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gp91^phox Contributes to NADPH oxidase activity in aortic fibroblasts but not smooth muscle cells

Departments of Internal Medicine and Free Radical and Radiation Biology Program, University of Iowa, Iowa City, Iowa 52242

Submitted 20 May 2003 ; accepted in final form 8 July 2003

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Reactive oxygen species (ROS) derived from vascular NADPH oxidase are important in normal and pathological regulation of vessel growth and function. Cell-specific differences in expression and function of the catalytic subunit of NADPH oxidase may contribute to differences in vascular cell response to NADPH oxidase activation. We examined the functional expression of gp91^phox on NADPH oxidase activity in vascular smooth muscle cells (SMC) and fibroblasts (FB). As measured by dihydroethidium fluorescence in situ, superoxide levels were greater in adventitial cells compared with medial SMC in wild-type aorta. In contrast, there was no difference in levels between adventitial cells and medial SMC in aorta from gp91^phox-deficient (gp91^phox KO) mice. Adventitial-derived FB and medial SMC were isolated from the aorta of wild-type and gp91^phox KO mice and grown in culture. Consistent with the observations in situ, basal and stimulated ROS levels were reduced in FB isolated from aorta of gp91^phox KO compared with FB from wild-type aorta, whereas ROS levels were similar in SMC derived from gp91^phox KO and wild-type aorta. There were no differences in expression of superoxide dismutase between gp91^phox KO and wild-type FB to account for these observations. Because gp91^phox is associated with membranes, we examined NADPH-stimulated production in membrane-enriched fractions of cell lysate. As measured by chemiluminescence, NADPH oxidase activity was markedly greater in wild-type FB compared with gp91^phox KO FB but did not differ among the SMCs. Confirming functional expression of gp91^phox in FB, antisense to gp91^phox decreased ROS levels in wild-type FB. Finally, deficiency of gp91^phox did not alter expression of the gp91^phox homolog NOX4 in isolated FB. We conclude that the neutrophil subunit gp91^phox contributes to NADPH oxidase function in vascular FB, but not SMC.

reactive oxygen species; blood vessel

Many of the effects of vascular cells appear to be mediated, in part, by cellular-derived reactive oxygen species (ROS). ROS have multiple effects in blood vessels. In addition to the well-described inactivation of nitric oxide and oxidation of lipoproteins, ROS participate in intracellular signaling and regulation of gene expression (7, 12). In this way, vascular cell-derived ROS may regulate vessel growth and function in atherosclerosis, hypertension, and diabetes mellitus (3, 6).

Although there are several potential sources of ROS in the blood vessel, a phagocyte-like NADPH oxidase is thought to be the major source of (6). In phagocytes, NADPH oxidase generates through the assembly of a multisubunit protein complex composed of the membrane-associated flavin and heme containing gp91^phox and the smaller p22^phox. Cytosolic components p47^phox and p67^phox are involved in activation of the oxidase after stimulation of the cell (2). It has recently been suggested that the phagocyte gp91^phox subunit is not involved in vascular cell NADPH oxidase activity (19). However, this study failed to examine individual cell types within the vessel wall. Gorlach et al. (5) have shown that gp91^phox is expressed and contributes to production and intracellular signaling in endothelial cells. Expression of gp91^phox protein has been reported in arteriolar SMC (20) but is not found in aortic SMC (6). The role of gp91^phox in FB is less well defined; however, gp91^phox is expressed in adventitial cells and appears to contribute to generation of adventitial (11, 13).

Complicating the characterization of the vascular NADPH oxidase is the finding that homologs of gp91^phox are expressed in vascular cells and may contribute to NADPH oxidase-derived ROS (6). The effect of gp91^phox deficiency on NADPH oxidase activity in isolated FB and SMC has not been studied. We hypothesized that deficiency of gp91^phox would reduce NADPH oxidase activity in isolated FB but not aortic SMC. We also examined whether expression of NOX4, the most abundant gp91^phox homolog in vascular cells, is altered by deficiency of gp91^phox.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Tissue culture. Adventitial FB and medial SMC were cultured from the thoracic aorta of wild-type and gp91^phox-deficient (gp91^phox KO) mice (Jackson Laboratory; Bar Harbor, ME). The loosely adhering perivascular tissue was trimmed from the aorta, and the endothelium was removed by scraping. Adhering adventitial cells were microdissected from the vessel. The adventitial layer and the medial layer were subjected to enzymatic digestion and culture as described (9). Three independent isolations of gp91^phox KO SMC and FB and three parallel isolations of wild-type control SMC and FB were obtained. Cells were grown in 10% fetal calf serum in DMEM containing 1% basal medium Eagle, 1% glutamine, 1% penicillin-streptomycin, 1% MEM 100x nonessential amino acids, and 2% HEPES. FB were confirmed by negative -actin staining and SMCs by positive -actin staining at second passage. Studies were performed at passages 4 to 9 and 70–90% confluence. All experimental protocols were approved by the University of Iowa Animal Care and Use Committee and conform to American Physiological Society guidelines for the use and care of laboratory animals.

Fluorescent dyes for detection of ROS. Immediately after harvest, aortic segments were rinsed in cold PBS and flash frozen in OCT mounting compound (Sakura). Perivascular tissue was not dissected away from the vessel so as to minimize trauma to the adventitial cells before measuring ROS levels. Aortas were sectioned (30 µm) and incubated in 5- and 6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (DCFH; 2 x 10^–6 M) or dihydroethidium (DHE; 2 x 10^–6 M) for 30 min at 37°C as previously described (13). Cultured SMC and FB were grown on chamber slides, serum-deprived (0.1% serum) overnight, washed with PBS, and incubated with DCHF or DHE for 30 min at 37°C. Cells were rinsed with PBS and coverslipped. Fluorescence was detected with a Bio-Rad laser scanning confocal microscope (excitation at 488 nm and detection at 585 nm by using a long-pass filter). Control wild-type and gp91^phox KO vessels or cells were analyzed in parallel under identical laser settings. Relative superoxide levels within vessel segments were quantitated by determining relative fluorescence per cell in the adventitia and media by using Scion Image (version 4.0).

In other experiments, SMC and FB were grown in 35-mm dishes, washed in PBS, and incubated in DCFH or DHE for 30 min in the dark at 37°C. Cells were washed with PBS, detached with trypsin, and collected by centrifugation. Fluorescence was measured by a Becton Dickinson (Franklin Lakes, NJ) FACScan, and data were analyzed with WinMDI flow cytometry software. These data are reported as relative fluorescent units per cell normalized to control cells. In some experiments, NADPH oxidase was activated by the addition of phorbol 12,13-dibutyrate (10^–6 M) 15 min after the addition of the fluorescent dye.

Chemiluminescence. SMC and FB were grown on 35-mm dishes, serum deprived overnight, and rinsed in PBS, and basal was measured by lucigenin (5 x 10^–6 M)- or coelenterazine (10^–5 M)-enhanced chemiluminescence in a luminometer (model FB12; Zylux) after 10 min of dark adaptation. In other experiments, cells were detached by trypsin, collected by centrifugation, and rinsed in PBS. Cells were suspended in ice-cold protease inhibitor buffer and sonicated on ice. The homogenate was centrifuged at 500 g for 10 min, the supernatant centrifuged at 30,000 g for 30 min, and then the supernatant centrifuged at 75,000 g for 1 h at 4°C. The pellet was suspended in protease inhibitor buffer and protein content was determined. NADPH oxidase activity of 20 µg protein was measured as the diphenyleneiodonium (DPI; 10^–4 M)-inhibited lucigenin (5 x 10^–6 M)- or methyl-cypridina-luciferin analog (MCLA; 10^–6 M)-enhanced chemiluminescence after the addition of NADPH (10^–4 M) (10). Chemiluminescence data are reported as relative light units per second normalized to cell number or protein concentration.

Western blot analysis. Cells were rinsed with PBS, harvested in SDS lysis buffer, and boiled for 5 min. Proteins were separated by using 4–20% Tris · HCl ready gel and transferred onto nitrocellulose membrane. The membrane was probed with mouse monoclonal anti-gp91^phox (1:1,000), sheep polyclonal anti-CuZn-SOD (1:1,000), or rabbit polyclonal anti-Mn-SOD antibodies (1:1,000) at 4°C overnight. Secondary anti-mouse, anti-sheep, or anti-rabbit antibodies (1:10,000) were added at room temperature for 1 h and detected after the addition of SuperSignal West Pico chemiluminescent substrate using Kodak film.

gp91^phox Antisense. Cells were grown to 40–50% confluence and treated with the single-stranded antisense oligonucleotide (ODN) mixed with Oligofectamine (Invitrogen; Carlsbad, CA) as per the manufacturer's instructions. Expression of gp91^phox and levels were measured after 24 h. The gp91^phox antisense ODN had the sequence 5'-AACTGGGCTGTGAATGAAG-3', and the scrambled control ODN had the sequence 5'-CATTGTGGAGTGACAGGAG-3'. The antisense targeted base pairs 7–25 of the coding sequence of gp91^phox mRNA (21).

NOX4 RT-PCR. Real-time PCR was used to measure mRNA expression of the gp91^phox homolog NOX4. Total mRNA was isolated from cultured SMC and FB by using Tri-Reagent (Molecular Research Center; Cincinnati, OH). Relative copy number of NOX4 was determined by using the QuantiTect SYBRgreen PCR kit (Qiagen; Valencia, CA) and ABI Prism 7000 (Applied Biosystems; Foster City, CA) detection system normalized to ribosomal 18S RNA. The primer pairs for NOX4 were sense 5'-CTGGTCTGACGGGTGTCTGCATGGTG-3' and antisense 5'-CTCCGCACAATAAAGGCACAAAGGTCCAG-3' (GenBank accession no. AB 041034).

Chemicals. Anti-gp91^phox (monoclonal antibody 54.1) was obtained by permission from Dr. A. J. Jesaitis, Department of Microbiology, Montana State University. Anti-CuZn-SOD was obtained from Calbiochem (San Diego, CA). Anti-Mn-SOD was a gift from Dr. Larry Oberley, University of Iowa. Anti-mouse, anti-sheep, and anti-rabbit secondary antibodies were obtained from Upstate Biotechnology (Waltham, MA). Coelenterazine, DCFH, and DHE were obtained from Molecular Probes (Eugene, OR). All other chemicals, unless indicated, were obtained from Sigma-Aldrich (St. Louis, MO).

Statistical analysis. Results are expressed as means ± SE. Statistical comparisons were performed by Student's two-tailed t-tests or ANOVA as appropriate. A value of P < 0.05 was considered significant.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Superoxide levels, as measured by DHE fluorescence, were greater in adventitial cells compared with medial SMC in wild-type aorta (Fig. 1). In contrast, levels were similar in the adventitial cells and medial SMC in gp91^phox KO aorta. This difference in relative levels between adventitial and medial cells resulted from increased in adventitial cells of wild-type aorta compared with cells within the adventitia of gp91^phox KO aorta. These observations suggest that gp91^phox contributes to the generation of in adventitial FB but not SMC.

View larger version (27K): [in this window] [in a new window]   Fig. 1. Basal levels in aorta from wild-type (wt) and gp91phox-deficient (gp91phox KO) mice. Aortic sections were incubated with dihydroethidium (DHE), and fluorescent images were obtained by confocal microscopy. Arrow indicates endothelium. Right, summary data of the ratio of adventitial cell to medial smooth muscle cells (SMC) fluorescence after DHE staining. Open symbols, data from individual aorta; closed symbols, means ± SE. RFU, relative fluorescent units (n = 7–9 aorta sections; P < 0.05).

To better evaluate the functional role of gp91^phox in NAPDH oxidase activity in vascular cells, we isolated SMC and FB from the aorta of wild-type and gp91^phox KO mice. Under quiescent conditions, levels were greater in FB compared with SMC cultured from wild-type aorta (Fig. 2A). In contrast, levels were similar in SMC and FB isolated from gp91^phox KO aorta. These observations were confirmed by measurement of by coelenterazine chemiluminescence (data not shown). Western blotting confirmed the absence of gp91^phox in FB cultured from the gp91^phox KO aorta (Fig. 2B).

View larger version (18K): [in this window] [in a new window]   Fig. 2. A: basal production in cultured cells. Fibroblasts (FB) and SMC were cultured from wt and gp91phox KO aorta. was measured by lucigenin-enhanced chemiluminescence and is reported as relative light units (RLU) · min–1 · 104 cells–1 (n = 6–8, *P < 0.05 vs. SMC). B: expression of gp91phox. Western blotting for gp91phox in spleen and FB from wt and gp91phox KO aorta is shown.

Intracellular levels of ROS were further characterized in FB with the fluorescent dyes DHE and DCFH. Superoxide (Fig. 3, A and C) and peroxide-derived (Fig. 3, B and C) radicals were greater in FB from wild-type aorta compared with FB from gp91^phox KO aorta. Differences in ROS levels between wild-type and gp91^phox KO FB were not due to differences in expression of intracellular superoxide dismutase (Fig. 3D).

View larger version (35K): [in this window] [in a new window]   Fig. 3. Reactive oxygen species (ROS) levels in cultured FB. A: basal intracellular levels of FB measured by DHE fluorescence using confocal microscopy. B: basal peroxide-derived radicals in FB measured by dichlorodihydrofluorescein (DCFH) fluorescence (RFU/cell) using flow cytometry. C: summary data of basal ROS levels in gp91phox KO FB measured by DCFH (solid bar) and DHE (crosshatched bar) by using flow cytometry. ROS levels are reported as RFU per cell normalized to wt (open bar) (n = 13 aorta sections, *P < 0.05 vs. wt). D: Western blotting for CuZn-SOD and Mn-SOD in wt- and gp91phox KO-cultured FB is shown. Data are representative of 3 separate experiments.

Activation of NADPH oxidase by phorbol ester increased intracellular ROS levels in wild-type-derived aortic FB, but not FB from gp91^phox KO aorta (Fig. 4A). In comparison, the mitochondrial electron chain uncoupler menadione increased levels similarly in wild-type and gp91^phox KO FB (2.0 ± 0.2 vs. 1.9 ± 0.3-fold normalized to untreated FB, n = 3).

View larger version (9K): [in this window] [in a new window]   Fig. 4. NADPH oxidase activity. A: cultured FB were stimulated with phorbol 12,13-dibutyrate (PDB; solid bars), and ROS levels were measured by DCFH fluorescence and flow cytometry. ROS levels are reported as RFU per cell normalized to untreated FB (open bars) (n = 4 aorta, *P < 0.05 vs. untreated). B: membrane-enriched fractions of cells cultured from wt (open bars) and gp91phox KO (filled bars) aorta were treated with NADPH (0.1 mM) and measured by lucigenin-enhanced chemiluminescence (n = 5 aorta sections, *P < 0.05 vs. wt FB).

Because gp91^phox is a membrane-associated subunit of NADPH oxidase, SMC and FB membrane-enriched fractions were treated with NADPH and measured by lucigenin-enhanced chemiluminescence (Fig. 4B). NADPH-stimulated production was similar in SMC isolated from both wild-type and gp91^phox KO aorta. In contrast, NADPH-stimulated was markedly reduced in membrane fractions of gp91^phox KO FB compared with wild-type FB. This finding of decreased NADPH oxidase activity in FB of gp91^phox KO aorta was confirmed by MCLA chemiluminescence (277 ± 2.1 vs. 210 ± 2.1 RLU · min^–1 · mg protein^–1, n = 3). The flavoenzyme inhibitor DPI reduced NADPH-stimulated generation by >70%.

Further confirming that gp91^phox contributes to NADPH oxidase activation and generation of in FB, wild-type FB were found to have decreased levels of after treatment with antisense to gp91^phox (Fig. 5). Western blotting verified reduction in gp91^phox protein levels in murine monocytes after antisense treatment (Fig. 5).

View larger version (29K): [in this window] [in a new window]   Fig. 5. Effect of gp91phox antisense. A: Western blotting of cultured murine monocytes after treatment with vehicle (veh) or gp91phox antisense (1.0 and 1.5 µM). B: levels as measured by DHE fluorescence using confocal microscopy in FB cultured from wt and gp91phox KO aorta after treatment with veh or gp91phox antisense (1.0 and 1.5 µM). Data are representative of 3 separate experiments.

NOX4 is a homolog of gp91^phox and is presumed to serve as a catalytic subunit for NADPH oxidase (6). Real-time PCR confirmed expression of NOX4 in wild-type SMC and FB (SMC, 1.5 ± 0. 4 and FB, 2.6 ± 0.8 NOX4/10³ copies 18S, n = 3). NOX4 levels in gp91^phox KO FB (2.3 ± 0.8 NOX4/10³ copies 18S, n = 3) were not different than NOX4 levels in wild-type FB.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
In this study, we clearly document the importance of gp91^phox in NADPH oxidase-derived in vascular FB and the functional absence of gp91^phox in vascular SMC. Our findings indicate that expression of the gp91^phox homolog NOX4 in FB is not altered by deficiency of gp91^phox. We also demonstrate that intracellular levels of are greater in adventitial FB compared with SMC, and that gp91^phox contributes <30% of ROS levels in the quiescent FB.

A major source of ROS in vascular cells is a phagocyte-type NADPH oxidase. Activation of a vascular NADPH oxidase has been associated with vascular dysfunction in disease (3, 6). The structure of NADPH oxidase is well characterized in neutrophils, consisting of a membrane-associated cytochrome b₅₅₈ composed of a p22^phox-gp91^phox heterodimer, and the cytosolic subunits p47^phox, p40^phox, p67^phox, and Rac (2). On activation, the cytosolic subunits bind to the membrane-bound complex to form a functional oxidase. In the blood vessel, expression of the specific NADPH oxidase subunits is dependent on cell type and vessel size. gp91^phox, p22^phox, p47^phox, and p67^phox are expressed in endothelial cells and FBs, whereas gp91^phox and p67^phox are not found in SMC of aorta (6). Interestingly, gp91^phox and p67^phox are expressed in SMC isolated from resistance arteries (20).

Consistent with previous reports, we were able to detect a small amount of gp91^phox mRNA by RT-PCR (data not shown) but unable to detect gp91^phox protein in SMC derived from aorta (18). In contrast to SMC and consistent with previous reports (11), gp91^phox protein was expressed in vascular FB. For the first time, our data show functional evidence of gp91^phox-dependent generation of in isolated FB. In addition, we confirm that gp91^phox does not contribute to NADPH oxidase-derived production in aortic SMC. These observations provide further evidence that the structure of NADPH oxidase differs among vascular cell types.

Despite the functional absence of gp91^phox, SMC demonstrate NADPH oxidase activity. This is evident by an increase in SMC ROS after stimulation with a phorbol ester and by NADPH oxidase-derived in membrane-enriched SMC homogenates. The observation that NADPH-stimulated is DPI inhibitable suggests that a -generating flavoenzyme is responsible for ROS production in SMC. Recently, a family of gp91^phox homologs, referred to as NOX, have been shown to be present in vascular cells (6). The most prevalent NOX isoform in SMC and FB is NOX4 (18). If the expression of NOX4 is dependent on gp91^phox, then NOX4 expression might be altered in FB deficient in gp91^phox. For example, NOX4 levels may be expected to undergo a compensatory increase in response to a decrease in gp91^phox. However, we found no difference in NOX4 levels between wild-type and gp91^phox KO FB. The homolog NOX1 could also potentially contribute to NADPH oxidase activity in SMC and FB, but we were unable to detect NOX1 in these cells.

Our data suggest that under quiescent culture conditions, gp91^phox contributes to <30% of superoxide generation in FBs (Figs. 2A and 3C). Other cellular sources, including gp91^phox homologs, mitochondria, cytochrome P-450, and arachidonate metabolism, would be expected to contribute to the remaining ROS generation. In contrast, gp91^phox contributed to the majority of NADPH oxidase activity in FBs (Fig. 4B). Homologs of gp91^phox may also contribute to NADPH oxidase activity, supported by our demonstration of NOX4 in FBs.

It has recently been suggested that the gp91^phox of blood vessels is distinct from that found in the phagocyte (19). However, this conclusion was derived from studies in vessel segments after removal of periadventitial tissue and may be limited in that the predominant cell type in this preparation would be the SMC, which we confirm in the present study does not contain gp91^phox. Our data, together with the findings of others (5, 22), confirm that the neutrophil gp91^phox subunit does contribute to ROS generation in vascular FB and endothelium.

In the isolated blood vessel, generation of by adventitial cells modulates nitric oxide-mediated responses (4). Angiotensin II activation of a gp91^phox-based NADPH oxidase in the adventitia impairs endothelium-dependent relaxation (13). In the isolated vessel segment, we cannot confirm that the FB and not other perivascular cells, including inflammatory cells or microvascular pericytes, are primarily responsible for generation of ROS. However, our findings in culture suggest that FB contribute to the reported adventitialderived in the blood vessel.

Adventitial FB may have an important role in the pathophysiology of vascular disease (8, 14). After balloon injury, there is migration of adventitial FB to the neointima (16, 17). Endoluminal injury activates adventitial FB, resulting in an NADPH oxidase-dependent increase in ROS (15). NADPH oxidase expression is also increased in adventitial FB in atherosclerosis (1). We demonstrate the functional importance of gp91^phox in vascular FB and propose that activation of a gp91^phox-based NADPH oxidase may contribute to the vessels response to injury. Differences in the expression of NADPH oxidase subunits may be important in defining vascular cell response to activation.

	DISCLOSURES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
The project described was supported by National Heart, Lung, and Blood Institute Grants HL-03669 and HL-62984 (to F. J. Miller). Study contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.

	ACKNOWLEDGMENTS

 
The authors appreciate the expert assistance of the University of Iowa Roy J. and Lucille A. Carver College of Medicine Flow Cytometry Facility, Central Microscopy Research Facility, and DNA Facility.

	FOOTNOTES

 

Address for reprint requests and other correspondence: F. J. Miller, Jr., 200 Hawkins Rd., Rm. E315 GH, Dept. of Internal Medicine, Univ. of Iowa Hospital, Iowa City, IA 52242 (E-mail: francis-miller{at}uiowa.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 

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