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. 2020 Dec 11;12(23):23609-23618.
doi: 10.18632/aging.103563. Epub 2020 Dec 11.

Inhibition of Exo-miR-19a-3p derived from cardiomyocytes promotes angiogenesis and improves heart function in mice with myocardial infarction via targeting HIF-1α

Lianping Gou  1 , Cheng Xue  1 , Xiaoyan Tang  2 , Zhiyuan Fang  3
Affiliations

Inhibition of Exo-miR-19a-3p derived from cardiomyocytes promotes angiogenesis and improves heart function in mice with myocardial infarction via targeting HIF-1α

Lianping Gou et al. Aging (Albany NY). .

Abstract

Background: Myocardial infarction (MI), a common presentation for cardiovascular disease, is caused by reduction of blood flow and oxygen supply and is one of the main causes of death worldwide. MicroRNAs participate in multiple physiological and pathological processed and play crucial role in myocardial infarction.

Results: qRT-PCR analysis showed that expression level of miR-19a-3p was increased in serum of patient with MI. In vitro study indicated that the miR-19a-3p level was upregulated in response to H2O2 treatment and transferred by exosome, and then, uptake occurred in endothelial cells. Furthermore, western blot and immunostaining showed that treatment of exosome enriched miR-19a-3p suppressed the proliferation of endothelial cells and induced cell death, which was inhibited by AMO-19 transfection. Administration of antagomiR-19a-3p promoted angiogenesis and improved heart function of MI mice. Moreover, miR-19a-3p overexpression downregulated the protein level of HIF-1α and transfection of si-HIF-1α reversed the promotion of endothelial cells proliferation caused by AMO-19 transfection. In addition, antagomiR-19a-3p treatment accelerated angiogenesis and infection of AAV5-shHIF-1α inhibited that effect in MI mice.

Conclusions: In conclusion, our finding indicated that miR-19a-3p inhibited endothelial cells proliferation and angiogenesis via targeting HIF-1α and attenuated heart function of mice after MI, and suggested a new mechanism of cell-to-cell communication between cardiomyocytes and endothelial cells.

Keywords: HIF-1α; angiogenesis; exosome; miR-19a-3p; myocardial infarction.

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Conflict of interest statement

CONFLICTS OF INTEREST: The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
Expression level of miR-19a-3p is upregulated in response to MI or H2O2.. (A–C) qRT-PCR analysis of miR-19a-3p level in patients’ serum, mice serum and culture medium of CM. U6 was used as the control. (D) Electron microscopy image of CM–derived exosomes, showing a size of approximately 30 to 150 nm in diameter. Scale bar: 100 nm. (E) Western blot analysis of the protein level of CD63. (F) Flow cytometry analysis of CD63 of CM-derived exosomes. CM-derived exosomes were immunostained against CD63 (red curve) and compared with the appropriate isotype control (gray curve). (G) qRT-PCR analysis of miR-19a-3p level in exosomes derived from patients’ serum, mice serum and culture medium of CM. U6 was used as the control. **P < 0.01 and ***P < 0.001. All experiments were performed more than 3 biological repeats.
Figure 2
Figure 2
Downregulation of miR-19a-3p promotes endothelial cells survival and proliferation. (A) qRT-PCR analysis of miR-19a-3p level was measured in endothelial cells. U6 was used as the control. (B) TUNEL staining of endothelial cells after treatment of exosomes derived from CM-culture medium. (C) Flow cytometry was performed to detect apoptosis of endothelial cells. (D) Western blot analysis of expression level of CD31. (E) Immunofluorescence staining of ki67 in endothelial cells. (F) EdU staining in endothelial cells to show the effect of miR-19a-3p on proliferation of endothelial cells. (G) Flow cytometry was performed to detect cell cycle of endothelial cells. Exosome was derived from CM-culture medium. AMO-19 was transfected to inhibit the level of miR-19a-3p. **P < 0.01. All experiments were performed more than 3 biological repeats. Scar bar = 50μm.
Figure 3
Figure 3
Inhibition of miR-19a-3p promotes angiogenesis and improves heart function in mice with MI. (A) Representative echocardiographic assessment images of heart function of mice with MI. (B) Ejection fractions (EF%) and fractional shortening (%). (C) Western blot analysis of expression level of CD31 in left ventricle of mice. (D) Immunofluorescence staining of ki67 in peri-infarct area of left ventricle. **P < 0.01. All experiments were performed more than 4 biological repeats. Scar bar = 50μm.
Figure 4
Figure 4
miR-19a-3p reduces endothelial cells proliferation and attenuates heart function by targeting HIF-1α. (A) The predicted binding site of miR-19a-3p on the 3’ UTR of HIF-1α gene. (B) Luciferase reporter activities of chimeric vectors carrying the luciferase gene and a fragment of the 3’ UTR of HIF-1α containing the wild type or mutant miR-19a-3p binding sites. (C) Western blot analysis of expression level of HIF-1α of endothelial cells transfected with miR-19a-3p or AMO-19. (D) Western blot analysis of expression level of HIF-1α of endothelial cells in response to H2O2 treatment with or without transfection of miR-19a-3p or AMO-19. (E) MTT assay of endothelial cells in response to H2O2 treatment with or without transfection of miR-19a-3p or AMO-19. (F) Immunofluorescence staining of ki67 in endothelial cells in response to H2O2 treatment with or without transfection of miR-19a-3p or AMO-19. (G) Western blot analysis of expression level of CD31 and HIF-1α in left ventricle of mice with or without MI. (H) Immunohistochemical staining of CD31 level in mice after MI. (I) TUNEL staining was performed to measure the level of endothelial cell death. (J) Immunofluorescence staining of ki67 in peri-infarct area of left ventricle. *P < 0.05 and **P < 0.01. All experiments were performed more than 4 biological repeats. Scar bar = 50μm.
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