Activin A promotes the development of acquired heterotopic ossification and is an effective target for disease attenuation in mice

Heterotopic ossification (HO) is a common, potentially debilitating, acquired pathology that is instigated by tissue damage or other insults and involves inflammation followed by chondrogenesis, osteogenesis and extraskeletal bone accumulation. Current standard of care are not very effective and have side effects, making the search for new treatments urgent. Activin A is a member of the transforming growth factor-b (TGF-b) superfamily, is produced by activated macrophages and other inflammatory cells and signals through activation of canonical SMAD2 and SMAD3 (SMAD2/3) intracellular effectors. Because HO starts with inflammation and because pSMAD2/3 activation is chondrogenic, here we tested whether activin A stimulates HO development. Using standard mouse models of acquired intramuscular and subdermal HO, we found that systemic administration of a neutralizing activin A antibody markedly reduced HO development and bone accumulation. Single-cell RNAseq and developmental trajectories showed that the antibody treatment had sharply reduced the number of Sox9+ skeletal progenitors, many of which also expressed the gene encoding activin A (Inhba), that were recruited to the HO site. In line with the latter finding, gain-of-function assays showed that treatment of progenitors with recombinant activin A enhanced their chondrogenic differentiation and did so through SMAD2/3 signaling, and inclusion of activin A to HO-inducing in vivo implants enhanced HO development. Together, our data reveal that activin A is a critical upstream signaling stimulator of HO in mice and could represent an effective therapeutic target against acquired forms of this pathology in patients. Overall design: Single cell RNA seq of cells from Ectopic masses were harvested on day 5 from mice implanted with Matrigel alone or Matrigel/rhBMP2 mixture, with the latter mice treated systemically with pre-immune control IgG or activin A neutralizing antibody