CD40-CD40L系统在急性脑梗死中的临床意义及其影响斑块稳定性的机制研究
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摘要
动脉粥样硬化是一种慢性炎性疾病,免疫调节起着至关重要的作用。CD40与CD40配体(CD40L,CD154)是一对互补跨膜糖蛋白,分别属于肿瘤坏死因子受体(tumor necrosis factor receptor,TNFR)和TNF超家族成员,是体液免疫和细胞免疫的一条重要的细胞信号转导途径,在抗原提呈和自身免疫性疾病中起重要作用。CD40L在体内主要以2种形式存在,即与相应细胞膜结合的膜结合型CD40L(membrane-bound CD40L,mCD40L)和游离状态的可溶性CD40L(soluble CD40L,sCD40L)。近年来的研究表明CD40-CD40L系统参与了动脉粥样硬化(artherosclerosis,AS)的起始、发展和斑块破溃后急性并发症。在动脉粥样硬化斑块中的上皮细胞、单核—巨噬细胞和平滑肌细胞上均有CD40和CD40L表达,而在正常动脉组织中没有或低表达。CD40L与CD40链接能诱导人类血管内皮细胞表达黏附分子,如E-选择素、血管细胞黏附分子(vascular cell adhesion molecule,VCAM)-1和细胞间黏附分子(intercellular adhesion molecule,ICAM)-1,并刺激释放趋化因子和细胞因子,如白细胞介素(interleukin,IL)-8、调节活化正常T细胞表达和分泌的趋化因子(regulated upon Activation Normal T-cell Expressed andSecreted,RANTES)或巨噬细胞炎性蛋白1a,这些分子参与了动脉粥样硬化斑块的形成。应用特异性抗CD40L抗体阻断CD40-CD40L之间的相互作用可明显抑制AS斑块的形成,并使病灶内成分改善,稳定性增加,巨噬细胞和脂质减少,平滑肌和胶原纤维增加。因此,CD40-CD40L系统不但参与动脉粥样硬化斑块的形成,更重要的是可能参与使斑块不稳定、破溃,导致血栓形成。
目前CD40-CD40L系统与急性冠脉综合征的关系研究已成为热点,动脉粥样硬化是脑梗死的基本病因,但该系统在急性脑梗死中的作用研究尚少,本研究着重观察不同病因类型脑梗死、单纯颈动脉狭窄及正常对照组周围血单核细胞CD40L表达和血清sCD40L水平,分析人颈动脉粥样硬化斑块中CD40表达与临床卒中事件的关系,研究CD40-CD40L系统在急性脑梗死中的临床意义及对动脉粥样硬化斑块稳定性的作用。通过观察颈动脉粥样硬化斑块中CD40、CD40L与MMP_9的关系,探讨CD40-CD40L系统影响斑块稳定性的机制。期望找到判断斑块稳定性、脑梗死病因和严重程度的简便生物学指标,并为治疗和预防动脉粥样硬化和脑梗死找到新的干预靶点。
第一部分急性脑梗死患者周围血单核细胞CD40L表达和血清sCD40L的临床意义
目的观察不同病因类型急性脑梗死患者周围血单核细胞CD40L表达和血清sCD40L水平的变化,探讨其在诊断大动脉粥样硬化型脑梗死和判断脑梗死病情中的价值,研究其对脑动脉粥样硬化及其斑块稳定性的作用。
方法入选我院神经内科的急性脑梗死患者82例,按SSS-TOAST分型分为大动脉粥样硬化型脑梗死组30例、小动脉闭塞型脑梗死组36例和心主动脉栓塞型脑梗死组16例,收集有颈动脉粥样硬化狭窄而无缺血性卒中事件患者17例为单纯颈动脉狭窄组,另设正常对照组20例。对急性脑梗死患者进行NIHSS评分、Barthel指数、改良Rankin评分,分别从神经功能、日常生活活动能力和残疾状况对患者病情进行全方面评估。采用直接免疫荧光流式细胞术检测各组周围血单核细胞CD40L表达,ELISA检测血清sCD40L水平,全自动生化仪以免疫散射比浊法检测各组血清hs-CRP水平。
结果1.大动脉粥样硬化脑梗死组血清hs-CRP水平高于单纯颈动脉狭窄组(P＜0.05)。2.周围血单核细胞CD40L表达,动脉粥样硬化患者高于无动脉粥样硬化患者(P＜0.01),其诊断动脉粥样硬化的敏感性和特异性高于血清hs-CRP(P＜0.01);大动脉粥样硬化型脑梗死组高于单纯颈动脉狭窄组(P＜0.01),其判断斑块稳定性的敏感性和特异性高于血清hs-CRP(P＜0.01);大动脉粥样硬化型脑梗死高于其他类型脑梗死(P＜0.01),其诊断大动脉粥样硬化型脑梗死的敏感性高于血清hs-CRP(P＜0.01),两者的特异性无显著性差异(P＞0.05)。3.血清sCD40L水平与NIHSS评分有正相关关系(r=0.537,P＜0.01),相关性高于hs-CRP(r=0.432,P＜0.01);sCD40L水平与Barthel指数有负相关关系(r=-0.515,P＜0.01),相关性高于hs-CRP(r=-0.510,P＜0.01);sCD40L与改良Rankin评分有正相关关系(r=0.589,P＜0.01),相关性高于hs-CRP(r=0.482,P＜0.01)。
结论血清hs-CRP水平与脑动脉粥样硬化斑块稳定性相关;周围血单核细胞CD40L表达与动脉粥样硬化、斑块稳定性和大动脉粥样硬化型脑梗死相关;单核细胞CD40L表达在判断动脉粥样硬化、斑块稳定性和诊断大动脉粥样硬化型脑梗死上比血清hs-CRP具有更高的敏感性和特异性;血清sCD40L水平与脑梗死病情的严重程度成正相关,与患者的日常生活活动能力成负相关,相关性高于血清hs-CRP,可作为反映脑梗死病情轻重的生物学指标。
第二部分CD40在人颈动脉粥样硬化及斑块稳定性中的作用
目的观察正常颈动脉和颈动脉粥样硬化斑块中CD40 mRNA水平和蛋白表达情况,分析其与临床卒中事件的关系,探讨其与动脉粥样硬化及斑块稳定性之间的关系。
方法收集颈动脉粥样硬化狭窄(＞70%)行颈动脉内膜切除术的颈动脉粥样硬化斑块标本28例,根据患者有无发生急性缺血性脑卒中事件分为有临床卒中事件组和无临床卒中事件组,另收集尸检正常颈动脉标本8例作为对照组。Realtime PCR检测各组CD40 mRNA水平,Western blotting检测各组CD40蛋白表达情况。
结果颈动脉粥样硬化斑块CD40 mRNA水平高于正常颈动脉(P＜0.01),有临床卒中事件组CD40 mRNA水平高于无临床卒中事件组(P＜0.01)。正常颈动脉中CD40基本不表达,无卒中事件组CD40表达明显高于正常颈动脉,有卒中事件组又明显高于无卒中事件组。
结论颈动脉粥样硬化斑块中CD40的转录和表达水平较正常颈动脉明显增高,有临床卒中事件的不稳定斑块增高更加显著,提示CD40参与了动脉粥样硬化的形成,并且与斑块稳定性相关。
第三部分CD40-CD40L系统影响动脉粥样硬化及斑块稳定性的机制研究
目的研究颈动脉粥样硬化斑块中CD40和MMP_9 mRNA水平及蛋白表达的关系,并观察CD40、CD40L和MMP_9表达及分布关系,探讨CD40-CD40L系统影响动脉粥样硬化及斑块稳定性的可能机制。
方法收集颈动脉粥样硬化狭窄(＞70%)行颈动脉内膜切除术患者的颈动脉粥样硬化斑块标本28例,根据患者有无发生急性缺血性脑卒中事件分为有临床卒中事件组和无临床卒中事件组,另收集尸检正常颈动脉标本8例作为对照组。RealtimePCR检测各组CD40和MMP_9 mRNA水平,Western blotting检测各组CD40和MMP_9蛋白表达情况。免疫组化检测各组CD40、CD40L和MMP_9表达及分布。
结果1.颈动脉粥样硬化斑块中CD40、MMP_9 mRNA水平高于正常颈动脉(P＜0.01),且有临床卒中事件组CD40、MMP_9 mRNA水平高于无临床卒中事件组(P＜0.01);2.CD40 mRNA与MMP_9 mRNA有正相关关系(r=0.964,P＜0.01);3.正常颈动脉中CD40和MMP_9基本不表达,无卒中事件组CD40和MMP_9的蛋白表达明显高于正常颈动脉,有卒中事件组又明显高于无卒中事件组;4.免疫组化显示CD40、CD40L主要表达于动脉粥样硬化斑块的内皮细胞和平滑肌细胞,MMP_9主要位于胶原蛋白和平滑肌细胞上,CD40、CD40L和MMP_9均在斑块肩部表达增多尤为明显,CD40、CD40L和MMP_9表达有卒中事件组高于无临床卒中事件组(P＜0.01),无卒中事件组高于正常颈动脉(P＜0.01);5.CD40、CD40L与MMP_9表达有正相关关系(P＜0.01)。
结论CD40-CD40L系统可能通过上调MMP_9导致动脉粥样硬化及斑块不稳定。
综上所述,CD40-CD40L系统可以成为判断脑动脉粥样硬化、斑块稳定性、脑梗死病情轻重和诊断大动脉粥样硬化型脑梗死的敏感的生物学指标,CD40-CD40L系统可能通过上调MMP_9导致动脉粥样硬化和斑块不稳定。进一步深入研究CD40-CD40L系统在动脉粥样硬化和斑块稳定性中的作用及相关机制,有望能为治疗和预防动脉粥样硬化和脑梗死找到干预的新靶点。
Atherosclerosis(AS) is a chronic inflammatory disorder,in which immune regulation plays a crucial role.CD40 is a receptor for CD40 ligand(CD40L,CD154).They are a pair of complemented transmembrane proteins and belong to the TNF receptor(TNFR) superfamily and the TNF superfamily,respectively.CD40-CD40L system is an important cellular signal transduction pathway of humoral and cellular immunity and plays an important role in the antigen presentation and autoimmune diseases.There are mainly two forms of CD40L in vivo,which are cellular membrane-bound CD40L(mCD40L) and soluble CD40L(sCD40L).Recent studies have demonstrated that CD40-CD40L system participates in initiation,evolution,and acute complications after the rupture of atherosclerotic lesions.CD40 and CD40L were expressed by the epithelial cells, monocytes,macrophages and smooth muscle cells in the atherosclerotic plaques. However,they were not expressed or low expressed in the normal arteries.CD40L linkage with CD40 can induce human vascular endothelial cells to express adhesion molecules,such as selectin-E,VCAM-1,ICAM-1,and stimulate the release of chemokines and cytokines,such as IL-8,RANTES or macrophage inflammatory protein 1 a,which take part in the formation of atherosclerotic plaques.Block the interaction of CD40-CD40L with specific CD40L antibody could inhibit the development of atherosclerotic plaques and change the composition of atheroma in manners thought to favor plaque stability,e.g.,reduced relative content of macrophages and lipid,as well as increased relative content of smooth muscle cells and collagen.Therefore,CD40-CD40L system not only participates in the initial events of atherogenesis,but also involves the instability and breakdown of established plaques and induces atherosclerotic thrombosis.
CD40-CD40L system related to atherosclerosis is the research focus in the acute coronal syndrome recently.Atherosclerosis is also the commen cause of acute cerebral infarctions.But there are few studies of CD40-CD40L system in the acute cerebral infarctions.Our researches focus on the expression of CD40L in the peripheral blood monocytes(PBMC) and serum sCD40L levels in different types of cerebral infarctions, pure carotid artery atherosclerotic stenosis and normal controls and analyzes the expression of CD40 in human carotid artery atherosclerotic plaques and its relationship with clinical stroke events.Therefore,the roles of CD40-CD40L system in acute cerebral infarction and plaque instability are evaluated.We further observe the expression and distributions of CD40 and CD40L in carotid artery atherosclerotic plaques and their relationship with MMP_9 and study the possible mechanisms of CD40-CD40L in the instability of plaques.We expect to find out a simple biomarker to evaluate plaque instability,etiology and severities of cerebral infarction and find out a new target for prevention and treatment of atherosclerosis and cerebral infarctions.
PartⅠThe clinical roles of the CD40L expression in peripheral blood monocytes and serum sCD40L levels in patients with acute cerebral infarctions
Objective The CD40L expression in peripheral blood monocytes(PBMC) and serum sCD40L levels were detected in patients with different types of acute cerebral infarctions and their roles in diagnosis of atherosclerotic cerebral infarctions and judgments of severity of cerebral infarctions were evaluated.
Methods 82 patients with acute cerebral infarctions in the Department of Neurology of Changhai Hospital were recruited.They are classified with SSS-TOAST classification as large artery atherosclerosis type 30 cases,small-artery occlusion type 36 cases, cardioaortic embolism type 16 cases.17 patients with carotid artery stenosis without stroke were recruited as pure carotid artery stenosis and 20 normal people as normal control.Patients with acute cerebral infarction were scored with NIHSS,Barthel index and modified Rankin scale to systemly comment their neurological function,activities of daily living and disability situation.Expression of CD40L on peripheral blood monocytes was detected by direct immunofluorescence with flow cytometry and sCD40L levels were measured by ELISA mothed.Serum hs-CRP level was tested by immune scattering ratio with automatic biochemistry.
Results 1.Serum hs-CRP levels in patients with large artery atherosclotic infarctions were higher than pure carotid artery stenosis(P＜0.05).2.The expression of CD40L on periphery blood monocytes in patients with atherosclerotic infarctions were higher than patients without atherosclerosis(P＜0.01).Sensitivity and specificity for its diagnosing atherosclerosis were higher than serum hs-CRP(P＜0.01).Expression of CD40L on periphery blood monocytes in patients with large artery atherosclerotic cerebral infarction was higher than pure carotid artery stenosis(P＜0.01).The sensitivity and specificity in judging atherosclerotic plaque instability were higher than serum hs-CRP (P＜0.01).CD40L expression on PBMC were higher in large artery atherosclerotic cerebral infarction than other types of cerebral infarction(P＜0.01).The sensitivity of CD40L expression on PBMC in diagnosing large artery atherosclerotic cerebral infarction was higher than hs-CRP(P＜0.01) and there were no significant differences in specificity(P＞0.05).3.There was positive correlation between the levels of serum sCD40L and NIHSS scores(r=0.537,P＜0.01) and their correlation was superior to hs-CRP(r=0.432,P＜0.01).There was inverse correlation between sCD40L levels and Barthel index(r=-0.515,P＜0.01) and their correlation was superior to hs-CRP(r=-0.510, P＜0.01).There was positive correlation between sCD40L levels and modified Rankin scales(r=0.589,P＜0.01) and its correlation was superior to hs-CRP(r=0.482,P＜0.01).
Conclusion In patients with acute cerebral infarction,serum hs-CRP was related to atherosclerotic plaque instability and CD40L expression on PBMC was related to atherosclerosis,plaque instability and large artery atherosclerotic cerebral infarctions. The sensitivity and specificity of CD40L expression on PBMC in judging atherosclerosis and plaque instability and diagnosing large artery atherosclerotic cerebral infarction were more senetive than serum hs-CRP.Serum sCD40L levels were related to the severity of cerebral infarctions and had an inverse correlation with patients' ability of daily living and its correlations were superior to serum hs-CRP.Serum sCD40L is a good biomarker of the severity of cerebral infarctions.
PartⅡThe roles of CD40 in human carotid atherosclerosis and plaque instability
Objective The CD40 mRNA and protein expression levels in human carotid artery atherosclerotic plaques and normal carotid artery were detected and its relationship with clinical stroke events was analyzed.The roles of CD40 in atherosclerosis and plaque instability were investigated.
Methods 28 specimen of carotid artery atherosclerotic plaques were collected from carotid endarterectomy due to carotid atherosclerotic stenosis(＞70%) performed in the Department of Vascular Surgery of our hospital.The plaques were further divided into clinical stroke events group(n=15) and no clinical stroke events group(n=13) according to whether the patients had ischemic cerebral strokes before surgery.Another 8 cases of normal carotid artery in autopsy were taken as normal control.CD40 mRNA levels were measured by real time PCR and CD40 protein levels were detected by Western blotting in each group.
Results CD40 mRNA levels in carotid artery atherosclerotic plaques were higher than normal carotid artery(P＜0.01) and higher in clinical stroke events group than no clinical stroke events group(P＜0.01).There was basically no expression of CD40 in normal carotid artery.The CD40 expression in carotid artery atherosclerosis with no clinical stroke events group was significant higher than normal carotid artery.The CD40 expression in carotid artery atherosclerosis with clinical stroke events group was higher than no clinical stroke events group.
Conclusion Transcription and expression levels of CD40 in carotid atherosclerotic plaques were higher than normal carotid artery and even more significantl in patients with clinical stroke events.The results suggested that CD40 may participate in formation of carotid atherosclerosis and be related to the plaque instability.
PartⅢThe investigations of possible mechanisms of CD40-CD40L system in affecting the carotid artery atherosclerosis and plaque instability
Objective CD40 and MMP_9 mRNA,protein expressions and their relationship in carotid artery atherosclerotic plaques were detected.Expression of CD40,CD40L and MMP_9 and their distributions in carotid atherosclerotic plaques were observed.The possible mechanisms of CD40-CD40L system in affecting carotid artery atherosclerosis and plaque instability were explored.
Methods 28 specimen of carotid artery atherosclerotic plaques were collected from carotid endarterectomy due to carotid atherosclerotic stenosis(＞70%) performed in the Department of Vascular Surgery of our hospital.The plaques were further divided into clinical stroke events group(n=15) and no clinical stroke events group(n=13) according to whether the patients had ischemic cerebral strokes before surgery.Another 8 cases of normal carotid artery in autopsy were used as normal control.CD40 and MMP_9 mRNA levels were measured by real time PCR and CD40 and MMP_9 protein levels were detected by Western blotting in each group.Expression and distribution of CD40,CD40L and MMP_9 in atherosclerotic plaques were observed by immunochemistry.
Results 1.CD40 and MMP_9 mRNA levels in carotid artery atherosclerotic plaques were higher than normal carotid artery(P＜0.01) and even more significant in clinical stroke events group than no clinical stroke events group(P＜0.01).2.There was positive correlation between CD40 and MMP_9 mRNA(r=0.964,P＜0.01).3.There was extremely rare expression of CD40 and MMP_9 protein in normal carotid artery.The expression of CD40 and MMP_9 protein was significant higher in carotid artery atherosclerosis with clinical stroke events than without clinical stroke events,and the expression of CD40 and MMP_9 protein in no clinical stroke events group higher than normal carotid artery.4. CD40 and CD40L were mainly expressed in edocthelial cells and smooth muscle cells. MMP_9 was located in the collagen and smooth muscle cells.CD40,CD40L and MMP_9 were more significantly expressed in the shoulder of the atherosclerotic plaques.The expressions of CD40,CD40L and MMP_9 were significantly higher in clinical event group than no clinical event group,and no clinical event group higher than normal controls.5.There were positive correlations between the expression of CD40 and MMP_9, CD40L and MMP_9(P＜0.01).
Conclusion CD40-CD40L system may induce atherosclerosis and plaque instability probably by up-regulation of MMP_9 in carotid artery atherosclerosis. In conclusion,CD40-CD40L system is a sensitive biomarker in judging cerebral atherosclerosis,plaque instability and the severity of cerebral infarctions and diagnosing large artery atherosclerotic cerebral infarctions.The mechanism of CD40-CD40L system in atherosclerosis and plaque instability may be related to up-regulation of MMP_9. Further investigation of the mechanisms of CD40-CD40L system in atherosclerosis and plaque instability will be a novel potential therapeutic target for atherosclerosis and cerebral infarctions.
目前CD40-CD40L系统与急性冠脉综合征的关系研究已成为热点,动脉粥样硬化是脑梗死的基本病因,但该系统在急性脑梗死中的作用研究尚少,本研究着重观察不同病因类型脑梗死、单纯颈动脉狭窄及正常对照组周围血单核细胞CD40L表达和血清sCD40L水平,分析人颈动脉粥样硬化斑块中CD40表达与临床卒中事件的关系,研究CD40-CD40L系统在急性脑梗死中的临床意义及对动脉粥样硬化斑块稳定性的作用。通过观察颈动脉粥样硬化斑块中CD40、CD40L与MMP_9的关系,探讨CD40-CD40L系统影响斑块稳定性的机制。期望找到判断斑块稳定性、脑梗死病因和严重程度的简便生物学指标,并为治疗和预防动脉粥样硬化和脑梗死找到新的干预靶点。
第一部分急性脑梗死患者周围血单核细胞CD40L表达和血清sCD40L的临床意义
目的观察不同病因类型急性脑梗死患者周围血单核细胞CD40L表达和血清sCD40L水平的变化,探讨其在诊断大动脉粥样硬化型脑梗死和判断脑梗死病情中的价值,研究其对脑动脉粥样硬化及其斑块稳定性的作用。
方法入选我院神经内科的急性脑梗死患者82例,按SSS-TOAST分型分为大动脉粥样硬化型脑梗死组30例、小动脉闭塞型脑梗死组36例和心主动脉栓塞型脑梗死组16例,收集有颈动脉粥样硬化狭窄而无缺血性卒中事件患者17例为单纯颈动脉狭窄组,另设正常对照组20例。对急性脑梗死患者进行NIHSS评分、Barthel指数、改良Rankin评分,分别从神经功能、日常生活活动能力和残疾状况对患者病情进行全方面评估。采用直接免疫荧光流式细胞术检测各组周围血单核细胞CD40L表达,ELISA检测血清sCD40L水平,全自动生化仪以免疫散射比浊法检测各组血清hs-CRP水平。
结果1.大动脉粥样硬化脑梗死组血清hs-CRP水平高于单纯颈动脉狭窄组(P＜0.05)。2.周围血单核细胞CD40L表达,动脉粥样硬化患者高于无动脉粥样硬化患者(P＜0.01),其诊断动脉粥样硬化的敏感性和特异性高于血清hs-CRP(P＜0.01);大动脉粥样硬化型脑梗死组高于单纯颈动脉狭窄组(P＜0.01),其判断斑块稳定性的敏感性和特异性高于血清hs-CRP(P＜0.01);大动脉粥样硬化型脑梗死高于其他类型脑梗死(P＜0.01),其诊断大动脉粥样硬化型脑梗死的敏感性高于血清hs-CRP(P＜0.01),两者的特异性无显著性差异(P＞0.05)。3.血清sCD40L水平与NIHSS评分有正相关关系(r=0.537,P＜0.01),相关性高于hs-CRP(r=0.432,P＜0.01);sCD40L水平与Barthel指数有负相关关系(r=-0.515,P＜0.01),相关性高于hs-CRP(r=-0.510,P＜0.01);sCD40L与改良Rankin评分有正相关关系(r=0.589,P＜0.01),相关性高于hs-CRP(r=0.482,P＜0.01)。
结论血清hs-CRP水平与脑动脉粥样硬化斑块稳定性相关;周围血单核细胞CD40L表达与动脉粥样硬化、斑块稳定性和大动脉粥样硬化型脑梗死相关;单核细胞CD40L表达在判断动脉粥样硬化、斑块稳定性和诊断大动脉粥样硬化型脑梗死上比血清hs-CRP具有更高的敏感性和特异性;血清sCD40L水平与脑梗死病情的严重程度成正相关,与患者的日常生活活动能力成负相关,相关性高于血清hs-CRP,可作为反映脑梗死病情轻重的生物学指标。
第二部分CD40在人颈动脉粥样硬化及斑块稳定性中的作用
目的观察正常颈动脉和颈动脉粥样硬化斑块中CD40 mRNA水平和蛋白表达情况,分析其与临床卒中事件的关系,探讨其与动脉粥样硬化及斑块稳定性之间的关系。
方法收集颈动脉粥样硬化狭窄(＞70%)行颈动脉内膜切除术的颈动脉粥样硬化斑块标本28例,根据患者有无发生急性缺血性脑卒中事件分为有临床卒中事件组和无临床卒中事件组,另收集尸检正常颈动脉标本8例作为对照组。Realtime PCR检测各组CD40 mRNA水平,Western blotting检测各组CD40蛋白表达情况。
结果颈动脉粥样硬化斑块CD40 mRNA水平高于正常颈动脉(P＜0.01),有临床卒中事件组CD40 mRNA水平高于无临床卒中事件组(P＜0.01)。正常颈动脉中CD40基本不表达,无卒中事件组CD40表达明显高于正常颈动脉,有卒中事件组又明显高于无卒中事件组。
结论颈动脉粥样硬化斑块中CD40的转录和表达水平较正常颈动脉明显增高,有临床卒中事件的不稳定斑块增高更加显著,提示CD40参与了动脉粥样硬化的形成,并且与斑块稳定性相关。
第三部分CD40-CD40L系统影响动脉粥样硬化及斑块稳定性的机制研究
目的研究颈动脉粥样硬化斑块中CD40和MMP_9 mRNA水平及蛋白表达的关系,并观察CD40、CD40L和MMP_9表达及分布关系,探讨CD40-CD40L系统影响动脉粥样硬化及斑块稳定性的可能机制。
方法收集颈动脉粥样硬化狭窄(＞70%)行颈动脉内膜切除术患者的颈动脉粥样硬化斑块标本28例,根据患者有无发生急性缺血性脑卒中事件分为有临床卒中事件组和无临床卒中事件组,另收集尸检正常颈动脉标本8例作为对照组。RealtimePCR检测各组CD40和MMP_9 mRNA水平,Western blotting检测各组CD40和MMP_9蛋白表达情况。免疫组化检测各组CD40、CD40L和MMP_9表达及分布。
结果1.颈动脉粥样硬化斑块中CD40、MMP_9 mRNA水平高于正常颈动脉(P＜0.01),且有临床卒中事件组CD40、MMP_9 mRNA水平高于无临床卒中事件组(P＜0.01);2.CD40 mRNA与MMP_9 mRNA有正相关关系(r=0.964,P＜0.01);3.正常颈动脉中CD40和MMP_9基本不表达,无卒中事件组CD40和MMP_9的蛋白表达明显高于正常颈动脉,有卒中事件组又明显高于无卒中事件组;4.免疫组化显示CD40、CD40L主要表达于动脉粥样硬化斑块的内皮细胞和平滑肌细胞,MMP_9主要位于胶原蛋白和平滑肌细胞上,CD40、CD40L和MMP_9均在斑块肩部表达增多尤为明显,CD40、CD40L和MMP_9表达有卒中事件组高于无临床卒中事件组(P＜0.01),无卒中事件组高于正常颈动脉(P＜0.01);5.CD40、CD40L与MMP_9表达有正相关关系(P＜0.01)。
结论CD40-CD40L系统可能通过上调MMP_9导致动脉粥样硬化及斑块不稳定。
综上所述,CD40-CD40L系统可以成为判断脑动脉粥样硬化、斑块稳定性、脑梗死病情轻重和诊断大动脉粥样硬化型脑梗死的敏感的生物学指标,CD40-CD40L系统可能通过上调MMP_9导致动脉粥样硬化和斑块不稳定。进一步深入研究CD40-CD40L系统在动脉粥样硬化和斑块稳定性中的作用及相关机制,有望能为治疗和预防动脉粥样硬化和脑梗死找到干预的新靶点。
Atherosclerosis(AS) is a chronic inflammatory disorder,in which immune regulation plays a crucial role.CD40 is a receptor for CD40 ligand(CD40L,CD154).They are a pair of complemented transmembrane proteins and belong to the TNF receptor(TNFR) superfamily and the TNF superfamily,respectively.CD40-CD40L system is an important cellular signal transduction pathway of humoral and cellular immunity and plays an important role in the antigen presentation and autoimmune diseases.There are mainly two forms of CD40L in vivo,which are cellular membrane-bound CD40L(mCD40L) and soluble CD40L(sCD40L).Recent studies have demonstrated that CD40-CD40L system participates in initiation,evolution,and acute complications after the rupture of atherosclerotic lesions.CD40 and CD40L were expressed by the epithelial cells, monocytes,macrophages and smooth muscle cells in the atherosclerotic plaques. However,they were not expressed or low expressed in the normal arteries.CD40L linkage with CD40 can induce human vascular endothelial cells to express adhesion molecules,such as selectin-E,VCAM-1,ICAM-1,and stimulate the release of chemokines and cytokines,such as IL-8,RANTES or macrophage inflammatory protein 1 a,which take part in the formation of atherosclerotic plaques.Block the interaction of CD40-CD40L with specific CD40L antibody could inhibit the development of atherosclerotic plaques and change the composition of atheroma in manners thought to favor plaque stability,e.g.,reduced relative content of macrophages and lipid,as well as increased relative content of smooth muscle cells and collagen.Therefore,CD40-CD40L system not only participates in the initial events of atherogenesis,but also involves the instability and breakdown of established plaques and induces atherosclerotic thrombosis.
CD40-CD40L system related to atherosclerosis is the research focus in the acute coronal syndrome recently.Atherosclerosis is also the commen cause of acute cerebral infarctions.But there are few studies of CD40-CD40L system in the acute cerebral infarctions.Our researches focus on the expression of CD40L in the peripheral blood monocytes(PBMC) and serum sCD40L levels in different types of cerebral infarctions, pure carotid artery atherosclerotic stenosis and normal controls and analyzes the expression of CD40 in human carotid artery atherosclerotic plaques and its relationship with clinical stroke events.Therefore,the roles of CD40-CD40L system in acute cerebral infarction and plaque instability are evaluated.We further observe the expression and distributions of CD40 and CD40L in carotid artery atherosclerotic plaques and their relationship with MMP_9 and study the possible mechanisms of CD40-CD40L in the instability of plaques.We expect to find out a simple biomarker to evaluate plaque instability,etiology and severities of cerebral infarction and find out a new target for prevention and treatment of atherosclerosis and cerebral infarctions.
PartⅠThe clinical roles of the CD40L expression in peripheral blood monocytes and serum sCD40L levels in patients with acute cerebral infarctions
Objective The CD40L expression in peripheral blood monocytes(PBMC) and serum sCD40L levels were detected in patients with different types of acute cerebral infarctions and their roles in diagnosis of atherosclerotic cerebral infarctions and judgments of severity of cerebral infarctions were evaluated.
Methods 82 patients with acute cerebral infarctions in the Department of Neurology of Changhai Hospital were recruited.They are classified with SSS-TOAST classification as large artery atherosclerosis type 30 cases,small-artery occlusion type 36 cases, cardioaortic embolism type 16 cases.17 patients with carotid artery stenosis without stroke were recruited as pure carotid artery stenosis and 20 normal people as normal control.Patients with acute cerebral infarction were scored with NIHSS,Barthel index and modified Rankin scale to systemly comment their neurological function,activities of daily living and disability situation.Expression of CD40L on peripheral blood monocytes was detected by direct immunofluorescence with flow cytometry and sCD40L levels were measured by ELISA mothed.Serum hs-CRP level was tested by immune scattering ratio with automatic biochemistry.
Results 1.Serum hs-CRP levels in patients with large artery atherosclotic infarctions were higher than pure carotid artery stenosis(P＜0.05).2.The expression of CD40L on periphery blood monocytes in patients with atherosclerotic infarctions were higher than patients without atherosclerosis(P＜0.01).Sensitivity and specificity for its diagnosing atherosclerosis were higher than serum hs-CRP(P＜0.01).Expression of CD40L on periphery blood monocytes in patients with large artery atherosclerotic cerebral infarction was higher than pure carotid artery stenosis(P＜0.01).The sensitivity and specificity in judging atherosclerotic plaque instability were higher than serum hs-CRP (P＜0.01).CD40L expression on PBMC were higher in large artery atherosclerotic cerebral infarction than other types of cerebral infarction(P＜0.01).The sensitivity of CD40L expression on PBMC in diagnosing large artery atherosclerotic cerebral infarction was higher than hs-CRP(P＜0.01) and there were no significant differences in specificity(P＞0.05).3.There was positive correlation between the levels of serum sCD40L and NIHSS scores(r=0.537,P＜0.01) and their correlation was superior to hs-CRP(r=0.432,P＜0.01).There was inverse correlation between sCD40L levels and Barthel index(r=-0.515,P＜0.01) and their correlation was superior to hs-CRP(r=-0.510, P＜0.01).There was positive correlation between sCD40L levels and modified Rankin scales(r=0.589,P＜0.01) and its correlation was superior to hs-CRP(r=0.482,P＜0.01).
Conclusion In patients with acute cerebral infarction,serum hs-CRP was related to atherosclerotic plaque instability and CD40L expression on PBMC was related to atherosclerosis,plaque instability and large artery atherosclerotic cerebral infarctions. The sensitivity and specificity of CD40L expression on PBMC in judging atherosclerosis and plaque instability and diagnosing large artery atherosclerotic cerebral infarction were more senetive than serum hs-CRP.Serum sCD40L levels were related to the severity of cerebral infarctions and had an inverse correlation with patients' ability of daily living and its correlations were superior to serum hs-CRP.Serum sCD40L is a good biomarker of the severity of cerebral infarctions.
PartⅡThe roles of CD40 in human carotid atherosclerosis and plaque instability
Objective The CD40 mRNA and protein expression levels in human carotid artery atherosclerotic plaques and normal carotid artery were detected and its relationship with clinical stroke events was analyzed.The roles of CD40 in atherosclerosis and plaque instability were investigated.
Methods 28 specimen of carotid artery atherosclerotic plaques were collected from carotid endarterectomy due to carotid atherosclerotic stenosis(＞70%) performed in the Department of Vascular Surgery of our hospital.The plaques were further divided into clinical stroke events group(n=15) and no clinical stroke events group(n=13) according to whether the patients had ischemic cerebral strokes before surgery.Another 8 cases of normal carotid artery in autopsy were taken as normal control.CD40 mRNA levels were measured by real time PCR and CD40 protein levels were detected by Western blotting in each group.
Results CD40 mRNA levels in carotid artery atherosclerotic plaques were higher than normal carotid artery(P＜0.01) and higher in clinical stroke events group than no clinical stroke events group(P＜0.01).There was basically no expression of CD40 in normal carotid artery.The CD40 expression in carotid artery atherosclerosis with no clinical stroke events group was significant higher than normal carotid artery.The CD40 expression in carotid artery atherosclerosis with clinical stroke events group was higher than no clinical stroke events group.
Conclusion Transcription and expression levels of CD40 in carotid atherosclerotic plaques were higher than normal carotid artery and even more significantl in patients with clinical stroke events.The results suggested that CD40 may participate in formation of carotid atherosclerosis and be related to the plaque instability.
PartⅢThe investigations of possible mechanisms of CD40-CD40L system in affecting the carotid artery atherosclerosis and plaque instability
Objective CD40 and MMP_9 mRNA,protein expressions and their relationship in carotid artery atherosclerotic plaques were detected.Expression of CD40,CD40L and MMP_9 and their distributions in carotid atherosclerotic plaques were observed.The possible mechanisms of CD40-CD40L system in affecting carotid artery atherosclerosis and plaque instability were explored.
Methods 28 specimen of carotid artery atherosclerotic plaques were collected from carotid endarterectomy due to carotid atherosclerotic stenosis(＞70%) performed in the Department of Vascular Surgery of our hospital.The plaques were further divided into clinical stroke events group(n=15) and no clinical stroke events group(n=13) according to whether the patients had ischemic cerebral strokes before surgery.Another 8 cases of normal carotid artery in autopsy were used as normal control.CD40 and MMP_9 mRNA levels were measured by real time PCR and CD40 and MMP_9 protein levels were detected by Western blotting in each group.Expression and distribution of CD40,CD40L and MMP_9 in atherosclerotic plaques were observed by immunochemistry.
Results 1.CD40 and MMP_9 mRNA levels in carotid artery atherosclerotic plaques were higher than normal carotid artery(P＜0.01) and even more significant in clinical stroke events group than no clinical stroke events group(P＜0.01).2.There was positive correlation between CD40 and MMP_9 mRNA(r=0.964,P＜0.01).3.There was extremely rare expression of CD40 and MMP_9 protein in normal carotid artery.The expression of CD40 and MMP_9 protein was significant higher in carotid artery atherosclerosis with clinical stroke events than without clinical stroke events,and the expression of CD40 and MMP_9 protein in no clinical stroke events group higher than normal carotid artery.4. CD40 and CD40L were mainly expressed in edocthelial cells and smooth muscle cells. MMP_9 was located in the collagen and smooth muscle cells.CD40,CD40L and MMP_9 were more significantly expressed in the shoulder of the atherosclerotic plaques.The expressions of CD40,CD40L and MMP_9 were significantly higher in clinical event group than no clinical event group,and no clinical event group higher than normal controls.5.There were positive correlations between the expression of CD40 and MMP_9, CD40L and MMP_9(P＜0.01).
Conclusion CD40-CD40L system may induce atherosclerosis and plaque instability probably by up-regulation of MMP_9 in carotid artery atherosclerosis. In conclusion,CD40-CD40L system is a sensitive biomarker in judging cerebral atherosclerosis,plaque instability and the severity of cerebral infarctions and diagnosing large artery atherosclerotic cerebral infarctions.The mechanism of CD40-CD40L system in atherosclerosis and plaque instability may be related to up-regulation of MMP_9. Further investigation of the mechanisms of CD40-CD40L system in atherosclerosis and plaque instability will be a novel potential therapeutic target for atherosclerosis and cerebral infarctions.
引文
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[22] Cipollone F, Chiarelli F and Davi G, et al. Enhanced soluble CD40 ligand contributes to endothelial cell dysfunction in vitro and monocyte activation in patients with diabetes mellitus: effect of improved metabolic control. Diabetologia, 2005, 48: 1216-1224.
[23] Kouwenhoven M, Carlstrom C and Ozenci V, et al. Matrix metalloproteinase and cytokine profiles in monocytes over the course of stroke. J Clin Immunol, 2001, 21:365-375.
[24] Di Napoli M, Papa F. Inflammation, hemostatic markers and antithrombotic agents in relation to long-term risk of new cardiovascular events in first-ever ischemic stroke patients. Stroke, 2002, 33:1763-1771.
[25] Freedman JE, Loscalzo J. Platelet-monocyte aggregates bridging thrombosis and inflammation. Circulation, 2002,105:2130-2132.
[26] van Kooten F, Ciabattoni G and Koudstaal PJ, et al. Increased platelet activation in the chronic phase after cerebral ischemia and intracerebral hemorrhage. Stroke, 1999, 30:546-549.
[27] Hermann A, Rauch BH and Braun M, et al. Platelet CD40 ligand (CD40L) subcellular localization, regulation of expression and inhibition by clopidogrel. Platelets, 2001,12:82.
[1]Russell R.Atherosclerosis-A inflammatory disease[J].N Engl J M,1999,340:115-126.
[2]Hansson GK,Robertson AK,and Soderberg-Naucler C.Inflammation and Atherosclerosis[J].Annu Rev Pathol Mech Dis,2006,1:297-329.
[3]Endarterectomy for asymptomatic carotid artery stenosis.Executive Committee for the Asymptomatic Carotid Atherosclerosis Study.JAMA.,1995,273:1421-1428.
[4]Risk of stroke in the distribution of an asymptomatic carotid artery.The European Carotid Surgery Trialists Collaborative Group.Lancet.,1995,345:209-212.
[5]Randomised trial of endarterectomy for recently symptomatic carotid stenosis:final results of the MRC European Carotid Surgery Trial(ECST) Lancet.,1998,351:1379-1387.
[6]Shah PK.Pathophysiology of plaque rupture and the concept of plaque stabilization[J].Cardiol Clin,1996,14:17-29.
[7]Fuessl RT,Kranenberg E,Kiausch U,et al.Vascular remodeling in atherosclerotic coronary arteries is affected by plaque composition[J].Coron Artery Dis,2001,12:91-97.
[8]Wiedermann CJ,Kiechl S,Dunzendorfer S,et al.Association of endotoxemia with carotid atherosclerosis and cardiovascular disease:prospective results from the Bruneck Study[J].J Am Coll Cardiol,1999,34:1975-1981.
[9]Ridker PM,Hennekens CH,Buring JE,et al.C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women[J].N Engl J Med,2000,342:836-843.
[10] Pearson TA, Mensah GA, Alexander RW, et al. Markers of inflammation and cardiovascular disease: application to clinical and public health practice: A statement for healthcare professionals from the Centers for Disease Control and Prevention and the American Heart Association[J]. Circulation, 2003,107:499-511.
[11] Ridker PM, Rifai N, Rose L, et al. Comparison of C-reactive protein and low-density lipoprotein cholesterol levels in the prediction of first cardiovascular events[J]. N Engl J Med, 2002, 347:1557-1565.
[12] Koenig W, Lowel H, Baumert J, et al. C-reactive protein modulates risk prediction based on the Framingham Score: implications for future risk assessment: results from a large cohort study in southern Germany[J]. Circulation, 2004, 109:1349-1353.
[13] Ballantyne CM, Hoogeveen RC, Bang H, et al. Lipoprotein-associated phospholipase A2, high-sensitivity C-reactive protein, and risk for incident ischemic stroke in middle-aged men and women in the Atherosclerosis Risk in Communities (ARIC) study[J]. Arch Intern Med, 2005,165:2479-2484.
[14] St-Pierre AC, Bergeron J, Pirro M, et al. Effect of plasma C-reactive protein levels in modulating the risk of coronary heart disease associated with small, dense, low-density lipoproteins in men (The Quebec Cardiovascular Study) [J]. Am J Cardiol, 2003, 91:555-558.
[15] Di Napoli M, Papa F, Bocola V. C-reactive protein in ischemic stroke: an independent prognostic factor[J]. Stroke, 2001, 32:917-924.
[16] Zwaka TP, Hombach V, Torzewski J. C-reactive protein-mediated low density lipoprotein uptake by macrophages: implications for atherosclerosis[J]. Circulation, 2001, 103:1194-1197.
[17] Nakagomi A, Freedman SB, Geczy CL. Interferon-gamma and lipopolysaccharide potentiate monocyte tissue factor induction by C-reactive protein: relationship with age, sex, and hormone replacement treatment[J]. Circulation, 2000, 101:1785-1791.
[18] Penn MS, Topol EJ. Tissue factor, the emerging link between inflammation, thrombosis, and vascular remodeling[J]. Circ Res, 2001, 89:1-2.
[19] Yasojima K, Schwab C, McGeer EG, et al. Generation of C-reactive protein and complement components in atherosclerotic plaques[J]. Am J Pathol, 2001, 158:1039-1051.
[20] Tomai F, Crea F, Gaspardone A, et al. Unstable angina and elevated C-reactive protein levels predict enhanced vasoreactivity of the culprit lesion[J]. Circulation, 2001, 104:1471-1476.
[21]Pasceri V,Willerson JT,Yeh ET.Direct proinflammatory effect of C-reactive protein on human endothelial cells.Circulation[J],2000,102:2165-2168.
[22]Nakajima T,Schulte S,warrington K J,et al.T-cell-mediated lysis of endothelial cells in acute coronary syndromes[J].Circulation,2002,105:570-575.
[23]Fichtlscherer S,Rosenberger G,Walter DH,et al.Elevated C-reactive protein levels and impaired endothelial vasoreactivity in patients with coronary artery disease[J].Circulation,2000,102:1000-1006.
[24]Mulvihill NT,Foley JB,Murphy R,et al.Evidence of prolonged inflammation in unstable angina and non-Q wave myocardial infarction[J].J Am Coll Cardiol,2000,36:1210-1216.
[25]Fisher M,Paganini-Hill A,Martin A,et al.Carotid plaque pathology:thrombosis,ulceration,and stroke pathogenesis[J].Stroke,2005,36:253-257.
[26]Luc G,Arveiler D,Evans A,et al.Circulating soluble adhesion molecules ICAM-1and VCAM-1 and incident coronary heart disease:the PRIME Study[J].Atherosclerosis,2003,170:169-176.
[27]Guray U,Erbay AR,Guray Y,et al.Levels of soluble adhesion molecules in various clinical presentations of coronary atherosclerosis[J].Int J Cardiol,2004,96:235-240.
[28]Doo YC,Han SJ,Lee JH,Cho GY,et al.Associations among oxidized low-density lipoprotein antibody,C-reactive protein,interleukin-6,and circulating cell adhesion molecules in patients with unstable angina pectoris[J].Am J Cardiol,2004,93:554-558.
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[33] Shah PK. Pathophysiology of plaque rupture and the concept of plaque stabilization. Cardiol Clin, 1996, 14:17-29.
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[1]Richard PP.Atherosclerosis:The emerging role of inflammation and the CD40-CD40 ligand system.Proc Natl Acad Sci USA,2000,97:6930-6932.
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[6]Hollenbaugh D,Mischel-Petty N and Edwards CP,et al.Expression of functional CD40 by vascular endothelial cells.J Exp Med,1995,182:33-40.
[7]Yellin MJ,Brett J and Baum D,et al.Functional interactions ofT cells with endothelial cells:the role of CD40L-CD40-mediated signals.J Exp Med,1995,182:1857-1864.
[8]Schonbeck U,Mach F and Sukhova G,et al.Expression of stromelysin-3 in atherosclerotic lesions:regulation via CD40-CD40 ligand signaling in vitro and in vivo.J Exp Med,1999,189:843-853.
[9]Schonbeck U,Sukhova G and Graber P,et al.CD40 ligation induces tissue factor expression in human vascular smooth muscle cells.Am J Pathol,1999,155:1281-1291.
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[12] Schonbeck U, Sukhova GK and Shimizu K et al. Inhibition of CD40 signaling limits evolution of established atherosclerosis in mice. Proc Natl Acad Sci USA, 2000, 97:7458-7463.
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[14] Zaverio MR. Platelets in atherothrombosis. Nat Med, 2002, 8:1227-1234.
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[16] Inwald DP, McDowall A and Peters MJ, et al. CD40 is constitutively expressed on platelets and provides a novel mechanism for platelet activation. Circ Res, 2003, 92:1041-1048.
[17] Chakrabarti S, Varghese S and Vitseva O, et al. CD40 Ligand influences platelet release of reactive oxygen intermediates. Arterioscler Thromb Vasc Biol, 2005, 25:2428-2434.
[18] Henn V, Steinbach S and Buchner K, et al. The inflammatory action of CD40 ligand (CD154) expressed on activated human platelets is temporally limited by coexpressed CD40. Blood, 2001, 98:1047-1054.
[19] Furman MI, Krueger LA and Linden MD, et al. Release of soluble CD40L from platelets is regulated by glycoprotein IIb/IIIa and actin polymerization. J Am Coll Cardiol, 2004, 43:2319-2325.
[20] Garlichs CD, Kozina S and Fateh-Moghadam, et al. Upregulation of CD40-CD40 ligand(CD154) in patients with acute cerebral ischemia. Stroke, 2003, 34:1412-1417.
[21] Henn V, Steinbach S and Buchner K et al. The inflammatory action of CD40 ligand (CD154) expressed on activated human platelets is temporally limited by coexpressed CD40. Blood, 2001, 98:1047-1054.
[22] Cipollone F, Chiarelli F and Davi G, et al. Enhanced soluble CD40 ligand contributes to endothelial cell dysfunction in vitro and monocyte activation in patients with diabetes mellitus: effect of improved metabolic control. Diabetologia, 2005, 48: 1216-1224.
[23] Kouwenhoven M, Carlstrom C and Ozenci V, et al. Matrix metalloproteinase and cytokine profiles in monocytes over the course of stroke. J Clin Immunol, 2001, 21:365-375.
[24] Di Napoli M, Papa F. Inflammation, hemostatic markers and antithrombotic agents in relation to long-term risk of new cardiovascular events in first-ever ischemic stroke patients. Stroke, 2002, 33:1763-1771.
[25] Freedman JE, Loscalzo J. Platelet-monocyte aggregates bridging thrombosis and inflammation. Circulation, 2002,105:2130-2132.
[26] van Kooten F, Ciabattoni G and Koudstaal PJ, et al. Increased platelet activation in the chronic phase after cerebral ischemia and intracerebral hemorrhage. Stroke, 1999, 30:546-549.
[27] Hermann A, Rauch BH and Braun M, et al. Platelet CD40 ligand (CD40L) subcellular localization, regulation of expression and inhibition by clopidogrel. Platelets, 2001,12:82.
[1]Russell R.Atherosclerosis-A inflammatory disease[J].N Engl J M,1999,340:115-126.
[2]Hansson GK,Robertson AK,and Soderberg-Naucler C.Inflammation and Atherosclerosis[J].Annu Rev Pathol Mech Dis,2006,1:297-329.
[3]Endarterectomy for asymptomatic carotid artery stenosis.Executive Committee for the Asymptomatic Carotid Atherosclerosis Study.JAMA.,1995,273:1421-1428.
[4]Risk of stroke in the distribution of an asymptomatic carotid artery.The European Carotid Surgery Trialists Collaborative Group.Lancet.,1995,345:209-212.
[5]Randomised trial of endarterectomy for recently symptomatic carotid stenosis:final results of the MRC European Carotid Surgery Trial(ECST) Lancet.,1998,351:1379-1387.
[6]Shah PK.Pathophysiology of plaque rupture and the concept of plaque stabilization[J].Cardiol Clin,1996,14:17-29.
[7]Fuessl RT,Kranenberg E,Kiausch U,et al.Vascular remodeling in atherosclerotic coronary arteries is affected by plaque composition[J].Coron Artery Dis,2001,12:91-97.
[8]Wiedermann CJ,Kiechl S,Dunzendorfer S,et al.Association of endotoxemia with carotid atherosclerosis and cardiovascular disease:prospective results from the Bruneck Study[J].J Am Coll Cardiol,1999,34:1975-1981.
[9]Ridker PM,Hennekens CH,Buring JE,et al.C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women[J].N Engl J Med,2000,342:836-843.
[10] Pearson TA, Mensah GA, Alexander RW, et al. Markers of inflammation and cardiovascular disease: application to clinical and public health practice: A statement for healthcare professionals from the Centers for Disease Control and Prevention and the American Heart Association[J]. Circulation, 2003,107:499-511.
[11] Ridker PM, Rifai N, Rose L, et al. Comparison of C-reactive protein and low-density lipoprotein cholesterol levels in the prediction of first cardiovascular events[J]. N Engl J Med, 2002, 347:1557-1565.
[12] Koenig W, Lowel H, Baumert J, et al. C-reactive protein modulates risk prediction based on the Framingham Score: implications for future risk assessment: results from a large cohort study in southern Germany[J]. Circulation, 2004, 109:1349-1353.
[13] Ballantyne CM, Hoogeveen RC, Bang H, et al. Lipoprotein-associated phospholipase A2, high-sensitivity C-reactive protein, and risk for incident ischemic stroke in middle-aged men and women in the Atherosclerosis Risk in Communities (ARIC) study[J]. Arch Intern Med, 2005,165:2479-2484.
[14] St-Pierre AC, Bergeron J, Pirro M, et al. Effect of plasma C-reactive protein levels in modulating the risk of coronary heart disease associated with small, dense, low-density lipoproteins in men (The Quebec Cardiovascular Study) [J]. Am J Cardiol, 2003, 91:555-558.
[15] Di Napoli M, Papa F, Bocola V. C-reactive protein in ischemic stroke: an independent prognostic factor[J]. Stroke, 2001, 32:917-924.
[16] Zwaka TP, Hombach V, Torzewski J. C-reactive protein-mediated low density lipoprotein uptake by macrophages: implications for atherosclerosis[J]. Circulation, 2001, 103:1194-1197.
[17] Nakagomi A, Freedman SB, Geczy CL. Interferon-gamma and lipopolysaccharide potentiate monocyte tissue factor induction by C-reactive protein: relationship with age, sex, and hormone replacement treatment[J]. Circulation, 2000, 101:1785-1791.
[18] Penn MS, Topol EJ. Tissue factor, the emerging link between inflammation, thrombosis, and vascular remodeling[J]. Circ Res, 2001, 89:1-2.
[19] Yasojima K, Schwab C, McGeer EG, et al. Generation of C-reactive protein and complement components in atherosclerotic plaques[J]. Am J Pathol, 2001, 158:1039-1051.
[20] Tomai F, Crea F, Gaspardone A, et al. Unstable angina and elevated C-reactive protein levels predict enhanced vasoreactivity of the culprit lesion[J]. Circulation, 2001, 104:1471-1476.
[21]Pasceri V,Willerson JT,Yeh ET.Direct proinflammatory effect of C-reactive protein on human endothelial cells.Circulation[J],2000,102:2165-2168.
[22]Nakajima T,Schulte S,warrington K J,et al.T-cell-mediated lysis of endothelial cells in acute coronary syndromes[J].Circulation,2002,105:570-575.
[23]Fichtlscherer S,Rosenberger G,Walter DH,et al.Elevated C-reactive protein levels and impaired endothelial vasoreactivity in patients with coronary artery disease[J].Circulation,2000,102:1000-1006.
[24]Mulvihill NT,Foley JB,Murphy R,et al.Evidence of prolonged inflammation in unstable angina and non-Q wave myocardial infarction[J].J Am Coll Cardiol,2000,36:1210-1216.
[25]Fisher M,Paganini-Hill A,Martin A,et al.Carotid plaque pathology:thrombosis,ulceration,and stroke pathogenesis[J].Stroke,2005,36:253-257.
[26]Luc G,Arveiler D,Evans A,et al.Circulating soluble adhesion molecules ICAM-1and VCAM-1 and incident coronary heart disease:the PRIME Study[J].Atherosclerosis,2003,170:169-176.
[27]Guray U,Erbay AR,Guray Y,et al.Levels of soluble adhesion molecules in various clinical presentations of coronary atherosclerosis[J].Int J Cardiol,2004,96:235-240.
[28]Doo YC,Han SJ,Lee JH,Cho GY,et al.Associations among oxidized low-density lipoprotein antibody,C-reactive protein,interleukin-6,and circulating cell adhesion molecules in patients with unstable angina pectoris[J].Am J Cardiol,2004,93:554-558.
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