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Division of Endocrinology, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7170
Address all correspondence and requests for reprints to: David R. Clemmons Division of Endocrinology, University of North Carolina, Chapel Hill, North Carolina 27599-7170. E-mail: dpm{at}med.unc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Vß3
integrin toward ligands and that occupancy of this integrin is
indispensable for IGF-I to stimulate cell migration. In this study, the
role of phosphatidylinositol 3-kinase (PI 3-kinase) and
mitogen-activated protein kinase (MAPK) pathways in IGF-I induced cell
motility and integrin activation was studied using porcine aortic
smooth muscle cells (pSMC). Two structurally different inhibitors of PI
3-kinase decreased IGF-I-stimulated pSMC migration in a dose-dependent
manner. The IC50 of wortmannin for inhibiting migration was
10 nM, and that of LY294002 was 0.3 µM. These
inhibitors also suppressed IGF-I-induced phosphorylation of protein
kinase B PKB/Akt at Ser437 using concentrations that also
inhibited cell motility. PD98059, an inhibitor of the MAPK pathway, was
somewhat less potent than PI 3-kinase inhibitors in blocking cell
migration that had been stimulated by IGF-I. When IGF-I increased
migration of pSMC 2.1-fold above control, 100 nM wortmannin
inhibited this response by 79%, 1 µM LY294002 inhibited
it by 58%, and 50 µM PD98059 caused a 34% reduction. In
comparison, 100 nM wortmannin inhibited IGF-I stimulated
DNA synthesis by 57%, 1 µM LY294002 inhibited it by
59%, whereas 50 µM PD98059 suppressed it completely.
Thus, activation of PI 3-kinase plays the major role in
IGF-I-stimulated migration and proliferation of pSMC. While the
activation of the MAPK pathway seems to be necessary for stimulation of
mitogenesis by IGF-I, the contribution of this pathway in IGF-I-induced
cell migration is limited in pSMC. Interestingly, neither PI 3-kinase
inhibitors nor PD98059 blocked the increase in Vß3 integrin
affinity that followed IGF-I treatment. Therefore, although both the PI
3-kinase and MAPK pathways were used by IGF-I to increase migration of
pSMC, Vß3 integrin activation did not depend on either PI 3-kinase
or MAPK activation, suggesting the possible importance of some other
signal transduction pathway to account for its full actions on pSMC. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Vß3 integrin toward ligands,
and specific inhibitors of ligand occupancy of the Vß3 integrin
block IGF-I-stimulated SMC migration (7). IGF-I elicits its actions on cells by binding to the insulin-like growth factor receptor and activating its intrinsic receptor tyrosine kinase (8). Tyrosine kinase activity of the receptor is indispensable for IGF-I actions on the target cells (9). When activated, the IGF-I receptor phosphorylates docking proteins, including the insulin receptor substrates (IRS 1–4), Shc, and Crk, and these molecules activate downstream signaling proteins that lead to biological responses, such as cell migration and proliferation (8, 10). Phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein kinase (MAPK) are two key enzymes that are activated by IGF-I, and each of them represents a distinct cascade of activation steps that result in the biological functions of IGF-I. PI 3-kinase can be activated through IRS-1, IRS-2, and Shc, whereas MAPK can be activated through IRS-1 or IRS-2, Shc, and Crk following IGF-I stimulation (8, 10). Both the PI 3-kinase and MAPK kinase pathways have been shown to be involved in many aspects of IGF-I action, including cell proliferation, protection of cells from apoptosis, and cell differentiation (11, 12, 13, 14). Evidence in other cell systems implies that activation of PI-3 kinase and MAPK play roles in cell motility. Expression of a constituitively activated form of PI 3-kinase increases the motility of mammary gland epithelial cells (15). PI 3-kinase is indispensable for platelet-derived growth factor (PDGF) to induce chemotaxis in NIH3T3 cells, TRMP cells, and CHO cells (16). PDGF-induced migration of mesangial cells depends on PI 3-kinase and MAPK activation (17). However, there is only limited information about the roles of PI 3-kinase and MAPK activation in IGF-I-stimulated migration of vascular SMC, and it is not known if either pathway is involved in controlling integrin activation.
The purpose of the present study was to determine the contribution of
the PI 3-kinase and MAPK pathways in IGF-I-stimulated cell migration,
proliferation, and Vß3 integrin activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Wounding assay
PSMC that had been grown to confluence in six-well culture
plates (Falcon no. 3046, Falcon Labware Division of Becton Dickinson and Co., Fairbanks, NJ) were subjected to wounding as
previously described (7). Cell layers were scraped with a single-edged
razor blade and rinsed with serum-free medium. The edges of the wound
were then observed under microscope to select straight and sharp edges.
For each treatment, 6–17 1-mm regions along the edge of the wound were
chosen and marked. Cells were incubated in DMEM containing 0.2% FBS
plus 0.1% DMSO with the addition of various concentrations of
wortmannin, LY294002, or PD98059 for 30 min. IGF-I (0 or 100 ng/ml) was
then added to the cultures. Following a 48 h incubation at 37 C,
the number of cells that migrated across the regions of the wound edge
that had been marked before the treatment was quantified by direct
counting of total cell number. Our previous analysis of the wounded
monolayer by [3H]thymidine autoradiography demonstrated
that the labeling index of pSMC at the wound edge was 7 ± 4% at
the basal level and 18 ± 7% after exposure to IGF-I (7).
Therefore, less than 10% of cells present in the denuded region at the
end of migration assay are considered to result from cell division
rather than cell migration. To measure the effect of the inhibitors on
cell detachment, confluent pSMC in six-well culture plates were also
incubated with DMEM containing 0.2% FBS plus 0.1% DMSO or the
inhibitors in the absence or presence of 100 ng/ml IGF-I for 48 h.
At the end of the incubation, the number of cells that remained
attached to the plate was counted by releasing the cells from the plate
with 0.02% EDTA plus 0.125% trypsin in PBS.
Detection of Akt phosphorylation at Ser473 and p44/p42
MAPK phosphorylation at Thr202 and Tyr204
pSMC were seeded using a density of 1 x
106/well onto six-well culture plates in DMEM with 1% FBS,
incubated for 24 h, and serum-starved in serum-free DMEM for
2 h. The medium was changed to DMEM with 0.1% DMSO plus the
indicated concentration of inhibitors, and the cells were incubated for
30 min at 37 C followed by stimulation with 100 ng/ml IGF-I for 30 min
at 37 C. The cells were solubilized in 100 µl of SDS sample buffer,
sonicated for 15 sec, and boiled for 10 min. Forty microliters of the
resulting lysate were loaded onto an 8% polyacrylamide gel, separated,
and transferred onto a polyvinylidine difluoride (PVDF) membrane, as
described previously (18). The membrane was blocked with 3% nonfat
milk in Tris-buffered saline (TBS), incubated with a 1:500 dilution of
Phospho-Akt (Ser473) antibody or a 1:1000 dilution of
phospho-p44/p42 MAPK (Thr202 and Tyr204)
antibody in TBS containing 3% BSA plus 0.2% Nonidet P40 at 4 C
overnight, washed, and treated with a 1:10,000 dilution of peroxidase
conjugated antirabbit IgG antibody. To determine the protein amount of
Akt or p44/p42 MAPK on the membranes, they were also probed with a
1:1000 dilution of Akt antibody or a 1:1000 dilution of p44/p42 MAPK
antibody.The bands were visualized with enhanced chemiluminescence, as
described previously (19). Densitometric analysis of the bands was
performed by scanning x-ray films with Scan Maker IV from Microtek
(Redondo Beach, CA) and analyzing the band density using NIH Image from
Scion Corp. (Frederick, MD).
[125I]kistrin binding
Kistrin is a small peptide, termed a disintegrin, with a high
affinity for Vß3. [125I]kistrin was prepared by
radiolabeling kistrin with [125I]NaI as described
previously (7). pSMC were grown to subconfluence on 48-well culture
plates. The cultures were washed three times with serum-free DMEM and
preincubated with DMEM containing 0.01% BSA plus 0.1% DMSO in the
presence or absence of inhibitors for 30 min at 37 C. IGF-I (0 or 100
ng/ml) was then added to the incubation medium that contained DMSO and
the inhibitors, and the plates were incubated for an additional 16
h at 37 C. The plates were placed on ice, washed twice with DMEM
containing 20 mM HEPES (pH 7.3), and then incubated with
[125I]kistrin (1 x 106 cpm/ml) in DMEM
containing 0.1% BSA plus 20 mM HEPES (pH 7.3) at 4 C for
5 h. The cell monolayers were washed three times with DMEM
containing 20 mM HEPES (pH 7.3) and solubilized in 0.1%
SDS plus 0.1 N NaOH. The bound radioactivity was determined
by 
counting.
Measurement of [3H]thymidine incorporation into
pSMC
pSMC were plated at a density of 2.5 x
104/cm2 in 96-well tissue culture plates and
grown for 5 days without a medium change. They were rinsed once with
serum-free DMEM and serum starved by incubating with DMEM plus 0.2%
platelet poor plasma (PPP) for 24 h (20). The medium was changed
to DMEM with 0.2% PPP plus 0.1% DMSO that contained varying
concentrations of inhibitors and IGF-I plus 0.5 µCi/well of
[3H]thymidine (specific activity 35 Ci/mmol). The cells
were incubated at 37 C for 24 h, and the amount of
[3H]thymidine incorporated into DNA was determined as
described previously (18).
Statistical analysis
Alternate Welch’s t test and the Mann Whitney test
were used to compare the differences between control and test groups.
P < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). LY294002, a reversible inhibitor of
PI 3-kinase, also suppressed IGF-I-stimulated migration of pSMC in a
dose-dependent manner. While IGF-I increased the cell migration to
228 ± 38% (mean ± SEM, n = 12), addition
of 5 µM LY294002 resulted in 98% inhibition of
IGF-I-stimulated cell migration (Fig. 1b). Therefore, the activation of
PI 3-kinase is required for IGF-I to increase the migration of pSMC
following wounding. The basal migration of the cells treated with 50
nM wortmannin was 158 ± 21% compared with migration
of the cells treated with 0.1% DMSO carrier expressed as 100%
(mean ± SEM, n = 17, NS, Fig. 1a).
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). Therefore, MAPK also participates in
the increase of pSMC migration after IGF-I treatment, but its
contribution is less than that of PI 3-kinase because 50
µM PD98059 was a high enough concentration to give
significant suppression of MAPK activity, as is shown below (Fig. 3). Treatment of cells with 100
nM wortmannin plus 50 µM PD98059 did not
significantly decrease the IGF-I-stimulated cell migration compared
with the effect of 100 nM wortmannin alone (Fig. 2). Thus,
wortmannin and PD98059 do not have an additive effect in inhibiting the
promotion of cell migration by IGF-I.
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PI 3-kinase inhibitors inhibit phosphorylation of Akt after IGF-I
stimulation
The finding that both wortmannin and LY294002 inhibited the effect
of IGF-I on cell migration strongly supports the hypothesis that both
inhibitors work by suppressing PI 3-kinase activity. To further
strengthen this hypothesis, we tested whether similar concentrations of
these inhibitors decreased the phosphorylation of Akt. After a 30-min
preincubation with or without inhibitors, pSMC were stimulated with 100
ng/ml of IGF-I for 30 min, and the extent of serine phosphorylation was
determined by immunoblotting the cell lysate using an antibody that
specifically recognizes Akt that has been phosphorylated at
Ser473. The intensity of immunoreactive bands was
considered to reflect the change in the phosphorylation status of Akt
because the treatment did not change the amount of Akt protein in pSMC
when it was analyzed by immunoblotting of cell lysates with an Akt
antibody that recognizes both phosphorylated and nonphosphorylated
forms of Akt (data not shown). While phosphorylation of Akt was
undetectable in the basal state, IGF-I stimulated phosphorylation of
Akt. 100 nM wortmannin completely inhibited the Akt
phosphorylation, and 1 µM LY294002 significantly
decreased the extent of Akt phosphorylation. On the other hand, 50
µM PD98059 did not affect Akt phosphorylation after IGF-I
stimulation (Fig. 4). Therefore, the
doses of wortmannin and LY294002 that were shown to suppress the cell
migration were sufficient to prevent PI 3-kinase dependent
phosphorylation of Akt after IGF-I stimulation. In contrast, the
concentration of PD98059 that inhibited cell migration did not suppress
PI 3-kinase activity in pSMC.
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). IGF-I stimulation clearly increased phosphorylation of MAPK
(Fig. 3). 50 µM PD98059 significantly reduced
phosphorylation of MAPK after IGF-I stimulation, indicating that it
effectively inhibited activation of MAPKK. Densitometric analysis of
the bands that correspond to p42 MAPK demonstrated that 50
µM PD98059 treatment decreased band intensity by 70%
compared with that obtained with cells treated with IGF-I alone (Fig. 3, lanes 2 and 3). 1 µM LY294002 did not reduce MAPK
phosphorylation after IGF-I treatment, whereas 100 nM
wortmannin seemed to decrease it slightly in repeated experiments.
Density of the immunoreactive band corresponding to p42 MAPK after 100
nM wortmannin treatment was reduced by 40% compared with
that after IGF-I treatment alone (Fig. 3, lanes 2 and 5). Therefore,
the majority of MAPK activation after IGF-I treatment seems to occur
independently of PI 3-kinase activation in pSMC. However, the partial
reduction of MAPK phosphorylation after 100 nM wortmannin
treatment indicates that the activation of PI 3-kinase may play a small
role in the full activation of MAPK pathway upon IGF-I stimulation. The
failure of LY294002 to suppress MAPK activation after IGF-I treatment
may be due to the fact that 1 µM of LY294002 is less
potent than 100 nM wortmannin in inhibiting PI 3-kinase
activity, as was demonstrated by their potency to inhibit Akt
phosphorylation (Fig. 4). Alternatively, it is possible that wortmannin
inhibits enzymes other than PI 3-kinase that activate MAPK pathway
after IGF-I treatment.
Neither wortmannin nor PD98059 inhibited activation of the Vß3
integrin by IGF-I in pSMC
Our previous studies have demonstrated that IGF-I increases
affinity of the Vß3 integrin toward ligands and that ligand
occupancy of the Vß3 integrin is required for IGF-I to stimulate
migration of pSMC (7). To determine whether the PI 3-kinase or MAPK
pathways are required for Vß3 integrin activation by IGF-I, we
determined the effect of these inhibitors on Vß3 activation. The
binding of [125I]kistrin, which is a specific ligand for
the Vß3 integrin, to pSMC surfaces was measured after cells were
treated with IGF-I and the indicated amounts of inhibitors. 100 ng/ml
IGF-I increased the [125I]kistrin binding by 42 ±
4% (mean ± SEM, n = 3) over control cultures
not exposed to IGF-I. Neither 100 nM wortmannin nor 50
µM PD98059 decreased the [125I]kistrin
binding after IGF-I stimulation significantly (Fig. 5). The treatment of cells with 100
nM wortmannin plus 50 µM PD98059 also did not
decrease [125I]kistrin binding after IGF-I stimulation
(data not shown). Therefore, the inhibition of PI 3-kinase and MAPK
diminished the IGF-I effect on cell migration by mechanisms other than
inhibition of Vß3 integrin activation, and Vß3 integrin
activation after IGF-I stimulation most likely involves pathways other
than PI 3-kinase or MAPK activation.
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, b and c) to 421 ± 62%
(Fig. 6a) (mean ± SEM, n = 3) when the value
without IGF-I was expressed as 100%. Increasing concentrations of
wortmannin inhibited the cellular response to IGF-I without changing
the basal level of [3H]thymidine incorporation
significantly. 1.0 µM wortmannin was required to
completely diminish the effect of IGF-I on DNA synthesis, and 100
nM wortmannin decreased [3H]thymidine
incorporation by 57% (Fig. 6a). 1 µM LY294002 suppressed
the maximum response to IGF-I (20 ng/ml) by 59%, and 10
µM of LY294002 was required to completely inhibit
[3H]thymidine incorporation after IGF-I stimulation (Fig. 6b). On the other hand, 50 µM PD98059, which decreased
IGF-I-stimulated cell migration by only 34%, inhibited
IGF-I-stimulated DNA synthesis completely (Fig. 6c). Therefore,
although MAPK plays a significant role in the cell replication response
to IGF-I, its contribution to IGF-I-stimulated cell migration is not as
important as PI 3-kinase pathway activation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although both the PI 3-kinase and MAPK pathways have been described as
distinctive signal transduction pathways, cross-talk between the two
pathways has been documented. This includes the activation of MAPK by
PI 3-kinase, especially by the PI 3 isoform (26, 27). The analysis
of IGF-I induced Akt phosphorylation in the presence of PD98059
indicated that the suppression of MAPK had little effect on Akt
phosphorylation after IGF-I treatment in our system. The IGF-I induced
MAPK phosphorylation was only partially suppressed by 100
nM wortmannin and was not suppressed at all by 1
µM LY294002. Therefore, the activation of PI 3 and MAPK
pathways seems to occur independently of each other after IGF-I
stimulation in pSMC. The discrepancy between wortmannin and LY294002 in
their effects on MAPK phosphorylation could be due to differences in
their potency, or to suppression of enzymes other than PI 3-kinase by
wortmannin. In either case, PI 3-kinase inhibitors effectively reduced
IGF-I induced cell migration at the doses that have little effect on
MAPK activation, indicating that the secondary activation of MAPK by PI
3-kinase is not the major mechanism responsible for IGF-I-dependent
cell migration in pSMC.
PD98059, an inhibitor of MAPKK, was only partially effective in suppressing IGF-I-stimulated migration of pSMC even at the dose that completely blocked IGF-I-stimulated DNA synthesis in the same cells. Because PD98059 decreased IGF-I-induced cell migration by 34% (P < 0.05), IGF-I apparently utilizes the MAPK pathway in stimulating the migration of pSMC. However, the contribution of the MAPK pathway for this IGF-I action is limited compared with that of PI 3-kinase, which is required for IGF-I to up-regulate cell motility. Several studies have analyzed the contribution of PI 3-kinase and MAPK pathways in the vascular SMC migration that occurs in response to PDGF (1). The results reported to date have placed variable relative importance on each pathway, depending on the source of SMC and on the conditions of the experiments (28, 29, 30, 31). To our knowledge, there has been only one other study that analyzed signal transduction pathways responsible for IGF-I-stimulated migration of vascular SMC. Pukac et al. (31) compared effects of signal transduction inhibitors on the migration of rat vascular SMC after IGF-I, PDGF, and phorbol 12-myristate 13-acetate (PMA) treatment. Our observation that the PI 3-kinase pathway is more involved than the MAPK pathway in IGF-I-stimulated pSMC migration is similar to their conclusion. However, unlike their speculation that the MAPK pathway was not activated after IGF-I treatment in their system, phosphorylation of MAPK was increased after IGF-I stimulation in pSMC, suggesting that, although the MAPK pathway is activated by IGF-I, this plays a minor role in IGF-I stimulation of pSMC migration.
Because occupancy of the Vß3 integrin is required for IGF-I to
increase migration of SMC, we hypothesized that activation of the PI 3
and MAP kinase pathways might be involved in increasing the affinity of
the Vß3 integrin after IGF-I treatment. The role of PI 3-kinase
activation in integrin functions has been reported in several other
systems. Wortmannin effectively inhibited maintenance of the active
state in the platelet specific integrin,
IIbß3, which is highly homologous to
Vß3 (32). In lymphocytes, CD2-induced activation of ß1 integrin,
measured as adhesion to fibronectin, was suppressed by wortmannin (33).
However, neither PI 3-kinase inhibitors nor a MAPKK inhibitor affected
the change in Vß3 integrin affinity upon IGF-I treatment of pSMC.
Although MAPK activation has been suggested to inactivate
constituitively activated integrins in CHO cells (34), the inhibition
of MAPK did not increase Vß3 integrin affinity upon IGF-I
treatment. We conclude that IGF-I probably activates pathways other
than PI 3-kinase and MAPK to induce activation of the Vß3 integrin
in pSMC. The possible roles of pathways other than PI 3-kinase and MAPK
in modulating IGF-I actions have been documented. Expression of mutant
IGF-I receptors with amino acid substitutions for specific tyrosine
phosphorylation sites results in a form of receptor that fails to
elucidate the full actions of IGF-I, even though these forms of the
receptor can activate maximal PI 3-kinase and MAPK responses (35, 36).
The activation of protein kinase C (PKC) is one possible pathway that
is responsible for the change in Vß3 integrin affinity, since it
has been known to be activated by IGF-I and is involved in integrin
function in mast and other cell types (37, 38).
There are several possible mechanisms by which activation of PI 3-kinase and MAPK increase cell migration. Although activation of MAPK played a limited role in stimulation of pSMC migration by IGF-I, it has been shown to play an important role for the increase in cell migration after PDGF treatment and H-Ras activation (17, 39). Phosphorylation of the myosin light chain by MAPK has been documented and may be one of the substrates used by this enzyme to regulate cell motility (40). As for PI 3-kinase, it has been well documented that its activation alters the cytoskeletal organization that changes dynamically when cells increase their motility. In neutrophils, PI 3-kinase is required for the cells to increase cytoskeletal actin and respond to chemoattractants (41). PI 3-kinase has been shown to mediate IGF-I and PDGF-stimulated membrane ruffling and lamellipodia formation, which are considered to be an important component of a set of responses that leads to cell migration (42, 43, 44). Cdc42 and Rac1 activation disrupt actin organization and increase cell motility through activation of PI 3-kinase in mammary epithelial cells (15). It will require further analysis to determine whether PI 3-kinase inhibitors change actin organization in pSMC and if this leads to a block in IGF-I-stimulated migration.
Both PI 3-kinase and MAPK pathways are involved in proliferation and migration of pSMC after IGF-I treatment, with PI 3-kinase bieng more involved in migration and MAPK being more important for proliferation. Considering the well established role of vascular SMC in neointima formation, knowledge about intracellular signaling pathways governing proliferation and migration of these cells may facilitate formulation of effective strategies against atherosclerosis.
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Acknowledgments |
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Footnotes |
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Received December 9, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-V-ß3 integrin is necessary for
smooth muscle cells to migrate in response to insulin-like growth
factor. Proc Natl Acad Sci USA 93:2482–2487 IR-3). J Biol Chem 268:2655–2661. Science 275:394–397This article has been cited by other articles:
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 D. Zhuang, Q. Pu, B. Ceacareanu, Y. Chang, M. Dixit, and A. Hassid Chronic insulin treatment amplifies PDGF-induced motility in differentiated aortic smooth muscle cells by suppressing the expression and function of PTP1B Am J Physiol Heart Circ Physiol, July 1, 2008; 295(1): H163 - H173. [Abstract] [Full Text] [PDF]
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G. M. Risinger Jr., T. S. Hunt, D. L. Updike, E. C. Bullen, and E. W. Howard Matrix Metalloproteinase-2 Expression by Vascular Smooth Muscle Cells Is Mediated by Both Stimulatory and Inhibitory Signals in Response to Growth Factors J. Biol. Chem., September 8, 2006; 281(36): 25915 - 25925. [Abstract] [Full Text] [PDF]
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M. Kwon, Y. Ling, L. A. Maile, J. Badley-Clark, and D. R. Clemmons Recruitment of the Tyrosine Phosphatase Src Homology 2 Domain Tyrosine Phosphatase-2 to the p85 Subunit of Phosphatidylinositol-3 (PI-3) Kinase Is Required for Insulin-Like Growth Factor-I-Dependent PI-3 Kinase Activation in Smooth Muscle Cells Endocrinology, March 1, 2006; 147(3): 1458 - 1465. [Abstract] [Full Text] [PDF]
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Y. Ling, L. A. Maile, J. Badley-Clarke, and D. R. Clemmons DOK1 Mediates SHP-2 Binding to the {alpha}V{beta}3 Integrin and Thereby Regulates Insulin-like Growth Factor I Signaling in Cultured Vascular Smooth Muscle Cells J. Biol. Chem., February 4, 2005; 280(5): 3151 - 3158. [Abstract] [Full Text] [PDF]
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P. Zahradka, G. Harding, B. Litchie, S. Thomas, J. P. Werner, D. P. Wilson, and N. 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, December 1, 2004; 287(6): H2861 - H2870. [Abstract] [Full Text] [PDF]
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, P < 0.05 when IGF-I (100 ng/ml) in the
absence of an inhibitor is compared with IGF-I (100 ng/ml) in the
presence of an inhibitor using the Mann Whitney test.
