Comparative proteomic analysis identifies differentially expressed proteins and reveals potential mechanisms of traumatic heterotopic ossification progression

Zhenyuan Wei; Shang Guo; Hongwei Wang; Yang Zhao; Jiren Yan; Chi Zhang; Biao Zhong

doi:10.1016/j.jot.2022.04.003

Comparative proteomic analysis identifies differentially expressed proteins and reveals potential mechanisms of traumatic heterotopic ossification progression

J Orthop Translat. 2022 May 14:34:42-59. doi: 10.1016/j.jot.2022.04.003. eCollection 2022 May.

Authors

Zhenyuan Wei¹, Shang Guo¹, Hongwei Wang², Yang Zhao¹, Jiren Yan¹, Chi Zhang¹, Biao Zhong¹

Affiliations

¹ Department of Orthopedic Surgery, And Shanghai Institute of Microsurgery on Extremities, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China.
² Department of Medicine, the University of Chicago. Chicago, IL 60637, USA.

Abstract

Background: Traumatic Heterotopic Ossification (tHO) is one of complications of elbow fractures to the detriment of patients' rehabilitation, and the severity of tHO corresponds to the size of ectopic bone. It has yet to be elucidated which proteins and pathways underlying the progression of tHO, and biomarkers to predict the severity of tHO at early stage of the disease also need further investigation.

Methods: In this study, a new rat model with distinct volume of ectopic bone was established first. Then a data-independent acquisition proteomics approach was used to investigate injured site tissues sequentially obtained from these rats (2, 7, 14, and 28 days post-injury). Differentially expressed analysis, functional annotation and co-expression analysis and protein-protein interaction network were performed to explore the pathways and hub proteins in the tHO progression. Clinical samples from a nest case-control study were used to validate the selected proteins for predicting the severity of tHO.

Results: The Achilles Tenotomy (AT) induced significantly larger sizes of ectopic bone compared to Partial Achilles Tenotomy (PAT) in rat models. A total of 3547 quantifiable proteins were screened for differential expression analysis among the AT, PAT and control groups. The hierarchical clustering and expression pattern analysis revealed more apparent difference in the pathways such as oxidative phosphorylation, mitochondrial function, and sirtuin signaling between AT and PAT group at the early stage (2 dpi) of tHO. The co-expression analysis identified five hub proteins, UBA1, EIF3E, RPL17, RPL27, and RPS28. qPCR assay, immunoblot assay and immunohistochemistry assay verified that these proteins had higher expression level in the tissue samples of clinically relevant HO patients and clinically irrelevant HO patients than HO negative patients.

Conclusion: The new established animal model and proteome profile could serve as a solid foundation for the comprehensive investigation of the progression of traumatic heterotopic ossification. And the identified 5 proteins (UBA1, EIF3E, RPL17, RPL27, and RPS28) may serve as potential biomarkers to predict the severity of tHO.

The translational potential of this article: The proteins identified in this study may be the potential biomarkers and therapeutic targets for predicting and treating the tHO at early stage.

Keywords: Biomarker; Heterotopic ossification; Proteomics; Rat model.