Tamara Searle1, Ujvalla Kalluri1, Sonia Ahmad2, Bahja Ahmed Abdi1, Sandra Lopez1, Shiwen Xu1, Teresa Collins1, Jennifer Cross3, George Martin4, Henry Lopez5, Clayton Yates6 and Richard Stratton1, 1University College London, London, United Kingdom, 2Holy Family Red Crescent Medical College, Dhaka, Bangladesh, 3Aurinia Pharmaceuticals Inc., Rockford, MD, 4Riptide Bioscience Inc., Bethesda, MD, 5Riptide Bioscience Inc., Valejo, CA, 6Tuskegee University, Tuskegee, AL
Background/Purpose: Calcinosis may result from localised trans-differentiation of tissue resident stem cells in the subcutaneous layer of affected skin in systemic sclerosis (SSc), as a severe disabling manifestation linked to ischaemia and local trauma.Beyond SSc, inherited defects in Activin A responsive pathway (ACTVAR1) result in widespread calcinosis, as seen in Fibrodysplasia Ossificans Progessiva, where macrophages are implicated as the source of excess tissue Activin A.We investigated the possible induction of SSc calcinosis in a tissue culture model generated by stimulation of adipose derived mesenchymal stem cells (MSCs) with SSc patients' macrophages and explored the relevance of the Activin A pathway.
Methods: Clinical associations were screened in a database of well-characterised SSc patients with symptomatic calcinosis (n=28) and SSc without calcinosis (n=51). Human subcutaneous fat derived MSCs were cultured in osteogenic media, with or without SSc patients' monocyte-derived macrophages, with or without inhibitors, AUR300 10µM (peptide inhibitor of M2 macrophages) or SB431542 10µM (antagonist of Activin/ALK5 pathway). Cultures were stained with Alizarin red for osteogenesis on day 21. Macrophage secreted levels of Activin A under basal and ATP-stimulated conditions, were assayed by ELISA (Biotechne), as were plasma levels in n= 57 diffuse SSc patients (n=26 anti-Scl70, n=22 anti-RNA polymerase and 9 anti-U3RNP subgroup) and 21 healthy controls (HC).
Results: Most clinical characteristics did not differ significantly between patients with calcinosis and those without, including clinical subset, age, gender, disease duration, organ involvement or autoantibody subtype (all P values NS), whereas digital ulceration (DU) was associated with calcinosis (DU in 16/28 calcinosis and 13/51 no calcinosis, chi square P< 0.008). Notably, Activin A levels were increased in SSc patients' plasma samples when compared to controls, only in diffuse anti-Scl70 patient subgroup (plasma Activin A healthy controls 147, 80-249, anti-Scl70 SSc 261, 136-590 pg/ml, median, IQR, P< 0.05 Mann Whitney) (Figure 1A). Moreover, anti-Scl70 SSc patients' macrophages, released Activin A in tissue culture, enhanced by stimulation with BzATP (0.1 µM) and inhibited by AUR300 (10µM) (Figure 1B), and stimulated calcinosis in the MSC model, where addition of SSc macrophages (M) to the MSC cultures induced Alizarin red positive osteogenic foci at 21 days, blocked by both AUR300 and SB431542 (MSCs 0.833+/-0.247, MSCs+M 1.33+/-0.307, MSCs+M+SB 0.5+/-0, MSCs+M+AUR300 0.33+/-0.105, Alizarin Red stain 0-4, P< 0.012 for AUR300 effect) (Figure 1C).
Conclusion: Calcinosis occurs in both diffuse and limited SSc subsets and across autoantibody subgroups and was associated with digital ulceration.Elevated plasma levels of Activin A, most notable in anti-Scl70 subgroup, is consistent with systemic upregulation of the Activin A pathway in SSc.Activated macrophages from SSc patients are a potential source of Activin A capable of stimulating MSCs via an osteogenic/calcinosis model.The AUR300 and SB431542 compounds as studied represent potential therapeutic inhibitors of this severe and currently resistant complication of SSc.
Figure 1 Role of activin A/ALK5 pathway in SSc macrophages induced calcinosis. (A) Plasma levels of Activin A are raised in diffuse SSc anti-Scl70 (ATA) group. (B) Release of Activin A by anti-Scl70 positive SSc patient’s macrophages under basal and BzATP stimulated conditions: effect of peptide drug AUR300. (C) Role of activin A in anti-Scl70 SSc macrophage-induced MSC calcinosis model; antagonism by Activin/Alk5 signalling by inhibitor SB431542 and by M2 macrophage inhibitor. (ATP=0.1 µM BzATP, AUR=AUR300 10µM, M=macrophages, MSC=mesenchymal stem cells, SB=SB431542 10 µM, ARA=anti-RNA polymerase, ATA=anti-Scl70 subgroup, U3RNP=anti-U3RNP, *=P<0.05).
T. Searle: None; U. Kalluri: None; S. Ahmad: None; B. Ahmed Abdi: None; S. Lopez: None; S. Xu: None; T. Collins: Aurinia Pharmaceuticals Inc., 3; J. Cross: Aurinia Pharmaceuticals Inc., 3; G. Martin: Riptide Bioscience Inc., 4; H. Lopez: Riptide Bioscience Inc., 4; C. Yates: Riptide Bioscience Inc., 4; R. Stratton: Aurinia Pharmaceuticals Inc., 5, Riptide Bioscience Inc., 5.
