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Regular Article |
a Departments of Biochemistry and
b Obstetrics and Gynecology, University of Missouri, Columbia, Missouri 65212
ABSTRACT
In vivo levels of mRNA and the specificity of the extrauterine environment on matrix metalloproteinase (MMP)-3, MMP-2, and tissue inhibitor of matrix metalloproteinase (TIMP)-1 were evaluated in eutopic and ectopic endometrial tissue during the establishment of endometriosis in a rat model. Uteri and endometriotic implants were collected and frozen at 36 h, 2 wk, and 4 wk postsurgery to study in vivo mRNA levels. Intact uteri, uterine tissues implanted in the peritoneum or under the skin, and peritoneal adipose implants were collected at 2 wk, halved, and either frozen or cultured. Gene-specific reverse transcriptase-polymerase chain reaction was performed to detect and quantify MMP-2, MMP-3, and TIMP-1 mRNA levels. The peritoneal endometriotic implants progressed from avascularized implants, to vascularized red lesions, to well-established encapsulated cysts. In vivo, MMP-3 mRNA was detectable at all times in ectopic tissues but not in eutopic uterine tissues, whereas MMP-2 and TIMP-1 were ubiquitously expressed at all times in both tissues. In vitro, only MMP-3 mRNA levels were elevated in endometrial tissues collected from the intact uterine and from under the skin, at levels similar to in vivo endometriotic implant MMP-3. In conclusion, ectopic endometrial MMP-3 may participate in the process of invasion and tissue remodeling that is hypothesized to occur in the pathogenesis of endometriosis.
female reproductive tract, gene regulation, menstrual cycle, progesterone, uterus
INTRODUCTION
Endometriosis is defined as the presence of endometrial tissue in locations other than the uterine cavity [1]. Endometriotic lesions, found naturally only in menstruating primates, are composed of endometrial glands and stroma, and can be commonly found on the ovaries and throughout the peritoneal cavity [1]. Although they are histologically similar, a wide variety of biochemical properties differ between ectopic and eutopic endometrial tissues [2].
Little is known of the pathogenic mechanisms of endometriosis. The prevailing hypothesis is that following retrograde menstruation, uterine endometrial tissue attaches, invades the peritoneal surface, and becomes vascularized [1, 3, 4]. Further, endometriosis is believed to be a progressive disease, with a transformation from initial attachment to red, brown, black, and fibrotic scar-like lesions [5, 6]. As the disease progresses, it is hypothesized that the endometriotic lesions become less biochemically active [6]. The most biochemically active lesions are the red or proposed early lesions [6].
It has been demonstrated that matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of matrix metalloproteinases (TIMPs), play a significant role in normal endometrial remodeling that accompanies menses [7, 8]. The MMP family consists of several structurally related Zn2+-dependent secreted endopeptidases [9–12]. The MMPs, as a group, are responsible for degrading the extracellular matrix [9–11]. In human endometrium, many MMPs, including MMP-1, MMP-3, and MMP-7, are hormonally controlled [13]. These MMPs are known to degrade a variety of extracellular matrix components, including several types of collagen, gelatins, proteoglycans, laminin, fibronectin, and elastin, as well as other MMPs, including MMP-1, MMP-8, and MMP-9 [13]. Several MMPs are expressed at maximum levels during the menstrual cycle, when progesterone withdrawal takes place preceding menses [13]. Another multimember family, the TIMPs, are natural inhibitors of MMPs [9–11, 13]. In combination, the TIMP family inhibits all of the MMPs [10, 12, 13]. TIMP-1, the first identified TIMP, can inhibit MMP-1, MMP-2, and MMP-3 in a 1:1 ratio [10, 12, 13].
Recent studies have provided evidence that MMPs and TIMPs may be actively involved in remodeling the peritoneal architecture in women with endometriosis [14, 15]. MMP-3, MMP-1, MMP-7, and TIMP-1 proteins have been found in endometriotic lesions [4, 14, 16–18]. MMP-7 protein is expressed in endometriotic lesions but is absent from the eutopic endometrium during the secretory stage of the menstrual cycle [8]. Our laboratory has found that although endometriotic lesions de novo synthesize and secrete TIMP-1 protein in vitro [17], interestingly, in vivo TIMP-1 protein concentrations are lower in the peritoneal fluid of women with endometriosis [19]. Addition of TIMP protein to the peritoneal cavity of nude mice prevents the establishment of endometriosis, implying that by blocking MMPs, attachment of endometrial fragments in ectopic sites can also be blocked [20]. The amino-terminal propeptide of type III collagen can be detected in the peritoneal cavity of women with endometriosis, suggesting active cleavage of collagen [15]. Messenger RNA of interstitial collagenase, the MMP responsible for type III collagen cleavage, has been found irrespective of menstrual cycle stage, in red, but not black, human endometriotic lesions [14]. Collectively, these studies support the hypothesis that endometriotic tissue is capable of degrading and remodeling peritoneal extracellular matrix in the establishment and remodeling of endometriotic lesions.
The goal of this study was to evaluate the pattern of MMP-3, MMP-2, and TIMP-1 mRNA expression in the establishment and progression of endometriotic lesions. MMP-3 was chosen because it is hormonally regulated during the menstrual cycle, with the highest levels of expression occurring during menses [13]. MMP-3 has not been well studied in endometriosis, however, studies suggest that retrogradely shed menstrual fragments, the putative precursors of endometriotic lesions, express high levels of MMP-3 [21]. Because MMP-2 is ubiquitously expressed, it was chosen as a comparative control [13]. MMP-2 has not been studied in endometriosis. TIMP-1 was chosen because it inhibits MMP-3 and MMP-2 and has been shown to prevent the establishment of endometriosis in nude mice [20]. Hence, these studies evaluated mRNA levels of MMP-2, MMP-3, and TIMP-1 at several time points after the surgical induction of endometriosis using a well-characterized rat model. Further, as our laboratory and others have shown, endometrial MMP expression changes in an extrauterine environment [22]; therefore, a culture model free from potentially confounding in vivo factors was used to help understand MMP and TIMP expression of shed endometrial fragments before they become established endometriotic lesions.
MATERIALS AND METHODS
Rat Model for Endometriosis
Because the need for repetitive invasive surgeries limits studies of the initiation and progression of endometriosis in women, we used an established rat model for endometriosis [23]. This model has been well characterized and allows a controlled experimental design, thereby reducing confounding results that may occur with studies of MMPs and TIMPs in human endometrium and endometriosis [24]. Because endometriosis is surgically induced in rats, this model offers the opportunity to study the events involved in the initiation and progression of the endometriotic implants, studies that are inaccessible in humans.
The Institutional Animal Care and Use Committee of the University of Missouri approved the use of rats for these studies. Mature female Sprague-Dawley rats (n = 12, 
250 g; Harlan, Madison, WI) were housed in an environmentally controlled room with a 14L:10D cycle. Rats were allowed a 2-wk period of acclimation to the vivarium before any procedure was performed. Vaginal cytology was examined daily at 0900 h to obtain an index of cyclicity and ovarian function. Only rats exhibiting regular 4-day estrous cycles were used in this study. Endometriosis was surgically induced as previously described by Vernon and Wilson [23]. Briefly, the distal third of the right uterine horn was ligated using a silk suture, removed from the peritoneal cavity, and cut into four equal size squares of uterine tissue (3-mm square) using aseptic technique. The endometrial surface of the uterine squares (implants) was attached to the arterial cascades of the intestinal mesentery using nonabsorbable sutures. To help eliminate any potential effect of estrous cycle stage, rats were killed at various stages of the estrous cycle (proestrus, estrus, diestrus I, and diestrus II) as determined by evaluation of vaginal cytology with tissues from each cycle stage represented at each time point.
Experiment 1: Eutopic and Ectopic Endometrial MMP/TIMP mRNA Levels In Vivo During the Establishment of Endometriosis
For experiment 1, rats with surgically induced endometriosis were killed by CO2 inhalation at 36 h, 2 wk, or 4 wk after surgery. At 36 h after surgery, we hypothesized that endometriotic implants would have initiated events associated with attachment and establishment of early endometriotic lesions. At 2 wk, we anticipated that endometriotic lesions would be well established and actively growing. The final time point of 4 wk was chosen because endometriotic lesions in the rat model have stopped growing exponentially in diameter at this point [23].
A ventral midline incision was made and the remaining uterine horn and endometriotic implants were collected aseptically. Approximately 125 mg of each type of tissue was immediately lysed by homogenization in a guadinium-isothiocyanate buffer with 1% 2-mercaptoethanol and frozen for subsequent RNA isolation. Therefore, these samples represent the in vivo MMP and TIMP mRNA expression of the tissues at the time of collection. Such uterine samples may receive signals from the uterine-ovarian vasculature, whereas the endometriotic implants may be influenced by factors in the peritoneal cavity.
Experiment 2: Effects of Extrauterine Environment on Endometrial MMP/TIMP mRNA Levels
For experiment 2, rats with surgically induced endometriosis (n = 12) were killed by CO2 inhalation 2 wk after surgery. To evaluate tissue specificity and the effects of the extrauterine environment, additional rats (n = 3) had either adipose tissue sutured to the mesentery, or uterine tissue squares inserted under the skin on the abdomen. In the rats with surgically induced peritoneal endometriosis, a ventral midline incision was made and the remaining uterine horn and endometriotic implants were collected aseptically. Approximately 125 mg of uterine tissue and endometriotic implant tissue were collected, one-half of each of the tissues was lysed and frozen as described above. The remaining half was placed in minimal essential medium (MEM; Gibco BRL, Grand Island, NY) for in vitro explant culture as previously described [25]. Prior to explant culture, the uterine horn and endometriotic implants were dissected to expose the luminal surface of the epithelial layer. From the additional control rats, the peritoneal adipose implants, peritoneal tissue distal to the lesions, and the uterine tissue squares inserted under the abdominal skin were also harvested aseptically and placed into explant culture.
Following 1 h of preincubation in MEM, tissue pieces were transferred to 3 ml of fresh MEM and cultured for 48 h at 37°C in a shaking water bath. The culture atmosphere consisted of a combination of 50% nitrogen, 45% oxygen, and 5% carbon dioxide, and was recirculated every 4 h. Following 48 h of explant culture, tissues were recovered and lysed as above. Subsequent to lysis, all samples were stored at -80°C until further analysis. These studies were conducted to help understand MMP and TIMP expression of shed endometrial fragments after withdrawal from the in vivo steroid milieu but before they established themselves as endometriotic lesions, and to understand the specificity of uterine and peritoneal interactions on MMP and TIMP expression. Culture of established endometriotic implants was performed as a control to compare in vitro mRNA expression with cultured uterine samples as well as to evaluate the effect of removal of the lesion from the peritoneal environment.
RNA Isolation and Semiquantitative Reverse Transcriptase-Polymerase Chain Reaction Analysis
Isolation of mRNA from lysed tissues was performed on both in vivo and in vitro samples using the oligo(dT) method. Final concentrations of mRNA were determined by spectrophotometric analysis at A260. Complementary DNAs were constructed from 250 ng of mRNA from all samples using reverse transcriptase (RT; Gibco BRL).
The polymerase chain reaction (PCR) analysis was subjected to validation and optimization steps to ensure that all reactions were linear relative to the amount of input cDNA and cycles of product amplification, as previously described [26]. These controls were performed by either varying the amount of initial cDNA added to the reaction or the number of PCR cycles. Amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with primers from two different exons was used as an internal PCR standard and genomic contamination control. Primers were designed using the Prime Program of the GCG package (Genetics Computer Group, Madison, WI). All primers were designed to represent a sequence from different exons, with intervening intron sequences, to ensure that PCR products were from cDNAs instead of contaminating genomic DNA. All PCR reactions used 20 mM Tris-HCl (pH 8.9 at 22°C); 50 mM KCl; 1.5 mM MgCl2; 0.2 mM each of dATP, dTTP, dGTP, and dCTP; 0.1 µM of each gene-specific primer; and 1.25 units of Taq DNA Polymerase (Gibco BRL). Primers and final PCR conditions are defined in Table 1.
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PCR products were resolved by ethidium bromide-stained agarose gel electrophoresis, photographed using a UV light source, and transferred to nylon membranes for Southern blot analysis. The ethidium bromide-stained DNA products in the agarose gel were quantified by densitometry (ImageQuant, Molecular Dynamics, Sunnyvale, CA). Results are expressed as the ratio between the densitometric values of MMP-3, MMP-2, or TIMP-1 and GAPDH.
Southern Blot Analysis
The specificity of all PCR reactions was tested by Southern blot analysis. Membranes were hybridized with the following internal probes: base pairs (bp) 231–442 of the rat GAPDH sequence (GenBank accession number AF106860), bp 195–396 of the rat TIMP-1 sequence (GenBank accession number L29512), bp 438–659 of the rat MMP-2 sequence (GenBank accession number U65656), and bp 877–1092 of the rat MMP-3 sequence (GenBank accession number X02601). Hybridization was carried out for 16 h at 65°C in the prehybridization solution (5x saline-sodium citrate [SSC], 5x Denhardt solution, 0.5% SDS, 100 µg/ml salmon sperm DNA) plus 50 ng of probe labeled with [32P]dCTP to a specific activity of at least 1 x 108 cpm/µg by random priming (Gibco BRL). Washes were carried out twice in 5x SSC and 0.5% SDS for 15 min at room temperature, and twice in 0.1x SSC and 1% SDS for 15 min at 65°C. Following the last wash, membranes were allowed to dry and were then exposed to x-ray film for 1 day.
Statistical Analyses
All statistical analyses were performed on the MMP/TIMP mRNA values divided by the level of GAPDH (ratio of MMP or TIMP to GAPDH). All data were normally distributed and passed equal variance testing. For experiment 1, two-way ANOVA was used to compare differences between in vivo mRNA levels for MMP-3, MMP-2, and TIMP-1. Model variables included tissue type (uterus vs. endometriotic implant), time of death (36 h, 2 wk, and 4 wk), plus a tissue x time interaction term. When statistical significance was detected by two-way ANOVA, post hoc analyses were performed using the Student-Newman-Keuls test. For experiment 2, differences between in vivo and in vitro tissue mRNA levels were analyzed using the paired t-test. Data from control tissues were examined using the Student t-test. The Student t-test was also used to compare data from cultured uterine tissue (in vitro) with data from frozen endometriotic implants (in vitro). Results from distal peritoneal tissue collected from one rat were not used in the statistical analysis. All statistical comparisons were limited to cDNAs that had been amplified in the same RT-PCR cycle.
RESULTS
Endometriotic Tissue Morphology
Figure 1A shows the uterine squares at the time of autotransplantation. Following ligation to the mesenteric arterial cascades, the uterine tissue implants appeared white and avascular. The peritoneal mesenteric arteries at each implant site were completely ligated and did not provide vascular support to the implants at this time. There was no evidence of any peritoneal adhesions.
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It is interesting that by 36 h after transplantation, the endometriotic implants appeared red and vascularized (Fig. 1B). Peritoneal adhesions were also observed. At 2 wk after transplantation, the endometriotic lesions were encapsulated by peritoneal tissue and were highly vascularized (not shown). This appearance was similar to the final observations at 4 wk after transplantation (Fig. 1C). At 4 wk after transplantation, none of the endometriotic lesions appeared fibrotic and all remained highly vascularized. Such changes were not observed in s.c. implanted endometrial tissues, nor in adipose tissue transplanted in the peritoneal cavity (not shown).
Eutopic and Ectopic Endometrial MMP-3, MMP-2, and TIMP-1 mRNA Levels In Vivo During the Establishment of Endometriosis
Differences in eutopic and ectopic endometrial MMP-3, MMP-2, and TIMP-1 mRNA levels in vivo during the establishment of endometriosis are shown in Figure 2 and Table 2. In vivo, endometriotic tissues expressed significantly more MMP-3 mRNA than their uterine counterparts. The largest difference was detected at 36 h, when endometriotic implant MMP-3 levels were almost threefold higher than corresponding uterine levels. Uterine MMP-3 levels, but not endometriotic implant MMP-3 levels, were significantly different, depending on the time of death, when the highest levels of uterine MMP-3 mRNA were observed at 2 wk after surgery. No significant interactions were found between tissue type and time point for MMP-3. Although differences in MMP-3 mRNA levels were observed, levels of MMP-2 mRNA and TIMP-1 mRNA were similar in endometriotic implants and uterine tissues at all times tested. Like MMP-3, no significant interactions were found between tissue type and time point for MMP-2 or TIMP-1.
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Effects of Extrauterine Environment on In Vitro mRNA Levels for MMP-3, MMP-2, and TIMP-1
Differences between in vitro mRNA levels for MMP-3, MMP-2, and TIMP-1 compared with in vivo uterine and endometriotic implants are shown in Table 3 and Figure 3. Significantly more MMP-3 mRNA was found in cultured uterine tissues than were found in vivo (Fig. 3A). Elevated levels of MMP-3 mRNA in cultured uterine tissues were, interestingly, similar to those detected in both the in vivo and in vitro endometriotic implants (Fig. 3, A and B; P = 0.262). Differences were not observed when in vitro uterine MMP-2 mRNA levels were compared with those in vivo or in uterine TIMP-1 mRNA levels (Fig. 3A), nor when in vitro endometriotic implant MMP-2 mRNA or TIMP-1 mRNA levels were compared with those in vivo (Fig. 2B). Southern blot analysis with internal probes confirmed the identity of all PCR transcripts (MMP-3 is shown, Fig. 3, A and B).
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MMP and TIMP mRNA Levels in Control Tissues
Uterine tissues recovered from under the skin and cultured as tissue explants expressed MMP-3 mRNA (MMP-3:GAPDH ratio = 1.076 ± 0.098, mean ± SEM), MMP-2 mRNA (1.407 ± 0.158) and TIMP-1 mRNA (2.420 ± 0.228). This expression was present as early as 36 h after surgery and continued throughout the sample collection period. Adipose tissue sutured in the peritoneal cavity and the peritoneal tissues collected from sites distal to the endometriotic implants that had been cultured as tissue explants did not express MMP-3 mRNA regardless of sampling time. By contrast, both MMP-2 (1.527 ± 0.118) and TIMP-1 (2.533 ± 0.173) were observed in the adipose tissue sutured into the peritoneal cavity and into distal peritoneal tissues at all sampling times. Because the mRNA from the control tissues was not amplified in the same RT-PCR cycles as the uterine and endometriotic implants, the data can not be statistically compared.
DISCUSSION
These data demonstrate that endometriotic implants differentially express MMP-3, but not MMP-2 or TIMP-1, compared with their parent endometrial tissues. Following initial establishment, we expected to see a decline in endometriotic implant MMP-3 mRNA levels. However, MMP-3 mRNA levels did not decline and remained elevated at both 2 and 4 wk after surgery. This phenomenon suggests that our initial hypothesis was not correct and that MMP-3 may be playing additional roles later in peritoneal remodeling and progression of lesions. The significant increase in MMP-3 mRNA in the implants at the 2-wk time point may be associated with postsurgical wound healing, with the pathogenesis of endometriosis, or both. Further studies are required to determine the mechanism of increased MMP-3 mRNA levels at this time. Studies of time periods longer than 4 wk will be required to determine whether MMP-3 mRNA levels decline, as has been suggested in humans for MMP-1 [14], as endometriotic lesions further progress and become more fibrotic. Overall, these data suggest that rat endometriotic lesions, like human lesions, express MMP-3 differently from the parental endometrium (unpublished observations).
In addition to describing differences between uterine and endometriotic tissue, these experiments have shown that following 48 h of in vitro tissue culture, uterine MMP-3 mRNA levels differ from in vivo MMP-3 mRNA levels in the uterus. These data parallel another study showing a similar effect of incubation on MMP-1, MMP-2, and MMP-9 protein expression by human tissues [22]. Collectively, these data suggest that care must be taken when evaluating certain MMP mRNA expression, protein expression, or both, using in vitro systems. Culturing endometrial tissue in the absence of steroid hormones, growth factors, or cytokines results in up-regulation of the MMP-3 message, thus providing a possible explanation for some of the discrepancies that have been found in control of MMPs in in vivo versus in vitro systems [20, 22, 27–29]. However, it must also be noted that the in vitro study of MMPs and TIMPs provides an attractive model, free from potentially confounding peritoneal factors, for studying the mechanisms of misexpression of these entities in shed endometrial tissues that are the purported precursors of endometriotic lesions.
We have confirmed that up-regulation of the MMP-3 message in cultured uterine tissue is a tissue-specific phenomenon, because neither cultured adipose tissue placed into the peritoneum nor cultured peritoneal tissue located distally to the endometriotic lesions contained MMP-3 message. The fact that MMP-3 mRNA was present both in cultured uterine horns as well as cultured uteri collected from under the skin suggests that MMP-3 up-regulation is not specific to the peritoneal cavity, but is a phenomenon of uterine tissues when removed from the normal uterine environment. We have previously shown that the presence of suture alone in the peritoneum does not elicit MMP-2, MMP-3, nor TIMP-1 production [25]. Hence, endometriotic implant MMP-3 expression appears to be dependent upon the uterine tissue component as opposed to the peritoneal tissue component of endometriotic lesions or the physical process of placing suturing in the peritoneal tissue.
In vivo, the ratio of active MMPs to TIMPs controls the degradation of the extracellular matrix [13]. Our results suggest that the ratio of mRNAs is being skewed in endometriosis to favor MMP-3 expression. Significant changes in MMP-3 expression but not MMP-2 nor TIMP-1 mRNA expression are observed in endometriotic tissue. Experiments performed in the rhesus monkey suggest that continual MMP-3 expression is probably not due to a loss of progesterone receptors in ectopic tissue [29]. A consequence of the up-regulation of MMP-3 mRNA may be a change in the ratio of MMP-3:TIMP, which contributes to matrix remodeling during endometriotic tissue establishment.
The process of tissue remodeling in the pathogenesis of endometriosis is not fully understood but may involve a variety of cellular events including proliferation, migration, differentiation, and apoptosis [13]. MMPs and TIMPs may participate in these cellular activities in numerous ways by altering cell-matrix and cell-cell interactions, change in cell shape, and release or activation of growth factors [13–21]. It has been shown that expression of MMP-3 mRNA and localization of MMP-3 protein occurs predominantly in the endometrial stroma around the time of menses, whereas expression of MMP-2, also produced by the endometrial stroma, is more ubiquitous throughout the menstrual cycle [7, 8]. This differential pattern of MMP-3 expression at the time of menses suggests that it may enhance the tissue remodeling capacity of retrogradely shed endometrial tissue fragments in the peritoneal cavity. Furthermore, the continued production of MMP-3 by established lesions may participate in lesion and peritoneal tissue remodeling and contribute to the variety of types of endometriotic lesions observed. As previously mentioned, MMP-3 acts on a broad spectrum of substrates, including collagens II, IV, and XI; gelatins; proteoglycans; laminin; and fibronectin [13], making it an excellent candidate for participation in tissue remodeling and invasion in the peritoneal cavity.
Previous studies that have shown that MMP-2 and TIMP-1 mRNA are expressed throughout the human menstrual cycle stage, with elevated expression noted around the time of menses [7, 8]. Because we did not see a significant change in the levels of MMP-2 or TIMP-1 within our study suggests that the patterns of expression of MMP-2 mRNA and TIMP-1 mRNA are similar in the rat uterus. These results do not, however, correlate with the results of our prior studies showing that rat endometriotic lesions, but not rat uteri, de novo synthesize and secrete TIMP-1 protein after 24 h of in vitro culture [25]. One explanation for these differences may be that although TIMP-1 mRNA is present in both eutopic and ectopic endometrium, the TIMP-1 protein was selectively localized within the uterine tissue explants, but not the endometriotic implants, and therefore was not found in the culture media. Another possibility is that in vivo TIMP-1 synthesis and secretion is subject to local regulation that is absent in the in vitro cultures. We have found that although human endometriotic lesions synthesize and secrete TIMP-1 in vitro [30], in vivo TIMP-1 protein concentrations are lower in the peritoneal fluid of women with endometriosis [19]. Further experimentation is needed to resolve these differences and to understand the role of TIMP-1 expression by endometriotic lesions in the peritoneal cavity.
Although the majority of literature concerning MMP and TIMP expression by the uterus describes endometrial expression, the myometrium also expresses MMP-1, MMP-3, TIMP-1, and TIMP-2 [31]. The studies performed in this project did not distinguish between endometrial and myometrial MMP or TIMP production. Nonetheless, these results provide an attractive hypothesis that endometrial tissue, in any ectopic site, is aberrantly generating MMP-3, suggesting that shed endometrial fragments possess a mechanism to facilitate invasion into the peritoneum following retrograde menstruation. These studies lay the foundation for subsequent studies to dissect the mechanisms for this altered expression and demonstrate that elevated MMP-3 expression by endometrial tissue leads to the establishment and progression of ectopic endometrial tissue growth. Ongoing studies in our laboratory are using this model to evaluate the possibility that this aberrant expression of MMP-3 may be the result of a positive MMP regulator such as interleukin-1 alpha or other cytokines.
ACKNOWLEDGMENTS
The authors thank Ms. Emily Ricke for her assistance with the animal surgeries and technical support and Ms. Kimberly Farinella for her aid in preparing the manuscript. The authors also thank Michael F. Smith, Ph.D., for his helpful comments.
FOOTNOTES
First decision: 7 September 2000.
1 This research was supported by a University of Missouri-Columbia Molecular Biology Fellowship, a National Institutes of Health predoctoral fellowship to K.E.C., and by support from TAP Holdings, Inc. to K.S.T. 
2 Correspondence: Kathy L. Sharpe-Timms, Department of Obstetrics and Gynecology, 1 Hospital Drive, N 625 Health Sciences Center, University of Missouri, Columbia, MO 65212. FAX: 573 882 9010; timmsk{at}health.missouri.edu
Accepted: June 4, 2001.
Received: August 10, 2000.
REFERENCES
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