Role of Complement in Vascular Diabetic Complications

描述(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的微血管和大血管疾病的主要原因。然而，高血糖导致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复制出人类所见的广泛的血管增生性并发症。因此，似乎无论是高血糖本身还是由高血糖激活的多种信号都不足以在动物中诱发类似人类的血管糖尿病并发症。这意味着一定有一个或多个基因和/或通路是人类糖尿病血管并发症发生所必需的，而动物对高血糖和/或糖基化的敏感性有很大不同。我们已经确定，编码补体调节膜蛋白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，由其H44残基在距K41约5A的角度形成。CD59编码补体调节膜蛋白CD59，它抑制膜攻击复合体(MAC)的形成。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。我们推测，hCD59由于其独特的H44残基导致的糖基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。在糖尿病患者的尿液、血浆和组织中始终发现高水平的糖化CD59；糖化CD59与糖尿病患者肾脏、神经和静脉中MAC沉积的增加是共定位的。靶组织中MAC沉积的增加会释放生长因子和细胞因子，刺激细胞增殖，并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将与其他高血糖诱导的途径协同作用，导致血管增生性糖尿病并发症。在这一应用中，我们建议使用可用的mCD59KO和转基因hCD59小鼠来进一步研究hCD59糖基化失活在糖尿病血管增生性并发症发病机制中的作用。我们将mCD59KO、mCD59KO/hCD59WT、mCD59KO/hCD59WT、mCD59KO/hCD59GIn44基因突变的糖尿病小鼠分型为人类糖尿病样血管病变，通过注射链脲佐菌素使小鼠糖尿病，并通过组织病理学和视网膜血流动力学及肾功能参数进行表型鉴定。我们预计，在mCD59KO和mCD59KO/hCD59WT小鼠中，高血糖将引发与人类糖尿病类似的血管增殖反应。相反，hCD59GIn44变体(对糖基化失活具有抵抗力)应该可以保护mCD59KO免受高血糖诱导的增殖性疾病的影响。我们期望这些实验将提供：1)糖基化失活hCD59在糖尿病增殖性血管并发症发病机制中的作用的明确证据；2)需要动物模型来研究糖尿病并发症的机制、治疗和预防；以及3)强有力的证据支持未来对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.