During the last three years, my laboratory has focused on the effects of hormones growth factors, and metabolic factors on the functions, metabolism, and growth of vascular cells from retina and arteries. Using these cultured cells from retinal capillaries and arteries of animals and humans, the receptors and functions of insulin and lGFs were extensively characterized and found that retinal capillary cells are responsive to these hormones, whereas arterial endothelial cells are not. Paradoxically, capillary cells cultured from diabetic animals lost their abilities to respond. Hyperglycemia was found to inhibit only the growth of retinal pericytes. This inhibitory effect was not due to sorbitol accumulation directly but alterations in the DNA levels. The effect of cell-cell interactions have been studied with regard to growth and vascular permeability using a specially designed cell culture chamber which mimics the cross-section of a capillary or artery. We are proposing to extend these studies to include vascular cells from renal glomeruli and diabetic animals and humans. Since the insulin resistance of the capillary endothelial cells from diabetic animals has been located at the tyrosine kinase subunit of the receptors, we will localize the change on the tyrosine kinase of the insulin receptor by peptide and mRNA analysis. The regulation of the synthesis or basement membrane components in retinal and renal glomerular cells by hyperglycemia, growth factors, and aldose reductase inhibitor will be assessed by measuring the mRNA levels of Type IV collagen, chondroitin sulfate, and laminin using ribosomal probes in Northern blot analysis. The roles of superoxide radicals as the mediators of hyperglycemia's toxicity on the fragmentation of the DNA of pericytes will be studied. Reversibility of hyperglycemic effects by nicotinamide, desferrioxamine, and agents affecting intracellular glutathione levels will be determined. Regulation or vascular permeability by hyperglycemia, cell-cell interaction, aldose reductase inhibitors, and prostaglandins will be determined. The role of growth factors secreted from retinal and arterial cells in causing excessive proliferation of retinal endothelial and aortic smooth muscle cells will also be evaluated. In summary, these proposed studies will provide a better understanding on the pathogenesis of three of the most prominent features in diabetic vascular disease: 1) increased vascular permeability: 2) basement membrane thickening; and 3) excessive cellular proliferation.
