Capacity of aflibercept to counteract VEGF-stimulated abnormal behavior of retinal microvascular endothelial cells

Abstract

Members of the vascular endothelial growth factor (VEGF) family differently regulate processes in retinal endothelial cells (REC) which are crucially involved in the pathogenesis of diabetic retinopathy: Both, VEGF-A and placenta growth factor (PlGF), stimulate proliferation of primary and immortalized bovine REC ((i)BREC) but only VEGF-A165 stimulates their migration. Diabetic macular edema is most likely a consequence of an elevated permeability of REC which can be induced by VEGF-A, but not by PlGF. Binding of VEGF-A by the antibody fragment ranibizumab is sufficient to completely restore or prevent VEGF-A-induced disturbance of the iBREC barrier or migration of these cells without affecting the basal processes. This was observed even in the presence of other growth factors when surplus proliferation was only partly blocked. The recombinant protein aflibercept (VEGF-trap) not only binds very strongly to VEGF-A, but – in contrast to ranibizumab – also recognizes PlGF. In this study, we investigated whether this additional targeting of PlGF also results in better inhibition of growth factor-induced proliferation and migration, and disturbance of the iBREC barrier. In addition, uptake of aflibercept by iBREC and potential functional consequences were examined. In accordance with its binding specificity, aflibercept strongly and specifically inhibited iBREC proliferation stimulated with VEGF-A, PlGF or a combination of these factors. By treatment with aflibercept at therapeutically achievable concentrations, VEGF-A-stimulated iBREC migration was reduced not only to normal values but driven below the basal level. However, the VEGF-A binding humanized antibody bevacizumab as well as the unrelated control antibody rituximab also inhibited basal or VEGF-A stimulated migration at clinically relevant concentrations, suggesting an effect of high amounts of IgG domain-containing proteins which does not depend on their binding specificity. However, aflibercept specifically blocked VEGF-A stimulated migration at lower concentration without influencing basal processes. Effects on permeability were determined by measuring transendothelial resistance (TER) of iBREC (±VEGF-A165) and their expression of the tight junction protein claudin-1. The VEGF-A-disturbed barrier was completely restored by treatment with ≤25 μg/ml aflibercept of which even much higher concentrations did not interfere with normal barrier function. Uptake of aflibercept by iBREC – analyzed by Western blot – was observed after 1 h of treatment and the amount further increased during prolonged incubation. Most of the internalized aflibercept was present in subcellular fractions of proteins assigned to the membranes and organelles, but it was also detected in the fraction consisting of cytoskeletal proteins. Co-immunofluorescence staining showed aflibercept absorbed by iBREC to be localized in or close to the Golgi apparatus. Aflibercept at high concentrations interferes with an important normal iBREC function, but prevents and restores VEGF-A-induced disturbances at considerably lower concentrations. Therefore, reduction of the doses administered in DR and DME therapy might be considered.