Clinical studies have indicated that glycemic control has to be initiated early if the goal is to prevent the onset of diabetic micro- and macroangiopathies. The causal factors of the vascular diseases are probably multiple, including cytokines, hormones and metabolites. Using cultured retinal and aortic cells, we have described a whole network of interactions involving cell to cell and hormone to cell. One of the most exciting findings is the detection of increases in DAG levels and PKC activities in vascular cells and tissues (retina, aorta and heart), when they are exposed to hyperglycemia or elevated glucose levels. We and others have identified that the DAG is derived from glucose via the de novo synthesis pathway. Immunoblotting studies have identified PKC betaII isoform to be preferentially increased as compared to other isoforms. Interestingly, the increases in DAG and PKC were not easily reversed since strict glycemic control by pancreatic islet cell transplants only normalized these values in the heart but not in the aorta, indicating chronic changes occurred after several weeks of diabetes. Correlation has also been made between changes in PKC and DAG with cellular targets of PKC and retinal hemodynamics. In the present proposal, we seek to understand the mechanism by which hyperglycemia is increasing PKC levels and preferentially activating PKC betaII isoform. Individual steps along the de novo synthesis pathways, such as glucose transport and hexokinase activities will be analyzed by enzyme activity, immunoblots and Northern blots. Chronic studies will be initiated to determine the persistence and the reversibility of the increases in DAG and PKC in the vascular tissue from diabetic rats. In addition, cellular and physiological consequences of increases in DAG and PKC will be characterized. In the cell, phosphorylation states of PKC targets such as MARCKS protein, calponin and growth factors will be determined. The relationship between these biochemical changes and retinal blood flow will be studied in vivo with PKC and DAG modulators. To prove this hypothesis, we are planning to over- express PKC betaII in cultured vascular cells and in mice in order to test the hypothesis that activation of PKC betaII is responsible for the retinal hemodynamics observed in diabetes and some of the pathologies observed in early diabetes. Lastly, we have identified a set of genes in pericytes which are glucose-regulated in parallel with glucose's growth inhibiting sects. The new proposal will be to characterize and fully sequence these new genes and determine their expression in vascular tissue of diabetic animals.
