Diabetic retinopathy is a disease of the retinal microvessels that is apparently a consequence of protracted high blood glucose levels. Multicenter trials, however, have shown that a subpopulation of diabetics develop retinopathy despite near normal glycemia suggesting the existence of other mechanisms in these patients. The principal investigator has found that a subpopulation of diabetics have autoantibodies to capillary pericytes and others have found autoantibodies to endothelial cells indicating a role for immune mechanisms in diabetic retinopathy. Immune mechanisms have been little studied, therefore the principal investigator is proposing to study the role of anti-endothelial cell autoantibodies in eliciting target cell dysfunction in diabetic retinopathy.
The specific aims of this pilot and feasibility proposal rely on expertise in both immunology and vascular cell biology to investigate target cell responses that are a consequence of anti-endothelial cell auto-antibody binding and the immunogenetic basis of the autoantibody response in diabetic retinopathy. We have three specific aims. The first seeks to identify endothelial cell auto-antigens by immuno-precipitation and N-terminal sequencing and also by the candidate antigen approach. The VEGF receptor is proposed as a candidate antigen based on published studies showing that anti-endothelial cell autoantibodies in systemic vasculitis also specifically bind to monocytes but not other cell types tested and that 50 percent of diabetic sera react similarly. The endothelial cell and monocyte both express Flt-1 (VEGF receptor 1) and endothelial cells also express FIk-1/KDR (VEGF receptor 2), consequently both are candidate antigens. VEGF receptors will be immuno-precipitated and analyzed by western blot for binding of diabetic serum antibodies. Additionally, western blot and ELISA assay on purified soluble receptors will be performed. The second specific aim examines the pathophysiological effects of antibody binding on target cells in vitro and in vivo. Autoantibody will be injected into the vitreous space in the eyes of normal rats that will be examined by fluorescein angiography for anomalies in retinal circulation times. The effect of autoantibody on endothelial cells in vitro will be examined by assay of various elements of signal transduction cascades as well as effects on cytoskeletal organization. The third specific aim will investigate the isotype spectrum of anti-endothelial cell autoantibodies. Isotype restriction to the IgG2 isotype will support an effector function based on the known association of diabetic retinopathy with the IgG2 specific allotype G2m(23). ELISA and western blot assay for anti-endothelial cell antigen G2m(23)+ autoantibodies will also be performed. These studies will determine whether G2m(23)+ IgG2 anti-endothelial cell auto-antibodies are the effector mechanism underlying the known association of diabetic retinopathy with this immunoglobulin allotype. This will be the first demonstration of an effector mechanism underlying an immunogenetic association with diabetic retinopathy.
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|Nayak, R C; Agardh, C D; Kwok, M G K et al. (2003) Circulating anti-pericyte autoantibodies are present in Type 2 diabetic patients and are associated with non-proliferative retinopathy. Diabetologia 46:511-3|