RESUMO
Various severe ocular diseases are associated with an elevated intravitreal expression of VEGF-A which increases the permeability of retinal endothelial cells (REC) or retinal pigment epithelial (RPE) cells in vivo and in vitro. Inhibition of VEGF receptor 2 (VEGFR2) is sufficient to completely prevent VEGF-A165-induced dysfunctions of barriers formed by long-term cultivated, immortal human ARPE-19 cells or immortalized bovine retinal endothelial cells (iBREC). Extended exposure to VEGF-A could result in additional activation of other growth factor receptors, potentially promoting synergistic effects of corresponding factors on various cellular processes including angiogenesis. Based on these observations, we investigated whether blocking of VEGFR2 is also sufficient to revert VEGF-A-induced changes of the barriers consisting of iBREC (i.e. inner blood-retina barrier) or ARPE-19 cells (i.e. outer blood-retina barrier) in vitro. Alterations of confluent monolayers' properties induced by treatment with VEGF-A165 for one day followed by addition of small molecule inhibitors of the VEGFR2 were determined by continuous cell index (CI) measurements using the microelectronic biosensor system for cell-based assays xCELLigence. VEGF-A165 induced a long-lasting drop of the otherwise high CI of iBREC accompanied by reduced expression of the tight junction (TJ) protein claudin-1 and subtle changes of the plasma membrane localizations of TJ-protein claudin-5 and of vascular endothelial cadherin. Blocking mainly VEGFR2 with 10 nM nintedanib, 10 nM tivozanib or 500 nM ZM323881 efficiently reverted these changes within one day; higher concentrations of nintedanib or additional inhibition of neuropilin-1 were not superior. Interestingly, the CI of short-term cultivated, confluent ARPE-19 cells slightly increased in the presence of VEGF-A165, but was not changed by nintedanib. In contrast, VEGF-A165 markedly reduced the transepithelial electrical resistance of ARPE-19 cells cultivated on porous membrane inserts for three weeks, which was also accompanied by a significant loss of the then strongly plasma membrane-expressed TJ-protein ZO-1. These alterations were completely reverted within one day by 10 nM nintedanib of which higher concentrations were not superior. None of the inhibitors tested diminished the strong barrier properties of iBREC or long-term cultivated ARPE-19 cells. Taken together, inhibition of VEGFR2 efficiently reverts VEGF-A165-induced barrier disturbances of both cell types forming and regulating the inner and outer blood-retina barrier. As synergistic actions of growth factors seem to play only a minor role in inducing a barrier dysfunction, specific inhibition of VEGFR2 could be an interesting option to treat VEGF-A-induced macular edema without obvious effects on vitality and functions of REC and RPE cells.