P155 Modelling calcinosis in systemic sclerosis through disease microenvironment-stem cell interactions: effect of novel therapeutic peptide RP832c
Authors: Coumbe Ben G T , Ahmad Sonia N , Thomas Gemma , Borukhson Liubov , Abdi Bahja Ahmed , Lopez Henry , Garvin Charles , Jaynes Jesse , Yates Clayton , Martin George , Denton Christopher , Abraham David , Stratton Richard J 
Published: April 2021

Abstract
Background/Aims
Systemic sclerosis (SSc) is a rare and progressive connective tissue disease that is more common in women in the third to fifth decades of life and is rare in children. Calcinosis cutis, the deposition of calcium deposits within the subcutaneous tissue, remains a challenging non-lethal complication of SSc. The development of calcinosis cutis is a poorly understood area and there are no functional mouse models or laboratory models for calcinosis in SSc. In this study we present clinical database analysis plus two potential in vitro models for examining calcinosis in the setting of SSc.
Methods
Clinical modelling through database analysis of n = 79 SSc patients with and without calcinosis was attempted. In tissue culture studies, the first model system utilised adipose-derived mesencyhmal stem cells (MSCs) stimulated with interstitial fluid from healthy controls or SSc patients (both n = 4). In a second model, macrophages from patients with SSc (n = 4 lines) were used to stimulate MSCs in co-culture. We examined the impact that this has on the deposition of calcium hydroxyapatite production after prolonged culture in osteogenic media, measured using a scale (scored 0-4) to describe the degree of alizarin red uptake. We also investigated the role of an inhibitor of TGFβ signalling, SB432542 (10µM), and RP832c,(10µM), a peptide therapy which targets and repolarises activated SSc macrophages via CD206, in reducing calcium hydroxyapatite production.
Results
Clinical database analysis demonstrated that 35% of patients had evidence of calcinosis, but the only significant factor between the calcinosis and non-calcinosis group was the presence of digital ulcerations (p = 0.0008). The tissue fluid based model of calcinosis demonstrated that on day 21 the blister fluid obtained from SSc patients induced a significant increase in calcium deposition as compared to the culture media alone (3.9 vs 2.5, p = 0.00071), whereas SB431542, reduced calcium production by SSc fluid treated cells (3.9 vs 3.0, p = 0.2147). In the cell based model, macrophages derived from SSc patients induced the production of mineralised deposits. Both the TGFβ inhibitor SB432542 and the RP832c peptide reduced calcinosis in this model (SB431542 0.5 vs 1.3 p = 0.0547, RP832c 0.2 vs 1.3 p = 0.0147).
Conclusion
Our understanding of the underlying pathogenesis of calcinosis within SSc remains poor leading to therapeutic challenges for clinicians. From this analysis, it appears that presence of digital ulceration is associated clinically with calcinosis, possibly implicating aberrant tissue repair or ischaemia. Both tissue culture models presented here may recreate certain aspects of calcinosis in patients with SSc and may provide a basis for further evaluation of therapeutic inhibitors. At present there are no therapies that have been consistently found to be effective but our data support a possible role for macrophage - mesenchymal stem cell interactions and for TGFβ.
Disclosure
B.G.T. Coumbe: None. S.N. Ahmad: None. G. Thomas: None. L. Borukhson: None. B. Ahmed Abdi: None. H. Lopez: None. C. Garvin: None. J. Jaynes: None. C. Yates: None. G. Martin: None. C. Denton: None. D. Abraham: None. R.J. Stratton: None.