Abstract
BackgroundMesenchymal 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.MethodsMicroarray 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.ResultsMicroarray 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.ConclusionOur 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.
Highlights
Myocardial infarction (MI) is a major cause of mortality and disability in the world [1]
Increased Vascular endothelial growth factor A (VEGFA) expression and production are observed in nonAC-human MSCs (hMSCs) We first used flow cytometry to identify Mesenchymal stem cells (MSCs) and found that hMSCs were positive for CD73, CD90, and CD105, whereas negative for CD11b, CD14, CD34, and CD45 (Additional file 1: Figure S1)
The expression of VEGFA in the nonAC-hMSCs was significantly higher than that in the AC-hMSCs at the two time points (Table 1). This increase in VEGFA mRNA in the nonAChMSCs was verified by Real-time quantitative polymerase chain reaction (RT-qPCR)
Summary
Myocardial infarction (MI) is a major cause of mortality and disability in the world [1]. MSCs have been commonly used in experimental research and clinical trials for treating MI Often for this application, a large amount of MSCs must be isolated from the same tissues and expanded in plastic adherent culturing containers [7, 8]. Compared to primary MSCs, cultured MSCs with high levels of CD44 displayed decreased targeting to the bone marrow [13]. Our previous research revealed that stem cell antigen 1 (Sca-1) is expressed at higher levels in adherent cultured mouse MSCs (AC-mMSCs) compared to mMSCs in nonadherent cultures maintained in ultra-low-adherent plates (nonAC-mMSCs) [14]. Mesenchymal stem cells (MSCs) can provide therapeutic benefits for myocardial infarction (MI) recovery; the molecular mechanism by which MSCs improve the heart function is unclear
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