[Basic in vitro studies on VEGF inhibition with aflibercept: similarities and differences to other VEGF-binding therapeutic proteins]. / Grundlegende In-vitro-Untersuchungen zur VEGF-Inhibition mit Aflibercept: Gemeinsamkeiten und Unterschiede zu anderen VEGF-bindenden therapeutischen Proteinen.

Patients suffering from various retinal diseases benefit from therapies directed against the vascular endothelial growth factor (VEGF). Aflibercept (Eylea) is another VEGF-binding protein available for intravitreal injection, in addition to the antibody bevacizumab (Avastin) and the F(ab) fragment ranibizumab (Lucentis). Aflibercept's distinct structure and broader binding specificity may have clinically relevant consequences, which is supported by basic in vitro studies and observations in animal eyes. All pathological processes involving neovascularisation are driven by the dominant action of VEGF, but other factors including placenta growth factor (PlGF), a mitogenic protein for retinal endothelial cells, potentially modulate its effects. Aflibercept is an inhibitor of both VEGF and PlGF and therefore may have superior therapeutic effects in some cases. However, whether or not aflibercept's broader binding specificity or different affinities for the different VEGF-binding proteins to VEGF result in substantially diverse therapeutic efficiencies has not yet been clarified. In vitro studies confirm that aflibercept efficiently prevents or normalises VEGF-stimulation of retinal cells and disturbance of their barrier function. These experiments also show that aflibercept is taken up by important retinal cell types and affects their normal function, i.e., migration of endothelial cells and phagocytosis of pigment epithelial cells. In accordance with a role of the Fc domains of aflibercept and bevacizumab, substantial amounts of both proteins are internalised, whereas only a small portion of ranibizumab enters the cells. Internalisation and storage by ocular cells, also observed in vivo after intravitreal injection into eyes of monkeys, may result in not yet recognised side effects during long-term treatment of patients with certain VEGF-binding proteins.

[Treatment of diabetic macular oedema with the VEGF inhibitors ranibizumab and bevacizumab: conclusions from basic in vitro studies]. / Therapie des diabetischen Makulaödems mit den VEGF-Inhibitoren Ranibizumab und Bevacizumab: Schlüsse aus grundlegenden In-vitro-Untersuchungen.

Diabetic macular oedema (DMO) which may occur at all stages of diabetic retinopathy (DR) is a severe vision-threatening complication. In most cases, laser treatment does not improve visual acuity. Therefore research in ophthalmology focuses on the improvement of the prognosis of DMO patients with a drug-based DMO therapy. Vascular endothelial growth factor (VEGF) is considered the most important therapeutic target because this growth factor also is the most potent permeability factor affecting the inner retinal barrier formed by endothelial cells (ECs). Compared to its angiogenic stimulation of proliferation and migration of ECs, effects of VEGF on permeability have not been studied in all details. In vitro investigations on the behaviour of primary or immortalised retinal endothelial cells confirmed the key role of VEGF in the regulation of the permeability of the inner retinal barrier. Despite the presence of a variety of other factors found to be elevated in DR, a VEGF-disrupted barrier can be completely restored with the VEGF-inhibiting ranibizumab (Lucentis®) and bevacizumab (Avastin®) when applied at clinically achievable concentrations. The antibody bevacizumab, but not the antibody fragment ranibizumab, accumulates in both retinal EC and pigment epithelial cells during prolonged treatment. This observation might be relevant because patients are often treated for several years and additional long-term side effects may be recognised in the future.

[Effect of VEGF165 and the VEGF aptamer pegaptanib (Macugen) on the protein composition of tight junctions in microvascular endothelial cells of the retina]. / Wirkung von VEGF165 und dem VEGF-Aptamer Pegaptanib (Macugen) auf die Zusammensetzung der Tight Junctions mikrovaskulärer Endothelzellen aus der Retina.

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 VEGF(165) with its receptors, are currently being discussed as therapeutic options in the treatment of diabetic retinopathy. VEGF(165) 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 VEGF(165) 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 VEGF(165), 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 microg/ml pegaptanib for 24 h to VEGF(165)-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.

[In vitro studies on the mechanism of action of VEGF and its inhibitors]. / In-vitro-Untersuchungen zum Wirkungsmechanismus von VEGF und seinen Inhibitoren.

VEGF expression and signalling are deregulated in diabetic retinopathy. Cellular processes like migration and proliferation as well as control of the permeability of the endothelium by VEGF are regulated as a consequence of its binding to the VEGF receptor 2. Proteins forming tight junctions between microvascular endothelial cells of the retina are delocated to the cytoplasm after treatment with VEGF(165), likely leading to the breakdown of the blood-retina barrier. VEGF-inhibitors such as a VEGF-aptamer (modified RNA-oligonucleotide; pegaptanib) or specific antibodies/antibody fragments (bevacizumab, ranibzumab) which directly interfere with the interaction of VEGF with its receptors are considered to be promising novel therapeutics to treat diabetic retinopathy and age-related macular degeneration. In vitro studies using microvascular endothelial cells will help to clarify the mechanisms of action of VEGF and its inhibitors. In particular, the influence of VEGF isoforms and the inhibitor ranibizumab on the proliferation and migration of bovine retinal microvascular endothelial cells was studied, as well as the rearrangement of tight-junction proteins after treatment of the cells with VEGF(165) and specific inhibitors. In addition to a review of recent publications in the field, primary data from our own studies are presented in this article.