丙丁酚对系膜细胞转录激活蛋白-1活性的影响

目的：研究氧化型低密度脂蛋白（ｏｘ－ＬＤＬ）诱导肾小球系膜细胞转录激活蛋白－１（ＡＰ－１）活性的改变及抗氧化剂丙丁酚对其影响,进一步探讨丙丁酚抗氧化作用的分子机制。方法：利用凝胶迁移率实验（Ｇｅｌ ｓｈｉｆｔ ａｓ－ｓａｙ）和超迁移率实验（Ｇｅｌ ｓｕｐｅｒｓｈｉｆｔ ａｓｓａｙ）检测不同浓度及不同时相ｏｘ－ＬＤＬ对大鼠肾小球系膜细胞ＡＰ－１活性的影响、ＡＰ－１二聚体中ｃ－Ｊｕｎ和ｃ－Ｆｏｓ成分的     

The splice variant LOXIN inhibits LOX-1 receptor function through hetero-oligomerization

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), encoded by the OLR1 gene, is a scavenger receptor that plays a central role in the pathogenesis of atherosclerosis. We have recently identified a truncated naturally occurring variant of the human receptor LOX-1, named LOXIN, which lacks part of the C-terminus lectin-like domain. In vivo and in vitro studies support that the new splicing isoform is protective against acute myocardial infarction. The mechanism by which LOXIN exerts its protective role is unknown. In this paper we report studies on the heterologous expression and functional characterization of LOXIN variant in mammalian fibroblasts and human endothelial cells. We found that LOXIN, when expressed in the absence of LOX-1, shows diminished plasma membrane localization and is deficient in ox-LDL ligand binding. When co-transfected with the full-length counterpart LOX-1, the two isoforms interact to form LOX-1 oligomers and their interaction leads to a decrease in the appearance of LOX-1 receptors in the plasma membrane and a marked impairment of ox-LDL binding and uptake. Co-immunoprecipitation studies confirmed the molecular LOX-1/LOXIN interaction and the formation of non-functional hetero-oligomers. Our studies suggest that hetero-oligomerization between naturally occurring isoforms of LOX-1 may represent a general paradigm for regulation of LOX-1 function by its variants.     

氧化低密度脂蛋白通过血凝素样氧化低密度脂蛋白受体1诱导血管内皮细胞粘附分子的表达

目的探讨血凝素样氧化低密度脂蛋白受体1（LOX-1）在氧化低密度脂蛋白（ox—LDL）诱导血管内皮细胞粘附分子表达中的作用。方法用不同浓度ox-LDL培养人脐静脉内皮细胞（HUVECs）,用RrealtimeRT—PCR测定LOX-1 mRNA的表达;RT—PCR测定细胞间粘附分子1（ICAM-1）、血管细胞粘附分子1（VCAM-1）以及E选择素的mRNA表达;用Westernblot测定LOX-1、ICAM-1、VCAM-1以及E选择素蛋白的表达,观察ox-LDL对血管内皮细胞粘附分子表达的影响。HUVECs预先用聚肌苷酸[poly（I）]和爱兰苔胶处理,再用ox—LDL培养,再分别测定上述粘附分子的表达水平,比较加入LOX-1阻断剂前后粘附分子表达的变化。结果ox-LDL各剂量组皆可上调LOX-1、ICAM-1、E选择素的mRNA和蛋白表达（P〈0．01）,在10～50μm/ml剂量范围内呈现明显的剂量一效应关系（P〈0．01）,但ox—LDL对VCAM-1的表达没影响。HUVECs预先与250μg／ml的poly（Ⅰ）或爱兰苔胶作用2h,然后加入50μg/ml的ox—LDL作用24h。poly（Ⅰ）和爱兰苔胶都能抑制ox—LDL诱导的LOX-1、ICAM-1和E选择素的mRNA和蛋白表达水平,与未阻断组相比,差异皆有统计学意义（P〈0．01）。结论ox—LDL可以上调血管内皮细胞粘附分子的表达,LOX-1受体阻断剂可以部分阻断ox—LDL的上调作用,ox-LDL诱导血管内皮细胞粘附分子的表过是通过LOX-1介导的。     

Surface binding,internalization and degradation by cultured human fibroblasts of low density lipoproteins isolated from Type 1 (insulin-dependent) diabetic patients: Changes with metabolic control

Summary A previous study of low density lipoprotein metabolism by cultured cells focused on the metabolism of normal lipoproteins in vitro by fibroblasts isolated from diabetic patients. No abnormalities were found. We have followed the opposite approach. Using normal human fibroblasts as test cells we compared the metabolism in vitro of low density lipoproteins isolated from diabetic patients before and after metabolic control. We found a significant decrease (p<0.02) in internalization and degradation of low density lipoproteins isolated from diabetic patients before metabolic control when compared with those isolated from normal control subjects or from the same patients after metabolic control. The observed changes were mainly apparent in intracellular degradation. To evaluate whether the observed differences in low density lipoprotein behaviour were correlated with lipid or apolipoprotein composition, we measured cholesterol, triglyceride, apolipoprotein B and total protein levels in the low density lipoproteins tested. A significant decrease (p<0.05) of the triglyceride/protein ratio was found in post-control low density lipoproteins suggesting that a high triglyceride content may interfere with low density lipoprotein metabolism. The present study represents the first observation that metabolic control in diabetes mellitus can alter low density lipoprotein-cell interaction and suggests a possible mechanism for the enhanced incidence of atherosclerosis in diabetic patients.     

氧化低密度脂蛋白诱导Zucker肥胖大鼠肾小球系膜细胞AP-1活性的变化

目的 :研究氧化低密度脂蛋白 (oxidizedlow densitylipoprotein ,Ox LDL)对体外培养的Zucker大鼠肾小球系膜细胞 (glomerularmesangialcell,GMC)中核转录因子激活蛋白 1(activatorprotein 1,AP 1)活化的影响 ,以及观察Ox LDL诱导的AP 1活性的变化与Zucker大鼠鼠龄以及基因型的相关性。　 方法 :①采用Zucker肥胖大鼠 (3月龄和 10月龄 )及Zucker瘦型大鼠 (3月龄和 10月龄 )的 4种GMC株 (O3m,O10m,L3m,L10m)进行传代培养。②利用凝胶迁移率实验 (EMSA)和超迁移率实验检测不同浓度及不同时相Ox LDL对Zucker大鼠GMCAP 1活性的影响 ,以及AP 1二聚体中c jun和c fos成分的变化。　 结果 :①经Ox LDL诱导后 ,4个组GMC内AP 1活性均较对照组明显增强 (F =177 84 ,P <0 0 1) ;②随着Ox LDL刺激浓度增加和时间的延长 ,GMC内AP 1活性相应增强 ,5 0mg/L的Ox LDL刺激 8h时 ,AP 1活性强度达最高峰 ;③Ox LDL主要激活AP 1二聚体成分中的c jun ;④O10m组AP 1的活性显著高于O3m组 (P <0 0 1) ,L10m组AP 1的活性显著高于L3m组 (P <0 0 1) ,O10m组显著高于L10m组 (P <0 0 1) ,O3m组显著高于L3m组 (P <0 0 1)。　 结论 :Ox LDL可诱导Zucker大鼠GMC内AP 1活化 ,其活化方式呈时间和剂量依赖 ;活化强度与大鼠的基因型及鼠龄     

Oxidized LDL induces serotonin release from blood platelets

Oxidized low-density lipoprotein (LDL) induces a release of serotonin from morphologically resting platelets and shape changed platelets. This suggests that oxidized LDL, a newly reported weak agonist, contributes to atherogenesis and thrombogenesis by stimulating platelets. © 1995 Wiley-Liss, Inc.     

Blood Pressure and Lipid Control Status in Japanese Hypertensive Patients

Strict blood pressure (BP) as well as lipid control is important to prevent cardiovascular events. The purpose of this study was to evaluate BP and lipid control status in hypertensive patients. Subjects were a total of 717 hypertensive patients who had been followed at National Kyushu Medical Center in FuKuoka, Japan. Goal BP was defined as < 130/85 mmHg (< 65 years) or < 140/90 mmHg (≥ 65 years). According to the Japanese guidelines, goal LDL cholesterol (LDL-C) levels were defined based on the patient category.

Average BP level and the number of anti-hypertensive drugs were 133 ± 12/74 ± 9 mmHg and 2.1 ± 1.1, respectively, and the LDL-C level was 119 ± 27 mg/dl. Goal BP was achieved in 40% of the patients of < 65 years and 67% of the elderly patients. Goal LDL-C was achieved in 65% of the patients. Even in the patients taking lipid-lowering agents (n = 178), 30% failed to achieve goal LDL-C levels. In the patients who achieved BP < 130/85 mmHg, 67% also achieved goal LDL-C, whereas 61% of the patients whose BP ≥ 140/90 mmHg achieved goal LDL-C. Both goal BP and LDL-C were achieved in 39% of the male and 36% of the female patients. In contrast, neither goal BP nor goal LDL-C was achieved in 16% of the male and 17% of the female patients.

Results suggest that intensive intervention should be required to achieve satisfactory BP and lipid control in hypertensive patients.     

Clinical significance of the physicochemical properties of LDL in type 2 diabetes

Atherosclerosis is the leading cause of death in type 2 diabetes. LDL cholesterol and atherosclerosis are related, both in healthy people and those with diabetes; however, people with diabetes are more prone to atheroma, even though their LDL cholesterol levels are similar to those in their non-diabetic peers. This is because LDL particles are modified in the presence of diabetes to become more atherogenic. These modifications include glycation in response to high plasma glucose levels; oxidative reactions mediated by increased oxidative stress; and transfer of cholesterol ester, which makes the particles smaller and denser. The latter modification is strongly associated with hypertriglyceridaemia. Oxidatively and non-oxidatively modified LDL is involved in plaque formation, and may thus contribute to the accelerated atherosclerosis. This review discusses the techniques currently used to determine the physicochemical properties of LDL, and examines the evidence that modification of these properties plays a role in the accelerated atherosclerosis associated with type 2 diabetes.     

对氧磷脂酶-1和氧化低密度脂蛋白在2型糖尿病肾病中的作用

目的 探讨2型糖尿病（DM0对氧磷脂酶－1（PON－1）和氧化低密度脂蛋白（ox-LDL)与内皮细胞和血小板功能的关系,以及对糖尿病肾病（DN）发生、发展的影响。方法 用酚乙酸酯为底物的速率法测定91例2型DM患者血清PON－1,用ELISA法测定ox-LDL,同时测定血清一氧化氮（NO）、血管性假血友病因子（VWF）及血浆颗粒膜蛋白（GMP140）,并与正常人作对照,以24h尿白蛋白排泄率（UAER）将DM患者分成三组。结果 血清PON－1活性在2型DM三组中与对照组比较显著降低（P＜0．01）,血浆ox-LDL浓度则显著升高（P＜0．01）,二者在DM三组之间的差别有显著性（P＜0．05）。PON－1与尿白蛋白呈显著性负相关（P＜0．01）,ox-LDL则与之呈显著性正相关（P＜0．01）,二者之间呈负相关（P＜0．01）。PON－1与血NO呈正相关,与GMP 140呈显著负相关;ox-LDL与血NO呈负相关,与VWF、GMP140呈正相关,经Logistic回归分析,提示ox-LDL是DN的危险因子。结论 DM的高血糖和脂代谢紊乱造成的PON－1酶活性下降以及脂质过氧化物的堆积,使内皮细胞的完整性及功能受损,同时血小板的活化程度明显增强。它们的共同作用与DN的发生、发展密切相关。     

Circulating oxidised low-density lipoprotein and intercellular adhesion molecule-1 and risk of type 2 diabetes mellitus: the Atherosclerosis Risk in Communities Study

Aims/hypothesis To evaluate the role of oxidative stress and inflammation in the aetiology of type 2 diabetes, we examined the association of oxidised LDL (ox-LDL) and soluble intercellular adhesion molecule-1 (sICAM-1) levels with type 2 diabetes incidence over 9 years in the Atherosclerosis Risk in Communities Study. Materials and methods In a large, prospective, case–cohort design, ox-LDL and sICAM-1 were measured in stored plasma samples collected at baseline in stratified samples of 581 diabetes cases and 572 non-cases selected from 10,275 middle-aged men and women without prevalent diabetes at baseline. Results Compared with non-cases, diabetes cases had significantly higher mean baseline levels of ox-LDL and sICAM-1. Elevated ox-LDL and sICAM-1 were both associated with increased risk of incident diabetes after adjustment for age, sex, race and centre, with hazard ratios for the highest vs lowest tertiles of 1.68 (95% CI 1.25–2.24) and 1.91 (95% CI 1.45–2.50), respectively. After additional adjustment for fasting glucose, waist circumference, HDL-cholesterol, triacylglycerol, hypertension and C-reactive protein, only sICAM-1 remained an independent predictor of incident diabetes (hazard ratio 1.50; 95% CI 1.02–2.23). Conclusions/interpretation In this community-based cohort of middle-aged US adults, elevated plasma ox-LDL and sICAM-1 levels were associated with increased risk of type 2 diabetes. Measurement of ICAM-1 or ox-LDL, or other measures related to inflammation or oxidative stress, may be helpful in identifying those patient populations in which to test whether novel therapies that inhibit specific pathways related to inflammation or oxidative stress are beneficial in the prevention of diabetes in humans.     

糖尿病患者过氧化低密度脂蛋白的测定及其临床意义

分别应用磷钨酸氯化镁分步沉淀法与超速离心法分离并测定了15例正常人的LDL-C、过氧化LDL(OX-LDL)、HDL-C水平,对两种方法的测得值进行了比较,发现两种方法之间的测定值相近,相关性良好。然后用磷钨酸氯化镁分步沉淀法测定了71例糖尿病患者和35例正常人LDL-C、OX-LDL、HDL-C水平,发现糖尿病患者OX-LDL高于正常人,而伴有血管病变者OX-LDL升高更为明显。     

氧化低密度脂蛋白对人红细胞左旋精氨酸转运的影响

目的 :观察氧化低密度脂蛋白对健康人红细胞左旋精氨酸 (L arg)转运的影响。方法 :用低密度脂蛋白和氧化低密度脂蛋白分别孵育正常人红细胞后将细胞分为对照组、低密度脂蛋白组和氧化低密度脂蛋白组 ,以3 H标记的L arg(3 H L arg)来测定红细胞的L arg转运。结果 :低密度脂蛋白对红细胞L arg转运无明显影响 (P >0 0 5 )。氧化低密度脂蛋白抑制红细胞L arg转运 :氧化低密度脂蛋白组氧化低密度脂蛋白 2 5、5 0、10 0mg/L 3个浓度总摄入的最大转运速率 (Vmax)值分别较对照组降低 3 3 %、2 7%、3 0 % (P均 <0 0 5 ) ,亲和力明显减小 (米氏常数值增大 ,P <0 0 5 ) ,均有显著性差异 ,未见明显的浓度—效应依赖关系 ;与对照组相比 ,氧化低密度脂蛋白组氧化低密度脂蛋白 2 5、5 0、10 0mg/L 3个浓度y +载体介导的L arg转运的最大转运速率值明显降低 ,分别降低 43 %、48%、45 % (P均 <0 0 5 ) ,均有显著性差异 ,亲和力明显减小 (米氏常数值增大 ) ;与对照组相比 ,氧化低密度脂蛋白组通过y +L载体介导的L arg转运 (最大转运速率值及米氏常数值 )均无明显改变 (P>0 0 5 )。结论 :低密度脂蛋白氧化成氧化低密度脂蛋白后抑制红细胞L arg的跨膜转运 ,推测氧化低密度脂蛋白是高脂血症患者红细胞L arg/一氧化氮途径     

老年男性代谢综合征患者血清抵抗素水平与各组分关系的研究

目的探讨老年男性血清抵抗素水平与代谢综合征(MS)各组分之间的关系。方法选择327例老年男性,分为MS组161例和无MS组166例。检测血清抵抗素、TG、TC、HDL-C、LDL-C、空腹血糖及C反应蛋白水平,并进行相关分析。结果 MS组血清抵抗素水平明显高于无MS组[(7.4±3.0)ng/L vs(6.7±3.3)ng/L,P<0.05]。血清抵抗素水平与TG、C反应蛋白和年龄呈正相关,与LDL-C呈负相关。血清抵抗素水平与LDL-C和C反应蛋白独立相关(P<0.01)。结论老年男性MS患者血清抵抗素水平明显升高,但抵抗素在MS中的作用有待于进一步研究。     

低密度脂蛋白胆固醇对人单核细胞膜联蛋白A1表达水平的影响

目的探讨LDL-C对人外周血单核细胞中膜联蛋白(annexin)A1表达水平的影响,为LDL-C致动脉粥样硬化机制提供新的视点。方法在体外分离培养人外周血单核细胞,随机分为对照组、不同浓度组(0.7、1.3、2.6、3.9 mmol/L LDL-C处理)和不同时间组(2、8、1 6、24 h用3.9 mmol/L LDL C处理),应用流式细胞仪检测annexinA1表达水平。结果与对照组比较,各浓度组annexin A1表达水平下降,且随浓度增加其抑制作用增强(P<0.01)。8 h后各时间组annexin A1表达水平明显下降,且随时间延长其抑制作用增强(P<0.05)。结论LDL-C可以通过降低人单核细胞annexin A1表达而影响单核细胞与内皮细胞黏附,进而启动动脉粥样硬化的发生发展,但具体机制有待进一步阐明。     

成人血低密度脂蛋白胆固醇水平升高及代谢综合征与卒中相关性研究

目的 分析血LDL-C水平升高伴或不伴代谢综合征(MS)对中国成人卒中发病的影响.方法 将2002年中国居民营养与健康状况调查中42 626例25～75岁成人,根据LDL-C水平分为<2.00 mmol/L组、2.00～2.50 mmol/L组、2.51～3.31 mmol/L组、≥3.32 mmol/L组.MS采用2005年国际糖尿病联盟的诊断定义.比较各组MS和卒中的患病率,以及伴或不伴MS时致卒中危险性.结果 (1)MS和卒中的患病率均随LDL-C水平升高而增加.与LDL-C<2.00 mmoL/L组相比,LDL-C≥3.32 mmol/L组MS和卒中的患病率分别增加了2.5倍(7.9%比20.1%)和4.2倍(0.5%比2.1%),P值均<0.01.(2)在同-LDL-C水平组,卒中患病率均为伴MS亚组高于不伴MS亚组,P值均<0.01.(3)logistic回归分析调整年龄、性别、吸烟后显示,LDL-C、MS与卒中发生正相关,致卒中的相对危险度(OR值)分别为2.35和3.15,P值均<0.0001.(4)与LDL-C<2.00mmol/L不伴MS亚组相比,LDL-C 2.00～2.50、2.51～3.31和≥3.32 mmol/L不伴MS各亚组发生卒中的OR值分别为1.03、1.89和2.08.LDL-C水平相似的伴MS亚组与不伴MS亚组相比,致卒中危险增加约3～4倍(OR值分别为4.38、5.23和6.15),P值均<0.0001.结论 LDL-C水平升高和MS均为卒中发生的独立危险因素,当二者并存时这种危险将进一步增加.对二者同时进行干预治疗对防治卒中十分重要.     

LDL的化学修饰及其致动脉粥样硬化作用

心血管疾病是危害人类健康的最主要疾病,是西方世界的第一死因〔1〕。美国心脏病协会估计结果显示5.7×107美国人受心血管病困扰,每年有9.54×105美国人因心血管病死亡〔2〕。WHO报告〔3〕显示,2000年全球因心血管疾病死亡人数高达1670万,一半以上发生在发展中国家。心血管病严重威胁着人类的生命和健康。据统计〔4〕,我国2003年城市居民主要疾病死亡率,脑血管病占73.12‰、心脏病占65.19‰,分别位居第二和第四;2003年农村居民主要疾病死亡率,脑血管病占83.71‰、心脏病占61.49‰,分别位居第三和第四。心脏病和脑血管病也是影响我国人民健…     

Associations of plasma homocysteine level with brachial-ankle pulse wave velocity, LDL atherogenicity, and inflammation profile in healthy men

Aims

To investigate the association of plasma total homocysteine (tHcy) with arterial stiffness, measured as brachial-ankle pulse wave velocity (baPWV), LDL atherogenicity, and inflammation profile in healthy men.

Methods and Results

In this cross-sectional study, 612 healthy men aged 31-79 years were classified into quartiles according to plasma tHcy concentration. In the total study population, tHcy concentration showed positive correlation with age (r = 0.083, P = 0.040), interleukin (IL)-1β (r = 0.249, P < 0.001), TNF-α (r = 0.150, P < 0.001), IL-6 (r = 0.154, P < 0.001), oxidized LDL (oxLDL) (r = 0.161, P = <0.001), and baPWV (r = 0.087, P = 0.032); and negative correlation with folate (r = −0.353, P < 0.001) and vitamin B₁₂ (r = −0.269, P < 0.001). In subgroup analysis based on plasma tHcy level, tHcy was associated with baPWV in men with high levels of tHcy (≥13.1 μmol/L, n = 153; r = 0.258, P = 0.001), but not in those with low-tHcy (<13.1 μmol/L, n = 459; r = −0.033, P = 0.478). The association between tHcy and baPWV in the high-tHcy group remained significant after adjustment for age, BMI, smoking, drinking, folate, and vitamin B₁₂. In the high-tHcy group, tHcy level was also positively correlated with IL-1β, TNF-α, oxLDL, and blood pressure; and negatively correlated with LDL particle size. In addition, baPWV showed negative correlation with LDL particle size and positive correlation with oxLDL in the high-tHcy group.

Conclusion

This study shows an association between high levels of plasma tHcy and more advanced arterial stiffness, smaller LDL particle size, and higher levels of oxLDL and cytokines in men with hyperhomocysteinemia. Enhanced arterial stiffness in hyperhomocysteinemia might be attributed, in part, to Hcy-related LDL atherogenicity.     

Determination of LOX-1-ligand activity in mouse plasma with a chicken monoclonal antibody for ApoB

Oxidized LDL (OxLDL) is implicated in endothelial dysfunction as well as the formation and progression of atherosclerosis. It has become evident that the atherogenic properties induced by OxLDL are mainly mediated via lectin-like OxLDL receptor-1 (LOX-1). Over the past decade, much research has been performed to investigate lipid metabolism and atherogenesis using genetically engineered mice. To understand the significance of OxLDL, methods to measure the levels of OxLDL in these experimental animals should be established. Utilizing a chicken monoclonal antibody technique, here, we generated anti-human ApoB antibodies that are able to recognize mouse VLDL/LDL. These antibodies were selected from single chain fragment of variable region (scFv) phage library constructed from chickens immunized with human LDL. One of these antibodies, HUC20, was reconstructed into IgY form. Immunohistochemical analysis revealed that this novel antibody specifically stains atherosclerotic lesions of ApoE-deficient mice, associated with Oil red O positive and macrophage-antigen-positive regions.Furthermore, in combination with recombinant LOX-1, a sandwich enzyme immunoassay was developed to measure the levels of LOX-1 ligands in mouse plasma. The sandwich enzyme immunoassay revealed a dramatic increase in the level of LOX-1 ligands in the plasma of ApoE-deficient mice fed high-fat diet, suggesting a link between the level of LOX-1-ligands and the progression of atherosclerosis in mice. Hence, the chicken anti-ApoB monoclonal antibody HUC20 developed here, could be a useful tool to analyze the role of ApoB-containing lipoprotein in atherogenesis in mice.     

Clinical effects of direct adsorption of lipoprotein apheresis: beyond cholesterol reduction

Direct adsorption of lipoproteins (DALI) from whole blood is the first LDL hemoperfusion technique for extracorporeal LDL and Lp(a) elimination without initial plasma separation. Thus, this technique is characterized by high user-friendliness. In a long-term multicenter study, LDL and lipoprotein (a) (Lp(a)) reductions were 69% and 64%, respectively, per session. Adverse effects were rare, as 95% of the sessions were uneventful. Biocompatibility studies showed only minor blood-adsorber interactions for most parameters; however, there was a significant bradykinin generation. After a single session, significant reductions of plasma viscosity, erythrocyte aggregation and adhesion molecules were documented. A retrospective analysis of 18 chronic DALI patients revealed that in the majority of patients, symptoms like angina and dyspnea as well as their general status and subjective well-being improved significantly. Moreover, the objective cardiovascular event rate (MACE) decreased from a total of 26 in the 3-year period prior to DALI to 6 during a mean follow-up of 3.8 years during chronic DALI therapy. Thus, the average event rate of 0.48 per patient year at baseline could be significantly reduced to 0.09 (P < 0.004) by DALI. This impressive improvement of symptoms and coronary events can hypothetically be related to the improvement of hemorheology and the transformation of unstable into stable plaques by DALI LDL apheresis.