The Role of the Wnt Pathway in VEGF/Anti-VEGF-Dependent Control of the Endothelial Cell Barrier.

Purpose: Investigate the contribution of the Wnt pathway to vascular endothelial growth factor (VEGF)/anti-VEGF-mediated control of endothelial cell permeability. Methods: High glucose-treated primary human retinal endothelial cells (HRECs) were exposed to either VEGF, or VEGF and then anti-VEGF. Changes in gene expression were assayed by RNAseq and qRT-PCR. Permeability was monitored by electrical cell-substrate impedance sensing (ECIS). Approaches to activate the Wnt pathway included treatment with LiCl and overexpression of constitutively activated β-catenin. β-catenin-dependent transcriptional activity was monitored in HRECs stably expressing a TCF/LEF-driven reporter. Results: VEGF/anti-VEGF altered expression of genes encoding many members of the Wnt pathway. A subset of these genes was regulated in a way that is likely to contribute to control of the endothelial cell barrier. Namely, the VEGF-induced alteration of expression of such genes was reversed by anti-VEGF, and such adjustments occurred at times corresponding to changes in barrier function. While pharmacological and molecular approaches to activate the Wnt pathway had no effect on basal permeability, they suppressed VEGF-induced relaxation. Furthermore, anti-VEGF-mediated restoration of barrier function was unaffected by activation of the Wnt pathway. Conclusions: VEGF/anti-VEGF engages multiple members of the Wnt pathway, and activating this pathway enforces the endothelial barrier by attenuating VEGF-induced relaxation. These data suggest that FDA-approved agents such as LiCl may be an adjuvant to anti-VEGF therapy for patients afflicted with blinding conditions including diabetic retinopathy.