INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

ENDOTHELIAL DYSFUNCTION with impaired nitric oxide production is a major contributor to pathogenesis of vascular diseases such as hypertension, hyperlipidemia, diabetes, vasospasm, and atherosclerosis (3, 8, 12). A recent advance in recombinant DNA technology has made it possible to increase local nitric oxide formation in the blood vessel wall (9, 20). Our previous ex vivo and in vivo studies demonstrated that adventitial fibroblasts transduced with recombinant endothelial (e) nitric oxide synthase (NOS) gene can restore production of nitric oxide in large cerebral arteries without endothelium and enable the blood vessels to relax in response to bradykinin (1, 2, 14, 17, 18).

Small arteries are an important target for vascular gene therapy because they regulate vascular resistance and therefore determine the distribution of blood flow (6). The effects of recombinant eNOS gene expression on functions of small blood vessels have not been investigated. Thus the present study was designed to determine whether expression of recombinant eNOS may affect vasomotor reactivity of small canine brain stem arteries.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Adenoviral vectors. Construction, propagation, purification, and evaluation of adenoviral vectors containing the eNOS gene used in the present study were described in detail previously (2). In brief, a replication-defective recombinant adenovirus vector encoding bovine eNOS gene, driven by cytomegalovirus immediate early promoter, was generated through homologous recombination. A full-length serotype 5 wild adenovirus was made replication-deficient by a deletion of the early region 1, followed by an insertion of the cDNA sequence encoding bovine aortic endothelial cell eNOS (generously provided by Dr. David G. Harrison, Emory University, Atlanta, GA). The shuttle vector, pACCMVpLpA, was a kind gift of Dr. Robert Gerard (University of Texas Southwestern Medical Center, Dallas, TX). A recombinant adenoviral vector encoding -galactosidase reporter gene driven by cytomegalovirus promoter, used in all experiments as a control, was a kind gift of Dr. James M. Wilson (University of Pennsylvania, Philadelphia, PA).

Gene transfer. All procedures were in accordance with the Institutional Animal Care and Use Committee guidelines of Mayo Clinic. Primary and secondary branches of basilar arteries (2-mm long rings, internal diameter 253 ± 2.5 µm, n = 231 rings) were taken from mongrel dogs (18-27 kg) anesthetized with 30 mg/kg pentobarbital sodium administered intravenously. Arterial rings were gently rinsed with Krebs-Ringer bicarbonate solution (in mmol/l: 118.3 NaCl, 4.7 KCl, 2.5 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, 25.0 NaHCO₃, 0.0026 calcium EDTA, and 11.1 glucose) to remove blood. In all rings, endothelium was removed mechanically. Surfaces of needles (28-30 gauge) were made rough with sandpaper, and the needles were fixed for denudation in a dish filled with Krebs-Ringer bicarbonate solution. Endothelial removal was accomplished by gently sliding an arterial segment over the needle with two pairs of fine forceps under a microscope (1, 14, 18). Next, the rings were randomly assigned for gene transfer. Arterial rings were transduced with an adenoviral vector in MEM (with Earle's salts, containing 0.1% BSA, 100 U/ml penicillin, and 100 µg/ml streptomycin) for 30 min at 37°C and then transferred to MEM and incubated for 24 h at 37°C in a CO₂ incubator (5% CO₂-95% air; Forma Scientific, Marietta, OH; see Refs. 2, 14, 18). Control arteries (nontransduced arteries) were incubated in MEM for 24 h in the same manner. Arterial rings taken from the same dogs were studied in parallel.

Immunohistochemical analyses of gene expression. For immunohistochemical staining of recombinant eNOS, arterial rings were frozen in optimum cutting temperature compound (10% polyvinyl alcohol, 4% polyethylene glycol, 86% nonreactive ingredients; Miles, Elkhart, IN), 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 the nonspecific protein binding sites. An eNOS monoclonal antibody (5 µg/ml, 1:50 of stock; Transduction Laboratory, Lexington, KY) was applied for 60 min at room temperature, followed by incubations with biotinylated rabbit anti-mouse F(ab')₂ (1:300, 20 min) secondary antibody and peroxidase-conjugated streptavidin (1:300, 20 min; Vector Laboratories, Burlingame, CA). After a 30-s immersion in 0.1 mol/l sodium acetate buffer (pH 5.2), eNOS immunoreactivity was visualized with 3-amino-9-ethylcarbazole and hematoxylin counterstaining.

For control studies, the specificity of eNOS immunolabeling was examined by omitting 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, Carpinteria, CA).

Vascular reactivity. Twenty-four hours after gene transfer, arterial rings were connected to isometric force-displacement transducers (Grass Instrument, Quincy, MA) using two stainless steel wires (75 µm) and were 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 h. The rings were then stretched progressively to optimal point (~1 g force), determined by repeated stimulation with 3 × 10⁶ mol/l UTP. Concentration-response curves to bradykinin (10¹¹ to 10⁸ mol/l), substance P (10¹¹ to 10⁸ mol/l), sodium nitroprusside (3 × 10⁹ to 3 × 10⁶ mol/l), diethylamine NONOate (10⁹ to 10⁷ mol/l), or forskolin (10⁹ to 10⁶ mol/l) were cumulatively obtained during submaximal contractions with an EC₅₀ of UTP (2 × 10⁷ to 10⁶ mol/l). To inhibit cyclooxygenase activity, all experiments were performed in the presence of indomethacin (10⁵ mol/l). The incubation time with indomethacin or N^G-nitro-L-arginine methyl ester (L-NAME; 300 µl/l) was 30 or 15 min, respectively. The relaxations were expressed as a percentage of maximal relaxations induced by papaverine (3 × 10⁴ mol/l). In all experiments, arterial rings taken from the same dogs were studied in parallel.

Intracellular cGMP levels. An RIA technique was used to determine the levels of cGMP, as reported previously (1, 2, 14, 18). Twenty-four hours after gene transfer, 10² mol/l indomethacin and 10³ mol/l 3-isobutyl-1-methylxanthine were added to the incubation medium for 30 min at 37°C to inhibit cyclooxygenase activity and the degradation of cGMP by phosphodiesterases, respectively. During the last 2 min of this incubation, some rings were stimulated with 3 × 10⁹ mol/l bradykinin. Next, rings were removed from the medium and quickly frozen in liquid nitrogen. After homogenization, cGMP levels were measured by a cGMP RIA kit (Amersham, Arlington Heights, IL). Total protein levels were determined by Lowry's (10) method. Arterial rings taken from the same dogs were studied in parallel.

Drugs. The following agents were used: UTP, bradykinin, substance P, sodium nitroprusside, diethylamine NONOate, forskolin, indomethacin, papaverine hydrochloride, L-NAME (all from Sigma Chemical, St. Louis, MO), FBS, MEM, and penicillin-streptomycin (GIBCO, Grand Island, NY). Indomethacin was dissolved with equal molar concentrations of Na₂CO₃. All concentrations are expressed as final molar concentrations.

Statistical analysis. The results are expressed as means ± SE. 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/Dunn's post hoc test (11). A value of P < 0.05 was considered to be statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Expression of recombinant eNOS gene. In canine brain stem artery, eNOS immunoreactivity (brown color) was seen predominantly in adventitia and partly in endothelial cells (Fig. 1). No staining was observed in smooth muscle cells.

View larger version (109K): [in this window] [in a new window]   Fig. 1.   Immunohistochemical staining of endothelial (e) nitric oxide synthase (NOS) in canine brain stem artery 24 h after eNOS gene transduction. Positive staining (brown color) was seen mainly in adventitia and sparsely in endothelial cells. Bar = 0.05 mm.

Effect of eNOS gene expression on microvascular tone. Expression of -galactosidase gene and eNOS gene did not affect the contractile responses to UTP (10⁸ to 10⁴ mol/l) in arteries with endothelium (Table 1). Relaxations to bradykinin were not altered in arteries with endothelium transduced with -galactosidase gene compared with control arteries with endothelium (Fig. 2A). However, in arteries with endothelium transduced with recombinant eNOS gene (10⁹-10¹⁰ plaque-forming units/ml), relaxations to bradykinin were significantly augmented (Fig. 2B; P < 0.05). Stimulation with 3 × 10⁹ mol/l bradykinin induced a significant increase in cellular cGMP levels in eNOS arteries with endothelium but not in control or -galactosidase arteries with endothelium (Table 2). The NOS inhibitor L-NAME (3 × 10⁴ mol/l) inhibited bradykinin-induced endothelium-dependent relaxations in control (Fig. 3A) and -galactosidase (Fig. 3B) arteries with endothelium. Augmentation of relaxations to bradykinin in eNOS arteries with endothelium was similarly blocked by L-NAME (3 × 10⁴ mol/l; Fig. 3C). Endothelium removal abolished relaxations to bradykinin in control and -galactosidase arteries (Fig. 4A). However, in eNOS-transduced arteries, even when endothelium was removed, stimulation with bradykinin caused prominent relaxations (Fig. 4A). L-NAME abolished the bradykinin-induced relaxations in the eNOS arteries without endothelium (Fig. 4B).

View this table: [in this window] [in a new window]   Table 1.   Effect of -galactosidase gene or eNOS gene transduction on contractions to UTP in canine brain stem arteries with endothelium

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Effect of -galactosidase (-Gal) gene (A) or eNOS gene transduction (B) on endothelium-dependent relaxations to bradykinin in canine brain stem arteries with endothelium. Relaxations were obtained during submaximal contractions induced by UTP. Data are shown as means ± SE and are expressed as percentage of maximal relaxation induced by papaverine [300 µmol/l; 100% = 0.9 ± 0.2, 0.7 ± 0.1, 0.6 ± 0.1, 0.7 ± 0.1, and 0.8 ± 0.1 g for control, 1010 plaque-forming units (PFU)/ml -galactosidase, and 108, 109, and 1010 PFU/ml eNOS, respectively]. n = No. of dogs.

View this table: [in this window] [in a new window]   Table 2.   Effect of -galactosidase gene or eNOS gene transduction on intracellular cGMP levels stimulated by bradykinin in canine brain stem arteries with endothelium

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Effect of NG-nitro-L-arginine methyl ester (L-NAME; 300 µmol/l) on endothelium-dependent relaxations to bradykinin in control (A), -galactosidase gene-transduced (B), or eNOS gene-transduced (C) canine brain stem arteries with endothelium. Relaxations were studied during submaximal contractions of UTP. Data are shown as means ± SE and are expressed as percentage of maximal relaxation induced by papaverine (300 µmol/l; 100% = 0.9 ± 0.2 and 1.0 ± 0.2 g for control and control + L-NAME, respectively; 100% = 0.7 ± 0.1 and 0.8 ± 0.1 g for -galactosidase and -galactosidase + L-NAME; 100% = 0.8 ± 0.1 and 0.8 ± 0.1 g for eNOS and eNOS + L-NAME, respectively). n = No. of dogs.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   A: effect of -galactosidase gene or eNOS gene transduction on relaxations to bradykinin in canine brain stem arteries without endothelium. Relaxations were obtained during submaximal contractions induced by UTP. Data are shown as means ± SE and are expressed as percentage of maximal relaxation induced by papaverine (300 mmol/l; 100% = 1.0 ± 0.1, 0.9 ± 0.1, and 0.9 ± 0.1 g for control, -galactosidase, and eNOS, respectively). B: effect of L-NAME on relaxations to bradykinin in eNOS gene-transduced canine brain stem arteries without endothelium. Relaxations were obtained during submaximal contractions induced by UTP. Data are shown as means ± SE and are expressed as percentage of maximal relaxation induced by papaverine (300 µmol/l; 100% = 0.9 ± 0.1 and 0.9 ± 0.1 g for eNOS and eNOS + L-NAME, respectively).

Relaxations to substance P were enhanced in eNOS arteries with endothelium but not in -galactosidase arteries with endothelium (Fig. 5). In endothelium-denuded arteries, only eNOS gene transduction evoked relaxations to substance P (Fig. 6A), and L-NAME inhibited the relaxations (Fig. 6B).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Effect of -galactosidase gene (A) or eNOS gene transduction (B) on endothelium-dependent relaxations to substance P in canine brain stem arteries with endothelium. Relaxations were obtained during submaximal contractions induced by UTP. Data are shown as means ± SE and are expressed as percentage of maximal relaxation induced by papaverine (300 µmol/l; 100% = 1.0 ± 0.2, 1.0 ± 0.2, and 1.1 ± 0.1 g for control, -galactosidase, eNOS, respectively). n = No. of dogs.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   A: effect of -galactosidase gene or eNOS gene transduction on relaxations to substance P in canine brain stem arteries without endothelium. Relaxations were obtained during submaximal contractions induced by UTP. Data are shown as means ± SE and are expressed as percentage of maximal relaxation induced by papaverine (300 µmol/l; 100% = 1.0 ± 0.1, 1.0 ± 0.1, and 0.9 ± 0.1 g for control, -galactosidase, and eNOS, respectively). B: effect of L-NAME on relaxations to substance P in eNOS gene-transduced canine brain stem arteries without endothelium. Relaxations were obtained during submaximal contractions induced by UTP. Data are shown as means ± SE and are expressed as percentage of maximal relaxation induced by papaverine (300 µmol/l; 100% = 0.9 ± 0.1 and 0.9 ± 0.1 g for eNOS and eNOS + L-NAME, respectively).

In control arteries, endothelium removal and resulting loss of basal nitric oxide release augmented relaxations to the nitric oxide donors sodium nitroprusside (Fig. 7A) and diethylamine NONOate (Fig. 7B). The augmentation was reversed by eNOS gene transduction but not by -galactosidase gene transduction (Fig. 8, A and B). These effects of eNOS gene transduction on relaxations to nitric oxide were abolished in the presence of L-NAME (Fig. 9, A and B). On the other hand, forskolin-induced, adenylate cyclase-mediated relaxations were unaffected by denudation of control arteries (Fig. 10A) or by transgene expression (Fig. 10B).

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Effect of endothelium removal on relaxations to sodium nitroprusside (A) and diethylamine NONOate (B) in control canine brain stem arteries. Data are shown as means ± SE and are expressed as percentage of maximal relaxation induced by papaverine (300 µmol/l; 100% = 1.0 ± 0.1 and 1.1 ± 0.2 g for with and without endothelium in A and 1.0 ± 0.1 and 1.0 ± 0.1 g for with and without endothelium in B, respectively). n = No. of dogs.

View larger version (16K): [in this window] [in a new window]   Fig. 8.   Effect of -galactosidase gene or eNOS gene transduction on relaxations to sodium nitroprusside (A) and diethylamine NONOate (B) in canine brain stem arteries without endothelium. Data are shown as means ± SE and are expressed as percentage of maximal relaxation induced by papaverine (300 µmol/l; 100% = 1.2 ± 0.1, 0.9 ± 0.1, and 0.8 ± 0.1 g for control, -galactosidase, and eNOS in A and 1.0 ± 0.1, 1.1 ± 0.1, and 0.9 ± 0.1 g for control, -galactosidase, and eNOS in B, respectively).

View larger version (14K): [in this window] [in a new window]   Fig. 9.   Effect of L-NAME on relaxations to sodium nitroprusside (A) and diethylamine NONOate (B) in canine brain stem arteries without endothelium transduced with eNOS. Data are shown as means ± SE and are expressed as percentage of maximal relaxation induced by papaverine (300 µmol/l; 100% = 1.2 ± 0.1 and 1.1 ± 0.1 g for control and eNOS + L-NAME in A and 1.0 ± 0.1 and 0.9 ± 0.1 g for control and eNOS + L-NAME in B, respectively).

View larger version (15K): [in this window] [in a new window]   Fig. 10.   A: effect of endothelium removal on relaxations to forskolin in control canine brain stem arteries. Data are shown as means ± SE and are expressed as percentage of maximal relaxation induced by papaverine (300 µmol/l; 100% = 1.1 ± 0.2 and 1.3 ± 0.1 for with and without endothelium, respectively). B: effect of -galactosidase gene or eNOS gene transduction on relaxations to forskolin in canine brain stem arteries without endothelium. Data are shown as means ± SE and are expressed as percentage of maximal relaxation induced by papaverine (300 µmol/l; 100% = 1.3 ± 0.1, 1.1 ± 0.1, and 1.2 ± 0.1 g for control, -galactosidase, and eNOS, respectively).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

This is the first study to examine the effect of expression of recombinant eNOS gene on vasomotor reactivity of small cerebral arteries. The major new findings are that expression of recombinant eNOS gene in canine brain stem arteries significantly augmented endothelium-dependent relaxations to bradykinin and substance P. Expression of recombinant eNOS in adventitia of arteries without endothelium restored production of nitric oxide in the cerebral arterial wall. This was shown by adventitia-dependent relaxations to bradykinin and substance P. Furthermore, expression of recombinant eNOS in arteries without endothelium decreased sensitivity to relaxations induced by exogenous nitric oxide.

In previous studies, we demonstrated that functional expression of recombinant eNOS gene in adventitia restores local nitric oxide production in large cerebral arteries without endothelium and enables the blood vessels to relax in response to bradykinin (1, 14, 17, 18). In the present study, these observations have been extended to small arteries of the brain stem. Both enhancement of endothelium-dependent relaxations and occurrence of adventitia-dependent relaxations were elicited in the arteries expressing recombinant eNOS gene in response to bradykinin and substance P. These results are consistent with previously reported findings on large cerebral arteries (1, 2, 14, 18). Augmentation of responses to bradykinin and substance P is best explained by increased local release of nitric oxide via activation of recombinant eNOS enzyme. This conclusion is further supported by the fact that stimulation with a low concentration of bradykinin (3 × 10⁹ mol/l) increased cellular levels of a major second messenger of nitric oxide, cGMP, in eNOS arteries but not in control or -galactosidase arteries. The vascular effects of eNOS gene transduction were abolished in the presence of the NOS inhibitor L-NAME, providing additional evidence that increased eNOS activity is a key mechanism underlying relaxations to bradykinin and substance P.

In control brain stem arteries, removal of endothelium augmented relaxations to the nitric oxide donors sodium nitroprusside and diethylamine NONOate. Augmentation of relaxations to nitric oxide donors was offset by expression of recombinant eNOS but not by expression of -galactosidase. These results suggest that restoration of nitric oxide formation in endothelium-denuded arteries is responsible for this reversal. Our conclusion is reinforced by the fact that the effect of eNOS gene transduction was attenuated by L-NAME. The selectivity of L-NAME was demonstrated in our previous studies reporting that L-NAME did not affect relaxations induced by the nitric oxide donor 3-morpholinosydnonimine (4).

Relaxations to forskolin, an activator of adenylate cyclase (13, 16), were unaffected by endothelial denudation of control arteries or by transgene expression. Contractions to UTP, an activator of smooth muscle cells (19), were also comparable among control arteries and arteries expressing recombinant proteins. It is therefore logical to conclude that eNOS gene transfer selectively augments relaxations mediated by increased production and release of endogenous nitric oxide.

In our previous study, we demonstrated that adenoviral-mediated gene transfer causes higher expression of recombinant -galactosidase in small than in large cerebral arteries (17). Based on this observation, we expected higher expression and biological activity of recombinant eNOS in small arteries. Indeed, recombinant eNOS expression caused significantly greater augmentation of endothelium-dependent relaxations to bradykinin in small arteries (maximal relaxation = 88 ± 2.0%, n = 6 and 71 ± 6.6%, n = 6, for small and large arteries, respectively; P < 0.05). Consistent with findings reported by Faraci (5), we did not detect significant differences between small and large arteries in response to bradykinin under control conditions, suggesting that, in dogs, stimulated production of nitric oxide is not different between the basilar artery and its branches.

Comparison of basal cGMP levels in small arteries with our previously reported values for cGMP in large cerebral arteries (2) indicates that significantly higher production of cGMP is present in small arteries (145 ± 38 pmol/mg protein, n = 6, and 43 ± 9 pmol/mg protein, n = 7, for small and large arteries, respectively; P < 0.05). This finding is best explained by higher basal production of nitric oxide in branches of basilar arteries than in the basilar artery itself. However, this conclusion is at variance with results of a previous pharmacological in vivo study on cerebral circulation of the rat (5). The NOS inhibitor N^G-monomethyl-L-arginine causes more pronounced vasoconstriction in large than in small arteries, suggesting that, in the rat, basal vascular tone in large arteries is more dependent on continuous release of nitric oxide than basal tone of small arteries. At the present time, the reason for apparent discrepancy between results obtained in vitro on cerebral arteries of dogs and in vivo on cerebral arteries of rats is unclear.

Intravascular administration of vectors in cerebral blood vessels in vivo requires interruption of cerebral blood flow and may damage the blood-brain barrier (7, 15). We demonstrated that functional expression of recombinant eNOS gene in adventitia of canine cerebral arteries, including small arteries, can be achieved by administration of vectors in cerebrospinal fluid via the cisterna magna (1). Immunogold labeling and electron microscopy indicated that expression of recombinant eNOS protein was limited to adventitial fibroblasts. Nitric oxide-dependent regulatory mechanisms are operative in resistance arteries and in large arteries (6). They are particularly important in resistance arteries because these vessels regulate vascular resistance and determine the distribution of the blood flow in the vascular bed (6). It is therefore likely that adventitial expression of recombinant eNOS gene into the small cerebral arteries may have beneficial effects in pathological conditions that require an increase in brain tissue perfusion.

In summary, the present study demonstrates that vasomotor reactivity of small cerebral arteries to bradykinin, substance P, and exogenous nitric oxide can be modulated by adenovirus-mediated eNOS gene transfer. Expression of recombinant eNOS in resistance arteries is a powerful experimental tool that can be used in studies designed to characterize vascular biology of nitric oxide in normal and diseased blood vessels.