Transient increase in plasma oxidized LDL during the progression of atherosclerosis in apolipoprotein E knockout mice.

BACKGROUND: Plasma level of oxidized low-density lipoprotein (OxLDL) is a risk marker for cardiovascular diseases. The behavior of plasma OxLDL before disease progression has not been studied previously. METHODS AND RESULTS: In this study, we developed a sensitive ELISA procedure for detecting mouse circulating OxLDL using a monoclonal antibody that recognizes oxidized phosphatidylcholine and a rabbit antimouse apolipoprotein B-48 polyclonal antibody. Apolipoprotein E knockout mice were fed on a chow diet for 40 weeks. Oil red O-positive lesions developed gradually by 20 weeks, and the percentage area covered by the lesions increased dramatically after 28 weeks; it covers 33.4% of the surface area by 40 weeks. The OxLDL level, measured after LDL fraction was isolated from each mouse, at 10 weeks was 0.015 ng/microg LDL. It increased 3-fold at 20 weeks of age and then decreased to the basal level by 40 weeks of age, suggesting that OxLDL appears before the development of atherosclerotic lesions. The occurrence of lipid peroxidation products, acrolein and oxidized phosphatidylcholines, in aortic tissue were revealed by immunohistochemical staining as early as 10 weeks. CONCLUSIONS: These results suggest that OxLDL might be involved in the early stages of progression of atherosclerotic lesions.

[Molecular pathology in atherosclerosis: the mechanism by which cholesteryl ester accumulates in atheromatous aorta].

To study how cholesterol accumulates in atheroma, novel monoclonal antibodies were developed, using crude homogenate of atheroma as immunogens. 212D monoclonal antibody recognizing extra cellular matrix with lipid-laden deposits was selected by histochemical staining. The antigen was deduced vitronectin from cDNA library. DLH3 monoclonal antibody recognizing oxidized LDL, epitope of which was 5- or 9-phosphatidylcholine. Significant correlations between oxidized LDL and coronary heart disease (CHD) patients were observed from clinical study. 256C monoclonal antibody recognizing atheromatous lesions in human aorta was selected. Epitope must be PC-cholesterol complex which may involve in foam cell rupture. Atherogenesis will be discussed from the aspects of these antibodies. Our working hypothesis is required to elucidate the mechanism. Denatured lipoproteins (either oxidized lipoprotein or ruptured foam cells) may induce atheroma. Oxidation of lipoprotein may be taken place both in foam cells and/or extra cellular matrix, and macrophage eliminate these denatured lipoproteins and become foam cells. The foam cells are ruptured by either apoptosis or necrosis afterward, and hydrophobic fragments of foam cells dispersed in extra cellular space, which destroys the function of biological membrane. Since biological function could be maintained by segregation of hydrophilic circumstances, macrophages uptake these fragmented material and oxidized lipoprotein to maintain the function. This vicious spiral may enhance chronically the atheroma.

In-vivo gene transfer into newly hatched chick brain by electroporation.

Newly hatched domestic chicks serve as ideal models for studies of the neural basis of behavioral plasticity, particularly for understanding the mechanisms of learning such as filial imprinting. To elucidate the molecular basis and gene functions involved in learning, we developed an in-vivo gene-transfer system in the brain of a living chick using electroporation. When green fluorescent protein-encoding plasmids were transfected to a chick brain, green fluorescence was clearly observed, and expression at the protein level was confirmed by immunoblotting. Most of the transfected brain cells were neuronal cells with dendrites. This neuron-selective electroporation system will facilitate the analysis of gene functions in the living chick brain and provide further clues as to the molecular mechanisms of avian learning.

Distribution of microsomal triglyceride transfer protein within sub-endoplasmic reticulum regions in human hepatoma cells.

Very low-density lipoprotein (VLDL) particles are formed in the endoplasmic reticulum (ER) through the association of lipids with apolipoprotein B (apoB). Microsomal triglyceride transfer protein (MTP), which transfers lipid molecules to nascent apoB, is essential for VLDL formation in ER. However, little is known of the distribution and interaction of MTP with apoB within ER. In this study, distribution patterns of apoB and MTP large subunit (lMTP) within ER were examined. Microsomes prepared from HuH-7 cells, a human hepatoma cell line, were further fractionated into rough ER (RER)-enriched subfractions (ER-I fraction) and smooth ER (SER)-enriched subfractions (ER-II fraction) by iodixanol density-gradient ultracentrifugation. ApoB was evenly distributed in the ER-I and the ER-II fractions, while 1.5 times more lMTP molecules were present in the ER-I fraction than in the ER-II fraction. lMTP and apoB were coprecipitated both in the ER-I and in the ER-II fractions by immunoprecipitation whenever anti-apoB or an anti-lMTP antibodies were used. ApoB-containing lipoprotein particles showed a lower density in the ER-II fraction than those in the ER-I fraction. From these results, it is suggested that MTP can function in both rough and smooth regions of ER in human hepatoma cells.

Identification of major proteins in the lipid droplet-enriched fraction isolated from the human hepatocyte cell line HuH7.

Recent studies have revealed the presence of intracellular lipid droplets in wide variety of species. In mammalian cells, there exist proteins specifically localize in lipid droplets. However, the protein profile in the droplet remains yet to be clarified. In this study, a fraction enriched with lipid droplets was isolated from a human hepatocyte cell line HuH7 using sucrose density gradient centrifugation, and 17 major proteins in the fraction were identified using nano LC-MS/MS techniques. Adipose differentiation-related protein (ADRP) was the most abundant protein in the fraction. The secondary abundant proteins were identified to be acyl-CoA synthetase 3 (ACS3) and 17beta-hydroxysteroid dehydrogenase 11 (17betaHSD11). Included in the identified proteins were five lipid-metabolizing enzymes as well as two lipid droplet-specific proteins. When HuH7 cell lysate was fractionated by a density gradient, most of 17betaHSD11 was found in the droplet-enriched fraction. In immunocytochemical analysis, 17betaHSD11 showed ring-shaped images which overlapped with those for ADRP. These results suggest that a specific set of proteins is enriched in the lipid droplet-enriched fraction and that 17betaHSD11 localizes specifically in the fraction.

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.

Minimally modified LDL is an oxidized LDL enriched with oxidized phosphatidylcholines.

The oxidative modification of low-density lipoprotein (LDL) is involved in atherogenesis. Among a variety of modified LDLs mentioned in the literature, so-called minimally modified LDL (MM-LDL) was reported to have pro-atherogenic properties despite minimal changes in its oxidative measures. After treatment of LDL with 1 micro M FeSO(4) at 4 degrees C for 96 h, the resulting MM-LDL showed a slight increase in thiobarbituric acid-reactive substances (TBARS) and little association with macrophages. On the other hand, heavily oxidized LDL, which was prepared by copper-induced oxidation of LDL at 37 degrees C, showed a sharp increase in TBARS and strong association with macrophages. By introducing a fluorometric procedure to detect aldehyde-containing phosphatidylcholines (aldehyde-PCs), we examined the amounts of aldehyde-PCs in modified LDL preparations. Aldehyde-PCs increased to 23.4 pmol/ microg protein in MM-LDL, which was more than four-fold higher than in the heavily oxidized LDL. We conclude that MM-LDL is a unique type of oxidized LDL enriched with aldehyde-PCs.

Serum high-density lipoprotein-cholesterol levels modify the association between plasma levels of oxidatively modified low-density lipoprotein and coronary artery disease in men.

We investigated the association among plasma levels of oxidatively modified low-density lipoprotein (Ox-LDL), high-density lipoprotein-cholesterol (HDL-C), and the prevalence of coronary artery disease (CAD) in a case-control study. Cases (n = 183, male [M]/female [F]:138/45, age: 64.9 +/- 10.6 years) were defined as patients with angiographically proven coronary atherosclerosis (>/=50% stenosis) and controls were subjects with normal coronary arteries (n = 74, M/F:36/38, age: 57.6 +/- 14.4 years). Plasma Ox-LDL levels were measured by a sensitive detection method using the monoclonal antibody DLH3. In women, both Ox-LDL and lipid variables were similar between cases and controls. In men, cases had significantly lower (P <.05) levels of HDL-C (39.1 +/- 10.3 v 42.8 +/- 10.9 mg/dL) and apolipoprotein (apo) A-I than controls, while the difference in Ox-LDL between cases and controls was not significant (1.05 +/- 0.79 and 0.83 +/- 0.65 ng/10 microg LDL protein, respectively). However, HDL-C levels interacted with the association between Ox-LDL levels and CAD in males: increased Ox-LDL levels were significantly associated with CAD after controlling for age when HDL-C levels were high, but were not associated with CAD when HDL-C levels were low, as assessed by a multiple logistic regression analysis. In addition, the combination of HDL-C and Ox-LDL levels was a better indicator for CAD in males than HDL-C levels alone (-2 log likelihood, 24.1 v 19.4) after controlling for age and conventional risk factors of CAD, while Ox-LDL levels were not significantly associated with CAD. HDL-C levels interact with the association between plasma Ox-LDL levels and CAD in men, and increased Ox-LDL levels are an indicator of CAD in male subjects with high HDL-C levels.

Gene expression profile in cerebrum in the filial imprinting of domestic chicks (Gallus gallus domesticus).

In newly hatched chicks, gene expression in the brain has previously been shown to be up-regulated following filial imprinting. By applying cDNA microarrays containing 13,007 expressed sequence tags, we examined the comprehensive gene expression profiling of the intermediate medial mesopallium in the chick cerebrum, which has been shown to play a key role in filial imprinting. We found 52 up-regulated genes and 6 down-regulated genes of at least 2.0-fold changes 3h after the training of filial imprinting, compared to the gene expression of the dark-reared chick brain. The up-regulated genes are known to be involved in a variety of pathways, including signal transduction, cytoskeletal organization, nuclear function, cell metabolism, RNA binding, endoplasmic reticulum or Golgi function, synaptic function, ion channel, and transporter. In contrast, fewer genes were down-regulated in the imprinting, coinciding with the previous data that the total RNA synthesis increased associated with filial imprinting. Our data suggests that the filial imprinting involves the modulation of multiple signaling pathways.

Up-regulation of microtubule-associated protein 2 accompanying the filial imprinting of domestic chicks (Gallus gallus domesticus).

Using cDNA microarrays, we have identified elsewhere the genes of microtubule-associated proteins as a group up-regulated in newly hatched chick brains after filial imprinting training. Here we show by in situ hybridization that the mRNA for the microtubule-associated protein 2 (MAP2) gene was enriched in the mesopallium and the hippocampus in the trained chick brain. The regionally specific enrichments of MAP2 mRNA were not observed in the brain of dark-reared or light-exposed chick as controls, implying an association between the degree of expression and the strength of the learned preference. In agreement with the gene expression, MAP2 protein was accumulated in the mesopallium of the trained chick brain, but not in the brains of the controls. The accumulation of MAP2 was found in the cytosol of neurons and co-localized with beta-tubulin, suggesting a change in microtubule assembly. Our results suggest a postnatal reorganization of cytoskeleton following filial imprinting.

Involvement of ACSL in local synthesis of neutral lipids in cytoplasmic lipid droplets in human hepatocyte HuH7.

Lipid droplets (LDs) function as intracellular storage depots of neutral lipids. Recently, we identified long-chain acyl-coenzyme A synthetase 3 (ACSL3) as a major LD-associated protein in the human hepatocyte cell line HuH7. In this study, we investigated whether droplet-associated ACSL is involved in lipid metabolism in LDs. Addition of oleic acid (OA) to culture medium was shown to enhance the intracellular accumulation of LDs in the cells, which was accompanied by an increase of droplet ACSL3. When LD-enriched cells induced by OA were further incubated without OA for 3 days, approximately 80% of LDs were retained in the cells. Conversely, cellular LD content was greatly decreased after the addition of an ACSL inhibitor, triacsin C. This was accompanied by a concomitant decrease of the droplet ACSL3. Incubation of isolated LD fractions with (14)C-labeled OA or palmitic acid resulted in [(14)C]acyl-CoA generation in vitro, indicating the presence of ACSL activity in LDs. The droplet ACSL activity varied according to the quantity of LDs in their emergence and disappearance in cells. Incubation of the LD fraction with [(14)C]oleoyl-CoA resulted in radioactive triacylglycerol and cholesteryl esters. These results suggest that LD ACSL activity is involved in local synthesis of neutral lipids and LD formation.

ADRP/adipophilin is degraded through the proteasome-dependent pathway during regression of lipid-storing cells.

Adipose differentiation-related protein (ADRP) is a major protein associated with lipid droplets in various types of cells, including macrophage-derived foam cells and liver cells. However, the role of ADRP in the processes of formation and regression of these cells is not understood. When J774 murine macrophages were incubated with either VLDL or oleic acid, their content of both ADRP and triacylglycerol (TG) increased 3- to 4-fold. Induction of ADRP during TG accumulation was also observed in oleic acid-treated HuH-7 human liver cells. Addition of triacsin C, a potent inhibitor of acyl-CoA synthase, for 6 h decreased the amount of TG in VLDL-induced foam cells and oleic acid-treated liver cells; it decreased the amount of ADRP protein in parallel, indicating the amount of ADRP reduced during regression of the lipid-storing cells. Addition of a proteasome inhibitor during triacsin C treatment abolished the ADRP decrease and accumulated polyubiquitinated ADRP. In addition, the proteasome inhibitor reversed not only the degradation of ADRP but also TG reduction by triacsin C. These results suggest that cellular amounts of ADRP and TG regulate each other and that the ubiquitin-proteasome system is involved in degradation of ADRP during regression of lipid-storing cells.

Transmembrane lipid transfer is crucial for providing neutral lipids during very low density lipoprotein assembly in endoplasmic reticulum.

Very low density lipoprotein (VLDL), a large particle containing apolipoprotein B (apoB) and large amounts of neutral lipids, is formed in the luminal space within the endoplasmic reticulum (ER) of hepatic cells. The assembly mechanism of VLDL particles is a tightly regulated process where apoB, associated with an insufficient amount of lipids, is selectively degraded intracellularly. In this study we found that treatment of HuH-7 human hepatoma cells with verapamil inhibited secretion of apoB-containing lipoprotein particles through increasing degradation of apoB. Addition of N-acetylleucyl-leucyl-norleucinal, an inhibitor of proteasome and other cysteinyl proteases that are responsible for apoB degradation, restored apoB recovery from verapamil-treated cells. De novo synthesis of lipids from [14C]acetate was increased in the presence of verapamil, suggesting that verapamil decreases lipid availability for apoB thus leading to the secretion of apoB-containing lipoprotein. We prepared cytosolic fractions from cells preincubated with [14C]acetate and used as a donor of radioactive lipids. When this cytosolic fraction was incubated with microsomes isolated separately, radioactive triglyceride (TG) accumulated in the luminal space of the microsomes. The transfer of radioactive TG from the cytosolic fraction to the microsomal lumen was inhibited in the presence of verapamil, suggesting that there is a verapamil-sensitive mechanism for TG transfer across ER membranes that is involved in formation of apoB-containing lipoprotein particles in ER. Verapamil showed no inhibitory effect on microsomal TG transfer protein, a well known lipid transfer protein in ER. We propose from these results that there is novel machinery for transmembrane movement of neutral lipids, which is involved in providing TG for apoB during VLDL assembly in ER.