Differential Effects of Isoxazole-9 on Neural Stem/Progenitor Cells,Oligodendrocyte Precursor Cells,and Endothelial Progenitor Cells

Adult mammalian brain can be plastic after injury and disease. Therefore, boosting endogenous repair mechanisms would be a useful therapeutic approach for neurological disorders. Isoxazole-9 (Isx-9) has been reported to enhance neurogenesis from neural stem/progenitor cells (NSPCs). However, the effects of Isx-9 on other types of progenitor/precursor cells remain mostly unknown. In this study, we investigated the effects of Isx-9 on the three major populations of progenitor/precursor cells in brain: NSPCs, oligodendrocyte precursor cells (OPCs), and endothelial progenitor cells (EPCs). Cultured primary NSPCs, OPCs, or EPCs were treated with various concentrations of Isx-9 (6.25, 12.5, 25, 50 μM), and their cell numbers were counted in a blinded manner. Isx-9 slightly increased the number of NSPCs and effectively induced neuronal differentiation of NSPCs. However, Isx-9 significantly decreased OPC number in a concentration-dependent manner, suggesting cytotoxicity. Isx-9 did not affect EPC cell number. But in a matrigel assay of angiogenesis, Isx-9 significantly inhibited tube formation in outgrowth endothelial cells derived from EPCs. This potential anti-tube-formation effect of Isx-9 was confirmed in a brain endothelial cell line. Taken together, our data suggest that mechanisms and targets for promoting stem/progenitor cells in the central nervous system may significantly differ between cell types.     

电磁辐射对大鼠内皮祖细胞和肾脏组织学的影响

目的:研究电磁辐射对体外培养骨髓来源的内皮祖细胞(EPCs)增殖、迁移、黏附能力的影响,并探讨其与肾脏疾病的可能关系。方法:密度梯度离心法获取大鼠骨髓单个核细胞(MNCs),接种至纤维连接素包被的培养板上,培养6d后进行免疫细胞化学和免疫荧光鉴定EPCs。采用MTT比色法、Transwell小室和黏附能力测定实验,观察平均功率密度为65mW/cm2,时间20min的电磁辐射对EPCs的增殖、迁移、黏附能力的影响;同等剂量全身照射大鼠,光镜和透射电镜观察大鼠肾脏组织学和超微结构的变化。结果:从大鼠骨髓能成功分离培养获得EPCs。EPCs的增殖、迁移、黏附能力较对照组显著下降;大鼠接受全身照射后各时相点无明显组织学改变,但超微结构显示在照射后3h后开始出现肾小球毛细血管袢足突肿胀,12h后出现部分足突融合。结论:电磁辐射导致EPCs生物功能显著减弱,肾小球超微结构改变,电磁辐射可能与起肾脏疾病的发生有关。     

Effects of Ezetimibe on Endothelial Progenitor Cells and Microparticles in High-Risk Patients

Imbalance on endothelial turnover can predict cardiovascular outcomes. We aimed at evaluating the effects of lipid-modifying therapies on circulating endothelial progenitor cells (EPCs), endothelial microparticles (EMPs), and platelet microparticles (PMPs) in high cardiovascular risk subjects with elevated C-reactive protein (CRP). Sixty-three individuals with coronary heart disease (CHD) or CHD risk equivalent on stable statin therapy, with LDL-cholesterol <100 mg/dL and CRP ≥2.0 mg/L were selected. After a 4-week run-in period with atorvastatin 10 mg, those with persistent CRP ≥2.0 mg/L were randomized to another 4-week treatment period with atorvastatin 40 mg, ezetimibe 10 mg or atorvastatin 40 mg/ezetimibe 10 mg. EPC (CD34⁺/CD133⁺/KDR⁺), EMP (CD51⁺), and PMP (CD42⁺/CD31⁺) were quantified by flow cytometry. Atorvastatin 40 mg and atorvastatin 40 mg/ezetimibe 10 mg reduced LDL-cholesterol (P < 0.001, paired T test, vs. baseline). Combined therapy, but not ezetimibe reduced CRP. CD34⁺/KDR⁺ EPC were reduced after ezetimibe alone (P = 0.011 vs. baseline, Wilcoxon test) or combined with atorvastatin (P = 0.016 vs. baseline, Wilcoxon test). In addition, ezetimibe increased CD51⁺ EMP (P = 0.017 vs. baseline, Wilcoxon test). No correlations between these markers and LDL-cholesterol or CRP were observed. These results contribute to understand the link between inflammation and vascular homeostasis and highlight the broader benefit of statins decreasing inflammation and preventing microparticles release, an effect not observed with ezetimibe alone.     

内皮祖细胞与血管重新内皮化

血管内皮是循环血液和血管壁组织间的一层天然屏障,在维持血管的正常形态和功能中起重要作用。内皮受损后可引起炎症反应、单核细胞浸润和血管平滑肌细胞增生,促发动脉粥样硬化和再狭窄。因此,直接修复受损血管内皮,促使血管重新内皮化已经成为防止动脉粥样硬化及再狭窄领域的重要课题。大量研究表明,内皮祖细胞(EPC)参与受损血管的重新内皮化。该文就内皮祖细胞的来源、鉴定、参与重新内皮化进行综述。     

Human Endothelial Progenitor Cells Internalize High-Density Lipoprotein

Endothelial progenitor cells (EPCs) originate either directly from hematopoietic stem cells or from a subpopulation of monocytes. Controversial views about intracellular lipid traffic prompted us to analyze the uptake of human high density lipoprotein (HDL), and HDL-cholesterol in human monocytic EPCs. Fluorescence and electron microscopy were used to investigate distribution and intracellular trafficking of HDL and its associated cholesterol using fluorescent surrogates (bodipy-cholesterol and bodipy-cholesteryl oleate), cytochemical labels and fluorochromes including horseradish peroxidase and Alexa Fluor® 568. Uptake and intracellular transport of HDL were demonstrated after internalization periods from 0.5 to 4 hours. In case of HDL-Alexa Fluor® 568, bodipy-cholesterol and bodipy-cholesteryl oleate, a photooxidation method was carried out. HDL-specific reaction products were present in invaginations of the plasma membrane at each time of treatment within endocytic vesicles, in multivesicular bodies and at longer periods of uptake, also in lysosomes. Some HDL-positive endosomes were arranged in form of “strings of pearl”- like structures. HDL-positive multivesicular bodies exhibited intensive staining of limiting and vesicular membranes. Multivesicular bodies of HDL-Alexa Fluor® 568–treated EPCs showed multilamellar intra-vacuolar membranes. At all periods of treatment, labeled endocytic vesicles and organelles were apparent close to the cell surface and in perinuclear areas around the Golgi apparatus. No HDL-related particles could be demonstrated close to its cisterns. Electron tomographic reconstructions showed an accumulation of HDL-containing endosomes close to the trans-Golgi-network. HDL-derived bodipy-cholesterol was localized in endosomal vesicles, multivesicular bodies, lysosomes and in many of the stacked Golgi cisternae and the trans-Golgi-network Internalized HDL-derived bodipy-cholesteryl oleate was channeled into the lysosomal intraellular pathway and accumulated prominently in all parts of the Golgi apparatus and in lipid droplets. Subsequently, also the RER and mitochondria were involved. These studies demonstrated the different intracellular pathway of HDL-derived bodipy-cholesterol and HDL-derived bodipy-cholesteryl oleate by EPCs, with concomitant.     

Adiponectin Protein Exists in Aortic Endothelial Cells

Aims

Inflammation is closely associated with the development of atherosclerosis and metabolic syndrome. Adiponectin, an adipose-derived secretory protein, possesses an anti-atherosclerotic property. The present study was undertaken to elucidate the presence and significance of adiponectin in vasculature.

Methods and Results

Immunofluorescence staining was performed in aorta of wild-type (WT) mice and demonstrated that adiponectin was co-stained with CD31. Thoracic aorta was cut through and then aortic intima was carefully shaved from aorta. Western blotting showed the existence of adiponectin protein in aortic intima, while there was no adiponectin mRNA expression. Adiponectin knockout (Adipo-KO) and WT mice were administered with a low-dose and short-term lipopolysaccharide (LPS) (1 mg/kg of LPS for 4 hours). The endothelium vascular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were highly increased in Adipo-KO mice compared to WT mice after LPS administration.

Conclusions

Adiponectin protein exists in aortic endothelium under steady state and may protect vasculature from the initiation of atherosclerosis.     

Nicotine Promotes Late Endothelial Progenitor Cells Functional Activity in a PI 3-Kinase-Dependent Manner

Recent studies have shown that endothelial progenitor cells (EPCs) participated in angiogenic effects of nicotine and nicotine dose dependently increased the functional activity of early EPCs. The effects of nicotine on late EPCs remain to be determined. Therefore, we investigated whether nicotine had influences on the functional activity of late EPCs. Late EPCs were isolated from human umbilical cord blood and characterized. Late EPCs of 3–5 passages were treated for 32 h with either vehicle or nicotine. The proliferative, migratory, and in vitro vasculogenesis activities of late EPCs were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, modified Boyden chamber assay, and in matrigel, respectively. Late EPCs adhesion assay was performed by replating cells on fibronectin-coated dishes, and then adherent cells were counted. Nicotine enhanced proliferative, migratory, adhesive, and in vitro vasculogenesis capacities of late EPCs. These effects were significantly reduced in the presence of phosphatidylinositol (PI) 3-kinase inhibitor.     

内皮祖细胞(EPCs)研究进展

组织工程血管以及组织工程化组织的血管化因目前内皮种子细胞扩增能力和生物活力的不足而受到限制。EPCs(内皮祖细胞)是内皮细胞的前体细胞。在胚胎期,内皮细胞系与造血细胞系来源于血岛内共同的祖先细胞;出生后,EPCs存在于骨髓,并可被转移至外周血,参与缺血组织的血管重建和血管的内膜化。因此EPCs有望成为今后组织工程内皮种子细胞的重要来源。     

高效诱导人胚胎干细胞成为内皮祖细胞

血管的发生和发育不仅对胚胎形成中各器官的发育分化十分重要,并且对成体的创伤修复和生殖功能也具有重要意义．血管内皮细胞是形成心血管封闭管道系统的形态基础,体外多种细胞可经诱导分化产生出内皮祖/内皮细胞(endothelial progenitor/endothelial cells,EPCs/ECs),但是存在一些不足．鉴于人类胚胎干细胞(human embryonic stem cells,hESCs)诱导分化的全能性和长期增殖能力,为EPCs/ECs提供了新的来源．现有文献报道,hESCs诱导分化为EPCs/ECs的比例较低,为了提高该诱导分化效率,我们使用分阶段的二维诱导方法,首先将细胞接种在超纯层纤连蛋白(Matrigel)上,之后通过在不同阶段添加不同的因子,最终获得CD31⁺KDR⁺细胞的比例可以达到16%．进一步内皮诱导分化的结果显示,获得的EPCs/ECs的比例可以达到约32%,这些细胞具有在Matrigel上形成血管样结构的能力,可结合植物凝集素．实时定量PCR的结果显示,诱导分化所得的细胞表达众多内皮相关基因,并且免疫荧光的结果也表明部分细胞表达内皮细胞特异性表面标志CD31．     

The Biphasic Effects of Oxidized-Low Density Lipoprotein on the Vasculogenic Function of Endothelial Progenitor Cells

Late-outgrowth endothelial progenitor cells (EPCs) are stress-resistant and responsible for reparative functions in the cardiovascular system. Oxidized-LDL (oxLDL) plays a critical role in cardiovascular disease pathogenesis. However, it is largely unknown what the impacts of oxLDL are on late-outgrowth EPCs. This study aimed to investigate the concentration-related effects of oxLDL on EPC functions and related angiogenesis, in vitro and in vivo. In this study, early and late-outgrowth EPCs were generated from circulating human mononuclear cells. oxLDL may regulate EPC vasculogenic function via the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). Lower concentrations (5 μg/mL) of oxLDL can potentiate EPC tube formation in vitro and in vivo by activating eNOS mechanisms, which are mediated by p38 MAPK- and SAPK/JNK-related pathways. Higher concentrations of oxLDL (10-50 μg/mL) impaired EPC function via the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase pathways and consequent inhibition of eNOS activity, which could be reversed by anti-oxidants (diphenylene iodonium and apocynin) and gp91^phox siRNA. In conclusion, oxLDL has concentration-dependent biphasic effects on human late-outgrowth EPC tube formation in vitro and in vivo.     

血脂康胶囊对高脂模型大鼠EPCs功能的影响

目的:高脂血症可增加心血管事件的发生率,本研究降脂红曲制剂血脂康胶囊对高脂模型大鼠的内皮祖细胞(endothelial progenitor cells,EPCs)生物学功能的影响。方法:给予Wistar大鼠高脂饲料30 d,造成高血脂大鼠模型,灌服血脂康胶囊。密度梯度离心法分别分离正常对照组,高脂模型组和血脂康治疗组大鼠骨髓单核细胞,应用EGM-2MV进行体外培养。以4~6代EPCs为靶细胞。采用Edu标记技术、CCK-8检测法、粘附能力测定试验、改良的Boyden小室、Matrigel法、荧光定量RT-PCR等方法分别检测EPCs增殖、粘附、迁移、体外成血管及单核细胞趋化蛋白-1(monocyte chemoattractant protein-1,MCP-1)等炎性因子的表达。结果:高血脂模型组大鼠EPCs的增殖、粘附、迁移及成血管能力均明显低于对照组,但炎症因子MCP-1表达则高于对照组;与高脂模型组EPCs比较,血脂康可明显促进EPCs的增殖、粘附、迁移及成血管,下调MCP-1的表达。结论:高脂状态下大鼠EPCs的增殖、粘附、迁移及成血管等生物学功能受损,血脂康能调整脂质代谢,改善高脂血症大鼠EPCs功能,从而起到保护血管内皮的功能,减少心血管疾病的发生。     

匹伐他汀对体外培养人脐静脉血内皮祖细胞数量及功能的影响

目的:通过体外培养人脐静脉血内皮祖细胞(endothelial progenitor cells,EPCs),观察他汀类新药(匹伐他汀)对EPCs数量及增殖、迁移和粘附功能的影响。方法:采用密度梯度离心法分离培养人脐静脉血单个核细胞,将其接种在包被有人纤维连接蛋白培养板上,培养7 d后,收集贴壁细胞,加入不同浓度匹伐他汀(分别为0.001μmol/L、0.01μmol/L、0.1μmol/L、1.0μmol/L)培养24h,用免疫荧光法观察EPCs吸收FITC-UEA-I和Dil-acLDL情况对EPCs进行鉴定,然后分别采用MTT比色法、改良的Boyden小室、粘附能力测定实验对各实验组测定,来观察匹伐他汀对EPCs数量及增殖、迁移和粘附功能影响。结果:匹伐他汀组与对照组相比,匹伐他汀显著提高了体外培养EPCs的数量及增殖、迁移与粘附能力(P0.05)。匹伐他汀浓度在0.1μmol/L时对EPCs影响达到最大。随着药物浓度的继续增大,EPCs的上述功能反呈下降趋势,但1.0μmol/L组仍高于对照组。结论:匹伐他汀能增加体外培养EPCs的数量及增殖、迁移和粘附能力,可作为EPCs培养的一种改良方法,为其更好的应用于临床具有重要的意义。     

FOXO3a沉默或过表达对内皮祖细胞增殖的影响

观察FOXO3a(forkhead box O3a)沉默或过表达对内皮祖细胞(endothelial progenitorcells,EPCs)增殖的影响。构建了沉默型Ad-shRNA-FOXO3a和突变型Ad-TM(triple mutant)-FOXO3a重组腺病毒载体。密度梯度离心法结合荧光激活细胞分选(fluorescence-activated cell sorting,FACS)分离人脐血来源的EPCs(CD133+CD34+),并进行体外培养及免疫荧光鉴定。将上述重组腺病毒载体转染EPCs并观察转染效率与细胞形态学改变;Western blot分析FOXO3a蛋白表达变化;细胞计数与MTT法分析FOXO3a沉默或过表达对EPCs增殖的影响。成功构建了Ad-shRNA-FOXO3a、Ad-TM-FOXO3a重组腺病毒载体并转染人脐血来源EPCs。Western blot提示,Ad-shRNA-FOXO3a转染EPCs后明显抑制了FOXO3a蛋白的表达;Ad-TM-FOXO3a转染EPCs后明显增加了FOXO3a蛋白的表达。结合细胞形态学改变、细胞计数与MTT法实验结果提示,有效沉默FOXO3a明显促进了EPCs增殖;FOXO3a过表达明显抑制了EPCs增殖。人脐血来源的EPCs中,FOXO3a参与了细胞增殖的调节。     

匹伐他汀对体外培养人脐静脉血内皮祖细胞数量及功能的影响

目的：通过体外培养人脐静脉血内皮祖细胞（endothelial progenitor cells,EPCs）,观察他汀类新药（匹伐他汀）对EPCs数量及增殖、迁移和粘附功能的影响。方法：采用密度梯度离心法分离培养人脐静脉血单个核细胞,将其接种在包被有人纤维连接蛋白培养板上,培养7 d后,收集贴壁细胞,加入不同浓度匹伐他汀（分别为0.001 μmol/L、0.01 μmol/L、0.1 μmol/L、1.0 μmol/L）培养24 h,用免疫荧光法观察EPCs 吸收FITC-UEA-I 和Dil-acLDL情况对EPCs 进行鉴定,然后分别采用MTT 比色法、改良的Boyden小室、粘附能力测定实验对各实验组测定,来观察匹伐他汀对EPCs 数量及增殖、迁移和粘附功能影响。结果：匹伐他汀组与对照组相比,匹伐他汀显著提高了体外培养EPCs的数量及增殖、迁移与粘附能力（P〈0.05）。匹伐他汀浓度在0.1 μmol/L 时对EPCs影响达到最大。随着药物浓度的继续增大,EPCs的上述功能反呈下降趋势,但1.0 μmol/L 组仍高于对照组。结论：匹伐他汀能增加体外培养EPCs的数量及增殖、迁移和粘附能力,可作为EPCs 培养的一种改良方法,为其更好的应用于临床具有重要的意义。     

血管内皮祖细胞与糖尿病微血管病变研究进展

糖尿病微血管病变严重影响了患者生活质量,是患者致死致残主要原因。微血管病变主要表现在视网膜、肾、神经、心肌组织。微血管病变的机制尚未完全清楚,近年越来越多研究发现血管内皮祖细胞(endothelial progenitor cells,EPCs)是该病发病重要原因。EPCs有分化为成熟的内皮细胞并且参与新血管形成和新生的能力。正常情况下内皮损失和EPCs对内皮的修复作用处于动态平衡状态,一旦EPCs受损,内皮损害和修复之间的平衡被打破,内皮层的完整性遭到破坏,必然参与糖尿病血管病变的发生发展。国内外大量研究证明糖尿病合并大血管病变EPCs数目功能改变,而糖尿病合并微血管病变EPCs的怎样变化?本文就EPCs与糖尿病微血管病变的关系进行系统综述。     

内皮祖细胞的生物学特性及其临床应用前景

内皮祖细胞（Endothelial Progenitor Cells,EPCs）是内皮细胞（endothelial cells,ECs）的前体细胞,即能分化为成熟ECs的祖细胞,它在血管内皮再生中发挥着重要作用。随着EPCs研究的深入,其在临床诊断、预后判断和各种缺血性疾病的治疗方面将会有广阔的应用前景。然而,关于EPCs的定义、来源、表面标记以及培养鉴定方法目前仍存在争议。     

内皮祖细胞的分离培养与鉴定

内皮祖细胞的分离方法有免疫磁珠分离法、淋巴细胞分离液分离法(1.077)和差速贴壁法,这3种方法已被人们广泛使用,均可分离到一定的目的细胞。分离到的目的细胞在培养过程中逐渐分化、成熟、发育为内皮细胞。在内皮细胞和内皮祖细胞的鉴别区分,使用CD34+/CD133+/KDR+鉴定为内皮祖细胞,同时使用内皮祖细胞吞噬D il-ac-LDLFITC-UEA双阳性的方法也可鉴定为内皮祖细胞。     

内皮祖细胞生物学特性及其进展

内皮祖细胞(Endothelial Progenitor Cells,EPCs)是血管内皮细胞的前体细胞,即能分化为成熟血管内皮细胞的祖细胞。随着对EPCs功能和影响其分化、生存、归巢和组织分布因素的了解,EPCs作为临床诊断、预后判断和治疗方法将有广阔的前景。本文就EPCs的的来源,EPCs的分离、培养、鉴定,EPCs的表面标志,EPCs的动员、分化和归巢等生物学特性及其进展展开综述。