Role of Complement in Vascular Diabetic Complications

描述（由申请人提供）：流行病学研究已经证实，高血糖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，高血糖引起血管并发症的机制尚不清楚，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，没有一种单一的糖尿病动物模型能再现人类所见的血管增生并发症的组合和强度。因此，似乎无论是高血糖本身还是高血糖激活的多重信号都不足以诱导动物的类人血管性糖尿病并发症。这意味着人类糖尿病血管并发症的发生必须有一个或多个基因和/或途径，而动物对高血糖和/或糖基化的敏感性有很大的不同。我们已经发现，编码抑制膜攻击复合物（MAC）形成的补体调节膜蛋白CD59的基因在人类和动物中在结构上是不同的，因为hCD59含有一个糖基化基序，由它的H44残基在距离K41约5A埃处形成。我们已经证明K41-H44基序使hCD59对糖基化失活敏感，并且H44残基不存在于其他物种的CD59中。我们假设，由于hCD59独特的H44残基，它的糖化失活可能代表了高血糖和人类糖尿病血管增生性并发症之间难以捉摸的联系。一贯地，在糖尿病患者的尿液、血浆和组织中发现高水平的糖化CD59；糖化CD59与糖尿病患者肾脏、神经和静脉中MAC沉积增加共定位。靶组织中MAC沉积增加，释放生长因子和细胞因子，刺激细胞增殖，诱导肾小球系膜细胞合成IV型胶原。糖尿病组织中mac诱导的有丝分裂信号的增加可能与其他高血糖诱导的途径协同作用，导致血管增殖性糖尿病并发症。在本应用中，我们拟利用现有的mCd59KO和转基因hCD59小鼠，进一步研究hCD59糖化失活在糖尿病血管增生性并发症发病机制中的致病作用。我们将对糖尿病mCd59KO、mCd59KO/hCD59WT和mCd59KO/hCD59GIn44变异体转基因小鼠进行表型分析，用于人类糖尿病样血管疾病的发生，通过注射链脲霉素使小鼠发生糖尿病，并通过组织病理学、视网膜血流动力学和肾功能参数进行表型分析。我们预计，在mCd59KO和mCd59KO/hCD59WT小鼠中，高血糖会引发与人类糖尿病相似的血管增殖反应。相反，hCD59GIn44变体（抵抗糖化失活）应该保护mCd59KO免受高血糖诱导的增生性疾病。我们期望这些实验能够提供：1)明确hCD59糖化失活在糖尿病增生性血管并发症发病机制中的作用；2)需要动物模型来研究糖尿病并发症的机制、治疗和预防；3)强有力的证据支持未来对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.