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H. L. Deissler, G. E. Lang; Ranibizumab but Not Protein Kinase C Inhibitors Reverted Long-Term Effects of VEGF165 on Tight Junction Protein Claudin-1. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5363.
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Elevated permeability of retinal endothelial cells (REC) induced by VEGF165 is most likely the reason for the break-down of the blood-retina-barrier leading to diabetic macular edema. Tight junction protein complexes are involved in the control of the paracellular signaling pathway of REC. We have recently shown that incubation with VEGF165 for upto 2 d induces delocalisation and loss of expression of the tight junction protein claudin-1 in immortalized bovine REC (iBREC) and that these effects could be completely reverted by the VEGF-inhbitor ranibizumab within 24 h. We now investigated whether VEGF-induced effects on claudin-1 are regulated by protein kinase C (PKC).
We studied the long-term effect of VEGF165 on the cellular localization and expression of claudin-1 in iBREC by immunofluorescence staining and Western-blot analyses in the presence and absence of several PKC inhibitors. Influence of VEGF and inhbitors on permeability of iBREC was measured by TER.
Treatment of iBREC with VEGF165 for 2 days resulted in an elevated permeability of iBREC, accompanied by delocalisation of claudin-1 from the plasma membrane as well as by its reduced expression. Pretreatment of iBREC with inhibitors for various PKC isoforms did not inhibit these effects of VEGF165 on claudin-1's cellular localization or expression. Similar results were obtained when PKC-inhibitors werde added after 2d-treatment of the cells with VEGF165. VEGF-induced permeability changes were also not inhibited by pretreatment with the general PKC-inhibitor GF109203X and claudin-1 was not localized in the plasma membrane of these cells whereas claudin-3, claudin-5 and ZO-1 were. In contrast, ranibizumab not only completely reverted VEGF165-induced delocalization and loss of expression of claudin-1, but also normalized permeability of VEGF165-treated iBREC within 24 h. All PKC inhibitors tested did not influence claudin-1 expression. However, activation of PKC by treatment with the phorbol-ester PMA led to elevated permeability of the cells as well as to delocalisation of claudin-1 and its decline in expression; these effects could be reverted by GF109203X.
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