【作者】 李吉林；

【导师】 许顶立； 孟宪忠；

【作者基本信息】 南方医科大学 ， 内科学， 2011， 博士

【摘要】 研究背景目前,已经有许多研究证实血管炎症与血管动脉粥样硬化的发生和发展密切相关,在心血管疾病病理过程中起了非常重要的作用。其中,作为血管的重要组成部分,内皮细胞在血管的免疫和炎症反应中起着重要作用,内皮细胞内的炎症激活是许多疾病发展的关键,比如其是组织缺血—再灌注损伤中单核细胞和巨噬细胞聚集和浸润的关键步骤,也是动脉粥样硬化发生的关键部分。Toll-like receptors (TLRs)是一种病原体功能识别受体,可以识别细菌,病毒以及其它一些病原体。TLR2或者TLR4被生物配体刺激后,可以激活细胞内的传导通路活化,诱导促炎因子的产生。因此,TLR2和TLR4在细胞内的免疫和炎症反应中起着关键作用。在体内,组织缺血—再灌注损伤时可以导致许多TLR受体激活物的释放,比如热休克蛋白(heat shock protein, HSP)和高迁移率蛋白(high mobility group box 1, HMGB1)。孟宪忠教授等的研究已经发现心肌组织的TLR2／4在缺血组织中的白细胞浸润起着非常关键的作用,与心肌的缺血—再灌注损伤的机制有关。目前对于TLRs在血管内皮细胞炎症中的作用,以及内皮细胞炎症在缺血心肌中作用尚缺乏研究。人类心血管疾病发病的性别差异已得到普遍认同。女性可以从雌激素的心血管保护作用中受益,但保护作用在妇女绝经后就终止了。这种心血管疾病的性别差异在动物中也被发现。几项研究证实雌性小鼠心肌梗死后比雄性小鼠有更好的心功能,同时早期的炎症反应减轻,纤维性修复加强,左室不良重构范围减小。而且,雌性小鼠对缺血中TNFR1通路的抵抗更强。雌激素明显具有抗炎症反应的作用,在雌性小鼠中具有心血管保护作用。TLR2明显在缺血后心肌的损伤和心功能减退中起着重要作用。但是,之前这些研究都是集中在雄性小鼠的TLR2作用上面,对TLR2在雌性小鼠中的作用缺乏研究。雌激素是调节组织和细胞损伤后炎症反应的重要因子。几项研究提示雌激素与TLRs之间有相互作用。LPS诱导的小鼠气道炎症反应在雌性小鼠中减轻,但在注射睾酮后增强。另外,17β-雌二醇可以通过雌激素受体,抑制TLR诱导的IL-8在囊性纤维化支气管内上皮细胞的释放。但是,在巨噬细胞中,去除内源性的雌激素后既可以减少促炎因子,也可以减少抗炎因子的产生,同时伴随细胞表面TLR4表达的减少。同样地,睾酮也可以减少巨噬细胞TLR4的表达。这些研究提示性激素可以改变TLRs的表达和TLRs介导的炎症反应,但对于缺血后TLR2介导的炎症反应是否有性别差异并不清楚。而在1型糖尿病人群中,动脉粥样硬化在年轻时已经发生,而且进展迅速,但是其中的机制尚不清楚。很多研究揭示TLR2和TLR4两种受体在动脉粥样硬化的发展机制中起着关键作用。另外,TLR2配体肽聚糖(peptidoglycan, PGN)和TLR4配体脂多糖(lipopolysaccharide, LPS)已经被发现存在于血管早期粥样硬化斑块中。当这些细菌抗原诱导单核细胞产生大量促炎因子时,它们对冠脉内皮细胞(coronary artery endothelial cells, CAECs)的炎症反应的作用尚未可知。因此,对于TLR2/4刺激时1型糖尿病对CAEC炎症反应的影响进行研究,将有助于了解这种疾病对于动脉粥样硬化影响的机制。目前已经有研究揭示TLR2和TLR4参与了1型糖尿病的病理生理机制中。在1型糖尿病模型中,TLR2参与了胰岛的自体免疫反应。在自发1型糖尿病的NOD小鼠中,骨髓来源的巨噬细胞中TLR2,以及TLR3-5的表达都升高了。胰岛素也被发现可以抑制单核细胞中TLR2转录水平的表达。另外,糖尿病病人中B细胞炎症反应中TLR4的功能也发生了改变,可能是由促炎因子IL-8的升高和抗炎因子IL-10的缺乏两种原因引起的。这些研究结果揭示了1型糖尿病中细胞TLR表达和反应发生变化与疾病有关,但是在1型糖尿病CAECs中TLR2/4的表达水平和炎症反应的改变尚有待研究。本实验中,我们假设TLR2和TLR4激活可以导致血管内皮细胞炎症因子表达,TLR2受体在雌雄小鼠中的作用不完全相同,1型糖尿病可以增强TLRs介导的冠状动脉内皮细胞内的炎症反应。我们利用TLR2受体激活剂PGN和TLR4受体激活剂LPS刺激内皮细胞内的TLRs受体,探讨TLRs介导的内皮细胞炎症反应,以及1型糖尿对其的影响和可能的机制。利用小鼠心肌梗死模型,探讨TLR2在心肌梗死后的炎症反应是否存在性别差异。第一章TLR2和TLR4激活诱导心脏微血管内皮细胞的炎症反应目的研究TLR2和TLR4激活诱导的心脏微血管内皮细胞内炎症反应情况和可能的机制。方法分离小鼠心脏微血管内皮细胞,分别予TLR2激活物PGN和TLR4激活物LPS进行干预。利用实时定量PCR方法分析ICAM-1 mRNA的表达,ELISA方法分析IL—6和IL—8的表达,免疫印迹分析ICAM-1蛋白表达和NF-κB活化的变化。结果PGN和LPS诱导小鼠内皮细胞ICAM-1蛋白的表达在TLR2敲除和TLR4突变时表达明显减弱。TLR2和TLR4激活可以诱导微血管内皮细胞ICAM-1 mRNA水平增加,ICAM-1蛋白,IL-6和IL-8表达增加,NF-κB活化增强。结论PGN和LPS分别通过TLR2和TLR4诱导小鼠心脏微血管内皮中炎症反应。TLR2和TLR4激活可以诱导微血管内皮细胞中的炎症反应,其机制可能是TLR的激活增强了通路中NF-κB的活化。第二章TLR2在心肌缺血后炎症反应和心力衰竭的作用具有性别差异目的研究TLR2在雄性和雌性小鼠心肌梗死后的炎症反应和左室早期不良重构中的作用。方法在雌性和雄性野生型‘(Wild type, WT)和TLR2基因敲除(Knock out, KO)小鼠中结扎左前降支冠脉,制造心肌梗死模型。部分小鼠3天后处死,ELISA和免疫印迹分别分析心脏组织内MCP-1和ICAM-1表达水平,组织切片利用免疫荧光方法检测中性粒细胞的浸润,部分小鼠7天后测量心脏功能,取心肌组织HE染色分析心肌梗死面积,免疫荧光分析单核细胞的聚集。结果与雄性野生型小鼠相比,雄性TLR2 KO小鼠缺血心肌组织中MCP-1和ICAM-1水平降低,白细胞聚集减少,伴随心肌梗死面积的减小和左室功能的改善。TLR2在雌性野生型小鼠心肌组织的表达没有改变,细胞对TLR2激活物PGN的炎症反应也相同,心肌梗死后的炎症反应和损伤有所减轻,但TLR2 KO并没有显示出进一步的保护作用。结论雄性小鼠TLR2基因敲除后可以抑制缺血后心肌炎症反应,减少心肌梗死面积和改善早期左室重构。但是,雌性小鼠并不能从TLR2基因敲除中受益。TLR2在心肌缺血后的炎症反应和损伤中具有性别差异,提示对TLR2通路的干预作用可能只对雄性有治疗意义。第三章TLR2/4诱导人冠状动脉内皮细胞炎症反应的机制目的TLR2/4介导的炎症反应在粥样硬化中起着重要作用,探讨1型糖尿病是否可以增强TLR2/4介导的冠脉内皮细胞中的炎症反应。方法非糖尿病和糖尿病CAECs分别予TLR2激活物PGN和TLR4激活物LPS进行干预。利用实时定量PCR方法分析ICAM-1,IL-6和IL-8mRNA的表达,免疫印迹分析ICAM-1蛋白表达,ELISA分析IL-6和IL-8小分子肽的表达,免疫印迹和免疫荧光的方法分析NF-κB的活化。另外,在TLR激活之前一个小时,予糖尿病细胞胰岛素处理,观察胰岛素缺乏是否为TLR2/4介导的1型糖尿病CAECs炎症反应变化的原因。结果TLR2和TLR4刺激可以诱导NF-κB活化,ICAM-1,IL-6和IL-8的表达。有意思的是,糖尿病细胞内炎症产物的表达明显增加,伴随NF-κB活化的增强,但TLR2和TLR4蛋白水平没有明显改变。利用胰岛素进一步的干预并没有抑制这种增强的炎症反应。结论糖尿病CAECs对TLR2和TLR4刺激时的炎症反应比非糖尿病细胞增强,单独使用胰岛素并不能抑制其高炎症反应。炎症反应增强的机制可能与促炎因子通路的增强有关,而不在于TLR2和TLR4水平的上调。这些发现提示糖尿病CAECs属于高炎症反应细胞,这种细胞表型的改变或许是冠状动脉粥样硬化易患性的原因。全文小结1.PGN和LPS是分别通过激活TLR2和TLR4受体诱导心脏微血管内皮细胞中炎症反应的发生；2.TLR2/4的激活引起了NF-κB的磷酸化,是其炎症反应的重要调控因子；3.心肌缺血后,TLR2 KO可以抑制雄性小鼠心肌炎症反应,减小心肌梗死面积和改善左室重构；4.雌性小鼠心肌梗死时心肌炎症反应和心肌损伤减轻,但不能从TLR2KO中受益；5.雌性小鼠TLR2 KO作用的消失与心肌TLR2的缺失无关；6.在1型糖尿病CAECs中,TLR2和TLR4的激活可以诱导IL-6,IL-8和ICAM-1的产生；7.糖尿病CAECs在TLR2和TLR4激活时的炎症反应比非糖尿病细胞增强,与NF-κB活化的增强有关,同时不伴随细胞TLR2和TLR4水平的改变；8.单独使用胰岛素不能抑制TLR2和TLR4激活所诱导的糖尿病CAECs内的炎症反应。更多还原

【Abstract】 BackgroundGrowing evidence supports an important role for vascular local inflammation in the development and progress of atherosclerosis. As the first cardiac cells that can come in contact with endogenous and exogenous agents, endothelial cells are important in immune and inflammatory responses, and inflammatory activation of the endothelial cells is a critical step in the development of many diseases. Further, inflammatory activation of the endothelial cells is a critical step in the recrument and infiltration of monocytes and macrophages in the tissue I/R injury, and the pathophysiological of atherosclerosis.Toll-like receptors (TLRs) are pathogen pattern recognition receptors that recognize bacterial and viral products, and other pathogens. Activation TLR2 or TLR4 by microbial ligands induce a cascade of intracellular signaling events, culminating in the production of pro-inflammatory mediators. Tissue I/R causes the release of endogenous TLR activators including heat shock protein and high mobility group box 1 (HMGB1), released from stressed and/or injured cells. Recent studies by our group and others indicate that myocardial tissue TLR2/4 are critical for leukocyte infiltration into the ischemic myocardium and contribute to the mechanisms of myocardial I/R injury. It is not determined that the roles of TLRs play in the inflammatory response in endothelial cells, and which plays in myocardial ischemia.Gender difference in cardiovascular diseases is well recognized in humans. Women are known to benefit from the cardioprotective actions of sex hormones which discontinue with the onset of menopause. This gender difference in cardiovascular conditions is also demonstrated in animal studies. Several studies confirmed that female mice have better cardiac function after myocardial infarction (MI) that is associated with attenuated early inflammation, augmented fibrotic response and less extent of LV adverse remodeling. Furthermore, female mice exhibit myocardial resistance to TNFR1 signaling during ischemia. Female sex hormones appear to be anti-inflammatory and are responsible for cardiovascular protection in females.Toll-like receptors (TLRs) are innate immune receptors that sense the presence of pathogen-associated molecular patterns. Accumulated evidence demonstrates that TLR2 and TLR4 play important roles in the signaling mechanisms that contribute to post-ischemic myocardial inflammatory response and injury. Several studies found that TLR2 and TLR4 also respond to endogenous proteins, such as heat shock proteins (HSP) and high mobility group box 1 (HMGB1), released from stressed and/or injured cells. TLR 2-deficient mice are also protected against post-ischemic coronary endothelial dysfunction. In addition, reduced myocardial fibrosis in TLR2 knock out (TLR2 KO) mice after ischemia indicates TLR2 is involved in maladaptive LV remodeling. Thus, TLR2 appears to play an important role in post-ischemic myocardial injury and cardiac dysfunction. However, these previous studies determined the role of TLR2 only in male mice. It is unknown whether TLR2 has a similar role in females. Sex hormones are important modifiers of tissue and cellular inflammatory response to injury. Several studies indicate the interaction of sex hormones with TLRs. LPS-induced inflammation in murine airway was reduced in female, but was exaggerated in females administered testosterone. In addition,17beta-estradiol inhibits IL-8 release induced by TLR agonists via ERbeta in cystic fibrosis bronchial epithelial cells. However, removal of endogenous estrogens has been shown to decrease the production of both pro- and anti-inflammatory cytokines, with a concomitant reduction in cell surface expression of TLR4 in macrophages. Similarly, testosterone is found to reduce macrophage expression of TLR4. Although these studies suggest that sex hormones could alter TLR expression and the TLR-mediated inflammatory responses, it remains unknown whether the TLR2-mediated myocardial inflammatory responses to ischemia are gender-dependent.In the Type 1 diabetes (T1D) population, atherogenesis occurs in younger ages and advances faster. However the underlying mechanisms are incompletely understood. A number of studies demonstrate that these two major innate immune receptors play a mechanistic role in the development of atherosclerosis. In addition, TLR2 ligand peptidoglycan (PGN) and TLR4 ligand lipopolysaccharide (LPS) have been found in vessels with early atherosclerotic lesions. While these bacterial agents induce the production of multiple pro-inflammatory mediators in mononuclear cells, their effects on the inflammatory responses in coronary artery endothelial cells (CAECs) remain to be determined. Investigation of the effect of T1D on CAEC inflammatory responses to TLR2/4 stimulation could provide insights into the mechanisms underlying the pro-atherogenic phenotype associated with this disease.TLR2 and TLR4 have also been implicated in the pathophysiology of T1D. In an experimental T1D model, TLR2 is involved in the autoimmune inflammation in the pancreatic islet. The expression of TLR2, as well as TLR3, TLR4 and TLR5 in bone marrow-derived macrophages is increased in diabetic NOD mice. Insulin is found to suppress the expression of TLR2 in mononuclear cells at the transcriptional level. In addition, altered TLR4 function is involved in the inflammation in B cells from diabetes mellitus patients by two mechanisms:elevation of pro-inflammatory IL-8 and lack of anti-inflammatory/protective IL-10 production. While these studies indicate altered cellular TLR expression and responses associated with T1D, it remains unclear whether TLR2/4 levels and the inflammatory responses to TLR2/4 agonists are altered in CAECs from T1D patients.In this study, we hypothized that TLR activation could induce inflammatory response in endothelial cells, the gender-disparity of TLR2, and T1D could enhance the inflammatory responses induced by TLR activation in endothelial cells. We determined the inflammatory response in the mouse MVECs and human coronary endothelial cells treated with PGN and LPS, the gender disparity of TLR2 in the ischemia myocardium in the LAD ligation model.Chapter 1:TLR activation induced inflammatory responses in microvascular endothelial cellsObjectiveTo determine the role and possible mechanism of TLR2 and TLR4 activation induce microvascular inflammatory responses induced by PGN and LPS, respectively.MethodsMicrovascular endothelial cells (MVECs) were isolated from mice and treated with TLR2 ligand PGN and TLR4 ligand LPS, respectively. ICAM-1 mRNA was analyzed with qPCR, IL-6 and IL-8 were assessed by ELISA, expression of ICAM-1 and NF- B activation by immunoblotting. ResultsPGN and LPS couldn’t induce ICAM-1 expression with TLR2 and TLR4 dysfunction, respectively. TLR2 and TLR4 activation could induce the expression of ICAM-1 mRNA and protein, IL-6 and IL-8 peptide, with increase of NF- B phosphorylation.ConclusionsPGN and LPS induce inflammatory response by activation of TLR2 and TLR4 in MVECs, respectively. TLR2 and TLR4 activation could induce the inflammatory response in MVECs, which maybe due to the NF- B phosphorylation.Chapter 2:Gender disparity in the role of Toll-like receptor 2 in post-ischemic myocardial inflammatory response and cardiac failureObjectiveThe main purpose was to determine whether TLR2 KO suppresses myocardial inflammatory response and improves early left ventricular remodeling after permanent ischemia in both male and female mice.MethodsPermanent ischemia was induced in male and female C57BL/6J (WT) and TLR2 KO mice. Infarct size and LV function were analyzed at day 7. Myocardial levels of MCP-1 and ICAM-1, as well as neutrophil infiltration, were assessed at day 3, and mononuclear cell accumulation was determined at day 7.ResultsWe found lower MCP-1 and ICAM-1 levels, and reduced leukocyte accumulation in male TLR2 KO mice. Suppression of myocardial inflammatory response by TLR2 KO in male mice correlated with smaller infarct size and improved LV function. Female WT mice exhibited attenuated myocardial inflammatory response and injury, and TLR2 KO in females did not provide a protective effect on either myocardial inflammatory response or injury although myocardial TLR2 levels in female WT mice is unaltered, and their cardiac cells respond to bacterial TLR2 agonist properly.ConclusionsThese results showed that TLR2 KO in male mice suppresses post-ischemic myocardial inflammatory response, reduces infarct size and improves early LV remodeling. However, TLR2 KO is not beneficial in female mice. The gender disparity in the role of TLR2 in post-ischemic myocardial inflammatory response and injury suggests that interception with TLR2 signaling may have therapeutic potentials only in males.Chapter 3:The mechanism underlying Toll-like receptor 2/4 stimulation in human coronary artery endothelial cellsBackgroundEndothelial inflammatory responses mediated by Toll-like receptors (TLRs), particularly TLR2 and TLR4, play an important role in atherogenesis. While Type 1 diabetes (T1D) promotes the development and progression of atherosclerosis, the effect of T1D on TLR2/4-mediated inflammatory responses in coronary artery endothelial cells (CAECs) remains unclear.MethodsWe tested the hypothesis that diabetic CAECs have enhanced inflammatory responses to TLR2/4 stimulation. Non-diabetic and diabetic CAECs were treated with TLR2 agonist peptidoglycan and TLR4 agonist lipopolysaccharide. The expression of ICAM-1, IL-6 and IL-8 were analyzed by real-time PCR, immunoblotting and ELISA, and NF-κB activation by immunoblotting and immunostaining. In additional experiments, insulin was added before TLR stimulation to determine whether insulin deficiency alone is responsible for the alteration of TLR2/4-mediated inflammatory responses.ResultsStimulation of TLR2 or TLR4 induced NF-κB activation, and the expression of ICAM-1, IL-6 and IL-8. Interestingly, the expression of inflammatory mediators was significantly enhanced in diabetic cells. The enhanced inflammatory responses correlated with augmented NF-κB activation in the absence of a change in TLR2 or TLR4 protein levels. Further, pretreatment of diabetic cells with insulin failed to suppress the enhanced inflammatory responses.ConclusionsDiabetic CAECs have enhanced inflammatory responses to stimulation of TLR2 or TLR4, and insulin alone is insufficient to correct the hyper-inflammatory responses. The mechanism underlying the enhanced inflammatory responses appears to be augmentation of pro-inflammatory signaling, rather than up-regulation of levels of TLR2 and TLR4. These findings suggest that diabetic CAECs adopt a hyper-inflammatory phenotype and that this endothelial phenotypic change may predispose coronary artery to atherogenesis.Total Conclusion1. TLR2 and TLR4 activation could induce inflammatory response in the myocardiac microvascular cells by PGN and LPS, respectively;2. TLR2 and TLR4 activation could activate NF- B phosphorylation which plays an important role in the inflammatory response;3. TLR2 KO suppresses myocardial inflammatory responses, reduces infarct size and improves early LV remodeling in male mice after permanent myocardial ischemia;4. Female mice have attenuated myocardial inflammatory responses and myocardial injury, and are not benefited from TLR2 KO;5. The lack of TLR2 KO in females effect is not due to myocardial TLR2 deficiency;6. Stimulation of TLR2 and TLR4 induces greater expression of IL-6, IL-8 and ICAM-1 in T1D CAECs;7. The enhanced inflammatory responses to TLR2 and TLR4 agonists in diabetic CAECs correlate with augmented NF-κB activation in the absence of an alteration of cellular TLR2 and TLR4 protein levels;8. Insulin alone is insufficient to suppress the hyper-inflammatory responses to both TLR2 and TLR4 agonists in diabetic CAECs.更多还原