Angiotensin II-Treated Cardiac Myocytes Regulate M1 Macrophage Polarization via Transferring Exosomal PVT1

Figures

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  Figure 1. Identification of HCM-derived exosomes. (a) PBS or Ang-II-treated HCM-derived vesicles were collected. Then, the expression of specific markers of exosomes like Alix, HSP70, and CD63 was measured by western blot. (b) TEM was used to ensure the morphology of vesicles derived from Ang-II-treated HCMs. (c) Flow cytometry was performed to detect the percentage of CD63-positive vesicle derived from Ang-II-treated HCMs. (d) The average diameter of the Ang-II-treated HCM-derived vesicles was measured by NTA. n = 3.

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  Figure 2. Ang-II-stimulated HCM-derived exosomes promoted M1 macrophage polarization. Exosomes were collected from the culture medium of HCM treated with PBS or Ang-II. Then, the exosomes were incubated with THP-1 cells. (a) qRT-PCR was carried out to detect the expression of iNOS and TNF-α mRNAs in THP-1 cells. (b) The expression levels of Arg-1 and IL-10 mRNAs were also measured by qRT-PCR. (c) The percentages of CD86-positive and CD163-positive macrophage were analyzed using flow cytometry. (d) The expression of PVT1 in PBS-Exo and Ang-II-Exo was measured by qRT-PCR. (e) The expression of PVT1 in macrophage was measured by qRT-PCR. PBS-Exo: PBS-treated HCM-derived exosomes; Ang-II-Exo: Ang-II-stimulated HCM-derived exosomes. n = 3. ∗P < 0.05 compared to the PBS-Exo group and ∗∗P < 0.01 compared to the PBS-Exo group. Student's t-test was performed to analyze the difference between two independent groups, and the value of P lower than 0.05 was considered as statistically significant difference.

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  Figure 3. Ang-II-treated HCM promoted M1 macrophage polarization through secreting exosomal PVT1. Lentivirus expressing shPVT1 or shCtrl was infected with Ang-II-stimulated HCM for 24 hours. (a) Exosomes were collected from the culture medium of HCM, and then, the expression of PVT1 in exosomes was measured by qRT-PCR. (b) These two types of exosomes were incubated with THP-1 cells. The expression of PVT1 in THP-1 cells was measured using qRT-PCR. (c) The gene expression of iNOS and TNF-α was detected using qRT-PCR. (d) qRT-PCR was carried out to examine the expression of Arg-1 and IL-10 mRNAs. (e) Flow cytometry was performed to ensure the percentage of M1 and M2 macrophage. Here, shCtrl-Exo was lentivirus-shCtrl-infected HCM-derived exosomes and shPVT1-Exo was lentivirus-shPVT1-infected HCM-derived exosomes. n = 3. ∗P < 0.05 and ∗∗P < 0.01. Student's t-test was performed to analyze the difference between two independent groups, and the value of P lower than 0.05 was considered as statistically significant difference.

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  Figure 4. PVT1 promoted IL-16 expression through acting as a miR-145-5p sponge. (a) Two different binding sequences between PVT1 and miR-145-5p were predicted using starBase. (b) The relationship between PVT1 and miR-145-5p was identified using luciferase reporter assay. n = 3. ∗P < 0.05. (c) qRT-PCR analysis of PVT1 and miR-145-5p enrichment by Ago2 and IgG antibody in RIP experiments. n = 3. ∗P < 0.05. (d) The binding sites between miR-145-5p and IL-16 3′-UTR region were predicted using starBase. (e) Luciferase activity of THP-1 cotransfected with the reporter plasmid inserted WT or MUT IL-16 3′-UTR sequences and miR-145-5p mimics, mimics NC, miR-145-5p inhibitor, or inhibitor NC. n = 3. ∗P < 0.05. (f) The expression of IL-16 gene was measured by qRT-PCR. n = 3. ∗P < 0.05 compared with the Ctrl group and #P < 0.05 compared with the PVT1 group. (g) The expression of IL-16 protein was determined using western blot. n = 3. ∗P < 0.05 compared with the Ctrl group and #P < 0.05 compared with the PVT1 group. (h) qRT-PCR was performed to detect the expression of IL-16 gene. n = 3. ∗P < 0.05 compared with the PBS group, and #P < 0.05 compared with the NC group. (i) Western blot was carried out to…

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  Figure 5. Ang-II-stimulated HCM-derived exosomes promoted M1 macrophage polarization via targeting the miR-145-5p/IL-16 signaling pathway. The expression of (a) iNOS mRNA, (b) TNF-α mRNA, (c) Arg-1 mRNA, and (d) IL-10 mRNA was measured by qRT-PCR. (e–g) The percentage of CD86-positive and CD163-positive THP-1 was detected using flow cytometry and analyzed. n = 3. ∗P < 0.05 and ∗∗P < 0.01 compared to the cells treated with nothing; #P < 0.05 and ##P < 0.05 contrasted with the cells treated with Ang-II. One-way ANOVA followed by Tukey's post hoc test was carried out to ensure the difference among multiple groups. For all assays, the value of P lower than 0.05 was considered as statistically significant difference.

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  Figure 6. Ang-II-treated HCM-derived exosomes facilitated atrial fibrosis via targeting macrophage polarization. (a) The expression of MMP-9, TIMP-1, collagen I, and collagen III was measured by western blot. (b) Analysis of MMP-9 and TIMP-1 expression. (c) Analysis of collagen I and collagen III expression. n = 3. ∗P < 0.05 contrasted with the PBS-Exo group and #P < 0.05 compared with the Ang-II-shCtrl-Exo group. One-way ANOVA followed by Tukey's post hoc test was carried out to ensure the difference among multiple groups. For all assays, the value of P lower than 0.05 was considered as statistically significant difference.