Activation of MMP-2 in response to vascular injury is mediated by
phosphatidylinositol 3-kinase-dependent expression of MT1-MMP
Peter Zahradka,1,3Greg Harding,1,2,3Brenda Litchie,3Shawn Thomas,3
Jeffrey P. Werner,1,3David P. Wilson,1,3and Natalia Yurkova3
Departments of1Physiology and2Surgery, University of Manitoba, and3lnstitute of Cardiovascular
Sciences, St. Boniface General Hospital Research Centre, Winnipeg, Manitoba, Canada R2H 2A6
Submitted 8 March 2004; accepted in final form 23 July 2004
Zahradka, Peter, Greg Harding, Brenda Litchie, Shawn
Thomas, Jeffrey P. Werner, David P. Wilson, and Natalia
Yurkova. Activation of MMP-2 in response to vascular injury is
mediated by phosphatidylinositol 3-kinase-dependent expression of
MT1-MMP. Am J Physiol Heart Circ Physiol 287: H2861–H2870,
2004. First published August 5, 2004; doi:10.1152/ajpheart.00230.
2004.—Phosphatidylinositol 3-kinase (PI3K) is required for smooth
muscle cell (SMC) proliferation. This study reports that inhibitors of
PI3K also prevent SMC migration and block neointimal hyperplasia
in an organ culture model of restenosis. Inhibition of neointimal
formation by LY-294002 was concentration and time dependent, with
10 ?M yielding the maximal effect. Continuous exposure for at least
the first 4–7 days of culture was essential for significant inhibition. To
assess the role of matrix metalloproteinases (MMPs) in this process,
we monitored MMP secretion by injured vessels in culture. Treatment
with LY-294002 selectively reduced active MMP-2 in media samples
according to zymography and Western blot analysis without concom-
itant changes in latent MMP-2. Parallel results with wortmannin
indicate that MMP-2 activation is PI3K dependent. Previous research
has shown a role for both furin and membrane-type 1 (MT1)-MMP
(MMP-14) in the activation of MMP-2. The furin inhibitor decanoyl-
Arg-Val-Lys-Arg-chloromethylketone did not prevent MMP-2 acti-
vation after balloon angioplasty. In contrast, balloon angioplasty
induced a significant increase in the levels of MT1-MMP, which was
suppressed by LY-294002. No change in MT1-MMP mRNA was
observed with LY-294002, because equivalent amounts of this mRNA
were present in both injured and noninjured vessels. These results
implicate PI3K-dependent regulation of MT1-MMP protein synthesis
and subsequent activation of latent MMP-2 as critical events in
neointimal hyperplasia after vascular injury.
matrix metalloproteinase; LY-294002; wortmannin; furin; restenosis
PHOSPHATIDYLINOSITOL 3-KINASE (PI3K) is a heteromeric protein
consisting of an 85-kDa (p85) regulatory subunit and a 110-
kDa (p110) catalytic subunit. PI3K functions as a lipid kinase
and phosphorylates phosphoinositides on the 3? position of the
inositol ring. The biological functions of PI3K can be grouped
into four distinct categories: mitogenic signaling, inhibition of
apoptosis, cell adherence and motility, and intracellular vesicle
trafficking (5). A role in cell motility and cell adherence was
indicated by evidence showing PDGF-dependent membrane
ruffling and chemotaxis requires an interaction between PI3K
and the PDGF receptor (25, 55). In addition, PI3K is involved
in microtubule reassembly in response to both insulin and
PDGF (21) and actin rearrangement by PDGF (59). The
involvement of PI3K in growth factor regulation of integrins
and cell adherence has also been established (16, 23). In
particular, PI3K has been shown to associate with focal adhe-
sion kinase (FAK) as well as participate in PDGF-mediated
phosphorylation of both FAK and paxillin (40). A recent study
(41) has also demonstrated that PI3K promotes cell migration
on fibronectin by facilitating the binding of FAK to Src and
p130Cas. In contrast, integrin-mediated migration of macro-
phages involves a PI3K-dependent, but FAK-independent,
PI3K is activated in vascular smooth muscle cells (SMCs) in
response to angiotensin II (ANG II) (44). Furthermore, inhibi-
tion of PI3K activity with LY-294002 or wortmannin prevents
the stimulation of DNA synthesis by ANG II, thus implicating
PI3K in the proliferation of SMCs. Interestingly, a similar
relationship between SMC migration and PI3K activation has
also been reported (56). However, it is recognized that cell
proliferation and migration are independent processes that are
controlled by distinct mechanisms (4). In fact, it is now evident
that matrix metalloproteinases (MMPs) have a major role in
SMC migration (22) and that their contribution is likely inde-
pendent of cell proliferation (38, 67).
The ability to regulate MMP activity is essential for the
maintenance of normal vascular architecture, and three levels
of control have been identified. MMP expression is transcrip-
tionally regulated by NF-?B (3, 10). MMPs are also produced
as zymogens, and activation requires proteolytic cleavage of
the proenzyme form. As well, secreted MMPs are found
complexed with tissue inhibitors of MMPs (TIMPs), which
suppress proteolytic activity. The mechanisms responsible for
MMP activation have not been identified in all cases; however,
membrane-type 1 (MT1)-MMP is one factor that has been
shown to cleave pro-MMP-2 to its active form (46). Interest-
ingly, expression of MT1-MMP, which is anchored on the cell
surface through a transmembrane domain, is also modulated by
tissue damage (39, 48). Indeed, a correlation between MT1-
MMP expression and neotintimal hyperplasia suggests this
protein may be involved in the vascular response to injury (34).
Although no connection between MMPs and PI3K has been
reported in SMCs, recent studies with tumour and endothelial
cells support the existence of such a link (9, 20). Furthermore,
these reports suggest that PI3K contributes to both MMP
activation and expression. We therefore investigated whether
PI3K modulates MMP-dependent migration by vascular
SMCs. A coronary artery organ culture model was also em-
ployed to examine the correlation between PI3K-dependent
Address for reprint requests and other correspondence: P. Zahradka, Insti-
tute of Cardiovascular Sciences, St. Boniface General Hospital Research
Centre, Winnipeg, Manitoba, Canada R2H 2A6 (E-mail: peterz@sbrc.
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.
Am J Physiol Heart Circ Physiol 287: H2861–H2870, 2004.
First published August 5, 2004; doi:10.1152/ajpheart.00230.2004.
0363-6135/04 $5.00 Copyright © 2004 the American Physiological Society http://www.ajpheart.orgH2861
MMP activation and the development of a neointimal lesion in
response to balloon angioplasty. Our study provides insight
into the role of PI3K in SMC migration and identifies a
mechanism by which PI3K may control MMP activation.
MATERIALS AND METHODS
SMC culture. Primary cultures of porcine coronary artery SMCs
were generated from the left anterior descending coronary artery by an
explant organ culture method (45). To obtain a quiescent cell popu-
lation, SMCs were grown to 70% confluence and then placed into
serum-free DMEM supplemented with 11 ?g/ml pyruvate, 5 ?g/ml
transferrin, 10?9M selenium, 2 ? 10?4M ascorbate, and 10?8M
insulin for 5 days. To maintain consistency between cultures, only
second passage cells with confirmed expression of smooth muscle
markers (?95% of cells staining for myosin) were used for all
Coronary artery organ culture. The left anterior descending coro-
nary artery of pig hearts obtained from the local abattoir was exposed
and flushed with PBS, and an angioplasty catheter (3.5 ? 20 mm) was
inserted into the vessel distal to the first major bifurcation. The
catheter was inflated to 5 atmospheres for 1 min. The vessel was
dissected free, and the damaged region of the vessel was cut into four
equal 5-mm segments. Control vessels were treated in an identical
manner with the exception of catheter insertion and inflation. One
vessel segment was randomly placed per well of a 24-well culture dish
containing 20% FBS in DMEM. Media, including treatments, were
changed every second day. Vessels harvested from culture were
placed into OCT embedding media and stored at ?70°C. A detailed
characterization of this model has been published elsewhere (58).
Boyden chamber migration. Proliferating SMCs were incubated in
DMEM (no FBS) for 48 h in a Boyden chamber, with chemoattractant
(10?6M ANG II) added to the lower compartment and antagonists
added to the upper compartment. Cells migrating to the underside of
the membrane separating these compartments were stained with
Giemsa and counted as described previously (63).
Immunohistochemistry. SMCs grown on Superfrost Plus glass
slides (Fisher Scientific) were fixed with 4% paraformaldehyde and
permeabilized with 0.1% Triton X-100 before antibody treatment. The
slides were blocked for 60 min at room temperature, rinsed with
Tris-buffered saline (TBS), and then incubated with MT1-MMP
antibody (MAb 3317, Chemicon, diluted 1:100 in 1% BSA-TBS-0.1%
Triton X-100) for 60 min at room temperature (44). The primary
antibody was detected with Cy3-coupled secondary antibody (diluted
1:200 with 1% BSA-TBS-Triton X-100). Nuclei were visualized with
Hoescht 33342 (0.5 mg/ml diluted 1:4,000 in TBS). Digital images
were captured with a DAGE-MTI charge-coupled device camera.
Western blot analysis. Western blotting of cellular proteins sepa-
rated by SDS-PAGE in a 7.5% gel and transferred to polyvinylidene
difluoride membrane was conducted as previously described (62).
Horseradish peroxidase-conjugated secondary antibody (1:10,000 di-
luted) was detected using the ECL chemiluminescent system (Amer-
sham). Band intensity was quantified by scanning densitometry.
Antibodies employed include MMP-2 (latent and active, Oncogene,
diluted 1:1,000), MMP-9 (latent and active, NeoMarkers, diluted
1:2,000), and MAb 3317 (diluted 1:1,000).
Gelatin zymography. Culture medium was diluted 1:1 in SDS
sample buffer [0.5 M Tris?HCl (pH 6.8), 2.0% SDS, 10% glycerol,
and 0.001% bromophenol blue] without reducing agent, and 7 ?l were
loaded per well of a 7.5% polyacrylamide gel containing 0.1% gelatin.
Gels were washed in glycine-Triton buffer [0.025 M glycine (pH 8.3)
and 2.5% Triton X-100] twice for 10 min at 4°C to remove the SDS
and permit partial renaturation of the protein. Gels were incubated in
buffer [0.05 M Tris?HCl (pH 8.0), 5 mM CaC12, and 0.1 M PMSF]
at 37°C for 12 h and then stained with Coomassie blue R-250. Protein
loading and incubation time were adjusted to ensure lytic activity
was in the linear range (10–200 pg). Gels were subsequently
scanned with an imaging densitometer, with transmittance corre-
lating to activity (57).
RT-PCR amplification. Coronary artery segments were flash frozen
in liquid nitrogen and pulverized with a mortar and pestle. Total RNA
was isolated from the powder with TRIzol. RNA was resuspended in
RNase-free water, and concentration was determined by spectropho-
tometic absorbance at 260 nm. Reverse transcription of 1 ?g RNA
was conducted (after removal of possible genomic DNA contamina-
tion with DNase I) according to the protocol (25 cycles of amplifica-
tion and 62°C annealing temperature) recommended for the Access
RT-PCR System (Promega). The specific forward and reverse oli-
godeoxynucleotide primers employed were as follows: MT1-MMP
(sense) 5?-AAGGCCAATGTTCGAAGGAA-3? and (antisense) 5?-
AAGAAGATCATGATGTCCT-3?; and L32 ribosomal protein (rP-
L32) (sense) 5?-TAAGCGAAACTGGCGGAAAC-3? and (antisense)
5?-GCTGCTCTTTCTACGATGGCTT-3?. Oligonucleotide primers
for MT1-MMP were designed with Primer3 (42) using a sequence
reported for Sus scrofa (GI:5051631). Amplification products were
analyzed by electrophoresis in 2% agarose gels, and ethidium bro-
mide-visualized bands were captured on Polaroid 667 black and white
instant film. Control reactions (without RNA, without RT, and without
primers) were used to demonstrate the specificity of the PCR.
Data analysis. Morphometric data, cell number, and band intensity
for both autoradiographic and zymography experiments were quanti-
fied and graphically represented as means ? SE. In most experiments,
the sample size was three to six; however, six to eight vessel segments
were used per treatment group in organ culture. All experiments were
replicated at least three times, with each replicate employing inde-
pendent cell or vessel isolations. Treatment means were compared
using ANOVA, whereas all other data were analyzed using the
unpaired Student’s t-test. Significance was set to P ? 0.05 in all cases.
Quantification of data obtained on film or autoradiograms was accom-
plished with a Bio-Rad model 670 Imaging Densitometer under
nonsaturating conditions. Background subtraction was achieved by
reading the absorbance of an equal-sized region directly adjacent
(above, below, or beside) to the band. Although multiple exposures
were acquired to ensure the absence of film saturation, the longest
exposures were typically selected for visual presentation and not used
for data analysis.
PI3K is required for SMC migration. ANG II is both a
mitogen and a chemoattractant for SMCs (44, 66). Although it
was previously established that PI3K is required for SMC
proliferation in response to ANG II (44), its role in SMC
migration was not examined. Migration of cells was measured
with a Boyden chamber. Basal migration over 48 h in the
absence of ANg II was set to 100% (Fig. 1A). ANG II
increased cell migration by 142%, a statistically significantly
change that was somewhat less than the 205% increase ob-
tained with serum. The addition of LY-294002 (10 ?M)
reduced migration to basal levels (Fig. lA). A requirement for
PI3K activation was confirmed by the concentration-dependent
decrease in migration obtained with both LY-294002 (Fig. 1B)
and wortmannin (Fig. 1C). EC50 values of 3.0 and 0.25 ?M
were calculated for LY-294002 and wortmannin, respectively.
Inhibition of PI3K prevents neointimal formation. The abil-
ity to block both SMC migration and proliferation suggests that
LY-294002 may be able to prevent neointimal formation (also
termed neointimal hyperplasia or restenosis) in response to a
vascular injury such as balloon angioplasty. We employed an
organ culture model of neointimal hyperplasia (58) in which
the left descending coronary artery of an isolated porcine heart
is subjected to balloon angioplasty in situ, and segments of the
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