Abstract
Purpose: :
The pathogenesis of many vascular diseases such as diabetic retinopathy involves increased vascular permeability. The present study aimed at (i) establishing a robust in vitro model of the retinal vascular endothelium constituting the inner blood–retinal barrier and (ii) assessing the molecular consequences of exposing these cells to various vasoactive and vasoprotective agents. Particular emphasis was put on changes in cell signaling, adherens and tight junctional architecture, as well as paracellular permeability.
Methods: :
Primary microvascular endothelial cell cultures were established from rat retinae (pRMVEC) or brain (pBMVEC). Subcellular localization of junctional proteins was determined using indirect immunofluorescence. Intracellular protein levels and phosphorylation states were determined by western blotting. Paracellular permeability was measured by fluorescein–dextran transport.
Results: :
The subcellular distribution of adherens and tight junction proteins such as VE–cadherin, catenins, occludin, claudins and ZO–1 was found to be very similar in pRMVEC and pBMVECs. Vascular endothelial growth factor, histamine, lysophosphatidic acid (LPA), bradykinin and thrombin affected the distribution of many junctional proteins. Although these vasoactive agents induced common signal transduction pathways, i.e. resulting in the rapid tyrosine phosphorylation of VE–cadherin, the spatio–temporal redistribution of junction proteins was unique for each molecule tested. Surprisingly, the vasoactive response, measured as an increase in paracellular permeability, to most of these molecules (with the exception of LPA) was mild or often completely absent in pRMVEC and pBMVEC, and did not correspond to junction protein redistribution. Hydrocortisone and even more so triamcinolone acted in a vasoprotective manner and decreased the paracellular permeability of pRMVEC and pBMVEC by an order of magnitude.
Conclusions: :
(1) Judged by the cellular localization of junctional proteins the endothelium lining the CNS and the inner neuroretina is morphologically very similar. (2) Both vascular endothelia are not only characterized by a high impermeability but also a relative insensitivity to classical vasoactive molecules. (3) Vascular hyperpermeability does not appear to be primarily mediated by redistribution of junctional proteins. (4) Glucocorticoids induce improved barrier function of MVEC which lends further support to their usefulness in alleviating clinical manifestations of vascular disease.
Keywords: cell adhesions/cell junctions • signal transduction • vascular cells