Identifying The Role of and Treatment Targeting Bone Progenitor Cell VEGF Secretion On The Niche Supporting Traumatic Heterotopic Ossification
Charles Hwang, BS1, Serra Ucer, PhD1, Michael Sorkin, MD1, Shawn Loder, MD2, Michael T. Chung, MD3, Chase Pagani, none4, John Li, MD1, Caitlin Priest, BS1, Christopher Breuler, BS1, Kaetlin Vasquez, MS1, Shuli Li, MD, PhD1, Jiajia Xu, PhD5, Aaron James, MD, PhD5, Shailesh Agarwal, MD1, Benjamin Levi, MD1.
1University of Michigan, Ann Arbor, MI, USA, 2University of Pittsburgh, Pittsburgh, PA, USA, 3Stanford University, Stanford, CA, USA, 4Colgate University, Hamilton, NY, USA, 5Johns Hopkins University, Baltimore, MD, USA.
PURPOSE: Patients who sustain mechanical trauma, spinal cord injury, burns, or extremity surgeries are at risk for developing heterotopic ossification (HO), the pathologic formation of extraskeletal bone. HO is formed through a process of endochondral ossification initiated by acute inflammation. Vascular endothelial growth factor (VEGF) has been shown to be critical for both normal bone development and for fracture repair. We hypothesized that VEGF plays a central role during ossification of the cartilaginous matrix present during pathologic HO formation and that therapeutic targeting of the vascular niche is sufficient to prophylax against traumatic HO.
METHODS: Male C57BL/6J mice underwent Achilles' tendon transection and 30% of total body surface area (TBSA) dorsal burn injury to induce HO. Mice underwent Microfil CT and near infra-red imaging with intravascular injection of Angiosense to survey local vascularity. CDH5 endogenous reporter lines were imaged with confocal microscope. Hindlimb sections from injured mice were immunostained and injury site was harvested for flow cytometry and PCR. To further validate these findings and define the source of VEGF, co-staining with PDGFRa and VEGF was performed. Mice with genetic loss of Vegf in cells of mesenchymal lineage (Vegf cKO: Prx-cre/Vegffl/fl) and their littermate controls underwent burn/tenotomy (n=4-7). Human histological sections from analogous early HO sites were also examined via immunohistochemistry to confirm the translational value of these findings. Finally, a subset of mice after burn/tenotomy were treated with bevacizumab (biweekly injections of drug 10mg/kg) or vehicle control and were analyzed by histology (3 weeks) and MicroCT (9 weeks, n=5-6) for therapeutic testing.
RESULTS: In wild-type mice, vascular density was intimately associated with the HO anlagen as demonstrated by near infrared imaging with Angiosense, Microfil imaging 5 weeks after burn/tenotomy, and endogenous CDH5 signal (Fig A). Immunostaining of early human HO specimens with VEGFa and PDGFRa confirmed co-localization (Fig B), corroborated by observations in mouse histology (Fig C). Mice treated with bevacizumab, a potent anti-VEGF antibody, formed significantly less HO when compared at 9 weeks with vehicle control (2.64 v. 6.85 mm3, p=.0013). Genetic targeting produced parallel findings, with Vegf cKO mice forming significantly less HO near the distal tibia when compared with littermate controls (2.52 v. 4.53 mm3/mm, normalized to tibial cortical thickness compared to littermate control)(Fig D).
CONCLUSIONS: Leveraging our knowledge of normal bone development, these findings suggest that VEGF from the mesenchymal niche plays a critical role in the formation of HO and can be successfully targeted to attenuate this process via bevacizumab, an FDA-approved pharmacologic agent used for its anti-VEGF properties. Given the importance of VEGF and angiogenesis during normal post-injury healing, future studies will focus on identifying the ideal treatment timing to prevent ossification of the cartilage anlagen and minimize off target effects.
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