Effects of oxidized low-density lipoproteins on the hepatic microvasculature

Oxidized low-density lipoproteins (oxLDLs) are involved in proinflammatory and cytotoxic events in different microcirculatory systems. The liver is an important scavenger organ for circulating oxLDLs. However, the interaction of oxLDL with the hepatic microcirculation has been poorly investigated. The present study was conducted to examine the effects of differently modified oxLDLs on the hepatic microvasculature. C57Bl/6J mice were injected intravenously with low-density lipoprotein (LDL), or LDL oxidized for 3 h (oxLDL(3)) or 24 h (oxLDL(24)), at doses resembling oxLDL plasma levels in cardiovascular disease patients. Radioiodinated ligands were used to measure blood decay and organ distribution, and nonlabeled ligands to evaluate microcirculatory responses, examined by in vivo microscopy 30-60 min after ligand injection, immunohistochemistry, and scanning and transmission electron microscopy. Mildly oxLDL (oxLDL(3)) was cleared from blood at a markedly slower rate than heavily oxLDL (oxLDL(24)), but significantly faster than LDL (P < 0.01). Injected oxLDLs distributed to liver. OxLDL effects were most pronounced in central areas of the liver lobules where oxLDL(3) elicited a significant (P < 0.05) reduction in perfused sinusoids, and both oxLDL(3) and oxLDL(24) significantly increased the numbers of swollen endothelial cells and adherent leukocytes compared with LDL (P < 0.05). OxLDL-treated livers also exhibited increased intercellular adhesion molecule (ICAM)-1 centrilobular staining. Electron microscopy showed a 30% increased thickness of the liver sinusoidal endothelium in the oxLDL(3) group (P < 0.05) and a reduced sinusoidal fenestration in centrilobular areas with increased oxidation of LDL (P for linear trend <0.05). In conclusion, OxLDL induced several acute changes in the liver microvasculature, which may lead to sinusoidal endothelial dysfunction.     

Effect of probucol on the physical properties of low-density lipoproteins oxidized by copper

Human plasma low-density lipoproteins (LDL) were incubated with 10 microM probucol for 1 h at 37 degrees C. Probucol incorporation into the LDL was complete as judged by filtration through a 0.2-micron filter, ultracentrifugation, and gel filtration. LDL with and without probucol were incubated for up to 24 h with 5 microM Cu2+ at 37 degrees C. Copper oxidation increased the content of random structure in the LDL protein from 30% to 36% at the expense of beta-structure (which decreased from 22% to 16%) without a change in alpha-helical content as measured by circular dichroism spectroscopy. This loss of beta-structure was prevented by the presence of probucol in the LDL during the copper incubation. Probucol reduced the rate of increase of fluorescence during copper oxidation at 37 degrees C. After 6 h, the fluorescence intensity at 360-nm excitation and 430-nm emission was 30% less in probucol-containing samples. Probucol had no effect on the circular dichroic spectrum of LDL and only minimal effects (less than 5%) on the fluorescence emission spectrum at wavelengths below 500 nm. Two fluorescence peaks, with emission at 420 nm and excitation at 340 and 360 nm, are resolved in three-dimensional fluorescence spectra of oxidized LDL. Probucol reduces the intensity of both peaks equally. The binding of a highly reactive heparin (HRH) fraction to LDL was measured by titration of LDL with HRH in the presence of fluoresceinamine-labeled HRH. The decrease in fluorescence anisotropy of the labeled HRH is proportional to the concentration of bound HRH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptors for modified low-density lipoproteins on human endothelial cells: different recognition for acetylated low-density lipoprotein and oxidized low-density lipoprotein

We examined the uptake pathway of acetylated low-density lipoprotein and oxidatively modified LDL (oxidized LDL) in human umbilical vein endothelial cells in culture. Proteolytic degradation of 125I-labeled Ac-LDL or Ox-LDL in the confluent monolayer of human endothelial cells was time-dependent and showed saturation kinetics in the dose-response relationship, which suggests that their incorporation is receptor-mediated. Cross-competition studies between acetylated LDL and oxidized LDL showed that the degradation of 125I-labeled acetylated LDL was almost completely inhibited by excess amount of unlabeled acetylated LDL, while only partially inhibited by excess unlabeled oxidized LDL. On the other hand, the degradation of 125I-labeled oxidized LDL was equally inhibited by excess amount of either acetylated or oxidized LDL. Cross-competition results of the cell-association assay paralleled the results shown in the degradation assay. These data indicate that human endothelial cells do not have any additional receptors specific only for oxidized LDL. On the contrary, they may have additional receptors, as we previously indicated on mouse macrophages, which recognize acetylated LDL, but not oxidized LDL.     

Time-resolved fluorometric assay for measuring cell binding and association of native and oxidized low-density lipoproteins to macrophages

Europium-labeled native and oxidized low-density lipoprotein (LDL) were used to measure their binding and cell association to mouse peritoneal macrophages, to suspended human monocyte cell line THP-1 cells, and to differentiated THP-1 macrophages. Cell binding and association were concentration dependent and saturable and showed the characteristics of ligand-receptor interaction. The validity of this assay was also supported by comparison with the method using 125iodine-labeled LDL. This nonradioactive assay proved to be specific, sensitive and simple and avoided any potential lipid peroxidation of LDL brought about by labeling lipoproteins with the widely used radioactive iodine. The latter fact is very important in studying lipoprotein-receptor interactions.     

Effect of low-density lipoprotein on the expression of high affinity Fc gamma receptors

A substrain of the human monocyte-like cell line U937, which is a cholesterol auxotroph, was used to study the effect of cellular cholesterol depletion on the expression of the type I Fc receptor for IgG (Fc gamma RI). Measurement of Fc gamma RI expression was performed by immunofluorescence and flow cytometry using the monoclonal antibody (mAb) 32.2, which is specific for an epitope on Fc gamma RI, and monomeric IgG2a, which binds to the ligand binding site of Fc gamma RI. Incubation of these cells for 24 h in growth medium containing delipidated fetal calf serum depletes cellular cholesterol without affecting growth or viability. While incubation of U937 cells with human interferon-gamma (IFN-gamma) increased Fc gamma RI expression, cholesterol depletion after cell growth in media containing delipidated serum and IFN-gamma resulted in reduced binding of both mAb 32.2 and IgG2a. A significant decrease in the number of cell surface binding sites, as measured by mean fluorescence intensity, was observed after cholesterol depletion. Supplementation of the delipidated serum medium with pure cholesterol in an ethanol/bovine serum albumin mixture, which replenished cellular cholesterol and supported growth, failed to restore antibody binding significantly. In contrast, low-density lipoprotein (LDL) which also delivered cholesterol to the cells restored binding both in terms of the number of the reactive cells and cell surface receptor density. High-density lipoprotein (HDL3), which does not deliver cholesterol to the cells, showed results similar to those obtained with pure cholesterol. This indicates that either LDL cholesterol is better utilized for membrane synthesis than pure cholesterol or that LDL provides another component, in addition to cholesterol, which is required for expression of Fc gamma RI, but not for growth. These studies indicate a role for LDL in regulating the expression of Fc gamma RI on the cell surface.     

Characterization of native and oxidized human low-density lipoproteins by the Z-scan technique

The nonlinear optical response of human normal and oxidized by Cu2+ low-density lipoproteins particles (LDL), were investigated by the Z-scan technique as a function of temperature and concentration of LDL particles. The Z-scan signals increase linearly with concentration of normal LDL particles, following the usual Beer-Lambert law in a broad range of concentrations. The oxidized LDL particles do not show nonlinear optical response. On the other hand, normal LDL increases its nonlinear optical response as a function of temperature. These behaviors can be attributed to an absorbing element that is modified by the oxidative process. Contrarily, changes in the physical state of the cores and conformation of the ApoB100 protein due to an increase in temperature seems to enhance their nonlinear optical properties. This tendency is not due to aggregation of particles. The main contribution to the nonlinear optical response of normal LDL particles comes from the phospholipid fraction of the particles.     

Human venous and arterial glycosaminoglycans have similar affinity for plasma low-density lipoproteins

We compared the glycosaminoglycan content of human venous and arterial walls. The most abundant glycosaminoglycan in human veins is dermatan sulfate whereas chondroitin 4/6-sulfate is preponderant in arteries. The concentrations of chondroitin 4/6-sulfate and heparan sulfate are approximately 4.8- and approximately 2.5-fold higher in arteries than in veins whereas dermatan sulfate contents are similar in the two types of blood vessels. Normal and varicose saphenous veins do not differ in their glycosaminoglycan contents. It is known that certain glycosaminoglycan species from the arterial wall, mainly high-molecular-weight fractions of dermatan sulfate+chondroitin 4/6-sulfate have greater affinity for plasma LDL. These types of glycosaminoglycans can be identified on a LDL-affinity column. We now demonstrated that a similar population of glycosaminoglycan also occurs in veins, although with a lower concentration than in the arteries due to less chondroitin 4/6-sulfate with affinity for LDL. The concentrations of dermatan sulfate species, which interact with LDL, are similar in arteries and veins. The presence of these glycosaminoglycans with affinity to plasma LDL in veins raises interesting questions concerning the role of these molecules in the pathogenesis of atherosclerosis. Possibly, the presence of these glycosaminoglycans in the vessel wall are not sufficient to cause retention of LDL and consequently endothelial dysfunction, but may require additional intrinsic factors and/or the hydrodynamic of the blood under the arterial pressure.     

Role of lysine residues of plasma lipoproteins in high affinity binding to cell surface receptors on human fibroblasts.

The low density lipoprotein (LDL) cell surface receptors on human fibroblasts grown in culture bind specific plasma lipoproteins, initiating a series of events which regulate intracellular cholesterol metabolism. Specificity for the interaction with the receptors resides with the protein moieties of the lipoproteins, specifically with the B and E apoproteins of LDL and certain high density lipoproteins (HDLc HDLl), respectively. It was previously established that the amino acid arginine is a functionally significant residue in or near the recognition sites on the B and E apoproteins and that modification of this residue abolishes the ability of these apolipoproteins to bind to the receptor. The present study indicates that lysine residues are also involved in the lipoprotein-receptor interaction. Chemical modification of 15% of the lysine residues of LDL by carbamylation with cyanate or 20% by acetoacetylation with diketene prevents the LDL from competitively displacing unmodified 125I-LDL from the high affinity receptor sites or from binding directly to the receptor. Moreover, quantitative reversal of the aceto-acetylation of the lysine residues of LDL by hydroxylamine treatment regenerates the lysyl residues and reestablishes greater than 90% of the original binding activity of the LDL. The reversibility of this reaction establishes that the loss of binding activity which follows lysine modification is not due to an irreversible alteration of the LDL or HDLc but is probably due to an alteration of a property of the recognition site associated with specific lysine residues. While acetoacetylation and carbamylation neutralize the positive charge on the epsilon-amino group of lysine, reductive methylation selectively modifies lysine residues of LDL and HDLc without altering the positive charge, yet abolishes their ability to bind to the receptor. Preservation of the charge but loss of binding activity following reductive methylation of the lipoproteins suggests that the specificity of the recognition site does not reside simply with the presence of positive charges but depends on other more specific properties of the site determined by the presence of a limited number of the lysine (and arginine) residues. The precise role of lysine remains to be defined, but its function may be to establish and maintain the conformation of the recognition site or the alignment of reactive residues, or both, or to chemically react, through its epsilon-amino group, with the receptor (hydrogen bond formation would be such a possibility).     

Influence of oxidized low-density lipoproteins (LDL) on the viability of osteoblastic cells

Cardiovascular diseases have recently been noted as potential risk factors for osteoporosis development. Although it is poorly understood how these two pathologies are related, it is a known fact that oxidized low-density lipoproteins (OxLDL) constitute potential determinants for both of them. The current study investigated the metabolism of OxLDL by osteoblasts and its effect on osteoblastic viability. The results obtained show that OxLDL are internalized but not degraded by osteoblasts while they can selectively transfer their CE to these cells. It is also demonstrated that OxLDL induce proliferation at low concentrations but cell death at high concentrations. This reduction of osteoblast viability was associated with lysosomal membrane damage caused by OxLDL as demonstrated by acridine orange relocalization. Accordingly, chloroquine, an inhibitor of lysosomal activity, accentuated cell death induced by OxLDL. Finally, we demonstrate that osteoblasts have the capacity to oxidize LDL and thereby potentially increase the local concentration of OxLDL. Overall, the current study confirms the potential role of OxLDL in the development of osteoporosis given its influence on osteoblastic viability.     

Gadolinium chloride-induced Kupffer cell blockade increases uptake of oxidized low-density lipoproteins by rat heart and aorta.

Oxidized low-density lipoproteins (LDL) play a key role in the formation of atherosclerotic lesions of arteries. We analyzed the effect of hepatic resident macrophage (Kupffer cell) blockade on oxidized [125I]LDL accumulation in different organs and tissues of the rat. Kupffer cell blockade was induced by gadolinium chloride (GdCl3) which was injected intravenously 24 h prior to injection of oxidized [125I]LDL into the rats. Ten minutes after administration to intact animals, oxidized [125I]LDL was accumulated in the liver (86.8% of the dose administered), muscles (4.7%), spleen (2.1%), lungs (0.8%), kidney (0.6%), adrenal glands (0.2%), heart (0.15%), and thymus (0.04%). Kupffer cell blockade significantly decreased the clearance rate of oxidized [125I]LDL from the blood. Specific radioactivity (per g tissue) decreased in the liver (1.3-fold compared to control), but increased in the aorta (2.5-fold), heart (2-fold), lungs (1.6-fold), and kidney (1.3-fold). The results indicate that the accumulation of oxidized LDL in heart and aorta significantly depends on the functional state of the mononuclear phagocyte system in the liver.     

Statins and foam cell formation: impact on LDL oxidation and uptake of oxidized lipoproteins via scavenger receptors

The uptake of oxidized lipoproteins via scavenger receptors and the ensuing formation of foam cells are key events during atherogenesis. Foam cell formation can be reduced by treatment with 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins). The efficacy of statins is evidently due not only to their cholesterol-lowering properties, but also to lipid-independent pleiotropic effects. This review focuses on lipid-independent pleiotropic effects of statins that influence foam cell formation during atherogenesis, with special emphasis on oxidative pathways and scavenger receptor expression.     

A zebrafish model for the rapid evaluation of pro-oxidative and inflammatory death by lipopolysaccharide, oxidized low-density lipoproteins, and glycated high-density lipoproteins

Oxidation and inflammation are leading causes of nearly all chronic metabolic disorders, and play major roles in cardiovascular disease, cancer, and chronic age-dependent disease. High-density lipoprotein (HDL) and apolipoprotein (apo) A-I have strong antioxidant and anti-inflammatory properties in the plasma. Fructose-induced non-enzymatic glycation of apoA-I can lead to the production of dysfunctional apoA-I and HDL. To compare the physiologic effects of dysfunctional apoA-I and HDL, reconstituted HDL containing native apoA-I (nA-I) or glycated apoA-I (gA-I) was injected into zebrafish embryos in the presence of inflammatory molecules. Co-injection of reconstituted HDL containing VLDL and LDL gA-I (gA-I-rHDL) and lipopolysaccaride (LPS) resulted in acute embryo deaths, while rHDL containing nA-I (nA-I-rHDL) and LPS resulted in significantly enhanced survival. Co-injection of oxidized LDL (oxLDL) and nA-I-rHDL improved embryo survival, while co-injection of oxLDL and gA-I-rHDL aggravated inflammatory deaths. Furthermore, co-injection of oxLDL and HDL(2) (5 ng of protein) or HDL(3) (15 ng of protein) from the young group (22 ± 2 years old) showed significantly increased embryo survival compared with the same co-injection of HDL from the elderly group (71 ± 4 years old). In conclusion, our assay system provides a rapid and economic method to screen antioxidant and anti-inflammatory agents using zebrafish embryos.     

Role of liver sinusoidal endothelial cells and stabilins in elimination of oxidized low-density lipoproteins

Atherogenesis is associated with elevated levels of low-density lipoprotein (LDL) and its oxidized form (oxLDL) in the blood. The liver is an important scavenger organ for circulating oxLDLs. The present study aimed to examine endocytosis of mildly oxLDL (the major circulating form of oxLDLs) in liver sinusoidal endothelial cells (LSECs) and the involvement of the scavenger receptors stabilin-1 and stabilin-2 in this process. Freshly isolated LSECs, Kupffer cells (KCs), and stabilin-1- and stabilin-2-transfected human embryonic kidney cells were incubated with fluorescently labeled or radiolabeled oxLDLs [oxidized for 3 h (oxLDL(3)), 6 h, or 24 h (oxLDL(24))] to measure endocytosis. The intracellular localization of oxLDLs and stabilins in LSECs was examined by immunofluorescence and immunogold electron microscopy. Whereas oxLDL(24) was endocytosed both by LSECs and KCs, oxLDL(3) (mildly oxLDL) was taken up by LSECs only. The LSEC uptake of oxLDLs was significantly inhibited by the scavenger receptor ligand formaldehyde-treated serum albumin. Uptake of all modified LDLs was high in stabilin-1-transfected cells, whereas stabilin-2-transfected cells preferentially took up oxLDL(24), suggesting that stabilin-1 is a more important receptor for mildly oxLDLs than stabilin-2. Double immunogold labeling experiments in LSECs indicated interactions of stabilin-1 and stabilin-2 with oxLDL(3) on the cell surface, in coated pits, and endocytic vesicles. LSECs but not KCs endocytosed mildly oxLDL. Both stabilin-1 and stabilin-2 were involved in the LSEC endocytosis of oxLDLs, but experiments with stabilin-transfected cells pointed to stabilin-1 as the most important receptor for mildly oxLDL.     

Effect of fish oils on rat plasma lipoproteins

Plasma high (HDL) and low (LDL) density lipoproteins were isolated from rats fed a diet supplemented with either fish (menhaden) oil or hydrogenated coconut oil (control). Fluorescence polarization and electron spin resonance of labelled fatty acid probe molecules incorporated into the outer amphiphilic monolayer of HDL indicate molecular motion is restricted in the upper portion of the acyl chain following the fish oil diet, which is consistent with a 'hook' conformation predicted by preliminary molecular model calculations for n-3 fatty acids (the predominant component of fish oil). Negligible dependence on diet was observed in LDL. Thus, a HDL specific effect of dietary fish oil on molecular fluidity and order in the outer monolayer of rat lipoproteins is suggested.     

Effect of gangliosides on the copper-induced oxidation of human low-density lipoproteins

The role of gangliosides in the copper-induced oxidative modification of human low-density lipoprotein (LDL) was studied focusing on the early stage of LDL oxidation in which the concentration of conjugated dienes increases only weakly. The changes in the protein and lipid component were followed using fluorescence spectroscopy. The results indicate that binding of gangliosides to LDL causes slower destruction of tryptophan fluorescence and suppresses cross-linking between the reactive groups of the protein and the products of lipid peroxidation. The protective role of gangliosides could be assigned to their interference with the lipid-protein interaction in the LDL particle, which might be important for the maintenance of the native plasma antioxidant status in vivo.     

Morphological studies on the binding of low-density lipoproteins and acetylated low-density lipoproteins to the plasma membrane of cultured monocytes

Binding of low density lipoproteins (LDL) and acetyl-LDL to the plasma membrane of cultured swine monocytes was investigated by immunofluorescent and immunoelectron microscopy. Binding sites for native LDL, visualized on both the light microscopical and the ultrastructural level, were found to be comparable to those of cultured human fibroblasts. These techniques, however, failed to reveal binding of acetyl-LDL to the cell surface. Biochemical experiments showed that both LDL and acetyl-LDL have specific receptors, the acetyl-LDL receptor being distinctly different from the LDL receptor. It is concluded that there are morphological differences in the binding of LDL and acetyl-LDL to cultured monocytes. These differences are supported by biochemical data.     

Unsaturated long-chain fatty acids inhibit the binding of oxidized low-density lipoproteins to a model CD36

Transmembrane protein CD36 binds multiple ligands, including oxidized low-density lipoproteins (oxLDLs) and long-chain fatty acids (LCFAs). Our aim was to determine whether LCFAs compete with oxLDLs for binding to CD36. We addressed this issue by examining the inhibitory effect of LCFAs against the binding of Alexa-fluor-labeled oxLDLs (AFL-oxLDL) to a synthetic peptide representing the oxLDL-binding site on CD36 (3S-CD36_150–168). All of the unsaturated LCFAs tested, inhibited the binding of AFL-oxLDL to 3S-CD36_150–168, albeit to varying degrees. For instance, the concentrations required for 50% inhibition of binding for oleic, linoleic, and α-linolenic acids were 0.25, 0.97, and 1.2?mM, respectively. None of the saturated LCFAs tested (e.g. stearic acid) exhibited inhibitory effects. These results suggest that at least unsaturated LCFAs can compete with oxLDLs for binding to CD36. The study also provides information on the structural requirements of LCFAs for inhibition of oxLDLs–CD36 binding.     

Detection of distinct receptors for native and acetylated low-density lipoproteins in human placental microvilli by ligand-immunoblotting

Ligand-immunoblotting was used to detect distinct receptors for native low-density lipoprotein and for acetylated low-density lipoprotein on microvillous membranes from human term placentas. Antisera directed against native and modified low-density lipoproteins were prepared in rabbits and their specificities were assessed by immunodiffusion and immunoelectrophoresis. The receptor for low-density lipoprotein was detected as a 160 kDa protein and that for acetylated low-density lipoprotein as a 200 kDa protein. These receptors were compared with their counterparts in cultured human skin fibroblasts, bovine adrenal cortex and J774 macrophage-like cells. This is the first investigation that visualizes the presence of receptors for both native and modified low-density lipoproteins in a steroidogenic tissue.     

Effect of the proteolysis of low-density serum lipoproteins on their interaction with macrophages

125I-labelled human serum low density lipoproteins (LDL) were incubated with cultured mouse peritoneal macrophages at 37 degrees C, with the following study of cellular uptake and 125I-LDL degradation by measuring the content of TCA-soluble products of LDL hydrolysis in the cultural medium. It was shown that limited pepsin proteolysis of LDL (10%) led to a more effective LDL uptake and degradation by macrophages. The data suggest that enzyme-induced modification of LDL may increase their atherogenicity.