Anti-inflammatory, Anti-fibrotic and Pro-cardiomyogenic Effects of Genetically Engineered Extracellular Vesicles Enriched in miR-1 and miR-199a on Human Cardiac Fibroblasts

Fig. 1. Generation of transgenic stem cell lines overexpressing miR-1 and miR-199a. (A) Scheme of cassette harbouring miR-1 or miR-199a coding sequence in lentiviral vector used for transduction. LTR, long terminal repeat; CMV, cytomegalovirus promoter; EF1α, elongation factor 1 alpha gene promoter; copGFP, variant of a green fluorescent protein derived from copepepod Pontellina plumata. (B) Representative microscopic images of hiPS and UC-MSC genetically modified cells overexpressing miR-1 or miR-199a (co-expressing copGFP in green). Scale bar = 100 μm. Relative miRNA expression levels of miR-1 or miR-199a in genetically engineered hiPS cells (C) and UC-MSCs (D). Expression levels of miRNAs co-induced after miR-1 or miR-199a overexpression in hiPS cells (E) and UC-MSCs (F). The data on panels C-F were normalized compared with the respective control (WT) cellular samples. Student’s t-test; comparison with copGFP-control cell lines; *p < 0.05, **p < 0.01

Fig. 2. Characterization of EVs from hiPS cells and UC-MSCs stably expressing miR-1 or miR-199a. (A) Particle size distribution in EV samples derived from control and genetically modified stem cells, measured by nanoparticle tracking analysis (NTA). Representative histograms are shown. (B) Western blot analysis of selected proteins in hiPS-miRs-EVs and UC-MSC-miRs-EVs. (C) Representative images of EVs obtained by transmission electron microscopy. Scale bars = 150 nm. (D) Relative levels of miR-1 and miR-199a in hiPS-miR-1-EVs and hiPS-miR-199a-EVs, respectively. (E) Relative levels of miR-1 and miR-199a in UC-MSC-miR-1-EVs and UC-MSC-miR-199a-EVs, respectively. (F) Other miRNAs found at elevated levels in hiPS-miR-1-EVs or hiPS-miR-199a-EVs, when compared with hiPS-WT control. (G) Other miRNAs found at elevated levels in UC-MSC-miR-1-EVs or UC-MSC-miR-199a-EVs, when compared with UC-MSC-WT control. The data on panels D-G were normalized compared with the respective control (WT) EV samples. Student’s t-test; comparison with copGFP-control EVs; *p < 0.05, **p < 0.01

Fig. 3. Treatment of cardiac fibroblasts with EVs derived from genetically modified stem cells. Expression levels of miR-1 and miR-199a in CFs after incubation with hiPS-miR-1-EVs and miR-199a-EVs, respectively (A); or UC-MSC-miR-1-EVs and miR-199a-EVs, respectively (B). Expression levels of pluripotency-associated miRNAs in CFs after incubation with EVs derived from genetically modified hiPS cells (C) and UC-MSCs (D). Expression levels of cardiomyogenic genes in CFs treated with hiPS-miRs-EVs (E) or UC-MSC-miRs-EVs (F) and subjected to induction of cardiac differentiation for 7 days. The data were normalized compared with control samples from CFs without EV treatment. ANOVA multiple comparisons and Tukey test; *p < 0.05, **p < 0.01

Fig. 4. Proliferation and metabolic activity of cardiac fibroblasts after EVs treatment. Analysis of proliferation capacity (A) and metabolic activity (B) of CFs following EV treatment for 24 h. The analyses were performed at 24, 48 and 96 h after incubation with EVs on CFs cultured in normoxic (upper panels) or hypoxic (1% O2; lower panels) conditions. The data were normalized compared with control CFs without EV treatment; red line. Student’s t-test; *p < 0.05

Fig. 5. Impact of hiPS-EVs on survival and activation of cardiomyocytic gene expression in CFs in vitro. (A) Flow cytometric analysis of CF viability and survival after incubation with iPS-EVs following (left graph) or followed by (right graph) treatment with cytotoxic agent (staurosporine) inducing cell apoptosis. The data were normalized compared with the control CFs without EV treatment; red line. Representative dot-plot from flow cytometric analysis is shown to demonstrate the gating strategy. Real time qPCR analysis of expression of selected genes indicative of cardiomyogenic differentiation and cardiac ion channels in CFs after treatment with EVs followed by cardiac differentiation (B) or without induction of differentiation (C) at 7 day of culture. The data were normalized compared with control CFs without EV treatment. (D) Quantitative (left panel) and qualitative (right panel) analysis of GATA4 protein expression in CFs after iPS-EV treatment. Representative fluorescence tile scan images are shown for CFs treated with hiPS-WT-EVs and hiPS-miR-199a-EVs. The composite of nine (3 × 3) title scans for every image are shown. Scale bar = 100μm. Student’s t-test; *p < 0.05

Fig. 6. Activation of molecular pathways in CFs after treatment with hiPS-miR-1-EVs. (A) Protein array analysis of selected apoptosis- related proteins after incubation with EVs and cytotoxic agent (staurosporin; S), in the presence/absence of following miRNA inhibitors (I): specific miR-1-3p inhibitor (1-3p-i) or control inhibitor (C-i). (B) Levels of activated (phosphorylated) kinases in CFs treated with EVs and effects of respective inhibitors on protein levels. (C) Real-time qPCR analysis of expression of selected proinflammatory genes after treatment with EVs and with/ without respective inhibitors. The data were normalized compared with control untreated CFs. (D) Protein array analysis of fibrosis related proteins in CFs treated with EVs and with/ without respective inhibitor. ANOVA multiple comparisons and Tukey test; *p < 0.05, **p < 0.01

Fig. 7. Activation of molecular pathways in CFs after treatment with hiPS-miR-199a-EVs. (A) RT-qPCR analysis of cardiomyogenic and cardiac ion channels-related genes in CFs treated with EVs and with/ without inhibitors (I): specific for miR-199a-3p (1991-3p-i), specific for miR-199a-5p (199a-5p-i) or control inhibitor (C-i). (B) Protein array analysis of activated kinases in CFs treated with EVs and with respective inhibitors. (C) Results of target scan analysis in CFs searching for candidate genes impacted by miR-199a-3p or miR-199a-5p, using RT-qPCR method. The data were normalized compared with control untreated CFs. ANOVA multiple comparisons and Tukey test; *p < 0.05, **p < 0.01