Oncostatin M-Enriched Small Extracellular Vesicles Derived from Mesenchymal Stem Cells Prevent Isoproterenol-Induced Fibrosis and Enhance Angiogenesis

Figures

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  Figure 1. Generation of OSM-loaded MSC-T-SEVs through OSM sequence modification and fusion to CD81TM. (a) Structure scheme of native and recombinant proteins with introduced variants in the OSM sequence. The PP domain sequence was removed in mature OSM (matOSM), and a nucleotide mutation was introduced to change an R for a G in CS2 to block excision of matOSM from SEV membranes. The entire OSM sequence was maintained in mutant OSM (mutOSM), but the same R-to-G mutation in CS2 was introduced. Both matOSM and mutOSM sequences are anchored to CD81TM to favor their presence on SEV membranes. (b) General experimental procedure to obtain engineered MSC-T and SEV samples. Step 1: Cell culture preparation for lentiviral transfection. Lentiviruses containing CD81, matOSM-CD81, or mutOSM-CD81 were used to generate engineered MSC-T. Step 2: MSC-T expansion and incubation with EVs-free medium containing doxycycline for 48 h for supernatant collection and protein isolation from cell lysates. Step 3: SEV isolation from cell supernatants by ultracentrifugation and filtration. Fusion of two proteins is represented with a dash (-). Scissors represent where OSM cleavage occurs, while red crosses represent…

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  Figure 2. SEVs derived from genetically modified MSC-T cells carry the recombinant proteins mat/mutOSM-CD81 on their surface but maintain the same physical characteristics in terms of morphology and size distribution. (a) OSM detection in protein samples of cell lysates or SEVs from MSC-T (Ctrl) (lane 1), MSC-T-CD81 (CD81) (lane 2), MSC-T-matureOSM-CD81 (matOSM-CD81) (lane 3), and MSC-T-mutantOSM-CD81 (mutOSM-CD81) (lane 4). OSM was identified at both native molecular weight (22–24 kDa) and its expected molecular weight after fusion to CD81 (45–47 kDa). SEV protein extracts, 10 µg/lane; cell lysate protein extracts, 30 µg/lane. (b) Representative images of SEVs obtained by transmission electron microscopy (scale bar = 100 nm). (c,d) Nanoparticle tracking analysis, showing average particles size (nm) and particles size distribution histograms. (e) ExoView analysis showing percentage of OSM-, CD81-, or CD9-positive SEVs in samples after capture with a CD63 antibody. (f) Both the proportion of SEVs double-positive for CD63 and other proteins (OSM, CD81 and CD9) and triple-positive for these markers was analyzed in all SEVs experimental groups and plotted as pie charts. Each experiment was…

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  Figure 3. Protein levels of the OSM receptors LIF-R/GP130 and IL-31RA are increased after HCF-V stimulation in vitro. (a) Representative Western blots for GP130, IL-31RA, and IL-31RB and β-tubulin (Tub) expression in HCF-V cells were measured under basal conditions (Ctrl), after starvation (Starved) and after starvation and stimulation with a pro-fibrotic cocktail containing L-ascorbic acid 2-phosphate, dextran sulphate, and recombinant TGFβ-1. Samples were collected both after starvation and after 0.5, 1, 2, 4, 24, and 48 h of starvation and fibrosis stimulation. Untreated cells in basal conditions were used as control. (b) GP130 protein expression quantification. (c) IL-31RB protein expression quantification. (d) IL-31RA protein expression quantification. Protein content was measured by densitometry in ImageJ 1.53t, using Tub as a loading control for each condition. Results were obtained from three independent experiments and were analyzed with ANOVA and Tukey’s post hoc test, using values from ctrl cells as a reference and represented as mean ± SEM (* p < 0.05; *** p < 0.001).

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  Figure 4. MutOSM-CD81-enriched SEVs have an enhanced ability to reduce HCF-V proliferation and telo-Collagen1α1 expression and secretion to the extracellular space. (a) Quantification of HCF-V proliferation rate under basal conditions (Ctrl), after starvation (starved), and after treatment with SEVs. The ratio between number of proliferative cells (Ki-67-positive nuclei, in green) and total number of cells (DAPI-positive nuclei, in blue) is shown. (b) Representative images of Ki-67 and DAPI nuclei staining for each experimental condition. (c,d) Quantification of telo-Collagen1α1 protein, which is shown in green. Mean fluorescence intensity (MFI) normalized to number of cells and extracellular telo-Collagen1α1 area are shown. (e) Representative images of telo-Collagen1α1 detection (shown in green) in samples under all experimental conditions. DAPI was used for nuclei staining (shown in blue). Scale bar = 200 µm. Both starved and stimulated cells were treated with equivalent volumes of vehicle (PBS). All results were obtained from three independent experiments and were analyzed with ANOVA and Tukey’s post hoc test. All experimental conditions were compared with both starved cells and starved…

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  Figure 5. MutOSM-CD81 SEVs reduce collagen deposition in an isoproterenol (ISO) in vivo model of myocardial infarction type 2. (a) Schematic of the experimental procedure. Three experimental groups were included: ISO (ctrl), ISO + CD81-SEVs, and ISO + mutOSM-CD81-SEVs. Fibrosis was induced by a daily ISO subcutaneous (SC) injection (dose: 150 mg/kg) during 5 consecutive days. Two intraperitoneal (IP) doses of SEVs (CD81 or mutOSM-CD81) were injected (day 1 and day 7). An equivalent volume of vehicle (PBS) was injected in the ISO-only experimental group. (b) Representative images of Picrosirius red staining. Scale bar: 200 µm. (c) Quantification of fibrotic area in Picrosirius red-stained heart sections. The percentage of collagen-covered area related to total heart area is represented for each animal. (d) Representative images of periostin (POSTN) and α-smooth muscle actin (αSMA) double staining. Scale bar: 50 µm. (e) Quantification of perivascular fibrosis by POSTN staining in pixels and mean fluorescence intensity. Five animals were included in each group. ImageJ was used for image analysis and colorimetric and fluorescence quantification. Data were analyzed with ANOVA and Tukey’s post…

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  Figure 6. MutOSM-CD81 SEVs reduce cardiac hypertrophy and increase angiogenesis in an isoproterenol (ISO) in vivo model of myocardial infarction type 2. (a) Representative images of wheat germ agglutinin (WGA) staining (shown in red) and nuclei staining (DAPI, shown in blue) for each experimental group. Scale bar: 50 µm. (b) Measurement of cardiomyocyte area, using WGA staining in heart sections of animals. (c) Immunodetection of F4/F80 (pan-macrophage marker; red) and CD274 (PD-L1; pro-inflammatory Mφ1 marker; green) or CD206 (pro-regenerative Mφ2 marker; green) in heart samples 21 days after ISO treatment. Scale bar: 50 μm. (d) Quantification of double-positive cells per mm2. Five sections of 0.14 mm2 per mouse were analyzed. (e) Measurement of microvasculature, using an antibody to CD31. Circular stained structures with an area up to 23.5 µm2 were counted. (f) Representative images of CD31 staining (shown in red) and nuclei staining (DAPI, shown in blue) for each experimental group. Scale bar: 50 µm. Five animals were included in each group. ImageJ was used for image analysis and colorimetric and fluorescence quantification. Data were analyzed with ANOVA and Tukey’s post hoc test, using…