Gene transfer of endothelial nitric oxide synthase but not Cu / Zn superoxide dismutase restores nitric oxide availability in the SHRSP

Objective: Previous studies from our group have shown a deﬁcit in nitric oxide (NO) bioavailability and an excess production of the 2 superoxide anion (O ) in the stroke prone spontaneously hypertensive rat (SHRSP) compared to the normotensive Wistar Kyoto (WKY) 2 strain. This present study has investigated whether adenoviral-mediated gene transfer of human eNOS or Cu/ZnSOD can alter the 2 NO/O balance, thereby improving endothelial function. Methods: A recombinant adenovirus, Ad/Hu/eNOS, containing the human 2 eNOS cDNA fragment was generated by homologous recombination in 293 cells. Ad/Hu/eNOS or Ad/Cu/ZnSOD was delivered into 9 10 SHRSP carotid arteries in vivo, using a titre of 2 3 10 –2 3 10 plaque forming units (pfu)/ml, and the effect on gene expression was observed 24 h later. Results: Western blotting conﬁrmed increased enzyme levels of eNOS and Cu/ZnSOD in the viral-infused vessels. Ex vivo, the pressor response to phenylephrine (PE) in the presence of L -NAME was increased in the eNOS-infused arteries relative to the contralateral controls, indicating restoration of basal NO availability to that observed in untreated control WKY rats. Infusion of the SOD virus produced a statistically insigniﬁcant increase in NO bioavailability. Conclusions: Our results support our previous ﬁndings obtained using a bovine eNOS recombinant adenovirus, that recombinant adenoviral gene transfer of human eNOS has a signiﬁcant effect on NO bioavailability. In contrast, AdCu/ZnSOD gene transfer does not elicit an effect in our model. These results indicate that short-term overexpression of a recombinant eNOS, but not Cu/ZnSOD gene, in carotid arteries of the SHRSP is an effective means of locally increasing NO bioavailability to improve endothelial function. (cid:211)


Introduction
angiotension II-induced hypertension [6] and in the strokeprone spontaneously hypertensive rat (SHRSP) [7,8], The endothelium is an important organ involved in the suggest that an increased production of the superoxide 2 control of vascular homeostasis through its ability to anion (O ) could play a role in the reduced bioavailability 2 produce a range of factors which induce contraction and of nitric oxide (NO) in these models of hypertension. . Endothelial dysfunction is the term used to Increased production of O may also contribute to de-2 describe the impaired endothelium-mediated vasodilation creased NO bioavailability in the SHR [9], hypercholeswhich occurs when the balance of these factors is dis-terolemic rabbits [10] and diabetic rats [11]. Endothelial rupted. It is a common characteristic of patients with NOS is a 140-kDa protein and its activity accounts for essential hypertension [2,3] and of many animal models of endothelium-dependent vasorelaxation by catalysing the hypertension, where it is thought that the main contributing conversion of arginine to citrulline with the release of NO factor is a deficit in NO availability [4,5]. Recent studies in and consequently is implicated in blood pressure regulation [1]. The deficit observed in the SHRSP may come about through a reduction in its production, which is unlikely, as it has been shown by northern and western analysis that was obtained by in vitro homologous recombination. eNOS mRNA and protein levels are in fact increased [4].
Individual Ad / Hu / eNOS plaques were expanded in 293 Alternatively, the reduction in bioavailability is more cells [24] and viral DNA was isolated as previously likely to be caused by an increased degradation, brought described [23] followed by restriction analysis, Southern 2 about by the excess O observed in this model. Since NO blotting and PCR using primers specific for the human 2 production has both beneficial and detrimental conse-eNOS and SOD cDNAs (Table 1) [25]. Before use, plaque quences, understanding the molecular mechanisms that isolates were evaluated for their potential to overexpress regulate NOS expression is critical to the control of NO NOS activity in endothelial cells using the Griess assay release in physiological and pathophysiological conditions.
[26]. 2 Cu / ZnSOD is an enzyme that dismutates O into The human Cu / ZnSOD viral vector was purchased from 2 oxygen and hydrogen peroxide, thereby providing a the Gene Transfer Core Group in University of Iowa. 2 protective effect on NO [12].  [14], characterised, maintained and infected with virus as previatherosclerosis [15][16][17], arterial vasospasm [18], vein graft ously described [23]. For in vivo studies, SHRSP males at failure [19], and coronary artery restenosis [20]. In this 12  expressing the E1 region of Ad2) [21], by conventional Hu / eNOS (2310 pfu / ml). The right contra-lateral carotid calcium phosphate precipitation [22] and AdCMVeNOS arteries from these animals were used as controls. At 24 h after infusion, rats were killed by an overdose of halothane hematoxylin and examined for positive staining of SOD and the carotid arteries were removed. Additional carotid (brown staining) by light microscopy. arteries from virus-infused animals were frozen in liquid nitrogen for western blot analysis, or taken for measure- Nitrite concentrations were determined at an optical dencarotid arteries were removed from the experimental sity of 554 nm by comparison to standard solutions of animals and embedded in OCT and 20-mm thick sections sodium nitrite. In the case of SOD, cells were removed were placed on coverslips and the dye topically applied.
from flasks by scraping, washed and pelleted. Then, 100 ml The sections were then incubated at 378C for 30 min water was added and samples were lysed by freeze / thawbefore visualising the fluorescence under a microscope.
ing. SOD activity was measured in 20-ml aliquots of Photomicrographs of both SHRSP and WKY animals were supernatant using a kit (Calbiochem, 574600). The assay is taken, and the intensity and localisation of the oxidised 2 based on the oxidation of 5,6,6a,11b-tetrahydro-3,9,10-HEt, which reflects O production, could be observed and 2 trihydroxybenzo[c]fluorene (THBF) and is measured as the compared between the rat strains.
rate of increase in optical density at 525 nm.  O generated by xanthine / hypoxanthine. Tissue O was 2 2 ously described [23]. Krebs buffer in which the carotid expressed as nmol / mg wet wt / min. artery rings were maintained contained indomethacin (0.02 mM) to inhibit any prostanoid-mediated effects. Isometric 2.6. Western blot and immunohistochemical analysis of tension studies were performed using a force transducer human eNOS and Cu /ZnSOD recombinant gene and recorded using a MacLab dedicated computer. expression Cumulative dose-response curves to phenylephrine (PE) 29 25 (10 to 10 mol / l) were constructed, first in the For western blotting, crude protein extracts from the absence, and again after washout, in the presence of 100 G harvested arteries were subjected to polyacrylamide gel mM N -nitro-L-arginine methylester (L-NAME) to inhibit electrophoresis and transferred to Hybond P membranes as NO synthase. The increase in tension in the presence of previously described [23]. Supernatant protein (10 mg) was L-NAME provided a measure of the effect of NO on basal separated on 7.5% SDS polyacrylamide gels in the case of tone [4]. The increase in tension in the presence of L-eNOS and 12% in the case of Cu / ZnSOD. For immuno-NAME was calculated for each ring over the full dosestaining, sections were treated as previously described response curve and expressed as an area under the curve [23]. In both cases, eNOS was detected by a mouse (AUC). anti-human monoclonal antibody (Transduction Laboratories) while Cu / ZnSOD was detected using a primary polyclonal sheep anti-human Cu / ZnSOD antibody (Cal-2.9. Statistical analysis biochem). The secondary antibody was a sheep anti-mouse IgG-peroxidase (Sapu) in the case of eNOS and donkey In each experiment, n refers to the number of animals anti-sheep IgG-peroxidase in the case of SOD. Enhanced from which the carotid arteries were harvested. The chemiluminescence (Amersham) was used to visualise the increase in tension in the presence of L-NAME in the protein bands for western analysis. For the histochemistry, infused rings (human eNOS or Cu / ZnSOD) was compared binding was visualised using 39,39-diaminobenzadine to that obtained in the contralateral untreated control rings (DAB) and 0.01% hydrogen peroxide as a chromogene.
using Student's t-test. Data are expressed as mean6S.E.M. Sections were briefly air-dried and counterstained with Difference among data sets were evaluated by performing analysis of variance (ANOVA). A level of P,0.05 was accepted as statistically significant.

Recombinant viral DNA analysis
A standard strategy was used to generate the recombinant human eNOS virus as described in the methods. To confirm the recombinant status and purity of the newly generated viral vector carrying the human eNOS cDNA, restriction analysis with HindIII was carried out on DNA sels and the contralateral control vessels, respectively (n5 the Griess assay [26]. Increased viral titre resulted in 6).
increased nitrate / nitrite production in three independent experiments (Fig. 4A). Infection of endothelial cells with 3.3. Superoxide localisation the SOD gene resulted in a dose-related increase in the rate of oxidation of THBF as illustrated in Fig. 4B experiment was carried out to compare levels and location Protein extracts were prepared from the infused arteries of superoxide in the SHRSP and WKY rat carotid arteries and subjected to western blot analysis using monoclonal and to add further strength to the results obtained in Fig. 2 and polyclonal antibodies reactive with human eNOS and using chemiluminescence. As can be seen in Fig. 3, tissue Cu / ZnSOD, respectively. In western immunoblots, shown  the western and immunohistochemical analysis, this result is not surprising, as a more specific block has been used in 3.6. Immunohistochemical analysis the latter to prevent cross-reactivity with the rat antigens.
The efficiency of adenoviral vector-mediated gene trans-3.7. Effect of gene transfer on basal NO bioavailability fer in rat carotid arteries was estimated on transverse in carotid arteries from SHRSP histological sections of the arteries transfected with Ad / CuZnSOD. No staining was found in the contralateral In the SHRSP, the pressor response to phenylephrine in uninfused or control-infused artery (Fig. 6A) indicating a the presence of L-NAME was increased in the eNOSlack of cross-reactivity between the rat and human isoform infused vessels relative to the contralateral controls; the  areas under the curve (AUC) for phenylephrine in treated NO availability which did not reach significance, illusand control carotid arteries were 2.9560.53 and trated in Fig. 7C, AUC for phenylephrine in treated and 1.7460.52, respectively, (n57), P50.0026; 95%CI 0.606-control carotids were 2.1460.41 and 1.6460.17, respec-9 1.788 at a titre of 2310 pfu / ml (Fig. 7B). In contrast, in tively (n58), P50.28; 95%CI 20.49 to 1.49. Titre is carotid arteries from WKY rats, the increase in the pressor usually a critical factor in determining gene expression response to phenylephrine in the presence of L-NAME was levels after gene transfer experiments. When no effect was 9 similar in the Ad / Hu / eNOS infused and contralateral observed in NO bioavailability at a titre of 2310 pfu / ml control vessel (Fig. 7A). AUC being 3.1260.51 and from the SOD virus, the titre of the SOD virus was 3.1960.64, respectively (n56). Infusion of the AdCu / increased by a log of 10 to determine if an improved ZnSOD gene in SHRSP produced a slight improvement in response could be obtained at this higher titre. However, a lack of response was still apparent at the higher titre of media, as well as the endothelium is required to reduce 10 oxidative stress and improve endothelial function in 2310 pfu / ml, (n53) (data not shown). Previous studies atherosclerosis. In contrast, Fang et al. [34] were able to have shown no effect on nitric oxide bioavailability when show a reduction in LDL oxidation in endothelial cells rat carotids are infused with a control b-galactosidase virus overexpressing SOD. However, these in vitro studies are [23].
not directly comparable to the in vivo situation. In vivo, the most likely site of oxidation of LDL within the vessel wall is the sub-endothelial space. Overexpression of SOD 4. Discussion within the endothelium has yet to be shown to inhibit LDL oxidation in vivo. The results of the present study support and confirm our Another aspect of our study which needs to be considprevious findings that in vivo eNOS gene transfer into ered is the choice of SOD isoform. Inhibition of Cu / intact endothelium of SHRSP-carotid arteries can success-ZnSOD has been shown to have deleterious effects on fully restore NO bioavailability to a level observed in the endothelial function [32,35], while lipid permeable Cu / WKY normotensive reference strain. In this study we used ZnSOD restored endothelium-dependent vasorelaxation in a human instead of a bovine eNOS gene as a first step cholesterol-fed rabbits [10] and enhanced vasorelaxation in towards future clinical applications.
B6C3F-1 mice [36] supporting our choice of Cu / ZnSOD We have previously reported that in the SHRSP, as in a for gene transfer. However, mitochondria are an important number of other models of hypertension, decreased NO 2 source of oxygen radicals generated as a by-product of bioavailability is associated with an increase in O levels 2 electron transport. Future studies will address the issue of [4,6,7,30]. In our previous studies using aortae of SHRSP, 2 2 the effect of MnSOD and extracellular (ec) SOD on O 2 it appeared that the O was of endothelial origin [8]. In 2 levels in carotid arteries from SHRSP although ecSOD is this study, we have confirmed the existence of increased not normally present in rats at significant levels [37]. The

2
O levels in carotid arteries from SHRSP compared to 2 relative importance of these two isoforms in this model of WKY. In the carotid arteries using dihydroethidine, we hypertension is uncertain. 2 have been able to show excess O throughout the arterial 2 Finally, overexpression of SODs alone may not improve wall. As the decreased NO bioavailability may be at least endothelial dysfunction as this could lead to increased 2 in part, if not primarily, a consequence of increased O 2 production of hydrogen peroxide. Hydrogen peroxide itself generation, we proposed that delivery of a recombinant may be harmful, it may promote vascular smooth muscle adenovirus carrying and expressing a SOD gene may also hypertrophy, alter gene expression and activate matrix be useful in the treatment of endothelial dysfunction by metalloproteinases [38]. Moreover, hydroxyl radicals 2 2 scavenging the excess O . In support of this hypothesis, 2 (OH ) may be produced from hydrogen peroxide via the 2 inhibition of bovine coronary artery superoxide dismutase Fenton reaction. OH is several orders of magnitude more has been shown to attenuate endothelium-dependent vasoreactive towards cellular contents than superoxide and is dilation [31], while treatment with membrane-permeable considered to be highly toxic. superoxide dismutase mimetics improves endothelial func-In conclusion, our data indicate that endothelial delivery tion in cholesterol-fed rabbits and SHR [9,10]. However, of the human eNOS gene to SHRSP carotid arteries can in our study, intraluminal delivery of AdCu / ZnSOD to correct the deficit in NO bioavailability in these animals. carotid arteries of SHRSP did not result in a significant Cu / ZnSOD gene transfer had little effect. This may relate improvement in NO bioavailability ex vivo, nor to a to the mode of gene delivery or could be a consequence of 2 reduction in O levels in the treated vessels. complexities in the SOD signalling pathways which have 2 There are a number of potential explanations for this. yet to be unravelled. 2 Increased O has been found throughout the vessel wall in 2 a number of models of atherosclerotic disease. In WHHL 2 rabbits, a marked increase in O levels in vascular smooth Acknowledgements 2 muscle has been reported [29], while in rats infused with 2 angiotensin, increases in adventitial production of O This work is supported by the British Heart Foundation 2 occurs [32,33]. Our results, showing intense fluorescence Programme RG / 97009 and Project PG / 97077 Grants to after hydroethidine staining in vascular smooth muscle of AFD. We would like to thank Professor Gwyn Gould for carotid arteries from SHRSP are consistent with these help with the confocal microscopy used for the hydro- sub-endothelial layers of the vessel and the virus is tance in the laboratory. delivered intraluminally, the SOD gene may be transferred primarily into endothelial cells, rather than vascular smooth muscle. Consistent with this hypothesis, Miller et References al. [29] found that ex vivo SOD virus infection of rings of aorta from WHHL rabbits failed to improve endothelial