Purpose: Exosomes containing proteins, microRNAs and mRNAs are found in extracellular spaces such as blood and other body fluids and function as messengers in cell-cell communication through transfer of their molecular content. We hypothesize that exosomes containing miRNAs function in a novel communication mechanism among joint cells in osteoarthritis (OA) pathogenesis. We previously observed that the balance of anabolic and catabolic gene expression in chondrocytes was impaired by exosomes derived from OAsynovial fibroblasts (SFB). Angiogenesis in various joint tissues, including synovium, ligaments, menisci and the osteochondral junction, is a recently recognized important factor in OA pathogenesis. The purpose of this study was to investigate potential angiogenic function of exosomes from OA-SFB. Methods: Human SFB were obtained from a normal, OA and rheumatoid arthritis (RA) knee joints, and cultured with or without interleukin-1b (IL-1b). Exosomes were isolated by ExoQuick from IL-1b stimulated medium and control medium. Isolated exosomes fraction was measured for protein content using BCA protein assay kit. To examine the angiogenic functions of exosomes derived from SFB, we examined whether migration and tube formation in human umbilical vein endothelial cells (HUVECs) were affected by normal-, OA-, RA-SFB derived exosomes. Migration assay was evaluated using HUVECs in a modified Boyden chamber. In the tube formation assay, HUVECs were seeded into plates coated with growth-factor-reduced Matrigel and tube length was measured. miRNA profiles in the exosome preparations were established using 3D-Gene miRNA microarray. Results: Migration and tube formation activity were significantly higher in endothelial cells treated with exosomes from OA and RA SFB as compared to exosomes from normal SFB. IL-1b stimulation of SFB enhanced the angiogenic activity in their exosomes. However, migration and tube formation were not induced in endothelial cells by cytokine IL-1b alone. miRNA array data showed that 349 miRNAs were changed in OA SFB exosomes as compared to normal SFB expsomes. OA-and RA-SFB exosomes shared 29 miRNAs that were up-regulated and 35 miRNAs that were down-regulated compared normal SFB exosomes. This includes several differentially expressed angiogenesis-related miRNAs. Conclusions: Exosomes from OA SFB accelerate angiogenic activity in HUVECs, and might be involved in OA development by promoting angiogenesis throughout the joint. miRNA array data suggest an OA-related miRNA profile in exosomes that may serve as a novel biomarker. These results support our hypothesis that exosomes containing miRNAs function in a novel regulatory network that contributes to various aspects of OA pathogenesis.

Objective. To determine the expression of suppressor of cytokine signaling 3 (SOCS-3) in human articular chondrocytes and its functional consequences.Methods. Chondrocytes were isolated from the cartilage of patients with osteoarthritis (OA), patients with rheumatoid arthritis (RA), and trauma patients and from the healthy cartilage of patients with a femoral neck fracture. The human chondrocyte cell line G6 and primary bovine chondrocytes were used in validation experiments. SOCS-3 messenger RNA (mRNA) expression was measured by quantitative polymerase chain reaction, and SOCS-3 protein levels were determined by Western blotting and immunohistochemical analysis. Conclusion. This study demonstrated that both SOCS-3 mRNA and SOCS-3 protein are expressed in human arthritic chondrocytes and affect cellular responses involved in cartilage pathology.

Chondrocytes in mice developing osteoarthritis (OA) exhibit an aberrant response to the secreted cytokine transforming growth factor (TGF)-β, consisting in a potentiation of intracellular signaling downstream of the transmembrane type I receptor kinase activin receptor-like kinase (ALK)1 against canonical TGF-β receptor ALK5-mediated signaling. Unfortunately, the underlying mechanisms remain elusive. In order to identify novel druggable targets for OA, we aimed to investigate novel molecules regulating the ALK1/ALK5 balance in OA chondrocytes. We performed gene expression analysis of TGF-β signaling modulators in joints from three different mouse models of OA and found an upregulated expression of the TGF-β co-receptor Cripto (Tdgf1), which was validated in murine and human cartilage OA samples at the protein level. In vitro and ex vivo, elevated expression of Cripto favors the hypertrophic differentiation of chondrocytes, eventually contributing to tissue calcification. Furthermore, we found that Cripto participates in a TGF-β-ALK1-Cripto receptor complex in the plasma membrane, thereby inducing catabolic SMAD1/5 signaling in chondrocytes. In conclusion, we demonstrate that Cripto is expressed in OA and plays a functional role promoting chondrocyte hypertrophy, thereby becoming a novel potential therapeutic target in OA, for which there is no efficient cure or validated biomarker.

Background The intracellular suppressor of cytokine signaling (SOCS)-proteins are inducible and negative regulators in receptor signaling pathways of several cytokines, toll-like receptor ligands and some growth factors. We previously have shown in mice that SOCS3 is expressed in chondrocytes during arthritis [1]. Furthermore, SOCS3 overexpression inhibits the insulin-like growth factor (IGF)-1 response, the main anabolic factor for chondrocytes. Objectives To determine the expression and functional consequnces of SOCS3 in human articular chondrocytes. Methods Chondrocytes were isolated from articular cartilage of patients undergoing surgical joint replacement. The human immortalized chondrocyte cell line G6, human mesenchymal stem cell (MSC)-differentiated chondrocytes, and primary bovine chondrocytes were used for comparison. SOCS3 mRNA and protein levels were measured by quantitative PCR, western blotting and immunohistochemistry, respectively. Regulation of SOCS3 expression was examined following incubation with different cytokines and Toll-like receptor (TLR) agonists. To determine the effect of SOCS3 on the chondrocyte response to various stimuli, SOCS3 was either reduced by an inhibitor of SP1 (Mithramycin) or with short interference RNA, and enhanced by adenoviral transduction. Results The expression of SOCS3 was significantly enhanced in chondrocytes obtained from cartilage of osteoarthritis (OA) (ΔCt 3.4±1.0, n=18 patients) and rheumatoid arthritis (RA) (ΔCt 3.4±1.4, n=6) as compared to and healthy cartilage from fractures of neck of femur (NOF) patients (ΔCt 5.3±1.2, n=8). The expression of SOCS3 correlated markedly with other genes known to be expressed in arthritic chondrocytes such as RUNX2 (r=0.341), MMP13 (r=0.511), ADAMTS4 (r=0.779), and ADAMTS5 (r=0.647). No correlation was found with aggrecan expression and an inverse relationship was found with the collagen-type II gene Col2A1 (r=0.577). Western blots and imunohistochemisty confirmed the enhanced expression of SOCS3 at the protein level in arthritic cartilage. The expression of SOCS1 in chondrocytes was low and similar between the different patient groups. The expression of SOCS3 in the immortalized human chondrocyte cell-line (G6) and MSC-derived chondrocytes could be enhanced by interleukin-1 and conditioned medium of OA synovium explants. This was dependent on the transcription factor SP1 as the specific inhibitor mithramycin prevented SOCS3 upregulation in these cells. Forced expression of SOCS3 in bovine chondrocytes impairs several aspects of chondrocyte function, including nitiric oxide production and proteoglycan synthesis. Interestingly, a similar impairment of function was found in OA chondrocytes and knockdown of SOCS3 in these chondrocytes partially restored human chondrocyte function. Conclusions This study demonstrates that SOCS3 is highly expressed in human articular chondrocytes and affects cellular responses, which may have important implications for cartilage pathology in humans. References Smeets RL, Veenbergen S, Arntz...

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