Construction and a preliminary study of paracrine effect of bone marrow-derived endothelial progenitor cell sheet

The release of paracrine factors from endothelial progenitor cell (EPC) sheet is a central mechanism of tissue repair. The purpose of this study was to constuct the rat bone marrow derived-endothelial progenitor cell (BM-EPCs) sheet and investigate invest the role of stromal cell-derived factor-1α (SDF-1α)/CXCR4 axis in the biological function of BM-EPCs sheet. BM-EPC cells were identified by the cell-surface markers-CD34/CD133/VE-cadherin/KDR using flow cytometry and dual affinity for acLDL and UEA-1. After 7 days of incubation, the BM-EPC single-cell suspensions were seeded on thermo-sensitive plate to harvest the BM-EPC cell sheets. The expression levels of SDF-1α/CXCR4 axis-associated genes and proteins were examined using RT-qPCR and western blot analysis, and enzyme-linked immunosorbent assay (ELISA) was applied to determine the concentration of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and SDF-1α in the cell culture medium. The BM-EPC cell sheets were successfully harvested. Moreover, BM-EPC cell sheets have superior migration and tube formation activity when compared with single cell suspension. When capillary-like tube were formed from EPCs sheets, the releasing of paracrine factors such as VEGF, EGF and SDF-1α were increased. To reveal the mechanism of tube formation of BM-EPCs sheets, our research showed that the activation of PI3K/AKT/eNOS pathway was involved in the process, because the phosphorylation of CXCR, PI3K, AKT and eNOS were increased. BM-EPC cell sheets have superior paracrine and tube formation activity than the BM-EPC single-cell. The strong ability to secrete paracrine factors was be potentially related to the SDF-1α/CXCR4 axis through PI3K/AKT/eNOS pathway.