J C E M
O N L I N E
Hot Topics in Translational Endocrinology—Endocrine Research
Fasudil Inhibits the Proliferation and Contractility and
Induces Cell Cycle Arrest and Apoptosis of Human
Endometriotic Stromal Cells: A Promising Agent for
the Treatment of Endometriosis
Akitoshi Tsuno, Kaei Nasu, Yukie Kawano, Akitoshi Yuge, Haili Li, Wakana Abe,
and Hisashi Narahara
Department of Obstetrics and Gynecology, Faculty of Medicine, Oita University, Oita 879-5593, Japan
Context: During the development of endometriotic lesions, excess fibrosis may lead to scarring and
to the alterations of tissue function that are the characteristic features of this disease. Enhanced
extracellular matrix contractility of endometriotic stromal cells (ECSC) mediated by the mevalonate-Ras homology (Rho)/Rho-associated coiled-coil-forming protein kinase (ROCK) pathway has
been shown to contribute to the pathogenesis of endometriosis.
Design: To assess the use of fasudil, a selective ROCK inhibitor, for the medical treatment of
endometriosis-associated fibrosis, the effects of this agent on the cell proliferation, apoptosis, cell
cycle, morphology, cell density, and contractility of ECSC were investigated. The effects of fasudil
on the expression of contractility-related, apoptosis-related, and cell cycle-related molecules in
ECSC were also evaluated.
Results: Fasudil significantly inhibited the proliferation and contractility of ECSC and induced the
cell cycle arrest in the G2/M phase and apoptosis of these cells. Morphological observation revealed
the suppression of ECSC attachment to collagen fibers and decrease of cell density by fasudil. The
expression of ␣-smooth muscle actin, RhoA, ROCK-I, and ROCK-II proteins was inhibited by fasudil
administration. The expression of the antiapoptotic factors, Bcl-2 and Bcl-XL, in two-dimensional
cultured ECSC were down-regulated by the addition of fasudil, whereas, the expression of p16INK4a
and p21Waf1/Cip1 was up-regulated by the addition of fasudil.
Conclusions: The present findings suggest that fasudil is a promising agent for the treatment
of endometriosis. The inhibition of cell proliferation, contractility, and myofibroblastic differentiation, the attenuation of attachment to collagen fibers, the decrease of cell density, and
the induction of cell cycle arrest and apoptosis of ECSC are involved in the active mechanisms
of fasudil. (J Clin Endocrinol Metab 96: E1944 –E1952, 2011)
E
ndometriosis, a benign estrogen-dependent disease affecting 3–10% of women of reproductive age, is
characterized by the ectopic growth of endometrial tissue
and is associated with pelvic pain, dysmenorrhea, and infertility (1). Histologically, this disease is characterized by
dense fibrous tissue surrounding the endometrial glands
and stroma (2). During the development and progression
of endometriotic lesions, excess fibrosis may lead to scar-
ring, chronic pain, and the alterations of tissue function,
all of which are characteristics of this disease (2, 3). Fibroblastic cells positive for ␣-smooth muscle actin (SMA)
are frequently detected in fibrotic areas associated with
endometriosis of the peritoneum, ovary, rectovaginal septum, and uterosacral ligaments (2, 4). Immunohistochemical analysis led Anaf et al. (4) to suggest that endometriotic stromal cells can differentiate into ␣-SMA-positive
ISSN Print 0021-972X ISSN Online 1945-7197
Printed in U.S.A.
Copyright © 2011 by The Endocrine Society
doi: 10.1210/jc.2011-1503 Received May 16, 2011. Accepted August 23, 2011.
First Published Online September 15, 2011
Abbreviations: BrdU, 5-Bromo-2⬘-deoxyuridine; 3-D, three-dimensional; ECSC, endometriotic stromal cells; FBS, fetal bovine serum; Rho, Ras homology; ROCK, Rho-associated
coiled-coil-forming protein kinase; SMA, ␣-smooth muscle actin; TBS-T, Tris-buffered saline with Tween 20.
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myofibroblasts. It has been suggested that type I collagen
is a major contributor to endometriosis-associated fibrosis
(3, 5). One approach to understanding the pathogenesis of
endometriosis is to investigate the mechanisms underlying
the fibrogenesis associated with this disease.
We have established a three-dimensional (3-D) collagen gel culture system with human endometriotic stromal
cells (ECSC) as a model of fibrosis formation in endometriosis (6 –9). In this system, ECSC are cultured in floating
collagen lattices to induce the reorganization and compaction of collagen fibers, resulting in the contraction of
collagen gels. This culture system provides a model of mechanically relaxed tissue with low tensile strength comparable to that in the early developmental stages of endometriotic lesions. Research on endometriotic stromal cell
biology in 3-D collagen matrices offers new opportunities
to gain a better understanding of the reciprocal and
adaptive interactions that take place between cells and
the surrounding matrix in a tissue-like environment.
Such interactions are integrated with the regulation of
endometriotic tissue morphogenesis and the dynamics
that characterize endometriosis-associated fibrosis. Using this model, we demonstrated that ECSC cultured in
floating 3-D collagen gel exhibit a more enhanced contractile profile and greater ability to differentiate into a
myofibroblastic phenotype than do normal endometrial
stromal cells (6 –9). Activation of the mevalonate-Ras
homology (Rho)/Rho-associated coiled-coil-forming
protein kinase (ROCK)-mediated pathway (Fig. 1) in
ECSC may be involved in this phenomenon (6 –10).
Therefore, it has been suggested that promising drugs
for the treatment of endometriosis may eventually be
derived from agents that suppress fibrosis formation by
targeting the Rho-ROCK-mediated pathway as well as
myofibroblastic differentiation. The pyridine derivative
Y-27632, the isoquinoline sulfonamide derivative fasudil
[hexahydro-1-(5-isoquinolinesulfony1)-1H-1,4-diazepine hydrochloride, HA1077], and its active metabolite hydroxyfasudil have recently been shown to be selective inhibitors of
ROCK (11). We have previously demonstrated that Y-27632
attenuated the contractility of ECSC (6).
Fasudil (Fig. 1) has been marketed since 1995 in Japan
for the treatment of ischemic stroke (12, 13), cerebral vasospasm (14), and subarachnoid hemorrhage (15). Fasudil
and hydroxyfasudil selectively inhibit ROCK by competing with ATP for binding to the kinase and show minimal
effects on other intracellular signaling pathways, such as
myosin light chain kinase, protein kinase A, protein kinase
C, or protein kinase G (11, 16, 17). In the present study,
we investigated the effects of fasudil on the proliferation,
apoptosis, and contractility of ECSC. We also discuss a
novel therapeutic strategy for endometriosis-associated
fibrosis.
FIG. 1. Mechanism of action of fasudil on the mevalonate-Rho/ROCK
pathways and the chemical structure of fasudil. Acetyl-CoA, Acetylcoenzyme A; ECM, extracellular matrix; FPP, farnesyl pyrophosphate;
GGPP, geranylgeranyl pyrophosphate; HMG-CoA, 3-hydroxy-3methylglutarylcoenzyme A.
Modified methylthiazoletetrazolium assay
Materials and Methods
ECSC isolation procedure and cell culture
conditions
Endometriotic tissues were obtained from premenopausal patients in the mid-to-late proliferative phase who had undergone
salpingo-oophorectomy or cystectomy for ovarian endometriotic cysts (n ⫽ 8, aged 26 –34 yr). None of the patients had
undergone any hormonal treatments for at least 12 months before the operation. This study was approved by the Institutional
Review Board of the Faculty of Medicine at Oita University.
ECSC were isolated from ovarian endometriotic tissues by enzymatic digestion as previously described (18). Isolated ECSC
were cultured in DMEM supplemented with 100 IU/ml penicillin
(Life Technologies, Inc.-BRL, Gaithersburg, MD), 50 mg/ml
streptomycin (Life Technologies, Inc.-BRL), and 10% heat-inactivated fetal bovine serum (FBS) (Life Technologies, Inc.-BRL)
at 37 C in 5% CO2 in air.
After the third passage, the ECSC in the monolayer culture
were more than 99% pure, as determined by immunocytochemical staining with antibodies to vimentin (V9; Dako, Copenhagen, Denmark), CD10 (SS2/36; Dako), cytokeratin (Dako), factor VIII (Dako), and leukocyte common antigen (2B11⫹PD7/26;
Dako). These ECSC were used for the following experiments
(18). Each experiment was performed in triplicate and repeated
at least four times.
The rate of proliferation of ECSC after fasudil treatment was
determined in 96-well plates by a modified methylthiazoletetrazolium assay system using WST-1 (Roche Diagnostics GmbH,
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Penzberg, Germany) according to the manufacturer’s protocol.
Then, 5 ⫻ 104 ECSC in DMEM supplemented with 1% BSA
(Sigma-Aldrich, St. Louis, MO) were distributed into each well
of a 96-well flat-bottomed microplate (Corning, New York,
NY), which was then incubated overnight. The medium was then
removed, and the cells were incubated for 48 h with 200 ␮l
experimental medium containing various concentrations of fasudil (0.1–100 ␮M) (BIOMOL International, L.P., Plymouth
Meeting, PA). Thereafter, 20 ␮l WST-1 dye was added to each
well. The wells were then incubated for an additional 4 h. Cell
proliferation was evaluated by measuring the light absorbance at
540 nm. Data were calculated as the ratio of values obtained for
the treated cells and for the untreated controls.
5-Bromo-2ⴕ-deoxyuridine (BrdU) incorporation
assay
The effects of fasudil on the DNA synthesis of ECSC were
also determined by BrdU incorporation using cell proliferation ELISA (Roche Diagnostics). ECSC (1 ⫻ 104) in DMEM
supplemented with 0.1% BSA were distributed into each well
of a 96-well flat-bottomed microplate. After overnight incubation, the medium was removed and the cells were incubated
for another 48 h with 100 ␮l experimental medium containing
various concentrations of fasudil (0.1–100 ␮M). Thereafter,
10 ␮l BrdU (10 mM) was added to each well, and the plates
were further incubated for 2 h. BrdU incorporation was then
evaluated by measuring the light absorbance at 450 nm according to the manufacturer’s protocols. Data were calculated
as the ratio of values obtained for the treated cells and those
for the untreated controls.
Assessment of fasudil-induced cell cycle arrest by
flow cytometry
The effects of fasudil on the cell cycle of ECSC were analyzed
by flow cytometry, as previously described (19). Briefly, ECSC
were cultured at less than 60% confluence for 48 h with or
without the presence of fasudil (100 ␮M). They were then
trypsinized, washed in PBS, fixed in 70% methanol, and incubated for 120 min at 4 C in the dark with a solution of 5 ␮g/ml
propidium iodide, 1 mg/ml ribonuclease (Sigma-Aldrich), and
0.1% Nonidet P-40 (Sigma-Aldrich). Flow cytometric analysis
of the cell cycle was performed after propidium iodide staining
using the CellFIT program (Becton Dickinson, Sunnyvale, CA),
in which the S-phase was calculated using a ModFit model.
Assessment of fasudil-induced apoptosis by a cell
death detection ELISA
Fasudil-induced apoptosis of ECSC was quantified by direct
determination of nucleosomal DNA fragmentation by cell death
detection ELISA (Roche Diagnostics) as previously described
(19). The assay used specific monoclonal antibodies directed
against histones from fragmented DNA, allowing the determination of mono- and oligonucleosomes in the cytoplasmic fraction of cell lysates. Briefly, 1 ⫻ 106 cells were plated on 24-well
culture plates (Corning) and cultured overnight. The supernatant was replaced with fresh culture medium containing various
amounts of fasudil (0.1–100 ␮M). Forty-eight hours after stimulation, the cells were lysed according to the manufacturer’s instructions, followed by centrifugation (200 ⫻ g for 5 min). The
mono- and oligonucleosomes contained in the supernatants were
J Clin Endocrinol Metab, December 2011, 96(12):E1944 –E1952
determined using an anti-histone-biotin antibody. The concentration of nucleosomes-antibody pairs was determined photometrically at a wavelength of 405 nm using 2,2⬘-azino-di(3-ethylbenzthiazoline-sulfonate) as a substrate. Data were calculated
as the ratio of the values obtained for the treated cells and for the
untreated controls.
Collagen gel contraction assay
To assess the effects of fasudil on the contractility of ECSC,
cellular collagen gel contraction assays were performed as previously described (6, 8, 20). A sterile solution of acid-soluble
collagen type I purified from porcine tendons (Cellmatrix type
I-A; Nitta Gelatin Inc., Osaka, Japan) was prepared according to
the manufacturer’s instructions. ECSC were embedded in collagen gel and cultured three-dimensionally. Briefly, the ECSC were
suspended in collagen solution (3.0 ⫻ 105 cells/ml), and this
collagen/cell mixture (2 ml/plate) was dispensed into 35-mm culture plates (Corning) coated with 0.2% BSA, after which the
mixture was allowed to polymerize at 37 C for 30 min. Immediately after polymerization, 1 ml culture medium containing
fasudil (final concentration, 0.1-100 ␮M) was added to each
plate. After incubation for 48 h, the collagen gels were photographed, and the area of the gel surface was measured with the
public domain image program ImageJ version 1.44 developed at
the National Institutes of Health (Bethesda, MD).
Morphology and cell density of ECSC in 3-D
collagen gel culture
Upon termination of the collagen gel contraction assay, the
contracted collagen gels treated with or without fasudil (100 ␮M)
were fixed with 4% paraformaldehyde, stained with Giemsa
solution, and dried overnight on the slide glass. The morphological features and the density of the cells were observed by light
microscopy.
Western blot analysis
The expression of ␣-SMA, Rho A, ROCK-I, and ROCK-II in
ECSC in the above-mentioned 3-D culture and Bcl-2, Bcl-XL,
p16INK4a, and p21Waf1/Cip1 in the conventional 2-D culture were
investigated by Western blotting analysis. ECSC in the 3-D and
2-D cultures were treated with fasudil (100 ␮M) for 48 h. The
contracted collagen gels were minced and incubated in 0.02%
collagenase type I (Sigma-Aldrich) in PBS for 40 min at 37 C. The
3-D cultured ECSC were isolated from collagen gels by centrifugation. Whole-cell extracts of 3-D- and 2-D-cultured ECSC
were prepared by lysing the cells in lysis buffer (50 mM Tris-HCl,
125 mM NaCl, 0.1% Nonidet P-40, 5 mM EDTA, 50 mM NaF,
and 0.1% phenylmethylsulfonyl fluoride). The suspension was
centrifuged at 15,000 rpm for 15 min at 4 C, and the supernatant
was collected. The total protein concentration was quantified
using the Coomassie protein assay reagent (Pierce, Rockford,
IL). The whole-cell protein extract was resolved with SDS-PAGE
using a 10% polyacrylamide gel under reduced conditions. After
transfer to Immobilon-P transfer membrane (Millipore, Bedford,
MA), the protein was stained with Ponceau S (Sigma-Aldrich) to
verify uniform loading and transfer. Membranes were blocked
with 5% skim milk (Becton Dickinson) in Tris-buffered saline
with Tween 20 (TBS-T) [50 mM Tris-HCl, 150 mM NaCl, 0.1%
Tween 20 (pH 7.4)] overnight and subsequently incubated with
primary antibodies [␣-SMA (1A4; R&D Systems, Minneapolis,
MN), Rho A and ROCK-I (Sigma-Aldrich), ROCK-II (Santa
J Clin Endocrinol Metab, December 2011, 96(12):E1944 –E1952
Cruz Biotechnology, Santa Cruz, CA), Bcl-2 (E17; Epitomics,
Inc., Burlingame, CA), Bcl-XL (54H6; Cell Signaling, Beverly,
MA), p16INK4a (EP4353Y; Epitomics), p21Waf1/Cip1 (12D1; Cell
Signaling), and glyceraldehydes-3-phosphate-dehydrogenase
(GAPDH) (Ambion, Austin, TX)] at appropriate dilutions for 1 h
at room temperature. The membrane was washed three times
with TBS-T and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Subsequently, the membrane was washed three times with
TBS-T and analyzed by enhanced chemiluminescence (Amersham Pharmacia Biotech, Chicago, IL). The relative expression
of these proteins in ECSC was analyzed using the public domain
Image program ImageJ version 1.44.
Statistical analysis
Data were calculated as percentages relative to the untreated
controls, presented as means ⫾ SD, and appropriately analyzed
by the Mann-Whitney U test and the t test with Bonferroni correction using Sigmaplot version 11.2 (Systat Software, Inc., San
Jose, CA). Values of P ⬍ 0.05 were considered to be statistically
significant.
Results
Effects of fasudil on the cell proliferation of ECSC
The effects of fasudil on the cell proliferation of ECSC
were assessed by the WST-1 assay. As shown in Fig. 2, the
cell number of viable ECSC was significantly decreased by
addition of increasing amounts of fasudil. Cell viability
decreased to 88.8 ⫾ 1.0% of the initial value after treatment with fasudil at 0.1 ␮M (P ⬍ 0.0005) and further
decreased to 51.7 ⫾ 1.5% after treatment with fasudil at
100 ␮M (P ⬍ 0.0001).
To further assess the effects of fasudil on cell proliferation, DNA synthesis of ECSC after fasudil treatment was
evaluated by the BrdU incorporation assay. As shown in
Fig. 3, DNA synthesis of ECSC was significantly decreased
by the addition of increasing amounts of fasudil. BrdU
incorporation decreased to 75.7 ⫾ 0.8% of the initial
value after treatment with fasudil at 0.1 ␮M (P ⬍ 0.0001)
FIG. 2. Effects of fasudil on the cell viability of ECSC as assessed by
the WST-1 assay. The cell viability of untreated ECSC was defined
as 100%. Data are presented as means ⫾ SD of representative
experiments. *, P ⬍ 0.0005; **, P ⬍ 0.0001 vs. untreated controls
(Bonferroni test).
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FIG. 3. Effects of fasudil on the DNA synthesis of ECSC as assessed by
the BrdU incorporation assay. The DNA synthesis of untreated ECSC
was defined as 100%. Data are presented as means ⫾ SD of
representative experiments. *, P ⬍ 0.0001 vs. untreated controls
(Bonferroni test).
and further decreased to 56.3 ⫾ 0.6% after treatment with
fasudil at 100 ␮M (P ⬍ 0.0001). The inhibitory effects of
fasudil on the cell proliferation of ECSC were diminished
by the addition of 10% FBS to the experimental media
(data not shown).
Effects of fasudil on the of the cell cycle of ECSC
The effects of fasudil on the cell cycle of ECSC were determined by flow cytometry. As shown in Fig. 4, culturing of
ECSC for 48 h in the presence of fasudil (100 ␮M) resulted in
an accumulation of these cells in the G2/M phase of the cell
cycle (4.3 ⫾ 2.1 to 19.2 ⫾ 1.9%, P ⬍ 0.0001), with a concomitant decrease in the proportion of these cells in the S
phase (45.6 ⫾ 0.8 to 25.0 ⫾ 3.5, P ⬍ 0.0001).
Effects of fasudil on the apoptosis of ECSC
The apoptotic effects of fasudil on ECSC were assessed
by evaluating the presence of internucleosomal DNA fragmentation with a cell death detection ELISA. As shown in
Fig. 5, apoptosis of ECSC was significantly induced by the
addition of increasing amounts of fasudil. The proportion
of apoptotic cells increased to 139.7 ⫾ 4.9% of the baseline value after treatment with fasudil at 1 ␮M (P ⬍ 0.0005)
and further increased to 223.3 ⫾ 5.8% after treatment
with fasudil at 100 ␮M (P ⬍ 0.0001). The inhibitory effects
of fasudil on the apoptosis of ECSC were diminished by
FIG. 4. The effects of fasudil on the cell cycle of ECSC. ECSC were
cultured for 48 h in the presence of fasudil (100 ␮M). Fasudil induced
the G2/M phase cell cycle arrest of ECSC, with a concomitant decrease
in the proportion of ECSC in the S phase. *, P ⬍ 0.0001 vs. untreated
controls (Bonferroni test).
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uated by fasudil in a dose-dependent manner (Relative surface area of ECSC treated with at 100 ␮M fasudil was 70.7 ⫾
3.4% that of 0-h controls, P ⬍ 0.0001) (Fig. 6B).
FIG. 5. Effects of fasudil on the apoptosis of ECSC as assessed by cell
death detection ELISA. The number of apoptotic cells of untreated
ECSC was defined as 100%. Data are presented as means ⫾ SD of
representative experiments. *, P ⬍ 0.0005; **, P ⬍ 0.0001 vs.
untreated controls (Bonferroni test).
the addition of 10% FBS to the experimental media (data
not shown).
Effects of fasudil on ECSC-mediated collagen gel
contraction
The effect of fasudil on the contractility of ECSC was
evaluated using a collagen gel contraction assay. The surface
area of the 35-mm culture dish was defined as 100%. In the
presence of 10% FBS, untreated ECSC showed significant
collagen gel contractility after 48 h of 3-D culture (relative
surface area was 20.8 ⫾ 0.6% compared with 0-h controls)
(Fig. 6A). The contractility of ECSC was significantly atten-
Effects of fasudil on the morphology and the cell
density of ECSC in 3-D collagen gel culture
When untreated ECSC were cultured in 3-D collagen
gels, the cells adhered to the collagen fibers, and the initially loose network contracted into a dense tissue-like
structure (Fig. 7A). The morphology was dendritic to stellate. In contrast, the contractile force of the fasudil-treated
ECSC was weak. Moreover, the cell morphology remained round to polygonal, and the cells did not adhere as
readily to the collagen fibers as did the untreated ECSC.
In addition to the morphological changes, the cell density of 3-D cultured ECSC was significantly decreased by
fasudil treatment at the concentration of 100 ␮M (Fig. 7B).
The morphology of 3-D cultured ECSC treated with fasudil at 0.1–10 ␮M was not examined.
Expression of contraction-related proteins,
apoptosis-related proteins, and cell cycle-related
proteins in ECSC
To analyze the underlying mechanisms of the action of
fasudil on the contractility of ECSC, the expressions of
␣-SMA, RhoA, ROCK-I, and ROCK-II proteins in ECSC
FIG. 6. Effects of fasudil on the contractility of ECSC as assessed by collagen gel contraction assay. ECSC were cultured in 3-D collagen gels for
48 h in the presence of fasudil (0.1–100 ␮M). A, The collagen gels were then photographed. The ECSC-mediated collagen gel contraction was
assessed by measuring the gel surface area using an image analysis program. The contractility of untreated ECSC was defined as 100%. B, The
relative contractility of the fasudil-treated ECSC was calculated. Data are presented as means ⫾ SD of representative experiments. *, P ⬍ 0.0001
vs. untreated controls (Bonferroni test).
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expressions of antiapoptotic factors Bcl-2 and
Bcl-XL and cell cycle modulators p16INK4a and
p21Waf1/Cip1 were evaluated. As shown in Fig.
8, the levels of Bcl-2 and Bcl-XL expression in
2-D cultured ECSC were strongly inhibited by
the addition of fasudil (100 ␮M), whereas the
levels of p16INK4a and p21Waf1/Cip1 expression
were strongly up-regulated by the addition of
fasudil (100 ␮M).
Discussion
In an effort to clarify the pathological tissue
remodeling in endometriosis and to establish
novel therapeutic strategies for this enigmatic
disease, our laboratory has been conducting an
ongoing investigation into the contractile profiles, proliferative activities, and gene expression of endometriotic cells. In the present
study, the following points were demonstrated: 1) fasudil inhibits the cell proliferation of
ECSC; 2) fasudil induces the cell cycle arrest of
ECSC at G2/M phase; 3) fasudil induces the
apoptosis of ECSC by down-regulating Bcl-2
and Bcl-XL expression; 4) fasudil inhibits the
contractility of ECSC in vitro; 5) fasudil inhibits the morphological changes of 3-D cultured
ECSC; 6) fasudil decreases the cell density of
3-D cultured ECSC; and 7) fasudil inhibits the
differentiation of ECSC into the myofibroblastic phenotype. All of these effects of fasudil
were suggested to be mediated by the inhibition
of mevalonate-Rho/ROCK-mediated signaling pathways and considered to be beneficial
FIG. 7. Morphological features (A) and cell density (B) of ECSC in 3-D collagen gel
for the treatment of endometriosis-associated
culture. A, Morphological features of the cells after collagen gel contraction assays
were observed after Giemsa staining. Untreated ECSC cultured in 3-D collagen
fibrosis.
gels attached to the surrounding collagen fibers, and the initially loose network
Fasudil is now used for a variety of cardiocontracted into a dense, tissue-like structure. Their cell morphology was dendritic to
vascular,
cerebrovascular, and neurovascular
stellate. In contrast, the contractile force of ECSC treated with 100 ␮M fasudil was
weak, and the cell morphology remained round to polygonal, unlike the untreated
diseases (12–15). This drug is considered to be
ECSC, suggesting that the fasudil-treated cells did not attach to the surrounding
effective without inducing severe side effects.
collagen fibers. B, Cell density of 3-D cultured ECSC after collagen gel contraction
Fasudil has been reported to inhibit ROCK at
assays were also calculated. The cell density of 3-D cultured ECSC was significantly
an inhibition constant (Ki) value of 0.33 mM
decreased by the addition of fasudil (100 ␮M). The cell density of untreated ECSC
was defined as 100%. Data are presented as the means ⫾ SD of a representative
(11), more efficiently than myosin light chain
experiment. *, P ⬍ 0.0025 vs. untreated controls (Bonferroni test).
kinase (17). It has also been reported that fasudil inhibits ROCK selectively with IC50 valwere evaluated. As shown in Fig. 8, the levels of ␣-SMA, ues of 801 and 325 nM for ROCK-I and ROCK-II, respecROCK-I, and ROCK-II protein in 3-D cultured ECSC were tively (21). It has been reported that the maximum drug
significantly inhibited by the addition of fasudil (100 ␮M), concentration (Cmax) for fasudil in 12 healthy elderly volwhereas RhoA expression was not affected by the addition of unteers after iv infusion of fasudil (60 mg/60 min) was
fasudil.
1.03 ⫾ 0.21 ␮M (299.2 ⫾ 62.0 ng/ml) (13). There was no
To analyze the underlying mechanisms of the action of serious adverse event during iv fasudil administration.
fasudil on the proliferation and apoptosis of ECSC, the Clinically, 90 mg/d iv administration of fasudil for 14 d is
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FIG. 8. The effects of fasudil (100 ␮M) on the expression of ␣-SMA, Rho A, ROCK-I, and ROCK-II proteins in ECSC in the 3-D culture and those
of Bcl-2, Bcl-XL, p16INK4a, and p21Waf1/Cip1 proteins in ECSC in the 2-D culture. A, Representative results of Western blot analysis; B–I, relative
expression of RhoA (B), ROCK-I (C), ROCK-II (D), ␣-SMA (E), Bcl-2 (F), Bcl-XL (G), p16INK4a (H), and p21Waf1/Cip1(I). The relative protein expression of
untreated controls was defined as 100%. Data are presented as the means ⫾ SD. *, P ⬍ 0.05 vs. untreated controls (Mann-Whitney U test).
considered a therapeutic dose. Our present data demonstrate that fasudil at the concentration of 0.1 ␮M may
affect the proliferation of ECSC, whereas a higher concentration of fasudil was required to induce apoptosis and
gel contraction of ECSC. In addition, fasudil is rapidly
metabolized in vivo to hydroxyfasudil, which possesses
more selective and stronger ROCK-inhibitory effects than
its parent drug (15). At 45 min after the initiation of iv
infusion, the concentration of hydroxyfasudil present in
plasma was approximately 80% that of the parent drug.
Hydroxyfasudil is eliminated more slowly than fasudil in
vivo. The inhibition activity against ROCK by 0.1 ␮M
fasudil, as used in this in vitro study, would be expected to
be close to inhibition by the serum concentration of fasudil
plus hydroxyfasudil at a clinical dose.
Members of the Rho family of small guanosine triphosphatases (GTPases) are known to regulate cell growth,
morphology, motility, adhesion, migration, and apoptosis, and extracellular matrix contraction through the reorganization of actin cytoskeletons (11, 22–28). The active form of Rho is GTP bound (22, 25), and many
polypeptides have been reported as targets of activated
Rho, ROCK-I, and ROCK-II. The substrates of the ROCK
proteins have been identified, including the myosin phosphatase target-1 subunit, ERN family proteins (ezrin, radixin, and moesin), claudins, adducin, troponin, intermediate filaments (e.g. vimentin and desmin), Na⫹-H⫹
exchangers, and the LIM kinases (Lin11, Isl1, and Mec3)
(29 –31). It is postulated that these target molecules of
fasudil might interact with other signaling pathways,
which are known to contribute to the pathogenesis of endometriosis (10).
It has been suggested that the mevalonate-Rho/ROCK
pathways play important roles in the formation of endometriosis-associated fibrosis. We demonstrated that simvastatin, which acts as a Rho inhibitor by inhibiting 3-hydroxy3-methylglutarylcoenzyme A (HMG-CoA) reductase (32),
reduced the proliferation and contractility of ECSC (7). Morphological observation of simvastatin-treated ECSC suggested that simvastatin attenuated the attachment of
ECSC to collagen fibers, thereby inhibiting the contraction of collagen gels. We have also demonstrated that
Y-27632, a pyridine derivative that acts as a specific inhibitor of ROCK-I and ROCK-II (11, 26), significantly
inhibited ECSC-mediated contractility (6). Although the
precise mechanisms are unknown, it has been demonstrated that heparin (9) and decidualization (8) attenuated
the contractility of ECSC. Based on these findings, it can
be concluded that agents targeting mevalonate-Rho/
ROCK-mediated pathways are promising candidates for
the treatment and prevention of endometriosis-associated
fibrosis.
Recently, in addition to fasudil and Y-27632, novel
ROCK inhibitors with improved potency and selectivity
have been developed for clinical use. These molecules include SAR407899 (33), SB-772077B (34), GSK269962A
(34), SR-715 (35), SR-899 (35), SLx-2119 (36), and
Y-32885 (37). However, fasudil is currently the only clinically available ROCK inhibitor with minimal adverse effects (38). Importantly, fasudil can be administered iv, ip,
J Clin Endocrinol Metab, December 2011, 96(12):E1944 –E1952
and orally (15, 39), which is considered to be beneficial for
the long-term treatment of endometriosis. Although additional animal and clinical studies are necessary, our present findings suggest that targeting Rho-ROCK inhibition
by fasudil is a promising therapeutic strategy for treating
endometriosis.
Acknowledgments
Address all correspondence and requests for reprints to: Kaei Nasu,
M.D., Ph.D., Department of Obstetrics and Gynecology, Faculty of
Medicine, Oita University, Idaigaoka 1-1, Hasama-machi, Yufushi, Oita 879-5593, Japan. E-mail: [email protected].
This work was supported by a Grant-in-Aid for Scientific
Research from the Japan Society for the Promotion of Science
(no. 20591920 to K.N.) and a Grant-in-Aid from the Japan Medical Association (to K.N.).
Disclosure Summary: A. T., K. N., Y. K., A. Y., H. L., W. A.,
and H. N. have nothing to declare.
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