Abstract
Purpose::
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.
Methods::
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.
Results::
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.
Conclusions::
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.
Keywords: cell adhesions/cell junctions • gene/expression • microscopy: light/fluorescence/immunohistochemistry