Abstract
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.
Highlights
The Endothelial Progenitor Cells (EPC) treatment strategy take advantages of preserving or regenerating tissue to develop towards clinical application (Li et al 2019; Guo et al 2020; Kim et al 2018)
Our groups focused on tissue repair for several years and we found tube formation of BM-EPCs sheets was associated with increasing secretion of SDF-1a, epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF)
Compared with traditional cell suspension injection methods, in which most of the cells are washed-out into blood vessels and can may die due to the locally harsh ischemic microenvironment, cell sheet technology has been used in surface injuries like wounds or some factor EGF (a); SDF-1a (b); VEGF (c) than did the BM-EPC single-cell group. (n = 6, per group; **p \ 0.001, *p \ 0.01)
Summary
The Endothelial Progenitor Cells (EPC) treatment strategy take advantages of preserving or regenerating tissue to develop towards clinical application (Li et al 2019; Guo et al 2020; Kim et al 2018). EPC derived from bone marrow (BMEPC) sheet are known to play an important role in angiogenesis by participating in the formation of vessels and in vessel repair and remodeling (Liman and Endres 2012; Esquiva et al 2018). It can release a wide array of paracrine factors to fabricate tissue repair. Accumulating evidence has indicated that the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is stimulated to participate in EPC cell proliferation and tube formation capacity. BM-EPCs cell sheet can not harvested with trypsin and cell-to-cell interactions and the integral structure of the BM-EPCs sheet were unknown (Zhou et al 2017; Lu et al 2019)
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