Myofibroblast-Derived Exosome Induce Cardiac Endothelial Cell Dysfunction

Figures

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  Figure 1. TGFβ treatment activates cardiac fibroblasts. TGFβ treatment significantly upregulated pro-fibrotic genes expression, including (A) collagen1α (Col1α), (B) periostin (Postn), and (C) fibronectin (Fn1) in NRVF cells. In addition, an increased phosphorylation of p38 and SMAD2 (D–F) was observed in TGFβ-activated NRVFs. GAPDH was used as loading control for both mRNA and protein expression analysis. (G) Immunostaining of BSA and TGFβ-treated fibroblasts with αSMA (red)/fibronectin (green)/DAPI (blue). Fibrotic markers were significantly upregulated upon TGFβ treatment. Images (G) were taken at 400X and scale bar is 100 μm. P < 0.05 was considered as statistical significance.

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  Figure 2. Characterization of exosomes from fibroblast-conditioned media after TGFβ treatments. (A) Size and number of exosomes were determined by nanoparticle tracking analysis. (B) Representative image by transmission electron microscopy (magnification 30,000X) revealed the presence of double-membrane bound, cup-shaped exosomes.

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  Figure 3. Exosomes derived from TGFβ-activated fibroblast reduced endothelial cell function and viability. (A,B) Tube formation assay for endothelial cell angiogenesis function; representative images are shown. For data analysis the length of branch points was measured using NIH ImageJ software, and the mean length is represented in the graph. Activated FB-derived exosome treatment significantly impaired tubulogenic potential of heart ECs. (C,D) Images of ECs with Giemsa stain for evaluating cell migration in a Boyden chamber. As anticipated, activated FB-derived exosomes significantly reduced EC migration as compared to control. (E) MTT assay to measure endothelial cell viability. Endothelial cell viability (%) was significantly reduced after treatment with activated FB-derived exosomes. P < 0.05 was considered as statistical significance.

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  Figure 4. Myofibroblasts-derived exosomes induced endothelial cell death (A,B). Primary cardiac endothelial cells were isolated from the C57BL/6 mice and were treated with exosomes derived from TGFβ-treated fibroblasts for 48 h. TUNEL staining was performed in 4% PFA fixed cells as per manufacturer's instruction. ECs were counterstained with CD31/PECAM (green; EC surface marker) antibody. Images (A) were taken at 400X and scale bar is 100 μm. P < 0.05 vs. control exosome and PBS treatment.

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  Figure 5. Myofibroblast-derived exosomes impaired EC function-related genes and proteins expression. The mRNA expression of (A) VEGF-A, (B) CD31, and (C) angiopoietin1 were downregulated in the primary endothelial cells after treatment with TGFβ-activated exosomes, as analyzed by real time qPCR. (D–G) Protein levels of HIF1α, eNOS, and Bcl2 were measured. Expression of eNOS and Hif1α were significantly altered by TGFβ-FB-exosomes as compared to control exosomes, but in opposite manner. Bcl2 expression was also decreased in TGFβ-FB-exosomes treated endothelial cells. P < 0.05 was considered as statistical significance.

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  Figure 6. Fibrosis miRNA array along with expression profile and validation of selected miRNAs in exosomes. (A) Fibrosis-miRNA array was performed in neonatal rat fibroblast-derived exosomes after TGFβ activation. Heat map of fibrotic miRNA array data suggest an altered expression of miRNAs in TGFβ-activated FB-derived exosomes as compared to control exosome. The change in expression of selected miRNAs was further confirmed by qPCR by comparing the expression of miRNAs (B) miR-132-3p, (C) miR-200a-3p, and (D) miR-125b-5p, in activated-FB-derived exosomes. P < 0.05 vs. control exosome and PBS treatment.

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  Figure 7. Altered miRNA (miRNA-200a-3p) packaging was seen in myofibroblast-derived exosomes. (A–C) Altered miRNA expression was found in ECs after treatments. Only miR-200a-3p expression showed a significant difference when treated with TGFβ-FB-exosomes. (D,E) Analysis of miR-200a-3p expression in exosomes (D) or EC (E) following miR200a-3p inhibition in parent cells. (E) miR-200a-3p expression was significantly reduced in endothelial cells treated with modified exosomes in comparison to Exo-TGFβ treatment alone. P < 0.05 vs. control exosome and PBS treatment. “ns”, not statistically significant.

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  Figure 8. miR-200a-3p packaged in fibroblast-derived exosomes altered expression of endothelial functional genes via PIGF growth factor inhibition. (A) ELISA-based angiogenesis proteomic profiling revealed a greatly reduced expression of PIGF. (B) PIGF expression was increased in ECs upon treatment with exosomes with inhibited miR-200a-3p. qPCR data showing an improved endothelial function, as assessed by (C) VEGF-A, (D) angiopoietin1 and (E) CD31 genes expression, upon treatment with miR-200a-3p inhibited exosomes. (F–K) Western blot analysis was performed in the cell lysate isolated from ECs treated with modified exosomes (inhibited miR-200a-3p). Cleaved caspase 3 expression (F,K) showed that endothelial cell death was also observed in FB- TGFβ-Exo treated cells which was significantly recovered after miR-200a-3p inhibition. P < 0.05 vs. control exosome and PBS treatment. “ns”, not statistically significant.

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  Figure 9. Schematic illustration of the proposed role of miR-200a-3p/ETS1/VEGF-A signaling cascade in endothelial dysfunction. Activated fibroblast-derived exosomes enriched with miR-200a-3p induce endothelial dysfunction by altering the VEGF-A/PIGF signaling. Alteration in miR-200a-3p expression using antimir therapy improved endothelial cell function. ETS1, E26 transformation-specific sequence factor-1; VEGF-A, vascular endothelial growth factor-A; PIGF, placental growth factor.