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I. Yamanaka, Y. Hata, K. Fujisawa, T. Ishibashi; Molecular Mechanism of VEGF-Induced Hyperpermeability in Vascular Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):347.
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Purpose: To ascertain the molecular mechanism by which VEGF regulates vascular permeability. Methods: After human umbilical vein endothelial cells (HUVECs) were treated with VEGF, the cells were harvested at various time points. With antibodies specific for tight junctions (TJs), we performed Western blot analysis of the cell lysates. For phosphorylation analysis, occludin (Oc) or zonula occludens-2 (ZO-2) was immunoprecipitated and then we performed Western blot analysis of the immunoprecipitated materials with anti-phosphoserine or anti-phosphotyrosine antibody. To examine the changes in the localization of TJs, we immunostained the cells with antibodies to TJs. We also stained F-actin with FITC-phalloidin. Results: Oc was detected as two distinct bands at 62 and 64 kDa. VEGF treatment caused an increase in the intensity of 64-kDa band with a concomitant decrease in the intensity of 62-kDa band. Alkaline phosphatase treatment of the immunoprecipitated Oc indicated that 64-kDa band represented the phosphorylation form of Oc. Phosphorylation analysis of immunoprecipitated materials demonstrated that VEGF induced serine phosphorylation of Oc and tyrosine phosphorylation of ZO-1. Immunohistochemistry showed that in stimulated cells Oc, claudin-5 and ZO-1 slightly diminished at cell-cell borders and F-actin disappeared from cell contacts. Conclusions: VEGF induces the phosphorylation of TJs and disrupts the linkage between TJs and actin cytoskelton, suggesting that VEGF may cause conformational changes of TJ architecture to regulate permeability in vascular endothelial cells.
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