Mechanisms of Diabetic Retinopathy: Oxidative Stress and Inflammation

描述(由申请人提供)： 这项研究的长期目标是描绘导致糖尿病视网膜病变的分子机制，并确定预防或逆转血管损伤的新策略。这项拟议的研究旨在确定调节失调的ROS的细胞和分子来源，确定它们在损伤视网膜血管中的具体作用，并评估阻断/逆转损伤的潜在治疗方法。研究人员已经证明，产生NADPH氧化酶的超氧化物歧化酶NOX2的激活是糖尿病和其他以视网膜炎症损伤为特征的疾病中血管炎症的关键特征。使用NOX2基因敲除小鼠的研究已经证明了NOX2的特殊病理作用。他们还表明，NOX2诱导的视网膜炎症涉及尿素循环酶精氨酸酶的激活，骨髓来源的细胞和视网膜细胞都参与其中。精氨酸酶活性升高通过减少eNOS共同底物L-精氨酸的供应而导致eNOS解偶联。未偶联的eNOS产生超氧化物，它与可利用的NO反应生成过氧亚硝酸盐。减少NO还原蛋白的S亚硝化，而过氧亚硝酸盐诱导蛋白酪氨酸硝化。这些翻译后修饰可以增强NADPH氧化酶和线粒体氧化酶的活性，并使关键的细胞抗氧化系统失活，从而进一步增加ROS的形成。一氧化氮合酶解偶联也与糖尿病引起的骨髓源性内皮祖细胞功能障碍有关。全球假说是，糖尿病引起的高血糖通过激活骨髓和视网膜细胞中的NOX2启动氧化应激循环，而NOX2激活精氨酸酶，导致NOS解偶联，进一步增加ROS的形成，导致慢性炎症和血管损伤。该实验将通过实现以下目标来测试和开发该模型：1)确定NOX2诱导的精氨酸酶表达在糖尿病诱导的炎症反应和骨髓来源细胞功能障碍中导致一氧化氮合酶解偶联的作用。2)检测精氨酸酶是否通过增加NADPH和线粒体氧化酶的活性，并通过NOS解偶联来抑制关键的线粒体抗氧化剂，从而促进视网膜细胞ROS的形成。3)测定和比较精氨酸酶阻断剂在预防BM来源细胞和视网膜细胞的ROS失调、炎症反应和功能障碍方面的作用，以及那些旨在释放NO、清除超氧阴离子和羟基自由基以及分解过氧亚硝酸盐的新型药物的效果。 公共卫生相关性： 该项目的目标是确定导致糖尿病视网膜病变的机制，并确定新的治疗策略。糖尿病视网膜病变是美国成人失明的主要原因。到目前为止，激光光凝是唯一推荐用于晚期糖尿病视网膜病变的治疗方法。这种治疗通常是有效的，但会损害视力，在一些患者中，视网膜病变会继续发展。抗血管内皮生长因子眼内注射的临床试验显示，在减少视网膜肿胀和限制病理性血管生长方面前景看好。然而，这些影响通常是暂时的，治疗不会修复受损的血管。因此，迫切需要治疗糖尿病视网膜病变的新方法。这项工作对退伍军人管理局的使命至关重要，因为接受退伍军人医疗保健的退伍军人中有近20%患有糖尿病，而且这一数字每天都在增加。此外，几乎所有的糖尿病患者都会发展成糖尿病视网膜病变。

DESCRIPTION (provided by applicant): The long term goal of this research is to delineate the molecular mechanisms that lead to diabetic retinopathy and identify novel strategies to prevent or reverse the vascular damage. The proposed research seeks to identify cellular and molecular sources of dysregulated ROS, define their specific role in damaging the retinal vessels and evaluate potential therapies for blocking/reversing the injury. The investigators have shown that activation of the superoxide generating NADPH oxidase enzyme NOX2 is a key feature of vascular inflammation during diabetes and other diseases characterized by retinal inflammatory injury. Studies using NOX2-knockout mice have demonstrated the specific pathological role of NOX2. They also have shown that the NOX2-induced retinal inflammation involves activation of the urea cycle enzyme arginase and that both bone marrow-derived and retinal cells are involved. Elevated arginase activity causes eNOS uncoupling by reducing the supply of their common substrate L-arginine. Uncoupled eNOS produces superoxide which reacts with available NO to form peroxynitrite. Decreases in NO reduce protein S-nitrosylation whereas peroxynitrite induces protein tyrosine nitration. These post-translational modifications can enhance the activity of NADPH oxidase and mitochondrial oxidases and deactivate key cellular anti-oxidant systems, which can further increase ROS formation. NOS uncoupling has also been associated with diabetes-induced dysfunction of bone marrow-derived endothelial progenitor cells. The global hypothesis is that diabetes-induced hyperglycemia initiates a cycle of oxidative stress by activating NOX2 in bone marrow-derived and retinal cells which activates arginase, causing uncoupling of NOS, further increasing ROS formation and resulting in chronic inflammation and vascular injury. The proposed experiments will test and develop this model by accomplishing the following aims: 1) Determine the role of NOX2-induced arginase expression in causing NOS uncoupling in diabetes-induced inflammatory reactions and dysfunction of BM-derived cells. 2) Test whether arginase amplifies ROS formation in retinal cells by increasing activities of NADPH and mitochondrial oxidases and deactivating key mitochondrial anti-oxidants via NOS uncoupling. 3) Determine and compare the effects of arginase blockade in preventing dysregulated ROS, inflammatory reactions and dysfunction of BM-derived and retinal cells with those of novel agents designed to release NO as well as scavenge superoxide and hydroxyl radicals and decompose peroxynitrite PUBLIC HEALTH RELEVANCE: The goal of this project is to define the mechanisms that lead to diabetic retinopathy and identify novel therapeutic strategies. Diabetic retinopathy is the leading cause of adult blindness in the USA. So far laser photocoagulation is the only recommended treatment for advanced diabetic retinopathy. This treatment is usually effective, but can impair vision and in some patients the retinopathy continues to progress. Clinical trials of anti-VEGF intraocular injections show promise in reducing retinal swelling and limiting pathological vascular growth. However, these effects are usually transient and the treatment does not repair the damaged vessels. Therefore, there is great need for new therapies for diabetic retinopathy. This work is fundamentally important for the mission of the VA because nearly 20% of veterans receiving VA health care are diabetic and this number is increasing every day. Furthermore, nearly all diabetic patients will develop diabetic retinopathy.