Combination of granulocyte colony-stimulating factor and CXCR4 antagonist AMD3100 for effective harvest of endothelial progenitor cells from peripheral blood and in vitro formation of primitive endothelial networks

Abstract

Endothelial progenitor cells (EPC) derived from the circulation may be used to enhance neovascularization. Since the combination of granulocyte colony-stimulating factor (GCSF) and CXCR4 antagonist AMD3100 efficiently mobilizes hematopoietic stem cells into peripheral circulation, it may increase the pool of endogenously circulating EPC. We tested this hypothesis by administering GCSF and AMD3100 to adult rabbits and rats, isolating mononuclear cells from peripheral blood by Ficoll density gradient centrifugation, and characterizing the blood-derived EPC based on morphology, immunophenotyping, gene expression and other functional analyses. These EPC showed clonal growth similar to that of human umbilical vein endothelial cells when cultured in complete EGM-2 medium on collagen I-precoated culture plates. The EPC exhibited a typical cobblestone-like morphology and were relatively homogeneous by the third passage. The cells expressed the typical endothelial marker CD31 based on flow cytometry and fluorescence microscopy, formed capillary-like structures when cultured in Matrigel, internalized DiI-acetylated low-density lipoprotein, bound Ulex europaeus agglutinin-1, and expressed CD31 and several other endothelial markers (VEGFR2, VE-cadherin, Tie-2, eNOS, vWF) at significantly higher levels than bone marrow-derived mesenchymal stem cells. These results suggest that the combination of GCSF and AMD3100 can efficiently release stem cells into peripheral circulation and generate EPC that show the desired morphological, immunophenotypic and functional characteristics. This minimally invasive approach may be useful for autologous cell transplantation for postnatal neovasculogenesis and tissue repair.

Transplantation of Endothelial Progenitor Cells Overexpressing miR-126-3p Improves Heart Function in Ischemic Cardiomyopathy

“…The upregulated cytokines of VEGF-A, 21 IL-10, 22 IL-3 23 and HGF 24 are effective pro-angiogenic or anti-inflammatory factors, and show anti-ischemic and vascular protection effects. Moreover, G-CSF may efficiently mobilize EPCs, 25 and IGF-1 can enhance EPC migration, invasion, and vessel formation. 26 Angiogenin can also promote EPC proliferation and colony formation.…”

Hemopexin promotes angiogenesis via up-regulating HO-1 in rats after cerebral ischemia-reperfusion injury

“…vWF, the carrier protein of coagulation factor VII, plays an important role in the process of blood platelet adhering to the subcutaneous tissue. Co-expression of CD31 and vWF were considered to be the specific marker to identify the endothelial cells [ 25 ]. Differentiation of endothelial progenitor cells in ischemic penumbra is observed and the density of neovascularization begins to increase at 3d after ischemia reperfusion.…”

AMD3100 and SDF‑1 regulate cellular functions of endothelial progenitor cells and accelerate endothelial regeneration in a rat carotid artery injury model

“…AMD3100 and SDF-1 are extensively used in regenerative medicine, including hematological disease ( 14 , 19 ), angiogenesis ( 17 , 20 ), intimal repairing ( 16 ), wound healing ( 21 , 22 ) and brain repair after ischemic stroke ( 18 , 23 ). Furthermore, previous studies have reported that AMD3100 and SDF-1 may be involved in mobilization and recruitment of EPCs ( 16 , 17 , 24 , 25 ). These studies indicated that AMD3100 or SDF-1 may be used for endothelial regeneration.…”

“…Intravenous or subcutaneous administration of AMD3100 has been reported to effectively induce mobilization of HSCs and EPCs ( 14 , 24 ). Furthermore, a single dose of AMD3100 may mobilize EPCs into peripheral blood ( 25 ).…”