Transgenic mice over-expressing endothelin-1 in endothelial cells have blood-retinal barrier breakdown and lower expression of glial fibrillary acidic protein

Abstract

Retinopathy, one of the microvascular complications of diabetes, is a major cause of blindness in adults. The breakdown of blood-retinal barrier (BRB) is a pathogenic feature of diabetic retinopathy (DR). The involvement of endothelin-1 (ET-1) in DR has been suggested by up-regulated ET-1 peptide expression in the retinal endothelial cells of diabetic rats. To investigate the contribution of ET-1 in retinal endothelial cells to morphological, biochemical and functional changes in DR, transgenic mice with endothelial ET-1 over-expression driven by mouse tie-(tyrosine kinase)-1 promoter (TET-1 mice) were generated. Semi-quantitative RT-PCR studies demonstrated that total ET-1 mRNA expression in transgenic mouse retina was increased at least 2-fold. Indeed, immunocytochemical studies showed up-regulation of ET-1 peptide specifically in retinal endothelial cells of TET-1 mice. Moreover, the blood vessel density in the ganglion cell layer of homozygous TET-1 mice was higher than that of non-transgenic mice, indicating that over-expression of ET-1 may lead to neovascularization in retina. On the other hand, the expression of glial fibrillary acidic protein (GFAP) was decreased in optic nerve, astrocytes and Müller cell processes, suggesting that over-expression of ET-1 may down-regulate the expression of GFAP, which is important in the maintenance of BRB. BRB integrity in TET-1 mice was examined by leakage of Evans blue in whole mount retina. Preliminary data showed that there was slightly more focal leakage in retinal blood vessels of TET-1 mice, raising the possibility of ET-1 affecting BRB functions. Our TET-1 mice will be an invaluable model to investigate the contribution of ET-1 in retinal endothelial cells to pathogenic effects of DR.