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Differential regulation of Nox1, Nox2 and Nox4 in vascular smooth muscle cells from WKY and SHR
Journal of the American Society of Hypertension 5(3) (2011) 137–153
Research Article
Differential regulation of Nox1, Nox2 and Nox4 in vascular smooth muscle cells from WKY and SHR Ana M. Briones, PhDa,b, Fatiha Tabet, PhDa,c, Glaucia E. Callera, PhDa, Augusto C. Montezano, PhDa, Alvaro Yogi, PhDa, Ying He, MSc, MDa, Mark T. Quinn, PhDd, Mercedes Salaices, PhDb, and Rhian M. Touyz, MD, PhDa,* b
a Kidney Research Centre, Ottawa Health Research Institute, University of Ottawa, Ontario, Canada; Department Farmacologıa y Terapeutica, Facultad de Medicina, Universidad Aut onoma de Madrid, Spain; c Heart Research Institute, Sydney, New South Wales, Australia; and d Department of Immunology and Infectious Diseases, Montana State University, Bozeman, USA Manuscript received December 13, 2010 and accepted February 3, 2011
Abstract The functional significance and regulation of NAD(P)H oxidase (Nox) isoforms by angiotensin II (Ang II) and endothelin-1 (ET-1) in vascular smooth muscle cells (VSMCs) from normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) was studied. Expression of Nox1, Nox2, and Nox4 (gene and protein) and NAD(P)H oxidase activity were increased in SHR. Basal NAD(P)H oxidase activity was blocked by GKT136901 (Nox1/4 inhibitor) and by Nox1 siRNA in WKY cells and by siNOX1 and siNOX2 in SHR. Whereas Ang II increased expression of all Noxes in WKY, only Nox1 was influenced in SHR. Ang II–induced NAD(P)H activity was inhibited by siNOX1 in WKY and by siNOX1 and siNOX2 in SHR. ET-1 upregulated Nox expression only in WKY and increased NAD(P)H oxidase activity, an effect inhibited by siNOX1 and siNOX2. Nox1 co-localized with Nox2 but not with Nox4, implicating association between Nox1 and Nox2 but not between Nox1 and Nox4. These data highlight the complexity of Nox biology in VSMCs, emphasising that more than one Nox member, alone or in association, may be involved in NAD(P)H oxidase-mediated O 2 production. Nox1 regulation by Ang II, but not by ET-1, may be important in O 2 formation in VSMCs from SHR. J Am Soc Hypertens 2011;5(3):137–153. Ó 2011 American Society of Hypertension. All rights reserved. Keywords: Redox signaling; gp91phox; Nox1; Nox4; NAD(P)H oxidase; vasculature.
Introduction In the vascular system, reactive oxygen species (ROS) play a physiological role in controlling endothelial function and vascular tone and a pathophysiological role in inflammation,
Funding/support: This study was funded by Grant 44018 from the Canadian Institute of Health Research (CIHR) and by MEC (SAF 2009-07201). A.M.B. was supported through a Beca de Posgrado from Fundacion Caja Madrid. F.T. received a Canada Graduate Scholarship Doctoral Award from the CIHR. Conflict of interest: none reported. *Corresponding author: Rhian M. Touyz, MD, PhD, Kidney Research Institute, Ottawa Health Research Institute, University of Ottawa, 304/451 Smyth Road, Ottawa (Ontario) K1H 8M5 Canada. Tel: 613-562–5800 ext 8241; fax: 613-562-5487. E-mail: [email protected]
hypertrophy, proliferation, apoptosis, migration, fibrosis, angiogenesis, and rarefaction, important in vascular remodeling and endothelial dysfunction associated with hypertension.13 Among the major sources of ROS in the vascular wall is nonphagocytic NAD(P)H oxidase (Nox),4,5 a multi-subunit enzyme that differs structurally and biochemically from phagocytic gp91phox (also termed Nox2)containing NAD(P)H oxidase, important for host defence.6 NAD(P)H oxidases are a family of enzymes with each member being distinguished by the specific Nox catalytic subunit. To date, 7 Nox isoforms have been identified in mammals (Nox1 to Nox7),7 of which many are present in the vascular wall.8,9 Vascular smooth muscle cells (VSMCs) express Nox1, Nox2, Nox4, and Nox5.1013 Nox2 is primarily membrane-associated, Nox1 co-localizes with cholesterolrich domains of the plasma membrane, and Nox4 is found in focal adhesions14,15 and the endoplasmic reticulum.1416
1933-1711/$ - see front matter Ó 2011 American Society of Hypertension. All rights reserved. doi:10.1016/j.jash.2011.02.001
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Nox5 (not found in rodents) has a perinuclear-endoplasmic reticular distribution.17 The functional significance of Nox isoforms in VSMCs is unclear, but considering their localization within specific subcellular compartments, ROS derived from different Nox isoforms may play distinct roles in modulating intracellular ROS-mediated signaling. For example, Nox4 appears to be important in maintaining 18,19 and hydrogen peroxide basal superoxide (O 2) 20 (H2O2) production, whereas Nox1 might be responsible for O 2 production and redox signaling after stimulation and in pathological conditions, such as atherosclerosis, diabetes, and hypertension.21 Increased expression and/or activity of vascular NAD(P)H oxidase has been suggested as a mechanism underlying 1,8,9,22 The role enhanced O 2 production in hypertension. of different Nox isoforms in this phenomenon is unclear. Li et al23 demonstrated greater mRNA expression of Nox1, Nox2, and Nox4 in aorta from spontaneously hypertensive rats (SHR) versus normotensive Wistar-Kyoto (WKY) rats; however, similar levels of Nox1 and Nox2 mRNA were found in aortas from WKY and stroke-prone SHR.24 In cerebral arteries from SHR, increased expression levels of Nox4 but not of Nox1 and Nox2 mRNA were described.25 Whereas Nox1 and Nox2 appear to be important in acute models of angiotensin (Ang) II–dependent hypertension and vascular remodeling,2628 they do not seem to play a major role in chronic Ang II–dependent hypertension.29,30 Many factors have been implicated in the regulation of NAD(P)H oxidase activity in vascular disease, including physical factors (stretch, flow, and pressure), vasoactive agents (Ang II, endothelin-1 [ET-1], and aldosterone), and growth factors (epidermal growth factor, insulin like growth factor, platelet-derived growth factor [PDGF]).1,8,9,31,32 However, it remains to be determined which Nox enzymes are involved and how these are differentially regulated in hypertension. Ang II seems to be particularly important. Lassegue et al33 reported that Ang II increases Nox1 and downregulates Nox4 mRNA in cultured rat aortic VSMCs, whereas Wingler et al34 demonstrated an upregulation of Nox1 and Nox4 mRNA levels by Ang II in A7r5m cells (cell line derived from rat aortic SMC). However, Hitomi et al35 did not find Ang II–dependent alterations of Nox1 expression in VSMCs. PDGF, basic fibroblast growth factor, prostaglandin F2a, and advanced glycation end products also increase Nox1 expression in VSMCs.8,9 Similar to Ang II, ET-1 stimulates ROS production in vascular cells. However, there is a paucity of information on the role of ET-1 in the regulation of vascular Nox isoforms. In rat aortic SMC, ET-1 had no effect on Nox1 or Nox4 expression34 but increased mRNA expression of Nox2 in human umbilical vein endothelial cells.36 In human aortic endothelial cells, ET-1 inhibited Nox1based NAD(P)H oxidase via ETB receptors.37 Whether ET-1 modulates vascular Nox isoforms in SHR is unclear.
To our knowledge, there are no studies that systematically assess effects of Ang II and ET-1 on the regulation and function of Nox isoforms in vascular cells from hypertensive animals. In the present study, we sought to determine whether expression of Nox family members in VSMC differs between WKY and SHR and whether Ang II and ET-1, potent vasoactive peptides important in the pathogenesis of hypertension, differentially regulate Nox expression and activity.
Methods Cell Culture The study was approved by the Animal Ethics Committee of the University of Ottawa and performed according to the recommendations of the Canadian Council for Animal Care. VSMC from mesenteric resistance arteries were obtained from 16-week-old WKY and SHR (Taconic Farms, Germantown, NY). Cells were isolated by enzymatic digestion and cultured and characterized as previously described.38 Briefly, arteries were cleaned of adipose and connective tissue, and VSMCs were dissociated by digestion of vascular arcades with enzymatic solution (collagenase, elastase, soybean trypsin inhibitor, and bovine serum albumin [BSA] type I; 60 minutes, 37 C). The tissue was filtered and the cell suspension centrifuged and resuspended in Dulbecco Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), 2 mmol/L glutamine, 20 mmol/L HEPES (pH 7.4), and antibiotics. Cells were used between passages 3 and 7. At subconfluence, the culture medium was replaced with serum-free medium for 24 hours to render the cells quiescent. Cells were incubated with vehicle or with Ang II (0.1 mmol/L) or ET-1 (0.1 mmol/L) for 2 to 24 hours. In some experiments, Ang II– and ET-1– (2–4 hours) stimulated cells were incubated with the Nox1/4 inhibitor GKT136901 (10 mmol/L; gift from Genkyotex, Geneva, Switzerland) with the specific Rac inhibitor EHT1864 (1 mmol/L) or with the antioxidants tiron (10 mmol/L) and tempol (1 mmol/L; 30 minutes pre-incubation).
Real-Time PCR Cellular mRNA levels of Nox1, Nox2, and Nox4 were studied by real-time polymerase chain reaction (PCR). Total RNA was isolated from WKY and SHR VSMCs using Trizol Reagent (GIBCO-BRL, Invitrogen, Burlington, ON, Canada). Reverse transcription was performed using 2 mg of RNA as previously described.39 Real-time PCR was performed with a Stratagene Mx4000 System (La Jolla, California, USA) for relative quantification of VSMC Nox1, Nox4, and Nox2 mRNA. Sense and antisense primers were designed to generate short amplification products (217 base pairs [bp] for Nox1 subunit, 110 bp for Nox4
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Figure 1. Basal mRNA and protein levels of Nox1 (A), Nox2 (B), and Nox4 (C) in WKY and SHR vascular smooth muscle cells. Bar graphs indicate relative quantification of Nox1, Nox2, and Nox4 mRNA or protein content in VSMCs from WKY and SHR. Results are expressed as the ratio between the gene or protein of interest and the gene of ribosomal protein S16 and b-actin respectively, and then normalized to the WKY group, taken as 100%. Results are presented as mean SEM of 4 to 8 experiments. *P < .05 and **P < .01 versus WKY.
subunit, 203 bp for Nox2, and 107 bp for ribosomal protein S16, used as an internal standard). Primers were as follows. For VSMC Nox1: sense 50 -CTTCCTCACTGGCTGG GATA-30 and antisense 50 -AGCCTGCGCAAATGCTGTC30 ; for VSMC Nox4 subunit: sense 50 -CAGTAATCAAT CATCCCTCAGA-30 and antisense 50 -TGTCCCAGTGTAT CAGCATTAG-30 ; for VSMC Nox2 subunit: sense 50 GTATTGTGGGAGACTGGACTGAG-30 and antisense 50 TGCCTCCATTCTCAAGTCTGTCT-30 ; and for S16: sense 50 -AGGAGCGATTTGCTGGTGTGG-30 and anti-sense 50 GCTACCAGGGCCTTTGAGATG-30 . To validate our realtime PCR protocol, gene-specific standard curves for Nox1, Nox4, Nox2, and S16 were generated from serial dilutions of the cDNA. Real-time PCR was conducted using the
Quantitect SYBR Green PCR Kit (Qiagen, Mississauga, Ontario, Canada) and a final 0.5 mmol/L concentration of primers. Data were calculated as the ratio between the gene of interest and S16 relative quantities. Levels of Nox1, Nox2, and Nox4 in SHR were calculated as the amount relative to that in the WKY group, taken as 100%.
Western Blot Analysis Protein levels for Nox1, Nox2, and Nox4 from WKY and SHR VSMCs were assessed by Western blotting. Quiescent VSMCs from WKY were stimulated with Ang II or ET-1 for 2 to 24 hours, and proteins were lysed and extracted for Western blotting, as previously described.40 Briefly, equal
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(Nox4 control; Santa Cruz Biotechnology). The immunocomplexes were captured by adding 30 mL of protein A agarose beads (Santa Cruz Biotechnology) and the mixture was incubated for 2 hours at 4 C. The agarose beads were collected by brief centrifugation (5 seconds, 14,000g) and the supernatant aspirated. The beads were washed 3 times with ice-cold cell lysis buffer. The proteins were eluted from the beads by the addition of 50 mL of 2X Laemmli sample buffer and boiling for 5 minutes. The supernatant was collected by brief centrifugation and run in an SDSpolyacrylamide gel. Western blot analysis for Nox1, Nox2, and Nox4 was performed as described previously. Figure 2. Basal NAD(P)H oxidase activity in vascular smooth muscle cells from WKY and SHR. The lucigenin-enhanced chemiluminescence assay was used to determine NAD(P)H oxidase–dependent superoxide production activity in total protein cell homogenates. Bar graphs represent mean SEM of 5 experiments. **P < .01 versus WKY.
amounts of proteins were separated by electrophoresis on a 10% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane (Boehinger, Mannheim, Laval, Quebec, Canada). Nonspecific binding sites were blocked with a 5% wt/vol nonfat dry milk blocking buffer containing Trisbuffered saline and 0.1% Tween-20 (TBS-T; 1 hour, room temperature). Membranes were incubated overnight at 4 C with goat anti-Nox1 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Nox4 (1:500 Santa Cruz Biotechnology), or mouse anti-Nox2 (1:500).41 Membranes were incubated with horseradish peroxidase–conjugated secondary antibodies for 1 hour at room temperature. After incubation with secondary antibodies, signals were detected by chemiluminescence and visualized by autoradiography. Blots were analyzed densitometrically using the imageQuant software (Molecular Dynamics, Sunnyvale, CA). Samples from vehicle-stimulated VSMCs were used as controls and variations of Nox1, Nox2, and Nox4 from Ang II– or ET-1–treated VSMCs were calculated as the amount relative to controls.
Co-immunoprecipitation Studies Immunoprecipitation analysis was performed in WKY VSMCs. Briefly, cells were lysed with an immunoprecipitation buffer containing 10 mmol/L HEPES (pH 7.6), 15 mmol/L KCl, 2 mmol/L MgCl2, 0.1% NP40, 1 mmol/L phenylmethyl sulfonyl fluoride (PMSF), 1 mg/mL aprotinin, 1 mg/mL leupeptin, and 1 mg/mL pepstatin A. The protein supernatant (500 mg) was incubated with 2 mg of Nox1, Nox2, or Nox4 antibodies overnight at 4 C. In the case of the control immunoprecipitates, the cell lysate was incubated with 2 mg of normal mouse immunoglobulin G (IgG) (Nox2 control), goat IgG (Nox1 control), and rabbit IgG
Immunofluorescence Studies Cells were plated on round glass coverslips and fixed with 4% phosphate-buffered paraformaldehyde (in 0.2 mol/L phosphate buffer, pH 7.2–7.4) for 15 minutes. Cells were then washed 3 times with PBS solution (pH, 7.5). Nonspecific binding sites were blocked with 1% BSA in PBS for 30 minutes. Cells were incubated with primary antibodies against Nox1 (1:50) or Nox2 (1:50) in PBS containing 1% BSA for 1 hour at 37 C. Cells were washed and incubated with either anti-goat IgG conjugated to Cy3 for Nox1 (Jackson Immuno Research Laboratories, West Grove, PA, USA) (1:300) or anti-mouse IgG labeled with Alexa 488 (1:1000) for Nox2, for a further 1 hour at 37 C (Jackson Immuno Research Laboratories). Cells were washed and incubated with the nuclear dye, Hoechst 33342 (0.01 mg/mL). The immunofluorescent signals were viewed with a confocal microscope (Leica TCS 4D, Leica, Barcelona, Spain) with a 63 oil objective. The specificity of the immunostaining was evaluated by omission of the primary antibody and processed as described previously. Under these conditions, no specific fluorescence staining was observed.
Measurement of NAD(P)H Oxidase Activity The lucigenin-enhanced chemiluminescence assay was used to determine the NAD(P)H oxidase activity in total protein cell homogenates. Cells were washed in ice-cold PBS and scraped off in lysis buffer containing 20 mmol/L KH2PO4, 1 mmol/L ethyleneglycoltetraacetic acid (EGTA), and protease inhibitors, pH 7.4. Lysates were transferred to Eppendorf tubes and sonicated for 3 seconds. The reaction was started by the addition of NAD(P)H (0.1 mmol/L) to the suspension (250 mL final volume) containing sample (50 mL), lucigenin (5 mmol/L), and assay phosphate buffer (50 mmol/L KH2PO4, 1 mmol/L EGTA, 150 mmol/l sucrose, pH 7.4). The luminescence was measured every 1.8 seconds for 3 minutes in a luminometer (Lumistar Galaxy; BMG Labtechnologies, Offenburg, Germany). Buffer blank was subtracted from each reading. Activity is expressed as arbitrary units/mg protein. Samples from vehicle-stimulated VSMCs were used as controls and variations of NAD(P)H
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Figure 3. Time course effect of angiotensin II (0.1 mmol/L) stimulation on Nox1 (A), Nox2 (B) and Nox4 (C) expression in vascular smooth muscle cells from WKY and SHR. Bar graphs indicate relative quantification of Nox1, Nox2 and Nox4 protein content expressed as the ratio between the protein of interest and b-actin and normalized to the vehicle (V) group, taken as 100%. Representative blots are displayed above. Data are presented as means SEM of 5 or more experiments. *P < .05 vs. control (C). A, Angiotensin II.
oxidase activity from Ang II– or ET-1–treated VSMCs were calculated as the amount relative to controls. In some experiments, samples from WKY VSMCs were used as controls and variations of NAD(P)H oxidase activity from SHR VSMCs were calculated as the amount relative to controls.
Fluorescence Detection of ROS Generation in Intact Cells Dihydroethidium Generation of intracellular O 2 in living cells was measured with dihydroethidium (DHE; excitation at 546
nm and emission at 610 nm, Molecular Probes, Invitrogen, Carlsbad, CA, USA). Growth-arrested VSMCs from WKY and SHR rats were incubated in Hank’s balanced salt solution (HBSS) containing 1.3 mmol/L CaCl2 and 5.5 mmol/L glucose supplemented with 2 mmol/L DHE in a lightprotected chamber at 37 C for 20 minutes. Cells were rinsed in HBSS and images were obtained before and after exposition to Ang II or ET-1. Cells were imaged on a wide-field epifluorescence microscope equipped with 40 oil immersion lens using the Stallion live-cell Digital Hi-Speed Multi-Channel Imaging System (Zeiss, Berlin, Germany).
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Figure 4. Time course effect of endothelin-1 (0.1 mmol/L) stimulation on Nox1 (A), Nox2 (B) and Nox4 (C) expression in vascular smooth muscle cells from WKY and SHR. Bar graphs indicate relative quantification of Nox protein content expressed as the ratio between the protein of interest and b-actin and normalized to the vehicle (V) group, taken as 100%. Representative blots are displayed above. Data are presented as means SEM of 5 or more experiments. *P < .05 and **P < 0.01 vs. control (C). ET, Endothelin-1.
RNA Interference and Cell Transfection WKY and SHR VSMCs were subjected to Nox1 and Nox2 gene knockdown with small interfering RNA (siRNA) before 0.1 mmol/L Ang II or ET-1 stimulation. High-performance purity-grade (>90% pure) siRNA was generated against rat Nox1 and Nox2 (Santa Cruz Biotechnology, Inc). Rat VSMCs were exposed to transfectant reagent alone or transfected with siRNA. A nonsilencing (NS) siRNA oligonucleotide sequence that does not recognize any known homology to mammalian genes was also generated as a negative control. Cells were seeded at
a density of 2.5 105 cells/well in 100-mm plates with DMEM containing 10% FBS and transfected with 20 nmol/L siRNA using HiPerFect Transfection Reagent (Qiagen Inc.) according to the manufacturer’s instructions. Optimum gene silencing was observed at 24 hours after transfection.
Statistical Analysis Data are presented as mean SEM. Groups were compared using 1-way analysis of variance (ANOVA) or
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Figure 5. (A) Representative fluorescent photomicrographs of microscopic sections of VSMCs from WKY and SHR stimulated with angiotensin II (0.1 mmol/L; 4 hours). Cells were stained with the fluorescent dye dihydroethidium. Images were captured with a wide-field epifluorescence microscope equipped with 40 oil immersion lens using the Stallion live-cell Digital Hi-Speed MultiChannel Imaging System. Magnification 640. (B) NAD(P)H oxidase activity in WKY and SHR cells stimulated with angiotensin II for 2 and 4 hours. (C) Representative fluorescent photomicrographs of microscopic sections of VSMCs from WKY stimulated with endothelin-1 (0.1 mmol/L; 4 hours). Cells were stained with the fluorescent dye dihydroethidium. Images were captured with a wide-field epifluorescence microscope equipped with 40 oil immersion lens using the Stallion live-cell Digital Hi-Speed MultiChannel Imaging System. Magnification 640. (D) NAD(P)H oxidase activity in WKY and SHR cells stimulated with endothelin-1 for 2 and 4 hours. The lucigenin-enhanced chemiluminescence assay was used to determine NAD(P)H oxidase–dependent superoxide production activity in total protein cell homogenates. Bar graphs represent mean SEM of 6 or more experiments. *P < .05 versus WKY. Ang II, angiotensin II; ET, endothelin-1.
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Figure 6. NAD(P)H oxidase activity in WKY and SHR vascular smooth muscle cells stimulated with angiotensin II (0.1 mmol/L) (A) and endothelin-1 (0.1 mmol/L) (B) for 2 and 10 min in the absence or in the presence of GKT136901 (10 mmol/L). The lucigenin-enhanced chemiluminescence assay was used to determine NAD(P)H oxidase-dependent superoxide production activity in total protein cell homogenates. Bar graphs represent mean SEM of 6 or more experiments. *P < .05 vs. WKY. Ang II, angiotensin II; ET, endothelin-1.
Student t test as appropriate. Newman-Keuls posttest was used to compensate for multiple testing procedures. A value of P less than .05 was significant.
Results mRNA and Protein Expression of Nox1, Nox2, and Nox4 and Basal NAD(P)H Oxidase Activity in WKY and SHR VSMC We compared the mRNA and protein expression of Nox2 and its homologues Nox1 and Nox4 in basal conditions in VSMC from WKY and SHR. As shown in Figure 1, higher mRNA and protein expression of Nox1, Nox2, and Nox4 was observed in SHR compared with WKY cells. Basal NAD(P)H oxidase activity was two fold greater in VSMCs from SHR compared with WKY (Figure 2).
Effects of Ang II and ET-1 on Protein Expression of Nox1, Nox2 and Nox4 in WKY and SHR VSMC As shown in Figure 3, expression of Nox1, Nox2, and Nox4 was increased by Ang II in a time-dependent manner in WKY VSMCs. In cells from SHR, Ang II increased Nox1 expression (Figure 3A) without affecting Nox2 or
Nox4 expression (Figure 3B, 3C). ET-1 also induced a time-dependent increase in Nox1, Nox2, and Nox4 protein expression in WKY cells (Figure 4). However, ET-1 did not significantly influence Nox1, Nox2, or Nox4 protein content in SHR cells (Figure 4).
Effects of Ang II and ET-1 on NAD(P)H Oxidase Activity in VSMCs Ang II induced a significant increase in O 2 production (Figure 5A) and NAD(P)H oxidase activity and at 2 and 4 hours in WKY and SHR cells (Figure 5B). ET-1 increased O 2 production (Figure 5C) and NAD(P)H oxidase activity in WKY cells, but had no significant effect in SHR cells (Figure 5D). Short-term stimulation with Ang II or ET-1 increased NAD(P)H oxidase activity in WKY cells (Figure 6). However, Ang II but not ET-1 increased NAD(P)H oxidase activity in SHR. Ang II and ET-1 effects were diminished by the NOX1/4 inhibitor GKT136901.
Inhibition of Nox Isoforms Block Activity of NAD(P)H Oxidase in VSMCs To evaluate the importance of different Nox isoforms in NAD(P)H oxidase activity, Nox1 and Nox2 were downregulated using an siRNA approach and Nox4 was inhibited
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Figure 7. Left panels: NAD(P)H oxidase activity in VSMCs from WKY and SHR incubated with GKT136901 (10 mmol/L) or in which Nox1 and Nox2 are downregulated by siRNA. Cells were transfected with Nox1 (siNox1), Nox2 (siNox2), or nonsilencing (NS) siRNAs and were stimulated or not with angiotensin II (0.1 mmol/L; 2 hours) or endothelin-1 (0.1 mmol/L; 4 hours). Vehicle cells were exposed to the transfectant without siRNA. Right panels: Representative blots of Nox1 and Nox2 expression in WKY and SHR vascular smooth muscle cells transfected with Nox1, Nox2, or NS siRNAs. Representative fluorescent photomicrographs of microscopic sections of VSMC from WKY and SHR transfected with Nox1, Nox2, or NS siRNAs and stimulated with angiotensin II or endothelin-1. Cells were stained with the fluorescent dye dihydroethidium. Images were captured with a wide-field epifluorescence microscope equipped with 40 oil immersion lens using the Stallion live-cell Digital Hi-Speed Multi-Channel Imaging System. Magnification 640. Data are represented as mean SEM of 6 or more experiments. *P < .05 versus vehicle (V). Ang II, angiotensin II; ET, endothelin-1. þ P .05 versus Ang II or ET-1.
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using the inhibitor GKT136901. Basal NAD(P)H oxidase activity was decreased by GKT and siNox1 in WKY cells and by siNox1 and siNox2 in SHR (Figure 7). In WKY, siNox1 induced a significant decrease in Ang II–induced NAD(P)H oxidase activity, whereas in SHR both siNox1 and siNox2 decreased Ang II–induced NAD (P)H oxidase activity (Figure 7). In VSMCs from WKY, both siNox1 and siNox2 decreased ET-1 induced activation of NAD(P)H oxidase activity (Figure 7).
Agonist-Stimulated ROS Generation Does Not Influence Nox Expression To determine the role of ROS on Nox expression, cells were exposed to tiron (ROS scavenger) and tempol (superoxide dismutase mimetic). Neither tiron nor tempol affected Ang II– or ET-1–induced expression of Nox1, Nox4 (Figure 8), or Nox2 (data not shown) in WKY. Similarly, these antioxidants did not affect Ang II–induced Nox1 expression in SHR (data not shown).
Rac Inhibition Does Not Influence Ang II– or ET-1–Induced NAD(P)H Oxidase Activity To determine the participation of Rac in the NAD(P)H oxidase activity induced by Ang II and ET-1, cells were preincubated with the selective Rac inhibitor EHT1864. After 2- or 4-hour stimulation, NAD(P)H oxidase activity was not affected by EHT1864 in WKY or SHR (Figure 9).
Analysis of Nox1 and Nox4 Co-immunoprecipitation with Nox2 Considering that VSMCs possess multiple Nox isoforms, we questioned the relationship among Nox1, Nox2, and Nox4 using a co-immunoprecipitation strategy. To study the association between Nox1 and Nox2, proteins were precipitated with Nox2 antibody and blotted with Nox1 antibody. As shown in Figure 10A, Nox2 co-immunoprecipitated with Nox1. In the reverse experiment, proteins were precipitated with Nox1 antibody and blotted with Nox2 antibody and, as shown in Figure 10B, Nox1 co-immunoprecipitated with Nox2. VSMC total protein lysate was used as positive control in these experiments. To study the association between Nox4 and Nox2 in VSMC, proteins were precipitated with Nox2 antibody and blotted with Nox4 antibody. As shown in Figure 10C, Nox2 did not co-immunoprecipitate with Nox4. In the reverse experiment, proteins were precipitated with Nox4 antibody and blotted with Nox2 antibody. As confirmed in this reverse experiment, Nox4 and Nox2 did not co-immunoprecipitate (Figure 10D). VSMC total protein lysate was used as positive control. These co-immunoprecipitation experiments suggest that Nox2 and Nox4 do not physically associate in resting VSMC (Figure 10).
Immunofluorescence of Nox1 and Nox2 Optical sectioning of VSMCs from WKY and SHR with laser scanning confocal microscopy revealed Nox1 and Nox2 labeling, essentially in a punctuate surface distribution along the cell margins, although some intracellular distribution was also detected (Figure 10E). In these studies, we found a close co-localization between Nox1 and Nox2, as shown by the yellow staining observed in the merged image. Interestingly, this co-localization pattern was seen in unstimulated cells, suggesting that the two proteins associate under basal conditions.
Discussion Vascular cells possess multiple Nox isoforms, the functional significance and regulatory mechanisms of which remain unclear, particularly in the setting of hypertension. Here, we provide novel data demonstrating that VSMCs from resistance arteries possess Nox1, Nox2, and Nox4; that Nox1 and Nox2 co-localize in the cell membrane; and that Nox homologues play an important role in NAD (P)H oxidase–driven ROS generation. Moreover, we show that vascular Nox expression and activation of NAD(P)H oxidase are augmented in SHR. Finally, we demonstrate differential regulation of the Nox isoforms by Ang II and ET-1 in WKY and SHR VSMCs. Whereas in WKY cells, all Nox isoforms are sensitive to Ang II and ET-1, in SHR cells, only Nox1 is influenced by Ang II with ET-1 having no effect on any of the Nox isoforms. These findings highlight the complexity of Nox-based NAD(P)H oxidase systems in VSMCs and suggest that increased ROS generation in SHR primarily involves Ang II–sensitive Nox1, with little influence by ET-1 (Figure 11). The expression of the catalytic Nox subunits varies among different cell types.9 Here we show that VSMCs from resistance arteries possess Nox1, Nox2, and Nox4 and that the content of these Nox isoforms is greater in VSMCs from hypertensive than normotensive rats. NAD (P)H oxidase activity was also greater in cells from SHR compared with WKY. Upregulation of Nox isoforms may contribute to increased NAD(P)H oxidase activity in SHR, at least in basal conditions. In fact, siRNA for Nox1 and Nox2 decreased basal NAD(P)H oxidase activity in SHR, suggesting that these isoforms may be responsible for the increased O 2 in SHR. In WKY, probably both Nox1 and Nox4 contribute to basal O 2 production as shown by the inhibitory effect induced by GKT136901 and by siRNA for Nox1. Several investigations have examined the expression of Nox family members in vascular tissues from hypertensive models. However, depending on the vascular bed and the experimental model studied, conflicting results have been found.2325,34,4244 Although the pattern of Nox expression may vary, increased vascular NAD(P)H oxidase–derived ROS is a common feature in
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Figure 8. Effect of the antioxidants tempol (1 mmol/L) and tiron (10 mmol/L) alone or in combination with angiotensin II (0.1 mmol/L) or endothelin-1 (0.1 mmol/L) on the expression of Nox1 and Nox4 in vascular smooth muscle cells from WKY. Bar graphs indicate relative quantification of Nox1 and Nox4 protein content expressed as the ratio between the protein of interest and b-actin and normalized to the vehicle (V) group, taken as 100%. Representative blots are displayed above. Data are presented as means SEM of 5 or more experiments. *P < .05 versus vehicle (V). Ang II, angiotensin II; ET, endothelin-1.
hypertension.45 In agreement with our results, other authors have shown that, similar to Nox2, Nox1 and Nox4 play a role in NAD(P)H oxidase–derived ROS production, as downregulation of these enzymes attenuated generation 10,27,30,33,34 of O 2. Both Ang II and ET-1 have been implicated in vascular processes underlying hypertension through redox-sensitive proinflammatory, mitogenic, fibrogenic, and vasoactive actions in the vascular wall.4648 Increased NAD(P)H oxidase activity and expression of Nox isoforms by Ang II have been demonstrated in vivo.43,49 Moreover, Ang II type 1 receptor antagonists normalized the increased expression of NAD(P)H oxidase subunits found in hypertension.42,43 In vitro studies using cultured VSMCs also
demonstrated that Ang II increases NAD(P)H oxidase activity; however, the Nox isoforms involved remain elusive.8,14,33,5052 Here we attempted to identify the catalytic subunits that may be responsible for increased NAD (P)H oxidase activation by Ang II in WKY and SHR cells. Ang II time-dependently increased Nox1, Nox2, and Nox4 expression in VSMCs from WKY. In agreement, we previously found that Ang II increased Nox2 expression in VSMCs from human resistance arteries.14 However, variable results have been reported by other investigators, with studies showing that Ang II increases, decreases, or has no effect on Nox1 and/or Nox4.8,3335 In this study, we demonstrate that in SHR VSMCs Nox1, but not Nox4 or Nox2, is upregulated by Ang II. Taken together, these
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Figure 9. NAD(P)H oxidase activity in WKY and SHR VSMCs stimulated with angiotensin II (0.1 mmol/L) and endothelin-1 (0.1 mmol/L) for 2 and 4 hours in the absence or in the presence of EHT1864 (1 mmol/L). The lucigeninenhanced chemiluminescence assay was used to determine NAD(P)H oxidase–dependent superoxide production activity in total protein cell homogenates. Bar graphs represent mean SEM of 6 or more experiments. *P < .05 versus WKY. Ang II, angiotensin II; ET, endothelin-1.
results suggest that all Nox isoforms might participate in Ang II–induced NAD(P)H oxidase activation in cells from normotensive animals, whereas Nox1 might be primarily responsible for Ang II–induced NAD(P)H oxidase activation in SHR. The participation of Nox1 in
Ang II–driven O 2 production seems especially important, because siRNA for Nox1 normalized Ang II–induced O 2 generation in both strains. Additionally, intriguing results were observed after Nox2 downregulation, as this isoform seems to participate in Ang II–induced O 2 production in SHR, despite a lack of effect of Ang II in Nox2 expression, but not in WKY, where we observed an increase in Nox2 expression by Ang II. Interestingly, GKT136901 did not affect Ang II–induced NAD(P)H oxidase activity in any strain, apparently excluding the participation of Nox4 in this effect. Similar to Ang II effects in WKY cells, ET-1 increased activation of NAD(P)H oxidase and augmented expression of Nox1, Nox2, and Nox4. It is well established that ET-1 increases ROS production, in part through vascular NAD(P)H oxidase.36,5355 However, the Nox subunits involved are unclear. Considering that ET-1 significantly modulated Nox1, Nox2, and Nox4, we suggest that all isoforms may be involved. In fact, siRNA for Nox1 and Nox2, but not GKT136901, decreased ET-1–induced NAD(P)H oxidase activation indicating that Nox1 and Nox2, but not Nox4, are responsible for ET-1–induced O 2 production. On the other hand, in cells from SHR, ET-1 did not alter protein expression of any of the Nox isoforms and did not increase activity of NAD(P)H oxidase, excluding ET-1 as a major regulator of NAD(P)H oxidase–derived ROS in SHR. This is not surprising, as ET-1 does not seem to play an important role in the SHR model.47 Both Ang II and ET-1 induced a rapid activation of NAD (P)H oxidase and hence O 2 production, which was sensitive to Nox1/4 inhibition by GKT136901. We tested the hypothesis that this ROS production might modulate Nox expression through a positive feed-forward mechanism, because Nox-derived ROS influences vascular function by modulating Nox activity and through redox-sensitive regulation of vascular smooth muscle cell growth, apoptosis, senescence, survival, and inflammatory responses.56,57 However, in our experimental conditions, ROS do not seem to modulate Nox expression, as neither tempol nor tiron affected Ang II– or ET-1–induced Nox expression. The subcellular location of ROS production seems to be important in dictating the cellular response.18 We found that Nox2 localizes in the cell membrane as well as intracellularly. We detected Nox1 in the cell membrane as described in rat and human VSMCs.15,58 Other authors have shown that Nox1 co-localizes with cholesterol-rich domains of the plasma membrane, whereas Nox4 is found in focal adhesions.14,15 However, whether Nox2 localizes in cholesterolrich domains is unknown. We did not address whether Nox1 and Nox2 co-localize in these regions in our experimental conditions, and this is a limitation of the present study. Nox1 immunoprecipited and co-localized with Nox2 but not with Nox4, implicating a close association between Nox1 and Nox2 but not between Nox1 and Nox4. The fact that Nox1 and Nox4 are located in distinct compartments
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Figure 10. Co-immunoprecipitation of Nox2 and Nox1 and of Nox2 and Nox4 in VSMCs. (A) Mouse anti-Nox2 antibody was used for immunoprecipitation and goat anti-Nox1 antibody was used for immunoblotting. In the case of the negative control immunoprecipitates, cell lysate was incubated with 2 mg of normal mouse IgG. (B) Goat anti-Nox1 antibody was used for immunoprecipitation and mouse anti-Nox2 antibody was used for immunoblotting. In the case of the negative control immunoprecipitates, cell lysate was incubated with 2 mg of normal goat IgG. (C) Anti-Nox2 antibody was used for immunoprecipitation and rabbit anti-Nox4 antibody was used for immunoblotting. (D) Anti-Nox4 antibody was used for immunoprecipitation and anti-Nox2 antibody was used for immunoblotting. In the case of the negative control immunoprecipitates, cell lysate was incubated with 2 mg of normal rabbit IgG. Total protein lysate from mesenteric VSMCs (Western blot sample) were used as positive control. Immunoblots are representative of 3 identical experiments. (E) Nox2 and Nox1 co-localization in WKY and SHR vascular smooth muscle cells. Double-labeling indirect immunofluorescence using antiNox1 antibody with Cy3-conjugated secondary antibody and anti-Nox2 antibody with Alexa 488-conjugated secondary antibody and optical sectioning with confocal microscopy show that Nox1 is co-localized with Nox2. Regions of co-localization appear as yellow. Nuclei appear in blue. Images were taken at the midplane level. Image size 238 238 mm. Representative images of 3 experiments.
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Rac at short stimulation times up to 30 minutes.63 In our experimental conditions (long stimulations of 2 or 4 hours), the specific Rac inhibitor EHT1864 did not affect Ang II– or ET-1–induced NAD(P)H oxidase activation in VSMCs from any strain apparently excluding the participation of Rac in long-term stimulations. In summary, our findings indicate that VSMCs from resistance arteries possess Nox1, Nox2, and Nox4; that Nox1 and Nox2 co-localize in the cell membrane; and that Nox homologues, through differential regulation, play an important role in NAD(P)H oxidase-driven ROS generation in WKY and SHR cells in basal and in Ang II–stimulated conditions. ET-1 also regulates Nox-based NAD(P)H oxidase, but such effects are present only in WKY cells. These data highlight the complexity of the Nox system in VSMCs, emphasizing that more than one Nox family member, alone or in association, may be involved in NAD(P)H oxidase–mediated ROS production and that Nox1 regulation by Ang II, but not by ET-1, may be important in ROS formation in SHR. Our findings provide new insights into redox mechanisms underlying vascular pathology in hypertension.
Figure 11. Regulation of Nox isoforms in VSMCs from normotensive (WKY) and spontaneously hypertensive rats (SHR). Vascular smooth muscle cells express 3 isoforms of the gp91phox (Nox2) subunit of the NADPH oxidase, including Nox1, Nox2, and Nox4. Expression and activity of Nox isoforms is higher in cells from SHR. Nox1 and probably Nox4 in cells from WKY and Nox1 and Nox2 in SHR contribute to basal ROS production. Upon stimulation with Ang II, all Nox isoforms are upregulated in cells from WKY but only Nox1 is upregulated in cells from SHR. This stimulation generates ROS production mainly through Nox1 in WKY and through Nox1 and Nox2 in SHR probably because of the interaction between the 2 Nox isoforms. ET-1 upregulates all Nox isoforms and increases NADPH oxidase activity in cells from WKY but had no effect in cells from SHR. Both Nox1 and Nox2 participate in ET-1–induced ROS generation in vascular smooth muscle cells from WKY. Nox1, Nox2, and Nox4 require p22phox (p22) for their activation. Solid traces indicate basal ROS production and dashed traces indicate agonist-induced ROS generation. AngII, angiotensin II; ET, endothelin-1.
suggests that they may influence specific cellular functions.15,5961 The functional significance of Nox1 and Nox2 acting in association is unclear, but the complex may provide an efficient system for NAD(P)H oxidase regulation and controlled ROS production. This awaits further clarification. The small G-protein Rac-1 is involved in Nox1-dependent 62 In addition, Ang II–induced stimulation O 2 production. of NAD(P)H oxidase has been reported to be dependent on
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Research Article
Differential regulation of Nox1, Nox2 and Nox4 in vascular smooth muscle cells from WKY and SHR Ana M. Briones, PhDa,b, Fatiha Tabet, PhDa,c, Glaucia E. Callera, PhDa, Augusto C. Montezano, PhDa, Alvaro Yogi, PhDa, Ying He, MSc, MDa, Mark T. Quinn, PhDd, Mercedes Salaices, PhDb, and Rhian M. Touyz, MD, PhDa,* b
a Kidney Research Centre, Ottawa Health Research Institute, University of Ottawa, Ontario, Canada; Department Farmacologıa y Terapeutica, Facultad de Medicina, Universidad Aut onoma de Madrid, Spain; c Heart Research Institute, Sydney, New South Wales, Australia; and d Department of Immunology and Infectious Diseases, Montana State University, Bozeman, USA Manuscript received December 13, 2010 and accepted February 3, 2011
Abstract The functional significance and regulation of NAD(P)H oxidase (Nox) isoforms by angiotensin II (Ang II) and endothelin-1 (ET-1) in vascular smooth muscle cells (VSMCs) from normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) was studied. Expression of Nox1, Nox2, and Nox4 (gene and protein) and NAD(P)H oxidase activity were increased in SHR. Basal NAD(P)H oxidase activity was blocked by GKT136901 (Nox1/4 inhibitor) and by Nox1 siRNA in WKY cells and by siNOX1 and siNOX2 in SHR. Whereas Ang II increased expression of all Noxes in WKY, only Nox1 was influenced in SHR. Ang II–induced NAD(P)H activity was inhibited by siNOX1 in WKY and by siNOX1 and siNOX2 in SHR. ET-1 upregulated Nox expression only in WKY and increased NAD(P)H oxidase activity, an effect inhibited by siNOX1 and siNOX2. Nox1 co-localized with Nox2 but not with Nox4, implicating association between Nox1 and Nox2 but not between Nox1 and Nox4. These data highlight the complexity of Nox biology in VSMCs, emphasising that more than one Nox member, alone or in association, may be involved in NAD(P)H oxidase-mediated O 2 production. Nox1 regulation by Ang II, but not by ET-1, may be important in O 2 formation in VSMCs from SHR. J Am Soc Hypertens 2011;5(3):137–153. Ó 2011 American Society of Hypertension. All rights reserved. Keywords: Redox signaling; gp91phox; Nox1; Nox4; NAD(P)H oxidase; vasculature.
Introduction In the vascular system, reactive oxygen species (ROS) play a physiological role in controlling endothelial function and vascular tone and a pathophysiological role in inflammation,
Funding/support: This study was funded by Grant 44018 from the Canadian Institute of Health Research (CIHR) and by MEC (SAF 2009-07201). A.M.B. was supported through a Beca de Posgrado from Fundacion Caja Madrid. F.T. received a Canada Graduate Scholarship Doctoral Award from the CIHR. Conflict of interest: none reported. *Corresponding author: Rhian M. Touyz, MD, PhD, Kidney Research Institute, Ottawa Health Research Institute, University of Ottawa, 304/451 Smyth Road, Ottawa (Ontario) K1H 8M5 Canada. Tel: 613-562–5800 ext 8241; fax: 613-562-5487. E-mail: [email protected]
hypertrophy, proliferation, apoptosis, migration, fibrosis, angiogenesis, and rarefaction, important in vascular remodeling and endothelial dysfunction associated with hypertension.13 Among the major sources of ROS in the vascular wall is nonphagocytic NAD(P)H oxidase (Nox),4,5 a multi-subunit enzyme that differs structurally and biochemically from phagocytic gp91phox (also termed Nox2)containing NAD(P)H oxidase, important for host defence.6 NAD(P)H oxidases are a family of enzymes with each member being distinguished by the specific Nox catalytic subunit. To date, 7 Nox isoforms have been identified in mammals (Nox1 to Nox7),7 of which many are present in the vascular wall.8,9 Vascular smooth muscle cells (VSMCs) express Nox1, Nox2, Nox4, and Nox5.1013 Nox2 is primarily membrane-associated, Nox1 co-localizes with cholesterolrich domains of the plasma membrane, and Nox4 is found in focal adhesions14,15 and the endoplasmic reticulum.1416
1933-1711/$ - see front matter Ó 2011 American Society of Hypertension. All rights reserved. doi:10.1016/j.jash.2011.02.001
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Nox5 (not found in rodents) has a perinuclear-endoplasmic reticular distribution.17 The functional significance of Nox isoforms in VSMCs is unclear, but considering their localization within specific subcellular compartments, ROS derived from different Nox isoforms may play distinct roles in modulating intracellular ROS-mediated signaling. For example, Nox4 appears to be important in maintaining 18,19 and hydrogen peroxide basal superoxide (O 2) 20 (H2O2) production, whereas Nox1 might be responsible for O 2 production and redox signaling after stimulation and in pathological conditions, such as atherosclerosis, diabetes, and hypertension.21 Increased expression and/or activity of vascular NAD(P)H oxidase has been suggested as a mechanism underlying 1,8,9,22 The role enhanced O 2 production in hypertension. of different Nox isoforms in this phenomenon is unclear. Li et al23 demonstrated greater mRNA expression of Nox1, Nox2, and Nox4 in aorta from spontaneously hypertensive rats (SHR) versus normotensive Wistar-Kyoto (WKY) rats; however, similar levels of Nox1 and Nox2 mRNA were found in aortas from WKY and stroke-prone SHR.24 In cerebral arteries from SHR, increased expression levels of Nox4 but not of Nox1 and Nox2 mRNA were described.25 Whereas Nox1 and Nox2 appear to be important in acute models of angiotensin (Ang) II–dependent hypertension and vascular remodeling,2628 they do not seem to play a major role in chronic Ang II–dependent hypertension.29,30 Many factors have been implicated in the regulation of NAD(P)H oxidase activity in vascular disease, including physical factors (stretch, flow, and pressure), vasoactive agents (Ang II, endothelin-1 [ET-1], and aldosterone), and growth factors (epidermal growth factor, insulin like growth factor, platelet-derived growth factor [PDGF]).1,8,9,31,32 However, it remains to be determined which Nox enzymes are involved and how these are differentially regulated in hypertension. Ang II seems to be particularly important. Lassegue et al33 reported that Ang II increases Nox1 and downregulates Nox4 mRNA in cultured rat aortic VSMCs, whereas Wingler et al34 demonstrated an upregulation of Nox1 and Nox4 mRNA levels by Ang II in A7r5m cells (cell line derived from rat aortic SMC). However, Hitomi et al35 did not find Ang II–dependent alterations of Nox1 expression in VSMCs. PDGF, basic fibroblast growth factor, prostaglandin F2a, and advanced glycation end products also increase Nox1 expression in VSMCs.8,9 Similar to Ang II, ET-1 stimulates ROS production in vascular cells. However, there is a paucity of information on the role of ET-1 in the regulation of vascular Nox isoforms. In rat aortic SMC, ET-1 had no effect on Nox1 or Nox4 expression34 but increased mRNA expression of Nox2 in human umbilical vein endothelial cells.36 In human aortic endothelial cells, ET-1 inhibited Nox1based NAD(P)H oxidase via ETB receptors.37 Whether ET-1 modulates vascular Nox isoforms in SHR is unclear.
To our knowledge, there are no studies that systematically assess effects of Ang II and ET-1 on the regulation and function of Nox isoforms in vascular cells from hypertensive animals. In the present study, we sought to determine whether expression of Nox family members in VSMC differs between WKY and SHR and whether Ang II and ET-1, potent vasoactive peptides important in the pathogenesis of hypertension, differentially regulate Nox expression and activity.
Methods Cell Culture The study was approved by the Animal Ethics Committee of the University of Ottawa and performed according to the recommendations of the Canadian Council for Animal Care. VSMC from mesenteric resistance arteries were obtained from 16-week-old WKY and SHR (Taconic Farms, Germantown, NY). Cells were isolated by enzymatic digestion and cultured and characterized as previously described.38 Briefly, arteries were cleaned of adipose and connective tissue, and VSMCs were dissociated by digestion of vascular arcades with enzymatic solution (collagenase, elastase, soybean trypsin inhibitor, and bovine serum albumin [BSA] type I; 60 minutes, 37 C). The tissue was filtered and the cell suspension centrifuged and resuspended in Dulbecco Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), 2 mmol/L glutamine, 20 mmol/L HEPES (pH 7.4), and antibiotics. Cells were used between passages 3 and 7. At subconfluence, the culture medium was replaced with serum-free medium for 24 hours to render the cells quiescent. Cells were incubated with vehicle or with Ang II (0.1 mmol/L) or ET-1 (0.1 mmol/L) for 2 to 24 hours. In some experiments, Ang II– and ET-1– (2–4 hours) stimulated cells were incubated with the Nox1/4 inhibitor GKT136901 (10 mmol/L; gift from Genkyotex, Geneva, Switzerland) with the specific Rac inhibitor EHT1864 (1 mmol/L) or with the antioxidants tiron (10 mmol/L) and tempol (1 mmol/L; 30 minutes pre-incubation).
Real-Time PCR Cellular mRNA levels of Nox1, Nox2, and Nox4 were studied by real-time polymerase chain reaction (PCR). Total RNA was isolated from WKY and SHR VSMCs using Trizol Reagent (GIBCO-BRL, Invitrogen, Burlington, ON, Canada). Reverse transcription was performed using 2 mg of RNA as previously described.39 Real-time PCR was performed with a Stratagene Mx4000 System (La Jolla, California, USA) for relative quantification of VSMC Nox1, Nox4, and Nox2 mRNA. Sense and antisense primers were designed to generate short amplification products (217 base pairs [bp] for Nox1 subunit, 110 bp for Nox4
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Figure 1. Basal mRNA and protein levels of Nox1 (A), Nox2 (B), and Nox4 (C) in WKY and SHR vascular smooth muscle cells. Bar graphs indicate relative quantification of Nox1, Nox2, and Nox4 mRNA or protein content in VSMCs from WKY and SHR. Results are expressed as the ratio between the gene or protein of interest and the gene of ribosomal protein S16 and b-actin respectively, and then normalized to the WKY group, taken as 100%. Results are presented as mean SEM of 4 to 8 experiments. *P < .05 and **P < .01 versus WKY.
subunit, 203 bp for Nox2, and 107 bp for ribosomal protein S16, used as an internal standard). Primers were as follows. For VSMC Nox1: sense 50 -CTTCCTCACTGGCTGG GATA-30 and antisense 50 -AGCCTGCGCAAATGCTGTC30 ; for VSMC Nox4 subunit: sense 50 -CAGTAATCAAT CATCCCTCAGA-30 and antisense 50 -TGTCCCAGTGTAT CAGCATTAG-30 ; for VSMC Nox2 subunit: sense 50 GTATTGTGGGAGACTGGACTGAG-30 and antisense 50 TGCCTCCATTCTCAAGTCTGTCT-30 ; and for S16: sense 50 -AGGAGCGATTTGCTGGTGTGG-30 and anti-sense 50 GCTACCAGGGCCTTTGAGATG-30 . To validate our realtime PCR protocol, gene-specific standard curves for Nox1, Nox4, Nox2, and S16 were generated from serial dilutions of the cDNA. Real-time PCR was conducted using the
Quantitect SYBR Green PCR Kit (Qiagen, Mississauga, Ontario, Canada) and a final 0.5 mmol/L concentration of primers. Data were calculated as the ratio between the gene of interest and S16 relative quantities. Levels of Nox1, Nox2, and Nox4 in SHR were calculated as the amount relative to that in the WKY group, taken as 100%.
Western Blot Analysis Protein levels for Nox1, Nox2, and Nox4 from WKY and SHR VSMCs were assessed by Western blotting. Quiescent VSMCs from WKY were stimulated with Ang II or ET-1 for 2 to 24 hours, and proteins were lysed and extracted for Western blotting, as previously described.40 Briefly, equal
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(Nox4 control; Santa Cruz Biotechnology). The immunocomplexes were captured by adding 30 mL of protein A agarose beads (Santa Cruz Biotechnology) and the mixture was incubated for 2 hours at 4 C. The agarose beads were collected by brief centrifugation (5 seconds, 14,000g) and the supernatant aspirated. The beads were washed 3 times with ice-cold cell lysis buffer. The proteins were eluted from the beads by the addition of 50 mL of 2X Laemmli sample buffer and boiling for 5 minutes. The supernatant was collected by brief centrifugation and run in an SDSpolyacrylamide gel. Western blot analysis for Nox1, Nox2, and Nox4 was performed as described previously. Figure 2. Basal NAD(P)H oxidase activity in vascular smooth muscle cells from WKY and SHR. The lucigenin-enhanced chemiluminescence assay was used to determine NAD(P)H oxidase–dependent superoxide production activity in total protein cell homogenates. Bar graphs represent mean SEM of 5 experiments. **P < .01 versus WKY.
amounts of proteins were separated by electrophoresis on a 10% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane (Boehinger, Mannheim, Laval, Quebec, Canada). Nonspecific binding sites were blocked with a 5% wt/vol nonfat dry milk blocking buffer containing Trisbuffered saline and 0.1% Tween-20 (TBS-T; 1 hour, room temperature). Membranes were incubated overnight at 4 C with goat anti-Nox1 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Nox4 (1:500 Santa Cruz Biotechnology), or mouse anti-Nox2 (1:500).41 Membranes were incubated with horseradish peroxidase–conjugated secondary antibodies for 1 hour at room temperature. After incubation with secondary antibodies, signals were detected by chemiluminescence and visualized by autoradiography. Blots were analyzed densitometrically using the imageQuant software (Molecular Dynamics, Sunnyvale, CA). Samples from vehicle-stimulated VSMCs were used as controls and variations of Nox1, Nox2, and Nox4 from Ang II– or ET-1–treated VSMCs were calculated as the amount relative to controls.
Co-immunoprecipitation Studies Immunoprecipitation analysis was performed in WKY VSMCs. Briefly, cells were lysed with an immunoprecipitation buffer containing 10 mmol/L HEPES (pH 7.6), 15 mmol/L KCl, 2 mmol/L MgCl2, 0.1% NP40, 1 mmol/L phenylmethyl sulfonyl fluoride (PMSF), 1 mg/mL aprotinin, 1 mg/mL leupeptin, and 1 mg/mL pepstatin A. The protein supernatant (500 mg) was incubated with 2 mg of Nox1, Nox2, or Nox4 antibodies overnight at 4 C. In the case of the control immunoprecipitates, the cell lysate was incubated with 2 mg of normal mouse immunoglobulin G (IgG) (Nox2 control), goat IgG (Nox1 control), and rabbit IgG
Immunofluorescence Studies Cells were plated on round glass coverslips and fixed with 4% phosphate-buffered paraformaldehyde (in 0.2 mol/L phosphate buffer, pH 7.2–7.4) for 15 minutes. Cells were then washed 3 times with PBS solution (pH, 7.5). Nonspecific binding sites were blocked with 1% BSA in PBS for 30 minutes. Cells were incubated with primary antibodies against Nox1 (1:50) or Nox2 (1:50) in PBS containing 1% BSA for 1 hour at 37 C. Cells were washed and incubated with either anti-goat IgG conjugated to Cy3 for Nox1 (Jackson Immuno Research Laboratories, West Grove, PA, USA) (1:300) or anti-mouse IgG labeled with Alexa 488 (1:1000) for Nox2, for a further 1 hour at 37 C (Jackson Immuno Research Laboratories). Cells were washed and incubated with the nuclear dye, Hoechst 33342 (0.01 mg/mL). The immunofluorescent signals were viewed with a confocal microscope (Leica TCS 4D, Leica, Barcelona, Spain) with a 63 oil objective. The specificity of the immunostaining was evaluated by omission of the primary antibody and processed as described previously. Under these conditions, no specific fluorescence staining was observed.
Measurement of NAD(P)H Oxidase Activity The lucigenin-enhanced chemiluminescence assay was used to determine the NAD(P)H oxidase activity in total protein cell homogenates. Cells were washed in ice-cold PBS and scraped off in lysis buffer containing 20 mmol/L KH2PO4, 1 mmol/L ethyleneglycoltetraacetic acid (EGTA), and protease inhibitors, pH 7.4. Lysates were transferred to Eppendorf tubes and sonicated for 3 seconds. The reaction was started by the addition of NAD(P)H (0.1 mmol/L) to the suspension (250 mL final volume) containing sample (50 mL), lucigenin (5 mmol/L), and assay phosphate buffer (50 mmol/L KH2PO4, 1 mmol/L EGTA, 150 mmol/l sucrose, pH 7.4). The luminescence was measured every 1.8 seconds for 3 minutes in a luminometer (Lumistar Galaxy; BMG Labtechnologies, Offenburg, Germany). Buffer blank was subtracted from each reading. Activity is expressed as arbitrary units/mg protein. Samples from vehicle-stimulated VSMCs were used as controls and variations of NAD(P)H
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Figure 3. Time course effect of angiotensin II (0.1 mmol/L) stimulation on Nox1 (A), Nox2 (B) and Nox4 (C) expression in vascular smooth muscle cells from WKY and SHR. Bar graphs indicate relative quantification of Nox1, Nox2 and Nox4 protein content expressed as the ratio between the protein of interest and b-actin and normalized to the vehicle (V) group, taken as 100%. Representative blots are displayed above. Data are presented as means SEM of 5 or more experiments. *P < .05 vs. control (C). A, Angiotensin II.
oxidase activity from Ang II– or ET-1–treated VSMCs were calculated as the amount relative to controls. In some experiments, samples from WKY VSMCs were used as controls and variations of NAD(P)H oxidase activity from SHR VSMCs were calculated as the amount relative to controls.
Fluorescence Detection of ROS Generation in Intact Cells Dihydroethidium Generation of intracellular O 2 in living cells was measured with dihydroethidium (DHE; excitation at 546
nm and emission at 610 nm, Molecular Probes, Invitrogen, Carlsbad, CA, USA). Growth-arrested VSMCs from WKY and SHR rats were incubated in Hank’s balanced salt solution (HBSS) containing 1.3 mmol/L CaCl2 and 5.5 mmol/L glucose supplemented with 2 mmol/L DHE in a lightprotected chamber at 37 C for 20 minutes. Cells were rinsed in HBSS and images were obtained before and after exposition to Ang II or ET-1. Cells were imaged on a wide-field epifluorescence microscope equipped with 40 oil immersion lens using the Stallion live-cell Digital Hi-Speed Multi-Channel Imaging System (Zeiss, Berlin, Germany).
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Figure 4. Time course effect of endothelin-1 (0.1 mmol/L) stimulation on Nox1 (A), Nox2 (B) and Nox4 (C) expression in vascular smooth muscle cells from WKY and SHR. Bar graphs indicate relative quantification of Nox protein content expressed as the ratio between the protein of interest and b-actin and normalized to the vehicle (V) group, taken as 100%. Representative blots are displayed above. Data are presented as means SEM of 5 or more experiments. *P < .05 and **P < 0.01 vs. control (C). ET, Endothelin-1.
RNA Interference and Cell Transfection WKY and SHR VSMCs were subjected to Nox1 and Nox2 gene knockdown with small interfering RNA (siRNA) before 0.1 mmol/L Ang II or ET-1 stimulation. High-performance purity-grade (>90% pure) siRNA was generated against rat Nox1 and Nox2 (Santa Cruz Biotechnology, Inc). Rat VSMCs were exposed to transfectant reagent alone or transfected with siRNA. A nonsilencing (NS) siRNA oligonucleotide sequence that does not recognize any known homology to mammalian genes was also generated as a negative control. Cells were seeded at
a density of 2.5 105 cells/well in 100-mm plates with DMEM containing 10% FBS and transfected with 20 nmol/L siRNA using HiPerFect Transfection Reagent (Qiagen Inc.) according to the manufacturer’s instructions. Optimum gene silencing was observed at 24 hours after transfection.
Statistical Analysis Data are presented as mean SEM. Groups were compared using 1-way analysis of variance (ANOVA) or
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Figure 5. (A) Representative fluorescent photomicrographs of microscopic sections of VSMCs from WKY and SHR stimulated with angiotensin II (0.1 mmol/L; 4 hours). Cells were stained with the fluorescent dye dihydroethidium. Images were captured with a wide-field epifluorescence microscope equipped with 40 oil immersion lens using the Stallion live-cell Digital Hi-Speed MultiChannel Imaging System. Magnification 640. (B) NAD(P)H oxidase activity in WKY and SHR cells stimulated with angiotensin II for 2 and 4 hours. (C) Representative fluorescent photomicrographs of microscopic sections of VSMCs from WKY stimulated with endothelin-1 (0.1 mmol/L; 4 hours). Cells were stained with the fluorescent dye dihydroethidium. Images were captured with a wide-field epifluorescence microscope equipped with 40 oil immersion lens using the Stallion live-cell Digital Hi-Speed MultiChannel Imaging System. Magnification 640. (D) NAD(P)H oxidase activity in WKY and SHR cells stimulated with endothelin-1 for 2 and 4 hours. The lucigenin-enhanced chemiluminescence assay was used to determine NAD(P)H oxidase–dependent superoxide production activity in total protein cell homogenates. Bar graphs represent mean SEM of 6 or more experiments. *P < .05 versus WKY. Ang II, angiotensin II; ET, endothelin-1.
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Figure 6. NAD(P)H oxidase activity in WKY and SHR vascular smooth muscle cells stimulated with angiotensin II (0.1 mmol/L) (A) and endothelin-1 (0.1 mmol/L) (B) for 2 and 10 min in the absence or in the presence of GKT136901 (10 mmol/L). The lucigenin-enhanced chemiluminescence assay was used to determine NAD(P)H oxidase-dependent superoxide production activity in total protein cell homogenates. Bar graphs represent mean SEM of 6 or more experiments. *P < .05 vs. WKY. Ang II, angiotensin II; ET, endothelin-1.
Student t test as appropriate. Newman-Keuls posttest was used to compensate for multiple testing procedures. A value of P less than .05 was significant.
Results mRNA and Protein Expression of Nox1, Nox2, and Nox4 and Basal NAD(P)H Oxidase Activity in WKY and SHR VSMC We compared the mRNA and protein expression of Nox2 and its homologues Nox1 and Nox4 in basal conditions in VSMC from WKY and SHR. As shown in Figure 1, higher mRNA and protein expression of Nox1, Nox2, and Nox4 was observed in SHR compared with WKY cells. Basal NAD(P)H oxidase activity was two fold greater in VSMCs from SHR compared with WKY (Figure 2).
Effects of Ang II and ET-1 on Protein Expression of Nox1, Nox2 and Nox4 in WKY and SHR VSMC As shown in Figure 3, expression of Nox1, Nox2, and Nox4 was increased by Ang II in a time-dependent manner in WKY VSMCs. In cells from SHR, Ang II increased Nox1 expression (Figure 3A) without affecting Nox2 or
Nox4 expression (Figure 3B, 3C). ET-1 also induced a time-dependent increase in Nox1, Nox2, and Nox4 protein expression in WKY cells (Figure 4). However, ET-1 did not significantly influence Nox1, Nox2, or Nox4 protein content in SHR cells (Figure 4).
Effects of Ang II and ET-1 on NAD(P)H Oxidase Activity in VSMCs Ang II induced a significant increase in O 2 production (Figure 5A) and NAD(P)H oxidase activity and at 2 and 4 hours in WKY and SHR cells (Figure 5B). ET-1 increased O 2 production (Figure 5C) and NAD(P)H oxidase activity in WKY cells, but had no significant effect in SHR cells (Figure 5D). Short-term stimulation with Ang II or ET-1 increased NAD(P)H oxidase activity in WKY cells (Figure 6). However, Ang II but not ET-1 increased NAD(P)H oxidase activity in SHR. Ang II and ET-1 effects were diminished by the NOX1/4 inhibitor GKT136901.
Inhibition of Nox Isoforms Block Activity of NAD(P)H Oxidase in VSMCs To evaluate the importance of different Nox isoforms in NAD(P)H oxidase activity, Nox1 and Nox2 were downregulated using an siRNA approach and Nox4 was inhibited
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Figure 7. Left panels: NAD(P)H oxidase activity in VSMCs from WKY and SHR incubated with GKT136901 (10 mmol/L) or in which Nox1 and Nox2 are downregulated by siRNA. Cells were transfected with Nox1 (siNox1), Nox2 (siNox2), or nonsilencing (NS) siRNAs and were stimulated or not with angiotensin II (0.1 mmol/L; 2 hours) or endothelin-1 (0.1 mmol/L; 4 hours). Vehicle cells were exposed to the transfectant without siRNA. Right panels: Representative blots of Nox1 and Nox2 expression in WKY and SHR vascular smooth muscle cells transfected with Nox1, Nox2, or NS siRNAs. Representative fluorescent photomicrographs of microscopic sections of VSMC from WKY and SHR transfected with Nox1, Nox2, or NS siRNAs and stimulated with angiotensin II or endothelin-1. Cells were stained with the fluorescent dye dihydroethidium. Images were captured with a wide-field epifluorescence microscope equipped with 40 oil immersion lens using the Stallion live-cell Digital Hi-Speed Multi-Channel Imaging System. Magnification 640. Data are represented as mean SEM of 6 or more experiments. *P < .05 versus vehicle (V). Ang II, angiotensin II; ET, endothelin-1. þ P .05 versus Ang II or ET-1.
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using the inhibitor GKT136901. Basal NAD(P)H oxidase activity was decreased by GKT and siNox1 in WKY cells and by siNox1 and siNox2 in SHR (Figure 7). In WKY, siNox1 induced a significant decrease in Ang II–induced NAD(P)H oxidase activity, whereas in SHR both siNox1 and siNox2 decreased Ang II–induced NAD (P)H oxidase activity (Figure 7). In VSMCs from WKY, both siNox1 and siNox2 decreased ET-1 induced activation of NAD(P)H oxidase activity (Figure 7).
Agonist-Stimulated ROS Generation Does Not Influence Nox Expression To determine the role of ROS on Nox expression, cells were exposed to tiron (ROS scavenger) and tempol (superoxide dismutase mimetic). Neither tiron nor tempol affected Ang II– or ET-1–induced expression of Nox1, Nox4 (Figure 8), or Nox2 (data not shown) in WKY. Similarly, these antioxidants did not affect Ang II–induced Nox1 expression in SHR (data not shown).
Rac Inhibition Does Not Influence Ang II– or ET-1–Induced NAD(P)H Oxidase Activity To determine the participation of Rac in the NAD(P)H oxidase activity induced by Ang II and ET-1, cells were preincubated with the selective Rac inhibitor EHT1864. After 2- or 4-hour stimulation, NAD(P)H oxidase activity was not affected by EHT1864 in WKY or SHR (Figure 9).
Analysis of Nox1 and Nox4 Co-immunoprecipitation with Nox2 Considering that VSMCs possess multiple Nox isoforms, we questioned the relationship among Nox1, Nox2, and Nox4 using a co-immunoprecipitation strategy. To study the association between Nox1 and Nox2, proteins were precipitated with Nox2 antibody and blotted with Nox1 antibody. As shown in Figure 10A, Nox2 co-immunoprecipitated with Nox1. In the reverse experiment, proteins were precipitated with Nox1 antibody and blotted with Nox2 antibody and, as shown in Figure 10B, Nox1 co-immunoprecipitated with Nox2. VSMC total protein lysate was used as positive control in these experiments. To study the association between Nox4 and Nox2 in VSMC, proteins were precipitated with Nox2 antibody and blotted with Nox4 antibody. As shown in Figure 10C, Nox2 did not co-immunoprecipitate with Nox4. In the reverse experiment, proteins were precipitated with Nox4 antibody and blotted with Nox2 antibody. As confirmed in this reverse experiment, Nox4 and Nox2 did not co-immunoprecipitate (Figure 10D). VSMC total protein lysate was used as positive control. These co-immunoprecipitation experiments suggest that Nox2 and Nox4 do not physically associate in resting VSMC (Figure 10).
Immunofluorescence of Nox1 and Nox2 Optical sectioning of VSMCs from WKY and SHR with laser scanning confocal microscopy revealed Nox1 and Nox2 labeling, essentially in a punctuate surface distribution along the cell margins, although some intracellular distribution was also detected (Figure 10E). In these studies, we found a close co-localization between Nox1 and Nox2, as shown by the yellow staining observed in the merged image. Interestingly, this co-localization pattern was seen in unstimulated cells, suggesting that the two proteins associate under basal conditions.
Discussion Vascular cells possess multiple Nox isoforms, the functional significance and regulatory mechanisms of which remain unclear, particularly in the setting of hypertension. Here, we provide novel data demonstrating that VSMCs from resistance arteries possess Nox1, Nox2, and Nox4; that Nox1 and Nox2 co-localize in the cell membrane; and that Nox homologues play an important role in NAD (P)H oxidase–driven ROS generation. Moreover, we show that vascular Nox expression and activation of NAD(P)H oxidase are augmented in SHR. Finally, we demonstrate differential regulation of the Nox isoforms by Ang II and ET-1 in WKY and SHR VSMCs. Whereas in WKY cells, all Nox isoforms are sensitive to Ang II and ET-1, in SHR cells, only Nox1 is influenced by Ang II with ET-1 having no effect on any of the Nox isoforms. These findings highlight the complexity of Nox-based NAD(P)H oxidase systems in VSMCs and suggest that increased ROS generation in SHR primarily involves Ang II–sensitive Nox1, with little influence by ET-1 (Figure 11). The expression of the catalytic Nox subunits varies among different cell types.9 Here we show that VSMCs from resistance arteries possess Nox1, Nox2, and Nox4 and that the content of these Nox isoforms is greater in VSMCs from hypertensive than normotensive rats. NAD (P)H oxidase activity was also greater in cells from SHR compared with WKY. Upregulation of Nox isoforms may contribute to increased NAD(P)H oxidase activity in SHR, at least in basal conditions. In fact, siRNA for Nox1 and Nox2 decreased basal NAD(P)H oxidase activity in SHR, suggesting that these isoforms may be responsible for the increased O 2 in SHR. In WKY, probably both Nox1 and Nox4 contribute to basal O 2 production as shown by the inhibitory effect induced by GKT136901 and by siRNA for Nox1. Several investigations have examined the expression of Nox family members in vascular tissues from hypertensive models. However, depending on the vascular bed and the experimental model studied, conflicting results have been found.2325,34,4244 Although the pattern of Nox expression may vary, increased vascular NAD(P)H oxidase–derived ROS is a common feature in
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Figure 8. Effect of the antioxidants tempol (1 mmol/L) and tiron (10 mmol/L) alone or in combination with angiotensin II (0.1 mmol/L) or endothelin-1 (0.1 mmol/L) on the expression of Nox1 and Nox4 in vascular smooth muscle cells from WKY. Bar graphs indicate relative quantification of Nox1 and Nox4 protein content expressed as the ratio between the protein of interest and b-actin and normalized to the vehicle (V) group, taken as 100%. Representative blots are displayed above. Data are presented as means SEM of 5 or more experiments. *P < .05 versus vehicle (V). Ang II, angiotensin II; ET, endothelin-1.
hypertension.45 In agreement with our results, other authors have shown that, similar to Nox2, Nox1 and Nox4 play a role in NAD(P)H oxidase–derived ROS production, as downregulation of these enzymes attenuated generation 10,27,30,33,34 of O 2. Both Ang II and ET-1 have been implicated in vascular processes underlying hypertension through redox-sensitive proinflammatory, mitogenic, fibrogenic, and vasoactive actions in the vascular wall.4648 Increased NAD(P)H oxidase activity and expression of Nox isoforms by Ang II have been demonstrated in vivo.43,49 Moreover, Ang II type 1 receptor antagonists normalized the increased expression of NAD(P)H oxidase subunits found in hypertension.42,43 In vitro studies using cultured VSMCs also
demonstrated that Ang II increases NAD(P)H oxidase activity; however, the Nox isoforms involved remain elusive.8,14,33,5052 Here we attempted to identify the catalytic subunits that may be responsible for increased NAD (P)H oxidase activation by Ang II in WKY and SHR cells. Ang II time-dependently increased Nox1, Nox2, and Nox4 expression in VSMCs from WKY. In agreement, we previously found that Ang II increased Nox2 expression in VSMCs from human resistance arteries.14 However, variable results have been reported by other investigators, with studies showing that Ang II increases, decreases, or has no effect on Nox1 and/or Nox4.8,3335 In this study, we demonstrate that in SHR VSMCs Nox1, but not Nox4 or Nox2, is upregulated by Ang II. Taken together, these
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Figure 9. NAD(P)H oxidase activity in WKY and SHR VSMCs stimulated with angiotensin II (0.1 mmol/L) and endothelin-1 (0.1 mmol/L) for 2 and 4 hours in the absence or in the presence of EHT1864 (1 mmol/L). The lucigeninenhanced chemiluminescence assay was used to determine NAD(P)H oxidase–dependent superoxide production activity in total protein cell homogenates. Bar graphs represent mean SEM of 6 or more experiments. *P < .05 versus WKY. Ang II, angiotensin II; ET, endothelin-1.
results suggest that all Nox isoforms might participate in Ang II–induced NAD(P)H oxidase activation in cells from normotensive animals, whereas Nox1 might be primarily responsible for Ang II–induced NAD(P)H oxidase activation in SHR. The participation of Nox1 in
Ang II–driven O 2 production seems especially important, because siRNA for Nox1 normalized Ang II–induced O 2 generation in both strains. Additionally, intriguing results were observed after Nox2 downregulation, as this isoform seems to participate in Ang II–induced O 2 production in SHR, despite a lack of effect of Ang II in Nox2 expression, but not in WKY, where we observed an increase in Nox2 expression by Ang II. Interestingly, GKT136901 did not affect Ang II–induced NAD(P)H oxidase activity in any strain, apparently excluding the participation of Nox4 in this effect. Similar to Ang II effects in WKY cells, ET-1 increased activation of NAD(P)H oxidase and augmented expression of Nox1, Nox2, and Nox4. It is well established that ET-1 increases ROS production, in part through vascular NAD(P)H oxidase.36,5355 However, the Nox subunits involved are unclear. Considering that ET-1 significantly modulated Nox1, Nox2, and Nox4, we suggest that all isoforms may be involved. In fact, siRNA for Nox1 and Nox2, but not GKT136901, decreased ET-1–induced NAD(P)H oxidase activation indicating that Nox1 and Nox2, but not Nox4, are responsible for ET-1–induced O 2 production. On the other hand, in cells from SHR, ET-1 did not alter protein expression of any of the Nox isoforms and did not increase activity of NAD(P)H oxidase, excluding ET-1 as a major regulator of NAD(P)H oxidase–derived ROS in SHR. This is not surprising, as ET-1 does not seem to play an important role in the SHR model.47 Both Ang II and ET-1 induced a rapid activation of NAD (P)H oxidase and hence O 2 production, which was sensitive to Nox1/4 inhibition by GKT136901. We tested the hypothesis that this ROS production might modulate Nox expression through a positive feed-forward mechanism, because Nox-derived ROS influences vascular function by modulating Nox activity and through redox-sensitive regulation of vascular smooth muscle cell growth, apoptosis, senescence, survival, and inflammatory responses.56,57 However, in our experimental conditions, ROS do not seem to modulate Nox expression, as neither tempol nor tiron affected Ang II– or ET-1–induced Nox expression. The subcellular location of ROS production seems to be important in dictating the cellular response.18 We found that Nox2 localizes in the cell membrane as well as intracellularly. We detected Nox1 in the cell membrane as described in rat and human VSMCs.15,58 Other authors have shown that Nox1 co-localizes with cholesterol-rich domains of the plasma membrane, whereas Nox4 is found in focal adhesions.14,15 However, whether Nox2 localizes in cholesterolrich domains is unknown. We did not address whether Nox1 and Nox2 co-localize in these regions in our experimental conditions, and this is a limitation of the present study. Nox1 immunoprecipited and co-localized with Nox2 but not with Nox4, implicating a close association between Nox1 and Nox2 but not between Nox1 and Nox4. The fact that Nox1 and Nox4 are located in distinct compartments
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Figure 10. Co-immunoprecipitation of Nox2 and Nox1 and of Nox2 and Nox4 in VSMCs. (A) Mouse anti-Nox2 antibody was used for immunoprecipitation and goat anti-Nox1 antibody was used for immunoblotting. In the case of the negative control immunoprecipitates, cell lysate was incubated with 2 mg of normal mouse IgG. (B) Goat anti-Nox1 antibody was used for immunoprecipitation and mouse anti-Nox2 antibody was used for immunoblotting. In the case of the negative control immunoprecipitates, cell lysate was incubated with 2 mg of normal goat IgG. (C) Anti-Nox2 antibody was used for immunoprecipitation and rabbit anti-Nox4 antibody was used for immunoblotting. (D) Anti-Nox4 antibody was used for immunoprecipitation and anti-Nox2 antibody was used for immunoblotting. In the case of the negative control immunoprecipitates, cell lysate was incubated with 2 mg of normal rabbit IgG. Total protein lysate from mesenteric VSMCs (Western blot sample) were used as positive control. Immunoblots are representative of 3 identical experiments. (E) Nox2 and Nox1 co-localization in WKY and SHR vascular smooth muscle cells. Double-labeling indirect immunofluorescence using antiNox1 antibody with Cy3-conjugated secondary antibody and anti-Nox2 antibody with Alexa 488-conjugated secondary antibody and optical sectioning with confocal microscopy show that Nox1 is co-localized with Nox2. Regions of co-localization appear as yellow. Nuclei appear in blue. Images were taken at the midplane level. Image size 238 238 mm. Representative images of 3 experiments.
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Rac at short stimulation times up to 30 minutes.63 In our experimental conditions (long stimulations of 2 or 4 hours), the specific Rac inhibitor EHT1864 did not affect Ang II– or ET-1–induced NAD(P)H oxidase activation in VSMCs from any strain apparently excluding the participation of Rac in long-term stimulations. In summary, our findings indicate that VSMCs from resistance arteries possess Nox1, Nox2, and Nox4; that Nox1 and Nox2 co-localize in the cell membrane; and that Nox homologues, through differential regulation, play an important role in NAD(P)H oxidase-driven ROS generation in WKY and SHR cells in basal and in Ang II–stimulated conditions. ET-1 also regulates Nox-based NAD(P)H oxidase, but such effects are present only in WKY cells. These data highlight the complexity of the Nox system in VSMCs, emphasizing that more than one Nox family member, alone or in association, may be involved in NAD(P)H oxidase–mediated ROS production and that Nox1 regulation by Ang II, but not by ET-1, may be important in ROS formation in SHR. Our findings provide new insights into redox mechanisms underlying vascular pathology in hypertension.
Figure 11. Regulation of Nox isoforms in VSMCs from normotensive (WKY) and spontaneously hypertensive rats (SHR). Vascular smooth muscle cells express 3 isoforms of the gp91phox (Nox2) subunit of the NADPH oxidase, including Nox1, Nox2, and Nox4. Expression and activity of Nox isoforms is higher in cells from SHR. Nox1 and probably Nox4 in cells from WKY and Nox1 and Nox2 in SHR contribute to basal ROS production. Upon stimulation with Ang II, all Nox isoforms are upregulated in cells from WKY but only Nox1 is upregulated in cells from SHR. This stimulation generates ROS production mainly through Nox1 in WKY and through Nox1 and Nox2 in SHR probably because of the interaction between the 2 Nox isoforms. ET-1 upregulates all Nox isoforms and increases NADPH oxidase activity in cells from WKY but had no effect in cells from SHR. Both Nox1 and Nox2 participate in ET-1–induced ROS generation in vascular smooth muscle cells from WKY. Nox1, Nox2, and Nox4 require p22phox (p22) for their activation. Solid traces indicate basal ROS production and dashed traces indicate agonist-induced ROS generation. AngII, angiotensin II; ET, endothelin-1.
suggests that they may influence specific cellular functions.15,5961 The functional significance of Nox1 and Nox2 acting in association is unclear, but the complex may provide an efficient system for NAD(P)H oxidase regulation and controlled ROS production. This awaits further clarification. The small G-protein Rac-1 is involved in Nox1-dependent 62 In addition, Ang II–induced stimulation O 2 production. of NAD(P)H oxidase has been reported to be dependent on
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