Roles of LOX-1 and Stress-Activated Kinases in Retinal Dysfunction during Early Diabetes

 描述(申请人提供)：视网膜病变是糖尿病的主要并发症，也是导致失明的主要原因。恢复视网膜功能的治疗方式相对无效。虽然正常的视网膜功能依赖于充足的血液供应，神经视网膜和血管视网膜的改变已有报道，但神经视网膜损伤和血管运动功能之间的机制和时间关系仍不清楚。因此，同时评估和揭示糖尿病早期视网膜血管和神经变化的机制，对于我们理解视网膜的发病机制以及开发早期治疗的新方法是至关重要的。尽管氧化应激与糖尿病视网膜病变的视网膜损伤有关，但抗氧化剂的临床治疗大多无效，这表明其他机制可能参与维持血管运动和神经视网膜功能障碍。此外，还缺乏与人类视网膜微循环及其病理生理学相关的糖尿病动物模型的开发。为了解决这些临床上的重要问题，我们在猪身上建立了链佐菌素诱导的1型糖尿病模型，我们已经证明这个动物模型在视网膜血管运动调节和调节失调方面与人类相似。我们的初步数据显示，在糖尿病的2周内，内皮依赖的一氧化氮(NO)介导的视网膜小动脉的扩张受到损害。可以观察到糖尿病视网膜小动脉内超氧化物的升高，但抗氧化剂并不能改善内皮依赖性的扩张。阻断凝集素样氧化型低密度脂蛋白受体-1(LOX-1)、蛋白酶体或c-jun氨基末端激酶-1(JNK1)的磷酸化可改善血管运动障碍。LOX-1和JNK1可能通过改变内皮细胞一氧化氮合酶活性参与心血管疾病，但它们与视网膜血管疾病相关的信号转导仍不清楚。由于暗视b波波幅在6周糖尿病期间也降低了，但在2周糖尿病期间没有降低，这表明血管舒缩功能障碍先于内神经视网膜损伤。因此，本研究的目的是阐明LOX-1和JNK1在视网膜内皮功能障碍中的联系，并确定这两个分子是否可以作为改善视网膜小动脉功能以及二次改善的新靶点 早期糖尿病患者的视网膜神经功能。我们将验证这一假说，即早期糖尿病启动超氧化物依赖的LOX-1上调以及随后下游的JNK1/泛素蛋白酶体信号，以持续降解视网膜小动脉内皮细胞中的SIRT1，从而导致视网膜神经功能障碍之前NO介导的扩张减少。我们将追求两个特定的目标：(1)在视网膜神经损伤之前，确定超氧化物歧化和LOX-1在糖尿病所致视网膜小动脉内皮功能障碍中的作用；(2)阐明JNK1依赖的磷酸化和SIRT1泛素蛋白酶体降解在糖尿病所致视网膜小动脉内皮功能障碍中的作用。基本发现的结果将转化为通过siRNA技术治疗早期糖尿病的视网膜血管疾病。

 DESCRIPTION (provided by applicant): Retinopathy is a major complication of diabetes mellitus and a leading cause of blindness. Treatment modalities for restoring retinal function are relatively ineffective. Although proper retinal function relies on a sufficient supply of blood flo and alterations of both neural and vascular retina have been reported, the mechanism and temporal relationship between neural retina damage and vasomotor function remains unclear. Therefore, simultaneous assessment and unveiling the mechanism of vascular and neural changes in the retina during early diabetes is vital to our understanding of the retinal pathogenesis as well as to development of new therapies for early treatment. Although oxidative stress is implicated in retinal damage in diabetic retinopathy, clinical therapy with antioxidants has been mostly ineffective, suggesting other mechanisms may be involved in sustaining vasomotor and neural retina dysfunction. Also, development of an animal model of diabetes relevant to the human retinal microcirculation and its pathophysiology is lacking. To address these clinically important issues, we have developed a streptozocin-induced type 1 diabetes model in the pig, an animal model that we have shown to resemble the human in retinal vasomotor regulation and dysregulation. Our preliminary data show that within 2 wk of diabetes, endothelium-dependent nitric oxide (NO)-mediated dilation of retinal arterioles is impaired. Elevation of superoxide within diabetic retinal arterioles was observed but antioxidants did not improve endothelium-dependent dilation. Vasomotor impairment was improved by blockade of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), proteasomes or phosphorylation of c-Jun N- terminal kinase-1 (JNK1). LOX-1 and JNK1 are implicated in cardiovascular diseases possibly by altering endothelial NO synthase activity, but their signaling associated with retinal vascular disease remains unknown. Since scotopic b-wave amplitude is also reduced during 6-wk but not 2-wk diabetes, it appears vasomotor dysfunction precedes inner neural retina damage. Thus, the goal of this study is to delineate the link between LOX-1 and JNK1 in the retinal endothelial dysfunction and to determine whether these two molecules can serve as novel targets for improving retinal arteriolar function, along with secondary amelioration of neural retina function, during early diabetes. We will test the hypothesis that early diabetes initiates superoxide-dependent upregulation of LOX-1 and subsequent downstream JNK1/ubiquitin-proteasome signaling for sustained degradation of SIRT1 in the retinal arteriolar endothelium, which leads to reduction of NO-mediated dilation prior to neural retina dysfunction. We will pursue two specific aims: (1) Determine the contributions of superoxide and LOX-1 to diabetes-induced endothelial dysfunction of retinal arterioles prior to neural retina damage; (2) Delineate the contribution of JNK1-dependent phosphorylation and ubiquitin-proteasome degradation of SIRT1 to diabetes-induced endothelial dysfunction of retinal arterioles. Outcomes from basic findings will be translated to therapeutic treatment of retinal vascular disease via siRNA technology in early diabetes.