Yasmine Sedjai, Augustin Latourte, Mathilde Pezot, Eric Hay, Pascal Bizot, Mylène Zarka, Pascal Richette, Hang Korng Ea
Affiliation(s):
Inserm U1132
Abstract: Osteoarthritis (OA) is the most common articular joint disease in adults, that affect more than 250 million people worldwide. The current medical treatments are limited to symptomatic treatment. Osteoarthritis is characterised by an articular destruction and a degradation of the cartilage, secondary to chondrocyte’s dysfunction.
The degradation of the cartilage and the loss of proteoglycans modify osmolarity of the extracellular matrix, causing water flows and volume cell variations. The regulation of cell volume depends on ionic channels, in particular the volume-regulated anion channel (VRAC), which is a hetero-hexamer composed of LRRC8 subunits (LRRC8A–E).
Aims: Evaluation of the role of osmolarity and VRAC channel in chondrocyte’s homeostasis and cartilage degradation during osteoarthritis.
Methods: In vivo, osteoarthritis was induced by partial medial meniscectomy (MNX) in wild type mice (WT) and conditional KO mice with the deletion of Lrrc8A in monocytes/macrophages lineage, designed as Lrrc8AΔMφ. In vitro, articular chondrocytes from WT new-born mice have been isolated from femoral heads and cultured in hypo-(100 mOsm/L), iso-(300 mOsm/L) or hyper-osmotic (400 mOsm/L) medium. Expression of genes encoding cartilage anabolism and catabolism, inflammatory mediators and different sub-unites of VRAC channel were analysed by RT-qPCR. Human chondrocytes were isolated from knee cartilages collected during total joint arthroplasty for OA and submitted to different osmotic conditions. Ex vivo, human knee OA cartilage explants, were cultured in hypo- and isotonic media for 24 hours and then embedded in paraffin. Proteoglycan degradation was assessed by safranin O staining and expression of LRRC8A sub-unit by immunostaining on both damaged and non-damaged area.
Results: In vivo, Lrrc8AΔMφ mice developed, 8 weeks after MNX, less severe OA than WT mice (OARSI score: Lrrc8AΔMφ 3,2 ± 0,9 vs WT 5,2 ± 0,6; p < 0,05). In vitro, the osmotic stress modulated chondrocyte’s homeostasis that favored chondrocyte catabolism. Hypotonic stress increased the expression of genes encoding VRAC channel sub-units (Lrrc8A, Lrrc8B, Lrrc8D). It decreased anabolic gene’s expression (Sox-9, Acan). In contrast, it increased the expression of catabolic (MMP13, MMP3) and inflammatory genes (TNF, Il-6). Interestingly, inhibition of VRAC channel by DCPIB diminished hypotonic-induced catabolic response. Ex vivo, expression of LRRCC8A was higher in human explants cultured in hypotonic medium than those cultured in isotonic medium. This hypotonic stress was associated with proteoglycan degradation.
Conclusion: Hypotonic stress, via VRAC, promotes a catabolic phenotype of chondrocytes. Complementary studies were necessary to characterise underlying mechanisms.
Keywords: Osteoarthritis, VRAC, LRRC8A, osmolarity, hypotonicity, metabolism.