IL-1β (10 ng/mL); PDGF (20 ng/mL). After 45 min preincubation at 37 °C, the preincubation solutions were replaced with 200 µL of a Krebs–HEPES assay solution containing lucigenin (5 µmol/L) and the appropriate drug treatment(s). Average photon emission per second per well was monitored over a 20-min period in a TopCount (Packard). In a subset of experiments, VSMCs were grown to confluence in 60-mm culture dishes, incubated in 5% FBS-containing media overnight and homogenised by incubation for 10 min in 200 µL of ice cold lysis buffer followed by sonication for 20 s. The lysate was centrifuged at 14,000 rpm for 10 min, and aliquots (20 µL) of the supernatant were added to the wells of a white 96-well Optiplate (Packard) containing 180 µL of Krebs–HEPES solution supplemented with lucigenin (5 µM) and, in some cases, either NADPH (100 µmol/L) or NADH (100 µmol/L). Average photon emission per second per well was monitored over a 20-min period and normalised to the amount of protein (assessed using a Protein Assay Kit, Bio-Rad, USA).

As an alternative measurement of cellular ROS production, the cell permeable dye, 2',7'-dichlorofluorescin-diacetate (DCF-DA; Molecular Probes, USA), was used. Upon entering cells, 2',7'-dichlorofluorescin-diacetate is deesterified to the nonfluorescent product, 2',7'-dichlorofluorescin. 2',7'-dichlorofluorescin is then reduced to the fluorescent compound, 2',7'-dichlorofluorescein, by cellular peroxides and peroxynitrite, providing a qualitative measure of intracellular oxidant stress. VSMCs were grown to ~80% confluence on plastic cover slips in 35-mm culture dishes. Prior to assaying, the medium was replaced with Krebs–HEPES containing 10 µmol/L DCF-DA. Some cells were further treated with NADPH (100 µmol/L) either alone or with DPI (100 nmol/L) in the dark for 60 min at 37 °C. Fluorescence was visualised under a fluorescence microscope.

2.4. Cell viability
After measuring superoxide production, VSMCs were washed with Krebs–HEPES and incubated for 3 h in 20% CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, USA) dissolved in Krebs–HEPES. Cell viability was assessed with this tetrazolium [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS]-based assay by measuring the absorbance of the supernatant at 490 nm. Note that none of the interventions used in this study had any significant effect on cell viability.

2.5. RNA extraction
RNA was extracted from cultured and freshly isolated VSMCs, and from whole aortas, using RNAwiz (Ambion, USA) or SV RNA Isolation Kit (Promega). RNA concentrations were determined spectrophotometrically by measuring absorbance at 260 nm.

2.6. Reverse transcription (RT) reaction
RNA (100–500 ng) was reverse-transcribed using TaqMan Reverse Transcription Reagents (Applied Biosystems, USA). As a control for genomic DNA contamination in subsequent real-time PCR, parallel RT reaction mixtures containing all reagents except the Reverse Transcriptase were prepared for all RNA samples.

2.7. Real-time PCR
Real-time PCR and the {Delta}{Delta}Ct method were used to examine mRNA expression of Nox4 and Nox1 relative to a ‘reference’ sample [25]. Primers and a 5'-carboxyfluorescein (FAM-)-labelled fluorescent probe for Nox4 were designed using Primer Express software (Applied Biosystems) and a published sequence for the mouse Nox4 gene (Table 1). For Nox1, SYBR® Green (Applied Biosystems) was used in place of a labelled probe. 18 S ribosomal RNA was used as the internal standard for each reaction and was detected with commercially available 18 S primers and a 5'-VIC-labelled probe (Applied Biosystems; Table 1).


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  		Table 1 Nox4 antisense, mismatch and scrambled oligonucleotide sequences

 
Nox4 was amplified in duplex with 18 S in PCR mixtures (25 µL final volume) containing 1 x TaqMan® Universal PCR master-mix (Applied Biosystems), cDNA template (5 ng) and optimised primer and probe concentrations for 18 S and Nox4 (Table 1). The PCR mixture for Nox1 contained 1 x SYBR® Green master-mix (Applied Biosystems), cDNA (20 ng) and optimised primer concentrations, in a final volume of 25 µL. PCR thermal cycle parameters were 2 min at 50 °C, 10 min at 95 °C and 40 cycles of 95 °C for 30 s and 60 °C for 1 min. Reactions were performed, and fluorescence was monitored in a Prism 7700 Sequence Detector (Applied Biosystems).

2.8. Antisense design and synthesis
Six antisense sequences were designed with Gene Runner Software (Hastings Software, USA) to complement various sites around the translation start codon of the native mouse Nox4 mRNA. Pilot experiments were performed to screen for efficacy of each sequence in which VSMCs were incubated with a range of concentrations of antisense (50–1000 nmol/L) for various times (12–72 h) and the effects on NADPH-dependent superoxide production measured. Of six molecules tested, only one spanning from 13 to 33 nucleotides upstream from the translation start codon (+13/+33) displayed efficacy, causing maximum inhibition of NADPH-dependent superoxide production at a concentration of 500 nmol/L and after 24-h incubation. To exclude potential nonspecific effects of the +13/+33 sequence, scrambled and mismatch control oligonucleotides were also designed. The scrambled sequence contained the same base composition as the antisense but in a random order, while the mismatch sequence differed from the antisense in three base positions (Table 2). All phosphorothioate oligonucleotides were commercially synthesised, and polyacrylamide gel purified (Sigma Genosys, Australia).


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  		Table 2 Primer and probe sequences and concentrations for real-time PCR

 
2.9. Antisense transfection
Mouse VSMCs were plated sparsely onto 96-well ViewPlates (for superoxide measurements) or 35-mm culture dishes (for RNA extraction) such that they were 30–50% confluent at the time of transfection 24 h later. At the time of transfection, cells were washed with serum- and antibiotic-free DMEM and were incubated in serum- and antibiotic-free DMEM containing 8 µL/mL Oligofectamine (Invitrogen, USA) complexed with antisense (0–1000 nmol/L), mismatch (500 nmol/L) or scrambled (500 nmol/L) oligonucleotides. After 4 h incubation at 37 °C, an equal volume of DMEM containing 10% FBS was added, and the cells were incubated at 37 °C for 24 h before RNA extraction or being assayed for superoxide production. To confirm that the above protocol resulted in significant cellular uptake of oligonucleotides, pilot studies were conducted in which VSMCs were incubated for 24 h with FITC-labelled antisense (500 nM) and oligofectamine (8 µl/mL). Visualisation of the VSMCs under an inverted fluorescent microscope (490 nm excitation; 520 nm emission) revealed that >90% of cells took up the antisense (data not shown). Importantly, fluorescence was concentrated within nucleus of the VSMCs, which is the major site of action of antisense via activation of ribonuclease H.

2.10. Statistical analysis
Results are expressed as mean ± standard error of the mean (S.E.M.) of n experiments. Superoxide production is expressed as counts per second per well, normalised to relative cell number and as a percentage of untreated or vehicle-treated control. Multiple comparisons were made by Tukey all-pairwise or Bonferroni t-tests after one-way repeated measures ANOVA. Differences were considered significant at P<0.05. pEC50 and pIC50 values for NADPH and DPI, respectively, were calculated after concentration-dependent superoxide generation and inhibition curves were computer-fitted with a sigmoidal regression (Graphpad Prism, USA).

2.11. Source of reagents
Unless otherwise stated in the manuscript, all reagents were purchased from Sigma-Aldrich (USA).


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. NADPH oxidase activity in mouse VSMCs
Superoxide generation was barely detectable in the absence of substrate in intact, cultured VSMCs. Incubation with NADPH caused a concentration-dependent increase in superoxide production (pEC50, 4.8 ± 0.4; Fig. 1A). Incubation with NADH (100 µmol/L) also appeared to increase superoxide production but much less than NADPH, and this was not significantly above control (Fig. 1B). To ensure that the above findings were not confounded by differences in uptake of NADPH and NADH into intact cells, we also compared the effects of the two pyridine nucleotides in VSMCs after homogenisation. Again, superoxide production was barely detectable above background in the absence of exogenously added substrate (6 ± 3 counts/s/mg; n=4) but was markedly increased in the presence of NADPH (100 µmol/L; 266 ± 33 counts/s/mg protein; P<0.001; n=4). Superoxide production was also elevated above basal levels by NADH (100 µmol/L; 82 ± 11 counts/s/mg protein; P<0.05; n=4), but again this response was significantly smaller (i.e., by 69%; P<0.001) than for NADPH.


Figure 1
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  		Fig. 1 Superoxide production in mouse VSMCs in response to different substrates. Superoxide production was measured via lucigenin-enhanced chemiluminescence in VSMCs after 45 min of incubation with (A) increasing concentrations of NADPH or (B) single concentrations of NADPH (100 µmol/L) and NADH (100 µmol/L). Values (mean ± S.E.M. from 4–8 experiments) are expressed as counts per second per relative cell number. ***P<0.001 vs. control.

 
In intact cells, NADPH-dependent superoxide production was inhibited in a concentration-dependent manner by the flavin antagonist and reputed NADPH oxidase inhibitor, diphenyleneiodonium (pIC50, 9.2 ± 0.2; Fig. 2A). Incubation with a structurally unrelated inhibitor of NADPH oxidase, apocynin, had no effect on NADPH-dependent superoxide production after 45 min, but it suppressed this response by ~50% after 24h (Fig. 2B). In contrast to the effect of DPI and apocynin, inhibitors of nitric oxide synthase (NOS, L-NAME), cyclooxygenase (indomethacin), xanthine oxidase (allopurinol), cytochrome P450 (17-ODYA) or mitochondrial respiration (rotenone) had no effect on NADPH-dependent superoxide production (Fig. 2C).


Figure 2
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  		Fig. 2 Effect of oxidase inhibitors and superoxide scavengers on NADPH-dependent superoxide production in mouse VSMCs. NADPH (100 µmol/L)-dependent superoxide production was measured via lucigenin-enhanced chemiluminescence in VSMCs after treatment with (A) increasing concentrations of DPI for 45 min, (B) increasing concentrations of apocynin for 24 h, (C) single concentrations of L-NAME, indomethacin, 17-ODYA, allopurinol or rotenone for 45 min or (D) single concentrations of SOD, tiron or M40403 for 45 min. Values (mean ± S.E.M. from 4–8 experiments) are expressed as a percentage of the counts per second per viable cell obtained in cells treated with NADPH alone. *P<0.05, ***P<0.001 vs. vehicle or control.

 
To ensure that the chemiluminescence signal obtained in the presence of NADPH was attributable to superoxide, we examined the effects of selective superoxide scavengers. NADPH-dependent chemiluminescence was inhibited by ~50% by superoxide dismutase (SOD) and virtually abolished by the cell permeable superoxide dismutase mimetics, tiron and M40403 (Fig. 2D). Confirming our lucigenin findings, VSMCs treated with DCF-DA displayed a low level of intracellular fluorescence (Fig. 3A), which was markedly increased after preincubation with NADPH (100 µM; Fig. 3B) and almost abolished by DPI (Fig. 3C). Collectively, these data provide strong evidence for the presence of a superoxide-producing NADPH oxidase in mouse VSMCs.


Figure 3
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  		Fig. 3 Fluorescent images of VSMCs incubated for 1 h with the fluorescent probe DCF-DA, either (A) alone or in the presence of (B) NADPH or (C) NADPH plus DPI.

 
3.2. Nox4 expression in mouse VSMCs
Nox4 mRNA appeared to be highly expressed in RNA extracts from cultured VSMCs ({Delta}Ct=12.3 ± 0.2 relative to 18 S; Fig. 4A). Importantly, this level of Nox4 expression relative to 18 S was similar to that observed in both whole aortas ({Delta}Ct=12.2 ± 0.5) and in VSMCs ({Delta}Ct=12.4 ± 0.3) freshly isolated from healthy 13-week-old mice (Fig. 4B). In contrast to Nox4, Nox1 was expressed at low levels in cultured VSMCs and could only be detected in some samples of freshly isolated VSMCs and whole aortas. Our primers were clearly effective because Nox1 was readily detectable in RNA obtained from mouse colon (data not shown) [26].


Figure 4
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  		Fig. 4 Nox4 mRNA expression in mouse VSMCs. Total RNA, extracted from cultured or freshly isolated mouse VSMCs or whole aortas, was reverse-transcribed and then used in real-time PCR to examine expression of Nox4. In panel (A), upper panel is a representative trace of FAM (Nox4)-dependent fluorescence intensity vs. PCR cycle number measured in a single VSMC culture (reaction performed in triplicate). The lower panel shows VIC (18 S)-dependent fluorescence vs. PCR cycle number in the same reactions. As a control for genomic DNA contamination, real-time PCR was also performed using, as a template, the same RNA sample that had not been reverse-transcribed (–RT). (B) Grouped data showing Nox4 expression relative to a ‘reference’ sample in cultured and freshly dissociated VSMCs and whole aortas. Values are mean ± S.E.M. from 4–8 experiments: there is no statistical difference between these means (P>0.05).

 
3.3. Nox4 antisense suppresses superoxide production
Incubation of VSMCs with a Nox4 specific antisense oligonucleotide, +13/+33, caused a 65% reduction in Nox4 mRNA, as measured by real-time RT-PCR (Fig. 5A). In contrast, neither the mismatch nor scrambled control oligonucleotides had any effect on Nox4 mRNA. Consistent with these findings, antisense also caused a significant 41% reduction in NADPH-dependent superoxide production, whereas the mismatch and scrambled sequences had no effect (Fig. 5B). Finally, Nox4 antisense did not cause a compensatory increase in mRNA expression of Nox1 (data not shown).


Figure 5
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  		Fig. 5 Effect of Nox4 antisense on (A) Nox4 mRNA expression and (B) NADPH-dependent superoxide production in mouse VSMCs. VSMCs were incubated for 24 h with oligofectamine alone (8 µL/mL) or in the presence of 500 nmol/L antisense, mismatch or scrambled oligonucleotides. Cells were then either (A) harvested and their RNA extracted and reverse-transcribed for measurement of Nox4 mRNA expression by real-time PCR, or (B) treated with NADPH (100 µmol/L) for 45 min and superoxide production measured using lucigenin-enhanced chemiluminescence. Values (mean ± S.E.M. from 8 experiments) are counts per second per viable cell number normalised as a percentage of the same values obtained in untreated cells. *P<0.05, ***P<0.001.

 
3.4. Effect of inflammatory mediators on Nox4 mRNA expression and NADPH-dependent superoxide production
The proinflammatory agents IL-1β, thrombin and PDGF each caused time-dependent decreases in Nox4 mRNA expression without altering expression of Nox1 (Fig. 6A and B) or the house keeping gene, 18 S (data not shown). Clearly, IL-1β, thrombin and PDGF did not induce a global suppression of VSMC gene expression. Nox4 mRNA remained suppressed after 6 h, although the effect began to wane after this time point (data not shown). Surprisingly, only IL-1β caused a transient reduction in NADPH-dependent superoxide production, and this occurred rapidly, within 30 min (Fig. 6C).


Figure 6
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  		Fig. 6 Time-dependent effect of proatherogenic stimuli IL-1β, thrombin and PDGF on mRNA expression of (A) Nox4 and (B) Nox1, and (C) NADPH-dependent superoxide production in mouse VSMCs. VSMCs were treated with either IL-1β, thrombin or PDGF for the indicated times. Cells were then either harvested and their RNA extracted and reverse-transcribed for use in real-time PCR experiments to measure mRNA expression of (A) Nox4 and (B) Nox1. Alternatively, cells (C) were treated with NADPH (100 µmol/L) for 45 min prior to measuring superoxide production via lucigenin-enhanced chemiluminescence. In panels (A) and (B), values (mean ± S.E.M. from 4–6 experiments) are expressed relative to 18 S and a ‘reference’ sample. In panel (C) values (mean ± S.E.M. from 5–6 experiments) are expressed as a percentage of the counts per second per viable cell obtained in cells treated with NADPH alone. *P<0.05 treatment vs. control.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
The major finding of this study is that Nox4 is a critical component of the superoxide-generating NADPH oxidase complex in cultured as well as freshly dissociated VSMCs from mice. Nox4 is the predominant FAD-containing subunit in mouse VSMCs and suppression of Nox4 by a specific antisense blocks superoxide production. However, Nox4 expression is down-regulated by proatherogenic stimuli IL-1β, thrombin and PDGF.

To confirm that mouse VSMCs express a functional NADPH oxidase, we examined the effects of known pharmacological modulators of this enzyme on superoxide production. ROS generation was barely detectable in untreated cells, either with lucigenin or DCF-DA, but addition of NADPH, the preferred substrate of NADPH oxidase, caused a marked increase in superoxide production. Superoxide appeared to be produced both intra- and extracellularly since native SOD, which is unable to cross plasma membranes, reduced the response by half, while the cell permeable SOD mimetics, tiron and M40403, essentially abolished superoxide production. NADPH-dependent superoxide production in mouse VSMCs was potently and completely inhibited by DPI, a flavin antagonist commonly used to inhibit NADPH oxidase activity. Apocynin, a structurally unrelated inhibitor of NADPH oxidase that binds to p47phox and prevents its association with the membrane-bound cytochrome b558 domain [27], also attenuated NADPH-dependent superoxide production but only after VSMCs had been exposed to the compound for an extended incubation period. This delay in suppressant action of apocynin suggests that vascular NADPH oxidases, unlike the phagocytic complex, are assembled constitutively to generate ROS continuously for use in cell signalling. Thus, while apocynin is unlikely to bind to p47phox subunits that are already complexed to cytochrome b558, the inhibitory effect of apocynin might be expected to grow as the pool of preassembled NADPH oxidase is slowly replaced during the regular cycle of cellular protein turnover.

The molecular structure of the NADPH oxidase complex responsible for superoxide production in vascular cells differs from that of phagocytes, and this may provide an explanation for the differences we and others have observed in the biochemical characteristics of the two enzymes. Thus, while p22phox and p47phox are critical for enzyme function in both cell types, gp91phox, which is required for NADPH binding and flavin-dependent electron transfer to molecular oxygen in phagocytes, appears to have a minimal role in superoxide production in VSMCs [14,16]. Rather, these cells express two homologues of gp91phox, Nox1 and Nox4, which also contain binding sites for NADPH, FAD and a heme moiety [18,28]. These characteristics make the Nox proteins strong candidates for the catalytic subunit of vascular NADPH oxidases. Indeed, Nox4 was readily detectable in unstimulated cultured VSMCs, but Nox1 mRNA could only be detected in some samples at very high PCR cycle numbers. Importantly, these levels of Nox4 and Nox1 were comparable to those in VSMCs that had been freshly isolated from healthy C57BL6/J mice, indicating that our basal culture conditions did not influence the Nox expression profile. Our results are thus in agreement with previous studies examining Nox expression in freshly isolated rat aortic tissue where the Nox1/Nox4 ratio was found to be extremely low (i.e., <0.5%) [19,29,30]. The same applies in human VSMCs, where Nox4 appears to be the most abundant gp91phox homologue while Nox1 levels are much lower [20].

To test directly whether Nox4 is critical for NADPH oxidase activity by VSMCs under resting conditions, we employed an antisense approach. Transfection of mouse VSMCs with sequence-specific antisense against Nox4 down-regulated mRNA expression of this subunit by ~65% and also attenuated NADPH-dependent superoxide production by ~40%, showing that Nox4 is indeed a major contributor to NADPH-dependent superoxide production in unstimulated VSMCs. While it is likely that the residual superoxide production remaining after treatment with Nox4 antisense was due to incomplete suppression of Nox4 mRNA expression, we cannot rule out the possibility that another flavin-containing (DPI-inhibitable) enzyme(s) contributed to the overall signal. Such an enzyme is unlikely to be NOS, cytochrome P450, xanthine oxidase, cyclooxygenase or a component of the respiratory transport chain, for inhibitors of each of these pathways had no effect on superoxide production.

Our finding that NADPH is the preferred substrate for Nox4 differs from previous reports in other cell types. Our observation is consistent with predictions based on the protein structure of Nox4 that reveal a canonical pyridine nucleotide binding domain (G–X–G–X–X–P–F; where X is any residue) characteristic of enzymes that bind NADPH in preference to NADH [26]. However, HEK293 cells express a Nox4-containing NADPH oxidase and displays a maximum rate of superoxide production in the presence of NADH that was two–threefold higher than for NADPH [23]. Furthermore, Ago et al. characterised a Nox4-containing NADPH oxidase in endothelial cells that appeared to have no preference for NADH or NADPH [24]. These two studies were both performed on disrupted cell preparations, whereas in our intact cells, the endpoint measure of superoxide production may be influenced by a variety of factors including cellular uptake, metabolism, endogenous levels of pyridine nucleotides and the presence of other enzyme systems that compete with NADPH oxidase for each of the substrates. In the present study, we obtained qualitatively similar findings whether we used intact cells or homogenates and can thus exclude cellular uptake as a reason for the differences. The explanation of the difference in substrate preference of Nox4 remains unclear, but it will only be resolved by comparing the effects of NADPH and NADH in a cell-free assay system containing purified recombinant proteins.

In rat-cultured VSMCs, Nox1 has been implicated in the stimulation of superoxide production by mitogenic stimuli. Lassegue et al. showed that treatment with angiotensin II or PDGF caused an increase in superoxide production in these cells that was mirrored by an increase in expression of the Nox1 subunit [18]. Conversely, Nox4 expression was down-regulated by these treatments. These authors also showed that adenoviral transfection of rat VSMCs with a full-length antisense against Nox1 abolished the angiotensin II-induced increase in superoxide production but failed to inhibit basal NADPH oxidase activity. These observations therefore support the concept that Nox1 is critical for stimulated, as opposed to basal, NADPH oxidase activity, at least in rat-cultured VSMCs. A surprising finding from the present study was that IL-1β, thrombin and PDGF each failed to influence Nox1 expression. However, all of these stimuli reduced Nox4 mRNA expression. The minimal effects on NADPH-dependent superoxide production could suggest that activity of another oxidase(s) is up-regulated to compensate for reduced Nox4 expression. Alternatively, the activity of expressed Nox1 or Nox4 might be increased following up-regulation of other subunits supporting Nox-dependent superoxide production [31], thus compensating for the down-regulation of Nox4 in the presence of proatherogenic mediators.

While the role of Nox4-derived ROS in mouse VSMC physiology remains to be determined, overexpression of Nox4 cDNA in NIH3T3 cells retarded their growth, suggesting that ROS, derived from Nox4, may be important for maintaining this fibroblastic cell line in a quiescent state [23]. In contrast, antisense against Nox4 inhibited the proliferation of melanoma cells [32]. In the present study, we did not detect any significant effect of Nox4 antisense on VSMC proliferation even up to 72 h after transfection (unpublished observations). However, these findings should be viewed with caution since the effect of the antisense appeared to wane after 24 h (presumably due to instability of oligonucleotides inside the cells). Nevertheless, in preliminary experiments, we did note that longer-term incubations (≥ 24 h) with both PDGF and thrombin, which down-regulated Nox4 mRNA, increased cell proliferation (data not shown), consistent with an antimitotic action of Nox4 in VSMCs.

In conclusion, we have demonstrated that Nox4 is an important component of the NADPH oxidase complex expressed under basal conditions in mouse VSMCs. The fact that Nox4 expression is diminished in the presence of mitogenic stimuli raises the possibility that ROS, derived from this subunit, play important signalling roles in quiescent rather than dividing VSMCs. Whether Nox4 is regulated in a similar manner in other vascular cell types (e.g., endothelial and fibroblastic) remains to be determined.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This work was supported by an Institute Block Grant from the National Health and Medical Research Council of Australia (NHMRC; 983001) and by a Grant-In-Aid from the National Heart Foundation of Australia (G00 M 0612). Ellmark was supported by the Swedish Foundation for Strategic Research via the National Network for Cardiovascular Research, and Drummond was supported by a NHMRC Peter Doherty Postdoctoral Fellowship (007044). We are grateful to Dr. Daniella Salvemini (Metaphore Pharmaceuticals) for providing us with M40403.


   Notes
 
1 Current address: Department of Pharmacology, Monash University, Victoria 3800, Australia. Tel.: +61 3 9905 4869; fax: +61 3 9905 5851. Back

Time for primary review 21 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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