Lysophosphatidylcholine potentiates the mitogenic activity of modified LDL for human monocyte-derived macrophages

The growth of murine peritoneal macrophages is induced by oxidized LDL (Ox-LDL), and lysophosphatidylcholine (lysoPC) plays an important role in its mitogenic activity. In the present++ study, Ox-LDL-induced macrophage growth was examined with human monocyte-derived macrophages. The cell growth of human macrophages was significantly induced by Ox-LDL but not by acetylated LDL (acetyl-LDL). The treatment of acetyl-LDL with phospholipase A2, however, led to a marked increase in its mitogenic activity, with a concomitant conversion of 75% of its phospholipids to lysoPC. The growth-stimulating activity became positive only when both acetyl-LDL and lysoPC were coincubated, although neither of them exhibited cell growth-promoting activity. These results suggest that Ox-LDL could stimulate the growth of human monocyte-derived macrophages, and lysoPC may play an essential role in the mitogenic activity of Ox-LDL.     

Minimally oxidized LDL is a potent inhibitor of lecithin:cholesterol acyltransferase activity

The oxidation of low density lipoproteins (LDL) has been implicated in the development of atherosclerosis. As a variety of highly reactive lipid peroxidation products can transfer from oxidized LDL to HDL, we evaluated the potential deleterious effects of LDL oxidation on HDL-cholesterol metabolism. To address this issue, we exposed the HDL-containing d > 1.063 g/ml fraction of human plasma to copperoxidized LDL and assessed lecithin:cholesterol acyltransferase (LCAT) activity and apolipoproteinA-I (apoA-I) structure. To determine whether LCAT was directly affected by oxidized LDL, independent of crosslinking of apoA-I, we used an exogenous, [14C]cholesterol-labeled proteoliposome substrate to measure plasma LCAT activity. We observed an inhibition of LCAT activity where copper-oxidized LDL possessing only 2.3 +/- 0.1 and 7.3 +/- 1.4 TBARS produced 24 +/- 3% and 47 +/- 10% reductions in [14C]cholesterol esterification by 1 h, respectively. Copper-oxidized LDL that had been passed through a GF-5 desalting column, while retaining only one-third of its original TBARS, possessed nearly all of its LCAT inhibitory capacity suggesting that the LCAT inhibitory factor(s) was a lipophilic oxidation product. Analysis of polarlipids isolated from copper-oxidized LDL indicated that phospholipid and sterol fractions effectively inhibited LCAT. Copper-oxidized LDL, with as little as 6.3 TBARS, also produced intermolecular crosslinking of apoA-I molecules. Taken together, these data suggest that products of LDL oxidation may adversely affect HDL-cholesterol metabolism by two separate mechanisms: 1) a direct inhibitory effect on LCAT activity and 2) through crosslinking of apoA-I. If occurring in vivo, minimally oxidized LDL may impair cholesteryl ester formation on HDL thereby limiting the ability of HDL to function efficiently in the putative antiatherogenic reverse cholesterol transport pathway.     

Endothelial cell apoptosis induced by oxidized LDL is associated with the down-regulation of the cellular caspase inhibitor FLIP

Fas (CD-95/APO-1) is a death receptor that initiates an apoptotic signal when activated by its ligand, FasL. Normal vascular endothelial cells are resistant to Fas-mediated apoptosis though they express both Fas and FasL. Oxidized low density lipoprotein (OxLDL) or lysophosphatidylcholine (LPC), a major component of OxLDL, induces endothelial cell suicide by sensitizing endothelial cells to Fas-mediated apoptosis. Here, we show that endothelial cell apoptosis by OxLDL and LPC-C16:0 was dose-dependent and correlated with down-regulation of FLICE-inhibitory protein (FLIP), an intracellular caspase inhibitor. FLIP down-regulation also occurred when endothelial cells were treated with toxic doses of LPC-C18:0 or minimally modified low density lipoprotein (LDL). In contrast, FLIP was not down-regulated by native LDL, acetylated LDL, LPC-C12:0, cholesterol, or 7-ketocholesterol, which are not toxic to endothelial cells. The cytotoxicity of oxidized lipids was reversed by transfecting endothelial cells with a FLIP expression plasmid. The results demonstrate, for the first time, FLIP regulation under conditions that lead to pathological tissue destruction.     

Phosphatidylinositol 3-kinase is involved in the induction of macrophage growth by oxidized low density lipoprotein

Early atherosclerotic lesions are characterized by the presence of cholesterol-rich, macrophage-derived foam cells. It has recently been shown that macrophage proliferation occurs during the development of early lesions and that oxidized low density lipoprotein (LDL) stimulates macrophage growth. Possible mechanisms for this induction of macrophage growth include potentiation of mitogenic signal transduction by a component of oxidized LDL following internalization and degradation, interaction with integral plasma membrane proteins coupled to signaling pathways, or direct or indirect activation of growth factor receptors on the cell surface (e.g. GM-CSF receptor) through an autocrine/paracrine mechanism. The present study was undertaken to characterize some of the early intracellular signaling events by which oxidized LDL mediates macrophage cell growth. Extensively oxidized LDL increased protein-tyrosine phosphorylation and caused a 2-fold increase in phosphatidylinositol (PI) 3-kinase activity in phorbol ester-pretreated THP-1 cells (a human monocyte-like cell line). Similar concentrations of native LDL had no effect. Oxidized LDL also stimulated growth of resident mouse peritoneal macrophages, and this effect was reduced by 40-50% in cells treated with PI 3-kinase inhibitors (100 nM wortmannin or 20 microM LY294002). These results suggest that PI 3-kinase mediates part of the mitogenic effect of oxidized LDL, but parallel pathways involving other receptors and signal transduction pathways are likely also involved.     

Role of macrophage glycosaminoglycans in the cellular catabolism of oxidized LDL by macrophages

We have previously developed an in vivo model of leukemogenesis utilizing mice reconstituted with genetically modified bone marrow cells. Based on those studies, a new single gene retroviral vector has been engineered which efficiently transfers v-myc into immature murine bone marrow cells. All reconstituted mice developed leukemia with a short latency period (5-11 weeks). In addition to hyperproliferation associated with elevated levels of PCNA, extensive apoptosis was also observed in all leukemic animals with p53 accumulating in the apoptotic cells. Whereas bax encoded protein, an effector of p53 apoptotic activity was detected in apoptotic cells, p21Waf1 protein, a potential mediator of p53 growth suppression was not detected in these cells suggesting that v-myc-induced apoptosis was independent of the ability of p53 to induce p21Waf1. These results indicate that apoptosis, a part of the cellular response to v-myc expression, does not prevent leukemia development and that hyperproliferation rather than abrogation of oncogene-induced apoptosis appears to be a critical event in v-myc-induced leukemia.     

A high concentration of melatonin inhibits in vitro LDL peroxidation but not oxidized LDL toxicity toward cultured endothelial cells

The pineal hormone, melatonin, was recently found to be a potent free scavenger for hydroxyl and peroxyl radicals. Melatonin also inhibits neuronal and thymocyte damage due to oxidative stress. Atherosclerosis development is mediated by low-density lipoprotein (LDL) oxidation and the endocytosis of oxidized LDL by resident macrophages in the subendothelial vascular wall. Furthermore, the cytotoxic effect of oxidized LDL increases atherogenicity. The goal of this study was to compare the antioxidant activities of melatonin and vitamin E against in vitro LDL oxidation and their cytoprotective actions against oxidized LDL-induced endothelial cell toxicity. An attempt at loading LDL with melatonin by incubating human plasma with an ethanolic melatonin solution gave only low protection against Cu2+-induced LDL oxidation in comparison with vitamin E and gave no detectable incorporation of melatonin into LDL, measured by high-performance liquid chromatography (HPLC) coupled to UV detection. High concentrations of melatonin (10-100 microM) added to the oxidative medium induced a clear inhibition of Cu2+-induced LDL oxidation, characterized as an increase in the lag-phase duration of conjugated diene formation and decreases in the maximal rate of the propagation phase and in the maximal amount of conjugated diene formation. Determination of the median efficacious dose (ED50) of melatonin and vitamin E by their ability to increase lag-phase duration showed that melatonin was less active than vitamin E (ED50, 79 vs. 10 microM, respectively). Melatonin was also less active than vitamin E in limiting the formation of thiobarbituric acid-reactive substances (TBARS) and LDL fluorescence intensity increase in the medium during Cu2+-induced LDL oxidation. Cu2+-induced LDL oxidation in the presence of 100 microM melatonin produced oxidized LDLs that were less recognizable for the scavenger receptors of J774 macrophages than were untreated LDLs. Vitamin E, 10 microM, was more active than 100 microM melatonin in inhibiting LDL oxidation and the resulting lipoprotein alterations leading to binding internalization and degradation by the J774 macrophages. Vitamin E, 100 microM, inhibited the pursuit of the oxidation of oxidized LDL mediated by bovine aortic endothelial cells (BAECs) in a culture medium containing Cu2+, whereas 100 microM melatonin had no antioxidant effect. Melatonin, 100 microM, as well as 100 microM vitamin E inhibited intracellular TBARS formation during the incubation of BAECs with highly oxidized LDL but had no influence on the increase in glutathione (GSH) concentration during this lengthy exposure (16 h) of BAECs to highly oxidized LDL. During this period, the same dose of vitamin E but not of melatonin tended to limit the decrease in adenosine triphosphate (ATP) concentration. Vitamin E, 100 microM, did not significantly reduce cellular lactate dehydrogenase (LDH) release in the culture medium during the incubation of oxidized LDL with BAECs, whereas 100 microM melatonin dramatically increased this release. These data show that melatonin is less active than vitamin E in inhibiting in vitro LDL oxidation and does not inhibit the cytotoxicity of oxidized LDL toward cultured endothelial cells. The concentrations necessary to inhibit LDL oxidation are far beyond those found in human plasma (100 microM vs. 100 pM). Therefore our results indicate that the pineal hormone melatonin per se appears to have little antiatherogenic property in the in vitro oxidation of LDL and the cytoprotective action against the toxicity of oxidized LDL. Nevertheless, in vivo LDL oxidation takes place in the subendothelium of the artery wall, and nothing is known about the concentration of melatonin or its catabolites in this environment.     

Chronic exposure of cultured bovine endothelial cells to oxidized LDL abolishes prostacyclin release

We investigated the effect of chronic exposure (3 days) with low-density lipoprotein (LDL) and oxidized (Ox)-LDL on the unstimulated and stimulated formation of prostacyclin (6-keto-prostaglandin [PG]F1 alpha) and total inositol phosphates (IPs) by cultured bovine aortic endothelial cells. Neither basal nor bradykinin-stimulated (1 to 10 nmol/L) formation of 6-keto-PGF1 alpha was affected by LDL, except at the highest concentration of bradykinin tested (100 nmol/L). In the presence of the antioxidants N-acetyl-L-cysteine (NAC, 10 mumol/L) or vitamin E (100 mumol/L), basal and bradykinin-stimulated formation of 6-keto-PGF1 alpha was potentiated by 20 micrograms protein/mL of LDL. Ox-LDL decreased unstimulated formation of the eicosanoid from 3.1 +/- 0.2 pg/micrograms protein in control cells to 1.6 +/- 0.1 and 0.5 +/- 0.1 pg/microgram protein after 3-day incubation with 5 and 20 micrograms protein/mL of Ox-LDL, respectively (P < .05). As in the basal state, Ox-LDL decreased bradykinin-induced 6-keto-PGF1 alpha formation. NAC or vitamin E did not influence Ox-LDL-induced endothelial cell changes in eicosanoid production. IPs formation by endothelial cells increased to a similar extent in the presence of 20 micrograms protein/mL of either LDL or Ox-LDL. However, no change was apparent in the bradykinin (10 mumol/L)-induced increase in total IPs formation after incubation with the lipoproteins. The data indicate that chronic exposure to Ox-LDL abolishes the production of prostacyclin by cultured endothelial cells. The oxidatively modified lipoprotein seems to more specifically affect the prostacyclin pathway.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rabbit and human atherosclerotic lesions contain IgG that recognizes epitopes of oxidized LDL

In the management of major cerebral artery obstruction, cerebral perfusion reserve is key to introducing cerebral revascularization surgery. The purpose of this study was to evaluate the feasibility of assessing cerebral perfusion reserve by consecutive 99mTc-hexamethyl-propyleneamine oxime (99mTc-HMPAO) SPECT with 5% carbon dioxide (CO2) inhalation. METHODS: The CO2 inhalation and consecutive 99mTc-HMPAO SPECT study was performed on 30 chronic ischemic cerebrovascular disease patients with unilateral major cerebral artery obstruction and on 27 patients without. CO2 reactivity was expressed as the percent increase of 99mTc-HMPAO accumulation from the baseline (%Change) and as a constant k' that was the ratio of 99mTc-HMPAO accumulation per 1 mmHg change of end-tidal CO2 tension by exponential curve fitting. RESULTS: The mean %Change and k' in the middle cerebral artery (MCA) territory on the side without an obstructive lesion or in the cerebellum ranged from 10.0% to 11.1% and from 0.98% to 1.13% per mmHg, respectively. In the MCA territory, an obstructive lesion was noted in 5.9% versus 0.54% per mmHg in the contralateral MCA territory (p < 0.01). Eleven of 30 patients with major cerebral artery obstruction revealed significant asymmetry in the k' value between bilateral MCA territories. CONCLUSION: The results showed compromised cerebral perfusion reserve in the obstructed major cerebral artery territory. The present method was proven clinically useful for evaluating cerebral perfusion reserve in patients with unilateral major cerebral artery obstruction.     

beta-VLDL hypercholesterolemia relative to LDL hypercholesterolemia is associated with higher levels of oxidized lipoproteins and a more rapid progression of coronary atherosclerosis in rabbits

The accumulation of the oxidized apolipoprotein, apoB-100, containing lipoproteins in the arterial wall and the progression of coronary atherosclerotic lesions in rabbits with beta-VLDL and LDL hypercholesterolemia was compared. In New Zealand White (NZW) rabbits on a 0.125% cholesterol diet, LDL cholesterol levels increased from 14 +/- 1 mg/dL (mean +/- SEM; n = 9) to 170 +/- 34 mg/dL (n = 10, P = .0002). On 0.5% cholesterol, LDL cholesterol levels were similar, but beta-VLDL cholesterol levels increased from 60 +/- 4 mg/dL (n = 10) to 550 +/- 75 mg/dL (n = 8; P < .0001). In Watanabe heritable hyperlipidemic (WHHL) rabbits, LDL cholesterol levels were 2.3-fold higher (n = 13; P < .0001) than in NZW rabbits on 0.5% cholesterol, whereas their beta-VLDL cholesterol levels were 3.7-fold lower (P < .0001), resulting in similar total cholesterol levels. At 2 months, mean intimal areas of lesions in the coronary arteries of NZW rabbits on 0.125% cholesterol were 0.13 +/- 0.045 mm2 (n = 4; mean +/- SEM) and were 5.8-fold, (n = 4; P = .016) and 2.0-fold (n = 6; P = NS versus 0.125% cholesterol and P = .014 versus 0.5% cholesterol) higher in NZW rabbits on 0.5% cholesterol and in WHHL rabbits, respectively. At 5 months, mean intimal areas were 0.47 +/- 0.088 mm2 (n = 6) in NZW rabbits on 0.125% cholesterol and were 4.5-fold (n = 4; P = .0001) and 2.0-fold (n = 7; P = .012 and P = .0019) higher in rabbits on 0.5% cholesterol and in WHHL rabbits, respectively. Levels of oxidized apoB-100 containing lipoproteins (both beta-VLDL and LDL) in the lesions correlated with mean intimal area (r = .88; n = 31; P < .0001) of those lesions and with the plasma levels of total beta-VLDL/LDL (r = .72; P < .0001). Levels of oxidized apoB-100 containing lipoproteins in the arterial wall correlate with progression of hypercholesterolemia-induced coronary atherosclerotic lesions. Plasma levels of beta-VLDL relative to similar increases in LDL result in a more pronounced accumulation of oxidized apoB-100 containing lipoproteins in the arterial wall and in the plasma and a more rapid progression of coronary atherosclerosis.     

Immune response to glycated and oxidized LDL in IDDM patients with and without renal disease

OBJECTIVE: To study autoantibodies to oxidized and glycated LDL in IDDM patients with and without diabetic nephropathy and in nephropathy-related macroangiopathy RESEARCH DESIGN AND METHODS: The study included 101 IDDM patients with a long duration of diabetes and 54 healthy subjects. Patients were divided into two groups according to their median urinary albumin excretion rate (AER); the normoalbuminuric group had AER <20 microg/min and the albuminuric group >200 microg/min. The groups were matched for age and BMI, and the two diabetic groups were matched for duration of diabetes and glycemic control. Antibodies against oxidized LDL (using malondialdehyde-modified LDL as the antigen) and against glycated LDL were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: The mean antibody levels against glycated LDL were higher in IDDM patients (0.305 +/- 0.399) than in healthy subjects (0.166 +/- 0.22 optical density [OD]; P = 0.019), but levels did not differ significantly between normoalbuminuric and albuminuric IDDM patients (0.258 +/- 0.354 vs. 0.388 +/- 0.459, respectively). Among the three groups, antibody levels to oxidized LDL did not differ. IDDM patients showed an inverse correlation between antibodies to oxidized LDL and HbA1 (r = -0.211, P = 0.04). The antibody levels to glycated and oxidized LDL did not differ among albuminuric IDDM patients with or without clinical macroangiopathy. CONCLUSIONS: Antibodies to glycated and oxidized LDL do not seem to associate with diabetic nephropathy or nephropathy-related macroangiopathy.     

Oxidized type IV hypertriglyceridemic VLDL-remnants cause greater macrophage cholesteryl ester accumulation than oxidized LDL

We have previously shown that very low density lipoproteins (VLDL, Sf 60-400) from subjects with type IV hyperlipoproteinemia (HTG-VLDL) will induce appreciable cholesteryl ester accumulation in cultured macrophages (J774A.1). The present study examined whether copper-mediated oxidative modification of HTG-VLDL and their remnants would further enhance cholesteryl ester accumulation in J774A.1 cells. Incubation with oxidized VLDL-remnants caused the greatest increase in cellular cholesteryl ester concentrations (54-fold) relative to control cells (P = 0.001). HTG-VLDL and VLDL-remnants each induced similar increases in cholesteryl ester levels (32.3- and 35.8-fold, respectively; both P = 0.001), whereas incubation with oxidized HTG-VLDL brought about only a 20.6-fold increase in cholesteryl ester concentrations (P = 0.014). The increase in cellular cholesteryl ester concentrations induced by oxidized VLDL-remnants was significantly higher (P < or = 0.04) than that induced by all other lipoproteins tested including low density lipoprotein (LDL) and oxidized LDL which caused a 6.7- and a 35.1-fold increase (P < or = 0.0002 for both), respectively. Unlike HTG-VLDL and to a lesser extent VLDL-remnants, uptake of oxidized VLDL and oxidized VLDL-remnants did not require catalytically active, cell secreted lipoprotein lipase. Co-incubation with polyinosine, which blocks binding to the type I scavenger receptor, completely inhibited the cholesteryl ester accumulation induced by oxidized HTG-VLDL, oxidized VLDL-remnants and oxidized LDL (P < or = 0.02). We conclude that oxidation of VLDL-remnants significantly enhances macrophage cholesteryl ester accumulation compared to either HTG-VLDL, VLDL-remnants, or oxidized LDL. Uptake of oxidized VLDL and oxidized VLDL-remnants does not require catalytically active lipoprotein lipase, and involves a receptor that can be competed for by polyinosine.     

Specific lipid protein interactions characterize 3 populations of clathrin coated vesicles involved in the LDL receptor traffic

We have previously isolated 3 different populations of clathrin coated vesicles (CCV) involved in the LDL-receptor traffic in bovine adrenal cortex. We now show that each CCV type contains the transferrin-R and the CI-MPR, therefore, they provide a good model for studying the membrane organization that may govern their targeting in one of the biosynthetic, endocytic and/or recycling pathways. Transferrin--prototype of recylcing ligand--, and alpha adaptin, dynamin and the 110 kDa phosphatidylinositol-3-kinase subunit--of the trafficking machinery--were mainly detected in only 2 of the vesicle populations which could be involved in the endocytic/recycling pathway. The third population which contained larger amounts of gamma adaptin and do not carry transferrin could be involved in the biosynthetic pathway. The vesicle lipid pattern and the saturation of their fatty acyl chains were analyzed and confirmed these results. The nature of the interactions between vesicle components was then determined using several classes of detergents. Only non ionic ones could solubilize the LDL-R in a complex with either alpha or gamma adaptin. In contrast, they dissociated clathrin or beta-beta' adaptins. Taken together these results prompt us to suggest an integrated model for targeting in membrane traffic. Besides specific targeting signals carried by cargo proteins and recognized by proteins from the coat and the cytosolic trafficking machinery, lipids would play a key modulatory role. At each step in the membrane traffic, the proteins which carry multiple targeting signals would interact transiently with a specific set of lipids. This would result in the exposure of the appropriate targeting signals which could now become recognized by the proper targeting machinery.     

Fragmented mitochondrial DNA is the predominant carrier of oxidized DNA bases

Previous analyses indicated a high level of oxidative base modification in mitochondrial DNA, the extent of which raised questions about the methodological validity and biological implications. In the present study DNA was isolated from rat liver mitochondria under carefully controlled conditions, and the extent of base oxidation, DNA fragmentation, and nuclear DNA contamination were analyzed. DNA isolated from intact mitochondria treated with DNase consisted of 16.3 kilobase pairs, mostly circular, mitochondrial DNA molecules and a mixture of nuclear and mitochondrial DNA fragments, as identified by agarose gel electrophoresis and hybridization. High-performance liquid chromatography in combination with electrochemical detection confirmed that the overall level of 8-hydroxy-2'-deoxyguanosine, a marker commonly used in the analysis of base oxidation, is higher in mitochondrial than in nuclear DNA. Importantly, 8-hydroxy-2'-deoxyguanosine is relatively scarce in the 16.3 kilobase pair mitochondrial DNA molecules (0.051 pmol/microgram) but is present in high levels in mitochondrial DNA fragments (0.741 pmol/microgram). The fragments constitute about 18% of total mitochondrial DNA. The antitumor agent bleomycin, which binds to DNA, forms an iron complex capable of transferring electrons from Fe2+ to molecular oxygen. Exposure of mitochondria to bleomycin and iron resulted in nicking but not in a significant increase in base oxidation of 16.3 kilobase pair mitochondrial DNA, whereas the amount and the oxidation level of fragmented mitochondrial DNA significantly increased. These findings are relevant for a better understanding of the role of mitochondria in aging and various diseases and are consistent with the notion that despite the overall high DNA oxidation level, mitochondria can faithfully proliferate.     

Lysophosphatidylcholine stimulates the release of arachidonic acid in human endothelial cells

Lysophosphatidylcholine (lyso-PC) is a product of phosphatidylcholine hydrolysis by phospholipase A2 (PLA2) and is present in cell membranes, oxidized lipoproteins, and atherosclerotic tissues. It has the ability to alter endothelial functions and is regarded as a causal agent in atherogenesis. In this study, the modulation of arachidonate release by lyso-PC in human umbilical vein endothelial cells was examined. Incubation of endothelial cells with lyso-PC resulted in an enhanced release of arachidonate in a time- and concentration-dependent manner. Maximum arachidonate release was observed at 10 min of incubation with 50 microM lyso-PC. Lyso-PC species containing palmitoyl (C16:0) or stearoyl (C18:0) groups elicited the enhancement of arachidonate release, while other lysolipids such as lysophosphatidylethanolamine, lysophosphatidylserine, lysophosphatidylinositol, or lysophosphatidate were relatively ineffective. Lyso-PC-induced arachidonate release was decreased by treatment of cells with PLA2 inhibitors such as para-bromophenacyl bromide and arachidonoyl trifluoromethyl ketone. Furthermore, arachidonate release was attenuated in cells grown in the presence of antisense oligodeoxynucleotides that specifically bind cytosolic PLA2 mRNA. Treatment of cells with lyso-PC resulted in a translocation of PLA2 activity from the cytosolic to the membrane fractions of cells. Lyso-PC induced a rapid influx of Ca2+ from the medium into the cells, with a simultaneous enhancement of protein kinase C (PKC) activity in the membrane fractions. The lyso-PC-induced arachidonate release was attenuated when cells were preincubated with specific inhibitors of PKC (staurosporine and Ro31-8220) or a specific inhibitor of mitogen-activated protein kinase/extracellular regulated kinase kinase (PD098059). Taken together, the results of this study show that lyso-PC caused the elevation of cellular Ca2+ and the activation of PKC, which stimulated cytosolic PLA2 in an indirect manner and resulted in an enhanced release of arachidonate.     

Characterization of a specific polyclonal antibody against 13-hydroperoxyoctadecadienoic acid-modified protein: formation of lipid hydroperoxide-modified apoB-100 in oxidized LDL

Lipid hydroperoxide may react with protein or amino phospholipid without secondary decomposition. We prepared a polyclonal antibody to lipid hydroperoxide-modified proteins using 13S-hydroperoxy-9Z, 11E-octadecadienoic acid-modified keyhole limpet hemocyanin (13-HPODE-KLH) as immunogen. The antibody recognized 13-HPODE-modified bovine serum albumin (BSA), but not aldehyde-modified proteins, such as malondialdehyde-modified BSA. The antibody also recognized adducts derived from 13-HPODE and 13S-hydroperoxy-9Z, 11E, 15Z-octadecatrienoic acid (13-HPOTRE(alpha)). The oxidized alpha-linolenic acid- and linoleate-protein adducts were recognized by the antibody. Oxidized phospholipid-protein adducts were scarcely recognized by the antibody. However, when ester bonds of phospholipids containing linoleic acid were hydrolyzed by alkaline treatment, the cross-reactivities appeared. The result suggests that a phospholipid hydroperoxide can react with a protein directly or indirectly, and a carboxyl terminal (COOH) of the lipid in an adduct was needed as an epitope. Oxidized LDL (ox-LDL) was prepared by the incubation of LDL with copper ion or 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), and the formation of lipid hydroperoxide-modified apolipoprotein was confirmed using the antibody. A slight immunoreactivity was observed in ox-LDL without alkaline treatment. When the ox-LDL was treated with alkali to hydrolyze the ester bonds of the lipid, enhanced antigenicity appeared with time-dependency. The results suggest that lipid hydroperoxide-modified apolipoprotein was formed during the oxidation of LDL.     

Induction of mitochondrial manganese superoxide dismutase in macrophages by oxidized LDL: its relevance in atherosclerosis of humans and heritable hyperlipidemic rabbits

The objective of the study was to analyze the intracellular antioxidative response of macrophages (Mphi) exposed to increased levels of low density lipoprotein (LDL). We studied manganese superoxide dismutase (MnSOD) and, in part, GSH in cultured human and rabbit Mphi, and in atheromatous arterial tissue of humans and heritable hyperlipidemic (HHL) rabbits. Incubation of human Mphi with oxidized-LDL (ox-LDL) resulted in an induction of MnSOD mRNA production as shown by RT-PCR. MnSOD immunoreactivity (IR) was found to be located in the mitochondria of Mphi. In HHL rabbits, MnSOD activity and GSH concentration were significantly increased in atherosclerotic intima compared to the media of the aorta, but significantly decreased (P<0.01) in larger plaques compared with smaller ones, resulting in a significant inverse correlation of MnSOD activity (r=-0.67, P<0.001) and GSH concentration (r=-0.57, P<0.01) with plaque size. Immunohistology of the atherosclerotic intima revealed MnSOD-IR in Mac-1 (CD 11b/CD 18)-immunoreactive (ir) Mphi of human arteries and, similarly, in RAM-11-ir Mphi of rabbit ones. The relation of MnSOD-ir Mphi decreased with plaque advancement, which is consistent with biochemical findings. Most MnSOD-ir Mphi in atherosclerotic plaques revealed TUNEL-positive nuclei, indicating DNA strand breaks, and p53-IR. We conclude that mitochondrial antioxidants such as MnSOD are induced in Mphi in vitro and in atherosclerotic arteries as a reply to increased mitochondrial oxidation. As normal consequences of an increased oxidative stress due to the exposure to ox-LDL nuclear DNA strand breaks occur, which are suggested to be a signal to increase p53 protein levels. Reactive oxygen species-mediated mitochondrial-dependent pathways are suggested as major contributing pathomechanisms to nuclear damage, which eventually may result in apoptosis. A common response to increased oxidative stress due to modified LDL is presumed in rabbit and human atherosclerotic plaques.     

Primary motor cortex is involved in bimanual coordination

Many voluntary movements involve coordination between the limbs. However, there have been very few attempts to study the neuronal mechanisms that mediate this coordination. Here we have studied the activity of cortical neurons while monkeys performed tasks that required coordination between the two arms. We found that most neurons in the primary motor cortex (MI) show activity specific to bimanual movements (bimanual-related activity), which is strikingly different from the activity of the same neurons during unimanual movements. Moreover, units in the supplementary motor area (SMA; the area of cortex most often associated with bimanual coordination) showed no more bimanual-related activity than units in MI. Our results challenge the classic view that MI controls the contralateral (opposite) side of the body and that SMA is responsible for the coordination of the arms. Rather, our data suggest that both cortical areas share the control of bilateral coordination.     

Mouse Zic1 is involved in cerebellar development

Zic genes encode zinc finger proteins, the expression of which is highly restricted to cerebellar granule cells and their precursors. These genes are homologs of the Drosophila pair-rule gene odd-paired. To clarify the role of the Zic1 gene, we have generated mice deficient in Zic1. Homozygous mice showed remarkable ataxia during postnatal development. Nearly all of the mice died within 1 month. Their cerebella were hypoplastic and missing a lobule in the anterior lobe. A bromodeoxyuridine labeling study indicated a reduction both in the proliferating cell fraction in the external germinal layer (EGL), from 14 d postcoitum, and in forward movement of the EGL. These findings suggest that Zic1 may determine the cerebellar folial pattern principally via regulation of cell proliferation in the EGL.