1、动脉粥样硬化性心脑血管病脂代谢相关性生物标记物 Biomarkers related to lipid metabolism,威海市立医院 李振光10-10-2015,动脉粥样硬化性心血管疾病(CVD),心血管疾病是全球的头号死因（约占31%）； 估计740万死于冠心病，670万人死于中风; 3/4以上CVD死亡发生在低、中等收入国家; 早期发现高危人群并予以管理是改善预后的关键 生物标记物在期中扮演重要角色。,TRADITIONAL CARDIOVASCULAR RISK FACTORS,Major, classic or traditional risk factors are those
2、 defined from the Framingham studies;The Framingham cohorts began in 1948, and are milestones in the identification of cardiovascular factors. Including age; sex; total cholesterol, LDL-C and HDL-C levels; systolic pressure; smoking; high blood glucose; body weight; dietary habits; and physical inac
3、tivity. Based on these findings, cardiovascular risk scales were developed in order to evaluate the risk of an event in a ten-year period.,Emerging biomarkers,It is estimated that about 10 to 20% of the individuals that suffer from an event do not present traditional risk factors, and that 60% of th
4、em present two or less factors. These findings emphasize the importance of searching for evidence that emerging biomarkers may better identify plaques that are more vulnerable to rupture.,Atherosclerosis develops,Activation of the endothelium and recruitment of immune cells; Monocyte differentiation
5、 and foam cell formation; Development of fibrotic plaques due to death of foam cells and migration and proliferation of smooth muscle cells (SMCs); Plaque rupture and thrombosis.,Atherosclerosis develops,Secrete extracellular matrix (ECM) proteins that stabilizes the plaques (fibrous cap).,Inflammat
6、ory markers for AS,Several inflammatory markers are affiliated with lipids level and the process of atherosclerosis. The most known of them are ：interleukin 6 and interleukin 1; C-reactive protein; tumor necrosis factor-alpha; pentraxin3(PTX-3); serum amyloid A; sCD40; adhesion molecules; monocyte c
7、hemoattractant protein-1; sEndoglin; PAPP-A; chemokine 16; insulin like growth factor; lipoprotein-associated phospholipase A2;galectin 3; Adiponectin (脂联素); myeloperoxidase (MPO,髓过氧化物酶),Biomarkers related to lipid metabolism,Biomarkers related to lipid metabolism（8）,1、 Modified LDL particles； 2、 Ap
8、olipoprotein AI (apo AI)； 3、 Apolipoprotein B； 4、 Lipoprotein (a) Lp (a)； 5、 Cholesteryl ester transfer protein (CETP)； 6、 Subtypes of LDL and HDL particles； 7、 Lipoprotein-associated phospholipase A2 (Lp-PLA2). 8、 Lysophosphatidic acid, LPA,主要与脂蛋白有关,脂蛋白：lipoproteins,Lipids are transported in the bl
9、oodstream inside lipoproteins, which are essentially made up by proteins (apolipoproteins), cholesteryl esters, cholesterol, TG, and phospholipids. The difference between the five most important lipoproteins is their composition: a) chylomicrons (CM): Rich in TG, made up of apolipoprotein C-I (apo C
10、-I), C-II (apo C-II), C-III (apo C-III), B-48 (apo B-48), and E (apo E); b) very low density lipoprotein (VLDL): Rich in TG, made up of apolipoprotein B-100 (apo B-100), apo E, apo C-I, apo C-II, and apo C-III; c) intermediate density lipoprotein (IDL): Rich in TG, made up of apo B-100; d) low densi
11、ty lipoprotein (LDL): Rich in TG, made up of apo B-100, apo C-I, and apo E; e) high density lipoprotein (HDL): Rich in cholesterol, made up of apolipoprotein A-I (apo A-I), A-II (apo A-II), apo C-I, apo C-II, apo C-III, and apo E.,LDL modified particles（1）,Modified LDL: oxidized LDL electronegative
12、LDL glycosylated LDLThese molecule are pro-inflammatory and highly atherogenic,Apolipoproteins （2）,As the concentration of apo B100 is high in atherogenic lipoproteins (VLDL, IDL and LDL); The apo B/ apo A-I ratio, which shows the balance between the atherogenic (apo B) and anti-atherogenic choleste
13、rol particles (apo A), has been employed in the evaluation of cardiovascular risk. The apo B/ apo A-I ratio was also predictive, with a risk of 1.86. The meta-analysis carried out with 23 studies showed that the greatest concentrations of apo B determined a relative risk of 1.99 for events, whereas
14、the lowest levels of apo A-I raised the risk in 62%.,Apolipoproteins （2）,It should be emphasized that only the apo B and the apo B/apo A-I ratio remained significantly associated after the adjustment for traditional risk factors; The indication for apo B determination is expected to be included in t
15、he next NCEP guidelines (ATP IV),Lipoprotein（a） （3）,Lipoprotein(a), Lp(a), is a particle with similar structure to LDL, containing one apo-B combined with an additional apo(a).Lp(a) concentrations are associated, therefore, with the atherogenic characteristics of the particles that contain apo B and
16、 the thrombogenic properties determined by apo(a).Lp(a) blood levels behave as independent risk factor for CVD. In most individuals, these values are lower than 30 mg/dL; those with values above 100 mg/dL present very high risk.,Lipoprotein（a） （3）,The third report of the National Cholesterol Educati
17、on Program for the Detection, Evaluation and Treatment of Hypercholesterolemia in Adults (NCEP-ATP III) stated that, in spite of the measurement limitations, Lp(a) dosage is an useful parameter. Its high concentration aids the identification of those individuals with even higher cardiovascular risk.
18、It is suggested that Lp(a) should be used as a second risk factor to support lower LDL cholesterol targets.,Apolipoprotein CIII,Apolipoprotein CIII (apoC-III) is an atherogenic protein found on HDL, VLDLand LDL. 荟萃分析表明： (1) Significantly higher levels of apoC-III in the non-HDL fraction of plasma (r
19、epresenting apoC-III in VLDL and LDL) in those with cardiovascular disease compared with controls; (2) No difference for apoC-III levels in HDL; (3) A trend toward higher total plasma apoC-III in the cases.,Apolipoprotein CIII,Apolipoprotein CIII,CETP and its lipoprotein subclasses （4）,Cholesteryl e
20、ster transfer protein (CETP) concentrations are increased in obesity, dyslipidemia and atherosclerosis, and are directly associated with inflammatory markers.However, their use as a risk biomarker is still controversial.,LDL颗粒大小与密度 （5）,Individuals with greater concentrations of small and dense LDL p
21、articles are carriers of phenotype B, which is associated with higher risk of atherosclerosis.Larger and less dense LDL particles (phenotype A) present opposite characteristics and, thus, lower cardiovascular risk.,CONCLUSION,Among them, promising ones are measurements of changes in LDL particles, L
22、p-PLA2, apolipoprotein (apo B and apo A-I), Lp(a), CETP, and LDL and HDL subclasses for a more rigorous evaluation of risk in specific population. Among thee markers, Lp(a) dosage is already contemplated in NCEP as a second risk factor to justify more strict lipid targets. The next guidelines of ATP
23、 IV are being determined, and possibly Lp(a) will be included as a reference measure for the treatment and monitoring of CVD. There are great expectations for apo B, non-HDL cholesterol, and total cholesterol/HDL-C ratio as aids in the evaluation of cardiovascular risk,LDL-C作为降脂治疗目标的局限性,LDL-C并不能反映导致
24、动脉粥样硬化的颗粒总量，即所有含有载脂蛋白B（ApoB）的颗粒，其中包括VLDL、中间密度脂蛋白（IDL）、LDL和Lpa。研究表明，评估这种所谓的“剩余风险”，如非高密度脂蛋白胆固醇（non-HDL-C，总胆固醇减HDL-C）、ApoB和LDL颗粒数（LDL-P）可较LDL-C更好地预测CVD风险;其中研究显示，ApoB和LDL-P比non-HDL-C有更好的预测表现。,目前的概念,流行病学研究发现，non-HDL-C较LDL-C可更好地预测心血管疾病的发病率和死亡率，该指标已被美国脂质协会（NLA）认可作为一个比LDL-C更好的目标。non-HDL-C可以通过从总胆固醇减去HDL-C计算出
25、来，而不需要额外的检测和费用。ApoB和LDL-P的水平可更好地反映导致动脉粥样硬化颗粒的负担。循环中约90%的ApoB包含在LDL颗粒中；LDL-P是通过核磁共振测量。NLA建议血脂异常管理中将non-HDL-C作为首要目标，LDL-C作为次要治疗靶点，ApoB作为一个“可选的次级治疗目标”，可考虑LDL-P作为ApoB的替代指标。,未来的脂质检测,未来标准脂质检测可能应包括： （1）传统脂质检测(如LDL-C）；（2）ApoB （apo B/apo A-I ratio ）；（3）LP（a）；（4） non-HDL-C ；（5）LDL-P ；（6）LDL（IDL、HDL）颗粒分类；（7）Lp
26、-PLA2；（8）LPA/S1P（溶血磷脂酸/鞘氨醇1-磷酸）。,脂蛋白：lipoproteins,脂蛋白：脂质与蛋白质结合在一起形成的脂质-蛋白质复合物。是血脂在血液中存在、转运及代谢的主要形式；分为乳糜微粒(CM)、极低密度脂蛋白(VLDL)、低密度脂蛋白(LDL)、中间密度脂蛋白（IDL）和高密度脂蛋白(HDL)； 成熟脂蛋白呈球形，中间是甘油三酯和胆固醇酯，表面组分为载脂蛋白、游离胆固醇和磷脂。LDL的氧化修饰(oxLDL)是动脉粥样硬化的基础。,脂蛋白（LDL）结构模式图,（磷脂酰胆碱）,磷脂酶（phospholipase, PLA）概述,根据磷脂酶对磷脂水解部位的不同可将磷脂酶分为
27、4类：磷脂酶A1（PLA1）: 作用于Sn-1位酰基; 磷脂酶A2（PLA2）：选择性作用于甘油磷脂sn-2位的酯键（-COO-基团，由羧基“-COOH”与羟基“-OH”脱水而成）;磷脂酶C（PLC）: 水解甘油和磷酸形成的酯键; 磷脂酶D（PLD）；将卵磷脂水解生成磷酯酸和乙酰胆碱;溶血磷脂酶D（Lyso-PLD, ATX）：产生溶血磷脂；,磷脂酶（phospholipase, PL）,甘油磷脂,磷脂是一类含有磷酸的脂类，机体中主要含有两大类磷脂，由甘油构成的磷脂称为甘油磷脂；由神经鞘氨醇构成的磷脂，称为鞘磷脂（S1P）。其结构特点是：具有由磷酸相连的取代基团(含氨碱或醇类)构成的亲水头和由
28、脂肪酸链构成的疏水头。在生物膜中磷脂的亲水头位于膜表面，而疏水尾位于膜内侧。,酯键,Sn-2,PLA2,取代基团,H,PLA2,溶血磷脂酰胆碱,胆碱,PA与LPA,PLA2作用位点，加氢变成OH，溶血磷脂,甘油,甘油三酯,磷脂酰胆碱（卵磷脂）,甘油骨架,脂肪酸,脂肪酸,（不含双键）,脂肪酸,甘油三酯,磷脂酶A2超家族,磷脂酶A2（Phospholipase A2， PLA2），即磷脂-2-酰基水解酶，是专一催化甘油磷脂Sn-2位酯键，酶解产物为溶血磷脂和脂肪酸。广泛参与人体细胞内外信号的传递及炎症、多种相关性疾病病理反应。其生理功能包括细胞信号传递及产生20多种类脂质介质，改造磷脂结构，促进机
29、体坏死组织自动消失，肺泡表面活性物质代谢等。,Phospholipase A2 ：structure/function,四种主要的磷脂酶A2 Secreted sPLA2：分泌型磷脂酶A2 ，与二十烷类产生、炎症及抗菌作用有关。Cytosolic cPLA2 ：胞浆型磷脂酶A2，花生四烯酸(AA)代谢具有优先选择性。 Calcium-independent iPLA2：钙不依赖磷脂酶A2，作为看家酶参与膜的重建 Platelet activating factor (PAF) acetyl hydrolase/oxidized lipid lipoprotein associated （Lp-
30、PLA2）：血小板活化因子乙酰水解酶（氧化脂蛋白相关性磷脂酶A2），以PAF和氧化磷脂为底物。,Atherosclerosis is a progressive inflammatory disease that develops over many years动脉粥样硬化是一种慢性进行性炎性疾病Inflammation has been shown to have a direct effect on plaque formation and plaque rupture炎症对斑块形成和斑块破裂发挥直接作用Inflammatory markers may help predict cardi
31、ovascular risk炎症标志物或有助于预测心血管疾病Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a novel inflammatory marker脂蛋白相关磷脂酶及其产物是一种新的炎症标志物,Inflammation and CHD Risk炎症和动脉粥样硬化,脂蛋白相关性磷脂酶A2,脂蛋白相关性磷脂酶A2（Lipoprotein-associated phospholipase A2，Lp-PLA2）又称血小板活化因子乙酰水解酶（PAF-AH），以PAF和氧化磷脂为底物。是近年来发现的一种与动脉粥样硬化和缺血性心脑血管
32、病相关的磷脂酶A2超家族（新的炎性标记物）。Lp-PLA2主要由炎症细胞（单核细胞/巨噬细胞、中性粒细胞、肥大细胞、T细胞、活化的血小板等）产生，其催化产生的促炎产物溶血磷脂酰胆碱 （LysoPC）和氧化非酯化脂肪酸（OxFA），具有促进动脉粥样硬化作用。,脂蛋白相关性磷脂酶A2,Lp-PLA2属于扩大的磷脂酶A2 超家族，其编码基因（PLA2G7）于1995年首次被克隆，它具有12个外显子，定位于6号染色体p21.212。Lp-PLA2为50kDa具有441个氨基酸残基的丝氨酸脂酶，其生物学活性不依赖于Ca2+，最初由于发现其可以降解血小板活化因子（PAF），因此它又被称为血小板活化因子乙酰
33、水解酶（PAF-AH）。The activity of Lp-PLA2 in the plasma is mainly associated with its presence in LDL (83%); a small amount of this enzyme is found in HDL (11%),LPA,LysoPLD,磷脂酰胆碱,氧化修饰磷脂酰胆碱,溶血磷脂酰胆碱,氧化脂肪酸oxFA,内皮细胞、单核细胞或平滑肌细胞产生的活性氧或自由基团使低密度脂蛋白氧化修饰而形成氧化修饰的低密度脂蛋白,LDL在氧化过程中，与LDL结合的Lp-PLA2活化，作用于LDL表面的甘油磷脂，产生具有促炎
34、作用的LysoPC和OxFA,甘油磷脂,LysoPC,OxFA,Lp-PLA2,活化,Lp-PLA2促动脉粥样硬化的机制,1、促进LDL的氧化修饰；2、作用于oxLDL，产生溶血磷脂酰胆碱（LysoPC）和自由脂肪酸（OxFA） ；后者活化炎症细胞，产生炎性细胞因子；3、促进吞噬细胞捕获oxLDL，形成泡沫细胞；4、Lp-PLA2在斑块坏死核心区显著表达，促进巨噬细胞凋亡，加快易损斑块破裂；5、 LysoPC抑制凋亡细胞清除，因而加剧血管壁的炎症反应，促进坏死核心的扩大；,脂蛋白 相关性 磷脂酶A2,“脂蛋白”：主要指LDL。 “相关性”：（1）约83%的Lp-PLA2与LDL结合；（2） L
35、DL在氧化过程中，与LDL结合的Lp-PLA2作用于LDL表面的甘油磷脂，产生具有促炎作用的产物LysoPC和OxFA；“磷脂酶A2”：作用于LDL表面的甘油磷脂sn-2位的酯键，产生溶血磷脂酰胆碱和自由脂肪酸。溶血磷脂酰胆碱和自由脂肪酸：具有促炎作用，促进动脉粥样硬化，加快易损斑块破裂；对冠心病、卒中有预警作用。,Lp-PLA2：心血管病预警分子,Lp-PLA2的两个主要来源，一是循环中与LDL结合的Lp-PLA2经动脉内膜直接进入内膜下动脉粥样硬化病灶中，二是动脉粥样硬化斑块中的炎性细胞的新合成。测定血浆中Lp-PLA2的含量或活性可作为独立危险因子，预警发生冠心病及缺血性卒中的危险性（F
36、DA批准）。Lp-PLA2抑制剂（他汀类、Darapladib）对降低高危心脑血管病患者的发病率具有重要意义。,LP-PLA2联合LPA检测的预警价值,临床流行病学研究及Meta分析结果表明, LP-PLA2 是评价冠心病和卒中风险的一个独立危险预测因子。局限性： LP-PLA2既具有抗炎活性，又具有促炎活性；其确切作用机制有争议。其主要代谢产物 LysoPC是溶血磷脂酸的主要底物来源。LP-PLA2是LPA的上游催化酶之一。 LP-PLA2联合LPA检测在动脉粥样硬化及冠心病、卒中的预警价值正在研究中。,Meta 分析,In all, the Lp-PLA2 Studies Collabor
37、ation report combined data for more than 79,000 subjects across 32 prospective studies in primary and secondary prevention and demonstrated that Lp-PLA2 activity and mass both have a continuous association with the risk of CHD and vascular death that is similar in magnitude to non-HDL cholesterol an
38、d systolic blood pressure and is independent of conventional risk factors.Thompson A, et al. Lipoproteinassociated phospholipase A(2) and risk of coronary disease, stroke, and mortality: collaborative analysis of 32 prospective studies. Lancet. 2010;375: 153644,Cross-sectional associations of Lp-PLA
39、2 activity,Cross-sectional associations of Lp-PLA2 activity,Risk ratios for CHD, ischaemic stroke, and vascular,Risk ratios for CHD, ischaemic stroke, and vascular,与其他危险因素比较（同等重要）,Lp-PLA2：含量与活性,Lp-PLA2 mass含量is measured by an enzyme immunoassay in human plasma (the PLAC test) (Dada et al 2002). In a
40、ddition, Lp-PLA2 activity活性can also be measured in human plasma (Tselepis et al 2002).,LP-PLA2 是评价冠心病和卒中风险的一个独立的危险预测因子,大量研究表明, LP-PLA2 是评价冠心病和卒中风险的一个独立的危险预测因子。Ballantyne等通过对Lp-PLA2和C反应蛋白（C-reactive protein, CRP）水平与传统危险因素进行评价以探讨其与缺血性卒中的关系。结果显示，升高的Lp-PLA2水平与卒中呈独立相关性，经68年的随访发现，Lp-PLA2水平升高者，卒中的发生率约是非升高者
41、的2倍。Lp-PLA2水平和CRP水平均升高者，较两者均正常者卒中的发生率增加10倍（提示脂质与炎症在AS中的协同作用）。,部分研究结果,Lysophosphatidic acid (LPA),溶血磷脂酸：是一种结构最为简单的甘油磷脂。,Atherosclerosis and thrombosis,Lysophosphatidic acid (LPA) has been found to accumulate in high concentrations in atherosclerotic lesions. 富含于粥样硬化斑块中； LPA is a bioactive phospholipi
42、d produced by activated platelets and formed during the oxidation of LDL. 由血小板活化或LDL轻度氧化过程中产生； Accumulating evidence suggests that this lipid mediator may serve as an important risk factor for development of atherosclerosis and thrombosis. 大量依据表明LPA是动脉粥样硬化和血栓形成的危险因素和标记物；,LPA在循环及心血管病领域研究历程,In 1963, V
43、ogt demonstrated that LPA has smooth muscle stimulating action. In 1978, Tokumura demonstrated that intravenous injection of LPA modulates blood pressure in rodents.In 1979, Schumacher KA and Gerrard JM demonstrated Influence on platelet aggregation.In 1986, Tokumura A, et al. lysophospholipase D in
44、 the production of lysophosphatidic acid in rat plasma. In 1989, Moolenaar WH. Lysophosphatidate-induced cell proliferation: identification and dissection of signaling pathways mediated by G proteins. In 1996, Chun J. Ventricular zone gene-1 (vzg-1) encodes a lysophosphatidic acid receptor. In 1999,
45、 Siess et al that LPA is a bioactive constituent in mildly oxLDL and atherosclerotic lesions has increased interest in the study of a role of LPA in atherosclerosis and thrombosis.,LPA synthesis,（1）血小板被活化后，在磷脂酶A1、A2、D等参与下，水解膜磷脂产生。（2）低密度脂蛋白（LDL）在轻度氧化过程中由lysoPLD产生，且产生的LPA位于轻度氧化LDL（moxLDL）分子的表面。（3）溶血磷脂
46、酰胆碱（LPC）在溶血磷脂酶D作用下产生。（4）除活化的血小板外，成纤维细胞、脂肪细胞、内皮细胞、某些炎症细胞及癌细胞、神经细胞及受损伤的细胞也可产生LPA。活化的血小板是血清中LPA的主要来源。,LPA受体与G蛋白偶联信号通路,目前在人类基因组中已克隆8个Edg基因（内皮分化基因），其中三个（Edg-2，Edg-4，Edg-7）编码LPA受体，分别称为LPA1、LPA2、LPA3；余下的五个编码神经鞘氨醇1-磷酸（sphingosine-1-phosphate，S1P）受体。LPA与跨膜G蛋白（Gi、Gq、G12/13）耦联受体结合，活化相应的信号传导通路，对血管内皮细胞、血小板、单核/巨噬
47、细胞等效应细胞发挥生物学效应。其主要生物学效应包括刺激细胞增殖与生存、促进血小板聚集、增加血管内皮细胞的通透性、血管平滑肌细胞(VSMCs)收缩、肿瘤细胞侵润等。,LPA处理使新内膜明显增生,在动脉粥样硬化斑块核心区LPA水平显著升高；当斑块破裂，暴露LPA，活化血小板，后者正反馈作用活化更多血小板，血栓扩大；,lysoPLD,LPA与肿瘤,Eur J Med Res. 2003 Sep 29;8(9):397-404.溶血磷脂酸是人类恶性肿瘤中激活血小板的唯一物质Sandmann G, Siess W, Essler M.结果表明：溶血磷脂酸在肿瘤患者血小板激活的病理过程中起着必需的作用。提
48、出：溶血磷脂酸受体拮抗剂可能有效地阻断由癌症导致的血小板激活，从而达到预防血栓形成的作用。,LPA与肿瘤,Lysophosphatidic acid as a potential biomarker for ovarian and other gynecologic cancers. Xu Y, Shen Z, Wiper DW, Wu M, Morton RE, Elson P, Kennedy AW, Belinson J, Markman JAMA. 1998 Aug 26;280(8):719-23.,LPA在血栓形成中的核心作用,Conclusions,Taken together,
49、 these accumulated in vitro and in vivo data strongly point to LPAs roles in promoting atherosclerosis and atherothrombosis. The selection of potent and specific inhibitors of LPA production and LPA degradation molecules, as well as LPA signaling effectors, are expected to prevent atherosclerosis and thrombosis.方法学的改良与结果的可信度是未来研究的重点！,
