Modulators of Nitric Oxide Synthase Activity in Sickle Cell Disease

镰状细胞病中一氧化氮合酶活性的调节剂

• 批准号: 7654865
• 负责人: Mary E Fabry
• 金额: $ 41.5万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7654865
• 关键词: Amino Acids; Animals; Antioxidants; Arginine; Benchmarking; Biological Availability; Biological Markers; Biological Preservation; Biopterin; Breeding; Cells; Citrulline; Dehydration; Dose; Enzymes; Erythrocytes; Event; Failure; Functional disorder; GTP Cyclohydrolase I; Hemoglobin; Hemolysis; Human; Hypoxia; Inflammation; Intervention; Lead; Metabolism; Modeling; Multienzyme Complexes; Mus; Nitric Oxide; Nitric Oxide Synthase; Ornithine; Oxidative Stress; Oxygen; Pathology; Pathway interactions; Patients; Phosphorylation; Pilot Projects; Plasma; Play; Polymers; Production; Rattus; Reaction; Response Elements; Role; Series; Serine; Severities; Sickle Cell; Sickle Cell Anemia; Superoxides; Supplementation; Testing; Threonine; Tissue Extracts; Tissues; Transgenic Mice; Transgenic Organisms; Vascular Diseases; base; blood perfusion; drinking water; improved; inhibitor/antagonist; mouse model; polymerization; prevent; sickling; tetrahydrobiopterin; vascular bed

The primary features of sickle cell disease are events resulting in low flow, poor perfusion and blood and tissue hypoxia that are both pre-disposing to and the consequence of vaso-occlusion. We will test the overall hypothesis that BH4 (tetra-hydrobiopterin, an essential co-factor of nitric oxide synthase (NOS)) levels are decreased in sickle cell disease and in sickle transgenic mice and treatment protective of tissue BH4 levels can have greater efficacy than arginine supplementation alone to interrupt the cycle of polymerization, vaso-occlusion and inflammation that is the origin of pathology in sickle cell disease. Depletion of either arginine or BH4 results in loss of NOS NO generating capacity and production shifts to superoxide, resulting in a vicious cycle in which BH4 is oxidized and more superoxide is generated. We have demonstrated that BH4 is depleted in sickle transgenic mice due in part to oxidative stress. NOS is up regulated by hypoxia via a HIF-sensitive, hypoxia-response element. We further speculate that failure to simultaneously up regulate BH4 will increase oxidative stress. Phosphorylation at serine 1177 and threonine 495 modulate NOS activity. We have demonstrated that the percent phosphorylation at serine 1177 is reduced in mild sickle transgenic mice when they are exposed to hypoxia. We speculate that simultaneous reduction of BH4 and serine phosphorylation will severely impact NOS activity. Hemolysis is thought to be a major factor in reduction of NO bioavailability, both through reaction of cell free hemoglobin with NO and through release of argininase that depletes arginine, the substrate of NOS. We have demonstrated that arginine supplementation reduces hemolysis and oxidative stress in sickle transgenic mice, in part, via inhibition of the Gardos channel. Inhibition of the Gardos channel prevents red cell dehydration and inhibits polymer formation and hemolysis that further reduces NO bioavailability. However, arginine supplementation does not completely correct the vasculopathy observed in sickle transgenic mice. We hypothesize that some of the benefit of arginine supplementation may be due to preservation of BH4. To further test this hypothesis, we are breeding mice that over-express GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme in BH4 synthesis into our sickle transgenic strains. These observations may be applicable to patient interventions.

镰状细胞病的主要特征是导致低流量、灌注不良以及血液和组织缺氧的事件，这些事件都是血管闭塞的诱因和后果。我们将测试以下总体假设：镰状细胞病和镰状转基因小鼠中BH 4（四氢生物蝶呤，一氧化氮合酶（NOS）的一种重要辅因子）水平降低，保护组织BH 4水平的治疗比单独补充精氨酸具有更大的功效，以中断聚合、血管阻塞和炎症的循环，这是镰状细胞病病理学的起源。精氨酸或BH4的消耗导致NOS NO产生能力的丧失，并且生产转移到超氧化物，导致BH4被氧化并且产生更多超氧化物的恶性循环。我们已经证明，BH4在镰状转基因小鼠中被耗尽，部分原因是氧化应激。NOS通过HIF敏感的缺氧反应元件被缺氧上调。我们进一步推测，未能同时上调BH4将增加氧化应激。丝氨酸1177和苏氨酸495的磷酸化调节NOS活性。我们已经证明，当轻度镰刀形转基因小鼠暴露于缺氧时，丝氨酸1177的磷酸化百分比降低。我们推测，同时减少BH4和丝氨酸磷酸化将严重影响NOS活性。溶血被认为是NO生物利用度降低的主要因素，通过无细胞血红蛋白与NO的反应以及通过释放消耗NOS底物精氨酸的精氨酸酶。我们已经证明，精氨酸补充减少溶血和氧化应激在镰状转基因小鼠，在某种程度上，通过抑制Gardos通道。Gardos通道的抑制可防止红细胞脱水，并抑制聚合物形成和溶血，从而进一步降低NO的生物利用度。然而，精氨酸补充剂不能完全纠正镰状转基因小鼠中观察到的血管病变。我们推测，精氨酸补充的一些益处可能是由于BH4的保存。为了进一步验证这一假设，我们正在将过度表达GTP环化水解酶I（GTPCH）（BH 4合成中的限速酶）的小鼠培育成我们的镰状转基因品系。这些观察结果可能适用于患者干预。