The human endometrium undergoes a complex process of vascular and glandular proliferation, differentiation, and regeneration with each menstrual cycle in preparation for implantation. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific angiogenic protein that appears to play an important role in both physiological and pathological neovascularization. To investigate whether VEGF may regulate human endometrial angiogenesis, we examined VEGF messenger ribonucleic acid (mRNA) and protein throughout the menstrual cycle and studied the regulation of VEGF by reproductive steroids in isolated human endometrial cells. By ribonuclease protection analysis, VEGF mRNA increased relative to early proliferative phase expression by 1.6-,2.0-, and 3.6-fold in midproliferative, late proliferative, and secretory endometrium, respectively. In histological sections, VEGF mRNA and protein were localized focally in glandular epithelial cells and more diffusely in surrounding stroma, with greatest VEGF expression in secretory endometrium. Consistent with these in vivo results, the treatment of isolated human endometrial cells with estradiol (E2), medroxyprogesterone acetate (MPA), or E2 plus MPA significantly increased VEGF mRNA expression over the control value by 3.1-, 2.8-, and 4.7-fold, respectively. The VEGF response to E2 was rapid, with steady state levels of VEGF mRNA reaching 85% maximum 1 h after the addition of steroid. E2 also caused a 46% increase in secreted VEGF protein, and the combination of E2 and MPA caused an 18% increase. VEGF expression in endometriosis, an angiogenesis-dependent, estrogen-sensitive disease was similar to that seen in eutopic endometrium. Peritoneal fluid concentrations of VEGF were significantly higher in women with moderate to severe endometriosis than in women with minimal to mild endometriosis or no disease. VEGF, therefore, may be important in both physiological and pathological angiogenesis of human endometrium, as it is an estrogen-responsive angiogenic factor that varies throughout the menstrual cycle and is elevated in women with endometriosis.

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-β (ER-β) isoform, the aim of this study was to investigate whether differences in the expression of ER-α and ER-β 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-α mRNA, however the relative concentrations of ER isoform mRNA transcripts differed between the two cell types. Steady-state ER-α:ER-β 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 K d (~0.9 nM) and densities (~24 500 binding sites/cell) respectively. Functional ER expression was indicated by an increase in progesterone receptor concentrations of~60% (P ⍧ 0.03) after incubation with 10 nM oestradiol. We postulate that differential transcript processing, ligand specificity and biological actions of the ER-α and -β isoforms may influence differential growth responses in normal and ectopic endometrium.

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Endometriosis is a common gynecological disorder of unclear pathogenesis. We have established an in vitro model to investigate phenotypic similarities and differences between normal endometrial and endometriosis cells. Highly purified cultures of epithelial and stromal cells were isolated from normal endometrium and endometriosis implants. Morphological features as well as immunocytochemical markers confirm these isolates as epithelial and stromal cells. Potential hormone responsiveness was established by the documentation of estrogen receptor mRNA in epithelial and stromal cells isolated from both tissue types. Expression of this receptor protein was verified in stromal cells by competitive radioligand binding, revealing comparable receptor numbers and dissociation constants. CA-125 is selectively secreted in similar concentrations by epithelial cells isolated from both tissue types. PRL secretion is selectively exhibited by progestin-stimulated stromal cells from both tissue types. Our findings demonstrate that highly purified epithelial and stromal cells cultured from normal endometrial and endometriosis tissues express the same phenotypic and functional markers as their in vivo counterparts. These cultures provide useful models to identify endometriosis-specific cell products that contribute to the pathogenesis of this disorder.

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We have demonstrated that sulindac exerts strong antiinflammatory effects by suppression of NF-kappaB translocation, inhibition of NF-kappaB-mediated gene transcription, RANTES gene expression, and protein secretion in normal and endometriotic stromal cells. These results suggest that drugs targeting the NF-kappaB pathway may be beneficial in the treatment of endometriosis-associated symptoms.

An in vitro model developed to compare human endometrial and endometriosis stromal cells was used to examine basal and stimulated expression of interleukin (IL-6). Stromal cells isolated from normal endometrium (NE) exhibited the lowest level of IL-6 secretion (84 pg/10(6) cells-48 h), whereas those cells isolated from endometriosis implants (EI) secreted the highest concentration of this inflammatory cytokine (46,284 pg/10(5) cells-48 h; P < 0.01). Eutopic endometrial stromal cells from women with endometriosis (EE) expressed an intermediate concentration of IL-6 (831 pg/10(6) cells-48 h). Stimulation of the various cultures with IL-1 beta dramatically augmented stromal cell production of IL-6. The mean concentrations of stimulated IL-6 secretion were 16,257, 37,800, and 264,290 pg/10(5) cells-48 h for NE, EE, and EI cells, respectively (P < 0.03). Exposure of the cell cultures to 10 nmol/L estradiol had little direct effect on IL-6 production. The results indicate that endometrial stromal cells isolated from tissues of women with and without endometriosis express IL-6 under basal and cytokine-stimulated conditions. Differential responsiveness among the three cell sources indicates that NE, EE, and EI cells have intrinsic quantitative differences in cytokine regulation.

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