Human Endometriotic Lesions Research Articles

Expression of membrane-type 5 matrix metalloproteinase in human endometrium and endometriosis

Background. The metalloproteinases (MMPs) are a family of proteolytic enzymes involved in tissue remodeling and cell migration. Endometrial tissue remodeling proceeds during the menstrual cycle and requires a temporary and spatially balanced expression of several different MMPs. Various members of the MMPs also seem to play an important role in the invasion process of endometriosis; however, so far only a limited number of studies have focused on membrane-associated MMPs.Methods. The present study investigated the expression of membrane-type 5 metalloproteinase (MT5-MMP) in the human endometrium and endometriotic lesions by microarray hybridization, real-time polymerase chain reaction (PCR) and immunofluorescence.Results. Both the gene chip expression analyses as well as PCR indicated expression of MT5-MMP in normal human endometrium and strongly elevated transcript levels in most peritoneal endometriosis lesions analyzed. Moreover we detected enhanced MT5-MMP expression in the eutopic endometrium from patients suffering from endometriosis, further supporting a role of MT5-MMP in the formation of endometriosis. Immunohistochemical analysis was used to determine the intracellular localization and tissue distribution of MT5-MMP. While the MT5-MMP antigen expression could be clearly attributed to the membrane of epithelial cells, a highly complex differential immunohistochemical staining of MT5-MMP in the various compartments of endometrial tissue was observed. The strongest staining was seen in luminal epithelial cells, whereas endometrial glands frequently showed partial expression of MT5-MMP.Conclusion. Our microarray analysis and real-time PCR of MT5-MMP transcripts may point to an elevated tissue remodeling and cell migration in endometrium from endometriosis patients as implied by the function of related MMPs.

P16, retinoblastoma (pRb), and cyclin D1 protein expression in human endometriotic and adenomyotic lesions

To investigate the expression of p16, retinoblastoma (pRb), and cyclin D1 oncoproteins in endometriomas and adenomyosis. Immunohistochemical study for p16, pRb, and cyclin D1 proteins in formalin-fixed paraffin-embedded endometriotic and adenomyotic tissues. University hospital. Tissues from 25 women with endometriomas and 31 women with adenomyosis were evaluated. Tissue samples were collected during gynecologic surgery and confirmed by histology to have endometriosis or adenomyosis. Nuclear expression of p16, pRb, and cyclin D1 proteins was examined by immunohistochemistry. Distribution and intensity of immunostaining. In the proliferative phase of the cycle, p16 was detected in 77% of adenomyosis tissues but in only 15% of endometriosis tissues. Moreover, in adenomyosis samples positive for p16, 100% of the adenomyotic cells expressed p16, whereas only 10%-20% of endometriosis cells from positive cases expressed p16. In contrast, pRb was detected in 28% of endometriosis cases but not in any adenomyotic tissues. Cyclin D1 was absent in both endometriotic and adenomyotic tissue samples. Differences in oncoprotein expression between endometriotic and adenomyotic tissues provide further evidence that the pathogenesis of endometriosis is different from that of adenomyosis.

Antiangiogenic Agents Are Effective Inhibitors of Endometriosis

Hull, M. Louise; Carnock-Jones, D. Stephen; Chan, Clement L. K.; Bruner-Tran, Kaylon L.; Osteen, Kevin G.; Tom, Brian D. M.; Fan, Tai-Ping D.; Smith, Stephen K.

Antiangiogenic agents are effective inhibitors of endometriosis.

Endometriosis is a disease in which the lining of the uterus (endometrium), shed at the time of menstruation, becomes established at sites such as the peritoneum and ovaries. These explants develop a rich blood supply that enables them to survive and grow. We hypothesized that inhibitors of angiogenesis would prevent this growth by disrupting sensitive vessels supplying endometriotic lesions. Vessels sensitive to angiogenic antagonism have few associations with pericyte cells. The vessels supplying human endometriotic lesions were immunohistochemically characterized and found to be predominantly pericyte free. A model in which human endometrium is implanted into nude mice was used to test the effects of two antagonists of the angiogenic growth factor, vascular endothelial cell growth factor A. Soluble truncated receptor (flt-1; P = 0.002) and an affinity-purified antibody to human vascular endothelial cell growth factor A (P = 0.03) significantly inhibited the growth of nude mouse explants. Pericyte-free vessels were shown to supply endometrial lesions in nude mice and were disrupted in lesions taken from soluble flt-1-treated mice. In summary, antiangiogenic agents inhibited the growth of explants in an in vivo model of endometriosis by disrupting the vascular supply, and this effect is likely to apply to the human disease. These findings suggest that antiangiogenic agents may provide a novel therapeutic approach for the treatment of endometriosis.

Identification of the cadherin subtypes present in the human peritoneum and endometriotic lesions: potential role for P-cadherin in the development of endometriosis.

Endometriosis is defined as endometrial tissue outside of the uterine cavity. The pathogenesis of this common disease remains poorly understood. However, the implantation and invasion of the viable cells from retrograde menstruation into the peritoneum is a widely accepted theory. To date, the mechanisms by which cell adhesion molecules mediate the development of human endometriosis remain unclear. Cadherins are a family of cell adhesion molecules that mediate cell-cell adhesion in a homophilic manner. In this study, the cadherins present in the peritoneum and endometriotic lesions were identified by RT-PCR using degenerate primers. In addition, differences in the levels of the cadherin mRNA transcripts present in eutopic endometrium and endometriotic lesions of the same patients were then compared by semiquantitative RT-PCR. Multiple cadherins were detected in the peritoneum and endometriotic lesions. Of these, P-cadherin appears to be the predominant cadherin subtype present in the peritoneum. Similarly, P-cadherin mRNA levels in endometriotic lesions were significantly greater than those observed in the corresponding eutopic endometrium. The expression of P-cadherin in both the human peritoneum and endometriotic lesions suggests that this cell adhesion molecule may play a central role in the development of endometriosis by mediating endometrial-peritoneal cell interactions in a homophilic manner.

Establishment of an improved mouse model of endometriosis allowing the non-invasive assessment of implantation, progression and regression of human endometriotic lesions

Objective: To develop an improved mouse model of endometriosis to allow the non-invasive and dynamic monitoring of human endometriotic lesion implantation and regression. Design: Develop a murine non-invasive experimental model for endometriosis by implanting green fluorescent protein (GFP) expressing fragments of human endometrium into immunocompromised mice. Materials/Methods: We developed an improved, non-invasive animal model for endometriosis. To this end, we introduced the GFP gene into 1–2 mm3 fragments of proliferative phase endometrium, by adenoviral infection in vitro. Ten fragments of GFP-expressing endometrial tissue were then injected either subcutaneously or intra-peritoneally into ovariectomized Nude mice, previously implanted with an estradiol-releasing pellet. Living transplanted mice were periodically examined using a light emitting box allowing GFP excitation. Image analysis allowed quantification of fluorescence intensity and size of endometriotic lesions. In a second set of experiments, monitoring of lesion evolution in untreated or progesterone-treated animals was performed by serial in vivo imaging of animals. Number of lesions, their size and fluorescence intensity, as well as time of complete disappearance were quantified and compared between groups. Results: One day after adenoviral infection, endometrial tissue was shown to be highly fluorescent both by microscopic observation and flow cytometric analysis. GFP-expressing endometrial tissue transplanted to Nude mice was able to form endometriotic lesions. Importantly, fluorescent endometriotic lesions were readily observed non-invasively in subcutaneous and intra-peritoneal sites. Lesion evolution could be followed in the same animal over a 3 week period, and their size and fluorescence intensity could be measured. These two parameters spontaneously decreased with time, likely due to GFP dilution among dividing cells. We postulated that decrease in size and fluorescence intensity of lesions would be more pronounced in mice undergoing endometriosis medication. To make the proof of concept that this model could indeed be used for the pre-clinical testing of new drugs targeting endometriosis, we treated Nude mice exhibiting fluorescent lesions with progesterone. Mice treated with progesterone had a faster and more intense decrease in the number and size of lesions, as assessed by non-invasive imaging. Conclusions: The easier identification of human endometriotic lesions in animals is a major strength of this improved model. Moreover, it allows the visualization of lesion evolution after treatment in the same animal, non-invasively, reducing time, cost and number of animals involved when assessing the efficacy of drugs directed against endometriosis. The dynamic and non-invasive characteristics of this improved mouse model should make it an unprecedented tool for compound evaluation in a pre-clinical setting of endometriosis. Furthermore, this animal model could have many other applications in endometriosis research, such as evaluating the effect of tested genes on lesion implantation and regression. Supported By: PROCREA BioSciences.

Gene expression of adhesion molecules and matrix metalloproteinases in endometriosis

Various types of cell adhesion molecules and matrix metalloproteinases (MMPs) seem to play an important role in the invasion process of endometriosis; however, limited investigation has focused on theirgene expression in human peritoneal endometriotic lesions. A total of 63 endometriotic tissues were surgically obtained from 35 women with endometriosis, which included 43 pigmented and 20 non-pigmentedlesions. Gene expression levels of E-cadherin, α- and β-catenin, MMP-2, MMP-9 and membrane-type 1 (MT1)-MMP in these endometriotic lesions were compared with those in normal eutopic endometriumobtained from 12 women without endometriosis. MMP-2, MMP-9 and MT1-MMP mRNA expression in pigmented lesions was significantly higher than that in normal endometrium (p < 0.05), whereas E-cadherin,α- and β-catenin mRNA expression was not suppressed in endometriotic lesions. There was a close correlation between MMP-2 or MT1-MMP and E-cadherin, α- or β-catenin gene expressionin 63 endometriotic tissues examined (p < 0.01). Immunohistochemical expression of E-cadherin, α- and β-catenin in glandular epithelial cells was positive not only for all of sevencases with normal eutopic endometrium but also for 9 of 11 with ovarian endometriosis. MMP expression in ectopic endometrium was much greater than that in eutopic endometrium. These results suggest thatendometriotic tissues expressing MMPs might be invasive and simultaneously possess cell-to-cell adhesion property in pelvic peritoneal foci.

Peritoneal endometriotic lesions differentially express a haptoglobin-like gene.

A unique glycoprotein, called endometriosis protein-I (ENDO-I), has previously been shown to be synthesized and secreted by rat and human endometriotic explants. Furthermore, the N-terminal amino acid sequence analysis showed that ENDO-I shares homology with haptoglobin. The present study was designed to determine this sequence of ENDO-I cDNA from human peritoneal endometriotic lesions and to determine the relative expression of the ENDO-I gene in several human tissues. Poly A-enriched RNA was isolated and reverse-transcribed. To determine the sequence of ENDO-I cDNA, a polymerase chain reaction was performed on cDNA from human endometriotic lesions and a gene-specific primer based on the human haptoglobin sequence. A similar procedure was followed to assess the relative expression of the ENDO-I gene in various tissues. Glyceraldehyde-3-phosphate-dehydrogenase was used as an internal control. ENDO-I gene expression was quantified by densitometry. Sequence analysis of ENDO-I cDNA identified 873 nucleotides that displayed 99.4% homology with the human haptoglobin beta-chain. Relative expression of ENDO-I mRNA by peritoneal endometriotic lesions was 19-fold greater than peritoneum, 28-fold greater than endometrioma and 37-fold greater than eutopic endometrium (P<0.01). Haptoglobin-like ENDO-I may be associated with localized angiogenesis and altered immune response involved with the aetiology/pathophysiology of endometriosis.

Expression of interstitial collagenase (matrix metalloproteinase-1) is related to the activity of human endometriotic lesions

Objective: To determine whether interstitial collagenase (matrix metalloproteinase-1), known to play a pivotal role in the initiation of menstruation, contributes to the pathogenesis of endometriosis. Design: Serial sections of peritoneal red and black endometriotic lesions, ovarian endometriotic cysts, and rectovaginal adenomyotic nodules were analyzed by in situ hybridization for the expression of matrix metalloproteinase-1 by silver staining for the integrity of the fibrillar extracellular matrix and by immunolabeling for the abundance of sex steroid receptors. Setting: Academic hospital and research laboratory. Patient(s): Premenopausal women undergoing laparoscopy for endometriosis. Intervention(s): Biopsy of endometriotic lesions, combined with endometrium whenever possible. Main Outcome Measure(s): Expression of matrix metalloproteinase-1 messenger RNA (mRNA). Result(s): Matrix metalloproteinase-1 mRNA was expressed focally in red peritoneal and ovarian endometriosis irrespective of the phase of the menstrual cycle but was not detectable in black peritoneal and rectovaginal lesions. Foci of matrix metalloproteinase-1 expression closely correlated with matrix breakdown and with the absence of P receptors in adjacent epithelial cells. Conclusion(s): Correlation of matrix metalloproteinase-1 expression with activity of endometriotic tissue suggests its involvement in tissue remodeling and bleeding, and possibly in the secondary shedding and reimplantation of endometriotic lesions.