Diabetic retinopathy (DR) is a severe complication of diabetes mellitus leading to vision loss in millions of patients. The main ocular target of DR is the retina that develops intravitreal hemorrhages, macular edema, retinal detachments and neovascularization. However, 70% of diabetic patients also suffer from corneal abnormalities. Clinical corneal problems include epithelial alterations (abnormal barrier function, defects and recurrent erosions, debridement after vitrectomy, delayed wound healing, keratitis, and edema), as well as neuropathy. Altered epithelial-stromal interactions and basement membrane (BM) structure likely contribute to diabetic epitheliopathy. In the previous funding period, we have validated human corneal organ culture model because diabetic animals do not fully reproduce human diabetic eye disease. Organ-cultured corneas from DR patients retain marker abnormalities and delayed wound healing as observed in diabetic patients. Organ-cultured corneas from DR patients maintain altered BM protein expression observed in intact donor corneas. Additionally, DR corneas have decreased levels of migratory growth factors/cytokines, such as HGF/c-met system and thymosin beta4, and simultaneously increased expression of proteinases, MMP-10 and cathepsin F. Changes in growth factor expression and increased proteolysis may trigger BM and wound healing alterations leading to clinically observed diabetic corneal disease. We propose the following hypothesis: Epithelial alterations found in diabetic corneas are due to decreased levels or activity of migration stimulating factors, such as HGF and thymosin beta4, accompanied by increased levels of proteinases, such as MMP-10 and cathepsin F, resulting in enhanced epithelial BM degradation and reduced cell adhesion. These changes will lead to delayed epithelial wound healing and persistent defects. Specific correction of these alterations by antisense inhibition or viral-mediated gene transfer may alleviate diabetic corneal epitheiiopathy. To test this mechanism, we propose the following Specific Aims:
Aim 1. To examine the role of growth factor alterations in diabetic corneal abnormalities using gene-based therapies in human corneal organ culture. Diabetic organ-cultured corneas will be treated with viral constructs harboring c-met or thymosin betap4 genes under a cornea-specific promoter to restore normal expression and HGF signaling through c-met receptor. Normal organ-cultured corneas will be treated with antisense oligonucleotides to cmet and thymosin beta4 to block their expression. With respect to marker patterns and wound healing rates, we expect treated DR corneas to become more similar to normal but normal corneas to become more similar to DR corneas.
Aim 2. To determine the role of proteinases in diabetic corneal alterations using human corneal organ culture model. DR organ-cultured corneas will be treated with antisense oligonucleotides to MMP-10 or cathepsin F, to inhibit their expression. Normal organ-cultured corneas will be treated with viral constructs harboring cathepsin F or MMP-10 genes to increase their expression. We expect that treated DR corneas will be similar to normal but treated normal corneas will be similar to DR by marker patterns and wound healing rates.
Aim 3. Demonstrate that diabetic alterations in wound healing and marker distribution result from a combined action of growth factors and proteinases. Growth factor and proteinase gene expression will be changed together towards normal in DR corneas using viral-based gene delivery and antisense approach. Expression of DR markers and wound healing rates will be determined and compared to single gene therapies. This approach will be also used in normal corneas in order to elicit delayed wound healing and DR-like changes in marker expression. These studies should facilitate the development of gene-based new therapy for diabetic corneal abnormalities.
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