Research article
Evaluation of the therapeutic efficacy of human bone marrow mesenchymal stem cells with COX-2 silence and TGF-β3 overexpression in rabbits with antigen-induced arthritis

https://doi.org/10.1016/j.yexcr.2021.112945Get rights and content

Abstract

Objective

Mesenchymal stem cells (MSCs), especially genetically modified MSCs, have become a promising therapeutic approach for the treatment of rheumatoid arthritis (RA) through modulating immune responses. However, most MSCs used in the treatment of RA are modified based on a single gene. In this study, we evaluated the therapeutic effects of human BMSCs (hBMSCs) with COX-2 silence and TGF-β3 overexpression in the treatment of RA in a rabbit model.

Materials and methods

hBMSCs were cotransfected with shCOX-2 and TGF-β3 through lentiviral vector delivery. After SPIO-Molday ION Rhodamine-B™ (MIRB) labeling, lenti-shCOX2-TGF-β3 hBMSCs, lenti-shCOX2 hBMSCs, lenti-TGF-β3 hBMSCs, hBMSCs without genetic modification, or phosphate-buffered saline (PBS) were injected into the knee joint of rabbits with antigen-induced arthritis (AIA). The diameter of the knee joint and soft-tissue swelling score (STS) were recorded, and the levels of inflammatory mediators, including interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and prostaglandin E2 (PGE2) were evaluated by ELISA. Clinical 3.0T MR imaging (MRI) was used to track the distribution and dynamic migration of hBMSCs in the joint. Histopathological and immunohistochemical assays were conducted to localize labeled hBMSCs and assess the alteration of synovial hyperplasia, inflammatory cell infiltration, and cartilage damage.

Results

COX-2 silencing and TGF-β3 overexpression in hBMSCs were confirmed through real-time PCR and Western blot analyses. Reduced joint diameter, soft-tissue swelling (STS) score, and PGE2, IL-1β, and TNF-α expression were detected 4 weeks after injection of MIRB-labeled lenti-shCOX2-TGF-β3 hBMSCs into the joint in rabbits with AIA. Eight weeks after hBMSC injection, reduced inflammatory cell infiltration, improved hyperplasia of the synovial lining, recovered cartilage damage, and increased matrix staining were observed in joints injected with lenti-shCOX2-TGF-β3 hBMSCs and lenti-shCOX2 hBMSCs. Slight synovial hyperplasia, no surface fibrillation, and strong positive expression of collagen II staining in chondrocytes and cartilage matrix were detected in the joints 12 weeks after injection of lenti-shCOX2-TGF-β3 hBMSCs. In addition, hBMSCs were detected by MRI imaging throughout the process of hBMSC treatment.

Conclusion

Intra-articular injection of hBMSCs with COX-2 silence and TGFβ3 overexpression not only significantly inhibited joint inflammation and synovium hyperplasia, but also protected articular cartilage at the early stage. In addition, intra-articular injection of hBMSCs with COX-2 silence and TGFβ3 overexpression promoted chondrocyte and matrix proliferation. This study provides an alternative therapeutic strategy for the treatment of RA using genetically modified hBMSCs.

Introduction

Rheumatoid arthritis (RA) is a chronic autoimmune disease that affects joints, connective tissues, and associated bone and blood vessels with comprehensive metabolic and psychological comorbidities. RA starts with inflammation of the synovial membrane, which then spreads to other tissues of the joint (cartilage/bone damage) primarily due to the invasive characteristics of fibroblast-like synoviocytes (FLS). Immune responses induced by the activation of joint fibroblasts and the release of numerous proinflammatory cytokines are the most important pathogenetic processes causing joint destruction in RA [1]. Proinflammatory cytokines, including TNF-α, IL-6, IL-1β, and IL-17, play a critical rule in immunological destruction of cartilage in RA [2].

Currently, drug treatment of RA can be classified into four categories: non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, non-biologic disease-modifying anti-rheumatic drugs (DMARDs), and biologic DMARDs. However, long-term use of these drugs may cause adverse effects and side effects in a significant number of RA patients. For these reasons, some RA patients are intolerant or resistant to the abovementioned therapies [[3], [4], [5]]. Therefore, the identification of other molecular targets and approaches for the treatment of RA is a great challenge.

Mesenchymal stem cells (MSCs), which are multipotent stem cells found in bone marrow, adipose tissue, cartilage tissue, and placenta, among others, are promising targets to address the unresolved issues in the treatment of RA. MSCs exhibit strong immunosuppression through modulating the proliferation and differentiation of T and B cells, the maturation of dendritic cells, and the activity of NK cells. MSCs have been applied to treat a number of autoimmune diseases due to their immunoregulatory properties [3,4]. For example, xenogeneic MSCs result in better outcomes than allogeneic or syngeneic MSCs [6]. Currently, adult bone marrow is the most prevalent source of MSCs because of their self-renewal and multipotent differentiation properties, and bone marrow-derived mesenchymal stem cells (BMSCs) possess immunoregulatory capacities that have been permissive to gene transplantation [4].

Two types of cyclooxygenase (COX) have been identified and characterized, including the constitutive COX-1, which mediates physiological functions, and COX-2, an inducible enzyme that converts arachidonic acids to prostaglandins. Prostaglandin is an important biological mediator generated from membrane phospholipids by phospholipase A2 and COX. COX-2 plays a critical role in the production of PGE2 during inflammation. The enzymatic activity of COX-2 can be inhibited by NSAIDs, which have been widely used to relieve the symptoms caused by RA [7,8]. Transforming growth factor-β (TGF-β) is involved in several rheumatic and autoimmune disorders. As a major cytokine in response to inflammation, TGF-β promotes the differentiation of T cells with suppressive and regulatory activities [9,10]. Three TGF-β isoforms, including TGF-β1, TGF-β2, and TGF-β3, have been characterized in mammals. Of them, TGF-β3 exhibits the strongest chondrogenic effects on BMSCs through increasing matrix deposition and chondrogenic differentiation [11,12].

In our previous study, we demonstrated that MIRB-labeled lenti-shCOX2 hBMSCs transplanted into nude rat models via tail vein injection could be detected and monitored in vivo for up to 14 days [13]. Here, we confirmed the successful downregulation of COX-2 and overexpression of TGF-β3 in hBMSC cells by lentivirus transduction, MRI for tracking magnetically labeled genetically modified hBMSCs in AIA models, real-time monitoring and evaluation of the potential therapeutic effects of genetically modified hBMSCs in AIA models [14].

Section snippets

Bone marrow donors

Human BMSCs were isolated from the bone marrow of 16 healthy donors (mean age 48 ± 8.5 years; 10 females and 6 males) without any infectious or blood diseases. This study was approved by the Ethics Committee of Yantai Yuhuangding Hospital affiliated with the Medical College of Qingdao University, China. Informed consent was provided by all subjects according to the latest version of the Helsinki Declaration.

Cell culture

According to the whole bone marrow adherent method [15], human BMSCs were cultivated

Robust transduction efficiency of lenti-shCOX2 and lenti-TGFβ3

GFP was detected in most hBMSCs transduced with lentiviral vector (lenti-shCOX2 or lenti-TGFβ3). The transduction efficiency was evaluated through fluorescence microscopy at 48 h after lentivirus transduction. The transduction efficiency of lenti-shCOX2 and lenti-TGFβ3 was 90% at a MOI of 50 and 80% at a MOI of 70, respectively (Fig. 1 A). Only a few transduced cells underwent apoptosis after lentiviral vector transduction; however, the transduction rate was not affected.

Expression of COX-2 and TGFβ-3

The mRNA levels of

Discussion

In the present study, we successfully downregulated COX-2 expression and upregulated TGF-β3 expression in hBMSCs through lentivirus transduction. After being injected into AIA rabbits, MIRB-labeled lenti-shCOX2-TGF-β3 hBMSCs could be traced for 12 weeks in vivo. Our results showed that reduced expression of COX2 and TGFβ3 overexpression in hBMSCs significantly improved joint inflammation and inhibited synovium hyperplasia in AIA rabbits. Moreover, lenti-shCOX2-TGF-β3 hBMSCs prevented cartilage

Conclusion

In summary, we demonstrated that MIRB-labeled lenti-shCOX2-TGFβ3 hBMSCs successfully inhibited inflammation and improved cartilage damage in AIA rabbit models. Genetically modified hBMSCs could be monitored in a real-time manner based on histopathological approaches and MR imaging after cell transplantation. Our study provides an alternative opportunity for the treatment of RA.

Credit author statement

Tian He: Validation, Formal analysis, Investigation, Resources, Data curation, Writing – original draft. Shui Sun: Conceptualization, Methodology, Writing – review & editing, Visualization, Supervision, Project administration.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Ethics approval and consent to participate

The present study was performed in compliance with Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The experimental protocol was approved by the Experimental Animal Ethics Committee of Yantai Yuhuangding Hospital.

Consent for publication

Not applicable

Declaration of competing interest

The authors declare that there are no conflicts of interest.

Acknowledgments

Not applicable.

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