Blocking of VEGF-A is not sufficient to completely revert its long-term effects on the barrier formed by retinal endothelial cells

Abstract

The VEGF-A-induced functional impairment of the barrier formed by retinal endothelial cells (REC) can be prevented and even - at least temporarily - reverted by trapping the growth factor in a complex with a VEGF-binding protein or by inhibiting the activity of the VEGF receptor 2 (VEGFR2). In an approach to emulate the clinically relevant situation of constant exposure to effectors, we investigated (1) whether prolonged exposure to VEGF-A₁₆₅ for up to six days results in a different type of disturbance of the barrier formed by immortalized bovine REC (iBREC) and (2) whether alterations of the barrier induced by VEGF-A₁₆₅ can indeed be sustainably reverted by subsequent treatment with the VEGF-A-binding proteins ranibizumab or brolucizumab. As a measure of barrier integrity, the cell index (CI) of iBREC cultivated on gold electrodes was monitored continuously. CI values declined shortly after addition of the growth factor and then remained low for more than six days over which considerable amounts of both extra- and intracellular VEGF-A were measured. Interestingly, the specific VEGFR2 inhibitor nintedanib normalized the lowered CI when added to iBREC pre-treated with VEGF-A₁₆₅ for one day, but failed to do so when cells had been exposed to the growth factor for six days. Expression of the tight junction (TJ) protein claudin-5 was unchanged early after addition of VEGF-A₁₆₅ but higher after prolonged treatment, whereas decreased amounts of the TJ-protein claudin-1 remained low, and increased expression of the plasmalemma vesicle-associated protein (PLVAP) remained high during further exposure. After two days, the characteristic even plasma membrane stainings of claudin-1 or claudin-5 appeared weaker or disordered, respectively. After six days the subcellular localization of claudin-5 was similar to that of control cells again, but claudin-1 remained relocated from the plasma membrane. To counteract these effects of VEGF-A₁₆₅, brolucizumab or ranibizumab was added after one day, resulting in recovery of the then lowered CI to normal values within a few hours. However, despite the VEGF antagonist being present, the CI declined again two days later to values that were just slightly higher than without VEGF inhibition during further assessment for several days. At this stage, neither the supernatants nor whole cell extracts from iBREC treated with VEGF-A₁₆₅ and its antagonists contained significant amounts of free VEGF-A. Treatment of VEGF-A₁₆₅-challenged iBREC with ranibizumab or brolucizumab normalized expression of claudin-1 and claudin-5, but not completely that of PLVAP. Interestingly, the characteristic VEGF-A₁₆₅-induced relocalization of claudin-1 from the plasma membrane was reverted within one day by any of the VEGF antagonists, but reappeared despite their presence after further exposure for several days. Taken together, barrier dysfunction induced by VEGF-A₁₆₅ results from deregulated para- and transcellular flow but the precise nature or magnitude of underlying changes on a molecular level clearly depend on the time of exposure, evolving into a stage of VEGF-A₁₆₅-independent barrier impairment. These findings also provide a plausible explanation for resistance to treatment with VEGF-A antagonists frequently observed in clinical practice.

Keywords: Aflibercept; Barrier stability; Brolucizumab; Cell adhesion; Paracellular flow; Plasmalemma vesicle-associated protein; Ranibizumab; Retinal endothelial cells; Tight junction; Transcellular flow; Transcytosis; VEGF-A.