SMSCs 来源的外泌体过表达 miR-433-3p 抑制由成纤维样滑膜细胞释放的外泌体在铁死亡触发下诱导的血管生成。
SMSCs-derived sEV overexpressing miR-433-3p inhibits angiogenesis induced by sEV released from synoviocytes under triggering of ferroptosis.
机构信息
Department of Rheumatology, The First Affiliated Hospital, Harbin Medical University, 23 Youzheng St., Nan Gang District, Harbin, China.
Department of Rheumatology, The First Affiliated Hospital, Harbin Medical University, 23 Youzheng St., Nan Gang District, Harbin, China.
出版信息
Int Immunopharmacol. 2023 Mar;116:109875. doi: 10.1016/j.intimp.2023.109875. Epub 2023 Feb 27.
BACKGROUND
Ferroptosis is characterized by accumulation of lipid peroxides that leads to oxidative stress. In progressive rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) suffered from oxidative stress induced by generation of excess reactive oxygen species (ROS) and survived from elevated lipid oxidation. However the phenomenon of abnormal synovial fibroblasts proliferation under ferroptotic stress remain to be explained and the effects of this event on disease progression of RA need to be investigated.
METHODS
FLS from RA patients (RA-FLS) were stimulated with LPS as an inflammatory model in vitro, and simultaneously treated with ferroptosis inducer Erastin/RSL3 or inhibitor ferrostatin-1. Besides, small extracellular vesicles (sEV) from the supernatant of RA-FLS culture under Erastin/RSL3 management were isolated. The degree of ferroptosis in cells were evaluated by Lipid-ROS detection via flowcytometry and ferroptosis marker protein expression determined by western bloting. The expression of core component of ESCRT-III CHMP4A and CHMP5 was determined by western bloting, and knockdown of CHMP4A was further performed to detect the influence of ESCRT-III complex on ferroptosis as well as LPS/Erastin induced sEV (LPS/Erastin-sEV) releasing. Moreover, miR-433-3p level in the isolated sEV was evaluated by RT-qPCR and interaction of miR-433-3p with FOXO1/VEGF axis were evaluated. MiR-433-3p was overexpressed in synovial mesenchymal stem cells (SMSCs) via miR-433-3p mimics transfection. RA-FLS was co-cultured with human dermal microvascular endothelial cells (HDMECs). LPS/Erastin-sEV or sEV derived from miR-433-3p-overexpressing SMSCs (miR-433-3p-SMSCs-sEV) were added to the co-culture system, and supernatants from co-culture without sEV were given to HDMECs. Angiogenic activity of HDMECs were identified by transwell test and endothelial tube formation analysis. Erastin-sEV and miR-433-3p-SMSCs-sEV were also administrated in collagen-induced arthritis (CIA) mouse model respectively, and progression of arthritis were evaluated.
RESULTS
Ferroptosis of RA-FLS was triggered by LPS/Erastin and accompanied with increased expression of ESCRT-III core components as well as elevated release of sEV from RA-FLS. HDMECs' migration and tube formation in vitro was significantly induced/suppressed by supernatants from co-culture under management of Erastin-sEV/miR-433-3p-SMSCs-sEV due to varied VEGF expression regulated by miR-433-3p targeting FOXO1. MiR-433-3p-SMSCs-sEV could inhibit the Erastin-sEV promoted VEGF expression and mitigated arthritis severity.
CONCLUSION
Erastin-sEV could aggravate synovial angiogenesis and promote arthritis progression. Administration of miR-433-3p-SMSCs-sEV may be a potential novel therapeutic method as significant antagonism to Erastin-sEV for RA treatment.
背景
铁死亡的特征是脂质过氧化物的积累，导致氧化应激。在进行性类风湿关节炎（RA）中，成纤维样滑膜细胞（FLS）遭受由过量活性氧（ROS）产生引起的氧化应激，并在脂质氧化升高的情况下存活。然而，铁死亡应激下异常滑膜成纤维细胞增殖的现象仍有待解释，需要研究这一事件对 RA 疾病进展的影响。
方法
用脂多糖（LPS）体外刺激 RA 患者的 FLS（RA-FLS）作为炎症模型，同时用铁死亡诱导剂 Erastin/RSL3 或抑制剂 Ferrostatin-1 处理。此外，从 Erastin/RSL3 管理下的 RA-FLS 培养物上清液中分离出小细胞外囊泡（sEV）。通过流式细胞术检测脂质-ROS 来评估细胞中的铁死亡程度，并通过 Western blot 测定铁死亡标志物蛋白的表达。Western blot 测定 ESCRT-III 核心成分 CHMP4A 和 CHMP5 的表达，进一步敲低 CHMP4A 以检测 ESCRT-III 复合物对铁死亡以及 LPS/Erastin 诱导的 sEV（LPS/Erastin-sEV）释放的影响。此外，通过 RT-qPCR 评估分离的 sEV 中的 miR-433-3p 水平，并评估 miR-433-3p 与 FOXO1/VEGF 轴的相互作用。通过 miR-433-3p 模拟物转染将 miR-433-3p 过表达于滑膜间充质干细胞（SMSCs）中。将 RA-FLS 与人真皮微血管内皮细胞（HDMECs）共培养。将 LPS/Erastin-sEV 或源自 miR-433-3p 过表达 SMSCs 的 sEV（miR-433-3p-SMSCs-sEV）添加到共培养系统中，并将无 sEV 的共培养上清液添加到 HDMECs 中。通过 Transwell 试验和内皮管形成分析鉴定 HDMECs 的血管生成活性。分别向胶原诱导性关节炎（CIA）小鼠模型中给予 Erastin-sEV 和 miR-433-3p-SMSCs-sEV，并评估关节炎的进展。
结果
LPS/Erastin 触发了 RA-FLS 的铁死亡，伴随着 ESCRT-III 核心成分表达的增加以及 RA-FLS 中 sEV 的释放增加。由于 miR-433-3p 靶向 FOXO1 调节的 VEGF 表达的变化，共培养上清液在 Erastin-sEV/miR-433-3p-SMSCs-sEV 处理下，显著诱导/抑制了体外 HDMECs 的迁移和管形成。miR-433-3p-SMSCs-sEV 可抑制 Erastin-sEV 促进的 VEGF 表达，并减轻关节炎严重程度。
结论
Erastin-sEV 可加重滑膜血管生成并促进关节炎进展。miR-433-3p-SMSCs-sEV 的给药可能是一种潜在的新型治疗方法，对 RA 治疗具有显著的拮抗作用，可对抗 Erastin-sEV。
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