BH4 Levels Research Articles

BH4-Mediated Enhancement of Endothelial Nitric Oxide Synthase Activity Reduces Hyperoxia-Induced Endothelial Damage and Preserves Vascular Integrity in the Neonate.

PurposeEndothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) has important vasoprotective functions that are compromised in the vasodegenerative phase of retinopathy of prematurity, owing to hyperoxia-induced depletion of the essential NOS cofactor BH4. Because modulating eNOS function can be beneficial or detrimental, our aim was to investigate the effect of BH4 supplementation on eNOS function and vascular regression in hyperoxia.MethodsEndothelial-specific eNOS-green fluorescent protein (GFP) overexpressing mice at postnatal day 7 (P7) were exposed to hyperoxia for 48 hours in the presence or absence of supplemental BH4, achieved by administration of sepiapterin, a stable BH4 precursor. Tissue was collected either for retinal flat mounts that were stained with lectin to determine the extent of vessel coverage or for analysis of BH4 by high-performance liquid chromatography, nitrotyrosine (NT) marker by Western blotting, VEGF expression by ELISA, and NOS activity by arginine-to-citrulline conversion. Primary retinal microvascular endothelial cells (RMEC) were similarly treated, and hyperoxia-induced damage was determined.ResultsSepiapterin effectively enhanced BH4 levels in hyperoxia-exposed retinas and brains, elevated NOS activity, and reduced NT-modified protein, leading to reversal of the exacerbated vasoregression observed in the presence of eNOS overexpression. In RMECs, hyperoxia-mediated depletion of BH4 dysregulated the redox balance by reducing nitrite and elevating superoxide and impaired proliferative ability. BH4 supplementation restored normal RMEC proliferation in vitro and also in vivo, providing a mechanistic link with the enhanced vascular coverage in eNOS-GFP retinas.ConclusionsThese results demonstrate that BH4 supplementation corrects hyperoxia-induced RMEC dysfunction and preserves vascular integrity by enhancing eNOS function.

Tetrahydrobiopterin contributes to the proliferation of mesangial cells and accumulation of extracellular matrix in early-stage diabetic nephropathy

Nitric oxide (NO) plays an important role in the progression of early-stage diabetic nephropathy (DN), which is found to contribute to extracellular matrix (ECM) accumulation in mesangial cells (MCs). As a cofactor for NO production, tetrahydrobiopterin (BH4 ), a folacin analogue, may be responsible for the ECM accumulation and proliferation of MCs. This study was to investigate the effects of BH4 on glomerulosclerosis in early-stage DN. In invitro studies with cultured mesangial cells and invivo studies with streptozotocin-induced diabetic rats, BH4 levels were assayed by HPLC; NO was determined by Griess agents; laminin and collagen IV were determined by enzyme-linked immunosorbent assay; the inducible NO synthase protein was determined by immunofluorescence staining and Western blot; and mesangial matrix expansion and MC proliferation in the renal cortex were observed by periodic acid-schiff staining and transmission electron microscopy, respectively. The invivo and invitro studies indicated that the increased BH4 resulted in the overproduction of NO, ECM accumulation and the proliferation of MCs in early-stage DN. Our results suggest that inhibiting excessive BH4 may be a potential approach to prevent glomerulosclerosis in early-stage DN.

Effects of tetrahydrobiopterin on the angiogenesis in hepatocellular carcinoma

Objective: To investigate the effect and mechanism of tetrahydrobiopterin (BH4) on the angiogenesis in hepatocellular carcinoma (HCC). Methods: BALB/c-nu mice were subcutaneously injected with HepG-2 cells and randomly divided into control and BH4 groups. The BH4 group and control group received 20 mg/kg BH4 or saline by intraperitoneal injection daily for two weeks, respectively. The level of BH4 was measured by high performance liquid chromatography (HPLC), the level of nitric oxide (NO) was measured by Griess test array, the transcriptional level of K-ras was measured by quantitative RT-PCR, and the protein expressions of guanosine triphosphate cyclohydrolase Ⅰ(GTPCH), endothelial nitric oxide synthase (eNOS), phospho-Akt and Akt were determined by Western blot. Results: BH4 level in the tumor tissues of BH4 group was (0.24±0.02) μg/ml, significantly higher than the (0.17±0.01) μg/ml in the control group (P<0.01). The level of NO in the tumor tissues of BH4 group was (51.44±2.90) mmol/L, significantly higher than the (24.77±0.54) mmol/L in the control group (P<0.01). The tumor volume of BH4 group was (191.05±8.70) mm3, significantly higher than the (103.10±5.03) mm3 in the control group (P<0.01). The expressions of CD34, K-ras, phospho-eNOS, phospho-Akt and GTPCH were significantly up-regulated in the tumor tissues of BH4 group when compared with those of the control group (P<0.01). Conclusions: BH4 recognized as an essential cofactor of eNOS can increase tumor-produced NO by activating the wild-type Ras-PI3K/Akt pathway, thus induces angiogenesis. This might provide a novel and promising way to control the progression of hepatocellular carcinoma through targeting BH4 synthesis pathway and inhibiting angiogenesis.

Tetrahydrobiopterin improves left ventricular diastolic function possibly through upregulating phosphorylated protein kinase B expression in hypertensive mice induced by deoxycorticosterone acetate

Objective: To investigate whether tetrahydrobiopterin (BH4) could improve left ventricular diastolic function through phosphoinositide-3 kinase/protein kinase B (PI3K/Akt) signaling pathway in hypertensive mice. Methods: Ten-week-old male C57BL/6 mice were used to establish the deoxycorticosterone acetate (DOCA)-salt hypertensive model, age matched Sham mice serve as the controls. Mice were divided into four groups: Sham(n=20), Sham+ BH4 (n=20), DOCA (n=22), and DOCA+ BH4 (n=22). On the 14 days after surgery, mice in Sham+ BH4 and DOCA+ BH4 groups received BH4 (0.1 ml/10 g) supplement for 7 days, while mice in Sham and DOCA groups were given equal volume of normal saline.The blood pressure measurements were performed 7 days later.Hemodynamic and echocardiographic parameters were used to assess left ventricular functions.High performance liquid chromatography (HPLC) analysis was used to measure cardiac biopterins BH4 and BH2.The phosphorylated phospholamban (p-PLB) was detected by immunohistochemical staining. PI3K, Akt and phosphorylated Akt were assayed with Western blot analysis. Results: (1) The systolic and diastolic blood pressure of DOCA group were significantly higher than control group (P<0.05). Compared with DOCA group, the systolic blood pressure was lower in DOCA+ BH4 mice (P=0.027). Diastolic blood pressure was similar between the groups. (2) Compared with Sham group, the left ventricular diastolic function indexes such as mitral annulus velocity (E') and E'/A'ratio were significantly lower, while the E/ E'ratio was significantly higher(P<0.05)in DOCA mice. The E/ E'ratio of DOCA+ BH4 group was significantly lower than that of DOCA group (P<0.05). Compared with Sham group, the left ventricular end-diastolic pressure (LVEDP), left ventricular end-diastolic pressure volumetric coefficient (EDPVR) and left ventricular relaxation time constant Tau index were significantly higher in DOCA mice (P=0.002, 0.011 and 0.016, respectively). The EDPVR and Tau index were significantly lower in DOCA+ BH4 group than in DOCA group (P<0.05). (3) Compared with Sham group, the myocardial contents of BH4 and BH2 were significantly lower in DOCA mice (P<0.05). The BH4 levels and BH4/BH2 ratio were significantly higher in Sham+ BH4 and DOCA+ BH4 groups than in the DOCA group (P<0.05), but the BH2 levels were similar between groups. (4) The cGMP content, SOD activity and NO content in the left ventricular myocardial tissue were significantly lower (P<0.05), while the MDA content was significantly higher in DOCA mice than in Sham mice.The NO content and SOD activity in DOCA+ BH4 groups were significantly higher than in the DOCA group (P<0.05). (5) Compared with DOCA group, the expression of p-PLB was significantly higher in Sham mice and lower in DOCA+ BH4 mice (P<0.05). (6) The expression of PI3K, Akt and p-Akt (Ser473 and Thr308) in DOCA mice were significantly lower than in Sham group (P<0.05). The expression of PI3K, Akt and p-Akt (Ser473) was significantly higher in DOCA+ BH4 group than in DOCA group (P<0.05). p-Akt (Thr308) expression was similar between DOCA + BH4 group and DOCA group (all P>0.05). Conclusion: Our results suggest that BH4 could improve left ventricular diastolic function in hypertensive mice, this effect might be mediated by reducing the oxidative stress in ventricular myocardium through modlating the expression of Akt and PLB phosphorylation.

OS 34-03 THE ASSOCIATION OF TETRAHYDROBIOPTERIN WITH A MARKER OF NITRIC OXIDE PRODUCTION IN A BLACK AND WHITE POPULATION

Objective: Tetrahydrobiopterin (BH4) is a cofactor for nitric oxide synthase (NOS) and may be a critical agent in endothelial function by improving nitric oxide (NO) mediated effects. It is reported that the NO synthesis capacity seems favourable in blacks despite their adverse cardiovascular profile. We therefore compared the concentrations of BH4, as well as blood pressure (BP) and arterial stiffness between black and white teachers. Furthermore, we tested the associations of BH4 with a product and inhibitor of NO synthesis, namely L-citrulline, and symmetric dimethylarginine (SDMA) respectively, in both ethnic groups. Design and Method: This cross-sectional sub-study is embedded in the SABPA study. The participants were stratified by race (black n = 194, white n = 204). Cardiovascular measurements included blood pressure (BP) and pulse wave velocity (PWV). Biochemical analyses of BH4, L-citrulline and SDMA were determined by recognised biochemical methods. Results: Although blacks had higher BP and PWV (p < 0.05), their BH4 levels also tended to be higher (p = 0.07) compared to whites. In whites positive correlations existed with single, partial (adjusted for age, sex and BMI) and multiple regression of BH4 with L-citrulline (adj.R2 = 0.20, β = 0.17, p = 0.01) and a negative correlation with SDMA (adj.R2 = 0.20, β = −0.15, p = 0.03). In single, partial and multiple regression analyses we found a positive correlation between BH4 and SDMA (adj.R2 = 0.12, β = 0.23, p = 0.003) in black participants. Conclusions: In white teachers BH4 independently associated with L-citrulline, a product of NO synthesis while no such correlation was found in black teachers. Instead an association of BH4 with SDMA, indicating an inhibition of NOS was indicated. Our result suggests a suppressed endothelial function in the black group and this is in line with the higher prevalence of hypertension known in this population.

Role of dihydrofolate reductase in tetrahydrobiopterin biosynthesis and lipid metabolism in the oleaginous fungus Mortierella alpina.

Mortierella alpina is a well-known polyunsaturated fatty acid-producing oleaginous fungus. Analysis of the Mort. alpina genome suggests that there is a putative dihydrofolate reductase (DHFR) gene playing a role in the salvage pathway of tetrahydrobiopterin (BH4), which has never been explored in fungi before. DHFR is the sole source of tetrahydrofolate and plays a key role in maintaining BH4 levels. Transcriptome data analysis revealed that DHFR was up-regulated by nitrogen exhaustion, when Mort. alpina starts to accumulate lipids. Significant changes were found in the fatty acid profile in Mort. alpina grown on medium containing DHFR inhibitors compared to Mort. alpina grown on medium without inhibitors. To explore the role of DHFR in folate/BH4 metabolism and its relationship to lipid biosynthesis, we expressed heterologously the gene encoding DHFR from Mort. alpina in Escherichia coli and we purified the recombinant enzyme to homogeneity. The enzymatic activity was investigated by liquid chromatography and MS and VIS-UV spectroscopy. The kinetic parameters and the effects of temperature, pH, metal ions and inhibitors on the activity of DHFR were also investigated. The transcript level of cytosolic NADPH-producing gene involved in folate metabolism is down-regulated by DHFR inhibitors, which highlights the functional significance of DHFR in lipid biosynthesis. The relationship between DHFR and lipid metabolism is thus of major importance, and folate metabolism may be an alternative NADPH source in fatty acid synthesis. To our knowledge, this study is the first to report the comprehensive characterization of a BH4salvage pathway in a fungus.

Established atherosclerosis might be a prerequisite for chicory and its constituent protocatechuic acid to promote endothelium-dependent vasodilation in mice.

Chicory (Cichorium intybus L. var. foliosum, Belgian endive), a typical Mediterranean vegetable, and its constituent protocatechuic acid (PCA) can inhibit established atherosclerosis progression. We thus investigated whether chicory can improve vascular relaxation, a critical pathway for combating atherosclerosis, and whether PCA is a contributor to a chicory-induced effect. Apolipoprotein E-deficient (ApoE-/- ) mice with established atherosclerosis and C57BL/6J mice without atherosclerosis were fed an AIN-93G diet, or AIN-93G plus 0.5% freeze-dried chicory or 0.003% PCA for 1 wk. In ApoE-/- mice, both chicory and PCA consumption increased endothelium-dependent vasodilation and endothelial nitric oxide synthase (eNOS) activity independent of eNOS and phospho-eNOS Ser1177 and Thr495 protein expression. Chicory- or PCA-induced eNOS activities were associated with increased vascular tetrahydrobiopterin (BH4 ) levels that result from reduced BH4 oxidation partially through preventing eNOS uncoupling. In C57BL/6J mice, neither chicory nor PCA consumption affected endothelium-dependent vasodilation and eNOS activity. Notably, in vitro studies showed that PCA increases eNOS activity in mouse aortic endothelial cells in co-culture with macrophage foam cells, but not in aortic endothelial cells alone. Chicory improves eNOS-mediated endothelium-dependent vasodilation by increasing BH4 levels in mice with established atherosclerosis, which might be partially ascribed to its constituent PCA.

Downregulation of tetrahydrobiopterin inhibits tumor angiogenesis in BALB/c-nu mice with hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a highly vascular tumor, and treatment options for patients of advanced-stage are limited. Nitric oxide (NO), which is derived from endothelial nitric oxide synthase (eNOS), provides crucial signals for angiogenesis in the tumor microenvironment. Tetrahydrobiopterin (BH4) is an essential cofactor eNOS and represents a critical determinant of NO production. To examine whether treatment of 2,4-diamino-6-hydroxypyrimidine (DAHP) inhibits angiogenesis of HCC, BALB/c-nu mice were injected with HepG-2 cells with DAHP. Supplemental DAHP treatment decreased K-ras mRNA transcripts, inhibition of phosphorylation of eNOS and Akt, inhibition of guanosine triphosphate cyclohydrolase (GTPCH), and decreased significantly NO synthesis, and then inhibited angiogenesis, compared with the results observed in the saline group. Histopathology demonstrated angiogenesis and tumor formation were significantly inhibited in HCC. DAHP downregulates GTPCH protein expression, corresponding to decreased levels of BH4 and the contents of NO. In addition, DAHP downregulates eNOS and Akt protein expression, corresponding to decreased eNOS phosphorylation at Ser1177 and Akt phosphorylation, compared with the saline control. We suggest that DAHP, recognized as a specific competitive inhibitor of GTPCH, can decrease tumor BH4 and NO by the inhibition of the wild-type Ras-PI3K/Akt pathway, and then inhibiting angiogenesis, and may provide a novel and promising way to target BH4 synthetic pathways to inhibit angiogenesis and to control potential progression of HCC. Whether DAHP has a therapeutic potential will require more direct testing in humans.

Physiological and behavioral responses in offspring mice following maternal exposure to sulfamonomethoxine during pregnancy

Sulfamonomethoxine (SMM), a veterinary antibiotic, is widely used in China. However, the impacts of maternal SMM exposure on neurobehavioral development in early life remain little known. In this study, we investigated the effects of maternal SMM exposure during pregnancy on behavioral and physiological responses in offspring mice. Pregnant mice were randomly divided into three SMM-treated groups, namely low-(10mg/kg/day), medium-(50mg/kg/day), and high-dose (200mg/kg/day), and a control group. The pregnant mice in the SMM-treated groups received SMM by gavage daily from gestational day 1–18, whereas those in the control received normal saline. On postnatal day (PND) 50, spatial memory was assessed using the Morris water maze test, and anxiety was measured using the elevated plus-maze and open field tests. The results showed significantly increased blood glucose in pups whose mothers received a high SMM dose. In addition, maternal SMM exposure increased anxiety-related activities among the offspring; spatial learning and memory were impaired more severely in the male offspring. The contents of tetrahydrobiopterin (BH4) and brain-derived neurotrophic factors (BDNF) on PND 22 were significantly reduced in the male offspring of the high-dose group compared with the controls. These findings indicate that SMM may be identified as a risk factor for cognitive and behavioral development on the basis of gender and that it may be associated with diminished BH4 and BDNF levels early in life.

Tetrahydrobiopterin Concentrations in Normal and Coronary Artery Diseased Heart Tissue

Background: Tetrahydrobiopterin (BH4) is a cofactor that plays a major role in cardiovascular health and disease. BH4 levels in the human heart have not been previously reported. Objective and Methods: Using a novel LC-MS/MS method we measured BH4 and BH2 levels in human heart tissue from subjects with (n=19) and without (n=19) coronary artery disease (CAD). Results: The concentration of BH4 was significantly lower in CAD subjects compared to controls (p=0.0006). There was a trend for a decrease in BH4/BH2 ratio (p=0.09). mRNA expression of GCH1, NOS3 and was not significantly different between CAD and controls. Conclusions: We conclude that the decrease in BH4 concentration in the left ventricular wall of subjects with CAD may be a causative factor or consequence of the failing heart.

Metformin attenuates fluctuating glucose-induced endothelial dysfunction through enhancing GTPCH1-mediated eNOS recoupling and inhibiting NADPH oxidase

AimsThe aim of this study was to investigate whether and how metformin ameliorated endothelial dysfunction induced by fluctuating glucose (FG) in human umbilical vein endothelial cells (HUVECs). MethodsHUVECs, which were exposed to FG to induce endothelial dysfunction, were incubated with nitric oxide synthase (NOS) inhibitor N-nitro-l-arginine-methyl ester (l-NAME), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin, metformin and/or adenosine monophosphate-activated protein kinase (AMPK) inhibitor compound C. The oxidative stress and endothelial NOS (eNOS) coupling were evaluated. ResultsFG induced endothelial dysfunction as indicated by increased reactive oxygen species (ROS) generation and decreased nitric oxide (NO) production. Although FG increased eNOS phosphorylation, uncoupled eNOS was evidenced by downregulated guanosine 5′-triphosphate cyclohydrolase 1 (GTPCH1) and tetrahydrobiopterin (BH4) levels. FG also upregulated the level of p47-phox, a subunit of NADPH oxidase. Similar to l-NAME and apocynin, metformin ameliorated the FG-induced endothelial dysfunction by decreasing ROS generation. Furthermore, metformin recoupled eNOS through upregulating GTPCH1 and BH4 levels, and attenuated the upregulation of p47-phox in FG-treated HUVECs. Addition of compound C abolished the above effects of metformin. ConclusionMetformin improves the FG-induced endothelial dysfunction in HUVECs. The protective effect of metformin may be mediated through activation of GTPCH1-mediated eNOS recoupling and inhibition of NADPH oxidase via an AMPK-dependent pathway.

Estrogen‐induced Superoxide Production in Coronary Artery Endothelial Cells via Uncoupled Nitric Oxide Synthase in Diabetes

Premenopausal women exhibit substantially lower risk for cardiovascular disease (CVD) than men of similar age; however, diabetes mellitus eliminates this sex‐related protection such that diabetic women have nearly double the risk of CVD as diabetic men. In fact, diabetes increases a woman's risk for coronary heart disease (CHD) by approximately 10‐fold. Although estrogens are believed to protect cardiovascular function in premenopausal women, potential effects of diabetes on how estrogen influences coronary arteries are largely unknown. To address this important gap in our knowledge, the present study has investigated the effects of estrogen on human coronary artery endothelial cells (HCAEC) obtained from normal female donors or donors with type 1 (HCAEC‐D1) diabetes. Human umbilical vein endothelial cells (HUVECS) were also employed as non‐diabetic controls. Production of nitric oxide was detected via fluorescence microscopy using 4‐amino‐5‐methylamino‐2′,7′‐difluorescein (DAF‐FM; 2 μM), whereas superoxide production was detected with dihydroethidium (DHE; 20μM). We found that treating control cells with 100nM 17®‐estradiol (17®‐E; 20–30 min) markedly increased DAF fluorescence by 13‐fold (n=42 cells; p&lt;0.0001). In contrast, 17β‐E decreased DAF fluorescence in HCAEC‐D1 by 75% (n=38 cells; p&lt;0.0001). In the presence of 100 μM L‐NAME, an inhibitor of nitric oxide synthase (NOS), 17®‐E had little effect on NO production. As expected, HCAEC‐D1 exhibited more than double the level of DHE fluorescence (n=16 cells; p&lt;0.001) compared to control cells, consistent with diabetes‐induced oxidative stress. Moreover, 17®‐E further increased DHE fluorescence by an average of 20% (n=55 cells; p&lt;0.0001) in HCAEC‐D1, but did not alter DHE fluorescence in control cells. Interestingly, L‐NAME inhibited the effect of 17®‐E on superoxide production in diabetic cells strongly suggesting uncoupled NOS as the primary source of estrogen‐stimulated superoxide. Finally, estrogen‐stimulated oxidant production in HCAEC‐D1 was prevented by “recoupling” NOS activity with 100μM sapropterin, an analog of tetrahydrobiopterin (BH4). Estrogen had no effect on DHE fluorescence in HCAEC‐D1 pretreated with sapropterin (n=16 cells). Taken together, these findings suggest that estrogens stimulate the release of NO from normal coronary artery endothelial cells, but in diabetes the primary effect of estrogen is enhanced superoxide production from these cells. Further, the fact that L‐NAME inhibits both NO and superoxide production in endothelial cells strongly suggests that NOS is the primary target of estrogen in these cells. Lastly, restoration of BH4 levels prevented estrogen from increasing oxidative stress in HCAEC‐D1. Thus, we conclude that diabetes uncouples NOS activity in coronary arteries, likely through depletion of BH4. The higher estrogen levels in premenopausal women would then stimulate NOS to produce predominately superoxide in diabetes to increase oxidative stress. We propose that these abnormal responses to estrogen contribute to the greater risk for CVD experienced by female diabetics. These findings now suggests a novel therapeutic target to alleviate endothelial dysfunction in women afflicted by this disease: recoupling of NOS activity via restoration of BH4 levels (which are known to be depleted by diabetes).Support or Funding InformationSupported by the Biomedical Sciences Program at GA‐PCOM and the PCOM Center for Chronic Disorders of Aging.

Tetrahydrobiopterin Attenuates DSS-evoked Colitis in Mice by Rebalancing Redox and Lipid Signalling.

Guanosine triphosphate cyclohydrolase [GCH1] governs the production of the enzyme cofactor tetrahydrobiopterin [BH4] which is essential for biogenic amine synthesis, lipid metabolism via alkylglycerol monooxygenase [AGMO], and redox coupling of nitric oxide synthases [NOSs]. Inflammation-evoked unequal regulation of GCH1 and NOS or AGMO may cause redox stress and lipid imbalances. The present study assessed potential therapeutic effects of rebalancing these systems with BH4 in experimental colitis in mice. Oral treatment with BH4 as a suspension of crushed tablets attenuated colitis, whereas inhibition of its production had opposite effects: aggravated weight loss, epithelial haemorrhages and ulcers, neutrophil infiltrates, production of reactive oxygen species, and unfavourable profile changes of endocannabinoids, ceramides, and lysophosphatidic acids. Conversely, oral BH4 normalised biopterin, reduced in vivo activity of oxidases and peroxidases in the inflamed gut, favoured nitric oxide over hydrogen peroxide, and maintained normal levels of lipid signalling molecules. BH4 favoured thereby resident CD3+CD8+ and regulatory CD3+CD25+ intraepithelial T cells that are important for epithelial integrity. BH4 protected against colitis in mice via two major pathways: [i] by reduction of oxidative stress; and [ii] by re-orchestration of alkyl- and acylglycerolipid signalling via AGMO. Oral treatment with BH4 is a safe approved supplementary therapy for genetic BH4 deficiency and did not excessively increase systemic BH4 levels. Therefore, one may consider repurposing of oral BH4 as an adjunctive treatment for colitis.

Increasing tetrahydrobiopterin in cardiomyocytes adversely affects cardiac redox state and mitochondrial function independently of changes in NO production

Tetrahydrobiopterin (BH4) represents a potential strategy for the treatment of cardiac remodeling, fibrosis and/or diastolic dysfunction. The effects of oral treatment with BH4 (Sapropterin™ or Kuvan™) are however dose-limiting with high dose negating functional improvements. Cardiomyocyte-specific overexpression of GTP cyclohydrolase I (mGCH) increases BH4 several-fold in the heart. Using this model, we aimed to establish the cardiomyocyte-specific responses to high levels of BH4. Quantification of BH4 and BH2 in mGCH transgenic hearts showed age-based variations in BH4:BH2 ratios. Hearts of mice (<6 months) have lower BH4:BH2 ratios than hearts of older mice while both GTPCH activity and tissue ascorbate levels were higher in hearts of young than older mice. No evident changes in nitric oxide (NO) production assessed by nitrite and endogenous iron–nitrosyl complexes were detected in any of the age groups. Increased BH4 production in cardiomyocytes resulted in a significant loss of mitochondrial function. Diminished oxygen consumption and reserve capacity was verified in mitochondria isolated from hearts of 12-month old compared to 3-month old mice, even though at 12 months an improved BH4:BH2 ratio is established. Accumulation of 4-hydroxynonenal (4-HNE) and decreased glutathione levels were found in the mGCH hearts and isolated mitochondria. Taken together, our results indicate that the ratio of BH4:BH2 does not predict changes in neither NO levels nor cellular redox state in the heart. The BH4 oxidation essentially limits the capacity of cardiomyocytes to reduce oxidant stress. Cardiomyocyte with chronically high levels of BH4 show a significant decline in redox state and mitochondrial function.

Genetic predisposition in patients with hypertension and normal ejection fraction to oxidative stress

The role of oxidative stress (OXS) due to myocardial nitric oxide synthase (NOS) uncoupling related to oxidative depletion of its cofactor tetrahydrobiopterin (BH4) emerged in the pathogenesis of heart failure with preserved ejection fraction. We determined the prevalence of six single nucleotide polymorphisms (SNPs) of genes encoding enzymes related to OXS, BH4 metabolism, and NOS function in ≥60-year-old 94 patients with hypertension and 18 age-matched controls with normal ejection fraction. Using echocardiography, 56/94 (60%) patients with hypertension had left ventricular (LV) diastolic dysfunction (HTDD+ group) and 38/94 (40%) patients had normal LV diastolic function (HTDD− group). Four SNPs (rs841, rs3783641, rs10483639, and rs807267) of guanosine triphosphate cyclohydrolase-1, the rate-limiting enzyme in BH4 synthesis, one (rs4880) of manganese superoxide dismutase, and one (rs1799983) of endothelial NOS genes were genotyped using real-time polymerase chain reaction method and Taqman probes. Protein carbonylation, BH4, and total biopterin levels were measured from plasma samples. No between-groups difference in minor allele frequency of SNPs was found. We calculated a genetic score indicating risk for OXS based on the minor allele frequencies of the SNPs. A high genetic risk for OXS was significantly associated with HTDD+ even after adjustment for confounding variables (odds ratio [95% confidence interval]:4.79 [1.12–20.54]; P = .035). In both patient groups protein carbonylation (P < .05 for both), plasma BH4 (P < .01 for both) and in the HTDD+ group total biopterin (P < .05) increased versus controls. In conclusion, in patients with hypertension and normal ejection fraction, a potential precursor of heart failure with preserved ejection fraction, a partly genetically determined increased OXS, seems to be associated with the presence of LV diastolic dysfunction.

Abstract 18446: Treatment With Tetrahydrobiopterin Decreases White Matter Injury in a Mouse Model for In Utero Hypoxia in Congenital Heart Disease

Introduction: Reduced oxygen delivery in complex congenital heart disease (CHD) can lead to brain white matter (WM) injury in utero. Currently, no treatment exists. Tetrahydrobiopterin (BH4) is a cofactor for neuronal nitric oxide synthase, and in its absence, toxic peroxynitrite production is favored. Hypoxia activates nitric oxide synthase, which reduces BH4 availability. Hypothesis: Decreased BH4 levels underlie WM injury in the fetus with CHD, and treatment with BH4 will reduce this injury. Methods: Mice were divided into three groups: normoxic controls (Nx), hypoxic (Hx), and hypoxic with BH4 treatment (Hx-BH4). Hx and Hx-BH4 mice were kept at 10.5% FiO2 from postnatal day 3 to 11--a period of WM development equivalent to the 3rd trimester in humans. Brain BH4 levels were quantified and compared between Nx (n=11) and Hx (n=12). Densities of cells expressing CNP (marker of oligodendrocytes--cells responsible for myelination) and Caspase3 (apoptosis marker) were quantified in three WM regions and compared between groups (n=3-6 each). Western blot detected myelin basic protein (myelin marker). Results: Brain BH4 levels were depleted in Hx compared to Nx (-38.4%, p=0.02). CNP+ oligodendrocytes increased after Hx compared to Nx (Fig.1a), consistent with hypoxia-induced proliferation seen previously. BH4 treatment did not limit this proliferation (Fig.1a). Hx had increased WM apoptosis (Fig.1b), which decreased with BH4 treatment (Fig.1b). Remarkably, there was no difference in WM caspase3+ cells between Nx and Hx-BH4 (Fig.1b). There was no difference in effect across WM region. Finally, loss of myelin with hypoxia was mitigated by BH4 treatment (Fig.1c). Conclusions: Our results show that suboptimal BH4 levels influence hypoxic WM injury. BH4 treatment of phenylketonuria is safe during pregnancy, thus maternal BH4 therapy is feasible. Our data demonstrate that repurposing BH4 for use during fetal brain development has potential to limit WM injury in CHD.

Folic Acid Promotes Recycling of Tetrahydrobiopterin and Protects Against Hypoxia-Induced Pulmonary Hypertension by Recoupling Endothelial Nitric Oxide Synthase.

Nitric oxide (NO) derived from endothelial NO synthase (eNOS) has been implicated in the adaptive response to hypoxia. An imbalance between 5,6,7,8-tetrahydrobiopterin (BH4) and 7,8-dihydrobiopterin (BH2) can result in eNOS uncoupling and the generation of superoxide instead of NO. Dihydrofolate reductase (DHFR) can recycle BH2 to BH4, leading to eNOS recoupling. However, the role of DHFR and eNOS recoupling in the response to hypoxia is not well understood. We hypothesized that increasing the capacity to recycle BH4 from BH2 would improve NO bioavailability as well as pulmonary vascular remodeling (PVR) and right ventricular hypertrophy (RVH) as indicators of pulmonary hypertension (PH) under hypoxic conditions. In human pulmonary artery endothelial cells and murine pulmonary arteries exposed to hypoxia, eNOS was uncoupled as indicated by reduced superoxide production in the presence of the nitric oxide synthase inhibitor, L-(G)-nitro-L-arginine methyl ester (L-NAME). Concomitantly, NO levels, BH4 availability, and expression of DHFR were diminished under hypoxia. Application of folic acid (FA) restored DHFR levels, NO bioavailability, and BH4 levels under hypoxia. Importantly, FA prevented the development of hypoxia-induced PVR, right ventricular pressure increase, and RVH. FA-induced upregulation of DHFR recouples eNOS under hypoxia by improving BH4 recycling, thus preventing hypoxia-induced PH. FA might serve as a novel therapeutic option combating PH.

Characterization of cerebral microvasculature in transgenic mice with endothelium targeted over-expression of GTP-cyclohydrolase I

Tetrahydrobiopterin (BH4) is a critical determinant of nitric oxide (NO) production by nitric oxide synthase (NOS) in the vascular endothelium and its biosynthesis is regulated by the enzymatic activity of GTP-cyclohydrolase I (GTPCH I). The present study was designed to determine the effects of endothelium-targeted overexpression of GTPCH I (eGCH-Tg) on murine cerebral vascular function. Endothelium targeted over-expression of GTPCH I was associated with a significant increase in levels of BH4, as well as its oxidized product, 7,8-dihydrobiopterin (7,8-BH2) in cerebral microvessels. Importantly, ratio of BH4 to 7,8-BH2, indicative of BH4 available for eNOS activation, was significantly increased in eGCH-Tg mice. However, expression of endothelial NOS, levels of nitrate/nitrite – indicative of NO production – remained unchanged between cerebral microvessels of wild-type and eGCH-Tg mice. Furthermore, increased BH4 biosynthesis neither affected production of superoxide anion nor expression of antioxidant proteins. Moreover, endothelium-specific GTPCH I overexpression did not alter intracellular levels of cGMP, reflective of NO signaling in cerebral microvessels. The obtained results suggest that, despite a significant increase in BH4 bioavailability, generation of endothelial NO in cerebral microvessels remained unchanged in eGCH-Tg mice. We conclude that under physiological conditions the levels of BH4 in the cerebral microvessels are optimal for activation of endothelial NOS and NO/cGMP signaling.

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.

Endothelial cell tetrahydrobiopterin deficiency attenuates LPS-induced vascular dysfunction and hypotension

Overproduction of nitric oxide (NO) is thought to be a key mediator of the vascular dysfunction and severe hypotension in patients with endotoxaemia and septic shock. The contribution of NO produced directly in the vasculature by endothelial cells to the hypotension seen in these conditions, vs. the broader systemic increase in NO, is unclear. To determine the specific role of endothelium derived NO in lipopolysaccharide (LPS)-induced vascular dysfunction we administered LPS to mice deficient in endothelial cell tetrahydrobiopterin (BH4), the essential co-factor for NO production by NOS enzymes. Mice deficient in endothelial BH4 production, through loss of the essential biosynthesis enzyme Gch1 (Gch1fl/flTie2cre mice) received a 24hour challenge with LPS or saline control. In vivo LPS treatment increased vascular GTP cyclohydrolase and BH4 levels in aortas, lungs and hearts, but this increase was significantly attenuated in Gch1fl/flTie2cre mice, which were also partially protected from the LPS-induced hypotension. In isometric tension studies, in vivo LPS treatment reduced the vasoconstriction response and impaired endothelium-dependent and independent vasodilatations in mesenteric arteries from wild-type mice, but not in Gch1fl/flTie2cre mesenteric arteries. Ex vivo LPS treatment decreased vasoconstriction response to phenylephrine in aortic rings from wild-type and not in Gch1fl/flTie2cre mice, even in the context of significant eNOS and iNOS upregulation. These data provide direct evidence that endothelial cell NO has a significant contribution to LPS-induced vascular dysfunction and hypotension and may provide a novel therapeutic target for the treatment of systemic inflammation and patients with septic shock.