2015講課脂類生物標記物

1、動脈粥樣硬化性心腦血管病脂代謝相關性生物標記物 Biomarkers related to lipid metabolism,威海市立醫(yī)院 李振光10-10-2015,,動脈粥樣硬化性心血管疾病(CVD),心血管疾病是全球的頭號死因（約占31%）； 估計740萬死于冠心病，670萬人死于中風; 3/4以上CVD死亡發(fā)生在低、中等收入國家; 早期發(fā)現高危人群并予以管理是改善預后的關鍵 生物標記物在期中扮演重要角色。

2、,,,,TRADITIONAL CARDIOVASCULAR RISK FACTORS,Major, classic or traditional risk factors are those defined from the Framingham studies;The Framingham cohorts began in 1948, and are milestones in the identification of car

3、diovascular factors. Including age; sex; total cholesterol, LDL-C and HDL-C levels; systolic pressure; smoking; high blood glucose; body weight; dietary habits; and physical inactivity. Based on these findings, cardiov

4、ascular 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 tr

5、aditional risk factors, and that 60% of them 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 t

6、o rupture.,,Atherosclerosis develops,Activation of the endothelium and recruitment of immune cells; Monocyte differentiation and foam cell formation; Development of fibrotic plaques due to death

7、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 th

8、e plaques (fibrous cap).,,,,,Inflammatory 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 1β;

9、 C-reactive protein; tumor necrosis factor-alpha; pentraxin3(PTX-3); serum amyloid A; sCD40; adhesion molecules; monocyte chemoattractant protein-1; sEndoglin; PAPP-A; chemokine 16; insulin like growth factor; lipoprotei

10、n-associated phospholipase A2;galectin 3; Adiponectin (脂聯(lián)素); myeloperoxidase (MPO,髓過氧化物酶),,,,Biomarkers related to lipid metabolism,Biomarkers related to lipid metabolism（8）,1、 Modified LDL particles； 2、 Apolipoprotei

11、n 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、 L

12、ysophosphatidic acid, LPA,,主要與脂蛋白有關,,,,脂蛋白：lipoproteins,Lipids are transported in the bloodstream inside lipoproteins, which are essentially made up by proteins (apolipoproteins), cholesteryl esters, cholesterol, TG, and

13、 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-I), C-II (apo C-II), C-III (apo C-III), B-48 (apo

14、 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, ma

15、de up of apo B-100; d) low density 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-I

16、I), apo C-I, apo C-II, apo C-III, and apo E.,,,LDL modified particles（1）,Modified LDL: oxidized LDL electronegative LDL glycosylated LDLThese molecule are pro-inflammatory and highly atherogenic,,Apolipoproteins （

17、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 cholesterol particles (

18、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 concentratio

19、ns of apo B determined a relative risk of 1.99 for events, whereas 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 remaine

20、d significantly associated after the adjustment for traditional risk factors; The indication for apo B determination is expected to be included in the next NCEP guidelines (ATP IV),,Lipoprotein（a） （3）,Lipoprotein(a), Lp

21、(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 contai

22、n apo B and 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 pre

23、sent very high risk.,,Lipoprotein（a） （3）,The third report of the National Cholesterol Education Program for the Detection, Evaluation and Treatment of Hypercholesterolemia in Adults (NCEP-ATP III) stated that, in spite o

24、f the measurement limitations, Lp(a) dosage is an useful parameter. Its high concentration aids the identification of those individuals with even higher cardiovascular risk.It is suggested that Lp(a) should be used as a

25、 second risk factor to support lower LDL cholesterol targets.,,Apolipoprotein CIII,Apolipoprotein CIII (apoC-III) is an atherogenic protein found on HDL, VLDLand LDL. 薈萃分析表明： (1) Significantly higher levels of apoC-II

26、I in the non-HDL fraction of plasma (representing 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 to

27、tal plasma apoC-III in the cases.,,Apolipoprotein CIII,,,,Apolipoprotein CIII,,,CETP and its lipoprotein subclasses （4）,Cholesteryl ester transfer protein (CETP) concentrations are increased in obesity, dyslipidemia and

28、atherosclerosis, and are directly associated with inflammatory markers.However, their use as a risk biomarker is still controversial.,,LDL顆粒大小與密度 （5）,Individuals with greater concentrations of small and dense LDL partic

29、les 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.,,CONCLUSI

30、ON,Among them, promising ones are measurements of changes in LDL particles, Lp-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 populati

31、on. 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 IV are being determined, and possibly Lp(a) will be included

32、 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作為降脂

33、治療目標的局限性,LDL-C并不能反映導致動脈粥樣硬化的顆?？偭?，即所有含有載脂蛋白B（ApoB）的顆粒，其中包括VLDL、中間密度脂蛋白（IDL）、LDL和Lp[a]。研究表明，評估這種所謂的“剩余風險”，如非高密度脂蛋白膽固醇（non-HDL-C，總膽固醇減HDL-C）、ApoB和LDL顆粒數（LDL-P）可較LDL-C更好地預測CVD風險;其中研究顯示，ApoB和LDL-P比non-HDL-C有更好的預測表現。,,目前的概念,

34、流行病學研究發(fā)現，non-HDL-C較LDL-C可更好地預測心血管疾病的發(fā)病率和死亡率，該指標已被美國脂質協(xié)會（NLA）認可作為一個比LDL-C更好的目標。non-HDL-C可以通過從總膽固醇減去HDL-C計算出來，而不需要額外的檢測和費用。ApoB和LDL-P的水平可更好地反映導致動脈粥樣硬化顆粒的負擔。循環(huán)中約90%的ApoB包含在LDL顆粒中；LDL-P是通過核磁共振測量。NLA建議血脂異常管理中將non-HDL-C作為首要目

35、標，LDL-C作為次要治療靶點，ApoB作為一個“可選的次級治療目標”，可考慮LDL-P作為ApoB的替代指標。,,未來的脂質檢測,未來標準脂質檢測可能應包括： （1）傳統(tǒng)脂質檢測(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）顆粒分類；（7）Lp-PLA2；（8）LPA/S1P（溶血磷脂酸/鞘氨醇1-磷酸

36、）。,,脂蛋白：lipoproteins,脂蛋白：脂質與蛋白質結合在一起形成的脂質-蛋白質復合物。是血脂在血液中存在、轉運及代謝的主要形式；分為乳糜微粒(CM)、極低密度脂蛋白(VLDL)、低密度脂蛋白(LDL)、中間密度脂蛋白（IDL）和高密度脂蛋白(HDL)； 成熟脂蛋白呈球形，中間是甘油三酯和膽固醇酯，表面組分為載脂蛋白、游離膽固醇和磷脂。LDL的氧化修飾(oxLDL)是動脈粥樣硬化的基礎。,,脂蛋白（LDL）結構模式圖,,

37、（磷脂酰膽堿）,,,磷脂酶（phospholipase, PLA）概述,根據磷脂酶對磷脂水解部位的不同可將磷脂酶分為4類：磷脂酶A1（PLA1）: 作用于Sn-1位?；? 磷脂酶A2（PLA2）：選擇性作用于甘油磷脂sn-2位的酯鍵（-COO-基團，由羧基“-COOH”與羥基“-OH”脫水而成）;磷脂酶C（PLC）: 水解甘油和磷酸形成的酯鍵; 磷脂酶D（PLD）；將卵磷脂水解生成磷酯酸和乙酰膽堿;溶血磷脂酶D（Lyso-PL

38、D, ATX）：產生溶血磷脂；,,,磷脂酶（phospholipase, PL）,,,,,甘油磷脂,磷脂是一類含有磷酸的脂類，機體中主要含有兩大類磷脂，由甘油構成的磷脂稱為甘油磷脂；由神經鞘氨醇構成的磷脂，稱為鞘磷脂（S1P）。其結構特點是：具有由磷酸相連的取代基團(含氨堿或醇類)構成的親水頭和由脂肪酸鏈構成的疏水頭。在生物膜中磷脂的親水頭位于膜表面，而疏水尾位于膜內側。,,,,酯鍵,Sn-2,,PLA2,,取代基團,,H,,PLA

39、2,,溶血磷脂酰膽堿,,膽堿,,,PA與LPA,PLA2作用位點，加氫變成OH，溶血磷脂,,,,,甘油,甘油三酯,磷脂酰膽堿（卵磷脂）,甘油骨架,,,,,,,,,脂肪酸,,脂肪酸,（不含雙鍵）,,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-?；饷?，是專一催化甘油磷脂Sn-2位酯鍵，酶解產物為溶血磷脂和脂肪酸。廣泛參與人體細胞內外信號的傳遞及炎癥、多種相關性疾病病

40、理反應。其生理功能包括細胞信號傳遞及產生20多種類脂質介質，改造磷脂結構，促進機體壞死組織自動消失，肺泡表面活性物質代謝等。,,Phospholipase A2 ：structure/function,四種主要的磷脂酶A2 Secreted sPLA2：分泌型磷脂酶A2 ，與二十烷類產生、炎癥及抗菌作用有關。Cytosolic cPLA2 ：胞漿型磷脂酶A2，花生四烯酸(AA)代謝具有優(yōu)先選擇性。 Calcium-indepen

41、dent iPLA2：鈣不依賴磷脂酶A2，作為看家酶參與膜的重建 Platelet activating factor (PAF) acetyl hydrolase/oxidized lipid lipoprotein associated （Lp-PLA2）：血小板活化因子乙酰水解酶（氧化脂蛋白相關性磷脂酶A2），以PAF和氧化磷脂為底物。,,,,,,,,Atherosclerosis is a progressive inflam

42、matory disease that develops over many years動脈粥樣硬化是一種慢性進行性炎性疾病Inflammation has been shown to have a direct effect on plaque formation and plaque rupture炎癥對斑塊形成和斑塊破裂發(fā)揮直接作用Inflammatory markers may help predict cardiov

43、ascular risk炎癥標志物或有助于預測心血管疾病Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a novel inflammatory marker脂蛋白相關磷脂酶及其產物是一種新的炎癥標志物,Inflammation and CHD Risk炎癥和動脈粥樣硬化,,脂蛋白相關性磷脂酶A2,脂蛋白相關性磷脂酶A2（Lipoprotein-associated p

44、hospholipase A2，Lp-PLA2）又稱血小板活化因子乙酰水解酶（PAF-AH），以PAF和氧化磷脂為底物。是近年來發(fā)現的一種與動脈粥樣硬化和缺血性心腦血管病相關的磷脂酶A2超家族（新的炎性標記物）。Lp-PLA2主要由炎癥細胞（單核細胞/巨噬細胞、中性粒細胞、肥大細胞、T細胞、活化的血小板等）產生，其催化產生的促炎產物溶血磷脂酰膽堿 （LysoPC）和氧化非酯化脂肪酸（OxFA），具有促進動脈粥樣硬化作用。,,脂蛋白相關

45、性磷脂酶A2,Lp-PLA2屬于擴大的磷脂酶A2 超家族，其編碼基因（PLA2G7）于1995年首次被克隆，它具有12個外顯子，定位于6號染色體p21.2~12。Lp-PLA2為50kDa具有441個氨基酸殘基的絲氨酸脂酶，其生物學活性不依賴于Ca2+，最初由于發(fā)現其可以降解血小板活化因子（PAF），因此它又被稱為血小板活化因子乙酰水解酶（PAF-AH）。The activity of Lp-PLA2 in the plasma i

46、s mainly associated with its presence in LDL (83%); a small amount of this enzyme is found in HDL (11%),,,,LPA,LysoPLD,,磷脂酰膽堿,氧化修飾磷脂酰膽堿,溶血磷脂酰膽堿,氧化脂肪酸oxFA,,,,,,,,,內皮細胞、單核細胞或平滑肌細胞產生的活性氧或自由基團使低密度脂蛋白氧化修飾而形成氧化修飾的低密度脂蛋白,LDL在氧化

47、過程中，與LDL結合的Lp-PLA2活化，作用于LDL表面的甘油磷脂，產生具有促炎作用的LysoPC和OxFA,,,,甘油磷脂,LysoPC,OxFA,Lp-PLA2,,,,,,,,,,,,,活化,,,,,,,Lp-PLA2促動脈粥樣硬化的機制,1、促進LDL的氧化修飾；2、作用于oxLDL，產生溶血磷脂酰膽堿（LysoPC）和自由脂肪酸（OxFA） ；后者活化炎癥細胞，產生炎性細胞因子；3、促進吞噬細胞捕獲oxLDL，形成泡沫細胞

48、；4、Lp-PLA2在斑塊壞死核心區(qū)顯著表達，促進巨噬細胞凋亡，加快易損斑塊破裂；5、 LysoPC抑制凋亡細胞清除，因而加劇血管壁的炎癥反應，促進壞死核心的擴大；,,,,,,,,脂蛋白 相關性 磷脂酶A2,“脂蛋白”：主要指LDL。 “相關性”：（1）約83%的Lp-PLA2與LDL結合；（2） LDL在氧化過程中，與LDL結合的Lp-PLA2作用于LDL表面的甘油磷脂，產生具有促炎作用的產物LysoPC和OxFA；

49、“磷脂酶A2”：作用于LDL表面的甘油磷脂sn-2位的酯鍵，產生溶血磷脂酰膽堿和自由脂肪酸。溶血磷脂酰膽堿和自由脂肪酸：具有促炎作用，促進動脈粥樣硬化，加快易損斑塊破裂；對冠心病、卒中有預警作用。,,,,,,,Lp-PLA2：心血管病預警分子,Lp-PLA2的兩個主要來源，一是循環(huán)中與LDL結合的Lp-PLA2經動脈內膜直接進入內膜下動脈粥樣硬化病灶中，二是動脈粥樣硬化斑塊中的炎性細胞的新合成。測定血漿中Lp-PLA2的含量或活性

50、可作為獨立危險因子，預警發(fā)生冠心病及缺血性卒中的危險性（FDA批準）。Lp-PLA2抑制劑（他汀類、Darapladib）對降低高危心腦血管病患者的發(fā)病率具有重要意義。,,LP-PLA2聯(lián)合LPA檢測的預警價值,臨床流行病學研究及Meta分析結果表明, LP-PLA2 是評價冠心病和卒中風險的一個獨立危險預測因子。局限性： LP-PLA2既具有抗炎活性，又具有促炎活性；其確切作用機制有爭議。其主要代謝產物 LysoPC是溶血磷脂酸

51、的主要底物來源。LP-PLA2是LPA的上游催化酶之一。 LP-PLA2聯(lián)合LPA檢測在動脈粥樣硬化及冠心病、卒中的預警價值正在研究中。,,Meta 分析,In all, the Lp-PLA2 Studies Collaboration report combined data for more than 79,000 subjects across 32 prospective studies in primary and sec

52、ondary 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 and systolic blood pressure

53、 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

54、. 2010;375: 1536–44,,Cross-sectional associations of Lp-PLA2 activity,,,,,,,,Cross-sectional associations of Lp-PLA2 activity,,,,,Risk ratios for CHD, ischaemic stroke, and vascular,,,Risk ratios for CHD, ischaemic strok

55、e, and vascular,,,與其他危險因素比較（同等重要）,,,Lp-PLA2：含量與活性,Lp-PLA2 mass含量is measured by an enzyme immunoassay in human plasma (the PLAC? test) (Dada et al 2002). In addition, Lp-PLA2 activity活性can also be measured in human plasma

56、 (Tselepis et al 2002).,,LP-PLA2 是評價冠心病和卒中風險的一個獨立的危險預測因子,大量研究表明, LP-PLA2 是評價冠心病和卒中風險的一個獨立的危險預測因子。Ballantyne等通過對Lp-PLA2和C反應蛋白（C-reactive protein, CRP）水平與傳統(tǒng)危險因素進行評價以探討其與缺血性卒中的關系。結果顯示，升高的Lp-PLA2水平與卒中呈獨立相關性，經6~8年的隨訪發(fā)現，Lp-PL

57、A2水平升高者，卒中的發(fā)生率約是非升高者的2倍。Lp-PLA2水平和CRP水平均升高者，較兩者均正常者卒中的發(fā)生率增加10倍（提示脂質與炎癥在AS中的協(xié)同作用）。,,部分研究結果,,,Lysophosphatidic acid (LPA),溶血磷脂酸：是一種結構最為簡單的甘油磷脂。,,Atherosclerosis and thrombosis,,Lysophosphatidic acid (LPA) has been found to

58、 accumulate in high concentrations in atherosclerotic lesions. 富含于粥樣硬化斑塊中； LPA is a bioactive phospholipid produced by activated platelets and formed during the oxidation of LDL. 由血小板活化或LDL輕度氧化過程中產生； Accumulating evide

59、nce suggests that this lipid mediator may serve as an important risk factor for development of atherosclerosis and thrombosis. 大量依據表明LPA是動脈粥樣硬化和血栓形成的危險因素和標記物；,LPA在循環(huán)及心血管病領域研究歷程,,In 1963, Vogt demonstrated that LPA has sm

60、ooth 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.

61、In 1986, Tokumura A, et al. lysophospholipase D in the production of lysophosphatidic acid in rat plasma. In 1989, Moolenaar WH. Lysophosphatidate-induced cell proliferation: identification and dissection of signaling

62、pathways mediated by G proteins. In 1996, Chun J. Ventricular zone gene-1 (vzg-1) encodes a lysophosphatidic acid receptor. In 1999, Siess et al that LPA is a bioactive constituent in mildly oxLDL and atherosclerotic l

63、esions 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等參與下，水解膜磷脂產生。（2）低密度脂蛋白（LDL）在輕度氧化過程中由lysoPLD產生，且產生的LPA位于輕度氧化LDL（moxLDL）分子的表面。（3）溶血磷脂酰膽

64、堿（LPC）在溶血磷脂酶D作用下產生。（4）除活化的血小板外，成纖維細胞、脂肪細胞、內皮細胞、某些炎癥細胞及癌細胞、神經細胞及受損傷的細胞也可產生LPA。活化的血小板是血清中LPA的主要來源。,LPA受體與G蛋白偶聯(lián)信號通路,,目前在人類基因組中已克隆8個Edg基因（內皮分化基因），其中三個（Edg-2，Edg-4，Edg-7）編碼LPA受體，分別稱為LPA1、LPA2、LPA3；余下的五個編碼神經鞘氨醇1-磷酸（sphingosin

65、e-1-phosphate，S1P）受體。LPA與跨膜G蛋白（Gi、Gq、G12/13）耦聯(lián)受體結合，活化相應的信號傳導通路，對血管內皮細胞、血小板、單核/巨噬細胞等效應細胞發(fā)揮生物學效應。其主要生物學效應包括刺激細胞增殖與生存、促進血小板聚集、增加血管內皮細胞的通透性、血管平滑肌細胞(VSMCs)收縮、腫瘤細胞侵潤等。,,LPA處理使新內膜明顯增生,,在動脈粥樣硬化斑塊核心區(qū)LPA水平顯著升高；當斑塊破裂，暴露LPA，活化血小板，

66、后者正反饋作用活化更多血小板，血栓擴大；,,,lysoPLD,LPA與腫瘤,Eur J Med Res. 2003 Sep 29;8(9):397-404.溶血磷脂酸是人類惡性腫瘤中激活血小板的唯一物質Sandmann G, Siess W, Essler M.結果表明：溶血磷脂酸在腫瘤患者血小板激活的病理過程中起著必需的作用。提出：溶血磷脂酸受體拮抗劑可能有效地阻斷由癌癥導致的血小板激活，從而達到預防血栓形成的作用。,,LP

67、A與腫瘤,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

68、.,,LPA在血栓形成中的核心作用,,Conclusions,Taken together, these accumulated in vitro and in vivo data strongly point to LPA's roles in promoting atherosclerosis and atherothrombosis. The selection of potent and specific inhibi