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A. Stahl, L. Paschek, G. Martin, H. T. Agostini; VEGF-Inhibition versus Disruption of Intracellular Signalling in an in vitro Assay of RPE-Induced Endothelial Cell Sprouting. Invest. Ophthalmol. Vis. Sci. 2008;49(13):854.
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© ARVO (1962-2015); The Authors (2016-present)
Formation of choroidal neovascularisation (CNV) in late stage age related macular degeneration (AMD) causes rapid vision loss in affected patients. Histologic data suggest retinal pigment epithelium (RPE) being involved in CNV formation. This study uses a co-culture assay of RPE and endothelial cells (ECs) to quantify the effect of different anti-angiogenic strategies. Clinically established anti-VEGF treatment is compared to inhibitors of intracellular signal transduction for their effect on RPE-induced EC-sprouting.
Spheroidal EC-clusters (HUVECs or HMVECs) in a 3D collagen matrix serve as focal starting points for in vitro angiogenesis. RPE monolayers seeded below the collagen gel provide a constant supply of angiogenic growth factors. This setting mimicks to a certain extent the morphological in vivo situation of choroidal capillaries in close proximity to an RPE monolayer. Different RPE isolates are used for induction of EC sprouting: a) ARPE cell line, b) normal human RPE (hRPE) and c) human RPE isolated from surgically extracted CNV-membranes (CNV-RPE). The anti-angiogenic effect of anti-VEGF and anti-FGF2 antibodies is compared to inhibitors of cell signalling (PTK787/ZK 222584, LY294002 and Rapamycin).
All RPE monolayers induced EC spheroid sprouting. Control monolayers of HeLa cells in contrast did not induce EC sprouting. Sprouting induced by hRPE monolayers could be blocked by VEGF-antibodies (bevacizumab, Avastin®) or receptor tyrosine kinase inhibitors (PTK787/ZK 222584). In CNV-RPE groups, anti-VEGF treatment alone showed only limited effect. Combination angiostatic therapy (anti-VEGF and anti-FGF2) inhibited EC-sprouting effectively in only one CNV-RPE group. EC-sprouting in all RPE-groups, however, could be blocked by targeting the signalling molecules PI3K or mTOR. Part of this inhibitory effect could be attributed to reduced VEGF-production in RPE cells.
VEGF-dependent as well as VEGF-independent pathways play a functional role in CNV-RPE-induced EC sprouting in vitro. The composition of the angiogenic factors involved differs between individual CNV-RPE membranes. Targeting downstream angiogenic signalling inhibits EC-sprouting irrespective of the RPE source by affecting both EC sprouting as well as growth factor expression in RPE cells. Protein kinase inhibitors may therefore prove beneficial for CNV patients who are not responding sufficiently to sole anti-VEGF treatment.
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