Diabetic Vasculopathy and Mitochondrial eNOS

一氧化氮（NO）由内皮NO合成酶（eNOS）产生，在维持血管健康和肾脏功能中起关键作用。长期暴露于高糖环境会触发四氢生物蝶呤（BH4）的氧化，这是一种必要的eNOS辅助因子，导致血管内皮中二氢生物蝶呤（BH2）的积累。在优异奖支持的初始阶段，我们发现BH2与eNOS的结合非常强烈，取代BH4并将eNOS产物从NO转变为超氧化物。研究表明，BH2与eNOS结合可以启动关键的前驱分子级联，驱动糖尿病血管中的氧化应激和NO不足，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。研究还证明了一种新的药理学方法的有效性，该方法可以通过与超氧化物（和/或衍生氧化剂）的有效反应释放一氧化氮，破坏糖尿病血管中一氧化氮不足和氧化应激的级联反应。值得注意的是，超氧化物依赖性NO释放是eNOS催化中间体N′¿-羟精氨酸（NOMA）的特性，这是一种内源性分子，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，给予NOMA可以选择性地靶向NO递送到氧化应激血管部位，增加BH4:BH2，恢复eNOS偶联和NO的产生。的确,

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.