Palovarotene action against heterotopic ossification includes the inhibition of local participating activin A-expressing cell populations
Heterotopic ossification (HO) consists of endochondral bone forming at extraskeletal anatomical sites. One form of HO is acquired and instigated by traumas or invasive surgery, and another form is genetic and severe and characterizes patients with Fibrodysplasia Ossificans Progressiva (FOP). Previously, we showed that the synthetic retinoid agonist Palovarotene inhibits both acquired and genetic HO, and we and others subsequently showed that systemic administration of a neutralizing activin A antibody reduces both HO forms as well. Here, we asked whether Palovarotene's action includes an interference with endogenous activin A expression and/or participation in HO. Using a standard mouse model of acquired HO, we found that activin A protein and encoding RNA (Inhba) were prominent in chondrogenic cells within developing HO masses in control untreated mice. Single cell RNAseq (scRNAseq) assays verified that Inhba expression characterized chondroprogenitors and chondrocytes in control HO, in addition to its expected expression in inflammatory cells and macrophages particularly. Palovarotene administration (4 mg/kg/day/gavage) caused a sharp inhibition of both HO and amounts of activin A and Inhba transcripts. Bioinformatic analyses of scRNAseq datasets indicated that the drug had reduced interactions and crosstalk amongst local cell populations. To determine if Palovarotene inhibited Inhba expression directly, we tested it in primary cultures of growth plate chondrocytes. Drug treatment inhibited cartilage phenotypic expression but not Inhba expression. Our data reveal that Palovarotene intersects with activin A during its anti-HO action and markedly reduces the number of local participating Inhba-expressing cell populations. The data broaden the spectrum of HO culprits against which Palovarotene acts, accounting for its therapeutic effectiveness. Overall design: Single cell RNA seq of cells from Ectopic masses were harvested on day 5  from mice implanted with Matrigel/rhBMP2 mixture, with the latter mice treated systemically with Palovarotene Single cell RNA seq of cells from Ectopic masses were harvested on day 5  from mice implanted with Matrigel/rhBMP2 mixture, with the mice treated systemically with Palovarotene Associated previously published scRNA seq files can be found in GSE157679