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.

Schlüsselwörter

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

Key words

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

Literatur

• 1 Aiello L P, Avery R L, Arrigg P G. et al . Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders.  N Engl J Med. 1994;  331 1480-1487
• 2 Antonetti D A, Barber A J, Hollinger L A. et al . Vascular endothelial growth factor induces rapid phosphorylation of tight junction proteins occludin and zonula occludens 1.  J Biol Chem. 1999;  274 23463-23467
• 3 Bazzoni G. Endothelial tight junctions: permeable barriers of the vessel wall.  Thromb Haemost. 2006;  95 36-42
• 4 Behzadian M A, Windsor L J, Ghaly N. et al . VEGF-induced paracellular permeability in cultured endothelial cells involves urokinase and its receptor.  FASEB J. 2003;  17 752-754
• 5 Boulton M, Foreman D, Williams G. et al . VEGF localisation in diabetic retinopathy.  Br J Ophthalmol. 1998;  82 561-568
• 6 Caldwell R B, Bartoli M, Behzadian M A. et al . Vascular endothelial growth factor and diabetic retinopathy: pathophysiological mechanisms and treatment perspectives.  Diabetes Metab Res Rev. 2003;  19 442-455
• 7 Caldwell R B, Bartoli M, Behzadian M A. et al . Vascular endothelial growth factor and diabetic retinopathy: role of oxidative stress.  Curr Drug Targets. 2005;  6 511-524
• 8 Deissler H, Deissler H, Lang G K. et al . Generation and characterization of iBREC: novel hTERT-immortalized bovine retinal endothelial cells.  Int J Mol Med. 2005;  16 65-70
• 9 Deissler H, Deissler H, Lang G K. et al . TGFβ induces transdifferentiation of iBREC to &alphaSMA-expressing cells.  Int J Mol Med. 2006;  18 577-582
• 10 Deissler H, Kuhn E M, Lang G E. et al . Tetraspanin CD 9 is involved in the migration of retinal microvascular endothelial cells.  Int J Mol Med. 2007;  20 643-652
• 11 Deissler H, Deissler H, Lang S. et al . VEGF-induced effects on proliferation, migration and tight junctions are restored by ranibizumab (Lucentis®) in microvascular retinal endothelial cells.  Br J Ophthalmol. 2008;  92 839-843
• 12 Ferrara N. Vascular endothelial growth factor: basic science and clinical progress.  Endocr Rev. 2004;  25 581-611
• 13 Ferrara N, Damico L, Shams N. et al . Development of ranibizumab, an anti-vascular endothelial growth factor antigen binding fragment, as therapy for neovascular age-related macular degeneration.  Retina. 2006;  26 859-870
• 14 Harhaj N S, Felinski E A, Wolpert E B. et al . VEGF activation of protein kinase C stimulates occludin phosphorylation and contributes to endothelial permeability.  Invest Ophthalmol Vis Sci. 2006;  47 5106-5115
• 15 Kowanetz M, Ferrara N. Vascular endothelial growth factor signaling pathways: therapeutic perspective.  Clin Cancer Res. 2006;  12 5018-5022
• 16 Lowe J, Araujo J, Yang J. et al . Ranibizumab inhibits multiple forms of biologically active vascular endothelial growth factor in vitro and in vivo.  Exp Eye Res. 2007;  85 425-430
• 17 Lutty G A, Mathews M K, Merges C. et al . Adenosine stimulates canine retinal microvascular endothelial cell migration and tube formation.  Curr Eye Res. 1998;  17 594-607
• 18 Malik R A, Li C, Aziz W. et al . Elevated plasma CD 105 and vitreous VEGF levels in diabetic retinopathy.  J Cell Mol Med. 2005;  9 692-697
• 19 Pan Q, Chathery Y, Wu Y. et al . Neuropilin-1 binds to VEGF121 and regulates endothelial cell migration and sprouting.  J Biol Chem. 2007;  282 24049-24056
• 20 Peters S, Julien S, Heiduschka P. et al . Antipermeability and antiproliferative effects of standard and frozen bevacizumab on choroidal endothelial cells.  Br J Ophthalmol. 2007;  91 827-831
• 21 Qaum T, Xu Q, Joussen A M. et al . VEGF-initiated blood-retinal barrier breakdown in early diabetes.  Invest Ophthalmol Vis Sci. 2001;  42 2408-2413
• 22 Raja T T, Grammas P. VEGF and VEGF receptor levels in retinal and brain-derived endlthelial cells.  Bioch Biophys Res Commun. 2002;  293 710-713
• 23 Sakakibara A, Furuse M, Saitou M. et al . Possible involvement of phosphorylation of occludin in tight junction formation.  J Cell Biol. 1997;  137 1393-1401
• 24 Spitzer M S, Wallenfels-Thilo B, Sierra A. et al . Antiproliferative and cytotoxic properties of bevacizumab on different ocular cells.  Br J Ophthalmol. 2006;  90 1316-1321
• 25 Spitzer M S, Yoeruek E, Sierra A. et al . Comparative antiproliferative and cytotoxic profile of bevacizumab (Avastin), pegaptanib (Macugen) and ranibizumab (Lucentis) on different ocular cells.  Graefe’s Arch Clin Exp Ophthalmol. 2007;  245 1837-1842
• 26 Suzuma K, Takagi H, Otani A. et al . Increased expression of KDR/Flk-1 (REGFR-2) in murine model of ischemia-induced retinal neovascularization.  Microvasc Res. 1998;  56 183-191
• 27 Tsukita S, Furuse M, Itoh M. Multifunctional strands in tight junctions.  Nat Rev Mol Cell Biol. 2001;  2 285-293
• 28 Vinores S A. Technology evaluation: pegaptanib, Eyetech/Pfizer.  Curr Opin Mol Ther. 2003;  5 673-679
• 29 Watanabe D, Suzuma K, Suzuma I. et al . Vitreous levels of angiopoietin 2 and vascular endothelial growth factor in patients with proliferative diabetic retinopathy.  Am J Ophthalmol. 2005;  139 476-481
• 30 Witmer A N, Blaauwgeers H G, Weich H A. et al . Altered expression patterns of VEGF receptors in human diabetic retina and in experimental VEGF induced retinopathy in monkey.  Invest Ophthalmol Vis Sci. 2002;  43 849-857
• 31 Wong V. Phosphorylation of occludin correlates with occludin localization and function at the tight junction.  Am J Physiol. 1997;  273 C1859-C1867
• 32 Wu L W, Mayo L D, Dunbar J D. et al . Utilization of distinct signaling pathways by receptor for vascular endothelial cell growth factor and other mitogens in the induction of endothelial cell proliferation.  J Biol Chem. 2000;  275 5096-5103
• 33 Xia P, Aiello L P, Ishii H. et al . Characterization of vascular endothelial growth factor’s effect on the activation of Protein Kinase C, its isoforms and endothelial cell growth.  J Clin Invest. 1996;  98 2018-2026