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Irusta G.,CONICET | Maidana C.P.,CONICET | Abramovich D.,CONICET | De Zuniga I.,Centro Medico PREGNA Medicina reproductiva | And 3 more authors.
Biology of Reproduction

Ovarian granulosa cell tumors (GCTs) represent 3%-5% of all ovarian malignancies. Treatments have limited proven efficacy and biologically targeted treatment is lacking. The aim of this study was to investigate the role of Notch signaling in the proliferation, steroidogenesis, apoptosis, and phosphatidylinositol 3-kinase (PI3K)/AKT pathway in a FOXL2-mutated granulosa tumor cell line (KGN) representative of the adult form of GCTs. When Notch signaling is initiated, the receptors expose a cleavage site in the extracellular domain to the metalloproteinase TACE and, following this cleavage, Notch undergoes another cleavage mediated by the presenilin-gamma-secretase complex. To achieve our goal, DAPT, an inhibitor of the gammasecretase complex, was used to investigate the role of the Notch system in parameters associated with cell growth and death, using a human granulosa cell tumor line (KGN) as an experimental model. We observed that JAGGED1, DLL4, NOTCH1, and NOTCH4 were highly expressed in KGN cells as compared to granulosa-lutein cells obtained from assisted reproductive techniques patients. The proliferation and viability of KGN cells, as well as progesterone and estradiol production, decreased in the presence of 20 μM DAPT. Apoptotic parameters like PARP and caspase 8 cleavages, BAX, and BCLXs increased in KGN cells cultured with DAPT, whereas others such as BCL2, BCLXl, FAS, and FAS ligand did not change. AKT phosphorylation decreased and PTEN protein increased when Notch signaling was inhibited in KGN cells. We conclude that the Notch system acts as a survival pathway in KGN cells, and might be interacting with the PI3K/AKT pathway. © 2013 by the Society for the Study of Reproduction, Inc. Source

Scotti L.,CONICET | Abramovich D.,CONICET | Pascuali N.,CONICET | Durand L.H.,CONICET | And 5 more authors.
Reproduction, Fertility and Development

Ovarian hyperstimulation syndrome (OHSS) is a complication of ovarian stimulation with gonadotrophins following human chorionic gonadotrophin (hCG) administration. The relationship between hCG and OHSS is partly mediated via the production of angiogenic factors, such as vascular endothelial growth factor A (VEGFA) and angiopoietins (ANGPTs). Here, we investigated the effect of ANGPT1 inhibition on ovarian angiogenesis in follicular fluid (FF) from women at risk of OHSS, using the chorioallantoic membrane (CAM) of quail embryos as an experimental model. We also analysed cytoskeletal changes and endothelial junction protein expression induced by this FF in the presence or absence of an ANGPT1-neutralising antibody in endothelial cell cultures. The presence of this antibody restored the number of vascular branch points and integrin αvβ3 levels in the CAMs to control values. ANGPT1 inhibition in FF from OHSS patients also restored the levels of claudin-5, vascular endothelial cadherin and phosphorylated β-catenin and partially reversed actin redistribution in endothelial cells. Our findings suggest that ANGPT1 increases pathophysiological angiogenesis in patients at risk of OHSS by acting on tight and adherens junction proteins. Elucidating the mechanisms by which ANGPT1 regulates vascular development and cell-cell junctions in OHSS will contribute to identifying new therapeutic targets for the treatment of human diseases with aberrant vascular leakage. © CSIRO 2016. Source

Scotti L.,CONICET | Abramovich D.,CONICET | Pascuali N.,CONICET | de Zuniga I.,Centro Medico PREGNA Medicina reproductiva | And 7 more authors.
Molecular and Cellular Endocrinology

Ovarian hyperstimulation syndrome (OHSS) is a disorder associated with ovarian stimulation. OHSS features are ovarian enlargement with fluid shifting to the third space. Disturbances in the vasculature are considered the main changes that lead to OHSS. Our aim was to analyze the levels of angiopoietins 1 and 2 (ANGPT1 and 2) and their soluble and membrane receptors (s/mTie-2) in follicular fluid (FF) and in granulosa-lutein cells culture (GLCs) from women at risk of developing OHSS. We also evaluated the effect of ANGPT1 on endothelial cell migration. In ovaries from an OHSS rat model, we analyzed the protein concentration of ANGPTs, their mTie-2 receptor, and platelet-derived growth factor PDGF-B, -D and PDGFR-β. ANGPT1 levels were increased in both FF and GLCs from women at risk of OHSS. Incubation of these FF with an ANGPT1 neutralizing antibody decreased endothelial cell migration. In the ovaries of OHSS rat model, mTie-2 protein levels increased and PDGF-B and -D decreased. In summary, these results suggest that ANGPT1 could be another mediator in the development of OHSS. © 2012 Elsevier Ireland Ltd. Source

Scotti L.,CONICET | Parborell F.,CONICET | Irusta G.,CONICET | De Zuniga I.,Centro Medico PREGNA Medicina reproductiva | And 5 more authors.
Molecular Reproduction and Development

SUMMARY: Polycystic ovary syndrome (PCOS) is the most common endocrinological pathology among women of reproductive age, and is characterized by abnormalities in ovarian angiogenesis, among other features. Consistent with this association, follicular fluid (FF) concentration and ovarian expression of vascular endothelial growth factor (VEGF) are increased in PCOS patients. In this study, we examined the protein levels of platelet-derived growth factor (PDGF) BB and DD (PDGFBB and PDGFDD), angiopoietin 1 and 2 (ANGPT1 and ANGPT2), and their soluble receptor sTIE2 in FF from PCOS and control patients undergoing assisted reproductive techniques. We also analyzed the effect of FF from PCOS and control patients on tight and adherens junction protein expression in an endothelial cell line. PDGFBB and PDGFDD were significantly lower whereas ANGPT1 concentration was significantly higher in FF from PCOS patients than from control patients. No changes were found in the concentration of ANGPT2 or sTIE2. Expression of claudin-5 was significantly increased in endothelial cells incubated for 24hr in the presence of FF from PCOS versus from control patients, while vascular-endothelial cadherin, β-catenin, and zonula occludens 1 expression were unchanged. The changes observed in the levels of PDGF isoforms and ANGPT1 may prevent VEGF-induced vascular permeability in the PCOS ovary by regulating endothelial-cell-junction protein levels. Restoring the levels of angiogenic factors may provide new insights into PCOS treatment and the prevention of ovarian hyperstimulation syndrome in affected women. Mol. Reprod. Dev. 81: 748-756, 2014. © 2014 Wiley Periodicals, Inc. Source

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