The goal of this project is to test the hypothesis that the most characteristic lesion of diabetic retinal vessel - thickened basement membranes - plays a role in the development of the serious lesions of diabetic retinopathy. The hypothesis is based on the recent finding that human retinal vessels manifest alterations that can be induced by the thickened basement membranes, and in turn, can compromise the integrity of the vessels inner lining. Having demonstrated that in diabetes there is increased production of the basement membrane proteins fironectin and collagen type IV, the aim is to interfere with such excess production. Because antisense technology offers a unique opportunity to reduce specific gene expression, the plan is to apply this technology to address the following issues: (i) establish whether antisense approach downregulates BM component synthesis in the retina, (ii) determine if downregulation of the specific BM genes alters other cellular processes in the retina, and (iii) determine whether decreased synthesis of basement membrane components reduces BM thickening in retinas of galactosemic rats. The longterm objective is to reduce BM thickening by down regulating FN and collagen IV synthesis and study whether such reduction in BM width will prevent the development of the retinal lesions in galactose-fed rats, an animal model of diabetic retinopathy. FN and collagen IV mRNA and protein levels in the retina will be monitored by RT-PCR, in situ hybridization, immunohistochemistry and western analysis. Retinal capillary BM width will be measured by morphometric analysis of electron micrographs. Since sustained down regulation of gene expression may be necessary to arrest BM thickening, a strategy using plasmid constructs that synthesize antisense RNA against firbronectin and collagen IVa1 transcripts will be tested. Plasmid constructs will be delivered intravitreously and studied for potential long-term inhibitory effect. This project will address the longstanding question of whether thickened vascular basement membranes play a role in diabetic microangiopathy. A successful study would provide the critical information that inhibition of excess synthesis of basement membranes prevents background diabetic retinopathy.
