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      Effect of treatment with angiopoietin-2 and vascular endothelial growth factor on the quality of xenografted bovine ovarian tissue in mice

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          Abstract

          Cryopreservation and transplantation of ovarian tissue (OT) represents a method for fertility preservation. However, as the transplantation is performed without vessel anastomosis, unavoidable ischemic damage occurs. To reduce this ischemic damage and improve outcomes after transplantation, we used two kind of angiogenic factors, angiopoietin-2 (ang-2) and vascular endothelial growth factor (VEGF). Fresh or vitrified-warmed bovine OTs were prepared for xenotransplantation (XT). Fresh OTs were immediately xenografted into nude mice (XT-Fresh). Vitrified-warmed OTs were xenografted into four subgroups of mice, which were injected intraperitoneally before XT with saline (XT-Vitri), Ang-2 (XT-Ang-2), VEGF (XT-VEGF), and a combination of Ang-2 and VEGF (XT-Combined). Seven or 28 days post-grafting, grafted OTs and blood samples were collected for evaluation. Follicle normality was higher in the angiogenic factor-treated groups than in the XT-Vitri group. The XT-VEGF and the XT-Combined showed higher (P<0.05) follicular density than the XT-Vitri group. The highest apoptotic follicle ratio was observed in the XT-Vitri group on day 7; this was decreased (P<0.05) in the XT-Combined group. Microvessel densities were higher in the angiogenic factor-treated groups than in the XT-Vitri group. The largest fibrotic area was showed in the XT-Vitri group on day 28, and it was decreased (P<0.05) in the XT-combined group. Based on these results, administration of Ang-2 and VEGF to recipients prior to XT appeared to alleviate ischemic damage by enhancing angiogenesis, which resulted in the maintenance of follicle integrity and density, and reduced follicle apoptosis and OT fibrosis.

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          Angiopoietin-2 sensitizes endothelial cells to TNF-alpha and has a crucial role in the induction of inflammation.

          The angiopoietins Ang-1 and Ang-2 have been identified as ligands of the receptor tyrosine kinase Tie-2 (refs. 1,2). Paracrine Ang-1-mediated activation of Tie-2 acts as a regulator of vessel maturation and vascular quiescence. In turn, the antagonistic ligand Ang-2 acts by an autocrine mechanism and is stored in endothelial Weibel-Palade bodies from where it can be rapidly released upon stimulation. The rapid release of Ang-2 implies functions of the angiopoietin-Tie system beyond its established role during vascular morphogenesis as a regulator of rapid vascular responses. Here we show that mice deficient in Ang-2 (encoded by the gene Angpt2) cannot elicit an inflammatory response in thioglycollate-induced or Staphylococcus aureus-induced peritonitis, or in the dorsal skinfold chamber model. Recombinant Ang-2 restores the inflammation defect in Angpt2(-/-) mice. Intravital microscopy showed normal TNF-alpha-induced leukocyte rolling in the vasculature of Angpt2(-/-)mice, but rolling cells did not firmly adhere to activated endothelium. Cellular experiments showed that Ang-2 promotes adhesion by sensitizing endothelial cells toward TNF-alpha and modulating TNF-alpha-induced expression of endothelial cell adhesion molecules. Together, these findings identify Ang-2 as an autocrine regulator of endothelial cell inflammatory responses. Ang-2 thereby acts as a switch of vascular responsiveness exerting a permissive role for the activities of proinflammatory cytokines.
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            VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells.

            Vascular endothelial growth factor (VEGF) has been shown to promote neovascularization in animal models and, more recently, in human subjects. This feature has been assumed to result exclusively from its direct effects on fully differentiated endothelial cells, i.e. angiogenesis. Given its regulatory role in both angiogenesis and vasculogenesis during fetal development, we investigated the hypothesis that VEGF may modulate endothelial progenitor cell (EPC) kinetics for postnatal neovascularization. Indeed, we observed an increase in circulating EPCs following VEGF administration in vivo. VEGF-induced mobilization of bone marrow-derived EPCs resulted in increased differentiated EPCs in vitro and augmented corneal neovascularization in vivo. These findings thus establish a novel role for VEGF in postnatal neovascularization which complements its known impact on angiogenesis.
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              Restoration of ovarian activity and pregnancy after transplantation of cryopreserved ovarian tissue: a review of 60 cases of reimplantation.

              Aggressive chemotherapy/radiotherapy and bone marrow transplantation can cure >90% of girls and young women affected by disorders requiring such treatment. However, the ovaries are very sensitive to cytotoxic drugs, especially to alkylating agents. Several options are currently available to preserve fertility in cancer patients. The present review reports the results of 60 orthotopic reimplantations of cryopreserved ovarian tissue performed by three teams, as well as 24 live births reported in the literature to date. Restoration of ovarian activity occurred in almost all cases in the three series. Among the 60 patients, eleven conceived and six of those had already delivered twelve healthy babies. In the future, we are looking to: 1) improve freezing techniques; and 2) enhance the "vascular bed" before reimplantation to increase pregnancy rates. On the other hand, cryopreservation of ovarian tissue may be combined with removal, via puncture, of small antral follicles, making it possible to freeze both ovarian tissue and isolated immature oocytes. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ResourcesRole: SoftwareRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: ValidationRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: Project administrationRole: SupervisionRole: ValidationRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Funding acquisitionRole: Project administrationRole: SupervisionRole: ValidationRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: ValidationRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                15 September 2017
                2017
                : 12
                : 9
                : e0184546
                Affiliations
                [1 ] Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, Gumi-dong, Bundang-gu, Seongnam, Korea
                [2 ] Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul, Korea
                [3 ] Department of Obstetrics and Gynecology, Seoul National University Hospital, Seoul, Korea
                Faculty of Animal Sciences and Food Engineering, University of São Paulo, BRAZIL
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                [¤]

                Current address: Department of Obstetrics and Gynecology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America

                Author information
                http://orcid.org/0000-0003-3743-2934
                Article
                PONE-D-16-48318
                10.1371/journal.pone.0184546
                5600380
                28915249
                04e5ccb8-0051-4ea5-b0fd-71afd1e63607
                © 2017 Kong et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 7 December 2016
                : 26 August 2017
                Page count
                Figures: 4, Tables: 2, Pages: 15
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100003625, Ministry of Health and Welfare;
                Award ID: HI12C0055
                Funded by: Seoul National University Bundang Hospital Research Fund
                Award ID: 02-2014-013
                Funded by: National Research Foundation of Korea (KR)
                Award ID: NRF-2017R1C1B2003897
                This work was supported by the Korea Healthcare Technology Research and Development Project, Ministry of Health and Welfare, Republic of Korea [grant number HI12C0055], National Research Foundation of Korea [grant number NRF-2017R1C1B2003897] and Seoul National University Bundang Hospital Research Fund [grant number 02-2014-013]. Ministry of Health and Welfare: http://www.mohw.go.kr/eng/index.jsp, Seoul National University Bundang Hospital Research Fund: https://www.snubh.org/dh/en/, National Research Foundation of Korea: http://www.nrf.re.kr/eng/main. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Cell Biology
                Cell Processes
                Cell Death
                Apoptosis
                Research and Analysis Methods
                Experimental Organism Systems
                Model Organisms
                Mouse Models
                Research and Analysis Methods
                Model Organisms
                Mouse Models
                Research and Analysis Methods
                Experimental Organism Systems
                Animal Models
                Mouse Models
                Biology and Life Sciences
                Physiology
                Cardiovascular Physiology
                Vasculogenesis
                Medicine and Health Sciences
                Physiology
                Cardiovascular Physiology
                Vasculogenesis
                Biology and Life Sciences
                Developmental Biology
                Morphogenesis
                Vasculogenesis
                Biology and Life Sciences
                Developmental Biology
                Fibrosis
                Biology and Life Sciences
                Cryobiology
                Cryopreservation
                Research and Analysis Methods
                Specimen Preparation and Treatment
                Specimen Preservation
                Cryopreservation
                Biology and Life Sciences
                Physiology
                Cardiovascular Physiology
                Angiogenesis
                Medicine and Health Sciences
                Physiology
                Cardiovascular Physiology
                Angiogenesis
                Biology and Life Sciences
                Developmental Biology
                Angiogenesis
                Biology and Life Sciences
                Anatomy
                Reproductive System
                Ovaries
                Medicine and Health Sciences
                Anatomy
                Reproductive System
                Ovaries
                Biology and Life Sciences
                Biochemistry
                Hormones
                Lipid Hormones
                Estradiol
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