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L. Wu, J. Cai, M. B. Grant, M. E. Boulton; A Regulatory Role for -Secretase in VEGF-Induced Retinal Vascular Permeability via Phosphorylation of the VE-Cadherin Complex. Invest. Ophthalmol. Vis. Sci. 2010;51(13):67.
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We have previously shown that γ-secretase controls vascular permeability, in part, via regulating the binding of VEGFR-1 to the VE-cadherin complex. The aim of this study was to determine if γ-secretase can regulate the phosphorylation state of the VE-cadherin complex.
Bovine retinal microvascular endothelial cells (BRMECs) were treated with VEGF, and/or PEDF (each at 100ng/ml) in the presence or absence of γ-secretase inhibitor for up to 12 hours. Protein samples were extracted from the different subcellular fractions and immunoprecipitated with an antibody against tyrosine phosphorylation (PY20). Total tyrosine phosphorylation of VE-cadherin and β-catenin was analyzed by Western blot. The proteins fractions were also subjected to Western blot using antibodies against VE-cadherin tyrosine phosphorylation sites (pY658 or pY731). In order to knockdown γ-secretase, BRMECs were transfected with siRNA against presenilin 1(PS1) or nicastrin (NTC) and the change in endothelial permeability after the treatment with VEGF and/or PEDF was determined.
VEGF-induced a rapid increase in tyrosine phosphorylation of VE-cadherin in the membrane fraction which could be prevented by PEDF. The effect of PEDF was reversed by addition of a γ-secretase inhibitor. This was confirmed by Western blot using antibodies against VE-cadherin pY658 and pY731. However, cytosolic and nuclear VE-cadherin was not phosphorylated at pY658 and pY731 even though immunoprecitation with PY20 and subsequent Western blot for VE-cadherin showed strong tyrosine phosphorylation of VE-cadherin within both the fractions. The total tyrosine phosphorylation of β-catenin showed a very similar pattern to that of VE-cadherin upon treatment with VEGF, PEDF and γ-secretase inhibitor. siRNA knockdown of PS1 and NTC was able to reduce the inhibitory effect of PEDF on VEGF-induced permeability of endothelial cells.
This study demonstrates that PEDF can block VEGF-induced permeability via γ-secretase modulating the tyrosine phosphoryation of the membrane bound VE-cadherin complex further supporting the regulatory role of γ-secretase in angiogenesis.
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