Diabetic Vasculopathy and Mitochondrial eNOS

一氧化氮(NO)是由内皮型一氧化氮合酶(ENOS)产生的，在维持 血管健康和肾功能。慢性高糖暴露引发四氢生物蝶呤氧化 (BH4)，一种重要的eNOS辅助因子，导致二氢生物蝶呤(BH2)在血管中积聚 内皮细胞。在荣誉奖支持的最初阶段，我们发现BH2将eNOS与高 亲和力，取代BH4，并将eNOS产品从NO转换为超氧化物。研究表明，BH2 与eNOS的结合可启动关键的FEED-FINIVARD分子级联反应，从而驱动氧化应激和NO 糖尿病血管功能不全，导致严重的糖尿病血管并发症，可导致 截肢、失明、肾衰竭和死亡。研究也证明了一部小说的功效 干扰糖尿病患者一氧化氮缺乏和氧化应激的药理学研究进展 血管，利用通过与超氧化物(和/或衍生的)有效反应释放NO的试剂 氧化剂)。值得注意的是，依赖于超氧化物的NO释放是eNOS催化中间体的一种特性， N‘-羟基精氨酸(NOMA)，一种内源性分子，在下午5-10点在血液中循环按并发方式 清除氧化剂和释放NO，NOMA可以选择性地将NO输送到血管 氧化应激部位，增加BH4：BH2，恢复eNOS偶联和NO产生。的确， Noma治疗遗传性糖尿病db/db可防止内皮功能障碍的发展， 而使用赋形剂的对照组则未出现高血压和其他功能不足。Noma(或相关的 羟基胍)可以通过为糖尿病提供靶向治疗来填补一个主要的未得到满足的临床需求 血管病变是一种一流的超氧化物依赖的NO释放剂。研究的总体目标 建议在本次功勋奖延期期间，是为了增强我们对生化作用的认识 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 将包括评估NOHA的药代动力学、代谢、反应机制、对代谢的影响 糖尿病损害伤口愈合、血管生成和肢体血液的啮齿动物模型的疗效 血流不足。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LC/MS/MS平台，用于全球非靶向代谢物分析(TO 调查数千个分子的表达变化，50-1000 m/z)和蛋白质组学方法 发现由非偶联eNOS导致的硝化蛋白质和位点，并可能导致血管病变。

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-fonivard 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.