骨髓间充质干细胞向动静脉内皮细胞的诱导及其机制的基础研究
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摘要
背景
血管系统包括动脉和静脉,动脉将血液从心脏送往全身各器官,组织,并为其提供了赖以生存的物质,包括营养物质和氧气,而静脉则将血液由全身各器官送回心脏,带走了细胞代谢的产物二氧化碳。血管由两类细胞组成:内皮细胞和外膜细胞,其中外膜细胞包括血管平滑肌细胞和外周细胞。和静脉内皮不同,动脉内皮周围由较厚的平滑肌细胞层和弹力纤维层包绕。血管的多样性主要是由内皮细胞的多样性决定。近来,很多特异性的动脉和静脉的标志物被发现,而且在发育阶段的早期血流开始之前就存在内皮细胞上。Eph受体是酪氨酸激酶受体家族(receptor tyrosine kinases RTKs)中最大的一个亚群,其配体主要表达于细胞表面,被命名为ephrin。Eph-ephrin具有非常重要的生理功能,参与胚胎发育中轴突导向、突触形成、细胞增殖和迁移、体节生成和血管发生,可以说其贯穿着脊椎动物和非脊椎动物的整个进化过程。最近研究表明,EphB/ephrinB在胚胎血管的发育中对动静脉的特异性分化起到关键作用。酪氨酸激酶配体ephrinB2和它的受体EphB4是第一个被报道可以分别作为动脉内皮细胞和静脉内皮细胞的分子标志物。在随后的几年,一系列其它的动脉和静脉的分子标志物被先后证实。酪氨酸激酶配体ephrinB1,神经纤维网蛋白—1(neuropilin 1)和Notch信号系统的配体Jagged-1,Jagged-2和Notch靶基因Hey2等在动脉上特异性表达,而神经纤维网蛋白—2(neuropilin 2)和COUP-TFll则特异性在静脉上表达。
最近,很多干细胞类型,包括胚胎干细胞,AC133~+内皮前体细胞,成体多潜能干细胞,都已被发现在体内和体外都能分化为成熟和具有功能的动脉和静脉内皮细胞。Notch通路是广泛存在于脊椎和非脊椎动物中的重要信号传导通路,与其他多个高度保守的细胞传导通路一起构筑起生物发展的信号骨架决定细胞的最终命运,影响器官形成及形态发生。研究发现Notch信号通路对血管的发生发展,包括细胞增殖、迁移、平滑肌分化、血管生成、动静脉分化等多个方面有重要调控功能。最早在斑马鱼的研究中发现,血管内皮细胞的动脉或静脉分化主要受Notch信号调控。Notch配体deltaC和Notch5受体在动脉上特异性表达,减弱Notch活性可以导致动脉标志物ephrinB2和Notch5的丢失,但在背弓主动脉上却激活了静脉标志物EphB4和Flt4。因此,Notch信号通路抑制静脉内皮的命运同时诱导了动脉的分化。对小鼠和斑马鱼的研究结果证实VEGF对动脉分化是必须和足够的,而且VEGF位于决定动脉化命运的Notch信号通路的上游。既往研究发现在皮肤感觉神经和小血管相伴行,近来一些研究进一步探讨了其中的分子机制。在小鼠胚胎肢体皮肤上,小动脉而不是静脉特异性地与外周感觉神经伴行。外周感觉神经或施旺氏细胞地缺失可以导致动脉生成障碍,同样在体外共培养时,这些细胞的存在也可以诱导胚胎内皮细胞表达动脉标志物。感觉神经元和胶质细胞分泌VEGF和体外实验中外源性VEGF也能够介导胚胎内皮细胞的动脉化。这些研究表明外周神经通过分泌VEGF能够提供皮肤上小动脉形成所需要的模板。
骨髓基质系统中的骨髓间充质干细胞(Mesenchymal stem cells MSCs)是一种具有多向分化潜能的干细胞,它可以向多种结缔组织以及部分来源于外胚层的组织分化,形成骨、软骨、骨骼肌、腱、韧带、真皮、脂肪、骨髓基质和神经。骨髓间充质干细胞具有黏附性,成纤维样和克隆样生长的特性,而且表达特定的细胞表型。因为骨髓间充质干细胞在体外具有高度扩增能力,能够分化为多系细胞,而且具有来源充足、细胞培养成活率高、无免疫排异等优点,其已经成为心脏病细胞移植治疗中最具潜力的种子细胞。最近研究发现人骨髓来源的间充质干细胞在体外能够诱导成内皮样细胞。脐血和胎膜来源的间充质干细胞在体外和体内都能分化为内皮细胞。然而,骨髓来源的间充质干细胞分化的内皮细胞的动静脉的特异性和其中的分子机制尚不清楚。
研究目的:
本研究旨在阐明在体外和体内实验中VEGF诱导入骨髓间充质干细胞分化的内皮样细胞的动静脉命运。
Notch信号通路在VEGF诱导人骨髓间充质干细胞分化的内皮样细胞的动静脉命运过程的作用。
研究方法
阜外心血管病医院伦理委员会批准这个研究协议,选择年龄6个月到12岁的心脏病患儿心脏开胸手术时从胸骨收取骨髓血,所有参与者的父母都签订书面赞同协议,研究协议遵守1975年赫尔新基宣言。
一、骨髓间充质干细胞的分离和鉴定
以Histopaque密度梯度离心法从人的胸骨骨髓血中分离人骨髓间充质干细胞,PBS洗两次后,细胞种植在T75cm~2塑料培养瓶中,培养液是含10%胎牛血清的IMDM,培养液中加入青霉素(100μg/mL)和链霉素(100mg/mL),在含5%CO_2,95%湿度的细胞培养箱。3天培养后,细胞换培养液,黏附的细胞继续生长,未贴壁的细胞抛弃。在10～14天培养后,黏附的细胞呈集落式生长,可达到70-80%的融合。0.25%胰蛋白酶消化收集细胞,以104个细胞/cm~2传代。所有实验均采用第一代细胞。生长曲线的制作。
骨髓间充质干细胞表面抗原检测。胰蛋白酶消化收集细胞,PBS洗两次,洗去残留培养液,制成10~6个/ml的细胞悬液100μl。加入相应抗体20μl,室温避光反应30min。1800rpm,10min室温离心,弃上清。加入500μl PBS重悬细胞,流式检测CD14-PE,CD34-PE,CD45-PE,CD90-PE,CD105-PE,和CD73-FITC。鼠IgG1-FITC和IgG1-PE作为同型对照。
骨髓间充质干细胞多向分化潜能测定
干细胞成脂肪细胞诱导。第一代骨髓间充质干细胞达60-70%融合后,加成脂肪细胞诱导液。每3天换液一次,继续加成脂肪细胞诱导液。两周后进行苏丹Ⅲ染色:PBS冲洗细胞两次,以洗去残留的培养液。加入苏丹Ⅲ染料,37℃静置30min。PBS洗去染料,光学显微镜下观察细胞中红色的脂肪颗粒。
干细胞成骨细胞诱导。第一代骨髓间充质干细胞达60-70%融合后,加成骨细胞诱导液。每3天换液一次,继续加成骨细胞诱导液。两周后进行茜素红染色:PBS冲洗细胞两次,以洗去残留的培养液。加入茜素红染料,室温静置5min。PBS洗去染料,光学显微镜下观察红色钙沉淀。
MSC向内皮细胞分化
以2×104cm~2密度种植细胞,IMDM培养基内加入50 ng/mL VEGF。
细胞免疫组化检测vWF,KDR,Flt-1,Tie-1,Tie-2。
实时荧光定量PCR检测vWF。
MSC向动静脉内皮的诱导
以2×104cm~2密度种植细胞,IMDM培养基内加入50ng/mL和100ng/mLVEGF。
细胞荧光免疫组化检测Ephrin-B2,和EphB4。二抗用FITC或TRITC标记。DAPI标记细胞核。
实时荧光定量检测不同浓度诱导的内皮细胞的内皮标致物KDR,Flt-1,Tie-2,vWF,动脉内皮标致物Ephrin-B2,Dll4和Notch4,静脉内皮标志物EphB4和COUP-TFⅡ。
内皮功能实验
Dil-ac-LDL吞噬实验。分化的hMSC种植在T 25 cm~2培养瓶,培养基加入10μg/mL Dil-ac-LDL,37℃下孵育4小时后,细胞用不含Dil-ac-LDL的培养基洗3次,然后在荧光显微镜下检查。
体外成血管实验。用成血管试剂盒(ECM625)分析微管状结构的形成。将ECM胶液和稀释液在0℃水浴箱内冻融,成液体状,每900微升ECM胶液加入100微升稀释液,混均,将上述溶液加入96孔板,每孔50微升,37摄氏度孵育1小时成胶。消化分化的MSC,并重新悬浮于IMDM培养液包含50ng/mL VEGF,调整细胞数目5×10~3ml,将细胞接种于ECM胶上,孵育4—24小时,在倒置显微镜下观察。
体内成血管实验。取10周龄的雄性裸鼠,1%戊巴比妥腹腔注射麻醉后,在皮下分别注射300微升Matrigel胶包含100ng/mL VEGF和MSCs或者100ng/mLVEGF但无MSCs细胞。MSCs用DAPI标记细胞核。2周后,处死裸鼠,取出Matrigel胶用OCT包埋。冰冻切片后行vWF,ephrinB2,EphB4和抗鼠CD31的免疫荧光染色。在荧光显微镜下检查。
Notch通路作用研究
抑制剂组:浓度1μM的γ-分泌酶抑制剂(L-685,458)加入到IMDM培养基内。hMSC加VEGF组。单纯hMSC组。
实时荧光定量PCR。检测各组Hey2,D114,ephrinB2,ephrinB1,COUP-TPⅡ和EphB4的表达。
结果
培养7天以后可见细胞呈集落式生长,集落中央细胞较为密集。细胞为成纤维细胞状,其间夹杂圆形的细胞。10-14天以后细胞可达到70—80%的融合。生长曲线显示1-7天为潜伏期,10天后进入平台生长期。表面抗原检测CD14,CD34,CD45阴性,超过90%的细胞表达CD90,CD105,CD73。骨髓间充质干细胞在一定的诱导条件下能分化为脂肪样细胞和骨样细胞类型,具有多向分化的潜能,。
50 ng/mL VEGF诱导MSC两周后细胞免疫组化染色结果Flt-1阳性率790%±4%,KDR阳性率87%±5%,Tie-1阳性率85%±8%,Tie-2阳性率830%±46%,vWF阳性率65%4±4%。实时荧光定量PCR显示vWF在2周和3周时显著升高。
100 ng/mL VEGF诱导MSC和50ng/mL VEGF诱导MSC组比较,实时荧光定量PCR显示100ng/mL VEGF组KDR,Flt-1,Tie-1,Tie-2,vWF较50ng/mLVEGF组升高,但差异没有显著性。荧光双染色显示50ng/mLVEGF组MSC诱导的ECs动脉标志物ephrinB2阳性率11%4±2%,静脉标志物EphB4阳性率65%±4%,100ng/mL VEGF组动脉标志物66%±5%,静脉标志物EphB4阳性率21%±3%。在诱导14天后提取mRNA,用实时荧光定量PCR测定显示100ng/mLVEGF组动脉标志物Ephrin-B2,D114,和Notch4显著升高(P＜0.01),静脉标志物COUP-TFⅡ和EphB4都下调,但只有COUP-TFⅡ有显著差异性(P＜0.01)。
体外实验中50ng/mLVEGF组诱导14天后能够摄取Dil-ac-LDL97%±3%,并且在Matrigel形成管状的网络结构。体内试验中,Matrigel胶含MSC组在14天后可以观察到许多新生的血管,对照组则几乎没有。DAPI标记的MSC显示MSC持续存在Matrgel栓内,免疫组化显示多数植入的细胞表达了vWF,ephrinB2和EphB4。用抗鼠的CD31染色证实Matrigel栓内的血管是鼠原性的。细胞组和非细胞植入组比较,血管密度分别为134±1.5和4.64±1.1每0.25mm~2,而且DAPI标记的MSC围绕在血管管腔周围,没有足够证据显示MSC分化的EC掺入了新生的血管,因此MSC诱导的ECs有助于宿主血管的新生但没有形成新生血管。
抑制Notch信号后,用实时荧光定量PCR检测动脉和静脉标志物显示,动脉标志物Hey2,D114,和ephrinB2显著降低(P＜0.01),但ephrinB1无显著差异性(P＞0.05),同时静脉标志物COUP-TPⅡ显著增加(P＜0.01),EphB4无显著差异性(P＞0.05)。
结论及意义
VEGF能诱导MSC分化为动脉和静脉内皮细胞,这种作用存在剂量依赖性。
高浓度的VEGF有助于MSC诱导的内皮细胞表达动脉标志物,低浓度的VEGF则有助于MSC诱导的内皮细胞表达静脉标志物。
抑制Notch通路后,VEGF诱导的动脉化分化被大部分阻止,转向分化为静脉内皮细胞。
Backgrounds
The 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 Objectives
We aimed to analyze the in vitro and in vivo arterial or venous endothelial differentiation of hMSCs
We 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 methods
Bone 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 cm~2 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% CO_2 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/cm~2 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 5×10~5 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 2×10~4/cm~2 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× 10~4/cm~2 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-cm~2 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 5×10~3 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.
Results
hMSCs 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 mm~2 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.
血管系统包括动脉和静脉,动脉将血液从心脏送往全身各器官,组织,并为其提供了赖以生存的物质,包括营养物质和氧气,而静脉则将血液由全身各器官送回心脏,带走了细胞代谢的产物二氧化碳。血管由两类细胞组成:内皮细胞和外膜细胞,其中外膜细胞包括血管平滑肌细胞和外周细胞。和静脉内皮不同,动脉内皮周围由较厚的平滑肌细胞层和弹力纤维层包绕。血管的多样性主要是由内皮细胞的多样性决定。近来,很多特异性的动脉和静脉的标志物被发现,而且在发育阶段的早期血流开始之前就存在内皮细胞上。Eph受体是酪氨酸激酶受体家族(receptor tyrosine kinases RTKs)中最大的一个亚群,其配体主要表达于细胞表面,被命名为ephrin。Eph-ephrin具有非常重要的生理功能,参与胚胎发育中轴突导向、突触形成、细胞增殖和迁移、体节生成和血管发生,可以说其贯穿着脊椎动物和非脊椎动物的整个进化过程。最近研究表明,EphB/ephrinB在胚胎血管的发育中对动静脉的特异性分化起到关键作用。酪氨酸激酶配体ephrinB2和它的受体EphB4是第一个被报道可以分别作为动脉内皮细胞和静脉内皮细胞的分子标志物。在随后的几年,一系列其它的动脉和静脉的分子标志物被先后证实。酪氨酸激酶配体ephrinB1,神经纤维网蛋白—1(neuropilin 1)和Notch信号系统的配体Jagged-1,Jagged-2和Notch靶基因Hey2等在动脉上特异性表达,而神经纤维网蛋白—2(neuropilin 2)和COUP-TFll则特异性在静脉上表达。
最近,很多干细胞类型,包括胚胎干细胞,AC133~+内皮前体细胞,成体多潜能干细胞,都已被发现在体内和体外都能分化为成熟和具有功能的动脉和静脉内皮细胞。Notch通路是广泛存在于脊椎和非脊椎动物中的重要信号传导通路,与其他多个高度保守的细胞传导通路一起构筑起生物发展的信号骨架决定细胞的最终命运,影响器官形成及形态发生。研究发现Notch信号通路对血管的发生发展,包括细胞增殖、迁移、平滑肌分化、血管生成、动静脉分化等多个方面有重要调控功能。最早在斑马鱼的研究中发现,血管内皮细胞的动脉或静脉分化主要受Notch信号调控。Notch配体deltaC和Notch5受体在动脉上特异性表达,减弱Notch活性可以导致动脉标志物ephrinB2和Notch5的丢失,但在背弓主动脉上却激活了静脉标志物EphB4和Flt4。因此,Notch信号通路抑制静脉内皮的命运同时诱导了动脉的分化。对小鼠和斑马鱼的研究结果证实VEGF对动脉分化是必须和足够的,而且VEGF位于决定动脉化命运的Notch信号通路的上游。既往研究发现在皮肤感觉神经和小血管相伴行,近来一些研究进一步探讨了其中的分子机制。在小鼠胚胎肢体皮肤上,小动脉而不是静脉特异性地与外周感觉神经伴行。外周感觉神经或施旺氏细胞地缺失可以导致动脉生成障碍,同样在体外共培养时,这些细胞的存在也可以诱导胚胎内皮细胞表达动脉标志物。感觉神经元和胶质细胞分泌VEGF和体外实验中外源性VEGF也能够介导胚胎内皮细胞的动脉化。这些研究表明外周神经通过分泌VEGF能够提供皮肤上小动脉形成所需要的模板。
骨髓基质系统中的骨髓间充质干细胞(Mesenchymal stem cells MSCs)是一种具有多向分化潜能的干细胞,它可以向多种结缔组织以及部分来源于外胚层的组织分化,形成骨、软骨、骨骼肌、腱、韧带、真皮、脂肪、骨髓基质和神经。骨髓间充质干细胞具有黏附性,成纤维样和克隆样生长的特性,而且表达特定的细胞表型。因为骨髓间充质干细胞在体外具有高度扩增能力,能够分化为多系细胞,而且具有来源充足、细胞培养成活率高、无免疫排异等优点,其已经成为心脏病细胞移植治疗中最具潜力的种子细胞。最近研究发现人骨髓来源的间充质干细胞在体外能够诱导成内皮样细胞。脐血和胎膜来源的间充质干细胞在体外和体内都能分化为内皮细胞。然而,骨髓来源的间充质干细胞分化的内皮细胞的动静脉的特异性和其中的分子机制尚不清楚。
研究目的:
本研究旨在阐明在体外和体内实验中VEGF诱导入骨髓间充质干细胞分化的内皮样细胞的动静脉命运。
Notch信号通路在VEGF诱导人骨髓间充质干细胞分化的内皮样细胞的动静脉命运过程的作用。
研究方法
阜外心血管病医院伦理委员会批准这个研究协议,选择年龄6个月到12岁的心脏病患儿心脏开胸手术时从胸骨收取骨髓血,所有参与者的父母都签订书面赞同协议,研究协议遵守1975年赫尔新基宣言。
一、骨髓间充质干细胞的分离和鉴定
以Histopaque密度梯度离心法从人的胸骨骨髓血中分离人骨髓间充质干细胞,PBS洗两次后,细胞种植在T75cm~2塑料培养瓶中,培养液是含10%胎牛血清的IMDM,培养液中加入青霉素(100μg/mL)和链霉素(100mg/mL),在含5%CO_2,95%湿度的细胞培养箱。3天培养后,细胞换培养液,黏附的细胞继续生长,未贴壁的细胞抛弃。在10～14天培养后,黏附的细胞呈集落式生长,可达到70-80%的融合。0.25%胰蛋白酶消化收集细胞,以104个细胞/cm~2传代。所有实验均采用第一代细胞。生长曲线的制作。
骨髓间充质干细胞表面抗原检测。胰蛋白酶消化收集细胞,PBS洗两次,洗去残留培养液,制成10~6个/ml的细胞悬液100μl。加入相应抗体20μl,室温避光反应30min。1800rpm,10min室温离心,弃上清。加入500μl PBS重悬细胞,流式检测CD14-PE,CD34-PE,CD45-PE,CD90-PE,CD105-PE,和CD73-FITC。鼠IgG1-FITC和IgG1-PE作为同型对照。
骨髓间充质干细胞多向分化潜能测定
干细胞成脂肪细胞诱导。第一代骨髓间充质干细胞达60-70%融合后,加成脂肪细胞诱导液。每3天换液一次,继续加成脂肪细胞诱导液。两周后进行苏丹Ⅲ染色:PBS冲洗细胞两次,以洗去残留的培养液。加入苏丹Ⅲ染料,37℃静置30min。PBS洗去染料,光学显微镜下观察细胞中红色的脂肪颗粒。
干细胞成骨细胞诱导。第一代骨髓间充质干细胞达60-70%融合后,加成骨细胞诱导液。每3天换液一次,继续加成骨细胞诱导液。两周后进行茜素红染色:PBS冲洗细胞两次,以洗去残留的培养液。加入茜素红染料,室温静置5min。PBS洗去染料,光学显微镜下观察红色钙沉淀。
MSC向内皮细胞分化
以2×104cm~2密度种植细胞,IMDM培养基内加入50 ng/mL VEGF。
细胞免疫组化检测vWF,KDR,Flt-1,Tie-1,Tie-2。
实时荧光定量PCR检测vWF。
MSC向动静脉内皮的诱导
以2×104cm~2密度种植细胞,IMDM培养基内加入50ng/mL和100ng/mLVEGF。
细胞荧光免疫组化检测Ephrin-B2,和EphB4。二抗用FITC或TRITC标记。DAPI标记细胞核。
实时荧光定量检测不同浓度诱导的内皮细胞的内皮标致物KDR,Flt-1,Tie-2,vWF,动脉内皮标致物Ephrin-B2,Dll4和Notch4,静脉内皮标志物EphB4和COUP-TFⅡ。
内皮功能实验
Dil-ac-LDL吞噬实验。分化的hMSC种植在T 25 cm~2培养瓶,培养基加入10μg/mL Dil-ac-LDL,37℃下孵育4小时后,细胞用不含Dil-ac-LDL的培养基洗3次,然后在荧光显微镜下检查。
体外成血管实验。用成血管试剂盒(ECM625)分析微管状结构的形成。将ECM胶液和稀释液在0℃水浴箱内冻融,成液体状,每900微升ECM胶液加入100微升稀释液,混均,将上述溶液加入96孔板,每孔50微升,37摄氏度孵育1小时成胶。消化分化的MSC,并重新悬浮于IMDM培养液包含50ng/mL VEGF,调整细胞数目5×10~3ml,将细胞接种于ECM胶上,孵育4—24小时,在倒置显微镜下观察。
体内成血管实验。取10周龄的雄性裸鼠,1%戊巴比妥腹腔注射麻醉后,在皮下分别注射300微升Matrigel胶包含100ng/mL VEGF和MSCs或者100ng/mLVEGF但无MSCs细胞。MSCs用DAPI标记细胞核。2周后,处死裸鼠,取出Matrigel胶用OCT包埋。冰冻切片后行vWF,ephrinB2,EphB4和抗鼠CD31的免疫荧光染色。在荧光显微镜下检查。
Notch通路作用研究
抑制剂组:浓度1μM的γ-分泌酶抑制剂(L-685,458)加入到IMDM培养基内。hMSC加VEGF组。单纯hMSC组。
实时荧光定量PCR。检测各组Hey2,D114,ephrinB2,ephrinB1,COUP-TPⅡ和EphB4的表达。
结果
培养7天以后可见细胞呈集落式生长,集落中央细胞较为密集。细胞为成纤维细胞状,其间夹杂圆形的细胞。10-14天以后细胞可达到70—80%的融合。生长曲线显示1-7天为潜伏期,10天后进入平台生长期。表面抗原检测CD14,CD34,CD45阴性,超过90%的细胞表达CD90,CD105,CD73。骨髓间充质干细胞在一定的诱导条件下能分化为脂肪样细胞和骨样细胞类型,具有多向分化的潜能,。
50 ng/mL VEGF诱导MSC两周后细胞免疫组化染色结果Flt-1阳性率790%±4%,KDR阳性率87%±5%,Tie-1阳性率85%±8%,Tie-2阳性率830%±46%,vWF阳性率65%4±4%。实时荧光定量PCR显示vWF在2周和3周时显著升高。
100 ng/mL VEGF诱导MSC和50ng/mL VEGF诱导MSC组比较,实时荧光定量PCR显示100ng/mL VEGF组KDR,Flt-1,Tie-1,Tie-2,vWF较50ng/mLVEGF组升高,但差异没有显著性。荧光双染色显示50ng/mLVEGF组MSC诱导的ECs动脉标志物ephrinB2阳性率11%4±2%,静脉标志物EphB4阳性率65%±4%,100ng/mL VEGF组动脉标志物66%±5%,静脉标志物EphB4阳性率21%±3%。在诱导14天后提取mRNA,用实时荧光定量PCR测定显示100ng/mLVEGF组动脉标志物Ephrin-B2,D114,和Notch4显著升高(P＜0.01),静脉标志物COUP-TFⅡ和EphB4都下调,但只有COUP-TFⅡ有显著差异性(P＜0.01)。
体外实验中50ng/mLVEGF组诱导14天后能够摄取Dil-ac-LDL97%±3%,并且在Matrigel形成管状的网络结构。体内试验中,Matrigel胶含MSC组在14天后可以观察到许多新生的血管,对照组则几乎没有。DAPI标记的MSC显示MSC持续存在Matrgel栓内,免疫组化显示多数植入的细胞表达了vWF,ephrinB2和EphB4。用抗鼠的CD31染色证实Matrigel栓内的血管是鼠原性的。细胞组和非细胞植入组比较,血管密度分别为134±1.5和4.64±1.1每0.25mm~2,而且DAPI标记的MSC围绕在血管管腔周围,没有足够证据显示MSC分化的EC掺入了新生的血管,因此MSC诱导的ECs有助于宿主血管的新生但没有形成新生血管。
抑制Notch信号后,用实时荧光定量PCR检测动脉和静脉标志物显示,动脉标志物Hey2,D114,和ephrinB2显著降低(P＜0.01),但ephrinB1无显著差异性(P＞0.05),同时静脉标志物COUP-TPⅡ显著增加(P＜0.01),EphB4无显著差异性(P＞0.05)。
结论及意义
VEGF能诱导MSC分化为动脉和静脉内皮细胞,这种作用存在剂量依赖性。
高浓度的VEGF有助于MSC诱导的内皮细胞表达动脉标志物,低浓度的VEGF则有助于MSC诱导的内皮细胞表达静脉标志物。
抑制Notch通路后,VEGF诱导的动脉化分化被大部分阻止,转向分化为静脉内皮细胞。
Backgrounds
The 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 Objectives
We aimed to analyze the in vitro and in vivo arterial or venous endothelial differentiation of hMSCs
We 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 methods
Bone 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 cm~2 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% CO_2 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/cm~2 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 5×10~5 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 2×10~4/cm~2 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× 10~4/cm~2 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-cm~2 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 5×10~3 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.
Results
hMSCs 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 mm~2 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.
引文
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