Proteomic analysis shows differential protein expression in endothelial progenitor cells between healthy subjects and ischemic stroke patients

Abstract

Objective: An increase in the circulating concentration of endothelial progenitor cells (EPCs) is associated with a better outcome in patients with acute ischemic stroke. Likewise, EPCs are heterogeneous cells, with functional differences and different protein expressions. Our objective was to compare protein expressions of EPCs from ischemic stroke patients and healthy subjects.

Methods: Eleven ischemic stroke patients and 11 healthy subjects, matched by age and gender, were included in this study. EPC colonies were defined as early outgrowth colony forming unit-endothelial cell. Cells were lysed and proteins were purified and separated on two-dimensional gels. Gel images were analyzed using the PDQuest software and protein differences between EPCs from ischemic stroke patients and healthy subjects were identified by mass spectrometry. Results were finally validated by western blot.

Results: Proteomic analysis revealed three qualitative differences between EPCs from healthy subjects and ischemic stroke patients. Two of them, endoplasmatic reticulum protein-29 and CdC-42, were only expressed in EPCs from healthy subjects, whereas elongation factor-2 was only identified in EPCs from ischemic stroke patients. Furthermore, we identified one protein, peroxiredoxin-1, whose expression was 10 times stronger in ischemic stroke patients than in healthy subjects. Western blot analysis showed greater expression of endoplasmatic reticulum protein-29 in EPCs from healthy subjects and elongation factor-2 and peroxiredoxin-1 in EPCs from ischemic stroke patients.

Conclusion: Proteomic analysis showed differences in protein expressions of EPCs from ischemic stroke patients and healthy subjects that may be involved in mechanisms related to functional impairment.     

丁苯酞软胶囊治疗脑小血管病的临床研究

目的探讨丁苯酞软胶囊治疗脑小血管病的临床疗效。方法选取2016年3月—2018年2月在天津中医药大学第一附属医院就诊的脑小血管病患者100例,随机分为对照组(50例)和治疗组(50例)。对照组给予常规治疗,治疗组在此基础上口服丁苯酞软胶囊,2粒/次,3次/d,持续治疗2个月。观察两组患者临床疗效,同时比较治疗前后两组患者NIHSS、Fugl-Meyer量表、MBI评分,反应性充血指数(RHI)和内皮祖细胞(EPCs)数以及细胞因子水平。结果治疗后,对照组临床有效率为60.0%,显著低于治疗组的76.0%,两组比较差异具有统计学意义(P0.01)。治疗后,两组NIHSS评分显著降低,FMA-UE、FMA-LE、MBI评分显著升高,同组治疗前后比较差异具有统计学意义(P0.05、0.01);且治疗后治疗组上述评分均显著优于对照组,两组比较差异具有统计学意义(P0.05、0.01)。两组RHI、EPCs均较治疗前明显升高,同组治疗前后比较差异具有统计学意义(P0.05);且治疗后治疗组RHI、EPCs明显高于对照组,两组比较差异具有统计学意义(P0.05)。治疗后,两组患者血清ET-1、v WF水平均显著降低,NO水平显著升高,同组治疗前后比较差异具有统计学意义(P0.05、0.01);且治疗后治疗组上述血清细胞因子水平显著优于对照组,两组比较差异具有统计学意义(P0.05、0.01)。结论丁苯酞软胶囊能显著改善脑小血管病患者的运动功能和生活自理能力,增加外周血EPCs数目、有效调节ET-1/NO平衡,具有一定的临床应用价值。     

Regulation of human endothelial progenitor cell maturation by polyurethane nanocomposites

The mobilization and homing of endothelial progenitor cells (EPCs) are critical to the development of an antithrombotic cardiovascular prosthesis. Polyurethane (PU) with superior elasticity may provide a mechanical environment resembling that of the natural vascular tissues. The topographical cues of PU were maximized by making nanocomposites with a small amount of gold nanoparticles (AuNPs). The nanocomposites of PU-AuNPs (“PU-Au”) with a favorable response of endothelial cells were previously established. In the current study, the effect of PU and PU-Au nanocomposites on the behavior of human peripheral blood EPCs was investigated in vitro and in vivo. It was found that PU-Au promoted EPCs to become differentiated endothelial cells in vitro, confirmed by the increased expressions of CD31 and VEGF-R2 surface markers. The increased maturation of EPCs was significantly more remarkable on PU-Au, probably through the stromal derived factor 1α (SDF-1α)/CXCR4 signaling pathway. In vivo experiments showed that EPCs seeded on PU-Au coated catheters effectively reduced thrombosis by differentiation into endothelial cells. Surface endothelialization with CD31 and CD34 expression as well as intimal formation with α-SMA expression was significantly accelerated in the group receiving EPC-seeded PU-Au catheters. Moreover, the analysis of collagen deposition revealed a reduction of fibrosis in the group receiving EPC-seeded PU-Au catheters as compared to the other groups. These results suggest that EPCs engineered with a proper elastic substrate may provide unique endothelialization and antithrombogenic properties that benefit vascular tissue regeneration.     

流体剪切应力对晚期内皮祖细胞生物学功能的影响

目的 研究流体剪切应力处理对晚期内皮祖细胞（endothelial progenitor cells,EPCs）体外及体内生物学功能的影响。 方法 密度梯度离心法分离大鼠骨髓单核细胞,应用EGM-2MV进行体外培养。以3~4代的EPCs,即晚期EPCs为靶细胞,对其施以1.2 Pa剪切应力处理。采用EdU标记技术、黏附能力测定实验、改良的Boyden小室、Annexin V/PI、β 半乳糖苷酶检测法、Matrigel法、荧光定量RT PCR等方法分别检测剪切应力对晚期EPCs增殖、黏附、迁移、凋亡、衰老、体外成血管及VEGF mRNA表达等生物学功能的影响。应用大鼠颈动脉损伤模型及细胞原位移植等实验手段检测剪切应力预处理对晚期EPCs修复受损内皮的影响。结果1.2 Pa剪切应力处理可不同程度提高晚期EPCs的增殖、黏附、迁移及成血管能力（P＜0.01）,上调VEGF的基因表达,抑制晚期EPCs的衰老及凋亡（P＜0.01）;移植经剪切应力预处理的晚期EPCs可加速损伤内皮的修复,减缓内膜的增生。结论 流体剪切应力可改善晚期EPCs的功能活性,提高晚期EPCs修复损伤血管内皮的能力, 这为EPCs的临床应用及剪切应力介导的细胞疗法提供了实验依据。     

Kinase inhibitors for cardiovascular disease

Over the last decade, there has been substantial progress toward understanding the pathophysiology and treatment of cardiovascular diseases (CVDs). Elucidating cellular responses to the extracellular environment and signal transduction mechanisms have provided the opportunity to explore novel molecular therapeutic approaches for the treatment of CVDs. Neurohormonal stimulation has been implicated in these diseases; blockade of the renin-angiotensin and beta-adrenergic systems are examples of therapeutic effectiveness. There are multiple cell signaling cascades, some of which are beneficial or compensatory and others deleterious. The balance between these pathways, which in large part is dictated by the cellular environment, determines the outcome as a diseased or non-diseased state. Selective targeting of signaling pathways using protein kinase inhibitors, would have a potential advantage over receptor blockers. We review potential protein kinase targets and recent evidence supporting therapeutic interventional value in CVDs.     

基质细胞衍生因子-1对糖尿病患者外周血内皮祖细胞功能影响的研究

目的 观察基质细胞衍生因子-1 （SDF-1）对糖尿病患者外周血内皮祖细胞（EPCs）功能的影响. 方法 选择糖尿病患者（DM组）20例和健康体检者（NC） 10名,获取外周血单个核细胞,培养4 d后鉴定EPCs.将两组随机分为NC1、NC2、DM1和DM2亚组.NC1、DM1亚组采用SDF-1进行干预,第7天检测EPCs迁移能力和血管形成能力. 结果 （1）DM2亚组EPCs迁移和血管形成能力均低于NC2亚组[（15.500±2.224） vs （21.200±1.304）个,（113.870±15.198） vs （181.800±9.202） μm,P＜0.001];与NC2亚组比较,NC1亚组EPCs迁移能力和血管形成能力均增强[（24.600土1.517）vs（21.200±1.304）个,（197.980±10.855） vs （181.800±9.202） μm,P＜0.05];与DM2亚组比较,DM1亚组EPCs迁移能力和血管形成能力增强[（19.300±1.337） vs （15.500±2.224）个,（140.520±10.622）vs （113.870±15.198） μm,P＜0.001];（2）加入SDF-1干预后,DM组EPCs迁移能力和血管形成能力增强的幅度均高于NC组,比较差异有统计学意义（P=0.036,0.006）;（3） SDF-1与EPCs迁移能力及血管形成能力呈正相关（r=0.488、0.428,P=0.006、0.018）. 结论 SDF-1可增强外周血EPCs迁移能力和血管形成能力,对于糖尿病患者效果更为明显.     

BMSc联合淫羊藿苷预处理的EPCs移植对AMI大鼠心功能的影响

目的：探讨中药淫羊藿苷预处理的内皮祖细胞（EPCs）与骨髓间充质干细胞（BMSCs）共移植后,对急性心肌梗死（AMI）大鼠心功能的影响。方法：密度梯度离心法和贴壁筛选法培养、纯化EPCs与BMSCs;免疫细胞化学法（CD34／CD133／CD44）分别鉴定两种细胞。结扎大鼠左冠状动脉,制作大鼠急性心肌梗死模型;淫羊藿苷最佳药物浓度处理的EPCs,与BMSCs混合,在大鼠心肌梗死区周边分5点注射。大鼠超声心动仪测定左室功能。结果：细胞移植4周后,EPCs与BMSCs共移植组LVDd、LVDs分别为6．925±0．848与4．323±1．193,EF、CO分别为76．71±9．78和0．111±0．029,与对照组差异具有统计学意义。结论：淫羊藿苷预处理的EPCs联合BMSc的移植促进了AMI大鼠左室功能的恢复。     

靶向S1PR3基因shRNA慢病毒表达载体构建及功能鉴定

目的：构建靶向小鼠S1PR3基因的shRNA慢病毒表达载体,并验证其对内皮祖细胞迁移能力的影响。方法依照shRNA设计原则,合成对应的shRNA序列,将其克隆到PHBLV载体中,使用三载体系统进行病毒包装,收集制备成功的病毒,并用荧光显微镜法测定病毒滴度,使用病毒感染EPC后,测定各组细胞中S1PR3蛋白表达情况,选择合适的病毒感染EPC后,测定其对EPC迁移能力的影响。结果成功将shRNA序列克隆到慢病毒表达载体PHBLV-U6-RFP,表达质粒与辅助质粒共转染293细胞后48 h就可以看到细胞中成功表达红色荧光。收集培养成功病毒感染小鼠EPC,可以不同程度干扰细胞中S1PR3蛋白表达,S1PR3蛋白表达受到抑制后,EPC的迁移能力明显减弱。结论我们成功构建了可以有效干扰S1PR3表达的慢病毒载体,并初步观察了其对EPC细胞的迁移能力的影响,为后续进一步调控S1PR3相关的细胞功能的研究奠定了基础。     

姜黄素对兔颈动脉斑块稳定性及EPCs活力的影响

目的:观察姜黄素对颈动脉斑块稳定性及内祖皮细胞(EPCs)的影响。方法:将30只新西兰兔随机分为空白对照组、模型组及姜黄素组,各10只,其中空白对照组予普通饲料喂养,模型组予喂养高脂饲料,姜黄素组予喂养高脂饲料+100 mg/d姜黄素。连续喂养12周后HE染色法观察3组西兰兔主动脉内膜斑块厚度、外周血总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白(LDL-C)、高密度脂蛋白(HDL-C)水平以及利用MTT法测定人纤维EPCs细胞迁徙及增殖活性。结果:1)血脂水平:与空白组比较,模型组及姜黄素组TC、TG及LDL-C均升高(P0.05),HDL-C降低(P0.05),其中姜黄素干预后上述指标有明显改善(P0.05)。2)形态学变化:空白对照组颈动脉壁厚薄均匀,结构均匀,模型组及姜黄素均出现动脉管腔狭窄,动脉内膜增厚,其中姜黄素组较模型组改善。3)EPCs增殖情况:与空白组比较,模型组及姜黄素OD值均降低(P0.05),与模型组比较,姜黄素OD有所上调(P0.05)。4)EPCs迁徙能力:空白对照组各时间点划痕愈合率均显著高于其余2组(P0.05),其中姜黄素组各时间点划痕愈合率均高于模型组(P0.05)。结论:姜黄素由明显抗动脉粥样硬化作用,其作用机制可能与促进EPCs增殖及迁徙有关。     

人外周血内皮前体细胞体外增殖规律的动态观察及其特性

目的：探讨人外周血内皮前体细胞（endothelial progenitor cells EPCs）体外诱导分化为内皮细胞（ECs）的生长增殖规律及其特性，以期证实人外周血是临床治疗缺血性疾病一个理想的内皮前体细胞来源。方法密度梯度离心提取单个核细胞，接种在人纤连蛋白包被的培养板上，培养于含有促血管生长因子VEGF、b—FGF、IGF、EGF等的内皮生长基质EGM-2 MV中。每天倒置显微镜观察，并记录。4d后去除未附着细胞，继续培养黏附细胞，同时，黏附细胞用Dn—AC—LDL和FITC—UEA—1进行免疫荧光染色，荧光显微镜和共聚焦激光扫描显微镜观察。收集第7d的黏附细胞，流式细胞仪检测细胞表面标志CD34和CD31。结果接种1d后，一些细胞变形；2d后，有细胞团形成；3d后，细胞团周围一些贴壁细胞开始出现；4d后，黏附细胞呈短梭形或多角形贴壁生长，荧光显微镜、共聚焦激光扫描显微镜下可观察到Dn—AC—LDL和FITC—UEA-1双阳性的黏附细胞；第6d、7d，黏附细胞呈长梭形；FACS分析，CD34和CD31阳性率分别为（14．13±2．79）％、（54．67±3．44）％。结论外周血中确实存在EPCs，并且在促血管生长因子VEGF、b—FGF、IGF、EGF等的刺激下能分化为成熟的内皮细胞。EPCS可望作为临床血管再生的细胞治疗