Peroxisome-proliferator activator receptor-gamma activation decreases attachment of endometrial cells to peritoneal mesothelial cells in an in vitro model of the early endometriotic lesion.

The aim of this study was to investigate whether peroxisome proliferator-activated receptor (PPAR)-gamma activation has an effect on the attachment of endometrial cells to peritoneal mesothelial cells in a well-established in vitro model of the early endometriotic lesion. The endometrial epithelial cell line EM42 and mesothelial cell line LP9 were used for this study. EM42 cells, LP9 cells or both were treated with the PPAR-gamma agonist ciglitazone (CTZ) at varying concentrations (10, 20 and 40 microM) x 48 h with subsequent co-culture of EM42 and LP9 cells. The rate of EM42 attachment and invasion through LP9 cells was then assessed and compared with control (EM42 and LP9 cells co-cultured without prior treatment with CTZ). Next, attachment of CTZ-treated and untreated EM42 cells to hyaluronic acid (HA), a cell adhesion molecule (CAM) on peritoneal mesothelial cells, were assessed. Although there was no difference in EM42 attachment when LP9 cells alone were treated with CTZ, treatment of EM42 cells with 40 microM CTZ decreased EM42 attachment to LP9 cells by 27% (P < 0.01). Treatment of both EM42 and LP9 cells with 40 microM CTZ decreased EM42 attachment to LP9 by 37% (P < 0.01). Treatment of EM42 cells with 40 microM CTZ decreased attachment to HA by 66% (P = 0.056). CTZ did not decrease invasion of EM42 cells through the LP9 monolayer. CTZ may inhibit EM42 cell proliferation. In conclusion, CTZ significantly decreased EM42 attachment to LP9 cells and HA in an in vitro model of the early endometriotic lesion.

Contemporary medical therapy for polycystic ovary syndrome.

Polycystic ovary syndrome is a multi-system endocrinopathy with long-term metabolic and cardiovascular health consequences. Patients typically present due to symptoms of irregular menstruation, hair growth, or infertility; however, recent management options are aimed at further treating underlying glucose-insulin abnormalities as well as androgen excess for proactive control of symptoms. By a 2003 international consensus conference, diagnosis is made by two out of three criteria: chronic oligoovulation or anovulation after excluding secondary causes, clinical or biochemical evidence of hyperandrogenism (but not necessarily hirsutism due to inter-patient variability in hair follicle sensitivity), and radiological evidence of polycystic ovaries. Traditional medical treatment options include oral contraceptive pills, cyclic progestins, ovulation induction, and anti-androgenic medications (aldosterone antagonist, 5alpha-reductase antagonist, and follicle ornithine decarboxylase inhibitor). Recent pharmacotherapies include insulin-sensitizing medications metformin and two thiazolidinediones (rosiglitazone/Avandia and pioglitazone/Actos), a CYP19 aromatase inhibitor (letrozole/Femara), and statins to potentially lower testosterone levels.

Endocrine and paracrine regulation of endometrial angiogenesis.

The human endometrium is a complex tissue comprised of different cell types, including epithelial, stromal, inflammatory, perivascular, and blood vessel cells. The hormonal receptivity and distribution of these cell populations change during the menstrual cycle. Cyclical endometrial growth is dependent on its ability to regenerate a vascular capillary network, which grows in parallel with the proliferation and differentiation of the endometrial lining. Natural hormonal effects on the endometrium and endocrine manipulation of this tissue, in response to the use of exogenous steroid therapies, can affect endometrial capillary proliferation and function, leading to clinical abnormalities of uterine bleeding. We propose that the regulation of endometrial angiogenesis is mediated indirectly via complex interactions among cell types. Our laboratory has focused on a prototypical member of the angiogenic proteins, vascular endothelial growth factor (VEGF)-A. In this paper we present data demonstrating that VEGF-A expression in normal endometrial epithelial and stromal cells and in Ishikawa adenocarcinoma cells is increased by an ovarian steroid, estradiol. Infiltrating immune cells, particularly polymorphonuclear granulocytes, also are sources of VEGF-A. In inflammatory conditions involving the endometrium (e.g., endometriosis), a proinflammatory cytokine, IL-1beta, can mediate neoangiogenesis by inducing VEGF-A gene transcription. Thus, endometrial vascularization is effected by both endocrine and paracrine pathways.

IL-1beta induction of RANTES (regulated upon activation, normal T cell expressed and secreted) chemokine gene expression in endometriotic stromal cells depends on a nuclear factor-kappaB site in the proximal promoter.

A complex network of cytokines mediates immunoregulatory responses in the pathogenesis of endometriosis. RANTES (regulated upon activation, normal T cell expressed and secreted) is a chemoattractant for monocytes and T cells. Endometriotic lesions express RANTES, and its concentration in peritoneal fluid correlates with the severity of endometriosis. We investigated the influence of IL-1beta, a potent macrophage cytokine, on RANTES production in endometriotic stromal cells and determined the region of the RANTES promoter responsible for IL-1beta action. RANTES mRNA was induced 5-fold in endometriotic stromal cells, and the conditioned medium RANTES protein concentrations were 12-fold higher in IL-1beta-treated endometriotic stromal cells vs. untreated controls (P < 0.05). IL-1beta activated the full-length (-940 bp) RANTES promoter as well as a truncated 456-bp 5'-flanking construct by 2-fold. Mutagenesis of a nuclear factor-kappaB response element at -30 bp abolished the IL-1beta effect, whereas mutation of a nearby TNF response element did not affect the IL-1beta induction. An IL-1beta time-course Western assay revealed a rapid diminution of IkappaB (endogenous inhibitor of nuclear factor-kappaB) in endometriotic stromal cells. Overexpression of IkappaB in endometriotic stromal cells inhibited the IL-1beta response of the RANTES gene promoter. Transcription of RANTES mRNA is up-regulated by IL-1beta via a nuclear factor-kappaB response element in the proximal RANTES gene promoter. These results demonstrate a feed-forward regulatory loop in the pathogenesis of endometriosis by which IL-1beta produced from activated macrophages can lead to further macrophage recruitment via RANTES production in endometriotic stromal cells.

Immunobiology of endometriosis.

OBJECTIVE: To provide a review of the humoral and cellular immunology of endometriosis and to discuss the rationale for future approaches to diagnosis and treatment. DESIGN: Literature survey. RESULT(S): Defective immunosurveillance in women who are destined to develop endometriosis may allow for the survival of ectopic endometrial tissue. The evidence includes endometrial cell resistance to apoptosis, perhaps through the secretion of proteins that interfere with implant recognition and/or FasL expression by stromal cells, inducing apoptosis of Fas-bearing immune cells. Although the immune response may be defective, aspects of it clearly are enhanced in endometriosis, as is seen by the generalized polyclonal B-cell autoimmune activation and secretion of immune proteins. Several cytokines, chemokines, and growth factors (including vascular growth factors) are increased in women with endometriosis. CONCLUSION(S): A complex network of locally produced cytokines modulate the growth and inflammatory behavior of ectopic endometrial implants. Proinflammatory proteins from endometriotic lesions and associated immune cells contribute to the enhanced inflammatory reaction associated with endometriosis that subserves the survival of these lesions instead of leading to their demise.

Ovarian steroid and cytokine modulation of human endometrial angiogenesis.

A key mechanism underlying the cyclical growth of the endometrium is its ability to regenerate a vascular capillary network. In normal cycling human endometrium, angiogenesis is influenced by both endocrine and paracrine factors. Hormonal manipulation of the endometrium, such as that occurring during the use of steroidal contraception, appears to result in capillary proliferation and fragility. As a consequence of these vascular changes, contraceptive users may be predisposed to unpredictable uterine bleeding, which is responsible for the high frequency of contraceptive discontinuation. In this paper we address mechanisms responsible for vascular endothelial cell proliferation in normal and contraceptive steroid-exposed endometria. We propose that regulation of endometrial angiogenesis is mediated indirectly, via steroid and cytokine actions on vascular endothelial growth factor (VEGF), and we present data indicating that VEGF expression in normal endometrial stromal cells is increased by oestrogens and progestins. Three proinflammatory cytokines with angiogenic effects in other systems (i.e. interleukin-1beta, tumour necrosis factor-alpha and interferon-gamma) do not appear to up-regulate VEGF expression in normal endometrial stromal cells. Well-characterized in-vitro models in conjunction with immunohistochemistry provide useful experimental systems to study endometrial neovascularization under physiological conditions and in those potentially perturbed via the use of contraceptive steroids.

Regulation of vascular endothelial growth factor (VEGF) gene transcription by estrogen receptors alpha and beta.

Vascular endothelial growth factor (VEGF) mediates angiogenic activity in a variety of estrogen target tissues. To determine whether estrogen has a direct transcriptional effect on VEGF gene expression, we developed a model system by transiently transfecting human VEGF promoter-luciferase reporter constructs into primary human endometrial cells and into Ishikawa cells, derived from a well-differentiated human endometrial adenocarcinoma. In primary endometrial epithelial cells, treatment with 17beta-estradiol (E(2)) resulted in a 3.8-fold increase in luciferase activity, whereas a 3. 2-fold induction was demonstrated for stromal cells. Our Ishikawa cells had less than 100 functional estrogen receptors (ER)/cell and were therefore cotransfected with expression vectors encoding either the alpha- or the beta-form of the human ER. In cells cotransfected with ERalpha, E(2) induced 3.2-fold induction in VEGF-promoter luciferase activity. A 2.3-fold increase was observed in cells cotransfected with ERbeta. Through specific deletions, the E(2) response was restricted to a single 385-bp PvuII-SstI fragment in the 5' flanking DNA. Cotransfection of this upstream region with a DNA binding domain ER mutant, or site-directed mutagenesis of a variant ERE within this fragment, resulted in the loss of the E(2) response. Electromobility shift assays demonstrated that this same ERE sequence specifically binds estradiol-ER complexes. These studies demonstrate that E(2)-regulated VEGF gene transcription requires a variant ERE located 1.5 kb upstream from the transcriptional start site. Site-directed mutagenesis of this ERE abrogated E(2)-induced VEGF gene expression.

Neutrophils infiltrating the endometrium express vascular endothelial growth factor: potential role in endometrial angiogenesis.

OBJECTIVE(S): To identify leukocytes within the human endometrium expressing vascular endothelial growth factor (VEGF). DESIGN(S): Prospective cohort study. SETTING(S): Healthy volunteers in an academic research environment. PATIENTS(S): Twenty-one normal cycling women without abnormal menstrual bleeding or infertility. INTERVENTION(S): Endometrial tissue collection by Pipelle de Cornier aspiration. MAIN OUTCOME MEASURES(S): Histologic, immunohistochemical (CD3, CD34, CD56, CD68, neutrophil elastase, estrogen and P receptors, VEGF), and simultaneous double immunoenzymatic labeling analysis of VEGF-positive cells within the human endometrium. RESULT(S): Ten endometrial samples were obtained in the proliferative (cycle days 5-10) and 11 samples in the secretory phase (cycle days 15-26). Immunohistochemical analyses showed the expected distribution of the different leukocyte cell types. Besides epithelial and stromal endometrial cells, the predominant cells that stained for VEGF were neutrophil granulocytes. Neutrophils were more abundant in the secretory phase but they expressed neither estrogen-a nor P receptors. CONCLUSION(S): Neutrophil granulocytes infiltrating the human endometrium express VEGF and regulate cyclical endometrial vascular proliferation. Ovarian steroids indirectly influence neutrophil migration.

Effects of progestins and relaxin on glycodelin gene expression in human endometrial cells.

OBJECTIVE: Glycodelin is an endometrial protein proposed to play an important role in embryonic implantation. We examined the effects of progestins and relaxin on glycodelin transcription, synthesis, and secretion. STUDY DESIGN: Northern blotting, metabolic labeling, and fluorography were used to assess glycodelin messenger ribonucleic acid and protein synthesis in endometrial tissue and cells. Luciferase reporter constructs transfected into endometrial adenocarcinoma cells (Ishikawa cells) were used to determine whether progestins or relaxin could activate the glycodelin gene promoter. RESULTS: Progestins but not relaxin stimulated glycodelin secretion in primary epithelial cell cultures. A 452-base pair fragment of the glycodelin gene promoter was activated 4.3 +/- 0.7 times normal by 10-nmol/L promegestone; however, addition of relaxin to the same construct repressed progestin-stimulate promoter activation by >30%. CONCLUSION: Glycodelin transcription, synthesis, and secretion by endometrial epithelial cells were stimulated by progestins. However, relaxin failed to stimulate production of this immunomodulatory protein and, in fact, repressed progestin-stimulated activation of the glycodelin gene promoter.

Induction of an angiogenic phenotype in endometriotic stromal cell cultures by interleukin-1beta.

Activated peritoneal macrophages are associated with endometriosis and may play a central role in its aetiology by releasing interleukin-1beta (IL-1beta) in response to refluxed endometrium. Pari passu with the establishment of endometriotic implants is the development of a vascular supply. In this study we investigated the angiogenic properties of two endometrial proteins, vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6), and assessed their production in response to IL-1beta stimulation in human stromal cells isolated from normal endometrium (NE) and endometriotic lesions (EI). Proliferation of bovine brain capillary endothelial cells (BBCE) with a [(3)H]-thymidine incorporation assay was observed when VEGF (2.1 +/- 0.2-fold; P < 0.05) or VEGF and IL-6 (1.8 +/- 0.1-fold; P < 0.05) were added in vitro, relative to saline-treated control cultures. Northern blot analysis showed induction of VEGF mRNA (2.6-fold; P < 0.05) and IL-6 mRNA (6.3-fold; P < 0.05) transcripts in EI cells, but not NE cells, exposed to IL-1beta. A similar induction was seen with VEGF and IL-6 protein secretion in the responsive EI cells. Reverse transcription-polymerase chain reaction (RT-PCR) for the IL-1 receptor type I (IL-1 RI) indicated that the differential effects of IL-1beta on NE and EI cells was associated with 2.4 +/- 0.1-fold more receptor mRNA in EI versus NE cells. We propose that the ability of IL-1beta to activate an angiogenic phenotype in EI stromal cells but not in NE cells, is mediated by the IL-1 RI.

Oestrogen receptor (ER)-alpha and ER-beta isoforms in normal endometrial and endometriosis-derived stromal cells.

Several investigators have noted that hormone-dependent development of endometriosis implants lags behind that of simultaneously analysed eutopic endometrium. With the recent discovery of the oestrogen receptor-beta (ER-beta) isoform, the aim of this study was to investigate whether differences in the expression of ER-alpha and ER-beta might explain this observation. mRNA transcripts from endometrial stromal cells isolated from normal endometrium (NE) and from endometriomas (EI) were analysed using a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) technique. RT-PCR and Southern blot analyses of the two ER isoforms indicated that NE and EI stromal cells predominantly express ER-alpha mRNA, however the relative concentrations of ER isoform mRNA transcripts differed between the two cell types. Steady-state ER-alpha:ER-beta mRNA ratios were 15.5 +/- 2.8 and 5.2 +/- 0.9 respectively for NE and EI cells (P = 0.02). NE and EI stromal cells expressed ER proteins with similar Kd ( approximately 0.9 nM) and densities ( approximately 24 500 binding sites/cell) respectively. Functional ER expression was indicated by an increase in progesterone receptor concentrations of approximately 60% (P = 0.03) after incubation with 10 nM oestradiol. We postulate that differential transcript processing, ligand specificity and biological actions of the ER-alpha and -beta isoforms may influence differential growth responses in normal and ectopic endometrium.

Vascular endothelial growth factor in reproductive biology.

The critical role of angiogenesis in embryology and tumor biology has been recognized for more than 20 years. However, the fact that neovascularization is essential to processes in mammalian female reproduction has only recently been appreciated widely. In this review we focus on a single angiogenic growth factor, vascular endothelial growth factor. As scientists have discovered in many aspects of cell biology, multiple and redundant signaling pathways have evolved in nature, presumably to protect essential biological functions from inactivating diseases or mutations. Despite this redundancy, some factors are of hierarchical importance. Vascular endothelial growth factor appears to be such a factor in the regulation of angiogenesis.

Vectorial secretion of vascular endothelial growth factor by polarized human endometrial epithelial cells.

OBJECTIVE: To analyze directional vascular endothelial growth factor (VEGF) secretion in polarized human endometrial epithelial cell cultures. VEGF has distinct distribution patterns in human endometrium. Stromal cells are diffusely positive for VEGF messenger RNA and protein, whereas glandular epithelium shows focal VEGF immunostaining at the apical surface. The epithelial distribution suggests that VEGF is secreted into gland lumina, potentially influencing the nutrition and/or apposition of the developing blastocyst. DESIGN: Controlled in vitro study of protein secretion by polarized endometrial epithelial cells established on polyethylene filters. SETTING: University hospital. PATIENT(S): Endometrial biopsies were obtained from healthy, ovulatory women undergoing elective surgery. INTERVENTION(S): Primary endometrial epithelial cells were cultured. MAIN OUTCOME MEASURE(S): VEGF mRNA and protein production were measured in polarized cells. The vectorial secretion of VEGF was determined. RESULT(S): VEGF production by endometrial epithelial cells was verified by Northern blotting and immunoassays of conditioned media. The mean (+/-SD) apical secretion of VEGF was 3.9 +/- 1.4 ng per 10(5) cells every 48 hours and the mean (+/-SD) basal secretion was 0.8 +/- 0.2 ng per 10(5) cells every 48 hours. In contrast, the apical and basal secretion of a soluble cellular isoform of fibronectin were 2.76 +/- 0.96 ng per 10(5) cells every 48 hours and 2.64 +/- 1.79 ng per 10(5) cells every 48 hours, respectively. CONCLUSION(S): VEGF is secreted preferentially into the lumina of endometrial glands. Apical VEGF may be an endometrial signal for blastocyst development or implantation.

Measurement of inducible proteins improves the precision of the local pigeon crop-sac bioassay for prolactin.

The pigeon crop-sac is a well-known target organ of prolactin (PRL). Pukac and Horseman (Endocrinology 114: 1718, 1984) reported that injections of the hormone caused changes in the expression of several specific proteins in that organ. We investigated the possibility that measuring the amounts of three of these proteins might improve the precision of the local pigeon crop-sac assay for PRL. Pigeons were given four injections of phosphate-buffered saline or different doses of ovine (o) PRL for 2 days and were killed on day 3. The DNA and total protein content of their crop-sac mucosal epithelial cells were measured and a supernatant of homogenized epithelial cells was processed by SDS-PAGE. The amounts of the three specific proteins in the stained gels were measured by densitometry. Treatment with oPRL increased the concentration of two of these proteins in the crop mucosal tissue (CP 14 and CP 25) and decreased that of the third one (CP 17). Measurement of total protein or of changes in the individual protein bands gave dose-response relationships with poor indices of precision (lambda greater than 0.3). Measurement of DNA content gave an assay endpoint with a reasonably good lambda value (0.16) but when the changes in the amounts of CP 14 and CP 25 were expressed as ratios to the changes occurring in CP 17, very precise assay endpoints (lambda = 0.06-0.08) were obtained.