内皮祖细胞与脑动脉粥样硬化相关性及脂联素提高内皮祖细胞活性的机制研究
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摘要
动脉粥样硬化是目前危害人类健康的主要病变之一,目前多项研究提示血管内皮功能障碍是动脉粥样硬化的起始步骤,且在动脉粥样硬化的发展中起着至关重要的作用。目前多项研究亦发现内皮祖细胞(Endothelial progenitor cells, EPCs)在动脉粥样硬化发生发展中起着重要作用,如促进损伤内皮修复等,Walter等研究发现血循环中的内皮祖细胞具有向球囊损伤血管部位归巢的特点,大量证据表明EPCs水平与血管危险因素有较强的相关性,甚至优于Framingham风险评分,临床研究也证实,机体内循环EPCs数量的增加和功能的改善对冠心病、心力衰竭等心血管疾病的治疗有重要意义。然而动脉粥样硬化的危险因素如高龄,高血压,高胆固醇血症,糖尿病等,都会导致机体内的EPCs数量减少和影响的EPCs活性。
脂联素主要是由白脂肪组织合成的分子量是30kDa蛋白,其具有胰岛素增敏、抗动脉粥样和抗炎特性。促进血管内皮祖细胞一氧化氮的产生以及减轻活性氧对血管内皮细胞的损伤。总之,脂联素通过激活多种细胞内信号级联反应在血管保护中发挥着举足轻重的作用。脂联素在循环中主要有三个亚型：低分子量的三聚体,中等分子量的六聚体和高分子量的多聚体。这三种亚型各有不同的功能,其中研究表明高分子量是主要的活性蛋白。在内皮细胞上主要有两种脂联素受体,即R1和R2受体。内皮是脂联素的主要靶点,脂联素通过其在内皮细胞上的受体可以抑制单核细胞粘附到内皮细胞,并最终抑制血管平滑肌的迁移和增殖,这有助于减轻血管损伤。
骨髓和外周血的内皮祖细胞是单核细胞前体细胞,能够分化成成熟的内皮细胞,当血管内皮受损时内皮祖细胞通过归巢定位到损伤部位加速再内皮化,促进血管修复,在维持血管内皮完整性,修复受损血管内皮细胞,促进新生血管形成及组织修复等方面起重要作用。EPCs主要来源于骨髓、外周血及脐血,脂肪中也存在少量的EPCS。外周血循环中EPCs的数量占外周血细胞总数的0.01%,占白细胞总数的0.05%,而骨髓中EPCs的含量是外周血的500倍。EPCs经药物刺激后可以提高至占白细胞总数的6%。EPCs促进血管的生成及修复的机制可能有两种：(1)直接分泌VEGF等细胞因子,通过旁分泌的方式促进局部缺血组织的血管新生；(2)通过归巢效应、整合于受损的血管,直接分化为内皮细胞,参与血管新生。
动脉粥样硬化的血管重塑及病理生理涉及多种细胞类型和级联反应。值得注意的是,PI3K/Akt信号通路是参与这些级联反应中的部分环节。磷酸肌醇3-激酶(PI3K)通过不同类型的膜受体(包括G-蛋白耦合受体和酪氨酸激酶受体)激活蛋白质和脂质。PI3K和Akt是内皮型一氧化氮合酶(eNOS)的重要的正调节子,通过L-精氨酸的依赖NADPH氧化产生NO。PI3K/Akt通路与内皮细胞功能,如调节血管张力等密切相关。
本研究旨在探讨内皮祖细胞降低是否是动脉硬化的危险因素?与脑动脉粥样硬化狭窄病变血管数量和程度的相关性,进一步探讨PI3K/Akt信号通路在脂联素促进内皮祖细胞增殖和迁移中的作用。
第一章内皮祖细胞与脑动脉粥样硬化程度的相关性研究
目的：
中风仍然是成人死亡和长期残疾的主要原因,而缺血性中风约占这些中风的87%。脑动脉粥样硬化疾病是缺血性中风的主要病因,血管内皮功能障碍在其发生和发展上占主导作用。动脉粥样硬化早期的一个重要特点是内皮功能障碍,而内皮功能障碍主要表现为内皮细胞逐渐丧失,促进斑块的发生和发展。但成熟的内皮细胞再生能力有限。因此,越来越多的证据表明血管内皮祖细胞(EPCs)可以向血管内皮层归巢、修复损伤的内皮并维护血管内皮功能,这是通过内皮祖细胞介导的新的内源性血管修复系统来实现的。
最近的研究表明,内皮祖细胞能够归巢到受损的内皮细胞,分化为成熟的内皮细胞和替换受损的内皮细胞,从而发挥抗动脉硬化的作用。因此,循环内皮祖细胞的数量是心血管健康状况的标志。最近的研究表明,循环内皮祖细胞的数目减少与不同程度的心血管疾病的危险因素密切相关,如吸烟、糖尿病、高脂血症和高血压等。Werner等研究发现内皮祖细胞水平与冠状动脉粥样硬化性疾病和外周闭塞性血管的严重程度密切相关。然而,脑动脉粥样硬化的严重性和内皮祖细胞水平之间的关系还未知。在此背景下,本研究试图探讨内皮祖细胞与脑动脉粥样硬化严重程度之间的关系,以及EPCs与传统的心血管危险因素(年龄,性别,吸烟,高脂血症,高血压,糖尿病,家族史)相关的程度。最后,进一步评估内皮祖细胞对脑动脉粥样硬化严重程度的预测价值是否优于传统的危险因素。
方法：
1.入组南京军区南京总医院2011年12月至2013年12月之间的接受全脑血管造影的患者,并获取人口统计学和基线资料。所有患者均接受常规心电图、心脏超声检查及数字减影血管造影(DSA)等检查。神经功能缺损采用NIHSS评分,心血管危险因素综合评分和下肢动脉硬化程度判断分别采用佛明汉评分(Framingham risk score, FRS)和踝肱指数(Ankle-brachial index, ABI)。
2.动脉硬化负荷评估：按照动脉硬化病变的位置分为单纯颅内动脉粥样硬化性狭窄(intracranial atherosclerotic stenosis, ICS)组、单纯颅外动脉粥样硬化性狭窄(extracranial atherosclerotic stenosis, ECS)组、(combined intracranial and extracranial atherosclerotic stenosis, CCS)组。颅外狭窄是使用北美症状性颈动脉内膜切除术试验(NASCET)标准计算,而颅内动脉狭窄(ICS)参考WASID标准。动脉粥样硬化负荷分别从两方面进行评估：1、数量：计算动脉硬化病变血管节段的数量(即狭窄≥50%的血管节段的总数目)；2、按照脑动脉粥样硬化狭窄程度来评估,0分代表<50%的狭窄,1分代表50%至99%的狭窄和血管闭塞是2分。在一个动脉段有多个病灶存在的情况下,按照最严重狭窄来计算。
3.统计方法：采用SPPS16.0软件进行统计学分析。计量资料以均数±标准差(x±s)表示。对连续变量采用独立样本t检验、Mann-Whitney U检验或方差分析,对于分类变量采用卡方检验或Fisher分类变量的精确检验。单因素分析P<0.2的变量做为候选入多因素logistic回归分析。动脉硬化严重程度和内皮祖细胞水平的数量之间的相关采用Spearman秩和相关分析。内皮祖细胞数量和动脉钙化或CAD严重程度之间的相关性进行了评估用多变量logistic回归。P<0.05为差异具有统计学意义。
结果：
1.基线资料特征
入组患者的年龄均值为60岁,29.4%的女性和12.2%无脑动脉粥样硬化者作为对照组。单纯颅外动脉狭窄54例(27.4%),单纯颅内动脉狭窄45例(22.8%). CD34+/CD309+, CD133+/CD309+和CD34+/CD133+/CD309+细胞中位值分别为0.172%(四分位距为0.066%至0.370%),0.131%(四分位距为0.041%至0.297%)和0.024%(四分范围,分别为0.005%至0.063%)。
2.内皮祖细胞与脑动脉粥样硬化负荷负相关
校正混杂因素(如年龄、吸烟、高血压、收缩压和纤维蛋白原)后,内皮祖细胞水平与脑动脉粥样硬化之间有统计学差异(CD45-/dimCD34+CD309+cells: B=-2.197, OR=0.111,95%CI:0.024to0.511, P=0.005; CD45-/dimCD133+CD309+cells:B=-2.172, OR=0.114,95%CI:0.023to0.556, P=0.007; CD45-/dim CD34+CD133+CD309+cells:B=-4.580, OR=0.010,95%CI:0.000to0.541,P=0.024)。进一步多元logistic辑回归显示,只有内皮祖细胞计数与颅外及混合脑动脉粥样硬化有统计学差异。然而,内皮祖细胞与颅内动脉粥样硬化无统计学差异(P>0.05)。
3.脑动脉粥样硬化负荷与EPCs和佛明汉评分相关性
受试者按动脉粥样硬化得分的三分位间距分为：低、中和高组。我们对EPCs的三种亚型通过顺序逐步回归分析提示,纤维蛋白原和高血压是脑动脉粥样硬化的危险因素,而皮祖细胞是保护因素。斯皮尔曼等级相关分析来分析动脉硬化负荷和EPCs的相关性：在78位颅外动脉硬化与对照组间相关分析提示CD133+/CD309+细胞与动脉粥样硬化负荷之间相关性有统计学差异(病变血管段数量和动脉硬化狭窄度评分：Spearman rho=-0.195和-0.197,P<0.05)。在69例颅内狭窄患者中CD133+/CD309+细胞与脑动脉粥样硬化负荷相关性有统计学差异(病变血管段数量：Spearman rho=-0.262, P=0.030;动脉硬化狭窄度评分：Spearman rho=-0.248, P=0.040)此外,在调整可能的混杂因素影响后二者相关性仍有统计学差异。然而,动脉粥样硬化的负荷与FRS、另外两个内皮祖细胞亚群之间相关性无统计学差异。
结论：
1.在校正年龄、吸烟、高血压、收缩压、纤维蛋白原和佛明汉评分后内皮祖细胞各个亚型计数与脑动脉粥样硬化相关性有统计学差异。
2.内皮祖细胞计数与颅外及混合脑动脉粥样硬化相关性有统计学差异,而与颅内动脉硬化无统计学差异。
第二章骨髓来源内皮祖细胞的分离、培养及鉴定
目的：
EPCs作为内皮细胞的前体细胞,它具有向内皮损伤部位分定向归巢并分化为成熟内皮细胞及分泌内皮生长因子等提高周围内皮细胞功能,在维持血管内皮完整性,修复受损血管内皮细胞,促进新生血管形成及组织修复等方面起重要作用,EPCs主要来源于骨髓、外周血,而骨髓中EPCs的含量是外周血的500倍。建立由C57BL/6小鼠骨髓单个核细胞诱导分化EPCs的培养体系。
方法：
选用4-6周龄的C57BL/6小鼠,PBS无菌条件下冲洗骨髓腔获得细胞悬液。使用淋巴细胞分离液经密度梯度离心法离心获得骨髓单核细胞层,收集骨髓单核细胞,用PBS洗涤后接种于由人纤维连接蛋白包被的细胞培养板中。细胞培养基选用EGM-2SingleQuots套装,培养条件为37℃,5%C02。接种后第4天首次更换培养液,去除未贴壁细胞。此后,每两天更换一次细胞培养液。细胞培养至第21天。
每日于光学倒置显微镜下观察细胞形态及生长状况,并拍摄照片。本研究选用培养至第21天的EPC进行细胞鉴定。使用免疫荧光法鉴定培养细胞表达EPC特异性表面抗原Sca-1及VEGFR2(CD309)'情况。使用流式细胞仪检测培养细胞双表达Sca-1及VEGFR2比例。同时,检测细胞吞噬DiI标记的乙酰化低密度脂蛋白(Dil-Ac-LDL)能力及结合异硫氰酸荧光素标记的荆豆凝集素能力(FITC-UEA-1)。
结果：
首次换液后,贴壁细胞生长加速,渐出现细胞集落。集落形态为中央小圆形细胞聚集,周围梭型细胞呈放射状生长状态。在细胞培养过程中,我们还观察到细胞可呈现线状、管状生长排列,提示该细胞具有成血管能力。大约2周后的细胞形态趋于一致,呈现“铺路石”样生长状态。
流式细胞仪检测结果也显示,本实验中培养至3周后的EPCs, CD34及VEGFR2双阳性率达到85%以上。培养细胞具备吞噬Dil-Ac-LDL和FITC-UEA-1的能力。
结论：
1.以上实验结果表明,本研究所采用密度梯度离心后贴壁筛选培养的方法可以从C57BL/6小鼠骨髓中成功分离培养EPCs。
2.在培养过程中,细胞呈“集落样”生长和“铺路石”样外观,并可见细胞呈现线、管状排列生长。且流式细胞检查及细胞功能检查均证明了所培养的细胞为内皮祖细胞。为后续的脂联素干预实验奠定了实验技术基础。
第三章脂联素提高内皮祖细胞增殖和迁移活性及其机制探讨
目的：
脂联素是一种由白色脂肪组织分泌的分子量30kDa蛋白。脂联素对血管保护的关键作用通过激活多种细胞内的多种信号途径。例如增强内皮细胞一氧化氮的生成和清除氧自由基。循环血液中低脂联素水平被认为是一种新的冠心病的独立危险因素,低脂联素血症将会导致血管内皮细胞功能紊乱。EPCs是一类能增殖并分化为血管内皮细胞的前体细胞,同时具有内皮细胞和祖细胞的一些生物学特性,EPCs是否也表达有脂联素受体,脂联素对EPCs的增殖和迁移功能有无影响及其具体的机制如何目前尚不清楚。
因此,我们提出假设：脂联素可能会影响EPCs的数量和功能,增强损伤内皮的修复能力,延缓动脉硬化病变的发生和发展。基于该假设我们拟采用第二部分成功建立的EPCs培养体系,初步探索脂联素是否影响EPCs的增殖和迁移活性及其可能的作用机制。
方法：
1.先做EPCs的增殖曲线,自C57BL/6、鼠骨髓提取骨髓单核细胞,按照不同的浓度接种到培养板中,以细胞计数试剂盒行细胞计数并绘制各组细胞生长曲线,检测不同接种浓度的EPCs的增殖活性；
2.进一步探索不同剂量的脂联素对EPCs增殖活性和迁移能力的影响情况,自C57BL/6小鼠骨髓提取骨髓单核细胞,计数后分为3组接种于培养板中。分别为：对照组、脂联素处理组(培养液中脂联素浓度分别为1和10ug/ml),以细胞计数试剂盒行细胞计数和Transwell做迁移能力测试；Western blot法检测各组细胞中Akt、p-Akt、p-eNOS. eNOS表达情况；
3.自C57BL/6小鼠骨髓提取骨髓单核细胞,计数后分为6组接种于培养板中。分别为对照组、脂联素(1ug/ml)组、NO供体组(sodium nitroprusside, SNP)、SNP+脂联素(1ug/ml)组、eNOS抑制剂组(L-NAME)和L-NAME+脂联素组。以细胞计数试剂盒行细胞计数了解各组细胞增殖能力；Transwell做迁移能力测试；Western blot法检测各组细胞蛋白Akt、p-Akt、eNOS和p-eNOS表达情况；NO检测试剂盒检测各组上清液中NO的表达量。
4.提取小鼠骨髓单核细胞后诱导分化为EPCs,分为4组接种于培养板中。分别为对照组、脂联素(1g/ml)组、LY294002组(Akt抑制剂)和LY294002+脂联素(1ug/ml)组。以细胞计数试剂盒行细胞计数了解各组细胞增殖能力；Transwell做迁移能力测试；Western blot法检测各组细胞蛋白Akt、p-Akt、eNOS和p-eNOS表达情况。
5.采用SPPS16.0软件进行统计学分析。计量资料以x±s表示。多组间细胞增殖能力、迁移能力及NO、Akt、p-Akt、eNOS和p-eNOS等各指标表达水平比较采用one-way ANOVA。P<0.05为差异具有统计学意义。
结果：
1.不同接种浓度的单核细胞的增殖曲线
接种约5-11X105cells/cm2单核细胞,培养2周左右细胞达到增殖高峰期,因此,用该浓度的单核细胞接种2周后即可用于下一步实验。
2.不同剂量的脂联素对EPCs增殖活性和迁移能力的影响
脂联素作用后EPCs的数量增加,该作用随脂联素剂量的增加而增强,呈量效依赖性。脂联素(1ug/ml)作用3天EPCs增殖活性和迁移能力既有统计学差异,且Western blot检测结果显示：Akt、p-Akt、p-eNOS、eNOS表达量脂联素组较对照组有统计学差异(P<0.05)。
3. eNOS抑制剂可以抑制脂联素对EPCs的作用
p-eNOS表达量增高可提高脂联素对EPCs的促增殖、迁移作用,但可被eNOS抑制剂L-NAME抑制。脂联素作用3天后脂联素组、SNP组、SNP+脂联素(1ug/ml)组较对照组细胞数量增加、细胞迁移能力提高和上清液NO表达量增高；Western blot检测结果显示：p-Akt、eNOS、p-eNOS表达量增高。L-NAME和L-NAME+脂联素组则与上述结果相反,细胞增殖、迁移受到抑制,p-Akt、eNOS、p-eNOS、NO表达量较对照组并无增高。
4.磷脂酰肌醇3激酶(PI3K)特异性抑制剂LY294002抑制脂联素对EPCs的作用
脂联素对EPCs的促增殖、抗凋亡作用与p-Akt、p-eNOS表达情况可被磷脂酰肌醇3激酶(PI3K)特异性抑制剂LY294002抑制。脂联素作用3天后脂联素(1ug/ml)组较对照组细胞数量增加、细胞迁移能力提高以及p-Akt、eNOS、 p-eNOS表达量增高,但与LY294002组和LY294002+脂联素组比细胞数量降低、细胞迁移能力降低以及p-Akt、eNOS、p-eNOS表达量降低(P<0.05)。
结论：
1.脂联素促进EPCs的增殖和迁移活性,该作用有一定的时间和剂量依赖性；
2.脂联素通过PI3K/Akt信号通路促进了EPCs的增殖和迁移活性。
Atherosclerosis is one of the major diseases threatening the human health, at present accumulating evidence suggests that endothelial dysfunction is an early step in the atherosclerotic process and plays a pivotal role in the development and progression of atherosclerosis. And several studies have also found that endothelial progenitor cells (EPCs) play an important role in the development of atherosclerosis, such as promoting the regeneration of damaged endothelium. Walter et al demonstrated that circulating endothelial progenitor cells has the characteristics of homing to denuded parts of the artery after balloon injury, a body of evidence is available demonstrating that EPC levels are very strong vascular risk markers, even better than the Framingham risk score. Most of the clinical studies also confirmed that increased number and function of EPCs may have important therapeutic implications for the management of cardiovascular disease, such as coronary heart disease, heart failure etc. Traditional risk factors such as elderly, hypertension, hyperlipidemia, and diabetes are major causes of reducing the number and function of circulating EPCs
Adiponectin is a30-kDa protein synthesized predominantly by white adipose tissue, which has insulin-sensitizing, antiatherogenic, and anti-inflammatory properties. Enhancement of nitric oxide generation and attenuation of reactive oxygen species production in endothelial cells along with stimulated endothelial progenitor cells (EPCs) survival, proliferation and differentiation constitute some of adiponectin's vasoprotective actions. Adiponectin exerts a pivotal role in vascular protection through activation of multiple intracellular signaling cascades and exists in the circulation in three isoforms:a low molecular weight trimer, a medium molecular weight hexamer, and a high molecular weight (HMW) multimer. The3isoforms might play different roles, but some arguments have suggested that the HMW isoform might be the major active protein. Both AdipoR1and AdipoR2, two receptors for adiponectin, are expressed in endothelial cells. The endothelium is a major target of adiponectin, where the adiponectin downregulates adhesion molecules, inhibits monocytes adhesion to endothelial cells and ultimately inhibits the migration and proliferation of vascular smooth muscle cells, which contribute to vascular damage.
Endothelial progenitor cells from bone marrow or peripheral blood are mononuclear precursor cells that can differentiate into mature endothelial cells. During wound repair EPCs home to sites of vascular injury where these cells can restore vascular integrity, and play a significant role in maintaining vascular endothelial integrity, repairing the damaged endothelial cells, promoting new blood vessel formation and tissue repair, etc. EPCs are mainly isolated from peripheral, umbilical cord, and bone marrow. There are also a small number of EPCs in adipose tissue. The number of immature progenitors from bone marrow is500-fold higher compared to peripheral blood. Drug-induced EPCs can be increased to6%of the total number of white blood cells. EPCs can remarkably promote new blood vessel formation and repair damaged vessels, the mechanisms underlying this phenomenon are likely to be explained in two ways:(1) EPCs improve neovascularization of ischemic tissues by secretion of cytokines such as vascular endothelial growth factor (VEGF) and so on.(2) EPCs have been found to directly incorporate into injured vessels and that participate in angiogenesis and reconstruction by differentiation into endothelial cells.
The atherosclerotic vascular remodelling and pathophysiology involve multiple cell types and a wide array of mediators and cascades. Of note, the PI3K/Akt signalling pathway impinges on several of them. Phosphoinositide3-kinases (PI3Ks) are protein and lipid kinases activated by G protein-coupled receptors and tyrosine kinase receptors. PI3K and Akt are important positive regulators of endothelial nitric oxide synthase (eNOS), which generates NO through the NADPH-dependent oxidation of L-arginine. The PI3K/Akt related pathways have a key role in regulating vascular tension, etc.
With this background, this study sought to investigate whether the reduction in EPCs numbers is an independent risk for arteriosclerosis, the relationships between numbers of EPCs with cerebral atherosclerotic severity, and further investigate whether the activation of PI3K/Akt is involved in adiponectin promoting endothelial progenitor cell proliferation and migration.
Chapter I Correlation of Different Subpopulations of Endothelial Progenitor Cells with Cerebral Arteriosclerosis Severity
Objective:
Stroke remains a leading cause of death and long-term disability in adults' worldwide, ischemic strokes (IS) account for approximately87%of these strokes. Cerebral atherosclerosis (CA) is one of the main causes of this type of obstruction and endothelial dysfunction also plays a critical role in its onset and progression. Endothelial dysfunction is characterized by a progressive loss of endothelial cell (ECs) and thought to be an important early event in the atherosclerotic disease process. It contributes to plaque initiation and progression. Mature ECs possess limited regenerative capacity. Thus, growing evidence suggests endothelial progenitor cells (EPCs), endothelial stem or precursor cells, may contribute to the restoration of the endothelial lining after injury and maintenance of endothelial function, which is a novel endogenous vascular repair system mediated by EPCs.
Recent studies have indicated that EPCs are immature cells capable of homing to the injured endothelium, differentiating into mature endothelial cells and replacing the injured endothelial cells, thereby exerting a protective effect against atherosclerosis. The number of circulating EPCs is now considered a mirror of cardiovascular health. Recent studies suggest that reduced number of circulating EPCs was closely related to the degree of endothelial dysfunction in humans at various degrees of cardiovascular risk, smoking, diabetes, hyperlipidemia; and hypertension. Werner et al. also identified levels of EPCs correlate inversely with the severity of several atherosclerotic vascular diseases such as coronary artery disease and peripheral occlusive vascular disease. However, the relationship between the severity of the CA and EPCs levels is not known. With this background, this study sought to investigate the relationships between the number of EPCs and the CA severity. We also examined the degree to which EPCs count were associated with traditional cardiovascular risk factors (age, gender, smoking, hyperlipidemia, hypertension, diabetes, and family history). Finally, we assessed whether EPCs provided significant diagnostic value beyond traditional risk factors for estimating CA severity.
Methods:
1. All subjects were enrolled from the consecutive inpatients who underwent cerebral angiography in the Department of Neurology at Jingling Hospital between December2011and December2013. They were thoroughly investigated for demographics and baseline clinical characteristics. All patients underwent conventional cardiac ultrasound and electrocardiogram examination. Neurological deficits were estimated using the National Institutes of Health Stroke Scale (NIHSS) score by two neurologists, who were blind of digital subtraction angiography (DSA) images and EPCs assay results. The Framingham risk score (FRS) was assessed at baseline for each subject's cardiovascular risk factors, and the Ankle-brachial index (ABI) is used to confirm the diagnosis and determine the severity of peripheral arterial disease (PAD).
2. Cerebral arteriosclerosis burden assessment:Locations of lesion were categorized as being in the intracranial, extracranial vessels or the combined extracranial and intracranial stenosis (IES). Intracranial lesions were defined as those at or above the precavernous or upper petrous carotid artery in the internal carotid artery. For the vertebral artery, the distinction was made at the point where the artery takes a turn and comes into the foramen magnum piercing the dura. Extracranial stenosis (ECS) was calculated using North American Symptomatic Carotid Endarterectomy Trial (NASCET) criteria; and intracranial stenosis (ICS) was measured by using Warfarin-Aspirin Symptomatic Intracranial Disease (WASID) criteria. The presence of arterial stenotic lesions was determined by the number of cerebral arteries showing steno-occlusion and the degree thereof. The extent of atherosclerotic burden (cerebral atherosclerotic burden, CAB) was graded according to the previous criteria (0indicating<50%stenosis,1indicating50%to99%stenosis, and2indicating occlusion). In the presence of multiple lesions within one arterial segment, data from the most significant stenosis were used. The total number of arterial segmental lesions ((≥50%stenosis) was also calculated (cerebral atherosclerotic number, CAN).
3. Statistical analysis was performed using SPSS software, version16.0(SPSS Inc., Chicago, IL). Comparisons were performed using independent sample t tests, Kruskal-Wallis tests, Mann-Whitney U tests, or analysis of variance for normal distributed continuous variables, and the chi-square test or fisher's exact test for categorical variables, as appropriate. Variables with a P value<0.2by univariate analysis were candidates for inclusion in multiple logistic regression analysis. A selection process was used to develop the final multivariable model, and adjusted odds ratios (OR) and95%confidence intervals (CI) were calculated as estimates of relative risk. Moreover, the relationship between number of arterial segmental lesions and EPCs levels was determined by Spearman rank test. Associations between EPCs level and arterial calcification or CA severity were assessed with logistic regression. A two-tailed P value<0.05was considered statistically significant.
Results:
1. Patients'Characteristics
The baseline characteristics of the197patients enrolled in this study are provided in Table1. Fifty-four patients (27.4%) had ECS and45patients (22.8%) had ICS. Mean age was60years, with29.4%women and12.2%no significant CA as a control group. The median values of were CD34+CD309+, CD133+CD309+and CD34+CD133+CD309+cells were0.172%(interquartile range,0.066%to0.370%),0.131%(interquartile range,0.041%to0.297%) and0.024%(interquartile range,0.005%to0.063%), respectively, in the total sample.
2. Circulating EPCs Are Reduced in the Presence of CA
In binary logistic regression analysis, the only EPCs level was significantly conversely associated with the prevalence of CA after adjustment for age, smoking, hypertension, SBP, fibrinogen and FRS (CD45-/dimCD34+CD309+cells:B=-2.197, OR=0.111,95%CI:0.024to0.511, P=0.005; CD45-/dimCD133+CD309+cells: B=-2.172, OR=0.114,95%CI:0.023to0.556,p=0.007; CD45-/dim CD34+CD133+CD309+cells:B=-4.580, OR=0.010,95%CI:0.000to0.541, P=0.024). The result of multivariate logistic regression showed that only EPCs count was significantly negatively associated with the location of CA (extracranial or combined extra-and intra-cranial artery stenosis). However, EPCs were not significantly associated with intracranial atherosclerosis (P>0.05).
3. Relationship of CA with EPCs, Calcification Score or Framingham Risk Score
The subjects were divided into three groups on the basis of their atherosclerosis score:low, intermediate and high group. In the ordinal regression analysis CA burden remained correlated conversely to EPCs and associated positively with fibrinogen and hypertension by controlling for confounders. We use Spearman's rank correlation analysis to analyze the correlation between CA burden and EPCs levels, there was also a significant inverse relationship between CD133+CD309+cells count and atherosclerotic burden in78patients with ECS and control subjects (CAN and CAB: Spearman rho=-0.195and-0.197, P<0.05). Additionally, the atherosclerotic burden was inversely related to CD133+CD309+cells count in69patients with ICS (CAN: Spearman rho=-0.262, P=0.030; CAB:Spearman rho=-0.248, P=0.040). There are still significant differences between CA burden and EPCs levels after adjustment for potential confounders. However, the atherosclerotic burden was not related to FRS or the other two progenitor cell subsets.
Conclusions:
1. EPCs level was significantly conversely associated with the prevalence of CA after adjustment for age, smoking, hypertension, SBP, fibrinogen and FRS.
2. There was a significant positive correlation between EPCs and atherosclerotic burden in patients with ECS. However, there was no statistically significant relationship of the atherosclerotic burden with EPCs in patients with ICS.
Chapter II Isolation, culture and characterization of bone marrow derived EPCs
Objective:
EPCs, endothelial precursor cells, are mobilized early after vascular injury. They can home to sites of vascular injury and differentiate into mature endothelial cells, and avidly secrete angiogenic factors encouraging resident endothelial cell proliferation and migration. EPCs may play a critical role in re-establishing the endothelial integrity, repairing damaged endothelial cells, promoting angiogenesis in tissue repair. EPCs can be mainly isolated from peripheral blood or bone marrow, which is500-fold higher compared to peripheral blood. We try to establish a culture system to induce the differentiation of C57BL/6mouse bone marrow-derived mononuclear cells into EPCs.
Methods:
Bone marrow of tibia and fibula from C57BL/6mice (4-6week old, male) was washed out with PBS and eentrifugated with Histopaque1077at400g for30minutes. The bone marrow mononuclear cells (BMMNCs) were re-suspended in EGM-2SingleQuots and washed twice. The BMMNCs were then seeded in the cell culture flasks coated with human fibronectin and cultured with EGM-2SingleQuots at37℃5%CO2. Nonadherent cells were removed4days after seeding and the culture medium was replaced every other day thereafter. EPCs cultured up to3weeks were used for later cell characterization and transplantation.
Cultured cells were observed and photographed by inverted microscope everyday. EPCs cultured up to3weeks were chosen for cell characterization. We identified the proportion of cultured cells which co-expressed CD34and VEGFR2by flow cytometer. Meanwhile, we also tested the abilities of of uptaking Dil-Ac-LDL and binding FITC-UEA-1of cultured cells.
Results:
After the first exchange of medium to remove adherent cells were growing rapidly. Then cell colonies began to appear on the5th day of culture. Colony morphology is small round cell aggregation center, surrounded by radial growth of cells. In cell culture, we also observed that cells can be present in linear and tubular growth, which suggested that the cells have the ability to grow new blood vessels. Since about2weeks of culture, the cells gradually changed to a cobblestone-like appearance.
After3weeks culture, cells were verified as EPCs by flow cytometer. And the result exhibited that more than85%of the cultured cells co-expressed CD34and VEGFR2. Finally, by testing the abilities of uptaking Dil-Ac-LDL and binding FITC-UEA-1of cultured cells, we found most cultured EPCs were able to uptake DiI-Ac-LDL and bind FITC-UEA-1.
Conclusions:
1. EPCs were separated from bone marrow with density gradient centrifugation, wall sticking screening and amplified in vitro. Our method of culturing EPC from BMMNCs of C57BL/6mice is feasible.
2. We observed a line-like and a tube-like appearance during the cell culture which indicating the vascular forming potential of cultured EPCs. The results of FACS and cell function tests all indicated that the cells we cultured were EPCs. This part of study provides the technological foundation for the later experiments.
Chapter Ⅲ Effects of Adiponectin on proliferation and migration activities of endothelial progenitor cells and its possible mechanism
Objective:
Adiponectin is a30-kDa protein synthesized predominantly by white adipose tissue. Adiponectin exerts a pivotal role in vascular protection through activation of multiple intracellular signaling cascades. Enhancement of nitric oxide generation and attenuation of reactive oxygen species production in endothelial cells along with stimulated endothelial progenitor cells (EPCs) survival, proliferation and differentiation constitute some of adiponectin's vasoprotective actions. Circulating adiponectin level is considered to be a new independent risk factor for coronary heart disease, hypoadiponectinemia will lead to vascular endothelial dysfunction. EPCs are the precursor cells that can proliferate and differentiate into endothelial cells and have some biological properties of endothelial cells and progenitor cells. Whether EPCs express adiponectin receptors, the effect of adiponectin on the proliferation and differentiation of EPCs and the possible mechanism are unclear.
Adiponectin promotes endothelial progenitor cell number and function, enhances the ability to repair damaged endothelium and delays the occurrence and development of atherosclerotic lesions. With this background, we intend to use EPCs culture system successfully established in the second part, explore preliminarilly effects of Adiponectin on proliferation and migration activities of endothelial progenitor cells and its possible mechanism.
Methods:
1. We intend to study cell growth curves of EPCs. Bone marrow mononuclear cells (BMMNCs) were harvested from C57BL/6mice, and plated on fibronection-coated culture dishes in different concentrations. Cell growth curves of each group were drawn with CCK-8assay, which was used to determine the proliferation of the cultured EPCs in different inoculation concentration.
2. To investigate the effects of adiponectin with different doses on proliferation and migration of EPCs. Obtain required amount of EPCs which were extracted from the bone marrow of C57BL/6mice, counted and seeded on the culture flasks. Namely, Control group and adiponectin-treated group (cell culture medium was supplemented with1and10ug/ml adiponectin respectively). EPCs'differentiation and migration were assessed with the CCK-8assay and Transwell respectively. The expression of Akt, p-Akt、p-eNOS、eNOS were measured by Western blot.
3. Bone marrow mononuclear cells (BMMNCs) were harvested from C57BL/6mice and divided into6groups for cell culture. The cell groups were as follows: control group, adiponectin treatment groups (cell culture medium was supplemented with lug/ml adiponectin), NO donor group (sodium nitroprusside, SNP), SNP and adiponectin treatment group (cell culture medium was supplemented with SNP and lug/ml adiponectin), eNOS inhibitor group (cell culture medium was supplemented with L-NAME), L-NAME and adiponectin treatment group (cell culture medium was supplemented with L-NAME and1ug/ml adiponectin). The expression of nitric oxide in supernatant was measured by nitric oxide assay kit.
4. Mouse BMMNCs were induced to differentiate into EPCs and divided into4groups for cell culture. The cell groups were as follows:control group, adiponectin treatment groups (cell culture medium was supplemented with lug/ml adiponectin), LY294002group (Akt inhibitor), LY294002and adiponectin treatment group (cell culture medium was supplemented with LY294002and lug/ml adiponectin). EPCs' differentiation and migration were assessed with the CCK-8assay and Transwell respectively. The expression of Akt、p-Akt、p-eNOS、eNOS were measured by Western blot.
5. Statistical analysis was performed using SPSS software, version16.0(SPSS Inc., Chicago, IL). Data are expressed as the mean value±standard deviation. Comparisons among more groups (such as levels of cell proliferation, migration, and NO, Akt, p-Akt, eNOS and p-eNOS, etc.) were performed by the analysis of variance. Statistical significance was accepted if the null hypothesis could be rejected at P≤0.05.
Results:
1. Monocyte proliferation curve in different inoculums concentrations
Monocytes cultured cells were seeded at a density of about5-11×105cells/cm2and reached its peak proliferation at2weeks. Thus, the concentration of mononuclear cells was used in the next experiment after2weeks of culture.
2. Different doses of Adiponectin influence the proliferation and migration of EPCs
Adiponectin inereased the number of EPCs with dose and time dependence. Adiponectin (lug/ml) signifieantly inereased proliferation and migration activities of EPCs after3days, Western blot analysis showed that there was statistically significant difference between adiponectin group and control group for Akt, p-Akt, p-eNOS and eNOS expression respectively (P<0.05).
3. The eNOS-inhibitor could effectively inhibit the effect of adiponectin on EPCs
Adiponectin increased the ability of migration and proliferation on cultured EPCs. And the properties revealed a dose-effect relationship and a positive relationship to p-eNOS expression which could be eliminated by eNOS inhibitor L-NAME. NO expression, the ability of migration and proliferation in adiponectin, SNP, SNP and adiponectin treatment group increased significantly compared with control group. Western blot analysis showed that there was statistically significant difference between adiponectin group and control group for p-Akt, eNOS and p-eNOS expression respectively. While cell proliferation and migration were inhibited and no significant differences of p-Akt, eNOS, p-eNOS and NO expressions existed among L-NAME, L-NAME and adiponectin treatment groups.
4. Phosphatidylinositol3-kinase (PI3K) inhibitor LY294002could effectively inhibit the effect of adiponectin on EPCs
Adiponectin exhibit pro-proliferative and anti-apoptotic properties on cultured EPCs, which could be eliminated by PI3K inhibitor LY294002.1ug/ml adiponectin treatment also significantly improved the number of cells, cell migration and the p-Akt, eNOS, p-eNOS expression levels. However, no significant differences of p-Akt expressions existed among the rest groups, the number of cells, cell migration and the p-Akt, eNOS, p-eNOS expression levels significantly decreased in LY294002group, and LY294002and adiponectin treatment group compared with control group (P<0.05).
Conclusions:
1. Adiponectin promote EPCs proliferation and migration activity in a time-and dose-dependent manner.
2. Adiponectin improves EPCs proliferation and migration activity through PI3K/Akt signaling pathway.
脂联素主要是由白脂肪组织合成的分子量是30kDa蛋白,其具有胰岛素增敏、抗动脉粥样和抗炎特性。促进血管内皮祖细胞一氧化氮的产生以及减轻活性氧对血管内皮细胞的损伤。总之,脂联素通过激活多种细胞内信号级联反应在血管保护中发挥着举足轻重的作用。脂联素在循环中主要有三个亚型：低分子量的三聚体,中等分子量的六聚体和高分子量的多聚体。这三种亚型各有不同的功能,其中研究表明高分子量是主要的活性蛋白。在内皮细胞上主要有两种脂联素受体,即R1和R2受体。内皮是脂联素的主要靶点,脂联素通过其在内皮细胞上的受体可以抑制单核细胞粘附到内皮细胞,并最终抑制血管平滑肌的迁移和增殖,这有助于减轻血管损伤。
骨髓和外周血的内皮祖细胞是单核细胞前体细胞,能够分化成成熟的内皮细胞,当血管内皮受损时内皮祖细胞通过归巢定位到损伤部位加速再内皮化,促进血管修复,在维持血管内皮完整性,修复受损血管内皮细胞,促进新生血管形成及组织修复等方面起重要作用。EPCs主要来源于骨髓、外周血及脐血,脂肪中也存在少量的EPCS。外周血循环中EPCs的数量占外周血细胞总数的0.01%,占白细胞总数的0.05%,而骨髓中EPCs的含量是外周血的500倍。EPCs经药物刺激后可以提高至占白细胞总数的6%。EPCs促进血管的生成及修复的机制可能有两种：(1)直接分泌VEGF等细胞因子,通过旁分泌的方式促进局部缺血组织的血管新生；(2)通过归巢效应、整合于受损的血管,直接分化为内皮细胞,参与血管新生。
动脉粥样硬化的血管重塑及病理生理涉及多种细胞类型和级联反应。值得注意的是,PI3K/Akt信号通路是参与这些级联反应中的部分环节。磷酸肌醇3-激酶(PI3K)通过不同类型的膜受体(包括G-蛋白耦合受体和酪氨酸激酶受体)激活蛋白质和脂质。PI3K和Akt是内皮型一氧化氮合酶(eNOS)的重要的正调节子,通过L-精氨酸的依赖NADPH氧化产生NO。PI3K/Akt通路与内皮细胞功能,如调节血管张力等密切相关。
本研究旨在探讨内皮祖细胞降低是否是动脉硬化的危险因素?与脑动脉粥样硬化狭窄病变血管数量和程度的相关性,进一步探讨PI3K/Akt信号通路在脂联素促进内皮祖细胞增殖和迁移中的作用。
第一章内皮祖细胞与脑动脉粥样硬化程度的相关性研究
目的：
中风仍然是成人死亡和长期残疾的主要原因,而缺血性中风约占这些中风的87%。脑动脉粥样硬化疾病是缺血性中风的主要病因,血管内皮功能障碍在其发生和发展上占主导作用。动脉粥样硬化早期的一个重要特点是内皮功能障碍,而内皮功能障碍主要表现为内皮细胞逐渐丧失,促进斑块的发生和发展。但成熟的内皮细胞再生能力有限。因此,越来越多的证据表明血管内皮祖细胞(EPCs)可以向血管内皮层归巢、修复损伤的内皮并维护血管内皮功能,这是通过内皮祖细胞介导的新的内源性血管修复系统来实现的。
最近的研究表明,内皮祖细胞能够归巢到受损的内皮细胞,分化为成熟的内皮细胞和替换受损的内皮细胞,从而发挥抗动脉硬化的作用。因此,循环内皮祖细胞的数量是心血管健康状况的标志。最近的研究表明,循环内皮祖细胞的数目减少与不同程度的心血管疾病的危险因素密切相关,如吸烟、糖尿病、高脂血症和高血压等。Werner等研究发现内皮祖细胞水平与冠状动脉粥样硬化性疾病和外周闭塞性血管的严重程度密切相关。然而,脑动脉粥样硬化的严重性和内皮祖细胞水平之间的关系还未知。在此背景下,本研究试图探讨内皮祖细胞与脑动脉粥样硬化严重程度之间的关系,以及EPCs与传统的心血管危险因素(年龄,性别,吸烟,高脂血症,高血压,糖尿病,家族史)相关的程度。最后,进一步评估内皮祖细胞对脑动脉粥样硬化严重程度的预测价值是否优于传统的危险因素。
方法：
1.入组南京军区南京总医院2011年12月至2013年12月之间的接受全脑血管造影的患者,并获取人口统计学和基线资料。所有患者均接受常规心电图、心脏超声检查及数字减影血管造影(DSA)等检查。神经功能缺损采用NIHSS评分,心血管危险因素综合评分和下肢动脉硬化程度判断分别采用佛明汉评分(Framingham risk score, FRS)和踝肱指数(Ankle-brachial index, ABI)。
2.动脉硬化负荷评估：按照动脉硬化病变的位置分为单纯颅内动脉粥样硬化性狭窄(intracranial atherosclerotic stenosis, ICS)组、单纯颅外动脉粥样硬化性狭窄(extracranial atherosclerotic stenosis, ECS)组、(combined intracranial and extracranial atherosclerotic stenosis, CCS)组。颅外狭窄是使用北美症状性颈动脉内膜切除术试验(NASCET)标准计算,而颅内动脉狭窄(ICS)参考WASID标准。动脉粥样硬化负荷分别从两方面进行评估：1、数量：计算动脉硬化病变血管节段的数量(即狭窄≥50%的血管节段的总数目)；2、按照脑动脉粥样硬化狭窄程度来评估,0分代表<50%的狭窄,1分代表50%至99%的狭窄和血管闭塞是2分。在一个动脉段有多个病灶存在的情况下,按照最严重狭窄来计算。
3.统计方法：采用SPPS16.0软件进行统计学分析。计量资料以均数±标准差(x±s)表示。对连续变量采用独立样本t检验、Mann-Whitney U检验或方差分析,对于分类变量采用卡方检验或Fisher分类变量的精确检验。单因素分析P<0.2的变量做为候选入多因素logistic回归分析。动脉硬化严重程度和内皮祖细胞水平的数量之间的相关采用Spearman秩和相关分析。内皮祖细胞数量和动脉钙化或CAD严重程度之间的相关性进行了评估用多变量logistic回归。P<0.05为差异具有统计学意义。
结果：
1.基线资料特征
入组患者的年龄均值为60岁,29.4%的女性和12.2%无脑动脉粥样硬化者作为对照组。单纯颅外动脉狭窄54例(27.4%),单纯颅内动脉狭窄45例(22.8%). CD34+/CD309+, CD133+/CD309+和CD34+/CD133+/CD309+细胞中位值分别为0.172%(四分位距为0.066%至0.370%),0.131%(四分位距为0.041%至0.297%)和0.024%(四分范围,分别为0.005%至0.063%)。
2.内皮祖细胞与脑动脉粥样硬化负荷负相关
校正混杂因素(如年龄、吸烟、高血压、收缩压和纤维蛋白原)后,内皮祖细胞水平与脑动脉粥样硬化之间有统计学差异(CD45-/dimCD34+CD309+cells: B=-2.197, OR=0.111,95%CI:0.024to0.511, P=0.005; CD45-/dimCD133+CD309+cells:B=-2.172, OR=0.114,95%CI:0.023to0.556, P=0.007; CD45-/dim CD34+CD133+CD309+cells:B=-4.580, OR=0.010,95%CI:0.000to0.541,P=0.024)。进一步多元logistic辑回归显示,只有内皮祖细胞计数与颅外及混合脑动脉粥样硬化有统计学差异。然而,内皮祖细胞与颅内动脉粥样硬化无统计学差异(P>0.05)。
3.脑动脉粥样硬化负荷与EPCs和佛明汉评分相关性
受试者按动脉粥样硬化得分的三分位间距分为：低、中和高组。我们对EPCs的三种亚型通过顺序逐步回归分析提示,纤维蛋白原和高血压是脑动脉粥样硬化的危险因素,而皮祖细胞是保护因素。斯皮尔曼等级相关分析来分析动脉硬化负荷和EPCs的相关性：在78位颅外动脉硬化与对照组间相关分析提示CD133+/CD309+细胞与动脉粥样硬化负荷之间相关性有统计学差异(病变血管段数量和动脉硬化狭窄度评分：Spearman rho=-0.195和-0.197,P<0.05)。在69例颅内狭窄患者中CD133+/CD309+细胞与脑动脉粥样硬化负荷相关性有统计学差异(病变血管段数量：Spearman rho=-0.262, P=0.030;动脉硬化狭窄度评分：Spearman rho=-0.248, P=0.040)此外,在调整可能的混杂因素影响后二者相关性仍有统计学差异。然而,动脉粥样硬化的负荷与FRS、另外两个内皮祖细胞亚群之间相关性无统计学差异。
结论：
1.在校正年龄、吸烟、高血压、收缩压、纤维蛋白原和佛明汉评分后内皮祖细胞各个亚型计数与脑动脉粥样硬化相关性有统计学差异。
2.内皮祖细胞计数与颅外及混合脑动脉粥样硬化相关性有统计学差异,而与颅内动脉硬化无统计学差异。
第二章骨髓来源内皮祖细胞的分离、培养及鉴定
目的：
EPCs作为内皮细胞的前体细胞,它具有向内皮损伤部位分定向归巢并分化为成熟内皮细胞及分泌内皮生长因子等提高周围内皮细胞功能,在维持血管内皮完整性,修复受损血管内皮细胞,促进新生血管形成及组织修复等方面起重要作用,EPCs主要来源于骨髓、外周血,而骨髓中EPCs的含量是外周血的500倍。建立由C57BL/6小鼠骨髓单个核细胞诱导分化EPCs的培养体系。
方法：
选用4-6周龄的C57BL/6小鼠,PBS无菌条件下冲洗骨髓腔获得细胞悬液。使用淋巴细胞分离液经密度梯度离心法离心获得骨髓单核细胞层,收集骨髓单核细胞,用PBS洗涤后接种于由人纤维连接蛋白包被的细胞培养板中。细胞培养基选用EGM-2SingleQuots套装,培养条件为37℃,5%C02。接种后第4天首次更换培养液,去除未贴壁细胞。此后,每两天更换一次细胞培养液。细胞培养至第21天。
每日于光学倒置显微镜下观察细胞形态及生长状况,并拍摄照片。本研究选用培养至第21天的EPC进行细胞鉴定。使用免疫荧光法鉴定培养细胞表达EPC特异性表面抗原Sca-1及VEGFR2(CD309)'情况。使用流式细胞仪检测培养细胞双表达Sca-1及VEGFR2比例。同时,检测细胞吞噬DiI标记的乙酰化低密度脂蛋白(Dil-Ac-LDL)能力及结合异硫氰酸荧光素标记的荆豆凝集素能力(FITC-UEA-1)。
结果：
首次换液后,贴壁细胞生长加速,渐出现细胞集落。集落形态为中央小圆形细胞聚集,周围梭型细胞呈放射状生长状态。在细胞培养过程中,我们还观察到细胞可呈现线状、管状生长排列,提示该细胞具有成血管能力。大约2周后的细胞形态趋于一致,呈现“铺路石”样生长状态。
流式细胞仪检测结果也显示,本实验中培养至3周后的EPCs, CD34及VEGFR2双阳性率达到85%以上。培养细胞具备吞噬Dil-Ac-LDL和FITC-UEA-1的能力。
结论：
1.以上实验结果表明,本研究所采用密度梯度离心后贴壁筛选培养的方法可以从C57BL/6小鼠骨髓中成功分离培养EPCs。
2.在培养过程中,细胞呈“集落样”生长和“铺路石”样外观,并可见细胞呈现线、管状排列生长。且流式细胞检查及细胞功能检查均证明了所培养的细胞为内皮祖细胞。为后续的脂联素干预实验奠定了实验技术基础。
第三章脂联素提高内皮祖细胞增殖和迁移活性及其机制探讨
目的：
脂联素是一种由白色脂肪组织分泌的分子量30kDa蛋白。脂联素对血管保护的关键作用通过激活多种细胞内的多种信号途径。例如增强内皮细胞一氧化氮的生成和清除氧自由基。循环血液中低脂联素水平被认为是一种新的冠心病的独立危险因素,低脂联素血症将会导致血管内皮细胞功能紊乱。EPCs是一类能增殖并分化为血管内皮细胞的前体细胞,同时具有内皮细胞和祖细胞的一些生物学特性,EPCs是否也表达有脂联素受体,脂联素对EPCs的增殖和迁移功能有无影响及其具体的机制如何目前尚不清楚。
因此,我们提出假设：脂联素可能会影响EPCs的数量和功能,增强损伤内皮的修复能力,延缓动脉硬化病变的发生和发展。基于该假设我们拟采用第二部分成功建立的EPCs培养体系,初步探索脂联素是否影响EPCs的增殖和迁移活性及其可能的作用机制。
方法：
1.先做EPCs的增殖曲线,自C57BL/6、鼠骨髓提取骨髓单核细胞,按照不同的浓度接种到培养板中,以细胞计数试剂盒行细胞计数并绘制各组细胞生长曲线,检测不同接种浓度的EPCs的增殖活性；
2.进一步探索不同剂量的脂联素对EPCs增殖活性和迁移能力的影响情况,自C57BL/6小鼠骨髓提取骨髓单核细胞,计数后分为3组接种于培养板中。分别为：对照组、脂联素处理组(培养液中脂联素浓度分别为1和10ug/ml),以细胞计数试剂盒行细胞计数和Transwell做迁移能力测试；Western blot法检测各组细胞中Akt、p-Akt、p-eNOS. eNOS表达情况；
3.自C57BL/6小鼠骨髓提取骨髓单核细胞,计数后分为6组接种于培养板中。分别为对照组、脂联素(1ug/ml)组、NO供体组(sodium nitroprusside, SNP)、SNP+脂联素(1ug/ml)组、eNOS抑制剂组(L-NAME)和L-NAME+脂联素组。以细胞计数试剂盒行细胞计数了解各组细胞增殖能力；Transwell做迁移能力测试；Western blot法检测各组细胞蛋白Akt、p-Akt、eNOS和p-eNOS表达情况；NO检测试剂盒检测各组上清液中NO的表达量。
4.提取小鼠骨髓单核细胞后诱导分化为EPCs,分为4组接种于培养板中。分别为对照组、脂联素(1g/ml)组、LY294002组(Akt抑制剂)和LY294002+脂联素(1ug/ml)组。以细胞计数试剂盒行细胞计数了解各组细胞增殖能力；Transwell做迁移能力测试；Western blot法检测各组细胞蛋白Akt、p-Akt、eNOS和p-eNOS表达情况。
5.采用SPPS16.0软件进行统计学分析。计量资料以x±s表示。多组间细胞增殖能力、迁移能力及NO、Akt、p-Akt、eNOS和p-eNOS等各指标表达水平比较采用one-way ANOVA。P<0.05为差异具有统计学意义。
结果：
1.不同接种浓度的单核细胞的增殖曲线
接种约5-11X105cells/cm2单核细胞,培养2周左右细胞达到增殖高峰期,因此,用该浓度的单核细胞接种2周后即可用于下一步实验。
2.不同剂量的脂联素对EPCs增殖活性和迁移能力的影响
脂联素作用后EPCs的数量增加,该作用随脂联素剂量的增加而增强,呈量效依赖性。脂联素(1ug/ml)作用3天EPCs增殖活性和迁移能力既有统计学差异,且Western blot检测结果显示：Akt、p-Akt、p-eNOS、eNOS表达量脂联素组较对照组有统计学差异(P<0.05)。
3. eNOS抑制剂可以抑制脂联素对EPCs的作用
p-eNOS表达量增高可提高脂联素对EPCs的促增殖、迁移作用,但可被eNOS抑制剂L-NAME抑制。脂联素作用3天后脂联素组、SNP组、SNP+脂联素(1ug/ml)组较对照组细胞数量增加、细胞迁移能力提高和上清液NO表达量增高；Western blot检测结果显示：p-Akt、eNOS、p-eNOS表达量增高。L-NAME和L-NAME+脂联素组则与上述结果相反,细胞增殖、迁移受到抑制,p-Akt、eNOS、p-eNOS、NO表达量较对照组并无增高。
4.磷脂酰肌醇3激酶(PI3K)特异性抑制剂LY294002抑制脂联素对EPCs的作用
脂联素对EPCs的促增殖、抗凋亡作用与p-Akt、p-eNOS表达情况可被磷脂酰肌醇3激酶(PI3K)特异性抑制剂LY294002抑制。脂联素作用3天后脂联素(1ug/ml)组较对照组细胞数量增加、细胞迁移能力提高以及p-Akt、eNOS、 p-eNOS表达量增高,但与LY294002组和LY294002+脂联素组比细胞数量降低、细胞迁移能力降低以及p-Akt、eNOS、p-eNOS表达量降低(P<0.05)。
结论：
1.脂联素促进EPCs的增殖和迁移活性,该作用有一定的时间和剂量依赖性；
2.脂联素通过PI3K/Akt信号通路促进了EPCs的增殖和迁移活性。
Atherosclerosis is one of the major diseases threatening the human health, at present accumulating evidence suggests that endothelial dysfunction is an early step in the atherosclerotic process and plays a pivotal role in the development and progression of atherosclerosis. And several studies have also found that endothelial progenitor cells (EPCs) play an important role in the development of atherosclerosis, such as promoting the regeneration of damaged endothelium. Walter et al demonstrated that circulating endothelial progenitor cells has the characteristics of homing to denuded parts of the artery after balloon injury, a body of evidence is available demonstrating that EPC levels are very strong vascular risk markers, even better than the Framingham risk score. Most of the clinical studies also confirmed that increased number and function of EPCs may have important therapeutic implications for the management of cardiovascular disease, such as coronary heart disease, heart failure etc. Traditional risk factors such as elderly, hypertension, hyperlipidemia, and diabetes are major causes of reducing the number and function of circulating EPCs
Adiponectin is a30-kDa protein synthesized predominantly by white adipose tissue, which has insulin-sensitizing, antiatherogenic, and anti-inflammatory properties. Enhancement of nitric oxide generation and attenuation of reactive oxygen species production in endothelial cells along with stimulated endothelial progenitor cells (EPCs) survival, proliferation and differentiation constitute some of adiponectin's vasoprotective actions. Adiponectin exerts a pivotal role in vascular protection through activation of multiple intracellular signaling cascades and exists in the circulation in three isoforms:a low molecular weight trimer, a medium molecular weight hexamer, and a high molecular weight (HMW) multimer. The3isoforms might play different roles, but some arguments have suggested that the HMW isoform might be the major active protein. Both AdipoR1and AdipoR2, two receptors for adiponectin, are expressed in endothelial cells. The endothelium is a major target of adiponectin, where the adiponectin downregulates adhesion molecules, inhibits monocytes adhesion to endothelial cells and ultimately inhibits the migration and proliferation of vascular smooth muscle cells, which contribute to vascular damage.
Endothelial progenitor cells from bone marrow or peripheral blood are mononuclear precursor cells that can differentiate into mature endothelial cells. During wound repair EPCs home to sites of vascular injury where these cells can restore vascular integrity, and play a significant role in maintaining vascular endothelial integrity, repairing the damaged endothelial cells, promoting new blood vessel formation and tissue repair, etc. EPCs are mainly isolated from peripheral, umbilical cord, and bone marrow. There are also a small number of EPCs in adipose tissue. The number of immature progenitors from bone marrow is500-fold higher compared to peripheral blood. Drug-induced EPCs can be increased to6%of the total number of white blood cells. EPCs can remarkably promote new blood vessel formation and repair damaged vessels, the mechanisms underlying this phenomenon are likely to be explained in two ways:(1) EPCs improve neovascularization of ischemic tissues by secretion of cytokines such as vascular endothelial growth factor (VEGF) and so on.(2) EPCs have been found to directly incorporate into injured vessels and that participate in angiogenesis and reconstruction by differentiation into endothelial cells.
The atherosclerotic vascular remodelling and pathophysiology involve multiple cell types and a wide array of mediators and cascades. Of note, the PI3K/Akt signalling pathway impinges on several of them. Phosphoinositide3-kinases (PI3Ks) are protein and lipid kinases activated by G protein-coupled receptors and tyrosine kinase receptors. PI3K and Akt are important positive regulators of endothelial nitric oxide synthase (eNOS), which generates NO through the NADPH-dependent oxidation of L-arginine. The PI3K/Akt related pathways have a key role in regulating vascular tension, etc.
With this background, this study sought to investigate whether the reduction in EPCs numbers is an independent risk for arteriosclerosis, the relationships between numbers of EPCs with cerebral atherosclerotic severity, and further investigate whether the activation of PI3K/Akt is involved in adiponectin promoting endothelial progenitor cell proliferation and migration.
Chapter I Correlation of Different Subpopulations of Endothelial Progenitor Cells with Cerebral Arteriosclerosis Severity
Objective:
Stroke remains a leading cause of death and long-term disability in adults' worldwide, ischemic strokes (IS) account for approximately87%of these strokes. Cerebral atherosclerosis (CA) is one of the main causes of this type of obstruction and endothelial dysfunction also plays a critical role in its onset and progression. Endothelial dysfunction is characterized by a progressive loss of endothelial cell (ECs) and thought to be an important early event in the atherosclerotic disease process. It contributes to plaque initiation and progression. Mature ECs possess limited regenerative capacity. Thus, growing evidence suggests endothelial progenitor cells (EPCs), endothelial stem or precursor cells, may contribute to the restoration of the endothelial lining after injury and maintenance of endothelial function, which is a novel endogenous vascular repair system mediated by EPCs.
Recent studies have indicated that EPCs are immature cells capable of homing to the injured endothelium, differentiating into mature endothelial cells and replacing the injured endothelial cells, thereby exerting a protective effect against atherosclerosis. The number of circulating EPCs is now considered a mirror of cardiovascular health. Recent studies suggest that reduced number of circulating EPCs was closely related to the degree of endothelial dysfunction in humans at various degrees of cardiovascular risk, smoking, diabetes, hyperlipidemia; and hypertension. Werner et al. also identified levels of EPCs correlate inversely with the severity of several atherosclerotic vascular diseases such as coronary artery disease and peripheral occlusive vascular disease. However, the relationship between the severity of the CA and EPCs levels is not known. With this background, this study sought to investigate the relationships between the number of EPCs and the CA severity. We also examined the degree to which EPCs count were associated with traditional cardiovascular risk factors (age, gender, smoking, hyperlipidemia, hypertension, diabetes, and family history). Finally, we assessed whether EPCs provided significant diagnostic value beyond traditional risk factors for estimating CA severity.
Methods:
1. All subjects were enrolled from the consecutive inpatients who underwent cerebral angiography in the Department of Neurology at Jingling Hospital between December2011and December2013. They were thoroughly investigated for demographics and baseline clinical characteristics. All patients underwent conventional cardiac ultrasound and electrocardiogram examination. Neurological deficits were estimated using the National Institutes of Health Stroke Scale (NIHSS) score by two neurologists, who were blind of digital subtraction angiography (DSA) images and EPCs assay results. The Framingham risk score (FRS) was assessed at baseline for each subject's cardiovascular risk factors, and the Ankle-brachial index (ABI) is used to confirm the diagnosis and determine the severity of peripheral arterial disease (PAD).
2. Cerebral arteriosclerosis burden assessment:Locations of lesion were categorized as being in the intracranial, extracranial vessels or the combined extracranial and intracranial stenosis (IES). Intracranial lesions were defined as those at or above the precavernous or upper petrous carotid artery in the internal carotid artery. For the vertebral artery, the distinction was made at the point where the artery takes a turn and comes into the foramen magnum piercing the dura. Extracranial stenosis (ECS) was calculated using North American Symptomatic Carotid Endarterectomy Trial (NASCET) criteria; and intracranial stenosis (ICS) was measured by using Warfarin-Aspirin Symptomatic Intracranial Disease (WASID) criteria. The presence of arterial stenotic lesions was determined by the number of cerebral arteries showing steno-occlusion and the degree thereof. The extent of atherosclerotic burden (cerebral atherosclerotic burden, CAB) was graded according to the previous criteria (0indicating<50%stenosis,1indicating50%to99%stenosis, and2indicating occlusion). In the presence of multiple lesions within one arterial segment, data from the most significant stenosis were used. The total number of arterial segmental lesions ((≥50%stenosis) was also calculated (cerebral atherosclerotic number, CAN).
3. Statistical analysis was performed using SPSS software, version16.0(SPSS Inc., Chicago, IL). Comparisons were performed using independent sample t tests, Kruskal-Wallis tests, Mann-Whitney U tests, or analysis of variance for normal distributed continuous variables, and the chi-square test or fisher's exact test for categorical variables, as appropriate. Variables with a P value<0.2by univariate analysis were candidates for inclusion in multiple logistic regression analysis. A selection process was used to develop the final multivariable model, and adjusted odds ratios (OR) and95%confidence intervals (CI) were calculated as estimates of relative risk. Moreover, the relationship between number of arterial segmental lesions and EPCs levels was determined by Spearman rank test. Associations between EPCs level and arterial calcification or CA severity were assessed with logistic regression. A two-tailed P value<0.05was considered statistically significant.
Results:
1. Patients'Characteristics
The baseline characteristics of the197patients enrolled in this study are provided in Table1. Fifty-four patients (27.4%) had ECS and45patients (22.8%) had ICS. Mean age was60years, with29.4%women and12.2%no significant CA as a control group. The median values of were CD34+CD309+, CD133+CD309+and CD34+CD133+CD309+cells were0.172%(interquartile range,0.066%to0.370%),0.131%(interquartile range,0.041%to0.297%) and0.024%(interquartile range,0.005%to0.063%), respectively, in the total sample.
2. Circulating EPCs Are Reduced in the Presence of CA
In binary logistic regression analysis, the only EPCs level was significantly conversely associated with the prevalence of CA after adjustment for age, smoking, hypertension, SBP, fibrinogen and FRS (CD45-/dimCD34+CD309+cells:B=-2.197, OR=0.111,95%CI:0.024to0.511, P=0.005; CD45-/dimCD133+CD309+cells: B=-2.172, OR=0.114,95%CI:0.023to0.556,p=0.007; CD45-/dim CD34+CD133+CD309+cells:B=-4.580, OR=0.010,95%CI:0.000to0.541, P=0.024). The result of multivariate logistic regression showed that only EPCs count was significantly negatively associated with the location of CA (extracranial or combined extra-and intra-cranial artery stenosis). However, EPCs were not significantly associated with intracranial atherosclerosis (P>0.05).
3. Relationship of CA with EPCs, Calcification Score or Framingham Risk Score
The subjects were divided into three groups on the basis of their atherosclerosis score:low, intermediate and high group. In the ordinal regression analysis CA burden remained correlated conversely to EPCs and associated positively with fibrinogen and hypertension by controlling for confounders. We use Spearman's rank correlation analysis to analyze the correlation between CA burden and EPCs levels, there was also a significant inverse relationship between CD133+CD309+cells count and atherosclerotic burden in78patients with ECS and control subjects (CAN and CAB: Spearman rho=-0.195and-0.197, P<0.05). Additionally, the atherosclerotic burden was inversely related to CD133+CD309+cells count in69patients with ICS (CAN: Spearman rho=-0.262, P=0.030; CAB:Spearman rho=-0.248, P=0.040). There are still significant differences between CA burden and EPCs levels after adjustment for potential confounders. However, the atherosclerotic burden was not related to FRS or the other two progenitor cell subsets.
Conclusions:
1. EPCs level was significantly conversely associated with the prevalence of CA after adjustment for age, smoking, hypertension, SBP, fibrinogen and FRS.
2. There was a significant positive correlation between EPCs and atherosclerotic burden in patients with ECS. However, there was no statistically significant relationship of the atherosclerotic burden with EPCs in patients with ICS.
Chapter II Isolation, culture and characterization of bone marrow derived EPCs
Objective:
EPCs, endothelial precursor cells, are mobilized early after vascular injury. They can home to sites of vascular injury and differentiate into mature endothelial cells, and avidly secrete angiogenic factors encouraging resident endothelial cell proliferation and migration. EPCs may play a critical role in re-establishing the endothelial integrity, repairing damaged endothelial cells, promoting angiogenesis in tissue repair. EPCs can be mainly isolated from peripheral blood or bone marrow, which is500-fold higher compared to peripheral blood. We try to establish a culture system to induce the differentiation of C57BL/6mouse bone marrow-derived mononuclear cells into EPCs.
Methods:
Bone marrow of tibia and fibula from C57BL/6mice (4-6week old, male) was washed out with PBS and eentrifugated with Histopaque1077at400g for30minutes. The bone marrow mononuclear cells (BMMNCs) were re-suspended in EGM-2SingleQuots and washed twice. The BMMNCs were then seeded in the cell culture flasks coated with human fibronectin and cultured with EGM-2SingleQuots at37℃5%CO2. Nonadherent cells were removed4days after seeding and the culture medium was replaced every other day thereafter. EPCs cultured up to3weeks were used for later cell characterization and transplantation.
Cultured cells were observed and photographed by inverted microscope everyday. EPCs cultured up to3weeks were chosen for cell characterization. We identified the proportion of cultured cells which co-expressed CD34and VEGFR2by flow cytometer. Meanwhile, we also tested the abilities of of uptaking Dil-Ac-LDL and binding FITC-UEA-1of cultured cells.
Results:
After the first exchange of medium to remove adherent cells were growing rapidly. Then cell colonies began to appear on the5th day of culture. Colony morphology is small round cell aggregation center, surrounded by radial growth of cells. In cell culture, we also observed that cells can be present in linear and tubular growth, which suggested that the cells have the ability to grow new blood vessels. Since about2weeks of culture, the cells gradually changed to a cobblestone-like appearance.
After3weeks culture, cells were verified as EPCs by flow cytometer. And the result exhibited that more than85%of the cultured cells co-expressed CD34and VEGFR2. Finally, by testing the abilities of uptaking Dil-Ac-LDL and binding FITC-UEA-1of cultured cells, we found most cultured EPCs were able to uptake DiI-Ac-LDL and bind FITC-UEA-1.
Conclusions:
1. EPCs were separated from bone marrow with density gradient centrifugation, wall sticking screening and amplified in vitro. Our method of culturing EPC from BMMNCs of C57BL/6mice is feasible.
2. We observed a line-like and a tube-like appearance during the cell culture which indicating the vascular forming potential of cultured EPCs. The results of FACS and cell function tests all indicated that the cells we cultured were EPCs. This part of study provides the technological foundation for the later experiments.
Chapter Ⅲ Effects of Adiponectin on proliferation and migration activities of endothelial progenitor cells and its possible mechanism
Objective:
Adiponectin is a30-kDa protein synthesized predominantly by white adipose tissue. Adiponectin exerts a pivotal role in vascular protection through activation of multiple intracellular signaling cascades. Enhancement of nitric oxide generation and attenuation of reactive oxygen species production in endothelial cells along with stimulated endothelial progenitor cells (EPCs) survival, proliferation and differentiation constitute some of adiponectin's vasoprotective actions. Circulating adiponectin level is considered to be a new independent risk factor for coronary heart disease, hypoadiponectinemia will lead to vascular endothelial dysfunction. EPCs are the precursor cells that can proliferate and differentiate into endothelial cells and have some biological properties of endothelial cells and progenitor cells. Whether EPCs express adiponectin receptors, the effect of adiponectin on the proliferation and differentiation of EPCs and the possible mechanism are unclear.
Adiponectin promotes endothelial progenitor cell number and function, enhances the ability to repair damaged endothelium and delays the occurrence and development of atherosclerotic lesions. With this background, we intend to use EPCs culture system successfully established in the second part, explore preliminarilly effects of Adiponectin on proliferation and migration activities of endothelial progenitor cells and its possible mechanism.
Methods:
1. We intend to study cell growth curves of EPCs. Bone marrow mononuclear cells (BMMNCs) were harvested from C57BL/6mice, and plated on fibronection-coated culture dishes in different concentrations. Cell growth curves of each group were drawn with CCK-8assay, which was used to determine the proliferation of the cultured EPCs in different inoculation concentration.
2. To investigate the effects of adiponectin with different doses on proliferation and migration of EPCs. Obtain required amount of EPCs which were extracted from the bone marrow of C57BL/6mice, counted and seeded on the culture flasks. Namely, Control group and adiponectin-treated group (cell culture medium was supplemented with1and10ug/ml adiponectin respectively). EPCs'differentiation and migration were assessed with the CCK-8assay and Transwell respectively. The expression of Akt, p-Akt、p-eNOS、eNOS were measured by Western blot.
3. Bone marrow mononuclear cells (BMMNCs) were harvested from C57BL/6mice and divided into6groups for cell culture. The cell groups were as follows: control group, adiponectin treatment groups (cell culture medium was supplemented with lug/ml adiponectin), NO donor group (sodium nitroprusside, SNP), SNP and adiponectin treatment group (cell culture medium was supplemented with SNP and lug/ml adiponectin), eNOS inhibitor group (cell culture medium was supplemented with L-NAME), L-NAME and adiponectin treatment group (cell culture medium was supplemented with L-NAME and1ug/ml adiponectin). The expression of nitric oxide in supernatant was measured by nitric oxide assay kit.
4. Mouse BMMNCs were induced to differentiate into EPCs and divided into4groups for cell culture. The cell groups were as follows:control group, adiponectin treatment groups (cell culture medium was supplemented with lug/ml adiponectin), LY294002group (Akt inhibitor), LY294002and adiponectin treatment group (cell culture medium was supplemented with LY294002and lug/ml adiponectin). EPCs' differentiation and migration were assessed with the CCK-8assay and Transwell respectively. The expression of Akt、p-Akt、p-eNOS、eNOS were measured by Western blot.
5. Statistical analysis was performed using SPSS software, version16.0(SPSS Inc., Chicago, IL). Data are expressed as the mean value±standard deviation. Comparisons among more groups (such as levels of cell proliferation, migration, and NO, Akt, p-Akt, eNOS and p-eNOS, etc.) were performed by the analysis of variance. Statistical significance was accepted if the null hypothesis could be rejected at P≤0.05.
Results:
1. Monocyte proliferation curve in different inoculums concentrations
Monocytes cultured cells were seeded at a density of about5-11×105cells/cm2and reached its peak proliferation at2weeks. Thus, the concentration of mononuclear cells was used in the next experiment after2weeks of culture.
2. Different doses of Adiponectin influence the proliferation and migration of EPCs
Adiponectin inereased the number of EPCs with dose and time dependence. Adiponectin (lug/ml) signifieantly inereased proliferation and migration activities of EPCs after3days, Western blot analysis showed that there was statistically significant difference between adiponectin group and control group for Akt, p-Akt, p-eNOS and eNOS expression respectively (P<0.05).
3. The eNOS-inhibitor could effectively inhibit the effect of adiponectin on EPCs
Adiponectin increased the ability of migration and proliferation on cultured EPCs. And the properties revealed a dose-effect relationship and a positive relationship to p-eNOS expression which could be eliminated by eNOS inhibitor L-NAME. NO expression, the ability of migration and proliferation in adiponectin, SNP, SNP and adiponectin treatment group increased significantly compared with control group. Western blot analysis showed that there was statistically significant difference between adiponectin group and control group for p-Akt, eNOS and p-eNOS expression respectively. While cell proliferation and migration were inhibited and no significant differences of p-Akt, eNOS, p-eNOS and NO expressions existed among L-NAME, L-NAME and adiponectin treatment groups.
4. Phosphatidylinositol3-kinase (PI3K) inhibitor LY294002could effectively inhibit the effect of adiponectin on EPCs
Adiponectin exhibit pro-proliferative and anti-apoptotic properties on cultured EPCs, which could be eliminated by PI3K inhibitor LY294002.1ug/ml adiponectin treatment also significantly improved the number of cells, cell migration and the p-Akt, eNOS, p-eNOS expression levels. However, no significant differences of p-Akt expressions existed among the rest groups, the number of cells, cell migration and the p-Akt, eNOS, p-eNOS expression levels significantly decreased in LY294002group, and LY294002and adiponectin treatment group compared with control group (P<0.05).
Conclusions:
1. Adiponectin promote EPCs proliferation and migration activity in a time-and dose-dependent manner.
2. Adiponectin improves EPCs proliferation and migration activity through PI3K/Akt signaling pathway.
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