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G. E. Lang, G. K. Lang, H. L. Deissler; Macugen Induces Relocation of Tight Junction Proteins ZO-1 and Occludin After Treatment of iBREC With VEGF165. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1357.
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VEGF signalling is deregulated in diabetic retinopathy (DR) and is therefore a promising target for therapeutical intervention. Modified RNA-oligonucleotides (VEGF-aptamers) which inhibit the interaction of VEGF with its receptors are currently used to treat AMD. Tight junction proteins ZO-1 and occludin, usually detected in the plasma membrane of microvascular endothelial cells of the retina (REC), are relocated to the cytoplasm after treatment with VEGF165 likely leading to the breakdown of the blood-retina barrier. We therefore studied whether the VEGF aptamer Macugen (Pegaptanib) can revert VEGF induced delocalization of tight junction proteins in immortalized microvascular endothelial cells of the bovine retina (iBREC) in vitro.
The protein composition of tight junctions in iBREC was studied by immunofluorescence staining in the presence and absence of VEGF165 and/or Macugen.
ZO-1, occludin and claudin-5 are strongly expressed at the plasma membrane in confluent iBREC, but are located in the cytoplasm in non-confluent cells. Low but specific expression at the plasma membrane of claudin-1 and claudin-3 was also detected. In the presence of 50 ng/ml VEGF165, ZO-1 and occludin were found in the cytoplasm after 1 to 2 days, whereas claudin-1, -3 or -5 were not influenced. However, after addition of 33 µg/ml Macugen for 24 h to VEGF165-treated iBREC, ZO-1 and occludin were again strongly expressed in the plasma membrane. In contrast, the plasma membrane localisation of the EC specific adherens junction protein VECadherin remained stable in the presence of VEGF165 and/or Macugen.
Macugen reverted VEGF165-induced delocalization of tight junction proteins ZO-1 and occludin in iBREC, confirming an important role of tight junction proteins in the mechanism of Macugen’s action on endothelial cells.
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