Wnt Signaling Regulates Blood Retinal Barrier Permeability by Suppressing MFSD2A-dependent Vascular Endothelial Transcytosis

Abstract

Purpose : The blood retinal barrier (BRB) is essential for retinal heath and characterized by a low rate of transcytosis, while it is impaired in Norrie disease and familial exudative vitreoretinopathy (FEVR) due to the Wnt signaling mutations in Norrin and low-density lipoprotein receptor-related protein 5 (LRP5). A novel suppressor of transcytosis in brain endothelial cells (EC), MFSD2A (major facilitator superfamily domain containing 2A), is down-regulated in Lrp5^-/- and Norrin^y/- retinas. We aim to study whether Wnt signaling regulates the BRB through controlling MFSD2A-dependent EC transcytosis.

Methods : Vascular leakage in Lrp5^-/-, Norrin^y/- and wild type (WT) retinas was evaluated with FITC-dextran (N=6). EC transcytosis in retinal capillaries was tested using electron microscopy (EM) (N=5). Effects of Wnt signaling on EC transcytosis were measured by transcytic transport of horseradish peroxidase (HRP) through human retinal microvascular endothelial cells (HRMEC) (N=3). MFSD2A expression levels affected by Wnt regulators were measured in HRMEC (N=3). Transcriptional control of MFSD2A promotor activity by Wnt signaling was determined via the luciferase reporter assay in HEK293 cells (N=3). Activation of Wnt signaling was achieved by Wnt3a conditional medium or active-β-catenin transfection and suppression by Wnt inhibitor XAV-939.

Results : Compared with WT, vascular leakage was increased in Lrp5^-/- (p=0.005) and Norrin^y/- (p=0.002) retinas; EC transcytotic vesicles were increased in Lrp5^-/- (2.8 fold, p<0.0001) and Norrin^y/- retinas (3.6 fold, p<0.0001). HRMEC transcytosis was reduced by Wnt signaling activation (by 71%, p=0.004), and increased by Wnt inhibition (5.1 fold, p=0.02). MFSD2A levels were enriched in retinal vessels (10 fold, p<0.0001), and decreased in whole retinas (by 50-80%, p<0.01) and microdissected vessels (by 70-90%, p<0.01) in Lrp5^-/- and Norrin^y/- vs. WT eyes. MFSD2A expression levels were increased by Wnt signaling activation, and decreased by Wnt inhibition in HRMEC. Induction of MFSD2A promoter-driven luciferase activity (>10 fold, p<0.0001) was found with co-transfection of β-catenin plasmid, and abolished by mutation of the TCF/LEF binding site.

Conclusions : Our findings demonstrate that Wnt/b-catenin signaling maintains healthy BRB through transcriptional control of the EC transcytosis suppressor, MFSD2A.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.