Journal: Bone Research

Article Title: FABP4 secreted by M1-polarized macrophages promotes synovitis and angiogenesis to exacerbate rheumatoid arthritis

doi: 10.1038/s41413-022-00211-2

Figure Lengend Snippet: The effect of FABP4 and M1-polarized macrophage supernatant on HUVECs, FLSs, and chondrocytes in vitro. a , b Tube formation assay and quantification of HUVECs cultured with vehicle (C), rhFABP4 (P), rhFABP4 + BMS309403 (PB), M1-polarized macrophage supernatant (S), or M1-polarized macrophage supernatant+BMS309403 (SB) ( n = 3 per group). Scale bar: 100 µm. c Immunoblot analysis of FABP4 and VEGFα in HUVECs cultured with C, P, PB, S, or SB for 24 h. d , g Representative images and quantification of BrdU (green) immunofluorescence ( d ) and Transwell assays ( g ) in HUVECs and FLSs treated with C, P, PB, S, or SB for 24 h ( n = 3 per group). Scale bar: 25 µm, 100 µm. e , f Immunoblot analysis of ERK1/2, p-ERK1/2, P65, and p-P65 in HUVECs ( e ) and FLSs ( f ) treated with C, P, PB, S, or SB for 1 h. h Toluidine blue staining of ATDC5 cells treated with C, P, PB, S, or SB for 24 h ( n = 3 per group). i Immunoblot analysis of FABP4, MMP13, Sox9, and Col2a1 in primary chondrocytes treated with C, P, PB, S, or SB for 24 h. j Immunoblot analysis of P65 and p-P65 in primary chondrocytes treated with C, P, PB, S, or SB for 1 h. One-way ANOVA and Tukey’s multiple comparison test. * P < 0.05, ** P < 0.01; # P < 0.05, ## P < 0.01 compared to the control. The data are shown as the mean ± SEM

Article Snippet: Macrophages and their supernatant were collected after being stimulated with 50, 200, or 500 ng·mL −1 LPS (Invitrogen, San Diego, CA, USA) for 12 h or 24 h. HUVECs, FLSs, and primary chondrocytes were treated with 0.4 μmol·L −1 recombinant human FABP4 (#RPB693Hu01, Cloud-Clone Corp., China), M1-polarized macrophage supernatant, or 20 μmol·L −1 BMS309403 (MedChemExpress, Shanghai, China) for 1 hour to examine pathway activation or 24 h to examine phenotypic alterations.

Techniques: In Vitro, Tube Formation Assay, Cell Culture, Western Blot, Immunofluorescence, Staining, Comparison, Control

Journal: Bone Research

Article Title: FABP4 secreted by M1-polarized macrophages promotes synovitis and angiogenesis to exacerbate rheumatoid arthritis

doi: 10.1038/s41413-022-00211-2

Figure Lengend Snippet: Activation of the mTORC1 pathway enhances the secretion of FABP4 by M1-polarized macrophages to exacerbate RA progression. a , b Representative images and quantification of F4/80 and pS6 coimmunostaining in the mouse ( n = 10 per group) ( a ) and human synovium ( n = 16 per group) ( b ) of the control and RA groups. Scale bar: 12.5 µm. c Western blot showing S6 and pS6 in human synovial tissue. d Western blot showing FABP4, NOS2, S6 and pS6 in BMDMs treated with LPS or rapamycin. e Representative immunofluorescence staining images and quantitative analysis of FABP4 in BMDMs stimulated with LPS or rapamycin ( n = 3 per group). Scale bar: 100 µm. f Western blot showing FABP4, NOS2, S6 and pS6 in BMDMs treated with MHY1485 or rapamycin. g Representative immunofluorescence staining images and quantitative analysis of FABP4 in BMDMs treated with MHY1485 or rapamycin ( n = 3 per group). Scale bar: 100 µm. h Western blot showing FABP4, NOS2, S6 and pS6 in BMDMs from control and TSC1KO mice. i Representative images and quantification of F4/80 and pS6 coimmunostaining in the synovium of wild-type and TSC1KO mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bar: 12.5 µm. j Representative images and quantification of FABP4 immunohistochemical staining and coimmunostaining of FABP4 with F4/80 or NOS2 in the synovium of control and TSC1KO mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bars: 12.5 µm and 100 µm. f–j Representative images ( f ) and quantification of Vimentin and MMP3 coimmunostaining ( g ), CD31 and EMCN coimmunostaining ( h ), Col2a1 immunofluorescence staining ( i ), and MMP13 immunohistochemical staining ( j ) in the knee joints of controls and TSC1KO mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bar: 12.5 µm, 25 µm, and 100 µm. Student’s t -test or one-way ANOVA and Tukey’s multiple comparison test. ** P < 0.01. The data are shown as the mean ± SEM

Article Snippet: Macrophages and their supernatant were collected after being stimulated with 50, 200, or 500 ng·mL −1 LPS (Invitrogen, San Diego, CA, USA) for 12 h or 24 h. HUVECs, FLSs, and primary chondrocytes were treated with 0.4 μmol·L −1 recombinant human FABP4 (#RPB693Hu01, Cloud-Clone Corp., China), M1-polarized macrophage supernatant, or 20 μmol·L −1 BMS309403 (MedChemExpress, Shanghai, China) for 1 hour to examine pathway activation or 24 h to examine phenotypic alterations.

Techniques: Activation Assay, Control, Western Blot, Immunofluorescence, Staining, Immunohistochemical staining, Comparison

Journal: Bone Research

Article Title: FABP4 secreted by M1-polarized macrophages promotes synovitis and angiogenesis to exacerbate rheumatoid arthritis

doi: 10.1038/s41413-022-00211-2

Figure Lengend Snippet: Inhibition of the mTORC1 pathway reduces FABP4 secretion by M1-polarized macrophages to attenuate RA progression. a Representative images and quantification of F4/80 and pS6 coimmunostaining in the synovium of controls and Rheb1KO mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bar: 12.5 µm. b – e Representative images ( b ) and quantitative analysis of FABP4 immunohistochemical staining ( c ) and coimmunostaining of FABP4 with F4/80 ( d ) or NOS2 ( e ) in the synovium of controls and Rheb1KO mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bars: 12.5 µm and 100 µm. f – j Representative images ( f ) and quantitative analysis of Vimentin and MMP3 coimmunostaining ( g ), CD31 and EMCN coimmunostaining ( h ), Col2a1 immunofluorescence staining ( i ), and MMP13 immunohistochemical staining ( j ) in the knee joints of controls and Rheb1KO mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bar: 12.5 µm, 25 µm, and 100 µm. Student’s t -test. ** P < 0.01. The data are shown as the mean ± SEM

Article Snippet: Macrophages and their supernatant were collected after being stimulated with 50, 200, or 500 ng·mL −1 LPS (Invitrogen, San Diego, CA, USA) for 12 h or 24 h. HUVECs, FLSs, and primary chondrocytes were treated with 0.4 μmol·L −1 recombinant human FABP4 (#RPB693Hu01, Cloud-Clone Corp., China), M1-polarized macrophage supernatant, or 20 μmol·L −1 BMS309403 (MedChemExpress, Shanghai, China) for 1 hour to examine pathway activation or 24 h to examine phenotypic alterations.

Techniques: Inhibition, Immunohistochemical staining, Staining, Immunofluorescence