旋覆花素抑制血管内皮损伤诱导的基质重构酶表达与活化

目的：观察旋覆花素对大鼠血管球囊损伤后内膜增生和基质金属蛋白酶-2（MMP-2）及组织金属蛋白酶抑制剂-2（TIMP-2）表达的影响,探讨旋覆花素防治血管再狭窄的可能作用和机制。方法：用球囊内皮剥脱法复制血管内膜增生模型 通过HE染色观察血管壁形态学变化 明胶酶图分析MMP-2的活性改变 Western blot和免疫组织化学检测MMP-2和TIMP-2的表达变化。结果：旋覆花素显著减轻血管损伤后内膜增生,抑制MMP-2的蛋白水解活性,降低MMP-2和TIMP-2的表达以及MMP-2/TIMP-2比值,并使其接近正常水平。结论：旋覆花素对球囊血管损伤后内膜增生的抑制作用与其对MMP-2/TIMP-2系统平衡调节有关。     

SM22α抑制球囊损伤诱导的新生内膜形成

观察外源性SM22α对球囊损伤诱导的大鼠颈总动脉新生内膜形成的影响,并探讨其机制。雄性SD大鼠经球囊剥脱颈总动脉内皮后随机分为3组:未感染组、pAd组和pAd-SM22α组。术后14天取颈总动脉标本,HE染色观察血管内膜增生情况,用Western blot和免疫组化方法检测外源性SM22α、PCNA和p27在血管壁中的表达水平,以及Raf-1、MEK1/2和ERK1/2的磷酸化水平。实验结果显示,外源性SM22α在血管壁中得到稳定表达;过表达SM22α可显著抑制球囊损伤诱导的血管新生内膜的增厚,与pAd组比较,内膜/中膜比值(I/M)降低70%;Western blot结果显示,在pAd-SM22α组中增殖标志物PCNA表达水平降低(P0.05),而增殖抑制蛋白p27表达水平增高(P0.05),同时伴有增殖相关信号转导分子Raf-1、MEK1/2和ERK1/2的磷酸化水平降低(P0.05)。结果提示,过表达SM22α可抑制球囊损伤诱导的血管内膜增生,其机制可能与阻断Raf-1-MEK1/2-ERK1/2通路的级联活化有关。     

骨桥蛋白13肽抑制球囊内皮剥脱术后血管狭窄的实验研究

目的：利用含有骨桥蛋白（OPN）多种功能位点的13肽（Gly^158-Lys^170）,从细胞和整体水平观察其对VSMC和单核巨噬细胞黏附、浸润以及内膜增生的影响,并初步探讨其作用机制。方法：用不同浓度OPN13肽（0,100,200,300mg／L）检测其对体外培养的平滑肌细胞（VSMc）与OPN黏附的抑制作用;以不合黏附序列的6肽分子为对照组,用以确定13肽抑制黏附特异性。用球囊内皮剥脱法建立大鼠内膜增生模型。实验动物分为4组：治疗组大鼠自术前1h及术后静脉滴注13肽,连续给药7d;对照组大鼠给予非特异性对照6肽分子;模型组大鼠给予相同剂量的生理盐水;正常对照组大鼠施假手术。并利用免疫组织化学染色和Western印迹分析方法,检测血管壁中OPN、FAK、ILK的表达变化。结果：OPN13肽能特异性的及浓度依赖性的抑制VSMC与OPN的相互作用,血管内膜剥脱后给予13肽治疗组血管壁单核／巨噬细胞浸润减少,OPN及其下游信号分子ILK,FAK表达下调,内膜增生被明显抑制。结论：含有OPN多功能位点的13肽可通过阻断OPN与膜受体的相互作用而抑制血管炎症的进展和内膜增生。     

AT2R载体可调控表达对大鼠颈动脉损伤后骨桥蛋白及新生内膜增生的影响

目的:血管紧张素Ⅱ 2型受体(AT2R)基因转染的骨髓间充质干细胞(MSC)在四环素可调控系统下作为载体,利用计算机图像分析系统研究新生内膜增生的情况,并探讨AT2R在体可调控表达对大鼠颈动脉损伤后骨桥蛋白(OPN)表达影响。方法:利用球囊损伤60只SD大鼠颈动脉,并随机将SD大鼠分为5组,分别为正常组(未行球囊扩张术)、对照组(球囊扩张术后注入PBS)、MSC组(球囊扩张术后注入常规MSC)、MSC转染组(球囊扩张术注入转染AT2R的MSC)、强力霉素(Dox)组(球囊扩张术后注入转染AT2R的MSC,术后当天至处死前三天通过尾静脉注射Dox 100μg/kg/d)。术后14及28天分别处死大鼠取材,光镜下观察血管内膜增生情况,Image pro plus 6.0计算机图像分析系统测量新生内膜面积(I/M),逆转录-多聚酶链反应(RT-PCR)检测AT2R及OPN在大鼠血管标本中的表达变化。结果:大鼠颈动脉AT2R在Dox组的表达显著增高,新的增生内膜面积较其它各损伤组显著降低(P≤0.01),并且OPN的表达显著低于其他各手术组。结论:AT2R基因在体可调控表达受到Dox的有效控制,AT2R基因可能抑制血管损伤后OPN的表达及新生内膜的过度增生。     

西洛他唑对大鼠颈总动脉球囊损伤后内膜增生及氧化应激的影响

目的:探讨西洛他唑对大鼠颈总动脉球囊损伤后内膜增生的抑制作用和血管壁氧化应激的影响。方法:SD大鼠24只,随机分组:假手术组、损伤组及西洛他唑治疗组。采用球囊损伤大鼠左侧颈总动脉,于术后2周处死大鼠,取损伤血管标本,进行HE染色、免疫组化染色及原位DHE染色,检测内膜增生、平滑肌细胞增殖及血管壁局部ROS水平。结果:球囊损伤2周后,血管壁内膜显著增生,西洛他唑治疗后内膜增生显著抑制,两组相比P<0.05。PCNA免疫组化染色:假手术组未见PCNA阳性细胞,损伤组PCNA阳性细胞面积百分比明显高于西洛他唑组,主要分布于新生内膜和内弹力膜区域(P<0.05)。原位DHE染色:球囊损伤后局部ROS水平显著升高,较假手术组差异显著P<0.05,西洛他唑干预后局部ROS水平显著降低(P<0.05)。结论:新型抗血小板制剂西洛他唑可显著抑制大鼠颈总动脉球囊损伤后内膜增生及局部氧化应激,抑制局部氧化应激可能是西洛他唑抑制内膜增生的机制之一。     

辛伐他汀干预鼠颈动脉损伤MCP-1的表达

应用鼠颈动脉结扎模型,采用原位杂交、免疫组化技术观察血管单核细胞趋化蛋白-1(monocyte chemoattractant protein-1, MCP-1)的表达及内膜增生情况,探讨辛伐他汀抗内膜增生机制.结果发现损伤血管内膜增生明显,MCP-1的表达增加;辛伐他汀干预可明显抑制血管MCP-1的表达及新生内膜形成.提示血管内膜增生可能与MCP-1表达上调有关,辛伐他汀抑制内膜增生也许通过MCP-1介导.     

Egr-1的诱骗性寡脱氧核苷酸抑制大鼠颈总动脉损伤后MMP-2的表达

目的观察局部转染早期生长反应因子-1(early growth response factor-1,Egr-1)的特异诱骗寡脱氧核苷酸(decoy oligodeoxynucleotides,decoy ODNs)对球囊损伤颈总动脉后基质金属蛋白酶-2(MMP-2)蛋白表达的影响及内膜增生的情况,初步探讨Egr-1,decoy ODNs抑制球囊损伤后内膜增生的机制。方法 96只健康雄性Wistar大鼠,随机分为4组,分别为假手术组、对照组、杂码组和诱骗组,每组24只。除假手术组外均应用2F球囊导管行颈总动脉球囊损伤术,术中采用转染试剂FuGENE6介导的Egr-1decoy ODNs转染至损伤后大鼠血管中,与假手术组、对照组、杂码组相比较。术后3、7、14、21d每组处死6只动物。应用HE染色和免疫组织化学方法观察大鼠颈总动脉球囊损伤后内膜增生情况和MMP-2蛋白的表达及转染Egr-1decoy ODNs后对它们的影响。结果 (1)、内膜损伤后3d内膜增厚不明显,7d内膜开始增厚,14、21d时内膜明显增厚。(2)、在假手术组近腔面中膜可见MMP-2有少量散在阳性表达;在对照组及杂码组动脉损伤后3d,在近腔面中膜,有少量阳性表达,与假手术组相比,阳性表达指数上升。7d时在新生内膜和靠近新生内膜处中膜表达明显,14d后表达逐渐下降。(3)转染decoy ODNs治疗后,在各个时间点内膜增厚程度减轻,MMP-2蛋白表达减少,与对照组比较差异有显著性(P<0.01)。结论血管球囊损伤后,内膜7d开始增生,14d、21d增生更明显,而MMP-2在7d时表达明显,之后逐渐下降,Egr-1decoy ODNs能抑制MMP-2的表达,从而减轻血管损伤后内膜的增生。     

早期生长反应因子-1的特异脱氧核酶对球囊损伤大鼠颈动脉后转化生长因子-β1表达的影响

目的观察局部转染早期生长反应因子-1(Egr-1)的特异脱氧核酶(ED5)对球囊损伤颈总动脉后内膜增生及转化生长因子-β1(TGF-β1)表达的影响,并初步探讨ED5抑制球囊损伤后内膜增生的机制。方法96只健康雄性Wistar大鼠,随机分为4组,每组24只。除假手术组外均应用2F球囊导管行颈总动脉内膜球囊损伤术,术中采用转染试剂FuGENE6介导ED5转染至损伤后大鼠血管中,以假手术组、单纯损伤组,FuGENE6组作为对照。术后3、7、14、21d处死动物每组6只。应用HE染色,RT-PCR,Western-blot和免疫组织化学方法观察大鼠颈动脉球囊损伤后内膜增生情况和TGF-β1的表达及ED5对它们的影响。结果(1)内皮损伤后3d内膜增厚不明显,7d内膜开始增厚,14、21d时内膜明显增厚。(2)TGF-β1mRNA于术后3d开始升高,7d达高峰,14d时下降。TGF-β1蛋白表达于术后3d开始升高,14d达高峰。(3)转染ED5治疗后,在各个时间点内膜增厚程度减轻,TGF-β1表达减少,与对照组比较差异有显著性(P<0·01)。结论血管内皮损伤后内膜增生过程中TGF-β1表达增加,ED5能抑制TGF-β1的表达,从而减轻血管损伤后内膜的增生。     

骨桥蛋白趋化和趋触作用对FAK和ILK磷酸化修饰的影响

为探讨可溶性(趋化作用)或锚定(趋触作用)形式的骨桥蛋白(OPN)与整合素相互作用对下游信号分子黏着斑激酶(FAK)和整合素偶联激酶(ILK)磷酸化修饰的影响,分别用包被于培养瓶上锚定型或加在培养液中的可溶性OPN刺激血管平滑肌细胞(VSMC)后,观察FAK和ILK的磷酸化及FAK与ILK相互作用的变化。结果显示,包被于培养瓶上的OPN通过趋触作用促进VSMC黏附和伸展,接种45min时,黏附细胞数达对照组的2.4倍(P<0.05);OPN的趋触及趋化作用均可诱导FAK磷酸化、ILK去磷酸化并抑制FAK与ILK结合;转染可表达整合素β3亚单位胞内区的表达质粒pEGFP-C3-β3CD能阻断OPN与整合素相互作用所引发的FAK磷酸化及ILK去磷酸化。研究结果表明,OPN的趋触和趋化作用对整合素下游信号分子FAK和ILK的影响是一致的,且这些作用是由整合素β3亚单位胞内区所介导的。     

Src介导骨桥蛋白诱导的血管平滑肌细胞黏附和迁移过程

为阐明酪氨酸激酶Src在整合素被骨桥蛋白(OPN)激活所触发的细胞黏附和迁移信号途径中所起的作用,应用Src特异性抑制剂PP2阻断Src,观察OPN诱导的血管平滑肌细胞(VSMC)黏附和迁移活性的改变,并利用免疫沉淀检查PP2对整合素下游信号分子黏着斑激酶(FAK)和整合素偶联激酶(ILK)磷酸化及其相互作用的影响。结果显示,PP2可明显抑制OPN诱导的VSMC黏附和伤口愈合(黏附和迁移活性分别为对照组的76.6%和33.8%);OPN可显著诱导FAK磷酸化(磷酸化水平达对照组的1.9倍),促进ILK去磷酸化,并使FAK与ILK的结合减少(降至对照组的46.4%)。10μmol/LPP2可明显抑制OPN诱导的FAK磷酸化、拮抗OPN诱导对ILK的去磷酸化作用、促进FAK与ILK之间的结合。研究结果表明,Src作为OPN-整合素-FAK信号途径中的信号分子,通过影响FAK和ILK的磷酸化以及两者之间的相互作用来调节VSMC的黏附和迁移活性。     

Nitric oxide modulates vascular inflammation and intimal hyperplasia in insulin resistance and the metabolic syndrome

Type 2 diabetes mellitus (DM) and the metabolic syndrome, both characterized by insulin resistance, are associated with an accelerated form of atherosclerotic vascular disease and poor outcomes following vascular interventions. These vascular effects are thought to stem from a heightened inflammatory environment and reduced bioavailability of nitric oxide (NO). To better understand this process, we characterized the vascular injury response in the obese Zucker rat by examining the expression of adhesion molecules, the recruitment of inflammatory cells, and the development of intimal hyperplasia. We also evaluated the ability of exogenous NO to inhibit the sequela of vascular injury in the metabolic syndrome. Obese and lean Zucker rats underwent carotid artery balloon injury. ICAM-1 and P-selectin expression were increased following injury in the obese animals compared with the lean rats. The obese rats also responded with increased macrophage infiltration of the vascular wall as well as increased neointima formation compared with their lean counterparts (intima/media = 0.91 vs. 0.52, P = 0.001). After adenovirus-mediated inducible NO synthase (iNOS) gene transfer, ICAM-1, P-selectin, inflammatory cell influx, and oxidized low-density lipoprotein (LDL) receptor expression were all markedly reduced versus injury alone. iNOS gene transfer also significantly inhibited proliferative activity (54% and 73%; P < 0.05) and neointima formation (53% and 67%; P < 0.05) in lean and obese animals, respectively. The vascular injury response in the face of obesity and the metabolic syndrome is associated with increased adhesion molecule expression, inflammatory cell infiltration, oxidized LDL receptor expression, and proliferation. iNOS gene transfer is able to effectively inhibit this heightened injury response and reduce neointima formation in this proinflammatory environment.     

Trauma induced by nontraumatic coronary devices and its impact on vascular reactivity and morphology

This study evaluated the impact of low-pressure balloon devices on coronary morphology and function. An active coronary perfusion catheter (2.5-mm balloon diameter, inflation with 1 bar for 30 min) was placed in the left anterior descending coronary artery of 12 German landrace pigs under general anesthesia. After 3 mo, coronary segments with balloon contact were compared with control segments taken from the right coronary artery as to histology, vascular reactivity, and expression of endothelial nitric oxide synthase. Thirty-three balloon treated segments were analyzed. Twenty of these segments (61%) showed neointima formation. In these segments endothelium-independent relaxation induced by sodium nitroprusside was preserved. However, endothelium-dependent bradykinin-induced relaxation was significantly attenuated compared with both the control segments and the balloon-treated segments without neointima formation. In >60% of the ballooned arterial segments examined, low-pressure balloon devices induced neointima formation accompanied by reduced endothelium-dependent relaxation. Thus interventions with so-called nontraumatic coronary devices can induce relevant vascular injury, with potential adverse clinical consequences.     

麦冬不同提取物对过氧化氢损伤人血管内皮细胞ICAM-1、VEGF、Bcl-2表达的影响

目的:观察麦冬不同提取物对过氧化氢诱导的人脐静脉内皮细胞(HUVEC)间黏附分子-1(ICAM-1)和VEGF、Bc(?)-2表达的影响。方法:体外培养HUVEC,用过氧化氢(H_2O_2)制造HUVEC损伤模型。以四甲基偶氮唑蓝(MTT)比色法检测细胞存活数量,用流式细胞仪检测HUVEC表面ICAM-1的表达量;免疫细胞化学方法检测HUVEC的VEGF、Bc(?)-2的分布情况。结果:模型组较正常对照组细胞增殖活性明显降低(P<0.01)。与模型组相比,经麦冬水提物、正丁醇提取物处理组细胞增殖活性明显增加(P<0.05,P<0.01)。流式细胞仪检测显示正丁醇提取物可降低过氧化氢增加的ICAM-1基因的表达。Bc(?)-2的表达,模型组明显低于正常对照组,而正丁醇组表达明显高于模型组(P<0.01)。VEGF的表达,模型组明显高于正常对照组,麦冬水提物、正丁醇提取物处理组高于模型组(P<0.05,P<0.01)。结论:麦冬提取物具有抗凋亡、促增殖、降低细胞间黏附分子-1表达的作用,尤以正丁醇提取物效果更为显著。     

Adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene inhibits vascular smooth muscle cell proliferation and neointima formation following balloon angioplasty of the rat carotid artery.

BACKGROUND: Vascular smooth muscle cell (VSMC) proliferation following arterial injury plays a critical role in a variety of vascular proliferative disorders, including atherosclerosis and restenosis after balloon angioplasty. In this study, we tested the hypothesis that localized arterial infection at the time of balloon angioplasty with an adenovirus (ADV-tk) encoding the herpes simplex virus thymidine kinase gene (HSV-tk), followed by systemic ganciclovir administration, can inhibit VSMC proliferation and neointima formation in a well-characterized model of arterial injury and restenosis. MATERIALS AND METHODS: The left carotid arteries of 31 male Sprague-Dawley rats were subjected to balloon angioplasty and immediately infected with 2 x 10(9) pfu of either ADV-tk or a control adenovirus that does not encode a recombinant protein (ADV-delta E1). Twenty-four hours after injury, animals from each experimental group were randomized to receive a course of systemic ganciclovir (ADV-tk/+GC, ADV delta E1/+GC) or saline (ADV-tk/-GC, ADV-delta E1/-GC). VSMC DNA synthesis was measured by 5'-bromodeoxuridine (BrdU) incorporation 2-4 days after balloon injury. The extent of restenosis, expressed as the neointima to media (I/M) area ratio was determined by digital planimetry 20 days after balloon injury in each of the four treatment groups. Immunohistochemistry using a mAb to von Willebrand factor (vWF) was used to determine the effects of ADV-tk infection and ganciclovir treatment on re-endothelialization of the carotid arteries 20 days following balloon angioplasty. RESULTS: Forty-one percent of the medial VSMCs in the ADV-tk/-GC arteries were labeled with BrdU 4 days after balloon injury. In contrast, ADV-tk infected animals that were treated with systemic ganciclovir (ADV-tk/+GC) displayed a 40% reduction in BrdU-staining medial VSMCs (p < 0.03). I/M area ratios of the three control groups were 1.17 +/- 0.18 (ADV-tk/-GC, n = 5), 1.15 +/- 0.10 (ADV-delta E1/+GC, n = 6), and 0.91 +/- 0.08 (ADV-delta E1/-GC, n = 6). These differences were not statistically significant (p > 0.05). In contrast, the ADV-tk/+GC animals (n = 6) displayed an I/M area ratio of 0.49 +/- 0.13 which was significantly lower than that seen in each of the three control groups (p < 0.02). None of the treated animals showed evidence of significant organ toxicity at autopsy. A regenerated endothelium was observed in the ADV-tk/+GC animals 20 days after balloon injury. CONCLUSIONS: Localized arterial infection with ADV-tk at the time of balloon angioplasty followed by systemic ganciclovir therapy reduces VSMC proliferation and neointimal expansion in the rat carotid artery injury model. Moreover, combined treatment with ADV-tk and systemic ganciclovir does not result in systemic toxicity and appears to selectively eliminate proliferating VSMCs, while preserving the capacity of the injured arterial segments to re-endothelialize within 3 weeks of injury. Taken together, these results support the feasibility of using this gene therapy approach for the treatment of human vascular proliferative disorders.     

Attenuation of neointima formation through the inhibition of DNA repair enzyme PARP-1 in balloon-injured rat carotid artery

Increased oxidative stress is a major characteristic of restenosis after angioplasty. The oxidative stress is mainly created by oxidants such as reactive oxygen species (ROS), which are assumed to play an important role in neointima formation after angioplasty. DNA is a sensitive target for oxidants; however, oxidative DNA damage remains a poorly examined field in the pathogenesis of restenosis. In the present study, we demonstrated that the expression of the oxidative DNA damage marker 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) was quickly increased in rat carotid arteries after balloon injury. It reached its peak at 14 days after injury and still kept high expression at 28 days after injury. The immunostaining of 8-oxo-dG was present predominantly in the neointima. In response to oxidative DNA damage, the DNA repair enzyme poly(ADP-ribose) polymerase-1 (PARP-1) was significantly increased after balloon injury. The time course change and location of PARP-1 is similar to that of 8-oxo-dG. Daily injections of the PARP-1 inhibitor PJ34 (5 mg.kg(-1).day(-1) ip) attenuated neointima formation by approximately 40% at 7, 14, and 28 days after balloon injury. Treatment with PJ34 inhibited leukocyte infiltration and improved both anatomic (reendothelialization) and functional (endothelial function) recovery of endothelial cells after balloon injury. In conclusion, levels of oxidative DNA damage and the DNA repair enzyme PARP-1 are increased in vessels after balloon injury. Inhibition of PARP-1 attenuates neointima formation through inhibition of leukocyte infiltration and improvement of endothelial cell recovery after balloon injury. Targeting of the DNA repair enzyme might be a therapeutic strategy for restenosis.     

Intramuscular gene transfer of CGRP inhibits neointimal hyperplasia after balloon injury in the rat abdominal aorta

CGRP is a well-known neuropeptide that has various protective effects on cardiovascular system. Our previous studies have shown that CGRP inhibits vascular smooth muscle cell (VSMC) proliferation in vitro. The present study aimed to explore the role of the CGRP in neointimal formation after balloon injury in the rat aortic wall and the underlying mechanism. Gene transfer of CGRP was performed with the use of intramuscular electroporation in a balloon-injured rat aorta model. Apoptosis in VSMCs was determined by electrophoresis assessment of DNA fragmentation and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay. Overexpression of the CGRP gene significantly inhibited the neointimal formation after balloon injury compared with the mock transfer, as assessed by the intima-to-media ratio 14 days after balloon injury (29.2 +/- 3.7% vs. 52.7 +/- 5.4%; n = 9-12, P < 0.05). In addition, CGRP gene expression increased the number of apoptotic cells in the neointima in vivo 14 days after balloon injury. Similarly, the addition of bioactive CGRP and the nitric oxide donor induced similar apoptosis in cultured VSMCs. The antagonist of the CGRP(1) receptor and inhibitors of cAMP-PKA and nitric oxide blocked CGRP-mediated apoptosis. Furthermore, CGRP gene transfer increased inducible nitric oxide synthase and p53 but decreased PCNA and Bcl-2 protein levels in balloon-injured rat aorta. Our data demonstrated that CGRP potently inhibited neointimal thickening in the rat aorta, at least in part through its distinct effects on apoptosis and proliferation of VSMCs both in vivo and in vitro. Therefore, delivery of the CGRP gene may have therapeutic implications in limiting vascular restenosis.     

CD40 is essential in the upregulation of TRAF proteins and NF-kappaB-dependent proinflammatory gene expression after arterial injury

Despite extensive investigations, restenosis, which is characterized primarily by neointima formation, remains an unsolved clinical problem after vascular interventions. A recent study has shown that CD40 signaling through TNF receptor associated factor 6 (TRAF6) plays a key role in neointima formation after carotid artery injury; however, underlying mechanisms are not clearly elucidated. Because neointima formation may vary significantly depending on the type of injury, we first assessed the effect of CD40 deficiency on neointima formation in 2 injury models, carotid artery ligation and femoral artery denudation injury. Compared with wild-type mice, CD40 deficiency significantly reduced neointima formation and lumen stenosis in two different models. Further, we investigated the mechanism by which CD40 signaling affects neointima formation after arterial injury. In wild-type mice, the expression levels of CD40, several TRAF proteins, including TRAF1, TRAF2, TRAF3, TRAF5, and TRAF6, as well as total NF-kB p65 and phospho-NF-kB p65, in the carotid artery were markedly upregulated within 3-7 days after carotid ligation. Deficiency of CD40 abolished the injury-induced upregulation of TRAFs including TRAF6 and NF-kB-p65 in the injured vessel wall. Further, CD40(-/-) mice showed a significant decrease in the recruitment of neutrophils (at 3, 7d) and macrophages (at 7, 21d) into injured artery; this effect was most likely attributed to inhibition of NF-kB activation and marked downregulation of NF-kB-related gene expression, including cytokines (TNFα, IL-1β, IL-6), chemokines (MCP-1), and adhesion molecules (ICAM-1, VCAM-1). Moreover, neutrophil recruitment in a model of thioglycollate-induced peritonitis is impaired in CD40-deficient mice. In vitro data revealed that CD40 deficiency blocked CD40L-induced NF-kB p65 nuclear translocation in leukocytes. Altogether, our data identified for the first time that CD40 is essential in the upregulation of TRAF6, NF-kB activation, and NF-kB-dependent proinflammatory genes in vivo. Our findings firmly established the role for CD40 in neointima formation in 2 distinct injury models.