Synergistic chondroprotective effect of chondroitin sulfate and glucosamine: a pharmacoproteomic study

V Calamia1, J Mateos1, P Fernández-Puente1, L Lourido1, E Montell0,2, J Vergés0,2, C Ruiz-Romero1, FJ Blanco1. 1INIBIC-Hospital Universitario A Coruña. ProteoRed/ISCIII. Proteomic Group, Rheumatology Division, Spain, 2Bioibérica, Basic Research Area, Scientific Medical Department, Spain

PURPOSE: To assess the synergistic chondroprotective effect of chondrotin sulphate (CS) and glucosamine hydrochloride (GH) in modifying cartilage extracellular matrix metabolism by chondrocytes secreted proteins analysis using iTRAQ technique.

METHODS

Cartilage obtained from patients undergoing joint replacement 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 osteoarthritic (OA) cartilage by enzymatic digestion were recovered and cultured in basic DMEM supplemented with antibiotics and 10% FBS. When confluence was reached, OA chondrocytes were treated with CS alone and in combination with GH (both at 200µg/mL). CS and GH were provided from Bioibérica, S.A. (Barcelona, Spain). 48 hours later, conditioned media were collected and their proteins were concentrated and quantified. Trypsin digestion and labelling with isobaric tags using iTRAQ reagents were performed. Then, peptides from the four conditions (untreated, CS-treated, GH-treated, and CS+GH-treated) were mixed, separated and analyzed by nanoscale reversed-phase liquid chromatography coupled to mass spectrometry (nLC-MALDI-MS/MS). The identification and quantification of proteins (by calculating the different iTRAQ tags intensities for each peptide) was carried out with ProteinPilot 3.0 software.

Each experiment was repeated at least three times. The statistical significance of the differences between mean values was determined using a two-tailed t test, considering significant p values ≤ 0.05. In the proteomic analyses, normalization tools and statistical package from ProteinPilot software were employed. We considered statistically significant only those changes with a p value of ≤0.05 and a ratio ≥1.2 (or ≤0.83), while exhibiting low error factor of the quantification (<2, in the ProteinPilot analysis).

RESULTS

Database search (UniprotKB/Swissprot) allowed us the identification of 143 different proteins in the OA chondrocyte secretome. For biological and functional analysis we considered only those proteins with a probability score higher than 95%, and a ratio ≥1.2 or ≤0.8. Finally, 17 secreted proteins presented statistically significant differences (p≤0.05) between untreated and treated samples: 13 were increased and 4 decreased (see table 1). CS alone modulated 3 proteins, while in combination with GH modulated 7 proteins. Most of the altered proteins are cartilage ECM components (59%), such as collagens and proteoglycans. Interestingly, we found increased LTBP2, thus suggesting a possible effect of the combined formulation in inhibiting osteophyte formation and fibrosis induced by high amounts of TGF-beta into OA joints.

CONCLUSIONS

We have carried out the first pharmacoproteomic study based on peptides labelling with 4 isobaric tags (iTRAQ) to study the effect of CS, alone and in combination with GH, on chondrocytes secretome. Our findings confirm the potential synergistic chondroprotective effect of chondrotin sulfate and glucosamine hydrochloride.

Table 1. Secreted proteins altered in OA treated chondrocytes compared to untreated cells.