Pathological Ossification of Spinal Ligament‐Derived Fibroblasts in an Inflammatory Micro‐Environment via Activation of the ECM / ITGA5 / FAK / AKT Pathway in Ankylosing Spondylitis

Chronic inflammation and heterotopic ossification (HO) were typical hallmarks of ankylosing spondylitis (AS). Although existing target inflammation inhibitors could reduce inflammation but failed to halt HO progression, highlighting the need for osteogenesis‐specific treatments. However, the role of fibroblasts (Fbs) responsible for extracellular matrix (ECM) remodelling in AS‐associated pathological ossification remained unclear. In this study, osteogenic capacity, pathological condition and underlying molecular pathways for Fbs derived from AS spinal ligaments were validated. Fbs from uncalcified spinal ligaments of age‐matched AS patients/controls were compared. AS‐Fbs were treated with a gradient concentration of TNF‐α during osteogenic induction. Meanwhile, specific inhibition of AKT phosphorylation was applied to confirm the role of PI3K/AKT in osteogenesis. Upstream signalling pathways were also explored via RNA‐sequencing. Additionally, in vivo, subcutaneous cell transplantation was performed to simulate ectopic ossification. AS‐Fbs possessed superior osteogenic capacity than controls. Moreover, osteogenic signs could be promoted by low concentration TNF‐α‐induced inflammatory micro‐environment. Functional analysis revealed a crucial role of the PI3K/AKT pathway. Mechanistically, RNA‐seq identified the ECM/integrin subunit alpha 5 (ITGA5)/focal adhesion kinase (FAK) upstream pathway. In detail, osteogenic induction increased ECM protein levels including thrombospondin‐1 (THBS1) and fibulin‐1 (FBLN1), activating ITGA5, subsequent FAK phosphorylation, osteogenic markers expression and eventually inducing Fb‐to‐osteocyte differentiation. ITGA5 knockdown or specific FAK inhibition attenuated osteogenic phenotypes and reduced osteogenic proteins expression. Regarding in vivo experiments, subcutaneous bone‐like tissue formation derived from transplantation of AS‐Fbs was identified and could be suppressed significantly via the AKT phosphorylation inhibition. In conclusion, the findings of this study revealed potential causes for HO progression derived from AS‐Fbs in spite of effective anti‐inflammation therapy. Furthermore, the crucial role of ECM/ITGA5/FAK/AKT axial signalling pathways in the aberrant osteogenesis of AS‐Fbs was identified. Combined targeted intervention at the aforementioned signals and application of anti‐inflammatory agents might be a novel potential strategy for the treatment of pathological ectopic ossification caused by AS.