Abstract
Purpose :
VEGFA165 induces a persistent dysfunction of the barrier formed by immortalized endothelial cells of the bovine retina (iBREC), completely prevented but only transiently reverted by inhibiting VEGF signaling. Here we investigated how duration of exposure to the growth factor affects the cells' response.
Methods :
Confluent iBREC were exposed to 1.2nM VEGFA165 for 1 to 9 days. As a measure of barrier function, we continuously determined the cell index (CI) of iBREC cultivated on gold electrodes. Levels of proteins regulating paracellular or transcellular flow, i.e. claudin-1, claudin-5, vascular endothelial cadherin, caveolin-1, plasma lemma vesicle associated protein (PLVAP), of adhesion proteins CD9/TSPAN29, integrins α5 and β1, and of nuclear β-catenin were assessed by Western-blotting; intra- and extracellular VEGFA levels by ELISA.
Results :
VEGFA treatment sginificantly reduced CI values still low after 9 days of exposure. Inhibition of VEGFR2 alone or in combination with PDGFRα/β or FGFR1/2/3 by tivozanib or nintedanib led to complete recovery of the low CI values when the inhibitor was added to iBREC pre-treated with VEGFA for 1 day; the effect was stable for 2 days. Reversion of the low CI was not successful when inhibitors were added to cells exposed to the growth factor for more than 3 days. VEGFA concentrations in the cells’ supernatant declined from 0.6nM to 0.1nM measured 2 or 9 days after addition, respectively, but its intracellular levels remained stable. Levels of tight junction (TJ)-protein claudin-1 remained low in VEGFA-exposed iBREC from days 1 to 9; those of TJ-protein claudin-5 were elevated, but only on day 6. VEGFA induced expression of PLVAP which was very high on days 2 and 6 after addition, but extremely low on day 9. Levels of integrin α5 - higher on day 2 - declined over time and were lower on day 9 compared to those of control cells; levels of integrin β1 remained slightly elevated at all time points. Only on day 2, VEGFA induced a significant increase of nuclear β-catenin. Levels of the other proteins investigated remained stable.
Conclusions :
Only barrier disturbances, i.e.deregulated para- and transcellular flow, observed early during VEGFA exposure depend on the growth factor’s action. Changes observed later include impaired TJs and adhesion, and are independent of the presence of the growth factor.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.