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J. Yang, E. J. Duh, R. B. Caldwell, M. A. Behzadian; PEDF Blockade of VEGF-induced Permeability Involves the Inhibition of p38 MAP Kinase and ERK1/2. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5385.
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To determine the role of p38 MAP kinase and ERK in PEDF’s inhibitory effect on the VEGF-induced vascular permeability. Two subtypes of MAP kinase, p38 MAP kinase and ERK (extracelluar signal-regulated protein kinase), regulate critical steps in the angiogenic process, including endothelial cell proliferation, migration and survival. Activation of p38 and ERK is induced by VEGF or high glucose and in retinas of diabetic animal models. We have previously shown that VEGF-induced endothelial-cell permeability is mediated by transcriptional activation of beta-catenin and uPAR (urokinase plaminogen activator receptor) gene expression. Here, we present data showing that p38 and ERK1/2 act upstream of GSK/beta-catenin and that inhibition of the MAP kinase by PEDF blocks VEGF-induced uPAR expression and endothelial cell permeability.
Bovine retinal endothelial (BRE) cells were treated with VEGF, PEDF and specific kinase inhibitors and processed for analysis of paracellular permeability by measuring transcellular electric resistance (TER) and tracer flux. Western blotting and cell fractionation were used to analyze treatment effects on phosphorylation of VEGF receptor 2 (VEGFR-2), GSK and MAP kinases and cellular distribution of beta-catenin. Quantitative real-time PCR was used to quantify uPAR expression.
PEDF did not block the VEGF-induced phosphorylation of VEGFR-2. However, VEGF-induced phosphorylation of GSK, p38 MAP kinase and ERK was blocked by PEDF and by inhibitors of p38 and MEK. PEDF also blocked the phosphorylation of MAPKAPK-2, a p38 specific substrate. VEGF-induced beta-catenin cytosolic accumulation and nuclear translocation were blocked by PEDF as well as by inhibitors of p38 and ERK. Moreover, blockade of p38 and ERK prevented VEGF-induced uPAR expression. Finally, the VEGF-induced permeability increase was also blocked both by PEDF and by inhibitors of p38 and ERK.
PEDF prevents VEGF-induced permeability by a process involving blockade of MAP kinase-mediated phosphorylation/deactivation of GSK. In the active form, GSK facilitates ubiquitination/degradation of beta-catenin, preventing transcriptional activity of beta-catenin and expression of uPAR. Understanding the mechanism of PEDF’s anti-permeability action at the intracellular signaling level is important for developing new PEDF-based targets for treatment of retinal disease.
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