This study was performed to compare the expression of CD44 in endometrial stromal cells (ESCs) of women with and without endometriosis and to evaluate the role of CD44 in the adherence of ESCs to peritoneal mesothelial cells (PMCs).
A PMC adherence assay was performed to evaluate the adherence of ESCs to PMCs in women with and without endometriosis. The expression of CD44 mRNA was measured by reverse transcription-polymerase chain reaction. CD44 protein was evaluated by Western blot analysis.
There were no significant differences in the expression of CD44 mRNA and protein in ESCs or in the binding of ESCs to PMCs between patients with endometriosis and controls. Although the expression of CD44 protein was decreased in both women with endometriosis and controls after anti-CD44 antibody treatment, there was no effect on binding of ESCs to PMCs. Treatment of ESCs with peritoneal fluid from endometriosis patients resulted in a significant increase in binding of ESCs to PMCs compared to untreated ESCs in the endometriosis group.
This study demonstrates that the expression of CD44 protein in ESCs from women with endometriosis might not be directly associated with adherence to PMCs.
Endometriosis is a chronic progressive disease inducing various symptoms such as infertility, pelvic pain, and menstrual pain due to the presence of endometrial tissues outside of the uterus. Even though endometriosis occurs in approximately 6% to 10% of women, the incidence increases to up to 35% to 50% for women with pelvic pain or infertility [
Lately, several researchers have demonstrated that CD44, the main receptor of hyaluronic acid (HA), was associated with the peritoneal adherence of gastric and ovarian carcinoma cells [
Therefore, the objectives of this study were to compare the expression of CD44 in ESCs between patients with endometriosis and controls and to investigate the roles of CD44 in the adherence of ESCs to PMCs.
For the endometriosis group, we recruited 13 patients who were confirmed to have endometriosis based on pathologic examinations after conservative surgery in the department of obstetrics and gynecology at Pusan National University Yangsan Hospital. The recruited patients had no history of medications such as gonadotropin releasing hormone agonist or steroids within the previous six months. The patients were not pregnant and had normal menstrual cycles. For the control group, we recruited 13 females with normal menstrual cycles who underwent hysterectomy because of benign gynecologic diseases or carcinoma in situ of the cervix. There were no gross lesions to consider the presence of endometriosis during surgery of the control group.
This study was approved by the Institutional Review Board at Pusan National University. Sufficient explanation regarding the study was provided to all subjects, and informed consent was obtained.
Endometrial aspiration biopsy using a Novak curette was performed during conservative surgery in the endometriosis group, whereas the endometrial tissues were obtained from the resected uterus after hysterectomy in the control group. The entire process of collecting tissues was conducted under aseptic conditions. The collected tissues were placed in conical tubes containing Dulbecco's modified Eagle's media F12 (DMEM:F12, Gibco, Grand Island, NY, USA) with 1% antibiotics/antimycotics (Gibco), and then the samples were transferred from the operation room to laboratory. The rest of the blood was removed by washing several times with phosphate-buffered saline (PBS, Gibco).
The endometrial tissues were placed in culture dishes with DMEM:F12 and then minced to 1-2 mm in size using sterile scissors. Minced tissues in the solution were transferred to conical tubes and treated with 0.1% collagenase type 1 (Gibco) and 0.05% DNAse (Boehringer Mannheim, Mannheim, Germany) followed by incubation in a waterbath at 37℃ for 1 hour 30 minutes while shaking [
The precipitated cells were reconstituted in media and filtered through a 40 µm nylon membrane (BD Falcon, Bedford, MA, USA) [
The LP9 human peritoneal cell line was purchased from NIA Aging Cell Culture Repository (USA). The cell line was maintained in MCDB131/M199 (1:1) media containing 20 ng/mL of epithelial growth factor (Sigma), 2 mM L-glutamin (Gibco), HEPES buffer (Sigma), 0.05 µg/mL of hydrocortisone (Sigma), 1% antibiotics/antimycotics, and 10% FBS [
SK-OV-3 human ovarian carcinoma cells were obtained from the Korean Cell Bank and utilized as the control group for adherence assays. SK-OV-3 cells were cultured in RPMI (Gibco) media with HEPES buffer, 10% FBS, and 1% antibiotics/antimycotics. Each cell line was cultured in an incubator at 37℃ and 5% CO2 with replacement of the media once every two to three days.
To perform an
The peritoneal fluids were centrifuged at 800 g for 10 minutes within 30 minutes after collection in the operating room, and then the supernatants were tightly sealed and stored in a deep freezer at -70℃. The mixture of the peritoneal fluids from the endometriosis group and the control group was used for the treatment of cells. The endometrial cells (1×106 cells) were aliquoted in 100 mm dishes. After incubation for 48 hours, the peritoneal fluids mixed with 10% of the culture media were added, and the cells were treated for 24 hours. The cells were then collected for further analysis.
The endometrial cells collected for the adherence assay with peritoneal cells (1×106 cells/mL) were treated with 10 µg/mL of anti-CD44 antibody (Lab vision corp., USA) at 37℃ for 30 minutes, and then the cells were collected for further analysis.
For RNA separation, the cells were dissolved by adding Trizol (Gibco, BRL) to the tissues, and chloroform (Sigma), 10% of total volume, was added prior to centrifugation to obtain supernatant. Then, the same amount of isopropanol (Sigma) was mixed to precipitate RNA. After washing the RNA precipitates with 75% ethanol, the supernatant was removed through centrifugation, and the precipitates were air-dried. The dried RNA was dissolved in diethylpyrocarbonate (DEPC, Sigma) and then subjected to absorbance and purity measurements at 260 and 280 nm, respectively. The dried RNA was stored in at -70℃ until needed for further analysis.
A total of 2 µg of RNA was added to the reaction solution containing 5×RNA PCR buffer, dNTP (2.5 mM), MgCl2 (25 mM), oligo dT primer, RNase inhibitor, and MMLV reverse transcriptase (Promega) and reacted at 42℃ for 60 minutes and at 95℃ for 5 minutes to synthesize cDNA. PCR was performed using the synthesized cDNA as templates. PCR was carried out utilizing CD44 primer (forward primer, 5' TCC CAG TAT GAC ACA TAT TGC 3'; reverse primer, 5' CAC CTT CTT CGA CTG TTG AC 3') [
Proteins were extracted from the cultured cells using cell extraction buffer (Pierce, Rockford, IL, USA) and then quantified utilizing Bio-Rad protein quantification reagent (Bio-Rad, Hercules, CA, USA).
The protein extracts, 20 µg of each experimental group, were loaded on 10% SDS-polyacrylamide gel to separate the proteins via gel electrophoresis, and the acrylamide gel with separated proteins was transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). The membrane was incubated in TBS with 0.1% TBS-T containing 5% non-fat dry milk for 1 hour in order to block non-specific binding. Then, the membrane was reacted with 1:1000 anti-CD44H (R&D Systems, Minneapolis, MN, USA) and β-actin (Sigma-Aldrich, St. Louis, MO, USA) at 4℃ overnight, followed by six ten-minute washes with TBS-T. Then, the membrane was reacted with 1:5,000 horseradish peroxidase (HRP)-conjugated anti-mouse goat Ig G antibody (Santa Cruz, San Diego, CA, USA) diluted by TBS-T at room temperature for 1 hour and washed six times for 5 minutes each. Lastly, the proteins were developed using an enhanced chemiluminescence (ECL) kit (Western blotting detection reagents, Amersham-Biosciences), and the X-ray film was exposed to light to determine protein expression.
All statistical analysis was performed using SPSS ver. 17.0 (SPSS Inc., Chicago, IL, USA). Mann-Whitney U-test was applied to compare the adherence of ESCs to the peritoneal cells between the two groups, while the Kruskall-Wallis test was utilized in the comparison of the three groups after the treatment with peritoneal fluids. A
The adherence of ESCs to peritoneal cells is shown in
RT-PCR was carried out using a primer from the study by Cannistra et al. [
The adherence of the ESCs to peritoneal cells after treatment with peritoneal fluids was not found to be significantly different among the control group, non-peritoneal fluid-treated group, normal peritoneal fluid-treated group, and endometriosis peritoneal fluid-treated group (
The attachment of endometrial cells to the peritoneum is an essential step in the development of endometriosis. Lucidi et al. [
In the analysis of the CD44 expression of ESCs in this study, the CD44 mRNA and protein were expressed, but the variant type of CD44 was not, without notable differences between the endometriosis group and the control group. As mentioned earlier, studies have investigated CD44 isoform expression in the endometrium, but the results were inconsistent. For normal endometrium, CD44 expression was increased in the secretory phase rather than proliferative phase [
Anti-CD44 antibody treatment of ESCs showed a notable decrease in the expression of CD44 proteins in endometriosis patients and the control group, while the adherence of peritoneal cells was not affected. In contrast, the adherence to peritoneal cells was remarkably increased when ESCs of the endometriosis patients were treated with endometriosis peritoneal fluids. CD44 adheres to extracellular matrix HA and induces cell-to-cell attachments and cell-to-matrix attachments. It is known that CD44 is particularly involved in the adherence of gastric carcinoma and ovarian carcinoma cells to peritoneal mesothelium [
In conclusion, CD44 expression in ESCs did not differ between the endometriosis group and the control group. Although a decrease of CD44 protein expression was observed in the endometriosis group and the control group after the CD44 antibody treatment, there were no differences in the attachment of ESCs to PMCs between the two groups even after the decrease in CD44 protein. Thus, CD44 expression does not seem to be directly associated with the attachment of ESCs to PMCs. However, since our study did not classify the ESCs based upon menstrual cycle, further studies are necessary to clarify if similar results are obtained when using endometrial cells in the secretory phase and during menstruation, where CD44 expression is known to be increased.
This study was supported by the Medical Research Institute Grant (2005-21), Pusan National University Hospital.
Endometrial stromal cell adherence to peritoneal mesothelial cells in the normal group and the endometriosis group, as measured by peritoneal mesothelial cells adherence assay.
Expression of CD44 mRNA in the control group (A) and the endometriosis group (B), as measured by reverse transcription-polymerase chain reaction.
Expression of CD44 protein in the control group (A) and the endometriosis group (B), as measured by Western blot analysis.
Expression of CD44 protein after anti-CD44 antibody treatment in the control group and the endometriosis group, as measured by Western blot analysis (A). Peritoneal mesothelial cells adherence assay after anti-CD44 antibody treatment in the control group and the endometriosis group (B).
Peritoneal mesothelial cells adherence assay after peritoneal fluid treatment in the control group (A) and the endometriosis group (B). Con, control; Ep, endometriosis peritoneal fluid; Np, normal peritoneal fluid. Significantly different *