Mechanism Of Effects Of Recombinant Human Endostatin On Fibroblast-like Synoviocyte In Rats With Adjuvant Arthritis

Rheumatoid arthritis(RA) is a chronic autoimmune disease affecting approximately 1% and 0.4%of the population worldwide and nationwide,respectively.RA presents clinically with joint swelling,pain,stiffness,deformity and is not healed.The major goals of treatment of RA are to reduce pain and discomfort,to prevent joint destruction and loss of joint function,and to maintain a productive and active lifestyle.Up to date, the mechanisms that contribute to the pathogenesis of RA are unknown.The affected RA joint is characterized by pronounced synovial hyperplasia composed of extensive proliferation of fibroblast-like synoviocytes(FLS) and inflammatory cell infiltration with neovascularization.Pannus tissue comprising the hyperplastic synovium leads to progressive joint destruction,deformity and functional incapacitation,which is manifested as bone erosion and cartilage destruction.Thus,the synovial hyperplasia has a critical role in the propagation of rheumatoid synovitis.It is generally accepted that there is a close relationship between cell proliferation and cell death,and that there may be an imbalance between the proliferation and death of FLS in RA,which leads to synovial hyperplasia.For this reason,the interruption of proliferation and induction of apoptosis in RA FLS have been proposed as one of the promising strategies for treating RA by way of reducing synovial hyperplasia in situ.Adjuvant arthritis(AA) is a model of experimental arthritis that is induced by injection of Freund's complete adjuvant(FCA).The similarities between the joint pathologies as well as the cellular and humoral immunities in AA and RA suggest that AA is a relevant animal model that acts as a useful test system for evaluating treatment of RA. Endostatin,a 20kD fragment of collagen XVIII,is a member of a group of powerfully endogenous antiangiogenic proteins that are activated by proteolytic processing.In fact, the antiangiogenic activity of endostatin is speculated to be specifically mediated by the inhibition of endothelial cell adhesion,migration and proliferation,and induction of apoptosis.Recently,endostatin has been studied not only for its inhibitory effects on vascular endothelial cell function but also its direct antitumor effects on cancer cell migration and proliferation,and induction of apoptosis.Fibroblast-like synoviocytes are the ultimate target cells of the pathologic changes in RA,also,they exhibit pre-neoplastic characteristic of transformed cells with active proliferation.The locally invasive tumourlike properties of RA synovial tissues suggest that approaches normally reserved for oncologic diseases might be useful in RA,including induction of apoptosis and antiangiogenesis agents.Studies on endostatin are mainly involved in its effects and mechanisms on anti-tumor now.There is few report on the effect of endostatin on upregulated angiogenesis in certain pathological conditions such as chronic inflammation. It is no reported that rhEndostatin has therapeutic effect on RA by targeting FLS. Previous studies have shown that subcutaneous administrations of recombinant human endostatin(rhEndostatin;(1.25,2.5,5.0 mg·kg^-1·d^-1,×7d) to the abdomen have therapeutic effect on AA rats,which is characterized by significantly inhibition of paw swelling, reduction of microvessel density(MVD) and the expression of vascular endothelial growth factor(VEGF) in the synovium of AA rats.Also,rhEndostatin(3.125,6.25,12.5, 25,50μg/ml) can directly have influence on AA FLS such as inhibition of proliferation and induction of apoptosis.However,the mechanisms of effects of rhEndostatin on AA FLS in rats remain unclear.The aim of the current study was to understand the potential mechanisms,and to provide a experimental data for the development of new therapeutic interventions and targets of RA.Objective:To clarify the molecular and ionic mechanisms of effect of rhEndostatin on AA FLS such as inhibition of proliferation and induction of apoptosis,and to provide a experimental data for the development of new therapeutic interventions and targets of RA,we investigatedâ‘ the effect of rhEndostatin on cell cycle of AA FLS,â‘¡the effect of rhEndostatin on the expression of the cell cycle regulatory genes such as p53,p21, CDK4,cyclinD₁,PCNA mRNA and CyclinD₁,PCNA protein in AA FLS,â‘¢the effect of rhEndostatin on the expression of the apoptosis regulatory genes such as fas,bcl-2, c-fos,c-jun mRNA and c-Jun,NFr,B,Caspase-3 protein in AA FLS,andâ‘£the effect of rhEndostatin on the cytosolic ionized free calcium(Ca²⁺) in AA FLS.Methods:â‘ Induction of the AA rat model:Male Sprague-Dawley(SD) rats weighing 140-160g were obtained from the Animal Center of Anhui Medical University.Rats were immunized on day 0 by intradermal injection of a 0.1-ml aliquot of FCA into the left hind paw.â‘¡Preparation of synovial tissues:The rats were randomly divided into normal,AA control,rhEndostatin(2.5mg/kg)-treated and methotrexate(MTX;(1mg/kg)-treated groups,and immunized to induce AA rat model in the groups except normal one. Subcutaneous administrations of rhEndostatin(2.5mg·kg^-1·d^-1) and MTX(1mg/kg twice weekly) to the abdomen were started on day 10 post-immunization and continued until day 16 post-immunization.Normal and AA control group received the same volumes of phosphate-buffered saline(PBS).On day 28,the animals were sacrificed and the synovial tissues were promptly removed for subsequent experiments.â‘¢Preparation of FLS:Synoviocytes were isolated from knee joints of the normal and AA rats,and cultured under permissive conditions.The synoviocytes were used in experiments from the second subculturing ones.The cells obtained from AA rats were identified by their expressed vascular cell adhesion molecule-1(VCAM-1) as evaluated by flow cytometry(FCM) analysis.â‘£Cell Cycle Analysis of FLS:The first subculturing synoviocytes obtained from normal and AA rats were plated in a 6-well plate.AA synoviocytes were divided into AA control,rhEndostatin(50μg/ml)-treated and MTX(1μg/ml)-treated groups.For synchronization,the cells were harvested in serum-free RPMI 1640 medium for 24h at 37℃.The cells were then placed in RPMI 1640 medium containing 20%(v/v) fetal calf serum(FCS).For rhEndostatin-treated and MTX-treated groups,the cells were treated with the designated doses of agents for 48 h at 37℃.The cells were collected and analysed by FCM.⑤Detection of cell cycle regulatory genes:Total RNA and protein was extracted from each frozen synovial tissue sample obtained from rats in various groups.The expression of p53,p21,cyclinD₁,PCNA,CDK₄ mRNA was quantified by real-time fluorescent quantitative polymerase chain reaction(RT-FQ-PCR).The expression of PCNA, CyclinD₁ proteins was detected by westem blot analysis.â‘¥Detection of apoptosis regulatory genes:The expression of fas,bcl-2,c-fos,c-jun mRNA in synovial tissue sample obtained from rats in various groups was quantified by RT-FQ-PCR.The expression of c-Jun,NFκB,Caspase-3 proteins was detected by westem blot analysis.⑦Measurements of the cytosolic Ca²⁺ in AA FLS:The cultured AA FLS were loaded with the calcium ion-sensitive fluorescent indicator fluo-3/AM,and perfused with rhEndostatin at the concentration of 50μg/ml.At the same time a confocal laser scanning microscope(CLSM) was applied to measure changes of cytosolic Ca²⁺ fluorescence intensity(FI) in AA FLS when cells were immersed in the buffer with or without Ca²⁺,and to investigate the effect of rhEndostatin on cytosolic free calcium concentration([Ca²⁺]_i) in AA FLS.Results:â‘ Effect of rhEndostatin on cell cycle of FLS:Flow cytometric analysis indicated that the cell cycle progression of FLS obtained from normal rats was arrested at G1 phase.The percentages of cells in G₀/G₁ phase,S phase,G₂/M phase were 68.3%, 23.65%,6.32%,respectively.The rate of AA FLS in G1 phase(10.0%) was significantly lower than that in normal control group.The cell cycle progression of AA FLS was mostly in S phase.The percentages of cells in S phase,G₂/M phase were 65.6%,19.5%, respectively.Recombinant human endostatin(50μg/ml) significantly increased G₁ phase cells as compared with those of AA FLS(74.1%versus 10.0%).The percentages of cells in S phase,G₂/M phase(12.6%,4.15%,) were significantly decreased compared with AA control group.â‘¡Effect of rhEndostatin on the expression of the cell cycle regulatory genes in synovial tissues obtained from AA rats:Analysis by RT-FQ-PCR showed that rhEndostatin(2.5mg/kg) could significantly decrease the expression of p53,p21, cyclinD₁,PCNA mRNA and increase the expression of CDK₄ mRNA in synovial tissues obtained from AA rats(P＜0.01).Westem Blot analysis indicated that the expression of PCNA and CyclinD₁ protein in synovial tissues treated with rhEndostatin were significantly decreased than that in AA control group(P＜0.01).â‘¢Effect of rhEndostatin on the expression of the apoptosis regulatory genes in synovial tissues obtained from AA rats:Real-time PCR analysis indicated that rhEndostatin(2.5mg/kg) could significantly increase the expression level of fas,bcl-2, c-fos and c-jun mRNA in synovial tissues obtained from AA rats(P＜0.01).The results of Western Blot analysis indicated that the expression of c-Jun protein(43kD,48kD) and Caspase-3 p20 in synovial tissues treated with rhEndostatin were significantly increased compared with that in AA control group(P＜0.01).But rhEndostatin could not significantly improve the expression of NFκB p65 in synovial tissues obtained from AA rats(P＞0.05).â‘£Effect of rhEndostatin on[Ca²⁺]_i in AA FLS:Analysis by CLSM showed that rhEndostatin(50μg/ml) did not change cytosolic Ca²⁺ FI in AA FLS when cells were immersed in non-calcium extracelluar solution.When cells were immersed in the buffer with Ca²⁺ and perfused with rhEndostatin at the concentration of 50μg/ml,the cytosolic Ca²⁺ FI in AA FLS was rapidly increased to maximum and then gradually declined.Conclution:â‘ Suppression of CyclinD₁ and PCNA expression by rhEndostatin can produce a virtual inhibition of S-phase entry and arrest the cell cycle progression of AA FLS in G₁ phase,which induces inhibition of AA FLS proliferation and is not associated with the expression of P53,P21,CDK₄.â‘¡Apoptosis induced by rhEndostatin in AA FLS is strongly associated with the increased expression and activation of fas,c-fos,c-jun,caspase-3,but not bcl-2,NFκB in AA FLS.â‘¢Recombinant human endostatin can elicit the extracellular calcium influx in intact AA FLS.Imbalance of Ca²⁺ steady state induced by rhEndostatin is the very early events occur prior to AA FLS damage.Cytoplasmic Ca²⁺ overload may occur prior to any other molecular events of apoptosis in AA FLS.