Mechanism of exosomes released from bone marrow stem cells regulating proliferation of cardiac microvascular endothelial cells

Abstract

Objective To investigate the mechanism of exosomes released from bone marrow stem cells (BMSCs) on the proliferation in cardiac microvascular endothelial cells (CMVECs) by miR-214 regulation. Methods Rat CMVECs were cultured by enzyme digestion combined with differential adherence. Ultra-high speed centrifugation was used to extract exosomes from normal-oxygen or hypoxic-pretreated BMSCs. Immunofluorescence assay was employed to track the internalization of exosomes in CMVECs. Then the BMSCs were divided into 4 groups: ①control group, ②free exosomes (Free-Exos), ③normal exosomes (Nor-Exos), and ④hypoxia exosomes (Hypoxia-Exos, 400 μg/μL exosome co-cultured with CMVECs for 24 h). The cell proliferation was measured by Edu assay and flow cytometry. The miR-214 levels in exosomes or CMVECs were assessed using qRT-PCR, and the levels were measured again after miR-214 in exosomes were inhibited. And the cell proliferation was observed again by the above methods. Finally, after liposome 2000 was employed to transfect miR-214 mimics into CMVECs, the cell proliferation ability was measured by the Edu assay. Results Transmission electron microscopy and immunoblotting indicated that the extracted vesicles were exosomes. Immunofluorescence assay revealed that DiI-labeled exosomes could be internalized by CMVECs. Edu assay and cell cycle assay showed that Hypoxic-Exos had stronger potential to promote cell proliferation than Nor-Exos in CMVECs. qRT-PCR results showed that miR-214 was highly expressed in Hypoxic-Exos compared with Nor-Exos (P < 0.05). In CMVECs, the expression of miR-214 in Hypoxia-Exos group was significantly higher than that in Control group and Nor-Exos group (P < 0.05). In addition, Hypoxia-Exos's ability to promote cell proliferation was partially blocked by down-regulation of miR-214 in Hypoxia-Exos. However, after overexpression of mir-214 in CMVECs, cell proliferation ability was enhanced. Conclusion Exosomes released from hypoxic-pretreated BMSCs can promote CMVECs proliferation by medicating miR-214.

Keywords

• bone marrow mesenchymal stem cells
• exosomes
• cardiac microvascular endothelial cells
• cell proliferation
• cell differentiation