Miwako Nagai

Dienogest reduces proliferation, aromatase expression and angiogenesis, and increases apoptosis in human endometriosis

Abstract Dienogest is a novel progestin that is highly selective for progesterone receptors and inhibits endometriosis. However, it remains unknown how the administration of dienogest to patients with endometriosis impacts on their lesion tissues. The aim of this study was to evaluate the in vivo effect of dienogest on endometriosis tissue. We collected endometrioma tissues from patients treated with dienogest (N = 7) or not treated (N = 11, controls). Cell proliferation, aromatase expression and blood vessel density were evaluated by staining for Ki67, aromatase and the von Willebrand factor, respectively. Apoptosis was detected using the TUNEL assay. The proportion of Ki67 and aromatase positive epithelial cells was significantly lower in the dienogest group than in controls (p < 0.05, respectively). The number of TUNEL positive cells was significantly higher in the dienogest group (p < 0.05). The density of blood vessels in endometrioma was marginally lower in the dienogest group compared with controls (p = 0.20). Our study demonstrates that endometrioma taken from patients treated with dienogest show remarkable histological features such as reduction of proliferation, aromatase expression and angiogenesis, and increase of apoptosis. This study clarified the impact of dienogest on local histological events that explain its therapeutic effect on endometriosis. Chinese abstract 地诺孕素是一种新型的孕激素，具有孕激素受体高度选择性，可抑制子宫内膜异位症。但是，地诺孕素作用于内异症患者受损组织的机理现在仍未明确。本研究的目的在于评估地诺孕素在体内对子宫内膜异位组织的影响。我们采集经地诺孕素治疗（N = 7）与未经治疗（N = 11，对照组）的患者的子宫内膜异位组织。通过对Ki67染色、芳香化酶及von Willebrand因子的测定，评估细胞增殖、芳香化酶表达及血管密度。细胞凋亡情况通过TUNEL检验测定。结果显示，地诺孕素组的Ki67与芳香化酶阳性上皮细胞比例显著低于对照组（均为p < 0.05）。地诺孕素组的TUNEL阳性细胞数目显著高于对照组（p < 0.05）。地诺孕素组子宫内膜异位组织中血管密度略低于对照组（p = 0.20）。本研究结果表明，经地诺孕素治疗的子宫内膜异位症患者可出现显著的组织学特征改变，如降低细胞增殖、芳香化酶表达及血管生成减少及细胞凋亡增加。研究明确了地诺孕素对局部组织的影响，证实了其治疗子宫内膜异位症的效果。

Effects of 1,25-Dihydroxy Vitamin D3 on Endometriosis

CONTEXT Endometriosis is an estrogen-dependent, chronic inflammatory disease. Recent studies have shown that vitamin D (VD) is an effective modulator of the immune system and plays an important role in controlling many inflammatory diseases. OBJECTIVE The objective of the study was to clarify the in vitro effects of 1,25-dihydroxy vitamin D3 (1,25[OH]2D3) on human endometriotic stromal cells (ESCs) and to determine the serum levels of VD in endometriosis patients. DESIGN, PATIENTS, AND MAIN OUTCOME MEASURES ESCs were isolated from ovarian endometrioma and cultured with 1,25(OH)2D3. Gene expression of IL-8, cyclooxygenase-2, microsomal prostaglandin E synthase-1, microsomal prostaglandin E synthase-2, cytosolic prostaglandin E synthase, 15-hydroxyprostaglandin dehydrogenase, matrix metalloproteinase (MMP)-2, and MMP-9 was examined using quantitative RT-PCR. The production of IL-8 and prostaglandin E2 was measured using an ELISA and an enzyme immunoassay. Viable cell number was assessed using a cell-counting assay, and DNA synthesis was assessed using the bromodeoxyuridine incorporation assay. Apoptosis was assessed using flow cytometry. The expression of inhibitory-κBα protein was detected using Western blotting. The serum levels of 25-hydroxyvitamin D3 and 1,25(OH)2D3 were measured by a RIA. RESULTS In vitro studies showed that 1,25(OH)2D3 significantly reduced IL-1β- or TNF-α-induced inflammatory responses, such as IL-8 expression and prostaglandin activity. 1,25(OH)2D3 also reduced viable ESC numbers and DNA synthesis but did not affect apoptosis. MMP-2 and MMP-9 expressions were reduced by 1,25(OH)2D3. 1,25(OH)2D3 inhibited nuclear factor-κB activation. The serum 25-hydroxyvitamin D3 levels were significantly lower in women with severe endometriosis than in the controls and women with mild endometriosis. Serum 1,25(OH)2D3 levels were not different between groups. CONCLUSIONS VD modulates inflammation and proliferation in endometriotic cells, and a lower VD status is associated with endometriosis. Taken together, VD supplementation could be a novel therapeutic strategy for managing endometriosis.

Interleukin-1β stimulates the secretion of thymic stromal lymphopoietin (TSLP) from endometrioma stromal cells: possible involvement of TSLP in endometriosis

STUDY QUESTION Is thymic stromal lymphopoietin (TSLP) involved in the pathophysiology of endometriosis? SUMMARY ANSWER TSLP is up-regulated by interleukin (IL)-1β and may be involved in the development of endometriosis. WHAT IS KNOWN ALREADY Endometriosis is a chronic inflammatory disease in which the Th2 immune response is activated and has been suggested to promote the disease. TSLP is a master cytokine that drive Th2 immune response. STUDY DESIGN, SIZE, DURATION A laboratory study. PARTICIPANTS/MATERIALS, SETTING, METHODS Primary cultures of endometrioma stromal cells (ESCs) were treated with IL-1β, a typical inflammatory cytokine associated with endometriosis. Gene expression of TSLP in ESCs and secretion of TSLP protein from ESCs were studied using quantitative PCR and a specific ELISA. Interferon γ (IFNγ), a typical Th1 cytokine, and IL-4, a typical Th2 cytokine, were added to the culture to evaluate their effect on the IL-1β-induced secretion of TSLP. Inhibitors of p38 mitogen-activated protein kinase (MAPK), p42/44 MAPK and stress-activated protein kinase/Jun amino-terminal kinase (SAPK/JNK) were added to the culture to examine intracellular signals involved in IL-1β-induced TSLP secretion. The expression of TSLP in endometrioma tissue was examined by immunohistochemistry. The concentration of TSLP in the serum and peritoneal fluid (PF) of women with or without endometriosis was measured with a specific ELISA. MAIN RESULTS AND THE ROLE OF CHANCE IL-1β stimulated the expression of TSLP mRNA and secretion of TSLP protein from ESCs. IL-4 enhanced the IL-1β-induced TSLP secretion from ESCs, while IFNγ reduced it. Inhibitors of p42/44 MAPK, p38 MAPK and SAPK/JNK suppressed the IL-1β-induced secretion of TSLP from ESCs. Positive immunostaining of TSLP was observed in the stroma of endometrioma tissue. TSLP concentrations in the serum and PF were both higher in women with endometriosis compared with those without endometriosis. LIMITATIONS, REASONS FOR CAUTION The present study was only in vitro. The samples used for culture were endometrioma tissues, not including other types of endometriosis. Therefore, the present findings should be interpreted with caution. WIDER IMPLICATIONS OF THE FINDINGS This study provided new insights in the Th2 immune response-related mechanism in endometriosis. STUDY FUNDING This study is partly supported by grants from the Ministry of Health, Labour and Welfare, and the Ministry of Education, Culture, Sports, Science and Technology. The authors have no conflicts of interest to declare.

Interleukin-11 alters placentation and causes preeclampsia features in mice

Significance Preeclampsia is an insidious disease, unique to humans, affecting ∼8% of pregnancies. There are no early detection tests or pharmacological treatments. Impaired placentation is widely accepted to contribute to the pathogenesis. However, the mechanisms remain elusive, given the complications of studying first-trimester placental development in women. A major limitation for the study of new treatments is the lack of available animal models that recapitulate the full spectrum of preeclampsia features. We have developed a mouse model characterized by elevated levels of the cytokine Interleukin-11 (IL11). This study provides evidence of a novel pathway causative of preeclampsia features in vivo. It also provides a novel in vivo mouse model that is useful for preclinical studies to test potential therapeutics. Preeclampsia (PE) is a pregnancy-specific disorder characterized by hypertension and proteinuria after 20 wk gestation. Abnormal extravillous trophoblast (EVT) invasion and remodeling of uterine spiral arterioles is thought to contribute to PE development. Interleukin-11 (IL11) impedes human EVT invasion in vitro and is elevated in PE decidua in women. We demonstrate that IL11 administered to mice causes development of PE features. Immunohistochemistry shows IL11 compromises trophoblast invasion, spiral artery remodeling, and placentation, leading to increased systolic blood pressure (SBP), proteinuria, and intrauterine growth restriction, although nonpregnant mice were unaffected. Real-time PCR array analysis identified pregnancy-associated plasma protein A2 (PAPPA2), associated with PE in women, as an IL11 regulated target. IL11 increased PAPPA2 serum and placental tissue levels in mice. In vitro, IL11 compromised primary human EVT invasion, whereas siRNA knockdown of PAPPA2 alleviated the effect. Genes regulating uterine natural killer (uNK) recruitment and differentiation were down-regulated and uNK cells were reduced after IL11 treatment in mice. IL11 withdrawal in mice at onset of PE features reduced SBP and proteinuria to control levels and alleviated placental labyrinth defects. In women, placental IL11 immunostaining levels increased in PE pregnancies and in serum collected from women before development of early-onset PE, shown by ELISA. These results indicate that elevated IL11 levels result in physiological changes at the maternal–fetal interface, contribute to abnormal placentation, and lead to the development of PE. Targeting placental IL11 may provide a new treatment option for PE.

Autoamputated adnexa presents as a peritoneal loose body

We report a case of unilateral adnexal absence with a peritoneal loose body. Laparoscopic findings and medical history suggested that she had adnexal torsion in her childhood followed by its calcification and autoamputation.

Interleukin-4 and Prostaglandin E2 Synergistically Up-Regulate 3β-Hydroxysteroid Dehydrogenase Type 2 in Endometrioma Stromal Cells

CONTEXT Endometriosis is a chronic inflammatory disease in which immune response and production of estrogen in endometriotic tissues are involved in the development of the disease. Prostaglandin E2 (PGE2) stimulates aromatase (P450arom) expression in endometrioma stromal cells (ESCs) and increases the production of estrogens. On the other hand, an accumulating amount of evidence suggests that IL-4, a typical Th2 cytokine, plays important roles in the disease. OBJECTIVE The objective of the investigation was to study the effect of IL-4 on the expression of 3β-hydroxysteroid dehydrogenase (HSD3B2), a pivotal enzyme for estrogen production, in ESCs. DESIGN, PATIENTS, AND MAIN OUTCOME MEASURES ESCs were isolated from ovarian endometrioma tissues and cultured with IL-4 and PGE2. CP-690550, a Janus protein tyrosine kinase 3 inhibitor, and HSD3B2 small interfering RNA were added to the culture. Gene expression of HSD3B2 and P450arom was examined by quantitative RT-PCR. Dehydroepiandrosterone (DHEA) was added to the culture, and then the combined enzyme activity of HSD3B2, which converts DHEA to androstenedione, and P450arom, which converts androstenedione to estrone, was examined by measuring estrone concentration in the supernatants with a specific enzyme immunoassay. RESULTS IL-4 increased the expression of HSD3B2 mRNA in a dose-dependent manner. CP-650550 inhibited the IL-4-induced increase in HSD3B2 mRNA expression. PGE2 also increased the expression of HSD3B2 mRNA, and the combination of IL-4 and PGE2 synergistically increased the expression of HSD3B2 mRNA. IL-4 had no effect on the expression of P450arom mRNA, whereas PGE2 increased the expression of P450arom mRNA. Although PGE2 alone increased the production of estrone from DHEA, the combination of IL-4 and PGE2 significantly augmented the production of estrone from DHEA. The enhanced production of estrone by the combination of IL-4 and PGE2 was inhibited by CP-690550 and HSD3B2 small interfering RNA. CONCLUSIONS IL-4 in combination with PGE2 may enhance estrogen production in endometriotic tissues, implying an elaborate mechanism that Th2 immune response augments inflammation-dependent progression of the disease.

Thrombin enhances soluble Fms-like tyrosine kinase 1 expression in trophoblasts; possible involvement in the pathogenesis of preeclampsia

OBJECTIVE To investigate the possible impact of thrombin on soluble Fms-like tyrosine kinase 1 (sFlt-1) expression in trophoblasts. DESIGN Experimental. SETTING University hospital laboratory. INTERVENTIONS(S) A trophoblast cell line (HRT-8/SVneo) was treated with thrombin, protease-activated receptor 1 (PAR-1)-specific agonist SFLLERN, and thrombin antagonist PPACK. MAIN OUTCOME MEASURE(S) mRNA expression of sFlt-1, vascular endothelial growth factor (VEGF), and placental growth factor (PlGF) in trophoblasts, with the use of real-time polymerase chain reaction; and the secretion of sFlt-1, VEGF, and PlGF protein from trophoblasts, with the use of ELISA. RESULT(S) Administration of thrombin (10 U/mL) and PAR-1-specific agonist SFLLRN (300 μmol/L) increased sFlt-1 mRNA expression (4.24 ± 0.74- and 4.21 ± 0.79-fold, respectively) and protein secretion (5.08 ± 0.42- and 1.89 ± 0.16-fold, respectively) in HRT-8/SVneo. The induction of sFlt-1 protein secretion by thrombin was dose dependent. The effect of thrombin was completely reduced by thrombin inhibitor PPACK. Thrombin increased mRNA expression of VEGF but did not change VEGF secretion and PlGF mRNA expression and secretion. CONCLUSION(S) During placental development, thrombin, generated in the local hemorrhage of the uteroplacenta increases trophoblast expression of sFlt-1. Consequently, thrombin may contribute to the pathogenesis of preeclampsia.

Mannose receptor is highly expressed by peritoneal dendritic cells in endometriosis

OBJECTIVES To characterize peritoneal dendritic cells (DCs) in endometriosis and to clarify their role in its etiology. DESIGN Experimental. SETTING University hospital. PATIENT(S) Sixty-three women (35 patients with endometriosis and 28 control women) who had undergone laparoscopic surgery. INTERVENTION(S) Peritoneal DCs from endometriosis and control samples were analyzed for the expression of cell surface markers. Monocyte-derived dendritic cells (Mo-DCs) were cultured with dead endometrial stromal cells (dESCs) to investigate changes in phagocytic activity and cytokine expression. MAIN OUTCOME MEASURE(S) Cell surface markers and cytokine expression and identification with the use of flow cytometry or reverse-transcription polymerase chain reaction (RT-PCR). Changes in cytokine expression and phagocytic activity of Mo-DCs cultured with dESCs and d-mannan were measured with the use of flow cytometry and RT-PCR. RESULT(S) The proportion of mannose receptor (MR)-positive myeloid DC type 1 was higher in endometriosis samples than in control samples. The blocking of MR reduced phagocytosis of dESCs by Mo-DCs. Mo-DCs cultured with dESCs expressed higher levels of interleukin (IL) 1β and IL-6 than control samples. CONCLUSION(S) Peritoneal DCs in endometriosis tissue express high levels of MR, which promotes phagocytosis of dead endometrial cells and thereby contributes to the etiology of endometriosis.

Cyclic Stretch Augments Production of Neutrophil Chemokines, Matrix Metalloproteinases, and Activin A in Human Endometrial Stromal Cells

The aim of this study was to test our hypothesis: Contractile activity that occurs in the uterus during menstruation induces biochemical factors that enhance remodeling of the endometrium.

Neutrophil depletion reduces endometriotic lesion formation in mice

Neutrophils are known to be accumulated in endometriosis; however, direct evidence of the impact of neutrophils in the development of endometriosis was lacking. To clarify the importance of neutrophils, we examined the effect of neutrophil depletion on the development of endometriosis.