Background Oxidative stress results in the disruption of normal physiologic signaling leading to inflammatory changes, cartilage degradation and osteoarthritis (OA) progression (1). Recent advances have revealed the role of cell-derived microvesicles (MV) as a new mechanism of cell-to-cell communication with potential therapeutic applications. We have shown previously the antiinflammatory effects of human adipose tissue-derived mesenchymal stem cells (AD-MSC) conditioned medium in OA chondrocytes (2).
Objectives We have isolated the MV fraction from the secretome of AD-MSC to investigate its activity on oxidative stress and inflammation in OA chondrocytes stimulated with interleukin (IL)-1β. Furthermore, we have characterized the MV protein content by proteomic analysis.
Methods AD-MSC were isolated from fat of patients who undergone abdominoplasty (without obesity). MV were isolated from AD-MSC conditioned medium by differential centrifugation with size filtration. MV size and concentration were determined by resistive pulse sensing. Proteomic analysis was performed by LC-MS/MS, with ProteinPilot and PeakView software and the bioinformatic tools UNIPROT and PANTHER. OA chondrocytes were isolated from knee specimens of advanced OA patients, stimulated with IL-1β (10 ng/mL) and treated with MV (3.6x107 particles/mL) for 24h. Accumulation of 4-hydroxy-2-nonenal (HNE)-modified proteins and cytokines were measured by ELISA, NO production and MMP activity by fluorometry. Expression of specific proteins was evaluated by confocal microscopy or immunostaining. The data were analysed by ANOVA followed by Dunnett's test.
Results MV reduced the accumulation of HNE-modified proteins, a biomarker of oxidative stress–induced lipid peroxidation, in OA chondrocytes stimulated with IL-1 β. The production of NO, IL-6 and TNFα, as well as MMP activity were also significantly reduced by MV treatment, whereas IL-10 and collagen II were enhanced. Proteomic analysis of MV showed high levels (5.89-fold upregulation) of peroxiredoxin 6 (Prdx6), a member of the peroxiredoxin family of antioxidant proteins which is downregulated in OA cartilage (3). MV treatment increased the expression of Prdx6 in OA chondrocytes suggesting a protective role against oxidative stress in these cells.
Conclusions MV from AD-MSC downregulate the production of oxidative stress and inflammatory mediators in OA chondrocytes. Prdx6, an antioxidant enzyme, may contribute to the protective effects of MV. Our data support the interest of these MV to develop new therapeutic approaches.
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Acknowledgements Funded by SAF2013–48724-R (MINECO, FEDER) and PROMETEOII/2014/071 (Generalitat Valenciana).
Disclosure of Interest None declared