(Circulation. 1996;94:1216-1225.)
© 1996 American Heart Association, Inc.
Articles |
Oxidation-Specific Epitopes in Human Coronary Atherosclerosis Are Not Limited to Oxidized Low-Density Lipoprotein
the Divisions of Cardiology (K.D.O'B.) and Metabolism, Endocrinology and Nutrition (J.E.H., S.W., A.C.), Department of Medicine; Department of Pathology (C.E.A.), School of Medicine; and Department of Epidemiology, School of Public Health and Community Medicine (J.E.H.), University of Washington, Seattle.
Correspondence to Kevin D. O'Brien, MD, Division of Cardiology, Box 356422, University of Washington, 1959 NE Pacific St, Seattle, WA 98195-6422. E-mail cardiac@u.washington.edu.
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Background Previous small studies have demonstrated positive immunohistochemical staining in rabbit and human atherosclerotic plaques by antibodies that recognize oxidized low-density lipoprotein (OxLDL), but none have examined a large number of human coronary arteries or evaluated whether epitopes recognized by these antibodies might be present on plaque proteins other than OxLDL.
Methods and Results Immunohistochemistry was performed on atherosclerotic (n=87) and nonatherosclerotic (n=51) coronary arterial segments from 20 patients by use of monoclonal antibodies that recognize epitopes on macrophages, smooth muscle cells, apolipoprotein (apo) B, and OxLDL. Staining with the OxLDL antibody (Ox5) was much more prevalent in atherosclerotic than in control segments. Extracellular Ox5 staining colocalized with apo B, but cell-associated Ox5 staining occurred in the absence of cell-associated apo B staining, which suggests that cell-associated epitopes for Ox5 were on proteins other than LDL. Epitopes for Ox5 formed in vitro on two readily available non–apo B proteins, human serum albumin and apo A-I, when these proteins were incubated under conditions of oxidant stress with polyunsaturated but not monounsaturated fatty acids; furthermore, an antioxidant inhibited Ox5 epitope formation. Thus, epitopes for Ox5 can form on proteins other than apo B. Also, phorbol ester–treated macrophages cultured in apo B–free medium developed epitopes for Ox5.
Conclusions These findings are consistent with the hypothesis that atherosclerosis is associated with oxidative modification of proteins in addition to LDL, particularly cell-associated proteins, and that the antiatherosclerotic effects of antioxidants seen in some studies may not be solely due to prevention of LDL oxidation.
Key Words: apolipoproteins • macrophages • fatty acids • immunohistochemistry • antioxidants
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Over the past several years, there has been substantial interest in the potential role of oxidatively modified LDL in the pathogenesis of atherosclerosis (reviewed in References 1 through 4). This interest has been based in part on in vitro studies that demonstrated (1) that minimally oxidized LDL stimulates endothelial expression of macrophage chemoattractants,5 adhesion molecules,6 7 and cytokines7 and (2) that extensively oxidized LDL is taken up by macrophage scavenger receptors in a manner that is unregulated by cell cholesterol content.1 8 9 Extensively oxidized LDL has a number of other potentially proatherogenic effects, including cytotoxicity10 11 ; expression of plasminogen activator inhibitor-1 by endothelial cells,12 expression of platelet-derived growth factor A-chain by smooth muscle cells (SMCs),13 14 and expression of interleukin-1ß,15 interleukin-8,16 and stress proteins17 by macrophages; and activation of T lymphocytes.18 However, although many studies19 20 21 22 23 have demonstrated that doses of antioxidants that effectively block LDL oxidation in vitro decrease atherosclerosis in hypercholesterolemic animals in vivo, other reports24 25 26 27 have found LDL oxidation-inhibiting doses of antioxidants to have no antiatherogenic effect.
Several studies have attempted to demonstrate the presence of OxLDL in atherosclerotic lesions. Lipoprotein particles with many of the characteristics of OxLDL can be eluted from human atherosclerotic lesions.28 29 Also, immunohistochemical studies have used a variety of antibodies that recognize epitopes on OxLDL to demonstrate the presence of these epitopes in rabbit30 31 32 33 34 and human35 36 atherosclerotic lesions. However, none of the immunohistochemical studies have examined the prevalence or distribution of oxidation epitopes in a large number of human arteries. Furthermore, these studies have been taken as strong evidence for the presence of OxLDL in lesions despite two substantial limitations. First, some of the "anti-OxLDL" antibodies used in these studies can recognize oxidation epitopes on proteins other than apo B,31 32 which demonstrates that these antibodies are not specific for OxLDL alone. Second, none of the immunohistochemical studies have attempted to resolve an important contradiction between in vitro and in vivo studies that use anti–apo B and "anti-OxLDL" antibodies. Specifically, in vitro, antibodies that recognize apo B, the protein component of LDL, also recognize OxLDL in Western blot and ELISA.33 37 In contrast, in vivo studies32 34 have shown that apo B immunostaining is absent from many atherosclerotic regions that contain prominent "anti-OxLDL" immunostaining. Thus, the present study was undertaken in a large number of human coronary arteries to determine the extent to which oxidation epitopes might represent OxLDL by comparison of immunostaining for oxidation epitopes with that for apo B and to determine the conditions under which these epitopes may be formed in vitro.
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Human Coronary Arterial Tissue
A total of 138 coronary arterial segments were obtained from 20 hearts removed at the time of cardiac transplantation for either ischemic (n=10) or idiopathic dilated cardiomyopathy (n=10). Patient age at the time of organ removal ranged from 30 to 66 years (median, 52.5 years) for all patients, from 43 to 66 years (median, 52 years) for those with ischemic cardiomyopathy, and from 30 to 57 years (median, 53 years) for those with idiopathic dilated cardiomyopathy. To minimize the possibility of ex vivo oxidation, coronary arterial tissue was fixed in methanol-Carnoy's fixative that contained both the lipid-soluble antioxidant BHT (Sigma Chemical Co) at a concentration of 25 µmol/L38 and the metal ion chelator DTPA (Sigma) at a concentration of 50 µmol/L. The coronary artery segments were classified according to conventional histological criteria as (1) atherosclerotic coronary segments, defined by the presence of typical features of luminal narrowing due to regional accumulation of cholesterol, foam cell, and non–foam cell macrophages and the presence of fibrous caps or (2) control coronary segments with diffuse intimal thickening, which consisted of intimal SMCs and matrix and represented the characteristic morphology of nonatherosclerotic adult human coronary arteries.39
Human Monocyte-Derived Macrophages
Human monocyte-derived macrophages were isolated from volunteer donors by the method of Boyum40 and cultured on eight-chamber glass slides in RPMI 1640 medium (BioWhittaker) with 2 mmol/L L-glutamine (BioWhittaker) in the presence of 20% autologous serum for 2 days before use.
Monoclonal Antibodies
The following mouse monoclonal antibodies were used:
1. Anti-CD68 (Dako Corp) was used at a titer of 1:1000 to identify macrophages.
2. Anti-
smooth muscle actin (Dako) was used at a titer of 1:2000 to identify SMCs.41
3. Antibody 9A was used to identify apo B. This antibody recognizes an epitope on apo B near the LDL receptor-binding site and has been demonstrated to inhibit cellular uptake and degradation of LDL in a dose-dependent manner.42 Also, antibody 9A recognizes OxLDL on both Western blot and in ELISA (data not shown).
4. Ox5 was used to identify oxidation epitopes. The Ox5 antibody was developed, as described previously, by immunization of mice with LDL that had been extensively oxidized in the presence of copper.37 Ox5 has been shown to be specific for OxLDL compared with other modified forms of LDL by fluid-phase radioimmunoassay, in that the binding of Ox5 to radiolabeled OxLDL was displaced only by either OxLDL or delipidated OxLDL but not by either native LDL or by LDL with modified lysine residues, including acetylated LDL and malondialdehyde-modified LDL.37
The 9A and Ox5 antibodies were kind gifts from Dr Santica Marcovina of the Northwest Lipid Research Laboratory, Seattle, Wash.
All four antibodies were used for immunohistochemistry, both on coronary arteries and on cultured human monocyte-derived macrophages. In addition, the apo B antibody 9A and the oxidation epitope antibody Ox5 were used in ELISAs as described below.
Immunohistochemistry
Single-label immunohistochemistry was performed as described previously.43 44 45 Briefly, tissue sections were deparaffinized with xylene and then rehydrated with graded alcohols. The slides were blocked with 3% H2O2 (Sigma), washed with PBS, incubated for 60 minutes with the primary antibody, and then washed again with PBS. A biotin-labeled anti-mouse secondary antibody then was applied for 30 minutes, followed by an avidin-biotin-peroxidase conjugate (ABC Elite, Vector Laboratories) for 30 minutes. Standard peroxidase enzyme substrate, 3,3'-diaminobenzidine (Sigma) with nickel chloride (Sigma) then was added to yield a black reaction product. The slides were counterstained with methyl green.
In theory, the application of H2O2 to block endogenous peroxidase could artifactually induce the formation of epitopes recognized by the Ox5 antibody. To exclude this possibility, 10 slides were not exposed to H2O2 but instead were treated with levamisole to block endogenous alkaline phosphatase, an avidin-biotin-alkaline phosphatase conjugate (Vector) was substituted for the avidin-biotin-peroxidase conjugate, and fuchsin red (Vector) was used as the alkaline phosphatase substrate.43 These 10 slides were counterstained with hematoxylin. The locations and patterns of Ox5 staining obtained with the immunoperoxidase and alkaline phosphatase techniques were superimposable.
Negative controls for each of the antibodies included substitution of the primary antibody with either PBS or isotype-matched, irrelevant monoclonal antibodies to abolish specific immunohistochemical staining.
Formation of Oxidation Epitopes In Vitro
To determine the conditions under which Ox5 epitopes might be formed on proteins in vitro, stock solutions of the fatty acids oleic acid (18:1), linoleic acid (18:2), and linolenic acid (18:3) were diluted to a final concentration of 6 mmol/L in PBS and emulsified in a vortex. Either HSA or delipidated human apo A-I then was added to a final concentration of 300 µg/mL. Fatty acids and HSA were obtained from Sigma and CuSO4 from JT Baker. Delipidated human apo A-I was isolated from healthy volunteer donors, as described previously.46
To initiate oxidation, CuSO4 (5 µmol/L final concentration) was added and the solutions were incubated overnight at 37°C. Control incubations included omission of the protein, fatty acid, and/or CuSO4 and to inhibit oxidation, addition of BHT (final concentration, 25 µmol/L) before the overnight incubation. The next morning, the oxidation reaction was stopped with the addition of BHT to a final concentration of 25 µmol/L, and samples then were used for ELISAs performed on the same day.
ELISA
ELISAs were performed by the addition of 2 µg protein to 96-well enzyme immunoassay plates (Costar Corp) followed by incubation at 37°C for 2 hours. Each well then was washed five times with PBS containing 25 µmol/L BHT. To each well, 300 µL of blocking buffer (3% BSA [Sigma] and 0.02% NaN3 [Sigma] in PBS) was added, followed by incubation at 37°C for 60 minutes. Each well was rinsed three times with blocking buffer; 200 µL of a 1:4000 dilution of primary antibody (either Ox5 or 9A) then was added, followed by incubation at 37°C for 90 minutes. The wells were rinsed five times with PBS; 200 µL of horseradish peroxidase–conjugated anti-mouse antibody (Boehringer Mannheim) then was added, followed by incubation at room temperature for 90 minutes. Each well then was rinsed five times with PBS, then 100 µL of freshly prepared OPD (Sigma) in citrate buffer with 0.7% H2O2 (Sigma) was added, followed by incubation in the dark at room temperature for 20 minutes. The reaction then was stopped by the addition of 25 µL 8N H2SO4 (JT Baker). Absorbance then was read at 490 nm on a Dynatec ELISA reader (Molecular Devices) with the background wavelength set at 405 nm.
Statistical Analyses
Categorical data were analyzed by use of 
2 analyses with Yates' correction when cell sizes were <5, and probability values were adjusted for multiple comparisons by use of Bonferroni's inequality. Pairwise quantitative data were analyzed by use of Student's t test. Kruskal-Wallis one-way ANOVA on ranks was used for comparisons between multiple groups, followed by the Student-Newman-Keuls method of multiple comparisons to isolate specific groups that differed. Analysis of categorical data was performed by use of Epi Info version 6.02 (Centers for Disease Control and Prevention and World Health Organization). Analysis of quantitative data was performed by use of Sigma Stat for Windows (Jandel Scientific). The level of significance was set at a value of P<.05.
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OxLDL Retains Immunoreactivity for Both the Oxidation-Specific Epitope Antibody Ox5 and the Apo B Antibody 9A After Uptake by Human Monocyte-Derived Macrophages
Because previous Western blot and ELISA studies that used the oxidation-specific epitope antibody Ox5 and the anti–apo B antibody 9A demonstrated that OxLDL not only was recognized by Ox5 but also retained immunoreactivity for the anti–apo B antibody 9A,37 it was crucial to determine whether OxLDL that had been internalized by cells retained immunoreactivity for both Ox5 and 9A. Human monocyte-derived macrophages were cultured for 2 days, exposed overnight to 50 µg/mL of either LDL (Fig 1
, left) or OxLDL (Fig 1, right), and fixed in cold methanol. Immunohistochemistry then was performed with either the anti–apo B antibody 9A (Fig 1, top) or the oxidation-specific epitope antibody Ox5 (Fig 1, bottom). Cells cultured in the presence of LDL (Fig 1, left) did not have detectable immunohistochemical staining for either the apo B antibody 9A (Fig 1, top left) or the oxidation-specific epitope antibody Ox5 (Fig 1, bottom left). The lack of detectable apo B in LDL-exposed cells is not surprising because the rate of uptake of native LDL by macrophages is relatively low. In contrast, in cells exposed to OxLDL (Fig 1, right), which is taken up rapidly by macrophages, immunohistochemical staining was found for both the anti–apo B antibody 9A (Fig 1, top right) and the oxidation-specific epitope antibody Ox5 (Fig 1, bottom right). Thus, it appears that OxLDL, when ingested by cultured cells, retains immunoreactivity for apo B.
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Extracellular and Cell-Associated Ox5 Immunostaining Are Present in Human Coronary Arteries
In human coronary arteries, two patterns of immunostaining were seen with the Ox5 antibody. The first was extracellular localization of Ox5 to areas of extracellular apo B staining, as detected with 9A. Extracellular staining with the Ox5 antibody (Fig 2, top) was restricted to a portion, not all, of the regions with extracellular staining for apo B (Fig 2, bottom). There were no regions in which extracellular Ox5 immunoreactivity was present without colocalized extracellular apo B immunoreactivity. Staining for apo B was almost exclusively extracellular in the specimens studied (Fig 2, bottom). Because 9A recognizes apo B on OxLDL as well as on LDL, colocalized staining for both Ox5 and 9A is consistent with the possibility that OxLDL is present in these areas. The prevalences of extracellular staining for both apo B and Ox5 were significantly higher in atherosclerotic plaques than in control segments (both P<.001) (Fig 3).
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The second pattern of Ox5 staining was cell-associated staining for Ox5 without associated staining for apo B. Areas frequently were found in which several cells with lipid inclusions (ie, foam cells) contained prominent cell-associated staining with the Ox5 antibody (Fig 4, top) but lacked cell-associated staining for apo B (Fig 4, bottom). However, striking cell-associated Ox5 staining also could be detected in nonfoam cells, particularly in those surrounding neovessels that infiltrated from the vasa vasorum,45 and in the adventitia as well. Cell-associated Ox5 immunoreactivity was detected primarily in macrophages, although Ox5-positive SMCs also were found. One particularly striking example of cell-associated Ox5 staining was observed on cells in the adventitia of a saphenous vein bypass graft (Fig 5). There was no staining for apo B in this region (data not shown). Similar to the results of Rosenfeld et al,32 punctate cell-associated immunostaining with Ox5 was observed occasionally; however, it was not associated with apo B immunostaining. Furthermore, diffuse, cell-associated Ox5 immunostaining was by far the most prevalent pattern of Ox5 cell-associated immunostaining identified in the present study (Figs 4 and 5).
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Both patterns of Ox5 staining, ie, the colocalization of extracellular Ox5 and apo B staining and the cell-associated Ox5 staining without apo B were relatively specific for atherosclerotic segments compared with nonatherosclerotic segments with only diffuse intimal thickening. Colocalization of extracellular Ox5 with apo B was detected in 34 (39%) of 87 atherosclerotic segments but in only 2 (4%) of 51 controls. The prevalence of cell-associated Ox5 without associated apo B was 59% (51/87) in atherosclerotic segments and was significantly higher (P<.001) than the 4% (2/51) prevalence in controls (Fig 6).
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The relationship of cell-associated and extracellular Ox5 to plaque macrophage density also was examined. Two independent observers scored the macrophage density of each atherosclerotic plaque on a semiquantitative scale, in which 1+ indicated no or scattered macrophages, 2+ indicated small foci of macrophages, and 3+ indicated large foci of macrophages. Interobserver agreement was 94%, and disagreements were resolved by consensus. The prevalences of extracellular and cell-associated Ox5 staining among plaques with each macrophage score are shown, respectively, in Fig 7A and 7B. 2 Analyses showed significant differences in the prevalence of positive immunostaining for both extracellular Ox5 and cell-associated Ox5 over the range of macrophage scores from 1+ to 3+ (both P<.01). However, the prevalence of extracellular Ox5 was not significantly increased until plaques reached the highest macrophage density (macrophage score of 3+) (P=.42 for comparison of 1+ versus 2+ and P<.01 for comparison of 1+ versus 3+), whereas the prevalence of positive cell-associated Ox5 staining was significantly increased in plaques with small foci of macrophages (macrophage scores of 2+) compared with those with only scattered macrophages (macrophage scores of 1+). There was no significant difference in the prevalence of positive intracellular Ox5 immunoreactivity in plaques with small foci of macrophages (2+) compared with those with large foci of macrophages (3+). In contrast, there was a significant difference in the prevalence of extracellular Ox5 immunoreactivity in plaques with macrophage scores of 2+ versus 3+. This suggests that oxidation of cell-associated proteins may actually occur at an earlier stage of atherogenesis than does oxidation of extracellular proteins such as apo B.
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Thus, OxLDL is recognized by both Ox5 and 9A in Western blot, in ELISA, and in immunohistochemical analysis after internalization by cultured human monocyte-derived macrophages, and in human coronary arteries, extracellular Ox5 colocalizes with apo B. However, in human atherosclerosis, cell-associated Ox5 occurs without concomitant cell-associated staining for apo B, which raises the important question of whether cell-associated Ox5 epitopes in human plaques are present on proteins other than LDL. To address this question, the conditions necessary to form Ox5 epitopes were investigated in vitro by use of combinations of an oxidant stress, two human proteins other than apo B, and fatty acid emulsions.
Oxidation-Specific Epitopes Can Be Formed on Human Proteins Exposed to Polyunsaturated Fatty Acid Emulsions
Ox5 epitopes were detected by ELISA on HSA and apo A-I after incubation of those proteins with an emulsion of polyunsaturated fatty acids and exposure to an oxidant stress. Fig 8 demonstrates the results of an ELISA with the Ox5 antibody on HSA or apo A-I that had been incubated in room air with or without 5 µm CuSO4, in the presence of the monounsaturated fatty acid oleic acid (18:1) or the polyunsaturated fatty acids linoleic acid (18:2) and linolenic acid (18:3). No Ox5 immunoreactivity was detectable on either HSA or apo A-I when 18:1 was incubated with copper. In contrast, highly significant increases in Ox5 immunoreactivity were detected on HSA (Fig 8A) and apo A-I (Fig 8B) when incubated with 18:2, with or without copper. Similarly, substantial immunoreactivity always was detected on either HSA or apo A-I incubated with 18:3, whether or not additional copper was present in the medium. Control ELISAs performed with the anti–apo B antibody 9A showed no immunoreactivity with HSA or apo A-I (data not shown). Thus, Ox5 epitopes could be formed on human plasma proteins other than apo B if those proteins were exposed to an emulsion of polyunsaturated fatty acids.
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P<.05 vs 18:1 only; 
P<.05 vs 18:1+Cu.
The formation of Ox5 epitopes on non–apo B proteins could be inhibited by the addition of the antioxidant BHT (Fig 9). The formation of detectable Ox5 epitopes on either HSA (Fig 9A) or apo A-I (Fig 9B) exposed to either 18:2 or 18:3 plus copper was inhibited by 70% to 100% by the addition of BHT (all P<.001 for comparison of incubations with BHT versus those without BHT). This finding demonstrates that the formation of Ox5 epitopes is oxidation dependent. Furthermore, no epitopes were present either in control wells that contained fatty acids without proteins or in control wells that contained proteins without fatty acids (data not shown), which indicates that these oxidation epitopes are formed on proteins by by-products or end products of fatty acid oxidation. Also, assays for TBARS confirmed that significant lipid peroxidation, evidenced by increases in TBARS, did occur in all incubations that contained the polyunsaturated fatty acids 18:2 and 18:3, regardless of whether or not HSA was present, but did not occur in incubations that contained the monounsaturated fatty acid 18:1 (data not shown). In contrast, formation of epitopes for Ox5 required the presence of both polyunsaturated fatty acids and proteins.
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Oxidation-Specific Epitopes Can Be Formed on Cellular Proteins of Cultured Macrophages in the Absence of LDL
Experiments were performed to determine whether Ox5 epitopes could be formed in cells not exposed to apo B–containing lipoproteins. Macrophages isolated 2 days previously were cultured overnight in serum-free medium, which therefore was devoid of apo B–containing lipoproteins, either with or without the phorbol ester PMA at a concentration of 100 nmol/L. Macrophages cultured in serum-free medium without PMA had no Ox5 immunoreactivity (Fig 10, left). In contrast, in macrophages cultured in serum-free medium with 100 nmol/L PMA, a concentration that induced superoxide anion production in separate experiments (data not shown), Ox5 immunoreactivity was present (Fig 10, right) in a pattern similar to that seen for atherosclerotic plaque cells. Furthermore, absence of immunostaining with the anti–apo B antibody 9A confirmed that LDL was not present in these cells (data not shown).
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The major findings of this study are that oxidation-specific epitopes are detected in cells in the majority of atherosclerotic plaques but not in control coronary segments, and in general, cell-associated Ox5 epitopes do not appear to be present on apo B, which suggests that these oxidation-specific epitopes have formed on other proteins. Several lines of evidence suggest that the cell-associated, oxidation-specific epitopes recognized by antibody Ox5 are not on apo B of OxLDL. First, there is a lack of colocalized cell-associated staining for apo B. Previous studies33 37 showed that OxLDL retains immunoreactivity for apo B antibodies on Western blot and in ELISA. The present study demonstrates that internalized OxLDL retains immunoreactivity for the apo B antibody 9A in cultured macrophages. Thus, the observation that cells in human coronary arteries with positive Ox5 staining do not stain for 9A strongly suggests that these cell-associated Ox5 epitopes are not on apo B of OxLDL. Second, the pattern of cell-associated Ox5 staining is nonlysosomal. As expected and as demonstrated in the present study by immunohistochemical studies of human monocyte-derived macrophages exposed to OxLDL, when OxLDL is ingested by cells, the Ox5 immunoreactivity attributable to OxLDL is present in a punctate distribution consistent with lysosomes. In contrast, much of the cell-associated Ox5 immunoreactivity detected in atherosclerotic plaques appears to be present on cell membranes rather than in lysosomes. Third, Ox5 immunoreactivity can be induced on proteins other than apo B in vitro. By use of ELISA, epitopes recognized by Ox5 were detected on two non–apo B proteins, HSA and apo A-I, when those proteins were incubated with polyunsaturated fatty acids. This demonstrates that although Ox5 does not recognize acetylated or malondialdehyde-modified LDL,37 it can recognize epitopes on other human proteins that have been exposed to polyunsaturated fatty acids and an oxidant stress. The specific requirement for polyunsaturated as opposed to monounsaturated fatty acids and the substantial inhibition of Ox5 epitope formation by the antioxidant BHT suggest that these epitopes are formed on proteins by by-products or end products of polyunsaturated fatty acid oxidation. Furthermore, no Ox5 immunoreactivity was detected by ELISA in wells that contained fatty acids but lacked proteins, which confirms that the epitopes detected by Ox5 are on proteins and not on oxidized fatty acids. Therefore, the epitopes detected on cell membranes in plaques most likely have formed directly on membrane proteins rather than having first formed on OxLDL lipids with subsequent transfer to cell membranes. Finally, epitopes recognized by Ox5 can be formed on human monocyte-derived macrophages in vitro. The presence of Ox5 epitopes on human monocyte-derived macrophages that had been cultured overnight in the absence of lipoproteins and exposed to a dose of phorbol ester, which stimulated superoxide anion production, also confirms that these epitopes can be formed on cellular proteins other than apo B. Furthermore, the pattern of distribution of these oxidation epitopes on cultured macrophages was similar to that of the cell-associated Ox5 staining present in lesions, which raises the possibility that similar types of oxidant stress might lead to formation of these epitopes on atherosclerotic plaque cells.
Although cellular localization of oxidation-specific epitopes by Ox5 predominantly was independent of apo B, extracellular Ox5 epitopes were confined to regions with colocalization of apo B. However, extracellular apo B was more widespread than extracellular Ox5. This suggests that some of the LDL bound to extracellular matrix in atherosclerotic lesions may be oxidized and is consistent with the findings of other studies28 29 that lipoproteins with many of the characteristics of OxLDL can be eluted from human lesions. It is highly unlikely that cell-associated OxLDL could be eluted intact from atherosclerotic lesions, because any internalized OxLDL would have been degraded rapidly by lysosomal enzymes. However, despite their colocalization with apo B epitopes, it cannot be concluded that all extracellular Ox5 epitopes present in lesions are on OxLDL, because these epitopes can be formed in vitro on other proteins, such as albumin, which has been shown by others47 to also be present in the extracellular matrix of human atherosclerotic lesions.
Several observations suggest that cell-associated oxidation epitopes may have formed on membranes of cells and their subcellular organelles. First, in vitro experiments demonstrated that inclusion of a polyunsaturated fatty acid emulsion was an absolute requirement for Ox5 epitope formation on non–apo B proteins. Because phospholipid fatty acids are in close apposition to one another and to proteins in cell membranes, peroxidation of membrane phospholipid polyunsaturated fatty acids could lead to the formation of oxidation-specific epitopes on membrane proteins. Second, the histological appearance of the cell-associated oxidation-specific epitopes, both in vitro in PMA-stimulated macrophages and in vivo in atherosclerotic plaques, suggests localization of the oxidation epitopes to membrane proteins of cells and their subcellular organelles. Interestingly, studies have demonstrated that diets enriched in 18:1 versus 18:2 improve resistance of LDL to oxidation in rabbits48 and humans.49 This raises the intriguing possibility that 18:1-enriched diets might be beneficial not only because they lower LDL and increase its resistance to oxidative modification48 49 but also because they lead to the formation of oxidation-resistant cell membranes.
One recent study50 reported similar findings to those of the present study by use of an antibody that recognizes oxidized phospholipids. In that study, the antibody, which had been generated by immunization of mice with human plaque homogenates, stained cell membranes of a subset of human atherosclerotic plaque macrophages, similar to the results of the present study. However, immunostaining with that antibody was not compared with immunostaining for apo B. The possibility of ex vivo oxidation is a concern in that study, not only because autopsy specimens were used but also because the fixative did not contain antioxidants. Furthermore, the antibody recognized an oxidized phospholipid epitope rather than a protein epitope, so it is possible that the oxidized phospholipids had transferred from oxidized lipoproteins. However, taken together, the demonstration that cellular and subcellular membranes of atherosclerotic plaque macrophage foam cells are recognized both by an antibody against oxidized phospholipids50 and by an antibody against epitopes formed on proteins by polyunsaturated fatty acid oxidation (present study) provides compelling evidence for lipoprotein-independent oxidative modification of cells in atherosclerosis.
Arterial wall cells have been shown recently to produce enzymes, including myeloperoxidase51 and lipoxygenases,35 36 that could induce formation of oxidation epitopes through the production of reactive oxygen species. In fact, previous studies in atherosclerotic human arteries35 36 and in normal rabbit arteries transfected with 15-lipoxygenase52 demonstrated that arterial wall cells that express the enzyme are stained positively with antibodies against malondialdehyde- and 4-hydroxynonenyl–modified LDL.35 36 52 However, these antibodies are known to recognize modified lysines on proteins other than apo B.31 In light of the findings of the present study, it is conceivable that these antibodies to malondialdehyde- and 4HNE-modified LDL recognized epitopes generated by active 15-lipoxygenase on other proteins in addition to apo B. Because 15-lipoxygenase typically is not detected in resident tissue macrophages in areas without active inflammation, its expression and the presence of oxidation-specific epitopes may be general features of active inflammation, in which reactive oxygen species are generated. Furthermore, oxidation-specific epitopes have been detected on non–apo B proteins in active inflammatory lesions, such as type IV collagen in Heymann nephritis53 and hepatic proteins in ethanol-induced liver damage,54 which demonstrates that inflammatory cells can induce formation of oxidation-specific epitopes on a variety of proteins.
Finally, the present study demonstrates that oxidation of proteins in addition to apo B of LDL, particularly cell-associated proteins, occurs in human atherosclerotic lesions. This observation might help to explain why administration of a variety of antioxidants to hypercholesterolemic animals in doses sufficient to protect LDL from oxidation does not uniformly inhibit atherosclerosis24 25 26 despite their ability to inhibit LDL oxidation in vitro. This suggests that in studies in which antioxidants decreased atherosclerosis, they may have done so through inhibition of oxidative processes central to atherogenesis in addition to LDL oxidation. For example, vitamin E treatment of endothelial cells inhibits cytokine-induced monocyte adhesion.55 Vitamin E also inhibits SMC proliferation, an effect shown to be independent of its antioxidant property.56 Probucol inhibits macrophage production of interleukin-1,57 whereas a probucol analogue, although effective for inhibition of LDL oxidation, does not retard atherosclerosis in a rabbit model.25 Importantly, a recent study27 showed that a dose of all-trans ß-carotene that has no effect on LDL oxidizability can effectively inhibit atherosclerosis in a cholesterol-fed rabbit model. In contrast, other antioxidants, although effective in protecting LDL from oxidation, may have failed to retard atherogenesis because of their inability to inhibit other cellular processes central to atherogenesis in which reactive oxygen species play a fundamental role. These processes could include adhesion molecule expression58 59 ; release of cytokines,13 14 15 growth factors,7 and chemoattractants5 ; and induction of cell proliferation60 and apoptosis.61 62 63 64
These results also suggest that monoclonal antibodies or circulating human antibodies that recognize OxLDL should not be referred to as "anti-OxLDL" antibodies, because none has the specificity for OxLDL that the term suggests. Moreover, particularly in the case of circulating antibodies, the term "anti-OxLDL" antibodies implies that OxLDL is the antigen, although it is quite conceivable that other oxidation epitopes on a variety of plaque proteins in addition to OxLDL may serve as antigens.
In summary, the present study demonstrates that many oxidation epitopes found in human atherosclerotic lesions, particularly cell-associated oxidation epitopes, are not present on LDL. These epitopes can be formed in vitro on a variety of human proteins when exposed to polyunsaturated fatty acids and oxidant stress and also in phorbol ester–stimulated cells cultured in media devoid of apo B. These results provide strong evidence of the involvement in atherosclerosis of oxidative processes in addition to lipoprotein oxidation and may help to explain why some agents with antioxidant properties may inhibit LDL oxidation in vitro but be ineffective in the reduction of atherosclerosis in vivo. Moreover, they raise the possibility that the ability to prevent in vitro LDL oxidation may not represent the most biologically relevant marker of the antiatherogenic potential of an antioxidant.
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Acknowledgments |
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The authors wish to thank Lisa Anne Billings for expert assistance in manuscript preparation and Thomas O. McDonald, Susan Rozell, and Kay Gurley for adroit technical assistance. This work was supported in part by grants HL-02788, HL-47151, and HL-30086 from the National Institutes of Health, Bethesda, Md, and by Grant-in-Aid WA-94-518R from the American Heart Association's Washington Affiliate, Seattle, Wash.
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Presented in part at the Annual Meetings of the Western Society for Clinical Investigation, Carmel, Calif, February 11, 1995, and the American Society for Clinical Investigation, San Diego, Calif, May 5, 1995.
Received November 8, 1995; revision received February 29, 1996; accepted March 12, 1996.
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