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. 2020 Jul 2;11(1):266.
doi: 10.1186/s13287-020-01780-x.

Nonadherent culture method promotes MSC-mediated vascularization in myocardial infarction via miR-519d/VEGFA pathway

Baoping Deng  1   2 , Xianlan Zhang  2 , Yi Liang  1 , Haiming Jiang  1 , Weizhao Huang  1 , Yinmeng Wu  1 , Weiping Deng  3
Affiliations

Nonadherent culture method promotes MSC-mediated vascularization in myocardial infarction via miR-519d/VEGFA pathway

Baoping Deng et al. Stem Cell Res Ther. .

Abstract

Background: Mesenchymal stem cells (MSCs) can provide therapeutic benefits for myocardial infarction (MI) recovery; however, the molecular mechanism by which MSCs improve the heart function is unclear.

Methods: Microarray analysis was performed to examine the expression profiling of human MSCs (hMSCs) grown as adherent cultures (AC-hMSCs) or nonadherent cultures on ultra-low-adherent plates (nonAC-hMSCs). Real-time quantitative polymerase chain reaction (RT-qPCR), western blotting, and enzyme-linked immunosorbent assays (ELISA) were used to assess VEGFA expression and secretion in the AC-hMSCs and nonAC-hMSCs. The paracrine effect of VEGFA-overexpressing AC-MSCs (AC-VEGFA-hMSCs) or VEGFA-knockdown nonAC-hMSCs (nonAC-shVEGFA-hMSCs) on the angiogenic ability of human umbilical vein endothelial cells (HUVECs) was evaluated using tube formation assay. AC-VEGFA-hMSCs or nonAC-shVEGFA-hMSCs were transplanted into myocardial infarction rats to investigate the therapeutic effect of AC-VEGFA-hMSCs or nonAC-shVEGFA-hMSCs. Luciferase reporter assay was used to confirm the association of VEGFA with miR-519d.

Results: Microarray analysis revealed that VEGFA is downregulated in AC-hMSCs compared to nonAC-hMSCs. Functional assays revealed that high levels of VEGFA produced from AC-VEGFA-hMSCs increased the tube formation capacity of HUVECs in vitro, improved angiogenesis and cardiac performance, and reduced infarct size in a rat MI model. Low levels of VEGFA secretion from nonAC-shVEGFA-hMSCs had the opposite effects. Mechanistically, we found that miR-519d directly targets VEGFA. High levels of VEGFA secreted from VEGFA-overexpressing nonAC-hMSCs abolished the repressive effect of miR-519d on HUVEC angiogenesis.

Conclusion: Our findings indicate that nonadherent culture-induced secretion of VEGFA plays an important role in MSCs via the miR-519d/VEGFA pathway and may provide a novel therapeutic strategy for MI treatment.

Keywords: Angiogenesis; Mesenchymal stem cells; Myocardial infarction; Nonadherent culture; VEGFA; miR-519d.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Increased VEGFA expression is observed in the nonAC-hMSCs at two different time points. a The 50 most downregulated and 50 most upregulated mRNAs in the AC-hMSCs and nonAC-hMSCs at 24 h and 72 h. b Venn diagram of upregulated and downregulated genes in the AC-hMSCs and nonAC-hMSCs at 24 h and 72 h. c VEGFA mRNA and d protein expression levels in AC-hMSCs and nonAC-hMSCs at 24 h and 72 h were measured by RT-qPCR and western blotting, respectively. e VEGFA secretion from hMSCs as determined by ELISA. Data are represented as mean ± SD (n = 3 per group). ∗P < 0.05
Fig. 2
Fig. 2
Decreased levels of VEGFA produced by VEGFA knockdown nonAC-hMSCs inhibits the angiogenesis of HUVECs. AC-hMSCs were infected with VEGFA or negative control (NC) lentiviruses and nonAC-hMSCs were infected with shVEGFA or the control lentiviruses. a RT-qPCR and b western blotting were performed to detect mRNA and protein levels of VEGFA, respectively. c ELISA analysis was performed to determine the levels of secreted VEGF. d Capillary-like structure formation of HUVECs cultured in CM from hMSCs with different genotypes was evaluated by tube formation assay. Data are represented as mean ± SD (n = 3 per group). ∗P < 0.05 compared to AC-hMSC-Ctrl or nonAC-hMSC-NC group. ▲P < 0.05 compared to AC-hMSC-Ctrl group
Fig. 3
Fig. 3
VEGFA production by VEGFA-overexpressing AC-hMSCs increases angiogenesis and reduces infarction size after MI. a RT-qPCR, b western blotting, and c ELISA were used to assess VEGFA expression and secretion in the heart tissues obtained from LADCA-ligated rats 48 h after transplantation with AC-VEGFA-hMSCs, nonAC-sh-VEGFA-hMSCs, or their corresponding control hMSCs. d Immunohistochemistry analysis for vWF expression in the heart tissues obtained from LADCA-ligated rats 28 days after transplantation with AC-VEGFA-hMSCs, nonAC-sh-VEGFA-h MSCs, or their corresponding control hMSCs. Red arrow indicates the small vessels. e Representative images and quantification of fibrosis area. Data are represented as mean ± SD (n = 8 per group). ∗P < 0.05 compared to AC-hMSC-Ctrl or nonAC-hMSC-NC group. #P < 0.05 compared to MI group. ▲P < 0.05 compared to AC-hMSC-Ctrl group
Fig. 4
Fig. 4
VEGFA secretion by VEGFA-overexpressing AC-hMSCs improves cardiac functions in a rat MI model. a Cardiac function was determined by echocardiography 28 days after MI induction and hMSC transplantation. b Quantification of LVEDD, c LVESD, d LVEF, and e LVFS. Data are represented as mean ± SD (n = 8 per group). ∗P < 0.05 compared to AC-hMSC-Ctrl or nonAC-hMSC-NC group. #P < 0.05 compared to MI group. ▲P < 0.05 compared to AC-hMSC-Ctrl group
Fig. 5
Fig. 5
VEGFA is a direct target of miR-519d. a The predicted binding site between VEGFA and miR-519d. b RT-qPCR analysis of miR-519d expression in AC-hMSCs and nonAC-hMSCs at 24 h and 72 h. c Luciferase activity in HEK293T cells co-transfected with miR-519d or control mimics and VEGFA-3′ UTR-WT or VEGFA-3′ UTR-Mut plasmids. d–f VEGFA expression and secretion in nonAC-hMSCs transfected with miR-519d or control mimics, and AC-hMSCs transfected with miR-519d or control inhibitors. Data are represented as mean ± SD (n = 3 per group). ∗P < 0.05 compared to miR-Ctrl or anti-miR-Ctrl group. P < 0.05 compared to miR-Ctrl group
Fig. 6
Fig. 6
The secretion of VEGFA in VEGFA-overexpressing nonAC-hMSCs attenuates miR-519d-mediated inhibition of tube formation of HUVECs. a The expression and secretion of VEGFA in the nonAC-hMSCs co-transfected with miR-519d mimics and VEGFA plasmid were measured using RT-qPCR, b western blotting, and c ELISA, respectively. d Tube formation assay was performed to detect the capillary-like tubules formation of HUVECs cultured in the presence of hMSC-conditioned medium. Data are represented as mean ± SD (n = 3 per group). ∗P < 0.05 compared to miR-Ctrl group. #P < 0.05 compared to miR-519d group
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