A preliminary study on the promotion of angiogenesis by NETs in rheumatoid arthritis / 天津医药

Objective To investigate the effect of neutrophil extracellular traps (NETs) on inflammation of rheumatoid arthritis (RA), especially angiogenesis. Methods The presence of NETs in synovial tissues of RA and osteoarthritis (OA) was observed by immunofluorescence assay. Neutrophils were isolated from peripheral blood of health volunteers. Neutrophils were cultured in vitro, the formation of NETs was observed. NETs were extracted as a stimulating agent. The effects of NETs on the proliferation of human umbilical vein endothelial cells (HUVECs) and synovial fibroblasts (RAFLS) were evaluated by MTT, and which were classified into two groups: HUVECs group and RAFLS group, with the following treatment: control and NETs (0.28 mg/L). Wound repair assay was employed to evaluate the effect on the cell migration stimulated with NETs. The experiment was divided into three groups:control, VEGF (40μg/L VEGF) and NETs (0.28 mg/L NETs). Results (1) Compared with OA, NETs were found more in the synovial tissue of RA. (2) NETs formation was induced by stimulator in vitro. The concentration of extracted NETs-DNA was 27.8 mg/L. (3) MTT assay showed that compared with the control groups, low concentration of NETs (0.28 mg/L) promoted the proliferation of HUVECs (0.499 ± 0.011 vs. 0.393 ± 0.009, P<0.05) and RAFLS (0.266 ± 0.007 vs. 0.192 ± 0.007, P<0.05). (4) It was showed that a significant wound closure induced by low concentration of NETs (0.28 mg/L) was found compared with control. Conclusion Our present study suggests that NETs are found more in the synovial tissue of RA, and low concentration of NETs can promote angiogenesis in RA.

Serum amyloid A induces the formation of neutrophil extracellular traps via Toll like receptor 4 in rheumatoid arthritis / 中华风湿病学杂志

Objective To investigate the neutrophil extracellular traps (NETs) formation and their molecular mechanisms induced by serum amyloid A (SAA) in rheumatoid arthritis (RA).Methods Neutrophils were isolated from peripheral blood of RA and healthy volunteers.① Neutrophils were cultured in vitro,the formation of NETs was observed and their percentage was calculated.② Neutrophils were cultured in vitro,divided into six groups:control,SAA,[SAA+anti-Toll like receptro4 (TLR4)-Ab],LPS,(LPS+anti-TLR4-Ab) and anti-TLR4-Ab.Appropriate stimulation was conducted for each group.NETs formation and their percentages were investigated.The concentration of DNA in supernatant was detected by fluorescent staining.F test and t test were used for statistical analysis.Results ① The purification of isolated neutrophils was higher than 95％.The network which was collocated with the spreading neutrophils nucleus and neutrophil elastase under the microscope,was NETs.In the RA group,the formation of NETs induced by SAA was significantly more than control [(19.1±0.8) vs (7.4±0.5),t=12.30,P＜0.05].② However,after pretreated with anti-TLR4 antibody,NETs formation was significantly less than the SAA group [(5.7±0.4) vs (14.7±1.1),t=7.825,P＜0.05].Moreover,the fluorescence intensity of DNA in supernatant was significantly higher in SAA group than that of anti-TLR4-Ab pretreatment group [(18.7 ±0.7) vs (12.9±0.8),t=5.552,P＜0.05].The concentration of DNA in supernatant of SAA group was higher than that of anti-TLR4-Ab pretreatment group [(36.9±1.3) μg/ml and (16.3±0.6) μ,g/ml,t=14.41,P＜0.05].Conclusion SAA can induce the formation of NETs from neutrophils by binding to TLR4 in RA.

Calreticulin-mediated upregulation of anti-apoptotic Bcl-XL and Mcl-1 promotes survival of fibroblast-like synoviocytes in rheumatoid arthritis / 中华风湿病学杂志

Objective Fibroblast-like synoviocytes (FLS) play important roles in the pathogenesis of rheumatoid arthritis (RA).The present study was undertaken to investigate the mechanism of calreticulin (CRT) to promote FLS survival in RA.Methods FLS were isolated by enzymatic digestion of synovial tissue specimens obtained from RA and osteoarthritis (OA) patients and cultured in vitro.The expression of Bcl-XL and Mcl-1 in FLS at mRNA and protein level was detected by quantitative-polymerase chain reaction (q-PCR),Western blotting and immunofluorescence respectively.RA and OA FLS were cultured with different concentrations of recombinant human CRT for 48-72 h,the expression of Bcl-XL and Mcl-1 was detected by q-PCR and Western blotting.The proliferation of RA FLS following CRT stimulation was determined by MTT assay.Results ① Compared with FLS from OA patients (1.00±0.39;1.00±0.46),the anti-apoptotic Bcl-XL and Mcl-l mRNA expression (14.51 ±2.20;12.82±1.80) was significantly higher in the FLS from RA patients (t=10.47,1 1.02;P＜0.01);Western blotting analysis also showed increased protein levels of Bcl-XL and Mcl-1 in RA FLS;Immunofluorescence results showed higher expression of Bcl-XL and Mcl-1 in RA at the single FLS level;② CRT up-regulated the expression of Bcl-XL and Mcl-1 in RA FLS:compared with the control group (0 ng/ml),CRT stimulation at 10 ng/ml and 50 ng/ml increased the levels of Bcl-XL mRNA (1.70±0.28 vs 1.00±0.20,q=4.58,P＜0.05;1.87±0.35 vs 1.00±0.20,q=5.69,P＜O.05) and Mcl-1 mRNA (1.85±0.36 vs 1.00±0.20,q=5.63,P＜0.05;1.72±0.26 vs 1.00±0.20,q=4.77,P＜0.05) in RA FLS,while no significant effects of CRT on Bcl-XL and Mcl-1 mRNA expression were observed in OA FLS (F=1.49,1.60;P＞0.05);Western blotting results showed elevated protein levels of both Bcl-XL and Mcl-1 in RA FLS after CRT treatment at a concentration dependent manner.However,neither Bcl-XL nor Mcl-1 expression was significantly changed in OA FLS.③ MTT assay showed that CRT had no significant effect on the proliferation of RA FLS (F=2.88,P＞ 0.05).Conclusion Our results indicate that CRT-mediated up-regulation of anti-apoptotic Bcl-XL and Mcl-1 may inhibit apoptosis and promote the survival of FLS from RA patients.

Serum amyloid A induces the formation of NETs / 天津医药

Objective To explore whether serum amyloid A (SAA) can induce the formation of neutrophil extracellular traps(NETs)in neutrophils in vitro. Methods A stable method for inducing NETs formation in vitro was established, in-cluding isolation of peripheral blood neutrophils, cell culture, and NETs formation and observation. The neutrophils were iso-lated from peripheral blood of healthy volunteers. And cells were cultured in vitro and classified into three groups:negative control (NC) group, SAA group and lipopolysaccharide (LPS) group. Following the distinct stimulation in three groups, NETs formation was observed and its percentage was calculated. The concentration of hinstone (h) 3 in supernatant was detected by ELISA. Results The purification and vitality of isolated neutrophils were both more than 95%. The nuclei of neutrophils lost their shape and spread, NETs formation was found. More NETs formation was found in SAA group than that in NC group (P < 0.05). Moreover, the concentration of h3 in supernatant was significantly higher in SAA group than that in NC group (P<0.05). Conclusion SAA can induce the formation of NETs in vitro.

Serum amyloid A-induced angiogenesis via scavenger receptor class B type 1 signaling pathway in rheumatoid arthritis / 中华风湿病学杂志

Objective To investigate the role of scavenger receptor class B type 1 (SR-B1) signaling pathway in serum amyloid A (SAA)-induced angiogenesis in rheumatoid arthritis (RA).Methods The expression and location of SR-B1 in RA and osteoarthritis (OA) synovial tissues were observed by immunohistochemistry.And SR-B1 expression in the resting human umbilical vein endothelial cells (HUVECs) was detected by immunoflourescence.Wound repair assessement and tube formation assessement were employed to evaluate the effect on cell migration and tube formation stimulated by SAA and/or anti-SR-B1 antibody.The t-test and one-way analysis of variance (ANOVA) were used for statistical analysis.Results ① SR-B1 was significantly highly expressed in RA tissue samples (A=6 788±819) when compared to the minimal expression in OA (A =31 849±6 977,t=3.567,P＜0.01).Positive staining of SR-B1 was observed in RA synovial vascular endothelial cells and perivascular areas.② Strong staining for SR-B1 was observed in all HUVECs tested.③ Significant wound healing induced by SAA (MI=2.50±0.17) was found compared with the untreated controls (MI=1.00±0.09,q=14.38,P＜0.01),and the effects were inhibited in the presence of anti-SR-B1 antibody (MI=1.16±0.14,q=13.02,P＜0.01).④ Compared to the untreated group (branch point number:6.6±0.8),there was an enhanced formation of branched and capillary-hke tube structure followed by SAA stimulation (branch point number:19.0±1.1,q=25.04,P＜0.01) after culturing for 72 h,whereas,tube formation decreased markedly upon pre-treated with anti-SR-B1 antibody (branch point number:7.6±1.3,vs SAA,q =23.32,P＜0.01).Conclusion Our present study suggests that serum amyloid A may induce angiogenesis via SR-B1 signaling pathway in RA.