Effects Of Osteoprotegerin Gene Therapy To Cartilage Destruction In Rheumatoid Arthritis:an Experimental Study

BackgroundRheumatoid arthritis (RA) is a chronic autoimmune disease that mainly affecting the synovial joints, characterized by synovial inflammation, progressive erosion of bone and cartilage. Although synovial hyperplasia leads to the swelling of joints, it is bone and cartilage destruction that ultimately result in life-long disability in function and work. Research on an effective approach to inhibit the joints destruction, therefore, is of great importance.Recent reports showed that osteoprotegerin (OPG) was able to prevent cartilage destruction in osteoarthritis model mice. Systematic OPG administration may completed inhibited the cartilage destruction. It was demonstrated that adjuvant-induced arthritis (AIA) model has disproportionally mild cartilage destruction relative to marked cartilage damage in another RA model, collagen-induced arthritis (CIA) model. Therefore, the effects of OPG in RA animals deserve further investigation. In addition, the pathological mechanism of OPG on RA cartilage remains unknown.Cartilage matrix is mainly composed of a fibrillar collagen network and aggregated proteoglycan. Chondrocytes are the only cell element in the cartilage, which are responsible for the metabolism of cartilage, including producing and maintaining the cartilaginous matrix. Chondrocytes also self-secrete several factors and to balance local environment and regulate the cartilage metabolism. Catabolic factors produced by chondrocytes, such as MMP-1,3,9,13, or ADAMTS-4,5can degrade the cartilage matrix. At the same time, chondrocytes also increase the secretion of TIMP-1,2,3,4, IGF-I, TGF-p, BMP-2, bFGF to inhibit the effect of MMPs, for the maintainace of cartilage self-renewal. Overloaded pressure on cartilage, inflammatory factors like IL-1, TNF-alpha may disturb the balance.It was generally accepted that cartilage erosion in RA was secondary to inflammatory pannus which attach to cartilage. To date, there is no effective therapy to suppress both synovium inflammation and joint destruction currently. Moreover, the association between advancing synovitis and erosive progression in hand joints of RA patients is weak. The pathophysiological courses of synovitis and cartilage erosions are not always the same. Alternatively, we should focus on the inherent mechanisms of cartilage destruction in RA, such as cell appotosis and proteoglycan loss, and then aiming at them as therapeutic targets. According to some previous researches, Kim et al. found that apoptosis of chondrocytes in RA joints was more prominent than those without RA. Also, it was reported that the expression of caspase3increased in cartilage of RA patients as well as animal models. Nevertheless, the regulating mechanisms of chondrocytes apoptosis in the progression and treatment of RA have not yet been fully elucidated. In addition, proteoglycan loss was significant in CIA model, as assessed by Safranin O staining. During the process of the development and progression of OA or RA, destruction of superficial aggrecan usually is the first step, and ADAMTS-4,5are two main factors contributing to the increased degradation of aggrecan.Thus, we are the first to perform this study to investigate the effects of OPG on the vitality and function of chondrocytes, and then to examine the effects of OPG gene therapy to RA model animals and elucidate the mechanisms. The efficacy was assessed in aspects of cellular, histology and pathology. To this end, we carried out experiment:1) to explore the effect of OPG on the proliferation of chondrocytes;2) to explore the cellular signal by which OPG affects chondrocytes;3) to explore the effect of OPG on the secretion function of chondrocytes;4) to establish the recombined adenovirus carrying OPG gene (Ad-OPG) and to established the collegan-induced arthritis model rats;5) to assess the cartilage destruction in CIA rats treated with or without Ad-OPG;6) to assess the apoptosis of chondroctyes in CIA rats treated with or without Ad-OPG;7) to assess the expression of ADAMTS-5.Part1:The effect of OPG to the proliferation and functions of chondrocytesObjectionThe role of OPG in the bone metabolism has been established, but it remains unknown whether OPG affects chondrocytes directly and whether OPG has any detailed cellular and molecular effects on chondrocytes. The present study is aiming to investigate the direct effect of OPG on chondrocyte viability, proliferation and functional consequences on chondrocytes.MethodsPrimary chondrocytes were isolated from knee of SD rats. Passage1chondrocytes were identified by toluidine blue staining, and then used in experiments. The cell proliferation induced by OPG at various concentrations was measured by Cell Counting kit-8(CCK-8) assay. After pretreatment with MEK inhibitor U0126, ERK inhibitor PD098059, P38MAPK inhibitor SB203580for30minutes, chondrocytes were then treated with OPG, and CCK-8was performed. The exact cellular signals of MAPKs including ERK, P38MAPK, and JNK, were investigated by western blotting after the treatment with OPG The functional consequences following treatment with soluble OPG were examined by Reverse Transcriptase-Poly-merase Chain Reaction(RT-PCR) and western blotting.ResultsOPG increased chondrocytes proliferation with maximal effect at concentration of lOng/ml, and induced the phosphorylation of MEK and ERK specifically, neither P38MAPK nor JNK. Suppression of ERK activity via PD098095inhibited OPG-induced chondrocytes proliferation. Administration of OPG significantly down-regulated ADAMTS-5and up-regulated TIMP-4production, but had no effect on the expression of TIMP-1,2,3, IGF-I, TGF-β,bFGF, BMP-2, collagen II, aggrecan and ADAMTS-4. Suppression of ERK activity via PD098095neutralizes alteration of ADAMTS-5and TIMP-4expression induced by OPGConclusionOPG regulates the proliferation of chondrocytes via MEK/ERK signaling, and directly affects chondrocytes by influencing the expression profile of ADAMTS-5and TIMP-4. Part2:Adenovirus-mediated osteoprotegerin for the cartilage destruction in collagen-induced arthritis model ratsObjectionOsteoprotegerin (OPG) was recently reported to be able to prevent cartilage degradation during osteoarthritis course, while it remains unclear whether OPG inhibits cartilage destruction in rheumatoid arthritis (RA). We employed adenovirus-mediated OPG gene therapy to examine the therapeutic potential and explore its mechanism of actions to cartilage in collagen-induced arthritis (CIA) model rats.Material and MethodsForty SD rats (8weeks, male) were injected with bovine type Ⅱ collagen subcutaneously into the tail base to establish the CIA model. The CIA rats were single injected with30ul adenovirus (Ad)-OPG intra-articularly either at the beginning of CIA (early OPG treatment) or one week after CIA established (late OPG treatment), while30ul vehicle or Ad-GFP was injected as control. The rats were sacrificed4weeks after treatment. The joint tissues were assessed histologically, and the serum samples were evaluated by ELISA.ResultsCIA was established and expression of GFP was confirmed by microscope. The proteoglycan loss was inhibited by both early and late Ad-OPG injection, while the overall cartilage destruction score and cartilage oligomeric matrix protein (COMP) level in serum was only decreased by early Ad-OPG treatment. TUNEL assay revealed that both early and late Ad-OPG treatment significantly inhibited the chondrocytes apoptosis in CIA rats and expression of caspase3decreased significantly after early Ad-OPG treatment. Furthermore, the number of ADAMTS-5positive cells was decreased significantly by both early and late OPG treatment. Findings also indicated that OPG protein was highly expressed in superficial zone cartilage from rats treated with Ad-OPG injection. However, swelling of ankle joints and IL-1β expression level in CIA rats were not significant altered by Ad-OPG treatment.ConclusionsOur results suggest that early Ad-OPG treatment had protective effect against the cartilage destruction during RA progression by inhibiting the chondrocytes apoptosis inherent in CIA cartilage. Moreover, OPG may exert a direct role on proteoglycan loss of CIA cartilage through suppressing the expression of ADAMTS-5.