Augmented BH4 by gene transfer restores nitric oxide synthase function in hyperglycemic human endothelial cells

OBJECTIVE: Endothelial dysfunction in diabetes is characterized by decreased nitric oxide (NO) bioactivity and increased superoxide (SO) production. Reduced levels of tetrahydrobiopterin (BH4), an essential cofactor of endothelial NO synthase (eNOS), appear to be associated with eNOS enzymatic uncoupling. We sought to investigate whether augmented BH4 biosynthesis in hyperglycemic human aortic endothelial cells (HAEC) by adenovirus-mediated gene transfer of GTP cyclohydrolase I (GTPCH, the rate-limiting enzyme for the de novo BH4 synthesis), would be sufficient to rescue eNOS activity and dimerization. HAEC were cultured in media with low glucose (5 mM) or high glucose (30 mM). METHODS: After 5 days, the cells with/without GTPCH gene transfer (AdeGFP as a control) were prepared for assays of (1) NO with electron paramagnetic resonance (EPR); (2) SO with cytochrome c reduction and dihydroethidine (DHE) fluorescence; (3) BH4 with high-performance liquid chromatography (HPLC); (4) eNOS expression and dimerization with immunoblotting. RESULTS: We found that high glucose decreased HAEC NO and increased SO production, in association with reductions in both total biopterin and BH4 levels. High glucose increased total eNOS protein levels in HAEC 1.5-fold, but this was present principally in the monomeric form. GTPCH gene transfer increased cellular biopterin levels and NO production but decreased SO production. Furthermore, augmenting BH4 increased the eNOS dimer:monomer ratio 2.6-fold. CONCLUSION: This study demonstrates a critical role for BH4 in regulating eNOS function, suggesting that GTPCH is a rational target to augment endothelial BH4 and recover eNOS activity in hyperglycemic endothelial dysfunction states.     

C-reactive protein decreases endothelial nitric oxide synthase activity via uncoupling

C-reactive protein (CRP), a cardiovascular risk marker, induces endothelial dysfunction. We have previously shown that CRP decreases endothelial nitric oxide synthase (eNOS) expression and bioactivity in human aortic endothelial cells (HAECs). In this study, we examined the mechanisms by which CRP decreases eNOS activity in HAECs. To this end, we explored different strategies such as availability of tetrahydrobiopterin (BH4)-a critical cofactor for eNOS, superoxide (O(2)(-)) production resulting in uncoupling of eNOS and phosphorylation/dephosphorylation of eNOS. CRP treatment significantly decreased levels of BH4 thereby promoting eNOS uncoupling. Pretreatment with sepiapterin, a BH4 precursor, prevented CRP-mediated effects on BH(4) levels, superoxide production as well as eNOS activity. The gene expression and enzymatic activity of GTPCH1, the first enzyme in the de novo biosynthesis of BH(4), were significantly inhibited by CRP. Importantly, GTPCH1 is known to be regulated by cAMP-mediated pathway. In the present study, CRP-mediated inhibition of GTPCH1 activity was reversed by pretreatment with cAMP analogues. Furthermore, CRP-induced O(2)(-) production was reversed by pharmacologic inhibition and siRNAs to p47 phox and p22 phox. Additionally, CRP treatment significantly decreased the eNOS dimer: monomer ratio confirming CRP-mediated eNOS uncoupling. The pretreatment of cells with NO synthase inhibitor (N-nitro-l-arginine methyl ester [l-NAME]) also prevented CRP-mediated O(2)(-) production further strengthening CRP-mediated eNOS uncoupling. Additionally, CRP decreased eNOS phosphorylation at Ser1177 as well as increased phosphorylation at Thr495. CRP appears to mediate these effects through the Fcgamma receptors, CD32 and CD64. To conclude, CRP uncouples eNOS resulting in increased superoxide production, decreased NO production and altered eNOS phosphorylation.     

C-reactive protein decreases endothelial nitric oxide synthase activity via uncoupling

C-reactive protein (CRP), a cardiovascular risk marker, induces endothelial dysfunction. We have previously shown that CRP decreases endothelial nitric oxide synthase (eNOS) expression and bioactivity in human aortic endothelial cells (HAECs). In this study, we examined the mechanisms by which CRP decreases eNOS activity in HAECs. To this end, we explored different strategies such as availability of tetrahydrobiopterin (BH4)-a critical cofactor for eNOS, superoxide (O₂⁻) production resulting in uncoupling of eNOS and phosphorylation/dephosphorylation of eNOS. CRP treatment significantly decreased levels of BH4 thereby promoting eNOS uncoupling. Pretreatment with sepiapterin, a BH4 precursor, prevented CRP-mediated effects on BH₄ levels, superoxide production as well as eNOS activity. The gene expression and enzymatic activity of GTPCH1, the first enzyme in the de novo biosynthesis of BH₄, were significantly inhibited by CRP. Importantly, GTPCH1 is known to be regulated by cAMP-mediated pathway. In the present study, CRP-mediated inhibition of GTPCH1 activity was reversed by pretreatment with cAMP analogues. Furthermore, CRP-induced O₂⁻ production was reversed by pharmacologic inhibition and siRNAs to p47 phox and p22 phox. Additionally, CRP treatment significantly decreased the eNOS dimer: monomer ratio confirming CRP-mediated eNOS uncoupling. The pretreatment of cells with NO synthase inhibitor (N-nitro-l-arginine methyl ester [l-NAME]) also prevented CRP-mediated O₂⁻ production further strengthening CRP-mediated eNOS uncoupling. Additionally, CRP decreased eNOS phosphorylation at Ser1177 as well as increased phosphorylation at Thr495. CRP appears to mediate these effects through the Fcγ receptors, CD32 and CD64. To conclude, CRP uncouples eNOS resulting in increased superoxide production, decreased NO production and altered eNOS phosphorylation.     

GTP环化水解酶I/四氢生物蝶呤通路对高血压内皮祖细胞体外活性和体内再内皮化能力的调节

目的研究GTP环化水解酶Ⅰ/四氢生物蝶呤信号通路对高血压患者循环内皮祖细胞功能的调节。方法分别纳入高血压患者和健康志愿者各19例,采集外周血进行内皮祖细胞分离、培养及鉴定,评估其迁移、增殖、黏附活性;建立裸鼠动脉损伤模型并移植高血压患者和健康志愿者内皮祖细胞,评估内皮祖细胞修复损伤血管内皮功能,并检测内皮祖细胞GTP环化水解酶Ⅰ/四氢生物蝶呤通路及一氧化氮、环磷酸鸟苷、凝血酶敏感蛋白1的mRNA表达水平。通过基因干扰、基因转染或药物阻滞干预,进一步证实该信号通路对内皮祖细胞功能的调节作用。结果高血压患者内皮祖细胞体外活性及体内再内皮化功能降低,且内皮祖细胞GTP环化水解酶Ⅰ/四氢生物蝶呤通路及其下游信号分子一氧化氮、环磷酸鸟苷的mRNA表达水平亦下降,而凝血酶敏感蛋白1 mRNA表达水平则升高。抑制该信号通路的表达可削弱内皮祖细胞的体外活性及再内皮化功能。结论研究证实GTP环化水解酶I/四氢生物蝶呤通路可通过凝血酶敏感蛋白1及可溶性鸟苷酸环化酶/环磷酸鸟苷系统调节高血压患者内皮祖细胞的体外及体内功能。     

Acute inhibition of guanosine triphosphate cyclohydrolase 1 uncouples endothelial nitric oxide synthase and elevates blood pressure

GTP cyclohydrolase 1 (GTPCH1) is the rate-limiting enzyme in de novo synthesis of tetrahydrobiopterin (BH4), an essential cofactor for endothelial NO synthase (eNOS) dictating, at least partly, the balance of NO and superoxide produced by this enzyme. The aim of this study was to determine the effect of acute inhibition of GTPCH1 on BH4, eNOS function, and blood pressure (BP) in vivo. Exposure of bovine or mouse aortic endothelial cells to GTPCH1 inhibitors (2,4-diamino-6-hydroxypyrimidine or N-acetyl-serotonin) or GTPCH1 small-interference RNA (siRNA) significantly reduced BH4 and NO levels but increased superoxide levels. This increase was abolished by sepiapterin (BH4 precursor) or N(G)-nitro-L-arginine methyl ester (nonselective NOS inhibitor). Incubation of isolated murine aortas with 2,4-diamino-6-hydroxypyrimidine or N-acetyl-serotonin impaired acetylcholine-induced endothelium-dependent relaxation but not endothelium-independent relaxation. Aortas from GTPCH1 siRNA-injected mice, but not their control-siRNA injected counterparts, also exhibited impaired endothelium-dependent relaxation. BH4 reduction induced by GTPCH1 siRNA injection was associated with increased aortic levels of superoxide, 3-nitrotyrosine, and adhesion molecules (intercellular adhesion molecule 1 and vascular cell adhesion molecule 1), as well as a significantly elevated systolic, diastolic, and mean BP in C57BL6 mice. GTPCH1 siRNA was unable to elicit these effects in eNOS(-/-) mice. Sepiapterin supplementation, which had no effect on high BP in eNOS(-/-) mice, partially reversed GTPCH1 siRNA-induced elevation of BP in wild-type mice. In conclusion, GTPCH1 via BH4 maintains normal BP and endothelial function in vivo by preserving NO synthesis by eNOS.     

Simvastatin Ameliorates Angiotensin II-Induced Endothelial Dysfunction Through Restoration of Rho-BH4-eNOS-NO Pathway

Endothelial dysfunction contributes to the initiation and development of hypertension. We previously found that simvastatin moderately decreases blood pressure in 2-kidney-2-clip (2k2c) renal hypertension, but the precise mechanisms are still unclear. The present study was designed to examine the protective actions of simvastatin in 2k2c-evoked endothelial dysfunction and also delineate the underlying mechanisms. Here we show that 2k2c-induced elevation in plasma angiotensin II impaired acetylcholine-induced endothelium-dependent vascular relaxation, suppressed endothelial NO synthase (eNOS) activity and reduced nitric oxide (NO) production. Additionally, the levels of tetrahydrobiopterin (BH4), an essential cofactor of eNOS, as well as the activity of GTP cyclohydrolase I (GTPCH I), the rate-limiting enzyme for BH4 synthesis, were markedly reduced. Administration of simvastatin significantly improved acetylcholine-induced endothelium-dependent carotid arteries relaxation at 9 weeks in reno-hypertensive rats. Notably, GTPCH I activity, BH4 production, p-eNOS expression and NO levels in the vascular endothelium were elevated as a result of simvastatin administration. In cultured rat arterial endothelial cells, simvastatin restored BH4, GTPCH I activity and NO release impaired by angiotensin II, and pretreatment with mevalonate (MVA) or geranylgeranyl pyrophosphate (GGPP) abolished the beneficial effects exerted by simvastatin. Moreover, RhoA inhibitor C3 exoenzyme, Rho kinase inhibitor Y-27632 and dominant negative mutant of RhoA prevented BH4 and NO loss due to Ang II treatment. Taken together, normalization of BH4-eNOS-NO pathway at least in part accounts for the beneficial actions of simvastatin on vascular endothelium during 2k2c hypertension, and RhoA-Rho kinase pathway is involved in regulation of BH4 production.     

GTP cyclohydrolase I/BH4 pathway protects EPCs via suppressing oxidative stress and thrombospondin-1 in salt-sensitive hypertension

Endothelial progenitor cells (EPCs) are both reduced and dysfunctional in hypertension that correlates inversely with its mortality, but the mechanisms are poorly understood. Endothelial nitric oxide synthase (eNOS) critically regulates EPC mobilization and function but is uncoupled in salt-sensitive hypertension because of the reduced cofactor tetrahydrobiopterin (BH4). We tested the hypothesis that GTP cyclohydrolase I (GTPCH I), the rate-limiting enzyme of BH4 de novo synthesis, protects EPCs and its function in deoxycorticosterone acetate (DOCA)-salt mice. EPCs were isolated from peripheral blood and bone marrow of wild-type (WT), WT DOCA-salt, endothelial-specific GTPCH transgenic (Tg-GCH), GTPCH transgenic DOCA-salt, and BH4-deficient hph-1 mice. In WT DOCA-salt and hph-1 mice, EPCs were significantly decreased with impaired angiogenesis and adhesion, which were restored in Tg-GCH DOCA-salt mice. Superoxide (O??) and nitric oxide (NO) levels in EPCs were elevated and reduced, respectively, in WT DOCA-salt and hph-1 mice; both were rescued in Tg-GCH DOCA-salt mice. eNOS(-/-)/GCH(+/-) hybrid mice demonstrated that GTPCH preserved the circulating EPC number, reduced intracellular O?? in EPCs, and ameliorated EPC dysfunction independent of eNOS in DOCA-salt hypertension. Secreted thrombospondin-1 (TSP-1; a potent angiogenesis inhibitor) from EPCs was elevated in WT DOCA-salt and hph-1 but not DOCA-salt Tg-GCH mice. In vitro treatment with BH4, polyethylene glycol-superoxide dismutase (PEG-SOD), or Nomega-nitro-L-arginine (L-NNA) significantly augmented NO and reduced TSP-1 and O?? levels from EPCs of WT DOCA-salt mice. These results demonstrated, for the first time, that the GTPCH/BH4 pathway critically regulates EPC number and function in DOCA-salt hypertensive mice, at least in part, via suppressing TSP-1 expression and oxidative stress.     

Cilostazol activates AMP-activated protein kinase and restores endothelial function in diabetes

BACKGROUND: Endothelial dysfunction plays a key role in atherogenesis. We investigated whether AMP-activated protein kinase (AMPK) activity is a downstream mediator of the beneficial effects of cilostazol on vascular endothelial cells and whether cilostazol might reverse endothelial dysfunction in diabetic rats. METHODS AND RESULTS: Treatment of human umbilical vein endothelial cells (HUVECs) with cilostazol resulted in time-dependent activation of AMPK, as monitored by phosphorylation of AMPK and its down-stream target, acetyl-CoA carboxylase (ACC). Activation of AMPK by cilostazol was through signaling pathway independent of cyclic AMP and caused phosphorylation of endothelial nitric oxide synthase (eNOS), leading to increased production of nitric oxide (NO), while inhibiting cytokine-induced nuclear factor-kappaB (NF-kappaB) activation, leading to suppression of VCAM-1 gene expression. Significantly reduced eNOS activity and NO production in response to cilostazol and attenuation of cilostazol-induced inhibition of NF-kappaB activation were observed in cells treated with AMPK siRNA. We also demonstrated that administration of cilostazol to diabetic rats significantly restored endothelium-dependent vasodilation. Furthermore, treatment of diabetic rats with cilostazol increased tetrahydrobiopterin (BH4) levels in the aorta. Thus, recovery of BH4 following administration of cilostazol might also contribute to restoration of endothelial function in diabetic rats. CONCLUSIONS: Our findings suggest that the beneficial effects of cilostazol on endothelial function may be due to AMPK activation. Restoration of endothelial dysfunction in diabetic rats by cilostazol is at least partly attributed to amelioration of biopterin metabolism in the aorta.     

Stoichiometric relationships between endothelial tetrahydrobiopterin, endothelial NO synthase (eNOS) activity, and eNOS coupling in vivo: insights from transgenic mice with endothelial-targeted GTP cyclohydrolase 1 and eNOS overexpression

Endothelial dysfunction in vascular disease states is associated with reduced NO bioactivity and increased superoxide (O2*-) production. Some data suggest that an important mechanism underlying endothelial dysfunction is endothelial NO synthase (eNOS) uncoupling, whereby eNOS generates O2*- 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 BH4-eNOS 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*- production was significantly increased in eNOS-Tg mice compared with wild type but was normalized after NOS inhibition with Nomega-nitro-L-arginine methyl ester hydrochloride (L-NAME), suggesting O2*- production by uncoupled eNOS. In contrast, in eNOS/GCH-Tg mice, O2*- 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 eNOS-BH4 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.     

非诺贝特促进人内皮细胞内皮一氧化氮合酶复偶联的作用研究

目的探讨非诺贝特能否对脂多糖（LPS）诱导的血管内皮一氧化氮合酶（eNOS）脱偶联发挥保护作用。方法体外培养人脐静脉内皮细胞（HUVECs）,用非诺贝特预处理HUVECs 2 h,再与LPS共孵育24 h,采用高效液相色谱法检测细胞四氢生物蝶呤（BH4）的表达水平,ELISA检测细胞eNOS表达水平和细胞上清一氧化氮（NO）浓度,利用Confocal方法检测细胞内活性氧（ROS）产生水平。结果与对照组比较,单纯LPS刺激组内皮细胞BH4表达水平降低,伴有eNOS表达下调和NO水平降低,而内皮细胞内ROS产生增加（P均〈0.05）。与单纯LPS刺激组比较,非诺贝特预处理组内皮细胞BH4表达水平升高,同时伴有eNOS表达上调和NO水平增加,而内皮细胞内ROS产生降低（P均〈0.05）。结论非诺贝特通过上调BH4水平,对LPS诱导的血管内皮细胞eNOS脱偶联有逆转作用,这可能是其发挥血管内皮保护作用的机制之一。     

eNOS gene therapy exacerbates hepatic ischemia-reperfusion injury in diabetes: a role for eNOS uncoupling

Previous studies indicate that endothelial nitric oxide synthase (eNOS) function is impaired in diabetes as a result of increased vascular generation of reactive oxygen species. We hypothesized that eNOS gene therapy would augment NO. bioavailability and protect against hepatic ischemia-reperfusion (I-R) injury in type 2 diabetes mellitus. We developed a transgenic (Tg) diabetic mouse in which eNOS is systemically overexpressed. We also examined the effects of hepatic eNOS adenovirus therapy in diabetic mice. Diabetic (db/db) and nondiabetic mice were subjected to hepatic I-R injury. In nondiabetic mice, genetic overexpression of eNOS (both eNOS-Tg and eNOS adenovirus) resulted in hepatoprotection. In contrast, hepatic I-R injury was significantly increased in the db/db eNOS-Tg mouse, as serum alanine aminotransaminase (ALT) levels were increased by 3.3-fold compared with diabetic controls. Similarly, eNOS adenovirus treatment resulted in a 3.2-fold increase in serum ALT levels as compared with diabetic controls. We determined that hepatic eNOS was dysfunctional in the db/db mouse and increased genetic expression of eNOS resulted in greater production of peroxynitrite. Treatment with the eNOS cofactor tetrahydrobiopterin (BH4) or the BH4 precursor sepiapterin resulted in a significant decrease in serum ALT levels following I-R injury. We present clear examples of the protective and injurious nature of NO. therapy in I-R. Our data indicate that eNOS exists in an "uncoupled" state in the setting of diabetes and that "recoupling" of the eNOS enzyme with cofactor therapy is beneficial.     

Endothelial nitric oxide synthase dysfunction in diabetic mice: importance of tetrahydrobiopterin in eNOS dimerisation

Aims/hypothesis Impaired nitric oxide (NO) bioactivity and increased superoxide (SO) production are characteristics of vascular endothelial dysfunction in diabetes. The underlying mechanisms remain unknown. In this regard, we investigated the role of tetrahydrobiopterin (BH4) bioavailability in regulating endothelial nitric oxide synthase (eNOS) activity, dimerisation and SO production in streptozotocin-induced diabetic mice.Methods Mouse aortas were used for assays of the following: (1) aortic function by isometric tension; (2) NO by electronic paramagnetic resonance; (3) SO by lucigenin-enhanced chemiluminescence and dihydroethidine fluorescence; (4) total biopterin and BH4 by high-performance liquid chromatography; and (5) eNOS protein expression and dimerisation by immunoblotting.Results In diabetic mouse aortas, relaxations to acetylcholine and NO levels were significantly decreased, but SO production was increased, in association with reductions in total biopterins and BH4. Although total eNOS levels were increased in diabetes, the protein mainly existed in monomeric form. Conversely, specifically augmented BH4 in diabetic endothelium preserved eNOS dimerisation, but the expression remained unchanged.Conclusions/interpretation Our results demonstrate that BH4 plays an important role in regulating eNOS activity and its functional protein structure, suggesting that increasing endothelial BH4 and/or protecting it from oxidation may be a rational therapeutic strategy to restore eNOS function in diabetes.     

Tetrahydrobiopterin, but not L-arginine, decreases NO synthase uncoupling in cells expressing high levels of endothelial NO synthase

Endothelial NO synthase (eNOS) produces superoxide when depleted of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) and L-arginine by uncoupling the electron flow from NO production. High expression of eNOS has been reported to have beneficial effects in atherosclerotic arteries after relatively short periods of time. However, sustained high expression of eNOS may have disadvantageous vascular effects because of uncoupling. We investigated NO and reactive oxygen species (ROS) production in a microvascular endothelial cell line (bEnd.3) with sustained high eNOS expression and absent inducible NOS and neuronal NOS expression using 4,5-diaminofluorescein diacetate and diacetyldichlorofluorescein as probes, respectively. Unstimulated cells produced both NO and ROS. After stimulation with vascular endothelial growth factor (VEGF), NO and ROS production increased. VEGF-induced ROS production was even further increased by the addition of extra L-arginine. Nomega-nitro-L-arginine methyl ester decreased ROS production. These findings strongly suggest that eNOS is a source of ROS in these cells. Although BH4 levels were increased as compared with another endothelial cell line, eNOS levels were >2 orders of magnitude higher. The addition of BH4 resulted in increased NO production and decreased generation of ROS, indicating that bEnd.3 cells produce ROS through eNOS uncoupling because of relative BH4 deficiency. Nevertheless, eNOS-dependent ROS production was not completely abolished by the addition of BH4, suggesting intrinsic superoxide production by eNOS. This study indicates that potentially beneficial sustained increases in eNOS expression and activity could lead to eNOS uncoupling and superoxide production as a consequence. Therefore, sustained increases of eNOS or VEGF activity should be accompanied by concomitant supplementation of BH4.     

非诺贝特通过上调三磷酸鸟苷环化水解酶Ⅰ促进eNOS复偶联

目的探讨非诺贝特发挥降脂外血管内皮保护作用的机制。方法体外培养人脐静脉内皮细胞（HU-VECs）,非诺贝特预处理HUVECs 2 h,再与脂多糖（LPS）共孵育24 h。采用Western blot检测三磷酸鸟苷环化水解酶Ⅰ（GTPCH-Ⅰ）的表达水平,高效液相色谱法检测四氢生物蝶呤（BH4）的表达水平,ELISA检测细胞上清一氧化氮（NO）浓度,利用Confocal方法检测细胞活性氧（ROS）产生水平。结果非诺贝特预处理后,较单纯LPS刺激组,细胞内BH4表达水平及细胞上清NO产生增多,伴有细胞内ROS产生减少（P均〈0.05）;此外,单独给予非诺贝特处理内皮细胞后,可见浓度依赖性上调细胞GTPCH-Ⅰ的表达,非诺贝特（10μmol/L）上调GTPCH-Ⅰ的表达在12 h达到高峰。结论非诺贝特通过上调内皮细胞GTPCH-Ⅰ的表达而增加BH4的水平,进而促进内皮型一氧化氮合酶的复偶联,改善血管内皮功能。     

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

OBJECTIVE: Previous studies from our group have shown a deficit in nitric oxide (NO) bioavailability and an excess production of the superoxide anion (O(2)(-)) in the stroke prone spontaneously hypertensive rat (SHRSP) compared to the normotensive Wistar Kyoto (WKY) strain. This present study has investigated whether adenoviral-mediated gene transfer of human eNOS or Cu/ZnSOD can alter the NO/O(2)(-) balance, thereby improving endothelial function. METHODS: A recombinant adenovirus, Ad/Hu/eNOS, containing the human eNOS cDNA fragment was generated by homologous recombination in 293 cells. Ad/Hu/eNOS or Ad/Cu/ZnSOD was delivered into SHRSP carotid arteries in vivo, using a titre of 2x10(9)-2x10(10) plaque forming units (pfu)/ml, and the effect on gene expression was observed 24 h later. RESULTS: Western blotting confirmed increased enzyme levels of eNOS and Cu/ZnSOD in the viral-infused vessels. Ex vivo, the pressor response to phenylephrine (PE) in the presence of L-NAME was increased in the eNOS-infused arteries relative to the contralateral controls, indicating restoration of basal NO availability to that observed in untreated control WKY rats. Infusion of the SOD virus produced a statistically insignificant increase in NO bioavailability. CONCLUSIONS: Our results support our previous findings obtained using a bovine eNOS recombinant adenovirus, that recombinant adenoviral gene transfer of human eNOS has a significant effect on NO bioavailability. In contrast, AdCu/ZnSOD gene transfer does not elicit an effect in our model. These results indicate that short-term overexpression of a recombinant eNOS, but not Cu/ZnSOD gene, in carotid arteries of the SHRSP is an effective means of locally increasing NO bioavailability to improve endothelial function.     

Regulation of endothelial nitric oxide synthase by tetrahydrobiopterin in vascular disease

Nitric oxide (NO), produced by endothelial nitric oxide synthase (eNOS), is a key signaling molecule in vascular homeostasis. Loss of NO bioavailability due to reduced synthesis and increased scavenging by reactive oxygen species is a cardinal feature of endothelial dysfunction in vascular disease states. The pteridine cofactor tetrahydrobiopterin (BH4) has emerged as a critical determinant of eNOS activity: when BH4 availability is limiting, eNOS no longer produces NO but instead generates superoxide. In vascular disease states, there is oxidative degradation of BH4 by reactive oxygen species. However, augmentation of BH4 concentrations in vascular disease by pharmacological supplementation, by enhancement of its rate of de novo biosynthesis or by measures to reduce its oxidation, has been shown in experimental studies to enhance NO bioavailability. Thus, BH4 represents a potential therapeutic target in the regulation of eNOS function in vascular disease.