4-hydroxy-2-nonenal and sapropterin

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.

Topics: Aldehydes; Animals; Biopterins; Dose-Response Relationship, Drug; Endothelium, Vascular; Glutathione; GTP Cyclohydrolase; Humans; Mice; Mice, Transgenic; Mitochondria; Myocytes, Cardiac; Nitric Oxide; Oxidation-Reduction; Oxidative Stress; Oxygen Consumption; Superoxides

Tetrahydrobiopterin (BH(4)), 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 BH(4), 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 BH(4) (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 BH(4) levels by 3-fold without changing protein levels of GTP cyclohydrolase, the enzyme that regulates de novo BH(4) 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-alpha and interferon-gamma 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.

Topics: Aldehydes; Animals; Apoptosis; Arginase; Biopterins; Cyclosporine; Cytokines; Echocardiography; Gene Expression; Graft Rejection; GTP Cyclohydrolase; Heart Transplantation; Heart Ventricles; Immunosuppressive Agents; Myocardium; Nitric Oxide; Nitric Oxide Synthase Type II; Protein Processing, Post-Translational; Pterins; Rats; Rats, Inbred Lew; Rats, Inbred WF; Transplantation, Homologous; Transplantation, Isogeneic; Tyrosine

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).. 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.. 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.

Topics: Aldehydes; Animals; Aorta; Biopterins; Cattle; Cells, Cultured; Endothelial Cells; GTP Cyclohydrolase; Nitric Oxide; Nitric Oxide Synthase Type III; Proteasome Endopeptidase Complex; Superoxides

To investigate endothelial function and levels of vascular oxidative stress in rat adjuvant-induced arthritis (AIA), in view of mounting evidence for an association between rheumatoid arthritis (RA) and accelerated vascular disease.. Thoracic aortic rings were prepared from AIA and control rats. After preconstriction by norepinephrine, the vasodilatory response to acetylcholine was determined. The amounts of 4-hydroxy-2-nonenal (HNE) and nitrotyrosine in AIA rat aortas were measured by Western blotting. Homogenates of the aortas were incubated with various substrates for superoxide-producing enzymes, and superoxide production was assessed by fluorogenic oxidation of dihydroethidium to ethidium. Expression of endothelial nitric oxide synthase (eNOS) in aortas was examined by real-time reverse transcriptase-polymerase chain reaction and Western blotting. Serum levels of tetrahydrobiopterin (BH4), a critical eNOS cofactor, were determined by high-performance liquid chromatography.. Endothelium-dependent relaxation of the aortic ring was significantly depressed in AIA rats compared with control rats. The amounts of HNE and nitrotyrosine were increased in AIA rat aortas, indicating overproduction of reactive oxygen species. Incubation of AIA rat aorta homogenates with NADH or L-arginine, a substrate of eNOS, resulted in a significant increase in superoxide production. Endothelial NOS was highly expressed in AIA rat aortas. Serum levels of BH4 were significantly lower in AIA. Treatment of AIA with BH4 reversed the endothelial dysfunction, suggesting that its deficiency may contribute to the uncoupling of eNOS.. Vascular dysfunction in RA can be partially modeled in animals. NAD(P)H oxidase and uncoupled eNOS are responsible for the increase in vascular oxidative stress, which is likely to be involved in the endothelial dysfunction in AIA.

Topics: Acetylcholine; Aldehydes; Animals; Aorta, Thoracic; Arthritis, Experimental; Biopterins; Endothelium, Vascular; In Vitro Techniques; Male; NADPH Oxidases; Nitric Oxide Synthase Type III; Rats; Rats, Inbred Lew; Reactive Oxygen Species; Superoxides; Tyrosine; Vasodilation