Abstract
BackgroundThe 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.Methodshuman 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.ResultsWe 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.ConclusionThese results suggest hucMSC-based miRNA-126-3p gene therapy may be a novel option for the treatment of vein graft disease after CABG.
Highlights
Coronary atherosclerotic heart disease (CHD) is considered one of the most serious diseases threatening human life and health
Primary human umbilical cord MSCs (hucMSCs) were transfected with lentiviral vectors carrying miRNA-126-3p at a multiplicity of infection (MOI) 75
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
Summary
Coronary atherosclerotic heart disease (CHD) is considered one of the most serious diseases threatening human life and health. Coronary artery bypass grafting (CABG) has become the main treatment for severe cases of CHD. For CABG, the autogenous great saphenous vein is the most widely used vascular graft [1]. More than half of vein grafts will be occluded during the first 10 years after CABG [2]. The restenosis caused by vein graft intimal hyperplasia has become a key factor influencing the efficacy of CABG. A method to increase long-term patency rate of the autogenous vein graft has become a hot topic of research [3]. The aim of this study was to determine whether the combination of MSC implantation with miRNA126-3p overexpression would further improve the surgical results after vein grafting
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