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.
OBJECTIVE
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 VEGF(165) 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 VEGF(165)-induced loss of claudin-1 is regulated by protein kinase C (PKC) and indeed affects the barrier function of iBRECs.


METHODS
The effects of various PKC inhibitors on claudin-1 expression and cellular localization in iBRECs treated with VEGF(165) 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.


RESULTS
Activation of PKC led to decreased expression of claudin-1, which was blocked by inhibitors of PKCdelta. However, none of the PKC inhibitors significantly affected VEGF(165)-induced effects on cellular localization or expression of claudin-1. Also VEGF(165)-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.


CONCLUSIONS
In iBRECs, PKC isoforms are not crucially involved in the VEGF(165)-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 VEGF(165). The results also confirmed that claudin-1 is a highly relevant component of functional tight junctions in retinal endothelial cells.
Deissler
Heidrun L
HL
Department of Ophthalmology, University of Ulm Medical School, Ulm, Germany. heidrun.deissler@uniklinik-ulm.de
Deissler
Helmut
H
Lang
Gabriele E
GE
D000818:Animals; D015153:Blotting, Western; D002199:Capillary Permeability; D002417:Cattle; D002452:Cell Count; D002478:Cells, Cultured; D062445:Claudin-1; D017097:Electric Impedance; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012171:Retinal Vessels / Q000166:cytology / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*
C568108:CLDN1 protein, human; D062445:Claudin-1; D008565:Membrane Proteins; D047428:Protein Kinase Inhibitors; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate