Cell-cell communication in the pathogenesis of diabetic retinopathy

• Main Author: Stottrup, Casey
• Language: en_US
• Published: Boston University 2015
• Online Access: https://hdl.handle.net/2144/12642

Thesis (M.A.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at [e-mail]. Thank you. === Purpose: The goal of this study is to test the hypothesis that high glucose, a prevalent characteristic of diabetes, induces vascular cell death associated with diabetic retinopathy by compromising gap junction intercellular communication. Methods: Cell culture models of hyperglycemia involving endothelial cells, pericytes, and Muller cells were used in this study. Cells were exposed to high glucose (HG: 30 mM) for 7 days and harvested for analysis of Cx43, ZO-1, rab20, and β-actin by Western blot and subsequent densitometric analyses using imageJ software. Immunoprecipitation was performed to identify rab20 by Western blot analysis. Localization and distribution of the tight junction and gap junction proteins were determined by immunostaining. Cells immunostained for Cx43 and ZO-1 were digitally photographed under immunofluorescence microscopy and assessed for protein localization and distribution. To determine the effect of reduced rab20, cells were transfected with rab20 siRNA in the presence of Lipofectin; scrambled siRNA was used as control. To identify cells undergoing apoptosis, TUNEL assay was performed. Results: Western blot analysis revealed that HG significantly reduced Cx43 protein expression in rMC-1 (64% of N) and cocultures of rMC-1 and BRPs (72% of N). Similarly, Cx43 and ZO-1 immunostaining were significantly reduced in these cells grown in HG condition. Western blot analysis also revealed that HG significantly upregulated rab20 (129% of N) protein expression. Under HG condition, an increased number of TUNEL+ cells was detected using TUNEL assay (7.3 vs. 1.5 TUNEL+ cells per 1000 cells). HG cells transfected with rab20 siRNA significantly reduced the number of apoptotic cells (1.5 vs. 7.3 TUNEL+ cells per 1000 cells). Conclusion: Findings from this study indicate that HG-induced disturbed gap junction intercellular communication may contribute to retinal vascular cell and Muller cell apoptosis. HG-induced overexpression of rab20 may play a significant role in reducing Cx43 expression and compromising cell-cell communication associated with the pathogenesis of diabetic retinopathy.