Adipose progenitor cell-derived extracellular vesicles suppress macrophage M1 program to alleviate midlife obesity

Figures

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  Fig. 1. The susceptibility of middle-aged mice to obesity is attributed to altered WAT function. a Body weight gain of young (3-month-old) and middle-aged (12-month-old) mice fed with a 4-week high-fat diet (HFD) (n = 14 per group). b, c Glucose tolerance test (GTT) (b) and insulin tolerance test (ITT) (c) results of young and middle-aged mice challenged with a 4-week HFD (n = 14 per group). d Flow cytometry analysis of the proportions of M1 (CD11b+/F4/80+/CD11c+/CD206-) and M2 (CD11b+/F4/80+/CD11c-/CD206+) macrophages in the epididymal WAT of young and middle-aged mice (n = 8 biological replicates per group). e RT‒qPCR analysis of the mRNA expression of proinflammatory genes (Tnf-α, Il-1β, and Il-6) in the epididymal WAT of young and middle-aged mice challenged with a 4-week HFD (n = 8 biological replicates per group). f Number of differentially expressed genes (DEGs) in the liver, muscle and WAT of C57BL/6 mice of different ages (9, 12, and 15 months). g–i Western blot analysis of p-AKTSer473/AKT, p-ACCSer79/ACC, p-HSLSer660/HSL and PGC1α in WAT (g), p-AKTSer473/AKT, PGC1α, SDHB and NDUFS1 in skeletal muscle (h), p-AKTSer473/AKT, PEPCK, G6Pase and p-GSK3βSer9/GSK3β in liver (i) from…

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  Fig. 2. Midlife APC-derived EVs exhibit a reduced capacity to maintain adipose immune homeostasis. a A t-SNE plot displaying single-nucleus RNA sequencing data of cells from WAT samples from young and middle-aged humans. b Cell identity annotation in human WAT samples. c Number of DEGs for each identified cell subgroup. d, e Western blot results of p19ARF and p53 in mouse APCs (d), P16Ink4a and P21 in human APCs (e) from the young (mice, 3-month-old; humans, aged 18-29), middle-aged (mice, 12-month-old; humans, aged 30-49) and aged (mice, 24-month-old; humans, aged 50-69) groups (n = 3 independent experiments). f, g Transcriptional levels of senescence-associated genes in the APCs of mice (p16Ink4a, p21, Il-6, and Igfbp3) (f) and humans (P16Ink4a, P21, IL-6, and IGFBP3) (g) from the indicated groups (n = 6 per group). h Number of DEGs annotated through gene ontology (GO) enrichment-based clustering based on bulk RNA-seq analysis of young and middle-aged APCs from mice or humans. i The engulfment of PKH26-labeled EVs (red) by APCs, adipocytes and macrophages was detected by confocal microscopy. The experiments were repeated independently three times. Scale bar, 20 µm. j The engulfment of…

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  Fig. 3. APC-derived EVs suppress WAT inflammaging to prevent obesity. a A schematic diagram of the mouse treatments. b Body weight gain of 12-month-old male mice fed a NCD or HFD and treated with PBS, yEVs or mEVs (n = 10 per group). c Energy expenditure of 12-month-old male mice treated with mEVs or yEVs (n = 4 per group). GTT (d) and ITT (e) of 12-month-old male mice fed a NCD or HFD and treated with PBS, yEVs or mEVs (n = 10 per group). f Western blot of p-AKTSer473/AKT in the eWAT, liver and skeletal muscle of 12-month-old male mice fed a NCD or HFD and treated with PBS, yEVs or mEVs. g Confocal images and quantitative analysis of crown-like structures (CLSs, arrows) in eWAT from 12-month-old male mice fed a NCD or HFD and treated with PBS, yEVs or mEVs (n = 10 biological replicates per group). Scale bar, 25 µm. h Flow cytometry analysis of M1 (CD11b+/F4/80+/CD11c+/CD206-) and M2 (CD11b+/F4/80+/CD11c-/CD206+) macrophage subsets in the eWAT of 12-month-old male mice fed a NCD or HFD and treated with PBS, yEVs or mEVs (n = 10 per group). i RT‒qPCR analysis of Tnf-α, Il-1β, and Il-6 in eWAT from 12-month-old male mice fed a NCD or HFD and treated with PBS, yEVs or mEVs (n = 10…

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  Fig. 4. EV-derived miR-145 serves as a critical factor in suppressing the ATM M1 program. a Heatmap of miRNA expression profiles in yEVs and mEVs (n = 3 per group). b RT‒qPCR analysis of the candidate miRNAs in yEVs and mEVs (n = 3 biological replicates per group). c Correlation analysis between age and relative miR-145 expression in EVs derived from APCs humans of different ages. d Relative miR-145 expression in APCs from mice of different aged mice (3, 6, 9, and 12 months old) (n = 3 biological replicates per group). Flow cytometry analysis of CD11c (e), Western blotting results for iNOS (f) and RT‒qPCR results for the proinflammatory genes Tnf-α, Il-1β, and Il-6 (g) in BMDMs treated with PBS, PBS + miR-145 mimics, LPS, LPS + scramble, LPS + miR-145 mimics, LPS + miR-145 mimics + miR-145 inhibitor (n = 3 biological replicates per group). Flow cytometry results of CD11c (h), Western blotting results for iNOS (i) and relative mRNA expression of Tnf-α, Il-1β, and Il-6 (j) in BMDMs treated with PBS, LPS, LPS + yEVs, LPS + miR-145 inhibitor + yEVs (n = 3 biological replicates per group). The data were presented as mean ± SEM. Statistical significance was assessed by two-sided Student’s…

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  Fig. 5. miR-145 attenuates the macrophage M1 program by inhibiting SELL expression. a Average FPKM values of the PBS-treated group and the miR-145 mimics-treated group (n = 3 per group). Box plots present the means (lines inside the boxes), the 1st and 3rd quartiles (bottom and top bounds of the boxes). b Volcano plot of DEGs (fold change >2, p value ≤ 0.05) between the PBS-treated group and the miR-145 mimics-treated group. c Potential target genes of miR-145 predicted by TargetScan. d Heatmap of potential differentially expressed target genes in the PBS- and miR-145 mimics-treated groups. e Correlation analysis between age and the relative mRNA expression of Sell in the WAT ATMs of humans of different ages. f Protein expression levels of iNOS in BMDMs treated with either scrambled or Sell siRNA (n = 3 independent experiments). g Protein expression levels of iNOS in BMDMs subjected to specific treatments (n = 3 independent experiments). h KEGG analysis of all genes in M1 macrophages treated with scrambled or Sell siRNA, with a focus on the top 20 categories that exhibited the most significant changes. i, j Western blot results for iNOS, p-NF-κB p65, NF-κB p65, p-IκBα, and IκBα in BMDMs…

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  Fig. 6. Construction of cationic liposomes for targeted delivery of miR-145. a Schematic illustration of miR-145@tar-lip via the binding of a peptide (CKGGRAKDC) to the surface of liposomes (miR-145@lip). b Hydrodynamic diameter, polydispersity index (PDI), zeta potential, and entrapment efficiency of the prepared liposomes. c Representative transmission electron microscopy (TEM) image of the liposomes. The experiments were repeated independently three times. Scale bar, 500 nm. d Hydrodynamic diameter distribution of the liposomes. e Colloidal stability of the liposomes within 24 h (n = 3 per group). f Biodistribution of liposomes at 0 h, 0.5 h, 2 h, 8 h, and 24 h tracked by an in vivo imaging system (IVIS) after intravenous injection of miR-145@lip or miR-145@tar-lip. g Accumulation of miR-145@lip and miR-145@tar-lip in iWATs and eWATs. h Flow cytometry analysis of miR-145@lip and miR-145@tar-lip uptake by SVF cells and ATMs. The data were presented as mean ± SEM (e). See also Supplementary Fig. 8. Source data are provided as a Source Data file.

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  Fig. 7. miR-145@tar-lip efficiently attenuates HFD-induced obesity in middle-aged mice. a Schematic diagram of the mouse treatments. b Body weight gain of middle-aged mice treated with PBS, NC miR@tar-lip, miR-145@lip or miR-145@tar-lip (n = 7 per group). c Energy expenditure of mice treated with miR-145@lip or miR-145@tar-lip (n = 4 per group). GTT (d) and ITT (e) of results for middle-aged mice treated with PBS, NC miR@tar-lip, miR-145@lip or miR-145@tar-lip (n = 7 per group). f Western blot results of p-AKTSer473/AKT in eWAT, liver and skeletal muscle from the indicated groups (n = 3 independent experiments). g Macrophage infiltration (green color) and crown-like structures (CLSs, arrows) in eWAT of mice treated with PBS, NC miR@tar-lip, miR-145@lip or miR-145@tar-lip and the statistics for the CLSs (n = 7 biological replicates per group). Scale bar, 25 µm. h Flow cytometry analysis of M1 (CD11b+/F4/80+/CD11c+/CD206-) and M2 (CD11b+/F4/80+/CD11c-/CD206+) macrophage subsets in eWAT of mice treated with PBS, NC miR@tar-lip, miR-145@lip or miR-145@tar-lip (n = 7 biological replicates per group). i RT‒qPCR results for the proinflammatory genes Tnf-α, Il-1β, and Il-6 in eWAT of mice…

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  Fig. 8. Scheme of miR-145 in APC-derived EVs modulating midlife obesity. Senescent adipose progenitor cells (APCs) in middle-aged individuals are the cause of midlife obesity. miR-145 in APC-derived extracellular vesicles improves inflammaging and insulin resistance in middle-aged subjects. miR-145 inhibits the M1 polarization of adipose tissue macrophages by suppressing the SELL‒NF‒κB axis. miR-145 is a proof-of-concept effective target for the intervention of midlife obesity (Created in BioRender. Wang, W. (2025) https://BioRender.com/k41c453).