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Heidrun L Deissler, Gerhard K Lang, Gabriele Elisabeth Lang; Aflibercept's potency to block or revert VEGF-stimulated behavior of retinal endothelial cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5837.
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Members of the VEGF family regulate processes in retinal endothelial cells (REC): VEGF-A and PlGF stimulate proliferation of immortalized bovine REC (iBREC) but only VEGF-A165 stimulates their migration and elevates their permeability. Binding of VEGF-A by ranibizumab is sufficient to completely restore or prevent VEGF-induced disturbance of the iBREC barrier or migration even in the presence of other growth factors when surplus proliferation was only partly blocked. Here, we investigated whether additional targeting of PlGF by aflibercept results in better inhibition of growth factor-induced proliferation/migration, and disturbance of the iBREC barrier. In addition, uptake of aflibercept and potential functional consequences were examined.
Effect of aflibercept on VEGF-A/PlGF-induced proliferation or migration was determined. Measurement of transendothelial resistance as well as presence of claudin-1 in iBREC treated with aflibercept (+/-VEGF) reflected its effect on iBREC barrier. VEGF-A content in culture supernatant was determined by ELISA. Localisation of internalized aflibercept was established by Western-Blot analysis and immunofluorescence.
Aflibercept strongly and specifically inhibited proliferation stimulated with VEGF-A, PlGF or a combination of these factors. By treatment of unstimulated iBREC with aflibercept at therapeutically achievable concentrations, gap closure was inhibited. However, the unrelated antibody rituximab also inhibited basal migration, suggesting an unspecific effect of high amounts of IgG domain-containing proteins. VEGF-A (2 nM) induced loss of claudin-1 was completely reverted by treatment with < 2 nM aflibercept or 20 nM ranibizumab. Under these conditions, VEGF-A in the culture supernatant was completely bound by aflibercept but not by ranibizumab whereas intracellular levels of free VEGF-A were comparable. Uptake of aflibercept was independent on fibronectin or VEGF-A, and most of the internalized protein was present in subcellular fractions of membrane/organelle proteins. Co-immunofluorescence staining showed aflibercept to be localized in or close to the Golgi apparatus.
Aflibercept efficiently prevents and restores VEGF-induced disturbances in iBREC. Whether its uptake results in deposits of the therapeutic protein in vivo which may be harmful to REC through unspecific inhibition of migration or otherwise, remains to be seen.
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