Inhibition of mTOR Signaling Reduces Mesenchymal Cell Migration To Sites Of Injury And Eliminates Heterotopic Ossification In A Mouse Model
David Cholok, BS, Michael Chung, MD, Shailesh Agarwal, MD, Shawn Loder, BS, Christopher Breuler, BS, Caitlin Priest, BS, Joseph Habbouche, BS, Arminder Kaura, BS, John Butts, BS, Jon Reimer, BS, John Li, MD, Hsiao H. Hsung, DDS, Kavitha Ranganathan, MD, David Fireman, BS, Shuli Li, PhD, Yuji Mishina, PhD, Benjamin Levi, MD.
University of Michigan, Ann Arbor, MI, USA.
PURPOSE: Patients with fibrodysplasia ossificans progressiva (FOP) carry a genetic mutation in the type I bone morphogenetic protein (BMP) receptor ACVR1 leading to hyperactivity. This mutation leads to pathologic cartilage and bone formation at extra-skeletal sites of even mild soft tissue injury which cause severe pain, restrict function and motion, and ultimately lead to early death. Until now, strategies to treat patients with FOP have focused on candidate small molecule agents with unproven safety profiles or translational potential. Here we show that an FDA-approved drug with a previously proven record of safety is able to completely inhibit ectopic lesions in a mouse model of FOP carrying the same mutation.
METHODS: Mice carrying the floxed FOP mutation (ACVR1 R206H) received a simultaneous hindlimb injection of Ad.cre to induce gene transformation and cardiotoxin to induce local injury (Ad.cre/CTX). Mice were treated with either daily vehicle control or rapamycin (5 mg/kg) administered i.p. (n=10/group). The presence of mesenchymal cells at the injury site was determined using immunofluorescent staining for PDGFRa and Sca-1 five days after injury. Ectopic cartilage and bone were determined using histology and microCT imaging 21 days after injury. PLGA microparticles were synthesized to deliver rapamycin as a slow-release; flow cytometry was used to quantify release time profile. Finally, a separate set of mice underwent Ad.cre/CTX injection with resection of formed HO 3 weeks after injury and subsequent treatment with or without rapamycin to eliminate recurrence.
RESULTS: While mice which received Ad.cre/CTX without rapamycin produced ectopic bone consistently, treatment with rapamycin nearly eliminated HO based on both microCT imaging (34.0 mm3 v. 1.0 mm3, p<0.01; Fig. 1A). Furthermore, histologic imaging showed elimination of ectopic cartilage with rapamycin treatment based on H&E, pentachrome staining (red arrows) and SOX9 immunofluorescence (Fig. 1B). Finally, rapamycin reduced the presence of mesenchymal cells (PDGFRa+ or aSMA+) at the injury site. PLGA microparticles released rapamycin during the first week after injury based on flow cytometry analysis (Fig. 1C). Mice that had resection of HO and were treated with rapamycin did not recur, while 100% of mice which had resection of HO without rapamycin developed new lesions at the resection site.
CONCLUSION: These findings demonstrate that rapamycin, an FDA-approved drug, eliminates ectopic cartilage and bone in a mouse model of FOP. This may occur through a reduction in the presence of activated mesenchymal cells at the injury site. These findings have prompted the initiation of clinical studies to assess efficacy of this potential therapeutic in humans. A slow-release microparticle may obviate repeated treatments in these patients. Rapamycin may also have a role in the management of FOP patients with ossified lesions, making surgical resection a reality for the first time.
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