Reduction Of BH4 Research Articles

Abstract 13625: Autophagy Plays a Key Role in the Loss of Nitric Oxide Signaling and Endothelial Dysfunction in Pulmonary Vascular Diseases

Introduction: Congenital heart defects (CHDs) are the most common type of birth defect. Children or infants with CHD often develop pulmonary vascular diseases (PVD) due to increased pulmonary blood flow and pressure. Unfortunately, there is no effective therapy to limit the shared pathophysiologic features of pulmonary endothelial dysfunction and vascular remodeling. Autophagy, a lysosome-dependent proteolytic pathway, enables cells to sequester portions of cytosolic proteins or damaged organelles into autophagosomes and degrade them in autolysosomes. Studies have found that autophagy participates in pulmonary hypertension, acute lung injury, lung cancer, and other pulmonary diseases. Hypothesis: Autophagy plays a key role in the development of pulmonary endothelial dysfunction in PVD associated with CHD. Methods and Results: We utilized lung endothelial cells isolated from an ovine CHD model, created by the fetal placement of an aortopulmonary graft (Shunt). First, we confirmed that autophagy was activated in Shunt pulmonary artery endothelial cells (PAEC), with increased LC3-II/LC3-I ratio (1.45 fold increase, n=3, p<0.05) and reduced p62 levels (64% reduction, n=3, p<0.05). Shunt PAECs were found hyperproliferative (BrdU Assay A 450 : control vs. Shunt: 2.36 ± 0.05 vs . 2.58 ± 0.05, n=9, p<0.01), which can be attenuated by pharmacologic autophagy inhibitor bafilomycin A1 (2.30 ± 0.05, n=9, p<0.01 compared with Shunt) or 3-MA (2.20 ± 0.03, n=9, p<0.001 compared with Shunt). Shunt PAECs exhibited significant endothelial Nitic Oxide Synthase (eNOS) uncoupling by BH4 reduction (31% reduction, n=3, p<0.01). Bafilomycin A1 reversed eNOS uncoupling in Shunt PAECs by BH4 restoration (2.1 fold increase, n=3, p<0.01), resulting in elevated cellular NOx level (1.8 increase fold, n=3, p<0.01). Mechanistically, this was dependent on increased levels of GTP cyclohydrolase I (GCH1) protein in Shunt PAEC. Conclusions: Enhanced autophagy plays a key role in the loss of NO signaling and endothelial dysfunction associated with CHD. We speculate that autophagy might be an effective therapeutic target for pulmonary vascular diseases.

Laser in situ synthesis of NiFe2O4 nanoparticle-anchored NiFe(OH)x nanosheets as advanced electrocatalysts for the oxygen evolution and urea oxidation reactions

The in situ growth of oxygen-vacancy-rich hybrid electrocatalysts with a high surface area is considered to be an effective strategy for enhancing the oxygen evolution reaction (OER) and urea oxidation reaction (UOR) performance. However, in situ synthesis is often difficult due to multistep procedures and harsh conditions. Herein, NiFe2O4 nanoparticles are successfully grown in situ on NiFe(OH)x nanosheets via using unique laser ablation in liquids technology. The quenching effect of the pulsed laser and the reduction of BH4− promote the generation of oxygen-vacancy-abundant NiFe(OH)x/NiFe2O4 nanocomposites, and the weak alkalinity of the NaBH4 solutions accelerates the production of NiFe(OH)x nanosheets. Profited from the affluent oxygen vacancies and enhancive surface areas, the NiFe(OH)x/NiFe2O4 nanocomposites reveal enhancement in electrocatalytic OER performance with small overpotential of 249 mV at 10 mA cm−2 and a Tafel slope of 40.1 mV dec−1, accompanied by superior stability (the current density retention after 150 h is 98.5% of the initial value). In addition, the NiFe(OH)x/NiFe2O4 hybrid electrocatalysts also exhibit excellent electrocatalytic performance for the urea oxidation reaction (a potential of 1.343 V at 10 mA cm−2 and a Tafel slope of 19.9 mV dec−1). This work can provide guidance for exploring other hydroxide/oxide materials with abundant oxygen vacancies and high surface areas.

Epigenetic silencing of GCH1promotes hepatocellular carcinoma growth by activating superoxide anion-mediated ASK1/p38 signaling via inhibiting tetrahydrobiopterin de novo biosynthesis

Metabolic reprogramming is a hallmark of cancer, including hepatocellular carcinoma (HCC). However, its role in HCC remains to be elucidated. Herein, we identified GTP cyclohydrolase 1 (GCH1), the first rate-limiting enzyme in tetrahydrobiopterin (BH4) de novo biosynthesis, as a novel metabolic regulator of HCC. GCH1 was frequently down-regulated in HCC tissues and cell lines by promoter methylation. Low GCH1 expression was associated with larger tumor size, increased tumor number, and worse prognosis in two independent cohorts of HCC patients. Functionally, GCH1 silencing promoted HCC growth in vitro and in vivo, while GCH1 overexpression exerted an opposite effect. The metabolite BH4 inhibited HCC growth in vitro and in vivo. GCH1 silencing exerted its growth-promoting effect through directly inhibiting BH4 de novo biosynthesis. Mechanistically, GCH1 silencing activated ASK1/p38 signaling; pharmacological or genetic inhibition of ASK1 or p38 abolished GCH1 silencing-induced growth-promoting effect. Further mechanistic studies found that GCH1 silencing-induced BH4 reduction resulted in an increase of intracellular superoxide anion levels in a dose-dependent manner, which mediated the activation of ASK1/p38 signaling. Collectively, our study reveals that epigenetic silencing of GCH1 promotes HCC growth by activating superoxide anion-mediated ASK1/p38 signaling via inhibiting BH4 de novo biosynthesis, suggesting that targeting GCH1/BH4 pathway may be a promising therapeutic strategy to combat HCC.

GTP-cyclohydrolase deficiency induced peripheral and deep microcirculation dysfunction with age

The present study assessed the impact of impaired tetrahydrobiopterin (BH4) production on vasoreactivity from conduit and small arteries along the vascular tree as seen during aging. For this purpose, the mutant hyperphenylalaninemic mouse (hph-1) was used. This model is reported to be deficient in GTP cyclohydrolase I, a rate limiting enzyme in BH4 biosynthesis. BH4 is a key regulator of vascular homeostasis by regulating the nitric oxide synthase 3 (NOS3) activity.In GTP-CH deficient mice, the aortic BH4 levels were decreased, by −77% in 12 week-middle-aged mice (young) and by −83% in 35–45 week-middle-aged mice (middle-aged). In young hph-1, the mesenteric artery ability to respond to flow was slightly reduced by 9%. Aging induced huge modification in many vascular functions. In middle-aged hph-1, we observed a decrease in aortic cGMP levels, biomarker of NO availability (−46%), in flow-mediated vasodilation of mesenteric artery (−31%), in coronary hyperemia response measured in isolated heart following transient ischemia (−27%) and in cutaneous microcirculation dilation in response to acetylcholine assessed in vivo by laser-doppler technic (−69%). In parallel, the endothelium-dependent relaxation in response to acetylcholine in conduit blood vessel, measured on isolated aorta rings, was unchanged in hph-1 mice whatever the age.Our findings demonstrate that in middle-aged GTP-CH depleted mice, the reduction of BH4 was characterized by an alteration of microcirculation dilatory properties observed in various parts of the vascular tree. Large conduit blood vessels vasoreactivity, ie aorta, was unaltered even in middle-aged mice emphasizing the main BH4-deletion impact on the microcirculation.

Pharmacological Assessment of Sepiapterin Reductase Inhibition on Tactile Response in the Rat.

There is an unmet medical need for nonopioid pain therapies in human populations; several pathways are under investigation for possible therapeutic intervention. Tetrahydrobiopterin (BH4) has received attention recently as a mediator of neuropathic pain. Recent reports have implicated sepiapterin reductase (SPR) in this pain pathway as a regulator of BH4 production. To evaluate the role of SPR inhibition on BH4 reduction, we developed analytical methods to monitor the relationship between the plasma concentration of test article and endogenous pterins and applied these in the rat spinal nerve ligation pain model. Sepiapterin is an endogenous substrate, which accumulates upon inhibition of SPR. In response to a potent inhibitor of SPR, plasma concentrations of sepiapterin increased proportionally with exposure. An indirect-effect pharmacokinetic/pharmacodynamic model was developed to describe the relationship between the plasma pharmacokinetics of test article and plasma sepiapterin levels in the rat, which was used to determine an in vivo SPR IC50 value. SPR inhibition and mechanical allodynia were assessed coordinately with pterin biomarkers in plasma and at the site of neuronal injury (i.e., dorsal root ganglion). Upon daily oral administration for 3 consecutive days, unbound plasma concentrations of test article exceeded the unbound in vivo rat SPR IC90 throughout the dose intervals, leading to a 60% reduction in BH4 in the dorsal root ganglion. Despite evidence for pharmacological modulation of the BH4 pathway, there was no significant effect on the tactile paw withdrawal threshold relative to vehicle-treated controls.

A novel GTPCH deficiency mouse model exhibiting tetrahydrobiopterin-related metabolic disturbance and infancy-onset motor impairments

BackgroundGTP cyclohydrolase I (GTPCH) deficiency could impair the synthesis of tetrahydrobiopterin and causes metabolic diseases involving phenylalanine catabolism, neurotransmitter synthesis, nitric oxide production and so on. Though improvements could be achieved by tetrahydrobiopterin and neurotransmitter precursor levodopa supplementation, residual motor and mental deficits remain in some patients. An appropriate GTPCH deficiency animal model with clinical symptoms, especially the motor impairments, is still not available for mechanism and therapy studies yet. Objectives and methodsTo investigate whether the heterozygous GTPCH missense mutation p.Leu117Arg identified from a patient with severe infancy-onset dopa-responsive motor impairments is causative and establish a clinical relevant GTPCH deficiency mouse model, we generated a mouse mutant mimicking this missense mutation using the CRISPR/Cas9 technology. Series of characterization experiments on the heterozygous and homozygous mutants were conducted. ResultsThe expressions of GTPCH were not significantly changed in the mutants, but the enzyme activities were impaired in the homozygous mutants. BH4 reduction and phenylalanine accumulation were observed both in the liver and brain of the homozygous mutants. Severer metabolic disturbance occurred in the brain than in the liver. Significant reduction of neurotransmitter dopamine, norepinephrine and serotonin was observed in the brains of homozygous mutants. Live-born homozygous mutants exhibited infancy-onset motor and vocalization deficits similar to the disease symptoms observed in the patient, while no obvious symptoms were observed in the young heterozygous mutant mice. With benserazide-levodopa treatment, survival of the homozygous mutants was improved but not completely rescued. ConclusionsThe GTPCH p.Leu117Arg missense mutation is deleterious and could cause tetrahydrobiopterin, phenylalanine and neurotransmitter metabolic disturbances and infancy-onset motor dysfunctions recessively. This is the first GTPCH deficiency mouse model which could be live-born and exhibits significant motor impairments. The different extents of BH4 reduction and phenylalanine accumulation observed between liver and brain in response to GTPCH deficiency gives potential new insights into the vulnerability of brain to GTPCH deficiency.

Heterozygous mutations in GTP-cyclohydrolase-1 reduce BH4 biosynthesis but not pain sensitivity.

Human studies have demonstrated a correlation between noncoding polymorphisms of "the pain protective" haplotype in the GCH1 gene that encodes for GTP cyclohydrolase I (GTPCH1)-which leads to reduced tetrahydrobiopterin (BH4) production in cell systems-and a diminished perception of experimental and clinical pain. Here, we investigate whether heterozygous mutations in the GCH1 gene which lead to a profound BH4 reduction in patients with dopa-responsive dystonia (DRD) have any effect on pain sensitivity. The study includes an investigation of GCH1-associated biomarkers and pain sensitivity in a cohort of 22 patients with DRD and 36 controls. The patients with DRD had, when compared with controls, significantly reduced levels of BH4, neopterin, biopterin, and GTPCH1 in their urine, blood, or cytokine-stimulated fibroblasts, but their pain response with respect to non-painful stimulation, (acute) stimulus-evoked pain, or pain response after capsaicin-induced sensitization was not significantly different. A family-specific cohort of 11 patients with DRD and 11 controls were included in this study. The patients with DRD were heterozygous for the pain protective haplotype in cis with the GCH1 disease-causing mutation, c.899T>C. No effect on pain perception was observed for this combined haplotype. In conclusion, a reduced concentration of BH4 is not sufficient to alter ongoing pain sensitivity or evoked pain responses.

Dexamethasone, tetrahydrobiopterin and uncoupling of endothelial nitric oxide synthase.

ObjectiveTo find out whether dexamethasone induces an uncoupling of the endothelial nitric oxide synthase (eNOS).Methods & ResultsA major cause of eNOS uncoupling is a deficiency of its cofactor tetrahydrobiopterin (BH4). Treatment of human EA.hy 926 endothelial cells with dexamethasone decreased mRNA and protein expression of both BH4-synthesizing enzymes: GTP cyclohydrolase I and dihydrofolate reductase. Consistently, a concentration- and time-dependent reduction of BH4, dihydrobiopterin (BH2) as well as BH4: BH2 ratio was observed in dexamethasone-treated cells. Surprisingly, no evidence for eNOS uncoupling was found. We then analyzed the expression and phosphorylation of the eNOS enzyme. Dexamethasone treatment led to a down-regulation of eNOS protein and a reduction of eNOS phosphorylation at serine 1177. A reduction of eNOS expression may lead to a relatively normal BH4: eNOS molar ratio in dexamethasone-treated cells. Because the BH4-eNOS stoichiometry rather than the absolute BH4 amount is the key determinant of eNOS functionality (i.e., coupled or uncoupled), the down-regulation of eNOS may represent an explanation for the absence of eNOS uncoupling. Phosphorylation of eNOS at serine 1177 is needed for both the NO-producing activity of the coupled eNOS and the superoxide-producing activity of the uncoupled eNOS. Thus, a reduction of serine 1177 phosphorylation may render a potentially uncoupled eNOS hardly detectable.ConclusionsAlthough dexamethasone reduces BH4 levels in endothelial cells, eNOS uncoupling is not evident. The reduction of NO production in dexamethasone-treated endothelial cells is mainly attributable to reduced eNOS expression and decreased eNOS phosphorylation at serine 1177.

Early replacement therapy in a first Japanese case with autosomal recessive guanosine triphosphate cyclohydrolase I deficiency with a novel point mutation

Autosomal recessive guanosine triphosphate cyclohydrolase I (GTPCH) deficiency is an inborn error of tetrahydrobiopterin (BH4) synthesis from GTP. GTPCH deficiency causes severe reduction of BH4, resulting in hyperphenylalaninemia (HPA) and decreased dopamine and serotonin synthesis. Without treatment, a patient with GTPCH deficiency develops complex neurological dysfunctions, including dystonia and developmental delays. The first Japanese patient with GTPCH deficiency was discovered by HPA during asymptomatic newborn screening. The phenylalanine level at the age of 5days was 1273μmol/L (cutoff value, 180.0μmol/L). The high serum phenylalanine level was decreased to normal after adequate BH4 oral supplementation. Serum and urinary pteridine examination revealed very low levels of neopterin and biopterin. Sequence analysis of GCH1 revealed compound heterozygous point mutations, including a novel point mutation (p.R235W). Replacement therapy with BH4 and L-dopa/carbidopa were started at the age of 1month, and 5-hydroxytryptophan (5-HTP) was started at the age of 5months. At 10months of age, the patient showed slight dystonia but no obvious developmental delay. Cerebrospinal fluid should be examined to determine the appropriate dosage of supplement drugs. In conclusion, it is important to control the serum phenylalanine level and perform early replacement of neurotransmitters to prevent neurological dysfunction.

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.

Endothelial Dysfunction of Coronary Resistance Arteries Is Improved by Tetrahydrobiopterin in Atherosclerosis

Tetrahydrobiopterin (BH4), an essential cofactor for the synthesis of NO, improves endothelial dysfunction after ischemia/reperfusion. Therefore, we hypothesized that reduction of BH4 is involved in the attenuation of endothelium-dependent vasodilation in atherosclerosis, and we investigated the effect of alterations of the BH4 level on the vasodilatory potential of coronary resistance vessels from humans and pigs with atherosclerosis. Coronary arterioles were obtained from patients undergoing CABG (atherosclerosis group) or valve replacement (control group) and from pigs fed either a standard diet (control group) or atherogenic diet (atherosclerosis group). After isolation, vessels were cannulated, pressurized, and placed on the stage of an inverted microscope. Dose-response curves were investigated in response to the endothelium-dependent agonists histamine, serotonin, and acetylcholine (for pigs, substance P) and to the endothelium-independent agonist sodium nitroprusside (SNP) under control conditions and before and after incubation of the vessels with sepiapterin (substrate for BH4 synthesis). In vessels from patients and from animals with atherosclerosis, compared with vessels from the control groups, there was a significant (P:<0.05) reduction of vasodilation to all tested endothelium-dependent agonists but not to SNP. After application of sepiapterin, the responses to the endothelium-dependent agonists but not to SNP were significantly improved in vessels from the atherosclerosis groups. Sepiapterin did not influence vascular reactivity in the control groups. Atherosclerosis severely compromises endothelial function of coronary resistance arteries. Administration of sepiapterin leads to a significant improvement of endothelium-dependent vasodilatation to different agonists in vessels from humans and pigs with atherosclerosis. Therefore, we conclude that a reduced availability of BH4 is involved in the development of endothelial dysfunction in atherosclerosis.

Identification of the phosphorylated intermediate of the Neurospora plasma membrane H+-ATPase as beta-aspartyl phosphate.

The chemical nature of the phosphoryl enzyme linkage of the electrogenic proton-translocating ATPase (ATP phosphohydrolase, EC 3.6.1.3) in the plasma membrane of Neurospora has been identified as a mixed anhydride between phosphate and the beta-carboxyl group of an aspartic acid residue in the polypeptide chain. Incubation of isolated Neurospora plasma membrane vesicles containing 32P-labeled ATPase in buffers of increasing pH followed by analysis of the hydrolysis products yielded a pH versus hydrolysis profile characteristic of an acyl phosphate linkage. Reaction of labeled membranes with hydroxylamine at pH 5.3 also released [32P]i from the ATPase. Amino acid analyses of the Na[3H]BH4 reduction products obtained from membranes containing phosphorylated and dephosphorylated ATPase identified [3H]homoserine, the expected reduction product of beta-aspartyl phosphate, as the only additional tritiated reduction product in the samples from phosphorylated membranes. Tritium was not found in alpha-amino-delta-hydroxyvaleric acid, the reduction product of gamma-glutamyl phosphate, nor in proline, the degradation product of alpha-amino-delta-hydroxyvaleric acid. These results indicate that the phosphorylated intermediate of the Neurospora plasma membrane ATPase is a beta-aspartyl phosphate identical with that already known to exist in the Na+:K+- and Ca2+-translocating ATPases of animal cell origin. A common model for the mechanisms of all 3 ion-translocating ATPases is presented.

Gene mapping and gene enrichment by the avidin-biotin interaction: use of cytochrome-c as a polyamine bridge

A modification of previously described methods of electron microscopic gene mapping and of gene enrichment based on the avidin-biotin interaction is presented. The modification consists of coupling cytochrome-c instead of pentane diamine to the oxidized 2', 3' terminus of an RNA by Schiff base formation and BH-4 reduction. The RNA-cytochrome-c conjugate is purified by a simple chromatographic procedure; several biotins are attached to the cytochrome moiety by acylation. The extended arm between biotin and RNA gives efficient electron microscopic gene mapping of DNA:RNA-biotin hybrids with avidin-ferritin and avidin-polymethacylate sphere labels and efficient gene enrichment by buoyant banding of DNA:RNA-biotin:avidin-spheres in CsCl. A 1400 fold enrichment (thus, 25% pure) and a 90% yield of long Drosophila DNA strands with 5S RNA genes is achieved.