通心络抑制血管内膜增生的作用和机制研究
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摘要
病理性血管重塑（vascular remodeling）是指血管壁结构对血流变化、机械负荷或者血管损伤所发生的失代偿性改变，进而可导致许多血管疾病的发生和发展，比如动脉粥样硬化、血管成形术后再狭窄以及血管旁路移植失败等。新生内膜形成是血管重塑的主要病理特征之一，病理学基础主要为局部炎性细胞的浸润和中膜平滑肌细胞的增殖和迁移。大量研究表明，炎症应答在引发和加剧血管重塑中起关键作用，表现为单核/巨噬细胞浸润以及炎性介质的释放，由此触发血管平滑肌细胞（vascular smoothmuscle cells, VSMCs）向内膜下迁移和增殖，最终导致管腔狭窄甚至闭塞。
MicroRNAs（miRNAs）是体内存在的一类非编码小RNA，长度通常不超过22个核苷酸，主要通过抑制翻译或促进mRNA降解在转录后水平调控基因表达。研究证实，许多miRNAs都参与了血管重塑过程。MicroRNA-155（miR-155）是一个炎症相关miRNA，在巨噬细胞和动脉粥样硬化斑块中表达上调。miR-155通过靶向抑制巨噬细胞炎症应答来发挥促炎或抗炎的作用。近期研究表明，miR-155在动脉粥样硬化中同样具有抗斑块形成和促斑块形成的双重作用，但其在血管重塑及新生内膜形成中的作用还尚不清楚。
通心络是一种中药复方提取物，由人参、赤芍、酸枣仁、檀香、降香、土鳖虫、蜈蚣、水蛭、蝉蜕、全蝎、乳香及冰片组成。该药已显示出多种血管保护样作用，包括降血脂、抗氧化、抗血栓形成、改善内皮细胞功能、促进血管新生以及抑制炎症反应等。抗炎是通心络发挥血管保护作用的主要机制之一，miRNA是否介导或参与通心络抑制血管炎症反应的过程目前还尚不清楚。因此，本研究利用小鼠颈动脉结扎模型诱导血管内膜增生，探讨通心络是否通过调控miR-155的表达而发挥抑制血管炎症及血管重塑的作用，旨在为阐明通心络的血管保护分子机制以及扩大临床应用范围提供实验依据。
第一部分通心络抑制颈动脉结扎诱导的内膜增生和血管炎症反应
目的：观察通心络对颈动脉结扎诱导的内膜增生和局部炎症应答中的抑制作用。
方法：1于左侧颈总动脉近分叉处结扎C57BL/6小鼠颈总动脉诱导内膜增生。2应用苏木精-伊红染色和Image-Pro Plus Analyzer version5.1软件观察和评价内膜增生的形态学改变。3qRT-PCR检测VEGF-α、FGF-b、TGF-β、PDGF-BB、TNF-α、IL-1β和IL-6的mRNA表达水平。4应用mac-2和SMA免疫荧光双重染色观察巨噬细胞浸润和血管平滑肌细胞增殖。5免疫组化法观察TNF-α和IL-1β的表达量。
结果：
1通心络抑制颈动脉结扎诱导的内膜增生
形态学分析显示，颈动脉结扎7天后既有新生内膜形成，到结扎后14天内膜增生进一步加重，到结扎后21天，新生内膜大量增加至血管壁厚度的70%以上。与结扎组相比，通心络低剂量、中剂量和高剂量灌胃组（于术前3天开始，每天灌胃给药一次）结扎血管的内膜面积和I/M比值在3个时间点（7、14和21天）均显著减少。同时，通心络中剂量和高剂量灌胃组对内膜增生的抑制作用比通心络低剂量灌胃组更显著。以上结果说明，通心络呈剂量依赖性抑制颈动脉结扎诱导的新生内膜形成。
2通心络抑制颈动脉结扎诱导的炎性因子的表达
qRT-PCR结果显示，在颈动脉结扎后21天，结扎血管中的TGF-β、PDGF-BB、TNF-α、IL-1β的mRNA水平均比对照组血管显著升高。尤其是TNF-α的mRNA水平达对照组的17倍。与结扎组相比，通心络中剂量灌胃组显著降低了PDGF-BB、TNF-α、IL-1β的mRNA水平，但没有影响TGF-β的mRNA水平。这些结果说明，通心络对颈动脉结扎诱导的炎性因子的上调有显著的抑制作用。
3通心络抑制颈动脉结扎诱导的巨噬细胞浸润和血管平滑肌细胞增殖，减少结扎血管中TNF-α和IL-1β的产生
免疫荧光结果显示，在颈动脉结扎后21天的血管新生内膜中有大量巨噬细胞浸润，同时伴有血管平滑肌细胞的大量增殖。然而，在通心络中剂量灌胃组的结扎血管中，几乎观察不到巨噬细胞的浸润，中膜平滑肌细胞的增殖和迁移也不明显。免疫组化显示，与对照组相比，损伤21天后结扎血管的新生内膜中有大量TNF-α、IL-1β的产生，而通心络中剂量灌胃显著抑制了结扎血管中TNF-α和IL-1β的表达。
4结扎后再给予通心络干预仍然可以显著抑制颈动脉结扎诱导的内膜增生
形态学结果显示，于颈动脉结扎后3天再给予通心络中剂量灌胃，与结扎组相比，在术后21天仍然抑制了血管内膜增生，但抑制作用与术前预干预组比较有所减弱。
小结：
通心络可以显著抑制小鼠颈动脉结扎诱导的内膜增生，其作用与减少巨噬细胞浸润从而减轻局部炎症反应有关。
第二部分通心络通过下调miR-155的表达发挥抑制内膜增生的作用
目的：观测miR-155是否参与颈动脉结扎诱导的新生内膜形成过程，以及通心络是否通过调节miR-155的表达来发挥抑制内膜增生和血管炎症反应的作用。
方法：1对miR-155基因敲除（miR-155-/-）小鼠和野生型（WT）小鼠行左侧颈总动脉结扎术。苏木精-伊红染色和Image-Pro Plus Analyzerversion5.1软件观察和评价内膜增生的形态学改变。2将腺病毒载体Ad-miR-155（空载体Ad-null作为对照）经尾静脉注射导入WT小鼠体内过表达miR-155。3qRT-PCR检测miR-155、TNF-α和IL-1β的表达水平。4mac-2免疫组化染色观察巨噬细胞浸润；免疫组化染色观察TNF-α的表达量。
结果：
1通心络抑制颈动脉结扎诱导的miR-155的表达
与未结扎的对照组相比，miR-155在颈动脉结扎14天和21天后的小鼠血管中均显著上调，通心络中剂量灌胃在结扎后14天和21天均明显抑制了结扎血管中miR-155的表达。在颈动脉结扎7天后的结扎组和通心络中剂量灌胃组的血管中miR-155的表达没有明显变化。
2miR-155缺失抑制颈动脉结扎诱导的内膜增生，而miR-155过表达部分拮抗了通心络对内膜增生的抑制作用
形态学分析显示，颈动脉结扎21天后，WT小鼠血管中有大量新生内膜形成，而miR-155-/-小鼠的结扎血管，无论是I/M比值还是内膜面积均显著低于WT小鼠，说明miR-155的缺失抑制了颈动脉结扎诱导的内膜增生。同时，在miR-155-/-小鼠给予通心络中剂量灌胃后，其结扎血管中的内膜增生进一步减少。
给通心络中剂量灌胃的WT小鼠尾静脉注射Ad-miR-155（于术前1天开始注射，每隔7天注射一次，共注射3次），与Ad-null注射组比较，在结扎21天后的血管中miR-155上调3.5倍。在通心络中剂量灌胃的WT小鼠中，颈动脉结扎21天后，与Ad-null注射组相比，Ad-miR-155注射组的结扎血管中I/M比值和内膜面积均显著增加，说明miR-155过表达部分拮抗了通心络对内膜增生的抑制作用。
3miR-155缺失抑制颈动脉结扎诱导的巨噬细胞浸润和炎性因子TNF-α和IL-1β的表达，而miR-155过表达部分拮抗了通心络对巨噬细胞浸润和TNF-α表达的抑制作用
免疫组化结果显示，颈动脉结扎21天后，WT小鼠的结扎血管的新生内膜中有大量巨噬细胞浸润，而miR-155-/-小鼠的结扎血管中巨噬细胞显著减少。给予通心络中剂量灌胃的miR-155-/-小鼠的结扎血管中几乎观察不到浸润的巨噬细胞。同时，在通心络中剂量灌胃的WT小鼠中，与尾静脉注射Ad-null组相比，尾静脉注射Ad-miR-155组的结扎血管中浸润的巨噬细胞明显增多。
qRT-PCR结果显示，在颈动脉结扎后21天，miR-155-/-小鼠结扎血管中TNF-α的mRNA水平减少到WT小鼠的11%。给予通心络中剂量灌胃的miR-155-/-小鼠的结扎血管中TNF-α的mRNA水平减少到给予通心络灌胃的WT小鼠的20%。此外，在通心络中剂量灌胃的WT小鼠中，与Ad-null注射组相比，Ad-miR-155注射组的结扎血管中TNF-α的mRNA水平升高了2.2倍。以上结果说明，miR-155介导了结扎血管中TNF-α的表达，而miR-155过表达拮抗了通心络对TNF-α表达的抑制作用。免疫组化显示，各组的TNF-α的表达水平与qRT-PCR结果一致。另外，与WT小鼠相比，miR-155-/-小鼠结扎血管中的IL-1β mRNA水平下降了50%，但给予通心络中剂量灌胃的miR-155-/-小鼠与给予通心络中剂量灌胃的WT小鼠组比较，IL-1β mRNA水平无明显变化，同时，在通心络中剂量灌胃的WT小鼠中，Ad-miR-155注射使miR-155过表达后并没影响结扎血管中IL-1β的mRNA水平。这些结果说明，通心络对IL-1β表达的抑制作用并不是通过调节miR-155的表达实现的。
4在颈动脉结扎后期过表达miR-155仍可部分拮抗通心络对内膜增生的抑制作用
在通心络中剂量灌胃的WT小鼠中，于颈动脉结扎术后第14天注射Ad-miR-155，与Ad-null注射组相比，Ad-miR-155注射组结扎血管的内膜增生加重，说明在血管重塑后期过表达miR-155仍可减弱通心络对内膜增生的抑制作用。
小结：
miR-155参与颈动脉结扎诱导的新生内膜的形成过程；通心络对颈动脉结扎诱导的内膜增生和血管炎症应答的抑制作用在一定程度上是通过抑制miR-155的表达实现的。
第三部分通心络通过阻断miR-155与TNF-α之间的反馈环路抑制巨噬细胞炎症应答
目的：揭示通心络调节miR-155表达以及抑制巨噬细胞炎症应答的分子机制。
方法：1用不同因素处理原代培养的小鼠骨髓来源巨噬细胞（BMMs）。2腺病毒感染实验用于在BMMs中过表达miR-155。3Westernblot检测p-Akt和Akt1的表达。4siRNA转染实验用于在BMMs中敲低Akt1。5qRT-PCR检测miR-155、TNF-α和IL-1β的表达水平。6ELISA检测BMMs培养基中TNF-α和IL-1β的含量。7伤口愈合实验。8细胞粘附实验。
结果：
1通心络通过阻断TNF-α和miR-155之间形成的正反馈环路而抑制巨噬细胞炎症应答
qRT-PCR结果显示，与对照组相比，TNF-α刺激使miR-155的表达水平上升2.5倍，通心络预孵育以剂量依赖性的方式抑制TNF-α诱导的miR-155表达上调。另一方面，在对照组、IL-1β刺激组和通心络预孵育+IL-1β刺激组之间miR-155的表达水平无明显差异。这些结果说明，TNF-α可以促进BMMs中miR-155的表达，但IL-1β对miR-155的表达无影响。与Ad-null感染组相比，Ad-miR-155感染的BMMs中TNF-α的mRNA水平升高6倍。来源于miR-155-/-小鼠的BMMs中TNF-α的mRNA水平比WT组显著降低。与qRT-PCR结果相一致，ELISA检测结果显示，BMMs中过表达miR-155显著增加培养基中TNF-α的含量，而miR-155-/-小鼠的BMMs培养基中TNF-α含量比WT组明显下降。另一方面，miR-155过表达或敲除均未影响BMMs及其培养基中IL-1β的表达水平。以上结果显示，TNF-α和miR-155之间形成正反馈环路促进巨噬细胞的炎症应答。
qRT-PCR和ELISA结果显示，与单纯过表达miR-155组比较，通心络预孵育减少了过表达miR-155诱导的TNF-α的表达上调，TNF-α的mRNA水平和培养基中的蛋白水平分别减少到过表达miR-155组的31%和78%。同时，miR-155-/-BMMs无论给予或不给予通心络预孵育，其TNF-α的表达均处于较低水平。这些结果表明，通心络可以阻断TNF-α和miR-155之间形成的正反馈环路，这可能是其抑制炎症应答发挥抗炎作用的机制之一。
2通心络通过上调Akt1抑制巨噬细胞中miR-155的表达
Western blot结果显示，BMMs被TNF-α刺激10、20和40分钟后p-Akt均轻微下调，通心络预孵育（2h）+TNF-α刺激组的p-Akt在三个时间点（10、20和40分钟）均显著上调。但是，通心络预孵育（2h）+TNF-α刺激组的总Akt1的蛋白水平在这三个时间点也显著上调。这些结果说明，通心络预孵育后p-Akt水平上升是由于上调了总的Akt1蛋白表达引起的。
qRT-PCR结果显示，在BMMs中敲低Akt1后，通心络对TNF-α诱导的miR-155表达上调的抑制被消除，说明通心络抑制BMMs中miR-155的表达是通过上调Akt1实现的。
3miR-155缺失抑制巨噬细胞的迁移，但不影响巨噬细胞的粘附
伤口愈合实验结果显示，来源于miR-155-/-小鼠的BMMs在划痕后迁移的距离比WT小鼠的BMMs有所下降，说明miR-155缺失在一定程度上抑制了巨噬细胞的迁移。
细胞粘附实验显示，与WT组相比，miR-155-/-BMMs与内皮细胞的粘附数量没有明显变化，说明miR-155缺失不影响巨噬细胞的粘附。
小结：
通心络通过阻断TNF-α和miR-155之间形成的正反馈环路而抑制巨噬细胞炎症应答；Akt1介导通心络对巨噬细胞中miR-155表达的抑制作用；miR-155缺失抑制巨噬细胞的迁移，但不影响巨噬细胞的粘附。
结论：
1通心络抑制颈动脉结扎诱导的内膜增生。
2通心络通过抑制炎性因子的表达和巨噬细胞的浸润而减轻血管炎症反应。
3miR-155缺失抑制颈动脉结扎诱导的内膜增生；通心络对内膜增生的抑制作用是通过抑制miR-155的表达实现的。
4TNF-α和miR-155之间形成正反馈环路，通心络阻断TNF-α和miR-155之间的正反馈环路，从而抑制巨噬细胞炎症应答。
5Akt1介导通心络对巨噬细胞miR-155表达的抑制。
Pathological vascular remodeling is defined as any maladaptivealterations of the vessel wall structure in response to altered blood flow,mechanical load, or after vascular injury. Neointima formation is one of thehallmarks of the remodeling process which occurs in several vascular diseasessuch as atherosclerosis, restenosis after angioplasty and bypass graft failuredriven by local infiltration of inflammatory cells and by migration andproliferation of resident medial vascular smooth muscle cells (VSMCs).Numerous studies have shown that inflammatory response is a key event forthe initiation and progression of vascular remodeling. The infiltration ofmonocytes/macrophages and production of cytokines trigger VSMC migrationand proliferation into the intima and cause arterial stenosis, even occlusions.
MicroRNAs (miRNAs) are a class of endogenous, small, noncodingRNAs of~22nucleotides that control gene expression by translationalrepression or mRNA degradation. Several miRNAs have been identified to bethat are involved in vascular remodeling. MiR-155, aninflammation-related-miRNA, which is upregulated in inflammatorymacrophages and in macrophages in atherosclerotic lesions, has both pro-andanti-inflammatory effect by targeting several mediators of inflammatorysignaling in macrophages. Recent studies have also shown that miR-155hasopposite effects on lesion formation in atherosclerosis. For the contrary effectsof miR-155on macrophage inflammatory reponse and atherosclerosis, the neteffect of miR-155on vascular remodeling and neointima formation remainsunclear.
Tongxinluo (TXL), a compound of traditional Chinese herbal medicine,is composed of ginseng, Hirudo, Buthus martensi, Eupolyphaga seusteleophaga, Periostracum cicadae, Scolopendra subspinipes, Radix paeoniae rubra, Lignum dalbergiae odoriferae, Semen ziziphi spinosae, Lignum santalialbi, and Borneolum syntheticum. Recent studies have shown that TXL haspleiotropic effects including lipid lowering, antioxidation, antithrombosis,improvement of endothelial function, enhancement of angiogenesis andanti-inflammation, which may be due to the synergistic effects of multiplecompounds present in the herbal extract. Although anti-inflammation is one ofthe major mechanisms underlying the beneficial effects of TXL, it remainsunclear whether anti-inflammation effects are mediated by miRNAs.Therefore, in this study, we used the carotid artery ligation model in mice toinvestigate whether and how TXL exerts a protective effect on carotidligation-induced vascular inflammation and remodeling by regulatingmiR-155expression.
PartⅠ TXL inhibits neointima hyperplasia and local inflammatoryresponse induced by carotid ligation
Objective: To observe the role of TXL in protecting against neointimahyperplasia and local inflammatory response induced by carotid ligation inmouse.
Methods:1The left common carotid artery was ligated near the carotidbifurcation to induce intima formation.2Morphology of neointimahyperplasia was assessed by hematoxylin/eosin staining and Image-Pro PlusAnalyzer version5.1software.3The expression of VEGF-α, FGF-b, TGF-β,PDGF-BB, TNF-α, IL-1β, and IL-6was observed at mRNA level by qRT-PCR.4Macrophage infiltration and VSMC proliferation were examined byimmunofluorescence histochemical double-staining for mac-2and SMA.5Immunohistochemical staining was performed to examine TNF-α and IL-1βproduction.
Results:
1TXL inhibits neointima hyperplasia induced by carotid ligation
Morphometric analysis showed that neointima hyperplasia was observedat days7after vessel ligation and well developed by14days. By21days,neointima accounted for over70%of the carotid arterial wall thickness. When compared with that of the ligated group, carotid arterial wall thickness wassignificantly decreased in the three TXL-treated groups (TXL power wasadministered by intragastric administration beginning3days before ligation),both I/M ratio and intima area were lower than that observed at the ligatedgroup at all three time points. Moreover, the inhibitory effect of TXL onneointima formation was more significant in the moderate-and high-doseTXL groups than that in low-dose TXL group. These results suggest that TXLdose-dependently inhibits neointima formation induced by carotid ligation.
2TXL inhibits inflammatory cytokine expression induced by carotidligation
qRT-PCR showed a significant increase in TGF-β, PDGF-BB, TNF-α andIL-1β mRNA in ligated vessels at21days after ligation. Especially, TNF-αmRNA in ligated vessels reached16.78-fold above the control levels.Moderate-dose TXL markedly suppressed carotid ligation-inducedup-regulation of PDGF-BB, TNF-α and IL-1β mRNA in ligated artery.Whereas，TXL had no effect on IL-1β mRNA expression after ligation. Theseresults show that TXL markedly suppressed carotid ligation-inducedup-regulation of inflammatory cytokine expression in ligated artery.
3TXL inhibits macrophage infiltration and VSMC proliferation inducedby carotid ligation, and reduces the production of TNF-α and IL-1β inligated artery
Macrophages infiltrated into the neointima of ligated arteries were readilydetectable at21days after carotid ligation with a large number of VSMCproliferation, whereas macrophages was barely observed in the neointima inmoderate-dose TXL-treated group similar to that in the unligated carotid artery.Consistent to the mRNA expression, the protein expression of TNF-α andIL-1β assessed by immunohistochemical staining was significantly elevated inthe neointima at21days after ligation compared with unligated arteries, butmoderate-dose TXL treatment inhibited carotid ligation-induced up-regulationof TNF-α and IL-1β production.
4TXL post-treatment still inhibits neointima hyperplasia induced by carotid ligation.
TXL was administrated intragastrically at moderate dose beginning3days after ligation injury and continuing for21days thereafter. TXLpost-treatment still inhibited neointima hyperplasia induced by carotid ligation,although the suppressive effect of TXL become less robust.
PartⅡ TXL exerts its inhibitory action on neointima formation partly bysuppressing miR-155expression
Objective: To examine whether miR-155is required for neointimaformation induced by carotid ligation and whether TXL exerts its inhibitoryaction on neointima formation by regulating miR-155expression.
Methods:1Left carotid artery ligation was performed in miR-155–/–andwild-type (WT) mice, and morphology of neointima hyperplasia was assessedby hematoxylin/eosin staining and Image-Pro Plus Analyzer version5.1software.2Ad-miR-155（Ad-null as a control） was introduced into WTmice through tail vein injection to overexpress miR-155.3qRT-PCR wasperformed to examine miR-155, TNF-α and IL-1β expression.4Immunohistochemical staining was performed to examine mac-2and TNF-αexpression.
Results:
1TXL suppresses miR-155expression induced by carotid ligation
MiR-155was up-regulated in ligated artery at14and21days comparedwith the unligated vessel. Moderate-dose TXL treatment significantlysuppressed the expression of miR-155induced by carotid ligation at14and21days. There was no significant difference between unligated, ligated andmoderate-dose TXL treatment group at7days after carotid ligation.
2MiR-155deletion protects against neointimal hyperplasia induced bycarotid ligation, and miR-155overexpression partly reverses theinhibitory effect of TXL on neointimal hyperplasia
Ligated carotid artery caused significant neointimal formation in WTmice after21days, whereas the I/M ratio and intima area in miR-155–/–micewere significantly lower than WT mice on21days after ligation，suggesting that deletion of miR-155protects against neointimal hyperplasia induced bycarotid ligation. Furthermore, neointima formation was further reduced in theligated carotid artery of miR-155–/–mice treated with TXL.
MiR-155expression was increased by3.5-fold after Ad-miR-155injection (every7days beginning1day before ligation injury until21days) inTXL-treated mice, and neointima thickness in the ligated carotid artery wassignificantly increased after Ad-miR-155was injected and overexpressedcompared with that in Ad-null-introduced mice treated with TXL，indicatingthat overexpression of miR-155partly reverses the inhibitory effect of TXL onneointimal hyperplasia
3MiR-155deletion inhibits macrophage infiltration and inflammatorycytokine production induced by carotid ligation, and miR-155overexpression partly reverses the inhibitory effect of TXL onmacrophage infiltration and TNF-α production
a marked macrophage infiltration was observed in the ligated carotidartery of WT mice on21days after ligation, which was strongly reduced inmiR-155–/–mice, and macrophage infiltration was barely observed in theligated carotid artery of miR-155–/–mice treated with TXL. Meanwhile, in theTXL-treated WT mice, Ad-miR-155-infected arteries showned increasedmacrophage infiltration compared with Ad-null-injected group. In addition,mRNA level of TNF-α in ligated arteries of miR-155–/–mice was reduced to11%of WT mice. In TXL-treated miR-155–/–mice, TNF-α mRNA levels werereduced to20%of TXL-treated WT mice. Importantly, TNF-α mRNA levelswere increased2.2-fold in the ligated arteries of Ad-miR-155-infected micecompared with that of Ad-null-injected mice regardless of TXL treatment，suggesting that miR-155might mediate TNF-α mRNA expression, and thatmiR-155overexpression declines the inhibitory effect of TXL on TNF-αexpression. In contrast, although a50%reduction of the IL-1β mRNA wasobserved in ligated arteries of miR-155–/–mice, no significant difference inIL-1β mRNA levels was detected between miR-155–/–and WT mice whenthese mice were treated with TXL. Moreover, after carotid artery ligation, miR-155overexpression did not affect IL-1β mRNA expression in the ligatedarteries regardless of TXL treatment. These results suggested that inhibitoryeffect of TXL on IL-1β expression is not through suppressing miR-155expression.
4Overexpression of miR-155in the late stage of carotid ligation alsopartly reverses the inhibitory effect of TXL on neointimal hyperplasia
Neointima thickness in the ligated carotid artery was increased afterAd-miR-155was injected on days14after carotid ligation compared with thatin Ad-null-introduced mice treated with TXL, indicating that Ad-miR-155injection initiated on days14after carotid ligation also partly reversed theinhibitory effect of TXL on neointimal hyperplasia.
Part Ⅲ TXL suppresses macrophage inflammatory response by blockingthe feedback loop between miR-155and TNF-α
Objective: To explore the molecular mechanisms whereby TXL regulatesmiR-155expression and the mechanism of the prevention effect of TXL onmacrophage inflammatory response.
Methods:1Primary cultured bone marrow-derived macrophages(BMMs) were used in vitro study.2Adenovirus infection experiments wereperformed in BMMs to overexpressing miR-155.3p-Akt and Akt1wereassessed by Western Blot.4siRNA transfection experiments were performedin BMMs to knocked down Akt1.5The expression of miR-155, TNF-α andIL-1β was examined by qRT-PCR.6The productions of TNF-α and IL-1βwere determined in BMMs cell culture medium by Elisa.7Wound Healingassay.8Cell adhesion assay.
Results:
1MiR-155and TNF-α form a positive feedback loop to promotemacrophage inflammatory response, and TXL blocks this feedbackpathway
qRT-PCR showed that miR-155expression was increased by2.5-foldafter TNF-α treatment, whereas TXL treatment suppressed the expression ofmiR-155induced by TNF-α in a dose-dependent manner. No significant up-regulation of miR-155expression was observed after IL-1β treatment, andTXL treatment did not significantly affect miR-155expression. These resultssuggested that TNF-α, but not IL-1β, can markedly induce miR-155expression in BMMs. On the other hand, overexpression of miR-155mediatedby an adenoviral vector in BMMs increased the expression of TNF-α by5-foldover that of Ad-null-infected BMMs, whereas BMMs from miR-155–/–micehad a much lower level of TNF-α expression compared with that of BMMsfrom WT mice. Similarly, overexpression of miR-155in BMMs significantlyincreased the release of TNF-α into the medium, but BMMs from miR-155–/–mice released less TNF-α into the culture medium compared with WT mice.However, overexpression or deletion of miR-155did not affect the expressionof IL-1β. TXL pretreatment reduced the up-regulation of TNF-α expressioninduced by miR-155overexpression, with TNF-α mRNA level being reducedto31%of the control and TNF-α protein level being decreased to78%of thecontrol. Moreover, BMMs from miR-155–/–mice showed also a low level ofTNF-α expression regardless TXL treatment. Together, these results suggestthat miR-155and TNF-α form a positive feedback loop to promotemacrophage inflammatory response in mice and that TXL has a inhibitoryeffect on this feedback pathway.
2TXL inhibits miR-155expression by up-regulating Akt1in BMMs
Western blot results showed that there was a slight decrease in the levelsof phospho-Akt upon TNF-α stimulation but TXL pretreatment markedlyincreased Akt phosphorylation. Meanwhile, the total Akt1also markedlyincreased by TXL pretreatment for2h, suggesting that the increasedphospho-Akt levels may be due to the up-regulation of total Akt1proteinlevels by TXL pretreatment. qRT-PCR revealed that knockdown of Akt1abrogated the inhibitory effect of TXL on miR-155expression induced byTNF-α. These data demonstrate that TXL inhibits miR-155expression byup-regulating Akt1in BMMs.
3miR-155deletion reduces macrophage migration, but has no effect onadhesion.
We have examined the functional effects of miR-155on macrophagemigration or adhesion. Wound healing assay and cell adhesion assay show thatmiR-155deletion reduced macrophage migration, but had no effect onadhesion.
Conclusions:
1TXL dose-dependently inhibited neointima formation induced bycarotid ligation.
2TXL reduced local inflammatory response in ligated vessels byinhibiting inflammatory cytokine production and macrophage infiltration.
3MiR-155deletion protected against neointimal hyperplasia induced bycarotid ligation, and TXL exerted its inhibitory action on neointima formationby suppressing miR-155expression.
4MiR-155and TNF-α formed a positive feedback loop to promotemacrophage inflammatory response, and TXL blocked this feedback pathway.
5Akt1mediated TXL-induced suppression of miR-155expression.
MicroRNAs（miRNAs）是体内存在的一类非编码小RNA，长度通常不超过22个核苷酸，主要通过抑制翻译或促进mRNA降解在转录后水平调控基因表达。研究证实，许多miRNAs都参与了血管重塑过程。MicroRNA-155（miR-155）是一个炎症相关miRNA，在巨噬细胞和动脉粥样硬化斑块中表达上调。miR-155通过靶向抑制巨噬细胞炎症应答来发挥促炎或抗炎的作用。近期研究表明，miR-155在动脉粥样硬化中同样具有抗斑块形成和促斑块形成的双重作用，但其在血管重塑及新生内膜形成中的作用还尚不清楚。
通心络是一种中药复方提取物，由人参、赤芍、酸枣仁、檀香、降香、土鳖虫、蜈蚣、水蛭、蝉蜕、全蝎、乳香及冰片组成。该药已显示出多种血管保护样作用，包括降血脂、抗氧化、抗血栓形成、改善内皮细胞功能、促进血管新生以及抑制炎症反应等。抗炎是通心络发挥血管保护作用的主要机制之一，miRNA是否介导或参与通心络抑制血管炎症反应的过程目前还尚不清楚。因此，本研究利用小鼠颈动脉结扎模型诱导血管内膜增生，探讨通心络是否通过调控miR-155的表达而发挥抑制血管炎症及血管重塑的作用，旨在为阐明通心络的血管保护分子机制以及扩大临床应用范围提供实验依据。
第一部分通心络抑制颈动脉结扎诱导的内膜增生和血管炎症反应
目的：观察通心络对颈动脉结扎诱导的内膜增生和局部炎症应答中的抑制作用。
方法：1于左侧颈总动脉近分叉处结扎C57BL/6小鼠颈总动脉诱导内膜增生。2应用苏木精-伊红染色和Image-Pro Plus Analyzer version5.1软件观察和评价内膜增生的形态学改变。3qRT-PCR检测VEGF-α、FGF-b、TGF-β、PDGF-BB、TNF-α、IL-1β和IL-6的mRNA表达水平。4应用mac-2和SMA免疫荧光双重染色观察巨噬细胞浸润和血管平滑肌细胞增殖。5免疫组化法观察TNF-α和IL-1β的表达量。
结果：
1通心络抑制颈动脉结扎诱导的内膜增生
形态学分析显示，颈动脉结扎7天后既有新生内膜形成，到结扎后14天内膜增生进一步加重，到结扎后21天，新生内膜大量增加至血管壁厚度的70%以上。与结扎组相比，通心络低剂量、中剂量和高剂量灌胃组（于术前3天开始，每天灌胃给药一次）结扎血管的内膜面积和I/M比值在3个时间点（7、14和21天）均显著减少。同时，通心络中剂量和高剂量灌胃组对内膜增生的抑制作用比通心络低剂量灌胃组更显著。以上结果说明，通心络呈剂量依赖性抑制颈动脉结扎诱导的新生内膜形成。
2通心络抑制颈动脉结扎诱导的炎性因子的表达
qRT-PCR结果显示，在颈动脉结扎后21天，结扎血管中的TGF-β、PDGF-BB、TNF-α、IL-1β的mRNA水平均比对照组血管显著升高。尤其是TNF-α的mRNA水平达对照组的17倍。与结扎组相比，通心络中剂量灌胃组显著降低了PDGF-BB、TNF-α、IL-1β的mRNA水平，但没有影响TGF-β的mRNA水平。这些结果说明，通心络对颈动脉结扎诱导的炎性因子的上调有显著的抑制作用。
3通心络抑制颈动脉结扎诱导的巨噬细胞浸润和血管平滑肌细胞增殖，减少结扎血管中TNF-α和IL-1β的产生
免疫荧光结果显示，在颈动脉结扎后21天的血管新生内膜中有大量巨噬细胞浸润，同时伴有血管平滑肌细胞的大量增殖。然而，在通心络中剂量灌胃组的结扎血管中，几乎观察不到巨噬细胞的浸润，中膜平滑肌细胞的增殖和迁移也不明显。免疫组化显示，与对照组相比，损伤21天后结扎血管的新生内膜中有大量TNF-α、IL-1β的产生，而通心络中剂量灌胃显著抑制了结扎血管中TNF-α和IL-1β的表达。
4结扎后再给予通心络干预仍然可以显著抑制颈动脉结扎诱导的内膜增生
形态学结果显示，于颈动脉结扎后3天再给予通心络中剂量灌胃，与结扎组相比，在术后21天仍然抑制了血管内膜增生，但抑制作用与术前预干预组比较有所减弱。
小结：
通心络可以显著抑制小鼠颈动脉结扎诱导的内膜增生，其作用与减少巨噬细胞浸润从而减轻局部炎症反应有关。
第二部分通心络通过下调miR-155的表达发挥抑制内膜增生的作用
目的：观测miR-155是否参与颈动脉结扎诱导的新生内膜形成过程，以及通心络是否通过调节miR-155的表达来发挥抑制内膜增生和血管炎症反应的作用。
方法：1对miR-155基因敲除（miR-155-/-）小鼠和野生型（WT）小鼠行左侧颈总动脉结扎术。苏木精-伊红染色和Image-Pro Plus Analyzerversion5.1软件观察和评价内膜增生的形态学改变。2将腺病毒载体Ad-miR-155（空载体Ad-null作为对照）经尾静脉注射导入WT小鼠体内过表达miR-155。3qRT-PCR检测miR-155、TNF-α和IL-1β的表达水平。4mac-2免疫组化染色观察巨噬细胞浸润；免疫组化染色观察TNF-α的表达量。
结果：
1通心络抑制颈动脉结扎诱导的miR-155的表达
与未结扎的对照组相比，miR-155在颈动脉结扎14天和21天后的小鼠血管中均显著上调，通心络中剂量灌胃在结扎后14天和21天均明显抑制了结扎血管中miR-155的表达。在颈动脉结扎7天后的结扎组和通心络中剂量灌胃组的血管中miR-155的表达没有明显变化。
2miR-155缺失抑制颈动脉结扎诱导的内膜增生，而miR-155过表达部分拮抗了通心络对内膜增生的抑制作用
形态学分析显示，颈动脉结扎21天后，WT小鼠血管中有大量新生内膜形成，而miR-155-/-小鼠的结扎血管，无论是I/M比值还是内膜面积均显著低于WT小鼠，说明miR-155的缺失抑制了颈动脉结扎诱导的内膜增生。同时，在miR-155-/-小鼠给予通心络中剂量灌胃后，其结扎血管中的内膜增生进一步减少。
给通心络中剂量灌胃的WT小鼠尾静脉注射Ad-miR-155（于术前1天开始注射，每隔7天注射一次，共注射3次），与Ad-null注射组比较，在结扎21天后的血管中miR-155上调3.5倍。在通心络中剂量灌胃的WT小鼠中，颈动脉结扎21天后，与Ad-null注射组相比，Ad-miR-155注射组的结扎血管中I/M比值和内膜面积均显著增加，说明miR-155过表达部分拮抗了通心络对内膜增生的抑制作用。
3miR-155缺失抑制颈动脉结扎诱导的巨噬细胞浸润和炎性因子TNF-α和IL-1β的表达，而miR-155过表达部分拮抗了通心络对巨噬细胞浸润和TNF-α表达的抑制作用
免疫组化结果显示，颈动脉结扎21天后，WT小鼠的结扎血管的新生内膜中有大量巨噬细胞浸润，而miR-155-/-小鼠的结扎血管中巨噬细胞显著减少。给予通心络中剂量灌胃的miR-155-/-小鼠的结扎血管中几乎观察不到浸润的巨噬细胞。同时，在通心络中剂量灌胃的WT小鼠中，与尾静脉注射Ad-null组相比，尾静脉注射Ad-miR-155组的结扎血管中浸润的巨噬细胞明显增多。
qRT-PCR结果显示，在颈动脉结扎后21天，miR-155-/-小鼠结扎血管中TNF-α的mRNA水平减少到WT小鼠的11%。给予通心络中剂量灌胃的miR-155-/-小鼠的结扎血管中TNF-α的mRNA水平减少到给予通心络灌胃的WT小鼠的20%。此外，在通心络中剂量灌胃的WT小鼠中，与Ad-null注射组相比，Ad-miR-155注射组的结扎血管中TNF-α的mRNA水平升高了2.2倍。以上结果说明，miR-155介导了结扎血管中TNF-α的表达，而miR-155过表达拮抗了通心络对TNF-α表达的抑制作用。免疫组化显示，各组的TNF-α的表达水平与qRT-PCR结果一致。另外，与WT小鼠相比，miR-155-/-小鼠结扎血管中的IL-1β mRNA水平下降了50%，但给予通心络中剂量灌胃的miR-155-/-小鼠与给予通心络中剂量灌胃的WT小鼠组比较，IL-1β mRNA水平无明显变化，同时，在通心络中剂量灌胃的WT小鼠中，Ad-miR-155注射使miR-155过表达后并没影响结扎血管中IL-1β的mRNA水平。这些结果说明，通心络对IL-1β表达的抑制作用并不是通过调节miR-155的表达实现的。
4在颈动脉结扎后期过表达miR-155仍可部分拮抗通心络对内膜增生的抑制作用
在通心络中剂量灌胃的WT小鼠中，于颈动脉结扎术后第14天注射Ad-miR-155，与Ad-null注射组相比，Ad-miR-155注射组结扎血管的内膜增生加重，说明在血管重塑后期过表达miR-155仍可减弱通心络对内膜增生的抑制作用。
小结：
miR-155参与颈动脉结扎诱导的新生内膜的形成过程；通心络对颈动脉结扎诱导的内膜增生和血管炎症应答的抑制作用在一定程度上是通过抑制miR-155的表达实现的。
第三部分通心络通过阻断miR-155与TNF-α之间的反馈环路抑制巨噬细胞炎症应答
目的：揭示通心络调节miR-155表达以及抑制巨噬细胞炎症应答的分子机制。
方法：1用不同因素处理原代培养的小鼠骨髓来源巨噬细胞（BMMs）。2腺病毒感染实验用于在BMMs中过表达miR-155。3Westernblot检测p-Akt和Akt1的表达。4siRNA转染实验用于在BMMs中敲低Akt1。5qRT-PCR检测miR-155、TNF-α和IL-1β的表达水平。6ELISA检测BMMs培养基中TNF-α和IL-1β的含量。7伤口愈合实验。8细胞粘附实验。
结果：
1通心络通过阻断TNF-α和miR-155之间形成的正反馈环路而抑制巨噬细胞炎症应答
qRT-PCR结果显示，与对照组相比，TNF-α刺激使miR-155的表达水平上升2.5倍，通心络预孵育以剂量依赖性的方式抑制TNF-α诱导的miR-155表达上调。另一方面，在对照组、IL-1β刺激组和通心络预孵育+IL-1β刺激组之间miR-155的表达水平无明显差异。这些结果说明，TNF-α可以促进BMMs中miR-155的表达，但IL-1β对miR-155的表达无影响。与Ad-null感染组相比，Ad-miR-155感染的BMMs中TNF-α的mRNA水平升高6倍。来源于miR-155-/-小鼠的BMMs中TNF-α的mRNA水平比WT组显著降低。与qRT-PCR结果相一致，ELISA检测结果显示，BMMs中过表达miR-155显著增加培养基中TNF-α的含量，而miR-155-/-小鼠的BMMs培养基中TNF-α含量比WT组明显下降。另一方面，miR-155过表达或敲除均未影响BMMs及其培养基中IL-1β的表达水平。以上结果显示，TNF-α和miR-155之间形成正反馈环路促进巨噬细胞的炎症应答。
qRT-PCR和ELISA结果显示，与单纯过表达miR-155组比较，通心络预孵育减少了过表达miR-155诱导的TNF-α的表达上调，TNF-α的mRNA水平和培养基中的蛋白水平分别减少到过表达miR-155组的31%和78%。同时，miR-155-/-BMMs无论给予或不给予通心络预孵育，其TNF-α的表达均处于较低水平。这些结果表明，通心络可以阻断TNF-α和miR-155之间形成的正反馈环路，这可能是其抑制炎症应答发挥抗炎作用的机制之一。
2通心络通过上调Akt1抑制巨噬细胞中miR-155的表达
Western blot结果显示，BMMs被TNF-α刺激10、20和40分钟后p-Akt均轻微下调，通心络预孵育（2h）+TNF-α刺激组的p-Akt在三个时间点（10、20和40分钟）均显著上调。但是，通心络预孵育（2h）+TNF-α刺激组的总Akt1的蛋白水平在这三个时间点也显著上调。这些结果说明，通心络预孵育后p-Akt水平上升是由于上调了总的Akt1蛋白表达引起的。
qRT-PCR结果显示，在BMMs中敲低Akt1后，通心络对TNF-α诱导的miR-155表达上调的抑制被消除，说明通心络抑制BMMs中miR-155的表达是通过上调Akt1实现的。
3miR-155缺失抑制巨噬细胞的迁移，但不影响巨噬细胞的粘附
伤口愈合实验结果显示，来源于miR-155-/-小鼠的BMMs在划痕后迁移的距离比WT小鼠的BMMs有所下降，说明miR-155缺失在一定程度上抑制了巨噬细胞的迁移。
细胞粘附实验显示，与WT组相比，miR-155-/-BMMs与内皮细胞的粘附数量没有明显变化，说明miR-155缺失不影响巨噬细胞的粘附。
小结：
通心络通过阻断TNF-α和miR-155之间形成的正反馈环路而抑制巨噬细胞炎症应答；Akt1介导通心络对巨噬细胞中miR-155表达的抑制作用；miR-155缺失抑制巨噬细胞的迁移，但不影响巨噬细胞的粘附。
结论：
1通心络抑制颈动脉结扎诱导的内膜增生。
2通心络通过抑制炎性因子的表达和巨噬细胞的浸润而减轻血管炎症反应。
3miR-155缺失抑制颈动脉结扎诱导的内膜增生；通心络对内膜增生的抑制作用是通过抑制miR-155的表达实现的。
4TNF-α和miR-155之间形成正反馈环路，通心络阻断TNF-α和miR-155之间的正反馈环路，从而抑制巨噬细胞炎症应答。
5Akt1介导通心络对巨噬细胞miR-155表达的抑制。
Pathological vascular remodeling is defined as any maladaptivealterations of the vessel wall structure in response to altered blood flow,mechanical load, or after vascular injury. Neointima formation is one of thehallmarks of the remodeling process which occurs in several vascular diseasessuch as atherosclerosis, restenosis after angioplasty and bypass graft failuredriven by local infiltration of inflammatory cells and by migration andproliferation of resident medial vascular smooth muscle cells (VSMCs).Numerous studies have shown that inflammatory response is a key event forthe initiation and progression of vascular remodeling. The infiltration ofmonocytes/macrophages and production of cytokines trigger VSMC migrationand proliferation into the intima and cause arterial stenosis, even occlusions.
MicroRNAs (miRNAs) are a class of endogenous, small, noncodingRNAs of~22nucleotides that control gene expression by translationalrepression or mRNA degradation. Several miRNAs have been identified to bethat are involved in vascular remodeling. MiR-155, aninflammation-related-miRNA, which is upregulated in inflammatorymacrophages and in macrophages in atherosclerotic lesions, has both pro-andanti-inflammatory effect by targeting several mediators of inflammatorysignaling in macrophages. Recent studies have also shown that miR-155hasopposite effects on lesion formation in atherosclerosis. For the contrary effectsof miR-155on macrophage inflammatory reponse and atherosclerosis, the neteffect of miR-155on vascular remodeling and neointima formation remainsunclear.
Tongxinluo (TXL), a compound of traditional Chinese herbal medicine,is composed of ginseng, Hirudo, Buthus martensi, Eupolyphaga seusteleophaga, Periostracum cicadae, Scolopendra subspinipes, Radix paeoniae rubra, Lignum dalbergiae odoriferae, Semen ziziphi spinosae, Lignum santalialbi, and Borneolum syntheticum. Recent studies have shown that TXL haspleiotropic effects including lipid lowering, antioxidation, antithrombosis,improvement of endothelial function, enhancement of angiogenesis andanti-inflammation, which may be due to the synergistic effects of multiplecompounds present in the herbal extract. Although anti-inflammation is one ofthe major mechanisms underlying the beneficial effects of TXL, it remainsunclear whether anti-inflammation effects are mediated by miRNAs.Therefore, in this study, we used the carotid artery ligation model in mice toinvestigate whether and how TXL exerts a protective effect on carotidligation-induced vascular inflammation and remodeling by regulatingmiR-155expression.
PartⅠ TXL inhibits neointima hyperplasia and local inflammatoryresponse induced by carotid ligation
Objective: To observe the role of TXL in protecting against neointimahyperplasia and local inflammatory response induced by carotid ligation inmouse.
Methods:1The left common carotid artery was ligated near the carotidbifurcation to induce intima formation.2Morphology of neointimahyperplasia was assessed by hematoxylin/eosin staining and Image-Pro PlusAnalyzer version5.1software.3The expression of VEGF-α, FGF-b, TGF-β,PDGF-BB, TNF-α, IL-1β, and IL-6was observed at mRNA level by qRT-PCR.4Macrophage infiltration and VSMC proliferation were examined byimmunofluorescence histochemical double-staining for mac-2and SMA.5Immunohistochemical staining was performed to examine TNF-α and IL-1βproduction.
Results:
1TXL inhibits neointima hyperplasia induced by carotid ligation
Morphometric analysis showed that neointima hyperplasia was observedat days7after vessel ligation and well developed by14days. By21days,neointima accounted for over70%of the carotid arterial wall thickness. When compared with that of the ligated group, carotid arterial wall thickness wassignificantly decreased in the three TXL-treated groups (TXL power wasadministered by intragastric administration beginning3days before ligation),both I/M ratio and intima area were lower than that observed at the ligatedgroup at all three time points. Moreover, the inhibitory effect of TXL onneointima formation was more significant in the moderate-and high-doseTXL groups than that in low-dose TXL group. These results suggest that TXLdose-dependently inhibits neointima formation induced by carotid ligation.
2TXL inhibits inflammatory cytokine expression induced by carotidligation
qRT-PCR showed a significant increase in TGF-β, PDGF-BB, TNF-α andIL-1β mRNA in ligated vessels at21days after ligation. Especially, TNF-αmRNA in ligated vessels reached16.78-fold above the control levels.Moderate-dose TXL markedly suppressed carotid ligation-inducedup-regulation of PDGF-BB, TNF-α and IL-1β mRNA in ligated artery.Whereas，TXL had no effect on IL-1β mRNA expression after ligation. Theseresults show that TXL markedly suppressed carotid ligation-inducedup-regulation of inflammatory cytokine expression in ligated artery.
3TXL inhibits macrophage infiltration and VSMC proliferation inducedby carotid ligation, and reduces the production of TNF-α and IL-1β inligated artery
Macrophages infiltrated into the neointima of ligated arteries were readilydetectable at21days after carotid ligation with a large number of VSMCproliferation, whereas macrophages was barely observed in the neointima inmoderate-dose TXL-treated group similar to that in the unligated carotid artery.Consistent to the mRNA expression, the protein expression of TNF-α andIL-1β assessed by immunohistochemical staining was significantly elevated inthe neointima at21days after ligation compared with unligated arteries, butmoderate-dose TXL treatment inhibited carotid ligation-induced up-regulationof TNF-α and IL-1β production.
4TXL post-treatment still inhibits neointima hyperplasia induced by carotid ligation.
TXL was administrated intragastrically at moderate dose beginning3days after ligation injury and continuing for21days thereafter. TXLpost-treatment still inhibited neointima hyperplasia induced by carotid ligation,although the suppressive effect of TXL become less robust.
PartⅡ TXL exerts its inhibitory action on neointima formation partly bysuppressing miR-155expression
Objective: To examine whether miR-155is required for neointimaformation induced by carotid ligation and whether TXL exerts its inhibitoryaction on neointima formation by regulating miR-155expression.
Methods:1Left carotid artery ligation was performed in miR-155–/–andwild-type (WT) mice, and morphology of neointima hyperplasia was assessedby hematoxylin/eosin staining and Image-Pro Plus Analyzer version5.1software.2Ad-miR-155（Ad-null as a control） was introduced into WTmice through tail vein injection to overexpress miR-155.3qRT-PCR wasperformed to examine miR-155, TNF-α and IL-1β expression.4Immunohistochemical staining was performed to examine mac-2and TNF-αexpression.
Results:
1TXL suppresses miR-155expression induced by carotid ligation
MiR-155was up-regulated in ligated artery at14and21days comparedwith the unligated vessel. Moderate-dose TXL treatment significantlysuppressed the expression of miR-155induced by carotid ligation at14and21days. There was no significant difference between unligated, ligated andmoderate-dose TXL treatment group at7days after carotid ligation.
2MiR-155deletion protects against neointimal hyperplasia induced bycarotid ligation, and miR-155overexpression partly reverses theinhibitory effect of TXL on neointimal hyperplasia
Ligated carotid artery caused significant neointimal formation in WTmice after21days, whereas the I/M ratio and intima area in miR-155–/–micewere significantly lower than WT mice on21days after ligation，suggesting that deletion of miR-155protects against neointimal hyperplasia induced bycarotid ligation. Furthermore, neointima formation was further reduced in theligated carotid artery of miR-155–/–mice treated with TXL.
MiR-155expression was increased by3.5-fold after Ad-miR-155injection (every7days beginning1day before ligation injury until21days) inTXL-treated mice, and neointima thickness in the ligated carotid artery wassignificantly increased after Ad-miR-155was injected and overexpressedcompared with that in Ad-null-introduced mice treated with TXL，indicatingthat overexpression of miR-155partly reverses the inhibitory effect of TXL onneointimal hyperplasia
3MiR-155deletion inhibits macrophage infiltration and inflammatorycytokine production induced by carotid ligation, and miR-155overexpression partly reverses the inhibitory effect of TXL onmacrophage infiltration and TNF-α production
a marked macrophage infiltration was observed in the ligated carotidartery of WT mice on21days after ligation, which was strongly reduced inmiR-155–/–mice, and macrophage infiltration was barely observed in theligated carotid artery of miR-155–/–mice treated with TXL. Meanwhile, in theTXL-treated WT mice, Ad-miR-155-infected arteries showned increasedmacrophage infiltration compared with Ad-null-injected group. In addition,mRNA level of TNF-α in ligated arteries of miR-155–/–mice was reduced to11%of WT mice. In TXL-treated miR-155–/–mice, TNF-α mRNA levels werereduced to20%of TXL-treated WT mice. Importantly, TNF-α mRNA levelswere increased2.2-fold in the ligated arteries of Ad-miR-155-infected micecompared with that of Ad-null-injected mice regardless of TXL treatment，suggesting that miR-155might mediate TNF-α mRNA expression, and thatmiR-155overexpression declines the inhibitory effect of TXL on TNF-αexpression. In contrast, although a50%reduction of the IL-1β mRNA wasobserved in ligated arteries of miR-155–/–mice, no significant difference inIL-1β mRNA levels was detected between miR-155–/–and WT mice whenthese mice were treated with TXL. Moreover, after carotid artery ligation, miR-155overexpression did not affect IL-1β mRNA expression in the ligatedarteries regardless of TXL treatment. These results suggested that inhibitoryeffect of TXL on IL-1β expression is not through suppressing miR-155expression.
4Overexpression of miR-155in the late stage of carotid ligation alsopartly reverses the inhibitory effect of TXL on neointimal hyperplasia
Neointima thickness in the ligated carotid artery was increased afterAd-miR-155was injected on days14after carotid ligation compared with thatin Ad-null-introduced mice treated with TXL, indicating that Ad-miR-155injection initiated on days14after carotid ligation also partly reversed theinhibitory effect of TXL on neointimal hyperplasia.
Part Ⅲ TXL suppresses macrophage inflammatory response by blockingthe feedback loop between miR-155and TNF-α
Objective: To explore the molecular mechanisms whereby TXL regulatesmiR-155expression and the mechanism of the prevention effect of TXL onmacrophage inflammatory response.
Methods:1Primary cultured bone marrow-derived macrophages(BMMs) were used in vitro study.2Adenovirus infection experiments wereperformed in BMMs to overexpressing miR-155.3p-Akt and Akt1wereassessed by Western Blot.4siRNA transfection experiments were performedin BMMs to knocked down Akt1.5The expression of miR-155, TNF-α andIL-1β was examined by qRT-PCR.6The productions of TNF-α and IL-1βwere determined in BMMs cell culture medium by Elisa.7Wound Healingassay.8Cell adhesion assay.
Results:
1MiR-155and TNF-α form a positive feedback loop to promotemacrophage inflammatory response, and TXL blocks this feedbackpathway
qRT-PCR showed that miR-155expression was increased by2.5-foldafter TNF-α treatment, whereas TXL treatment suppressed the expression ofmiR-155induced by TNF-α in a dose-dependent manner. No significant up-regulation of miR-155expression was observed after IL-1β treatment, andTXL treatment did not significantly affect miR-155expression. These resultssuggested that TNF-α, but not IL-1β, can markedly induce miR-155expression in BMMs. On the other hand, overexpression of miR-155mediatedby an adenoviral vector in BMMs increased the expression of TNF-α by5-foldover that of Ad-null-infected BMMs, whereas BMMs from miR-155–/–micehad a much lower level of TNF-α expression compared with that of BMMsfrom WT mice. Similarly, overexpression of miR-155in BMMs significantlyincreased the release of TNF-α into the medium, but BMMs from miR-155–/–mice released less TNF-α into the culture medium compared with WT mice.However, overexpression or deletion of miR-155did not affect the expressionof IL-1β. TXL pretreatment reduced the up-regulation of TNF-α expressioninduced by miR-155overexpression, with TNF-α mRNA level being reducedto31%of the control and TNF-α protein level being decreased to78%of thecontrol. Moreover, BMMs from miR-155–/–mice showed also a low level ofTNF-α expression regardless TXL treatment. Together, these results suggestthat miR-155and TNF-α form a positive feedback loop to promotemacrophage inflammatory response in mice and that TXL has a inhibitoryeffect on this feedback pathway.
2TXL inhibits miR-155expression by up-regulating Akt1in BMMs
Western blot results showed that there was a slight decrease in the levelsof phospho-Akt upon TNF-α stimulation but TXL pretreatment markedlyincreased Akt phosphorylation. Meanwhile, the total Akt1also markedlyincreased by TXL pretreatment for2h, suggesting that the increasedphospho-Akt levels may be due to the up-regulation of total Akt1proteinlevels by TXL pretreatment. qRT-PCR revealed that knockdown of Akt1abrogated the inhibitory effect of TXL on miR-155expression induced byTNF-α. These data demonstrate that TXL inhibits miR-155expression byup-regulating Akt1in BMMs.
3miR-155deletion reduces macrophage migration, but has no effect onadhesion.
We have examined the functional effects of miR-155on macrophagemigration or adhesion. Wound healing assay and cell adhesion assay show thatmiR-155deletion reduced macrophage migration, but had no effect onadhesion.
Conclusions:
1TXL dose-dependently inhibited neointima formation induced bycarotid ligation.
2TXL reduced local inflammatory response in ligated vessels byinhibiting inflammatory cytokine production and macrophage infiltration.
3MiR-155deletion protected against neointimal hyperplasia induced bycarotid ligation, and TXL exerted its inhibitory action on neointima formationby suppressing miR-155expression.
4MiR-155and TNF-α formed a positive feedback loop to promotemacrophage inflammatory response, and TXL blocked this feedback pathway.
5Akt1mediated TXL-induced suppression of miR-155expression.
引文
1Cowan DB, Langille BL. Cellular and molecular biology of vascularremodeling. Curr Opin Lipidol,1996,7:94-100
2Schwartz SM, deBlois D, O’Brien ER. The intima: soil for atherosclerosisand restenosis. Circ Res,1995,77:445-465
3Libby P, Tanaka H. The molecular bases of restenosis. Prog Cardiovasc Dis,1997,40:97-106.
4Pasterkamp G, de Kleijn DP, Borst C. Arterial remodeling inatheroscle-rosis, restenosis and after alteration of blood flow: potentialmechanisms and clinical implications. Cardiovasc Res,2000,45:843-852
5Vo¨ lker W, Dorszewski A, Unruh V, et al. Copper-induced inflammatoryreactions of rat carotid arteries mimic restenosis/arteriosclerosis-likeneointima formation. Atherosclerosis,1997,130:29-36
6Owens GK, Kumar MS, Wamhoff BR. Molecular regulation ofvascularsmooth muscle cell differentiation in development and disease.Physiol Rev,2004,84:767-801
7Bonta PI, Pols TW, van Tiel CM, et al. Nuclear receptor Nurr1is expressedin and is associated with human restenosis and inhibits vascular lesionformation in mice involving inhibition of smooth muscle cell proliferationand inflammation. Circulation,2010,121:2023-2032
8Bose D, Leineweber K, Konorza T, et al. Release of TNF-alpha duringstent implantation into saphenous vein aortocoronary bypass grafts and itsrelation to plaque extrusion and restenosis. Am J Physiol Heart CircPhysiol,2007,292: H2295-2299
9Moore KJ, and Tabas I. Macrophages in the pathogenesis of atherosclerosis.Cell,2011,145:341-355
10Wu T, Harrison RA, Chen X, et al. Tongxinluo (tong xin luo ortong-xin-luo) capsule for unstable angina pectoris. Cochrane Database SystRev,2006, CD004474
11Li Z, Yang YJ, Qin XW, et al. Effects of tongxinluo and simvastatin on thestabilization of vulnerable atherosclerotic plaques of aorta in aorticatherosclerosis and molecular mechanism thereof: a comparative studywith rabbits. Zhonghua Yi Xue Za Zhi,2006,86:3146-3150
12Liu JX, Shang XH, Wang G. Effect of tongxinluo capsule on experi-mentalmyocardial ischemia, arrhythmia and hyperlipidemia. Zhongguo Zhong XiYi Jie He Za Zhi,1997,17:425-428
13Wu YL, You JH, Yuan GQ, et al. The effects of tongxinluo supermicropowder on nuclear factor-kappaB, inter-cellular adhesion molecule-1andvascular cell adhesion molecule-1ex-pression in aorta of rabbits fed withhigh-lipid diet. Zhonghua Xin Xue Guan Bing Za Zhi,2007,35:271-274
14Zhang L, Wu YL, Jia ZH, et al. Protective effects of a compound herbalextract (tong xin luo) on free fatty acid induced endothelial injury:implications of antioxidant system. BMC Complement Altern Med,2008,8:39
15黄峻,李春坚，杨国平等。通心络对兔外周血管球囊损伤后血栓形成及内膜增生的影响。中国临床药理学与治疗学,2001,6(1):35-37
16熊清萍,张社兵，吕新华等。通心络对兔髂动脉球囊损伤后内膜增生的影响。中国动脉硬化杂志,2004,12(6):727-728
17Kumar A, Lindner V. Remodeling with neointima formation in the mousecarotid artery after cessation of blood flow. Arterioscler Thromb Vasc Biol,1997,17:2238-2244
18Cheng KS, Mikhailidis DP, Hamilton G, et al. A review of the carotid andfemoral intima-media thickness as an indicator of the presence ofperipheral vascular disease and cardiovascular risk factors. Cardiovascularresearch,2002,54:528-538
19Ando J, and Yamamoto K. Effects of shear stress and stretch on endothelialfunction. Antioxidants&redox signaling,2011,15:1389-1403
20Su W, Sun A, Xu D, et al. Tongxinluo inhibits oxidized low-densitylipoprotein-induced maturation of human dendritic cells via activatingperoxisome proliferator-activated receptor gamma pathway. Journal ofcardiovascular pharmacology,2010,56:177-183
21Chen WQ, Zhong L, Zhang L, et al. Chinese medicine tongxinluosignificantly lowers serum lipid levels and stabilizes vulnerable plaques ina rabbit model. Journal of ethnopharmacology,2009,124:103-110
22Zhang L, Liu Y, Lu XT, et al. Traditional Chinese medication Tongxinluodose-dependently enhances stability of vulnerable plaques: a comparisonwith a high-dose simvastatin therapy. Am J Physiol Heart Circ Physiol,2009,297: H2004-2014
23Rectenwald JE, Moldawer LL, Huber TS, et al. Direct evidence forcytokine involvement in neointimal hyperplasia. Circulation,2000,102:1697-1702
24Jovinge S, Hultgardh-Nilsson A, Regnstrom J, et al. Tumor necrosisfactor-alpha activates smooth muscle cell migration in culture and isexpressed in the balloon-injured rat aorta. Arterioscler Thromb Vasc Biol,1997,17:490-497
25Yu K, Zheng B, Han M, et al. ATRA activates and PDGF-BB represses theSM22alpha promoter through KLF4binding to, or dissociating from, itscis-DNA elements. Cardiovascular research,2011,90:464-474
26Danenberg HD, Fishbein I, Gao J, et al. Macrophage depletion byclodronate-containing liposomes reduces neointimal formation afterballoon injury in rats and rabbits. Circulation,2002,106:599-605
1Faraoni I, Antonetti FR, Cardone J, et al. miR-155gene: a typicalmultifunctional microRNA. Biochim Biophys Acta,2009,1792:497-505
2O’Connell RM, Rao DS, Chaudhuri AA, et al. Physiological andpathological roles for microRNAs in the immune system. Nat RevImmunol,2010,10:111-122
3O’Connell RM, Taganov KD, Boldin MP, et al. MicroRNA-155is inducedduring the macrophage inflammatory response. Proc Natl Acad Sci USA,2007,104:1604-1609
4Androulidaki A, Iliopoulos D, Arranz A, et al. The kinase Akt1controlsmacrophage response to lipopolysaccharide by regulating microRNAs.Immunity,2009,31:220-231
5Ceppi M, Pereira PM, Dunand-Sauthier I, et al. MicroRNA-155modulatesthe interleukin-1signaling pathway in activated human monocyte-deriveddendritic cells. Proc Natl Acad Sci USA,2009,106:2735-2740
6Louafi F, Martinez-Nunez RT, and Sanchez-Elsner T. MicroRNA-155targets SMAD2and modulates the response of macrophages totransforming growth factor-{beta}. J Biol Chem,2010,285:20492-20498
7McCoy CE, Sheedy FJ, Qualls JE, et al. IL-10inhibits miR-155inductionby toll-like receptors. J Biol Chem,2010,285:20492-20498
8Fichtlscherer S, De Rosa S, Fox H, et al. Circulating microRNAs inpatients with coronary artery disease. Circ Res,2010,107:677-684
9Rayner KJ, Sheedy FJ, Esau CC, et al. Antagonism of miR-33in micepromotes reverse cholesterol transport and regression of atherosclerosis. JClin Invest,2011,121:2921-2931
10Donners MM, Wolfs IM, Stoger LJ, et al. Hematopoietic miR155deficiency enhances atherosclerosis and decreases plaque stability inhyperlipidemic mice. PloS One,2012,7: e35877
11Nazari-Jahantigh M, Wei Y, Noels H, et al. MicroRNA-155promotesatherosclerosis by repressing Bcl6in macro-phages. J Clin Invest,2012,122:4190-4202
12陈育尧，黄雪玲等。小鼠尾静脉注射法。毒理学杂志，2008，22(4):311-312
13余绍兰，马跃荣等。实验用小鼠尾静脉穿刺的几点体会。沪州医学院学报，2007，30(4):330
14蒋兰兰，玛俊涛，罗百灵，等,硝酸廿汕干预法对小鼠尾静脉穿刺效果影响。中国医学工程，2004,12(4):92-93
15Moore KJ, Tabas I. Macrophages in the pathogenesis of atherosclerosis.Cell,2011,145:341-355
16Schober A, Weber C. Mechanisms of monocyte recruitment in vascularrepair after injury. Antioxid Redox Signal,2005,7:1249-1257
17Weber C, Noels H. Atherosclerosis: current pathogenesis and therapeuticoptions. Nat Med,2011,17:1410-1422
18Graff JW, Dickson AM, Clay G, et al. Identifying functional microRNAs inmacrophages with polarized pheno-types. J Biol Chem,2012,287:21816-21825
19Martin MM, Buckenberger JA, Jiang J, et al. The human angiotensin IItype1receptor&1166A/C polymorphism attenuates microrna-155binding.J Biol Chem,2007,282:24262-24269
20Zhu N, Zhang D, Chen S, et al. Endothelial enriched microRNAs regulateangiotensin II-induced endothelial inflammation and migration.Atherosclerosis,2011,215:286-293
21Taganov KD, Boldin MP, Chang KJ, et al. NF-kappaB-dependent inductionof microRNA miR-146, an inhibitor targeted to signaling proteins of innateimmune responses.Proc Natl Acad Sci USA,2006,103:12481-12486
22Ruggiero T, Trabucchi M, De Santa F, et al. LPS induces KH-type splicingregulatory protein-dependent processing of microRNA-155precursors inmacrophages. FASEB J,2009,23:2898-2908
23Raitoharju E, Lyytikainen LP, Levula M, et al. miR-21, miR-210, miR-34a,and miR-146a/b are up-regulated in human atherosclerotic plaques in theTampere Vascular Study. Atherosclerosis,2011,219:211-217
24Yang K, He YS, Wang XQ, et al. MiR-146a inhibits oxidized low-densitylipoprotein-induced lipid accu-mulation and inflammatory response viatargeting toll-like receptor4. FEBS Lett,2011,585:854-860
25Drechsler M, Megens RT, Zandvoort M, et al. Hyperlipidemiatriggeredneutrophilia promotes early atherosclerosis. Circulation,2010,122:1837-1845
1Shimizu N, Suzuki H, Wakabayashi K, et al. Expression of intercellularadhesion molecule-1and vascular cell adhesion molecule-1in the pigcoronary artery injury model: comparison of plain old balloon angioplastyand stent implantation. Cardiol,2004,43(3):131-139
2Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. NEngl J Med,2005,352(16):1685-1695
3Ogawa H. Coronary artery disease and inflammation. Nihon Naika GakkaiZasshi,2008,97(3):524-532
4Sbarsi I, Falcone C, Boiocchi C, et al. Inflammation and atherosclerosis:the role of TNF and TNF receptors polymorphisms in coronary arterydisease. Int J Immunopathol Pharmacol,2007,20(1):145-154
5Drachman DE, Simon DI. Inflammation as a mechanism and therapeutictarget for in-stent restenosis. Curr Atheroscler Rep,2005,7(1):44-49
6Chu WM. Tumor necrosis factor. Cancer Lett,2013,328(2):222-225
7Bradley JR. TNF-mediated inflammatory disease. J Pathol,2008,214(2):149-160
8Androulidaki A, Iliopoulos D, Arranz A, et al. The kinase Akt1controlsmacrophage response to lipopolysaccharide by regulating microRNAs.Immunity,2009,31:220-231
9Lee CW, Lin CC, Lin WN, et al. TNF-alpha induces MMP-9expressionvia activation of Src/EGFR, PDGFR/PI3K/Akt cascade and promotion ofNF-kappaB/p300binding in human tracheal smooth muscle cells. Am JPhysiol Lung Cell Mol Physiol,2007,292(3):L799-812
10Van Buuren F, Dahm JB, Horskotte D. Stent restenosis and thrombosis:etiology,treatment, and outcomes. Minerva Med,2012,103(6):503-511
11O'Connell RM, Taganov KD, Boldin MP, et al. MicroRNA-155is inducedduring the macrophage inflammatory response. Proc Natl Acad Sci USA,2007,104:1604-1609
12Sun HX, Zeng DY, Li RT, et al. Essential role of microRNA-155inregulating endothelium-dependent vasorelaxation by targeting endothelialnitric oxide synthase. Hypertension,2012,60:1407-1414
13Bala S, Marcos M, Kodys K, et al. Up-regulation of microRNA-155inmacrophages contributes to increased tumor necrosis factor {alpha}(TNF{alpha}) production via increased mRNA half-life in alcoholic liverdisease. J Biol Chem,2011,286:1436-1444
14O’ Connell RM, Chaudhuri AA, Rao DS, et al. Inositol phosphatase SHIP1is a primary target of miR-155. Proc Natl Acad Sci USA,2009,106:7113–7118
15Arranz A, Doxaki C, Vergadi E, et al. Akt1and Akt2protein kinasesdifferentially contribute to macrophage polarization. Proc Natl Acad SciUSA,2012,109:9517-9522
16Fernandez-Hernando C, Ackah E, Yu J, et al. Loss of Akt1leads to severeatherosclerosis and occlusive coronary artery disease. Cell Metab,2007,6:446-457
17Wei Y, Nazari-Jahantigh M, Chan L, et al. The microRNA-342-5p fostersinflammatory macrophage activation through an Akt1-andmicroRNA-155-dependent pathway during atherosclerosis. Circulation,2013,127:1609-1619
18Bluhm F, Bemtgen X, Zeiser R, et al. MicroRNA-155exertsanti-angiogenic but pro-arteriogenic effects in the regulation of adaptiveneovascularization in mice by cell specific regulation of divergent targetgenes. Eur. Heart J,2013,34:3675
1Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med,2000,6:389-395
2Gibbons G H and Dzau V J. The emerging concept of vascular remodeling.N Engl JMed,1994,330:1431-1438
3Schober A. Chemokines in vascular dysfunction and remodeling.Arterioscler Thromb Vasc Biol,2008,28:1950-1959
4Morrell N W, Adnot S, Archer S L, et al. Cellular and molecular basis ofpulmonary arterial hypertension. J Am Coll Cardiol,2009,54: S20-31
5Nazari-Jahantigh M, Wei Y, Noels H, et al. MicroRNA-155promotesatherosclerosis by repressing Bcl6in macrophages. J Clin Invest,2012,122:4190-4202
6Weber C and Noels H. Atherosclerosis: current pathogenesis andtherapeutic options. Nat Med,2011,17:1410-1422
7Rensen S S, Doevendans P A and van Eys G J. Regulation andcharacteristics of vascular smooth muscle cell phenotypic diversity. NethHeart J,2007,15:100-108
8Owens G K, Kumar M S and Wamhoff B R. Molecular regulation ofvascular smooth muscle cell differentiation in development and disease.Physiol Rev,2004,84:767-801
9Doran A C, Meller N and McNamara C A. Role of smooth muscle cells inthe initiation and early progression of atherosclerosis. Arterioscler ThrombVasc Biol,2008,28:812-819
10Hu Y and Xu Q. Adventitial biology: differentiation and function.Arterioscler Thromb Vasc Biol,2011,31:1523-1529
11Li G, Chen S J, Oparil S, et al. Direct in vivo evidence demonstratingneointimal migration of adventitial fibroblasts after balloon injury of ratcarotid arteries. Circulation,2000,101:1362-1365
12Chatzizisis Y S, Coskun A U, Jonas M, et al. Role of endothelial shearstress in the natural history of coronary atherosclerosis and vascularremodeling: molecular, cellular, and vascular behavior. J Am Coll Cardiol,2007,49:2379-2393
13Savoia C and Schiffrin E L. Inflammation in hypertension. Curr OpinNephrol Hypertens,2006,15:152-158
14Schober A and Weber C. Mechanisms of monocyte recruitment in vascularrepair after injury. Antioxid Redox Signal,2005,7:1249-1257
15Schober A and Zernecke A. Chemokines in vascular remodeling. ThrombHaemost,2007,97:730-737
16Yan Z Q and Hansson G K. Innate immunity, macrophage activation, andatherosclerosis. Immunol Rev,2007,219:187-203
17Fichtlscherer S, De Rosa S, Fox H, et al. Circulating microRNAs inpatients with coronary artery disease. Circ Res,2010,107:677-684
18Ji R, Cheng Y, Yue J, et al. MicroRNA expression signature andantisense-mediated depletion reveal an essential role of MicroRNA invascular neointimal lesion formation. Circ Res,2007,100:1579-1588
19Raitoharju E, Lyytikainen L P, Levula M, et al. miR-21, miR-210,miR-34a, and miR-146a/b are up-regulated in human atheroscleroticplaques in the Tampere Vascular Study. Atherosclerosis,2011,219:211-217
20Yang G, Pei Y, Cao Q, et al. MicroRNA-21represses human cystathioninegamma-lyase expression by targeting at specificity protein-1in smoothmuscle cells. J Cell Physiol,2012,227:3192-3200
21Davis B N, Hilyard A C, Lagna G, et al. SMAD proteins controlDROSHA-mediated microRNA maturation. Nature,2008,454:56-61
22Wang F, Zhao X Q, Liu J N, et al. Antagonist of microRNA-21improvesballoon injury-induced rat iliac artery remodeling by regulatingproliferation and apoptosis of adventitial fibroblasts and myofibroblasts. JCell Biochem,2012,113:2989-3001
23Yang S, Banerjee S, Freitas A, et al. miR-21regulates chronichypoxia-induced pulmonary vascular remodeling. Am J Physiol Lung CellMol Physiol,2012,302: L521-529
24Horita H N, Simpson P A, Ostriker A, et al. Serum response factorregulates expression of phosphatase and tensin homolog through amicroRNA network in vascular smooth muscle cells. Arterioscler ThrombVasc Biol,2011,31:2909-2919
25Kuehbacher A, Urbich C, Zeiher A M, et al. Role of Dicer and Drosha forendothelial microRNA expression and angiogenesis. Circ Res,2007,101:59-68
26Voellenkle C, Rooij J, Guffanti A, et al. Deep-sequencing of endothelialcells exposed to hypoxia reveals the complexity of known and novelmicroRNAs. RNA,2012,18:472-484
27Weber M, Baker M B, Moore J P, et al. MiR-21is induced in endothelialcells by shear stress and modulates apoptosis and eNOS activity. BiochemBiophys Res Commun,2010,393:643-648
28Zhou J, Wang K C, Wu W, et al. MicroRNA-21targets peroxisomeproliferators-activated receptor-alpha in an autoregulatory loop to modulateflow-induced endothelial inflammation. Proc Natl Acad Sci USA,2011,108:10355-10360
29Ando J and Yamamoto K. Effects of shear stress and stretch on endothelialfunction. Antioxid Redox Signal,2011,15:1389-1403
30Parikh V N, Jin R C, Rabello S, et al. MicroRNA-21integrates pathogenicsignaling to control pulmonary hypertension: results of a networkbioinformatics approach. Circulation,2012,125:1520-1532
31Harris T A, Yamakuchi M, Ferlito M, et al. MicroRNA-126regulatesendothelial expression of vascular cell adhesion molecule1. Proc NatlAcad Sci USA,2008,105:1516-1521
32Wang S, Aurora A B, Johnson B A, et al. The endothelial-specificmicroRNA miR-126governs vascular integrity and angiogenesis. Dev Cell,2008,15:261-271
33Harris T A, Yamakuchi M, Kondo M, et al. Ets-1and Ets-2regulate theexpression of microRNA-126in endothelial cells. Arterioscler ThrombVasc Biol,2010,30:1990-1997
34Nicoli S, Standley C, Walker P, et al. MicroRNA-mediated integration ofhaemodynamics and Vegf signalling during angiogenesis. Nature,2010,464:1196-1200
35Hergenreider E, Heydt S, Treguer K, et al. Atheroprotectivecommunication between endothelial cells and smooth muscle cells throughmiRNAs. Nat Cell Biol,2012,14:249-256
36Fish J E, Santoro M M, Morton S U, et al. miR-126regulates angiogenicsignaling and vascular integrity. Dev Cell,2008,15:272-284
37van Solingen C, de Boer H C, Bijkerk R, et al. MicroRNA-126modulatesendothelial SDF-1expression and mobilization of Sca-1(+)/Lin(-)progenitor cells in ischaemia. Cardiovasc Res,2011,92:449-455
38Asgeirsdottir S A, van Solingen C, Kurniati N F, et al. MicroRNA-126contributes to renal microvascular heterogeneity of VCAM-1proteinexpression in acute inflammation. Am J Physiol Renal Physiol,2012,302:F1630-1639
39Zernecke A, Bidzhekov K, Noels H, et al. Delivery of microRNA-126byapoptotic bodies induces CXCL12-dependent vascular protection. SciSignal,2009,2: ra81
40Zampetaki A, Kiechl S, Drozdov I, et al. Plasma microRNA profilingreveals loss of endothelial miR-126and other microRNAs in type2diabetes. Circ Res,2010,107:810-817
41De Rosa S, Fichtlscherer S, Lehmann R, et al. Transcoronary concentrationgradients of circulating microRNAs. Circulation,2011,124:1936-1944
42Chen J, Yin H, Jiang Y, et al. Induction of microRNA-1by myocardin insmooth muscle cells inhibits cell proliferation. Arterioscler Thromb VascBiol,2011,31:368-375
43Cordes K R, Sheehy N T, White M P, et al. miR-145and miR-143regulatesmooth muscle cell fate and plasticity. Nature,2009,460:705-710
44Jiang Y, Yin H and Zheng X L. MicroRNA-1inhibits myocardin-inducedcontractility of human vascular smooth muscle cells. J Cell Physiol,2010,225:506-511
45Xin M, Small E M, Sutherland L B, et al. MicroRNAs miR-143andmiR-145modulate cytoskeletal dynamics and responsiveness of smoothmuscle cells to injury. Genes Dev,2009,23:2166-2178
46Boucher J M, Peterson S M, Urs S, et al. The miR-143/145cluster is anovel transcriptional target of Jagged-1/Notch signaling in vascular smoothmuscle cells. J Biol Chem,2011,286:28312-28321
47Yamaguchi S, Yamahara K, Homma K, et al. The role of microRNA-145in human embryonic stem cell differentiation into vascular cells.Atherosclerosis,2011,219:468-474
48Cheng Y, Liu X, Yang J, et al. MicroRNA-145, a novel smooth muscle cellphenotypic marker and modulator, controls vascular neointimal lesionformation. Circ Res,2009,105:158-166
49Boettger T, Beetz N, Kostin S, et al. Acquisition of the contractilephenotype by murine arterial smooth muscle cells depends on theMir143/145gene cluster. J Clin Invest,2009,119:2634-2647
50Elia L, Quintavalle M, Zhang J, et al. The knockout of miR-143and-145alters smooth muscle cell maintenance and vascular homeostasis in mice:correlates with human disease. Cell Death Differ,2009,16:1590-1598
51Olson E N, Arnold H H, Rigby P W, et al. Know your neighbors: threephenotypes in null mutants of the myogenic bHLH gene MRF4. Cell,1996,85:1-4
52Pham C T, MacIvor D M, Hug B A, et al. Long-range disruption of geneexpression by a selectable marker cassette. Proc Natl Acad Sci USA,1996,93:13090-13095
53Fouillade C, Monet-Lepretre M, Baron-Menguy C, et al. Notch signallingin smooth muscle cells during development and disease. Cardiovasc Res,2012,95:138-146
54Gomez D and Owens G K. Smooth muscle cell phenotypic switching inatherosclerosis. Cardiovasc Res,2012,95:156-164
55Trang P, Wiggins J F, Daige C L, et al. Systemic delivery of tumorsuppressor microRNA mimics using a neutral lipid emulsion inhibits lungtumors in mice. Mol Ther,2011,19:1116-1122
56Zeiffer U, Schober A, Lietz M, et al. Neointimal smooth muscle cellsdisplay a proinflammatory phenotype resulting in increased leukocyterecruitment mediated by P-selectin and chemokines. Circ Res,2004,94:776-784
57Lovren F, Pan Y, Quan A, et al. MicroRNA-145targeted therapy reducesatherosclerosis. Circulation,2012,126: S81-90
58Liu X, Cheng Y, Zhang S, et al. A necessary role of miR-221and miR-222in vascular smooth muscle cell proliferation and neointimal hyperplasia.Circ Res,2009,104:476-487
59Zhu N, Zhang D, Chen S, et al. Endothelial enriched microRNAs regulateangiotensin II-induced endothelial inflammation and migration.Atherosclerosis,2011,215:286-293
60Galardi S, Mercatelli N, Farace M G, et al. NF-kB and c-Jun induce theexpression of the oncogenic miR-221and miR-222in prostate carcinomaand glioblastoma cells. Nucleic Acids Res,2011,39:3892-3902
61Stinson S, Lackner M R, Adai A T, et al. TRPS1targeting by miR-221/222promotes the epithelial-to-mesenchymal transition in breast cancer. SciSignal,2011,4: ra41
62Zhang H, Sunnarborg S W, McNaughton K K, et al. Heparin-bindingepidermal growth factor-like growth factor signaling in flow-inducedarterial remodeling. Circ Res,2008,102:1275-1285
63Liu X, Cheng Y, Yang J, et al. Cell-specific effects of miR-221/222invessels: molecular mechanism and therapeutic application. J Mol CellCardiol,2012,52:245-255
64Davis B N, Hilyard A C, Nguyen P H, et al. Induction of microRNA-221by platelet-derived growth factor signaling is critical for modulation ofvascular smooth muscle phenotype. J Biol Chem,2009,284:3728-3738
65Versari D, Lerman L O and Lerman A. The importance ofreendothelialization after arterial injury. Curr Pharm Des,2007,13:1811-1824
66Dentelli P, Rosso A, Orso F, et al. microRNA-222controlsneovascularization by regulating signal transducer and activator oftranscription5A expression. Arterioscler Thromb Vasc Biol,2010,30:1562-1568
67Martin M M, Buckenberger J A, Jiang J, et al. The human angiotensin IItype1receptor+1166A/C polymorphism attenuates microRNA-155binding. J Biol Chem,2007,282:24262-24269
68Moore K J and Tabas I. Macrophages in the pathogenesis of atherosclerosis.Cell,2011,145:341-355
69Graff J W, Dickson A M, Clay G, et al. Identifying functional microRNAsin macrophages with polarized phenotypes. J Biol Chem,2012,287:21816-21825
70Faraoni I, Antonetti F R, Cardone J, et al. miR-155gene: a typicalmultifunctional microRNA. Biochim Biophys Acta,2009,1792:497-505
71O'Connell R M, Rao D S, Chaudhuri A A, et al. Physiological andpathological roles for microRNAs in the immune system. Nat RevImmunol,2010,10:111-122
72Taganov K D, Boldin M P, Chang K J, et al. NF-kappaB-dependentinduction of microRNA miR-146, an inhibitor targeted to signalingproteins of innate immune responses. Proc Natl Acad Sci USA,2006,103:12481-12486
73Ceppi M, Pereira P M, Dunand-Sauthier I, et al. MicroRNA-155modulatesthe interleukin-1signaling pathway in activated human monocyte-deriveddendritic cells. Proc Natl Acad Sci USA,2009,106:2735-2740
74McCoy C E, Sheedy F J, Qualls J E, et al. IL-10inhibits miR-155induction by toll-like receptors. J Biol Chem,2010,285:20492-20498
75Luyendyk J P, Schabbauer G A, Tencati M, et al. Genetic analysis of therole of the PI3K-Akt pathway in lipopolysaccharide-induced cytokine andtissue factor gene expression in monocytes/macrophages. J Immunol,2008,180:4218-4226
76Androulidaki A, Iliopoulos D, Arranz A, et al. The kinase Akt1controlsmacrophage response to lipopolysaccharide by regulating microRNAs.Immunity,2009,31:220-231
77Louafi F, Martinez-Nunez R T and Sanchez-Elsner T. MicroRNA-155targets SMAD2and modulates the response of macrophages totransforming growth factor-{beta}. J Biol Chem,2010,285:41328-41336
78Yang K, He Y S, Wang X Q, et al. MiR-146a inhibits oxidized low-densitylipoprotein-induced lipid accumulation and inflammatory response viatargeting toll-like receptor4. FEBS Lett,2011,585:854-860
79Donners M M, Wolfs I M, Stoger L J, et al. Hematopoietic miR155deficiency enhances atherosclerosis and decreases plaque stability inhyperlipidemic mice. PLoS One,2012,7: e35877
80Drechsler M, Megens R T, van Zandvoort M, et al.Hyperlipidemia-triggered neutrophilia promotes early atherosclerosis.Circulation,2010,122:1837-1845
81Heeneman S, Sluimer J C and Daemen M J. Angiotensin-convertingenzyme and vascular remodeling. Circ Res,2007,101:441-454
82Tehler D, Hoyland-Kroghsbo N M and Lund A H. The miR-10microRNAprecursor family. RNA Biol,2011,8:728-734
83Garzon R, Pichiorri F, Palumbo T, et al. MicroRNA fingerprints duringhuman megakaryocytopoiesis. Proc Natl Acad Sci USA,2006,103:5078-5083
84Han L, Witmer P D, Casey E, et al. DNA methylation regulates MicroRNAexpression. Cancer Biol Ther,2007,6:1284-1288
85Weiss F U, Marques I J, Woltering J M, et al. Retinoic acid receptorantagonists inhibit miR-10a expression and block metastatic behavior ofpancreatic cancer. Gastroenterology,2009,137:2136-2145e2131-2137
86Huang H, Xie C, Sun X, et al. miR-10a contributes to retinoid acid-inducedsmooth muscle cell differentiation. J Biol Chem,2010,285:9383-9389
87Ma L, Teruya-Feldstein J and Weinberg R A. Tumour invasion andmetastasis initiated by microRNA-10b in breast cancer. Nature,2007,449:682-688
88Qin X, Wang X, Wang Y, et al. MicroRNA-19a mediates the suppressiveeffect of laminar flow on cyclin D1expression in human umbilical veinendothelial cells. Proc Natl Acad Sci USA,2010,107:3240-3244
89Fang Y, Shi C, Manduchi E, et al. MicroRNA-10a regulation ofproinflammatory phenotype in athero-susceptible endothelium in vivo andin vitro. Proc Natl Acad Sci USA,2010,107:13450-13455
90Chen K, Fan W, Wang X, et al. MicroRNA-101mediates the suppressiveeffect of laminar shear stress on mTOR expression in vascular endothelialcells. Biochem Biophys Res Commun,2012,427:138-142
91Fingar D C, Richardson C J, Tee A R, et al. mTOR controls cell cycleprogression through its cell growth effectors S6K1and4E-BP1/eukaryotictranslation initiation factor4E. Mol Cell Biol,2004,24:200-216
92Ni C W, Qiu H and Jo H. MicroRNA-663upregulated by oscillatory shearstress plays a role in inflammatory response of endothelial cells. Am JPhysiol Heart Circ Physiol,2011,300: H1762-1769
93Hamik A, Lin Z, Kumar A, et al. Kruppel-like factor4regulatesendothelial inflammation. J Biol Chem,2007,282:13769-13779
94Krutzfeldt J, Rajewsky N, Braich R, et al. Silencing of microRNAs in vivowith 'antagomirs'. Nature,2005,438:685-689
95Elmen J, Lindow M, Schutz S, et al. LNA-mediated microRNA silencingin non-human primates. Nature,2008,452:896-899
96Obad S, dos Santos C O, Petri A, et al. Silencing of microRNA families byseed-targeting tiny LNAs. Nat Genet,2011,43:371-378
97Rayner K J, Sheedy F J, Esau C C, et al. Antagonism of miR-33in micepromotes reverse cholesterol transport and regression of atherosclerosis. JClin Invest,2011,121:2921-2931
98Zhang Y, Liu D, Chen X, et al. Secreted monocytic miR-150enhancestargeted endothelial cell migration. Mol Cell,2010,39:133-144
2Schwartz SM, deBlois D, O’Brien ER. The intima: soil for atherosclerosisand restenosis. Circ Res,1995,77:445-465
3Libby P, Tanaka H. The molecular bases of restenosis. Prog Cardiovasc Dis,1997,40:97-106.
4Pasterkamp G, de Kleijn DP, Borst C. Arterial remodeling inatheroscle-rosis, restenosis and after alteration of blood flow: potentialmechanisms and clinical implications. Cardiovasc Res,2000,45:843-852
5Vo¨ lker W, Dorszewski A, Unruh V, et al. Copper-induced inflammatoryreactions of rat carotid arteries mimic restenosis/arteriosclerosis-likeneointima formation. Atherosclerosis,1997,130:29-36
6Owens GK, Kumar MS, Wamhoff BR. Molecular regulation ofvascularsmooth muscle cell differentiation in development and disease.Physiol Rev,2004,84:767-801
7Bonta PI, Pols TW, van Tiel CM, et al. Nuclear receptor Nurr1is expressedin and is associated with human restenosis and inhibits vascular lesionformation in mice involving inhibition of smooth muscle cell proliferationand inflammation. Circulation,2010,121:2023-2032
8Bose D, Leineweber K, Konorza T, et al. Release of TNF-alpha duringstent implantation into saphenous vein aortocoronary bypass grafts and itsrelation to plaque extrusion and restenosis. Am J Physiol Heart CircPhysiol,2007,292: H2295-2299
9Moore KJ, and Tabas I. Macrophages in the pathogenesis of atherosclerosis.Cell,2011,145:341-355
10Wu T, Harrison RA, Chen X, et al. Tongxinluo (tong xin luo ortong-xin-luo) capsule for unstable angina pectoris. Cochrane Database SystRev,2006, CD004474
11Li Z, Yang YJ, Qin XW, et al. Effects of tongxinluo and simvastatin on thestabilization of vulnerable atherosclerotic plaques of aorta in aorticatherosclerosis and molecular mechanism thereof: a comparative studywith rabbits. Zhonghua Yi Xue Za Zhi,2006,86:3146-3150
12Liu JX, Shang XH, Wang G. Effect of tongxinluo capsule on experi-mentalmyocardial ischemia, arrhythmia and hyperlipidemia. Zhongguo Zhong XiYi Jie He Za Zhi,1997,17:425-428
13Wu YL, You JH, Yuan GQ, et al. The effects of tongxinluo supermicropowder on nuclear factor-kappaB, inter-cellular adhesion molecule-1andvascular cell adhesion molecule-1ex-pression in aorta of rabbits fed withhigh-lipid diet. Zhonghua Xin Xue Guan Bing Za Zhi,2007,35:271-274
14Zhang L, Wu YL, Jia ZH, et al. Protective effects of a compound herbalextract (tong xin luo) on free fatty acid induced endothelial injury:implications of antioxidant system. BMC Complement Altern Med,2008,8:39
15黄峻,李春坚，杨国平等。通心络对兔外周血管球囊损伤后血栓形成及内膜增生的影响。中国临床药理学与治疗学,2001,6(1):35-37
16熊清萍,张社兵，吕新华等。通心络对兔髂动脉球囊损伤后内膜增生的影响。中国动脉硬化杂志,2004,12(6):727-728
17Kumar A, Lindner V. Remodeling with neointima formation in the mousecarotid artery after cessation of blood flow. Arterioscler Thromb Vasc Biol,1997,17:2238-2244
18Cheng KS, Mikhailidis DP, Hamilton G, et al. A review of the carotid andfemoral intima-media thickness as an indicator of the presence ofperipheral vascular disease and cardiovascular risk factors. Cardiovascularresearch,2002,54:528-538
19Ando J, and Yamamoto K. Effects of shear stress and stretch on endothelialfunction. Antioxidants&redox signaling,2011,15:1389-1403
20Su W, Sun A, Xu D, et al. Tongxinluo inhibits oxidized low-densitylipoprotein-induced maturation of human dendritic cells via activatingperoxisome proliferator-activated receptor gamma pathway. Journal ofcardiovascular pharmacology,2010,56:177-183
21Chen WQ, Zhong L, Zhang L, et al. Chinese medicine tongxinluosignificantly lowers serum lipid levels and stabilizes vulnerable plaques ina rabbit model. Journal of ethnopharmacology,2009,124:103-110
22Zhang L, Liu Y, Lu XT, et al. Traditional Chinese medication Tongxinluodose-dependently enhances stability of vulnerable plaques: a comparisonwith a high-dose simvastatin therapy. Am J Physiol Heart Circ Physiol,2009,297: H2004-2014
23Rectenwald JE, Moldawer LL, Huber TS, et al. Direct evidence forcytokine involvement in neointimal hyperplasia. Circulation,2000,102:1697-1702
24Jovinge S, Hultgardh-Nilsson A, Regnstrom J, et al. Tumor necrosisfactor-alpha activates smooth muscle cell migration in culture and isexpressed in the balloon-injured rat aorta. Arterioscler Thromb Vasc Biol,1997,17:490-497
25Yu K, Zheng B, Han M, et al. ATRA activates and PDGF-BB represses theSM22alpha promoter through KLF4binding to, or dissociating from, itscis-DNA elements. Cardiovascular research,2011,90:464-474
26Danenberg HD, Fishbein I, Gao J, et al. Macrophage depletion byclodronate-containing liposomes reduces neointimal formation afterballoon injury in rats and rabbits. Circulation,2002,106:599-605
1Faraoni I, Antonetti FR, Cardone J, et al. miR-155gene: a typicalmultifunctional microRNA. Biochim Biophys Acta,2009,1792:497-505
2O’Connell RM, Rao DS, Chaudhuri AA, et al. Physiological andpathological roles for microRNAs in the immune system. Nat RevImmunol,2010,10:111-122
3O’Connell RM, Taganov KD, Boldin MP, et al. MicroRNA-155is inducedduring the macrophage inflammatory response. Proc Natl Acad Sci USA,2007,104:1604-1609
4Androulidaki A, Iliopoulos D, Arranz A, et al. The kinase Akt1controlsmacrophage response to lipopolysaccharide by regulating microRNAs.Immunity,2009,31:220-231
5Ceppi M, Pereira PM, Dunand-Sauthier I, et al. MicroRNA-155modulatesthe interleukin-1signaling pathway in activated human monocyte-deriveddendritic cells. Proc Natl Acad Sci USA,2009,106:2735-2740
6Louafi F, Martinez-Nunez RT, and Sanchez-Elsner T. MicroRNA-155targets SMAD2and modulates the response of macrophages totransforming growth factor-{beta}. J Biol Chem,2010,285:20492-20498
7McCoy CE, Sheedy FJ, Qualls JE, et al. IL-10inhibits miR-155inductionby toll-like receptors. J Biol Chem,2010,285:20492-20498
8Fichtlscherer S, De Rosa S, Fox H, et al. Circulating microRNAs inpatients with coronary artery disease. Circ Res,2010,107:677-684
9Rayner KJ, Sheedy FJ, Esau CC, et al. Antagonism of miR-33in micepromotes reverse cholesterol transport and regression of atherosclerosis. JClin Invest,2011,121:2921-2931
10Donners MM, Wolfs IM, Stoger LJ, et al. Hematopoietic miR155deficiency enhances atherosclerosis and decreases plaque stability inhyperlipidemic mice. PloS One,2012,7: e35877
11Nazari-Jahantigh M, Wei Y, Noels H, et al. MicroRNA-155promotesatherosclerosis by repressing Bcl6in macro-phages. J Clin Invest,2012,122:4190-4202
12陈育尧，黄雪玲等。小鼠尾静脉注射法。毒理学杂志，2008，22(4):311-312
13余绍兰，马跃荣等。实验用小鼠尾静脉穿刺的几点体会。沪州医学院学报，2007，30(4):330
14蒋兰兰，玛俊涛，罗百灵，等,硝酸廿汕干预法对小鼠尾静脉穿刺效果影响。中国医学工程，2004,12(4):92-93
15Moore KJ, Tabas I. Macrophages in the pathogenesis of atherosclerosis.Cell,2011,145:341-355
16Schober A, Weber C. Mechanisms of monocyte recruitment in vascularrepair after injury. Antioxid Redox Signal,2005,7:1249-1257
17Weber C, Noels H. Atherosclerosis: current pathogenesis and therapeuticoptions. Nat Med,2011,17:1410-1422
18Graff JW, Dickson AM, Clay G, et al. Identifying functional microRNAs inmacrophages with polarized pheno-types. J Biol Chem,2012,287:21816-21825
19Martin MM, Buckenberger JA, Jiang J, et al. The human angiotensin IItype1receptor&1166A/C polymorphism attenuates microrna-155binding.J Biol Chem,2007,282:24262-24269
20Zhu N, Zhang D, Chen S, et al. Endothelial enriched microRNAs regulateangiotensin II-induced endothelial inflammation and migration.Atherosclerosis,2011,215:286-293
21Taganov KD, Boldin MP, Chang KJ, et al. NF-kappaB-dependent inductionof microRNA miR-146, an inhibitor targeted to signaling proteins of innateimmune responses.Proc Natl Acad Sci USA,2006,103:12481-12486
22Ruggiero T, Trabucchi M, De Santa F, et al. LPS induces KH-type splicingregulatory protein-dependent processing of microRNA-155precursors inmacrophages. FASEB J,2009,23:2898-2908
23Raitoharju E, Lyytikainen LP, Levula M, et al. miR-21, miR-210, miR-34a,and miR-146a/b are up-regulated in human atherosclerotic plaques in theTampere Vascular Study. Atherosclerosis,2011,219:211-217
24Yang K, He YS, Wang XQ, et al. MiR-146a inhibits oxidized low-densitylipoprotein-induced lipid accu-mulation and inflammatory response viatargeting toll-like receptor4. FEBS Lett,2011,585:854-860
25Drechsler M, Megens RT, Zandvoort M, et al. Hyperlipidemiatriggeredneutrophilia promotes early atherosclerosis. Circulation,2010,122:1837-1845
1Shimizu N, Suzuki H, Wakabayashi K, et al. Expression of intercellularadhesion molecule-1and vascular cell adhesion molecule-1in the pigcoronary artery injury model: comparison of plain old balloon angioplastyand stent implantation. Cardiol,2004,43(3):131-139
2Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. NEngl J Med,2005,352(16):1685-1695
3Ogawa H. Coronary artery disease and inflammation. Nihon Naika GakkaiZasshi,2008,97(3):524-532
4Sbarsi I, Falcone C, Boiocchi C, et al. Inflammation and atherosclerosis:the role of TNF and TNF receptors polymorphisms in coronary arterydisease. Int J Immunopathol Pharmacol,2007,20(1):145-154
5Drachman DE, Simon DI. Inflammation as a mechanism and therapeutictarget for in-stent restenosis. Curr Atheroscler Rep,2005,7(1):44-49
6Chu WM. Tumor necrosis factor. Cancer Lett,2013,328(2):222-225
7Bradley JR. TNF-mediated inflammatory disease. J Pathol,2008,214(2):149-160
8Androulidaki A, Iliopoulos D, Arranz A, et al. The kinase Akt1controlsmacrophage response to lipopolysaccharide by regulating microRNAs.Immunity,2009,31:220-231
9Lee CW, Lin CC, Lin WN, et al. TNF-alpha induces MMP-9expressionvia activation of Src/EGFR, PDGFR/PI3K/Akt cascade and promotion ofNF-kappaB/p300binding in human tracheal smooth muscle cells. Am JPhysiol Lung Cell Mol Physiol,2007,292(3):L799-812
10Van Buuren F, Dahm JB, Horskotte D. Stent restenosis and thrombosis:etiology,treatment, and outcomes. Minerva Med,2012,103(6):503-511
11O'Connell RM, Taganov KD, Boldin MP, et al. MicroRNA-155is inducedduring the macrophage inflammatory response. Proc Natl Acad Sci USA,2007,104:1604-1609
12Sun HX, Zeng DY, Li RT, et al. Essential role of microRNA-155inregulating endothelium-dependent vasorelaxation by targeting endothelialnitric oxide synthase. Hypertension,2012,60:1407-1414
13Bala S, Marcos M, Kodys K, et al. Up-regulation of microRNA-155inmacrophages contributes to increased tumor necrosis factor {alpha}(TNF{alpha}) production via increased mRNA half-life in alcoholic liverdisease. J Biol Chem,2011,286:1436-1444
14O’ Connell RM, Chaudhuri AA, Rao DS, et al. Inositol phosphatase SHIP1is a primary target of miR-155. Proc Natl Acad Sci USA,2009,106:7113–7118
15Arranz A, Doxaki C, Vergadi E, et al. Akt1and Akt2protein kinasesdifferentially contribute to macrophage polarization. Proc Natl Acad SciUSA,2012,109:9517-9522
16Fernandez-Hernando C, Ackah E, Yu J, et al. Loss of Akt1leads to severeatherosclerosis and occlusive coronary artery disease. Cell Metab,2007,6:446-457
17Wei Y, Nazari-Jahantigh M, Chan L, et al. The microRNA-342-5p fostersinflammatory macrophage activation through an Akt1-andmicroRNA-155-dependent pathway during atherosclerosis. Circulation,2013,127:1609-1619
18Bluhm F, Bemtgen X, Zeiser R, et al. MicroRNA-155exertsanti-angiogenic but pro-arteriogenic effects in the regulation of adaptiveneovascularization in mice by cell specific regulation of divergent targetgenes. Eur. Heart J,2013,34:3675
1Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med,2000,6:389-395
2Gibbons G H and Dzau V J. The emerging concept of vascular remodeling.N Engl JMed,1994,330:1431-1438
3Schober A. Chemokines in vascular dysfunction and remodeling.Arterioscler Thromb Vasc Biol,2008,28:1950-1959
4Morrell N W, Adnot S, Archer S L, et al. Cellular and molecular basis ofpulmonary arterial hypertension. J Am Coll Cardiol,2009,54: S20-31
5Nazari-Jahantigh M, Wei Y, Noels H, et al. MicroRNA-155promotesatherosclerosis by repressing Bcl6in macrophages. J Clin Invest,2012,122:4190-4202
6Weber C and Noels H. Atherosclerosis: current pathogenesis andtherapeutic options. Nat Med,2011,17:1410-1422
7Rensen S S, Doevendans P A and van Eys G J. Regulation andcharacteristics of vascular smooth muscle cell phenotypic diversity. NethHeart J,2007,15:100-108
8Owens G K, Kumar M S and Wamhoff B R. Molecular regulation ofvascular smooth muscle cell differentiation in development and disease.Physiol Rev,2004,84:767-801
9Doran A C, Meller N and McNamara C A. Role of smooth muscle cells inthe initiation and early progression of atherosclerosis. Arterioscler ThrombVasc Biol,2008,28:812-819
10Hu Y and Xu Q. Adventitial biology: differentiation and function.Arterioscler Thromb Vasc Biol,2011,31:1523-1529
11Li G, Chen S J, Oparil S, et al. Direct in vivo evidence demonstratingneointimal migration of adventitial fibroblasts after balloon injury of ratcarotid arteries. Circulation,2000,101:1362-1365
12Chatzizisis Y S, Coskun A U, Jonas M, et al. Role of endothelial shearstress in the natural history of coronary atherosclerosis and vascularremodeling: molecular, cellular, and vascular behavior. J Am Coll Cardiol,2007,49:2379-2393
13Savoia C and Schiffrin E L. Inflammation in hypertension. Curr OpinNephrol Hypertens,2006,15:152-158
14Schober A and Weber C. Mechanisms of monocyte recruitment in vascularrepair after injury. Antioxid Redox Signal,2005,7:1249-1257
15Schober A and Zernecke A. Chemokines in vascular remodeling. ThrombHaemost,2007,97:730-737
16Yan Z Q and Hansson G K. Innate immunity, macrophage activation, andatherosclerosis. Immunol Rev,2007,219:187-203
17Fichtlscherer S, De Rosa S, Fox H, et al. Circulating microRNAs inpatients with coronary artery disease. Circ Res,2010,107:677-684
18Ji R, Cheng Y, Yue J, et al. MicroRNA expression signature andantisense-mediated depletion reveal an essential role of MicroRNA invascular neointimal lesion formation. Circ Res,2007,100:1579-1588
19Raitoharju E, Lyytikainen L P, Levula M, et al. miR-21, miR-210,miR-34a, and miR-146a/b are up-regulated in human atheroscleroticplaques in the Tampere Vascular Study. Atherosclerosis,2011,219:211-217
20Yang G, Pei Y, Cao Q, et al. MicroRNA-21represses human cystathioninegamma-lyase expression by targeting at specificity protein-1in smoothmuscle cells. J Cell Physiol,2012,227:3192-3200
21Davis B N, Hilyard A C, Lagna G, et al. SMAD proteins controlDROSHA-mediated microRNA maturation. Nature,2008,454:56-61
22Wang F, Zhao X Q, Liu J N, et al. Antagonist of microRNA-21improvesballoon injury-induced rat iliac artery remodeling by regulatingproliferation and apoptosis of adventitial fibroblasts and myofibroblasts. JCell Biochem,2012,113:2989-3001
23Yang S, Banerjee S, Freitas A, et al. miR-21regulates chronichypoxia-induced pulmonary vascular remodeling. Am J Physiol Lung CellMol Physiol,2012,302: L521-529
24Horita H N, Simpson P A, Ostriker A, et al. Serum response factorregulates expression of phosphatase and tensin homolog through amicroRNA network in vascular smooth muscle cells. Arterioscler ThrombVasc Biol,2011,31:2909-2919
25Kuehbacher A, Urbich C, Zeiher A M, et al. Role of Dicer and Drosha forendothelial microRNA expression and angiogenesis. Circ Res,2007,101:59-68
26Voellenkle C, Rooij J, Guffanti A, et al. Deep-sequencing of endothelialcells exposed to hypoxia reveals the complexity of known and novelmicroRNAs. RNA,2012,18:472-484
27Weber M, Baker M B, Moore J P, et al. MiR-21is induced in endothelialcells by shear stress and modulates apoptosis and eNOS activity. BiochemBiophys Res Commun,2010,393:643-648
28Zhou J, Wang K C, Wu W, et al. MicroRNA-21targets peroxisomeproliferators-activated receptor-alpha in an autoregulatory loop to modulateflow-induced endothelial inflammation. Proc Natl Acad Sci USA,2011,108:10355-10360
29Ando J and Yamamoto K. Effects of shear stress and stretch on endothelialfunction. Antioxid Redox Signal,2011,15:1389-1403
30Parikh V N, Jin R C, Rabello S, et al. MicroRNA-21integrates pathogenicsignaling to control pulmonary hypertension: results of a networkbioinformatics approach. Circulation,2012,125:1520-1532
31Harris T A, Yamakuchi M, Ferlito M, et al. MicroRNA-126regulatesendothelial expression of vascular cell adhesion molecule1. Proc NatlAcad Sci USA,2008,105:1516-1521
32Wang S, Aurora A B, Johnson B A, et al. The endothelial-specificmicroRNA miR-126governs vascular integrity and angiogenesis. Dev Cell,2008,15:261-271
33Harris T A, Yamakuchi M, Kondo M, et al. Ets-1and Ets-2regulate theexpression of microRNA-126in endothelial cells. Arterioscler ThrombVasc Biol,2010,30:1990-1997
34Nicoli S, Standley C, Walker P, et al. MicroRNA-mediated integration ofhaemodynamics and Vegf signalling during angiogenesis. Nature,2010,464:1196-1200
35Hergenreider E, Heydt S, Treguer K, et al. Atheroprotectivecommunication between endothelial cells and smooth muscle cells throughmiRNAs. Nat Cell Biol,2012,14:249-256
36Fish J E, Santoro M M, Morton S U, et al. miR-126regulates angiogenicsignaling and vascular integrity. Dev Cell,2008,15:272-284
37van Solingen C, de Boer H C, Bijkerk R, et al. MicroRNA-126modulatesendothelial SDF-1expression and mobilization of Sca-1(+)/Lin(-)progenitor cells in ischaemia. Cardiovasc Res,2011,92:449-455
38Asgeirsdottir S A, van Solingen C, Kurniati N F, et al. MicroRNA-126contributes to renal microvascular heterogeneity of VCAM-1proteinexpression in acute inflammation. Am J Physiol Renal Physiol,2012,302:F1630-1639
39Zernecke A, Bidzhekov K, Noels H, et al. Delivery of microRNA-126byapoptotic bodies induces CXCL12-dependent vascular protection. SciSignal,2009,2: ra81
40Zampetaki A, Kiechl S, Drozdov I, et al. Plasma microRNA profilingreveals loss of endothelial miR-126and other microRNAs in type2diabetes. Circ Res,2010,107:810-817
41De Rosa S, Fichtlscherer S, Lehmann R, et al. Transcoronary concentrationgradients of circulating microRNAs. Circulation,2011,124:1936-1944
42Chen J, Yin H, Jiang Y, et al. Induction of microRNA-1by myocardin insmooth muscle cells inhibits cell proliferation. Arterioscler Thromb VascBiol,2011,31:368-375
43Cordes K R, Sheehy N T, White M P, et al. miR-145and miR-143regulatesmooth muscle cell fate and plasticity. Nature,2009,460:705-710
44Jiang Y, Yin H and Zheng X L. MicroRNA-1inhibits myocardin-inducedcontractility of human vascular smooth muscle cells. J Cell Physiol,2010,225:506-511
45Xin M, Small E M, Sutherland L B, et al. MicroRNAs miR-143andmiR-145modulate cytoskeletal dynamics and responsiveness of smoothmuscle cells to injury. Genes Dev,2009,23:2166-2178
46Boucher J M, Peterson S M, Urs S, et al. The miR-143/145cluster is anovel transcriptional target of Jagged-1/Notch signaling in vascular smoothmuscle cells. J Biol Chem,2011,286:28312-28321
47Yamaguchi S, Yamahara K, Homma K, et al. The role of microRNA-145in human embryonic stem cell differentiation into vascular cells.Atherosclerosis,2011,219:468-474
48Cheng Y, Liu X, Yang J, et al. MicroRNA-145, a novel smooth muscle cellphenotypic marker and modulator, controls vascular neointimal lesionformation. Circ Res,2009,105:158-166
49Boettger T, Beetz N, Kostin S, et al. Acquisition of the contractilephenotype by murine arterial smooth muscle cells depends on theMir143/145gene cluster. J Clin Invest,2009,119:2634-2647
50Elia L, Quintavalle M, Zhang J, et al. The knockout of miR-143and-145alters smooth muscle cell maintenance and vascular homeostasis in mice:correlates with human disease. Cell Death Differ,2009,16:1590-1598
51Olson E N, Arnold H H, Rigby P W, et al. Know your neighbors: threephenotypes in null mutants of the myogenic bHLH gene MRF4. Cell,1996,85:1-4
52Pham C T, MacIvor D M, Hug B A, et al. Long-range disruption of geneexpression by a selectable marker cassette. Proc Natl Acad Sci USA,1996,93:13090-13095
53Fouillade C, Monet-Lepretre M, Baron-Menguy C, et al. Notch signallingin smooth muscle cells during development and disease. Cardiovasc Res,2012,95:138-146
54Gomez D and Owens G K. Smooth muscle cell phenotypic switching inatherosclerosis. Cardiovasc Res,2012,95:156-164
55Trang P, Wiggins J F, Daige C L, et al. Systemic delivery of tumorsuppressor microRNA mimics using a neutral lipid emulsion inhibits lungtumors in mice. Mol Ther,2011,19:1116-1122
56Zeiffer U, Schober A, Lietz M, et al. Neointimal smooth muscle cellsdisplay a proinflammatory phenotype resulting in increased leukocyterecruitment mediated by P-selectin and chemokines. Circ Res,2004,94:776-784
57Lovren F, Pan Y, Quan A, et al. MicroRNA-145targeted therapy reducesatherosclerosis. Circulation,2012,126: S81-90
58Liu X, Cheng Y, Zhang S, et al. A necessary role of miR-221and miR-222in vascular smooth muscle cell proliferation and neointimal hyperplasia.Circ Res,2009,104:476-487
59Zhu N, Zhang D, Chen S, et al. Endothelial enriched microRNAs regulateangiotensin II-induced endothelial inflammation and migration.Atherosclerosis,2011,215:286-293
60Galardi S, Mercatelli N, Farace M G, et al. NF-kB and c-Jun induce theexpression of the oncogenic miR-221and miR-222in prostate carcinomaand glioblastoma cells. Nucleic Acids Res,2011,39:3892-3902
61Stinson S, Lackner M R, Adai A T, et al. TRPS1targeting by miR-221/222promotes the epithelial-to-mesenchymal transition in breast cancer. SciSignal,2011,4: ra41
62Zhang H, Sunnarborg S W, McNaughton K K, et al. Heparin-bindingepidermal growth factor-like growth factor signaling in flow-inducedarterial remodeling. Circ Res,2008,102:1275-1285
63Liu X, Cheng Y, Yang J, et al. Cell-specific effects of miR-221/222invessels: molecular mechanism and therapeutic application. J Mol CellCardiol,2012,52:245-255
64Davis B N, Hilyard A C, Nguyen P H, et al. Induction of microRNA-221by platelet-derived growth factor signaling is critical for modulation ofvascular smooth muscle phenotype. J Biol Chem,2009,284:3728-3738
65Versari D, Lerman L O and Lerman A. The importance ofreendothelialization after arterial injury. Curr Pharm Des,2007,13:1811-1824
66Dentelli P, Rosso A, Orso F, et al. microRNA-222controlsneovascularization by regulating signal transducer and activator oftranscription5A expression. Arterioscler Thromb Vasc Biol,2010,30:1562-1568
67Martin M M, Buckenberger J A, Jiang J, et al. The human angiotensin IItype1receptor+1166A/C polymorphism attenuates microRNA-155binding. J Biol Chem,2007,282:24262-24269
68Moore K J and Tabas I. Macrophages in the pathogenesis of atherosclerosis.Cell,2011,145:341-355
69Graff J W, Dickson A M, Clay G, et al. Identifying functional microRNAsin macrophages with polarized phenotypes. J Biol Chem,2012,287:21816-21825
70Faraoni I, Antonetti F R, Cardone J, et al. miR-155gene: a typicalmultifunctional microRNA. Biochim Biophys Acta,2009,1792:497-505
71O'Connell R M, Rao D S, Chaudhuri A A, et al. Physiological andpathological roles for microRNAs in the immune system. Nat RevImmunol,2010,10:111-122
72Taganov K D, Boldin M P, Chang K J, et al. NF-kappaB-dependentinduction of microRNA miR-146, an inhibitor targeted to signalingproteins of innate immune responses. Proc Natl Acad Sci USA,2006,103:12481-12486
73Ceppi M, Pereira P M, Dunand-Sauthier I, et al. MicroRNA-155modulatesthe interleukin-1signaling pathway in activated human monocyte-deriveddendritic cells. Proc Natl Acad Sci USA,2009,106:2735-2740
74McCoy C E, Sheedy F J, Qualls J E, et al. IL-10inhibits miR-155induction by toll-like receptors. J Biol Chem,2010,285:20492-20498
75Luyendyk J P, Schabbauer G A, Tencati M, et al. Genetic analysis of therole of the PI3K-Akt pathway in lipopolysaccharide-induced cytokine andtissue factor gene expression in monocytes/macrophages. J Immunol,2008,180:4218-4226
76Androulidaki A, Iliopoulos D, Arranz A, et al. The kinase Akt1controlsmacrophage response to lipopolysaccharide by regulating microRNAs.Immunity,2009,31:220-231
77Louafi F, Martinez-Nunez R T and Sanchez-Elsner T. MicroRNA-155targets SMAD2and modulates the response of macrophages totransforming growth factor-{beta}. J Biol Chem,2010,285:41328-41336
78Yang K, He Y S, Wang X Q, et al. MiR-146a inhibits oxidized low-densitylipoprotein-induced lipid accumulation and inflammatory response viatargeting toll-like receptor4. FEBS Lett,2011,585:854-860
79Donners M M, Wolfs I M, Stoger L J, et al. Hematopoietic miR155deficiency enhances atherosclerosis and decreases plaque stability inhyperlipidemic mice. PLoS One,2012,7: e35877
80Drechsler M, Megens R T, van Zandvoort M, et al.Hyperlipidemia-triggered neutrophilia promotes early atherosclerosis.Circulation,2010,122:1837-1845
81Heeneman S, Sluimer J C and Daemen M J. Angiotensin-convertingenzyme and vascular remodeling. Circ Res,2007,101:441-454
82Tehler D, Hoyland-Kroghsbo N M and Lund A H. The miR-10microRNAprecursor family. RNA Biol,2011,8:728-734
83Garzon R, Pichiorri F, Palumbo T, et al. MicroRNA fingerprints duringhuman megakaryocytopoiesis. Proc Natl Acad Sci USA,2006,103:5078-5083
84Han L, Witmer P D, Casey E, et al. DNA methylation regulates MicroRNAexpression. Cancer Biol Ther,2007,6:1284-1288
85Weiss F U, Marques I J, Woltering J M, et al. Retinoic acid receptorantagonists inhibit miR-10a expression and block metastatic behavior ofpancreatic cancer. Gastroenterology,2009,137:2136-2145e2131-2137
86Huang H, Xie C, Sun X, et al. miR-10a contributes to retinoid acid-inducedsmooth muscle cell differentiation. J Biol Chem,2010,285:9383-9389
87Ma L, Teruya-Feldstein J and Weinberg R A. Tumour invasion andmetastasis initiated by microRNA-10b in breast cancer. Nature,2007,449:682-688
88Qin X, Wang X, Wang Y, et al. MicroRNA-19a mediates the suppressiveeffect of laminar flow on cyclin D1expression in human umbilical veinendothelial cells. Proc Natl Acad Sci USA,2010,107:3240-3244
89Fang Y, Shi C, Manduchi E, et al. MicroRNA-10a regulation ofproinflammatory phenotype in athero-susceptible endothelium in vivo andin vitro. Proc Natl Acad Sci USA,2010,107:13450-13455
90Chen K, Fan W, Wang X, et al. MicroRNA-101mediates the suppressiveeffect of laminar shear stress on mTOR expression in vascular endothelialcells. Biochem Biophys Res Commun,2012,427:138-142
91Fingar D C, Richardson C J, Tee A R, et al. mTOR controls cell cycleprogression through its cell growth effectors S6K1and4E-BP1/eukaryotictranslation initiation factor4E. Mol Cell Biol,2004,24:200-216
92Ni C W, Qiu H and Jo H. MicroRNA-663upregulated by oscillatory shearstress plays a role in inflammatory response of endothelial cells. Am JPhysiol Heart Circ Physiol,2011,300: H1762-1769
93Hamik A, Lin Z, Kumar A, et al. Kruppel-like factor4regulatesendothelial inflammation. J Biol Chem,2007,282:13769-13779
94Krutzfeldt J, Rajewsky N, Braich R, et al. Silencing of microRNAs in vivowith 'antagomirs'. Nature,2005,438:685-689
95Elmen J, Lindow M, Schutz S, et al. LNA-mediated microRNA silencingin non-human primates. Nature,2008,452:896-899
96Obad S, dos Santos C O, Petri A, et al. Silencing of microRNA families byseed-targeting tiny LNAs. Nat Genet,2011,43:371-378
97Rayner K J, Sheedy F J, Esau C C, et al. Antagonism of miR-33in micepromotes reverse cholesterol transport and regression of atherosclerosis. JClin Invest,2011,121:2921-2931
98Zhang Y, Liu D, Chen X, et al. Secreted monocytic miR-150enhancestargeted endothelial cell migration. Mol Cell,2010,39:133-144