Background: Pathologic calcification (PC) is a process characterized by the deposition of calcium-containing crystals in soft tissues that typically do not calcify, such as cartilage. In cartilage, crystals trigger chondrocyte apoptosis, induce oxidative stress (ROS) and inflammatory molecules (IL-6, IL-1β,…), and catabolic pathways (MMPs, ADAMTs) that play a pathogenic role in osteoarthritis (OA) [1, 2]. The gasotransmitter H ₂ S regulates multiple pathophysiological processes [3]. H ₂ S is produced in cells by three enzymes including CSE. H ₂ S levels can also be increased by H ₂ S-donors such as sodium thiosulfate (STS). We have previously demonstrated that the degree of cartilage PC in OA, is negatively correlated with the expression of CSE. Moreover, surgically-induced OA was attenuated by administration of STS. Finally, in the same murine OA model, we showed that CSE deficiency led to reduced H ₂ S levels and inhibition of chondrocyte calcification [4, 5].

Objectives: To test if increased cellular H ₂ S production by positive allosteric modulators of CSE (CSE-PAMs) may alleviate OA features in chondrocyte.

Methods: Murine ATDC5 chondrocytes and primary murine chondrocytes, isolated from 6-day old CSE-deficient mice and WT littermates, were used. Human TC28 chondrocytes, and primary human chondrocytes, isolated from undamaged OA cartilage were also studied. Cells were cultured in DMEM + 10% FBS. To induce calcification, cells were cultured in calcifying medium (calciprotein particles CPP2), in presence of CSE positive allosteric modulators (CSE-PAMs, initially described in [5], at 50 µM), or vehicle. Cells were stained with Alizarin red and crystals were quantified. Alkaline phosphatase (Alp) activity was measured in cell lysate by the p-Nitrophenyl Phosphate assay, mitochondrial ROS by MitoSOX, and murine or human IL-6 in cell supernatants by ELISA kit. H ₂ S production capacity was measured in cell/tissue homogenate by the lead acetate method or by the AzMC probe. Persulfidation of cellular proteins in TC28 chondrocytes stimulated with CPP2 for 1h in presence or absence of CSE-PAMs was performed by the maleimide assay. Calcifying TC28 chondrocytes, in presence or absence of CSE-PAMs were lysed and 3′-end bulk RNA barcoding and sequencing (BRB-seq) libraries was performed by Alithea Genomics SA (Lausanne, Switzerland).

Results: A dose-dependent increase in H ₂ S was observed in TC28 chondrocytes incubated with CSE-PAMs (SAN111, SAN115 and SAN401, MW ~ 300 Da, 0.3 - 50 µM). Inhibition of chondrocyte calcification by 50 µM SAN111 and SAN401 was of about 70% in human chondrocytes. Importantly, chondrocytes from CSE KO mice exhibited higher calcification compared to WT cells, which was not influenced by addition of SAN111, indicating its specificity for CSE. Mechanistically, SAN401 at 50 µM significantly inhibited ALP activity, ROS generation and IL-6 secretion, all key triggers of chondrocytes calcification. As most of the biological effects of H₂S can be explained by protein persulfidation, a post-translational modification which converts cysteine thiol (-SH) groups into persulfide (-SSH) groups, we assessed persulfidation levels in TC28 chondrocytes. The CSE-PAMs SAN115 and SAN401 increased persulfidation in cells after a 2-hour incubation. Finally, we analyzed whether SAN401 could modulate the transcriptional profile of TC28 chondrocytes undergoing calcification (CPP2-treated). Using BRB-seq we determined the top 50 differential expressed genes. Amongst these, we identified four genes ( INHBA , SERPINE1 , CCND1 , and SGK1 ) that were upregulated by CPP2 and simultaneously downregulated upon treatment with SAN401, and for which a role in calcification is established.

Conclusion: We developed a novel strategy to enhance H₂S levels by activating CSE activity. Our small molecules show potential as therapeutic agents by simultaneously boosting H₂S synthesis and inhibiting pathological cartilage calcification. These findings suggest that CSE-PAMs could represent a groundbreaking approach for treating cartilage PC-related conditions, such as OA.

REFERENCES: [1] I. Bernabei, A. So, N. Busso, S. Nasi, Cartilage calcification in osteoarthritis: mechanisms and clinical relevance, Nat Rev Rheumatol 19(1) (2023) 10-27.

[2] S. Nasi, A. So, C. Combes, M. Daudon, N. Busso, Interleukin-6 and chondrocyte mineralisation act in tandem to promote experimental osteoarthritis, Ann Rheum Dis 75(7) (2016) 1372-9.

[3] G. Cirino, C. Szabo, A. Papapetropoulos, Physiological roles of hydrogen sulfide in mammalian cells, tissues, and organs, Physiol Rev 103(1) (2023) 31-276.

[4] S. Nasi, H.K. Ea, F. Liote, A. So, N. Busso, Sodium Thiosulfate Prevents Chondrocyte Mineralization and Reduces the Severity of Murine Osteoarthritis, PLoS One 11(7) (2016) e0158196.

[5] S. Nasi, D. Ehirchiou, J. Bertrand, M. Castelblanco, J. Mitchell, I. Ishii, A. So, N. Busso, The Gasotransmitter Hydrogen Sulfide (H(2)S) Prevents Pathologic Calcification (PC) in Cartilage, Antioxidants (Basel) 10(9) (2021).

Acknowledgements: NIL.

Disclosure of Interests: None declared.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.