Bioinspired therapeutic platform based on extracellular vesicles for prevention of arterial wall remodeling in hypertension

Figures

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  Fig. 1. Transfer of EC derived EVs to the artery. (A) Transmission electron microscopy of mouse aorta. Robust vesicles secretion could be seen in the basal side of the endothelium in the artery. Scale bars represents 1 μm or 200 nm. (B) Schematic illustration of the procedure how the HUVEC derived EVs were isolated. (C) Representative image of transmission electron microscopy of the EV released from HUVEC. Scale bars represents 100 nm. (D) Size distribution of EVs as analyzed by NTA. (E) Western blot analysis of EV inclusive and exclusive marker expression, including GM130, TSG101 and CD9. Representative data of at least three independent experiments. (F) Representative images of DiR-labeled EVs distributed in different tissues. About 150 μg (at protein level) in 100 μL of EV from EC labeled with DiR was injected via tail vein. About 4 h after the intravenous injection, in vivo and ex vivo fluorescence imaging were performed. Accumulation of EVs could be seen in the aorta, especially the branched region. n = 3 mice. (G) Quantification data of the relative fluorescence intensity of distributed EVs in different tissues. n = 3 mice.

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  Fig. 2. Uptake of the endothelial cell-derived EVs by the SMC. (A) Schematic illustration of the setup of the cell model subjected to compressive forces and the procedure how the derived EVs were harvested. (B) Representative electron microscopy of the EV released from control or weight loaded HUVECs. Scale bars represents 100 nm. (C) Size distributions of EVctrl and EV2g as analyzed by NTA. (D) Western blot analysis of EV inclusive and exclusive marker expression, including GM130, TSG101 and CD9. Representative data of at least three independent experiments. (E) Schematic representation of the animal experiment procedure. EVs were labeled with DiI (Red) followed by tail vein injection. (F) EV distribution in the artery. SMCs were stained with anti-α-SMA (Green) and the nuclei were counterstained with Hoechst (Blue). Images were taken under confocal microscope. Arrow indicates the SMC distribution while arrowhead indicates the possible endothelium localization of the EVs. (G) Quantification data of the relative fluorescence intensity of DiI labeled EV distribution in the artery. Data are presented as mean ± SEM. n = 3 mice. *P < 0.05 as determined by t-test.

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  Fig. 3. The effects of EVctrl and EV2g on arterial remodeling. (A) Schematic representation of the experimental procedure. (B) Representative images showing how PWV was acquired. The pulse wave velocity spectra were acquired at the ascending aorta and abdominal aorta, with the simultaneous ECG. The time lagging of peak velocity in the two positions was used for PWV calculation. (C) The PWV of the EVctrl and EV2g treated mice. Data are means ± SEM. n = 5, **P < 0.01 by t-test. (D) Vascular change as revealed by Hematoxylin/eosin staining. (E-G) Wall thickness (E), internal diameter (F) and wall/lumen ratio (G) of the arteries of mice receiving EVctrl and EV2g treatment for 4 weeks. n = 5. n.s., no significance, **P < 0.01, ***P < 0.001 as determined by t-test. (H) Masson's trichrome staining of the aorta in mice with indicated treatment. Representative images of at least 3 mice of each group. Scale bars represent 50 μm. (I–K) Expression of Col3a1 (I), Col1a1 (J) and Spp1 (K) in the arteries of EVctrl and EV2g treated mice. Expression of mRNA candidates were normalized to Gapdh expression. Data are expressed as mean ± SEM of at least 3 biological replicates. *P < 0.05, **P < 0.01 by t-test.

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  Fig. 4. Profiling of the miRNA abundance in the EVs from hypertension patients. (A) Schematic illustration of the procedure of blood harvesting and EV-miRNA sequencing in normal volunteers and hypertension patients. (B) Representative electron microscopy of the EVNBP and EVHBP. Scale bars represents 100 nm. (C) Size distributions of EVs from blood samples were compared by NTA. (D) Heat map analysis based on the 19 most significant miRNAs of EVs from control group or hypertension group. (E-F) Pearson correlations were performed to investigate the correlation between mean blood pressure and the expression levels of EV-miR-320d (E) or EV-miR-423-5p (F). Expression of miRNA candidates were normalized to U6 expression. Mean blood pressure represents 1/3SBP +2/3DBP.

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  Fig. 5. EVmiR-320d-mimics and EVmiR-423-5p-mimics treatments recapitulate the arterial remodeling of hypertension. (A) Schematic representation of the experimental procedure. (B) The PWV of mice receiving the EVNC-mimics, EVmiR-320d-mimics and EVmiR-423-5p-mimics. Data are means ± SEM. n = 5, **P < 0.01 by one-way ANOVA. (C) Vascular change as revealed by Hematoxylin/eosin staining. (D-F) Wall thickness (D), internal diameter (E) and wall/lumen ratio (F) of the arteries of mice receiving EVNC-mimics, EVmiR-320d-mimics and EVmiR-423-5p-mimics treatment for 4 weeks. n = 5. n.s., no significance, *P < 0.05, **P < 0.01, ***P < 0.001 as determined by one-way ANOVA. (G) Masson's trichrome staining of the aorta in mice with indicated treatments. Representative images of at least 3 mice of each group. Scale bars represent 50 μm. (H-J) Expression of Col3a1 (H), Col1a1 (I) and Spp1 (J) in the arteries of mice receiving EVNC-mimics, EVmiR-320d-mimics and EVmiR-423-5p-mimics treatment. Expression of mRNA candidates were normalized to Gapdh expression. Data are expressed as mean ± SEM of at least 3 biological replicates. *P < 0.05, **P < 0.01 by one-way ANOVA.

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  Fig. 6. EVmiR-320d-inhibitor and EVmiR-423-5p-inhibitor treatments alleviate the arterial remodeling in SHR rats. (A) Schematic illustration of the experimental procedure. EVs encapsulating miRNA inhibitors were injected into SHR rats once a week for 4 weeks. (B) The PWV of SHR rats receiving EVNC-inhibitor, EVmiR-320d-inhibitor and EVmiR-423-5p-inhibitor. Data were expressed as mean ± SEM. n = 5, *P < 0.05 by one-way ANOVA. (C) Vascular change as revealed by Hematoxylin/eosin staining. (D-F) Wall thickness (D), internal diameter (E) and wall/lumen ratio (F) of the arteries of rats receiving EVNC-inhibitor, EVmiR-320d-inhibitor and EVmiR-423-5p-inhibitor for 4 weeks. n = 5. n.s., no significance, *P < 0.05, **P < 0.01 as determined by one-way ANOVA. (G) Masson's trichrome staining of the aorta in rats with indicated treatments. Scale bars represent 50 μm. (H-J) Expression of Col3a1 (H), Col1a1 (I) and Spp1 (J) in the arteries of SHR rats receiving EVNC-inhibitor, EVmiR-320d-inhibitor and EVmiR-423-5p-inhibitor injection. Expression of mRNA candidates were normalized to Gapdh expression. Data were expressed as mean ± SEM of at least 3 biological replicates. *P < 0.05, **P < 0.01 by…

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  Fig. 7. Mechanical force induces EV enrichment of miR-320d and miR-423-5p in an ROS dependent manner. (A) Confocal microscope images of the DHE staining of the ROS (red) in HUVECs with different treatments. Nuclei were counterstained with Hoechst. Scale bars represent 30 μm. (B) Quantification of the DHE fluorescence intensity in Figure A. Data were presented as means ± SEM. ***P < 0.001 as determined by one-way ANOVA. (C-D) qPCR analysis of the abundance of miRNAs in cells with control or 2 g weight loading or 2 g weight loading plus NAC treatment (C) or the derived EVs (D). Data were expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 as determined by one-way ANOVA.