Abstract
Purpose :
Because retinal endothelial cells (REC) are involved in the clearance of therapeutic IgG in the eye, we investigated the transport and intracellular localization of the VEGF-binding protein aflibercept in immortalized bovine REC (iBREC). The neonatal Fc receptor (FcRn) which is necessary for transport and recycling of IgG in EC, is a potential receptor for shuttling proteins containing an Fc-terminus like aflibercept. FcRn is located in early endosomes; it is expressed in iBREC and down regulated in the absence of fetal bovine serum (FBS).
Methods :
Intracellular localization of aflibercept was assessed by western blot analyses of subcellular protein fractions. To study transport, iBREC were grown on membrane inserts placed in multi-well plates. After addition of 250 µg/ml (~2 µM) aflibercept to the lower chamber, aliquots of the supernatant in the upper chamber were analyzed at 2 h to 2 d. Uptake and transport of aflibercept was studied under various conditions affecting interaction with FcRn: a) presence or absence of FBS (→up/down regulation of FcRn), b) presence of 10 µM protein A or G (→prevention of aflibercept’s binding to FcRn) or c) presence of 100 nM wortmannin (→inhibition of early endosome formation).
Results :
Aflibercept was transported through an iBREC monolayer when added to the lower chamber in excess, but it was also recycled into an aflibercept-free environment at the same side of the cell. The rates of both processes were strongly enhanced by 5% FBS in the culture medium. The amount of internalized aflibercept slightly increased over time, and it was homogenously distributed in the fractions of membrane proteins (plasma membrane/organelles) or cytoskeletal proteins when iBREC were cultivated with 5% FBS. In the absence of FBS, aflibercept was more strongly associated with proteins of the cytoskeleton. In the presence of protein A or G, association of aflibercept only with cytoskeletal proteins was observed, and its transport was delayed. Uptake but not transport of aflibercept was decreased by wortmannin.
Conclusions :
Absence of FcRn (→induced by serum deprivation) or inhibition of aflibercept’s binding to FcRn (→presence of protein A/G) correlate with aflibercept’s delayed transcytosis and increased association with cytoskeletal proteins. Our results therefore suggest that FcRn is indeed involved in transport and processing of aflibercept in REC in vitro.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.