doi: 10.1161/hq0701.092145
© 2001 American Heart Association, Inc.
Vascular Biology |
Apoptotic Death of Inflammatory Cells in Human Atheroma
From the Division of Pathology II, Faculty of Health Sciences, Linköping University, Linköping, Sweden (W.L., X.-M.Y.); the Department of Pathology, Gade Institute, University of Bergen, Bergen, Norway (H.D.); and the James Graham Brown Cancer Center, University of Louisville, Louisville, Ky (J.W.E.).
Correspondence to Wei Li, MD, PhD, Division of Pathology II, Linköping University Hospital, S-581 85 Linköping, Sweden. E-mail weili{at}pat.liu.se
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Abstract |
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Abstract—Although the accumulation of cholesterol and other lipidic material is unquestionably important in atherogenesis, the reasons why this material progressively accumulates, rather than being effectively cleared by phagocytic cells such as macrophages, are not completely understood. We hypothesize that atheromatous lesions may represent "death zones" that contain toxic materials such as oxysterols and in which monocytes/macrophages become dysfunctional and apoptotic. Indeed, cathepsins B and L, normally confined to the lysosomal compartment, are present in the cytoplasm and nuclei of apoptotic (caspase-3–positive) macrophages within human atheroma. The possible involvement of oxysterols is suggested by experiments in which cultured U937 and THP-1 cells exposed to 7-oxysterols similarly undergo marked lysosomal destabilization, caspase-3 activation, and apoptosis. Like macrophages within atheroma, intralysosomal cathepsins B and L are normally present in the cytoplasm and nuclei of these oxysterol-exposed cells. Lysosomal destabilization, cathepsin release, and apoptosis may be causally related, because inhibitors of cathepsins B and L suppress oxysterol-induced apoptosis. Thus, toxic materials such as 7-oxysterols in atheroma may impair the clearance of cholesterol and other lipidic material by fostering the apoptotic death of phagocytic cells, thereby contributing to further development of atherosclerotic lesions.
Key Words: atherosclerosis • apoptosis • lysosomal enzymes • oxysterols • plaque instability
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Introduction |
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The most obvious characteristic of atherosclerotic lesions is an abundance of cholesterol and other lipidic material that, in advanced lesions, is often referred to as "gruel." It is not fully understood, however, why this material persists rather than being cleared by the professional phagocytes such as monocytes/macrophages that typically abound in such lesions. Our investigations have centered on the idea—certainly not original with us—that components of these atherosclerotic lesions may be cytotoxic and, in particular, cause dysfunction and death of phagocytic cells recruited to the site. Our particular focus has been on oxidized lipids, including oxidized LDL (oxLDL) and associated oxysterols. These are known to be abundant in atherosclerotic lesions and to be toxic to a variety of vascular cells.1 2 3 4 5 6 Furthermore, increased plasma concentrations of 7ß-hydroxycholesterol (7ß-OH) are related to enhanced risk for cardiovascular diseases.7 Among the oxysterols, 7ß-OH and 7-ketocholesterol (7-keto) are major components of oxLDL.8
Apoptosis is a prominent feature of atherosclerotic lesions and may be associated with instability and remodeling of atheroma plaques.9 10 Previous investigations have suggested that destabilization of lysosomes might be an important early event in apoptosis triggered by a variety of agonists. This has led us to the idea that minor leakage of lysosomal proteases and other hydrolytic enzymes into the cytosol of cells may actually be an initiating event in apoptosis.11 12 13 14 15 Two lysosomal cysteine proteases, cathepsins B and L, have been identified as caspase-processing enzymes and inducers of apoptosis.16 17
Therefore, in the present investigations, we examined macrophages within human atherosclerotic plaque for evidence of the release of lysosomal enzymes, in particular cathepsins B and L. We also sought to determine whether the release of cathepsins might coincide with the occurrence of typical signs of apoptosis in these cells. Finally, we tested the effects of one potential class of toxins, 7-oxysterols, on macrophage cell lines to determine whether the features found in macrophages within atherosclerotic lesions might be reproduced by one known toxic component of the atherosclerotic plaque. Overall, the results support the concept that the interior of atheroma lesions may be a "death zone" in which various cytotoxic plaque components cause the dysfunction and, ultimately, death of phagocytes that would otherwise be capable of clearing the debris and resolving the lesion.
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Methods |
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Immunohistochemistry
Segments from normal human mammary arteries (n=4), atherosclerotic coronary arteries (n=5), and thoracic aortas (n=5) were collected from clinical autopsy cases. For visualization of cathepsins B and L, serial sections were overlaid with goat polyclonal anti–human cathepsins B– and L–reactive primary antibodies (Santa Cruz Biotechnology, Inc), respectively, followed by an alkaline phosphatase–conjugated swine anti-goat antibody. The alkaline phosphatase activity was visualized with 4-benzoylamino-2,5-diethoxybenzenediazonium chloride (Sigma Chemical Co). Immunoreactivity of the active form of caspase-3 was detected by incubating the samples with polyclonal rabbit anti–human active caspase-3 (Pharmingen) followed by a goat anti–rabbit peroxidase conjugated antibody (DAKO Corp). The peroxidase activity was localized by use of 3-amino-9-ethylcarbazole (Sigma).
To determine the relationship between caspase-3 and lysosomal cathepsins within the same atherosclerotic lesions, double immunostaining of cathepsins B and L and caspase-3 was performed. The immunodetection of macrophages with monoclonal anti-CD 68 antibodies (DAKO) was carried out as previously described.10
As a negative control, the primary antibody was omitted. In all cases, endogenous peroxidase was blocked by preincubation with 3% H2O2. To avoid nonspecific antibody adsorption, preimmune serum of the same origin as the secondary antibody was used before primary antibody application. All antibodies were diluted with 1% BSA.
Cells and Culture Conditions
Cells from 2 human monocytic cell lines, U-937 and
THP-1, were cultured in RPMI-1640 culture medium containing 10% FCS.
For the experiments, cells were directly exposed to either 7ß-OH or
7-keto (0 to 56 µmol/L), dissolved in ethanol for 6 to 48 hours or
pretreated with 75 µg/mL E64
[trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane,
Sigma] for 14 hours, and then exposed to 7ß-OH and 7-keto for
another 24 hours. A selective inhibitor of cathepsins B and
L, Z-Phe-Ala-fluoromethylketone (Z-FAFMK) (Enzyme Systems
Products), was applied (50 µmol/L) for 24 hours together with
7ß-OH and 7-keto in some experiments. The solvent, ethanol (maximum
0.4%), had no toxic effect. Cell membrane integrity was determined by
trypan blue dye exclusion test, lactate dehydrogenase (LDH) assay, and
propidium iodide (PI) staining.
Assessment of Apoptosis
For morphological assessment, cultured cells were
stained with Wright-Giemsa and examined by light microscopy as
previously described.14 The
cells were also examined by transmission electron microscopy as
previously
described.15
Caspase-3 activity of the cultured cells was assayed by spectrofluorometry. The cells were incubated with 20 µmol/L Ac-DEVD-AMC (Pharmingen) at 37°C for 2 hours. The fluorescence intensity of the liberated 7-amino-4-methylcoumarin was measured at excitation 380/emission 435 nm.
Apoptosis of both cultured cells and sections from normal and atherosclerotic human arteries was detected by the terminal dUTP nick end-labeling (TUNEL) technique using ApopTag in situ apoptosis detection kit according to the manufacturer’s instructions (Oncor Inc). Apoptotic and necrotic cells were simultaneously detected by flow cytometry by use of an annexin V–PI kit according to the manufacturer’s instruction (Roche Diagnostics GmbH).
Determination of Lysosomal Stability
Lysosomal stability was assessed by the acridine
orange (AO) vital uptake technique as described
previously.11 12
The intensity of AO-induced red fluorescence was measured by
static cytofluorometry (50 cells per sample) or by FACScan
(Becton-Dickinson) flow cytofluorometer (10 000
cells per sample) fitted with Lysis II
software.12
Assessment of Cathepsin B and L
Activity
Control and E64-treated cells were collected by
centrifugation and washed twice with PBS. Cell pellets
were lysed with 0.1% Triton X-100, and the
activities of the lysosomal cysteine proteinases, cathepsins B and L,
were measured with Z-Arg-Arg-7-amido-4-methylcoumarin
and Z-Phe-Arg-7-amido-4-methylcoumarin as the
respective substrates.
Statistics
Statistical comparisons were made by use of the
Mann-Whitney U test.
Differences were considered significant at values of
P
0.05.
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Results |
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Arterial Cathepsins B and L and Caspase-3 Immunohistochemistry
Normal human arteries contain few or no cathepsin B– and L–positive cells. In atherosclerotic lesions, there is a lesion-dependent enhanced expression of cathepsin B (Figure
I, see
online at http://atvb.ahajournals.org), mainly in
macrophages and smooth muscle cells in the lesions (cathepsin L
showed the same pattern). In support of a possible causal relationship
between lysosomal leakage and cellular apoptosis, in the same
plaques the presence of immunoreactive cathepsins B and L
(Figure 1, middle) colocalized with apoptotic
macrophages, as assessed by both CD68 and TUNEL staining
(Figure 1, top). Interestingly, immunoreactive cathepsins B
and L were seen not only in the cytoplasm of arterial
macrophages and in extracellular spaces but also in the nuclei
of some apoptotic macrophages
(Figure 1, bottom right). This finding is in accord with
earlier reports that translocation of cathepsin B may participate in
apoptosis by proteolytic attack on nuclear
contents.18 19 By
double immunohistochemistry, both cathepsins B and L were found
colocalized with caspase-3–positive areas in the lesions, especially
in advanced fibrous plaques
(Figure 1, bottom).
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Lysosomal Destabilization and 7-Oxysterol
Cytotoxicity
The in vivo findings reported above support the general
concept that the contents of atherosclerotic plaque might be cytotoxic,
perhaps by engendering lysosomal instability, release of lysosomal
material such as cathepsins, and subsequent apoptosis. As a
model system for these events, we used the human monocytic cell lines
U937 and THP-1, exposing them to varying doses of the oxysterols
7ß-OH and 7-keto.
Lysosomal integrity was assayed by static and flow
cyto-fluorometry after AO staining. AO is a lysosomotropic weak
base and a metachromatic fluorophore that accumulates in the lysosomal
compartment as a result of proton trapping. In normal cells it induces
red granular lysosomal fluorescence on green light excitation.
Cells with ruptured lysosomes, on the contrary, appear
"pale" (diminished red fluorescence due to fewer intact
lysosomes). Lysosomal destabilization caused by 7ß-OH and
7-keto (28 µmol/L) was clearly time-dependent and occurred before
apoptotic cell death. The number of pale cells increased to
more than 3 times (7ß-OH) and 2 times (7-keto) that of untreated
control cells after 12 hours of exposure
(Figure 2, top row), whereas there was no increased number of
apoptotic or necrotic cells
(Figure 2, bottom row). After an exposure period of 24 hours,
an even greater lysosomal rupture was evident, together with increased
numbers of apoptotic and necrotic cells
(Figure 2). Static cytofluorometry showed similar results.
Exposure to cholesterol did not change red lysosomal
fluorescence.
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3 independent experiments.
Apoptosis was also confirmed by the activation of
caspase-3 (
250% and 150% of control values in 7ß-OH and 7-keto
exposed cells, respectively) and the occurrence of DNA fragmentation as
determined by TUNEL (500% and 300% of control values in 7ß-OH–
and 7-keto–exposed cells, respectively). As assessed by transmission
electron microscopy, exposure to 28 µmol/L 7ß-OH for 24 hours
caused many U937 cells to exhibit typical apoptotic morphology,
with condensation and margination of the nuclear chromatin, pyknosis,
and/or nuclear fragmentation
(Figure 3, B and C). The apoptotic cells also
exhibited other alterations, including increased cytosolic
multivesicular inclusions
(Figure 3, B and C), which have been described earlier for
apoptosis induced by oxysterols in endothelial
cells,18 in cultured
plaque-derived vascular smooth muscle
cells,19 and in
macrophages.20 These
multivesicular inclusions in the cytosol of apoptotic cells are
most likely formed by autophagocytosis of cellular organelles, such as
mitochondria, during frustrated reparative attempts.
Postapoptotic necrotic cells with fragmented or pyknotic nuclei
and permeabilized membranes also were observed
(Figure 3D). Similar ultrastructural alterations, caused by
oxysterols, also were seen in THP-1 cells under the same conditions
(data not shown).
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Cellular plasma membranes remained intact during the first
12 hours of oxysterol exposure, and 80% (7ß-OH) and 95% (7-keto)
of the cells were still intact after 24 hours of exposure to 28
µmol/L of 7-oxysterols
(Figure 2
, bottom row). The trypan blue exclusion test and
LDH assay gave similar results. These cytotoxic effects were specific
for oxysterols, inasmuch as native cholesterol (28 to 56
µmol/L) or <0.4% ethanol had no effect on either cell membrane
integrity or growth over a 48-hour period. Almost identical results
were obtained with both cell lines.
Role of Cathepsins B and L in Apoptosis
Induced by 7-Oxysterols
The expression and localization of lysosomal cathepsins
were examined in U937 cells after exposure to 7-oxysterols. Normally,
the cells display a granular immunoreactivity of cathepsins B and L,
reflecting the lysosomal localization of these proteases. In contrast,
oxysterol treatment caused enhanced cathepsin B and L immunoreactivity,
no longer of a granular nature and dispersed throughout the cytoplasm
of the cells. The apoptotic cells also showed pronounced
cathepsins B and L, which localized with the condensed and shrunken
nuclei
(Figure 4). These findings correspond well to the above
observations in human atheroma, indicating that release of
lysosomal cathepsins and their dispersion throughout the cell is
associated with apoptotic cell death in these lesions. The
activities of cathepsins B and L were significantly increased after 6
hours of exposure to 7-oxysterols and then gradually decreased (as
assayed after 12 and 24 hours). The early increase of the lysosomal
enzymes may represent induction of autophagocytotic repair,
whereas the later decline may be due to autodegradation when the
enzymes are released to the cytosol during the apoptotic
process.
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To determine whether the release of lysosomal cathepsins and
apoptotic cell death might be causally associated, we
investigated the effects of E64 (inhibits mainly the cysteine proteases
cathepsins B, H, and L)21
and the selective inhibitor of cathepsins B and L, Z-FAFMK,
on 7-oxysterol–induced apoptosis. Because E64 is relatively
membrane-impermeable, a high concentration (75 µg/mL) was applied.
Neither inhibitor caused any changes in cell growth or
viability, but cathepsin B activity was reduced to 30% (E64) and
50% (Z-FAFMK), and cathepsin L activity to 12% (E64) and 40%
(Z-FAFMK) of that of control cells. Both E64 and Z-FAFMK provided
remarkable protection against oxysterol-induced apoptosis and
necrosis. The inhibition of oxysterol-induced cell death by E64 was
stronger than that of Z-FAFMK, which may be a result of the more
extensive inhibition of cathepsins B and L by the former. As shown in
Figure 5, both E64 and Z-FAFMK significantly reduced the
numbers of apoptotic cells, as well as the activation of
caspase-3. These results may lend further support to the idea that
leakage of lysosomal cysteine proteinases may play a crucial role in
caspase-3 activation. Similar activation of caspase-3 and its
prevention by E64 also were observed in THP-1 cells exposed to 7ß-OH
(data not shown). Importantly, we did conduct control in vitro assays
of the activity of caspase-3 (using both active caspase-3 and lysates
of 7ß-OH–treated cells). The results showed that unlike caspases
inhibitor Z-VAD, neither E64 nor Z-FAFMK had direct
inhibitory effect on caspase-3
activity.
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Discussion |
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The experiments reported above were focused on the question of why atheromatous lesions not only persist but increase in size despite the presence of cells, such as macrophages, that should ordinarily be capable of catabolizing the material within these lesions. Our overall hypothesis was that cytotoxic substances within atheroma lesions (such as, in this case, oxysterols) might not only suppress the catabolism of plaque material by macrophages but also predispose these cells, which are constantly recruited to atherosclerotic lesions, to apoptotic death. In fact, it was reported earlier that the catabolism of oxLDL and 7-oxysterols by macrophages is impaired, resulting in accumulation of unesterified or esterified cholesterol, cholesteryl hydroperoxide, and oxy- sterols22 23 24 25 and that the degradation of acetylated LDL by lysosomal enzymes is inhibited in the presence of added 7-keto.25
Here, we report, as have others,9 10 20 that human atherosclerotic lesions contain large numbers of apoptotic monocytes/macrophages. Furthermore, we find that these same cells show evidence of lysosomal destabilization. Two lysosomal proteases in particular, cathepsins B and L, are present within the cytoplasm and nuclei of these apoptotic cells. Others4 5 6 have suggested that the progressive cell death within atheroma lesions might result from oxLDL cytotoxicity. OxLDL (which contains oxidized apoproteins as well as oxidized fatty acids and oxysterols) is present in substantial amounts in human and rabbit atheroma.26 At least in in vitro models, both oxLDL and oxysterols have been found to be cytotoxic.4 5 6 8 18
We therefore elected to determine whether the cytotoxic effects that one of these components of oxLDL, 7-oxysterols, might have on the human monocytic cell lines U937 and THP-1 are similar to those observed in macrophages within human atherosclerotic lesions. Indeed, we find that micromolar concentrations of both 7ß-OH and 7-keto induce apoptotic and necrotic death of these cultured cells, in agreement with an earlier report.9 Furthermore, as was the case for apoptotic macrophages within atherosclerotic lesions, we find that apoptosis induced by 7ß-OH and 7-keto is associated with lysosomal rupture, apparent release to the cytosol of cathepsins B and L, and activation of caspase-3. The possibility that released lysosomal enzymes might initiate the apoptotic process is strengthened by our observations that E64 (which mainly inhibits lysosomal cathepsins B, H, and L, but also the nonlysosomal proteases papain and calpain) and Z-FAFMK (a relatively selective inhibitor of cathepsins B and L) increase cell survival and partially suppress both apoptosis and activation of caspase-3 after exposure to 7-oxysterols. E64 also has been found to prevent cell death in other cell culture models,27 28 as well as neuronal death in an ischemic animal model.29 Z-FAFMK effectively protects against apoptosis induced by P53 and several cytotoxic agents.30 A growing body of evidence thus supports the role of released lysosomal cathepsins in the initiation of apoptosis: (1) apoptosis triggered by several different agonists is associated with lysosomal enzyme release11 12 13 14 15 ; (2) leakage of lysosomal proteinases may lead to caspase activation16 17 ; and (3) cathepsin B and other lysosomal proteases are initiating agents for apoptosis, promoting the cleavage of Bid and resulting in the release of cytochrome c from mitochondria.31 32
Matrix degradation by proteolytic enzymes secreted from macrophages has been suggested to be an important factor in atherosclerotic plaque disruption.33 Enzymes involved in the process include serine proteases, cysteine proteases, and matrix metalloproteinases.34 Here, we report, for the first time, the occurrence of extensive immunoreactivity of cathepsins B and L in the nuclei of apoptotic cells within the atherosclerotic plaque and 7-oxysterol–treated apoptotic U937 cells. The mechanism(s) involved in the increase of cathepsins B and L immunoreactivity in apoptotic nuclei is not yet clear. The importance of the cysteine protease cathepsin B in matrix degradation and its association with matrix metalloproteinases was recently demonstrated.35 The authors reported that, in addition to direct participation in tissue destruction, cathepsin B enhances the activity of matrix metalloproteinases by destroying their inhibitors. These findings, together with our results, indicate that lysosomal cathepsins may play an important role in the formation and destabilization of atherosclerotic plaques.
It should be emphasized that the concentrations of oxy- sterols used in the present experiments are substantially lower than those reported to exist in human atherosclerotic plaque. Therefore, there is good reason to suspect that cells that enter into atheroma will be damaged or killed by these and similar toxic plaque materials. If the interior of atheroma lesions does, indeed, represent a death zone for inflammatory cells such as monocytes/macrophages, this has implications for the overall disease process. First, this may lead to the accumulation of large numbers of dead and dying macrophages that ultimately release degradative enzymes, resulting in matrix degradation. Second, it is conceivable that products of these intoxicated cells will serve to attract further inflammatory cells to the lesion, thereby amplifying the inflammatory component and, perhaps, contributing to the overall growth of the plaque. Finally, these observations suggest one possible reason for the evident impotence of lesion-associated macrophages in clearance of the plaque material itself. These findings may open new perspectives for understanding the progression of atherosclerotic disease and, perhaps, generate new molecular targets for pharmaceutical control of plaque development.
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Acknowledgments |
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This study was supported by grants from the Swedish Medical Research Council (4481 and 3490), the Norwegian Medical Research council (129557/310), the Swedish National Network for Cardiovascular Research (project No. B6 3039/96), the Commonwealth of Kentucky Research Challenge Trust Fund (to J.W.E.), and the Landstinget in Östergötland (project No. 99069) (to X.M.Y.). H.D. and J.W.E. were recipients of visiting professor scholarships from the Linköping University Hospital.
Received November 10, 2000; accepted March 23, 2001.
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