Hyperglycemia has been shown to be the major causal factor in the initiation of and the progression of diabetic microvascular disease. We have proposed that hyperglycemia mediates its adverse effects through the activation of diacylglycerol-protein kinase C (DAG-PKC) pathway involving predominately PKC beta isoform which has been reported to increase extracellular matrix production, growth factor expression, and enzyme activation. We have designed a specific PKC beta isoform inhibitor (LY333531) that can prevent many of the vascular changes described above and changes in retinal blood flow, renal glomerular filtration rate and albuminurea in the diabetic rat. Thus, we propose to determine which of the abnormal vascular functions derived in the retina, renal glomeruli, and cardiovascular system in rodent models of diabetes are due to the activation of DAG-PCD eta isoform. To accomplish this goal, we plan to delineate (1) the cellular effects of glucose induced activation of DAG-PKC beta isoform pathway in the retinal capillary and other vascular cells by measuring the various signal transduction pathways such as MAP kinase cascase and cPLA2 cell functions as assessed by the expression of extracellular matrix proteins and growth factors, production of prostanoids and cell growth. The specific role of PKC beta isoform will be determined by using PKC beta isoform inhibitor, LY333531, antisense to beta isoform and vascular cells cultured from PCK isoform beta knockout or overexpressed transgenic mice. (2) to assess the in vivo and pathological consequences of PKC beta isoform activation in the retina, renal glomeruli, and heart of rodent models of diabetes by using LY333531, PKC beta isoform knockout mouse with induced diabetes and in transgenic mice overexpressing PKC beta isoform in the heart and endothelial cells; (3) To characterize the regulation and functions of a pericyte specific gene which could be regulated by glucose and growth factors. The results from the use of LY333531 and the transgenic mice models will provide direct evidence to determine the roles of PKC beta isoform activation in the development of vascular complications of diabetes.
