LDL受体对清道夫受体活性的影响

应用经ＰＭＡ诱导衍生的ＴＨＰ－１巨噬细胞为模型,以单克隆抗体Ｃ７Ｂ封闭ｏｘＬＤＬ上的ＬＤＬ受体结合位点,结果发现,正常细胞在摄取ｏｘＬＤＬ时ＬＤＬ受体与清道夫受体起协同作用;但Ｃ７Ｂ作用于蓄积了脂质的ＴＨＰ－１巨噬细胞时,对细胞脂质蓄积程度无明显影响,清道夫受体活性不但不降低反而有所升高,说明由于脂质蓄积ＬＤＬ受体的作用减弱。     

DMT 1在THP-1氧化LDL过程中对基因表达的影响

采用RT-PCR法和Western blot法对二价拿属转运蛋白1(divalent metal transporter 1, DMT 1)在人单核细胞系THP-1氧化低密度脂蛋白(low density lipoprotein,LDL)的过程中所起到的作用进行了验证。结果表明,DMT 1参与了THP-1氧化LDL的过程,且DMT 1的表达水平显著升高,为临床心血管疾病的抗氧化治疗提供了新的思路。     

人血浆HDL对动脉粥样硬化家兔肝细胞膜LDL受体活性影响的研究

高胆固醇饲料喂养造成的动脉粥样硬化(As)模型家兔通过静脉注射人血浆HDL制剂,观察HDL对As家兔肝细胞膜LDL受体活性的影响.结果发现,摄取高胆固醇饲料的As家兔,其肝细胞膜LDL受体K_d值虽无明显变化但B_max值显著减小（P＜0.01,与正常对照组比较);注射HDL制剂后,As家兔肝细胞膜LDL受体K_d值仍无明显改变,但B_max值却显著回升（P＜0.01,与高脂组比较).表明人血浆HDL具有增加As家兔肝细胞膜LDL受体活性的作用.     

人血浆HDL对动脉粥样硬化家兔肝细胞膜 LDL受体活性影响的研究

高胆固醇饲料喂养造成的动脉粥样硬化（Ａｓ） 模型家兔通过静脉注射人血浆ＨＤＬ 制剂, 观察ＨＤＬ 对Ａｓ家兔肝细胞膜ＬＤＬ受体活性的影响． 结果发现, 摄取高胆固醇饲料的Ａｓ 家兔, 其肝细胞膜ＬＤＬ 受体 Ｋｄ 值虽无明显变化但Ｂｍａｘ 值显著减小（ Ｐ＜ ０－０１ , 与正常对照组比较） ; 注射ＨＤＬ 制剂后, Ａｓ 家兔肝细胞膜ＬＤＬ受体Ｋｄ 值仍无明显改变, 但Ｂｍａｘ 值却显著回升（ Ｐ＜ ０－０１ , 与高脂组比较） ． 表明人血浆ＨＤＬ 具有增加Ａｓ 家兔肝细胞膜ＬＤＬ 受体活性的作用．     

LDL亚组分研究进展

根据低密度脂蛋白（low density lipoprotein,LDL）颗粒的不均一性,可以利用密度梯度超速离心法和梯度凝胶电泳法将其分成若干亚组分。近年来,对于LDL亚组分分离方法的研究取得了显著进展。除对上述两种基本实验方法进行改进外,有实验室采用Western印迹法对LDL颗粒进行分离。LDL亚组分分离方法的进步,使对LDL亚组分的认识更加深入：LDL亚组分的高度不均一性、氧化易感性及电负性等不同特性与动脉粥样硬化（atherosclerosis,AS）关系密切。LDL亚组分的研究为认识动脉粥样硬化及其相关疾病提供了重要的理论依据。     

2型糖尿病患者血清LDL，CysC与动脉粥样硬化的相关性研究

目的:分析2型糖尿病(T2DM)患者血清低密度脂蛋白(LDL)、胱抑素C(CysC)与动脉粥样硬化(AS)的相关性。方法:选取2型糖尿病患者300名,根据颈动脉内膜中膜厚度分为非动脉粥样硬化斑块组(n=109)和动脉粥样硬化斑块组(n=191),并对动脉粥样硬化斑块的相关危险因素进行多因素Logistic回归分析。结果:(1)Pearson相关分析显示,LDL、CysC水平与IMT值呈正相关(P0.05)。(2)单因素分析示,非AS组和AS组两组间LDL(t=8.876,P0.05)、CysC(t=7.985,P0.05)、HbA1c(t=9.912,P0.05)、Hs-CRP(t=12.461,P0.05)、年龄(t=7.114,P0.05)、UA((t=8.618,P0.05)间差异有统计学意义;(3)多因素Logistic回归分析示,LDL、CysC、HbA1c、年龄是T2DM并AS的独立危险因素(P0.05);结论:LDL与CysC水平是T2DM并AS的独立危险因素。     

天然属性抗LDL 及oxLDL IgM 亚类抗体的制备与鉴定

目的:制备天然属性抗低密度脂蛋白(LDL)及抗氧化低密度脂蛋白(oxLDL)IgM亚类抗体。方法:给予Babl/c小鼠高胆固醇饮食,4周后取脾细胞直接与SP2/0细胞融合,以纯化的LDL及oxLDL为抗原,对阳性杂交瘤细胞生长孔进行间接ELISA筛选。鉴定杂交瘤上清的免疫球蛋白亚类,进而采用免疫沉淀和免疫印迹法对获得的抗体进行免疫学反应性鉴定。结果:杂交瘤细胞分泌的抗LDL及抗oxLDL的天然抗体通过ELISA法被筛选出来,可以与LDL或oxLDL发生高亲和力结合,经过4次克隆化,最终获得2株稳定分泌天然抗LDL的抗体,命名为5G8和2H7,及1株稳定抗oxLDL的抗体,命名为3A6,3株抗体均属于IgM亚类,无交叉反应,可以满足免疫印迹、免疫沉淀等实验要求。结论:成功制备了抗LDL及抗oxLDL IgM亚类抗体,为研究天然抗体在体内脂质代谢和相关心脑血管疾病如动脉粥样硬化等发生发展中的作用提供了重要的研究工具。     

天然属性抗LDL及oxLDL IgM亚类抗体的制备与鉴定

目的：制备天然属性抗低密度脂蛋白（LDL）及抗氧化低密度脂蛋白（oxLDL）IgM亚类抗体。方法：给予Babl/c小鼠高胆固醇饮食,4周后取脾细胞直接与SP2/0细胞融合,以纯化的LDL及oxLDL为抗原,对阳性杂交瘤细胞生长孔进行间接ELISA筛选。鉴定杂交瘤上清的免疫球蛋白亚类,进而采用免疫沉淀和免疫印迹法对获得的抗体进行免疫学反应性鉴定。结果：杂交瘤细胞分泌的抗LDL及抗oxLDL的天然抗体通过ELISA法被筛选出来,可以与LDL或oxLDL发生高亲和力结合,经过4次克隆化,最终获得2株稳定分泌天然抗LDL的抗体,命名为5G8和2H7,及1株稳定抗oxLDL的抗体,命名为3A6,3株抗体均属于IgM亚类,无交叉反应,可以满足免疫印迹、免疫沉淀等实验要求。结论：成功制备了抗LDL及抗oxLDL IgM亚类抗体,为研究天然抗体在体内脂质代谢和相关心脑血管疾病如动脉粥样硬化等发生发展中的作用提供了重要的研究工具。     

氧化修饰低密度脂蛋白与动脉粥样硬化

氧化修饰低密度脂蛋白与动脉粥样硬化周永列（浙江省人民医院,杭州３１００１４）关键词氧化修饰低密度脂蛋白,动脉粥样硬化在动脉粥样硬化（ＡＳ）与血浆脂质关系的研究中,血浆低密度脂蛋白（ＬＤＬ）浓度升高被认为是引起ＡＳ的重要危险因素而受到广泛的重视。但是脂...     

Retention of aggregated LDL by cultured human coronary artery endothelial cells

Aggregated LDL (AgLDL) accumulates within the subendothelial space of developing atherosclerotic lesions. We were interested to learn whether endothelial cells can interact with AgLDL. Incubation of endothelial cells with AgLDL resulted in apparent cholesterol retention. Microscopic examination revealed that cholesterol retention resulted mainly from endothelial cell surface attachment of AgLDL. Little AgLDL entered endothelial cells consistent with the small amount of endothelial cell degradation of AgLDL. Although endothelial cell retention of AgLDL was inhibited by LDL, AgLDL retention was not blocked by lactoferrin, C7 anti-LDL receptor monoclonal antibody, or receptor-associated protein, suggesting that LDL receptor family members did not mediate this retention. Surface retention of AgLDL depended on microtubule function and could be regulated by the protein kinase C activator, PMA. Treatment of endothelial cells with PMA either before or during, but not after incubation with AgLDL, inhibited retention of AgLDL. Our findings show that endothelial cells can retain AgLDL but internalize and metabolize little of this AgLDL. Thus, it is unlikely that endothelial cells can transport AgLDL out of atherosclerotic lesions, but it is likely that retention of AgLDL affects endothelial function.     

LDL受体对清道夫受体活性的影响

应用经ＰＭＡ诱导衍生的ＴＨＰ－１巨噬细胞为模型,以单克隆抗体Ｃ７Ｂ封闭ｏｘＬＤＬ上的ＬＤＬ受体结合位点,结果发现,正常细胞在摄取ｏｘＬＤＬ时ＬＤＬ受体与清道夫受体起协同作用;但Ｃ７Ｂ作用于蓄积了脂质的ＴＨＰ－１巨噬细胞时,对细胞脂质蓄积程度无明显影响,清道夫受体活性不但不降低反而有所升高,说明由于脂质蓄积ＬＤＬ受体的作用减弱．     

Oxidized LDL induces phosphorylation of non-muscle myosin IIA heavy chain in macrophages

Oxidized LDL (oxLDL) performs critical roles in atherosclerosis by inducing macrophage foam cell formation and promoting inflammation. There have been reports showing that oxLDL modulates macrophage cytoskeletal functions for oxLDL uptake and trapping, however, the precise mechanism has not been clearly elucidated. Our study examined the effect of oxLDL on non-muscle myosin heavy chain IIA (MHC-IIA) in macrophages. We demonstrated that oxLDL induces phosphorylation of MHC-IIA (Ser1917) in peritoneal macrophages from wild-type mice and THP-1, a human monocytic cell line, but not in macrophages deficient for CD36, a scavenger receptor for oxLDL. Protein kinase C (PKC) inhibitor-treated macrophages did not undergo the oxLDL-induced MHC-IIA phosphorylation. Our immunoprecipitation revealed that oxLDL increased physical association between PKC and MHC-IIA, supporting the role of PKC in this process. We conclude that oxLDL via CD36 induces PKC-mediated MHC-IIA (Ser1917) phosphorylation and this may affect oxLDL-induced functions of macrophages involved in atherosclerosis. [BMB Reports 2015; 48(1): 48-53]     

Three different pathways for human LDL oxidation are inhibited in vitro by water extracts of the medicinal herb Achyrocline satureoides

In this study we investigated the antioxidant properties of one herbal preparation widely used in complementary and alternative medicine in large areas of the world: Achyrocline satureoides (AS), popularly known as "marcela". Although rich in flavonoids, the ethnopharmacological uses of this plant do not include atherosclerosis prevention. Furthermore, no study had been conducted so far exploring the antioxidant activity of Achyrocline satureoides vis-à-vis human LDL oxidation, which is the compelling issue in pinpointing potential cardioprotective new uses for a traditional remedy. We explored the effects of AS extracts on human LDL oxidation, employing 3 different systems which are thought to play a role in oxidation of LDL in the arterial wall: copper, peroxynitrite, and lipoxygenase. Oxidation was monitored by conjugate dienes, TBARS formation and aggregation of apoB using SDS-PAGE. In copper-initiated oxidation a dose dependent inhibition of the initiation and propagation of lipid oxidation is shown by an increase in the lag phase for conjugate diene production which was 60 +/- 15 min in the absence and 120 +/- 20 min in the presence of 4 microg/ml AS extracts (p < 0.001). TBARS production was reduced by 95% after 3 h incubation at 5 microg/ml. Aggregation of apoB was abolished at the same concentrations. SIN-1 (3-morpholinosydnonimine) produces peroxynitrite via generation of NO and O2-. When LDL was incubated in its presence, a milder oxidation was observed as compared with Cu2+, and AS produced over 70% inhibition. Finally, we show a striking dose-dependent inhibitory effect of lipoxygenase conjugate diene production, which is over 95% at AS concentrations of 5 microg/ml. When compared with other antioxidants, AS effect is greater but in the same order of magnitude than that of ascorbic acid and similar to the popular herbal tea Ilex paraguariensis. In all three systems employed an effect is already substantiated at a concentration of the AS extract of 4 microg/ml, which corresponds to a 1/100 dilution of the preparations usually drunk.     

Immunological responses to oxidized LDL

Considerable evidence now points to an important role for the immune system in experimental models of atherosclerosis. We have reviewed the growing body of evidence that oxidation of LDL generates a wide variety of neoself determinants that lead to cellular and humoral immune responses. In particular, we have demonstrated that at least some of the oxidation-specific epitopes generated on the oxidized LDL particle, such as oxidized phospholipid epitopes, are also generated on apoptotic cells and are also present on the surface of some bacteria. Many of these same epitopes serve as important ligands mediating the binding and clearance of oxidatively damaged lipoprotein particles and apoptotic cells, and the innate immune response to these epitopes can be seen as a conserted response to effect their removal. In addition, other epitopes of OxLDL also undoubtedly play a role in the immune activation that characterizes the progressive atherosclerotic plaque. It will be of great importance to define the importance of the role of these responses and to understand which are beneficial and which deleterious. Such information could lead one day to novel therapeutic approaches to inhibit atherogenesis that take advantage of the ability to manipulate the immune response.     

Simulation of Low Density Lipoprotein (LDL) permeation into multilayer coronary arterial wall: Interactive effects of wall shear stress and fluid-structure interaction in hypertension

Due to increased atherosclerosis-caused mortality, identification of its genesis and development is of great importance. Although, key factors of the origin of the disease is still unknown, it is widely believed that cholesterol particle penetration and accumulation in arterial wall is mainly responsible for further wall thickening and decreased rate of blood flow during a gradual progression. To date, various effective components are recognized whose simultaneous consideration would lead to a more accurate approximation of Low Density Lipoprotein (LDL) distribution within the wall. In this research, a multilayer Fluid-Structure Interaction (FSI) model is studied to simulate the penetration of LDL into the arterial wall. Distention impact on wall properties is taken into account by considering FSI and Wall Shear Stress (WSS) dependent endothelium properties. The results show intensified permeation of LDL whilst the FSI approach is applied. In addition, luminal distension prompted by FSI reduces WSS along lumen/wall interface, especially in hypertension. This effect leads to a lowered endothelial resistance against LDL permeation, comparing to the case in which WSS effect is overlooked. The results are in an acceptable consistency with the clinical researches on WSS effect on atherosclerosis development.     

Endothelial Chlamydia pneumoniae infection promotes oxidation of LDL

The bacterium Chlamydia pneumoniae chronically infects atheromatous lesions and is linked to atherosclerosis by modifying inflammation, proliferation, and the lipid metabolism of blood monocytes. As continuous LDL modification in the vascular intima is crucial for atherogenesis we investigated the impact of endothelial infection on LDL oxidation. HUVEC were infected with a vascular C. pneumoniae strain. Supernatants of infected cells but not cell lysates increased lipid peroxidation products (6.44 vs 6.14 nmol/ml, p<0.05) as determined by thiobarbituric acid reacting substances assay. Moreover, supernatants rendered human LDL more susceptible to oxidation as shown in a copper-ion catalysed LDL oxidation assay by a 16% reduction of LDL resistance against pro-oxidative stimuli (p<0.05). Chlamydial infection of vascular endothelial cells releases acellular components that convert LDL to its proatherogenic form and reduce its resistance against oxidation. Foci of chronic endothelial chlamydial infection may thus continuously contribute to the dysregulated lipid metabolism that promotes atherogenesis.     

Regulation of cell growth by oxidized LDL

The first reports of the influences of oxidized LDL (oxLDL) on cell function pertained to negative effects on cell growth—growth arrest, injury, and toxicity. Since these studies, it has become apparent that sublethal levels of oxLDL cause some, but not all, cells to proliferate. This review highlights the growth-promoting effects of oxLDL rather than its inhibitory or injurious effects. Smooth muscle cells (SMCs) and monocyte-macrophages proliferate after exposure to oxLDL; endothelial cells do not. Scavenger receptors are involved in the proliferative effects on monocyte-macrophages, whereas the effects of oxLDL on SMCs appear to be receptor independent. Lysophosphatidylcholine (lysoPC), and structurally related lipids are among the growth-promoting constituents of oxLDL. OxLDL exerts at least a part of its effects by inducing expression or causing the release of growth factors. OxLDL (or lysoPC) can cause the release of basic fibroblast growth factor (bFGF) from SMCs; oxLDL (or lysoPC) can induce heparin binding EGF-like growth factor (HB-EGF) synthesis and release from macrophages. An imposing array of changes in cytokine and growth factor expression and/or release can be imposed by oxLDL on a wide variety of cell types. These effects and the studies probing the cell signaling events leading to them are described.     

Cell-mediated oxidation of LDL: Comparison of different cell types of the atherosclerotic lesion

The three major cell types of the human atherosclerotic lesion — macrophages (Mø), smooth muscle cells (SMC) and endothelial cells (EC) — were compared for their ability to oxidise low density lipoprotein (LDL) in vitro under identical conditions. Near-confluent cultures were incubated for up to 48 h with 50 μg protein/ml LDL in Ham's F10 medium supplemented with 7 μM Fe²⁺. All three cell types oxidised LDL readily using our culture conditions. After 24 and 48 h, the degree of LDL oxidation was in the order: Mø > SMC > EC when based on cell growth area and EC > SMC > Mø when based on cellular DNA content. However, LDL oxidation in vitro progressed more slowly between 24 and 48 h, probably due to increasing toxicity to the cells and/or depletion of polyunsaturated fatty acids. We therefore compared the time of onset of LDL oxidation. The earliest increase in LDL oxidation was always apparent with SMC. Gas chromatography revealed that LDL oxidation by all three cell types followed a similar pattern. The polyunsaturated fatty acids linoleic acid (18:2) and arachidonic acid (20:4) were depleted (to 10.3–18.1% and 4.5–24.7% respectively, compared to native LDL), whereas the content of stearic acid (18:0) and oleic acid (18:1) remained unchanged. Cholesterol was depleted (to 54.1–75.6% of native LDL) with a concomitant rise in 7β-hydroxycholesterol (to 60.6–128.1 μg/mg LDL). This corresponds to a conversion of 4.9, 9.5 and 10.4% of LDL cholesterol in EC-, SMC- and Mø-modified LDL respectively. All three cell types showed significant toxicity in the oxidising culture after 24 h. The possible relevance to LDL oxidation in atherosclerosis is discussed.