The role of Tbdn in retinal endothelial cell permeability and retinal homeostasis

Whelan, Maria A. (Maria Anne) (2011) The role of Tbdn in retinal endothelial cell permeability and retinal homeostasis. Masters thesis, Memorial University of Newfoundland.

[English] PDF - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. Download (106MB)

Abstract

Tubedown (Tbdn) is a cortactin-binding acetyltransferase subunit which controls retinal endothelial permeability and promotes retinal vascular homeostasis. The Paradis-Gendron laboratory has generated a binary antisense Tbdn (Astbdn) transgenic mouse model (TIE2/rtTA/Enh-TRE/ASTBDN-1) that facilitates the conditional knockdown of Tbdn protein expression in retinal endothelial cells. The suppression of Tbdn expression in mouse retinal endothelium, facilitated by the introduction of doxycycline (Dox) to the binary system, results in several pathologic features that are also observed in human retinopathies. Tbdn protein expression is reestablished once doxycycline is removed from the system. -- In study one, it was hypothesized that reexpression of Tbdn would occur in retinal blood vessels of TIE2/rtTA/Enh-TRE/ASTBDN mice upon Dox withdrawal and that Tbdn reexpression would correlate with decreased retinal pathology. In study one it was found through immunohistochemistry and morphological assessment that reexpression of Tbdn in retinal blood vessels resulted in a reduction of albumin leakage across the retinal blood vessel endothelium, along with a decrease in the extent of retinal vascularization and retinal pathologies compared to Tbdn knockdown mice. These results suggest that restoration of Tbdn protein expression after knockdown reduces retinal pathology at the morphological and functional level. -- In this study it was shown that the proliferative retinal neovascularization and thickening resulting from induction of Tbdn knockdown in endothelium in transgenic mice is associated with a significant increase in extravasation or leakage of albumin from abnormal retinal blood vessels in vivo. These results provide evidence that Tbdn is involved in the regulation of retinal endothelial cell permeability to albumin and implicates a functional role for Tbdn in blood vessel permeability dynamics. Study two explored the relationship between Tbdn and other known regulators of vascular endothelial permeability. Tbdn suppression in endothelial cells correlates with an increase in transcellular permeability pathway. Recent experimental evidence points to the importance of Src family protein tyrosine kinase (SFK) signaling in regulation of microvascular barrier function and vascular endothelial permeability. -- In study two it is hypothesized that Tbdn expression regulates retinal endothelial cell permeability through regulation of the Src pathway. The Paradis-Gendron laboratory have previously generated RF/6A endothelial cell clones in which Tbdn expression had been suppressed by stable expression of the antisense TBDN cDNA construct ASTBDN. Src activation in retinal endothelial cell clones was measured by western blot and immunoprecipitation analysis. It was shown that when Tbdn is suppressed in endothelial cells, there is a higher level of activated Src compared to parental and negative control clones. This high level of activated Src suggests that Tbdn may regulate the transcellular permeability pathway for albumin transport through suppression of the Src pathway. -- Based on the cumulative results of both studies I propose a mechanism whereby Tbdn plays an important role in regulating the permeability of retinal endothelial cells to albumin by interacting with other proteins in the transcellular albumin pathway.

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