Abstract
Because rare, but severe adverse effects, i.e. retinal vasculitis or retinal vein occlusion, have been observed after repetitive intravitreal injections of VEGF-A-binding single-chain variable fragment brolucizumab (Beovu), we investigated its possible impact on the barrier formed by immortalized bovine retinal endothelial cells (iBREC) in comparison to that of the VEGF-A-binding Fab fragment ranibizumab (Lucentis). As a measure of stability of the barrier formed by a confluent monolayer of iBREC, we determined the cell index over seven days by continuous electric cell-substrate impedance measurements: Beovu but not Lucentis indeed significantly lowered the cell index, evident about 1.5 days after its addition, pointing to barrier impairment. Early after addition of Beovu, amounts of the integrins α5 and β1-subunits of the fibronectin receptor-had changed in opposite ways, suggesting an effect on cell adhesion due to hindered dimer formation. After exposure for eight days to Beovu, levels of claudin-1-an essential part of the iBREC barrier-were significantly lower, less claudin-1 was located at the plasma membrane after exposure to the VEGF-A antagonist for five days. Beovu did not induce secretion of inflammatory cytokines or VEGF-A. Interestingly, polysorbate-80-component of Beovu-but not polysorbate-20-in Lucentis-slightly, but significantly lowered the cell index, also associated with reduced claudin-1 expression. In summary, our results indicate that Beovu changes the behavior of retinal endothelial cells, thus providing an alternative "non-immunological" explanation for the most relevant of observed side effects.