1. Investigación

Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes progressive joint destruction. Despite the advances in the treatment of this condition there remains a clinical need for safe therapies leading to clinical remission. Mesenchymal stem/stromal cells (MSCs) play immunomodulatory and regenerative roles which can be partly mediated by their secretome. In recent years, the important contribution of extracellular vesicles (EVs) to MSC actions has received an increasing interest as a new therapeutic approach. We provide an extensive overview of the immunomodulatory properties of MSC EVs and their effects on articular cells such as fibroblast-like synoviocytes that play a central role in joint destruction. This review discusses the anti-arthritic effects of MSC EVs in vitro and in animal models of RA as well as their potential mechanisms. Recent preclinical data suggest that transfer of non-coding RNAs by MSC EVs regulates key signaling pathways involved in the pathogenesis of RA. We also examine a number of EV modifications for improving their anti-arthritic efficacy and carrier ability for drug delivery.

Parathyroid hormone-related protein (PTHrP) C-terminal peptides regulate the metabolism of bone cells. PHTrP [107–111] (osteostatin) promotes bone repair in animal models of bone defects and prevents bone erosion in inflammatory arthritis. In addition to its positive effects on osteoblasts, osteostatin may inhibit bone resorption. The aim of this study was to determine the effects of osteostatin on human osteoclast differentiation and function. We used macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor B ligand (RANKL) to induce the osteoclast differentiation of adherent human peripheral blood mononuclear cells. Tartrate-resistant acid phosphatase (TRAP) staining was performed for the detection of the osteoclasts. The function of mature osteoclasts was assessed with a pit resorption assay. Gene expression was evaluated with qRT-PCR, and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) nuclear translocation was studied by immunofluorescence. We observed that osteostatin (100, 250 and 500 nM) decreased the differentiation of osteoclasts in a concentration-dependent manner, but it did not modify the resorptive ability of mature osteoclasts. In addition, osteostatin decreased the mRNA levels of cathepsin K, osteoclast associated Ig-like receptor (OSCAR) and NFATc1. The nuclear translocation of the master transcription factor in osteoclast differentiation NFATc1 was reduced by osteostatin. Our results suggest that the anti-resorptive effects of osteostatin may be dependent on the inhibition of osteoclastogenesis. This study has shown that osteostatin controls human osteoclast differentiation in vitro through the downregulation of NFATc1.

Background: Osteoarthritis (OA) is a joint disease characterized by cartilage degradation, low-grade synovitis and subchondral bone alterations. In the damaged joint, there is a progressive increase of oxidative stress leading to disruption of chondrocyte homeostasis. The modulation of oxidative stress could control the expression of inflammatory and catabolic mediators involved in OA. We have previously demonstrated that extracellular vesicles (EVs) present in the secretome of human mesenchymal stem cells from adipose tissue (AD-MSCs) exert antiinflammatory and anti-catabolic effects in OA chondrocytes. In the current work, we have investigated whether AD-MSC EVs could regulate oxidative stress in OA chondrocytes as well as the possible contribution of peroxiredoxin 6 (Prdx6). Methods: Microvesicles (MV) and exosomes (EX) were isolated from AD-MSC conditioned medium by differential centrifugation with size filtration. The size and concentration of EVs were determined by resistive pulse sensing. OA chondrocytes were isolated from knee articular cartilage of advanced OA patients. 4-Hydroxynonenal adducts, IL-6 and MMP-13 were determined by enzyme-linked immunosorbent assay. Expression of Prdx6 and autophagic markers was assessed by immunofluorescence and Western blotting. Prdx6 was downregulated in AD-MSCs by transfection with a specific siRNA. Results: MV and to a lesser extent EX significantly reduced the production of oxidative stress in OA chondrocytes stimulated with IL-1β. Treatment with MV resulted in a dramatic upregulation of Prdx6. MV also enhanced the expression of autophagy marker LC3B. We downregulated Prdx6 in AD-MSCs by using a specific siRNA and then MV were isolated. These Prdx6-silenced MV failed to modify oxidative stress and the expression of autophagy markers. We also assessed the possible contribution of Prdx6 to the effects of MV on IL-6 and MMP-13 production. The reduction in the levels of both mediators induced by MV was partly reverted after Prdx6 silencing. Conclusion: Our results indicate that EVs from AD-MSCs regulate the production of oxidative stress in OA chondrocytes during inflammation. Prdx6 may mediate the antioxidant and protective effects of MV. The translational potential of this article: This study gives insight into the protective properties of EVs from AD-MSCs in OA chondrocytes. Our findings support the development of novel therapies based on EVs to prevent or treat cartilage degradation.

Adipose tissue represents an abundant source of mesenchymal stem cells (MSC) for therapeutic purposes. Previous studies have demonstrated the anti-inflammatory potential of adipose tissue-derived MSC (ASC). Extracellular vesicles (EV) present in the conditioned medium (CM) have been shown to mediate the cytoprotective effects of human ASC secretome. Nevertheless, the role of EV in the anti-inflammatory effects of mouse-derived ASC is not known. The current study has investigated the influence of mouse-derived ASC CM and its fractions on the response of mouse-derived peritoneal macrophages against lipopolysaccharide (LPS). CM and its soluble fraction reduced the release of pro-inflammatory cytokines, adenosine triphosphate and nitric oxide in stimulated cells. They also enhanced the migration of neutrophils or monocytes, in the absence or presence of LPS, respectively, which is likely related to the presence of chemokines, and reduced the phagocytic response. The anti-inflammatory effect of CM may be dependent on the regulation of toll-like receptor 4 expression and nuclear factor- B activation. Our results demonstrate the antiinflammatory effects of mouse-derived ASC secretome in mouse-derived peritoneal macrophages stimulated with LPS and show that they are not mediated by EV.

Mesenchymal stem/stromal cells (MSCs) represent a promising therapy for musculoskeletal diseases. There is compelling evidence indicating that MSC e ects are mainly mediated by paracrine mechanisms and in particular by the secretion of extracellular vesicles (EVs). Many studies have thus suggested that EVs may be an alternative to cell therapy with MSCs in tissue repair. In this review, we summarize the current understanding of MSC EVs actions in preclinical studies of (1) immune regulation and rheumatoid arthritis, (2) bone repair and bone diseases, (3) cartilage repair and osteoarthritis, (4) intervertebral disk degeneration and (5) skeletal muscle and tendon repair. We also discuss the mechanisms underlying these actions and the perspectives of MSC EVs-based strategies for future treatments of musculoskeletal disorders.

The inflammatory process is an essential phenomenon in the induction of immune responses. Monocytes are key effector cells during the inflammatory process. A wide range of evidence indicates that mesenchymal stem cells from adipose tissue (ASC) are endowed with immunomodulatory capacity. However, the interaction between ASC and monocytes in the innate immune response is not well understood. The aim of this work was to investigate the possible paracrine anti-inflammatory effects of ASC in human monocytes. Monocytes were isolated from buffy coats and ASC from fat of non-obese patients. Conditioned medium (CM) from ASC in primary culture was used. We have assessed the effects of CM on the production of inflammatory mediators, degranulation, migration, phagocytic activity, senescence, oxidative stress, mitochondrial membrane potential and macrophage polarization. We have shown that ASC exert paracrine antiinflammatory actions on human monocytes. CM significantly reduced the production of TNFa, NO and PGE2 and the activation of NF-kB. In addition, we observed a significant reduction of degranulation, phagocytic activity and their migratory ability in the presence of the chemokine CCL2. The senescence process and the production of oxidative stress and mitochondrial dysfunction were inhibited by CM which also reduced the production of TNFa by M1 macrophages while enhanced TGFb1 and IL-10 release by M2 macrophages. This study have demonstrated relevant interactions of ASC with human monocytes and macrophages which are key players of the innate immune response. Our results indicate that ASC secretome mediates the anti-inflammatory actions of these cells. This paracrine mechanism would limit the duration and amplitude of the inflammatory response.

Background/Aims: Chronic inflammation contributes to cartilage degeneration during the progression of osteoarthritis (OA). Adipose tissue-derived mesenchymal stem cells (ADMSC) 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. Methods: 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. Results: 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. Conclusions: Our data support the interest of AD-MSC extracellular vesicles to develop new therapeutic approaches in joint conditions.