| Objective:Rheumatoid arthritis is a chronic,synovial inflammation-based autoimmune disease which can cause swelling and pain in peripheral joints and can also damage extra-articular important organs.Although the significant advances of biological therapy and targeted therapy have improved the prognosis,a large proportion of patients show no or little response to the therapies,resulting in progressively inflammation,dysfunction and progressive disability of joints.The early factor in the development of RA is the activation of endothelial cells in the synovium.A majority of new vascular network in the inflammatory synovium can make the immune cells and inflammatory factors in the blood migrate to the synovium to induce an inflammatory response.Meanwhile,it can also make the normal synovial cells proliferate and transform into invasive pannus.This is similar to a solid tumor,also is the main cause and pathological basis of causing joint disease and cartilage destruction.The difference between these new blood vessels and normal blood vessels,is the main direction we will explore in this experiment.End MT（Endothelial to mesenchymal transition）is activated by transcription factors and increased expression of a series of mesenchymal cell markers,resulting in endothelial cells gradually losing their endothelial phenotype and gradually acquiring mesenchymal characteristics.After End MT,endothelial cells have acquired the ability to induce a series of diseases,including inflammation,fibrosis,and tumorigenesis and metastasis.However,whether End MT occurs in vascular endothelial cells in RA has not been reported.Syndecan-1（SDC-1）is a member of heparan sulfate proteoglycan family and belongs to type I transmembrane proteoglycan.SDC-1 is mainly located on the surface of endothelial cells and epithelial cells.Studies have shown that SDC-1 reduction can induce epithelial-mesenchymal transformation,but it has not been reported whether SDC-1 can regulate End MT.Current studies have found SDC-1 is involved in acute brain injury,acute lung injury,COVID-19 and other diseases,SDC-1 can be shed from the surface of endothelial cells into the blood,further leading to organ damage.But RA is a chronic disease,only one study has detected a significant increase in soluble SDC-1 in the serum of RA patients by ELISA at present,but the specific mechanism and the relationship of RA have never been clarified in detail.This study focuses on exploring the role of SDC-1 in the development of RA and whether SDC-1 can regulate End MT.Transforming growth factorβ（TGF-β）is an important cytokine that can regulate changes in cell phenotype and structure.The TGF-βsignaling pathway is initiated by TGF-β1 binding to its receptor（TGFβR2）,and then Smad2/3 is activated to cause endothelial cells to End MT,which leads to the development of multiple diseases（tumors,fibrosis,immune diseases,etc.）.The latest research is focused on blocking TGF-βpathway to inhibit End MT.So how to block TGF-βpathway is a hot spot in current research.Our experiments explore whether the binding of SDC-1 to TGF-β1 prevents TGF-β1from binding to its receptors,thereby blocking the Smad2/3 pathway and inhibiting the occurrence of End MT.The focus is on exploring the interrelationship between SDC-1 and TGF-β1 and the mechanism of action of RA.Methods:1.Synovial tissue samples from patients with RA and knee osteoarthritis who underwent knee replacement surgery at Shengjing Hospital of China Medical University from January 2021 to January 2022 were retained for human experiments.The expression of endothelial proteins（CD31,CDH5）,mesenchymal proteins（FN1,α-SMA,VIM）,snail,Smad2/3 and p-Smad2/3 was detected by Western blot technology.The expression of SDC-1 and p-Smad2/3 in the vascular wall was observed by immunofluorescence technology.The colocalization of endothelial protein（CD31）and mesenchymal protein（VIM）,as well as SDC-1 and TGF-β1 were also observed by immunofluorescence technology.Blood samples were collected from patients with active rheumatoid arthritis and healthy patients.The expression level of soluble SDC-1 in the blood of these people was detected by ELISA.2.C57BL/6 mice and SDC-1 gene knockout C57BL/6 mice were selected for animal experiment and CAIA arthritis models were established after genotype identification.The experimental animals were divided into four groups:the wild-type mouse control group（WT NC group）,the wild-type mouse CAIA arthritis model group（WT CAIA group）,the SDC-1 knockout mouse control group（SDC-1 KO NC group）,and the SDC-1 knockout mouse CAIA arthritis model group（SDC-1 KO CAIA group）,Observing the joint swelling degree and arthritis index score of the above groups,Western blot detected the expression of endothelial protein（CD31,CDH5）,mesenchymal protein（FN1,α-SMA,VIM）,snail,SDC-1,p-Smad2/3 and Smad2/3 in the above groups.3.HUVEC and HUMVEC lentivirus transfected cell line were selected for cell experiment and divided into control group and RA group.DMEM medium containing 20%HFLS-RA conditioned medium is added to HUVEC as the stimulation factor of RA.The cell experiment was divided into six groups:HUVEC control group（WT NC group）,HUVEC treated with RA stimulation group（WT RA group）,SDC-1 knockout HUVEC control group（SDC-1 KO NC group）,SDC-1knockout HUVEC treated with RA stimulation group（SDC-1 KO RA group）,SDC-1over-expressed HUVEC control group（SDC-1 OE NC group）,SDC-1 over-expressed HUVEC treated with RA stimulation group（SDC-1 OE RA group）.Western blot technology was applied to detect the expression changes of SDC-1,p-Smad2/3 and Smad2/3 at different time points after RA stimulation,as well as the expression of endothelial proteins（CD31,CDH5）,mesenchymal proteins（FN1,α-SMA,VIM）,snail,TGF-β1 and TGFβR2.The binding of SDC-1 and TGF-β1 was detected by co-immunoprecipitation assay,as well as the binding of SDC-1 and TGF-β1 was re-detected in the SDC-1 knockout HUVEC cell line and SDC-1 overexpression cell line,to determine whether the binding of SDC-1 and TGF-β1 affects the binding of TGF-β1and TGFβR2.Results:1.In clinical samples,compared with OA group,the expression of endothelial protein（CD31,CDH5）in synovial tissue vascular endothelium of RA group was reduced（P<0.0001,both）,the expression of mesenchymal protein（FN1,α-SMA,VIM）was increased（P<0.0001,all）,and snail was significantly increased（P<0.0001）;In addition,co-localization of endothelial protein（CD31）and mesenchymal protein（VIM）was found in the synovial vascular wall of patients with RA.In animal experiments,compared with WT NC group,the expression of endothelial protein（CD31,CDH5）in synovial tissue of WT CAIA group was reduced（P<0.01,both）,the expression of mesenchymal protein（FN1,α-SMA,VIM）was increased（P<0.001,<0.05 and<0.05,respectively）and snail was significantly increased（P<0.01）;After RA stimulation in HUVEC,endothelial proteins（CD31,CDH5）decreased significantly after 48h（P<0.01 and<0.05,respectively）,mesenchymal proteins（FN1,α-SMA,VIM）increased significantly after48h（P<0.001,<0.01 and<0.0001,respectively）,and snail was significantly increased after 48h（P<0.001）.Therefore,48h was selected as the stimulation time point for the subsequent cell experiment.2.In clinical samples,the level of soluble SDC-1 in the serum of patients with RA was significantly higher than that in healthy people（89.879±15.084 ng/ml vs 40.218±9.391 ng/ml,P<0.0001）The fluorescence intensity of SDC-1in the vascular wall of synovial tissue of RA group was significantly lower than that of OA group（P<0.01）.In animal experiments,compared with NC group,the expression of SDC-1 in synovial tissue of C57BL/6 mice CAIA group was reduced（P<0.01）,In HUVEC,SDC-1 protein began to decline significantly after 12 hours of RA stimulation（P<0.0001）,and the most obvious decline was at 48 hours（P<0.0001）.3.Compared with the WT CAIA group,SDC-1-/-CAIA group had more obvious joint swelling,higher AI score（11.8±0.837 vs 9.4±0.548,P<0.0001）and decreased expression of endothelial protein（CD31,CDH5）（P<0.01and<0.05,respectively）,increased expression of mesenchymal protein（FN1,α-SMA,VIM）（P<0.0001,<0.01 and<0.05,respectively）,and increased of snail（P<0.0001）in synovial tissue.In HUVEC,compared with WT NC group,the expression of endothelial protein（CD31,CDH5）of SDC-1 KO NC group was reduced（P<0.05 and<0.01,respectively）,the expression of mesenchymal protein（FN1,α-SMA,VIM）was increased（P<0.01,all）and snail was significantly increased（P<0.001）.While compared with WT NC group,the expression of endothelial protein（CD31,CDH5）of SDC-1 OE NC group was increased（P<0.01 and<0.0001,respectively）,the expression of mesenchymal protein（FN1,α-SMA）was reduced（P<0.05 and<0.01,respectively）and snail was significantly reduced（P<0.05）,the change of VIM is not obvious（P>0.99）.Similarly,compared with WT RA group,the expression of endothelial protein（CD31,CDH5）of SDC-1 KO RA group was reduced（P<0.05,both）,the expression of mesenchymal protein（FN1,α-SMA,VIM）was increased（P<0.0001,<0.01 and<0.001,respectively）and snail was significantly increased（P<0.0001）.While compared with WT RA group,the expression of endothelial protein（CD31,CDH5）of SDC-1 OE RA group was increased（P<0.05,both）,the expression of mesenchymal protein（FN1,α-SMA,VIM）was reduced（P<0.01,<0.001and<0.01,respectively）and snail was significantly reduced（P<0.001）.4.In clinical samples,compared with OA group,the expression of p-Smad2/3 in synovial tissue vascular endothelium of RA group was increased（P<0.05）,while there was no significant change in Smad2/3.（P=0.73 and 0.99 respectively）.In HUVEC,p-Smad2/3began to increase significantly after 6 hours of RA stimulation（P<0.0001）,and the most obvious increase was at 24 hours（P<0.0001,both）.However,Smad2/3 did not change significantly until 24h（P=0.896 and 0.903 respectively）.5.In cell experiment,SDC-1and TGF-β1 are colocalization in the vascular endothelium of RA synovial tissue and can also be combined with each other in co-IP experiment.The expression of TGFβR2 was the same in WT group,SDC-1 KO group and SDC-1 OE group.However,compared with WT group,the binding of TGFβR2 to TGF-β1 increased（P<0.0001）in SDC-1 KO group（P<0.0001）and the binding of TGFβR2 to TGF-β1 decreased（P<0.05）in SDC-1OE group.6.In cell experiment,compared with WT NC group,the expression of p-Smad2/3 in SDC-1 KO NC group increased（P<0.01 and 0.05,respectively）,while the expression of Smad2/3 did not change significantly.Compared with WT NC group,the expression of p-Smad2/3 in SDC-1 OE NC group decreased（P<0.05,both）,while the expression of Smad2/3 did not change significantly.Compared with WT RA group,the expression of p-Smad2/3 in SDC-1 KO RA group increased（P<0.0001 and<0.001,respectively）,while the expression of Smad2/3 did not change significantly.Compared with WT RA group,the expression of p-Smad2/3 in SDC-1 OE RA group decreased（P<0.05,both）,while the expression of Smad2/3 did not change significantly.In animal experiments,compared with WT NC group,the expression of p-Smad2/3 in synovial tissue of SDC-1-/-NC group was increased（P<0.01,both）,while there was no significant change in Smad2/3.;compared with WT CAIA group,the expression of p-Smad2/3 in synovial tissue of SDC-1-/-CAIA group was increased（P<0.05 and<0.01,respectively）,while there was no significant change in Smad2/3.Conclusion:1.Endothelial mesenchymal transformation（End MT）can occur in synovial vascular endothelium in patients with RA.2.When RA occurs,SDC-1 falls off the surface of endothelial cells and enters into the blood,which reduces the expression of SDC-1 in synovial vessels and increases the expression of soluble SDC-1 in blood.3.End MT is regulated by SDC-1.SDC-1 is a protective factor for endothelial cells of RA.When SDC-1 is absent,RA stimulation will cause more serious damage to endothelial cells,and End MT is more likely to occur,When SDC-1 is over-expressed,the damage of RA stimulation to endothelial cells is weakened,which can prevent the occurrence of End MT.4.The TGF-β/Smad2/3 pathway is activated during RA,and activation of the TGF-β/Smad2/3 pathway is regulated by SDC-1.5.SDC-1,as a transmembrane protein,can competitively bind to TGF-β1 with TGFβR2,thereby reducing TGF-β1 binding to TGFβR2 and suppress TGF-β/Smad pathway.When SDC-1 is knocked out,the combination of TGF-β1 and TGFβR2 increases,and TGF-β/Smad pathway is activated.However,when SDC-1 is over-expressed,the combination of TGF-β1 and TGFβR2decrease,and TGF-β/Smad pathway is inhibited.This is a new mode of SDC-1regulating TGF-β/Smad pathway,and can also bring new insights for the mechanism study and therapeutic exploration of RA. |
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