Abstract
Purpose :
Retinal endothelial cells (REC) are involved in clearing of Fc terminus-containing proteins from the vitreous. After uptake by unchallenged immortalized bovine REC (iBREC), aflibercept is either transcytosed - mediated by the neonatal Fc receptor (FcRn) - or degraded. Under physiological conditions, REC are exposed to serum proteins and aflibercepts's transcytosis is indeed enhanced in the presence of fetal bovine serum (FBS) due to stronger expression of transport proteins like FcRn, Rab11 and dynein. VEGFA, target protein of aflibercept, not only enhances the permeability of (iB)REC thereby allowing paracellular flow but may also change aflibercept’s binding to the FcRn. Therefore, we investigated transport of aflibercept through VEGFA165-challenged iBREC.
Methods :
In all experiments, we exposed confluent iBREC in the presence of 5% FBS to 1.3nM VEGFA165 for 1d before 2µM aflibercept was added. As a measure of barrier function, we continuously determined the cell index (CI) of iBREC cultivated on gold electrodes. To study transport, iBREC were grown on membrane inserts placed in multi-well plates, aflibercept added to the lower chamber and the supernatant taken from the upper chamber was analyzed for presence of the Fc fusion protein.
Results :
iBREC expressed a strong and stable barrier in the presence of FBS indicated by a high cell index. VEGFA treatment strongly reduced CI for up to 3d associated with loss of tight junction-protein claudin-1; reversion to normal values was complete 1d after aflibercept's addition. Transcytosis assays with VEGFA-pretreated iBREC revealed that more aflibercept was present in the upper chamber even 1d after its addition to the lower chamber. Free VEGFA was no longer detected in the upper chamber already 2h after putting aflibercept in the lower chamber. More aflibercept was internalized by VEGFA-pretreated iBREC exposed to aflibercept for 6h, VEGFA was then no longer detectable in cell extracts. Pre-treating of VEGFA-challenged iBREC with an FcRn-inhibiting antibody did neither change transport rates nor internalized amounts of aflibercept 4h after its addition.
Conclusions :
Aflibercept's transport through a leaky iBREC monolayer is enhanced at least partly due to increased paracellular flow. In contrast to unchallenged iBREC, FcRn seems to play a minor role under these conditions. Intracellular stability of VEGFA is significantly reduced in the presence of aflibercept.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.