Fig 1: Effect of exosomes isolated from aneurysmal telocytes (TCs) on proliferation, metabolism, and phenotype specific mRNA expression of aortic vascular smooth muscle cell (vSMC)-cell culture. Exosomes were isolated from isolated TCs from healthy aortic tissue (HTA, n = 10) or thoracic ascending aneurysm (TAA, n = 10). (A) Morphological changes shown by light microscopy and α-SMA reengagement by immunofluorescence in SMCs treated with TC-exosomes compared to SMC control in isolated aortic cell culture containing vSMCs and fibroblasts (FB). CD90 (FB-marker), red; α-SMA (vSMC-marker), green; DAPI (cell nucleus), blue. Scale bar of light microscopy 20 µm. (B) Quantitative RT-PCR of SMC-phenotype specific mRNA expression after HTA-exosomes or TAA-exosomes compared to vSMC-control. Synthetic phenotype mRNA expression (COL1A1, VIM, and KLF4) were increased, whereby contractile phenotype mRNA expression (MYH11, ACTA, and CNN1) showed no differences after treatment with TAA-exosomes. Bars indicate the relative expression of each mRNA normalized to GAPDH and RPLP0. Data are mean ± SD of two independent experiments. **, p < 0.01. (C) Effect of HTA- and TAA-exosomes compared to vSMC-control tested in aortic vSMCs by MTT assay after 1 and 4 days of treatment. PDGF-BB treatment was used as positive control. ns, non-significant. Data are mean ± SD of four independent experiments. (D) Dilution-depended exosome-induced cell proliferation tested in aortic SMCs. OD values resulting from CCK-8 assay, HTA- and TAA-exosomes were compared to vSMC control. OD, optical density. Data are mean ± SD of four independent experiments. *, p < 0.05; **, p < 0.01. (E) Collagen-I measurements after different exosome or control treatments after 3 days tested in vSMCs with PDGF-BB as positive control. Data are mean ± SD of three independent experiments. (F,G) Cell migration (scratch wound healing assay). (F) Values of percentage wound closure ± SEM (n = 3). Different exosome treatment or TC-conditioned medium (TCM) treatment were compared to vSMC medium as control group 24 h after treatment start. (G) Representative images are shown from three independent experiments at time points beginning (0 h), 12 h, or 24 h. Blue area defines the areas lacking cells, initial scratch line shown by dashed lines (wound area, ImageJ). Scale bar, 20 µm.
Fig 2: Effects of fEVs from MASLD patients on the mRNA expression of different genes in HepG2 cells (n = 4). (A) Receptors involved in bacterial recognition (TLR4 and TLR5). (B) Genes related to the immune response (IL1B, IL6 and IL10). (C) Secretion of soluble collagen into the culture medium and mRNA expression of genes involved in fibrogenesis (COL1A1 and TGFB1). (D) Genes related to matrix degradation (MMP9 and TIMP1). (E) Genes involved in lipid synthesis (SREBF1, SREBF2, FASN, CD36, DGAT1, PLIN5 and PNPLA3). (F) Genes involved in fatty acid β‐oxidation (CPT1A) and oxidative stress (GPX1). (G) Analysis of the mitochondrial membrane potential: image from HepG2 cells incubated with a control fEVs sample and results from all conditions tested. (H) Receptors involved in mitochondrial dysfunction (OPA1 and DNM1L). (I) Gene involved in apoptosis (CASP3). PA/OA: Palmitic acid/oleic acid; M F≤2: MASLD F≤2 patients; M F≥3: MASLD F≥3 patients. Data are expressed as means ± SEM. Groups with different letters statistically differ (p < 0.05).
Fig 3: Effects of fEVs from DILI patients on the mRNA expression of different genes in HepG2 cells (n = 4). (A) Receptors involved in bacterial recognition (TLR4 and TLR5). (B) Genes related to the immune response (IL1B, IL6 and IL10). (C) Secretion of soluble collagen into the culture medium and mRNA expression of genes involved in fibrogenesis (COL1A1 and TGFB1). (D) Genes related to matrix degradation (MMP9 and TIMP1). (E) Genes involved in lipid synthesis (SREBF1, SREBF2, FASN, CD36, DGAT1, PLIN5 and PNPLA3). (F) Genes involved in fatty acid β‐oxidation (CPT1A) and oxidative stress (GPX1). (G) Analysis of the mitochondrial membrane potential: image from HepG2 cells incubated with a DILI fEVs sample and results from all conditions tested. (H) Receptors involved in mitochondrial dysfunction (OPA1 and DNM1L). (I) Gene involved in apoptosis (CASP3). PA/OA: Palmitic acid/oleic acid; DILI: DILI patients. Data are expressed as means ± SEM. Groups with different letters statistically differ (p < 0.05).
Fig 4: Comparison between the effects of fEVs from patients with MASLD and DILI on the mRNA expression of different genes in HepG2 cells (n = 4). (A) Receptors involved in bacterial recognition (TLR4 and TLR5). (B) Genes related to the immune response (IL1B, IL6 and IL10). (C) Secretion of soluble collagen into the culture medium and mRNA expression of genes involved in fibrogenesis (COL1A1 and TGFB1). (D) Genes related to matrix degradation (MMP9 and TIMP1). (E) Genes involved in lipid synthesis (SREBF1, SREBF2, FASN, CD36, DGAT1, PLIN5 and PNPLA3). (F) Genes involved in fatty acid β‐oxidation (CPT1A) and oxidative stress (GPX1). (G) Analysis of the mitochondrial membrane potential. (H) Receptors involved in mitochondrial dysfunction (OPA1 and DNM1L). (I) Gene involved in apoptosis (CASP3). M F≤2: MASLD F≤2 patients; M F≥3: MASLD F≥3 patients; DILI: DILI patients. Data are expressed as means ± SEM. Groups with different letters statistically differ (p < 0.05).
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