Keywords endometriosis - cell differentiation - cell proliferation - tissue invasion
Palavras-chave endometriose - diferenciação celular - proliferação celular - invasão tecidual
Introduction
The etiopathogenesis of endometriosis is controversial.[1 ] Several theories have been proposed, such as the presence of retrograde menstrual
flow associated with an immunological predisposition in the peritoneal microenvironment
that facilitates the implantation of viable endometrial cells and has the potential
for implantation.[2 ] Recent studies suggest that the endometria of patients with endometriosis have an
invasive and aggressive behavior, and higher expression levels of substances related
to cellular invasion, cellular differentiation, and proliferation, such as metalloproteases
(MMPs) and tissue inhibitors of MMPs (TIMPs),[3 ]
[4 ]
[5 ]
[6 ]
[7 ]
[8 ]
[9 ]
[10 ]
[11 ] p63,[12 ]
[13 ]
[14 ]
[15 ]
[16 ] and metallothionein (MT)[17 ]
[18 ]
[19 ] have been described.
Metalloproteases are a family of endopeptidases that play a role in degrading and
remodeling the extracellular matrix. They are zinc dependent and include collagenase,
gelatinase, and stromal enzymes. Their activities are regulated by TIMPs.[20 ] The production of MMPs and of TIMPs occurs in the endometrial stroma and in the
epithelium, as well as in polymorphonuclear leucocytes. Another important source of
these enzymes are macrophages, neutrophils, and eosinophils, activated in response
to a certain degree of inflammation present in the peritoneal cavity of women with
endometriosis.[21 ]
[22 ]
[23 ]
The membrane protein p63 is a marker of cell differentiation, homologous to the tumor
protein suppressor p53, and is expressed in basal squamous and subcolumnar reserve
cells in the uterine cervix, in the breasts, in the salivary glands, and in the prostate.[24 ] It regulates proliferation and epithelial differentiation.[25 ]
Metallothionein is a low-molecular-weight protein that performs functions in cell
growth, repair, and proliferation.[26 ] The perinuclear location of MT is known to be important in the protection against
DNA damage and apoptosis induced by external stressors.[27 ]
[28 ]
The use of female rabbits in experimental models of endometriosis is characterized
by the development of homogeneous lesions, generally solid hemorrhagic masses, which
are easily produced through an autotransplant of endometrial fragments or through
the opening and exposure of the endometrial cavity.[29 ]
[30 ] In addition, rabbits were chosen as the experimental animals because of their low
infection rate, which makes antibiotic administration unnecessary.[31 ]
In the experimental model conducted in our service,[32 ] the development of lesions after endometrial tissue implantation was 100% after
4 and 8 weeks, with the presence of stroma and gland on histological observation,
a fact also reported in other experimental studies.[31 ]
[33 ]
The objective of the present study was to characterize the proliferation, differentiation,
and invasion behavior in eutopic and ectopic endometria in rabbits submitted to the
induction of endometriosis lesions by using a known experimental model, 4 and 8 weeks
after the endometrial implantation procedure.
Methods
Animals
The present study was performed in the experimental surgery sector of the department
of surgery and anatomy of the Hospital das Clínicas, Ribeirão Preto, State of São
Paulo and in the department of pathology of the Faculty of Medicine of Riberão Preto
of the Universidade de São Paulo, state of São Paulo, Brazil. It was approved by the
ethics committee for animal experimentation by the same institution. After the sample
calculation, the size of the study group was set at 10 animals. However, considering
the possibility of losses, 15 adult animals were included per group, with one loss
(death) during the experiment, therefore totaling 29 adult New Zealand female virgin
rabbits from the vivarium of the Faculty of Medicine of Ribeirão Preto. The rabbits
were kept in appropriate cages under the same conditions for 3 days before the induction
of the lesions. All of the rabbits were submitted to a laparotomy under general anesthesia
with intravenous administration of 3 mL of thionembutal (2.5%) along with 1 mL of
xylestesin (2%).
Induction Technique for Endometriotic Lesions
The pelvic cavity was opened with a median longitudinal incision of ∼ 2 cm in length,
2 cm from the pubis of the animal. Then, ∼ 4 cm of the right uterine horn was resected
and then the horn was closed. The portion of the uterine that was resected was immersed
in 0.9% saline solution for ∼ 2 minutes for tissue cleaning and then cut longitudinally,
resecting a 5 × 5-mm fragment. This endometrial tissue fragment was sutured to the
peritoneum near the reproductive tract of the rabbits by using 2 simple Vicryl 6.0
(Ethicon Inc., Sommerville, NJ, USA) sutures, with the endometrium was facing inward
facing the abdominal cavity, with posterior closing of the abdominal surgical incision.
No hormonal supplements were administered before or after the laparotomy. The same
observer performed all of the procedures.[32 ]
Removal of Lesions for Histological Analysis
The rabbits were divided into two groups, namely group 1, which consisted of 14 animals
whose lesion evolution time was 4 weeks, and group 2, which consisted of 14 animals
with a lesion evolution time of 8 weeks. After pelvic inspection, identification,
and documentation of the lesion, the rabbits were sacrificed. The lesion, along with
the left uterine horn (contralateral), was excised for histological analysis. The
excised tissues were set in 10% formaldehyde and processed for inclusion in paraffin.
After preparing the slides, the tissues were stained with hematoxylin and eosin (H&E)
stain for histological analysis. The analysis results indicated that 100% of the samples
in both groups were composed of active endometrial (gland and stroma) tissues, with
similar morphological characteristics. The lesions were characterized by thin-walled
cysts located on the striated muscle of the abdominal wall, projecting toward the
abdominal cavity. The cyst walls were formed by a thin layer of connective tissue,
rich in cells, and covered by simple squamous epithelium. The stroma is rich in fibroblasts,
and contains some macrophages and eosinophils in addition to a large quantity of typical
endometrial glands. The evaluation of the lesions in two stages has the purpose of
verifying the presence of any tissue modification during the progression of the lesions,
as well as their growth.
Immunohistochemical Techniques
Histological sections (4–5 mm) were submitted for histochemical analysis with the
antigen-antibody reaction. The reaction was developed by using a marker visible under
the microscope. The deparaffinized and hydrated sections were recovered antigenically
by incubation in a buffered medium in a steam pot for 40 minutes. After cooling, the
endogenous tissue peroxidases were removed by adding hydrogen peroxide, and horse
serum was added to prevent nonspecific binding of the primary antibody. The slides
were then incubated with primary antibodies obtained from Novocastra Laboratories
Ltd. (Newcastle upon Tyne, United Kingdom). The samples were then evaluated regarding
the following markers: MT (1:100; clone E9; Dako North America Inc., Carpinteria,
CA, USA) and p63 (1:500; clone BC4A4; Biocare Medical, Concord, CA, USA), which are
related to cell proliferation and differentiation, and MMP 9 (1:100 clone 15W2; Leica
Biosystems, Wetzlar, Germany) and TIMP-2 (1:100 clone 3A4; Novocastra Laboratories,
United Kingdom), which are involved with invasive capacity. The materials were then
incubated with the secondary antibody and submitted to the avidin-biotin step. The
reaction was developed by treatment with 3.30-diaminobenzidine (Sigma-Aldrich Inc.,
St. Louis, MO, USA) for 5 minutes. Thereafter, the materials were counterstained with
Harris H&E stain and mounted on slides. The immunohistochemical markers were analyzed
quantitatively and manually, counting the number of marked cells in thousands, and
divided into 4 quadrants of 250 cells each. All of the slides were evaluated by two
pathologists experienced in immunohistochemistry who were blinded to the type of tissue
to be analyzed.
Statistical Analysis
A statistical analysis was performed by using the GraphPad Prism 5.0 32-bit executable
software (GraphPad Software Inc., San Diego, CA, USA). The paired Student t -test was used for variables with a normal distribution and for comparison of the
paired data. The non-paired Student t -test was used for variables with normal distribution and non-paired data. Finally,
the Mann-Whitney U-test was used for non-paired variables with non-normal distribution.
The level of statistical significance was set at 5%.
Results
A significantly more intense stain for MMP9, TIMP-2, MT, and p63 were observed in
the ectopic endometria than in the eutopic endometria. ([Table 1 ] and [Fig. 1 ]). However, regarding the ectopic lesions that were compared at 4 and 8 weeks, no
significant differences were observed, with the exception of the p63 indicator, which
was more evident after 8 weeks of ectopic endometrial tissue progression. ([Table 2 ] and [Fig. 1 ]).
Fig. 1 Immunostaining by: metalloprotease in the eutopic (A) and ectopic endometria (B);
tissue inhibitor of metalloprotease in the eutopic (C) and ectopic endometria (D);
metallothionein in the eutopic (E) and ectopic endometria (F); and p63 in the eutopic
(G) and ectopic endometria (H). 40x enlargement.
Table 1
Cellular immunostaining according to endometrial types
Topic
mean ± SD
Ectopic
mean ± SD
p -value
MMP-9
0.273 ± 0.147
0.364 ± 0.223
0.0003
TIMP-2
0.249 ± 0.126
0.274 ± 0.152
0.02
Metallothionein
0.277 ± 0.141
0.361 ± 0.220
0.0003
p63
0.218 ± 0.080
0.276 ± 0.095
< 0.0001
Abbreviations: MMP-9, metalloprotease; SD, standard deviation; TIMP-2, tissue inhibitor
of metalloprotease.
Table 2
Cellular immunostaining in ectopic endometrium according to progression time
4 weeks
mean ± SD
8 weeks
mean ± SD
p -value
MMP-9
0.418 ± 0.209
0.314 ± 0.231
0.21
TIMP-2
0.288 ± 0.152
0.262 ± 0.157
0.66
Metallothionein
0.428 ± 0.221
0.298 ± 0.208
0.11
p63
0.240 ± 0.058
0.309 ± 0.113
0.05
Abbreviations: MMP-9, metalloprotease; SD, standard deviation; TIMP-2, tissue inhibitor
of metalloprotease.
Discussion
In the present study, we have demonstrated that the ectopic endometrial lesions showed
a higher number of immune stained cells expressing cellular invasion markers (MMP-9
and its inhibitor TIMP-2) and of molecules involved in cellular proliferation and
differentiation (MT and p63) than the eutopic endometrial lesions, using a rabbit
experimental model of endometriosis. However, when 4- and 8-week ectopic lesions were
compared, there was no significant difference except for the p63 marker, which was
more evident after 8 weeks of evolution of the ectopic endometrial tissue. This suggests
that this marker increases the longer the period of lesion evolution, demonstrating
a greater capacity of differentiation of the ectopic endometriotic foci.[34 ]
The MMP system is formed by an enzymatic component, MMP, and by an inhibiting component,
the TIMPs. It has been well established that this system performs a critical role
during the normal development and growth of the endometrium, as well as many other
physiological processes.[35 ]
Metalloproteases have been implicated in the pathogenesis of endometriosis, the greater
part of which are synthesized during the proliferative phase and stimulated by estrogen
expression. Inversely, progesterone levels reduce MMP transcription and secretion.
In an endometriosis model using mice and human endometria, endometrial treatment with
estrogen led to an increase in MMP production and in endometrial ectopic implantation.
In contrast, treatment with progesterone, which inhibits the production of MMP[36 ] or TIMP-1, decreased the success rate of ectopic implantation.[37 ]
[38 ]
[39 ]
Metalloproteases work on the dynamics of the degradation and remodeling process of
the extracellular matrix, stimulating cellular proliferation and apoptosis, as well
as inducing cellular migration. Several previous studies suggest that eutopic and
ectopic endometria in women with endometriosis present altered levels of the MMP/TIMP
system, indicating that these two enzymes play a role in the pathogenesis of the illness.
In general, the irregular synthesis and secretion of MMPs by endometriosis lesions,
combined with aberrant quantities of TIMP-1 in the peritoneal fluid, could disturb
the normal proteolytic environment of the peritoneal cavity, thus inducing a more
aggressive behavior and facilitating invasion by ectopic cells. The exact mechanisms
that lead to the aberrant expressions of MMPs and their tissue inhibitors in endometriosis
have yet to be defined.[3 ]
[36 ]
[40 ]
[41 ]
[42 ]
[43 ]
[44 ]
[45 ]
[46 ]
[47 ]
Meanwhile, studies inferred that the increased expression levels of MMPs in the peritoneal
fluid and in ectopic lesions in endometriosis patients could be a secondary event
that results from an innate difference in peritoneal and systemic factors instead
of from alterations in the endometrium, causing an abnormal peritoneal response to
the menstrual reflux, which in turn facilitates ectopic implantation.[48 ]
Vinatier et al (2000)[49 ] suggested that the characteristics of endometrial cells differ between women with
and without endometriosis. The endometrium in women with endometriosis has an increased
capacity for proliferation and implantation in ectopic locations. Therefore, the interpretation
of the results should consider that eutopic endometrial cells and refluxed cells in
the peritoneal cavity are not regulated by the same environment. Refluxed cells in
the peritoneal cavity are regulated by the microenvironment of the peritoneal fluid,
contrary to eutopic endometrial cells, which are regulated by blood flow factors.
This concept of a different environment is rarely considered when eutopic and ectopic
endometria are compared. The differences that are observed should be interpreted not
only as differences in tissue characteristics, but also as a result of environmental
differences. The type of cellular modification along with local factors such as the
microenvironment of the peritoneal fluid or the intraovarian environment will determine
if they will develop typical lesions, deep endometriosis, or ovarian cysts.[49 ]
[50 ]
The present study observed a higher immunostaining intensity of MMP-9 and its inhibitor,
TIMP-2, in ectopic endometria, which was related to a higher ability to proliferate
and to the invasive capacity of these tissues, as shown in previous studies.[3 ]
[4 ]
[5 ]
[6 ]
[7 ]
[8 ]
[9 ]
[10 ]
[11 ]
Although the progression time of these lesions did not interfere in a statistically
significant way in the MMP-9 (p = 0.214) and in the TIMP-2 staining (p = 0.66), our results suggest that the evaluation of MMP-9 and TIMP-2 could be used
as a prognostic indicator of endometrial invasion; therefore, increasing the proteolytic
activity would be one of the many factors that contribute to the invasive properties
of the endometrium, resulting in the development of endometriosis.
The molecular alterations observed in the human endometrium during the menstrual cycle
could be crucial to the reproductive function. The accumulation of cytotoxic cells
increases the exposure of endometrial cells to apoptosis, and protection against this
process could be reached by self-regulating cellular mechanisms, suggesting that MT
participates in this context. Significant differences in MT expression observed in
the endometrium regarding alterations in the menstrual cycle could suggest the participation
of MT in the protection against apoptosis in endometrial cells.[51 ]
Metalloprotease takes part in the detoxification process of organisms, being found
in benign and malignant neoplasms, among others, in animals and humans, mainly in
the S phase of the cellular cycle. It is considered as an index for cell proliferation
and tumor progression. During proliferation, epithelial cells have a higher expression
level of MT, indicating an increase in the number of dividing cells, particularly
during the S phase of the cell cycle. This is why MT could be considered as a marker
of endometriosis.[18 ]
The perinuclear location of MT is known to be important for protection against DNA
damage and apoptosis induced by external stressors.[27 ] Therefore, the MT expression in endometrial cells could favor their persistence
in ectopic localization, as reported by Wicherek et al (2006).[19 ]
We have observed a higher-intensity stain in ectopic endometrial tissue in comparison
with eutopic tissue; however, the difference was not statistically significant regarding
the progression time of the lesion in the ectopic tissue.
Data suggest that MT expression seems to be under hormonal control in normal endometrium
and that MT could modify the p53 expression and be used as a biological marker of
aggressive behavior in endometrial lesions.[17 ] The ability of the endometrium to distinguish cytotoxic activity from increased
protection against DNA damage (MT expression), as well as concomitant changes in the
number of cells in the immune system and its activity, which are observed in normal
endometrium during the phases of the menstrual cycle, seems to be fundamental for
the pathological characteristics of endometriosis.[52 ]
The p63 protein has been described as a marker of basal and reserve cells in the female
genital tract,[53 ]
[54 ]
[55 ] being strongly related with altered differentiation, including metaplasia, either
isolated or in combination with neoplasms.[56 ]
[57 ] Some clinical and laboratory data provide evidence that suggests that ectopic endometrial
lesions result in the dislocation of basal endometrial cells.[58 ]
Studies showed that endometriotic lesions express p63 differently; however, whether
the lack of p63 expression in some lesions is related to the extent of the illness,
to its clinical behavior, or to the exacerbation of the symptoms that accompany it
is unclear.[13 ]
We have noted in our experimental model that the stain was more evident in ectopic
endometria than in eutopic endometria (p < 0.0001), inferring a greater potential for differentiation in eutopic tissue, favoring
the establishment of endometriosis, as it has already been suggested that p63-positive
cells in normal endometria represent cells with a stem cell phenotype that have the
potential for multidirectional differentiation.[12 ]
[13 ]
[14 ]
[15 ]
[16 ]
Another relevant fact was the interference of the progression time of the lesion regarding
the staining intensity, which could suggest that the longer the progression, the greater
the ability of endometriotic foci in the ectopic tissue to differentiate.
Conclusion
Upon analyzing the different markers of cell proliferation and differentiation, as
well as of tissue invasion in eutopic and ectopic endometria in the rabbits submitted
to the induction of endometriotic lesions by the experimental model, 4 and 8 weeks
after the endometrial implantation procedure, we conclude that the ectopic lesions
seem to express a greater ability for cell proliferation and differentiation, as well
as for tissue invasion when compared with eutopic endometria. This is evident in the
greater intensity of the immunostaining for the proteins involved in the capacity
to invade tissues (MMP-9 and TIMP-2) in ectopic endometria compared to the eutopic
endometrium and in the higher number of cells of molecules involved in cell proliferation
and differentiation (MT and p63) that were stained in ectopic endometria, which was
most evident by the p63 stain in the endometrium after 8 weeks of progression. The
ectopic endometrial lesions seem to express a greater ability for cell differentiation
and tissue invasion than eutopic endometrial lesions, characterizing endometriosis
as a potentially invasive, progressive, and heterogeneous disease in its presentation.
However, more studies are necessary to better clarify the participation of these markers
in the complex pathophysiological mechanism of endometriosis.
