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: 7918872
• 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 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7918872
• 关键词: Acute; Adult; Affect; Amino Acids; Animals; Antioxidants; Arginine; Benchmarking; Biological Availability; Biological Markers; Biological Preservation; Biopterin; Blood Vessels; Breeding; Cells; Childhood; Citrulline; Clinic; Clinical Trials; Collaborations; Dehydration; Diabetes Mellitus; Dose; Enzymes; Erythrocytes; Event; Failure; Folate; Functional disorder; GTP Cyclohydrolase I; Hemoglobin; Hemolysis; Human; Hypoxia; Individual; Inflammation; Intervention; Laboratories; Lead; Mediating; Metabolism; Modeling; Multienzyme Complexes; Mus; Nitric Oxide; Nitric Oxide Synthase; Ornithine; Oxidative Stress; Oxygen; PTGS2 gene; Pathology; Pathway interactions; Patients; Peroxonitrite; Phosphorylation; Plasma; Play; Polymers; Preparation; Production; Protocols documentation; Pulmonary Hypertension; 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; arginine treatment; base; blood perfusion; drinking water; experience; improved; inhibitor/antagonist; mouse model; polymerization; prevent; research study; response; restoration; sepiapterin; 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、血液和组织缺氧的事件，这些事件既是血管闭塞的易感因素，也是血管闭塞的后果。我们将测试BH4（四氢生物蝶呤，一氧化氮合酶（NOS）的重要辅助因子）水平在镰状细胞病和镰状转基因小鼠中降低的总体假设，并且治疗保护组织BH4水平比单独补充精氨酸更有效地中断聚合、血管闭塞和炎症循环，这是镰状细胞病的病理起源。精氨酸或BH4的消耗都会导致nosno生成能力的丧失，生产转向超氧化物，从而导致BH4被氧化和生成更多超氧化物的恶性循环。我们已经证明，由于氧化应激的部分原因，镰刀转基因小鼠的BH4被耗尽。缺氧通过hif敏感、缺氧反应元件上调NOS。我们进一步推测BH4不能同时上调会增加氧化应激。丝氨酸1177和苏氨酸495的磷酸化调节NOS活性。我们已经证明，当轻度镰状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BH4和丝氨酸磷酸化同时减少会严重影响NOS活性。溶血被认为是降低一氧化氮生物利用度的一个主要因素，这既通过游离血红蛋白与一氧化氮的反应，也通过精氨酸酶的释放消耗一氧化氮的底物精氨酸。我们已经证明，补充精氨酸可以减少镰刀转基因小鼠的溶血和氧化应激，部分原因是通过抑制Gardos通道。抑制Gardos通道可防止红细胞脱水，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，补充精氨酸并不能完全纠正在镰刀转基因小鼠中观察到的血管病变。我们假设补充精氨酸的一些好处可能是由于BH4的保存。为了进一步验证这一假设，我们正在培育过表达GTP环水解酶I （GTPCH）的小鼠，GTPCH是我们镰刀转基因菌株BH4合成的限速酶。这些观察结果可能适用于患者干预。