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S. Peters, I.A. Cree, P.G. Hykin, S.E. Moss; Angiopoietin–2 multiplies the permeability–enhancing effect of Vascular Endothelial Growth Factor on retinal endothelial cells . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1901.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:: Increased vascular permeability is the major problem in a diabetic retina. Yet, no standardized therapy is available to spare patients from laser treatment or to improve the outcome of the latter. Developing agents to prevent vascular leakage requires understanding of basic mechanisms and the cross talk of growth factors involved in neovascularization. The aim of our study was to investigate the cross talk between Angiopoietin–2 (Ang–2) and Vascular Endothelial Growth Factor (VEGF), acting on retinal endothelial cells. Methods: Retinal endothelial cells (PREC) were isolated from porcine eyes and seeded onto membranes of permeable insert systems. When monolayers were confluent, growth medium was replaced by assay medium (1% FCS). 24 hours later, PREC were treated with different concentrations of VEGF and/or Ang–2, corresponding to physiological concentrations found in human diabetic eyes: VEGF 10–20 ng/ml; Ang–2 4–15 ng/ml. Permeability of the PREC monolayer was assessed in certain time intervals using fluorescent dextrans with molecular weights of 4 and 70 kD. Results: Ang–2 added once in a concentration of 4 ng/ml or 15 ng/ml showed no increased permeability in PREC, 10–20 ng/ml VEGF added once only led to a mild, up to 50% increase in permeability, preferably 5 hours after treatment. Growth factors added with 15 ng/ml Ang–2 and 20 ng/ml VEGF every 2 hours over an 8–hour period, increased permeability of PREC significantly. The increase compared to untreated PREC was on average: a) 50% with Ang–2 only, preferably 5–6 hrs after treatment; b) 100% with VEGF only, from 3 hrs after treatment; c) up to 300% with VEGF and Ang–2 together, preferably 7–24 hrs after treatment. Whereas the 70 kD dextran was suitable to detect changes in permeability, the 4 kD dextran did not well reflect permeability changes in PREC.Conclusions: Permeability of a confluent porcine retinal endothelial cell monolayer is increased in the presence of VEGF and Ang–2. VEGF alone is twice as potent in interrupting the endothelial cell tight junctions as Ang–2 on its own. Both growth factors acting together, however, increase permeability three times as much as VEGF would do alone. The effect of single growth factors cannot simply be added to estimate the effect they will have in angiogenesis, because the cross talk between these factors may multiply their effect. A 70 kD dextran is more suitable to measure endothelial cell permeability than a 4 kD dextran.
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