Protein markers and differentiation in culture for Schlemm's canal endothelial cells

The two cell types that populate the human conventional outflow pathway, Schlemm's canal (SC) and trabecular meshwork (TM) regulate intraocular pressure. In culture, SC and TM cells have been useful tools toward understanding their respective roles in conventional outflow homeostasis. Unfortunately, currently available protein markers that distinguish SC from TM cells are limited, motivating the present study. Antibodies that specifically recognize different vascular endothelial markers were used to probe lysates from mature cell monolayers subjected to SDS-PAGE followed by western blot analyses. Results show that SC and TM cells both expressed many of the endothelial candidate proteins investigated, such as Robo1/4, Tie2/TEK, VEGF-R1/R2, VCAM-1, eNOS and neuropilin-1. In contrast, all SC cell strains tested (n=11) expressed two proteins, fibulin-2 and vascular endothelial (VE) cadherin, not expressed by TM cells. To examine changes in VE-cadherin expression and cell-cell junction formation, indicated by transendothelial electrical resistance (TEER), SC cells were seeded onto filters at confluence and growth factors were withdrawn. Culturing cells in media containing adult bovine serum rather than fetal bovine serum resulted in a 75% mean increase in TEER and 67% corresponding average increase in VE-cadherin expression (p<0.05). While both TM and SC cells form monolayers, are contact inhibited, share some endothelial responsibilities and several endothelial protein markers, SC cells uniquely express at least two proteins which likely reflect a distinction in cellular responsibilities in vivo. One of these responsibilities, maintenance of the blood-aqueous barrier, can be modeled in culture upon withdrawal of growth factors from SC cell monolayers.