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INTRODUCTION |
Closure of cutaneous wounds involves three processes:
epithelialization, connective tissue deposition, and contraction. Wound contraction, which brings the margins of open wounds together (1, 2),
is believed to be mediated by specialized fibroblasts called
myofibroblasts because of their content of actin stress fibers and
-smooth muscle actin (3). The myofibroblast phenotype can occur
early or late during the wound contraction process depending on the
mechanical resistance of surrounding tissue (4). Myofibroblasts have
also been implicated in the pathology of wound contractures and
fibrotic disease (5, 6).
Using several different culture models, we and others have studied the
ability of fibroblasts to reorganize and contract collagen matrices
in vitro. In the "floating" model (7), a freshly
polymerized collagen matrix containing fibroblasts is released from the
culture dish and allowed to float in culture medium, and contraction
occurs in the absence of external mechanical load and without
appearance of actin stress fibers in the cells (8). In the
"attached" model, a polymerized collagen matrix containing
fibroblasts remains attached to the culture dish during contraction. In
this case, mechanical load (i.e. isometric tension) develops
during contraction, and cellular stress fibers assemble (9-11).
Finally, the two-step "stressed" model combines an initial period
of attached matrix contraction leading to mechanical loading, followed
by release of the matrices, resulting in mechanical unloading and
further contraction as mechanical stress dissipates (i.e.
stress-relaxation) (12).
Contraction of collagen matrices depends on cell binding to collagen
through
2
1 integrins (13-15) and
requires stimulation by serum factors (16-18). Otherwise, the signal
transduction mechanisms that regulate contraction are poorly
understood. Moreover, previous studies have assumed for the most part
interchangeability between different growth factors used to stimulate
contraction and different model systems used to measure contraction.
Platelet-derived growth factor was identified as the putative factor in
serum that stimulates contraction of floating matrices (13, 19). In a
previous study (20), we found that
PDGF1 attenuated the ability
of sodium vanadate to inhibit serum-dependent contraction
of floating matrices, but enhanced the ability of vanadate to inhibit
contraction of mechanically stressed matrices. These opposing responses
to PDGF raised the possibility that different signaling pathways
regulate contraction in floating and stressed matrices.
In this study, we examined the above possibility by comparing floating
and stressed matrix contraction stimulated by platelet-derived growth
factor and lysophosphatidic acid (21, 22). The results of our studies
suggest that collagen matrix contraction is determined by complex
interrelationships between different growth factors, signal
transduction mechanisms, and the mechanical state of the cells,
i.e. mechanically loaded or unloaded. Details are reported herein.
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EXPERIMENTAL PROCEDURES |
Materials--
PDGF (BB isotype) was obtained from Upstate
Biotechnology, Inc. (Lake Placid, NY). Bovine serum albumin (fatty
acid-free), collagenase type I, soybean trypsin inhibitor, and
lysophosphatidic acid were obtained from Sigma. Pertussis toxin was
obtained from Calbiochem. C3 exotransferase was obtained from List
Biological Laboratories Inc. (Campbell, CA). KT5926 was obtained from
BIOMOL Research Labs Inc. (Plymouth Meeting, PA). Dulbecco's modified Eagle's medium (DMEM), trypsin/EDTA solution, and Lipofectin were obtained from Life Technologies, Inc. Fetal bovine serum (FBS) was
obtained from Intergen Co. (Purchase, NY). Vitrogen 100 collagen was
obtained from Collagen Corp. (Palo Alto, CA).
Collagen Matrix Contraction--
Fibroblasts from human foreskin
specimens (<10 passages) were maintained in Falcon 75-cm2
tissue culture flasks in DMEM supplemented with 10% FBS. Fibroblasts were harvested from monolayer culture with 0.25% trypsin and 1 mM EDTA. Trypsin was neutralized with soybean trypsin
inhibitor (3.3 mg/ml) or 10% serum-containing medium. To harvest
fibroblasts from collagen matrices (see below), the matrices were
incubated at 37 °C for 10 min with 0.05% trypsin and 0.53 mM EDTA (200 µl/matrix), followed by treatment in a
shaking incubator for 20 min with 5 mg/ml collagenase (250 µl/matrix)
and then 10 mg/ml soybean trypsin inhibitor (100 µl).
The procedure for studying contraction of floating and stressed
collagen matrices has been described previously (20, 23) and is
outlined in Fig. 1. Hydrated collagen
matrices were prepared from Vitrogen 100 collagen. Neutralized collagen
solutions (1.5 mg/ml) contained fibroblasts in DMEM without serum.
Aliquots (0.2 ml, 2 × 105 cells) of the cell/collagen
mixtures were prewarmed to 37 °C for 3-4 min and then placed in
Corning 24-well culture plates. Each aliquot occupied an area outlined
by an 11-mm diameter circular score within a well. Polymerization of
collagen matrices required 60 min at 37 °C. Subsequently, the
polymerized matrices were used immediately or cultured with 1.0 ml of
DMEM and 10% FBS and 50 µg/ml ascorbic acid for 1-2 days to allow
the cells to develop mechanical stress.
To initiate matrix contraction, freshly polymerized or mechanically
stressed matrices were gently released from the underlying culture dish
with a spatula into ~0.5 ml of serum-free DMEM containing 5 mg/ml
bovine serum albumin and growth factors as indicated, after which the
matrices were incubated at 37 °C. For convenience in carrying out
the experimental protocols, growth factors usually were added to
floating collagen matrices immediately after release and to stressed
collagen matrices immediately before release. Reversing the procedure
did not change the results, however.
To determine the extent of floating or stressed matrix contraction,
samples were fixed with 3% paraformaldehyde in phosphate-buffered saline (150 mM NaCl, 3 mM KCl, 1 mM
KH2PO4, and 6 mM
Na2HPO4, pH 7.2) for 10 min at 22 °C. The
matrices were washed, placed on a flat surface, and measured with a
ruler. For quantitative purposes, contraction data are presented as the
change in diameter (starting
final) measured in millimeters.
All experiments were carried out in duplicate, and every experiment was
repeated two or more times. Data points and error bars in the figures
represent averages and S.D. Where error bars cannot be seen, the data
points overlapped.
Loading with Pertussis Toxin (PTx) and C3 Exotransferase--
To
load fibroblasts in monolayer culture or mechanically stressed collagen
matrices with pertussis toxin, the cells were incubated overnight in
DMEM and 10% FBS containing PTx at the concentrations indicated. To
load fibroblasts in monolayer cultures with C3 exotransferase (C3),
Lipofectin was used as a delivery system (24). Lipofectin/C3 was
prepared in 120 µl of antibiotic/antimycotic-free DMEM and diluted
with additional antibiotic/antimycotic-free DMEM after 1 h at
22 °C to give a final concentration of 10 µg/ml Lipofectin and C3
as indicated. Subsequently, the cells were incubated with the
Lipofectin/C3 mixture or Lipofectin prepared identically except without
C3 for 30 min at 37 °C. Following treatment with Lipofectin/C3 or
Lipofectin alone, the cells were rinsed and further incubated with DMEM
and 10% FBS for 60 min at 37 °C before harvesting. To load
fibroblasts in collagen matrices with C3 exotransferase, cells in
stressed matrices were rinsed and incubated with DMEM containing C3 as
indicated for 30 min at 37 °C. Subsequently, the matrices were
released, allowing C3 exotransferase to enter through ~4-nm plasma
membrane passages that open for <5 s during basal contraction (23).
Afterward, the cells were incubated for an additional 1 h at
37 °C. An analogous method was used for loading C3 into cells during
embryonic wounding (25).
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RESULTS |
Contraction of Floating Collagen Matrices in Response to LPA and
PDGF and Selective Inhibition of PDGF Stimulation by KT5926--
Fig.
2 shows the typical appearance of
floating collagen matrices after contraction for 4 h
(FMC). The diameter of the matrices was 10.5 mm at the time
contraction was initiated. In DMEM alone, little contraction of the
matrix occurred, but the matrix diameter decreased markedly when
contraction was carried out in the presence of 100 ng/ml PDGF or 10 µM LPA. Fig. 3 shows a
typical time course of matrix contraction in the presence of LPA or
PDGF. The rates of contraction were relatively linear over 4 h,
although there was some variation from experiment to experiment whether
PDGF or LPA was the better stimulator of contraction.

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Fig. 2.
Appearance of floating and stressed matrices
after contraction. Contraction of floating (FMC) and
stressed (SMC) matrices was carried out for the times shown
with PDGF or LPA added to the medium as indicated.
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Fig. 3.
Time course of contraction of floating
matrices. Contraction of floating matrices was carried out for the
time periods shown with PDGF (100 ng/ml) or LPA (10 µM)
added as indicated.
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Mixing experiments with LPA and PDGF were carried out, and typical
results are shown in Fig. 4. We
consistently observed faster contraction with LPA and PDGF combined
than with saturating concentrations of either growth factor alone,
suggesting that complementary signaling mechanisms could activate
contraction. One way to distinguish these mechanisms was with the
protein kinase inhibitor KT5926. As shown in Fig.
5, KT5926 completely blocked
PDGF-stimulated contraction of floating matrices, but had little effect
on LPA-stimulated contraction. Another protein kinase inhibitor,
staurosporine, also selectively blocked PDGF-stimulated contraction,
but appeared to do so by interfering with PDGF receptor phosphorylation
(26). KT5926, on the other hand, had no effect on PDGF receptor
phosphorylation (data not shown).

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Fig. 4.
Contraction of floating matrices stimulated
by PDGF and LPA. Contraction of floating matrices was carried out
for the times shown with PDGF and LPA added at the concentrations
indicated.
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Fig. 5.
Effect of KT5926 on contraction of floating
matrices stimulated by PDGF and LPA. Contraction of floating
matrices was carried out for 4 h in the presence of PDGF (100 ng/ml) or LPA (10 µM) with KT5926 added at the
concentrations indicated 10 min prior to adding the growth
factors.
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Differences in the Time Course of Contraction of Stressed Collagen
Matrices in Response to LPA and PDGF--
Fig. 2 also shows the
typical appearance of stressed collagen matrices undergoing contraction
(SMC). In this case, a substantial decrease in matrix
diameter occurred even in DMEM without serum or growth factors
(i.e. basal contraction). During the first hour, the extent
of contraction was stimulated further by the addition of LPA, but not
by PDGF. Fig. 6 shows the time course for
contraction of stressed matrices. The basal component of contraction
(no growth factor) was complete within 5 min. LPA-stimulated
contraction increased rapidly up to 1 h. On the other hand,
stimulation of contraction by PDGF was undetectable until after an
~1-h lag period. The lag period in PDGF stimulation of stressed
matrices was observed at PDGF concentrations ranging from 12.5 to 200 ng/ml, whereas PDGF at 50 ng/ml was sufficient to maximally stimulate
contraction of floating matrices (data not shown).

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Fig. 6.
Time course of contraction of stressed
matrices. Contraction of stressed matrices was carried out for the
time periods shown with PDGF (100 ng/ml) or LPA (10 µM)
added as indicated.
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The faster rate of LPA-stimulated contraction of stressed collagen
matrices compared with floating matrices might have occurred because
cells in stressed matrices have actin stress fibers (12, 17). To
examine this possibility, the matrices were released in DMEM alone,
allowing stress fibers to disassemble in the absence of growth
factor-stimulated contraction (23), and then LPA was added. LPA
stimulated rapid contraction of stressed matrices even if added 2 h after release (data not shown), suggesting that the presence of the
stress fibers at the time of LPA addition was unnecessary for rapid contraction.
LPA Stimulates Contraction of Floating and Stressed Collagen
Matrices by Pertussis Toxin-sensitive and -insensitive Pathways,
Respectively--
Besides the differences in rate of contraction,
floating and stressed collagen matrices were observed to respond to
markedly different concentrations of LPA. The typical dose-response
curve in Fig. 7 shows that 100 nM LPA was sufficient to maximally stimulate contraction of
floating collagen matrices, but had little effect on stressed matrices.
The latter required 10-100 µM LPA for maximal contraction.

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Fig. 7.
Contraction of floating and stressed matrices
as a function of LPA concentration. Contraction of floating
(FMC) and stressed (SMC) matrices was carried out
for the times shown with LPA added at the concentrations
indicated.
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The difference in the LPA concentration dependence and rate of
contraction of floating versus stressed collagen matrices
raised the possibility that different LPA receptors or receptor-linked signaling pathways were involved. LPA interacts with receptors linked
to pertussis toxin-sensitive (Gi) and -insensitive
(Gq and G12/13) heterotrimeric G-proteins (27).
Therefore, experiments were carried out to determine the effects of
pertussis toxin on fibroblast contraction of collagen matrices.
Fig. 8 shows that preloading fibroblasts
with pertussis toxin almost completely blocked the ability of LPA to
stimulate contraction of floating matrices. Under the same conditions,
PDGF-stimulated contraction was only slightly inhibited. In marked
contrast, as shown by the example in Fig.
9, preloading cells with pertussis toxin
did not inhibit LPA-stimulated contraction of stressed matrices (SMC). Even when the cells were incubated with 100 ng/ml
PTx, there was little effect on contraction of stressed matrices (data not shown). Fig. 9 also shows that when fibroblasts were harvested from
stressed collagen matrices and retested for contraction in floating
matrices (FMC), contraction of PTx-treated cells was inhibited ~50% compared with controls. These results suggest that LPA-stimulated contraction of floating matrices depends on LPA receptors linked to pertussis toxin-sensitive heterotrimeric
G-proteins, whereas LPA-stimulated contraction of stressed matrices
depends on receptors linked to pertussis toxin-insensitive
heterotrimeric G-proteins.

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Fig. 8.
Effect of pertussis toxin on contraction of
floating matrices stimulated by PDGF and LPA. Fibroblasts in
monolayer culture were incubated overnight with 25 ng/ml PTx
(PT) as indicated. Subsequently, the cells were harvested
and tested for contraction of floating matrices for 4 h in the
presence of PDGF (100 ng/ml) or LPA (10 µM) as
shown.
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Fig. 9.
Effect of pertussis toxin on LPA-stimulated
contraction of stressed and floating matrices. Fibroblasts in
stressed matrices were incubated overnight with 25 ng/ml PTx
(PT) as indicated. Subsequently, contraction of stressed
matrices (SMC) was tested with LPA (10 µM)
added as indicated for 1 h. Alternatively, cells were harvested
from the stressed matrices and then tested for contraction of floating
matrices (FMC) for 4 h with LPA (10 µM)
added as indicated.
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The Rho Inhibitor C3 Exotransferase Blocks Contraction of Floating
or Stressed Collagen Matrices--
LPA has been identified as the
principal factor in serum that promotes assembly of stress fibers and
focal adhesions (28) through a contractile process that depends on the
small G-protein Rho (29, 30). This effect of LPA is pertussis
toxin-insensitive (31) and probably involves LPA receptors linked to
the heterotrimeric G-protein G13 (32). Experiments were
therefore carried out to test whether collagen matrix contraction was
affected by the toxin C3 exotransferase, a specific inhibitor of Rho
activity (33).
Fig. 10 shows that releasing stressed
matrices in the presence of 20 µg/ml C3 exotransferase blocked
subsequent LPA stimulation of contraction. As would be predicted (23),
C3 had no effect if it was added after the matrices were released (data
not shown). In control experiments, C3-loaded cells were harvested from
collagen matrices and tested for cell spreading on fibronectin-coated
substrata after 2 and 24 h. After 2 h, C3-loaded cells spread
in an arborized morphology on fibronectin-coated substrata (31, 34),
but the cells attained normal morphology within 24 h (data not
shown).

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Fig. 10.
Effect of C3 exotransferase on LPA-simulated
contraction of stressed matrices. Fibroblasts in stressed matrices
were incubated for 30 min with antibiotic/antimycotic-free DMEM
containing 20 µg/ml C3 as indicated. Subsequently, matrices were
released in the same medium and incubated for an additional 1 h
(No Growth Factor; basal contraction).
Then, LPA (10 µM) was added as shown, and the extent of
contraction of stressed matrices was measured after an additional
1 h.
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To study the effect of C3 exotransferase on contraction of floating
collagen matrices, C3 was loaded into fibroblasts in monolayer culture
using Lipofectin. Fibroblasts treated with Lipofectin/C3, but not
Lipofectin or C3 alone, showed arborized spreading morphology on
fibronectin-coated substrata (data not shown). Fig.
11 presents a typical experiment
showing that cells treated with Lipofectin/C3 were unable to contract
floating collagen matrices regardless of whether LPA or PDGF was used
to stimulate contraction, whereas cells treated with Lipofectin or C3
alone were unaffected. Therefore, the small G-protein Rho appeared to
be required not only for contraction of stressed collagen matrices, but
also for contraction of floating collagen matrices.

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Fig. 11.
Effect of C3 exotransferase on contraction
of floating collagen matrices stimulated by PDGF and LPA.
Fibroblasts in monolayer culture were incubated for 30 min at 37 °C
with Lipofectin alone (LF; 10 µg/ml) or Lipofectin (10 µg/ml) mixed with C3 (2 µg/ml) as indicated. Subsequently, the
cells were tested for contraction of floating matrices for 4 h
with PDGF (100 ng/ml) or LPA (10 µM) added as
indicated.
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DISCUSSION |
The signal transduction pathways that regulate contraction of
collagen matrices by fibroblasts are poorly understood. To learn more
about these pathways, we compared the ability of LPA and PDGF to
stimulate contraction of floating and stressed collagen matrices. The
results suggest that contraction of floating and stressed collagen
matrices depends on different signaling mechanisms.
PDGF and LPA appeared to stimulate contraction of floating collagen
matrices by complementary signaling pathways since contraction obtained
by the combination of growth factors exceeded that observed with
saturating concentrations of either alone. One way to distinguish these
pathways was with the protein kinase inhibitor KT5926. Staurosporine also selectively blocked PDGF-stimulated contraction, but appeared to
do so by interfering with PDGF receptor phosphorylation (26). Previous
studies demonstrated that serum-stimulated contraction could be blocked
by inhibitors of protein kinase C (35, 36), protein-tyrosine kinase
(36-38), phospholipase C (39), and phosphatidylinositol 3-kinase (38).
None of the other inhibitors used previously, however, was found to
preferentially inhibit PDGF- versus LPA-stimulated contraction.
Although we have not determined the precise site at which KT5926
inhibits PDGF-stimulated contraction, it is worth noting other studies
suggesting that KT5926 selectively inhibits myosin light chain (MLC)
kinase (40), and phosphorylation of MLC has been implicated in matrix
contraction, albeit indirectly (21, 41). Therefore, preferential
inhibition of PDGF-stimulated contraction by KT5926 may reflect a
requirement for MLC kinase in MLC phosphorylation during PDGF- but not
LPA-stimulated contraction. Consistent with this possibility, LPA can
cause MLC phosphorylation without MLC kinase through the activity of
Rho kinase (42, 43). We cannot exclude the possibility, however, that
preferential inhibition of PDGF-stimulated contraction by KT5926 occurs
at the level of some other protein kinase, and future studies will be
required to analyze this point.
Surprisingly, LPA-stimulated contraction of floating matrices was
pertussis toxin-sensitive, whereas LPA-stimulated contraction of
stressed matrices was pertussis toxin-insensitive. One interpretation of this result is that LPA stimulates contraction of fibroblasts in
floating matrices through receptors coupled to Gi and
stimulates contraction of cells in stressed matrices through receptors
coupled to G13 (although Gq cannot be ruled out
at present).
LPA receptors coupled to different heterotrimeric G-proteins have been
implicated in different aspects of cell motility. LPA stimulation of
fibroblast migration and chemotaxis was shown to depend on LPA
receptors coupled to Gi (44, 45). On the other hand, LPA
stimulation of stress fiber formation and focal adhesion assembly
requires activation of the small G-protein Rho (29, 30), a pertussis
toxin-insensitive process (31) in which LPA receptors are coupled to
G13 (32). The latter pathway has also been implicated in
neurite rounding and retraction (46).
Contraction of floating and stressed collagen matrices may also reflect
different aspects of cell motility. Fibroblasts in floating collagen
matrices are round to begin with and spread during contraction (Ref. 8;
see also Ref. 11), whereas cells in stressed matrices begin in a spread
morphology with prominent stress fibers and withdraw their extensions
during contraction (12, 17, 47). In the former case, contraction
probably depends on tractional forces that accompany the protrusion of
cell extensions (48). In the latter case, the stress fibers themselves
can contract once there is no longer a rigid substratum to maintain
isometric tension (49). Given this interpretation, it is not surprising that the Rho inhibitor C3 exotransferase blocks contraction of both
floating and stressed collagen matrices since inhibition of Rho with C3
exotransferase can prevent normal spreading of freshly plated cells
(34) as well as actin filament assembly and focal adhesion formation in
already spread cells (50).
The observation of different regulatory mechanisms for contraction of
stressed and floating collagen matrices opens the possibility for
future studies to characterize in more detail the signaling mechanisms
involved and the interrelationships between different growth factors,
signal transduction mechanisms, and the mechanical state of the cells,
i.e. mechanically loaded or unloaded. In addition, our
results have potentially important clinical implications. In the
absence of complications, the process of wound contraction leads to
wound closure with little scarring or loss of function. In large wounds
and some pathological situations such as hypertrophic scars, however,
the consequences of contraction can result in loss of joint motion or
major body deformations referred to as contractures (51-53).
Contraction of floating collagen matrices resembles more closely the
initial phase of wound contraction dependent on cell motility (8, 54),
whereas the myofibroblast-like cells in mechanically stressed matrices
are more typical of the late phase of wound contraction and
contractures (3, 4). Consequently, wound contraction and wound
contracture may be under the control of different growth factors and
signal transduction regulatory mechanisms in vivo and
therefore subject to different pharmacological means of clinical intervention.