Inhibition of Protein Kinase C Is not Sufficient to Prevent or Revert Effects of VEGF165 on Tight Junction Protein Claudin-1 and Permeability in Microvascular Retinal Endothelial Cells
Heidrun L Deissler 1*,
Helmut Deissler 2,
and
Gabriele E. Lang 3
1 Department of Ophthalmology, University of Ulm Medical School, Ulm, Germany
2 Department of Obstetrics and Gynecology, University of Ulm Medical School, Ulm, Germany
3 Ophthalmology, University Eye Hospital, Ulm, Germany; Department of Ophthalmology, University of Ulm Medical School, Ulm, Germany
* To whom correspondence should be addressed. E-mail: heidrun.deissler{at}uniklinik-ulm.de.
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Abstract |
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Purpose: Pathogenesis of diabetic macular edema is driven by deregulated expression of VEGF. Studying long-term exposure of immortalized bovine retinal endothelial cells (iBREC) to VEGF165 clearly confirmed the role of the tight junction protein claudin-1 which almost completely disappeared within 24 h, an effect that could be completely reverted by addition of the VEGF-binding Fab fragment ranibizumab. In this study, we investigated if this VEGF165-induced loss of claudin-1 is regulated by protein kinase C (PKC) and indeed affects the barrier function of iBREC.
Methods: The effects of various PKC inhibitors on claudin-1 expression and cellular localization in iBREC treated with VEGF165 for up to 2 days were studied by Western blot analyses and immunofluorescence microscopy. Permeability of cell layers was determined by transendothelial electrical resistance measurements.
Results: Activation of PKC led to decreased expression of claudin-1 which could be blocked by inhibitors of PKC 
. 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 reverted 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.
Conclusion: We showed that in iBREC PKC isoforms are not crucially involved in VEGF165-initiated signal transduction affecting permeability and expression of claudin-1. This is in contrast to published results concerning only short-term effects of VEGF165. We also confirmed that claudin-1 is a highly relevant component of functional tight junctions in retinal endothelial cells.
Key Words:
diabetic retinopathy, tight junction, immortalization, retinal endonthelial cell, protein kinase C, VEGF inhibitor
