Incubation Of Low Density Lipoprotein Research Articles

Discrimination between minimally modified LDL and fully oxidized LDL using monoclonal antibodies

Low density lipoprotein (LDL) can be oxidized in a stepwise process that leads to the production of minimally modified low density lipoprotein (mm-LDL), in which only the lipid component is oxidized, and then of fully oxidized LDL (oxLDL), in which both the lipids and the protein are oxidized. The thiobarbituric acid-reactive substances (TBARS) assay is a recognized method for determination of oxidized LDL, however this method is unable to distinguish between mm-LDL and oxLDL. In this study, seven specific monoclonal antibodies (mAbs) against human LDL were generated and selectively bound to the apolipoprotein B-100 (apoB-100) component of LDL. Oxidized LDL was produced by incubation of human LDL with 10 μM CuSO4 for various times. The TBARS assay revealed that the optimal incubation time to achieve maximal lipid oxidation was 9 h. Indirect ELISA using the newly generated mAbs was implemented to differentiate between mm-LDL and oxLDL and it was found that binding of the mAbs to oxLDL was significantly decreased after 48 h of incubation, reflecting the oxidative modification of apoB-100. Our results suggest that the optimal times for incubation of LDL with CuSO4 for generation of mm-LDL and oxLDL were 9 h and 48 h, respectively.

Lipids and Their Effects in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is an incurable common B-cell malignancy with a spectrum of clinical outcomes. Over the past decade, our increasing understanding of the drivers of CLL progression has led to the development and use of novel therapeutics. For example, B-cell receptor (BCR) signaling was shown to be overactive in CLL, and subsequently the kinase inhibitors ibrutinib (BTK inhibitor) and idelalisib (PI3K delta inhibitor) were found to have clinical efficacy in this malignancy (Byrd et al., 2013, Furman et al., 2014). Despite these new treatments, CLL remains incurable and there remains a need to identify new therapeutic targets.

Lipid Peroxide-Mediated Oxidative Rearrangement of the Pyrazinone Carboxamide Core of Neutrophil Elastase Inhibitor AZD9819 in Blood Plasma Samples.

This study focused on the mechanistic interpretation of ex vivo oxidation of a candidate drug in blood plasma samples. An unexpected lipid peroxide-mediated epoxidation followed by a dramatic rearrangement led to production of a five-membered oxazole derivative from the original six-membered pyrazinone-carboxamide core of a human neutrophil elastase inhibitor, 6-(1-(4-cyanophenyl)-1H-pyrazol-5-yl)-N-ethyl-5-methyl-3-oxo-4-(3-(trifluoromethyl)phenyl)-3,4-dihydropyrazine-2-carboxamide (AZD9819). The rearranged oxidation product 2-(1-(4-cyanophenyl)-1H-pyrazol-5-yl)-5-(N-ethylacetamido)-N-(3-(trifluoromethyl)phenyl)oxazole-4-carboxamide was characterized by accurate-mass tandem mass spectrometry fragmentations, by two-dimensional NMR and X-ray crystallography of an authentic standard, and by incorporation of an (18)O atom from molecular (18)O2 to the location predicted by our proposed mechanism. The lipid peroxide-mediated oxidation was demonstrated by using human low-density lipoprotein (LDL) in pH 7.4 phosphate buffer and by inhibiting the oxidation with ascorbic acid or l-glutathione, two antioxidants effective in both plasma and the LDL incubation. A nucleophilic mechanism for the epoxidation of AZD9819 by lipid hydroperoxides explains the prevention of its ex vivo oxidation by acidification of the plasma samples. The discovery of the lipid peroxide-dependent oxidation of an analyte and the means of prevention could provide valuable information for biotransformation and bioanalysis.

Evaluation of Antioxidant Activity of Natural and Synthetic Compounds Against LDL Oxidation Using CNT Electrode

In this paper, a novel method for evaluating antioxidant activity against low density lipoprotein (LDL) oxidation (anti-LDLox) using a carbon nanotube (CNT) based electrode was investigated. Although ORAC and DPPH are known methods to evaluate antioxidant activity, they do not directly reflect the capacities of anti-LDLox. A method has been reported for measuring oxidized LDL using a CNT electrode. We evaluated anti-LDLox by measuring the amount of oxidized LDL using the electrode after incubation of LDL with antioxidants during copper-mediated oxidation. Thiobarbituric acid reactive substances (TBARS) were also determined for comparison with the CNT electrode. There was a correlation of the CNT electrode with the TBARS and the ORAC, whereas no correlation was found between the CNT electrode and the DPPH. We demonstrate here that a CNT electrode method has a potential for the determination of the antioxidant activity of various natural and synthetic compounds by detecting oxidized LDL.

Downregulation of p38 MAPK involved in inhibition of LDL-induced proliferation of mesangial cells and matrix by curcumin

Curcumin, as a main pharmacological component in the traditional Chinese medicine-turmeric, has shown anti-inflammatory, anti-oxidation, anti-tumor and anti-fibrotic effects. This study aimed to investigate the possible underlying signaling pathway which was involved in the inhibition of LDL-induced proliferation of mesangial cells and matrix by curcumin. Rat mesangial cells in vitro were incubated with low-density lipoprotein (LDL) and different concentrations of curcumin (0, 6.25, 12.5, 25.0 μmol/L) or p38 MAPK inhibitor, SB203580 (10 μmol/L). Under LDL incubation, mesangial cells proliferated, the expression of MMP-2 mRNA and protein was decreased, the expression of COX-2 mRNA and protein was increased, reactive oxygen species (ROS) generation was increased and p38 MAPK was activated significantly (P<0.05). When LDL-induced cells were treated with curcumin in the concentration of 12.5 or 25.0 μmol/L, LDL-induced proliferation of mesangial cells was suppressed, the expression of MMP-2 mRNA and protein increased, the expression of COX-2 mRNA and protein downregulated, the production of ROS inhibited and p38 MAPK inactivated (P<0.05). In conclusion, curcumin can inhibit the LDL-induced proliferation of mesangial cells and up-regulate the expression of MMP-2, which may be related with the inhibitory effect of curcumin on COX-2 expression, ROS production and p38 MAPK.

Acrolein-conjugated low-density lipoprotein induces macrophage foam cell formation

ObjectiveAcrolein-conjugated lysine residues in proteins are present in human atherosclerotic lesions, and are detected in human low-density lipoprotein (LDL). These findings suggest that acrolein may contribute to macrophage foam cell formation and atherogenesis through modification of LDL. The purpose of this study is to determine whether acrolein-conjugated LDL (Acro-LDL) induces macrophage conversion to form foam cells. MethodsAcro-LDL was prepared by incubation of LDL with acrolein. Characteristics of Acro-LDL were examined by agarose gel electrophoresis and western blotting. Cholesterol contents of THP-1 macrophages incubated with Acro-LDL were determined by enzymatic method. Pathway of Acro-LDL uptake by THP-1 macrophages was determined using neutralizing antibody against scavenger receptors. Delivery of Acro-LDL into lysosome and formation of lipid droplet by incubation with Acro-LDL were demonstrated by confocal microscopy. ResultsThe mobility of Acro-LDL determined by agarose gel electrophoresis was increased by modification with acrolein, and the shift of mobility was dependent on the concentration of acrolein. Acrolein interacted with apolipoprotein B in LDL and Acro-LDL uptake by THP-1 macrophage was a more effective inducer of cholesterol accumulation than oxidized LDL uptake. Acro-LDL uptake was mediated by scavenger receptor class A type 1 (SR-A1), but not by CD36. As a result of Acro-LDL uptake, cholesterol ester accumulated in lipid droplets of macrophages, converting them to foam cells. ConclusionsThe results show that Acro-LDL uptake via SR-A1 receptors can mediate macrophage foam cell formation.

Telmisartan and N-acetylcysteine Suppress Group V Secretory Phospholipase A2 Expression in TNFα-stimulated Human Endothelial Cells and Reduce Associated Atherogenicity

Group V secretory phospholipase A2 (sPLA2-V) hydrolyzes phosphatidylcholine in low-density lipoprotein (LDL) to increase lysophosphatidylcholine (LPC) content. Because in human umbilical vein endothelial cells (HUVEC), tumor necrosis factor alpha (TNFα)-induced sPLA2-V expression, and LPC content in LDL and monocyte chemoattractant protein-1 mRNA were enhanced by incubation of LDL with TNFα-stimulated HUVEC, we investigated whether an angiotensin II receptor type 1 blocker, telmisartan, or an antioxidant drug, N-acetylcysteine (NAC), suppressed TNFα-induced sPLA2-V expression. Telmisartan or NAC administered before and during TNFα stimulation diminished the increase of sPLA2-V mRNA in HUVEC and reduced TNFα-induced sPLA2-V protein at 3 days after TNFα stimulation. Angiotensin II did not induce sPLA2-V mRNA, and a peroxisome proliferator-activated receptor-γ antagonist, GW3335, did not influence the inhibitory effect of telmisartan on TNFα-induced sPLA2-V mRNA. At 3 days after TNFα stimulation, 30 μM telmisartan or 20 mM NAC administered before and during TNFα stimulation prevented the enhancement of LPC content in LDL and monocyte chemoattractant protein-1 mRNA by LDL incubation with TNFα-stimulated HUVEC. A 2-month treatment with telmisartan in 29 hypertensive type 2 diabetic patients significantly reduced LPC content in circulating LDL. Telmisartan's suppressive effect on TNFα-induced sPLA2-V expression may have beneficial effects in preventing proatherogenic changes of LDL.

2D-NMR reveals different populations of exposed lysine residues in the apoB-100 protein of electronegative and electropositive fractions of LDL particles

Several potentially atherogenic LDL subfractions present low affinity for the LDL receptor, which result in impaired plasma clearance. Electronegative LDL [LDL(-)] is one of these minor subfractions and the molecular basis for its reduced receptor affinity is not well understood. In the present study, high-resolution 2D-NMR spectroscopy has been employed to characterize the surface-exposed lysine residues of the apolipoprotein (apo)B-100 protein in both LDL(-) and LDL(+) subfractions. LDL(+) showed two populations of lysine residues, similar to those previously described in total LDL. "Normal" Lys have a pk(a) of 10.4 whereas "active" Lys have a pk(a) of 8.8 and have been suggested to be involved in receptor binding. In contrast to LDL(+), the LDL(-) subfraction presented a third type of Lys, named as "intermediate" Lys, with a different microenvironment and higher basicity (pk(a) 10.7). These intermediate Lys cannot be reliably identified by 1D-NMR. Because the abundance of normal Lys is similar in LDL(+) and LDL(-), the intermediate Lys in the apoB-100 molecule of LDL(-) should come from a group of active Lys in LDL(+) particles that have a less basic microenvironment in the LDL(-) particle. These differences between LDL(+) and LDL(-) are indicative of a distinct conformation of apoB-100 that could be related to loss of affinity of LDL(-) for the LDL receptor.

The protective effect of Opuntia dillenii Haw fruit against low-density lipoprotein peroxidation and its active compounds

The antioxidant activity and inhibitory effect of extracts from Opuntia dillenii Haw fruit (ODHF) and its active compounds on low-density lipoprotein (LDL) peroxidation were investigated. The results indicated that the antioxidant activity of methanolic extracts of ODHF in Trolox equivalent antioxidant capacity and oxygen-radical absorbance capacity assays were in the order of seed > peel > pulp. The lag time of conjugated diene formation in Cu 2+-induced LDL oxidation was increased by incubation of LDL with various methanolic extracts of ODHF. The methanolic extracts from seed, peel and pulp prolonged the lag time compared to control (154.1 min) to 514.8, 163.9 and 190.2 min, respectively, at a concentration of 10 μg/ml. Among the extracts, seed extracts of ODHF (10 μg/ml) possessed the highest inhibitory effect on the formation of thiobarbituric acid reactive substances and relative electrophoretic mobility. The results also demonstrated that seeds of ODHF contained the highest amounts of polyphenols and flavonoids (212.8 and 144.1 mg/100 g fresh seed, respectively), such as gallic acid, catechin, sinapinic acid, epicatechin, p-coumaric acid, quercetin and ferulic acid, but no betanin, isobetanin and ascorbic acid as determined by HPLC. However, the peel and pulp contained high amounts of betanin, isobetanin and ascorbic acid, but with lower contents of phenolics and flavonoids as compared to the seed. These findings suggest that phenolics and flavonoids may directly contribute to the antioxidant activity of the seeds of ODHF.

Phenolic-extract from argan oil ( Argania spinosa L.) inhibits human low-density lipoprotein (LDL) oxidation and enhances cholesterol efflux from human THP-1 macrophages

Argan oil is rich in unsaturated fatty acids, tocopherol and phenolic compounds. These protective molecules make further study of its cardiovascular diseases (CVDs) action interesting. Furthermore, no previous study has explored the antioxidant activity of argan oil in comparison with olive oil. The present study was conducted to evaluate the beneficial properties of Virgin argan oil phenolic extracts (VAO-PE) towards CVD by: (A) protecting human (low-density lipoprotein, LDL) against lipid peroxidation and (B) promoting high-density lipoprotein (HDL)-mediated cholesterol efflux. Human LDLs were oxidized by incubation with CuSO 4 in the presence of different concentrations of VAO-PE (0–320 μg/ml). LDL lipid peroxidation was evaluated by conjugated diene and MDA formation as well as Vitamin E disappearance. Incubation of LDL with VAO-PE significantly prolonged the lag-phase and lowered the progression rate of lipid peroxidation ( P < 0.01) and reduced the disappearance of Vitamin E in a concentration-dependent manner. Incubation of HDL with VAO-PE significantly increased the fluidity of the HDL phospholipidic bilayer ( P = 0.0004) and HDL-mediated cholesterol efflux from THP-1 macrophages. These results suggest that Virgin argan oil provides a source of dietary phenolic antioxidants, which prevent cardiovascular diseases by inhibiting LDL-oxidation and enhancing reverse cholesterol transport. These properties increase the anti-atherogenic potential of HDL.

Mechanism of in vitro heme-induced LDL oxidation: effects of antioxidants.

Heme protein toxicity, owing to generation of reactive oxygen species most likely by direct interaction between heme iron and hydrogen peroxide (H2O2), may be involved in various pathologies, including atherogenesis and pigmentary acute renal failure. The aim of this study was to investigate the mechanism of heme cytotoxicity and the effects of antioxidant therapies in an in vitro model of heme-induced low-density lipoprotein (LDL) oxidation. Human LDLs were exposed to heme, iron (Fe), protoporphyrin (PPIX) and PPIX-Zinc (Zn) with or without H2O2. Lipid peroxidation was monitored by measurement of conjugated diene formation (at the 234-nm absorbance). The effect of various antioxidants, such as vitamin E and vitamin C, reduced glutathione (GSH), and oxidized glutathione (GSSG), mannitol and desferoxamine (DFO) was further investigated in the established in vitro model of LDL oxidation. Incubation of LDLs in the presence of heme/H2O2 induced lipid peroxidation with the optimal oxidation rate being at 5 microm heme/100 microm H2O2 doses. By contrast, incubation of LDL with H2O2, Fe, Fe/H2O2, PPIX, PPIX/H2O2, heme or PPIX-Zn did not initiate any LDL oxidation. In vitro, the vitamin E load protected native LDLs against heme/H2O2 oxidative modifications. Incubation of LDLs with increasing doses of vitamin C, GSH and DFO conferred a dose-dependent protection, while mannitol and GSSG had no effect. Initiation and propagation of heme-induced lipid peroxidation is not mediated by a Fenton reaction but depends on specific interactions between heme and H2O2. It may result from the generation of ferryl and perferryl radicals derived from hemic Fe and H2O2 interactions. A protective effect of vitamins E, C, GSH and DFO was demonstrated in this model.

Modification induced by homocysteine and low-density lipoprotein on human aortic endothelial cells: an in vitro study.

The aim of the present study was to investigate the effect exerted by low-density lipoprotein (LDL) modified by homocysteine (Hcy)-thiolactone (Hcy-LDL) on functional properties on human endothelial cells. Hcy-thiolactone, a reactive product formed in human cells from enzymatic conversion of Hcy, was hypothesized to play an important role in Hcy-induced vascular damages. Using endothelial cultured cells [human aortic endothelial cells (HAEC)] as cellular model, we evaluated nitric oxide (NO) production, cytoplasmic Ca(2+) levels, Na(+)/K(+)-ATPase activity, and peroxynitrite production in cells incubated in the presence of control LDL or Hcy-LDL. Homocysteinylation of LDL was carried out by incubation of LDL, isolated from plasma of healthy subjects, with 100 microm Hcy-thiolactone. A significant increase in cytoplasmic Ca(2+) levels and peroxynitrite production and a decrease in Na(+)/K(+)-ATPase and NO production in HAEC incubated with Hcy-LDL compared with HAEC incubated with control LDL were observed. Moreover, a positive correlation was found between Na(+)/K(+)-ATPase activity and cytoplasmic Ca(2+) content and between peroxynitrite activity and cytoplasmic Ca(2+) content. In conclusion, our results demonstrated that LDL homocysteinylated in vitro induced alterations of functional properties and NO metabolism of human endothelial cells.

Differential antioxidant properties of red wine in water soluble and lipid soluble peroxyl radical generating systems.

Red wine and its components have been shown to possess cardioprotective and anti-atherogenic effects. Additionally, red wine and many of its components like catechin, epicatechin, rutin, transresveratrol and quercetin possess antioxidant properties. Oxidized low density lipoprotein (LDL) is involved in the development of an atherosclerotic lesion. Red wine, therefore, may be anti-atherogenic because of its antioxidant effects on LDL modification. This study examined the antioxidant effects of catechin, epicatechin, rutin, transresveratrol, quercetin and Merlot wines on LDL oxidation. Merlot was chosen because although other red wines have been tested, limited information exists for this variety. Oxidation was carried out with AAPH (2,2'-Azo-bis(2-amidinopropane) dihydrochloride) and AMVN (2,2'-Azo-bis(2,4-dimethylvaleronitrile)), as water and lipid soluble peroxyl radical generating systems (FRGS), respectively. This allowed us to determine the lipophilic antioxidant characteristics of the wine and its components. Conjugated diene assays were used to measure LDL oxidation over 6 hrs. In an AAPH system, all polyphenolic compounds except transresveratrol displayed an antioxidant effect. LDL oxidation by AAPH was also inhibited by aliquots of Merlot wine. No antioxidant effects were observed in an AMVN environment except for a mild antioxidant effect by quercetin. Surprisingly, incubation of LDL with Merlot wine strongly protected against oxidation by AMVN. In summary, the five phenolic compounds displayed antioxidant effects in a water soluble free radical generating system, but only quercetin showed this in a lipid soluble one. However, red wine inhibited LDL oxidation by both water and lipid soluble free radical generating systems. Our data suggest, therefore, that red wines contain unidentified antioxidants that provide protection against LDL oxidation within a lipid soluble environment. (Mol Cell Biochem 263: 211-215, 2004).

Thyroid hormone (T 3) and its acetic derivative (TA 3) protect low-density lipoproteins from oxidation by different mechanisms

Triiodothyronine (T 3) and triiodothyroacetic acid (TA 3) are thyroid compounds that similarly protect low-density lipoprotein (LDL) against oxidation induced by the free radical generator 2,2′-azobis-[2-amidinopropane] dihydrochloride (AAPH). However, TA 3 is more antioxidant than T 3 on LDL oxidation induced by copper ions (Cu 2+), suggesting that these compounds act by different mechanisms. Here we measured conjugated diene production kinetics during in vitro human LDL (50 mg LDL-protein per l) oxidation induced by various Cu 2+ (0.5–4 μM) or AAPH (0.25–2 mM) concentrations in the presence of T 3, TA 3, butylated hydroxytoluene (BHT) (a free radical scavenger) or ethylenediaminetetracetic acid (EDTA) (a metal chelator). From the kinetics were estimated: length of the lag phase ( T lag), maximum velocity of conjugated diene production ( V max), and maximum amount of generated dienes ( D max). Thyroid compound effects on these oxidation parameters were compared to those of the controls BHT and EDTA. In addition we measured by atomic absorption spectrometry copper remaining in LDL after a 30 min incubation of LDL with Cu 2+ and the compounds followed by extensive dialysis, i.e. copper bound to LDL. As expected, LDL-copper was decreased by EDTA in a concentration-dependent manner, whereas it was not affected by BHT. T 3 increased LDL-copper whereas TA 3 slightly decreased it. The whole data suggest that T 3 and TA 3 are free radical scavengers that also differently disturb LDL-copper binding, an essential step for LDL lipid peroxidation. The most likely mechanisms are that T 3 induces new copper binding sites inside the LDL particle, increasing the LDL-copper amount but in a redox-inactive form, whereas TA 3 blocks some redox-active copper binding sites highly implicated in the initiation and the propagation of lipid peroxidation. Alternatively, we also found that a little amount of copper is tightly bound in LDL, which may be essential for the propagation of lipid peroxidation induced by free radical generators.

Mesangial matrix‐activated mononuclear cells express functional scavenger receptors and accumulate intracellular lipid

Introduction Monocyte recruitment into the mesangium and foam cell formation are recognized features of glomerular injury. External signals encountered by these infiltrating cells may determine their behaviour and thereby potentially influence disease outcomes. Our previous studies indicate that activation of monocytes by mesangial matrix stimulates the production of a variety of mediators including inflammatory cytokines and matrix degrading enzymes.Methods Using expression of peroxisome proliferator activator receptor‐γ (PPAR‐γ) and scavenger receptor (ScR) as differentiation markers, we examined whether matrix activation was associated with the expression of monocyte characteristics usually associated with a macrophage phenotype. THP‐1 mononuclear cells were incubated for 7 days with 500 µg/ml solublized matrix extracted from cultured human mesangial cells.Results Using phorbol methyl ester (PMA) (125 nm) and albumin (500 µg/ml) as positive and negative controls, respectively, we demonstrated that matrix activation of monocytes led to intracellular lipid accumulation as demonstrated by oil red O staining. Matrix activation was also associated with a concentration‐dependent increase in the expression of both ScR and PPAR‐γ mRNA and a corresponding increase in PPAR‐γ protein expression on Western blotting. The presence of functional ScR was confirmed using FACS analysis in which incubation of matrix‐activated monocytes with Dil‐labelled acetylated low‐density lipoprotein (LDL) led to an increase in mean fluorescent intensity of 373% (P &lt; 0.001) as compared to albumin (100%) and PMA (423%). This could be inhibited by addition of excess unlabelled ligand, suggesting specific binding to the ScR. Furthermore, incubation of LDL with mesangial matrix in the absence of cells led to enhanced electrophoretic mobility of recovered lipoprotein on agarose gel. A similar shift was seen when LDL was incubated with Cu2+, a powerful lipoprotein oxidant.Discussion These results demonstrate that interactions with mesangial matrix induce expression of monocyte characteristics associated with a macrophage phenotype and promote oxidation of LDL, thereby converting it to an ScR ligand. Such observations may help to explain foam cell formation in the mesangium in the context of glomerular disease.

Peroxynitrite-mediated oxidative modification of low-density lipoprotein by aqueous extracts of cigarette smoke and the preventive effect of fluvastatin

Cigarette smoking is known to promote atherosclerosis, possibly through enhanced oxidative stress. The aim of the present study was to elucidate the possible involvement of peroxynitrite in oxidative modification of low-density lipoprotein (LDL) induced by aqueous extract of cigarette smoke (CSE) and the preventive effect of fluvastain, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor with antioxidative activity, in vitro and in vivo. Modification of LDL was monitored by LDL subfraction analysis using anion-exchange HPLC, TBARS formation and 3-nitrotyrosine production. Incubation of LDL with CSE caused a marked increase in oxidative modification of LDL and nitration of tyrosine residues in the apolipoprotein B. These modifications were prevented by treatment with fluvastatin as well as Vitamin E in a concentration-related manner. Fluvastatin was equal to or more effective than Vitamin E for preventing protein nitration, but weaker for preventing oxidative modification. When CSE was injected daily into the ear vein of Watanabe heritable hyperlipidemic rabbits for 5 months, both oxidative modification and nitration of the plasma LDL noticeably occurred. These changes induced by CSE could be effectively prevented by the simultaneous oral administration of fluvastatin (10 and 30 mg/kg) or Vitamin E (150 mg/kg). Fluvastatin prevented the LDL nitration more effectively than Vitamin E. These results suggest that peroxynitrite in CSE is involved in oxidative modification of LDL and that fluvastatin can efficiently prevent LDL modification by scavenging peroxynitrite. Fluvastatin may be potentially beneficial to hypercholesterolemic patients with oxidative stress such as smoking.

Low-density lipoprotein modification by normal, myeloperoxidase-deficient and NADPH oxidase-deficient granulocytes and the impact of redox active transition metal ions

The modification of low-density lipoprotein (LDL) by normal, myeloperoxidase (MPO)-deficient and NADPH oxidase-deficient granulocytes was investigated using the monoclonal antibody (mAb) OB/04, which was originally generated against copper-oxidized LDL. Incubation of LDL with normal granulocytes increased the reactivity of LDL with mAb OB/04. These effects were even more pronounced using MPO-deficient granulocytes. Inhibitors of oxidative reactions (the NADPH oxidase inhibitor diphenyleneiodonium chloride [DPI], catalase, superoxide dismutase [SOD]) did not significantly reduce LDL oxidation by normal granulocytes. Furthermore, granulocytes of a patient with NADPH oxidase deficiency were almost equally effective as normal granulocytes, indicating that oxidative burst-derived reactive oxygen species are of only minor importance in the generation of mAb OB/04-detectable new epitopes on LDL in vitro. In contrast, incubation of LDL with iron and copper prior to and during incubation with normal granulocytes markedly enhanced the generation of OB/04-detectable epitopes. It is supposed that, besides superoxide (in normal and MPO-deficient granulocytes) or instead of superoxide (in NADPH oxidase-deficient granulocytes), lytic enzymes released by activated granulocytes may enhance the availability of transition metals for oxidation of LDL. Our results support the concept that transition-metal-dependent pathways of LDL oxidation in combination with degranulation products of granulocytes are important.

Participation of peroxynitrite in oxidative modification of LDL by aqueous extracts of cigarette smoke

Aqueous extracts of cigarette smoke (CSE) can oxidatively modify plasma low-density lipoprotein (LDL). The aim of the present study was to elucidate the participation of peroxynitrite in LDL oxidation. When LDL was incubated with CSE, its oxidative modification was dependent on time and concentration. It could be effectively prevented by vitamin E, partially by superoxide dismutase, but hardly by catalase, mannitol and metal chelators. CSE also increased the 3-nitrotyrosine content in LDL. A similar increase of 3-nitrotyrosine occurred after incubation of LDL with a peroxynitrite generating agent, 3-morpholinosydnonimine, thus suggesting that prominent pro-oxidants in CSE are peroxynitrite-generating species.

Human plasma trans-sialidase causes atherogenic modification of low density lipoprotein

In earlier studies we have found that incubation of low density lipoprotein (LDL) with autologous blood plasma-derived serum leads to a loss of sialic acid from lipoprotein particles. In this study we demonstrated that sialic acid removed from LDL was transferred to glycoconjugates of lipoproteins, glycoproteins and sphingolipids of human serum. This showed that human serum contained the trans-sialidase activity. Gel-filtration chromatography of human blood serum demonstrated the presence of trans-sialidase activity in lipoprotein subfractions as well as in lipoprotein-deficient serum. Trans-sialidase (about 65 kDa) was isolated from lipoprotein-deficient serum using affinity chromatography carried out with Neu5Acα2-8Neu5Ac-sepharose FF-6. Optimal pH values for the trans-sialidase were 3.0, 5.0 and 7.0. Calcium and magnesium ions stimulated the enzyme activity at millimolar concentrations. Isolated enzyme can remove sialic acid from LDL, IDL, VLDL, and HDL particles (in decreasing rate order). Serum trans-sialidase transferred sialic acid from glycoconjugates of plasma proteins (fetuin, transferrin) and gangliosides (GM3, GD3, GM1, GD1a, GD1b). Sialylated glycoconjugates of human blood erythrocytes also served as substrate for serum trans-sialidase. We have found that sialic acid can also be removed from N- and O-linked glycans, sialylated Le x and Le a, oligosialic acids, and sphingolipid carbohydrate chains. The rate of sialic acid release decreased in the following order: α2,6>α2,3≫α2,8. Transferred molecule of sialic acid can form α2,6, α2,3 and to a lesser degree α2,8 linkage with galactose, N-acetyl-galactosamine or sialic acid of acceptors. The glycoconjugates of erythrocytes, lipoprotein particles, plasma proteins, neutral sphingolipids and gangliosides may serve as acceptors of transferred sialic acid. Trans-sialidase-treated native LDL becomes desialylated and then can induce cholesteryl ester accumulation in human aortic intimal smooth muscle cells. Thus, trans-sialidase may be involved in the early stages of atherogenesis characterized by foam cell formation.

Changes in expression of antioxidant enzymes affect cell-mediated LDL oxidation and oxidized LDL-induced apoptosis in mouse aortic cells.

Transgenic mice overexpressing Cu/Zn superoxide dismutase (hSod1Tg(+/0)) or catalase (hCatTg(+/0)) and knockout mice underexpressing manganese superoxide dismutase (Sod2(+/)(-)) or glutathione peroxidase-1 (Gpx1(-/-)) were used to study the effect of antioxidant enzymes on cell-mediated low density lipoprotein (LDL) oxidation and oxidized LDL (oxLDL)-induced apoptosis. Incubation of LDL with mouse aortic segments or smooth muscle cells (SMCs) resulted in a significant increase in LDL oxidation. However, LDL oxidation was significantly reduced when LDL was incubated with aortic segments and SMCs obtained from hSod1Tg(+/0) and hCatTg(+/0) mice compared with those obtained from wild-type mice. In contrast, LDL oxidation was significantly increased when LDL was incubated with aortic segments and SMCs obtained from Sod2(+/)(-) and Gpx1(-/-) mice. CuSO(4)-oxidized LDL increased DNA fragmentation and caspase activities in the primary cultures of mouse aortic SMCs. However, oxLDL-induced DNA fragmentation and caspase activities were reduced 50% in SMCs obtained from hSod1Tg(+/0) and hCatTg(+/0) mice compared with wild-type control mice. In contrast, oxLDL-induced DNA fragmentation and caspase activities were significantly increased in SMCs obtained from Sod2(+/)(-) and Gpx1(-/-) mice. These findings suggest that overexpression of Cu/Zn superoxide dismutase or catalase reduces cell-mediated LDL oxidation and oxLDL-induced apoptosis, whereas underexpression of manganese superoxide dismutase or glutathione peroxidase-1 increases cell-mediated LDL oxidation and oxLDL-induced apoptosis.