Jennifer Whitsett

Altered Tetrahydrobiopterin Metabolism in Atherosclerosis. Implications for Use of Oxidized Tetrahydrobiopterin Analogues and Thiol Antioxidants

Objective—Tetrahydrobiopterin (BH4) is of fundamental importance for the normal function of endothelial NO synthase. The purpose of this study was to investigate the effects of hyperlipidemia on vascular BH4 levels and the effect of supplementation with sepiapterin in the presence and absence of N-acetylcysteine (NAC). Methods and Results—New Zealand White rabbits were fed normal chow (normocholesterolemic [NC] group) or hyperlipidemic chow (hyperlipidemic [HL] group) for 8 to 10 weeks. Mean cholesterol levels were 1465±333 and 53±17 mg/dL in the HL and NC group, respectively. Markedly diminished BH4 levels were found in the HL group compared with the NC group, but these levels could be restored after 6 hours of incubation with sepiapterin. Peak relaxations to acetylcholine and A23187 were impaired in the HL group. Supplementation with sepiapterin resulted in a further diminution of relaxation in the HL but not NC group. Incubation with NAC for 6 hours failed to raise BH4 levels, whereas NAC in conjunction with sepiapterin raised BH4 levels ≈221-fold. However, this increase did not improve relaxations to A23187 and acetylcholine. Conclusions—Prolonged exposure to sepiapterin impairs vasorelaxation in hyperlipidemia despite repletion of endogenous BH4. Antioxidant thiols do not correct this impairment. These studies have implications for the use of sepiapterin in the correction of vasomotor tone in atherosclerosis.

Reaction of tetrahydrobiopterin with superoxide: EPR-kinetic analysis and characterization of the pteridine radical

It has been shown that BH(4) ameliorates endothelial dysfunction associated with conditions such as hypertension, cigarette smoking, and diabetes. This effect has been proposed to be due to a superoxide scavenging activity of BH(4). To examine this possibility we determined the rate constant for the reaction between BH(4) and superoxide using electron paramagnetic resonance (EPR) spin trapping competition experiments with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO). We calculated a rate constant for the reaction between BH(4) and superoxide of 3.9 +/- 0.2 x 10(5) M(-1)s(-1) at pH 7.4 and room temperature. This result suggests that superoxide scavenging by BH(4) is not a major reaction in vivo. HPLC product analysis showed that 7,8-BH(2) and pterin are the stable products generated from the reaction. The formation of BH(4) cation radical (BH(4)(*+)) was demonstrated by direct EPR only under acidic conditions. Isotopic substitution experiments demonstrated that the BH(4)(*+) is mainly delocalized on the pyrazine ring of BH(4). In parallel experiments, we investigated the effect of ascorbate on 7,8-BH(2) reduction and eNOS activity. We demonstrated that ascorbate does not reduce 7,8-BH(2) to BH(4), nor does it stimulate nitric oxide release from eNOS incubated with 7,8-BH(2). In conclusion, it is likely that BH(4)-dependent inhibition of superoxide formation from eNOS is the mechanism that better explains the antioxidant effects of BH(4) in the vasculature.

4-Hydroxy-2-Nonenal Increases Superoxide Anion Radical in Endothelial Cells via Stimulated GTP Cyclohydrolase Proteasomal Degradation

Objective—4-Hydroxy-2-nonenal (4-HNE) is an abundant electrophilic lipid that mediates oxidative stress in endothelium by mechanisms that remain controversial. This study examines the effects of 4-HNE on nitric oxide (NO) and superoxide levels in bovine aorta endothelial cells (BAECs). Methods and Results—Exposure of BAECs to 4-HNE caused a dose-dependent inhibition of NO that correlated with losses of hsp90 and phosphorylated eNOS-serine1179 but not eNOS protein levels. 4-HNE failed to inhibit NO production in sepiapterin and ascorbate supplemented cells suggesting that tetrahydrobiopterin (BH4) is a limiting factor in non supplemented cells. This was verified by quantification of BH4 by high-performance liquid chromatography analysis with electrochemical detection and by examining GTP cyclohydrolase I (GTPCH) protein levels and activity all of which were diminished by 4-HNE treatment. Analysis of 2-hydroxyethidium indicated that 4-HNE increased superoxide release in BAECs. The effects of 4-HNE on GTPCH and hsp90 were efficiently counteracted by proteasomal inhibition, indicating that depletion of BH4 by 4-HNE is attributable to specific mechanisms involving protein degradation. Conclusions—4-HNE by altering BH4 homeostasis mediates eNOS-uncoupling and superoxide generation in BAECs. By also decreasing phosphorylation of eNOS-serine 1179 4-HNE may specifically regulate NO/reactive oxygen species fluxes in the endothelium with important consequences to redox signaling.

Deficient BH4 production via de novo and salvage pathways regulates NO responses to cytokines in adult cardiac myocytes

Adult rat cardiac myocytes typically display a phenotypic response to cytokines manifested by low or no increases in nitric oxide (NO) production via inducible NO synthase (iNOS) that distinguishes them from other cell types. To better characterize this response, we examined the expression of tetrahydrobiopterin (BH4)-synthesizing and arginine-utilizing genes in cytokine-stimulated adult cardiac myocytes. Intracellular BH4 and 7,8-dihydrobiopterin (BH2) and NO production were quantified. Cytokines induced GTP cyclohydrolase and its feedback regulatory protein but with deficient levels of BH4 synthesis. Despite the induction of iNOS protein, cytokine-stimulated adult cardiac myocytes produced little or no increase in NO versus unstimulated cells. Western blot analysis under nonreducing conditions revealed the presence of iNOS monomers. Supplementation with sepiapterin (a precursor of BH4) increased BH4 as well as BH2, but this did not enhance NO levels or eliminate iNOS monomers. Similar findings were confirmed in vivo after treatment of rat cardiac allograft recipients with sepiapterin. It was found that expression of dihydrofolate reductase, required for full activity of the salvage pathway, was not detected in adult cardiac myocytes. Thus, adult cardiac myocytes have a limited capacity to synthesize BH4 after cytokine stimulation. The mechanisms involve posttranslational factors impairing de novo and salvage pathways. These conditions are unable to support active iNOS protein dimers necessary for NO production. These findings raise significant new questions about the prevailing understanding of how cytokines, via iNOS, cause cardiac dysfunction and injury in vivo during cardiac inflammatory disease states since cardiac myocytes are not a major source of high NO production.

Tetrahydrobiopterin in the Prevention of Hypertonia in Hypoxic Fetal Brain

Tetrahydrobiopterin (BH4) deficiency is a cause of dystonia at birth. We hypothesized that BH4 is a developmental factor determining vulnerability of the immature fetal brain to hypoxic‐ischemic injury and subsequent motor deficits in newborns.

Neuronal nitric oxide synthase inhibition prevents cerebral palsy following hypoxia-ischemia in fetal rabbits: comparison between JI-8 and 7-nitroindazole.

Cerebral palsy and death are serious consequences of perinatal hypoxia-ischemia (HI). Important concepts can now be tested using an animal model of cerebral palsy. We have previously shown that reactive oxygen and nitrogen species are produced in antenatal HI. A novel class of neuronal nitric oxide synthase (nNOS) inhibitors have been designed, and they ameliorate postnatal motor deficits when administered prior to the hypoxic-ischemic insult. This study asks how the new class of inhibitors, using JI-8 (Ki for nNOS: 0.014 µM) as a representative, compare with the frequently used nNOS inhibitor 7-nitroindazole (7-NI; Ki: 0.09 ± 0.024 µM). A theoretical dose equivalent to 75 Ki of JI-8 or equimolar 7-NI was administered to pregnant rabbit dams 30 min prior to and immediately after 40 min of uterine ischemia at 22 days gestation (70% term). JI-8 treatment resulted in a significant decrease in NOS activity (39%) in fetal brain homogenates acutely after HI, without affecting maternal blood pressure and heart rate. JI-8 treatment resulted in 33 normal kits, 2 moderately and 13 severely affected kits and 5 stillbirths, compared with 8 normal, 3 moderately affected and 5 severely affected kits and 10 stillbirths in the 7-NI group. In terms of neurobehavioral outcome, 7-NI was not different from saline treatment, while JI-8 was superior to saline and 7-NI in its protective effect (p < 0.05). In the surviving kits, JI-8 significantly improved the locomotion score over both saline and 7-NI scores. JI-8 was also significantly superior to saline in preserving smell, muscle tone and righting reflex function, but 7-NI did not show significant improvement. Furthermore, a 100-fold increase in the dose (15.75 µmol/kg) of 7-NI significantly decreased systolic blood pressure in the dam, while JI-8 did not. The new class of inhibitors such as JI-8 shows promise in the prevention of cerebral palsy and is superior to the previously more commonly used nNOS inhibitor.

Sepiapterin Decreases Acute Rejection and Apoptosis in Cardiac Transplants Independently of Changes in Nitric Oxide and Inducible Nitric-Oxide Synthase Dimerization

Tetrahydrobiopterin (BH4), a cofactor of inducible nitric-oxide synthase (iNOS), is an important post-translational regulator of NO bioactivity. We examined whether treatment of cardiac allograft recipients with sepiapterin [S-(-)-2-amino-7,8-dihydro-6-(2-hydroxy-1-oxopropyl)-4-(1H)-pteridinone], a precursor of BH4, inhibited acute rejection and apoptosis in cardiac transplants. Heterotopic cardiac transplantation was performed in Wistar-Furth donor to Lewis recipient strain rats. Recipients were treated daily after transplantation with 10 mg/kg sepiapterin. Grafts were harvested on post-transplant day 6 for analysis of BH4 (high-performance liquid chromatography), expression of inflammatory cytokines (reverse transcription- and real-time polymerase chain reaction), iNOS (Western blots), and NO (Griess reaction and NO analyzer). Histological rejection grade was scored, and graft function was determined by echocardiography. Apoptosis, protein nitration, and oxidative stress were determined by immunohistochemistry. Treatment of allografts with sepiapterin increased cardiac BH4 levels by 3-fold without changing protein levels of GTP cyclohydrolase, the enzyme that regulates de novo BH4 synthesis. Sepiapterin decreased inflammatory cell infiltrate and significantly inhibited histological rejection scores and apoptosis similar in magnitude to cyclosporine. Sepiapterin also decreased nitrative and oxidative stress. Sepiapterin caused a smaller increase in left ventricular mass versus untreated allografts but without improving fractional shortening. Sepiapterin did not alter tumor necrosis factor-α and interferon-γ expression, whereas it decreased interleukin (IL)-2 expression. Sepiapterin did not change total iNOS protein or monomer levels, or plasma and tissue NO metabolites levels. It is concluded that the mechanism(s) of antirejection are due in part to decreased apoptosis, protein nitration, and oxidation of cardiomyocytes, which seems to be mediated at the immune level by limiting inflammatory cell infiltration via decreased IL-2-mediated T-lymphocyte expansion.

Human endothelial dihydrofolate reductase low activity limits vascular tetrahydrobiopterin recycling

Tetrahydrobiopterin (BH₄) is required for NO synthesis and inhibition of superoxide release from endothelial NO synthase. Clinical trials using BH₄ to treat endothelial dysfunction have produced mixed results. Poor outcomes may be explained by the rapid systemic and cellular oxidation of BH₄. One of the oxidation products of BH₄, 7,8-dihydrobiopterin (7,8-BH₂), is recycled back to BH₄ by dihydrofolate reductase (DHFR). This enzyme is ubiquitously distributed and shows a wide range of activity depending on species-specific factors and cell type. Information about the kinetics and efficiency of BH4 recycling in human endothelial cells receiving BH₄ treatment is lacking. To characterize this reaction, we applied a novel multielectrode coulometric HPLC method that enabled the direct quantification of 7,8-BH₂ and BH₄, which is not possible with fluorescence-based methodologies. We found that basal untreated BH₄ and 7,8-BH₂ concentrations in human endothelial cells (ECs) are lower than in bovine and murine endothelioma cells. Treatment of human ECs with BH₄ transiently increased intracellular BH₄ while accumulating the more stable 7,8-BH₂. This was different from bovine or murine ECs, which resulted in preferential BH₄ increase. Using BH₄ diastereomers, 6S-BH₄ and 6R-BH₄, the narrow contribution of enzymatic DHFR recycling to total intracellular BH₄ was demonstrated. Reduction of 7,8-BH₂ to BH₄ occurs at very slow rates in cells and needs supraphysiological levels of 7,8-BH₂, indicating this reaction is kinetically limited. Activity assays verified that human DHFR has very low affinity for 7,8-BH₂ (DHF7,8-BH₂) and folic acid inhibits 7,8-BH₂ recycling. We conclude that low activity of endothelial DHFR is an important factor limiting the benefits of BH4 therapies, which may be further aggravated by folate supplements.

Cytokines and lipopolysaccharides induce inducible nitric oxide synthase but not enzyme activity in adult rat cardiomyocytes

There is evidence that nitric oxide (NO) formation in adult cardiomyocytes stimulated with lipopolysaccharide (LPS) is not commensurate with iNOS levels. Tetrahydrobiopterin (BH(4)) is a key factor in the stabilization and NO production by iNOS homodimer. Thus we hypothesized that BH(4) is a limiting factor for NO production in adult cardiomyocytes in response to LPS and cytokines (TNF-alpha, IL-1, IFN-gamma alone, or mixed). It was verified that LPS and cytokines induced iNOS expression which did not translate into increased nitrite or [(14)C]citrulline production. This response coincided with defective BH(4) synthesis and low GTP cyclohydrolase activity. Furthermore, supplementation with BH(4) and ascorbate failed to increase iNOS activity. This effect was related to preferential accumulation of BH(2) rather than BH(4) in these cells. Uncoupled iNOS activity in stimulated cells was examined using mitochondrial aconitase activity as an endogenous marker of superoxide anion radical (O(2)(-)) formation, and found not to be significantly inhibited. 2-Hydroxyethidium also was not significantly increased. We conclude that adult cardiomyocytes are an unlikely source of NO and O(2)(-) in inflammatory conditions. This finding adds a new and unexpected layer of complexity to our understanding of the responses of the adult heart to inflammation.

NAD(P)H oxidase and eNOS play differential roles in cytomegalovirus infection-induced microvascular dysfunction.

Primary cytomegalovirus (CMV) infection promotes oxidative stress and reduces nitric oxide (NO) bioavailability in endothelial cells. These events are among the earliest vascular responses to cardiovascular risk factors. We assessed the roles of NAD(P)H oxidase and NO bioavailability in microvascular responses to persistent CMV infection alone or with hypercholesterolemia. Wild-type (WT) or gp91(phox) (NAD(P)H oxidase subunit) knockout mice received mock inoculum or 3×10(4) PFU murine CMV (mCMV) ip 5 weeks before placement on a normal or high-cholesterol diet (HC) for 4 weeks before assessment of arteriolar function and venular blood cell recruitment using intravital microscopy. Some WT groups received sepiapterin (a precursor of the nitric oxide synthase cofactor tetrahydrobiopterin) or apocynin (NAD(P)H oxidase inhibitor/antioxidant). Endothelium-dependent vasodilation was impaired in mCMV vs mock WT, regardless of diet. This was not affected by sepiapterin, and pharmacological inhibition of nitric oxide synthase reduced dilation similarly in mock and mCMV mice. Apocynin or deficiency of total, but not blood cell or vascular wall only (tested using bone marrow chimeras), gp91(phox) protected against arteriolar dysfunction. Blood cell recruitment was induced by mCMV-HC. Sepiapterin, but not NAD(P)H oxidase deficiency/apocynin, reduced leukocyte accumulation, whereas platelet adhesion was reduced by sepiapterin, apocynin, or total, platelet-specific, or vascular wall gp91(phox) deficiency. These data implicate activation of both hematopoietic and vessel wall NAD(P)H oxidase in mCMV-induced arteriolar dysfunction and platelet and vascular NAD(P)H oxidase in the thrombogenic phenotype induced by mCMV-HC. In contrast, findings with sepiapterin suggest that eNOS dysfunction, perhaps uncoupling, mediates venular, but not arteriolar, responses to mCMV-HC, thus indicating that NAD(P)H oxidase and eNOS differentially regulate microvascular responses to mCMV.