Résumé
Ischemic dysfunction is an important global health problem. Vascular endothelial cells(VECs) play a key role in angiogenesis, and insufficient vascular remodeling may lead to chronic nonhealing wounds. Therefore, effective VEC generation strategies of exploration help improve angiogenesisin damaged tissues. Embryonic stem cells (ESCs) are widely used in the study of tissueendothelialization, and endothelial progenitor cells (EPCs) are indispensable parts of the development ofVECs. The aims of this study were to find a rapid, easily screened and reproducible method for thederivation of EPCs from mouse embryonic stem cells (mESCs), and obtain VECs with high survival ratesand strong functions from the directed differentiation of the EPCs. The results showed that mESCs weredifferentiated into " stepping stone" -like progenitor cells with active proliferative ability by 10 ng / mLVEGF and 5 ng / mL bFGF. At the same time, the method of differential adherence was helpful for theselection of EPCs, and EPCs induced high expression of CD133 and CD34 (The relative expressionlevels were 0. 88 ± 0. 04 and 2. 12 ± 0. 02, respectively) for 3 days. Then EPCs were digested withacctuse enzymes, and induced to differentiate into vascular endothelial-like cells by 50 ng / mL VEGF and25 ng / mL bFGF for 7 days. The endothelial cells not only expressed endothelial marker genes (CD31, CD144, LAMA5, Tek, KDR and vWF),and marker proteins CD31, CD144 and LAMA5 (The relativeexpression levels were 1. 07 ± 0. 03, 0. 60 ± 0. 02 and 0. 70 ± 0. 02, respectively), but also had thegood ability of migration, tubulogenesis and formation of W-P bodies. Moreover, PBS, EPC and VECwere used to treat wounds of the same size. Both EPC and VEC could accelerate the degree of tissuehealing (The relative healing rates were 78. 93 ± 75. 35%, 95. 57 ± 83. 73% and 100. 00 ± 0. 00%, respectively), and VEC significantly enhanced the ability of wound angiogenesis and inflammatoryresponses. In consequence, this study preliminarily confirmed that mESC-derived EPCs coulddifferentiate into VECs after directional induction for 7 days, which had good function of tissue repair. The physiological pathway on stem cells by stimulating angiogenesis is expected to become a new target fortissue remodeling.
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Objective:To study the effects of endothelial progenitor cells(EPCs)on the osteogenesis of bone marrow mesenchymal stem cell (BMSCs)sheet-implant complex.Methods:EPCs were added to the BMSC sheets,and the expression of osteogenesis-relat-ed genes was examined by real time PCR.Cell sheets were wrapped around implants to construct cell sheet-implant complexes and the complexes were subcutaneously transplanted into SCID mice.The complexes were harvested 8 weeks after operation and observed by micro-CT and histological examination.Results:The BMSC sheet with EPCs showed higher expression of Runx2,ALP,BMP2 and VEGF in the in vitro test;higher bone volume ratio,greater amount of new bone tissue and higher expression of Runx2 and BMP2 in the in vivo test.Conclusion:EPCs can improve the osteogenesis of BMSC sheet-implant complex.
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Objective To investigate the effects of shear stress on late endothelial progenitor cells (EPCs) functions in vitro and in vivo. Methods Density gradient centrifugation-isolated rat bone marrow mononuclear cells were cultured in EGM-2MV and induced into EPCs. The 3rd~4th generation of EPCs, namely late EPCs, were treated with shear stress (1.2 Pa). Then cell biological functions, such as proliferation, adhesion, migration and ability of tube formation, were assayed with EdU incorporation assay, adhesion testing, Boyden chamber assay and Matrigel, respectively. The gene expression of VEFG was analyzed by real time RT-PCR. The apoptosis and aging situation of late EPCs were assayed by FACS and senescence-associated β-galactosidase (SA-β-gal) staining. The reendothelialization capacity of late EPCs treated by shear stress was evaluated by establishing models of freshly balloon-injured carotid arteries of rats and cell transplantation in situ. Results Shear stress could increase proliferation, adhesion, migration and tube formation of late EPCs (P＜0.05), upregulate the gene expression of VEGF, inhibit EPC apoptosis and delayed EPC aging (P＜0.05). Transplantation of late EPCs treated by shear stress facilitated in vivo reendothelialization in the injured arterial segment and inhibited neointima formation. Conclusions Shear stress within the physiological range can improve the functions of late EPCs and enhance their therapeutic ability of repairing vascular endothelial injury, which provides experimental basis for the clinic application of EPCs and shear stress-mediated cell therapy.
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Objective To investigate effects of F-actin cytoskeleton on differentiation of endothelial progenitor cells (EPCs) under laminar shear stress. MethodsEPCs isolated from rat bone marrow were treated with laminar shear stress (1.2 Pa). Then the gene and protein expressions of the endothelial cell differentiation markers, such as vWF and CD31, were assayed with real time RT-PCR and Flow Cytometry. The effects of laminar shear stress on F-actin cytoskeleton and Ras activity were investigated by immunofluorescence technique and Pull-down assay. Results Compared with the untreated group, the expressions of vWF and CD31 were obviously increased in the group treated with laminar shear stress (P<0.05). Moreover, exposure of EPCs to laminar shear stress led to the reorganization of cytoskeleton and enhanced the activity of Ras in EPCs. The treatment to EPCs with either F-actin stabilizer jasplakinolide or depolymerizers cytochalasin D inhibited the cytoskeleton reorganization induced with laminar shear stress, the activity of Ras and the up-regulation of the vWF and CD31 genes. However, over-expression of Ras augmented the up-regulation of the vWF and CD31 genes induced by laminar shear stress in EPCs.Conclusions The mechanism that laminar shear stress accelerates the differentiation of EPCs may be related with the laminar shear stress-induced cytoskeleton rearrangement and Ras activation. This study is of significance in revealing the mechanism of vascular endothelial repair which could be useful for the prevention and treatment of atherosclerosis.
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Objective To study the effect of endothelial progenitor cells(EPCs) transplantation on chronic deep venous thrombosis.Methods Bone marrow-derived mouonuclear cells (BMMNCs) were isolated from rat bone marrow by ficoll and cultured with EGM-2MV medium.A rat model of chronic deep vein thrombosis was established by partial ligation of the inferior vena cava and intravenous injection of thrombosin.Model rats were randomly divided into three groups:A(n =25),EPCs group,1 ml 10~6 EPCs transplantation;B(n = 25),EGM-2MV medium group,1 ml EGM-2MV medium transplantation;C (n =25),control group,without any treatment.After transplantation,HE staining and immunohistochemical staining was conducted to detect recanalization of the inferior vena cava.Western blotting of inferior vena cava thrombosis was used to detect VEGF,bFGF protein expression changes.SPSS13.0 software was used for analysis.Results Compared with group B and C,VEGF,bFGF protein significantly increased in group A.The recanalization capillary density was significantly higher in group A than that in group B,and C (P ＜0.05).The neovascularization was identified by immunohistochemical staining using vWF antibody,as endothelial cells.Conclusions EPCs were the precursor of endothelial cells,when transplanted into the deep vein thrombos,initiating angiogenesis and accelerating organization and recanalization of vein thrombus.
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Endothelial progenitor cells (EPCs) can differentiate into mature endothelial cells and participate in postnatal vascular regeneration and impaired endothelium repair.Rearches in recent years on use of EPCs as seed cells in promoting angiogenesis,maintaining the integrity of endothelial function and constructing tissue engineered blood vessels are reviewed.
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Objective:To culture and identify ratendo the lialprogen it or cells,and too bserve the expression of recombin anthuman vascular endothelial growth fator-165(VEGF165)in endothelial progenitor cells transfected with pcDNA3.1(+)/VEGF165 plasmid.Methods:Endothelial progenitor cells were cultured in vitro,and immunocytochemical staining was used to detect the expression of surface antigen(CD34,CD133,KDR).After transfection of VEGF165,VEGF protein in the culture supernatant of endothelial progenitor cells was detected by enzyme-linked immunosorbent assay(ELISA),and VEGF165 mRNA in endothelial progenitor cells was detected by reverse transcription polymerase chain reaction(RT-PCR).Results:The cultured cells were identified to be endothelial progenitor cells by immunocytochemical analysis.Gel electrophoresis showed that a 576 bp product was amplified by RT-PCR in VEGF165-transfected cells,while a weak expression of VEGF165 was observed in mock-transfected and non-transfected cells.The protein level of VEGF165 in the supernatant of VEGF165-transfected,mock-transfected and non-transfected cells were 175.8?10.7 pg/ml,10.5?1.6 pg/ml and 9.3?1.3 pg/ml,respectively.Conclusion:There is small amount of VEGF165 expression in endothelial progenitor cells in vitro,and the endothelial progenitor cells transfected with pcDNA3.1(+)/VEGF165 plasmid have an increased expression of VEGF165.