(Circulation. 1999;100:533-540.)
© 1999 American Heart Association, Inc.
Basic Science Reports |
Adenovirus-Mediated Overexpression of Tissue Inhibitor of Metalloproteinase-1 Reduces Atherosclerotic Lesions in Apolipoprotein E–Deficient Mice
From Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 321 "Lipoproteins and Atherogenesis," Hôpital de la Pitié-Salpétrière, Paris, France (M.R., P.L., M.M., P.M.L., C.D., M.J.C.); Rhône Poulenc-RORER, Département de Cardiologie, 94403 Vitry sur Seine, France (N.D., F.E., J.M.C.); and Laboratoire mixte Rhône-Poulenc-RORER/CNRS C9923, Génétique Moléculaire de la Neurotransmission et des Processus Dégénératifs, CERVI, Hôpital de la Pitié-Salpêtrière, Paris, France (P.H.).
Correspondence to M. Rouis, PhD, Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 321 "Lipoproteins and Atherogenesis" Hôpital de la Pitié-Salpétrière, 83 Bd de l'Hôpital, 75651 Paris Cedex 13, France. E-mail rouis{at}infobiogen.fr
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Abstract |
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Background—To define the role of metalloproteinases (MMPs) in the development of lipid-rich atherosclerotic lesions in relation to the balance between proteolytic and antiproteolytic activities, we investigated the impact of adenovirus-mediated elevation in the circulating levels of human tissue inhibitor of MMP (TIMP-1) in atherosclerosis-susceptible apolipoprotein E–deficient (apoE-/-) mice.
Methods and Results—Infusion of apoE-/- mice fed a
lipid-rich diet with rAd.RSV.TIMP-1 (1x1011 viral
particles) resulted in high hepatic expression of TIMP-1. At 2
weeks after injection, plasma TIMP-1 levels ranged from 7 to 24 µg/mL
(mean 14.8±6.8). Marked overexpression of TIMP-1 was transient, with
levels of TIMP-1 decreasing to 2.5 to 8 µg/mL (mean 4.3±2.1) at 4
weeks. Plasma lipid and lipoprotein levels in mice treated with
rAd.RSV.TIMP-1 were similar to those treated with rAd.RSV.ßGal.
However, rAd.RSV.TIMP-1–infused mice displayed a marked reduction
(
32%; P<0.05) in mean lesion area per section
(512±121 µm2x103; n=12 sections from 4
animals) as compared with rAd.RSV.ßGal-infused mice (750±182
µm2x103; n=12 sections from 4 animals).
Similarly, marked reduction in macrophage deposition as well as
MMP-2, MMP-3, and MMP-13 antigens was observed.
Conclusions—Histological and immunohistologic
analyses of atherosclerotic lesions revealed increases in
collagen, elastin, and smooth muscle 
-actin content in mice treated
with rAd.RSV.TIMP-1. These qualitative and quantitative features were
the consequence of TIMP-1 infiltration from plasma to
arterial intima, as immunohistochemical analyses
revealed an abundance of TIMP-1 specifically in lesions of
rAd.RSV.TIMP-1–treated mice.
Key Words: atherosclerosis • metalloproteinases • apolipoproteins • genes
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Atherosclerosis is a multifactorial, complex pathological process. Cell-cell and cell-matrix interactions involving monocyte-derived macrophages, vascular smooth muscle cells (SMC), endothelial cells, and T-lymphocytes are involved. Extracellular matrix degradation is a key factor in clinical thromboembolic events. Indeed, sudden death caused by acute myocardial infarction frequently results from the rupture of coronary atheroma, particularly at sites of thinning of the lesion's fibrous cap; such rupture results in hemorrhage into the plaque, resulting in a prothrombotic response followed by rapid occlusion of the artery.1 The accumulation of macrophage-derived foam cells in rupture-prone areas, such as the vulnerable shoulder regions of plaques,2 3 correlates with increased local release of matrix-degrading metalloproteinases (MMPs) and weak fibrous cap tissue.4 These findings suggest a potential role of macrophage-derived MMPs in the fragilization and ultimate rupture of plaque structure.5 6
MMPs are a family of enzymes secreted in a latent zymogen form. Interactions between cysteine residues in the propeptide domain and the zinc atom present at the catalytic site of all MMPs are responsible for maintaining the propeptide over the catalytic site. Metalloproteinase activation can occur in vitro or in vivo when the propeptide is cleaved by proteases7 8 9 10 or when the zinc-cysteine bond is disrupted.11
The proteolytic capacity of these MMPs together facilitates degradation of many if not all extracellular components present within the fibrous areas of atheromatous plaques. The activity of MMPs can be controlled by specific tissue inhibitors or TIMPs. Three TIMPs (TIMP-1, TIMP-2, and TIMP-3) have been characterized12 13 14 and a fourth (TIMP-4) has been cloned.15
TIMP-1 is produced by virtually all mesenchymal cells; its cDNA has
been cloned and sequenced.16 TIMP-1 is a secreted
glycoprotein with a molecular weight of 28 kDa
stabilized by 6 disulfide bridges.17 TIMP-1 inhibits all
MMPs but has high affinity for MMP-1, MMP-2, MMP-3, and
MMP-9.18
Increased expression of MMPs has been detected in injured arteries and atherosclerotic plaques. Indeed, stromelysin mRNA localize to focal areas enriched in macrophages in human lesions,19 whereas immunoreactive interstitial collagenase (MMP-1), the 72-kDa gelatinase (MMP-2), stromelysin-1 (MMP-3), and the 92-kDa gelatinase (MMP-9) colocalize to lesional macrophages.20 In addition, human lipid-laden macrophages prominently express matrilysin (MMP-7) and metalloelastase (MMP-12).21 These MMPs have a potent capacity to degrade proteoglycans, insoluble elastin, and fibronectin,22 23 suggesting that such matrix components are major targets of macrophage-mediated tissue destruction.
Apolipoprotein E (apoE)-deficient mice (apoE-/-) are hypercholesterolemic and develop atherosclerosis spontaneously even when fed a chow diet. Both marked hypercholesterolemia and atherosclerosis are manifest more rapidly in these mice when fed a high-fat, Western-type diet.24 In such animals, the atherosclerotic lesions present at advanced stages of progression display necrotic cores and are located at the same sites of predilection as human lesions.25 ApoE-/- mice are therefore a relevant animal model in which to identify the complex molecular and cellular mechanisms involved in plaque progression and fragilization.
To define the role of MMPs in relation to the balance between proteolytic and antiproteolytic activities in atherosclerotic lesions, we investigated the impact of elevated circulating levels of human TIMP-1 on the severe atherosclerosis characteristic of hypercholesterolemic apoE-/- mice. Overexpression of human TIMP-1 was achieved by adenovirus-mediated gene transfer.
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Methods |
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Generation of Recombinant Adenovirus
Recombinant vectors were constructed on the basis of the pAd.RSV.ßGal vector by the use of standard techniques.26 The full-length TIMP-1 cDNA was generated by reverse transcription–polymerase chain reaction from the total RNA of the HL-60 cell line. To optimize initiation of translation, the hybrid intron isolated from the pCI vector (Promega), the untranslated sequence derived from the Xenopus ß-globin gene, and the Kozak consensus sequence were linked downstream of TIMP-1 cDNA. This fragment was then cloned under the control of the RSV-LTR promoter into the pAd.RSV. vector, into which the bovine growth hormone polyadenylation site from the pRC/CMV plasmid (Invitrogen) had been inserted. The recombinant adenovirus was constructed by in vivo homologous recombination in 293 cells between shuttle plasmids and Ad.RSV.ßGal and plaque purified. Ad.Empty was constructed with homologous recombination in Escherichia coli.27 Ad.Empty contains the cytomegalovirus (CMV) promoter in place of E1 adenovirus sequences but encodes no transgene. High-titer stocks of viral particles (vp) (5.8x1012, 1.2x1012, 1.8x1012) were produced in 293 cells for rAd.RSV.TIMP-1, rAd.RSV.ßGal, and rAd.CMV.Empty, respectively, and were purified on CsCl gradients.
Cell Transfection Analysis
Human 293 kidney cells (American Type Culture Collection,
Rockville, Md) of low passage number were transfected with
rAd.RSV.TIMP-1 by use of the calcium phosphate procedure, and TIMP-1
was identified in the culture media by use of an ELISA kit (Amersham)
and by immunoblotting with a TIMP-1–monospecific
antibody. In addition, use of SDS-PAGE zymography showed human TIMP-1
to inhibit murine MMP activity.
SDS-PAGE Zymography
Tissue from 6-month-old atherosclerotic mouse aortas were used
as a source of MMPs. Mouse aortas were homogenized in 1 mL
cold buffer containing 0.2% Triton X-100, 50 mmol/L Tris, 10
mmol/L CaCl2, and 2 mol/L guanidine
hydroxychloride (pH 7.5). The homogenate was
centrifuged for 5 minutes at 14 000g. The
supernatant was extensively dialyzed overnight at 4°C against 0.2%
Triton X-100 and 50 mmol/L Tris (pH 7.5) and protein concentration
determined. The homogenate (10 µg protein) was
solubilized in buffer lacking reducing agent and loaded without heating
onto a 10% SDS-PAGE containing either 1 mg/mL bovine skin type II
gelatin (Sigma) or 1 mg/mL -casein (bovine milk, Sigma) as
substrate. The gels were incubated initially in 2.5% Triton-X-100
(2x15 minutes) and subsequently overnight at 37°C in the presence or
in the absence of 300 µg/mL of recombinant human TIMP-1 (a kind gift
from Dr G. Murphy) in 50 mmol/L Tris-HCl, pH 7.4, containing
10 µmol/L ZnCl2 and 0.05% Brij. The gels
were then fixed and stained with colloidal brilliant blue G.
Animals
Ten-week-old female C57BL/6 apoE-deficient mice
(Transgenic Alliance) were bred and housed at the animal facility of
Rhône-Poulenc Rorer (Vitry-sur-Seine, France) under protocols in
accordance with French legislation. These mice were fed a regular chow
diet and allowed to acclimatize for 3 weeks and then fed a chow diet
supplemented with 0.2% cholesterol and 10% coconut oil.
After 6 weeks, the mice were divided into 4 groups. Either
rAd.RSV.TIMP-1 (1x1011 vp), rAd.RSV.ßGal
(1x1011 vp), rAd.CMV.Empty
(1x1011 vp), or PBS (200 µL) was injected into
the tail vein of each group of mice. Blood was drawn once per
week from the retro-orbital plexus of each mouse for assay of TIMP-1,
lipids, and lipoproteins. Four weeks after treatment with either
adenoviruses or PBS, the mice were killed and tissues
analyzed.
Plasma Lipid and Lipoprotein Analyses
Total cholesterol and triglyceride
levels were assayed on fasting plasma (10 µL) with the use of
enzymatic kits (Biomerieux); HDL cholesterol was measured
with dextran sulfate precipitation (Biomerieux) according to the
manufacturer's specifications. Plasma lipoproteins were
analyzed by fast protein liquid chromatography
with 2 Superose 6 columns in series (Pharmacia Biotechnology).
Lipoproteins were eluted at a constant flow rate (0.4 mL/min) with PBS
buffer containing 1 mmol/L EDTA and 0.02% sodium
azide.28
Quantification of Plasma TIMP-1 in Mice by ELISA Procedure
Human TIMP-1 in mouse plasma was quantified by a sandwich ELISA
kit (Amersham) containing 2 monoclonal antibodies specific to human
TIMP-1 antigen.
Quantitative and Qualitative Analysis of
Atherosclerotic Plaques
Mice were killed, and freshly-removed hearts were fixed in 10%
buffered formalin and then either embedded in paraffin for
histological analysis (collagen and elastin
staining) or frozen for immunohistochemical studies. Lesion
quantification was performed on paraffin sections.
Histological Analysis
Serial paraffin-embedded sections (5 µm) were stained
with Sirius red or orcein for collagen and elastin visualization,
respectively, or stained with hematoxylin, eosin, and saffron for
lesion area measurement and the mean lesion area per animal quantified
(three 5-µm aortic sections, each separated by 100
µm).29
Immunohistochemistry
Frozen serial sections (7 µm) were treated with 0.3%
H2O2 in PBS to block
endogenous peroxidase activity, followed by blocking in 4%
BSA (Sigma). Slides were incubated (2 hours at 20°C) with either
purified rat monoclonal antibody against mouse macrophages
(CD11b antigen=Mac-1) (Valbiotech) at 1:25 dilution (20 µg/mL), goat
polyclonal antibody against mouse MMP-3 (a generous gift of Dr C.
Peeters-Joris, Louvain, Belgium) at 1:300 dilution (40 µg/mL), goat
polyclonal antibody against mouse MMP-13 (a generous gift of Prof Y.
Eeckhout, Louvain, Belgium) at 1:50 dilution (40 µg/mL), sheep
polyclonal antibody against human MMP-2, which cross-reacts with mouse
MMP-2 (Valbiotech) at 1:100 dilution (82 µg/mL), and a rabbit
polyclonal antibody against human TIMP-1 (Euromedex) at 1:1000 dilution
(1 µg/mL); for -smooth muscle actin studies, a kit (Sigma, ref.
IMMH-2) was used. As a negative control, we used either a purified
nonimmune IgG from rat, goat, sheep, or rabbit. A second biotinylated
antibody was added, either anti-rat at 1:50 dilution, anti-goat,
anti-sheep, or anti-rabbit at 1:100, respectively. Thereafter,
peroxidase-labeled or alkaline phosphatase–labeled
streptavidin was added. The activity of peroxidase was revealed with
diaminobenzidine (Merck) as a substrate, yielding a yellow-brown
deposit; the substrate for alkaline phosphatase was the fast red
substrate system (Dako) yielding an insoluble red reaction
product.
Statistical Analysis
The mean surface area of lesions was compared between groups by
ANOVA.
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Results |
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Plasma lipoprotein profiles were determined by fast protein liquid chromatography and revealed that HDL predominated with minor amounts of VLDL and LDL in control mice. In contrast, VLDL and IDL were prominent in apoE-/- mice; HDL levels were significantly diminished (
5-fold). A similar profile was
observed in apoE-/- mice when fed the
cholesterol-rich diet (data not shown), thereby confirming
earlier data.30
Infusion of apoE-/- mice fed the lipid-rich
diet (Figure 1
) with
1x1011 vp of rAd.RSV.TIMP-1 (Figure 2) resulted in a high level of expression
of TIMP-1 (Figure 3). At 2 weeks after
injection, plasma TIMP-1 levels ranged from 7 to 24 µg/mL (mean
14.8±6.8). Marked overexpression of TIMP-1 was transient, with levels
of TIMP-1 decreasing to 2.5 to 8 µg/mL (mean 4.3±2.1) by 4 weeks.
Human TIMP-1 expressed in mice efficiently inhibited murine
metalloproteinases in vitro (Figure 4).
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The recombinant ßGal adenovirus construct was used to determine the tissue distribution of adenovirus-mediated gene delivery. Our results (data not shown) confirmed that the liver was the major organ of ßGal transgene expression.31
Plasma lipid and lipoprotein levels in the
rAd.RSV.TIMP-1–treated apoE-/- mice fed an
atherogenic diet as compared with the same mice injected with
rAd.RSV.ßGal, Ad.CMV.Empty, or PBS are summarized in the
Table. All parameters increased over the time period
but were not statistically different between each group of treated
mice.
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To determine the effect of TIMP-1 gene expression on the capacity
of the cholesterol-rich diet to induce
atherosclerosis in apoE-/-
mice, we infused a subset of 5-month-old mice with either
rAd.RSV.TIMP-1, rAd.RSV.ßGal, rAd.CMV.Empty, or PBS and then killed
the animals 4 weeks later (Figure 1
). The rAd.RSV.ßGal-,
rAd.CMV.Empty-, and PBS-infused mice displayed a mean lesion area per
section of 750±182
µm2x103 (n=12 sections
from 4 animals), 780±91
µm2x103 (n=9 sections
from 3 animals), and 753±121
µm2x103 (n=9 sections
from 3 animals), respectively. However, the rAd.RSV.TIMP-1–infused
mice exhibited a mean aortic lesion area that was significantly smaller
(512±121 µm2x103;
n=12 sections from 4 animals) than that in animals infused with either
rAd.RSV.ßGal, rAd.CMV.Empty, or PBS P<0.05 (Figure 5).
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To evaluate the impact of TIMP-1 overexpression on the
qualitative features of aortic lesions in
apoE-/- mice fed the lipid-rich diet, we
investigated the integrity of collagen, elastin, and -smooth muscle
actin, the major constituents of the extracellular matrix of the
arterial wall. Four 5-µm paraffin sections per animal,
starting from the aortic root, each separated by 100 µm, were
taken from a subset of mice infused with rAd.RSV.TIMP-1,
rAd.RSV.ßGal, rAd.CMV.Empty, or PBS and stained with Sirius red for
collagen or with orcein for elastin. For -actin analysis, we
used a kit (Sigma, ref. IMMH-2). The results indicated that these 3
matrix proteins were degraded to a lesser degree in the subset of
animals treated with rAd.RSV.TIMP-1 as compared with control animals
(Figure 6, A through F).
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The media of the aorta in rAd.RSV.TIMP-1–treated mice was devoid of
infiltrating Mac-1–immunoreactive macrophages despite their
abundance at the base of the plaque (Figure 7B); by contrast, analyses of
rAd.ßGal-treated mice revealed an accumulation of Mac-1–positive
macrophages in an intimal-to-adventitial gradient (Figure 7A).
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MMP-3 and MMP-13 were detectable in all sections of
rAd.RSV.TIMP-1–treated mice as well as in control mice and appeared to
colocalize with Mac-1–positive cells (Figure 7, C through F).
In contrast, TIMP-1 antigen was detectable only in
rAd.RSV.TIMP-1–treated mice and was uniformly distributed from the
intima to the adventitia (Figure 7, G and H).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The present findings reveal, for the first time, that elevated circulating levels of human TIMP-1 significantly reduce atherosclerotic lesions in atherosclerosis-susceptible hypercholesterolemic apoE-/- mice.
A rapid (4 weeks) and significant reduction of lesion area (32% vs
control; P<0.05) occurred in rAd.RSV.TIMP-1–treated mice
(Figure 5). This reduction appears to result from the local
inhibition of the activity of MMPs by human TIMP-1, a protein detected
only in the lesions of mice treated with rAd.RSV.TIMP-1 (Figure 7H). Human TIMP-1 inhibited murine MMPs in vitro (Figure 4), a finding consistent with previous work showing that
murine MMPs were inhibited in transgenic mice overexpressing human
TIMP-1.32 33 In addition, MMP-3, MMP-13 (Figure 7, C through F), and MMP-2 (data not shown) have been
immunolocalized in lesions of our apoE-/- mice:
one consequence of MMP inhibition might be the limitation of the
proteolysis of elastin, collagen, and -actin (Figure 6, A
through F). Indeed, inhibition of MMPs by local overexpression of
TIMP-1 increases elastin accumulation in rat carotid artery
intima.34
Overexpression of TIMPs in neoplastic human35 and murine36 cells is associated with decreased MMP activity and reduction in the malignant behavior of these cells. Indeed, MMP overexpression might lead to proliferation and migration, whereas overexpression of TIMP-1 might serve to maintain cells in the quiescent state. Inhibition of TIMP-1 expression with antisense oligonucleotides37 or through targeted gene disruption by homologous recombination38 enhances the invasive properties of tumor cells in vitro and their metastatic potential in vivo. In addition, adenovirus-mediated overexpression of TIMP-1 in cultured rat SMC inhibits SMC chemotaxis and invasion without affecting the number of cells.39 Similarly, adenovirus-mediated gene transfer of TIMP-1 inhibits SMC migration and neointimal formation in human saphenous vein segments in organ culture without effect on SMC proliferation.40 In this context, our data suggest that the beneficial effect resulting from the overexpression of TIMP-1 might also arise from the inhibition of SMC invasion. In addition, increase in lipid levels with time in rAd.RSV.TIMP-1–treated mice occurred in a manner similar to that in controls. This finding suggests that high levels of human TIMP-1 in murine plasma do not alter lipid metabolism in such a way as to inhibit progression or facilitate lesion regression. Because mice were continuously fed with a cholesterol-rich diet during the adenoviral treatment, the increasing level of TIMP-1 may inhibit the progression of arterial lesions rather than inducing their regression. The reduced number of foam cell–derived macrophages in the lesions of rAd.RSV.TIMP-1–treated mice, in addition to decrease in the dilation of the aortic area as a result of extracellular matrix remodeling, could account for this beneficial effect. A similar observation was reported recently by Carmeliet et al,10 who established that deficiency of urokinase-type plasminogen in apoE-/- mice protects against media destruction and aneurysm formation, probably by means of reduced plasmin-dependent activation of pro-MMPs. Equally, TIMP-1 may contribute to lesion regression through MMP-induced reduction in HDL-mediated cholesterol efflux from human macrophage foam cells after truncation of the carboxyl terminus of apoA-I.41 Therefore MMP inhibition by TIMP-1 may protect HDL particles and promote efficient reverse cholesterol transport.
The efficiency of intravascular gene delivery to atherosclerotic lesions may be low because lesions that are frequently rich in connective tissue contain limited numbers of transfectable cells.42 Nevertheless, high levels of TIMP-1 were observed in lesions treated by rAd.RSV.TIMP-1 in our animal model, and this despite the expression of the irrelevant ßGal gene mainly in the liver (data not shown). The localization of TIMP-1 protein in lesions is unlikely to result from transfection of vascular cells but more likely to be attributed to TIMP-1 infiltration across the endothelium. Indeed, TIMP-1 is a stable protein of low molecular weight that may readily infiltrate into the intimal space. Indeed, overexpression of apoIV in murine plasma exerts a protective effect against plaque formation by a mechanism that does not involve increase in HDL cholesterol,43 suggesting that apoIV infiltrates into the intimal space, where it exerts a protective effect. In addition, adenovirus-mediated overexpression of apoE induces regression of the atherosclerotic lipid core in plaques of apoE-/- mice by a mechanism implicating its infiltration and association with lipid present within the intimal area (Duverger et al, personal communication).
In conclusion, our data prompt the hypothesis that local progression of established atherosclerotic lesions or vulnerable lipid-rich plaques can be retarded by gene transfer techniques. The adenovirus-mediated overexpression of human TIMP-1 in apoE-/- mice, an animal model of atherosclerotic plaque formation, provides a convincing example of this therapeutic approach.
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Acknowledgments |
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These studies were supported by a grant from the Ministère de la Recherche, France (ACC-SV9, No. 1A036A), and by INSERM. The authors are most grateful to I. Hugou for excellent technical assistance.
Received October 23, 1998; revision received April 8, 1999; accepted April 9, 1999.
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