Role of the kallikrein-kinin system in diabetic retinopathy

描述(申请人提供)：过度的视网膜血管通透性导致糖尿病黄斑水肿(DME)的发病机制，这是导致工作年龄成年人视力丧失的主要原因。我们最近报道，通过玻璃体内注射碳酸酐酶1(CA-I)激活激肽释放酶-激动素系统(KKS)，导致视网膜血管通透性(RVP)迅速增加，随后在注射后48小时，荧光素标记的2x106 Dal葡聚糖结合物出现局部血管渗漏增加(Gao等人)。自然药物2007，附录1)。此外，我们发现，在糖尿病存在的情况下，CA-I导致视网膜内增厚，这是通过3D光学相干断层扫描(OCT)测量的，在这个时间点。据我们所知，这是第一个糖尿病啮齿动物视网膜增厚的报告，类似于临床上明显的视网膜水肿。我们报道了补体1抑制剂、中和抗体可减轻CA-I诱导的视网膜水肿。 前激肽释放酶和缓激肽受体拮抗剂。我们证明血浆激肽释放酶、XII因子和高分子激肽原在糖尿病视网膜病变患者的玻璃体中以它们的激活形式存在。在初步研究中，我们已经发现，玻璃体内注射激活的纯化血浆激肽释放酶，激肽释放酶(ACT)在30min时可引起急性弥漫性RVP，48h后可引起RVP和视网膜白质淤滞。此外，我们还发现，与非糖尿病对照组相比，激肽释放酶(ACT)在糖尿病大鼠和小鼠对荧光素标记的2x106 Dal葡聚糖渗漏的局部区域的发展的影响都增加了。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激肽释放酶的作用机制。虽然KKS已被认为是卒中和血管水肿中血管炎症和血管源性水肿的关键途径，但对KKS在视网膜上的作用及其在糖尿病视网膜病变中的潜在作用知之甚少。这笔赠款将调查KKS对视网膜血管功能的这些影响，并检查该系统在糖尿病视网膜水肿中的作用。我们的研究将验证这样一种假设，即糖尿病增加了激肽释放酶对视网膜的作用，并且激肽释放酶作用的增加通过B2-R激活和激肽释放酶对细胞外蛋白分解的直接作用相结合而导致视网膜血管通透性和水肿的增加。我们将通过对糖尿病和非糖尿病啮齿动物的体内研究，以及使用视网膜微血管内皮细胞和星形胶质细胞的体外研究，来研究缓激肽受体依赖和非依赖的血浆激肽释放酶对视网膜的作用机制。与麻省理工学院的James Fujimoto博士合作，我们将使用OCT来表征KKS和糖尿病对视网膜超微结构的影响。这项资助将提供有关血浆激肽释放酶在视网膜中的作用的关键新信息，这些作用有助于RVP的弥漫性和局灶性损害、炎症和视网膜增厚，这些已被认为是DME的主要原因和特征。公共卫生相关性：这项拨款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。这笔赠款是基于令人兴奋的新数据，该数据确定了一种在糖尿病啮齿动物模型中触发视网膜浮肿的激素系统。这些研究可能会揭示治疗糖尿病黄斑水肿的新治疗策略，糖尿病黄斑水肿是导致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.