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. 2017 Aug 3;8(43):73529–73546. doi: 10.18632/oncotarget.19867

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Copyright: © 2017 Zhang et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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RA-FLS and HDMECs co-cultures were respectively treated with As₂O₃ alone or together with TNF-α (100ng/ml) for 48 h. A. TSP-1, TGF-β1, CTGF and VEGF protein expression in the supernatants from RA-FLS and HDMECs co-cultures were analyzed by ELISA. Results showed that concentrations of TSP-1, TGF-β1, CTGF and VEGF increased significantly after treatment with TNF-α (100 ng/ml) (n = 3) compared to vehicle control group (n = 3; & p < 0.05), and then significantly decreased concentrations of TSP-1, TGF-β1, CTGF and VEGF were observed after the treatment of As₂O₃ at doses of 1.0 μM and 2.0 μM (n = 3, respectively; #p < 0.05, ##p < 0.01). TSP-1, TGF-β1, CTGF and VEGF protein expression in the supernatants from RA-FLS and HDMECs co-cultures without TNF-α addition also decreased significantly after treatment of As₂O₃ at doses of 1.0 μM (n = 3) and 2.0 μM (n = 3) compared to vehicle control (n = 3; *p < 0.05). B. mRNA levels of TSP1, TGF-β1, CTGF and VEGF expression in RA-FLS co-cultured were performed by real-time PCR. Results showed similar changes of TSP-1, TGF-β1, CTGF and VEGF mRNA expression as demonstrated in protein regulation after treatment of As₂O₃ alone (n = 3) or together with TNF-α (100ng/ml) (n = 3; *p < 0.05, **p < 0.01, #p < 0.05, ##p < 0.01). C. and D. Transwell assay and tube formation test were performed by applying supernatants from RA-FLS and HDMECs co-culture to HDMECs for 6 h respectively. Results showed that migration and capillary-like structure formation of HDMECs significantly increased after TNF-α (100ng/ml) stimulation (n = 3, respectively; *p < 0.05), while As₂O₃ at doses of 1.0 μM (n = 3) and 2.0 μM (n = 3) played a significantly opposite role in migration and tube formation with or without TNF-α (*p < 0.05, **p < 0.01, #p < 0.05, ##p < 0.01), which were also in a dose dependent manner. E. Ex vivo aortic ring angiogenesis assay showed similar changes of microvessel sprouting as demonstrated in migration and tube formation of HDMECs in the transwell assay and tube formation test above (n = 3, respectively; *p < 0.05, **p < 0.01, #p < 0.05). Bars = 300μm. Original magnification = ×5. Results are expressed as the mean ± S.E.M. & p < 0.05 versus Veh. *p < 0.05, **p < 0.01 versus Veh, #p < 0.05, ##p < 0.01 versus TNF-α. Veh = vehicle control under treatment of 1% FBS DMEM alone. TNF-α = control group under treatment of TNF-α (100ng/ml).

RA-FLS and HDMECs co-cultures were respectively treated with As₂O₃ alone or together with TNF-α (100ng/ml) for 48 h. A. TSP-1, TGF-β1, CTGF and VEGF protein expression in the supernatants from RA-FLS and HDMECs co-cultures were analyzed by ELISA. Results showed that concentrations of TSP-1, TGF-β1, CTGF and VEGF increased significantly after treatment with TNF-α (100 ng/ml) (n = 3) compared to vehicle control group (n = 3; & p < 0.05), and then significantly decreased concentrations of TSP-1, TGF-β1, CTGF and VEGF were observed after the treatment of As₂O₃ at doses of 1.0 μM and 2.0 μM (n = 3, respectively; #p < 0.05, ##p < 0.01). TSP-1, TGF-β1, CTGF and VEGF protein expression in the supernatants from RA-FLS and HDMECs co-cultures without TNF-α addition also decreased significantly after treatment of As₂O₃ at doses of 1.0 μM (n = 3) and 2.0 μM (n = 3) compared to vehicle control (n = 3; *p < 0.05). B. mRNA levels of TSP1, TGF-β1, CTGF and VEGF expression in RA-FLS co-cultured were performed by real-time PCR. Results showed similar changes of TSP-1, TGF-β1, CTGF and VEGF mRNA expression as demonstrated in protein regulation after treatment of As₂O₃ alone (n = 3) or together with TNF-α (100ng/ml) (n = 3; *p < 0.05, **p < 0.01, #p < 0.05, ##p < 0.01). C. and D. Transwell assay and tube formation test were performed by applying supernatants from RA-FLS and HDMECs co-culture to HDMECs for 6 h respectively. Results showed that migration and capillary-like structure formation of HDMECs significantly increased after TNF-α (100ng/ml) stimulation (n = 3, respectively; *p < 0.05), while As₂O₃ at doses of 1.0 μM (n = 3) and 2.0 μM (n = 3) played a significantly opposite role in migration and tube formation with or without TNF-α (*p < 0.05, **p < 0.01, #p < 0.05, ##p < 0.01), which were also in a dose dependent manner. E. Ex vivo aortic ring angiogenesis assay showed similar changes of microvessel sprouting as demonstrated in migration and tube formation of HDMECs in the transwell assay and tube formation test above (n = 3, respectively; *p < 0.05, **p < 0.01, #p < 0.05). Bars = 300μm. Original magnification = ×5. Results are expressed as the mean ± S.E.M. & p < 0.05 versus Veh. *p < 0.05, **p < 0.01 versus Veh, #p < 0.05, ##p < 0.01 versus TNF-α. Veh = vehicle control under treatment of 1% FBS DMEM alone. TNF-α = control group under treatment of TNF-α (100ng/ml).