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Cardiovascular Research 1997 35(3):536-546; doi:10.1016/S0008-6363(97)00147-8
© 1997 by European Society of Cardiology
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Copyright © 1997, European Society of Cardiology

Percutaneous delivery of the gax gene inhibits vessel stenosis in a rabbit model of balloon angioplasty

Luc Maillarda, Eric Van Bellea, Roy C Smitha, Aude Le Rouxb, Patrice Denéfleb, Gabriel Stegc, James J Barryd, Didier Branellecb, Jeffrey M Isnera and Kenneth Walsha,*

aDivision of Cardiovascular Research, St. Elizabeth's Medical Center and Tufts University School of Medicine, Boston, MA, USA
bRhône-Poulenc Rorer Gencell, Centre de Recherche de Vitry-Alfortville, Vitry-sur-Seine, France
cHôpital Bichat, Service de Cardiologie, Paris, France
dBoston Scientific Corp., Natick, MA, USA

* Corresponding author. Division of Cardiovascular Research, St. Elizabeth's Medical Center, 736 Cambridge St., Boston, MA 02135, USA. Tel.: +1 (617) 562-7501; fax: +1 (617) 562-7506.

Received 7 February 1997; accepted 23 May 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: The expression of gax, an anti-proliferative homeobox gene, is rapidly downregulated in vascular smooth muscle cells (VSMCs) following arterial injury. Here we performed percutaneous adenovirus-mediated gene transfer into the iliac arteries of normal rabbits using a channel balloon catheter to assess the effects of gax overexpression on neointima formation, lumen diameter, reendothelialization and functional vasomotion. Methods: A channel balloon catheter was used to perform both the arterial injury and local gene delivery. In each animal both iliac arteries were randomly assigned to receive either an adenovirus expressing the gax gene (Ad-Gax) or the β-galactosidase gene (Ad-βgal). In a second group of animals arteries were randomly assigned to receive either Ad-βgal or saline. Results: At one month, angiography revealed 36% less luminal narrowing in the Ad-Gax-treated arteries relative to the Ad-βgal-treated control arteries. Histological analysis revealed that the intimal/medial ratio (I/M) was reduced by 56% in the Ad-Gax group. Endothelium-dependent vasomotion was not affected by the gax gene transfer. In the second group, no statistically significant differences were found between the saline and the Ad-βgal-treated vessels for any of these parameters. Conclusions: Percutaneous adenovirus delivery of the gax gene to rabbit iliac arteries following endothelial denudation and vessel wall injury reduces neointimal hyperplasia and luminal stenosis, but does not affect endothelium-dependent vasomotion. This study demonstrates that a VSMC transcription factor can potentially be utilized for the development of a molecular therapy for vascular disorders.

KEYWORDS Ad-βgal, replication-defective adenoviral vector expressing the β-galactosidase gene; Ad-Gax, replication-defective adenoviral vector expressing the gax gene; PBS, phosphate-buffered saline; VSMC, vascular smooth muscle cell; pfu, plaque-forming unit; RSV LTR, Rous sarcoma virus long terminal repeat; CMV, cytomegalovirus


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The excessive proliferation of phenotypically modulated vascular smooth muscle cells (VSMCs) in response to vessel wall injury contributes to the restenosis that occurs in 30–50% of patients undergoing balloon angioplasty [1]and is the major cause of in-stent restenosis [2–4]and bypass graft occlusion [5]. A number of growth factor-regulatable transcription factors have been identified in VSMCs [6, 7]. Presumably these transcription factors function to coordinate the expression of muscle-specific and cell cycle-regulatory genes in response to the activation of receptor-mediated signaling pathways following vascular injury. The homeobox transcription factor gene gax (growth arrest homeobox) is expressed in quiescent VSMCs in vitro and in the normal rat carotid artery, but its expression is rapidly down-regulated when cultured VSMCs are stimulated to proliferate with mitogens or when vessels are subjected to balloon injury [8, 9]. Members of the homeobox gene family have been shown to encode transcription factors that function to model the body plan and they control growth, differentiation, and migration at a cellular level [10, 11]. Thus, we reasoned that the forced expression of gax might inhibit the fibroproliferative response to balloon injury and result in a decrease in neointima formation. To test this hypothesis we performed adenovirus-mediated gax gene transfer to balloon angioplasty-injured rabbit iliac arteries. In this study recombinant adenovirus was delivered percutaneously using a channel balloon without surgical exposure or branch ligature of the target vessel. These results demonstrate that adenovirus-mediated gax gene expression can inhibit neointima formation and luminal narrowing without affecting endothelium-dependent vasomotor responses.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Cell culture
Human vascular smooth muscle cell (VSMC) lines were obtained as described [12]from unused saphenous vein segments excised at the time of coronary bypass surgery at St. Elizabeth's Medical Center. Human VSMC cultures containing 15% FBS were maintained in high glucose DMEM (Gibco BRL, Gaithersburg, MD). Human umbilical vein endothelial cells (HUVEC) were isolated from post-natal material obtained at St. Elizabeth's Medical Center (Boston, MA) according to the method of Jaffe [13]and were maintained in M199 medium (Gibco BRL Gaithersburg, MD) supplemented with 20% fetal bovine serum (FBS) and 100 µg/ml of endothelial cell growth supplement (ECGS, Sigma, St. Louis, MO). Primary cultures were used before passage 10.

2.2 Recombinant adenoviral vectors
Replication-defective recombinant adenoviral vectors, based on human Ad5 serotype, were produced as previously described [14–16]. The rat gax gene cDNA was inserted between the XbaI and BamHI sites of the pCGN vector [17]resulting in an in-frame fusion of the gax gene, starting at codon 2 of the putative open reading frame [8], to the N-terminal sequence of the influenza virus hemagglutinin (HA) epitope that is downstream from the cytomegalovirus (CMV) early promoter and herpes simplex virus thymidine kinase gene 5' untranslated region (UTR). The XmnI-SfiI fragment from pCGN-gax was then inserted at the EcoRV site of the pCO1 vector containing the Ad5 adenoviral sequence required for homologous recombination. The resulting plasmid was linearized by XmnI and cotransfected in 293 cells with large fragment of the Ad5 d1324 viral DNA [15]. The resulting replication-defective recombinant adenoviruses were purified from isolated plaques and viral DNA was prepared. Recombinant adenoviruses containing the gax cDNA were identified by restriction fragment analysis and amplified in 293 cells. The viral preparations used for both in vivo studies were purified by two CsCl gradient centrifugations, dialysed against buffer containing 10 mM Tris-HCl pH 7.5, 1 mM MgCl2 and 135 mM NaCl and stored at –80°C in 10% glycerol. Viral titer was determined by plaque assay on 293 cells as previously described [18]and expressed as plaque-forming units (pfu) per ml. The construction of the control Ad-βgal used in this work has been previously described [15]. This construct expresses the β-galactosidase coding sequence, fused to a nuclear localization sequence, under control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter.

2.3 Adenovirus infection in vitro and analyses of cell proliferation
Cell number per well was determined by counting with a hemacytometer. Viral dilutions were prepared in DMEM containing 0.5% FBS and infections were conducted for 24 h. At the end of the infection period the virus containing medium was removed and the culture was transferred to growth medium. Cultures were incubated for 24 h to allow entry into S-phase after which cell proliferation was determined by [3H]thymidine incorporation in medium containing 3 µCi/ml of [3H]thymidine (6.7 Ci/mmol, DuPont NEN, Boston, MA) for 12 h. Wells were washed twice with PBS after which cold 10% TCA was added for 1 h. The wells were then rinsed twice with water and precipitated material was solubilized with 0.25 M sodium hydroxide. Tritium content of the sodium hydroxide solution was determined by liquid scintillation counting in Scintiverse II (Fisher Scientific, Pittsburgh, PA) utilizing a Beckman LS 5000TD scintillation counter.

2.4 Percutaneous arterial gene transfer and balloon angioplasty in vivo
Animal protocols were approved by St. Elizabeth's Medical Center Institutional Animal Care and Use Committee. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985). New Zealand White rabbits (3.0–3.5 kg) (Pine Acre Rabbitry, Norton, MA) were anesthetized with ketamine (10 mg/kg) and acepromazine (0.2 mg/kg) following premedication with xylazine (2 mg/kg). In each rabbit a 2.0 cm long channel balloon catheter (Boston Scientific, Watertown, MA) was introduced via the right common carotid and used to perform balloon angioplasty and arterial gene transfer. Balloon diameter was chosen to approximate a 1.3:1.0 balloon/artery ratio based on caliper measurement of magnified angiographic frames of nitroglycerin-treated vessels.

The angioplasty catheter was advanced to the lower abdominal aorta using a 0.014 inch guidewire (Hi-Torque Floppy II, Advanced Cardiovascular Systems, Temecula, CA) under fluoroscopic guidance following reference angiography with 200 µg of nitroglycerin. The balloon catheter was then advanced into the external iliac artery and positioned at the mid-point between the origin of the internal iliac artery and the origin of the common femoral artery. Balloon inflation was performed 3 times for 1 min each at 6 atm. The catheter was then inflated at nominal pressure (6 atm), and 200 µl of viral solution was instilled through the infusion port of the catheter and chased with a volume of saline solution equivalent to the predetermined dead space of the catheter (typically 500–600 µl). Infusion time was 60 s. After 30 min incubation, the balloon was deflated and the catheter was removed.

In each animal, iliac arteries were randomly assigned to be treated with either Ad-Gax (4x109 pfu) or the β-galactosidase gene (Ad-βgal, 4x109 pfu) (group 1, n = 9). Alternatively, animals were treated with Ad-βgal or saline (group 2, n = 8). After treatment of one artery, a new balloon was used to treat the contralateral iliac artery. Before the procedure, heparin sodium (200 USP units, Elkins-sinn, Cherry Hill, NJ) was administered intra-arterially. All animals received aspirin in water, approximately 50 mg daily, from 3 days prior to the procedure until sacrifice.

2.5 Angioplasty and in vivo vasomotor reactivity
The angiographic luminal diameter of the iliac artery was determined prior to gene transfer as well as both before and after drug infusion using an automated edge-detection system [19, 20]. Vasomotor reactivity of the arterial segment subjected to balloon angioplasty and arterial gene transfer was evaluated on the day of sacrifice. A 3 Fr. end-hole infusion catheter (Tracker-18, Target Therapeutics, San Jose, CA) was inserted into the left carotid artery and advanced to the origin of the transfected iliac artery using a 0.018 inch guidewire (Hi-Torque Floppy II) under fluoroscopic guidance. This catheter was used for both infusion of vasoactive drugs and selective angiography of the iliac artery. Angiography was performed immediately before and after each drug administration using 1 ml of non-ionic contrast media (Isovue-370, Squibb Diagnostics, New Brunswick, NJ). Serial angiographic images were recorded on 105-mm spot film at a rate of 2 films per second for 4 s. To assess endothelium-dependent vasomotor reactivity, acetylcholine chloride (Ach) or serotonin creatine sulfate (5-HT) were delivered from a constant infusion pump (1 ml/min) via the 3 Fr. catheter at doses of 5 µg/kg/min for 2 min. Five minutes were allowed to elapse following each dose of agent to re-establish basal blood flow conditions. After administration of Ach and 5-HT, respectively, was completed an identical protocol was employed to evaluate the contralateral artery. Finally, a single intra-aorta injection of 200 µg nitroglycerin was administered to assess endothelium-independent vasodilatation. The extent of the tone response was calculated as the percent of maximal lumen diameter as induced by nitroglycerin. For these analyses, minimal lumen diameter was assessed by an automatic edge detection system. The operator taking the measurements was blinded to the outcome of the analysis.

2.6 Evaluation of reendothelialization and intimal hyperplasia
Following angiographic analysis and 30 min prior to sacrifice, all rabbits received an intravenous injection of 5 ml 0.5% Evans blue dye (Sigma) delivered via the ear vein [21]. A cannula was inserted into the lower abdominal aorta and used to perfuse a total of 100 ml of 10 units/ml heparin, 0.9% saline solution in situ, followed by 100 ml of 100% methanol. The baseline angiogram recorded prior to balloon injury and pilot radiographic recording of the angioplasty balloon were used to identify the arterial segment to be harvested. The injured segment of iliac artery was then dissected and incised longitudinally. The harvested arterial segment was pinned to a cork board, further fixed in 100% methanol, and photographed for planimetric analysis of reendothelialization. Tissues were immersed in 100% methanol, embedded on longitudinal edge in paraffin, and cut in 5 µm sections onto slides coated with 3-aminopropyl-triethoxy-silane. The area of the intimal surface stained blue following application of Evans blue dye was interpreted to identify the portion of the arterial segment which remained endothelium-deficient [21]. A computerized sketching program (MacMeasure version 1.9; NIMH, Bethesda, MD) interfaced with a digitizing board (Summagraphics, Fairfield, CT) was used to outline the Evans blue positive and negative areas, respectively. The extent of endothelialized area was calculated as a percent of the total intimal area encompassed within the 2 cm length of artery. Longitudinal histologic sections obtained from the 20 mm length of injured artery and stained with an elastic tissue trichrome stain were projected onto the digitizing board, and the area of the intima and media, respectively, was measured using the computerized sketching program described above. The thickness of the native media of the artery wall is variable, reflecting, in part, the dimensions (diameter) of the individual rabbit iliac artery. Accordingly, thickness of the media was used to index the extent of neointimal thickening and is thus stated as the ratio of intima to media area (I/M). The means of 5 regularly spaced histological sections were analyzed to assess I/M ratio.

2.7 Analysis of gene expression
Five animals were sacrificed at 3 days after bilateral injury and treated with Ad-Gax in one iliac artery and saline in the contralateral vessel. Expression of gax in the vessel wall and at remote sites was determined by reverse transcription-polymerase chain reaction (RT-PCR). Tissue samples from transfected vessels, contralateral saline-treated vessels, liver, spleen, brain, testes, heart, lungs, ileum, kidneys and ipsilateral skeletal muscle were retrieved and immediately frozen in liquid nitrogen. Remote site tissue samples were also retrieved at 1 month post-treatment from 3 of the 9 Ad-Gax-transfected rabbits (group 1).

RNA was extracted from tissues using the Ultraspec RNA system. Reverse transcription and DNA amplification were carried out in a thermal cycler (MJ Research, PTC-100) with oligodeoxynucleotide primers designed to selectively amplify the Ad-Gax-encoded mRNA. The sense primer was designed to anneal to the expression vector sequence while the antisense primer was designed to anneal to the protein coding region of gax (5'-CCTTATGACGTGCCTGACTATGCC-3' and 5'-TGTGATGCTGGCTGGCAAACATGC-3', respectively). The predicted amplification product was 238 base pairs. In each set of experiments, 1 µg of total RNA was denatured at 65°C for 10 min and reverse transcription was carried out at 42°C for 15 min. The PCR reactions were performed as follows: initially 95°C for 105 s, then 35 cycles at 95°C for 15 s, 60°C for 30 s, and a final extension at 72°C for 7 min. Amplification products were detected following electrophoresis on 2% agarose gels by staining with ethidium bromide. A plasmid containing the identical expression cassette as Ad-Gax was used as a positive control for RT-PCR analysis.

Ten rabbits were analyzed for β-galactosidase expression in iliac arteries at 3 days after Ad-βgal infection. Excised arterial segments were treated with X-gal [22]. These segments were then mounted, cut into 5 µm sections and counterstained with hematoxylin and eosin. Expression of β-galactosidase in the nuclei of the vessel wall was determined by microscopic examination.

2.8 Statistical analysis
All results are expressed as mean±standard error (S.E.). Statistical significance was evaluated using a two-tailed paired Student's t-test for comparisons between two means in the same animal. A value of p<0.05 was interpreted to denote statistical significance.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Ad-Gax inhibits vascular cell proliferation
The Ad-Gax and Ad-βgal constructs were assayed for their effects on cell cycle activity in human VSMC and HUVEC cultures (Fig. 1). The Ad-Gax construct produced a dose-dependent inhibition of DNA synthesis, as determined by [3H]thymidine incorporation, in both cell types. Similar dose–response curves were observed for the human VSMC and HUVEC cultures. In either cell type, the Ad-βgal construct did not exhibit any inhibitory activity within this dose range indicating that the inhibition observed was not due to a non-specific viral toxicity.


Figure 1
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  		Fig. 1 The effect of adenoviral constructs on cell proliferation in vitro. Primary cultures of human vascular smooth muscle cells (hVSMC) (A) or human umbilical vein endothelial cells (HUVEC) (B) were made quiescent in low mitogen media prior to addition of virus. Cells were transduced with adenovirus at the indicated multiplicities of infection for 24 h. Following viral transduction, growth medium was added for 24 h to stimulate proliferation after which [3H]thymidine was added for 12 h to measure DNA synthesis. Incorporated label was determined by TCA precipitation. The graph depicts the effect of adenoviral constructs on the serum-stimulated growth by Ad-Gax (circles) or Ad-βgal (triangles). Error bars represent standard error of the mean.

 
3.2 Ad-Gax effects on the vessel wall
A total of 54 iliac arteries from 32 rabbits were analyzed in this study. Nine animals (group 1) underwent bilateral balloon injury and randomized transfection with Ad-Gax in one iliac artery and with Ad-βgal in the contralateral artery using a channel balloon catheter. Under identical conditions, a second group of 8 animals underwent bilateral injury and opposing arteries received either Ad-βgal or saline (group 2). Iliac arteries were examined 1 month later for I/M ratio, lumen diameter, functional vasomotion and reendothelialization. A third group of 5 animals underwent bilateral balloon injury and treatment with Ad-Gax in one artery and saline in the contralateral artery. These animals were sacrificed at 3 days to analyze gax transgene expression in the arterial wall and at remote sites by RT-PCR. A fourth group of 10 rabbits was injured and treated with Ad-βgal in a single iliac artery. At 3 days post-treatment these vessels were assayed for β-galactosidase expression in tissue sections.

3.2.1 Neointimal thickening
The effect of Ad-Gax and Ad-βgal on neointimal thickening was evaluated by light microscopic examination and quantitative morphometric analyses on longitudinal sections (Fig. 2). In Ad-Gax-treated arteries, the area of the intima to that of the media (I/M ratio) was 56% less than the I/M ratios in the contralateral Ad-βgal-treated arteries (Ad-Gax=0.35±0.15; Ad-βgal=0.80±0.18; p<0.02). In contrast, no statistically significant differences between the Ad-βgal and the contralateral saline-treated animals was observed in the second group (Ad-βgal=0.81±0.19; saline=0.84±0.21; p = ns).


Figure 2
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  		Fig. 2 Ad-Gax-infected vessels display less intimal hyperplasia. Longitudinal cross-sections of Ad-Gax- (A and C) and Ad-βgal-infected (B and D) contralateral arteries from the same animal harvested 28 days after angioplasty. (A and B) Hematoxylin and eosin staining; (C and D) elastic trichrome staining. A smaller neointima was found in all Ad-Gax-treated vessels relative to the Ad-βgal-treated contralateral vessels. The vessels shown here displayed one of the greatest relative differences within the same animal. The arrowheads point to the internal and external elastic lamina. N: neointima; star: media; bar=100 µm. (E) Ratios of intimal/medial areas in the Ad-Gax- versus the Ad-βgal- and in the Ad-βgal- versus saline-treated arteries. Results from quantitative morphologic analysis demonstrate that the Ad-Gax-treated arteries had I/M ratios that were 56% less than that of the I/M ratios in the contralateral Ad-βgal-treated arteries (p<0.02). No statistically significant differences were observed between the Ad-βgal-treated and the contralateral saline-treated animals in the second group.

 
3.2.2 Reendothelialization
Planimetric analysis was performed with Evans blue dye to evaluate the extent of reendothelialization at 28 days post-injury (Fig. 3). In the group 1 animals, a 17% reduction in the extent of reendothelialization was detected in the Ad-Gax-treated vessels relative to the Ad-βgal-treated vessels (Ad-Gax=50±11.6%; Ad-βgal=60.6±10.7%), but this difference was not statistically significant. No differences in the extent of reendothelialization were found in the group 2 animals comparing arteries treated with Ad-βgal or saline (Ad-βgal=59.8±6.9%; saline=61±5.5).


Figure 3
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  		Fig. 3 Summary of extents of reendothelialization at 1 month in the Ad-Gax- vs. Ad-βgal- and in the Ad-βgal- vs. saline-treated arteries. No significant difference was found in the extent of reendothelialization between contralateral injured arteries infected with either Ad-Gax vs. Ad-βgal (group 1) or Ad-βgal vs. saline (group 2).

 
3.2.3 Angiographic analyses of lumen diameter
The impact of Ad-Gax and Ad-βgal infection was also evaluated by angiographic luminal diameter measurements after maximum dilatation was induced by nitroglycerin in animals at 28 days post-injury in groups 1 and 2 (Fig. 4). The Ad-βgal-treated arteries were significantly more narrow (1.39±0.16 mm) than the corresponding Ad-Gax-treated arteries (1.84±0.14 mm) after dilation with nitroglycerin in the group 1 animals (p = 0.006). No significant differences in luminal narrowing were detected in group 2 animals between vessels treated with Ad-βgal (1.49±0.1 mm) and saline (1.46±0.09 mm).


Figure 4
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  		Fig. 4 Ad-Gax-infected vessels display larger lumen diameters in angiographic analysis. (A) Representative angiograms obtained after maximal vasodilatation induced by nitroglycerin of the Ad-Gax- and the contralateral Ad-βgal-treated arteries from the same rabbit 1 month after balloon angioplasty and adenoviral infection. (B) Average lumen diameters at 1 month in the Ad-Gax- versus Ad-βgal-treated arteries and in the Ad-βgal- versus the saline-treated arteries obtained after maximal vasodilatation induced by nitroglycerin. The Ad-βgal-treated arteries were significantly more narrow than the corresponding Ad-Gax-treated arteries in the group 1 animals (p = 0.006). No significant difference in luminal narrowing was detected in the second group between vessels treated with Ad-βgal or saline.

 
3.2.4 Vasomotor reactivity
The vasomotor response to endothelium-dependent agonists was determined using quantitative angiography. Rabbit iliac arteries treated bilaterally with either Ad-Gax vs. Ad-βgal (group 1) or with Ad-βgal vs. saline (group 2) demonstrated no differences in vasomotor response to the endothelium-dependent agents acetylcholine (Fig. 5A) or serotonin (Fig. 5B) at 28 days post-injury.


Figure 5
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  		Fig. 5 Arteries treated bilaterally with Ad-Gax and Ad-βgal or with Ad-βgal and saline demonstrated persistent impairment in endothelium-dependent vasomotor response to acetylcholine and serotonin at 28 days post-injury. Bar graphs showing the percentage loss in lumen diameter induced by acetylcholine (A) or serotonin (B). No significant differences were found between the different sets of vessels in each group.

 
3.2.5 Detection of gene expression and dissemination
Five animals were investigated for gax transgene expression at 3 days following bilateral injury and treatment (either Ad-Gax or saline) (Fig. 6A and B). RT-PCR analyses revealed gax transgene expression in all Ad-Gax-infected vessels. No expression was detected from any animal in brain, heart, testis, ileum, kidney, skeletal muscle, nor in the contralateral saline-treated vessel. In 2 of 5 animals, gax transgene transcripts were detected in liver and spleen. One month following bilateral injury and transfection (group 1 from above), gax transgene mRNA was not detected at any of the remote sites examined in 3 animals.


Figure 6
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  		Fig. 6 Transgene expression following percutaneous adenovirus delivery with a channel balloon. (A and B) gax transgene expression in Ad-Gax-transfected arteries and remote sites at 3 days following injury/infection. Exogenous gax gene expression was detected by RT-PCR using primers that were specific for the virally encoded gene. RNA transcripts specific for the virally encoded gax were detectable in all Ad-Gax-treated arteries. (A) Representative DNA gel demonstrating gax transgene expression in the transduced iliac artery (lane 6) and no expression in brain (lane 1), ileum (lane 2), the contralateral saline-treated artery (lane 5), liver (lane 8), heart (lane 9), spleen (lane 10), lung (lane 11), testes (lane 12), kidney (lane 13) or ipsilateral skeletal muscle (lane 14). Also shown are a plasmid (pCGN-gax) positive control (lane 3) and DNA size markers (lane 7). Lane 4 is blank. (B) Ad-Gax dissemination was detected in 2 of 5 animals. In this representative gel recombinant gax expression was detected in the Ad-Gax-transduced artery (lane 8), and in spleen (lane 2) and liver (lane 3), but not in brain (lane 5), testes (lane 6), or kidney (lane 7). Also shown are DNA size markers (lane 1) and a plasmid (pCGN-gax) positive control (lane 4). (C) Representative section showing β-galactosidase expression in Ad-βgal-transfected arteries. Nuclear β-galactosidase expression was detected by X-gal staining of transfected vessels that were subsequently sectioned and stained with hematoxylin and eosin.

 
Ten separate animals we evaluated for β-galactosidase expression in transfected vessels at 3 days post-injury and treatment with the Ad-βgal construct. Light microscopic analyses revealed numerous nuclei stained with X-gal (β-galactosidase positive) that tended to be localized to near the luminal surface of the medial layer (Fig. 6C). X-gal-stained nuclei were always confined to the arterial segment that was denuded of endothelium and delimited by the length of the balloon. Quantitative analysis of 40 sections reveled that 6.5±0.7% of the medial nuclei were stained positive for β-galactosidase expression.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Full-length gax cDNA was initially isolated from adult rat aorta and analyses of gax expression properties revealed that it is rapidly downregulated by mitogen stimulation of quiescent VSMCs and slowly upregulated by conditions that result in growth arrest [8]. This pattern of expression is reminiscent of the growth arrest-specific (gas) and growth arrest and DNA damage-inducible (gadd) genes that have been shown to encode a diverse group of proteins that can inhibit cell growth when overexpressed [23–25]. gax is unique as a gas/gadd family member in that it is the only homeobox transcription factor to be identified with these expression characteristics. Furthermore, gax expression is rapidly downregulated in rat carotid arteries following vascular injury [9]. Since other homeobox genes have been shown to function as regulators of cellular differentiation and growth, we tested whether forced gax expression could modulate the injury-induced stenosis of rabbit iliac arteries.

In this study a replication-defective adenovirus encoding gax was delivered percutaneously using a channel balloon catheter. In one group of animals, a randomly assigned vessel received Ad-Gax while the contralateral injured vessel received the control virus Ad-βgal. In a second set of animals a bilateral comparison was made between Ad-βgal and saline. Analyses of longitudinal tissue sections of the iliac arteries harvested at 28 days post-injury revealed a 56% reduction in the I/M ration of the Ad-Gax-treated arteries relative to the Ad-βgal-treated arteries. The inhibitory action of Ad-Gax is likely to derive, at least in part, from its ability to inhibit cellular proliferation, via its ability to upregulate the cyclin-dependent kinase inhibitor p21 [26]. However, homeodomain transcription factors are widely recognized to function as pleiotropic regulators and gax overexpression may also affect VSMC differentiation, migration or viability. In contrast to the Ad-Gax-treated arteries, no discernible difference was seen in the extent of neointima formation between the Ad-βgal-treated and saline-treated arteries in the control group of animals. The ability of gax overexpression to inhibit neointima formation compares favorably with recent results reported by other groups using adenovirus-encoded genes for wild-type and mutant forms of Rb in rat and porcine models [27, 28], the herpes simplex virus thymidine kinase gene in rat, rabbit and porcine models [28–32], p21 [33]in the rat model and hirudin in the rat model [34]. Overall, the reduction in I/M ratio achieved with these agents ranged from 35 to 50%.

In our study, in vivo analysis of luminal narrowing was performed using quantitative angiography to obviate any concerns that morphological studies could be altered by methodological aspects of tissue retrieval, fixation, or morphometry. Ad-Gax-treated vessels displayed significantly larger lumen diameters than the contralateral Ad-βgal-treated vessels at 28 days. In contrast, no differences were detected in the lumen diameters between the Ad-βgal- and saline-treated animals.

Evans blue staining at 28 days revealed incomplete reendothelialization in all experimental groups. No statistically significant differences were seen between the Ad-Gax-treated and the Ad-βgal-treated vessels nor between the Ad-βgal- and saline-treated vessels. Of note, Ad-Gax appeared equally effective in inhibiting the proliferation of VSMCs and endothelial cells in vitro. Thus we propose that the selective effect of Ad-Gax in vivo on VSMCs results from the local delivery of this agent to VSMCs of the denuded artery. Subsequently, the endothelium may regenerate from non-infected cells that migrate in from either adjacent endothelium or from circulating endothelial precursor cells [35]. Previous investigations on reendothelialization in a variety of animal models have demonstrated that restoration of anatomic integrity and recovery of physiologic function do not proceed simultaneously [36–38]. Accordingly, we analyzed vasomotor reactivity following adenovirus-mediated gene transfer or saline treatment. All injured vessels demonstrated persistent impairment in response to acetylcholine and serotonin at 4 weeks. No differences in these vasomotor responses were detected between the Ad-Gax-treated and Ad-βgal-treated arteries nor the Ad-βgal- and saline-treated vessels.

These findings demonstrate that a channel balloon can deliver sufficient gax-encoding adenovirus to the injured vessel wall to achieve a significant biological effect on lesion formation. Evidence of gax transgene expression at the site of delivery was provided by RT-PCR analysis using primers designed to specifically amplify the virally encoded gax gene transcript. gax transcript was not detected in the injured contralateral artery nor in brain, testes, heart, lung, ileum, or ipsilateral skeletal muscle. However, gax transcripts were detected in the liver and spleen of 2 of 5 test animals, presumably as a result of viral dissemination. Analysis of Ad-βgal delivery by X-gal staining revealed that 6.5±0.7% of medial cells stained positive for β-galactosidase activity at the site of virus delivery.

A number of possible explanations can account for the apparent discrepancy between the magnitude of the Ad-Gax biological effect (56% reduction in I/M ratio) and the transduction efficiency estimated from β-galactosidase activity (6.5%). First, it is likely that staining for β-galactosidase activity underestimates transfection efficiency. In support of this hypothesis, it has been reported that adenovirus-infected cultures of VSMCs harbor latent β-galactosidase activity that can be detected only when cells are exposed to agents that boost transgene expression from the CMV promoter [39]. This issue may be particularly relevant to the current study since β-galactosidase was expressed from the RSV LTR, a weaker promoter in VSMCs than the CMV promoter. Second, the VSMCs that are most likely to migrate and proliferate, i.e. those at the sites of greatest injury, may be preferentially exposed to infection by virus. Third, gax-transduced cells may affect the activity of adjacent cells through a bystander effect, as has been described for p53 [40]. Along these lines it should also be noted that homeodomain transcription factors regulate the expression of cell surface proteins that can influence the behavior of neighboring cells [41].

A number of conclusions can be drawn from these findings. We have found that a control adenovirus construct (Ad-βgal) at a dose of 4x109 pfu has no discernible effects on neointimal thickening, angiographic luminal stenosis, extent of re-endothelialization or vasomotor reactivity to nitroglycerin, serotonin or acetylcholine. Furthermore, we have found that the percutaneous delivery of a gax expression cassette can reduce neointimal thickening and luminal stenosis, but does not alter endothelium-dependent vasomotion. The data presented herein validate that gax can function to inhibit injury-induced alterations in vessel wall morphology. These findings suggest that Ad-Gax gene therapy may have utility for the treatment of proliferative vessel wall disorders.

Time for primary review 28 days.


   Acknowledgements
 
This work was supported by NIH grants AR40197 and HL50692 to K.W. and NIH grants HL02824 and HL53354 to J.M.I. L.M. was supported by the French Federation of Cardiology. Our thanks to Valerie Conard for excellent technical assistance and Jean-Marc Guillaume for viral supply/stock production.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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 SEMIN CARDIOTHORAC VASC ANESTHHome page
S. L. Meyerson, C. L. Skelly, M. A. Curi, and L. B. Schwartz
Gene Therapy for Cardiovascular Disease
Seminars in Cardiothoracic and Vascular Anesthesia, November 1, 2000; 4(4): 289 - 300.
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