J. Mateos

Comparative proteomic analysis of bone marrow mesenchymal stem cells from OA and healthy patients undergoing chondrogenic differentiation

Average 3D cartilage thickness (Cg.Th) was calculated for the medial and lateral tibial plateaux. Joints were dissected, fixed in buffered formalin, and embedded in paraffin for histology processing with Safranin O-Fast Green. Registration of histology and microCT images allowed identification of the corresponding microCT slice. 2D Cg.Th from both images was calculated and correlated. Results: A sagittal view of a typical healthy knee joint (Fig. 1) shows the clearly defined cartilage between bone and joint space (contrast agent). The correlation between Cg.Th obtained from histology and microCT is depicted in figure 2a. Correlations were similar for OA-induced and healthy rat knees, therefore the data was pooled to give a correlation, R 1⁄4 0.89, p < 0.001. Additional 3D assessment of the cartilage was also achieved. The central medial load-bearing surface of a typical healthy 3D sample (Fig. 2b) shows regions of thicker cartilage compared to the same region, subject to erosion, in the OA model (Fig. 2c). With the segmentation of joint components, sample specific images can be generated (Fig. 3). Conclusions: The results clearly show a very good correlation of 2D Cg.Th from microCT and histology. The high-resolution, 3D data additionally allows whole-joint analysis, and permits direct investigation of local bone adaptation with cartilage degeneration. Validated individual models can be used to study load bearing of articulating surfaces, and the role of microstructural changes in the progression of OA. Reliable measures of cartilage combined with the regional analysis of bone provide a powerful analysis tool, and gives new insight into the progression and monitoring of disease models. Acknowledgement: Swiss Commission for Technology & Innovation, No. 9853.1. 87 COMPARATIVE PROTEOMIC ANALYSIS OF BONE MARROW MESENCHYMAL STEM CELLS FROM OA AND HEALTHY PATIENTS UNDERGOING CHONDROGENIC DIFFERENTIATION

504 COMPREHENSIVE ANALYSIS OF THE INTERLEUKIN-1-BETA-MEDIATED MODULATION OF CHONDROCYTE INTRACELLULAR AND EXTRACELLULAR PROTEOMES BY METABOLIC LABELLING

chondrocyte secreted proteins using the stable isotope labelling by amino acids in cell culture (SILAC) technique. Methods: Cartilage obtained from patients undergoing joint replacement, or from patients with no history of joint disease was provided by the Tissue Bank and the Autopsy Service at CHU A Coruña. The study was approved by the local Ethics Committee. Chondrocytes released from cartilage by enzymatic digestion were recovered and plated at low density in basic SILAC medium (Silantes) supplemented with antibiotics and 10% FBS dialyzed. In the case of light media, standard L-lysine (146 mg/L) and L-arginine (28 mg/L) were used, while in the heavy media isotope-labelled L-lysine (C6), and isotope-labelled L-arginine (C6,N4) were used. When complete incorporation of the heavy isotope was achieved in the cells (2-3 weeks), normal (N) chondrocytes were treated with CS 200μg/mL and then stimulated with IL-1β 5ng/mL, while osteoarthritic (OA) chondrocytes were treated with CS 200μg/mL alone. 48 hours later, conditioned media were collected and their proteins were concentrated and quantified. Heavy and light samples were mixed 1:1, and 4 μg of each mixed sample were in-solution reduced, alkylated and digested with trypsin. Separation and analysis of the resulting tryptic peptide mixtures was performed by nanoscale reversed-phase-LC-MS/MS. The identification and quantification of proteins was carried out with Protein Pilot software, which detects the heavy/light peak pairs and calculates the heavy/light ratios based on the peak areas. Identifications with a probability score higher than 95% and quantifications with a p value ≤0.05 were included in the results list. Results: Database search (UniprotKB/Swissprot) allowed us the identification of 39 different proteins in the OA chondrocyte secretome and 70 in N chondrocyte secretome. Interestingly, in both cases the most abundant protein was cartilage glycoprotein 39, which has been previously related with OA pathogenesis. For biological and functional analysis we considered only those proteins detected in all replicates with a heavy/light ratio ≥1.2 or ≤0.8. In OA chondrocytes, chondroitin sulfate mainly improves the anabolic/catabolic balance of the extracellular cartilage matrix, by increasing the level of structural proteins like collagens, decorin, lumican, vimentin and fibronectin. In N chondrocytes stimulated with IL1β, CS appears to act primarily as an anti-inflammatory drug. We show in this work how CS reduces inflammation by two mechanisms: directly, by decreasing the presence of potent inflammatory mediators like IL6 (ratio=0.6), and also indirectly, by increasing proteins such as tumor necrosis factor α-induced protein (TSG6, ratio=3). TSG6 plays a crucial role in extracellular matrix formation, inflammatory cell migration and cell proliferation. It’s a key component of a negative feedback loop operating through the protease network which reduces matrix degradation during OA process. The mechanism driven by TSG6 leads to a decrease in proMMPs activation, which might protect cartilage from extensive degradation even in the presence of acute inflammation (represented in our case by a high level of IL1β). Conclusions: We have carried out the first pharmacoproteomic study using a quantitative proteomics approach (SILAC), based on the metabolic labelling of the cells, to study the effect of CS on chondrocyte secretome. Our findings provide novel information about the mechanisms that may exert the in vivo beneficial effects of CS on the OA disease process. This work also illustrates that chondrocyte secreted proteins are an attractive sub-proteome for the discovery of new targets of CS in OA therapy.

049 PROTEOMICS APPROACH FOR THE SEARCH OF OA BIOMARKERS IN SERUM

bone. This supposition is supported by these data as concentrations of uCTXII were highly correlated to the severity of OA as measured by osteophyte formation, particularly of the medial compartment. The significant association of concentrations of collagenous urinary markers, ALPHA CTX and CTXII, to knee bone turnover by bone scintigraphy suggests that these markers may be useful non-invasive surrogates for active bone turnover in knee osteoarthritis.

A27 DECREASED LEVELS OF MITOCHONDRIAL SUPEROXIDE DISMUTASE IN OSTEOARTHRITIC HUMAN ARTICULAR CHONDROCYTES REVEALED BY MITOCHONDRIAL PROTEOMIC ANALYSIS

by the C/EBPb dificiency in the chondrocytes. p57 was co-localized with C/EBPb in late proliferative and pre-hypertrophic chondrocytes of the growth plate, which was dramatically decreased by the C/EBPb deficiency. The promoter activity of p57 was enhanced by C/EBPb transfection. Deletion, mutagenesis, and tandem-repeat analyses of the luciferase assay identified the core responsive element to be between the −150 and −130 bp region containing a putative C/EBP-binding motif. EMSA revealed the binding of C/EBPb-overexpressed nuclear extracts with the oligonucleotide including this region, whose specificity was verified by the C/EBPb antibody supershift. The knockdown of p57 by the siRNA inhibited the C/EBPb-induced hypertrophic differentiation in cultured chondrocytes. Conclusions: C/EBPb directly transactivates p57 at a specific C/EBP motif to promote transition from proliferation to hypertrophic differentiation of chondrocytes during skeletal growth, indicating the essential role of the C/EBPb/p57 signal during endochondral ossification.

476 GLUCOSAMINE SULFATE AND CHONDROITIN SULFATE: THEIR EFFECT ON CHONDROCYTE PROTEOME

and connective tissue growth factor. Il-1 stimulated release of the cartilage degeneration marker MMP-3, as well as proteins with uncharacterized roles in cartilage pathology, such as neutrophil gelatinase-associated lipocalin. RetA stimulated release of extracellular matrix proteins COMP, link protein and matrilin-3 into the media, accompanied by dramatic reduction in corresponding mRNA transcripts levels. Gelsolin, implicated in cytoskeletal reorganization in arthritic synovial fibroblasts but not previously associated with cartilage pathology, was regulated by Il-1 and RetA. Conclusions: This first analysis of mouse cartilage degradation and protein release using proteomics has identified proteins and fragments, some of which represent novel candidate biomarkers for cartilage degradation. Applying these proteomic techniques to wild-type and geneticallymodified mouse cartilage will provide insights into mechanisms of cartilage degeneration.