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G. E. Lang, H. L. Deissler; Composition of Tight Junction Complexes of Retinal Endothelial Cells in the Presence of VEGF165 and Ranibizumab. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5367.
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We have recently shown that long-term treatment of retinal endothelial cells (REC) with VEGF165 reduces the expression of the tight junction protein claudin-1. This effect was reverted by the VEGF-inhibitor ranibizumab within 24 h. Proteins of tight junction protein complexes are localised in the plasma membrane and are involved in the control of the paracellular signaling pathway of REC. VEGF165-induced changes might be responsible for the break-down of the blood-retina-barrier observed in diabetic retinopathy. Here we studied the protein composition of tight junctions in immortalised bovine REC (iBREC) in the presence or absence of VEGF165 and its inhibitor.
iBREC were incubated with VEGF165 (and ranibizumab) for 2 d and the composition tight junction complexes was studied by double-immunofluorescence staining and (co)-immunoprecipitation.
Double-immunofluoresence staining of iBREC revealed strong co-localisation for claudin-5 and ZO-1, claudin-5 and claudin-3, occludin and ZO-1 or claudin-5. Only part (<10%) of claudin-1 was co-localised with claudin-5 or ZO-1. In contrast, none of these proteins co-localised with the adherens junction protein VECad. After addition of VEGF165 for 2 days, claudin-3 and claudin-5 or ZO-1, as well as ZO-1 and claudin-5 were still co-localised in the plasma membrane, whereas no co-staining was found for claudin-1 and claudin-5, or occludin and claudin-5. After addition of ranibizumab, claudin-1 was again present in the plasma membrane but was again only partly co-localised with claudin-5. Claudin-1 or claudin-5 could be precipitated from the cell lysate using specific antibodies, whereas the other antibodies were not suitable for immunoprecipitation. Only claudin-5 but not claudin-1 was precipitated from cells treated with VEGF165 whereas both proteins could be precipitated form iBREC treated with VEGF165 and ranibizumab. In addition, only claudin-3 was detected in the preciptate of the claudin-5-antibody, indicating that these two proteins can indeed be found in the same complex. None of the tight junction proteins tested was found in the precipitate of the claudin-1-antibody.
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