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Neuronal cell dysfunction plays an important role in neurodegenerative diseases. Oxidative stress can disrupt the redox balance within neuronal cells and may cause neuronal nitric oxide synthase (nNOS) to uncouple, contributing to the neurodegenerative processes. Experimental studies and clinical trials using nNOS cofactor tetrahydrobiopterin (BH 4) and antioxidants in neuronal cell dysfunction have shown inconsistent results. A better mechanistic understanding of complex interactions of nNOS activity and oxidative stress in neuronal cell dysfunction is needed. In this study, we developed a computational model of neuronal cell using nNOS biochemical pathways to explore several key mechanisms that are known to influence neuronal cell redox homeostasis. We studied the effects of oxidative stress and BH 4 synthesis on nNOS nitric oxide production and biopterin ratio (BH 4 /total biopterin). Results showed that nNOS remained coupled and maintained nitric oxide production for oxidative stress levels less than 230 nM/s. The results showed that neuronal oxidative stress above 230 nM/s increased the degree of nNOS uncoupling and introduced instability in the nitric oxide production. The nitric oxide production did not change irrespective of initial biopterin ratio of 0.05–0.99 for a given oxidative stress. Oxidative stress resulted in significant reduction in BH 4 levels even when nitric oxide production was not affected. Enhancing BH 4 synthesis or supplementation improved nNOS coupling, however the degree of improvement was determined by the levels of oxidative stress and BH 4 synthesis. The results of our mechanistic analysis indicate that there is a potential for significant improvement in neuronal dysfunction by simultaneously increasing BH 4 levels and reducing cellular oxidative stress. [Display omitted] • Computational model of nNOS was developed to understand the role of oxidative stress. • nNOS remained coupled & maintained NO production for oxidative stress level <230 nM/s. • NO production decreased and caused nNOS to uncouple at high oxidative stress levels. • BH 4 deficiency contributed to uncoupling of nNOS and reduced NO production. • BH 4 supplementation/synthesis enhanced neuronal function by restoring nNOS coupling. [ABSTRACT FROM AUTHOR]

Abstract: Endothelial dysfunction causes an imbalance in endothelial NO and O2 − production rates and increased peroxynitrite formation. Peroxynitrite and its decomposition products cause multiple deleterious effects including tyrosine nitration of proteins, superoxide dismutase (SOD) inactivation, and tissue damage. Studies have shown that peroxynitrite formation during endothelial dysfunction is strongly dependent on the NO and O2 − production rates. Previous experimental and modeling studies examining the role of NO and O2 − production imbalance on peroxynitrite formation showed different results in biological and synthetic systems. However, there is a lack of quantitative information about the formation and biological relevance of peroxynitrite under oxidative, nitroxidative, and nitrosative stress conditions in the microcirculation. We developed a computational biotransport model to examine the role of endothelial NO and O2 − production on the complex biochemical NO and O2 − interactions in the microcirculation. We also modeled the effect of variability in SOD expression and activity during oxidative stress. The results showed that peroxynitrite concentration increased with increase in either O2 − to NO or NO to O2 − production rate ratio (Q O2 −/Q NO or Q NO/Q O2 −, respectively). The peroxynitrite concentrations were similar for both production rate ratios, indicating that peroxynitrite-related nitroxidative and nitrosative stresses may be similar in endothelial dysfunction or inducible NO synthase (iNOS)-induced NO production. The endothelial peroxynitrite concentration increased with increase in both Q O2 −/Q NO and Q NO/Q O2 − ratios at SOD concentrations of 0.1–100μM. The absence of SOD may not mitigate the extent of peroxynitrite-mediated toxicity, as we predicted an insignificant increase in peroxynitrite levels beyond Q O2 −/Q NO and Q NO/Q O2 − ratios of 1. The results support the experimental observations of biological systems and show that peroxynitrite formation increases with increase in either NO or O2 − production, and excess NO production from iNOS or from NO donors during oxidative stress conditions does not reduce the extent of peroxynitrite mediated toxicity. [Copyright &y& Elsevier]

Abstract: Endothelial dysfunction is associated with increase in oxidative stress and low NO bioavailability. The endothelial NO synthase (eNOS) uncoupling is considered an important factor in endothelial cell oxidative stress. Under increased oxidative stress, the eNOS cofactor tetrahydrobiopterin (BH4) is oxidized to dihydrobiopterin, which competes with BH4 for binding to eNOS, resulting in eNOS uncoupling and reduction in NO production. The importance of the ratio of BH4 to oxidized biopterins versus absolute levels of total biopterin in determining the extent of eNOS uncoupling remains to be determined. We have developed a computational model to simulate the kinetics of the biochemical pathways of eNOS for both NO and O2 •− production to understand the roles of BH4 availability and total biopterin (TBP) concentration in eNOS uncoupling. The downstream reactions of NO, O2 •−, ONOO−, O2, CO2, and BH4 were also modeled. The model predicted that a lower [BH4]/[TBP] ratio decreased NO production but increased O2 •− production from eNOS. The NO and O2 •− production rates were independent above 1.5μM [TBP]. The results indicate that eNOS uncoupling is a result of a decrease in [BH4]/[TBP] ratio, and a supplementation of BH4 might be effective only when the [BH4]/[TBP] ratio increases. The results from this study will help us understand the mechanism of endothelial dysfunction. [Copyright &y& Elsevier]

Low levels of ascorbate (Asc) are observed in cardiovascular and neurovascular diseases. Asc has therapeutic potential for the treatment of endothelial dysfunction, which is characterized by a reduction in nitric oxide (NO) bioavailability and increased oxidative stress in the vasculature. However, the potential mechanisms remain poorly understood for the Asc mitigation of endothelial dysfunction. In this study, we developed an endothelial cell based computational model integrating endothelial cell nitric oxide synthase (eNOS) biochemical pathway with downstream reactions and interactions of oxidative stress, tetrahydrobiopterin (BH 4) synthesis and biopterin ratio ([BH 4 ]/[TBP]), Asc and glutathione (GSH). We quantitatively analyzed three Asc mediated mechanisms that are reported to improve/maintain endothelial cell function. The mechanisms include the reduction of •BH 3 to BH 4 , direct scavenging of superoxide (O 2 •−) and peroxynitrite (ONOO−) and increasing eNOS activity. The model predicted that Asc at 0.1–100 μM concentrations improved endothelial cell NO production, total biopterin and biopterin ratio in a dose dependent manner and the extent of cellular oxidative stress. Asc increased BH 4 availability and restored eNOS coupling under oxidative stress conditions. Asc at concentrations of 1–10 mM reduced O 2 •− and ONOO− levels and could act as an antioxidant. We predicted that glutathione peroxidase and peroxiredoxin in combination with GSH and Asc can restore eNOS coupling and NO production under oxidative stress conditions. Asc supplementation may be used as an effective therapeutic strategy when BH 4 levels are depleted. This study provides detailed understanding of the mechanism responsible and the optimal cellular Asc levels for improvement in endothelial dysfunction. [Display omitted] • Investigation of mechanisms of vitamin C in treatment of endothelial dysfunction. • Computational modeling of eNOS pathway, oxidative stress, biopterins & ascorbate. • 0.1–100 μM ascorbate improves NO production, biopterin ratio & endothelial function. • Above 1 mM ascorbate scavenges O 2.•− and ONOO− and reduces oxidative stress. • Combination of Asc & BH 4 supplementation may considerably improve biopterin ratio. [ABSTRACT FROM AUTHOR]

Introduction Oxidative stress and endothelial dysfunction have been associated in the development and progression of the cardiovascular diseases (CVDs). Oxidative stress is characterized by significant increase in the reactive oxygen species (predominantly superoxide (O 2 •‒ )) and the decrease in the nitric oxide (NO) bioavailability and antioxidant system of the cell. The major antioxidant system affected in endothelial dysfunction are enzymatic system such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and non-enzymatic systems such as glutathione (GSH) and ascorbate ((ASC). Studies on the therapeutic potential of increasing the antioxidants capacity to improve endothelial dysfunction have reported mixed results. This is because we have very little quantitative information on the complex interplay between the reactive species and antioxidant system involved in the disease progression. The role of antioxidants in oxidative stress is a complicated problem and can be addressed by computational modeling approaches. Material and Methods In the present study, we developed a computational model that involves eNOS biochemical pathway, downstream reactions involving NO and various reactive oxygen and nitrogen species, cellular oxidative stress and antioxidant systems. The role of GSH, GPX, ASC, SOD and CAT in the NO and O 2 •‒ co-generated system was analyzed to investigate the changes in NO and O 2 •- production rates, biopterin ratio with respect to changes in cellular oxidative stress. Results and Discussions The model results show that oxidative stress decreased the NO production and biopterin ratio and increased O 2 •‒ production. The upregulation of ASC, GSH, SOD improved the NO production rates and biopterin ratio but the decrease in O 2 •‒ production was insignificant when higher cellular oxidative stress was present. ASC supplementation improved endothelial dysfunction by improving biopterin ratio and making tetrahydrobiopterin (BH 4 ) available. The extent of improvement in endothelial dysfunction by antioxidants was dependent on extent of cellular oxidative stress. Conclusion The present study provides quantitative insights on how the up- or down- regulation of key antioxidant enzymes affects the cellular ROS levels and endothelial function. The thorough mechanistic analysis of each antioxidant performed under endothelial dysfunction reveals the important antioxidant systems relevant for oxidative stress. [ABSTRACT FROM AUTHOR]

Introduction Ascorbate (Asc) plays a critical role in maintaining of endothelial cell function through inhibition of apoptosis, stimulation of cell proliferation and improvement of endothelial derived NO bioavailability. A number of mechanisms including scavenging of free radicals, increase of tetrahydrobiopterin (BH 4 ) levels and improving endothelial nitric oxide synthase (eNOS) coupling state have been proposed for Asc related NO bioavailability. Increase in cellular oxidative stress is associated with low cellular and plasma Asc levels. However, it is not certain which mechanisms and what optimal ascorbate levels are involved in the improvement of endothelial cell function in oxidative stress. Material and Methods In the present study, we developed a computational model that involves eNOS biochemical pathway, downstream reactions involving NO and various reactive oxygen and nitrogen species, cellular oxidative stress, BH 4 synthesis and Asc supplementation. The model was simulated to investigate the changes in NO production and BH 4 concentration with respect to Asc supplementation. Results and Discussions The model result shows that oxidative stress decreased the NO production. As shown in the below figure, supplementation of 50 µM Asc improved the NO production ~90 % under oxidative stress conditions, whereas Asc did not change NO production in control conditions (Figure A). Asc increased BH 4 concentration in both control and oxidative stress condition (Figure B). This suggests Asc role in increasing BH 4 bioavailability and keeping eNOS in coupled state. Experimental studies also confirm the role of ascorbate in replenishing the total antioxidant system of endothelial cells. Image 1 Conclusion Our model result provides proof for the suggested role of Asc in replenishing BH 4 and making it available for eNOS coupling. The results from this study provide quantitative understating of the role of ascorbate in the endothelial dysfunction and vascular diseases. [ABSTRACT FROM AUTHOR]