BH2 Ratio Research Articles

Endothelial cell-derived tetrahydrobiopterin prevents aortic valve calcification.

Tetrahydrobiopterin (BH4) is a critical determinant of the biological function of endothelial nitric oxide synthase. The present study was to investigate the role of valvular endothelial cell (VEC)-derived BH4 in aortic valve calcification. Plasma and aortic valve BH4 concentrations and the BH4:BH2 ratio were significantly lower in calcific aortic valve disease patients than in controls. There was a significant decrease of the two key enzymes of BH4 biosynthesis, guanosine 5'-triphosphate cyclohydrolase I (GCH1) and dihydrofolate reductase (DHFR), in calcified aortic valves compared with the normal ones. Endothelial cell-specific deficiency of Gch1 in Apoe-/- (Apoe-/-Gch1fl/flTie2Cre) mice showed a marked increase in transvalvular peak jet velocity, calcium deposition, runt-related transcription factor 2 (Runx2), dihydroethidium (DHE), and 3-nitrotyrosine (3-NT) levels in aortic valve leaflets compared with Apoe-/-Gch1fl/fl mice after a 24-week western diet (WD) challenge. Oxidized LDL (ox-LDL) induced osteoblastic differentiation of valvular interstitial cells (VICs) co-cultured with either si-GCH1- or si-DHFR-transfected VECs, while the effects could be abolished by BH4 supplementation. Deficiency of BH4 in VECs caused peroxynitrite formation increase and 3-NT protein increase under ox-LDL stimulation in VICs. SIN-1, the peroxynitrite generator, significantly up-regulated alkaline phosphatase (ALP) and Runx2 expression in VICs via tyrosine nitration of dynamin-related protein 1 (DRP1) at Y628. Finally, folic acid (FA) significantly attenuated aortic valve calcification in WD-fed Apoe-/- mice through increasing DHFR and salvaging BH4 biosynthesis. The reduction in endothelial-dependent BH4 levels promoted peroxynitrite formation, which subsequently resulted in DRP1 tyrosine nitration and osteoblastic differentiation of VICs, thereby leading to aortic valve calcification. Supplementation of FA in diet attenuated hypercholesterolaemia-induced aortic valve calcification by salvaging BH4 bioavailability.

Metastatic Melanoma Progression Is Associated with Endothelial Nitric Oxide Synthase Uncoupling Induced by Loss of eNOS:BH4 Stoichiometry.

Melanoma is the most aggressive type of skin cancer due to its high capability of developing metastasis and acquiring chemoresistance. Altered redox homeostasis induced by increased reactive oxygen species is associated with melanomagenesis through modulation of redox signaling pathways. Dysfunctional endothelial nitric oxide synthase (eNOS) produces superoxide anion (O2−•) and contributes to the establishment of a pro-oxidant environment in melanoma. Although decreased tetrahydrobiopterin (BH4) bioavailability is associated with eNOS uncoupling in endothelial and human melanoma cells, in the present work we show that eNOS uncoupling in metastatic melanoma cells expressing the genes from de novo biopterin synthesis pathway Gch1, Pts, and Spr, and high BH4 concentration and BH4:BH2 ratio. Western blot analysis showed increased expression of Nos3, altering the stoichiometry balance between eNOS and BH4, contributing to NOS uncoupling. Both treatment with L-sepiapterin and eNOS downregulation induced increased nitric oxide (NO) and decreased O2• levels, triggering NOS coupling and reducing cell growth and resistance to anoikis and dacarbazine chemotherapy. Moreover, restoration of eNOS activity impaired tumor growth in vivo. Finally, NOS3 expression was found to be increased in human metastatic melanoma samples compared with the primary site. eNOS dysfunction may be an important mechanism supporting metastatic melanoma growth and hence a potential target for therapy.

In vivo vascular rarefaction and hypertension induced by dexamethasone are related to phosphatase PTP1B activation not endothelial metabolic changes

Glucocorticoids have important anti-inflammatory and immunomodulatory activities. Dexamethasone (Dex), a synthetic glucocorticoid, induces insulin resistance, hyperglycemia, and hypertension. The hypertensive mechanisms of Dex are not well understood. Previously, we showed that exercise training prior to Dex treatment significantly decreases blood vessel loss and hypertension in rats. In this study, we examined whether the salutary effects of exercise are associated with an enhanced metabolic profile. Analysis of the NAD and ATP content in the tibialis anterior muscle of trained and non-trained animals indicated that exercise increases both NAD and ATP; however, Dex treatment had no effect on any of the experimental groups. Likewise, Dex did not change NAD and ATP in cultured endothelial cells following 24 h and 48 h of incubation with high concentrations. Reduced VEGF-stimulated NO production, however, was verified in endothelial cultured cells. Reduced NO was not associated with changes in survival or the BH4 to BH2 ratio. Moreover, Dex had no effect on bradykinin- or shear-stress-stimulated NO production, indicating that VEGF-stimulated eNOS phosphorylation is a target of Dex's effects. The PTP1B inhibitor increased NO in Dex-treated cells in a dose-dependent fashion, an effect that was replicated by the glucocorticoid receptor inhibitor, RU486. In combination, these results indicate that Dex-induced endothelial dysfunction is mediated by glucocorticoid receptor and PTP1B activation. Moreover, since exercise reduces the expression of PTP1B and normalized insulin resistance in aging rats, our findings indicate that exercise training by reducing PTP1B activity counteracts Dex-induced hypertension in vivo.

NOS cofactor tetrahydrobiopterin contributes to anesthetic preconditioning induced myocardial protection in the isolated exvivo rat heart.

Anesthetic preconditioning (APC) may decrease the myocardium injury nearly 50% following ischemia/reperfusion (I/R) by enhancing recovery of cardiac function, reducing myocardial enzyme release and lowering infarct size when utilized as pretreatment or posttreatment agents. I/R increases nitric oxide (NO) production through endothelial NO synthase (NOS3) and heat shock protein 90 (HSP90). The present study aimed to observe the role of BH4 availability and the association of HSP90 with NOS3 in APC‑mediated cardioprotection against I/R injury. Isolated rat hearts were subjected to no‑flow ischemia for 30min and reperfusion for 120min. Sevoflurane (3.5%) was administered for 15min followed by a 15min washout prior to ischemia. 2,4-Diamino-6-hydroxypyrimidine(DAHP) or sepiapterin(SP) was administered for 40min until the onset of ischemia. The results revealed that compared with pre‑ischemic basal levels, BH4 levels decreased and BH2 levels increased following I/R. BH4 levels were significantly increased and BH2 levels were significantly decreased in the APC+I/R hearts compared with the I/R group hearts. The BH4:BH2 ratio in the APC‑treated hearts was also increased compared with that in the I/R group hearts. SP increased the recovery of contractile function and the production of NO, and decreased the production of superoxide anion (O2·‑) in I/R heart, but did not elicit these effects in APC‑treated hearts. DAHP treatment inhibited the APC‑mediated recovery of contractile function, increased O2·‑ levels and decreased NO production, but had no effect in I/R hearts. The cardioprotection of APC was demonstrated to be modulated by the BH4 precursor SP, which increased BH4 levels, or DAHP, which inhibited GTP cyclohydrolase I. Both APC and SP treatments increased the combination of HSP90 and NOS3, which improved the NOS3 activity and function. The results suggested that BH4, which servesas a cofactor for NOS, mediated the resistance of APC to I/R injury by promoting the binding of HSP90 and NOS3.

Imbalance between nitric oxide and superoxide anion induced by uncoupled nitric oxide synthase contributes to human melanoma development.

Melanoma is the most aggressive type of cutaneous tumors due to its metastatic potential and high mortality. Increased levels of reactive oxygen species, including superoxide anion (O2-), and the consequent installation of a pro-oxidant environment are associated with melanoma development. The enzyme nitric oxide synthase (NOS), responsible for the production of nitric oxide (NO), when uncoupled is as a source of O2-, for example by the absence of its cofactor tetrahydrobiopterin (BH4). Western blot analysis showed increased expression of endothelial and inducible NOS in human melanoma cells, altering the stoichiometry between NOS levels and BH4 concentration and together with decreased BH4:BH2 ratio are contributing to NOS uncoupling. The treatment of melanoma cells with exogenous BH4 increased NO concentration and decreased O2- levels, leading to NOS coupling, which in turn reduced cell viability, cell proliferation and the ability of melanoma cells to form melanoma spheroids. Moreover, BH4 level restoration rendered melanoma cells more sensitive to apoptosis, demonstrating the role of dysfunctional NOS in melanoma genesis.

145 - Imbalance between nitric oxide and superoxide anion induced by uncoupled nitric oxide synthase contributes to human metastatic melanoma development

Melanoma is the most aggressive type of cutaneous tumors due to its metastatic potential and high mortality. Increased levels of reactive oxygen species, including superoxide anion (O2–), and the consequent installation of a pro-oxidant environment are associated with melanoma development. The enzyme nitric oxide synthase (NOS), responsible for the production of nitric oxide (NO), when uncoupled is as a source of O2–, for example by the absence of its cofactor tetrahydrobiopterin (BH4). The present work shows that BH4 synthesis is compromised in melanoma cell lines. Analysis through qPCR showed that the expression of the transcripts for proteins involved in the de novo, salvage and recycling BH4 pathways are altered in melanoma cells when compared to melanocytes, resulting in decreased BH4:BH2 ratio. Western blot analysis showed increased expression of endothelial and inducible NOS in human melanoma cells, altering the stoichiometry between NOS levels and BH4 concentration and contributing to NOS uncoupling. The treatment of melanoma cells with exogenous BH4 increased NO concentration and decreased O2– levels, leading to NOS coupling, which in turn reduced the cell viability, cell proliferation and the ability of melanoma cells to form tumorspheres. Moreover, BH4 level restoration rendered melanoma cells more sensitive to apoptosis, demonstrating the role of dysfunctional NOS in melanoma genesis.

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.

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.

Abstract 1210: Biopterin metabolism drives tumor progression

Abstract Previous studies demonstrate that nitric oxide synthases (NOS’s) can become uncoupled during inflammatory disease states. A hallmark of this uncoupling is superoxide production from NOS in lieu of nitric oxide (NO) resulting from a reduced tetrahydrobiopterin:dihydrobiopterin ratio (BH4:BH2 ratio). As BH4 is a necessary cofactor for NO production from NOS, when this ratio is reduced, NOS becomes a peroxynitrite synthase enhancing the pro-inflammatory/pro-survival tumor environment. Our lab demonstrated that NOS is uncoupled in a variety of tumor cells both in vitro and in vivo, as well as in paired human colorectal samples due to a reduced BH4:BH2 ratio and that recoupling NOS in via exogenous BH4 both in vitro and in vivo leads to decreased tumor cell proliferation. To examine the mechanism behind the reduced BH4:BH2 ratio we used the DSS/AOM model of colitis and colon tumorigenesis. Here we demonstrate that BH4:BH2 ratio is reduced in mouse colon tumors vs mouse normal colon tissue, 1.36±0.35 vs 7.1±0.6. We determined that this reduced ratio is due at least in part to direct oxidation of BH4 to BH2, as treatment of colon cells with an NADPH Oxidase inhibitor increased the BH4:BH2 two-fold; however, this is unable to account for the degree of uncoupling associated with tumor cells. Given that inflammation appears to drive NOS uncoupling, we extracted colons from DSS/AOM treated animals and preferentially harvested colonic epithelial cells and colonic tumor cells at different stages of inflammation to assess the molecular and biochemical changes in molecules important in the regulation and production of BH4. The molecules we focused on were: GTP cyclohydrolase-1 (GCH1, rate limiting enzyme in de novo BH4 synthesis), GTP cyclohydrolase 1 feedback regulatory protein (GCHFRP, regulates GCH1 expression), quinoiddihydropteridine reductase (QDPR, necessary for salvage pathway) and sepiapterin reductase (SR, necessary for both de novo synthesis and salvage of BH4). Transcript levels of these molecules in DSS treated epithelial cells showed that GCH1 and SR were upregulated but QDPR was significantly downregulated when compared with untreated cells, potentially leading to an increase in total BH4 but once oxidized to BH2, a decrease in the salvage back to BH4. Our current research investigates the protein levels as well biochemical activity of these molecules in DSS treated epithelial cells, as well as tumor cells compared with normal cells to further understand the molecular component of the decrease in BH4:BH2 ratio in tumors. Note: This abstract was not presented at the meeting. Citation Format: Christopher Rabender, Asim Alam, Mike Waters, Ross Mikkelsen. Biopterin metabolism drives tumor progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1210. doi:10.1158/1538-7445.AM2015-1210

The Role of Nitric Oxide Synthase Uncoupling in Tumor Progression.

Here, evidence suggests that nitric oxide synthases (NOS) of tumor cells, in contrast with normal tissues, synthesize predominantly superoxide and peroxynitrite. Based on high-performance liquid chromatography analysis, the underlying mechanism for this uncoupling is a reduced tetrahydrobiopterin:dihydrobiopterin ratio (BH4:BH2) found in breast, colorectal, epidermoid, and head and neck tumors compared with normal tissues. Increasing BH4:BH2 and reconstitution of coupled NOS activity in breast cancer cells with the BH4 salvage pathway precursor, sepiapterin, causes significant shifts in downstream signaling, including increased cGMP-dependent protein kinase (PKG) activity, decreased β-catenin expression, and TCF4 promoter activity, and reduced NF-κB promoter activity. Sepiapterin inhibited breast tumor cell growth in vitro and in vivo as measured by a clonogenic assay, Ki67 staining, and 2[18F]fluoro-2-deoxy-D-glucose-deoxyglucose positron emission tomography (FDG-PET). In summary, using diverse tumor types, it is demonstrated that the BH4:BH2 ratio is lower in tumor tissues and, as a consequence, NOS activity generates more peroxynitrite and superoxide anion than nitric oxide, resulting in important tumor growth-promoting and antiapoptotic signaling properties. The synthetic BH4, Kuvan, is used to elevate BH4:BH2 in some phenylketonuria patients and to treat diseases associated with endothelial dysfunction, suggesting a novel, testable approach for correcting an abnormality of tumor metabolism to control tumor growth.

Efecto de atorvastatina sobre los niveles de biopterinas y la relación bh4/bh2 en pacientes diabéticos:

ResumenEl objetivo de este estudio es evaluar el efecto de atorvastatina sobre los niveles de biopterinas y el índice BH4/BH2 en pacientes con diabetes tipo 2.Materiales y Métodos. 20 pacientes con diabetes tipo 2, sin tratamiento previo con estatinas ni inhibidores del eje renina-angiotensina, recibieron atorvastatina 10 mg una vez al día por 3 meses. En todos ellos BH4 y BH2 se midieron por cromatografía líquida de fase reversa con fluorescencia al inicio y el final del estudio. Los pacientes fueron evaluados mensualmente y se registró presión arterial, glucemia de ayuno, perfil de lípidos y efectos adversos. El análisis estadístico se realizó con las pruebas de Shapiro-Wilk y Wilcoxon. Resultados: El manejo con atorvastatina aumentó los niveles de BH4 (5.4 a 9.1, p=0.047) y redujo los de BH2 (7.4 a 6.99, p= 0.60), además de mejorar el índice BH4/BH2 (0.83 a 1.85 p= 0.016). Ningún paciente presentó efectos adversos.Conclusión: Nuestros resultados muestran que atorvastatina brinda un beneficio sobre los niveles de las biopterinas. Esto explica, al menos en parte, los efectos favorables de las estatinas en pacientes diabéticos cardiovascular.

Abstract 15954: Both Tetrahydrobiopterin Depletion and eNOS S-Glutathionytion Contribute to eNOS Uncoupling in Coronary Disease Patients

Background: Vascular endothelial dysfunction (VED) is associated with increased oxidative stress due to eNOS uncoupling with increased superoxide (•O2-) production and has important prognostic clinical implications for subsequent cardiovascular events. Our recent studies in hypertensive rats revealed a novel redox-regulated pathway through which eNOS is uncoupled due to S-glutathionylation. However, its role in human vascular disease remains unclear. Hence, we studied eNOS S-glutathionylation and uncoupling and in aortic tissue from patients undergoing cardiovascular surgery and heart transplantation (N=25). Methods: Studies were performed using vessels from spontaneously hypertensive rats (SHRs), control WKY rats and aortic tissue from patients undergoing cardiovascular surgery. S-glutathionylation was determined with anti-glutathione and anti-eNOS, and an antibody specific for eNOS S-glutathionylation at C689 (anti-eNOS Cys689-SG). •O2- was measured using fluorescent probe DHE in the absence/presence of the NOS inhibitor, L-NAME, DTT, or the SOD mimetic, MnTBAP. The NOS cofactor, BH4, was measured by HPLC. Results &amp; Conclusion: S-glutathionylation of eNOS (eNOS-SG) was evident by immunohistology in SHR vessels compared to respective WKY control rat vessels. Similarly, immune assays of the aortic samples from vascular disease patients (VD) showed an increase in eNOS-SG protein when compared to no-disease control tissues (ND) and increased •O2- production was detected. Addition of DTT or β-ME decreased the levels of eNOS-SG with decreased •O2- generation. BH4 content was markedly decreased in the vessels of patients with coronary disease. BH4:BH2 ratio was decreased by over 5-fold also contributing to eNOS uncoupling. This provides the first evidence for an association of both eNOS S-glutathionylation and BH4 depletion leading to eNOS uncoupling with loss of endothelial dependent vasodilation in vessels of coronary disease patients.

Tetrahydrobiopterin- and glutathionylation-dependent regulation of eNOS coupling: Relationships revealed by eNOS glutathionylation mutants in cells with tet-regulated GTP cyclohydrolase I expression

Endothelial nitric oxide synthase (eNOS) is critical in the regulation of vascular homeostasis and generates nitric oxide (NO) by a process that is dependent on the binding of the cofactor tetrahydrobiopterin (BH4). When BH4 availability is limiting, electron transfer from NOS flavins becomes ‘uncoupled’ from l -arginine oxidation and superoxide is produced in place of NO. This process of eNOS ‘uncoupling’ is associated with many pathophysiologic conditions including atherosclerosis and hypercholesterolemia, although BH4 repletion only partially restores NOS activity and NOS-dependent vasodilation. Recent evidence suggests that eNOS uncoupling can also be induced by S-glutathionylation, however the exact mechanisms remain unknown. To address a possible role for BH4 in S-glutathionylation-induced eNOS uncoupling, we developed novel cell lines with tet-regulated expression of human GTP cyclohydrolase I (GTPCH; the rate-limiting enzyme in BH4 synthesis) and either WT or mutant eNOS rendered resistant to S-glutathionylation by mutation of two critical cysteine residues (C689S and C908S). We reveal that S-glutathionylation of eNOS by exposure of endothelial cells to 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) or glutathione reductase (GR)-specific siRNA results in diminished NO production and elevated eNOS-derived superoxide production, along with a concomitant reduction in BH4 levels and BH4:BH2 ratio. In our model of BH4-dependent eNOS uncoupling, BCNU exposure further exacerbated superoxide production, BH4 oxidation and eNOS activity, effects that were totally abolished following mutation of either C689S or C908S. These data provide the first evidence that BH4 deficiency- and S-glutathionylation-induced mechanisms of eNOS uncoupling occur independently and their effects are additive.

Endothelial nitric oxide synthase impairment is restored by clofibrate treatment in an animal model of hypertension

Adequate production of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) requires eNOS coupling promoted by tetrahydrobiopterin (BH4). Under pathological conditions such as hypertension, BH4 is diminished, avoiding eNOS coupling. When eNOS is “uncoupled”, it yields a superoxide anion instead of NO. Peroxisome proliferator activated receptors (NR1C) are a family of nuclear receptors activated by ligand. Clofibrate, a member of a hypolipidemic class of drugs, acts by activating the alpha isoform of NR1C. To determine the participation of NR1C1 activation in BH4 and dihydrobiopterin (BH2) metabolism and its implications on eNOS coupling in hypertension, we performed aortic coarctation (AoCo) at inter-renal level on male Wistar rats in order to have a hypertensive model. Rats were divided into the following groups: Sham+vehicle (Sham-V); AoCo+vehicle (AoCo-V); Sham+clofibrate (Sham-C), and AoCo+clofibrate (AoCo-C). Clofibrate (7days) increased eNOS coupling in the AoCo-C group compared with AoCo-V. Clofibrate also recovered the BH4:BH2 ratio in control values and prevented the rise in superoxide anion production, lipoperoxidation, and reactive oxygen species production. In addition, clofibrate increased GTP cyclohydrolase-1 (GTPCH-1) protein expression, which is related with BH4 recovered production. NR1C1 stimulation re-establishes eNOS coupling, apparently through recovering the BH4:BH2 equilibrium and diminishing oxidative stress. Both can contribute to high blood pressure attenuation in hypertension secondary to AoCo.

Abstract 2057: Tumor suppressor effects of tetrahydrobiopterin are mediated through beta-catenin in breast cancer cells

Abstract Nitric Oxide (NO) can promote apoptosis in some cells, whereas it inhibits apoptosis in others. This complexity is an indication of the importance of NO source (NO donors vs. endogenous), location of that source, NO concentration and cell type in understanding the role of NO. Current experiments examine NO-dependent pathways involved in tumor cell survival from a new perspective of Nitric Oxide synthase (NOS) dysregulation. Tetrahydrobiopterin (BH4) is a necessary cofactor of NOS and can be readily oxidized to BH2 in an inflammatory environment, such as that found in solid tumors. When BH4 levels are low, electron transfer in the active site of the enzyme becomes uncoupled from L-arginine oxidation resulting in the production of O2− instead of NO. The uncoupled enzyme therefore becomes a generator of peroxynitrite (ONOO−), which is produced rapidly by the reaction of O2− with NO. In normal tissue the ratio of BH4 to BH2 (which when utilized by NOS the enzyme is uncoupled) is 40-70:1 and sometimes greater. We examined BH4 levels by HPLC in MCF7 and MDA-231 cells in tissue culture and in flank tumor xenografts. Our experiments showed that the BH4:BH2 ratio in tissue culture and flank tumor xenografts is around 1. These findings show that NOS in tumor cells as compared to normal cells is uncoupled. Subsequent experiments focused on the ability to manipulate BH4 levels through exogenous sepiapterin (SP), converted to BH4 through sepiapterin reductase and dihydrofolate reductase, or through overexpression of GTP cyclohydrolase I, the rate-limiting enzyme in BH4 production. Both tissue culture cells and flank tumor xenografts showed a 10-15-fold increase in BH4:BH2 when given SP in the tissue culture media or in the drinking water of mice. To ensure that increasing the BH4:BH2 ratio leads to enhanced NOS coupling, the production of NO was measured by cGMP formation and O2− levels were assessed by HPLC. As hypothesized NO levels went up and O2− levels went down. To directly examine the effects of manipulating the BH4:BH2 ratio cells were treated with varying concentrations of SP. MCF7 cells when exposed to SP showed a 2-fold decrease in colony formation and after 3 days of treatment had no viable cells, which was assessed by the MTT assay. Effects of SP on MDA 231 cells were not as dramatic, but by using the MTT assay we were able to show that the growth rate of the cells was cut in half. To assess the effects of SP on xenografts, we used an ex-vivo clonogenic assay. This assay also showed that colony formation is significantly reduced upon treatment of the animals with SP. Previous studies in colon cancer cells have shown that when PKG, the major pathway activated by NO, is activated β-catenin levels are reduced. In our experiments MCF7 xenografts showed a significant decrease in β-catenin levels after treatment with SP. These results represent a potential new therapeutic strategy in the treatment of solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2057. doi:1538-7445.AM2012-2057

Abstract 4062: Nitric Oxide synthase dysregulation in solid tumors: The therapeutic potential of tetrahydrobiopterin

Abstract Nitric Oxide (NO), in terms of cancer therapeutics, can be viewed as a double edge sword. Both anti-NO and NO-based anticancer treatments appear effective in several preclinical models. NO can promote apoptosis in some cells, whereas it inhibits apoptosis in others. This complexity is an indication of the importance of NO source (NO donors vs. endogenous), location of that source, NO concentration and cell type in understanding the role of NO. Current experiments examine NO-dependent pathways involved in tumor cell survival from a new perspective of Nitric Oxide synthase (NOS) dysregulation. Tetrahydrobiopterin (BH4) is a necessary cofactor of NOS and can be readily oxidized to BH2 in an inflammatory environment, such as that found in solid tumors. When BH4 levels are low, electron transfer in the active site of the enzyme becomes uncoupled from L-arginine oxidation resulting in the production of O2- instead of NO. The uncoupled enzyme therefore becomes a generator of peroxynitrite (ONOO-), which is produced rapidly by the reaction of O2- with NO, and is a highly reactive species that can modify proteins, lipids and DNA. In normal tissue the ratio of BH4 to BH2 (which when utilized by NOS the enzyme is uncoupled) is 40-70:1 and sometimes greater. We examined BH4 levels by HPLC in MCF7, Fadu, and A431 cells in vitro and Fadu and A431 cells in flank tumor xenografts. Our experiments showed that the BH4:BH2 ratio in tissue culture and flank tumor xenografts is less than 1. These findings show that NOS in tumor cells as compared to normal cells is uncoupled. Subsequent experiments focused on the ability to manipulate BH4 levels through exogenous sepiapterin (SP), converted to BH4 through sepiapterin reductase and dihydrofolate reductase, or through overexpression of GTP cyclohydrolase I, the rate-limiting enzyme in BH4 production. Both tissue culture cells and flank tumor xenografts showed a 10-15-fold increase in BH4:BH2 when given SP in the tissue culture media or in the drinking water of mice. Increasing the BH4:BH2 ratio leads to enhanced NO production as evidenced by an increase in cGMP formation. To directly examine the effects of manipulating the BH4:BH2 ratio, MCF7 and MDA cells were treated with varying concentrations of SP. MCF7 cells when exposed to SP showed a 2-fold decrease in colony formation and after 3 days of treatment had no viable cells, which was assessed by the MTT assay. Effects of SP on MDA 231 cells were not as dramatic, but by using the MTT assay we were able to show that the growth rate of the cells was cut in half. Current experiments focus on SP's effects in vivo and elucidating the mechanism of cell death and the signaling pathways involved. These results represent a potential new therapeutic strategy in the treatment of solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4062. doi:10.1158/1538-7445.AM2011-4062

Dihydrofolate reductase protects endothelial nitric oxide synthase from uncoupling in tetrahydrobiopterin deficiency

Tetrahydrobiopterin (BH4) is a required cofactor for the synthesis of NO by endothelial nitric oxide synthase (eNOS), and endothelial BH4 bioavailability is a critical factor in regulating the balance between NO and superoxide production (eNOS coupling). Biosynthesis of BH4 is determined by the activity of GTP-cyclohydrolase I (GTPCH). However, BH4 levels may also be influenced by oxidation, forming 7,8-dihydrobiopterin (BH2), which promotes eNOS uncoupling. Conversely, dihydrofolate reductase (DHFR) can regenerate BH4 from BH2, but whether DHFR is functionally important in maintaining eNOS coupling remains unclear. To investigate the mechanism by which DHFR might regulate eNOS coupling in vivo, we treated wild-type, BH4-deficient (hph-1), and GTPCH-overexpressing (GCH-Tg) mice with methotrexate (MTX), to inhibit BH4 recycling by DHFR. MTX treatment resulted in a striking elevation in BH2 and a decreased BH4:BH2 ratio in the aortas of wild-type mice. These effects were magnified in hph-1 but diminished in GCH-Tg mice. Attenuated eNOS activity was observed in MTX-treated hph-1 but not wild-type or GCH-Tg mouse lung, suggesting that inhibition of DHFR in BH4-deficient states leads to eNOS uncoupling. Taken together, these data reveal a key role for DHFR in regulating the BH4 vs BH2 ratio and eNOS coupling under conditions of low total biopterin availability in vivo.

B Chronic oral tetrahydrobiopterin treatment in patients with coronary artery disease elevates total biopterin levels but does not improve biopterin redox status or vascular function: a randomised placebo-controlled trial

Background Tetrahydrobiopterin (BH4), a critical endothelial nitric oxide synthase (eNOS) cofactor, is an important determinant of endothelial function. Oral BH4 therapy has been proposed as a treatment for vascular disease. We investigated the pharmacokinetics and pharmacodynamics of oral BH4 in both plasma and vascular tissue to determine whether oral BH4 therapy would improve vascular function in patients with established atherosclerosis and oxidative stress. Methods In a double-blind, randomised trial, 49 patients with coronary artery disease (CAD) awaiting coronary artery bypass graft surgery received oral BH4 700 mg/d (n=16) or 400 mg/d (n=14), or placebo (n=19), for a mean duration of 30 days prior to surgery. Biopterin levels were measured in plasma at baseline and after the treatment period, and in saphenous vein (SV) and internal mammary artery (IMA) tissue collected at the time of surgery. Vascular superoxide and endothelial function were measured ex vivo. Cardiovascular MRI was used to determine brachial artery flow-mediated dilatation and indices of arterial stiffness before and after treatment. Results Plasma BH4 levels were elevated threefold by both low- and high-dose BH4 treatment compared to placebo (p ex vivo , or in MRI indices of vascular function. Conclusion Although absolute BH4 levels in plasma and SV are increased, chronic oral BH4 therapy does not alter biopterin redox status (BH4:BH2 ratio) in either plasma or vascular tissue. Accordingly, oral BH4 does not improve vascular function or oxidative stress in patients with established CAD.

Critical Role for Tetrahydrobiopterin Recycling by Dihydrofolate Reductase in Regulation of Endothelial Nitric-oxide Synthase Coupling: RELATIVE IMPORTANCE OF THE DE NOVO BIOPTERIN SYNTHESIS VERSUS SALVAGE PATHWAYS

Tetrahyrobiopterin (BH4) is a required cofactor for the synthesis of nitric oxide by endothelial nitric-oxide synthase (eNOS), and BH4 bioavailability within the endothelium is a critical factor in regulating the balance between NO and superoxide production by eNOS (eNOS coupling). BH4 levels are determined by the activity of GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in de novo BH4 biosynthesis. However, BH4 levels may also be influenced by oxidation, forming 7,8-dihydrobiopterin (BH2), which promotes eNOS uncoupling. Conversely, dihydrofolate reductase (DHFR) can regenerate BH4 from BH2, but the functional importance of DHFR in maintaining eNOS coupling remains unclear. We investigated the role of DHFR in regulating BH4 versus BH2 levels in endothelial cells and in cell lines expressing eNOS combined with tet-regulated GTPCH expression in order to compare the effects of low or high levels of de novo BH4 biosynthesis. Pharmacological inhibition of DHFR activity by methotrexate or genetic knockdown of DHFR protein by RNA interference reduced intracellular BH4 and increased BH2 levels resulting in enzymatic uncoupling of eNOS, as indicated by increased eNOS-dependent superoxide but reduced NO production. In contrast to the decreased BH4:BH2 ratio induced by DHFR knockdown, GTPCH knockdown greatly reduced total biopterin levels but with no change in BH4:BH2 ratio. In cells expressing eNOS with low biopterin levels, DHFR inhibition or knockdown further diminished the BH4:BH2 ratio and exacerbated eNOS uncoupling. Taken together, these data reveal a key role for DHFR in eNOS coupling by maintaining the BH4:BH2 ratio, particularly in conditions of low total biopterin availability.

Quantitative Regulation of Intracellular Endothelial Nitric-oxide Synthase (eNOS) Coupling by Both Tetrahydrobiopterin-eNOS Stoichiometry and Biopterin Redox Status: INSIGHTS FROM CELLS WITH TET-REGULATED GTP CYCLOHYDROLASE I EXPRESSION

Tetrahydrobiopterin (BH4) is a critical determinant of endothelial nitric-oxide synthase (eNOS) activity. In the absence of BH4, eNOS becomes "uncoupled" and generates superoxide rather than NO. However, the stoichiometry of intracellular BH4/eNOS interactions is not well defined, and it is unclear whether intracellular BH4 deficiency alone is sufficient to induce eNOS uncoupling. To address these questions, we developed novel cell lines with tet-regulated expression of human GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in BH4 synthesis, to selectively induce intracellular BH4 deficiency by incubation with doxycycline. These cells were stably co-transfected to express a human eNOS-green fluorescent protein fusion protein, selecting clones expressing either low (GCH/eNOS-LOW) or high (GCH/eNOS-HIGH) levels. Doxycycline abolished GTPCH mRNA expression and GTPCH protein, leading to markedly diminished total biopterin levels and a decreased ratio of BH4 to oxidized biopterins in cells expressing eNOS. Intracellular BH4 deficiency induced superoxide generation from eNOS, as assessed by N-nitro-L-arginine methyl ester inhibitable 2-hydroxyethidium generation, and attenuated NO production. Quantitative analysis of cellular BH4 versus superoxide production between GCH/eNOS-LOW and GCH/eNOS-HIGH cells revealed a striking linear relationship between eNOS protein and cellular BH4 stoichiometry, with eNOS uncoupling at eNOS:BH4 molar ratio >1. Furthermore, increasing the intracellular BH2 concentration in the presence of a constant eNOS:BH4 ratio was sufficient to induce eNOS-dependent superoxide production. This specific, reductionist approach in a cell-based system reveals that eNOS:BH4 reaction stoichiometry together with the intracellular BH4:BH2 ratio, rather than absolute concentrations of BH4, are the key determinants of eNOS uncoupling, even in the absence of exogenous oxidative stress.