Inhibition of Single Routes of Intracellular Signaling is Not Sufficient to Neutralize the Biphasic Disturbance of a Retinal Endothelial Cell Barrier Induced by VEGF-A165

Abstract

Background/Aims: Hallmark of diabetic macular edema is the enhanced permeability of retinal endothelial cells (REC) induced by vascular endothelial growth factor (VEGF-A₁₆₅), which acts through activating specific receptors. To improve the predictability of inhibitors' potentials to block harmful effects of VEGF-A₁₆₅, we investigated if its signaling pathways triggered in REC are redundant. Methods: Immortalized bovine REC monolayers were treated with inhibitors specific for various protein kinases in combination with VEGF-A₁₆₅. Permeability was monitored continuously by measurements of the cell index (CI) to reveal even subtle and transient changes. Expression of tight junction (TJ) proteins was determined as additional indicator of barrier stability. Results: After a sharp but transient CI drop caused by VEGF-A₁₆₅ early after its addition, further exposure resulted in a continuous CI decline over several days associated with loss of TJ protein claudin-1. Both phases were blocked by inhibition of VEGF receptor 2. Tested inhibitors of intracellular kinases had a limited or no effect, or were efficient only in certain phases of exposure to VEGF-A₁₆₅, e.g. inhibiting protein kinase C only prevented the early response. High concentrations of some inhibitors even resulted in VEGF-independent barrier destabilization. Conclusions: Specific kinase inhibitors differently affect VEGF-A₁₆₅-triggered processes in distinct phases of its action. VEGF-A₁₆₅-initiated signaling is redundant and blocking of key proteins of single pathways is not sufficient to suppress REC barrier breakdown.

Imatinib Ameliorated Retinal Neovascularization by Suppressing PDGFR-α and PDGFR-β

“…In addition, pathological angiogenesis comprises abnormal proliferation and migration of ECs, pericytes (PCs), and smooth muscle cells (SMCs). The latter two are not typical cellular targets of VEGF and show limited response to anti-VEGF therapy [6][7][8][9]. Therefore, novel anti-angiogenesis strategies that target PCs and SMCs are strongly desired.…”

Bradykinin signaling regulates solute permeability and cellular junction organization in lymphatic endothelial cells

“…Our observations in the HDMEC model possibly reflect this compensatory mechanism, in which BK induces lymphangiogenesis through VEGF‐C to resolve the edema. However, it is also known that an excessive amount of VEGF triggers the abnormal growth and leakage of blood vessels . Additional experiments are needed to clarify whether these findings predispose a lymphatic breakdown or whether they will improve AE resolution in the long‐term.…”

“…However, it is also known that an excessive amount of VEGF triggers the abnormal growth and leakage of blood vessels. 75,76 Additional experiments are needed to clarify whether these findings predispose a lymphatic breakdown or whether they will improve AE resolution in the long-term.…”

Fate of the Fc fusion protein aflibercept in retinal endothelial cells: competition of recycling and degradation

“…Pericytes or other cells expressing α-smooth muscle actin are not present in iBREC cultures [ 21 ]. To ensure authenticity and optimal conditions of iBREC in the experiments, we routinely recorded their characteristic proliferation profile by electric cell-substrate impedance measurements using the microelectronic biosensor system for cell-based assays xCELLigence RTCA DP (Acea, OLS, Bremen, Germany) [ 27 ]. All experiments—repeated at least twice—were performed with confluent monolayers of iBREC formed after cultivation for 4 days.…”

“…To assess the effects of the protease inhibitors MG-101 or MG-132 (Selleckchem, Absource Diagnostics, Munich, Germany) on barrier stability, confluent iBREC grown on gold electrodes (E-Plates 16 PET, Acea) were treated with these substances at concentrations ranging from 20 nM to 5 μM or to 20 μM, respectively [ 27 – 30 ]. The cell index as a measure of permeability was recorded every 5 min for at least 24 h [ 27 ].…”