Evaluation Of The Therapeutic Efficacy Of Human Bone Marrow Mesenchymal Stem Cells With COX-2 Silence And TGF-β3 Over-expression In Rabbits With Antigen-induced Arthritis

BackgroundRheumatoid arthritis(RA)is a chronic autoimmune disease that affects joints,connective tissues,and associated bone and blood vessels with numerous metabolic and psychological comorbidities.Currently,drugs used for the 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,a substantial number of RA patients are resistant to those drugs.Patients with severe RA have to undergone joint replacement surgery,but clinical outcomes are not acceptable.Therefore,identification of other molecular targets and approaches for the treatment of RA is a great challenge.Mesenchymal stem cells(MSCs)are multipotent stem cells found in bone marrow.Theoretically,cell therapy using MSCs is a promising approach for the treatment of RA due to their capacity of differentiation into three dermal lineages,including osteoblasts,chondrocytes and adipocytes.In addition,MSCs can be easily harvested from the bone marrow,peripheral blood,adipose tissue,synovium and other mesodermal tissues.Furthermore,MSCs exhibit strong immunosuppression through regulating the proliferation and differentiation of T and B cells,promoting the maturation of dendritic cells,and activating NK cells.MSCs have been applied to the treatment of a number of auto-immune diseases based on their immunoregulatory properties.Significant advances have been made in recent years in RA treatment in animal models using gene therapy approaches.Delivery of multiple genes in target-specific or inducible manner for the continuous intra-articular expression of therapeutic products is highlighted in gene therapy.Two types of cyclooxygenase(COX)have been identified and characterized.Constitutive COX-1 mediates physiological functions.Inducible COX-2 converts arachidonic acids to prostaglandins,which is an important biological mediator generated from membrane phospholipids by phospholipase A2 and COX.COX-2 plays a critical role in the production of prostaglandin E2(PGE2)during inflammation.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.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.In the present study,we simultaneously downregulated the expression of COX-2 and upregulated the expression of TGF-β3 in hBMSCs to investigate the potential therapeutic effects of genetically modified hBMSCs in antigen-induced arthritis(AIA)model.PartⅠThe differentiation of MIRB labeled human bone marrow-derived mesenchymal stem cells(hBMSCs)with COX-2 silence and TGF-β3 over-expressionObjectiveTo evaluate the differentiation of MIRB-labeled hBMSCs with lentivirus-mediated COX-2 silence and TGF-β3 over-expression in vitro.Methods1.hBMSCs were co-transfected with shCOX-2 and TGF-β3 through lentivirus vectors.2.The expression COX-2 and TGF-β3 in hBMSCs were assessed by real-time PCR and Western blotting assays.3.Compare the Differentiation capacity of MIRB-labeled Lenti-hBMSCs between different groups.Results1.The transduction efficiency was evaluated with fluorescence microscopy at 48h after lentivirus transduction.The highest transduction efficiency of Lenti-shCOX2 and Lenti-TGFβ3 was at MOI of 50 and 70,respectively.Only few transduced cells undergone apoptosis,suggesting successful lentivirus transduction.2.The mRNA levels of PGE2,SOX9,and ACAN in hBMSCs after lentivirus transduction were assessed by real-time PCR.A significantly reduced mRNA level of PGE2 was detected in the Lenti-shCOX2 and co-transduction groups(P<0.01).The mRNA levels of SOX9 and AC AN were significantly increased in the Lenti-TGFβ3 and co-transduction groups.The average optical density of COX-2 protein in the the Lenti-shCOX2 infection and co-transduction groups was significantly lower than that in the control groups(P<0.05).The TGFβ3 protein level in the Lenti-TGFβ3 and co-transduced groups was significantly increased(P<0.05).3.EGFP-stained green fluorescence was observed in both the cytoplasm and nucleus under fluorescence microscopy.Red fluorescence was detected in the cytoplasm of MIRB-labeled lenti-hBMSCs.Similar results were observed in the cytoplasm of hBMSCs with Fe based on Prussian Blue staining.Regarding osteogenic and chondrogenic differentiation,significantly higher density of nodules and alcian blue staining was detected in MIRB-labeled lenti-TGFβ3-hBMSCs and lenti-shCOX-2-TGFβ3 hBMSCs compared with other groups.Conclusions1.Transduction of Lenti-shCOX2 and Lenti-TGFβ3 was successfully achieved in hBMSCs with high transduction efficiency.Lentivirus transduction resulted in significant down-regulation of COX-2 and over-expression of TGF-β3 in hBMSC cells.2.The differentiation of genetically modified hBMSCs was not significantly affecgted by MIRB labeling.TGFβ3 transfection improved osteogenic and chondrogenic differentiation in hBMSCs.Part ⅡTreatment of antigen-induced arthritis in rabbits through intra-articular delivery of human bone marrow mesenchymal stem cells with COX-2 silence and TGF-β3 over-expressionObjectiveTo evaluate the therapeutic efficacy of hBMSCs with COX-2 silence and TGF-β3 over-expression in a rabbit RA model.Methods1.Establishment of the antigen-induced arthritis(AIA)model in rabbits.2.Tracking MIRB-labeled hBMSCs with COX-2 silence and TGF-β3 over-expression in the knee joint cavity using magnetic resonance imaging MRI.3.Evaluation of the levels of a number of inflammatory mediators using ELISA.4.Examination of the joints under macroscopic.5.Iron detection in synovial membrane and infrapatellar fat pad tissues using Prussian blue staining and Laser Scanning Confocal Microscopy System.6.Histopathological evaluation of synovial membrane and infrapatellar fat pad stained with hematoxylin and eosin.7.The joints were cut and stained with Safranin O/Fast green to assess cartilage alterations.The modified Mankin’s score was used to microscopically evaluate the cartilage status.8.Cartilage was incubated with anti-collagen Ⅱ antibodies to evaluate cartilage damage based on the percentage of positively stained collagen Ⅱ area.Results1.The diameter of knee joints and STS scores in other groups were significantly lower than those in the control group.At 4 weeks,the knee joint diameter of the lenti-shCOX2 hBMSCs group and lenti-shCOX2-TGFβ3 hBMSCs groups were significantly smaller than the single hBMSCs group and lenti-TGFβ3 hBMSCs groups(P<0.05).However,no significant difference in the diameter of knee joints was identified between the lenti-shCOX2 hBMSCs group and lenti-shCOX2-TGFβ3 hBMSCs groups(P>0.05).2.Under GRE T2-weighted MR images,joint effusion and synovium hyperplasia of various degrees in the right knees of rabbits were observed in all groups one week after AIA.In the control group,the joint effusion and synovium hyperplasia became gradually worse.Destruction of articular cartilage,change of periarticular soft tissues,and progressing joint flexion contracture were detected 12 weeks after the establishment of AIA model.Effusion and synovial hyperplasia in the lenti-shCOX2-TGFβ3 hBMSCs group were further improved compared with the single hBMSCs group at 4 weeks.Inflammation was rarely detected at both 8 and 12 weeks after the transplantation of lenti-TGFβ3-shCOX2 hBMSCs.3.The expression of inflammatory cytokines,including IL-1β,TNF-α,and PGE2,in the serum and synovial fluid of the lenti-shCOX2-TGFβ3 hBMSCs group was significantly lower than that in the control and single gene transfection groups.The expression of these inflammatory cytokines was gradually inhibited after lenti-TGFβ3-shCOX2 hBMSCs transfection at 12 weeks.4.Brown discoloration was observed in infrapatellar fat pad in most joints treated with MIRB-labeled hBMSCs of different groups.No cartilage damage was observed in any groups at the fourth week.The lenti-shCOX2-TGFβ3 hBMSCs group demonstrated the best outcomes and showed generally intact surfaces,although minor surface fibrillation was observed after 12 weeks.5.Iron particles were detected between cell layers of hyperplasia of synovial lining,around adipose cell under hyperplasia of synovial from infrapatellar fat pad,and in proliferating fibroblasts of infrapatellar fat pad,which is consistent with MRI imaging.6.H&E staining and histopathological scoring suggest improved inflammation in single hBMSCs group and lenti-shCOX2 hBMSCs group compared with the control group at 8 and 12 weeks(P<0.01).Only synovial hyperplasia was observed at 12 weeks in lenti-shCOX2-TGFβ3 hBMSCs group,the histopathological score of synovium in lenti-shCOX2-TGFβ3 hBMSCs group was lower than that in lenti-shCOX2 hBMSCs(P<0.05).7.After 12 weeks,Safranin O/Fast green staining demonstrated progressive cartilage damage with high Mankin’s score in the control and single hBMSCs group.Nevertheless,non-surface fibrillation and increased chondrocytes and matrix hyperchromatism were observed in lenti-shCOX2-TGFβ3 hBMSCs group during the entire treatment process.8.Regarding immunohistochemical staining,the lenti-shCOX2-TGFβ3 hBMSCs group exhibited stronger straining of collagen Ⅱ in chondrocytes and cartilage matrix compared with other groups.The percentage of positively stained collagen Ⅱ area in the lenti-shCOX2-TGFβ3 hBMSCs group was statistically higher than other groups(P<0.01).Conclusions1.MIRB-labeled hBMSCs,which were successfully transplanted into AIA models,could be detected and monitored in vivo using GRE T2*WI sequence.2.HBMSCs were detected in the synovial lining and infrapatellar fat pad tissues after transplantation.3.Transplantation of lenti-TGFβ3-shCOX2 with hBMSCs not only significantly inhibited the hyperplasia of synovial and the infiltration of inflammatory cells but also protected articular cartilage at the early stage and promoted chondrocyte and matrix proliferation in AIA rabbits.