We describe a modification of transverse pore gradient gel electrophoresis in which pore size is regulated by crosslinker proportion (%C) rather than by total monomer concentration (%T). We electrophoresed plasma lipoprotein [a] transversely across linear N,N'-methylenebisacrylamide gradients and measured mobility as a function of %C. This method allows for the simultaneous assessment of pore sizes generated over a wide range of crosslinker proportions.

Acrylamides↗

PubMedDate not supplied

Modification of human serum low density lipoprotein by oxidation--characterization and pathophysiological implications.

Plasma low density lipoprotein (LDL) can undergo free radical oxidation either catalyzed by divalent cations, such as Cu2+ or Fe2+ or promoted by incubation with cultured cells such as endothelial cells, smooth muscle cells and monocytes. The content of vitamin E, beta-carotene and unsaturated fatty acids is decreased in oxidized LDL. A breakdown of apolipoprotein-B (apoB), hydrolysis of the phospholipids, an increase of thiobarbituric acid reactive substances and the generation of aldehydes also occur. Changes in the ratio of lipid to protein, the electrophoretic mobility and the fluorescent properties have also been reported to accompany oxidation of this lipoprotein. The functional changes of oxidized LDL include its recognition by the scavenger receptor on macrophages, its cytotoxicity especially to proliferating cells, its chemotactic properties with respect to monocyte-macrophages and its regulation of platelet-derived growth factor-like protein (PDGFc) production by endothelial cells. In this article we summarize some of the contributions to this topic and present speculations relating oxidized LDL to pathological conditions such as atherosclerosis.

Free Radicals↗

PubMedDate not supplied

Stimulation of cholesteryl ester synthesis in macrophages by extracts of atherosclerotic human aortas and complexes of albumin/cholesteryl esters.

Cholesteryl ester-rich particles extracted from human atherosclerotic plaques were shown to increase the rate of incorporation of [14C]oleate into cholesteryl [14C]oleate and to cause massive accumulation of cholesteryl esters in monolayers of mouse peritoneal macrophages. This stimulation showed saturation kinetics and susceptibility to competition by polyanions (polyinosinic acid, fucoidin, dextran sulfate), suggesting that cell surface binding was required. Cellular uptake and lysosomal hydrolysis of the cholesteryl esters were also required, as indicated by the finding that stimulation of cholesteryl ester formation was prevented by the lysosomal inhibitor, chloroquine. The cholesterol esterification-stimulating activity of the aortic extracts was excluded on a 2% agarose column and floated in the density range of 1.006 to 1.063 g/ml. Cholesterol-rich extracts from human adrenal glands and liver did not stimulate cholesteryl ester formation in macrophages. The aortic extracts did not stimulate cholesteryl ester synthesis in human fibroblasts. Complexes of 125I-labeled albumin and cholesteryl linoleate formed in vitro were taken up and degraded in macrophages, but not in fibroblasts, by a process resembling the uptake of the aortic extracts. The current data suggest that macrophages express mechanisms for internalizing certain types of cholesteryl ester-rich lipid/protein complexes, including those present in atherosclerotic plaques.

Adult↗

PubMedDate not supplied

Lesion-derived low density lipoprotein and oxidized low density lipoprotein share a lability for aggregation, leading to enhanced macrophage degradation.

In this study we assessed whether low density lipoproteins (LDL) isolated from minced aortic atherosclerotic plaques obtained at autopsy (A-LDL) shared structural and functional properties with LDL oxidized by incubation with Cu2+ for 8-18 hours at 20 degrees C (Ox-LDL). Although both A-LDL and Ox-LDL represented monomeric particles about the size of LDL, both differed from LDL in that they showed an increase in electrophoretic mobility relative to LDL, an increase in cholesterol to protein ratio, and an increase in reactivity with a monoclonal antibody that recognizes epitopes on malondialdehyde (MDA)-modified proteins. In addition, both showed an increase in fluorescence at 360 nm excitation, 430 nm emission, an increase in fragmentation of apolipoprotein B with patterns that were quite similar, and an increase in recognition by the scavenger receptor on mouse peritoneal macrophages (MPMs) based on competition of 125I-A-LDL and 125I-Ox-LDL degradation by excess acetylated LDL. In addition, inhibition of degradation by MPMs of 125I-A-LDL and 125I-Ox-LDL by excess unlabeled Ox-LDL and A-LDL were similar. When MDA was added in increasing amounts to labeled LDL and A-LDL, less MDA was required to modify A-LDL than LDl to obtain ligands that were degraded by MPMs to the same degree. Finally, both A-LDL and Ox-LDL but not LDL underwent aggregation (increased metastability) when concentrated to levels exceeding 1 mg protein/ml and showed enhanced macrophage uptake via phagocytosis (inhibition by cytochalasin D). These results demonstrate that A-LDL and Ox-LDL share properties additional to those previously reported, suggesting that oxidation may be a major mode of modification of LDL accumulating in atherosclerotic lesions. This could lead to lipid loading of macrophages induced by phagocytosis of aggregated particles, in addition to unregulated uptake via the scavenger receptor of monomeric particles.

Acetylation↗

PubMedDate not supplied

Cholesterol esterification in macrophages. Stimulation by lipoproteins containing apo B isolated from human aortas.

A lipoprotein fraction possessing many of the characteristics of plasma low density lipoproteins (P-LDL) was isolated from homogenates of lesioned human aortas by affinity chromatography. In contrast to P-LDL, this fraction, termed A-LP, was found to be more electronegative than P-LDL and to stimulate cholesterol esterification in mouse peritoneal macrophages (MPM). This stimulation tended toward saturation at approximately 100 micrograms/ml lipoprotein cholesterol. Cholesterol esterification was partially inhibited by fucoidin, a competitive inhibitor of the scavenger receptor on MPM. These results suggest that A-LP is recognized by a high affinity binding site on MPM. Stimulation of cholesterol esterification in MPM by A-lP was inhibited by the lysosomotropic agent, chloroquine, indicating that degradation in lysosomes was a prerequisite for cholesterol esterification. Substantial degradation of apo B within the intact A-LP was demonstrated by SDS-PAGE and immunoblotting. This degradation could be responsible for the increased negative charge on A-LP, and the enhanced recognition of A-LP by a receptor on MPM. Stimulation of cholesterol esterification increased linearly over a 48-hour time period, suggesting that the receptor on MPM recognizing A-LP was not down-regulated. This unabated increase in cholesterol esterification resulted in massive accumulation of intracellular cholesteryl esters and a gradual increase in oil red O-positive droplets, giving the MPM the characteristic morphology of foam cells. If these mechanisms demonstrated in vitro also occur in the intact artery, they could explain how foam cells are formed within the fatty streak lesion.

Animals↗

PubMedDate not supplied

Modification of low density lipoprotein with 4-hydroxynonenal induces uptake by macrophages.

There is indirect evidence that the oxidation of low density lipoprotein (LDL) may be involved in the development of atherosclerosis. Modification of LDL by oxidation may lead to its unregulated uptake by intimal macrophages to form foam cells. Because of the complexity of events occurring during LDL oxidation, we have tested whether LDL modified directly with 4-hydroxynonenal (HNE), a major propagation product formed during lipid peroxidation and known to be present in oxidized LDL, could bring about lipid loading of macrophages. Modification was accomplished by incubating LDL with various concentrations of HNE up to 7.5 mM. When LDL was derivatized with lower concentrations of HNE, concentration-dependent increases were observed in the covalent binding of HNE to apolipoprotein B (apo B), the blockage of the epsilon-amino groups on lysine residues of apo B, and the relative electrophoretic mobility of LDL. Decreases were observed in degradation of the modified LDL by the J774 cell line, mouse peritoneal macrophages, and smooth muscle cells. Modification of LDL by incubation with the higher concentrations of HNE resulted in LDL aggregation. This modification was associated with marked increases in the macrophage degradation of LDL. Degradation of aggregated HNE-modified LDL increased linearly with incubation time, leading to lipid loading of these cells as observed by oil red O staining and cholesterol accumulation. Uptake appeared to occur by phagocytosis, since cytochalasin D, an inhibitor of phagocytosis, quantitatively inhibited uptake and degradation of labeled HNE LDL. Uptake did not appear to be mediated by either the LDL receptor or the scavenger receptor, since competition with excess amounts of LDL or acetyl LDL failed to inhibit degradation of labeled, aggregated HNE LDL. Saturation of degradation of HNE LDL by macrophages could be attributed, in part, to steric hindrance, since both excess HNE LDL and other particulate ligands could inhibit this degradation. These studies suggest that interaction of LDL with HNE formed during lipid peroxidation could be responsible for structural modifications leading to unregulated uptake of the lipoprotein by tissue macrophages. This could partially explain lipid loading or foam cell formation in atherosclerosis.

Aldehydes↗

PubMedDate not supplied

Localization of apo-lipoproteins in human carotid artery plaques.

Apoproteins from plasma lipoproteins were localized by immunofluorescence techniques in human carotid artery atherosclerotic lesions. These studies were performed in light of the possible importance of these apoproteins in both lipid metabolism and the pathogenesis of atherosclerosis. ApoA-I from high density lipoproteins, apoB from low density lipoproteins, and apoC-III from very low density lipoproteins were localized also as markers for their respective lipoproteins, since the latter cross-react immunologically. The three apoproteins were localized to the same regions of lesions as neutral lipids and, to some extent, fibrinogen. These regions consisted of bands of collagen fibers, usually deeper within the lesion, and the lipid core or atheroma of such advanced lesions. Although the superposition of localization for the three apoproteins and lipid was only 53%, it was suggested that deviation from complete superposition was due to the abrupt changes in lesion structure resulting from the focal nature of the atherosclerotic process. These results suggest that there is a broader specificity than previously implied of the interaction between such lesion components as connective tissue and extracellular lipid accumulations, and apoproteins from plasma lipoproteins. This interaction is believed to result in a net retention within atherosclerotic lesions of human extracranial arteries of these plasma-derived factors, either as free apoproteins or as native lipoproteins.

Apoproteins↗

PubMedDate not supplied

Apolipoprotein localization in human cranial arteries, coronary arteries, and the aorta.

Apolipoproteins from human plasma high density (HDL), low density (LDL) and very low density lipoproteins (VLDL) were visualized in human arteries employing immunofluorescence techniques. Comparison between the localization patterns in extracranial and intracranial arteries and those in coronary arteries and the aorta was made. ApoA-I from HDL, apoB from LDL, and apoC-III from VLDL, as well as neutral lipid, were all localized to connective tissue and extracellular lipid pools in atherosclerotic lesions, and only to areas of intimal thickening in grossly "uninvolved" arteries. The degree of superposition of localizations was similiar in each vascular bed, and within the error resulting from the structural changes due to the focal nature of the atherosclerotic process. These results suggest a broad specificity in localization of apolipoproteins in most arterial lesions, and suggest that no differences in apolipoprotein accumulation exist between extracranial and intracranial arteries, coronary arteries, or the aorta. Variations in prevalence for atherosclerosis in each arterial bed must be accounted for on other bases.

Adolescent↗

PubMedDate not supplied

Apolipoprotein B (apoB) retention in atherosclerotic intracranial arteries.

Low (LDL) and very low (VLDL) density lipoproteins retained in grossly normal and atherosclerotic human intracranial arteries have been quantitated using an electro-immunoassay directed against apolipoprotein B (apoB), the major protein of these two lipoprotein fractions. Buffer-homogenates of grossly normal arteries contained apoB amounts ranging from less than 0.04 to 1.58 microng/mg tissue dry weight, while those of atherosclerotic plaques gave values ranging from 0.80 to 3.9 microng per mg tissue dry weight. These results were consistent with immunofluorescence studies localizing apoB in these arteries. Plaques also contained a remaining fraction of tightly-bound apoB as evidenced by positive immunofluorescence in sections of pellets from buffer homogenates. This was in contrast to the negative results from grossly normal arteries. These results would suggest that retention of apoB by intracranial arteries correlates positively with vessel lesions. Arterial apoB is present in both grossly normal regions and plaques in a loosely-bound form, possibly representing intact lipoprotein. ApoB is also present in a tightly-bound form in plaques.

Adolescent↗