Regulation of mycobacterial infection by macrophage Gch1 and tetrahydrobiopterin.

Inducible nitric oxide synthase (iNOS) plays a crucial role in controlling growth of Mycobacterium tuberculosis (M.tb), presumably via nitric oxide (NO) mediated killing. Here we show that leukocyte-specific deficiency of NO production, through targeted loss of the iNOS cofactor tetrahydrobiopterin (BH4), results in enhanced control of M.tb infection; by contrast, loss of iNOS renders mice susceptible to M.tb. By comparing two complementary NO-deficient models, Nos2-/- mice and BH4 deficient Gch1fl/flTie2cre mice, we uncover NO-independent mechanisms of anti-mycobacterial immunity. In both murine and human leukocytes, decreased Gch1 expression correlates with enhanced cell-intrinsic control of mycobacterial infection in vitro. Gene expression analysis reveals that Gch1 deficient macrophages have altered inflammatory response, lysosomal function, cell survival and cellular metabolism, thereby enhancing the control of bacterial infection. Our data thus highlight the importance of the NO-independent functions of Nos2 and Gch1 in mycobacterial control.

Aging modifies the effect of GCH1 RS11158026 on DAT uptake and Parkinson's disease clinical severity.

Novel single nucleotide polymorphisms within Parkinson's disease (PD) can predict disease risk, but their influence on clinical, cognitive, and neurobiological indices remains unexplored. We investigated differences between functional polymorphisms at RS11158026 coding for guanosine triphosphate cyclohydrolase-1 (GCH1), an essential enzyme for dopamine production in nigrostriatal cells. Among newly diagnosed, untreated PD subjects and age-matched controls from the Parkinson's Progression Markers Initiative, T allele carriers showed higher PD risk (odds ratio = 1.23, p = 0.048), earlier age of onset by 5 years (p = 0.003), and lower striatal dopamine reuptake transporter uptake (p = 0.003). Carriers also had increased cerebrospinal fluid α-synuclein (p = 0.016), worse motor function (p = 0.041), anxiety (p = 0.038), and executive function (p < 0.001). Strikingly, these effects were only in younger T carriers (<50 years), where aging quells the effects of these genetic factors. This suggests GCH1 variants affect early PD risk through altered dopamine uptake, and aging alters how genetic factors contribute to disease development. Future studies should investigate how aging modifies genotypes' contributions on PD risk and sequelae.

Role of GTP-CHI links PAH and TH in melanin synthesis in silkworm, Bombyx mori.

In insects, pigment patterns are formed by melanin, ommochromes, and pteridines. Here, the effects of pteridine synthesis on melanin formation were studied using 4th instar larvae of a wild-type silkworm strain, dazao (Bombyx mori), with normal color and markings. Results from injected larvae and in vitro integument culture indicated that decreased activity of guanosine triphosphate cyclohydrolase I (GTP-CH I, a rate-limiting enzyme for pteridine synthesis), lowers BH4 (6R-l-erythro-5,6,7,8-tetrahydrobiopterin, a production correlated with GTP-CH I activity) levels and eliminates markings and coloration. The conversion of phenylalanine and tyrosine to melanin was prevented when GTP-CH I was inhibited. When BH4 was added, phenylalanine was converted to tyrosine, and the tyrosine concentration increased. Tyrosine was then converted to melanin to create normal markings and coloration. Decreasing GTP-CH I activity did not affect L-DOPA (3,4-l-dihydroxyphenylalanine). GTP-CH I affected melanin synthesis by generating the BH4 used in two key reaction steps: (1) conversion of phenylalanine to tyrosine by PAH (phenylalanine hydroxylase) and (2) conversion of tyrosine to L-DOPA by TH (tyrosine hydroxylase). Expression profiles of BmGTPCH Ia, BmGTPCH Ib, BmTH, and BmPAH in the integument were consistent with the current findings.

Cell-autonomous role of endothelial GTP cyclohydrolase 1 and tetrahydrobiopterin in blood pressure regulation.

Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS) function and NO generation. Augmentation of BH4 levels can prevent eNOS uncoupling and can improve endothelial dysfunction in vascular disease states. However, the physiological requirement for de novo endothelial cell BH4 biosynthesis in eNOS function remains unclear. We generated a novel mouse model with endothelial cell-specific deletion of GCH1, encoding GTP cyclohydrolase 1, an essential enzyme for BH4 biosynthesis, to test the cell-autonomous requirement for endothelial BH4 biosynthesis in vivo. Mice with a floxed GCH1 allele (GCH1(fl/fl)) were crossed with Tie2cre mice to delete GCH1 in endothelial cells. GCH1(fl/fl)Tie2cre mice demonstrated virtually absent endothelial NO bioactivity and significantly greater O2 (•-) production. GCH1(fl/fl)Tie2cre aortas and mesenteric arteries had enhanced vasoconstriction to phenylephrine and impaired endothelium-dependent vasodilatations to acetylcholine and SLIGRL. Endothelium-dependent vasodilatations in GCH1(fl/fl)Tie2cre aortas were, in part, mediated by eNOS-derived hydrogen peroxide (H2O2), which mediated vasodilatation through soluble guanylate cyclase. Ex vivo supplementation of aortic rings with the BH4 analogue sepiapterin restored normal endothelial function and abolished eNOS-derived H2O2 production in GCH1(fl/fl)Tie2cre aortas. GCH1(fl/fl)Tie2cre mice had higher systemic blood pressure than wild-type littermates, which was normalized by NOS inhibitor, NG-nitro-L-arginine methyl ester. Taken together, these studies reveal an endothelial cell-autonomous requirement for GCH1 and BH4 in regulation of vascular tone and blood pressure and identify endothelial cell BH4 as a pivotal regulator of NO versus H2O2 as alternative eNOS-derived endothelial-derived relaxing factors.

GTP cyclohydrolase I prevents diabetic-impaired endothelial progenitor cells and wound healing by suppressing oxidative stress/thrombospondin-1.

Endothelial progenitor cell (EPC) dysfunction is a key contributor to diabetic refractory wounds. Endothelial nitric oxide synthase (eNOS), which critically regulates the mobilization and function of EPCs, is uncoupled in diabetes due to decreased cofactor tetrahydrobiopterin (BH4). We tested whether GTP cyclohydrolase I (GTPCH I), the rate-limiting enzyme of BH4 synthesis, preserves EPC function in type 1 diabetic mice. Type 1 diabetes was induced in wild-type (WT) and GTPCH I transgenic (Tg-GCH) mice by intraperitoneal injection of streptozotocin (STZ). EPCs were isolated from the peripheral blood and bone marrow of WT, Tg-GCH, and GTPCH I-deficient hph-1 mice. The number of EPCs was significantly lower in STZ-WT mice and hph-1 mice and was rescued in STZ Tg-GCH mice. Furthermore, GTPCH I overexpression improved impaired diabetic EPC migration and tube formation. EPCs from WT, Tg-GCH, and STZ-Tg-GCH mice were administered to diabetic excisional wounds and accelerated wound healing significantly, with a concomitant augmentation of angiogenesis. Flow cytometry measurements showed that intracellular nitric oxide (NO) levels were reduced significantly in STZ-WT and hph-1 mice, paralleled by increased superoxide anion levels; both were rescued in STZ-Tg-GCH mice. Western blot analysis revealed that thrombospondin-1 (TSP-1) was significantly upregulated in the EPCs of STZ-WT mice and hph-1 mice and suppressed in STZ-treated Tg-GCH mice. Our results demonstrate that the GTPCH I/BH4 pathway is critical to preserve EPC quantity, function, and regenerative capacity during wound healing in type 1 diabetic mice at least partly through the attenuation of superoxide and TSP-1 levels and augmentation of NO level.

The crystal structure reveals the molecular mechanism of bifunctional 3,4-dihydroxy-2-butanone 4-phosphate synthase/GTP cyclohydrolase II (Rv1415) from Mycobacterium tuberculosis.

The enzymes 3,4-dihydroxy-2-butanone 4-phosphate synthase (DHBPS) and GTP cyclohydrolase II (GCHII) catalyze the initial steps of both branches of the bacterial riboflavin-biosynthesis pathway. The structures and molecular mechanisms of DHBPS and GCHII as separate polypeptides are known; however, their organization and molecular mechanism as a bifunctional enzyme are unknown to date. Here, the crystal structure of an essential bifunctional DHBPS/GCHII enzyme from Mycobacterium tuberculosis (Mtb-ribA2) is reported at 3.0 Šresolution. The crystal structure revealed two conformationally different molecules of Mtb-ribA2 in the asymmetric unit that form a dimer via their GCHII domains. Interestingly, analysis of the crystal packing revealed a long `helical-like oligomer' formed by DHBPS and GCHII functional homodimers, thus generating an `open-ended' unit-cell lattice. However, size-exclusion chromatography studies suggest that Mtb-ribA2 exists as a dimer in solution. To understand the discrepancy between the oligomerization observed in solution and in the crystal structure, the DHBPS (Mtb-DHBPS) and GCHII (Mtb-GCHII) domains of Mtb-ribA2 have been cloned, expressed and purified as His-tagged proteins. Size-exclusion chromatography studies indicated that Mtb-GCHII is a dimer while Mtb-DHBPS exists as a monomer in solution. Moreover, kinetic studies revealed that the GCHII activities of Mtb-ribA2 and Mtb-GCHII are similar, while the DHBPS activity of Mtb-ribA2 is much higher than that of Mtb-DHBPS alone. Taken together, the results strongly suggest that Mtb-ribA2 exists as a dimer formed through its GCHII domains and requires full-length Mtb-ribA2 for optimal DHBPS activity.

Regulation of ß-adrenergic control of heart rate by GTP-cyclohydrolase 1 (GCH1) and tetrahydrobiopterin.

AIMS: Clinical markers of cardiac autonomic function, such as heart rate and response to exercise, are important predictors of cardiovascular risk. Tetrahydrobiopterin (BH4) is a required cofactor for enzymes with roles in cardiac autonomic function, including tyrosine hydroxylase and nitric oxide synthase. Synthesis of BH4 is regulated by GTP cyclohydrolase I (GTPCH), encoded by GCH1. Recent clinical studies report associations between GCH1 variants and increased heart rate, but the mechanistic importance of GCH1 and BH4 in autonomic function remains unclear. We investigate the effect of BH4 deficiency on the autonomic regulation of heart rate in the hph-1 mouse model of BH4 deficiency. METHODS AND RESULTS: In the hph-1 mouse, reduced cardiac GCH1 expression, GTPCH enzymatic activity, and BH4 were associated with increased resting heart rate; blood pressure was not different. Exercise training decreased resting heart rate, but hph-1 mice retained a relative tachycardia. Vagal nerve stimulation in vitro induced bradycardia equally in hph-1 and wild-type mice both before and after exercise training. Direct atrial responses to carbamylcholine were equal. In contrast, propranolol treatment normalized the resting tachycardia in vivo. Stellate ganglion stimulation and isoproterenol but not forskolin application in vitro induced a greater tachycardic response in hph-1 mice. ß1-adrenoceptor protein was increased as was the cAMP response to isoproterenol stimulation. CONCLUSION: Reduced GCH1 expression and BH4 deficiency cause tachycardia through enhanced ß-adrenergic sensitivity, with no effect on vagal function. GCH1 expression and BH4 are novel determinants of cardiac autonomic regulation that may have important roles in cardiovascular pathophysiology.

H(2)O(2) increases de novo synthesis of (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin via GTP cyclohydrolase I and its feedback regulatory protein in vitiligo.

Patients with vitiligo accumulate up to 10(-3) mol/L concentrations of H(2)O(2) in their epidermis, which in turn affects many metabolic pathways in this compartment, including the synthesis and recycling of the cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH(4)). De novo synthesis of 6BH(4) is dependent on the rate-limiting enzyme GTP cyclohydrolase I (GTPCHI) together with its feedback regulatory protein (GFRP). This step is controlled by 6BH(4) and the essential amino acid L-phenylalanine. In the study presented here we wanted to investigate whether H(2)O(2) affects the GTPCHI/GFRP cascade in these patients. Our results demonstrated concentration-dependent regulation of rhGTPCHI where 100 micromol/L H(2)O(2) was the optimum concentration for the activation of the enzyme and >300 micromol/L resulted in a decrease in activity. Oxidation of GFRP and GTPCHI does not affect feedback regulation via L-phenylalanine and 6BH(4). In vitiligo a constant upregulation of 6BH(4) de novo synthesis results from epidermal build up of L-phenylalanine that is not controlled by H(2)O(2). Taking the results together, 6BH(4) de novo synthesis is controlled by H(2)O(2) in a concentration-dependent manner, but H(2)O(2)-mediated oxidation does not affect the functionality of the GTPCHI/GFRP complex.

Hsp27 decreases inclusion body formation from mutated GTP-cyclohydrolase I protein.

GTP cyclohydrolase I (GCH), an oligomeric protein composed of 10 identical subunits, is required for the synthesis of neurotransmitters; mutations in GCH are associated with dopa-responsive dystonia (DRD) and hyperphenylalaninemia. Mutated GCH proteins are unstable and prone to dominant-negative effect. We show herein that expression of the GCH mutant GCH-201E or the splicing variant GCH-II caused intracellular inclusion bodies. When Hsp27 was expressed together with the GCH mutants, Hsp27 expression decreased the formation of inclusion bodies by GCH (as assessed by immunofluorescence) and decreased the amount of insoluble GCH mutant proteins (as assessed by Western blot). Transfection of pcDNA-Hsp27-S3D, a phosphorylation-mimicry Hsp27 mutant, was more effective at the mutated GCH proteins than transfection with pcDNA-Hsp27, but okadaic acid, a phosphatase inhibitor, enhanced the effect of pcDNA-Hsp27. Hsp27-S3D also abolished the dominant-negative action of GCH-II. The mutated GCH proteins interacted with the wild-type GCH protein; the inclusion bodies were positive for lysosomal marker LAMP1, soluble in 2% SDS, and were not ubiquitinated. Phophorlyated Hsp27 also decreased the inclusion body formation by the huntingtin polyglutamines. Therefore, diseases involving mutated oligomeric proteins would be manageable by chaperone therapies.

Understanding functional divergence in proteins by studying intragenomic homologues.

Studies of intragenomic homologues in bacterial genomes can provide valuable insights into functional divergence. Three GTP cyclohydrolase II homologues in the Streptomyces coelicolor genome have been shown to catalyze two related but distinct reactions [Spoonamore, J. E., Dahlgran, A. L., Jacobsen, N. E., and Bandarian, V. (2006) Biochemistry 45, 12144-12155]. Two of the homologues, SCO 1441 and 2687, convert GTP to 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (APy); one of the homologues (SCO 6655) produces 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (FAPy). We show herein that the differences in the fate of GTP in SCO 6655 relative to SCO 1441 and 2687 results from a single amino acid substitution in the active site of the protein: a Tyr residue in the active sites of SCO 1441 and SCO 2687 is replaced with a Met in SCO 6655. Site-directed interchange of this residue in the three S. coelicolor intragenomic homologues is necessary and sufficient for interconversion of catalytic function which, except for SCO 1441, occurs with little loss of catalytic efficiency. Furthermore, we show that of 14 additional site-directed variants at this position of SCO 6655, His confers catalytic efficiency within 1 order of magnitude of that of the wild type and supports conversion of GTP to both FAPy and APy. The results demonstrate a clear set of mutational events that permit GCH II to produce either FAPy or APy. These results highlight a mechanism whereby functional divergence can be achieved in enzymes that catalyze multistep transformations.

Endothelium-targeted transgenic GTP-cyclohydrolase I overexpression inhibits neointima formation in mouse carotid artery.

1. Tetrahydrobiopterin (BH(4)) is an essential cofactor that maintains the normal function of endothelial nitric oxide (NO) synthase. Restenosis is a key complication after transluminal angioplasty. Guanosine 5'-triphosphate-cyclohydrolase I (GTPCH) is the first rate-limiting enzyme for de novo BH(4) synthesis. However, the role of GTPCH in restenosis is not fully understood. The present study tested the hypothesis that endothelial-targeted GTPCH overexpression retards neointimal formation, a hallmark of restenosis, in mouse carotid artery. 2. Transluminal wire injury was induced in the left carotid arteries of adult male wild-type C57BL/6 (WT) and endothelial GTPCH transgenic (Tg-GCH) mice. Re-endothelialization was confirmed with in vivo Evans blue staining. Endothelium-dependent and -independent relaxations were measured using isometric tension recording. Morphological analysis was performed 2 and 4 weeks after carotid injury to assess neointimal formation. Fluorescence-based high-performance liquid chromatography (HPLC) was used to determine GTPCH activity and BH(4) levels. Basal NO release following carotid injury was assessed by N(G)-nitro-L-arginine methyl ester-induced vascular contraction. 3. The endothelium was completely removed upon transluminal wire injury and full re-endothelialization was achieved at Day 10. Endothelium-dependent relaxation was impaired 10 days and 4 weeks after carotid injury, whereas endothelium-independent relaxation remained unaffected. Morphological analysis revealed that the endothelial-specific overexpression of GTPCH reduced neointimal formation and medial hypertrophy 2 and 4 weeks after carotid injury. Both arterial GTPCH enzyme activity and BH(4) levels were significantly elevated in Tg-GCH mice compared with WT mice and basal NO release of the injured carotid artery tended to increase in Tg-GCH mice. 4. These findings suggest that the endothelial overexpression of GTPCH increased endothelial BH(4) synthesis and played a preventive role in neointimal formation induced by endothelium denudation.

GTP cyclohydrolase and tetrahydrobiopterin regulate pain sensitivity and persistence.

We report that GTP cyclohydrolase (GCH1), the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis, is a key modulator of peripheral neuropathic and inflammatory pain. BH4 is an essential cofactor for catecholamine, serotonin and nitric oxide production. After axonal injury, concentrations of BH4 rose in primary sensory neurons, owing to upregulation of GCH1. After peripheral inflammation, BH4 also increased in dorsal root ganglia (DRGs), owing to enhanced GCH1 enzyme activity. Inhibiting this de novo BH4 synthesis in rats attenuated neuropathic and inflammatory pain and prevented nerve injury-evoked excess nitric oxide production in the DRG, whereas administering BH4 intrathecally exacerbated pain. In humans, a haplotype of the GCH1 gene (population frequency 15.4%) was significantly associated with less pain following diskectomy for persistent radicular low back pain. Healthy individuals homozygous for this haplotype exhibited reduced experimental pain sensitivity, and forskolin-stimulated immortalized leukocytes from haplotype carriers upregulated GCH1 less than did controls. BH4 is therefore an intrinsic regulator of pain sensitivity and chronicity, and the GTP cyclohydrolase haplotype is a marker for these traits.

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.

Pivotal role for endothelial tetrahydrobiopterin in pulmonary hypertension.

BACKGROUND: Pulmonary hypertension is a fatal disease characterized by vasoconstriction and vascular remodeling. Loss of endothelial nitric oxide bioavailability is implicated in pulmonary hypertension pathogenesis. Recent evidence suggests that the cofactor tetrahydrobiopterin (BH4) is an important regulator of nitric oxide synthase enzymatic function. METHODS AND RESULTS: In the hph-1 mouse with deficient BH4 biosynthesis, BH4 deficiency caused pulmonary hypertension, even in normoxic conditions, and greatly increased susceptibility to hypoxia-induced pulmonary hypertension. In contrast, augmented BH4 synthesis in the endothelium, by targeted transgenic overexpression of GTP-cyclohydrolase I (GCH), prevented hypoxia-induced pulmonary hypertension. Furthermore, specific augmentation of endothelial BH4 in hph-1 mice by crossing with GCH transgenic mice rescued pulmonary hypertension induced by systemic BH4 deficiency. Lung BH4 availability controlled pulmonary vascular tone, right ventricular hypertrophy, and vascular structural remodeling in a dose-dependent manner in both normoxia and hypoxia. Furthermore, BH4 availability had striking effects on the immediate vasoconstriction response to acute hypoxia. These effects of BH4 were mediated through the regulation of nitric oxide compared with superoxide synthesis by endothelial nitric oxide synthase. CONCLUSIONS: Endothelial BH4 availability is essential for maintaining pulmonary vascular homeostasis, is a critical mediator in the pathogenesis of pulmonary hypertension, and is a novel therapeutic target.

Reduction of GTP cyclohydrolase I feedback regulating protein expression by hydrogen peroxide in vascular endothelial cells.

We examined the effect of H(2)O(2) on the expression of GTP cyclohydrolase I (GTPCH) feedback regulating protein (GFRP). Addition of H(2)O(2) to endothelial cells decreased GFRP mRNA levels, in contrast to the increase of tetrahydrobiopterin (BH(4)) content and GTPCH mRNA levels. The inhibitors of nitric oxide (NO) synthase and GTPCH had no influence on the decrease of GFRP mRNA levels in H(2)O(2)-treated cells. It is suggested that H(2)O(2) induces BH(4) synthesis through not only induction of GTPCH but also reduction of GFRP. The decrease of GFRP mRNA level appears to be independent of the produced NO and BH(4).

Cytokine-stimulated GTP cyclohydrolase I expression in endothelial cells requires coordinated activation of nuclear factor-kappaB and Stat1/Stat3.

Endothelial production of nitric oxide (NO) is dependent on adequate cellular levels of tetrahydrobiopterin (BH4), an important cofactor for the nitric oxide synthases. Vascular diseases are often characterized by vessel wall inflammation and cytokine treatment of endothelial cells increases BH4 levels, in part through the induction of GTP cyclohydrolase I (GTPCH I), the rate-limiting enzyme for BH4 biosynthesis. However, the molecular mechanisms of cytokine-mediated GTPCH I induction in the endothelium are not entirely clear. We sought to investigate the signaling pathways whereby cytokines induce GTPCH I expression in human umbilical vein endothelial cells (HUVECs). Interferon-gamma (IFN-gamma) induced endothelial cell GTPCH I protein and BH4 modestly, whereas high-level induction required combinations of IFN-gamma and tumor necrosis factor-alpha (TNF-alpha). In the presence of IFN-gamma, TNF-alpha increased GTPCH I mRNA in a manner dependent on nuclear factor-kappaB (NF-kappaB), as this effect was abrogated by overexpression of a dominant-negative IkappaB construct. HUVEC IFN-gamma treatment resulted in signal transducer and activator of transcription 1 (Stat1) activation and DNA binding in a Jak2-dependent manner, as this was inhibited by AG490. Conversely, overexpression of Jak2 effectively substituted for IFN-gamma in supporting TNF-alpha-mediated GTPCH I induction. The role of IFN-gamma was also Stat1-dependent as Stat1-null cells exhibited no GTPCH I induction in response to cytokines. However, Stat1 activation with oncostatin M failed to support TNF-alpha-mediated GTPCH I induction because of concomitant Stat3 activation. Consistent with this notion, siRNA-mediated Stat3 gene silencing allowed oncostatin M to substitute for IFN-gamma in this system. These data implicate both NF-kappaB and Stat1 in endothelial cell cytokine-stimulated GTPCH I induction and highlight the role of Stat3 in modulating Stat1-supported gene transcription. Thus, IFN-gamma and TNF-alpha exert distinct but cooperative roles for BH4 biosynthesis in endothelium that may have important implications for vascular function during vascular inflammation.

Increased endothelial tetrahydrobiopterin synthesis by targeted transgenic GTP-cyclohydrolase I overexpression reduces endothelial dysfunction and atherosclerosis in ApoE-knockout mice.

OBJECTIVE: Increased production of reactive oxygen species and loss of endothelial nitric oxide (NO) bioactivity are key features of vascular disease states such as atherosclerosis. Tetrahydrobiopterin (BH4) is a required cofactor for NO synthesis by endothelial nitric oxide synthase (eNOS); pharmacologic studies suggest that reduced BH4 availability may be an important mediator of endothelial dysfunction in atherosclerosis. We aimed to investigate the importance of endothelial BH4 availability in atherosclerosis using a transgenic mouse model with endothelial-targeted overexpression of the rate-limiting enzyme in BH4 synthesis, GTP-cyclohydrolase I (GTPCH). METHODS AND RESULTS: Transgenic mice were crossed into an ApoE knockout (ApoE-KO) background and fed a high-fat diet for 16 weeks. Compared with ApoE-KO controls, transgenic mice (ApoE-KO/GCH-Tg) had higher aortic BH4 levels, reduced endothelial superoxide production and eNOS uncoupling, increased cGMP levels, and preserved NO-mediated endothelium dependent vasorelaxations. Furthermore, aortic root atherosclerotic plaque was significantly reduced in ApoE-KO/GCH-Tg mice compared with ApoE-KO controls. CONCLUSIONS: These findings indicate that BH4 availability is a critical determinant of eNOS regulation in atherosclerosis and is a rational therapeutic target to restore NO-mediated endothelial function and reduce disease progression.

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.