【作者】 姜玫秀；

【导师】 韩际宏；

【作者基本信息】 南开大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 心血管疾病已经成为人类健康与生命的重大威胁。冠心病是心血管疾病中最常见的一种,而动脉粥样硬化(atherosclerosis, AS)则是造成冠心病的主要病理之一。动脉粥样硬化的发生与多种因素有关,如血管内皮损伤,单核细胞和巨噬细胞聚集,血管平滑肌细胞增生以及多种趋化因子和细胞因子的产生等。其中,单核细胞衍生的巨噬细胞转化成泡沫细胞是动脉粥样硬化形成的起始与关键步骤。脂肪酸结合蛋白(Fatty acid binding proteins, FABPs)是一组同源小分子细胞胞浆蛋白,分子量为14-15kD,能够结合疏水性脂类并对其进行细胞内亚组织转运。脂肪细胞型脂肪酸结合蛋白(adipocyte fatty acid binding protein, FABP4,又称为A-FABP或aP2)是细胞内FABP家族成员之一。FABP4广泛存在于各种正常组织、细胞中,但在脂肪组织和巨噬细胞中高表达。FABP4能够可逆性结合饱和及不饱和长链脂肪酸,促进脂肪酸代谢和转运。此外,FABP4在其他生物学过程中尤其是代谢综合征的许多方面具有重要作用。FABP4基因敲除小鼠可以抑制由高脂肪饮食诱导的动脉粥样硬化发展,在巨噬细胞中诱导FABP4高表达会促进泡沫细胞形成,表明FABP4在动脉粥样硬化的发展中也具有重要作用。他莫昔芬(tamoxifen)是一种选择性雌激素受体调节剂(selective estrogen receptor modulator, SERM),同时也是抗血管再生因子和蛋白激酶C的抑制剂。在体内细胞色素P4502D6(CYP2D6)的作用下,tamoxifen可以被羟基化为四羟他莫昔芬(4-hydroxytamoxifen或4-OH-tamoxifen),四羟他莫昔芬具有更强的SERM活性。过去20多年中,作为化疗药tamoxifen在临床上用于治疗早期和晚期高表达雌激素受体的乳腺癌,同时大量的临床前和临床试验证明tamoxifen具有心血管保护功能。例如，tamoxifen可以降低绝经后妇女心肌梗死的发生率以及降低颈总动脉的内膜厚度；还可以增加男性晚期动脉粥样硬化内皮依赖性血流调控的血管扩张；在动物模型中,tamoxifen可以降低雄性野生型小鼠和apoE-/-小鼠以及通过手术绝经的雌猴体内由高脂肪饮食诱导的动脉损伤与动脉粥样硬化的发生。Tamoxifen虽具有心血管保护功能,但其作用的具体分子机制尚未完全清楚。因此本论文研究tamoxifen和4-OH-tamoxifen能否通过抑制FABP4表达以达到减少泡沫细胞形成从而抑制动脉粥样硬化发展的目的,同时我们将深入研究tamoxifen作用于FABP4表达的具体分子机制。首先,我们分离来自于雌性和雄性野生型小鼠腹腔的原代巨噬细胞,在tamoxifen和4-OH-tamoxifen作用下,发现tamoxifen和4-OH-tamoxifen抑制小鼠原代巨噬细胞FABP4蛋白表达且不存在性别依赖现象。我们前期研究成果表明地塞米松(dexamethasone, Dex)、匹伐他汀(pitavastatin)与dexamethasone结合、激活过氧化物酶体增殖物激活受体Y(PPARy)等皆诱导FABP4表达,为此,我们研究tamoxifen和4-OH-tamoxifen对上述诱导作用的拮抗效果。通过Western blot检测我们证明了tamoxifen和4-OH-tamoxifen能有效的拮抗上述诱导作用。在转录水平上,我们通过Northern blot发现tamoxifen和4-OH-tamoxifen抑制巨噬细胞FABP4mRNA表达,进一步我们对FABP4启动子活性进行分析,探究tamoxifen和4-OH-tamoxifen下调FABP4表达的具体分子机制。通过分析,我们发现FABP4启动子区域存在糖皮质激素受体(GR)的正负调节元件(pGRE和nGRE)以及PPARγ的两个潜在结合元件(PPRE1和PPRE2)。双荧光素酶活性实验和EMSA实验结果证明,tamoxifen和4-OH-tamoxifen通过激活nGRE活性并增加核转录因子GR与nGRE结合同时抑制PPRE1活性以及核转录因子PPARγ与PPRE结合,从而下调FABP4启动子活性,抑制FABP4转录与表达。之后,我们利用Western blot结合荧光标记定位实验证明,tamoxifen和4-OH-tamoxifen不影响GR表达,但是可以抑制由dexamethasone诱导的GR核转移。此外,我们检测tamoxifen和4-OH-tamoxifen对FABP4表达的抑制作用在小鼠体内和人血单核细胞衍生的巨噬细胞中是否存在。我们分别喂食雌性和雄性apoE-/-小鼠含有tamoxifen的高脂食物(0.5%的胆固醇和21%的脂肪)一个月,之后分别收集小鼠腹腔巨噬细胞和脂肪组织,检测FABP4蛋白表达的变化情况。结果与体外实验一致,tamoxifen以非性别依赖的方式抑制巨噬细胞和脂肪组织中FABP4蛋白表达。同时,我们从志愿者身上获取血液并提取白细胞,其衍生的巨噬细胞中FABP4蛋白表达同样被tamoxifen和4-OH-tamoxifen所抑制,与小鼠体内外实验结果一致。为研究tamoxifen是否可以抑制动脉粥样硬化斑块的形成,我们喂食雌性apoE-/.小鼠含有tamoxifen的高脂食物(雄性小鼠已有报道)16周后,完整分离出其主动脉进行油红O染色后观察斑块的形成情况,发现tamoxifen确实可以抑制动脉粥样硬化斑块的形成。此外,我们通过FABP4RNA干扰实验证明tamoxifen减少泡沫细胞形成并起到抑制动脉粥样硬化作用是与其抑制FABP4表达密切相关。综上所述,本文首次证明了tamoxifen和4-OH-tamoxifen通过GR和PPARy两条信号通路相结合来抑制巨噬细胞FABP4表达从而减少FABP4诱导的泡沫细胞形成,最终起到心血管保护作用。我们的研究为临床上治疗动脉粥样硬化等疾病提供了一定的理论基础。更多还原

【Abstract】 Cardiovascular disease is the biggest threat to our health and life. Coronary heart disease (CHD) is a major disease in cardiovascular system. The atherosclerotic lesion is the one of major pathogens for CHD. Formation of atherosclerosis is a complex process including endothelial injury, monocyte adhesion, macrophage differentiation and accumulation, smooth muscle cells proliferation and production of a variety of chemotactic factors and cytokines in arteries. During the development of atherosclerosis, monocyte-derived macrophages express macrophage scavenger receptors which bind and internalize large amounts of low-density lipoprotein (LDL) and modified LDL. The accumulation of lipids in macrophages drives the formation of foam cell, the major component in lesions. Thus, the formation of foam cells is the initial and critical step in the lesion development.Fatty acid binding proteins (FABPs) is a family of cytoplasmic proteins with small molecular-weight (14-15kD) and capable of binding hydrophobic fatty acids with high affinity. Adipocyte fatty acid binding protein (FABP4, also known as A-FABP or aP2), is a member of the FABP family. FABP4is widely expressed by various tissues and cells with the highest leves in adipose tissues and macrophages. In addition to fatty aicd transport and modulation of intracellular lipid metabolism, FABP4also plays an important role in other biological processes, particularly in many aspects of metabolic syndrome. Genetic deletion of FABP4expression inhibits the high fat diet-induced atherosclerosis in mice, and over-expressed FABP4facilitates foam cell formation in macrophages. Thus, FABP4plays an important role in the development of atherosclerosis.Tamoxifen is a selective estrogen receptor modulator. It also functions as an anti-angiogenesis factor and protein kinase C inhibitor. By the action of cytochrome P4502D6(CYP2D6), tamoxifen is hydroxylated into4-hydroxytamoxifen, which has more potent modulator activity. Clinically, tamoxifen has been used for more than two decades to treat both the early and advanced ER-positive breast cancer. However, both pre-clinical and clinical studies also have suggested the cardioprotective effects of tamoxifen. For example, tamoxifen reduces the incidence of fatal myocardial infarction and the intima-media thickness of the common carotid artery in postmenopausal women. It also increases the endothelium-dependent flow-mediated dilation (ED-FMD) in men with advanced atherosclerosis. In animal models, tamoxifen reduces the high-fat diet induced atherosclerotic lesions in both male wild type and apoE deficient (apoE-/-) mice and surgically postmenopausal monkeys. In spite of the cardioprotective effects, molecular mechanisms by which tamoxifen functions have not been fully elucidated. In the study, we determined if tamoxifen inhibits the development of atherosclerosis is completed through the inhibition on macrophage FABP4expression. We also disclosed the involved mechanism of tamoxifen-inhibited macrophage FABP4expression.Firstly, we isolated peritoneal macrophages from both male and female mice. We treated the cells with tamoxifen and4-hydroxytamoxifen at different concentrations overnight. The results of Western blot indicate that tamoxifen or4-hydroxtamoxifen inhibited FABP4protein expression in macrophages isolated either from male or female mice incidating the inhibition of macrophage FABP4expression by tamoxifen is in a sex-independent manner. Our previous studies suggest that dexamethasone or dexamethasone plus pitavastatin or PPARy activation can induce macrophage FABP4expression. In this study, we observed that tamoxifen and4-hudroxytamoxifen are able to antagonize the above inductions. FABP4mRNA expression was also inhibited by tamoxifen and4-hydroxytamoxifen indicating the. regulation is at the transcriptional level. In deed, we found that tamoxifen and4-hydroxytamoxifen inhibited FABP4promoter activity. We further disclosed the GRE (nGRE/pGRE) and putative PPRE (PPRE1/PPRE2) in FABP4promoter and our study indicated that tamoxifen and4-hydroxytamoxifen inhibited FABP4transcriptuon by activating nGRE and inhibiting PPRE1activity. EMSA.indicated that tamoxifen and4-hydroxytamoxifen enhanced the binding of nGRE with nuclear proteins but reduced the affinity of PPRE1to nuclear proteins. In addition, we determined tamoxifen did not affecet GR expression but reduced dexamethasone-induced GR nuclear translocation by Western Blot and image analysis. To disclose the physiological relavence of inhibition of macrophage FABP4expression by tamoxifen, we initially determined the inhibitory effect of tamoxifen on FABP4expression in vivo. We fed apoE-/-female and male mice a high-fat diet (0.5%cholesterol and21%fat) or the diet containing tamoxifen (2mg/100g food) for one month. After treatment, the peritoneal macrophages and adipose tissuses were individually collected and used to determine changes in FABP4protein expression. We observed the similar inhibitory effects on macrophage or adipose FABP4expression in vivo between male and female mice further suggesting the regulation of FABP4expression by tamoxifen is sex-independent. We also determined the effects of tamoxifen and4-hydroxytamoxifen on FABP4protein expression in human monocyte-derived macrophages from a healthy donor’s blood. Similar to mouse macrophages and in vivo, tamoxifen and4-hydroxytamoxifen inhibited human macrophage FABP4protein expression.Finally, to disclose if the inhibition of FABP4expression by tamoxifen would impact the development of atherosclerosis in apoE-/-female mice (the effect on male mice has been reported), we fed the mice a high-fat diet or the diet containing tamoxifen (2mg/100g food) for16weeks. We observed that tamoxifen inhibited the development of atherosclerosis in en face aortas induced by the Western diet. By using FABP4siRNA, we observed that the inhibition of foam cells by tamoxifen was completed by inhibiting macrophage FABP4expression that can be attributed to the inhibitory properties of tamoxifen on atherosclerosis.Taken together, for the first time, our study suggests that tamoxifen inhibits macrophage FABP4expression by a GR and PPARy combinational pathway. Tamoxifen inhibits the development of atherosclerosis is partially related to its inhibition of macrophage FABP4expression. Also, our study may have implication for the clinical treatment of atherosclerosis.更多还原