Gene transfer of endothelial nitric oxide synthase

Gene transfer of endothelial nitric oxide synthase (eNOS) in eNOS-deficient mice KRISTY D. LAKE-BRUSE,1 FRANK M. FARACI,1 EDWARD G. SHESELY,2 NOBUYO MAEDA,3 CURT D. SIGMUND,1 AND DONALD D. HEISTAD1 (With the Technical Assistance of Kara L. Brown) 1Departments of Internal Medicine, Pharmacology, and Physiology, Cardiovascular Center and Center on Aging, University of Iowa College of Medicine, Iowa City, Iowa, 52242; 2Division of Hypertension and Vascular Research, Henry Ford Hospital, Detroit, Michigan 48202; and 3Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599 Lake-Bruse, Kristy D., Frank M. Faraci, Edward G. Shesely, Nobuyo Maeda, Curt D. Sigmund, and Donald D. Heistad. Gene transfer of endothelial nitric oxide synthase (eNOS) in eNOS-deficient mice. Am. J. Physiol. 277 (Heart Circ. Physiol. 46): H770–H776, 1999.—Relaxation to acetylcholine (ACh) and calcium ionophore (A-23187) is absent in aortas from endothelial nitric oxide synthase (eNOS)deficient (eNOS -/-) mice. We hypothesized that gene transfer of eNOS would restore relaxation to ACh and A-23187 in eNOS -/- mice. Aortic rings from eNOS -/- and eNOS ⫹/⫹ mice were exposed in vitro to vehicle or adenoviral vectors encoding ␤-galactosidase (lacZ) or eNOS. Histochemical staining for ␤-galactosidase and eNOS demonstrated transduction of endothelial cells and adventitia. Vehicle-treated vessels from eNOS -/- mice did not relax to ACh or A-23187 compared with eNOS ⫹/⫹ mice. In contrast, relaxation to nitroprusside (NP) was significantly greater in eNOS -/- mice than in eNOS ⫹/⫹ mice. Gene transfer of eNOS, but not lacZ, to vascular rings of eNOS -/- mice restored relaxation to ACh and A-23187. In vessels from eNOS -/- mice that were transduced with eNOS, N ␻-nitro-L-arginine (10⫺4 M) inhibited relaxation to ACh and A-23187 but not NP. Thus vascular function can be significantly improved by gene transfer in vessels where a major relaxation mechanism is genetically absent. adenovirus; aorta; knockout mice

replication-deficient recombinant adenovirus containing the eNOS transgene to distinguish between gene deficiency vs. embryological and developmental anomalies. We hypothesized that vascular abnormalities caused by simple loss of eNOS would be complemented by ex vivo gene transfer, whereas abnormalities caused by life-long deficiency and compensatory changes in other vasoactive systems would not be corrected. Gene complementation has been used previously in gene-targeted mice. For example, gene transfer of apolipoprotein E or low-density lipoprotein receptor to the liver of mice deficient in those proteins reduces plasma cholesterol (12, 27, 29). Several studies have demonstrated gene transfer to blood vessels (2, 13, 21, 22, 24, 31), but gene transfer to blood vessels of gene-targeted mice has not been reported. In the present study, we modified the ex vivo gene transfer method that we have used in other species for use in mice (19). We evaluated effects of overexpression of the eNOS transgene in control mice (C57BL/6 and eNOS ⫹/⫹ mice) and eNOS -/- mice, to answer the question, Does overexpression of recombinant eNOS in aorta of eNOS -/- mice improve relaxation to acetylcholine and A-23187? MATERIALS AND METHODS

of the endothelial isoform of nitric oxide synthase (eNOS) in vascular pathophysiology is difficult to evaluate with pharmacological approaches because most NOS inhibitors affect all three isoforms (endothelium derived, neuronal, and inducible; Refs. 17, 26). Mice with targeted disruption of the gene provide a new tool to study the role of eNOS in regulation of vasomotor tone (10). Aorta and carotid artery from eNOS-deficient (eNOS -/-) mice exhibit impaired endothelium-dependent vasodilatation to acetylcholine (6, 10). It is not known, however, whether this abnormal vascular phenotype is the result of eNOS gene disruption per se or embryological and developmental abnormalities that result from life-long eNOS deficiency. We therefore performed an ex vivo complementation study (replacement of a disrupted gene) with a THE ROLE

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. H770

Four-month-old male and female C57BL/6 mice (Harlan, 18–30 g) were used to establish a method for gene transfer to aortas of mice. Four-month-old littermate male and female eNOS -/- and eNOS ⫹/⫹ mice (18–30 g), originally generated at the University of North Carolina, were used for gene complementation studies. Generation of eNOS -/- mice has been described previously (25). Animals were maintained in the Animal Care Facility at the University of Iowa, which is American Association for Accreditation of Laboratory Animal Care approved. Experiments were conducted in accordance with guiding principles of the American Physiological Society and the University of Iowa Institutional Animal Care and Use Committee. Adenoviral vectors. Two replication-deficient recombinant adenovirus vectors were used. Ad-CMVntLacZ (AdlacZ; generated at the University of Iowa Vector Core) encoding the reporter gene for nuclear-targeted bacterial ␤-galactosidase was used as the control virus. Bovine eNOS [Ad-CMVeNOS (AdeNOS)] (kindly provided by Dr. Zvonimir Katusic) was used to overexpress eNOS. These viral vectors were constructed with methods similar to those described previously (3, 4, 5). Viral titer was determined by plaque assay on human embryonic kidney 293 cells that complement the E1 early

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viral promoters. Wild-type virus concentration was ⬍3 ⫻ 104 plaque-forming units (PFU)/ml as determined by plaque assay on human airway carcinoma A549 cells. After purification, the virus was suspended in phosphate-buffered saline with 3% sucrose added for stabilization of virus particles and stored at ⫺80°C. Gene transfer to aorta. The descending thoracic aorta was removed from the mice and placed in a dish containing cold, oxygenated Krebs bicarbonate solution of the following composition (mmol/l): 118 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4 ·7H2O, 11.1 D-glucose, 25 NaHCO3, and 2.54 CaCl2H2O. Loose fat and connective tissue were gently dissected away without disruption of the adventitia, and the vessel was cut into four rings 3 mm in length. With the use of a 96-well cell culture dish, each ring was incubated in a 200-µl volume of vehicle (PBS with 3% sucrose) or virus (3 ⫻ 108 PFU/200 µl of AdlacZ or AdeNOS) for 3 h. Stock virus titers (1010 PFU/ml) were diluted with Eagle’s minimal essential medium (MEM) containing 100 g/ml of penicillin per 100 U/ml of streptomycin. We selected the final viral titer on the basis of preliminary studies with viral titers ranging from 107 to 109 PFU/200 µl and exposure times to virus ranging from 2 to 5 h. After incubation with virus, the vessels were transferred into MEM to remove nonadherent virus particles. Vessels were then placed in 1 ml of MEM and incubated at 37°C with 95% O2-5% CO2 for 24 h. Vessel segments were then evaluated for vasomotor function or histochemical and biochemical analysis of transgene expression. Histochemical and biochemical analysis of expression of reporter transgene. We examined expression of ␤-galactosidase in blood vessels that were transduced by AdlacZ, washed with PBS, lightly fixed for 10 min in 2% paraformaldehyde and 0.25% gluteraldehyde, and incubated with 5-bromo-4chloro-3-indolyl-␤-D-galactopyranoside (X-Gal) solution for 2 h at room temperature. After incubation with X-gal, vessels were rinsed in PBS and fixed in 7% Formalin. Tissues were embedded in paraffin, sectioned, and counterstained with hematoxylin. Transgene expression in the vessel was examined (but not quantified) by identifying blue nuclei in each cell layer (intima, media, and adventitia). Expression of ␤-galactosidase was quantitated with a chemiluminescent assay (Galacto-Light Plus, Tropix, Bedford, MA). Vessels were minced and soaked in 90 µl of lysis buffer containing 0.2% Triton X-100 and 100 mmol/l potassium phosphate, pH 7.8. After 60 min, the tissue suspension was centrifuged at 10,000 g for 10 min and the supernatant was assayed for ␤-galactosidase. Light emission was measured with a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA) and calibrated to a standard curve that was generated with purified Escherichia coli ␤-galactosidase. Enzyme activity was normalized to tissue protein concentration with the Bradford assay (BioRad protein assay, Hercules, CA). Light emission was measured with a Molecular Devices Thermomax microplate reader, and values were calibrated to a standard curve that was generated with bovine albumin. Data are presented as ␤-galactosidase (mU/mg protein). Immunohistochemical analysis of eNOS expression. Arterial rings were fresh-frozen in Tissue-Tek embedding medium (Miles). Serial 11-µm thick sections were cut, adhered to poly-L-lysine-coated slides, and stored in a cryostat at 4°C over night. Before being stained, slides were allowed to dry in room air for 1 h. Horse serum (5%) was applied for 60 min to block nonspecific binding of protein. Mouse anti-eNOS antibody (1:50; kit no. 30020, Transduction Laboratories, Lexington, KY) was applied for 1 h. After sections of vessels were

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washed for 15 min in PBS, biotinylated goat anti-mouse immunoglobulin G (kit no. 4002, Vector Laboratories, Burlington, CA) was applied for 30 min. Sections were rinsed for 15 min in PBS, and then a complex of avidin and biotinylated horseradish peroxidase (Vector Laboratories, Burlingame, CA) was applied for 30 min. After a 15-min rinse with PBS, a 3,38-diaminobenzidine tetrahydrochloride substrate kit (no. SK-4100; Vector Laboratories, Burlingame, CA) for peroxidase was further diluted fourfold and applied for 2 min and then washed with water for 5 min. Vessel sections were counterstained with hematoxylin and examined for positive staining of eNOS (dark purple color) by light microscopy. Vasomotor function. Rings of aorta were suspended in an organ bath containing 25 ml of oxygenated Krebs buffer maintained at 37°C. The rings were connected to a force transducer to measure isometric tension (contraction and relaxation). Resting tension was increased stepwise to reach a final optimal tension of 0.5 g, and rings were allowed to equilibrate for at least 30 min. Krebs solution was changed before and twice after each curve (approximately every 30 min). We measured vascular responses to acetylcholine (receptormediated agonist), calcium ionophore A-23187 (non-receptormediated activation of eNOS), and nitroprusside (a NO donor, endothelium-independent agonist). Cumulative concentration response curves (10⫺10 –10⫺5 M for nitroprusside and 10⫺8 –10⫺5 M for acetylcholine and A-23187) were generated after precontraction of vessels with 9,11-dideoxy-11␣,9␣epoxymethanoprostaglandin F2␣ (U-46619). Vessels were precontracted to 30–50% of maximal contraction. Preliminary studies were performed to determine the average maximal contraction elicited in aortic rings. In each experiment, acetylcholine and nitroprusside were examined first. The order was alternated between acetylcholine and nitroprusside. The final intervention was A-23187 or maximal contraction to U-46619. Because A-23187 has prolonged effects and interfered with maximal contraction, vessels could not be evaluated for both relaxation to A-23187 and to U-46619 maximal contraction. In studies of vasomotor function in ex vivo transduced aorta, vessels were incubated with a relatively low concentration of nifedipine (3 ⫻ 10⫺7 M) for 25 min before examination of vascular responses (see DISCUSSION). Nifedipine was rinsed from the organ bath before contracting the vessels. In some experiments, N ␻-nitro-L-arginine (10⫺4 M) was added to the Krebs solution after the vessels were stretched to the resting tension and maintained in the organ bath for the duration of the assay. Drugs. Acetylcholine, nitroprusside, N ␻-nitro-L-arginine, calcium ionophore A-23187, U-46619, and X-Gal were obtained from Sigma (St. Louis, MO). Nifedipine was obtained from Research Biochemicals International (Natick, MA). U-46619 was obtained from Cayman Chemical (Ann Arbor, MI). A-23187 was dissolved in dimethyl sulfoxide and diluted with distilled water. Nifedipine was dissolved in absolute ethanol and diluted with isotonic saline. Dimethyl sulfoxide and ethanol were diluted so that the final bath concentration was ⱕ0.1%. Vehicles for nifedipine and A-23187 did not alter vasomotor tone. N ␻-nitro-L-arginine was warmed and dissolved in Krebs solution. Acetylcholine and sodium nitroprusside were dissolved in isotonic saline. All concentrations are expressed as the final concentration of each drug in the tissue bath. Calculation and statistical analysis. All data are presented as means ⫾ SE; n indicates the number of animals. Relaxation to acetylcholine, A-23187, and nitroprusside was expressed as percent relaxation from the amount of precontrac-


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tion produced by U-46619. The response was recorded as cumulative relaxation at each dose, expressed as a percentage of the initial precontraction tension. Experiments were performed in duplicate segments of vessels, and values are an average from both vascular segments. The EC50 values for nitroprusside were calculated with a linear regression analysis program (Cricket Graph III version 1.5.3, Computer Associates). To determine drug effect, or drug and treatment effects, comparisons were made with a one-way or two-way ANOVA with repeated measures, respectively, followed by adjusted Bonferroni’s post hoc test. Statistical significance was accepted at P ⬍ 0.05. RESULTS

Preliminary studies. We evaluated activity of ␤-galactosidase in C57BL/6 mice aortic segments that were exposed to vehicle or three concentrations of AdlacZ (final viral titer concentration: 108, 3 ⫻ 108, 109 PFU/ 200 µl) for 2, 3, or 5 h followed by incubation in media for a total of 24 h. After gene transfer of ␤-galactosidase, enzyme activity was related to both the viral titer and duration of incubation with virus. After incubation with AdlacZ for 2 h, enzyme activity in vessels increased from 0 ⫾ 2 (control) to 4 ⫾ 1, 10 ⫾ 5, and 50 ⫾ 7 mU/mg protein (n ⫽ 4–6 duplicate segments) after incubation with 108, 3 ⫻ 108, 109 PFU/200 µl, respectively. After incubation with AdlacZ for 3 h, enzyme activity in vessels increased from 0 ⫾ 2 (control) to 6 ⫾ 1, 20 ⫾ 5, and 130 ⫾ 7 mU/mg protein (n ⫽ 4–6 duplicate segments after incubation with the same concentrations of virus used above). Incubation with 3 ⫻ 108 PFU/200 µl AdlacZ for 5 h increased enzyme activity in vessels to 408 ⫾ 106 mU/mg protein (n ⫽ 4). In preliminary studies of vasomotor function, we found that relaxation to acetylcholine and nitroprusside was impaired after incubation with 3 ⫻ 108 and 109 PFU/ 200 µl AdlacZ for 5 h as well as 109 PFU/200 µl AdlacZ for 3 h (data not shown). On the basis of these data, we exposed vessel segments to a viral titer of 3 ⫻ 108 PFU/200 µl for 3 h. This protocol provided moderate transduction of AdlacZ but retained intact vasomotor function. Immunohistochemistry. Immunostaining of aorta from C57BL/6 and eNOS ⫹/⫹ mice confirmed the presence of endogenous eNOS only in endothelial cells (Fig. 1A). After transduction by AdeNOS, staining for eNOS was evident in both endothelium and many cells in adventitia (Fig. 1B). Staining of endothelial cells for eNOS was absent in aorta from eNOS -/- mice (Fig. 1C). In eNOS -/- mice after transduction with eNOS, staining was observed in some endothelial cells and many cells in adventitia (Fig. 1D). No eNOS staining was observed in aorta from eNOS -/- mice transduced with AdlacZ (data not shown). Vasomotor function after incubation with virus. After incubation in media, vehicle, 3 ⫻ 108 PFU/200 µl, or 109 PFU/200 µl of AdlacZ or AdeNOS for 2 or 3 h, vessels often developed irregular oscillations in vascular tone, which were not observed in freshly harvested vessels. Incubation of vessels with other types of culture media (Krebs, medium 199, and DMEM), polymyxin B, or addition of indomethacin (10⫺5 M) did not prevent these

spontaneous contractions or relaxations. However, incubation of the vessels with nifedipine (3 ⫻ 10⫺7 M) for 25 min before precontraction with U-46619 (in the absence of nifedipine) greatly reduced or prevented spontaneous changes in vascular tone. This approach was used previously in human epicardial coronary artery rings to inhibit phasic activity and allow quantification of vasodilator responses in vitro (26). To determine if vasomotor function was altered by pretreatment with nifedipine, freshly harvested vessels from C57BL/6 mice were studied. Relaxation and contraction were examined after pretreatment with vehicle or nifedipine (3 ⫻ 10⫺7 M). Contraction to U-46619 (3 ⫻ 10⫺7 M) tended to be less in nifedipine-pretreated vessels than in untreated vessels (1.29 ⫾ 0.14 vs. 1.58 ⫾ 0.10 g of tension; n ⫽ 9, P ⬎ 0.05). Nevertheless, pretreatment with nifedipine did not alter relaxation to acetylcholine or nitroprusside and only slightly reduced relaxation to low (10⫺7 M) concentrations of A-23187. On the basis of these findings, all vessels in subsequent gene transfer studies were pretreated for 25 min with nifedipine, and then the nifedipine was washed out of the organ bath before vascular responses were examined. Vasomotor function after gene transfer. Studies were performed in C57BL/6 mice and eNOS ⫹/⫹ and eNOS -/- mice obtained from an interbreeding of eNOS ⫹/mice. Relaxation to A-23187, acetylcholine, and nitroprusside in vessels from eNOS ⫹/⫹ mice transduced with AdlacZ or AdeNOS was not significantly different from that in vehicle-treated vessels (Fig. 2). Pretreatment of vessels incubated with AdlacZ or AdeNOS with N ␻-nitro-L-arginine (10⫺4 M) inhibited relaxation to A-23187 and acetylcholine but not nitroprusside (data not shown). Relaxation to A-23187, acetylcholine, and nitroprusside in vessels from C57BL/6 mice transduced with lacZ or eNOS was not significantly different from that in eNOS ⫹/⫹ mice (data not shown). In eNOS -/- mice, there was minimal relaxation of the aorta to A-23187 or acetylcholine in vehicle- or AdlacZtreated vessels (Fig. 3). Responses to A-23187 and acetylcholine were significantly different from responses of eNOS ⫹/⫹ mice (Fig. 2 vs. Fig. 3). Compared with eNOS ⫹/⫹ mice, relaxation to nitroprusside was enhanced in vehicle- and AdlacZ-treated eNOS -/- mice aorta [half-maximal effective dose (ED50 ) ⫽ 1 ⫻ 10⫺8 M and 2 ⫻ 10⫺9 M, respectively; P ⬍ 0.05]. No significant differences in maximum contraction were observed between treatment groups from C57BL/6, eNOS ⫹/⫹, and eNOS -/- mice (3 ⫻ 10⫺7 M U-46619 average, 1.50 ⫾ 0.06 g of tension, n ⫽ 30, P ⬎ 0.05). In aorta from eNOS -/- mice transduced with eNOS, there was pronounced relaxation to both A-23187 and acetylcholine (Figs. 3 and 4). Relaxation to 10⫺6 M A-23187 (73 ⫾ 8%) was greater (P ⬍ 0.05) in eNOStransduced vessels from eNOS -/- mice than in eNOStransduced eNOS ⫹/⫹ vessels (49 ⫾ 12%). Relaxation to 10⫺5 M acetylcholine (47 ⫾ 8%) in eNOS-transduced eNOS -/- vessels was similar to responses in vessels from vehicle-treated eNOS ⫹/⫹ mice (48 ⫾ 13%). N ␻-nitro-L-arginine (10⫺4 M) inhibited (P ⬍ 0.05) relaxation to A-23187 and acetylcholine in eNOS-transduced


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Fig. 1. Immunohistochemical staining for endothelial nitric oxide synthase (eNOS) protein (dark purple) in endothelium of vehicletreated vessels from control (A) but not eNOSdeficient (C) mice. Staining for eNOS protein is apparent in endothelial cells and many adventitial cells of eNOS-transduced vessels from control mice (B). In eNOS-transduced vessels from eNOS-deficient mice, a few endothelial cells were stained for eNOS protein, and many cells in adventitia were stained positive for eNOS (D). Magnification A–D, ⫻400. Sections counterstained with hematoxylin.

vessels from eNOS -/- mice without inhibition of relaxation to sodium nitroprusside (Fig. 4). These data provide evidence that restoration of relaxation was a result of eNOS transgene expression. DISCUSSION

Two novel tools, targeted disruption of a selected gene and gene transfer, offer unique opportunities to study the role of a gene in vascular biology. These

approaches can be combined to assess effects of replacement of genes in genetically altered mice. In normal mouse aorta, relaxation to acetylcholine is mediated by NO. However, in eNOS -/- mice, the aorta does not relax to acetylcholine (10). The present study confirms that relaxation of aorta in response to acetylcholine and A-23187 is mediated by eNOS. The novel finding in this study is that gene replacement (complementation) restores vascular function toward normal. This study


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Fig. 2. Relaxation to acetylcholine (A), calcium ionophore A-23187 (B), and nitroprusside (C) in aorta from wild-type control mice. Vessels were treated with vehicle (s) or were incubated with AdCMVntLacZ (AdlacZ; j) or AdCMVeNOS (AdeNOS; l). Data are means ⫾ SE; n ⫽ 6–9. P ⬎ 0.05 for comparison between groups (2-way ANOVA with adjusted Bonferroni’s post hoc).

demonstrates that adenovirus-mediated transduction of eNOS into aorta in eNOS -/- mice results in restoration of NO-mediated relaxation to both acetylcholine and A-23187. Gene transfer to aorta of mice ex vivo. Ex vivo gene transfer has been used for large vessels (aorta and carotid arteries) of rabbits (16, 19) and basilar arteries of dogs (2), wherein gene transfer to vessels from mice has not been described. In contrast to carotid and basilar arteries from rabbits (16), ex vivo gene transfer of AdlacZ to mice aortas resulted in less ␤-galactosidase enzyme expression, suggesting that efficiency of transduction is lower in mice aortas. Compared with rabbits, a 10-fold higher viral titer and a longer viral exposure time were needed to obtain similar enzyme expression of ␤-galactosidase in mice aorta (unpublished data). Unlike the larger vessels in rabbits (20), mice aorta exposed to viral titers that were higher than 3 ⫻ 108 PFU/200 µl or exposure times to virus longer than 3 h resulted in vasomotor dysfunction. Schulick et al. (24) have found that endothelial and smooth muscle cell damage occurs after intra-arterial infusions of high concentrations of adenoviral vector in balloon-injured rat carotid arteries. This tissue damage was avoided by reducing the concentration of virus in the infusion

Fig. 3. Relaxation to acetylcholine (A), A-23187 (B), and nitroprusside (C) in aorta from eNOS-deficient mice. Vessels were treated with vehicle (s) or were incubated with AdlacZ (j) or AdeNOS (l). Data are means ⫾ SE; n ⫽ 5–13. * P ⬍ 0.05 eNOS-transduced vessels vs. vehicle-treated and lacZ-transduced vessels (2-way ANOVA with adjusted Bonferroni’s post hoc).

Fig. 4. Relaxation of eNOS-transduced aorta from eNOS-deficient mice in response to acetylcholine (A), calcium ionophore A-23187 (B), and nitroprusside (C). Vessels were evaluated in the absence (k) or presence (j) of a NOS inhibitor, N ␻-nitro-L-arginine (10⫺4 M; n ⫽ 4–13). Data are means ⫾ SE; n ⫽ 5–13. * P ⬍ 0.05 without vs. with NOS inhibitor (2-way ANOVA with adjusted Bonferroni’s post hoc).

solution. In our ex vivo studies, we found attenuation of relaxation of mice aorta in response to acetylcholine and A-23187 (but not nitroprusside) after a high viral titer (109 PFU/200 µl) or a long exposure time to virus. Thus we used a submaximal titer that did not cause detectable vascular dysfunction but nevertheless improved vasculature responses. Spontaneous oscillation in tone was observed in vessels incubated for 24 h in either the absence or the presence of virus. Spontaneous phasic contractile activity in isolated coronary arteries from humans has been reported by many researchers over the past 20 years (7, 8, 11, 23). In human coronary arteries, the mechanisms controlling tone are complex; however, the phasic contractions have been shown to be dihydropyridinesensitive, voltage-operated Ca2⫹ channels (7, 28). Pretreatment with nifedipine inhibited spontaneous phasic activity in human coronary arteries and allowed quantitative analysis of vasoconstrictor and vasodilator agents (28). We also found that nifedipine was useful in preventing phasic activity of isolated segments of mice aorta. Pretreatment with nifedipine and removal of nifedipine before examination of vascular response did not prevent contraction with U-46619, nor did it alter maximal relaxation to acetylcholine, A-23187, and nitroprusside in vessels. Thus quantitative analysis of responses to vasodilator agents is feasible in mice aorta after ex vivo gene transfer. Relaxation of eNOS-transduced aorta in eNOS -/mice. Overexpression of eNOS in aorta of eNOS -/- mice produced histochemical evidence of eNOS staining of endothelial cells and adventitia and significantly improved relaxation to acetylcholine and A-23187. In contrast, overexpression of eNOS in aorta of control mice produced similar eNOS staining but without altering relaxation to acetylcholine and A-23187. We cannot explain the lack of enhanced relaxation in eNOS transduced vessels from eNOS ⫹/⫹ mice. It is possible that insufficient concentrations of cofactors or substrate in the in vitro vasomotor assay could account for these results. Inhibition of relaxation to acetylcholine and A-23187 with a NOS inhibitor provided evidence


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that restoration of relaxation in eNOS -/- mice was mediated by NO via recombinant eNOS. In eNOS -/- mice, relaxation of the aorta in response to low concentrations of nitroprusside was augmented, perhaps as a compensatory response to the absence of eNOS (6). Overexpression of eNOS in these eNOS -/vessels did not significantly alter the hypersensitivity to nitroprusside but restored maximal relaxation to acetylcholine and A-23187, which was minimal in aortic segments treated with vehicle or AdlacZ. Adventitial transduction of eNOS alters vascular function. Gene transfer of eNOS and lacZ to adventitia by adenoviral vectors has been demonstrated by our lab (20–22) and others (1–3, 14, 15). Intracisternal administration of AdeNOS resulted in transduction of adventitial fibroblasts in cerebral arteries (2). Electron microscopy with immunogold labeling demonstrated recombinant eNOS cellular localization (caveoli) in adventitial fibroblasts. In the present study, both immunohistochemistry for eNOS and X-Gal staining for ␤-galactosidase showed transduction of both endothelial cells (eNOS -/- mice transduced with eNOS) and many fibroblast-like cells in the adventitia (control and eNOS -/- mice). It was not surprising to find that endothelial cells from eNOS -/- mice could be transduced with eNOS, but it is of interest that restoration of relaxation to acetylcholine resulted from transduction of what appears to be only a relatively limited number of endothelial cells in eNOS -/- mice. We do not know why such an apparently low level of transduction resulted in significant improvement of acetylcholine relaxation in aorta from eNOS -/- mice. One possible explanation for this restored relaxation to acetylcholine relates to increased sensitivity of vessels to NO. We observed a significant increase in sensitivity to the NO donor nitroprusside in aorta from eNOS -/- mice. A second possibility is that eNOS-transduced fibroblasts in the adventitia contributed to acetylcholine-induced relaxation. There is evidence that fibroblasts, in culture, contain muscarinic receptors (9, 13), and thus it is possible that adventitial fibroblasts express muscarinic receptors that could be coupled to recombinant eNOS. Previous studies in other species have shown that recombinant eNOS in fibroblasts can be activated after stimulation of fibroblast receptors (18, 30). Thus we cannot exclude the possibility that activation of eNOS in adventitia contributed to relaxation in response to acetylcholine in eNOS -/- mice after gene transfer with eNOS. Immunohistochemistry suggested that adventitia was transduced with eNOS in both control (eNOS ⫹/⫹ and C57BL/6) and eNOS -/- mice. In common carotid arteries of rabbits, with adventitia transduced with eNOS and after denudation of endothelium, A-23187 but not acetylcholine produces vascular relaxation (21). Thus, in rabbit carotids, endothelium is required to obtain relaxation to acetylcholine, even after gene transfer of eNOS. Enhanced responses to A-23187 in eNOStransduced vessels from eNOS -/- mice, in conjunction with immunohistochemical staining of adventitia, are compatible with the possibility that adventitial gene

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transfer contributed to improvement of vascular responses to A-23187. Our goal in this study was to determine if gene transfer to blood vessels of eNOS -/- mice would improve relaxation. We observed significant improvement in relaxation to acetylcholine and A-23187. It is uncertain what percentage of vasomotor improvement was contributed by eNOS-transduced cells in endothelium and adventitia. We have not studied endotheliumdenuded vessels from eNOS -/- mice after gene transfer. Because of the small size of the mouse aorta, it is difficult to completely remove endothelium without damage to smooth muscle and adventitia. For example, rolling of vascular rings, which is a common approach to denuding vessels, may damage transduced adventitia. Previous studies (30) have shown that endotheliumdenuded canine basilar, coronary, and femoral arteries transduced with eNOS relax to low concentrations of bradykinin. Electron microscopy confirmed that fibroblasts in the adventitia expressed the recombinant eNOS protein (30). We speculate, on the basis of our previous studies in eNOS-transduced endotheliumdenuded rabbit carotid (21), that relaxation to acetylcholine was a result of transduced endothelium and that relaxation to A-23187 was at least in part due to the transduction of cells in the adventitia. In summary, we have demonstrated, first, that ex vivo gene transfer to mice aorta is feasible and that functional consequences can be evaluated. Second, eNOS transduction results in restoration of relaxation to acetylcholine and A-23187 in aorta of eNOS -/- mice. This restoration is mediated by NOS, as demonstrated with a NOS inhibitor. Finally, this is the first study to our knowledge that demonstrates improvement of vascular function in gene-targeted mice with gene transfer to blood vessels. This study indicates that, even in vessels in which a major relaxation mechanism is genetically absent, vascular function can be significantly improved by gene transfer. We thank Zvonimir Katusic for providing AdCMVeNOS; Beverly L. Davidson for providing AdCMVntLacZ; Pamela K. Tompkins for technical assistance, and Arlinda LaRose for secretarial assistance. We also thank the University of Iowa Gene Transfer Vector Core and Richard D. Anderson for preparation of the viruses and Lisa Hancox from the Transgenic Animal Facility for genotyping the mice. Genetically deficient mice generated at the University of North Carolina and maintained at the University of Iowa Transgenic Animal Facility are supported in part by the College of Medicine and the Diabetes and Endocrinology Research Center. This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-24621 and National Heart, Lung, and Blood Institute Grants HL-16066, HL-14388, and HL-38901. K. D. Lake-Bruse is supported by Institutional Training Grant DK07690. F. M. Faraci and C. D. Sigmund are Established Investigators of the American Heart Association. Address for reprint requests and other correspondence: D. D. Heistad, Dept. of Internal Medicine, Univ. of Iowa College of Medicine, Iowa City, IA 52242 (E-mail: [email protected]). Received 12 November 1998; accepted in final form 13 April 1999. REFERENCES 1. Cable, D. G., T. O’Brien, I. J. Kullo, R. S. Schartz, H. V. Schaff, and V. J. Pompili. Expression and function of a recombinant endothelial nitric oxide synthase gene in porcine coronary arteries. Cardiovasc. Res. 35: 553–559, 1997.


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