Norrin Increases Vessel Integrity upon VEGF Induced Permeability

Abstract

Purpose: The purpose of this study was to examine the effect of norrin on VEGF induced vessel permeability. VEGF signaling is induced by ischemic retina and it reduces the cell-to-cell adherence of vessel endothelial cells. Norrin is a growth factor that is necessary for normal retinal vascular development and maintenance.

Methods: Confluent human retinal microvascular endothelial cells (HRMEC) were treated with VEGF (or TGF-β) alone or in combination with Norrin. After 24 hours, changes in morphology and junction protein localization were assessed by immunstaining with antibodies to an adherens junction protein, VE-cadherin (VE-Cad), or a tight junction protein, claudin-5 (Cln5). To determine expression levels, RT-PCR was performed after the above treatments using a Cln5 TaqMan assay. To correlate the disruption in cell junction proteins with an increase in permeability, norrin was injected into the vitreous of OIR mice at P14. At P17, the mice were injected (IP) with Evans Blue dye and sacrificed two hours later. The retinas were immediately fixed, flat mounted and examined by fluorescence microscopy to visualize dye leakage from vessels.

Results: Treatment with VEGF and TGF-β had a similar effect upon cell morphology and junction protein localization. The cells became elongated and there was a decrease in Cln5 and VE-Cad localization at the cell borders. Cells treated in combination with Norrin, however, maintained their cobblestone appearance and had more junction protein staining. Expression assays revealed an increase in Cln5 mRNA in the cells treated in combination with norrin compared to VEGF or TGF-β treatment alone. Finally, Norrin seemed to reduce the amount of permeability caused by the OIR model. Norrin treated eyes generally had leakage only at the terminal endothelial cell buds whereas control OIR eyes had a more diffuse leakage pattern along the length of vessels.

Conclusions: This investigation provides evidence that Norrin overcomes the loss of endothelial cell-to-cell junction proteins that occurs upon VEGF stimulation and thereby reduces VEGF induced vessel permeability. By focusing on the initial BRB breakdown, treatment with norrin may be able to prevent VEGF induced pathology.

Keywords: 446 cell adhesions/cell junctions • 609 neovascularization