June O'Neil

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.

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.

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.

Hydroxynonenal inactivates cathepsin B by forming Michael adducts with active site residues

Oxidation of plasma low‐density lipoprotein (oxLDL) generates the lipid peroxidation product 4‐hydroxy‐2 nonenal (HNE) and also reduces proteolytic degradation of oxLDL and other proteins internalized by mouse peritoneal macrophages in culture. This leads to accumulation of undegraded material in lysosomes and formation of ceroid, a component of foam cells in atherosclerotic lesions. To explore the possibility that HNE contributes directly to the inactivation of proteases, structure‐function studies of the lysosomal protease cathepsin B have been pursued. We found that treatment of mouse macrophages with HNE reduces degradation of internalized maleyl bovine serine albumin and cathepsin B activity. Purified bovine cathepsin B treated briefly with 15 μM HNE lost ∼76% of its protease activity and also developed immunoreactivity with antibodies to HNE adducts in Western blot analysis. After stabilization of the potential Michael adducts by sodium borohydride reduction, modified amino acids were localized within the bovine cathepsin B protein structure by mass spectrometric analysis of tryptic peptides. Michael adducts were identified by tandem mass spectrometry at cathepsin B active site residues Cys 29 (mature A chain) and His 150 (mature B chain). Thus, covalent interaction between HNE and critical active site residues inactivates cathepsin B. These results support the hypothesis that the accumulation of undegraded macromolecules in lysosomes after oxidative damage are caused in part by direct protease inactivation by adduct formation with lipid peroxidation products such as HNE.

Phospholipid Hydroxyalkenals Biological and Chemical Properties of Specific Oxidized Lipids Present in Atherosclerotic Lesions

Objective—Phosphatidylcholine hydroxyalkenals (PC-HAs) are a class of oxidized PCs derived from lipid peroxidation of arachidonate or linoleate at the sn-2 position to form terminal &ggr;-hydroxy, &agr;-, and &bgr;-unsaturated aldehydes. The aim of this study was to characterize some of their biological properties, ascertain the mechanism of their action, and assess whether they have in vivo relevance. Methods and Results—Combinations of cell biological approaches with radiolabels, mass spectroscopy, and immunochemical as well as immunohistochemical techniques were used to show that PC-HAs reduce the proteolytic degradation by mouse peritoneal macrophages (MPMs) of internalized macromolecules, such as maleylated bovine serum albumin, and that the activity of the lysosomal protease, cathepsin B, in MPMs form Michael adducts with MPM proteins and with N-acetylated cysteine in vitro form pyrrole adducts with MPM proteins and reduce the maturation of Rab5a, thereby impairing phagosome-lysosome fusion (maturation) in phagocytes; they are present unbound and as pyrrole adducts in human atherosclerotic lesions. Conclusions—PC-HAs are present in vivo and possess multiple functions characteristic of oxidized LDL and 4-hydroxynonenal.

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.

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.

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.

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)