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From the Department of Pathology,*
Centre Médical
Universitaire, University of Geneva, Geneva; and the Department
of Surgery,
Geneva University Hospital,
Geneva, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-smooth muscle
actin (-SMA) appeared earlier in splinted than in unsplinted wounds.
Myofibroblast marker expression decreased in control wounds starting at
10 days after wounding as expected, but persisted in splinted
wounds. In the second model, granuloma pouches were induced by
subcutaneous croton oil injection; pouches were either left intact or
released from tension by evacuation of the exudate at 14 days. The
expression of myofibroblast markers was reduced after tension release
in the following sequence: F-actin (2 days), -SMA (3
days), and ED-A fibronectin (5 days); cell density was not
affected. In both models, isometric contraction of tissue
strips was measured after stimulation with smooth muscle agonists.
Contractility correlated always with the level of -SMA
expression, being high when granulation tissue had been
subjected to tension and low when it had been relaxed. Our results
support the assumption that mechanical tension is crucial for
myofibroblast modulation and for the maintenance of their contractile
activity.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-smooth muscle actin (-SMA),5
the actin isoform
typical of vascular smooth muscle cells.6
Incorporation of
-SMA into microfilament bundles or stress fibers has been suggested
to mediate granulation tissue contraction in
vivo,3
and was shown to promote myofibroblast
contraction in vitro.7-9
Stress fibers have
been proposed to function as contractile organelles10,11
and their presence has been correlated with the production of isometric
tension.12
The factors regulating the transition between
fibroblastic and myofibroblastic phenotypes are not fully identified.
Transforming growth factor-ß1 (TGF-ß1) is accepted to be the major
cytokine involved in -SMA expression and myofibroblast
differentiation13,14
through the specific binding to
TGF-ß-receptor type II (TGF-ß-RII).15,16
This TGF-ß
effect depends on the presence of the fibronectin (FN) splice variant
ED-A FN in the extracellular matrix.17
Isometric tension has been shown to be
important in vitro for the development of fibroblast
contractile features such as stress fiber formation18-20
and -SMA expression.21
Mechanical tension alters FN
fiber assembly22
and fibroblast response to growth
factors;23,24
it also prevents fibroblast
apoptosis.19
An altered mechanical load of the matrix
stimulates fibroblasts to adjust their contractile activity to achieve
tensional homeostasis.25
Moreover, isometric tension
modulates signaling mechanisms regulating fibroblast
contraction.26
Most previous studies on the effect of
mechanical tension on fibroblastic cells have been restricted to the
in vitro use of collagen substrates with different
mechanical properties.27
In this work, we have used an
in vivo approach using two different rat models in view of
evaluating: the role of mechanical load in myofibroblast modulation
(-SMA expression in particular) and in granulation tissue
contractility. In the first model, the contraction of full thickness
wound granulation tissue was prevented by splinting with a plastic
frame. In the second model, the wall of rat granuloma
pouches4
was released from mechanical tension by
evacuating the pouch exudate. In both models isometric contraction and
expression of the myofibroblast markers F-actin, ED-A FN, -SMA,
TGF-ß-RII, and TGF-ß1 were evaluated. Our results indicate that
mechanical tension is crucial for differentiation and maintenance of
the myofibroblast phenotype. In addition, we show that tissue
contractility correlates with the expression of -SMA.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A total of 96 female Wistar rats (200 to 220 g) was used. After shaving the skin, full-thickness 25 x 25-mm wounds, including the cutaneous muscle, were made using surgical scissors in the middle of the dorsum on the first day of the experiments. Wounds were either allowed to heal spontaneously (unsplinted) or were subjected to mechanical tension (splinted) by fixing their edges on a 25 x 25-mm plastic frame using surgical thread (Supramid USP 2-0 DS 19; B. Braun-SSC AG, Emmenbrügge, Switzerland). Rats were sacrificed by CO2 anesthesia and tissues were dissected daily for 2 to 12 days after wounding. In a second series of experiments that investigated the effect of tension release, wounds were splinted for 7 days, the plastic frame was then removed and animals were sacrificed at 1 and 2 days thereafter. Granulation tissue from 8-day and 9-day unsplinted and splinted wounds, served as controls. Granulation tissues were cleaned from the scab and transferred to ice-cold Krebs-Henseleit solution (118.7 mmol/L NaCl, 4.7 mmol/L KCl, 1.17 mmol/L KH2PO4, 2.41 mmol/L MgSO4, 5.05 mmol/L CaCl2, 11.1 mmol/L D-glucose, 25.0 mmol/L NaHCO3), bubbling with 95% O2 and 5% CO2.
Granuloma pouches were induced in rats by injecting 15 ml of air into
the dorsal subcutaneous tissue, followed by 1 ml of 1% croton oil in
maize oil.28,29
This treatment produces the formation of
an abscess-like structure whose wall is rich in
myofibroblasts4,29,30
and has been used to study the
effects of several smooth muscle contraction-inducing and -inhibiting
agents on the isometric force development by a myofibroblastic
tissue.29,30
Pouches were either left intact or released
from internal tension by evacuating 
3 ml of the exudate after 14
days using an 18-gauge needle. Evacuation was repeated every 2 days to
prevent new development of tension. To control whether the removal of
exudate has other effects than tension release, we replaced it with an
equal volume of physiological saline in an additional group. To study
the role of TGF-ß1 in granuloma pouch wall development, we injected
daily 14-day-old granuloma pouches with 1 ml of soluble TGF-ß-RII
(final, 1 µg/ml muTGF-ß-sR; gift of Dr. V. Koteliansky, Biogen
Inc., Cambridge, MA)31,32
for 7 days in another group.
Tension-released granuloma pouches were dissected after 15, 16, 17, 18,
and 21 days, intact pouches after 14 days and 21 days. Dissected
pouches were transferred to ice-cold Krebs-Henseleit solution, bubbling
with 95% O2 and 5% CO2,
and thoroughly washed. Only tissue from the dorsal portion of granuloma
pouches was used to standardize experiments. As control we used normal
dermis and dorsal fascia covering the muscular layer. Fascia was
exposed after full-thickness incision of skin and subcutaneous tissue
and removed using fine forceps and scissors. Dermis was prepared by
removing the epidermis and subcutaneous tissue with a scalpel blade.
Fascia and normal dermis were further treated as described for
granulation tissue and granuloma pouch.
Isometric Force Measurement
Dissected tissues were cut into strips of 5 x 10 mm in transversal orientation to the animal body axis. These were transferred into an organ bath containing 5 ml of Krebs-Henseleit solution bubbling with 95% O2 and 5% CO2 at 37°C and were mounted on the levers of an isometric force displacement transducer (FT03; Grass Instrument Co., West Warwick, RI). A resting tension of 1.5 g was applied in steps of 0.5 g with a 30-minute interval followed by 2 hours of equilibration. Tissue contraction was documented on an oscillograph writer (7WC16H; Grass Instrument Co.) and quantified by converting the analogue data with a MacLab 8/Chart digitization system (AD Instruments Pty. Ltd., Castle Hill, Australia). In a first series of experiments maximal tension development was determined after cumulative stimulation with various agonists of granulation tissue contraction in concentrations ranging from 10-10 mol/L to 10-4 mol/L. For all subsequent experiments agonists were applied at the following concentrations: angiotensin-II (AT-II; Bachem AG, Bubendorf, Switzerland), 10-5 mol/L; endothelin-1 (ET-1; Bachem), 10-7 mol/L; serotonin (Sigma Chemical Co., St. Louis, MO), 10-4 mol/L; histamine (Sigma Chemical Co.), 10-4 mol/L; KCl, 4 x 10-3 mol/L. Tension peaks were registered and mean values were calculated from four tissue strips per animal and three to five animals per experimental condition.
Immunohistochemistry, Confocal Laser-Scanning Microscopy, and Quantification of F-Actin by Fluorescence Image Analysis
Strips from granuloma pouch and wound granulation tissues were fixed in 4% neutral-buffered formalin and embedded in paraffin. Sections of 4 µm were stained with hematoxylin and eosin or with Masson’s trichrome. For immunohistochemistry deparaffinized sections were immersed in methanol containing 0.5% H2O2 for 10 minutes. Sections were incubated with primary antibodies either overnight at 4°C (anti-TGF-ß-RII, rbAb; Santa Cruz Biotechnology Inc., Heidelberg, Germany) or 60 minutes at room temperature (anti-TGF-ß1, rbAb; Santa Cruz), followed by 30 minutes of incubation with a secondary biotinylated goat anti-rabbit antibody (DAKO, Copenhagen, Denmark). Presence of the specific protein was evaluated by means of the streptavidin-biotin complex peroxidase method (DAKO) and peroxidase activity was visualized with 3-amino-9-ethylcarbazole (Sigma). Slides were counterstained with hematoxylin and mounted in Eukit. Pictures were acquired using a Zeiss Axiophot microscope equipped with Plan-Neofluar x20/0.50 objective (Carl Zeiss Inc., Oberkochen, Germany) and with a digital color camera (Coolview; Photonic Science, London, UK).
For confocal laser-scanning microscopy tissue strips were snap-frozen
in precooled liquid isopentane and embedded in OTCTM resin
(Miles Scientific, Naperville, IL). Cryosections of 4 µm were stained
for -SMA (anti-SM-1, IgG2a mAb),6
F-actin
(phalloidin-Alexa 488; Molecular Probes, Eugene, OR), ED-A FN (IST-9,
IgG1 mAb; gift from Dr. L. Zardi, National Institute for Cancer
Research, Laboratory of Cell Biology, Genoa, Italy),33,34
desmin (D33, IgG1 mAb; DAKO), smooth muscle myosin heavy chain (SMMHC,
rb polyclonal Ab35
or BT-562, rb polyclonal Ab; Biomedical
Technologies, Stoughton, MA), and non-muscle myosin heavy chain (NMMHC,
rb polyclonal Ab)35
and DNA [4,6-diamidino-2-phenylindole
(DAPI)]. As secondary antibodies goat anti-mouse IgG2a
tetramethylrhodamine B isothiocyanate-conjugated, goat anti-mouse IgG1
fluorescein isothiocyanate-conjugated, and goat anti-rabbit
tetramethylrhodamine B isothiocyanate-conjugated (Jackson
ImmunoResearch Laboratories, West Grove, PA) were used. All slides were
mounted in buffered polyvinyl alcohol. Images were taken by means of
confocal laser-scanning microscopy (LSM 410, Zeiss) using a
Plan-Neofluar x10/0.50 objective or a x63/1.4 Plan-Neofluar oil
immersion objective with a digitally extended focus depth of 1 µm,
reconstructed from five optical sections at 0.2-µm
z-resolution. All digital images were processed for printing
using Adobe Photoshop and printed with a digital Fujifilm Pictrography
4000 printer (Fujifilm, Tokyo, Japan).
To quantify F-actin by fluorescence image analysis 4-µm sections of granulation tissue strips were fixed and triple-stained for F-actin (red), desmin (green), and cell nuclei (blue) as described above. For a given region of interest digital images with a resolution of 515 x 512 pixels were taken at red-, green-, and UV-light excitation, respectively, using a Plan-Neofluar x20/0.50 objective (Zeiss), color-camera (Coolview, Photonic Science Ltd.), and image grabber software (ImageAccess V2.04K; Imagic Bildverarbeitung AG, Glattbrugg, Switzerland). Constant gain values and integration times were used for the digital camera to obtain similar exposure times for all specimens. To determine the level of F-actin, images were then automatically processed by KS400 software (Zeiss). First, the three 24-bit Red · Green · Blue-images per region were converted to 8-bit gray value images with pixel intensity values ranging from 0 (black) to 255 (white). Desmin staining, specific for vascular smooth-muscle cells, was automatically selected by a thresholding process and corresponding pixel values were set to 255. To exclude vessels from F-actin quantification, desmin-positive areas were then subtracted from F-actin and DAPI images. Second, the background of the desmin-corrected F-actin image was calculated from a manually chosen F-actin-negative area and subtracted from the mean pixel intensity of the whole image, thereby quantifying the level of F-actin expression in the region of interest. Finally, the F-actin expression level was related to the number of cells in the region, represented by the number cell nuclei calculated from the desmin-corrected DAPI image. Tissue was collected from three animals per experimental condition, five sections were prepared per animal, and five regions of interest were analyzed per section.
Western Blot Analysis
Dissected tissues were immediately frozen in liquid nitrogen,
crushed, and dissolved in sample buffer: 10 mmol/L EGTA (Sigma), 1
mmol/L N-tosyl-L-arginine methyl
ester hydrochloride (TAME; Fluka, Buchs, Switzerland), 1 mmol/L
phenylmethyl sulfonyl fluoride (Fluka), 4% aprotinin (Bayer AG,
Zurich, Switzerland), 0.5 mmol/L benzamidine (Sigma), and 1 mmol/L
di-isopropylfluorophosphate (Fluka). Samples were sonicated (Branson
Sonic Power Company, Danbury, CT), boiled for 3 minutes, and
protein concentration was determined according to Bradford as described
previously.36
Equal amounts of total protein (5 to 20
µg) were loaded to 10 or 7% sodium dodecyl sulfate minigels (Bio-Rad
Laboratories AG, Glattbrugg, Switzerland), separated by
polyacrylamide gel electrophoresis, and transferred to nitrocellulose
membrane (Protran; Schleicher & Schuell, Dassel, Germany). Membranes
were then probed with the primary antibodies: anti--SMA, (SM-1),
ß-actin (ß74, rbAb),37
total-actin (ß74+AL-20), ED-A
FN (IST-9), desmin (DAKO), SMMHC, NMMHC,35
and TGF-ß-RII
(Santa Cruz). Secondary goat anti-mouse and anti-rabbit antibodies,
conjugated with horseradish-peroxidase (Jackson ImmunoResearch
Laboratories) were used and signals were detected by ECL
chemiluminescence (Amersham, Rahn AG, Zürich, Switzerland).
Subsequently, membranes were stripped of bound antibodies and reprobed
for vimentin (mAb, clone V9; DAKO). Bands were digitized with a scanner
(Arcus II; Agfa, Köln, Germany) and the ratio between all band
densities of one blot was calculated by commercial computer software
(ImageQuant V3.3l Molecular Dynamics, Sunnyvale, CA). Relative protein
expression was normalized to the respective values for vimentin.
Samples from three different animals per experimental condition were
tested and protein/vimentin mean ratio was calculated from three
scanned Western blots per animal sample.
Statistical Analysis
Quantitative results are presented as mean values ± SD. Mean
values were tested for the likelihood to belong to the same numeric
population by means of a two-tailed heteroscedastic Student’s
t-test. Differences were considered to be statistically
significant at values of P 
0.01. In graphs,
differences with statistical significance are indicated by filled
symbols. In column charts P values 0.01 are
indicated by an asterisk (*) and P 0.005 by a double
asterisk (**). Positive linear correlation between isometric tissue
contraction and expression of myofibroblast markers, quantified by
Western blot analysis was statistically tested by calculating the
square of the Pearson correlation product
(r2
value).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been shown that fibroblasts subjected to mechanical tension
develop a contractile phenotype in vitro.20,21
To test whether mechanical tension induces similar effects on
fibroblasts in vivo, we have first increased the mechanical
tension of rat granulation tissue by splinting wounds with plastic
frames and compared the contractility of tissues from splinted and
unsplinted wounds (Figure 1)
. Isometric
contraction was stimulated with various smooth muscle agonists in the
following order of efficiency: ET-1 (10-7
mol/L), AT-II (10-5 mol/L), and serotonin
(10-4 mol/L). Wound tissue contracted only
weakly after stimulation with histamine (10-4
mol/L) and potassium (4 x 10-3 mol/L).
Control tissues (0 days) did not show any significant response after
stimulation with all agonists (not shown). The contraction of
unsplinted and splinted granulation tissue increased from 3-day-old to
9-day-old tissue after stimulation with ET-1 (Figure 1A)
; moreover,
splinted tissues contracted more strongly at 6 days (266%) and 8 days
(185%) compared to unsplinted tissues. Surprisingly, splinted and
unsplinted tissues exhibited comparable contraction after 9 days. At 10
days the contractile activity of unsplinted tissues decreased (67%
compared to 9 days), whereas the contractile activity of splinted
tissues was unchanged compared to 9 days. At 12 days unsplinted tissues
showed a further decrease in contractility (71% compared to 10 days);
splinted wounds also decreased their contractility compared to 10 days
(78%) but maintained a higher contractility (168%) compared to
unsplinted tissues. Results obtained after stimulation with AT-II
(Figure 1B)
and serotonin (not shown) were similar to those obtained
after ET-1 stimulation.
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To assess whether mechanical tension stimulates myofibroblast
differentiation in vivo, we compared the expression of
myofibroblast markers such as F-actin, ED-A FN, -SMA, TGF-ß-RII,
TGF-ß1, desmin, SMMHC, and NMMHC in the different situations. F-actin
was not present in fibroblasts of normal dermis (not shown), started to
be visible in 3-day-old unsplinted wounds (Figure 2Aa
) and gradually increased (Figure 2,
Ab to Ad
). Splinting induced noticeable accumulation of F-actin already
at 1 day (not shown) and importantly enhanced F-actin levels throughout
the following days (Figure 2, Ae to Ah
). Moreover, splinting improved
the alignment of F-actin along the lines of mechanical tension, ie,
parallel to the wound surface. F-actin bundles were devoid of -SMA
up until day 6 in unsplinted and until day 4 in splinted tissue, as
evaluated by double staining (not shown). Quantification of Alexa
488-phalloidin fluorescence intensity confirmed higher levels of
F-actin in splinted compared to unsplinted granulation tissues at any
wound age (Figure 2B
, between 376 and 226% increase). No significant
changes of marker distribution were observed in the different wound
locations. Quantification of granulation tissue cellularity by means of
DAPI staining demonstrated a linear increase in cell number from 3- to
12-day-old granulation tissue (120%) and no difference between
unsplinted and splinted tissue. ED-A FN was detected in low levels in
3-day-old unsplinted granulation tissue (Figure 2Aa
), expression
increased between day 6 and day 9 (Figure 2, Ab and Ac
), and was
reduced in 12-day-old granulation tissue (Figure 2Ad
). Splinting
induced ED-A FN expression starting from day 1 (not shown) and enhanced
its expression compared to unsplinted granulation tissue at all stages
of wound healing (Figure 2, Ae to Ah
). No differences in vascularity
were observed in splinted versus unsplinted wounds.
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-SMA was not expressed in fibroblasts of normal dermis (not shown)
and of 3-day-old granulation tissue (Figure 3, A and E)
, and was not detected in
fibroblasts of 6-day-old unsplinted granulation tissue (Figure 3B)
. In
unsplinted wounds, it was first detected after 7 days (not shown), was
strongly expressed after 9 days (Figure 3C)
, and expression was clearly
reduced after 12 days (Figure 3D)
. In contrast, splinting induced
-SMA expression by granulation tissue fibroblasts already at 4 to 6
days after wounding (Figure 3F)
; the level of -SMA expression
increased in splinted tissue to a maximum at 9 days (Figure 3; E to G
)
and remained at an apparently similar level at 12 days (Figure 3H)
.
Three-day-old to 12-day-old splinted wounds exhibited increased
expression of both TGF-ß1 and TGF-ß-RII; however, no clear
differences were observed between splinted and unsplinted wound tissue
by means of immunohistochemistry (not shown). Desmin and SMMHC were not
expressed by granulation tissue fibroblasts, and expression of NMMHC
did not change in any condition as tested by means of
immunofluorescence and Western blotting (not shown).
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essentially confirmed immunolocalization results. In unsplinted tissue
ED-A FN expression was continuously increased from 3 days to 9 days
(630%) and exhibited a reduction by 160% between 9 days and 12 days
(Figure 4, A and B)
. Splinting enhanced the amount of ED-A FN (Figure 4A)
with the largest difference at 3 days (Figure 4B
, 347%).
Expression of -SMA moderately increased from 3-day to 6-day
unsplinted tissue (195%), steeply increased from 6 days to 9 days
(400%), and was reduced by 145% between 9 days and 12 days (Figure 4, A and C)
. Splinted tissue exhibited increased -SMA expression levels
at 6 days (Figure 4C
, 420%) and showed similar -SMA expression at 9
days compared to unsplinted granulation tissue. Splinting maintained
high -SMA expression levels at 12 days in contrast to the decreased
expression in unsplinted wound tissue (Figure 4C)
. TGF-ß-RII
expression increased from 0 to 12 days after wounding with no
significant difference between splinted and unsplinted granulation
tissue (Figure 4, A and D)
. To relate changes in myofibroblast marker
expression to enhanced tissue contractility during the evolution of
unsplinted and splinted wound granulation tissue, we calculated
correlation coefficients. Highest correlation was observed between the
temporal course of ET-1-stimulated tissue contraction (Figure 1A)
and
expression of -SMA (r2
=
0.98). Lower correlation was calculated for TGF-ß-RII
(r2
= 0.92), ED-A FN
(r2
= 0.90), and F-actin
(r2
= 0.84), no correlation for
ß-actin (r2
= 0.69), NMMHC
(r2
= 0.61), desmin
(r2
= 0.57), and vimentin
(r2
= 0.55).
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Because increased mechanical tension seems to induce myofibroblast
differentiation and increase granulation tissue contractility, we
sought to investigate whether reduction of tension may have the
opposite effect. First, wounds were splinted for 7 days, then released
from tension for 1 or 2 days and compared to granulation of the same
age but still splinted. One-day-released granulation tissue contracted
similarly compared to 8-day splinted tissue (96 ± 7%).
However, tension release for 2 days significantly reduced contraction
compared to 9-day splinted tissue to 63 ± 3% after stimulation
with AT-II and to 63 ± 2% after stimulation with ET-1.
Contraction of 2-day-released granulation tissue was also lower
(71 ± 4%) when compared to 9-day-unsplinted granulation tissue.
Characterization of released tissue by immunostaining and Western
blotting revealed no changes after 1 day, but a decrease of F-actin and
-SMA after 2 days. Expression levels of ED-A FN, TGF-ß-RII,
TGF-ß1, NMMHC, desmin, and vimentin were not significantly changed 2
days after release (not shown).
To investigate further the chronological relationship between tissue
contraction and myofibroblast modulation after release of mechanical
tension, we used the granuloma pouch model.29
Tension was
released in granuloma pouch tissue at 14 days by evacuating the exudate
and isometric contraction was measured 1 to 7 days after release
(Figure 5)
. Contraction of 21-day control
pouch tissue was maximally stimulated by ET-1
(10-7 mol/L) and by the following agonists in
percentage of ET-1-response: AT-II (10-5 mol/L),
60%; serotonin (10-4 mol/L), 48%; potassium (4
x 10-3 mol/L), 45%; and histamine
(10-4 mol/L), 6%. Results are presented for
ET-1 (Figure 5A)
and AT-II (Figure 5B)
. Tissue contractility was higher
in intact 21-day-old compared to 14-day-old pouches (Figure 5
, dashed
lines), confirming previous observations.30,38
Release of
mechanical tension at 14 days decreased pouch tissue contraction
beginning 2 days after release, exhibited a minimum at 3 days after
release (14d+3dR), and kept this level until 7 days.
Compared to intact pouches of the same age (21 days),
tension release resulted in a significantly lower tissue contraction
(38% for ET-1 and 46% for AT-II). As a control, we substituted the
evacuated exudate with an identical volume of physiological saline for
7 days (Figure 5
, 14+7dS). Saline-injected pouch tissue contraction did
not differ from intact pouches of the same age (21 days).
|
, insets)
revealed an intense F-actin staining in 14-day-old granuloma pouch
fibroblasts, that was clearly decreased already 2 days after release
(Figure 6B)
; this decrease was more important at 7 days after release
(Figure 6C
, inset). ED-A FN and -SMA were co-localized and their
expression increased in 21-day-old compared to 14-day-old intact
granuloma pouch tissue (Figure 6, A and D)
. Three days after tension
release -SMA expression was reduced (Figure 6B)
; 7 days after
tension release, ED-A FN expression was also reduced and -SMA was
hardly detectable (Figure 6C)
. TGF-ß1, previously shown to be
expressed in granuloma pouch tissue,39,40
and TGF-ß-RII
were both predominantly expressed in the external collagen-rich layer
of the pouch tissue. TGF-ß1 and TGF-ß-RII expression patterns in
7-day-released pouches did not differ significantly from 1- to
5-day-released tissue or 21-day intact pouch tissue, but the thickness
of the collagen-rich layer decreased progressively during the 7-day
period of tension release to 70% compared to 21-day intact pouches
(not shown).
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-SMA was significantly reduced 2
to 3 days after release, compared to 14-day-old intact pouch tissue,
whereas decrease of ED-A FN expression started 4 days after release
(Figure 7)
. TGF-ß-RII expression was
only moderately diminished throughout the 7-day period after release
and no change was observed for ß-actin and vimentin expression
(Figure 7A)
. Compared to 21-day-old intact pouch tissue, 7-day tension
release reduced -SMA expression to 51%, ED-A FN to 47%, and
TGF-ß-RII to 81% (Figure 7B)
. In contrast to wound granulation
tissue fibroblasts, which did not express desmin and SMMHC, low
expression of both proteins was observed in fibroblastic cells of
14-day and 21-day intact granuloma pouches. Tension release did not
change desmin and NMMHC expression and only moderately diminished
expression of SMMHC 7 days after release as evaluated by Western
blotting and immunofluorescence (not shown). When removed exudate was
replaced by physiological saline, no changes in protein expression were
detected (Figure 7A
, 14d+7dS).
|
, 14d+7dT) and to 48% after AT-II stimulation (Figure 5B
,
14d+7dT) compared to 21-day-old control pouch tissue. It reduced the
expression levels of TGF-ß, TGF-ß-RII, ED-A FN, F-actin, and
-SMA significantly compared to 21-day-old control pouch tissue as
demonstrated by Western blotting (Figure 7A)
and immunofluorescence
(not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-SMA.27
Here, we demonstrate that mechanical tension
also influences myofibroblast differentiation in vivo.
Fibroblasts populating the granulation tissue of splinted wounds
exhibit earlier formation of stress fibers, expression of ED-A FN and
of -SMA compared to nonsplinted wounds. Inversely, release of
tension in wound granulation tissue and in granuloma pouch leads to a
sequential loss of stress fibers, -SMA, and ED-A FN. Moreover, our
study indicates a correlation between the level of -SMA expression
and contractility of the tissues obtained during the different
experimental situations. The acquisition of contractile activity by
granulation tissue on stimulation with smooth muscle agonists is a
well-accepted phenomenon and has been suggested to represent a useful
indication of the retractile potential of granulation
tissue.29,30,41
Acquisition of an increased contractile
activity by wound splinting42
has been correlated to the
induction of microfilament bundles in fibroblasts.18
Our
results corroborate these observations and establish a correlation
between changes in tissue contractility and cytoskeletal protein
expression and/or organization after mechanical load modulation.
In both splinted and unsplinted wounds, the granulation tissue strip
contractile activity evolves according to three phases, as depicted
schematically in Figure 8
. During the
first phase (red) the contractility of granulation tissue increases
slowly, whereas during the second phase (blue), the increase is steep;
the third phase (green) corresponds to a decrease of contractile
activity. In the first phase there is an increase of F-actin and of
ED-A FN expression that is clearly more important in splinted wounds.
The onset of high contractile activity correlates always with the
de novo expression of -SMA, assembled into stress fibers.
It is noteworthy that the first phase is shorter and the second phase
is longer in splinted compared to unsplinted wounds and corresponds to
the maximal level of -SMA expression. Interestingly, the maximal
level of contractile capacity is similar in both situations. Finally, a
decrease in contractile activity takes place more slowly in splinted
than in unsplinted wounds. The observed correlation between -SMA
expression and granulation tissue contractility is in agreement with
the previously reported correlation between -SMA expression and
contraction of fibroblast-populated anchored collagen
gels.8,9
More recently we have shown that 3T3 fibroblasts
exhibit a significantly higher contractile activity after transient
and/or stable transfection with -SMA cDNA compared to fibroblasts
transfected with ß-actin, 
-actin, or even -cardiac actin cDNA
in the absence of any change of myosin heavy chain
expression.7
All these data are compatible with the
assumption that -SMA expression alone plays a role in the
enhancement of fibroblast contractility in vitro. Our
results allow to hypothesize that also in vivo -SMA
expression plays a crucial role in the development of granulation
tissue contractile capacity. In all situations, increased tension
results in a greater expression of -SMA and an increased force
production, whereas decreased tension has the opposite
effect.2,21,43,44
Although in vivo and
in vitro data indicate that -SMA expression is stimulated
by mechanical stress, it remains unclear how fibroblasts translate the
physical signal into protein expression. In cells such as
cardiomyocytes, endothelial and smooth muscle cells, mechanical stress
was shown to stimulate the production and secretion of growth factors
that mediate stress-induced cell responses.45
In
particular, expression of TGF-ß1, the major inducer of -SMA
expression13,14
and contractility in
myofibroblasts,8,21
is up-regulated in mechanically
stressed mesangial cells.46
Under our conditions the
expression of TGF-ß1 and of its receptor TGF-ß-RII was gradually
increased, but similarly in splinted tissue and unsplinted tissues.
Nevertheless, blocking TGF-ß1 by an excess of soluble receptor
without changing the mechanical load reduced -SMA expression and
tissue contraction in our experimental conditions, implying a role of
TGF-ß1. Enhanced mechanical stress increases growth factor
sensitivity23,24,47
and loss of mechanical tension reduces
the growth factor response of cultured fibroblasts.48,49
It remains to be examined whether TGF-ß1 activation changes in our
experimental situations.
|
-SMA-positive myofibroblasts and is
essential to mediate TGF-ß1-induced -SMA
expression.17
Splinting induces ED-A FN expression in
granulation tissue, suggesting a possible regulatory function of this
protein during stress-induced myofibroblast differentiation. Regulation
may occur at the level of FN expression,55
alternative splicing,56,57
fibril assembly,22
or accessibility of functional residues in the ED-A domain of
FN58
as it has been demonstrated for plasma
FN.59
Integrins of focal adhesions are potential receptors
and transducers of these extracellular signals.60
When myofibroblasts were released from mechanical stress by removing
the wound splint or by evacuating the granuloma pouch, disappearance of
-SMA preceded the decrease of ED-A FN and TGF-ß1 levels. Thus,
ED-A FN and TGF-ß1 are crucial for the induction of -SMA
expression but are not sufficient to maintain myofibroblast
differentiation in the absence of a mechanical stimulus. Taking into
account the early formation of F-actin during wound healing and the
rapid loss of actin stress fibers after load release, we suggest that
the physical integrity of stress fibers61
provides an
important prerequisite for -SMA expression and myofibroblast
contraction. Mechanical stress induces actin polymerization and stress
fiber formation when applied externally62
or by increasing
intracellular tension.10,63
The ratio between
G-actin/F-actin was shown to directly influence ß-actin
synthesis.64,65
However, the role of the G-actin/F-actin
ratio for the expression of other actin isoforms, such as -SMA, has
not been evaluated. It is conceivable that mechanical tension induces
actin expression without discriminating among isoforms whereas factors
such as TGF-ß and ED-A FN exert a specific stimulation of
-SMA.17
A similar phenomenon has been shown for the
stress-dependent expression of -skeletal actin by cardiomyocytes in
the presence of AT-II.45
In conclusion our results show that mechanical tension is a prerequisite for the development and maintenance of myofibroblast differentiation and hence of granulation tissue contraction. Given the reciprocal relationship between fibroblast contractility and the mechanical state of the matrix, the modulation of extracellular and intracellular tension may help to influence wound healing and development of fibrocontractive diseases.
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Acknowledgements |
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Footnotes |
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Supported by the Swiss National Science Foundation Grants 31-61.336.00 and 31-54048.98.
Accepted for publication June 1, 2001.
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References |
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