Identification of the Role of Sensory Nerve TrkA Signaling on Progenitor Cell Fate after Extremity Trauma
Charles Hwang, BS1, David M. Stepien, MD, PhD1, Carrie Kubiak, MD1, Carolyn A. Meyers, BS2, Seungyong Lee, PhD2, Michael Sorkin, MD1, Chase A. Pagani, BA1, Talis Rehse, BS1, Noelle D. Visser, MS1, Mohamed A. Garada1, Zaid N. Khatib, BS1, Prasanth Kotha1, Jeffrey Lisiecki, MD1, Kaetlin Vasquez, MS1, Paul S. Cederna, MD1, Stephen W.P. Kemp, PhD1, Thomas L. Clemens, PhD2, Aaron W. James, MD, PhD2, Benjamin Levi, MD1.
1University of Michigan, Ann Arbor, MI, USA, 2Johns Hopkins University, Baltimore, MD, USA.
PURPOSE: Heterotopic ossification (HO) is a painful, debilitating formation of ectopic bone, often found after severe polytrauma, burn and spinal cord injury. Nerve growth factor (NGF) is a well-documented neurotrophic factor that has been correlated with phenotypes of chronic inflammation and pain. Recent literature has implicated neurotrophic signals including NGF as a crucial signal in normal bone development and fracture healing. Given the similar progression through endochondral ossification between fracture and post-traumatic heterotopic ossification, we hypothesized that neurogenic/neurotrophic signaling via sensory innervation is crucial for pathologic stem cell differentiation and HO.
METHODS: Microarray data of human ligament cells harvested from excisions of ossification of the posterior longitudinal ligament (GEO dataset GSE5464) were analyzed using linear modeling with empirical Bayes method for differential expression of genes. Using a proven mouse burn/tenotomy (B/T) HO model with bisection of Achilles tendon and concomitant 30% TBSA dorsal burn, mRNA was harvested from injured and uninjured tendon (n=3/group) and hybridized to Affymetrix microarray (1 week). To assess the requirement for TrkA+ nerves in HO genesis, TrkAF592A mice were used which are homozygous for knockin alleles rendering TrkA activity sensitive to specific inhibition by the membrane-permeable, small molecule 1NMPP1. Analysis of HO formation was performed by in TrkAF592A mice or littermate controls via ÁCT at 9 weeks (n=5-14/group). Imaging of nerve fibers was examined by confocal microscopy at 1 and 3 weeks using Thy1-YFP reporter sections.
RESULTS: Human spinal ligament cells subjected to cyclic strain exhibited robust upregulation of NGF and BRDF (Fig. A); a loading shown to be associated with ossification of connective tissues. Mouse BT (Fig. B, right) induced upregulation of characteristic genes of inflammation (Il6, Ptgs2, Ptger1, Il1a, Tacr1) vs. ininjured tendon at 1 week (left), as well as NGF. Immunolabeling of the injury site 1 week after surgery demonstrated NGF expression in close proximity to the native tendon, with regions of overlap with PDGFRα+ mesenchymal progenitor cells (top) vs. the uninjured contralateral tendon (bottom). Additionally, immunofluorescent labels on endogenous Thy1 reporters showed nerve fibers in close proximity to F4/80+ macrophages and PDGFRα+ progenitors, a cellular niche that facilitates HO formation. Interruption of TrkA signaling via the introduction of 1NMPP1 drug to homozygous F592A mutant mice demonstrated a significant reduction in the volume of floating, intramuscular HO.
CONCLUSIONS: HO induces upregulation of NGF, concordant with upregulation seen in strained human cells from patients with aberrant ossification of soft tissues. HO tissue demonstrated robust labeling of NGF at the injury site, exhibiting regions of colocalization with infiltrating mesenchymal progenitor cells. These signals are found in similar regions where Thy1+ nerves are present, whose TrkA receptors facilitate NGF signaling. Selective interruption of NGF-TrkA coupling attenuated ectopic bone volume suggesting neural signals modulate aberrant wound healing and the mesenchymal cell programming heralding HO. These data suggest that inhibition of neurogenic signaling through NGF-TrkA represents a potential therapeutic target to prevent post-traumatic HO.
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