Role of Complement in Vascular Diabetic Complications

描述（由申请人提供）：流行病学研究已经确定，高血糖症是导致人类糖尿病特征性并发症的微血管和大血管疾病的原因。然而，高血糖症引起血管并发症的机制知之甚少，部分原因是已知缺乏足够的糖尿病并发症动物模型。事实上，没有单一的糖尿病动物模型能再现在人类中所见的广泛的血管增殖并发症的组合和强度。因此，似乎高血糖症本身或由高血糖症激活的多种信号都不足以在动物中诱导类人血管糖尿病并发症。这意味着必须有一个或多个基因和/或途径是人类糖尿病血管并发症发展所必需的，这些基因和/或途径在动物中对高血糖症和/或糖化的敏感性有很大差异。我们已经鉴定出，编码补体调节膜蛋白CD 59的基因在人类和动物中在结构上是不同的，因为hCD 59含有由其H44残基在距离K41约5A埃处形成的糖基化基序，所述补体调节膜蛋白CD 59抑制膜攻击复合物（MAC）的形成。我们已经表明，K41-H44基序使hCD 59对糖化失活敏感，并且H44残基不存在于来自其他物种的CD 59中。我们推测，由于其独特的H44残基，hCD 59的糖基化失活可能代表了高血糖症和人类糖尿病血管增生并发症之间难以捉摸的联系。一致地，在糖尿病患者的尿液、血浆和组织中发现高水平的糖化CD 59;并且糖化CD 59与来自糖尿病受试者的肾脏、神经和静脉中增加的MAC沉积共定位。在靶组织中增加的MAC沉积释放刺激细胞增殖并诱导肾小球系膜细胞合成IV型胶原的生长因子和细胞因子。糖尿病组织中MAC诱导的促有丝分裂信号增加将与其他高血糖诱导的途径协同作用，引起血管增生性糖尿病并发症。在本申请中，我们建议使用可用的mCd 59 KO和转基因hCD 59小鼠，以进一步研究糖化失活的hCD 59在糖尿病血管增生并发症的发病机制中的致病作用。我们将对糖尿病mCd 59 KO、mCd 59 KO/hCD 59 WT和mCd 59 KO/hCD 59 GIn 44变体转基因小鼠进行表型分析，以发展人糖尿病样血管疾病。通过注射链脲佐菌素使小鼠患糖尿病，并通过组织病理学和视网膜血流动力学和肾功能参数进行表型分析。我们预期在mCd 59 KO和mCd 59 KO/hCD 59 WT小鼠中，高血糖将引发与人类糖尿病中所见相当的血管增殖反应。相反，hCD 59 GIn 44变体（对糖基化失活具有抗性）应保护mCd 59 KO免受高血糖诱导的增殖性疾病。我们期望这些实验将提供：1）明确的证据表明hCD 59的糖基化失活在糖尿病增殖性血管并发症的发病机制中的作用; 2）研究糖尿病并发症的机制、治疗和预防所需的动物模型;以及3）强有力的证据支持未来关于人类补体和糖尿病的研究。

DESCRIPTION (provided by applicant): Epidemiological studies have established that hyperglycemia is responsible for the micro- and macrovascular disease that characteristically complicates human diabetes. However, the mechanism by which hyperglycemia causes vascular complications is poorly understood, in part because of the known absence of adequate animal models of diabetic complications. Indeed, no single animal model of diabetes reproduces the extensive vascular proliferative complications in the combination and intensity seen in humans. Thus, it appears that neither hyperglycemia itself nor the multiple signals activated by hyperglycemia are sufficient to induce in animals human-like vascular diabetic complications. This implies that there must be one or more genes and/or pathways necessary for the development of the diabetic vascular complications in humans that in animals differ substantially in their sensitivity to hyperglycemia and/or glycation. We have identified that the gene encoding for the complement regulatory membrane protein CD59, which inhibits formation of the membrane attack complex (MAC), is structurally different in humans and animals because hCD59 contains a glycation motif, formed by its H44 residue at approximately 5A angstroms from K41. We have shown that the K41-H44 motif makes hCD59 sensitive to inactivation by glycation, and that the H44 residue is not present in CD59 from other species. We postulate that glycation-inactivation of hCD59 due to its unique H44 residue could represent the elusive link between hyperglycemia and vascular proliferative complications of human diabetes. Consistently, high levels of glycated CD59 are found in diabetic urine, plasma and tissues; and glycated CD59 co-localizes with increased MAC deposition in kidneys, nerves and veins from diabetic subjects. Increased MAC deposition in the target tissues releases growth factors and cytokines that stimulate cell proliferation and induce synthesis of collagen type IV by glomerular mesangial cells. Increased MAC-induced mitogenic signals in diabetic tissues would act synergistically with other hyperglycemia-induced pathways causing vascular proliferative diabetic complications. In this application, we propose to use available mCd59KO and transgenic hCD59 mice to further investigate the causative role of glycation-inactivation of hCD59 in the pathogenesis of vascular proliferative complications of diabetes. We will phenotype diabetic mCd59KO, mCd59KO/hCD59WT, and mCd59KO/hCD59GIn44 variant transgenic mice for the development of human diabetic-like vascular disease, Mice will be made diabetic by injection of streptozotocin, and phenotyped by histopathology and parameters of retinal hemodynamic and renal function. We expect that in mCd59KO and in mCd59KO/hCD59WT mice, hyperglycemia will trigger a vascular proliferative response comparable to that seen in human diabetes. Instead, the hCD59GIn44 variant (resistant to glycation-inactivation) should protect mCd59KO from hyperglycemia-induced proliferative disease. We expect that these experiments will provide: 1) clear evidence for the role of glycation-inactivation of hCD59 in the pathogenesis of the proliferative vascular complications of diabetes; 2) needed animal models to study mechanism, therapy and prevention of diabetic complications; and 3) strong evidence to support future studies on complement and diabetes in humans.