EMMPRIN is secreted by human uterine epithelial cells in microvesicles and stimulates metalloproteinase production by human uterine fibroblast cells.

Endometrial remodeling is a physiological process involved in the gynecological disease, endometriosis. Tissue remodeling is directed by uterine fibroblast production of matrix metalloproteinases (MMPs). Several MMPs are regulated directly by the protein extracellular matrix metalloproteinase inducer (EMMPRIN) and also by proinflammatory cytokines such as interleukin (IL)1-α/ß. We hypothesized that human uterine epithelial cells (HESs) secrete intact EMMPRIN to stimulate MMPs. Microvesicles from HES cell-conditioned medium (CM) expressed intact EMMPRIN protein. Treatment of HES cells with estradiol or phorbyl 12-myristate-13-acetate increased the release of EMMPRIN-containing microvesicles. The HES CM stimulated MMP-1, -2, and -3 messenger RNA levels in human uterine fibroblasts (HUFs) and EMMPRIN immunodepletion from HES-cell concentrated CM reduced MMP stimulation (P < .05). Treatment of HUF cells with low concentrations of IL-1ß/α stimulated MMP production (P < .05). These results indicate that HES cells regulate MMP production by HUF cells by secretion of EMMPRIN, in response to ovarian hormones, proinflammatory cytokines as well as activation of protein kinase C.

Extracellular matrix metalloproteinase inducer expression in the baboon endometrium: menstrual cycle and endometriosis.

Extracellular matrix metalloproteinase inducer (EMMPRIN; BSG) regulates tissue remodeling through matrix metalloproteinases (MMPs). In human and non-human primates, endometrial remodeling is important for menstruation and the pathogenesis of endometriosis. We hypothesized that as in humans, BSG and MMPs are expressed in the endometrium of cycling baboons, and their expression is hormonally regulated by ovarian hormones, but endometriosis disrupts this regulation. BSG expression was evaluated in the baboon endometrium by q-PCR and immunohistochemistry. In the endometrium of control cycling animals, BSG mRNA levels were highest in late secretory stage tissue. BSG protein localized to glandular epithelial cells during the proliferative phase; whereas, secretory stage tissues expressed BSG in glandular and luminal epithelia with weak stromal staining. Several MMPs were differentially expressed throughout the menstrual cycle with the highest levels found during menstruation. In ovariectomized animals, BSG endometrial mRNA levels were highest with treatment of both estrogen and progesterone than that with only estrogen. Estrogen alone resulted in BSG protein localization primarily in the endometrial glandular epithelia, while estrogen and progesterone treatment displayed BSG protein localization in both the glandular and stromal cells. Exogenous hormone treatment resulted in differential expression patterns of all MMPs compared with the control cycling animals. In the eutopic endometrium of endometriotic animals, BSG mRNA levels and protein were elevated early but decreased later in disease progression. Endometriosis elevated the expression of all MMPs except MMP7 compared with the control animals. In baboons, BSG and MMP endometrial expression is regulated by both ovarian hormones, and their expression patterns are dysregulated in endometriotic animals.

Changes in small intestinal nutrient transport and barrier function after lipopolysaccharide exposure in two pig breeds.

Specific pig breeds with unique characteristics have been developed, and the current study sought to characterize some of these differences. Using modified Ussing chambers, electrophysiological mucosal transport of D-glucose, L-Gln, L-Pro, L-Arg, L-Thr, and glycylsarcosine was assessed in small intestinal tissues (duodenum, jejunum, ileum) taken from Yorkshire-based hybrid (BW = 142.4 +/- 2.0 kg; mean age = 8 mo) and Meishan (BW = 65.8 +/- 0.8 kg; mean age = 6 mo) female pigs after 4 h of lipopolysaccharide (LPS) exposure. Gilts were randomly assigned to control (saline infusion; n = 6 Yorkshires, n = 5 Meishans) or LPS (n = 7 Yorkshires, n = 5 Meishans) groups. Therefore, treatments were arranged in a 2 (breed) x 2 (LPS infusion) factorial. Four hours after infusions, pigs were euthanized, and intestinal segment samples were removed. Glucose transport in the ileum was decreased (P < 0.001) in Yorkshires with LPS but was increased (P < 0.001) by over 2-fold in Meishans with LPS. After LPS infusion, Pro transport was increased in duodenum (over 5-fold; P = 0.04) and ileum (over 10-fold; P < 0.001) of Meishans but was unaffected in Yorkshires. Arginine transport in the ileum of control Meishans was greater (P = 0.05) than Arg transport in control Yorkshires. Glycylsarcosine transport was greater (P = 0.02) in Meishans than Yorkshires (nearly 2-fold), regardless of LPS provision. Glycylsarcosine transport was increased (P = 0.003) over 2-fold by LPS, regardless of pig breed. Resistance (barrier function) was increased (P = 0.03) by LPS in Yorkshires but was unaffected in Meishans. The current study indicates that small intestinal function responded differently to LPS in Yorkshire and Meishan gilts and that these effects were nutrient- and segment-dependent.

Extracellular matrix metalloproteinase inducer regulates metalloproteinases in human uterine endometrium.

CONTEXT: Endometrial remodeling occurs during each menstrual cycle in women and also during the establishment of endometriosis. Both processes involve the production of metalloproteinases (MMPs) by uterine endometrial cells. OBJECTIVE: The objective of this study was to determine whether tissue remodeling and endometrial invasion involve activation of MMPs by extracellular matrix metalloproteinase inducer (EMMPRIN). MAIN OUTCOME MEASURES: EMMPRIN expression was examined by immunohistochemistry and real-time PCR in ectopic and eutopic endometria. For functional assays, human uterine fibroblasts were treated in the absence or presence of IL-1beta (10 ng/ml) or purified native EMMPRIN (0.5 or 1 microg/ml) for 24 h. Cellular RNA and conditioned medium were assayed by real-time PCR or immunoblotting. RESULTS: EMMPRIN protein localized to epithelial and fibroblast cells of eutopic and ectopic endometria. The pattern of localization was regulated by ovarian hormones. EMMPRIN mRNA levels varied throughout the menstrual cycle in parallel with the cyclic changes in estradiol. EMMPRIN treatment (0.5 microg/ml) of human uterine fibroblast cells stimulated MMP-1 (5.23-fold) and MMP-2 (8.55-fold), but not MMP-3, mRNA levels over levels in control cells (P < 0.05). EMMPRIN treatment (1 microg/ml) stimulated endogenous EMMPRIN (1.6-fold) mRNA levels (P > 0.05). IL-1beta stimulated MMP-1 (5.6-fold), MMP-2 (2.8-fold), and MMP-3 (75-fold) gene expression, but not EMMPRIN, over levels in control cells (P < 0.05). Both EMMPRIN and IL-1beta treatments stimulated MMP-1, -2, and -3, but not EMMPRIN protein secretion, with 0.5 microg/ml producing the greatest response. CONCLUSIONS: The ability of EMMPRIN to stimulate MMP secretion by endometrial fibroblasts indicates its potential role in uterine remodeling and the pathogenesis of endometriosis.

Adenomyosis demonstrates increased expression of the basic fibroblast growth factor receptor/ligand system compared with autologous endometrium.

OBJECTIVES: Basic fibroblast growth factor (bFGF) is an angiogenic growth factor present in human endometrium and myometrium. Women with leiomyoma-related abnormal uterine bleeding have local dysregulation of bFGF and its type 1 receptor (FGF-R). This study was designed to evaluate if adenomyosis expresses bFGF and FGF-R, and if present, to compare bFGF and FGF-R expression in adenomyosis and autologous endometrium. DESIGN: Menopausal uteri containing endometrium and adenomyosis were analyzed using immunohistochemistry with monoclonal antibodies specific for bFGF, FGF-R, and proliferating cell nuclear antigen (PCNA), a marker of cellular proliferation. The expression and intensity of staining for bFGF, FGF-R, and PCNA were evaluated in the glandular epithelium and stroma of adenomyosis and endometrium. RESULTS: Glandular epithelial staining was significantly greater in adenomyosis compared with autologous endometrium for bFGF and FGF-R. Stromal staining for bFGF and PCNA was significantly increased in adenomyosis compared with autologous endometrium. CONCLUSIONS: Upregulation of the bFGF receptor/ligand system and increased cellular proliferation in adenomyosis may contribute to the pathogenesis of abnormal uterine bleeding associated with adenomyosis.

Identification of new therapies for leiomyomas: what in vitro studies can tell us.

Leiomyomas are a significant problem in women's health. An understanding of the biology of these tumors and how their growth is regulated is emerging from in vitro studies using tissue specimens and cultured cells. These studies have clarified how the ovarian steroid hormones regulate growth of uterine SMCs and how the ovarian steroid ligand-receptor system has been altered in leiomyomas. Such information will allow investigators to identify steroid hormone antagonists and steroid hormone receptor modulators that may be useful for treatment of leiomyomas. We are now also developing a much better understanding of the growth factors that are produced by SMCs of leiomyoma tumors. These growth factors not only regulate the proliferation, apoptosis, and extra-cellular matrix production of the SMCs but also regulate proliferation and migration of vascular endothelial cells. Targeting these growth factors and their receptors can reduce leiomyoma growth through two different mechanisms. One targets the SMCs and the other targets the vascular system that supports the growth of the tumor. Another important lesson that can be learned from reading the scientific literature is that there are striking similarities between the biology of uterine leiomyomas and other pathologic diseases that involve mesenchymally derived cells. These include benign keloids, other fibrotic diseases such as pulmonary fibrosis, and vascular diseases such as atherosclerosis. Compounds that are developed to treat these conditions may also be beneficial for treatment of uterine leiomyomas. The next few years will undoubtedly yield many new drug discoveries for these diseases.

Human leiomyoma smooth muscle cells show increased expression of transforming growth factor-beta 3 (TGF beta 3) and altered responses to the antiproliferative effects of TGF beta.

Transforming growth factor-betas (TGF betas) are multifunctional peptides that regulate growth and differentiation in a variety of cells. The goals of this study were to compare expression of the TGF beta isoforms in normal myometrium and benign leiomyoma tumors of the uterus and to examine the effects of TGF betas on cell proliferation and collagen production by these cells in vitro. Myometrium and leiomyoma tissues were obtained from patients undergoing elective hysterectomies. Tissues were processed for ribonucleic acid (RNA) and were also established as primary cell cultures. Northern blot analysis showed that the levels of TGF beta 1 messenger RNAs (mRNAs) were similar between leiomyoma and myometrium, whereas leiomyoma showed 5-fold higher levels of expression of TGF beta 3 mRNA than autologous myometrium. Expression of TGF beta 3 protein detected by immunohistochemistry was much more intense in leiomyoma tissues than in corresponding myometrium. Levels of both TGF beta 1 and TGF beta 3 increased with increasing cell density for leiomyoma and myometrium smooth muscle cells cultured in vitro. Effects of TGF beta 1 and TGF beta 3 on cell proliferation were assessed by measuring changes in DNA synthesis with the tritiated thymidine incorporation assay. The doses of TGF betas tested were 0, 0.1, 1.0, and 10.0 ng/mL. All three doses of TGF beta 1 and TGF beta 3 inhibited DNA synthesis in myometrium smooth muscle cells by 31--54%. Concomitant treatment with an immunoneutralizing antibody to TGF beta 1--3 reversed this inhibitory effect. In contrast, TGF beta 1 had no effect on leiomyoma smooth muscle cells, whereas TGF beta 3 increased DNA synthesis by leiomyoma cells. Combined treatment with the immunoneutralizing antibody prevented this increase. Treatment of leiomyoma and myometrial cells with the TGF beta immunoneutralizing antibody for 24 h caused a 45--60% reduction in collagen type I and type III mRNA levels, suggesting that endogenous TGF betas are important for collagen production. These results support the hypothesis that alterations in the TGF beta system produce loss of sensitivity to the antiproliferative effects of TGF beta, and increased expression of TGF beta 3 may contribute to the growth of these tumors.

Novel therapeutic strategies for leiomyomas: targeting growth factors and their receptors.

Leiomyomas (fibroids) are benign smooth-muscle cell (SMC) tumors of the uterus and are the most common pelvic tumors in women. These tumors occur primarily during the reproductive years and are the most common indication for hysterectomy in women. Unfortunately the only effective treatments for leiomyomas and the associated abnormal uterine bleeding are surgical, involving either hysterectomy, myomectomy, or hysteroscopic removal of the tumors. The goal of this paper is to discuss recent research findings that support the idea of using therapeutic compounds that block the actions of specific growth factors as therapeutic agents for treatment of leiomyomas and abnormal uterine bleeding. Most of the studies were carried out using cell cultures of leiomyoma or myometrial SMCs. Primary cultures of SMCs provide a system for investigation of the roles of growth factors and their receptors in proliferation of normal myometrial and leiomyoma SMCs. Several growth factors have been shown to be present and to have regulatory roles in the proliferation of uterine SMCs. Bioassay and Western blotting of fast protein liquid chromatography fractions of tissue extracts identified platelet-derived growth factor, heparin-binding epidermal growth factor, hepatoma-derived growth factor, and basic fibroblast growth factor in normal myometrium and fibroid tumors. The presence of heparin-binding growth factors suggests a possible focus for therapeutic agents. RG13577 (a heparinlike compound) and halofuginone (an alkyloid) reversibly inhibited DNA synthesis of normal myometrial and leiomyoma cells without toxic effects. Pirfenidone, a known antifibrotic drug, inhibited DNA synthesis and synthesis of collagen type I mRNA in normal and fibroid cells, and decreased collagen type III mRNA only in normal myometrial cells. Another hopeful therapeutic candidate, interferon-Alpha, significantly inhibited growth factor-stimulated proliferation in both normal and leiomyoma cells. These results suggest that future nonsurgical treatments for leiomyomas may include compounds that block the actions of specific growth factors that regulate proliferation and collagen production by uterine SMCs.

Prolactin is an autocrine or paracrine growth factor for human myometrial and leiomyoma cells.

OBJECTIVE: To test the hypothesis that prolactin (PRL) acts as a mitogenic growth factor for human leiomyoma and myometrial cells. METHODS: To test this hypothesis, we performed three different types of experiments. First, we assessed whether exogenous PRL acted as a mitogen for cultured uterine smooth muscle cells. Second, we examined the role of endogenous PRL by assessing the cell number after exposure of the cultures to a neutralizing antibody to PRL. Finally, we examined both fresh tissues and cultured cells for expression of the PRL receptor messenger ribonucleic acid using the techniques of reverse-transcriptase polymerase chain reaction and Southern blotting. RESULTS: A significant suppression in cell number was seen after 5 days of culture for leiomyoma cells but not for myometrial cells after treatment with exogenous PRL. Both cell types showed a significant decrease in cell number after treatment with anti-PRL antibody. A 893-bp segment consistent with the cytoplasmic domain of the long form of the PRL receptor was amplified from both fresh and cultured tissues and confirmed by Southern blotting and sequencing. CONCLUSIONS: PRL appears to be an autocrine or paracrine growth factor for both leiomyoma and myometrial cells. However, there are some differences between tissues in their sensitivity to this growth factor.

HMGIC expression in human adult and fetal tissues and in uterine leiomyomata.

The high-mobility-group (HMG) protein gene, HMGIC, is localized to chromosome 12, band q15, a region often rearranged in benign mesenchymal tumors, including uterine leiomyomata. Although some evidence suggests a role in regulation of cell proliferation, the precise function of HMGIC in the development or progression of these tumors remains unclear. We investigated HMGIC expression in 17 fetal tissues (adrenal, aorta, bone, brain, heart, intestine, kidney, liver, lung, muscle, ovary, placenta, skin, spleen, stomach, testis, and uterus) and 10 adult tissues (aorta, brain, cerebellum, fat, kidney, liver, lung, lymph node, myometrium, and spinal cord) by Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) assays. Comparisons between HMGIC gene expression in tumor samples from 11 uterine leiomyomata and 7 normal matched myometrium or in vitro cell cultures (chorionic villi, placenta, myometrium, leiomyoma, and skin) were also performed. The gene was expressed in all fetal tissues tested but only in adult lung and kidney. HMGIC was also expressed in leiomyoma tumor samples containing t(12;14) and in all in vitro cell cultures. The pattern of HMGIC expression suggests that this gene is important in rapidly proliferating human fetal tissues. Restoration of expression in leiomyomata required dysregulation of HMGIC. Transcripts of HMGIC can also be detected after in vitro cell culture, suggesting that HMGIC expression may be affected by factors present in culture media and serum. Genes Chromosomes Cancer 25:316-322, 1999.

Onapristone suppresses prolactin production in explant cultures of leiomyoma.

OBJECTIVE: To assess the action of onapristone, a type I antiprogestin, on prolactin (PRL) production by explant cultures of leiomyoma and myometrium. DESIGN: Explant cultures of myometrium and leiomyomas from 3 premenopausal women undergoing hysterectomy in the proliferative phase of the menstrual cycle. MAIN OUTCOME MEASURES: PRL secretion measured by radioimmunoassay. RESULTS: PRL secretion was decreased in leiomyomas by onapristone. There was no effect in the myometrium. There was no additional effect with the addition of the type II antiprogestin mifepristone (RU 486). CONCLUSION: PRL production is suppressed in leiomyomas but not in myometrium after treatment with onapristone in vitro. This suppression may serve as a marker for the clinical effectiveness of agents used in the treatment of leiomyomas.

Messenger ribonucleic acid for the gonadal luteinizing hormone/human chorionic gonadotropin receptor is not present in human endometrium.

OBJECTIVE: To determine whether messenger RNA for the gonadal LH/hCG receptor is present in human endometrium with the use of reverse-transcriptase polymerase chain reaction. DESIGN: In vitro experiment. SETTING: Academic medical center. PATIENT(S): Premenopausal women who were not receiving hormonally active medications and who were undergoing hysterectomy for uterine leiomyomas, menorrhagia, pelvic pain, or uterine prolapse. INTERVENTION(S): Tissue from hysterectomy specimens was processed for RNA and treated with deoxyribonuclease where appropriate, and RNA was reverse-transcribed to complementary DNA. MAIN OUTCOME MEASURE(S): An appropriately sized band after reverse-transcriptase polymerase chain reaction, followed by sequencing to confirm the results. RESULT(S): A primer pair that spanned the extracellular domain was unable to amplify receptor complementary DNA from human endometrial tissue. For a primer pair that spanned transmembrane regions 2-6 of the receptor and was contained wholly in exon 11, a 552-base pair fragment was amplified successfully in 19 of 25 human endometrial samples. CONCLUSION(S): The traditional gonadal LH/hCG receptor does not appear to be present in human endometrial tissue. The presence of a portion of the transmembrane part of the molecule suggests that human endometrium may express a truncated or variant form of the receptor.

Transforming growth factor-beta stimulates mouse blastocyst outgrowth through a mechanism involving parathyroid hormone-related protein.

The goals of this study were 1) to compare the effects of transforming growth factor-beta (TGF-beta) and parathyroid hormone-related protein (PTHrP) on mouse blastocyst attachment and outgrowth in vitro, 2) to determine whether TGF-beta acts through a mechanism involving PTHrP, 3) to examine effects of PTHrP on preimplantation mouse embryo development, and 4) to determine the pattern of expression of PTHrP protein in the uterus of the mouse during early gestation. In the first set of experiments, hatched blastocysts were placed in fibronectin-coated wells. Cultures were treated with PTHrP or TGF-beta1 and assessed at 24, 48, and 72 h for attachment and surface area of blastocyst outgrowth. Results showed that both PTHrP and TGF-beta1 increased blastocyst outgrowth significantly. A PTHrP-neutralizing antibody blocked the stimulatory effect of both PTHrP and TGF-beta1, suggesting that TGF-beta1 acts to increase endogenous production of PTHrP by the blastocyst. Immunoassay of conditioned medium from blastocysts treated with either TGF-beta1 or PTHrP 1-34 confirmed a 3- to 4-fold increase in levels of PTHrP 1-141. In the second series of experiments, pronuclear zygotes were cultured in various concentrations of PTHrP for 96 h. Blastocysts then were subjected to differential fluorescent staining of inner cell mass and trophectoderm cells. Treatment of mouse embryos with the various concentrations of PTHrP altered neither the number developing to the blastocyst stage nor the number of inner cell mass or trophectoderm cells in the resulting blastocysts. In the third experiment, pregnant mice were killed at Days 3, 4, 5, 6, and 7 of gestation, and uterine horns were processed for immunohistochemistry. Uterine sections were stained with antibodies to PTHrP, desmin, and laminin. On Days 3, 4, and 5, uterine luminal and glandular epithelial cells stained intensely for PTHrP, while stromal cells were negative. By Days 6 and 7, decidualized stromal cells stained positively for PTHrP, desmin, and laminin. These results support the hypothesis that TGF-beta and PTHrP play an important role in the process of implantation.

Fibroids: pathophysiology and current medical treatment.

Uterine leiomyomas, or fibroids, are a major cause of abnormal uterine bleeding in women. These benign tumours develop during the reproductive years and their growth has been shown to be dependent on the ovarian steroid hormones oestradiol and progesterone. The growth promoting effects of these steroid hormones appear to be mediated through the local production of specific growth factors. Traditional treatment for leiomyomas has been surgical removal through either hysterectomy or myomectomy. Newer surgical techniques, such as hysteroscopic removal of leiomyomas, endometrial ablation, or uterine arterial embolization, are now being tested as effective but less invasive methods of treatment. Non-surgical treatment of leiomyomas has been primarily through the use of gonadotrophin-releasing hormone agonists which suppress circulating oestradiol and progesterone levels by shutting down the pituitary-ovarian axis. The suppression in steroid hormone levels results in significant fibroid shrinkage, but long-term use of these compounds is not recommended because patients suffer significant bone loss. New antisteroidal compounds, such as the antiprogestin RU 486 and the selective oestrogen-receptor modulator raloxifene, are now being tested as possible therapeutic agents for fibroids.

New concepts in the treatment of uterine leiomyomas.

Uterine leiomyomas are a common clinical occurrence for gynecologists. The current approach to treating these neoplasms is shaped by classic surgical principles and the knowledge that these tumors are responsive to the gonadal steroids estrogen and progesterone. As knowledge of leiomyomas advances through the techniques of molecular biology and molecular genetics, new concepts are developed that go beyond just myomas as steroid-responsive tumors. Understanding the molecular events involved in the transformation of a normal myometrial cell into a neoplastic cell and the subsequent growth of these leiomyoma cells will be important in determining the pathogenesis of these tumors and providing new targets for treatment. Knowing the role of peptide growth factors, including basic fibroblast growth factor and transforming growth factor-beta, in the pathogenesis of leiomyoma-related symptoms might lead to new treatments targeting these molecules or their receptors. As the effects of genes, including HMGIC and HMGI(Y), are determined; new treatments to prevent leiomyoma formation or growth may be developed. As we gain understanding of the molecular events that cause benign gynecologic conditions such as leiomyomas, safer and more effective treatments might be found as we enter the 21st century.

Increased expression of stromelysin 3 mRNA in leiomyomas (uterine fibroids) compared with myometrium.

OBJECTIVE: To determine the level of expression of selected matrix metalloproteinases in uterine leiomyoma compared with unaffected myometrium in an effort to explain the abnormal accumulation of extracellular matrix in the leiomyoma. METHODS: The levels of matrix metalloproteinase (MMP) mRNA in leiomyoma and myometrium were measured in samples from 22 patients during either proliferative (n = 6) or secretory phases (n = 16) of the menstrual cycle. Relative amounts of collagenase (MMP-1) and stromelysin (MMP-3) mRNAs were measured by Northern blot analysis, and amounts of stromelysin 3 (MMP-11) and matrilysin (MMP-7) mRNA from each sample were determined by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and normalized to beta-actin mRNA. RESULTS: The levels of MMP-1 and MMP-3 mRNAs were similar in both leiomyoma and unaffected myometrium. The levels of MMP-11 mRNA were twofold greater in leiomyoma compared with myometrium throughout the menstrual cycle, and the differences in the levels of MMP-11 were significantly different during the secretory phase. The level of MMP-7 mRNA expression was similar in leiomyoma, myometrium, and endometrium. CONCLUSIONS: Among the metalloproteinases examined in this study, only the levels of MMP-11 mRNA were elevated in leiomyoma compared with myometrium. The increased expression of MMP-11 in uterine leiomyoma compared with myometrium is analagous to previously reported elevations of MMP-11 mRNA in dermatofibromas compared with unaffected skin. The increased expression of MMP-11 mRNA in fibroid tumors suggests that this MMP may be involved in the formation of a more fibrous extracellular matrix in leiomyoma relative to unaffected myometrium.

Interferon-alpha is a potent inhibitor of basic fibroblast growth factor-stimulated cell proliferation in human uterine cells.

PROBLEM: Abnormal uterine bleeding is a significant health problem for many women and is the number-one reason for performing hysterectomy in the United States. Leiomyomas (uterine fibroids) are benign neoplasms that are a frequent cause of abnormal uterine bleeding. The goal of this study was to assess the effects of the anti-angiogenic cytokine, interferon (INF)-alpha, on the proliferation of both leiomyoma and normal uterine cells. METHOD OF STUDY: Primary cultures of leiomyoma, myometrial, and endometrial stromal cells were established for in vitro study. The effects of INF-alpha (10, 100, and 1000 U/ml) were tested on serum-stimulated and basic fibroblast growth factor-stimulated cell proliferation using the [3H]thymidine incorporation assay. RESULTS: INF-alpha was a potent inhibitor of cell proliferation for all three cell types, with endometrial stromal cells showing the greatest sensitivity. The antiproliferative effect did not appear to result from toxic effects on the cells. CONCLUSION: INFs may prove to be useful therapeutic agents for the treatment of leiomyoma-related abnormal uterine bleeding.

Leuprolide acetate-treated leiomyomas retain their relative overexpression of collagen type I and collagen type III messenger ribonucleic acid.

OBJECTIVE: To evaluate the levels of mRNA for the extracellular matrix proteins collagen type I, collagen type III, and fibronectin in leiomyomas and myometrium obtained from women treated with GnRH-agonist (GnRH-a) and to examine steroid hormone regulation of these proteins using an in vitro explant culture system. METHODS: Northern blot analysis of mRNA was obtained from hysterectomy specimens at the time of surgery or after 48 hours of in vitro steroid treatment. A portion of the tissue was processed for RNA, and the remaining tissue was used to establish explant cultures. Extracellular matrix/alpha-tubulin ratio was computed for each band, and within each experiment the lowest ratio was standardized to a value of one densitometry unit to allow for comparison among experiments. RESULTS: There is a relative overexpression of both collagen type I and collagen type III mRNAs but not fibronectin in leiomyomas compared to myometrium from the same uteri of women treated with GnRH-a. CONCLUSION: Leiomyomas obtained from women treated with GnRH-a show an up-regulation of collagens type I and type III similar to that seen in leiomyomas obtained from women in the proliferative phase of their menstrual cycles.

Pirfenidone: a novel pharmacological agent that inhibits leiomyoma cell proliferation and collagen production.

There are currently no effective, long-term drug therapies for the treatment of leiomyomas. Pirfenidone (Marnac, Inc.) is an antifibrotic agent that is being tested for use in patients with pulmonary fibrosis. Because leiomyomas are characterized also by increased cell proliferation and tissue fibrosis, we examined the effects of pirfenidone on cell proliferation and collagen expression in cultured myometrial and leiomyoma smooth muscle cells. Effects of pirfenidone on proliferation of myometrial and leiomyoma cells were measured using tritiated thymidine incorporation assays and changes in actual cell numbers. Possible cytotoxic effects were examined using lactate dehydrogenase assays and trypan blue exclusion. Effects on collagen type I and type III production were assessed by Northern blotting. Doses of pirfenidone tested were: 0, 0.01, 0.1, 0.3, and 1.0 mg/mL. Serum-stimulated increases in DNA synthesis and cell proliferation by myometrial and leiomyoma cells were significantly inhibited in a dose-dependent manner by pirfenidone. Densitometric analysis of Northern blots showed significantly decreased expression of collagen type I and type III messenger RNAs in both leiomyoma and myometrial cells. Lactate dehydrogenase assays and trypan blue exclusion measurements showed no cytotoxic effect of pirfenidone at concentrations that inhibited cell proliferation and collagen production. Pirfenidone is an effective inhibitor of myometrial and leiomyoma cell proliferation in vitro and reduces the messenger RNA levels of collagen types I and III in a dose-dependent manner. This compound may prove to be an effective nonsteroidal therapy for treatment of uterine leiomyomas.

Expression of the fibroblast growth factor receptor in women with leiomyomas and abnormal uterine bleeding.

Basic fibroblast growth factor (bFGF) is a regulator of angiogenesis which is overexpressed in leiomyomas compared with matched myometrium. To understand the physiological significance of this finding we characterized the expression of the type 1 receptor for this ligand (FGFR1). Utilizing reverse transcription-polymerase chain reaction (RT-PCR) we identified the complete and alternatively spliced transmembrane forms and two secreted forms of the FGFR1 in endometrium, myometrium and leiomyomas from all patients. This is the first report of secreted forms in uterine tissue. Proteins consistent with each of these isoforms were identified by Western blot analysis in all three tissues. Immunohistochemistry revealed menstrual cycle-specific regulation of FGFR1 protein in the endometrial stroma of normal women but not in women with leiomyomas and abnormal uterine bleeding. Stromal FGFR1 expression is suppressed in the early luteal phase in normal women, but not in women with leiomyoma-related bleeding. These findings support the role of the bFGF ligand-receptor system in the pathogenesis of leiomyoma-related bleeding and may have implications for fertility and contraception since the differential FGFR1 expression occurs in the peri-implantation period of the early luteal phase.