Liu Shuting, Chen Hua, Xie Huatao, Liu Xin, Zhang Mingchang
Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Senior Department of Ophthalmology, The Third Medical Center of PLA General Hospital, Beijing, China.
Invest Ophthalmol Vis Sci. 2024 Mar 5;65(3):15. doi: 10.1167/iovs.65.3.15.
To explore the role of substrate stiffness and the mechanism beneath corneal endothelial cells' (CECs') stemness maintenance and differentiation.
CECs were divided into central zone (8 mm trephined boundary) and peripheral zone (8 mm trephined edge with attached limbal). Two zones were analyzed by hematoxylin-eosin staining and scanning electron microscopy for anatomic structure. The elastic modulus of Descemet's membrane (DM) was analyzed by atomic force microscopy. Compressed type I collagen gels with different stiffness were constructed as an in vitro model system to test the role of stiffness on phenotype using cultured rabbit CECs. Cell morphology, expression and intracellular distribution of Yes-associated protein (YAP), differentiation (ZO-1, Na+/K+-ATPase), stemness (FOXD3, CD34, Sox2, Oct3/4), and endothelial-mesenchymal transition (EnMT) markers were analyzed by immunofluorescence, quantitative RT-PCR, and Western blot.
The results showed that the peripheral area of rabbit and human DM is softer than the central area ex vivo. Using the biomimetic extracellular matrix collagen gels in vitro model, we then demonstrated that soft substrate weakens the differentiation and EnMT in the culture of CECs. It was further proved by the inhibitor experiment that soft substrate enhances stemness maintenance via inhibition of paxillin-YAP signaling, which was activated on a stiff substrate.
Our findings confirm that substrate stiffness modulates the stemness maintenance and differentiation of CECs and suggest a potential strategy for CEC-based corneal tissue engineering.
探讨基质硬度在角膜内皮细胞（CECs）干性维持和分化中的作用及其潜在机制。
将CECs分为中央区（8mm环钻边界）和周边区（8mm环钻边缘及附着的角膜缘）。通过苏木精-伊红染色和扫描电子显微镜分析两个区域的解剖结构。用原子力显微镜分析后弹力层（DM）的弹性模量。构建不同硬度的压缩I型胶原凝胶作为体外模型系统，用培养的兔CECs测试硬度对细胞表型的作用。通过免疫荧光、定量逆转录-聚合酶链反应和蛋白质印迹分析细胞形态、Yes相关蛋白（YAP）的表达和细胞内分布、分化标志物（紧密连接蛋白1、钠钾ATP酶）、干性标志物（叉头框蛋白D3、CD34、性别决定区Y框蛋白2/OCT3/4）以及内皮-间充质转化（EnMT）标志物。
结果显示，离体兔和人DM的周边区域比中央区域更柔软。利用体外仿生细胞外基质胶原凝胶模型，我们证明了软基质会削弱CECs培养中的分化和EnMT。抑制剂实验进一步证明，软基质通过抑制桩蛋白-YAP信号通路增强干性维持，该信号通路在硬基质上被激活。
我们的研究结果证实基质硬度调节CECs的干性维持和分化，并为基于CECs的角膜组织工程提供了一种潜在策略。
