Abstract
Purpose :
VEGFA-binding single-chain variable fragment brolucizumab is intravitreally injected to successfully treat retinal diseases associated with deregulated expression of VEGFA, but severe adverse effects, i.e. retinal vasculitis, have been reported. Using the well-established in vitro-model of immortalized bovine REC (iBREC) we studied brolucizumab’s effects on unchallenged or VEGFA-exposed iBREC.
Methods :
Confluent iBREC were exposed to PBSd or VEGFA165 (final concentration: 50ng/ml) for 1d before brolucizumab (Beovu®, final concentration of 1mg/ml reached by intravitreal injection) was added for up to 5d. As a measure of barrier function, we continuously determined the cell index (CI) of iBREC cultivated on gold electrodes. Expression or subcellular localization of tight-junction proteins claudin-1 or claudin-5 or of adhesion proteins VEcadherin or CD9 were assessed. Secretion of VEGFA, IL-6, IL-8 or TNFα by iBREC was determined by ELISA.
Results :
VEGFA treatment strongly reduced CI within 12h and persistent for up to 6d accompanied by the loss of claudin-1 and (subtle) changes of claudin-5’s and CD9’s subcellular localization; VEcadherin was not affected. Beovu reverted these disturbances within 1d, but 5d after its addition, the CI started to decline again although claudin-1 was still strongly expressed. VEGFA was not detected by competitive ELISA in the cell culture supernatant of unchallenged iBREC or those exposed to the growth factor and Beovu, and less VEGFA was then internalized by the cells. None of the inflammatory relevant cytokines studied was detected in the culture supernatant of iBREC at 2h or 30h after Beovu’s addition to unchallenged or VEGFA-exposed iBREC. When added to a confluent monolayer formed by iBREC, Beovu induced a transient decline of the CI between 2h and 6h associated with subtle changes of CD9’s subcellular localization.
Conclusions :
As expected, Beovu strongly counteracts VEGFA-induced dysfunction of the barrier formed by iBREC. However, Beovu might interfere with the adhesion of unchallenged iBREC to the extracellular matrix.
This is a 2021 ARVO Annual Meeting abstract.