SMOC2 Expression In Knee Osteoarthritis Patients And The Role Of SMOC2 In The Pathogenesis Of Osteoarthritis

BackgroundOsteoarthritis(OA)is a kind of degenerative disease which mainly involves musculoskeletal system and is characterized by cartilage destruction,pathological degeneration and hyperosteogeny.The pathogenesis of osteoarthritis originates from the disruption of balance among synthesis and degradation of cartilage cells,extracellular matrix and subchondral bone.Though all joints of human body may be affected by osteoarthritis,the most commonly involved joints are knee,hip and hand joints.Osteoarthritis patients often suffer from joint pain and restricted movement.The serious cases cannot work normally and may eventually become disabled.According to estimated data in developed countries,osteoarthritis is the number two risk factor which leads to incapacity in male over 50 years following cardiovascular diseases.The unemployment will futher add to economic burden and lead to more severe social problems.According to statistics by epidemiologists,in 2017 more than 300 million people suffered from osteoarthritis in the whole world and the prevalence rate in people over 40 years in China is about 10%to 17%.The articular cartilage which is composed of cartilage cells and extracellular matrix(ECM)belongs to hyaline cartilage.The extracellular matrix is essential for maintaining the stability of extracellular environment and it is the major component of cartilage.It is the material basis which is responsible for the physiological function and mechanical property.The cartilage ECM proteins can bond to fibril to assist in building the scaffold and maintain the strength of cartilage.Besides,the cartilage ECM proteins are not only the components of cartilage but also act like ligands which can bind to specific receptors to coordinate the mobility,proliferation and differentiation of cartilage cells.Recent studies have reported that after the onset of osteoarthritis,ECM proteins could mediate inflammation by coordinating certain signal pathways.SPARC(secreted protein acidic and rich in cysteine)family proteins,also known as osteonectins or BM-40 proteins,which originate from bone,are the most studied ECM proteins.They play a pivotal role in bone mineralization,the interaction between cells and ECM and bone remodeling.One of SPACR family proteins,SMOC(SPARC related modular calcium-binding protein),consists of 2 isoforms,SMOC1 and SMOC2.SMOC2,located in chromosome 6q27,is widely expressed in different tissues and closely related to skeletal development.The expression of Smoc2 can be detected in mouse embryonic limb and especially in the growth plate.SMOC2 knock-out mice show dysplasia in skull.Patients with SMOC2-mutant have microdontia and oligodontia.When upregulated in osteoprogenitor cells,SMOC2 can inhibit osteogenic differentiation and ECM mineralization.Our research team has identified a mutant site in SMOC2 may lead to hereditary multiple epiphyseal dysplasia(MED)and one of typical clinical manifestations of MED is early-onset osteoarthritis.Given above clues,we speculate that SMOC2 may play a role in the process of skeletal diseases,especially osteoarthritis.The progressive degradation of cartilage matrix,of which the main components are collagen and aggrecan,is a representative pathological symbol of osteoarthritis.In the process of osteoarthritis,the degradation of cartilage matrix is far faster than synthesis,due to the inhibited synthesis of matrix components of chondrocyte and the increased expression of matrix metalloproteinase(MMP)and a disintegrin and metalloproteinase with thrombospondin motifs(ADAMTS).Inflammation factors like interleukin(IL)and tumor necrosis factor(TNF)also play a crucial role in osteoarthritis.The stimulation of chondrocytes by inflammation factors would downregulate the synthesis of matrix components,upregulate the synthesis of proteases and inflammation factors and induce apoptosis.Previous studies showed that stimulated by certain ECM proteins,the expression of matrix components in chondrocyte would reduce and the expression of inflammation factors and proteases would increase.NF-?B pathway is involved in the pathogenesis of multiple inflammatory diseases and rheumatoid diseases like osteoarthritis,rheumatoid arthritis,asthma and atherosclerosis.NF-?B pathway is not only able to induce pro-inflammatory factors such as inflammation factors,chemokines and proteases,but also capable of coordinating the proliferation and apoptosis of chondrocyte.It is an important therapeutic target for osteoarthritis.The inflammation provoked by NF-?B is inhibited in the SMOC2 knock-out mice,indicating that SMOC2 can regulate NF-?B pathway.SMOC2,as an ECM protein existing in cartilage,plays an important role in bone growth and inflammation.In the present study,we speculate that SMOC2 is involved in the development of osteoarthritis and pose following questions:first,whether there is a difference in the expression of SMOC2 between osteoarthritis patients and healthy people;second,how is SMOC2 involved in the process of osteoarthritis.Objectives1.To investigate the expression of SMOC2 in healthy and osteoarthritic cartilage and detect whether the expression of SMOC2 in osteoarthritic cartilage is related with the degree of degeneration of articular cartilage.2.To investigate the expression of SMOC2 in synovial flood and serum of different people.The tested objects comprising knee osteoarthritis patients,patients with tendon or meniscus injuries and healthy people.To detect whether the SMOC2 level in the serum is related with specific clinical indexes.3.To investigate the effect of SMOC2 on inflammation of chondrocytes and the development of osteoarthritis in mice.Material and Methods1.Cartilage from osteoarthritis patients and healthy people was collected.Total RNA was extracted from cartilage to undergo Real-time PCR to detect the expression of SMOC2.The cartilage specimens were also prepared as paraffin sections.H&E,Safranin O/fast green staining as well as immunohistochemical analysis of SMOC2 were performed.Osteoarthritis Research Society International(OARSI)scoring system was used for accessing the degeneration of cartilage.Quantification of positive staining was performed by Image J software.2.Synovial flood was collected from osteoarthritis patients and patients with tendon or meniscus injuries.Serum was collected from osteoarthritis patients,patients with tendon or meniscus injuries and healthy people.The volume of SMOC2 and osteoarthritic biomarkers was detected by enzyme-linked immunoabsorbent assay(Elisa).Correlation analyses were performed between SMOC2 level and other clinical indexes.3.Chondrocytes were treated with rhSMOC2.Total RNA and protein were extracted from chondrocytes to undergo Real-time PCR and Western blot for detecting the expression of matrix components,inflammation factors and proteases as well as the activation of NF-?B pathway.Also the nuclear translocation of P65 was detected by immunofluorescence.4.Destabilization of medial meniscus(DMM)was performed in C57BL/6 mice to induce osteoarthritic model.Intra-articular Injection of rhSMOC2 was performed after DMM surgeries.The knee joints were collected for histological analysis.ResultsPart 1 SMOC2 expression in cartilage and body fluid of osteoarthritis patientsIn total,cartilage was collected from 28 osteoarthritis patients underwent total knee arthroplasties and 3 normal people underwent amputations due to car accident.The mRNA level was higher in osteoarthritis cartilage compared with healthy cartilage.Forty-one cartilage specimens were graded from GO to G4 according to OARSI scoring system.The results of Spearman's rank correlation showed that the expression of SMOC2 was positively correlated with OARSI score(r=0.816,p<0.001).The volume of SMOC2 in synovial flood of osteoarthritis patients was significantly higher than patients with tendon or meniscus injuries.The SMOC2 level in serum was similar between osteoarthritis patients and patients with tendon or meniscus injuries but the levels of both groups were significantly higher than healthy people.The SMOC2 level in serum of osteoarthritis patients was positively correlated with COMP level(r=0.328,p=0.045),WOMAC index(r=0.328,p=0.044)and VAS(r=0.366,p=0.024).When applying a multiple stepwise regression analysis considering biomarkers together with clinical parameters,results indicated that COMP level(p=0.003)and VAS(p=0.004)might be risk factors for high SMOC2 level.The SMOC2 level in serum might be potential biomarker in osteoarthritis.Part 2 SMOC2 promotes inflammation and degradation of articular cartilageThe primary murine chondrocytes(PMC)which were isolated from the ribs of new-born mice and human immortalized chondrocytes(C28/I2)were treated with rhSMOC2 by different concentrations.In PMCs,the expression of type ? collagen and aggrecan was downregulated by rhSMOC2 while expression of inflammation factors like IL-1?,IL-6,iNos,Cox2,chemokine Ccl5 as well as proteases such as Mmp3,Mmp13 and Adamts4 was induced by rhSMOC2 in a dose-dependent manner(0?g/ml,0.1?g/ml,1?g/ml,5?g/ml,25?g/ml and 50?g/ml for 24h).Also the expression of IL-Ira was inhibited after rhSMOC2 stimulation.SMOC2 promoted inflammation and degradation of articular cartilage.Stimulated by rhSMOC2 in incremental concentrations and times(0?g/ml,0.1?g/ml,1?g/ml and 25?g/ml for 15min,30min and 60min),enhanced phosphorylation of P65 was shown in C28/I2.Also the activation of P65 was shown in PMC when treated with rhSMOC2.Increased nuclear translocation of P65 was shown in C28/I2 and PMC after stimulation of rhSMOC2 with 1?g/ml for 60min.SMOC2 might promote inflammation in osteoarthritis by inducing NF-?B pathway.Part 3 SMOC2 promotes the process of osteoarthritis in miceEighy-week-old WT C58BL/6 male mice were performed DMM surgeries and SHAM surgeries.The expression of Smoc2 was higher in the articular cartilage and synovial membrane of DMM mice than of SHAM mice in 8 weeks after the surgeries.Mice were intra-articularly injected with rhSMOC2 or PBS following DMM surgeries.Injections were performed once a week,totally 4 times.The mice injected with rhSMOC2 showed higher severity of knee joint destruction as well as higher expression of hypertrophy marker,type X collagen and Runx2,compared with those injected with PBS.SMOC2 was able to promote osteoarthritis process in mice.Conclusions1.SMOC2 expression is increased in osteoarthritic cartilage.The expression level of SMOC2 is positively correlated with the degenerative grade of cartilage.2.The volume of SMOC2 is increased in the synovial flood and serum of osteoarthritis patients.SMOC2 level in serum is positively correlated with COMP level,WOMAC index and VAS.The SMOC2 level in serum might be potential biomarker in osteoarthritis.3.SMOC2 is able to accelerate inflammation process and degradation of cartilage probably through activating NF-?B pathway.SMOC2 may act as a pro-inflammatory factor in osteoarthritis.