Corneal endothelial cells (CECs) are essential for maintaining corneal transparency and hydration through their barrier and pump functions. The COL8A2 gene encodes a component of the extracellular matrix of the cornea, which is crucial for the normal functioning of these cells. Mutations in COL8A2 are linked to corneal dystrophies, emphasizing the gene's importance in corneal health. The purpose of this research is to explore the effects of COL8A2 activation within CECs, to understand its contribution to cellular behavior and health. COL8A2 CRISPR/dCas9 activation system (aCOL8A2) was used to activate the COL8A2. In rats, wound healing and mitochondrial function were assessed after COL8A2 activation. As a result, aCOL8A2 promoted wound healing of rat corneal endothelium by increasing mitochondrial membrane potential. In cultured human CECs, proteomic analysis was performed to screen and identify the differential protein profiles between control and aCOL8A2 cells. Western blot was used to validate the differential proteins from both cells. Mitochondrial function and intracellular distribution were assessed by measuring ATP production and mitochondrial membrane potential. In cultured human CECs, aCOL8A2 increased COL8A2 and phospho-YAP levels. Transendothelial electrical resistance (TEER) was increased and actin cytoskeleton was attenuated by aCOL8A2. Gene ontology analysis revealed that the proteins were mainly involved in the regulation of folate biosynthesis, ECM-receptor interaction, cell differentiation, NADP activity and cytoskeleton. ATP production was increased, mitochondrial membrane potential was polarized and mitochondrial distribution was widespread in the aCOL8A2 group. In conclusion, aCOL8A2 induces a regulatory cascade affecting mitochondrial positioning and efficiency, mediated by alterations in the cytoskeletal architecture and the YAP signaling pathway. This sequence of events serves to bolster the functional capacities of corneal endothelial cells, including their pump and barrier functions, essential for corneal health and transparency.

中文翻译：

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COL8A2 激活通过 HIPPO 信号传导/线粒体通路增强角膜内皮细胞的功能


角膜内皮细胞 （CEC） 通过其屏障和泵功能维持角膜透明度和水合作用至关重要。COL8A2 基因编码角膜细胞外基质的一个成分，这对这些细胞的正常功能至关重要。COL8A2 突变与角膜营养不良有关，强调了该基因在角膜健康中的重要性。本研究的目的是探索 CEC 中 COL8A2 激活的影响，以了解其对细胞行为和健康的贡献。COL8A2 CRISPR/dCas9 激活系统 （aCOL8A2） 用于激活 COL8A2。在大鼠中，评估 COL8A2 激活后伤口愈合和线粒体功能。因此，aCOL8A2 通过增加线粒体膜电位促进大鼠角膜内皮的伤口愈合。在培养的人 CEC 中，进行蛋白质组学分析以筛选和鉴定对照细胞和 aCOL8A2 细胞之间的差异蛋白质谱。Western blot 用于验证来自两个细胞的差异蛋白。通过测量 ATP 产生和线粒体膜电位来评估线粒体功能和细胞内分布。在培养的人 CEC 中，aCOL8A2 增加了 COL8A2 和磷酸化 YAP 水平。跨内皮电阻 （TEER） 增加，肌动蛋白细胞骨架被 aCOL8A2 减弱。基因本体分析显示，这些蛋白主要参与叶酸生物合成、ECM-受体相互作用、细胞分化、NADP 活性和细胞骨架的调节。ATP 生成增加，线粒体膜电位极化，线粒体分布广泛在 aCOL8A2 组中。 总之，aCOL8A2 诱导影响线粒体定位和效率的调节级联反应，由细胞骨架结构和 YAP 信号通路的改变介导。这一系列事件有助于增强角膜内皮细胞的功能能力，包括它们对角膜健康和透明度至关重要的泵和屏障功能。