ABSTRACT
Objective:To explore the trans-membrane signaling mechanism of interleukin-6 (IL-6)-induced osteogenic differentiation and calcification of human umbilical artery smooth muscle cells (HUASMCs).Methods:HUASMCs were primarily cultured in vitro and were stimulated with IL-6, IL-6+solutable IL-6 receptor (sIL-6R), IL-6+sIL-6R+solutable gp130 (sgp130), or vehicle (blank control). Alizarin red and Von Kossa staining were used for detecting cell calcification, Western blot was used to test the protein expression of tissue-nonspecific alkaline phosphatase (TNAP), osteopontin (OPN), bone morphogenetic protein-2 (BMP-2) and Runt related transcription factor 2 (Runx2), and immunofluorescence was used to examine the mIL-6R expression of HUASMCs. The comparison of measurement date between the two groups was conducted by t-test. The comparison of measurement date between multiple groups was conducted by one-way analysis of variance (ANOVA). Results:The intensity severity of calcification stain was IL-6+sIL-6R group >IL-6+sIL-6R+sgp130 group>IL-6 group=blank control. After stimulated for 12 hours, the TNAP expression in blank control, IL-6 group, IL-6+sIL-6R group, IL-6+sIL-6R+sgp130 group were (0.44±0.08), (0.52±0.14), (0.84±0.16) and (0.55±0.10) respectively ( F=290.96, P<0.001). After stimulated for 3 days, the OPN expression in blank control, IL-6 group, IL-6+sIL-6R group, IL-6+sIL-6R+sgp130 group were (0.61±0.84), (0.95±0.16), (1.65±0.24) and (0.99±0.10) respectively ( F=507.72, P<0.001). After stimulated for 12 hours, the BMP-2 expression in blank control, IL-6 group, IL-6+sIL-6R group, IL-6+sIL-6R+sgp130 group were (0.77±0.05), (1.69±0.16), (2.81±0.26) and (0.57±0.12) respectively ( F=959.09, P<0.001). After stimulated for 3 days, the Runx2 expression in blank control, IL-6 group, IL-6+sIL-6R group,IL-6+sIL-6R+sgp130 group were (0.57±0.03) , (0.92±0.10), (1.31±0.13) and (0.66±0.06) respectively ( F=1141.27, P<0.001). Comparing with Jurkat cells (positive control) and CEM cells (negative control), HUASMCs limited expressed mIL-6R. Conclusion:IL-6 may induce HUASMCs osteogenic differentiation and calcification mainly via the sIL-6R-mediated trans-signaling pathway.
ABSTRACT
Objective To observe the aortic calcification level in patients with rheumatoid arthritis (RA),and to analyze the relationships between aortic calcification and some RA disease related presentations.Methods RA patients (RA group) were all in-patients consecutively recruited from the Department of Rheumatology in one single tertiary hospital,and healthy subjects (control group) were individuals for check-up from the same hospital at the same time.Subjects with long-term smoking and drinking history,diabetes,hypertension,coronary heart disease,cancer,active or chronic infection,other autoimmune diseases and liver or kidney dysfunction were excluded in both groups.The aortic calcification scores (including ascending aorta,arcus aorta and aorta thoracica) were obtained automatically by 256-slice spiral CT scanner using the Heart Beat-CS program.Statistical package from Soci-science (SPSS) 17.0 software was used for data analysis.Student's t test,Mann-Whitney U test,Spearman test and x2 test were used.Results One hundred RA patients and 60 healthy subjects were selected,and there were no differences of age [(53±10) vs (51 ±8),t=1.031,P=0.304) and gender compositions [male 40(40％) vs 25(41％),x2=0.430,P=0.869) between the two groups.The aortic calcification score in the RA group was higher than that in the control group [19.4(3.3,190.0) vs 2.1 (1.9,18.0),U=1 579.5,P＜0.01].In RA group,the calcification score was positively correlated with age (r=0.729,P＜0.01),course of disease (r=0.227,P=0.023),C-reactive protein (CRP) (r=0.229,P=0.022),total cholesterol (TC) (r=0.220,P=0.028) and low density lipoprotein cholesterol (LDL-C) (r=0.224,P=0.014),but not related with treatment duration,number of tender joints and swollen joints,erythrocyte sedimentation rate,rheumatoid factor,anti-CCP antibody,DAS-28 (CRP),DAS-28 (ESR),triglyceride (TG) and high density lipoprotein cholesterol (HDL-C).The aortic calcification was also positively correlated with age in control group (r=0.465,P＜0.01),but not related with TC,TG,HDL-C,LDL-C.Conclusion RA patients have more severe aortic calcification than the matched general population.Aortic calcification degree is related to disease course,CRP,TC and LDL-C,which indicates that chronic systemic inflammation is essential to aortic calcification in RA.
