Oxidatively modified low density lipoproteins: a potential role in recruitment and retention of monocyte/macrophages during atherogenesis.

Previous studies in this laboratory established that low density lipoprotein (LDL) incubated with cultured endothelial cells, smooth muscle cells, or macrophages undergoes free radical-catalyzed oxidative modification that generates lipid peroxides and extensive structural changes in the LDL molecule. The oxidatively modified LDL strongly inhibited chemotactic responses of the mouse resident peritoneal macrophage. The present studies show that this oxidized LDL does not inhibit the motility of mouse monocytes and actually exhibits a chemotactic activity for human monocytes; the chemotactic activity of the oxidized LDL resides in the lipid fraction. These findings allow us to propose a pathogenetic sequence by which elevated plasma LDL levels, followed by oxidative modification in the arterial wall, could sufficiently account for the generation of the lipid-laden foam cells and the initiation of the fatty streak, the earliest well-defined lesion in atherogenesis.     

氧化低密度和极低密度脂蛋白对单核细胞的趋化性研究

用改良的Boyden小室微孔滤膜法检测低密度脂蛋白、极低密度脂蛋白及氧化低密度脂蛋白和极低密度脂蛋白对人血单核细胞的趋化作用。结果表明,低密度脂蛋白对单核细胞具有趋化活性,而且当其被氧化后,对单核细胞的趋化活性明显增强。极低密度脂蛋白对单核细胞无趋化活性,而氧化极低密度脂蛋白对单核细胞有明显的趋化活性。     

Endothelial cell-derived chemotactic activity for mouse peritoneal macrophages and the effects of modified forms of low density lipoprotein.

Cultured rabbit and bovine aortic endothelial cells generated chemotactic activity for mouse resident peritoneal macrophages, demonstrable in the conditioned medium. This chemotactic activity was heat stable and was not extracted into chloroform/methanol. It was inhibited by addition of endothelial cell-modified low density lipoprotein (EC-modified LDL), a form of LDL shown previously to contain peroxidized lipids, increased lysophosphatidylcholine, and partially degraded apoprotein B. The chemotactic activity was also inhibited by LDL previously oxidized in the absence of cells with 5 microM Cu2+. Inhibitory activity was present in the lipid extract of EC-modified LDL but not in that of native LDL, presumably representing peroxidized lipid components. EC-modified LDL also inhibited the chemotactic activity of zymosan-activated serum. Because EC-modified LDL is taken up in part by way of the acetyl-LDL receptor, the effects of acetyl-LDL were tested. Rather than inhibiting chemotaxis, acetyl LDL showed intrinsic positive chemotactic activity as did also fucoidin and polyinosinic acid, both of which also interact with the acetyl-LDL receptor. These studies suggest mechanisms by which macrophages may be recruited into the subendothelial space by endothelial cell-derived chemotactic factors or by natural polyanions structurally related to fucoidin or polyinosinic acid and then become "trapped" there because of the inhibitory effects of peroxidized lipid components in modified forms of LDL.     

Oxidized low density lipoprotein induces differentiation and adhesion of human monocytes and the monocytic cell line U937.

Hypercholesterolemia is a major risk factor for development of atherosclerosis. In experimental animals fed a high-cholesterol diet, monocytes adhere to the arterial endothelium and penetrate into the intima where they differentiate into macrophages and ingest lipids thus giving rise to fatty streaks, the earliest type of atherosclerotic plaque. Macrophages express few receptors for normal low density lipoprotein (LDL) but can take up oxidized LDL by way of a scavenger receptor. The present study was designed to investigate the possible role of oxidized LDL in recruitment of resident intimal macrophages. We found that oxidized LDL induced enhanced expression of major histocompatibility complex class II molecules on human monocytes and U937 cells, a well-established system for studies of monocytic differentiation. Oxidized LDL also induced enhanced expression of the surface antigen LeuM3 but caused decreased expression of CD4 antigen, a pattern compatible with expression of a more-differentiated macrophage-like phenotype. Oxidized LDL also initiated aggregation of monocytes and U937 cells and stimulated adhesion of U937 cells to cultured endothelial cells. The results indicate that oxidized LDL may contribute to development of atherosclerosis by inducing adhesion of monocytes to the arterial intima and by stimulating intimal monocytes to differentiate into resident macrophages.     

Stimulation of phospholipase D activity by oxidized LDL in mouse peritoneal macrophages

Oxidation of LDL is an important factor in the development of atherosclerosis. However, the mechanisms by which oxidized LDL exerts its atherogenic actions are poorly understood. In the present work, we show that oxidized LDL stimulates phospholipase D (PLD) activity in mouse peritoneal macrophages and that this effect increases with the degree of LDL oxidation. Oxidative modification of LDL results in the production of lipid peroxides and the conversion of phosphatidylcholine to lysophosphatidylcholine. Although we found that lysophosphatidylcholine alone activates PLD, the stimulation of this enzyme activity by oxidized LDL is independent of lysophosphatidylcholine formation. Also, 7-ketocholesterol, the major oxysterol in oxidized LDL, failed to stimulate PLD activity. To determine the mechanism(s) whereby oxidized LDL activates PLD, the possible involvements of protein kinase C and tyrosine phosphorylation were investigated. Pretreatment of macrophages with the protein kinase C inhibitor Ro-32-0432 or downregulation of protein kinase C activity by prolonged incubation with 100 nmol/L 4beta-phorbol 12-myristate 13-acetate did not alter the stimulatory effect of oxidized LDL on PLD activation. However, oxidized LDL stimulated tyrosine phosphorylation of several macrophage proteins, and preincubation of the macrophages with genistein, a tyrosine kinase inhibitor, blocked the activation of PLD by oxidized LDL. In addition, pretreatment with orthovanadate, which inhibits tyrosine phosphatases, enhanced basal and oxidized LDL-stimulated PLD activity. Pretreatment of macrophages with pertussis toxin decreased the stimulatory effect of oxidized LDL, indicating that GTP-binding proteins may also be involved in the activation of PLD by oxidized LDL. We also found that the platelet-activating factor receptor antagonists WEB 2086 and L-659,989 inhibit the oxidized LDL stimulation of PLD, suggesting a role for platelet-activating factor receptor in this process. The stimulation of the PLD pathway by oxidized LDL may be of importance in atherogenesis, because PLD activation leads to generation of important second messengers such as phosphatidate, lysophosphatidate, and diacylglycerol, which are known to regulate many cellular functions.     

Effects of cardiac natriuretic peptides on oxidized low-density lipoprotein- and lysophosphatidylcholine-induced human mesangial cell migration

The objectives of the present study were (1) to determine whether oxidized LDL and lysophosphatidylcholine (lyso-PtdCho), a major phospholipid component of oxidized LDL, stimulate the migration of cultured human mesangial cells and (2) to investigate the possible effects on mesangial cell migration of the cardiac natriuretic peptides atrial and brain natriuretic peptide (ANP and BNP). Oxidized LDL (10 and 100 microg/mL) and lyso-PtdCho (10(-7) to 10(-5) mol/L) stimulated migration in a concentration-dependent manner. In contrast, the effects of native LDL and phosphatidylcholine were modest or nonexistent. Protein kinase C (PKC) inhibitor and downregulation of PKC activity by phorbol ester inhibited oxidized LDL- and lyso-PtdCho-induced migration. Human ANP(1-28) and human BNP-32 significantly inhibited oxidized LDL- and lyso-PtdCho-induced migration in a concentration-dependent manner. C-ANF (des-[Glu(18),Ser(19),Gly(20),Leu(21),Gly(22)]ANP(4-23)), a specific ligand for ANP clearance receptors, could not inhibit oxidized LDL- and lyso-PtdCho-induced migration. Inhibition by ANP and BNP of lyso-PtdCho-induced migration was paralleled by an increase in the cellular level of GMP. Oxidized LDL- and lyso-PtdCho-induced migrations were inhibited by 8-bromo-cGMP. The results suggest that oxidized LDL and lyso-PtdCho stimulate the migration of human mesangial cells, at least in part, through a PKC-dependent process and that ANP and BNP inhibit this stimulated migration, probably through a cGMP-dependent process.     

Ligands for peroxisome proliferator-activated receptor inhibit monocyte CCR2 expression stimulated by plasma lipoproteins

Substantial evidence supports a causal role for monocyte chemoattractant protein-1 (MCP-1) and its receptor, CCR2, in the recruitment of monocytes from the circulation into atherosclerotic lesions. MCP-1 is produced and secreted by virtually every cellular component of the vessel wall. It generally is assumed that the magnitude of the monocyte chemotactic activity, which is initiated by the functional activation of CCR2 by MCP-1, is directly proportional to the concentration of the chemoattractant. However, we recently demonstrated that an inflammatory response of monocytes is finely regulated and also depends on the expression levels of CCR2. We identified plasma low-density lipoprotein (LDL) as a positive regulator and showed that it greatly increased monocyte CCR2 gene expression. In contrast, activation of peroxisome proliferator-activated receptor by synthetic ligands or components of oxidized LDL reduces monocyte CCR2 expression and blocks chemotaxis mediated by MCP-1. We hypothesized that the excessive monocyte accumulation in the vessel wall during atherogenesis may result in part from an enhanced chemotactic response. These findings suggest CCR2 gene expression in circulating monocytes as a potential additional target for intervention and prevention of atherosclerosis.     

Phospholipase A2 activity of low density lipoprotein: evidence for an intrinsic phospholipase A2 activity of apoprotein B-100.

During oxidative modification of low density lipoprotein (LDL) there is extensive degradation of phosphatidylcholine (PtdCho) to lysophosphatidylcholine (lyso-PtdCho), with the removal of fatty acids from the 2 position. The phospholipase A2 (PLA2) activity responsible for hydrolysis is closely associated with LDL. By use of lipoxygenase-oxidized 2-[1-14C]linoleoyl PtdCho as the substrate and delipidated apoprotein B (apo-B), evidence is presented to show that (i) the activity is destroyed progressively during the oxidative modification of LDL; (ii) p-bromophenacyl bromide (pBPB), a histidine modifier that inhibits the oxidative modification of LDL, also substantially inhibits the PLA2 activity; and (iii) photooxidation of LDL in the presence of Rose Bengal completely inactivates the enzyme with concomitant loss of apo-B histidine residues. High molecular weight proteins from delipidated LDL, separated by polyacrylamide gel electrophoresis, showed PLA2 activity. It is suggested that apo-B itself may possess PLA2 activity.     

Identification of cyclophilin as a proinflammatory secretory product of lipopolysaccharide-activated macrophages.

We have isolated an 18-kDa peptide (designated sp18, for 18-kDa secreted protein) from the conditioned medium of lipopolysaccharide-stimulated RAW 264.7 murine macrophages. Purified sp18 had in vivo inflammatory activity and in vitro chemotactic activity for human peripheral blood polymorphonuclear leukocytes and monocytes. Surprisingly, N-terminal sequencing and tryptic mapping studies revealed that sp18 and cyclophilin, an intracellular protein that binds the immunosuppressive drug cyclosporin A, are highly homologous. The in vitro chemotactic activity of sp18 on monocytes was blocked by cyclosporin A but not by cyclosporin H, a structural analog of cyclosporin A that does not bind cyclophilin. Like purified porcine cyclophilin, mouse sp18 exhibited peptidyl-prolyl cis-trans isomerase activity. Medium conditioned by lipopolysaccharide-stimulated resident peritoneal exudate macrophages isolated from C57BL/6 mice contained substantially higher levels of sp18/cyclophilin than medium conditioned by nonstimulated macrophages. The observation that sp18/cyclophilin exhibits proinflammatory activity and is secreted by macrophages in response to endotoxin suggests that this protein may function as a cytokine, and invites the hypothesis that the immunosuppressive action of cyclosporin A results in part from interaction with an extracellular form of cyclophilin released as a mediator of immune and inflammatory functions.     

Relationship between atherosclerosis and the aging process

On this paper, author reviews studies on the relationship between atherogenesis and aging. An increase in serum lipid peroxide level increased the number of circulating vascular endothelial cells. This indicates that the free radical is an injury factor for endothelial cells. In addition macrophages discharged the active oxygen upon stimulation and incorporated oxidized low density lipoprotein. These results suggested that macrophages play a main role in the initiation of atheroma formation. A very small dose of H2O2 induced platelet aggregation in vitro. Thrombin has chemotactic and chemokinetic activity for the smooth muscle cell as does fibrinogen, fibronectin and oxidized LDL. Conclusion. These results suggest close association through free radicals between the aging process and atherogenesis.     

Lovastatin decreases the receptor-mediated degradation of acetylated and oxidized LDLs in human blood monocytes during the early stage of differentiation into macrophages.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors are used therapeutically to upregulate the LDL receptor-mediated removal of plasma cholesterol by the liver. Several lines of evidence indicate that these drugs also exert direct effects on the metabolism of native and modified LDL in extrahepatic cells. We studied the effects of lovastatin (LOV) on the degradation of native, acetylated, and oxidized LDL, and on levels of mRNA encoding for the LDL, types I and II class A macrophage scavenger, and CD36 receptors in human blood monocytes at different stages of their maturation into adherent macrophages. LOV (10 micromol/L) reduced the degradation of acetylated LDL when added to freshly isolated cells cultured for 2 (81+/-4% of control, P<0.05) and 5 (76+/-6%, of control, P<0.05) days. The degradation of oxidized LDL was also reduced in cells treated with LOV for 2 days after seeding (51+/-3% of control, P<0. 001) but not in 5-day-old cells. LOV had no significant effect on the degradation of either acetylated or oxidized LDL when added to fully matured macrophages allowed to differentiate under control conditions for 7 days before incubations with 10 micromol/L LOV for an additional 2 days. In contrast, LOV increased the degradation of native LDL in these cells at all 3 stages of cell differentiation. LOV also reduced class A types I and II macrophage scavenger receptor and CD36 mRNA levels in 2- and 5-day-old cells but not in the more mature macrophages. These data suggest that 3-hydroxy-3-methylglutaryl-coenzyme A inhibitors may reduce the expression and function of the class A types I and II macrophage scavenger receptor and CD36 in monocytes, during the early stages of their differentiation into adherent macrophages. These effects, if operative in vivo, may slow down the development of the atherosclerotic plaque and thus contribute to the beneficial effects of these drugs.     

Enhanced procoagulatory activity (PCA) of human monocytes/macrophages after in vitro stimulation with chemically modified LDL

In cultured human monocytes/macrophages, surface expression of procoagulatory activity (PCA) was induced by chemically modified LDL (acetyl-LDL and MDA-LDL) in a dose- and time-dependent manner. Maximum PCA (30-fold increase) was detected after 24 h of culture with modified LDL at doses of 25-750 micrograms protein/ml. Using factor VII deficient human plasma and phospholipase C this PCA was identified as tissue thromboplastin activity (factor III). These results suggest a further atherogenic potential for modified LDL through stimulation of the conversion of fibrinogen to fibrin in the atheromatous lesion.     

Colocalization of 15-lipoxygenase mRNA and protein with epitopes of oxidized low density lipoprotein in macrophage-rich areas of atherosclerotic lesions.

Oxidation of low density lipoprotein (LDL) enhances its atherogenicity, and inhibition of such oxidation decreases the rate of progression of atherosclerotic lesions. The mechanism of LDL oxidation in vivo remains uncertain, but in vitro studies have suggested that cellular lipoxygenases may play a role by initiating lipid peroxidation in LDL. In situ hybridization studies using a 15-lipoxygenase riboprobe and immunostaining using antibodies against 15-lipoxygenase showed strongly positive reactivity largely confined to macrophage-rich areas of atherosclerotic lesions. Polymerase chain reaction with 15-lipoxygenase-specific oligonucleotides and restriction enzyme digestions of the amplified fragment were used to confirm the presence of 15-lipoxygenase message in the reverse-transcribed lesion mRNA. Immunostaining with antibodies reactive with oxidized LDL (but not with native LDL) indicated that the lipoxygenase colocalizes with epitopes of oxidized LDL, compatible with a role for macrophage lipoxygenase in the oxidation of LDL in vivo. Since oxidized LDL is chemotactic for blood monocytes, early lesions might progress at a markedly accelerated rate because of further recruitment of more monocytes which, in turn, would increase further the rate of oxidation of LDL. These data suggest that therapy targeted to block macrophage lipoxygenase activity might decrease the rate of development of atherosclerotic lesions.     

Monocyte migration explains the changes in macrophage arachidonate metabolism during the immune response.

The profile of arachidonic acid metabolites in resident peritoneal macrophages is distinctly different from the profile of macrophages isolated after an acute bacterial infection. The latter produce decreased prostaglandins E2 and I2 and leukotriene C4 while conserving the synthesis of thromboxane A2. We show here that the initial changes in peritoneal macrophage arachidonate metabolism during the immune response appear to be the result of the large influx of blood monocytes, which have a characteristic metabolism distinct from resident macrophages. We demonstrate that the initial decrease in peritoneal macrophage arachidonate metabolism and the increase in macrophage numbers occur simultaneously after infection with Listeria monocytogenes. Also the macrophage arachidonate metabolism seen at the height of the peritoneal cellular influx is the same as that of purified blood monocytes. Both Listeria peritoneal macrophages and blood monocytes produce equal or greater quantities of thromboxane A2 relative to prostaglandins I2 and E2 or leukotriene C4 whereas resident cells produce 1/10 to 1/25 as much thromboxane A2 compared to the other products. Furthermore, the changes in peritoneal macrophage arachidonate metabolism in response to Listeria infection do not occur if the influx of blood monocytes is stopped by irradiating the mice prior to infection implying that the cellular influx is necessary to see the changes in arachidonate metabolism. Finally, activation of peritoneal macrophages, measured as an increase in Ia expression, occurs 36 hr after the influx of monocytes from the blood and the resultant shift in arachidonate metabolism during Listeria infection.     

脂蛋白相关磷脂酶A2在颈动脉粥样硬化和缺血性卒中中的作用

脂蛋白相关磷脂酶A2(lipoprotein-associated phospholipase A2,Lp-PLA2)是磷脂酶A2超家族的一个亚型,主要由巨噬细胞和淋巴细胞产生.Lp-PLA2选择性水解低密度脂蛋白颗粒表面的氧化型磷脂,产生溶血磷脂胆碱和氧化型游离脂肪酸.Lp-PLA2表达于动脉粥样硬化斑块和不稳定斑块纤维帽内的巨噬细胞.研究表明,缺血性卒中患者血浆Lp-PLA2活性显著增高,而Lg-PLA2可能成为预测缺血性脑血管事件的独立危险因素.选择性LP-PLA2抑制剂可减轻炎症反应,增强斑块稳定性,抑制动脉粥样硬化斑块形成,有可能成为抗动脉粥样硬化斑块形成的一类新型药物.     

心肌缺血预适应对缩小心肌梗塞范围的临床研究

用培养的兔腹腔巨噬细胞条件培养基作为趋化因子的来源，用改良的Ｂｏｙｄｅｎ小室微孔滤膜法进行单核细胞趋化试验，观察了兔腹腔巨噬细胞条件培养基对单核细胞的趋化作用和抗单核细胞趋化蛋白－１（ｍｏｎｏｃｙｔｅｃｈｅｍｏａｔｔｒａｃｔａｎｔｐｒｏｔｅｉｎ－１，ＭＣＰ－１）抗体对单核细胞迁移的影响。同时用Ｎｏｒｔｈｅｒｎｂｌｏｔ分析方法检测了ＭＣＰ－１ｍＲＮＡ在该巨噬细胞的表达。结果显示，该条件培养基对单核细胞有明显的趋化作用，并被抗ＭＣＰ－１抗体所抑制。同时该巨噬细胞也能表达ＭＣＰ－１ｍＲＮＡ。这提示，巨噬细胞能分泌ＭＣＰ－１，并招引单核细胞迁入内皮下间隙，从而在动脉粥样硬化的发病过程中发挥重要的作用。     

家兔腹腔巨噬细胞能产生单核细胞趋化蛋白-1

用培养的兔腹有空巨噬细胞条件培养基作为趋化因子的来源，用改良的Ｂｏｙｄｅｎ小室微孔滤膜法进行单核细胞趋化试验，观察了兔腹腔巨噬细胞条件培养基对单核细胞的趋化作用和抗单核细胞趋化蛋白－１抗体对单核细胞迁移的影响，同时用Ｎｏｒｔｈｅｒｎ ｂｌｏｔ分析方法检测了ＭＣＰ－１ ｍＲＮＡ在该巨噬细胞的表达。结果显示，该条件培养基对单核细胞有明显的趋化作用，并被抗ＭＣＰ－１抗体所抑制，同时该巨噬细胞也能表达ＭＣ     

心肌缺血预适应对缩小心肌梗塞范围的临床研究

近年动物试验证明心肌缺血预适应有一种保护作用。本组分析了近３年我院急性前壁心肌梗塞１１０例病人缺血预适应对心肌梗塞的影响。其中男７７例，女３３例。心肌梗塞前有预缺血者６９例（Ａ组），无预缺血者４１例（Ｂ组）。资料分析显示：Ｂ组心肌梗塞时心肌坏死范围、心肌酶峰值、心功能损害、室壁瘤形成及死亡率均高于Ａ组，两组差异有显著性。还就心肌缺血预适应发生时间及其对心肌保护作用之间的关系，缺血预适应的可能机制及临床意义进行了讨论。