| BackgroundsThe vascular system is a bipolar complex network of arteries that transport oxygen-rich blood to all tissues and veins that bring oxygendeprived blood back to the heart.Blood vessels consist of two cell types,endothelial cells(ECs) and mural cells (vascular smooth muscle cells and pericytes) that provide a wide variety of vascular diversity.Because of this bipolar set-up,arteries and veins feature anatomic and physiological differences.Unlike venous endothelium,arterial endothelium is surrounded by several layers of smooth muscle cells(SMCs),separated by elastic laminae,and embedded in a thick layer of fibrillar collagen.EC diversity is a main determinant of the vascular diversity.Recently,specific markers for arteries and veins were discovered,which labeled ECs from early development stages onwards,before the onset of blood flow.The Eph receptors are the largest of the 14 subfamilies of receptor tyrosine kinases(RTKs),and are activated by membrane-tethered ligands of the equally large ephrin family.It appears that Eph/ephrin signaling play important functional roles in the vasculature.Eph/ephrin signaling regulate a variety of morphogenetic processes in different tissues,including segmentation of the vertebrate hindbrain and paraxial mesoderm,repulsive axonal guidance and fasciculation during the formation of topographic maps in the vertebrate embryonic nervous system,and cell movement in both vertebrates and invertebrates.Recent work has implicated EphB/ephrin B signaling in embryonic vascular development and revealed its critical role in arterial-venous vascular differentiation.A transmembrane ligand,Ephrin-B2, and its receptor,the tyrosine kinase EphB4,were the first reported molecular markers for arterial and venous ECs,respectively.During the last several years,additional molecular markers specific for arterial and venous ECs have been identified.For arterial ECs,ephrinB1,Hey2,Jagged-1 and -2,neuropilin 1,and members of the Notch pathway appear to play critical roles.Other molecules such as COUP-TFll or neuropilin-2 are specifically expressed in the venous system.Recently,many stem-cell types,including embryonic stem cells,AC133+ endothelial progenitor cells,and multipotent adult progenitor cells,have been found to differentiate in vitro and in vivo into mature and functional arterial and venous ECs. Notch signaling is an evolutionarily conserved pathway that is essential for a variety of developmental processes,including asymmetric cell-fate decisions,boundary formation and cell proliferation.The Notch signaling pathway has been considered important in regulating arterial-venous cell specification,as was first shown in zebrafish.A Notch ligand,deltaC,and the notch5 receptor are both expressed specifically in arteries.Reduction of Notch activity results in a loss of arterial markers such as ephrinB2 and notch5 and activation of venous markers such as EphB4 and flt4 in the dorsal aorta.Taken together,Notch signaling suppresses venous endothelial cell fate and induces arterial differentiation.The studies in mice and in the zebrafish demonstrating that VEGF is necessary and sufficient for arterial differentiation.The zebrafish studies also demonstrated that VEGF acts upstream of Notch signaling in arterial fate determination.Descriptive studies performed some time ago showed that nerves and larger vessels coalign in the skin,and another recent study explored the molecular basis for this phenomenon.Arteries,but not veins,specifically align with peripheral nerves in embryonic mouse limb skin.Loss of peripheral sensory nerves or Schwann cells leads to defects in arteriogenesis,while these same cells can induce arterial marker expression in isolated embryonic endothelial cells when they are cocultured in vitro.Sensory neurons and glia both express VEGF,and additional in vitro experiments demonstrated that VEGF is necessary and sufficient to mediate the arterial induction effects of these cells.Together,these data suggest that peripheral nerves provide a template for the formation of arteries in the skin via local secretion of VEGF.Mesenchymal stem cells(MSC) are derived from the mesoderm,distributed in many somatic tissues during fetal development and later confined to BM and a number of connective tissues in the adult.They exhibit adherent,fibroblastic and clonal properties and express a specific cell-surface phenotype.Because the cells are highly expandable ex vivo and capable of differentiating along a variety of different cell lineages,they are regarded as one of the potential resources for stem cellbased therapy and transplantation.Recent studies have shown that mesenchymal stem cells (MSCs) derived from human bone marrow(hMSCs) could be induced to differentiate into endothelial-like cells.Umbilical cord blood-derived and amniotic membrane-derived hMSCs were also found to differentiate into ECs in vitro and in vivo.However,a precise analysis of the arterial-venous specification and the molecular mechanism in hMSC differentiation into endothelial-like cells remains unknown.Research ObjectivesWe aimed to analyze the in vitro and in vivo arterial or venous endothelial differentiation of hMSCsWe aimed to investigate the mechanism of VEGF in hMSCs inducing an arterial or venous fate in endothelial-like cells and the role of Notch signaling in the process.Materials and methodsBone marrow was obtained from the sternum of children(age 6 months to 12 years) who were undergoing congenital heart disease surgery,after approval by the ethics committee at Fu Wai Hospital.Informed consent was obtained from a parent of each donor,and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki.Isolation and culture of hMSCs Bone marrow aspirates of 3 to 5 ml were placed in a tube containing 5 ml of phosphate-buffered saline(PBS) and 1250μheparin.The marrow sample was loaded onto an equal volume of 1.073 g/mL Histopque solution (Sigma-Aldrich) and centrifuged at room temperature at 900g for 30 min. Mononuclear cells were collected and washed twice in PBS.The cells were seeded into 75 cm2 flasks containing Iscove's modified Dulbecco's medium(IMDM) supplemented with 10%fetal bovine serum(FBS)(Gibco Grand Island,NY), penicillin(100μg/mL),and streptomycin(100 mg/mL) and incubated at 37℃in 5% CO2 and 95%humidity.After 3 days of culture,adherent cells were allowed to continue to culture in medium that was changed every 3 days.After 10 to 14 days of primary cultivation,the adherent cells were 80%-90%confluent as visualized by phase contrast microscope.Cells were dissociated with use of 0.25%trypsin and 1 mM EDTA,replated in T-25 flasks at 104 cells/cm2 and grown to near confluence for passaging,hMSCs were used at first passage for all experiments.The cell growth curve was constructed according to daily mean values measured by the MTT method from the second to the sixteen day.Flow cytometric analysis About 5×105 cells per 100μl were labeled with primary antibodies against CD14-PE,CD34-PE,CD45-PE,CD90-PE,CD105-PE, and CD73-FITC(Becton Dickinson).Cells were incubated at 4℃for 30 min and washed;mouse IgG1-FITC and IgG1-PE(Becton Dickinson) were used as isotype controls.Multipotent differentiation potential of hMSC Differentiation of the MSC into adipocytes or osteocytes was initiated by further incubation of the cells in medium with adipogenic or osteogenic supplements,respectively.Adipocytes or osteocytes were analyzed according to the supplier's instructions.Differentiation of hMSCs into ECs Human MSCs were plated at 2×104/cm2 in endothelial differentiation medium containing Iscove's modified Dulbecco's medium(IMDM),50 ng/mL VEGF(R&D Systems,Minneapolis,MN) and 5%FBS.Cells underwent staining for von Willebrand factor(vWF;Dako,Carpinteria,CA),KDR,Flt-1,Tie-1,Tie-2.Images were acquired by use of a microscope.The expression of vWF mRNA was calculated by real time PCR.Arterial-venous differentiation of hMSCs Human MSCs were plated at 2× 104/cm2 in endothelial differentiation medium containing Iscove's modified Dulbecco's medium(IMDM),50 or 100 ng/mL VEGF(R&D Systems,Minneapolis, MN) and 5%FBS.Ephrin-B2 and EphB4 were stained in fluorescent staining.For immunofluorescent staining,secondary antibodies were coupled to FITC or TRITC (Sigma-Aldrich,USA) and cells were stained with 4',6-diamidino-2-phenylindole (DAPI).mRNA expression of arterial and venous genes in hMSC-derived ECs with 50 ng/mL and 100 ng/mL VEGF at day 14.In vitro EC functional tests Incorporation of Dil-ac-LDL To observe the uptake of 1,1'-dioctadecyl-1-3,3,3,3-tetramethyl-indo-carbocyanine perchlorate conjugated to acetylated LDL(Dil-ac-LDL)(Biomedical Technologies,Stoughton,MA), differentiated hMSCs were seeded onto T-25-cm2 flasks.The medium was replaced by fresh medium with 10μg/mL Dil-ac-LDL.After incubation for 4 hr at 37℃,cells were washed several times with probe-free media and observed on fluorescence microscopy.In Vitro Angiogenesis Analysis of tubular structure formation involved use of the in vitro angiogenesis kit(ECM625) according to the manufacturer's instructions(Chemincon,Temecula,CA,USA).An amount of 50μl of Matrigel was applied to one well of a 96-well plate and incubated for 1 h at 37℃.Cells were trypsinized,and 5×103 cells were suspended in 100μL IMDM containing 50 ng/mL VEGF,plated onto the gel matrix and incubated at 37℃.After 4- to 24-h incubation, the formation of a cord- or tube-like network was examined and recorded on phase-contrast microscopy.In vivo Angiogenesis Ten-week-old male nude mice(BALB/c-nu/nu) were used in this experiment.The mice were anesthetized with use of 1%pentobarbital(80 mg/kg,given intraperitoneally) and injected subcutaneously in the back with 300μL Matrigel containing 100 ng/mL VEGF and hMSCs or VEGF and no cells.The injected cells were pre-labeled with 4',6-diamidino-2-phenylindole(DAPI) as described.Two weeks later,all mice were sacrificed,and Matrigel plugs were removed and processed for OCT embedding.Tissue sections were immunostained for human-specific vWF,ephrinB2,EphB4 and mouse-specific CD31 and viewed on fluorescence microscopy.The role of Notch signaling in Arterial-venous differentiation of hMSCs To inhibit Notch signaling,γ-secretase inhibitor(L-685,458;Bachem King of Prussia, PA) was added at a concentration of 1 Mm.Arterial and venous genes were analyzed by quantitative RT-PCR with VEGF(100 ng/mL) with or withoutγ-secretase inhibitor.ResultshMSCs displayed no expression of hematopoietic markers CD14,CD34 and CD45,but more than 90%expressed typical MSC markers CD90,CD105,CD73. hMSC had a faster proliferative period from the 7 days to 14 days.hMSC were able to differentiate to osteoblasts,adipocytes in vitro differentiating conditions.Afer 14 days of exposure to 50 ng/mL,most hMSCs expressed VEGF receptors 1(79%±4%) and 2(87%±5%)(Flt-1 and KDR,respectively),angiopoietin receptors Tie-1(85%±8%) and Tie-2(83%±6%),and vWF(65%±4%),which suggested differentiation to ECs.ECs were shown to be functional by acetylated LDL uptake (97%±3%) and had tube-forming potential.As a negative control,about(23%±2%) cells among undifferentiated hMSCs took up red fluorescence.The expression of vWF mRNA was remarkably enhanced in hMSC-derived ECs.Low transcript levels of the arterial markers ephrinB2,D114 and Notch4 and the venous markers EphB4 and COUP-TFⅡwere detected in hMSCs before differentiation.With VEGF treatment(50 ng/mL),ECs showed expression of the venous marker EphB4 but little of the arterial marker ephrinB2 on immunostaining. With increased VEGF dosage(100 ng/mL),the expression of the arterial markers ephrinB2 increased and the venous EphB4 decreased at day 14.Meanwhile,the mRNA expression of the arterial genes Ephrin-B2,D114,and Notch4 was strongly upregulated in ECs.In contrast,the venous genes EphB4 and COUP-TFⅡwere downregulated as compared with their level at 50 ng/mL VEGF.Thus,arterial specification in hMSC-derived ECs depends on VEGF dosage.Matrigel plugs with hMSCs recovered for observation of the formation of functional vessels 14 days later showed many new blood vessels as compared with few blood vessels in controls(Figure 5A,B).Fluorescent-labeled hMSCs were observed on tissue sections,which indicated that implanted cells persisted in Matrigel plugs(Figure 5C).Most implanted cells expressed human-specific vWF as well as ephrinB2 or EphB4,which indicated their arterial-venous EC identity(Figure 5D-F). Using mouse-specific CD31 antibodies we stained blood vessels in Matrigel plugs that show the vascular structure were of mouse origin.(Figure 5G,H).We quantified blood vessel density in Matrigel plugs by counting CD31-positive vessels;average vessels density was 13±1.5 and 4.6±1.1 vessels per 0.25 mm2 for hMSCs- or no hMSCs-seeded plugs,respectively.(Figure 5G,H).However,we observed the DAPI-labeled hMSC surrounded the lumen and we did not find any evidence of integration of hMSC-derived ECs into the endothelium of host growing vessels (Figure 5G).So,the hMSCs-derived ECs contributed to growing of host vessels and did not significantly contributed to the structure of a vessel-like lumen.We prevented Notch signaling with theγ-secretase inhibitor L-685,458 and found significantly decreased expression of the arterial markers D114(P＜0.05),Hey2, and ephrinB2(P＜0.01) but not ephrinB1(P＞0.05)(Figure 4).In contrast,the venous marker COUP-TFⅡwas significantly increased in expression(P＜0.01) and that of EphB4 slightly increased but not significantly.Conclusions and significance our data show that VEGF is required for arterial-venous EC differentiation of hMSCs and the effect is dose dependent. hMSC-derived ECs remain plastic in terms of arterial-venous differentiation.High VEGF concentration led to the expression of arterial marker genes,whereas low VEGF concentration contributed to venous differentiation.After inhibition of Notch signaling,this VEGF-induced arteriogenesis was largely blocked,which resulted in a shift from arterial to venous cell fate.
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