The dynamics of macrophage infiltration into the arterial wall during atherosclerotic lesion development in low-density lipoprotein receptor knockout mice

Atherosclerosis is a progressive disease in which macrophages play an essential role. Macrophage infiltration into the arterial wall induces the development of an early atherosclerotic lesion. However, the dynamics of macrophage infiltration into the arterial wall during lesion progression remain poorly understood. In this study, low-density lipoprotein receptor knockout mice were fed a Western-type diet for 3, 6, 9, and 12 weeks to induce the formation of atherosclerotic lesions with different degrees of complexity. Subsequently, these mice underwent transplantation with bone marrow-overexpressing enhanced green fluorescent protein to track donor-derived cells, including macrophages. After 8 weeks of Western-type diet feeding after transplantation, macrophage infiltration was evaluated by immunohistochemical staining of donor-derived macrophages (enhanced green fluorescent protein-positive F4/80(+)) in the aortic roots. We found that the growth of pre-existing initial lesions was mainly caused by continued recruitment of donor-derived macrophages into the arterial wall. Interestingly, macrophage infiltration into pre-existing more advanced lesions was largely impaired, likely because of the formation of fibrous caps. In addition, interference with the expression of macrophage ATP-binding cassette transporter 1, an ATP-binding cassette transporter involved in cellular cholesterol efflux and macrophage recruitment into tissues, affects the infiltration of macrophages into pre-existing early lesions but not into advanced lesions. In conclusion, our data suggest that the dynamics of macrophage infiltration into the arterial wall vary greatly during atherogenesis and, thus, may affect the efficiency of pharmaceutical interventions aimed at targeting macrophage infiltration into the arterial wall.     

血管平滑肌细胞凋亡在血管再狭窄中作用的研究进展

血管平滑肌细胞构成新生内膜增生的重要部分,且在血管腔内治疗术后再狭窄的心血管疾病的发生和发展中具有重要作用。血管平滑肌细胞凋亡能有效抑制血管球囊损伤和血管旁路移植术后新生内膜增生,从而可为血管术后再狭窄提供治疗手段。     

Effects of native and oxidized low-density lipoproteins on macrophage chemiluminescence

Effects of low-density lipoproteins (LDL) obtained from healthy donors and patients with hypercholesterolemia on spontaneous luminol-dependent and zymosan-induced chemiluminescence of rat macrophages were studied. Unlike LDL from healthy donors, native LDL from patients with hypercholesterolemia inhibited spontaneous chemiluminescence of macrophages. Simultaneous incubation with endotheliocytes from the umbilical vein led to the appearance of inhibitory effect of LDL from healthy donors (incubation for 24 h) and potentiated this effect of LDL from patients with hypercholesterolemia (incubation for 6 and 24 h). The inhibitory effect was more pronounced in LDL incubated with umbilical endotheliocytes under ischemic conditions then after aerobic incubations. This corresponded to higher oxidation of LDL confirmed by accumulation of thiobarbituric acid-reactive substances, increased fluorescence, and high electrophoretic mobility in agarose gel. These data suggest that the model system of spontaneous and zymosan-induced chemiluminescence of macrophages can be used for evaluating the degree of oxidation and potential atherogenicity of LDL. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 11, pp. 514–517, November, 1999     

Oxidized phospholipids in oxidized low-density lipoprotein reduce the activity of tissue factor pathway inhibitor through association with its carboxy-terminal region

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that inhibits the initial reactions of blood coagulation. In this study, we explored the nature of active components that reduce the anticoagulant activity of TFPI in oxidized low-density lipoprotein (ox-LDL). The organic solvent-soluble fraction obtained from ox-LDL was fractionated by normal-phase HPLC. The binding profile of each fraction to TFPI showed a single peak eluting near purified oxidized phospholipid. To explore further the components in oxidized phospholipid that inhibit TFPI activity, we used oxidized phospholipids that mimic the biological activity of ox-LDL. The oxidation products of 1- and/or 2-oleoyl phosphatidylcholine or phosphatidylethanolamine were the most potent inhibitors of TFPI activity, whereas those of arachidonyl phosphatidylcholine possessed only a weak inhibitory effect on the TFPI activity. These oxidized phospholipids mainly associated with the C-terminal basic region of the TFPI molecule. The results indicate that oxidation products of delta-9 unsaturated phospholipids are candidate active components of ox-LDL that impair the function of TFPI through specific association with its C-terminal basic region.     

Plasma oxidized low-density lipoprotein levels and risk of Alzheimer's disease

This study examines the association of plasma oxidized low-density lipoprotein (OxLDL) levels with all-cause dementia, including Alzheimer's disease (AD) and vascular dementia. Data are taken from the Canadian Study of Health and Aging, a population-based study of a representative sample of persons aged more than 65 years conducted from 1991 to 2002. The present study sample included 670 subjects of which, 155 developed all-cause dementia with 109 cases of AD and 32 of vascular dementia. In Cox regression models, no association between OxLDL and risks of dementia or subtypes was found. A triple interaction between OxLDL, sex, and history of cardiovascular disease on the risk of AD (p = 0.0077) was found. Increased levels of OxLDL were significantly associated with an increased risk of AD in men with a history of cardiovascular disease (hazard ratio = 1.11; 95% confidence interval 1.04–1.19); no association in women was found. These findings suggest that increased levels of OxLDL are not associated with the risk of dementia, AD, and vascular dementia. The association of OxLDL with AD in men with a history of cardiovascular disease merits further investigation.     

雷帕霉素对氧化低密度脂蛋白刺激血管平滑肌细胞信号转导的抑制作用

目的 探讨氧化低密度脂蛋白(oxLDL)促血管平滑肌细胞(VSMC)增殖及分泌功能改变的细胞内信号转导机制及雷帕霉素(rapamycin)干预作用。方法 培养兔血管平滑肌细胞分为7组处理。以直接细胞计数及噻唑盐(MTT)比色法测定细胞增殖能力；酶联免疫法检测肿瘤坏死因子α(TNF-α)、白细胞介素-6(IL-6)、白细胞介素-8(IL-8)水平；Western免疫印迹法定量蛋白激酶B(PKB)表达水平；免疫沉淀、特异底物组蛋白H2Bγ^32P掺入量测定PKB活性。结果 oxLDL使细胞增殖指标增加1．62～3．2倍，细胞因子分泌增加3～5倍，PKB活性增加11．8倍。而相同浓度的低密度脂蛋白对细胞增殖及细胞因子分泌无明显影响。磷脂酰肌醇3-激酶(PI3K)特异性抑制剂沃漫青霉素(Wortrmannin．WT)及雷帕霉素均可显著抑制VSMC增殖、细胞因子分泌及PKB活性，并完全逆转oxLDL的上述作用。结论 PI3K／PKB是oxLDL促VSMC增殖及分泌功能的信号通路之一。雷帕霉素可能通过抑制PI2K／PKB对VSMC生物学功能发挥较全面调控效用。     

Serum antibodies to oxidized low-density lipoprotein and ceroid in chronic periaortitis

The incidence of serum antibodies to human low-density lipoprotein, to oxidized low-density lipoprotein, and to ceroid extracted from human atheroma was assessed in 100 subjects using an adaptation of the enzyme-linked immunosorbent assay technique. Patients with chronic periaortitis, subclinical chronic periaortitis, and ischemic heart disease, and "elderly control" individuals were compared with young, healthy adults. Provided that precautions were taken to prevent oxidation of the low-density lipoprotein during the assay, antibodies were not found to native human low-density lipoprotein. Antibodies to oxidized low-density lipoprotein or ceroid, usually both, were detected in all 20 patients with clinical chronic periaortitis, in 17 of 20 patients with subclinical chronic periaortitis, in 12 of 20 patients with ischemic heart disease, and in 10 of 20 elderly control subjects. Binding inhibition studies showed cross-reactions between oxidized low-density lipoprotein and ceroid. Western blotting after sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that in some patients with clinical chronic periaortitis, these antibodies were directed against breakdown products of apolipoprotein B that resulted from oxidation of low-density lipoprotein. Antibodies to oxidized low-density lipoprotein or ceroid were not detected in healthy young adults. These findings show that chronic periaortitis is accompanied by autoallergy to ceroid, which is probably at least partly composed of low-density lipoprotein oxidized within the human atherosclerotic plaque, and that a number of middle-aged and elderly people without chronic periaortitis also have such antibodies.     

Impairment of endothelium-dependent aorta relaxation by phospholipid components of oxidized low-density lipoprotein.

Oxidized low-density lipoprotein (LDL) is a major component in the pathophysiology of atherosclerosis and plays a role in the changes of vascular reactivity observed in this disease. Herein the authors investigate the potential involvement of platelet-activating factor (PAF)-like phospholipid components of oxidized LDL in rabbit aorta reactivity. Aortic rings were precontracted with noradrenaline (0.5 microM) and relaxation was induced by subsequent stimulation with sequential additions of acetylcholine (1 nM to 3 microM). High-performance liquid chromatography (HPLC) fractions (6- and 7-min) obtained from phospholipids extracted from oxidized LDL inhibited relaxation evoked by acetylcholine, but not the relaxation induced by sodium nitroprusside. This effect was not antagonized either by incubation of the fractions with PAF acetylhydrolase or by incubation of the aortic rings with a PAF receptor antagonist. Authentic PAF or C4-PAF, a PAF mimetic previously found in fractions 6 and 7 did not inhibit acetylcholine-induced relaxation. In contrast, lyso-PAF inhibited acetylcholine, but not sodium nitroprusside-induced relaxation. The authors conclude that phospholipids of oxidized LDL impair vascular reactivity to endothelium-dependent agonists. This effect is not due to oxidatively generated proinflammatory PAF mimetics, but rather to a metabolite of these phospholipids, lysoPAF.     

Rethinking oxidized low-density lipoprotein, its role in atherogenesis and the immune responses associated with it

Atherosclerosis is a chronic inflammatory disease, resulting from hyperlipidemia and a complex interplay of many environmental, metabolic, and genetic risk factors. The unregulated macrophage uptake of cholesterol and lipids through modified forms of low-density lipoprotein (LDL), such as "OxLDL", transforms macrophages into "foam cells" to form the initial morphological lesion (the fatty streak). The modification of LDL not only enhances its uptake by macrophages, but also changes the natural structures of these otherwise ubiquitous molecules to generate a variety of modified lipids and proteins that represent highly immunogenic neo-determinants. For example, in ApoE-/- mice, autoantibody titers to epitopes on OxLDL are correlated with the extent of atherosclerosis. Similarly, oxidative stress on cellular membranes could also give rise to "oxidation-specific" epitopes and common autoantibodies. However, OxLDL is not uniform, but rather contains complex structures, ranging from a small conformational change in surface lipids to the breakdown of the peptide chain. Therefore, the immune responses to the variety of OxLDL and their association to atherosclerosis progression are very different. For example, phosphorylcholine (PC) is a natural component of phospholipids and exists in LDL and plasma membranes. "Natural" antibodies against PC can distinctively react to PC on bacteria, OxLDL and apoptotic cells, but not to those on unoxidized phospholipids, native LDL and viable cells, which suggests the broader role of such autoantibodies in maintaining the homeostasis of the host. While malondialdehyde-modified structures resemble more the exogenous changes and associate with advanced stage of lesion, they are more likely to associate with adaptive immunity.     

氧化低密度脂蛋白诱导巨噬细胞泡沫化氧化损伤后血管紧张素转化酶2的表达变化及意义

目的观察氧化低密度脂蛋白(ox-LDL)诱导巨噬细胞泡沫化后血管紧张素转化酶2(ACE2)的表达变化与氧化应激损伤的关系,探讨ACE2的抗氧化作用。方法用不同浓度ox-LDL诱导小鼠巨噬细胞RAW 264.7泡沫化,油红O染色观察巨噬细胞内脂质堆积变化,并定量分析细胞内脂滴含量,通过检测细胞上清中丙二醛(MDA)含量鉴定细胞氧化损伤程度,实时定量聚合酶链反应(Real-time PCR)检测ACE2及MAS m RNA表达,化学比色法检测超氧化物歧化酶(SOD)活性与还原型谷胱甘肽(GSH)的含量。结果不同浓度(20、40、60、80mg/L)ox-LDL处理巨噬细胞24 h,油红O染色可见巨噬细胞变大、变圆,胞质内脂滴含量呈剂量依赖性增加;用60mg/L ox-LDL诱导巨噬细胞泡沫化模型组中MDA含量较对照组明显升高(P0.01);Real-time PCR结果显示,模型组ACE2和MAS m RNA表达水平较对照组显著减少(P0.05);与对照组比较,模型组中SOD活性与GSH含量显著降低(P0.05或P0.01)。结论巨噬细胞泡沫化氧化损伤进程与ACE2及其下游受体MAS表达下调密切相关,提示ACE2具有一定的抗氧化作用,其机制可能与SOD及GSH有关。     

Minimally oxidized low-density lipoprotein induces tissue factor expression in cultured human endothelial cells.

Oxidatively modified low-density lipoprotein is present in atherosclerotic lesions and has been proposed to play an important role in atherogenesis through its biologic effects on vascular cells. This study examined the effects of minimally oxidized preparations of LDL (MM-LDL) on tissue factor (TF) expression by cultured human endothelial cells. Low-density lipoprotein purified from normal donors was modified by exposure to iron or by prolonged storage, resulting in levels of thiobarbituric acid-reacting substances of approximately 2.5 to 4 nmoles/mg cholesterol. Preparations had less than 2.5 pg of endotoxin per microgram LDL and had no intrinsic procoagulant activity. This form of modified but not native LDL induced TF expression in endothelial cells in a time- and dose-dependent manner. Peak TF coagulant activity in cells exposed to 40 micrograms/ml MM-LDL were observed at 4 to 6 hours, and ranged from 50 to 500 pg/10(5) cells, compared with less than 10 pg/10(5) cells exposed to native LDL. Northern blot analysis showed TF mRNA levels to increase approximately 30-fold with exposure to MM-LDL for 2 hours. Induction of TF activity was dependent on the concentration of MM-LDL from 1 microgram/ml to 80 micrograms/ml, a range in which cell viability and morphology were unaffected. The findings suggest that minimally oxidized LDL may be a local mediator promoting thrombosis in atherosclerotic lesions.     

The influence of C57B1/6J mice immunization by oxidized low-density lipoprotein on the intensity of atherosclerotic changes

ApoE knockout C57B1/6J mice fed Western type diet were immunized by MDA-modified human LDL. After 8 weeks in 50% immunized mice the atherosclerotic lesions in aorta was absent whereas both in control group and in 50% immunized mice the region of atherosclerotic lesions was in average 6,6%. A positive correlation between area of lesions and level of antilipoprotein antibodies IgG2b was founded.     

Serum oxidized low-density lipoprotein level and risk of cognitive impairment in older women

We investigated the association between serum level of oxidized low-density lipoprotein (oxLDL) and risk of cognitive impairment (dementia or mild cognitive impairment) among 572 nondemented community-dwelling women from a prospective cohort study of aging. After 5 years of follow-up, 228 (39.9%) developed cognitive impairment; and this did not differ by tertile of baseline oxLDL level (highest compared with lowest tertile 38.2% vs. 39.5%; odds ratio, 0.90; 95% confidence interval, 0.63–1.43). Multivariate adjustment produced similar results (odds ratio, 0.91; 95% confidence interval, 0.60–1.39). These findings suggest that increased levels of serum oxLDL are not associated with a greater risk of incident cognitive impairment in older women.     

Foam cell replication and smooth muscle cell apoptosis in human saphenous vein grafts

Occlusion of saphenous vein grafts is a major problem after coronary artery bypass grafting. Segments of occluded and suboccluded implanted aortocoronary grafts were obtained during re-intervention bypass grafting in 47 patients yielding a total of 80 vein grafts. The grafts were studied by immunohistochemistry for smooth muscle cells (ÉL-SMC actin), macrophages (HAM56), cell replication (PCNA, Ki-67) and transmission and scanning electronmicroscopy (TEM, SEM). In 81% of the examined grafts the (sub)occlusion was due to a myo-intimal thickening and an associated luminal accumulation of foam cells and mural thrombi. The foam cells were constantly found at the luminal site of the myo-intimal thickening and within the luminal part of adherent thrombi. Transmission electronmicroscopy demonstrated phagocytosis of platelets and platelet fragments by the foam cells. A significant fraction of the foam cells demonstrated nuclear immunoreactivity for Ki-67 and PCNA. The myo-intimal thickening of the vein grafts was composed of smooth muscle cells lying in a fibrous tissue matrix. The smooth muscle cells were surrounded by prominent basal lamina and showed ultrastructural features of apoptosis. Our results support the hypothesis that phagocytosis of lipid rich platelets by monocytes set up a mechanism for foam cell formation and replication in human saphenous vein grafts. The transformation of a smooth muscle cell rich myo-intimal thickening towards a fibrous, cell poor intimal thickening could be induced by progressive smooth muscle cell loss through apoptosis.     

The inhibitory effects of isoflavonoids and resveratrol on oxdized low-density lipoprotein uptake in macrophage cell line J774.1

The interaction between oxidized low-density lipoprotein (LDL) and macrophages are known to be important in the development of arteriosclerosis. Macrophages take up oxidized LDL, becoming foam cells, which contribute to the thickening of the blood vessel wall. The antioxidant properties of polyphenols found in vegetables and other foods have been shown to have a protective effect against arteriosclerosis. To elucidate the effect of flavonoids from fruits, vegetables and cereals on oxidized LDL uptake in macrophages, the inhibitory activity of various flavonoids on DiI-ac-LDL uptake reaction in mouse macrophage cell line J774.1 was measured. We found significant uptake of DiI-ac-LDL, but not DiI-LDL, into the J774.1 cells. In addition, polyinosin and dextran sulphate inhibited uptake, but apigenin, kaempferol, and naringenin, did not. Isoflavones and resveratrol significantly inhibit uptake of DiI-ac-LDL into macrophages, and have a protective effect against arteriosclerosis.     

Focal toxicity of oxysterols in vascular smooth muscle cell culture. A model of the atherosclerotic core region.

Cell necrosis and reactive cellular processes in and near the atherosclerotic core region might result from short-range interactions with toxic lipids. To model these interactions in cell culture, focal crystalline deposits of cholestane-3 beta,5 alpha,6 beta-triol, 25-OH cholesterol, and cholesterol were overlaid by a collagen gel, on which canine aortic smooth muscle cells were seeded. Oxysterols, but not cholesterol, caused focally decreased plating efficiency and cell death, leading to the formation of a persistent circular gap in the cell culture. Cholestanetriol was largely removed from the culture dishes over 3 to 4 weeks, whereas cholesterol and 25-OH cholesterol were largely retained. Smooth muscle cells were motile even in proximity to oxysterol crystals, with occasional suicidal migration toward the crystals. Chemoattraction, however, could not be demonstrated. Despite toxicity, cholestanetriol did not appear to alter the fraction of cells exhibiting 3H-thymidine uptake, even in areas close to the crystals. Thus, oxysterols may be toxic to some cells, without causing major impairment of the migration and proliferation of nearby cells. This would allow the simultaneous occurrence of cell death and proliferation evident in atherosclerosis.     

Impact of lipophilic antioxidants and level of antibodies against oxidized low-density lipoprotein in Polish children with phenylketonuria

The treatment of phenylketonuria (PKU) patients constitutes a phenylalanine (Phe) intake restriction in their diet, which is achieved by adding a special Phe-free amino acid mixture to the diet. It has been reported that this diet could have some micronutrient deficiency. Several authors have also reported an increased oxidative stress or impaired antioxidant status in human and experimental PKU. Our project assessed the concentrations of retinol, alpha-tocopherol, coenzyme Q10, and anti-oxidized low-density lipoprotein (ox-LDL) antibodies in PKU children's plasma. It was found that retinol concentration in PKU children remains within the norm despite a low intake. The lower plasma alpha-tocopherol concentration in PKU children compared with normal children was associated with the lower level of antibodies against ox-LDL. This raises the question whether higher than observed circulatory alpha-tocopherol is indeed beneficial to lower plasma ox-LDL levels. Further studies are needed to explain the genetic factor in PKU patients (e.g., CD36/FAT polymorphism gene). The open clinical question is whether daily supplementation of alpha-tocopherol changes the PKU patients' level of antibodies against ox-LDL.     

Inhibitory effects of fucose-related sugar compounds on oxidised low-density lipoprotein uptake in macrophage cell line J774.1

The interaction between oxidised low-density lipoprotein (LDL) and macrophages is known to be important in the development of arteriosclerosis. Macrophages take up oxidised LDL and then become foam cells, which contribute to the thickening of the blood vessel wall. In our previous paper, some flavonoids found in vegetables and fruits were shown to have a protective effect against arteriosclerosis. In this communication, to elucidate the effect of fucose-related compounds on oxidised LDL uptake in macrophages, the inhibitory activity of various fucose-related compounds on the 1,1-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate-acetylated-LDL (DiI-ac-LDL) uptake reaction in mouse macrophage cell line J774.1 was measured. Fucoidan, 4-deoxy-fucose and fucose-galactose-N-acetyl-glucose significantly inhibited uptake of DiI-ac-LDL into macrophages. The IC₅₀ of 4-deoxy-fucose was 108.4 µM, the highest value of the fucose-related compounds used in the experiments. The protective effects of the fucose-related compounds on atherosclerosis can be used as new health foods.