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1Hypertension and Vascular Research Division, 2Division of Vascular Surgery, and 3Biostatistics and Research Epidemiology Department, Henry Ford Health System, Detroit, Michigan; and 4Myocardial Biology Unit, Boston University Medical Center, Boston, Massachusetts
Submitted 27 June 2005 ; accepted in final form 31 October 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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NADPH oxidase; p47phox; adventitia; restenosis
NADPH oxidase is a well-characterized ROS-generating system that catalyzes the one-electron reduction of O2 to O2–, a precursor of a variety of other ROS. The classical phagocyte NADPH oxidase is known to be a multicomponent enzyme complex that includes the two membrane-spanning polypeptide subunits p22phox and anchoring component gp91phox, which are associated with the plasma membrane cytoskeleton (and together comprise flavocytochrome b558), along with three cytoplasmic polypeptide subunits, p40phox, p47phox, and p67phox (8, 43). The cytosolic guanine nucleotide-binding protein p21rac, a member of the Ras family of proteins, is required for oxidase activation (20). Exposure of the cell to agonists induces interaction of cytosolic and membrane-associated components and activates the dormant oxidase (8). Numerous studies have suggested a similar process of oxidase activation in the vasculature (16, 23, 26, 29, 43).
Recently, NADPH oxidases in VSMCs and adventitial fibroblasts have been implicated in the proliferation and migration of cells from the vascular wall to the neointima (13, 18). VSMCs in large arteries express p22phox, p47phox, and homologs of gp91phox but do not appear to express p67phox (21, 22, 36). In contrast, we reported that the vascular adventitia expresses four major components of the oxidase and showed that classical p67phox is involved in adventitial fibroblast O2– production (27, 40). In smooth muscle cells and adventitial fibroblasts, critical components of NADPH oxidase are upregulated in response to vascular injury and atherosclerosis, and ROS derived from NADPH oxidase appear to be involved in cell proliferation and migration (2, 38). We recently reported that delivery of a peptide targeting the interaction of p47phox and gp91phox (gp91ds) attenuates neointimal hyperplasia in vivo (18). Thus we postulated that therapies targeting another essential component of the NADPH oxidase system, p67phox, would prove useful in treating vascular injury. Moreover, in light of the growing interest in migrating adventitial fibroblasts playing an important role in neointimal hyperplasia, we examined the influence of p67phox targeting on fibroblast proliferation in vitro.
In this study, we examined for the first time the ability of dominant-negative p67phox (p67dn) to attenuate neointimal hyperplasia in response to balloon angioplasty and compared it with the effects of gp91ds. Focusing on the ability of adventitial fibroblasts to participate in this response, we also compared the dose-dependent effects of each inhibitor on fibroblast proliferation in vitro. Our data suggest that p67dn attenuates angioplasty-induced increases in vascular neointima formation, but not to the same degree as gp91ds. Interestingly, p67dn had no effect on adventitial fibroblast proliferation in vitro, whereas gp91ds inhibited proliferation. These data appear to suggest that p47phox plays a greater role in adventitial fibroblast proliferation and neointimal hyperplasia than p67phox.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Ad-gp91ds.
We designed a bicistronic adenoviral expression vector, Ad-PDGF
R-gp91ds-IRES-eGFP (Ad-gp91ds) to coexpress gp91ds inhibitory peptide and enhanced green fluorescent protein (eGFP) under the control of the platelet-derived growth factor- receptor (PDGFR). The pPDGFR-luciferase plasmid (3) was generously provided by Dr. K. Funa (Göteborg University, Göteborg, Sweden). A 1,468-bp portion of the pPDGFR-luciferase plasmid was removed by Sac I digestion (3) (PDGFR promoter sequence) and inserted into a replication-deficient human adenovirus serotype 5 (Ad5) vector. This 1,468-bp portion of the pPDGFR-luciferase vector was placed upstream from a Kozac sequence (GCC-ACC-ATG), followed by the nucleotide sequence (TGC-TCG-ACA-AGG-ATT-CGA-AGA-CAA-CTG) encoding the gp91ds peptide, an internal ribosome entry site (IRES) sequence, a 719-bp sequence encoding eGFP, and a sequence for simian virus 40 pRep8 poly(A).
Ad-p67dn. Adenovirus expressing p67dn (Ad-p67dn) was constructed at the Myocardial Biology Unit, Boston University, by placement of a cytomegalovirus (CMV) promoter upstream from the transcript encoding p67dn (substitution of alanine for valine at amino acid 204) as described by Han et al. (15).
Ad-control. Ad-CMV-eGFP (University of Iowa Gene Transfer Core) was used as a control. eGFP expression was under the control of the CMV promoter.
Animals and Viral Infection
Male Sprague-Dawley rats (10–12 wk old, 300–400 g; Charles River) were anesthetized with ketamine (80 mg/kg ip) and xylazine (7 mg/kg ip), and the neck was dissected to expose the common carotid artery (CCA) as well as the internal and external carotid arteries. Ad-control, Ad-p67dn, or Ad-gp91ds in 15% pluronic gel [Poloxamer 407 NF, BASF; 3.5 x 108 plaque-forming units (pfu)/ml] was gently spread around the outside of the left CCA. The incision was closed and the animals were allowed to recover, with free access to water and food. All protocols were approved by the Institutional Animal Care and Use Committee of Henry Ford Hospital and are consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
In Vivo Balloon Injury and Morphometric Analysis of Neointima Formation
At 2 days after viral infection, the animals were anesthetized and the incision was reopened. A 4-0 silk ligature was tied around the distal portion of the left external carotid artery, and another was passed around the proximal portion but not tied. A small transverse arteriotomy was made between the ligatures, and a 2-Fr (Fogarty) embolectomy catheter (Edwards Life Sciences) was passed through the left CCA into the thoracic aorta. The balloon was distended with 0.9% saline to increase CCA diameter by 
100% and withdrawn through the CCA to cause arterial injury. Distension and withdrawal were repeated five times. After CCA injury, the balloon catheter was removed and the proximal external carotid artery suture was tied. After the incision was closed, the animals were allowed to recover, with free access to water and food. After 14 days, the vessels were perfusion-fixed with 10 ml of PBS followed by 10% formaldehyde in PBS under pressure (120 mmHg). CCAs were harvested (with care taken not to disrupt the adventitia) and embedded in paraffin, and the middle third was serially cut into 6-µm sections. Masson's trichrome staining (Sigma), performed following the manufacturer's instructions, revealed several layers of elastic laminae. Digital morphometric analysis was performed using image analysis software (Spot Diagnostics) by an examiner with no knowledge of the treatment group. When cross-sectional area was measured, medial area was determined by subtraction of the area defined by the internal elastic lamina (IEL) from the area defined by the external elastic lamina (EEL). Intimal area was determined by subtraction of the luminal area from the area defined by the IEL. We compared elastolysis visually at high magnification by examining whether the IEL and EEL were intact. Radial thicknesses of the neointima and media were measured at four points around the circumference and averaged. Myointimal proliferation of the injured carotid artery was expressed as the ratio of neointimal area and thickness to medial area and thickness, respectively. The circumferences of the IEL and EEL were measured to examine the remodeling response. Each section was examined several times, and the values were averaged.
Immunohistochemistry for Carotid Artery eGFP and p67phox Expression
Monoclonal mouse anti-eGFP (Clontech) was used to detect eGFP expression, and monoclonal mouse anti-p67phox (generously supplied by Dr. Mark Quinn) (7) was used to detect p67phox expression. Sections were heated at 58°C for 2 h, deparaffinized and hydrated using standard techniques, and then boiled in 10 mM citric acid buffer for 10 min for antigen retrieval. The sections were incubated in 0.3% H2O2 in 80% methanol for 30 min. Immunostaining was performed using a mouse IgG kit (Vector Laboratories). The sections were blocked with 10% normal horse serum for 30 min and then incubated overnight at 4°C with primary antibody (1:250 dilution for anti-eGFP and 1:20 dilution for anti-p67phox in PBS containing 2% normal horse serum). Negative controls were incubated with the same concentration of matching IgG isotype (IgG2a). Sections were incubated with biotinylated secondary antibody (1:400 dilution in PBS containing 2% normal horse serum) for 30 min and then with ABC reagent for 30 min (Vector). The reaction was visualized using diaminobenzidine tetrahydrochloride (Vector Laboratories). The sections were counterstained with hematoxylin (Sigma), and the slides were dehydrated with alcohol, cleared in xylene, and mounted.
Preparation of Rat Aortic Adventitial Fibroblasts
Aortic fibroblasts were prepared as described previously (27). Briefly, aortas from anesthetized rats were removed using sterile techniques and placed in DMEM containing 100 U/ml penicillin and 100 µg/ml streptomycin. Vessels were cleaned of perivascular adipose tissue and cut longitudinally, and the endothelium was scraped free. The tissue was incubated in DMEM containing 1 mg/ml collagenase and 0.125 mg/ml elastase for 10–15 min at room temperature. Medial smooth muscle cells were peeled from the adventitia, which was then digested in DMEM containing 1 mg/ml collagenase and 0.125 mg/ml elastase for 4 h at 37°C. The resulting solution was centrifuged, and the pellet was resuspended in DMEM containing 20% fetal bovine serum (FBS) and then seeded onto 100-mm culture dishes. These cells were considered passage 0.
Comparison of the Effect of Ad-p67dn and Ad-gp91ds on Adventitial Fibroblast Proliferation
Fibroblasts at passage 7 were seeded in six-well plates (Corning) at a density of 50,000 cells/well and grown to 60% confluence. The cells were washed extensively with Hanks' balanced salt solution to ensure total removal of serum and placed in 0% FBS-DMEM for 20 h (quiescence). Ad-control, Ad-p67dn, or Ad-gp91ds was added to individual wells for the dose-response assay (3.5 x 107, 1.0 x 108, and 3.5 x 108 pfu/ml) and allowed to transfect the cells for 4 h. The medium was removed, and 0% FBS-DMEM or 10% FBS-DMEM was added to the wells. The cells were harvested at 66 h and counted in a Coulter counter. Cell number for each treatment group was calculated as a percentage and compared with Ad-control-treated cells.
Effect of Ad-p67dn on Adventitial Fibroblast NADPH Oxidase Activity
Fibroblasts at passage 7 were seeded in 60-mm culture plates (Corning), grown to 60% confluence, and brought to quiescence, and Ad-control or Ad-p67dn (3.5 x 108 pfu/ml) was added for 4 h. The medium was replaced with 0% FBS-DMEM for 72–96 h. Vehicle (0.01 N acetic acid in saline) or ANG II (10–7 M) was added, and the plates were incubated at 37°C for 3 h. NADPH oxidase activity was measured as described previously (30) by comparison of ANG II-stimulated NADPH-dependent lucigenin (5 µM) chemiluminescence in Ad-control with that in Ad-p67dn-transfected cells.
Statistical Analyses
Values are means ± SE, with n indicating the number of experiments in vivo. Morphometric measurements were compared for differences between groups by one-way ANOVA. Body weight, IEL and EEL circumference, and cell proliferation were compared by ANOVA with repeated measures. To determine differences, pairwise testing was implemented using Hochberg's method to set the criteria for significance and adjust for multiple testing. NADPH oxidase activity was examined using Student's two-sample t-test.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Body weight rose over the course of the study in all treatment groups. However, we observed no difference in body weight among groups at the beginning or end of the study.
Transgene Expression by Adenoviral Constructs
Figure 1A shows eGFP in a cross section of the rat CCA treated with Ad-control. Staining was prominent in both adventitia and neointima. Figure 1B shows a negative control from the Ad-control group in which primary antibody for eGFP was replaced by IgG2a. In Fig. 1C, eGFP staining in a CCA treated with Ad-gp91ds demonstrates substantially lighter staining, primarily in the adventitia. At higher magnification (Fig. 1D), staining was associated primarily with fusiform adventitial fibroblasts. We also confirmed an effect of the p67dn construct by immunohistochemistry using monoclonal p67phox (7). As shown in Fig. 1, E and F, reactivity for p67phox is reduced in Ad-p67dn-treated (n = 4) compared with Ad-control-treated carotid arteries (n = 5). A negative control, in which primary antibody was replaced with IgG2a, showed no reactivity (not shown). These data are consistent with reduced expression of the wild-type protein. Alternatively, reduced specific binding of the antibody as a consequence of overexpressed mutated p67dn may have resulted in lower overall reactivity. In both cases, p67phox activity would be reduced, resulting in lower ROS production.
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Figure 2 shows representative cross sections of injured CCAs transfected with Ad-gp91ds and Ad-p67dn vs. Ad-control. At 14 days after injury, in the control group, growth in neointimal and medial areas was approximately equal (Fig. 2A). Neointimal-to-medial area ratio appeared partially reduced in CCAs treated with Ad-p67dn (Fig. 2B) and markedly reduced in those treated with Ad-gp91ds (Fig. 2C).
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Contrary to the changes in neointimal area, neither inhibitor construct had any effect on medial area. Digital quantification of medial area (Fig. 5A) and medial thickness (Fig. 5B) did not vary across treatment groups.
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To examine whether either treatment had any effect on vessel size, we digitally determined IEL and EEL circumference. Figure 6, A and B, suggests a slight trend toward larger IEL and EEL circumference in the p67dn and gp91ds groups than in the control group; however, these data did not reach significance.
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To examine the ability of each construct to inhibit cell proliferation more thoroughly, we tested the effect of a range of concentrations of each virus on adventitial fibroblast proliferation in vitro. We did not observe an inhibitory effect of either virus on fibroblast proliferation at 42 h after 10% serum treatment at any concentration tested. However, we observed a 35% reduction in cell number at 66 h of serum treatment with 3.5 x 108 pfu/ml Ad-gp91ds (Fig. 7), the same concentration that proved effective in vivo. Interestingly, Ad-p67dn did not affect adventitial fibroblast proliferation at any concentration.
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We previously established that gp91ds inhibits whole vessel and adventitial fibroblast ROS production (9, 18, 25, 30). Thus we examined whether Ad-p67dn inhibits fibroblast NADPH oxidase activity, despite its lack of effect on proliferation. Figure 8 shows that ANG II-stimulated NADPH oxidase activity in adventitial fibroblasts was inhibited by 47% by treatment with 3.5 x 108 pfu/ml Ad-p67dn vs. Ad-control.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Immunohistochemistry for eGFP revealed that adenoviral constructs delivered the marker gene to the perivascular cells and neointima of the rat CCA. Because there are no available means of immunologically distinguishing endogenous p67phox from p67dn (a 1-amino acid substitution from the endogenous form), we were unable to assess p67dn delivery directly. Thus, as a control, we chose a vector expressing eGFP under the direction of the same CMV promoter, allowing us to use eGFP to determine p67dn distribution. When we performed immunohistochemistry for p67phox, we found a marked decrease in p67phox reactivity in Ad-p67dn- vs. Ad-control-treated carotid arteries. These data are consistent with reduced expression of the wild-type p67phox. Alternatively, our data may suggest reduced specific binding of the antibody to the overexpressed mutated p67dn, resulting in decreased reactivity under the stringency of the immunohistochemical assay. In either case, p67phox activity would be reduced in the Ad-p67dn group, resulting in lower ROS production.
Expression of eGFP in CCAs infected with Ad-gp91ds seems to be localized primarily to adventitial fibroblasts but is expected to include other proliferating cells. Limited adventitial eGFP expression early after injury (1 day) is consistent with the presence of its fibroblast-active PDGFR promoter, which has been reported to direct expression in cells of a proliferative phenotype (3). In fact, in our recent report examining the mouse carotid artery, we showed that Ad-gp91ds caused the inhibitor to be expressed primarily in fibroblasts; however, a subpopulation of macrophage-like cells also appeared to express the inhibitor (25).
Practically speaking, the lower overall eGFP expression in Ad-gp91ds vs. Ad-control raised the question of whether higher eGFP expression in the controls may have contributed artificially to heightened neointimal growth and thus enhanced the effect of the inhibitory construct. This does not appear to be the case, because neointimal size and neointimal-to-medial area ratios were lower in Ad-eGFP-treated CCAs than in non-virus-treated controls (18).
Neointimal hyperplasia is characterized by VSMC proliferation and migration to the neointima during injury. Recent studies suggest that adventitial myofibroblasts derived from fibroblasts also proliferate and migrate to the site of injury (24, 42). Because fibroblast proliferation is ROS dependent (17), inhibition of ROS production in adventitial fibroblasts may be an important means of attenuating the direct contribution of fibroblasts to injury- and atherosclerosis-related hyperplasia. Perhaps more importantly, perivascular oxidases appear to have an indirect paracrine effect on the vascular wall via the release of tissue-permeant ROS such as H2O2, which can stimulate medial smooth muscle cells to undergo a change from the contractile to the proliferative and migratory phenotype (31). Regardless of a direct or indirect effect on proliferation, adventitial NADPH oxidase is increasingly being seen as an early mediator in this response (9). Our present findings are consistent with both a paracrine and a direct effect of fibroblast oxidase inhibition on vascular cell hyperplasia.
Several reports have shown an association between various forms of vascular NADPH oxidase and neointimal growth (28, 38). Moreover, studies have clearly shown that phosphorylation and translocation of the classical cytosolic component p47phox to the plasma membrane participate in the process of vascular NADPH oxidase activation (23, 29). We recently confirmed that interaction between p47phox and gp91phox in NADPH oxidase is involved in the production of vascular O2– and neointimal growth by showing that gp91ds-tat (a novel peptide inhibitor of p47-gp91 interaction and precursor of Ad-gp91ds) inhibits stretch-induced O2– production and neointimal growth of the rat CCA (18). We infused gp91ds-tat or vehicle control into rats for 2 days and then examined O2– after stretch. We observed a significant reduction in ROS and neointimal proliferation by gp91ds-tat (18); in subsequent studies, we demonstrated that the active sequence gp91ds was capable of markedly reducing whole vessel and fibroblast ROS levels (9, 25) and neointimal proliferation (9).
In the present study, on the basis of substantial evidence showing that vascular NADPH oxidase is a complex system composed of at least five essential components (31), we compared the antihyperplastic effect of delivering Ad-gp91ds vs. Ad-p67dn to the adventitia. We and others showed that classical NADPH oxidase components gp91phox, p47phox, and p67phox are present in the aortic adventitia of the rabbit and rat (27, 40) and postulated that adventitial oxidase plays a role in the promotion of neointimal growth (31). For this reason, we examined the potential contribution of adventitial p67phox per se to vascular neointimal growth compared with the p47phox-gp91phox interaction. We previously showed that p67phox is involved in adventitial fibroblast NADPH oxidase activity (21, 22, 36). However, consistent with a lesser role of adventitial p67phox in stretch-induced neointimal growth, the present morphometric data suggest a reduction in neointimal growth in adventitially applied Ad-p67dn-treated CCAs compared with viral controls, as changes closely approached statistical significance (45%, P = 0.058). On the other hand, Ad-gp91ds caused statistically significant inhibition compared with control (67%, P = 0.001). These data indicate that adventitial p47phox is critically involved in NADPH oxidase-mediated neointimal growth and suggest a role for classical p67phox, particularly because gp91ds expression seemed more limited than p67dn expression (Fig. 1B vs. 1A). Moreover, the lesser effect of Ad-p67dn may be attributed in part to the presence of the p67phox homolog NOXA1 (12). It is unclear whether NOXA1 is important for adventitial or medial NADPH oxidase activity. Because inhibitors of this homolog are unavailable, we are currently unable to address the contribution of NOXA1 relative to that of p67phox.
Because a number of previous studies suggested a role for migrating adventitial fibroblasts per se in neointimal hyperplasia (35), we compared the effectiveness of Ad-p67dn vs. Ad-gp91ds to reduce fibroblast proliferation in vitro. Interestingly, we observed no effect of Ad-p67dn over a range of concentrations, including the highest concentration that showed a partial effect in vivo. In contrast, Ad-gp91ds caused a 35% reduction in fibroblast proliferation (compared with a 67% reduction in neointimal hyperplasia in vivo). Indeed, the greater effect of these constructs in vivo is consistent with the expected participation of multiple cell types in neointimal hyperplasia. That is, the enhanced effect of both adenoviral constructs in vivo appears to suggest that ROS derived from adventitial cell types other than fibroblasts play an important role in vascular cell hyperplasia and migration in this model. It is also noteworthy that Ad-p67dn did not inhibit fibroblast proliferation, despite its ability to decrease NADPH oxidase activity. Thus it is tempting to speculate that these findings suggest a difference in the function of Ad-p67dn and gp91ds; i.e., their antiproliferative actions may depend on the particular isoforms and/or subcellular localizations of NADPH oxidase that are affected.
In conclusion, our data are consistent with the involvement of a multicomponent adventitial phagocyte-like NADPH oxidase in neointimal growth. The data indicate that classical p47phox is critically involved in this response, whereas they appear to suggest only partial involvement of classical p67phox. However, future studies examining the relative contribution of their homologs, as well as other vascular cells, are necessary. Our findings also shed light on the possible clinical utility of perivascular application of viral inhibitors to reverse deleterious processes involving vascular oxidases.
Perspectives
NADPH oxidases are increasingly being recognized as important mediators in a complex signaling cascade leading to vascular growth. Studies show that the vascular growth response involves c-Src-mediated NADPH oxidase assembly and includes activation of a host of tyrosine and serine/threonine kinases, leading to activation of transcription factors, cell hypertrophy, and proliferation (5, 10, 14, 39). With the discovery of homologs of the various NADPH oxidase components in the vasculature, including gp91phox homologs nox1 and nox4, as well as interaction with classical p22phox (1) and novel cytosolic components (4), clearly the cellular effects of oxidase activation are expected to be multifactorial. Thus a multifaceted, combinatorial pharmacological approach to oxidase inhibition will likely be necessary to attenuate cell growth.
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ACKNOWLEDGMENTS |
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R promoter, as well as Dr. Mark Quinn for the supply of the p67phox antibody. |
FOOTNOTES |
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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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REFERENCES |
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-receptor promoter. Biochem Biophys Res Commun 210: 165–173, 1995.[CrossRef][ISI][Medline]This article has been cited by other articles:
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