INOS/REACTIVE OXYGEN SPECIES IN DIABETIC RETINOPATHY

糖尿病视网膜病变中的 INOS/活性氧

• 批准号: 6621447
• 负责人: Maria Bartolomeo Grant
• 金额: $ 36.25万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-30 至 2005-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6621447
• 关键词: diabetic retinopathy; electron spin resonance spectroscopy; electrophysiology; enzyme activity; fluorescence spectrometry; free radical oxygen; gene expression; genetically modified animals; glucose; high performance liquid chromatography; human tissue; immunocytochemistry; laboratory rat; molecular cloning; nitric oxide; nitric oxide synthase; organ culture; oxidative stress; pathologic process; polymerase chain reaction; retina circulation disorder; ribozymes; vascular endothelium; vasoconstriction; western blottings

The long-term goal of this work is to elucidate the molecular mechanisms responsible for the altered retinal vascular function associated with diabetic retinopathy., the leading cause of adult blindness. We propose the following hypothesis: the loss of retinal auto regulation observed in diabetic retinopathy is due to an early phenotypic switch in the retinal arteries whereby increased levels of inducible nitric oxide synthetase (iNOS) replace the constitutively expressed, calcium- regulated endothelial nitric oxide synthase (eNOS). This calcium- independent increase in iNOS results in increased and continuous generation of nitric oxide (NO) and vasodilation. In healthy vessels and early on in diabetic vessels, free radical scavenging systems eliminate glucose-induced, oxygen-derived free radicals. However, if antioxidant defense systems fail, oxygen-derived free radicals combine with NO to form the toxic peroxynitrite, resulting in retinal vessel vasoconstriction. We will use a unique combination of experimental approaches to integrate information obtained at both the subcellular and cellular levels in single endothelial cells with observations and measurements made in intact small, pressurized retinal arteries from diabetic rats. Our main experimental tools will be rat and human endothelial cell cultures, fluorescence spectroscopy and electron spin trap resonance spectroscopy (for measurement of NO and reactive oxygen species), and immunohistochemistry. Advanced electrophysiology and imaging methodologies will be used to measure redox state in living cells and intact vessel function. We put forth 3 Specific Aims to test our hypothesis. In AIM 1 (cell culture studies), we will test the prediction that the cellular redox state and the availability of substrate and co- factors regulate the amount and the type of reactive oxygen formed by NOS in endothelial cells. In AIM 2 )studies using intact vessel preparations) we will examine the changes in vascular reactivity in intact pressurized retinal vessels from rats with type II diabetes in relation to increased iNOS activity and peroxynitrite formation. We will carefully map the initial and progressive changes in vascular reactivity in these animals by quantifying the "time course of change" in vascular reactivity and oxidative stress in vessels of these rats and then correlate these changes to those observ4ed in diabetic retinal arteries. In AIM 3 (whole animal studies), we will test whether mitigating the effects of iNOS in the BBZ/Wor diabetic rat improves blood retinal barrier and vascular dysfunction. A reduction in iNOS bioactivity will be accomplished either by cleaving iNOS mRNA using a ribozyme or by reducing enzymatic activity with pharmacological inhibitors. Results from these studies should suggest new therapeutic interventions for prevention of diabetic retinopathy that lead towards restoration of both retinal cell redox balance and retinal vascular function.

这项工作的长期目标是阐明与糖尿病视网膜病变相关的视网膜血管功能改变的分子机制。这是导致成年人失明的主要原因。我们提出以下假设：在糖尿病视网膜病变中观察到的视网膜自身调节的丧失是由于视网膜动脉的早期表型转换，由此诱导型一氧化氮合成酶（iNOS）水平的增加取代了组成性表达的、钙调节的内皮型一氧化氮合成酶（eNOS）。这种不依赖钙的iNOS增加导致一氧化氮（NO）的增加和持续产生和血管舒张。在健康血管和早期糖尿病血管中，自由基清除系统消除葡萄糖诱导的氧源自由基。然而，如果抗氧化防御系统失效，氧源性自由基与NO结合形成有毒的过氧亚硝酸盐，导致视网膜血管收缩。我们将使用一种独特的实验方法组合来整合在单个内皮细胞亚细胞和细胞水平上获得的信息，并对来自糖尿病大鼠的完整的小加压视网膜动脉进行观察和测量。我们的主要实验工具将是大鼠和人内皮细胞培养，荧光光谱和电子自旋阱共振光谱（用于测量NO和活性氧），以及免疫组织化学。先进的电生理学和成像方法将用于测量活细胞的氧化还原状态和完整的血管功能。我们提出了3个具体目标来检验我们的假设。在AIM 1（细胞培养研究）中，我们将验证细胞氧化还原状态和底物及辅助因子的可用性调节内皮细胞中NOS形成的活性氧的数量和类型的预测。在AIM（使用完整血管制剂的研究）中，我们将研究II型糖尿病大鼠完整加压视网膜血管中血管反应性的变化与iNOS活性增加和过氧亚硝酸盐形成的关系。我们将通过量化这些大鼠血管反应性和氧化应激的“时间变化过程”，仔细绘制这些动物血管反应性的初始和渐进变化，然后将这些变化与糖尿病视网膜动脉中观察到的变化联系起来。在AIM 3（全动物研究）中，我们将测试减轻iNOS对BBZ/Wor糖尿病大鼠的影响是否能改善血液视网膜屏障和血管功能障碍。iNOS生物活性的降低可以通过核酶切割iNOS mRNA或通过药物抑制剂降低酶活性来实现。这些研究的结果应该为预防糖尿病视网膜病变提供新的治疗干预措施，从而恢复视网膜细胞氧化还原平衡和视网膜血管功能。