背景:新生血管性年龄相关性黄斑变性(nAMD),占高达90%的AMD相关视力丧失,最终导致黄斑区纤维化瘢痕的形成。nAMD视网膜下纤维化的发病机制涉及视网膜色素上皮(RPE)中发生的上皮-间质转化(EMT)过程。这里,我们的目的是研究RPE细胞EMT过程中Wnt信号传导的潜在机制,以及nAMD继发视网膜下纤维化的病理过程.
方法:体内,通过激光光凝对雄性C57BL/6J小鼠进行视网膜下纤维化的诱导。FH535(β-连环蛋白抑制剂)或Box5(Wnt5a抑制剂)在同一天或激光诱导后14天玻璃体内施用。收集RPE-Bruch膜脉络膜复合体(RBCC)组织,并进行Western印迹分析和免疫荧光以检查纤维血管和Wnt相关标志物。体外,将转化生长因子β1(TGFβ1)处理的ARPE-19细胞与或不与FH535,Foxy-5(Wnt5a模拟肽)共孵育,Box5或Wnt5ashRNA,分别。EMT和Wnt相关信号分子的变化,以及使用qRT-PCR评估细胞功能,核-细胞质分级分离试验,蛋白质印迹,免疫荧光,划痕试验或transwell迁移试验。使用细胞计数试剂盒(CCK)-8测定ARPE-19细胞的细胞活力。
结果:体内分析显示Wnt5a/ROR1,但未显示Wnt3a,与正常对照组相比,激光诱导的CNV小鼠的RBCC上调。玻璃体内注射FH535可有效降低Wnt5a蛋白表达。FH535和Box5均可有效减轻视网膜下纤维化和EMT,以及激光诱导的CNV小鼠中β-catenin的激活,纤连蛋白阳性区域的显著减少证明了这一点,α-平滑肌肌动蛋白(α-SMA),胶原蛋白I,和活性β-连环蛋白标记。体外,Wnt5a/ROR1,活性β-连环蛋白,和一些其他Wnt信号分子在TGFβ1诱导的EMT细胞模型中使用ARPE-19细胞上调。与FH535、Box5或Wnt5ashRNA共同处理显著抑制了Wnt5a的激活,活性β-连环蛋白的核易位,以及TGFβ1处理的ARPE-19细胞中的EMT。相反,用Foxy-5处理独立地导致上述分子的活化和随后在ARPE-19细胞中诱导EMT。
结论:我们的研究揭示了Wnt5a和β-catenin之间的相互激活介导EMT是nAMD视网膜下纤维化的关键驱动因素。这种正反馈回路为治疗nAMD患者视网膜下纤维化的潜在治疗策略提供了有价值的见解。
BACKGROUND: Neovascular age-related macular degeneration (nAMD), accounts for up to 90% of AMD-associated vision loss, ultimately resulting in the formation of fibrotic scar in the macular region. The pathogenesis of subretinal fibrosis in nAMD involves the process of epithelial-mesenchymal transition (EMT) occurring in retinal pigment epithelium (RPE). Here, we aim to investigate the underlying mechanisms involved in the Wnt signaling during the EMT of RPE cells and in the pathological process of subretinal fibrosis secondary to nAMD.
METHODS: In vivo, the induction of subretinal fibrosis was performed in male C57BL/6J mice through laser photocoagulation. Either FH535 (a β-catenin inhibitor) or Box5 (a Wnt5a inhibitor) was intravitreally administered on the same day or 14 days following laser induction. The RPE-Bruch\'s membrane-choriocapillaris complex (RBCC) tissues were collected and subjected to Western blot analysis and immunofluorescence to examine fibrovascular and Wnt-related markers. In vitro, transforming growth factor beta 1 (TGFβ1)-treated ARPE-19 cells were co-incubated with or without FH535, Foxy-5 (a Wnt5a-mimicking peptide), Box5, or Wnt5a shRNA, respectively. The 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. The cell viability of ARPE-19 cells was determined using Cell Counting Kit (CCK)-8.
RESULTS: The in vivo analysis demonstrated Wnt5a/ROR1, but not Wnt3a, was upregulated in the RBCCs of the laser-induced CNV mice compared to the normal control group. Intravitreal injection of FH535 effectively reduced Wnt5a protein expression. Both FH535 and Box5 effectively attenuated subretinal fibrosis and EMT, as well as the activation of β-catenin in laser-induced CNV mice, as evidenced by the significant reduction in 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 some other Wnt signaling molecules were upregulated in the TGFβ1-induced EMT cell model using ARPE-19 cells. Co-treatment with FH535, Box5, or Wnt5a shRNA markedly suppressed the activation of Wnt5a, nuclear translocation of active β-catenin, as well as the EMT in TGFβ1-treated ARPE-19 cells. Conversely, treatment with Foxy-5 independently resulted in the activation of abovementioned molecules and subsequent induction of EMT in ARPE-19 cells.
CONCLUSIONS: Our study reveals a reciprocal activation between Wnt5a and β-catenin to mediate EMT as a pivotal driver of subretinal fibrosis in nAMD. This positive feedback loop provides valuable insights into potential therapeutic strategies to treat subretinal fibrosis in nAMD patients.