Leveraging lymphatics to reverse heterotopic ossification

Project Summary Heterotopic ossification (HO) is the pathologic formation of extra-skeletal bone that occurs in ~20% of patients after hip arthroplasty, burns or severe musculoskeletal injury. HO forms almost exclusively at sites rich in immune cells, and where we recently discovered a robust lymphatic network. Currently, no therapeutics exist to reverse HO once it has formed, and surgical intervention rarely restores baseline function and often results in recurrence. In this regard, there is a void in our understanding of the causative immune and lymphatic pathways behind this debilitating process and ways we can leverage this to resorb existing HO. Our compelling preliminary data demonstrate that lymphatic signaling is activated during HO progression in tandem with changes in T-cell RANKL expression and myeloid to osteoclast cell fate. Our unbiased transcriptional profiles in mouse cells recovered from traumatic HO regions in combination with magnetic resonance lymphangiography, tissue clearing and immunostaining of mouse and human HO sites revealed that a series of lymphatic-linked pathways, including VEGFD-VEGFR3 which are highly upregulated in tandem with observed changes in T-cell RANKL and myeloid cell osteoclast differentiation. Together, these observations have led to our hypothesis that MPC-derived VEGFD stimulates VEGFR3-expressing lymphatic ingrowth into HO and that T-cell Rankl stimulates myeloid cell to osteoclast fate, thereby providing a window of opportunity that can be leveraged to resorb existing HO. Aim 1: Determine lymphatic localization in skeletal and heterotopic bone post-injury and elucidate the role of VEGFR3 and lymphangiogenesis in regulating HO formation. Hypothesis: After injury, VEGFR3- stimulated lymphatics surround skeletal and heterotopic bone where they serve to partially limit ectopic bone deposition such that interfering with lymphangiogenic responses aggravates HO disease progression. Aim 2: Define the extent to which augmented VEGFR3-induced lymphangiogenesis causes osteolysis of heterotopic bone through T-cell Rankl signaling and myeloid cell-to-osteoclast differentiation. Hypothesis: MPC specific Vegfd overexpression and T-cell-derived Rankl expression stimulate myeloid cells at the injury site to differentiate into osteoclasts which resorb HO while sparing native skeletal bone which lack these osteoclast precursor myeloid cells. Aim 3: Evaluate the ability of pharmacologic activation of VEGFR3 to promote lymphangiogenesis and subsequent resorption of heterotopic bone while preserving normal skeletal bone. Hypothesis: Sustained stimulation of lymphangiogenesis resorbs HO without damaging skeletal bone. Impact: These studies will provide a comprehensive and mechanistic understanding of how VEGFD-VEGFR3 and lymphatics regulate T-cell signaling, myeloid cell fate and HO resorption offering a new therapy.