| Background and objective Rheumatoid arthritis(RA)is a chronic systemic autoimmune disease characterized by aggressive pannus formation and synovial inflammation.Angiogenesis is the basis of pannus formation.Serum amyloid A(SAA)is one of the most important acute phase reactive proteins,which plays a key role in inflammatory injury and pannus formation in RA.Recently,toll-like receptor 4(TLR4)was found to be an endogenous receptor of SAA.In addition,SAA can participate in angiogenesis in RA by binding to TLR4 and inducing the formation of neutrophils extracellular traps(NETs).However,the exact mechanism of SAA/TLR4 driving this process has not yet been elucidated,and the role of SAA/TLR4 and NETs in RA angiogenesis still needs further investigation.Therefore,we conducted a series of studies to reveal the effects of SAA/TLR4 and NETs on endothelial cells and the molecular mechanism of promoting angiogenesis in RA.Methods1.The levels of SAA in the synovial fluid and SAA,MP0 and NE levels in the serum were detected by ELISA.2.Immunohistochemical staining was used to detect the expression of SAA and TLR4 in synovium of RA patients and control group.3.Disease activity in RA patients was assessed using the Rheumatoid Arthritis Disease Activity Score Scale(DAS-28).4.The proliferation of HUVECs was measured by MTT assay.5.The migration ability of HUVECs was analyzed by scratch test.6.The vascular tubule forming test was used to detect angiogenesis.7.Western blotting was used to detect the expression level of the target protein in HUVECs.8.Immunofluorescence staining was used to assess the formation of NETs.9.Pico Green DNA was used to detect the level of DNA in the supernatant of cell culture.10.Neutrophils were isolated from peripheral blood by density gradient centrifugation.-Finally,11.SPSS 17.0 software package,and Graph Pad Prism 5 software were used for data analysis and graphing,respectively.Results1.SAA was correlated with disease activity-The serum data analysis showed that compared to the other groups,the levels of inflammatory markers such as ESR and CRP were significantly increased in RA patients(p<0.05).-ELISA results indicated that serum levels of SAA in RA were significantly increased compared to those of the SLE,other autoimmune diseases,OA and HC groups(p<0.05).In the synovial fluids,SAA levels were significantly increased in the RA group(compared to those of the OA group(p<0.05).-Immunohistochemical staining revealed higher expression of SAA in all tissue samples from RA patients compared to those of OA.-The correlation results analysis showed a significant correlation of SAA levels in RA sera with DAS28,CRP,and ESR(p<0.001).ESR(p<0.001)and CRP(p=0.004)levels were demonstrated to be positively correlated with DAS28.-No significant correlation was identified between serum anti-CCP levels and DAS28(p=0.250).However,RA patients positive for anti-CCP showed significantly increased levels of SAA compared to those of patients negative for anti-CCP(p<0.001).2.SAA/TLR4 promoted angiogenesis-Immunohistochemistry results showed that TLR4 expression was remarkably detected in all RA tissue samples compared to a minimal expression in all OA tissue sections.Besides,ELISA assay results revealed that there was a statistically significant difference among the serum levels of SAA in RA and OA groups(p<0.001).-MTT assay,scratch cell assay,and tube formation assay results showed significant increased proliferation,migration,and vascular tube formation of HUVECs under SAA stimulation(p<0.001).Whereas,there was a significant decrease once TLR4 was inhibited(SAA+TAK-242)(p<0.001).-ELISA was used to measure the expression levels of pro-angiogenic factors.VEGF expression levels in the experimental group(5μg/ml SAA)was significantly increased(p<0.05).While,the VEGF expression significantly decreased compared to its expression in the experimental group,after the endothelial cells were pre-incubated with TAK-242(SAA+TAK-242),p<0.05.-Western blot was used to investigate the NF-κB signaling pathway activation in SAA/TLR4-induced migration,proliferation and tube formation.The results showed that the expression level of total NF-κB was relatively constant over time.Nonetheless,the phosphorylated NF-κB(p NF-κB)expression level ranged according to the duration of stimulation(SAA),with a maximum expression reached at 120 minutes.In contrast,the expression was markedly suppressed after the endothelial cells were pre-incubated with TAK-242(SAA+ TAK-242).The involvement of the NF-κB signaling pathway in angiogenesis was further demonstrated.Its inhibition using BAY11-7082(SAA+BAY11-7082)showed a significant decrease of HUVECs’ proliferation(p<0.001),migration(p<0.05),and vascular tube formation.3.SAA/TLR4 promotes angiogenesis by inducing NETs formation through NADPH dependent pathway-Serum MPO and NE levels were measured by ELISA.Compared to healthy controls,the serum levels of MPO and NE in RA patients were significantly higher than those in healthy controls(p<0.0001).In the same batch of serum,the relative fluorescence quantitative results of DNA showed that the absolute free DNA content in serum of RA group was significantly lower than that in healthy controls group(p<0.01).-Afterwards,the results showed that MPO was positively correlated with DAS28 and ESR(p<0.0001),whereas it was positively and slightly correlated with CRP(p=0.006).As well,NE was positively correlated with DAS28(p=0.0015).-Furthermore,The results showed that SAA was positively correlated with MPO and NE(p<0.0001).-Immunohistochemistry results showed that obvious NETs formation was present in RA synovial tissue,whereas no NETs release was observed in OA synovial tissue.-The immunofluorescence staining was performed to assess the NETs formation after SAA stimulation.The results showed that the spontaneous NETs formation was higher in the RA patients group compared to the HC group(p<0.05).Likewise,in SAA stimulated neutrophils group,the amounts of NETs formed in the RA patients group were significantly elevated than those of the HC group(p<0.05).-Afterwards,It could be seen under the microscope that the isolated neutrophils accounted for more than 95% of all cells.-Immunofluorescence staining was performed to determinate the percentage of formed NETs between groups.Compared with the control group,the NETs formed in the SAA stimulation group were significantly increased(p<0.05).But after pretreatment with TAK-242,and despite the same stimulation by SAA,the formation of NETs was significantly reduced(p<0.05).Then,the Pico Green DNA quantification was used to determine the DNA concentration in the supernatants of neutrophils.The results showed that the SAA stimulation group after adding TAK-242 compared with the SAA group had significantly lower DNA concentration in the culture supernatant(p<0.05);and similar results were found in the LPS group and(LPS+TAK-242)group(p<0.05).-ROS generation analysis and DNA staining were used to determine whether SAA/TLR4 induced NETs formation was independent or dependent on NADPH pathway.The results showed that there was a significant decreased NETs formation when neutrophils were stimulated with SAA in presence of NADPH oxidase inhibitors(SAA+apocynin or SAA+VAS2870;p<0.0001).Similarly,there was a significant decreased serum DNA levels when neutrophils were stimulated with SAA in presence of NADPH oxidase inhibitors(p<0.0001).-MTT assay,scratch cell assay,and tube formation assay were performed to determine the angiogenic effect of NETs on HUVECs.The results showed significant increased proliferation,migration,and vascular tube formation of HUVECs under NETs stimulation(p<0.05).Whereas,there was a significant decrease once pretreatment of NETs with DNase I(p<0.05).Conclusion Our current study highlighted the pivotal role of SAA/TLR4 in the direct and indirect induction of angiogenesis.Indeed,TLR4 is expressed on both endothelial cells and neutrophils and its interaction with SAA could induce angiogenic morphological changes on endothelial cell,and promote the NETs formation,which in turn induce angiogenesis by activating TLR4.This pivotal role of SAA/TLR4 may lead to a synergism of action,and we are the first to report this molecular pattern.Therefore,this study offers new perspectives in the understanding of RA pathogenicity and its management. |
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