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 (\u3c6 \u3emonths) 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

Tetrahydrobiopterin analogues with NO-dependent pulmonary vasodilator properties

Reduced NO levels due to the deficiency of tetrahydrobiopterin (BH4) contribute to impaired vasodilation in pulmonary hypertension Due to the chemically unstable nature of BH4 it was hypothesised that oxidatively stable analogues of BR, would be able to support NO synthesis to improve Endothelial dysfunction in pulmonary hypertension Two analogues of BH4 namely 6-hydroxymethyl pterin (HMP) and 6-acetyl 7 7-dimethyl 7 8-dihydropterin (ADDP) were evaluated for vasodilator activity on precontracted rat pulmonary artery rings ADDP was administered to pulmonary hypertensive rats followed by measurement of pulmonary vascular resistance in perfused lungs and eNOS expression by immunohistochemistry ADDP and HMP caused significant relaxation in vitro in rat pulmonary arteries depleted of BH4 with a maximum relaxation at 0 3 mu M (both P<005) Vasodilator activity of ADDP and HMP was completely abolished following preincubation with the NO synthase inhibitor L-NAME ADDP and HMP did not alter relaxation induced by carbachol or spermine NONOate BH4 Itself did not produce relaxation In rats receiving ADDP 141 mg/kg/day pulmonary vasodilation induced by calcium ionophore A23187 was augmented and eNOS immunoreactivity was increased In conclusion ADDP and HMP are two analogues of BH4 which can act as oxidatively stable alternatives to BH4 in causing NO-mediated vasorelaxation Chronic treatment with ADDP resulted in Improvement of NO-mediated pulmonary artery dilation and enhanced expression of eNOS in the pulmonary vascular endothelium Chemically stable analogue, of BH4 may be able to limit endothelial dysfunction in the pulmonary vasculatur

Studies on the regulation of endothelial nitric oxide synthase in endothelial dysfunction

Ischaemic heart disease and cerebrovascular disease are the leading causes of morbidity and mortality in the world. The underlying progression of the disease is linked to a reduction in the bioavailability of nitric oxide. One factor contributing to this is an increase in the production of superoxide radicals. A combination of increased oxidative stress, inappropriate lipid metabolism and cell death sets the stage for what will subsequently develop into atherosclerosis. The process of atherogenesis can slow down if patients at risk are identi ed early, receive the necessary pharmacological treatment and change to a healthier lifestyle. The aim of the following studies was to identify whether the uptake, synthesis and recycling of tetrahydrobiopterin (BH4), the essential co-factor of endothelial nitric oxide synthase (eNOS), could in uence oxidative stress in human vasculature. We also sought to elucidate whether endothelin-1 (ET-1), a potent vasoconstrictor, played an important role in oxidative stress in human vasculature and the potential mechanisms underlying this in uence. In Study I, 49 patients with coronary artery disease took part in a placebo-controlled clinical trial with the aim of determining the mechanisms of exogenous BH4 in relation to vascular function. Oral BH4 treatment signi cantly elevates the levels of BH4 in blood, but this effect is limited by the rapid systemic oxidation of exogenous BH4. The ratio of reduced to oxidised biopterins in blood and vascular tissue is unchanged by exogenous BH4 treatment, resulting in no net effect on vascular superoxide production or endothelial function. In Study II, the aim was to explore the regulation of endogenous BH4 and subsequent effects on endothelial function in patients with coronary artery disease. In three clinical models and one in vitro model, involving 465 subjects, we observed that an inability to increase vascular BH4 synthesis leads to signi cant impairment of endothelial function. In Study III, the aim was to explore the role of ET-1 in endothelial dysfunction, speci cally with regard to superoxide production. ET-1 increases superoxide production in human coronary artery bypass grafts via a receptor-driven mechanism involving, the largest contributor of superoxide in the vascular wall, nicotine amide dinucleotide phosphate (NADPH) oxidase. In Study IV, I applied what I had learned from Study I and Study II and sought to further delineate whether endothelin-1 in uences biopterin homeostasis in both human and animal tissues. ET-1 did not have any effect on BH4 in human coronary artery bypass grafts or resistance arteries, endothelial cells and mice with an over-expression of ET-1 in the endothelium (ET-transgenic mice)

Long-term correction of murine phenylketonuria by viral gene transfer: liver versus muscle

Current therapy for phenylketonuria (PKU) consists of life-long dietary restriction of phenylalanine (Phe), which presents problems of adherence for patients. Alternative therapies under investigation include, among others, the use of gene therapy to provide copies of wild-type, non-mutant, phenylalanine hydroxylase (PAH) enzyme. Expression of PAH in both liver (the usual metabolic source of this enzyme) and skeletal muscle is under investigation. Liver gene therapy, using a viral vector based on the adeno-associated viruses (AAVs), provided effective clearance of serum Phe that was sustained for 1year in some mice. In order for PAH expression to be effective in skeletal muscle, the essential metabolic cofactor, tetrahydrobiopterin (BH4), must also be provided, either by supplementation or gene therapy. Both these approaches were effective. When transgenic PKU mice that constitutively expressed PAH in muscle were given intraperitoneal supplementation with BH4, this produced (transient) effective clearance of Phe to normal levels. In addition, use of an AAV vector containing the genes for PAH, and for two key synthetic enzymes for BH4, provided substantial and long-lasting correction (more than 1year) of blood Phe levels when injected into skeletal muscle of PKU mice. These two strategies provide promising treatment alternatives for the management of PKU in patient

Inflammation and endothelial function: Direct vascular effects of human C-reactive protein on nitric oxide bioavailability

Background - Circulating concentrations of the sensitive inflammatory marker C-reactive protein (CRP) predict future cardiovascular events, and CRP is elevated during sepsis and inflammation, when vascular reactivity may be modulated. We therefore investigated the direct effect of CRP on vascular reactivity. Methods and Results - The effects of isolated, pure human CRP on vasoreactivity and protein expression were studied in vascular rings and cells in vitro, and effects on blood pressure were studied in rats in vivo. The temporal relationship between changes in CRP concentration and brachial flow-mediated dilation was also studied in humans after vaccination with Salmonella typhi capsular polysaccharide, a model of inflammatory endothelial dysfunction. In contrast to some previous reports, highly purified and well-characterized human CRP specifically induced hyporeactivity to phenylephrine in rings of human internal mammary artery and rat aorta that was mediated through physiological antagonism by nitric oxide (NO). CRP did not alter endothelial NO synthase protein expression but increased protein expression of GTP cyclohydrolase-1, the rate-limiting enzyme in the synthesis of tetrahydrobiopterin, the NO synthase cofactor. In the vaccine model of inflammatory endothelial dysfunction in humans, increased CRP concentration coincided with the resolution rather than the development of endothelial dysfunction, consistent with the vitro findings; however, administration of human CRP to rats had no effect on blood pressure. Conclusions - Pure human CRP has specific, direct effects on vascular function in vitro via increased NO production; however, further clarification of the effect, if any, of CRP on vascular reactivity in humans in vivo will require clinical studies using specific inhibitors of CRP. © 2005 American Heart Association, Inc

Mutationen im PTS-Gen und mögliche Auswirkungen auf Funktion und Struktur der 6-Pyruvoyl-Tetrahydropterin-Synthase

Background: Research on Juvenile Idiopathic Arthritis (JIA) should support patients, caregivers/parents (carers) and clinicians to make important decisions in the consulting room and eventually to improve the lives of patients with JIA. Thus far these end-users of JIA-research have rarely been involved in the prioritisation of future research. Main body: Dutch organisations of patients, carers and clinicians will collaboratively develop a research agenda for JIA, following the James Lind Alliance (JLA) methodology. In a 'Priority Setting Partnership' (PSP), they will gradually establish a top 10 list of the most important unanswered research questions for JIA. In this process the input from clinicians, patients and their carers will be equally valued. Additionally, focus groups will be organised to involve young people with JIA. The involvement of all contributors will be monitored and evaluated. In this manner, the project will contribute to the growing body of literature on how to involve young people in agenda setting in a meaningful way. Conclusion: A JIA research agenda established through the JLA method and thus co-created by patients, carers and clinicians will inform researchers and research funders about the most important research questions for JIA. This will lead to research that really matters.</p

Diagnosis of tetrahydrobiopterin deficiency using filter paper blood spots: further development of the method and 5 years experience

In every newborn with even mild hyperphenylalaninemia (HPA) tetrahydrobiopterin (BH4) deficiencies need to be excluded as soon as possible. Differential diagnosis is most commonly performed by analysis of urinary neopterin and biopterin. In 2005 a new method for the measurement of neopterin, biopterin and other pterins in dried blood spot (DBS) on filter paper was introduced. In order to evaluate the usefulness of this method as a standard tool for differential diagnosis of HPAs we analyzed neopterin, biopterin, pterin and dihydropteridine reductase activity in DBS from 362 patients with HPA over the period of five years. Age-dependent reference values were established for the HPA population. Sixty-four patients with BH4 deficiency (27 patients with 6-pyruvoyl-tetrahydropterin synthase deficiency, seven with GTP cyclohydrolase I deficiency, and 30 with dihydropteridine reductase) were identified. Reference values for neopterin and biopterin in DBS were calculated for each of the variants. 6-pyruvoyl-tetrahydropterin synthase and GTP cyclohydrolase I deficiency can be diagnosed by neopterin and biopterin analysis alone, while for diagnosis of dihydropteridine reductase deficiency additional determination of enzyme activity from the same DBS is essential. Regarding test sensitivity, the interpretation of neopterin and biopterin concentration per hemoglobin is more valid than the interpretation of neopterin and biopterin per liter. Percentage of biopterin, of the sum of neopterin and biopterin should always be calculated. In addition, determination of hemoglobin concentration is essential as a measure for efficient extraction of neopterin and biopterin. Although the measurement of neopterin and biopterin in urine is more sensitive due to the higher concentrations present, our data prove the usefulness of their measurement from DBS for the routine diagnosis of BH4 deficiencie

Tetrahydrobiopterin Deficiency: From Phenotype to Genotype

As a result of the selective screening worldwide during the last 18 years, approximately 250 patients with tetrahydrobiopterin deficiency were discovered. Most patients suffer from 6-pyruvoyl tetrahydropterin synthase deficiency (58%), followed by dihydropteridine reductase deficiency (35%), GTP cyclohydrolase I deficiency (3%), and "primapterinuria” (4%). The patients can be treated with neurotransmitter precursors, as well as with tetrahydrobiopterin. However, data on long term treatment are still scarce and it is therefore of great value to investigate all newborns with even mild hyperphenylalaninemia. Cloning of the enzymes involved in the biosynthesis and regeneration of tetrahydrobiopterin makes them to be easily accessible for biochemical and biological studies. So far, all proteins expressed heterologous are active in E. coli. Cloning of the wild type gene and mutant analysis of patients allow the rapid identification of the defective gene on the molecular leve