DESCRIPTION (provided by applicant): Chronic high blood sugar levels (hyperglycemia) are responsible for diabetic complications including nephropathy, retinopathy, neuropathy and accelerated atherosclerosis, all major causes of morbidity and mortality in the adult population. However, how hyperglycemia causes diabetic complications is still poorly understood. Extensive clinical studies by us and others indicate that the complement system, a major effector of the immune system and mediator of inflammation, may play a significant role in the pathogenesis of these complications. In particular, we have established that human CD59, a key complement regulatory protein that protects cells from complement-mediated damage, is inhibited by glycation, the non-enzymatic attachment of glucose to proteins. This is because human CD59 contains a glycation motif, formed by its H44 residue at H 5A0 from its K41 residue in the active site of the protein. Interestingly, this glycation motif is not present in CD59 from other species. Our hypothesis is that glycation-inactivation of hCD59 due to its unique H44 residue could represent the elusive link between hyperglycemia and the vascular proliferative complications of human diabetes. However, practical and ethical considerations preclude conducting clinical studies to test this hypothesis comprehensively in humans. Therefore, our strategy to determine the cellular and molecular mechanism by which complement damages the target organs of diabetic complications has focused on developing molecular engineered mice that are now available in our laboratory. We will make these mice diabetic by STZ injection (type 1 diabetes model), and investigate the role of glycation-inactivation of hCD59 in diabetic cardiovascular disease taking advantage of our previous generation of mCD59KO mice backcrossed into the Apoe-/- background. Specifically, we will compare diabetic hCD59 wild type (glycation-inactivation sensitive) transgenic mice with diabetic hCD59Gln44 mutant (glycation- inactivation resistant) transgenic mice, both in a mCd59KO and Apoe-/-background. We will also use an available anti-mC5 antibody to block the terminal complement cascade and provide direct mechanistic evidence for the contribution of complement to diabetic vascular diseases. Successful accomplishment of this work will provide: 1) clear evidence for the role of complement and glycation-inactivation of hCD59 in the pathogenesis of the cardiovascular 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.

The major goal of this proposal is to test our hypothesis that glycation-inactivation of the complement regulatory protein CD59 plays a critical role in the pathogenesis of diabetic complications with particular focus on the cardiovascular disease commonly seen in diabetic individuals. To this end, we will use STZ-induced diabetic mice that lack mCD59 (mCD59KO), are backcrossed into an Apoe deficient background, and transgenetically express human CD59 in either the glycation-senstive wild type form or as a glycation-insensitive mutant, all generated and available in our laboratory. The proposed studies are highly translational; their relevance is highlighted by the following facts: 1) Diabetes and its complications are a leading cause of morbidity and mortality in the adult population, 2) vascular pathology causing ischemic heart disease, stroke, and peripheral vascular disease account for more than 50% of the mortality rate in the diabetic population, 3) treatment of diabetic cardiovascular disease accounts for close to 65% of the more than 100 billion dollars annually spent in the US to treat chronic diabetic complications (H 1 out 10 dollars of the overall US health budget), and 4) successful accomplishment of our aims will provide a much needed animal model for pre-clinical studies on the pathogenesis, treatment and prevention of diabetic complications.