The overall goal of our research is to characterize mechanisms underlying the vascular complications of diabetes that can be probed toward an understanding of the different susceptibility to complications of individual patients and guide new approaches to prevention and management. We propose the hypothesis that elevated glucose concentrations may be toxic to vascular endothelium through mechanisms involving damage to cellular DNA. We plan at this time to pursue the observations that disturbed DNA synthesis and DNA damage occur in endothelial cells cultured in high glucose and to define the relevance of these observations to the in vivo situation. We will study temporal pattern and nature of disturbed DNA synthesis along the growth curve (BrdU flow-cytometry, pulse-chase experiments) and establish whether a causal relationship exists between disturbed DNA synthesis and DNA damage (single strand breaks by FADU after exposure to high glucose during replication or in the postconfluent state). As potential mechanisms for the DNA abnormalities we will investigate non-enzymatic glycosylation of nucleotides (HPLC) and of proteins associated with DNA synthesis (RIA/Western blots employing antibodies against glucose-lysine adducts), formation of activated oxygen species, excess cellular glucose entry and fate of intracellular glucose. In order to assess the biologic importance of DNA damage-repair induced by high glucose we will study the effects of inhibition of repair on cell survival. The relevance of the in vitro findings to the in vivo diabetic state will be sought by investigating 1) biosynthetic aspects of endothelial cells cultured in high glucose (amount and composition of messenger RNA transcripts for von Willebrand factor, proteoglycan, fibronectin, PDGF-like protein studied with cDNA probes and S1 nuclease treatment for homology vs partial homology), and 2) the occurrence of DNA lesions in aortic endothelium of diabetic rabbits (calf thymus polymerase on """"""""en face"""""""" preparations of endothelium). The information derived from these studies should provide a novel perspective on the pathophysiology of diabetic complications and possibly on the teratogenic effect of high glucose. In addition, since the DNA repair mechanisms are under genetic control, the occurrence of DNA damage-repair may be envisioned to play a role in the different individual susceptibility to diabetic vascular complications.
