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the Department of Cardiovascular Medicine (J.K.B., N.J.A., N.W., S.C., D.A., K.M.C.), University of Oxford, John Radcliffe Hospital, United Kingdom
Childhood Infection Group (K.R.), Wellcome Trust Centre for Human Genetics, University of Oxford, United Kingdom
Kobe University School of Medicine (M.Y., S.K.), Japan.
Abstract
Endothelial dysfunction in vascular disease states is associated with reduced NO bioactivity and increased superoxide (O2·eC) production. Some data suggest that an important mechanism underlying endothelial dysfunction is endothelial NO synthase (eNOS) uncoupling, whereby eNOS generates O2·eC rather than NO, possibly because of a mismatch between eNOS protein and its cofactor tetrahydrobiopterin (BH4). However, the mechanistic relationship between BH4 availability and eNOS coupling in vivo remains undefined because no studies have investigated the regulation of eNOS by BH4 in the absence of vascular disease states that cause pathological oxidative stress through multiple mechanisms. We investigated the stoichiometry of BH4eCeNOS interactions in vivo by crossing endothelial-targeted eNOS transgenic (eNOS-Tg) mice with mice overexpressing endothelial GTP cyclohydrolase 1 (GCH-Tg), the rate-limiting enzyme in BH4 synthesis. eNOS protein was increased 8-fold in eNOS-Tg and eNOS/GCH-Tg mice compared with wild type. The ratio of eNOS dimer:monomer was significantly reduced in aortas from eNOS-Tg mice compared with wild-type mice but restored to normal in eNOS/GCH-Tg mice. NO synthesis was elevated by 2-fold in GCH-Tg and eNOS-Tg mice but by 4-fold in eNOS/GCH-Tg mice compared with wild type. Aortic BH4 levels were elevated in GCH-Tg and maintained in eNOS/GCH-Tg mice but depleted in eNOS-Tg mice compared with wild type. Aortic and cardiac O2·eC production was significantly increased in eNOS-Tg mice compared with wild type but was normalized after NOS inhibition with N-nitro-L-arginine methyl ester hydrochloride (L-NAME), suggesting O2·eC production by uncoupled eNOS. In contrast, in eNOS/GCH-Tg mice, O2·eC production was similar to wild type, and L-NAME had no effect, indicating preserved eNOS coupling. These data indicate that eNOS coupling is directly related to eNOSeCBH4 stoichiometry even in the absence of a vascular disease state. Endothelial BH4 availability is a pivotal regulator of eNOS activity and enzymatic coupling in vivo.
Key Words: endothelial nitric oxide synthase tetrahydrobiopterin nitric oxide superoxide
Introduction
Nitric oxide (NO), produced by endothelial NO synthase (eNOS) in the vascular endothelium, is a critical signaling molecule in vascular homeostasis.1 NO serves as an endothelium-derived relaxing factor, regulates vasomotor tone and blood pressure,1,2 and has multiple antiatherogenic roles by inhibiting vascular smooth muscle cell proliferation, platelet aggregation, and leukocyte adhesion.1 Loss of NO bioavailability is a key feature of endothelial dysfunction in vascular disease states such as hypertension, diabetes, and atherosclerosis. Furthermore, impaired NO-mediated endothelial function is an independent risk factor for cardiovascular disease.3eC5 Several factors contribute to loss of NO bioavailability, including reduced NO synthesis and NO scavenging by reactive oxygen species (ROS).6 Under physiological conditions, there is a balance between endothelial NO and ROS production. However, vascular diseases are associated with increased ROS generation.6 Several oxidase systems contribute to the increased oxidative stress, notably the NADPH oxidases.7,8
Increasing evidence suggests that eNOS itself can generate superoxide (O2·eC) under certain pathophysiological conditions.9 Ozaki et al10 reported recently that transgenic overexpression of eNOS in apolipoprotein E knockout mice paradoxically increases vascular O2·eC production because of enzymatic uncoupling of increased eNOS protein levels. Recent data indicate that the pterin cofactor tetrahydrobiopterin (BH4) is a major determinant of whether eNOS produces NO or O2·eC.11,12 When BH4 levels are insufficient, there is a shift toward the production of O2·eC as electron transfer within the active site of eNOS becomes uncoupled from L-arginine oxidation, and molecular oxygen is instead reduced to form O2·eC.11 O2·eC generated by eNOS has been implicated in endothelial dysfunction associated with a number of vascular disease states, including diabetes, smoking, hypertension, and atherosclerosis,10,12eC16 and BH4 supplementation improves endothelium-dependent vasodilatation under these conditions.16 However, the effects of systemic pharmacological BH4 supplementation in these studies may be mediated in part by nonspecific antioxidant properties of acute high-dose BH4,17 which can increase NO bioavailability indirectly by reducing its scavenging by ROS.
Recent studies have focused on the potential role of BH4 oxidation, to dihydrobiopterin (BH2) and other biopterin species, in reducing BH4 bioavailability in preatherosclerotic disease states.16eC18 In particular, the interaction of BH4 with peroxynitrite (generated from the reaction between NO and O2·eC) rapidly oxidizes BH4 and can provoke eNOS uncoupling and endothelial dysfunction.12,19eC21 Indeed, eNOS uncoupling may exacerbate the process by contributing to BH4 oxidation. However, it is unclear whether eNOS uncoupling alone is sufficient to initiate BH4 oxidation and exacerbate eNOS uncoupling in vivo because all in vivo studies to date have evaluated BH4-dependent eNOS regulation in complex vascular disease states in which multiple inflammatory and redox pathways are implicated. Other previous studies of the role of BH4 in eNOS function have relied on purified recombinant proteins in reconstituted cell-free systems.9,11,22,23
Accordingly, we sought to investigate the importance of BH4 in regulating eNOS activity in vivo in healthy animals without vascular disease. We used a transgenic mouse model with endothelial-targeted overexpression of GTP cyclohydrolase 1 (GTPCH), the rate-limiting enzyme in BH4 synthesis, in which endothelial BH4 levels are specifically increased.24 We crossed this transgenic mouse with a mouse overexpressing eNOS in the endothelium to generate mouse models with graded alterations in endothelial BH4 and eNOS levels to investigate the mechanistic relationships between BH4 and eNOS coupling in vivo.
Materials and Methods
Animals
All studies involving laboratory animals were conducted in accordance with the UK Home Office Animals (Scientific Procedures) Act 1986 (HMSO, UK). eNOS transgenic (eNOS-Tg) mice, in which bovine eNOS transgene overexpression is targeted to the vascular endothelium under the control of the murine preproendothelin-1 promoter in a C57BL/6 background, were produced as described previously.25 GTPCH transgenic (GCH-Tg) mice, in which human GTPCH transgene overexpression is targeted to the endothelium under control of the murine Tie-2 promoter, were generated in a C57BL/6 background as described previously.26 Heterozygote eNOS-Tg mice were mated with heterozygote GCH-Tg mice to produce experimental eNOS/GCH-Tg, eNOS-Tg, GCH-Tg, and wild-type littermates in a 1:1:1:1 ratio. Mice (between 13 and 20 weeks of age in all experiments) were housed in individually ventilated cages with 12-hour light/dark cycle and controlled temperature (20°C to 22°C) and fed normal chow and water ad libitum.
Western Blot Analysis
Lung samples (n4 per group) were homogenized on ice for 20 seconds in lysis buffer (50 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 0.1% SDS, 0.5% deoxycholate, 1% Nonidet P-40) containing protease inhibitors (Complete; Boehringer Mannheim) and 1 mmol/L phenylmethylsulfonyl fluoride. Protein lysates (8 e) were resolved using SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were incubated with a 1:2000 dilution of mouse anti-eNOS monoclonal antibody (Transduction Laboratories), which recognizes murine and bovine eNOS, followed by a 1:2500 dilution of rabbit anti-mouse horseradish peroxidaseeCconjugated secondary antibody (Promega). Protein bands were visualized by chemiluminescence. To investigate the ratio of eNOS homodimer to monomer, Western blots were performed as above using nonboiled aortic lysates and low-temperature SDS-PAGE as described previously.27
Primary Cultures of Murine Lung Endothelial Cells
Lungs were harvested into culture medium (35% DMEM, 35% Ham’s F-10 nutrient mixture, 20% FBS, 2 mmol/L L-glutamine, 100 U/100 e/mL penicillineCstreptomycin, 100 e/mL heparin, and 50 e/mL endothelial mitogen ), cut into 1- to 2-mm pieces and digested using 0.1% collagenase type I for 1 hour at 37°C. The lung digest was passed through a 100-e cell strainer. Cells were centrifuged, resuspended in culture medium, and plated onto 0.1% gelatin-coated cover slips. Cultures were maintained at 37°C in humidified 5% CO2/95% air atmosphere for 72 hours before fixation with 4% paraformaldehyde.
Immunocytochemistry
Fixed cultures were permeabilized with PBS containing 0.5% Triton X-100, and nonspecific staining was reduced by blocking with 10% normal goat serum. Cultures were incubated with a polyclonal rabbit anti-eNOS primary antibody (Transduction Laboratories) followed by goat anti-rabbit secondary antibody (Alexa Fluor 488; Molecular Probes). Cells were mounted with cover slips using Vectashield containing propidium iodide (Vector Laboratories) and imaged using a Bio-Rad MRC-1024 laser-scanning confocal microscope.
Measurement of Biopterins and Neopterin
Biopterins, such as BH4, BH2 and biopterin, and neopterin were measured in aortic homogenates by high-performance liquid chromatography (HPLC) analysis after iodine oxidation in acidic or alkaline conditions as described previously.24,28 In brief, thoracic aortas (n=6 to 8 per group) were homogenized for 20 seconds in ice-cold extract buffer (50 mmol/L Tris-HCl, pH 7.4, 1 mmol/L dithiothreitol, and 1 mmol/L EDTA) containing 0.1 eol/L neopterin as an internal recovery standard. Samples were deproteinated before undergoing oxidation with 1% iodine/2% potassium iodide under either acidic or basic conditions. Biopterin content was assessed using HPLC in 5% methanol/95% water using an ACE 5 C18 column (ACT) and fluorescence detection (350 nm excitation and 450 nm emission). BH4 concentration was calculated as picomoles per milligram of protein by subtracting BH2 and biopterin from total biopterin content.
Arginine-to-Citrulline Conversion
NOS enzymatic activity, and indirectly NO synthesis, was measured by the conversion of 14C L-arginine to 14C L-citrulline in fresh intact aorta (n=5 to 8 per group) and lung homogenate (n=6 per group) as described previously.24,29 The integrals of citrulline peaks were expressed as a proportion of total 14C counts for each sample.
Electron Paramagnetic Resonance Spectroscopy
Electron paramagnetic resonance (EPR) spectroscopy was used to quantify vascular NO production according to previously described and validated methods.30 In brief, freshly harvested aortas (n=8 to 11 per group) were stimulated with calcium ionophore (A23187; 1 eol/L) in 100 e蘈 KrebseCHEPES buffer, then incubated with colloid iron (II) diethyldithiocarbamate [Fe(DETC)2] (285 eol/L) at 37°C for 90 minutes. After incubation, aortas were snap-frozen in a column of KrebseCHEPES buffer in liquid nitrogen, and EPR spectra were obtained using an X-band EPR spectrometer (Miniscope MS 200; Magnettech). Signals were quantified by measuring the total amplitude, after correction of baseline, and after subtracting background signals from incubation with colloid Fe(DETC)2 alone.
Lucigenin-Enhanced Chemiluminescence Detection of Superoxide in Heart Lysates
Basal O2·eC production was measured in left ventricular (LV) homogenates (n=7 to 10 per group) using the technique of lucigenin (5 eol/L) chemiluminescence according to methods described previously.14,31 In brief, hearts were flushed with ice-cold KrebseCHEPES buffer, the LV excised, and snap-frozen in liquid nitrogen. Samples were homogenized in KrebeCHEPES buffer containing protease inhibitors (Complete; Boehringer Mannheim) at pH 7.4. Chemiluminescence was measured in a FB12 luminometer (Berthold Detection Systems) at 37°C. Chemiluminescence of 200 e LV protein was recorded every minute for 8 minutes. The NOS inhibitor N-nitro-L-arginine methyl ester hydrochloride (L-NAME; 1 mmol/L) was subsequently added and chemiluminescence recorded for an additional 5 minutes. Background readings were subtracted from sample readings and results expressed as counts per second.
Lucigenin-Enhanced Chemiluminescence Detection of Superoxide in Intact Aorta
Basal O2·eC production was measured in intact aorta (n=8 to 12 per group) according to methods described previously.14,32 In brief, freshly cleaned and harvested thoracic aortas were opened longitudinally, cut into 2, and transferred to ice-cold KrebseCHEPES buffer. Vessels were equilibrated in KrebseCHEPES buffer gassed with 95% oxygen/5% carbon dioxide for 30 minutes at 37°C, with one half of each vessel being incubated in the presence of L-NAME (1 mmol/L). Lucigenin (20 eol/L) chemiluminescence was then recorded every minute for 10 minutes as above. Background readings were subtracted from sample readings and results expressed as counts per second per milligram dry weight of aorta.
Oxidative Fluorescent Microtopography
O2·eC production in tissue sections of mouse aorta (n=5 to 7 per group) was detected using the fluorescent probe dihydroethidium (DHE), as described previously.14,24,33 Fresh segments of thoracic aorta were frozen in optimal cutting temperature compound. Cryosections (30 e) were incubated with KrebseCHEPES buffer with or without L-NAME (1 mmol/L; to inhibit eNOS) for 30 minutes at 37°C, then for an additional 5 minutes with DHE (2 eol/L; Molecular Probes). Images were obtained using a Bio-Rad laser-scanning confocal microscope, equipped with a krypton/argon laser, using identical acquisition settings for each section. DHE fluorescence was quantified by automated image analysis using Image-Pro Plus software (Media Cybernetics). DHE fluorescence from high power (x60) images was measured only on the luminal side of the internal elastic lamina to quantify endothelial cell fluorescence. For each vessel, mean fluorescence was calculated from 4 separate high-power fields taken in each quadrant of the vessel to produce n=1, and all experiments were performed in a batch design.
Statistical Analysis
One-way ANOVA tests were used to compare data sets, with appropriate post hoc correction for multiple comparisons. P<0.05 was considered significant. Data are expressed as means and SEM.
Results
eNOS Protein Levels and Subcellular Localization
Western blot analysis confirmed that eNOS protein levels were elevated 8-fold in eNOS-Tg compared with wild-type animals (P<0.001; Figure 1A). Overexpression of endothelial GTPCH, the rate-limiting enzyme in BH4 synthesis, in GCH-Tg mice did not significantly alter eNOS protein levels compared with wild type. However, as for eNOS-Tg mice, eNOS protein levels were elevated 8-fold in double-transgenic eNOS/GCH-Tg mice.
We used low-temperature SDS-PAGE and immunoblotting to investigate eNOS homodimerization and the ratio of eNOS dimer to monomer in aortas. In eNOS-Tg aortas, eNOS dimer:monomer was significantly depleted compared with wild type (P<0.05) but unchanged in GCH-Tg mice (Figure 1B). Importantly, the reduced eNOS dimer:monomer ratio in the eNOS-Tg group was restored to wild-type levels in double- transgenic eNOS/GCH-Tg mice.
We investigated the subcellular localization of eNOS in primary cultures of lung endothelial cells using immunocytochemistry. eNOS appeared to be localized mainly to plasma membranes and the Golgi apparatus in endothelial cells from all 4 groups (Figure 1C). However, in accordance with the immunoblotting data, the intensity of eNOS immunostaining, unchanged in GCH-Tg mice, was markedly increased in endothelial cells from eNOS-Tg and eNOS/GCH-Tg animals compared with wild type.
Aortic BH4 Levels
We next measured vascular BH4 levels in homogenates of snap-frozen aorta using iodine oxidation and HPLC. Surprisingly, BH4 levels were significantly depleted in eNOS-Tg mice compared with wild type, suggesting oxidative degradation of BH4 (P<0.05; Figure 2). We then sought to confirm that increased endothelial GTPCH expression led to increased BH4 levels in aortic homogenates of GCH-Tg and eNOS/GCH-Tg mice. As reported previously,24 aortic BH4 levels were significantly elevated by >2-fold in GCH-Tg mice compared with wild type (P<0.05). Importantly, aortic BH4 levels were also elevated in eNOS/GCH-Tg mice and were not significantly different between GCH-Tg and eNOS/GCH-Tg mice.
eNOS Enzymatic Activity and NO Production
To determine the relationship between eNOS protein levels and eNOS enzymatic activity, we measured conversion of 14C L-arginine to 14C L-citrulline by eNOS in intact aorta using HPLC with online scintillation detection. Citrulline production was increased only 2-fold in eNOS-Tg aortas compared with wild type (P<0.05; Figure 3A and 3B), despite eNOS protein levels being elevated 8-fold in these animals. Indeed, the ratio of eNOS enzymatic activity to eNOS protein was 0.6 in eNOS-Tg mice compared with 2.0 in wild-type animals. A similar pattern of results was obtained when using lung tissue lysates (Figure 3C). To further investigate the stoichiometric relationship between eNOS and endothelial BH4 in vivo and to determine whether increasing endothelial BH4 in eNOS-Tg mice could augment eNOS enzymatic activity, we next measured eNOS enzymatic activity in GCH-Tg and eNOS/GCH-Tg mice. NOS activity was increased 2-fold in GCH-Tg aorta and lung compared with wild type (P<0.05; Figure 3A through 3C). Indeed, eNOS enzymatic activity was similar in GCH-Tg and eNOS-Tg mice despite eNOS protein levels being considerably higher in eNOS-Tg animals. Critically, eNOS enzymatic activity was further elevated in eNOS/GCH-Tg mice compared with eNOS-Tg animals (P<0.05): augmented levels of endothelial BH4 in eNOS/GCH-Tg mice resulted in an &4-fold increase in eNOS enzymatic activity in aorta and lung compared with wild-type mice (P<0.01). These data suggest that eNOS activity is exquisitely dependent on endothelial BH4 levels even in the absence of vascular disease.
In complementary experiments, we used Fe-DETC EPR to directly measure NO bioavailability in mouse aortas. In accordance with measures of enzymatic activity, net NO levels were increased &2-fold in eNOS-Tg aortas compared with wild type (Figure 4). These results demonstrate that there was a striking discordance between eNOS protein levels, eNOS enzymatic activity, and NO production in eNOS-Tg mice. We then determined the effects of increased endothelial BH4 using the GCH-Tg and eNOS/GCH-Tg mice and observed a similar pattern of results as for NOS enzymatic activity. Aortic NO bioavailability was elevated almost 2-fold in GCH-Tg mice compared with wild type and not significantly different from eNOS-Tg mice (Figure 4). Critically, net NO bioavailability was further elevated (&3-fold compared with wild type) in eNOS/GCH-Tg mice.
eNOS Uncoupling: Effect of eNOS and GTPCH Overexpression In Vivo
To investigate whether eNOS uncoupling results from discordance between eNOS and BH4, we measured O2·eC production and, more specifically, eNOS-derived O2·eC production using the NOS inhibitor L-NAME. We first measured O2·eC production in tissue lysates using lucigenin chemiluminescence. Chemiluminescence was increased 2-fold in eNOS-Tg mice compared with wild-type animals (P<0.05; Figure 5A) but was unchanged in GCH-Tg mice. Critically, O2·eC production was restored in eNOS/GCH-Tg mice. The proportion of O2·eC production attributable to uncoupled NOS, assessed by quantifying L-NAMEeCinhibitable chemiluminescence, was significantly increased in eNOS-Tg lysates compared with wild type, indicating increased NOS uncoupling (P<0.05; Figure 5B). L-NAMEeCinhibitable chemiluminescence was unchanged in GCH-Tg mice. The presence of the GTPCH transgene in eNOS/GCH-Tg mice restored the enhanced L-NAMEeCinhibitable chemiluminescence of the eNOS-Tg group back to wild-type levels. We also investigated O2·eC production in intact aorta under basal conditions and after incubation with L-NAME using lucigenin chemiluminescence and saw a similar pattern of results. Basal chemiluminescence was significantly increased in eNOS-Tg aortas compared with wild type (P<0.05; Table). Importantly, basal O2·eC production in GCH-Tg and eNOS/GCH-Tg aortas was similar to wild type. Incubation of aortas with L-NAME caused a significant reduction in the O2·eC signal in eNOS-Tg mice (P<0.05), indicating NOS uncoupling. However, L-NAME had little effect in wild-type, GCH-Tg, and eNOS/GCH-Tg aortas, suggesting that NOS coupling is preserved in these mice. Together, these observations suggest that in eNOS-Tg mice elevated O2·eC production is at least partly attributable to uncoupled NOS, likely resulting from discordance between eNOS protein and endothelial BH4 because NOS coupling is preserved by increasing endothelial BH4 in association with elevated eNOS levels in eNOS/GCH-Tg animals.
To investigate O2·eC production specifically from the aortic endothelium, we quantified endothelial DHE fluorescence using oxidative confocal microtopography. Endothelial DHE fluorescence was increased 2-fold in eNOS-Tg mice compared with wild-type and GCH-Tg mice (Figure 6). Importantly, endothelial fluorescence was restored to wild-type levels in eNOS/GCH-Tg mice. Fluorescence from the other layers of the vessel wall was not significantly different between groups. Incubation with L-NAME had little effect in wild-type aortas but reversed the elevated DHE signal in eNOS-Tg endothelium back to wild-type levels, again indicating that the source of O2·eC was likely uncoupled eNOS. In contrast, L-NAME significantly increased the endothelial O2·eC signal in GCH-Tg mice, indicating that in these aortas, eNOS was predominantly coupled and producing NO. Critically, as in wild-type aortas, NOS inhibition with L-NAME had little effect in eNOS/GCH-Tg mice, indicating restored eNOS coupling compared with eNOS-Tg animals. In accordance with the data for O2·eC production measured by chemiluminescence, these results suggest that increased eNOS uncoupling in eNOS-Tg aortas increases eNOS-derived O2·eC, but that eNOS coupling is, at least in part, preserved by increased endothelial BH4 synthesis in eNOS/GCH-Tg mice.
Discussion
In this study, we describe a new double-transgenic mouse model in which endothelial-targeted overexpression of GTPCH leads to increased endothelial BH4 levels in mice with endothelial-targeted eNOS overexpression. We used this model to investigate the role of BH4 in the regulation of eNOS coupling in vivo, specifically in the absence of pathological oxidative stress associated with vascular disease states.6 The major findings in this study are as follows. First, eNOS protein levels are markedly elevated in eNOS-Tg and eNOS/GCH-Tg mice but not in GCH-Tg animals, although the proportion of eNOS dimer to monomer is depleted only in eNOS-Tg aortas. Second, endothelial-specific overexpression of GTPCH is sufficient to increase vascular BH4 levels in GCH-Tg and in eNOS/GCH-Tg aortas, whereas BH4 levels are depleted in eNOS-Tg aortas. Third, this increase in BH4 is sufficient to augment vascular eNOS enzymatic activity even in GCH-Tg mice, which have unchanged eNOS protein levels. Indeed, eNOS activity is similar between GCH-Tg and eNOS-Tg mice despite eNOS-Tg mice having 8-fold more eNOS protein. Importantly, the increase in endothelial BH4 in eNOS/GCH-Tg mice further enhances eNOS activity and NO bioavailability compared with eNOS-Tg mice. Fourth, the discordance between endothelial BH4 and eNOS protein in eNOS-Tg mice results in uncoupled eNOS and increased NOS-derived O2·eC production in tissue lysates and intact aorta. However, increased vascular BH4 in eNOS/GCH-Tg mice is sufficient, at least in part, to restore eNOS coupling, increase NO production, and reduce eNOS-dependent O2·eC production.
These findings provide important insights into the role of endothelial BH4 synthesis in regulating eNOS activity and eNOS coupling even in the absence of vascular oxidative stress. Previous studies have reported that endothelial dysfunction in vascular diseases, such as hypertension,12 diabetes,24 and atherosclerosis,26 is associated with increased O2·eC production deriving principally from NADPH oxidases.7,8 Landmesser et al12 demonstrated that the increase in NADPH oxidaseeCderived O2·eC in deoxycorticosterone acetateeCsalt hypertensive mice led to enhanced oxidation of BH4, resulting in eNOS uncoupling, increased eNOS-derived O2·eC production, and reduced NO formation, thereby exacerbating oxidative stress. Oral supplementation with BH4, or a reduction in NADPH oxidase activity (using p47phoxeC/eC mice), reversed eNOS uncoupling. However, the mechanistic relationship between eNOS and its cofactor BH4 has not been investigated in vivo in the absence of pathological oxidative stress. We now show that a stoichiometric discordance between eNOS protein and BH4 levels is alone sufficient to cause eNOS uncoupling, and that eNOS uncoupling in the absence of vascular disease is sufficient to deplete BH4 levels by oxidation. Laursen et al19 demonstrated that peroxynitrite may be the principal ROS involved in oxidation of BH4.
NO, constitutively produced by eNOS in the vascular endothelium, is a potent vasodilator and exerts numerous vasoprotective antiatherogenic effects. Reduced NO bioactivity is an early feature of a number of vascular diseases, including atherosclerosis.5 Short-term in vivo gene transfer of eNOS or neuronal NOS can improve NO-mediated vascular relaxation in atherosclerotic arteries.34 However, previous studies investigating the possible vasoprotective effects of chronic eNOS overexpression in eNOS-Tg mice have yielded conflicting results. Kawashima et al35 demonstrated reduced lesion formation after carotid artery ligation in eNOS-Tg mice. In contrast, Ozaki et al,10 using the same strain of eNOS-Tg mice as used in the present study, found that eNOS overexpression accelerated rather than reduced atherosclerosis in apolipoprotein E knockout mice, at least in part, because of eNOS uncoupling and O2·eC generation. In the present study, using a mouse model not exposed to pathological vascular oxidative stress, we also show that eNOS-derived O2·eC production is enhanced in eNOS-Tg mice, in cardiac tissue lysates and intact aorta, indicating increased eNOS uncoupling in these animals. We performed additional experiments, using quantitative RT-PCR, to confirm that the increased O2·eC in eNOS-Tg animals is not a result of a concomitant increase in the expression of the NADPH oxidase system, a major source of vascular O2·eC generation (data not shown). These findings agree with those from Ohashi et al25 using nondiseased eNOS-Tg mice. This enhanced vascular oxidative stress may account for the depleted aortic BH4 levels observed in the eNOS-Tg mice because BH4 is readily oxidized by ROS to BH2 that is inactive for eNOS cofactor function. In accordance with having increased uncoupled eNOS and depleted BH4 levels, specific NOS enzymatic activity is markedly attenuated in eNOS-Tg mice (elevated only 2-fold compared with wild type) relative to eNOS protein levels (elevated 8-fold compared with wild type). Indeed, the ratio of eNOS enzymatic activity to eNOS protein was only 0.6 in eNOS-Tg mice compared with 2.0 in wild-type animals.
Several previous studies have established that BH4 is a required cofactor for NOS activity.9,28,36 Recent studies, including those in atherosclerotic eNOS-Tg mice,10 have demonstrated that NOS uncoupling can be reversed and NOS enzymatic activity increased by augmenting BH4 levels.10,12 However, an advantage of the present study is that by targeting overexpression of GTPCH, the rate-limiting enzyme in BH4 biosynthesis, to the vascular endothelium, we avoid the potential confounding antioxidant effects of high-dose pharmacological BH4 supplementation used in other studies.10,12 Furthermore, we have been able to specifically evaluate the role of endothelial BH4, as opposed to systemic BH4, in the regulation of eNOS activity. Importantly, using 2 methods to measure O2·eC production in cardiac tissue and intact aorta, as well as specifically in aortic endothelium, we demonstrate that NOS-dependent O2·eC generation, elevated in eNOS-Tg mice, is normalized in eNOS/GCH-Tg mice. These data support the hypothesis that discordance between eNOS protein and endothelial BH4 levels is sufficient to cause eNOS uncoupling even in the absence of pathological oxidative stress. In support of this conclusion, NOS enzymatic activity and the ratio of enzymatic activity relative to eNOS protein levels were increased in eNOS/GCH-Tg compared with eNOS-Tg mice. Interestingly, GCH-Tg mice also had increased NOS enzymatic activity compared with wild type, indicating that even in the absence of either enhanced eNOS protein or disease, BH4 levels may limit eNOS enzymatic activity in vivo. These data therefore suggest that eNOS activity (to generate NO) can be augmented by modestly increasing BH4 levels, specifically in the endothelium, even under normal physiological conditions.
Previous data have shown that eNOS dimerization is an important aspect of eNOS activation and NO production.28 BH4 has been suggested to increase the stability of the eNOS homodimer such that the ratio of dimer to monomer is increased.28,37 In eNOS-Tg mice, the discordance between high levels of eNOS protein and depleted aortic BH4 levels may account for the relative decrease in homodimeric eNOS protein that we observed. In support, the increased production of endothelial BH4 in eNOS/GCH-Tg mice was sufficient to maintain the ratio of eNOS dimer to monomer. These in vivo findings corroborate previous in vitro studies suggesting that an important action of BH4, in addition to its direct contribution to electron transport within the eNOS active site, is to maintain eNOS in its homodimeric conformation.
We conclude that eNOS uncoupling is an independent and direct consequence of a stoichiometric discordance between enzyme and its cofactor BH4. BH4 is critical for regulating eNOS activity and its production of NO, as opposed to O2·eC, even in the absence of increased oxidative stress associated with vascular disease states. Thus, strategies to increase eNOS protein without a concomitant augmentation of endothelial BH4 levels may lead to eNOS uncoupling and paradoxically exacerbate oxidative stress and the progression of vascular diseases. Although reduced biosynthesis of BH4 may not be the principal mechanism of BH4 loss in vascular disease, strategies aimed at increasing BH4 synthesis or reducing BH4 oxidation may be valid therapeutic approaches in vascular disease states.
Acknowledgments
This work was supported by the British Heart Foundation (RG02/007) and the Wellcome Trust.
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