© 1995 American Heart Association, Inc.
Articles |
Inflammation and Matrix Metalloproteinases in the Enlarging Abdominal Aortic Aneurysm
From the Departments of Biochemistry (T.F., R.J.T., J.T.P.), Histopathology (A.C.), and Surgery (D.J.H., R.M.G., J.T.P.), Charing Cross and Westminster Medical School, London, UK.
Correspondence to Dr J.T. Powell, Department of Surgery, Charing Cross Hospital, Fulham Palace Rd, London W6 8RF, UK.
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Abstract The risk of rupture of an abdominal aortic aneurysm increases with aortic diameter. To obtain insight into the pathological processes associated with the vascular remodeling that accompanies aortic dilatation, we compared the histological features and the activity of matrix metalloproteinases (MMPs) in biopsies from 21 small (4.0 to 5.5 cm in diameter) and 45 larger abdominal aortic aneurysms. The histological feature most clearly associated with enlarging aneurysm diameter was a higher density of inflammatory cells in the adventitia, P=.018. This inflammation was nonspecific, principally macrophages and B lymphocytes. Fibrosis of the adventitia provided compensatory thickening of the aortic wall as the aneurysm diameter increased. A combination of zymography and immunoblotting identified gelatinase A (MMP-2) as the principal metallogelatinase in small aneurysms, whereas zymography indicated an increasing activity of gelatinase B (MMP-9) in large aneurysms. Homogenates prepared from both small and large aneurysms had similar total activity against gelatin or type IV collagen. However, the concentration of gelatinase A, determined by immunoassay, was highest for small aneurysms: median concentrations, 385, 244, and 166 ng/mg protein for small aneurysms, large aneurysms, and atherosclerotic aorta, respectively. Immunolocalization studies indicated that gelatinase A was concentrated along fibrous tissue of both the acellular media and the atherosclerotic plaque. The recruitment of inflammatory cells into the adventitia, with subsequent elaboration of metalloproteinases, including gelatinase B, may contribute to the rapid growth and rupture of larger aneurysms.
Key Words: aortic aneurysm • gelatinase A • inflammation • matrix remodeling
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Introduction |
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Abdominal aortic aneurysm is a common condition, often asymptomatic until catastrophic aneurysm rupture. The pathogenesis of this condition appears to be multifactorial, with aging, environmental and genetic history interacting to cause the dilatation and weakening of the least elastic portion of the aorta, proximal to the iliac bifurcation.1 The dilatation is accompanied by another phenotypic change, the accelerated loss of the elastic properties of the abdominal aorta, this phenotype being associated with the declining elastin content of the aortic media.2 3 Destruction and fragmentation of the elastin fibers in the media have been proposed as the principal pathological process underlying aneurysmal dilatation of the aorta, although both atherosclerosis and inflammation have been proposed as important contributory processes.4 5 6 7 8 All of these processes are evident in the aneurysmal aorta, where the dilatation is accompanied by extensive vascular remodeling. The MMPs are a family of tightly regulated enzymes that play an important role in the remodeling of the extracellular matrix components: in the aorta principally collagen and elastin. Several members of this family of enzymes have been identified in aneurysmal aorta, including gelatinase A (MMP-2), stromelysin (MMP-3), and gelatinase B (MMP-9).9 10 Each of these enzymes has a different spectrum of activity to hydrolyze specific components of the extracellular matrix. Gelatinase A, which is synthesized constitutively by smooth muscle cells, is the best elastase.11 12
In this study we focus on pathological differences between biopsies of small and large aneurysms to increase our understanding of how both the disruption of elastic fibers and inflammation contribute to the process of aneurysmal dilatation and rupture.
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Methods |
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Materials
All reagents were of the best available quality and were obtained from either Merck or Sigma Chemical Co. Antibodies were purchased from Dako, the radiochemicals sodium borotritide and tritiated acetic anhydride from Amersham, and solid-phase supports for coupling antibodies and Coomassie reagent for protein quantification from Pierce Chemical Co. Cyanogen bromide–activated Sepharose 4B was obtained from Pharmacia and Titermax from Stratatech. Peptides and oligonucleotides were synthesized by the Advanced Biotechnology Centre at Charing Cross Hospital.
Aortic Biopsies and Their Pathological Grading
Aortic biopsies were obtained from 64 patients undergoing
elective repair of an abdominal aortic aneurysm. The maximum
anterior-posterior aneurysm diameters, measured by
ultrasonography, ranged from 4 to 10 cm (median, 6.2 cm). The biopsy
(
1x2 cm) was obtained from the anterior aortic wall, opposite the
origin of the inferior mesenteric artery, close to the
point of maximum dilatation. The biopsy was subdivided for histology
and biochemical studies. For biochemical studies, all adherent thrombus
was removed before the tissue was stored at -80°C. The histology was
reported by a single pathologist (A.C.), who described the specimens on
a standard reporting form that included degree of
atherosclerosis (graded 0 to 3), wall thickness (in
millimeters), plaque cholesterol, plaque and vessel
calcification, hemosiderin, degree of medial and
adventitial inflammation (density of inflammatory cells, both graded 0
to 3), fibrosis (graded 0 to 3), vascularity of media and adventitia
(both graded 0 to 3), and presence of bacteria. The grading system for
inflammatory cells was similar to that previously described by Koch et
al.8 Lymphocytes were identified by immunohistochemistry
as either of T-cell lineage (CD 3–positive) or B-cell lineage (CD
19/22–positive). Adventitial fibrosis was assessed from the mean of
three measurements of the thickness of the fibrotic tissue, with
embedded blood vessels, in the adventitia. The thickness of the
fibrotic tissue ranged from 0 to 7 mm: 0 mm (grade 0), 1 to 2 mm (grade
1), 2 to 4 mm (grade 2), and >4 mm (grade 3), and was measured with a
Vernier microscope scale. To obtain reassurance about the objectivity
of the pathological grading, 6 biopsies were divided and sent for
duplicate pathological descriptions. Wall thickness (in millimeters)
was measured from the base of the atherosclerotic plaque to the layer
of adventitial fat with a microscope Vernier scale. Thickness was
measured at three points with four separate sections, and the mean
thickness was recorded (to the nearest millimeter). Biopsies also
were available from patients undergoing aortic grafting for occlusive
aortic disease and from brain-dead renal donors. The local ethical
committee approved the use of all biopsy material.
Polyclonal Antibodies to Gelatinase A (MMP-2)
The synthetic peptide DPMGPLLVATFWPELPEK (as encoded by
exon 10 in the hemopexin domain of gelatinase A) was coupled to rabbit
serum albumin with 0.25% glutaraldehyde. Amino
acid analysis indicated that the resulting conjugate contained
20 to 25 mol peptide/mol albumin. Conjugate (100 µg in 200
µL PBS) was emulsified with Titermax (200 µL) before intramuscular
injection into New Zealand White rabbits. Animals received boosters of
half the initial dose after 1 week and 3 weeks, and immune serum was
collected after 5 weeks. The specificity of the serum was assessed by
ELISA and Western blotting against both purified gelatinase and aortic
homogenates: the antiserum recognized purified gelatinase
(72 kD) and in aortic homogenates recognized bands at
>250, 72, and 65 kD. Previously, dot blotting had demonstrated that
the aortic homogenates contained both gelatinase B (MMP-9)
and stromelysin (MMP-3),9 but the new antiserum did not
react with bands of molecular weight corresponding to either of these
MMPs (92 to 95 kD for MMP-9 and 55 kD for MMP-3). The antiserum was
purified by ion exchange chromatography on DE52 to
yield an IgG fraction.13 The IgG fraction was purified
further by affinity chromatography on a column of the
peptide-albumin conjugate coupled to cyanogen
bromide–activated Sepharose 4B. Affinity-purified IgG was
eluted with either 3 mol/L KSCN containing 10% polyethylene glycol
6000 or with 0.1 mol/L ammonium formate, pH 3.0.
ELISA for Monitoring Antibody Production and Quantifying
Gelatinase A
Microtiter plates (Dynatech M129B) were coated with the
synthetic peptide DPMGPLLVATFWPELPEK at a concentration of 5 µg/mL in
50 mmol/L sodium carbonate, pH 9.6 (100 µL) before blocking with BSA
in the same buffer. For assessing antibody production, serial
dilutions of antiserum (or purified IgG fractions) were added to the
prepared microtiter plates in PBS, 200 µL/well, and left for 2 hours
at room temperature before emptying and washing with PBS containing
0.5% Tween 20. Plates then were incubated for 2 hours at room
temperature with swine anti-rabbit IgG peroxidase conjugate diluted
1/500 in PBS. After a washing with PBS containing 0.5% Tween 20, the
plates were developed with o-phenylenediamine (3.7 mmol/L)
and 0.01 mmol/L hydrogen peroxide in 0.1 mol/L phosphate-citrate buffer
pH 5.0, the assay was terminated after 20 minutes with 2.5 mol/L
sulfuric acid, and the absorbance at 492 nm was recorded.
Gelatinase A concentrations were determined by competition for
affinity-purified antibody (1/50 dilution, 5 µg/mL) in PBS containing
rabbit IgG (100 µg/mL) and 0.1% BSA in the first step; the assay was
calibrated with gelatinase A purified from human aortic biopsies.
Alternatively, gelatinase A was determined by allowing fractions
containing this enzyme to bind at 4°C for 1 hour to microtiter plates
precoated with type IV collagen at a concentration of 10 µg/mL at
37°C. Following binding to collagen-coated wells, the plates were
washed with PBS containing 0.5% Tween 20 before further incubation at
4°C for 2 hours with rabbit anti-gelatinase IgG (1/100 dilution in
PBS). Further washing and incubation with a second antibody,
peroxidase-conjugated swine anti-rabbit IgG, preceded development of
the assay with o-phenylenediamine as above.
Preparation of Aortic Homogenates
Frozen biopsy samples were weighed, finely diced, and agitated
in PBS (10 mL) at 4°C for 30 minutes to remove blood and serum
contamination. The washing was repeated until the washes were
colorless. The diced tissue was homogenized with a polytron
in 6 volumes of ice-cold 1 mol/L NaCl containing 2 mol/L urea, 0.1%
EDTA, 0.1% Brij 35, 0.2 mmol/L phenylmethylsulfonyl
fluoride, and 50 mmol/L Tris-Cl (TBS), pH 7.6. The tissue was
usually calcified, and the yield of protein and the MMPs (gelatinase A
and gelatinase B) was increased markedly by the inclusion of EDTA in
the homogenizing buffer: the free calcium concentration
in the homogenates ranged from 0.02 to >3 mmol/L (atomic
absorption spectrometry) for the different biopsies. The
homogenate was centrifuged at 3000g for
10 minutes, debris was removed, and the remainder of the
homogenate was centrifuged at 100 000g
for 1 hour at 4°C. Proteins were precipitated from the supernatant
with ammonium sulfate (65 g/100 mL), and the pellet was collected by
centrifugation at 10 000g for 30 minutes,
dissolved in 50 mmol/L TBS, pH 7.6, containing 10 mmol/L calcium
chloride, 0.1% Brij 35, and 0.2 mmol/L phenylmethylsulfonyl
fluoride, and dialyzed against the same buffer overnight at
4°C. For analysis of metalloproteinase activity, the dialyzed
fraction was treated further with 3 mol/L KSCN to destroy
2-macroglobulin.9 14 To abolish the
inhibitory effects of TIMPs, the dialyzed fraction was
reduced with 2.5 mmol/L dithiothreitol (40 minutes, 37°C) and then
alkylated by the addition of iodoacetamide (5 mmol/L); after 30 minutes
at 37°C, the reagents were removed by dialysis against 50 mmol/L TBS,
pH 7.6, containing 10 mmol/L calcium chloride, 0.1% Brij 35, and 0.2
mmol/L phenylmethylsulfonyl fluoride at 4°C overnight. These
treatments to abolish the inhibitory effect of
2-macroglobulin and TIMP have previously been validated
in the quantification of proteinases in tibial
cartilage.15
Purification of Gelatinase From Aortic Biopsies
The dialyzed supernatant, prepared as described above, was
absorbed onto a column of DE52 (20 mL) equilibrated with 50 mmol/L TBS,
pH 7.6, containing 10 mmol/L calcium chloride, 0.04% Brij 35, and 0.2
mmol/L phenylmethylsulfonyl fluoride. Zymograms revealed that
fractions eluted with 0.1 mol/L NaCl had
gelatinolytic activities of 
92 kD molecular
weight; these fractions did not react with the antiserum prepared to
gelatinase A. Fractions eluted with 0.25 mol/L NaCl had
gelatinolytic activities at 72 and 65 kD,
which reacted with the antiserum to gelatinase A. The principal
elastinolytic activity resided in fractions eluted with 0.25 mol/L
NaCl. Gelatinase A was purified further from the fractions eluted with
0.25 mol/L NaCl by affinity chromatography over a
column of gelatin-Sepharose (1x5 cm); gelatinase was eluted with 0.25
mol/L NaCl containing 10% DMSO, 0.04% Brij 35, and 50 mmol/L TBS, pH
7.6.16 The final product was estimated to be 90%
pure by SDS acrylamide gel electrophoresis, and on a
gelatin zymogram, it yielded lytic zones at 72 and 65 kD.
Zymography
Zymography of homogenates (10 µg protein load) was
performed as described previously using gelatin, type IV collagen, or
-elastin incorporated at concentrations of 1 mg/mL into the SDS
polyacrylamide gel.9 Samples were
activated with 1 mmol/L p-aminophenyl mercuric
acetate at 37°C for 1 hour before electrophoresis. The gels were
developed in the presence and absence of 1 mmol/L 1,10-phenanthroline
or 0.2 mmol/L phenylmethylsulfonyl fluoride to confirm
metalloproteinase activity.
Metalloproteinase Assays
Tritium-labeled macromolecular substrates were prepared as
described previously: elastin by borohydride reduction and type IV
collagen by acetylation.9 The specific
activity of the elastin was 12 500 dpm/µg, and the specific activity
of the type IV collagen was 1050 dpm/µg. Controls contained 1 mmol/L
1,10-phenanthroline to selectively inhibit metalloproteinases.
Immunocytochemistry
Formal saline-fixed biopsies were embedded in paraffin, and
8-µm sections were taken. These sections were dewaxed, and
endogenous peroxidase activity was blocked with 5%
hydrogen peroxide for 10 minutes. Rinsed sections were prewarmed to
37°C and treated with a 0.01% solution of protease (Sigma type XXIV)
in 0.14 mol/L NaCl containing 50 mmol/L TBS, pH 7.6, for 1 minute.
After the protease was rinsed off, nonspecific immunoglobulin binding
sites were blocked by incubation for 15 minutes in a 5% solution of
normal swine serum (Dako) in TBS. The swine serum was removed, and
sections were incubated for 1 hour with either rabbit IgG at 10 µg/mL
(controls) or affinity-purified rabbit anti-gelatinase A IgG at 10
µg/mL. Additional controls were performed by omitting primary or
secondary antibody. Sections were washed with TBS before incubation
with peroxidase-conjugated swine anti-rabbit immunoglobulins and
development of bound peroxidase activity with diaminobenzidine as the
chromogenic substrate. After washing with water, the
sections were counterstained with hematoxylin for 1 minute,
differentiated with acid alcohol, rinsed, and dehydrated through graded
alcohols before mounting.
Statistical Analyses
Multivariate regression analysis was
used to discern which histological features of the
aneurysm wall were associated with wall thickness and maximum
anterior-posterior aneurysm diameter. Associations between
graded characteristics and aneurysm diameter or thickness also
were assessed by 
2 tests. The concentrations of
gelatinase A in different aortic homogenates were compared
by the Mann-Whitney U test. The variation of
metalloproteinase activity with grade of adventitial inflammation and
other univariate relationships were assessed by one-way
ANOVA.
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Results |
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Histological Analysis of Aneurysm Biopsies
The aneurysm biopsies showed evidence of extensive atherosclerosis, graded either 2 or 3, in all biopsies. The blind reporting of thickness and other pathological characteristics in the six biopsies sent in duplicate was closely similar in the duplicates. The infiltration of both the media and the adventitia by inflammatory cells was very variable, and the full extent of the grading range, 0 to 3, was used. Representative examples of the extent of adventitial inflammation are shown in Fig 1
. This adventitial infiltrate was usually nonspecific,
predominantly a mixture of macrophages and lymphocytes:
polymorphonuclear and other inflammatory cells were not
prominent. Immunohistochemistry showed that the majority of adventitial
lymphocytes were of B-cell lineage (CD 19/22–positive) in all cases.
Aneurysm diameter was associated significantly with the density
of inflammatory cells in the adventitia (ANOVA, P=.011), the
largest aneurysms having the most intense inflammatory
infiltrate. The distribution of grading for adventitial inflammation
according to tertile of aneurysm diameter is shown in Table 1: diameter was associated with the density of
inflammatory cells in the adventitia, 2 trend
8.61, P=.018. After adjustment for the degree of
atherosclerosis, medial inflammation, vascularity, and
adventitial fibrosis, adventitial inflammation remained as the only
histological variable associated with
aneurysm diameter, P=.022. There was no evidence of
bacteria or other unusual findings in any of the biopsies.
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The thickness (media plus adventitia) of the aortic wall ranged from
0.2 to 0.8 mm. The tendency for the larger aneurysms to have
thicker walls, shown in Fig 2, did not achieve
statistical significance on univariate analysis,
P=.089. On multivariate analysis,
the thickness of the aneurysmal wall was more closely
associated with the amount of adventitial fibrosis, P=.005,
than with aneurysm diameter (Table 2).
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Gelatin Zymography of Aortic Homogenates
The pattern of metalloproteinase activity of aortic
homogenates, described on gelatin zymograms, varied with
each sample and was intensified greatly after pretreatment of
homogenates to abolish the inhibitory effect of
TIMPs or 2-macroglobulin (Fig 3). From
small aneurysms, diameter 
5.5 cm, with adventitial
inflammation grade 0 or 1, the principal gelatinase activities resided
in bands in the molecular weight range of 65 to 72 kD (Fig 4). From larger aneurysms, with adventitial
inflammation graded as 2 or 3, a spectrum of gelatinase activities in
the molecular weight range of 60 to 100 kD was evident (Fig 4). The
intense lytic band at 65 kD corresponded to the protein recognized by
anti–gelatinase A IgG on a parallel Western blot in which the
acrylamide gel did not contain gelatin (Fig 4).
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Hydrolysis of Type IV Collagen, Gelatin, and Elastin by
Aortic Homogenates
Aortic homogenates are likely to contain both TIMPs
(TIMP-1 and TIMP-2) together with the plasma metalloproteinase
inhibitor 2-macroglobulin. The potential for
2-macroglobulin and TIMPs to inhibit the proenzymes
following activation by aminophenyl mercuric acetate was investigated
by the selective inactivation of these inhibitors.
Metalloproteinase activity against several macromolecular substrates
was calculated after subtraction of background activity in the presence
of 1 mmol/L 1,10-phenanthroline. Treatment of the
homogenates with KSCN to abolish
2-macroglobulin inhibition effected an increase in the
ability of homogenates to hydrolyze type IV collagen,
gelatin, and elastin: the increase in activity was highest in
homogenates prepared from aneurysms with little
adventitial inflammation (Table 3). Treatment of the
homogenates to remove the potential for TIMP inhibition
effects a much greater increase in the ability of
homogenates to hydrolyze type IV collagen, elastin, and
gelatin, and this can be visualized in gelatin zymograms (Fig 3
). The
ability of homogenates, classified according to the degree
of adventitial inflammation, to hydrolyze type IV collagen and elastin
is shown in Table 3. The basal metalloproteinase activity was low and
not associated with aneurysm diameter or any pathological
characteristic of the aortic biopsy. The highest activities against all
three macromolecular substrates were recorded after the potential
for TIMP inhibition was abolished; activities against type IV collagen
and gelatin tended to be highest in homogenates deriving
from aneurysm walls with the greatest degree of adventitial
inflammation, but there was no significant independent association of
metalloproteinase activity with this or any other pathological
characteristic or aneurysm diameter. The ability to hydrolyze
elastin, after abolition of TIMP inhibition, was highest in
aneurysms with least inflammation, P=.042 (Table 3).
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Determination of Gelatinase A (MMP-2) Concentration by
Immunoassay
The two immunoassays for gelatinase A were in good agreement for
concentrations of purified gelatinase ranging from 50 ng/mL to 1
µg/mL. For the determination of gelatinase A in aortic
homogenates, the agreement between the two methods was good
only at low concentrations of gelatinase A (<200 ng/mL). At higher
concentrations, the agreement was poor, and serial dilution experiments
demonstrated that the assay using binding to type IV collagen as the
first step underestimated higher concentrations of gelatinase A.
Consequently, the competition assay, with peptide-coated wells, was
used for the determination of gelatinase A in aortic
homogenates. Since there was no evidence that the
anti-gelatinase A immunoglobulins recognized gelatinase-TIMP complexes,
homogenates treated by reduction and alkylation were used
to quantify gelatinase A. There was a significant inverse association
between total gelatinase concentration (per milligram protein) and
aneurysm diameter, P<.001. The protein
concentrations of homogenates tended to increase with
aneurysm diameter and adventitial inflammation, but these
associations did not achieve statistical significance. In
homogenates prepared from small aneurysms (<5.6 cm
in diameter), the median gelatinase A concentration was 385 ng/mg
protein compared with 244 ng/mg protein in homogenates
prepared from larger aneurysms, P=.018 (Mann-Whitney
U test), Fig 5. The concentration of
gelatinase A also was determined in homogenates prepared
from normal aorta and atherosclerotic aorta. In the
homogenates prepared from normal aorta (n=4), the total
gelatinase A concentration was <50 ng/mg protein; in the
homogenates prepared from atherosclerotic aorta (n=5), the
median gelatinase A concentration was 166 ng/mg protein (range, 106 to
202 ng/mg), Fig 5.
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Immunolocalization of Gelatinase A in Aneurysm
Biopsies
The wall of an abdominal aortic aneurysm is characterized
by atherosclerosis at the luminal aspect, a thinned,
fibrous, acellular media, and a thickened adventitia with a
variable inflammatory infiltrate. All these features can be
observed in Fig 6a, in which a section of
aneurysmal aorta has been stained with hematoxylin and eosin.
Gelatinase A is localized in two principal regions: the fibrous
connective tissue of the luminal plaque and the acellular media. In the
media, the staining was patchy and diffusely associated with connective
tissue fibrils (Fig 6b). The most cellular areas, the adventitia and
foci of inflammatory cells, showed little positive staining for
gelatinase A. Sections from more than a dozen different
aneurysms were studied, and the staining pattern was similar,
gelatinase A being localized in the media and associated with fibrous
tissue of the atherosclerotic plaque.
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We also attempted to identify the cellular origin of gelatinase A by in situ hybridization studies, but the acellular and fatty nature of the tissue mitigated against producing satisfactory results.
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Discussion |
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Inspection of any biopsy from an abdominal aortic aneurysm reveals the extent of degeneration and vascular remodeling that has accompanied the dilatation of the aorta to two to six times the normal aortic diameter. This pathological picture is just one snapshot in time and may not always provide insight into the mechanism of disease. Comparison of the changes that occurred when the aorta dilated from its normal diameter (range, 1.5 to 2.2 cm) to two to six times this diameter is an alternative approach that we exploited here. We benefited from access to biopsies of small aneurysms (4.0 to 5.5 cm in diameter) from patients randomized to elective surgery in the UK Small Aneurysm Trial17 ; in many centers, only patients with larger aneurysms proceed to elective surgery.
As the aorta enlarges and the risk of rupture increases, it might be assumed that, like a balloon, the aortic wall would become thinner. Our studies show that this premise is untrue. The larger aneurysms have thicker, but probably weaker, walls than the smaller aneurysms. These observations, even in the absence of detailed histology, underscore the extent of vascular remodeling that takes place as the aorta dilates. More particularly, as the aorta dilates, the media is attenuated, and the increased thickness of aneurysmal walls results from compensatory adventitial remodeling and fibrosis. At a fixed arterial pressure load, this thickening of the aortic wall will act to reduce wall tension (Laplace's law). The fibrotic thickening of the adventitia appears to arise principally from the deposition of new collagen fibers; this new collagen in the walls of large aneurysms appears to be more soluble than normal aortic collagen.18 Such soluble collagen may be particularly susceptible to degradation by the family of MMPs.
The cells of the atherosclerotic plaque express a complement of MMPs, including gelatinases A and B (MMP-2, MMP-9) and stromelysin (MMP-3).19 20 Zymography and Western blotting indicated the presence of gelatinase A (MMP-2), stromelysin (MMP-3), and gelatinase B (MMP-9) in homogenates prepared from abdominal aortic aneurysm biopsies.9 10 Gelatinase B is immunolocalized principally in the adventitial macrophages of abdominal aortic aneurysms.21 22 We used a polyclonal antibody to gelatinase A, which does not appear to cross-react with other MMPs, and observed that gelatinase A (MMP-2) was localized preferentially in the atherosclerotic plaque and the media, with medial localization being particularly evident in the biopsies obtained from smaller aneurysms.
The interaction of MMPs with their inhibitors in tissue homogenates may not reflect the tissue interactions of these proteins in vivo. In the homogenates, substantial metalloproteinase activity was revealed only after chemical abolition of potential TIMP inhibition: in this circumstance, total gelatinase activity was similar in all aneurysms. However, gelatin zymography revealed qualitative differences between small, noninflamed and large, inflammatory aneurysms. Small aneurysms have the most prominent lytic zones at 65 to 72 kD, attributed, from Western blotting, to gelatinase A (MMP-2). In contrast, with increasing adventitial inflammation and aneurysm diameter, there is an increasing intensity of lytic zones at 92 kD, attributable to gelatinase B (MMP-9). These findings are in keeping with the observations concerning the MMP activity of homogenates after treatment to abolish TIMP inhibition. Gelatinases A and B have comparable activities against gelatin, but gelatinase A has much higher activity against elastin than gelatinase B.12 The ratio of metalloproteinase activity against elastin and gelatin was highest in homogenates prepared from smaller aneurysms, those with least adventitial inflammation, indicating an increased proportional activity of gelatinase A compared with gelatinase B. The lower ratio of metalloproteinase activity (elastin/gelatin) from large aneurysms indicates an increasing activity of gelatinase B. Moreover, when gelatinase A was quantified by ELISA, there was a significantly higher concentration of this enzyme in homogenates prepared from the smaller aneurysms, these concentrations being severalfold higher than in homogenates prepared from normal aorta. In the zymograms, there was scant evidence of stromelysin (MMP-3) activity at molecular weights of 55 kD and lower. This is discordant with the results of Newman et al,10 who report both MMP-3 and MMP-9 activity in homogenates prepared from aortic aneurysms. However, there are substantial differences in the preparation of homogenates, and Newman et al did not describe the aneurysms from which the homogenates were obtained. Our homogenization procedure was designed to maximize the yield of metalloelastase activity. These results emphasize the problem of using tissue homogenates, which may not reflect the tissue biology.
Our findings, the increased concentration of gelatinase A (MMP-2) in small aneurysms together with its localization in the media and the plaque, provide circumstantial evidence that the increased expression and activation of this enzyme may, through its ability to degrade elastin, contribute to early aneurysmal dilatation. Since larger aneurysms appear to dilate faster than small aneurysms,23 as the aneurysm dilates, the pace of vascular remodeling is likely to increase. Our study indicates that this phase is associated with an increased concentration of inflammatory cells in the adventitia, principally macrophages and B cells. These inflammatory cells are likely to be the source of both the increased amount of gelatinase B (MMP-9) and a cascade of cytokines involved in the vascular remodeling. Our results showing a very significant association between aneurysm diameter and the extent of adventitial inflammation are mirrored by animal experiments, in which nonspecific activation of the immune system potentiates the growth of elastase-induced aneurysms in rats.24
Larger aneurysms are at much higher risk of rupture than the smaller aneurysms. This description of the differences between large and small aneurysms highlights the possibility that the recruitment of inflammatory cells into the adventitia and their subsequent elaboration of cytokines and metalloproteinases, including gelatinase B, are the important cellular processes underlying the transformation of a slowly growing small aneurysm to a dangerous, fast-growing aneurysm.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by the British Heart Foundation.
Received December 16, 1994; accepted May 3, 1995.
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References |
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Z. S. Galis and J. J. Khatri Matrix Metalloproteinases in Vascular Remodeling and Atherogenesis: The Good, the Bad, and the Ugly Circ. Res., February 22, 2002; 90(3): 251 - 262. [Abstract] [Full Text] [PDF]
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T. W.G. Carrell, K. G. Burnand, G. M.A. Wells, J. M. Clements, and A. Smith Stromelysin-1 (Matrix Metalloproteinase-3) and Tissue Inhibitor of Metalloproteinase-3 Are Overexpressed in the Wall of Abdominal Aortic Aneurysms Circulation, January 29, 2002; 105(4): 477 - 482. [Abstract] [Full Text] [PDF]
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S. Saito, N. Zempo, A. Yamashita, H. Takenaka, K. Fujioka, and K. Esato Matrix Metalloproteinase Expressions in Arteriosclerotic Aneurysmal Disease Vascular and Endovascular Surgery, January 1, 2002; 36(1): 1 - 7. [Abstract] [PDF]
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Y.-X. Wang, B. Martin-McNulty, A. D. Freay, D. A. Sukovich, M. Halks-Miller, W.-W. Li, R. Vergona, M. E. Sullivan, J. Morser, W. P. Dole, et al. Angiotensin II Increases Urokinase-Type Plasminogen Activator Expression and Induces Aneurysm in the Abdominal Aorta of Apolipoprotein E-Deficient Mice Am. J. Pathol., October 1, 2001; 159(4): 1455 - 1464. [Abstract] [Full Text]
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G. Sangiorgi, R. D'Averio, A. Mauriello, M. Bondio, M. Pontillo, S. Castelvecchio, S. Trimarchi, V. Tolva, G. Nano, V. Rampoldi, et al. Plasma Levels of Metalloproteinases-3 and -9 as Markers of Successful Abdominal Aortic Aneurysm Exclusion After Endovascular Graft Treatment Circulation, September 18, 2001; 104 (2009): I-288 - I-295. [Abstract] [Full Text] [PDF]
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J.-B. Michel Contrasting Outcomes of Atheroma Evolution: Intimal Accumulation Versus Medial Destruction Arterioscler. Thromb. Vasc. Biol., September 1, 2001; 21(9): 1389 - 1392. [Full Text] [PDF]
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B. Zhang, S. Dhillon, I. Geary, W. M. Howell, F. Iannotti, I. N.M. Day, and S. Ye Polymorphisms in Matrix Metalloproteinase-1, -3, -9, and -12 Genes in Relation to Subarachnoid Hemorrhage Stroke, September 1, 2001; 32(9): 2198 - 2202. [Abstract] [Full Text] [PDF]
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P. Schoenhagen, K. M. Ziada, D. G. Vince, S. E. Nissen, and E. M. Tuzcu Arterial remodeling and coronary artery disease: the concept of "dilated" versus "obstructive" coronary atherosclerosis J. Am. Coll. Cardiol., August 1, 2001; 38(2): 297 - 306. [Abstract] [Full Text] [PDF]
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S. Goodall, M. Crowther, D. M. Hemingway, P. R. Bell, and M. M. Thompson Ubiquitous Elevation of Matrix Metalloproteinase-2 Expression in the Vasculature of Patients With Abdominal Aneurysms Circulation, July 17, 2001; 104(3): 304 - 309. [Abstract] [Full Text] [PDF]
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K.G. Jones, D.J. Brull, L.C. Brown, M. Sian, R.M. Greenhalgh, S.E. Humphries, and J.T. Powell Interleukin-6 (IL-6) and the Prognosis of Abdominal Aortic Aneurysms Circulation, May 8, 2001; 103(18): 2260 - 2265. [Abstract] [Full Text] [PDF]
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Y. L. Song, J. W. Ford, D. Gordon, and C. J. Shanley Regulation of Lysyl Oxidase by Interferon-{gamma} in Rat Aortic Smooth Muscle Cells Arterioscler. Thromb. Vasc. Biol., April 1, 2000; 20(4): 982 - 988. [Abstract] [Full Text] [PDF]
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G. Plenz, A. Dorszewski, W. Volker, Y. S. Ko, N. J. Severs, G. Breithardt, and H. Robenek Cholesterol-Induced Changes of Type VIII Collagen Expression and Distribution in Carotid Arteries of Rabbit Arterioscler. Thromb. Vasc. Biol., October 1, 1999; 19(10): 2395 - 2404. [Abstract] [Full Text] [PDF]
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L. J. Walton, I. J. Franklin, T. Bayston, L. C. Brown, R. M. Greenhalgh, G. W. Taylor, and J. T. Powell Inhibition of Prostaglandin E2 Synthesis in Abdominal Aortic Aneurysms : Implications for Smooth Muscle Cell Viability, Inflammatory Processes, and the Expansion of Abdominal Aortic Aneurysms Circulation, July 6, 1999; 100(1): 48 - 54. [Abstract] [Full Text] [PDF]
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L. E. P. Rohde, L. H. Arroyo, N. Rifai, M. A. Creager, P. Libby, P. M. Ridker, and R. T. Lee Plasma Concentrations of Interleukin-6 and Abdominal Aortic Diameter Among Subjects Without Aortic Dilatation Arterioscler. Thromb. Vasc. Biol., July 1, 1999; 19(7): 1695 - 1699. [Abstract] [Full Text] [PDF]
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 R.W. Thompson, S. Liao, and J.A. Curci Therapeutic Potential of Tetracycline Derivatives to Suppress the Growth of Abdominal Aortic Aneurysms Advances in Dental Research, November 1, 1998; 12(1): 159 - 165. [Abstract] [PDF]
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R. Mohan, W. B. Rinehart, P. Bargagna-Mohan, and M. E. Fini Gelatinase B/lacZ Transgenic Mice, a Model for Mapping Gelatinase B Expression during Developmental and Injury-related Tissue Remodeling J. Biol. Chem., October 2, 1998; 273(40): 25903 - 25914. [Abstract] [Full Text] [PDF]
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V. Davis, R. Persidskaia, L. Baca-Regen, Y. Itoh, H. Nagase, Y. Persidsky, A. Ghorpade, and B. T. Baxter Matrix Metalloproteinase-2 Production and Its Binding to the Matrix Are Increased in Abdominal Aortic Aneurysms Arterioscler. Thromb. Vasc. Biol., October 1, 1998; 18(10): 1625 - 1633. [Abstract] [Full Text] [PDF]
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U. Schonbeck, F. Mach, and P. Libby Generation of Biologically Active IL-1{beta} by Matrix Metalloproteinases: A Novel Caspase-1-Independent Pathway of IL-1{beta} Processing J. Immunol., October 1, 1998; 161(7): 3340 - 3346. [Abstract] [Full Text] [PDF]
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D. R. Todor, I. Lewis, G. Bruno, and D. Chyatte Identification of a Serum Gelatinase Associated With the Occurrence of Cerebral Aneurysms as Pro-Matrix Metalloproteinase-2 Stroke, August 1, 1998; 29(8): 1580 - 1583. [Abstract] [Full Text] [PDF]
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M.J. Davies Aortic Aneurysm Formation : Lessons From Human Studies and Experimental Models Circulation, July 21, 1998; 98(3): 193 - 195. [Full Text] [PDF]
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Z. S. Galis, K. Asanuma, D. Godin, and X. Meng N-Acetyl-Cysteine Decreases the Matrix-Degrading Capacity of Macrophage-Derived Foam Cells : New Target for Antioxidant Therapy? Circulation, June 23, 1998; 97(24): 2445 - 2453. [Abstract] [Full Text] [PDF]
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J. Juvonen, H.-M. Surcel, J. Satta, A.-M. Teppo, A. Bloigu, H. Syrjala, J. Airaksinen, M. Leinonen, P. Saikku, and T. Juvonen Elevated Circulating Levels of Inflammatory Cytokines in Patients With Abdominal Aortic Aneurysm Arterioscler. Thromb. Vasc. Biol., November 1, 1997; 17(11): 2843 - 2847. [Abstract] [Full Text]
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P. K. Shah Inflammation, Metalloproteinases, and Increased Proteolysis : An Emerging Pathophysiological Paradigm in Aortic Aneurysm Circulation, October 7, 1997; 96(7): 2115 - 2117. [Full Text]
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W. D. McMillan, N. A. Tamarina, M. Cipollone, D. A. Johnson, M. A. Parker, and W. H. Pearce Size Matters : The Relationship Between MMP-9 Expression and Aortic Diameter Circulation, October 7, 1997; 96(7): 2228 - 2232. [Abstract] [Full Text]
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C. E. Ruiz, H. P. Zhang, A. I. Butt, and P. Whittaker Percutaneous Treatment of Abdominal Aortic Aneurysm in a Swine Model : Understanding the Behavior of Aortic Aneurysm Closure Through a Serial Histopathological Analysis Circulation, October 7, 1997; 96(7): 2438 - 2448. [Abstract] [Full Text]
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W. D. Coats, P. Whittaker, D. T. Cheung, J. W. Currier, B. Han, and D. P. Faxon Collagen Content Is Significantly Lower in Restenotic Versus Nonrestenotic Vessels After Balloon Angioplasty in the Atherosclerotic Rabbit Model Circulation, March 4, 1997; 95(5): 1293 - 1300. [Abstract] [Full Text]
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J. B. Knox, G. K. Sukhova, A. D. Whittemore, and P. Libby Evidence for Altered Balance Between Matrix Metalloproteinases and Their Inhibitors in Human Aortic Diseases Circulation, January 7, 1997; 95(1): 205 - 212. [Abstract] [Full Text]
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T. Freestone, R.J. Turner, D.J. Higman, M.J. Lever, and J.T. Powell Influence of Hypercholesterolemia and Adventitial Inflammation on the Development of Aortic Aneurysm in Rabbits Arterioscler. Thromb. Vasc. Biol., January 1, 1997; 17(1): 10 - 17. [Abstract] [Full Text]
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S. W. Galt, S. Lindemann, D. Medd, L. L. Allen, L. W. Kraiss, E. S. Harris, S. M. Prescott, T. M. McIntyre, A. S. Weyrich, and G. A. Zimmerman Differential Regulation of Matrix Metalloproteinase-9 by Monocytes Adherent to Collagen and Platelets Circ. Res., September 14, 2001; 89(6): 509 - 516. [Abstract] [Full Text] [PDF]
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F. J. Miller Jr, W. J. Sharp, X. Fang, L. W. Oberley, T. D. Oberley, and N. L. Weintraub Oxidative Stress in Human Abdominal Aortic Aneurysms: A Potential Mediator of Aneurysmal Remodeling Arterioscler. Thromb. Vasc. Biol., April 1, 2002; 22(4): 560 - 565. [Abstract] [Full Text] [PDF]
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