This Article Abstract Full Text (PDF) All Versions of this Article: 90/8/897    most recent 01.RES.0000016501.56641.83v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Silence, J. Articles by Lijnen, H.R. Search for Related Content PubMed PubMed Citation Articles by Silence, J. Articles by Lijnen, H.R. Pubmed/NCBI databases Gene GEO Profiles HomoloGene UniGene Substance via MeSH Related Collections Animal models of human disease Pathophysiology

(Circulation Research. 2002;90:897.)
© 2002 American Heart Association, Inc.

Integrative Physiology

Reduced Atherosclerotic Plaque but Enhanced Aneurysm Formation in Mice With Inactivation of the Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Gene

J. Silence, D. Collen, H.R. Lijnen

From the Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium.

Correspondence to H.R. Lijnen, Center for Molecular and Vascular Biology, University of Leuven, Campus Gasthuisberg, O&N, Herestraat 49, B-3000 Leuven, Belgium. E-mail roger.lijnen{at}med.kuleuven.ac.be

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Development and progression of atherosclerotic lesions and aneurysm formation were investigated in mice with single or combined deficiency of apolipoprotein E (ApoE) and tissue inhibitor of metalloproteinase-1 (TIMP-1) kept on a cholesterol-rich diet for 30 weeks. Atherosclerotic lesions throughout the thoracic aorta were significantly (P<0.001) larger in mice wild-type for TIMP-1 (ApoE^-/-:TIMP-1^+/+) than in mice deficient in TIMP-1 (ApoE^-/-:TIMP-1^-/-). Aneurysms in the thoracic and abdominal aortas were less frequent in ApoE^-/-:TIMP-1^+/+ mice than in ApoE^-/-:TIMP-1^-/- mice (11±3.0 versus 23±5.1 aneurysms per 100 sections analyzed, mean±SD, P<0.001). Immunocytochemistry revealed enhanced accumulation of Oil red O–stained lipids, colocalizing with macrophages in atherosclerotic lesions of ApoE^-/-:TIMP-1^-/- mice (P<0.05). In situ zymography using a casein substrate showed enhanced lysis in plaques of ApoE^-/-:TIMP-1^-/- mice as compared with ApoE^-/-:TIMP-1^+/+ mice (P<0.01). MMP activity was most pronounced at sites where degradation of the elastic lamina occurred. These data suggest that enhanced MMP activity, as a result of TIMP-1 deficiency, contributes to a reduction of atherosclerotic plaque size but promotes aneurysm formation.

Key Words: matrix metalloproteinases • tissue inhibitor of metalloproteinase-1 • apolipoprotein E • atherosclerosis • aneurysm

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
A potential role for matrix metalloproteinase (MMP)–mediated proteolysis in neovascularization and rupture of atherosclerotic plaques or in ulceration and rupture of aneurysms has been suggested.^1,2 Several MMP system components (MMP-1, MMP-2, MMP-3, MMP-9, MMP-11, and MMP-14) are expressed in atherosclerotic tissue.^3–8

Studies in mice with combined deficiency of apolipoprotein E (ApoE) and urokinase (u-PA) revealed that u-PA deficiency protects against media destruction and aneurysm formation, probably by means of reduced plasmin-dependent activation of proMMPs secreted by infiltrating macrophages.⁹ Similar studies with mice deficient for both ApoE and stromelysin-1 (MMP-3) indicated that MMP-3 activity contributes to a reduction of plaque size, possibly by degradation of matrix components, and promotes aneurysm formation by degradation of the elastic lamina.¹⁰

In vivo, MMPs are inhibited by endogenous tissue inhibitors (TIMPs), of which 4 different types have been identified.¹¹ TIMP-1, which is synthesized by most types of connective tissue cells as well as macrophages, acts against most members of the collagenase, stromelysin, and gelatinase classes of MMPs.¹² Regulated expression of TIMPs was shown to counteract MMP activity in human atheroma and to influence plaque stability.¹³ Overexpression of TIMP-1 reduced atherosclerotic lesions in ApoE^-/- mice¹⁴ and prevented degeneration and rupture of the elastic lamina in a rat model,¹⁵ further substantiating a functional role of MMPs.

In this study, we investigated a potential contribution of TIMP-1 to the development and progression of atherosclerosis with the use of mice with single or combined deficiency of ApoE and TIMP-1, kept on a cholesterol-rich diet for up to 30 weeks.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of Mice Colonies and Experimental Protocol
TIMP-1–deficient (TIMP-1^-/-) and wild-type (TIMP-1^+/+) mice were obtained from the Roswell Park Cancer Institute (Buffalo, NY) and characterized as described.^16,17 They were maintained as outcrossed animals arising from F1 (C57/Bl6x129 SvJ) founders. Because of the X-linked inheritance pattern, female homozygous-deficient mice are TIMP-1^-/-, whereas males are hemizygous, TIMP-1^-/0. Apolipoprotein E–deficient (ApoE^-/-) mice (50% C57/Bl6:50% 129 SvJ)¹⁸ and TIMP-1^-/- mice were rederived by back-crossing onto a C57/Bl6 background, yielding 75% C57/Bl6:25% 129 SvJ. Genomic DNA was extracted from the tail tips for genotyping of offspring by Southern blotting for TIMP-1 and by polymerase chain reaction for ApoE (data not shown).

Mice were kept in microisolation cages on a 12-hour day/night cycle and fed a high-fat cholesterol-rich diet from the age of 5 weeks on (wt/wt: 47% sucrose, 20% casein, 19% butter, 1% corn oil, 1.25% cholesterol, 0.5% cholic acid, 0.5% NaCl, 5% -cellulose, 5% mineral mix, 1% vitamin mix, 1% choline chloride, 0.3% DL-methionine, and 0.13% -tocopherol).

Following overnight fasting, the mice were anesthetized by IP injection of 60 mg/kg Nembutal (Abbott Laboratories). Blood was collected from the vena cava in 1:10 volume EDTA, pH 6.8, and centrifuged at 3000 rpm for 10 minutes; the plasma was stored at -20°C and used for cholesterol determination. The arterial system was perfused at physiological pressure with 4% paraformaldehyde in PBS, dissected, and incubated in 1% paraformaldehyde (3 hours for aortas, overnight for the hearts). After rinsing with PBS and storage in 20% sucrose, samples were embedded in ornithine carbamyl transferase (Tissue-Tek, Laborimpex), snap frozen in precooled 2-methylbutane, and stored at -80°C. Sections (8 µm thick) were made around the cardiac valves and at 80-µm–spaced distances throughout the aorta. The aortic arch was dissected free of tissue and frozen at -80°C. Gonadal, retroperitoneal, and subcutaneous fat pads were removed and weighed.

All animal experiments were approved by the local ethical committee and were performed in accordance with the guiding principles of the American Physiological Society and the International Society on Thrombosis and Hemostasis.¹⁹

Zymographic Analysis
The aortic arch was pulverized after submersion in liquid nitrogen and incubated for 1.5 hours at 4°C with 150 µL extraction buffer (10 mmol/L sodium phosphate buffer, pH 7.2, containing 150 mmol/L NaCl, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, and 0.2% sodium azide). After extensive vortexing and centrifugation (13 000 rpm for 5 minutes), the protein concentration of the supernatant was determined (BCA protein assay; Pierce). Equivalent amounts of total protein were subjected to zymography on gelatin-containing gels²⁰ using a sample of fibroblast medium containing the different molecular forms of MMP-2 and MMP-9 for calibration. Casein-containing gels with addition of 5 µg/mL human plasminogen were calibrated with purified murine t-PA or u-PA.²¹

Histology and Immunocytochemistry
Sections (8 µm thick) were stained with hematoxylin-eosin, Oil red O, Verhoeff-van Gieson’s, or Sirius red under standard conditions. Plaque sizes and stained areas were quantified by computer-assisted image analysis using KS300 imaging software (Zeiss). For each animal, 10 sections were analyzed throughout the thoracic aorta at 80-µm–spaced intervals. Therefore, 8-µm thick sections were applied consecutively on 10 microscopic slides (slide 1 contains the 1st, 11th...section; slide 2 contains the 2nd, 12th...section; and so on). These 10 slides, each containing sections separated 80 µm from each other, were analyzed. The site where the cardiac valves are first visible is used as a reference point. Data are reported as mean±SD of 10 animals. Statistical analysis was performed by unpaired Student’s t test.

In situ zymography on cryosections using casein-containing gels without plasminogen was performed essentially as described.²² The substrate gel (0.5% agarose) contained 1.0 mg/mL resorufin-labeled casein (Boehringer Mannheim). Overlays were analyzed by computer-assisted image analysis (Zeiss, Axioplan 2) after incubation for 48 hours in a moist chamber at 37°C and lysis is expressed as percentage of the total section area. Macrophages were detected using a rat monoclonal anti-mouse Mac-3 antigen (clone M3/84; Pharmingen), and smooth muscle cells were detected with biotinylated mouse anti-human smooth muscle -actin (clone A14; Sigma Chemical Co), using the appropriate negative controls.²³

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of Mice Colonies
Male mice with ApoE gene deficiency (ApoE^-/-:TIMP-1^+/0) were crossed with female mice with TIMP-1 gene deficiency (ApoE^+/+:TIMP-1^-/-) to generate male ApoE^+/-:TIMP-1^-/0 and female ApoE^+/-:TIMP-1^+/- offspring. Further crosses generated littermate male ApoE^-/-:TIMP-1^+/0 (4.2% of 142 offspring) and ApoE^-/-:TIMP-1^-/0 (6.3%) mice, and female ApoE^-/-:TIMP-1^+/- (5.6%) and ApoE^-/-:TIMP-1^-/- (5.6%) mice. The sex ratio was 48% male versus 52% female. This distribution of genotypes is in agreement with the expected Mendelian ratio (6.25% for each genotype). Further crossing of ApoE^-/-:TIMP-1^+/0 males with ApoE^-/-:TIMP-1^+/- females yielded ApoE^-/-:TIMP-1^+/+ females (25% of offspring).

Mice used in this study thus were all ApoE^-/- and TIMP-1^+/+ or TIMP-1^-/- for females and TIMP-1^+/0 or TIMP-1^-/0 for males, with genetic background 75% C57/Bl6:25% 129SvJ; for convenience they are referred to as ApoE^-/-:TIMP-1^+/+ and ApoE^-/-:TIMP-1^-/- mice. The mice all appeared healthy, and no macroscopic abnormalities were observed. Ten ApoE^-/-:TIMP-1^+/+ (6 males, 4 females) or ApoE^-/-:TIMP-1^-/- (5 males, 5 females) mice were kept on the cholesterol-rich diet for 30 weeks. At the time of euthanasia, body weights were not different between both strains (26±1.8 versus 26±2.5 g, mean±SD). Also, the weight of the different fat pads was comparable: 99±31 versus 89±44 mg for retroperitoneal, 190±51 versus 178±32 mg for subcutaneous, and 263±77 versus 248±87 mg for gonadal adipose tissue. The plasma cholesterol levels were also comparable for ApoE^-/-:TIMP-1^+/+ and ApoE^-/-:TIMP-1^-/- mice: 1460±370 versus 1400±420 mg/dL.

Analysis of Atherosclerotic Lesions
ApoE^-/-:TIMP-1^+/+ as well as ApoE^-/-:TIMP-1^-/- mice developed extensive atherosclerotic lesions throughout the aortic root, as revealed by hematoxylin-eosin (not shown) and Oil red O staining (Figures 1a and 1e) of transverse cryosections. Computer-assisted image analysis of sections taken at regularly spaced distances (80 µm) throughout the thoracic aorta revealed that the plaque size was significantly smaller in ApoE^-/-:TIMP-1^-/- than in ApoE^-/-:TIMP-1^+/+ mice (Table). Separate analysis of males and females (6 males/4 females in the ApoE^-/-:TIMP-1^+/+ and 5 males/5 females in the ApoE^-/-:TIMP-1^-/- group) did not reveal significant differences between lumen areas or plaque areas, measured throughout the thoracic aorta (all P>0.1 by unpaired 2-tailed t test). Oil red O staining also indicated a higher lipid content of the plaques in ApoE^-/-:TIMP-1^-/- than in ApoE^-/-:TIMP-1^+/+ sections (Figures 1a and 1e). Immunostaining for Mac-3 indicated the more abundant presence of macrophages in atherosclerotic plaques of ApoE^-/-:TIMP-1^-/- aortas, and comparison of Mac-3 and Oil red O staining patterns of adjacent sections (Figures 2a and 2c) revealed colocalization of lipids with macrophages, suggesting that macrophages occur predominantly as foam cells. Quantification by image analysis of the Mac-3–stained area throughout the thoracic aorta (Figure 3A) and of the lipid content, defined as the percentage of plaque area that is stained with Oil red O (Figure 3B), confirmed the more abundant presence of macrophages/foam cells in the ApoE^-/-:TIMP-1^-/- sections. These are mainly located at the plaque surface but locally also infiltrate the plaque (Figures 2a and 2c). Staining of fibrillar collagen with Sirius red (Figures 1b and 1f) suggested that the atherosclerotic lesions of ApoE^-/-:TIMP-1^+/+ mice contained somewhat more collagen than those of ApoE^-/-:TIMP-1^-/- mice; quantification of the stained area, normalized to plaque size, however, did not consistently reveal statistically significant differences throughout the thoracic aorta (Figure 3C).

View larger version (78K): [in this window] [in a new window]   Figure 1. Light microscopic analysis of atherosclerotic aortas from ApoE-/-:TIMP-1-/- (a through d) or ApoE-/-:TIMP-1+/+ (e through h) mice. Staining was performed with Oil red O (a and e), Sirius red (b and f), or Verhoeff-van Gieson’s (c and g) stain. d and h, In situ zymography with a casein-containing gel. Arrows indicate the cardiac valves (CV) in a and e; P, location of atherosclerotic plaque; and E, the elastic lamina. Dark spots in d indicate lytic activity. Scale bars=100 µm.

View this table: [in this window] [in a new window]   Table 1. Quantification of Atherosclerotic Plaque Size in the Aortas

View larger version (99K): [in this window] [in a new window]   Figure 2. Light microscopic analysis of adjacent sections of atherosclerotic aortas from ApoE-/-:TIMP-1-/- mice. Staining was performed with an antiserum against Mac-3 (c and e) or against -actin (d and f), or with Oil red O (a) or Verhoeff-van Gieson’s (b) stain. P indicates location of atherosclerotic plaque; E, elastic lamina. Scale bars=100 µm.

View larger version (35K): [in this window] [in a new window]   Figure 3. Quantification of macrophage (Mac-3 staining; A), lipid (Oil red O staining; B), and fibrillar collagen (Sirius red staining; C) content in atherosclerotic lesions in the aortas of ApoE-/-: TIMP-1+/+ (open bars) or ApoE-/-:TIMP-1-/- (solid bars) mice. The stained area (in percentage of the plaque area) is shown as a function of location in the aorta. Sections were taken at fixed 80-µm distances around the cardiac valves (0). Data are mean±SD. *P<0.05, **P<0.01, and ***P<0.001.

In situ zymography with casein-containing gels showed more lytic activity in sections of ApoE^-/-:TIMP-1^-/- mice (20±8.3% versus 7.7±2.8% lysis of the total section area, n= 8, P<0.01). Addition of a mixture of EDTA (final concentration 25 mmol/L) and 1.10 phenanthroline (final concentration 5 mmol/L) to the agarose gel resulted in a reduction of the lytic activity to 1.8±1.8% or 1.9±1.6% in ApoE^-/-:TIMP-1^+/+ or ApoE^-/-:TIMP-1^-/- sections, respectively, confirming that it is largely MMP-dependent.

Zymographic analysis of aortic extracts on gelatin-containing gels (Figure 4A) revealed lower levels of proMMP-2 and MMP-2 molecular forms in samples of ApoE^-/-:TIMP-1^+/+ aortas (140±18 arbitrary units (AU) of lysis/mg protein versus 860±70 for 58-kDa MMP-2, 44±13 versus 680±100 for 65-kDa proMMP-2, whereas 70-kDa proMMP-2 was undetectable in ApoE^-/-:TIMP-1^+/+ sections versus 180±39 AU of lysis/mg protein in ApoE^-/-:TIMP-1^-/- sections; mean±SD, n= 4, P<0.001). ProMMP-9 (90 to 94 kDa) was detected in only low concentration. These data indicate lower total gelatinolytic activity in the wild-type as compared with TIMP-1^-/- samples. Zymography on casein gels containing plasminogen (Figure 4B) indicated comparable u-PA activity in atherosclerotic tissues from ApoE^-/-:TIMP-1^+/+ and ApoE^-/-:TIMP-1^-/- mice. Furthermore, u-PA antigen levels in the extracts as determined by ELISA²⁴ were comparable: 0.49±0.18 ng/mg protein for ApoE^-/-:TIMP-1^+/+ and 0.54±0.15 ng/mg for ApoE^-/-:TIMP-1^-/- aortas (mean±SEM, n=5 or 4).

View larger version (36K): [in this window] [in a new window]   Figure 4. Zymographic analysis on gelatin-containing (A) or casein-containing (B) gels of aortic extracts obtained from ApoE-/-:TIMP-1+/+ (lanes 1) or ApoE-/-:TIMP-1-/- (lanes 2) mice.

Analysis of Aneurysms
Verhoeff-van Gieson’s staining of elastin (Figures 1c and 1g) revealed that the elastic lamina showed more frequent aneurysms in the thoracic and abdominal aortas of ApoE^-/-:TIMP-1^-/- mice. Aneurysms are characterized by thinning of the aortic wall and fragmentation and rupture of elastic membranes across the media.

Analysis of 220 to 230 equally spaced (80 µm) sections per animal (taken from the thoracic aorta between the aortic arch and the split of the femoral aorta, covering a total distance of about 1.8 cm) revealed 23±5.1 aneurysms per 100 ApoE^-/-:TIMP-1^-/- sections analyzed versus only 11±3.0 aneurysms per 100 ApoE^-/-:TIMP-1^+/+ sections analyzed (10 animals each, P<0.001). Further analysis indicated that of all sections showing aneurysm the occurrence of 2 or 3 aneurysms per section was more frequent in ApoE^-/-:TIMP-1^-/- than in ApoE^-/-:TIMP-1^+/+ mice (2 aneurysms in 16% of all sections with at least one aneurysm versus 7.4% in ApoE^-/-:TIMP-1^+/+ mice, or 3 aneurysms in 2.6% versus 0.7% in ApoE^-/-: TIMP-1^+/+ mice). Also, the percentage of aneurysms extending over 3 consecutive sections (240 µm) was higher in the ApoE^-/-:TIMP-1^-/- mice (6.0% versus 3.7%).

In both genotypes, comparison of the Verhoeff-van Gieson’s and -actin staining patterns of adjacent sections (Figures 2b and 2d) indicated colocalization of smooth muscle cells with the elastic lamina. At the site of an aneurysm, virtually no -actin staining was observed, indicating that smooth muscle cells are depleted at the site of media destruction (Figure 2f). Immunostaining for Mac-3 revealed the consistent presence of macrophages at sites of aneurysm (Figure 2e).

Caseinolytic activity (appearing as black spots) was most pronounced at sites with thinning of the elastic lamina and aneurysm formation, whereas it was less pronounced at sites with intact elastic lamina (Figures 1d and 1h).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Atherosclerosis is an inflammatory process in which plaques are formed in the intimal layer of the vessel wall as a result of accumulation of lipid-rich macrophages, smooth muscle cells, and lipids and deposition of extracellular matrix. Clinical complications of atherosclerosis are often triggered by rupture of unstable plaques, triggering intravascular thrombosis and tissue ischemia.^25–27 Alternatively, thinning of the atherosclerotic vessel wall due to elastin and collagen degradation and matrix necrosis may result in aneurysm formation and bleeding.^28,29 The MMP system has been implicated in the pathogenesis of atherosclerosis and aneurysm formation,^12,26 largely based on in situ expression of several of its components. In addition, in animal models overexpression of TIMP-1 prevented aortic aneurysm formation and rupture^14,15 and targeted gene disruption of MMP-9 (gelatinase B) suppressed development of experimental abdominal aortic aneurysms.³⁰ Impaired MMP activity may thus contribute to the growth and stabilization of atherosclerotic plaques, whereas increased activity may be associated with plaque rupture and aneurysm formation. In agreement with this hypothesis, it was previously shown in an atherosclerosis model in mice that stromelysin-1 (MMP-3), which can activate proMMP-9, contributes to reduction of plaque size but promotes aneurysm formation.¹⁰ In the present study, the role of TIMP-1, a major MMP inhibitor, in these phenomena was investigated by using mice with a combined deficiency of ApoE (which are susceptible to atherosclerosis) and TIMP-1, kept on a cholesterol-rich diet. All mice used in this study were of the same genetic background. Pronounced differences in atherosclerotic lesion formation were indeed observed in mice with different genetic backgrounds.^31–33 Furthermore, for morphometric analysis paraformaldehyde-fixed cryosections were used in both experimental groups.

This study confirmed a potential dual effect of MMPs. Indeed, deficiency of TIMP-1 in mice with an atherosclerosis-susceptible genetic background resulted in a reduction of the size of atherosclerotic lesions throughout the thoracic aorta, but in a higher incidence of more severe aortic aneurysms. In situ zymography with casein-containing gels confirmed the presence of higher MMP-related proteolytic activity in atherosclerotic lesions of ApoE^-/-:TIMP-1^-/- mice, compatible with deficiency of the MMP inhibitor. Furthermore, the collagen content of the plaques was somewhat, but not consistently significantly, lower in the ApoE^-/-:TIMP-1^-/- sections. These findings suggest that the observed phenomena are at least in part related to higher MMP activity as a result of TIMP-1 deficiency. Due to the lack of a specific and sensitive antiserum against murine TIMP-1, we have not directly confirmed its presence in plaque of ApoE^-/-:TIMP-1^+/+ mice. Also, reverse gelatin zymography with extracts of the aortic arch appeared not sensitive enough for detection of TIMP-1 activity (data not shown). Several other studies have, however, previously reported TIMP-1 expression in atherosclerotic lesions. Also, expression of TIMP-2 and TIMP-3 was observed,¹³ but their potential contribution in our model is unclear. Higher MMP activity in ApoE^-/-:TIMP-1^-/- lesions may not only be due to the absence of inhibitory activity. Indeed, our data seem to indicate the more abundant presence of macrophages/foam cells in lesions of ApoE^-/-:TIMP-1^-/- mice. These may contribute to higher secretion levels of MMPs. It is conceivable that TIMP-1 deficiency allows more macrophage accumulation in the plaque. TIMP-1 may indeed impair cellular migration, as previously shown for murine smooth muscle cells.³⁴ In a vascular injury model, neointima formation was found to be enhanced in TIMP-1^-/- as compared with wild-type mice. In this model, migration of smooth muscle cells from the borders of the injury into the necrotic center to populate the neointima is impaired by TIMP-1.³⁴ The present model, however, focuses on macrophages/foam cells, which accumulate more in the TIMP-1–deficient mice, whereas smooth muscle cells are predominantly present at the site of the elastic lamina, but are depleted at the sites of aneurysm (Figure 2f). Also, after thioglycollate injection in TIMP-1^-/- mice, somewhat, although not significantly, more infiltration of macrophages into the peritoneal space was observed, as compared with wild-type mice.³⁵ Thus, the enhanced MMP activity observed in plaques of ApoE^-/-:TIMP-1^-/- mice may be related to the absence of inhibitory activity, but also to more pronounced macrophage infiltration and enhanced MMP secretion/activation. Smooth muscle cells may also contribute to the enhanced MMP-2 levels observed on zymography of aortic extracts of ApoE^-/-:TIMP-1^-/- mice. A previous study in mice with combined deficiency of ApoE and u-PA suggested that u-PA–mediated plasmin generation contributes to activation of proMMP-3, -9, -12, and -13, which are secreted by macrophages.⁹ Our data do not show different u-PA levels in plaques of ApoE^-/-:TIMP-1^-/- or ApoE^-/-:TIMP-1^+/+ animals, suggesting that differential u-PA–mediated proMMP activation does not play a role. Reduced plaque size in the ApoE^-/-:TIMP-1^-/- mice may thus be related to enhanced matrix degradation and to macrophage accumulation.

Rouis et al¹⁴ have previously reported that adenovirus-mediated overexpression of TIMP-1 reduces atherosclerotic lesions in ApoE^-/- mice. They suggested that this effect might be mediated by inhibition of smooth muscle cell invasion. In these studies, mice were kept on a high cholesterol diet for 6 weeks before injection of the adenovirus and were analyzed 4 weeks later. The effect of TIMP-1 may thus be in inhibition of lesion progression and not in induction of regression. It should also be kept in mind that TIMP-1 overexpression in this model is only transient. In our study, both ApoE^-/-:TIMP-1^-/- and ApoE^-/-:TIMP-1^+/+ mice were kept on a cholesterol-rich diet for 30 weeks; we are thus analyzing established and more complex atherosclerotic lesions versus the earlier lesions in the study of Rouis et al.¹⁴ Furthermore, enhanced MMP activity in TIMP-1^-/- mice in our study contributes to a higher frequency of aneurysms; this is in agreement with the observation by Rouis et al that TIMP-1 overexpression reduces elastin degradation, although aneurysms were not quantified.

Taken together, it appears that TIMP-1, through its effect on MMP activity, may play a dual role in atherosclerosis. On the one hand, it reduces aneurysm formation, but on the other hand, it promotes development of more advanced lesions. However, we have not studied very late lesions, which were recently shown to be prone to hemorrhagic necrosis and plaque rupture in the ApoE^-/- mouse model.³⁶ As several MMPs are expressed in the atherosclerotic plaque,^3–8 our data do not allow to conclude if any specific MMP is targeted, but nevertheless support the concept that MMPs play a functional role in atherosclerosis.

	Acknowledgments

 
This study was supported by grants from the "Fonds voor Wetenschappelijk Onderzoek Vlaanderen" (FWO, contract G.0293.98) and from the Interuniversity Attraction Poles (IUAP). J.S. was the recipient of a fellowship from the "Vlaams instituut voor de bevordering van het wetenschappelijk-technologisch onderzoek in de industrie (IWT)." Skillful technical assistance by A. Dewolf, L. Frederix, and B. Van Hoef is gratefully acknowledged.

Received November 1, 2001; revision received March 14, 2002; accepted March 15, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Newman KM, Jean Claude J, Li H, Scholes JV, Ogata Y, Nagase H, Tilson MD. Cellular localization of matrix metalloproteinase in the abdominal aortic aneurysm wall. J Vasc Surg. 1994; 20: 814–820.
2. Sakalihasan N, Delvenne P, Nusgens BV, Limet R, Lapiere CM. Activated forms of MMP2 and MMP9 in abdominal aortic aneurysms. J Vasc Surg. 1996; 24: 127–133.
3. 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.
4. Henney AM, Wakely 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.
5. Galis SZ, Muszynski M, Sukhova GK, Simon-Morrisey E, Libby P. Enhanced expression of vascular matrix metalloproteinases induced in vitro by cytokines and in regions of human atherosclerotic lesions. Ann N Y Acad Sci. 1995; 748: 501–507.
6. Schonbeck J, Mach F, Sukhova GK, Atkinson E, Levesque E, Herman M, Graber P, Basset P, Libby P. Expression of stromelysin-3 in atherosclerotic lesions: regulation via CD40-CD40 ligand signaling in vitro and in vivo. J Exp Med. 1999; 189: 843–853.
7. Rajavashisth TB, Xu XP, Jovinge S, Meisel S, Xu XO, Chai NN, Fischbein MC, Kaul S, Cercek B, Sharifi B, Shah PK. Membrane type 1 matrix metalloproteinase expression in human atherosclerotic plaques: evidence for activation by proinflammatory mediators. Circulation. 1999; 99: 3103–3109.
8. Hong BK, Kwon HM, Lee BK, Kim D, Kim IJ, Kang SM, Jang Y, Cho SH, Kim HK, Jang BC, Cho SY, Kim HS, Kim MS, Kwon HC, Lee N. Coexpression of cyclooxygenase-2 and matrix metalloproteinases in human aortic atherosclerotic lesions. Yonsei Med J. 2000; 41: 82–88.
9. Carmeliet P, Moons L, Lijnen HR, Baes M, Lemaitre V, Tipping P, Drew A, Eeckhout Y, Shapiro S, Lupu F, Collen D. Urokinase-generated plasmin is a candidate activator of matrix metalloproteinases during atherosclerotic aneurysm formation. Nature Genet. 1997; 17: 439–444.
10. Silence J, Lupu F, Collen D, Lijnen HR. Persistence of atherosclerotic plaque but reduced aneurysm formation in mice with stromelysin-1 (MMP-3) gene inactivation. Arterioscler Thromb Vasc Biol. 2001; 21: 1440–1445.
11. Brew K, Dinakarpandian D, Nagase H. Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochim Biophys Acta. 2000; 1477: 267–283.
12. Dollery CM, McEwan JR, Henney AM. Matrix metalloproteinases and cardiovascular disease. Circ Res. 1995; 77: 863–868.
13. Fabunmi RP, Sukhova GK, Sugiyama S, Libby P. Expression of tissue inhibitor of metalloproteinases-3 in human atheroma and regulation in lesion-associated cells: a potential protective mechanism in plaque stability. Circ Res. 1998; 83: 270–278.
14. Rouis M, Adamy C, Duverger N, Lesnik P, Horellou P, Moreau M, Emmanuel F, Caillaud JM, Laplaud PM, Dachet C, Chapman MJ. Adenovirus-mediated overexpression of tissue inhibitor of metalloproteinase-1 reduces atherosclerotic lesions in apolipoprotein E–deficient mice. Circulation. 1999; 100: 533–540.
15. Allaire E, Forough R, Clowes M, Starcher B, Clowes AW. Local overexpression of TIMP-1 prevents aortic aneurysm degeneration and rupture in a rat model. J Clin Invest. 1998; 102: 1413–1420.
16. Soloway PD, Alexander CM, Werb Z, Jaenisch R. Targeted mutagenesis of TIMP-1 reveals that lung tumor invasion is influenced by TIMP-1 genotype of the tumor but not by that of the host. Oncogene. 1996; 13: 2307–2314.
17. Nothnick WB, Soloway P, Curry TE. Assessment of the role of tissue inhibitor of metalloproteinase-1 (TIMP-1) during the periovulatory period in female mice lacking a functional TIMP-1 gene. Biol Reprod. 1997; 56: 1181–1188.
18. Plump AS, Smith JD, Hayek T, Aalto-Setala K, Walsh A, Verstuyft JG, Rubin EM, Breslow JL. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E–deficient mice created by homologous recombination in ES cells. Cell. 1992; 71: 343–353.
19. Giles AR. Guidelines for the use of animals in biomedical research. Thromb Haemost. 1987; 58: 1078–1084.
20. Kleiner DE, Stetler Stevenso WG.n Quantitative zymography: detection of picogram quantities of gelatinases. Anal Biochem. 1994; 218: 325–329.
21. Heussen C, Dowdle EB. Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal Biochem. 1980; 102: 196–202.
22. Galis ZS, Sukhova GK, 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.
23. Lijnen HR, Van Hoef B, Lupu F, Moons L, Carmeliet P, Collen D. Function of the plasminogen/plasmin and matrix metalloproteinase systems after vascular injury in mice with targeted inactivation of fibrinolytic system genes. Arterioscler Thromb Vasc Biol. 1998; 18: 1035–1045.
24. Declerck PJ, Verstreken M, Collen D. Immunoassay of murine t-PA, u-PA, and PAI-1 using monoclonal antibodies raised in gene-inactivated mice. Thromb Haemost. 1995; 74: 1305–1309.
25. Davies MJ, Richardson PD, Wolf N, Katz DR, Mann J. Risk of thrombosis in human atherosclerotic plaques: role of extracellular lipid, macrophage and smooth muscle cell content. Br Heart J. 1993; 69: 377–381.
26. Libby P. Molecular basis of the acute coronary syndromes. Circulation. 1995; 91: 2844–2850.
27. Ross R Atherosclerosis: an inflammatory disease. N Engl J Med. 1999; 340: 115–26.
28. Halloran BG, Baster BT. Pathogenesis of aneurysms. Semin Vasc Surg. 1995; 8: 85–92.
29. Patel MI, Hardman DT, Fisher CM, Appleberg M. Current views on the pathogenesis of abdominal aortic aneurysms. J Am Coll Surg. 1995; 181: 371–382.
30. Pyo R, Lee JK, Shipley JM, Curci JA, Mao D, Ziporin SJ, Ennis TL, Shapiro SD, 2 Senior RM, Thompson RW. Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms. J Clin Invest. 2000; 105: 1641–1649.
31. Paigen B, Ishida BY, Verstuyft J, Winters RB, Albee D. Atherosclerosis susceptibility differences among progenitors of recombinant inbred strains of mice. Arteriosclerosis. 1990; 10: 316–323.
32. Paigen B. Genetics of responsiveness to high-fat and high-cholesterol diets in the mouse. Am J Clin Nutr. 1995; 62: 458S–462S.
33. Qiao J-H, Xie P-Z, Fishbein MC, Kreuzer J, Drake TA, Demer LL, Lusis AJ. Pathology of atheromatous lesions in inbred and genetically engineered mice: genetic determination of arterial calcification. Arterioscler Thromb. 1994; 14: 1480–1497.
34. Lijnen HR, Soloway P, Collen D. Tissue inhibitor of matrix metalloproteinases-1 impairs arterial neointima formation after vascular injury in mice. Circ Res. 1999; 85: 1186–1191.
35. Lijnen HR, Van Hoef B, Soloway P, Collen D. Plasminogen/plasmin system function in mice deficient in stromelysin-1 (MMP-3) or in tissue inhibitor of metalloproteinases type 1 (TIMP-1). Fibrinolysis Proteolysis. 1998; 12: 1–8.
36. Rosenfeld ME, Polinsky P, Virmani R, Kauser K, Rubanyi G, Schwartz SM. Advanced atherosclerotic lesions in the innominate artery of the ApoE knockout mouse. Arterioscler Thromb Vasc Biol. 2000; 20: 2587–2592.

This article has been cited by other articles:

				C. Franco, G. Hou, P. J. Ahmad, E. Y.K. Fu, L. Koh, W. F. Vogel, and M. P. Bendeck Discoidin Domain Receptor 1 (Ddr1) Deletion Decreases Atherosclerosis by Accelerating Matrix Accumulation and Reducing Inflammation in Low-Density Lipoprotein Receptor-Deficient Mice Circ. Res., May 23, 2008; 102(10): 1202 - 1211. [Abstract] [Full Text] [PDF]

				G. Gavazzi, C. Deffert, C. Trocme, M. Schappi, F. R. Herrmann, and K.-H. Krause NOX1 Deficiency Protects From Aortic Dissection in Response to Angiotensin II Hypertension, July 1, 2007; 50(1): 189 - 196. [Abstract] [Full Text] [PDF]

				J. D. Schmoker, K. J. McPartland, E. K. Fellinger, J. Boyum, L. Trombley, F. P. Ittleman, C. Terrien III, A. Stanley, and A. Howard Matrix metalloproteinase and tissue inhibitor expression in atherosclerotic and nonatherosclerotic thoracic aortic aneurysms J. Thorac. Cardiovasc. Surg., January 1, 2007; 133(1): 155 - 161. [Abstract] [Full Text] [PDF]

				J. L. Johnson, R. Fritsche-Danielson, M. Behrendt, A. Westin-Eriksson, H. Wennbo, M. Herslof, M. Elebring, S. J. George, W. L. McPheat, and C. L. Jackson Effect of broad-spectrum matrix metalloproteinase inhibition on atherosclerotic plaque stability Cardiovasc Res, August 1, 2006; 71(3): 586 - 595. [Abstract] [Full Text] [PDF]

				M. Kuzuya, K. Nakamura, T. Sasaki, X. Wu Cheng, S. Itohara, and A. Iguchi Effect of MMP-2 Deficiency on Atherosclerotic Lesion Formation in ApoE-Deficient Mice Arterioscler. Thromb. Vasc. Biol., May 1, 2006; 26(5): 1120 - 1125. [Abstract] [Full Text] [PDF]

				S. Janssens and H. R. Lijnen What has been learned about the cardiovascular effects of matrix metalloproteinases from mouse models? Cardiovasc Res, February 15, 2006; 69(3): 585 - 594. [Abstract] [Full Text] [PDF]

				C. M. Dollery and P. Libby Atherosclerosis and proteinase activation Cardiovasc Res, February 15, 2006; 69(3): 625 - 635. [Abstract] [Full Text] [PDF]

				E. Lubos, R. Schnabel, H. J. Rupprecht, C. Bickel, C. M. Messow, S. Prigge, F. Cambien, L. Tiret, T. Munzel, and S. Blankenberg Prognostic value of tissue inhibitor of metalloproteinase-1 for cardiovascular death among patients with cardiovascular disease: results from the AtheroGene study Eur. Heart J., January 2, 2006; 27(2): 150 - 156. [Abstract] [Full Text] [PDF]

				J.-O Deguchi, E. Aikawa, P. Libby, J. R. Vachon, M. Inada, S. M. Krane, P. Whittaker, and M. Aikawa Matrix Metalloproteinase-13/Collagenase-3 Deletion Promotes Collagen Accumulation and Organization in Mouse Atherosclerotic Plaques Circulation, October 25, 2005; 112(17): 2708 - 2715. [Abstract] [Full Text] [PDF]

				K. Lee, F. Forudi, G. M. Saidel, and M. S. Penn Alterations in Internal Elastic Lamina Permeability As a Function of Age and Anatomical Site Precede Lesion Development in Apolipoprotein E-Null Mice Circ. Res., September 2, 2005; 97(5): 450 - 456. [Abstract] [Full Text] [PDF]

				H. Achneck, B. Modi, C. Shaw, J. Rizzo, G. Albornoz, D. Fusco, and J. Elefteriades Ascending Thoracic Aneurysms Are Associated With Decreased Systemic Atherosclerosis Chest, September 1, 2005; 128(3): 1580 - 1586. [Abstract] [Full Text] [PDF]

				A. Garcia-Touchard, T. D. Henry, G. Sangiorgi, L. G. Spagnoli, A. Mauriello, C. Conover, and R. S. Schwartz Extracellular Proteases in Atherosclerosis and Restenosis Arterioscler. Thromb. Vasc. Biol., June 1, 2005; 25(6): 1119 - 1127. [Abstract] [Full Text] [PDF]

				E. T. Choi, E. T. Collins, L. A. Marine, M. G. Uberti, H. Uchida, J. E. Leidenfrost, M. F. Khan, K. P. Boc, D. R. Abendschein, and W. C. Parks Matrix Metalloproteinase-9 Modulation by Resident Arterial Cells Is Responsible for Injury-Induced Accelerated Atherosclerotic Plaque Development in Apolipoprotein E-Deficient Mice Arterioscler. Thromb. Vasc. Biol., May 1, 2005; 25(5): 1020 - 1025. [Abstract] [Full Text] [PDF]

				A. C. Newby Dual Role of Matrix Metalloproteinases (Matrixins) in Intimal Thickening and Atherosclerotic Plaque Rupture Physiol Rev, January 1, 2005; 85(1): 1 - 31. [Abstract] [Full Text] [PDF]

				Y. Fukumoto, J.-o Deguchi, P. Libby, E. Rabkin-Aikawa, Y. Sakata, M. T. Chin, C. C. Hill, P. R. Lawler, N. Varo, F. J. Schoen, et al. Genetically Determined Resistance to Collagenase Action Augments Interstitial Collagen Accumulation in Atherosclerotic Plaques Circulation, October 5, 2004; 110(14): 1953 - 1959. [Abstract] [Full Text] [PDF]

				J. Sundstrom, J. C. Evans, E. J. Benjamin, D. Levy, M. G. Larson, D. B. Sawyer, D. A. Siwik, W. S. Colucci, P. W.F. Wilson, and R. S. Vasan Relations of plasma total TIMP-1 levels to cardiovascular risk factors and echocardiographic measures: the Framingham heart study Eur. Heart J., September 1, 2004; 25(17): 1509 - 1516. [Abstract] [Full Text] [PDF]

				A. Luttun, E. Lutgens, A. Manderveld, K. Maris, D. Collen, P. Carmeliet, and L. Moons Loss of Matrix Metalloproteinase-9 or Matrix Metalloproteinase-12 Protects Apolipoprotein E-Deficient Mice Against Atherosclerotic Media Destruction but Differentially Affects Plaque Growth Circulation, March 23, 2004; 109(11): 1408 - 1414. [Abstract] [Full Text] [PDF]

				A. Daugherty and L. A. Cassis Mouse Models of Abdominal Aortic Aneurysms Arterioscler. Thromb. Vasc. Biol., March 1, 2004; 24(3): 429 - 434. [Abstract] [Full Text]

				C. Whatling, W. McPheat, and E. Hurt-Camejo Matrix Management: Assigning Different Roles for MMP-2 and MMP-9 in Vascular Remodeling Arterioscler. Thromb. Vasc. Biol., January 1, 2004; 24(1): 10 - 11. [Full Text] [PDF]

				E. Lutgens, R.-J. van Suylen, B. C. Faber, M. J. Gijbels, P. M. Eurlings, A.-P. Bijnens, K. B. Cleutjens, S. Heeneman, and M. J.A.P. Daemen Atherosclerotic Plaque Rupture: Local or Systemic Process? Arterioscler. Thromb. Vasc. Biol., December 1, 2003; 23(12): 2123 - 2130. [Abstract] [Full Text] [PDF]

				W. Shi, M. D. Brown, X. Wang, J. Wong, D. F. Kallmes, A. H. Matsumoto, G. A. Helm, T. A. Drake, and A. J. Lusis Genetic Backgrounds but Not Sizes of Atherosclerotic Lesions Determine Medial Destruction in the Aortic Root of Apolipoprotein E-Deficient Mice Arterioscler. Thromb. Vasc. Biol., October 1, 2003; 23(10): 1901 - 1906. [Abstract] [Full Text] [PDF]

				C. B Jones, D. C Sane, and D. M Herrington Matrix metalloproteinases: A review of their structure and role in acute coronary syndrome Cardiovasc Res, October 1, 2003; 59(4): 812 - 823. [Abstract] [Full Text] [PDF]

				T. L. Medley, B. A. Kingwell, C. D. Gatzka, P. Pillay, and T. J. Cole Matrix Metalloproteinase-3 Genotype Contributes to Age-Related Aortic Stiffening Through Modulation of Gene and Protein Expression Circ. Res., June 13, 2003; 92(11): 1254 - 1261. [Abstract] [Full Text] [PDF]

				M. W. Manning, L. A. Cassis, and A. Daugherty Differential Effects of Doxycycline, a Broad-Spectrum Matrix Metalloproteinase Inhibitor, on Angiotensin II-Induced Atherosclerosis and Abdominal Aortic Aneurysms Arterioscler. Thromb. Vasc. Biol., March 1, 2003; 23(3): 483 - 488. [Abstract] [Full Text] [PDF]