HIF-1α dependent RhoA as a novel therapeutic target to regulate rheumatoid arthritis fibroblast-like synoviocytes migration in vitro and in vivo

Jianhai Chen; Jingqin Chen; Jianwei Tan; Jian Li; Wenxiang Cheng; Liqing Ke; Qijing Wang; Anqiao Wang; Sien Lin; Gang Li; Peng Zhang; Benguo Wang

doi:10.1016/j.jot.2023.05.004

HIF-1α dependent RhoA as a novel therapeutic target to regulate rheumatoid arthritis fibroblast-like synoviocytes migration in vitro and in vivo

J Orthop Translat. 2023 Jun 6:40:49-57. doi: 10.1016/j.jot.2023.05.004. eCollection 2023 May.

Authors

Jianhai Chen^{1

2

3}, Jingqin Chen³, Jianwei Tan³, Jian Li², Wenxiang Cheng², Liqing Ke², Qijing Wang¹, Anqiao Wang¹, Sien Lin⁴, Gang Li⁴, Peng Zhang², Benguo Wang¹

Affiliations

¹ Rehabilitation Department of The Second Affiliated Hospital, School of Medicine, The Chinese University of Hong Kong, Shenzhen & Longgang District People's Hospital of Shenzhen, Shenzhen, Guangdong, 518172, China.
² Center for Translational Medicine Research and Development, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China.
³ Research Center for Biomedical Optics and Molecular Imaging, CAS Key Laboratory of Health Informatics, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China.
⁴ Department of Orthopaedics & Traumatology, Stem Cells and Regenerative Medicine Laboratory, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong, China.

Abstract

Objective: The purpose of this work is to investigate how the Rho family of GTPases A (RhoA) mediates the pathogenesis of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS).

Methods: The expression of RhoA in the synovial tissues of RA and Healthy people (Control) was detected using immunohistochemistry methods. The expression of RhoA and hypoxia-inducible factor-1α (HIF-1α) is inhibited by small interfering RNAs (siRNAs). The inhibition effect on RA-FLS migration was further investigated. The protein expression level of HIF-1α, RhoA, focal adhesion kinase (FAK), and myosin light chain (MLC) was also analysed using western blotting (WB). DBA1 mice were immunised with the mixture of bovine type II collagen and Freund's adjuvant to establish collagen induced arthritis (CIA) mouse model. Lip-siRhoA is administered through joint injection every two days. Micro-computed tomography (micro-CT) was used to detect mouse ankle joint destruction and evaluate the bone loss of the periarticular side. Destruction of the ankle articular cartilage was tested by histology. Expressions of P-RhoA, P-FAK and P-MLC in the ankle joint was detected by immunohistochemistry assay.

Results: The expression level of RhoA in the synovial tissues of RA patients was significantly higher than that in control group. Hypoxia was able to up-regulate the expression of RhoA. Whereas, HIF-1α siRNA (siHIF-1α) could down-regulate the expression of RhoA. Additionally, both of siHIF-1α and RhoA siRNA (siRhoA) delivered by liposome (Lip-siHIF-1α and Lip-siRhoA) were found to suppress FAK and MLC phosphorylation in vitro. In CIA mouse model, Lip-siRhoA was demonstrated to ameliorate the destruction of ankle joint and reduce the severity of ankle joint cartilage damage by micro-CT and histological staining, respectively. Therefore, inhibition of FLS cell migration can protect articular bone from destruction. Furthermore, the expression of P-RhoA, P-FAK and P-MLC was evaluated and found to be down-regulated by Lip-siRhoA in vivo.

Conclusion: The results demonstrated that under hypoxic environment, HIF-1α dependent RhoA pathway played an important role on cytoskeleton remodelling and RA-FLS migration. Through down-regulating RhoA expression, it could effectively treat RA in vitro and in vivo.

The translational potential of this article: Our study provides new evidence for the potential clinical application of RhoA as a candidate for the treatment of RA.

Keywords: Hypoxia; Migration; Rheumatoid arthritis; RhoA.