Wirkung von VEGF₁₆₅ und dem VEGF-Aptamer Pegaptanib (Macugen®) auf die Zusammensetzung der Tight Junctions mikrovaskulärer Endothelzellen aus der Retina

Zusammenfassung

Hintergrund: Die von VEGF abhängigen Signaltransduktionswege sind bei der diabetischen Retinopathie dereguliert. Daher werden VEGF-Inhibitoren, wie das modifizierte RNA-Oligonukleotid Pegaptanib (VEGF-Aptamer, Macugen®), das die Interaktion von VEGF₁₆₅ mit seinem Rezeptor beeinflusst, derzeit als Therapeutika für die Behandlung der diabetischen Retinopathie diskutiert. VEGF₁₆₅ stimuliert nicht nur Proliferation und Migration von Endothelzellen, sondern kann auch die Lokalisation des an der Bildung von Tight Junctions in retinalen Endothelzellen beteiligten Proteins Occludin verändern, wodurch es zum Zusammenbruch der Blut-Retina-Schranke kommen könnte. Material und Methoden: Um den Wirkungsmechanismus von VEGF₁₆₅ und dem VEGF-Aptamer Pegaptanib besser verstehen zu können, haben wir den Einfluss von VEGF₁₆₅ und/oder Pegaptanib auf die Proteinzusammensetzung der Tight Junctions in immortalisierten Endothelzellen aus der Rinderretina (iBREC) durch spezifische Immunfluoreszenzfärbungen untersucht. Ergebnisse: Die Tight-Junction-Proteine ZO-1, Occludin und Claudin-5 werden in konfluenten iBREC stark in der Plasmamembran exprimiert, während sie in subkonfluenten Zellen intrazellulär vorkommen. Nach 1- bis 2-tägiger Inkubation mit VEGF₁₆₅ (50 ng/ml), war Occludin nicht mehr in der Plasmamembran, sondern intrazellulär lokalisiert. Dagegen wurden Claudin-5 nicht und ZO-1 nur schwach beeinflusst. Durch Zugabe von 33 μg/ml Pegaptanib waren alle untersuchten Tight-Junction-Proteine nach ca. 24 h wieder an der Plasmamembran lokalisiert. Schlussfolgerungen: Diese In-vitro-Untersuchungen bestätigen eine wichtige Rolle von Tight-Junction-Proteinen für den Wirkungsmechanismus von VEGF₁₆₅ und Pegaptanib in retinalen Endothelzellen.

Abstract

Background: VEGF signalling is deregulated in diabetic retinopathy. Therefore, VEGF inhibitors like the modified RNA-oligonucleotide pegaptanib (VEGF aptamer, Macugen®) which inhibits the interaction of VEGF₁₆₅ with its receptors, are currently being discussed as therapeutic options in the treatment of diabetic retinopathy. VEGF₁₆₅ does not only stimulate the proliferation and migration of endothelial cells but also induces delocalization of occludin which is part of the so-called tight junctions of endothelial cells likely associated with the breakdown of the blood-retina barrier. Methods and Material: To further investigate the mechanisms of action of VEGF and its inhibitor, we studied the influence of VEGF₁₆₅ and/or pegaptanib on the protein composition of tight junctions in immortalised endothelial cells of the bovine retina (iBREC) by immunofluorescence staining. Results: The tight junction proteins ZO-1, occludin and claudin-5 are strongly expressed at the plasma membrane in confluent iBREC, but are located in the cytoplasm in non-confluent cells. In the presence of 50 ng/ml VEGF₁₆₅, occludin was found in the cytoplasm after 1 to 2 days, whereas claudin-5 was not and ZO-1 was only weakly influenced. However, after addition of 33 μg/ml pegaptanib for 24 h to VEGF₁₆₅-treated iBREC, all tight junction proteins tested were again strongly expressed in the plasma membrane. Conclusion: These results confirm an important role of tight junction proteins in the mechanisms of action of VEGF and pegaptanib on endothelial cells.

diabetische Retinopathie - retinale mikrovaskuläre Endothelzellen - VEGF - VEGF-Inhibitoren - Pegaptanib

diabetic retinopathy - retinal microvascular endothelial cells - VEGF - VEGF inhibitors - pegaptanib

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