Novel Cartilage-Protective Activities in Pro-Resolving Exudates

MK Kaneva1, P Mori2, A Moore2, M Perretti1. 1William Harvey Research Institute, Barts' and The London School of Medicine and Dentistry, QMUL, London, UK, 2UCB Pharma, Slough, UK

Introduction: We hypothesized that tissue protective activities might be produced during the resolution phase of an experimental acute response. Application of an integrated approach with experimental exudates, proteomic analyses and bioactions with human chondrocytes led to the identification of novel tissue protective mediators – αα1-Antitrypsin (α1-AT), Hemopexin (Hx), and Gelsolin (GSN).

Methods: Rat pleural exudates (24h post-carrageenan injection) were subjected to gel filtration chromatography on HiPrep S200 16/60 SuperdexTM column (Tricorn), using AKTA FPLC system and run for mass spectrometry using ion-trap mass analyzer (MS/MS LTQ Orbitrap XL, Thermofisher Scientific). C28/I2 chondrocytes were grown in 3D high-density micromass1, incubated with the fractions (optimal dilution 1:30) in the presence or absence of interleukin(IL)-1ββ (20 ng/ml) or osteoarthritis synovial fluids (OA SF; 1:100) for 48h. α1-AT, GSN or Hx (0 – 30µg/ml) were tested against catabolic stimulation. Sulphated glycosaminoglycans (GAGs) deposition was assessed using Alcian Blue (AB) staining1. Gene expression was quantified by qPCR using QuantiTect Primer Assay (Qiagen) and PowerSYBR Green Mastermix (Promega) GAPDH – internal control. Results are based on calculation of 2-ΔΔCtmethod – relative amount of target genes were normalized to GAPDH and to untreated controls, with expression set to 1.0. Experiments were run 3 times. One-way ANOVA was used for statistical analyses.

Results: Whilst large molecular weight (MW) fractions inhibited (≤60%) GAG deposition, low MW fractions were strongly anabolic with >60% increase in GAG deposition mirrored by ~25% increase in COL2A1 and ACAN gene expression (p<0.05). Low MW fractions reverted the catabolic effect of IL1β. Proteome analysis of rat exudates revealed 66 proteins including hemopexin, gelsolin and α1-AT. Exogenous α1-AT abrogated the effect of IL1β on MMP13 (8-fold increase over control; p<0.001). Gelsolin and Hemopexin also afforded anabolic properties.

Incubation of chondrocyte micromasses with OA SF augmented IL6 and MMP13, with concomitant downregulation of COL2A1 and ACAN, gene products (p<0.01). α1-AT, and GSN, at 10 µg/ml, and GSN at 0.3 µg/ml, all attenuated these effects (p<0.01). The AB assay for GAG deposition confirmed the chondroprotection afforded by these proteins, with GAG deposition being inhibited with 80% by OA SF and marked reversal to control levels was observed with α1-AT, GSN and Hx (p<0.01).

Conclusion: The strategy for identification of novel chondroprotective activities in resolving exudates elicited new mediators like α1-AT, gelsolin and hemopexin as opportunities for new discovery programs.

Funded by a collaborative project between UCB Celltech and QMUL.

1. Greco et al., PMID: 21946086