miRNA-126-3p carried by human umbilical cord mesenchymal stem cell enhances endothelial function through exosome-mediated mechanisms in vitro and attenuates vein graft neointimal formation in vivo

Abstract

Background: The aim of this study was to determine whether the combination of MSC implantation with miRNA-126-3p overexpression would further improve the surgical results after vein grafting.

Methods: human umbilical cord MSCs (hucMSCs) and human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cords and characterized by a series of experiments. Lentivirus vector encoding miRNA-126-3p was transfected into hucMSCs and verified by PCR. We analyzed the miRNA-126-3p-hucMSC function in vascular endothelial cells by using a series of co-culture experiments. miRNA-126-3p-hucMSCs-exosomes were separated from cell culture supernatants and identified by WB and TEM. We validated the role of miRNA-126-3p-hucMSCs-exosomes on HUVECs proliferative and migratory and angiogenic activities by using a series of function experiments. We further performed co-culture experiments to detect downstream target genes and signaling pathways of miRNA-126-3p-hucMSCs in HUVECs. We established a rat vein grafting model, CM-Dil-labeled hucMSCs were injected intravenously into rats, and the transplanted cells homing to the vein grafts were detected by fluorescent microscopy. We performed historical and immunohistochemical experiments to exam miRNA-126-3p-hucMSC transplantation on vein graft neointimal formation and reendothelialization in vitro.

Results: We successfully isolated and identified primary hucMSCs and HUVECs. Primary hucMSCs were transfected with lentiviral vectors carrying miRNA-126-3p at a MOI 75. Co-culture studies indicated that overexpression of miRNA-126-3p in hucMSCs enhanced HUVECs proliferation, migration, and tube formation in vivo. We successfully separated hucMSCs-exosomes and found that miRNA-126-3p-hucMSCs-exosomes can strengthen the proliferative, migratory, and tube formation capacities of HUVECs. Further PCR and WB analysis indicated that, SPRED-1/PIK3R2/AKT/ERK1/2 pathways are involved in this process. In the rat vein arterialization model, reendothelialization analysis showed that transplantation with hucMSCs modified with miRNA-126-3p had a higher reendothelialization of the vein grafts. The subsequent historical and immunohistochemical examination revealed that delivery with miRNA-126-3p overexpressed hucMSCs significantly reduced vein graft intimal hyperplasia in rats.

Conclusion: These results suggest hucMSC-based miRNA-126-3p gene therapy may be a novel option for the treatment of vein graft disease after CABG.

Keywords: Exosomes; Mesenchymal stem cell; Neointimal hyperplasia; Reendothelialization; Vein graft; miRNA-126-3p.

Fig. 1

Characterization of hucMSCs and HUVECs. a hucMSCs formed cell colony 3 days following isolation. b hucMSCs exhibited a fibroblast-like shape at passage 3. c hucMSCs were stained with Oil Red O to assess their adipogenic ability. d hucMSCs were stained with Alizarin Red to assess their osteogenic ability. e Flow cytometry analysis suggested that cultivated cells expressed strongly expressed CD29, CD44, CD90, CD105, and CD166 and HLA-DR but did not express CD34 and CD45. f Immunostaining of hucMSCs showing CD31 (red) and vWF (green) g staining. h Cell nuclei were stained with DAPI. i The merged images

Fig. 2

miRNA-126-3p transduction by lentiviral vectors. a Primary hucMSCs were transfected with lentiviral vectors carrying miRNA-126-3p-GFP. b CM-DiI-labeled MSCs showed red fluorescence under fluorescence microscope. c GFP image of hucMSCs at 72 h after transduction by lentiviral vectors. d The merged images. e The transfection efficiency of miRNA-126-3p was evaluated by RT-PCR. f Secretion of miRNA-126-3p in the cell culture supernatants of hucMSCs and GFP-hucMSCs and miRNA-126-3p-hucMSCs

Fig. 3

Overexpression of miRNA-126-3p in hucMSCs regulates HUVECs proliferation, migration, and tube formation. a The images of the EDU incorporation assay of HUVECs co-cultured with hucMSCs. b The images of cell scratching assay of HUVECs co-cultured with hucMSCs. c The images of cell Transwell migration assay of HUVECs co-cultured with hucMSCs. d The images of the tube formation assay of HUVECs co-cultured with hucMSCs. e Quantification of the proliferation rates of HUVECs. f Quantification of the migration area of HUVECs. g Quantification of the migrated cells numbers of HUVECs. Quantification of the tubes numbers (h) and tube length (i) of HUVECs

Fig. 4

The role and mechanisms of miRNA-126-3p-hucMSCs-exosomes in the promotion of HUVECs proliferation, migration, and tube formation. a The morphology of hucMSCs-exosomes. b Western blotting analysis of exosome markers. c DiI-labeled hucMSCs-exosomes (red) could be internalized by DAPI-labeled HUVECs (blue). d The expression of miRNA-126-3p in exosomes derived from hucMSCs and GFP-hucMSCs and miRNA-126-3p-hucMSCs. e Quantification of the proliferation rates of HUVECs. f Quantification of the migration area of HUVECs. g Quantification of the migrated cell numbers of HUVECs. Quantification of the h tube length and tubes numbers (i) of HUVECs

Fig. 5

a SPRED-1and PIK3R2 mRNA levels were measured in HUVECs co-cultured with hucMSCs and GFP-hucMSCs and miRNA-126-3p-hucMSCs. b Western blots of PIK3R2 and SPRED-1 protein expression in HUVECs co-cultured with hucMSCs and GFP-hucMSCs and miRNA-126-3p-hucMSCs. c SPRED-1 and PIK3R2 protein levels were higher in the miRNA-126-3p-hucMSCs group than in the hucMSCs or GFP-hucMSCs group. d Representative western blots of AKT phosphorylation and ERK1/2 phosphorylation in HUVECs co-cultured with hucMSCs and GFP-hucMSCs and miRNA-126-3p-hucMSCs. e Quantification of the proliferation rates of HUVECs. f Quantification of the migration area of HUVECs. g Quantification of the migrated cell numbers of HUVECs. i Quantification of the tube length and tubes numbers of HUVECs

Fig. 6

Treatment with hucMSCs overexpressing miRNA-126-3p accelerated endothelialization of the vein grafts in an arterialized rat model. Rat vein graft model: external jugular vein into infrarenal aorta (a). The luminal diameter (b) and peak systolic velocity (c) were measured by serial ultrasound studies during the whole study period. d Representative gross morphology of vein grafts at 4 weeks after transplantation. e Representative images of hucMSCs recruitment in the vein grafts, hucMSCs were predominantly found at the intraluminal site of the blood vessel as indicated by CM-Dil and GFP fluorescence. f Vein grafts harvested 2 weeks after hucMSC transplantation were analyzed by CD34 immunohistochemical staining to assess vascular reendothelialization in each group

Fig. 7

Treatment with hucMSCs overexpressing miRNA-126-3p attenuated vein graft intimal hyperplasia and vascular inflammation in a rat model. a Representative images of the vein graft stained with HE. b Representative images of the vein graft stained with Masson’s trichrome. c Representative images of the vein graft stained with Ki67. d Representative images of the vein graft stained with TNF-α. e Representative sections of the vein graft stained with CD68

Fig. 8

Schematic representation of in vivo and vitro experimental design and relevant morphology