PDF(Pubmed)
Abstract：
UNASSIGNED: Primary open-angle glaucoma (POAG) is a leading cause of blindness, and its primary risk factor is elevated intraocular pressure (IOP) due to pathologic changes in the trabecular meshwork (TM). We previously showed that there is a cross-inhibition between TGFβ and Wnt signaling pathways in the TM. In this study, we determined if activation of the Wnt signaling pathway using small-molecule Wnt activators can inhibit TGFβ2-induced TM changes and ocular hypertension (OHT).
UNASSIGNED: Primary human TM (pHTM) cells and transduced SBE-GTM3 cells were treated with or without Wnt and/or TGFβ signaling activators and used for luciferase assays; for the extraction of whole-cell lysate, conditioned medium, cytosolic proteins, and nuclear proteins for Western immunoblotting (WB); or for immunofluorescent staining. Human donor eyes were perfusion cultured to study the effect of Wnt activators on IOP.
UNASSIGNED: We found that the small-molecule Wnt activators (GSK3β inhibitors) (BIO, SB216763, and CHIR99021) activated canonical Wnt signaling in pHTM cells without toxicity at tested concentrations. This activation inhibited TGFβ signaling as well as TGFβ2-induced extracellular matrix deposition and formation of cross-linked actin networks in pHTM cells or SBE-GTM3 cells. We also observed nuclear translocation of both Smad4 and β-catenin in pHTM cells, which suggested that the cross-inhibition between the TGFβ and Wnt signaling pathways may occur in the nucleus. Using our ex vivo model, we found that CHIR99021 inhibited TGFβ2-induced OHT in perfusion-cultured human eyes.
UNASSIGNED: Our results showed that small-molecule Wnt activators have the potential for treating TGFβ signaling-induced OHT in patients with POAG.
UNASSIGNED: Primary human TM (pHTM) cells and transduced SBE-GTM3 cells were treated with or without Wnt and/or TGFβ signaling activators and used for luciferase assays; for the extraction of whole-cell lysate, conditioned medium, cytosolic proteins, and nuclear proteins for Western immunoblotting (WB); or for immunofluorescent staining. Human donor eyes were perfusion cultured to study the effect of Wnt activators on IOP.
UNASSIGNED: We found that the small-molecule Wnt activators (GSK3β inhibitors) (BIO, SB216763, and CHIR99021) activated canonical Wnt signaling in pHTM cells without toxicity at tested concentrations. This activation inhibited TGFβ signaling as well as TGFβ2-induced extracellular matrix deposition and formation of cross-linked actin networks in pHTM cells or SBE-GTM3 cells. We also observed nuclear translocation of both Smad4 and β-catenin in pHTM cells, which suggested that the cross-inhibition between the TGFβ and Wnt signaling pathways may occur in the nucleus. Using our ex vivo model, we found that CHIR99021 inhibited TGFβ2-induced OHT in perfusion-cultured human eyes.
UNASSIGNED: Our results showed that small-molecule Wnt activators have the potential for treating TGFβ signaling-induced OHT in patients with POAG.
摘要：
■原发性开角型青光眼（POAG）是导致失明的主要原因，其主要危险因素是由于小梁网（TM）的病理变化引起的眼内压（IOP）升高。我们先前表明在TM中TGFβ和Wnt信号通路之间存在交叉抑制。在这项研究中,我们确定了使用小分子Wnt激活剂激活Wnt信号通路是否可以抑制TGFβ2诱导的TM变化和高眼压（OHT）。
■用或不用Wnt和/或TGFβ信号传导激活剂处理原代人TM（pHTM）细胞和转导的SBE-GTM3细胞，并用于荧光素酶测定；用于提取全细胞裂解物，条件培养基,胞浆蛋白，和用于Western免疫印迹(WB)的核蛋白;或用于免疫荧光染色。灌注培养人供体眼以研究Wnt激活剂对IOP的影响。
■我们发现小分子Wnt激活剂（GSK3β抑制剂）（BIO，SB216763和CHIR99021）在测试浓度下激活pHTM细胞中的经典Wnt信号而无毒性。这种激活抑制了pHTM细胞或SBE-GTM3细胞中TGFβ信号传导以及TGFβ2诱导的细胞外基质沉积和交联肌动蛋白网络的形成。我们还观察到Smad4和β-catenin在pHTM细胞中的核易位，这表明TGFβ和Wnt信号通路之间的交叉抑制可能发生在细胞核中。使用我们的离体模型，我们发现CHIR99021在灌注培养的人眼中抑制TGFβ2诱导的OHT。
■我们的结果表明，小分子Wnt激活剂具有治疗POAG患者中TGFβ信号传导诱导的OHT的潜力。
■用或不用Wnt和/或TGFβ信号传导激活剂处理原代人TM（pHTM）细胞和转导的SBE-GTM3细胞，并用于荧光素酶测定；用于提取全细胞裂解物，条件培养基,胞浆蛋白，和用于Western免疫印迹(WB)的核蛋白;或用于免疫荧光染色。灌注培养人供体眼以研究Wnt激活剂对IOP的影响。
■我们发现小分子Wnt激活剂（GSK3β抑制剂）（BIO，SB216763和CHIR99021）在测试浓度下激活pHTM细胞中的经典Wnt信号而无毒性。这种激活抑制了pHTM细胞或SBE-GTM3细胞中TGFβ信号传导以及TGFβ2诱导的细胞外基质沉积和交联肌动蛋白网络的形成。我们还观察到Smad4和β-catenin在pHTM细胞中的核易位，这表明TGFβ和Wnt信号通路之间的交叉抑制可能发生在细胞核中。使用我们的离体模型，我们发现CHIR99021在灌注培养的人眼中抑制TGFβ2诱导的OHT。
■我们的结果表明，小分子Wnt激活剂具有治疗POAG患者中TGFβ信号传导诱导的OHT的潜力。
