Gene alterations identified by expression profiling in tumor-associated endothelial cells from invasive ovarian carcinoma.

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.

Metronomic chemotherapy enhances the efficacy of antivascular therapy in ovarian cancer.

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.

The periphery of the human fetal adrenal gland is a site of angiogenesis: zonal differential expression and regulation of angiogenic factors.

CONTEXT: Although the inner fetal zone (FZ) of the mid-gestation human fetal adrenal (HFA) produces dehydroepiandrosterone sulfate, the function of the outer definitive zone (DZ) remains less clear. We have proposed that the DZ phenotype is that of a pool of progenitor cells, many of which are mitotically active. Recently, we studied HFA expression of a family of vascular endothelial cell-specific angiogenic factors, the angiopoietins (Angs), and demonstrated that Ang2 was localized predominantly in the periphery of the gland. Ang1 stabilizes, whereas Ang2 destabilizes, vessels, increasing responsiveness to angiogenic stimuli such as vascular endothelial growth factor (VEGF)-A and fibroblast growth factor (FGF)-2. OBJECTIVE: Our objective was to test the hypothesis that the periphery of the HFA is a site of angiogenesis. DESIGN: Studies were conducted involving RNA, frozen sections, and primary cell cultures from midgestation HFAs. MAIN OUTCOME MEASURES: Immunofluorescence, laser capture microdissection, and real-time quantitative RT-PCR were used. RESULTS: Double immunostaining demonstrated that proliferating endothelial cells were limited to the DZ and DZ/FZ border. Ang2 mRNA was primarily expressed in the DZ, whereas Ang1 mRNA was primarily in the FZ. VEGF-A and FGF-2 mRNA levels were higher in the DZ. FGF-2 (10 ng/ml) induced Ang2 mRNA by 4-fold in both zones of cells (P < 0.01, at 24 h), but not Ang1 or VEGF-A mRNA. CONCLUSION: Data suggest that angiogenesis occurs at the periphery of the HFA. The DZ-predominant expression of Ang2 may be explained, in part, by the parallel pattern of FGF-2 expression.

A challenge for the 21st century: whither physician-scientists in obstetrics, gynecology, and the reproductive sciences?

As we begin a new century, research in obstetrics and gynecology and its subspecialties face a crisis. Federal support to academic departments of obstetrics and gynecology through the National Institutes of Health is distressingly low in relation to that for other major specialties. In addition, academic departments face a shortage of clinically trained investigators and physician-scientists who will respond to the challenge of contributing to a greater understanding of the reproductive sciences and to the amelioration of diseases of women.

Impact of vessel maturation on antiangiogenic therapy in ovarian cancer.

OBJECTIVE: The purpose of this study was to examine the functional and therapeutic significance of pericytes in ovarian cancer vasculature. STUDY DESIGN: Tumor vessel morphologic condition and efficacy of endothelial and pericyte targeting were examined with the use of in vivo ovarian cancer models. The expression of platelet-derived growth factor (PDGF) ligands and receptors was examined in endothelial, pericyte-like, and ovarian cancer cells. RESULTS: Relative to normal vessels, tumor vasculature was characterized by loosely attached pericytes in reduced density. PDGF-BB was expressed predominantly by the endothelial and cancer cells, whereas PDGFRbeta was present in pericyte-like cells. PDGF-BB significantly increased the migration of and VEGF production by pericyte-like cells; PDGFRbeta blockade abrogated these effects. Dual VEGF (VEGF-Trap) and PDGF-B (PDGF-Trap) targeted therapy was more effective in inhibiting in vivo tumor growth than either agent alone. CONCLUSION: Aberrations in the tumor microenvironment contribute to endothelial cell survival. Strategies that target both endothelial cells and pericytes should be considered for clinical trials.

Dual targeting of endothelial cells and pericytes in antivascular therapy for ovarian carcinoma.

PURPOSE: Pericytes are known to provide a survival advantage for endothelial cells. We hypothesize that strategies aimed at dual targeting of tumor-associated endothelial cells and pericytes will be highly efficacious. EXPERIMENTAL DESIGN: Paclitaxel-sensitive (HeyA8 and SKOV3ip1) or paclitaxel-resistant (HeyA8-MDR) orthotopic tumors in mice were examined for therapeutic efficacy by targeting the endothelial cells (using a vascular endothelial growth factor receptor inhibitor, AEE788) and pericytes (using STI571) alone or in combination. Additional therapy and survival studies in combination with paclitaxel were also done. Following therapy, tumors were examined for endothelial cell apoptosis, pericyte coverage, microvessel density, and proliferation. RESULTS: AEE788 inhibited tumor growth by 45% and 59% in the HeyA8 and SKOV3ip1 models, respectively, whereas STI571 alone was not effective. AEE788 plus STI571 resulted in 69% to 84% inhibition of tumor growth in both models. Moreover, combination of these agents with paclitaxel was even more effective, resulting in up to 98% inhibition of tumor growth. The triple combination was even effective in the HeyA8-MDR model. Remarkably, this triple combination also resulted in improved survival compared with all other groups (P<0.001) and caused regression of formed tumors. Pericyte coverage was significantly decreased in the STI571 treatment groups, and microvessel density was significantly reduced in the AEE788 treatment groups. AEE788 induced endothelial cell apoptosis, which was further enhanced by the addition of STI571. CONCLUSIONS: Strategies targeting both endothelial cells and pericytes are highly effective for in vivo treatment of ovarian carcinoma. This antiangiogenic effect may be partially due to decreased pericyte coverage, thus increasing the sensitivity of tumor vasculature to therapy. These encouraging data support the development of clinical trials based on this strategy.

Selective estrogen receptor modulators: an update on recent clinical findings.

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.

Multiple transcription factor elements collaborate with estrogen receptor alpha to activate an inducible estrogen response element in the NKG2E gene.

Estrogen receptors (ERs) regulate transcription by interacting with regulatory elements in target genes. However, known ER regulatory elements cannot explain the expression profiles of genes activated by estradiol (E2) and selective estrogen receptor modulators (SERMs). We previously showed that the killer cell lectin-like receptor (NKG2E) gene is regulated by E2, tamoxifen, and raloxifene. Here we used the NKG2E gene as a model to investigate the mechanism whereby target genes are regulated by E2 and SERMs with ERalpha. The ER regulatory element in the NKG2E promoter was mapped to the -1825 and -1686 region. Full activation of the NKG2E promoter required the collaboration between a transcription factor cluster containing c-jun, heat-shock factor 2, and CCAAT/enhancer-binding protein beta and a unique variant estrogen response element (ERE) that has only a two nucleotide spacer between half sites. The cluster elements and the variant ERE were inactive on their own, but the regulation by E2 and SERMs was restored when the c-jun, heat-shock factor-2, and CCAAT/enhancer-binding protein beta cluster was placed upstream of the variant ERE. The activation of the NKG2E gene by E2 and selective ER modulators was associated with the recruitment of the p160 coactivators glucocorticoid receptor-interacting protein 1 and amplified in breast cancer 1 but not steroid receptor coactivator 1. These studies identified one of the most complex ER regulatory units thus far reported and demonstrate that a cluster of flanking transcription factors collaborate with ER to induce a functional ERE in the NKG2E promoter.

Adrenocorticotropin preferentially up-regulates angiopoietin 2 in the human fetal adrenal gland: implications for coordinated adrenal organ growth and angiogenesis.

CONTEXT: ACTH is the key tropic hormone for the human fetal adrenal (HFA). Because vascular development must be coordinated with organ growth, ACTH may regulate local angiogenic factors, thereby influencing HFA angiogenesis. We previously demonstrated that ACTH up-regulates vascular endothelial growth factor in HFA cortical cells. A newer angiogenic factor family, the angiopoietins (Angs), also plays critical roles. Ang1 stabilizes, whereas Ang2 destabilizes vessels, increasing responsiveness to angiogenic stimuli. OBJECTIVE: The objective of this study was to investigate expression and ACTH regulation of Angs and their receptor Tie2 in the HFA. DESIGN, SETTING, AND PATIENTS: Studies were conducted involving RNA, frozen sections, and primary cell cultures from HFAs (14-24 wk) and human adult adrenal RNA. MAIN OUTCOME MEASURES: Angs and Tie2 mRNA levels were determined by real-time quantitative RT-PCR, Ang2 and Tie2 were localized by immunostaining, and ACTH regulation of Angs was investigated by real-time quantitative RT-PCR, Western blot, and ELISA. RESULTS: Mean HFA Ang2 to Ang1 mRNA ratio was 6.3-fold higher than adult adrenals (P < 0.001). Ang2 was localized predominantly in the HFA periphery, whereas Tie2 demonstrated endothelial localization. In isolated HFA cortical cells, ACTH up-regulated Ang mRNA levels in a time- and dose-dependent manner, with the balance favoring Ang2. Ang2 protein levels were elevated in ACTH-stimulated HFA cortical cells and conditioned medium. 8-Bromoadenosine cAMP and forskolin mimicked ACTH effects on the Angs. CONCLUSIONS: Higher HFA Ang2 to Ang1 ratios may reflect greater vascular remodeling than in the adult. Angs, particularly Ang2, in concert with vascular endothelial growth factor, may mediate ACTH tropic action, ensuring coordination of HFA growth, steroidogenesis, and angiogenesis.

Midkine, a heparin-binding growth factor, selectively stimulates proliferation of definitive zone cells of the human fetal adrenal gland.

CONTEXT: In the human fetal adrenal gland (HFA), the inner fetal zone (FZ) secretes dehydroepiandrosterone sulfate. The function of the outer definitive zone (DZ) is less clear; however, the DZ phenotype is that of a reservoir of progenitor cells, many of which are mitotically active. Midkine (MK) is a heparin-binding growth factor with various bioactivities. OBJECTIVE: The objective of this study was to investigate expression, proliferative effects, and ACTH regulation of MK in the HFA. DESIGN AND SETTING: RNA, cryosections, and primary cell cultures from HFAs (14-24 wk) and adult adrenal RNA were used. MAIN OUTCOME MEASURES: The main outcome measures were MK mRNA levels (measured by quantitative real-time RT-PCR); MK localization (measured by immunostaining); MK proliferative effects and mechanism (measured by proliferation assays, flow cytometry, pharmacological interventions); and ACTH regulation (measured by quantitative real-time RT-PCR). RESULTS: HFA MK mRNA levels were 4-fold higher than in adult adrenals (P < 0.05) and were comparable to levels in fetal and adult brains (positive controls). MK immunoreactivity was abundant throughout the HFA. Exogenous MK caused proliferation of isolated DZ cells but not FZ cells (72 h, P < 0.05). In contrast, basic fibroblast growth factor induced proliferation of cells from both zones. Pharmacological interventions indicated that MK-induced DZ cell proliferation may be mediated by phosphatidylinositol 3-kinase, MAPK kinase, and Src family kinases. ACTH (1 nm) increased MK mRNA by 3.5-fold (48 h, P < 0.01) in isolated FZ cells. CONCLUSIONS: MK likely plays a key role in HFA development. MK's selective in vitro mitotic effects on DZ cells may provide insights into the mechanism underlying the distinct in vivo differences in mitotic activity between the DZ and FZ.

Differential zonal expression and adrenocorticotropin regulation of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein, in the midgestation human fetal adrenal gland: implications for adrenal development.

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.

Paracrine VEGF/VE-cadherin action on ovarian cancer permeability.

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.

Phosphatidylinositol 3-kinase mediates angiogenesis and vascular permeability associated with ovarian carcinoma.

PURPOSE: To assess the role of phosphatidylinositol-3 kinase (PI3K) inhibition in vascular permeability, angiogenesis, and vascular remodeling in tumor vessels and peritoneal lining in an athymic mouse model of i.p. human ovarian carcinoma. EXPERIMENTAL DESIGN: Mice were inoculated i.p. with cells from the human ovarian cancer cell line, OVCAR-3. Fourteen days after inoculation, mice were treated with or without the PI3K inhibitor LY294002, 3 days weekly for 4 weeks. At the end of the experiment, some mice were anesthetized and injected via the tail vein with FITC-labeled lycopersicon lectin and perfused through the aorta before sacrifice. The peritoneal wall and tumor from all mice were removed and embedded in 10% agarose. Tumor sections were visualized by fluorescence microscopy. RESULTS: Ascites in the LY294002-treated group (0.69 +/- 0.27 mL) was reduced by 72.4% compared with the control group (2.5 +/- 1.2 mL). Tumor burden in the LY294002-treated group (0.62 +/- 0.32 g) was reduced by 47.3% compared with the control group (1.18 +/- 0.41 g). LY294002 inhibited peritoneal and tumor vascularization resulting in numerous leaky, irregular, tortuous vessels in scant, straight, relatively impermeable vessels. CONCLUSIONS: The data indicate that LY294002 inhibits ascites formation in our mouse model of human ovarian cancer by inhibiting tumor and peritoneal neovascularization as well as vascular permeability. The data also show that LY294002 directly inhibits vascular endothelial growth factor (VEGF) protein expression and release from ovarian carcinoma and suggest that LY294002 blocks the VEGF signaling pathway involved in angiogenesis and vascular permeability.

Vascular endothelial growth factor trap combined with paclitaxel strikingly inhibits tumor and ascites, prolonging survival in a human ovarian cancer model.

Ovarian cancer is characterized by i.p. carcinomatosis and massive ascites. Vascular endothelial growth factor (VEGF) plays a pivotal role in tumor angiogenesis and vascular leakage leading to ascites. We assessed the efficacy of a soluble decoy receptor (VEGF Trap) combined with paclitaxel, in a mouse model of human ovarian cancer. Tumor burden after VEGF Trap plus paclitaxel was reduced by approximately 98% versus controls. No measurable ascites developed in the treated group. Morphologic studies showed that most residual tumor had degenerative changes. Diaphragmatic and hepatic tumors were not found in the VEGF Trap plus paclitaxel group in contrast to controls, indicating lack of metastasis. In vivo FITC-lectin tumor vessel imaging showed sparse, short, straight vessels in treated mice as compared to controls, in which vessels were numerous, irregular, tortuous, and leaky. In a survival study, all controls underwent euthanasia between 29 and 58 days after tumor cell inoculation (cachexia, extensive ascites, and tumor masses). In the VEGF Trap plus paclitaxel group, mice were ambulating and eating normally with no signs of disease for at least 81 days after tumor cell inoculation, and survival occurred for 129.9 +/- 38.88 days with no further treatment. We conclude that combination therapy with VEGF Trap plus paclitaxel may provide a novel, long-lasting therapeutic strategy for treatment of patients with ovarian cancer associated with ascites.

Inhibition of phosphatidylinositol 3'-kinase increases efficacy of paclitaxel in in vitro and in vivo ovarian cancer models.

Phosphatidylinositol 3'-kinase (PI3k) is implicated in a wide array of biological and pathophysiological responses. Thus, inhibiting molecules involved in its signal transduction pathway is a possible means of treating cancer. Our previous studies demonstrated that LY294002, a potent and selective PI3k inhibitor, decreases growth of ovarian carcinoma and ascites formation in an athymic mouse xenogeneic transplant model of ovarian cancer. However, the dose of LY294002 used to decrease tumor growth resulted in significant dermatological toxicity. We demonstrate herein that introduction of an active catalytic subunit of PI3k into an ovarian cancer cell line, and thus activation of the PI3k/AKT pathway, confers resistance to the effects of paclitaxel, one of the major drugs used in ovarian cancer therapy. The resistance to paclitaxel can be reversed in vitro by inhibition of PI3k. Therefore, we evaluated whether combined therapy with paclitaxel and LY294002 would result in increased efficacy and allow utilization of doses of LY294002 that do not induce dermatological toxicity. Two weeks after i.p. inoculation with OVCAR-3 ovarian cancer cells, mice were treated i.p. with LY294002 plus paclitaxel, each three times weekly on alternate days, for 4 weeks. Tumor burden in the LY294002 + paclitaxel, LY 294002 alone, and paclitaxel alone groups was reduced by 80% (P < 0.01), 38% (P < 0.05), and 51% (P < 0.05), respectively, compared with controls. Virtually no ascites developed in the combined treatment group; mean volume of ascites in the controls was 3.7 ml. Treatment with LY294002 alone reduced ascites by 70% (P < 0.01), whereas paclitaxel alone reduced ascites slightly but not significantly. No dermatological lesions or weight loss were observed in any treatment group. In vivo and in vitro morphological studies demonstrated that inhibition of PI3k enhanced paclitaxel-induced apoptosis in the human ovarian cancers. Our data suggest that a combination of a PI3k inhibitor and conventional chemotherapy may provide an effective approach to inhibiting tumor growth and ascites production in ovarian cancer with acceptable side effects.

Dll4 Inhibition plus Aflibercept Markedly Reduces Ovarian Tumor Growth.

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.

Management of postmenopausal hot flushes with venlafaxine hydrochloride: a randomized, controlled trial.

OBJECTIVE: To examine the efficacy of extended-release venlafaxine for the treatment of postmenopausal hot flushes. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Vascular endothelial growth factor-trap decreases tumor burden, inhibits ascites, and causes dramatic vascular remodeling in an ovarian cancer model.

Ovarian cancer is the most lethal gynecological malignancy and the fifth most common cause of cancer in women. It is characterized by diffuse peritoneal carcinomatosis and often by large volumes of i.p. ascites. Because vascular endothelial growth factor (VEGF), also known as vascular permeability factor, increases vascular permeability and stimulates endothelial cell growth, its role in ovarian cancer has been evaluated in a number of studies. However, questions remain regarding the ability of VEGF alone to cause ascites formation and the ability of VEGF blockade to inhibit the growth of disseminated cancer. We have used retroviral technology to create cell populations that overproduce VEGF and report that enforced expression of VEGF by ovarian carcinoma cells dramatically reduces the time to onset of ascites formation. In fact, even tumor-free peritoneal overexpression of VEGF, created by using adenoviral vectors, is sufficient to cause ascites to accumulate. We have found that systemic administration of the VEGF-Trap, a recently described high-affinity soluble decoy receptor for VEGF, prevents ascites accumulation and also inhibits the growth of disseminated cancer. Remarkably, much as is observed in s.c. tumor models, VEGF blockade results in dramatic remodeling of the blood vessels in disseminated ovarian carcinoma. The potent effects of the VEGF-Trap in reducing both ascites and tumor burden suggest that it will be of value in a regimen for treatment of women with ovarian cancer and ascites.

Identification of definitive and fetal zone markers in the human fetal adrenal gland reveals putative developmental genes.

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.

Human placental vascular development: vasculogenic and angiogenic (branching and nonbranching) transformation is regulated by vascular endothelial growth factor-A, angiopoietin-1, and angiopoietin-2.

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.