Consistent with increasing findings, extracellular vesicles (EVs), consider as a major constituents of the aqueous humor, have a role as signaling mediators in glaucoma. Following secretion, EVs hold immense promise for utilization as bio-therapeutics and drug delivery vehicles due to their nature as biological nanoparticles that facilitate intercellular molecular transport. Yet, the specific pathway utilizing for transferring signals by EVs in the ocular drainage system is not fully understood. Hence, the objective of this study was to examine internalization mechanisms by which Non-Pigmented Ciliary Epithelium (NPCE)-derived EVs deliver their signals to the Trabecular Meshwork (TM) cells. EVs were isolated and size and concentration were determined. Internalization study of treated EVs with Proteinase-K to achieve removal of surface membrane proteins on EVs was conducted. Energy dependent uptake mechanism was examined under various temperatures. Using uptake inhibitors endocytosis, phagocytosis, and Wnt-TGFβ2 signaling were investigated. TM cells exposed to NPCE EVs demonstrate a significant decrease in the levels of two proteins in two Wnt-TGFb2 signaling proteins levels: p-GSK3β and β-catenin. A significant decrease in the uptake by TM cells of Proteinase-K-treated EVs was found, followed by attenuation of the Wnt-TGFβ2 proteins expression. Energy dependent uptake revealed a significant decrease in EVs internalization. The exposure of TM cells to endocytosis uptake inhibitors abolished the decrease of the Wnt-TGFβ2 proteins levels. Exposure to phagocytosis uptake inhibitor resulted in a partial inhibition of NPCE EVs effect in TM cells. The attenuation of proteins expression levels following uptake inhibitors treatment or EVs membrane proteins removal indicates that Wnt-TGFβ2 signaling in TM cells is mediated through NPCE EVs surface proteins in an active manner that involves endocytosis-dependent routes.

中文翻译：

---

无色素睫状上皮衍生的细胞外囊泡通过小梁网摄取机制

与越来越多的发现一致，细胞外囊泡 (EV) 被认为是房水的主要成分，在青光眼中起着信号传导介质的作用。分泌后，EVs 作为生物治疗剂和药物输送载体具有巨大的应用前景，因为它们作为生物纳米颗粒，促进细胞间分子运输。然而，眼部引流系统中用于通过 EV 传递信号的具体途径尚不完全清楚。因此，本研究的目的是检查无色素睫状上皮 (NPCE) 衍生的 EV 向小梁网 (TM) 细胞传递信号的内化机制。EVs 被隔离并确定大小和浓度。进行了用蛋白酶-K 处理的 EV 的内化研究，以实现去除 EV 上的表面膜蛋白。在各种温度下检查了能量依赖性吸收机制。研究了使用摄取抑制剂的内吞作用、吞噬作用和 Wnt-TGFβ2 信号传导。暴露于 NPCE EV 的 TM 细胞表明两种 Wnt-TGFb2 信号蛋白水平中的两种蛋白质水平显着降低：p-GSK3β 和 β-连环蛋白。发现TM细胞对蛋白酶K处理的EV的摄取显着减少，随后Wnt-TGFβ2蛋白表达减弱。能量依赖的吸收显示电动汽车的内化显着下降。TM 细胞暴露于内吞摄取抑制剂消除了 Wnt-TGFβ2 蛋白水平的降低。暴露于吞噬作用摄取抑制剂导致 TM 细胞中 NPCE EV 效应的部分抑制。摄取抑制剂处理或 EVs 膜蛋白去除后蛋白质表达水平的减弱表明 TM 细胞中的 Wnt-TGFβ2 信号传导通过 NPCE EVs 表面蛋白以涉及内吞作用依赖性途径的主动方式介导。