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Abstract：
During the progression of knee osteoarthritis (OA), the synovium and infrapatellar fat pad (IFP) can serve as source for Substance P (SP) and calcitonin gene-related peptide (CGRP), two important pain-transmitting, immune, and inflammation modulating neuropeptides. Our previous studies showed that infrapatellar fat pad-derived mesenchymal stem/stromal cells (MSC) acquire a potent immunomodulatory phenotype and actively degrade Substance P via CD10 both in vitro and in vivo. On this basis, our hypothesis is that CD10-bound IFP-MSC sEVs can be engineered to target CGRP while retaining their anti-inflammatory phenotype. Herein, human IFP-MSC cultures were transduced with an adeno-associated virus (AAV) vector carrying a GFP-labelled gene for a CGRP antagonist peptide (aCGRP). The GFP positive aCGRP IFP-MSC were isolated and their sEVs\' miRNA and protein cargos were assessed using multiplex methods. Our results showed that purified aCGRP IFP-MSC cultures yielded sEVs with cargo of 147 distinct MSC-related miRNAs. Reactome analysis of miRNAs detected in these sEVs revealed strong involvement in the regulation of target genes involved in pathways that control pain, inflammation and cartilage homeostasis. Protein array of the sEVs cargo demonstrated high presence of key immunomodulatory and reparative proteins. Stimulated macrophages exposed to aCGRP IFP-MSC sEVs demonstrated a switch towards an alternate M2 status. Also, stimulated cortical neurons exposed to aCGRP IFP-MSC sEVs modulate their molecular pain signaling profile. Collectively, our data suggest that yielded sEVs can putatively target CGRP in vivo, while containing potent anti-inflammatory and analgesic cargo, suggesting the promise for novel sEVs-based therapeutic approaches to diseases such as OA.
摘要：
在膝骨关节炎（OA）的进展过程中，滑膜和髌下脂肪垫（IFP）可以作为P物质（SP）和降钙素基因相关肽（CGRP）的来源，两个重要的疼痛传递，免疫，和炎症调节神经肽。我们先前的研究表明，髌下脂肪垫衍生的间充质干细胞/基质细胞（MSC）获得了有效的免疫调节表型，并在体外和体内通过CD10主动降解P物质。在此基础上,我们的假设是,CD10结合的IFP-MSCsEV可以被设计为靶向CGRP,同时保留其抗炎表型.在这里，使用携带GFP标记的CGRP拮抗剂肽(aCGRP)基因的腺相关病毒(AAV)载体转导人IFP-MSC培养物。分离GFP阳性aCGRPIFP-MSC，并使用多重方法评估其sEV的miRNA和蛋白质货物。我们的结果表明，纯化的aCGRPIFP-MSC培养物产生的sEV具有147个不同的MSC相关miRNA的货物。在这些sEV中检测到的miRNAs的反应组分析显示，在控制疼痛的途径中涉及的靶基因的调节中强烈参与，炎症和软骨稳态。sEV货物的蛋白质阵列证明了关键免疫调节和修复蛋白的高度存在。暴露于aCGRPIFP-MSCsEV的刺激巨噬细胞显示向替代M2状态的转换。此外，暴露于aCGRPIFP-MSCsEV的刺激皮质神经元调节其分子疼痛信号传导谱。总的来说，我们的数据表明，产生的sEV可以在体内推定靶向CGRP，同时含有有效的抗炎和镇痛药物，这表明了基于sEV的新型治疗方法对OA等疾病的前景。
