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Delta-like ligand 4 (Dll4), one of the Notch ligands, is overexpressed in ovarian cancer, especially in tumors resistant to anti-VEGF therapy. Here, we examined the biologic effects of dual anti-Dll4 and anti-VEGF therapy in ovarian cancer models. Using Dll4-Fc blockade and anti-Dll4 antibodies (murine REGN1035 and human REGN421), we evaluated the biologic effects of Dll4 inhibition combined with aflibercept or chemotherapy in orthotopic mouse models of ovarian cancer. We also examined potential mechanisms by which dual Dll4 and VEGF targeting inhibit tumor growth using immunohistochemical staining for apoptosis and proliferation markers. Reverse-phase protein arrays were used to identify potential downstream targets of Dll4 blockade. Dual targeting of VEGF and Dll4 with murine REGN1035 showed superior antitumor effects in ovarian cancer models compared with either monotherapy. In the A2780 model, REGN1035 (targets murine Dll4) or REGN421 (targets human Dll4) reduced tumor weights by 62% and 82%, respectively; aflibercept alone reduced tumor weights by 90%. Greater therapeutic effects were observed for Dll4 blockade (REGN1035) combined with either aflibercept or docetaxel (P < 0.05 for the combination vs. aflibercept). The superior antitumor effects of REGN1035 and aflibercept were related to increased apoptosis in tumor cells compared with the monotherapy. We also found that GATA3 expression was significantly increased in tumor stroma from the mice treated with REGN1035 combined with docetaxel or aflibercept, suggesting an indirect effect of these combination treatments on the tumor stroma. These findings identify that dual targeting of Dll4 and VEGF is an attractive therapeutic approach. Mol Cancer Ther; 15(6); 1344–52. ©2016 AACR.

Supplementary Figure 1. Representative OVCAR3-inoculated mice after aflibercept, Dll4-Fc, and the combination. Supplementary Figure 2. In vivo study of Dll4 blockade, aflibercept, and docetaxel, alone and in combinations, in A2780 and HeyA8 models. Supplementary Figure 3. Immunohistochemical staining of CX3CL1 in the tumor stroma of mice inoculated with A2780 ovarian cancer cells. Supplementary Figure 4. Quantitative real-time PCR of CX3CL1 expression in A2780 cells cultured with primary dendritic cells isolated from C57/BL6 mice. Supplementary Figure 5. ELISA analysis of plasma IFN-gamma in the mice inoculated with A2780 ovarian cancer cells. Supplementary Figure 6. RT-PCR analysis of E-cadherin expression in tumors (tumor cells and stroma) obtained from A2780 mice treated with a single agent or a combination.

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Supplementary Table 1 from Gene Alterations Identified by Expression Profiling in Tumor-Associated Endothelial Cells from Invasive Ovarian Carcinoma

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Emerging evidence suggests that the Notch/Delta-like ligand 4 (Dll4) pathway may offer important new targets for antiangiogenesis approaches. In this study, we investigated the clinical and biological significance of Dll4 in ovarian cancer. Dll4 was overexpressed in 72% of tumors examined in which it was an independent predictor of poor survival. Patients with tumors responding to anti-VEGF therapy had lower levels of Dll4 than patients with stable or progressive disease. Under hypoxic conditions, VEGF increased Dll4 expression in the tumor vasculature. Immobilized Dll4 also downregulated VEGFR2 expression in endothelial cells directly through methylation of the VEGFR2 promoter. RNAi-mediated silencing of Dll4 in ovarian tumor cells and tumor-associated endothelial cells inhibited cell growth and angiogenesis, accompanied by induction of hypoxia in the tumor microenvironment. Combining Dll4-targeted siRNA with bevacizumab resulted in greater inhibition of tumor growth, compared with control or treatment with bevacizumab alone. Together, our findings establish that Dll4 plays a functionally important role in both the tumor and endothelial compartments of ovarian cancer and that targeting Dll4 in combination with anti-VEGF treatment might improve outcomes of ovarian cancer treatment. Cancer Res; 71(18); 6030–9. ©2011 AACR.

Continuous efforts have been devoted to unraveling the biophysiology and development of the human fetal adrenal cortex, which is structurally and functionally unique from other species. It plays a pivotal role, mainly through steroidogenesis, in the regulation of intrauterine homeostasis and in fetal development and maturation. The steroidogenic activity is characterized by early transient cortisol biosynthesis, followed by its suppressed synthesis until late gestation, and extensive production of dehydroepiandrosterone and its sulfate, precursors of placental estrogen, during most of gestation. The gland rapidly grows through processes including cell proliferation and angiogenesis at the gland periphery, cellular migration, hypertrophy, and apoptosis. Recent studies employing modern technologies such as gene expression profiling and laser capture microdissection have revealed that development and/or function of the fetal adrenal cortex may be regulated by a panoply of molecules, including transcription factors, extracellular matrix components, locally produced growth factors, and placenta-derived CRH, in addition to the primary regulator, fetal pituitary ACTH. The role of the fetal adrenal cortex in human pregnancy and parturition appears highly complex, probably due to redundant and compensatory mechanisms regulating these events. Mounting evidence indicates that actions of hormones operating in the human feto-placental unit are likely mediated by mechanisms including target tissue responsiveness, local metabolism, and bioavailability, rather than changes only in circulating levels. Comprehensive study of such molecular mechanisms and the newly identified factors implicated in adrenal development should help crystallize our understanding of the development and physiology of the human fetal adrenal cortex.

Purpose: Metronomic chemotherapy regimens have shown anti-tumor activity by anti-angiogenic mechanisms, however, the efficacy of metronomic topotecan in ovarian cancer is not known and the focus of the current study. Experimental Design: In vivo dose-finding and therapy experiments with oral metronomic topotecan were performed in an orthotopic model of advanced ovarian cancer. Tumor vascularity (MVD: CD31), proliferation (PCNA), and apoptosis (TUNEL) were examined among treatment arms. In vitro experiments including MTT and western blot analysis were performed to identify specific anti-angiogenic mechanisms of topotecan. Results: Compared to controls, metronomic (0.5, 1.0 and 1.5 mg/kg; daily) and maximum tolerated therapy (MTD; 7.5 and 15 mg/kg; weekly) dosing regimens reduced tumor growth in dose-finding experiments, but significant morbidity and mortality was observed with higher doses. Metronomic and MTD topotecan therapy significantly reduced tumor growth in both HeyA8 and SKOV3ip1 models: 41-74% (metronomic), and 64-86% (MTD dosing) (p

Recent clinical data on selective estrogen receptor modulators (SERMs) have provided the basis for reassessment of the SERM concept. The molecular basis of SERM activity involves binding of the ligand SERM to the estrogen receptor (ER), causing conformational changes which facilitate interactions with coactivator or corepressor proteins, and subsequently initiate or suppress transcription of target genes. SERM activity is intrinsic to each ER ligand, which accomplishes its unique profile by specific interactions in the target cell, leading to tissue selective actions. We discuss the estrogenic and anti-estrogenic effects of early SERMs, such as clomiphene citrate, used for treatment of ovulation induction, and the triphenylethylene, tamoxifen, which has ER antagonist activity in the breast, and is used for prevention and treatment of ER-positive breast cancer. Since the development of tamoxifen, other triphenylethylene SERMs have been studied for breast cancer prevention, including droloxifene, idoxifene, toremifene, and ospemifene. Other SERMs have entered clinical development more recently, including benzothiophenes (raloxifene and arzoxifene), benzopyrans (ormeloxifene, levormeloxifene, and EM-800), lasofoxifene, pipendoxifene, bazedoxifene, HMR-3339, and fulvestrant, an anti-estrogen which is approved for breast cancer treatment. SERMs have effects on tissues containing ER, such as the breast, bone, uterine and genitourinary tissues, and brain, and on markers of cardiovascular risk. Current evidence indicates that each SERM has a unique array of clinical activities. Differences in the patterns of action of SERMs suggest that each clinical end point must be evaluated individually, and conclusions about any particular SERM can only be established through appropriate clinical trials. Target Audience: Obstetricians & Gynecologists, Family Physicians Learning Objectives: After completion of this article, the reader should be able to summarize the complex function and interactions of selective estrogen receptor modulators (SERMs); recall their suppression or initiation of target genes leading them to their actions; and explain their important roles in breast cancer treatment and possible side effects on bone, the genitourinary system, and on markers of cardiovascular risk.

Therapeutic strategies based on antiangiogenic approaches are beginning to show great promise in clinical studies. However, full realization of these approaches requires identification of key differences in gene expression between endothelial cells from tumors versus their normal counterparts. Here, we examined gene expression differences in purified endothelial cells from 10 invasive epithelial ovarian cancers and 5 normal ovaries using Affymetrix U133 Plus 2.0 microarrays. More than 400 differentially expressed genes were identified in tumor-associated endothelial cells. We selected and validated 23 genes that were overexpressed by 3.6- to 168-fold using real-time reverse transcription-PCR and/or immunohistochemistry. Among these, the polycomb group protein enhancer of Zeste homologue 2 (EZH2), the Notch ligand Jagged1, and PTK2 were elevated 3- to 4.3-fold in tumor-associated endothelial cells. Silencing these genes individually with small interfering RNA blocked endothelial cell migration and tube formation in vitro. The present study shows that tumor and normal endothelium differ at the molecular level, which may have significant implications for the development of antiangiogenic therapies. [Cancer Res 2007;67(4):1757–68]

Metronomic chemotherapy is the frequent administration of low doses of chemotherapeutic agents targeting tumor-associated endothelial cells. We examined the efficacy of metronomic taxanes alone and in combination with AEE788—a dual epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) inhibitor—in an orthotopic mouse model of ovarian cancer. Growth-modulating effects of metronomic and maximum tolerated dose (MTD) regimens on overall survival were tested in vivo using both chemotherapy-sensitive (HeyA8 and SKOV3ip1) and chemotherapy-resistant (HeyA8-MDR) models. Treated tumors were stained for microvessel density (CD31), proliferation index (proliferating cell nuclear antigen), and apoptosis (terminal deoxyribonucleotide transferase–mediated nick-end labeling). The cytotoxic effects of MTD and metronomic dosing were tested with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Effects of metronomic regimens on circulating endothelial precursors (CEP) and tumor-specific cell-free DNA levels were assessed. In vivo, metronomic docetaxel resulted in significant reduction of tumor growth in the taxane-sensitive cell lines, whereas metronomic docetaxel plus AEE788 had an additive effect resulting in significant prolongation in survival. Combination therapy was effective even in the taxane-resistant model. Metronomic chemotherapy alone and combined with AEE788 resulted in a decrease in the proliferative index and microvessel density of treated tumors, whereas combination therapy increased the apoptotic index (P < 0.001). In vitro, metronomic taxanes caused endothelial cell toxicity at 10- to 100-fold lower concentrations compared with MTD dosing. Metronomic regimens inhibited mobilization of CEPs (P < 0.05) and led to a decrease in cell-free DNA levels (P < 0.05). Our results suggest that metronomic taxane chemotherapy with dual EGFR and VEGFR inhibition is highly efficacious and should be considered for future clinical trials. [Cancer Res 2007;67(1):281–8]

Delta-like ligand 4 (Dll4), one of the Notch ligands, is overexpressed in ovarian cancer, especially in tumors resistant to anti-VEGF therapy. Here, we examined the biologic effects of dual anti-Dll4 and anti-VEGF therapy in ovarian cancer models. Using Dll4-Fc blockade and anti-Dll4 antibodies (murine REGN1035 and human REGN421), we evaluated the biologic effects of Dll4 inhibition combined with aflibercept or chemotherapy in orthotopic mouse models of ovarian cancer. We also examined potential mechanisms by which dual Dll4 and VEGF targeting inhibit tumor growth using immunohistochemical staining for apoptosis and proliferation markers. Reverse-phase protein arrays were used to identify potential downstream targets of Dll4 blockade. Dual targeting of VEGF and Dll4 with murine REGN1035 showed superior antitumor effects in ovarian cancer models compared with either monotherapy. In the A2780 model, REGN1035 (targets murine Dll4) or REGN421 (targets human Dll4) reduced tumor weights by 62% and 82%, respectively; aflibercept alone reduced tumor weights by 90%. Greater therapeutic effects were observed for Dll4 blockade (REGN1035) combined with either aflibercept or docetaxel (P < 0.05 for the combination vs. aflibercept). The superior antitumor effects of REGN1035 and aflibercept were related to increased apoptosis in tumor cells compared with the monotherapy. We also found that GATA3 expression was significantly increased in tumor stroma from the mice treated with REGN1035 combined with docetaxel or aflibercept, suggesting an indirect effect of these combination treatments on the tumor stroma. These findings identify that dual targeting of Dll4 and VEGF is an attractive therapeutic approach. Mol Cancer Ther; 15(6); 1344–52. ©2016 AACR.

Ascites formation associated with neoplasms is most likely due to increased vascular permeability, a process in which vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) plays a pivotal role. We hypothesized that tumor-derived VEGF/VPF modulates ascites formation through a paracrine effect on both tumor and peritoneal vessels. We investigated human vascular endothelial permeability using a newly developed dual-chamber permeability assay by co-culturing human umbilical vein cells with and without ovarian cancer cell lines (OVCAR-3, Hey-A8, and OCC-1) in the presence or absence of a human VEGF monoclonal antibody and VE-cadherin function–blocking antibody. This method permits determination of mechanisms by which substances released from neoplasms and other sources of vascular endothelial cell secretagogues modulate vascular permeability and likely other pathologic states.

Context: Matricellular proteins are a group of secreted, multifunctional extracellular matrix glycoproteins that includes thrombospondins (TSPs), tenascin-C, and secreted protein acidic and rich in cysteine (SPARC). They may be implicated in the dynamic developmental processes of the human fetal adrenal (HFA) in which the outer, definitive zone (DZ) cells are postulated to proliferate, migrate centripetally, differentiate, and populate the inner, steroidogenic fetal zone (FZ).Objective: The objective of the study was to identify a matricellular molecule that likely plays a major role in HFA development.Design: Studies involved RNA, cryosections, and cell cultures from 14- to 23-wk HFAs and human adult adrenal RNA.Main Outcome Measures: Measures included transcripts encoding matricellular proteins, using real-time quantitative RT-PCR; SPARC localization by immunostaining; and ACTH regulation of SPARC expression and secretion by quantitative RT-PCR and Western blot.Results: SPARC HFA mRNA was 100-, 700-, and 300-fold higher than TSP-1, TSP-2, and tenascin-C mRNA, respectively. HFA SPARC mRNA was 3-fold higher than adult adrenals (P < 0.005), comparable with levels in adult brain (positive control), whereas mRNAs encoding TSP-1 and TSP-2 were lower in fetal than adult adrenals. SPARC immunoreactivity was detected exclusively in the FZ, not DZ. ACTH, a key regulator of HFA growth and function, increased SPARC mRNA (by 1.7-fold at 1 nm, 48 h, P < 0.05) in isolated FZ cells but not DZ cells. ACTH up-regulation of SPARC protein was also detected in FZ cell lysates and culture medium.Conclusions: Results suggest a possible role for SPARC in development of functional and/or structural zonation of the HFA.

To examine the efficacy of extended-release venlafaxine for the treatment of postmenopausal hot flushes.Eighty postmenopausal women with more than 14 hot flushes per week were randomized to receive treatment with extended-release venlafaxine or placebo. Participants received 37.5 mg daily for 1 week, followed by 75 mg daily for 11 weeks. Daily hot flush severity scores and adverse effects were recorded by subjects. Baseline and monthly follow-up questionnaires assessed patient-perceived hot flush score, quality of life, and sexual function. Participants were treated for 12 weeks.Of the 80 subjects who enrolled in the study, 40 were in the treatment group and 40 in the control group. Of these, 61 completed the study (treatment, n = 29; control, n = 32). Subjective assessment at monthly visits of the effects of hot flush symptoms on daily living were significantly improved in the treatment group (P.001). Hot flush severity scores based on daily diaries were somewhat lower in the treatment group, but the between-group difference did not reach statistical significance (P = .25). Three side effects, dry mouth, sleeplessness, and decreased appetite, were significantly more frequent in the venlafaxine group, but others, including dizziness, tremors, anxiety, diarrhea, and rash, were significantly less frequent. Ninety-three percent of participants in the venlafaxine group chose to continue treatment at the conclusion of the study.Extended-release venlafaxine, 75 mg per day, is an effective treatment for postmenopausal hot flushes in otherwise healthy women, based on a significant decrease in patient-perceived hot flush score.

Modulation of the signaling pathways that are aberrant in cancer cells has the potential to provide an effective nontoxic approach to patient management in a broad range of cancers. This quest has taken a major leap forward with the demonstration that STI-571 (imatinib mesylate) induces clinical and molecular remissions in the majority of patients with interferon-refractory chronic myelogenous leukemia and gastrointestinal stromal tumors through inhibition of the Bcr/Abl fusion protein required for the initiation and progression of chronic myelogenous leukemia and inhibition of a mutant, activated c-kit present in gastrointestinal stromal tumors. Support for the concept of targeting products of fusion genes found in specific cancers was first provided by the efficacy of all-trans retinoic acid in acute promyelocytic leukemia where the RARalpha all-trans retinoic acid target is the target of multiple different chromosomal rearrangements. In breast cancer, trastuzumab, which alters the function of the HER2 proto-oncogene overexpressed in a portion of breast cancers, provides an additional example of targeting specific molecular aberrations present in cancer cells. Although the target for these signal transduction modulators is functional in normal cells, acceptable therapeutic indices sufficient to prevent tumor growth without unacceptable toxicities have been observed. Whether STI-571 and other signal transduction modulators also target the stroma, and specifically the neovasculature, in addition to the tumor remains an open question. The presence of the target in the cancer cells or in the surrounding stroma appears to be required but not sufficient for the action of molecular therapeutics. Thus, linking molecular diagnostics to identify patients where the target is amplified or activated and driving the pathophysiology of the patients' tumor to effective molecular therapeutics will be necessary to translate these concepts into approaches that will alter the outcome for breast cancer patients. This review will focus on the phosphatidylinositol 3-kinase pathway and novel molecules targeting this pathway to illustrate the questions and challenges underlying the implementation of molecular therapeutics in breast cancer.

Organogenesis is a coordinated process involving cell replication, differentiation, adhesion, and migration. We seek to understand the complex developmental signals involved in the ontogeny of the human fetal adrenal gland. The gland is comprised initially of two zones, the definitive and fetal zones. A third zone, the transitional zone, develops between them after midgestation. We have suggested that the definitive zone is comprised of a pool of progenitor cells that proliferate and differentiate into cells of the transitional and fetal zones. However, it has not been possible to demonstrate that definitive zone cells have this capacity because of the absence of protein markers unique to these cells; thus, they could not be purified or positively identified. We sought to identify definitive and fetal zone markers to facilitate cell sorting and identify molecules of biological interest in adrenal development. We performed subtractive hybridization, in situ hybridization, and immunofluorescence to identify unique markers of definitive zone cells. NovH and metallopanstimulin were identified by subtraction hybridization, primarily in the definitive zone. P-Glycoprotein, also principally on definitive zone cells, and the low density lipoprotein (LDL) receptor, predominantly on fetal zone cells, were identified by immunofluorescence. Identification of cellular markers unique to each zone of the fetal adrenal gland will enhance the ability to characterize the proliferative potential of definitive zone cells and assess their capacity to differentiate into cells of the transitional and fetal zones. Purified cells also will permit detailed molecular and mechanistic studies of regulation of human fetal adrenal development.

Background: Lysophosphatidic acid (LPA), at concentrations present in ascitic fluid, indirectly stimulates the growth of malignant ovarian tumors by increasing the expression of vascular endothelial growth factor (VEGF) in ovarian cancer cells. We investigated whether LPA could also directly promote ovarian tumor growth by increasing the level of cyclin D1, a key G1-phase checkpoint regulator, which thereby increases cell proliferation. Methods: Expression of cyclin D1 and LPA receptors (EDG4 and EDG7) was determined in six ovarian cancer cell lines (including OVCAR-3 cells) and immortalized ovarian surface epithelial cells (IOSE-29). Cyclin D1 promoter activity was measured in LPA-treated OVCAR-3 cells cotransfected with cyclin D1 promoter-driven luciferase constructs and cDNA expression plasmids for IBM (a nuclear factor B [NFB] superrepressor). Results: Four of six cancer cell lines, including OVCAR-3, overexpressed cyclin D1 protein relative to levels in IOSE-29 cells. LPA treatment increased cyclin D1 protein in a dose- and time-dependent manner in OVCAR-3 cells but not in IOSE-29 cells. LPA stimulated cyclin D1 promoter activity (3.0-fold, 95% confidence interval [CI] = 2.7-fold to 3.3-fold). Mutation of the NFB-binding site in the cyclin D1 promoter to block NFB binding and expression of IBM, which binds NFB and inhibits its binding to the promoter, markedly diminished LPA stimulation of cyclin D1 promoter activity (activity stimulated only 1.4-fold, 95% CI = 1.1-fold to 1.7-fold, and 0.7-fold, 95% CI = 0.6-fold to 0.8fold, respectively). EDG4 was overexpressed in all cancer cell lines studied relative to that in IOSE-29 cells, but EDG7 was overexpressed in only two lines. Conclusions: Dual mechanisms are probably involved in LPA stimulation of ovarian tumor growth in vivo. In addition to the previously characterized indirect mechanism that increases angiogenesis via VEGF, LPA may directly increase the level of cyclin D1 in ovarian cancer cells, increasing their proliferation. [J Natl Cancer Inst 2003;95:733–40]

Ovarian cancer is characterized by rapid growth of solid intraperitoneal tumors and production of large volumes of ascites. Our previous studies of intraperitoneal ovarian carcinoma in an athymic mouse model demonstrated that a monoclonal antibody (mAb) to human vascular endothelial growth factor (VEGF) could prevent ascites formation. Although ascites was almost completely inhibited, tumor burden was variably reduced. To develop more effective therapy, we assessed the combination of a human VEGF mAb plus paclitaxel. Four groups of female athymic nude mice were inoculated intraperitoneally with OVCAR3 cells. Two weeks after inoculation, one group was treated with a human VEGF mAb intraperitoneally twice weekly plus paclitaxel intraperitoneally three times weekly for 6 weeks. The second group was treated with VEGF mAb alone. The third group was treated with paclitaxel alone. The remaining group was treated with vehicle only. Tumor burden in the VEGF mAb plus paclitaxel and paclitaxel alone groups was reduced by 83.3% and 85.7% and 58.5% and 59.5%, respectively, in two separate experiments, compared to controls. VEGF mAb alone caused no significant decrease in tumor burden, nor did treatment of mice inoculated intraperitoneally with HEY-A8 cells, a non-VEGF-secreting ovarian cell line. Virtually no ascites developed in the combined treatment group or the group treated with VEGF mAb alone. Paclitaxel alone reduced ascites slightly, but not significantly. Morphological studies demonstrated that VEGF immunoneutralization enhanced paclitaxel-induced apoptosis in these human ovarian cancers. Thus, combination therapy with inhibitors of VEGF plus paclitaxel may be an effective way to markedly reduce tumor growth and ascites in ovarian carcinoma.

During placental development, vessel formation occurs initially by vasculogenesis and subsequently by branching and nonbranching angiogenesis. We investigated vascular endothelial growth factor (VEGF)-A, angiopoietin (Ang)-1 and -2 transcript profiles, and the protein products that they encode in placentas from normotensive pregnancies throughout pregnancy. In addition, we compared these genes in placentas from normotensive women and those with preeclampsia during the third trimester. Quantitative real-time PCR analysis demonstrated that VEGF-A and Ang1 mRNA increased in a linear pattern by 2.5 (not significant) and 2.8%/wk (P = 0.034), respectively, whereas Ang2 decreased logarithmically by 3.5%/wk (P = 0.0003). Ang2 mRNA was 400- and 100-fold higher than Ang1 and VEGF-A, respectively, in the first trimester and declined to 20-fold and 7-fold in the third. Ang2 protein (ELISA) decreased by 4.7%/wk (P = 0.0001), whereas Ang1 and VEGF-A were undetectable. In preeclampsia compared with normotensive pregnancy, only VEGF-A mRNA increased significantly, by 3-fold (P = 0.006). This increase may be related to low oxygen tension, as VEGF-A is up-regulated by hypoxia. In situ hybridization and immunohistochemical studies revealed that VEGF-A was localized in cyto- and syncytiotrophoblast and perivascular cells, whereas Ang1 and Ang2 were only in syncytiotrophoblast and perivascular cells in the immature intermediate villi during the first and second trimesters, and mature intermediate and terminal villi during the third trimester. These data suggest that these molecules may play important roles in placental biology and chorionic villus vascular development and remodeling in an autocrine/paracrine manner. The tight correlation between Ang2 mRNA and protein indicates that regulation of placental vascular development occurs at the transcriptional, and not translational, level.

Lysophosphatidic acid (LPA) is a naturally occurring phospholipid that exhibits pleiotrophic biological activities, ranging from rapid morphological changes to long-term cellular effects such as induction of gene expression and stimulation of cell proliferation and survival on a wide spectrum of cell types. LPA binds and activates distinct members of the Edg/LP subfamily of G protein-coupled receptors that link to multiple G proteins including G(i), G(q) and G(12/13) to elicit cellular responses. LPA plays a critical role as a general growth, survival and pro-angiogenic factor, in the regulation of physiological and pathophysiological processes in vivo and in vitro. Our previous work indicates that abnormalities in LPA metabolism and function in ovarian cancer patients may contribute to the initiation and progression of the disease. Thus, LPA could be a potential target for cancer therapy. This review summarizes evidence that implicates LPA in the pathophysiology of human ovarian cancer and likely other types of human malignancies.

Vascular endothelial growth factor (VEGF) is a mitogen in physiological and pathological angiogenesis. Understanding the expression of different VEGF isoforms might be important for distinguishing angiogenesis in tissue development, vascular remodelling and tumour formation. We examined its expression and noted the presence of the isoforms VEGF121 and VEGF165 (121 and 165 residues long respectively) in fetal heart, lung, ovary, spleen, placenta and ovarian tumours. Unexpectedly, a 47kDa species predominated in fetal intestine and muscle. The presumed initiation site in VEGF is an AUG codon (AUG1039), 1039nt from its main transcriptional start site. AUG1039 is preceded in the 5′ untranslated region by an in-frame CUG at nt 499 (CUG499), which could produce the 47kDa form with a 180-residue N-terminal extension. We therefore assessed whether CUG499 functions as an initiator. CUG499 initiation produced the 47kDa VEGF165 precursor, which was processed at two sites to yield VEGF and three N-terminal fragments. When CTG499 was mutated to CGC, the precursor and N-terminal fragments were barely detectable. Although the precursor form was predominant in VEGF165, both CUG499 and AUG1039 forms were found in VEGF121. VEGF precursor induced neither the proliferation of human umbilical vein endothelial cells nor the expression of angiopoietin 2, which can be induced by, and act with, VEGF to induce tumour angiogenesis. The precursor also adheres to the extracellular matrix (ECM), suggesting that it might be a storage form for generating active VEGF in the cell or ECM. Alternate CUG499 and AUG1039 initiation and processing of the inactive precursor and its products might be important in regulating angiogenesis.

Objectives: Estrogen is often prescribed for symptoms and sequelae of ovarian estrogen loss after menopause. Methods: To assess efficacy and acceptability of a new, highly soluble estrogen–calcium preparation, we formulated a water-soluble powdered combination of estrogen (0.625 mg estrone piperazine sulfate) and calcium (1 g, ions) as the highly soluble glycerophosphate salt (Estrosol®). Effects of once-daily administration on bone mineral turnover of Estrosol® dissolved in water (n=11) was compared with 0.625 mg conjugated estrogens (Premarin®) + 1 g calcium (Tums® 500 Calcium Supplement) (n=8). All women had had a previous hysterectomy, were between the ages 40 and 75, within 25% of ideal body weight, and had not taken hormonal preparations for at least 3 months. Assessment of bone mineral turnover was by monitoring N-telopeptides and bone specific alkaline phosphatase (BSAP) on 5 occasions: pretreatment and once during each of the 4 months of treatment. Results: Mean N-telopeptide values decreased (p=.005) in both groups: Estrosol®, 29.2% (40 → 29 mmol bone collagen equivalents (BCE)/mmol creatinine), and Premarin® + calcium, 44.8% (33 → 18 mmol). Mean BSAP values also decreased (p=0.007) in both groups: Estrosol®, 12.6% (12.06 → 10.54 mg/l), Premarin® + calcium, 19.1% (11.57 → 9.36 mg/l). The difference between groups for both N-telopeptides and BSAP was not significant, although sample size was small. Symptoms (hot flashes, vaginal dryness) improved similarly in both groups. Symptoms during treatment (breast or nipple tenderness, bloating) also were similar in both groups. Both preparations were well-tolerated. There were no changes in CBC, liver function tests, electrolytes or urinalyses in either group. Conclusions: This pilot study indicates that the combined, highly water-soluble preparation of estrogen and calcium is effective in reducing bone mineral turnover, acceptable and well-tolerated. Use of this single aqueous preparation may lead to better compliance than using two separate pills.

In humans, placental corticotropin-releasing hormone (CRH) production has been linked to the determination of gestational length, and a late gestational fall in CRH-binding protein (CRH-BP) has been linked to the onset of parturition. Expression of placental CRH mRNA is limited to primates, and only in man has a circulating CRH-BP been described. As the fall in CRH-BP in late gestation has been associated with parturition in humans, we sought to determine whether a CRH-BP circulated in the plasma of other primates. It is unclear whether maternal plasma CRH concentrations are elevated in New World monkeys and prosimians. We have therefore performed CRH plasma measurements in the blood of pregnant marmosets, in several species of lemur, and in pregnant and fetal rhesus monkeys as a positive control. Using gel chromatography, CRH-BP was detected in the human, gorilla, chimpanzee, orangutan, gibbon, macaque, squirrel monkey, and marmoset, but was absent in the mandrill, spider monkey, and lemur. CRH was detected in the plasma of pregnant marmosets and rhesus monkeys. CRH was also detected in the fetal rhesus monkey, but at lower concentrations than in maternal plasma. CRH immunoreactivity was not detectable in the plasma of pregnant lemurs or in extracts of lemur placenta. In conclusion, a circulating binding protein for CRH exists in all species of apes but occurs variably among New World and Old World monkeys and is absent in lemurs. The variable occurrence of the CRH-BP does not support a role for this protein in the mechanism of parturition in primates. Maternal CRH is elevated in the pregnant marmoset and rhesus, and may play a role in the pregnancy of New and Old World monkeys.

Objective: Magnetic resonance imaging enhanced with macromolecular contrast medium was used to monitor effects of angiogenesis inhibition on tumor microvascular permeability and ascites volume in an athymic rat model of human ovarian cancer. Study Design: Groups of 6 athymic rats implanted intraperitoneally with SKOV-3, a human ovarian cancer cell line, were treated through a 14-day course with antibody to vascular endothelial growth factor or with saline solution for control animals. Dynamic magnetic resonance imaging was performed with a 92,000-d contrast agent, albumin-(gadolinium-diethylenetriaminepentaacetic acid)30. Vascular permeability was estimated from dynamic enhancement data that were analyzed with a unidirectional 2-compartment kinetic model. Results: Animals treated with vascular endothelial growth factor antibody accumulated significantly smaller volumes of peritoneal ascites (P < .05) and showed significantly lower magnetic resonance imaging–assayed tumor microvascular permeabilities (P < .05) than did control animals. Conclusion: Magnetic resonance imaging enhanced with a macromolecular contrast agent in an athymic rat model of human ovarian cancer treated with anti–vascular endothelial growth factor antibody can be used to measure a reduction in tumor microvascular permeability, corresponding to a reduction in ascites production. (Am J Obstet Gynecol 2000;183:956-63.)

Objective: To review the current literature on the role of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in ovarian physiology and pathology. Design: A computerized search was conducted to identify relevant in vitro and in vivo studies published in English. MEDLINE, Current Contents, and the Index Medicus were searched for studies published before January 2000. Result(s): VEGF/VPF is an angiogenic factor and a potent mitogen for vascular endothelium. During reproductive life, VEGF/VPF plays a role in the cyclic growth of ovarian follicles and corpus luteum development and maintenance, mediating ovarian angiogenesis. VEGF/VPF expression and secretion are induced by both FSH and LH/hCG receptor-activated pathways. Conclusion(s): VEGF/VPF expression and production within the ovary are critical for normal reproductive function. Defects in angiogenesis may contribute to a variety of disorders including anovulation and infertility, pregnancy loss, ovarian hyperstimulation syndrome, and ovarian neoplasms.

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.

Phytoestrogens influence a variety of biological processes. As 17beta-estradiol alters adrenocortical cell function, we examined whether the dietary phytoestrogens, genistein and daidzein, have related effects. In cultured human fetal and postnatal adrenal cortical cells, genistein and daidzein (both 0.4-40 micromol/L) decreased ACTH-stimulated cortisol production to basal levels (ED50, 1-4 micromol/L). In the adult adrenocortical cell line, H295, genistein, daidzein, and 17beta-estradiol (10 micromol/L) decreased cAMP-stimulated cortisol synthesis in a similar fashion. Neither genistein nor daidzein altered basal or ACTH-stimulated dehydroepiandosterone sulfate (DHEA-S) production in fetal adrenocortical cells, whereas in postnatal adrenocortical cells, DHEA and DHEA-S were markedly increased (ED50, 1-4 micromol/L). In H295 cells, basal and cAMP-stimulated DHEA production were similarly increased by the phytoestrogens and 17beta-estradiol. Genistein and daidzein did not affect the expression of steroid-metabolizing enzymes. However, genistein and daidzein specifically inhibited the activity of 21-hydroxylase (P450c21); the activities of other steroidogenic enzymes were not affected. Thus, phytoestrogens may decrease cortisol synthesis by suppressing the activity of P450c21 and, as a consequence, increase DHEA/DHEA-S synthesis by shunting metabolites away from the glucocorticoid synthetic pathway. Therefore, consumption of foods containing phytoestrogens may alter adrenocortical function by decreasing cortisol and increasing androgen production.

After 10–15 weeks of gestation, the human fetal adrenal cortex undergoes rapid growth due to enlargement of a specialized cortical compartment known as the fetal zone (FZ). Soon after birth, the FZ regresses and the adult zonation pattern develops at least in part from cells derived from the persistent definitive zone (DZ), a thin layer of tightly packed cells surrounding the FZ. We postulated that growth of the fetal adrenal cortex involves zone-specific cellular hyperplasia, whereas the postnatal involution of the FZ is due to apoptosis. Therefore, we investigated the pattern of cellular proliferation and death in the FZ and DZ of the human fetal and postnatal adrenal cortex using immunohistochemical staining for proliferating cell nuclear antigen as a marker of mitosis and in situ detection of DNA fragmentation as a marker of apoptosis. Between 10–14 weeks’ gestation, the mitotic indexes (percentage of proliferating cell nuclear antigen-positive cells) in the DZ (26.46 ± 2.95%) and in the FZ (21.26 ± 2...

Ovarian cancer is characterized by the rapid growth of solid intraperitoneal tumors and large volumes of ascitic fluid. Vascular endothelial growth factor (VEGF) augments tumor growth by inducing neovascularization and may stimulate ascites formation by increasing vascular permeability. We examined the role of VEGF in ovarian carcinoma using in vivo models in which intraperitoneal or subcutaneous tumors were induced in immunodeficient mice using the human ovarian carcinoma cell line SKOV-3. After tumor engraftment (7 to 10 days), some mice were treated with a function-blocking VEGF antibody (A4.6.1) specific for human VEGF. A4.6.1 significantly (P < 0.05) inhibited subcutaneous SKOV-3 tumor growth compared with controls. However, tumor growth resumed when A4.6.1 treatment was discontinued. In mice bearing intraperitoneal tumors (IP mice), ascites production and intraperitoneal carcinomatosis were detected 3 to 7 weeks after SKOV-3 inoculation. Importantly, A4.6.1 completely inhibited ascites production in IP mice, although it only partially inhibited intraperitoneal tumor growth. Tumor burden was variable in A4.6.1-treated IP mice; some had minimal tumor, whereas in others tumor burden was similar to that of controls. When A4.6.1 treatment was stopped, IP mice rapidly (within 2 weeks) developed ascites and became cachectic. These data suggest that in ovarian cancer, tumor-derived VEGF is obligatory for ascites formation but not for intraperitoneal tumor growth. Neutralization of VEGF activity may have clinical application in inhibiting malignant ascites formation in ovarian cancer.

Estrogens produced by the placenta play a pivotal role in the endocrine control of pregnancy and induce many of the key changes involved in parturition. The placentae of humans and higher primates use the C19 androgen dehydroepiandrosterone sulfate (DHEA-S) supplied by the fetal adrenals as the principal substrate for estrogen synthesis. Thus, secretion of androgens by the fetal adrenals may be central to the process of primate parturition. The timing of human parturition also is correlated with placental CRH concentrations in the maternal circulation. Because the mechanisms that regulate DHEA-S production by the fetal adrenals are incompletely understood, we examined whether there is a functional relationship between CRH and steroid production by human fetal adrenal cortical cells. Using Northern blot analysis, we detected messenger RNA transcripts (2.7 kb) encoding the type-1 CRH receptor in total RNA extracted from midgestation human fetal adrenals, suggesting that the fetal adrenal cortex may be directly responsive to CRH. To test this, primary cultures of human fetal adrenal cortical cells were exposed to human CRH. Human CRH increased DHEA-S production by cultured human fetal adrenal cortical cells in a dose-dependent fashion, with an ED50 of 10-100 pmol/L. Human CRH was as effective as ACTH at stimulating DHEA-S production; however, it was 70% less potent than ACTH at stimulating cortisol production, indicating that its actions were preferentially directed toward increasing DHEA-S synthesis. Consistent with this thesis, we found that CRH increased abundance of messenger RNA encoding cytochrome P450 cholesterol side-chain cleavage and 17alpha-hydroxylase/17,20 lyase but not 3beta-hydroxysteroid dehydrogenase in adrenal cells. CRH did not alter cell number, indicating that it is not mitogenic for fetal adrenal cortical cells. These data demonstrate a direct functional interaction between CRH and the fetal adrenal. Therefore, placental CRH production, which rises exponentially during human pregnancy, may play a key role in promoting DHEA-S production by the fetal adrenals, which could lead to increasing placental estrogen synthesis and contribute to the process of parturition in humans.

The human adrenal cortex has a complex vasculature that is essential for growth, organ maintenance, and access of secreted hormones to the circulation. Growth and function of the adrenal cortex are regulated by corticotropin (ACTH), the actions of which are in part mediated by locally produced growth factors. As cortical growth and vascularization must increase in a coordinated manner, we hypothesized that ACTH also influences adrenal cortical angiogenesis by stimulating the local expression of specific angiogenic factors. Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific angiogenic peptide, the expression of which has been detected in adrenal cortical cells. Therefore, we examined the localization of VEGF expression in the midgestation (16–20 weeks) human fetal adrenal cortex and determined whether VEGF expression and secretion by isolated human fetal adrenal cortical cells are regulated by ACTH. By immunohistochemical analysis, strong cytoplasmic staining for VEGF was detected in scattered clusters of fetal zone (inner cortical compartment) cells. In contrast, cells in the outer, definitive zone of the cortex stained only weakly for VEGF. The predominant staining for VEGF in the fetal zone correlated with the extensive vasculature of this zone as detected by immunohistochemical staining for von Willebrand factor, which is specific for endothelial cells. In primary cultures of human fetal adrenal cortical cells, ACTH (1 nmol/L) and forskolin (10 μmol/L) increased the abundance of messenger ribonucleic acid transcripts encoding VEGF, as assessed by Northern and slot blot analyses. The stimulatory effect of ACTH and forskolin on VEGF gene expression occurred within 2 h of agonist exposure and persisted for at least 24 h. ACTH and forskolin also increased VEGF protein secretion by fetal adrenal cortical cells, as assessed by enzyme-linked immunosorbent assay for VEGF in fetal adrenal cortical cell-conditioned medium. A significant (P< 0.05) increase in VEGF secretion was detected as early as 8 h after ACTH or forskolin treatment. By 24 h after the addition of ACTH or forskolin, VEGF secreted from isolated human fetal adrenal cells was increased 5- to 6-fold. These data demonstrate that the human fetal adrenal cortex, particularly the cells of the inner fetal zone, express VEGF and that VEGF expression and secretion by these cells are directly regulated by ACTH and the activation of adenylate cyclase. Thus, VEGF may be a local regulator of adrenal cortical angiogenesis and an important mediator of the tropic action of ACTH, ensuring the coordination of ACTH-stimulated cortical growth and vascularization.

Prebeta-1 HDL is a molecular species of plasma HDL of approximately 67 kDa mass that contains apolipoprotein A-I, phospholipids, and unesterified cholesterol. It participates in a cyclic process involved in the retrieval of cholesterol from peripheral tissues. In this cycle, unesterified cholesterol from cells is incorporated into prebeta-1 HDL, providing a substrate for esterification of cholesterol by lecithin:cholesterol acyltransferase. Prebeta-1 HDL then becomes incorporated into larger HDL species of alpha mobility as esterification proceeds and is regenerated during the transfer of cholesteryl esters from alpha HDL particles to acceptor lipoproteins. Thus the steady state level of prebeta-1 HDL in plasma reflects the relative efficiencies of the major metabolic processes involved in its generation and removal. We have used an isotope dilution technique to measure prebeta-1 HDL levels in the plasmas of 136 normolipidemic individuals (46 M, 90 F). The mean absolute concentration of prebeta-1 HDL as apolipoprotein A-I was 68 ± 40 μg/ml for women, and 84 ± 49 m/ml for men. Prebeta-1 HDL represented 5.5 ± 3.3% of total apolipoprotein A-I in women, and 7.2 ± 4.0% in men. The distributions of both absolute and percent prebeta-1 HDL are highly asymmetric, with skew toward higher values. However, the skew appears not to be attributable to either plasma cholesterol or triglyceride levels which are also skewed in population samples. The percent prebeta-1 HDL was negatively correlated with HDL cholesterol levels (P < 0.0001), whereas absolute levels of prebeta-1 HDL were positively correlated with apolipoprotein A-I and negatively correlated with HDL cholesterol (P, for both, < 0.0001). Multiple linear regression analysis revealed effects of age and gender, but no association with lipoprotein fractions other than HDL. Lower levels of prebeta-1 HDL were associated with female gender in all models.—O'Connor, P. M., B. R. Zysow, S. A. Schoenhaus, B. Y. Ishida, S. T. Kunitake, J. M. Naya-Vigne, P. N. Duchateau, R. F. Redberg, S. J. Spencer, S. Mark, M. Mazur, D. C. Heilbron, R. B. Jaffe, M. J. Malloy, and J. P. Kane. Prebeta-1 HDL in plasma of normolipidemic individuals: influences of plasma, age, and gender.

The rapid growth of the human fetal adrenal gland, which is primarily a reflection of the growth of the unique fetal zone, is regulated by ACTH acting indirectly to stimulate the expression of locally produced growth factors, of which IGF-II and bFGF appear to play key roles. Through most of gestation, the outer definitive zone appears to function as a reservoir of progenitor cells which move centripetally to populate the rest of the gland. At the end of pregnancy, the fetal zone undergoes senescence through an apoptotic process. Activin and TGF-beta are capable of inducing apoptosis in the fetal zone. Corticotropin-releasing hormone, which is produced by the placenta in markedly increased amounts at the end of gestation, may orchestrate a variety of processes, including direct stimulation of fetal adrenal steroidogenesis, culminating in the initiation of parturition.

Insulin-like growth factor (IGF)-binding proteins (IGFBPs) antagonize IGF and gonadotropin actions on granulosa cells. Human atretic follicles express IGFBP-2 in granulosa cells more strongly and contain higher levels of IGFBP-2 and IGFBP-4 than healthy follicles. We studied the effects of interferon-γ (IFNγ) and activin A, which decrease progesterone accumulation, on granulosa cell IGFBP production and apoptosis. Conditioned media from luteinizing granulosa cells cultured with IFNγ or activin A and/or LH were subjected to ligand blotting; northern blots of total ribonucleic acid (RNA) from these cells were probed for IGFBP-2 and -4. Apoptosis was measured by in situ DNA end labeling. LH decreased medium IGFBP-2 to 21% of the control value. Although IFNγ did not alter basal medium IGFBP-2, in the presence of LH it increased IGFBP-2 3.4-fold, with parallel changes in messenger RNA levels. Activin A also tended to increase medium IGFBP-2 in LH-treated cultures. In conditioned medium, IGFBP-4 was consistently decreased by LH, whereas both IFNγ and activin A increased IGFBP-4 and decreased IGFBP-4 protease activity. Both LH and IFNγ modestly stimulated IGFBP-4 messenger RNA levels. Follistatin antagonized the action of activin A, but not that of IFNγ. IFNγ, but not activin A, increased granulosa cell apoptosis. In conclusion, IFNγ produced by activated lymphocytes may decrease endogenous IGF activity through stimulation of IGFBPs and may promote apoptosis of granulosa-lutein cells in vivo and, thus, luteal regression. Activin A similarly promotes IGFBP accumulation, but it does not promote apoptosis. (J Clin Endocrinol Metab 83: 179–186, 1998)

Previous studies of raloxifene conducted in animal models and postmenopausal women have demonstrated antiestrogenic action on the endometrium. The purpose of this first study of raloxifene in women with normal menstrual cycles was to determine its reproductive endocrine and endometrial effects. In part I, raloxifene (400 mg) was administered for 5 days in the follicular, periovulatory, or luteal phase of the menstrual cycle (n = 12). In part II, women were randomized to receive raloxifene (100 or 200 mg) for 28 days beginning on day 3 of the cycle (n = 19). All women ovulated in both parts of the study. Raloxifene did not alter the length of the menstrual cycle or the day of the LH surge. A 5-day course of raloxifene administered in any phase of the cycle elevated FSH area under the curve (AUC) for the entire cycle and estradiol AUC for the second half of the cycle compared with those in control cycles. In part II, raloxifene also appeared to increase the FSH AUC and estradiol AUC. Raloxifene decreased the number of gland mitoses in follicular phase endometrial biopsies. Subtle effects suggestive of gland-stromal dysynchrony were noted in a limited number of the secretory phase endometrial biopsies. This study has demonstrated that 1) raloxifene does not prevent ovulation in women with normal menstrual cycles; 2) ovarian estrogen production will continue, and in some cases increase, in response to raloxifene; and 3) antiestrogenic effects of raloxifene on the endometrium are subtle in the endocrine milieu of normal to high circulating estradiol concentrations.

The unique characteristics of the primate (particularly human) fetal adrenal were first realized in the early 1900s when its morphology was examined in detail and compared with that of other species. The unusual architecture of the human fetal adrenal cortex, with its unique and disproportionately enlarged fetal zone, its compact definitive zone, and its dramatic remodeling soon after birth captured the interest of developmental anatomists. Many detailed anatomical studies describing the morphology of the developing human fetal adrenal were reported between 1920 and 1960, and these morphological descriptions have not changed significantly. More recently, it has become clear that fetal adrenal cortical growth involves cellular hypertrophy, hyperplasia, apoptosis, and migration and is best described by the migration theory, i.e. cells proliferate in the periphery, migrate centripetally, differentiate during their migration to form the functional cortical zones, and then likely undergo apoptosis in the center of the cortex. Consistent with this model, cells of intermediate phenotype, arranged in columnar cords typical of migration, have been identified between the definitive and fetal zones. This cortical area has been referred to as the transitional zone and, based on the expression of steroidogenic enzymes, we consider it to be a functionally distinct cortical zone. Elegant experiments during the 1950s and 1960s demonstrated the central role of the primate fetal adrenal cortex in establishing the estrogenic milieu of pregnancy. Those findings were among the first indications of the function and physiological role of the human fetal adrenal cortex and led Diczfalusy and co-workers to propose the concept of the feto-placental unit, in which DHEA-S produced by the fetal adrenal cortex is used by the placenta for estrogen synthesis. Tissue and cell culture techniques, together with improved steroid assays, revealed that the fetal zone is the primary source of DHEA-S, and that its steroidogenic activity is regulated by ACTH. In recent years, function of the human and rhesus monkey fetal adrenal cortical zones has been reexamined by assessing the localization and ontogeny of steroidogenic enzyme expression. The primate fetal adrenal cortex is composed of three functionally distinct zones: 1) the fetal zone, which throughout gestation does not express 3 beta HSD but does express P450scc and P450c17 required for DHEA-S synthesis; 2) the transitional zone, which early in gestation is functionally identical to the fetal zone but late in gestation (after 25-30 weeks) expresses 3 beta HSD, P450scc, and P450c17, and therefore is the likely site of glucocorticoid synthesis, and 3) the definitive zone, which lacks P450c17 throughout gestation but late in gestation (after 22-24 weeks) expresses 3 beta HSD and P450scc, and therefore is the likely site of mineralocorticoid synthesis. Indirect evidence, based on effects of P450c21 deficiency and maternal estriol concentrations, indicate that the fetal adrenal cortex produces cortisol and DHEA-S early in gestation (6-12 weeks). However, controversy exists as to whether cortisol is produced de novo or derived from the metabolism of progesterone, as data regarding the expression of 3 beta HSD in the fetal adrenal cortex early in gestation are conflicting. During the 1960s, Liggins and colleagues demonstrated that in the sheep, cortisol secreted by the fetal adrenal cortex late in gestation regulates maturation of the fetus and initiates the cascade of events leading to parturition. Those pioneering discoveries provided insight into the mechanism underlying the timing of parturition and therefore were of particular interest to obstetricians and perinatologists confronted with the problems of preterm labor. However, although cortisol emanating from the fetal adrenal cortex promotes fetal maturation in primates as it does in sheep, its role in the regulation of primate parturition, unlike that in sheep

Objective: To determine if the GnRH antagonist (GnRH-a) Ganirelix (Syntex Research, Palo Alto, CA), administered by intranasal (IN) spray to normal women, is absorbed into the systemic circulation and suppresses LH secretion. Design: A single center, open label, nonrandomized, dose-escalation study. Setting: Academic research environment. Patient(s): Normal female volunteers ages 23 to 43 years. Intervention(s): Ganirelix was administered as a single dose by IN spray. The administered doses and the number of women receiving each of them were 0.1 mg (n = 1), 0.3 mg (n = 1), 1 mg (n = 2), 3 mg (n = 5), and 6 mg (n = 5). Blood samples were collected from −15 minutes to 24 hours after dosing. Main Outcome Measure(s): Serum concentrations of Ganirelix and LH. Result(s): Ganirelix was absorbed rapidly. The mean time to maximal serum levels in the 3- and 6-mg groups was 0.67 and 0.53 hour, respectively. Mean serum LH levels were suppressed by ≥35% relative to baseline from 2 to 12 hours after dosing in both groups. The mean maximal percent decrease in serum LH was −62% (at 8 hours after dosing) and −74% (at 6 hours after dosing) in the 3- and 6-mg groups, respectively. Conclusion(s): Single dose IN administration of 3 or 6 mg of Ganirelix suppressed serum LH levels in women, further enhancing the potential clinical utility of this potent GnRH-a. This is the first clinical report of a GnRH-a reducing the secretion of a pituitary gonadotropin when administered by an IN delivery system. Based on the duration and extent of LH suppression observed in this study, Ganirelix, administered by twice daily IN spray, may be effective for the treatment of gonadal hormone-dependent disorders in women.

Development of the human fetal adrenals is characterized by rapid growth and high levels of steroidogenic activity during the latter two-thirds of pregnancy. By midgestation, the human fetal adrenals are composed of two distinct cortical zones: the predominant fetal zone, which occupies 80-90% of the cortical volume and produces large amounts of the delta 5-steroid dehydroepiandrosterone sulfate, and the narrow definitive zone, which surrounds the fetal zone. Late in gestation, the peripheral portion of the fetal zone develops into a third, functionally distinct compartment, the transitional zone, which is the likely site of cortisol synthesis. Soon after birth, the adrenal cortex is remodeled and the fetal zone disappears. The adult cortical zones are thought to arise from the definitive zone, which persists postnatally. Development of the human fetal adrenals is regulated primarily by corticortropin (ACTH) secreted from the fetal pituitary. However, as ACTH is not a mitogen per se, its proliferative actions on human fetal adrenal cortical cells are thought to be mediated by autocrine/paracrine growth factors produced by adrenal cortical cells in response to ACTH. In addition, these growth factors appear to modulate the functional response of fetal adrenal cortical cells to ACTH. The roles of several growth factors, including the insulin like growth factors I and II (IGF-I and IGF-II), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), activin, inhibin, and the transforming growth factors alpha and beta (TGF-alpha and TGF-beta) have been examined. In cultured human fetal adrenal cortical cells, EGF, bFGF, and IGF-I and -II are mitogenic, whereas activin and TGF-beta inhibit proliferation. IGF-II, activin, and TGF-beta also modulate ACTH-stimulated steroidogenesis. Human fetal adrenal cortical cells express IGF-II, bFGF and the activin/inhibin subunits, and the abundance of mRNAs for each of these factors is up-regulated by ACTH, suggesting that these growth factors are autocrine/paracrine mediators of ACTH action. Thus, although human adrenal development is primarily regulated by ACTH, its actions appear to be mediated/modulated by a cohort of locally expressed growth factors, the net effect of which results in the unique growth and steroidogenic activity of the human fetal adrenal cortex.

In this report, we describe abrupt onset of secondary amenorrhea in a woman with history of chronic systemic sarcoidosis. Endocrinologic evaluation of her hypothalamic-pituitary axis revealed abnormally low levels of follicle-stimulating hormone, luteinizing hormone, and insulinlike growth factor 1 and elevated prolactin. Urine osmolality was low, and serum osmolality was high. Magnetic resonance imaging revealed diffuse extensive leptomeningeal enhancement, with involvement of the hypothalamus, pituitary stalk, and the optic chiasm. Clinical diagnosis was consistent with neurosarcoidosis with hypothalamic-pituitary infiltration resulting in clinical hypogonadotropic hypogonadism, hyper-prolactinemia, and diabetes insipidus. In our report, we provide an overview of basic reproductive neuroendocrinology and discuss salient concepts of the pathogenesis, clinical manifestations, evaluation, and management of hypogonadotropic hypogonadism. The current literature on neurosarcoidosis with involvement of the hypothalamic-pituitary axis is summarized. The possibility of infiltrative process should be considered in patients with new diagnosis of hypogonadotropic hypogonadal amenorrhea.

The rapid growth of the primate fetal adrenal from midgestation until term is regulated by ACTH secreted by the fetal pituitary. Previous studies suggest that the trophic actions of ACTH are mediated by insulin-like growth factor II (IGF-II) synthesized by fetal adrenal cortical cells. To characterize further the role of IGF-II in the regulation of fetal adrenal growth, we investigated the expression of the messenger RNAs (mRNAs) encoding IGF-I, IGF-II, IGF-I receptor (IGF-IR) and IGF binding protein (IGFBP) 1-6 in the fetal rhesus monkey adrenal in vivo from 109 days of gestation until term (165 +/- 5 days) using in situ hybridization. To assess the role of ACTH in the regulation of expression of the IGF system in vivo, we administered metyrapone (3-7 days) to late gestation fetal rhesus monkeys (n = 4) in utero to increase fetal pituitary ACTH secretion. IGF-II mRNA was abundant in the definitive, transitional and fetal zones of the adrenal cortex from 109 days until term. IGF-IR mRNA was expressed in the definitive, transitional and fetal zones and decreased to nondetectable levels at term. IGFBP-2 and IGFBP-6 mRNAs were expressed in the definitive, transitional, and fetal zones, whereas IGFBP-1, -3, -4, and -5 were not detected in adrenal cells. The effects of increasing ACTH secretion on the growth of the specific zones of the adrenal were determined using morphometric techniques. Metyrapone treatment approximately doubled adrenal weight, which was due to an increase in the area of the definitive, transitional, and fetal zones with decreased cell density of the definitive, transitional, and fetal zones compared with controls and not due to a change in total cell number. Therefore, the increase in adrenal weight after metyrapone treatment was due to hypertrophy of the three cortical zones; there was no effect on adrenal medullary growth. The relative abundance of the mRNAs encoding IGF-II and the IGF-IR was increased after metyrapone treatment, whereas the localization and relative abundance of IGFBP 1-6 mRNAs were not altered by metyrapone treatment. We conclude that the ontogenetic increase in adrenal growth may be regulated, at least in part, by locally synthesized IGF-II, and the cessation of adrenal growth that occurs at term may be mediated by the decrease in the IGF-IR. The adrenal cortical expression of IGFBP-2 and IGFBP-6 suggests that these IGFBPs may modulate the IGF-IGF-IR interaction. Metyrapone treatment, which likely increased fetal pituitary ACTH secretion, causes a coordinated increase in expression of IGF-II and IGF-IR in fetal adrenal cortical cells, which may be an important mechanism of regulation of fetal adrenal cortical growth.

The long term efficacy and safety of transsphenoidal resection for preoperative diagnosis of PRL-secreting pituitary adenomas in a large series of women have not been described. Four hundred and nine consecutive women at this university tertiary referral center undergoing transsphenoidal resection for preoperative diagnosis of PRL-secreting pituitary adenoma were followed for a minimum of 4 yr. The objective was to determine the efficacy and morbidity of this procedure and to identify features correlating with the resolution of hyperprolactinemia. Outcome measures included referral, preoperative, surgical, postoperative hospitalization, and long-term follow-up information, including recent PRL concentration. Follow-up was ascertained in 83% of patients who were followed for a mean of 9.2 yr. Recurrence of hyperprolactinemia occurred in 47% of total patients, but in only 16% with a single surgical procedure, histological diagnosis of prolactinoma, and postoperative PRL concentration of 5 ng/mL or less. The best single predictor of cure was postoperative day 1 PRL concentration of 5 ng/mL or less. Eighty-eight percent of women desiring conception conceived within 1 yr. Glucocorticoid-dependent hypopituitarism occurred in 23% of patients undergoing postoperative radiotherapy. There was no operative mortality. Operative morbidity was low. Our experience demonstrates that women undergoing transsphenoidal surgery for diagnosis of PRL-secreting adenoma form a heterogeneous patient population. The best long term results are achieved in the pure prolactinoma group, for whom transsphenoidal resection is generally safe and effective.