Department of Ophthalmology, Mater Misericordiae University Hospital, Dublin, Ireland.
Clinical Research Centre, School of Medicine, University College Dublin, Dublin, Ireland.
Invest Ophthalmol Vis Sci. 2022 Jan 3;63(1):16. doi: 10.1167/iovs.63.1.16.
Extracellular matrix stiffening is characteristic of both aging and glaucoma, and acts as a promoter and perpetuator of pathological fibrotic remodeling. Here, we investigate the role of a mechanosensitive transcriptional coactivator, Yes-associated protein (YAP), a downstream effector of multiple signaling pathways, in lamina cribrosa (LC) cell activation to a profibrotic, glaucomatous state.
LC cells isolated from glaucomatous human donor eyes (GLC; n = 3) were compared to LC cells from age-matched nonglaucomatous controls (NLC; n = 3) to determine differential YAP expression, protein levels, and proliferation rates. NLC cells were then cultured on soft (4 kPa), and stiff (100 kPa), collagen-1 coated polyacrylamide hydrogel substrates. Quantitative real-time RT-PCR, immunoblotting, and immunofluorescence microscopy were used to measure the expression, activity, and subcellular location of YAP and its downstream targets, respectively. Proliferation rates were examined in NLC and GLC cells by methyl thiazolyl tetrazolium salt assays, across a range of incrementally increased substrate stiffness. Endpoints were examined in the presence or absence of a YAP inhibitor, verteporfin (2 µM).
GLC cells show significantly (P < 0.05) increased YAP gene expression and total-YAP protein compared to NLC cells, with significantly increased proliferation. YAP regulation is mechanosensitive, because NLC cells cultured on pathomimetic, stiff substrates (100 kPa) show significantly upregulated YAP gene and protein expression, increased YAP phosphorylation at tyrosine 357, reduced YAP phosphorylation at serine 127, increased nuclear pooling, and increased transcriptional target, connective tissue growth factor. Accordingly, myofibroblastic markers, α-smooth muscle actin (α-SMA) and collagen type I, alpha 1 (Col1A1) are increased. Proliferation rates are elevated on 50 kPa substrates and tissue culture plastic. Verteporfin treatment significantly inhibits YAP-mediated cellular activation and proliferation despite a stiffened microenvironment.
These data demonstrate how YAP plays a pivotal role in LC cells adopting a profibrotic and proliferative phenotype in response to the stiffened LC present in aging and glaucoma. YAP provides an attractive and novel therapeutic target, and its inhibition via verteporfin warrants further clinical investigation.
细胞外基质的变硬与衰老和青光眼都有关，并且是病理性纤维化重塑的促进和延续因素。在这里，我们研究了机械敏感转录共激活因子 Yes 相关蛋白（YAP）的作用，YAP 是多种信号通路的下游效应物，在层粘连 cribrosa（LC）细胞向致纤维化、青光眼状态的激活中发挥作用。
从青光眼供体眼（GLC；n=3）中分离 LC 细胞，并与年龄匹配的非青光眼对照（NLC；n=3）的 LC 细胞进行比较，以确定 YAP 的差异表达、蛋白水平和增殖率。然后，将 NLC 细胞培养在软（4kPa）和硬（100kPa）胶原 1 包被的聚丙烯酰胺水凝胶底物上。使用定量实时 RT-PCR、免疫印迹和免疫荧光显微镜分别测量 YAP 及其下游靶标的表达、活性和亚细胞定位。通过甲基噻唑基四唑盐测定法在 NLC 和 GLC 细胞中测定增殖率，范围从逐渐增加的底物刚度。在存在或不存在 YAP 抑制剂维替泊芬（2µM）的情况下检查终点。
与 NLC 细胞相比，GLC 细胞的 YAP 基因表达和总 YAP 蛋白显著增加（P<0.05），增殖明显增加。YAP 调节是机械敏感的，因为在病理模拟硬底物（100kPa）上培养的 NLC 细胞显示出显著上调的 YAP 基因和蛋白表达、YAP 酪氨酸 357 磷酸化增加、YAP 丝氨酸 127 磷酸化减少、核池增加和转录靶标结缔组织生长因子增加。相应地，肌成纤维标记物，α-平滑肌肌动蛋白（α-SMA）和胶原蛋白 I，α1（Col1A1）增加。在 50kPa 底物和组织培养塑料上，增殖率升高。尽管微环境变硬，维替泊芬治疗仍能显著抑制 YAP 介导的细胞激活和增殖。
这些数据表明，YAP 在 LC 细胞对老化和青光眼存在的变硬 LC 产生致纤维化和增殖表型的反应中起着关键作用。YAP 提供了一个有吸引力的新的治疗靶点，其通过维替泊芬的抑制值得进一步的临床研究。
