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(Circulation. 1997;96:2254-2261.)
© 1997 American Heart Association, Inc.

Articles

Adventitial Gene Transfer of Recombinant Endothelial Nitric Oxide Synthase to Rabbit Carotid Arteries Alters Vascular Reactivity

Iftikhar J. Kullo, MD; Geza Mozes, MD; Robert S. Schwartz, MD; Peter Gloviczki, MD; Thomas B. Crotty, MD; Dustan A. Barber, PhD; Zvonimir S. Katusic, MD, PhD; ; Timothy O'Brien, MD

From the Divisions of Cardiovascular Disease (I.J.K., R.S.S.), Surgical Research (D.A.B.), Vascular Surgery (G.M., P.G.), Anatomic Pathology (T.B.C.), Anesthesiology and Pharmacology (Z.S.K.), and Endocrinology and Metabolism (T.O.), Mayo Clinic and Foundation, Rochester, Minn.

Correspondence to Timothy O'Brien, MD, Senior Associate Consultant, Department of Endocrinology and Metabolism, Mayo Clinic, 200 First St SW, Rochester, MN 55905. E-mail obrien.timothy{at}mayo.edu

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background Adventitial gene transfer may serve as a tool to study vascular biology and may have therapeutic potential. We investigated the hypothesis that adenovirus-mediated transfer of the gene for endothelial nitric oxide synthase (eNOS) to the adventitia would alter vascular reactivity.

Methods and Results Rabbit carotid arteries were surgically isolated and adenoviral vectors encoding eNOS (AdeNOS) or ß-galactosidase instilled into the periarterial sheath at a concentration of 1x10¹⁰ pfu/mL. Arteries were harvested 4 days later for immunostaining, NOS enzymatic assay, measurement of cGMP, and vasomotor studies. Transgene expression in the adventitia was confirmed by histochemistry for ß-galactosidase and immunostaining for eNOS with a monoclonal antibody. Calcium-dependent NOS enzymatic activity and cGMP levels were significantly greater in the AdeNOS-transduced arteries. Maximal contractions to phenylephrine (10^-5 mol/L) were diminished in the AdeNOS-transduced arteries (4.6±0.2 versus 5.6±0.2 g; P<.05), but in the presence of the eNOS inhibitor N^G-monomethyl-L-arginine (3x10^-4 mol/L) there was no difference between the two groups (7.1±0.2 versus 7.5±0.3 g; P=NS). Relaxations to calcium ionophore obtained during submaximal contractions to phenylephrine were significantly enhanced in the AdeNOS-transduced arteries (-log EC₅₀, 7.77±0.08 versus 7.45±0.07; P<.02).

Conclusions We conclude that eNOS gene transfer to the adventitia alters vascular reactivity, as demonstrated by diminished contractile responses to phenylephrine and enhanced relaxations to calcium ionophore. This may represent a therapeutic strategy for vascular diseases characterized by decreased bioavailability of NO.

Key Words: genes • nitric oxide • viruses

	Introduction

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The adventitia anchors the vessel wall to the surrounding connective tissue and contains vasa vasorum, lymphatic channels, and autonomic nerve fibers.^{1 2} Recent investigations suggest that the adventitia may have additional functions and play an important role in the biology of both the normal and diseased vessel wall.^{3 4} In the injured state, human and experimental data demonstrate that the adventitia plays a key role in remodeling and neointimal formation.^{3 4 5 6} Adventitial myofibroblasts may contribute to the effects of vascular injury by causing chronic adventitial constriction and migrating into the neointima.³ The adventitia may also participate in atherogenesis⁷ and vascular hypertrophy secondary to hypertension.⁸ The adventitia is therefore a target for site-specific vascular wall therapy. Adventitial gene transfer offers a mode of delivering recombinant proteins to the vessel wall, avoiding interruption of blood flow or disruption of the endothelium.⁹ Such a strategy may have therapeutic potential as well as help investigate the role of the adventitia in vascular biology.

A potential candidate for therapeutic adventitial gene transfer is the enzyme eNOS. This enzyme produces NO constitutively in a calcium calmodulin–dependent reaction. NO is a pleiotropic effector molecule in the vessel wall, modulating vascular tone,^{10 11} platelet adhesion/aggregation,¹² leukocyte adhesion,¹³ and smooth muscle growth.¹⁴ Expression of recombinant eNOS in the adventitia may be a unique mode of increasing local NO production in the vessel wall. Because of its diffusibility, NO produced in the adventitia may permeate toward the lumen and affect vascular function. The present study was designed to evaluate the effects of adenovirus-mediated adventitial gene transfer of eNOS on vascular reactivity. Specifically, we sought to determine the effects of adventitial expression of eNOS on the response to a contractile agonist, phenylephrine, and vascular relaxations to calcium ionophore, a known stimulant of eNOS, in rabbit carotid arteries.

	Methods

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Construction, Propagation, and Purification of Adenoviral Vector
A recombinant adenovirus encoding the eNOS gene driven by a cytomegalovirus promoter was generated as described.¹⁵ Briefly, bovine eNOS cDNA (provided by Dr David Harrison, Emory University, Atlanta, Ga) was cloned into the pACCMVpLpA vector (provided by Dr Robert Gerard, University of Texas Southwestern Medical Center, Dallas). The resulting plasmid was linearized with Nru I and cotransfected with dl309 into 293 cells by calcium phosphate/DNA coprecipitation. dl309 is a biologically selected, restriction enzyme–site-loss variant of wild-type adenovirus type 5, which retains only a single Xba I site at nucleotide 1339.¹⁶ 293 cells are human embryonic kidney carcinoma cells that have been transformed with the left end of human adenovirus type 5 DNA.¹⁷ Recombinant adenoviral vectors were generated by homologous recombination.¹⁵ Viral plaques were selected and propagated in 293 cells. Viral DNA was enriched by Hirt extraction¹⁸ and screened by restriction mapping and polymerase chain reaction for the presence of eNOS cDNA. Positive plaques underwent two more rounds of plaque purification in 293 cells. Stocks were prepared from positive plaques, and these were used to generate high-titer preparations. Viral preparations were generated by infecting confluent monolayers of 293 cells in 175-cm² flasks with viral stock at an MOI of 1 to 10. Virus was purified by double cesium chloride gradient ultracentrifugation and was dialyzed against 10 mmol/L Tris, 1.0 mmol/L MgCl₂, 1.0 mmol/L HEPES, and 10% glycerol for 4 hours at 4°C. Viral titer was determined by plaque assay.¹⁵ eNOS activity was confirmed by positive NADPH diaphorase staining^{19 20} in confluent 293 cells transduced with AdeNOS. The defectiveness of AdeNOS for replication was tested by adding the virus (10⁷ to 10⁸ pfu/mL) to a monolayer of diploid human embryonic lung fibroblasts (60-mm dish). Replication-competent viruses at an MOI of 10 produce a cytopathic effect and destroy the monolayer in <3 days. Infection with AdeNOS at a comparable MOI produced no observable cytopathic effect after 5 days. The sensitivity for detection of replication-competent virus was 10⁷ to 10⁸ pfu/mL. A recombinant replication-defective adenoviral vector encoding the Escherichia coli ß-galactosidase gene (AdßGal) driven by the cytomegalovirus promoter²¹ was obtained from Dr James Wilson (University of Pennsylvania, Philadelphia) and used as a control. It was propagated, isolated, and quantified as described above. Viral stocks were stored at -70°C. Before use, viral preparations were tested in vitro by histochemical detection of NADPH diaphorase and ß-galactosidase activity in transduced porcine coronary artery smooth muscle cells.

Gene Delivery
Male New Zealand White rabbits weighing 3.0 to 3.5 kg (n=27) were used in these experiments. The animals were housed individually in stainless steel, wire-bottomed cages in a room with a 12-hour light-dark cycle. All experimental protocols were approved by the Institutional Animal Care and Use Committee and were performed in accordance with the recommendations of the American Association for the Accreditation of Laboratory Animal Care. Sedation and anesthesia were obtained with an intramuscular injection of a cocktail containing ketamine (65 mg/kg), xylazine (13 mg/kg), and acepromazine (22 mg/kg). Paramedian cervical incisions were made in the anterior neck, and the common carotid arteries were exposed bilaterally by blunt dissection. The periarterial sheath was exposed near the distal end of the artery, and an incision was made in this sheath after a purse-string suture was placed. A 24-gauge plastic vascular cannula (Jelco, Critikon) was inserted through this incision, and the adenoviral vector–containing solution (200 µL of a 1x10¹⁰-pfu/mL concentration) was instilled into the sheath via the cannula. The cannula was removed, and the purse-string suture was closed around the defect in the periarterial sheath. This method resulted in layering of the vector solution around a length of the artery equal to the length of the cannula (2 cm). The purse-string suture at the entry point of the cannula served as a mark to identify the transduced segment of the artery. An identical concentration of AdßGal was placed in the periarterial sheath of the contralateral vessel. To assess the effects of AdßGal on vascular reactivity, in 6 animals the periarterial sheaths of carotid arteries were instilled with AdßGal vector on one side and vehicle on the other. The cervical incisions were closed with subcuticular sutures, and the animal was allowed to recover. Four days later, carotid arteries were isolated as above. A 2-cm segment of each carotid artery measured from the entry point of the cannula was harvested, and the animal was then euthanized. Four animals were used for confirmation of gene transfer by histochemistry for ß-galactosidase and immunohistochemistry for eNOS, 5 animals were used for NOS enzymatic activity, and 18 animals were used for measurement of cGMP and vascular reactivity studies.

Efficiency of Adventitial Gene Transfer
The efficiency of adventitial gene transfer was determined by X-Gal staining. Segments of rabbit carotid arteries transduced with AdßGal or AdeNOS were fixed for 30 minutes at 4°C in 2% formaldehyde and 0.2% glutaraldehyde in PBS, pH 7.4. An X-Gal solution (1 mL) was added to the rings, and these were incubated at 37°C for 2 hours. Vessels were then photographed, rinsed with PBS, cut into 3-mm rings, and embedded in paraffin. Cross sections 5 µm thick spaced at least 100 µm apart were cut from each segment and counterstained with nuclear fast red. The sections were examined under a light microscope. The efficiency of gene transfer to the adventitia was determined by the number of stained cells within a distance of 100 to 150 µm from the outer extent of the media and expressed as a percentage of total cells counted.

eNOS Immunoreactivity
For immunohistochemical detection of eNOS, arterial rings were fresh-frozen in O.C.T. compound (Miles, Inc), and serial 5-µm sections were cut. After immersion fixation in acetone (4°C) and 1% paraformaldehyde/EDTA, the sections were incubated in 0.1% sodium azide/0.3% hydrogen peroxide and then incubated with 5% goat serum/PBS–Tween 20 to block nonspecific protein binding sites. An eNOS monoclonal antibody (5 µg/mL, 1:50 of stock, Transduction Laboratory) was applied for 60 minutes at room temperature, followed by incubations with biotinylated rabbit anti-mouse F(ab')₂ (1:300, 20 minutes) secondary antibody and peroxidase-conjugated streptavidin (1:300, 20 minutes) (Vector Laboratories, Inc). After a 30-second immersion in 0.1 mol/L sodium acetate buffer (pH 5.2), eNOS immunoreactivity was visualized with 3-amino-9-ethylcarbazole and hematoxylin counterstaining.

Quantification of eNOS immunoreactivity in the adventitia was done by two of the authors in a blinded fashion. A score of 0 was given for no staining, 1 for minimal staining, 2 for moderate staining, and 3 for intense staining. The score assigned was determined by consensus of the observers. For control studies, the specificity of eNOS immunolabeling was examined by (1) omission of the primary antiserum in the incubation medium and (2) immunostaining of AdeNOS-transduced arteries with an isotype-matched primary antibody of eNOS, a mouse monoclonal IgG1 against anti-human CD4 antiserum (OPD4, 1:50 dilution, Dako).

NOS Enzymatic Assay
NOS enzymatic activity was assayed in transduced arteries with L-arginine–to–L-citrulline conversion as described previously.²² Briefly, protein homogenates prepared as described above were incubated for 1 hour at 27°C in a solution containing 1 mmol/L NADPH, 10 µmol/L tetrahydrobiopterin, 0.83 mmol/L CaCl₂, 14.7 nmol/L [³H]-L-arginine, 5 µmol/L L-arginine, 54 mmol/L l-valine, 1.2 mmol/L MgCl₂, 2 µmol/L FAD, and 50 U/mL calmodulin. Calcium-independent NOS activity was measured by replacement of CaCl₂ with EGTA (1 mmol/L) in replicate samples. Incubations were terminated by addition of 1.5 mL ice-cold stop buffer (20 mmol/L HEPES, 8 mmol/L EDTA, pH 5.5). The reaction mix was then passed through Poly-Prep chromatography columns (Bio-Rad) containing 1 mL of Dowex AG50W-X8 resin and eluted with 2 mL of distilled water. [³H] activity in the effluent is due to [³H]-L-citrulline and was measured in a scintillation counter. A small aliquot of tissue homogenate was used to determine protein concentration with bicinchoninic acid protein assay reagent (Pierce), and NOS activity is expressed as pmol [³H]-L-citrulline produced · mg protein^-1 · h^-1.

Measurement of Arterial cGMP Levels
From each harvested artery, a ring was immersed immediately in a solution of 3-isobutyl-L-methylxanthine (IBMX, 1 mmol/L) and incubated at 37°C for 30 minutes before being snap-frozen in liquid nitrogen and stored at -70°C until the time of assay. cGMP levels were determined by a radioimmunoassay kit (Amersham) as previously described.²³

Vascular Reactivity Studies
Rings (4 mm long) from each carotid artery were used for assessing vascular reactivity. Rings were connected to isometric force displacement transducers (Grass Instruments) and suspended in organ chambers filled with 25 mL of gassed (95% O₂/5% CO₂) modified Krebs-Ringer bicarbonate solution (pH 7.4, temperature 37°C; composition in mmol/L: NaCl 118.3, KCl 4.7, CaCl₂ 2.5, MgSO₄ 1.2, KH₂PO₄ 1.2, NaHCO₃ 25.0, calcium sodium EDTA 0.026, and glucose 11.1). The rings were allowed to equilibrate for 1 hour and then were stretched to the optimal point on the length-tension curve as determined by repeated exposure to 20 mmol/L KCl. After three washes with the control solution, a concentration-response curve to phenylephrine (3x10^-8 to 10^-5 mol/L) was obtained. After three washouts, the concentration-response to phenylephrine was repeated in the presence of L-NMMA (3x10^-4 mol/L). All concentration responses were done in the presence of indomethacin (10^-5 mol/L) to block any effects of prostanoids. In a separate group of rabbits (n=6), concentration responses to calcium ionophore, acetylcholine, and SNP were examined during a submaximal contraction obtained with 3x10^-7 to 10^-6 mol/L phenylephrine. The concentration response to calcium ionophore was repeated in the presence of the NOS enzyme inhibitor L-NAME (3x10^-4 mol/L). Care was taken to match the contractions in the two groups before the concentration responses were obtained. To test the effects of AdßGal on vascular reactivity, in 6 animals responses to phenylephrine and calcium ionophore were compared in arteries exposed to AdßGal or vehicle.

The following drugs were used: acetylcholine, calcium ionophore (A23187), L-NMMA, L-NAME, indomethacin, SNP, and phenylephrine bitartrate (all from Sigma Chemical Co). Drugs were made up immediately before study, and the concentrations are reported as the final molar concentration in the organ chamber. Relaxation is expressed as percent reduction of the phenylephrine-induced contraction.

Statistics
Data are presented as mean±SEM. Immunoreactivity scores, cGMP levels, and maximal contractions to phenylephrine were analyzed by unpaired t test. NOS enzymatic assay results were analyzed by one-way ANOVA followed by Scheffé's post hoc test. Vascular reactivity data were analyzed as follows: The EC₅₀ and AUC were compared by unpaired t test; the concentration-response curves were analyzed by two-way ANOVA. Statistical significance was accepted at a value of P<.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Efficiency and Localization of Gene Transfer
In arteries exposed to AdßGal at 1x10¹⁰ pfu/mL and harvested 4 days later, expression of ß-galactosidase in the adventitia was confirmed by X-Gal staining (Fig 1A). Transgene expression was limited to the adventitial layer (Fig 1C). The cells expressing the transgene included those with a characteristic elongated morphology suggestive of a fibroblast phenotype. There was no staining in the AdeNOS-transduced arteries (Fig 1B and 1D). The efficiency of gene transfer to the adventitia was quantified at 18±5% of total cells counted. No inflammatory infiltrate was present in the transduced arteries.

View larger version (78K): [in this window] [in a new window]   Figure 1. X-Gal staining of rabbit carotid arteries 4 days after instillation of 200 µL of a 1x1010-pfu/mL concentration of AdßGal or AdeNOS vector in periarterial sheath. Segments of rabbit carotid arteries transduced with AdßGal or AdeNOS were fixed and incubated in a solution of X-Gal at 37°C for 2 hours. Blue staining is seen on adventitial surface of artery transduced with AdßGal (A) but not in artery transduced with AdeNOS (B). Bar=5 mm. C and D, High-power cross-sectional view of arteries after staining with nuclear fast red. Adventitia-specific expression of ß-galactosidase gene is seen in AdßGal-transduced artery (C) but not in AdeNOS-transduced artery. Morphology of transduced cells is suggestive of fibroblasts. Magnification C and D x400.

eNOS Immunoreactivity
Strongly positive eNOS immunoreactivity was detected in the adventitia of AdeNOS-transduced arteries (Fig 2B and 2D), whereas in the AdßGal-transduced arteries, such immunoreactivity was localized to the endothelium of vasa vasorum and capillaries (Fig 2A and 2C). The immunoreactivity intensity score was significantly greater in the AdeNOS-transduced arteries (3.0 versus 1.3±0.3; P<.05, n=3). No immunoreactivity was observed when AdeNOS-transduced arteries were immunostained with an isotype-matched primary antibody of eNOS, thereby confirming the specificity of eNOS immunolabeling (data not shown). The endothelium of the arteries served as a positive control for the eNOS immunoreactivity.

View larger version (120K): [in this window] [in a new window]   Figure 2. Immunohistochemical localization of eNOS in rabbit carotid arteries 4 days after transduction in vivo with AdßGal (A and C) or AdeNOS (B and D). Positive immunostaining is seen in endothelium of both arteries, and a marked increase in immunoreactivity is seen in adventitia of AdeNOS-transduced arteries (B and D). Magnification A and B x40; C and D x400.

NOS Enzymatic Assay
NOS enzymatic activity was more than a log-fold increased in AdeNOS-transduced arteries compared with AdßGal-transduced arteries (Fig 3). More than 90% of this activity was calcium dependent, indicating that iNOS was not responsible for this increase. NOS enzymatic activity detected in AdßGal-transduced arteries is probably a result of endogenous eNOS in the endothelium, because this activity was predominantly Ca⁺² dependent.

View larger version (10K): [in this window] [in a new window]   Figure 3. Total and Ca2+-independent NOS enzymatic activity in rabbit carotid arteries transduced with AdeNOS or AdßGal 4 days earlier; n=5 rabbits. *P<.01.

Arterial cGMP Levels
Biological activity of recombinant eNOS in the adventitia was evaluated by measurement of arterial cGMP levels. cGMP levels were significantly elevated in the AdeNOS-transduced arteries compared with AdßGal-transduced arteries (Fig 4). cGMP levels in the AdßGal-transduced arteries were not significantly different from arteries exposed to vehicle alone (5.7±1.6 versus 6.6±3.0 pmol/mg protein; n=4).

View larger version (8K): [in this window] [in a new window]   Figure 4. cGMP levels in arterial rings from rabbit carotid arteries transduced with AdeNOS or AdßGal 4 days earlier; n=8 rabbits. *P<.05.

Contractile Responses to Phenylephrine
Maximal contractions to phenylephrine were significantly reduced in the AdeNOS-transduced arteries (4.6±0.2 versus 5.6±0.2 g, P<.05; Fig 5). When maximal contractions to phenylephrine were repeated after rings were incubated with L-NMMA (3x10^-4 mol/L), no difference was observed in the two groups (7.1±0.2 versus 7.5±0.3 g, P=NS; Fig 5). L-NMMA caused a significant increase in the maximal contractions in both groups, with a greater mean percentage increase seen in the AdeNOS-transduced arteries (53±7% versus 34±3%, P<.05). When responses to phenylephrine in AdßGal-transduced arteries were compared with those of arteries exposed to vehicle alone, no significant differences were observed (maximal contractions, 6.3±0.4 versus 6.6±0.5 g, n=6; P=NS).

View larger version (13K): [in this window] [in a new window]   Figure 5. Maximal contractions to phenylephrine (10-5 mol/L) in rabbit carotid arteries transduced with AdßGal or AdeNOS in absence and presence of L-NMMA (3x10-4 mol/L). *P<.05 AdßGal vs AdeNOS; #P<.05 for maximal contractions in presence of L-NMMA compared with maximal contractions in the absence of L-NMMA. Data are mean±SEM; n=6 rabbits.

Relaxations to Calcium Ionophore, Acetylcholine, and SNP
Relaxations to calcium ionophore were significantly enhanced in the AdeNOS-transduced arteries compared with AdßGal-transduced arteries. Both EC₅₀ and AUC were significantly greater in the AdeNOS group (Fig 6A and Table). Submaximal contractions to phenylephrine (2.4±0.2 g in the AdeNOS group and 2.7±0.13 g in the AdßGal group) were not significantly different. When the concentration response to calcium ionophore was repeated in the presence of the NOS enzyme inhibitor L-NAME (3x10^-4 mol/L), the relaxations obtained were significantly inhibited, and no differences were observed in the two groups (Fig 6B). Enhanced relaxations in AdeNOS-transduced arteries were not due to a nonspecific effect of the vector, because relaxations to calcium ionophore were similar in arteries transduced with AdßGal compared with arteries instilled with vehicle alone (Fig 6C). Relaxations to acetylcholine were significantly enhanced in the AdeNOS-transduced arteries (Table and Fig 7). Relaxations to SNP were not different in the two groups (Fig 8).

View larger version (9K): [in this window] [in a new window]   Figure 6. Concentration-response curves to A23187 in rabbit carotid arteries during submaximal contractions to phenylephrine. A, Relaxations to A23187 in arteries transduced with AdßGal or AdeNOS; B, relaxations to A23187 in arteries transduced with AdßGal or AdeNOS in presence of L-NAME (3x10-4 mol/L); C, relaxations to A23187 in arteries transduced with AdßGal or exposed to vehicle alone. Relaxations are expressed as percent reduction of phenylephrine-induced contractions. Submaximal contractions obtained with 3x10-7 to 10-6 mol/L phenylephrine were not different in the groups (data not shown). Data are mean±SEM; n=6 rabbits for each panel.

View this table: [in this window] [in a new window]   Table 1. Relaxations of Isolated Carotid Artery Rings to Calcium Ionophore (A23187) and Acetylcholine 4 Days After Adventitial Gene Transfer With AdßGal or AdeNOS at a Concentration of 1x1010 pfu/mL

View larger version (15K): [in this window] [in a new window]   Figure 7. Concentration-response curves demonstrating relaxations to acetylcholine in rabbit carotid arteries transduced with AdßGal or AdeNOS. Submaximal contractions were obtained with phenylephrine and were not significantly different (3.4±0.2 g in AdeNOS group and 3.2±0.2 g in AdßGal group). Data are mean±SEM; n=4 rabbits; *P<.05.

View larger version (15K): [in this window] [in a new window]   Figure 8. Concentration-response curves to SNP in rabbit carotid arteries transduced with AdßGal and AdeNOS obtained during submaximal contractions to phenylephrine. Data are mean±SEM; n=5 rabbits.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This study demonstrates for the first time that expression of a recombinant protein in the adventitia by in vivo adenovirus-mediated gene transfer can result in a biological effect. Our results indicate that delivery of adenoviral vectors encoding the ß-galactosidase and eNOS genes to the periarterial sheath of rabbit carotid arteries results in adventitia-specific gene transfer and expression. Expression of recombinant eNOS in the adventitia resulted in marked increase in calcium-dependent NOS activity, an elevation of cGMP levels, and a diminished sensitivity to phenylephrine. Furthermore, the recombinant eNOS is responsive to stimulation by calcium ionophore and acetylcholine, as manifested by enhanced relaxations to these agents.

Gene transfer to the adventitia by adenoviral vectors has been demonstrated by Rios and coworkers⁹ in monkey femoral and carotid arteries. We obtained similar adventitia-specific gene transfer in rabbit carotid arteries by instilling adenoviral vectors into the periarterial sheath. The exact nature of cells transduced is not clear from the present study but is likely to include adventitial fibroblasts, on the basis of their characteristic morphology. Preliminary evidence from immunogold electron microscopy suggests that after ex vivo adventitial gene transfer of the AdeNOS vector to the canine basilar artery, recombinant eNOS is localized to the membrane and cytoplasm of adventitial fibroblasts (Z.S.K., unpublished observations). The concentration of adenoviral vectors used in the present study has yielded relatively efficient vascular gene transfer after luminal or adventitial delivery.^{9 24} An inflammatory cell infiltrate in response to the vectors was not observed, although the possibility of an inflammatory infiltrate developing at a later time point cannot be excluded. The method of gene delivery described in this study may serve as a useful model for testing the effects of gene transfer to the adventitia using small amounts of vector in an inexpensive laboratory animal.

NO mediates several of its effects by activating a soluble cytoplasmic guanylate cyclase, an enzyme that catalyzes formation of cGMP from GTP.²⁵ Increased levels of cGMP in arteries transduced with the AdeNOS vector are probably a result of the increased production of NO in the adventitia, leading to increased cGMP levels in the underlying smooth muscle cells. Adenovirus-mediated gene transfer has been associated with increased production of cytokines²⁶ and may, in theory, lead to expression of iNOS. In the present study, a marked (greater than a log-fold) elevation was seen in the NOS enzymatic activity in AdeNOS-transduced arteries compared with AdßGal-transduced arteries. Nearly all of this activity was Ca²⁺ dependent, suggesting that expression of the recombinant eNOS rather than the iNOS isoform led to the increase in enzyme activity. Furthermore, significant elevations of cGMP or hyporeactivity to contractile agonists were not demonstrated in AdßGal-transduced arteries compared with arteries exposed to vehicle alone. These observations help to exclude the possibility of iNOS expression secondary to adenovirus-mediated gene transfer to the arterial wall.

Maximal contractions to phenylephrine were diminished in the AdeNOS-transduced arteries. This is probably secondary to increased NO production in these arteries due to expression of recombinant eNOS. The specificity of these findings was confirmed by use of the eNOS inhibitor L-NMMA. After preincubation with L-NMMA, a significant increase in maximal contractions to phenylephrine occurred in both groups, with a proportionately greater increase in the AdeNOS-transduced arteries, thereby abolishing the difference in the two groups. Although phenylephrine is not a physiological or pathophysiological mediator of arterial vasoconstriction, these findings serve as a proof of the concept that responses to local or circulating vasoconstrictors may be attenuated by activity of recombinant eNOS. Our results are in agreement with other studies that report a decrease in contractile responses as a result of increased NO production in the vascular wall.^{27 28} The diminished sensitivity to contractile agonists is not due to a toxic effect of AdeNOS on smooth muscle, because responses to SNP in the two groups were similar. SNP is a nitrovasodilator that produces arterial relaxations by spontaneous release of NO.²⁹

The enhancement of relaxations to calcium ionophore in arteries transduced with AdeNOS is a novel finding. Calcium ionophore causes endothelium-dependent relaxations by increasing calcium entry into the endothelium, resulting in stimulation of endogenous eNOS. Because it acts in a receptor-independent manner, it may result in increased intracellular calcium in adventitial cells, thereby stimulating recombinant eNOS in cells expressing the transgene. In AdeNOS-transduced arteries, the concentration-response curve to calcium ionophore was significantly shifted to the left, indicating an enhanced response. Although maximal relaxations were the same in both groups (100%), the EC₅₀ and AUC were significantly different in the AdeNOS-transduced arteries. This effect was abolished in the presence of the NOS inhibitor L-NAME. Furthermore, relaxations to calcium ionophore were not different in AdßGal-transduced arteries compared with arteries exposed to vehicle alone. Therefore, an enhanced response to calcium ionophore is an effect specific to AdeNOS transduction and increased expression of recombinant eNOS in the arterial wall. This effect is not due to an enhanced sensitivity to NO, because relaxations to SNP were similar in the AdeNOS and AdßGal arteries. Interestingly, relaxations to acetylcholine were also enhanced in AdeNOS-transduced arteries. This observation suggests that muscarinic receptors may be present in adventitial cells expressing recombinant eNOS. Stimulation of these receptors by acetylcholine may increase intracellular Ca²⁺ and thereby increase the activity of recombinant eNOS, resulting in enhanced relaxations.

The adventitia may be an important target for site-specific vascular wall therapy, in particular to influence the arterial response to injury. The adventitial response to arterial injury may be an important determinant of restenosis by influencing remodeling as well as neointimal formation.³⁰ Recent studies demonstrated that balloon injury results in an increased rate of replication in the adventitia.^{3 5} Adventitial fibroblasts differentiate into myofibroblasts, migrate into the intima, and importantly, may cause a chronic adventitial constriction and therefore result in restenosis.^{5 3} An increase in local NO production in the adventitia and media by recombinant eNOS may limit restenosis by inhibiting cellular proliferation and vasoconstriction. The limited duration of transgene expression after adenoviral gene transfer may be enough to alter critical pathways in this process. Adventitial delivery of adenoviral vectors may be easily achieved in large peripheral arteries or cerebral arteries by direct application during surgery or instillation into the subarachnoid space.³¹ Adventitialdelivery to the coronary arteries may be possible by such novel strategies as instillation of vectors into the pericardial space³² or percutaneous intervention by special catheters.³³

A short half-life, high reactivity, and limited solubility in aqueous media make local delivery of NO to the vessel wall difficult.³⁴ Our results demonstrate that adenovirus-mediated gene transfer of eNOS to the adventitia results in a functionally active recombinant enzyme. Because NO is an easily and rapidly diffusible substance, adventitial expression of eNOS may result in effects on the medial smooth muscle. This technique is therefore a feasible method of local delivery of NO. A recent study demonstrated that adenovirus-mediated transfer of the eNOS gene to the lungs reduced acute hypoxic pulmonary vasoconstriction in rats.³⁵ Recombinant adenovirus was delivered by aerosol and localized in part to the adventitia of the pulmonary arteries.

The present study illustrates a strategy of gene transfer to the adventitia in vivo, yielding expression of a functional recombinant protein and a resulting biological effect on vasomotor function. Expression of recombinant eNOS in the adventitia results in a marked increase in Ca²⁺-dependent NOS enzyme activity in the arterial wall, elevated levels of cGMP, diminished contractile responses to phenylephrine, and enhanced relaxations to calcium ionophore and acetylcholine. These findings imply that expression of recombinant eNOS in the adventitia may be beneficial in vascular disorders characterized by vasoconstriction and excessive cell proliferation. Such conditions could include coronary, cerebral, and peripheral arterial spasm; pulmonary hypertension; and restenosis after balloon angioplasty.

	Selected Abbreviations and Acronyms

 

AdßGal = recombinant adenovirus encoding Escherichia coli ß-galactosidase gene driven by cytomegalovirus promoter AdeNOS = recombinant adenovirus encoding bovine eNOS gene driven by cytomegalovirus promoter AUC = area under the curve EC50 = concentration causing 50% decrease of phenylephrine-induced contraction expressed as negative logarithm of molar concentration eNOS = endothelial nitric oxide synthase iNOS = inducible isoform of nitric oxide synthase L-NAME = NG-monomethyl-L-arginine ester L-NMMA = NG-monomethyl-L-arginine MOI = multiplicity of infection NOS = nitric oxide synthase pfu = plaque-forming units SNP = sodium nitroprusside X-Gal = 5-bromo-4-chloro-3-indoyl-ß-D-galactopyranoside

	Acknowledgments

 
This work was supported by Mayo Clinic intramural research grants (Drs O'Brien and Schwartz), the J. Holden DeHaan Foundation (Dr Schwartz), and in part by National Institutes of Health grants HL-44116 and HL-53542 (Dr Katusic). The authors would like to thank Sharon Guy and Leslie Smith for invaluable technical assistance.

Received September 24, 1996; revision received April 1, 1997; accepted April 18, 1997.

	References

Top Abstract Introduction Methods Results Discussion References

 
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