© 1997 American Heart Association, Inc.
Articles |
Expression of Matrix Metalloproteinases and Their Inhibitor TIMP-1 in the Rat Carotid Artery After Balloon Injury
Correspondence to Karen Webb, PhD, The Centre for Genetics of Cardiovascular Disorders, University College London Medical School, 5 University Street, London WC1E 6JJ, United Kingdom. E mail k.webb{at}med.ucl.ac.uk
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Abstract |
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Abstract The temporal relationship of matrix metalloproteinases (MMPs) and a specific tissue inhibitor (TIMP-1) has been examined by reverse transcription-polymerase chain reaction and substrate zymography, after balloon catheter angioplasty of the rat carotid artery. The contralateral uninjured carotid artery was used as a comparative control. Of the MMPs examined, only MMP-2 (72-kDa gelatinase) was produced constitutively by normal uninjured arteries. After injury, MMP-2 mRNA levels fell compared with the uninjured arteries; by 24 hours, levels had increased 2-fold. Zymography showed that the inactive form of MMP-2 predominated in uninjured vessels, but after injury, the level of the active form was increased. MMP-9 (92-kDa gelatinase) mRNA levels and activity peaked at 6 hours after injury and were still detectable at 7 days. MMP-3 (stromelysin) expression was detectable at low levels as early as 2 hours after injury and showed an approximate 2-fold increase of expression at 7 days. The presence of the active protein paralleled the mRNA expression. The inhibitor TIMP-1 mRNA was first detected 6 hours after injury and showed a marked peak of expression at 24 hours; however, no expression was detected by 7 days. The presence of a constitutively expressed, low molecular weight caseinolytic enzyme (27 kDa) was observed, and the induction of a caseinolytic enzyme (30 kDa) was noted that was induced as early as 2 hours after injury, peaked at 6 hours, and was barely detectable by 7 days. These results demonstrate that the process of extracellular matrix breakdown by MMPs after balloon catheter-induced injury is controlled by a tightly regulated temporal response by the genes responsible for the production of these enzymes and their inhibitor and by post-translational activation of the proenzymes.
Key Words: matrix metalloproteinases • RT-PCR • rat • carotid artery • injury
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Introduction |
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Percutaneous transluminal coronary angioplasty can be used to improve angiographic lumen diameter at the sites of discrete arterial stenoses. The remodeling of the vessel after balloon catheter dilation involves proliferation of medial smooth muscle cells, migration of cells to the intima, and proliferation of intimal smooth muscle cells to form a thickened neointima. In about 30% of patients, this tissue remodeling process is so exuberant that it produces restenosis of the vessel and a recurrence of related symptoms.1
Vascular smooth muscle cells of the large arteries are normally enmeshed in a dense matrix composed principally of elastin, collagens, and proteoglycans; because migration of the cells is essential for the formation of the neointima, altered metabolism of the extracellular matrix of the blood vessels must occur. Despite the information generated by many research groups, the precise mechanisms governing the proteolytic events remain obscure. Medial smooth muscle cell proliferation begins immediately after injury and peaks early, reaching a maximum at 2 days. Smooth muscle cells have been shown to express mRNA for urokinase plasminogen activator and tissue-type plasminogen activator after balloon injury in the rat model of angioplasty.2 Plasminogen activators convert plasminogen to plasmin, which can degrade several matrix molecules. However, they are also involved in the activation of MMPs, a family of proteinases capable of degrading all constituents of the extracellular matrix.3,4 Members of this group of enzymes are divided into three main groups: interstitial collagenases, type IV collagenases or gelatinases, and stromelysins. Studies of human atherosclerotic lesions have demonstrated that stromelysin (MMP-3) and other MMPs are expressed in macrophages and individual smooth muscle cells.5–9 A 72-kDa form of gelatinase (MMP-2; gelatinase A) has been shown to be produced constitutively in rat carotid arteries, whereas the 92-kDa gelatinase (MMP-9; gelatinase B) was induced after balloon catheter injury during the period of migration of smooth muscle cells from the media to the intima.10
The expression and activity of the MMPs are tightly controlled at several levels. First, the secreted latent proenzymes must undergo proteolytic activation, and second, ubiquitous endogenous tissue inhibitors can interfere with MMP proteolytic activation and enzymatic activity. Changes in the temporal expression of these enzymes and their inhibitors may regulate the local accumulation and degradation of the matrix and could be involved in the process of vascular remodeling that results in restenosis. Previous studies have looked at production10,11 and expression10 of MMPs in the rat after injury, using Northern analysis and substrate zymography. The study presented herein looked at the temporal relationship of several of the metalloproteinases, and also the inhibitor TIMP-1, in the rat carotid artery after balloon catheter injury using the more sensitive technique of reverse transcription-polymerase chain reaction (RT-PCR) and substrate zymography from as early as 2 hours after injury, to elucidate further the pattern of metalloproteinase expression and activity after vessel injury. Results confirmed that injury induced the expression of MMP-2 and MMP-9 and for the first time demonstrated the induction of MMP-3 and the TIMP-1, both important in the regulation of MMP activity.
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Methods |
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Surgery
Male Sprague-Dawley rats (300 to 350 g) were anaesthetized by intraperitoneal injection of fentanyl (Jansson) and midazolam (Roche). A midline incision was made in the neck to expose the left external carotid artery. A 2F Fogarty balloon catheter (Baxter Healthcare Corp) was introduced through the left external carotid artery and passed into the common carotid artery. Injury was induced by distending the balloon with 20 µL of saline until a slight resistance was felt and then rotating while pulling it back through the common carotid artery.12,13 The procedure was repeated three times, and the catheter was removed. The external carotid artery was ligated, and the wound was closed. Uninjured right carotid arteries were used as controls. At various time points after injury, rats were anesthetized by an injection of fentanyl and midazolam and exsanguinated via an aortic cannula. For RT-PCR analysis, the vessels were snap-frozen in liquid nitrogen and stored at -70°C. For protein extraction, the vessels were cleaned of adhering connective tissue, opened longitudinally, and any blood clots removed. The endothelium from the uninjured right carotid arteries was removed with a sterile cotton wool bud. The samples were then snap-frozen in liquid nitrogen.
Northern Analysis
Vessels were ground under liquid nitrogen in a pestle and
mortar, and RNA was extracted using TRIzolTM Reagent (Gibco
BRL) according to the manufacturer's instructions. RNA was extracted
from one carotid artery at each time point and denatured in an equal
volume of RNA loading buffer (Sigma) at 55°C for 10 minutes. After
electrophoresis through a 1.2% agarose gel containing 1%
formaldehyde, samples were transferred to a Hybond N+
membrane (Amersham) and fixed by baking at 80°C for 2 hours.
Membranes were prehybridized in 50% formamide, 1% SDS, 5xSSC (0.3
mol/L trisodium citrate and 3 mol/L NaCl), 1xDenhardt's
solution [1% Ficoll, polyvinyl pyrrolidone, and Pentax Fraction V of
BSA] containing 100 µg/mL denatured salmon sperm DNA for 5
hours at 42°C. The membrane was probed in hybridization solution
containing denatured probe for 20 hours at 42°C. The
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe
was generated by random labeling (Boehringer) of a 348-bp PCR
fragment of rat cDNA using the primers listed in Table 1
. After hybridization, the filter was washed in 2xSSC
containing 0.1% SDS for 15 minutes, 1xSSC containing 0.1% SDS for 15
minutes, and 0.1% SSC containing 0.1% SDS for 2 minutes at room
temperature. Results were visualized and analyzed using a Fuji
bas1000 phospho-imager and its accompanying software (Fuji).
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RNA Extraction and RT-PCR
Vessels were ground under liquid nitrogen in a pestle and
mortar, and RNA was extracted using RNAzolBTM according to
the manufacturer's instructions (Biogenesis Ltd). RNA (20 µg) was
reverse transcribed with 1,500 U of Moloney murine leukemia virus
reverse transcriptase (Gibco-BRL) in the buffer provided, which
contained 15 µg random hexanucleotide primers (Pharmacia
LKB Biotechnology Inc), 0.02 mol/L DTT, 2.5 mmol/L
each dNTP (Pharmacia) for each 150 µL reaction for 2 hours at 37°C
in the presence of 1 U of the specific RNase inhibitor
Inhibit-ACETM (5 Prime 
3 Prime Inc, supplied
by CP Laboratories). cDNA was purified and concentrated by
centrifugation through an Microcon-100 concentrator
(Amicon Inc.) for 20 minutes at 1,000g at 4°C. This was
followed by a second centrifugation with 500 µL TE
(10 mmol/L Tris-HCl, pH 8, 1 mmol/L EDTA), and
a third centrifugation for 30 minutes with 500 µL
sterile distilled water (dH2O). The cDNA was collected by
inverting the filter and centrifuging at 1,000g for 3
minutes and the volume made up to 50 µL with dH2O.
PCR amplification was carried out on 1 µL of each cDNA using an
OmniGene Temperature Cycler (Hybaid Ltd, UK) on block control
temperature. The primers (from Genosys) were chosen so that they
spanned intron/exon boundaries (except TIMP-1) and, thus, only
amplified products from cDNA. TIMP-1 primer pairs were designed to
cross several exon/intron borders and, thus, distinguished PCR
products amplified from cDNA and genomic DNA. The sequences of the
oligonucleotide primers, and the expected PCR
product size, are listed in Table 1
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PCR reactions were carried out in a standard buffer, as recommended by
Gibco-BRL (10x = 500 mmol/L KCl, 100 mmol/L
Tris-HCl, pH 8.3, 2 mmol/L each dNTP, 0.01% gelatin) with
200 ng each primer per reaction, and final concentrations of 5% W-1
detergent and 1.5 mmol/L MgCl2 (2.5
mmol/L for MMP-2). Taq polymerase (Gibco-BRL) was
used at a concentration of 1 U per 50 µL reaction, and
-[32P]dCTP (Amersham International Plc) was used at a
concentration of 0.5 µCi per reaction. After 10 or 15 cycles of
denaturation at 94°C for 45 seconds (except for an initial
denaturation for 5 minutes), annealing at 58°C for 1 minute and
extension at 72°C for 1 minute for MMP-2 and TIMP-1, and MMP-3 and
MMP-9, respectively, 200 ng of each (GAPDH) primer was added to each
reaction and the PCR reaction continued until a total of 30 cycles had
been completed. PCR products were visualized by ethidium bromide
staining after separation on a 6% polyacrylamide gel in Tris
borate buffer (TBE: 0.089 mol/L Tris borate, 0.089 mol/L
boric acid, 0.002 mol/L EDTA) and exposure to preflashed X-ray
film.
Films were analyzed using the GelBlot-Pro 1.01 Gel Documentation System (UVP Ltd). Because cDNA synthesis varies,14 the amount of cDNA to be amplified by PCR was normalized using mRNA from a "housekeeping" gene whose mRNA is present in every cell and whose expression is not expected to vary greatly. GAPDH was used as an internal control for the concentration of starting cDNA in each PCR reaction and for equal loading on to the acrylamide gel. Values were adjusted for the GAPDH signal in each PCR reaction and expressed in an arbitrary scale. Results are given as the mean±SEM of 1 to 3 PCR reactions from three sets of animals.
Zymography
Proteins were extracted from tissue, as described
previously.15 Briefly, samples were ground under liquid
nitrogen in a pestle and mortar. The samples were vortexed in lysis
buffer containing 1% SDS, 1 µmol/L phenylmethylsulfonyl
fluoride (PMSF), and 10 µg/mL leupeptin in 50
mmol/L Tris buffer, pH 7.6. Insoluble matter was removed by
centrifugation at 13,500g for 5 minutes.
Total protein concentration for each sample was determined by the
Bradford reagent method.16 Five microliters of each sample
was diluted into 495 µL of dH2O and an equal volume of
Bradford reagent (0.06% Coomassie brilliant blue G-250, 3% perchloric
acid) added. The absorbance of each sample was read at 595 nm and
compared with BSA standards.
Proteins with gelatinolytic or caseinolytic
activity were identified as described previously.17 Gelatin
(Bio-Rad) and -casein (Sigma Chemical Co) were incorporated into
10% SDS-polyacrylamide gels to a final concentration of 1
mg/mL. After electrophoresis, the proteins in the gel were
renatured by incubation for 30 minutes (2x15 minutes) in 2.5% Triton
X-100. Gels were subsequently incubated overnight at 37°C in 50
mmol/L Tris-HCl, pH 7.4, containing 10 mmol/L
calcium chloride and 0.05% Brij 35 (Sigma). Bands of lytic activity
were visualized as zones of clearing after staining with Coomassie
brilliant blue G-250. To verify MMP activity, identical gels were
incubated overnight in the presence of 20 mmol/L EDTA, an
inhibitor of MMPs, 2 mmol/L PMSF, a serine
protease inhibitor, or 1 µmol/L
trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane
(E64; Sigma), a cysteine protease inhibitor.18
The MMP inhibitor TIMP-1 was visualized by reverse
zymography. MMP-2 (University Technologies International Inc.,
University of Calgary) was incorporated into the polyacrylamide
gel at a concentration of 0.3 µg/mL. Electrophoresis was
performed at 4°C and TIMP-1 activity visualized as undigested bands
after Coomassie blue staining. Serum-free medium from stably
transfected BHK-1 cells that overexpress TIMP-1, TIMP-2, and TIMP-3
(University Technologies International Inc, University of Calgary) was
used as a positive control.
Statistics
RT-PCR data are presented as mean±SEM of one to three
PCR experiments from three separate sets of animals. Results were
assessed using the Student's t test for paired samples,
with a value of P
.05 considered significant.
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Results |
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Effect of Injury on GAPDH mRNA Expression
Northern analysis of RNA from uninjured and injured rat carotid arteries was used to assess whether GAPDH was affected by the ballooning procedure. Total RNA was extracted from one carotid artery for each time point studied. The variation of loading was measured from the ethidium bromide-stained gel using the GelBlot-Pro 1.01 Gel Documentation System and compared with the Northern Blot using the Fuji bas1000 phospho-imager (Fuji) and software provided. The adjusted intensities were all between 0.77 (uninjured artery after 72 hours) and 0.94 (uninjured artery after 7 days), with no increase over time or after injury, indicating that GAPDH mRNA levels were not affected by the balloon injury over the time points used in the subsequent RT-PCR experiments (results not shown).
Metalloproteinase mRNA Expression
MMP mRNA expression was studied using RT-PCR. The results given in
Fig 1 are mean values (±SEM) obtained
from between one and three PCR experiments from three different sets of
animals standardized to the GAPDH signal.
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Fig 1A shows the expression of MMP-2 in arbitrary units in both
uninjured and balloon catheter-injured rat carotid arteries up to 7
days after treatment. MMP-2 is expressed constitutively in normal
uninjured rat carotid arteries. This expression drops after balloon
injury and, although some recovery of expression is observed, remains
low until 24 hours, when the expression of MMP-2 is increased
(~2-fold) when compared with control levels in uninjured vessels at
the same time point. MMP-2 expression peaks with an approximate 3-fold
increase over control levels 72 hours after injury, and it is still
significantly elevated at 7 days.
MMP-9, MMP-3, and TIMP-1 expression are not seen in uninjured control
vessels (Fig 1B, 1C, and 1D). However, balloon injury induces the
expression of MMP-9 as early as 6 hours after injury (Fig 1B), and this
expression gradually falls, although it is still detected 7 days after
injury. By contrast, MMP-3 expression was detected in the injured
carotid artery at low levels as early as 2 hours after injury (Fig 1D).
This expression remained low but showed an approximate 2-fold increase
by 7 days after injury. TIMP-1 expression, which is absent in uninjured
arteries, was seen at very low levels 6 hours after injury, but it
showed a marked increase of expression 24 hours after injury, and
expression disappeared by 7 days (Fig 1D).
Metalloproteinase Activity
When protein extracts from both normal and injured rat carotid
arteries were subjected to gelatin zymography, several bands of lytic
activity were observed (Fig 2). The two
major bands seen in extracts from uninjured arteries are most likely to
correspond to the inactive (70 kDa) form and the active (62 kDa) form
of MMP-2, according to size and inhibition of activity on incubation
with EDTA. The inactive form is predominant in the uninjured samples.
Injury increases the level of active protein with respect to the
inactive form, and this increase is maximal 7 days after injury.
Balloon catheter injury induced the production of extra bands
with molecular masses of 92 kDa, 105 kDa, and two of >200 kDa. The
92-kDa and 105-kDa bands are likely to represent the active and
inactive forms of MMP-9, respectively, according to size and inhibition
by EDTA. The two forms are seen as early as 2 hours after injury (lane
2), peak at 6 hours (lane 4), and have almost entirely disappeared by 7
days. The production of the >200-kDa bands paralleled that
of the 92-kDa and 105-kDa bands. All these lytic bands were inhibited
in the presence of 20 mmol/L EDTA (not shown).
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Balloon catheter injury also induced the production of several
bands of caseinolytic activity (Fig 3);
the two larger bands (88 kDa and <200 kDa) are likely to
represent stromelysin-like proteins or possibly stromelysin
complexed to other proteins because they are inhibited by incubation
with EDTA. Very faint 55-kDa lytic bands are seen in all the injured
samples (Fig 3, lanes 2, 4, 6, 8, and 10), which are assigned as MMP-3
with respect to size and inhibition by EDTA. On casein zymography,
uninjured arteries produced one small predominant band at approximately
27 kDa (Fig 3). This band is also seen in injured samples, with the
addition of an extra band at ~30 kDa. All of the larger bands (55
kDa, 88 kDa, and 200 kDa) disappear on incubation with 20
mmol/L EDTA. However, the smaller bands (27 kDa and 30 kDa) are
only partially inhibited by chelation of heavy metals by EDTA,
are more strongly inhibited by the serine protease
inhibitor, PMSF, but are not inhibited at all by a cysteine
protease inhibitor (E64), which suggests that they may not
be metalloproteinases or cysteine proteinases but may be serine
proteases (Fig 4).
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TIMP-1 Activity
TIMP-1 (~30 kDa), TIMP-2 (~21 kDa), and TIMP-3 (~24 kDa)
were seen in the media obtained from stably transfected BHK-1 cells
that overexpress TIMP-1, TIMP-2, and TIMP-3, with a minimal detection
level of 0.125 µg total protein. No TIMP activity could be
reproducibly demonstrated in either the uninjured or injured carotid
arteries at any of the time points studied (results not shown).
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Discussion |
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In the normal vascular system, the extracellular matrix (ECM), a complex network of various proteins and proteoglycans, is maintained by an intricate balance between synthesis and degradation of its structural components. Maintenance of tissue integrity during normal development and growth is achieved by programmed remodeling of the matrix, involving numerous enzymes as well as specific inhibitors that keep their activity in check. The MMPs are a family of at least 12 zinc-dependent endoproteinases that function at neutral pH and cooperatively hydrolyze most of the proteins in the ECM. In this study, the temporal expression and activity of members of the MMP family and one specific inhibitor, TIMP-1, have been examined using the balloon catheter-induced injury model in the rat carotid artery.
Unlike in the human, there are no smooth muscle cells (SMCs) in the intima of a normal rat carotid artery.19,20 SMCs resident in the media are surrounded by a basal lamina and anchored to the ECM. After balloon injury, the SMCs must modulate this matrix to migrate to the intima to form the intimal lesion. The migration requires that SMCs cross major extracellular barriers, including the intimal elastic lamina and a dense mesh of interstitial collagens and proteoglycans. Although the rat model used in this study does not accurately represent human restenosis, it does provide a convenient model of cell proliferation and migration after balloon-induced injury.
The expression of genes encoding the MMPs is cell type specific and is regulated by growth factors,21,22 oncogenes,23–25 mediators of inflammation,26–28 and tumor promoters.22,29–31 Macrophages have been shown to produce MMPs,9 and activated macrophages secrete cytokines and growth factors,32 which in turn induce neighboring cells, such as SMCs, to produce MMPs. It is thought that the production of these enzymes, along with others that break down the ECM, by both the macrophages and the SMCs allows the migration of the medial SMC from the media into the intima to form the intimal lesion associated with restenosis. By following the temporal expression of MMPs, it should be possible to gain insight into the processes allowing the migration of SMCs, in itself a repair mechanism, which in 30% to 50% of patients undergoing angioplasty contributes to the reocclusion of the vessel, leading to reoccurrence of symptoms. However, because MMPs are produced as inactive zymogens, a high level of expression of mRNA does not necessarily result in high enzyme activity. Therefore, to assess the role of the enzymes and the possible control mechanisms involved, it was necessary to look for the presence of active proteins as well as the expression of mRNA. Lytic activity was studied by running protein extracts through a polyacrylamide gel in the presence of SDS containing either gelatin, to visualize the gelatinases (MMP-2 and MMP-9), or casein, to visualize stromelysin (MMP-3). Because of the presence of SDS within the gel, both active and inactive forms of the proteins are seen as zones of clearing. However, because activation is achieved by proteolytic cleavage of the proenzymes, the different forms can be distinguished easily as they migrate at different rates. TIMP-1 activity was studied by adding activated MMP-9 to the polyacrylamide gel and was visualized as bands where TIMP-1 had inhibited the action of the enzyme.
We have used RT-PCR to look at the expression of the mRNAs as they are expressed at relatively low levels. Previous workers10 looked at these enzymes using Northern analysis. The results obtained in this study substantiated their data for MMP-2 and MMP-9, but by using the more sensitive method of RT-PCR, we were able to detect the presence of MMP-3 and TIMP-1 mRNA. As a quantitative procedure, RT-PCR is still highly controversial. However, as a qualitative procedure, RT-PCR is a powerful technique. In this study, we used it as a semiquantitative technique using an invariant constitutively expressed housekeeping gene, GAPDH, as a control (reviewed in Refs 33 and 34). Results obtained unequivocally show that injury induces the expression of MMP-3, MMP-9, and TIMP-1 and affects the expression of the constitutively expressed MMP-2. However, the relative levels of mRNA between samples at different time points are open to interpretation, and the results represent reproducible trends seen over a series of different PCR experiments using three different sets of animals.
In this study, it was observed that both the mRNA expression and the activity of the MMPs studied responded to balloon catheter-induced injury in a precise and controlled manner. The only MMP to be produced in detectable amounts by normal uninjured vessels was MMP-2, the expression and activity of which are known not to be controlled in the same way as other MMPs (reviewed in Ref 35). The immediate response to injury involved a fall in expression of the constitutively expressed MMP-2 and the induction of low levels of MMP-3. This occurs as SMCs start to proliferate within the media.36 The results of this study concur with those of Bendeck et al10 who, using Northern analysis, showed that MMP-2 mRNA levels dropped rapidly after injury in the rat model, although recovery of expression was slower than in this study. Previous studies identified the production of an active 92-kDa protein10,11 24 hours after injury and the expression of MMP-9 6 hours after injury.10 In this study, we showed that this 92-kDa gelatinolytic protein is present in the rat carotid artery as early as 2 hours after injury and that after 6 hours there is a surge in both MMP-9 mRNA expression and 92-kDa gelatinolytic activity; this sugggests that MMP-9 activity is associated with SMC migration, because medial SMC are seen in the intima 4 days after injury.
Stromelysin (MMP-3) has a broad substrate specificity, the principal substrate being proteoglycan core protein, but it is also capable of degrading procollagens I and III, nonhelical regions of types II, IV, and IX collagen, laminin, fibronectin, and gelatin. Stromelysin has been shown to be expressed in some isolated cells in atherosclerotic plaques5 and was located to the shoulders of the lesions but was never seen in normal nonatherosclerotic arteries.37 A common polymorphism in the promoter of the stromelysin gene has been shown to affect the progression of angiographic disease.38 These findings indicate that MMP-3 plays a role in the connective tissue remodeling associated with atherogenic diseases. With the more sensitive technique of RT-PCR, we were able to consistently detect MMP-3 mRNA at low levels in injured arteries as early as 2 hours after injury, although it was never detected in uninjured arteries. MMP-3 mRNA levels are greatest 7 days after injury, at a time when SMC within the intima proliferate at a maximal rate.13 The levels of expression were low and the presence of an active protein, of the size predicted for MMP-3, was only just visible. However, the fact that the protein is only produced after injury strongly suggests that it does play a pathophysiological role in the response to balloon catheter-induced injury in vivo. Its role may not only be in the degradation of matrix components. The early production (2 hours after injury) suggests that it may also be involved in the activation of other members of the MMP family.39
As indicated by the mRNA results, the protein studies showed that the
inactive (70 kDa) form of MMP-2 was produced constitutively in both
uninjured and injured samples, and the amount of proenzyme did not
change with injury. It was also noted that the active form of the
enzyme was increased after injury from 2 hours after injury (Fig 2, lane 2), but levels were greatest between 72 hours and 7 days after
injury (Fig 2, lanes 8 and 9). This indicates that MMP-2 is regulated
at both the transcriptional and activational levels.
MMP-9 mRNA expression and activity changed in parallel, indicating that there is regulation of the enzyme at the transcriptional level. MMP-9 can be activated by MMP-3, which is present as early as 2 hours after injury, suggesting an additional role for MMP-3 after balloon catheter-induced injury. The production of the large (>200 kDa) bands seen on gelatin substrate gels in injured arteries parallels that of MMP-9 and may be a multimer of smaller gelatinolytic molecules.40 Other larger gelatinases have been reported in other tissues,41–44 although none correspond exactly to the size seen here, which supports the multimer theory.
The production of two smaller (~27 kDa and ~30 kDa) caseinolytic proteins was observed. The smaller of the two proteins is produced constitutively and appears to be down-regulated by balloon catheter-induced injury, whereas the 30-kDa protein is induced after injury. Both of the proteins are partially inactivated by the presence of EDTA, but their activity is markedly reduced on incubation in the presence of PMSF, suggesting that they may be serine proteases rather than metalloproteinases. Both of the serine proteases, neutrophil elastase and cathepsin G, are located in azurophil granules of mature circulating polymorphonuclear leukocytes45 and have molecular masses of 29 kDa and 22 kDa, respectively. Together, they are very active against all components of the tissue matrix at neutral pH values.46,47 It is unlikely that the 30-kDa caseinolytic enzyme induced after balloon catheter injury is either of these serine proteases, because it does not seem to be active against gelatin under the conditions used in this study. Zempo et al11 also reported the production of two small molecular mass proteinases of 24.5 kDa and 27 kDa that were active against both casein and gelatin. In contrast to our observations on the 27-kDa and 30-kDa proteinases described here, they found that their proteinases were inhibited by 10 mmol/L EDTA but were not affected by other proteinase inhibitors including E-64 and PMSF, which indicated that the proteinases were metalloproteinases. Despite similar sizes, the 27-kDa and 30-kDa proteins reported in the present study seem to be distinct from those described by Zempo et al.11 Further studies are needed to fully characterize these proteins.
The regulation of proteolytic activity in the connective tissue requires the precisely regulated interaction of cellular processes. These include the modulation of the synthesis rate of the inactive zymogens, the activation of these zymogens, the binding of the active enzymes to their respective substrates, and the prevention or termination of the activity of the enzymes by their interaction with inhibitors. A growing literature emphasizes the biological importance of TIMPs as modulators of proteolytic activity in ECM in a variety of physiological and pathological processes (reviewed in Refs 48 and 49). To assess the role of the active MMPs in the response to balloon catheter-induced injury, it is important to look also at the production and activity of the MMP inhibitors. The inhibitors are found in a tight-binding 1:1 complex with the active forms of any of the members of the MMP family or with the latent forms of MMP-2 (TIMP-2) or MMP-9 (TIMP-1).34,50 Therefore, the expression of TIMP-1, which binds to the inactive form of MMP-9, was examined. Expression occurs in a distinct peak 24 hours after injury, after the peak of MMP-9 expression (6 hours). Reverse zymography did not show any increase in extractable TIMP-1 activity after balloon injury at any time point studied. Recent evidence indicates that this may be because tissue TIMP-1 remains tightly bound to its substrate, latent MMP-9, and being effectively employed is unable to inhibit new endogenously added enzyme.51 In addition, many other protein bands mask the inhibition of gelatin digestion when larger concentrations of total protein are loaded on to the gel. Reverse zymography is, therefore, less sensitive than Western analysis. Unfortunately, no rat specific antibodies are available to these proteins, and no cross-reactivity was seen using human antibodies.
In summary, the production and activation of various MMPs are affected by the procedure of balloon angioplasty in the rat model of restenosis. The enzymes studied are capable of degrading components of the ECM, allowing the proliferation and migration of medial SMC into the intima, where they form the major component of the restenotic lesion.34 The expression and activity of these enzymes is tightly controlled at the level of expression, of activation, and also of inhibition. These stringent controls allow the vessel to respond to the injury by the proliferation and migration of medial SMCs into the media and remodeling of all vessel wall components. It is when this repair mechanism becomes disordered that restenosis occurs. By understanding the processes taking place in the vessel wall, it may be possible to produce therapies to prevent the common and expensive problem of restenosis.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by The Wellcome Trust and by the British Heart Foundation (grant RG 16).
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Footnotes |
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The Centre for Genetics of Cardiovascular Disorders, University College London Medical School, The Rayne Institute, University Street, London ; The Wellcome Trust Centre for Human Genetics, Windmill Road, Oxford (A.M.H.); and the Hatter Institute and Centre for Cardiology, University College London Medical School, Grafton Way, London, United Kingdom (S.A., J.R.M.).
Received August 7, 1996; accepted January 14, 1997.
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References |
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