Hu Di, Jiang Junhong, Ding Baiyang, Xue Kang, Sun Xinghuai, Qian Shaohong
Department of Ophthalmology & Visual Science, Eye & ENT Hospital, Shanghai Medical College, Fudan University, Shanghai, China.
NHC Key Laboratory of Myopia, Chinese Academy of Medical Sciences, and Shanghai Key Laboratory of Visual Impairment and Restoration (Fudan University), Shanghai, China.
Front Physiol. 2021 Sep 30;12:712509. doi: 10.3389/fphys.2021.712509. eCollection 2021.
Scleral extracellular matrix (ECM) remodeling is thought to play a critical role in the pathogenesis of glaucoma. Mechanical strain induced by elevated intraocular pressure can promote myofibroblast differentiation of fibroblasts and result in scleral ECM remodeling; however, the underlying mechanism remains poorly understood. Yes-associated protein (YAP) is a mechanosensory protein and the key downstream transcriptional effector of the Hippo signaling pathway. Here, we investigated the role of YAP in mechanical strain-induced myofibroblast transformation during glaucoma scleral ECM remodeling. Integrative bioinformatics analyses were performed to identify the key pathways for the ECM remodeling of the sclera in glaucoma. Sprague-Dawley rats were used to establish a chronic ocular hypertension model, and the expression of collagen type I (COL1) and YAP in the sclera was analyzed by immunohistochemical analysis and Western blotting. Furthermore, human scleral fibroblasts (HSFs) were cultured and subjected to mechanical strain. In groups with or without the YAP siRNA or YAP inhibitor, cell proliferation, migration capacity, and the expression levels of YAP, COL1, and α-smooth muscle actin (α-SMA) were evaluated by Cell Counting Kit-8 assay, scratch assay, and Western blotting. The interactions between YAP and Smad3 were demonstrated by coimmunoprecipitation, and the expression levels of COL1 and α-SMA were evaluated in groups treated with or without the Smad3 inhibitor. We first revealed that the Hippo signaling pathway may be involved in mechanical strain-induced scleral ECM remodeling through bioinformatics analysis. Furthermore, the study showed upregulated YAP, COL1, and α-SMA expression in the hypertensive sclera of rats. , mechanical strain increased YAP and COL1 expression in HSFs and promoted myofibroblast differentiation. After YAP knockdown or inhibition with verteporfin, mechanical strain-induced fibrotic changes in HSFs were markedly suppressed. Additionally, YAP showed a protein interaction with Smad3, and the upregulation of a-SMA and COL1 in response to mechanical strain was also significantly downregulated following the inhibition of Smad3. In conclusion, mechanical strain activated scleral myofibroblast differentiation YAP. The YAP pathway may play an important role in regulating scleral myofibroblast differentiation and ECM remodeling of the sclera in glaucoma.
巩膜细胞外基质（ECM）重塑被认为在青光眼的发病机制中起关键作用。眼内压升高引起的机械应变可促进成纤维细胞向肌成纤维细胞分化，并导致巩膜ECM重塑；然而，其潜在机制仍知之甚少。Yes相关蛋白（YAP）是一种机械感受蛋白，也是Hippo信号通路的关键下游转录效应因子。在此，我们研究了YAP在青光眼巩膜ECM重塑过程中机械应变诱导的肌成纤维细胞转化中的作用。进行综合生物信息学分析以确定青光眼巩膜ECM重塑的关键途径。使用Sprague-Dawley大鼠建立慢性高眼压模型，并通过免疫组织化学分析和蛋白质印迹法分析巩膜中I型胶原（COL1）和YAP的表达。此外，培养人巩膜成纤维细胞（HSF）并使其承受机械应变。在有或没有YAP siRNA或YAP抑制剂的组中，通过细胞计数试剂盒-8检测、划痕试验和蛋白质印迹法评估细胞增殖、迁移能力以及YAP、COL1和α-平滑肌肌动蛋白（α-SMA）的表达水平。通过免疫共沉淀证明YAP与Smad3之间的相互作用，并在有或没有Smad3抑制剂处理的组中评估COL1和α-SMA的表达水平。我们首先通过生物信息学分析揭示Hippo信号通路可能参与机械应变诱导的巩膜ECM重塑。此外，研究表明大鼠高血压巩膜中YAP、COL1和α-SMA表达上调。机械应变增加了HSF中YAP和COL1的表达并促进了肌成纤维细胞分化。在用维替泊芬敲低或抑制YAP后，机械应变诱导的HSF纤维化变化被明显抑制。此外，YAP与Smad3存在蛋白质相互作用，在抑制Smad3后，机械应变引起的α-SMA和COL1上调也显著下调。总之，机械应变激活了巩膜肌成纤维细胞分化中的YAP。YAP途径可能在调节青光眼巩膜肌成纤维细胞分化和巩膜ECM重塑中起重要作用。
