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Generation of an anti-angiogenic endothelial progenitor cell line via endostatin gene transfer

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      Abstract

      The viability of endothelial progenitor cells (EPCs) as a therapeutic treatment for neovascularization (NV) was subject to investigation in the present study. Furthermore, endostatin has previously been demonstrated to be an inhibitor of angiogenesis and a suppressant of vascular leakage. The aim of the present study was to generate transgenic EPCs with anti-angiogenic effects for the treatment of ocular NV. EPCs were obtained from rat peripheral blood samples and then verified. A lentiviral-endostatin-green fluorescent protein recombinant construct was generated and used to infect EPCs. Transfected cells were then subjected to puromycin selection. Reverse transcription-quantitative polymerase chain reaction and a western blot assay were then applied in order to determine both the endostatin mRNA and protein expression levels, respectively. In addition, vascular endothelial growth factor (VEGF) expression levels were also detected in order to observe the anti-angiogenic effect of the endostatin-transfected EPCs. Following puromycin (1 µg/ml) selection for 4 days, a stable endostatin-transfected EPC line was generated. In this stable endostatin-transfected EPC line, the expression levels of endostatin increased; whereas the expression levels of VEGF decreased. The results of the present study revealed that EPCs can be genetically modified to overexpress endostatin, which may provide the cells with an anti-angiogenic effect via increased expression of endostatin and decreased expression of VEGF. Thus, EPCs genetically modified to overexpress endostatin may serve as a potential therapeutic agent for ocular NV treatment.

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      Most cited references 36

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      Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.

       K Livak,  T Schmittgen (2001)
      The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data. Copyright 2001 Elsevier Science (USA).
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        Isolation of putative progenitor endothelial cells for angiogenesis.

        Putative endothelial cell (EC) progenitors or angioblasts were isolated from human peripheral blood by magnetic bead selection on the basis of cell surface antigen expression. In vitro, these cells differentiated into ECs. In animal models of ischemia, heterologous, homologous, and autologous EC progenitors incorporated into sites of active angiogenesis. These findings suggest that EC progenitors may be useful for augmenting collateral vessel growth to ischemic tissues (therapeutic angiogenesis) and for delivering anti- or pro-angiogenic agents, respectively, to sites of pathologic or utilitarian angiogenesis.
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          Endostatin: an endogenous inhibitor of angiogenesis and tumor growth.

           Y Shing,  N Fukai,  G Vasios (1997)
          We previously identified the angiogenesis inhibitor angiostatin. Using a similar strategy, we have identified endostatin, an angiogenesis inhibitor produced by hemangioendothelioma. Endostatin is a 20 kDa C-terminal fragment of collagen XVIII. Endostatin specifically inhibits endothelial proliferation and potently inhibits angiogenesis and tumor growth. By a novel method of sustained release, E. coli-derived endostatin was administered as a nonrefolded suspension. Primary tumors were regressed to dormant microscopic lesions. Immunohistochemistry revealed blocked angiogenesis accompanied by high proliferation balanced by apoptosis in tumor cells. There was no toxicity. Together with angiostatin data, these findings validate a strategy for identifying endogenous angiogenesis inhibitors, suggest a theme of fragments of proteins as angiogenesis inhibitors, and demonstrate dormancy therapy.
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            Author and article information

            Affiliations
            [1 ]Eye Centre, The Second Affiliated Hospital of The School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310009, P.R. China
            [2 ]Key Laboratory of Combined Multi-Organ Transplantation, Ministry of Public Health, The First Affiliated Hospital of The School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, P.R. China
            Author notes
            Correspondence to: Professor Ke Yao, Eye Centre, The Second Affiliated Hospital of The School of Medicine, Zhejiang University, 88 Jiefang Road, Hangzhou, Zhejiang 310009, P.R. China, E-mail: xlren@ 123456zju.edu.cn
            [*]

            Contributed equally

            Journal
            Mol Med Rep
            Mol Med Rep
            Molecular Medicine Reports
            D.A. Spandidos
            1791-2997
            1791-3004
            April 2018
            21 February 2018
            21 February 2018
            : 17
            : 4
            : 5814-5820
            29484399 5866025 10.3892/mmr.2018.8623 mmr-17-04-5814
            Copyright: © Ai et al.

            This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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