Abstract

SO.08.04

In vitro studies on the mechanism of VEGF165 action and its inhibition

Deissler H. L., Lang G. E.
Universitätsklinikum Ulm, Augenklinik, Forschungslabor, Ulm

Objective: Expression and signalling of VEGF165 are deregulated in diabetic retinopathy (DR). Inhibitors of VEGF165 like modified RNA oligonucleotides (VEGF aptamers) and specific antibodies are promising agents to treat AMD or DR. Breakdown of the blood-retina-barrier induced by VEGF165 might be associated with delocalization of tight junction proteins. Based on these observations, we studied in vitro whether VEGF165-specific inhibitors can revert VEGF-induced delocalization of tight junction proteins in immortalized microvascular endothelial cells of the bovine retina (iBREC).
Methods: The homogenous cell line iBREC (microvascular endothelial cells of the bovine retina immortalized by ectopic expression of hTERT) were used as an in vitro model to study the protein composition of tight junctions by immunofluorescence staining and western blot analysis in the presence and absence of VEGF165 and/or its inhibitors.
Results: Expression of tight-junction proteins ZO-1 and occludin in iBREC was shown by western blot analysis. Both proteins and claudin-5 were strongly expressed in the plasma membranes of confluent iBREC in prescence of hydrocortisol. Only weak expression of claudin-1 and claudin-3 was detected in the plasma membrane whereas claudin-2 and claudin-4 were not expressed. After VEGF-treatment both ZO-1 and occludin were partly found in the cytoplasm and reversion of protein localization was achieved by addition of VEGF165 inhibitors, whereas claudin-5 and claudin-1 were not affected. Interestingly, when iBREC were cultivated in high glucose medium, intracellular localization of claudin-5 was occasionally observed after treatment with VEGF165 and this delocalization was reverted by addition of VEGF165 inhibitors.
Conclusions: iBREC proved to be a suitable in vitro-modell to study action of VEGF165 and its inhibitors. Delocalization of tight junction proteins ZO-1 and occludin was reverted by its inhibitors, supporting an important role of tight junction proteins in this process.

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