Abstract
Purpose::
Choroidal neovascularization (CNV) is the major sight-threatening complication of age-related macular degeneration, a leading cause of blindness. Knockout of the very low-density lipoprotein receptor (Vldlr) gene in mice has been shown to cause subretinal neovascularization (NV) with an unknown mechanism. The purpose of this study was to establishes that the wnt signaling pathway is involved in the subretinal NV observed in Vldlr knockout (Vldlr-/-) mice.
Methods::
Retinal vasculature was examined by fluorescein angiography and staining of endothelial markers on retinal sections. Expression and phosphorylation levels of wnt pathway components were determined by Western blot analysis and immunohistochemistry. VEGF function was blocked by intravitreal injection of a neutralizing antibody against VEGF receptor (VEGFR). VLDLR expression was knocked down in cultured cells by a specific siRNA.
Results::
Vldlr-/- mice have early onset of sub-retinal NV. NV initiated in the choroid and progressed to penetrate Bruch’s membrane and the RPE layer, proliferating in the subretinal space. This phenotype recapitulated what is seen in wet AMD, suggesting that this mouse is a model for CNV. In addition to the proliferative response, the neovasculature lacked pericyte coverage, suggesting delayed maturation. The CNV correlated with increased levels of VEGF, hypoxia-inducible factor-1α and VEGFR2 in the eyecups and was effectively blocked by a neutralizing antibody against VEGFR2. While the wnt receptor Fz4 was not changed, the wnt co-receptor LRP5/6 was significantly elevated in the Vldlr-/- mice, compared to that in wild-type control mice. Significantly, the Vldlr-/- mice showed impaired phosphorylation of downstream effectors of the wnt signaling pathway, GSK-3ß and ß-catenin, concomitant with increased levels of free GSK-3ß and ß-catenin, suggesting an increased activation. Down-regulation of VLDLR by siRNA resulted in activation of the wnt pathway in cultured endothelial cells, suggesting that VLDLR functions as a negative regulator of the wnt signaling.
Conclusions::
The Vldlr-/- mouse is a genetic model of early onset CNV. As the wnt signaling pathway is known to up-regulate VEGF expression, the activated wnt signaling pathway may play an important role in the VEGF up-regulation and subsequent CNV in Vldlr-/- mice.
Keywords: choroid • age-related macular degeneration • neovascularization