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Cardiovascular Research 1999 43(3):798-807; doi:10.1016/S0008-6363(99)00146-7
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Gene transfer of endothelial nitric oxide synthase improves nitric oxide-dependent endothelial function in a hypertensive rat model

M.Yvonne Alexander1,a,*, M.Julia Brosnan1,a, Carlene A Hamiltona, Paul Downiea, Alison M Devlina, Fiona Dowellb, William Martinb, Howard M Prenticeb, Timothy O’Brienc and Anna F Dominiczaka

aDepartment of Medicine and Therapeutics, University of Glasgow, Glasgow, G11 6NT, UK
bInstitute of Biomedical and Life Sciences, University of Glasgow, Glasgow, G11 6NT, UK
cDepartment of Endocrinology and Metabolism, Mayo Clinic, Rochester, MN 55905, USA

* Corresponding author. Tel.: +44-141-211-2111; fax: +44-141-211-1763 yalexander{at}clinmed.gla.ac.uk

Received 2 December 1998; accepted 30 March 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: We have shown previously that there is a relative nitric oxide deficiency at the level of vascular endothelium in the stroke-prone spontaneously hypertensive rat (SHRSP), a model of human essential hypertension, as compared to its normotensive reference strain Wistar Kyoto (WKY) rat. The aim of the current study was to investigate whether adenoviral-mediated gene transfer of an endothelial nitric oxide synthase (eNOS) cDNA (AdCMVeNOS) into carotid arteries of the SHRSP may improve endothelial function. Methods: Enzyme activity of the recombinant eNOS protein encoded by AdCMVeNOS was tested using a Griess assay in endothelial cells in culture. Left carotid arteries of SHRSP were surgically isolated and exposed to either the AdCMVeNOS or control β-galactosidase-containing virus, (2x109 pfu/ml) ex vivo and in vivo. The vessels were harvested 24 h after surgery and analysed by Western blotting, immunohistochemistry and by examining endothelial function ex vivo. Results: Cultured endothelial cells showed almost 100% transduction with both viruses and a dose response of eNOS expression showed a five-fold increase in nitrite production for AdCMVeNOS with no change for β-galactosidase-containing virus. Western blotting demonstrated a significant increase of eNOS expression in vessels infused with AdCMVeNOS when compared to controls. Immunohistochemistry showed highly positive staining with monoclonal antibodies against eNOS in the intact endothelial cells of the AdCMVeNOS infused vessels. The areas under the curve of the concentration responses to phenylephrine (10–9 to 3x10–6M) in the absence and presence of NG-nitroarginine methyl ester (100 µM) showed increased basal nitric oxide bioavailability in the carotid arteries infused with AdCMVeNOS compared to the control (n=6 for each; P=0.0069; 95% CI, 0.864 to 3.277). Conclusions: Our results show that AdCMVeNOS is an effective tool for vascular gene transfer and that it can improve endothelial NO availability in the SHRSP, a genetic model of essential hypertension and endothelial dysfunction.

KEYWORDS Hypertension; Nitric oxide; Gene therapy; Gene expression


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Gene transfer technology, which enables expression of recombinant genes in the vasculature, holds promise both to understand and modify vessel wall pathophysiology. This topic is comprehensively reviewed by O’Brien [1]. Since endothelial cells are notoriously difficult to transfect [2] replication-defective adenoviruses display several properties that could be useful for expressing recombinant genes in the vasculature; (i), their ability to rapidly and efficiently infect smooth muscle and endothelial cells in vitro [3] and in vivo [4]; (ii) high titre stocks (up to 1011 pfu/ml) of replication defective adenovirus can be prepared, thus allowing for high efficiency gene transfer following infusion of minimal volume of virus in vivo [5] and (iii), their ability to accommodate a relatively large insert of DNA of up to 8.0 Kb [6]. Furthermore, adenoviruses are common human pathogens that cause relatively low level morbidity and have not been associated with human malignancies [7]. Thus, adenovirus would appear to be a relatively safe vector for vascular gene therapy.

Nitric oxide (NO) is a potent vasodilator which is generated by the action of the enzyme nitric oxide synthase (NOS), which catalyses the conversion of L-arginine to L-citrulline with the release of NO [8]. We are using the endothelial isoform, also called NOS III, which is widely expressed in endothelial cells throughout the vasculature. It is generally accepted that endothelium-derived NO is an important factor in the control of basal vascular tone, as well as other diverse physiological functions, such as platelet adhesion and aggregation, and smooth muscle cell migration and proliferation [9]. Moreover, eNOS knock-out mice display significantly increased blood pressure and no endothelium-dependent relaxation [10]. It seems therefore, that the overexpression of eNOS gene in the vasculature of animal models of hypertension and endothelial dysfunction may provide the necessary first step to therapeutic strategies.

Several studies attempted to transfer eNOS gene into the vessel wall in various animal models [11–16]. However, with a few exceptions, [17,18], the majority of these studies used intact arteries [12,15] or balloon-injury models in normal animals [11,13,14,16]. The only previous study which used a genetically hypertensive rat model, attempted a systemic delivery of human eNOS cDNA in an AdCMV expression vector [17]. The vector was delivered by tail vein injection, and the hypotensive effect was seen for up to 6 weeks [17]. The precise mechanism of blood pressure reduction in this experiment is not fully explained so far. The second study which used a disease model has been published by Channon et al. [18] who chose to study neuronal NOS (nNOS) delivery to carotid arteries of cholesterol fed rabbits. Their studies resulted in improved acetylcholine-mediated vasodilation in these vessels but the rationale of choosing nNOS rather than eNOS isoform remains unclear. Furthermore basal, tonic NO bioavailability has not been studied in these experiments.

The present study was undertaken to determine whether adenoviral-mediated gene transfer of the eNOS cDNA could modify arterial function in the carotid arteries of the SHRSP, a genetic model of hypertension which demonstrates endothelial dysfunction and a markedly reduced NO bioavailability [19].


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Propagation of the recombinant adenoviral vectors
The nuclear-targeted Lac-Z reporter virus Ad5/CMV/nt-LacZ was purchased from the Gene Transfer Core Group in the University of Iowa. AdCMVeNOS contains bovine eNOS cDNA which is under the control of the CMV IE promoter and terminated by pA signals from the SV40 genome at the 3' end of the multiple cloning sequence, adjacent to the 3' flanking regions of Ad5 [20]. Individual bovine eNOS plaques were expanded and tested for eNOS-specific sequences by PCR and Southern blotting [21] in HEK 293 cells (human embryo kidney cells expressing the E1 region of Ad2) [22]. The bovine eNOS primers used are shown in Table 1. Adenovirus stocks were prepared by infection of 293 cells at a multiplicity of infection of 10 pfu/cell. Cells and media were harvested 48 h after infection when a cytopathic effect (cpe) was visible. Infected cell pellets were resuspended in Tris saline (20 mM Tris pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2) and the virus was released by three cycles of freezing and thawing, followed by a spin at 6000 g to remove cell debris. Viral preparations were purified by ultracentrifugation on a 1.3 to 1.4 g/ml CsCl step gradient at 100 000 g for 2.5 h at 4°C [6] and desalted by dialysis. Serial dilutions of the lysates were titred on HEK293 cells [23]. The 293 cells were grown in MEM (Gibco) and 5 mM glutamine containing 10% foetal calf serum (FCS) (v/v) and 100 µg/ml penicillin and 100 U/ml streptomycin. Virus concentrations were initially estimated by absorbance at 260 nm (pfu/ml=A260xdilutionx1010) and ranged from 1x1010 to 1x1011 pfu/ml. Infectious titre of all viral stocks were determined by duplicate plaque assays on 293 cells using standard techniques [23]. Before use, plaque isolates were evaluated for their potential to overexpress NOS activity in endothelial cells using the Griess assay [24].


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  		Table 1
 
2.2 Screening of viral lysates to exclude wild-type contamination
PCR procedures were used to verify that the recombinant adenovirus retained the E1A deletion and that the infection (cpe observed) was not due to wild-type Ad5 contamination. Primers were designed to amplify a 560 bp product corresponding to the Ad5 base pairs 690 to 1250 relative to the transcription start site and are illustrated in Table 1. As a positive control, Ad5 DNA was used as template in the PCR.

2.3 Small-scale isolation of viral DNA
The 293 cells were grown and overlayed with virus (108 pfu/ml), incubated with the virus suspension for 45 min. MEM (Gibco) containing 5 mM glutamine, 8% fetal calf serum and 100 µg/ml penicillin/100 U/ml streptomycin was added and cells incubated at 37°C with 95% air–5% CO2 atmosphere until the appearance of a cpe was evident. The cells were pelleted and resuspended in 800 µl of Tris saline and freeze–thawed 3–4 times to lyse the cells and release the virus. An aliquot of cell lysate containing virus was digested with proteinase-K (Sigma) at 1 mg/ml in the presence of 5 mM EDTA, pH 8.0 and 0.5% SDS at 37°C for 3 h. The digest mixture was phenol–chloroform extracted once, chloroform extracted and then ethanol precipitated. DNA was resuspended in 39 µl of TE and the solution RNase treated with 1 µl of RNase (10 mg/ml). A 34-µl volume of the DNA was used for restriction analysis, typically by HindIII digestion and the remaining 6 µl used for PCR.

2.4 PCR analysis of viral DNA
For screening viral recombinants, the viral DNA was subjected to an appropriate thermal cycling program in a Hybaid Omni-gene thermal cycler. A typical PCR reaction was carried out in a total volume of 50 µl using Promega PCR kit with 1x PCR buffer, 5 mM Mg2+, 200 µM of each dNTPs, 100 ng of template DNA and 1 nmol each of the two primers. Thermal cycling programs were designed with an initial denaturing step at 94°C for 4 min. This was followed by 30 cycles of denaturing at 94°C for 45 s, 30 s primer annealing at 62°C and then an extension step at 72°C, followed by a final extension for 15 min at 72°C.

2.5 Experimental animals, rat aortic endothelial cell culture
SHRSP and WKY rats were obtained from Glasgow colonies which have been maintained by brotherxsister mating as previously described [25] and systolic blood pressures were measured by tail-cuff plethysmography according to the standard protocol [25]. For in vivo studies, we used twelve SHRSP males, at 12 weeks of age, (mean systolic BP of 171±17 mmHg) and six age-matched WKY males, (mean systolic BP of 130±10 mmHg); for cell culture studies, we used fourteen SHRSP (mean BP 196±5 mmHg) and seventeen WKY (mean BP 130±15 mmHg) male rats at 16 weeks of age. For the endothelial cell preparation, rats were sacrificed and the full length of the thoracic aorta was removed under sterile conditions. Endothelial cells from the thoracic aorta were isolated and cultured using the primary explant method of McGuire and Orkin [26,27]. Briefly, the vessel was carefully stripped of adherent fat and connective tissue and the adventitia was carefully removed; gentle manipulation of the aorta being essential to avoid injury to the endothelial lining, thereby assuring outgrowth of endothelial cells. The vessel was cut into rings approximately 2 mm in width and placed on basement membrane components derived from Engelbreth–Holm–Swarm (EHS) sarcoma [28] (Matrigel, Collaborative Research). These explants were incubated in complete medium (RPMI 1640, containing 20% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin and 50 µg/ml ECGS, Gibco/Life Technologies), at 37°C in a 95% air–5% CO2 atmosphere. Endothelial cells grew out onto the Matrigel and the aortic explants were removed after 4–7 days depending upon the degree of outgrowth. The endothelial cells that had grown out onto the Matrigel were recovered by passaging with 2% dispase. Cells were characterised as endothelial cells on the basis of positive immunostaining with antibodies specific for Von Willebrand factor and also on the basis of morphology [29,30]. For the transfection experiments, endothelial cells were subcultured using 0.05% trypsin–EDTA, counted using an electronic Coulter counter (Model ZM, Coulter Electronics, UK) and inoculated in small (25 cm2) tissue culture flasks at a density of 0.05x106 cells/ml. The endothelial cells were grown until approximately 90% confluent before transfection with the virus and cells between passages four to eight were used in all experiments.

2.6 Viral infection and measurement of nitrite concentration in supernatant of cultured endothelial cells
AdCMVeNOS infection was carried out for 1 h with different titres of viral suspensions diluted in 100 µl Tris–saline. The flasks were then incubated at 37°C in culture medium (as above). Supernatant was removed 48 h after infection, for the Griess assay and the remaining cells were fixed for immunocytochemical analysis [31]. Activity of the NOS enzyme was assessed according to the Griess method [24] which measures nitrite concentration after conversion of nitrate to nitrite with nitrate reductase. Nitrite concentrations were determined at an optical density of 554 nm by comparison to standard solutions of sodium nitrite.

2.7 Carotid artery gene transfer ex vivo
Animals were killed by an overdose of halothane and the carotid arteries were dissected free, excised and washed in PBS. These vessels were then cut into two 4-mm rings and each ring incubated with either Tris–saline, AdCMVeNOS or Ad5/CMV/nt-LacZ (100 µl of 2x109 pfu/ml) for 4 h. The vessels were then rinsed in Dulbecco’s minimal essential media (DMEM) to remove the virus and incubated for 24 h in DMEM containing 100 µg/ml penicillin–100 U/ml streptomycin at 37°C with 95% air–5% CO2 atmosphere. The vessels were then fixed for X-gal histochemistry or eNOS immunohistochemical analysis.

2.8 Carotid artery gene transfer in vivo
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the National Institute of Health (NIH Publication No. 85-23, revised 1996) and carried out under the project licence from the UK Home Office. Rats were anaesthetised using halothane, and underwent gene transfer to the left carotid artery with the right contralateral artery acting as a control vessel. The left artery was carefully exposed through a mid-line neck incision, and high-titre stock, (2x109 pfu/ml) was infused, using PE-10 polyethylene catheter tubing (Becton Dickinson) previously tapered to 0.008 in. and inserted through a proximal arteriotomy after isolating the exposed artery between microvascular clamps. The lumen of the vessel was cleared of blood by gently flushing through the virus solution, and clamping the distal portion of the artery. Approximately 30 µl of virus solution was instilled into the vessel lumen and incubated for 20 min. The left carotid artery was then ligated and the incision repaired by suturing. Rats were then allowed to recover from the anaesthetic. Vessels were harvested 24 h after surgery. Animals were killed by an overdose of halothane and the carotid arteries were dissected free, excised and washed in PBS. From each vessel, two rings of 4 mm each were used for organ bath experiments. The remaining segments were frozen at –80°C for protein extraction or processed for immunohistochemistry.

2.9 Histological analysis of β-galactosidase activity
Carotid arteries were examined 24 h after viral infusion (20 min with 2x109 pfu/ml) of recombinant Ad5/CMV/nt-LacZ in the in vivo experiments and after a 48-h incubation (following 4 h viral exposure) in the ex vivo experiments to assess transgene expression for β-galactosidase. The artery rings were fixed in 2% paraformaldehyde and 0.2% glutaraldehyde in PBS for 30 min and stained with X-gal as previously described [32]. Sections (5 µm) were collected on glass slides and lightly counterstained with hematoxylin and eosin and examined for positive staining for β-galactosidase by light microscopy.

2.10 Western blotting
Carotid arteries from male SHRSP and WKY animals were infected with 30 µl viral lysate, Ad5/CMV/nt-LacZ or AdCMVeNOS at a titre of 2x109 pfu/ml and incubated for 20 min. The animal was sacrificed 24 h after surgery and the arteries were harvested and protein extracted for western analysis. Carotid arteries were homogenized in 2x extraction buffer (250 mM Tris, pH 6.8, 4% SDS, 10% glycerol, 0.006% bromophenol blue and 2% β-mercaptoethanol) and insoluble material removed by centrifugation for 5 min at 14 000 g. A 10-µg amount of protein was heated at 100°C for 5 min and electrophoresed on an 8% polyacrylamide gel and electroblotted to Hybond P membrane (Amersham). Endothelial NOS was detected using a 1 in 2500 dilution of a monoclonal antibody (Transduction Laboratories) according to the manufacturer’s instructions. Bands were detected using a 1:500 goat anti-mouse IgG peroxidase secondary antibody followed by the enhanced chemiluminescence (ECL) detection system (Amersham).

2.11 Immunohistochemistry
The method of Cattel and Mosley [31] was followed with minor modifications. Briefly, sections were dehydrated through alcohols, followed by the inhibition of endogenous peroxidase with 3% H2O2 in phosphate buffered saline (PBS) for 30 min. Sections were rinsed twice in Tris buffered saline (TBS) (50 mM Tris–HCl, pH 7.4, 0.9% NaCl). Non-specific binding of protein was blocked by incubation in TBS containing 4% goat serum and 0.2% BSA. For immunostaining, sections were incubated with primary monoclonal mouse anti-eNOS antibody directed against the C-terminal end of human eNOS (Transduction Laboratories), diluted 1:100 with blocking buffer for 24 h at 4°C. This procedure is designed to block the endogenous eNOS signal. Sections were rinsed three times for 5 min in TBS, and binding of primary antibody was visualised with a secondary mouse IgG labelled with HRP and stained for 10 min using 3',3'-diaminobenzadine (DAB) and 0.01% hydrogen peroxide as a chromogene. Sections were briefly air-dried and counterstained with hematoxylin and examined for positive staining of eNOS (brown staining) by light microscopy. Aortic endothelial primary cell lines were established in vitro from SHRSP and WKY and infected with AdCMVeNOS at an MOI of 100. Cells were fixed 48 h later and transgene expression of eNOS was analysed using a monoclonal anti-eNOS antibody as described for tissue sections.

2.12 Vascular studies
Carotid arteries from twelve SHRSP rats and six WKY rats were infused with 30 µl viral lysate at a viral titre of 2x109 pfu/ml. Vessels were harvested 24 h after surgery by removing fat and connective tissue, cut into 2-mm rings, and suspended between stainless steel hooks in 10-ml organ baths containing Kreb’s buffer (130 mM NaCl, 4.7 mM KCl, 1.6 mM CaCl2.2H2O, 1.17 mM MgSO4.7H2O, 1.18 mM KH2PO4, 14.9 mM NaHCO3, 0.03 mM CaNa2EDTA and 5.5 mM glucose, pH 7.4) maintained at 37°C and oxygenated with 95% air–5% CO2. The baths also contained indomethacin (0.02 mM) to inhibit any prostanoid-mediated effects. Vessels were equilibrated for 60 min, with changes of bathing fluid every 15 min. Isometric tension studies were performed using a Grass FT03 force transducer and displayed using a MacLab dedicated computer. The contractile response to 0.1 M KCl was examined and then cumulative dose–response curves to phenylephrine (PE) (10–9 to 3x10–6 M) were constructed, first in the absence and again after washout, in the presence of 100 µM NG-nitroarginine methyl ester (L-NAME) to inhibit nitric oxide synthase. Responses to phenylephrine were expressed relative to the response to KCl. The increase in tension in the presence of L-NAME provided a measure of the effect of NO on basal tone [19]. The increase in tension in the presence of L-NAME was calculated for each ring over the full dose–response curve and expressed as an area under the curve (AUC). The increase in tension in the infused rings (Ad5/CMV/nt-LacZ or AdCMVeNOS) was compared to that obtained in the contralateral untreated control rings using Student’s t-test.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Viral DNA analysis
DNA was extracted from 293 cells infected with AdCMVeNOS, and after digestion with HindIII, run on an 0.8% agarose gel together with wild-type d1309 DNA, Fig. 1A. The band observed at 2.8 Kb in lane 1, Fig. 1A, represents the E1 region present in the wild-type adenoviral genome, and since it is absent in the AdCMVeNOS DNA in lane 2, suggests that the recombinant virus lacks this region and is indeed replication-defective (Fig. 1A). These DNAs were subsequently transferred to nylon membrane and probed with an eNOS cDNA which confirms the presence of the eNOS fragment at 3.6 Kb in lane 2 but is absent in the wild-type DNA as seen in lane 1 (Fig. 1B). PCR analysis was also used to confirm the presence of the eNOS cDNA in the recombinant viral vector, using eNOS-specific primers (Table 1) and amplifying a fragment of 804 bp which can then be visualised on an agarose gel as shown in Fig. 1C, lane 2, with lane 1 showing size markers.


Figure 1
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  		Fig. 1 Restriction analysis of DNA extracted from virally-infected cells. (A) Recombinants were screened by extracting the adenoviral DNA from the virus-infected cells, digesting it with HindIII followed by fractionation on a 0.8% agarose gel. Lane 1, wild-type dl309 DNA with a doublet at 2.8 Kb, indicating the presence of the E1 region. Lane 2, the recombinant has lost one of the two bands but has the eNOS insert as shown. (B) These DNAs were subsequently transferred to a Hybond N+nylon membrane and probed with an eNOS cDNA which is present in the recombinant (lane 2) but absent in wild-type Ad5dl309 DNA (lane 1). The Ad5dl309 and eNOS viral DNA was extracted from CsCl gradient-purified viral stocks. (C) DNA extracted from cells infected with AdCMVeNOS, lane 2, was subjected to PCR analysis using eNOS-specific primers (Table 1) as described in Section 2, and positive plaques yielded an 804 bp band (lane 2) with a 1 Kb ladder (Gibco) shown in lane 1.

 
3.2 Western blot analysis of recombinant eNOS protein
In the western immunoblot shown in Fig. 2, lane 1, endothelial cell extract (Transduction Laboratories) was used as a positive control and expression of eNOS was confirmed by the presence of a single band at 140 KD, corresponding to the expected Mr of the eNOS protein. Endogenous eNOS protein was observed in vessel lysate from the right contralateral control carotid arteries (lane 2), however, after AdCMVeNOS infection into the left carotid arteries of these animals, eNOS expression was clearly enhanced, as shown in lane 3. No cross-reactivity with other NOS isoforms was observed using this eNOS-specific antibody.


Figure 2
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  		Fig. 2 Western blot analysis of protein extracted from carotid arteries infected with a bovine eNOS recombinant virus. Lanes shown are: endothelial cell extract, which acts as a positive control: uninfected control arteries; and the left carotid arteries infected with a bovine eNOS recombinant virus. A 10-µg amount of protein extract was electrophoresed on an 8% polyacrylamide gel and transferred to a Hybond P membrane. Endothelial NOS was detected using a monoclonal antibody. The 140 KD eNOS band is indicated.

 
3.3 Griess assay for the determination of nitrite production in endothelial cells
SHRSP and WKY aortic endothelial cells were infected with Ad5/CMV/nt-LacZ and AdCMVeNOS at various viral titres (multiplicity of infection, MOI, 0–500). Nitrite concentration in the supernatant was assessed using the Griess assay [24]. Both SHRSPs and WKYs showed similar expression patterns with nitrite production being increased as viral titre was increased as shown in Fig. 3.


Figure 3
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  		Fig. 3 Nitrite production in endothelial cells from SHRSP and WKY after eNOS infusion measured by the Griess assay. NO accumulation in culture supernatants of aortic endothelial cells from SHRSP and WKY animals was determined by chemiluminescence after reduction of nitrate to nitrite. Nitrite concentrations were determined for WKY and SHRSP cells after AdCMVeNOS infusion. Viral titer ranged from a multiplicity of infection (MOI) 0–500.

 
3.4 Foreign gene expression in vitro in aortic endothelial cells infected with AdCMVeNOS
Abundant cytoplasmic eNOS immunoreactivity was observed in AdCMVeNOS-infected endothelial cells (Fig. 4A) but not in the cells mock-infected with saline as control (Fig. 4B). The cells showed a similar pattern of transduction with Ad5/CMV/nt-LacZ compared to controls and there was no difference in transduction efficiency between the cells harvested from SHRSP and WKY, indicating that, at least in vitro, these cells have a similar capacity to be infected with an adenoviral vector and express a foreign gene.


Figure 4
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  		Fig. 4 Histological and immunocytochemical analysis of transgene expression in rat endothelial cells and carotid arteries. SHRSP endothelial cells (ECs) were infected with either (A) AdCMVeNOS at an MOI of 100, or (B) vehicle for 1 h and incubated for a further 24 h in culture media as described in Section 2. Cells were fixed, immunocytochemical staining was performed with eNOS-specific antibodies and counterstained with haemotoxylin. Rat carotid arteries were infected in vivo with (C) Ad5/CMV/nt-LacZ (2x109 pfu/ml) or D), 30 µl vehicle; and stained for β-galactosidase expression. β-gal activity, as assessed by blue staining of nuclei on infected cells, was detected in endothelium and adventitia of the arteries which were infused with Ad5/CMV/nt-LacZ, (C) but not in the contralateral control artery (D). Distribution of adenoviral transgene expression in vivo after adenoviral delivery of AdCMVeNOS via a catheter and the eNOS infused vessels showed brown staining in both the endothelial layer and adventitia as shown in (E), while the contralateral control vessels show no brown staining (F).

 
3.5 Transgene expression in SHRSP carotid arteries ex vivo
After AdCMVeNOS and Ad5/CMV/nt-LacZ viral infusion of the carotid arteries from SHRSP ex vivo and incubation for 48 h, the segments of carotid arteries were stained for β-gal activity or eNOS expression. Histochemical analysis of these sections confirmed localised dark-blue staining in the luminal endothelial cells and also in the adventitia, while no β-gal staining was observed in the eNOS-infused or contralateral control arteries, identical to the in vivo data presented in Fig. 4C and D, respectively. Immunocytochemical analysis also revealed eNOS expression in the endothelium and adventitia with no detectable staining in the control vessels identical to the in vivo data presented in Fig. 4E and F, respectively.

3.6 Transgene expression in the SHRSP carotid arteries in vivo
Adenovirus-mediated gene transfer was achieved in vivo as described in Section 2. To determine tissue integrity after surgery and to localise the expression of the adenoviral-delivered genes, immunohistochemical analyses was carried out. Marked eNOS immunoreactivity was evident in the adventitia and the luminal endothelial layer (Fig. 4E). In the β-gal infused and control uninfused arteries, no eNOS immunoreactivity was detected (Fig. 4F). In the Ad5/CMV/nt-LacZ infused vessels, segments were also harvested 24 h after infection and after X-gal staining, nuclear blue staining was observed in the endothelium and the adventitia, shown in Fig. 4C, while in the segments from the carotid arteries infected with AdCMVeNOS or in the right contralateral control vessel, no β-gal expression was apparent, Fig. 4D.

3.7 Effect of gene transfer on basal NO bioavailability in carotid arteries from the SHRSP
Three sets of animals were used, six SHRSP rats and six WKY rats underwent infusion of the AdCMVeNOS virus into the left carotid artery with the right carotid artery serving as control; six SHRSP rats were infused with a Ad5/CMV/nt-LacZ virus according to the same protocol. Rats were sacrificed 24 h after infusion, and isometric tension studies were performed as described in Section 2, to examine the response of the vessel segments to the contractile agonist phenylephrine (PE) in the absence and presence of L-NAME. The increase in tension in the presence of L-NAME was similar in rings from the Ad5/CMV/nt-LacZ -infused and their control vessels, Fig. 5C, the area under the curve (AUC) for the two groups being 2.21±0.75 and 2.16±0.48, respectively, (P=0.87). In contrast, there was a significantly greater increase in tension after L-NAME treatment in rings from SHRSP AdCMVeNOS-infused vessels compared to their control rings, Fig. 5B. The AUC for rings for AdCMVeNOS infused vessels from SHRSP was 3.64±0.44 and for their control rings 1.56±0.46 (P=0.0069; 95% CI, 0.864, 3.277). The AUC for rings of AdCMVeNOS infused vessels from WKY was increased in five out of six cases compared to their respective controls. However, in WKY, the difference was smaller than in SHRSP and did not reach significance, the AUC for AdCMVeNOS infused and control rings being 2.36±0.44 and 1.94±0.48, respectively, P=0.17; Fig. 5A.


Figure 5
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  		Fig. 5 Basal nitric oxide availability after AdCMVeNOS infusion into the SHRSP model. Carotid rings were removed from the SHRSP and WKY 24 h after infusion of virus. Increase in tension (g) in response to phenylephrine in the presence of L-NAME in carotid arteries infected with adenovirus containing the cDNA for either β-galactosidase or eNOS and their respective contralateral controls. (A) WKY infected with eNOS (bullet) and contralateral controls ({circ}), n=6; (B) SHRSP eNOS infected vessels (bullet) and contralateral controls ({circ}) n=6; (C) SHRSP infected with β-gal (bullet) and contralateral controls ({circ}). Results are expressed as response to PE relative to KCl in the presence of L-NAME–response to PE relative to KCl in the absence of L-NAME.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The hypothesis that a reduced bioavailability of endothelial NO contributes to the pathogenesis of vascular dysfunction led us to examine whether AdCMVeNOS gene transfer would elevate NO availability in the SHRSP. The SHRSP is one of the best existing models of human essential hypertension [33]. Previous studies from our laboratory [19,34] suggested that SHRSP exhibits endothelial dysfunction due to reduced NO bioavailability compared with the normotensive reference strain WKY [19]. A similar finding has been made in human essential hypertension [35]. In the current study, we used 12-week-old male SHRSP rats to ascertain the potential of recombinant adenovirus as a means of introducing and expressing the eNOS gene and elevating basal NO bioavailability in these animals. A recombinant adenovirus that carries the bovine eNOS cDNA under the control of the CMV promoter or the Ad5/CMV/nt-LacZ virus was infused into the left carotid artery of these rats. Expression of the foreign genes was analysed 24 h after infusion. We have shown β-gal expression in both the endothelium and in the adventitial layer of the carotid artery. We have also shown expression of the eNOS protein in cell lysates by Western blotting, and localised recombinant eNOS protein in both the endothelium and adventitia in the carotid arteries infused with the AdCMVeNOS in vivo. Data on the endothelial contractile function of carotid arteries following infection with the eNOS and β-gal viruses showed that basal NO availability can be elevated by infusion of the eNOS virus. It may be argued that increasing NO production could lead to an increased production of superoxide and peroxynitrite, which could in turn cause vascular damage. Indeed, our own studies show an imbalance of NO and the superoxide anion being a major cause of endothelial dysfunction observed in the SHRSP model and we suggest the overproduction of superoxide in the SHRSP could explain the reduced bioavailability of NO in this model [36].

Our future studies will address this problem of the imbalance of NO/O2– by using a combination gene transfer strategy aimed at coexpressing protective genes encoding eNOS and one of the SOD isoforms. The experiments reported here document the feasibility of using adenovirus for the direct delivery in vivo of a gene to improve endothelial function. Since recombinant protein expression has been shown in sheep arteries up to 4 weeks after viral infusion [37] and expression of genes introduced into cells by Ad vectors can last up to 60 days following infection into skeletal muscle [38,39], they may prove highly efficient vectors for vascular gene transfer strategies.

Several limitations are apparent with vascular gene transfer and its putative therapeutic application. The need to obtain long-term gene expression has to be addressed as does the chronic inflammatory response which has been reported to be induced by adenoviral gene transfer [40]. Channon et al. [41] provided evidence to show that endothelial inflammation was dependent on time after infection by the virus, and neutrophil inflammation caused functional endothelial injury progressing from 6 to 72 h at titres of 4x109 pfu/ml. We, like others [12], chose a 24-h time-point for our in vivo studies to avoid an endothelial inflammatory cell infiltrate interfering with transgene expression. We therefore need to address the question of optimal viral titre to be delivered and duration of exogenous gene expression, since Channon et al. [41] also found a threshold for viral titre, above which foreign gene expression was unaffected, but showed significant effect on the inflammatory response in New Zealand White rabbits.

Adenoviral-mediated vascular gene transfer has been achieved in animal models, with a successful modification of vasomotor function. Davies et al. [42] showed alteration of arterial vasomotor function in vitro by gene transfer with adenovirus. Channon et al. [18] delivered AdCMVnNOS into the carotid arteries of cholesterol-fed rabbits while Chen et al. [15] used an ex vivo approach with AdCMVeNOS to show functional expression in the adventitia and endothelium of canine basilar arteries. The current study constitutes the first report of increasing basal NO availability in the SHRSP model after catheter-based local gene transfer. This study shows (i), that the catheter approach allows highly efficient adenoviral-mediated gene transfer into an intact endothelium of non-injured carotid arteries; (ii) intra-carotid gene transfer results in expression of significant levels of functional recombinant protein; and (iii) adenovirus-mediated eNOS gene transfer elevates NO availability in these hypertensive animals and has functional consequences locally.

The methodology for in vivo infusion of the foreign gene into the rat carotid artery may require further refinement since the adventitial expression observed from both genes could be due to slight leakage of the viral lysate after removal of the catheter during surgery. However, other groups have shown it may not be absolutely critical to achieve endothelial-specific expression of eNOS in order to achieve an alteration in vasomotor tone. Ooboshi et al. [12] have shown that after endothelial denudation in rabbit carotid arteries the adventitial expression of eNOS resulted in alteration of NO-mediated endothelial function. This work is further supported by the work of Tsutsui et al. [43] where they also find adventitial expression of recombinant eNOS in canine basilar arteries affects vascular tone.

Recent findings suggest an overproduction of superoxide in the hypertensive rat model [36,44] which would counter the effects of elevated NO achieved in this study. The findings in this report allow us to manipulate NO availability, however, given the multifactorial nature of endothelial dysfunction in hypertension, for this to become a successful therapeutic modality, additional studies will be necessary to examine the more complex events associated with the balance between NO production and superoxide levels and to develop a strategy that would enable us to manipulate that balance.

In conclusion, intraluminal eNOS gene transfer restores local NO production in the SHRSP model. This constitutes a useful strategy to study molecular events in the diseased vessel wall and may lead to potential new therapeutic approaches for vascular disorders.

Time for primary review 40 days.


   Acknowledgements
 
This work was supported by the British Heart Foundation Programme RG/97009 and Project PG/97077 Grants to AFD. We would like to thank Karen Jess, Emma Jardine, Elisabeth Beattie and Danny McSharry for technical assistance.


   Notes
 
1 The first two authors contributed equally to this work. Back


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

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