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Hypoxia Signaling in Macrophages Orchestrates Endochondral Ossification in Fractures
David Cholok, BS, Kavitha Ranganathan, MD, Joe Habbouche, BS, Arminder Kaura, BS, Jonathan Butts, BS, Hsiao Sung, DDS, Caitlin Priest, BS, Michael Chung, MD, Chris Breuler, BS, John Li, MD, Shuli Li, MD,PhD, Serra Ucer, PhD, Steven R. Buchman, MD, Ben Levi, MD.
University of Michigan, Ann Arbor, MI, USA.

PURPOSE: The hypoxia-signaling pathway regulates endochondral ossification, and is critically important for proper modulation of inflammation, angiogenesis, and osteogenesis following trauma. Although hypoxia inducible factor-1α (Hif-1α) and hypoxia inducible factor-2α (Hif-2α) are critical mediators of this pathway, the role of these factors in fracture healing remains unclear, particularly with regards to the regulation of inflammation after injury. In this study, we define the role of hypoxia signaling in fracture healing, with the ultimate goal of identifying novel pharmacologic targets to improve fracture healing in a targeted manner.
METHODS: To identify the population of cells that most directly contributes to fracture healing, we performed femoral fractures with intramedullary rod placement in mice expressing a lineage restricted reporter switch in cells derived from limb-bud mesenchyme (Prx1Cre-mTmG). Confocal imaging was used to evaluate the fracture callus at 1, 3, and 6 weeks post-injury. Next, transgenic mice that conditionally lacked Hif-1α or Hif-2α within this contributory lineage (Prx1Cre-Hif2αf/f or Prx1CreER-Hif1αf/f) underwent femoral fractures. After 1, 3, and 6 weeks post-injury, microCT, biomechanical testing, and histology were performed. At 5 and 7 days post-injury, tissue from the fracture site was harvested, digested, and stained to quantify the contribution of macrophages to callus formation via flow cytometry. Immunostaining for macrophage markers (F4/80) with Hif-1α and HIF-2α was also performed to define hypoxia signaling specific to resident macrophages. Quantitative PCR and western blot were performed on macrophages and tissue from the fracture site to quantify the extent of local Hif-1α and Hif-2α expression.
RESULTS: Cells of the paraxial mesodermal lineage contributed directly to fracture healing. (Fig. 1A). Immunofluorescence imaging at the fracture site at 1 and 3 weeks after injury demonstrated persistent Hif-1α and Hif-2α co-staining with F4/80+ positive macrophages within the maturing callus (Fig.1B). Within macrophages specifically, inflammatory macrophages polarized to an M1 phenotype demonstrate increased Hif-1α expression compared to M2 macrophages (0.135 vs. 0.092, p<0.05), and VEGF (0.095 v. 0.008, p<0.05) Tissue collected from the fracture site was significantly enriched for Hif-1α protein at 1 and 3 weeks following injury as compared to uninjured femurs (25.3 v. 82.7, p<0.05) (Fig. 1D).
CONCLUSION: Our findings demonstrate that Hif-1α and Hif-2α are important mediators of fracture healing that are upregulated immediately following injury. This upregulation is necessary for macrophage recruitment to the fracture site. Given this data, we can now expand our pharmacologic armamentarium to include targeted modulation of hypoxia signaling as a means of improving fracture healing in vulnerable patient populations.


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