Ectopic endometrial stromal cell-derived extracellular vesicles encapsulating PKM2 inhibit autophagy inducing endometrial collagen I deposition in endometriosis

Endometriosis (EMs) is a common infertility-related disease in women of reproductive age. Impaired endometrial decidualization is one of the most important factors contributing to the embryo implantation failure EMs patients. However, the exact mechanism remains unclear. Previous studies have shown collagen I deposition in the eutopic endometrium of EMs patients, which may lead to impaired endometrial decidualization. The level of collagen I in eutopic endometrium of EMs was analyzed. We performed a proteomic analysis of ectopic endometrial stromal cell-derived extracellular vesicles (EMs-EVs) and extracellular vesicles derived from endometrial stromal cells in the endometrium of control patients (CTL-EVs). An endometrial transcriptional profiles of EMs patients and normal controls in the mid-secretory phase of menstrual cycle was compared. Endometrial stromal cells (ESCs) were stimulated with chloroquine or rapamycin to evaluate the association between autophagy and collagen I. The expression of PKM2 in EMs-EVs and serum extracellular vesicles from EMs patients were examined by western blotting. PKM2 was overexpressed or knockdown in ESCs to investigate the level of autophagy and collagen I. ESCs were treated with ectopic ESC-derived extracellular vesicles with highly expressed PKM2 protein, and the potential molecular mechanisms were further confirmed through western blotting and immunohistochemical analysis. We found that endometrial collagen I expression during the mid-secretory phase was increased in the EMs group. We demonstrated that autophagy is defective in eutopic endometrial stromal cells (ESCs) of EMs patients. In ESCs, pharmacological inhibition of autophagy by chloroquine (CQ) promoted collagen I deposition. Ectopic endometrial stromal cell-derived extracellular vesicles (EMs-EVs) inhibited autophagy of ESCs and promoted collagen I deposition in vivo and in vitro. Mechanistically, EMs-EVs encapsulating PKM2 impair eutopic endometrial autophagy via Akt/mTOR signaling pathway. Together, we demonstrated that EMs-EVs encapsulating PKM2 impaired endometrial autophagy inducing collagen I deposition in EMs, which provided a potential target for therapeutic implications. Overall design: The level of collagen I in eutopic endometrium of EMs was analyzed. We performed a proteomic analysis of ectopic endometrial stromal cell-derived extracellular vesicles (EMs-EVs) and extracellular vesicles derived from endometrial stromal cells in the endometrium of control patients (CTL-EVs). An endometrial transcriptional profiles of EMs patients and normal controls in the mid-secretory phase of menstrual cycle was compared. Endometrial stromal cells (ESCs) were stimulated with chloroquine or rapamycin to evaluate the association between autophagy and collagen I. The expression of PKM2 in EMs-EVs and serum extracellular vesicles from EMs patients were examined by western blotting. PKM2 was overexpressed or knockdown in ESCs to investigate the level of autophagy and collagen I. ESCs were treated with ectopic ESC-derived extracellular vesicles with highly expressed PKM2 protein, and the potential molecular mechanisms were further confirmed through western blotting and immunohistochemical analysis.

子宫内膜异位症（Endometriosis，EMs）是育龄女性常见的不孕症相关疾病。子宫内膜蜕膜化受损是子宫内膜异位症患者胚胎着床失败的关键致病因素之一，但其确切分子机制尚未明确。既往研究显示，子宫内膜异位症患者的在位子宫内膜存在I型胶原沉积，这可能导致子宫内膜蜕膜化功能受损。本研究首先分析了子宫内膜异位症患者在位子宫内膜的I型胶原水平；随后对异位子宫内膜间质细胞来源的细胞外囊泡（EMs-EVs）及对照患者子宫内膜间质细胞来源的细胞外囊泡（CTL-EVs）开展蛋白质组学分析；同时比较了月经周期分泌中期的子宫内膜异位症患者与正常对照的子宫内膜转录组谱。为探究自噬与I型胶原的关联，本研究使用氯喹或雷帕霉素处理子宫内膜间质细胞（Endometrial Stromal Cells，ESCs）；通过蛋白免疫印迹检测了EMs-EVs及子宫内膜异位症患者血清细胞外囊泡中丙酮酸激酶M2（PKM2）的表达水平；在ESCs中过表达或敲低PKM2，以检测自噬水平及I型胶原的表达情况；用高表达PKM2的异位ESC来源细胞外囊泡处理ESCs，并通过蛋白免疫印迹与免疫组织化学分析进一步验证潜在分子机制。研究结果显示：分泌中期的子宫内膜异位症患者在位子宫内膜的I型胶原表达水平显著升高；子宫内膜异位症患者的在位子宫内膜间质细胞（ESCs）存在自噬功能缺陷；在ESCs中，通过氯喹（CQ）药理学抑制自噬可促进I型胶原沉积；EMs-EVs可在体内外抑制ESCs的自噬并促进I型胶原沉积。机制上，EMs-EVs包裹的PKM2通过Akt/mTOR信号通路损伤在位子宫内膜的自噬功能。综上，本研究证实子宫内膜异位症患者的EMs-EVs通过包裹PKM2损伤子宫内膜自噬，进而诱导I型胶原沉积，为临床治疗提供了潜在靶点。总体实验设计：本研究首先分析了子宫内膜异位症患者在位子宫内膜的I型胶原水平；随后对异位子宫内膜间质细胞来源的细胞外囊泡（EMs-EVs）及对照患者子宫内膜间质细胞来源的细胞外囊泡（CTL-EVs）开展蛋白质组学分析；同时比较了月经周期分泌中期的子宫内膜异位症患者与正常对照的子宫内膜转录组谱。为探究自噬与I型胶原的关联，本研究使用氯喹或雷帕霉素处理子宫内膜间质细胞（ESCs）；通过蛋白免疫印迹检测了EMs-EVs及子宫内膜异位症患者血清细胞外囊泡中PKM2的表达水平；在ESCs中过表达或敲低PKM2，以检测自噬水平及I型胶原的表达情况；用高表达PKM2的异位ESC来源细胞外囊泡处理ESCs，并通过蛋白免疫印迹与免疫组织化学分析进一步验证潜在分子机制。