Abstract
Purpose:
Vascular endothelial growth factor (VEGF) induced permeability of retinal endothelial cells (REC) is considered causative of diabetic macular edema (DME) that may appear at any stage of DR and can be treated with the VEGF-binding antibody fragment ranibizumab or with the recombinant protein VEGF-trap/aflibercept. In addition, the VEGF-binding antibody bevacizumab is used. In contrast to ranibizumab and bevacizumab, which binds to all VEGF-A isoforms, aflibercept also inhibits other members of the VEGF family. We have recently shown that bevacizumab is internalized by immortalized bovine REC (iBREC) and associates with the cytoskeleton. Here we investigated whether aflibercept is taken up by iBREC and whether the endothelial barrier of the cells is influenced thereby.
Methods:
Uptake of 250 µg/ml aflibercept (Zaltrap) by iBREC was measured by Western-blot analyses of fractionated cellular extracts and immunofluorescence staining. Presence of claudin-1 as a marker for functional tight junctions in iBREC was determined to evaluate the effect of the VEGF-inhibitor on barrier function.
Results:
The concentration of 250 µg/ml aflibercept used in this study reflects therapeutical achievable values. Uptake of aflibercept was detected within 4 h and up to several days after treatment. The recombinant protein was mainly present in the plasma membrane and organelle fraction, but considerable amounts were also detected in the cytoskeleton fraction after 4 d incubation. Under these conditions, expression of claudin-1 and VECad but not of claudin-5 was slightly reduced. After extended incubation, aflibercept mainly co-localized with the cytoskeleton and expression of claudin-1 and VECad normalized. VEGF165 induced loss of claudin-1 was prevented by 25 µg/ml aflibercept.
Conclusions:
Because intracellular aflibercept at clinical relevant concentrations may influence vital functions of iBREC, lower concentrations should be used.
Keywords: 499 diabetic retinopathy •
748 vascular endothelial growth factor •
446 cell adhesions/cell junctions