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Heidrun L. Deissler, Helmut Deissler, Gabriele E. Lang; Inhibition of Protein Kinase C Is Not Sufficient to Prevent or Reverse Effects of VEGF165 on Claudin-1 and Permeability in Microvascular Retinal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(1):535-542. doi: 10.1167/iovs.09-3917.
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Pathogenesis of diabetic macular edema is driven by deregulated expression of VEGF. A study of long-term exposure of immortalized bovine retinal endothelial cells (iBRECs) to VEGF165 clearly confirmed the role of the tight junction protein claudin-1, which almost completely disappeared within 24 hours, an effect that was completely reversed by addition of the VEGF-binding Fab fragment ranibizumab. This study was conducted to investigate whether the VEGF165-induced loss of claudin-1 is regulated by protein kinase C (PKC) and indeed affects the barrier function of iBRECs.
The effects of various PKC inhibitors on claudin-1 expression and cellular localization in iBRECs treated with VEGF165 for up to 2 days were studied by Western blot analyses and immunofluorescence microscopy. The permeability of the cell layers was determined by transendothelial electrical resistance measurements.
Activation of PKC led to decreased expression of claudin-1, which was blocked by inhibitors of PKCδ. However, none of the PKC inhibitors significantly affected VEGF165-induced effects on cellular localization or expression of claudin-1. Also VEGF165-induced higher permeability of iBREC layers could be reversed or prevented by ranibizumab but not by PKC inhibitors. In addition, low claudin-1 expression and its delocalization from the plasma membrane were significantly associated with elevated permeability.
In iBRECs, PKC isoforms are not crucially involved in the VEGF165-initiated signal transduction that affects permeability and expression of claudin-1. This finding is in contrast to published results concerning only short-term effects of VEGF165. The results also confirmed that claudin-1 is a highly relevant component of functional tight junctions in retinal endothelial cells.
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