Jean-Louis Vigne

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.

Progestins activate vascular endothelial growth factor gene transcription in endometrial adenocarcinoma cells

OBJECTIVE To determine whether progestins activate vascular endothelial growth factor (VEGF) gene transcription in endometrial adenocarcinoma cells. DESIGN In vitro study. SETTING University reproductive biology laboratories. PATIENT(S) None. INTERVENTION(S) Ishikawa cells were transfected with VEGF promoter-luciferase reporter constructs and expression vectors encoding human progesterone receptors (hPR) A or B. The cells were treated with different progestins and antiprogestins, and luciferase activity was compared with controls. MAIN OUTCOME MEASURE(S) Three functional progesterone response elements (PREs) in the VEGF promoter were identified by electrophoretic mobility-shift assay, and different constructs were created to assess each PRE. RESULT(S) In cells expressing hPRA or B, treatment with 10 nM R5020 or 100 nM medroxyprogesterone acetate statistically significantly increased luciferase activity (3.3- to 4.8-fold). Pretreatment with 100 nM RU486 blunted the effect of 10 nM R5020, resulting only in a slight, statistically nonsignificant increase in luciferase activity (1.3- to 1.7-fold). Although three different functional PREs could be identified, no single PRE accounted for the preponderance of the luciferase activity. Full VEGF promoter activation required all three PREs. CONCLUSION(S) Progestins have a direct effect on VEGF gene transcription. However, hPR-mediated transcriptional regulation of the VEGF promoter is complex and cannot be localized to confined PRE sequences. Other response element motifs are likely to play a contributory role.

Thiazolidinedione inhibition of peritoneal inflammation

Chemoattraction of macrophages into the peritoneal cavity is one of the important characteristics in patients with endometriosis. An inflammatory response is postulated to be responsible for infertility and pelvic pain associated with this syndrome. The present in vivo studies were designed to test if thiazolidinediones (TZDs), activators of peroxisome proliferator activated receptor gamma, could inhibit monocyte chemotaxis in a murine model. TZDs were first used as orally bioavailable insulin-sensitizing agents. They are currently under investigation in the treatment of inflammatory diseases, including arthritis or colitis. Intraperitoneal injection of thioglycollate was used to elicit high numbers of activated peritoneal macrophages in female mice. Concomitant peritoneal injection of ciglitazone, a member of the TZD family, significantly reduced the number of macrophages. When cultured and stimulated by tumor necrosis factor alpha, these peritoneal macrophages also secreted less RANTES and less IL-1β protein. This animal model suggests that treatment of endometriosis patients with TZDs may diminish symptoms associated with intraperitoneal inflammation.

Developmental Gonadal Expression of the Transcription Factor SET and Its Target Gene, P450c17 (17α-Hydroxylase/c17,20 Lyase)

Cytochrome P450c17 catalyzes the 17alpha-hydroxylase/17,20 lyase activity needed for sex steroid synthesis. We recently characterized the nuclear phosphoprotein SET as a novel transcriptional regulator that binds to the -447/-399 region of the rat P450c17 gene, along with the transcription factors COUP-TF II, NGF-IB, and SF-1. Gel shift studies localized SET binding to nucleotides -410/-402. We have shown that SET activates transcription of the rat P450c17 gene in neuronal precursor cells and now show that it also activates transcription from the -418/-399 region of the rat P450c17 gene in mouse Leydig MA-10 cells. Studying the ontogenic expression of SET and P450c17 in the rodent gonad, we found that SET expression preceded P450c17 expression in the embryonic genital ridge, suggesting that SET may be important for initiating P450c17 expression in this region. Expression of SET also preceded P450c17 expression in the testis and ovary, and its expression was much greater during embryogenesis than in the adult gonad. In the adult rat testis, P450c17 was expressed only in Leydig cells, while SET was expressed in Leydig cells and in spermatocytes. In the adult rat ovary, P450c17 was expressed only in theca cells, while SET was expressed in theca cells and also in oocytes. Because SET is expressed early in development in the genital ridge and in the testis and ovary, and because SET has many functions in addition to its activity as a transcription factor, we determined whether SET acts a transcription factor in oocytes. The SET protein was detected by Western blots in Xenopus oocytes from stages II through VI and in mature oocytes. Using extracts of Xenopus oocytes in gel shift assays, we detected a protein that bound to the -418/-399 region of the rat P450c17 gene, to which SET binds. Nuclear injection of either a -418/-399TK32LUC wildtype reporter construct or a construct containing a mutant SET site into Xenopus oocytes from stages III through VI resulted in activation of luciferase activity with the wildtype but not the mutant construct in all stages. These data suggest that Xenopus SET is able to bind to specific DNA sequences to activate transcription at all stages of Xenopus oogenesis. These data indicate that SET is an evolutionarily conserved transcription factor that participates in the early ontogenesis of the gonadal system, regulates P450c17 gene transcription in Leydig cells, and may also activate other genes expressed in immature oocytes, thus playing a role in oocyte development.

Histocompatibility leukocyte antigen-G is not expressed by endometriosis or endometrial tissue

OBJECTIVE The immunological mechanisms that support persistence and proliferation of ectopic endometrial implants within the peritoneal cavity of women with endometriosis are unknown. Inhibition of natural killer (NK) and cytotoxic T-cell function has been proposed as a mechanism. We tested the hypothesis that expression of a nonclassical major histocompatibility antigen, HLA-G, might explain the local immunosuppression associated with ectopic endometrium. DESIGN Nested case-control study of women with and without laparoscopic evidence of endometriosis. SETTING Reproductive endocrinology clinic at a university hospital. PATIENT(S) Peritoneal fluid specimens from 10 women with revised AFS stage I-IV endometriosis and from 10 age-matched normal controls without laparoscopic evidence of endometriosis were tested for the presence of HLA-G protein. Endometriosis and normal endometrial biopsies from four patients were used to prepare stromal cell cultures directly evaluated for HLA-G protein. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) The expression of HLA-G in peritoneal fluid, tissue, and cell cultures was determined by immunoblotting with a specific monoclonal antibody. RESULT(S) HLA-G protein was not detectable in peritoneal fluid specimens of endometriosis patients or controls. Moreover, ectopic and normal endometrial tissues and stromal cells did not express HLA-G. CONCLUSION(S) Immune cell inhibition in endometriosis must be mediated by factors other than HLA-G.

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.

Natural and recombinant human glycodelin activate a proapoptotic gene cascade in monocyte cells

Glycodelin‐A (GdA) is a member of the superfamily of lipocalins and the predominant glycoprotein secreted by human and primate endometrium in the secretory and early pregnancy phases. GdA can inhibit NK cell activity, T cell proliferation, and chemotaxis of monocytes. Its physiological function is thought to mediate immunotolerance at the fetomaternal interface. In the present studies, we engineered recombinant Gd (rGd) in yeast and tested its biological effects on monocyte viability. rGd, like the natural, purified endometrial GdA, is glycosylated and secreted, and they both induced apototic changes in monocytic U937 cells and primary human monocytes. Trypan blue exclusion, nucleosome release, DNA laddering, and immunocytochemistry to detect free 3′‐OH DNA ends were used to characterize the effects of GdA and rGd. Using U937 cells as a model, cDNA microarray analyses revealed several pro‐ and antiapoptotic genes that were up‐ and down‐regulated, respectively, in accordance with the kinetics of rGd‐induced monocyte cell death. Real‐time RT‐PCR confirmed that Bad, Bax, and TNF‐R1 gene expression were increased, whereas Bcl‐2A1 and a proliferation‐inducing ligand (APRIL) were reduced by rGd. Transfection assays in U937 cells indicated that the immunomodulatory actions of rGd were associated with NF‐κB inhibition. Western blotting of U937 and primary monocyte lysates demonstrated that rGd activated caspase‐8, ‐2, and ‐3 to execute programmed cell death in these cells. We postulate that infiltrating monocytes and potentially other innate immune cells of the decidua might be manipulated by this glycoprotein to enhance embryonic implantation rates or conversely, to develop novel contraceptive strategies.

Properties of Lipoproteins in Blood Plasma and Liver Perfusates of Rats With Cholestasis

We have shown that perfused livers of rats with experimental cholestasis, produced by obstruction of the common bile duct, secrete lipoprotein X and an abnormal, triglyceride-rich, low-density lipoprotein. This report describes changes in very-lowdensity and high-density lipoproteins secreted by perf used livers of cholestatic rats. Because these animals ate little and lost weight during 42 h of cholestasis, the alterations of lipoproteins were compared with those in fasted and sham-operated control rats. Fasting (42 h) resulted in a marked reduction (>50%) in the recovery of all lipoproteins from perfusates. Accumulation of very-low-density lipoproteins in liver perf usates and the amount of nascent very-low-density lipoproteins in cisternae and secretory vesicles of the Golgi apparatus of hepatocytes were similarly reduced by fasting and cholestasis. Perfusate very-low-density lipoproteins of cholestatic as well as fasted rats were smaller (~350 A mean diameter) than those from sham-operated (~480 A) and contained a smaller proportion of nonpolar (core) lipid components, with an increased proportion of apolipoprotein B. The total amount of high-density lipoprotein-protein recovered from perf usates was unaffected by cholestasis. However, perf usate high-density lipoproteins from cholestatic rats contained: (a) more discoidal particles; (b) more polar lipids and less cholesteryl esters; (c) more apolipoprotein B; and (d) much less apolipoprotein A-IV than perf usate high-density lipoproteins from sham-operated and fasted control rats. The total amount of apolipoprotein A-I in perfusate highdensity lipoproteins was also unaffected by cholestasis. In experiments in which [3H]lysine was added to perf usates, however, specific activity of apolipoprotein A-I was twofold higher in high-density lipoproteins from the livers of cholestatic rats than from the livers of sham-operated controls. The rate of accumulation of apolipoprotein A-I in whole perf usates of cholestatic livers was 2.5-fold higher than in sham-operated controls, whereas the rate of secretion of apolipoprotein E and serum albumin was not significantly changed. The alterations of lipoproteins in blood plasma of cholestatic rats were remarkably similar to those observed in liver perfusates. These studies suggest that reduced food intake during cholestasis in the rat contributes to changes in plasma and perf usate very-low-density lipoproteins. However, the more pronounced changes in low-density lipoprotein and high-density lipoprotein fractions in this disorder appear to be directly related to the intrahepatic effects of bile duct obstruction. Many of the abnormalities of perfusate lipoproteins from cholestatic rat livers resemble those found in plasma lipoproteins of humans with obstructive liver diseases.

Eicosanoid secretion by human endothelial cells exposed to normal pregnancy and preeclampsia plasma in vitro

Eicosanoids play an important role in the pathogenesis of preeclampsia. The major eicosanoid metabolite reported to be secreted by endothelial cells, the vasodilator prostacyclin, is generally reduced in preeclampsia. By contrast, it was shown previously that prostacyclin secretion by cultured human umbilical vein endothelial (HUVE) cells is increased significantly after exposure to blood from preeclamptic women. In the current study, eicosanoid profiles in conditioned media from HUVE cells incubated with pregnancy plasma were analyzed by high-performance liquid chromatography, thin layer chromatography and quantitative radio- and enzyme immunoassays. More prostaglandin F2alpha, prostacyclin and 8-isoprostane were secreted after exposure to plasma from preeclamptic women than plasma from matched, normal pregnant patients. Predominant secretion of the vasoconstrictor prostaglandin F2alpha by HUVE cell cultures and a stimulatory effect of preeclampsia plasma on eicosanoid biosynthesis underscore the importance of bioactive lipids in the vasospasm associated with clinical preeclampsia.

A Botanical Extract from Channel Flow Inhibits Cell Proliferation, Induces Apoptosis, and Suppresses CCL5 in Human Endometriotic Stromal Cells

Abstract Growing evidence suggests that medicinal herbs have direct actions on endometrial cells. By screening multiple herbs using an in vitro model of endometriosis, we found that a commonly used herbal formula exerted considerable antiproliferative effects. Our purpose was to investigate the effects of this antiendometriosis herbal mixture on cell proliferation, apoptosis, and CCL5 expression and secretion in endometriotic stromal cells in vitro. Isolated normal endometrial, eutopic, and ectopic endometriotic stromal cells were cultured under established conditions. Cell proliferation, apoptosis, and CCL5 gene expression protein secretion was evaluated after incubation with different concentrations of an antiendometriosis herbal mixture extract. Cell proliferation was assessed by cell counting, 3H-thymidine incorporation, and MTS assays. Apoptosis was determined by blotting using anti-cleaved caspase 3 antibodies and by a TUNEL assay. CCL5 gene expression and protein secretion were determined by transient transfection of gene promoter reporters and ELISAs in cell supernatants. Extracts of a traditional herbal mixture dose-dependently decreased cell proliferation in normal, eutopic, and ectopic endometriotic stromal cells. 3H-Thymidine uptake and MTS confirmed these findings. The herbal extracts induced apoptosis, as evidenced by activation of caspase 3 and the presence of TUNEL-positive cells after treatment. The herbal extracts also suppressed CCL5 gene transcription and protein secretion in endometriotic stromal cells, even when corrected for cell number. Extracts from a medicinal herbal mixture have direct effects on cell proliferation, apoptosis, and CCL5 production in endometriotic stromal cells. Our findings support the further investigation of novel, potentially safe and well-tolerated botanical products as future endometriosis treatments.