Endothelial Dysfunction and Hypertension in Rats Transduced With CYP4A2 Adenovirus
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Ji-Shi Wang, Harpreet Singh, Frank Zhang
参见附件。
the Departments of Pharmacology and Physiology, New York Medical College, Valhalla, New York.
Abstract
Vascular cytochrome P450 (CYP) 4A enzymes catalyze the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE), an eicosanoid which participates in the regulation of vascular tone by sensitizing the smooth muscle cells to constrictor and myogenic stimuli. This study was undertaken to investigate the consequences of CYP4A overexpression on blood pressure and endothelial function in rats treated with adenoviral vectors carrying the CYP4A2 construct. Intravenous injection of Adv-CYP4A2 increased blood pressure (from 114±1 to 133±1 mm Hg, P<0.001), and interlobar renal arteries from these rats displayed decreased relaxing responsiveness to acetylcholine, which was offset by treatment with an inhibitor of CYP4A. Relative to data in control rats, arteries from Adv-CYP4A2–transduced rats produced more 20-HETE (129±10 versus 97±7 pmol/mg protein, P<0.01) and less nitric oxide (NO; 4.2±1.6 versus 8.4±1 nmol nitrite+nitrate/mg; P<0.05). They also displayed higher levels of oxidative stress as measured by increased generation of superoxide anion and increased expression of nitrotyrosine and gp91phox. Collectively, these findings demonstrate that augmentation in vascular 20-HETE promotes the development of hypertension and causes endothelial dysfunction, a condition characterized by decreased NO synthesis and/or bioavailability, imbalance in the relative contribution of endothelium-derived relaxing and contracting factors, and enhanced endothelial activation.
Key Words: arachidonic acid 20-HETE nitric oxide eNOS superoxide anion
Introduction
The endothelium plays a critical role in the short- and long-term regulation of the cardiovascular system. It serves as a protective barrier between tissues and circulating blood and functions to maintain vascular homeostasis by releasing bioactive factors in response to hemodynamic changes and blood borne signals. Such endothelial cell functions are impaired in many disease processes, including, diabetes, atherosclerosis, and hypertension.
The expression of the cytochrome P450 (CYP) 4A and its catalytic activity as arachidonic acid -hydroxylase which produces 20-hydroxyeicosatetraenoic acid (20-HETE) has been linked to the regulation of vascular reactivity and tone and to the development of hypertension. This notion is substantiated by observations that 20-HETE promotes vasoconstriction,1,2 that CYP4A expression and/or 20-HETE production is increased in vascular tissues of hypertensive animals,3 and that interventions that decrease CYP4A expression and/or activity cause blood pressure to fall in animal models of hypertension.4–11
The vasoconstrictor action of 20-HETE is primarily attributed to inhibition of the large conductance Ca2+-activated K+ channel (KCa) in vascular smooth muscle cells leading to depolarization and elevation of cystolic [Ca2+].12 This inhibitory action of 20-HETE is believed to enhance constrictor responsiveness of arterial vessels to increased pressure,13,14 oxygen,15,16 phenylephrine,2 and endothelin17 as well as inhibition of NO synthesis.18 Although the regulatory action of 20-HETE on the vascular smooth muscle function is well documented, little is known about the regulatory actions on endothelial function. A study by Frisbee et al19 alluded to the possibility that 20-HETE affects vascular reactivity by promoting endothelial dysfunction. Their study demonstrated that 20-HETE attenuates acetylcholine-induced dilation in cremasteric arterioles of normotensive rats on a low salt diet and that inhibition of 20-HETE synthesis enhances arteriolar dilation to acetylcholine in hypertensive rats on a high salt diet. More recently, 20-HETE has been shown to functionally antagonize EDHF-mediated relaxation in small porcine coronary arteries.20
In the present study, we examined the consequences of overexpressing CYP4A in the vasculature on blood pressure and endothelial function in rats. We demonstrated that intravenous injection of Adv-CYP4A2 caused hypertension and that renal arteries from these rats featured increased vascular CYP4A expression and 20-HETE production, and displayed endothelial dysfunction exemplified by reduced acetylcholine-induced relaxations, reduced levels of NO, and increased levels of superoxide anion. These findings indicate that vascular overexpression of CYP4A fosters prohypertensive mechanisms and that vascular 20-HETE may be a determinant of endothelial dysfunction and activation.
Materials and Methods
Construction of Recombinant Adenoviruses
CYP4A2 cDNA (1720 bp), originally isolated from the Lewis-Wistar rat kidney,21 was subcloned into the adenovirus vector pAd5CMV-NpA.22 The pAd5CMV-NpA-CYP4A2 (Adv-4A2) was propagated, purified, and titrated to 1x1012 pfu/mL by the Gene Transfer Vector Core, University of Iowa, as described.22 Recombinant adenovirus containing the coding region of the enhanced green fluorescent protein (GFP) (pAd5CMV-NpA-eGFP) was constructed in the same manner, designated as Adv-GFP and used as the control adenovirus.
In Vitro Transduction
COS-1 and EA.hy926 cells23 were used to evaluate functional transgene expression. Cells were infected with the adenoviruses; GFP and CYP4A2 protein levels were measured by Western blot and CYP catalytic activity was evaluated by either measuring -hydroxylation of the CYP4A-preferred substrate, lauric acid,24 or by quantifying endogenous 20-HETE levels.25
Animal Experimentation
All experimental protocols were performed following an IACUC approved protocol in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Adenoviruses were injected into the tail vein (200 μL of 1012 pfu/mL in saline) of Sprague-Dawley rats (9- to 10-week-old, 250 to 275 g body weight) under anesthesia with sodium pentobarbital (65 mg/kg body weight, ip). At day 6 after injection, rats were placed in metabolic cages for 24 hour urine collection. Urinary electrolytes were measured by flame photometry. Blood pressure was measured by the tail cuff method before and 7 days after adenovirus administration. At day 7, rats were euthanized and arterial vessels including renal interlobar arteries were dissected as previously described.26
Immunohistochemistry and Western Blotting
Arteries were embedded in OCT (Sakura) and sectioned at 5 μm thick. Sections were processed for immunohistochemistry using Vectastain ABC elite kit (Vector Laboratories, Inc), stained with Vector-DAB, and counterstained with hematoxylin. Western blotting was performed on homogenized arterial segments using mouse anti-EGFP monoclonal antibody and goat anti-rat CYP4A1 polyclonal antibody, which cross-reacts with all CYP4A isoforms, ie, CYP4A2, CYP4A3, and CYP4A8.24
Measurements of HETE and Epoxyeicosatrienoic Acid Levels
Renal interlobar arteries (4 segments per tube) were placed in Krebs bicarbonate buffer (pH 7.4) for 1 hour at 37°C with gentle shaking. At the end of the incubation, [2H2]-20-HETE (0.5 ng) and a mixture of [2H8]-epoxyeicosatrienoic acid (EETs) (3 ng) were added as internal standards. HETEs and EETs were extracted, separated by HPLC, derivatized, and quantified by gas chromatography/mass spectrometry as described previously.26
Agonist-Induced Vasorelaxation
Renal interlobar arteries were dissected and mounted on wires in myograph chambers (J.P. Trading) for measurement of isometric tension as described.27 Vasorelaxation responses of phenylephrine-constricted arteries to cumulative increments in the acetylcholine (10–9 to 10–4 mol/L) or DETA-NONOate (10–9 to 10–5 mol/L) concentrations were examined in the presence of indomethacin (10 μmol/L) with and without L-NAME (1 mmol/L). The response to acetylcholine was also assessed in arteries exposed to the CYP4A inhibitor DDMS (30 μmol/L) in the absence and presence of 20-HETE (0.1 to 10 μmol/L).
Measurement of NO and cGMP
NOx concentration in urine was measured by the Griess reaction. NO production in blood vessels was measured according to the method established by Zeballos et al.28 Production of cGMP in the presence IBMX (0.5 mmol/L) and L-arginine (1 mmol/L) with and without carbachol (100 μmol/L) was measured as described by Pucci et al.29
Measurement of O2·–
Arterial segments were placed in scintillation vials (2 per vial) containing 1 mL of Krebs-HEPES buffer, pH 7.4, and lucigenin (5 μmol/L). Superoxide anion levels were measured by lucigenin chemiluminescence method as previously described.30
Statistical Analysis
Data are expressed as mean±SE. Concentration-response data derived from each vessel were fitted separately to a logistic function by non-linear regression. Maximum asymptote of the curve (Rmax) and the concentration of agonist producing 50% of the maximal response (EC50) were calculated using commercially available software (Prism 2.01, GraphPAD software). Concentration-response data were analyzed by a two-way analysis of variance followed by a Duncan multiple range test. Other data were analyzed by a Student t test for paired or unpaired observations as appropriate. The null hypothesis was rejected at P<0.05.
Results
Functional Expression of CYP4A2 and GFP Adenoviruses In Vitro
Functional transgene expression of the recombinant adenoviruses was evaluated in vitro in COS-1 cells (please see the online data supplement, available at http://circres.ahajournals.org) and in EA.hy926 cells. Cells infected with Adv-GFP displayed GFP immunoreactivity and basal levels of CYP4A immunoreactivity; in contrast, cells infected with Adv-4A2 showed a strong CYP4A immunoreactivity (Figure 1A). Moreover, CYP4A catalytic activity measured as 20-HETE production was only present in Adv-4A2–infected EA.hy926 cells (Figure 1B).
CYP4A2 Gene Transfer Increases Blood Pressure
Recombinant adenoviruses (Adv-4A2, Adv-GFP) were administered intravenously to 9-week-old rats. Systolic blood pressure increased from 114.4±4.3 to 133.1±4.4 mm Hg one week after injection of Adv-4A2 (n=10; P<0.001) but was not affected by Adv-GFP injection (114.8±3.6 and 116.7±4.0 mm Hg before and after Adv-GFP administration, respectively; n=10). Urinary potassium excretion showed no change (from 3.99±0.52 to 4.47±0.54 meq per 24 hours; n=8), whereas urinary excretion of sodium significantly increased 7 days after injection of Adv-4A2 (from 1.85±0.21 versus 2.63±0.33 meq per 24 hour; n=8; P<0.01). Neither urinary potassium excretion (3.48±0.45 versus 4.11±0.24 meq per 24 hours; n=8) nor sodium (1.92±0.26 versus 2.31±0.17 meq per 24 hours; n=8) were changed in rats injected with the Adv-GFP.
Intravenous Administration of Recombinant Adenoviruses Transduces the Vasculature
Transgene expression carried by the adenoviral vectors used in this study was not tissue-selective and followed a pattern previously shown for the adenoviral-mediated HO-1 gene transfer.31 Nevertheless, with systemic administration, the vasculature was sufficiently transduced with the transgenes (online data supplement). Immunohistochemical analysis of interlobar arteries taken from rats injected with the Adv-GFP indicated a positive CYP4A staining (Figure 2A and 2B). The intensity of CYP4A staining was greatly enhanced in arteries taken from Adv-4A2–treated rats and appeared to localize to the intima and the media (Figure 2C and 2D). Western blot analysis verified GFP immunoreactivity only in vessels from rats injected with Adv-GFP (Figure 3A) and increased CYP4A immunoreactivity in Adv-4A2–infected arteries as compared with arteries infected with the Adv-GFP (Figure 3B and 3C). Importantly, as seen in Figure 3D, interlobar arteries from rats transduced with Adv-4A2 produced more 20-HETE than arteries from Adv-GFP transduced rats. The levels of 19-HETE, another CYP4A-derived arachidonate metabolite,24 also increased (P<0.05) from 20.9±3.4 pmol/mg in Adv-GFP-infected arteries to 39.1±3.7 pmol/mg in Adv-4A2 infected arteries (mean±SE, n=4). EET production in interlobar arteries appeared (P=0.066) to increase (from 39.03±15.60 to 111.35±53.21 pmol/mg in Adv-GFP and Adv-4A2, respectively; n=7; P<0.05), possibly reflecting the capacity of CYP4A2 to catalyze arachidonate epoxidation in addition to -hydroxylation.21
Adv-4A2 Transduced Rats Display Impaired Vasorelaxing Responsiveness to Acetylcholine
Relaxing responses to acetylcholine (10–9 to 10–4 mol/L) or NONOate (10–9 to 5x10–5 mol/L) were examined in renal interlobar arteries from Adv-4A2– and Adv-GFP–transduced rats. Arteries were relaxed in a concentration-dependent manner by both agonists. At the maximally effective concentration, the response to acetylcholine in arteries from Adv-4A2–transduced rats was significantly lower (54±4% relaxation) than in arteries from Adv-GFP-transduced rats (87±2% relaxation) (Figure 4A). After addition of L-NAME, the residual (NO-independent) relaxing effect of acetylcholine was similar in arteries of rats transduced with Adv-4A2 (30±5% relaxation) or Adv-GFP (39±5% relaxation) (Figure 4A, lower panel). DDMS (30 μmol/L), a CYP4A inhibitor, enhanced (P<0.05) acetylcholine-induced relaxation in arteries of rats transduced with Adv-4A2 (from 54±±4 to 74±8% relaxation) but not with Adv-GFP (from 87±2 to 92±4% relaxation). 20-HETE (10 μmol/L) reduced (P<0.02) the response to acetylcholine in DDMS-treated arteries of rats transduced with Adv-4A2 (from 74±8 to 47±2% relaxation) or Adv-GFP (from 92±4 to 47±9% relaxation) (Figure 4B). Relaxing responses to DETA-NONOate were comparable in arteries of rats transduced with Adv-4A2 or Adv-GFP (online data supplement). These data suggest that vascular overexpression of CYP4A2 attenuates acetylcholine-induced dilation of renal interlobar arteries, presumably via a mechanism involving interference by 20-HETE with the NO-dependent component of the vasorelaxing action of acetylcholine. Pertinently, urinary NOX measured by the Griess reaction was significantly lower in rats 7 days after transduction with Adv-4A2 (13.76±1.32 versus 8.00±2.66μmol per 24 hours; n=5; P<0.05), whereas urinary NOX was not significantly altered in rats transduced with Adv-GFP (11.86±2.29 versus 8.62±1.29 μmol per 24 hours; n=5; P=0.12).
NO, cGMP, and O2– Production in Renal Arteries of Adv-4A2 Transduced Rats
The observation of a reduced NO-dependent acetylcholine-induced relaxation in arteries of rats transduced with Adv-4A2 suggested that NO production/bioavailability is impaired in those vessels. Accordingly, we contrasted rats treated with Adv-GFP and Adv-4A2 in terms of vascular production of NO, cGMP, and O2–. As seen in Figure 5A, renal arteries of rats transduced with Adv-4A2 produced significantly less NO than arteries of Adv-GFP transduced rats. Similarly, basal and carbachol-induced cGMP levels in Adv-4A2 treated rats were less than those in Adv-GFP rats (Figure 5B). In contrast, as seen in Figure 5C, production of O2– was significantly higher in renal arteries of rats transduced with Adv-4A2 as compared with arteries from Adv-GFP transduced rats.
These results further suggest a potential link between CYP4A expression, NO generation, and endothelial dysfunction. To this end, measurements of eNOS proteins in blood vessels taken from rats transduced with Adv-4A2 indicated no change in eNOS immunoreactivity (protein levels) as compared with rats transduced with Adv-GFP. As seen in Figure 6A and 6C, eNOS protein levels in aorta and renal arteries from Adv-4A2 and Adv-GFP transduced rats were similar, suggesting that the reduced NO-dependent vasorelaxation in Adv-4A2–transduced rats was not associated with downregulation of eNOS expression. On the other hand, Western blot of aortic homogenates and densitometry analysis revealed a significant increase in immunoreactive nitrotyrosine, a marker of peroxynitrite-induced oxidative stress in Adv-4A2 rats (Figure 6B and 6C). Western blot and densitometry analysis of gp91phox, a major component of the NADPH oxidase in endothelial cells, showed increased levels in aortic tissue of Adv-4A2 rats (Figure 6B and 6C). As for the renal arteries, aortas from rats injected with Adv-4A2 displayed reduced acetylcholine-induced relaxations as compared with aortas from Adv-GFP-injected rats (viz., EC50s to acetylcholine were 1.75±0.92 and 7.05±2.39 μmol/L for Adv-GFP and Adv-4A2 infected aortas, respectively; mean±SE; n=5; P<0.05).
Discussion
The primary finding of this study is that treatment of rats with adenovirus carrying the rat CYP4A2 full-length cDNA promotes augmentation in blood pressure accompanied by impairment of acetylcholine-induced relaxation of renal interlobar arteries. These animals also display increased vascular production of 20-HETE, the major product of arachidonic acid metabolism by the CYP4A -hydroxylases. Accordingly, the development of hypertension and impaired vasorelaxing responsiveness to acetylcholine in rats transduced with adenoviral vectors carrying the CYP4A cDNA may be the functional manifestation of increased vascular synthesis of 20-HETE.
Increased 20-HETE production in renal interlobar arteries of rats treated with recombinant CYP4A adenoviruses is in line with observations that COS-1 and EA.hy 926 cells infected with Adv-4A2 manufacture catalytically active CYP4A2. It is also consistent with the finding of increased CYP4A immunoreactivity in arterial vessels from rats treated with Adv-4A2. Notably, immunohistochemistry of arterial vessels of rats treated with Adv-4A2 suggested that the intima is a primary site of expression of the transgene. By extension, the intima may be expected to be the major site of vascular 20-HETE overproduction in rats treated with Adv-4A2. Pertinent to this point, recent reports have identified the intima and endothelial cells as a major site of CYP4A protein expression and a target for 20-HETE bioactions,32–36 questioning the early notion that the vascular CYP4A-20-HETE pathway of arachidonic acid metabolism is limited to the smooth muscle.12,37 To this end, our immunohistochemical analysis of CYP4A in arterial vessels taken from Adv-GFP rats indicated the presence of CYP4A positive signals not only in the media but also in the intima where CYP4A immunopositivity was greatly increased after Adv-4A2 administration. Certainly, extensive studies are needed to substantiate the possibility of constitutive CYP4A expression in the endothelium of vascular beds other than the pulmonary circulation.
20-HETE sensitizes the smooth muscle of resistance vessels to myogenic or hormonal constrictor stimuli.13,14,16,17,38 This action of 20-HETE has been attributed to its ability to inhibit Ca2+-activated K+ channels,12 increase the conductance of Ca2+ channels,39 promote the activation of Rho-kinase,40 and/or inhibit vascular Na+-K+-ATPase activity.20 Because of reports that interventions that increase and decrease vascular production of 20-HETE elicit vasoconstriction and vasodilation, respectively, 20-HETE of vascular origin has gained recognition as a major contributor to the implementation of vasoconstrictor mechanisms in physiological and pathophysiological settings.37 In like manner, reports that interventions that decrease 20-HETE synthesis bring about lowering of blood pressure in experimental models of hypertension have given rise to the concept that vascular 20-HETE subserves a prohypertensive function.37 Two recent reports further strengthened this concept and substantiated the link between CYP4A, 20-HETE, and hypertension: Holla et al41 demonstrated that in Cyp4a14 knockout mice, hypertension is more severe in the male and is associated with increased Cyp4a12 expression (the mouse homologue of CYP4A8) and 20-HETE synthesis; Nakagawa et al3 showed that rats treated with 5a-dihydrotestosterone displayed increased vascular CYP4A8 expression and 20-HETE synthesis that may contribute to the hypertension seen in these rats. Our present study provides critical support to this concept by documenting that vascular transduction of CYP4A in rats injected with Adv-4A1 or Adv-4A2 is accompanied by increased vascular 20-HETE production along with sustained elevation of blood pressure. In line with the preceding discussion, the development of hypertension in rats so treated may be caused by 20-HETE–induced amplification of the vasoconstrictor influence of various neurohormonal systems, eg, the sympathetic, endothelin, and renin-angiotensin systems. Equally possible, as judged by the results of the present study, the development of hypertension in rats transduced with CYP4A oxygenases may be caused by 20-HETE–induced interference with endothelial functions that foster vasodilation.
The endothelium is a rich source of vascular smooth muscle relaxing mediators, viz., prostacyclin, NO, and EETs, which implement vasodilator responses to agonists and shear stress. Rat renal interlobar arteries are relaxed by muscarinic agonists such as acetylcholine, the response of which is endothelium-dependent and involves both NO-dependent and -independent mechanisms. The vasorelaxing response to acetylcholine is impaired in hypertension and other cardiovascular diseases, and the impairment is taken as an indication of endothelium dysfunction. Studies in different animal models of arterial hypertension42 and in patients with both essential and secondary hypertension43 have demonstrated an association between elevated systemic blood pressure and impaired endothelium-dependent vascular relaxation. Moreover, a diminished bioavailability of NO has been identified as a mechanism responsible for endothelial dysfunction in hypertensive patients.43 A most striking finding in the present study is that acetylcholine-induced relaxation of renal interlobar arteries is greatly attenuated in Adv-4A2–transduced rats. Importantly, when the assessment of vasorelaxing responsiveness was conducted in the presence of L-NAME to inhibit NO synthase (NOS) and thus eliminate the NO-dependent component of the acetylcholine response, the muscarinic agonist was equally effective in relaxing renal interlobar arteries taken from rats injected with Adv-GFP (control) or Adv-4A2. This implies that only the NO-dependent component of acetylcholine-induced vasorelaxation is impaired in vessels taken from Adv-4A2–transduced rats. The NO-independent component in renal arterioles, which was unaltered by Adv-4A2 administration, has been associated with EETs.44 Indeed, the levels of EETs in renal arteries of Adv-4A2–treated rats were not significantly different than those in renal arteries of Adv-GFP–treated rats and, in fact, appeared to increase, likely reflecting the capacity of CYP4A2 to catalyze arachidonate epoxidation.21
According to the present study, the impairment in acetylcholine-induced relaxation displayed by renal arteries of Adv-4A2–transduced rats was nearly offset by ex vivo treatment of vessels treated with DDMS, a known inhibitor of 20-HETE synthesis.45 Because the application of exogenous 20-HETE reinstated impaired vasorelaxing responsiveness to acetylcholine in DDMS-treated vessels of Adv-4A2–transduced rats and attenuated the responsiveness to the muscarinic agonist in vessels of Adv-GFP–transduced rats, it may be concluded that the NO-dependent component of acetylcholine-induced relaxation is suppressed in settings in which 20-HETE levels are increased. This conclusion is in keeping with reports that 20-HETE minimizes the vasodilator effect of acetylcholine in cremasteric arterioles.19
Because the NO donor, NONOate, was equally effective in relaxing renal interlobar arteries from rats transduced with GFP and CYP4A2, neither a defect in NO signaling nor a generalized defect in responsiveness to relaxing stimuli can account for the attenuated relaxing effect of the muscarinic agonist in vessels from Adv-4A2–transduced rats. Rather, the impaired responsiveness to acetylcholine in such vessels may be ascribed to decreased availability of NO to molecular targets in vascular smooth muscle. In keeping with this interpretation, we found that the urinary excretion of nitrates/nitrites and the vascular production of NO ex vivo are depressed in rats treated with Adv-4A2 relative to the control Adv-GFP–treated rats. Furthermore, both basal and carbachol-stimulated cGMP levels in arteries of Adv-4A2–treated rats were exceeded by corresponding values in arteries of rats treated with Adv-GFP.
A decrease in bioactive NO within the endothelium may arise from either an inactivation or uncoupling of the eNOS46 or accelerated degradation of bioactive NO as a result of rapid activation of oxidant generation which, in turn, scavenge NO.47 It has been shown that microsomes enriched with CYP enzymes produce large amounts of superoxide anion.48 For example, the endothelial CYP2C9 is a functionally significant source of reactive oxygen species in coronary arteries.49 Whether CYP4A possesses the ability to produce superoxide is yet to be determined, as is the possibility that 20-HETE directly affects superoxide-generating systems. In all, it is possible that increased production of O2– brought about by increased CYP4A expression contributes to the decreased NO availability and, consequently, endothelial dysfunction in arteries overexpressing CYP4A2 and overproducing 20-HETE. That increased oxidative stress may contribute to increased endothelial dysfunction is further substantiated by observations of increased immunoreactive nitrotyrosine, a marker of peroxynitrate-induced oxidative stress, and gp91phox protein, a major component of the NADPH oxidase in endothelial cells50 and a determinant of endothelial dysfunction in hypertension,51 in arteries of rats transduced with Adv-4A2. However, although eNOS immunoreactivity was not different between arteries from Adv-4A2 and Adv-GFP rats, we cannot exclude, at this point, the possibility that eNOS inactivation or uncoupling occurred, leading to decreased NO production and that such uncoupling also contributed to the increase in O2– levels.46 To this end, preliminary experiments indicated that addition of 20-HETE (5 nmol/L) to cultured bovine aortic endothelial cells reduced ionophore-stimulated NO production by 50%. Moreover, experiments in collaboration with Dr K. Pritchard (Medical College of Wisconsin), showed that addition of 20-HETE (5 nmol/L) had no effect on eNOS phosphorylation after addition of the calcium ionophore A23187 in these cells; however, 20-HETE markedly inhibited HSP90 association with eNOS (Pritchard K.A. and Laniado-Schwartzman M., unpublished data, 2005). Association of HSP90 with eNOS is critical for eNOS activation52 and removal of this association by 20-HETE may underlie the mechanism, at least in part, by which increased CYP4A expression and activity cause endothelial dysfunction.
In summary, the present study demonstrates that rats injected with adenoviral vectors carrying CYP4A constructs feature increased vascular production of 20-HETE associated with elevation of blood pressure, impaired expression of the NO-dependent component of the vasorelaxing action of acetylcholine, and elevated vascular production of superoxide anion. Based on this study and as depicted in Figure 7, we reason that upregulation of vascular 20-HETE production brings about development of hypertension along with endothelium dysfunction in a mechanism that may include interference with eNOS activation, reduction in NO bioavailability, and amplification of O2–-producing systems including the NADPH (gp91phox) oxidase pathway. Hence, the increase in blood pressure and the endothelium dysfunction may be a manifestation of oxidative stress. The significance of these findings is further underscored by reports documenting association between elevated urinary excretion of 20-HETE, endothelial dysfunction, and oxidative stress in hypertensive subjects.53,54
Acknowledgments
This study was supported by National Institutes of Health grants HL34300, HL50142, and HL31069.
Footnotes
Both authors contributed equally to this study.
Original received May 25, 2005; resubmission received January 31, 2006; revised resubmission received February 24, 2006; accepted March 2, 2006.
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the Departments of Pharmacology and Physiology, New York Medical College, Valhalla, New York.
Abstract
Vascular cytochrome P450 (CYP) 4A enzymes catalyze the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE), an eicosanoid which participates in the regulation of vascular tone by sensitizing the smooth muscle cells to constrictor and myogenic stimuli. This study was undertaken to investigate the consequences of CYP4A overexpression on blood pressure and endothelial function in rats treated with adenoviral vectors carrying the CYP4A2 construct. Intravenous injection of Adv-CYP4A2 increased blood pressure (from 114±1 to 133±1 mm Hg, P<0.001), and interlobar renal arteries from these rats displayed decreased relaxing responsiveness to acetylcholine, which was offset by treatment with an inhibitor of CYP4A. Relative to data in control rats, arteries from Adv-CYP4A2–transduced rats produced more 20-HETE (129±10 versus 97±7 pmol/mg protein, P<0.01) and less nitric oxide (NO; 4.2±1.6 versus 8.4±1 nmol nitrite+nitrate/mg; P<0.05). They also displayed higher levels of oxidative stress as measured by increased generation of superoxide anion and increased expression of nitrotyrosine and gp91phox. Collectively, these findings demonstrate that augmentation in vascular 20-HETE promotes the development of hypertension and causes endothelial dysfunction, a condition characterized by decreased NO synthesis and/or bioavailability, imbalance in the relative contribution of endothelium-derived relaxing and contracting factors, and enhanced endothelial activation.
Key Words: arachidonic acid 20-HETE nitric oxide eNOS superoxide anion
Introduction
The endothelium plays a critical role in the short- and long-term regulation of the cardiovascular system. It serves as a protective barrier between tissues and circulating blood and functions to maintain vascular homeostasis by releasing bioactive factors in response to hemodynamic changes and blood borne signals. Such endothelial cell functions are impaired in many disease processes, including, diabetes, atherosclerosis, and hypertension.
The expression of the cytochrome P450 (CYP) 4A and its catalytic activity as arachidonic acid -hydroxylase which produces 20-hydroxyeicosatetraenoic acid (20-HETE) has been linked to the regulation of vascular reactivity and tone and to the development of hypertension. This notion is substantiated by observations that 20-HETE promotes vasoconstriction,1,2 that CYP4A expression and/or 20-HETE production is increased in vascular tissues of hypertensive animals,3 and that interventions that decrease CYP4A expression and/or activity cause blood pressure to fall in animal models of hypertension.4–11
The vasoconstrictor action of 20-HETE is primarily attributed to inhibition of the large conductance Ca2+-activated K+ channel (KCa) in vascular smooth muscle cells leading to depolarization and elevation of cystolic [Ca2+].12 This inhibitory action of 20-HETE is believed to enhance constrictor responsiveness of arterial vessels to increased pressure,13,14 oxygen,15,16 phenylephrine,2 and endothelin17 as well as inhibition of NO synthesis.18 Although the regulatory action of 20-HETE on the vascular smooth muscle function is well documented, little is known about the regulatory actions on endothelial function. A study by Frisbee et al19 alluded to the possibility that 20-HETE affects vascular reactivity by promoting endothelial dysfunction. Their study demonstrated that 20-HETE attenuates acetylcholine-induced dilation in cremasteric arterioles of normotensive rats on a low salt diet and that inhibition of 20-HETE synthesis enhances arteriolar dilation to acetylcholine in hypertensive rats on a high salt diet. More recently, 20-HETE has been shown to functionally antagonize EDHF-mediated relaxation in small porcine coronary arteries.20
In the present study, we examined the consequences of overexpressing CYP4A in the vasculature on blood pressure and endothelial function in rats. We demonstrated that intravenous injection of Adv-CYP4A2 caused hypertension and that renal arteries from these rats featured increased vascular CYP4A expression and 20-HETE production, and displayed endothelial dysfunction exemplified by reduced acetylcholine-induced relaxations, reduced levels of NO, and increased levels of superoxide anion. These findings indicate that vascular overexpression of CYP4A fosters prohypertensive mechanisms and that vascular 20-HETE may be a determinant of endothelial dysfunction and activation.
Materials and Methods
Construction of Recombinant Adenoviruses
CYP4A2 cDNA (1720 bp), originally isolated from the Lewis-Wistar rat kidney,21 was subcloned into the adenovirus vector pAd5CMV-NpA.22 The pAd5CMV-NpA-CYP4A2 (Adv-4A2) was propagated, purified, and titrated to 1x1012 pfu/mL by the Gene Transfer Vector Core, University of Iowa, as described.22 Recombinant adenovirus containing the coding region of the enhanced green fluorescent protein (GFP) (pAd5CMV-NpA-eGFP) was constructed in the same manner, designated as Adv-GFP and used as the control adenovirus.
In Vitro Transduction
COS-1 and EA.hy926 cells23 were used to evaluate functional transgene expression. Cells were infected with the adenoviruses; GFP and CYP4A2 protein levels were measured by Western blot and CYP catalytic activity was evaluated by either measuring -hydroxylation of the CYP4A-preferred substrate, lauric acid,24 or by quantifying endogenous 20-HETE levels.25
Animal Experimentation
All experimental protocols were performed following an IACUC approved protocol in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Adenoviruses were injected into the tail vein (200 μL of 1012 pfu/mL in saline) of Sprague-Dawley rats (9- to 10-week-old, 250 to 275 g body weight) under anesthesia with sodium pentobarbital (65 mg/kg body weight, ip). At day 6 after injection, rats were placed in metabolic cages for 24 hour urine collection. Urinary electrolytes were measured by flame photometry. Blood pressure was measured by the tail cuff method before and 7 days after adenovirus administration. At day 7, rats were euthanized and arterial vessels including renal interlobar arteries were dissected as previously described.26
Immunohistochemistry and Western Blotting
Arteries were embedded in OCT (Sakura) and sectioned at 5 μm thick. Sections were processed for immunohistochemistry using Vectastain ABC elite kit (Vector Laboratories, Inc), stained with Vector-DAB, and counterstained with hematoxylin. Western blotting was performed on homogenized arterial segments using mouse anti-EGFP monoclonal antibody and goat anti-rat CYP4A1 polyclonal antibody, which cross-reacts with all CYP4A isoforms, ie, CYP4A2, CYP4A3, and CYP4A8.24
Measurements of HETE and Epoxyeicosatrienoic Acid Levels
Renal interlobar arteries (4 segments per tube) were placed in Krebs bicarbonate buffer (pH 7.4) for 1 hour at 37°C with gentle shaking. At the end of the incubation, [2H2]-20-HETE (0.5 ng) and a mixture of [2H8]-epoxyeicosatrienoic acid (EETs) (3 ng) were added as internal standards. HETEs and EETs were extracted, separated by HPLC, derivatized, and quantified by gas chromatography/mass spectrometry as described previously.26
Agonist-Induced Vasorelaxation
Renal interlobar arteries were dissected and mounted on wires in myograph chambers (J.P. Trading) for measurement of isometric tension as described.27 Vasorelaxation responses of phenylephrine-constricted arteries to cumulative increments in the acetylcholine (10–9 to 10–4 mol/L) or DETA-NONOate (10–9 to 10–5 mol/L) concentrations were examined in the presence of indomethacin (10 μmol/L) with and without L-NAME (1 mmol/L). The response to acetylcholine was also assessed in arteries exposed to the CYP4A inhibitor DDMS (30 μmol/L) in the absence and presence of 20-HETE (0.1 to 10 μmol/L).
Measurement of NO and cGMP
NOx concentration in urine was measured by the Griess reaction. NO production in blood vessels was measured according to the method established by Zeballos et al.28 Production of cGMP in the presence IBMX (0.5 mmol/L) and L-arginine (1 mmol/L) with and without carbachol (100 μmol/L) was measured as described by Pucci et al.29
Measurement of O2·–
Arterial segments were placed in scintillation vials (2 per vial) containing 1 mL of Krebs-HEPES buffer, pH 7.4, and lucigenin (5 μmol/L). Superoxide anion levels were measured by lucigenin chemiluminescence method as previously described.30
Statistical Analysis
Data are expressed as mean±SE. Concentration-response data derived from each vessel were fitted separately to a logistic function by non-linear regression. Maximum asymptote of the curve (Rmax) and the concentration of agonist producing 50% of the maximal response (EC50) were calculated using commercially available software (Prism 2.01, GraphPAD software). Concentration-response data were analyzed by a two-way analysis of variance followed by a Duncan multiple range test. Other data were analyzed by a Student t test for paired or unpaired observations as appropriate. The null hypothesis was rejected at P<0.05.
Results
Functional Expression of CYP4A2 and GFP Adenoviruses In Vitro
Functional transgene expression of the recombinant adenoviruses was evaluated in vitro in COS-1 cells (please see the online data supplement, available at http://circres.ahajournals.org) and in EA.hy926 cells. Cells infected with Adv-GFP displayed GFP immunoreactivity and basal levels of CYP4A immunoreactivity; in contrast, cells infected with Adv-4A2 showed a strong CYP4A immunoreactivity (Figure 1A). Moreover, CYP4A catalytic activity measured as 20-HETE production was only present in Adv-4A2–infected EA.hy926 cells (Figure 1B).
CYP4A2 Gene Transfer Increases Blood Pressure
Recombinant adenoviruses (Adv-4A2, Adv-GFP) were administered intravenously to 9-week-old rats. Systolic blood pressure increased from 114.4±4.3 to 133.1±4.4 mm Hg one week after injection of Adv-4A2 (n=10; P<0.001) but was not affected by Adv-GFP injection (114.8±3.6 and 116.7±4.0 mm Hg before and after Adv-GFP administration, respectively; n=10). Urinary potassium excretion showed no change (from 3.99±0.52 to 4.47±0.54 meq per 24 hours; n=8), whereas urinary excretion of sodium significantly increased 7 days after injection of Adv-4A2 (from 1.85±0.21 versus 2.63±0.33 meq per 24 hour; n=8; P<0.01). Neither urinary potassium excretion (3.48±0.45 versus 4.11±0.24 meq per 24 hours; n=8) nor sodium (1.92±0.26 versus 2.31±0.17 meq per 24 hours; n=8) were changed in rats injected with the Adv-GFP.
Intravenous Administration of Recombinant Adenoviruses Transduces the Vasculature
Transgene expression carried by the adenoviral vectors used in this study was not tissue-selective and followed a pattern previously shown for the adenoviral-mediated HO-1 gene transfer.31 Nevertheless, with systemic administration, the vasculature was sufficiently transduced with the transgenes (online data supplement). Immunohistochemical analysis of interlobar arteries taken from rats injected with the Adv-GFP indicated a positive CYP4A staining (Figure 2A and 2B). The intensity of CYP4A staining was greatly enhanced in arteries taken from Adv-4A2–treated rats and appeared to localize to the intima and the media (Figure 2C and 2D). Western blot analysis verified GFP immunoreactivity only in vessels from rats injected with Adv-GFP (Figure 3A) and increased CYP4A immunoreactivity in Adv-4A2–infected arteries as compared with arteries infected with the Adv-GFP (Figure 3B and 3C). Importantly, as seen in Figure 3D, interlobar arteries from rats transduced with Adv-4A2 produced more 20-HETE than arteries from Adv-GFP transduced rats. The levels of 19-HETE, another CYP4A-derived arachidonate metabolite,24 also increased (P<0.05) from 20.9±3.4 pmol/mg in Adv-GFP-infected arteries to 39.1±3.7 pmol/mg in Adv-4A2 infected arteries (mean±SE, n=4). EET production in interlobar arteries appeared (P=0.066) to increase (from 39.03±15.60 to 111.35±53.21 pmol/mg in Adv-GFP and Adv-4A2, respectively; n=7; P<0.05), possibly reflecting the capacity of CYP4A2 to catalyze arachidonate epoxidation in addition to -hydroxylation.21
Adv-4A2 Transduced Rats Display Impaired Vasorelaxing Responsiveness to Acetylcholine
Relaxing responses to acetylcholine (10–9 to 10–4 mol/L) or NONOate (10–9 to 5x10–5 mol/L) were examined in renal interlobar arteries from Adv-4A2– and Adv-GFP–transduced rats. Arteries were relaxed in a concentration-dependent manner by both agonists. At the maximally effective concentration, the response to acetylcholine in arteries from Adv-4A2–transduced rats was significantly lower (54±4% relaxation) than in arteries from Adv-GFP-transduced rats (87±2% relaxation) (Figure 4A). After addition of L-NAME, the residual (NO-independent) relaxing effect of acetylcholine was similar in arteries of rats transduced with Adv-4A2 (30±5% relaxation) or Adv-GFP (39±5% relaxation) (Figure 4A, lower panel). DDMS (30 μmol/L), a CYP4A inhibitor, enhanced (P<0.05) acetylcholine-induced relaxation in arteries of rats transduced with Adv-4A2 (from 54±±4 to 74±8% relaxation) but not with Adv-GFP (from 87±2 to 92±4% relaxation). 20-HETE (10 μmol/L) reduced (P<0.02) the response to acetylcholine in DDMS-treated arteries of rats transduced with Adv-4A2 (from 74±8 to 47±2% relaxation) or Adv-GFP (from 92±4 to 47±9% relaxation) (Figure 4B). Relaxing responses to DETA-NONOate were comparable in arteries of rats transduced with Adv-4A2 or Adv-GFP (online data supplement). These data suggest that vascular overexpression of CYP4A2 attenuates acetylcholine-induced dilation of renal interlobar arteries, presumably via a mechanism involving interference by 20-HETE with the NO-dependent component of the vasorelaxing action of acetylcholine. Pertinently, urinary NOX measured by the Griess reaction was significantly lower in rats 7 days after transduction with Adv-4A2 (13.76±1.32 versus 8.00±2.66μmol per 24 hours; n=5; P<0.05), whereas urinary NOX was not significantly altered in rats transduced with Adv-GFP (11.86±2.29 versus 8.62±1.29 μmol per 24 hours; n=5; P=0.12).
NO, cGMP, and O2– Production in Renal Arteries of Adv-4A2 Transduced Rats
The observation of a reduced NO-dependent acetylcholine-induced relaxation in arteries of rats transduced with Adv-4A2 suggested that NO production/bioavailability is impaired in those vessels. Accordingly, we contrasted rats treated with Adv-GFP and Adv-4A2 in terms of vascular production of NO, cGMP, and O2–. As seen in Figure 5A, renal arteries of rats transduced with Adv-4A2 produced significantly less NO than arteries of Adv-GFP transduced rats. Similarly, basal and carbachol-induced cGMP levels in Adv-4A2 treated rats were less than those in Adv-GFP rats (Figure 5B). In contrast, as seen in Figure 5C, production of O2– was significantly higher in renal arteries of rats transduced with Adv-4A2 as compared with arteries from Adv-GFP transduced rats.
These results further suggest a potential link between CYP4A expression, NO generation, and endothelial dysfunction. To this end, measurements of eNOS proteins in blood vessels taken from rats transduced with Adv-4A2 indicated no change in eNOS immunoreactivity (protein levels) as compared with rats transduced with Adv-GFP. As seen in Figure 6A and 6C, eNOS protein levels in aorta and renal arteries from Adv-4A2 and Adv-GFP transduced rats were similar, suggesting that the reduced NO-dependent vasorelaxation in Adv-4A2–transduced rats was not associated with downregulation of eNOS expression. On the other hand, Western blot of aortic homogenates and densitometry analysis revealed a significant increase in immunoreactive nitrotyrosine, a marker of peroxynitrite-induced oxidative stress in Adv-4A2 rats (Figure 6B and 6C). Western blot and densitometry analysis of gp91phox, a major component of the NADPH oxidase in endothelial cells, showed increased levels in aortic tissue of Adv-4A2 rats (Figure 6B and 6C). As for the renal arteries, aortas from rats injected with Adv-4A2 displayed reduced acetylcholine-induced relaxations as compared with aortas from Adv-GFP-injected rats (viz., EC50s to acetylcholine were 1.75±0.92 and 7.05±2.39 μmol/L for Adv-GFP and Adv-4A2 infected aortas, respectively; mean±SE; n=5; P<0.05).
Discussion
The primary finding of this study is that treatment of rats with adenovirus carrying the rat CYP4A2 full-length cDNA promotes augmentation in blood pressure accompanied by impairment of acetylcholine-induced relaxation of renal interlobar arteries. These animals also display increased vascular production of 20-HETE, the major product of arachidonic acid metabolism by the CYP4A -hydroxylases. Accordingly, the development of hypertension and impaired vasorelaxing responsiveness to acetylcholine in rats transduced with adenoviral vectors carrying the CYP4A cDNA may be the functional manifestation of increased vascular synthesis of 20-HETE.
Increased 20-HETE production in renal interlobar arteries of rats treated with recombinant CYP4A adenoviruses is in line with observations that COS-1 and EA.hy 926 cells infected with Adv-4A2 manufacture catalytically active CYP4A2. It is also consistent with the finding of increased CYP4A immunoreactivity in arterial vessels from rats treated with Adv-4A2. Notably, immunohistochemistry of arterial vessels of rats treated with Adv-4A2 suggested that the intima is a primary site of expression of the transgene. By extension, the intima may be expected to be the major site of vascular 20-HETE overproduction in rats treated with Adv-4A2. Pertinent to this point, recent reports have identified the intima and endothelial cells as a major site of CYP4A protein expression and a target for 20-HETE bioactions,32–36 questioning the early notion that the vascular CYP4A-20-HETE pathway of arachidonic acid metabolism is limited to the smooth muscle.12,37 To this end, our immunohistochemical analysis of CYP4A in arterial vessels taken from Adv-GFP rats indicated the presence of CYP4A positive signals not only in the media but also in the intima where CYP4A immunopositivity was greatly increased after Adv-4A2 administration. Certainly, extensive studies are needed to substantiate the possibility of constitutive CYP4A expression in the endothelium of vascular beds other than the pulmonary circulation.
20-HETE sensitizes the smooth muscle of resistance vessels to myogenic or hormonal constrictor stimuli.13,14,16,17,38 This action of 20-HETE has been attributed to its ability to inhibit Ca2+-activated K+ channels,12 increase the conductance of Ca2+ channels,39 promote the activation of Rho-kinase,40 and/or inhibit vascular Na+-K+-ATPase activity.20 Because of reports that interventions that increase and decrease vascular production of 20-HETE elicit vasoconstriction and vasodilation, respectively, 20-HETE of vascular origin has gained recognition as a major contributor to the implementation of vasoconstrictor mechanisms in physiological and pathophysiological settings.37 In like manner, reports that interventions that decrease 20-HETE synthesis bring about lowering of blood pressure in experimental models of hypertension have given rise to the concept that vascular 20-HETE subserves a prohypertensive function.37 Two recent reports further strengthened this concept and substantiated the link between CYP4A, 20-HETE, and hypertension: Holla et al41 demonstrated that in Cyp4a14 knockout mice, hypertension is more severe in the male and is associated with increased Cyp4a12 expression (the mouse homologue of CYP4A8) and 20-HETE synthesis; Nakagawa et al3 showed that rats treated with 5a-dihydrotestosterone displayed increased vascular CYP4A8 expression and 20-HETE synthesis that may contribute to the hypertension seen in these rats. Our present study provides critical support to this concept by documenting that vascular transduction of CYP4A in rats injected with Adv-4A1 or Adv-4A2 is accompanied by increased vascular 20-HETE production along with sustained elevation of blood pressure. In line with the preceding discussion, the development of hypertension in rats so treated may be caused by 20-HETE–induced amplification of the vasoconstrictor influence of various neurohormonal systems, eg, the sympathetic, endothelin, and renin-angiotensin systems. Equally possible, as judged by the results of the present study, the development of hypertension in rats transduced with CYP4A oxygenases may be caused by 20-HETE–induced interference with endothelial functions that foster vasodilation.
The endothelium is a rich source of vascular smooth muscle relaxing mediators, viz., prostacyclin, NO, and EETs, which implement vasodilator responses to agonists and shear stress. Rat renal interlobar arteries are relaxed by muscarinic agonists such as acetylcholine, the response of which is endothelium-dependent and involves both NO-dependent and -independent mechanisms. The vasorelaxing response to acetylcholine is impaired in hypertension and other cardiovascular diseases, and the impairment is taken as an indication of endothelium dysfunction. Studies in different animal models of arterial hypertension42 and in patients with both essential and secondary hypertension43 have demonstrated an association between elevated systemic blood pressure and impaired endothelium-dependent vascular relaxation. Moreover, a diminished bioavailability of NO has been identified as a mechanism responsible for endothelial dysfunction in hypertensive patients.43 A most striking finding in the present study is that acetylcholine-induced relaxation of renal interlobar arteries is greatly attenuated in Adv-4A2–transduced rats. Importantly, when the assessment of vasorelaxing responsiveness was conducted in the presence of L-NAME to inhibit NO synthase (NOS) and thus eliminate the NO-dependent component of the acetylcholine response, the muscarinic agonist was equally effective in relaxing renal interlobar arteries taken from rats injected with Adv-GFP (control) or Adv-4A2. This implies that only the NO-dependent component of acetylcholine-induced vasorelaxation is impaired in vessels taken from Adv-4A2–transduced rats. The NO-independent component in renal arterioles, which was unaltered by Adv-4A2 administration, has been associated with EETs.44 Indeed, the levels of EETs in renal arteries of Adv-4A2–treated rats were not significantly different than those in renal arteries of Adv-GFP–treated rats and, in fact, appeared to increase, likely reflecting the capacity of CYP4A2 to catalyze arachidonate epoxidation.21
According to the present study, the impairment in acetylcholine-induced relaxation displayed by renal arteries of Adv-4A2–transduced rats was nearly offset by ex vivo treatment of vessels treated with DDMS, a known inhibitor of 20-HETE synthesis.45 Because the application of exogenous 20-HETE reinstated impaired vasorelaxing responsiveness to acetylcholine in DDMS-treated vessels of Adv-4A2–transduced rats and attenuated the responsiveness to the muscarinic agonist in vessels of Adv-GFP–transduced rats, it may be concluded that the NO-dependent component of acetylcholine-induced relaxation is suppressed in settings in which 20-HETE levels are increased. This conclusion is in keeping with reports that 20-HETE minimizes the vasodilator effect of acetylcholine in cremasteric arterioles.19
Because the NO donor, NONOate, was equally effective in relaxing renal interlobar arteries from rats transduced with GFP and CYP4A2, neither a defect in NO signaling nor a generalized defect in responsiveness to relaxing stimuli can account for the attenuated relaxing effect of the muscarinic agonist in vessels from Adv-4A2–transduced rats. Rather, the impaired responsiveness to acetylcholine in such vessels may be ascribed to decreased availability of NO to molecular targets in vascular smooth muscle. In keeping with this interpretation, we found that the urinary excretion of nitrates/nitrites and the vascular production of NO ex vivo are depressed in rats treated with Adv-4A2 relative to the control Adv-GFP–treated rats. Furthermore, both basal and carbachol-stimulated cGMP levels in arteries of Adv-4A2–treated rats were exceeded by corresponding values in arteries of rats treated with Adv-GFP.
A decrease in bioactive NO within the endothelium may arise from either an inactivation or uncoupling of the eNOS46 or accelerated degradation of bioactive NO as a result of rapid activation of oxidant generation which, in turn, scavenge NO.47 It has been shown that microsomes enriched with CYP enzymes produce large amounts of superoxide anion.48 For example, the endothelial CYP2C9 is a functionally significant source of reactive oxygen species in coronary arteries.49 Whether CYP4A possesses the ability to produce superoxide is yet to be determined, as is the possibility that 20-HETE directly affects superoxide-generating systems. In all, it is possible that increased production of O2– brought about by increased CYP4A expression contributes to the decreased NO availability and, consequently, endothelial dysfunction in arteries overexpressing CYP4A2 and overproducing 20-HETE. That increased oxidative stress may contribute to increased endothelial dysfunction is further substantiated by observations of increased immunoreactive nitrotyrosine, a marker of peroxynitrate-induced oxidative stress, and gp91phox protein, a major component of the NADPH oxidase in endothelial cells50 and a determinant of endothelial dysfunction in hypertension,51 in arteries of rats transduced with Adv-4A2. However, although eNOS immunoreactivity was not different between arteries from Adv-4A2 and Adv-GFP rats, we cannot exclude, at this point, the possibility that eNOS inactivation or uncoupling occurred, leading to decreased NO production and that such uncoupling also contributed to the increase in O2– levels.46 To this end, preliminary experiments indicated that addition of 20-HETE (5 nmol/L) to cultured bovine aortic endothelial cells reduced ionophore-stimulated NO production by 50%. Moreover, experiments in collaboration with Dr K. Pritchard (Medical College of Wisconsin), showed that addition of 20-HETE (5 nmol/L) had no effect on eNOS phosphorylation after addition of the calcium ionophore A23187 in these cells; however, 20-HETE markedly inhibited HSP90 association with eNOS (Pritchard K.A. and Laniado-Schwartzman M., unpublished data, 2005). Association of HSP90 with eNOS is critical for eNOS activation52 and removal of this association by 20-HETE may underlie the mechanism, at least in part, by which increased CYP4A expression and activity cause endothelial dysfunction.
In summary, the present study demonstrates that rats injected with adenoviral vectors carrying CYP4A constructs feature increased vascular production of 20-HETE associated with elevation of blood pressure, impaired expression of the NO-dependent component of the vasorelaxing action of acetylcholine, and elevated vascular production of superoxide anion. Based on this study and as depicted in Figure 7, we reason that upregulation of vascular 20-HETE production brings about development of hypertension along with endothelium dysfunction in a mechanism that may include interference with eNOS activation, reduction in NO bioavailability, and amplification of O2–-producing systems including the NADPH (gp91phox) oxidase pathway. Hence, the increase in blood pressure and the endothelium dysfunction may be a manifestation of oxidative stress. The significance of these findings is further underscored by reports documenting association between elevated urinary excretion of 20-HETE, endothelial dysfunction, and oxidative stress in hypertensive subjects.53,54
Acknowledgments
This study was supported by National Institutes of Health grants HL34300, HL50142, and HL31069.
Footnotes
Both authors contributed equally to this study.
Original received May 25, 2005; resubmission received January 31, 2006; revised resubmission received February 24, 2006; accepted March 2, 2006.
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