Beijie Qi,1,* Qi Wang,1,* Shi Shi,1,* Qian Wang,1,* Weihao Jiang,1 Chenxu Wang,2 Dejian Li,1 Yaying Sun,3 Chengqing Yi1 

1Department of Orthopedics, Shanghai Pudong Hospital, Fudan University Pudong Medical Center, Shanghai, People’s Republic of China; 2Department of Orthopedics, The First Affiliated Hospital of Henan University, Kaifeng, Henan, People’s Republic of China; 3Department of Sports Medicine, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Jiao Tong University, Shanghai, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Chengqing Yi, Email chengqingyi100@163.com Yaying Sun, Email yaying.sun@shgh.cn

Background: Adhesive capsulitis of shoulder (ACS) is a common shoulder disease with pain, joint stiffness, and capsule fibrosis. The pathogenesis of ACS remains unclear, and long-term efficacy of current treatment for ACS is not satisfactory. Given the absence of targeted therapy, proteomic analysis is conducted on the capsules of ACS patients, in an effort to identify the potential target for ACS treatment. The liposome-based siRNA delivery system was utilized for in vivo experiments due to its non-immunogenicity, stability, and low off-target effects compared to other siRNA delivery methods. We aimed to provide a potential approach for targeted therapy of ACS.
Methods: Capsules from rotator cuff tear (RCT) patients with or without ACS were collected. Proteomic analysis was performed to detect differentially expressed proteins. For in vitro experiments, EdU assay, wound healing assay, CCK-8, quantitative polymerase chain reaction, immunofluorescence staining and western blot was conducted. Histological, immunofluorescence and biomechanical assessments were utilized in a mouse model of ACS. The properties of siRNA-loaded liposomes were determined using laser particle size analyzer and electron microscopy.
Results: Proteomic analysis identified integrin α 6 (ITGA6) as a significantly upregulated protein in the capsules of RCT patients with ACS compared to those without ACS. ITGA6 promoted cell proliferation, migration ability, and pro-fibrotic proteins expression, while the blockade of ITGA6 exhibited anti-fibrotic effect. The regulatory effect of ITGA6 was dependent on FAK/PI3K/Akt signaling pathway. Finally, liposome was prepared to carry si-ITGA6, and this si-ITGA6-loaded liposomes demonstrated a potent therapeutic effect in a mouse model of ACS. After si-ITGA6-loaded liposomes injection, the capsule thickness was decreased from 120.3 ± 14.3 μm to 73.3 ± 12.6 μm (p < 0.01), and the ROM also improved from 81.0 ± 9.0 to 112.2 ± 8.1 degrees (p < 0.01). These results indicated that si-ITGA6-loaded liposomes significantly ameliorated capsule fibrosis and joint mobility without indication of toxicity.
Conclusion: This research elucidated that ITGA6 was upregulated in the capsules of patients with ACS and is capable of significantly activating fibroblasts through the FAK/PI3K/Akt signaling pathway. By targeting ITGA6, si-ITGA6-loaded liposomes could effectively attenuate capsule fibrosis. In this study, we identified ITGA6 as a key regulator in ACS and demonstrated that targeting ITGA6 with siRNA-loaded liposomes effectively attenuated capsule fibrosis and improved joint mobility, offering a promising approach for targeted therapy of ACS.

Keywords: adhesive capsulitis, integrin α 6, fibrosis, liposome