【作者】 戈程；

【导师】 张梅； 张利宁；

【作者基本信息】 山东大学 ， 内科学（专业学位）， 2014， 博士

【摘要】 研究背景动脉粥样硬化斑块多发生在大血管的分叉和弯曲处等涡流及病理震荡剪切力作用区域,而在大血管直行区域,平稳血流对血管壁产生单向的、大小高于震荡剪切力的生理脉冲剪切力。血管内皮细胞是在血管对剪切力的应答过程中起最重要作用的细胞。大量研究表明,紊乱的血流会通过增强内皮细胞的氧化应激反应和炎症反应、促进细胞周期进程和细胞增殖等促进动脉粥样硬化斑块的发生发展,而脉冲剪切力则使内皮细胞保持较低的增殖水平、较低的氧化应激反应和炎症反应水平,维持其相对稳定的状态,抑制动脉粥样硬化斑块的发生发展。程序性细胞死亡4(Programmed Cell Death4, PDCD4)主要通过抑制肿瘤蛋白的翻译过程起到抑制肿瘤发生发展的作用。PDCD4蛋白可直接与靶基因(MYB/c-MYB)的mRNA编码区域结合或通过与真核细胞翻译起始因子(Eukaryotic Translation Initiation Factor, eIF)4G竞争性结合eIF4A抑制靶蛋白的翻译起始过程。通过该机制,PDCD4可以影响多种蛋白的表达,从而影响肿瘤细胞的增殖、凋亡、转化、侵袭以及自噬等生物学行为。PDCD4抑制肿瘤发生发展的具体作用及机制因肿瘤和细胞种类的不同而有所差异。近期研究发现,PDCD4还影响心血管系统各类细胞的生物学功能。但是,在血管内皮细胞稳态的维持中PDCD4发挥怎样的作用尚无研究报道。在对PDCD4和载脂蛋白E (Apolipoprotein E, ApoE)双基因敲除小鼠(PDCD4-/-ApoE-/-)的研究中发现,PDCD4基因的敲除可明显减少血管壁的动脉粥样硬化斑块,说明PDCD4有促进动脉粥样硬化发生发展的作用,具体机制尚不明确。研究目的(1)体内、外实验明确不同剪切力对血管内皮细胞PDCD4表达的影响；(2)明确PDCD4对血管内皮细胞增殖和凋亡的影响；(3)明确PDCD4在不同剪切力引起的血管内皮细胞增殖和凋亡中的作用.研究方法1.小鼠主动脉的en face免疫荧光染色C57BL/6、鼠经腹腔注射深度麻醉后,灌注,固定,分离主动脉弓及胸腹主动脉,去除周围结缔组织,纵向剖开主动脉弓,封闭后,两种一抗共同孵育4℃过夜,不同荧光标记的两种相应二抗共同37℃避光孵育,封片,共聚焦显微镜下观察。2.人脐静脉内皮细胞(HUVECs)的提取和培养无菌条件下获取剖宫产新生儿脐带,用胰酶消化法获取HUVECs,用20%M199培养基(20%胎牛血清+400μl/ml贝复济+青-链霉素)于37℃、5%CO2培养箱中培养,一代之后换10%M199培养基(10%胎牛血清+400μl/ml贝复济+青-链霉素)培养。3.体外剪切力干预体外培养的4-8代HUVECs用于实验。将HUVECs接种至铺有Ⅰ型鼠尾胶原的培养载片上,经完全培养基培养24小时后,继续静止培养或给予0、1、3、6、9、12小时的脉冲或震荡剪切力刺激,观察剪切力对HUVECs中PDCD4表达以及细胞功能的影响,不同剪切力描述如下：(1)脉冲剪切力：12dyne/cm2大小,频率为1Hz；(2)震荡剪切力：0±4dyne/cm2大小,频率为1Hz。4.蛋白质印迹(Western Blotting,WB)提取细胞蛋白,煮沸,经SDS-PAGE电泳,湿转法转膜,封闭,一抗4℃C孵育过夜,相对应的二抗孵育,化学发光法发光后,分析蛋白条带灰度值。5.免疫荧光染色给予培养载片上培养的HUVECs以12小时的脉冲剪切力、12小时的震荡剪切力或静止培养作对照,经固定、打孔、封闭、一抗4℃孵育过夜、荧光标记二抗37℃避光孵育、染核、封片后,共聚焦显微镜下观察不同组内皮细胞PDCD4的表达差异。6.细胞转染p-EGFP-C1-PDCD4和p-EGFP-C1-Mock质粒来自纽约大学Olubunmi Afonja博士的友情馈赠,分别用于HUVECs中过表达PDCD4和阴性对照。PDCD4的小干扰核糖核酸(Small Interfering RNA, siRNA)和Negative Control购自上海吉玛公司,序列如下：质粒和siRNA的转染均采用脂质体转染法,步骤遵照Lipo-2000说明书进行。7.5-溴脱氧尿嘧啶核苷(BrdU)法检测PDCD4在剪切力调节HUVECs增殖中的作用HUVECs的增殖功能检测运用BrdU法,所用试剂盒为美国罗氏公司的BrdU标记及检测试剂盒I (Cat. No.11296736001),实验步骤遵照试剂盒说明书进行：将HUVECs种在培养载片上进行不同刺激和处理,在刺激结束前6小时以1：1000的比例向培养基中加入BrdU labeling medium,刺激结束后用95%乙醇固定,经anti-BrdU4℃孵育过夜、Anti-mouse-Ig-fluorescei或nAlexa flour647标记的山羊抗小鼠免疫荧光二抗37℃避光孵育、4’,6-二脒基-2-苯基吲哚(4’,6-Diamidino-2-Phenylindole, DAPI)染核后封片,共聚焦显微镜下观察不同组HUVECs增殖功能的差异,计数并计算BrdU阳性细胞率。8.脱氧核苷末端转移酶介导的缺口末端标记(TUNEL)法检测PDCD4在剪切力调节HUVECs凋亡中的作用HUVECs的凋亡情况检测运用TUNEL法,所用试剂盒为美国Millipore公司的ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Cat. No. S7101),实验步骤遵照试剂盒说明书进行：将HUVECs种在培养载片上培养,予以不同刺激和处理,之后依次经免疫染色固定液固定、预冷的乙醇／乙酸液(乙醇：乙酸=2：1)-20℃C孵育、TdT酶37℃孵育、抗异羟基洋地黄毒苷过氧化物酶结合液室温孵育、二氨基联苯胺(3,3N-Diaminobenzidine Tertrahydroch-loride, DAB)显色、甲基绿染核、脱水封片后镜下观察不同组HUVECs凋亡的差异,计数并计算TUNEL阳性细胞率。9.统计学分析数据均以均数±均数标准误(mean±SEM)的形式表示,使用SPSS统计学软件进行数据分析,两组之间的比较采用非配对t检验,多组之间的比较运用单因素方差分析,P<0.05为统计学有显著性差异。研究结果1.小鼠体内促动脉粥样硬化血流作用区域内皮细胞PDCD4的表达量高于抗动脉粥样硬化血流作用区域胸主动脉直段的内皮细胞受抗动脉粥样硬化的脉冲剪切力作用,呈长梭状,沿血管长轴规律排列,而主动脉弓小弯侧的内皮细胞受促动脉粥样硬化的震荡剪切力及低剪切力作用,呈多向不规则状排列。主动脉弓小弯侧的内皮细胞PDCD4的表达量(以荧光强度表示)高于胸主动脉直段内皮细胞(P<0.05),说明在体内,抗动脉粥样硬化的剪切力下调内皮细胞PDCD4的表达,而促动脉粥样硬化的剪切力则上调内皮细胞PDCD4的表达。2.脉冲剪切力下调HUVECs中PDCD4的表达,震荡剪切力上调HUVECs中PDCD4的表达脉冲剪切力作用下的内皮细胞PDCD4蛋白水平随时间延长而逐渐下调,与静止对照组(0小时组)比较,脉冲剪切力作用3小时至12小时PDCD4蛋白水平下调有统计学意义(P<0.05)；在震荡剪切力作用下,PDCD4蛋白水平随时间延长而逐渐上调,与静止对照组(0小时组)比较,震荡剪切力作用6小时至12小时PDCD4蛋白上调有统计学意义(P<0.05)。免疫荧光染色结果也显示,脉冲剪切力作用后HUVECs中PDCD4的含量明显降低(P<0.05),而震荡剪切力作用后HUVECs中PDCD4的含量明显升高(P<0.05)。3. PDCD4促进HUVECs的增殖和凋亡与空载体对照组比较,转染pEGFP-C1-PDCD4以过表达PDCD4下调p21Wafl/cip1的表达(P<0.05),促进细胞的增殖(P<0.05)；而转染siRNA将PDCD4沉默,与阴性对照组比较,则上调p21Wafl/Cip1的表达(P<0.05),抑制细胞的增殖(P<0.05),表明PDCD4促进内皮细胞的增殖。与对照组比较,过表达PDCD4上调激活的Caspase-3(P<.05),促进细胞的凋亡(P<0.05),而沉默PDCD4,与阴性对照组比较,则抑制激活的Caspase-3(P<0.05),抑制细胞的凋亡(P<0.05),表明PDCD4促进内皮细胞的凋亡。4. PDCD4参与不同剪切力对内皮细胞增殖的调节,不参与其对凋亡的调节与静止对照组比较,脉冲剪切力上调p21Wafl/Cipl的表达(P<0.05),抑制内皮细胞的增殖(P<0.05)；而过表达PDCD4,与空载体阴性对照组比较,能够阻断脉冲剪切力对p21Waf1/cip1的促进(P<0.05)和对增殖的抑制作用(P<0.05)。与静止对照组比较,震荡剪切力抑制21Wafl/Cipl的表达(P<0.05),促进内皮细胞的增殖(P<0.05)；而沉默PDCD4,与阴性对照组比较,能够阻断震荡剪切力对p2iWaf1/Cipl的抑制(P<0.05)和对增殖的促进作用(P<0.05)。与静止对照组比较,脉冲剪切力抑制激活的Caspase-3(P<0.05),抑制内皮细胞的凋亡(P<0.05)；但过表达PDCD4,与空载体阴性对照组比较,不能阻断脉冲剪切力对对激活的Caspase-3的抑制及对凋亡的抑制。与静止对照组比较,震荡剪切力促进激活的Caspase-3(P<0.05),促进内皮细胞的凋亡(P<0.05)；但沉默PDCD4,与阴性对照组比较,不能阻断震荡剪切力对激活的Caspase-3及对凋亡的促进。以上结果表明,PDCD4参与不同剪切力对内皮细胞增殖的调节,但是不参与其对内皮细胞凋亡的调节。结论(1)脉冲剪切力下调血管内皮细胞PDCD4的表达,而震荡剪切力上调其表达；(2) PDCD4促进血管内皮细胞的增殖和凋亡；(3)PDCD4参与不同剪切力对血管内皮细胞增殖的调节作用,不参与剪切力对血管内皮细胞凋亡的调节作用。研究背景血流流经血管管腔产生剪切力作用于血管壁内表面的内皮细胞,激活血管内皮细胞胞膜上以及周围细胞外基质中的相关受体,改变细胞内的信号转导通路,影响多种基因表达和蛋白活性,维持血管内皮细胞在体内的相对稳定状态。在血管弯曲、分叉及狭窄处,血流受到干扰,剪切力由脉冲和层流剪切力变为震荡的低剪切力,引起内皮细胞功能障碍,促进动脉粥样硬化斑块形成,使这些部位成为动脉粥样硬化斑块的好发部位。近期研究发现,抑癌基因程序性细胞死亡4(Programmed Cell Death4, PDCD4)在心血管系统中同样发挥重要作用,如抑制血管平滑肌细胞、心肌细胞和成纤维细胞的增殖并促进其凋亡等。我们的前期工作发现PDCD4能同时促进血管内皮细胞的增殖和凋亡。我们在体内和体外实验中均证实剪切力能够调节血管内皮细胞PDCD4的蛋白含量,但其调节机制尚不明了。对众多肿瘤细胞和一些心血管系统细胞的研究发现,微小核糖核酸-21(microRNA-21, miR-21)与PDCD4的表达负相关。在PDCD4基因的3’非编码区有一个保守的miR-21结合位点,miR-21可直接与PDCD4的信使核糖核酸(Messenger Ribonucleic Acid, mRNA)结合抑制其表达。同时,其他研究发现肿瘤刺激物12-O-十四烷酰佛波醇-13-醋酸酯(12-O-tetradecanoylphorbol-13-acetate, TPA)以及给饥饿处理的细胞重新添加血清均可引起PDCD4蛋白的泛素化降解。以上两条途径均在转录后水平对PDCD4进行调节,而PDCD4在转录水平是如何被调节的鲜有报道。有研究发现小鼠的pdcd4基因序列中有一个可能的核转录因子κB (Nuclear Factor kappa B, NF-κB)结合位点,但该位点是否发挥作用尚不清楚。目前为止,对内皮细胞PDCD4表达调节机制的研究很少且存在争议。剪切力调节血管内皮细胞PDCD4表达的机制尚不明确,而niR-21、泛素-蛋白酶体系统及其他途径是否参与介导剪切力的作用也有待研究证实。研究目的(1)明确泛素-蛋白酶体系统在不同剪切力调节HUVECs PDCD4表达中的作用；(2)明确miR-21在HUVECs中对PDCD4表达的调节作用；(3)明确NF-κB在震荡剪切力上调HUVECs PDCD4表达中的作用。研究方法1.人脐静脉内皮细胞(Human Umbilical Vein Endothelial Cells, HUVECs)的提取和培养无菌条件下获取剖宫产新生儿脐带,胰酶消化法获取HUVECs,20%培养基(20%胎牛血清+400μl/ml贝复济+青-链霉素)置于37℃、5%CO2培养箱中培养。一代之后更换10%培养基(10%胎牛血清+400μl/ml贝复济+青-链霉素)培养。2.体外剪切力干预体外培养的4-8代HUVECs用于实验。将HUVECs接种至铺有Ⅰ型鼠尾胶原的Flexcell培养载片上,经10%培养基培养24小时后,予以适当处理,继续静止培养或给予脉冲或震荡剪切力刺激,观察剪切力对HUVECs中目的蛋白表达的影响,不同剪切力描述如下：(1)脉冲剪切力：12dyne/cm2大小,频率为1Hz；(2)震荡剪切力：0±4dyne/cm2大小,频率为1Hz。3.蛋白质印迹(Western Blotting, WB)提取细胞蛋白,煮沸,经SDS-PAGE电泳,湿转法转膜,封闭,一抗4℃孵育过夜,相对应的二抗孵育,化学发光法发光后,分析蛋白条带灰度值。4.实时荧光定量聚合酶链反应(Polymerase Chain Reaction, PCR)分析PDCD4mRNA的表达给予种在培养载片上的HUVECs以0、1、3、6、9、12小时的脉冲剪切力或震荡剪切力后,运用TRIzol法提取细胞总核糖核酸(Ribonucleic Acid, RNA),运用PrimeScript RT reagent Kit Perfect Real Time (TaKaRa Code:DRR037A)互补脱氧核糖核酸(Complementary Deoxyribonucleic Acid, cDNA)合成试剂盒将总RNA逆转录成cDNA,再使用SYBR Premix Ex Taq GC (Perfect Real Time)实时定量PCR试剂盒进行实时荧光定量PCR以测定各组内皮细胞PDCD4mRNA的表达。5.细胞转染β转导素重复包含蛋白(β-Transducin Repeat-Containing Protein, β-TrCP)和p65的小干扰核糖核酸(Small Interfering RNA, siRNA)、microRNA的模拟物(mimics)和抑制物(inhibitor)以及Negative Control (N.C)均购自上海吉玛公司,序列如下：siRNA及microRNA的模拟物和抑制物的转染均采用脂质体转染法,步骤遵照Lipo-2000说明书进行。6.免疫共沉淀对种在培养载片上的HUVECs予以刺激后,用Western及IP细胞裂解液提取细胞总蛋白,取出少量煮沸保存留作对照,向剩余提取液中加入Protein A/G PLUS Agarose和兔抗PDCD4单抗4℃C孵育过夜后收集琼脂糖凝胶珠,清洗后加入2×SDS上样缓冲液煮沸,取上清,进行Western Blotting检测其中相应的蛋白相对含量。7.统计学分析数据均以均数±均数标准误(mean±SEM)表示,使用SPSS统计学软件进行数据分析,多组之间的比较采用单因素方差分析,两组之间的比较采用非配对t检验,P<0.05为统计学有显著性差异。研究结果1.脉冲剪切力在转录后水平下调HUVECs中PDCD4的表达检测脉冲剪切力作用时间梯度下PDCD4的蛋白及mRNA水平,结果显示：与未经脉冲剪切力作用的0小时组比较,在脉冲剪切力作用下,PDCD4蛋白水平随时间延长而逐渐下调(P<0.05),但是其mRNA水平无明显变化(P>0.05),提示脉冲剪切力对HUVECs中PDCD4表达的下调作用发生在转录后水平。2.脉冲剪切力通过泛素-蛋白酶体通路下调PDCD4蛋白在剪切力刺激前1小时向培养基中加入蛋白酶体抑制剂MG-132(10μM)或乳胞素(10μM),二甲基亚砜(Dimethylsulfoxide, DMSO)作阴性对照；将E3配体β-TrCP的siRNA转染入HUVECs中将其沉默。结果显示：与DMSO组比较,蛋白酶体抑制剂和(3-TrCP的干扰下调均可阻断脉冲剪切力对PDCD4的下调(P<0.05),但在静止培养条件下均不影响PDCD4的表达(P>0.05)。免疫共沉淀结果显示：与静止对照组和震荡剪切力组比较,脉冲剪切力明显增加PDCD4的泛素化(P<0.05)。在细胞中转染miR-21的模拟物或抑制物(PTEN为HUVECs中miR-21的靶基因,作阳性对照),与转染阴性对照比较,并不影响PDCD4蛋白的表达(P>0.05)。3.磷脂酰肌醇3-激酶(Phosphatidyl Inositol3-Kinase, PI3K)/Akt通路介‘导脉冲剪切力对PDCD4的降解在进行剪切力刺激之前,用P13K抑制剂ⅠY294002(10μM)预处理细胞1小时,结果显示在LY294002作用下,和DMSO组比较,脉冲剪切力引起的Akt磷酸化被阻断(P<0.05),p70-S6K的激活及PDCD4磷酸化也被阻断(P<0.05),从而PDCD4的蛋白水平稳定并有上调趋势(P<0.05)。4.震荡剪切力在转录水平上调HUVECs中PDCD4的表达检测震荡剪切力作用时间梯度下PDCD4的蛋白及mRNA水平,结果显示：与未经震荡剪切力作用的0小时组比较,在震荡剪切力作用下,PDCD4蛋白水平随时间延长而逐渐上调(P<0.05),其mRNA水平也上调,在6小时达到峰值(P<0.05),提示震荡剪切力对HUVECs中PDCD4表达的上调作用发生在转录水平。5.泛素-蛋白酶体系统引起的PDCD4降解不参与震荡剪切力对PDCD4的上调作用在剪切力刺激前1小时向培养基中加入蛋白酶体抑制剂MG-132(10μM), DMSO作阴性对照,结果显示：与DMSO组比较,蛋白酶体抑制剂并不使PDCD4蛋白表达量进一步增加,反而阻断震荡剪切力引起的PDCD4上调(P<0.05),提示震荡剪切力可能不引起PDCD4的泛素化降解。免疫共沉淀结果显示,和静止对照组比较,震荡剪切力未明显改变PDCD4的泛素化(P>0.05)。另外,震荡剪切力不抑制反而上调Akt磷酸化(P<0.05),上调程度与脉冲剪切力比较无明显差别(P>0.05),该结果提示PI3K/Akt通路不参与震荡剪切力对血管内皮细胞PDCD4的上调。6. NF-κB参与震荡剪切力对PDCD4的上调作用在震荡剪切力刺激前1小时向培养基中加入NF-κB抑制剂MG-132(10μM), DMSO作阴性对照；将p65的siRNA转染入HUVECs中将其沉默,结果显示：与阴性对照组比较, NF-κB抑制剂和p65的干扰下调均阻断震荡剪切力对PDCD4的上调(P<0.05),表明NF-κB参与震荡剪切力对PDCD4的上调作用。结论(1)脉冲剪切力通过PI3K/Akt通路介导的泛素化降解下调血管内皮细胞PDCD4的蛋白；(2)震荡剪切力在转录水平上调血管内皮细胞PDCD4的表达,且NF-κB参与其中。更多还原

【Abstract】 BackgroundAtherosclerotic plaques preferentially develop at arterial branches and curvatures where endothelial cells bear low and oscillatory shear stress induced by disturbed blood flow, as opposed to straight parts which feature protective unidirectional pulsatile shear stress. Vascular endothelial cells, a monolayer in direct contact with the flowing blood, bear the most of the wall shear stresses and play an important role in maintaining homeostasis in response to stress. Ample evidence shows that pro-atherosclerotic disturbed flow induces sustained activation of atherogenic genes in endothelial cells to promote their oxidation, inflammation, cell cycle progression and proliferation, whereas pulsatile shear stress tends to maintain endothelial cells in a quiescent and less proliferative state with a low level of oxidation and inflammation.Programmed cell death4(PDCD4) is an important tumor suppressor in the development of various human cancers and inhibits translation rather than transcription. Specifically, the PDCD4protein combines directly with the mRNA coding region of the target gene (MYB/c-MYB) to block translation. It can also compete with eukaryotic translation initiation factor (eIF)4G and ribonucleic acid (RNA) for eIF4A binding and trap eIF4A in an inactive conformation to inhibit translation initiation via its two highly conserved MA3domains. Thus, PDCD4regulates molecules functioning during tumor cell proliferation, apoptosis, transformation, invasion and autophagy. Although PDCD4in general suppresses the development and progression of tumors, its specific biological functions differ by cell type.PDCD4also plays a role in cardiovascular cell biology by inhibiting proliferation and inducing apoptosis of most cardiovascular cells, including vascular smooth muscle cells, cardiac myocytes and fibroblasts, and repressing contractile gene expression in vascular smooth muscle cells. However, little is known about the action of PDCD4in endothelial cells.Our recent study of PDCD4and ApoE double-deficient mice (PDCD4-/-ApoE-/-) fed a high-cholesterol diet found that knockout of PDCD4was associated with reduced atherosclerosis plaque areas, so PDCD4downregulation might protect arteries against atherosclerosis. To determine whether PDCD4plays a role in the response of vascular endothelial cells to shear stresses, we examined PDCD4expression in endothelial cells under different type of shear stresses both in vivo and in vitro and performed gain and loss of function experiments to investigate the role of PDCD4in modulating turnover of vascular endothelial cells.ObjectivesWe investigated whether atheroprotective unidirectional pulsatile shear stress affects the expression of PDCD4in endothelial cells in vivo and in vitro and the role of PDCD4in modulating turnover of vascular endothelial cells.Materials and Methods MaterialsRabbit monoclonal antibody (mAb) for PDCD4, rabbit polyclonal antibody (pAb) for Cleaved Caspase-3and Caspase-3, rabbit mAb for p21Waf1/Cip1and mouse mAb for β-actin were from Cell Signaling Technology (Danvers, MA, USA). Rat mAb for PECAM-1were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse mAb for GAPDH and FITC-conjugated goat anti-rat IgG secondary antibodies were from ZSGB-BIO (Beijing). Alexa Fluor647-labeled goat anti-rabbit IgG secondary antibodies were from Beyotime Institute of Biotechnology (Haimen, Jiangsu, China). Rat tail collagen I was from BD Biosciences (San Jose, CA, USA). All other chemicals of reagent grade were from Invitrogen (Life Technologies, Carlsbad, CA, USA), unless otherwise noted.En Face Staining of Mice AortasFive C57BL/6mice (male,8-10weeks old,20-25g) were purchased from Peking University (Beijing). All animal experiments were performed in accordance with the Animal Management Rules of the Chinese Ministry of Health (document No55,2001) and with the approval of the Animal Care Committee of Shandong University.C57BL/6mice were deeply anesthetized with0.8%(wt/vol) pentobarbital sodium and transcardially perfused with30mL lukewarm saline, followed by100mL of4%paraformaldehyde. After perfusion, aortas were harvested and postfixed in this fixative solution for4hr and then subjected to en face immunostaining. Briefly, the adventitia was removed carefully, aortas were longitudinally dissected, blocked with5%(vol/vol) bovine serum albumin, then incubated with specific primary antibodies rat anti-CD31mAb and rabbit anti-PDCD4mAb at4℃overnight, then FITC-conjugated anti-rat IgG and Alexa Fluor647-conjugated anti-rabbit IgG secondary antibodies. Samples were counterstained with4’,6-diamidino-2-phenylindole (DAPI) for nuclei, mounted and photographed under a laser-scanning confocal microscope.Cell CultureHuman umbilical vein endothelial cells (HUVECs) were isolated from fresh human umbilical cords by trypsin perfusion. The cell pellet was resuspended in a culture medium consisting of medium199supplemented with20%(vol/vol) fetal bovine serum (FBS),2ng/ml fibroblast growth factor-2, and1%(vol/vol) penicillin/streptomycin. HUVECs (from passage4to7) were cultured in M199containing10%FBS with5%CO2at37℃for3days and then seeded onto glass slides precoated with collagen I.The study protocol conformed to the ethical guidelines of the1975Declaration of Helsinki with the approval of the Institutional Medical Ethics Committee of Qilu Hospital, Shandong University. All donors provided written informed consent.Flow ApparatusCells plated on collagen-coated slides were exposed to unidirectional pulsatile shear stress (1Hz) of12dynes/cm2or oscillatory shear stress (1Hz) of0±4dynes/cm2for0,1,3,6,9,12hr in a Streamer (Flexcell International Corp.), the parallel plate flow chamber, which was incorporated into a closed loop containing culture medium and kept in an incubator with5%CO2at37℃. A MasterFlex peristaltic pump (Cole Parmer, Vernon Hills, IL, USA) was applied to run the medium in the loop, and the type of shear stress was determined by the computer-controlled osci-flow apparatus (Flexcell International Corp.).Western Blot AnalysisCells were lysed with Cell lysis buffer for Western and IP and1mM phenylmethanesulfonyl fluoride. The total cell lysates were separated by SDS-PAGE and after incubation overnight at4℃with the designated antibodies, protein levels were analyzed by use of the LAS-4000luminescent image analyzer. Band densities were analyzed by use of Adobe Photoshop CS3.ImmunofluorescenceCells were fixed with4%paraformaldehyde and permeabilized with0.1%Triton X-100. After incubation overnight at4℃with anti-PDCD4antibodies, cells were incubated for1hr with Alexa647-conjugated secondary antibodies. Nuclei were stained with DAPI. Cells were examined under a laser-scanning confocal microscope.Transient TransfectionpEGFP-C1-Mock and pEGFP-C1-PDCD4plasmids were constructed and kindly provided by Dr. Olubunmi Afonja (New York University, New York). Transfection of vectors involved the Lipofectamine2000method.The sequence for PDCD4small interfering RNA (siRNA) was5’-GUGUUGGCAGUAUCCUUAG-3’. siRNA interference involved the Lipofectamine2000method. Bromodeoxyuridine (BrdU) Incorporation AssayProliferation was detected using the5-Bromo-2’-deoxy-uridine Labeling and Detection Kit I (Roche Diagnostics Corp, Indianapolis, IN, USA). Cells were treated with BrdU (10μM) for6hr before harvesting and were fixed at-20℃with Ethanol fixative, stained with Anti-BrdU working solution at4℃overnight, incubated with Anti-mouse-Ig-fluorescein or Alexa Fluor647-conjugated anti-mouse antibody for1hr at37℃and DAPI for8min at room temperature as a counterstain for the nucleus. The stained cells were examined using a laser-scanning confocal microscope. Proliferation was assessed based on the percentage of nuclei exhibiting BrdU incorporation.Apoptosis Detection (terminal deoxynucleotidyl transferase-mediated UTP nick end labeling, TUNEL)Apoptosis was detected using the ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Millipore, Billerica, MA, USA). Cells were fixed in1%paraformaldehyde and permeabilized in precooled ethanol:acetic acid2:1at-20℃. They were incubated with the Working Strength TdT Enzyme at37℃and then with the Anti-Digoxygenin Peroxidase Conjugate and Peroxidase Substrate to detect signs of apoptosis, staining brown. Counterstaining was carried out with methyl green. Apoptosis was assessed based on the percentage of TUNEL positive nuclei.Statistical AnalysisData are expressed as mean±SEM. Statistical analysis involved independent Student t test for comparing2groups and one-way ANOVA for multiple comparisons. P<0.05was considered statistically significant.ResultsPDCD4expression is maintained at a low level in areas of atheroprotective flow and induced in areas of atheroprone flow in vivoEndothelial cells were observed elongated and aligned with the longitudinal axis of the vessel in the straight segments of thoracic aortas, where shear stress is high and pulsatile; they showed a polygonal morphology in the inner curvature of aortic arches, where disturbed flow occurs with relatively low and oscillating shear stress or even under static conditions. Level of PDCD4was high in endothelial cell nuclei in the inner curvature of aortic arches, but was low in the straight segments of thoracic aortas. Thus, pulsatile shear stress in the native circulation maintains PDCD4expression at a low level in endothelial cells and oscillatory shear stress induces PDCD4expression in vivo.Pulsatile shear stress downregulates whereas oscillatory shear stress induces PDCD4protein level in HUVECsThe protein level of PDCD4was reduced with pulsatile shear stress since3hr (P<0.05) and induced with oscillatory shear stress since6hr (P<0.05) as compared with static control cells. Immunofluorescence of HUVECs revealed decreased PDCD4level in nuclei of cells under pulsatile shear stress (P<0.05) and induced under oscillatory shear stress (P<0.05) as compared with static conditions.PDCD4induces turnover (proliferation and apoptosis) of HUVECsOverexpression of PDCD4by transfecting cells with pEGFP-C1-PDCD4downregulated p21Waf1/Cip1protein level and increased proliferation as compared with mock treatment but upregulated that of Cleaved Caspase-3and induced apoptosis. Downregulation of PDCD4by siRNA did the opposite. Therefore, PDCD4induces turnover of HUVECs.Downregulation of PDCD4by pulsatile shear stress rescues p21Waf1/Cip1and reduces proliferation to slow the turnover of HUVECsHUVECs transfected with pEGFP-C1-PDCD4or pEGFP-C1-Mock were subjected to pulsatile shear stress or kept under static conditions. Pulsatile shear stress induced p21Waf1/Cip1protein expression and reduced proliferation. Overexpression of PDCD4blocked the induction of p21Waf1/Cip1and partly rescued proliferation reduction with pulsatile shear stress.HUVECs transfected with pEGFP-C1-PDCD4or pEGFP-C1-Mock were subjected to pulsatile shear stress or kept under static conditions. Pulsatile shear stress suppressed Cleaved Caspase-3and reduced apoptosis. Overexpression of PDCD4had no effect on the suppression of Cleaved Caspase-3or reduction of apoptosis with pulsatile shear stress.Upregulation of PDCD4by oscillatory shear stress reduces p21Waf1/Cip1and upregulates proliferation to maintain the turnover of HUVECsHUVECs transfected with PDCD4siRNA or negative control were subjected to oscillatory shear stress or kept under static conditions. Oscillatory shear stress reduced p21Wafl/Cipl protein expression and upregulated proliferation. Silense of PDCD4induced p21Waf1/Cipl and partly suppressed proliferation induction with oscillatory shear stress.HUVECs transfected with PDCD4siRNA or negative control were subjected to oscillatory shear stress or kept under static conditions. Oscillatory shear stress induced Cleaved Caspase-3and upregulated apoptosis. Silense of PDCD4had no effect on the induction of Cleaved Caspase-3or apoptosis with oscillatory shear stress.ConclusionsPulsatile shear stress reduces whereas oscillatory shear stress induces PDCD4protein expression in endothelial cells. PDCD4induces turnover (proliferation and apoptosis) of HUVECs. PDCD4level is associated with modulation of endothelial cell proliferation but not apoptosis under shear stresses. BackgroundVascular endothelial cells form a monolayer along the inner wall of blood vessels. Blood flows inside the tubes and generates shear stress against vessel wall surface. Shear stress activates receptors on the membrane of endothelial cells or in the extracellular matrix around and subsequently activates or suppresses specific signal pathways, modulates various gene expression and protein activity to keep the homeostasis of vascular endothelial cells. However, at bifurcations and curvatures of vessels or where the lumen is restricted, blood flow is disturbed and shear stress changes from laminar and pulsatile to low and oscillatory, which results in endothelial cell dysfunction and promotes onset of atherosclerotic plaques.Tumor suppressor programmed cell death4(PDCD4) was recently found important in cardiovascular cell biology, which inhibits proliferation and induces apoptosis of vascular smooth muscle cells, cardiac myocytes and fibroblasts, and represses contractile gene expression in vascular smooth muscle cells. Our above work showed that PDCD4promotes both proliferation and apoptosis of vascular endothelial cells. Chemical stimuli as well as mechanical stresses alter PDCD4expression. We have confirmed that shear stresses modulate protein level of PDCD4in vascular endothelial cells both in vivo and in vitro. However, the mechanisms involved remain unclear.Studies of tumor cells and several cardiovascular cells confirmed a negative correlation between microRNA-21(miR-21) and PDCD4. A conserved target site for miR-21was found within the3’untranslated region of PDCD4gene and miR-21blocked PDCD4expression by directly binding to the PDCD4mRNA. However, several other studies showed that refueling the starved cells with serum and tumor promoter12-O-tetradecanoylphorbol-13-acetate induced degradation of PDCD4protein via the ubiquitin-proteasome pathway. Both ways above suppress PDCD4expression post-transcriptionally. Little is known about modulation of PDCD4expression at the transcription stage. A putative binding site for nuclear factor kappaB (NF-κB) was found in the pdcd4gene in mice, but whether the sequence functions is unclear.The mechanism of PDCD4modulation by shear stresses is still under cover. Whether miR-21and the ubiquitin-proteasome system or other pathways are involved in the modulation remains to be clarified.ObjectivesWe investigated whether the reduction of PDCD4under pulsatile shear stress was via the ubiquitin-proteasome pathway and if NF-κB was involved in the induction of PDCD4under oscillatory shear stress.MethodsMaterialsRabbit monoclonal antibody (mAb) for PDCD4, Akt,(3-transducin repeat-containing protein (β-TrCP) and p65, rabbit polyclonal antibody (pAb) for ubiquitin, mouse mAb for phospho-Akt (Ser473),(3-actin and phosphatase and tensin homologue deleted on chromosome ten (PTEN), MG-132and LY294002were from Cell Signaling Technology (Danvers, MA, USA). Rabbit pAbs for70-kDa ribosomal protein S6kinase (p70-S6K) and phospho-p70-S6K (T412) were from Immunoway (Newark, NJ, USA). Protein A/G plus agarose were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit pAb for PDCD4(phospho S67) were from Abcam (Cambridge, UK). Mouse mAb for reduced glyceraldehyde-phosphate dehydrogenase (GAPDH) were from ZSGB-BIO (Beijing). Negative control microRNA inhibitor, miR-21inhibitor, negative control microRNA mimics and miR-21mimics were from GenePharma (Shanghai). Rat tail collagen I was from BD Biosciences (San Jose, CA, USA). Lactacystin was from Sigma (St. Louis, MO, USA). All other chemicals of reagent grade were from Invitrogen (Life Technologies, Carlsbad, CA, USA), unless otherwise noted.Cell CultureHuman umbilical vein endothelial cells (HUVECs) were isolated from fresh human umbilical cords by trypsin perfusion. The cell pellet was resuspended in a culture medium consisting of medium199supplemented with20%(vol/vol) fetal bovine serum (FBS),2ng/ml fibroblast growth factor-2, and1%(vol/vol) penicillin/streptomycin. HUVECs (from passage4to7) were cultured in M199containing10%FBS with5%CO2at37℃for3days and then seeded onto glass slides precoated with collagen I.The study protocol conformed to the ethical guidelines of the1975Declaration of Helsinki with the approval of the Institutional Medical Ethics Committee of Qilu Hospital, Shandong University. All donors provided written informed consent.Flow ApparatusCells plated on collagen-coated slides were exposed to unidirectional pulsatile shear stress (1Hz) of12dynes/cm2or oscillatory shear stress (1Hz) of0±4dynes/cm2for0,1,3,6,9,12hr in a Streamer (Flexcell International Corp.), the parallel plate flow chamber, which was incorporated into a closed loop containing culture medium and kept in an incubator with5%CO2at37℃. A MasterFlex peristaltic pump (Cole Parmer, Vernon Hills, IL, USA) was applied to run the medium in the loop, and the type of shear stress was determined by the computer-controlled osci-flow apparatus (Flexcell International Corp.). Western Blot AnalysisCells were lysed with Cell lysis buffer for Western and IP and1mM phenylmethanesulfonyl fluoride. The total cell lysates were separated by SDS-PAGE and after incubation overnight at4℃with the designated antibodies, protein levels were analyzed by use of the LAS-4000luminescent image analyzer. Band densities were analyzed by use of Adobe Photoshop CS3.RNA Isolation and Real-time PCRTotal RNA was extracted by use of TRIzol reagent. The first-strand cDNA was synthesized from2μg total RNA with use of random primers and the PrimeScript RT reagent kit (Takara Bio Inc.; Otsu, Shiga, Japan). Real-time PCR involved the SYRB Premix Ex Taq kit (Takara Bio Inc.). Primers for PDCD4were forward,5’-TGAGCACGGAGATACGAACGA-3’and reverse,5’-GCTAAGGACACTGCCAACACG-3’; and p-actin forward,5’-CGTGCGTGACATTAAGGAGA-3’and reverse,5’-CACCTTCACCGTTCCAGTTT-3’.β-actin was used as housekeeping gene. The relative mRNA expression level was assessed by the2-ΔΔCt method.ImmunoprecipitationCells were lysed with Cell lysis buffer for Western and IP and1mM phenylmethanesulfonyl fluoride. The same amount of protein from each sample was incubated with cognate antibodies for2hr at4℃, then with protein A/G plus agarose overnight at4℃with gentle rotation. The agarose-bound immunoprecipitates were rinsed and collected by centrifugation and incubated with the SDS-PAGE sample loading buffer (2×)(Beyotime Institute of Biotechnology), and subjected to SDS-PAGE and western blot analysis.Transient TransfectionThe sequence for the miR-21inhibitor was5’-UCAACAUCAGUCUGAUAAGCUA-3’and those for miR-21mimics were5’-UAGCUUAUCAGACUGAUGUUGA-3’and5’-AACAUCAGUCUGAUAAGCUAUU-3’. The sequence for β-TrCP siRNA were5’-GCACUUGCGUUUCAAUAAUTT-3’ and 5’-AUUAUUGAAACGCAAGUGCTT-3’; those for p65were5’-CGGAUUGAGGAGAAACGUATT-3’. MicroRNA inhibitor, mimics and siRNA interference involved the Lipofectamine2000method (Invitrogen).Statistical AnalysisData are expressed as mean±SEM. Statistical analysis involved independent Student t test for comparing2groups and one-way ANOVA for multiple comparisons. P<0.05was considered statistically significant.ResultsPulsatile shear stress post-transcriptionally reduces PDCD4expression in HUVECsWe examined both the protein and mRNA level of PDCD4and found that PDCD4protein level decreased gradually over time with pulsatile shear stress (P<0.05), however, with only a slight and non-significant increase in mRNA level (P<0.05). Therefore, pulsatile shear stress decreases PDCD4expression post-transcriptionally in HUVECs.Pulsatile shear stress reduces PDCD4protein level via the ubiquitin-proteasome pathwayThe proteasome inhibitor MG-132(10μM) or lactacystin (10μM) was added into the medium1hr before and during the shearing process with HUVECs, with dimethyl sulfoxide (DMSO) as a control. We also transfected the cells with β-TrCP siRNA. β-TrCP is an ubiquitin ligase involved in degradation of PDCD4. Either proteasome inhibitors or downregulation of β-TrCP completely rescued the PDCD4level downregulated with pulsatile shear stress (P<0.05), but neither affected PDCD4level under static conditions (P>0.05). Results of coimmunoprecipitation assays confirmed that pulsatile shear stress significantly induced ubiquitination of PDCD4as compared with oscillatory shear stress or static conditions (P<0.05). Hence, pulsatile shear stress reduces PDCD4protein level via the ubiquitin-proteasome pathway. We also transfected HUVECs with miR-21mimics or miR-21inhibitor (PTEN as a positive control, which is confirmed to be a target of miR-21in HUVECs), with no alteration of PDCD4expression (P>0.05), so the ubiquitin-proteasome pathway is more implied in the mechanism.Phosphatidyl inositol3-kinase (PI3K)/Akt pathway mediates the degradation of PDCD4by pulsatile shear stressHUVECs were pretreated with the specific PI3K inhibitor LY294002(10μM) or vehicle control for1hr, then were subjected to pulsatile shear stress for various times. Along with inhibition of Akt phosphorylation (P<0.05), activation of p70-S6K and phosphorylation of PDCD4were blocked (P<0.05), whereas PDCD4level was retained or even upregulated (P<0.05), which indicates that the PI3K/Akt pathway mediates the degradation of PDCD4by pulsatile shear stress.Oscillatory shear stress transcriptionally induces PDCD4expression in HUVECsWe examined both the protein and mRNA level of PDCD4and found that PDCD4protein level increased gradually over time with oscillatory shear stress, and PDCD4mRNA was also upregulated with a peak at6hr. Therefore, oscillatory shear stress increases PDCD4expression transcriptionally in HUVECs.Ubiquitin-proteasome-mediated degradation of PDCD4is not involved in the induction of PDCD4under oscillatory shear stressThe proteasome inhibitor MG-132(10μM) was added into the medium1hr before and during the shearing process with HUVECs, with DMSO as control. The proteasome inhibitor blocked but not promoted the PDCD4level induced by oscillatory shear stress (P<0.05), which indicates that oscillatory shear stress may not induce ubiquitin-proteasome degradation of PDCD4. Coimmunoprecipitation assays showed that oscillatory shear stress did not alter ubiquitination of PDCD4compared with static control (P>0.05). Phosphorylation of Akt was also induced but not suppressed by oscillatory shear stress (P<0.05) and no obvious difference was observed between Akt phosphorylation by oscillatory shear stress and that by pulsatile shear stress (P>0.05), which indicates that PI3K/Akt pathway is not involved in the induction of PDCD4by oscillatory shear stress. NF-κB is involved in the induction of PDCD4under oscillatory shear stressWe added NF-κB innibitor MG-132(10μM) into the medium1hr before and during the shearing process with HUVECs, with DMSO as control. We also transfected the cells with p65siRNA. We found that either NF-κB inhibitor or downregulation of p65blocked the PDCD4level induced by oscillatory shear stress (P<0.05), which indicates that NF-κB is involved in the induction of PDCD4under oscillatory shear stress.ConclusionsPulsatile shear stress induces ubiquitin-proteasome-mediated degradation of PDCD4via PI3K/Akt pathway in HUVECs. NF-κB is involved in the induction of PDCD4in HUVECs under oscillatory shear stress.更多还原