Research

Chondrocyte dedifferentiation and fibrosis

Osteoarthritis (OA) is a degenerative joint disease characterized by progressive cartilage degradation but also synovial membrane inflammation, osteophyte formation and subchondral bone sclerosis. Medical care is mainly based on alleviating pain symptoms, but to date, no effective drug can stop the disease progression. A better comprehension of OA physiopathology is therefore a prerequisite to this purpose.

Cartilage is a tissue composed of only one cell type, chondrocytes, wrapped in a collagen rich extracellular matrix that they synthesize. Chondrocytes can adopt different phenotypes in vivo and in vitro, defined by the collagen type they produce. The acquisition of a hypertrophic phenotype (producing aberrant type X collagen and catabolic MMP-13 protease) by chondrocytes is well documented and contributes to OA development. However, it is increasingly believed that chondrocytes rather acquire a variety of other degenerated phenotypes at the onset of OA, including a “dedifferentiated-like” phenotype. With OA onset, chondrocytes undergo multiple changes, in terms of proliferation, viability, but also secretory profile.

In the lab, we contribute to a better characterization of dedifferentiated chondrocyte phenotype that might transdifferentiate into chondro-myo-fibroblasts inducing fibrosis and contributing to OA progression. Isolated from their matrix, chondrocytes present the particularity to dedifferentiate, producing fibroblastic type I and III collagens as well as pro-fibrotic proteins a-SMA and Cemip (1-3) .

Regarding to newly highlighted features, we proposed an active participation of dedifferentiated chondrocytes in OA physiopathology (4), through its capacity to proliferate, generate fibrocartilage (3) and produce some catabolic molecules. 

Further, we set up an in vitro model to study the modulation of fibrosis markers in synovial fibroblasts. Accordingly, we observed the anti-fibrotic impact of prednisolone in the presence of PGJ2, a PPAR-γ agonist (5).

References

1. Withofs N. et al., Ann. Nucl. Med. 2015Dec;29(10):839-47. [PMID:26254227]

2. Charlier E. et al., Osteoarthritis Cartilage. 2016 Feb; 24(2):315-24. [PMID:26318657]

3. Deroyer C. et al., Cell Death Dis. 2019 Feb 4;10(2):103. [PMID:30718510]

4. Charlier Edith, Deroyer Céline, Ciregia Federica, Malaise Olivier, Neuville Sophie, Plener Zelda, Malaise Michel, de Seny Dominique. Chondrocytes dedifferentiation and Osteoarthritis. Biochem. Pharmacol. 2019 Mar 29.Review. [PMID:30853397]

5. Vaamonde-Garcia C. et al. Biochem. Pharmacol. 2019 Mar 29. [PMID:30936016]

 

Human synovial fibroblast survival depends on autophagy

The basic wish in rheumatology is chondrocytes survival. Chondrocyte survival is very much influenced by inflammation, proliferation and apoptosis of synovial tissue. Accordingly, chondrocyte (6) and synovial fibroblast survival are extensively studied. Chondrocyte and synovial fibroblast survival are one of the basic bioassays in the lab that allowed us to search for protective substances (7-10). One of those is PPAR-γ agonist 15d-PGJ2. This prostaglandin has a lasting protective effect against both chondrocytes and synovial fibroblast cell death induced by NF-κB inhibitor BAY 11-7085 (10). This cell death was followed by intensive and persistent ERK phosphorylation that was efficiently downregulated by 15d-PGJ2. Furthermore, BAY 11-7085 induced PPAR-γ  dephosphorylation and degradation (11). In addition, 15d-PGJ2, especially when applied together with glucocorticoids, induces synovial fibroblast adipogenesis (12). Of interest, glucocorticoids induce marked leptin and Ob-R expressions in synovial fibroblasts, in the absence or presence of adipogenesis (12,13). Among PPAR-γ agonists, the plant flavonoid genistein has very interesting properties. While it increased adipogenesis, it suppresses endogenous and glucocorticoid-induced leptin and Ob-R.

Human synovial fibroblast survival is dependent on autophagy (14,15). Increased autophagy by BAY 11-7085 (14) and serum starvation (15) decreased synovial fibroblast survival. In contrast, autophagy inhibition has cell protective effects (14,15). Of interest, BAY 11-7085 induced GR phosphorylation on Ser211 as well as GR degradation (14). In addition, similar activation of GR receptor was observed during synovial fibroblast autophagy induced by serum deprivation. These results showed the importance of GR in synovial fibroblast autophagy.

Proteasome inhibition has marked cell protective effect against BAY 11-7085-induced synovial fibroblast death (11, 15). Proteasome inhibitors revealed p62 phosphorylated on Serine 349 while starvation increased its turnover. Of interest, phospho-mimetic mutant of p62 (S349E) has protective on synovial fibroblasts (15). These results showed that autophagy modulation is crucial for human synovial fibroblast survival.

References

6. Charlier Edith et al., Osteoarthritis.Int J Mol Sci. 2016 Dec 20;17(12). Review. [PMID:27999417]

7. Relic B et al., J Immunol. 2001 Feb 15;166(4):2775-82. [PMID: 11160344]

8. Guicheux J et al., J Cell Physiol. 2002 Jul;192(1):93-101.[PMID: 12115740]

9. Relić B et al., Lab Invest. 2002 Dec;82(12):1661-72. [PMID: 12480916]

10. Relic B et al., J Biol Chem. 2004 May 21;279(21):22399-40. [PMID: 15004016]

11. Relic B et al. J Biol Chem. 2006 Aug 11;281(32):22597-604. [PMID: 16766531]

12. Relic B et al., Lab Invest. 2009 Jul;89(7):811-22. [PMID: 19434061]

13. Zeddou M et al. Stem Cells Dev. 2012 Jul 20;21(11):1948-55. [PMID: 22087763]

14. Relic B et al. Oncotarget. 2016 Apr 26;7(17):23370-82. [PMID: 26993765]

15. Relic B et al.,Oncotarget. 2018 Nov 9;9(88):35830-35843. [PMID: 30533198]

 

Senescence- therapeutic intervention in osteoarthritis

Our laboratory has been for long interested in the action of glucocorticoids in osteoarthritis (OA). In the absence of curative treatment, management of OA remains symptomatic, including widely used intra-articular GC injections. The literature remains controversial on this subject. GCs appear initially useful for chondrogenic differentiation and protect against joint degradation in post-traumatic OA model. However, they can also generate well-known adverse effects (mainly metabolic ones) and become toxic for the joint by inhibiting the production of extracellular matrix by chondrocytes. At the clinical level, recent studies question their effectiveness on synovitis as they can aggravate cartilage damage without improving pain. Accordingly, we studied the role of glucocorticoids on the adipokines secretion (16) and biological pathways involved in synovial fibroblasts and chondrocytes. We also analyzed the role of new selective agonist of the glucocorticoid receptor (SEGRAs) such as Compound A (CpdA) as new therapeutic agent with better risk-benefit ratio (17).

Up to now, there is no validated curative treatment of OA in human. Recent progresses were done in the physiopathological understanding of OA with the involvement of cellular senescence. Cellular senescence is characterized by the irreversible arrest of proliferation in response to a genotoxic stress and secretion of molecules, representing a phenotype called SASP (senescence-associated secretory phenotype). SASP is made of cytokines, chemokines, growth factors and metalloproteases leading to tissue destruction. Our current work in the laboratory is to better understand the presence, but also the involvement of cellular senesence, in the OA pathogeny in order to find new target for emerging and innovating OA treatment.

The third therapeutic intervention developed in our laboratory is the study of mesenchymal stem cell and organels (especially exosome). Recently, a major therapeutic step was achieved with the therapeutic use of mesenchymal stem cells. Therapeutic autologous injection (“cell-therapy”) of MSCs preserved cartilage from degeneration in OA patients but several concerns about MSCs long-term efficiency appeared. We currently studied alternatives to MSCs with the use of exosome (that is called cell-free therapy). Exosomes are small vesicles that are secreted into biofluids such as synovial fluid by several cell types, especially MSCs and are under investigation to analyze their potential therapeutic effect in osteoarthritis.

References

16. Malaise O et al. Arthritis Res. Ther. 2016 Oct 4 ;18(1) :219. [PMID:27716396]

17. Malaise O et al. Rheumatology (Oxford). 2015 Jun;54(6):1087-92. [PMID:25389358]

Proteomic studies for biomarkers discovery

Over the last years, cutting-edge proteomics techniques and the acquisition of large cohorts of patients led us to identify new biomarkers in rheumatic diseases such as osteoarthritis and rheumatoid arthritis, besides confirming the presence of inflammation proteins (e.g. S100 and SAA proteins) (18-23). In rheumatoid arthritis we have also detected the alteration of protein glycosylation and future studies are foreseen to understand the role of this post-translational modification thanks to advances in MS-based methods. In addition, we have demonstrated the presence of specific peptides in osteoarthritis (C3f complement factor and a fragment of vitronectin) for which we have developed a new approach of absolute quantification by mass spectrometry (24,25). Both peptides can now be simultaneously quantified by ion trap microfluidic mass spectrometry in serum samples. Moreover, in vitro primary cells and affinity binding competition assays are using to help in shedding light upon the biological roles in inflammation and fibrosis of identified biomarkers.

References

18. de Seny D et al. Arthritis Rheum. 2005;52(12):3801-12. [PMID: 16320331]

19. de Seny D et al., Clin Chem. 2008;54(6):1066-75. [PMID: 18436720]

20. de Seny D et al, Ann Rheum Dis. 2011;70(6):1144-52. [PMID: 21362709]

21. de Seny D et al., PLoS One. 2013;8(6):e66769. [PMID: 23776697]

22. de Seny D et al. PLoS One. 2015;10(4):e0122904. [PMID: 25849372]

23. de Seny D et al.  J Transl Med. 2016;14:258. [PMID: 27599571]

24. Ourradi K et al. PLoS One. 2017;12(7):e0181334. [PMID: 28715494]

25.Cobraiville G et al. Talanta. 2017;169:170-180. [PMID: 28411808