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Endothelin 1 contributes to the effect of transforming growth factor 1 on wound repair and skin fibrosis.

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ARTHRITIS & RHEUMATISM
Vol. 62, No. 3, March 2010, pp 878–889
DOI 10.1002/art.27307
© 2010, American College of Rheumatology
Endothelin 1 Contributes to the Effect of
Transforming Growth Factor ␤1 on
Wound Repair and Skin Fibrosis
David Lagares,1 Rosa Ana Garcı́a-Fernández,2 Clara López Jiménez,1 Noemi Magán-Marchal,1
Oscar Busnadiego,1 Santiago Lamas,1 and Fernando Rodrı́guez-Pascual1
Objective. To characterize the pathways induced
by transforming growth factor ␤1 (TGF␤1) that lead to
the expression of endothelin 1 (ET-1) in human dermal
fibroblasts, and to study the effects of TGF␤1 and ET-1
on the acquisition of a profibrotic phenotype and assess
the contribution of the TGF␤1/ET-1 axis to skin wound
healing and fibrosis in vivo.
Methods. The mechanism of induction of ET-1
expression by TGF␤1 and its effect on the expression of
␣-smooth muscle actin and type I collagen were studied
in human dermal fibroblasts, in experiments involving the TGF␤ receptor inhibitor GW788388 and the
ET receptor antagonist bosentan, by real-time reverse
transcription–polymerase chain reaction (RT-PCR),
enzyme-linked immunosorbent assay, immunofluorescence, Western blotting, and promoter/reporter transient transfection analyses. Experiments assessing dermal wound healing in mice were performed with
adenovirus-driven overexpression of active TGF␤1 and
ET-1, with or without treatment with bosentan. The
contributions of TGF␤1 and ET-1 to the fibrotic response were also assessed in a mouse model of
bleomycin-induced skin fibrosis, by histologic, immunohistochemical, RT-PCR, and protein analyses.
Results. TGF␤1 induced ET-1 expression in human dermal fibroblasts through Smad- and activator
protein 1/JNK–dependent signaling. The ability of
TGF␤1 to induce the expression of profibrotic genes was
dependent on ET-1. Adenovirus-mediated overexpression of TGF␤1 and ET-1 in mouse skin was associated
with accelerated wound closure, increased fibrogenesis,
and excessive scarring. Treatment with bosentan prevented the effects of TGF␤1. In the bleomycin-induced
fibrosis model, treatment with GW788388 and bosentan
prevented the fibrotic response.
Conclusion. Our results strongly support the notion that the TGF␤1/ET-1 axis has a role in wound
repair and skin fibrosis. ET-1 receptor antagonists,
such as bosentan, may represent a useful therapeutic
tool in the treatment of excessive scarring and fibrosisrelated diseases.
Supported by the Ministerio de Ciencia e Innovación, Plan
Nacional de I⫹D⫹I (Network of Excellence for Research on Oxidative
Stress in Spain grants SAF2006-02410 and Consolider CSD-2007-0020),
the Comunidad Autónoma de Madrid (CARDIOVREP Consortium),
the Sociedad Española de Nefrologı́a (Senefro grant), the Fundación
Médica Mutua Madrileña (FundacionMM grant), the Fundación
Genoma España (Meica Project), and Actelion Pharmaceuticals España, Barcelona, Spain.
1
David Lagares, BSc, Clara López Jiménez, Noemi MagánMarchal, Oscar Busnadiego, BSc, Santiago Lamas, MD, PhD, Fernando Rodrı́guez-Pascual, PhD: Laboratorio Mixto Fundación Renal
“Iñigo Alvárez de Toledo,” FRIAT-CSIC, Madrid, Spain; 2Rosa Ana
Garcı́a-Fernández, VetD: Universidad Complutense de Madrid, Madrid, Spain.
Address correspondence and reprint requests to Fernando
Rodrı́guez-Pascual, PhD, Centro de Investigaciones Biológicas (CSIC),
Ramiro de Maeztu 9, E-28040 Madrid, Spain. E-mail: frodriguez@
cib.csic.es.
Submitted for publication July 23, 2009; accepted in revised
form November 3, 2009.
Wound healing is an evolutionarily conserved,
dynamic, and complex process aimed at the restoration
of organ function. Upon injury to the skin, a tightly
orchestrated repertoire of cellular responses is called
into play, and normally the wound is rapidly and efficiently repaired (1,2). Under some conditions, repair is
excessive, resulting in pathologic scar formation, a process that may eventually lead to progressive distortion of
the tissue structure, with consequent dysfunction and
ultimate failure of fibrotic organs. In fact, excessive
scarring occurs not only as a defective tissue repair
process in the skin, but also as an important feature of a
number of chronic diseases in which any part of the body
is involved, as, for instance, in systemic sclerosis (SSc;
scleroderma).
878
TGF␤1/ET-1 AXIS IN WOUND REPAIR AND SKIN FIBROSIS
Fibroblasts are generally considered to be the
major effector cells in excessive scarring and fibrotic
diseases, since they contribute to the increased synthesis
and contraction of the extracellular matrix (ECM),
which is typical of these disorders. The transformation
of the fibroblast from a form that displays a quiescent
phenotype to one that displays a proliferating, matrixproducing, contractile phenotype, referred to as the
myofibroblast, has been shown to be a key process in
wound healing (3). The myofibroblast differentiation
process is regulated by a complex repertoire of profibrotic and antifibrotic cytokines, among which transforming growth factor ␤ (TGF␤) family members play
a preeminent role, particularly the TGF␤1 isoform.
TGF␤1 induces fibrotic responses in vivo and promotes
the ability of fibroblasts to synthesize and contract the
ECM in vitro (4).
Blocking the action of TGF␤1 is an approach that
has been shown to ameliorate experimental fibrosis and
scarring (5–10). On this basis, pharmaceutical companies are currently developing and testing various TGF␤targeting therapeutic strategies. However, because
TGF␤1 plays many roles in normal physiology, indiscriminate targeting of TGF␤ signaling is anticipated to
be a potential problem. Thus, there is much interest in
the need to identify appropriate TGF␤-downstream
targets, such as connective tissue growth factor or endothelin 1 (ET-1), that could serve as the basis for selective
antifibrotic therapies.
Several lines of evidence indicate that ET-1 may
be a mediator in the development of excessive scarring
and fibrosis. The binding of ET-1 has been demonstrated by autoradiography in skin and lung biopsy
samples from SSc patients, suggesting an overall increase in ET receptor expression in this fibrotic disease
(11). ET-1 is normally produced by endothelial cells, but
it has also been shown to be overexpressed by fibroblasts
in patients with SSc (12,13). In cell culture, ET-1 promotes myofibroblast differentiation as well as ECM
production and contraction (14). Therefore, ET-1 has
become recognized as a profibrotic factor that functions
both in vivo and in vitro, encouraging the potential
intervention of ET-1 signaling as a therapeutic approach
for fibrosis. ET receptor antagonists, either ETA-specific
(ambrisentan and sitaxentan) or mixed ETA/ETBspecific (bosentan), are currently in use in clinical practice for the treatment of pulmonary arterial hypertension. Bosentan is also approved for the prevention of the
formation of digital ulcers in patients with SSc (15,16).
Despite the preeminent role of ET-1 in fibrogenesis, the
879
mechanisms underlying the links between pathologic
healing and fibrosis remain unknown.
The aim of the present study was to characterize
the TGF␤1-induced pathways leading to ET-1 expression in human dermal fibroblasts and to analyze the
contribution of ET-1 to TGF␤1 responses in vivo. We
found that TGF␤1 induces ET-1 through Smad- and
activator protein 1 (AP-1)/JNK-dependent signaling,
and that the ability of TGF␤1 to induce the expression
of profibrotic genes is dependent on ET-1. Using the
punch model of skin wound healing in mice, we found
that adenovirus-mediated TGF␤1 overexpression is associated with accelerated wound closure and excessive
scarring, and that the antagonism of ET receptors with
bosentan prevents these effects. Accordingly, ET-1 overexpression results in enhanced wound closure and collagen deposition.
In addition, using a mouse model of bleomycininduced skin fibrosis, we corroborated that blockade of
TGF␤ signaling with the use of a new TGF␤ receptor
type I/type II inhibitor, GW788388, prevents skin fibrosis. Moreover, we observed that bleomycin-induced skin
fibrosis not only develops in association with increased
TGF␤1 levels, but also is associated with elevated ET-1
levels. Finally, our results indicated, for the first time,
that treatment with bosentan can block the development
of fibrosis in this model. Therefore, these results
strongly support a role for the TGF␤1/ET-1 axis in
wound repair and skin fibrosis, both in vitro and in vivo.
MATERIALS AND METHODS
Cell culture. Primary human dermal fibroblasts as well
as embryonic fibroblasts from Smad3-knockout and wild-type
mice were grown and maintained in culture medium using
standard procedures (expanded details on the methods are
available in the supplementary Materials and Methods text,
available on the Arthritis & Rheumatism Web site at http:
www3.interscience.wiley.com/journal/76509746/home). All animal handling was carried out in accordance with European and
Spanish laws and regulations governing the use and protection of
vertebrate mammals for experimentation and other sicentific
purposes.
Determination of messenger RNA (mRNA) expression
by real-time reverse transcription–polymerase chain reaction
(RT-PCR). For RNA experiments, primary human dermal
fibroblasts and Smad3-knockout and wild-type mouse embryonic fibroblasts were cultured in 6-well plates and treated with
10 ng/ml TGF␤1 (R&D Systems, Minneapolis, MN) or 100 nM
ET-1 (Bachem AG, Weil am Rhein, Germany). In some
experiments, cells were pretreated with the mixed ETA/ETB
receptor antagonist bosentan (10 ␮M final concentration;
Actelion Pharmaceuticals, Allschwil, Switzerland) or with the
TGF␤ receptor type II/type III inhibitor GW788388 (10 ␮M
880
LAGARES ET AL
Figure 1. Induction of the expression of endothelin 1 (ET-1) by transforming growth factor ␤1 (TGF␤1) in human dermal fibroblasts.
Cells were incubated without or with TGF␤1 (10 ng/ml), and changes in the expression of ET-1 at the level of mRNA expression (A),
peptide production (B), and promoter activity (C) were determined. The involvement of TGF␤ receptors was analyzed in cells by
incubation with the TGF␤ receptor type I/type II inhibitor GW788388 (10 ␮M). A, Time course of the induction of ET-1 mRNA
expression, as analyzed by quantitative reverse transcription–polymerase chain reaction. Results are the mean and SD fold induction in
3 samples per group (untreated [basal] or treated with TGF␤1 up to 24 hours without the inhibitor or with the inhibitor [24 ⫹
GW788388]). ⴱ ⫽ P ⬍ 0.01 versus basal conditions at the corresponding time point; # ⫽ P ⬍ 0.01 versus TGF␤1 cultures at 24 hours
in the absence of GW788388. B, ET-1 peptide levels, as detected by specific enzyme-linked immunosorbent assay. Results are the mean
and SD pg/ml in 3 samples. C, Transcriptional activation of the human ET-1 gene, as assessed by luciferase activity on luminometry in cells
transfected with a ⫺650/⫹172–bp fragment of the human ET–1 promoter. Results are the mean and SD normalized units (n.u.) in 6 samples.
In B and C, ⴱ ⫽ P ⬍ 0.05 versus basal conditions without the inhibitor; # ⫽ P ⬍ 0.05 versus TGF␤1 cultures without the inhibitor.
final concentration; GlaxoSmithKline, Les-Ulis, France). Cells
were then processed for RNA isolation by guanidium
thiocyanate–phenol–chloroform extraction. Essentially the
same procedure was used to isolate RNA from mouse skin
biopsy samples, except that sections of tissue were deep-frozen
in liquid nitrogen. Relative mRNA expression, normalized to
the levels of GAPDH, was determined by RT-PCR (17), with
results calculated using the difference in threshold cycle (2–
⌬⌬Ct) method.
Western blotting analysis. For analyses of protein
expression, proteins were isolated from human dermal fibroblasts, Smad3-knockout and wild-type mouse embryonic
fibroblasts, and skin biopsy samples, followed by Western
blotting as previously described (17). To detect antigens, blots
were probed with anti–␣-smooth muscle actin (anti–␣-SMA)
(1:2,000; Master Diagnostica, Granada, Spain), anti–␤-actin
(1:20,000; Sigma, St. Louis, MO), and anti-JNK and anti–
phospho-JNK antibodies (1:2,000; Cell Signaling Technology,
Beverly, MA).
Reporter plasmids and cell transfection. Luciferase
reporters driven by ⫺650-bp and ⫺193-bp fragments (representing the wild-type and mutated Smad binding element and
AP-1 site, respectively) of the human ET-1 promoter were
transiently transfected into human dermal fibroblasts and wildtype or Smad3-knockout mouse embryonic fibroblasts, as
described previously (18). In some experiments, the selective
JNK inhibitor SP600123 (Sigma) was included at a concentration of 25 ␮M.
Adenoviral infection for assessment of skin wound
healing. Skin wound healing was analyzed using the dermal
punch-wound model in female, 6–8-week-old, pathogen-free
C3H/HeJ mice. Briefly, animals (8 per experimental group)
were anesthetized and their backs were shaved and disinfected.
Two full-thickness (6-mm–diameter) skin wounds were then
generated using a dermal biopsy punch (Stiefel Biopsy Punch,
Stiefel, Germany). Wound diameter was quantified using
ImageJ software (NIH, Bethesda, MD).
To induce overexpression of TGF␤1 and ET-1,
adenovirus-mediated gene transfer was performed. An active
form of rat TGF␤1 or ET-1 was transferred as adenovirus
(Adv-TGF␤1 or Adv–Et-1, respectively) into the skin tissue 2
days prior to dermal biopsy punch, as described previously
(19). After adenoviral infection, biopsy samples of the wound
tissue were collected for analysis of TGF␤1 or ET-1 expression
by enzyme-linked immunosorbent assay (ELISA), as well as
for histologic examination and hydroxyproline assay for estimation of collagen content.
Mouse model of bleomycin-induced dermal fibrosis.
Skin fibrosis was induced in mice by local injection of bleomycin, in accordance with previously published protocols (20).
Groups of mice (6 per group) were injected with either bleomycin
or phosphate buffered saline (PBS) or DMSO as a vehicle
control, and additional groups were also treated with bosentan
or GW788388. Mice were killed by cervical dislocation, and the
back skin was removed and processed for histologic examination. In addition, skin biopsy samples were frozen for myofibroblast RNA and protein analyses.
Statistical analysis. Experimental data were analyzed
using Student’s unpaired t-test, in the case of normal distribution of the data, or using nonparametric tests, as appropriate.
P values less than or equal to 0.05 were considered statistically
significant.
RESULTS
Induction of ET-1 expression by TGF␤1 through
activation of the JNK and Smad signaling pathways in
human dermal fibroblasts. Incubation of human dermal
fibroblasts in culture with TGF␤1 induced a time-
TGF␤1/ET-1 AXIS IN WOUND REPAIR AND SKIN FIBROSIS
881
Figure 2. Involvement of JNK and Smad signaling pathways in the TGF␤1 induction of the ET-1 gene. A, Induction of Smad3 nuclear translocation
by TGF␤1 in the presence or absence of the TGF␤ receptor inhibitor GW788388 (10 ␮M) or the JNK inhibitor SP600125 (25 ␮M) (original
magnification ⫻ 400). Green ⫽ Smad3; blue ⫽ nuclei. B, Smad-specific CAGA-luc reporter activity in human dermal fibroblasts. Results are the
mean and SD normalized units in 4 samples. ⴱ ⫽ P ⬍ 0.05 versus basal conditions without inhibitors; # ⫽ P ⬍ 0.05 versus TGF␤1 cultures without
GW788388. C, JNK activation by TGF␤1 in human dermal fibroblasts, as analyzed by Western blotting using anti–phospho-JNK or anti–total JNK
antibodies (n ⫽ 4). Values above the blots refer to the ratio of the signal for phospho-JNK to that for total JNK after densitometric analysis. D and
E, Effect of JNK inhibition by SP600125 on basal and TGF␤1-induced ET-1 expression in human dermal fibroblasts (D) and on Smad-specific
CAGA-luc reporter activity (E, left) and ET-1 mRNA expression (E, right) in wild-type and Smad3-knockout (KO) mouse embryonic fibroblasts
(MEFs). Results are the mean and SD in 3 samples. ⴱ ⫽ P ⬍ 0.05 versus basal conditions without the inhibitor; # ⫽ P ⬍ 0.05 versus TGF␤1 cultures
without the inhibitor. F, JNK activation by TGF␤1 in wild-type and Smad3-knockout mouse embryonic fibroblasts, and inhibition with GW788388
or SP600124. G, TGF␤1 inducibility of luciferase reporter genes under the control of Smad-and/or activator protein 1 (AP-1)–mutated ET-1
promoter fragments. Results are the mean and SD normalized units in 6 samples per group (untreated [basal; open bars] or treated with TGF␤1
[solid bars], without [X] or with [solid and open boxes] promoter sites). ⴱ ⫽ P ⬍ 0.05 versus basal conditions. See Figure 1 for other definitions.
dependent increase in the steady-state ET-1 mRNA
levels as compared with that in cells incubated under
basal conditions (Figure 1A). The effect of GW788388,
a new inhibitor of TGF␤ receptor type I (activin
receptor–like kinase 5 [ALK-5]) and receptor type II
(21,22), on TGF␤1 induction of ET-1 mRNA was assessed in human dermal fibroblasts. The effects of
TGF␤1 on ET-1 expression and its inhibition by
GW788388 were also observed at the level of ET-1
peptide accumulation in the extracellular medium, as
882
Figure 3. Contribution of the TGF␤1/ET-1 axis to the induction
of the profibrotic phenotype in human dermal fibroblasts. The effect of TGF␤1 and ET-1 on the expression of ␣-smooth muscle
actin (␣-SMA) was analyzed by incubation of human dermal fibroblasts with TGF␤1 (10 ng/ml) (A) or ET–1 (100 nM) (B) in the
presence or absence of GW788388 or bosentan (10 ␮M), respectively. Changes in the expression of ␣-SMA mRNA were determined
by reverse transcription–polymerase chain reaction. Bars show the
mean ⫾ SD fold induction in 3 samples per group. ⴱ ⫽ P ⬍ 0.05
versus basal conditions in the absence of stimulus; # ⫽ P ⬍ 0.05
versus TGF␤1 or ET-1 cultures without inhibitor. C, Levels of ␣SMA protein, as analyzed by Western blotting in cells incubated
with TGF␤1 or ET-1 in the presence or absence of GW788388 or
bosentan, respectively (n ⫽ 4). Values above the blots refer to the
results normalized to the values from densitometric detection of
␤-actin. D, Results of immunofluorescence microscopy analysis to
confirm the induction of the expression of ␣-SMA and the effect of
GW788388 and bosentan on TGF␤1/ET-1 effects (original magnification ⫻ 200). Primary anti–␣-SMA was coupled to Alexa 488–labeled
secondary antibody (green signal). Nuclei were stained with 4⬘,6diamidino-2-phenylindole (blue signal). See Figure 1 for other
definitions.
LAGARES ET AL
measured by specific ELISA (Figure 1B), and also at the
level of transcription of the ET–1 gene, as determined by
transfection of a luciferase reporter driven by a ⫺650/
⫹172-bp fragment of the promoter of the human ET-1
gene (Figure 1C).
In addition to the classic Smad-dependent signaling pathway linking TGF␤ activation to transcriptional
regulation, TGF␤ utilizes a number of regulatory
mechanisms to control cellular functions. The bestcharacterized non-Smad pathway is the JNK signaling
cascade. We therefore investigated whether both the
Smad and JNK signaling pathways are activated by
TGF␤1 in human dermal fibroblasts, and also determined to what extent their activation is required for the
induction of expression of the ET-1 gene. As shown in
Figure 2A, incubation of human dermal fibroblasts with
TGF␤1 induced the translocation of Smad3 from the
cytosol to the nucleus, an observation that has been
extensively correlated with its activation. Preincubation
with the TGF␤ receptor inhibitor GW788388 forced
Smad3 to remain in the cytosol, whereas treatment with
the selective JNK inhibitor SP600125 (25 ␮M) did not
alter the effect of TGF␤1.
Functional assessment of the activation of the
Smad signaling pathway was done with a Smad-specific
luciferase reporter (CAGA-luc). As shown in Figure 2B,
TGF␤1-induced luciferase expression was blocked by
GW788388, but not by SP600125. In contrast, the
TGF␤1-induced significant increase in the levels of
phospho-JNK was blocked by both inhibitors (Figure
2C). These results indicate that TGF␤1 independently
activates both the Smad and JNK signaling cascades in
human dermal fibroblasts. We then assessed the effect
of JNK inhibition on the induction of ET-1 expression by
TGF␤1. As shown in Figure 2D, pretreatment with
SP600125 blocked the TGF␤1 induction of ET-1 expression, an effect that was monitored at the level of mRNA
expression, peptide production, and promoter activity.
To analyze whether an intact Smad signaling
cascade is necessary for the regulation of ET-1 transcription, we performed experiments in mouse embryonic
fibroblasts from wild-type and Smad3-knockout animals.
As expected, the response of the Smad-specific luciferase reporter to TGF␤1 was impaired in embryonic
fibroblasts from Smad3-knockout mice (Figure 2E).
Accordingly, TGF␤1 induced the expression of the ET-1
gene in wild-type, but not in Smad3-knockout, mouse
embryonic fibroblasts. However, the action of TGF␤1
on ET-1 expression was blocked by SP600125, whereas
the JNK inhibitor was unable to prevent the induction of
the Smad-specific reporter in wild-type mouse embry-
TGF␤1/ET-1 AXIS IN WOUND REPAIR AND SKIN FIBROSIS
onic fibroblasts. JNK signaling was equally activated by
TGF␤1 in both mouse embryonic fibroblast types, and
was inhibitable by SP600125 and GW788388, as shown
in Figure 2F.
Finally, we used ET-1 promoter luciferase reporters with specific mutations in the Smad and AP-1
transcription factor binding sites to confirm the contribution of these elements to the effect of TGF␤1 in
human dermal fibroblasts. As shown in Figure 2G,
specific alteration of the Smad binding element or the
AP-1 site significantly abolished the TGF␤1 induction of
the ET-1 promoter. Thus, these results indicate that the
Smad and JNK signaling pathways are independently
activated by TGF␤1 in human dermal fibroblasts and
converge to induce the expression of the ET-1 gene.
Role of the TGF␤1/ET-1 axis in the acquisition of
the profibrotic phenotype. We then investigated the
effect of TGF␤1 and ET-1 on the expression of ␣-SMA,
the most reliable marker of myofibroblast differentiation. As shown in Figure 3A, TGF␤1 induced an increase in the steady-state levels of ␣-SMA mRNA in
human dermal fibroblasts, an effect that was abolished
by inhibition with GW788388. Incubation with 100 nM
ET-1 also induced a time-dependent increase in ␣-SMA
expression. This effect occurred through the activation
of ET receptors, since it was blocked by preincubation
with the mixed ETA/ETB antagonist bosentan (10 ␮M)
(Figure 3B). These actions were also corroborated at the
level of protein expression, as confirmed by Western
blotting (Figure 3C) and immunofluorescence analysis
(Figure 3D).
Since ET-1 is a direct downstream target of
TGF␤1 in human dermal fibroblasts, we examined
whether TGF␤1-induced ␣-SMA expression could be
partially mediated by ET-1. As shown in Figures 3C and
D, the ETA/ETB antagonist bosentan reduced the capacity of TGF␤1 to induce the expression of ␣-SMA.
Myofibroblast differentiation is accompanied by
an increased capacity of the cells to synthesize and
contract ECM components. TGF␤1 and ET-1 induced
the expression of type I collagen (results shown in
Supplementary Figure 1, available on the Arthritis &
Rheumatism Web site at http://www3.interscience.
wiley.com/journal/76509746/home), and also promoted
the contraction of the collagen gel matrix (results shown
in Supplementary Figure 2, available on the Arthritis &
Rheumatism Web site), and both effects were inhibited
by pretreatment with GW788388 and bosentan, respectively. Preincubation with bosentan significantly reduced
the effects of TGF␤1 on type I collagen expression and
gel contraction. These results suggest that the TGF␤1/
883
ET-1 axis promotes the differentiation of fibroblasts to
profibrotic and contractile myofibroblasts, and therefore, it constitutes a candidate signaling pathway for
tissue repair and fibrogenesis in vivo.
Association of overexpression of TGF␤1 with
accelerated skin wound healing and increased collagen
deposition, in an ET-1–dependent manner. Normal skin
wound healing involves the migration of adjacent fibroblasts to the wound and their differentiation to myofibroblasts, which play an active role in the synthesis and
contraction of ECM components. TGF␤1, due to its
ability to drive this transition, has become a fundamental
target for scar-reducing therapeutic strategies (23,24).
Nevertheless, TGF␤1 is an archetype of pleiotropy, a
feature that hampers the use of TGF␤-targeting approaches. In this regard, therapeutic strategies acting on
targets downstream of TGF␤1, such as ET-1, could be of
benefit in clinical practice.
To study the contribution of the TGF␤1/ET-1
axis to the process of skin wound healing in vivo, we
studied mouse skin biopsy samples using the dermal
punch-wound model, and we monitored wound closure
over a 15-day time period either in control conditions or
in cultures with induced overexpression of active TGF␤1
by subcutaneous injection of TGF␤1-overexpressing
adenoviruses (1010 plaque-forming units of Adv-TGF␤1).
This experimental technique was performed in mice
receiving bosentan (100 mg/kg by daily oral gavage) or
vehicle control. As shown in Figure 4A, subcutaneous
injection of Adv-TGF␤1 resulted in significant increases
in the levels of TGF␤1, as detected by specific ELISA, in
wound biopsy tissue on days 3 and 15 after punching,
when compared with control untreated animals. Interestingly, the quantification of levels of ET-1 also revealed significantly higher levels of the peptide in
TGF␤1-overexpressing wounds (Figure 4A).
Macroscopic examination of wound closure
showed a marked acceleration in skin wound healing in
mice with adenovirus-driven TGF␤1 overexpression as
compared with control animals (Figure 4B). Mice
treated with empty backbone adenoviruses did not differ
from control untreated animals (results not shown). As
shown in Figure 4B, cotreatment of the animals with the
mixed ETA/ETB receptor antagonist bosentan prevented
this acceleration of wound closure. Treatment with
bosentan alone did not show any significant effect on
wound closure in control animals (results not shown).
Histologic examination using Masson’s trichrome
staining of tissue sections from mice on days 7 and 15
after wounding further confirmed the effect of the
overexpression of TGF␤1 (see representative sections
884
.
Figure 4. Enhancement of wound closure and collagen deposition in
mouse skin by adenovirus-induced overexpression of TGF␤1, in an
ET-1–dependent manner. Skin wound healing was analyzed using the
dermal punch-wound model in control mice (open bars) or mice
receiving adenovirus for the overexpression of active rat TGF␤1
(Adv-TGF␤1) (solid bars) without or with treatment with the inhibitor
bosentan (100 mg/kg/day by oral gavage in 5% gummi arabicum) (gray
shaded bars). A, Protein levels of TGF␤1 (left) and ET-1 (right), as
determined by specific enzyme-linked immunosorbent assay. Bars
show the mean and SD results in 10 mice per group. ⴱ ⫽ P ⬍ 0.05 versus
controls at the corresponding time point. B, Kinetics of wound closure.
Representative photographs (left) and analysis of wound diameter (right)
are shown for each group. Bars show the mean and SD in 8 skin samples
per group. ⴱ ⫽ P ⬍ 0.05 versus control; # ⫽ P ⬍ 0.05 versus TGF␤1
overexpression without inhibitor. Scale bar ⫽ 3 mm. C, Histologic
examination of wound closure with hematoxylin and eosin and Masson’s
trichrome staining of wound-tissue sections. Representative data from 6
animals per group are shown. Arrows indicate the leading edges of
wounded dermis. Higher-magnification insets (original magnification
⫻ 200) show the density of dermis collagen networks for each experimental treatment. Scale bar ⫽ 200 ␮m. D, Collagen determination by
hydroxyproline assay. Bars show the mean and SD ␮g hydroxyproline per
mg tissue in 8 samples per group. ⴱ ⫽ P ⬍ 0.05 versus control; # ⫽ P ⬍
0.05 versus TGF␤1 overexpression without inhibitor. See Figure 1 for
other definitions.
LAGARES ET AL
and insets in Figure 4C) and also revealed a more
intense staining indicative of an increased presence of
collagen in the newly synthesized dermal layer of animals with adenovirus-driven TGF␤1 overexpression.
Furthermore, these qualitative changes, i.e., diminished
wound diameter and enhanced collagen content, were
prevented by treatment with bosentan. As shown in the
higher-magnification insets in Figure 4C, a significantly
more disorganized collagen fiber network was observed
in the dermis of animals treated with Adv-TGF␤1 alone
compared with those that also received bosentan or
compared with control untreated animals. Quantitative
analysis of wound collagen synthesis using the hydroxyproline assay showed enhanced collagen production on days 3, 7, and 15 in mice receiving Adv-TGF␤1
compared with controls (Figure 4D). Again, the administration of bosentan prevented the increase in collagen
content.
Consistent with the findings in previous reports,
our results indicate that TGF␤1 accelerates wound
closure, and that this effect is correlated with a marked
increase in collagen deposition (25). By antagonizing ET
receptors with bosentan, we found that ET-1 is a downstream mediator of the actions of TGF␤1 on skin wound
healing. To further confirm the involvement of ET-1 in
the observed effects, we induced the overexpression of
ET-1 by application of Adv–ET-1. As shown in Figure
5A, ELISA-specific detection of ET-1 in wound biopsy
tissue showed increased ET-1 levels in adenovirustreated animals compared with control untreated mice.
On macroscopic examination of the wound-closure kinetics, we found that ET-1–overexpressing wounds
closed faster than control wounds (Figures 5B and C). In
addition, Masson’s trichrome staining revealed a significantly higher collagen content in wounds from mice
treated with Adv–ET-1 compared with that in control
wounds (see representative sections and insets in Figure
5D), an observation that was quantitatively corroborated
with the hydroxyproline assay (Figure 5E).
Taken together, these results indicate that increased levels of TGF␤1 prompt the acceleration of skin
wound closure and increase collagen deposition, and
that the actions of ET-1, a downstream target of TGF␤1,
may account for these effects. Enhanced pathologic
healing and synthesis of collagen are features of excessive scarring and fibrogenesis. Therefore, activation of
the TGF␤1/ET-1 axis might contribute to the formation
of fibrotic scars in pathologic wound healing.
Involvement of the TGF␤1/ET-1 axis in the fibrotic response in the mouse model of bleomycininduced skin fibrosis. To further analyze the contribution of TGF␤1 and ET-1 to the development of fibrosis
TGF␤1/ET-1 AXIS IN WOUND REPAIR AND SKIN FIBROSIS
Figure 5. Acceleration of wound closure and increase in collagen deposition in mouse skin by overexpression of ET-1. Wound closure was
analyzed in control mice (open bars) relative to mice receiving adenovirus
for the overexpression of ET-1 (Adv–ET-1) (solid bars). A, Peptide ET-1
levels from wound biopsy samples at the corresponding times after
wounding, as determined by specific enzyme-linked immunosorbent
assay. Bars show the mean and SD in 6 samples per group. ⴱ ⫽ P ⬍ 0.05
versus control at the corresponding time point. B, Kinetics of wound
closure. Representative photographs of wounds obtained on days 0, 7, and
10 after wounding are shown. Scale bar ⫽ 3 mm. C, Analysis of wound
diameter. Bars show the mean and SD in 10 samples per group. ⴱ ⫽ P ⬍
0.05 versus control at the corresponding time point. D, Histologic examination of wound closure with hematoxylin and eosin and Masson’s
trichrome staining of wound-tissue sections. Arrows indicate the leading
edges of wounded dermis. Higher-magnification insets (original magnification ⫻ 200) show the density of dermis collagen networks for each
experimental treatment. Scale bar ⫽ 200 ␮m. E, Collagen determination
by hydroxyproline assay. Bars show the mean and SD in 6 samples per
group. ⴱ ⫽ P ⬍ 0.05 versus control at the corresponding time point. See
Figure 1 for other definitions.
885
in vivo, we used a mouse model of bleomycin-induced
skin fibrosis (20,26). According to previous reports in
the literature, TGF␤1 plays a key role in the development of skin fibrosis in this model (5–8). To confirm the
involvement of TGF␤1 and to analyze the contribution
of ET-1, we tested the effect of GW788388 and bosentan
in the fibrosis induced by bleomycin.
Subcutaneous injections of bleomycin administered daily for 4 weeks resulted in dermal fibrosis in
C3H/HeJ mice. Histologic examination of the lesional
skin demonstrated a considerable increase in dermal
thickness, with a significant accumulation of densely
packed parallel collagen bundles and replacement of
subcutaneous fat by connective tissue, as revealed by
hematoxylin and eosin and Masson’s trichrome staining
(Figure 6A). Quantitative analysis of the dermal thickness of bleomycin-injected skin showed a 2-fold increase
in thickness as compared with that of PBS-injected skin
(Figure 6B). Determination of the hydroxyproline content of skin biopsy samples also showed a 2-to 3-fold
increase in bleomycin-injected skin as compared with
PBS-injected skin (Figure 6C).
The administration of GW788388 (dissolved in
20% DMSO and administered via intraperitoneal injection at 2 mg/kg/day) to bleomycin-injected animals reduced the fibrotic response compared with that in
animals treated with bleomycin alone, as shown by the
diminished dermal thickness and hydroxyproline content
in the bleomycin-injected GW788388-treated group.
The treatment of animals with GW788388 alone or with
the vehicle DMSO did not produce any significant effect
(Figures 6B and C).
We also assayed the effect of bosentan in the
bleomycin-induced skin fibrosis model. As shown in
Figure 6, the administration of bosentan partially prevented the fibrosis induced by bleomycin. These results
point to an essential role for TGF␤1 and ET-1 in the
development of fibrosis in this model. In fact, RT-PCR
determination of the levels of TGF␤1 and ET-1 mRNA
revealed an increased expression of both factors in skin
biopsy tissue from bleomycin-injected mice compared
with PBS-injected mice. Interestingly, the administration
of GW788388 and bosentan significantly downregulated the TGF␤1 mRNA levels to values found in
untreated animals. These inhibitors also showed a tendency to reduce ET-1 mRNA levels toward control
levels, but, in this particular case, the effect was not
statistically significant (Figure 6D).
We also examined the expression of ␣-SMA as a
bona fide marker of the differentiation of fibroblasts
to myofibroblasts, which act to synthesize and contract
the ECM. Bleomycin treatment was accompanied by
886
LAGARES ET AL
Figure 6. Prevention of the fibrotic response by GW788388 and bosentan in the mouse model of bleomycin-induced skin fibrosis. Skin fibrosis
was induced in mice by subcutaneous injection of bleomycin (100 ␮l, or 0.5 mg/ml, in phosphate buffered saline [PBS] daily for 4 weeks). A,
Representative tissue sections stained for assessment of skin structure with hematoxylin and eosin (upper panels) or for estimation of collagen
content with Masson’s trichrome (lower panels) in skin samples from mice left untreated (control), bleomycin-injected (bleomycin),
bleomycin-injected and treated with bosentan (Bleo⫹Bos), or bleomycin-injected and treated with GW788388 (intraperitoneally at 2 mg/kg/day)
(Bleo⫹GW). Scale bar ⫽ 100 ␮m. Higher-magnification insets (original magnification ⫻ 200) show the density of dermis collagen networks
for each experimental treatment. B–D, Analysis of dermal thickness of tissue sections (B), hydroxyproline content (C), and mouse ET-1 and
TGF␤1 mRNA levels (D) determined in skin biopsy tissue obtained from the indicated groups of mice. Control groups included untreated mice
or mice injected with PBS or DMSO as vehicle control. Bars show the mean and SD in 6 samples per group. ⴱ ⫽ P ⬍ 0.05 versus all controls;
# ⫽ P ⬍ 0.05 versus bleomycin treatment without inhibitor. See Figure 1 for other definitions.
an increased expression of ␣-SMA (see Supplementary
Figure 3, available on the Arthritis & Rheumatism Web
site at http://www3.interscience.wiley.com/journal/
76509746/home). The concomitant administration of
bleomycin with GW788388 or bosentan resulted in a
reduction in the expression of ␣–SMA as compared with
that in the group of animals injected only with bleomycin. Taken together, these results are consistent with the
notion that TGF␤1 and ET-1 play a fundamental role in
the development of fibrosis in vivo in this mouse model.
DISCUSSION
Myofibroblasts are responsible for the synthesis
and contraction of ECM components during the process
of wound healing (3). In normal wound repair, myofibroblasts disappear and organ function is restored.
However, fibrotic diseases are characterized by the
persistence of myofibroblasts, resulting in the accumulation of a highly contracted matrix, which is a feature of
scar tissue. These events are regulated by a complex set
of profibrotic and antifibrotic factors, among which,
TGF␤ family members have been proposed to be key
mediators, and TGF␤1 is the most relevant. TGF␤1
exerts its action through the induction (or repression) of
an important number of genes that function as downstream targets and contribute to its effects in a coordinated way (4,27). Herein we have shown that TGF␤1
induces the expression of ET–1 in human dermal fibroblasts, and this induction involves a transcriptional activation of the ET-1 gene. We have also explored the
signal transduction pathways responsible for this action
of TGF␤1. Human dermal fibroblasts in culture exhibited an active Smad- and AP-1/JNK–dependent signaling pathway that was utilized by TGF␤1 to increase the
expression of the ET-1 gene, a result which is consistent
with previous observations from our group in vascular
endothelial cells (18). This finding is in contrast to that
observed in lung fibroblasts, in which the action of
TGF␤1 on ET-1 expression relied only on the JNK/AP-1
signaling pathway, a fact suggesting a cell-type–specific
phenomenon (13).
Having demonstrated that human dermal fibro-
TGF␤1/ET-1 AXIS IN WOUND REPAIR AND SKIN FIBROSIS
blasts possess an active TGF␤1/ET-1 axis, and considering that both factors have been proposed to be mediators of fibrogenic responses, we also assessed their role
in the acquisition of a profibrotic phenotype in these
cells. Our results confirmed those of previous reports
showing that TGF␤1 is an inducer of the expression of
␣-SMA and type I collagen (28,29). Herein we have
shown that ET-1 is also able to increase the expression
of both profibrotic proteins. Interestingly, we also observed that bosentan impairs the capacity of TGF␤1 to
induce the expression of both ␣-SMA and type I collagen in human dermal fibroblasts, an observation suggesting that ET-1 is a downstream mediator of these
actions of TGF␤1. Previous reports have already shown
that ET-1 promotes a profibrotic phenotype in human
dermal fibroblasts, and that the antagonism of ET
receptors, using either ETA-specific or mixed ETA/ETB–
specific antagonists, blocks this action (13,14).
Another important issue addressed was how
ET-1 signaling contributes to the effect of TGF␤1, since
TGF␤1 itself is able to induce the expression of most
fibrotic genes. In this respect, subcutaneous injection of
TGF␤1 alone resulted in transient fibrosis that was
dependent on the continuous application of ligand,
whereas a sustained fibrotic response to TGF␤1 requires
an additional stimulus (30,31). Consistent with this
notion, recent studies have shown that TGF␤1 and ET-1
act in cooperation to induce myofibroblast formation
and collagen synthesis (28,32). Therefore, these observations and our own data suggest a role for ET-1 in the
persistence of the fibrotic phenotype.
Our findings and those from other groups point
to a fundamental role for the TGF␤1/ET-1 axis in
wound healing and fibrosis. In order to validate this
hypothesis in vivo, we examined its contribution to these
processes in the punch model of skin wound healing.
Adenovirus-induced overexpression of active TGF␤1
resulted in accelerated pathologic healing and increased
collagen content. This result is consistent with previous
observations showing that exogenous TGF␤1 prompted
wound closure and collagen deposition (25). Accordingly, it has also been reported that experimental approaches targeting TGF␤1 using neutralizing antibodies
or antisense RNA were effective in reducing matrix
deposition and scar formation (9,10). By antagonizing
ET-1 receptors with bosentan, we found that an important portion of the effect of TGF␤1 was mediated by
ET-1.
Considering that treatment with bosentan might
have actions unrelated to ET-1 signaling, we also examined the effect of the adenovirus-induced overexpression
887
of ET–1. Similar to the findings on TGF␤1, our results
demonstrated that increased ET-1 levels are associated
with accelerated wound closure and increased matrix
deposition. The present study is the first to describe a
fundamental role for ET-1 in the process of skin wound
healing, and the results support the notion that the
actions of TGF␤1 in vitro and in vivo are mediated by
ET-1. Interestingly, treatment with bosentan did not
alter the wound healing response in the absence of
overexpression of TGF␤1. Therefore, it seems that ET-1
blockade does not appreciably affect the process of
normal healing, but instead, ET-1 appears to contribute
to pathologic wound healing responses. This finding is in
concordance with previous observations indicating that
ET-1–mediated profibrotic actions occur only in pathologic settings (13,33). Regarding the potential therapeutic use of ET-1 antagonists, although the time window of
our animal studies did not allow us to specifically assess
the process of scarring, the observation that bosentan
treatment prevents collagen deposition suggests that this
drug may be effective in diminishing the formation of
scar tissue.
We also confirmed the profibrotic potential of
the TGF␤1/ET-1 axis in the mouse model of bleomycininduced skin fibrosis. Previous reports have already
demonstrated that TGF␤1 plays a key role in the
development of fibrosis in this model (5–8). Our study
supports this notion, in that our findings indicated
1) increased expression of TGF␤1 in skin biopsy tissue
from treated animals, and 2) prevention of the fibrotic
response by coadministration of the inhibitor
GW788388 to bleomycin-injected mice. This molecule,
which first was identified as an inhibitor of the ALK-5
form of the TGF␤ type I receptor and then also shown
to have inhibitory effects on the type II receptor, has
been shown to decrease renal fibrosis in the db/db mouse
model of spontaneous diabetic nephropathy (21,22).
Our results also indicated that this model of fibrosis is
characterized by enhanced ET–1 expression, and that
the antagonism of ET receptors with bosentan attenuates the fibrotic response. These data extend previous
observations of the effectiveness of bosentan in a model
of bleomycin-induced pulmonary fibrosis, and indeed
point to a fundamental role of the TGF␤1/ET-1 axis in
the molecular mechanisms leading to fibrosis (34).
Collectively, the results of the present study
support the notion of a role for ET-1 in the pathogenesis
of tissue repair and fibrosis, and also support the use of
bosentan for the prevention of scar formation or the
treatment of fibrotic diseases in clinical practice. In fact,
bosentan (Tracleer; Actelion Pharmaceuticals) was the
888
LAGARES ET AL
first orally active ET receptor antagonist compound to
be used clinically for the treatment of pulmonary arterial
hypertension and to prevent formation of new digital
ulcers in scleroderma patients (15,16).
However, use of bosentan for the indication of
SSc-associated fibrosis is still controversial. The first
clinical trials performed for the treatment of idiopathic
pulmonary fibrosis and SSc-associated interstitial lung
disease, known as the Bosentan versus Placebo in Interstitial Lung Disease Secondary to Systemic Sclerosis
phase I (BUILD-1) and BUILD-2 trials, respectively,
yielded negative results (35,36). In contrast, specific
studies addressing the performance of bosentan in SSc
patients with digital ulceration (the Randomized
Placebo-Controlled Study on the Prevention of Ischemic
Digital Ulcers Secondary to Systemic Sclerosis phase I
[RAPIDS-1] and RAPIDS-2 trials) have shown a significant reduction in the occurrence of new digital ulcers.
However, its use was not associated with improved
healing of existing active ulcers (15,37). Additional
studies performed recently for the evaluation of bosentan in the treatment of cutaneous fibrosis in connective
tissue diseases showed a significant improvement in
specific skin-related symptoms (38–40). For final conclusions to be drawn, more experimental and clinical
studies are needed.
In summary, the results of the present study
strongly support a role for the TGF␤1/ET–1 axis in
tissue repair and fibrosis, both in vitro and in vivo. The
finding that bosentan significantly abrogated the fibrotic
response suggests it may be a useful therapeutic tool in
the treatment of fibrosis-related diseases.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Rodrı́guez-Pascual had full access
to all of the data in the study and takes responsibility for the integrity
of the data and the accuracy of the data analysis.
Study conception and design. Lagares, Lamas, Rodrı́guez-Pascual.
Acquisition of data. Lagares, Garcı́a-Fernández, López Jiménez,
Magán-Marchal, Busnadiego.
Analysis and interpretation of data. Lagares, Garcı́a-Fernández,
Lamas, Rodrı́guez-Pascual.
ROLE OF THE STUDY SPONSOR
Actelion Pharmaceuticals supplied the ET receptor antagonist bosentan. Actelion Pharmaceuticals had no role in the study
design, collection, analysis, or interpretation of the data or writing of
the manuscript. The study sponsor has not revised the content of the
submitted manuscript, and publication of this article was not contingent on approval by the study sponsor.
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