Effects Of Endothelin-1 On The Functional Activity Of Endothelial Progenitor Cells From Peripheral Blood And Its Molecular Mechanisms

Objective:Vascular endothelial progenitor cells(EPCs)are the precursor of endothelial cells.Increasing evidence suggests that circulating progenitor cells contribute to postnatal neovascularization.These cells home to sites of ischemia,adopt an endothelial phenotype,and contribute to new blood vessel formation,the identity of the circulating cells that contribute to neovascularization is not entirely clear. Bone-marrow derived hematopoietic progenitor cells can give rise to endothelial progenitor cells and contribute to endothelial recovery and new capillary formation after ischemia.Endothelin-1(ET-1)is a 21-amino acid peptide releasede by a variety of cells, including endothelial cells,with potent vasoconstrictor activity and mitogenic properties.ET-1 has been causatively implicated in the structural and functional abnormalities,such as arterial hypertension,atherosclerosis,and glomerulosclerosis. Its concentration is elevated in hypertension,atherosclerosis and many tumor patients, ET-1 production is increased in atherosclerotic plaques.Interestingly,ET-1 has dual vasoactive effects,mediating vasoconstriction and cell proliferation via ETA receptor activation of vascular smooth muscle cells;and vasorelaxation and NO, PGI₂ production via ETB receptor activation of endothelial cells.The effects of ET-1 on EPCs are unknown.So the aim of our study was to investigated the role of ET-1 in the vasculogenic process by evaluating the effects of ET-1 on number and activity of endothelial progenitor cells from peripheral blood.Furthermore,we investigated the signaling pathway downstream of ETB receptor activation by evaluating the expression of phosphorylated-Akt and phosphorylated-eNOS in ET-1 pretreated EPC.Methods:Total mononuclear cells(MNCs)were isolated from peripheral blood by Ficoll density gradient centrifugation,and then the cells were plated on fibronectin-coated culture dishs.After 7 days cultured,attached cells were stimulated with different concentration ET-1 or ET-1 1nM with Ly294002,or with BQ788for 24 hours.EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding by direct fluorescent staining.EPCs proliferation,migration were assayed with MTT assay and modified Boyden chamber assay,respectively.EPCs adhesion assay was performed by replating them on fibronectin-coated dishes,and then adherent cells were counted.In vitro vasculogenesis activity was assayed by in vitro vasculogenesis kit.phosphorylated-Akt and phosphorylated-eNOS were assayed by western blot.Results:1.Characteristics of human EPCs:Total MNCs isolated and cultured for 7 days resulted in a spindle-shaped, endothelial cells like morphology.EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding under a laser scanning confocal microscope.We and other investigators have previously demonstrated that endothelial progenitor cells isolated in this fashion also exhibit many other endothelial characteristics,including expression of CD31,Von Willebrand factor(vWF),and vascular endothelial growth factor receptor 2(VEGFR-2),and so on.2.Effect of ET-1 on EPCs numbers:Incubation of isolated human MNCs with ET-1 increased the number of EPCs in a concentration and time dependent manner.Ly294002,or BQ788 can significantly inhibit ET-1 induced EPCs number increase.3.Effects of ET-1 on the proliferative capacity of isolated EPCs:Incubation of isolated human MNCs with ET-1 increased EPCs proliferative capacity in a concentration dependent manner.Ly294002,or BQ788 can significantly inhibit ET-1 induced EPCs proliferation.4.Effects of ET-1 on the migratory capacity of isolated EPCs:Incubation of isolated human MNCs with ET-1 increased EPCs migratory capacity in a concentration dependent manner.Ly294002,or BQ788 can significantly inhibit ET-1 induced EPCs migration.5.Effect of ET-1 on the adhesive capacity of isolated EPCs:Incubation of isolated human MNCs with ET-1 increased EPCs adhesive capacity in a concentration dependent manner.Ly294002,or BQ788 can significantly inhibit ET-1 induced EPCs adhesion.6.Effects of ET-1 on EPCs in vitro vasculogenesis:Incubation of isolated human MNCs with ET-1 increased EPCs in vitro vasculogenesis capacity.Ly294002,or BQ788 can significantly inhibit ET-1 induced EPCs in vitro vasculogenesis.7.Effects of ET-1 on EPCs phosphorylated-Akt and phosphorylated-eNOS:Western blot analysis showed that the expression of phosphorylated-Akt and phosphorylated-eNOS was significantly increased in the cells treated with ET-1 in a concentration dependent manner,Ly294002,or BQ788 can significantly inhibit ET-1 induced phosphorylated-Akt and phosphorylated-eNOS expression.Conclusions:1.EPCs can be isolated and cultured from mononuclear cells from peripheral blood. 2.ET-1 increase the number,proliferative,migratory,adhesive and in vitro vasculogenesis capacity of EPCs.The effects of ET-1 can be inhibited by Ly294002, or BQ788.3.ET-1 also increase phosphorylated-Akt and phosphorylated-eNOS expression in EPCs.These protein expression induced by ET-1 can be inhibited by Ly294002,or BQ788.4.The effects of ET-1 on functional activities of EPCs are mediated by ETB,and are related with PI3K/Akt/eNOS signaling pathway.