The role of the Ard1 protein in retinal endothelial cell permeability

Morrissey, Karla Anne (2014) The role of the Ard1 protein in retinal endothelial cell permeability. Masters thesis, Memorial University of Newfoundland.

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Abstract

Tubedown (Tbdn) is a retinal homeostatic factor which is normally highly expressed in the retinal blood vessels which associates with the N-acetyltransferase Ard1. Tbdn also associates with Cortactin, a c-Src tyrosine kinase substrate. Downregulation of Tbdn correlates with neovascular retinal pathology in neovascular retinopathies. These diseases involve growth and hyperpermeability of retinal blood vessels leading to leakage of plasma proteins such as Albumin, inflammation and tissue damage. Knockdown of Tbdn in retinal endothelial cells results in co-suppression of Ard1, activation of c-Src/Fyn tyrosine kinase and increased permeability to Albumin. The focus of this study is to determine how Ard1 might regulate signaling pathways mediating the permeability of endothelial cells to Albumin as well as levels of phospho-Cortactin Tyr421 and activated c-Src/Fyn. Ard1 was either knocked down using siRNA or overexpressed in RF/6A primate retinal endothelial cell line and the permeability to Albumin assayed. In parallel experiments, the activation status of components of the Albumin permeability pathway (phospho-Cortactin Tyr 421 and activated c-Src/Fyn) was monitored. Ard1 knockdown reduced the permeability of the cells to FITC-Albumin but did not change the levels of phospho-Cortactin Tyr 421 or activated c-Src/Fyn when compared to controls. Cells transfected with the Ard1 expression vector overexpressed Ard1 and showed increased permeability of Albumin and increased levels of activated c-Src/Fyn compared to controls. Our results suggest that Ard1-mediated increase of activated c-Src/Fyn does not require Tbdn.

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