Bernabei I.1, Kronenberg D.2, Stange R.2, Bertrand J.3, Hügle T1., So A.1, Busso N.1, Nasi S.1
1 Department Of Musculoskeletal Medicine, Service Of Rheumatology, Centre Hospitalier Universitaire Vaudois And University Of Lausanne, Switzerland 2 Department Of Regenerative Musculoskeletal Medicine, Institute Of Musculoskeletal Medicine, Westfaelische Wilhelms University Muenster, Muenster, Germany 3 Department Of Orthopaedic Surgery, Otto-Von-Guericke University, Magdeburg, Germany
Objective: Pathological (or heterotopic) calcification is the deposition of calcium-containing crystals in soft tissues that normally do not calcify. The deposition of these crystals in tendons such as the rotator cuff and the Achilles tendon is known as calcific tendinopathy (CT). CT is a painful condition, which increases tendon rupture rate and leads to disability. Understanding what inhibits calcification may provide new strategies to treat a condition for which existing therapies are ineffective.
Methods: We investigated the role of the gasotransmitter hydrogen sulfide (H2S), and in particular of the H2S-producing enzyme cystathionine γ-lyase (CSE) in CT. In vitro, we induced calcification in tenocytes from WT and CSE KO mice or we treated WT tenocytes with different H2S donors. In vivo, calcification was assessed in a surgery-induced murine model of CT (tenotomy of the Achilles tendon) and in a spontaneous model of CT (aging). Samples obtained from patients with rotator cuff or Achilles tendon CT were also analyzed. To investigate the underlying mechanisms of the CSE-H2S effect, we focused on the bone morphogenic proteins (BMPs) pathway. We also explored if altered extracellular matrix (ECM) organization, due to lysyl oxidase (LOX) activity and aberrant collagen-crosslinks, could also be involved in CT. Then, we studied if H2S production could affect this deleterious process.
Results: In vitro, tenocyte calcification was inhibited by exogenous H2S-donors, while it was exacerbated in CSE KO tenocytes. The protective role of CSE-H2S was confirmed in vivo. In aged mice, microtomography analysis revealed exacerbated Achilles tendon calcification in CSE KO mice compared to WT. In the surgery-induced model of CT, an inverse correlation between calcification and CSE expression in operated Achilles tendon was seen over time. Similarly, inversed correlation between calcification and CSE expression was found in human CT samples. 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). On the contrary, BMPs expression and BMPs-pathway activation were exacerbated in CSE KO tenocytes compared to WT tenocytes. We next investigated whether ECM disorganization could play a role in CT. Tenocytes cultured in calcification media and treated with the pan-inhibitor of lysyl oxidases (LOX, LOXL1-4) β-aminopropionitrile (BAPN) showed decreased calcification. This pointed to a potential beneficial role of LOX inhibition, therefore decreased collagen-crosslinks, in CT. By analysis of LOXs gene expression in WT and CSE KO tenocytes cultured in calcifying condition, we found much higher expression (4-fold) of LOX, LOL2 and LOXL4 in CSE KO tenocytes. Altogether, these results suggest that decreased H2S could lead to aberrant LOX expression and activity, excessive collagen cross-links in the ECM, and ultimately calcification. Further experiments are ongoing to prove these hypotheses.
Conclusions: We suggest targeting H2S production by CSE, or supplying an H2S-donor, is of therapeutic relevance to pathological calcification in the context of CT and can modify its disease course. The anti-mineralizing effect of H2S in tendons could be due to both inhibition of the BMPs pathway and suppression of abnormal LOXs activity.