Role of the kallikrein-kinin system in diabetic retinopathy

描述（由申请人提供）：视网膜血管通透性过高导致糖尿病性黄斑水肿（DME）的发病机制，DME是工作年龄成人视力丧失的主要原因。我们最近报道，在大鼠中通过玻璃体内注射碳酸酐酶1（CA-I）激活激肽释放酶-激肽系统（KKS），导致视网膜血管通透性（RVP）快速增加，随后在注射后48小时出现荧光素标记的2x 106 Dal葡聚糖缀合物的血管渗漏增加的局灶性区域（Gao et al. Nature Medicine 2007，附录1）。此外，我们发现，在糖尿病的存在下，CA-I引起视网膜内增厚，在这个时间点通过3D光学相干断层扫描（OCT）测量。据我们所知，这是第一次报告视网膜增厚的糖尿病啮齿动物，类似于临床上明显的视网膜水肿。我们报道了CA-1诱导的视网膜水肿可被补体1抑制剂，中和抗体， 前激肽释放酶和缓激肽受体拮抗作用。我们证明了血浆激肽释放酶，因子XII和高分子量激肽原，是目前在玻璃体中的人与糖尿病视网膜病变的活化形式。在初步研究中，我们已经表明，玻璃体内注射活化的纯化血浆激肽释放酶，激肽释放酶（act），诱导急性弥漫性RVP在30分钟和局灶性区域的RVP和视网膜白细胞停滞在48小时后注射。此外，我们发现，与非糖尿病对照组相比，糖尿病大鼠和小鼠中激肽释放酶（act）对荧光素标记的2x 106 Dal葡聚糖渗漏的焦点区域的发展的影响增加。利用培养的视网膜微血管内皮细胞和星形胶质细胞，我们已经确定了缓激肽依赖和非依赖的激肽释放酶的作用机制。虽然KKS已被确定为中风和血管性水肿中血管炎症和血管源性水肿的关键途径，但关于KKS对视网膜的作用及其在糖尿病视网膜病变中的潜在作用知之甚少。该基金将研究KKS对视网膜血管功能的影响，并检查该系统对糖尿病视网膜水肿的贡献。我们的研究将检验糖尿病增加激肽释放酶对视网膜的作用以及增加的激肽释放酶作用通过B2-R活化和激肽释放酶对细胞外蛋白水解的直接作用的组合导致视网膜血管通透性增加和水肿的假设。我们将调查的贡献缓激肽受体依赖性和非依赖性的血浆激肽释放酶的作用机制对视网膜使用糖尿病和非糖尿病啮齿动物的体内研究和体外研究，使用视网膜微血管内皮细胞和星形胶质细胞。与James Fujimoto博士（马萨诸塞州理工学院）合作，我们将使用OCT表征KKS和糖尿病对视网膜超微结构的影响。该资助将提供有关视网膜中血浆激肽释放酶作用的关键新信息，这些作用有助于弥漫性和局灶性RVP病变、炎症和视网膜增厚，这些病变被认为是DME的主要致病因素和特征。公共卫生相关性：该基金将描述血浆激肽释放酶诱导的炎症在糖尿病视网膜病变中的作用。这项资助是基于令人兴奋的新数据，这些数据已经确定了一种激素系统，该系统在糖尿病啮齿动物模型中引发视网膜水肿。这些研究可能揭示治疗糖尿病黄斑水肿的新治疗策略，这是视力丧失的主要原因。

DESCRIPTION (provided by applicant): Excessive retinal vascular permeability contributes to the pathogenesis of diabetic macular edema (DME), a leading cause of vision loss in working-age adults. We recently reported that activation of the kallikrein-kinin system (KKS) by intravitreal injection of carbonic anhydrase 1 (CA-I) in rats resulted in a rapid increase in retinal vascular permeability (RVP) followed by the development of focal areas of increased vascular leakage to fluorecein-labeled 2x106 Dal dextran conjugate at 48 hrs post injection (Gao et al. Nature Medicine 2007, Appendix 1). In addition, we showed that in the presence of diabetes, CA-I caused intraretinal thickening, measured by 3D optical coherence tomography (OCT), at this time point. To our knowledge, this is the first report of retinal thickening in a diabetic rodent that is similar to clinically evident retinal edema. We reported that CA-I-induced retinal edema was decreased by complement 1 inhibitor, neutralizing antibody to prekallikrein, and bradykinin receptor antagonism. We demonstrated that plasma kallikrein, factor XII, and high molecular weight kininogen, are present in their activated forms in vitreous from people with diabetic retinopathy. In preliminary studies, we have shown that intravitreal injection with activated purified plasma kallikrein, kallikrein(act), induced both acute diffuse RVP at 30 min and focal areas of RVP and retinal leukostasis at 48 h post injection. In addition, we show that the effects of kallikrein(act) on the development of focal areas of leakage to fluorecein-labeled 2x106 Dal dextran are increased in both rats and mice with diabetes compared with nondiabetic controls. Using cultured retinal microvessel endothelial cells and astrocytes, we have identified both bradykinin-dependent and -independent mechanisms of kallikrein action. Although the KKS has been identified as a key pathway of vascular inflammation and vasogenic edema in stroke and angioedema, little is known regarding the actions of the KKS on the retina and its potential role in diabetic retinopathy. This grant will investigate these effects of the KKS on retinal vascular function and examine the contribution of this system to diabetic retinal edema. Our studies will examine the hypothesis that diabetes increases the actions of kallikrein on the retina and that increased kallikrein action leads to increased retinal vascular permeability and edema via a combination of B2-R activation and kallikrein's direct effect on extracellular proteolysis. We will investigate the contributions of both bradykinin receptor-dependent and -independent mechanisms of plasma kallikrein action on the retina using both in vivo studies on diabetic and nondiabetic rodents and in vitro studies using retinal microvessel endothelial cells and astrocytes. In collaboration with Dr. James Fujimoto (Massachusetts Institute of Technology), we will characterize the effects of the KKS and diabetes on retinal ultrastructure using OCT. This grant will provide critical new information on plasma kallikrein actions in the retina that contribute to both the diffuse and focal lesions of RVP, inflammation, and retinal thickening, which have been implicated as primary causal factors and characteristics of DME. PUBLIC HEALTH RELEVANCE: This grant will characterize the role of plasma kallikrein-induced inflammation in diabetic retinopathy. This grant is based on exciting new data that has identified a hormone system that triggers retinal edema in diabetic rodent models. These studies could reveal new therapeutic strategies to treat diabetic macular edema, a leading cause of vision loss.