N-Acetyl-Cysteine Decreases the Matrix-Degrading Capacity of Macrophage-Derived Foam Cells
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© 1998 American Heart Association, Inc.
Basic Science Reports |
N-Acetyl-Cysteine Decreases the Matrix-Degrading Capacity of Macrophage-Derived Foam Cells
New Target for Antioxidant Therapy?
From the Division of Cardiology, Emory University School of Medicine, Atlanta, Ga.
Correspondence to Zorina S. Galis, PhD, Emory University School of Medicine, Division of Cardiology, 1639 Pierce Dr, WMB #319, Atlanta, GA 30322. E-mail zgalis@emory.edu
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Abstract |
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Background—Atherosclerotic
plaque destabilization triggers clinical
cardiovascular disease and thus represents an
attractive therapeutic target. Weakening of
tissue through the action of
matrix-degrading enzymes, called matrix
metalloproteinases (MMPs), released by
resident macrophages was previously implicated in
unstable vascular syndromes.
Methods and Results—We used a hypercholesterolemic rabbit model of atherosclerosis to investigate the gelatinolytic activity associated with macrophage-derived foam cells (FCs). Gelatinolytic activity and expression of MMP-9 but not of MMP-2 cosegregated with macrophage FCs in aortic lesions. Macrophage-derived gelatinases were further investigated in vitro. MMP-9 was identified as the main macrophage-derived gelatinase in cells isolated from aortic lesions and from granuloma induced in the same rabbits to increase cell yield. Importantly, detection of activated MMP-9 in the FC culture medium supports the notion that these cells can independently initiate processing of secreted MMP zymogens to active enzymes. We further examined whether FC gelatinolytic activity is dependent on the presence of reactive oxygen species (ROS). We found that treatment (1 to 5 days) with 1 to 10 mmol/L N-acetyl-L-cysteine (NAC), an ROS scavenger, decreased not only gelatinolytic activity but also gelatinase expression by FCs. Similarly, NAC treatment of explanted lesions abolished in situ gelatinolytic activity and MMP-9 expression.
Conclusions—Macrophage FCs are an abundant source of gelatinolytic activity that can be inhibited in vitro and in situ by NAC. This newly described action of antioxidant therapy might prove useful to inhibit matrix degradation and to improve vascular stability.
Key Words: atherosclerosis
• metalloproteinases • free radicals • antioxidants
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Macrophage-derived FCs, which figure
prominently in areas of atherosclerotic
plaques prone to rupture, are a likely source
of plaque instability.1
2 3
Many recent studies focusing on various
aspects of the active macrophage FC involvement in the
weakening of the vascular matrix scaffold
suggest that it is a major factor
determining plaque vulnerability. Activated macrophages
release cytokines, which increase the
repertoire of matrix-degrading enzymes,
called MMPs, secreted in vitro by vascular cells4
5
6 and produce their own
MMPs.7 Furthermore, we and
others have shown that macrophage-derived
FCs resident in human and experimental
atherosclerotic lesions are also associated with
matrix-degrading activity due to active MMPs.8
9 10
11 To digest matrix
components, however, latent cell-secreted MMPs
require posttranslational processing to
active forms, a step acting as a key regulatory
mechanism of matrix degradation by MMPs.12
In our search for MMP activation mechanisms
relevant to atherosclerosis, we recently found
that ROSs can trigger activation of MMP
precursors released by vascular SMCs.13
Similar ROSs are produced by macrophage-derived
FCs; thus, in addition to modulating vascular MMP
gene transcription via release of
stimulatory cytokines, these cells may regulate
MMP enzymatic activation via release of ROSs. In
addition, macrophage-derived FCs most likely
contribute with their own MMPs to matrix degradation.
Certain conditions enhance production of MMPs by
monocytic cells and cell lines,7
and the intracellular lipid accumulation characteristic
of monocyte-derived macrophages residing in
atheroma could be one of them.14
We found previously that the macrophage-derived
FCs isolated from aortic lesions of
hypercholesterolemic rabbits secrete in
vitro precursors of the inducible MMPs interstitial
collagenase and stromelysin.14
However, we did not detect generation of
their active forms, the only ones capable of matrix
degradation. We also did not investigate the
expression of the macrophage-derived
gelatinases MMP-9 and MMP-2. These MMPs, specialized in
digestion of basement membrane collagens and
elastin,15 have since
been implicated in weakening of vascular
tissue in unstable coronary syndromes10
and in aortic aneurysms.16
Thus, in the present study, we sought to
characterize gelatinase production by in vivo
differentiated macrophage FCs, which to the
best of our knowledge has not yet been
investigated. We also explored the hypothesis that
gelatinase activity in atheroma areas rich
in macrophage-derived FCs is ROS-dependent
and thus inhibitable by ROS scavengers. For this
purpose, we used an experimental
hypercholesterolemic rabbit model that
develops macrophage FC–rich aortic lesions
and allows isolation of in vivo differentiated
macrophage FCs for in vitro studies.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Animal Model of Atherosclerosis
Experimental atherosclerotic lesions were induced in
the double-injury rabbit model, in which
aortic intimal lesions are rich in lipid-laden
macrophages.17 We
induced aortic lesions in New Zealand White
rabbits (n=8), as previously described in detail,14
by balloon angioplasty 1 week after
initiating a hypercholesterolemic diet (0.5%
cholesterol and 4.5% coconut oil added to Purina chow).
The diet was maintained for the following 8
weeks. Three weeks before the animals were
killed, subcutaneous granulomas were induced
in the same rabbits by implantation of 2 or 3 sterile
polyurethane sponges (Baxter Scientific) per
animal under the dorsal skin. A similar
procedure was used simultaneously to induce
subcutaneous granuloma formation in
normocholesterolemic New Zealand White rabbits
on regular Purina chow. All animals were
euthanized with 100 mg/mL pentobarbital.
Aortas were harvested for morphological processing,
biochemical analysis, and isolation of FCs.
Sponges were removed under sterile
conditions and used for isolation of macrophages.
The protocol for animal use was approved by the
Emory University Committee on Institutional
Animal Care and Use.
Isolation of
Macrophages
Aortic lipid-laden macrophages were isolated from
rabbit atheromas as described previously.14
The aortic intima was separated and minced
in ice-cold sterile HBSS. Tissue was incubated with
agitation at 37°C in sterile HBSS containing
collagenase (type I, Worthington), elastase,
and soybean trypsin inhibitor (Sigma
Chemical Co), then filtered through sterile nylon mesh.
Granuloma macrophages were collected by gentle
squeezing of sponges. Macrophages were
further isolated by metrizamide-density
centrifugation as previously described and plated in
Opti-MEM (Gibco-BRL). Macrophage purity was
assessed by immunocytochemical staining as
described below.
Cell Culture
Experiments
Macrophages and macrophage-derived FCs were maintained
in culture in serum-free Opti-MEM for up to
5 days. In some experiments, NAC (100 µmol/L
to 10 mmol/L) was added to the culture
medium of granulomatous macrophages. We harvested
cell-conditioned culture media and cell
lysates obtained by use of ice-cold 10 mmol/L
phosphate buffer/150 mmol/L sodium chloride containing
1% Triton X-100, 0.1% SDS, 0.5% sodium
deoxycholate, and 0.2% sodium azide. Cell
viability was assessed at the end of various
treatments by the "Live-dead" fluorescent kit
(Molecular Probes). Computer-assisted image
analysis was performed with ImagePro Plus
2.0 software (Media Cybernetics).
In Situ
Treatment With ROS Scavengers
Paired aortic rings (of abdominal, thoracic, or aortic
arch) were incubated with or without 10 mmol/L
NAC for up to 4 days. Tissues were then
processed for immunocytochemistry or extracted with the
lysis buffer and analyzed for gelatinolytic
activity by SDS-PAGE as previously
described.6 Conditioned
culture media were harvested and compared
for expression and activity of secreted gelatinases.
SDS-PAGE
Zymography
In this method, a gelatin substrate was included in the
composition of the polyacrylamide/SDS gels,
and samples were separated according to
their apparent molecular weight by electrophoresis.
Areas of lysis appeared as white after
renaturing and staining of gels with
colloidal Brilliant Blue G-250 (Fisher Scientific).
MMPs with gelatinolytic activity were identified
and compared in samples of culture media
harvested from explanted lesions or cultured
macrophages. An increase in the intensity of
gelatinolytic bands with lower apparent
molecular weight and/or generation of new
gelatinolytic bands relative to bands representing the
zymogens is interpreted as gelatinase activation.18
Gelatinolytic bands were quantified after
scanning densitometry with NIH Image 1.57 software.
Statistical significance was investigated by
Student's t test with one-tailed
distribution by use of Microsoft Excel 5.0 software.
Western Blotting
Culture media were separated on 10% SDS-PAGE minigels
and transferred onto nitrocellulose (Bio-Rad
Laboratories). Incubation with anti–MMP-2 or
anti–MMP-9 monoclonal antibodies (Oncogene Science),
recognizing both the latent and active
forms, was followed by incubation with
secondary antibodies coupled to horseradish peroxidase
and development of a chemiluminescent
reaction (ECL kit from Amersham International).
Signals (positive bands) were quantified and
analyzed as described above.
Histological
Characterization of Aortic Tissue and Isolated Cells
Immunostaining was performed on frozen tissue specimens
embedded in O.C.T. compound (Miles) to
identify macrophages (anti–RAM-11, Dako
Corp) and to detect MMP-9 and MMP-2 (Oncogene).
Staining was developed with the LSAB
staining kit (Dako), and sections were
counterstained with Gill's hematoxylin (Sigma).
Isolated cells were stained by single or
double immunofluorescence using
species-specific secondary antibodies coupled to
fluorochromes (FITC or Texas Red, Jackson
ImmunoResearch). Nuclei were counterstained
for 2 minutes with 0.5 µg/mL bisbenzamide (Calbiochem).
Intracellular lipid accumulation was revealed by
staining of cells with 5 µg/mL Nile red
(Molecular Probes).
In Situ
Zymography
In situ gelatinolytic activity was detected in frozen
aortic tissue specimens as previously
described,9 with gelatin
coupled to a green fluorochrome. Briefly,
tissues were processed for obtaining unfixed
frozen sections, which were placed on microscope
slides previously coated with fluorescent
gelatin. The specimens were then incubated
at 37°C for 2 days and examined with a Zeiss
fluorescence microscope to reveal areas of active lysis
of the gelatin substrate.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Gelatinolytic
Activity Is Associated With Increased MMP-9 Expression
and Macrophage-Derived FCs in Experimental Atheroma
Neointimal lesions developed in the double-injury model
of atherosclerosis were composed
predominantly of macrophage-derived FCs (Figure
1
)
identified by immunohistochemistry with a
macrophage-specific antibody. The
gelatinolytic activity, examined by in situ zymography,
was restricted to the macrophage-rich intimal
area. Immunostaining for the two possible
sources of gelatinolytic activity, MMP-2 (gelatinase
A) and MMP-9 (gelatinase B), showed that expression
of MMP-9 was specifically associated with the
neointimal lesion, whereas MMP-2 staining
was diffuse. This observation suggested that
the macrophage-derived gelatinolytic activity was due
mainly to the presence of macrophage-derived
MMP-9.
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Characterization
of Lipid-Laden Macrophages
Further experiments were performed to confirm that the
lipid-laden macrophage was the source of
MMP-9 and gelatinolytic activity. However,
to further investigate expression and activation of
gelatinases in culture, we had to find an
alternative source of macrophage-derived FCs,
the yield of aortic FCs being very limited (3x105
to 106 cells
per aorta). We therefore produced and isolated in vivo
differentiated macrophage-derived FCs from
subcutaneous granulomas of
hypercholesterolemic rabbits. To be able to assess
possible differences associated with the FC
phenotype, we also produced and studied, in
parallel, non–lipid-laden macrophages by
implanting subcutaneous sponges in normocholesterolemic
rabbits. Isolated cells were positively
identified as being of macrophage origin by
immunofluorescence with the RAM-11 antibody (Figure
2)
and were used for the in vitro experiments. Macrophage
purity and viability were >98%. Nile red
staining confirmed intracellular lipid
accumulation in macrophages from hypercholesterolemic
rabbits. Granuloma FC yield was an order of
magnitude higher than aorta (2x107
to 3x107 per rabbit).
These FCs produced MMP-3 (Figure 2),
similar to macrophage-derived FCs isolated
from aortic lesions.14
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)
from normocholesterolemic rabbits. Bottom,
Macrophage-derived FCs of granuloma express
stromelysin (MMP-3). Nuclei are counterstained
with bisbenzamide. Magnification x400.
Macrophage-Derived FCs Release and Activate MMP-9
Analysis of gelatinolytic activity released in culture
by different segments of atherosclerotic
rabbit aorta by SDS-PAGE zymography showed
that the aortic arch consistently produced the highest
level of total gelatinolytic activity. The
activity released by thoracic aorta was also
higher than that of abdominal aorta (Figure
3).
The gradual increase in gelatinolytic activity
paralleled the extent of macrophage-rich
lesions. Migration of higher-molecular-weight
gelatinolytic activity was consistent with
the presence of MMP-9, whereas gelatinolytic
activity running at lower molecular weight was
most likely due to MMP-2, the main MMP produced
by vascular SMCs. Analysis of gelatinolytic
activity released in culture by isolated
aortic or granulomatous FCs showed that these cells
produce copious amounts of the gelatinolytic
activity associated with MMP-9. Because
gelatinolytic profiles were identical for
the FCs isolated from either source, we used granuloma
macrophages for further in vitro
experiments. Both lipid-laden and non–lipid-laden
macrophages were maintained in culture up to 5
days.
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Effect of NAC on
Protein Expression and Activity of Macrophage-Derived
MMP-9
Because our recent experiments showed that ROSs may
function as activators of SMC-derived latent
gelatinases,13 we
hypothesized that activation of FC-derived
pro–MMP-9 may be related to concomitant
production of ROSs. To test ROS contribution,
we treated macrophages with NAC, an ROS
scavenger. Gelatinolytic activity in the
culture media of untreated and NAC-treated cells
was analyzed by SDS-PAGE zymography. We found
that 24 hours of treatment with NAC reduced
both the gelatinolytic activity consistent
with migration of the MMP-9 zymogen and that of the
faster band migrating at the expected position
for active MMP-9 (Figure 4).
To confirm the identity of MMP-9 and to further
assess effects of treatment, we also analyzed the
effect of NAC on MMP-9 protein level by
immunoblotting (Figure 5).
Comparing non–lipid-laden and lipid-laden
macrophages, we identified the MMP-9
precursor in culture media conditioned by either
macrophage population. Interestingly, only
the culture media conditioned by lipid-laden
macrophages contained the fully activated MMP-9,
migrating around 66 kDa as previously
reported.19
20 The same anti–MMP-9
antibodies did not recognize SMC-derived MMP-2,
which has a similar apparent molecular
weight (not shown), confirming that this
faster band was indeed generated through processing of
latent MMP-9. Culture media from macrophage FCs
treated with 10 mmol/L NAC for 48 hours had
significantly lower levels of the precursor
and active forms of MMP-9. NAC treatment also
inhibited expression of the MMP-9 precursor by
non–lipid-laden macrophages. We also
confirmed that the decreased gelatinase
production was not due to a cytotoxic effect of NAC.
Viability tests performed at the end of each
experiment showed that after 5 days in
culture, FC viability was 79.3±6.8% live cells
in untreated versus 72.4±11.9% live cells treated
with 10 mmol/L NAC (n=8, P=NS).
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NAC Reduces the
Gelatinolytic Activity and In Situ Expression of MMP-9
in Experimental Atherosclerotic Lesions
The results obtained with cultured macrophage-derived
FCs and non–lipid-laden macrophages
suggested the possibility of reducing
production of active gelatinase by resident macrophages
through NAC treatment. This effect was tested by
incubating segments of atherosclerotic
rabbit aorta with NAC in organ culture
conditions. We found that the NAC treatment abolished
the gelatinolytic activity released by
aortic tissue and significantly decreased
the level of MMP-9 protein detected by Western blotting
(Figure 6).
The fact that the effect of NAC was not restricted to
inhibition of gelatinolytic activity but
rather also affected FC MMP-9 expression was
confirmed by the disappearance of MMP-9 immunopositive
staining in the FC-rich lesions maintained in
culture with NAC (Figure 7).
This effect was specific to the signal associated
with the presence of MMP-9, since it did not
affect expression of macrophage markers also
detected by immunostaining. In addition,
detection of MMP-9 was not affected in paired untreated
specimens that were maintained in culture
and processed simultaneously (Figure 7).
Thus, lack of MMP-9 detection in lesions after
treatment with NAC suggests that the action
of NAC is not restricted to inhibition of
MMP-9 activity or cellular secretion but rather
also involves suppression of macrophage MMP-9
synthesis.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Recently, the action of MMPs has emerged as an
important component of the natural history
of atherosclerosis8
11 21
and of the vascular response to injury.22
23 24
Macrophage-derived FCs associate clinically
with unstable human plaques1
2 3
and microscopically with vulnerable areas
and active MMPs8 and thus
may be responsible for compromising vascular
tissue integrity through matrix degradation.
Macrophages are also thought to be a major source of
the redox stress that characterizes
atherosclerotic vessels.25
26 27
In the present study, we investigated production and
activation of gelatinases produced by
macrophage FCs of hypercholesterolemic rabbits,
a good model for study of macrophage-derived FCs
resident in human atheroma. We also
hypothesized that macrophage-derived FCs
have a built-in redox-dependent mechanism leading to
activation of their own MMP zymogens. We
found that gelatinolytic activity in rabbit
atherosclerotic lesions is restricted to the
macrophage-rich areas and colocalizes with
expression of MMP-9. Study of isolated cells
confirmed that macrophage-derived FCs were a major
source of MMP-9 and detected the presence of
active MMP-9. As mentioned, increases in
MMP-9 activity were previously reported in vascular
disease10
28 29
and a variety of other pathological situations with an
inflammatory component, such as rheumatoid
arthritis, as well as metastasis.30
To obtain sufficient material for meaningful
in vitro studies with macrophage-derived FCs, we also
induced formation of these cells in vivo in
granulomas of hypercholesterolemic rabbits.
Implantation of subcutaneous sponges triggers formation
of granulomas whose macrophages accumulate
lipid in hypercholesterolemic conditions.
These macrophage-derived FCs from subcutaneous
granulomas had the same MMP profile as the
aortic FCs.
Observations made on isolated macrophages in culture showed the presence of active gelatinase, although all MMPs known so far are reportedly secreted by cells as zymogens only. This in vitro observation supports the notion that MMP-9 activation can occur independently of the classic plasmin-mediated pathway.12 The possibility that macrophage gelatinolytic activity is redox-dependent is suggested by our previous studies showing activation of gelatinase zymogens by ROSs know to be produced by macrophage FCs,13 as well as by our present detection of active MMP-9 in an isolated cell system along with inhibition of macrophage-derived gelatinolytic activity by treatment with NAC, an ROS scavenger. Activated macrophages, especially those of atherosclerotic lesions, are a major source of ROSs; thus, such an activation mechanism would result in activation of MMP zymogens secreted by the macrophages themselves as well as by the neighboring cells. However, our observations do not exclude the possibility that activation of MMP-9 might occur through other cell-dependent mechanisms, including the action of a group of cell membrane molecules called MT-MMPs. It was reported that these molecules activate the zymogens of MMP-2 and MMP-13,31 but it is not yet known whether monocytes/macrophages express MT-MMPs and whether pro–MMP-9, the gelatinase whose activity was investigated in this study, is a substrate for MT-MMPs.
NAC is widely used as
an antioxidant on the basis of its capacity
to scavenge ROSs32; however, its inhibitory
effect on gelatinolytic activity might also
involve a direct interaction with the enzyme. Although
further biochemical studies will be necessary to
differentiate between these two possible
components of gelatinase inhibition, the capacity
of NAC to inhibit gelatinolysis that we found is
unquestionable. This is important because
regulation of gelatinolytic activity
ultimately determines the level of matrix degradation
by vascular gelatinases. However, our
results suggesting that NAC may also affect
prior events in the pathway leading to secretion of
MMP-9 are also interesting. For instance, we
found that NAC treatment effectively
inhibited the level of MMP-9 protein secreted in
culture by macrophages. Findings from in situ
observations of macrophage-derived FCs of
NAC-treated experimental atheroma support
NAC inhibition of MMP-9 expression. The hypothesis that
redox stress modulates cellular MMP-9 expression
is novel; thus, at present, the mechanism is
still unknown. Possibly relevant information
is provided by studies showing MMP-9 induction by
cytokines and phorbol esters in a variety of
cells, including vascular endothelial cells
and SMCs.5
6 MMP-9 induction may require
cooperation between AP-1–, NF-
B–,
or SP-1–responsive elements.33
Conversely, monocyte/macrophages constitutively
express pro–MMP-9, but its expression was
shown to be enhanced by cellular differentiation,7
concanavalin A, or LPS stimulation.34
Interestingly, the effects of LPS,
cytokines, and phorbol esters may occur via generation
of ROS intermediates. In transformed cells,
multiple pathways leading to activation of
redox-sensitive transcription factors,35
such as NF-B,
SP-1, Ets, and AP-1, have been shown to increase
expression of MMP-9.36
These additional considerations and the
present results support the hypothesis that MMP-9 gene
expression could be redox-regulated. As they
become available, further experiments using
species-specific molecular probes will be performed to
confirm our observations at the messenger
RNA level.
Beneficial
cardiovascular effects of various antioxidant therapies
were reported in human and experimental
atherosclerosis or restenosis, and efforts
to understand its mechanism of action are under way.37
Treatments with probucol and vitamins E and C38
39 40
have been shown to reduce intimal lesions
after balloon injury in hypercholesterolemic
animals. In addition to decreasing the lipid
content and lesion area, antioxidants also appear to
alter lesion cellularity, reducing primarily
the monocyte-macrophage content.41
In addition to potentially reducing the
extracellular effects of ROSs (eg, oxidative
modification of lipoproteins and activation
of MMPs), scavenging ROSs may prevent or diminish
intracellular activation of redox-sensitive
genes. Recently published results showed
that after dietary supplementation with
-tocopherol,
peripheral blood monocytes harvested from healthy
volunteers produced fewer ROSs and
interleukin-1 and displayed decreased
adherence to endothelium when stimulated with LPS.42
Also, NAC was found to inhibit vascular cell
adhesion molecule-1 expression in vitro43
and in vivo.44 Abatement
of these effects of oxidative stress in
incipient stages of atherosclerosis probably diminishes
stimuli leading to monocyte recruitment and
formation of macrophage-derived FCs. NAC was
also found to inhibit the chemotactic and invasive
activities of human tumor cells, most likely
through inhibition of gelatinase activity,45
and it is currently considered to be a promising
antioxidant and anticancer chemopreventive agent.32
Our study shows a new potential use for NAC
as an inhibitor of MMP activity. Inhibition
of MMP activity by antioxidants early in the course
of atherosclerotic lesion development may limit
inflammatory cell infiltration, cell
movement, and proliferation, events that all
require participation of active MMPs. Importantly,
by showing the possibility of inhibiting the
matrix-degrading capacity of macrophage FCs
prevailing in advanced plaques, the present
results suggest that treatment with ROS scavengers may
be effective in late stages of atherosclerosis.
This would contribute to restricting the
weakening of vascular matrix, thought to be
a major factor precipitating plaque destabilization.
Although we extensively examined and report
here the effects of NAC, we believe that the
results are applicable to other antioxidants,
such as vitamin E and probucol, as suggested by
our preliminary experiments. Future in vivo
experiments need to be undertaken to confirm
the possibility of using antioxidant therapy as a
way to improve the stability of atherosclerotic
plaques.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported through funds provided by a
Grant-in-Aid from the American Heart
Association and a faculty development award
from the Beda Whitaker Foundation.
Received January 13, 1998; revision received April 13, 1998; accepted April 22, 1998.
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