Coronary Serum Exosomes Derived from Patients with Myocardial Ischemia Regulate Angiogenesis through the miR-939-mediated Nitric Oxide Signaling Pathway

Figures

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  Figure 1. Characterization and internalization pathways of coronary serum exosomes. (A) The particle diameter distribution ratio measured by Nanosight analysis of isc-Exo (blue) and con-Exo (red), which indicated that most of the particles were between 30-100 nm. (B, C) The transmission electron microscopy appearance of exosomes. Bars indicate 200 nm and 50 nm, respectively. (D) The CD9, CD63 and Flotillin expression levels in both con-Exo and isc-Exo. (E) Representative horizontal and vertical images of a 3D Z-stack reconstruction showing labeled exosomes (green) were located in HUVEC cytoplasm (red). Bars indicate 10 μm.

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  Figure 2. Effects of isc-Exo and con-Exo on in vitro angiogenesis. (A, B) The isc-Exo group showed greater proliferative effects than con-Exo at both 200 μg/mL and 400 μg/mL doses in EdU-positive cells measured by CCK8 absorbance (C) and manual cell count (D). (E) The isc-Exo group showed better vascular formation ability than con-Exo in tube formation analysis measured by number of nodes (F) and tube length (G). isc-Exo promoted endothelial cell migration, as evaluated by scratch wound assay (H) and analyzed by the ratio of non-migrated area divided by the baseline wound area (I). (J) The Transwell assay also revealed the same trend as the wound assay and was analyzed by the number of migrated cells (K). Bars indicate 200 μm in all pictures. * indicates P < 0.05.

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  Figure 3. Healing effects of isc-Exo and con-Exo on mouse ischemic injury after hind-limb ischemia. (A) Blood flow recovery evaluated by Laser-Doppler perfusion imaging 0, 7, 14, 21 days after surgery. The left side was the ischemia limb and the right side was the normal control limb. Bars indicate 1 cm. (B) The healing effect of blood perfusion recovery was analyzed by the perfusion ratio of the ischemia side divided by that of the normal control side. The isc-Exo group showed better blood perfusion recovery effects compared to the con-Exo group 21 days after surgery. (C) The ischemic limb skin appearance 21 days after surgery. The PBS group showed scarring on the left ischemia limb and the con-Exo group showed swelling in the whole ischemia limb. Meanwhile, the isc-Exo group showed a better recovery. Muscle sections stained by hematoxylin and eosin (D), CD31 immunohistochemistry (E) and CD31 and α-SMA immunofluorescence (F). The arrows indicate the microvessels in the muscle and the bars indicate 100 μm in the pictures. red: α-SMA, green: CD31, blue: DAPI. (G) The capillary density in the ischemia muscles was analyzed by the number of microvessels per cm2. The isc-Exo group showed higher…

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  Figure 4. Exosome biogenesis related proteins under hypoxia stress.The expression of exosome biogenesis-related proteins Alix, Tsg101 and Rab11a in ischemic myocardium (A) primary cardiomyocytes (B) primary endothelial cells (C) and fibroblasts (D). Alix showed a higher expression in the ischemic myocardium and hypoxic cardiomyocytes. Rab11a was also higher in hypoxic cardiomyocytes. However, the hypoxic endothelial cells and fibroblasts did not show an increase in any of Alix, Tsg101 or Rab11a. * indicates P < 0.05 and NS indicates P > 0.05. The protein levels are shown as the experiment group divided by the control group.

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  Figure 5. The microarray analysis of isc-Exo and con-Exo. (A) volcano plot and heat map (B) of isc-Exo and con-Exo (the red dots indicate up-regulated miRNAs and the green dots indicate the down-regulated miRNAs). For the regulated miRNAs, the GO analysis of biological processes involved in endothelial (C) and cardiomyocytes (D). The columns indicate the related GO terms depending on the enrichment factor and the numbers in the parentheses indicate the number of genes. (E) qPCR verification of the regulated miRNAs and the results are shown as fold changes. (F) Overexpression analysis of selected miRNAs (miR-939-5p, miR-1246, miR-4787-3p) for their proliferative effect on endothelial cells. miR-939-5p and miR-4787-3p repressed endothelial cell proliferation. * indicates P < 0.05 and NS indicates P > 0.05).

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  Figure 6. The effect of miR-939-5p on angiogenesis and down-stream targets. The effect of the miR-939-5p mimic or inhibitor on endothelial cell proliferation (A) and tube formation (B-D). The bar indicated 200 μm. miR-939-5p mimic strikingly repressed endothelial cell proliferation and tube formation and miR-939-5p inhibitor reserved the trend. (E) Plasmid vectors of human NOS2 (iNOS) 3′-UTR or its mutation were transfected into HEK293 cells followed by miR-939-5p mimic or control transfection. Dual luciferase activity assay showed that miR-939-5p mimic targeted iNOS 3'-UTR but not its mutation. The effect of miR-939-5p mimic or inhibitor on iNOS mRNA (F) and protein expression (G, H). miR-939-5p mimic significantly repressed iNOS mRNA and protein, while miR-939-5p inhibitor removed the suppression effects. The miR-939-5p mimic impaired iNOS activity (I) and the NO levels (J), while miR-939-5p inhibitor reserved the trend. * indicates P < 0.05 and NS indicates P > 0.05. The mRNA and protein expressions are shown as fold change vs. NC group.

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  Figure 7. miR-939 and iNOS levels after isc-Exo and con-Exo treatment. (A) After treatment by isc-Exo and con-Exo, the miR-939 expression of con-Exo-treated cells was 3-fold higher than that of isc-Exo-treated cells. (B) Meanwhile, the iNOS mRNA level was 5-fold higher in the isc-Exo group. (C, D) The iNOS protein level in the isc-Exo-treated cells was also higher than that of con-Exo-treated cells. * indicates P < 0.05.