Effect Of Sodium Arsenite On Human Endothelial Progenitor Cells And The Molecular Mechanisms Involved

Endemic arsenism is a serious threat to human health,the arsenic pollution of rice caused by arsenic-containing water irrigation has become a global problem.Peripheral vascular disease caused by arsenic is the thorny problem in its prevention and treatment.Endothelial progenitor cells(EPCs)play a key role in the maintenance of vascular integrity and in the repair and reconstitution of injured blood vessels.However,to our knowledge,the effects of arsenic on the EPCs have not been elucidated.Early EPCs expressed VEGFR2(KDR),CD34 and CD133 three surface marker molecules.Currently purified EPCs are mainly derived from bone marrow and peripheral blood.Bone marrow-derived EPCs are early EPCs,but t the number of harvested cells is limited.Although EPCs can be isolated from peripheral blood,but these EPCs are more advanced EPCs,and their function is far less than the early EPCs.Therefore,there is still a need to find new ways to obtain enough early EPCs.In this study,human EPCs were isolated from human umbilical vein endothelial cells by using magnetic micro-beads coated with an antibody against human CD34.Flow cytometric assay were used to analysis three molecular markers of early EPCs.A bioreactor micro-carrier cell culture system was used to expand the EPCs in vitro.And then we investigated the effects of sodium arsenite on human EPCs in terms of proliferation,apoptosis,migration and angiogenesis and the molecular mechanisms involved systematacially.To explore alternative source of human EPCs,we studied whether the expression of OCT4 alone might change the phenotype of HUVECs to EPCs.The EPCs from HUVECs might be a novel resource of cells for regenerative medicine and that a bio-MCCS culture system described here might be broadly used for in vitro expansion of EPCs.And the results provided new evidence for elucidating the pathogenesis of arsenic vascular injury and a new pathway for the prevention.1 Isolation,purification and high quality in vitro expansion of human endothelial progenitor cells of human umbilical vein originObjective: To isolate EPCs from human umbilical vein endothelial cells(HUVECs)and in vitro expand cells with high yields.Methods: EPCs were separated from primary HUVECs by CD34+ immunomagnetic microbeads.Bioreactor microcarrier cell culture system(Bio-MCCS)was used to expand EPCs in vitro.Flow cytometry analysis and matrix-gel based in vitro angiogenesis assay of cells were assessed to evaluated the cell properties and function after expansion in vitro.Results: we isolated human EPCs from HUVECs by using magnetic micro-beads coated with an antibody against human CD34.Flow cytometric assay showed that majority of these cells expressed VEGFR2(KDR),CD34 and CD133,three molecular markers for early EPCs.It was also found that a bioreactor micro-carrier cell culture system was superior to dish culture for in vitro expansion of EPCs.It expanded more EPCs which were in the early stage,as shown by the expression of characteristic molecular markers and had better angiogenic potential,as shown by matrix-gel based in vitro angiogenesis assay.Conclusions: These results suggest that HUVECs might be a novel promising resource of EPCs for regenerative medicine and that a bio-MCCS cell culture system might be broadly used for in vitro expansion of EPCs.2 The effect of sodium arsenite on cellular functions of human endothelial progenitor cells and the molecular mechanisms involvedObjective: The aim of this study was to investigate the effect of sodium arsenite on the proliferation,apoptosis,migration and angiogenesis of EPCs and its molecular mechanism involved.Methods: We used CCK-8 assay and flow cytometry analysis to detect cell proliferation and apoptosis of sodium arsenite on EPCs separately,and determined cell migration and in vitro angiogenesis by using the scratch assay and matrix-gel based in vitro angiogenesis assay separately.The gene expression profile was analyzed by an oligonucleotide probe-based gene array chip and validated by RT-QPCR.Results: The arsenite suppressed the cell proliferation,cell migration and in vitro angiogenesis of EPCs in a dose dependent manner,and promote the cell apoptosis in a dose dependent manner.We found that arsenite broadly changed the gene expression profile and particularly down-regulated the expression of CYP1A1,AGT and IGF2 which were representive marker molecules for endothelial proliferation,meanwhile up-regulated the expression of SFRP1,RPS6KA2 and HTRA2;as well as AXL,COL1A1 and TPM1 which were representive marker molecules for endothelial apoptosis and oxidative response stress,respectively.We also found that arsenite down-regulated the expression of BMP2,CCL20 and VTN;as well as C3,AGT and APOH which were representive marker molecules for endothelial migration and angiogenesis ability,respectively.All of the m RNAs were analyzed showed the same pattern of change in expression by the two techniques.Conclusions: These results suggest that sodium arsenite inhibited the proliferation,promoted the apoptosis,inhibited the migration and reduced angiogenesis of human EPCs,by down-regulating CYP1A1,AGT and IGF2,by up-regulation of SFRP1,RPS6KA2 and HTRA2,by down-regulation of BMP2,CCL20,and VTN and by down-regulation of BMP2,C3,AGT and APOH,respectively.These findings suggest novel potential mechanisms for arsenite-induced injury of vascular EPCs.3 OCT4 remodels the phenotype and promotes angiogenesis of HUVECs by changing the gene expression profileObjective: To explore whether the expression of OCT4 alone might change the phenotype of HUVECs to EPCs and,if so,to examine the possible mechanism involved.Methods: A Matrigel-based in vitro angiogenesis assay was used to evaluate the angiogenesis of the cells;the gene expression profile was analyzed by an oligonucleotide probe-based gene array chip and validated by RT-QPCR.The cellular functions of the m RNAs altered by OCT4 were analyzed with Gene Ontology.Results: We found that induced ectopic expression of mouse OCT4 in HUVECs significantly enhanced angiogenesis of the cells,broadly changed the gene expression profile and particularly increased the expression of CD133,CD34,and VEGFR2(KDR)which are characteristic marker molecules for EPCs.Furthermore by analyzing the cellular functions that were targeted by the m RNAs altered by OCT4 we found that stem cell maintenance and cell differentiation were among the top functional response targeted by up-regulated and down-regulated m RNAs upon forced expression of OCT4.Conclusions: These results support the argument that OCT4 remodels the phenotype of HUVECs from endothelial cells to EPCs by up-regulating the genes responsible for stem cell maintenance and down-regulating the genes for cell differentiation.