小窝蛋白-1在实验性糖尿病视网膜病变中对血-视网膜屏障完整性的调控机制

The blood-retinal barrier (BRB) selectively and tightly regulates the local environment of the neural retina. Hyperpermeability of the BRB is a the major cause of blindness in diabetic retinopathy. Recent evidence from our laboratory indicates that caveolin-1 (Cav-1), an integral protein component of specialized lipid microdomains called caveolae, is essential for BRB integrity and for normal retinal function. We have data demonstrating the loss of BRB integrity in mice in which the Cav-1 gene has been deleted (Cav-1 null mice) resulting in abnormal accumulation of serum proteins in the eye. In addition, we have observed acute extravasation of systemically-injected tracers into the eyes of Cav-1 null mice. This increased permeability likely results in both reduced retinal function as measured electroretinography. Our results clearly indicate that Cav-1 expression/function is essential for BRB integrity. A major complication of diabetes is retinopathy that results from the abnormal growth of fragile, leaky blood vessels within and around the retina. The increased BRB permeability in diabetic, neovascular retinas results in retinal edema, swelling and detachment which can lead to blindness. The role of Cav-1 in diabetic retinopathy is largely unknown although caveolae are the site of accumulation of advanced glycation endproducts (AGEs) in retinal vascular cells. Cav-1 is a known negative regulator of NOS activity and excessive activation of nitric oxide synthase (NOS) is a key pathological component of diabetic retinopathy. Our preliminary data demonstrate that the levels of both nitrate/nitrite and nitrated proteins are increased in retinas from Cav-1 null mice suggesting production of nitrix oxide (NO). Furthermore, Cav-1 protein levels are reduced in retinas from mice in which diabetes has been induced by STZ injection. Given that glycated proteins accumulate in caveolae and that Cav-1 is important for normal maintenance of the BRB, we hypothesize that Cav-1 is damaged in the diabetic retina by oxidative and/or glycoxidative modification and that loss of Cav-1 function results in enhanced NO production and subsequent damage to the BRB. The studies proposed here are designed to test the hypotheses that Cav-1 is damaged in the diabetic retina by oxidative and/or glycoxidative modification and loss of Cav-1 function results in enhanced generation of RNS and subsequent damage to the BRB.

血-视网膜屏障（BRB）的破坏是糖尿病视网膜病变（DR）致盲的主要原因之一。DR中BRB的渗漏与一氧化氮合成酶（NOS）过度激活有关,但具体机制不明。Cav-1是NOS的负性调控因子，我们前期工作显示Cav-1基因敲除鼠BRB通透性增强伴NOS活性增高，提示Cav-1可通过NOS调控BRB的通透性。新近报道显示小窝蛋白-1（Cav-1）的缺失与糖尿病及并发症的发生密切相关，由此，我们进一步推测DR持续的高血糖状态使Cav-1氧化或糖化氧化受损，导致NOS活性增强并引起BRB的破坏。为验证该假说，我们拟通过荧光示踪、免疫印迹、比色定量、示踪电镜、药物干预、基因敲除鼠模型等方法揭示Cav-1在DR中对BRB完整性的调控机制，并检测人工合成Cav-1衍生肽在DR中对BRB的保护作用。本研究结果将明确Cav-1与DR的相关性，完善视网膜血管渗漏的机理，为寻找DR治疗靶点奠定理论和实验基础。

血-视网膜屏障（BRB）的破坏是糖尿病视网膜病变（DR）致盲的主要原因之一。有报道表明携带纯合Cav-1突变的人群易患糖尿病及并发症。我们发现Cav-1蛋白在STZ小鼠视网膜中表达明显降低，荧光示踪、比色定量及辣根过氧化酶（HRP）结合示踪电镜结果显示Cav-1敲除小鼠BRB通透性增强。Cav-1是NOS的负性调控因子，有报道显示DR中BRB的渗漏与一氧化氮合成酶（NOS）过度激活有关。因此,我们给予Cav-1 敲除小鼠注射NOS广谱抑制剂L-NAME。然而,L-NAME的干预并不降低Cav-1依赖的BRB渗漏，并且Cav-1&eNOS双敲小鼠也不减轻Cav-1依赖的BRB渗漏。这些实验结果表明Cav-1缺失导致的血管通透性增强并不依赖于NOS活性。采用Western Blot、冰冻切片结合免疫荧光的方法检测Cav-1-敲除小鼠与对照鼠视网膜血管紧密连接蛋白，发现紧密连接蛋白claudin-5, ZO-1, and occludin均无改变。但有趣的是，当给予Cav-1 敲除小鼠注射Cav-1衍生肽“AP-CAV”时，其有改善BRB渗漏的功效。本着转化医学的宗旨，我们将把科研成果转化为科技产品。目前，Cav-1功能片段为基础的多肽AP-Cav将用于研发滴眼剂，应用于临床糖尿病视网膜病的治疗。本研究结果明确了Cav-1与DR的相关性，完善了视网膜血管渗漏的机理，并为寻找DR治疗靶点奠定了理论和实验基础。