Robert B. Jaffe

Ovarian steroid regulation of vascular endothelial growth factor in the human endometrium: implications for angiogenesis during the menstrual cycle and in the pathogenesis of endometriosis.

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

Role of Vascular Endothelial Growth Factor in Ovarian Cancer: Inhibition of Ascites Formation by Immunoneutralization

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.

Role of vascular endothelial growth factor in ovarian physiology and pathology

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.

Lysophosphatidic acid is a bioactive mediator in ovarian cancer

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 Gi, Gq and G12/13 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.

Regulation of Tumor Angiogenesis by EZH2

Although VEGF-targeted therapies are showing promise, new angiogenesis targets are needed to make additional gains. Here, we show that increased Zeste homolog 2 (EZH2) expression in either tumor cells or in tumor vasculature is predictive of poor clinical outcome. The increase in endothelial EZH2 is a direct result of VEGF stimulation by a paracrine circuit that promotes angiogenesis by methylating and silencing vasohibin1 (vash1). Ezh2 silencing in the tumor-associated endothelial cells inhibited angiogenesis mediated by reactivation of VASH1, and reduced ovarian cancer growth, which is further enhanced in combination with ezh2 silencing in tumor cells. Collectively, these data support the potential for targeting ezh2 as an important therapeutic approach.

Lysophospholipid Growth Factors in the Initiation, Progression, Metastases, and Management of Ovarian Cancer

Abstract: Levels of lysophosphatidic acid (LPA) and lysophosphatidylcholine (LPC) are elevated in the plasma and ascites of ovarian cancer patients, but not in most other tumor types. LPA increases cell proliferation, cell survival, resistance to cisplatin, cell shrinkage, and production of vascular endothelial growth factor, urokinase plasminogen activator, and LPA itself in ovarian cancer cells, but not in normal ovarian surface epithelial cells. PSP24 and members of the endothelial differentiation gene (EDG) family (EDG1, EDG2, EDG4, and EDG7) of G protein‐coupled receptors mediate LPA signaling. Ovarian cancer cell lines do not express EDG1 mRNA, have variable EDG2 mRNA and protein levels, and frequently exhibit elevated levels of EDG4 mRNA and protein, suggesting that EDG4 may contribute to the deleterious effects of LPA in ovarian cancer. In contrast, activation of the EDG2 LPA receptor on ovarian cancer cells may lead to apoptosis and counter the effects of other LPA receptors. Thus, the development of agonists and antagonists for the appropriate spectrum of LPA receptors may alter proliferation, apoptosis, or response to therapy of ovarian cancer cells. Indeed, over 60% of all current drugs target the G protein‐coupled family of receptors, making the LPA receptor family a “drugable” target. LPC, although not as thoroughly studied, increases cellular proliferation and mediates multiple other functions through unique signaling pathways.

Vascular endothelial growth factor immunoneutralization plus Paclitaxel markedly reduces tumor burden and ascites in athymic mouse model of ovarian cancer.

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.

Development and Function of the Human Fetal Adrenal Cortex: A Key Component in the Feto-Placental Unit

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.

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.

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.