Small extracellular vesicles (sEV) derived from synovial fibroblasts (SF) represent a novel molecular mechanism regulating cartilage erosion in osteoarthritis (OA). However, a comprehensive evaluation using disease relevant cells has not been undertaken. The aim of this study was to isolate and characterise sEV from OA SF and to look at their ability to regulate OA chondrocyte effector responses relevant to disease. Profiling of micro (mi) RNA signatures in sEV and parental OA SF cells was performed. SF and chondrocytes were isolated from OA synovial membrane and cartilage respectively (n = 9). sEV were isolated from OA SF (± IL-1β) conditioned media by ultracentrifugation and characterised using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Particle size was confirmed by nanoparticle tracking analysis (NTA). sEV regulation of OA chondrocyte and cartilage effector response was evaluated using qPCR, ELISA and sulphated glycosaminoglycan assay (sGAG). RNA-sequencing was used to establish miRNA signatures in isolated sEV from OA SF. OA SF derived sEV were readily taken up by OA chondrocytes, with increased expression of the catabolic gene MMP 13 (p < 0.01) and decreased expression of the anabolic genes aggrecan and COL2A1 (p < 0.01) observed. Treatment with sEV derived from IL-1β stimulated OA SF significantly decreased expression of aggrecan and COL2A1 (p < 0.001) and increased SOX 9 gene expression (p < 0.05). OA chondrocytes cultured with sEV from either non-stimulated or IL-1β treated OA SF, resulted in a significant increase in the secretion of IL-6, IL-8 and MMP-3 (p < 0.01). Cartilage explants cultured with sEV from SF (± IL-1β) had a significant increase in the release of sGAG (p < 0.01). miRNA signatures differed between parental SF cells and isolated sEV. The recently identified osteoclastogenic regulator miR182, along with miR4472-2, miR1302-3, miR6720, miR6087 and miR4532 were enriched in sEV compared to parental cells, p < 0.01. Signatures were similar in sEVs derived from non-stimulated or IL-1β stimulated SF. OA SF sEV regulate chondrocyte inflammatory and remodelling responses. OA SF sEV have unique signatures compared to parental cells which do not alter with IL-1β stimulation. This study provides insight into a novel regulatory mechanism within the OA joint which could inform future targeted therapy.

中文翻译：

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源自滑膜成纤维细胞的小细胞外囊泡含有独特的 miRNA 谱，有助于骨关节炎中的软骨细胞损伤


源自滑膜成纤维细胞（SF）的小细胞外囊泡（sEV）代表了调节骨关节炎（OA）软骨侵蚀的新分子机制。然而，尚未使用疾病相关细胞进行综合评估。本研究的目的是从 OA SF 中分离和表征 sEV，并研究它们调节与疾病相关的 OA 软骨细胞效应反应的能力。对 sEV 和亲本 OA SF 细胞中的 micro (mi) RNA 特征进行了分析。 SF 和软骨细胞分别从 OA 滑膜和软骨中分离出来 (n = 9)。通过超速离心从 OA SF (± IL-1β) 条件培养基中分离出 sEV，并使用扫描电子显微镜 (SEM) 和透射电子显微镜 (TEM) 进行表征。通过纳米颗粒跟踪分析（NTA）确认颗粒尺寸。使用 qPCR、ELISA 和硫酸化糖胺聚糖测定 (sGAG) 评估 sEV 对 OA 软骨细胞和软骨效应器反应的调节。 RNA 测序用于在从 OA SF 分离的 sEV 中建立 miRNA 特征。 OA SF 衍生的 sEV 很容易被 OA 软骨细胞摄取，观察到分解代谢基因 MMP 13 的表达增加 (p < 0.01)，而合成代谢基因聚集蛋白聚糖和 COL2A1 的表达降低 (p < 0.01)。使用源自 IL-1β 的 sEV 治疗刺激 OA SF，显着降低聚集蛋白聚糖和 COL2A1 的表达 (p < 0.001)，并增加 SOX 9 基因表达 (p < 0.05)。用未刺激或经 IL-1β 处理的 OA SF 的 sEV 培养 OA 软骨细胞，导致 IL-6、IL-8 和 MMP-3 的分泌显着增加 (p < 0.01)。用来自 SF 的 sEV (± IL-1β) 培养的软骨外植体中 sGAG 的释放显着增加 (p < 0.01)。 亲本 SF 细胞和分离的 sEV 之间的 miRNA 特征不同。与亲代细胞相比，最近鉴定的破骨细胞生成调节因子 miR182 以及 miR4472-2、miR1302-3、miR6720、miR6087 和 miR4532 在 sEV 中富集，p < 0.01。来自未刺激或 IL-1β 刺激的 SF 的 sEV 的特征相似。 OA SF sEV 调节软骨细胞炎症和重塑反应。与亲本细胞相比，OA SF sEV 具有独特的特征，不会因 IL-1β 刺激而改变。这项研究深入了解了 OA 关节内的一种新型调节机制，可为未来的靶向治疗提供信息。