Effects of Endothelial Cell-Specific Over-Expression of Endothelin-1 on Diabetic and Ischemic Retinopathy

This dissertation, "Effects of Endothelial Cell-specific Over-expression of Endothelin-1 on Diabetic and Ischemic Retinopathy" by Shiu-fai, Cheung, 張劭暉, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled "Effects of Endothelial Cell-specific Over-expression of Endothelin-1 on Diabetic and Ischemic Retinopathy" Submitted by Cheung Shiu Fai for the degree of Doctor of Philosophy at The University of Hong Kong in September 2006 Diabetic retinopathy (DR) is characterized by a number of cellular and pathological changes, including blood retinal barrier (BRB) breakdown, glial cell activation leading to neuronal death, endothelial cell dysfunction, pericyte loss and edema. Endothelin-1 (ET-1), a potent vasoconstrictor, has been suggested to participate in the pathogenesis of DR, including reduced retinal blood flow and increased intraocular pressure. However, there is no suitable transgenic rodent model to study the effects of endothelial ET-1 in DR. Therefore, we generated mice with over-expression of ET-1 specifically in vascular endothelial cells using mouse Tie-1 promoter (TET-1 mice). Over-expressions of ET-1 mRNA and peptide in the endothelial cells of TET-1 mouse retina were confirmed by RT-PCR and immunocytochemistry, respectively. Under normal physiological conditions, homozygous (Hm) TET-1 mice displayed slightly more leakage sites in retinal capillaries as detected by Evans blue extravasation, and down-regulation of glial fibrillary acidic protein (GFAP) in astrocytes. Altered GFAP expression may be linked with inability to maintain the BRB in endothelial cells. However, there was no sign of edema, cell loss, and decrease in thickness of inner nuclear layers. To determine whether BRB breakdown and changes in GFAP expression would be further exacerbated in TET-1 mice by diabetes, TET-1 mice were induced to become diabetic by intraperitonal injection of streptozotocin. Greater severity of diabetes was observed in Hm TET-1 mice, with higher blood glucose level, and a greater rate of decrease in body weight followed by a smaller rate of increase during the duration of diabetes, compared to the corresponding non-transgenic (NTg) diabetic mice. GFAP expression was increased in retina from 5-month diabetic NTg mice compared to NTg non-diabetic mice. Hm TET-1 diabetic mice displayed decreased GFAP expression in the astrocytes and increased Evans blue extravasation, when compared with NTg diabetic mice. Increased vascular permeability and glial cell changes developed in Hm diabetic TET-1 mice, but they did not lead to neuronal or pericyte loss. Since DR was thought to be the consequence of hypoxia and ischemia, possible effects of endothelial ET-1 over-expression on ischemic retinal injury was investigated by 2-hour occlusion and 22-hour of reperfusion of middle cerebral artery, which transiently blocked the ophthalmic and central retinal arteries. Morphological changes was shown in retinal sections of NTg ipsilateral retina compared with NTg contralateral retina, including less than 80% of cells in ganglion cell layer (GCL) containing pyknotic nuclei, and up-regulation of GFAP and glutamine synthetase (GS) peptides in Muller cells. Ipsilateral retinas of Hm TET-1 mice demonstrated more severe changes compared to ipsilateral retina of NTg mice and contralateral retina of Hm TET-1 mice, including more than 80% of cells in GCL containing pyknotic nuclei, up-regulation of GS and aquaporin-4 immunoreactivities in the Muller cell processes, and increased inner retinal thickness. These data suggested that more severe glutamate-induced neur