Heterotopic ossification vs. fracture healing: Extracellular vesicle cargo proteins shed new light on bone formation

Stela Hrkac; Rudjer Novak; Grgur Salai; Simeon Grazio; Tomislav Vlahovic; Lovorka Grgurevic

doi:10.1016/j.bonr.2022.101177

Heterotopic ossification vs. fracture healing: Extracellular vesicle cargo proteins shed new light on bone formation

Bone Rep. 2022 Feb 28:16:101177. doi: 10.1016/j.bonr.2022.101177. eCollection 2022 Jun.

Authors

Stela Hrkac^{1

2}, Rudjer Novak¹, Grgur Salai^{1

3}, Simeon Grazio⁴, Tomislav Vlahovic⁵, Lovorka Grgurevic^{1

6}

Affiliations

¹ Center for Translational and Clinical Research, Department of Proteomics, School of Medicine, University of Zagreb, Zagreb, Croatia.
² Department of Emergency Medicine, University Hospital Centre Zagreb, Croatia.
³ Teaching Institute of Emergency Medicine of the City of Zagreb, Zagreb, Croatia.
⁴ University Department of Rheumatology, Physical and Rehabilitation Medicine, University Hospital Center "Sestre Milosrdnice", Zagreb, Croatia.
⁵ Clinic of Traumatology, University Hospital Center "Sestre Milosrdnice", Zagreb, Croatia.
⁶ Department of Anatomy, "Drago Perovic", School of Medicine, University of Zagreb, Zagreb, Croatia.

Abstract

Fibrodysplasia ossificans progressiva (FOP) is an extremely rare disease in which bone tissue forms in extraskeletal sites, which is known as heterotopic ossification (HO). Extracellular vesicles (EVs) are small phospholipid-enclosed particles released by various cells which have an emerging, but not completely understood role in various (patho)physiological processes. In order to further study the pathophysiology of FOP we conducted a small observational study comparing the proteomic profiles of EV cargo, derived from pooled plasma of four patient groups: FOP patient (N = 1) during active disease phase (flare-up), FOP patients during remission (N = 2), patients after long bone fracture (N = 20) and healthy controls (N = 10). After isolation of EVs - their protein cargo was determined using liquid chromatography / mass spectrometry, after which a functional gene enrichment analysis was performed. Our results show a sizeable difference of the proteomics profiles in which EVs from the bone fracture group show significant activity of integrin interactions, Wnt, VEGF, IGF-1 and PDGF pathways; conversely, FOP patients' EVs indicate that HO occurs via processes of innate immunity and the Ephrin B signaling pathway. We hypothesize that the Ephrin B signaling (expressed in EVs) contributes to HO by aiding in mesenchymal stem cell recruitment and osteogenic differentiation, as well as by contributing to the inflammatory response, including macrophage chemotaxis and activation. This is, to our knowledge, the first published analysis of EV protein cargo in FOP.

Keywords: Bone fractures; Ephrin B signaling; Extracellular vesicles; Fibrodysplasia ossificans progressiva; Heterotopic ossification; Microvesicles; Myositis ossificans.