Gene Transfer of Human Guanosine 5′-Triphosphate Cyclohydrolase I Restores Vascular Tetrahydrobiopterin Level and Endothelial Function in Low Renin Hypertension
Background— We recently reported that arterial superoxide (O2−) is augmented by increased endothelin-1 (ET-1) in deoxycorticosterone acetate (DOCA)-salt hypertension, a model of low renin hypertension. Tetrahydrobiopterin (BH4), a potent reducing molecule with antioxidant properties and an essential cofactor for endothelial nitric oxide synthase, protects against O2−–induced vascular dysfunction. However, the interaction between O2− and BH4 on endothelial function and the underlying mechanisms are unknown.
Methods and Results— The present study tested the hypothesis that BH4 deficiency due to ET-1–induced O2− leads to impaired endothelium-dependent relaxation and that gene transfer of human guanosine 5′-triphosphate (GTP) cyclohydrolase I (GTPCH I), the first and rate-limiting enzyme for BH4 biosynthesis, reverses such deficiency and endothelial dysfunction in carotid arteries of DOCA-salt rats. There were significantly increased arterial O2− levels and decreased GTPCH I activity and BH4 levels in DOCA-salt compared with sham rats. Treatment of arteries of DOCA-salt rats with the selective ETA receptor antagonist ABT-627, NADPH oxidase inhibitor apocynin, or superoxide dismutase (SOD) mimetic tempol abolished O2− and restored BH4 levels. Basal arterial NO release and endothelium-dependent relaxations were impaired in DOCA-salt rats, conditions that were improved by apocynin or tempol treatment. Gene transfer of GTPCH I restored arterial GTPCH I activity and BH4 levels, resulting in reduced O2− and improved endothelium-dependent relaxation and basal NO release in DOCA-salt rats.
Conclusions— These results indicate that a BH4 deficiency resulting from ET-1–induced O2− via an ETA/NADPH oxidase pathway leads to endothelial dysfunction, and gene transfer of GTPCH I reverses the BH4 deficiency and endothelial dysfunction by reducing O2− in low renin mineralocorticoid hypertension.
Original received June 26, 2003; revision received July 17, 2003; accepted July 17, 2003.
Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS).1–4 It is also a potent reducing molecule and has antioxidant activity for scavenging reactive oxygen species (ROS).2 On the other hand, because BH4 is a potent reducing molecule, it is also susceptible to oxidation under oxidative stress. Under physiological conditions, there is a balance between endothelial nitric oxide (NO) and superoxide (O2−) production. However, when the BH4 levels are insufficient, there is a shift of balance toward the production of O2−.1,2 Loss of BH4 availability causes formation of O2− instead of NO after eNOS activation and is an important contributor to endothelial dysfunction in cardiovascular diseases, including hypertension.1–6 A BH4 deficiency results in endothelial dysfunction in hypertension,5,6 diabetes,7,8 and atherosclerosis,9 and BH4 supplementation improves endothelium-dependent vasodilation under these conditions. It has been hypothesized that either an increase in oxidative stress or a decrease of expression of guanosine 5′-triphosphate (GTP) cyclohydrolase I (GTPCH I), the first and rate-limiting enzyme for BH4 biosynthesis, results in reduced BH4 levels. However, the cellular mechanisms responsible for BH4 deficiency remain to be delineated.
Arterial O2− levels are markedly increased in deoxycorticosterone acetate (DOCA)-salt hypertension10–15 (a model of low renin hypertension), resulting in endothelial dysfunction in carotid arteries.16,17 Our recent studies indicate that endothelin-1 (ET-1) plays an important role in NADPH oxidase-derived O2− production in this model.13–15 However, the interaction between ET-1–induced O2− and BH4 on endothelial dysfunction and the underlying mechanisms are unknown. In the present study, we tested the hypothesis that ET-1–induced O2− reduces BH4 and endothelium-dependent relaxation in carotid arteries of DOCA-salt rats, and gene transfer of human GTPCH I reverses the BH4 deficiency and endothelial dysfunction by reducing O2− levels. Our results indicate that a BH4 deficiency due to ET-1-induced NADPH oxidase-derived O2− results in endothelial dysfunction, and that gene transfer of GTPCH I reverses the BH4 deficiency and endothelial dysfunction by reducing O2−. These findings demonstrate a critical interaction between O2− and BH4 on endothelial function, which may provide a mechanistic basis for regulating BH4 levels as novel therapeutic strategies in cardiovascular disease.
DOCA-Salt Hypertensive Rats
DOCA-salt hypertension was created in adult male Sprague-Dawley rats as previously described.13–15 Briefly, rats (250 to 275 g, Charles River, Portage, Mich) underwent uninephrectomy (flank incision, left side) and a silicon rubber DOCA implant (Sigma) (200 mg/kg) was placed subcutaneously between the shoulder blades. Sham rats were also uninephrectomized but received no implant. DOCA rats received 1.0% NaCl and 0.2% KCl in water to drink and sham rats received tap water. All animals were fed standard rat chow and had ad libitum access to both food and water. Hypertension develops gradually in this model, with arterial pressure slowly rising over a 4-week period. Blood pressure was measured using noninvasive tail cuff measurements in conscious but restrained and pre-warmed rats. Carotid arteries were collected between weeks 4 to 5 after DOCA implantation. All animal procedures were in accordance with institutional guidelines of Michigan State University.
In Vitro Pharmacological Interventions
Carotid arteries from normal rats were cut into ring segments (3 mm) and incubated at 37°C for 4 hours with or without ET-1 at increasing concentrations (0.1 to 10 nmol/L), or pretreated with the ETA receptor antagonist ABT-627 (30 nmol/L), the NADPH oxidase inhibitor apocynin (APO, 0.1 mmol/L), the xanthase oxidase inhibitor allopurinol (ALLO, 1 μmol/L), the NOS inhibitor Nω-l-arginine methylester (L-NAME, 0.1 mmol/L), or the superoxide dismutase (SOD) mimetic tempol (TEM, 1 mmol/L) for 1 hour, respectively, before ET-1 (10 nmol/L, 4 hours) treatment.13–15 Some arterial segments from sham and DOCA-salt rats were incubated at 37°C for 4 hours with or without the respective agents before O2−, BH4, and GTPCH I measurements.
Ex Vivo Gene Transfer
Arterial segments from sham and DOCA-salt rats were transduced with adenoviral vectors encoding human GTPCH I gene (AdGCH, 1x1010 pfu/mL, Gen Vec, Inc) or β-gal reporter gene (AdLacZ) as previously described.13–15,18 The rings were then transferred to fresh Eagle minimal essential medium (EMEM) with 0.1% bovine serum albumin and incubated for 24 hours at 37°C. Some vessels from sham and DOCA-salt rats were incubated for 24 hours without transduction and served as normal negative controls. Transgene expression and function were assessed by Western blot analysis, measurements of BH4 level, GTPCH I activity, and O2− levels, as well as vascular reactivity.
Western Blot Analysis for GTPCH I Protein
Western blots were performed with a rabbit anti-human antibody (1:5000 dilution),19 and a goat anti-rabbit immunoglobulin G secondary antibody (1:10 000 dilution, Oncogene Research Products, San Diego, Calif) as previously described.18–20
HPLC Detection of BH4 Level and GTPCH I Activity
Arterial BH4 levels and GTPCH I activities were measured by high-performance liquid chromatography (HPLC) with florescence detection after iodine oxidation in acidic or alkaline conditions as previously described.19,21–25 Tissue was homogenized in cold extract buffer (50 mmol/L Tris-HCl of pH 7.4, 1 mmol/L DTT, 1 mmol/L EDTA), and centrifuged at 16 000g for 15 minutes at 4°C. Protein was removed by adding 10 μL of a 1:1 mixture of 1.5 mol/L HClO4 and 2 mol/L H3PO4 to 90 μL of extracts, followed by centrifugation. To determine total biopterin (BH4, dihydropterin [BH2], and oxidized biopterin) by acid oxidation, 10 μL of 1% iodine in 2% KI solution was added to the 90 μL protein-free supernatant. To determine BH2 and oxidized biopterin by alkali oxidation, 10 μL of 1 mol/L NaOH was added to 80 μL of extract, then 10 μL of 1% iodine in 2% KI solution was added. Samples were incubated at room temperature for 1 hour in the dark. Alkaline-oxidation samples were then acidified with 20 μL of 1 mol/L H3PO4. Iodine was reduced by adding 5 μL of fresh ascorbic acid (20 mg/mL). Samples of 50 μL were injected into a 250-mm long, 4.6-mm–inner diameter Spherisorb ODS-1 column (5 μm particle size; Alltech Associates, Inc) isocraticallyeluted with a methanol-water (5:95, v/v) mobile phase running at a flow rate of 1.0 mL/min. Fluorescence detection (350 nm excitation, 450 nm emission) was performed using a fluorescence detector (RF10AXL, Shimadzu Co). BH4 concentrations, expressed as pmol/mg protein, were calculated by subtracting BH2 plus oxidized biopterin from total biopterins. Similarly, GTPCH I activities were assayed according to the same HPLC method with measurement of neopterin, which was released from dihydroneopterin triphosphate after oxidation and phosphatase treatment.22
Vascular O2− Levels
Arterial O2− levels were assayed by oxidative dihydroethidium (DHE) fluorescence confocal microscopy and lucigenin (5 μmol/L) chemiluminescence as described previously.13–15 The oxidative fluorescent dye dihydroethidine was used to evaluate in situ O2− production. Unfixed frozen rings of carotid arteries were cut into 30 μm sections and placed on a glass slide. Slides were incubated with dihydroethidium (1 μmol/L) in a light-protected, humidified chamber at 37°C for 60 minutes and cover slipped. Tissue sections were then visualized with a Zeiss 210 confocal microscope with fluorescence detected with a 590 nm long-pass filter, and images were collected and stored digitally. Red signal in tissue section represents O2− level.
Basal NO Levels
Basal NO release was estimated by nitrite (a stable NO metabolite) accumulation in EMEM after 24-hour incubation of carotid arteries with Griess Reagent (Sigma).26 EMEM incubation media without blood vessels were used as negative controls. To determine the calcium-dependent NO release, both an extracellular calcium chelator (EGTA, 1 mmol/L, Sigma) and an intracellular calcium chelator (BAPTA/AM, 20 μmol/L, Calbiochem) were added to EMEM.18,20 Calcium-dependent NO release was estimated as the difference between samples incubated with and without calcium chelators. After 24 hours incubation, EMEM was subjected to determination of nitrite with NaNO2 as standard.
Vascular reactivity studies were performed in arterial ring segments as we previously described.18,20 In preliminary studies, the optimal tension of the carotid arteries from both sham and DOCA-salt rats in response to KCl stimulation was determined to be 1.5 g. Rings were connected to isometric transducers (Radnoti Grass Technology Inc) and suspended in organ chambers filled with 15 mL of gassed (95% O2, 5% CO2) Krebs solution (pH 7.4, 37°C) composed of (in mmol/L) 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.0 NaHCO3, 0.0026 EDTA, and 11.1 glucose. Isometric tension was recorded continuously. Arteries were allowed to equilibrate for 30 minutes and were then gradually stretched to 1.5 g tension over 40 minutes. After 2 challenges with 60 mmol/L KCl, 10 μmol/L of indomethacin was added to eliminate the influence of endogenous cyclooxygenase before the submaximal contractions induced by thromboxane A2 analogue 9, 11-dideoxy-11a,9a-epoxy-methanoprostaglandin F2 (U46619, 1 to 100 nmol/L). After a stable contraction plateau was reached, acetylcholine (endothelium-dependent, 1 nmol to 10 μmol/L) or sodium nitroprusside (endothelium- independent, 1 nmol to 10 μmol/L)-induced relaxations were performed. The relaxations were expressed as a percentage of maximal relaxations induced by papaverine (0.1 mol/L). In some experiments, arteries were incubated with NOS inhibitor Nω-nitro-l-arginine (L-NNA, 0.1 mmol/L, 30 minutes), tempol (1 mmol/L, 15 minutes), or apocynin (0.1 mmol/L, 30 minutes) before the relaxation experiments. All the relaxation responses in control arteries were obtained after a 24-hour incubation period in minimal essential medium (EMEM), which was needed to compare with the responses in 24-hour transduced arteries.
Acetylcholine, indomethacin, sodium nitroprusside, and papaverine were purchased from Sigma and dissolved in saline. U46619 was purchased from Cayman Chemical and dissolved in 100% ethanol. Subsequent dilutions were made with saline. Dihydroethidium was purchased from Molecular Probes and dissolved in 0.1 mol/L DMSO.
Data were expressed as mean ± SEM. Repeated measures ANOVA was used for comparison of multiple values obtained from the same subject, whereas factorial ANOVA was used for comparing data obtained from 2 independent samples of subjects. Bonferroni’s procedure was used to control type I error. A value of P<0.05 was considered significant.
ET-1 Increases Arterial O2− Levels via an ETA/NADPH Oxidase Pathway
O2− levels in carotid arteries of normal Sprague-Dawley rats were significantly increased in a concentration-dependent manner after treatment with ET-1 for 4 hours (Figure 1A, n=6; P<0.01). The effect of ET-1 treatment (10 nmol/L) was abolished by pretreatment with the selective ETA receptor antagonist ABT-627 (n=5; P<0.05), the NADPH oxidase inhibitor apocynin (n=5; P<0.05), or the SOD mimetic tempol (n=5; P<0.05), but not by the NOS inhibitor L-NAME (n=5; P>0.05) or the xanthine oxidase inhibitor allopurinol (n=5; P>0.05) (Figure 1B). Treatment of carotid arteries of normal rats with ABT-627, apocynin, tempol, L-NAME, or allopurinol did not alter basal O2− levels (data not shown). There were also significantly increased arterial O2− levels (n=6; P<0.01) in carotid arteries of DOCA-salt rats compared with sham rats (Figure 1C). Treatment of carotid arteries of DOCA-salt rats with ABT-627, apocynin, or tempol abolished O2− increase (n=5 to 6; P<0.05). In contrast, neither L-NAME nor allopurinol had any effects on O2− levels (n=5 to 6; P>0.05). Treatment of carotid arteries of sham rats with ABT-627, apocynin, tempol, L-NAME, or allopurinol did not alter basal O2− levels (data not shown).
ET-1-Induced O2− Decreases Arterial BH4 Levels
ET-1 treatment of carotid arteries of normal rats (10 nmol/L, 4 hours) significantly reduced BH4 levels (Figure 2A, n=5; P<0.01), an effect that was prevented by pretreatment with ABT-627 (n=5; P<0.05), apocynin (n=5; P<0.05), or tempol (n=5; P<0.05), but not by L-NAME (n=5; P>0.05) or allopurinol (n=5; P>0.05) (Figure 2A). Treatment of carotid arteries of normal rats with only ABT-627, apocynin, tempol, L-NAME, or allopurinol did not alter basal BH4 levels (data not shown). There were significantly decreased arterial BH4 levels (n=5; P<0.05) in carotid arteries of DOCA-salt rats as compared with sham rats (Figure 2B). Treatment of carotid arteries of DOCA-salt rats with ABT-627, apocynin, or tempol restored BH4 levels (n=5 to 6; P<0.05). L-NAME and allopurinol had no effect on BH4 levels (n=5 to 6; P>0.05). Treatment of carotid arteries of sham rats with ABT-627, apocynin, tempol, L-NAME, or allopurinol did not alter basal BH4 levels (data not shown).
Effect of Gene Transfer of Human GTPCH I on Arterial GTPCH I, BH4, and O2− Levels
To demonstrate transgene expression, carotid arteries of sham and DOCA-salt rats were analyzed by Western blotting for human GTPCH I protein. A rabbit anti-human GTPCH I primary antibody (1:5000 dilution)19 was used to recognize recombinant protein expressed by human GTPCH I gene that had been integrated into the AdGCH viral vectors. Typical immunoblot images revealed significant GTPCH I bands in AdGCH-transduced arteries but not in AdLacZ-transduced or non-transduced controls (Figure 3A).
There were significantly decreased GTPCH I activities (Figure 3B) and BH4 levels (Figure 3C) in carotid arteries of DOCA-salt rats compared with those of sham rats. After gene transfer of GTPCH I, arteries of both DOCA-salt (n=5; P<0.01) and sham rats (n=5; P<0.01) demonstrated a significant elevation of GTPCH I activities (Figure 3B) and BH4 levels (Figure 3C) compared with AdLacZ-transduced or non-transduced control arteries.
In situ detection of O2− indicated that the dihydroethidine signal in arteries of DOCA-salt rats was markedly more prominent than that in sham rats, which was suppressed after AdGCH but not AdLacZ transduction (Figure 4A). Quantitatively, gene transfer of AdGCH but not AdLacZ resulted in significantly diminished O2− levels in arteries of DOCA-salt rats (n=6; P<0.05) (Figure 4B).
Treatment With Apocynin and Tempol, or Gene Transfer of GTPCH I Improves Endothelium-Dependent Relaxations in Carotid Arteries of DOCA-Salt Rats
Endothelium-dependent relaxations to acetylcholine (1 nmol to 10 μmol/L) were significantly reduced in carotid arteries of DOCA-salt rats compared with those of sham rats (n=5 to 6; P<0.05) (Figure 5A). Acetylcholine produced 68.32±6.01% of maximal relaxations in sham rats compared with 40.5±3.7% of maximal relaxations in DOCA-salt rats (P<0.05), which were attenuated by preincubation with NOS inhibitor L-NNA. Preincubation with the NADPH oxidase inhibitor apocynin or SOD mimetic tempol significantly improved endothelium-dependent relaxations in arteries of DOCA-salt rats (n=5 to 6; P<0.05) (Figure 5A). There was no significant difference in the relaxation in arteries of DOCA-salt rats compared with those of sham rats when treated with sodium nitroprusside (data not shown).
Gene transfer of AdGCH but not AdLacZ significantly improved endothelium-dependent relaxations in DOCA-salt but not sham rats (Figure 5B and 5C, n=5 to 6; P<0.05). The relaxations of AdGCH-transduced arteries of either DOCA-salt or sham rats to sodium nitroprusside were not different compared with those of AdLacZ-transduced controls (data not shown). It is important to note that all relaxation responses in control arteries were obtained after a 24-hour incubation period in minimal essential medium, which was needed to compare with the responses in 24-hour transduced arteries. As a result, the acetylcholine-induced maximal relaxation in control arteries was around 60%, which has been shown to be due to the effect of 24-hour incubation in minimal essential medium.18
Gene Transfer of GTPCH I Increases Basal NO Release in Arteries of DOCA-Salt Rats
Basal NO levels, detected as calcium-dependent NO2 production, were shown as the difference between NO2 levels with and without calcium chelator treatment. Basal NO production was significantly lower in arteries of DOCA-salt rats than in sham rats (n=5; P<0.05) (Figure 6). AdGCH-transduced arteries of DOCA-salt rats exhibited significantly elevated NO levels compared with AdLacZ-transduced or non-transduced controls (n=6; P<0.01). There were no significant elevations of basal NO levels in AdGCH-transduced arteries of sham rats compared with those of AdLacZ-transduced or non-transduced controls (n=5 to 6; P>0.05).
In the present study, there are 3 major new findings. First, arterial GTPCH I activity was decreased and BH4 was deficient in DOCA-salt rats. Second, arterial BH4 levels were restored after inhibition of ETA-receptors, NADPH oxidase, or O2− itself. Inhibition of NADPH oxidase or O2− also improved endothelium-dependent relaxations, which were impaired in carotid arteries of DOCA-salt rats. Finally, gene transfer of human GTPCH I restored arterial GTPCH I activity and BH4 levels, with a subsequent reduction of O2− levels, augmentation of basal NO levels, and improvement of endothelium-dependent relaxations.
Several recent studies have shown that arterial O2− levels are markedly increased in salt-sensitive hypertension of both human and animal models, including DOCA-salt hypertension,10–15 a model with suppressed plasma renin level in which ET-1 has been implicated in the development and maintenance of hypertension.27–29 Our recent study14 has demonstrated that the ET-1 levels in carotid arteries of DOCA-salt rats are markedly increased, which contributes to O2− production via an ETA/NADPH oxidase pathway. The present study extends further these findings that (1) ET-1 increases O2− levels in a concentration-dependent manner in carotid arteries of normal rats, (2) the selective ETA receptor antagonist ABT-627, NADPH oxidase inhibitor apocynin, and SOD mimetic tempol, but not xanthine oxidase inhibitor allopurinol or NOS inhibitor L-NAME, suppressed the O2− levels in both ET-1-stimulated arteries of normal rats and arteries of DOCA-salt rats, and (3) inhibition of either NADPH oxidase or O2− itself led to improved endothelium-dependent NO-mediated relaxations.
An arterial BH4 deficiency exists in cardiovascular diseases, including hypertension,5,6 diabetes,7,8 hypercholesterolemia and therosclerosis,9,30–32 and chronic smoking,33 resulting in impaired endothelium-dependent NO-mediated vasodilations. However, the cellular mechanisms underlying BH4 deficiency are not well understood.1,2 In mammalian cells, BH4 is synthesized through 2 distinct pathways.34 One is the de novo synthetic pathway in which the precursor GTP is catalyzed by the first and rate-limiting enzyme GTPCH I to dihydroneopterin triphosphate, whereby BH4 is then generated by further steps involving 6-pyruvoltetrahydropterin synthase and sepiapterin reductase. Recently, reduced expression and activity of GTPCH I have been shown in coronary endothelial cells of diabetic rats and aortas of insulin-resistant rats, resulting in diminished BH4 levels.7,8 The other pathway of BH4 synthesis is the regeneration of BH4 from BH2 through a pterin salvage pathway, which is independent of GTPCH I but depends on a normal cellular redox state. It has been reported that BH4 can rapidly be oxidized to BH2 by peroxynitrite, and regeneration of BH2 to BH4 is impaired by oxidative stress.5,7,9 Chronic treatment with antioxidants restores arterial BH4 levels in apolipoprotein-E knockout mice.24 An important question that remains to be answered is how the DOCA-salt hypertensive state affects BH4 levels. The present study demonstrated that arterial BH4 levels were reduced by ET-1–induced O2− and inhibition of ETA receptors, NADPH oxidase, or removal of O2− restored BH4 levels in DOCA-salt hypertensive rats. Furthermore, the present study is the first to show that arterial GTPCH I activities were significantly decreased in this model. As illustrated in Figure 7, our findings indicate that both decreased GTPCH I activity and increased O2− contribute to the observed BH4 deficiency in DOCA-salt hypertensive rats. These results extend a recent report that peroxynitrite oxidizes BH4 in DOCA-salt mice.5 These data are also consistent with the observations obtained in BH4-deficient mice that increased oxidative stress is SOD-sensitive and contributes to a hypertensive phenotype,33 and they illustrate the complex mechanisms underlying reduced BH4 levels in cardiovascular disease.
Relative deficiency of BH4 has been suggested to be a major cause of impaired endothelium-dependent relaxation,2 and BH4 supplementation has been shown to improve endothelium-dependent relaxations by preserving endogenous NO production in vitro and in vivo.3,7,23,30–33 In agreement with this concept, the present study demonstrates that ex vivo gene transfer of human GTPCH I resulted in recombinant GTPCH I protein expression and restoration of both GTPCH I enzymatic activities and BH4 levels in carotid arteries of DOCA-salt rats. Most importantly, O2− levels were diminished in GTPCH I-transduced arteries with a concomitant increase of calcium-sensitive NO production and improvement of endothelium-dependent relaxations. Together, these findings indicate a demonstrable functional effect of GTPCH I gene transfer. Because the carotid arteries of DOCA-salt rats exhibited a sensitivity to sodium nitroprusside (a NO donor)-induced endothelium-independent relaxation similar to that of sham rats, the smooth muscle sensitivities to NO of carotid arteries of DOCA-salt and sham rats are not different. Thus, the observed endothelial dysfunction was most likely due to the loss of endothelium-derived NO. The latter may result from a decreased NO biosynthesis as a result of O2− oxidation of BH4 and/or an inactivation of NO by O2−. Because pterins (including BH4) possess antioxidant property by virtue of their scavenging activity for reactive oxygen species,32,35 decreased levels of BH4 may lead to increased O2− formation and endothelial dysfunction. By restoring the BH4 levels, gene transfer of GTPCH I may exert its beneficial effects via increasing the direct antioxidant effect of BH4 and thereby shifting the imbalance between O2− and NO and/or enhancing physiological eNOS function, which is known to preserve endothelium-dependent relaxations.
In conclusion, our findings demonstrate that (1) a BH4 deficiency resulting from ET-1-induced O2− via an ETA/NADPH oxidase pathway contributes to endothelial dysfunction and (2) gene transfer of human GTPCH I reverses BH4 deficiency and suppresses O2− levels, with a subsequent restoration of endothelial function in low renin mineralocorticoid hypertension. These results may provide a mechanistic basis for therapeutic strategies aimed at increasing vascular BH4 levels for therapy of endothelial dysfunction in cardiovascular disease, including hypertension.
This work was supported in part by the National Institutes of Health grant 1 P01, HL70687–01 (to Drs Fink and Chen); the American Heart Association grants 9806347X, 0130537Z, and 0225408Z; the American Diabetes Association Research Award 7–01-RA-10; the Juvenile Diabetes Research Foundation Innovative Grant 5–2001–311; and the Michigan State University Intramural Research Grants Program grant #41140 (all to Dr. Chen).
This article originally appeared Online on August 18, 2003 (Circulation. 2003;108:r53–r60).
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