PI3K对IL-8／Rac1信号通路介导的内皮细胞迁移的影响

目的研究磷脂酰肌醇-3激酶（PI3K）对IL-8／Rac1信号通路所介导的内皮细胞迁移的影响。方法选择P13K抑制剂wortmannin及划痕创伤愈合修复分析,研究不同wortmannin的作用浓度和不同时间对IL-8／Rac1信号通路诱导的内皮细胞迁移的影响。结果不同wortmannin的作用浓度和不同时间预处理均可抑制IL-8／Rac1信号通路介导的内皮细胞迁移,其中wortmannin预处理浓度为100nmol／L,预处理时间为20min时IL-8诱导的内皮细胞迁移水平最低。结论P13K能够影响IL-8／Rac1信号通路介导的内皮细胞迁移;P13K抑制剂wortmannin的最佳作用浓度为100nmol／L,最佳作用时间为20min。     

基因突变和转基因技术评价TLR-4信号受体在流体切应力诱导血管内皮细胞IL-8基因转录激活中的作用

用基因突变和转基因技术，评价TLR-4信号受体在层流低切应力刺激诱导血管内皮细胞IL-8基因转录激活中的作用，RT-PCR，Northern杂交和免疫荧光细胞化学染色均显示脐静脉血管内皮细胞表达TLR-4，同时RT-PCR和Northern杂交显示，层流切应力刺激1h后血管内皮细胞TLR-4表达增强，用RT-PCR技术从血管内皮细胞扩增出胞内区段缺失突变TLR-4cDNA，用PCR技术从其基因组DNA中扩增出IL-8上游调控序列，分别克隆于真核表达质粒pcDNA3和绿色荧光增强蛋白报告基因pEGFP1质粒，构建出重组TLR-4缺失突变基因真核表达质粒pcDNA3-mTLR4和IL-8报告基因表达质粒pEGFP1-IL8USCS。用pEGFP1-IL8USCS转染或pcD-NA3-mTLR4和pEGFP1-IL8USCS共转染ECV304细胞，4.2dyne/cm^2层流切应力刺激3h后，流式细胞仪观察荧光蛋白表达强度变化，用pEGFP1-IL8USCS转染细胞，经层流切应力刺激3h后荧光蛋白表达增强（1.06:2.71)，同样用pcDNA3-mTLR4和pEGFP1-IL8USCS共转染细胞，层流切应力刺激3h后荧光蛋白表达未明显增强，提示TLR4/NF-кB信号传导通路可能介导层流切应力诱导血管内皮细胞IL-8基因的表达。     

层流剪应力对内皮细胞IL-8受体CXCR1表达的影响

为研究内皮细胞在不同时间、不同大小层流剪应力作用下IL-8受体CXCR 1的表达规律,通过体外培养人脐静脉内皮细胞株EA.Hy926细胞,分别施加5.56、10.02、15.27 dyn/cm2三种剪应力,选取剪切1、2、4和8 h等4个时间点进行观测,Western blot检测IL-8受体CXCR 1表达变化。结果表明,在5.56 dyn/cm2剪应力作用下,与静止组相比,CXCR 1表达随作用时间的增加而显著升高(P<0.01),至4h达到最大值,为对照组的2.2倍。在10.02dyn/cm2剪应力作用下,CXCR 1的表达随剪应力作用时间而相对缓慢增加,但仍高于对照组(P<0.05)。在15.27dyn/cm2剪应力作用下,其CXCR 1的表达随时间呈现较显著性降低(P<0.01),当持续作用8 h,其CXCR 1的表达为对照组62.59%。以上结果表明,不同强度、作用时间的层流剪应力参与调节IL-8受体CXCR 1的表达。     

Rac1参与调节IL-8诱导的内皮细胞迁移

研究Rac1是否参与调节IL-8诱导的内皮细胞迁移.采用Transwell小室迁移率分析法,考察不同Matrigel稀释比例以及IL-8不同作用时间对内皮细胞迁移的影响;同时用RT-PCR法检测Rac1 mRNA的表达.结果表明,Matrigel稀释比例为1:2时与比例为1:3、1:8时细胞迁移数量有显著性差异,而比例为1:4、1:5和1:6时与其他各组没有显著性差异;故选用Matrigel稀释比例1:4进行后期实验,随着IL-8作用时间的增加,内皮细胞迁移数量也逐渐增加;IL-8作用6h后,Rac1 mRNA的表达较其他各时间组稍强.以上结果提示,Rac1参与调节IL-8诱导的内皮细胞迁移,本实验为进一步研究Rac1在IL-8诱导内皮细胞迁移中的作用奠定基础.     

粘着斑激酶在血管内皮细胞黏附和迁移中的作用

目的 采用粘着斑激酶（focal adhesion kinases,FAK)抑制剂抑制FAK在Y397位点的酪氨酸磷酸化,测定不同浓度的FAK抑制剂的对内皮细胞黏附、迁移及下游信号Rac1蛋白表达的影响,探索粘着斑激酶在内皮细胞黏附和迁移中的作用。方法 运用内皮损伤模型（划痕法）测定FAK抑制剂在2、4、8、24 h各时间点对EA.hy 926细胞迁移的影响。Western blot结合免疫荧光测定不同浓度的0~250 nmol/mL的FAK抑制剂的加入对Rac1蛋白分布和表达的影响。结果 随着FAK抑制剂浓度的增加,细胞迁移距离减少,Rac1蛋白表达逐渐减弱。结论 抑制FAK的磷酸化将抑制内皮细胞的黏附和迁移的生物学行为,下游Rac1蛋白表达降低。内皮细胞的黏附和迁移与FAKRho GTPases 信号轴相关。     

流体剪应力对EA.Hy926细胞IL-8受体mRNA表达的影响

为研究EA．Hy926细胞在不同时间、不同大小流体剪应力作用下IL-8受体CXCR1、CXCR2 mRNA的表达规律,通过体外培养人脐静脉内皮细胞株EA．Hy926细胞,分别施加5．56、10．02、15．27 dyn／cm2三种剪应力,选取剪切5 min、10 min、15 min、20 min、25 min和30 min、1、2、4和8 h等10个时间点进行观测,以半定量RT- PCR方法检测IL-8受体mRNA表达变化。结果表明,在5．56 dyn／cm2剪应力作用下,与静止组相比,各时间点CXCR1 mRNA与CXCR2 mRNA表达均显著升高(P<0．05)。在10．02 dyn／cm2剪应力作用下,CXCR1 mRNA表达随时间相对缓慢下降;而CXCR2 mRNA表达在30min出现短暂的升高,然后随着作用时间的延长开始缓慢下降。在15．27 dyn／cm2剪应力作用下,其CXCR1、CXCR2 mRNA的表达随时间呈现较显著性降低(P<0．01),当持续作用4 h以上其CXCR2 mRNA表达极低。以上结果表明,不同强度、作用时间的流体剪应力参与调节IL-8受体表达。     

IL-38拮抗RhoA/ROCK信号抑制PDGF-BB诱导的血管平滑肌细胞增殖和迁移

目的探究白细胞介素(IL)-38对血小板源性生长因子-BB(PDGF-BB)诱导的血管平滑肌细胞增殖和迁移的作用和机制。方法将人主动脉血管平滑肌细胞(HA-VSMC)随机分成4组:对照组(不进行任何处理)、PDGF-BB组(加入20 ng/ml重组人PDGF-BB蛋白处理24 h)、PDGF-BB+空质粒组(转染pcDNA3.1空质粒48 h后加入20 ng/ml重组人PDGF-BB蛋白处理24 h)和PDGF-BB+IL-38组(转染pcDNA3.1-IL-38质粒48 h后加入20 ng/ml重组人PDGF-BB蛋白刺激24 h)。MTT法检测细胞活力、Transwell检测细胞迁移、Western blot检测增殖细胞核抗原(PCNA)、RhoA、Rho相关蛋白激酶(ROCK)1、ROCK2、肌球蛋白磷酸酶靶标亚基1(MYPT1)和磷酸化MYPT(p-MYPT)蛋白水平的表达。结果与对照组相比,PDGF-BB组中细胞活力,迁移细胞数目,PCNA、RhoA、ROCK1、ROCK2的表达和p-MYPT/MYPT1的值均增加,差异均具有统计学意义(均P0.05);与PDGFBB组相比,PDGF-BB+空质粒组中这些指标变化无统计学意义(均P0.05);与PDGF-BB+空质粒组相比,PDGF-BB+IL-38组中细胞活力,迁移细胞数目,PCNA、RhoA、ROCK1、ROCK2的表达和p-MYPT/MYPT1的值均降低,差异均具有统计学意义(均P0.05)。结论 IL-38能够减弱PDGF-BB诱导的血管平滑肌细胞增殖和迁移,这种作用可能是通过抑制RhoA/ROCK通路活化发挥作用的。     

ERK1/2信号转导途径在低切应力上调人脐静脉内皮细胞IL-8基因表达中的作用

血管内皮细胞位于血流和血管壁之间,除受化学因素的调节外还受力学因素的影响。切应力可通过刺激相应的力学感受系统来调节内皮细胞某些基因的表达,其中包括诱导内皮细胞表达IL- 8。为了阐明MAPK信号途径中的ERK1/ 2信号通路是否参与调控低切应力上调人脐静脉内皮细胞IL- 8基因表达,采用Western blot分析了低切应力(4.2 0 dyne/ cm2 )处理不同时间内皮细胞ERK1/ 2的磷酸化水平及TPK抑制剂Genistein,MEK抑制剂PD980 5 9对其磷酸化的影响;采用定量RT- PCR检测内皮细胞经低切应力刺激或给予阻断剂后再行切应力刺激等处理后IL- 8基因的表达。结果显示:(1)低切应力处理可引起内皮细胞ERK1/ 2蛋白磷酸化水平上调,其磷酸化水平与切应力作用时间有关,具有快速、双向性的特点(在刺激10 min时达到高峰,2 h左右降至未刺激水平) ,阻断剂Genistein和PD980 5 9处理后,ERK1/ 2磷酸化水平与低切应力刺激10 min比较明显降低;(2 )阻断剂Genistein,PD980 5 9可显著抑制低切应力所致的内皮细胞IL- 8m RNA上调。结果表明低切应力可通过ERK1/ 2信号途径上调人脐静脉内皮细胞IL- 8基因的表达。     

TLR-4信号转导通路介导层流切应力诱导血管内皮细胞IL-8基因表达的研究

目的: 观察TLR-4信号转导通路在层流低切应力诱导血管内皮细胞IL-8基因表达中的作用。方法: 设计突变引物, 用RT-PCR技术从血管内皮细胞扩增出胞内区段缺失突变TLR4 cDNA, 用PCR技术从其DNA中扩增出IL-8上游调控序列(IL-8USCS), 分别克隆于真核表达质粒pcDNA3及绿色荧光增强蛋白报告基因pEGFP1质粒,构建出重组TLR-4缺失突变基因真核表达质粒pcDNA3-mTLR4和IL-8报告基因表达质粒pEGFP1-IL8USCS。用pEGFP1-IL8USCS转染或pcDNA3-mTLR4和pEGFP1-IL8USCS共转染ECV304细胞, 用4.2 dyne/cm²层流切应力刺激3 h, 流式细胞仪观察荧光蛋白表达强度变化。细胞裂解物IκB免疫印迹和NF-κB p65免疫荧光细胞化学染色检测IκB的磷酸化及降解和NF-κB的活化。结果: 用pEGFP1-IL8USCS转染细胞, 经层流切应力刺激3 h后荧光蛋白表达增强(1.06:2.71), 同样用pcDNA3-mTLR4和pEGFP1-IL8USCS共转染细胞, 层流切应力刺激3 h后荧光蛋白表达未明显增强。细胞裂解物免疫印迹显示, 刺激10 min时磷酸化IκB即显著增强, 1 h后磷酸化IκB印迹强度几乎降到基线水平, 而IκB随刺激时间延长而逐渐降低, 1 h后几乎测不到IκB。NF-κB p65免疫荧光细胞化学染色显示, 切应力刺激0.5 h, 胞核即出现阳性反应, 刺激1.5 h、2 h后, 胞核呈强阳性染色。结论: 本研究提示, TLR4/NF-κB信号转导通路可能介导层流切应力诱导血管内皮细胞IL-8基因的表达。     

IL-8对肺癌NCI-H157细胞增殖和迁移的影响

目的 研究IL-8对肺癌细胞增殖和迁移的影响,初步探讨IL-8调控肺癌细胞的分子机制.方法 体外培养肺癌NCI-H157细胞,用不同浓度的IL-8刺激肺癌细胞,分别用MTT法检测IL-8对肺癌细胞的增殖作用;划痕损伤实验和Transwdl小室两种方法检测肺癌细胞的迁移能力;Western blot检测Rac1和Cdc 42蛋白的表达变化.结果 IL-8促进NCI-H157细胞增殖,但随浓度增加,细胞增殖活性差异无统计学意义;细胞划痕损伤和Transwell实验均表明IL-8可诱导NCI-H157细胞迁移,且具有浓度依赖性;Western blot结果显示,随着IL-8浓度增加,Rac1和Cdc 42的表达水平逐渐升高,以Cdc42变化最为显著.结论 IL-8可促进肺癌细胞增殖和迁移,可能与Rac1和Cdc42表达有关.     

Shear stress protects against endothelial regulation of vascular smooth muscle cell migration in a coculture system.

Vascular endothelial cells (ECs) are constantly exposed to blood flow-induced shear stress; these forces strongly influence the behaviors of neighboring vascular smooth muscle cells (VSMCs). VSMC migration is a key event in vascular wall remodeling. In this study, the authors assessed the difference between VSMC migration in VSMC/EC coculture under static and shear stress conditions. Utilizing a parallel-plate coculture flow chamber system and Transwell migration assays, they demonstrated that human ECs cocultured with VSMCs under static conditions induced VSMC migration, whereas laminar shear stress (1.5 Pa, 15 dynes/cm2) applied to the EC side for 12 h significantly inhibited this process. The changes in VSMC migration is mainly dependent on the close interactions between ECs and VSMCs. Western blotting showed that there was a consistent correlation between the level of Akt phosphorylation and the efficacy of shear stress-mediated EC regulation of VSMC migration. Wortmannin and Akti significantly inhibited the EC-induced effect on VSMC Akt phosphorylation and migration. These results indicate that shear stress protects against endothelial regulation of VSMC migration, which may be an atheroprotective function on the vessel wall.     

流体切应力对人内皮细胞迁移和整合素基因表达的影响

目的研究流体切应力空间梯度和均匀切应力对内皮细胞（ECs）迁移和整合素基因表达的影响，为阐明应力诱导血管重建的分子机制提供一些实验依据。方法将脐静脉内皮细胞置于平行平板流动腔系统，分别施加11dyn／cm^2的均匀切应力（矩形组）和5～14dyn／cm^2的切应力梯度（梯度组）为受力组，以静态条件下培养的ECs为对照组（静止组）。Transwell方法检测切应力对ECs迁移能力的影响；半定量RT-PCR测定ECs整合素α3β1、α5β1mRNA3、6和12h的表达情况。结果①同静止组相比，切应力梯度组ECs3h的迁移（88±4 vs 23±3，P〈0．01，n=3）能力显著提高；而均匀切应力组ECs3h迁移（21±3VS23±3）同静止组相比，差异无统计学意义（P〉0．05，n=3）；②ECs受切应力梯度作用3h即可诱导ECs整合素α3β1、α5β1mRNA表达（P〈0．01，P〈0．05）；均匀切应力于6h激活ECs整合素理，亚型表达（P〈0．01），α3 、β1亚型表达延迟到12h激活（P〈0．01）。结论切应力通过上调整合素基因表达促进了ECs迁移，提示整合素信号通路参与了切应力条件下ECs迁移过程的信号转导。     

NADPH oxidase-dependent signaling in endothelial cells: role in physiology and pathophysiology

Reactive oxygen species (ROS) including superoxide (O(2)(.-)) and hydrogen peroxide (H(2)O(2)) are produced endogenously in response to cytokines, growth factors; G-protein coupled receptors, and shear stress in endothelial cells (ECs). ROS function as signaling molecules to mediate various biological responses such as gene expression, cell proliferation, migration, angiogenesis, apoptosis, and senescence in ECs. Signal transduction activated by ROS, "oxidant signaling," has received intense investigation. Excess amount of ROS contribute to various pathophysiologies, including endothelial dysfunction, atherosclerosis, hypertension, diabetes, and acute respiratory distress syndrome (ARDS). The major source of ROS in EC is a NADPH oxidase. The prototype phagaocytic NADPH oxidase is composed of membrane-bound gp91phox and p22hox, as well as cytosolic subunits such as p47(phox), p67(phox) and small GTPase Rac. In ECs, in addition to all the components of phagocytic NADPH oxidases, homologues of gp91(phox) (Nox2) including Nox1, Nox4, and Nox5 are expressed. The aim of this review is to provide an overview of the emerging area of ROS derived from NADPH oxidase and oxidant signaling in ECs linked to physiological and pathophysiological functions. Understanding these mechanisms may provide insight into the NADPH oxidase and oxidant signaling components as potential therapeutic targets.     

IL-8-mediated angiogenic responses of endothelial cells to lipid antigen activation of iNKT cells depend on EGFR transactivation

iNKT cells are a unique T cell subset, which is CD1d-restricted and specific for glycolipid antigens. In advanced atherosclerotic plaques, focal collections of inflammatory cells correlate with areas of intraplaque neovascularization. We reported recently that iNKT cells might facilitate intraplaque neovascularization by enhancing EC migration and sprouting in an IL-8-dependent manner. This study investigated the participating effector mechanisms. In ECs, CM, derived from antigen-stimulated human iNKT cells (CM+), induced up-regulation of IL-8R CXCR2 and the phosphorylation of EGFR and of multiple intracellular signaling effectors, including FAK, Src, Erk, Jnk, p38-MAPK, and STAT1 and -3. We found that a cascade of events, which were IL-8-dependent and involved EGFR activation, was responsible for signaling through FAK and Src kinases and necessary for acquisition of angiogenic morphology, migration in a two-dimensional wound assay, and sprout outgrowth in a three-dimensional model of angiogenesis in vitro. The data support that IL-8-dependent activation of angiogenic behavior in ECs, in response to activated iNKT, involves CXCR2, transactivation of EGFR, and subsequent FAK/Src signaling. We found too that activated iNKT increased VEGFR2 expression in ECs. Functional studies confirmed that EGF is the motogenic-enhancing factor in CM+ and is necessary, together with an exogenous source of VEGF, for iNKT-promoted sprout formation. EGFR inhibition may represent a novel therapeutic modality aimed at plaque stabilization through control of neovascularization within developing atherosclerotic plaques.     

流体切应力作用时间对内皮细胞IL-8 基因表达的影响

内皮细胞对力学环境变化敏感，流体切应力可以直接调节内皮细胞基因的表达。为阐明内皮细胞白细胞介素-8（IL-8）基因的表达除受化学因子的调节外还受力学因素的影响，本文用流体切应力（2.23、4.20、6.08dyne/cm^2）处理培养的人脐静脉内皮细胞，然后采用定量RT-PCR的方法检测内皮细胞IL-8基因的表达情况。结果显示：未用切应力处理的内皮细胞没有IL-8基因的表达；切应力处理内皮细胞后，1h IL-8mRNA表达增加，2hIL-8mRNA表达量至最高值，3hIL-8mRNA表达量开始下降，4h后IL-8mRNA持续低表达；各实验组（2.23、4.20、6.08dyne/cm^2）均表现出相同的IL-8mRNA随时间的变化规律。提示流体切应力确可诱导内皮细胞表达IL-8，而且IL-8的表达量与切应力作用时间有关，呈双相性变化。流体切应力诱导内皮细胞表达IL-8，可能在急性炎症和动脉粥样硬化的发生、发展过程中具有重要作用。     

流体低切应力对内皮细胞IL—8mRNA表达的影响

为证实内皮细胞IL-8表达不仅受化学因子的调节,而且还受力学因素的影响。我们用低层流切应力（4.2dyne/cm^2）处理培养的人脐静脉内皮细胞后采用RT-PCR法检测内皮细胞IL-8基因表达,并用免疫细胞化学染色法检测内皮细胞内NF-kB激活的影响,结果发现：①未用切应力处理和切应力作用0.5h后内皮细胞IL-8mRNA表达量很少,切应力作用1h后内皮细胞IL-8 mRNA表达增加,2h进一步增加。②未用切应力处理和切应力作用0.5h内皮细胞核内NF-kBp65染色阴性,切应力作用1h呈弱阳性,2h呈阳性。提示低层流切应力可诱导内皮细胞表达增加,IL-8表达量与切应力作用时间有关。流体切应力可诱导内皮细胞内NF-kB的激活,激活程度与作用时间有关。低切应力诱导内皮细胞表达IL-8,可能在急性炎症和动脉粥样硬化发生、发展过程中具有重要作用。     

Gene response in endothelial cells cultured on engineered surfaces is regulated by shear stress

In vitro endothelialization of small-diameter synthetic vascular prostheses confluently lined with cultured autologous endothelial cells (ECs) before implantation has been shown to increase their patency. Many authors have studied the effects of shear stress on EC gene response seeded on various substrates showing different gene expression profiles according to cell type, flow times, or shear type with different molecular biology techniques, but few studies have reported any EC gene response to shear stress when cells are seeded on vascular grafts. The purpose of this in vitro study was to investigate whether ECs were able to transduce shear stress at the level of the nucleus. Human saphenous vein ECs were seeded on glass slides coated with gelatin or fibrin glue or on 6-mm fibrin-glue-coated grafts. Then cells were exposed to 12 dyn/cm(2) for 4 h and ribonucleic acid (RNA) were extracted. The relative messenger RNA (mRNA) expression was studied using real-time quantitative polymerase chain reaction for the following mRNAs: von Willebrand Factor, tissue-plasminogen activator, CD31, vascular endothelial (VE)-cadherin, beta(1) integrin, and vascular endothelial growth factor receptor type 2. From parallel flow chambers, results have shown similar EC gene response on gelatin and fibrin glue under laminar shear stress with downregulation of prothrombotic genes, as well as upregulation of nonthrombotic genes and upregulation of adhesion molecules such as VE-cadherin, but some discrepancies are noted, with a downregulation of CD31 and kinase insert domain receptor (KDR) for the former, without significant variation for the latter. In comparison, results show upregulation of tissue type plasminogen activator gene and downregulation of KDR, VE-cadherin, and beta(1) integrin genes in ECs lining grafts. To conclude, the major finding of our study is to show that human saphenous vein ECs seeded on fibrin glue (in planar flow chambers or in tubular grafts) can be regulated using shear stress via gene expression changes in a nonthrombotic way.