Fig 1: M2 macrophage-secreted exosomes promote pulmonary interstitial fibroblast proliferation. a Arg-1 activity of M0 and M2 macrophages, *p < 0.05 vs. M0 macrophages. b Positive expression rates of CD9, CD63 and CD81 in M0 and M2 macrophages, * p < 0.05 vs. M0 macrophages. c Exosome morphology observed by TEM. D, Exosome particle size analysis. e The protein levels of the exosomal markers CD9, CD63 and CD81 examined by western blot analysis. f The internalization of M2 macrophage-secreted exosomes by pulmonary interstitial fibroblasts at different time points; green indicates PKH67-labeled exosomes, and red represents pulmonary interstitial fibroblasts, bar = 25 μm. g, Edu labeling to examine the proliferation of pulmonary interstitial fibroblasts after a coculture with exosomes (200 × ). H, Quantification of the results in G. * p < 0.05 vs. pulmonary interstitial fibroblasts. I, Expression of Collagen 1 A, Collagen 3 A and α-SMA in pulmonary interstitial fibroblasts after a coculture with exosomes, as measured by RT-qPCR. * p < 0.05 vs. pulmonary interstitial fibroblasts. Arg-1, arginase 1; EdU, 5-ethynyl-2’-deoxyuridine; α-SMA, α-smooth muscle actin; and RT-qPCR, reverse transcription quantitative polymerase chain reaction. The results were measurement data and were analyzed using an unpaired t test. The results are expressed as the mean ± standard deviation. The experiment was conducted in triplicate
Fig 2: Characterization of small‐EVs isolated from human ovarian ascites fluid. (A) Representative liquid Atomic Force Microscopy (AFM) micrographs of small‐EVs from ascites‐derived tumor cells (A) and small‐EVs from bulk fluid ascites (B), the length of both scale bars is 1 µm. On the right: Contact Angle vs Diameter scatterplot of small‐EVs. Each circle represents one individual exosome as measured via AFM imaging in liquid. (B) CD63, CD9, and TSG101 were detected by western blot in small‐EVs extracted from ascites‐derived tumor cell cultures (1) and bulk fluid ascites small‐EVs (2). For each set of analyses, representative results out of three independent experiments are shown.
Fig 3: Albumin promoted exosomes secretion via Rab27a and induced TECs injury as a paracrine or autocrine signal.a, b Rab27a knockdown by siRNA transfection in the presence of BSA. Rab27a was efficiently knockdown at mRNA and protein level. *p < 0.05 vs TECs transfected with NC. c, d Quantification of exosomes isolated from cultured supernatant of TECs. Knockdown of Rab27a significantly reduced exosome secretion as detected by western blotting analysis of exosomal markers (Alix, CD63, and CD81) (c) and EXOCET assay (d). **p < 0.01, ****p < 0.0001 vs TECs transfected with NC. e Exosomes isolated from TECs exposed to different doses of BSA were applied to naïve TECs. The mRNA expression of inflammatory cytokine (MCP-1, TNF-α, and IL-6) and tubular injury markers (KIM-1 and LCN2) in exosome-treated TECs are normalized to GAPDH and compared with Ctrl-exosomes-treated TECs (represented by 1-fold). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs TECs treated with Ctrl TEC-exo. f Expression of inflammatory cytokine and tubular injury marker in Rab27a knockdown-TECs in the presence of BSA are normalized to GAPDH and compared with NC-transfected TECs with BSA treatment (represented by 1-fold). **p < 0.01, ***p < 0.001 vs TECs transfected with NC. NC, negative control. Data presented as mean ± S.E.M. of three independent experiments.
Fig 4: Rab27a-dependent exosome secretion provides an alternative pathway to lysosome for albumin handling in TECs.a, b Detection of BSA in purified exosome fraction from cultured TECs. After incubation with or without BSA for 24 h, the medium was replaced with albumin-free medium and cells were cultured for another 24 h allowing the secretion of exosomes. BSA was readily detected by western blot analysis in the cell and exosome fractions isolated by differential ultracentrifugation (a). BSA was detected in exosome fractions with density of 1.11 g/ml which was purified from OptiPrep density gradient centrifugation to exclude the possibility of protein contaminant (b). c Representative confocal co-localization of the MVB marker CD63 (red) and FITC-BSA (green). FITC-BSA was applied to TECs for 24 h and cells were fixed and immunostained with anti-CD63 antibody. Scale bars: 20 μm. d Western blotting of lysosome cathepsin B and D in cell lysates from TECs with Rab27a siRNA at different dose of BSA. e Representative confocal microscopy analysis of DQ-BSA (red) to show albumin degradation in TECs. DQ-Red BSA were added after BSA treatment for 20 h, cells were fixed and stained with DAPI (blue). Significant amount of degraded albumin (red) was detected in BSA overload TECs, while increased albumin degradation was observed with Rab27a siRNA group. Scale bars: 20 μm. f–g Exosome secretion and inflammation response in TECs with Lysosome inhibitor, Baf. Exosome producing (f) as well as inflammatory cytokine expression were enhanced by Baf treatment (g). Data presented as mean ± S.E.M. of three independent experiments.
Fig 5: Urinary small EVs derived CCL21 mRNA increases in biopsy-proven DN patients. A Representative micrographs of transmission electron microscopy showed the typical size and shape of urinary small EVs from DN patients with higher and lower magnification. (scale bar: up-100 nm, down-200 nm). B Western blot and analysis of exosome (Alix, CD81 and CD63) and tubular marker AQP1 and AQP2 in urinary small EVs fraction from healthy individuals, patients with T2DM and biopsy-proven DN and the quantification was normalized to urine creatinine. C Size distribution and particle number of urinary small EVs analyzed by nanoparticle tracking analysis (NTA) and the quantification was normalized to urine creatinine. D Fourteen cytokines and chemokines as well as receptors were screened in DN (n = 4 for each group). The relative expression of CCL21 mRNA in urinary small EVs showed significant increase in DN patients compared with healthy controls and T2DM patients. E The expression of CCL21 mRNA in urinary small EVs of DN patients (n = 28) was significantly higher than healthy controls (n = 16) and patients with T2DM (n = 15) in validation cohorts. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with healthy controls. #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001, compared with patients with T2DM. HC healthy control; T2DM type 2 diabetes; DN diabetic nephropathy. F CCL21 mRNA expression in urinary small EVs isolated from DN patients with or without RNase and Triton X treatment. ****p < 0.0001, compared with urinary small EVs treated with RNase and TritonX
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