Journal: The American journal of pathology

Article Title: Macrophages are critical inducers of bleomycin-induced fibrosis in a systemic scleroderma model

doi: 10.1016/j.ajpath.2025.09.009

Figure Lengend Snippet: (A) Histology sections of mouse skin and lungs stained with the antibody specific for cleaved caspase-3 (CC3, brown, some indicated by arrows) and counterstained with hematoxylin (blue). Quantification is per 1 cm 2 of skin or per 1 lung. Data from one experiment is shown as mean ± SEM. n=10 mice/group. *p<0.05, t-test nonparametric test and Mann-Whitney test. Scale bar, 100 μm. ( B ) Primary thymocytes were induced to undergo apoptosis by irradiation and labeled with pHrodo red, and efferocytosis by Macro BLM or Macro CTRL was analyzed by flow cytometry. Efferocytosis measurements of the percent of all pHrodo red + (Effero All ) and pHrodo red High (Effero High ) macrophages and their geometric mean of pHrodo red fluorescence are shown. Data from one experiment are shown as mean ± SEM, n=3/group. *p<0.05; **p<0.01, ****p<0.0001, Student’s t-test. ( C ) Transcript expression in Macro CTRL and Macro BLM . Data shown is pooled from 3 independent experiments and presented as mean ± SEM, n=4 to 6/group. *p<0.05; **p<0.01, Mann-Whitney tests. ( D ) CTGF and TGFβ1 quantification in Macro BLM and Macro CTRL supernatants. Data from one experiment shown as mean ± SEM, n=4/group. *p<0,05, Student’s t-test. Fibroblast collagen deposit ( E ) and scratch “wound” closure ( F ) were measured after culture with the conditioned media from vehicle (Mac CM ) or bleomycin (BleoMac CM ) treated macrophages for 48 hours. Mitomycin (5 μg/ml) was added in some conditions to inhibit cell proliferation. Data shown is pooled from 2 independent experiments with 2 mice/group and is presented as mean ± SEM. *p<0.05, ****p<0.0001, 1 way ANOVA. ( G ) Apoptotic human Jurkat T cells were labeled with pHrodo red and efferocytosis by human monocyte-derived macrophages was analyzed by flow cytometry as in 5B. Data shown is pooled from 2 independent experiments (each symbol represents a different human donor) and is presented as mean ± SEM. *p<0.05; **p<0.01, Student’s t-test. ( H ) Primary human fibroblasts were cultured in standard medium or in supernatants of vehicle- (Mac CM ) or bleomycin-treated (BleoMac CM ) human macrophages and the collagen content, scratch wound closure and cell viability analyzed after 48 hours. Data shown is pooled from 2 independent experiments with n=3/group and is presented as mean ± SEM. *p<0.05, Mann-Whitney tests.

Article Snippet: Primers were obtained from Thermo Fisher: Tgf1 (Mm01227699_m1), MMP2 (Mm00439498_m1), MMP9 (Mm00442991_m1), TIMP1 (Mm01341361_m1), TIMP2 (Mm00441825_m1), Arg1 (Mm00475988_m1), Lyve1 (Mm00475056_m1), Spp1 (Mm00436767_m1), Ctgf (Mm01192933_g1), Vegf (Mm00437306_m1), Egf (Mm00438696_m1), Tnf (Mm00443258_m1), Il1b (Mm00434228_m1), Ccl2 (Mm00441242_m1), Mertk (Mm00434920_m1), Sirpa (Mm00455928_m1), Slc2a1 (Mm00441480_m1), Il6 (Mm00446190_m1), Il10 (Mm01288386_m1), Adam17 (Mm00456428_m1), Lrp1 (Mm00464608_m1), Cd36 (Mm01135198_m1), Axl (Mm00437221_m1), Tyro3 (Mm00444547_m1), Gas6 (Mm00490378_m1), Elmo1 (Mm00519109_m1), Elmo2 (Mm01248046_m1), and Rac1 (Mm01201653_m1).

Techniques: Staining, MANN-WHITNEY, Irradiation, Labeling, Flow Cytometry, Fluorescence, Expressing, Derivative Assay, Cell Culture

Journal: World Journal of Stem Cells

Article Title: Extracellular vesicles derived from human amniotic fluid stem cells improve bladder dysfunction in rat model of diabetic atherosclerosis

doi: 10.4252/wjsc.v18.i1.113614

Figure Lengend Snippet: Relative mRNA expressions of tumor necrosis factor-α, interleukin-6, transforming growth factor-β1, Smad3, connective tissue growth factor, and fibronectin in the bladder of control and diabetic groups. A-F: Compared with the control group, diabetes mellitus and diabetic atherosclerosis (DMA) bladders showed significant increases in the mRNA expression of tumor necrosis factor α ( TNF-α ), interleukin-6 ( IL-6 ), transforming growth factor β1 ( TGF-β1 ), Smad3 , connective tissue growth factor ( CTGF ), and fibronectin at 4 weeks and 12 weeks after induction. Compared with the diabetes mellitus and DMA groups, the mRNA expression of TNF-α , TGF-β1 , and CTGF in DMA rats treated with human amniotic fluid stem cell-derived extracellular vesicles were significantly decreased at 4 weeks after induction, while the mRNA expression of IL-6 and Smad3 were significantly decreased 12 weeks. a P < 0.05 vs normal control (4 weeks and 12 weeks), b P < 0.05 vs diabetes mellitus (4 weeks and 12 weeks), c P < 0.05 vs diabetic atherosclerosis (4 weeks and 12 weeks). TNF: Tumor necrosis factor; IL: Interleukin; TGF: Transforming growth factor; CTGF: Connective tissue growth factor; DM: Diabetes mellitus; DMA: Diabetic atherosclerosis; hAFSC-EVs: Human amniotic fluid stem cell-derived extracellular vesicles.

Article Snippet: The assay codes of TNF-α, IL-6, TGF-β1, Smad3, CTGF, and fibronectin were Rn01525859_g1, Rn01410330_m1, Rn00572010_m1, Rn00565331_m1, Rn01537279_g1 and Rn00569575_m1, respectively (Applied Biosystems, Oster City, CA, United States).

Techniques: Control, Expressing, Derivative Assay