Wnt5a/β-catenin-mediated epithelial-mesenchymal transition: a key driver of subretinal fibrosis in neovascular age-related macular degeneration
Background: Neovascular age-related macular degeneration (nAMD) accounts for up to 90% of AMD-related vision loss, leading to the formation of fibrotic scars in the macular region. The pathogenesis of subretinal fibrosis in nAMD involves epithelial-mesenchymal transition (EMT) in retinal pigment epithelium (RPE) cells. This study aims to investigate the mechanisms underlying Wnt signaling during the EMT of RPE cells and the development of subretinal fibrosis secondary to nAMD.
Methods: Subretinal fibrosis was induced in male C57BL/6J mice via laser photocoagulation. Intravitreal administration of FH535 (a β-catenin inhibitor) or Box5 (a Wnt5a inhibitor) was performed on the same day or 14 days post-laser induction. The RPE-Bruch’s membrane-choriocapillaris complex (RBCC) tissues were collected for Western blot analysis and immunofluorescence to examine fibrovascular and Wnt-related markers. In vitro, ARPE-19 cells treated with transforming growth factor beta 1 (TGFβ1) were co-incubated with or without FH535, Foxy-5 (a Wnt5a-mimicking peptide), Box5, or Wnt5a shRNA. Changes in EMT- and Wnt-related signaling molecules, as well as cell functions, were assessed using qRT-PCR, nuclear-cytoplasmic fractionation assay, Western blot, immunofluorescence, scratch assay, or transwell migration assay. Cell viability of ARPE-19 cells was determined using the Cell Counting Kit (CCK)-8.
Results: In vivo analysis showed that Wnt5a/ROR1, but not Wnt3a, was upregulated in the RBCCs of laser-induced CNV mice compared to controls. Intravitreal injection of FH535 significantly reduced Wnt5a protein expression. Both FH535 and Box5 effectively attenuated subretinal fibrosis and EMT, as well as β-catenin activation in laser-induced CNV mice, demonstrated by reduced areas positive for fibronectin, alpha-smooth muscle actin (α-SMA), collagen I, and active β-catenin labeling. In vitro, Wnt5a/ROR1, active β-catenin, and other Wnt signaling molecules were upregulated in the TGFβ1-induced EMT model using ARPE-19 cells. Co-treatment with FH535, Box5, or Wnt5a shRNA markedly suppressed Wnt5a activation, nuclear translocation of active β-catenin, and EMT in TGFβ1-treated ARPE-19 cells. Conversely, Foxy-5 treatment independently activated these molecules and induced EMT in ARPE-19 cells.
Conclusions: Our study reveals a reciprocal activation between Wnt5a and β-catenin that mediates EMT, serving as a pivotal driver of subretinal fibrosis in nAMD. This positive feedback loop provides valuable insights into potential therapeutic strategies for treating subretinal fibrosis in nAMD patients.