Adventitial Expression of Recombinant eNOS Gene Restores NO Production in Arteries Without Endothelium

From the Departments of Anesthesiology and Pharmacology (M.T., A.F.Y.C., Z.S.K.) and the Divisions of Endocrinology and Metabolism (T.O'B.) and Anatomic Pathology (T.B.C.), Mayo Clinic, Rochester, Minn.

Correspondence to Zvonimir S. Katusic, MD, PhD, Associate Professor, Departments of Anesthesiology and Pharmacology, Mayo Clinic, 200 First St SW, Rochester, MN 55905. E-mail Katusic.Zvonimir{at}mayo.edu

	Abstract

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Abstract—The current study was designed to determine the effect of recombinant endothelial nitric oxide synthase (eNOS) gene expression on endothelium-dependent relaxations to bradykinin in isolated canine basilar, coronary, or femoral arteries. Arterial rings were exposed ex vivo (30 minutes at 37°C) to an adenoviral vector encoding either the eNOS gene (AdCMVeNOS) or the ß-galactosidase reporter gene (AdCMVß-Gal). Twenty-four hours after transduction, transgene expression was evident mainly in the adventitia. Expression of recombinant proteins was much higher in basilar arteries than in coronary or femoral arteries. Rings of control, AdCMVß-Gal, and AdCMVeNOS arteries with and without endothelium were suspended for isometric tension recording. Levels of cGMP were measured by radioimmunoassay. In AdCMVeNOS basilar arteries with endothelium, relaxations to low concentrations of bradykinin (3x10^-11 to 10^-9 mol/L) were significantly augmented. In contrast, in coronary and femoral arteries with endothelium, AdCMVeNOS transduction did not affect relaxations to bradykinin. Removal of the endothelium abolished bradykinin-induced relaxations in control and AdCMVß-Gal basilar arteries. However, in basilar arteries transduced with AdCMVeNOS even when the endothelium was removed, stimulation with bradykinin (3x10^-11 to 10^-9 mol/L) caused relaxations as well as increases in cGMP production. The relaxations to bradykinin were completely blocked by an NOS inhibitor, N^G-nitro-L-arginine methyl ester. Electron microscopic analysis revealed that recombinant eNOS protein was expressed in fibroblasts of the basilar artery adventitia. These results suggest that genetically modified adventitial fibroblasts may restore production of NO in cerebral arteries without endothelium. Our findings support a novel concept in vascular biology that fibroblasts in the adventitia may play a role in the regulation of vascular tone after successful transfer and expression of recombinant eNOS gene.

Key Words: fibroblasts • adenoviral vector • gene transfer • gene therapy • bradykinin

	Introduction

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Nitric oxide is a potent vasodilator produced by activation of eNOS. NO mediates vascular relaxation in response to shear stress and a number of endogenous vasoactive substances.^{1 2 3 4 5} NO has been implicated in the regulation of blood pressure and regional blood flow,^{3 6} and NO also inhibits vascular smooth muscle cell proliferation,⁷ platelet aggregation,⁸ and leukocyte adhesion.⁹ Impaired endothelial production of NO has been reported in blood vessels exposed to hypertension, hyperlipidemia, diabetes, and atherosclerosis.^{10 11} In the clinical setting, NO donors such as nitroglycerin or nitrates have been used to restore normal levels of NO in the vascular wall. However, it is difficult to administer these compounds locally, and there are several problems associated with their systemic administration, including headache, hypotension, and tolerance. To restore impaired production of NO, site-specific delivery of recombinant eNOS gene has been proposed as an alternative new therapeutic approach.¹²

Recent studies have reported that in rat carotid arteries, direct transfer of eNOS cDNA with the use of the Sendai virus/liposome complex prevents neointimal formation after balloon injury,¹² and that in rat lungs, adenovirus-mediated transfer of eNOS gene by aerosol delivery reduces hypoxia-induced pulmonary vasoconstriction.¹³ Our previous study demonstrated that adenovirus-mediated transfer of recombinant eNOS gene increases local NO production and formation of cGMP in canine cerebral arteries.¹⁴ The current study was designed to determine whether expression of recombinant eNOS gene may affect endothelium-dependent relaxations to bradykinin in isolated canine cerebral and peripheral arteries. During our preliminary studies, we noticed that after eNOS gene transfer into basilar arteries, bradykinin caused relaxations even in arteries without endothelium. This finding suggests that expression of recombinant eNOS in the adventitia may restore production of NO. Therefore, we also attempted to characterize the target cells expressing recombinant eNOS in the adventitia and to analyze the mechanisms of endothelium-independent relaxations to bradykinin in eNOS gene–transduced cerebral arteries.

	Methods

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Construction, Propagation, and Purification of Adenoviral Vectors
Recombinant adenovirus containing the cDNA encoding eNOS was generated as described.¹⁵ In brief, the shuttle vector pACCMVpLpA, a kind gift of Dr Robert Gerard (University of Texas Southwestern Medical Center, Dallas, Tex), was used. The plasmid containing cDNA for bovine aortic endothelial cell eNOS was generously provided by Dr David G. Harrison (Emory University, Atlanta, Ga). The eNOS cDNA was inserted into pACCMVpLpA. The resulting plasmid was linearized with NruI and cotransduced 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 XbaI site at nucleotide 1339.¹⁶ The 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 picked 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 2 further 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 performed by infecting a confluent monolayer of 293 cells in T175 flasks with viral stock at a multiplicity of infection of 1 to 10. Virus was purified by double cesium gradient ultracentrifugation and 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 measured in cultured porcine coronary smooth muscle cells transduced for 48 hours with AdCMVeNOS at a multiplicity of infection of 200.¹⁹ The cells acquired eNOS enzymatic activity as quantified by measuring [³H]L-citrulline formation from [³H]L-arginine.^{2 19} The activity of eNOS was almost abolished by EGTA (10^-3 mol/L), a calcium chelator, or L-NAME (10^-4 mol/L), an NOS inhibitor.¹⁹ Enzymatic activity was also confirmed by positive NADPH diaphorase staining^{20 21} in 293 cells transduced with AdCMVeNOS. The replication defectiveness of AdCMVeNOS was tested by adding the virus (10⁷ to 10⁸ pfu/mL) to a monolayer of diploid human embryonic lung fibroblasts (60-mm dish). Infection with AdCMVeNOS at a multiplicity of infection 10 produced no observable cytopathic effect after 5 days. Replication-competent viruses at a comparable multiplicity of infection produced a cytopathic effect and destroyed the monolayer in <3 days. The sensitivity for detection of replication-competent virus was 10⁷ to 10⁸ pfu/mL. AdCMVß-Gal, used in all experiments as a control, was a kind gift of Dr James M. Wilson (University of Pennsylvania, Philadelphia). It was propagated, isolated, and quantified as described above.

Gene Transfer
Rings (3 mm long) of basilar, left anterior descending coronary, and femoral arteries were taken from mongrel dogs (18 to 27 kg) anesthetized with 30 mg/kg sodium pentobarbital administered intravenously. All procedures were in accordance with Institutional Animal Care and Use Committee guidelines of Mayo Clinic. To remove blood, arterial rings were gently rinsed with Krebs-Ringer bicarbonate solution (in mmol/L: NaCl 118.3, KCl 4.7, CaCl₂ 2.5, MgSO₄ 1.2, KH₂PO₄ 1.2, NaHCO₃ 25.0, calcium EDTA 0.0026, and glucose 11.1). Loose perivascular tissue was removed carefully. In certain rings of basilar arteries, the endothelium was removed mechanically. The surfaces of needles (19 to 22 gauge) were made rough by abrasion with sandpaper, and the needles were fixed in a dish filled with Krebs-Ringer bicarbonate solution. Denudation was accomplished by sliding an arterial segment over the needle with 2 pairs of forceps under microscopic guidance. Successful removal of endothelial cells was confirmed by light microscopy and polychromatic staining.²² Then the rings were randomly assigned for gene transfer. Arterial rings were transduced with an adenoviral vector in minimal essential medium (with Earle's salts, containing 0.1% BSA, 100 U/mL penicillin, and 100 µg/mL streptomycin) for 30 minutes at 37°C, transferred to minimal essential medium, and incubated for 24 hours at 37°C in a CO₂ incubator (5% CO₂–95% air, Forma Scientific, Inc).

Histochemical and Immunohistochemical Analyses of Gene Expression
For histochemical staining of ß-gal, the vessels were fixed for 30 minutes in 2% paraformaldehyde and 0.2% glutaraldehyde in PBS. They were then rinsed with PBS and placed in 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside reagent for 2 hours.²³ The stained vessels were dehydrated through a serial graduation of ethyl alcohol–xylene washes and embedded in paraffin. Serial 5-µm sections were lightly counterstained with eosin. For immunohistochemical staining of recombinant eNOS, arterial rings were frozen in OCT compound (10% polyvinyl alcohol, 4% polyethylene glycol, and 86% nonreactive ingredients; Miles), and serial 5-µm sections were cut. After immersion-fixation in acetone (4°C) and 1% paraformaldehyde–EDTA, the sections were incubated in a mixture of 0.1% NaN₃ and 0.3% H₂O₂ and then incubated with 5% goat serum–PBS-T to block the nonspecific protein-binding sites. An eNOS monoclonal antibody (5 µg/mL, 1:50 of stock; Transduction Laboratories) 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. Arterial rings taken from the same dogs were used and stained in the same manner.

For control studies, the specificity of eNOS immunolabeling was examined by omission of the primary eNOS antiserum from the incubation medium and staining with an isotype-matched primary antibody of eNOS, a mouse IgG1 monoclonal anti-human CD4 antiserum (OPD-4, 1:50 dilution; Dako).

Quantification of ß-Gal Protein
An ELISA technique was used to quantify ß-gal protein. Twenty-four hours after gene transfer, arterial rings were homogenized in buffer containing 0.2% Triton X-100, 1 mmol/L PMSF, and 100 mmol/L K₂HPO₄, pH 7.8, at 4°C. Tissue debris was cleared by centrifugation at 14 000g for 10 minutes at 4°C. ß-Gal protein levels in the supernatant were determined by using a ß-gal ELISA kit (5 Prime 3 Prime, Inc). Total protein levels in the supernatant were measured by the method of Lowry et al.²⁴ In some experiments, the external diameter of the vessels was measured microscopically with a stage micrometer. Arterial rings taken from the same dogs were studied in parallel.

Analyses of Vascular Reactivity
Twenty-four hours after gene transfer, arterial rings were connected to isometric force-displacement transducers (Grass Instruments) and suspended in an organ chamber filled with 25 mL of Krebs-Ringer bicarbonate solution (pH 7.4, 37°C) gassed with 94% O₂–6% CO₂. Isometric tension was recorded continuously. Arteries were allowed to stabilize for 1 hour. The rings were then stretched progressively to optimal tension (3 g tension in basilar arteries,²⁵ 8 g tension in coronary arteries,²⁶ or 9 g tension in femoral arteries,²⁶ as determined by repeated stimulation with 10 µmol/L UTP in basilar arteries or with 20 mmol/L KCl in coronary and femoral arteries). Concentration-response curves to bradykinin were obtained cumulatively during submaximal contractions with the EC₅₀ of each contractile agonist (UTP in basilar arteries, U46619 in coronary arteries, and phenylephrine in femoral arteries). To inhibit cyclooxygenase activity, endothelium-dependent relaxations to bradykinin were performed in the presence of indomethacin (10 µmol/L). The incubation time with indomethacin or L-NAME was 30 or 15 minutes, respectively. The relaxations were expressed as a percentage of maximal relaxations induced by papaverine (300 µmol/L).

Measurement of Intracellular cGMP
A radioimmunoassay technique was used to determine the levels of cGMP, as reported previously.²⁷ Twenty-four hours after gene transfer, 10 µmol/L indomethacin and 1 mmol/L 3-isobutyl-1-methylxanthine were added to the incubation medium for 30 minutes at 37°C to inhibit cyclooxygenase activity and the degradation of cGMP by phosphodiesterases, respectively. During the last 2 minutes of the 30-minute incubation, certain rings were stimulated with 1 nmol/L bradykinin. Then the rings were removed from the medium and quickly frozen in LN₂. After homogenization, cGMP levels were measured by a cGMP radioimmunoassay kit (Amersham). Total protein levels were determined by the method of Lowry et al.²⁴ Arterial rings taken from the same dogs were studied in parallel.

Electron Microscopy
Localization of eNOS protein was examined in ultrathin sections of the specimens (on uncoated nickel grids) with the use of the immunogold labeling technique.²⁸ After fixation, specimens were dehydrated in a graded series of ethyl alcohol, infiltrated, and embedded in LR White resin, which was allowed to polymerize at 50°C to 55°C for 2 to 3 days. Cross sections of the vessel were thin sectioned and mounted on 300-mesh nickel grids. Grids were preincubated for 1 hour in PBS-T plus 5% BSA, incubated for 2 hours in mouse monoclonal antibodies to eNOS (Transduction Laboratories) diluted 1:50 in PBS-T, rinsed thoroughly in PBS-T, incubated for 60 minutes in goat anti-mouse IgG-serum, conjugated to 15-nm colloidal gold, and rinsed again in PBS-T. All incubations were performed at room temperature. After they were dry, the grids were stained with uranyl acetate and lead citrate. Examination and photomicrographs of labeled fibroblasts were obtained on a CM-10 transmission electron microscope. Control specimens were exposed to the same procedure after omission of the primary antibody.

Drugs
The following agents were used: indomethacin, UTP, bradykinin, papaverine hydrochloride, EDTA, L-NAME, phenylephrine bitartrate, BSA (fraction V), PMSF (Sigma Chemical Co), U46619 (Cayman Chemical Co), minimal essential medium, and penicillin-streptomycin (GIBCO BRL). Indomethacin was dissolved with equal molar concentrations of Na₂CO₃. All concentrations are expressed as final molar concentration in medium or solution.

Statistical Analysis
The results are expressed as mean±SEM. In each set of experiments, n refers to the number of animals studied. Statistical evaluation of the data was performed by ANOVA, followed by Bonferroni correction and Dunnett's post hoc test.²⁹ A value of P<0.05 was considered statistically significant.

	Results

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Expression of ß-Gal Reporter Gene and eNOS Gene
Twenty-four hours after AdCMVß-Gal transduction, ß-gal activity (blue) was seen in canine basilar, coronary, and femoral arteries (Figure 1). Quantitative analysis showed titer-dependent increases in ß-gal protein levels in those arteries transduced with AdCMVß-Gal (Figure 2). Control and AdCMVeNOS-transduced vessels lacked expression of recombinant ß-gal protein (Figure 2). eNOS immunoreactivity (brown) was observed in canine basilar, coronary, and femoral arteries transduced with AdCMVeNOS (Figure 3). The activity was localized predominantly in the adventitia. Endothelial cells were also stained positively (Figure 3). On the other hand, no staining was found in smooth muscle cells (Figure 3). eNOS immunoreactivity was observed not only in endothelial cells of AdCMVeNOS-transduced arteries but also in those of control and AdCMVß-Gal–transduced arteries (data not shown), suggesting the presence of endogenous eNOS.

View larger version (107K): [in this window] [in a new window]   Figure 1. ß-Gal staining in isolated canine basilar (A), coronary (B), and femoral (C) arteries 24 hours after ß-gal gene transduction. ß-Gal activity (blue) was seen on the adventitial side of basilar, coronary, and femoral arteries. Intensity of ß-gal staining was much higher in basilar than in coronary and femoral arteries. Arterial segments taken from the same dog were used for gene transfer (AdCMVß-Gal 108 pfu/mL) and were stained simultaneously.

View larger version (19K): [in this window] [in a new window]   Figure 2. ß-Gal protein levels in canine femoral, coronary, or basilar arteries transduced with eNOS gene or ß-gal gene. Arterial rings taken from the same dogs were used for gene transfer (AdCMVeNOS 1010 pfu/mL or AdCMVß-Gal 108 to 1010 pfu/mL) and were measured for ß-gal protein in parallel. Data are shown as mean±SEM. ß-Gal indicates AdCMVß-Gal; eNOS, AdCMVeNOS; and n, number of dogs. Statistical evaluation of data was performed by 2-way ANOVA followed by Bonferroni correction and Dunnett's test. *Significantly different from femoral and coronary arteries (P<0.05).

View larger version (92K): [in this window] [in a new window]   Figure 3. Immunohistochemical staining of eNOS in canine basilar (A, B), coronary (C, D), and femoral (E, F) arteries 24 hours after eNOS gene transduction. Positive staining (brown) was seen mainly in the adventitia and partly in endothelial cells. Intensity of eNOS staining was much higher in basilar than in coronary and femoral arteries. Arterial segments taken from the same dog were used for gene transfer (AdCMVeNOS 1010 pfu/mL) and were stained simultaneously. Bar=0.2 mm for A and F; 0.05 mm for B; 0.5 mm for C; 0.1 mm for D; and 1 mm for E.

To assess the specificity of the antibody against eNOS, the antibody either was omitted from the incubation medium or was replaced by the same dilution factor of an isotype-matched primary antibody of eNOS (ie, monoclonal mouse IgG1 against OPD4), and no positive staining was observed in AdCMVeNOS-transduced vessels (data not shown).

Heterogeneity of Transgene Expression
The intensity of ß-gal activity and eNOS immunoreactivity was much higher in basilar arteries than in coronary and femoral arteries (Figures 1 and 3). ß-Gal protein levels were also significantly higher in basilar arteries than in coronary and femoral arteries (Figure 2).

Vessel Size and Heterogeneity of Gene Transfer Efficiency
To elucidate why the efficiency of gene transfer was different among basilar, coronary, and femoral arteries, the effect of vessel size on ß-gal expression was examined in basilar arteries (external diameter, 1.19±0.03 mm) and their secondary branches (external diameter, 0.29±0.01 mm). When those vessels were incubated with 10¹⁰ pfu/mL of AdCMVß-Gal, the ß-gal protein level was significantly higher in the secondary branches than in basilar arteries (Table 1).

Effect of Expression of the eNOS Gene on Functions of Arteries With Endothelium
Expression of the ß-gal and eNOS genes did not affect the contractile responses to UTP (10^-8 to 10^-3 mol/L) in basilar arteries, to U46619 (10^-10 to 10^-6 mol/L) in coronary arteries, and to phenylephrine (10^-8 to 10^-4 mol/L) in femoral arteries (Table 2). Endothelium-dependent relaxations to bradykinin were not altered in basilar arteries transduced with AdCMVß-Gal compared with control (Figure 4). However, in basilar arteries transduced with AdCMVeNOS (10⁹ to 10¹⁰ pfu/mL), relaxations to low concentrations of bradykinin were significantly augmented (Figure 4 and Table 3). In contrast, in coronary and femoral arteries, expression of the eNOS gene (Figure 5) as well as that of the ß-gal gene (data not shown) did not affect endothelium-dependent relaxations to bradykinin, although in coronary arteries the relaxations tended to be augmented (statistically, P=0.07 between control and AdCMVeNOS 10¹⁰ pfu/mL by 2-way repeated-measures ANOVA with Bonferroni correction and Dunnett's test; Figure 5).

View larger version (15K): [in this window] [in a new window]   Figure 4. Effect of ß-gal (A) or eNOS (B) gene transduction on endothelium-dependent relaxations to bradykinin in canine basilar arteries with endothelium. eNOS gene transduction (109 to 1010 pfu/mL) significantly augmented relaxation to bradykinin (P<0.05). Relaxations were obtained during submaximal contractions induced by UTP (3 to 30 mmol/L). Arterial rings taken from the same dogs were studied in parallel. Data are shown as mean±SEM and are expressed as percent of maximal relaxation induced by papaverine (300 mmol/L; 100%=3.5±0.7, 3.6±0.8, 4.4±1.1, 4.6±1.1, and 3.1±0.9 g for control; AdCMVß-Gal 1010 pfu/mL; and AdCMVeNOS 108, 109, and 1010 pfu/mL, respectively). ß-Gal indicates AdCMVß-Gal; eNOS, AdCMVeNOS; and n, number of dogs. Statistical evaluation of data was performed by 2-way repeated-measures ANOVA followed by Bonferroni correction and Dunnett's test.

View larger version (18K): [in this window] [in a new window]   Figure 5. Effect of eNOS gene transduction on endothelium-dependent relaxations to bradykinin in canine coronary (A) or femoral (B) arteries with endothelium. eNOS gene transduction did not significantly affect relaxation to bradykinin in these arteries. Relaxations were obtained during submaximal contraction to U46619 (20 to 60 nmol/L) or phenylephrine (0.6 to 1 mmol/L) in coronary or femoral arteries, respectively. In each artery, rings taken from the same dogs were studied in parallel. Data are shown as mean±SEM and are expressed as percent of maximal relaxation induced by papaverine (300 mmol/L; for coronary arteries, 100%=4.4±0.6, 5.9±0.8, 3.9±0.7, and 5.1±1.2 g for control and for AdCMVeNOS 108, 109, and 1010 pfu/mL, respectively; for femoral arteries, 100%=11.4±0.9, 12.9±0.8, 12.1±0.5, and 12.3±0.8 g for control and AdCMVeNOS 108, 109, and 1010 pfu/mL, respectively). eNOS indicates AdCMVeNOS; n, number of dogs. Statistical evaluation of data was performed by 2-way repeated-measures ANOVA followed by Bonferroni correction and Dunnett's test.

An NO synthase inhibitor, L-NAME (300 µmol/L), abolished bradykinin-induced endothelium-dependent relaxations in control, AdCMVß-Gal–, and AdCMVeNOS-transduced basilar arteries (Figure 6).

View larger version (15K): [in this window] [in a new window]   Figure 6. Effect of L-NAME (300 mmol/L) on endothelium-dependent relaxations to bradykinin in control (left), ß-gal gene–transduced (middle), or eNOS gene–transduced (right) canine basilar arteries with endothelium. L-NAME completely blocked relaxation in these arteries (P<0.05). Relaxations were studied during submaximal contraction to UTP (3 to 30 mmol/L). Arterial rings taken from the same dogs were studied in parallel. Data are shown as mean±SEM and are expressed as percent of maximal relaxation induced by papaverine (300 mmol/L; 100%=4.0±0.7 and 5.0±0.8 g for control and control plus L-NAME, respectively; 100%=4.1±0.9 and 4.9±1.1 g for AdCMVß-Gal and AdCMVß-Gal plus L-NAME, respectively; 100%=3.5±0.3 and 4.9±0.5 g for AdCMVeNOS and AdCMVeNOS plus L-NAME, respectively). ß-Gal indicates AdCMVß-Gal; eNOS, AdCMVeNOS; and n, number of dogs. Statistical evaluation of data was performed by 2-way repeated-measures ANOVA followed by Bonferroni correction and Dunnett's test.

Effect of eNOS Gene Expression on Reactivity of Arteries Without Endothelium to Bradykinin
In control and AdCMVß-Gal–transduced basilar arteries without endothelium, relaxations to bradykinin were abolished (Figures 7 and 8A). However, in AdCMVeNOS-transduced basilar arteries, even after endothelial removal, stimulation with bradykinin caused prominent relaxations (Figures 7 and 8A). Maximum relaxation reached >40% at 1 nmol/L bradykinin (Figures 7 and 8A). The relaxations were blocked by L-NAME (Figure 8B). Significant increases in cGMP level were also noted in AdCMVeNOS-transduced basilar arteries without endothelium stimulated with 1 nmol/L bradykinin (Figure 9). There was no significant difference in basal cGMP levels among control, AdCMVß-Gal–transduced, and AdCMVeNOS-transduced arteries without endothelium (Figure 9).

View larger version (13K): [in this window] [in a new window]   Figure 7. Original tracing demonstrating relaxations to bradykinin in canine basilar arteries without endothelium transduced with eNOS gene. Relaxations to bradykinin were obtained during submaximal contractions to UTP (10 mmol/L). Papaverine (PPV, 300 mmol/L) was added at the end of the experiment to produce complete relaxation of arteries.

View larger version (17K): [in this window] [in a new window]   Figure 8. Effect of ß-gal or eNOS gene transduction on relaxations to bradykinin in canine basilar arteries without endothelium (A). Basilar arteries transduced with eNOS but not with ß-gal gene caused remarkable relaxations in response to bradykinin (P<0.05 compared with control and AdCMVß-Gal). Relaxations were obtained during submaximal contractions induced by UTP (3 to 30 mmol/L). Arterial rings taken from the same dogs were studied in parallel. Data are shown as mean±SEM and are expressed as percent of maximal relaxation induced by papaverine (300 mmol/L; 100%=3.6±0.7, 3.7±0.5, and 3.4±0.7 g for control, AdCMVß-Gal 1010 pfu/mL, and AdCMVeNOS 1010 pfu/mL, respectively). Effect of L-NAME on relaxations to bradykinin in eNOS gene–transduced canine basilar arteries without endothelium (B). L-NAME inhibited the relaxations completely (P<0.05). Relaxations were obtained during submaximal contractions induced by UTP (3 to 30 mmol/L). Arterial rings taken from the same dogs were studied in parallel. Data are shown as mean±SEM and are expressed as percent of maximal relaxation induced by papaverine (300 mmol/L; 100%=3.9±0.6 and 4.4±0.7 g for AdCMVeNOS and AdCMVeNOS plus L-NAME, respectively). E- indicates without endothelium; ß-Gal, AdCMVß-Gal; eNOS, AdCMVeNOS; and n, number of dogs. Statistical evaluation of data was performed by 2-way repeated-measures ANOVA followed by Bonferroni correction and Dunnett's test.

View larger version (34K): [in this window] [in a new window]   Figure 9. Effect of ß-gal or eNOS gene transduction on intracellular cGMP production stimulated by 1 nmol/L bradykinin in canine basilar arteries without endothelium. A significant increase in cGMP production was observed in eNOS gene–transduced canine basilar arteries only (*P<0.05 compared with basal level). Twenty-four hours after gene transfer, 10 mmol/L indomethacin and 1 mmol/L 3-isobutyl-1-methylxanthine were added to the incubation medium for 30 minutes at 37°C. During the last 2 minutes of the 30-minute incubation, rings were stimulated with 1 nmol/L bradykinin. Then cGMP levels were assayed as described in Methods. Arterial rings taken from the same dogs were studied in parallel. Data are shown as mean±SEM. ß-Gal indicates AdCMVß-Gal; eNOS, AdCMVeNOS; and n, number of dogs. Statistical evaluation of data was performed by 2-way ANOVA followed by Bonferroni correction and Dunnett's test.

Localization of Recombinant eNOS Protein
Electron microscopic analysis revealed that eNOS protein was localized in fibroblasts of the basilar artery adventitia transduced with AdCMVeNOS (Figure 10). Fibroblasts are flat, elongated, connective-tissue cells with cytoplasmic processes at each end and have a flat, oval, vesicular nucleus³⁰ (Figure 10A). Immunogold particles were localized mainly in the membrane region, although eNOS protein was present in the cytoplasm as well (see arrows in Figure 10B). Interestingly, it appeared that microdomains of the plasma membrane, caveolae, were present in transduced fibroblasts and that recombinant eNOS protein could be detected in these membrane invaginations (Figure 10B). Control specimens, which were processed according to the same procedure but with the omission of primary antibody, showed an absence of any gold particles in adventitial fibroblasts (Figure 10C).

View larger version (96K): [in this window] [in a new window]   Figure 10. Electron photomicrographs of recombinant eNOS in adventitia of canine basilar arteries transduced with eNOS gene (A, low-power magnification, bar=2 µm; B and C, high-power magnification, bar=0.5 µm). eNOS protein was observed in fibroblasts of basilar artery adventitia. Immunogold particles were localized mainly in the membrane region, although eNOS protein was present in the cytoplasm as well (see arrows in B). Immunogold particles were not detected in adventitial fibroblasts of nontransduced control basilar arteries (C).

	Discussion

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The current study revealed 4 novel findings. First, the efficiency of ex vivo adenovirus-mediated gene transfer was markedly higher in cerebral arteries than in peripheral arteries. Second, expression of recombinant eNOS gene in cerebral arteries with endothelium significantly augmented relaxations to bradykinin. Third, even after removal of the endothelium, relaxations and increases in cGMP production in response to bradykinin were detected in eNOS transgene arteries. Fourth, expression of functional recombinant eNOS protein was localized in adventitial fibroblasts of cerebral arteries.

Successful gene transfer with subsequent expression of recombinant proteins was assessed by ß-gal histochemistry and eNOS immunohistochemistry, respectively. Positive staining of recombinant proteins was detected predominantly in the adventitia, consistent with our previous findings.¹⁴ eNOS immunoreactivity in endothelial cells was observed not only in eNOS gene–transduced arteries but also in nontransduced (control) and ß-gal gene–transduced arteries, thus confirming the presence of endogenous eNOS.

Our results demonstrated that transgene expression was remarkably higher in canine cerebral than in coronary or femoral arteries. The observed heterogeneity is in agreement with a previous report indicating that efficiencies of adenovirus-mediated transduction with ß-gal or luciferase were significantly higher in rat renal arteries than in the thoracic aorta.³¹ Heterogeneous transgene expression of recombinant proteins in rat arteries was explained by the difference in proliferative activity assessed by [³H]thymidine uptake.³¹ However, we were unable to detect any difference in proliferation rates between cerebral and peripheral arteries in experiments with proliferating cell nuclear antigen and Ki-67 antigen labeling or with [³H]thymidine incorporation (M.T., unpublished observations, 1997). Adenovirus can transfer foreign genes to both replicating and nonreplicating cells, and cell proliferation is not required for expression of the recombinant gene because adenovirus does not integrate into host cell DNA.³² In the current study, the efficiency of ß-gal expression by transduction with 10¹⁰ pfu/mL of AdCMVß-Gal was significantly higher in small brainstem arteries than in basilar arteries. These results suggest that vessel size may be in part responsible for the differences in efficiency of adenovirus-mediated gene transfer. Anatomic difference or heterogeneous distribution of adenovirus receptors³³ also may explain these findings. However, further studies are needed to characterize the mechanisms underlying heterogeneous sensitivity of different arteries to adenovirus.

In basilar arteries with endothelium transduced with the ß-gal gene, endothelium-dependent relaxations to bradykinin were not altered, suggesting that adenovirus-mediated gene transfer itself does not affect endothelial function. However, in eNOS gene–transduced basilar arteries with endothelium, relaxations to low concentrations of bradykinin were significantly augmented. Thus, eNOS gene transduction decreased the threshold concentration of bradykinin needed for relaxations of cerebral arteries. In contrast, in coronary and femoral arteries, transduction with the eNOS gene did not affect relaxations to bradykinin. These findings are best explained by very low expression of the transgene in coronary and femoral arteries obtained in the current study.

Augmentation of relaxations to bradykinin in basilar arteries with endothelium transduced with the eNOS gene was completely blocked by the NOS inhibitor L-NAME. In our previous study, the selectivity of this inhibitor was confirmed by the fact that L-NAME did not affect relaxation elicited by the NO donor 3-morpholinosydnonimine.³⁴ More importantly, the inhibitory effect of L-NAME could be corrected by L-arginine.³⁴ It is therefore logical to conclude that this augmentation is mediated by activation of recombinant eNOS.

Further analysis of relaxation to bradykinin in eNOS gene–transduced arteries revealed that removal of endothelial cells did not abolish the vasodilator effect of bradykinin. In contrast, endothelial denudation abolished relaxation to bradykinin in control arteries or arteries transduced with the ß-gal gene. These findings represent the first demonstration that adventitial expression of recombinant eNOS may restore bradykinin-induced formation of NO in arteries without endothelium. The role of NO in mediating relaxations to bradykinin was further supported by the fact that in eNOS gene–transduced arteries without endothelium, an increase in cGMP production was detected in the presence of bradykinin. Furthermore, the relaxations to bradykinin were completely blocked by L-NAME. It is important to emphasize that arterial rings without endothelium were randomly exposed to adenoviral vectors, excluding the possibility that endothelium-independent relaxations to bradykinin may be due to incomplete endothelial removal. Furthermore, cerebral arteries are devoid of vasa vasorum,³⁵ and it is therefore unlikely that the endothelium of these nourishing vessels could be activated by bradykinin.

Expression of recombinant proteins in the adventitia has been reported after perivascular gene delivery in cerebral^{14 36 37} and peripheral³⁸ arteries. However, the nature of target cells expressing recombinant proteins in the adventitia has not been identified. In the current study, electron microscopic analysis revealed that recombinant eNOS protein was expressed in fibroblasts of basilar artery adventitia. Interestingly, immunogold particles were detected predominantly in membrane regions associated with structures reminiscent of caveolae. Previous studies demonstrated that in the vascular endothelium, eNOS localizes mainly in the membrane invaginations, caveolae.³⁹ More importantly, fibroblasts are rich in caveolae,^{39 40} indicating that these cells may have mechanisms needed for trafficking of proteins from the cytosol to the cell membrane. This situation in turn may provide optimal conditions for formation and release of NO toward smooth muscle cells. Taken together, these results suggest that fibroblasts in the adventitia acquired eNOS enzymatic activity after recombinant eNOS gene transduction and became capable of producing NO in response to bradykinin. Indeed, adenovirus-mediated eNOS gene transfer in cultured rat fibroblasts has been reported,¹³ and it is well known that bradykinin receptors exist in fibroblasts, as indicated by the fact that cloning of bradykinin receptors was carried out by using fibroblast cell lines.^{41 42} Furthermore, it is well established that activation of bradykinin receptors on fibroblasts is coupled to an increase in intracellular calcium levels.^{43 44} This certainly may provide powerful stimulus for activation of recombinant eNOS and release of NO.^{1 4}

Previous studies have demonstrated that cerebral vasospasm that develops after subarachnoid hemorrhage is associated with impaired function of the L-arginine–NO pathway^{45 46} and that nitrovasodilators or NO may reverse cerebral vasospasm after subarachnoid hemorrhage.^{47 48 49} Cerebral vasospasm usually occurs between 4 and 12 days after the onset of subarachnoid hemorrhage,⁵⁰ whereas transgene expression in the vasculature could be maintained 7 to 14 days after adenoviral transduction.⁵¹ Furthermore, successful perivascular expression of the recombinant ß-gal gene in cerebral arteries in vivo has been shown by infusing the adenoviral vectors into cerebrospinal fluid.³⁶ Therefore, expression of the recombinant eNOS gene may provide a new therapeutic approach to the treatment of cerebrovascular diseases, including vasospasm.

Since the critical importance of the endothelium in vascular relaxation was discovered by Furchgott and Zawadzki in 1980,⁵² accumulating evidence has clearly indicated that the endothelium and endothelium-derived NO play a key role in the regulation of vascular tone and that endothelial dysfunction with impaired NO production is a major contributor to the pathogenesis of vascular diseases such as hypertension, hyperlipidemia, diabetes, and atherosclerosis.^{1 4 10} The current study has demonstrated that adventitial fibroblasts transduced with the eNOS gene can restore production of NO in arteries without endothelium and enable the blood vessels to relax in response to bradykinin. These results therefore provide a novel concept in vascular biology, demonstrating that fibroblasts in the adventitia may play a role in the regulation of vascular tone after successful transfer and expression of recombinant eNOS gene.

	Selected Abbreviations and Acronyms

 

AdCMVeNOS = recombinant adenovirus encoding endothelial nitric oxide synthase gene driven by cytomegalovirus promoter AdCMVß-Gal = recombinant adenovirus encoding ß-galactosidase gene driven by cytomegalovirus promoter ß-gal = ß-galactosidase L-NAME = NG-nitro-L-arginine methyl ester PBS-T = PBS containing 0.05% Tween 20 pfu = plaque-forming unit

	Acknowledgments

 
This work was supported in part by National Heart, Lung, and Blood Institute grant HL-53524 (to Z.S.K.), Mayo Clinic intramural grants (to T.O.), funds from the Bruce and Ruth Rappaport Program in Vascular Biology, and the Mayo Foundation. The authors would like to thank Leslie Smith, Adele Stelter, and Sharon Guy for their invaluable technical assistance; Steve Ziesmer for help with the immunohistochemistry of eNOS; and Janet Beckman for typing the manuscript. We would also like to thank Dr Jeffrey Salisbury and Margaret Springett for performing electron microscopy.

Received June 19, 1997; accepted February 19, 1998.

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