Study on the mechanisms of alternative mRNA splicing that regulates the regulation of angiogenesis and diabetic complication susceptibility

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In this research, we studied the mechanisms of alternative pre-mRNA splicing/processing by which mRNAs for soluble RAGE and soluble VEGF receptor are produced, and their roles in the regulation of diabetic vascular complications and angiogenesis. Soluble RAGE has a protective activity against AGE-induced vascular cell injury and soluble VEGF receptor acts as a potent anti-angiogenic factor.(1) We isolated the marine equivalent of soluble RAGE by RT-PCR cloning. This study will provide an animal orthologue of soluble RAGE to clarify its roles in health and disease.(2) We investigated the expression of soluble RAGE protein in human organs and tissues by immunohistochemical analysis, and found that soluble RAGE was widely distributed in various organs and tissues including vascular endothelium, neurons, pancreatic β cells, macrophages/monocytes, bile ducts, salivary glands, digestive tracts, renal tubules, prostate, skin, and thyroid.(3) We developed enzyme-linked immunosorbent assay (ELISA) for human soluble RAGE and examined the association of plasma soluble RAGE level with atherosclerosis, and found that it inversely correlated with carotid or femoral atherosclerosis.(4) We established an assay system for RAGE alternative splicing using a human RAGE mini-gene and HEK293T cells. Transfection experiments with various mutant RAGE mini-genes identified cis-acting elements on RAGE pre-mRNA, which regulated the alternative splicing of soluble RAGE mRNA. We also found the involvement of hnRNP-H in the regulation of soluble RAGE mRNA production.(4) We established an assay system for alternative 3'-end processing of VEGF receptor-1 (Flt-1) mRNA using a human Flt-1 mini-gene and primary cultured human vascular endothelial cells, and identified a cis-acting element on Flt-1 pre-mRNA, which regulated the production of soluble Flt-1 mRNA.