Journal Article

POS0617 THE GASOTRANSMITTER HYDROGEN SULFIDE PROTECTS AGAINST CALCIFIC TENDINOPATHY VIA INHIBITION OF LYSYL OXIDASES

Bernabei, I., Ehirchiou, D., Hügle, T., Stange, R., Kronenberg, D., Faure, E., Busso, N., Nasi, S., Bertrand, J.

2023

Overview

BackgroundPathological calcification ((PC), i.e. the deposition of calcium-containing crystals) in tendons is a hallmark of calcific tendinopathy (CT), a disease associated with pain, tendon rupture, and disability. Currently, only symptomatic treatments exist for CT and none of them target specifically calcification. Intriguingly, the gasotransmitter H2S has recently emerged as a potential anti-calcifying molecule [1].ObjectivesTo establish in CT the potential anti-calcifying effect of hydrogen sulfide (H2S) and of the H2S-producing enzyme cystathionine gamma lyase (CSE), and the underlying mechanisms involved.MethodsWild type (WT) and CSE knock-out (KO) tenocytes were isolated from adult mice Achilles’ tendons. Tenocyte were cultured in αMEM + 10% FBS + 50μg/ml ascorbic acid (Control medium) in presence of 10% calciprotein particles (calcification medium, CM). Spontaneous Achilles’ tendon CT in old mice (35 weeks old) and surgery-induced Achilles’ tendon CT in8-12 weeks old mice were quantified by micro computed tomography. Dynamic Young’s modulus was measured in 35 weeks old mice as previously described [2]. Immunohistochemistry was performed on human and murine tendon sections with anti-CSE and anti-LOX and anti-LOXL2rabbit polyclonal antibodies. LOX expression was analyzed by qPCR and by Western-blot. LOX(L) activity was quantified in cells supernatants and lysates.ResultsIn vitro, tenocyte calcification (in CM) was inhibited by exogenous H2S-donors, while it was exacerbated in CSE KO tenocytes, producing as expected less H2S than WT. Reduced calcification in tenocytes exposed to H2S was accompanied by decreased expression of genes coding for BMP2, BMP4 and decreased activation of the BMP signaling pathway (pSMAD1/5/8). Accordingly, BMPs expression and BMPs-pathway activation were exacerbated in CSE KO tenocytes compared to WT tenocytes.The protective role of CSE-H2S was confirmed in vivo. Indeed, in aged mice, micro computed tomography revealed exacerbated Achilles’ tendon calcification in CSE KO mice compared to WT. Interestingly, CSE deficiency led to reduced biomechanical strength, as the dynamic Young’s modulus was significantly decreased upon increased tendon displacement. Furthermore, using a tenotomy model of CT, we found an inverse correlation between CSE expression and calcification in tendons. This was confirmed in human tendons from CT patients, which exhibited decreased CSE expression where calcification was present.In parallel experiments, we found that calcification in tenocytes was significantly reduced by addition of BAPN, a pan-inhibitor of lysyl oxidases (LOX(L)) enzymes. LOX(L) family includes 5 enzymes LOX and LOX1-4, catalysing elastin and collagen cross-links. We next investigated if the anti-calcfiying effect of H2S in CT could be mediated by inhibition of LOX(L). Indeed, in CM-stimulated tenocytes we found that H2S impacts both LOX/LOX2 expression (as CSE deficiency increased significantly LOX/LOX2 gene expression) and LOX(L) activity (as CSE deficiency increased LOX(L) activity and conversely H2S dose-dependently inhibited LOX(L) activity in WT tenocytes. Finally, in vivo we discovered that, CSE was inversely correlated with calcification and LOX and LOXL2 expression in mouse and human tendons.ConclusionAltogether, our results suggest that increasing H2S levels in tenocytes could represent a future strategy to prevent or decrease calcification in CT, likely via down-modulation of LOX(L) expression and activity.References[1]Castelblanco, M., et al., The role of the gasotransmitter hydrogen sulfide in pathological calcification. Br J Pharmacol, 2020. 177(4): p. 778-792.[2]Kronenberg, D., et al., Increased Collagen Turnover Impairs Tendon Microstructure and Stability in Integrin alpha2beta1-Deficient Mice. Int J Mol Sci, 2020. 21(8).Acknowledgements:NIL.Disclosure of InterestsNone Declared.

• Publisher: BMJ Publishing Group Ltd and European League Against Rheumatism,Elsevier B.V,Elsevier Limited
• Subject: Animal models / Ascorbic acid / Calcification / Cell biology / Collagen / Computed tomography / Cross-linking / Crystals / Cystathionine g-lyase / Elastin / Enzymes / Gene expression / Hydrogen sulfide / Immunohistochemistry / LOX2 gene / Lysates / Mechanical properties / Medical treatment / Polyclonal antibodies / Scientific Abstracts / Signal transduction / Tendons / Tomography