Inactivation of lysosomal proteases by oxidized low density lipoprotein is partially responsible for its poor degradation by mouse peritoneal macrophages.

Deficient processing of apo B in oxidized LDL (ox-LDL) by macrophage lysosomal proteases has been documented and attributed to modifications in apo B. We have investigated whether direct inactivation of lysosomal proteases by ox-LDL could also be responsible for this deficient degradation. When mouse peritoneal macrophages (MPM) were preincubated for 21 h at 37 degrees C with ox-LDL, LDL, or vortex-aggregated LDL, only ox-LDL inhibited the subsequent degradation of 125I-labeled forms of the above lipoproteins. Uptake of labeled lipoproteins was not appreciably affected by preincubation with ox-LDL, suggesting that the inhibition was at the level of lysosomal degradation. Thiol protease activity of cell extracts at pH 4.0, was reduced in MPM preincubated with ox-LDL relative to cells preincubated with LDL or medium alone. Extracts from untreated MPM, or mixtures of cathepsin B and D, showed a reduced ability to degrade 125I-LDL at pH 4.5 and reduced cathepsin B activity, after incubation with ox-LDL relative to incubation with LDL. Thus, the reduced degradation of lipoproteins in MPM pretreated with ox-LDL could be due to direct inactivation of the lysosomal protease, cathepsin B.

Myeloperoxidase-generated reactive nitrogen species convert LDL into an atherogenic form in vitro.

Oxidized LDL is implicated in atherosclerosis; however, the pathways that convert LDL into an atherogenic form in vivo are not established. Production of reactive nitrogen species may be one important pathway, since LDL recovered from human atherosclerotic aorta is enriched in nitrotyrosine. We now report that reactive nitrogen species generated by the MPO-H2O2-NO2- system of monocytes convert LDL into a form (NO2-LDL) that is avidly taken up and degraded by macrophages, leading to massive cholesterol deposition and foam cell formation, essential steps in lesion development. Incubation of LDL with isolated MPO, an H2O2-generating system, and nitrite (NO2-)-- a major end-product of NO metabolism--resulted in nitration of apolipoprotein B 100 tyrosyl residues and initiation of LDL lipid peroxidation. The time course of LDL protein nitration and lipid peroxidation paralleled the acquisition of high-affinity, concentration-dependent, and saturable binding of NO2-LDL to human monocyte-derived macrophages and mouse peritoneal macrophages. LDL modification and conversion into a high-uptake form occurred in the absence of free metal ions, required NO2-, occurred at physiological levels of Cl-, and was inhibited by heme poisons, catalase, and BHT. Macrophage binding of NO2-LDL was specific and mediated by neither the LDL receptor nor the scavenger receptor class A type I. Exposure of macrophages to NO2-LDL promoted cholesteryl ester synthesis, intracellular cholesterol and cholesteryl ester accumulation, and foam cell formation. Collectively, these results identify MPO-generated reactive nitrogen species as a physiologically plausible pathway for converting LDL into an atherogenic form.

Targeted disruption of the class B scavenger receptor CD36 protects against atherosclerotic lesion development in mice.

Macrophage scavenger receptors have been implicated as key players in the pathogenesis of atherosclerosis. To assess the role of the class B scavenger receptor CD36 in atherogenesis, we crossed a CD36-null strain with the atherogenic apo E-null strain and quantified lesion development. There was a 76.5% decrease in aortic tree lesion area (Western diet) and a 45% decrease in aortic sinus lesion area (normal chow) in the CD36-apo E double-null mice when compared with controls, despite alterations in lipoprotein profiles that often correlate with increased atherogenicity. Macrophages derived from CD36-apo E double-null mice bound and internalized more than 60% less copper-oxidized LDL and LDL modified by monocyte-generated reactive nitrogen species. A similar inhibition of in vitro lipid accumulation and foam cell formation after exposure to these ligands was seen. These results support a major role for CD36 in atherosclerotic lesion development in vivo and suggest that blockade of CD36 can be protective even in more extreme proatherogenic circumstances.

Macrophage scavenger receptor CD36 is the major receptor for LDL modified by monocyte-generated reactive nitrogen species.

The oxidative conversion of LDL into an atherogenic form is considered a pivotal event in the development of cardiovascular disease. Recent studies have identified reactive nitrogen species generated by monocytes by way of the myeloperoxidase-hydrogen peroxide-nitrite (MPO-H(2)O(2)-NO(2)(-)) system as a novel mechanism for converting LDL into a high-uptake form (NO(2)-LDL) for macrophages. We now identify the scavenger receptor CD36 as the major receptor responsible for high-affinity and saturable cellular recognition of NO(2)-LDL by murine and human macrophages. Using cells stably transfected with CD36, CD36-specific blocking mAbs, and CD36-null macrophages, we demonstrated CD36-dependent binding, cholesterol loading, and macrophage foam cell formation after exposure to NO(2)-LDL. Modification of LDL by the MPO-H(2)O(2)-NO(2)(-) system in the presence of up to 80% lipoprotein-deficient serum (LPDS) still resulted in the conversion of the lipoprotein into a high-uptake form for macrophages, whereas addition of less than 5% LPDS totally blocked Cu(2+)-catalyzed LDL oxidation and conversion into a ligand for CD36. Competition studies demonstrated that lipid oxidation products derived from 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine can serve as essential moieties on NO(2)-LDL recognized by CD36. Collectively, these results suggest that MPO-dependent conversion of LDL into a ligand for CD36 is a likely pathway for generating foam cells in vivo. MPO secreted from activated phagocytes may also tag phospholipid-containing targets for removal by CD36-positive cells.

Formation of nitric oxide-derived oxidants by myeloperoxidase in monocytes: pathways for monocyte-mediated protein nitration and lipid peroxidation In vivo.

Protein nitration and lipid peroxidation are implicated in the pathogenesis of atherosclerosis; however, neither the cellular mediators nor the reaction pathways for these events in vivo are established. In the present study, we examined the chemical pathways available to monocytes for generating reactive nitrogen species and explored their potential contribution to the protein nitration and lipid peroxidation of biological targets. Isolated human monocytes activated in media containing physiologically relevant levels of nitrite (NO(2)(-)), a major end product of nitric oxide ((*)NO) metabolism, nitrate apolipoprotein B-100 tyrosine residues and initiate LDL lipid peroxidation. LDL nitration (assessed by gas chromatography-mass spectrometry quantification of nitrotyrosine) and lipid peroxidation (assessed by high-performance liquid chromatography with online tandem mass spectrometric quantification of distinct products) required cell activation and NO(2)(-); occurred in the presence of metal chelators, superoxide dismutase (SOD), and scavengers of hypohalous acids; and was blocked by myeloperoxidase (MPO) inhibitors and catalase. Monocytes activated in the presence of the exogenous (*)NO generator PAPA NONOate (Z-[N-(3-aminopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2- diolate) promoted LDL protein nitration and lipid peroxidation by a combination of pathways. At low rates of (*)NO flux, both protein nitration and lipid peroxidation were inhibited by catalase and peroxidase inhibitors but not SOD, suggesting a role for MPO. As rates of (*)NO flux increased, both nitrotyrosine formation and 9-hydroxy-10,12-octadecadienoate/9-hydroperoxy-10,12-octadecadieno ic acid production by monocytes became insensitive to the presence of catalase or peroxidase inhibitors, but they were increasingly inhibited by SOD and methionine, suggesting a role for peroxynitrite. Collectively, these results demonstrate that monocytes use distinct mechanisms for generating (*)NO-derived oxidants, and they identify MPO as a source of nitrating intermediates in monocytes.

Lipoproteins containing apolipoprotein A-I extracted from human aortas.

Apolipoprotein A-I was quantitated by electroimmunoassay in buffer-soluble fractions of both grossly normal intima and raised atherosclerosis lesions of the human aorta. The mean value for apolipoprotein A-I content in microgram/mg tissue dry weight of normal intima (12 cases) was 0.71 +/- 0.10 S.E. and of aortic plaques (19 cases) was 0.64 +/- 0.40 S.E. When compared to the buffer-extractable apolipoprotein B content measured in these same cases from both regions, the ratio of apolipoprotein B to apolipoprotein A-I was approximately 6. No apolipoprotein A-I was measurable in tunica media. Following differential ultracentrifugation into d less than 1.063, d 1.063-1.21 and d greater than 1.21 fractions, the distributions of recovered apolipoprotein A-I were, respectively: 1, 94 and 5% for normal intima, 19, 31 and 50% for plaques and 1, 89 and 10% for plasma. Characterization of a chromatographically purified d 1.063-1.21 or HDL density fraction from fatty-fibrous plaques demonstrated particles of between 60 and 120 A diameter, a characteristic apolipoprotein A-I band by SDS-polyacrylamide gel electrophoresis, and a precipitin peak closely migrating with that for plasma HDL by two-dimensional immunoelectrophoresis. The d greater than 1.21 density fraction from plaques isolated by affinity chromatography on a Sepharose-anti-apolipoprotein A-I column contained small amounts of phospholipid but no measurable cholesterol. The d 1.063-1.21 density fraction from plaques showed a significant increase in percent free cholesterol and phospholipid contents and decrease in cholesteryl ester content relative to plasma HDL. This increase in free cholesterol could represent evidence for an anti-atherogenic mechanism wherein infiltrated HDL removes cholesterol together with phospholipid from the arterial wall.

Non-conventional modification of low density lipoproteins: chemical models for macrophage recognition of oxidized LDL.

To define the structural and chemical criteria governing recognition of oxidized LDL (oxLDL) by mouse peritoneal macrophages (MPM), we exposed LDL to novel chemical modification agents that induce defined neutralizing and non-neutralizing alterations of lysine as models for distinct apoB adducts present in oxLDL. We found some exceptions to the usual notion that neutralization of lysine positive charges is the principal determinant governing MPM recognition. In addition, competitive binding experiments using chemically modified 125I-LDL preparations revealed that, whereas some modifications engendered recognition principally by the classical scavenger receptor class A (SRA), as seen for acetylated LDL (acLDL), chemical models of advanced aldehydic modifications of LDL led instead to MPM uptake mainly by oxLDL receptors distinct from SRA.

Macrophage recognition of LDL modified by levuglandin E2, an oxidation product of arachidonic acid.

Levuglandin (LG) E2, a secoprostanoic acid levulinaldehyde derivative, is a product of free radical oxidation that forms covalent adducts with lysyl residues on proteins. Treatment of LDL with LGE2 leads to uptake and degradation by mouse peritoneal macrophages. Oxidized LDL, but not acetyl LDL efficiently competed for binding and uptake of LGE2-modified 125I-LDL. This result suggests that LGE2-modified LDL was recognized by a class of scavenger receptor that demonstrated ligand specificity for oxidized LDL but not for acetyl LDL.

Characterization of low density lipoprotein-like particle in the human aorta from grossly normal and atherosclerotic regions.

Physical and chemical criteria of lipoproteins containing apolipoprotein B, extracted from human aortic intima, were compared with those of plasma low density lipoproteins (LDL). Homogenates of grossly normal intima and advanced atherosclerotic lesions were subjected to differential ultracentrifugation to isolate a d = 1.006--1.063 g/ml density fraction which was extensively characterized. By electroimmunoassay, over 90% of the recovered apolipoprotein B immunological reactivity was found in isolates from both plaques and normal intima. In isolates of plaque and normal intima, particles of the same size as LDL were found, although a small population of very large structures was also present in plaque fractions. Apolipoprotein composition was similar to that of plasma LDL except for the presence of human serum albumin in aortic isolates. Fractions from aorta demonstrated greater electrophoretic mobilities than LDL. The lipid composition of isolates from normal intima was similar to that of LDL. The lipid composition of plaque fractions showed a significant decrease in the cholesteryl ester to free cholesterol ratio and in the triglyceride content in comparison to LDL and to fractions from normal intima. The fatty acid pattern of the cholesteryl ester fraction from isolates of both normal and plaque aortic homogenates demonstrated a significant decrease in the linoleate to oleate ratio as compared to LDL. Our initial studies suggest that althought aortic fractions are similar to LDL by certain criteria, some differences observed are more pronounced in fractions from lesions than from normal intima.

Inactivation of cathepsin B by oxidized LDL involves complex formation induced by binding of putative reactive sites exposed at low pH to thiols on the enzyme.

We recently showed that the poor degradation of apo B in oxidized (ox-) LDL by mouse peritoneal macrophages could be attributed to the inactivation of cathepsin B by ox-LDL. In this current study, we show that enzyme inactivation involves complex formation of ox-LDL with cathepsin B rather than the diffusion of reactive components from ox-LDL to the enzyme. Complex formation between ox-LDL and cathepsin B was far greater at pH 4.5 than at pH 7.4 and far greater with ox-LDL than with LDL. Even though complexes were also formed between ox-LDL and other proteins such as BSA, insulin, and LDL, ox-LDL bound up to 30 times more cathepsin B than BSA, when compared on a molar level and under the same conditions. Unlike ox-LDL alone, complexes of ox-LDL and BSA were unable to inactive cathepsin B, suggesting that BSA was sequestering reactive sites on ox-LDL. The interaction of ox-LDL with proteins such as cathepsin B appears to represent aldehydic modifications of apo B, since treatment of ox-LDL with the reductant NaBH4, which stabilizes such adducts, greatly decreased the binding of ox-LDL to BSA and prevented ox-LDL from inactivating cathepsin B. It is likely that thiols on cathepsin B or other proteins interact with reactive groups on ox-LDL, since BSA in which thiols were blocked with N-ethylmaleimide (NEM), failed to bind to ox-LDL. Moreover, NEM-treated BSA had no effect on the ability of ox-LDL to inactivate cathepsin B. Similar results were obtained with LDL modified with 4-hydroxynonenal (HNE). These data suggest that aldehydic adducts on ox-LDL that are unreactive at neutral pH, possibly HNE bound to apo B, become exposed at acidic pH and then covalently bind thiols on neighboring proteins such as cathepsin B in lysosomes, inducing crosslinking of proteins and enzyme inactivation.

APO low density lipoprotein localization. Intracranial and extracranial atherosclerotic lesions from human normolipoproteinemics and hyperlipoproteinemics.

APO low density lipoprotein (apoB), the major protein in plasma low (LDL) and very low (VLDL) density lipoproteins, was localized in extracranial and intracranial arteries from normolipoproteinemics and hyperlipoproteinemics to determine if apoB extensiveness and localization varied with plasma lipoprotein profile. Specimens of carotid bifureation, internal carotid, basilar, and middle cerebral arteries from 23 subjects with normal lipoprotein levels, four with elevated LDL (type II), and 13 with elevated VLDL (type IV) values were studied with the employment of immunofluorescence techniques. Although the apoB localization pattern was identical in each group, extensiveness of positive localization was greatest in lesions from type II cases and the same in lesions from type IV and normolipoproteinemics. This suggests that sites of apoB retention are dependent on the chemical and structural changes in atherosclerotic arteries, whereas extensiveness correlates with the apoB concentration gradient between plasma and tissue.

Extracts of human atherosclerotic lesions can modify low density lipoproteins leading to enhanced uptake by macrophages.

Plasma low density lipoproteins (LDL) and/or other lipoproteins containing apo B that accumulate in atherosclerotic lesions of human aortas exhibit structural changes that are associated with enhanced uptake in an unregulated fashion by macrophages in culture, resulting in the formation of foam cells in vitro. In an attempt to better characterize the structure-function modifications, we have incubated plasma LDL with extracts of human atherosclerotic plaques obtained at surgery, and determined whether such plaque-modified LDL also demonstrates enhanced uptake by cultured mouse peritoneal macrophages (MPM). Enhanced uptake was found which was linear over a concentration range of 100 micrograms lipoprotein protein/ml, as assessed by enhanced degradation of [125I]LDL and by stimulation of cholesterol esterification. Extracts of non-arterial human tissue were unable to induce this modification, suggesting tissue specificity. When delipidated apo B from tissue-treated [125I]LDL was subjected to SDS-PAGE, autoradiograms demonstrated, in addition to the B-100 band of apo B, a doublet of higher molecular weight than B-100 and a band just entering the gel, both at the expense of the B-100 band. No lower molecular weight bands suggestive of apo B degradation were seen. Modest increases in LDL electrophoretic mobility and thiobarbituric acid reactive substances were found following the incubation of LDL with plaque extracts. These changes could be inhibited by butylated hydroxytoluene (BHT), suggesting that free radical-induced lipid peroxidation was responsible for these modifications. However, since BHT did not inhibit the uptake of the tissue-incubated LDL by macrophages, the actual modification responsible for enhanced macrophage recognition did not appear to be free radical-induced. Uptake of plaque-modified [125I]LDL was inhibited by only 22% by a 20-fold excess of acetyl LDL or plaque-modified LDL. If the latter did not represent a mixture of modified and unmodified particles, this result would suggest that uptake was not mediated by the scavenger receptor. It is possible that foam cells are formed in vivo when LDL particles, which have been modified by interacting with components of the arterial wall, are taken up by tissue macrophages.

Plasma low density lipoprotein accumulation in aortas of hypercholesterolemic swine correlates with modifications in aortic glycosaminoglycan composition.

Arterial wall sulfated glycosaminoglycans (GAG) of matrix proteoglycans have been implicated in the retention of plasma low density lipoproteins in the early stages of atherosclerosis. We have studied modifications in porcine aortic GAG composition after 4 and 11 weeks of diet-induced hypercholesterolemia. After these time intervals no grossly visible atherosclerotic lesions were discerned. GAG changes were correlated with tissue LDL accumulation estimated by quantification of immunochemically-identifiable apolipoprotein B (apoB). Values of apoB ranged from less than 10 to 250 ng/mg wet weight of aorta, and correlated significantly with tissue total cholesterol contents. Although total GAG concentrations did not differ between a normolipemic control and the two diet groups, apoB showed a significantly positive correlation with the percent of total GAG that was chondroitin sulfate and a significantly negative correlation with the percent of total GAG that was dermatan sulfate. Total tissue cholesterol likewise demonstrated similar correlations with GAG. Since areas of the aorta were chosen that were devoid of intimal thickening, these metabolic changes may occur in the inner part of the arterial tunica media. The results suggest that the accumulation of plasma LDL in the arterial wall following hypercholesterolemia may induce alterations in arterial GAG composition, presumably by affecting GAG synthesis by medial smooth muscle cells.

Reduction in tissue LDL accumulation during coronary artery regression in cynomolgus macaques.

The aim of this study was to determine whether the amount of LDL that had accumulated in the coronary arteries of cynomolgus monkeys during an 18-month period on a hypercholesterolemic (H) diet was reduced during subsequent periods of 6 and 12 months on a normolipemic (N) diet. This was performed by assessing the accumulation of LDL in the left anterior descending (LAD) branch of the left coronary artery within an area 4 mm from its origin, since this region contained the largest lesions in the LAD. LDL accumulation was estimated by measuring the percent cross-sectional area of artery occupied by reaction product depicting apo B by an immunoperoxidase procedure. The following reduction in mean (+/- SD) percent cross-sectional area occupied by reaction product was found in 8 animals on the progression diet (group I), 7 animals on the 6-month regression diet (group II), and 9 animals on the 12-month regression diet (group III), respectively: 21.7 +/- 4.7, 6.9 +/- 5.5, and 2.3 +/- 1.3. Differences between group I and either group II or III were statistically significant (using the Wilcoxon signed-ranks test). In group I, LDL was localized primarily in the necrotic core and around pools of foam cells. In groups II and III fewer foam cells and smaller pools of extracellular debris were qualitatively evident, and LDL was localized closer to the lumen and along collagen fibers. These results suggest that lowering of the plasma LDL level following termination of a hypercholesterolemic diet also induces a decrease in the LDL content in coronary artery lesions, even without significant reductions in lesion size, and that this decrease might be responsible for the decrease in foam cells.

Correlation in the human aorta of APO B fractions with tissue cholesterol and collagen content.

The amounts of buffer- and Triton-extracted apo B (LDL-protein), as well as the sum of these two fractions, were correlated with the total tissue cholesterol and hydroxyproline content (as a measure of collagen) in grossly normal intima, fatty streaks, and fibrous plaques of human aortas obtained at autopsy. Quantitative values of buffer- and Triton-extracted apo B were obtained by sequentially extracting homogenates of aortic intima with an aqueous buffer and one containing Triton X-100, and measuring the apo B content in each extract by an electroimmunoassay relative to plasma LDL or Triton-treated LDL. Significant positive correlations were obtained between the following: tissue cholesterol and both buffer-extracted and total-extracted apo B in grossly normal intima; tissue cholesterol and Triton-extracted apo B in microdissected fibrotic caps and cores of fibrous plaques, as well as in whole plaques. A positive correlation was also obtained between tissue cholesterol and total-extracted apo B in the necrotic core. A significant negative correlation was found between Triton-extracted apo B and collagen in whole plaques. The calculated mean percent of total tissue cholesterol in the different aortic regions that could be present as part of an intact LDL particle were: 100% in grossly normal intima, 16% in fatty streaks, and 11% in fibrous plaques. The positive correlation between Triton-extracted apo B and cholesterol in plaques suggests one or both of the following: the extracellular pool of cholesterol or some material increasing concurrently with cholesterol interacts with apo B or another part of the LDL particle; or the apo B containing lipoprotein is trapped in the hydrophobic environment of extracellular lipid. Both possibilities would render the particle less soluble in aqueous buffers. The negative correlation between Triton-extracted apo B and tissue collagen and the lack of a significant correlation between buffer-extracted apo B and collagen content suggests that collagen is probably not responsible for apo B retention in the aortic intima.

Oxidized low density lipoprotein-induced inhibition of processing of photoreceptor outer segments by RPE.

PURPOSE: To examine the effects of oxidized low-density lipoproteins (oxLDL) on phagocytosis and processing of photoreceptor outer segments (OS) by retinal pigment epithelial (RPE) cells. METHODS: Confluent cultures of RPE-J cells were pretreated with oxLDL or LDL, and the effects of such treatment on the processing of added OS was determined. Processing was determined either by the degradation of 125I-labeled OS to trichloroacetic acid-soluble label or by the cleavage of rhodopsin observed on Western blot analysis of cell lysates separated by sucrose density gradient fractionation. Binding to and uptake of OS by RPE-J cells was assessed by determining the fluorescence of FITC-labeled OS before and after quenching with trypan blue. RESULTS: OxLDL induced a significant decrease in the degradation of 125I-OS in RPE-J cells but no reductions in either binding or uptake, when a 24-hour recovery period was inserted between treatment with oxLDL and challenge with OS. Rhodopsin cleavage increased in a time-dependent manner after phagocytosis of OS by RPE-J cells. The small guanosine triphosphatase (GTPase), Rab5, was first found in phagosome fractions containing rhodopsin and its cleavage products, and at later times of challenge, in more dense fractions representing phagolysosomes. These were assessed by the colocalization of rhodopsin cleavage products in density fractions with cathepsin D, a marker of lysosomes. OxLDL induced a reduction in rhodopsin cleavage product formation and in phagosome-lysosome fusion (maturation). It also reduced the time-dependent shift of rhodopsin to higher density fractions containing more cathepsin D without any detectable reduction in the expression of cathepsin D or in acid protease activity. CONCLUSIONS: OxLDL induces a reduction in the processing of OS by RPE by perturbing the fusion of lysosomes with phagosomes.

Lipoproteins containing apo B extracted from human aortas. Structure and function.

We have isolated, by anti-LDL affinity chromatography, apo B-containing lipoproteins from homogenates of atherosclerotic plaques excised from the human aorta. This fraction, called A-LP, has similarities with plasma LDL, such as having similar size and relative lipid composition, along with containing apo B. However, the fraction also contains some particles larger than LDL, it is more electronegative than LDL, the relative protein content is less than in LDL, and its apo B is highly degraded. A-LP is recognized by a high affinity binding site on mouse peritoneal macrophages (MPM), as suggested by dose-response curves of stimulation of cholesterol esterification. The interaction is inhibited by negatively-charged carbohydrates such as fucoidin, but excess A-LP did not inhibit the degradation of labeled acetyl-LDL by MPM, suggesting that the binding site recognizing A-LP may not be the scavenger receptor. Finally, stimulation of cholesterol esterification by A-LP in MPM is unregulated over a 48 hr time interval, leading to massive accumulations of cholesteryl esters and a transition of these MPM to a morphology characteristic of foam cells. It is possible that when monocytes enter the arterial intima at specific sites and become tissue macrophages, they internalize A-LP in an unregulated fashion. This, in turn, would make the monocyte-macrophage lipid-laden, and could explain the etiology of foam cells in fatty streak lesions. The modification in A-LP relative to P-LDL responsible for the enhanced recognition still needs to be elucidated.

Localization of LDL in arteries: improvements in immunofluorescence procedures.

We have described the development of procedures used to localize LDL in arteries with atherosclerotic lesions from humans and experimental animal models. We have first illustrated data obtained from earlier cryostat studies, described the procedures, given examples of results of LDL localization in sections of paraffin-embedded blocks, and finally outlined the procedures used and examples for LDL localization in epoxy-embedded blocks. Future improvements in resolution of LDL localization will probably require performing electron microscopy on ultrathin sections of epoxy-embedded arteries followed by immunocytochemistry.

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.

Immunochemically detectable lipid-free apo(a) in plasma and in human atherosclerotic lesions.

Although Lp(a) is an independent risk factor for cardiovascular diseases in humans, the precise pathogenetic mechanisms are still unknown. We have shown that Lp(a) accumulates in human atherosclerotic lesions, and some particles undergo oxidation. Since, following agarose electrophoresis of both plaque extracts and plasma, a region close to the origin immunostained intensely for apo(a) but was lipid-free, we sought to identify whether such samples contained lipid-free apo(a), as previously reported to occur in plaque extracts. Immunochemically identifiable apo(a) was found following density-gradient ultracentrifugation both in the 1.05 < d < 1.09 and the d > 1.21 density fraction from both plasma and plaque extracts. However, because in a competitive binding RIA, displacement curves of apo(a) in plasma and the d > 1.21 were not parallel, it is premature to ascribe a relative amount of total apo(a) to this fraction. Whereas apo(a) immunoblots of SDS-PAGE under reducing conditions of the d > 1.21 fraction of a plaque extract with high apo(a) content showed high molecular weight bands consistent with apo(a) isoforms, the corresponding d > 1.21 fraction showed multiple low molecular weight bands characteristic of fragmentation. Since the d > 1.21 of arterial extracts contained all the material immunostaining for apo(a) migrating towards the cathode, characteristic of immunoglobulins (IgG), we asked whether fragments of apo(a) might have associated with human IgG both in plasma and tissue extracts, or whether our anti-apo(a) reacted with epitopes on human IgG. Immunoblotting with our anti-apo(a) of samples of plasma and plaque extracts run on agarose electrophoresis or SDS-PAGE further demonstrated intense staining of multiple bands in the molecular weight range of human IgG. Furthermore, a fraction of plasma and tissue extracts that bound to a protein G affinity column demonstrated immunostaining for apo(a) and was in the size range of IgG. Although one polyclonal anti-apo(a) provided by another laboratory showed the same findings as our antibody, two other polyclonal anti-apo(a) failed to demonstrate immunostaining of human IgG, either on agarose electrophoresis or SDS-PAGE. We speculate that the Lp(a) immunogen used to prepare our anti-apo(a) may have undergone modest oxidation, thus exposing epitopes not normally expressed on apo(a) in native Lp(a). Either antibodies to these epitopes could be recognizing apo(a) fragments, possibly released during oxidation, which are then covalently bound to IgG, or oxidation of apo(a) creates epitopes on apo(a) that are homologous with IgG, thereby leading to cross-reactivity with IgG.(ABSTRACT TRUNCATED AT 400 WORDS)