Accumulation of oxidized lipid-protein complexes alters phagosome maturation in retinal pigment epithelium.

Lipid peroxidation has been implicated in many age-associated disorders including macular degeneration of the retina. We sought to elucidate the mechanism by which accumulation of oxidized LDL (oxLDL) reduces the ability of retinal pigment epithelium (RPE) to process photoreceptor outer segments (OS) as a model of peroxidation-induced disruption of phagocytosis. OxLDL did not reduce the lysosomal hydrolytic capacity of the RPE, but efficiently inhibited processing of various internalized proteins. OxLDL caused a delay in the acquisition of late lysosomal markers by newly formed phagosomes. At the same time, an excessive accumulation of markers of early phagosomal compartments was also observed. The activity of phosphatidylinositol 3-kinase (PI3K) was reduced in phagosomes of the RPE treated with oxLDL. These results suggest that accumulation of oxidized lipid-protein complexes in the RPE impedes phagosome maturation by blocking PI3K recruitment to the phagosomal membrane, leading to delayed processing of internalized OS.

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.

Macrophage receptors responsible for distinct recognition of low density lipoprotein containing pyrrole or pyridinium adducts: models of oxidized low density lipoprotein.

Oxidation of low density lipoproteins (LDL) induced by incubation with Cu(2+) ions results in the formation of a heterogeneous group of aldehydic adducts on lysyl residues (Lys) of apolipoprotein B (apoB) that are thought to be responsible for the uptake of oxidized LDL (oxLDL) by macrophages. To define the structural and chemical criteria governing such cell recognition, we induced two modifications of lysines in LDL that mimic prototypic adducts present in oxLDL; namely, epsilon-amino charge-neutralizing pyrrolation by treatment with 2,5-hexanedione (hdLDL), and epsilon-amino charge-retaining pyridinium formation via treatment with 2,4,6-trimethylpyrylium (tmpLDL). Both modifications led to recognition by receptors on mouse peritoneal macrophages (MPM). To assess whether the murine scavenger receptor class A-I (mSR-A) was responsible for recognition of hdLDL or tmpLDL in MPM, we measured binding at 4 degrees C and degradation at 37 degrees C of these modified forms of (125)I-labeled LDL by mSR-A-transfected CHO cells. Although uptake and degradation of hdLDL by mSR-A-transfected CHO cells was quantitatively similar to that of the positive control, acLDL, tmpLDL was not recognized by these cells. However, both tmpLDL and hdLDL were recognized by 293 cells that had been transfected with CD36. In the human monocytic cell line THP-1 that had been activated with PMA, uptake of tmpLDL was significantly inhibited by blocking monoclonal antibodies to CD36, further suggesting recognition of tmpLDL by this receptor. Macrophage uptake and degradation of LDL oxidized by brief exposure to Cu(2+) was inhibited more effectively by excess tmpLDL and hdLDL than was more extensively oxidized LDL, consistent with the recognition of the former by CD36 and the latter primarily by SR-A.Collectively, these studies suggest that formation of specific pyrrole adducts on LDL leads to recognition by both the mSR-A and mouse homolog of CD36 expressed on MPM, while formation of specific pyridinium adducts on LDL leads to recognition by the mouse homolog of CD 36 but not by mSR-A. As such, these two modifications of LDL may represent useful models for dissecting the relative contributions of specific modifications on LDL produced during oxidation, to the cellular uptake of this heterogeneous ligand.

HNE-derived 2-pentylpyrroles are generated during oxidation of LDL, are more prevalent in blood plasma from patients with renal disease or atherosclerosis, and are present in atherosclerotic plaques.

Free radical oxidation of human plasma low-density lipoprotein (LDL) produces 2-pentylpyrrole epitopes that are generated by reaction of 4-hydroxy-2-nonenal (HNE), a product of lipid oxidation, with protein lysyl residues. The HNE-derived 2-pentylpyrrole ("HNE-pyrrole") epitopes were detected with an enzyme-linked immunosorbent assay (ELISA) using antibodies (ON-KLH) raised against protein-bound 2-pentylpyrrole obtained by the reaction of 2-oxononanal (ON) with keyhole limpet hemocyanin (KLH). HNE-pyrrole epitopes in human plasma are not associated primarily with LDL protein, apolipoprotein (apo) B, since only 15% of the total HNE-pyrrole immunoreactivity is removed by immunoprecipitation of apo B. The levels of ON-KLH immunoreactivity detected in human plasma were found to be significantly elevated in renal failure and atherosclerosis patients when compared to those in healthy volunteers. HNE-pyrrole immunoreactivity was also detected in atherosclerotic plaques. The highest levels were associated with extracellular connective tissue. Levels of ON-KLH immunoreactivity in human plasma far exceed levels of free HNE, presumably because of the rapid clearance of free relative to protein-bound HNE. Therefore, HNE-pyrrole epitopes provide a more indelible marker of oxidative injury than levels of free HNE.

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.

Protein adducts of iso[4]levuglandin E2, a product of the isoprostane pathway, in oxidized low density lipoprotein.

Levuglandin (LG) E2, a cytotoxic seco prostanoic acid co-generated with prostaglandins by nonenzymatic rearrangements of the cyclooxygenase-derived endoperoxide, prostaglandin H2, avidly binds to proteins. That LGE2-protein adducts can also be generated nonenzymatically is demonstrated by their production during free radical-induced oxidation of low density lipoprotein (LDL). Like oxidized LDL, LGE2-LDL, but not native LDL, undergoes receptor-mediated uptake and impaired processing by macrophage cells. Since radical-induced lipid oxidation produces isomers of prostaglandins, isoprostanes (isoPs), via endoperoxide intermediates, we postulated previously that a similar family of LG isomers, isoLGs, is cogenerated with isoPs. Now iso[4]LGE2-protein epitopes produced by radical-induced oxidation of arachidonic acid in the presence of protein were detected with an enzyme-linked immunosorbent assay. Iso[4]LGE2-protein epitopes are also generated during free radical-induced oxidation of LDL. All of the LGE2 isomers generated upon oxidation of LDL are efficiently sequestered by covalent adduction with LDL-based amino groups. The potent electrophilic reactivity of iso-LGs can be anticipated to have biological consequences beyond their obvious potential as markers for specific arachidonate-derived protein modifications that may be of value for the quantitative assessment of oxidative injury.

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.

Measurement of oxidation in plasma Lp(a) in CAPD patients using a novel ELISA.

BACKGROUND: LGE2 is produced by the cyclooxygenase- or free radical-mediated modification of arachidonate and is formed during the oxidation of low density lipoprotein (LDL) with subsequent adduction to lysine residues in apo B. We have developed a sensitive enzyme-linked sandwich immunosorbent assay (ELISA) for detection and measurement of LGE2-protein adducts as an estimate of oxidation of plasma LDL and Lp(a). METHODS: The assay employs rabbit polyclonal antibodies directed against LGE2-protein adducts that form pyrroles, and alkaline phosphatase-conjugated polyclonal antibodies specific for apo B or apo (a). It demonstrates a high degree of specificity, sensitivity and validity. RESULTS: Epitopes characteristic for LGE2-pyrroles were quantified in patients with end-stage renal disease (ESRD) that had undergone continuous ambulatory peritoneal dialysis (CAPD) and in a gender- and age-matched control population. In addition to finding that both LDL and Lp(a) levels were elevated in CAPD patients, we also found that plasma Lp(a) but not LDL was more oxidized in CAPD patients when compared to corresponding lipoproteins from healthy subjects. Using density gradient ultra-centrifugation of plasma samples, we found that modified Lp(a) floats at the same density as total Lp(a). CONCLUSIONS: The results of this study demonstrate that oxidation of plasma Lp(a) is a characteristic of ESRD patients undergoing CAPD. This ELISA may be useful for further investigations on oxidation of lipoproteins in the circulation of specific patient populations.

Oxidation products of cholesteryl linoleate are resistant to hydrolysis in macrophages, form complexes with proteins, and are present in human atherosclerotic lesions.

Accumulation of the insoluble lipid-protein complex, ceroid, is a characteristic of atherosclerotic plaques. To determine whether deficient processing of cholesteryl esters in oxidized (ox) low density lipoprotein (LDL) contributes to ceroid formation, we studied the hydrolysis of internalized [3H] cholesteryl linoleate (CL) in oxLDL by mouse peritoneal macrophages (MPM). The hydrolysis by MPM of [3H]CL incorporated into oxLDL or LDL did not differ, suggesting that products of lipid and/or apoB oxidation had no impact on the lysosomal hydrolysis of [3H]CL. To evaluate the hydrolysis of oxCL by MPM, we subjected extensively ox[3H]CL to fractionation by TLC. The predominant fraction (D) consisted of sterols and oxysterols esterified to scission products of oxidized fatty acids containing terminal carbonyl groups, i.e., lipid core aldehydes. The extent of hydrolysis of [3H]-fraction D by MPM cultures, as well as by MPM extracts at pH 4.0, was significantly reduced when compared to the hydrolysis of intact [3H]CL. Fraction D also formed complexes with serum proteins, and the purified core aldehyde, cholesteryl 9-oxononanoate reacted with epsilon-amino group of lysines. Finally, several cholesteryl ester aldehydes were detected in lipid extracts of human atheroma. These results suggest that decomposition products of extensively oxidized cholesteryl linoleate that are also present in atherosclerotic lesions, are not adequately degraded by mouse peritoneal macrophage lysosomes and could interact with proteins to form ceroid.

Oxidation of low-density lipoproteins produces levuglandin-protein adducts.

Free-radical oxidation of human low-density lipoprotein (LDL) produces levuglandin (LG)-protein adducts that were detected with an enzyme-linked immunosorbent assay using LGE2-KLH antibodies which recognize LGE2-derived pyrroles. The level of immunoreactivity increases with time of oxidation and reaches a maximum by 8 h. The yield of pyrrole varies nonlinearly with the level of LG adduction to LDL. At low LG:LDL ratios, such as those detected in oxidized LDL, the reaction of primary amino groups with LGE2 produces mostly non-pyrrole adducts that are not immunoreactive. Concomitant phospholipolysis must occur if the generation of immunoreactive epitopes in LDL involves oxidation of arachidonyl phospholipids. Thus, since a protein adduct prepared from synthetic LGE2-2-lysophosphatidylcholine ester showed, at most, only 0.5% cross-reactivity with the LGE2-KLH antibodies, the epitopes detected in oxidized LDL are almost certainly not protein adducts of LG-phospholipid esters. As expected, hydrolysis of the carboxylic ester in the protein adduct of LGE2-2-lysophosphatidylcholine ester by treatment with phospholipase A2 produced a fully immunoreactive LGE2-protein adduct.

Levuglandin E2-protein adducts in human plasma and vasculature.

The prostaglandin endoperoxide PGH2 rearranges nonenzymatically to generate prostaglandins and secoprostanoic acid levulinaldehyde derivatives such as PGE2 and levuglandin (LG) E2, respectively. Direct detection of LGE2 in biological samples is complicated because it is rapidly sequestered by covalent adduction to endogenous nucleophiles including proteins, which produces LGE2-derived protein-bound pyrroles. Therefore, to detect LGE2-protein adducts in vivo, antibodies were raised against a covalent adduct of LGE2 with keyhole limpet hemocyanin (KLH). This antigen enabled the production of high-titer antibodies that exhibit minimal cross-specificity and are sensitive for detecting LGE2-derived pyrroles. Although pyrrole yields are low at LG/protein ratios found in vivo, an enzyme-linked immunosorbent assay with the LGE2-KLH antibodies detects LGE2-derived protein-bound pyrrole immunoreactivity in human plasma from specific patient populations. Furthermore, prominent immunocytochemical staining of human brain thin sections revealed the presence of LGE2-derived pyrrole immunoreactivity, especially in the meningeal vessels of some patients. This demonstration of LG-protein adducts in human plasma and vasculature provides the first evidence for the biological occurrence of levuglandins in vivo and further suggests that these antibodies might prove useful in diagnostic and mechanistic studies of various disease conditions.

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.

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.

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.

(Carboxyalkyl)pyrroles in human plasma and oxidized low-density lipoproteins.

Free-radical oxidation of human plasma low-density lipoprotein (LDL) produces (carboxyalkyl)pyrrole (CAP) epitopes that were detected with enzyme-linked immunosorbent assays using antibodies raised against keyhole limpet hemocyanin (KLH)-bound 2-(omega-carboxyheptyl)-pyrrole (CHP) and 2-(omega-carboxypropyl)pyrrole (CPP). These antibodies exhibit high structural selectivity (< 0.5% cross-reactivity) in competitive binding inhibition assays with the corresponding human serum albumin (HSA)-bound pyrroles. No cross-reactivity was detected for HSA-bound 2-pentylpyrrole, an epitope that is generated by a reaction of 4-hydroxy-2-nonenal (HNE) with protein lysyl residues. Oxidation of either arachidonic or linoleic acid in the presence of HSA produced an HNE-derived 2-pentylpyrrole epitope. However, only oxidation of linoleic acid formed HSA-bound CHP, while only oxidation of arachidonic acid generated HSA-bound CPP. Since ester hydrolysis with KOH markedly elevated levels of immunoreactive epitopes detected in oxidized LDL, the CAPs are presumably generated by reactions of oxidized cholesteryl esters, triglycerides, and phospholipids with LDL protein, and only some of these oxidized esters are hydrolyzed, e.g., by phospholipase activity associated with LDL. Protein-bound CHP immunoreactivity was detected in human plasma, and levels are significantly elevated in renal failure and atherosclerosis patients compared with healthy volunteers. This provides the first evidence for the biological occurrence of protein-bound CAPs in vivo and further suggests that free-radical oxidation of polyunsaturated lipids produces hydroxyalkenal carboxylate esters whose gamma-hydroxy-alpha,beta-unsaturated aldehyde functionality and reactivity resemble that of HNE.

Structure of cholesterol-containing particles accumulating in atherosclerotic lesions and the mechanisms of their derivation.

Structural and chemical modifications of plasma lipoproteins retained in atherosclerotic lesions, especially LDL, are a characteristic of atherogenesis. The major cholesterol-containing structures believed to be derived primarily from LDL are monomeric or aggregated native or modified LDL particles, cholesteryl ester droplets, liposomes rich in unesterified cholesterol, and ceroid-lipofuscin. They are suggested to be formed primarily from LDL by combinations of oxidation, hydrolysis by proteases and esterases, fusion of neutral lipid components, and covalent interactions between lipid and protein components of oxidized LDL in lysosomes. Although many of these structures appear to be refractory to removal by reverse cholesterol transport mechanisms, they may possess functional properties that still need to be elucidated.