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S. B. Zanello, A. Perry, W. D. Stamer, R. Heimark; Regulation of Junctional Proteins in Schlemm’s Canal Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5908.
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The lining cells of the inner wall of the Schlemm’s canal (SC) constitute a unique subtype of endothelial cells. Since the actin architecture and associated cell-cell adhesion sites in cells within the outflow tract has a potential role in modulating aqueous outflow resistance, the aim of the present study was to characterize the expression of components of adherens and tight junctions in SCE cells.
SCE cells were isolated /cultured in vitro as described previously and analysis of cell-cell adhesion proteins was performed by immunofluorescence, Western blotting and semi-quantitative RT/PCR. Frozen histological sections comprising the outflow pathway tissues were obtained from perfusion-fixed cadaveric eyes and used for immunohistochemistry.
In vivo, the inner wall of SC expressed vascular endothelial cadherin (VE-cadherin or cadherin 5) which colocalized with platelet endothelial cell adhesion molecule-1 (PECAM1) at sites of cell-cell contact. In vitro, immunofluorescence microscopy, Western blot analysis and quantitative RT/PCR demonstrated the expression of adherens junction and tight junction proteins in SCE monolayers. These included VE-cadherin, N-cadherin, associated catenins, and occludin. Western blotting and comparative quantitation by real time RT/PCR evidenced decreased levels of VE-cadherin protein and mRNA in SCE cells compared to vascular endothelial cells (HUVEC). In contrast, the E/VE-cadherin transcriptional repressor SLUG (SNAI2) was significantly expressed in SCE cells in culture and inversely correlated with mRNA levels of VE-cadherin. Monolayers of SCE cells transfected with 50 nM SLUG siRNA oligonucleotides resulted in an 80% reduction of SLUG mRNA compared to control-siRNA transfected cells and a concomitant increase in VE-cadherin mRNA, with no change in p120catenin mRNA.
Together these results suggest that VE-cadherin gene expression is under transcriptional regulation by the zinc-finger transcription factor SLUG in SCE cells. The coordinated expression and subcellular localization of cell-cell adhesion proteins in SCE cells likely facilitate responses of the inner wall to changes in the dynamic environment in the conventional outflow pathway.
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