Tofiño-Vian Miguel, Guillén Maria Isabel, Pérez Del Caz María Dolores, Silvestre Antonio, Alcaraz María José
Instituto Interuniversitario de Investigación de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Universitat Politècnica de València, Universitat de València, Valencia, Spain.
Department of Pharmacy, Cardenal Herrera-CEU University, Valencia, Spain.
Cell Physiol Biochem. 2018;47(1):11-25. doi: 10.1159/000489739. Epub 2018 May 9.
BACKGROUND/AIMS: Chronic inflammation contributes to cartilage degeneration during the progression of osteoarthritis (OA). Adipose tissue-derived mesenchymal stem cells (AD-MSC) show great potential to treat inflammatory and degradative processes in OA and have demonstrated paracrine effects in chondrocytes. In the present work, we have isolated and characterized the extracellular vesicles from human AD-MSC to investigate their role in the chondroprotective actions of these cells.
AD-MSC were isolated by collagenase treatment from adipose tissue from healthy individuals subjected to abdominal lipectomy surgery. Microvesicles and exosomes were obtained from conditioned medium by filtration and differential centrifugation. Chondrocytes from OA patients were used in primary culture and stimulated with 10 ng/ml interleukin(IL)-1β in the presence or absence of AD-MSC microvesicles, exosomes or conditioned medium. Protein expression was investigated by ELISA and immunofluorescence, transcription factor-DNA binding by ELISA, gene expression by real-time PCR, prostaglandin E2 (PGE2) by radioimmunoassay, and matrix metalloproteinase (MMP) activity and nitric oxide (NO) production by fluorometry.
In OA chondrocytes stimulated with IL-1β, microvesicles and exosomes reduced the production of inflammatory mediators tumor necrosis factor-α, IL-6, PGE2 and NO. The downregulation of cyclooxygenase-2 and microsomal prostaglandin E synthase-1 would lead to the decreased PGE2 production while the effect on NO could depend on the reduction of inducible nitric oxide synthase expression. Treatment of OA chondrocytes with extracellular vesicles also decreased the release of MMP activity and MMP-13 expression whereas the production of the anti-inflammatory cytokine IL-10 and the expression of collagen II were significantly enhanced. The reduction of inflammatory and catabolic mediators could be the consequence of a lower activation of nuclear factor-κB and activator protein-1. The upregulation of annexin A1 specially in MV may contribute to the anti-inflammatory and chondroprotective effects of AD-MSC.
Our data support the interest of AD-MSC extracellular vesicles to develop new therapeutic approaches in joint conditions.
背景/目的：慢性炎症在骨关节炎（OA）进展过程中促使软骨退变。脂肪组织来源的间充质干细胞（AD-MSC）在治疗OA的炎症和退变过程中显示出巨大潜力，并已在软骨细胞中表现出旁分泌作用。在本研究中，我们分离并鉴定了人AD-MSC的细胞外囊泡，以研究它们在这些细胞软骨保护作用中的角色。
通过胶原酶处理从接受腹部脂肪切除术的健康个体的脂肪组织中分离AD-MSC。通过过滤和差速离心从条件培养基中获得微囊泡和外泌体。将OA患者的软骨细胞进行原代培养，并在存在或不存在AD-MSC微囊泡、外泌体或条件培养基的情况下用10 ng/ml白细胞介素（IL）-1β刺激。通过ELISA和免疫荧光研究蛋白质表达，通过ELISA研究转录因子与DNA的结合，通过实时PCR研究基因表达，通过放射免疫测定法研究前列腺素E2（PGE2），并通过荧光测定法研究基质金属蛋白酶（MMP）活性和一氧化氮（NO）产生。
在用IL-1β刺激的OA软骨细胞中，微囊泡和外泌体减少了炎症介质肿瘤坏死因子-α、IL-6、PGE2和NO的产生。环氧合酶-2和微粒体前列腺素E合酶-1的下调会导致PGE2产生减少，而对NO的影响可能取决于诱导型一氧化氮合酶表达的降低。用细胞外囊泡处理OA软骨细胞也降低了MMP活性的释放和MMP-13的表达，而抗炎细胞因子IL-10的产生和胶原蛋白II的表达则显著增强。炎症和分解代谢介质的减少可能是核因子-κB和活化蛋白-1活化降低的结果。膜联蛋白A1特别是在微囊泡中的上调可能有助于AD-MSC的抗炎和软骨保护作用。
我们的数据支持AD-MSC细胞外囊泡在开发关节疾病新治疗方法方面的价值。
