The Role Of Young Plasma On HUVECs Senescence And The Underlying Mechanism

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Young Plasma attenuated senescent phenotype of vascular endothelial cells ObjectiveVascular endothelial cell senescence companied with the aging process and play an important role in the initiation and development of vascular aging and age-related diseases.Young blood could restore multiplies younger phenotypes in old mice evidenced by parabiotic experiment,while its effects on endothelial senescence remain largely unknown.This study was intended to explore the influence of young plasma on HUVECs senescence.MethodsPrimary HUVECs were isolated from segments of fresh newborn human umbilical cords which was digested by 0.1% collagenase II and confirmed by morphology and histochemical staining with a monoclonal anti-human vWF antibody.The replicative senescence of HUVECs was obtained by sustaining cell passage and population doubling levels were calculated.Blood plasma were collected from healthy young male patients(15-30 years)and pre-elderly male patients(45-60 years)in the morning after an overnight fast.Equal volume of each plasma sample was pooled into a young plasma(YP)pool and a pre-elderly plasma(PEP)pool.At PDL 28,cells were cultivated in the medium containing 20% PEP for 48 hours before they were trypsinized and divided into 2 groups.One group of cells was continuously cultivated in the 20% PEP-containing medium as the control group,while the other group of cells was cultivated in a medium containing 20% YP.After 5 days cultivation,both groups of cells were collected and subjected for the following experiments.Endothelial cell function and senescent status were evaluated by angiogenesis,wound healing,senescence-associated ?-gal(SA-?-gal)stain and cell cycle related RNA and protein expression.ResultsPrimary HUVECs isolated from the fetal umbilical cords were identified and confirmed by vWF factors.The purity of HUVECs was more than 95%,and the cells were used in the following experiments.HUVECs shown senescent phenotypes at PDL 28.The proliferation rate was decreased and population doubling time was extended.The percentage of positive SA-?-gal stained cells significantly increased in the senescent cells(54.3% ± 6.0% in the later-passage cells,~PDL28)compared to the younger cells(13.3% ± 3.1% in the earlier-passage cells,~PDL8)(P < 0.05).The level of RNA and protein SIRT1 decreased during HUVEC senescence,while the levels of RNA and protein P53 and P21 increased during cell senescence.Young plasma “rejuvenated” HUVECs senescence phenotypes.Under our experimental conditions,YP treatment significantly enhanced HUVECs tube formation as revealed by increased number of branch points(80.0 ± 14.8 in YP vs 38.8 ± 21.3 in PEP,p < 0.05)and enhanced cell proliferation and migration as assessed by the wound healing assay(65.4% ± 1.2% in YP vs 44.1% ± 8.0% in PEP,p < 0.05),accompanied with lowered the percentile of SA-?-gal positively stained HUVECs as compared to the PEP group(58.2% ± 7.9% in YP vs 74.8% ± 5.0% in PEP,p < 0.05).Moreover,YP treatment significantly lowered the protein levels of P53,P21,as compared to the PEP group? ConclusionHUVECs replicative senescence model obtained by sustain passage shown significantly senescence phenotype and could be used in the following studies.Young plasma rejuvenated senescent HUVECs younger phenotypes,accompanied with increased angiogenesis and wound healing potential and decreased percentage of SA-b-gal statin positive cells and the cell cycle related proteins expression.Part ? Mechanism of Young Plasma on improving senescence phenotype of vascular endothelial cellsObjective Cell senescence is a complicated biological process which were mainly determined by genes and processed by environment.Vascular endothelial cells senescence was mainly influenced by blood environment due to its specific location.Our precious studies found that the phenotype of senescent endothelial cells could be restored by young plasma intervention,while its mechanism need further investigation.This study was intended to explore the mechanisms by using RNA-sequence and si RNA knockdown technology.Methods The HUVECs used in this study were come from three independent umbilical cords.At PDL 28,senescent HUVECs were incubated by pre-elderly plasma and then part of them were change cultured with young plasma for another 48 h.Then,cell RNA was obtained and sent to Illumulia Hiseq 2000 for RNA-sequence.The differentially expressed genes was identified as the RPKM>2 at least one sample and Fold change ?1.25.The differentially expressed genes were verified by RT-PCR.The expression of differentially expressed genes(DEGs)also detected in HUVECs replicative models.The senescence associated differentially expressed genes(SADEGs)were picked out and its function in endothelial cell senescence were explored by si RNA transiently knockdown.Result 36 DEGs were obtained by overlay three group sequence data,within 20 DEGs were up-regulated and 16 DEGs were down-regulated by young plasma intervention.14 DEGs were found to be differentially expressed during HUVEC senescence,and 7 of them the expression tendency during replicative senescence could be transcriptionally reversed by YP treatment.DHCR24 and MCM10 were identified as anti-senescence gene by si RNA transiently knockdown technology and UNC5 B,CYP26B1,WBP1 were as pro-senescence genes,while ACAT2 and CYP26B1 have no significantly effect on endothelial cell senescence.Conclusion DHCR24 and MCM10 were identified as anti-senescence while UNC5 B,CYP26B1,WBP1 were as pro-senescence genes.Young plasma could restore younger phenotype in senescent HUVECs by increasing proliferation,migration,angiogenesis potential and modulate cell cycle,DNA damage,inflammation and oxidative stress process through up-regulate DHCR24,MCM10 and down-regulate UNC5 B,CYP26B1 and WBP1.