Diabetic Vasculopathy and Mitochondrial eNOS

一氧化氮(NO)由内皮型一氧化氮合酶(ENOS)产生，在维持血管健康和肾功能方面起着关键作用。长期暴露在高糖环境中会引发eNOS必需辅因子四氢生物蝶呤(BH4)的氧化，导致二氢生物蝶呤(BH2)在血管内皮细胞中积聚。在荣誉奖支持的最初阶段，我们发现BH2以高亲和力结合eNOS，取代了BH4，并将eNOS产品从NO转换为超氧化物。研究表明，BH2与eNOS的结合可以启动一个关键的前馈分子级联反应，导致糖尿病血管中的氧化应激和一氧化氮不足，导致严重的糖尿病血管并发症，可能导致截肢、失明、肾功能衰竭和死亡。研究还证明了一种新的药理学方法的有效性，该方法利用通过与超氧化物(和/或衍生氧化剂)的有效反应释放NO的试剂来破坏糖尿病血管中NO缺乏和氧化应激的级联反应。值得注意的是，依赖超氧化物的NO释放是eNOS催化中间体N‘-羟精氨酸(NOMA)的一种特性，NOMA是一种内源性分子，在下午5-10点在血液中循环通过同时清除氧化剂和释放NO，NOMA可以选择性地将NO输送到氧化应激的血管部位，增加BH4：BH2，恢复eNOS偶联和NO的产生。的确， Noma对遗传性糖尿病db/db的慢性治疗可防止内皮功能障碍、高血压的发展，而在赋形剂治疗的对照组中则没有发生血管内皮功能不全。NOMA(或相关的羟基胍)可以作为一种一流的超氧化物依赖NO释放剂，为糖尿病血管病变提供靶向治疗，从而填补一个主要的未得到满足的临床需求。在这次功勋奖延期期间提出的研究的总体目标是增强我们对这种作用的生化理解 并延长我们对NOHA用于糖尿病血管病变的潜在治疗的评估。这将包括在糖尿病损伤伤口愈合、血管生成和肢体血流不足的啮齿动物模型中评估NOHA的药代动力学、代谢、反应机制、对代谢的影响和治疗效益。研究将依赖于在初始阶段建立的新的研究方法和分析 优秀奖获得期-包括一个强大的全球非靶向代谢物图谱的LC/MS/MS平台(以调查数千个分子的表达变化，50-1000 m/z)，以及一个蛋白质组学方法，用于发现由解偶联eNOS引起的可能导致血管病变的硝化蛋白质和位点。

Nitric oxide (NO) is produced by endothelial NO synthase (eNOS) and plays a key role in maintaining vascular health and renal function. Chronic exposure to high glucose triggers oxidation of tetrahydrobiopterin (BH4), an essential eNOS cofactor, resulting in accumulation of dihydrobiopterin (BH2) in the vascular endothelium. During the initial period of Merit Award support, we discovered that BH2 binds eNOS with high avidity, replacing BH4 and switching the eNOS product from NO to superoxide. Studies suggest that BH2 binding to eNOS can initiate a pivotal feed-forward molecular cascade that drives oxidative stress and NO insufficiency in diabetic blood vessels, responsible for severe diabetic vascular complications that can lead to amputations, blindness, kidney failure and death. Research also demonstrates the efficacy of a novel pharmacological approach for disrupting the cascade of NO insufficiency and oxidative stress in diabetic blood vessels, utilizing agents that release NO via efficient reaction with superoxide (and/or derived oxidants). Remarkably, superoxide-dependent NO release is a property of the eNOS catalytic intermediate, N'¿-hydroxyarginine (NOMA), an endogenous molecule that circulates in blood at 5-10 pM. By concurrently scavenging oxidants and releasing NO, administered NOMA can selectively target NO delivery to vascular sites of oxidative stress, increasing BH4:BH2 and restoring eNOS coupling and NO production. Indeed, chronic NOMA treatment of genetically-diabetic db/db prevented development of endothelial dysfunction, hypertension and NO insufficiency that othenwise occurred in vehicle-treated controls. NOMA (or a related hydroxyguanidine) could fill a major unmet clinical need, by providing targeted therapy for diabetic vasculapathies as a first-in-class superoxide-dependent NO-releasing agent. The overall goal of studies proposed during this Merit Award extension period is to enhance our biochemical understanding of the role of NO in diabetes and extend our assessment of NOHA for potential therapy of diabetic vasculopathies. This will include evaluation of NOHA pharmacokinetics, metabolism, reaction mechanisms, effects on metabolism and therapeutic benefit in rodent models of diabetes-impaired wound healing, angiogenesis and limb blood flow insufficiency. Studies will rely on new research approaches and assays, established during the initial Merit Award period - including a powerful LC/MS/MS platform for global untargeted metabolite profiling (to survey expression changes in thousands of molecules, 50 - 1000 m/z) and a proteomic approach for discovering nitrated proteins and sites that result from uncoupled eNOS and may contribute to vasculopathy.