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Am J Physiol Heart Circ Physiol 288: H946-H953, 2005. First published September 23, 2004; doi:10.1152/ajpheart.00413.2004
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Perivascular gene transfer of NADPH oxidase inhibitor suppresses angioplasty-induced neointimal proliferation of rat carotid artery

Hector M. Dourron,1 Gary M. Jacobson,1 James L. Park,2 Jianhua Liu,2 Daniel J. Reddy,1 Maria L. Scheel,3 and Patrick J. Pagano2

1Division of Vascular Surgery and 2Hypertension and Vascular Research Division, Henry Ford Health System, Detroit, Michigan; and 3Gene Transfer Vector Core, University of Iowa, Iowa City, Iowa

Submitted 30 April 2004 ; accepted in final form 16 September 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Vascular stretch induces NADPH oxidase-derived superoxide anion (O2–), which has been implicated in hypertrophy and cell proliferation. We hypothesized that targeted delivery of an NADPH oxidase inhibitor to the adventitia would reduce stretch-induced vascular O2– and attenuate neointima formation. We designed a novel replication-deficient adenovirus containing a fibroblast-active promoter driving expression of NADPH oxidase inhibitory sequence gp91ds (Ad-PDGF{beta}R-gp91ds/eGFP). 1) We characterized the specificity of this promoter using pPDGF{beta}R-luciferase by showing induction of luciferase in cultured rat aortic fibroblasts but not in vascular smooth muscle cells. 2) Using RT-PCR, we observed expression of gp91ds and the reporter gene in fibroblasts after infection with Ad-PDGF{beta}R-gp91ds/eGFP. 3) Using Ad-CMV-eGFP as a control, we delivered Ad-PDGF{beta}R-gp91ds/eGFP to the adventitia of the rat common carotid artery (CCA). Immunohistochemistry confirmed localized delivery of the inhibitor to the adventitia. After CCAs were injured with an embolectomy catheter, we observed a significant increase in neointima-to-media area ratio in control CCAs, which was significantly attenuated in CCAs treated with the gp91ds-expressing virus. In a second group of rats, we detected a 10-fold increase in distension-stimulated O2–, which was significantly reduced in CCAs infected with gp91ds-expressing virus. These data demonstrate that localized adventitial delivery of an NADPH oxidase inhibitor is effective in reducing overall vascular O2– and neointima formation, suggesting that adventitial NADPH oxidase plays a functional role in development of neointimal hyperplasia.

superoxide; adventitia; stenosis

NEOINTIMAL HYPERPLASIA is a major response to vascular injury (27). Proliferation and migration of vascular smooth muscle cells (VSMCs) and, more recently, fibroblasts have been implicated in narrowing of the arterial lumen in response to injury, representing one of the hallmark characteristics of atherosclerosis (8, 26, 29, 31, 41). This process of vascular hyperplasia and migration is promoted by protooncogenes (22, 28) and various growth factors (9, 10, 15, 34, 39). Recent studies have implicated vascular NADPH oxidase-derived superoxide anion (O2–) in vascular cell proliferation and neointimal growth (11, 13, 18, 33). In addition, studies have suggested a role for elevated levels of reactive oxygen species (ROS) in a number of growth-related signaling pathways (12). In smooth muscle cells, critical components of NADPH oxidase are upregulated in response to vascular injury and atherosclerosis, and ROS derived from this oxidase may be involved in cell proliferation and migration (1, 35).

Increasing attention has been given to the role of the vascular adventitia in a variety of vascular diseases, including hypertension and atherosclerosis (6, 30, 36, 42). We and others have reported that the vascular adventitia is a substantial source of ROS (1, 19, 20, 40), and previous studies suggest that adventitial NADPH oxidase plays a role in vascular remodeling after injury. It has been postulated that adventitial fibroblasts proliferate in response to the formation of ROS by NADPH oxidase, which precedes phenotypic modulation to myofibroblasts and neointima formation (14, 30, 31, 33, 43). It is also plausible that activation of adventitial NADPH oxidase has a paracrine effect, stimulating medial proliferation and migration. Thus we tested the hypothesis that targeted inhibition of NADPH oxidase in the adventitia reduces stretch-induced O2– and attenuates neointima formation.

We previously described a chimeric peptide inhibitor (gp91ds-tat) that interferes with the interaction of oxidase components gp91phox and p47phox and, in particular, its ability to specifically inhibit angiotensin II-induced aortic NADPH oxidase activity in vitro and in vivo (14, 23). We also demonstrated that systemic infusion of this inhibitor attenuated balloon angioplasty-induced neointimal hyperplasia of the carotid artery (14). In the present study, we delivered the gp91ds portion of the inhibitor locally to the adventitia to examine whether perivascular NADPH oxidase plays an important role in angioplasty-induced O2– levels and neointima formation. This involved perivascular application of a replication-deficient adenovirus containing a fibroblast-active promoter that drives expression of gp91ds. To increase the likelihood of localized expression, we designed an adenoviral construct to limit the expression of gp91ds to adventitial cells, choosing the platelet-derived growth factor (PDGF)-{beta} receptor (PDGF{beta}R) promoter, since it is active in fibroblasts (2, 38), which are abundant in large-vessel adventitia. Our data suggest that targeted perivascular delivery of a specific NADPH oxidase inhibitor effectively reduces angioplasty-induced increases in vascular O2– and neointima formation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Preparation of rat aortic fibroblasts and VSMCs. Aortic fibroblasts and VSMCs were prepared as described previously (19). Briefly, aortas from anesthetized rats were removed using sterile technique and placed in DMEM containing penicillin (100 U/ml) and streptomycin (100 µg/ml). Vessels were cleaned of perivascular adipose tissue and cut longitudinally, and the endothelium was scraped free using a sterile cotton swab. The tissue was incubated in DMEM containing collagenase (1 mg/ml) and elastase (0.125 mg/ml) for 10–15 min at room temperature. Medial smooth muscle cells were peeled from the adventitia with forceps, and the adventitia and smooth muscle were digested in DMEM containing collagenase (1 mg/ml) and elastase (0.125 mg/ml) for 4 h at 37°C. The resulting solution was centrifuged, and the pellet was resuspended in DMEM containing 20% FBS and then seeded onto 100-mm culture dishes. These cells were considered passage 0. Cells between passages 5 and 8 were used.

Luciferase reporter assay for determination of differential cell expression of the PDGF{beta}R promoter. A luciferase reporter assay using the pPDGF{beta}R-luciferase plasmid was performed to compare PDGF{beta}R promoter activity in cultured aortic smooth muscle cells and adventitial fibroblasts. FuGENE, a nonliposomal transfection reagent (Roche Applied Science), was added to 14 µg of plasmid DNA of pPDGF{beta}R-luciferase to give a 6:1 FuGENE-to-DNA ratio. The pPDGF{beta}R-luciferase plasmid (2) was generously provided by Dr. K. Funa (Göteborg University, Göteborg, Sweden). The complex was added to serum-free DMEM to give a total volume of 1 ml and added to cells grown to 80% confluence in a ratio of 7 ml of medium to 1 ml of the designated DNA complex. After cells were incubated for 8 h, fresh DMEM containing 10% FBS was added, and the cells were incubated for another 16 h. They were washed twice with sterile PBS and incubated with 1.5 ml of cell lysis buffer (Promega). Cells were stored at –70°C to precipitate out proteins and then centrifuged at 12,000 g for 10 min. Aliquots of the supernatants were used for luminescence measurements (Opticomp I) in the presence of luciferase substrate (Luciferase Assay System, Promega). Protein concentration was quantified by the Bradford assay, and data are reported as relative light units per microgram of protein.

Adenoviral constructs. Ad-PDGF{beta}R-gp91ds/eGFP virus was constructed at the University of Iowa Gene Transfer Vector Core. A 1,468-bp portion of the pPDGF{beta}R-luciferase vector was removed by SacI digestion (2) and transferred to a replication-deficient human adenovirus serotype 5 (Ad5) vector. This 1,468-bp portion of the pPDGF{beta}R-luciferase vector was placed upstream of a Kozac sequence (GCC-ACC-ATG), followed by the nucleotide sequence (TGC-TCG-ACA-AGG-ATT-CGA-AGA-CAA-CTG) encoding the gp91ds peptide, a stop sequence (TAA) and a simian virus 40 poly(A) tail. The PDGF{beta}R promoter drives expression of gp91ds. On the viral backbone, the Rous sarcoma virus promoter drives expression of the marker protein, enhanced green fluorescence protein (eGFP). As a control, we used Ad-CMV-eGFP in which eGFP expression is under the control of the cytomegalovirus (CMV) promoter (purchased from the University of Iowa Gene Transfer Core).

RT-PCR determination of gp91ds expression in rat aortic adventitial fibroblasts. Aortic adventitial fibroblasts were grown to 80% confluence in DMEM containing 10% FBS. Cells were infected with Ad-PDGF{beta}R-gp91ds/eGFP (n = 4) or control virus Ad-CMV-eGFP (n = 2) at a multiplicity of infection (MOI) of 1,000 for 8 h in DMEM containing 0.67% FBS; then the medium was replaced with DMEM + 10% FBS for 5 days. Total RNA was extracted with TRIzol reagent (GIBCO) according to the manufacturer's protocol. Amplification-grade DNase I (Invitrogen) was used to remove residual genomic DNA from the samples. cDNA was synthesized from total RNA using Superscript II reverse transcriptase and oligo(dT) primers (Invitrogen). The RT reaction was carried out at 42°C for 50 min. The products were PCR amplified using primers targeting the gp91ds coding sequence: 5'-ATGTGCTCGACAAGGATTCG-3' (forward gp91for4) and 5'-CAAATAAAGCAATAGCATCAC-3' [reverse poly(A)rev4]. When the gp91for4 and poly(A)rev4 primers are used, the predicted PCR product is 183 bp. Primers used to detect eGFP were forward eGFP-(628–645) (5'-AAAGACCCCAACGAGAAG-3') and reverse poly(A)rev4. The predicted PCR product using the eGFP-(628–645) and poly(A)rev4 primers is 208 bp when Ad-CMV-eGFP is used to infect cells or 300 bp when Ad-PDGF{beta}R-gp91ds/eGFP is used. The difference in length between the two products is a function of differential subcloning of the eGFP gene into each vector in conjunction with its poly(A) coding sequence. GAPDH was used as an internal control. Primers were forward GAPDH (forGAPDH: 5'-CCATGGAGAAGGCTGGGG-3') and reverse GAPDH (revGAPDH: 5'-CAAAGTTGTCATGGATGACC-3'). The conditions used for PCR were 94°C for 30 s (denaturing), 50°C for 30 s (annealing), and 72°C for 1 min (extension) for 35 cycles plus a hot start. PCR products were separated on a 2.0% (wt/vol) agarose gel containing ethidium bromide and visualized with ultraviolet transillumination.

Fibroblast proliferation assay. Rat vascular adventitial fibroblasts between passages 5 and 8 were used. Fibroblasts were seeded in 12-well dishes (Corning) at a density of 15,000 cells/well. Day 0 and day 1 cells were seeded in separate wells. Cells were synchronized (made quiescent) by total serum withdrawal for 48 h. At the time of serum withdrawal, cells were infected with Ad-CMV-eGFP or Ad-PDGF{beta}R-gp91ds/eGFP at an MOI of 1,000. Day 0 was considered 48 h after initial serum withdrawal, just before fibroblast stimulation with 10% FBS. Day 0 cells were trypsinized and quantified in a Coulter counter. After stimulation with 10% FBS, fibroblasts were allowed to proliferate for 24 h (day 1). Day 1 cells were trypsinized and counted in a Coulter counter. Data are expressed as percent change from day 0.

Animals and viral infection. Male Sprague-Dawley rats (9–10 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-PDGF{beta}R-gp91ds/eGFP or the control virus Ad-CMV-eGFP (University of Iowa Gene Transfer Core) in 15% pluronic gel (3.5 x 108 plaque-forming units/ml; Poloxamer 407 NF, BASF) 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 Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.

Measurement of distension-induced O2– production in vitro. Basal and distension-induced vascular O2– production were measured in isolated rat carotid arteries 2 days after viral infection, as previously described (14). Briefly, 2 days after viral infection, CCAs were harvested in ice-cold Krebs-HEPES buffer, flushed with cold buffer, cut in half, and equilibrated at 37°C for 45 min. Rings were transferred to fresh buffer at 37°C and 1) distended with a 2-F Fogarty embolectomy catheter (Baxter) to increase diameter by 100% for a sustained 60 s or 2) not distended and left untouched. Segments were transferred to a luminometer for detection of lucigenin-dependent chemiluminescence, as previously described (14).

In vivo balloon injury and morphometric analysis of neointima formation. In a separate set of rats, 2 days after viral infection with Ad-CMV-eGFP or Ad-PDGF{beta}R-gp91ds/eGFP, 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 ligatures, and a 2-F 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 injury. Distension and withdrawal were repeated five times. After injury, the balloon catheter was removed and the proximal external carotid artery suture was tied. The incision was closed, and the animals were allowed to recover, with free access to water and food. At 14 days after carotid injury, vessels were perfusion fixed with 10 ml of PBS and then 10% formaldehyde in PBS under pressure (~120 mmHg). CCAs were harvested (with care taken not to disrupt the adventitia) and embedded in paraffin. The middle third of the CCA was serially sectioned (6-µm sections). Hematoxylin-and-eosin staining 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, the medial area was determined by subtraction of the area defined by the internal elastic lamina (IEL) from that defined by the external elastic lamina (EEL). The intimal area was tabulated by subtracting the luminal area from that defined by the IEL. Elastolysis was compared visually at high magnification by examining whether the IEL and EEL were intact. Radial thickness of the neointima and media was also measured at four points around the vessel 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. Each section was examined several times, and the values were averaged.

Immunohistochemistry for carotid artery eGFP expression. Monoclonal mouse anti-eGFP (Clontech) was used to detect eGFP. The sections were heated at 58°C for 2 h, deparaffinized, hydrated, and boiled in citric acid buffer (10 mmol/l) for 10 min for antigen retrieval and then rinsed in PBS. The marker protein, eGFP, was detected using a combination of an Animal Research Kit and Catalyzed Signal Amplification Kit (DAKO) following the manufacturer's protocols to reduce cross-reaction from the secondary antibody and enhance sensitivity, respectively. Peroxidase blocking reagent (Animal Research Kit) and DAKO Biotin Blocking System were applied to the sections. Primary antibody was diluted 1:500 with DAKO antibody diluent containing background-reducing components. Primary and secondary antibody were mixed in vitro to form a biotinylated complex. Excess secondary antibody was removed using blocking reagent containing normal mouse serum. Biotinylated complex was applied to the sections and incubated for 15 min, and streptavidin-biotin complex, amplification reagent, streptavidin peroxidase, and substrate chromogen were applied according to the manufacturer's protocol (Catalyzed Signal Amplification Kit). Nuclei were visualized with hematoxylin staining.

Statistical analysis. Values are means ± SE. Morphometric results and O2– levels were compared by t-test. Multiple comparisons in the fibroblast proliferation assay were made using ANOVA with Hochberg's adjustment for multiple comparisons. Data were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Expression of PDGF{beta}R promoter-driven luciferase in adventitial fibroblasts compared with VSMCs. Luciferase activity was detected in fibroblasts transfected with PDGF{beta}R-luciferase, demonstrating PDGF{beta}R promoter activity in rat adventitial fibroblasts (68.15 ± 38.25 relative light units/µg protein). On the other hand, luciferase activity was undetectable in three preparations of VSMCs transfected with PDGF{beta}R-luciferase, suggesting that the promoter is not active in rat aortic VSMCs.

PDGF{beta}R promoter-driven expression of gp91ds in cultured adventitial fibroblasts. RT-PCR confirmed gp91ds mRNA expression in cultured adventitial fibroblasts (Fig. 1). PCR amplification of reverse-transcribed total RNA from adventitial fibroblasts infected with Ad-PDGF{beta}R-gp91ds/eGFP using gp91for4 and poly(A)rev4 primers resulted in a single band just below the 200-bp marker, indicative of gp91ds expression in cultured fibroblasts. However, cells infected with Ad-CMV-eGFP did not show such a band (Fig. 1A). Use of eGFP-(628–645) and poly(A)rev4 primers in the PCR amplification resulted in a single ~300-bp product from cells infected with Ad-PDGF{beta}R-gp91ds/eGFP or 200 bp with Ad-CMV-eGFP, consistent with eGFP expression by both constructs (Fig. 1B). Use of forward and reverse primers for GAPDH (an internal control) in the PCR amplification resulted in a single ~200-bp product of similar intensity in all samples from cells infected with Ad-PDGF{beta}R-gp91ds/eGFP or Ad-CMV-eGFP (Fig. 1C).



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  		Fig. 1. RT-PCR characterization of platelet-derived growth factor (PDGF)-{beta} receptor (PDGF{beta}R) promoter-driven expression of gp91ds in cultured rat adventitial fibroblasts. A: detection of gp91ds mRNA expression by RT-PCR in cultured rat adventitial fibroblasts infected with Ad-PDGF{beta}R-gp91ds/eGFP, but not in cells infected with Ad-CMV-eGFP. B: enhanced green fluorescent protein (eGFP) mRNA was detected in cells infected with Ad-CMV-eGFP or Ad-PDGF{beta}R-gp91ds/eGFP. C: when GAPDH was used as an internal control, it was detected in cells infected with Ad-CMV-eGFP or Ad-PDGF{beta}R-gp91ds/eGFP. Each lane represents a separate population of adventitial fibroblasts infected with Ad-CMV-eGFP or Ad-PDGF{beta}R-gp91ds/eGFP.

 
Ad-PDGF{beta}R-gp91ds/eGFP inhibited serum-induced fibroblast proliferation. To examine potential antiproliferative effects of Ad-PDGF{beta}R-gp91ds/eGFP, fibroblast growth was studied after infection with Ad-CMV-eGFP or Ad-PDGF{beta}R-gp91ds/eGFP (Fig. 2). In response to 10% FBS, cells infected with the control virus Ad-CMV-eGFP or the inhibitor-expressing virus Ad-PDGF{beta}R-gp91ds/eGFP exhibited proliferation at day 1. Proliferation was significantly inhibited by 25.7% in cells infected with Ad-PDGF{beta}R-gp91ds/eGFP.



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  		Fig. 2. Effect of Ad-PDGF{beta}R-gp91ds/eGFP on proliferation in quiescent cultured fibroblasts. Cells were infected at a multiplicity of infection (MOI) of 1,000 with Ad-CMV-eGFP (open bars) or Ad-PDGF{beta}R-gp91ds/eGFP (solid bars). Fibroblasts were stimulated to proliferate with 10% FBS. Values are means ± SE; n = 3 per group. *P < 0.05, day 1 Ad-PDGF{beta}R-gp91ds/eGFP vs. day 0 Ad-PDGF{beta}R-gp91ds/eGFP. **P < 0.05, day 1 Ad-CMV-eGFP vs. day 0 Ad-CMV-eGFP. {dagger}P < 0.05, day 1 Ad-PDGF{beta}R-gp91ds/eGFP vs. day 1 Ad-CMV-eGFP.

 
PDGF{beta}R promoter-driven expression of gp91ds in the adventitia. Application of Ad-PDGF{beta}R-gp91ds/eGFP to CCAs in vivo resulted in eGFP expression in the adventitia (Fig. 3). Immunohistochemistry for eGFP in a representative cross section of the left CCA 1 day after injury (3 days after infection) demonstrated eGFP expression primarily throughout the adventitia but not the media (Fig. 3A). Similarly, in CCAs treated with Ad-CMV-eGFP, most immunostaining was seen in the adventitia (Fig. 3B). Interestingly, expression was also evident in cells adhering to the intimal surface (Fig. 3A). Little or no background staining was evident in the negative controls, in which a control isotype IgG2a was used as primary antibody instead of the monoclonal anti-eGFP (data not shown).



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  		Fig. 3. Detection of eGFP expression in rat common carotid arteries (CCAs) using monoclonal anti-eGFP. A: representative cross section of left CCA 1 day after injury (3 days after treatment with Ad-PDGF{beta}R-gp91ds/eGFP). B: representative cross section of left CCA 1 day after injury (3 days after treatment with Ad-CMV-eGFP). Positive staining for eGFP appears brown. M, media; A, adventitia; IEL, internal elastic lamina; EEL, external elastic lamina. Original magnification x1,000.

 
Ad-PDGF{beta}R-gp91ds/eGFP, but not Ad-CMV-eGFP, reduced distension-induced carotid artery O2– production. In CCAs treated with the control virus Ad-CMV-eGFP, distension caused a 10-fold increase in O2– compared with nondistended controls (Fig. 4A). Vascular O2– was reduced by 75% (p < 0.05) in distended CCAs infected with Ad-PDGF{beta}R-gp91ds/eGFP compared with Ad-CMV-eGFP (Fig. 4B).



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  		Fig. 4. Effect of vascular distension on O2– levels in CCAs treated with Ad-CMV-eGFP and Ad-PDGF{beta}R-gp91ds/eGFP. A: O2– in CCA rings infected with Ad-CMV-eGFP under basal conditions (nondistended) or immediately after distension in rings from CCAs infected with Ad-CMV-eGFP. B: O2– immediately after distension in CCA rings treated with Ad-CMV-eGFP and Ad-PDGF{beta}R-gp91ds/eGFP. Differences between average chemiluminescence values in the presence and absence of an O2– scavenger are expressed as milliunits of chemiluminescence (mU)·min–1·mg blotted tissue wt–1. Values are means ± SE; n = 5–7 rats per group. *P < 0.05 vs. nondistended (A) and vs. Ad-CMV-eGFP (B).

 
Ad-PDGF{beta}R-gp91ds/eGFP reduced neointimal growth in the carotid artery after balloon injury. Figure 5 shows representative cross sections of injured CCAs infected with Ad-PDGF{beta}R-gp91ds/eGFP or Ad-CMV-eGFP. At 14 days after injury, neointimal growth was significantly decreased in CCAs treated with Ad-PDGF{beta}R-gp91ds/eGFP (Fig. 5B) compared with Ad-CMV-eGFP (Fig. 5A). Neointimal area, as calculated by digital quantitative analysis, was decreased fourfold in the injured CCAs infected with Ad-PDGF{beta}R-gp91ds/eGFP compared with Ad-CMV-eGFP (Fig. 6A). Similarly, the neointima-to-media area ratio was significantly decreased in CCAs infected with Ad-PDGF{beta}R-gp91ds/eGFP (Fig. 6B). In addition, neointimal thickness and neointima-to-media ratio were significantly decreased in injured CCAs infected with Ad-PDGF{beta}R-gp91ds/eGFP compared with Ad-CMV-eGFP (Fig. 6, C and D). The attenuation in neointimal thickness and neointima-to-media thickness ratio was equivalent to the changes in neointimal area and neointima-to-media area ratio in the injured CCAs infected with Ad-PDGF{beta}R-gp91ds/eGFP compared with Ad-CMV-eGFP. No difference in elastolysis was observed between groups (data not shown).



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  		Fig. 5. Cross sections of CCAs infected with Ad-CMV-eGFP (A) and Ad-PDGF{beta}R-gp91ds/eGFP (B) obtained 14 days after balloon injury. Hematoxylin-and-eosin-stained images represent cross sections from 10 rats in each group. Magnification x40. Arrows indicate external (EEL) and internal elastic lamina (IEL).

 


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  		Fig. 6. Effect of Ad-CMV-eGFP and Ad-PDGF{beta}R-gp91ds/eGFP on neointima formation. A: neointimal area (µm2 x 103) in CCAs 14 days after balloon injury. B: neointima-to-media area ratio. C: neointimal thickness. D: neointima-to-media thickness ratio. Values are means ± SE; n = 10. *P < 0.05 vs. Ad-CMV-eGFP.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our present findings indicate that perivascular delivery of an adenoviral vector expressing a novel NADPH oxidase inhibitor in the rat CCA adventitia reduced acute rises in vascular O2– in response to stretch and significantly attenuated neointimal area and neointima-to-media ratio after balloon angioplasty. Moreover, our data appear to demonstrate the ability of this vector to target a specific NADPH oxidase inhibitor to vascular adventitial fibroblasts, but not to medial smooth muscle cells. A number of reports have suggested that vascular NADPH oxidase activation is associated with proliferation of vascular cells and migration to the neointima (14, 30, 35, 41). Our data appear to confirm this association between NADPH oxidase and vascular cell proliferation and suggest that perivascular delivery of an NADPH oxidase inhibitor is an effective means of attenuating vascular cell hyperplasia and neointimal growth.

One goal of this study was to demonstrate that the PDGF{beta}R promoter provides relatively specific targeting of gp91ds expression to the adventitia, inasmuch as the PDGF{beta}R promoter is primarily active in fibroblasts, particularly those with a proliferative phenotype (38). In the present study, we first demonstrated that the PDGF{beta}R promoter is active in fibroblasts and completely inactive in smooth muscle cells, suggesting that it acts in a cell-specific manner to target fibroblasts, but not smooth muscle cells. Second, we illustrated the ability of the Ad-PDGF{beta}R-gp91ds/eGFP, but not Ad-CMV-eGFP, to cause expression of the gp91ds mRNA sequence, confirming the ability of the construct to produce gp91ds in adventitial fibroblasts. We also demonstrated that gp91ds expression in the adventitial fibroblasts resulted in attenuated fibroblast proliferation in vitro, suggesting that NADPH oxidase plays a role in fibroblast growth and proliferation. Third, after in vivo application of Ad-PDGF{beta}R-gp91ds/eGFP to the carotid artery, we confirmed viral infection via expression of eGFP primarily in the adventitia, again suggesting specific targeting of gp91ds with this viral construct.

We believe that our adenoviral construct allows specific adventitial fibroblast-targeted expression of gp91ds under the conditions of our study for several reasons. 1) The PDGF{beta}R promoter is expected to be most active in vascular fibroblasts at the time of application and infection. Contrary to this notion, previous studies suggest that VSMCs in culture derived from uninjured arteries express PDGF-like activity and the PDGF{beta} receptor. However, the PDGF-like activity in VSMCs from uninjured arteries is fivefold less than that of VSMCs from injured arteries, suggesting that injury is needed to increase the mitogenic effects of the PDGF{beta} receptor (39). Moreover, we believe that the timing of our application strategy optimizes targeting the fibroblasts; that is, when we apply the virus before injury, in vivo fibroblasts are in a more proliferative state than medial VSMCs. In the uninjured state, the VSMCs are of the contractile, not proliferative, phenotype. Therefore, the PDGF{beta}R promoter would be expected to target expression of gp91ds to proliferative fibroblasts of the adventitia before induction of balloon-mediated injury. 2) There is abundant evidence that perivascular delivery of the adenovirus limits infection to adventitial cells (7, 16, 25, 37). Numerous attempts to transfect the media from the endothelial or adventitial side of large blood vessels have failed because of the impervious nature of the IEL and EEL. Thus we cannot rule out the possibility that this barrier aided in selective adventitial expression of the inhibitor. The similar limited adventitial expression patterns of Ad-CMV-eGFP and Ad-PDGF{beta}R-gp91ds/eGFP in these studies are consistent with this anatomic limitation in the rat carotid artery.

Nevertheless, in most cross sections from the Ad-PDGF{beta}R-gp91ds/eGFP and Ad-CMV-eGFP treatment groups, we also observed positively stained intimal cells lining the inner surface of the IEL at day 1, with little evidence of medial smooth muscle expression. The origin of these positively stained cells is unclear. One possibility is that they migrated from the adventitia, as suggested by a number of studies (17, 31). Another is that the adenovirus penetrated to the intima and infected residual endothelial cells remaining after injury. Careful temporal analysis of eGFP expression will be necessary to determine the origin of the positive cells. Taken together, the fact that eGFP expression appeared to predominate in the adventitia suggests that localized delivery of our novel NADPH oxidase inhibitor to this vascular area may be an effective means to attenuate neointima formation.

Although our data appear consistent with primary expression of gp91ds in adventitial fibroblasts, the construct used for this study in vivo did not allow us to rule out other adventitial cells as sources of NADPH oxidase-derived ROS, of which infiltrating macrophages and neutrophils are potential prime candidates. In our in vivo studies, eGFP expression was under the control of the Rous sarcoma virus promoter, whereas gp91ds expression was under the control of the PDGF{beta}R promoter; thus generalized adventitial eGFP expression would not be expected to indicate gp91ds expression.

In addition to demonstrating targeting specificity with our adenoviral delivery system, we confirmed the ability of perivascular infection of the NADPH oxidase-inhibitory sequence, gp91ds, to suppress O2– in injured rat CCAs. We observed a 10-fold increase in O2– as a result of angioplasty-induced stretch, which was inhibited by 62% when the carotid artery was infected with Ad-PDGF{beta}R-gp91ds/eGFP. Souza et al. (33) demonstrated that stretching arteries with a balloon catheter causes an immediate and significant increase in ROS, largely attributable to NADPH oxidase. ROS such as O2– can rapidly activate signaling pathways that increase transcription factor expression and growth responses (12, 33). Thus our data suggest that adventitial NADPH oxidase is fundamental to neointimal hyperplasia after vascular injury.

Neointimal hyperplasia is characterized by VSMC proliferation and migration to the neointima during injury (8, 26, 29, 31, 41). Recent studies suggest that adventitial myofibroblasts derived from fibroblasts may also proliferate and migrate to the site of injury (17, 31, 33, 43). Although some studies have suggested that dissection of the media is necessary before fibroblasts migrate to the neointima (31, 32), other reports suggest that this is not necessary, inasmuch as direct perivascular injury can cause neointimal lesions without mechanical injury (3–5, 21). Adventitial NADPH oxidase has been suggested as an early mediator in this response (30). Because fibroblast proliferation is ROS dependent (13, 18), ROS production in adventitial fibroblasts is a potential target for treatment of injury- and atherosclerosis-related hyperplasia. In addition, perivascular oxidase may cause an indirect paracrine effect via the release of ROS such as H2O2, which can stimulate medial smooth muscle cells to change from the contractile phenotype to the proliferative and migratory phenotype (24).

In a previous study, we demonstrated that systemic infusion of gp91ds-tat was highly effective at attenuating neointima formation (14). However, because of the broad distribution of the cell-permeant peptide, it was unclear whether systemic or vascular inhibition of NADPH oxidase contributed to the attenuated neointima formation. In the present study, delivery of a gp91ds-expressing virus to the perivascular space caused localized adventitial expression of the inhibitor and similar suppression of neointima formation, suggesting that the vascular adventitia plays an important paracrine role in neointimal growth. There is, however, one important difference between the two strategies that limits such an interpretation: in the present study, adenovirus was applied to the outside of the vessel 2 days before injury; in our previous study, no such application was made. Although using Ad-CMV-eGFP controlled for the technique, the basal state of the carotid artery in each study (and, therefore, its growth response) may have differed. Thus direct comparison of the degree of inhibition achieved with each strategy is not possible.

Our present data demonstrate that targeted perivascular delivery of a novel NADPH oxidase inhibitor effectively attenuates neointima formation. Perivascular application of Ad-PDGF{beta}R-gp91ds/eGFP significantly reduced the induction of arterial O2– in response to balloon angioplasty, consistent with a fundamental role of perivascular oxidase in neointimal hyperplasia. Studies targeting other vascular cells and isoforms of NADPH oxidase will likely lead to clarification of their relative contributions to injury-induced neointima formation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-55425 and HL-28982 and by American Heart Association Grants 95011900 and 9808086W.


   ACKNOWLEDGMENTS
 
We thank Dr. Keiko Funa for the generous supply of the PDGF{beta}R promoter and Dr. Beverly Davidson and the Gene Transfer Vector Core for assistance in construction of the virus.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. J. Pagano, Hypertension and Vascular Research Div., Rm. 7044, E & R Bldg., Henry Ford Hospital, 2799 West Grand Blvd., Detroit, MI 48202-2689 (E-mail: ppagano1{at}hfhs.org)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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