Fig 2: miR-146b-5p is upregulated after myocardial infarction. (A) Levels of miR-146b-5p in the plasma of patients with chronic total occlusion (CTO) (n = 8 per group). (B) Representative images of infarcted mouse heart. Myocardium samples from different anatomic locations were harvested for the following analysis. (C and D) Expression levels of fibrotic genes Col1 (COL1A1) and αSMA (ACTA2) in myocardium samples (3 hearts, 5 samples per heart). (E) Expression levels of miR-146b-5p in mouse myocardium from different locations and time points, as measured by qPCR (n = 5 per group). Data are expressed as the mean ± SD. *P < 0.05 vs. control; **P < 0.01 vs. control. Data in panel (A), (C), and (D) were analyzed using Student’s unpaired t-test. Data in panel (E) was analyzed using one-way ANOVA followed by Tukey’s post-hoc analysis. Remo, remote zone; Infar, infarction zone.

Fig 3: Exogenous H2S ameliorated TGF-β1–induced AF phenotype switching, proliferation, and migration. AFs were pretreated with or without NaHS (100 μM) or vehicle for 1 hour, then were stimulated with TGF-β1 (10 ng/mL) for 24 hours. A, B, Quantification by cell counting Kit-8 assay. C, AF phenotype switching indicated by the expression of a-SMA was determined by immunofluorescence staining. Scale bar = 20 μm. D, The expression of collagen I and α-SMA was determined by western blotting. E, Quantitative analysis for collagen I. F, Quantitative analysis for α-SMA. G, H, Quantification by cell counting and cell counting Kit-8 assay. I, Representative images of transwell migration assay. Scale bar =100 μm. J, Quantification of migrated cells. Values are represented as means ± standard error of the mean (n = 3); *P < 0.05 versus control group; #P < 0.05 versus TGF-β1 group.

Fig 4: Hypoxia-induced miR-146b-5p expression acts as a phenotypic modulator in fibroblasts, endothelial cells, and macrophages. (A) The expression profile of miR-146b-5p in primary cultured cardiac cells under hypoxia was studied using qPCR (n = 3 per group, 3 replicates). Multiple phenotypic studies were performed after 24 h of treatment with the miR-146b-5p mimic (miR-146b) or control (Ctrl) (B–H). (B) Representative images for EdU staining and EdU+ cell quantification of cardiac fibroblasts (n = 7 samples per group, 5 random fields per sample). Bar = 200 μm. (C and D), Representative migration images and quantification of data from Transwell and scratch assays in mouse fibroblasts (NIH3T3, n = 5 per group, 5 random fields per sample). Bar = 200 μm. (E) Representative images for immunostaining and quantification of expression of the fibroblast-to-myofibroblast transition genes Col1 (COL1A1) and αSMA (ACTA2) in cardiac fibroblasts (n = 3 per group, 7 fields per sample); bar = 5 μm. (F) Western blot analysis of the FMT genes in cardiac fibroblasts after treatment (n = 5). (G) Tube-formation assays of cardiac microvascular endothelial cells (CMVECs), with representative images on the left and quantification data on the right (n = 3 samples per group, 5 random fields per samples, 3 replicates). Bar = 200 μm. (H) ELISA of macrophage-secreted inflammatory factors (n = 5 per group). Data are expressed as the mean ± SD for at least three independent assays, unless otherwise noted. *P < 0.05; **P < 0.01; ns, not significant. Data were analyzed using Student’s t-test. CM, cardiomyocyte; FB, fibroblast; EC, endothelial cell; Mø, macrophage; IL, interleukin; TNF, tumor necrosis factor; MCP, monocyte chemotactic protein.

Fig 5: The distribution of GDNF-AMSCs in the injured kidney and GDNF-AMSCs inhibits EndMT progression in the obstructed kidney. a Intravenously delivered GFP-labeled AMSCs (green) were detected in the obstructed kidneys at days 3 and 7, and GFP-labeled GDNF-AMSCs (green) were more detected by laser confocal microscopy. Nuclei were stained with DAPI (blue). b There was no evident two-color immunofluorescence that identifies EndMT cells that coexpress endothelial cells (CD31, red) and myofibroblast cells (a-SMA, green) shown in the sham group. EndMT progression was increased in the UUO group and decreased in the AMSC and GDNF-AMSC group, especially in the GDNF-AMSC group, indicating by the coexpression of CD31+a-SMA+. White arrows indicate positive coexpressing staining. Nuclei were stained with DAPI in blue. Bar = 20 μm