From the Atherosclerosis Research Center, Division of
Cardiology, Cedars-Sinai Medical Center, UCLA School of Medicine, Los
Angeles, California 90048, the § Harbor-UCLA Medical Center,
Torrance, California 90502, and the ¶ Vascular Medicine and
Atherosclerosis Unit, Brigham and Women's Hospital, Harvard Medical
School, Boston, Massachusetts 02115
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ABSTRACT |
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We investigated whether inflammatory cytokines or
oxidized low density lipoproteins (Ox-LDL) present in human atheroma
modulate extracellular matrix degradation by inducing membrane type
1-matrix metalloproteinase (MT1-MMP) expression. Cultured human
endothelial cells (EC) constitutively expressed MT1-MMP mRNA and
protein with enzymatic activity. Tumor necrosis factor- The early events in the initiation of atherosclerosis include the
migration of blood-borne cells into the subendothelial space and the
migration of medial smooth muscle cells into intimal layer of arteries
(1, 2). Localized degradation of extracellular matrix components by
matrix metalloproteinases
(MMPs)1 of endothelial cells
(EC) may facilitate the migration of cells observed in early as well as
late stages of atherosclerosis. In addition, many episodes of coronary
thrombosis, particularly in women and diabetics, occur when endothelial
cells slough from the surface of plaques, causing a superficial erosion
(3). Severing the tethers of endothelial cells to the underlying
extracellular matrix may favor endothelial desquamation and acute
coronary syndromes. Indeed, accumulating evidence indicates that matrix
degradation mediated by locally produced MMPs may contribute to the
genesis and progression of atherosclerotic lesions (4, 5) and to the
development of intimal lesions in experimental arterial injury (6,
7).
Matrix metalloproteinases require activation from a latent zymogen form
to attain their enzymatic activity. Cultured EC constitutively secrete
precursor MMP-2 (8), which when activated can participate in the
degradation of interstitial collagen, an important constituent of the
vascular extracellular matrix. Collagenolysis by endothelial cells may
contribute to infiltration of inflammatory cells and angiogenesis
during evolution of the atherosclerotic plaque. Importantly, MMP-2
(also known as collagenase IV) can degrade type IV collagen, an
important component of the basement membrane to which endothelial cells
attach. The mechanism underlying the activation of MMP-2 remains
uncertain. The recently discovered MT1-MMP (9) seems an important
candidate because it can activate latent MMP-2 in vitro. In
addition, as a membrane-bound rather than -soluble molecule, MT1-MMP
might participate in activation of precursor MMP-2 in a localized
manner. EC covering atherosclerotic lesions are exposed to both
inflammatory cytokines and oxidized low density lipoproteins (10, 11).
To understand the nature and regulation of the factor(s) that promote
activation of MMP-2 from its precursor, this study investigated whether
cytokines and Ox-LDL regulate expression of MT1-MMP by cultured
vascular EC and whether they endow EC with the capacity to activate
pro-MMP-2.
Materials--
All tissue culture medium and supplements were
purchased from Life Technologies, Inc. Fetal calf serum was from
Hyclone Laboratories (Logan, UT). Human cytokines TNF- Cell Culture--
Human EC (HSVEC) were harvested enzymatically
from saphenous vein using type II collagenase as described (12). Cells
were grown in Medium 199 containing 20 mM HEPES, 50 µg/ml
endothelial cell growth factor, 5 mM glutamine, 5% fetal
calf serum, and an antibiotic mixture of penicillin (100 units/ml),
streptomycin (100 µg/ml), and fungizone (1.25 µg/ml). Primary
cultures of human aortic EC were kindly provided to us by Dr. Mahamad
Navab (Division of Cardiology, UCLA). The isolation and
characterization of human aortic EC were previously described (13). EC
were routinely characterized by phase contrast microscopy (Zeiss ICM
405, × 40 objective) and expression of von Willebrand factor antigen
(12). EC within three passages were used throughout the experiments. Cells were studied at confluence in all treatment conditions. Cellular
viability was assessed by Trypan blue exclusion. Treatment of EC with
cytokines was performed essentially as described (14). Preparation and
treatment of Ox-LDL were performed essentially as described (14, 15).
The amount of Ox-LDL to which EC were exposed in certain experiments
was reduced from 100 to 50 µg/ml when treating EC for 24 h to
avoid toxicity to the cells. All reagents in our tissue culture studies
were verified for the absence of endotoxin by a commercially available
assay kit (BioWhittaker, Walkersville, MD) that has a sensitivity
detection level of 1 pg/ml. The final concentration of endotoxin in
lipoprotein preparations was less than 20 pg/ml of the culture medium used.
Amplification of Human MT1-MMP-specific cDNA
Sequence--
Two primers (MT-2, TCGATGGTGAGGGCGGCTTCCTGGCCCATGC
(nucleotides 686-716); MT-3, GCTCGAGCCCCAGGGCATGGCCCAGCTCGTG
(nucleotides 826-856)), each 31 nucleotides long, were
synthesized corresponding to the published cDNA sequence of
human MT1-MMP (9). Reverse transcriptase-polymerase chain reaction was
performed to amplify a 171-nucleotide-long cDNA sequence using
total RNA prepared from EC as substrate (16). A diagnostic restriction
enzyme digestion was performed. The amplified product was subcloned in
pCRII vector (Invitrogen, San Diego, CA) and sequenced by following
standard sequencing protocol (17) using Sequenase (U.S. Biochemical
Corp./Amersham Pharmacia Biotech).
Preparation of RNA and Northern Blot Analyses--
Total
cellular RNA was isolated by lysis of EC in guanidinium isothiocyanate,
phenol-chloroform extraction and ethanol precipitation (18). Each RNA
preparation (20 µg) was denatured and electrophoresed through a 1.2%
formaldehyde agarose gel followed by blotting onto nylon filters and
ultraviolet (UV) cross-linking. Filters were hybridized with isolated
and radiolabeled MT1-MMP-specific cDNA probe (19, 20). The blots
were washed, autoradiographed, and then rehybridized with either
tubulin or actin cDNA probe as an internal control. Quantitative
results of the assays were obtained by densitometry of autoradiograms.
Ribonuclease Protection Assays--
The linearized plasmids
containing human MT1-MMP or Nuclear Run-on Assays--
The nuclear run-on transcription
assays were performed according to a published procedure (21). Nuclei
from untreated and cytokine or Ox-LDL-treated cells were incubated in a
reaction mixture containing 10 mM Tris, pH 8.0, 20%
glycerol, 0.15 M KCl, 1.5 mM MgCl2,
5 mM dithiothreitol, and 250 units RNAsin (Promega, Madison, WI) supplemented with 0.5 mM each of CTP, ATP, and
GTP and 0.250 mCi of SDS-Polyacrylamide Gel Electrophoresis and
Immunoblotting--
Extracts of partially purified plasma membrane
fractions of EC treated with TNF- Flow Cytometry--
HSVEC grown in absence or presence of
TNF- Immunoprecipitation and Gelatin Zymography--
The
immunoprecipitation of the MT1-MMP was performed in the presence of a
mixture of protease inhibitors (Roche Molecular Biochemicals) using a
standard protocol as described previously (23). Equal amounts of
extracts of partially purified plasma membrane fractions of EC
untreated or treated with TNF- Data Analysis--
Intensities of experimental bands from the
RNA and protein assays were measured by computer-assisted densitometry.
Results are expressed as mean ± S.E. Statistical analyses were
performed by Student's t test to determine the significance
of change in the densitometric measurements. A significance difference
was considered for p values equal to or less than 0.05.
Cultured Human EC Constitutively Express MT1-MMP--
To assess
whether EC express MT1-MMP mRNA or a related sequence, we performed
reverse transcriptase-polymerase chain reaction on total RNA prepared
from cultured but unstimulated EC using a primer set designed on the
basis of the human MT1-MMP sequence published by Sato et al.
(9). A polymerase chain reaction product of the expected size (171 base
pairs) was identified and cloned into the pCRII vector. The DNA
sequence of the amplified fragment revealed a complete identity to the
human MT1-MMP cDNA sequence (GenBankTM accession number
D26512) and showed homologies to other published MT-MMP cDNA
sequences (25-28). Northern blotting assays using this cloned cDNA
fragment as a probe showed that EC contain a single mRNA species of
4.5 kb (Figs. 1 and
2), a size similar to that of MT1-MMP
mRNA observed in normal lung tissue and tumor cells.
Inflammatory Cytokines or Ox-LDL Increase Expression of MT1-MMP
mRNA in EC--
Northern analyses and RNase protection assays
revealed that exposure of cultured EC to TNF- Effects of TNF- Stimulated EC Show Increased Immunoreactive MT1-MMP--
To
determine whether the mRNA levels corresponded to the amount of
translated MT1-MMP protein and to assess whether this protein was
membrane-bound, we performed immunoblot analysis on the protein lysates
of the plasma membrane extracts of EC stimulated with TNF-
To establish that HSVEC expressed membrane-anchored immunoreactive
MT1-MMP protein, we performed flow cytometric analysis on the HSVEC
grown in the absence and presence of TNF- Stimulated EC Show Increased MMP-2 Activation--
To examine
whether increased levels of MT1-MMP mRNA and immunoreactive protein
correspond to augmented enzymatic activity, we analyzed plasma membrane
extracts of EC stimulated with TNF- This study showed that cultured human EC constitutively express
MT1-MMP, a membrane-anchored MMP that activates the zymogen form of
MMP-2. We found that exposure of EC to Ox-LDL appreciably increases the
steady-state levels of MT1-MMP mRNA. Cytokines released by
inflammatory cells or induced in vascular cells by Ox-LDL (1, 29-32),
mediators relevant to vascular pathology, progressively increased
MT1-MMP transcription by cultured EC. The augmented MT1-MMP mRNA
correlated with increased plasma membrane-associated immunoreactive
protein and catalytic function of precursor MMP-2, an activity ascribed
to this enzyme. Since endothelial cells also secrete latent MMP-2 (8),
the pathways described here in vitro probably operate
in vivo.
Recent work has shown that the MT-MMP family includes at least four
members (9, 25-28). Our reverse transcriptase-polymerase chain
reaction fragment corresponds to MT1-MMP, and the affinity-purified anti-MT1-MMP antibody specifically blocked a substantial portion of the
membrane-associated proteolytic activity that catalyzed the conversion
of 72-kDa pro-MMP-2 to MMP-2. Since MT1-MMP belongs to a family of
enzymes containing at least four members, the residual activity that
partially converts pro-MMP-2 to MMP-2 after immunodepletion of MT1-MMP
in our assays may derive from other members of the family or from
related enzymes present in endothelial membrane extracts.
Activated MT1-MMP acts on several substrates of potential relevance to
vascular pathobiology (33-37). The catalytic domain of MT1-MMP
activates pro-MMP-2 and pro-MMP-2·tissue inhibitor of
metalloproteinase-2 complex (34). The trimolecular complex of
MT1-MMP·tissue inhibitor of metalloproteinase-2·pro-MMP-2 functions as an activated form of MT1-MMP and thus provides a mechanism for
spatially regulated matrix degradation (35, 36). MT1-MMP can also
process pro-MMP-13 (procollagenase-3) to the fully active enzyme (37).
Precursor MMP-2 potentiates activation of pro-MMP-13 by MT1-MMP, and
active MMP-13 can in turn activate MMP-2 and MMP-g zymogens (38),
thereby indicating an activation cascade of three members of the MMP
family (33, 37-39). Thus, increased expression of MT1-MMP can favor
digestion of native interstitial collagens by MMP-13, continued
degradation of partially degraded collagens due to gelatinase activity
of MMP-2, and proteolysis of basement membrane collagen (type IV) and
elastin by active MMP-2.
The ability of endothelial cells to produce MT1-MMP, and hence activate
the spectrum of proteases described above may have several important
functional consequences in vascular pathophysiology, including
angiogenesis, leukocyte transmigration, and plaque disruptions that
cause thrombosis, the dreaded complication of atherosclerotic vascular
diseases. Proteolysis plays an important role in migration of EC inside
tissues during vasculogenesis and angiogenesis (40, 41). Transmigration
of leukocytes through the endothelium and subjacent basement membrane
is a key process in normal host defenses, in inflammatory conditions,
and in many vascular diseases including atherosclerosis (1, 2, 42).
Activation of MMP-2 by endothelial MT1-MMP may promote these processes,
since MMP-2 degrades basement membrane collagen type IV (29, 39). In
addition, active MMP-2 may promote local endothelial desquamation by
lysing contacts with the basement membrane, causing superficial erosion
of atherosclerotic plaques, now thought often to provoke coronary
thrombosis and sudden death, particularly in women and diabetics (3,
43). Indeed, areas of such superficial erosion of atheroma exhibit augmented expression of MMP-2 and tissue inhibitor of
metalloproteinase-2, which combine with MT1-MMP to form the active
ternary complex (4). Another common pathway of atherosclerotic plaque
disruption and thrombosis involves rupture of the atheroma's cap which
depends on interstitial collagen for its tensile strength (44).
Activation of pro-MMP-13 by endothelial MT1-MMP may contribute to this
mechanism of plaque disruption and consequent thrombosis.
In conclusion, the ability of endothelial cells to express MT1-MMP and
its augmented expression in response to pathobiologically relevant
stimuli such as inflammatory cytokines and Ox-LDL highlight a novel
proteolytic pathway. This mechanism may contribute to normal host
defenses and to important pathological conditions including atherosclerosis.
(TNF-
),
interleukin-1
, or interleukin-1
caused a
time-dependent increase in the steady-state MT1-MMP
mRNA levels within 4 h of exposure, peaking about 4-fold by
6 h, and remaining elevated for 12 h. Increased MT1-MMP
mRNA correlated with a 2.5-fold increase in MT1-MMP protein in EC
membranes. Ox-LDL also increased MT1-MMP mRNA levels that varied
with the duration of exposure and degree of LDL oxidation. The increase in MT1-MMP mRNA occurred within 6 h of exposure to Ox-LDL and peaked over 3-fold by 6 h. Ox-LDL, but not native LDL, increased MT1-MMP protein by 2-fold in EC membranes. A combination of TNF-
and
Ox-LDL was additive in increasing MT1-MMP expression. Nuclear run-on
assays showed that TNF-
or Ox-LDL augmented steady-state mRNA
levels by increased transcription of the MT1-MMP gene. These findings
indicate that activation of EC by inflammatory cytokines and/or Ox-LDL
increase MT1-MMP expression. Since MT1-MMP promotes matrix degradation
by activating pro-MMP-2, these results suggest a novel mechanism
whereby cytokines or Ox-LDL may influence extracellular matrix remodeling.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
interleukin-1
or interleukin-1
were purchased from R & D Systems
(Minneapolis, MN) or provided by Hoffmann-La Roche. Purified human
native and oxidized low density lipoprotein (Ox-LDL) were kindly
provided by Dr. Judith Berliner (UCLA). The endothelial cell marker von
Willebrand factor antibody was purchased from Dako (Carpenteria, CA).
Nylon transfer membranes were purchased from Oncor (Gaithersburg, MD).
Radioisotopes were purchased either from NEN Life Science Products or
Amersham Pharmacia Biotech. Purified mouse monoclonal antibodies to
human MT1-MMP were purchased from Oncogene Research Products
(Cambridge, MA). The peroxidase-conjugated rabbit anti-mouse IgG was
obtained from Zymed Laboratories Inc. (San Francisco,
CA). Goat IgG used to block unspecific binding in the flow cytometric
analysis was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA). Phycoerythrin-conjugated anti-mouse goat IgG was purchased from
Caltag Laboratories (Burlingame, CA).
-actin cDNA (0.5 µg, 0.2 pM) were used as template to synthesize radiolabeled antisense MT1-MMP and
-actin sequences using the MAXIscript in vitro transcription kit (Ambion, Austin, TX). Briefly, template DNA was incubated in a total volume of 10 µl containing 1×
transcription buffer. Radiolabeled probes corresponding to MT1-MMP and
-actin were gel-purified and used in the ribonuclease protection
assays. Hybridization of probes to 10 µg of total RNA, prepared from
unstimulated EC or EC stimulated with cytokines or Ox-LDL, was
performed essentially as described in the ribonuclease protection
manual of Ambion, Inc. RNase digestion of hybridized probe was carried
out using a mixture of RNase A and RNase T1. Protected fragments were
ethanol-precipitated and recovered by centrifugation. Pellets were
dissolved in gel loading buffer and electrophoresed through a 5%
polyacrylamide, 8 M urea gel. The gel was dried and
autoradiographed. Quantitative results of the assays were obtained by
densitometry of autoradiograms.
-[32P]UTP (NEN Life Science
Products). Radiolabeled nuclear RNA was purified by DNase I and
proteinase K digestion, phenol-chloroform extraction, and ethanol
precipitation. Relative amounts of incorporation of label into specific
RNAs were estimated by DNA-excess filter hybridization. Linearized and
denatured plasmids carrying human MT1-MMP and
-tubulin DNAs and
their corresponding vectors were slot-blotted onto nylon filters.
Filters were probed with an equal amount of radiolabeled RNA probes as
described (15, 19). The blots were washed and autoradiographed.
Quantitative results of the assays were obtained both by counting of
individual hybridized slots and densitometry of autoradiograms.
and/or Ox-LDL were isolated as
described (22). Proteins of EC membranes (50 µg) and known molecular
weight markers were separated by SDS-polyacrylamide gel
electrophoresis. Proteins were electrophoretically transferred onto
Western polyvinylidene difluoride membranes and incubated overnight at
4 °C with blocking solution (5% skimmed milk in PBS).
Affinity-purified mouse monoclonal antibodies (10 µg of IgG/ml) to
human MT1-MMP were incubated with the blots overnight at 4 °C in PBS
buffer containing 0.1% Tween 20 (23). The blots were washed twice with
PBS buffer and then treated with rabbit anti-mouse antibody (1:4000
dilution) coupled to horseradish peroxidase. Immunodetection was
accomplished using the Enhance Chemiluminescence Kit (Amersham
Pharmacia Biotech)
and/or Ox-LDL were harvested by treating the culture with
Hanks' solution containing 3 mM EDTA for 30 min on ice and
then scraping the cells from the wells. The cells were pelleted by
centrifugation and incubated with 20 µg of goat IgG in PBS containing
1% fetal calf serum and 0.1% sodium azide on ice for 15 min. Primary
mouse monoclonal antibodies to human MT1-MMP were added to cells, 0.5 µg/sample, to a total volume of 50 µl and incubated on ice for 30 min. After two washes with PBS containing 1% fetal calf serum and
0.1% sodium azide, cells were incubated with saturating concentrations
of phycoerythrin-conjugated goat anti-mouse IgG for 30 min on ice. After two more washes, cells were fixed with 1% paraformaldehyde in
PBS. Analysis was performed using FACScan (Becton Dickinson, Mountain
View, CA). Cell populations were gated according to forward and side scattering.
, interleukin-1
, or Ox-LDL were
incubated with the purified monoclonal antibodies to either human
MT1-MMP or membrane associated human c-FMS protein. Antigen-antibody
complexes were precipitated with protein G- and protein A-coupled
agarose beads (Oncogene Research Products, Cambridge, MA) by
centrifugation. Equal amounts of the supernatants were added to culture
media harvested from human smooth muscle cells containing pro-MMP-2 and
assayed for gelatinolytic activity essentially as described by Galis
et al. (24). Proteins were electrophoresed in the presence
of SDS in discontinuous 10% SDS-polyacrylamide gels containing 1 mg/ml
gelatin (Novex, San Diego, CA). Gels were processed to renature the
protein by exchanging SDS to Triton X-100 (two changes of 2.5% Triton
X-100 for a total of 30 min). Gels were subsequently incubated for
18 h at 37 °C in 50 mM Tris-HCl, pH 7.4, containing
10 mM CaCl2 and 0.05% Brij 3 and stained with Colloidal Brilliant Blue G (Sigma) followed by destaining in 5% methanol and 7% acetic acid.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A, Northern analysis showing the
induction of MT1-MMP mRNA in human saphenous vein EC in response to
treatment with TNF- and interleukin-1
or interleukin-1
.
Primary cultures of EC were treated for 6 h with TNF-
(10 ng/ml) or interleukin-1
or interleukin-1
(10 ng/ml).
B, dose-dependent effect of TNF-
on the
expression of MT1-MMP in human aortic endothelial cells
(HAEC).
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Fig. 2.
A, RNase protection assays showing the
time-dependent effects of TNF- on the induction of
MT1-MMP mRNA. B, quantitative results of MT1-MMP
mRNA were obtained by densitometry of the autoradiograms. Results
represent the mean ± S.E. of three separate RNase protection
assays. *, p < 0.05 compared with untreated
control.
, interleukin-1, or
Ox-LDL results in the accumulation of MT1-MMP mRNA (Figs. 1-4).
Both cytokines and Ox-LDL caused a time-dependent
progressive increase in the steady state levels of MT1-MMP mRNA in
stimulated EC (Figs. 2 and 4). MT1-MMP mRNA levels increased within
4 h of exposure to TNF-
, reached a peak level of about 4-fold
above control by 6 h, and remained elevated for at least 12 h
(Fig. 2, A and B). The effect of Ox-LDL on the
levels of MT1-MMP mRNA depended on the degree of LDL oxidation as
measured by presence of the thiobarbituric acid-reactive substances
(TBARS). Ox-LDL with TBARS ranging from 2.6 to 13.4 nmol/mg of LDL
protein caused increase in steady-state MT1-MMP mRNA levels as
compared with native LDL (TBARS = 0.2 nmol/mg). Ox-LDL with higher
TBARS up to 24.2 nmol/mg LDL protein also increased the levels of
MT1-MMP mRNA in EC, although to a lesser extent than Ox-LDL with
7.3 TBARS (Fig. 3B). The time
course for the induction of MT1-MMP mRNA in response to Ox-LDL
appeared similar to TNF-
. MT1-MMP mRNA levels increased and
peaked at 6 h of exposure and remained elevated for at least
24 h (Fig. 4, A and
B). Treatment of EC for 8 h with a combination of
TNF-
(10 ng/ml) and Ox-LDL (100 µg/ml) increased the levels of
MT1-MMP mRNA in an additive manner (data not shown).
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Fig. 3.
A, Northern analysis showing the
induction of MT1-MMP mRNA in human saphenous vein EC in response to
treatment with Ox-LDL. Primary cultures of EC were treated for 6 h
with various LDL preparations (100 µg/ml) as indicated. B,
densitometric analysis of Northern blots showing the effects of native
LDL (n-LDL) and Ox-LDL (50 µg/ml) on MT1-MMP expression at
24 h with respect to oxidative modification (TBARS). The effect of
TNF- on MT1-MMP expression at 6 h is shown for comparison.
Results represent the mean ± S.E. of three independent
experiments. *, p < 0.05 compared with untreated
control.
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Fig. 4.
A, RNase protection assays showing the
time-dependent effects of Ox-LDL on the induction of
MT1-MMP mRNA. B, quantitative results of MT1-MMP
mRNA were obtained by densitometry of the autoradiograms. Results
represent the mean ± S.E. of three separate RNase protection
assays. *, p < 0.05 compared with untreated
control.
or Ox-LDL on MT1-MMP Gene
Transcription--
Increased levels of MT1-MMP mRNA in TNF-
or
Ox-LDL-stimulated EC could result from enhanced transcription and RNA
processing or reduced degradation. To examine whether an increase in
steady state levels of MT1-MMP mRNA in response to TNF-
or
Ox-LDL was associated with the increased rate of MT1-MMP gene
transcription, we performed run-on assays on isolated nuclei. As shown
in Fig. 5A, the basal level of
MT1-MMP gene transcription in EC increased about 2-3-fold when cells
were stimulated with TNF-
(10 ng/ml). Ox-LDL (100 µg/ml,
TBARS = 7.3 nmol/mg) also caused about similar increase in the
rate of MT1-MMP gene transcription (Fig. 5B). The
transcription of
-tubulin, a constitutively expressed cytoskeletal protein gene, remained unchanged in response to either TNF-
or Ox-LDL.
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Fig. 5.
Induction of MT1-MMP gene transcription by
TNF- (A) and Ox-LDL
(B). Nuclei from EC grown in absence or presence
of TNF-
(10 ng/ml) for 6 h or Ox-LDL (100 µg/ml) for 6 h
were isolated for preparation of radiolabeled nuclear RNA. Equal
amounts of linearized plasmids carrying vector DNA,
-tubulin DNA,
and human MT1-MMP cDNA were slot-blotted onto nylon filters.
Filters were then probed with equal amounts of 32P-labeled
nuclear RNA.
and/or
Ox-LDL. TNF-
-mediated increase in MT1-MMP mRNA correlated with a
2.5-fold increase in MT1-MMP protein levels in EC membranes (Fig.
6A). Treatment of cells with
Ox-LDL also increased (2.0-fold) the levels of MT1-MMP proteins in EC
membranes. The levels of MT1-MMP immunoreactive protein were increased
about 3.1-fold in EC treated with a combination of TNF-
and Ox-LDL
(Fig. 6A).
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Fig. 6.
A, densitometric analyses of immunoblots
showing increases in MT1-MMP protein levels (mean ± S.E.) by
TNF- or Ox-LDL alone or in combination (*, p < 0.05; **, p < 0.02; n = 3). The blot
on top represents three separate immunoblotting experiments
of partially purified EC membrane extracts (50 µg) using MT1-MMP
antibody from untreated cells (control) and cells treated with Ox-LDL
(50 µg/ml, TBARS = 7.3 nmol/mg), TNF-
(10 ng/ml), or Ox-LDL
(50 µg/ml) and TNF-
(10 ng/ml) together for 24 h.
B, flow cytometric analysis of HSVEC using purified mouse
monoclonal antibody to human MT1-MMP. Plots represent the distribution
of fluorescence arbitrary units (mean ± S.E.) in untreated HSVEC
(control) or treated with TNF-
or Ox-LDL alone or in combination as
indicated on the abscissas (*, p < 0.05; **,
p < 0.02; n = 4).
and/or Ox-LDL. HSVEC
expressed constitutively a membrane-associated protein that reacted
with human MT1-MMP-specific antibody. In untreated cells, 60% of the
population showed binding of the primary MT1-MMP antibody (data not
shown). The background level, as determined when no primary antibody
was included, had 2% of cells positive. Stimulation of EC with TNF-
and/or Ox-LDL for 24 h increased the number of MT1-MMP-bearing
cells to about 1.8-fold (Ox-LDL), 1.7-fold (TNF-
), or 2.5-fold
(TNF-
and Ox-LDL together) as compared with unstimulated cells (Fig.
6B).
, interleukin-1
, or Ox-LDL by
SDS-polyacrylamide gel electrophoresis gelatin zymography. Incubation
of medium conditioned by human smooth muscle cells that contained
pro-MMP-2, with plasma membrane extracts prepared from EC treated with
TNF-
, interleukin-1
, or Ox-LDL, increased the proteolytic
conversion of 72-kDa pro-MMP-2 to new gelatinolytic bands of 70- and
68-kDa corresponding to the processed active MMP-2 (Fig.
7A). Purified mouse monoclonal antibody to human MT1-MMP immunoprecipitated a 64-kDa protein of the
size of MT1-MMP (not shown). Proteolytic processing of pro-MMP-2 was
reduced in membrane extracts incubated with anti-MT1-MMP antibody (Fig.
7A and B).
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Fig. 7.
Increased processing of pro-MMP-2 by plasma
membrane preparations of EC stimulated with TNF-
(10 ng/ml), interleukin-1
(10 ng/ml), or
Ox-LDL (50 µg/ml) for 24 h.
Proteolytic processing of 72-kDa pro-MMP-2 from media conditioned by
unstimulated human smooth muscle cells. Note that immunodepletion of
MT1-MMP by specific antibody reduces the processing of pro-MMP-2.
Positions of molecular mass markers are indicated in kDa. B,
densitometric analyses of gelatin zymogram showing increased levels of
activated MMP-2 in response to TNF-
, interleukin-1
, or Ox-LDL.
Immunodepletion of MT1-MMP from the EC membrane preparations reduces
the proteolytic conversion of pro-MMP-2 to MMP-2. Bars indicate the
levels of activated MMP-2 before (
) and after (
) immunodepletion
of MT1-MMP.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Drs. Judith Berliner and Mahamad Navab for providing native and oxidized LDL preparations and for human aortic endothelial cells and Aatish Kumar for expert technical assistance. We thank Dr. Maria Muszynski, DVM, for help with human saphenous vein endothelial cell cultures.
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FOOTNOTES |
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* This work was supported by NHLBI, National Institutes of Health, Grants HL51980 and HL58555 (to T. B. R.), HL52233 (to J. K. L.), and HL34636 (to P. L.) and generous grants from the Grand Foundation of Los Angeles (to P. K. S), the United Hostesses Charities of Los Angeles (to P. K. S.), and the Henry (Sam) Wheeler Research Fund (to P. K. S).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence and reprint requests should be
addressed: Tripathi B. Rajavashisth, Atherosclerosis Research Center, Division of Cardiology, D1062, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048. Tel.: 310-855-8070; Fax: 310-652-8131; E-mail: rajavashisth{at}cshs.org.
** Supported by the Dunitz family fellowship in cardiology.
Supported by a fellowship from the Johann and Throne Holst
Foundation and the Swedish Heart and Lung Foundation.
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ABBREVIATIONS |
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The abbreviations used are:
MMP, matrix
metalloproteinase;
MT1-MMP, membrane type 1-MMP;
LDL, low density
lipoprotein;
Ox-LDL, oxidized LDL;
TBARS, thiobarbituric acid-reactive
substance;
TNF-, tumor necrosis factor-
;
EC, endothelial cell(s);
HSVEC, human saphenous vein EC;
PBS, phosphate-buffered saline.
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REFERENCES |
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