Inflammatory platelet-activating factor-like phospholipids in oxidized low density lipoproteins are fragmented alkyl phosphatidylcholines.

Oxidation of human low density lipoprotein (LDL) generates proinflammatory mediators and underlies early events in atherogenesis. We identified mediators in oxidized LDL that induced an inflammatory reaction in vivo, and activated polymorphonuclear leukocytes and cells ectopically expressing human platelet-activating factor (PAF) receptors. Oxidation of a synthetic phosphatidylcholine showed that an sn-1 ether bond confers an 800-fold increase in potency. This suggests that rare ether-linked phospholipids in LDL are the likely source of PAF-like activity in oxidized LDL. Accordingly, treatment of oxidized LDL with phospholipase A(1) greatly reduced phospholipid mass, but did not decrease its PAF-like activity. Tandem mass spectrometry identified traces of PAF, and more abundant levels of 1-O-hexadecyl-2-(butanoyl or butenoyl)-sn-glycero-3-phosphocholines (C(4)-PAF analogs) in oxidized LDL that comigrated with PAF-like activity. Synthesis showed that either C(4)-PAF was just 10-fold less potent than PAF as a PAF receptor ligand and agonist. Quantitation by gas chromatography-mass spectrometry of pentafluorobenzoyl derivatives shows the C(4)-PAF analogs were 100-fold more abundant in oxidized LDL than PAF. Oxidation of synthetic alkyl arachidonoyl phosphatidylcholine generated these C(4)-PAFs in abundance. These results show that quite minor constituents of the LDL phosphatidylcholine pool are the exclusive precursors for PAF-like bioactivity in oxidized LDL.     

Antibodies against oxidized low density lipoproteins in pregnant women

Oxidized low density lipoproteins (oxLDL) formed in vivo induce a humoral immune response. Oxidative modification of LDL renders it immunogenic and a heterogeneous population of specific anti-oxLDL antibodies is produced. These antibodies could represent a biological marker of oxidative stress and serve as markers of atherosclerosis. Autoantibodies against oxLDL (oLAb) have been detected in human subjects practically of every age. oLAb also appear in the blood of pregnant women. Some studies have shown that the levels of antibodies to oxLDL were elevated in women with established preeclampsia. The present study was aimed to estimate the oLAb IgG levels in the first and second trimester of pregnancy. Furthermore, we estimated the correlation between maternal serum (MS) levels of oLAb and alpha-1-fetoprotein (MS AFP), human chorionic gonadotrophin (MS HCG) and trophoblast-specific-beta-1-glycoprotein (MS SP1), because these proteins are determined as a part of prenatal biochemical screening for fetal congenital abnormalities. Our study deals with the oLAb changes in women with pregnancy-induced hypertension. We also investigated the correlation between oLAb IgG and anticardiolipin antibodies IgG (ACA) in the serum of pregnant women. We examined 40 pregnant women attending Institute for Mother and Child Care for their antenatal care as outpatients. Routine blood samplings between the 9-13th week of pregnancy and 16-18th week of pregnancy were performed as a part of biochemical prenatal screening for fetal congenital abnormalities (Group 1). Their mean age was 27 +/- 4.1 years. Furthermore, we examined 26 women in the second or third trimester with pregnancy-induced hypertension (Group 2). Group 2 was compared with 49 pregnant women in the second or third trimester who were normotensive (Group 3). We used commercial standardized ELISA kits for determination of oLAb IgG, ACA IgG, MS AFP and MS HCG, MS SP1 was analyzed by single radial immunodiffusion. We did not find any differences in the levels of oLAb IgG in the first and second trimester in the women of Group 1. The correlation between oLAb and ACA IgG was not statistically significant (Spearman coefficient r=0.22, p=0.1). The correlation between oLAb IgG with MS AFP, MS HCG and MS SP1 was not statistically significant. Weak negative correlation for AFP and HCG was suggested both in the first and in the second trimester. The levels of oLAb IgG in the group of women with pregnancy-induced hypertension were significantly lower than in the group of normotensive women (348 +/- 388 U/ml v.s. 579 +/- 400 mU/ml, p<0.01). We can conclude that the levels of oLAb do not differ in the first and second trimester of gravidity. However, we cannot exclude the possible influence of an inverse relationship between oLAb IgG titers and the synthesis of fetoplacental antigens. This finding is important especially in the context of the results of prenatal biochemical screening. Pregnancy-induced hypertension is associated with lower levels of oLAb. Weak cross-reactivity between oLAb and anticardiolipin antibodies may exist but there is a possibility that there are two different populations of antibodies reacting with various antigens.     

Multiple receptors for modified low density lipoproteins in mouse peritoneal macrophages: different uptake mechanisms for acetylated and oxidized low density lipoproteins

Receptor-mediated incorporations of two modified low density lipoproteins (LDL), acetylated LDL (acetyl-LDL) and oxidized LDL were compared in vitro in mouse peritoneal macrophages by cross-competition experiments. Excess amount of oxidized LDL inhibits the binding of [125I]acetyl-LDL only partially, and excess amount of acetyl-LDL inhibits that of [125I]oxidized LDL also only partially, suggesting that the uptake of the two LDL by macrophages is mediated by partially overlapped yet different mechanisms. Scatchard analysis of [125I]acetyl-LDL binding showed a linear plot and addition of excess amount of oxidized LDL partially displaced the binding sites without changing the affinity, suggesting that there are two classes of receptors with similar affinity; one is specific for acetyl-LDL and the other is common. And the plot of [125I]oxidized LDL binding showed a curvilinear plot and excess amount of acetyl-LDL partially displaced the binding sites of the low affinity, suggesting that there are two classes of binding sites with different affinities and the low affinity one is shared with acetyl-LDL. These results indicate that macrophage receptors for modified LDL consist of at least three receptors, two of which are specific for each LDL and the rest is a common receptor.     

Studies on oxidized low density lipoproteins. Controlled oxidation and a prostaglandin artifact

Low density lipoproteins (LDL), isolated by ultracentrifugal flotation, were oxidized (LDLOXID) slowly during dialysis against 0.15 M NaCl and subsequent incubation in 96% air-4% CO2 at 37 degrees C. Butylated hydroxytoluene prevented LDL oxidation. LDL preparations from different sera were oxidized at different rates and the degree of lipid peroxidation was controlled by varying the incubation time. Mild oxidation did not alter the electrophoretic mobility of the LDLOXID preparations. LDLOXID contained lipid peroxides in neutral lipids, had increased amounts of lysophosphatidylcholine, and contained a number of complex oxidation products that were generated from the oxidation of free fatty acids. These oxidation products included large amounts of soluble material that cross-reacted with antibodies to PGE2 but not 6-keto-PGF1 alpha. The amount of cross-reacting material was proportional to the degree of lipid peroxidation. Cross-reacting material in LDLOXID preparations was evidently formed from the oxidation of free fatty acids released from LDL, since cross-reacting material was also formed when a synthetic fat emulsion was oxidized in the presence of free arachidonic acid.     

Effects of oxidized low density lipoproteins on arachidonic acid metabolism in smooth muscle cells

The role of oxidized plasma lipoproteins in modifying arachidonic acid (AA) metabolism was studied in smooth muscle cells (SMC). Very low density lipoproteins (VLDL), unoxidized low density lipoproteins (LDLBHT) isolated with butylated hydroxytoluene (BHT), and oxidized LDL (LDLOXID) were separated from human serum. Thiobarbituric acid reactant (TBAR) levels were adjusted by saline incubations. Prostanoids in guinea pig SMC cultures were measured either by radioimmunoassay (RIA) or the isolation by high performance liquid chromatography (HPLC) of labeled prostanoids from SMC prelabeled with [14C]AA. Cell morphology and viability were studied by staining with Giemsa, nile red, and propidium iodide. VLDL and LDLBHT had little effect on prostanoid synthesis. Low-TBAR-LDLOXID enhanced total prostanoid levels and diminished the release of labeled prostanoids. Similar effects were found with exogenous free AA (unlabeled). Low-TBAR-LDLOXID did not affect the release of endogenous phospholipid AA as free AA. Synergism occurred between LDLOXID and exogenous free AA in prostanoid synthesis. Low-TBAR-LDLOXID evidently enhanced prostanoid levels in SMC both by supplying AA and by stimulating cyclooxygenase. High-TBAR-LDLOXID blocked prostanoid synthesis and enhanced cell death but time and pulse-recovery experiments showed that these effects were unrelated. High-TBAR-LDLOXID stimulated prostanoid synthesis when BHT was added to the incubation media. High-TBAR-LDLOXID also caused massive free AA release and the formation of many nonprostanoid derivatives. High-TBAR-LDLOXID evidently diminished overall prostanoid levels in SMC by inhibiting cyclooxygenase and at the same time stimulating AA release and the formation of other AA derivatives.     

氧化修饰高密度脂蛋白的致动脉粥样硬化作用

高密度脂蛋白（ｈｉｇｈ ｄｅｎｓｉｔｙ ｌｉｐｏｐｒｏｔｅｉｎ ,ＨＤＬ）具有抗动脉粥样硬化（ａｔｈｅｒｏｓｃｌｅｒｏｓｉｓ,ＡＳ）作用,一旦发生氧化修饰后,即具致ＡＳ作用。氧化修饰ＨＤＬ（ｏｘｉｄｉｚｅｄ ＨＤＬ,ＯＸ－ＨＤＬ）通过减少胆固酥因管平滑肌细胞（ｓｍｏｏｔｈ ｍｕｓｃｌｅ ｃｅｌｌ,ＳＭＣ）,内皮细胞及巨噬细胞流出,抑制胆固醇逆向转运;     

Interactions of high density lipoproteins with very low and low density lipoproteins during lipolysis

Interactions of high density lipoproteins (HDL) with very low (VLDL) and low (LDL) density lipoproteins were investigated during in vitro lipolysis in the presence of limited free fatty acid acceptor. Previous studies had shown that lipid products accumulating on lipoproteins under these conditions promote the formation of physical complexes between apolipoprotein B-containing particles (Biochim. Biophys. Acta, 1987. 919: 97-110). The presence of increasing concentrations of HDL or delipidated HDL progressively diminished VLDL-LDL complex formation. At the same time, association of HDL-derived apolipoprotein (apo) A-I with both VLDL and LDL could be demonstrated by autoradiography of gradient gel electrophoretic blots, immunoblotting, and apolipoprotein analyses of reisolated lipoproteins. The LDL increased in buoyancy and particle diameter, and became enriched in glycerides relative to cholesterol. Both HDL2 and HDL3 increased in particle diameter, buoyancy, and relative glyceride content, and small amounts of apoA-I appeared in newly formed particles of less than 75 A diameter. Association of apoA-I with VLDL or LDL could be reproduced by addition of lipid extracts of lipolyzed VLDL or purified free fatty acids in the absence of lipolysis, and was progressively inhibited by the presence of increasing amounts of albumin. We conclude that lipolysis products promote multiple interactions at the surface of triglyceride-rich lipoproteins undergoing lipolysis, including physical complex formation with other lipoprotein particles and transfers of lipids and apolipoproteins. These processes may facilitate remodeling of lipoproteins in the course of their intravascular metabolism.     

Endothelin-converting enzyme-1 increases in atherosclerotic mice: potential role of oxidized low density lipoproteins

The aim of our study was to analyze the relationships between atherosclerosis and endothelin-converting enzyme-1 (ECE-1). Four-week-old C57BL/6J [wild-type (WT)] and apolipoprotein E-deficient (apoE) mice were fed with a standard or Western-type fat diet for 8 wks. ApoE showed atherosclerotic lesions in the aorta, higher blood pressure and vascular lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) protein content than WT. ApoE showed a significant increase in ECE-1 protein content and mRNA expression in aorta, lung, and kidney, without changes in heart. When an ECE-1 inhibitor, FR-901533, was administered to them, blood pressure decreased in apoE on fat diet versus apoE on normal diet and WT. ECE-1 and LOX-1 protein content were elevated in peripheral blood mononuclear cells (PBMC) from hypercholesterolemic patients. In order to study the mechanism involved in this ECE-1 up-regulation, bovine aortic endothelial cells (BAEC) were treated with oxidized-low density lipoproteins (oxLDL). OxLDL, but not LDL, increased ECE-1 protein content, mRNA expression and promoter activity. Our results demonstrate that ECE-1 increases in different atherosclerosis situations. Up-regulation of ECE-1 could contribute, at least partially, to the development of hypertension seen in apoE mice, because FR-901533 avoided it. Probably, atherosclerotic situations course with an increase of oxLDL, which is able to induce ECE-1 expression with the subsequent potential pathological effects.     

A macrophage Fc receptor for IgG is also a receptor for oxidized low density lipoprotein.

The internalization of oxidized low density lipoprotein (OxLDL) by macrophages is hypothesized to contribute to foam cell formation and eventually to atherosclerotic lesion formation. OxLDL is a ligand for the acetylated low density lipoprotein (AcLDL) receptor, however, our data show that this receptor accounts for less than half of OxLDL uptake by mouse macrophages, suggesting additional receptors for OxLDL. We have developed a novel expression cloning strategy in order to isolate clones encoding OxLDL receptors. In addition to the AcLDL receptor, we isolated a molecular clone for a structurally unrelated receptor capable of mediating the high affinity uptake of OxLDL following transfection into cells. This receptor has been identified as the mouse Fc gamma RII-B2, a member of a family of receptors known to mediate immune complex uptake through recognition of the Fc region of IgG. The uptake of OxLDL by cells transfected with the Fc gamma RII-B2 clone is not blocked by AcLDL but is blocked by the anti-Fc gamma RII monoclonal antibody, 2.4G2.     

Postprandial low-density lipoproteins in type 2 diabetes are oxidized more extensively than fasting diabetes and control samples

This study examined the kinetics of low-density lipoprotein (LDL) oxidation in the fasting and postprandial states of diabetic and control subjects to determine if LDL oxidation may contribute to accelerated atherosclerosis in diabetes. We compared in vitro oxidation of LDL from 12 control and 13 Type 2 diabetic subjects in the fasting and postprandial states. The extent of oxidation was assessed by length of lag phase, formation of conjugated dienes (CD), lipid peroxides, thiobarbituric acid reactive substances (TBARS), and percentage reduction in free amine groups. Diabetic subjects were significantly older and heavier. Comparisons between control and diabetic subjects in the postprandial state showed that the lag phase was significantly shorter in diabetic subjects than controls (P = 0.005), TBARS were significantly higher (P = 0.006), and levels of CD were higher at 60, 65, and 70 min (P < 0.01). In the fasting state, however, these comparisons were not significant. In diabetic subjects, postprandial samples had a significantly shorter lag phase (P = 0.003), higher TBARS (P = 0.006), and higher levels of CD at 60, 65 (P < 0.001), and 70 min (P = 0.0013) compared to fasting samples. Elevated levels of serum triglycerides in diabetic subjects were negatively correlated to lag phase, in fasting (P = 0.06) and postprandial states (P = 0.002). We conclude that accelerated oxidation of LDL seen in postprandial states in diabetes may be a critical contributor to cardiovascular risks. Elevated levels of serum triglycerides may contribute to the rapid oxidation of LDL seen in diabetic subjects.     

Cholesteryl ester hydroperoxides are biologically active components of minimally oxidized low density lipoprotein

Oxidation of low density lipoprotein (LDL) occurs in vivo and significantly contributes to the development of atherosclerosis. An important mechanism of LDL oxidation in vivo is its modification with 12/15-lipoxygenase (LO). We have developed a model of minimally oxidized LDL (mmLDL) in which native LDL is modified by cells expressing 12/15LO. This mmLDL activates macrophages inducing membrane ruffling and cell spreading, activation of ERK1/2 and Akt signaling, and secretion of proinflammatory cytokines. In this study, we found that many of the biological activities of mmLDL were associated with cholesteryl ester (CE) hydroperoxides and were diminished by ebselen, a reducing agent. Liquid chromatography coupled with mass spectroscopy demonstrated the presence of many mono- and polyoxygenated CE species in mmLDL but not in native LDL. Nonpolar lipid extracts of mmLDL activated macrophages, although to a lesser degree than intact mmLDL. The macrophage responses were also induced by LDL directly modified with immobilized 12/15LO, and the nonpolar lipids extracted from 12/15LO-modified LDL contained a similar set of oxidized CE. Cholesteryl arachidonate modified with 12/15LO also activated macrophages and contained a similar collection of oxidized CE molecules. Remarkably, many of these oxidized CE were found in the extracts of atherosclerotic lesions isolated from hyperlipidemic apoE(-/-) mice. These results suggest that CE hydroperoxides constitute a class of biologically active components of mmLDL that may be relevant to proinflammatory activation of macrophages in atherosclerotic lesions.     

Effect of garlic supplementation on oxidized low density lipoproteins and lipid peroxidation in patients of essential hypertension

Reactive oxygen species (ROS) has been implicated in the pathogenesis of many diseases including hypertension. Therefore, certain compounds with antioxidative capacity are believed to be protective against such diseases. Some components of garlic are known to possess antioxidative properties. Therefore, in the present study we investigated the effect of short-term garlic supplementation in essential hypertensive patients (EH) on indices of oxidative stress. Twenty patients of EH as diagnosed by JNC VI criteria (Group I) and 20 age and sex-matched normotensive controls were enrolled for the study. Both groups were given garlic pearls (GP) in a dose of 250 mg per day for 2 months. Baseline samples were obtained at start of the study, i.e. 0 day, and thereafter, 2 months (follow-up). Lipids and lipoprotein subfractions, plasma-oxidized low-density lipoproteins (ox-LDL), plasma and urinary concentration of 8-iso-Prostaglandin F2alpha (8-iso-PGF2alpha) as a biomarker of oxidative stress in vivo, and the total antioxidant status (TOS) of these individuals were determined. We observed a moderate hypercholesterolemia and a significantly raised blood pressure in hypertensive patients as compared to the controls. The indices of oxidative stress, i.e. plasma ox-LDL and plasma and urinary concentration of 8-iso-PGF2alpha were significantly increased in EH group. Further, hypertensive patients had a significantly low TOS as compared to the control group. With in 2 months of GP supplementation, there was a significant decline in both systolic (SBP) and diastolic blood pressures (DBP) and a significant reduction in ox-LDL and 8-iso-PGF2alpha levels in Group I patients. Further, a moderate increase in the TOS was also observed in this group as compared to their control counterparts. These findings suggest that dietary supplementation of garlic may be beneficial in reducing blood pressure and oxidative stress in hypertensive individuals.     

Role of oxidized human plasma low density lipoproteins in atherosclerosis: effects on smooth muscle cell proliferation

The effects of oxidized human plasma low density lipoproteins (Ox-LDL) on the proliferation of cultured aortic smooth muscle cells was studied, employing viable cell counting, [³H] thymidine incorporation into DNA, and the release of lactate dehydrogenase (LDH) into the medium. Oxidized LDL (prepared by incubation of LDL with copper sulfate) exerted a concentration-dependent stimulation (2 fold, compared to control) of aortic smooth muscle cell proliferation at low concentrations (0.1 µg – 10 µg/ml medium). On the other hand, at high concentrations (25–200 µg/ml), Ox-LDL produced a pronounced decrease in viable cells, a decrease in the incorporation of [³H] thymidine into DNA, and an increase in the release of LDH in the medium. In this report, the previously postulated biological roles of oxidized-LDL in atherosclerosis are discussed in view of these findings.Abbreviations Ox-LDL Oxidized human plasma Low Density Lipoproteins - SMC Smooth Muscle Cells - LDH Lactate Dehydrogenase - LPC Lysophosphatidycholine - PC Phosphatidylcholine - TNF Tumor Necrosis Factor     

Flotation equilibrium of serum low density lipoproteins

The molecular weight of human serum low density lipoprotein (LDL) was determined for the first time by sedimentation equilibrium or, more accurately, flotation equilibrium, in high concentration of KBrNaBr containing Tris-Cl buffer plus EDTA (density = 1.20 – 1.49 g/ml). Assuming both the molecular weight and the partial specific volume were unknown, the results at different densities gave a value of 2.87 ± 0.12 × 10⁶ for the molecular weight and 0.965 ± 0.014 ml/g for the partial specific volume.     

Nascent very low density lipoproteins from rat hepatocytic Golgi fractions are enriched in phosphatidylethanolamine

The phospholipid composition of nascent very low density lipoproteins (VLDL) of rat hepatocytic Golgi fractions differs greatly from that of plasma VLDL. The phospholipids of nascent VLDL contain about four times more phosphatidylethanolamine (PE) than plasma VLDL, whereas plasma VLDL contain considerably more sphingomyelin. Thus, the ratio of PE to sphingomyelin differs by a factor of about 12 between nascent Golgi VLDL and circulating plasma VLDL. It is evident from these data that the PE/sphingomyelin ratio of VLDL can be used to estimate endosomal contamination of hepatocytic Golgi fractions.     

Guinea pig very low density lipoproteins are a good substrate for lipoprotein lipase.

In contrast to plasma from most other animals, guinea pig plasma causes little or no stimulation of lipoprotein lipase activity. Very low density lipoproteins (VLDL) isolated by ultracentrifugation of guinea pig serum caused a definite stimulation of lipase activity, whereas the infranatant inhibited the activity. Gel filtration in 5 M guanidinium hydrochloride of delipidated VLDL demonstrated that the activation was caused by a low molecular weight protein. The VLDL themselves were hydrolized at similar rates as human VLDL both by guinea pig and by bovine lipoprotein lipases. Thus, guinea pig VLDL contain an activator for lipoprotein lipase analogous to that in other animals and there is enough of the activator to support rapid hydrolysis of the VLDL lipids by the lipase.     

Synergistic interaction between thromboxane A2 and mildly oxidized low density lipoproteins on vascular smooth muscle cell proliferation

Low density lipoprotein (LDL) and mildly oxidized low density lipoprotein (mox-LDL) are known mitogens for vascular smooth muscle cell (VSMC). Since aggregating platelets at sites of atherosclerotic injury release thromboxane A2(TXA2), a known mitogen for VSMC, we examined whether TXA2 can act synergistically with mox-LDL or its oxidative components in inducing VSMC proliferation. Growth arrested primary aortic rabbit VSMCs in 1st or 2nd passage were incubated with different concentrations of LDL or mox-LDL or lysophosphatidylcholine (LPC) or H2O2 or 4-hydroxy-2-nonenel (HNE) for 24 h followed by incubation with TXA2 mimetic U46619 for another 24 h. The amount of 3[H]-thymidine incorporated into the DNA was measured. Both LDL and mox-LDL at a concentration of 120 microg/ml induced proliferation of VSMC (168% or 184% respectively) when compared to the control. U46619 induced VSMC proliferation was observed at a concentration of 5 microm/L. As compared to native LDL, the mitogenic effect of mox-LDL on VSMC proliferation was markedly potentiated by U46619 to 301% or 316% at 0.5 or 5 microm/L U46619 respectively. LPC, H2O2 and HNE induced DNA synthesis was also marked by enhanced by U46619. These results suggest that even low concentration of TXA2 released from aggregating platelets may potentiate the mitogenic effect of mox-LDL at sites of vascular damage. The mitogenic effect of mox-LDL may be mediated via its oxidation products LPC, H2O2 (reactive oxygen species donor), and HNE.     

The various effects of fractionated oxidized low density lipoproteins on the growth of smooth muscle cells in culture

The effects of fractionated oxidized low density lipoproteins (oxidized LDL) on the growth of vascular smooth muscle cells (VSMC) and their relationship to the formation of lysophosphatidylcholine (lyso-PC) as well as the activation of protein kinase C (PKC) were studied. VSMC were isolated from porcine aorta by explant culture. LDL was isolated from porcine blood by sequential ultracentrifugation and oxidized LDL was obtained by incubating LDL with 5 µM CuSO₄ at 37° C for various lengths of time. Our results showed that LDL oxidized for 12 h and eluted from fast protein liquid chromatography at 43 min inhibited the growth of VSMC, and that LDL oxidized for longer than 48 h and eluted at 48 min stimulated the growth of VSMC. The formation of lyso-PC in the oxidized LDL correlated well with its stimulatory effect, suggesting that lyso-PC is responsible for the mitogenic effect of oxidized LDL. This stimulatory effect of oxidized LDL was inhibited by staurosporine, a PKC inhibitor. Treatment with oxidized LDL increased the activity of membrane PKC, but it decreased that of cytosolic PKC, suggesting the translocation of PKC from cytosol to the membrane in the presence of oxidized LDL. These results suggested that the oxidized LDL-stimulated VSMC growth was mediated by the formation of lyso-PC and the activation of PKC.