Efficient inhibition of intimal hyperplasia by adenovirus-mediated inducible nitric oxide synthase gene transfer to rats and pigs in vivo

Efficient Inhibition of Intimal Hyperplasia by Adenovirus-Mediated Inducible Nitric Oxide Synthase Gene Transfer to Rats and Pigs In Vivo Larry L Shears II, MD, Melina R Kibbe, MD, Alan D Murdock, MD, Timothy R Billiar, MD, FACS, Alena Lizonova, PhD, Imre Kovesdi, PhD, Simon C Watkins, PhD, and Edith Tzeng, MD

Background: Inadequate nitric oxide (NO) availability may underlie vascular smooth muscle overgrowth that contributes to vascular occlusive diseases including atherosclerosis and restenosis. NO possesses a number of properties that should inhibit this hyperplastic healing response, such as promoting reendothelialization, preventing platelet and leukocyte adherence, and inhibiting cellular proliferation.

PFU/pig) to injured porcine iliac arteries in vivo was also efficacious, reducing intimal hyperplasia by 51.8%. Conclusions: These results indicate that shortterm overexpression of the iNOS gene initiated at the time of vascular injury is an effective method of locally increasing NO levels to prevent intimal hyperplasia. (J Am Coll Surg 1998;187:295–306. © 1998 by the American College of Surgeons)

Study Design: We proposed that shortterm but sustained increases in NO synthesis achieved with inducible NO synthase (iNOS) gene transfer at sites of vascular injury would prevent intimal hyperplasia. We constructed an adenoviral vector, AdiNOS, carrying the human iNOS cDNA and used it to express iNOS at sites of arterial injury in vivo.

Atherosclerosis contributes to approximately 50% of all mortality in the United States and Europe, being the predominant process underlying myocardial ischemia, cerebral ischemia, and peripheral arterial insufficiency.1 Current therapies, such as angioplasty or surgical bypass, directed at the regional complications of atherosclerosis are limited by the very nature of vascular healing, which involves exuberant smooth muscle cell (SMC) proliferation that often leads to intimal hyperplasia and restenosis.1 While the pathogenesis of intimal hyperplasia is multifactorial, the most common initiating event appears to be endothelial disruption.1,2 The endothelium is a normal source of regulatory molecules that modulates its own behavior as well as that of the underlying SMC.2 One such molecule is nitric oxide (NO), which is synthesized in low levels by a calcium-dependent, constitutively expressed endothelial NO synthase (ecNOS)3-5 in response to agonist stimulation. This NO has been shown to relax SMC, inducing a state of tonic vasorelaxation,3,4 and confers anti-platelet6 and antiinflammatory7 properties to the endothelium. NO can also inhibit cellular proliferation.8,9 There is evidence that suggests the loss of NO synthetic capacity at a site of vascular injury may be pivotal to neointimal lesion formation and perhaps atherogenesis.10,11 The vascular healing response is comprised of

Results: AdiNOS-treated cultured vascular smooth muscle cells produced up to 100-fold more NO than control cells. In vivo iNOS gene transfer, using low concentrations of AdiNOS (2 3 106 plaque forming units [PFU]/rat) to injured rat carotid arteries, resulted in a near complete (>95%) reduction in neointima formation even when followed longterm out to 6 weeks postinjury. This protective effect was reversed by the continuous administration of an iNOS selective inhibitor L-N6-(1-iminoethyl)-lysine. However, iNOS gene transfer did not lead to regression of preestablished neointimal lesions. In an animal model more relevant to human vascular healing, iNOS gene transfer (5 3 108 This work was supported by National Institute of Health grants GM44100, GM-37753, and GM-16645. TRB is the recipient of the George HA Clowes Jr, MD FACS, Memorial Research Career Development Award of the American College of Surgeons. Edith Tzeng is the recipient of a Competitive Medical Research Fund Award of the University of Pittsburgh Medical Center. Received September 9, 1997; Revised April 8, 1998; Accepted May 1, 1998. From the University of Pittsburgh, Departments of Surgery (Shears, Kibbe, Murdock, Billiar, Tzeng) and Cell Biology and Physiology (Watkins) Pittsburgh, PA, and GenVec Corporation (Lizonova, Kovesdi), Rockville, MD. Correspondence address: Edith Tzeng, MD, 497 Scaife Hall, University of Pittsburgh, Pittsburgh, PA 15261. © 1998 by the American College of Surgeons Published by Elsevier Science Inc.

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several phases.12,13 First, disruption of the endothelium initiates platelet and leukocyte adhesion with the release of numerous chemotactic and mitogenic factors. Following this initial phase, medial SMCs begin to proliferate and migrate to form a neointima. This proliferative phase lasts 2–4 weeks. Subsequent increases in neointimal lesion size occur through continued up-regulation of extracellular matrix synthesis and deposition, which can persist for months.14 Because NO has the capacity to attenuate essentially every aspect of the intimal hyperplasia response, therapeutic modalities based on reestablishing or augmenting NO availability hold great promise for the prevention of restenosis. Several groups15-18 have provided evidence that increasing NO availability systemically or locally, as accomplished with NO donor compounds, inhaled NO, and ecNOS gene transfer, reduces balloon-injury induced arterial neointima formation. These studies, however, have examined only the shortterm consequences of augmenting NO levels in normal vessels and have been restricted to rodent models that have limited value in predicting therapeutic outcomes in humans.1,19 We have previously reported that gene transfer using the human inducible NO synthase (iNOS) isoform to porcine arteries in an ex vivo organ culture system can eliminate intimal hyperplasia in response to injury.20 In contrast to ecNOS, iNOS is characterized by a greater specific activity, producing much larger quantities of NO in a calcium- and agonist-independent fashion.21 The dramatic effect of iNOS gene transfer was achieved with low levels of iNOS transgene expression, suggesting that the high enzymatic activity of iNOS may be advantageous for in vivo gene delivery. In this report, we further characterize iNOS gene transfer as a source of sustained local NO production in in vivo rodent and porcine models of injuryinduced intimal hyperplasia. We have selected an adenoviral vector because of the ability of this vector to mediate efficient shortterm in vivo gene transfer.22,23 Although others have shown the benefit of NObased approaches in rodent models, we propose that iNOS gene transfer warrants further and separate consideration because of the unique characteristics of iNOS gene transfer as a local source of NO. Our results show that iNOS gene transfer performed using low concentrations of adenoviral vector at the time of vascular injury provides sustained protection against intimal hyperplasia.

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METHODS Adenoviral vectors The human iNOS cDNA was previously cloned from cytokine-stimulated human hepatocytes.24 An E1 and E3 deleted vector carrying the human iNOS cDNA (AdiNOS) was designed and constructed. An adenovirus transfer plasmid with the cytomegalovirus promoter and an artificial splice sequence was designed to drive the transcription of the iNOS cDNA. To generate infectious virus, this plasmid was co-transfected into 293 cells (ATCC CRL. 1573) with the large ClaI fragment of AdlacZ DNA.25 Intracellular recombination of the plasmid with the ClaI fragment carrying adenoviral sequences generated a full-length recombinant adenoviral genome.26 Recombinant AdiNOS virus was double plaque purified and the expression of the iNOS transgene was determined by screening for nitrite (NO22) release from infected cells using the Griess reaction.27 Viral stocks were purified by triple banding on a cesium chloride gradient. Concentrations of AdiNOS and the control adenovirus, AdlacZ, which carries the bacterial b-galactosidase gene, were determined by plaque assay. The titers of AdiNOS and AdlacZ preparations were 109 and 1010 plaque forming units/mL (PFU/mL), respectively. Cell culture Rat aortic SMCs were cultured from explanted thoracic aorta from Sprague-Dawley rats as previously described.28 The cells had the characteristic hills and valleys appearance and were routinely .95% pure by SMC a-actin staining. SMCs were used for experiments between passages 2 and 6. The cells were grown in Dulbecco’s modified Eagle’s medium (low glucose)/Ham’s F12 (1:1 vol:vol) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 mg/mL streptomycin, and 4 mmol/L L-glutamine, and maintained in a 37°C, 95% air/5% CO2 incubator. In vitro iNOS gene transfer Rat aortic SMCs were plated onto 12 well plates and adenoviral infections were performed the following day. Virus was diluted in Optimem I (Gibco, Grand Island, NY) to the desired multiplicity of infection (MOI 5 0–100) and incubated with the cells for 90 minutes at 37°C. To measure NO synthesis following adenoviral infection, SMCs were then cultured in medium 6 10% FBS for 24 hours and NO22 accumulation was assayed using the Griess reaction.27 Because SMCs do not express the biosyn-

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thetic pathway for the essential NOS cofactor, tetrahydrobiopterin (BH4),29,30 exogenous BH4 (10 mmol/L) was also added to some cells to optimize iNOS activity. Total cellular protein was quantified using the BCA protein assay (Pierce, Rockford, IL). Gene transfer efficiency was estimated by staining for b-galactosidase activity in AdlacZ infected cells. Cells were fixed with 0.5% glutaraldehyde and stained with X-gal as described.31 RNA isolation, Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was isolated from SMC 48 hours after adenoviral infection using RNAzol B as described.27 Aliquots (20 mg) of RNA were electrophoresed on a 0.9% agarose gel and blotted to GeneScreen (DuPont-NEN, Boston, MA). Membranes were probed with a 2.3 kb fragment of the human hepatocyte iNOS cDNA.24 18S rRNA was also probed and served as a control for relative RNA loading. For RT-PCR, cDNA synthesis was performed on 250 ng of RNA, and PCR was performed using 25 ng of cDNA with reactions carried out for 30 cycles of 92°C denaturation for 1 minute, 57°C annealing for 2 minutes, and 72°C elongation for 3 minutes.20 PCR primers that recognize only human iNOS sequences were used to detect adenovirally transferred iNOS expression. The 59 primer sequence is 59-AGGACATCCTGCGGCAGC-39 and the 39 primer sequence is 59-GCTTTAACCCCTC CTGTA-39, which amplify the human hepatocyte iNOS sequence (bp 3376-3691) with a 316 bp product. To control for RNA quality and cDNA synthesis, b-actin mRNA was also amplified. Western blot analysis Cultured SMCs were collected by scraping and were resuspended in 20 mmol/L Tris, 100 mmol/L phenylmethylsulfonylflouride. Samples (100 mg) were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis on 8% gels and transferred to nitrocellulose membranes (Schleicher & Schuell; Keene, NH) as described.20 Membranes were blocked with 5% milk/phosphate buffered saline/0.1% Tween-20 and hybridized with a monoclonal anti-mouse macrophage iNOS antibody (1: 2000, Transduction Laboratories, Lexington, KY) that detects human iNOS, followed by horseradish peroxidase-linked goat anti-mouse IgG (1:2000, Schleicher & Schuell). Proteins were visualized using ECL reagents (DuPont-NEN).

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Animals and surgical procedure All animal procedures were performed using aseptic technique in accordance with the Institutional Animal Care and Use Committee of the University of Pittsburgh. Adult male Sprague-Dawley rats weighing 400–450 gms were anesthetized with intraperitoneal nembutal (45 mg/kg) and supplemental inhalational metaphane. The left common carotid artery was exposed through a collar incision and proximal and distal vascular control was obtained with noncrushing vascular clamps. A 2 Fr arterial embolectomy balloon catheter (Applied Vascular, Laguna Hills, CA) was inserted into the common carotid artery through the external carotid branch, and a uniform injury was created by inflating the balloon to 5 atmospheres of pressure for 5 minutes. AdiNOS or AdlacZ viral solution (107 PFU/mL diluted in Optimem I, ;200 mL/rat) was then instilled through an angiocatheter inserted through the external carotid artery. The viral solution was maintained in the artery for 60 minutes. After the incubation period, the viral solution was evacuated, the external carotid artery was ligated, and blood flow through the common carotid artery was reestablished. In some experiments, the balloon injury was created and viral infection was delayed until 2 weeks postinjury. The partially selective iNOS inhibitor L-N6(1-iminoethyl)-lysine (NIL) (Cayman Chemical, Ann Arbor, MI),32 was administered as a continuous infusion (20 mmol/kg/d) to some animals using Alzet microosmotic pumps (Alza, Palo Alto, CA) placed in the contralateral external jugular vein at the time of angioplasty. Male domestic pigs, 12–15 kg (Walter Whippo, Enon Valley, PA), were placed under general anesthesia and bilateral common iliac arteries were exposed through a low-midline laparotomy incision. After obtaining distal and proximal vascular control with noncrushing vascular clamps, common iliac arterial injury was created with a 4 Fr embolectomy catheter inserted through a side branch inflated to 2 atmospheres for 5 minutes. Adenovirus solution (109 PFU/mL diluted in Optimem I, ;500–750 mL/iliac artery) was instilled into the common iliac artery and allowed to incubate for 30 minutes. After the incubation period, the adenovirus solution was evacuated, the side branch ligated, and blood flow reestablished. In all pigs, AdiNOS was instilled into the right common iliac artery while AdlacZ was instilled into the left. No anticoagulation or antiplatelet agents were administered at any point during the preoperative, operative, or postoperative periods.

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Tissue processing and morphometric analysis At various intervals after viral transduction, animals were euthanized and arteries were collected for morphometric analysis. Vessels were fixed in 2% paraformaldehyde for 1 hour at 4°C and cryoprotected in 30% sucrose overnight at 4°C. Vessels were then quick-frozen with HistoFreeze-2000 (Fisher, Pittsburgh, PA) and 5 mm cryosections cut. Semiserial sections were stained with a modified VerhoeffVan Giesen stain33 or with a hematoxylin-eosin (H&E) stain. Intima/media ratios (I/M) were quantified with the Optimas program (Optimas Corp., Seattle, WA) for six sites for each of six different vessel sections per artery in rat experiments and calculated as the mean of all the measurements. Pig vessels were examined in cross-section and intimal and medial thicknesses were performed on 16 different sections and vessels. For PCR analysis, tissues were snapfrozen and then homogenized with a polytron. RNA was extracted from vessels with RNAzol B and RTPCR was carried out as described earlier. Some pig vessels were collected 3 days post-viral transduction with AdlacZ, fixed in 2% paraformaldehyde and then stained with X-gal as described.31 Statistical analysis NO22 levels and arterial measurements are expressed as mean 6 SEM. The significance of differences were determined using analysis of variance (ANOVA) or Fisher’s PLSD test. RESULTS Generation of recombinant AdiNOS An adenoviral vector containing the human hepatocyte iNOS cDNA from bp 47-4125 was constructed. The generation of recombinant AdiNOS virus was complicated by the sensitivity of 293 cells to the very high concentrations of NO synthesized (.150 mmol/L NO22) during virus propagation. In addition, virus replication in 293 cells was also adversely affected by the arginine analogs commonly used to inhibit NOS activity such as NGmonomethyl-L-arginine (NMA). Both these factors limited the preparation of high titer AdiNOS stocks. The growth condition that permitted optimal AdiNOS production included the addition of 0.2 mM NMA. Titers of AdiNOS stocks were 109 PFU/mL. In vitro testing of AdiNOS SMC were infected with various MOI of AdiNOS and AdlacZ, ranging between 0 and 100. Cells

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Table 1. Nitrite Accumulation by Small Muscle Cells after AdiNOS Infection AdiNOS concentration Multiplicity of infection 0 1 10 100

Nitrite (nmol/mg protein per 24 h) 2BH4

1BH41

2.1 6 1.1* 9.9 6 0.6 74.8 6 1.8‡ 176.7 6 20.2‡

2.2 6 0.9 19.7 6 3.1 228.0 6 11.9‡ 545.5 6 32.5‡

*Values 5 mean 6 SEM (n 5 3 per group, representative of 6 experiments). † BH4 added at 10 mmol/L concentration. ‡ p . 0.01 versus MOI 0 with or without BH4. BH4, tetrahydrobiopterin; MOI, multiplicity of infection.

treated with AdiNOS at an MOI $10 released high levels of NO22 (Table 1). We have previously reported that resting SMC do not express GTP cyclohydrolase I (GTPCH),29,30 the rate limiting enzyme for BH4 biosynthesis, and are unable to support maximal activity of a genetically transferred iNOS enzyme.20 BH4 is an essential cofactor required for the activity of all NOS enzymes, functioning by promoting the dimerization of inactive NOS subunits.34,35 In the absence of serum, SMCs infected with AdiNOS demonstrated minimal NO synthesis despite high levels of iNOS expression (Fig. 1). The addition of serum increased NO22 three-fold. Serum is known to contain biopterins,34 which may account for this increase in NO synthesis. However, the addition of exogenous BH4 to AdiNOS infected SMCs, in the absence or presence of serum, further increased NO22 accumulation (Fig. 1A). AdiNOSinfected SMCs produced the greatest amount of NO when both serum and BH4 were provided, suggesting that serum may contain additional elements that are required for optimal iNOS enzymatic activity not supported by BH4 alone. AdlacZ-treated SMCs released only background levels of NO22 (,34 nmol/ mg/24 h 6 BH4) at all MOI tested. An MOI of 10 was associated with a gene transfer efficiency of approximately 10–20% as estimated by X-gal staining of AdlacZ-infected cells (data not shown). Expression of the adenoviral iNOS transgene was examined by Northern and Western blot analyses that confirmed the presence of high levels of recombinant iNOS mRNA and protein, respectively (Fig. 1B and 1C). AdlacZ-treated SMCs showed no evidence of iNOS mRNA or protein, indicating that adenovirus infection in itself did not induce endogenous iNOS expression. Levels of iNOS mRNA and protein in AdiNOS infected cells were unaffected by the addition of either FBS or BH4.

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Figure 1. Adenoviral-mediated iNOS gene transfer in vitro. (A) Cultured rat aortic SMCs were infected with AdiNOS or AdlacZ (multiplicity of infection 5 10) and NO synthesis was quantitated as NO22 accumulation using the Griess reaction in the presence of 0% or 10% FBS 6 10 mM BH4. Results are the mean 6 SEM of 3 values/group, each experiment was repeated 6 times. *p , 0.01 versus all AdlacZ groups and AdiNOS 1 0%, †p , 0.01 versus all other AdiNOS groups. (B) Northern blot analysis for iNOS mRNA in AdlacZ or AdiNOS (MOI 10) infected SMC grown in 0% or 10% FBS 6 BH4. A 2.3 kb fragment of the human iNOS cDNA was used to probe for iNOS mRNA. 18s rRNA was probed as a control for equal loading. Representative of 3 experiments. (C) Western blot analysis for iNOS protein in AdiNOS or AdlacZ infected SMCs using a monoclonal murine iNOS antibody that also detects human iNOS. The iNOS protein measures ;131 kDa in size. Representative of three experiments. (BH4, tetrahydrobiopterin; FBS, fetal bovine serum; iNOS, inducible NO synthase; SMC, smooth muscle cell.)

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Figure 2. Effect of iNOS gene transfer on intimal hyperplasia in injured rat carotid arteries. (A) Representative photomicrographs (310) of cross-sections of rat carotid arteries collected 14 days after balloon injury and AdlacZ or AdiNOS infection (;2 3 106 plaque-forming units/artery) as well as arteries treated with AdiNOS with continuous infusion of NIL. Sections are stained with hematoxylin and eosin or Verhoeff-Van Giesen stain. Arrows indicate the internal elastic lamina. (B) The intima/media ratios are shown as the mean 6 SEM (N 5 5 for AdlacZ, 8 for AdiNOS, 3 for AdiNOS 1 NIL). *p 5 0.002 versus AdlacZ, p 5 0.0055 vs AdiNOS 1 NIL. (C) RT-PCR for human iNOS expression in injured rat carotid arteries 72 h following AdiNOS or AdlacZ infection. Human iNOS specific PCR primers were used to amplify the expression of the iNOS transgene. The positive control for human iNOS was RNA prepared from cytokine stimulated human hepatocytes (2nd lane). RT-PCR for b-actin controls for RNA and cDNA quality. Representative of two experiments. iNOS, inducible NO synthase; NIL, L-N6-(iminoethyl)-lysine; PCR, polymerase chain reaction; RT-PCR, reverse-transcription polymerase chain reaction.

Inhibition of injury-induced intimal hyperplasia in vivo in rat carotid arteries by iNOS gene transfer Balloon injury of the common carotid artery in rats creates a reproducible injury response with the development of a thick neointimal lesion by 14 days post-injury that measures approximately 1.5 times the depth of the medial layer. Treatment of these arteries immediately post-injury with low concentra-

tions of AdlacZ (2 3 106 PFU/carotid artery) did not alter neointima formation (I/M 5 1.53 6 0.27, Fig. 2A and 2B) as compared with animals treated with arterial injury alone (data not shown). However, intimal hyperplasia was almost completely inhibited in rats whose carotid arteries were treated with AdiNOS at a similar concentration (I/M 5 0.05 6 0.03, p 5 0.002 versus AdlacZ, Fig. 2A and 2B). To demon-

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strate that the AdiNOS effect was mediated through the production of NO, the partially selective iNOS inhibitor, NIL,32 was administered in a continuous fashion via Alzet pumps to rats following carotid injury and gene transfer. NIL was used to avoid any local or systemic effects associated with inhibition of ecNOS activity with a less selective inhibitor. The administration of NIL completely reversed the protective effect of iNOS gene transfer; animals receiving both AdiNOS and NIL developed neointimal lesions almost identical to those observed in AdlacZtreated animals (I/M 5 1.29 6 0.14, p 5 0.0055 versus AdiNOS alone, Fig. 2A and 2B). In addition, NIL administration slightly increased intimal hyperplasia in rats subjected to injury alone (n 5 3). Quantification of medial and intimal thicknesses revealed that iNOS gene transfer did not alter the media as compared to AdlacZ-treated arteries but reduced neointima formation by 96.7%. To confirm iNOS expression, RT-PCR was used to detect hu-

Figure 3. Long term effects and effects on pre-formed neointimal lesions of iNOS gene transfer in rats. (A) Representative photomicrographs (320) of cross-sections of rat carotid arteries collected 6 weeks following injury and gene transfer as well as arteries that were subjected to AdiNOS infection 2 weeks after the initial injury and collected 2 weeks later (Delay). Sections were stained with the Verhoeff-Van Giesen stain. Arrows mark the internal elastic lamina. (B) Intima/media ratios represented as mean 6 SEM (n 5 4 for AdlacZ-6 wk and AdiNOS-6 wk, 3 for Delay). *p 5 0.052 versus AdlacZ-6 wk, †one animal with complete arterial occlusion by intimal hyperplasia and was not included in the data.

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man iNOS mRNA in carotid arteries 72 hours after injury and adenoviral infection. Human iNOS mRNA could be found only in AdiNOS-treated vessels (Fig. 2C). Characterization of the timing and duration of the beneficial effects of iNOS gene transfer in rat carotid arteries A group of rats subjected to carotid artery injury and gene transfer were examined 6 weeks post-injury to determine the durability of the effect of transient iNOS gene transfer on intimal hyperplasia. Neointimal lesions in AdlacZ-treated animals continued to progress between 2 and 6 weeks after injury (I/M 5 2.58 6 0.99 at 6 weeks, Fig. 3A and 3B). However, AdiNOS-treated animals still exhibited minimal intimal hyperplasia 6 weeks post-injury (I/M 5 0.24 6 0.02, p 5 0.052), indicating the sustained effect of early iNOS over-expression on inhibiting intimal hyperplasia in the rodent model (Fig. 3A and 3B). To

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Figure 4. Effect of iNOS gene transfer on intimal hyperplasia in injured pig iliac arteries. Representative photomicrographs (34) of cross-sections (hematoxylin and eosin stain) of pig iliac arteries collected 21 days post-balloon injury and AdlacZ (A) or AdiNOS (B) infection (5 3 108 plaque-forming units/artery). Internal elastic lamina is marked with arrows and external elastic lamina is marked with arrowheads. (C) Intima/media ratios represented as mean 6 SEM (n 5 11/group). *p 5 0.0002 versus AdlacZ.

determine if iNOS gene transfer could mediate the regression of a pre-formed neointimal lesion, adenoviral infection was delayed until 14 days after carotid artery injury, when significant neointima formation had already occurred, and the arteries were examined 14 days thereafter (a total of 28 days after carotid artery injury). In these animals, neointimal lesions resembled those found in arteries from AdlacZtreated animals at 2 weeks (I/M 5 1.65 6 0.47, Fig. 3A and 3B). Therefore, with the low concentrations of AdiNOS used in this study, iNOS gene transfer could not mediate the regression of a pre-established lesion.

Effect of iNOS gene transfer in a porcine model of arterial injury Rodent models of arterial injury have many limitations in predicting the efficacy of various treatments in human disease.1,19 For this reason, we also evaluated the efficacy of iNOS gene transfer for preventing intimal hyperplasia in a pig model of vascular injury.36 Balloon catheter injury of common iliac arteries with a 4 Fr Fogarty catheter inflated to 2 ATM for 5 minutes resulted in the development of uniform and reproducible neointima lesions by 3 weeks postinjury. Treatment with 5 3 108 PFU/artery of AdlacZ immediately after injury did not alter neointi-

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iNOS gene transfer are mediated through local NO production as supported by the capacity of an iNOSselective inhibitor32 to reverse these beneficial effects. Adenovirus-mediated gene transfer results in transient gene expression,22,23 typically lasting several days to 2 weeks. Our data indicate that AdiNOS

Figure 5. Estimate of gene transfer efficiency to pig iliac arteries. Pig iliac arteries were subjected to balloon injury followed by AdlacZ infection (5 3 108 plaque-forming units/pig) and animals were sacrificed 3 days later. Iliac arteries were collected and stained for b-galactosidase activity using X-gal. A representative photomicrograph (34) shows the lumenal surface of the iliac artery and dots are cells staining positive of b-galactosidase activity. Experiment was repeated four times.

mal lesions with an I/M of 0.79 6 0.08 (Fig. 4A, 4B, 4C). In contrast, treatment with the same concentration of AdiNOS significantly reduced intimal hyperplasia with a resultant I/M of 0.38 6 0.04 (n 5 11, p 5 0.0002), a reduction in intimal hyperplasia by 51.8%. Staining of pig arteries 3 days after AdlacZ infection showed a patchy distribution of SMC on the lumenal surface expressing the transgene (Fig. 5). These cells represent a gene transfer efficiency of approximately 5–10% of the arterial surface. Evaluation of the duration of iNOS gene expression following AdiNOS infection was performed using RT-PCR in pig vessels collected on days 3 and 8 post-gene transfer (n 5 2 per time point). Human iNOS mRNA expression was detectable in vessels collected on day 3 (Fig. 6) but was no longer present by 8 days after gene transfer (data not shown). These results indicate that NO delivery for the prevention of intimal hyperplasia is important in the early time period following vascular injury. DISCUSSION Intimal hyperplasia develops through a complex cascade of events that involves the local activation of platelets, inflammatory cells, and vascular SMCs leading to the release of chemotactic and mitogenic factors that stimulate SMC migration and proliferation.1,2,19 We have demonstrated here that adenovirus-mediated iNOS gene transfer can effectively inhibit this complex process in both rodent and porcine models of arterial injury. These actions of

Figure 6. Expression of human iNOS mRNA in injured pig iliac arteries 72 hours following AdiNOS or AdlacZ infection as determined by RT-PCR. Human iNOS specific PCR primers were used to amplify the expression of the iNOS transgene. RT-PCR for b-actin controls for RNA and cDNA quality. Representative of two experiments. iNOS, inducible NO synthase; PCR, polymerase chain reaction; RT-PCR, reverse-transcription polymerase chain reaction.

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does successfully deliver the iNOS transgene to arteries. iNOS gene expression was easily detected 3 days post-gene transfer in both rat and pig models of arterial injury. In agreement with the literature, iNOS expression was no longer detectable by day 8. Despite transient expression, iNOS gene transfer suppressed neointima formation up to 6 weeks postinjury. However durable the prophylaxis, the level of iNOS gene transfer achieved could not eliminate a pre-established neointimal lesion. These findings suggest that iNOS expression and NO synthesis function by inhibiting the initiation of the injury response or the proliferative phase of healing, or both. Possible targets include interfering with platelet and leukocyte associated interactions, blocking SMC signal transduction, and/or suppressing SMC response to promitogenic and chemotactic stimuli. We have shown that iNOS gene transfer blocks SMC proliferation in vitro via a cGMP-dependent pathway (unpublished data, 1997), which is consistent with the findings of others using NO donors.8,37 The ineffectiveness of iNOS gene transfer on an established lesion makes it unlikely that NO is functioning through cytotoxic pathways. It is also interesting to note that the known pro-inflammatory effects of adenovirus22,23 did not adversely influence the benefit of vascular iNOS gene transfer. Inflammation associated with adenovirus gene transfer is dependent, in part, on the viral titer with significant inflammation typically seen at viral titers of 109 PFU or greater.38 Effective iNOS gene transfer required only 106 PFU in rats and 108 PFU in pigs. This suggests that another benefit of gene therapies accomplished with minimal virus load such as can be achieved with iNOS would be the reduction in pro-inflammatory stimuli. In addition, a consistent observation in AdiNOS-treated arteries was a greatly diminished amount of extralumenal adhesion formation as compared with AdlacZ-treated or untreated arteries (data not shown). Adhesion formation is a common sequela of surgical dissection of tissues and is the result of inflammation and fibrin deposition. Normal tissues heal with minimal adhesion formation, which is typically exacerbated by tissue injury and ischemia. The capacity of iNOS gene transfer to reduce adhesions suggests that the NO can reduce inflammation, improve fibrinolysis, or reduce fibroblast in-growth. Vascular injury induces iNOS expression in the arterial wall, beginning shortly after injury and persisting for several days.39 iNOS induction has also been reported in another form of vascular injury, that of transplantation arteriosclerosis.40,41 The role of

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this endogenous iNOS has not been previously determined, but we propose that it may serve to attenuate intimal hyperplasia and accelerate normal healing. In transplantation arteriosclerosis, we demonstrated that inhibiting endogenous iNOS activity with NIL accelerated the progression of vasculopathy.41 Similarly, we showed that inhibiting endogenous iNOS activity with NIL exacerbated injuryinduced intimal hyperplasia. While endogenous iNOS expression may be important in controlling vascular healing following minor perturbations, it is not sufficient to eliminate the proliferative process associated with severe vascular injury. iNOS gene transfer may be viewed as an extension of the evolved injury response and may be an useful approach to optimize NO availability. Unstimulated SMCs do not express GTP cyclohydrolase I (GTPCH),29,30 and therefore, do not produce BH4. SMCs were unable to support maximal iNOS activity after in vitro gene transfer unless BH4 was supplied.20,29 However, iNOS gene transfer into rodents and pigs was biologically effective in the absence of additional BH4. There are several possible explanations for this. Our data indicate that serum carries biopterins that can support iNOS activity in SMCs, likely through a salvage pathway that converts biopterins to BH4.42 Another explanation may be that cells synthesizing BH4 can supply cofactor to adjacent cells. We have previously demonstrated that biopterins can diffuse in vitro to activate iNOS in an adjacent cell.30 Finally, GTPCH may be upregulated by vascular injury in vivo. Indeed, GTPCH mRNA appears to be increased in injured rat carotid arteries (unpublished data, 1996). We have not shown, however, that iNOS activity has been optimized in these in vivo studies. Supplemental BH4 could increase NO synthesis and thereby reduce the concentration of virus or the duration of viral exposure required to achieve the therapeutic effect. We have reported that co-transfer of GTPCH and iNOS cDNA into BH4deficient cells30 can support maximal iNOS activity, suggesting that a co-transfer strategy may be applied in vivo if BH4 levels require optimization. Similar to our results with iNOS gene transfer, other modes of delivering NO have been successful in rodents and rabbits.15-18 However, unlike these other studies, we have demonstrated the efficacy of this NO-based therapy for the prevention of intimal hyperplasia in a more relevant animal model, namely the pig model. While the pig represents another animal model, vascular healing following injury in pigs resembles that in humans and is an excellent model

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to evaluate potential therapies directed at preventing intimal hyperplasia, a vascular injury response. In addition, NO donor therapy could require continuous delivery15-17 or result in systemic toxicity (eg, hypotension). To provide sustained delivery of NO, iNOS gene transfer may be the ideal source. Although iNOS is regulated predominantly at the transcriptional level,21 this regulatory step is bypassed during gene transfer. iNOS is typically expressed during physiologic perturbation for antimicrobial or antioxidant functions, and therefore, synthesizes supraphysiologic amounts of NO. Because the greatest limitation of in vivo gene delivery is gene transfer efficiency, iNOS may still yield adequate NO synthesis despite low transfer efficiencies. A theoretical concern about iNOS overexpression is the potential for cytotoxicity as a result of high level NO synthesis. While it had previously been implicated that NO possesses many cytotoxic properties,43 it has become more evident that NO is innocuous to a variety of cell types at the concentrations that would be generated endogenously. Products of reactions between NO and other reactive radicals are more likely the cytotoxic agents.44 It has recently been reported that NO may be cytoprotective to some cell types such as hepatocytes.45 In our studies, there was no detectable cytotoxicity to SMCs infected with AdiNOS at an MOI of 10 with or without BH4 supplementation. Instead, we recently reported that iNOS gene transfer to vascular endothelial cells conferred cytoprotection to these cells by rendering them resistant to endotoxin-induced apoptosis.46 This suggests that iNOS gene therapy may accelerate vascular healing by preventing intimal hyperplasia and protecting the regenerating endothelium. Other gene therapy approaches for restenosis using the cytotoxic herpes simplex thymidine kinase gene,47 the retinoblastoma tumor suppressor gene,48 or cell cycle inhibitors49 have also been proposed and evaluated. However, the majority of these therapies will be limited by the number of cells that can be targeted. Since the products of these genes are not secreted, only cells expressing the transgene will be affected, which may be prohibitively inefficient. In contrast, iNOS elicits its actions through its highly diffusible product, NO. Although only a few cells may express iNOS following gene transfer, NO can access many more cells through its diffusional properties.50 This is supported by the effectiveness of AdiNOS using 100–10,000-fold less virus than other gene therapy studies.47-49 Given these properties,

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