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(Circulation. 1998;97:2445-2453.)
© 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?

Zorina S. Galis, PhD; Kazuhiko Asanuma, MD; Denis Godin, PhD; ; Xiaoping Meng, MD

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{at}emory.edu

	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

	Introduction

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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 cells^{4 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.

	Methods

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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.

	Results

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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.

View larger version (88K): [in this window] [in a new window]   Figure 1. Experimental atheroma in aorta of hypercholesterolemic rabbits. Detection of macrophages, gelatinolytic activity, and expression of gelatinases MMP-2 and MMP-9. Top four panels illustrate consecutive sections of lesions in abdominal aorta; bottom four panels, in thoracic aorta. Top, Staining with cell-specific antibody (RAM-11) identifies numerous macrophages in intimal region. Same region contains gelatinolytic activity, as shown by processing of a consecutive section by in situ zymography (gelatinolytic activity is indicated by disappearance of green fluorescent gelatin substrate used to coat microscope slide). Immunostaining for MMP-2 and MMP-9 gelatinases shows colocalization of macrophage accumulations with MMP-9 expression but not with MMP-2. Bottom, Macrophages, MMP-9 expression, and gelatinolytic activity colocalize in a thoracic aorta lesion. Control specimens were processed without primary antibody (Ab). Bars=100 µm.

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

View larger version (72K): [in this window] [in a new window]   Figure 2. Macrophage-derived FCs isolated from subcutaneous granuloma. Top, Cells isolated from subcutaneous granuloma of hypercholesterolemic rabbits are identified by immunofluorescence as macrophages (RAM-11 antibody). Inset, Negative control for immunostaining in which primary antibody (Ab) was eliminated. Middle, Intracellular lipid in macrophages from hypercholesterolemic rabbits is revealed by staining with Nile red. Inset, Nile red staining of macrophages (M) 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.

View larger version (99K): [in this window] [in a new window]   Figure 3. Detection of gelatinolytic activity released by aortic segments and by isolated macrophage-derived FCs. Gelatinolytic activity released by abdominal aorta (Abd), thoracic aorta (Thr), and aortic arch (Arch) was detected by SDS-PAGE zymography (white areas indicate lysis of gelatin substrate, denoting presence of gelatinases). Levels of total gelatinolytic activity, as well as that of higher-molecular-weight activity indicative of MMP-9, were consistently dependent on aortic region (Abd<Thr<Arch) and paralleled extent of macrophage-rich lesions. The great majority of gelatinolytic activity released in culture by FCs isolated from aorta (aFC) and granuloma (gFC) run at apparent molecular weight consistent with MMP-9. Same amount of protein (20 µg) was loaded on each lane. Simultaneous migration of prestained molecular weight markers (MWM) is indicated on left in kilodaltons (kDa, kD).

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).

View larger version (31K): [in this window] [in a new window]   Figure 4. NAC reduces gelatinolytic activity in culture media of macrophage-derived FCs. A, SDS-PAGE gelatin zymography. Culture media were harvested from macrophage FCs maintained in culture for up to 4 days in absence (-) or presence (+) of 10 mmol/L NAC. Incubation of FCs with NAC reduces gelatinolytic activity running at both high apparent molecular weight (MW) and low MW. Migration of gelatinolytic activity with high MW is consistent with pro–MMP-9, while faster gelatinolytic band is consistent with both processed (active) form of MMP-9 and pro–MMP-2. B, Densitometric analysis of gelatinolytic activity (high and low MW) released by FCs shows a significant reduction after 48 hours of treatment with 10 mmol/L NAC. Values were normalized to matched untreated control ±SEM (n=4 independent experiments, *P<0.05).

View larger version (51K): [in this window] [in a new window]   Figure 5. NAC inhibits level of MMP-9 released by macrophage-derived FCs. A, Western blotting. MMP-9 in cell culture media was identified by monoclonal antibodies recognizing both latent and active forms. FC culture media contain both forms, while culture media of non–lipid-laden macrophages contain only zymogen. MMP-9 signal is decreased in culture media of NAC-treated FCs. B, NAC treatment decreases level of FC-derived pro–MMP-9 expression in a dose-dependent fashion, as quantified by laser densitometry of immunoblots. Graph represents data from 4 independent experiments in which FCs were incubated in presence of 1 or 10 mmol/L NAC for 48 hours. *P<0.05.

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.

View larger version (32K): [in this window] [in a new window]   Figure 6. NAC treatment abolishes gelatinolytic activity and decreases MMP-9 protein level released in vitro by aortic lesion explants. Aortic segments were maintained in culture for up to 4 days with or without NAC. A, Gelatinolytic activity released by tissue explants of abdominal or thoracic aorta of hypercholesterolemic rabbits in an experiment in which aortic explants were treated with 10 mmol/L for 24 hours (loading of all lanes was normalized by protein). B, NAC treatment significantly diminished MMP-9 protein level released by aortic explants, detected by immunoblotting of culture media conditioned by segments of aortic arch, thoracic, and abdominal atherosclerotic aorta. Tissues were incubated in presence (+) or absence (-) of 10 mmol/L NAC for 24 hours. Bars represent average values obtained by normalizing signal of culture media conditioned by treated tissues to those of control (untreated, -) specimens ±SEM in 4 independent experiments. *P<0.05, **P<0.01.

View larger version (94K): [in this window] [in a new window]   Figure 7. NAC treatment reduces expression of MMP-9 in rabbit atherosclerotic lesions. Atherosclerotic lesion specimens were processed for immunodetection of macrophages and MMP-9 after incubation in culture without or with NAC. Images illustrate segments of abdominal aorta (top 2 panels), thoracic aorta (second 2 panels), and aortic arch (bottom 4 panels) incubated for 48 hours with 10 mmol/L NAC. Top panels show that after NAC treatment, expression of MMP-9 became undetectable in macrophage-derived FC–rich lesions. This effect was specific, because immunodetection of macrophage markers (RAM-11) was not affected. Loss of MMP-9 signal was not due to maintaining tissues in culture, because MMP-9 immunostaining was not affected in matched untreated segments of atherosclerotic aorta (bottom panels). Bar=100 µm.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Recently, the action of MMPs has emerged as an important component of the natural history of atherosclerosis^{8 11 21} and of the vascular response to injury.^{22 23 24} Macrophage-derived FCs associate clinically with unstable human plaques^{1 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 disease^{10 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 C^{38 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.

	Selected Abbreviations and Acronyms

 

FC = foam cell LPS = lipopolysaccharide MMP = matrix metalloproteinase MT-MMP = membrane-type MMP NAC = N-acetyl-L-cysteine NF = nuclear factor ROS = reactive oxygen species SMC = smooth muscle cell

	Acknowledgments

 
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.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Lendon CL, Davies MJ, Born GV, Richardson PD. Atherosclerotic plaque caps are locally weakened when macrophages density is increased. Atherosclerosis. 1991;87:87–90.[Medline] [Order article via Infotrieve]
   
2. Moreno PR, Falk E, Palacios IF, Newell JB, Fuster V, Fallon JT. Macrophage infiltration in acute coronary syndromes: implications for plaque rupture. Circulation. 1994;90:775–778.[Abstract/Free Full Text]
   
3. van der Wal AC, Becker AE, van der Loos CM, Das PK. Site of intimal rupture or erosion of thrombosed coronary atherosclerotic plaques is characterized by an inflammatory process irrespective of the dominant plaque morphology. Circulation. 1994;89:36–44.[Abstract/Free Full Text]
   
4. Partridge CA, Jeffrey JJ, Malik AB. A 96-kDa gelatinase induced by TNF-alpha contributes to increased microvascular endothelial permeability. Am J Physiol. 1993;265:L438–L447.[Abstract/Free Full Text]
   
5. Hanemaaijer R, Koolwijk P, le Clercq L, de Vree WJ, van Hinsbergh VW. Regulation of matrix metalloproteinase expression in human vein and microvascular endothelial cells: effects of tumour necrosis factor alpha, interleukin 1 and phorbol ester. Biochem J. 1993;296:803–809.
   
6. Galis ZS, Muszynski M, Sukhova GK, Simon-Morrissey E, Unemori EN, Lark MW, Amento E, Libby P. Cytokine-stimulated human vascular smooth muscle cells synthesize a complement of enzymes required for extracellular matrix digestion. Circ Res. 1994;75:181–189.[Abstract/Free Full Text]
   
7. Welgus HG, Senior RM, Parks WC, Kahn AJ, Ley TJ, Shapiro SD, Campbell EJ. Neutral proteinase expression by human mononuclear phagocytes: a prominent role of cellular differentiation. Matrix Suppl. 1992;1:363–367.[Medline] [Order article via Infotrieve]
   
8. Galis ZS, Sukhova GK, Lark MW, Libby P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest. 1994;94:2493–2503.
   
9. Galis Z, Sukhova G, Libby P. Microscopic localization of active proteases by in situ zymography: detection of matrix metalloproteinase activity in vascular tissue. FASEB J. 1995;9:974–980.[Abstract]
   
10. Brown DL, Hibbs MS, Kearney M, Loushin C, Isner JM. Identification of 92-kD gelatinase in human coronary atherosclerotic lesions: association of active enzyme synthesis with unstable angina. Circulation. 1995;91:2125–2131.[Abstract/Free Full Text]
    
11. Shah PK, Falk E, Badimon JJ, Fernandez-Ortiz A, Mailhac A, Villareal-Levy G, Fallon JT, Regnstrom J, Fuster V. Human monocyte-derived macrophages induce collagen breakdown in fibrous caps of atherosclerotic plaques: potential role of matrix-degrading metalloproteinases and implications for plaque rupture. Circulation. 1995;92:1565–1569.
    
12. Murphy G, Willenbrock F, Crabbe T, O'Shea M, Ward R, Atkinson S, O'Connell J, Docherty A. Regulation of matrix metalloproteinase activity. Ann N Y Acad Sci. 1994;732:31–41.[Medline] [Order article via Infotrieve]
    
13. Rajagopalan S, Meng XP, Ramasamy S, Harrison DG, Galis ZG. Reactive oxygen species produced by macrophage-derived foam cells regulate the activity of vascular matrix metalloproteinases in vitro: implications for atherosclerotic plaque stability. J Clin Invest. 1996;98:2572–2579.[Medline] [Order article via Infotrieve]
    
14. Galis ZS, Sukhova GK, Kranzhöfer R, Clark S, Libby P. Macrophage foam cells from experimental atheroma constitutively produce matrix-degrading proteinases. Proc Natl Acad Sci U S A. 1995;92:402–406.[Abstract/Free Full Text]
    
15. Senior RM, Griffin GL, Fliszar CJ, Shapiro SD, Goldberg GI, Welgus HG. Human 92- and 72-kilodalton type IV collagenases are elastases. J Biol Chem. 1991;266:7870–7875.[Abstract/Free Full Text]
    
16. McMillan WD, Patterson BK, Keen RR, Shively VP, Cipollone M, Pearce WH. In situ localization and quantification of mRNA for 92-kD type IV collagenase and its inhibitor in aneurysmal, occlusive, and normal aorta. Arterioscler Thromb Vasc Biol. 1995;15:1139–1144.[Abstract/Free Full Text]
    
17. Rosenfeld ME, Khoo JC, Miller E, Parthasarathy S, Palinski W, Witztum JL. Macrophage-derived foam cells freshly isolated from rabbit atherosclerotic lesions degrade modified lipoproteins, promote oxidation of low-density lipoproteins, and contain oxidation-specific lipid-protein adducts. J Clin Invest. 1991;87:90–99.
    
18. Kleiner DE, Stetler-Stevenson WG. Quantitative zymography: detection of picogram quantities of gelatinases. Anal Biochem. 1994;218:325–329.[Medline] [Order article via Infotrieve]
    
19. Sang QX, Birkedal-Hansen H, Van Wart HE. Proteolytic and non-proteolytic activation of human neutrophil progelatinase B. Biochim Biophys Acta. 1995;1251:99–108.[Medline] [Order article via Infotrieve]
    
20. Shapiro SD, Fliszar CJ, Broekelmann TJ, Mecham RP, Senior RM, Welgus HG. Activation of the 92-kDa gelatinase by stromelysin and 4-aminophenylmercuric acetate: differential processing and stabilization of the carboxyl-terminal domain by tissue inhibitor of metalloproteinases (TIMP). J Biol Chem. 1995;270:6351–6356.[Abstract/Free Full Text]
    
21. Henney AM, Wakeley PR, Davies MJ, Foster K, Hembry R, Murphy G, Humphries S. Localization of stromelysin gene expression in atherosclerotic plaques by in situ hybridization. Proc Natl Acad Sci U S A. 1991;88:8154–8158.[Abstract/Free Full Text]
    
22. Southgate KM, Davies M, Booth RF, Newby AC. Involvement of extracellular-matrix-degrading metalloproteinases in rabbit aortic smooth-muscle cell proliferation. Biochem J. 1992;288:93–99.
    
23. Bendeck MP, Zempo N, Clowes AW, Galardy RE, Reidy MA. Smooth muscle cell migration and matrix metalloproteinase expression after arterial injury in the rat. Circ Res. 1994;75:539–545.[Abstract/Free Full Text]
    
24. Zempo N, Kenagy RD, Au YPT, Bendeck M, Clowes MM, Reidy MA, Clowes AW. Matrix metalloproteinases of vascular wall cells are increased in balloon-injured rat carotid artery. J Vasc Surg. 1994;20:209–217.[Medline] [Order article via Infotrieve]
    
25. Halliwell B. The role of oxygen radicals in human disease, with particular reference to the vascular system. Haemostasis. 1993;23:118–126.
    
26. Ohara Y, Peterson TE, Harrison DG. Hypercholesterolemia increases endothelial superoxide anion production. J Clin Invest. 1993;91:2546–2551.
    
27. White CR, Brock TA, Chang LY, Crapo J, Briscoe P, Ku D, Bradley WA, Gianturco SH, Gore J, Freeman BA, Tarpey MM. Superoxide and peroxynitrite in atherosclerosis. Proc Natl Acad Sci U S A. 1994;91:1044–1048.[Abstract/Free Full Text]
    
28. Freestone T, Turner RJ, Coady A, Higman DJ, Greenhalgh RM, Powell JT. Inflammation and matrix metalloproteinases in the enlarging abdominal aortic aneurysm. Arterioscler Thromb Vasc Biol. 1995;15:1145–1151.[Abstract/Free Full Text]
    
29. Nikkari ST, Hoyhtya M, Isola J, Nikkari T. Macrophages contain 92-kd gelatinase (MMP-9) at the site of degenerated internal elastic lamina in temporal arteritis. Am J Pathol. 1996;149:1427–1433.[Abstract]
    
30. Ueda Y, Imai K, Tsuchiya H, Fujimoto N, Nakanishi I, Katsuda S, Seiki M, Okada Y. Matrix metalloproteinase 9 (gelatinase B) is expressed in multinucleated giant cells of human giant cell tumor of bone and is associated with vascular invasion. Am J Pathol. 1996;148:611–622.[Abstract]
    
31. d'Ortho MP, Will H, Atkinson S, Butler G, Messent A, Gavrilovic J, Smith B, Timpl R, Zardi L, Murphy G. Membrane-type matrix metalloproteinases 1 and 2 exhibit broad-spectrum proteolytic capacities comparable to many matrix metalloproteinases. Eur J Biochem. 1997;250:751–757.[Medline] [Order article via Infotrieve]
    
32. Annex BH, Denning SM, Channon KM, Sketch MH Jr, Stack RS, Morrissey JH, Peters KG. Differential expression of tissue factor protein in directional atherectomy specimens from patients with stable and unstable coronary syndromes. Circulation. 1995;91:619–622.[Abstract/Free Full Text]
    
33. Sato H, Kita M, Seiki M. v-Src activates the expression of 92-kDa type IV collagenase gene through the AP-1 site and the GT box homologous to retinoblastoma control elements: a mechanism regulating gene expression independent of that by inflammatory cytokines. J Biol Chem. 1993;268:23460–23468.[Abstract/Free Full Text]
    
34. Xie B, Dong Z, Fidler IJ. Regulatory mechanisms for the expression of type IV collagenases/gelatinases in murine macrophages. J Immunol. 1994;152:3637–3644.[Abstract]
    
35. Barchowsky A, Munro SR, Morana SJ, Vincenti MP, Treadwell M. Oxidant-sensitive and phosphorylation-dependent activation of NF-kappa B and AP-1 in endothelial cells. Am J Physiol. 1995;269:L829–L836.[Abstract/Free Full Text]
    
36. Gum R, Lengyel E, Juarez J, Chen JH, Sato H, Seiki M, Boyd D. Stimulation of 92-kDa gelatinase B promoter activity by ras is mitogen-activated protein kinase kinase 1-independent and requires multiple transcription factor binding sites including closely spaced PEA3/ets and AP-1 sequences. J Biol Chem. 1996;271:10672–10680.[Abstract/Free Full Text]
    
37. Gaziano JM. Antioxidant vitamins and coronary artery disease risk. Am J Med. 1994;97:18S–21S; discussion 22S–28S.[Medline] [Order article via Infotrieve]
    
38. Ferns GA, Forster L, Stewart-Lee A, Konneh M, Nourooz-Zadeh J, Anggard EE. Probucol inhibits neointimal thickening and macrophage accumulation after balloon injury in the cholesterol-fed rabbit. Proc Natl Acad Sci U S A. 1992;89:11312–11316.[Abstract/Free Full Text]
    
39. Lafont AM, Chai YC, Cornhill JF, Whitlow PL, Howe PH, Chisolm GM. Effect of alpha-tocopherol on restenosis after angioplasty in a model of experimental atherosclerosis. J Clin Invest. 1995;95:1018–1025.
    
40. Nunes GL, Sgoutas DS, Redden RA, Sigman SR, Gravanis MB, King SB III, Berk BC. Combination of vitamins C and E alters the response to coronary balloon injury in the pig. Arterioscler Thromb Vasc Biol. 1995;15:156–165.[Abstract/Free Full Text]
    
41. Baumann DS, Doblas M, Schonfeld G, Sicard GA, Daugherty A. Probucol reduces the cellularity of aortic intimal thickening at anastomotic regions adjacent to prosthetic grafts in cholesterol-fed rabbits. Arterioscler Thromb. 1994;14:162–167.[Abstract/Free Full Text]
    
42. Devaraj S, Li D, Jialal I. The effects of alpha tocopherol supplementation on monocyte function: decreased lipid oxidation, interleukin 1 beta secretion, and monocyte adhesion to endothelium. J Clin Invest. 1996;98:756–763.[Medline] [Order article via Infotrieve]
    
43. Marui N, Offermann MK, Swerlick R, Kunsch C, Rosen CA, Ahmad M, Alexander RW, Medford RM. Vascular cell adhesion molecule-1 (VCAM-1) gene transcription and expression are regulated through an antioxidant-sensitive mechanism in human vascular endothelial cells. J Clin Invest. 1993;92:1866–1874.
    
44. Fruebis J, Gonzalez V, Silvestre M, Palinski W. Effect of probucol treatment on gene expression of VCAM-1, MCP-1, and M-CSF in the aortic wall of LDL receptor-deficient rabbits during early atherogenesis. Arterioscler Thromb Vasc Biol. 1997;17:1289–1302.[Abstract/Free Full Text]
    
45. Albini A, D'Agostini F, Giunciuglio D, Paglieri I, Balansky R, De Flora S. Inhibition of invasion, gelatinase activity, tumor take and metastasis of malignant cells by N-acetylcysteine. Int J Cancer. 1995;61:121–129.Plaque destabilization triggers clinical events, representing an attractive therapeutic target. Tissue weakening by matrix metalloproteinases (MMPs) of resident macrophage foam cells (FCs) is considered key. Gelatinase activity investigated in aortic lesions of a rabbit model cosegregated with FC and MMP-9 expression. To examine whether gelatinolytic activity was redox-mediated, we tested N-acetyl-cysteine (NAC) effects on isolated FCs and on explanted aortic lesions. NAC treatment decreased both gelatinase activity and expression in vitro and in situ. Such new action of antioxidant therapy might prove useful to inhibit matrix degradation and to improve vascular stability.[Medline] [Order article via Infotrieve]
    

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