Abstract
Purpose :
Expression of tight-junction (TJ) protein claudin-1 was higher after exposure of immortalized bovine retinal endothelial cells (iBREC) over several days to the inhibitor of dipeptidyl-peptidase-IV sitagliptin, but that of the subunit of the fibronectin receptor CD29 and the tetraspanin CD9 - both important for adhesion of iBREC - was lower. CD9 was also translocated from the plasma membrane to the cytosol, suggesting that sitagliptin weakens interaction of iBREC with the extracellular matrix. Here we investigated whether sitagliptin changes the barrier-impairing effects of VEGFA.
Methods :
Confluent iBREC were exposed to 10-1000nM sitagliptin after cells had been pretreated with 50ng/ml VEGFA165 for 1d. The cell index (CI) of iBREC grown on gold electrodes was continuously followed to evaluate changes in paracellular flow and adhesion of the cells. We analyzed expression or localization of proteins involved in a stable barrier (→claudin-1, -5, VECadherin), transcellular transport (→caveolin-1) or interaction with the extracellular matrix (→CD29, CD9) by Western blotting or immunofluorescence staining.
Results :
VEGFA induced a persistent decline of the CI which was completely reverted by the inhibitor of VEGF receptor 2 tivozanib. Sitagliptin neither prevented nor reverted the VEGFA-induced reduction of the CI, but also did not change the protective effect of tivozanib. Expression of claudin-1 reduced by VEGFA exposure of the cells was significantly higher after sitagliptin treatment but did not reach normal values. Interestingly, expression of CD9 was significantly lower under these conditions whereas that of the other proteins investigated was not significantly changed. Spindle-like morphology of VEGFA-pretreated iBREC reverted to cobble-stone morphology after addition of tivozanib but not of sitagliptin. CD9 was localized at the plasma membrane in untreated or tivozanib-rescued VEGFA-challenged iBREC but it was concentrated at the foci of iBREC exposed to VEGFA or VEGFA plus sitagliptin.
Conclusions :
Sitagliptin neither enhanced nor reverted the detrimental action of VEGFA on the stability of the iBREC barrier despite increasing the amount of the TJ-protein claudin-1. The low CI rather correlated with a low expression and a changed subcellular localization of the tetraspanin CD9 confirming the hypothesis that sitagliptin might interfere with interaction of REC with the extracellular matrix.
This is a 2020 ARVO Annual Meeting abstract.