Effect Of Endothelin-1 On Proliferation, Cell Cycle, NO Secreting Function, And Apoptosis Of Endothelial Progenitor Cells Derived From Rat Bone Marrow

Background and AimsEndothelins are a family of peptides, which comprises endothelin-1 (ET-1), endothelin-2 (ET-2) and endothelin-3 (ET-3), each containing 21 amino-acids. ET-1 is a peptide secreted mostly by vascular endothelial cells, the predominant isoform expressed in vasculature and the most potent vasoconstrictor currently known. ET-1 is a multifunctional regulatory peptide, which has extensive biological effects. It mediates inflammation, stimulates the secretion of other hormones, facilitates caryomitosis, and stimulates cell proliferation, cell migration and cell adhesion by autocrine and paracrine through its recepters. Through numerous clinical and experimental investigations, it has become evident that ET-1, beyond its function as a vasoactive peptide, also plays a seminal role in the angiogenic process. ET-1, by acting directly on endothelial cells via the ETB receptor, modulates different stages of neovascularization, including proliferation, migration, protease production and morphogenesis, and also stimulates neovascularization in vivo.Endothelial progenitor cells (EPC) are a cell population of endothelial precursors that have the capacity to circulate, proliferate, and differentiate into mature endothelial cells, but have not yet acqueired characteristic mature endothelial markers. Recent studies demonstrated that EPC participated not only during embryonic neovascularization, but also in postnatal neovascularization and reendothelialization. When vessel injury occurs, EPCs are released from bone marrow, migrate to the site of injury, incorporate into the vessel wall, differentiate into mature endothelial cells, repair the injured vessel, participate the neovascularization and restore or improve the blood flow of the ischemic tissue.The present study intends to explore the effects of ET-1 on EPC proliferation, cell cycle, NO secretion, and apoptosis. The results may help to understand the cardiovascular effects of ET-1 from a new aspect.MethodsPartâ… : EPC culture and identification.1. Harvest rat bone marrow, collect mononuclear cells through density gradient centrifugation, and culture them in Medium 199 supplemented with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF).2. Immunofluorescent co-stain with Diâ… -ac-LDL and FITC-UEA-â… to identify EPC.Partâ…¡: Effect of ET-1 on proliferation, cell cycle, NO secreting function, and apoptosis of EPC.1. Collect EPCs at the 5th day of culture, then add different doses of ET-1 to the medium and incubate for different time.2. Test the proliferative ability of EPC by MTT assay.3. Test the cell cycle of EPC by flow cytometry.4. Measure the secretion of NO by modified Griess reaction method.5. Test the apoptosis rate of EPC by flow cytometry.Statistical analysis was performed with SPSS version 11.0. One-way analysis of variation and post hoc t (LSD-t) test were employed.Results1. The cells obtained showed double positive for Diâ… -ac-LDL and FITC-UEA-â… , indicating their endothelial lineage and progenitor property, i.e., they are EPCs.2. ET-1 dose- and time-dependently improved EPCs proliferation activity. The proliferative capacity of EPCs improved at 48h (P<0.05), with a peak at 96h (P<0.01) and at 10-6mol/L concentration (P<0.01).3. ET-1 dose-dependently transforms EPCs from G0/G1 phrase to S and G2/M phrase, with a maximal effect at 10-6mol/L concentration (P<0.01).4. ET-1 dose- and time-dependently promoted NO production of EPC. The effect is significance at 48h (P<0.05), with a peak at 96h (P<0.01) and at 10-6mol/L concentration (P<0.01).5. ET-1 significantly improve the rate of EPC apoptosis, with a maximal effect at 48h (P<0.01). After then, the rate of apoptosis decrease.Conclusion1. ET-1 dose- and time-dependently improved EPCs proliferation activity.2. ET-1 dose-dependently transforms EPCs from G0/G1 phrase to S and G2/M phrase.3. ET-1 dose- and time-dependently promoted NO production of EPC.4. ET-1 improves the rate of EPC apoptosis. At the 48th hour, it has the strongest effect. But after the 48th hour, its effect decrease.5. ET-1 can significantly stimulated proliferation and apoptosis of EPC. Because of the extent of proliferation more than apoptosis, it leaded unbalance between proliferation and apoptosis on EPC.