The TrkA Antagonist AR786 Prevents Heterotopic Ossification

Abstract

Soft tissue trauma can result in the pathologic formation of cartilage and bone outside of the skeleton, termed heterotopic ossification (HO). Therapeutic options for treatment of HO are lacking. We recently recognized the role of sensory nerve fibers in trauma-induced HO through nerve growth factor (NGF) and tropomyosin receptor kinase A (TrkA) signaling whereby the deletion of NGF-TrkA signaling impairs axonal invasion and blunts osteochondral differentiation after HO induction in a mouse model. Given this observation, we hypothesized that utilization of a small molecule antagonist for TrkA in clinical development, AR786, would demonstrate therapeutic benefit in the prevention of HO. Association of NGF and innervation with human HO of the posterior longitudinal ligament (HOPLL) was analyzed by microarray and immunohistochemistry. In a mouse model, trauma-induced HO was generated by the combination of cutaneous burn and Achilles tenotomy in 10 week old C57BL/6J mice, which causes HO over a well characterized 9 week time period. NGF expression was examined by single cell sequencing, immunohistochemistry, and western blot. Dorsal root ganglion (DRG) from the innervating lumbar tracts were examined for pTrkA expression by immunohistochemistry. The phenotypic consequences of TrkA inhibition by AR786 on HO formation was next assessed (Vehicle, n=5 and AR786, n=5). Nerve innervation within the injury site was visualized using immunohistochemistry stained with Beta III Tubulin (TUBB3) and confocal microscopy. Abnormal chondrogenesis was assessed by cartilage histomorphometry, and immunohistochemical detection (SOX9 and COL2). Data were presented as Means ± SD. Results showed that NGF and associated downstream NGF signaling were increased among HOPLL patient cells. NGF immunoreactivity was present in perivascular cells adjacent to HO, fibroblastic cells, and bone-associated cells. Increased nerve fibers were present surrounding HO and axonal sprouting was confirmed by neural antigen S100 and neurofilament SM31 in human HO tissue sections. During HO progression in mice, NGF, proNGF, and pTrkA were upregulated following HO induction. Nerve frequency was significantly reduced with AR786 treated animals compared to vehicle control in uninjured (p<0.001, Vehicle; 548.2 ± 118.0 vs. AR786; 104.2 ± 47.9, µm3) and 3-weeks post-injury (p<0.001, Vehicle; 3255.6 ± 1405.1 vs. AR786; 462.8 ± 201.1, µm3). Consequently, reduced axonal ingrowth significantly delays in ectopic cartilage formation (p<0.01, Vehicle; 0.33 ± 0.07 vs. AR786; 0.07 ± 0.06, mm2). In conclusion, NGF-TrkA signaling mediates crucial functions in human and rodent HO, and inhibition of TrkA-expressing neural inputs by AR786 abrogates HO progression. These findings suggest that NGF-TrkA signaling inhibition can be a potential therapeutic use in HO patients as negative regulator of HO disease progression.