Systemic Inhibition of Tgfβ Signaling Attenuates Trauma-induced Heterotopic Ossification
Nicole Patel, BS1, Michael Sorkin, MD1, Charles Hwang, BS1, John Li, MD1, Serra Ucer, PhD1, Sidra Kader, BS1, Kaetlin Vasquez, MS1, Shuli Li, MD, PhD1, Ravi Kumar, PhD2, Yuji Mishina, PhD1, Benjamin Levi, MD1.
1University of Michigan, Ann Arbor, MI, USA, 2Acceleron Pharma, Inc., Cambridge, MA, USA.
PURPOSE: Heterotopic ossification (HO) can commonly occur after severe trauma, burn injuries, and is a debilitating consequence of the congenital disease fibrodysplasia ossificans progressive (FOP). The etiology remains poorly understood, however, it is presumed that inflammation plays a critical role with several inflammatory cell types being recruited to the site of HO development. While the role of aberrant BMP signaling is established in HO formation, there is recent indication that macrophage secreted transforming growth factor beta 1 (TGFβ1) is also involved through propagation of chondrogenesis and endochondral ossification. Here we explore the effect of Tgfβ1 inhibition utilizing a near clinical Tgfβ1 receptor ligand trap to attenuate HO formation.
METHODS: Bone marrow derived macrophages were isolated and polarized into M2 phenotype in-vitro. Secreted TGFβ was measured in conditioned medium using ELISA. A model of traumatic heterotopic ossification involving a 30% dorsal burn and Achilles tenotomy was utilized in-vivo and 6-week old male C57BL/6 mice were randomized into receiving treatment with the pre-clinical pharmaceutical grade TGFβR-Fc ligand trap (n=10) or PBS control (n=10). This was administered subcutaneously twice weekly for 3 weeks. At 3 weeks, histology samples were collected, decalcified and stained with Safranin O to assess formation of HO anlagen (n=3/group). Volume of mature formed HO was quantified using micro CT analysis and imaging reconstruction at 9 weeks (n=6/group).
RESULTS: Recruited macrophages are a known source of cytokines that influence the inflammatory microenvironment. We therefore initially assessed the secretion of Tgfβ1 in cultured macrophages. Interestingly, we observed that while M0 macrophages secrete only minimal TGFβ1, the regenerative M2 polarized macrophages, which are known to be recruited to inflammatory sites, had a 500-fold increase in Tgfβ1 secretion. Furthermore, this increase in TGFβ1 levels was completely abrogated when the TGFβR-Fc ligand trap was present in the culture media (Fig A). We next aimed to assess the effect of systemically administered TGFβR-Fc on HO formation. Following treatment for 3 weeks, we observed a substantially attenuated development of HO anlagen in mice treated with TGFβR-Fc with decreased osseous deposition and marrow space formation on histologic examination (Fig B). Furthermore, the volume of mature HO was significantly decreased in the treatment group (Fig C).
CONCLUSIONS: Heterotopic ossification, from trauma or congenital FOP, severely limits mobility, function and quality of life of affected patients and currently, no prevention strategies exist. In this study, we demonstrate that Tgfβ1 signaling plays a critical role in HO formation and treatment with TGFβR-Fc is effective in attenuating the early stages of ectopic bone deposition resulting in decreased HO volume. This therapeutic approach reveals a novel avenue for the treatment of this condition that may translate into effective clinical applications.
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