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Sample records for recombinant vegf bioconjugates

  1. Establishment of a recombinant adeno-associated virus expressing hVEGF165

    Institute of Scientific and Technical Information of China (English)

    Xianghui Huang; Zhibin Shi; Xiaoqian Dang; Chen Zhang; Pengbo Yu; Kunzheng Wang

    2008-01-01

    BACKGROUND: Because certain gene vectors could have deleterious effects in the central nervous system, the choice of a safe and effective vector system has become more important for gene therapy of nerve regeneration. OBJECTIVE: To construct a non-pathogenic, recombinant adeno-associated virus (AAV) simultaneously expressing human vascular endothelial growth factor 165 (hVEGF165) and green fluorescent protein (GFP). DESIGN, TIME AND SETTING: A randomized controlled experiment was performed at the Virology Laboratory of Shaanxi Provincial Center for Disease Control and Prevention between March and September 2007. MATERIALS: AAV helper-free system, AAV-293 packaging cell line, and AAV HT-1080 cells were purchased from Stratagene, USA. E. Coli DH5α was a stocked strain from Centers for Disease Control and Prevention of Shaanxi, China. Plasmid pUC18-hHVEGF165 was a gift from Zhibin Shi. METHODS: The hVEGF165 gene was amplified by PCR from pUC18-hHVEGF165 and inserted into plasmid pAAV-IRES-hrGFP to construct recombinant plasmid pAAV-hVEGF165-IRES-hrGFP. Subsequently pAAV-hVEGF165-IRES-hrGFP, pAAV-RC (the rep/cap-gene containing plasmid), and pHelper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hrGFP packaging through homologous recombination. The efficiency of AAV packaging was monitored under a fluorescent microscope, and the recombinant viral particles were harvested from infected AAV-293 cells, and further concentrated and purified. AAV HT-1080 cells were infected with the recombinant virus AAV-hVEGF165-IRES-hrGFP. MAIN OUTCOME MEASURES: Recombinant virus titer was measured by fluorescent cell counting, and infection efficiency was detected by Fluorescence Activated Cell Sorter (FACS) upon infecting AAV-HT1080 cells. The recombination with the exogenous gene was verified by PCR. RESULTS: The PCR amplified products were verified as hVEGF165 gene by DNA sequencing, and the recombinant pAAV-hVEGF165-IRES-GFP was confirmed by double digestion

  2. Bioconjugation of recombinant tissue plasminogen activator to magnetic nanocarriers for targeted thrombolysis

    Directory of Open Access Journals (Sweden)

    Yang HW

    2012-10-01

    Full Text Available Hung-Wei Yang,1,* Mu-Yi Hua,1,* Kun-Ju Lin,2,* Shiaw-Pyng Wey,3 Rung-Ywan Tsai,4 Siao-Yun Wu,5 Yi-Ching Lu,5 Hao-Li Liu,6 Tony Wu,7 Yunn-Hwa Ma5 1Chang Gung Molecular Medicine Research Center, Department of Chemical and Materials Engineering, 2Molecular Imaging Center, Department of Nuclear Medicine, Chang Gung Memorial Hospital, Kuei-Shan, Tao-Yuan, Taiwan, Republic of China; 3Department of Medical Imaging and Radiological Sciences, 4Electronics and Optoelectronics Research Laboratories, Industrial Technology Research Institute, Hsin-chu, Taiwan, Republic of China; 5Department of Physiology and Pharmacology and Healthy Aging Research Center, 6Department of Electrical Engineering, Chang Gung University, Kuei-Shan, Tao-Yuan, Taiwan, Republic of China; 7Department of Neurology, Chang Gung University College of Medicine and Memorial Hospital, Tao-Yuan, Taiwan, Republic of China*These authors contributed equally to this workAbstract: Low-toxicity magnetic nanocarriers (MNCs composed of a shell of poly [aniline-co-N-(1-one-butyric acid aniline] over a Fe3O4 magnetic nanoparticle core were developed to carry recombinant tissue plasminogen activator (rtPA in MNC-rtPA for targeted thrombolysis. With an average diameter of 14.8 nm, the MNCs exerted superparamagnetic properties. Up to 276 µg of active rtPA was immobilized per mg of MNCs, and the stability of the immobilized rtPA was greatly improved during storage at 4°C and 25°C. In vitro thrombolysis testing with a tubing system demonstrated that magnet-guided MNC-rtPA showed significantly improved thrombolysis compared with free rtPA and reduced the clot lysis time from 39.2 ± 3.2 minutes to 10.8 ± 4.2 minutes. In addition, magnet-guided MNC-rtPA at 20% of the regular rtPA dose restored blood flow within 15–25 minutes of treatment in a rat embolism model without triggering hematological toxicity. In conclusion, this improved system is based on magnetic targeting accelerated thrombolysis and is

  3. Rapid Construction of EGFP Labled Recombinant Adenovirus Containing hVEGF165 and Its Expression in Haematopoietic Cells

    Institute of Scientific and Technical Information of China (English)

    仲照东; 邹萍; 黄士昂; 胡中波; 刘凌波; 卢运萍

    2003-01-01

    By using AdEasy system, which is based on the homologous recombination in bacteria, an EGFP labeled recombinant adenovirus vector containing hVEGF165 was constructed quickly and efficiently expressed in mouse haematopoietic cells. First, hVEGF165 coding sequence was subcloned into shuttle plasmid pAdTrack-CMV, then cotransformed with adenoviral backbone vector pAdEasy-1 into E. coli strain BJ5183. The recombinant adenoviral plasmid was transfected into HEK293 cells to assembly replication-defective adenovirus Ad-EGFP/hVEGF165. The expression of EGFP could be easily detected. The rate of EGFP positive mouse bone marrow mononuclear cells by flow cytometric analysis was 27.3 % (MOI= 100), and the expression of hVEGF165 protein in the supernatant was (1385+332) pg/106 cells. These results suggest that the construction of adenovirus vector by homologous recombination in bacteria features high efficiency and simplicity. The prepared high titer AdEGFP/hVEGF165 can be used an efficient helpful vector to infect hematopoietic cells.

  4. Transfection of bone marrow mesenchymal stem cells using green fluorescence protein labeled hVEGF165 recombinant plasmid mediated by liposome

    Institute of Scientific and Technical Information of China (English)

    Tao Wang; Tian-An Liao; Shao-Bo Zhong

    2013-01-01

    Objective:To study the role of bone marrow mesenchymal stem cells (BMSCs) in construction of vascularized engineered tissue. Methods: hVEGF165 was amplified via RT-PCR before recombinant with pShuttle-green fluorescence protein;green fluorescent protein (GFP)-CMV. Then the recombinant shuttle plasmid was transfected into BMSCs with LipofectamineTM 2000 for packaging and amplifying. hVEGF165 mRNA expression in BMSCs cells was tested. Results:The sequence of hVEGF165 in pShuttle-GFP-hVEGF165 plasmid was confirmed by double-enzyme cleavage method and sequencing. hVEGF165 was highly expressed in BMSCs. Conclusions:The GFP/hVEGF165 recombinant plasmid vector was constructed successfully and expressed effectively in host cells, which may be helpful for discussing the possibility of the application of VEGF165-BMSCs in tissue engineering and ischemic disease cure.

  5. Blocking αvβ3 integrin by a recombinant RGD disintegrin impairs VEGF signaling in endothelial cells.

    Science.gov (United States)

    Montenegro, Cyntia F; Salla-Pontes, Carmen L; Ribeiro, Juliana U; Machado, Aline Z; Ramos, Rafael F; Figueiredo, Camila C; Morandi, Verônica; Selistre-de-Araujo, Heloisa S

    2012-08-01

    Vascular endothelial growth factor (VEGF) and αvβ3 integrin are key molecules that actively participate in tumor angiogenesis and metastasis. Some integrin-blocking molecules are currently under clinical trials for cancer and metastasis treatment. However, the mechanism of action of such inhibitors is not completely understood. We have previously demonstrated the anti-angiogenic and anti-metastatic properties of DisBa-01, a recombinant His-tag RGD-disintegrin from Bothrops alternatus snake venom in some experimental models. DisBa-01 blocks αvβ3 integrin binding to vitronectin and inhibits integrin-mediated downstream signaling cascades and cell migration. Here we add some new information on the mechanism of action of DisBa-01 in the tumor microenvironment. DisBa-01 supports the adhesion of fibroblasts and MDA-MB-231 breast cancer cells but it inhibits the adhesion of these cells to type I collagen under flow in high shear conditions, as a simulation of the blood stream. DisBa-01 does not affect the release of VEGF by fibroblasts or breast cancer cells but it strongly decreases the expression of VEGF mRNA and of its receptors, vascular endothelial growth factor receptors 1 and 2 (VEGFR1 and VEGFR2) in endothelial cells. DisBa-01 at nanomolar concentrations also modulates metalloprotease 2 (MMP-2) and 9 (MMP-9) activity, the latter being decreased in fibroblasts and increased in MDA-MB-231 cells. In conclusion, these results demonstrate that αvβ3 integrin inhibitors may induce distinct effects in the cells of the tumor microenvironment, resulting in blockade of angiogenesis by impairing of VEGF signaling and in inhibition of tumor cell motility.

  6. Enhanced Mitogenic Activity of Recombinant Human Vascular Endothelial Growth Factor VEGF121 Expressed in E. coli Origami B (DE3) with Molecular Chaperones

    Science.gov (United States)

    Kaplan, Ondřej; Zárubová, Jana; Mikulová, Barbora; Filová, Elena; Bártová, Jiřina; Bačáková, Lucie; Brynda, Eduard

    2016-01-01

    We describe the production of a highly-active mutant VEGF variant, α2-PI1-8-VEGF121, which contains a substrate sequence for factor XIIIa at the aminoterminus designed for incorporation into a fibrin gel. The α2-PI1-8-VEGF121 gene was synthesized, cloned into a pET-32a(+) vector and expressed in Escherichia coli Origami B (DE3) host cells. To increase the protein folding and the solubility, the resulting thioredoxin-α2-PI1-8-VEGF121 fusion protein was co-expressed with recombinant molecular chaperones GroES/EL encoded by independent plasmid pGro7. The fusion protein was purified from the soluble fraction of cytoplasmic proteins using affinity chromatography. After cleavage of the thioredoxin fusion part with thrombin, the target protein was purified by a second round of affinity chromatography. The yield of purified α2-PI1-8-VEGF121 was 1.4 mg per liter of the cell culture. The α2-PI1-8-VEGF121 expressed in this work increased the proliferation of endothelial cells 3.9–8.7 times in comparison with commercially-available recombinant VEGF121. This very high mitogenic activity may be caused by co-expression of the growth factor with molecular chaperones not previously used in VEGF production. At the same time, α2-PI1-8-VEGF121 did not elicit considerable inflammatory activation of human endothelial HUVEC cells and human monocyte-like THP-1 cells. PMID:27716773

  7. Overcoming inefficient secretion of recombinant VEGF-C in baculovirus expression vector system by simple purification of the protein from cell lysate.

    Science.gov (United States)

    Klaus, Tomasz; Kulesza, Małgorzata; Bzowska, Monika; Wyroba, Barbara; Kilarski, Witold W; Bereta, Joanna

    2015-06-01

    The first reports about successfully expressed recombinant proteins with the use of a baculovirus vector were published over 30years ago. Despite the long time of refining this expression system, early problems with the production of baculovirus-derived secretory proteins are still not satisfactorily solved. The high expression level driven by baculoviral promoters often does not result in the desired yield of secreted recombinant proteins, which frequently accumulate inside insect cells and are only partially processed. During our attempts to produce vascular endothelial growth factor C (VEGF-C) with the use of a baculovirus vector we also faced an inefficient secretion of the recombinant protein to culture medium. We were not able to improve the outcome and obtain an acceptable concentration of VEGF-C in the medium by changing the culture conditions or utilizing different signal peptides. However, as a significant amount of native VEGF-C was detected inside the baculovirus-infected cells, we developed a simple method to purify recombinant, glycosylated VEGF-C from a lysate of the cells. The presented results indicate that the lack of a secretory protein in the insect cell culture medium after baculovirus infection does not necessarily signify failure in the production of the protein. As demonstrated by us and contrary to generally accepted views, the lysate of baculovirus-infected cells may constitute a valuable source of the biologically active, secretory protein.

  8. A versatile approach for the site-specific modification of recombinant antibodies using a combination of enzyme-mediated bioconjugation and click chemistry.

    Science.gov (United States)

    Alt, Karen; Paterson, Brett M; Westein, Erik; Rudd, Stacey E; Poniger, Stan S; Jagdale, Shweta; Ardipradja, Katie; Connell, Timothy U; Krippner, Guy Y; Nair, Ashish K N; Wang, Xiaowei; Tochon-Danguy, Henri J; Donnelly, Paul S; Peter, Karlheinz; Hagemeyer, Christoph E

    2015-06-22

    A unique two-step modular system for site-specific antibody modification and conjugation is reported. The first step of this approach uses enzymatic bioconjugation with the transpeptidase Sortase A for incorporation of strained cyclooctyne functional groups. The second step of this modular approach involves the azide-alkyne cycloaddition click reaction. The versatility of the two-step approach has been exemplified by the selective incorporation of fluorescent dyes and a positron-emitting copper-64 radiotracer for fluorescence and positron-emission tomography imaging of activated platelets, platelet aggregates, and thrombi, respectively. This flexible and versatile approach could be readily adapted to incorporate a large array of tailor-made functional groups using reliable click chemistry whilst preserving the activity of the antibody or other sensitive biological macromolecules.

  9. ScVEGF-PEG-HBED-CC and scVEGF-PEG-NOTA conjugates: comparison of easy-to-label recombinant proteins for [{sup 68}Ga]PET imaging of VEGF receptors in angiogenic vasculature

    Energy Technology Data Exchange (ETDEWEB)

    Eder, Matthias [German Cancer Research Center (DKFZ), 69120 Heidelberg (Germany)], E-mail: m.eder@dkfz.de; Krivoshein, Arcadius V.; Backer, Marina; Backer, Joseph M. [SibTech, Inc., Brookfield, CT 06804 (United States); Haberkorn, Uwe [Department of Nuclear Medicine, University of Heidelberg, 69120 Heidelberg (Germany); Eisenhut, Michael [German Cancer Research Center (DKFZ), 69120 Heidelberg (Germany)

    2010-05-15

    Introduction: VEGF receptors play a key role in angiogenesis and are important targets for several approved and many experimental drugs. Imaging of VEGF receptor expression in malignant tumors would provide important information, which can influence patient management. The aim of this study was the development of an easy-to-label positron-emitting tracer for imaging VEGF receptors. The tracer is based on engineered single-chain VEGF (scVEGF), expressed with cysteine-containing fusion tag (Cys-tag) for site-specific conjugation of PEGylated bifunctional chelating agents, HBED-CC or NOTA, suitable for labeling with {sup 68}Ga at ambient temperature. Methods: scVEGF-PEG-HBED-CC was synthesized by activating a single carboxyl group of the [Fe(HBED-CC)]{sup -} complex with N-hydroxysuccinimide. Reaction of the activated complex with NH{sub 2}-PEG-maleimide was followed by site-specific conjugation of PEGylated chelator to a thiol group in Cys-tag of scVEGF. The scVEGF-PEG-NOTA conjugate was synthesized using NHS-PEG-maleimide and p-NH{sub 2}-Bn-NOTA. {sup 68}Ga complexation was performed in HEPES buffer (pH 4.2) at room temperature. The functional activity after labeling was tested by radioligand cell binding assays. Biodistribution and PET studies in tumor-bearing mice were performed after 1, 2, 3 and 4 h postinjection. Results: The radiolabeling of scVEGF-PEG-HBED-CC proved more efficient than scVEGF-PEG-NOTA allowing to stop the reaction after 4 min (>97% radiochemical yield). Radioligand cell binding assays performed on HEK-293 cells overexpressing VEGFR-2 revealed no change in the binding properties of {sup 68}Ga-radiolabeled scVEGF relative to other scVEGF-based tracers. Both tracers showed comparable results in biodistribution, such as tumor accumulation and low liver uptake. The tracers were stable in 50% human serum for at least 72 h. Conclusions: The conjugates scVEGF-PEG-HBED-CC and scVEGF-PEG-NOTA revealed comparable in vivo characteristics and allowed easy

  10. Recombinant snake venom cystatin inhibits tumor angiogenesis in vitro and in vivo associated with downregulation of VEGF-A165, Flt-1 and bFGF.

    Science.gov (United States)

    Xie, Qun; Tang, Nanhong; Wan, Rong; Qi, Yuanlin; Lin, Xu; Lin, Jianyin

    2013-05-01

    Previous studies have shown that recombinant snake venom cystatin (sv-cystatin) inhibits the invasion and metastasis of tumor cells in vitro and in vivo. The purpose of this study was to investigate the ability of recombinant sv-cystatin to inhibit tumor angiogenesis in vitro and in vivo, and the mechanisms underlying this effect. Recombinant sv-cystatin inhibited proliferation of human umbilical vein endothelial cells (HUVECs) at 100 and 200 μg/mL after 72, 96 and 120 h. Recombinant sv-cystatin also inhibited tumor-endothelial cell adhesion at 25, 50, 100 and 200 μg/mL. Recombinant sv-cystatin inhibited capillary-like tube formation by HUVECs at 10, 25, 50, 100 and 200 μg/mL following 12, 24 and 36 h incubation. Furthermore, recombinant sv-cystatin significantly suppressed microvessel density (MVD) of lung tumor colonies in C57BL/6 mice inoculated in the lateral tail vein with B16F10 melanoma cells. Administration of recombinant sv-cystatin significantly decreased MVD of primary tumor tissues in nude mice implanted subcutaneously with human hepatocellular carcinoma cells (MHCC97H). Exposure of B16F10 and MHCC97H cells to increasing doses of recombinant sv-cystatin suppressed secretion of vascular endothelial growth factor (VEGF)-A165 and basic fibroblast growth factor (bFGF) into the surrounding medium (P cystatin (P cystatin inhibits tumor angiogenesis associated with downregulation of VEGF-A165, Flt-1 and bFGF. This suggests that recombinant sv-cystatin may have potential pharmaceutical applications as an antiangiogenic and antimetastatic therapeutic agent.

  11. Subretinal recombinant tissue plasminogen activator and pneumatic displacement for the management of subretinal hemorrhage occurring after anti-VEGF injections for wet AMD.

    Science.gov (United States)

    Tognetto, Daniele; Skiadaresi, Eirini; Cecchini, Paolo; Ravalico, Giuseppe

    2011-01-01

    We describe three cases of submacular hemorrhage that occurred two to four days after anti-VEGF intravitreal injection for occult choroidal neovascularisation in age-related macular degeneration and their management with 25 gauge pars plana vitrectomy with injection of subretinal recombinant tissue plasminogen activator (rTPA) followed by fluid-air exchange and postoperative prone position. Vitrectomy, subretinal rTPA injection and fluid-gas exchange apply as a safe and effective treatment in these cases. Functional results seem to be positive especially if surgical treatment is promptly performed.

  12. VEGF121b and VEGF165b are weakly angiogenic isoforms of VEGF-A

    Directory of Open Access Journals (Sweden)

    Pio Ruben

    2010-12-01

    Full Text Available Abstract Background Different isoforms of VEGF-A (mainly VEGF121, VEGF165 and VEGF189 have been shown to display particular angiogenic properties in the generation of a functional tumor vasculature. Recently, a novel class of VEGF-A isoforms, designated as VEGFxxxb, generated through alternative splicing, have been described. Previous studies have suggested that these isoforms may inhibit angiogenesis. In the present work we have produced recombinant VEGF121/165b proteins in the yeast Pichia pastoris and constructed vectors to overexpress these isoforms and assess their angiogenic potential. Results Recombinant VEGF121/165b proteins generated either in yeasts or mammalian cells activated VEGFR2 and its downstream effector ERK1/2, although to a lesser extent than VEGF165. Furthermore, treatment of endothelial cells with VEGF121/165b increased cell proliferation compared to untreated cells, although such stimulation was lower than that induced by VEGF165. Moreover, in vivo angiogenesis assays confirmed angiogenesis stimulation by VEGF121/165b isoforms. A549 and PC-3 cells overexpressing VEGF121b or VEGF165b (or carrying the PCDNA3.1 empty vector, as control and xenotransplanted into nude mice showed increased tumor volume and angiogenesis compared to controls. To assess whether the VEGFxxxb isoforms are differentially expressed in tumors compared to healthy tissues, immunohistochemical analysis was conducted on a breast cancer tissue microarray. A significant increase (p xxxb and total VEGF-A protein expression in infiltrating ductal carcinomas compared to normal breasts was observed. A positive significant correlation (r = 0.404, p = 0.033 between VEGFxxxb and total VEGF-A was found. Conclusions Our results demonstrate that VEGF121/165b are not anti-angiogenic, but weakly angiogenic isoforms of VEGF-A. In addition, VEGFxxxb isoforms are up-regulated in breast cancer in comparison with non malignant breast tissues. These results are to be taken

  13. Construction and identification of recombinant adenovirus vector co-expressing VEGF121 and BMP2 genes and its expression in HEK293 cells%VEGF121和BMP2双基因共表达重组腺病毒载体的构建及其在HEK293中的表达

    Institute of Scientific and Technical Information of China (English)

    栗刚; 吴秀成; 钟声; 王巍; 李媛; 刘丹平

    2011-01-01

    目的 构建人血管内皮生长因子121(VEGF121)与人骨形态发生蛋白2(BMP2)双基因共表达腺病毒载体Adv-BMP2-IRES-VEGF121,并观察其在人胚肾细胞株(HEK293)中的表达情况.方法 对腺病毒质粒pShuttle-CMV-BMP2的目的基因BMP2进行PCR扩增.腺病毒质粒pShuttle-CMV-VEGF121-IRES-hrGFP-1经Kpn I/Xba I酶切后,将BMP2片段定向导入pShuttle-CMV-VEGF121-IRES,构建pShuttle-CMV-V EGF121-IRES-BMP2,并注入大肠杆菌DH5a中扩增,提取质粒.通过酶切分析、PCR检测和序列分析进行鉴定.将构建所得的质粒转染HEK293,采用RT-PCR法检测HEK293中的BMP2、VEGF121 mRNA,Western blot法检测其蛋白.结果 成功构建了Adv-BMP2-IRES-VEGF121.酶切分析及DNA序列测定证实重组质粒构建正确.质粒转染后的HEK293 BMP2和VEGF121表达阳性.结论 成功构建了Adv-BMP2-IRES-VEGF121,其转染HEK293后,VEGF121、BMP2在HEK293中共表达阳性.%Objective To construct and identify the adenovirus shuttle plasmid pShuttle-CMV-VEGF121-IRES-BMP2 and its express in HEK293 cells.Methods The DNA fragments of human BMP2 gene were changed restriction sites and subcloned by PCR.The human BMP2 genes and pShuttle-CMV-VEGF121-IRES were ligated into the plasmid by directional cloning method.The inserted target genes in the plasmid were verified by restriction enzyme digestion and nucleotide sequencing.The correct recombinant express plasmid was transfected to HEK293 cells.The expression of VEGF121, BMP2 mRNA were detected by RT-PCR, the VEGF121, BMP2 protein were detected by Western blotting.Results The adenovirus shuttle plasmid was constructed correctly.The VEGF121, BMP2 mRNA and protein were expressed in HEK293 cells.Conclusion The adenovirus shuttle plasmid is constructed, VEGF121, BMP2 mRNA and protein are successfully expressed in HEK293 cells.

  14. Bio-Conjugates for Nanoscale Applications

    DEFF Research Database (Denmark)

    Villadsen, Klaus

    Bio-conjugates for Nanoscale Applications is the title of this thesis, which covers three different projects in chemical bio-conjugation research, namely synthesis and applications of: Lipidated fluorescent peptides, carbohydrate oxime-azide linkers and N-aryl O-R2 oxyamine derivatives. Lipidated...

  15. Bio-Conjugates for Nanoscale Applications

    DEFF Research Database (Denmark)

    Villadsen, Klaus

    Bio-conjugates for Nanoscale Applications is the title of this thesis, which covers three different projects in chemical bio-conjugation research, namely synthesis and applications of: Lipidated fluorescent peptides, carbohydrate oxime-azide linkers and N-aryl O-R2 oxyamine derivatives. Lipidated...

  16. Investigating bioconjugation by atomic force microscopy

    Science.gov (United States)

    2013-01-01

    Nanotechnological applications increasingly exploit the selectivity and processivity of biological molecules. Integration of biomolecules such as proteins or DNA into nano-systems typically requires their conjugation to surfaces, for example of carbon-nanotubes or fluorescent quantum dots. The bioconjugated nanostructures exploit the unique strengths of both their biological and nanoparticle components and are used in diverse, future oriented research areas ranging from nanoelectronics to biosensing and nanomedicine. Atomic force microscopy imaging provides valuable, direct insight for the evaluation of different conjugation approaches at the level of the individual molecules. Recent technical advances have enabled high speed imaging by AFM supporting time resolutions sufficient to follow conformational changes of intricately assembled nanostructures in solution. In addition, integration of AFM with different spectroscopic and imaging approaches provides an enhanced level of information on the investigated sample. Furthermore, the AFM itself can serve as an active tool for the assembly of nanostructures based on bioconjugation. AFM is hence a major workhorse in nanotechnology; it is a powerful tool for the structural investigation of bioconjugation and bioconjugation-induced effects as well as the simultaneous active assembly and analysis of bioconjugation-based nanostructures. PMID:23855448

  17. Peptide linkers for the assembly of semiconductor quantum dot bioconjugates

    Science.gov (United States)

    Boeneman, Kelly; Mei, Bing C.; Deschamps, Jeffrey R.; Delehanty, James B.; Mattoussi, Hedi; Medintz, Igor

    2009-02-01

    The use of semiconductor luminescent quantum dots for the labeling of biomolecules is rapidly expanding, however it still requires facile methods to attach functional globular proteins to biologically optimized quantum dots. Here we discuss the development of controlled variable length peptidyl linkers to attach biomolecules to poly(ethylene) glycol (PEG) coated quantum dots for both in vitro and in vivo applications. The peptides chosen, β-sheets and alpha helices are appended to polyhistidine sequences and this allows for control of the ratio of peptide bioconjugated to QD and the distance from QD to the biomolecule. Recombinant DNA engineering, bacterial peptide expression and Ni-NTA purification of histidine labeled peptides are utilized to create the linkers. Peptide length is confirmed by in vitro fluorescent resonance energy transfer (FRET).

  18. The effect of recombinant human erythropoietin on the expression of VEGF in acute spinal cord injury rat%重组人促红细胞生成素对大鼠脊髓损伤后VEGF表达的影响

    Institute of Scientific and Technical Information of China (English)

    霍岩; 沈兆亮; 王冬; 王巍; 高爽

    2012-01-01

    目的 研究重组人促红细胞生成素(recombinant human erythropoietin,rHuEPO)对急性脊髓损伤大鼠血管内皮生长因子(vascular endothelial growth factor,VEGF)表达的影响.方法 参照Nystrom's压迫方法制作大鼠脊髓压迫损伤模型,成年健康Wistar大鼠72只,雌雄不限,按随机数字表法分为正常对照组8只、损伤组32只、重组人促红细胞生成素治疗组32只.免疫组化和Western blot检测各组大鼠VEGF的表达.结果 免疫组化结果显示:损伤组VEGF阳性产物的平均光密度值(mean optic density,MOD)显著高于正常对照组(P<0.01),rHuEPO治疗组与损伤组相比MOD值明显升高(P<0.01).Western blot结果显示:与正常对照组比较,损伤组VEGF积分光密度值(integrated density value,IDV)与内参照IDV的比值明显升高(P<0.01),而rHuEPO治疗组则明显高于损伤组(P<0.01).结论 rHuEPO参与脊髓继发性损伤修复,可能与上调VEGF的表达相关.%Objective To study the expression of vascular endothelial growth factor (VECF) in acute spinal cord injury rat after recombinant human erythropoietin (rHuEPO)was injected. Methods The spinal cord injury was induced with Nystrom's way. The healthy adult Wistar rats (72) were randomly divided into normal control group, spinal cord injury group, recombinant human erythropoietin treated group on average. The expression of VEGF was observed by immunochemistry and Western blot methods in all groups. Results The mean optic density (MOD) of VECF positive product increased significantly in acute spinal cord injury group than in normal control (P<0.01), and increased in the recombinant human erythropoietin treated group than in acute spinal cord injury group (P<0.01) by immunochemistry. The integrated density value (IDV) for VEGF protein band increased significantly in the spinal cord injury group than in normal control (P< 0.01), and increased in recombinant human erythropoietin group than in spinal cord injury group (P

  19. Bioconjugation of antibodies to horseradish peroxidase (hrp)

    Science.gov (United States)

    The bioconjugation of an antibody to an enzymatic reporter such as horseradish peroxidase (HRP) affords an effective mechanism by which immunoassay detection of a target antigen can be achieved. The use of heterobifunctional cross—linkers to covalently link antibodies to HRP provides a simple and c...

  20. 电穿孔介导的pIRES-hVEGF165-EGFP转染对牵引成骨过程中早期血管生成的影响%Effect of electroporation mediated transfecting recombinant plasmid pIRES-hVEGF165-EGFP on angiogene-sis of distraction area during early mandibular distraction osteogenesis

    Institute of Scientific and Technical Information of China (English)

    吴国平; 黎德平; 何小川; 李盛华; 杨智慧; 廖毅; 郭力

    2010-01-01

    目的 探讨电穿孔介导的基因治疗对下领骨牵引成骨过程中早期血管生成的影响.方法 32只新西兰大白兔随机分为4组:质粒+电穿孔组(A组),质粒组(B组),生理盐水+电穿孔组(C组),空白对照组(D组).各组动物分别于注射后1、3、7、14 d处死,取牵引区组织进行组织学检查、电镜观察、CD34免疫组织化学染色及微血管密度检测.结果 A、B组血管内皮细胞呈增殖活跃状态;C、D组多数血管内皮细胞部分呈现退变及凋亡早期改变.免疫组化染色发现,转染后第1天血管壁内皮细胞浆CD34表达较弱;第3、7、14天,牵引区肉芽组织血管内皮细胞均出现CD34阳性表达.A组CD34阳性表达较B组强,A、B组的CD34表达持续阳性且呈上升趋势;C、D组表达最弱,CD34阳性表达维持在第1天水平上平稳波动.结论 电穿孔介导的pIRES-hVEGF165-EGFP重组质粒体内转染能够促进牵引区早期微血管的生成,使局部血管增生、渗入,增加骨断端的血流量.对调节和促进骨的生长和修复过程具有重要作用.%Objective To explore the effect of electroporation mediated gene therapy on angiogene-sis of the distraction area during early mandibular distraction osteogenesis (DO). Methods Thirty-two New-Zeland rabbits were randomly divided into 4 groups: group A: recombinant plasmid pIRES-VEGF165-EGFP and electroporation; group B: recombinant plasmid pIRES-VEGF165-EGFP; group C: normal saline (NS) and electroporation and group D: control group. The rabbits were sacrificed at 1d, 3d, 7d and 14d after injection, respectively. The distraction area tissue was removed for histological examination and electron microscopy, and immunohistochemical stain for CD34 was performed to detect the microvessel density. Results Generation of vascular endothelial cells (VEC) in the group A and group B were active, and majority of VEC in groups C and D took on early change of cataplasia and apoptosis. The

  1. Development of Bioorthogonal Reactions and Their Applications in Bioconjugation

    Directory of Open Access Journals (Sweden)

    Mengmeng Zheng

    2015-02-01

    Full Text Available Biomolecule labeling using chemical probes with specific biological activities has played important roles for the elucidation of complicated biological processes. Selective bioconjugation strategies are highly-demanded in the construction of various small-molecule probes to explore complex biological systems. Bioorthogonal reactions that undergo fast and selective ligation under bio-compatible conditions have found diverse applications in the development of new bioconjugation strategies. The development of new bioorthogonal reactions in the past decade has been summarized with comments on their potentials as bioconjugation method in the construction of various biological probes for investigating their target biomolecules. For the applications of bioorthogonal reactions in the site-selective biomolecule conjugation, examples have been presented on the bioconjugation of protein, glycan, nucleic acids and lipids.

  2. Cloning, large-scale production, and purification of active dimeric rat vascular endothelial growth factor (rrVEGF-164).

    NARCIS (Netherlands)

    Geutjes, P.J.; Nillesen, S.T.M.; Lammers, G.; Daamen, W.F.; Kuppevelt, A.H.M.S.M. van

    2010-01-01

    Large-scale production of recombinant rat vascular endothelial growth factor (rrVEGF-164) is desirable for angiogenic studies. In this study, biologically active recombinant rat vascular endothelial growth factor (rrVEGF-164) was cloned and expressed in the yeast Pichia pastoris, and large-scale pro

  3. Relationship between Expression of beta-catenin and VEGFs(VEGFA,VEGF-C),VEGF Receptors-2(VEGFR-2)in Medulloblastoma

    Institute of Scientific and Technical Information of China (English)

    ZHANG Hong-mei; ZHANG Xiong; LI Yu; MI Can

    2008-01-01

    Objective:To investigate the expression of beta-catenin and VEGFs(VEGF-A,VEGF-C)and VEGF receptor-2(VEGFR-2)protein in medulloblastoma.Methods:Immunohistochemical staining with SP method Was conducted to determine the expression of beta-eatenin and VEGFs(VEGF-A,VEGF-C)and VEGFR-2 in 33 cases of medulloblastoma and 10 normal cerebellar tissues. Results:The expression rate of beta-catenin,and VEGFs (VEGF-A,VEGF-C)and VEGFR-2 in medulloblastoma were significantly higher than that in normal tissue.A significant positive correlation was found between beta-catenin and VEGFs(VEGF-A,VEGF-C)and VEGFR-2 protein in medulloblastoma. Conclusion:There was a correlation between beta-catenin and VEGFs(VEGF-A,VEGF-C)and VEGFR-2 in medulloblastoma,which may play a role in the pathogenesis and development of medulloblastoma.

  4. General Dialdehyde Click Chemistry for Amine Bioconjugation.

    Science.gov (United States)

    Elahipanah, Sina; O'Brien, Paul J; Rogozhnikov, Dmitry; Yousaf, Muhammad N

    2017-05-17

    -conjugation strategy, we designed a straightforward scheme to synthesize a suite of dialdehyde reagents. The dialdehyde molecules were used for applications in cell-surface engineering and for tailoring surfaces for material science applications. We anticipate the broad utility of the general dialdehyde click chemistry to primary amines in all areas of chemical research, ranging from polymers and bioconjugation to material science and nanoscience.

  5. Photochemical Synthesis of the Bioconjugate Folic Acid-Gold Nanoparticles

    DEFF Research Database (Denmark)

    León, John Jairo Castillo; Bertel, Linda; Páez-Mozo, Edgar

    2013-01-01

    In this paper we present a rapid and simple onepot method to obtain gold nanoparticles functionalized with folic acid using a photochemistry method. The bioconjugate folic acid-gold nanoparticle was generated in one step using a photo-reduction method, mixing hydrogen tetrachloroaurate with folic...

  6. Engineering an arginine catabolizing bioconjugate: Biochemical and pharmacological characterization of PEGylated derivatives of arginine deiminase from Mycoplasma arthritidis.

    Science.gov (United States)

    Wang, Maoliang; Basu, Amartya; Palm, Thomas; Hua, Jack; Youngster, Stephen; Hwang, Lisa; Liu, Hsien-Ching; Li, Xiguang; Peng, Ping; Zhang, Yue; Zhao, Hong; Zhang, Zhihua; Longley, Clifford; Mehlig, Mary; Borowski, Virna; Sai, Prakash; Viswanathan, Manickam; Jang, Eun; Petti, Gerald; Liu, Sam; Yang, Karen; Filpula, David

    2006-01-01

    Arginine is an important metabolite in the normal function of several biological systems, and arginine deprivation has been investigated in animal models and human clinical trials for its effects on inhibition of tumor growth, angiogenesis, or nitric oxide synthesis. In order to design an optimal arginine-catabolizing enzyme bioconjugate, a novel recombinant arginine deiminase (ADI) from Mycoplasma arthritidis was prepared, and multi-PEGylated derivatives were examined for enzymatic and biochemical properties in vitro, as well as pharmacokinetic and pharmacodynamic behavior in rats and mice. ADI bioconjugates constructed with 12 kDa or 20 kDa monomethoxy-poly(ethylene glycol) polymers with linear succinimidyl carbonate linkers were investigated via intravenous, intramuscular, or subcutaneous administration in rodents. The selected PEG-ADI compounds have 22 +/- 2 PEG strands per protein dimer, providing an additional molecular mass of about 0.2-0.5 x 10(6) Da and prolonging the plasma mean residence time of the enzyme over 30-fold in mice. Prolonged plasma arginine deprivation was demonstrated with each injection route for these bioconjugates. Pharmacokinetic analysis employed parallel measurement of enzyme activity in bioassays and enzyme assays and demonstrated a correlation with the pharmacodynamic analysis of plasma arginine concentrations. Either ADI bioconjugate depressed plasma arginine to undetectable levels for 10 days when administered intravenously at 5 IU per mouse, while the subcutaneous and intramuscular routes exhibited only slightly reduced potency. Both bioconjugates exhibited potent growth inhibition of several cultured tumor lines that are deficient in the anabolic enzyme, argininosuccinate synthetase. Investigations of structure-activity optimization for PEGylated ADI compounds revealed a benefit to constraining the PEG size and number of attachments to both conserve catabolic activity and streamline manufacturing of the experimental therapeutics

  7. Identification and function analysis of a novel vascular endothelial growth factor, LvVEGF3, in the Pacific whiteleg shrimp Litopenaeus vannamei.

    Science.gov (United States)

    Wang, Zhiwei; Li, Shihao; Li, Fuhua; Xie, Shijun; Xiang, Jianhai

    2016-10-01

    VEGF signaling pathway is first discovered in mammals and proved to play important roles in the biological processes of angiogenesis, tumor migration, cell differentiation, apoptosis, host-virus interaction etc. Three members in the VEGF signaling pathway, including LvVEGFR, LvVEGF1 and LvVEGF2 in shrimp have been proved to be related with WSSV infection in our previous studies. Currently, another member of VEGF family, LvVEGF3, was isolated and its function during the WSSV infection of shrimp was studied. The deduced amino acid sequence of LvVEGF3 contained a signal peptide, a typical PDGF/VEGF domain and a cysteine-knot motif (CXCXC). Tissue distribution analysis showed that LvVEGF3 was predominantly expressed in hemocytes. The transcriptional level of LvVEGF3 in hemocytes was apparently up-regulated during WSSV infection. Silencing of LvVEGF3 with double-stranded RNA caused a reduction of the cumulative mortality rate of shrimp during WSSV infection. The expression of LvVEGFR was apparently down-regulated after LvVEGF3 silencing and up-regulated after injection of recombinant LvVEGF3 protein, suggesting an interaction between LvVEGF3 and LvVEGFR. Furthermore, the interaction between LvVEGFR and LvVEGF3 was confirmed using the yeast two-hybrid system. The results provided new insights into understanding the role of VEGF signaling pathway during virus infection.

  8. Synthesis of Bioconjugate Sesterterpenoids with Phospholipids and Polyunsaturated Fatty Acids

    Directory of Open Access Journals (Sweden)

    Ana Gil-Mesón

    2015-12-01

    Full Text Available A series of sesterterpenoid bioconjugates with phospholipids and polyunsaturated fatty acids (PUFAs have been synthesized for biological activity testing as antiproliferative agents in several cancer cell lines. Different substitution analogues of the original lipidic ether edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine are obtained varying the sesterterpenoid in position 1 or 2 of the glycerol or a phosphocholine or PUFA unit in position 3. Simple bioconjugates of sesterterpenoids and eicosapentaenoic acid (EPA have been obtained too. All synthetic derivatives were tested against the human tumour cell lines HeLa (cervix and MCF-7 (breast. Some compounds showed good IC50 (0.3 and 0.2 μM values against these cell lines.

  9. HER-2 Targeted Nanoparticle-Affibody Bioconjugates for Cancer Therapy

    Science.gov (United States)

    Alexis, Frank; Basto, Pamela; Levy-Nissenbaum, Etgar; Radovic-Moreno, Aleksandar F.; Zhang, Liangfang; Pridgen, Eric; Wang, Adrew Z.; Marein, Shawn L.; Westerhof, Katrina; Molnar, Linda K.; Farokhzad, Omid C.

    2010-01-01

    Affibodies are a class of polypeptide ligands that are potential candidates for cell- or tissue-specific targeting of drug-encapsulated controlled release polymeric nanoparticles (NPs). Here we report the development of drug delivery vehicles comprised of polymeric NPs that are surface modified with Affibody ligands that bind to the extracellular domain of the trans-membrane human epidermal growth factor receptor 2 (HER-2) for targeted delivery to cells which over express the HER-2 antigen. NPs lacking the anti-HER-2 Affibody did not show significant uptake by these cells. Using paclitaxel encapsulated NP-Affibody (1 wt% drug loading), we demonstrated increased cytotoxicity of these bioconjugates in SK-BR-3 and SKOV-3 cell lines. These targeted, drug encapsulated NPAffibody bioconjugates may be efficacious in treating HER-2 expressing carcinoma. PMID:19012296

  10. Nanoshell bioconjugates for integrated imaging and therapy of cancer

    Science.gov (United States)

    Loo, Christopher H.; Lee, Min-Ho; Hirsch, Leon R.; West, Jennifer L.; Halas, Naomi J.; Drezek, Rebekah A.

    2004-06-01

    Currently, separate diagnostic and therapeutic modalities are required for the diagnosis and treatment of cancer. In many cases, the present standard of care requires invasive surgical procedures and/or other treatments associated with significant side effect profiles, high cost, and poor clinical outcome. A single technology with dual diagnostic/therapeutic capabilities would potentially yield significant savings in the time and cost associated with diagnosing and treating many cancers. In this paper, we discuss gold nanoshell bioconjugates and their role in the development of an integrated cancer imaging and therapy application. Nanoshells are a novel class of nanomaterials that have unique properties including continuous and broad wavelength tunability, far greater scattering and absorption coefficients, increased chemical stability, and improved biocompatibility. Here, we describe the development of an integrated cancer imaging and therapy application using near-infrared (NIR) gold nanoshell bioconjugates.

  11. VEGF Inhibitors for Cancer Therapy

    OpenAIRE

    Prakash S. Sukhramani; Maulik P. Suthar

    2010-01-01

    Despite significant advances in systemic therapies, radiation oncology, and surgical techniques, many patients with cancer are still incurable. A novel therapeutic approach has been to target the vascular endothelial growth factors (VEGFs) which are often mutated and/or over-expressed in many tumors. The ligands and receptors of VEGF family are well established as key regulators of angiogenesis and vasculogenesis processes. VEGF is a homodimeric, basic, 45 kDa glycoprotein specific for vascul...

  12. Thymosin beta 10 Prompted the VEGF-C Expression in Lung Cancer Cell

    Directory of Open Access Journals (Sweden)

    Zixuan LI

    2014-05-01

    Full Text Available Background and objective Our previous study found that thymosin β10 overexpressed in lung cancer and positively correlated with differentiation, lymph node metastasis and stage of lung cancer. In this reasearch we aim to study the effects and mechanism of exogenous human recombinant Tβ10 on the expression of VEGF-C on non-small cell lung cancer. Methods After SPC, A549 and LK2 cells were treated with 100 ng/mL recombinant human Tβ10, the mRNA level of VEGF-C were detected by RT-PCR. The mean while the protein expression of VEGF-C, P-AKT and AKT were determined by Western blot assay. Results Exogenous recombinant human Tβ10 were significantly promote the expression levels of VEGF-C mRNA and protein while promoting the phosphorylation of AKT. Exogenous Tβ10 can promote the expression of VEGF-C mRNA and protein in lung cancer cell lines A549 and LK2 (P<0.05, and this effect can be inhibited by use AKT inhibitor LY294002 (P<0.05. Conclusion Tβ10 human recombinant proteins can promote the expression of VEGF-C by activating AKT phosphorylation in lung cancer cell lines.

  13. Labeling of mesenchymal stem cells by bioconjugated quantum dots.

    Science.gov (United States)

    Shah, Bhranti S; Clark, Paul A; Moioli, Eduardo K; Stroscio, Michael A; Mao, Jeremy J

    2007-10-01

    Long-term labeling of stem cells during self-replication and differentiation benefits investigations of development and tissue regeneration. We report the labeling of human mesenchymal stem cells (hMSCs) with RGD-conjugated quantum dots (QDs) during self-replication, and multilineage differentiations into osteogenic, chondrogenic, and adipogenic cells. QD-labeled hMSCs remained viable as unlabeled hMSCs from the same subpopulation. These findings suggest the use of bioconjugated QDs as an effective probe for long-term labeling of stem cells.

  14. Imaging Pancreatic Cancer Using Bioconjugated InP Quantum Dots

    OpenAIRE

    2009-01-01

    In this paper, we report the successful use of non-cadmium based quantum dots (QDs) as highly efficient and non-toxic optical probes for imaging live pancreatic cancer cells. Indium phosphide (core)-zinc sulphide (shell), or InP/ZnS, QDs with high quality and bright luminescence were prepared by a hot colloidal synthesis method in non-aqueous media. The surfaces of these QDs were then functionalized with mercaptosuccinic acid to make them highly dispersible in aqueous media. Further bioconjug...

  15. Imaging pancreatic cancer using bioconjugated InP quantum dots.

    Science.gov (United States)

    Yong, Ken-Tye; Ding, Hong; Roy, Indrajit; Law, Wing-Cheung; Bergey, Earl J; Maitra, Anirban; Prasad, Paras N

    2009-03-24

    In this paper, we report the successful use of non-cadmium-based quantum dots (QDs) as highly efficient and nontoxic optical probes for imaging live pancreatic cancer cells. Indium phosphide (core)-zinc sulfide (shell), or InP/ZnS, QDs with high quality and bright luminescence were prepared by a hot colloidal synthesis method in nonaqueous media. The surfaces of these QDs were then functionalized with mercaptosuccinic acid to make them highly dispersible in aqueous media. Further bioconjugation with pancreatic cancer specific monoclonal antibodies, such as anticlaudin 4 and antiprostate stem cell antigen (anti-PSCA), to the functionalized InP/ZnS QDs, allowed specific in vitro targeting of pancreatic cancer cell lines (both immortalized and low passage ones). The receptor-mediated delivery of the bioconjugates was further confirmed by the observation of poor in vitro targeting in nonpancreatic cancer based cell lines which are negative for the claudin-4-receptor. These observations suggest the immense potential of InP/ZnS QDs as non-cadmium-based safe and efficient optical imaging nanoprobes in diagnostic imaging, particularly for early detection of cancer.

  16. Preparation and immunogenicity-evaluation of typhoid O-specific polysaccharides bio-conjugate vaccines.

    Science.gov (United States)

    Zhehui, Peng; Chao, Pan; Peng, Sun; Erling, Feng; Jun, Wu; Li, Zhu; Qingzhong, Peng; Hengliang, Wang

    2015-05-01

    Typhoid fever caused by Salmonella Typhi is still a major public health problem in developing countries. In this study, we constructed a genetically modified Salmonella Typhi strain expressing O-specific polysaccharides (OPS) antigen conjugated to a carrier, recombinant Pseudomonas aeruginosa exotoxin A(rEPA N29). The conjugates (OPS-rEPA N29) were further purified and evaluated for their immunogenicity. The results of ELISA showed that the conjugates evoked higher titers of IgG than OPS, suggesting that rEPAN29 increased immunogenicity of OPS significantly as a carrier. Moreover, three injections with 3-week interval evoked slightly higher titers of IgG than three injections with 2-week interval. However, injection of excess conjugates could not evoke higher titers of IgG against lipid polysaccharide (LPS). In summary, our study provides a new strategy for preparing polysaccharides-protein conjugate vaccines as well as similar bio-conjugate vaccines of other Gram-negative pathogens.

  17. Construction and characterization of replication-deficient adenoviral vector containing the cDNA for human VEGF165 in an antisense orientation%反义VEGF165腺病毒重组体的构建及鉴定

    Institute of Scientific and Technical Information of China (English)

    王家宁; 黄永章; 王俊峰; 王卫民; 李瑞明; 张群林

    2001-01-01

    objective To construct the recombinant adenovirus vectorcontaining the cDNA for hVEGF165 in an antisense orientation for future study of tumor treatment by antisense hVEGF165 RNA strategy.Methods The VEGF165 cDNA has been extracted from pUCCAGGS/hVEGF165 with EcoRI and then inserted in an antisense orientation into the E1-deleted expression plasmid pHCMVsp1A shuttle vector, called pAd-ahVEGF165. pAd-ahVEGF165 was cotransfected with the plasmid pJM17 into the transformed human embryonic kidney cell line 293 cells by liposome-mediated method. Homologous recombination of the pAd-ahVEGF165 and pJM17 in 293 cells replaced the E1 region with the expression cassette from pAd-ahVEGF165. Ad-ahVEGF165 was confirmed by PCR.Ad-ahVEGF165 was propagated in 293 cells and then underwent CsCl density purification. subsequently, the preparations were didalyzed in dialysis buffer. The titer of each viral stock was determined by measuring the absorbance at 260nm. Results VEGF165 cDNA was successfully inserted into the shuttle vector pHCMVSPIA. pAd-ahVEGF165 was confirmed by NcoI and XhoI digestion. Ad-ahVEGF165 was characterized by PCR coamplification. The virus titer was 5.6×1011pfu/ml. Conclusions Ad-ahVEGF165 containing the antisense VEGF165 sequence was successfully constructd.This investigation provides the basis for future study of tumor treatment based on antisense VEGF RNA strategy.%目的:构建含人反义血管内皮生长因子165(VEGF165)基因的重组腺病毒载体,为采用反义VEGF165RNA防治肿瘤的研究奠定基础。方法:将人VEGF165 cDNA反向插入到穿梭质粒pHCMVSP1A的CMV启动子之下,即pAd-ahVEGF165。后者与pJM17通过脂质体共转染293细胞,经同源重组获得含人反义VEGF165基因的重组腺病毒Ad-ahVEGF165。通过PCR共扩增法鉴别Ad-ahVEGF165的正确与否。根据260nm的紫外光吸收值计算病毒滴度。结果:VEGF165 cDNA成功地反向插入了pHCMVSP1A载体,以重组病毒基因组DNA

  18. Construction of adeno-associated virus coexpression system for human angiopoietin-1 and VEGF gene

    Institute of Scientific and Technical Information of China (English)

    陈德杰; 谭最; 谢友利; 刘芳

    2004-01-01

    Background Ischemic disease is one of the leading causes of death in the world. In order to further study gene therapy for ischemic disease, we constructed a recombinant plasmid for co-expression of human angiopoietin-1 and vascular endothelial growth factor 165 (VEGF165) gene in adeno-associated virus (AAV) gene delivery system.Methods Human angiopoietin 1 and VEGF165 gene were obtained using PCR. The upstream of angiopoietin 1 contained restriction enzyme site Hind Ⅲ, and the downstream of angiopoietin 1contained restriction enzyme site BamH Ⅰ. The upstream of VEGF165 contained restriction enzyme site Bgl Ⅱ, and the downstream of VEGF165 contained restriction enzyme site BamH Ⅰ . Using the multiple cloning sites (MCS) in plasmid pZero ++ such as BamH Ⅰ , Bgl Ⅱ, Hind Ⅲ, Not Ⅰ , XhoⅠ,Xba Ⅰ , Sal Ⅰ , BspH Ⅰ , Ksp Ⅰ and the corresponding MCS in plasmid pAAV-MCS, angiopoietin 1 and VEGF165 gene were subcloned into pAAV-MCS.Results DNA sequencing revealed that the PCR- amplified angiopoietin 1 and VEGF165 were consistent with NCBI Gene Bank. The recombinant plasmid was identified using PCR and digestion,which proved to be consistent with our hypothesis. In recombinant plasmid, angiopoietin1 and VEGF possessed a CMV promoter and polyA terminator system respectively, thus assuring co-expression of the two genes.Conclusion Successful construction of AAV co-expression system for human angiopoietin 1 and VEGF165 gene will provide the foundation for gene therapy to cure severe ischemic disease.

  19. Impaired VEGF Signaling in Lungs with Hypoplastic Esophageal Atresia and Effects on Branching Morphogenesis

    Directory of Open Access Journals (Sweden)

    Xiaomei Liu

    2016-07-01

    Full Text Available Background/Aims: Patients with esophageal atresia (EA and tracheoesophageal fistula (TEF often suffer chronic respiratory tract disease. We previously reported that primary lung maldevelopment caused by deficient branching of embryonal airways in experimental EA-TEF was induced by Adriamycin. In this study, we investigated the Vascular endothelial growth factor (VEGF pathway in the developing lung in an EA-TEF rat model. We further analyzed the effect of recombinant VEGF treatment in vitro on branching morphogenesis of embryo lungs in experimental EA-TEF. Methods: Pregnant rats received either Adriamycin or vehicle on E7, E8 and E9. Lungs were recovered at E15, E18 and E21. Expression of VEGF and receptors (Flk-1 and Flt-1 were assessed by quantitative PCR, immunohistochemistry and immunoblotting. E13 lungs were cultured for 72 hours with 50 ng/mL of recombinant rat VEGF in serum-free medium. The rates of increase in bud count and airway contour were evaluated. Results: Our results showed a significant downregulation of VEGF during pseudoglandular and canalicular stages. In contrast, there were significantly higher levels of the Flt-1 receptor in the canalicular stage, which may represent a compensatory response to decreased VEGF. However, both variables returned to normal levels at the saccular stage. Exogenous VEGF treatment enhanced hypoplastic lung growth, evidenced by the increase in bud count and airway contour. Conclusions: A VEGF signaling defect possibly plays an important role in defective embryonic airway branching. Additionally, VEGF treatment may accelerate lung growth in EA-TEF lungs.

  20. Bioconjugates of PAMAM dendrimers with trans-retinal, pyridoxal, and pyridoxal phosphate

    Directory of Open Access Journals (Sweden)

    Filipowicz A

    2012-09-01

    Full Text Available A Filipowicz, S WołowiecDepartment of Cosmetology, University of Information Technology and Management in Rzeszów, Rzeszów, PolandBackground: Bioconjugates of a polyamidoamine (PAMAM G3 dendrimer and an aldehyde were synthesized as carriers for vitamins A and B6, and the bioavailability of these vitamins for skin nutrition was investigated.Methods: Nuclear magnetic resonance (NMR and ultraviolet-visible methods were used to characterize the structure of the bioconjugates and for monitoring release of pyridoxal (Pyr and pyridoxal phosphate (PLP from these bioconjugates in vitro. A skin model permeation of bioconjugates was also studied in a Franz chamber.Results: A transdermal G3 PAMAM dendrimer was used to synthesize bioconjugates with trans-retinal (Ret, pyridoxal (Pyr, or PLP. These nanomolecules, containing up to four covalently linked Ret, Pyr, or PLP (G34Ret, G34Pyr, and G34PLP, were able to permeate the skin, as demonstrated in vitro using a model skin membrane. PLP and Pyr bound to a macromolecular vehicle were active cofactors for glutamic pyruvic transaminase, as shown by 1H NMR spectral monitoring of the progress of the L-alanine + α-ketoglutarate → glutamic acid + pyruvic acid reaction.Conclusion: PAMAM-PLP, PAMAM-Pyr, and PAMAM-Ret bioconjugates are able to permeate the skin. PLP and Pyr are available as cofactors for glutamic pyruvic transaminase.Keywords: PAMAM, trans-retinal, pyridoxal phosphate, pyridoxal, transamination

  1. MMP-2 Alters VEGF Expression via αVβ3 Integrin-Mediated PI3K/AKT Signaling in A549 Lung Cancer Cells

    OpenAIRE

    2010-01-01

    Vascular endothelial growth factor (VEGF) is one of the most important angiogenic growth factors for tumor angiogenesis. Here, we sought to explore whether RNA interference (RNAi) targeting Matrix metalloproteinase-2 (MMP-2) could disrupt VEGF mediated angiogenesis in lung cancer. MMP-2 siRNA inhibited lung cancer cell-induced tube formation of endothelial cells in vitro; addition of recombinant human-MMP-2 restored angiogenesis. MMP-2 transcriptional suppression decreased VEGF, PI3K protein ...

  2. Antiangiogenic VEGF Isoform in Inflammatory Myopathies

    Directory of Open Access Journals (Sweden)

    Nila Volpi

    2013-01-01

    Full Text Available Objective. To investigate expression of vascular endothelial growth factor (VEGF antiangiogenic isoform A-165b on human muscle in idiopathic inflammatory myopathies (IIM and to compare distribution of angiogenic/antiangiogenic VEGFs, as isoforms shifts are described in other autoimmune disorders. Subjects and Methods. We analyzed VEGF-A165b and VEGF-A by western blot and immunohistochemistry on skeletal muscle biopsies from 21 patients affected with IIM (polymyositis, dermatomyositis, and inclusion body myositis and 6 control muscle samples. TGF-β, a prominent VEGF inductor, was analogously evaluated. Intergroup differences of western blot bands density were statistically examined. Endomysial vascularization, inflammatory score, and muscle regeneration, as pathological parameters of IIM, were quantitatively determined and their levels were confronted with VEGF expression. Results. VEGF-A165b was significantly upregulated in IIM, as well as TGF-β. VEGF-A was diffusely expressed on unaffected myofibers, whereas regenerating/atrophic myofibres strongly reacted for both VEGF-A isoforms. Most inflammatory cells and endomysial vessels expressed both isoforms. VEGF-A165b levels were in positive correlation to inflammatory score, endomysial vascularization, and TGF-β. Conclusions. Our findings indicate skeletal muscle expression of antiangiogenic VEGF-A165b and preferential upregulation in IIM, suggesting that modulation of VEGF-A isoforms may occur in myositides.

  3. Prokaryotic Soluble Overexpression and Purification of Human VEGF165 by Fusion to a Maltose Binding Protein Tag.

    Directory of Open Access Journals (Sweden)

    Minh Tan Nguyen

    Full Text Available Human vascular endothelial growth factor (VEGF is a key regulator of angiogenesis and plays a central role in the process of tumor growth and metastatic dissemination. Escherichia coli is one of the most common expression systems used for the production of recombinant proteins; however, expression of human VEGF in E. coli has proven difficult because the E. coli-expressed VEGF tends to be misfolded and forms inclusion bodies, resulting in poor solubility. In this study, we successfully produced semi-preparative amounts of soluble bioactive human VEGF165 (hVEGF. We created seven N-terminal fusion tag constructs with hexahistidine (His6, thioredoxin (Trx, glutathione S-transferase (GST, maltose-binding protein (MBP, N-utilization substance protein A (NusA, human protein disulfide isomerase (PDI, and the b'a' domain of PDI (PDIb'a', and tested each construct for soluble overexpression in E. coli. We found that at 18°C, 92.8% of the MBP-tagged hVEGF to be soluble and that this tag significantly increased the protein's solubility. We successfully purified 0.8 mg of pure hVEGF per 500 mL cell culture. The purified hVEGF is stable after tag cleavage, contains very low levels of endotoxin, and is 97.6% pure. Using an Flk1+ mesodermal precursor cell (MPC differentiation assay, we show that the purified hVEGF is not only bioactive but has similar bioactivity to hVEGF produced in mammalian cells. Previous reports on producing hVEGF in E. coli have all been based on refolding of the protein from inclusion bodies. To our knowledge, this is the first report on successfully expressing and purifying soluble hVEGF in E. coli.

  4. The correlation analysis of the expression of VEGF and the DCs’ infiltration density of gastric cancer tissues

    Institute of Scientific and Technical Information of China (English)

    Feng Yang; Ai-Mei Li; De-Huai Jing

    2016-01-01

    Objective:To investigate the correlation of the expression of vascular endothecial growth factor (VEGF) and the dendritic cells (DCs) infiltration density of tumor tissues in patients with gastric cancer and the correlation of the frequency of DCs of VEGF in gastric cancer tissues and the clinic stages; and to analyze the expressions of HLA-DR and CD86 on peripheral blood monocyte-derived DCs.Methods:Immuno fluorescence method was applied to test the expressions of CD11c and VEGF in gastric cancer tissues and para-carcinoma tissues and enzyme-linked immune sorbent assay was used to detect the concentration of serum VEGF. Peripheral blood mononuclear cells of the gastric cancer patients and healthy people were separated and induced DCs by GM-CSF and IL-4in vitro. Then, the expressions of HLA-DR and CD86 on DCs were tested by flow cytometry. Finally, the recombinant VEGF protein was added into DCs cultures to explore how VEGF affected the expression of CD86. Results: There was a negative correlation between the expression intensity of VEGF on gastric cancer cells and the tumor-infiltrating density of DCs; while there was a certain positive correlation between the frequency of DCs of VEGF and the development of the disease. The concentration of serum VEGF had no association with the density of tumor-infiltrating DCs. As for peripheral blood mononuclear cell, it had a certain induced effect on the decrease of DCs, the expressions of HLA-DR and CD86 and the expression of CD86 on DCs’ cell surface by VEGF.Conclusions:The activities of DCs were inhibited by VEGF secretion of gastric cancer tissues so as to mediate immune escape of the cancer cells. In addition, DCs infiltrated in gastric cancer tissues may secrete VEGF to participate the development of the disease.

  5. Detection of Salmonella typhi utilizing bioconjugated fluorescent polymeric nanoparticles

    Science.gov (United States)

    Jain, Swati; Chattopadhyay, Sruti; Jackeray, Richa; Abid, Zainul; Singh, Harpal

    2016-05-01

    Present work demonstrates effective utilization of functionalized polymeric fluorescent nanoparticles as biosensing probe for the detection of Salmonella typhi bacteria on modified polycarbonate (PC) filters in about 3 h. Antibody modified-PC membranes were incubated with contaminated bacterial water for selective capturing which were detected by synthesized novel bioconjugate probe. Core-shell architecture of polymeric nanoparticles endows them with aqueous stabilization and keto-enolic functionalities making them usable for covalently linking S. typhi antibodies without any crosslinker or activator. Bradford analysis revealed that one nanoparticle has an average of 3.51 × 10-19 g or 21 × 104 bound S. typhi Ab molecules. Analysis of the regions of interest (ROI) in fluorescent micrographs of modified fluoroimmunoassay showed higher detection sensitivity of 5 × 102 cells/mL due to signal amplification unlike conventional naked dye FITC-Ab conjugate. Fluorescence of pyrene dye remained same on immobilization of biomolecules and nanoparticles showed stable fluorescent intensity under prolong exposure to laser owing to protective polymeric layer allowing accurate identification of bacteria. Surface-functionalized PC matrix and fluorescent label NPs permit covalent interactions among biomolecules enhancing signal acquisitions showing higher detection efficiency as compared to conventional microtiter plate-based system. Our novel immunoassay has the potential to be explored as rapid detection method for identifying S. typhi contaminations in water.

  6. Experimental Study of Adenovirus Vector Mediated-hVEGF165 Gene on Prevention of Restenosis after Angioplasty

    Institute of Scientific and Technical Information of China (English)

    刘启功; 陆再英; 岳远坤; 林立; 张卫东; 颜进

    2004-01-01

    This study evaluated the effects of adenovirus vector mediated human vascular endothelial growth factor-165 (hVEGF165) gene on prevention of restenosis after angioplasty. Rabbit models of bilateral carotid artery injury were established by balloon denudation. The recombinant adenoviruses containing hVEGF165 cDNA was directly injected into left side of the injured carotid arteries.On day 3 and week 3 after transfection the expression of VEGF was observed by RT-PCR and immunohistochemistry. The thrombokinesis, reendothelialization (rET) and intimal hyperplasia in carotid arteries were evaluated by computerized image analysis system 3 weeks after gene transfer.The changes in the VEGF gene-treated side were compared with the control side. Our results showed that 3 days and 3 weeks after hVEGF165 gene transfer the VEGF mRNA and antigen expression were detected in vivo. 3 weeks after the transfer, the carotid artery rET was markedly better in the VEGF gene-treated group compared with the control. The thrombokinesis, intima area/media area (I/M), maximal intimal and medial thicknesses (ITmax and MTmax) demonstrated a statistically significant decrease in arteries treated with VEGF gene as compared with the control group. It is concluded that VEGF gene transfer could be achieved by intra-arterial injection of recombinant adenoviruses. It might accelerate the restoration of endothelial integrity, inhibit thrombokinesis and attenuate intimal hyperplasia in the injured arteries after VEGF gene transfer. This procedure could be useful in preventing restenosis after angioplasty.

  7. Reinstate the Damaged VEGF Signaling Pathway with VEGF-activating Transcription Factor

    Institute of Scientific and Technical Information of China (English)

    Yao-guo Yang; Heng Guan; Chang-wei Liu; Yong-jun Li

    2009-01-01

    Objective To investigate the role of vascular endothelial growth factor-activating transcriptional factor(VEGF-ATF)on the VEGF signaling pathway in diabetes mellitus.Methods Totally,20 C57BL/6 mice fed with high fat diet was induced into diabetes mellitus.Ten diabetes mellitus mice received a lower limb muscle injection with VEGF-ATF plasmid,and another ten were as control.VEGF-ATF is an engineered transcription factor designed to increase VEGF expression.Three days later,mice were sacrificed and the injected gastrocnemius was used for analysis.VEGF mRNA and protein expressions were examined by real-time PCR and ELISA respectively.VEGF receptor 2 mRNA expression was tested with RT-PCR.Phosphorylated Akt,Akt,endothelial nitric oxide synthase(eNOS),and phosphorylated eNOS were assessed by western blot.Results At 3 days post-injection,in mice with diabetes mellitus,VEGF gene transfer increased VEGF mRNA copies and VEGF protein expression in injected muscles compared with control;and reinstated the impaired VEGF signaling pathway with increasing the ratios of phosphorylated Akt/Akt and phosphorylated eNOS/eNOS.However,it did not affect the expression of VEGF receptor 2 mRNA.Conclusion Gene transfer with VEGF-ATF is able to reinstate the impaired VEGF downstream pathway,and potentially promote therapeutic angiogenesis in mice with diabetes mcllitus.

  8. Analytical bioconjugates, aptamers, enable specific quantitative detection of Listeria monocytogenes.

    Science.gov (United States)

    Lee, Sang-Hee; Ahn, Ji-Young; Lee, Kyeong-Ah; Um, Hyun-Ju; Sekhon, Simranjeet Singh; Sun Park, Tae; Min, Jiho; Kim, Yang-Hoon

    2015-06-15

    As a major human pathogen in the Listeria genus, Listeria monocytogenes causes the bacterial disease listeriosis, which is a serious infection caused by eating food contaminated with the bacteria. We have developed an aptamer-based sandwich assay (ABSA) platform that demonstrates a promising potential for use in pathogen detection using aptamers as analytical bioconjugates. The whole-bacteria SELEX (WB-SELEX) strategy was adopted to generate aptamers with high affinity and specificity against live L. monocytogenes. Of the 35 aptamer candidates tested, LMCA2 and LMCA26 reacted to L. monocytogenes with high binding, and were consequently chosen as sensing probes. The ABSA platform can significantly enhance the sensitivity by employing a very specific aptamer pair for the sandwich complex. The ABSA platform exhibited a linear response over a wide concentration range of L. monocytogenes from 20 to 2×10(6) CFU per mL and was closely correlated with the following relationship: y=9533.3x+1542.3 (R(2)=0.99). Our proposed ABSA platform also provided excellent specificity for the tests to distinguish L. monocytogenes from other Listeria species and other bacterial genera (3 Listeria spp., 4 Salmonella spp., 2 Vibrio spp., 3 Escherichia coli and 3 Shigella spp.). Improvements in the sensitivity and specificity have not only facilitated the reliable detection of L. monocytogenes at extremely low concentrations, but also allowed for the development of a 96-well plate-based routine assay platform for multivalent diagnostics. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. The Schlemm's canal is a VEGF-C/VEGFR-3-responsive lymphatic-like vessel.

    Science.gov (United States)

    Aspelund, Aleksanteri; Tammela, Tuomas; Antila, Salli; Nurmi, Harri; Leppänen, Veli-Matti; Zarkada, Georgia; Stanczuk, Lukas; Francois, Mathias; Mäkinen, Taija; Saharinen, Pipsa; Immonen, Ilkka; Alitalo, Kari

    2014-09-01

    In glaucoma, aqueous outflow into the Schlemm's canal (SC) is obstructed. Despite striking structural and functional similarities with the lymphatic vascular system, it is unknown whether the SC is a blood or lymphatic vessel. Here, we demonstrated the expression of lymphatic endothelial cell markers by the SC in murine and zebrafish models as well as in human eye tissue. The initial stages of SC development involved induction of the transcription factor PROX1 and the lymphangiogenic receptor tyrosine kinase VEGFR-3 in venous endothelial cells in postnatal mice. Using gene deletion and function-blocking antibodies in mice, we determined that the lymphangiogenic growth factor VEGF-C and its receptor, VEGFR-3, are essential for SC development. Delivery of VEGF-C into the adult eye resulted in sprouting, proliferation, and growth of SC endothelial cells, whereas VEGF-A obliterated the aqueous outflow system. Furthermore, a single injection of recombinant VEGF-C induced SC growth and was associated with trend toward a sustained decrease in intraocular pressure in adult mice. These results reveal the evolutionary conservation of the lymphatic-like phenotype of the SC, implicate VEGF-C and VEGFR-3 as critical regulators of SC lymphangiogenesis, and provide a basis for further studies on therapeutic manipulation of the SC with VEGF-C in glaucoma treatment.

  10. Evidence for Pro-angiogenic Functions of VEGF-Ax.

    Science.gov (United States)

    Xin, Hong; Zhong, Cuiling; Nudleman, Eric; Ferrara, Napoleone

    2016-09-22

    The VEGF-A isoforms play a crucial role in vascular development, and the VEGF signaling pathway is a clinically validated therapeutic target for several pathological conditions. Alternative mRNA splicing leads to the generation of multiple VEGF-A isoforms, including VEGF165. A recent study reported the presence of another isoform, VEGF-Ax, arising from programmed readthrough translation. Compared to VEGF165, VEGF-Ax has a 22-amino-acid extension in the COOH terminus and has been reported to function as a negative regulator of VEGF signaling in endothelial cells, with potent anti-angiogenic effects. Here, we show that, contrary to the earlier report, VEGF-Ax stimulates endothelial cell mitogenesis, angiogenesis, as well as vascular permeability. Accordingly, VEGF-Ax induces phosphorylation of key tyrosine residues in VEGFR-2. Notably, VEGF-Ax was less potent than VEGF165, consistent with its impaired binding to the VEGF co-receptor neuropilin-1.

  11. Miniaturizing VEGF: Peptides mimicking the discontinuous VEGF receptor-binding site modulate the angiogenic response.

    Science.gov (United States)

    De Rosa, Lucia; Finetti, Federica; Diana, Donatella; Di Stasi, Rossella; Auriemma, Sara; Romanelli, Alessandra; Fattorusso, Roberto; Ziche, Marina; Morbidelli, Lucia; D'Andrea, Luca Domenico

    2016-08-08

    The angiogenic properties of VEGF are mediated through the binding of VEGF to its receptor VEGFR2. The VEGF/VEGFR interface is constituted by a discontinuous binding region distributed on both VEGF monomers. We attempted to reproduce this discontinuous binding site by covalently linking into a single molecular entity two VEGF segments involved in receptor recognition. We designed and synthesized by chemical ligation a set of peptides differing in length and flexibility of the molecular linker joining the two VEGF segments. The biological activity of the peptides was characterized in vitro and in vivo showing a VEGF-like activity. The most biologically active mini-VEGF was further analyzed by NMR to determine the atomic details of its interaction with the receptor.

  12. Development of bioconjugated dye-doped poly(styrene-co-maleimide) nanoparticles as a new bioprobe

    CSIR Research Space (South Africa)

    Swanepoel, A

    2015-02-01

    Full Text Available -1 Journal of Materials Chemistry B Development of bioconjugated dye-doped poly(styrene-co- maleimide) nanoparticles as a new bioprobe A. Swanepoel, I. du Preez, T. Mahlangu, A. Chetty and B. Klumperman Abstract Fluorescent dye-doped poly...

  13. Synthesis and antiproliferative activity of new bioconjugates of Salinomycin with amino acid esters.

    Science.gov (United States)

    Antoszczak, Michał; Sobusiak, Maria; Maj, Ewa; Wietrzyk, Joanna; Huczyński, Adam

    2015-09-01

    New Salinomycin (SAL) bioconjugates with amino acid methyl esters were obtained and their antiproliferative activity against cancer cell lines including drug-resistant ones was studied. New compounds exhibit antiproliferative activity towards leukemia and doxorubicin-resistant colon adenocarcinoma cell line and are more effective and less toxic than the commonly currently used anticancer drugs.

  14. Microstructured, functional PVA hydrogels through bioconjugation with oligopeptides under physiological conditions.

    Science.gov (United States)

    Chong, Siow-Feng; Smith, Anton A A; Zelikin, Alexander N

    2013-03-25

    In this work, bioconjugation techniques are developed to achieve peptide functionalization of poly(vinyl alcohol), PVA, as both a polymer in solution and within microstructured physical hydrogels, in both cases under physiological conditions. PVA is unique in that it is one of very few polymers with excellent biocompatibility and safety and has FDA approval for clinical uses in humans. However, decades of development have documented only scant opportunities in bioconjugation with PVA. As such, materials derived thereof fail to answer the call for functional biomaterials for advanced cell culture and tissue engineering applications. To address these limitations, PVA is synthesized with terminal thiol groups and conjugated with thiolated peptides using PVA in solution. Further, microstructured, surface-adhered PVA physical hydrogels are assembled, the available conjugation sites within the hydrogels are quantified, and quantitative kinetic data are collected on peptide conjugation to the hydrogels. The success of bioconjugation in the gel phase is quantified through the use of a cell-adhesive peptide and visualization of cell adhesion on PVA hydrogels as cell culture substrates. Taken together, the presented data establish a novel paradigm in bioconjugation and functionalization of PVA physical hydrogels. Coupled with an excellent safety profile of PVA, these results deliver a superior biomaterial for diverse biomedical applications.

  15. Heterologous expression of a truncated form of human recombinant vascular endothelial growth factor-A and its biological activity in wound healing

    Directory of Open Access Journals (Sweden)

    Mohsen Khaki

    2017-07-01

    Full Text Available Objective(s: Vascular endothelial growth factor (VEGF is one of the most effective proteins in angiogenesis, mesenchymal stem cells (MSCs differentiation and wound healing. These abilities are therapeutic potential of VEGF in diabetic retinopathy, nephropathy and other tissue damage circumstances. In this study, recombinant VEGF was produced in Escherichia coli (E. coli system and then biological activity of this protein was evaluated in animal wound healing. Materials and Methods: E. coli BL21 (DE3 competent cells were transformed with pET32a-VEGF clone and induced by isopropyl-β-D-thio-galactoside (IPTG. The recombinant protein was purified byaffinity chromatography. Recombinant VEGF-A-based ointment (VEGF/Vaseline 0.8 mg/100 w/w was used for external wound (25×15mm thickness healing in animal model. In vivo activity of ointment was evaluated by clinical evidences and cytological microscopic assessment. Results: The recombinant protein with molecular weight of 45 kilodaltons (kDa and concentration of 0.8 mg/ml was produced.Immunoblotting data showed that the antigenic region of VEGF can be expressed in E. coli and the recombinant protein has similar epitopes with close antigenic properties to the natural form. Macroscopic findings and microscopic data showed that the recombinant VEGF-A ointment was effective on excisional wound healing. Conclusion: Recombinant VEGF-A produced by pET32a in E. coli, possesses acceptable structure and has wound healing capability.

  16. 重组人VEGF和前列腺素E1对移植脂肪早期血管生成的作用%The effct of Recombinant human VEGF and prostaglandin E1 on early angiogenesis of the fat transplanted

    Institute of Scientific and Technical Information of China (English)

    梁杰; 赵坤; 吴浩俊; 罗少军

    2009-01-01

    目的 探讨重组人血管内皮细胞生长因子165(hVEGF165)基因质粒转染移植脂肪组织和脂微球化前列腺素E1(Lipo-PGE1)局部应用对移植脂肪颗粒早期血管生成的作用.方法 在颗粒脂肪移植的动物模型中,分别应用PCD-VEGF165质粒、Lipo-PGE1、PCD-VEGF165质粒+Lipo-PGE1.Western blot检测实验组和对照组移植组织的VEGF表达,免疫组化法测定各组移植组织的微血管密度值.结果 各实验组组织中VEGF蛋白表达量及微血管密度值明显高于对照组.结论 VEGF165基因质粒能在移植脂肪组织中表达外源性VEGF蛋白,诱导移植脂肪血管的新生,Lipo-PGE1能有效促进移植脂肪早期血管生成,VEGF165基因质粒和Lipo-PGE1有协同作用.

  17. The Invasion and Reproductive Toxicity of QDs-Transferrin Bioconjugates on Preantral Follicle in vitro

    Directory of Open Access Journals (Sweden)

    Gaixia Xu, Suxia Lin, Wing-Cheung Law, Indrajit Roy, Xiaotan Lin, Shujiang Mei, Hanwu Ma, Siping Chen, Hanben Niu, Xiaomei Wang

    2012-01-01

    Full Text Available The toxicity of QD has been extensively studied over the past decade. However, the potential toxicity of QDs impedes its use for clinical research. In this work, we established a preantral follicle in vitro culture system to investigate the effects of QD-Transferrin (QDs-Tf bioconjugates on follicle development and oocyte maturation. The preantral follicles were cultured and exposed to CdTe/ZnTe QDs-Tf bioconjugates with various concentrations and the reproductive toxicity was assessed at different time points post-treatment. The invasion of QDs-Tf for oocytes was verified by laser scanning confocal microscope. Steroid production was evaluated by immunoassay. C-band Giemsa staining was performed to observe the chromosome abnormality of oocytes. The results showed that the QDs-Tf bioconjugates could permeate into granulosa cells and theca cells, but not into oocyte. There are no obvious changes of oocyte diameter, the mucification of cumulus-oocyte-complexes and the occurrence of aneulpoidy as compared with the control group. However, delay in the antrum formation and decrease in the ratio of oocytes with first polar body were observed in QDs-Tf-treated groups. The matured oocytes with first polar body decreased significantly by ~16% (from 79.6±10 % to 63±2.9 % when the concentration of QDs-Tf bioconjugates exceeded 2.89 nmol·L-1 (P < 0.05. Our results implied that the CdTe/ZnTe QDs-Tf bioconjugates were reproductive toxic for follicle development, and thus also revealed that this in vitro culture system of preantral follicle is a highly sensitive tool for study on the reproductive toxicity of nanoparticles.

  18. VEGF internalization is not required for VEGFR-2 phosphorylation in bioengineered surfaces with covalently linked VEGF

    Science.gov (United States)

    Anderson, Sean M.; Shergill, Bhupinder; Barry, Zachary T.; Manousiouthakis, Eleana; Chen, Tom T.; Botvinick, Elliot; Platt, Manu O.; Iruela-Arispe, M. Luisa; Segura, Tatiana

    2011-01-01

    Vascular endothelial growth factor (VEGF) is known to activate proliferation, migration, and survival pathways in endothelial cells through phosphorylation of VEGF receptor-2 (VEGFR-2). VEGF has been incorporated into biomaterials through encapsulation, electrostatic sequestration, and covalent attachment, but the effect of these immobilization strategies on VEGF signaling has not been thoroughly investigated. Further, although growth factor internalization along with the receptor generally occurs in a physiological setting, whether this internalization is needed for receptor phosphorylation is not entirely clear. Here we show that VEGF covalently bound through a modified heparin molecule elicits an extended response of pVEGFR-2 in human umbilical vein endothelial cells (HUVECs) and that the covalent linkage reduces internalization of the growth factor during receptor endocytosis. Optical tweezer measurements show that the rupture force required to disrupt the heparin-VEGF-VEGFR-2 interaction increases from 3–8 pN to 6–12 pN when a covalent bond is introduced between VEGF and heparin. Importantly, by covalently binding VEGF to a heparin substrate, the stability (half-life) of VEGF is extended over three-fold. Here, mathematical models support the biological conclusions, further suggesting that VEGF internalization is significantly reduced when covalently bound, and indicating that VEGF is available for repeated phosphorylation events. PMID:21826315

  19. Myc regulates VEGF production in B cells by stimulating initiation of VEGF mRNA translation.

    Science.gov (United States)

    Mezquita, Pau; Parghi, Sean S; Brandvold, Kimberly A; Ruddell, Alanna

    2005-01-27

    Deregulated c-myc gene expression is associated with many human and animal cancers. Myc overexpression promotes the growth of blood and lymphatic vessels, which is due in part to induction of growth factors including vascular endothelial growth factor (VEGF). We determined that the P493-6 human B-cell line increases VEGF production 10-fold upon Myc overexpression. Myc overexpression in avian B cells similarly resulted in high level VEGF production. Real-time RT-PCR analyses showed that Myc did not alter the VEGF mRNA content of these cell lines, indicating that a post-transcriptional mechanism regulates VEGF production. VEGF mRNA translation was examined by RT-PCR analysis of monosome and polysome sucrose gradient fractions from Myc-on and Myc-off P493-6 cells. Myc increased VEGF mRNA translation initiation, as VEGF mRNA loading onto polysomes increased 14-fold in Myc-on cells, and the number of ribosomes loaded per VEGF mRNA increased threefold. This translational regulation is specific to VEGF mRNA, as total polysomes show the same sucrose gradient profile in Myc-on and Myc-off cells, with no change in the percent ribosomes in polysomes, or in the number of ribosomes per polysomal mRNA. Myc stimulates VEGF production by a rapamycin- and LY294002-sensitive pathway, which does not involve alteration of eIF4E activity.

  20. Direct electrochemistry and electrocatalysis of a glucose oxidase-functionalized bioconjugate as a trace label for ultrasensitive detection of thrombin.

    Science.gov (United States)

    Bai, Lijuan; Yuan, Ruo; Chai, Yaqin; Yuan, Yali; Wang, Yan; Xie, Shunbi

    2012-11-18

    For the first time, a glucose oxidase-functionalized bioconjugate was prepared and served as a new trace label through its direct electrochemistry and electrocatalysis in a sandwich-type electrochemical aptasensor for ultrasensitive detection of thrombin.

  1. Inhibiting angiogenesis with human single-chain variable fragment antibody targeting VEGF.

    Science.gov (United States)

    Hosseini, Hossien; Rajabibazl, Masoumeh; Ebrahimizadeh, Walead; Dehbidi, Gholamreza Rafiei

    2015-01-01

    Vascular endothelial growth factor (VEGF) is a highly specific angiogenesis factor which has crucial roles in the angiogenesis of tumors. Anti-angiogenesis agents can inhibit growth and metastasis of tumor cells. Single-chain variable fragments (scFv) have the same affinity as whole antibodies and smaller size, thus result in more tissue permeability and higher production yield. In this research we aim to isolate a human scFv antibody against VEGF that inhibits angiogenesis. For that, we have used human scFv phage library to isolate a specific scFv antibody against binding site of VEGF. The human scFv phage library was amplified according to the manufacture protocol and panned against recombinant VEGF. ScFv antibody was isolated after five rounds of panning. Phage ELISA was used for detection of the highest affinity binder (HR6). Soluble HR6 scFv was expressed in non-suppressor strain of Escherichia coli HB2151 and purified using Ni-NTA chromatography. In vivo and in vitro function of the HR6 scFv was analyzed by chorioallantoic membrane assay and endothelial cell proliferation assay on VEGF stimulated HUVECs. Result of the cross reactivity showed that HR6 scFv specifically bounds to VEGF. The affinity was calculated to be 1.8×10(-7)M. HR6 could stop HUVEC proliferation in a dose dependent manner and anti-angiogenesis activity was observed using 10μg of HR6 in chorioallantoic membrane assay. In this work, we demonstrate that a HR6 scFv selected from human library phage display specifically blocks VEGF signaling, furthermore, this scFv has an anti-angiogenesis effect and because of its small size has more tissue diffusion. The HR6 antibody was isolated form a human library thus, it is not immunogenic for humans and could serve as a potential therapeutic agent in cancer.

  2. Detection of biomolecules and bioconjugates by monitoring rotated grating-coupled surface plasmon resonance

    CERN Document Server

    Szalai, Aniko; Somogyi, Aniko; Szenes, Andras; Banhelyi, Balazs; Csapo, Edit; Dekany, Imre; Csendes, Tibor; Csete, Maria

    2016-01-01

    Plasmonic biosensing chips were prepared by fabricating wavelength-scaled dielectric-metal interfacial gratings on thin polycarbonate films covered bimetal layers via two-beam interference laser lithography. Lysozyme (LYZ) biomolecules and gold nanoparticle (AuNP-LYZ) bioconjugates with 1:5 mass ratio were seeded onto the biochip surfaces. Surface plasmon resonance spectroscopy was performed before and after biomolecule seeding in a modified Kretschmann-arrangement by varying the azimuthal and polar angles to optimize the conditions for rotated grating-coupling. The shift of secondary and primary resonance peaks originating from rotated grating-coupling phenomenon was monitored to detect the biomolecule and bioconjugate adherence. Numerical calculations were performed to reproduce the measured reflectance spectra and the resonance peak shifts caused by different biocoverings. Comparison of measurements and calculations proved that monitoring the narrower secondary peaks under optimal rotated-grating coupling ...

  3. Synthesis and Bioconjugation of Gold Nanoparticles as Potential Molecular Probes for Light-Based Imaging Techniques

    Directory of Open Access Journals (Sweden)

    Raja Gopal Rayavarapu

    2007-01-01

    Full Text Available We have synthesized and characterized gold nanoparticles (spheres and rods with optical extinction bands within the “optical imaging window.” The intense plasmon resonant driven absorption and scattering peaks of these nanoparticles make them suitable as contrast agents for optical imaging techniques. Further, we have conjugated these gold nanoparticles to a mouse monoclonal antibody specific to HER2 overexpressing SKBR3 breast carcinoma cells. The bioconjugation protocol uses noncovalent modes of binding based on a combination of electrostatic and hydrophobic interactions of the antibody and the gold surface. We discuss various aspects of the synthesis and bioconjugation protocols and the characterization results of the functionalized nanoparticles. Some proposed applications of these potential molecular probes in the field of biomedical imaging are also discussed.

  4. Construction and identification of recombinant adenovirus-mediated gene transfer system for rat vascular endothelial growth factor

    Institute of Scientific and Technical Information of China (English)

    Hongyu Yang; Hong Qi; Junjie Zou; Xiwei Zhang

    2008-01-01

    Objective: To construct the recombinant adenovirus vector carrying rat vascular endothelial growth factor(VEGF), as preparation for genetic transfection that follows. Methods: Rat VEGF was obtained by using RT-PCR amplification and then cloned into the shutter plasmid pDC316. Subsequently, this newly constructed plasmid pDC316-VEGF, after identification by nuclease digestion analysis and sequencing analysis, was transfected into human embryonic kidney cells HEK293 by Lipofectamine 2000 mediation, together with adenovirus-packaging plasmid pBHGE3. Based on the homologous recombination of the two plasmids within HEK293 cells, the recombinant adenovirus vector carrying VEGF and VDC316-VEGF was created. VDC316-VEGF was subsequently identified using PCR, purified using repeated plaque passages, proliferated using freezing and melting within HEK293 cells, and titrated using 50% Tissue Culture Infective Dose(TCID50) assay. Results:The newly constructed recombinant adenovirus was confirmed to carry rat VEGF based on PCR results, and its titration value determined based on TCID50 assay was 3×109 pfu/ml. Conclusion:The recombinant adenovirus carrying rat VEGF was successfully constructed. The newly constructed adenovirus can produce a sufficiently high titration value within HEK293 cells, providing a reliable tool for genetic transfection in further gene therapy researches.

  5. In vitro cytotoxicity of the ternary PAMAM G3–pyridoxal–biotin bioconjugate

    Science.gov (United States)

    Uram, Łukasz; Szuster, Magdalena; Gargasz, Krzysztof; Filipowicz, Aleksandra; Wałajtys-Rode, Elżbieta; Wołowiec, Stanisław

    2013-01-01

    A third-generation polyamidoamine dendrimer (PAMAM G3) was used as a macromolecular carrier for pyridoxal and biotin. The binary covalent bioconjugate of G3, with nine molecules of biotin per one molecule of G3 (G39B), and the ternary covalent bioconjugate of G3, with nine biotin and ten pyridoxal molecules (G39B10P), were synthesized. The biotin and pyridoxal residues of the bioconjugate were available for carboxylase and transaminase enzymes, as demonstrated in the conversion of pyruvate to oxaloacetate and alanine to pyruvate, respectively, by in vitro monitoring of the reactions, using 1H nuclear magnetic resonance spectroscopy. The toxicity of the ternary bioconjugate (BC-PAMAM) was studied in vitro on BJ human normal skin fibroblasts and human squamous cell carcinoma (SCC-15) cell cultures in comparison with PAMAM G3, using three cytotoxicity assays (XTT, neutral red, and crystal violet) and an estimation of apoptosis by confocal microscopy detection. The tests have shown that BC-PAMAM has significantly lower cytotoxicity compared with PAMAM. Nonconjugated PAMAM was not cytotoxic at concentrations up to 5 μM (NR) and 10 μM (XTT), and BC-PAMAM was not cytotoxic up to 50 μM (both assays) for both cell lines. It has been also found that normal fibroblasts were more sensitive than SCC to both PAMAM and BC-PAMAM. The effect of PAMAM and BC-PAMAM on the initiation of apoptosis (PAMAM in fibroblasts at 5 μM and BC-PAMAM at 10 μM in both cell lines) corresponded with cytotoxicity assays for both cell lines. We concluded that normal fibroblasts are more sensitive to the cytotoxic effects of the PAMAM G3 dendrimer and that modification of its surface cationic groups by substitution with biologically active molecules significantly decreases that effect, confirming that PAMAM G3 is a useful candidate as a carrier for active biocompound delivery. PMID:24376351

  6. Peculiarities of Raman scattering in bioconjugated CdSe/ZnS quantum dots

    Energy Technology Data Exchange (ETDEWEB)

    Diaz Cano, A; Jimenez Sandoval, S; Vorobiev, Y; Rodriguez Melgarejo, F [CINVESTAV-IPN, Santiago de Queretaro, Queretaro 76230 (Mexico); Torchynska, T V [ESFM-Instituto Politecnico Nacional, Mexico DF 07738 (Mexico)

    2010-04-02

    The article presents the results of analysis of Raman scattering spectra of non-conjugated and bioconjugated CdSe/ZnS core-shell quantum dots (QDs). Commercial CdSe/ZnS QDs used covered by polymer are characterized by color emission with the maxima at 605-610 nm (2.03-2.05 eV). The bioconjugation process is performed to biomolecules-the antihuman Interleukin 10 (IL10) antibodies (mab). Raman scattering spectra measured at room temperature with excitation by a He-Ne laser line (632.8 nm) demonstrate two groups of peaks: (1) related to the Si substrate at 230-460, 522, 610, 670, 940-1040 cm{sup -1} and (2) to the PEG polymer on the QD surface in the range of 837-3320 cm{sup -1}. It is revealed that the CdSe/ZnS QD bioconjugation to the antihuman Interleukin 10 antibodies is accompanied with the dramatic changes in the intensity of the Raman lines of both types: the intensity of the Si related line increases six- or ten-fold, but the intensity of the polymer related line decreases ten-fold. The models explaining the mentioned effects in Raman scattering spectra have been discussed.

  7. Chemical modifications and bioconjugate reactions of nanomaterials for sensing, imaging, drug delivery and therapy.

    Science.gov (United States)

    Biju, Vasudevanpillai

    2014-02-07

    As prepared nanomaterials of metals, semiconductors, polymers and carbon often need surface modifications such as ligand exchange, and chemical and bioconjugate reactions for various biosensor, bioanalytical, bioimaging, drug delivery and therapeutic applications. Such surface modifications help us to control the physico-chemical, toxicological and pharmacological properties of nanomaterials. Furthermore, introduction of various reactive functional groups on the surface of nanomaterials allows us to conjugate a spectrum of contrast agents, antibodies, peptides, ligands, drugs and genes, and construct multifunctional and hybrid nanomaterials for the targeted imaging and treatment of cancers. This tutorial review is intended to provide an introduction to newcomers about how chemical and bioconjugate reactions transform the surface of nanomaterials such as silica nanoparticles, gold nanoparticles, gold quantum clusters, semiconductor quantum dots, carbon nanotubes, fullerene and graphene, and accordingly formulate them for applications such as biosensing, bioimaging, drug and gene delivery, chemotherapy, photodynamic therapy and photothermal therapy. Nonetheless, controversial reports and our growing concerns about toxicity and pharmacokinetics of nanomaterials suggest the need for not only rigorous in vivo experiments in animal models but also novel nanomaterials for practical applications in the clinical settings. Further reading of original and review articles cited herein is necessary to buildup in-depth knowledge about the chemistry, bioconjugate chemistry and biological applications of individual nanomaterials.

  8. Cytochrome P450 Bioconjugate as a Nanovehicle for Improved Chemotherapy Treatment.

    Science.gov (United States)

    Quester, Katrin; Juarez-Moreno, Karla; Secundino, Isamel; Roseinstein, Yvonne; Alejo, Karla P; Huerta-Saquero, Alejandro; Vazquez-Duhalt, Rafael

    2016-11-28

    Cancer is still a growing public health problem, especially breast cancer that is one of the most important cancers in women. Chemotherapy, even though a successful treatment, is accompanied by severe side effects. Moreover, most of the drugs used for chemotherapy are administered as prodrugs and need to be transformed to the active form by cytochromes P450 (CYPs). In addition, increasing numbers of cancer tissues show lower CYP activity than the surrounding healthy tissues in which prodrugs are preferentially activated causing cytotoxicity. Here, the design of a functionalized cytochrome P450 bioconjugate is reported as nanovehicle for the enzyme direct delivery to the tumor tissue in order to improve the local drug activation. MCF-7 breast cancer cells are treated with CYP-polyethylene glycol bioconjugate functionalized folic acid, where it activates the prodrug tamoxifen and significantly reduces the dose of tamoxifen needed to kill the tumor cells. The CYP bioconjugate covered with polyethylene glycol shows no immunogenic activity. The advantages of increasing the site-specific CYP activity in tumor tissues are discussed.

  9. VEGF regulates TRPC6 channels in podocytes

    DEFF Research Database (Denmark)

    Thilo, Florian; Liu, Ying; Loddenkemper, Christoph;

    2012-01-01

    BACKGROUND: Both, increased plasma concentrations of vascular endothelial growth factor (VEGF) and increased expression of transient receptor potential canonical type 6 (TRPC6) channels in podocytes have been associated with proteinuric kidney diseases. Now, we investigated the hypothesis that VE...

  10. Expression of vascular endothelial growth factor (VEGF) and VEGF-C in serum and tissue of Wilms tumor

    Institute of Scientific and Technical Information of China (English)

    WANG Lei; ZHANG Da; CHEN Xin-rang; FAN Yu-xia; WANG Jia-xiang

    2011-01-01

    Background Angiogenesis and lymphogenesis which were promoted by vascular endothelial growth factor (VEGF)and VEGF-C are important in the growth and metastasis of solid tumors.The high level of VEGF and VEGF-C were distributed in numerous types of cancers,but their distribution and expression in Wilms tumor,the most common pediatric tumor of the kidney,was unclear.Methods To learn about the distribution,mass spectroscopy and immunohistochemistry were used to measure the level of VEGF and VEGF-C in serum and tissue of Wilms tumor.Results The expression level of VEGF in serum of Wilms tumor was the same as in pre-surgery and control,so it was the same case of VEGF-C.Both of these factors were chiefly located in Wilms tumor tissue,but not in borderline and normal.In addition,the higher clinical staging and histopathologic grading were important elements in high expression of VEGF and VEGF-C.Gender,age and the size of tumor have not certainly been implicated in expression level of VEGF and VEGF-C.Conclusions The lymph node metastasis and growth of tumors resulted from angiogenesis and lymphogenesis which were promoted by VEGF and VEGF-C in Wilms tumor.The autocrine and paracrine process of VEGF and VEGF-C were the principal contributor to specific tissues of Wilms tumor but not to the entire body.

  11. VEGF: a potential target for hydrocephalus.

    Science.gov (United States)

    Shim, Joon W; Sandlund, Johanna; Madsen, Joseph R

    2014-12-01

    Growth factors are primarily responsible for the genesis, differentiation and proliferation of cells and maintenance of tissues. Given the central role of growth factors in signaling between cells in health and in disease, it is understandable that disruption of growth factor-mediated molecular signaling can cause diverse phenotypic consequences including cancer and neurological conditions. This review will focus on the specific questions of enlarged cerebral ventricles and hydrocephalus. It is also well known that angiogenic factors, such as vascular endothelial growth factor (VEGF), affect tissue permeability through activation of receptors and adhesion molecules; hence, recent studies showing elevations of this factor in pediatric hydrocephalus led to the demonstration that VEGF can induce ventriculomegaly and altered ependyma when infused in animals. In this review, we discuss recent findings implicating the involvement of biochemical and biophysical factors that can induce a VEGF-mimicking effect in communicating hydrocephalus and pay particular attention to the role of the VEGF system as a potential pharmacological target in the treatment of some cases of hydrocephalus. The source of VEGF secretion in the cerebral ventricles, in periventricular regions and during pathologic events including hydrocephalus following hypoxia and hemorrhage is sought. The review is concluded with a summary of potential non-surgical treatments in preclinical studies suggesting several molecular targets including VEGF for hydrocephalus and related neurological disorders.

  12. VEGF and prostatic cancer: a systematic review.

    Science.gov (United States)

    Botelho, Francisco; Pina, Francisco; Lunet, Nuno

    2010-09-01

    Elevated vascular endothelial growth factor (VEGF) blood concentration reflects its prostatic production, making this a potentially interesting tumour marker to support the decision of submitting a patient for prostatic biopsy. The objective was to review systematically the evidence on the role of VEGF blood concentration in prostate cancer detection. Published studies addressing the relation between serum or plasma VEGF levels and prostate cancer were identified by searching Pubmed, ISI Web of Knowledge, SCOPUS and LILACS up to January 2010, and reviewed following a standardized protocol. Three studies reported higher plasma VEGF (pg/ml) in patients with localized prostate cancer than in healthy controls (7.0 vs. 0.0, 9.9 vs. 2.2, and 210 vs. 26.5, Pprostate cancer patients than in patients with benign prostate hypertrophy (518.9 vs. 267.9, Pbenign prostate hypertrophy, localized or metastatic prostate cancer. The three studies that used controls with previous suspicion of prostatic cancer but a negative biopsy reported non-statistically significant difference in VEGF serum levels (pg/ml) between controls and localized prostate cancer patients (241 vs. 206; 69.5 vs. 55; 215.2 vs. 266.4). Higher VEGF plasma levels are observed in prostatic cancer patients compared with healthy controls, but serum levels do not appear to be useful in differentiating benign from malignant prostatic disease using, as controls, individuals with high risk of prostate cancer and negative biopsy.

  13. LoVo colon cancer cells resistant to oxaliplatin overexpress c-MET and VEGFR-1 and respond to VEGF with dephosphorylation of c-MET.

    Science.gov (United States)

    Mezquita, Belén; Pineda, Estela; Mezquita, Jovita; Mezquita, Pau; Pau, Montserrat; Codony-Servat, Jordi; Martínez-Balibrea, Eva; Mora, Conchi; Maurel, Joan; Mezquita, Cristóbal

    2016-05-01

    Oxaliplatin-resistant LoVo colon cancer cells overexpressing c-MET and VEGFR-1 were selected to study several signaling pathways involved in chemoresistance, as well as the effect of increasing amounts of VEGF in the regulation of c-MET. In comparison with chemosensitive LoVo colon cancer cells, oxaliplatin-resistant cells (LoVoR) overexpress and phosphorylate c-MET, upregulate the expression of transmembrane and soluble VEGFR-1 and, unexpectedly, downregulate VEGF. In addition, LoVoR cells activate other transduction pathways involved in chemoresistance such as Akt, β-catenin-TCF4 and E-cadherin. While c-MET is phosphorylated in LoVoR cells expressing low levels of VEGF, c-MET phosphorylation decreases when recombinant VEGF is added into the culture medium. Inhibition of c-MET by VEGF is mediated by VEGFR-1, since phosphorylation of c-MET in the presence of VEGF is restored after silencing VEGFR-1. Dephosphorylation of c-MET by VEGF suggests that tumors coexpressing VEGFR-1 and c-MET may activate c-MET as a result of anti-VEGF therapy. © 2015 Wiley Periodicals, Inc.

  14. In vitro cytotoxicity of the ternary PAMAM G3–pyridoxal–biotin bioconjugate

    Directory of Open Access Journals (Sweden)

    Uram Ł

    2013-12-01

    Full Text Available Łukasz Uram, Magdalena Szuster, Krzysztof Gargasz, Aleksandra Filipowicz, Elżbieta Wałajtys-Rode, Stanisław Wołowiec Cosmetology Department, University of Information Technology and Management in Rzeszów, Rzeszów, Poland Abstract: A third-generation polyamidoamine dendrimer (PAMAM G3 was used as a macromolecular carrier for pyridoxal and biotin. The binary covalent bioconjugate of G3, with nine molecules of biotin per one molecule of G3 (G39B, and the ternary covalent bioconjugate of G3, with nine biotin and ten pyridoxal molecules (G39B10P, were synthesized. The biotin and pyridoxal residues of the bioconjugate were available for carboxylase and transaminase enzymes, as demonstrated in the conversion of pyruvate to oxaloacetate and alanine to pyruvate, respectively, by in vitro monitoring of the reactions, using 1H nuclear magnetic resonance spectroscopy. The toxicity of the ternary bioconjugate (BC-PAMAM was studied in vitro on BJ human normal skin fibroblasts and human squamous cell carcinoma (SCC-15 cell cultures in comparison with PAMAM G3, using three cytotoxicity assays (XTT, neutral red, and crystal violet and an estimation of apoptosis by confocal microscopy detection. The tests have shown that BC-PAMAM has significantly lower cytotoxicity compared with PAMAM. Nonconjugated PAMAM was not cytotoxic at concentrations up to 5 µM (NR and 10 µM (XTT, and BC-PAMAM was not cytotoxic up to 50 µM (both assays for both cell lines. It has been also found that normal fibroblasts were more sensitive than SCC to both PAMAM and BC-PAMAM. The effect of PAMAM and BC-PAMAM on the initiation of apoptosis (PAMAM in fibroblasts at 5 µM and BC-PAMAM at 10 µM in both cell lines corresponded with cytotoxicity assays for both cell lines. We concluded that normal fibroblasts are more sensitive to the cytotoxic effects of the PAMAM G3 dendrimer and that modification of its surface cationic groups by substitution with biologically active molecules

  15. Human recombinant vascular endothelial growth factor reduces necrosis and enhances hepatocyte regeneration in a mouse model of acetaminophen toxicity.

    Science.gov (United States)

    Donahower, Brian C; McCullough, Sandra S; Hennings, Leah; Simpson, Pippa M; Stowe, Cindy D; Saad, Ali G; Kurten, Richard C; Hinson, Jack A; James, Laura P

    2010-07-01

    We reported previously that vascular endothelial growth factor (VEGF) was increased in acetaminophen (APAP) toxicity in mice and treatment with a VEGF receptor inhibitor reduced hepatocyte regeneration. The effect of human recombinant VEGF (hrVEGF) on APAP toxicity in the mouse was examined. In early toxicity studies, B6C3F1 mice received hrVEGF (50 microg s.c.) or vehicle 30 min before receiving APAP (200 mg/kg i.p.) and were sacrificed at 2, 4, and 8 h. Toxicity was comparable at 2 and 4 h, but reduced in the APAP/hrVEGF mice at 8 h (p toxicity and increased hepatocyte regeneration in APAP toxicity in the mouse. Attenuation of sinusoidal cell endothelial dysfunction and changes in neutrophil dynamics may be operant mechanisms in the hepatoprotection mediated by hrVEGF in APAP toxicity.

  16. The Schlemm’s canal is a VEGF-C/VEGFR-3–responsive lymphatic-like vessel

    Science.gov (United States)

    Aspelund, Aleksanteri; Tammela, Tuomas; Antila, Salli; Nurmi, Harri; Leppänen, Veli-Matti; Zarkada, Georgia; Stanczuk, Lukas; Francois, Mathias; Mäkinen, Taija; Saharinen, Pipsa; Immonen, Ilkka; Alitalo, Kari

    2014-01-01

    In glaucoma, aqueous outflow into the Schlemm’s canal (SC) is obstructed. Despite striking structural and functional similarities with the lymphatic vascular system, it is unknown whether the SC is a blood or lymphatic vessel. Here, we demonstrated the expression of lymphatic endothelial cell markers by the SC in murine and zebrafish models as well as in human eye tissue. The initial stages of SC development involved induction of the transcription factor PROX1 and the lymphangiogenic receptor tyrosine kinase VEGFR-3 in venous endothelial cells in postnatal mice. Using gene deletion and function-blocking antibodies in mice, we determined that the lymphangiogenic growth factor VEGF-C and its receptor, VEGFR-3, are essential for SC development. Delivery of VEGF-C into the adult eye resulted in sprouting, proliferation, and growth of SC endothelial cells, whereas VEGF-A obliterated the aqueous outflow system. Furthermore, a single injection of recombinant VEGF-C induced SC growth and was associated with trend toward a sustained decrease in intraocular pressure in adult mice. These results reveal the evolutionary conservation of the lymphatic-like phenotype of the SC, implicate VEGF-C and VEGFR-3 as critical regulators of SC lymphangiogenesis, and provide a basis for further studies on therapeutic manipulation of the SC with VEGF-C in glaucoma treatment. PMID:25061878

  17. Functional relevance of the switch of VEGF receptors/co-receptors during peritoneal dialysis-induced mesothelial to mesenchymal transition.

    Directory of Open Access Journals (Sweden)

    María Luisa Pérez-Lozano

    Full Text Available Vascular endothelial growth factor (VEGF is up-regulated during mesothelial to mesenchymal transition (MMT and has been associated with peritoneal membrane dysfunction in peritoneal dialysis (PD patients. It has been shown that normal and malignant mesothelial cells (MCs express VEGF receptors (VEGFRs and co-receptors and that VEGF is an autocrine growth factor for mesothelioma. Hence, we evaluated the expression patterns and the functional relevance of the VEGF/VEGFRs/co-receptors axis during the mesenchymal conversion of MCs induced by peritoneal dialysis. Omentum-derived MCs treated with TGF-β1 plus IL-1β (in vitro MMT and PD effluent-derived MCs with non-epithelioid phenotype (ex vivo MMT showed down-regulated expression of the two main receptors Flt-1/VEGFR-1 and KDR/VEGFR-2, whereas the co-receptor neuropilin-1 (Nrp-1 was up-regulated. The expression of the Nrp-1 ligand semaphorin-3A (Sema-3A, a functional VEGF competitor, was repressed throughout the MMT process. These expression pattern changes were accompanied by a reduction of the proliferation capacity and by a parallel induction of the invasive capacity of MCs that had undergone an in vitro or ex vivo MMT. Treatment with neutralizing anti-VEGF or anti-Nrp-1 antibodies showed that these molecules played a relevant role in cellular proliferation only in naïve omentum-derived MCs. Conversely, treatment with these blocking antibodies, as well as with recombinant Sema-3A, indicated that the switched VEGF/VEGFRs/co-receptors axis drove the enhanced invasion capacity of MCs undergoing MMT. In conclusion, the expression patterns of VEGFRs and co-receptors change in MCs during MMT, which in turn would determine their behaviour in terms of proliferation and invasion in response to VEGF.

  18. Nano-graphene oxide carboxylation for efficient bioconjugation applications: a quantitative optimization approach

    Energy Technology Data Exchange (ETDEWEB)

    Imani, Rana; Emami, Shahriar Hojjati, E-mail: semami@aut.ac.ir [Amirkabir University of Technology, Department of Biomedical Engineering (Iran, Islamic Republic of); Faghihi, Shahab, E-mail: shahabeddin.faghihi@mail.mcgill.ca, E-mail: sfaghihi@nigeb.ac.ir [National Institute of Genetic Engineering and Biotechnology, Tissue Engineering and Biomaterials Division (Iran, Islamic Republic of)

    2015-02-15

    A method for carboxylation of graphene oxide (GO) with chloroacetic acid that precisely optimizes and controls the efficacy of the process for bioconjugation applications is proposed. Quantification of COOH groups on nano-graphene oxide sheets (NGOS) is performed by novel colorimetric methylene blue (MB) assay. The GO is synthesized and carboxylated by chloroacetic acid treatment under strong basic condition. The size and morphology of the as-prepared NGOS are characterized by scanning electron microscopy, transmission electron microscopy (TEM), and atomic force microscopy (AFM). The effect of acid to base molar ratio on the physical, chemical, and morphological properties of NGOS is analyzed by Fourier-transformed infrared spectrometry (FTIR), UV–Vis spectroscopy, X-ray diffraction (XRD), AFM, and zeta potential. For evaluation of bioconjugation efficacy, the synthesized nano-carriers with different carboxylation ratios are functionalized by octaarginine peptide sequence (R8) as a biomolecule model containing amine groups. The quantification of attached R8 peptides to graphene nano-sheets’ surface is performed with a colorimetric-based assay which includes the application of 2,4,6-Trinitrobenzene sulfonic acid (TNBS). The results show that the thickness and lateral size of nano-sheets are dramatically decreased to 0.8 nm and 50–100 nm after carboxylation process, respectively. X-ray analysis shows the nano-sheets interlaying space is affected by the alteration of chloroacetic acid to base ratio. The MB assay reveals that the COOH groups on the surface of NGOS are maximized at the acid to base ratio of 2 which is confirmed by FTIR, XRD, and zeta potential. The TNBS assay also shows that bioconjugation of the optimized carboxylated NGOS sample with octaarginine peptide is 2.5 times more efficient compared to bare NGOS. The present work provides evidence that treatment of GO by chloroacetic acid under an optimized condition would create a functionalized high

  19. Characterization of gold nanoparticle bioconjugation by resonance light scattering correlation spectroscopy

    Institute of Scientific and Technical Information of China (English)

    Xin Qiu; Li Chuan Tang; Chao Qing Dong; Ji Cun Ren

    2010-01-01

    Gold nanoparticles(GNPs)have been widely used as probes and nanomaterials in certain biological and biomedical fields thanks to its special physical and chemical properties.However,it is still difficult to characterize GNPs-bioconjugates in solution,which has greatly limited further bioapplications of GNPs.In this study,we reported a single particle method for characterizing GNPsbiomolecules in solution using resonance light scattering correlation spectroscopy(RLSCS).The interaction of GNPs with bovine serum albumin(BSA)and thiol-modified oligonucletides were investigated.

  20. Synthesis and use of 2-[18F]fluoromalondialdehyde, an accessible synthon for bioconjugation

    Energy Technology Data Exchange (ETDEWEB)

    Hooker, Jacob M. [Massachusetts General Hospital, Boston, MA (United States)

    2017-03-16

    We proposed methods for the synthesis and purification of 2-[18F]fluoromalondialdehyde, which will be a readily accessible synthon for bioconjugation. Our achievements in these areas will specifically address a stated goal of the DOE providing a transformational technology for macromolecule radiolabeling. Accomplishment of our aims will serve both DOE mission-related research as well as nuclear medicine research supported by the NIH and industry. At the heart of our proposal is the aim to “improve synthetic methodology for rapidly and efficiently incorporating radionuclides into a wide range of organic compounds.”

  1. The influence of bio-conjugation on photoluminescence of CdSe/ZnS quantum dots

    Energy Technology Data Exchange (ETDEWEB)

    Torchynska, Tetyana V. [ESFM Instituto Politécnico Nacional, Av. Instituto Politécnico Nacional, México, D.F. 07738 (Mexico); Vorobiev, Yuri V. [Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV) Querétaro, Libramiento Norponiente 2000, Fracc. Real de Juriquilla, 76230 Querétaro (Mexico); Makhniy, Victor P. [Yuri Fedkovych Chernivtsi National University, 2 Kotsyubynsky Str., 58012 Chernivtsi (Ukraine); Horley, Paul P., E-mail: paul.horley@cimav.edu.mx [Centro de Investigación en Materiales Avanzados, S.C. (CIMAV), Chihuahua/Monterrey, 120 Avenida Miguel de Cervantes, 31109 Chihuahua (Mexico)

    2014-11-15

    We report a considerable blue shift in the luminescence spectra of CdSe/ZnS quantum dots conjugated to anti-interleukin-10 antibodies. This phenomenon can be explained theoretically by accounting for bio-conjugation as a process causing electrostatic interaction between a quantum dot and an antibody, which reduces effective volume of the dot core. To solve the Schrödinger equation for an exciton confined in the quantum dot, we use mirror boundary conditions that were successfully tested for different geometries of quantum wells.

  2. Cellular trafficking of quantum dot-ligand bioconjugates and their induction of changes in normal routing of unconjugated ligands

    DEFF Research Database (Denmark)

    Tekle, Christina; van Deurs, Bo; Sandvig, Kirsten

    2008-01-01

    :Qdots were internalized by clathrin-dependent endocytosis as fast as Tf, but their recycling was blocked. Unlike Shiga toxin, the Shiga:Qdot bioconjugate was not routed to the Golgi apparatus. The internalized ricin:Qdot bioconjugates localized to the same endosomes as ricin itself but could...... not be visualized in the Golgi apparatus. Importantly, we find that the endosomal accumulation of ricin:Qdots affects endosome-to-Golgi transport of both ricin and Shiga toxin: Transport of ricin was reduced whereas transport of Shiga toxin was increased. In conclusion, the data reveal that, although coupling...

  3. Comparing protein VEGF inhibitors: In vitro biological studies

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Lanlan; Liang, Xiao Huan [Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080 (United States); Ferrara, Napoleone, E-mail: nf@gene.com [Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080 (United States)

    2011-05-06

    Highlights: {yields} VEGF is a mediator of angiogenesis. {yields} VEGF inhibitors have clinical applications in cancer and eye disorders. {yields} Five protein VEGF inhibitors were compared for their ability to inhibit. {yields} VEGF-induced activities in cultured endothelial cells. -- Abstract: VEGF inhibitors are widely used as a therapy for tumors and intravascular neovascular disorders, but limited and conflicting data regarding their relative biological potencies are available. The purpose of the study is to compare different protein VEGF inhibitors for their ability to inhibit VEGF-stimulated activities. We tested ranibizumab, the full-length variant of ranibizumab (Mab Y0317), bevacizumab, the VEGF-TrapR1R2 and Flt(1-3)-IgG in bioassays measuring VEGF-stimulated proliferation of bovine retinal microvascular endothelial cells or chemotaxis of human umbilical vein endothelial cells (HUVEC). The inhibitors were also compared for their ability to inhibit MAP kinase activation in HUVECs following VEGF addition. Ranibizumab, VEGF-TrapR1R2 and Flt(1-3)-IgG had very similar potencies in the bioassays tested. Bevacizumab was over 10-fold less potent than these molecules. Mab Y0317 was over 30-fold more potent than bevacizumab. The findings reported in this manuscript describe important intrinsic characteristics of several VEGF inhibitors that may be useful to design and interpret preclinical or clinical studies.

  4. Potentiometric urea biosensor based on an immobilised fullerene-urease bio-conjugate.

    Science.gov (United States)

    Saeedfar, Kasra; Heng, Lee Yook; Ling, Tan Ling; Rezayi, Majid

    2013-12-06

    A novel method for the rapid modification of fullerene for subsequent enzyme attachment to create a potentiometric biosensor is presented. Urease was immobilized onto the modified fullerene nanomaterial. The modified fullerene-immobilized urease (C60-urease) bioconjugate has been confirmed to catalyze the hydrolysis of urea in solution. The biomaterial was then deposited on a screen-printed electrode containing a non-plasticized poly(n-butyl acrylate) (PnBA) membrane entrapped with a hydrogen ionophore. This pH-selective membrane is intended to function as a potentiometric urea biosensor with the deposition of C60-urease on the PnBA membrane. Various parameters for fullerene modification and urease immobilization were investigated. The optimal pH and concentration of the phosphate buffer for the urea biosensor were 7.0 and 0.5 mM, respectively. The linear response range of the biosensor was from 2.31 × 10-3 M to 8.28 × 10-5 M. The biosensor's sensitivity was 59.67 ± 0.91 mV/decade, which is close to the theoretical value. Common cations such as Na+, K+, Ca2+, Mg2+ and NH4+ showed no obvious interference with the urea biosensor's response. The use of a fullerene-urease bio-conjugate and an acrylic membrane with good adhesion prevented the leaching of urease enzyme and thus increased the stability of the urea biosensor for up to 140 days.

  5. Bioconjugated PLGA-4-arm-PEG branched polymeric nanoparticles as novel tumor targeting carriers

    Science.gov (United States)

    Ding, Hong; Yong, Ken-Tye; Roy, Indrajit; Hu, Rui; Wu, Fang; Zhao, Lingling; Law, Wing-Cheung; Zhao, Weiwei; Ji, Wei; Liu, Liwei; Bergey, Earl J.; Prasad, Paras N.

    2011-04-01

    In this study, we have developed a novel carrier, micelle-type bioconjugated PLGA-4-arm-PEG branched polymeric nanoparticles (NPs), for the detection and treatment of pancreatic cancer. These NPs contained 4-arm-PEG as corona, and PLGA as core, the particle surface was conjugated with cyclo(arginine-glycine-aspartate) (cRGD) as ligand for in vivo tumor targeting. The hydrodynamic size of the NPs was determined to be 150-180 nm and the critical micellar concentration (CMC) was estimated to be 10.5 mg l - 1. Our in vitro study shows that these NPs by themselves had negligible cytotoxicity to human pancreatic cancer (Panc-1) and human glioblastoma (U87) cell lines. Near infrared (NIR) microscopy and flow cytometry demonstrated that the cRGD conjugated PLGA-4-arm-PEG polymeric NPs were taken up more efficiently by U87MG glioma cells, over-expressing the αvβ3 integrin, when compared with the non-targeted NPs. Whole body imaging showed that the cRGD conjugated PLGA-4-arm-PEG branched polymeric NPs had the highest accumulation in the pancreatic tumor site of mice at 48 h post-injection. Physical, hematological, and pathological assays indicated low in vivo toxicity of this NP formulation. These studies on the ability of these bioconjugated PLGA-4-arm-PEG polymeric NPs suggest that the prepared polymeric NPs may serve as a promising platform for detection and targeted drug delivery for pancreatic cancer.

  6. Investigation of novel quantum dots/proteins/cellulose bioconjugate using NSOM and photoluminescence

    Science.gov (United States)

    Zhang, Peng; Ding, Shi-You; Xu, Qi; Smith, Steve; Rumbles, Garry; Himmel, Michael E.

    2004-10-01

    We investigated the engineered bioconjugate of cadmium selenide core/zinc sulfide shell, (CdSe)ZnS, quantum dots (QDs) with genetically modified proteins using near-field scanning optical microscopy (NSOM). A genetically engineered protein polymer was expressed and purified from E. coli. The protein polymer was allowed to self-assemble to the bacterial microcrystalline cellulose surface through the cellulosic binding domain. QDs were then conjugated to the protein/cellulose assembly through interaction with the 6x-histidine tag on the protein. The transmitted near-field optical signals are collected and detected by both a PMT (near-field scanning optical microscopy, NSOM) and a spectrometer (near-field scanning optical spectroscopy, NSOS). Results from the sample containing the QDs/protein/cellulose assemblies suggest that QDs were arrayed along the cellulose surface. The near-field spectroscopic study also showed that the slight change of spectroscopic properties of the QDs upon bioconjugation, indicating the strong interaction between the constructed protein and QDs.

  7. Quantum dot bioconjugates: uptake into cells and induction of changes in normal cellular transport

    Science.gov (United States)

    Iversen, Tore-Geir; Frerker, Nadine; Sandvig, Kirsten

    2009-02-01

    Can quantum dots (QDs) act as relevant intracellular probes to investigate routing of ligands in live cells? To answer this question we studied intracellular trafficking of QDs that were coupled to the plant toxin ricin, Shiga toxin or the ligand transferrin (Tf) by confocal fluorescence microscopy in three different cell lines. The Tf:QDs were internalized but instead of being recycled they accumulated within endosomes in all cell lines. However, for the HEp-2 and SW480 cells a higher fraction colocalized with a lysosomal marker as compared with HeLa cells. The Shiga:QD bioconjugate was internalized slowly and with poor efficiency in the HEp-2 and SW480 cells as compared with HeLa cells, and was not routed to the Golgi apparatus in any of the cell lines. The internalized ricin:QD bioconjugates localized to the same endosomes as ricin itself, but could in contrast to ricin not be visualized in the Golgi apparatus. Importantly, we find that the endosomal accumulation of either ricin:QDs or transferrin:QDs affects endosome-to-Golgi transport of both ricin and Shiga toxin: Transport of ricin was reduced whereas transport of Shiga toxin was increased. In conclusion, the data from different cells reveal that in general these ligand-coupled QD nanoparticles are arrested within endosomes, and somehow perturb the normal endosomal sorting in cells.

  8. Development of bioconjugate from Streptomyces tyrosinase and gold nanoparticles for rapid detection of phenol constituents.

    Science.gov (United States)

    Zainab, Mazhari Bi Bi; Madhusudhan, D N; Raghavendra, H; Dastager, Syed G; Dayanand, Agsar

    2014-11-01

    Most of the phenol compounds are toxic and have been considered as hazardous pollutants. Several physicochemical and biological methods are available to detect and monitor the phenol pollutants in water and soil. In the present study, phenol constituents of winery, paper and plastic industrial effluents were successfully detected employing tyrosinase-gold nanoparticles bioconjugate. The synthesis of extracellular tyrosinase and gold nanoparticles was achieved by a single isolate of Streptomyces sp. DBZ-39. Enhanced production (369.41 IU) of tyrosinase was produced in submerged bioprocess employing response surface method with central composite design. Extracellular gold nanoparticles synthesized (12-18 nm) by Streptomyces sp. DBZ-39 were characterized with TEM, EDAX and FTIR analysis. A rapid detection (within 10 min) of phenol constituents from winery effluents was achieved by bioconjugate, when compared to tyrosinases and gold nanoparticles independently. Streptomyces tyrosinase could exhibit relatively a better performance than commercially available mushroom tyrosinase in the detection of phenol constituents. Winery effluent has shown much higher content (0.98 O.D) of phenol constituents than paper and plastic effluents based on the intensity of color and U.V absorption spectra.

  9. Overview of the main methods used to combine proteins with nanosystems: absorption, bioconjugation, and encapsulation

    Directory of Open Access Journals (Sweden)

    Mariagrazia Di Marco

    2009-12-01

    Full Text Available Mariagrazia Di Marco1, Shaharum Shamsuddin2, Khairunisak Abdul Razak3, Azlan Abdul Aziz4, Corinne Devaux1, Elsa Borghi1, Laurent Levy1, Claudia Sadun51Nanobiotix, Paris, France; 2School of Health Sciences, Health Campus Universiti Sains Malaysia, Kelantan, Malaysia; 3School of Materials and Mineral Resources Engineering, Engineering Campus, 4School of Physics, Universiti Sains Malaysia, Penang, Malaysia; 5Department of Chemistry, Sapienza, University of Rome, Rome, ItalyAbstract: The latest development of protein engineering allows the production of proteins having desired properties and large potential markets, but the clinical advances of therapeutical proteins are still limited by their fragility. Nanotechnology could provide optimal vectors able to protect from degradation therapeutical biomolecules such as proteins, enzymes or specific polypeptides. On the other hand, some proteins can be also used as active ligands to help nanoparticles loaded with chemotherapeutic or other drugs to reach particular sites in the body. The aim of this review is to provide an overall picture of the general aspects of the most successful approaches used to combine proteins with nanosystems. This combination is mainly achieved by absorption, bioconjugation and encapsulation. Interactions of nanoparticles with biomolecules and caveats related to protein denaturation are also pointed out. A clear understanding of nanoparticle-protein interactions could make possible the design of precise and versatile hybrid nanosystems. This could further allow control of their pharmacokinetics as well as activity, and safety.Keywords: nanoparticles, drug delivery, proteins, polypeptides, absorption, bioconjugation, encapsulation

  10. Cardiomyocyte VEGF Regulates Endothelial Cell GPIHBP1 to Relocate Lipoprotein Lipase to the Coronary Lumen During Diabetes Mellitus.

    Science.gov (United States)

    Chiu, Amy Pei-Ling; Wan, Andrea; Lal, Nathaniel; Zhang, Dahai; Wang, Fulong; Vlodavsky, Israel; Hussein, Bahira; Rodrigues, Brian

    2016-01-01

    Lipoprotein lipase (LPL)-mediated triglyceride hydrolysis is the major source of fatty acid for cardiac energy. LPL, synthesized in cardiomyocytes, is translocated across endothelial cells (EC) by its transporter glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1). Previously, we have reported an augmentation in coronary LPL, which was linked to an increased expression of GPIHBP1 following moderate diabetes mellitus. We examined the potential mechanism by which hyperglycemia amplifies GPIHBP1. Exposure of rat aortic EC to high glucose induced GPIHBP1 expression and amplified LPL shuttling across these cells. This effect coincided with an elevated secretion of heparanase. Incubation of EC with high glucose or latent heparanase resulted in secretion of vascular endothelial growth factor (VEGF). Primary cardiomyocytes, being a rich source of VEGF, when cocultured with EC, restored EC GPIHBP1 that is lost because of cell passaging. Furthermore, recombinant VEGF induced EC GPIHBP1 mRNA and protein expression within 24 hours, an effect that could be prevented by a VEGF neutralizing antibody. This VEGF-induced increase in GPIHBP1 was through Notch signaling that encompassed Delta-like ligand 4 augmentation and nuclear translocation of the Notch intracellular domain. Finally, cardiomyocytes from severely diabetic animals exhibiting attenuation of VEGF were unable to increase EC GPIHBP1 expression and had lower LPL activity at the vascular lumen in perfused hearts. EC, as the first responders to hyperglycemia, can release heparanase to liberate myocyte VEGF. This growth factor, by activating EC Notch signaling, is responsible for facilitating GPIHBP1-mediated translocation of LPL across EC and regulating LPL-derived fatty acid delivery to the cardiomyocytes. © 2015 American Heart Association, Inc.

  11. Preparation and in vitro characterization of vascular endothelial growth factor (VEGF)-loaded poly(D,L-lactic-co-glycolic acid) microspheres using a double emulsion/solvent evaporation technique.

    Science.gov (United States)

    Karal-Yılmaz, Okşan; Serhatlı, Müge; Baysal, Kemal; Baysal, Bahattin M

    2011-01-01

    Biodegradable Poly(lactic-co-glycolic acid; PLGA), microspheres encapsulating the angiogenic protein recombinant human vascular endothelial growth factor (rhVEGF) were formed to achieve VEGF release in a sustained manner. These microspheres are a promising delivery system which can be used for therapeutic angiogenesis. The PLGA microspheres incorporating two different initial loading amounts of rhVEGF have been prepared by a modified water-in-oil-in-water (w/o/w) double emulsion/solvent evaporation technique. The microspheres have been characterized by particle size distribution, environmental scanning electron microscopy (ESEM), light microscopy, encapsulation efficiency and their degradation was studied in vitro. The rhVEGF released from microspheres was quantified by the competitive enzyme-linked immunosorbent assay (ELISA) and human umbilical vein endothelial cell (HUVEC) proliferation assay was used to assess biological activity of the released VEGF. The microspheres were spherical with diameters of 10-60 µm and the encapsulation efficiency was between 46% and 60%. The release kinetics of rhVEGF was studied for two different amounts: 5 µg VEGF (V5) and 50 µg VEGF (V50) per 500 mg starting polymer. The total protein (VEGF:BSA) release increased up to 4 weeks for two rhVEGF concentrations. The ELISA results showed that the burst release for V5 and V50 microspheres were 4 and 27 ng/mL, respectively. For V5, the microspheres showed an initial burst release, followed by a higher steady-state release until 14 days. VEGF release increased up to 2 weeks for V50 microsphere. HUVEC proliferation assay showed that endothelial cells responded to bioactive VEGF by proliferating and migrating.

  12. Protein-nanoparticle interaction in bioconjugated silver nanoparticles: A transmission electron microscopy and surface enhanced Raman spectroscopy study

    Science.gov (United States)

    Reymond-Laruinaz, Sébastien; Saviot, Lucien; Potin, Valérie; Marco de Lucas, María del Carmen

    2016-12-01

    Understanding the mechanisms of interaction between proteins and noble metal nanoparticles (NPs) is crucial to extend the use of NPs in biological applications and nanomedicine. We report the synthesis of Ag-NPs:protein bioconjugates synthesized in total absence of citrates or other stabilizing agents in order to study the NP-protein interaction. Four common proteins (lysozyme, bovine serum albumin, cytochrome-C and hemoglobin) were used in this work. Transmission electron microscopy (TEM) and surface enhanced Raman spectroscopy (SERS) were mainly used to study these bioconjugated NPs. TEM images showed Ag NPs with sizes in the 5-40 nm range. The presence of a protein layer surrounding the Ag NPs was also observed by TEM. Moreover, the composition at different points of single bioconjugated NPs was probed by electron energy loss spectroscopy (EELS). The thickness of the protein layer varies in the 3-15 nm range and the Ag NPs are a few nanometers away. This allowed to obtain an enhancement of the Raman signal of the proteins in the analysis of water suspensions of bioconjugates. SERS results showed a broadening of the Raman bands of the proteins which we attribute to the contribution of different configurations of the proteins adsorbed on the Ag NPs surface. Moreover, the assignment of an intense and sharp peak in the low-frequency range to Ag-N vibrations points to the chemisorption of the proteins on the Ag-NPs surface.

  13. The VEGF signaling pathway in cancer: the road ahead

    Institute of Scientific and Technical Information of China (English)

    Steven A.Stacker; Marc G.Achen

    2013-01-01

    The vascular endothelial growth factor (VEGF) family of soluble protein growth factors consists of key mediators of angiogenesis and lymphangiogenesis in the context of tumor biology.The members of the family,VEGF-A (also known as VEGF),VEGF-B,VEGF-C,VEGF-D,and placenta growth factor (PIGF),play important roles in vascular biology in both normal physiology and pathology.The generation of a humanized neutralizing antibody to VEGF-A (bevacizumab,also known as Avastin) and the demonstration of its benefit in numerous human cancers have confirmed the merit of an anti-angiogenesis approach to cancer treatment and have validated the VEGF-A signaling pathway as a therapeutic target.Other members of the VEGF family are now being targeted,and their relevance to human cancer and the development of resistance to anti-VEGF-A treatment are being evaluated in the clinic.Here,we discuss the potential of targeting VEGF family members in the diagnosis and treatment of cancer.

  14. Autocrine VEGF isoforms differentially regulate endothelial cell behavior

    Directory of Open Access Journals (Sweden)

    Hideki Yamamoto

    2016-09-01

    Full Text Available Vascular endothelial growth factor A (VEGF is involved in all the essential biology of endothelial cells, from proliferation to vessel function, by mediating intercellular interactions and monolayer integrity. It is expressed as three major alternative spliced variants. In mice, these are VEGF120, VEGF164, and VEGF188, each with different affinities for extracellular matrices and cell surfaces, depending on the inclusion of heparin-binding sites, encoded by exons 6 and 7. To determine the role of each VEGF isoform in endothelial homeostasis, we compared phenotypes of primary endothelial cells isolated from lungs of mice expressing single VEGF isoforms in normoxic and hypoxic conditions. The differential expression and distribution of VEGF isoforms affect endothelial cell functions, such as proliferation, adhesion, migration and integrity, which are dependent on the stability of and affinity to VEGF receptor 2 (VEGFR2. We found a correlation between autocrine VEGF164 and VEGFR2 stability, which is also associated with increased expression of proteins involved in cell adhesion. Endothelial cells expressing only VEGF188, which localizes to extracellular matrices or cell surfaces, presented a mesenchymal morphology and weakened monolayer integrity. Cells expressing only VEGF120 lacked stable VEGFR2 and dysfunctional downstream processes, rendering the cells unviable. Endothelial cells expressing these different isoforms in isolation also had differing rates of apoptosis, proliferation, and signaling via nitric oxide (NO synthesis. These data indicate that autocrine signaling of each VEGF isoform has unique functions on endothelial homeostasis and response to hypoxia, due to both distinct VEGF distribution and VEGFR2 stability, which appears to be, at least partly, affected by differential NO production. This study demonstrates that each autocrine VEGF isoform has a distinct effect on downstream functions, namely VEGFR2-regulated endothelial cell

  15. COX-2, VEGF and tumour angiogenesis.

    LENUS (Irish Health Repository)

    Toomey, D P

    2009-06-01

    Epidemiological evidence suggests a protective effective of regular NSAID use against developing cancer. Cyclooxygenase-2, a target of NSAIDs, is upregulated in many cancers and has been associated with increased VEGF production and angiogenesis. Angiogenesis is the formation of new vessels from existing vasculature and as an essential process for tumour development represents an important therapeutic target. Following an extensive review of the literature this article details the current knowledge on the role of COX-2 in tumorigenesis focusing on its relationship to angiogenesis and VEGF production by tumour cells. While COX-2 is clearly detrimental to prognosis and NSAIDs have a beneficial effect, the possibility of COX-2 independent effects being partly or wholly responsible for this benefit cannot be excluded.

  16. Expression of VEGF(xxx)b, the inhibitory isoforms of VEGF, in malignant melanoma.

    Science.gov (United States)

    Pritchard-Jones, R O; Dunn, D B A; Qiu, Y; Varey, A H R; Orlando, A; Rigby, H; Harper, S J; Bates, D O

    2007-07-16

    Malignant melanoma is the most lethal of the skin cancers and the UK incidence is rising faster than that of any other cancer. Angiogenesis - the growth of new vessels from preexisting vasculature - is an absolute requirement for tumour survival and progression beyond a few hundred microns in diameter. We previously described a class of anti-angiogenic isoforms of VEGF, VEGF(xxx)b, that inhibit tumour growth in animal models, and are downregulated in some cancers, but have not been investigated in melanoma. To determine whether VEGF(xxx)b expression was altered in melanoma, PCR and immunohistochemistry of archived human tumour samples were used. In normal epidermis and in a proportion of melanoma samples, VEGF(xxx)b staining was seen. Some melanomas had much weaker staining. Subsequent examination revealed that expression was significantly reduced in primary melanoma samples (both horizontal and vertical growth phases) from patients who subsequently developed tumour metastasis compared with those who did not (analysis of variance (ANOVA) Pxxx)b expression appears to predict metastatic spread in patients with primary melanoma. These results suggest that there is a switch in splicing as part of the metastatic process, from anti-angiogenic to pro-angiogenic VEGF isoforms. This may form part of a wider metastatic splicing phenotype.

  17. Recombination instability

    DEFF Research Database (Denmark)

    D'Angelo, N.

    1967-01-01

    A recombination instability is considered which may arise in a plasma if the temperature dependence of the volume recombination coefficient, alpha, is sufficiently strong. Two cases are analyzed: (a) a steady-state plasma produced in a neutral gas by X-rays or high energy electrons; and (b) an af...

  18. VEGF Expression in Patellar Tendinopathy: A Preliminary Study

    Science.gov (United States)

    Lian, Øystein; Bahr, Roald; Hart, David A.; Duronio, Vincent

    2008-01-01

    Vascular function and angiogenesis are regulated by vascular endothelial growth factor-A (VEGF). The purpose of this preliminary study was to address the following questions: Is VEGF expression in the patellar tendon more prevalent in patients with patellar tendinopathy than in individuals with normal, pain-free patellar tendons? Which cell populations express VEGF in normal and tendinopathic tendon? Is there a difference in symptom duration between VEGF+ and VEGF− tendons? We collected patellar tendon tissue from 22 patients undergoing open débridement of the patellar tendon and from 10 patients undergoing intramedullary nailing of the tibia. VEGF expression was assessed immunohistochemically. Relevant inflammatory and repair cell types were immunolabeled. VEGF expression was absent from control tendons, but was present in a subset of patients with histopathological evidence of angiofibroblastic tendinosis. VEGF was expressed in the intimal layer of tendon vessels, but was absent in other cell types. Patients demonstrating VEGF expression in the patellar tendon had a shorter symptom duration (12 ± 7.8 months) than patients with no detectable VEGF (32.8 ± 23.5 months). VEGF may contribute to the vascular hyperplasia that is a cardinal feature of symptomatic tendinosis, particularly in cases with more recent onset. PMID:18459027

  19. Angiogenic Type I Collagen Extracellular Matrix Integrated with Recombinant Bacteriophages Displaying Vascular Endothelial Growth Factors.

    Science.gov (United States)

    Yoon, Junghyo; Korkmaz Zirpel, Nuriye; Park, Hyun-Ji; Han, Sewoon; Hwang, Kyung Hoon; Shin, Jisoo; Cho, Seung-Woo; Nam, Chang-Hoon; Chung, Seok

    2016-01-21

    Here, a growth-factor-integrated natural extracellular matrix of type I collagen is presented that induces angiogenesis. The developed matrix adapts type I collagen nanofibers integrated with synthetic colloidal particles of recombinant bacteriophages that display vascular endothelial growth factor (VEGF). The integration is achieved during or after gelation of the type I collagen and the matrix enables spatial delivery of VEGF into a desired region. Endothelial cells that contact the VEGF are found to invade into the matrix to form tube-like structures both in vitro and in vivo, proving the angiogenic potential of the matrix.

  20. Effects of lentivirus-mediated co-transfection of BMP2 gene and VEGF165 gene on osteogenic differentiation of bone marrow-derived mesenchymal stromal cells%慢病毒介导BMP2和VEGF165基因共转染对骨髓基质干细胞成骨分化的影响

    Institute of Scientific and Technical Information of China (English)

    蒋佳; 范存义; 曾炳芳

    2011-01-01

    Objective To investigate the effects of lentivirus-mediated co-transfection of bone morphogenetic protein 2 (BMP2) gene and vascular endothelial growth factor 165 ( VEGF165) gene on osteogenic differentiation of bone marrow-derived mesenchymal stromal cells ( MSCs). Methods The expression lentivirus vectors carrying VEGF165, BMP2 or green fluorescent protein (GFP) gene were constructed respectively, and recombinant lentivirus carrying VEGF165 ( Lv-VEGF), BMP2 ( Lv-BMP) or GFP ( Lv-GFP) were packaged and produced respectively. Rat bone marrow-derived MSCs were isolated and cultured in vitro, and were transfected with Lv-VEGF (VEGF group), Lv-BMP ( BMP group) or Lv-GFP (GFP group), co-transfected with Lv-VEGF and Lv-BMP (BMP + VEGF group), or transfected with no virus ( control group). The expression of VEGF165 and BMP2 mRNA 7 d after transfection and that of osteocalcin (OCN) mRNA 1, 2 and 4 weeks after transfection in MSCs was detected by RT-PCR in each group. The expression of VEGF165 and BMP2 protein 1, 4 and 8 weeks after transfection and that of OCN protein 1, 2 and 4 weeks after transfection in supernatant fluid of culture was detected by ELISA in each group. Alkaline phosphatase (ALP) staining was conducted, and ALP activity of MSCs was measured 14 d after transfection in each group. Results VEGF165 and BMP2 mRNA and protein effectively co-expressed in BMP + VEGF group. There was no significant difference in the expression of BMP2 mRNA and protein between BMP + VEGF group and BMP group (P > 0. 05), and there was also no significant difference in the expression of VEGF165 mRNA and protein between BMP + VEGF group and VEGF group (P >0.05). There was no expression of BMP2 mRNA and protein in control group, GFP group and VEGF group, and there was also no expression of VEGF165 mRNA and protein in control group, GFP group and BMP group. The expression of OCN mRNA and protein in BMP + VEGF group was significantly higher than that in the other groups (P <0.01). The

  1. PRINT: A Protein Bioconjugation Method with Exquisite N-terminal Specificity

    Science.gov (United States)

    Sur, Surojit; Qiao, Yuan; Fries, Anja; O'Meally, Robert N.; Cole, Robert N.; Kinzler, Kenneth W.; Vogelstein, Bert; Zhou, Shibin

    2015-12-01

    Chemical conjugation is commonly used to enhance the pharmacokinetics, biodistribution, and potency of protein therapeutics, but often leads to non-specific modification or loss of bioactivity. Here, we present a simple, versatile and widely applicable method that allows exquisite N-terminal specific modification of proteins. Combining reversible side-chain blocking and protease mediated cleavage of a commonly used HIS tag appended to a protein, we generate with high yield and purity exquisitely site specific and selective bio-conjugates of TNF-α by using amine reactive NHS ester chemistry. We confirm the N terminal selectivity and specificity using mass spectral analyses and show near complete retention of the biological activity of our model protein both in vitro and in vivo murine models. We believe that this methodology would be applicable to a variety of potentially therapeutic proteins and the specificity afforded by this technique would allow for rapid generation of novel biologics.

  2. Chemically robust fluoroalkyl phthalocyanine-oligonucleotide bioconjugates and their GRP78 oncogene photocleavage activity.

    Science.gov (United States)

    Patel, Pradeepkumar; Patel, Hemantbhai H; Borland, Emily; Gorun, Sergiu M; Sabatino, David

    2014-06-18

    The first representative of functionalized fluoroalkyl phthalocyanines, F48H7(COOH)PcZn, is reported. The complex generates (1)O2 affording long-lasting photooxidation of an external substrate without self-decomposition. The carboxylic group couples with an antisense oligonucleotide targeting GRP78 oncogenes, resulting in the F48H7PcZn-cancer targeting oligonucleotide (CTO). The bioconjugated fluorophthalocyanine effectively hybridizes complementary GRP78 DNA and mRNA sequences. Piperidine cleavage assays reveal desired photochemical oligonucleotide oxidative degradation for both F48H7PcZn-CTO:DNA and F48H7PcZn-CTO:mRNA hybrids. This new materials strategy could be extended to other functional fluorinated phthalocyanines-antisense oligonucleotide combinations for long-lasting oncogene-targeting photodynamic therapy.

  3. Stable Poly(methacrylic acid Brush Decorated Silica Nano-Particles by ARGET ATRP for Bioconjugation

    Directory of Open Access Journals (Sweden)

    Marcello Iacono

    2015-08-01

    Full Text Available The synthesis of polymer brush decorated silica nano-particles is demonstrated by activator regeneration by electron transfer atom transfer radical polymerization (ARGET ATRP grafting of poly(tert-butyl methacrylate. ATRP initiator decorated silica nano-particles were obtained using a novel trimethylsiloxane derivatised ATRP initiator obtained by click chemistry. Comparison of de-grafted polymers with polymer obtained from a sacrificial initiator demonstrated good agreement up to 55% monomer conversion. Subsequent mild deprotection of the tert-butyl ester groups using phosphoric acid yielded highly colloidal and pH stable hydrophilic nano-particles comprising approximately 50% methacrylic acid groups. The successful bio-conjugation was achieved by immobilization of Horseradish Peroxidase to the polymer brush decorated nano-particles and the enzyme activity demonstrated in a conversion of o-phenylene diamine dihydrochloride assay.

  4. Design, Synthesis and Applications of Hyaluronic Acid-Paclitaxel Bioconjugatesâ€

    Directory of Open Access Journals (Sweden)

    Rinaldo Marini Bettolo

    2008-02-01

    Full Text Available Paclitaxel (1a, a well known antitumor agent adopted mainly for the treatmentof breast and ovarian cancer, suffers from significant disadvantages such as low solubility,certain toxicity and specific drug-resistance of some tumor cells. To overcome theseproblems extensive research has been carried out. Among the various proposed strategies,the conjugation of paclitaxel (1a to a biocompatible polymer, such as hyaluronic acid(HA, 2, has also been considered. Coupling a bioactive compound to a biocompatiblepolymer offers, in general, many advantages such as better drug solubilization, betterstabilization, specific localization and controlled release. Hereafter the design, synthesisand applications of hyaluronic acid-paclitaxel bioconjugates are reviewed. An overview ofHA-paclitaxel combinations is also given.

  5. Evaluation of bicinchoninic acid as a ligand for copper(I)-catalyzed azide-alkyne bioconjugations.

    Science.gov (United States)

    Christen, Erik H; Gübeli, Raphael J; Kaufmann, Beate; Merkel, Lars; Schoenmakers, Ronald; Budisa, Nediljko; Fussenegger, Martin; Weber, Wilfried; Wiltschi, Birgit

    2012-09-07

    The Cu(I)-catalyzed cycloaddition of terminal azides and alkynes (click chemistry) represents a highly specific reaction for the functionalization of biomolecules with chemical moieties such as dyes or polymer matrices. In this study we evaluate the use of bicinchoninic acid (BCA) as a ligand for Cu(I) under physiological reaction conditions. We demonstrate that the BCA-Cu(I)-complex represents an efficient catalyst for the conjugation of fluorophores or biotin to alkyne- or azide-functionalized proteins resulting in increased or at least equal reaction yields compared to commonly used catalysts like Cu(I) in complex with TBTA (tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine) or BPAA (bathophenanthroline disulfonic acid). The stabilization of Cu(I) with BCA represents a new strategy for achieving highly efficient bioconjugation reactions under physiological conditions in many application fields.

  6. Quantum dot-DNA bioconjugates for fluorescence-resonance-energy-transfer-based biosensing

    Science.gov (United States)

    Medintz, Igor L.; Berti, Lorenzo; Pons, Thomas; Mattoussi, Hedi

    2007-02-01

    Semiconductor quantum dots (QDs) have unique photophysical properties which make them excellent fluorescence resonance energy transfer donors. However, lack of facile methods for conjugating biomolecules such as DNA, proteins and peptides to QDs have limited their applications. In this report, we describe a general procedure for the preparation of a synthetic peptide that can be covalently attached to DNA segments and used to facilitate the self-assembly of the modified DNA onto water soluble QDs. To characterize this conjugation strategy, dye-labeled DNA is first reacted with the synthetic peptide and the resulting peptide-DNA then self-assembled onto QDs. QD attachment is verified by monitoring resonance energy transfer efficiency from the QD donor to the dye-labeled DNA acceptor. QD-DNA bioconjugates assembled using this method may find applications as molecular beacons and hybridization probes.

  7. Potentiometric Urea Biosensor Based on an Immobilised Fullerene-Urease Bio-Conjugate

    Directory of Open Access Journals (Sweden)

    Kasra Saeedfar

    2013-12-01

    Full Text Available A novel method for the rapid modification of fullerene for subsequent enzyme attachment to create a potentiometric biosensor is presented. Urease was immobilized onto the modified fullerene nanomaterial. The modified fullerene-immobilized urease (C60-urease bioconjugate has been confirmed to catalyze the hydrolysis of urea in solution. The biomaterial was then deposited on a screen-printed electrode containing a non-plasticized poly(n-butyl acrylate (PnBA membrane entrapped with a hydrogen ionophore. This pH-selective membrane is intended to function as a potentiometric urea biosensor with the deposition of C60-urease on the PnBA membrane. Various parameters for fullerene modification and urease immobilization were investigated. The optimal pH and concentration of the phosphate buffer for the urea biosensor were 7.0 and 0.5 mM, respectively. The linear response range of the biosensor was from 2.31 × 10−3 M to 8.28 × 10−5 M. The biosensor’s sensitivity was 59.67 ± 0.91 mV/decade, which is close to the theoretical value. Common cations such as Na+, K+, Ca2+, Mg2+ and NH4+ showed no obvious interference with the urea biosensor’s response. The use of a fullerene-urease bio-conjugate and an acrylic membrane with good adhesion prevented the leaching of urease enzyme and thus increased the stability of the urea biosensor for up to 140 days.

  8. Multidentate Polymer Coatings for Compact and Homogeneous Quantum Dots with Efficient Bioconjugation.

    Science.gov (United States)

    Ma, Liang; Tu, Chunlai; Le, Phuong; Chitoor, Shweta; Lim, Sung Jun; Zahid, Mohammad U; Teng, Kai Wen; Ge, Pinghua; Selvin, Paul R; Smith, Andrew M

    2016-03-16

    Quantum dots are fluorescent nanoparticles used to detect and image proteins and nucleic acids. Compared with organic dyes and fluorescent proteins, these nanocrystals have enhanced brightness, photostability, and wavelength tunability, but their larger size limits their use. Recently, multidentate polymer coatings have yielded stable quantum dots with small hydrodynamic dimensions (≤10 nm) due to high-affinity, compact wrapping around the nanocrystal. However, this coating technology has not been widely adopted because the resulting particles are frequently heterogeneous and clustered, and conjugation to biological molecules is difficult to control. In this article we develop new polymeric ligands and optimize coating and bioconjugation methodologies for core/shell CdSe/Cd(x)Zn(1-x)S quantum dots to generate homogeneous and compact products. We demonstrate that "ligand stripping" to rapidly displace nonpolar ligands with hydroxide ions allows homogeneous assembly with multidentate polymers at high temperature. The resulting aqueous nanocrystals are 7-12 nm in hydrodynamic diameter, have quantum yields similar to those in organic solvents, and strongly resist nonspecific interactions due to short oligoethylene glycol surfaces. Compared with a host of other methods, this technique is superior for eliminating small aggregates identified through chromatographic and single-molecule analysis. We also demonstrate high-efficiency bioconjugation through azide-alkyne click chemistry and self-assembly with hexa-histidine-tagged proteins that eliminate the need for product purification. The conjugates retain specificity of the attached biomolecules and are exceptional probes for immunofluorescence and single-molecule dynamic imaging. These results are expected to enable broad utilization of compact, biofunctional quantum dots for studying crowded macromolecular environments such as the neuronal synapse and cellular cytoplasm.

  9. Exploring the flexible chemistry of 4-fluoro-3-nitrophenyl azide for biomolecule immobilization and bioconjugation.

    Science.gov (United States)

    Kumar, Saroj; Kumar, Dileep; Ahirwar, Rajesh; Nahar, Pradip

    2016-10-01

    Bioconjugation and functionalization of polymer surfaces are two major tasks in materials chemistry which are accomplished using a variety of coupling agents. Immobilization of biomolecules onto polymer surfaces and the construction of bioconjugates are essential requirements of many biochemical assays and chemical syntheses. Different linkers with a variety of functional groups are used for these purposes. Among them, the benzophenones, aryldiazirines, and arylazides represent the most commonly used photolinker to produce the desired chemical linkage upon their photo-irradiation. In this review, we describe the versatile applications of 4-fluoro-3-nitrophenyl azide, one of the oldest photolinkers used for photoaffinity labeling in the late 1960s. Surprisingly, this photolinker, historically known as 1-fluoro-2-nitro-4-azidobenzene (FNAB), has remained unexplored for a long time because of apprehension that FNAB forms ring-expanded dehydroazepine as a major product and hence cannot activate an inert polymer. The first evidence of photochemical activation of an inert surface by FNAB through nitrene insertion reaction was reported in 2001, and the FNAB-activated surface was found to conjugate a biomolecule without any catalyst, reagent, or modification. FNAB has distinct advantages over perfluorophenyl azide derivatives, which are contemporary nitrene-generating photolinkers, because of its simple, single-step preparation and ease of thermochemical and photochemical reactions with versatile polymers and biomolecules. Covering these aspects, the present review highlights the flexible chemistry of FNAB and its applications in the field of surface engineering, immobilization of biomolecules such as antibodies, enzymes, cells, carbohydrates, oligonucleotides, and DNA aptamers, and rapid diagnostics. Graphical Abstract An overview of the FNAB-engineered activated polymer surfaces for covalent ligation of versatile biomolecules.

  10. Bioconjugation of lipase and cholesterol oxidase with graphene or graphene oxide

    Energy Technology Data Exchange (ETDEWEB)

    Silva, Rubens A.; Souza, Michele L.; Bloisi, Georgia D.; Corio, Paolo; Petri, Denise F. S., E-mail: dfsp@iq.usp.br [Universidade de São Paulo, Instituto de Química (Brazil)

    2015-04-15

    The catalytic behavior of lipase and cholesterol oxidase (ChOx) in the absence and in the presence of graphene (G) or graphene oxide (GO) was investigated at 24 ± 1 °C and pH 6.5. GO flat sheets (0.5–2 μm) were ∼2-nm thick, while G formed aggregates. The maximum reaction velocity (V{sub max}) values and turnover numbers (k{sub cat}) determined for reactions catalyzed by physical mixtures of lipase (at 0.01 g l{sup −1}) or ChOx (at 0.03 g l{sup −1}) and G (0.012 g l{sup −1}) increased six-fold or doubled, respectively, in comparison to neat enzymes. Circular dichroism (CD) and photoluminescence (PL) spectroscopic measurements revealed the preservation of native secondary structures of enzymes and bioconjugation driven by hydrophobic interaction and energy transfer (redshift) between lipase or ChOx and G, corroborating with the enhanced catalytic behavior. On the other hand, the interactions between GO, which has hydrophilic moieties on the basal plane, and ChOx caused enzyme deactivation, as evidenced by the absence of typical CD signal. At low GO concentration (<0.012 g l{sup −1}), bioconjugates of lipases with GO led to V{sub max} and k{sub cat} values four-fold lower than their counterparts with G, but the GO hydrophilic groups probably favored the affinity for the substrate, because the Michaelis constant (K{sub m}) values decreased in comparison to that of neat lipase. Upon increasing the GO concentration, lipases lost secondary structure and the typical lipase PL bands disappeared.

  11. Growing applications of "click chemistry" for bioconjugation in contemporary biomedical research.

    Science.gov (United States)

    Nwe, Kido; Brechbiel, Martin W

    2009-06-01

    This update summarizes the growing application of "click" chemistry in diverse areas such as bioconjugation, drug discovery, materials science, and radiochemistry. This update also discusses click chemistry reactions that proceed rapidly with high selectivity, specificity, and yield. Two important characteristics make click chemistry so attractive for assembling compounds, reagents, and biomolecules for preclinical and clinical applications. First, click reactions are bio-orthogonal; neither the reactants nor their product's functional groups interact with functionalized biomolecules. Second, the reactions proceed with ease under mild nontoxic conditions, such as at room temperature and, usually, in water. The copper-catalyzed Huisgen cycloaddition, azide-alkyne [3 + 2] dipolar cycloaddition, Staudinger ligation, and azide-phosphine ligation each possess these unique qualities. These reactions can be used to modify one cellular component while leaving others unharmed or untouched. Click chemistry has found increasing applications in all aspects of drug discovery in medicinal chemistry, such as for generating lead compounds through combinatorial methods. Bioconjugation via click chemistry is rigorously employed in proteomics and nucleic research. In radiochemistry, selective radiolabeling of biomolecules in cells and living organisms for imaging and therapy has been realized by this technology. Bifunctional chelating agents for several radionuclides useful for positron emission tomography and single-photon emission computed tomography imaging have also been prepared by using click chemistry. This review concludes that click chemistry is not the perfect conjugation and assembly technology for all applications, but provides a powerful, attractive alternative to conventional chemistry. This chemistry has proven itself to be superior in satisfying many criteria (e.g., biocompatibility, selectivity, yield, stereospecificity, and so forth); thus, one can expect it will

  12. Bioconjugated PLGA-4-arm-PEG branched polymeric nanoparticles as novel tumor targeting carriers

    Energy Technology Data Exchange (ETDEWEB)

    Ding Hong; Yong, Ken-Tye; Roy, Indrajit; Hu Rui; Zhao Lingling; Law, Wing-Cheung; Ji Wei; Liu Liwei; Bergey, Earl J; Prasad, Paras N [Department of Chemistry, Institute for Lasers, Photonics and Biophotonics, University at Buffalo, State University of New York, Buffalo, NY 14260 (United States); Wu Fang [Department of Pharmaceutical Sciences, University at Buffalo, State University of New York, Buffalo, NY 14260 (United States); Zhao Weiwei, E-mail: bergeye@buffalo.edu, E-mail: pnprasad@buffalo.edu [Department of Microbiology and Immunology, University at Buffalo, State University of New York, Buffalo, NY 14215 (United States)

    2011-04-22

    In this study, we have developed a novel carrier, micelle-type bioconjugated PLGA-4-arm-PEG branched polymeric nanoparticles (NPs), for the detection and treatment of pancreatic cancer. These NPs contained 4-arm-PEG as corona, and PLGA as core, the particle surface was conjugated with cyclo(arginine-glycine-aspartate) (cRGD) as ligand for in vivo tumor targeting. The hydrodynamic size of the NPs was determined to be 150-180 nm and the critical micellar concentration (CMC) was estimated to be 10.5 mg l{sup -1}. Our in vitro study shows that these NPs by themselves had negligible cytotoxicity to human pancreatic cancer (Panc-1) and human glioblastoma (U87) cell lines. Near infrared (NIR) microscopy and flow cytometry demonstrated that the cRGD conjugated PLGA-4-arm-PEG polymeric NPs were taken up more efficiently by U87MG glioma cells, over-expressing the {alpha}{sub v{beta}3} integrin, when compared with the non-targeted NPs. Whole body imaging showed that the cRGD conjugated PLGA-4-arm-PEG branched polymeric NPs had the highest accumulation in the pancreatic tumor site of mice at 48 h post-injection. Physical, hematological, and pathological assays indicated low in vivo toxicity of this NP formulation. These studies on the ability of these bioconjugated PLGA-4-arm-PEG polymeric NPs suggest that the prepared polymeric NPs may serve as a promising platform for detection and targeted drug delivery for pancreatic cancer.

  13. Expression of VEGF, b-FGF, and their receptors after injection of VEGF on ischemic heart muscle

    Institute of Scientific and Technical Information of China (English)

    XU Xun-yu; SUN Lin; HAN Tao; CHEN Wen-hong; CUI Yong; LI Yan

    2004-01-01

    Objective: To study the expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) and their receptors after injection of external VEGF on ischemic heart muscle and to investigate the mechanism of therapeutic myocardial angiogenesis of VEGF. Methods: Standard experimental pigs underwent placement of a left circumflex artery ameroid occluder. Six weeks later, the animals in the experimental group were treated with VEGF (20μg) by direct epicardial injection ( n = 8) and other animals in the control group did not receive any treatment ( n = 8).Four weeks after therapy, the animals were evaluated with regard to mRNA and protein expression of VEGF and b-FGF and their receptors by RT-PCR and Western blotting. Results: The mRNA expression of VEGF and b-FGF and their receptors by RT-PCR expressing as percentage of density ratio to the GAPDH control was increased in experimental group versus control group. The protein expression of VEGF and b-FGF and their receptors by Western blot expressing as percentage of density ratio to the Commassie Blue control was increased in experimental group versus control Group. Conclusion: Exogenous VEGF can induce the expression of endogenous VEGF, b-FGF, and their receptors; b-FGF may play a role in the angiogenesis of VEGF.

  14. Deproteinized bone with VEGF gene transfer to facilitate the repair of early avascular necrosis of femoral head of rabbit

    Institute of Scientific and Technical Information of China (English)

    CAO Kai; HUANG Wei; AN Hong; JIANG Dian-ming; SHU Yong; HAN Zhi-min

    2009-01-01

    Objective: To explore a new method for early avascular necrosis of femoral head (AVNFH) therapy.Methods: Sixty-nine AVNFH New Zealand adult rabbits were randomly divided into three groups with equal number. In Group A, deproteinized bone (DPB) that absorbed with recombinant plasmid pcDNA3.1-hVEGF165 was implanted into the drilled tunnel of necrotic femoral head. In Group B, only DPB was implanted. In Group C, only tunnel was drilled without DPB or plasmid implanted. Femoral head specimens were obtained at postoperative 1, 2, 4, 8, 16 weeks. The expression of VEGF165 and collagen I was detected by immunohistochemistry. Bone formation was detected generally by X-ray. Angiogenesis and the repair of the femoral head were observed histologically.Results: The expression of VEGF 165 could be detected 2 weeks after implantation in Group A, but it was not observed in other groups. The result of collagen I expression had a significantly difference 2, 4 and 8 weeks after operation in Group A from those in other groups (P<0.01).X-ray results indicated that there was more bone formation in Group A than in other groups. The regenerated capillary vessels staining result of necrotic femoral head in Group A was significantly different from those in other groups at postoperative 2 and 4 weeks (P<0.01).Conclusions: Transfection ofhVEGF165 gene enhances local angiogenesis and DPB-VEGF compound improves the repair of necrotic femoral head. Deproteinized bone grafting with VEGF gene transfer provides a potential method for the treatment of osteonecrosis.

  15. VEGF Deficit is Involved in Endothelium Dysfunction in Preeclampsia

    Institute of Scientific and Technical Information of China (English)

    周琼; 刘海意; 乔福元; 吴媛媛; 徐京晶

    2010-01-01

    This study examined the association of expression of vascular endothelial growth factor(VEGF),a promoter of angiogenesis,with endothelium dysfunction in preeclampsia.The level of VEGF protein and mRNA in the placenta and peripheral blood samples of 30 preeclampsia patients and 30 normotensive pregnant women was measured by immunohistochemistry,real-time reverse transcriptase-polymerase chain reaction(RT-PCR) and enzyme-linked immunosorbent assay(ELISA),respectively.VEGF expression in the human umbilical vei...

  16. Anti-VEGF Cancer Therapy in Nephrology Practice

    Directory of Open Access Journals (Sweden)

    Hassan Izzedine

    2014-01-01

    Full Text Available Expanded clinical experience with the antivascular endothelial growth factor (VEGF agents has come with increasing recognition of their renal adverse effects. Although renal histology is rarely sought in antiangiogenic-treated cancer patients, kidney damage related to anti-VEGF is now established. Its manifestations include hypertension, proteinuria, and mainly glomerular thrombotic microangiopathy. Then, in nephrology practice, should we continue to perform kidney biopsy, and what should be done with the anti-VEGF agents in case of renal toxicity?

  17. Hypoxia promotes adipose-derived stem cell proliferation via VEGF

    Directory of Open Access Journals (Sweden)

    Phuc Van Pham

    2016-01-01

    Full Text Available Adipose-derived stem cells (ADSCs are a promising mesenchymal stem cell source with therapeutic applications. Recent studies have shown that ADSCs could be expanded in vitro without phenotype changes. This study aimed to evaluate the effect of hypoxia on ADSC proliferation in vitro and to determine the role of vascular endothelial growth factor (VEGF in ADSC proliferation. ADSCs were selectively cultured from the stromal vascular fraction obtained from adipose tissue in DMEM/F12 medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic. ADSCs were cultured under two conditions: hypoxia (5% O2 and normal oxygen (21% O2. The effects of the oxygen concentration on cell proliferation were examined by cell cycle and doubling time. The expression of VEGF was evaluated by the ELISA assay. The role of VEGF in ADSC proliferation was studied by neutralizing VEGF with anti-VEGF monoclonal antibodies. We found that the ADSC proliferation rate was significantly higher under hypoxia compared with normoxia. In hypoxia, ADSCs also triggered VEGF expression. However, neutralizing VEGF with anti-VEGF monoclonal antibodies significantly reduced the proliferation rate. These results suggest that hypoxia stimulated ADSC proliferation in association with VEGF production. [Biomed Res Ther 2016; 3(1.000: 476-482

  18. [VEGF gene expression in transfected human multipotent stromal cells].

    Science.gov (United States)

    Smirnikhina, S A; Lavrov, A V; Bochkov, N P

    2011-01-01

    Dynamics of VEGF gene expression in transfected multipotent stromal cells from adipose tissue was examined using electroporation and lipofection. Differences in the potency and dynamics of plasmid elimination (up to day 9) between cell cultures were observed. All cultures were divided into fast and slow plasmid-eliminating ones. Interculture differences in VEGF expression were detected. The possibility of a 5-6-fold increase of VEGF expression was shown. There were no differences in transfection potency, plasmid elimination dynamics, and VEGF expression after transfection by both nonviral methods.

  19. Genetic variations in VEGF and VEGFR2 and glioblastoma outcome

    DEFF Research Database (Denmark)

    Sjöström, S; Wibom, C; Andersson, U

    2010-01-01

    Vascular endothelial growth factor (VEGF) and its receptors (VEGFR) are central components in the development and progression of glioblastoma. To investigate if genetic variation in VEGF and VEGFR2 is associated with glioblastoma prognosis, we examined blood samples from 154 glioblastoma cases...... collected in Sweden and Denmark between 2000 and 2004. Seventeen tagging single nucleotide polymorphisms (SNPs) in VEGF and 27 in VEGFR2 were genotyped and analysed, covering 90% of the genetic variability within the genes. In VEGF, we found no SNPs associated with survival. In VEGFR2, we found two SNPs...

  20. Genetic variations in VEGF and VEGFR2 and glioblastoma outcome

    DEFF Research Database (Denmark)

    Sjöström, S; Wibom, C; Andersson, U

    2011-01-01

    Vascular endothelial growth factor (VEGF) and its receptors (VEGFR) are central components in the development and progression of glioblastoma. To investigate if genetic variation in VEGF and VEGFR2 is associated with glioblastoma prognosis, we examined blood samples from 154 glioblastoma cases...... collected in Sweden and Denmark between 2000 and 2004. Seventeen tagging single nucleotide polymorphisms (SNPs) in VEGF and 27 in VEGFR2 were genotyped and analysed, covering 90% of the genetic variability within the genes. In VEGF, we found no SNPs associated with survival. In VEGFR2, we found two SNPs...

  1. Recombination monitor

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, S. Y. [Brookhaven National Lab. (BNL), Upton, NY (United States); Blaskiewicz, M. [Brookhaven National Lab. (BNL), Upton, NY (United States)

    2017-02-03

    This is a brief report on LEReC recombination monitor design considerations. The recombination produced Au78+ ion rate is reviewed. Based on this two designs are discussed. One is to use the large dispersion lattice. It is shown that even with the large separation of the Au78+ beam from the Au79+ beam, the continued monitoring of the recombination is not possible. Accumulation of Au78+ ions is needed, plus collimation of the Au79+ beam. In another design, it is shown that the recombination monitor can be built based on the proposed scheme with the nominal lattice. From machine operation point of view, this design is preferable. Finally, possible studies and the alternative strategies with the basic goal of the monitor are discussed.

  2. Determination of vascular endothelial growth factor (VEGF) in circulating blood: significance of VEGF in various leucocytes and platelets

    DEFF Research Database (Denmark)

    Werther, K; Christensen, Ib Jarle; Nielsen, Hans Jørgen

    2002-01-01

    . In corresponding blood samples, automated complete blood count was performed, and the number of each cell type was correlated to VEGF concentrations in plasma, serum and lysed whole blood. Finally, the impact of increasing clotting time on the release of VEGF to serum was analysed. RESULTS: Isolated neutrophils......AIM: The sources of increased vascular endothelial growth factor (VEGF) concentrations in peripheral blood from cancer patients are not known in detail. The aim of the present study was to evaluate correlations between the VEGF content in isolated leucocyte subpopulations and VEGF concentrations...... in plasma, serum and lysed whole blood. METHODS: In 51 colorectal cancer (CRC) patients, circulating T-lymphocytes, B-lymphocytes, monocytes, and granulocytes were isolated by means of immunomagnetic separation. Subsequently, the isolated cells were lysed and VEGF contents in the lysates were determined...

  3. The endogenous anti-angiogenic VEGF isoform, VEGF165b inhibits human tumour growth in mice.

    NARCIS (Netherlands)

    Rennel, E.; Waine, E.; Guan, H.; Schuler, Y.; Leenders, W.P.J.; Woolard, J.; Sugiono, M.; Gillatt, D.; Kleinerman, E.; Bates, D.; Harper, S.

    2008-01-01

    Vascular endothelial growth factor-A is widely regarded as the principal stimulator of angiogenesis required for tumour growth. VEGF is generated as multiple isoforms of two families, the pro-angiogenic family generated by proximal splice site selection in the terminal exon, termed VEGFxxx, and the

  4. Bio-Conjugated Gold Nanoparticle Based SERS Probe for Ultrasensitive Identification of Mosquito-Borne Viruses Using Raman Fingerprinting

    OpenAIRE

    Amber M Paul; Fan, Zhen; Sinha, Sudarson S.; Shi, Yongliang; Le, Linda; Bai, Fengwei; Ray, Paresh C

    2015-01-01

    Dengue virus (DENV) and West Nile virus (WNV) are two well-documented mosquito-borne flaviviruses that cause significant health problems worldwide. Driven by this need, we have developed a bio-conjugated gold nanoparticle (AuNP)-based surface enhanced Raman spectroscopy (SERS) probe for the detection of both DENV and WNV. Reported data demonstrate anti-flavivirus 4G2 antibody conjugated gold nanoparticle (GNP) SERS probe can be used as a Raman fingerprint for the ultrasensitive detection of D...

  5. Novel VEGF decoy receptor fusion protein conbercept targeting multiple VEGF isoforms provide remarkable anti-angiogenesis effect in vivo.

    Directory of Open Access Journals (Sweden)

    Qin Wang

    Full Text Available VEGF family factors are known to be the principal stimulators of abnormal angiogenesis, which play a fundamental role in tumor and various ocular diseases. Inhibition of VEGF is widely applied in antiangiogenic therapy. Conbercept is a novel decoy receptor protein constructed by fusing VEGF receptor 1 and VEGF receptor 2 extracellular domains with the Fc region of human immunoglobulin. In this study, we systematically evaluated the binding affinity of conbercept with VEGF isoforms and PlGF by using anti-VEGF antibody (Avastin as reference. BIACORE and ELISA assay results indicated that conbercept could bind different VEGF-A isoforms with higher affinity than reference. Furthermore, conbercept could also bind VEGF-B and PlGF, whereas Avastin showed no binding. Oxygen-induced retinopathy model showed that conbercept could inhibit the formation of neovasularizations. In tumor-bearing nude mice, conbercept could also suppress tumor growth very effectively in vivo. Overall, our study have demonstrated that conbercept could bind with high affinity to multiple VEGF isoforms and consequently provide remarkable anti-angiogenic effect, suggesting the possibility to treat angiogenesis-related diseases such as cancer and wet AMD etc.

  6. VEGF and colon cancer growth beyond angiogenesis: does VEGF directly mediate colon cancer growth via a non-angiogenic mechanism?

    Science.gov (United States)

    Ahluwalia, Amrita; Jones, Michael K; Matysiak-Budnik, Tamara; Tarnawski, Andrzej S

    2014-01-01

    In this article we review the role of vascular endothelial growth factor (VEGF) in colon cancer growth and the underlying mechanisms. Angiogenesis, the growth of new capillary blood vessels in the body, is critical for tissue injury healing and cancer growth. In 1971, Judah Folkman proposed the concept that tumor growth beyond 2 mm is critically dependent on angiogenesis. Tumors including colon cancers release angiogenic growth factors that stimulate blood vessels to grow into the tumors thus providing oxygen and nutrients that enable exponential growth. VEGF is the most potent angiogenic growth factor. Several studies have highlighted the role of VEGF in colon cancer, specifically in the stimulation of angiogenesis. This role of VEGF is strongly supported by studies showing that inhibition of VEGF using the blocking antibody, bevacizumab, results in decreased angiogenesis and abrogation of cancer growth. In the United States, bevacizumab in combination with chemotherapy is FDA approved for the treatment of metastatic colon cancer. However, the source of VEGF in colon cancer tissue, the mechanisms of VEGF generation in colon cancer cells and the molecular pathways involved in VEGF mediated angiogenesis in colon cancer are not fully known. The possibility that VEGF directly stimulates cancer cell growth in an autocrine manner has not been explored in depth.

  7. Expression of Vascular Endothelial Growth Factor (VEGF) in Human Osteosarcoma Cells Transfected with Adeno-associated Virus-antisense VEGF

    Institute of Scientific and Technical Information of China (English)

    徐卫国; 陈安民; 张衣北; 易成腊

    2004-01-01

    Summary: The expression of protein vascular endothelial growth factor (VEGF) in osteosarcoma cells transfected with adeno-associated virus (rAAV)-antisense VEGF was studied to provide the foundation of osteosarcoma treatment through antivascularization. The rAAV-antisense VEGF at different doses (0, 20, 50, 100, 200, 240 μl) was transfected into osteosarcoma MG-63 cell. The cells and culture supernatants were collected before and after tansfection. The expression of VEGF protein was detected by using immunohistochemical staining (SP) and Western blot. SP and Western-blot tests revealed that the MG-63 Cells transfected with rAAV-antisense VEGF had less staining than those without transfection with rAAV-antisense VEGF, and the staining intensity was negatively correlated with the doses of genes. The corresponding A values of transfected genes with different doses of rAAV-antisense VEGF (0, 20, 50, 100, 200, 240 μA) were 86 614±13 776, 73 245±15 414, 61 078±12 124, 54 657±10 953, 39 802±11 308, 32 014±15 057 respectively,w ith the difference being significant (P<0.05). It was concluded that the expression of VEGF protein in MG-63 cells could be inhibited by rAAV-antisense VEGF.

  8. Synthesis and Photophysical and Photocatalytic Properties of a Highly Fluorinated and Durable Phthalocyanine-Peptide Bioconjugate for Potential Theranostic Applications.

    Science.gov (United States)

    Carrión, Erik N; Santiago, Jenyffer; Sabatino, David; Gorun, Sergiu M

    2017-06-19

    The functionalized, asymmetric fluoro-fluoroalkyl scaffold F48H7COOHPcZn (3) was used to prepare F48H7COOPcZn-6-amino-hexanoate-CTVALPGGYVRVC (5), a Pep42 peptide bioconjugate envisioned for photodynamic therapy, which can specifically target the GRP78 chaperone protein overexpressed and exclusively localized on some cancer cell surfaces. The analogous F48H7COOHPcCu (4) has also been prepared, and its single-crystal X-ray structure was elucidated. Despite reduced steric hindrance relative to the nonfunctionalized, single-site complexes of the F64Pc scaffold, no aggregation was detected in solution via UV-vis spectroscopy, for either 3, 4, or 5, consistent with the lack of π stacking observed for the crystalline 4. The 6-aminohexanoic acid-Pep42 moiety diminishes the fluorescence efficiency of 5, relative to 3, but for singlet oxygen ((1)O2) generation, photochemical hydroperoxidation of β-(-)-citronellol using 5 and 3 occurs with comparable substrate turnover efficiency, albeit at a slower initial triplet oxygen uptake for 5. The bioconjugate 5 is durable; it does not decompose under (1)O2 photoreaction conditions. These results suggest a synthetic coupling pathway for obtaining diverse biotargeting polypeptide-fluorinated phthalocyanine bioconjugates of potential utility as both fluorescence reporters and photocatalysts and highlight the importance of fluorinated scaffolds for generating chemically resilient, catalytic, theranostic materials.

  9. Clinical significance of the VEGF level in urinary bladder carcinoma.

    Science.gov (United States)

    Sankhwar, Monica; Sankhwar, Satya Narayan; Abhishek, Amar; Rajender, Singh

    2015-01-01

    To investigate the correlation of Vascular Endothelial Growth Factor (VEGF) and micro-vessel density (MVD) with urinary bladder tumor and its stage. The study was conducted between January 2010 and December 2012. The study included screening of 122 patients at elevated risk for bladder cancer, of which 35 patients were finally enrolled in the study. Diagnosis was made on the basis of urine cytology, radiological investigation (ultrasound KUB, and CT-scan) and histopathology. Thirty-five normal cancer-free individuals were enrolled as controls. Human VEGF levels were measured using an enzyme linked immunoassay and protein content (pg/mg protein) by Lowry method. SPSS for Windows version 10.0.7 (SPSS, Chicago, IL, USA) was used for statistical analysis of the data. Mean urine VEGF level in the cases was significantly higher in comparison to the control group. There was a direct correlation between VEGF level and tumor stage. Mean urine VEGF values were minimum in the control group (22.75 ± 15.41 pg/mg creatinine) and maximum in stage IV patients (180.15 ± 75.93 pg/mg creatinine). Tissue VEGF levels also showed a similar trend of increase with increase in stage. Urine VEGF level also showed a correlation with tissue VEGF level. Similarly, MVD showed a significant increase with increase in tumor stage. A correlation between bladder cancer and MVD and VEGF suggest that the latter can serve as markers for therapeutic guidance. This is the first study from India on clinical and pathological correlation among urine VEGF, tumor tissue VEGF levels, and Micro Vessel Density (MVD) in urinary bladder cancer patients.

  10. Novel transcriptional regulation of VEGF in inflammatory processes.

    Science.gov (United States)

    Tang, Xiaoren; Yang, Yu; Yuan, Huaiping; You, Jian; Burkatovskaya, Marina; Amar, Salomon

    2013-03-01

    Vascular endothelial growth factor (VEGF) is a critical angiogenic factor affecting endothelial cells, inflammatory cells and neuronal cells. In addition to its well-defined positive role in wound healing, pathological roles for VEGF have been described in cancer and inflammatory diseases (i.e. atherosclerosis, rheumatoid arthritis, inflammatory bowel disease and osteoarthritis). Recently, we showed that transcription factors LITAF and STAT6B affected the inflammatory response. This study builds upon our previous results in testing the role of mouse LITAF and STAT6B in the regulation of VEGF-mediated processes. Cells cotransfected with a series of VEGF promoter deletions along with truncated forms of mLITAF and/or mSTAT6B identified a DNA binding site (between -338 and -305 upstream of the transcription site) important in LITAF and/or STAT6B-mediated transcriptional regulation of VEGF. LITAF and STAT6B corresponding protein sites were identified. In addition, siRNA-mediated knockdown of mLITAF and/or mSTAT6B leads to significant reduction in VEGF mRNA levels and inhibits LPS-induced VEGF secretion in mouse RAW 264.7 cells. Furthermore, VEGF treatment of mouse macrophage or endothelial cells induces LITAF/STAT6B nuclear translocation and cell migration. To translate these observations in vivo, VEGF164-soaked matrigel were implanted in whole-body LITAF-deficient animals (TamLITAF(-/-) ), wild-type mice silenced for STAT6B, and in respective control animals. Vessel formation was found significantly reduced in TamLITAF(-/-) as well as in STAT6B-silenced wild-type animals compared with control animals. The present data demonstrate that VEGF regulation by LITAF and/or STAT6B is important in angiogenesis signalling pathways and may be a useful target in the treatment of VEGF diseases. © 2013 The Authors. Published by Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

  11. Anti-VEGF Treatment Strategies for Wet AMD

    Directory of Open Access Journals (Sweden)

    Jaclyn L. Kovach

    2012-01-01

    Full Text Available Over the past few years, antivascular endothelial growth factor (VEGF therapy has become a standard treatment for neovascular age-related macular degeneration (AMD. During this time, treatment strategies have evolved from a monthly dosing schedule to individualized regimens. This paper will review the currently available anti-VEGF agents and evidence-based treatment strategies.

  12. Anti-VEGF Treatment Strategies for Wet AMD.

    Science.gov (United States)

    Kovach, Jaclyn L; Schwartz, Stephen G; Flynn, Harry W; Scott, Ingrid U

    2012-01-01

    Over the past few years, antivascular endothelial growth factor (VEGF) therapy has become a standard treatment for neovascular age-related macular degeneration (AMD). During this time, treatment strategies have evolved from a monthly dosing schedule to individualized regimens. This paper will review the currently available anti-VEGF agents and evidence-based treatment strategies.

  13. Anti-VEGF Treatment Strategies for Wet AMD

    Science.gov (United States)

    Kovach, Jaclyn L.; Schwartz, Stephen G.; Flynn, Harry W.; Scott, Ingrid U.

    2012-01-01

    Over the past few years, antivascular endothelial growth factor (VEGF) therapy has become a standard treatment for neovascular age-related macular degeneration (AMD). During this time, treatment strategies have evolved from a monthly dosing schedule to individualized regimens. This paper will review the currently available anti-VEGF agents and evidence-based treatment strategies. PMID:22523653

  14. Changing paradigms of anti-VEGF in the Indian scenario

    Directory of Open Access Journals (Sweden)

    P Mahesh Shanmugam

    2014-01-01

    Full Text Available Anti-vascular endothelial growth factors (VEGF agents have revolutionized the treatment of retinal diseases. Use of anti-VEGF agents in the Indian Scenario present some unique challenges considering the absence of compounding pharmacies, poor penetrance of health insurance and limited affordability of the citizens of a developing economy. To study the changing paradigms of anti-VEGF use in the Indian scenario, all articles published by Indian authors, data from web-based surveys amongst Indian vitreo-retinal specialists were reviewed. In the paucity of compounding pharmacies in India, fractionation and injection techniques differ from those of developed countries. Frequent anti-VEGF monotherapy offers the best anatomical and visual results, but economics of scale do not allow the same in the Indian scenario, resulting in PRN dosing and combination of anti-VEGF with laser photocoagulation, being the commonly employed treatment protocols.

  15. Design, synthesis, and evaluation of cisplatin-containing EGFR targeting bioconjugates as potential therapeutic agents for brain tumors

    Directory of Open Access Journals (Sweden)

    Barth RF

    2016-05-01

    Full Text Available Rolf F Barth,1 Gong Wu,1 W Hans Meisen,2 Robin J Nakkula,1 Weilian Yang,1 Tianyao Huo,1 David A Kellough,1 Pravin Kaumaya,3–5 Claudia Turro,6 Lawrence M Agius,7 Balveen Kaur2 1Department of Pathology, 2Department of Neurological Surgery, 3Department of Obstetrics and Gynecology, 4Department of Molecular and Cellular Biochemistry, 5Department of Microbiology, 6Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, USA; 7Department of Pathology, Mater Dei Hospital, University of Malta Medical School, Msida, Malta Abstract: The aim of this study was to evaluate four different platinated bioconjugates containing a cisplatin (cis-diamminedichloroplatinum [cis-DDP] fragment and epidermal growth factor receptor (EGFR-targeting moieties as potential therapeutic agents for the treatment of brain tumors using a human EGFR-expressing transfectant of the F98 rat glioma (F98EGFR to assess their efficacy. The first two bioconjugates employed the monoclonal antibody cetuximab (C225 or Erbitux® as the targeting moiety, and the second two used genetically engineered EGF peptides. C225-G5-Pt was produced by reacting cis-DDP with a fifth-generation polyamidoamine dendrimer (G5 and then linking it to C225 by means of two heterobifunctional reagents. The second bioconjugate (C225-PG-Pt employed the same methodology except that polyglutamic acid was used as the carrier. The third and fourth bioconjugates used two different EGF peptides, PEP382 and PEP455, with direct coordination to the Pt center of the cis-DDP fragment. In vivo studies with C225-G5-Pt failed to demonstrate therapeutic activity following intracerebral (ic convection-enhanced delivery (CED to F98EGFR glioma-bearing rats. The second bioconjugate, C225-PG-Pt, failed to show in vitro cytotoxicity. Furthermore, because of its high molecular weight, we decided that lower molecular weight peptides might provide better targeting and microdistribution within the tumor. Both PEP

  16. VEGF and Pleiotrophin Modulate the Immune Profile of Breast Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Lynn, Kristi D.; Roland, Christina L. [Division of Surgical Oncology, Department of Surgery, Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX, 75390-8593 (United States); Brekken, Rolf A., E-mail: rolf.brekken@utsouthwestern.edu [Division of Surgical Oncology, Department of Surgery, Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX, 75390-8593 (United States); Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, 75390-8593 (United States)

    2010-05-26

    Angiogenesis, the sprouting of the existing vascular network to form new vessels, is required for the growth of solid tumors. For this reason, the primary stimulant of angiogenesis, vascular endothelial growth factor-A (VEGF), is an attractive target for tumor therapy. In fact, there are currently numerous anti-VEGF therapies in clinical development for the treatment of various cancers, including breast cancer. VEGF signals through two primary VEGF receptors, VEGFR1 and VEGFR2. VEGFR2 is the primary angiogenic receptor, and VEGFR1 has been implicated in macrophage chemotaxis and tumor cell survival and invasion. It has only been appreciated recently that the VEGFRs are expressed not only on endothelial cells and tumor cells but also on many host immune cells. Therefore, to better understand the effects of anti-VEGF therapy it is important to consider the effects of VEGF on all cells in the tumor microenvironment, including immune cells. Bevacizumab (Avastin{sup ®}, Genetech), which binds VEGF and inhibits interaction with VEGFR1 and VEGFR2, was approved for the treatment of metastatic HER2/NEU-negative breast cancer in 2008, however, the majority of human mammary tumors are either innately resistant or will acquire resistance to anti-VEGF therapy. This suggests that these tumors activate alternate angiogenesis pathways. Pleiotrophin (PTN) is an important angiogenic cytokine in breast cancer and is expressed at high levels in approximately 60% of human breast tumors. PTN functions as an angiogenic factor and promotes remodeling of the tumor microenvironment as well as epithelial-mesenchymal transition (EMT). In addition, PTN can have profound effects on macrophage phenotype. The present review focuses on the functions of VEGF and PTN on immune cell infiltration and function in breast cancer. Furthermore, we will discuss how anti-VEGF therapy modulates the immune cell profile.

  17. Single-Chain VEGF/Cy5.5 Targeting VEGF Receptors to Indicate Atherosclerotic Plaque Instability

    NARCIS (Netherlands)

    Lam, Ming Kai; Al-Ansari, Sali; van Dam, Gooitzen M.; Tio, Rene A.; Breek, Jan-Cees; Slart, Riemer H. J. A.; Hillebrands, Jan-Luuk; Zeebregts, Clark J.

    2013-01-01

    Unstable plaques may cause clinical events. Plaque destabilization results from the synergy between intraplaque angiogenesis and inflammation. Vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) are considered to be involved in these processes. We investigated the efficacy of the a

  18. Design of Novel Relaxase Substrates Based on Rolling Circle Replicases for Bioconjugation to DNA Nanostructures

    Science.gov (United States)

    Sagredo, Sandra; de la Cruz, Fernando; Moncalián, Gabriel

    2016-01-01

    During bacterial conjugation and rolling circle replication, HUH endonucleases, respectively known as relaxases and replicases, form a covalent bond with ssDNA when they cleave their target sequence (nic site). Both protein families show structural similarity but limited amino acid identity. Moreover, the organization of the inverted repeat (IR) and the loop that shape the nic site differs in both proteins. Arguably, replicases cleave their target site more efficiently, while relaxases exert more biochemical control over the process. Here we show that engineering a relaxase target by mimicking the replicase target, results in enhanced formation of protein-DNA covalent complexes. Three widely different relaxases, which belong to MOBF, MOBQ and MOBP families, can properly cleave DNA sequences with permuted target sequences. Collaterally, the secondary structure that the permuted targets acquired within a supercoiled plasmid DNA resulted in poor conjugation frequencies underlying the importance of relaxase accessory proteins in conjugative DNA processing. Our results reveal that relaxase and replicase targets can be interchangeable in vitro. The new Rep substrates provide new bioconjugation tools for the design of sophisticated DNA-protein nanostructures. PMID:27027740

  19. Synthesizing and modifying peptides for chemoselective ligation and assembly into quantum dot-peptide bioconjugates.

    Science.gov (United States)

    Algar, W Russ; Blanco-Canosa, Juan B; Manthe, Rachel L; Susumu, Kimihiro; Stewart, Michael H; Dawson, Philip E; Medintz, Igor L

    2013-01-01

    Quantum dots (QDs) are well-established as photoluminescent nanoparticle probes for in vitro or in vivo imaging, sensing, and even drug delivery. A critical component of this research is the need to reliably conjugate peptides, proteins, oligonucleotides, and other biomolecules to QDs in a controlled manner. In this chapter, we describe the conjugation of peptides to CdSe/ZnS QDs using a combination of polyhistidine self-assembly and hydrazone ligation. The former is a high-affinity interaction with the inorganic surface of the QD; the latter is a highly efficient and chemoselective reaction that occurs between 4-formylbenzoyl (4FB) and 2-hydrazinonicotinoyl (HYNIC) moieties. Two methods are presented for modifying peptides with these functional groups: (1) solid phase peptide synthesis; and (2) solution phase modification of pre-synthesized, commercial peptides. We further describe the aniline-catalyzed ligation of 4FB- and HYNIC-modified peptides, in the presence of a fluorescent label on the latter peptide, as well as subsequent assembly of the ligated peptide to water-soluble QDs. Many technical elements of these protocols can be extended to labeling peptides with other small molecule reagents. Overall, the bioconjugate chemistry is robust, selective, and modular, thereby potentiating the controlled conjugation of QDs with a diverse array of biomolecules for various applications.

  20. Bioconjugated fluorescent silica nanoparticles for the rapid detection of Entamoeba histolytica.

    Science.gov (United States)

    Hemadi, Ahmad; Ekrami, Alireza; Oormazdi, Hormozd; Meamar, Ahmad Reza; Akhlaghi, Lame; Samarbaf-Zadeh, Ali Reza; Razmjou, Elham

    2015-05-01

    Rapid detection of Entamoeba histolytica based on fluorescent silica nanoparticle (FSNP) indirect immunofluorescence microscopy was evaluated. Silica nanoparticles were synthesized using Stöber's method, with their surface activated to covalently bind to, and immobilize, protein A. For biolabeling, FSNP was added to conjugated E. histolytica trophozoites with monoclonal anti-E. histolytica IgG1 for microscopic observation of fluorescence. Fluorescent silica nanoparticle sensitivity was determined with axenically cultured E. histolytica serially diluted to seven concentrations. Specificity was evaluated using other intestinal protozoa. Fluorescent silica nanoparticles detected E. histolytica at the lowest tested concentration with no cross-reaction with Entamoeba dispar, Entamoeba moshkovskii, Blastocystis sp., or Giardia lamblia. Visualization of E. histolytica trophozoites with anti-E. histolytica antibody labeled with fluorescein isothiocyanate (FITC) was compared with that using anti-E. histolytica antibody bioconjugated FSNP. Although FITC and FSNP produced similar results, the amount of specific antibody required for FITC to induce fluorescence of similar intensity was fivefold that for FSNP. Fluorescent silica nanoparticles delivered a rapid, simple, cost-effective, and highly sensitive and specific method of detecting E. histolytica. Further study is needed before introducing FSNP for laboratory diagnosis of amoebiasis.

  1. The Chemistry of Bioconjugation in Nanoparticles-Based Drug Delivery System

    Directory of Open Access Journals (Sweden)

    Karolina Werengowska-Ciećwierz

    2015-01-01

    Full Text Available Nanomedicine is, generally, the application of nanotechnology to medicine. The term nanomedicine includes monitoring, construction of novel drug delivery systems, and any possible future applications of nanotechnology and nanovaccinology. In this review, the most important ligand-nanocarrier and drug-nanocarrier bioconjugations are described. The detailed characterizations of covalently formed bonds between targeted ligand and nanocarrier, including amide, thioether, disulfide, acetyl-hydrazone and polycyclic groups, are described. Also, the coupling of small elements and heteroatoms in the form of R-X-R the “click chemistry” groups is shown. Physical adsorption and chemical bonding of drug to nanocarrier surface involving drug on the internal or external surfaces of nanocarriers are described throughout possibility of the formation of the above-mentioned functionalities. Moreover, the most popular nanostructures (liposomes, micelles, polymeric nanoparticles, dendrimers, carbon nanotubes, and nanohorns are characterized as nanocarriers. Building of modern drug carrier is a new method which could be effectively applied in targeted anticancer therapy.

  2. Targeted delivery of doxorubicin to breast cancer cells by magnetic LHRH chitosan bioconjugated nanoparticles.

    Science.gov (United States)

    Varshosaz, Jaleh; Hassanzadeh, Farshid; Aliabadi, Hojat Sadeghi; Khoraskani, Fatemeh Rabbani; Mirian, Mina; Behdadfar, Behshid

    2016-12-01

    The novel dual targeted nanoparticles loaded with doxorubicin (DOX) and magnetic nanoparticles (MNPs) were prepared for treatment of breast cancer. Nanoparticles were produced by a layer-by-layer technique and functionalized with a bioconjugate of chitosan-poly(methyl vinyl ether maleic acid)(PMVMA)-LHRH to target LHRH receptors. The successful production of chitosan-PMVMA copolymer and its conjugation to LHRH was confirmed by FTIR and (1)HNMR spectroscopy. Capillary electrophoresis analysis showed 72.51% LHRH conjugation efficiency. Transmission electron microscopy and thermogravimetric analysis showed the entrapment of the MNPs in the core of the nanoparticles and vibrating sample magnetometery confirmed their paramagnetic properties. The iron content of nanoparticles determined by inductively coupled plasma optical emission spectrometry showed to be between 3.5-84%. Particle size, zeta potential, drug entrapment and release efficiency of the nanoparticles were 88.1-182.6nm, 10-30mV, 62.3-87.6% and 79.8-83.4%, respectively. No significant protein binding was seen by nanoparticles. The MTT assay showed in LHRH positive cells of MCF-7 the IC50 of the drug reduced to about 2 fold compared to the free drug. By saturation of LHRH receptors the viable MCF7 cells increased significantly after exposure with the targeted nanoparticles. Therefore, the cellular uptake of the nanoparticles might be done by active endocytosis through the LHRH receptors. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Targeted disinfection of E. coli via bioconjugation to photoreactive TiO2.

    Science.gov (United States)

    Ye, Lu; Pelton, Robert; Brook, Michael A; Filipe, Carlos D M; Wang, Haifeng; Brovko, Luba; Griffiths, Mansel

    2013-03-20

    The selective control of pathogenic bacteria is an ongoing challenge. A strategy is proposed that combines targeted binding of the bacterium, using antibodies, with their photoactivated oxidative destruction. Photoactive colloidal TiO2 was first derivatized with E. coli antibodies (EA-TiO2). When mixtures of the organisms E. coli and Pseudomonas putida ( P. putida ) were exposed to modified EA-TiO2, the particles preferentially selected E. coli for surface binding. Two consequences arose from surface bioconjugation: bacteria were found to flocculate upon mixing at appropriate ratios of EA-TiO2/ E. coli , and EA-TiO2-bound E. coli underwent cell death after exposure to UV light. In the former case, flocculation of the bacteria was optimal at ~50 EA-TiO2 particles per E. coli . Selective flocculation provides an alternative strategy for pathogen removal. With respect to UV disinfection, as few as 26 EA-TiO2 particles per E. coli gave a 10 000-fold decrease in viable bacteria. Thus, it is possible to selectively target and kill one type of bacteria in a mixture of pathogens. The results give support to the proposal that photocatalytic TiO2 most effectively delivers an oxidizing agent when the titania is bound to the bacterial surface.

  4. Bioconjugation of Oligodeoxynucleotides Carrying 1,4-Dicarbonyl Groups via Reductive Amination with Lysine Residues.

    Science.gov (United States)

    Yang, Bo; Jinnouchi, Akiko; Usui, Kazuteru; Katayama, Tsutomu; Fujii, Masayuki; Suemune, Hiroshi; Aso, Mariko

    2015-08-19

    We evaluated the efficacy of bioconjugation of oligodeoxynucleotides (ODNs) containing 1,4-dicarbonyl groups, a C4'-oxidized abasic site (OAS), and a newly designed 2'-methoxy analogue, via reductive amination with lysine residues. Dicarbonyls, aldehyde and ketone at C1- and C4-positions of deoxyribose in the ring-opened form of OAS allowed efficient reaction with amines. Kinetic studies indicated that reductive amination of OAS-containing ODNs with a proximal amine on the complementary strand proceeded 10 times faster than the corresponding reaction of an ODN containing an abasic site with C1-aldehyde. Efficient reductive amination between the DNA-binding domain of Escherichia coli DnaA protein and ODNs carrying OAS in the DnaA-binding sequence proceeded at the lysine residue in proximity to the phosphate group at the 5'-position of the OAS, in contrast to unsuccessful conjugation with abasic site ODNs, even though they have similar aldehydes. Theoretical calculation indicated that the C1-aldehyde of OAS was more accessible to the target lysine than that of the abasic site. These results demonstrate the potential utility of cross-linking strategies that use dicarbonyl-containing ODNs for the study of protein-nucleic acid interactions. Conjugation with a lysine-containing peptide that lacked specific affinity for ODN was also successful, further highlighting the advantages of 1,4-dicarbonyls.

  5. Design of Novel Relaxase Substrates Based on Rolling Circle Replicases for Bioconjugation to DNA Nanostructures.

    Science.gov (United States)

    Sagredo, Sandra; de la Cruz, Fernando; Moncalián, Gabriel

    2016-01-01

    During bacterial conjugation and rolling circle replication, HUH endonucleases, respectively known as relaxases and replicases, form a covalent bond with ssDNA when they cleave their target sequence (nic site). Both protein families show structural similarity but limited amino acid identity. Moreover, the organization of the inverted repeat (IR) and the loop that shape the nic site differs in both proteins. Arguably, replicases cleave their target site more efficiently, while relaxases exert more biochemical control over the process. Here we show that engineering a relaxase target by mimicking the replicase target, results in enhanced formation of protein-DNA covalent complexes. Three widely different relaxases, which belong to MOBF, MOBQ and MOBP families, can properly cleave DNA sequences with permuted target sequences. Collaterally, the secondary structure that the permuted targets acquired within a supercoiled plasmid DNA resulted in poor conjugation frequencies underlying the importance of relaxase accessory proteins in conjugative DNA processing. Our results reveal that relaxase and replicase targets can be interchangeable in vitro. The new Rep substrates provide new bioconjugation tools for the design of sophisticated DNA-protein nanostructures.

  6. Polymerization of Ethylene Oxide, Propylene Oxide, and Other Alkylene Oxides: Synthesis, Novel Polymer Architectures, and Bioconjugation.

    Science.gov (United States)

    Herzberger, Jana; Niederer, Kerstin; Pohlit, Hannah; Seiwert, Jan; Worm, Matthias; Wurm, Frederik R; Frey, Holger

    2016-02-24

    The review summarizes current trends and developments in the polymerization of alkylene oxides in the last two decades since 1995, with a particular focus on the most important epoxide monomers ethylene oxide (EO), propylene oxide (PO), and butylene oxide (BO). Classical synthetic pathways, i.e., anionic polymerization, coordination polymerization, and cationic polymerization of epoxides (oxiranes), are briefly reviewed. The main focus of the review lies on more recent and in some cases metal-free methods for epoxide polymerization, i.e., the activated monomer strategy, the use of organocatalysts, such as N-heterocyclic carbenes (NHCs) and N-heterocyclic olefins (NHOs) as well as phosphazene bases. In addition, the commercially relevant double-metal cyanide (DMC) catalyst systems are discussed. Besides the synthetic progress, new types of multifunctional linear PEG (mf-PEG) and PPO structures accessible by copolymerization of EO or PO with functional epoxide comonomers are presented as well as complex branched, hyperbranched, and dendrimer like polyethers. Amphiphilic block copolymers based on PEO and PPO (Poloxamers and Pluronics) and advances in the area of PEGylation as the most important bioconjugation strategy are also summarized. With the ever growing toolbox for epoxide polymerization, a "polyether universe" may be envisaged that in its structural diversity parallels the immense variety of structural options available for polymers based on vinyl monomers with a purely carbon-based backbone.

  7. VEGF Correlates with Inflammation and Fibrosis in Tuberculous Pleural Effusion

    Directory of Open Access Journals (Sweden)

    Mauo-Ying Bien

    2015-01-01

    Full Text Available Objective. To investigate the relationship among angiogenic cytokines, inflammatory markers, and fibrinolytic activity in tuberculous pleural effusion (TBPE and their clinical importance. Methods. Forty-two patients diagnosed with TBPE were studied. Based on chest ultrasonography, there were 26 loculated and 16 nonloculated TBPE patients. The effusion size radiological scores and effusion vascular endothelial growth factor (VEGF, interleukin- (IL- 8, plasminogen activator inhibitor type-1 (PAI-1, and tissue type plasminogen activator (tPA were measured. Treatment outcome and pleural fibrosis, defined as radiological residual pleural thickening (RPT, were assessed at 6-month follow-up. Results. The effusion size and effusion lactate dehydrogenase (LDH, VEGF, IL-8, PAI-1, and PAI-1/tPA ratio were significantly higher, while effusion glucose, pH value, and tPA were significantly lower, in loculated than in nonloculated TBPE. VEGF and IL-8 correlated positively with LDH and PAI-1/tPA ratio and negatively with tPA in both loculated and nonloculated TBPE. Patients with higher VEGF or greater effusion size were prone to develop RPT (n=14; VEGF, odds ratio 1.28, P=0.01; effusion size, odds ratio 1.01, P=0.02, and VEGF was an independent predictor of RPT in TBPE (receiver operating characteristic curve AUC=0.985, P<0.001. Conclusions. Effusion VEGF correlates with pleural inflammation and fibrosis and may be targeted for adjunct therapy for TBPE.

  8. Correlation between the expression of vegf and survival in osteosarcoma.

    Science.gov (United States)

    Baptista, André Mathias; Camargo, André Ferrari De França; Filippi, Renée Zon; Oliveira, Cláudia Regina Gomes Cardim Mendes De; Azevedo Neto, Raymundo Soares De; Camargo, Olavo Pires De

    2014-01-01

    To present a series of 50 consecutive patients with non-metastatic extremity osteosarcoma, and attempt to correlate expression of the vascular endothelial growth factor (VEGF) protein in biopsy tissue to their prognosis regarding overall survival, disease-free survival and local recurrence. Fifty cases of non-metastatic osteosarcoma of the extremities treated between 1986 and 2006 at Instituto de Ortopedia e Traumatologia da Universidade de São Paulo, São Paulo, Brasil, were evaluated regarding expression of the VEGF protein. There were 19 females and 31 males. The mean age was 16 years old (range 5-28 years old) and the mean follow-up was 60.6 months (range 25-167 months). The variables studied were age, gender, anatomic location, type of surgery, surgical margins, tumor size, post chemotherapy necrosis, local recurrence, pulmonary metastasis and death. Thirty-six patients showed VEGF expression on 30% or less cells (low), and the remaining 14 cases had VEGF expression above 30% (high). Among the 36 patients with low VEGF expression, nine developed pulmonary metastasis and four died (11.1%). Among the 14 patients with high VEGF expression, six developed pulmonary metastasis and three died (21.4%). There was no statistically significant correlation between the expression of VEGF and any of the variables studied. Level of Evidence IV, Therapeutic Study.

  9. BMP-non-responsive Sca1+ CD73+ CD44+ mouse bone marrow derived osteoprogenitor cells respond to combination of VEGF and BMP-6 to display enhanced osteoblastic differentiation and ectopic bone formation.

    Science.gov (United States)

    Madhu, Vedavathi; Li, Ching-Ju; Dighe, Abhijit S; Balian, Gary; Cui, Quanjun

    2014-01-01

    Clinical trials on fracture repair have challenged the effectiveness of bone morphogenetic proteins (BMPs) but suggest that delivery of mesenchymal stem cells (MSCs) might be beneficial. It has also been reported that BMPs could not increase mineralization in several MSCs populations, which adds ambiguity to the use of BMPs. However, an exogenous supply of MSCs combined with vascular endothelial growth factor (VEGF) and BMPs is reported to synergistically enhance fracture repair in animal models. To elucidate the mechanism of this synergy, we investigated the osteoblastic differentiation of cloned mouse bone marrow derived MSCs (D1 cells) in vitro in response to human recombinant proteins of VEGF, BMPs (-2, -4, -6, -9) and the combination of VEGF with BMP-6 (most potent BMP). We further investigated ectopic bone formation induced by MSCs pre-conditioned with VEGF, BMP-6 or both. No significant increase in mineralization, phosphorylation of Smads 1/5/8 and expression of the ALP, COL1A1 and osterix genes was observed upon addition of VEGF or BMPs alone to the cells in culture. The lack of CD105, Alk1 and Alk6 expression in D1 cells correlated with poor response to BMPs indicating that a greater care in the selection of MSCs is necessary. Interestingly, the combination of VEGF and BMP-6 significantly increased the expression of ALP, COL1A1 and osterix genes and D1 cells pre-conditioned with VEGF and BMP-6 induced greater bone formation in vivo than the non-conditioned control cells or the cells pre-conditioned with either VEGF or BMP-6 alone. This enhanced bone formation by MSCs correlated with higher CADM1 expression and OPG/RANKL ratio in the implants. Thus, combined action of VEGF and BMP on MSCs enhances osteoblastic differentiation of MSCs and increases their bone forming ability, which cannot be achieved through use of BMPs alone. This strategy can be effectively used for bone repair.

  10. The pathophysiologic role of VEGF in hematologic malignancies: therapeutic implications.

    Science.gov (United States)

    Podar, Klaus; Anderson, Kenneth C

    2005-02-15

    Besides its role as an essential regulator of physiologic and pathologic angiogenesis, vascular endothelial growth factor (VEGF) triggers growth, survival, and migration of leukemia and multiple myeloma cells; plays a pivotal role in hematopoiesis; inhibits maturation of dendritic cells; and increases osteoclastic bone-resorbing activity as well as osteoclast chemotaxis. Dysregulation of VEGF expression and signaling pathways therefore plays an important role in the pathogenesis and clinical features of hematologic malignancies, in particular multiple myeloma. Direct and indirect targeting of VEGF and its receptors therefore may provide a potent novel therapeutic approach to overcome resistance to therapies and thereby improve patient outcome.

  11. Extracellular biosynthesis of gadolinium oxide (Gd2O3 nanoparticles, their biodistribution and bioconjugation with the chemically modified anticancer drug taxol

    Directory of Open Access Journals (Sweden)

    Shadab Ali Khan

    2014-03-01

    Full Text Available As a part of our programme to develop nanobioconjugates for the treatment of cancer, we first synthesized extracellular, protein-capped, highly stable and well-dispersed gadolinium oxide (Gd2O3 nanoparticles by using thermophilic fungus Humicola sp. The biodistribution of the nanoparticles in rats was checked by radiolabelling with Tc-99m. Finally, these nanoparticles were bioconjugated with the chemically modified anticancer drug taxol with the aim of characterizing the role of this bioconjugate in the treatment of cancer. The biosynthesized Gd2O3 nanoparticles were characterized by UV–vis spectroscopy, transmission electron microscopy (TEM, X-ray diffraction (XRD and X-ray photoemission spectroscopy (XPS. The Gd2O3–taxol bioconjugate was confirmed by UV–vis spectroscopy and fluorescence microscopy and was purified by using high performance liquid chromatography (HPLC.

  12. Extracellular biosynthesis of gadolinium oxide (Gd2O3) nanoparticles, their biodistribution and bioconjugation with the chemically modified anticancer drug taxol.

    Science.gov (United States)

    Khan, Shadab Ali; Gambhir, Sanjay; Ahmad, Absar

    2014-01-01

    As a part of our programme to develop nanobioconjugates for the treatment of cancer, we first synthesized extracellular, protein-capped, highly stable and well-dispersed gadolinium oxide (Gd2O3) nanoparticles by using thermophilic fungus Humicola sp. The biodistribution of the nanoparticles in rats was checked by radiolabelling with Tc-99m. Finally, these nanoparticles were bioconjugated with the chemically modified anticancer drug taxol with the aim of characterizing the role of this bioconjugate in the treatment of cancer. The biosynthesized Gd2O3 nanoparticles were characterized by UV-vis spectroscopy, transmission electron microscopy (TEM), X-ray diffraction (XRD) and X-ray photoemission spectroscopy (XPS). The Gd2O3-taxol bioconjugate was confirmed by UV-vis spectroscopy and fluorescence microscopy and was purified by using high performance liquid chromatography (HPLC).

  13. In vivo and in vitro toxicity and anti-inflammatory properties of gold nanoparticle bioconjugates to the vascular system.

    Science.gov (United States)

    Uchiyama, Mayara Klimuk; Deda, Daiana Kotra; Rodrigues, Stephen Fernandes de Paula; Drewes, Carine Cristiane; Bolonheis, Simone Marques; Kiyohara, Pedro Kunihiko; Toledo, Simone Perche de; Colli, Walter; Araki, Koiti; Farsky, Sandra Helena Poliselli

    2014-12-01

    Gold nanoparticle (AuNP) bioconjugates have been used as therapeutic and diagnostic tools; however, in vivo biocompatibility and cytotoxicity continue to be two fundamental issues. The effect of AuNPs (20 nm) conjugated with antibody [immunoglobulin G (IgG)], albumin, protein A, PEG4000, and citrate (cit) were evaluated in vitro using primary human cells of the vascular system. AuNP bioconjugates did not cause lysis of human erythrocytes, apoptosis or necrosis of human leukocytes, and endothelial cells in vitro, although AuNPs had been internalized and detected in the cytoplasm. Moreover, the influence of AuNPs on rheological parameters, blood and vessel wall characteristics was investigated in vivo by intravital microscopy assay using male Wistar rats mesentery microcirculation as model. Intravenous injection of AuNP-IgG or cit-AuNP did not cause hemorrhage, hemolysis or thrombus formation, instead suppressed the leukocyte adhesion to postcapillary vessel walls, an early stage of an inflammatory process. Furthermore, AuNP-IgG abrogated the expression of platelet-endothelial cell adhesion molecule-1, chemotaxis, and oxidative burst activation on neutrophils after leukotriene B4 stimulation, a membrane receptor-dependent stimulus, thus confirming their anti-inflammatory effects in vitro. The expression of oxidative burst activation was also suppressed after stimulating AuNP-IgG-treated neutrophils with lipid-soluble phorbol myristate acetate (PMA), confirming the direct intracellular action of AuNP-IgG on the inflammatory process in vitro. Our in vitro and in vivo experimental approaches highlighted the great potentiality of AuNP bioconjugates for therapeutic and diagnostic applications by parenteral routes. © The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  14. Nanoparticle bioconjugates as "bottom-up" assemblies of artifical multienzyme complexes

    Science.gov (United States)

    Keighron, Jacqueline D.

    2010-11-01

    The sequential enzymes of several metabolic pathways have been shown to exist in close proximity with each other in the living cell. Although not proven in all cases, colocalization may have several implications for the rate of metabolite formation. Proximity between the sequential enzymes of a metabolic pathway has been proposed to have several benefits for the overall rate of metabolite formation. These include reduced diffusion distance for intermediates, sequestering of intermediates from competing pathways and the cytoplasm. Restricted diffusion in the vicinity of an enzyme can also cause the pooling of metabolites, which can alter reaction equilibria to control the rate of reaction through inhibition. Associations of metabolic enzymes are difficult to isolate ex vivo due to the weak interactions believed to colocalize sequential enzymes within the cell. Therefore model systems in which the proximity and diffusion of intermediates within the experiment system are controlled are attractive alternatives to explore the effects of colocalization of sequential enzymes. To this end three model systems for multienzyme complexes have been constructed. Direct adsorption enzyme:gold nanoparticle bioconjugates functionalized with malate dehydrogenase (MDH) and citrate synthase (CS) allow for proximity between to the enzymes to be controlled from the nanometer to micron range. Results show that while the enzymes present in the colocalized and non-colocalized systems compared here behaved differently overall the sequential activity of the pathway was improved by (1) decreasing the diffusion distance between active sites, (2) decreasing the diffusion coefficient of the reaction intermediate to prevent escape into the bulk solution, and (3) decreasing the overall amount of bioconjugate in the solution to prevent the pathway from being inhibited by the buildup of metabolite over time. Layer-by-layer (LBL) assemblies of MDH and CS were used to examine the layering effect of

  15. Phenylboronic acid-salicylhydroxamic acid bioconjugates. 2. Polyvalent immobilization of protein ligands for affinity chromatography.

    Science.gov (United States)

    Wiley, J P; Hughes, K A; Kaiser, R J; Kesicki, E A; Lund, K P; Stolowitz, M L

    2001-01-01

    Phenylboronic acid bioconjugates prepared from alkaline phosphatase by reaction with either 2,5-dioxopyrrolidinyl 3-[N-[3-(1,3,2-dioxaboran-2-yl)phenyl]carbamoyl]propanoate (PBA-XX-NHS) or 2,5-dioxopyrrolidinyl 6-[[3,5-di-(1,3,2-dioxaboran-2-yl)phenyl]carbonylamino]hexanoate (PDBA-X-NHS) were compared with respect to the efficiency with which they were immobilized on salicylhydroxamic acid-modified Sepharose (SHA-X-Sepharose) by boronic acid complex formation. When immobilized on moderate capacity SHA-X-Sepharose (5.4 micromol of SHA/mL of gel), PDBA-alkaline phosphatase conjugates were shown to be stable with respect to both the alkaline (pH 11.0) and acidic (pH 2.5) buffers utilized to recover anti-alkaline phosphatase during affinity chromatography. Boronic acid complex formation was compared to covalent immobilization of alkaline phosphatase on Affi-Gel 10 and Affi-Gel 15. PDBA-AP.SHA-X-Sepharose was shown to afford superior performance to both Affi-Gel 10 and Affi-Gel 15 with respect to immobilization of alkaline phosphatase, retention of anti-alkaline phosphatase and recovery of anti-alkaline phosphatase under alkaline conditions. High capacity SHA-X-Sepharose (> or = 7 micromol of SHA/mL of gel) was shown to afford superior performance to moderate capacity SHA-X-Sepharose (4.5 micromol of SHA/mL of gel) with respect to stability at pH 11.0 and pH 2.5 when a PDBA-alphaHuman IgG conjugate with a low incorporation ratio of only 1.5:1 was immobilized on SHA-X-Sepharose and subsequently utilized for affinity chromatography of Human IgG. The results are interpreted in terms of either a bivalent or trivalent interaction involving boronic acid complex formation.

  16. Gene Expression of VEGF-A and VEGF-C in Peripheral Blood Mononuclear Cells of Iranian Patients with Acute Myeloid Leukemia

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    Mohammad Reza Aliparasti

    2013-06-01

    Full Text Available OBJECTIVE: The crucial role of angiogenesis in the pathophysiology of acute myeloid leukemia (AML has been proposed. One of the key regulators of angiogenesis is the vascular endothelial growth factor (VEGF. Among the VEGF family, it has been observed that VEGF-A and VEGF-C are expressed by AML cells and mediate leukemic cell proliferation, survival, and resistance to chemotherapy. Emerging evidence, however, suggests that elevated levels of VEGF or a proangiogenic phenotype may impede, rather than promote, early tumor development and progression. As the significance of VEGF-A and VEGF-C levels in the pathogenesis of AML has not been clarified well, the aim of this study is to evaluate gene expression of these angiogenesis promoters and its possible prognostic value in peripheral blood mononuclear cells of Iranian patients with AML. METHODS: We investigated the mRNA expression of VEGF-A and VEGF-C in peripheral blood mononuclear cells of 27 patients with newly diagnosed AML and 28 healthy controls by quantitative real-time PCR. RESULTS: Expression of VEGF-C mRNA was significantly lower in AML patients than in healthy controls (p<0.001. However, there was no significant decrement in expression of VEGF-A mRNA of AML patients compared to the control group (p=0.861. VEGF-A and VEGF-C expression were not able to predict clinical outcome. CONCLUSION: Our data showed that AML is associated with a decreased expression of VEGF-C mRNA. However, expression levels did not influence the clinical outcome in our study. It seems that angiogenesis is affected by different cytokines other than VEGF-C or VEGF-A, and VEGF is also affected by different cytokines. Taken together, these findings help to provide new insights into the investigation of other angiogenic factors and cytokines that may play roles in the pathogenesis of AML.

  17. VEGF in hepatocellular carcinoma and surrounding cirrhotic liver tissues

    Institute of Scientific and Technical Information of China (English)

    Muriel Mathonnet; Bernard Descottes; Denis Valleix; Francois Labrousse; Yves Denizot

    2006-01-01

    @@ TO THE EDITOR We read with a great interest the recent work of Deli and colleagues.[1] in the World Journal of Gastroenterology reporting vascular endothelial growth factor (VEGF) expression in hepatocellular carcinoma (HCC) and cirrhotic liver tissues.

  18. Analysis of diabetic retinopathy biomarker VEGF gene by computational approaches

    OpenAIRE

    Jayashree Sadasivam; Ramesh, N.; K. Vijayalakshmi; Vinni Viridi; Shiva prasad

    2012-01-01

    Diabetic retinopathy, the most common diabetic eye disease, is caused by changes in the blood vessels of the retina which remains the major cause. It is characterized by vascular permeability and increased tissue ischemia and angiogenesis. One of the biomarker for Diabetic retinopathy has been identified as Vascular Endothelial Growth Factor ( VEGF )gene by computational analysis. VEGF is a sub-family of growth factors, the platelet-derived growth factor family of cystine-knot growth factors...

  19. Vascular endothelial growth factor (VEGF and prostate pathology

    Directory of Open Access Journals (Sweden)

    Francisco Botelho

    2010-08-01

    Full Text Available PURPOSE: Previous studies suggest that vascular endothelial growth factor (VEGF circulating levels might improve identification of patients with prostate cancer but results are conflicting. Our aim was to compare serum VEGF levels across different prostate pathologies (including benign prostatic hyperplasia, prostatitis, high grade prostate intraepithelial neoplasia and prostate cancer in patients at high risk of prostate cancer. MATERIALS AND METHODS: We consecutively enrolled 186 subjects with abnormal digital rectal examination and/or total PSA (tPSA = 2.5 ng/mL. Blood was collected before diagnostic ultrasound guided trans-rectal prostate biopsy, or any prostate oncology treatment, to measure PSA isoforms and VEGF. Unconditional logistic regression was used to compute age-, tPSA- and free/total PSA-adjusted odds ratios (OR and respective 95% confidence intervals (95% CI for the association between serum VEGF and different prostatic pathologies. RESULTS: Prostate biopsy main diagnoses were normal or benign prostatic hyperplasia (27.3%, prostatitis (16.6%, and prostatic cancer (55.0%. The median VEGF levels (ng/mL in these groups were 178.2, 261.3 and 266.4 (p = 0.029, respectively, but no significant differences were observed for benign vs. malignant pathologies (215.2 vs. 266.4, p = 0.551. No independent association was observed between VEGF (3rd vs. 1st third and prostate cancer, when compared to benign conditions (adjusted OR = 1.44; CI 95%: 0.64-3.26. CONCLUSIONS: In patients at high risk of prostate cancer, circulating VEGF levels have no clinical role in deciding which patients should be submitted to prostate biopsy. Prostatitis patients, often with higher PSA levels, also present high serum levels of VEGF, and their inclusion in control groups might explain the heterogeneous results in previous studies.

  20. Thrombospondin-1 and VEGF in inflammatory bowel disease

    Directory of Open Access Journals (Sweden)

    Canan Alkim

    2012-01-01

    Full Text Available Angiogenesis is an important process in the pathogenesis of chronic inflammation. We aimed to study the angiogeneic balance in inflammatory bowel disease (IBD by evaluating the expression of vascular endothelial growth factor (VEGF and thrombospondin-1 (TSP-1 on colonic epithelial cells, together with the expression of inducible nitric oxide synthase (iNOS.Twenty-one ulcerative colitis (UC, 14 Crohn's disease (CD, 11 colorectal cancer patients, and 11 healthy controls colonic biopsy samples were evaluated immunohistochemically.The expressions of TSP-1, VEGF, and iNOS in UC and CD groups were higher than expression in healthy control group, all with statistical significance. However, in colorectal cancer group, VEGF and iNOS expressions were increased importantly, but TSP-1 expression was not statistically different from healthy control group's expression. Both TSP-1 and VEGF expressions were correlated with iNOS expression distinctly but did not correlate with each other.Both pro-angiogeneic VEGF and antiangiogeneic TSP-1 expressions were found increased in our IBD groups, but in colorectal cancer group, only VEGF expression was increased. TSP-1 increases in IBD patients as a response to inflammatory condition, but this increase was not enough to suppress pathologic angiogenesis and inflammation in IBD.

  1. Electrochemical detection of vascular endothelial growth factors (VEGFs) using VEGF antibody fragments modified Au NPs/ITO electrode.

    Science.gov (United States)

    Kim, Gang-Il; Kim, Kyung-Woo; Oh, Min-Kyu; Sung, Yun-Mo

    2010-03-15

    A new electrochemical technique for the detection of vascular endothelial growth factors (VEGFs) as a cancer-related biomarker is presented in this paper. Gold nanoparticles (Au NPs) were self-assembled onto an indium tin oxide (ITO) electrode to prepare a modified sandwich type electrochemical immunoassay platform. VEGF antibodies were cleaved into two half-fragments by 2-mercaptoethylamine-HCl (2-MEA) and the fragments were immobilized onto the Au NP substrates by their thiol groups. Through this strategy, randomly oriented attachment of antibodies was prevented which frequently occurs in a general use of whole antibody and reduces the number of available sites for the attachment of target molecules. VEGF target molecules were applied to the immunoelectrodes and they combined with the antibody fragments covering the Au NP electrode, forming antigen-antibody complexes. Then, ferrocene-tagged antibodies, which release electrons under a proper applied potential, were added to the system and they combined with the VEGF molecules pre-attached to the antibody fragments. The redox current of ferrocene measured by the differential pulse voltammetry (DPV) increased almost linearly from 1.27 x 10(-4) to 4.17 x 10(-4)A according to the increase in the concentration of the VEGF target molecules from 100 to 600 pg/ml. The measured current values represent the concentration of the VEGF since they are proportional to the number of ferrocene molecules which is in turn proportional to the concentration of VEGF target molecules. Using this modified sandwich immunoassay with the Au NP/ITO electrode, VEGFs as low as 100 pg/ml were detected with high specificity.

  2. Bioconjugation of gold-polymer core-shell nanoparticles with bovine serum amine oxidase for biomedical applications.

    Science.gov (United States)

    Venditti, I; Hassanein, T F; Fratoddi, I; Fontana, L; Battocchio, C; Rinaldi, F; Carafa, M; Marianecci, C; Diociaiuti, M; Agostinelli, E; Cametti, C; Russo, M V

    2015-10-01

    Core-shell gold nanoparticles [AuNPs], stabilized with a hydrophilic polymer, poly(3-dimethylammonium-1-propyne hydrochloride) [PDMPAHCl], have been used for the immobilization of bovine serum amine oxidase [BSAO]. The functionalized surface of the hybrid nanoparticles is pH responsive, due to the presence of aminic groups that carry out a double role: on one hand they act as ligands for the gold nanoparticle surface, allowing the colloidal stabilization and, on the other hand, they give a hydrophilic characteristic to the whole colloidal suspension. The core-shell nanoparticles [Au@PDMPAHCl] have been characterized by using UV-vis and X-ray photoelectron spectroscopy, DLS, ζ-potential measurements and by FE-TEM microscopy. BSAO enzyme can be loaded by non-covalent immobilization onto Au@PDMPAHCl nanoparticles up to 70% in weight, depending on the pH values of the environmental medium. Activity tests on Au@PDMPAHCl-BSAO bioconjugates confirm an enzymatic activity up to 40%, with respect to the free enzyme activity. Moreover, our results show that loading and enzymatic activity are rather interrelated characteristics and that, under appropriate polymer concentration and pH conditions, a satisfactory compromise can be reached. These results, as a whole, indicate that Au@PDMPAHCl-BSAO bioconjugate systems are promising for future biomedical applications.

  3. Efficient synthesis of diverse heterobifunctionalized clickable oligo(ethylene glycol) linkers: potential applications in bioconjugation and targeted drug delivery.

    Science.gov (United States)

    Goswami, Lalit N; Houston, Zachary H; Sarma, Saurav J; Jalisatgi, Satish S; Hawthorne, M Frederick

    2013-02-21

    Herein we describe the sequential synthesis of a variety of azide-alkyne click chemistry-compatible heterobifunctional oligo(ethylene glycol) (OEG) linkers for bioconjugation chemistry applications. Synthesis of these bioorthogonal linkers was accomplished through desymmetrization of OEGs by conversion of one of the hydroxyl groups to either an alkyne or azido functionality. The remaining distal hydroxyl group on the OEGs was activated by either a 4-nitrophenyl carbonate or a mesylate (-OMs) group. The -OMs functional group served as a useful precursor to form a variety of heterobifunctionalized OEG linkers containing different highly reactive end groups, e.g., iodo, -NH(2), -SH and maleimido, that were orthogonal to the alkyne or azido functional group. Also, the alkyne- and azide-terminated OEGs are useful for generating larger discrete poly(ethylene glycol) (PEG) linkers (e.g., PEG(16) and PEG(24)) by employing a Cu(I)-catalyzed 1,3-dipolar cycloaddition click reaction. The utility of these clickable heterobifunctional OEGs in bioconjugation chemistry was demonstrated by attachment of the integrin (α(v)β(3)) receptor targeting peptide, cyclo-(Arg-Gly-Asp-D-Phe-Lys) (cRGfKD) and to the fluorescent probe sulfo-rhodamine B. The synthetic methodology presented herein is suitable for the large scale production of several novel heterobifunctionalized OEGs from readily available and inexpensive starting materials.

  4. Size control of laser-fabricated surfactant-free gold nanoparticles with highly diluted electrolytes and their subsequent bioconjugation.

    Science.gov (United States)

    Rehbock, Christoph; Merk, Vivian; Gamrad, Lisa; Streubel, René; Barcikowski, Stephan

    2013-03-07

    Size control of laser-fabricated surfactant-free gold nanoparticles is a challenging endeavor. In this work, we show that size control can be achieved by adding ions with low salinity during synthesis. In addition, this approach offers the opportunity to fundamentally study ion interactions with bare nanoparticle surfaces and can help to elucidate the nanoparticle formation mechanism. The studies were carried out in a flow-through reactor and in the presence of NaCl, NaBr and sodium phosphate buffer at minimal ionic strengths. A significant size quenching effect at ionic strengths from 1-50 μM was found, which allowed surfactant-free nanoparticle size control with average diameters of 6-30 nm. This effect went along with low polydispersity and minimal aggregation tendencies and was confirmed by UV-vis spectroscopy, TEM, SEM and analytical disk centrifugation. Our findings indicate that size quenching originates from an anionic electrostatic stabilization depending on the nanoparticle surface area, which may be caused by specific ion adsorption. By subsequent delayed bioconjugation in liquid-flow using bovine serum albumin as a stabilizing agent, nano-bioconjugates with good stability in cell culture media were obtained, which are applicable in toxicology and cell biology.

  5. Smad4 Inhibits VEGF-A and VEGF-C Expressions via Enhancing Smad3 Phosphorylation in Colon Cancer.

    Science.gov (United States)

    Li, Xuemei; Li, Xinlei; Lv, Xiaohong; Xiao, Jianbing; Liu, Baoquan; Zhang, Yafang

    2017-09-01

    Smad4 is a critical factor in the TGF-β pathway and is involved in tumor progression and metastasis, but the role of Smad4 in colon cancer cells is unclear. The aim of this study is to explore the effect and the underlying mechanism of Smad4 on the growth, migration and apoptosis of colon cancer cells as well as vascular endothelial growth factor (VEGF)-A and VEGF-C secreted by these cells. In this study, we showed that Smad4, VEGF-A, and VEGF-C are independent prognostic factors of colon cancer, and Smad4 expression was negatively correlated with VEGF-A and -C in samples. We found that Smad4 mRNA and protein levels in colon cancer cells, particularly in HCT-116 cells, were significantly lower than those in the human intestinal epithelial cell line (HIEC). Smad4 overexpression promoted tumor cell apoptosis, inhibited VEGF-A and -C expression in vitro and in vivo, but had no effect on cell proliferation and migration. Tail vein injection of the virus inhibited xenograft growth in nude mice. Importantly, we also demonstrated that Smad4 could increase the phosphorylation level of Smad3, but not Smad2, which may be one of the mechanisms underlying these effects of Smad4 in colon cancer. Therefore, Smad4 may be a new target for the treatment of colon cancer. Anat Rec, 300:1560-1569, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  6. CXCL7-Mediated Stimulation of Lymphangiogenic Factors VEGF-C, VEGF-D in Human Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Minghuan Yu

    2010-01-01

    Full Text Available Increased expression of lymphangiogenesis factors VEGF-C/D and heparanase has been correlated with the invasion of cancer. Furthermore, chemokines may modify matrix to facilitate metastasis, and they are associated with VEGF-C and heparanase. The chemokine CXCL7 binds heparin and the G-protein-linked receptor CXCR2. We investigated the effect of CXCR2 blockade on the expression of VEGF-C/D, heparanase, and on invasion. CXCL7 siRNA and a specific antagonist of CXCR2 (SB225002 were used to treat CXCL7 stably transfected MCF10AT cells. Matrigel invasion assays were performed. VEGF-C/D expression and secretion were determined by real-time PCR and ELISA assay, and heparanase activity was quantified by ELISA. SB225002 blocked VEGF-C/D expression and secretion (P<.01. CXCL7 siRNA knockdown decreased heparanase (P<.01. Both SB225002 and CXCL7 siRNA reduced the Matrigel invasion (P<.01. The MAP kinase signaling pathway was not involved. The CXCL7/CXCR2 axis is important for cell invasion and the expression of VEGF-C/D and heparanase, all linked to invasion.

  7. Detection of aqueous VEGF concentrations before and after intravitreal injection of anti-VEGF antibody using low-volume sampling paper-based ELISA.

    Science.gov (United States)

    Hsu, Min-Yen; Hung, Yu-Chien; Hwang, De-Kuang; Lin, Shang-Chi; Lin, Keng-Hung; Wang, Chun-Yuan; Choi, Hin-Yeung; Wang, Yu-Ping; Cheng, Chao-Min

    2016-10-11

    Intraocular vascular endothelial growth factor (VEGF) levels play an important role in the pathogenesis of blindness-related diseases, such as age-related macular degeneration (AMD). Here, we aimed to develop a paper-based enzyme-linked immunosorbent assay (P-ELISA) to analyze the suppression of aqueous VEGF concentrations following intravitreal injection (IVI) of anti-VEGF antibody (bevacizumab or ranibizumab). A total of 25 eyes with wet AMD, one with myopic neovascularization, and one with polypoidal choroidal vasculopathy were enrolled in this study. The limit of detection using P-ELISA was 0.03 pg/mL. Forty-six consecutive samples of aqueous humor were acquired. From all samples, 66.67% (10/15) achieved complete VEGF suppression (below the detection limit) within 5 weeks of receiving IVI of anti-VEGF antibody. Only 13.33% of samples (2/15) achieved complete VEGF suppression 5 weeks after receiving treatment. In some patients, elevated VEGF was still detected 5 weeks after receipt of anti-VEGF antibody, and all samples (10/10) were found to have elevated VEGF levels 49 days after treatment. Thus, we suggest that monthly IVI of anti-VEGF antibody may be required to ensure durable VEGF inhibition. Ultrasensitive P-ELISA can detect elevated VEGF at an earlier time point and may facilitate decision-making regarding appropriate treatment strategies.

  8. VEGF111b, a new member of VEGFxxxb isoforms and induced by mitomycin C, inhibits angiogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Gu, Fang; Li, Xiuli [Department of Obstetrics and Gynecology, Chinese PLA General Hospital, Beijing (China); Kong, Jian [Department of Hepatobiliary Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing (China); Pan, Bing [The Institute of Cardiovascular Sciences, Peking University Health Science Center, Key Laboratory of Molecular Cardiovascular Sciences of Education Ministry, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides of Health Ministry, Beijing (China); Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Key Laboratory of Molecular Cardiovascular Sciences of Education Ministry, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides of Health Ministry, Beijing (China); Sun, Min [Department of Obstetrics and Gynecology, Tangdu Hospital, Fourth Military Medical University, Xian (China); Zheng, Lemin, E-mail: zhengl@bjmu.edu.cn [The Institute of Cardiovascular Sciences, Peking University Health Science Center, Key Laboratory of Molecular Cardiovascular Sciences of Education Ministry, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides of Health Ministry, Beijing (China); Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Key Laboratory of Molecular Cardiovascular Sciences of Education Ministry, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides of Health Ministry, Beijing (China); Yao, Yuanqing, E-mail: yqyao@126.com [Department of Obstetrics and Gynecology, Chinese PLA General Hospital, Beijing (China)

    2013-11-08

    Highlights: •We discovered a new member of VEGFxxxb family-VEGF111b. •We found VEGF111b mRNA and protein can be induced by mitomycin C. •We confirmed VEGF111b over-expression inhibits angiogenesis. •VEGF111b inhibits angiogenesis through inhibiting VEGF-R2/PI3K/Akt and VEGF-R2/ERK1/2 phosphorylation. -- Abstract: Vascular endothelial growth factor (VEGF-A) stimulating angiogenesis is required for tumor growth and progression. The conventional VEGF-A isoforms have been considered as pro-angiogenic factors. Another family of VEGF-A isoforms generated by alternative splicing, termed VEGFxxxb isoforms, has anti-angiogenic property, exemplified by VEGF165b. Here, we identify a new number of VEGFxxx family-VEGF111b induced by mitomycin C, although not detected in mitomycin C-unexposed ovarian cancer cells. SKOV3 cells were transfected with pcDNA{sub 3.1} empty vector, pcDNA{sub 3.1}-VEGF111b or pcDNA{sub 3.1}-VEGF165b to collect conditioned mediums respectively. VEGF111b overexpression inhibits proliferation, migration and tube formation of endothelial cell by inhibiting VEGF-R2 phosphorylation and its downstream signaling, similar to VEGF165b but slightly lower than VEGF165b. The anti-angiogenic property depends on the six amino acids of exon 8b of the VEGFxxxb isoforms. Our results show that VEGF111b is a novel potent anti-angiogenic agent that can target the VEGF-R2 and its signaling pathway to inhibit ovarian tumor growth.

  9. Formation of VEGF isoform-specific spatial distributions governing angiogenesis: computational analysis

    Directory of Open Access Journals (Sweden)

    Mac Gabhann Feilim

    2011-05-01

    Full Text Available Abstract Background The spatial distribution of vascular endothelial growth factor A (VEGF is an important mediator of vascular patterning. Previous experimental studies in the mouse hindbrain and retina have suggested that VEGF alternative splicing, which controls the ability of VEGF to bind to heparan sulfate proteoglycans (HSPGs in the extracellular matrix (ECM, plays a key role in controlling VEGF diffusion and gradients in tissues. Conversely, proteolysis notably by matrix metalloproteinases (MMPs, plays a critical role in pathological situations by releasing matrix-sequestered VEGF and modulating angiogenesis. However, computational models have predicted that HSPG binding alone does not affect VEGF localization or gradients at steady state. Results Using a 3D molecular-detailed reaction-diffusion model of VEGF ligand-receptor kinetics and transport, we test alternate models of VEGF transport in the extracellular environment surrounding an endothelial sprout. We show that differences in localization between VEGF isoforms, as observed experimentally in the mouse hindbrain, as well as the ability of proteases to redistribute VEGF in pathological situations, are consistent with a model where VEGF is endogenously cleared or degraded in an isoform-specific manner. We use our predictions of the VEGF distribution to quantify a tip cell's receptor binding and gradient sensing capacity. A novel prediction is that neuropilin-1, despite functioning as a coreceptor to VEGF165-VEGFR2 binding, reduces the ability of a cell to gauge the relative steepness of the VEGF distribution. Comparing our model to available in vivo vascular patterning data suggests that vascular phenotypes are most consistently predicted at short range by the soluble fraction of the VEGF distributions, or at longer range by matrix-bound VEGF detected in a filopodia-dependent manner. Conclusions Isoform-specific VEGF degradation provides a possible explanation for numerous examples

  10. Bioconjugation of trypsin onto gold nanoparticles: Effect of surface chemistry on bioactivity

    Energy Technology Data Exchange (ETDEWEB)

    Hinterwirth, Helmut; Lindner, Wolfgang [Department of Analytical Chemistry, University of Vienna, Waehringerstrasse 38, 1090 Vienna (Austria); Laemmerhofer, Michael, E-mail: michael.laemmerhofer@uni-tuebingen.de [Department of Analytical Chemistry, University of Vienna, Waehringerstrasse 38, 1090 Vienna (Austria)

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer Size and spacer affect bioactivity of nanoparticulate trypsin reactor. Black-Right-Pointing-Pointer Increase of GNP's size increases activity of bound trypsin. Black-Right-Pointing-Pointer Increase of spacer length increases amount and activity of immobilized enzyme by factor 6. Black-Right-Pointing-Pointer Decrease of digestion time up to less than 1 h when trypsin immobilized onto GNPs. Black-Right-Pointing-Pointer Reduced auto-digestion compared to trypsin in-solution. - Abstract: The systematic study of activity, long-time stability and auto-digestion of trypsin immobilized onto gold nanoparticles (GNPs) is described in this paper and compared to trypsin in-solution. Thereby, the influence of GNP's size and immobilization chemistry by various linkers differing in lipophilicity/hydrophilicity and spacer lengths was investigated with regard to the bioactivity of the conjugated enzyme. GNPs with different sizes were prepared by reduction and simultaneous stabilization with trisodium citrate and characterized by UV/vis spectra, dynamic light scattering (DLS), {zeta}-potential measurements and transmission electron microscopy (TEM). GNPs were derivatized by self-assembling of bifunctional thiol reagents on the nanoparticle (NP) surface via dative thiol-gold bond yielding a carboxylic acid functionalized surface. Trypsin was either attached directly via hydrophobic and ionic interactions onto the citrate stabilized GNPs or immobilized via EDC/NHS bioconjugation onto the carboxylic functionalized GNPs, respectively. The amount of bound trypsin was quantified by measuring the absorbance at 280 nm. The activity of bound enzyme and its Michaelis Menten kinetic parameter K{sub m} and v{sub max} were measured by the standard chromogenic substrate N{sub {alpha}}-Benzoyl-DL-arginine 4-nitroanilide hydrochloride (BApNA). Finally, digestion of a standard protein mixture with the trypsin-conjugated NPs followed by analysis with

  11. Facile and stabile linkages through tyrosine: bioconjugation strategies with the tyrosine-click reaction.

    Science.gov (United States)

    Ban, Hitoshi; Nagano, Masanobu; Gavrilyuk, Julia; Hakamata, Wataru; Inokuma, Tsubasa; Barbas, Carlos F

    2013-04-17

    The scope, chemoselectivity, and utility of the click-like tyrosine labeling reaction with 4-phenyl-3H-1,2,4-triazoline-3,5(4H)-diones (PTADs) is reported. To study the utility and chemoselectivity of PTAD derivatives in peptide and protein chemistry, we synthesized PTAD derivatives possessing azide, alkyne, and ketone groups and studied their reactions with amino acid derivatives and peptides of increasing complexity. With proteins we studied the compatibility of the tyrosine click reaction with cysteine and lysine-targeted labeling approaches and demonstrate that chemoselective trifunctionalization of proteins is readily achieved. In particular cases, we noted that PTAD decomposition resulted in formation of a putative isocyanate byproduct that was promiscuous in labeling. This side reaction product, however, was readily scavenged by the addition of a small amount of 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) to the reaction medium. To study the potential of the tyrosine click reaction to introduce poly(ethylene glycol) chains onto proteins (PEGylation), we demonstrate that this novel reagent provides for the selective PEGylation of chymotrypsinogen, whereas traditional succinimide-based PEGylation targeting lysine residues provided a more diverse range of PEGylated products. Finally, we applied the tyrosine click reaction to create a novel antibody-drug conjugate. For this purpose, we synthesized a PTAD derivative linked to the HIV entry inhibitor aplaviroc. Labeling of the antibody trastuzumab with this reagent provided a labeled antibody conjugate that demonstrated potent HIV-1 neutralization activity demonstrating the potential of this reaction in creating protein conjugates with small molecules. The tyrosine click linkage demonstrated stability to extremes of pH, temperature, and exposure to human blood plasma indicating that this linkage is significantly more robust than maleimide-type linkages that are commonly employed in bioconjugations. These

  12. Controlled branching of polyglycidol and formation of protein-glycidol bioconjugates via a graft-from approach with "PEG-like" arms.

    Science.gov (United States)

    Spears, Benjamin R; Waksal, Julian; McQuade, Caitlin; Lanier, Laura; Harth, Eva

    2013-03-25

    The control of the branching in polyglycidols as semibranched alternatives to traditional polyglycidols is presented. The relative abundance of dendritic carbons is lowered by five-fold compared to hyperbranched polyglycidols. It is the first example of tailoring the branching in polyglycidol and creating protein-glycidol bioconjugates as alternatives to pegylated biostructures.

  13. Protein expression profile of HT-29 human colon cancer cells after treatment with a cytotoxic daunorubicin-GnRH-III derivative bioconjugate.

    Directory of Open Access Journals (Sweden)

    Verena Natalie Schreier

    Full Text Available Targeted delivery of chemotherapeutic agents is a new approach for the treatment of cancer, which provides increased selectivity and decreased systemic toxicity. We have recently developed a promising drug delivery system, in which the anticancer drug daunorubicin (Dau was attached via oxime bond to a gonadotropin-releasing hormone-III (GnRH-III derivative used as a targeting moiety (Glp-His-Trp-Lys(Ac-His-Asp-Trp-Lys(Da  = Aoa-Pro-Gly-NH2; Glp = pyroglutamic acid, Ac = acetyl; Aoa = aminooxyacetyl. This bioconjugate exerted in vitro cytostatic/cytotoxic effect on human breast, prostate and colon cancer cells, as well as significant in vivo tumor growth inhibitory effect on colon carcinoma bearing mice. In our previous studies, H-Lys(Dau = Aoa-OH was identified as the smallest metabolite produced in the presence of rat liver lysosomal homogenate, which was able to bind to DNA in vitro. To get a deeper insight into the mechanism of action of the bioconjugate, changes in the protein expression profile of HT-29 human colon cancer cells after treatment with the bioconjugate or free daunorubicin were investigated by mass spectrometry-based proteomics. Our results indicate that several metabolism-related proteins, molecular chaperons and proteins involved in signaling are differently expressed after targeted chemotherapeutic treatment, leading to the conclusion that the bioconjugate exerts its cytotoxic action by interfering with multiple intracellular processes.

  14. Correction: New synthesis of phenyl-isothiocyanate C-functionalised cyclams. Bioconjugation and (64)Cu phenotypic PET imaging studies of multiple myeloma with the te2a derivative.

    Science.gov (United States)

    Halime, Zakaria; Frindel, Mathieu; Camus, Nathalie; Orain, Pierre-Yves; Lacombe, Marie; Bernardeau, Karine; Chérel, Michel; Gestin, Jean-François; Faivre-Chauvet, Alain; Tripier, Raphaël

    2016-04-12

    Correction for 'New synthesis of phenyl-isothiocyanate C-functionalised cyclams. Bioconjugation and 64Cu phenotypic PET imaging studies of multiple myeloma with the te2a derivative' by Zakaria Halime et al., Org. Biomol. Chem., 2015, 13, 11302-11314.

  15. Synthesis of the copper chelator TGTA and evaluation of its ability to protect biomolecules from copper induced degradation during copper catalyzed azide-alkyne bioconjugation reactions.

    Science.gov (United States)

    Ekholm, F S; Pynnönen, H; Vilkman, A; Koponen, J; Helin, J; Satomaa, T

    2016-01-21

    One of the most successful bioconjugation strategies to date is the copper(I)-catalyzed cycloaddition reaction (CuAAC), however, the typically applied reaction conditions have been found to degrade sensitive biomolecules. Herein, we present a water soluble copper chelator which can be utilized to protect biomolecules from copper induced degradation.

  16. The expression of VEGF and its receptors in the human ductus arteriosus.

    Science.gov (United States)

    Weber, Sven C; Rheinlaender, Cornelia; Sarioglu, Nanette; Peiser, Christian; Rüdiger, Mario; Obladen, Michael; Koehne, Petra S

    2008-10-01

    Programmed proliferative degeneration of the human fetal ductus arteriosus (DA) preceding definite postnatal closure has a large developmental variability and is controlled by several signaling pathways. Among these vascular endothelial growth factor (VEGF) and its receptors (VEGF-Rs) play an important role. Until now, gestational age dependent expression of VEGF and its receptors has not been investigated in a large number of human DA tissue specimens. We examined protein expression of VEGF and the three VEGF-Rs immunohistochemically in 63 human fetal autopsy DA specimens of 11-38 wk gestation. Specimens were classified into different maturity stages according to their histologic appearance. VEGF and VEGF-Rs-staining was detected in all maturity stages. VEGF-staining was localized perinuclearly in all vascular layers and did not change during development. VEGF-R1 and VEGF-R3 expression was marked in the endothelium in early maturity stages and decreased during development. In contrast, -R2 predominated in the media in later developmental stages. Our results emphasize the importance of VEGF as a mediator during programmed proliferative degeneration of fetal DA and support the hypothesis that VEGF-R1 and VEGF-R3 are required for normal blood vessel development during embryogenesis. In contrast, VEGF-R2 is the predominant receptor in later angiogenic signaling.

  17. Anti-VEGF Agents for Ocular Angiogenesis and Vascular Permeability

    Directory of Open Access Journals (Sweden)

    Kenichi Kimoto

    2012-01-01

    Full Text Available We review articles describing intravitreal injection of anti-VEGF drug trials, while discussing the mechanisms of the action of anti-VEGF antibodies, and also evaluating their outcomes. Intraocular injections of anti-VEGF drug are considered to be an effective treatment for macular edema after retinal vein occlusion, however, recurrent/persistent edema is common. The recent reports may lead to a shift in treatment paradigm for DME, from laser photocoagulation, to newer approaches using anti-VEGF drugs. There have been several well-publicized prospective, randomized studies that demonstrated the efficacy of intravitreal injection of anti-VEGF drugs for patients with AMD. Adjuvant bevacizumab for neovascular glaucoma may prevent further PAS formation, and it is likely to open up a therapeutic window for a panretinal photocoagulation and trabeculectomy. Intravitreal injection of bevacizumab (IVB results in a substantial decrease in bleeding from the retinal vessels or new vessels during a standard vitrectomy. IVB has also been reported to be effective for inducing the regression of new vessels in proliferative diabetic retinopathy. The use of bevacizumab in stage 4 or 5 retinopahty of permaturity (ROP is to reduce the plus sign to help reduce hemorrhage during the subsequent vitrectomy. Some authors reported cases of resolution of stage 4 A ROP after bevacizumab injection.

  18. SREBP inhibits VEGF expression in human smooth muscle cells.

    Science.gov (United States)

    Motoyama, Koka; Fukumoto, Shinya; Koyama, Hidenori; Emoto, Masanori; Shimano, Hitoshi; Maemura, Koji; Nishizawa, Yoshiki

    2006-03-31

    Sterol regulatory element-binding proteins (SREBPs) are transcription factors that regulate expression of genes encoding enzymes for lipid biosynthesis. SREBPs are activated by HMG-CoA reductase inhibitors (statins). Statins have been also reported to suppress vascular endothelial growth factor (VEGF) expression in vascular smooth muscle cells (VSMCs). Therefore, we hypothesized that SREBPs are involved in statin-mediated regulation of VEGF production in VSMCs. SREBP1 was robustly expressed, and was activated by atorvastatin in VSMCs, as demonstrated by increased levels of the mature nuclear form of SREBP1, and increased promoter activities of a reporter containing sterol regulatory elements by atorvastatin. Moreover, overexpression of SREBP1a dose-dependently suppressed VEGF promoter activity. Site-specific mutation or deletion of the proximal Sp1 sites reduced the inhibitory effects of SREBP1a on VEGF promoter activity. These data demonstrated that SREBP1, activated by atorvastatin, suppressed VEGF expression through the indirect interaction with the proximal tandem Sp1 sites in VSMCs.

  19. Antiangiogenic VEGF-Ax: A New Participant in Tumor Angiogenesis.

    Science.gov (United States)

    Eswarappa, Sandeepa M; Fox, Paul L

    2015-07-15

    The transcript of the angiogenic factor vascular endothelial growth factor A (VEGF-A) is subject to a multitude of stimulus-dependent, posttranscriptional regulatory events, consistent with its unusually long 3' untranslated region. We have recently reported translational readthrough of VEGFA mRNA whereby translating ribosomes traverse the canonical stop codon to a conserved, downstream stop codon, generating VEGF-Ax ("x" for extended), a novel, extended isoform with an additional 22 amino acids appended at the C-terminus. This event is the first vertebrate example of protein-regulated, programmed translational readthrough that generates a protein with a known function. Remarkably, VEGF-Ax exhibits potent antiangiogenic activity, both in vitro and in vivo, thus raising profound clinical implications, particularly with respect to cancer treatment. In this review, we discuss the potential of VEGF-Ax as a therapeutic agent and drug target, as well as its possible role in the failure of, or resistance to, conventional anti-VEGF therapies in many types of cancers.

  20. VEGF Spliced Variants: Possible Role of Anti-Angiogenesis Therapy

    Directory of Open Access Journals (Sweden)

    Caroline Hilmi

    2012-01-01

    Full Text Available Angiogenesis has been targeted in retinopathies, psoriasis, and a variety of cancers (colon, breast, lung, and kidney. Among these tumour types, clear cell renal cell carcinomas (RCCs are the most vascularized tumours due to mutations of the von Hippel Lindau gene resulting in HIF-1 alpha stabilisation and overexpression of Vascular Endothelial Growth Factor (VEGF. Surgical nephrectomy remains the most efficient curative treatment for patients with noninvasive disease, while VEGF targeting has resulted in varying degrees of success for treating metastatic disease. VEGF pre-mRNA undergoes alternative splicing generating pro-angiogenic isoforms. However, the recent identification of novel splice variants of VEGF with anti-angiogenic properties has provided some insight for the lack of current treatment efficacy. Here we discuss an explanation for the relapse to anti-angiogenesis treatment as being due to either an initial or acquired resistance to the therapy. We also discuss targeting angiogenesis via SR (serine/arginine-rich proteins implicated in VEGF splicing.

  1. BMP2、VEGF165双基因修饰的小鼠BMSCs复合人工骨的体内诱导成骨%In vivo osteogenic potential of calcium phosphate cement seeded with BMP2-and VEGF165-transduced mice BMSCs

    Institute of Scientific and Technical Information of China (English)

    孙立; 姜小峰; 田晓滨; 田家亮; 杨先腾; 胡如印; 张一; 韩伟; 陈涛

    2014-01-01

    目的 观察BMP2、VEGF165双基因转染的小鼠BMSCs复合自固化磷酸钙人工骨后的异位诱导成骨能力. 方法 脂质体介导下将含BMP2、VEGF165双基因的真核表达质粒pIRES转染小鼠BMSCs,转染空白质粒作阴性对照.用免疫组织化学和Western blot方法观察BMP2和VEGF165双基因在小鼠BMSCs内的表达;以自固化磷酸钙人工骨为支架,建立真核表达质粒pIRES-BMP2-VEGF165双基因修饰的组织工程骨植入小鼠右侧股骨肌袋内;将复合转染空白质粒小鼠BMSCs的自固化磷酸钙人工骨植入小鼠左侧股骨肌袋内.于术后第3天、第2,4,6周进行X线摄影对比后处死小鼠,取肌袋内组织行病理切片染色,观察成骨情况. 结果 小鼠BMSCs在转染BMP2、VEGF165基因后相关蛋白显著表达,由真核表达质粒pIRES-BMP2-VEGF165双基因修饰的组织工程骨在X线和病理学检查中相对于复合转染空白质粒小鼠BMSCs的组织工程骨具有更明显的异位成骨能力. 结论 由BMP2、VEGF165双基因质粒构建的组织工程骨有明显的异位成骨能力.%Objective To evaluate the ectopic osteogenic potential of calcium phosphate cement seeded with BMP2-and VEGF165-transduced mice BMSCs.Methods BMSCs transfected with recombinant plasmid pIRES-BMP2-VEGF165 using a liposome method were implanted into the muscle pouches of right thigh of the mice.BMSCs transfected only with pIRES served as a negative control and implanted into the muscle pouches of right thigh of the mice.Expressions of BMP2 and VEGF165 genes were assayed by immunohistochemical analysis and Western blot.Ectopic bone formation was evaluated by radiography and histology at 3 days and 2,4,and 6 weeks after implantation.Results Expressions of BMP2 and VEGF165 proteins were highly improved in BMP2-and VEGF165-transduced BMSCs.Calcium phosphate cement with BMSCs transfected with pIRES-BMP2-VEGF165 provided better ectopic osteogenesis compared to control group.Conclusion Tissue

  2. DNA methylation regulates expression of VEGF-C, and S-adenosylmethionine is effective for VEGF-C methylation and for inhibiting cancer growth

    Energy Technology Data Exchange (ETDEWEB)

    Da, M.X. [Department of Surgical Oncology, Gansu Provincial Hospital, Lanzhou (China); Zhang, Y.B. [Department of Surgery, Ningxia Medical University, Yinchuan (China); Yao, J.B. [Department of Surgical Oncology, Gansu Provincial Hospital, Lanzhou (China); Duan, Y.X. [Department of Surgery, Ningxia Medical University, Yinchuan (China)

    2014-09-30

    DNA hypomethylation may activate oncogene transcription, thus promoting carcinogenesis and tumor development. S-adenosylmethionine (SAM) is a methyl donor in numerous methylation reactions and acts as an inhibitor of intracellular demethylase activity, which results in hypermethylation of DNA. The main objectives of this study were to determine whether DNA hypomethylation correlated with vascular endothelial growth factor-C (VEGF-C) expression, and the effect of SAM on VEGF-C methylation and gastric cancer growth inhibition. VEGF-C expression was assayed by Western blotting and RT-qPCR in gastric cancer cells, and by immunohistochemistry in tumor xenografts. VEGF-C methylation was assayed by bisulfite DNA sequencing. The effect of SAM on cell apoptosis was assayed by flow cytometry analyses and its effect on cancer growth was assessed in nude mice. The VEGF-C promoters of MGC-803, BGC-823, and SGC-7901 gastric cancer cells, which normally express VEGF-C, were nearly unmethylated. After SAM treatment, the VEGF-C promoters in these cells were highly methylated and VEGF-C expression was downregulated. SAM also significantly inhibited tumor growth in vitro and in vivo. DNA methylation regulates expression of VEGF-C. SAM can effectively induce VEGF-C methylation, reduce the expression of VEGF-C, and inhibit tumor growth. SAM has potential as a drug therapy to silence oncogenes and block the progression of gastric cancer.

  3. Placenta growth factor-1 antagonizes VEGF-induced angiogenesis and tumor growth by the formation of functionally inactive PIGF-1/VEGF heterodimers

    DEFF Research Database (Denmark)

    Eriksson, A.; Cao, R.; Pawliuk, R.

    2002-01-01

    , the biological function of its related homolog, placenta growth factor (PlGF), is poorly understood. Here we demonstrate that PlGF-1, an alternatively spliced isoform of the PlGF gene, antagonizes VEGF-induced angiogenesis when both factors are coexpressed in murine fibrosarcoma cells. Overexpression of PlGF-1...... in VEGF-producing tumor cells results in the formation of PlGF-1/VEGF heterodimers and depletion of the majority of mouse VEGF homodimers. The heterodimeric form of PlGF-1/VEGF lacks the ability to induce angiogenesis in vitro and in vivo. Similarly, PlGF-1/VEGF fails to activate the VEGFR-2-mediated...... signaling pathways. Further, PlGF-1 inhibits the growth of a murine fibrosarcoma by approximately 90% when PlGF-1-expressing tumor cells are implanted in syngeneic mice. In contrast, overexpression of human VEGF in murine tumor cells causes accelerated and exponential growth of primary fibrosarcomas...

  4. Recombinant vascular endothelial growth factor 121 injection for the prevention of fetal growth restriction in a preeclampsia mouse model.

    Science.gov (United States)

    Sulistyowati, Sri; Bachnas, Muhammad Adrianes; Anggraini, Nuri Dyah; Yuliantara, Eric Edwin; Prabowo, Wisnu; Anggraini, Nutria Widya Purna; Pramono, Mochammad Besari Adi; Adityawarman; Dachlan, Erry Gumilar; Andonotopo, Wiku

    2017-02-01

    To discover the potential role of recombinant VEGF121 (rVEGF121) injection for the prevention of fetal growth restriction in a preeclampsia (PE) mouse model (Mus musculus). This is an experimental study of 30 pregnant mice that were randomly divided into three groups: normal, PE, and PE with rVEGF121 injection. The PE mouse model was created by injecting anti Qa-2 10 ng iv, which is deleterious to Qa-2 expression (homologous to HLA-G), from the first to the fourth day of gestation. PE was validated by measuring serum levels of soluble fms-like tyrosine kinase-1 (sFlt-1) and placental growth factor(PIGF) and also by kidney histopathology. Recombinant VEGF121 was given on the ninth day until the 11th day of pregnancy; mice were terminated on the 16th day. Fetal weights were acquired with a Denver analytical balance. Serum levels of sFlt-1 and PlGF were measured using enzyme-linked immunosorbent assay (ELISA). The data were statistically analyzed via analysis of variance (ANOVA). On average, fetal birth weight was 0.7150 g in the normal group, 0.4936 g in the PE group, and 0.6768 g in the PE with rVEGF121 injection group. ANOVA showed significant growth restriction in the PE group (P=0.006), confirming the use of anti Qa-2 as a suitable PE model. Kidney histopathology results, sFlt-1 levels, and PlGF levels also demonstrated that anti Qa-2 consistently conferred hallmarks of PE in mice. Vascular endothelial growth factor (VEGF) injection prevented fetal growth restriction; comparable fetal weights were observed between the PE model with VEGF treatment and the normal group (P=0.610) but differed from the untreated PE group (P=0.021). Injection of rVEGF121 has the potential to prevent fetal growth restriction in a newly proposed PE mouse model.

  5. An experimental study of VEGF induced changes in vasoactivity in pig retinal arterioles and the influence of an anti-VEGF agent

    Directory of Open Access Journals (Sweden)

    Su Er-Ning

    2012-07-01

    Full Text Available Abstract Background Vascular endothelial growth factor (VEGF plays an important role in ocular physiology. Anti-VEGF agents are now used for treatment of common retinal diseases. This study characterises the vasoactive properties of VEGF in isolated perfused pig retinal arterioles under normal tone or endothelin-1 (ET-1 pre-contracted conditions and determines the influence of an anti VEGF agent on VEGF induced vasoactivity. Methods An isolated perfused retinal arteriole preparation was used. The outer diameter of retinal vessels was monitored at 2 second intervals in response to VEGF and the anti VEGF agent, bevacizumab. The effect of intraluminal delivery of VEGF was determined over a wide concentration range (10-16 to 10-7 M both with and without pre-contraction with ET-1 (3 x 10-9 M. Bevacizumab (0.35 mg mL-1 was applied extraluminally to determine the influence of bevacizumab on VEGF induced vasoactive changes on ET-1 pre-contracted vessels. Results In retinal arterioles with normal tone, VEGF induced a concentration dependent contraction at low concentrations, reaching 93.5% at 10-11 M and then contraction was reduced at higher concentrations, recovering to 98.1% at 10-7 M. VEGF produced a potent concentration dependent vasodilatation in arterioles pre-contracted with ET-1. VEGF induced vasodilatation in arterioles pre-contracted with ET-1 was significantly inhibited by bevacizumab. Conclusions VEGF induced vasoactive changes in pig retinal arterioles are dependent on concentration and vascular tone. Bevacizumab inhibits VEGF-induced vasodilatation in pre-contracted arterioles.

  6. VEGF promotes gastric cancer development by upregulating CRMP4

    Science.gov (United States)

    Peng, Jianjun; Zhai, Ertao; He, Yulong; Wu, Hui; Chen, Chuangqi; Ma, Jinping; Wang, Zhao; Cai, Shirong

    2016-01-01

    This study aimed to investigate the precise role of CRMP4 in gastric tumor growth and patient survival. The mRNA and protein expression levels of CRMP4, VEGF and VEGFR2 were validated by qRT-PCR and immunohistochemistry. We investigated the effects on tumor growth of overexpression and knockdown of CRMP4 both in vitro and in vivo by constructing stable gastric cell lines using lentiviral-mediated transduction and shRNA interference-mediated knockdown of CRMP4 expression. We further validated the role of the ERK/AKT signaling pathways in VEGF and CRMP4 expression using ERK and PI3K inhibitors. Increased expression of VEGF and CRMP4 were observed in gastric cancer tissues compared with tumor-adjacent tissue. We found that higher CRPM4 expression was associated with lymph node metastasis, TNM stage, tumor differentiation and poorer prognosis in gastric cancer patients. In HGC27 and SGC7901 gastric cancer cells, VEGF upregulated CRMP4 in time and dose-dependent manners. Overexpression of CRMP4 increased cell proliferation, migration and invasion, whereas knockdown of CRMP4 expression had opposite effects. VEGF activated CRMP4 expression in gastric cancer cells, and this effect was significantly inhibited by MAPK and PI3K inhibitors (PD98059 and LY294002). In mice, CRMP4 overexpression also resulted in increased tumor growth. These results suggest that increased CRMP4 expression mediated by the activation of VEGF signaling facilitates gastric tumor growth and metastasis, which may have clinical implications associated with a reduced survival rate in gastric cancer patients. PMID:26934554

  7. Study of VEGF transfection on rabbit mesenchymal stem cells%VEGF基因修饰兔骨髓间充质干细胞的研究

    Institute of Scientific and Technical Information of China (English)

    徐松柏; 赵刚; 赵红光; 许侃; 于洪泉; 候宜

    2008-01-01

    ELISA method.The proliferation capability of human umbilical vein endothelial cells (HUVEC) stimulated by the supernatant was measured with MTT methods, untreated MSCs and pcDNA3.0 transfected MSCs were used as control groups.MAIN OUTCOME MEASURES: ① Result of restriction enzyme digestion and DNA sequencing of the recombinant plasmid pcDNA3.0-VEGF165;② the secretion of human VEGF165 proteins of the transfected MSCs analyzed by ABC-ELISA; ③ MTT method was used to detect the effects of MSCs culture supematant transfected with VEGF165 on HUVEC cells proliferation ability.RESULTS: ①Result of restriction enzyme digestion and DNA sequencing of the recombinant plasmid: The constructed plasmid was digested with Hind Ⅲ and XHol Ⅰ, and then two pieces fragments were isolated with agarose gel electrophoresis, which was accordance with expected results. And sequencing results showed that PeDNA3.0-VEGF165 eukaryotic expression vector was successfully constructed. ② ABC-ELISA method: Compared with the control group, concentration of human VEGF protein in the supernatant of the cultured cells increased significantly after the MSCs were transfected with pcDNA3.0-VEGF165 for 24, 48, 72 hours (P<0.05).③ MTT method was used to detect the effects of MSCs culture supernatant transfected with VEGF165 on HUVEC cells proliferation ability. The results showed MSCs supematant transfected with VEGF165 (2%, 4%,8%, 16%, and 32%) had statistical significance in promoting HUVEC cells proliferation rate compared with the normal control (P<0.05).CONCLUSION: Human VEGF gene can be successfully transfected into MSCs and expressed effectively.%背景:血管内皮生长因子的应用是组织工程组织血管化的有效手段.但是,血管内皮生长因子价格昂贵,并且半衰期非常短.很难在体内保持有效的作用浓度.故本实验将其转入组织工程的种子细胞--骨髓间充质干细胞中,使骨髓间充质干细胞能够有效地分泌血管内皮生长

  8. Click-generated triazole based ferrocene-carbohydrate bioconjugates: A highly selective multisignalling probe for Cu(II) ions

    Indian Academy of Sciences (India)

    Arunabha Thakur; Sinjinee Sardar; Sundargopal Ghosh

    2012-11-01

    Two Cu2+-specific colorimetric sensors, based on ferrocene-carbohydrate bioconjugates, 2, C46H56O20N6Fe and 3, C28H33O10N3Fe were designed and synthesized in good yields. Both the compounds, 2 and 3, behave as very selective and sensitive chromogenic and electrochemical chemosensor for Cu2+ ion in aqueous environment (CH3CN/H2O (2:8, /). The analytical detection limit (ADL) for receptor 2 was 7.5 × 10−7 M. The considerable changes in their absorption spectra of 2 and 3 are accompanied by the appearance of a new low energy (LE) peak at 630 nm (2: = 1600 M-1 cm-1 and 3: 822 M-1 cm-1). This is further accompanied by a strong colour change from yellow to dark green that allows the prospective for `naked eye’ detection of Cu2+ ion.

  9. Design of brain imaging agents for positron emission tomography: do large bioconjugates provide an opportunity for in vivo brain imaging?

    Science.gov (United States)

    Schirrmacher, Ralf; Bernard-Gauthier, Vadim; Reader, Andrew; Soucy, Jean-Paul; Schirrmacher, Esther; Wängler, Björn; Wängler, Carmen

    2013-09-01

    The development of brain imaging agents for positron emission tomography and other in vivo imaging modalities mostly relies on small compounds of low MW as a result of the restricted transport of larger molecules, such as peptides and proteins, across the blood-brain barrier. Besides passive transport, only a few active carrier mechanisms, such as glucose transporters and amino acid transporters, have so far been exploited to mediate the accumulation of imaging probes in the brain. An important question for the future is whether some of the abundant active carrier systems located at the blood-brain barrier can be used to shuttle potential, but non-crossing, imaging agents into the brain. What are the biological and chemical constrictions toward such bioconjugates and is it worthwhile to persue such a delivery strategy?

  10. Energy Transfer with Semiconductor Quantum Dot Bioconjugates: A Versatile Platform for Biosensing, Energy Harvesting, and Other Developing Applications.

    Science.gov (United States)

    Hildebrandt, Niko; Spillmann, Christopher M; Algar, W Russ; Pons, Thomas; Stewart, Michael H; Oh, Eunkeu; Susumu, Kimihiro; Díaz, Sebastian A; Delehanty, James B; Medintz, Igor L

    2017-01-25

    Luminescent semiconductor quantum dots (QDs) are one of the more popular nanomaterials currently utilized within biological applications. However, what is not widely appreciated is their growing role as versatile energy transfer (ET) donors and acceptors within a similar biological context. The progress made on integrating QDs and ET in biological configurations and applications is reviewed in detail here. The goal is to provide the reader with (1) an appreciation for what QDs are capable of in this context, (2) how this field has grown over a relatively short time span, and, in particular, (3) how QDs are steadily revolutionizing the development of new biosensors along with a myriad of other photonically active nanomaterial-based bioconjugates. An initial discussion of QD materials along with key concepts surrounding their preparation and bioconjugation is provided given the defining role these aspects play in the QDs ability to succeed in subsequent ET applications. The discussion is then divided around the specific roles that QDs provide as either Förster resonance energy transfer (FRET) or charge/electron transfer donor and/or acceptor. For each QD-ET mechanism, a working explanation of the appropriate background theory and formalism is articulated before examining their biosensing and related ET utility. Other configurations such as incorporation of QDs into multistep ET processes or use of initial chemical and bioluminescent excitation are treated similarly. ET processes that are still not fully understood such as QD interactions with gold and other metal nanoparticles along with carbon allotropes are also covered. Given their maturity, some specific applications ranging from in vitro sensing assays to cellular imaging are separated and discussed in more detail. Finally a perspective on how this field will continue to evolve is provided.

  11. The relationship of VEGF polymorphisms with serum VEGF levels and progression-free survival in patients with epithelial ovarian cancer

    DEFF Research Database (Denmark)

    Steffensen, K D; Waldstrøm, M; Brandslund, Ivan

    2010-01-01

    -linked immunosorbent assay (ELISA). VEGF gene polymorphisms (-2578 C/A, -1154 G/A, -460 T/C, +405 G/C and +936C/T) were determined by real time PCR using genomic DNA extracted from whole blood samples. RESULTS: VEGF serum levels were significantly higher in carriers of the 2578C, 460T and 405C, alleles compared to non......-carriers (p=0.003, p=0.003 and p=0.001, respectively). There was no significant correlation between VEGF SNP genotypes and progression-free survival. In haplotype analysis, the multivariate survival analysis showed that progression-free survival (PFS) for the patients with the AGCGC haplotype...

  12. Enhanced Vascularization in Hybrid PCL/Gelatin Fibrous Scaffolds with Sustained Release of VEGF

    National Research Council Canada - National Science Library

    Wang, Kai; Chen, Xuejiao; Pan, Yiwa; Cui, Yun; Zhou, Xin; Kong, Deling; Zhao, Qiang

    2015-01-01

    .... In this study, fibrous scaffolds that consist of PCL and gelatin fibers were fabricated. The gelatin fibers were further functionalized by heparin immobilization, which provides binding sites for VEGF and thus enables the sustained release of VEGF...

  13. Relationship of VEGF/VEGFR with immune and cancer cells:staggering or forward?

    Institute of Scientific and Technical Information of China (English)

    Yu-Ling Li; Hua Zhao; Xiu-Bao Ren

    2016-01-01

    Vascular endothelial growth factor (VEGF) is primarily known as a proangiogenic factor and is one of the most important growth and survival factors affecting the vascular endothelium. However, recent studies have shown that VEGF also plays a vital role in the immune environment. In addition to the traditional growth factor role of VEGF and VEGF receptors (VEGFRs), they have a complicated relationship with various immune cells. VEGF also reportedly inhibits the differentiation and function of immune cells during hematopoiesis. Dendritic cells (DCs), macrophages, and lymphocytes further express certain types of VEGF receptors. VEGF can be secreted as well by tumor cells through the autocrine pathway and can stimulate the function of cancer stemness. This review will provide a paradigm shift in our understanding of the role of VEGF/VEGFR signaling in the immune and cancer environment.

  14. Extracellular Matrix Stiffness Controls VEGF Signaling and Processing in Endothelial Cells.

    Science.gov (United States)

    Sack, Kelsey D; Teran, Madelane; Nugent, Matthew A

    2016-09-01

    Vascular endothelial growth factor A (VEGF) drives endothelial cell maintenance and angiogenesis. Endothelial cell behavior is altered by the stiffness of the substrate the cells are attached to suggesting that VEGF activity might be influenced by the mechanical cellular environment. We hypothesized that extracellular matrix (ECM) stiffness modifies VEGF-cell-matrix tethering leading to altered VEGF processing and signaling. We analyzed VEGF binding, internalization, and signaling as a function of substrate stiffness in endothelial cells cultured on fibronectin (Fn) linked polyacrylamide gels. Cell produced extracellular matrices on the softest substrates were least capable of binding VEGF, but the cells exhibited enhanced VEGF internalization and signaling compared to cells on all other substrates. Inhibiting VEGF-matrix binding with sucrose octasulfate decreased cell-internalization of VEGF and, inversely, heparin pre-treatment to enhance Fn-matrix binding of VEGF increased cell-internalization of VEGF regardless of matrix stiffness. β1 integrins, which connect cells to Fn, modulated VEGF uptake in a stiffness dependent fashion. Cells on hard surfaces showed decreased levels of activated β1 and inhibition of β1 integrin resulted in a greater proportional decrease in VEGF internalization than in cells on softer matrices. Extracellular matrix binding is necessary for VEGF internalization. Stiffness modifies the coordinated actions of VEGF-matrix binding and β1 integrin binding/activation, which together are critical for VEGF internalization. This study provides insight into how the microenvironment may influence tissue regeneration and response to injury and disease. J. Cell. Physiol. 231: 2026-2039, 2016. © 2016 Wiley Periodicals, Inc.

  15. Recombinant Technology and Probiotics

    Directory of Open Access Journals (Sweden)

    Icy D’Silva

    2011-09-01

    Full Text Available Recombinant technology has led the way to monumental advances in the development of useful molecules, including the development of safe probiotics. The development of novel approaches using recombinant technology and probiotics that allow accurate targeting of therapeutics to the mucosa is an interesting area of research. The creation and use of recombinant probiotics expressing recombinantovalbumin, recombinant ovalbumin mutants and yet-to-be-designed recombinant hypo/non-allergenic molecules offer the opportunity to further investigate their effects for food, nutrition, environment andhealth. This review highlights advances in native probiotics and recombinant probiotics expressing native and recombinant molecules for food, nutrition, environment and health.

  16. Pharmacokinetics and tolerability of cediranib, a potent VEGF signalling inhibitor, in cancer patients with hepatic impairment

    DEFF Research Database (Denmark)

    van Herpen, Carla M L; Lassen, Ulrik; Desar, Ingrid M E

    2013-01-01

    Vascular endothelial growth factor (VEGF) signalling plays a key role in tumour angiogenesis. Cediranib (AZD2171) is a small-molecule VEGF signalling inhibitor with potent activity against all three VEGF receptors. In this phase I, open-label, parallel-group study, adults with advanced solid tumo...

  17. Biological variations in plasma VEGF and VEGFR-1 may compromise their biomarker value in colorectal cancer

    DEFF Research Database (Denmark)

    Svendsen, Mads N.; Brunner, Nils; Christensen, Ib Jarle

    2010-01-01

    Vascular Endothelial Growth Factor (VEGF) plays a prominent role in tumor angiogenesis and plasma VEGF concentration may carry prognostic information in colorectal cancer. The VEGF receptor 1 (VEGFR-1) is a regulatory receptor which is shredded into plasma of patients with colorectal cancer. For ....... For both molecules, large biological variation and lack of standardization of assay procedures are major challenges....

  18. In vivo VEGF imaging with radiolabeled bevacizumab in a human ovarian tumor xenograft

    NARCIS (Netherlands)

    Nagengast, Wouter B.; Hospers, Geke A.; Mulder, Nanno H.; de Jong, Johan R.; Hollema, Harry; Brouwers, Adrienne H.; van Dongen, Guns A.; Perk, Lars R.; Lub-de Hooge, Marjolijn N.

    Vascular endothelial growth factor (VEGF), released by tumor cells, is an important growth factor in tumor angiogenesis. The humanized monoclonal antibody bevacizumab blocks VEGF-induced tumor angiogenesis by binding, thereby neutralizing VEGF. Our aim was to develop radiolabeled bevacizumab for

  19. Pharmacokinetics and tolerability of cediranib, a potent VEGF signalling inhibitor, in cancer patients with hepatic impairment

    NARCIS (Netherlands)

    Herpen, C.M.L. van; Lassen, U.; Desar, I.M.E.; Brown, K.H.; Marotti, M.; Jonge, M.J. de

    2013-01-01

    Vascular endothelial growth factor (VEGF) signalling plays a key role in tumour angiogenesis. Cediranib (AZD2171) is a small-molecule VEGF signalling inhibitor with potent activity against all three VEGF receptors. In this phase I, open-label, parallel-group study, adults with advanced solid tumours

  20. Opiate receptor blockade on human granulosa cells inhibits VEGF release.

    Science.gov (United States)

    Lunger, Fabian; Vehmas, Anni P; Fürnrohr, Barbara G; Sopper, Sieghart; Wildt, Ludwig; Seeber, Beata

    2016-03-01

    The objectives of this study were to determine whether the main opioid receptor (OPRM1) is present on human granulosa cells and if exogenous opiates and their antagonists can influence granulosa cell vascular endothelial growth factor (VEGF) production via OPRM1. Granulosa cells were isolated from women undergoing oocyte retrieval for IVF. Complementary to the primary cells, experiments were conducted using COV434, a well-characterized human granulosa cell line. Identification and localization of opiate receptor subtypes was carried out using Western blot and flow cytometry. The effect of opiate antagonist on granulosa cell VEGF secretion was assessed by enzyme-linked immunosorbent assay. For the first time, the presence of OPRM1 on human granulosa cells is reported. Blocking of opiate signalling using naloxone, a specific OPRM1 antagonist, significantly reduced granulosa cell-derived VEGF levels in both COV434 and granulosa-luteal cells (P opiate receptors and opiate signalling in granulosa cells suggest a possible role in VEGF production. Targeting this signalling pathway could prove promising as a new clinical option in the prevention and treatment of ovarian hyperstimulation syndrome.

  1. Programmed translational readthrough generates antiangiogenic VEGF-Ax.

    Science.gov (United States)

    Eswarappa, Sandeepa M; Potdar, Alka A; Koch, William J; Fan, Yi; Vasu, Kommireddy; Lindner, Daniel; Willard, Belinda; Graham, Linda M; DiCorleto, Paul E; Fox, Paul L

    2014-06-19

    Translational readthrough, observed primarily in less complex organisms from viruses to Drosophila, expands the proteome by translating select transcripts beyond the canonical stop codon. Here, we show that vascular endothelial growth factor A (VEGFA) mRNA in mammalian endothelial cells undergoes programmed translational readthrough (PTR) generating VEGF-Ax, an isoform containing a unique 22-amino-acid C terminus extension. A cis-acting element in the VEGFA 3' UTR serves a dual function, not only encoding the appended peptide but also directing the PTR by decoding the UGA stop codon as serine. Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 binds this element and promotes readthrough. Remarkably, VEGF-Ax exhibits antiangiogenic activity in contrast to the proangiogenic activity of VEGF-A. Pathophysiological significance of VEGF-Ax is indicated by robust expression in multiple human tissues but depletion in colon adenocarcinoma. Furthermore, genome-wide analysis revealed AGO1 and MTCH2 as authentic readthrough targets. Overall, our studies reveal a novel protein-regulated PTR event in a vertebrate system.

  2. Recombinant Technology and Probiotics

    OpenAIRE

    Icy D’Silva

    2011-01-01

    Recombinant technology has led the way to monumental advances in the development of useful molecules, including the development of safe probiotics. The development of novel approaches using recombinant technology and probiotics that allow accurate targeting of therapeutics to the mucosa is an interesting area of research. The creation and use of recombinant probiotics expressing recombinantovalbumin, recombinant ovalbumin mutants and yet-to-be-designed recombinant hypo/non-allergenic molecule...

  3. Plasmid construction and idetification of human vascular endothelial growth factor receptor(VEGF-R3) extracellular domain 1-3Loop cDNA abstract%人类血管内皮生长因子受体VEGF-R3胞外区1-3环cDNA基因的研究

    Institute of Scientific and Technical Information of China (English)

    解宝童; 李涛

    2007-01-01

    Objective To insert VEGF-R3(FIT-4)gene fragment into cloning vector and identify it.Methods By T-A cloning,the VEGF-R3 gene fragment amplified by RT-PCR was cloned on the PGEM-T Easy plasmid vector and transfected into E.coli JM109.the recombinant clones were screened by"white-blue plaque selection" and tested by the methods of single restrictional enzyme.Results The VEGF-R3 gene fragment amplified by RT-PCR showed a smear after the electrophoresis on agarose Gel and obtained white clones by"white-blue plaque selection".The electrophoresis result of the recombinant clones tested by the single restrictional enzyme was accordant with expectant result.Conclusion The recombinant clones through the methods T-A may be used in the further gene expression and gene therapy studies.%目的 将VEGF-R3(FIT-4)基因片断插入克隆载体并进行分子生物学鉴定,对肿瘤细胞通过淋巴管转移到局部淋巴结的机制进行研究.方法 用T-A克隆的方法 将RT-PCR产物克隆入质粒载体PGEM-T Easy,转染大肠杆菌JM109,进行蓝白斑筛选,获取重组体单酶切电泳图谱.结果 经过RT-PCR扩增,得到VEGF-R3的cDNA,琼脂糖凝胶电泳显示为一条带,经蓝白斑筛选获得基因VEGF-R3的白色克隆,重组质粒单酶切电泳结果 与预期结果 一致.结论 RT-PCR产物可采用T-A克隆,获得的阳性重组体可用于进一步基因表达及基因治疗等研究.

  4. The relationship of the angiogenesis regulators VEGF-A, VEGF-R1 and VEGF-R2 to p53 status and prognostic factors in epithelial ovarian carcinoma in FIGO-stages I-II.

    Science.gov (United States)

    Skirnisdottir, Ingiridur; Seidal, Tomas; Åkerud, Helena

    2016-03-01

    The aim of this study was to evaluate prognostic effect of the angiogenesis regulators VEGF-R1, VEGF-R2 and VEGF-A for recurrent disease and disease-free survival (DFS), and their relation to the apoptosis regulator p53, in 131 patients with FIGO-stages I-II with epithelial ovarian cancer. For the detection of positivity of the markers the techniques of tissue microarrays and immunohistochemistry (IHC) were used. In tumors the frequency of positive staining for VEGF-R1 was 19%, for VEGF-R2 and VEGF-A, it was 77 and 70%, respectively. Positivity for p53 was detected in 25% of tumors. The total number of recurrences in the complete series was 34 out of 131 (26%) and 5-year disease-free survival (DFS) was 68%. Positive staining for VEGF-A (P=0.030), VEGF-R2 (P=0.011) and p53 (P=0.015) was found more frequently in type II tumors than in type I tumors. Patients with VEGF-R1 negative tumors had worse (P=0.021) DFS compared to patients with VEGF-R1 positive tumors. In two multivariate Cox analyzes with DFS as endpoint, FIGO-stage (HR=3.8), VEGF-R2 status (HR=0.4) and p53 status (HR=2.3), all were significant and independent prognostic factors. When the variables VEGF-R2 and p53 were replaced with the new variable VEGF-R2+p53-/other three combinations in one group, it was found that patients from that subgroup had 86% reduced risk of dying in disease (HR=0.24). Findings above, confirmed relationship between VEGF-R2 and VEGF-A and p53, respectively, with regard to recurrent disease and survival. Some findings from the present study are different from results from previous studies on the regulation of angiogenesis. Despite many trials with anti-angiogenic agents in the front line of ovarian cancer have shown to be positive for progression-free survival, no one has demonstrated an impact on overall survival. Therefore, one of the greatest challenges in ovarian cancer research, is to discover predictive and prognostic biomarkers.

  5. Detection of aqueous VEGF concentrations before and after intravitreal injection of anti-VEGF antibody using low-volume sampling paper-based ELISA

    OpenAIRE

    Min-Yen Hsu; Yu-Chien Hung; De-Kuang Hwang; Shang-Chi Lin; Keng-Hung Lin; Chun-Yuan Wang; Hin-Yeung Choi; Yu-Ping Wang; Chao-Min Cheng

    2016-01-01

    Intraocular vascular endothelial growth factor (VEGF) levels play an important role in the pathogenesis of blindness-related diseases, such as age-related macular degeneration (AMD). Here, we aimed to develop a paper-based enzyme-linked immunosorbent assay (P-ELISA) to analyze the suppression of aqueous VEGF concentrations following intravitreal injection (IVI) of anti-VEGF antibody (bevacizumab or ranibizumab). A total of 25 eyes with wet AMD, one with myopic neovascularization, and one with...

  6. VEGF regulates hippocampal neurogenesis and reverses cognitive deficits in immature rats after status epilepticus through the VEGF R2 signaling pathway.

    Science.gov (United States)

    Han, Wei; Song, Xiaojie; He, Rong; Li, Tianyi; Cheng, Li; Xie, Lingling; Chen, Hengsheng; Jiang, Li

    2017-02-10

    Epilepsy is the most common chronic disease in children, who exhibit a higher risk for status epilepticus (SE) than adults. Hippocampal neurogenesis is altered by epilepsy, particularly in the immature brain, which may influence cognitive development. Vascular endothelial growth factor (VEGF) represents an attractive target to modulate brain function at the neurovascular interface and is a double-edged sword in seizures. We used the lithium-pilocarpine-induced epilepsy model in immature Sprague-Dawley rats to study the effects of VEGF on hippocampal neurogenesis in the acute phase and on long-term cognitive behaviors in immature rats following status epilepticus (SE). VEGF correlates with cell proliferation in the immature brain after SE. By preprocessing VEGF in the lateral ventricles prior to the induction of the SE model, we found that VEGF increased the proliferation of neural stem cells (NSCs) and promoted the migration of newly generated cells via the VEGF receptor 2 (VEGFR2) signaling pathway. VEGF also inhibited cell loss and reversed the cognitive deficits that accompany SE. Based on our results, VEGF positively contributes to the initial stages of neurogenesis and alleviates cognitive deficits following seizures; moreover, the VEGF/VEGFR2 signaling pathway may provide a novel treatment strategy for epilepsy.

  7. The prognosis was poorer in colorectal cancers that expressed both VEGF and PROK1 (No correlation coefficient between VEGF and PROK1).

    Science.gov (United States)

    Goi, Takanori; Nakazawa, Toshiyuki; Hirono, Yasuo; Yamaguchi, Akio

    2015-10-06

    The angiogenic proteins vascular endothelial growth factor (VEGF) and prokineticin1 (PROK1) proteins are considered important in colorectal cancer, the relationship between their simultaneous expression and prognosis was investigated in the present study. VEGF and PROK1 expression in 620 primary human colorectal cancer lesions was confirmed via immunohistochemical staining with anti-VEGF and anti-PROK1 antibodies, and the correlation between the expression of these 2 proteins and recurrence/prognosis were investigated. VEGF protein was expressed in 329 (53.1%) and PROK1 protein was expressed in 223 (36.0%). PROK1 and VEGF were simultaneously expressed in 116 (18.7%) of the 620 cases. The correlation coefficient between VEGF expression and PROK1 expression was r = 0.11, and therefore correlation was not observed. Clinical pathology revealed that substantially lymphnode matastasis, hematogenous metastasis, or TMN advanced-stage IV was significantly more prevalent in cases that expressed both VEGF and PROK1 than in the cases negative for both proteins or those positive for only 1 of the proteins. Also the cases positive for both proteins exhibited the worst recurrence and prognosis. In the Cox proportional hazards model, VEGF and PROK1 expression was an independent prognostic factor. The prognosis was poorer in colorectal cancers that expressed both PROK1 and VEGF relative to the cases that expressed only 1 protein, and the expression of both proteins was found to be an independent prognostic factor.

  8. Site-specific bioconjugation of a murine dihydrofolate reductase enzyme by copper(I-catalyzed azide-alkyne cycloaddition with retained activity.

    Directory of Open Access Journals (Sweden)

    Sung In Lim

    Full Text Available Cu(I-catalyzed azide-alkyne cycloaddition (CuAAC is an efficient reaction linking an azido and an alkynyl group in the presence of copper catalyst. Incorporation of a non-natural amino acid (NAA containing either an azido or an alkynyl group into a protein allows site-specific bioconjugation in mild conditions via CuAAC. Despite its great potential, bioconjugation of an enzyme has been hampered by several issues including low yield, poor solubility of a ligand, and protein structural/functional perturbation by CuAAC components. In the present study, we incorporated an alkyne-bearing NAA into an enzyme, murine dihydrofolate reductase (mDHFR, in high cell density cultivation of Escherichia coli, and performed CuAAC conjugation with fluorescent azide dyes to evaluate enzyme compatibility of various CuAAC conditions comprising combination of commercially available Cu(I-chelating ligands and reductants. The condensed culture improves the protein yield 19-fold based on the same amount of non-natural amino acid, and the enzyme incubation under the optimized reaction condition did not lead to any activity loss but allowed a fast and high-yield bioconjugation. Using the established conditions, a biotin-azide spacer was efficiently conjugated to mDHFR with retained activity leading to the site-specific immobilization of the biotin-conjugated mDHFR on a streptavidin-coated plate. These results demonstrate that the combination of reactive non-natural amino acid incorporation and the optimized CuAAC can be used to bioconjugate enzymes with retained enzymatic activity.

  9. Polydopamine-Based Surface Modification of Novel Nanoparticle-Aptamer Bioconjugates for In Vivo Breast Cancer Targeting and Enhanced Therapeutic Effects

    OpenAIRE

    Tao, Wei; Zeng,Xiaowei; Wu, Jun; De Zhu, Xi; Yu,Xinghua; Zhang, Xudong; Zhang, Jinxie; Liu,Gan; Mei, Lin

    2016-01-01

    In this study, we reported a simple polydopamine (pD)-based surface modification method to prepare novel nanoparticle-aptamer bioconjugates (Apt-pD-DTX/NPs) for in vivo tumor targeting and enhanced therapeutic effects of breast cancer. With simple preparation procedures, the new functionalized Apt-pD-DTX/NPs could maximumly increase the local effective drug concentration on tumor sites, achieving enhanced treatment effectiveness and minimizing side effects. The dopamine polymerization and apt...

  10. Site-specific bioconjugation of a murine dihydrofolate reductase enzyme by copper(I)-catalyzed azide-alkyne cycloaddition with retained activity.

    Science.gov (United States)

    Lim, Sung In; Mizuta, Yukina; Takasu, Akinori; Kim, Yong Hwan; Kwon, Inchan

    2014-01-01

    Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is an efficient reaction linking an azido and an alkynyl group in the presence of copper catalyst. Incorporation of a non-natural amino acid (NAA) containing either an azido or an alkynyl group into a protein allows site-specific bioconjugation in mild conditions via CuAAC. Despite its great potential, bioconjugation of an enzyme has been hampered by several issues including low yield, poor solubility of a ligand, and protein structural/functional perturbation by CuAAC components. In the present study, we incorporated an alkyne-bearing NAA into an enzyme, murine dihydrofolate reductase (mDHFR), in high cell density cultivation of Escherichia coli, and performed CuAAC conjugation with fluorescent azide dyes to evaluate enzyme compatibility of various CuAAC conditions comprising combination of commercially available Cu(I)-chelating ligands and reductants. The condensed culture improves the protein yield 19-fold based on the same amount of non-natural amino acid, and the enzyme incubation under the optimized reaction condition did not lead to any activity loss but allowed a fast and high-yield bioconjugation. Using the established conditions, a biotin-azide spacer was efficiently conjugated to mDHFR with retained activity leading to the site-specific immobilization of the biotin-conjugated mDHFR on a streptavidin-coated plate. These results demonstrate that the combination of reactive non-natural amino acid incorporation and the optimized CuAAC can be used to bioconjugate enzymes with retained enzymatic activity.

  11. Extracellular biosynthesis of gadolinium oxide (Gd2O3) nanoparticles, their biodistribution and bioconjugation with the chemically modified anticancer drug taxol

    OpenAIRE

    Khan, Shadab Ali; Gambhir, Sanjay; Ahmad, Absar

    2014-01-01

    As a part of our programme to develop nanobioconjugates for the treatment of cancer, we first synthesized extracellular, protein-capped, highly stable and well-dispersed gadolinium oxide (Gd2O3) nanoparticles by using thermophilic fungus Humicola sp. The biodistribution of the nanoparticles in rats was checked by radiolabelling with Tc-99m. Finally, these nanoparticles were bioconjugated with the chemically modified anticancer drug taxol with the aim of characterizing the role of this bioconj...

  12. Angiogenesis and bone regeneration of porous nano-hydroxyapatite/coralline blocks coated with rhVEGF165 in critical-size alveolar bone defects in vivo.

    Science.gov (United States)

    Du, Bing; Liu, Weizhen; Deng, Yue; Li, Shaobing; Liu, Xiangning; Gao, Yan; Zhou, Lei

    2015-01-01

    To improve the regenerative performance of nano-hydroxyapatite/coralline (nHA/coral) block grafting in a canine mandibular critical-size defect model, nHA/coral blocks were coated with recombinant human vascular endothelial growth factor(165) (rhVEGF) via physical adsorption (3 μg rhVEGF165 per nHA/coral block). After the nHA/coral blocks and VEGF/nHA/coral blocks were randomly implanted into the mandibular box-shaped defects in a split-mouth design, the healing process was evaluated by histological observation and histomorphometric and immunohistological analyses. The histological evaluations revealed the ingrowth of newly formed blood vessels and bone at the periphery and cores of the blocks in both groups at both 3 and 8 weeks postsurgery, respectively. In the histomorphometric analysis, the VEGF/nHA/coral group exhibited a larger quantity of new bone formation at 3 and 8 weeks postsurgery. The percentages of newly formed bone within the entire blocks in the VEGF/nHA/coral group were 27.3% ± 8.1% and 39.3% ± 12.8% at 3 weeks and 8 weeks, respectively, and these values were slightly greater than those of the nHA/coral group (21.7% ± 3.0% and 32.6% ± 10.3%, respectively), but the differences were not significant (P>0.05). The immunohistological evaluations revealed that the neovascular density in the VEGF/nHA/coral group (146 ± 32.9 vessel/mm(2)) was much greater than that in the nHA/coral group (105 ± 51.8 vessel/mm(2)) at the 3-week time point (P0.05). The present study indicated that nHA/coral blocks might be optimal scaffolds for block grafting in critical-size mandibular defects and that additional VEGF coating via physical adsorption can promote angiogenesis in the early stage of bone healing, which suggests that prevascularized nHA/coral blocks have significant potential as a bioactive material for bone regeneration in large-scale alveolar defects.

  13. Use of 5'-γ-ferrocenyl adenosine triphosphate (Fc-ATP) bioconjugates having poly(ethylene glycol) spacers in kinase-catalyzed phosphorylations.

    Science.gov (United States)

    Martić, Sanela; Rains, Meghan K; Freeman, Daniel; Kraatz, Heinz-Bernhard

    2011-08-17

    The 5'-γ-ferrocenyl adenosine triphosphate (Fc-ATP) bioconjugates (3 and 4), containing the poly(ethylene glycol) spacers, were synthesized and compared to a hydrophobic analogue as co-substrates for the following protein kinases: sarcoma related kinase (Src), cyclin-dependent kinase (CDK), casein kinase II (CK2α), and protein kinase A (PKA). Electrochemical kinase assays indicate that the hydrophobic Fc-ATP analogue was an optimal co-substrate for which K(M) values were determined to be in the 30-200 μM range, depending on the particular protein kinase. The luminescence kinase assay demonstrated the kinase utility for all Fc-ATP conjugates, which is in line with the electrochemical data. Moreover, Fc-ATP bioconjugates exhibit competitive behavior with respect to ATP. Relatively poor performance of the polar Fc-ATP bioconjugates as co-substrates for protein kinases was presumably due to the additional H-bonding and electrostatic interactions of the poly(ethylene glycol) linkers of Fc-ATP with the kinase catalytic site and the target peptides. Phosphorylation of the full-length protein, His-tagged pro-caspase-3, was demonstrated through Fc-phosphoamide transfer to the Ser residues of the surface-bound protein by electrochemical means. These results suggest that electrochemical detection of the peptide and protein Fc-phosphorylation via tailored Fc-ATP co-substrates may be useful for probing protein-protein interactions.

  14. VEGF-A isoforms program differential VEGFR2 signal transduction, trafficking and proteolysis

    Science.gov (United States)

    Fearnley, Gareth W.; Smith, Gina A.; Abdul-Zani, Izma; Yuldasheva, Nadira; Mughal, Nadeem A.; Homer-Vanniasinkam, Shervanthi; Kearney, Mark T.; Zachary, Ian C.; Tomlinson, Darren C.; Harrison, Michael A.; Wheatcroft, Stephen B.; Ponnambalam, Sreenivasan

    2016-01-01

    ABSTRACT Vascular endothelial growth factor A (VEGF-A) binding to the receptor tyrosine kinase VEGFR2 triggers multiple signal transduction pathways, which regulate endothelial cell responses that control vascular development. Multiple isoforms of VEGF-A can elicit differential signal transduction and endothelial responses. However, it is unclear how such cellular responses are controlled by isoform-specific VEGF-A–VEGFR2 complexes. Increasingly, there is the realization that the membrane trafficking of receptor–ligand complexes influences signal transduction and protein turnover. By building on these concepts, our study shows for the first time that three different VEGF-A isoforms (VEGF-A165, VEGF-A121 and VEGF-A145) promote distinct patterns of VEGFR2 endocytosis for delivery into early endosomes. This differential VEGFR2 endocytosis and trafficking is linked to VEGF-A isoform-specific signal transduction events. Disruption of clathrin-dependent endocytosis blocked VEGF-A isoform-specific VEGFR2 activation, signal transduction and caused substantial depletion in membrane-bound VEGFR1 and VEGFR2 levels. Furthermore, such VEGF-A isoforms promoted differential patterns of VEGFR2 ubiquitylation, proteolysis and terminal degradation. Our study now provides novel insights into how different VEGF-A isoforms can bind the same receptor tyrosine kinase and elicit diverse cellular outcomes. PMID:27044325

  15. VEGF-A isoforms program differential VEGFR2 signal transduction, trafficking and proteolysis

    Directory of Open Access Journals (Sweden)

    Gareth W. Fearnley

    2016-05-01

    Full Text Available Vascular endothelial growth factor A (VEGF-A binding to the receptor tyrosine kinase VEGFR2 triggers multiple signal transduction pathways, which regulate endothelial cell responses that control vascular development. Multiple isoforms of VEGF-A can elicit differential signal transduction and endothelial responses. However, it is unclear how such cellular responses are controlled by isoform-specific VEGF-A–VEGFR2 complexes. Increasingly, there is the realization that the membrane trafficking of receptor–ligand complexes influences signal transduction and protein turnover. By building on these concepts, our study shows for the first time that three different VEGF-A isoforms (VEGF-A165, VEGF-A121 and VEGF-A145 promote distinct patterns of VEGFR2 endocytosis for delivery into early endosomes. This differential VEGFR2 endocytosis and trafficking is linked to VEGF-A isoform-specific signal transduction events. Disruption of clathrin-dependent endocytosis blocked VEGF-A isoform-specific VEGFR2 activation, signal transduction and caused substantial depletion in membrane-bound VEGFR1 and VEGFR2 levels. Furthermore, such VEGF-A isoforms promoted differential patterns of VEGFR2 ubiquitylation, proteolysis and terminal degradation. Our study now provides novel insights into how different VEGF-A isoforms can bind the same receptor tyrosine kinase and elicit diverse cellular outcomes.

  16. Cancer-derived VEGF plays no role in malignant ascites formation in the mouse

    Institute of Scientific and Technical Information of China (English)

    Bayasi Guleng; Tsuneo Ikenoue; Yasushi Fukushima; Keita Morikane; Makoto Miyagishi; Kazunari Taira; Takao Kawabe; Masao Omata; Keisuke Tateishi; Fumihiko Kanai; Amarsanaa Jazag; Miki Ohta; Yoshinari Asaoka; Hideaki Ijichi; Yasuo Tanaka; Jun Imamura

    2005-01-01

    AIM: Vascular endothelial growth factor (VEGF) is a potent mediator of peritoneal fluid accumulation following tumor progression. This study investigated the role of VEGF secreted by cancerous cells in the formation of malignant ascites.METHODS: VEGF expression was eliminated byknockdown in the pancreas cancer cell-line PancO2 using vector-based short-hairpin type RNA interference (RNAi).Malignant ascites formation in the mouse was analyzed by intraperitoneal injection of PancO2 cells expressing VEGF or with expression knockdown.RESULTS: The VEGF knockdown PancO2 cell was successfully established. Knockdown of VEGF did not affect cancer cell proliferation in vitro or in vivo. The volume of ascites following peritoneal expansion of the tumor in VEGF knockdown cells and control cells did not differ statistically in this in vivo study. Moreover, the VEGF concentration in the ascites did not differ statistically.CONCLUSION: Malignant ascites formation might be mediated by VEGF production in noncancerous tissues,such as stromal compartments. An anti-VEGF strategy against malignant ascites could be applied to various tumors regardless of whether they secrete VEGF.

  17. Angiogenic Effects of Collagen/Mesoporous Nanoparticle Composite Scaffold Delivering VEGF165

    Directory of Open Access Journals (Sweden)

    Joong-Hyun Kim

    2016-01-01

    Full Text Available Vascularization is a key issue for the success of tissue engineering to repair damaged tissue. In this study, we report a composite scaffold delivering angiogenic factor for this purpose. Vascular endothelial growth factor (VEGF was loaded on mesoporous silica nanoparticle (MSN, which was then incorporated within a type I collagen sponge, to produce collagen/MSN/VEGF (CMV scaffold. The CMV composite scaffold could release VEGF sustainably over the test period of 28 days. The release of VEGF improved the cell proliferation. Moreover, the in vivo angiogenesis of the scaffold, as studied by the chick chorioallantoic membrane (CAM model, showed that the VEGF-releasing scaffold induced significantly increased number of blood vessel complexes when compared with VEGF-free scaffold. The composite scaffold showed good biocompatibility, as examined in rat subcutaneous tissue. These results demonstrate that the CMV scaffold with VEGF-releasing capacity can be potentially used to stimulate angiogenesis and tissue repair.

  18. The expression and function of VEGF at embryo implanta- tion "window" in the mouse

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that plays a critical role in angiogenesis. Recent reports indicated that VEGF was closely involved in embryo implantation and embryonic vasculogenesis. However, very little information is available about the detailed expression and function of VEGF at implantation "window". In this work, VEGFs were primarily present on uterine epithelial cell monolayer and blastocysts including the outgrew trophoblasts at implantation window. VEGF antibodies decreased the number of mice embryos implanted and the percentage of blastocysts with attachment and outgrowth in a co-culture model in a dose-dependant manner. These findings demonstrate that VEGF is one of the essential cytokines for embryo implantation in mouse. VEGF may act as a local mediator to regulate the maternal-fetal interaction, and facilitate blastocyst implantation.

  19. Vertical bone regeneration using rhBMP-2 and VEGF.

    Science.gov (United States)

    Schorn, Lara; Sproll, Christoph; Ommerborn, Michelle; Naujoks, Christian; Kübler, Norbert R; Depprich, Rita

    2017-06-07

    Sufficient vertical and lateral bone supply and a competent osteogenic healing process are prerequisities for the successful osseointegration of dental implants in the alveolar bone. Several techniques including autologous bone grafts and guided bone regeneration are applied to improve quality and quantity of bone at the implantation site. Depending on the amount of lacking bone one- or two-stage procedures are required. Vertical bone augmentation has proven to be a challenge particularly in terms of bone volume stability. This study focuses on the three dimensional vertical bone generation in a one stage procedure in vivo. Therefore, a collagenous disc-shaped scaffold (ICBM = Insoluble Collagenous Bone Matrix) containing rhBMP-2 (Bone Morphogenetic Protein-2) and/or VEGF (Vascular Endothelial Growth Factor) was applied around the coronal part of a dental implant during insertion. RhBMP-2 and VEGF released directly at the implantation site were assumed to induce the generation of new vertical bone around the implant. One hundred eight titanium implants were inserted into the mandible and the tibia of 12 mini pigs. Four experimental groups were formed: Control group, ICBM, ICBM + BMP-2, and ICBM + BMP-2 + VEGF. After 1, 4 and 12 weeks the animals were sacrificed and bone generation was investigated histologically and histomorphometrically. After 12 weeks the combination of ICBM + rhBMP2 + VEGF showed significantly more bone volume density (BVD%), a higher vertical bone gain (VBG) and more vertical bone gain around the implant (PVBG) in comparison to the control group. By using collagenous disc-shaped matrices in combination with rhBMP-2 and VEGF vertical bone can be generated in a one stage procedure without donor site morbidity. The results of the presenting study suggest that the combination of rhBMP-2 and VEGF applied locally by using a collagenous carrier improves vertical bone generation in vivo. Further research is needed to establish whether this

  20. Conditional deletion of epithelial IKKβ impairs alveolar formation through apoptosis and decreased VEGF expression during early mouse lung morphogenesis

    Directory of Open Access Journals (Sweden)

    Li Changgong

    2011-10-01

    Full Text Available Abstract Background Alveolar septation marks the beginning of the transition from the saccular to alveolar stage of lung development. Inflammation can disrupt this process and permanently impair alveolar formation resulting in alveolar hypoplasia as seen in bronchopulmonary dysplasia in preterm newborns. NF-κB is a transcription factor central to multiple inflammatory and developmental pathways including dorsal-ventral patterning in fruit flies; limb, mammary and submandibular gland development in mice; and branching morphogenesis in chick lungs. We have previously shown that epithelial overexpression of NF-κB accelerates lung maturity using transgenic mice. The purpose of this study was to test our hypothesis that targeted deletion of NF-κB signaling in lung epithelium would impair alveolar formation. Methods We generated double transgenic mice with lung epithelium-specific deletion of IKKβ, a known activating kinase upstream of NF-κB, using a cre-loxP transgenic recombination strategy. Lungs of resulting progeny were analyzed at embryonic and early postnatal stages to determine specific effects on lung histology, and mRNA and protein expression of relevant lung morphoreulatory genes. Lastly, results measuring expression of the angiogenic factor, VEGF, were confirmed in vitro using a siRNA-knockdown strategy in cultured mouse lung epithelial cells. Results Our results showed that IKKβ deletion in the lung epithelium transiently decreased alveolar type I and type II cells and myofibroblasts and delayed alveolar formation. These effects were mediated through increased alveolar type II cell apoptosis and decreased epithelial VEGF expression. Conclusions These results suggest that epithelial NF-κB plays a critical role in early alveolar development possibly through regulation of VEGF.

  1. Response to anti-VEGF-A treatment of endothelial cells in vitro.

    Science.gov (United States)

    Puddu, Alessandra; Sanguineti, Roberta; Traverso, Carlo Enrico; Viviani, Giorgio L; Nicolò, Massimo

    2016-05-01

    This study was conducted to compare the effects of two anti-VEGF-A drugs, Ranibizumab and Aflibercept, on the expression and secretion of VEGFs family members, and on their influence in proliferation and migration of endothelial cells (HECV) in vitro. HECV cells were exposed 24 h (T1), 4 days (T2) and 6 days (T3) to Ranibizumab or Aflibercept at pharmacodynamically relevant concentrations (Ranibizumab: 12.5 μg/ml and 125 μg/ml; Aflibercept: 50 μg/ml and 500 μg/ml). Cell viability and then expression and secretion of VEGF-A, VEGF-B, VEGF-C and PlGF were evaluated respectively by Real Time-PCR and ELISA. Intracellular signaling activated by VEGF-A and VEGF-C was investigated evaluating phosphorylation of VEGFR2. Influence in would healing was evaluated through scratch assay. In general no differences were observed among the tested concentrations of anti-vegf drugs. Ranibizumab and Aflibercept did not affect HECV cell viability in all experimental times. At T1, Ranibizumab decreased mRNA levels of VEGF-A, induced VEGF-C secretion, abrogated phosphorylation of VEGFR2 stimulated by VEGF-A, and impaired ability of HECV cells to repair wound healing. Aflibercept decreased mRNA levels of VEGF-A, -B and PlGF; slightly increased basal level of phVEGFR2, and completely abrogated phosphorylation stimulated by VEGF-A and VEGF-C. No effects on secretion of VEGF-B and on would healing were observed after exposure to Aflibercept. Prolonged exposure to anti-VEGFs decreased expression and secretion of VEGF-A and VEGF-B, up-regulated VEGF-C mRNA levels and its secretion, and increased basal phosphorylation of VEGFR2. Acute treatment with Ranibizumab or Aflibercept evoked different responses on endothelial cells, however these differences were lost after prolonged exposure. Scratch test results suggest that treatment with Ranibizumab may be more effective than Aflibercept in reducing angiogenic potential of endothelial cells in vitro.

  2. Ad-hVEGF165 reverses homocysteine-induced endothelial dysfunction by regulating nitric oxide system%Ad-hVEGF165通过调控一氧化氮系统逆转同型半胱氨酸对内皮细胞的损伤

    Institute of Scientific and Technical Information of China (English)

    赵莉莉; 毛用敏; 张莹; 师莹; 宋衍秋

    2015-01-01

    ,0.05,1.00 mmol/L) for 24 h.The same concentration of Hey,Ad-Track and Ad-hVEGF165 were added to the cells in the following groups:blank group,Hcy0.05 group,Hcy1.00 group,Ad-Track group,Hey0.05 + Ad-Track group,Hcy1.00 + Ad-Track group,Ad-hVEGF165 group,Hey0.05 + Ad-hVEGF165 group,Hcy1.00 + AdhVEGF165 group for 48 h.The mRNA and protein expressions of eNOS and DDAH2 were detected by realtime PCR and Western blot.The correlations of mRNA and protein expressions between endothelial nitric oxide synthase (eNOS) and dimethylarginine dimthylaminohydrolase (DDAH)2 were evaluated by Pearson correlation analysis.Results Compared with blank group and Ad-hVEGF465 group,the mRNA and protein expressions of eNOS were decreased in Hcy0.05 group and Hcy0.05 + Ad-hVEGF165 group (both P < 0.05),and the mRNA and protein expressions of DDAH2 in cells treated with 0.05 mmol/L and 1.00 mmol/L Hcy were reduced as well(all P < 0.05).DDAH2 mRNA and protein expression are increased (all P < 0.05) in Ad-hVEGF165 group compared with the blank group and Ad-Track,Hcy0.05 + Ad-hVEGF165 and Hcy0.05 group compared with Hcy0.05 + Ad-Track group,Hcy1.00 + Ad-hVEGF165 and Hcy1.00 group compared with Hcy1.00 + Ad-Track group.The mRNA and protein expressions of eNOS and DDAH2 were uncorrelated under the effect of Hcy(r =0.057 and 0.449,both P >0.05) and VEGF (r =0.284 and 0.432,both P > 0.05).Conclusion Recombinant adenovirus Ad-hVEGF165 could reverse Hcy-induced endothelial cells dysfunction via upregulating the expressions of eNOS and DDAH2.

  3. Defining response to anti-VEGF therapies in neovascular AMD.

    Science.gov (United States)

    Amoaku, W M; Chakravarthy, U; Gale, R; Gavin, M; Ghanchi, F; Gibson, J; Harding, S; Johnston, R L; Kelly, S P; Kelly, S; Lotery, A; Mahmood, S; Menon, G; Sivaprasad, S; Talks, J; Tufail, A; Yang, Y

    2015-06-01

    The introduction of anti-vascular endothelial growth factor (anti-VEGF) has made significant impact on the reduction of the visual loss due to neovascular age-related macular degeneration (n-AMD). There are significant inter-individual differences in response to an anti-VEGF agent, made more complex by the availability of multiple anti-VEGF agents with different molecular configurations. The response to anti-VEGF therapy have been found to be dependent on a variety of factors including patient's age, lesion characteristics, lesion duration, baseline visual acuity (VA) and the presence of particular genotype risk alleles. Furthermore, a proportion of eyes with n-AMD show a decline in acuity or morphology, despite therapy or require very frequent re-treatment. There is currently no consensus as to how to classify optimal response, or lack of it, with these therapies. There is, in particular, confusion over terms such as 'responder status' after treatment for n-AMD, 'tachyphylaxis' and 'recalcitrant' n-AMD. This document aims to provide a consensus on definition/categorisation of the response of n-AMD to anti-VEGF therapies and on the time points at which response to treatment should be determined. Primary response is best determined at 1 month following the last initiation dose, while maintained treatment (secondary) response is determined any time after the 4th visit. In a particular eye, secondary responses do not mirror and cannot be predicted from that in the primary phase. Morphological and functional responses to anti-VEGF treatments, do not necessarily correlate, and may be dissociated in an individual eye. Furthermore, there is a ceiling effect that can negate the currently used functional metrics such as >5 letters improvement when the baseline VA is good (ETDRS>70 letters). It is therefore important to use a combination of both the parameters in determining the response.The following are proposed definitions: optimal (good) response is defined as when there

  4. Anticancer therapy-induced vascular toxicity: VEGF inhibition and beyond.

    Science.gov (United States)

    Di Lisi, Daniela; Madonna, Rosalinda; Zito, Concetta; Bronte, Enrico; Badalamenti, Giuseppe; Parrella, Paolo; Monte, Ines; Tocchetti, Carlo Gabriele; Russo, Antonio; Novo, Giuseppina

    2017-01-15

    Cardiotoxicity induced by chemotherapeutic agents and radiotherapy is a growing problem. In recent years, an increasing number of new drugs with targeted action have been designed. These molecules, such as monoclonal antibodies and tyrosine kinase inhibitors, can cause different type of toxicities compared to traditional chemotherapy. However, they can also cause cardiac complications such as heart failure, arterial hypertension, QT interval prolongation and arrhythmias. Currently, a field of intense research is the vascular toxicity induced by new biologic drugs, particularly those which inhibit vascular endothelial growth factor (VEGF) and its receptor (VEGF-R) and other tyrosine kinases. In this review, we aim at focusing on the problem of vascular toxicity induced by new targeted therapies, chemotherapy and radiotherapy, and describe the main mechanisms and emphasizing the importance of early diagnosis of vascular damage, in order to prevent clinical complications.

  5. Gene therapy for cardiovascular disease: the potential of VEGF.

    Science.gov (United States)

    Tiong, Alice; Freedman, Saul Benedict

    2004-04-01

    The quest for new therapeutic options and the recent exponential explosion in our knowledge of genetics have led to active interest and research into gene therapy. One area of gene therapy that has generated much debate and controversy is the use of vascular endothelial growth factor (VEGF) for therapeutic angiogenesis for palliative intent, and for the prevention of restenosis following percutaneous revascularization in coronary and peripheral arterial disease. This review highlights the development in VEGF gene therapy in the last 12 to 18 months, particularly the results from randomized, double-blind, placebo-controlled phase I and II studies that have evolved from encouraging results from animal models and early pilot studies in humans.

  6. Anti-VEGF Anticancer Drugs: Mind the Hypertension.

    Science.gov (United States)

    Katsi, Vasiliki; Zerdes, Ioannis; Manolakou, Stavroula; Makris, Thomas; Nihoyannopoulos, Petros; Tousoulis, Dimitris; Kallikazaros, Ioannis

    2014-01-01

    The introduction of therapies that inhibit tumor angiogenesis and particularly target to vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor (VEGFR) (VEGF inhibitors/VEGFi) have revolutionized the treatment of various cancer types. Although their clinical benefit can be optimal for cancer-affected patients, the safety of these targeted agents is of special concern especially for longer-term adjuvant or maintenance treatment. Importantly, VEGFi therapy has been significantly associated with hypertension (HTN) as an adverse effect and therefore the control of blood pressure (BP) after the administration of these drugs remains a challenging matter to be faced. The aim of this review is to summarize studies which investigate the association of VEGFi agents with HTN manifestation and the possible risks associated with this complication. Additionally, given that the optimal management of HTN caused by VEGFi remains obscure, this review will focus on prevention strategies including BP monitoring plans and propose potential therapeutic approaches.

  7. Therapeutic Recombinant Monoclonal Antibodies

    Science.gov (United States)

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  8. Therapeutic Recombinant Monoclonal Antibodies

    Science.gov (United States)

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  9. The remote effects of intravitreal anti-VEGF therapy.

    Science.gov (United States)

    Balta, F; Merticariu, M; Taban, C; Neculau, G; Merticariu, A; Muresanu, D; Badescu, D; Jinga, V

    2016-01-01

    Objective: To study the effects of intravitreal anti-Vascular Endothelial Growth Factor (VEGF) therapy with Avastin for wet Age-Related Macular Degeneration (AMD) on Benign Prostatic Hyperplasia (BPH)-related symptoms. Methods: An exploratory trial was conducted from August 1, 2013 to February 1, 2014, that included 14 male patients previously diagnosed with BPH, who were aged between 59 and 69 years. The trial was performed in Bucharest and involved two medical institutions: the Clinical Hospital of Eye Emergencies and the "Prof. Dr. Theodor Burghele" Hospital. This prospective study utilized both objective and subjective indicators to analyze the link between intravitreal anti-VEGF therapy for wet AMD and BPH. The evaluations consisted of uroflowmetry and International Prostate Symptom Score (I-PSS) assessments. Results: The maximum flow rate (Qmax) improved by an average of 5.05 ml/ sec in 9 patients, whereas the remaining 5 patients showed a slight decrease in Qmax (mean 1.6 ml/ sec). The I-PSS score improved, with an overall decrease of 1.18 points at follow-up compared to the initial score (mean initial score = 2.42; mean follow-up score = 1.24). Conclusion: The analysis revealed that anti-VEGF therapy for wet AMD had a significant positive effect on all BPH-related symptoms; patients reported improved urinary streams and decreased nocturia. Abbreviations: BPH = benign prostatic hyperplasia, AMD = age-related macular degeneration, VEGF = vascular endothelial growth factor, I-PSS = international prostate symptom score, Qmax = maximum flow rate, TSP-1 = thrombospondin-1, FGF-2 = fibroblast growth factor, mRNA = precursor messenger ribonucleic acid, PSA = prostate-specific antigen, DRE = digital rectal examination, AUR = acute urinary retention, COX2 = cyclooxygenase 2, QoL = quality of life.

  10. VEGF Promotes Malaria-Associated Acute Lung Injury in Mice

    Science.gov (United States)

    Carapau, Daniel; Pena, Ana C.; Ataíde, Ricardo; Monteiro, Carla A. A.; Félix, Nuno; Costa-Silva, Artur; Marinho, Claudio R. F.; Dias, Sérgio; Mota, Maria M.

    2010-01-01

    The spectrum of the clinical presentation and severity of malaria infections is broad, ranging from uncomplicated febrile illness to severe forms of disease such as cerebral malaria (CM), acute lung injury (ALI), acute respiratory distress syndrome (ARDS), pregnancy-associated malaria (PAM) or severe anemia (SA). Rodent models that mimic human CM, PAM and SA syndromes have been established. Here, we show that DBA/2 mice infected with P. berghei ANKA constitute a new model for malaria-associated ALI. Up to 60% of the mice showed dyspnea, airway obstruction and hypoxemia and died between days 7 and 12 post-infection. The most common pathological findings were pleural effusion, pulmonary hemorrhage and edema, consistent with increased lung vessel permeability, while the blood-brain barrier was intact. Malaria-associated ALI correlated with high levels of circulating VEGF, produced de novo in the spleen, and its blockage led to protection of mice from this syndrome. In addition, either splenectomization or administration of the anti-inflammatory molecule carbon monoxide led to a significant reduction in the levels of sera VEGF and to protection from ALI. The similarities between the physiopathological lesions described here and the ones occurring in humans, as well as the demonstration that VEGF is a critical host factor in the onset of malaria-associated ALI in mice, not only offers important mechanistic insights into the processes underlying the pathology related with malaria but may also pave the way for interventional studies. PMID:20502682

  11. Functional and pharmacological characterization of a VEGF mimetic peptide on reparative angiogenesis.

    Science.gov (United States)

    Finetti, Federica; Basile, Anna; Capasso, Domenica; Di Gaetano, Sonia; Di Stasi, Rossella; Pascale, Maria; Turco, Caterina Maria; Ziche, Marina; Morbidelli, Lucia; D'Andrea, Luca Domenico

    2012-08-01

    Vascular endothelial growth factor (VEGF) is the main regulator of physiological and pathological angiogenesis. Low molecular weight molecules able to stimulate angiogenesis have interesting medical application for example in regenerative medicine, but at present none has reached the clinic. We reported that a VEGF mimetic helical peptide, QK, designed on the VEGF helix sequence 17-25, is able to bind and activate the VEGF receptors, producing angiogenesis. In this study we evaluate the pharmacological properties of peptide QK with the aim to propose it as a VEGF-mimetic drug to be employed in reparative angiogenesis. We show that the peptide QK is able to recapitulate all the biological activities of VEGF in vivo and on endothelial cells. In experiments evaluating sprouting from aortic ring and vessel formation in an in vivo angiogenesis model, the peptide QK showed biological effects comparable with VEGF. At endothelial level, the peptide up-regulates VEGF receptor expression, activates intracellular pathways depending on VEGFR2, and consistently it induces endothelial cell proliferation, survival and migration. When added to angiogenic factors (VEGF and/or FGF-2), QK produces an improved biological action, which resulted in reduced apoptosis and accelerated in vitro wound healing. The VEGF-like activity of the short peptide QK, characterized by lower cost of production and easier handling compared to the native glycoprotein, suggests that it is an attractive candidate to be further developed for application in therapeutic angiogenesis.

  12. Clinicopathological and prognostic significance of HER-2/neu and VEGF expression in colon carcinomas

    Directory of Open Access Journals (Sweden)

    Li Jing

    2011-06-01

    Full Text Available Abstract Background HER-2/neu and VEGF expression is correlated with disease behaviors in various cancers. However, evidence for their expression in colon cancer is rather contradictory both for the protein expression status and prognostic value. HER-2/neu is found to participate in VEGF regulation, and has known correlation with VEGF expression in some tumors. In this study, we investigated HER-2/neu and VEGF expression in Chinese colon patients and explored whether there was any correlation between their expression patterns. Methods HER-2/neu and VEGF were investigated immunohistochemically using tumor samples obtained from 317 colon cancer patients with all tumor stages. Correlation of the degree of staining with clinicopathological parameters and survival was investigated. Results Positive expression rates of HER-2/neu and VEGF in colon cancer were 15.5% and 55.5% respectively. HER-2/neu expression was significantly correlated with tumor size and distant metastases (P (P > 0.05. Expression of VEGF was significantly correlated with tumor size, tumor stage, lymph node metastases, and distant metastases (P (P = 0.146. No correlation between HER-2/neu and VEGF expression was detected (P = 0.151. Conclusions HER-2/neu and VEGF are not important prognostic markers of colon cancer. The present results do not support any association between HER2/neu and VEGF expression in this setting.

  13. The significance of VEGF expression in stage II carcinoma of uterine cervix treated with definitive radiotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Park, Won; Choi, Yoon La; Huh, Seung Jae; Yoon, Sang Min; Park, Young Je; Nam, Hee Rim; Ahn, Yong Chan; Lim, Do Hoon; Park, Hee Chul [Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of)

    2006-03-15

    We wanted to determine the clinical characteristics and prognosis according to the VEGF expression in stage II cervical carcinoma patients treated with definitive radiotherapy. We enrolled 31 patients who were diagnosed with cervical cancer from 1995 to 2003 at Samsumg Medical Center and their paraffin block tissue samples were available for study. The median age of the patients was 65 years. The mean tumor size was 4.1 cm (range: 1.2 {approx}8.2 cm). Seven patients (22.6%) were suspected of having pelvic lymph node metastasis. An external beam irradiation dose of 45-56.4 Gy was administered to the whole pelvis with a 15 MV linear accelerator, and an additional 24 Gy was given to point A by HDR intracavitary brachytherapy. VEGF staining was defined as positive when more than 10% of the tumor cells were stained. The median follow-up duration was 58 months. A positive VEGF expression was observed in 21 patients (67.7%). There was no significant correlation between the VEGF expression and pelvic lymph node metastasis, tumor size and the response of radiotherapy. During follow-up, 7 patients had recurrence. The complete response rate was not significant between the VEGF (-) and VEGF(+) tumors. However, the VEGF(+) tumors showed a significantly higher recurrence rate in comparison with the VEGF(-) tumors ({rho} = 0.040). The three year disease-free survival rates were 100% and 66.7%, respectively, for patients with VEGF(-) or VEGF(+) tumor ({rho} = 0.047). The VEGF expression was a significant factor for recurrence and disease-free survival. However, the significance of the VEGF expression is still controversial because of the various definitions of VEGF expression and the mismatches of the clinical data in the previous studies.

  14. VEGF incorporated into calcium phosphate ceramics promotes vascularisation and bone formation in vivo

    Directory of Open Access Journals (Sweden)

    E Wernike

    2010-02-01

    Full Text Available Bone formation and osseointegration of biomaterials are dependent on angiogenesis and vascularization. Angiogenic growth factors such as vascular endothelial growth factor (VEGF were shown to promote biomaterial vascularization and enhance bone formation. However, high local concentrations of VEGF induce the formation of malformed, nonfunctional vessels. We hypothesized that a continuous delivery of low concentrations of VEGF from calcium phosphate ceramics may increase the efficacy of VEGF administration.VEGF was co-precipitated onto biphasic calcium phosphate (BCP ceramics to achieve a sustained release of the growth factor. The co-precipitation efficacy and the release kinetics of the protein were investigated in vitro. For in vivo investigations BCP ceramics were implanted into critical size cranial defects in Balb/c mice. Angiogenesis and microvascularization were investigated over 28 days by means of intravital microscopy. The formation of new bone was determined histomorphometrically. Co-precipitation reduced the burst release of VEGF. Furthermore, a sustained, cell-mediated release of low concentrations of VEGF from BCP ceramics was mediated by resorbing osteoclasts. In vivo, sustained delivery of VEGF achieved by protein co-precipitation promoted biomaterial vascularization, osseointegration, and bone formation. Short-term release of VEGF following superficial adsorption resulted in a temporally restricted promotion of angiogenesis and did not enhance bone formation. The release kinetics of VEGF appears to be an important factor in the promotion of biomaterial vascularization and bone formation. Sustained release of VEGF increased the efficacy of VEGF delivery demonstrating that a prolonged bioavailability of low concentrations of VEGF is beneficial for bone regeneration.

  15. Angiogenic functionalisation of titanium surfaces using nano-anchored VEGF – an in vitro study

    Directory of Open Access Journals (Sweden)

    H Schliephake

    2012-03-01

    Full Text Available The aim of the present study was to test the hypothesis that sandblasted and acid etched titanium surfaces can be functionalised with vascular endothelial growth factor (VEGF using oligonucleotides for anchorage and slow release. rhVEGF165 molecules were conjugated to strands of 30-mer non-coding DNA oligonucleotides (ODN and hybridised to complementary ODN anchor strands which had been immobilised to the surface of sandblasted/acid etched (SAE Ti specimens. Specimens with non-conjugated VEGF adsorbed to ODN anchor strands and to blank SAE surfaces served as controls. Specific binding of conjugated VEGF exhibited the highest percentage of immobilised VEGF (71.0 %, whereas non-conjugated VEGF only achieved 53.2 and 30.7 %, respectively. Cumulative release reached 54.0 % of the immobilised growth factor in the group of specifically bound VEGF after 4 weeks, whereas non-conjugated VEGF adsorbed to ODN strands released 78.9% and VEGF adsorbed to SAE Ti surfaces released 97.4 %. Proliferation of human umbilical vein endothelial cells (HUVECs was significantly increased on the surfaces with specifically bound VEGF compared to the control surfaces and SAE Ti surfaces without VEGF. Moreover, the released conjugated VEGF exhibited biological activity by induction of von Willebrand Factor (vWF in mesenchymal stem cells. It is concluded that the angiogenic functionalisation of SAE titanium surfaces can be achieved by conjugation of VEGF to ODN strands and hybridisation to complementary ODN strands that are anchored to the titanium surface. The angiogenic effect is exerted both through the immobilised and the released portion of the growth factor.

  16. Novel aptamer-nanoparticle bioconjugates enhances delivery of anticancer drug to MUC1-positive cancer cells in vitro.

    Directory of Open Access Journals (Sweden)

    Chenchen Yu

    Full Text Available MUC1 protein is an attractive target for anticancer drug delivery owing to its overexpression in most adenocarcinomas. In this study, a reported MUC1 protein aptamer is exploited as the targeting agent of a nanoparticle-based drug delivery system. Paclitaxel (PTX loaded poly (lactic-co-glycolic-acid (PLGA nanoparticles were formulated by an emulsion/evaporation method, and MUC1 aptamers (Apt were conjugated to the particle surface through a DNA spacer. The aptamer conjugated nanoparticles (Apt-NPs are about 225.3 nm in size with a stable in vitro drug release profile. Using MCF-7 breast cancer cell as a MUC1-overexpressing model, the MUC1 aptamer increased the uptake of nanoparticles into the target cells as measured by flow cytometry. Moreover, the PTX loaded Apt-NPs enhanced in vitro drug delivery and cytotoxicity to MUC1(+ cancer cells, as compared with non-targeted nanoparticles that lack the MUC1 aptamer (P<0.01. The behavior of this novel aptamer-nanoparticle bioconjugates suggests that MUC1 aptamers may have application potential in targeted drug delivery towards MUC1-overexpressing tumors.

  17. Enhanced Biosensor Platforms for Detecting the Atherosclerotic Biomarker VCAM1 Based on Bioconjugation with Uniformly Oriented VCAM1-Targeting Nanobodies

    Directory of Open Access Journals (Sweden)

    Duy Tien Ta

    2016-07-01

    Full Text Available Surface bioconjugation of biomolecules has gained enormous attention for developing advanced biomaterials including biosensors. While conventional immobilization (by physisorption or covalent couplings using the functional groups of the endogenous amino acids usually results in surfaces with low activity, reproducibility and reusability, the application of methods that allow for a covalent and uniformly oriented coupling can circumvent these limitations. In this study, the nanobody targeting Vascular Cell Adhesion Molecule-1 (NbVCAM1, an atherosclerotic biomarker, is engineered with a C-terminal alkyne function via Expressed Protein Ligation (EPL. Conjugation of this nanobody to azidified silicon wafers and Biacore™ C1 sensor chips is achieved via Copper(I-catalyzed azide-alkyne cycloaddition (CuAAC “click” chemistry to detect VCAM1 binding via ellipsometry and surface plasmon resonance (SPR, respectively. The resulting surfaces, covered with uniformly oriented nanobodies, clearly show an increased antigen binding affinity, sensitivity, detection limit, quantitation limit and reusability as compared to surfaces prepared by random conjugation. These findings demonstrate the added value of a combined EPL and CuAAC approach as it results in strong control over the surface orientation of the nanobodies and an improved detecting power of their targets—a must for the development of advanced miniaturized, multi-biomarker biosensor platforms.

  18. Capillary electrophoresis for the characterization of quantum dots after non-selective or selective bioconjugation with antibodies for immunoassay

    Directory of Open Access Journals (Sweden)

    Lai Edward PC

    2008-10-01

    Full Text Available Abstract Capillary electrophoresis coupled with laser-induced fluorescence was used for the characterization of quantum dots and their conjugates to biological molecules. The CE-LIF was laboratory-built and capable of injection (hydrodynamic and electrokinetic from sample volumes as low as 4 μL via the use of a modified micro-fluidic chip platform. Commercially available quantum dots were bioconjugated to proteins and immunoglobulins through the use of established techniques (non-selective and selective. Non-selective techniques involved the use of EDCHCl/sulfo-NHS for the conjugation of BSA and myoglobin to carboxylic acid-functionalized quantum dots. Selective techniques involved 1 the use of heterobifunctional crosslinker, sulfo-SMCC, for the conjugation of partially reduced IgG to amine-functionalized quantum dots, and 2 the conjugation of periodate-oxidized IgGs to hydrazide-functionalized quantum dots. The migration times of these conjugates were determined in comparison to their non-conjugated QD relatives based upon their charge-to-size ratio values. The performance of capillary electrophoresis in characterizing immunoconjugates of quantum dot-labeled IgGs was also evaluated. Together, both QDs and CE-LIF can be applied as a sensitive technique for the detection of biological molecules. This work will contribute to the advancements in applying nanotechnology for molecular diagnosis in medical field.

  19. Controlled Release of Damascone from Poly(styrene-co-maleic anhydride-based Bioconjugates in Functional Perfumery

    Directory of Open Access Journals (Sweden)

    Andreas Herrmann

    2013-02-01

    Full Text Available Poly(styrene-co-maleic anhydrides were modified with poly(propylene oxide (PO-co-ethylene oxide (EO side chains (Jeffamine® with different EO/PO molar ratios, varying between 0.11 and 3.60. These copolymers were then further functionalized with a β-mercapto ketone of δ-damascone. The obtained poly(maleic acid monoamide-based β-mercapto ketones were then studied as delivery systems for the controlled release of δ-damascone by retro 1,4-addition. The release of δ-damascone, a volatile, bioactive molecule of the family of rose ketones, was studied by dynamic headspace analysis above a cotton surface after deposition of a cationic surfactant containing fabric softening formulation, as a function of the ethylene oxide (EO/propylene oxide (PO molar ratio of the grafted copolymer side chains. The polarity of the EO/PO side chain influenced the release efficiency of the damascone in a typical fabric softening application. PO-rich copolymers and the corresponding poly(styrene-co-maleic anhydride without Jeffamine® side chains were found to be less efficient for the desired fragrance release than the corresponding bioconjugate with a EO/PO ratio of 3.60 in the side chain. This copolymer conjugate seemed to represent a suitable balance between hydrophilicity and hydrophobicity to favor the release of the δ-damascone and to improve the deposition of the conjugate from an aqueous environment onto a cotton surface.

  20. Designer Leptin Receptor Antagonist Allo-aca Inhibits VEGF Effects in Ophthalmic Neoangiogenesis Models

    Science.gov (United States)

    Coroniti, Roberta; Fario, Rafal; Nuno, Didier J.; Otvos, Laszlo; Scolaro, Laura; Surmacz, Eva

    2016-01-01

    Experimental and clinical data suggest that pro-angiogenic, pro-inflammatory and mitogenic cytokine leptin can be implicated in ocular neovascularization and other eye pathologies. At least in part, leptin action appears to be mediated through functional interplay with vascular endothelial growth factor (VEGF). VEGF is a potent regulator of neoangiogenesis and vascular leakage with a proven role in conditions such as proliferative diabetic retinopathy, age-related macular degeneration and diabetic macular edema. Accordingly, drugs targeting VEGF are becoming mainstream treatments for these diseases. The crosstalk between leptin and VEGF has been noted in different tissues, but its involvement in the development of eye pathologies is unclear. Leptin is coexpressed with VEGF during ocular neovascularization and can potentiate VEGF synthesis and angiogenic function. However, whether or not VEGF regulates leptin expression or signaling has never been studied. Consequently, we addressed this aspect of leptin/VEGF crosstalk in ocular models, focusing on therapeutic exploration of underlying mechanisms. Here we show, for the first time, that in retinal (RF/6A) and corneal (BCE) endothelial cells, VEGF (100 ng/mL, 24 h) stimulated leptin mRNA synthesis by 70 and 30%, respectively, and protein expression by 56 and 28%, respectively. In parallel, VEGF induced RF/6A and BCE cell growth by 33 and 20%, respectively. In addition, VEGF upregulated chemotaxis and chemokinesis in retinal cells by ~40%. VEGF-dependent proliferation and migration were significantly reduced in the presence of the leptin receptor antagonist, Allo-aca, at 100–250 nmol/L concentrations. Furthermore, Allo-aca suppressed VEGF-dependent long-term (24 h), but not acute (15 min) stimulation of the Akt and ERK1/2 signaling pathways. The efficacy of Allo-aca was validated in the rat laser-induced choroidal neovascularization model where the compound (5 μg/eye) significantly reduced pathological

  1. Up-regulation of VEGF-C secreted by cancer cells and not VEGF-A correlates with clinical evaluation of lymph node metastasis in esophageal squamous cell carcinoma (ESCC).

    Science.gov (United States)

    Krzystek-Korpacka, Malgorzata; Matusiewicz, Malgorzata; Diakowska, Dorota; Grabowski, Krzysztof; Blachut, Katarzyna; Banas, Teresa

    2007-05-01

    Tissue expression of VEGF-C correlates with lymph node involvement (LNI) in ESCC and serum VEGF-C (sVEGF-C) in a non-small cell lung cancer has been more accurate marker of LNI than chest CT. Despite LNI importance in ESCC, the usefulness of serum VEGF-C (sVEGF-C) as a disease and LNI marker in ESCC has not been investigated yet. We found elevated sVEGF-C in ESCC (17.40 vs. 10.57 ng/ml in controls, pmarker than described elsewhere: CEA, CA19-9 and SCC-Ag, with: sensitivity--70%, specificity--81%, accuracy--83.7%. Analysis of sVEGF-C correlation with clinico-pathological cancer features revealed relation to LNI (N0: 15.77 vs. N1: 21.78 ng/ml, p=0.02), especially in advanced cancers. Serum VEGF-C as a marker of LNI was characterized by: sensitivity--76%, specificity--58%, accuracy--64.4%. No relation was observed between LNI and sVEGF-A or sVEGF-A/platelets (PLT). Because sVEGF-C was higher in N0 cancers (ptumor presence also up-regulates sVEGF-C. We found sVEGF-C correlation with PLT and WBC: R=0.36 and R=0.32 (pcancer features implies that elevation of sVEGF-C in N1 cancers is not related to them.

  2. Vascular endothelial growth factor (VEGF) gene polymorphisms may influence the efficacy of thalidomide in multiple myeloma

    DEFF Research Database (Denmark)

    Andersen, Niels Frost; Vogel, Ulla Birgitte; Klausen, Tobias W;

    2012-01-01

    Vascular endothelial growth factor (VEGF) is a potent proangiogenic factor. Several single nucleotide polymorphisms (SNPs) in the VEGF gene with influence on VEGF expression have been described. In multiple myeloma, VEGF stimulates angiogenesis which is correlated with disease progression...... and prognosis. In this study, we evaluated the association between genetic variations in the VEGF gene in patients with multiple myeloma and time to treatment failure (TTF) after high-dose melphalan and stem cell support (HDT), overall survival (OS) and efficacy of the anti-angiogenic drug thalidomide....... Retrospectively, the SNPs -2,578C>A (rs699947), -460C>T (rs833061), +405G>C (rs2010963) and +936C>T (rs3025039) in the VEGF gene were examined in 348 patients with newly diagnosed multiple myeloma initially treated with HDT, where 176 patients were treated with thalidomide at relapse. None of the examined geno...

  3. Contraction induced secretion of VEGF from skeletal muscle cells is mediated by adenosine

    DEFF Research Database (Denmark)

    Høier, Birgitte; Olsen, Karina; Nyberg, Michael Permin

    2010-01-01

    The role of adenosine and contraction for secretion of VEGF in skeletal muscle was investigated in human subjects and rat primary skeletal muscle cells. Microdialysis probes were inserted into the thigh muscle of seven male subjects and dialysate was collected at rest, during infusion of adenosine...... and contraction caused secretion of VEGF (pcontraction induced secretion of VEGF protein was abolished by the A(2B) antagonist enprofyllin and markedly reduced by inhibition of PKA or MAPK. The results demonstrate that adenosine causes secretion of VEGF from human skeletal muscle cells...... and that the contraction induced secretion of VEGF is partially mediated via adenosine acting on A(2B) adenosine receptors. Moreover, the contraction induced secretion of VEGF protein from muscle is dependent on both PKA and MAPK activation, but only the MAPK pathway appears to be adenosine dependent....

  4. Construction of recombinant adenovirus co-expression vector carrying the human transforming growth factor-β1 and vascular endothelial growth factor genes and its effect on anterior cruciate ligament fibroblasts

    Institute of Scientific and Technical Information of China (English)

    WEI Xue-lei; LIN Lin; HOU Yu; FU Xin; ZHANG Ji-ying; MAO Ze-bin; YU Chang-long

    2008-01-01

    Background Remodeling of the anterior cruciate ligament (ACL) graft usually takes longer than expected. Gene therapy offers a radical different approach to remodeling of the graft. In this study, the internal ribosome entry site (IRES) sequence was used to construct a new recombinant adenovirus which permits co-expression of transforming growth factor-β1 (TGFβ1) and vascular endothelial growth factor 165 (VEGF165) genes (named Ad-VEGF165-1RES-TGFβ1). We investigated the effects of the new adenovirus on the migration of and matrix synthesis by ACL fibroblasts.Methods Adenoviral vector containing TGFβ1 and VEGF165 genes was constructed. ACL fibroblasts were obtained from New Zealand white rabbits. After ACL fibroblasts were exposed to Ad-VEGF165-1RES-TGFβ1, the expression of VEGF165 and TGFβ1 proteins were assessed by enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis. Bioassay of VEGF165 and TGFβ1 proteins were assessed by Western blotting analysis. Proliferation and migration of ACL fibroblasts were assessed by in vitro wound closure assay. Gene expression of collagen type I, collagen type Ⅲ, and fibronectin mRNA among matrix markers were assessed by real-time PCR.Results The results showed the successful construction of a recombinant co-expression adenovirus vector containing TGFβI and VEGF165 genes. Co-expression of TGFβ1 and VEGF165 can induce relatively rapid and continuous proliferation of ACL fibroblasts and high gene expression of collagen type Ⅰ, collagen typeⅢ, and fibronectin mRNA among matrix markers.Conclusion Co-expression of TGFβ1 and VEGF165 genes has more powerful and efficient effects on the migration of and matrix synthesis by ACL fibroblasts.

  5. Direct evidence that the VEGF-specific antibody bevacizumab has antivascular effects in human rectal cancer

    OpenAIRE

    2004-01-01

    The effects of vascular endothelial growth factor (VEGF) blockade on the vascular biology of human tumors are not known. Here we show here that a single infusion of the VEGF-specific antibody bevacizumab decreases tumor perfusion, vascular volume, microvascular density, interstitial fluid pressure and the number of viable, circulating endothelial and progenitor cells, and increases the fraction of vessels with pericyte coverage in rectal carcinoma patients. These data indicate that VEGF block...

  6. VEGF secretion during hypoxia depends on free radicals-induced Fyn kinase activity in mast cells

    Energy Technology Data Exchange (ETDEWEB)

    Garcia-Roman, Jonathan; Ibarra-Sanchez, Alfredo; Lamas, Monica [Departamento de Farmacobiologia, Centro de Investigacion y de Estudios Avanzados del IPN (Cinvestav, IPN) (Mexico); Gonzalez Espinosa, Claudia, E-mail: cgonzal@cinvestav.mx [Departamento de Farmacobiologia, Centro de Investigacion y de Estudios Avanzados del IPN (Cinvestav, IPN) (Mexico)

    2010-10-15

    Research highlights: {yields} Bone marrow-derived mast cells (BMMCs) secrete functional VEGF but do not degranulate after Cobalt chloride-induced hypoxia. {yields} CoCl{sub 2}-induced VEGF secretion in mast cells occurs by a Ca{sup 2+}-insensitive but brefeldin A and Tetanus toxin-sensitive mechanism. {yields} Trolox and N-acetylcysteine inhibit hypoxia-induced VEGF secretion but only Trolox inhibits Fc{epsilon}RI-dependent anaphylactic degranulation in mast cells. {yields} Src family kinase Fyn activation after free radical production is necessary for hypoxia-induced VEGF secretion in mast cells. -- Abstract: Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conduce to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl{sub 2}) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl{sub 2} promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl{sub 2}-induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl{sub 2}-induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl{sub 2} in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free radicals

  7. Circulating VEGF as a biological marker in patients with rheumatoid arthritis? Preanalytical and biological variability in healthy persons and in patients

    DEFF Research Database (Denmark)

    Hetland, Merete Lund; Christensen, Ib Jarle; Lottenburger, Tine

    2008-01-01

    and contamination of plasma with cellular elements lead to significant increases in VEGF levels, whereas storage for up to 2 years at -80 degrees C or up to 10 freeze/thaw cycles did not affect VEGF levels. Serum VEGF levels were 7-10 fold higher than plasma VEGF levels. Reference intervals for VEGF (plasma: 45 pg...

  8. Circulating VEGF as a biological marker in patients with rheumatoid arthritis? Preanalytical and biological variability in healthy persons and in patients

    DEFF Research Database (Denmark)

    Hetland, Merete Lund; Christensen, Ib Jarle; Lottenburger, Tine

    2008-01-01

    and contamination of plasma with cellular elements lead to significant increases in VEGF levels, whereas storage for up to 2 years at -80 degrees C or up to 10 freeze/thaw cycles did not affect VEGF levels. Serum VEGF levels were 7-10 fold higher than plasma VEGF levels. Reference intervals for VEGF (plasma: 45 pg...

  9. Suppression of Human Tenon Fibroblast Cell Proliferation by Lentivirus-Mediated VEGF Small Hairpin RNA

    Directory of Open Access Journals (Sweden)

    Zhongqiu Li

    2017-01-01

    Full Text Available Purpose. The functions of vascular endothelial growth factor (VEGF in scar formation after trabeculectomy were investigated in a human Tenon fibroblast cell line from glaucoma patients using lentivirus-mediated VEGF shRNA. Methods. Human Tenon fibroblast (HTF cells were isolated from scar tissue of glaucoma patients during secondary surgery. Lentivirus-VEGF-shRNA was constructed and transfected into HTF cells. Subsequently, VEGF mRNA and protein expression were analyzed using quantitative RT-PCR and western blotting, respectively, and the effects of VEGF knockdown were analyzed. The inhibition of HTF proliferation was monitored according to total cell numbers using ScanArray. Results. Both mRNA and protein levels of VEGF were reduced by lentivirus-mediated VEGF-shRNA, and proliferation of HTF cells was inhibited. Conclusions. Primary cultures of human Tenon fibroblast (HTF were established, and proliferation was decreased following inhibition of VEGF. VEGF may be a suitable therapeutic target for reducing scar tissue formation in glaucoma patients after filtration surgery.

  10. Suppression of Human Tenon Fibroblast Cell Proliferation by Lentivirus-Mediated VEGF Small Hairpin RNA.

    Science.gov (United States)

    Li, Zhongqiu; Hua, Wen; Li, Xuedong; Wang, Wei

    2017-01-01

    Purpose. The functions of vascular endothelial growth factor (VEGF) in scar formation after trabeculectomy were investigated in a human Tenon fibroblast cell line from glaucoma patients using lentivirus-mediated VEGF shRNA. Methods. Human Tenon fibroblast (HTF) cells were isolated from scar tissue of glaucoma patients during secondary surgery. Lentivirus-VEGF-shRNA was constructed and transfected into HTF cells. Subsequently, VEGF mRNA and protein expression were analyzed using quantitative RT-PCR and western blotting, respectively, and the effects of VEGF knockdown were analyzed. The inhibition of HTF proliferation was monitored according to total cell numbers using ScanArray. Results. Both mRNA and protein levels of VEGF were reduced by lentivirus-mediated VEGF-shRNA, and proliferation of HTF cells was inhibited. Conclusions. Primary cultures of human Tenon fibroblast (HTF) were established, and proliferation was decreased following inhibition of VEGF. VEGF may be a suitable therapeutic target for reducing scar tissue formation in glaucoma patients after filtration surgery.

  11. AlphaB-crystallin is involved in oxidative stress protection determined by VEGF in skeletal myoblasts.

    Science.gov (United States)

    Mercatelli, Neri; Dimauro, Ivan; Ciafré, Silvia Anna; Farace, Maria Giulia; Caporossi, Daniela

    2010-08-01

    Recent studies suggest that the effects of VEGF-A, the prototype VEGF ligand, may extend to a variety of cell types other than endothelial cells. The expression of VEGF-A and its main receptors, Flt-1/VEGFR-1 and KDR/Flk-1/VEGFR-2, was indeed detected in several cell types, including cardiac myocytes and regenerating myotubes. In addition to its proangiogenic activity, evidence indicates that VEGF-A can sustain skeletal muscle regeneration by enhancing the survival and migration of myogenic cells and by promoting the growth of myogenic fibers. In this study, our aim was to investigate whether VEGF could protect skeletal muscle satellite cells from apoptotic cell death triggered by reactive oxygen species and to identify the main molecular mechanisms. C2C12 mouse myoblasts, cultured in vitro in the presence of exogenous VEGF or stably transfected with a plasmid vector expressing VEGF-A, were subjected to oxidative stress and analyzed for cell growth and survival, induction of apoptosis, and molecular signaling. The results of our study demonstrated that VEGF protects C2C12 myoblasts from apoptosis induced by oxidative or hypoxic-like stress. This protection did not correlate with the modulation of the expression of VEGF receptors, but is clearly linked to the phosphorylation of the KDR/Flk-1 receptor, the activation of NF-kappaB, and/or the overexpression of the antiapoptotic protein alphaB-crystallin. Copyright 2010 Elsevier Inc. All rights reserved.

  12. A Biomimic Reconstituted High Density Lipoprotein Nanosystem for Enhanced VEGF Gene Therapy of Myocardial Ischemia

    Directory of Open Access Journals (Sweden)

    Xiaotian Sun

    2015-01-01

    Full Text Available A biomimic reconstituted high density lipoprotein (rHDL based system, rHDL/Stearic-PEI/VEGF complexes, was fabricated as an advanced nanovector for delivering VEGF plasmid. Here, Stearic-PEI was utilized to effectively condense VEGF plasmid and to incorporate the plasmid into rHDL. The rHDL/Stearic-PEI/VEGF complexes with diameter under 100 nm and neutral surface charge demonstrated enhanced stability under the presence of bovine serum albumin. Moreover, in vitro cytotoxicity and transfection assays on H9C2 cells further revealed their superiority, as they displayed lower cytotoxicity with much higher transfection efficiency when compared to PEI 10K/VEGF and Lipos/Stearic-PEI/VEGF complexes. In addition, in vivo investigation on ischemia/reperfusion rat model implied that rHDL/Stearic-PEI/VEGF complexes possessed high transgene capacity and strong therapeutic activity. These findings indicated that rHDL/Stearic-PEI/VEGF complexes could be an ideal gene delivery system for enhanced VEGF gene therapy of myocardial ischemia, which might be a new promising strategy for effective myocardial ischemia treatment.

  13. High VEGF with Rapid Growth and Early Metastasis in a Mouse Osteosarcoma Model

    Directory of Open Access Journals (Sweden)

    Shang-You Yang

    2007-01-01

    Full Text Available A murine model of osteosarcoma was developed to investigate the association between the expression of VEGF and the progression of osteosarcoma. Two human osteosarcoma cell lines with distinct VEGF expressions were introduced into proximal tibiae of immuno-deficient SCID mice, either by direct injection through the cortical bone or surgical exposing and drilling on the tibial metaphysis to seed tumor cells. Bone tumors were obvious on microCT within 4 weeks following osteosarcoma cell inoculation through surgical delivery. In contrast, direct injection without drilling often resulted in periosteal tumors. Although neoplasms were developed regardless of VEGF levels, orthotopic tumors derived from high VEGF-expressing cells were detected 2 weeks earlier on CT images than the ones from VEGF negative cells. At sacrifice, high VEGF tumors were distinctively larger in size and more frequently invaded the adjacent bone tissue. Multiple metastatic lesions were found in all the lung tissues at 8 weeks from high VEGF group, whereas only 1 of 7 VEGF negative tumors exhibited pulmonary metastasis. Overall, this model developed with the surgical tumor cell delivery results in histological and radiographic features more consistent with primary osteosarcoma. Interestingly, VEGF expression correlates with the early establishment, rapid tumor growth, and the development of pulmonary metastasis.

  14. EXPERIMENTAL STUDY ON THE GENE THERAPY OF MALIGNANT GLIOMA WITH ANTISENSE VEGF RNA

    Institute of Scientific and Technical Information of China (English)

    浦佩玉; 王建桢; 黄强; 张敬; 张云亭

    2003-01-01

    Objective: To study the effect of antisense VEGF RNA on rat C6 gliomas in vivo and find out the feasibility of antiangiogenesis therapy with antisense VEGF RNA for malignant gliomas. Methods: Parental rat C6 glioma cells and C6 cells transfected with antisense VEGF cDNA were implanted intracerebrally and subcutaneously into SD rats as control and transfected group. Rats bearing cerebral and subcutaneous C6 gliomas were treated with antisense VEGF cDNA as treated group and sense VEGF cDNA and empty vector as control of treated group. The general manifestation, survival time, MRI and histopathological changes of all rats were observed. The volume of subcutaneously implanted tumors was determined regularly. In situ hybridization and immunohistochemical staining were used for detection of VEGF gene expression of gliomas while PCNA immunostaining and TUNEL method for examination of proliferation activity and apoptosis of gliomas, respectively. Results: The survival of the rats in transfected and treated group was prolonged. There were two rats surviving over 90 d in the treated group and their tumors disappeared. The VEGF gene expression, the number of microvessels and the proliferation activity were decreased and a large amount of apoptotic cells could be found in cerebral and subcutaneous gliomas in treated and transfected groups. Conclusion: VEGF is one of the candidate genes for gene therapy of malignant gliomas. Antisense VEGF RNA combined with other therapies should be studied further for enhancing the therapeutic effect of malignant gliomas.

  15. VEGF-A gene promoter polymorphisms and microvascular complications in patients with essential hypertension.

    Science.gov (United States)

    Palmirotta, Raffaele; Ferroni, Patrizia; Ludovici, Giorgia; Martini, Francesca; Savonarola, Annalisa; D'Alessandro, Roberta; Raparelli, Valeria; Proietti, Marco; Scarno, Antongiulio; Riondino, Silvia; Basili, Stefania; Guadagni, Fiorella

    2010-09-01

    We investigated the possible involvement of vascular endothelial growth factor (VEGF-A) gene promoter polymorphisms in essential hypertension (EH). 1225bp of the VEGF-A gene promoter were screened for polymorphisms using PCR amplification and direct DNA sequence analysis in 62 EH and 62 normotensive (HS) individuals. Circulating VEGF-A levels were determined by immunoassay. -152G/A (p=0.009) and -116G/A (p=0.016) polymorphisms were correlated to hypertension (p<0.05). Median platelet VEGF-A load in EH was 2.10fg/plt. Patients with microvascular complications (MC) had higher platelet VEGF-A load than those without (p=0.005). Multivariate analyses showed that -116 A allele was an independent predictor of microalbuminuria (p=0.014) and increased platelet VEGF-A load (p=0.009) in EH. Platelet VEGF-A load independently predicted MC (p=0.049) in addition to -116G/A polymorphism (p=0.035). Abnormal regulation of VEGF-A due to polymorphism at position -116 might represent a genetic factor for increased VEGF-A production and MC in EH. Copyright (c) 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  16. Modified VEGF targets the ischemic myocardium and promotes functional recovery after myocardial infarction.

    Science.gov (United States)

    Yang, Yun; Shi, Chunying; Hou, Xianglin; Zhao, Yannan; Chen, Bing; Tan, Bo; Deng, Zongwu; Li, Qingguo; Liu, Jianzhou; Xiao, Zhifeng; Miao, Qi; Dai, Jianwu

    2015-09-10

    Vascular endothelial growth factor (VEGF) promotes angiogenesis and improves cardiac function after myocardial infarction (MI). However, the non-targeted delivery of VEGF decreases its therapeutic efficacy due to an insufficient local concentration in the ischemic myocardium. In this study, we used a specific peptide to modify VEGF and determined that this modified VEGF (IMT-VEGF) localized to the ischemic myocardium through intravenous injection by interacting with cardiac troponin I (cTnI). When IMT-VEGF was used to mediate cardiac repair in a rat model of ischemia-reperfusion (I-R) injury, we observed a decreased scar size, enhanced angiogenesis and improved cardiac function. Moreover, an alternative treatment using the repeated administration of a low-dose IMT-VEGF also promoted angiogenesis and functional recovery. The therapeutic effects of IMT-VEGF were further confirmed in a pig model of MI as the result of the conserved properties of its interacting protein, cTnI. These results suggest a promising therapeutic strategy for MI based on the targeted delivery of IMT-VEGF. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Erythropoietin attenuates renal and pulmonary injury in polymicrobial induced-sepsis through EPO-R, VEGF and VEGF-R2 modulation.

    Science.gov (United States)

    Heitrich, Mauro; García, Daiana Maria de Los Ángeles; Stoyanoff, Tania Romina; Rodríguez, Juan Pablo; Todaro, Juan Santiago; Aguirre, María Victoria

    2016-08-01

    Sepsis remains the most important cause of acute kidney injury (AKI) and acute lung injury (ALI) in critically ill patients. The cecal ligation and puncture (CLP) model in experimental mice reproduces most of the clinical features of sepsis. Erythropoietin (EPO) is a well-known cytoprotective multifunctional hormone, which exerts anti-inflammatory, anti-oxidant, anti-apoptotic and pro-angiogenic effects in several tissues. The aim of this study was to evaluate the underlying mechanisms of EPO protection through the expression of the EPO/EPO receptor (EPO-R) and VEGF/VEF-R2 systems in kidneys and lungs of mice undergoing CLP-induced sepsis. Male inbred Balb/c mice were divided in three experimental groups: Sham, CLP, and CLP+EPO (3000IU/kg sc). Assessment of renal functional parameters, survival, histological examination, immunohistochemistry and/or Western blottings of EPO-R, VEGF and VEGF-R2 were performed at 18h post-surgery. Mice demonstrated AKI by elevation of serum creatinine and renal histologic damage. EPO treatment attenuates renal dysfunction and ameliorates kidney histopathologic changes. Additionally, EPO administration attenuates deleterious septic damage in renal cortex through the overexpression of EPO-R in tubular interstitial cells and the overexpression of the pair VEGF/VEGF-R2. Similarly CLP- induced ALI, as evidenced by parenchymal lung histopathologic alterations, was ameliorated through pulmonary EPO-R, VEGF and VEGF-R2 over expression suggesting and improvement in endothelial survival and functionality. This study demonstrates that EPO exerts protective effects in kidneys and lungs in mice with CLP-induced sepsis through the expression of EPO-R and the regulation of the VEGF/VEGF-R2 pair.

  18. Combined inhibition of IL1, CXCR1/2, and TGFβ signaling pathways modulates in-vivo resistance to anti-VEGF treatment.

    Science.gov (United States)

    Carbone, Carmine; Tamburrino, Anna; Piro, Geny; Boschi, Federico; Cataldo, Ivana; Zanotto, Marco; Mina, Maria M; Zanini, Silvia; Sbarbati, Andrea; Scarpa, Aldo; Tortora, Giampaolo; Melisi, Davide

    2016-01-01

    Resistance of tumors to antiangiogenic therapies is becoming increasingly relevant. We recently identified interleukin-1 (IL1), CXC receptors (CXCR)1/2 ligands, and transforming growth factor β (TGFβ) among the proinflammatory factors that were expressed at higher levels in murine models resistant to the antivascular endothelial growth factor (anti-VEGF) antibody bevacizumab. Here, we hypothesized that the combined inhibition of these proinflammatory signaling pathways might reverse this anti-VEGF resistance. Bevacizumab-resistant FGBR pancreatic cancer cells were treated in vitro with bevacizumab, the recombinant human IL1 receptor antagonist anakinra, the monoclonal antibody against TGFβ receptor type II TR1, and a novel recombinant antibody binding CXCR1/2 ligands. The FGBR cells treated with these agents in combination had significantly higher levels of E-cadherin and lower levels of vimentin, IL6, phosphorylated p65, and SMAD2, and showed significantly lower migration rates than did their controls treated with the same agents without bevacizumab or with a single agent bevacizumab as a control. Consistently, the combination of these agents with bevacizumab reduced the FGBR tumor burden and significantly prolonged mice survival compared with bevacizumab in monotherapy. Tumors from mice receiving the combination treatment showed significantly lower expression of IL6 and phosphorylated SMAD2, higher expression of E-cadherin and lower levels of vimentin, and a significantly lower infiltration by CD11b cells compared with bevacizumab-treated controls. This study suggests that inhibition of IL1, CXCR1/2, and TGFβ signaling pathways is a potential therapeutic approach to modulate the acquired resistance to anti-VEGF treatment by reversing epithelial-mesenchymal transition and inhibiting CD11b proangiogenic myeloid cells' tumor infiltration.

  19. 4-Trifluoromethyl-substituted coumarins with large Stokes shifts: synthesis, bioconjugates, and their use in super-resolution fluorescence microscopy.

    Science.gov (United States)

    Schill, Heiko; Nizamov, Shamil; Bottanelli, Francesca; Bierwagen, Jakob; Belov, Vladimir N; Hell, Stefan W

    2013-12-02

    Bright and photostable fluorescent dyes with large Stokes shifts are widely used as sensors, molecular probes, and light-emitting markers in chemistry, life sciences, and optical microscopy. In this study, new 7-dialkylamino-4-trifluoromethylcoumarins have been designed for use in bioconjugation reactions and optical microscopy. Their synthesis was based on the Stille reaction of 3-chloro-4-trifluoromethylcoumarins and available (hetero)aryl- or (hetero)arylethenyltin derivatives. Alternatively, the acylation of 2-trifluoroacetyl-5-dialkylaminophenols with available (hetero)aryl- or (hetero)arylethenylacetic acids followed by intramolecular condensation afforded coumarins with 3-(hetero)aryl or 3-[2-(hetero)aryl]ethenyl groups. Hydrophilic properties were provided by the introduction of a sulfonic acid residue or by phosphorylation of a primary hydroxy group attached at C-4 of the 2,2,4-trimethyl-1,2-dihydroquinoline fragment fused to the coumarin fluorophore. For use in immunolabeling procedures, the dyes were decorated with an (activated) carboxy group. The positions of the absorption and emission maxima vary in the ranges 413-480 and 527-668 nm, respectively. The phosphorylated dye, 9,CH=CH-2-py,H, with the 1-(3-carboxypropyl)-4-hydroxymethyl-2,2-dimethyl-1,2-dihydroquinoline fragment fused to the coumarin fluorophore bearing the 3-[2-(2-pyridyl)ethenyl] residue (absorption and emission maxima at 472 and 623 nm, respectively) was used in super-resolution light microscopy with stimulated emission depletion and provided an optical resolution better than 70 nm with a low background signal. As a result of their large Stokes shifts, good fluorescence quantum yields, and adequate photostabilities, phosphorylated coumarins enable two-color imaging (using several excitation sources and a single depletion laser) to be combined with subdiffractional optical resolution.

  20. Effect of acute exercise and exercise training on VEGF splice variants in human skeletal muscle.

    Science.gov (United States)

    Jensen, Lotte; Pilegaard, Henriette; Neufer, P Darrell; Hellsten, Ylva

    2004-08-01

    The present study investigated the effect of an acute exercise bout on the mRNA response of vascular endothelial growth factor (VEGF) splice variants in untrained and trained human skeletal muscle. Seven habitually active young men performed one-legged knee-extensor exercise training at an intensity corresponding to approximately 70% of the maximal workload in an incremental test five times/week for 4 wk. Biopsies were obtained from the vastus lateralis muscle of the trained and untrained leg 40 h after the last training session. The subjects then performed 3 h of two-legged knee-extensor exercise, and biopsies were obtained from both legs after 0, 2, 6, and 24 h of recovery. Real-time PCR was used to examine the expression of VEGF mRNA containing exon 1 and 2 (all VEGF isoforms), exon 6 or exon 7, and VEGF(165) mRNA. Acute exercise induced an increase (P < 0.05) in total VEGF mRNA levels as well as VEGF(165) and VEGF splice variants containing exon 7 at 0, 2, and 6 h of recovery. The increase in VEGF mRNA was higher in the untrained than in the trained leg (P < 0.05). The results suggest that in human skeletal muscle, acute exercise increases total VEGF mRNA, an increase that appears to be explained mainly by an increase in VEGF(165) mRNA. Furthermore, 4 wk of training attenuated the exercise-induced response in skeletal muscle VEGF(165) mRNA.

  1. Detection of VEGF-A(xxx)b isoforms in human tissues.

    Science.gov (United States)

    Bates, David O; Mavrou, Athina; Qiu, Yan; Carter, James G; Hamdollah-Zadeh, Maryam; Barratt, Shaney; Gammons, Melissa V; Millar, Ann B; Salmon, Andrew H J; Oltean, Sebastian; Harper, Steven J

    2013-01-01

    Vascular Endothelial Growth Factor-A (VEGF-A) can be generated as multiple isoforms by alternative splicing. Two families of isoforms have been described in humans, pro-angiogenic isoforms typified by VEGF-A165a, and anti-angiogenic isoforms typified by VEGF-A165b. The practical determination of expression levels of alternative isoforms of the same gene may be complicated by experimental protocols that favour one isoform over another, and the use of specific positive and negative controls is essential for the interpretation of findings on expression of the isoforms. Here we address some of the difficulties in experimental design when investigating alternative splicing of VEGF isoforms, and discuss the use of appropriate control paradigms. We demonstrate why use of specific control experiments can prevent assumptions that VEGF-A165b is not present, when in fact it is. We reiterate, and confirm previously published experimental design protocols that demonstrate the importance of using positive controls. These include using known target sequences to show that the experimental conditions are suitable for PCR amplification of VEGF-A165b mRNA for both q-PCR and RT-PCR and to ensure that mispriming does not occur. We also provide evidence that demonstrates that detection of VEGF-A165b protein in mice needs to be tightly controlled to prevent detection of mouse IgG by a secondary antibody. We also show that human VEGF165b protein can be immunoprecipitated from cultured human cells and that immunoprecipitating VEGF-A results in protein that is detected by VEGF-A165b antibody. These findings support the conclusion that more information on the biology of VEGF-A165b isoforms is required, and confirm the importance of the experimental design in such investigations, including the use of specific positive and negative controls.

  2. Monitoring PAI-1 and VEGF Levels in 6 Human Squamous Cell Carcinoma Xenografts During Fractionated Irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Bayer, Christine, E-mail: christine.bayer@yahoo.com [Department of Radiation Oncology, Klinikum rechts der Isar der Technischen Universitaet Muenchen, Munich (Germany); Kielow, Achim [Department of Radiation Oncology, Klinikum rechts der Isar der Technischen Universitaet Muenchen, Munich (Germany); Schilling, Daniela [Department of Radiation Oncology, Klinikum rechts der Isar der Technischen Universitaet Muenchen, Munich (Germany); HelmholtzZentrum Muenchen, German Research Center for Environmental Health, Department of Radiation Oncology, University Hospital and Medical Faculty Carl Gustav Carus University of Technology, Dresden (Germany); Maftei, Constantin-Alin [Department of Radiation Oncology, Klinikum rechts der Isar der Technischen Universitaet Muenchen, Munich (Germany); Zips, Daniel; Yaromina, Ala; Baumann, Michael [OncoRay Center for Radiation Research, Department of Radiation Oncology, University Hospital and Medical Faculty Carl Gustav Carus University of Technology, Dresden (Germany); Molls, Michael [Department of Radiation Oncology, Klinikum rechts der Isar der Technischen Universitaet Muenchen, Munich (Germany); Multhoff, Gabriele [Department of Radiation Oncology, Klinikum rechts der Isar der Technischen Universitaet Muenchen, Munich (Germany); HelmholtzZentrum Muenchen, German Research Center for Environmental Health, Department of Radiation Oncology, University Hospital and Medical Faculty Carl Gustav Carus University of Technology, Dresden (Germany)

    2012-11-01

    Purpose: Previous studies have shown that the plasminogen activator inhibitor type-1 (PAI-1) and vascular endothelial growth factor (VEGF) are regulated by hypoxia and irradiation and are involved in neoangiogenesis. The aim of this study was to determine in vivo whether changes in PAI-1 and VEGF during fractionated irradiation could predict for radiation resistance. Methods and Materials: Six xenografted tumor lines from human squamous cell carcinomas (HSCC) of the head and neck were irradiated with 0, 3, 5, 10, and 15 daily fractions of 2 Gy. The PAI-1 and VEGF antigen levels in tumor lysates were determined by enzyme-linked immunosorbent assay kits. The amounts of PAI-1 and VEGF were compared with the dose to cure 50% of tumors (TCD{sub 50}). Colocalization of PAI-1, pimonidazole (hypoxia), CD31 (endothelium), and Hoechst 33342 (perfusion) was examined by immunofluorescence. Results: Human PAI-1 and VEGF (hVEGF) expression levels were induced by fractionated irradiation in UT-SCC-15, UT-SCC-14, and UT-SCC-5 tumors, and mouse VEGF (msVEGF) was induced only in UT-SCC-5 tumors. High hVEGF levels were significantly associated with radiation sensitivity after 5 fractions (P=.021), and high msVEGF levels were significantly associated with radiation resistance after 10 fractions (P=.007). PAI-1 staining was observed in the extracellular matrix, the cytoplasm of fibroblast-like stroma cells, and individual tumor cells at all doses of irradiation. Colocalization studies showed PAI-1 staining close to microvessels. Conclusions: These results indicate that the concentration of tumor-specific and host-specific VEGF during fractionated irradiation could provide considerably divergent information for the outcome of radiation therapy.

  3. Semaphorin 6A regulates angiogenesis by modulating VEGF signaling

    Science.gov (United States)

    Segarra, Marta; Maric, Dragan; Salvucci, Ombretta; Hou, Xu; Kumar, Anil; Li, Xuri; Tosato, Giovanna

    2012-01-01

    Formation of new vessels during development and in the mature mammal generally proceeds through angiogenesis. Although a variety of molecules and signaling pathways are known to underlie endothelial cell sprouting and remodeling during angiogenesis, many aspects of this complex process remain unexplained. Here we show that the transmembrane semaphorin6A (Sema6A) is expressed in endothelial cells, and regulates endothelial cell survival and growth by modulating the expression and signaling of VEGFR2, which is known to maintain endothelial cell viability by autocrine VEGFR signaling. The silencing of Sema6A in primary endothelial cells promotes cell death that is not rescued by exogenous VEGF-A or FGF2, attributable to the loss of prosurvival signaling from endogenous VEGF. Analyses of mouse tissues demonstrate that Sema6A is expressed in angiogenic and remodeling vessels. Mice with null mutations of Sema6A exhibit significant defects in hyaloid vessels complexity associated with increased endothelial cell death, and in retinal vessels development that is abnormally reduced. Adult Sema6A-null mice exhibit reduced tumor, matrigel, and choroidal angiogenesis compared with controls. Sema6A plays important roles in development of the nervous system. Here we show that it also regulates vascular development and adult angiogenesis. PMID:23007403

  4. Novel Recombinant Sapovirus

    Science.gov (United States)

    Katayama, Kazuhiko; Miyoshi, Tatsuya; Uchino, Kiyoko; Oka, Tomoichiro; Tanaka, Tomoyuki; Takeda, Naokazu

    2004-01-01

    We determined the complete genome sequences of two sapovirus strains isolated in Thailand and Japan. One of these strains represented a novel, naturally occurring recombinant sapovirus. Evidence suggested the recombination site was at the polymerase-capsid junction within open reading frame one. PMID:15504283

  5. VEGF-C Is a Thyroid Marker of Malignancy Superior to VEGF-A in the Differential Diagnostics of Thyroid Lesions.

    Directory of Open Access Journals (Sweden)

    Kosma Woliński

    Full Text Available Thyroid nodular goiter is one of the most common medical conditions affecting even over a half of adult population. The risk of malignancy is rather small but noticeable-estimated by numerous studies to be about 3-10%. The definite differentiation between benign and malignant ones is a vital issue in endocrine practice. The aim of the current study was to assess the expression of vascular endothelial growth factor A (VEGF-A and VEGF-C on the mRNA level in FNAB washouts in case of benign and malignant thyroid nodules and to evaluate the diagnostic value of these markers of malignancy.Patients undergoing fine-needle aspiration biopsy (FNAB in our department between January 2013 and May 2014 were included. In case of all patients who gave the written consent, after ultrasonography (US and fine-needle aspiration biopsy (FNAB performed as routine medical procedure the needle was flushed with RNA Later solution, the washouts were frozen in -80 Celsius degrees. Expression of VEGF-A and VEGF-C and GADPH (reference gene was assessed in washouts on the mRNA level using the real-time PCR technique. Probes of patients who underwent subsequent thyroidectomy and were diagnosed with differentiated thyroid cancer (DTC; proved by post-surgical histopathology were analyzed. Similar number of patients with benign cytology were randomly selected to be a control group.Thirty one DTCs and 28 benign thyroid lesions were analyzed. Expression of VEGF-A was insignificantly higher in patients with DTCs (p = 0.13. Expression of VEGF-C was significantly higher in patients with DTC. The relative expression of VEGF-C (in comparison with GAPDH was 0.0049 for DTCs and 0.00070 for benign lesions, medians - 0.0036 and 0.000024 respectively (p<0.0001.Measurement of expression VEGF-C on the mRNA level in washouts from FNAB is more useful than more commonly investigated VEGF-A. Measurement of VEGF-C in FNAB washouts do not allow for fully reliable differentiation of benign and

  6. Discontinuation of anti-VEGF cancer therapy promotes metastasis through a liver revascularization mechanism

    DEFF Research Database (Denmark)

    Yang, Yunlong; Zhang, Yin; Iwamoto, Hideki

    2016-01-01

    The impact of discontinuation of anti-VEGF cancer therapy in promoting cancer metastasis is unknown. Here we show discontinuation of anti-VEGF treatment creates a time-window of profound structural changes of liver sinusoidal vasculatures, exhibiting hyper-permeability and enlarged open-pore size...... for uninterrupted and sustained antiangiogenic therapy for treatment of human cancers....

  7. Platelet Activation Determines Angiopoietin-1 and VEGF Levels in Malaria : Implications for Their Use as Biomarkers

    NARCIS (Netherlands)

    Brouwers, Judith; Noviyanti, Rintis; Fijnheer, Rob; de Groot, Philip G.; Trianty, Leily; Mudaliana, Siti; Roest, Mark; Syafruddin, Din; van der Ven, Andre; de Mast, Quirijn

    2013-01-01

    Introduction: The angiogenic proteins angiopoietin (Ang)-1, Ang-2 and vascular endothelial growth factor (VEGF) are regulators of endothelial inflammation and integrity. Since platelets store large amounts of Ang-1 and VEGF, measurement of circulation levels of these proteins is sensitive to platele

  8. A nanobody directed to a functional epitope on VEGF, as a novel strategy for cancer treatment

    DEFF Research Database (Denmark)

    Farajpour, Zahra; Rahbarizadeh, Fatemeh; Kazemi, Bahram

    2014-01-01

    Compelling evidence suggests that vascular endothelial growth factor (VEGF), due to its essential role in angiogenesis, is a critical target for cancer treatment. Neutralizing monoclonal antibodies against VEGF are important class of drugs used in cancer therapy. However, the cost of production...

  9. Pulmonary Large Cell Carcinoma Displays High Expression of EMMPRIN and VEGF

    Institute of Scientific and Technical Information of China (English)

    Yushuang Zheng; Miao Yu; Huachuan Zheng; Yifu Guan; Yasuo Takano

    2008-01-01

    OBJECTIVE To investigate the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and vascular endothelial growth factor (VEGF) in lung carcinomas,and to clarify their roles in carcinoma progression.METHODS Expression of EMMPRIN and VEGF was examined with tissue microarrays (TMAs) of lung carcinomas (n = 181),and their suppression in adjacent normal lung samples (n = 40) were determined by immunohistochemistry.The results were compared with clinicopathological findings for the same tumors.RESULTS Both EMMPRIN and VEGF were occasionally expressed in pseudostratified columnar epithelium and frequently in lung carcinomas.Histologically,EMMPRIN and VEGF displayed higher levels in large (LCC) cell carcinomas than adenocarcinoma (AD),squamous (SQ) and small cell carcinomas (SCC) (P < 0.05).EMMPRIN was more highly expressed in SQ as compared with AD (P < 0.05),while the converse was true for VEGF (P < 0.05).Binding was generally more intense for EMMPRIN in samples from male compared to female patients (P < 0.05),whereas the latter tended to exhibit more VEGF expression (P < 0.05).Positive associations of VEGF expression with the TNM stage and amounts of EMMPRIN were noted in the lung carcinomas (P < 0.05).CONCLUSION EMMPRIN and VEGF possibly contribute to physiological repair of normal lung and histogenesis of lung carcinoma.Both proteins might be involved in the molecular basis for differences in the incidence of lung carcinoma between men and women.

  10. Prolonged presence of VEGF promotes vascularization in 3D bioprinted scaffolds with defined architecture

    NARCIS (Netherlands)

    Poldervaart, Michelle T; Gremmels, Hendrik; van Deventer, Kelly; Fledderus, Joost O; Oner, F Cumhur; Verhaar, Marianne C; Dhert, Wouter J A; Alblas, Jacqueline

    2014-01-01

    Timely vascularization is essential for optimal performance of bone regenerative constructs. Vascularization is efficiently stimulated by vascular endothelial growth factor (VEGF), a substance with a short half-life time. This study investigates the controlled release of VEGF from gelatin microparti

  11. Correlation of VEGF and COX-2 Expression with VM in Malignant Melanomas

    Institute of Scientific and Technical Information of China (English)

    BaocunSun; ShiwuZhang; XiulanZhao; YanxueLiu; ChunshengNi; DanfangZhang; HongQi; ZhiyongLiu; XishanHao

    2004-01-01

    OBJECTIVE To investigate the relationship between vascular epithelial growth factor (VEGF) and cyclooxygenase-2 (COX-2) in melanomas and the expressive difference of VEGF and COX-2 between melanomas with and without vasculogenic mimicry(VM).METHODS Sixty cases of malignant melanomas emoeaaea In paraffin were studied. The tumors were divided into a high-grade malignant group and a low-grade malignant group based on their tumor type, atypia and survival time of the patient. Then tissue microarrays were produced from these paraffin-embedded tumor tissues which were stained for VEGF, COX-2 and PAS. The difference in expression between VEGF and COX-2 in the malignant melanomas was compared using a grid-count. In addition, the tumors were also divided into mimicry and non-mimicry groups based on their PAS staining. Then the differences between the PAS positive and negative areas of the 2 groups were compared.RESULTS In malignant melanomas with VM, VEGF and COX-2 expression was less in tumors in which VM was absent, but VEGF, COX-2 expression in high-grade malignant melanomas was higher than that in low-grade grade malignant melanomas. Expression of VEGF was correlated with COX-2 expression.CONCLUSION VM exists in some high-grade malignant melanomas. The differences and relations between VEGF and COX-2 showed that some high-grade malignant melanomas possess a unique molecular-mechanism of tumor metastasis and blood supply.

  12. Anti-VEGF agents in metastatic colorectal cancer (mCRC: are they all alike?

    Directory of Open Access Journals (Sweden)

    Saif MW

    2013-06-01

    Full Text Available Muhammad Wasif Saif GI Oncology Program, Tufts University School of Medicine, Boston, MA, USA Abstract: Bevacizumab is a monoclonal antibody that binds and neutralizes vascular endothelial growth factor (VEGF-A, a key player in the angiogenesis pathway. Despite benefits of bevacizumab in cancer therapy, it is clear that the VEGF pathway is complex, involving multiple isoforms, receptors, and alternative ligands such as VEGF-B, and placental growth factor, which could enable escape from VEGF-A-targeted angiogenesis inhibition. Recently developed therapies have targeted other ligands in the VEGF pathway (eg, aflibercept, known as ziv-aflibercept in the United States, VEGF receptors (eg, ramucirumab, and their tyrosine kinase signaling (ie, tyrosine kinase inhibitors. The goal of the current review was to identify comparative preclinical data for the currently available VEGF-targeted therapies. Sources were compiled using PubMed searches (2007 to 2012, using search terms including, but not limited to: “bevacizumab,” “aflibercept,” “ramucirumab,” and “IMC-18F1.” Two preclinical studies were identified that compared bevacizumab and the newer agent, aflibercept. These studies identified some important differences in binding and pharmacodynamic activity, although the potential clinical relevance of these findings is not known. Newer antiangiogenesis therapies should help further expand treatment options for colorectal and other cancers. Comparative preclinical data on these agents is currently lacking. Keywords: aflibercept, antiangiogenesis, metastatic colorectal cancer (mCRC, tyrosine kinase inhibitor (TKI, vascular endothelial growth factor (VEGF

  13. [Measurement and correlation analysis of plasma VEGF level in the patients of hyperthyroidism].

    Science.gov (United States)

    Zhao, Xiaoni; Wang, Guangrong; You, Jinhui

    2013-04-01

    Vascular endothelial growth factor (VEGF) is a glycoprotein that promotes endothelial regeneration, stimulates formation of collateral blood vessels and increases vascular permeability. The purpose of this study was to measure the peripheral blood plasma level of VEGF and FT3, FT4, TSH and to analyze the correlation of the level of VEGF and TSH, FT3, FT4, age and gender in the patients of hyperthyroidism. The relationship between hyperthyroidism and VEGF was investigated as well. The plasma level of VEGF in 45 hyperthyroidism patients and 27 healthy persons were measured by enzyme-linked immunosorbent assay (ELISA), while plasma FT3, FT4, TSH were detected by chemiluminescence. The result showed that the plasma level of VEGF in hyperthyroidism patients [(92.53 +/- 62.38) pg/mL] was significantly lower than that in the control group [(158.28 +/- 77.15) pg/mL] (P hyperthyroidism patients (P > 0.05). These results suggested that the peripheral blood plasma level of VEGF in hyperthyroidism patients was significantly lower than that in the control group. Further experimental investigations are needed to estimate the relationship between VEGF and hyperthyroidism.

  14. RNA interference inhibits expression of vascular endothelial growth factor (VEGF) in human retinal pigment epithelial cells

    Institute of Scientific and Technical Information of China (English)

    CAI Chun-mei; SUN Bao-chen; LIU Xu-yang; WANG Jin-jin; LI Jun-fa; HAN Song; WANG Ning-li; LU Qing-jun

    2005-01-01

    @@ Choroidal neovascularization (CNV), a major cause of vision loss, is the result of the increased vascular endothelial growth factor (VEGF) expression in human retinal pigment epithelial (RPE) cells. It is important to inhibit the expression of VEGF protein in RPE cells.

  15. Modulation of age-related insulin sensitivity by VEGF-dependent vascular plasticity in adipose tissues.

    Science.gov (United States)

    Honek, Jennifer; Seki, Takahiro; Iwamoto, Hideki; Fischer, Carina; Li, Jingrong; Lim, Sharon; Samani, Nilesh J; Zang, Jingwu; Cao, Yihai

    2014-10-14

    Mechanisms underlying age-related obesity and insulin resistance are generally unknown. Here, we report age-related adipose vascular changes markedly modulated fat mass, adipocyte functions, blood lipid composition, and insulin sensitivity. Notably, VEGF expression levels in various white adipose tissues (WATs) underwent changes uninterruptedly in different age populations. Anti-VEGF and anti- VEGF receptor 2 treatment in different age populations showed marked variations of vascular regression, with midaged mice exhibiting modest sensitivity. Interestingly, anti-VEGF treatment produced opposing effects on WAT adipocyte sizes in different age populations and affected vascular density and adipocyte sizes in brown adipose tissue. Consistent with changes of vasculatures and adipocyte sizes, anti-VEGF treatment increased insulin sensitivity in young and old mice but had no effects in the midaged group. Surprisingly, anti-VEGF treatment significantly improved insulin sensitivity in midaged obese mice fed a high-fat diet. Our findings demonstrate that adipose vasculatures show differential responses to anti-VEGF treatment in various age populations and have therapeutic implications for treatment of obesity and diabetes with anti-VEGF-based antiangiogenic drugs.

  16. VEGFR1-mediated pericyte ablation links VEGF and PlGF to cancer-associated retinopathy

    DEFF Research Database (Denmark)

    Cao, Renhai; Xue, Yuan; Hedlund, Eva-Maria

    2010-01-01

    , and adenoviral vectors ablates pericytes from the mature retinal vasculature through the VEGF receptor 1 (VEGFR1)-mediated signaling pathway, leading to increased vascular leakage. In contrast, we demonstrate VEGF receptor 2 (VEGFR2) is primarily expressed in nonvascular photoreceptors and ganglion cells...

  17. Tumor-associated fibroblasts as "Trojan Horse" mediators of resistance to anti-VEGF therapy.

    Science.gov (United States)

    Francia, Giulio; Emmenegger, Urban; Kerbel, Robert S

    2009-01-06

    While targeting VEGF has shown success against a number of human cancers, drug resistance has resulted in compromised clinical benefits. In this issue of Cancer Cell, Crawford et al. (2009) report that tumors resistant to anti-VEGF therapy stimulate tumor-associated fibroblasts to express proangiogenic PDGF-C, implicating it as a potential therapeutic target.

  18. Tumour cells expressing single VEGF isoforms display distinct growth, survival and migration characteristics.

    Directory of Open Access Journals (Sweden)

    Chryso Kanthou

    Full Text Available Vascular endothelial growth factor-A (VEGF is produced by most cancer cells as multiple isoforms, which display distinct biological activities. VEGF plays an undisputed role in tumour growth, vascularisation and metastasis; nevertheless the functions of individual isoforms in these processes remain poorly understood. We investigated the effects of three main murine isoforms (VEGF188, 164 and 120 on tumour cell behaviour, using a panel of fibrosarcoma cells we developed that express them individually under endogenous promoter control. Fibrosarcomas expressing only VEGF188 (fs188 or wild type controls (fswt were typically mesenchymal, formed ruffles and displayed strong matrix-binding activity. VEGF164- and VEGF120-producing cells (fs164 and fs120 respectively were less typically mesenchymal, lacked ruffles but formed abundant cell-cell contacts. On 3D collagen, fs188 cells remained mesenchymal while fs164 and fs120 cells adopted rounded/amoeboid and a mix of rounded and elongated morphologies respectively. Consistent with their mesenchymal characteristics, fs188 cells migrated significantly faster than fs164 or fs120 cells on 2D surfaces while contractility inhibitors accelerated fs164 and fs120 cell migration. VEGF164/VEGF120 expression correlated with faster proliferation rates and lower levels of spontaneous apoptosis than VEGF188 expression. Nevertheless, VEGF188 was associated with constitutively active/phosphorylated AKT, ERK1/2 and Stat3 proteins. Differences in proliferation rates and apoptosis could be explained by defective signalling downstream of pAKT to FOXO and GSK3 in fs188 and fswt cells, which also correlated with p27/p21 cyclin-dependent kinase inhibitor over-expression. All cells expressed tyrosine kinase VEGF receptors, but these were not active/activatable suggesting that inherent differences between the cell lines are governed by endogenous VEGF isoform expression through complex interactions that are independent of tyrosine

  19. Inhibition of VEGF signaling pathways in multiple myeloma and other malignancies.

    Science.gov (United States)

    Podar, Klaus; Anderson, Kenneth C

    2007-03-01

    Due to its direct effects on endothelial cells, circulatory endothelial progenitor cells, hematopoietic stem cells, immune cells, osteoclasts, osteoblasts and neurons, vascular endothelial growth factor (VEGF) is linked to tumor cell development, progression, metastatic osteolysis and drug resistance, as well as clinical features such as metastatic osteolysis. Importantly, recent advances in the understanding of mechanisms of action of antiangiogenic drugs/VEGF-inhibitors have fundamentally changed treatment regimens in cancer. VEGF plays a key role not only in solid tumors but also in hematologic malignancies, including multiple myeloma (MM). Despite recent advances in our understanding of MM pathogenesis and novel therapies (bortezomib and lenalidomide), it remains incurable. Our own and others' work suggest that VEGF-inhibitors e.g., the small molecule VEGF receptor inhibitor pazopanib, may also improve patient outcome in MM.

  20. Metformin inhibits development of diabetic retinopathy through inducing alternative splicing of VEGF-A

    Science.gov (United States)

    Yi, Quan-Yong; Deng, Gang; Chen, Nan; Bai, Zhi-Sha; Yuan, Jian-Shu; Wu, Guo-Hai; Wang, Yu-Wen; Wu, Shan-Jun

    2016-01-01

    Previous studies have shown that metformin, an AMP-activated protein kinase activator widely prescribed for type 2 diabetes, is especially beneficial in cases of diabetic retinopathy (DR) with undetermined mechanisms. Here, we used a streptozotocin-induced diabetes model in mice to study the effects of metformin on the development of DR. We found that 10 weeks after STZ treatment, DR was induced in STZ-treated mice, regardless treatment of metformin. However, metformin alleviated the DR, seemingly through attenuating the retina neovascularization. The total vascular endothelial cell growth factor A (VEGF-A) in eyes was not altered by metformin, but the phosphorylation of the VEGF receptor 2 (VEGFR2) was decreased, which inhibited VEGF signaling. Further analysis showed that metformin may induce VEGF-A mRNA splicing to VEGF120 isoform to reduce its activation of the VEGFR2. These findings are critical for generating novel medicine for DR treatment.

  1. Polydopamine-Based Surface Modification of Novel Nanoparticle-Aptamer Bioconjugates for In Vivo Breast Cancer Targeting and Enhanced Therapeutic Effects.

    Science.gov (United States)

    Tao, Wei; Zeng, Xiaowei; Wu, Jun; Zhu, Xi; Yu, Xinghua; Zhang, Xudong; Zhang, Jinxie; Liu, Gan; Mei, Lin

    2016-01-01

    In this study, we reported a simple polydopamine (pD)-based surface modification method to prepare novel nanoparticle-aptamer bioconjugates (Apt-pD-DTX/NPs) for in vivo tumor targeting and enhanced therapeutic effects of breast cancer. With simple preparation procedures, the new functionalized Apt-pD-DTX/NPs could maximumly increase the local effective drug concentration on tumor sites, achieving enhanced treatment effectiveness and minimizing side effects. The dopamine polymerization and aptamer conjugation barely changed the characters of NPs. Both in vitro cell experiments (i.e. endocytosis of fluorescent NPs, in vitro cellular targeting and cytotoxicity assays) and in vivo animal studies (i.e. in vivo imaging, biodistribution and antitumor effects of NPs) demonstrated that the Apt-pD-DTX/NPs could achieve significantly high targeting efficiency and enhanced therapeutic effects compared with clinical Taxotere(®) and NPs without functional modification. Above all, the Apt-pD-DTX/NPs showed great potential as a promising nanoformulation for in vivo breast cancer therapy and the construction of pD-modified NP-aptamer bioconjugates could be of great value in medical use.

  2. Polydopamine-Based Surface Modification of Novel Nanoparticle-Aptamer Bioconjugates for In Vivo Breast Cancer Targeting and Enhanced Therapeutic Effects

    Science.gov (United States)

    Tao, Wei; Zeng, Xiaowei; Wu, Jun; Zhu, Xi; Yu, Xinghua; Zhang, Xudong; Zhang, Jinxie; Liu, Gan; Mei, Lin

    2016-01-01

    In this study, we reported a simple polydopamine (pD)-based surface modification method to prepare novel nanoparticle-aptamer bioconjugates (Apt-pD-DTX/NPs) for in vivo tumor targeting and enhanced therapeutic effects of breast cancer. With simple preparation procedures, the new functionalized Apt-pD-DTX/NPs could maximumly increase the local effective drug concentration on tumor sites, achieving enhanced treatment effectiveness and minimizing side effects. The dopamine polymerization and aptamer conjugation barely changed the characters of NPs. Both in vitro cell experiments (i.e. endocytosis of fluorescent NPs, in vitro cellular targeting and cytotoxicity assays) and in vivo animal studies (i.e. in vivo imaging, biodistribution and antitumor effects of NPs) demonstrated that the Apt-pD-DTX/NPs could achieve significantly high targeting efficiency and enhanced therapeutic effects compared with clinical Taxotere® and NPs without functional modification. Above all, the Apt-pD-DTX/NPs showed great potential as a promising nanoformulation for in vivo breast cancer therapy and the construction of pD-modified NP-aptamer bioconjugates could be of great value in medical use. PMID:26941841

  3. Aptamer-hybrid nanoparticle bioconjugate efficiently delivers miRNA-29b to non-small-cell lung cancer cells and inhibits growth by downregulating essential oncoproteins

    Directory of Open Access Journals (Sweden)

    Perepelyuk M

    2016-07-01

    Full Text Available Maryna Perepelyuk, Christina Maher, Ashakumary Lakshmikuttyamma, Sunday A Shoyele Department of Pharmaceutical Science, College of Pharmacy, Thomas Jefferson University, Philadelphia, PA, USA Abstract: MicroRNAs (miRNAs are potentially attractive candidates for cancer therapy. However, their therapeutic application is limited by lack of availability of an efficient delivery system to stably deliver these potent molecules intracellularly to cancer cells while avoiding healthy cells. We developed a novel aptamer-hybrid nanoparticle bioconjugate delivery system to selectively deliver miRNA-29b to MUC1-expressing cancer cells. Significant downregulation of oncoproteins DNMT3b and MCL1 was demonstrated by these MUC1 aptamer-functionalized hybrid nanoparticles in A549 cells. Furthermore, downregulation of these oncoproteins led to antiproliferative effect and induction of apoptosis in a superior version when compared with Lipofectamine 2000. This novel aptamer-hybrid nanoparticle bioconjugate delivery system could potentially serve as a platform for intracellular delivery of miRNAs to cancer cells, hence improving the therapeutic outcome of lung cancer. Keywords: aptamer, nanoparticles, microRNA, lung cancer, targeted delivery

  4. Assessment of the systemic distribution of a bioconjugated anti-Her2 magnetic nanoparticle in a breast cancer model by means of magnetic resonance imaging

    Science.gov (United States)

    Huerta-Núñez, L. F. E.; Villanueva-Lopez, G. Cleva; Morales-Guadarrama, A.; Soto, S.; López, J.; Silva, J. G.; Perez-Vielma, N.; Sacristán, E.; Gudiño-Zayas, Marco E.; González, C. A.

    2016-09-01

    The aim of this study was to determine the systemic distribution of magnetic nanoparticles of 100 nm diameter (MNPs) coupled to a specific monoclonal antibody anti-Her2 in an experimental breast cancer (BC) model. The study was performed in two groups of Sprague-Dawley rats: control ( n = 6) and BC chemically induced ( n = 3). Bioconjugated "anti-Her2-MNPs" were intravenously administered, and magnetic resonance imaging (MRI) monitored its systemic distribution at seven times after administration. Non-heme iron presence associated with the location of the bioconjugated anti-Her2-MNPs in splenic, hepatic, cardiac and tumor tissues was detected by Perl's Prussian blue (PPB) stain. Optical density measurements were used to semiquantitatively determine the iron presence in tissues on the basis of a grayscale values integration of T1 and T2 MRI sequence images. The results indicated a delayed systemic distribution of MNPs in cancer compared to healthy conditions with a maximum concentration of MNPs in cancer tissue at 24 h post-infusion.

  5. Sustained delivery of VEGF from designer self-assembling peptides improves cardiac function after myocardial infarction

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Hai-dong [Department of Anatomy, School of Basic Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 201203 (China); Cui, Guo-hong; Yang, Jia-jun [Department of Neurology, Shanghai No. 6 People' s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200233 (China); Wang, Cun [Institutes of Biomedical Sciences, Fudan University, Shanghai 200032 (China); Zhu, Jing; Zhang, Li-sheng; Jiang, Jun [Department of Anatomy, School of Basic Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 201203 (China); Shao, Shui-jin, E-mail: shaoshuijin@163.com [Department of Anatomy, School of Basic Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 201203 (China)

    2012-07-20

    Highlights: Black-Right-Pointing-Pointer The designer peptide LRKKLGKA could self-assemble into nanofibers. Black-Right-Pointing-Pointer Injection of LRKKLGKA peptides could promote the sustained delivery of VEGF. Black-Right-Pointing-Pointer Injection of VEGF with LRKKLGKA peptides lead to sufficient angiogenesis. Black-Right-Pointing-Pointer Injection of VEGF with LRKKLGKA peptides improves heart function. -- Abstract: Poor vascularization and insufficient oxygen supply are detrimental to the survival of residual cardiomyocytes or transplanted stem cells after myocardial infarction. To prolong and slow the release of angiogenic factors, which stimulate both angiogenesis and vasculogenesis, we constructed a novel self-assembling peptide by attaching the heparin-binding domain sequence LRKKLGKA to the self-assembling peptide RADA16. This designer self-assembling peptide self-assembled into nanofiber scaffolds under physiological conditions, as observed by atomic force microscopy. The injection of designer self-assembling peptides can efficiently provide the sustained delivery of VEGF for at least 1 month. At 4 weeks after transplantation, cardiac function was improved, and scar size and collagen deposition were markedly reduced in the group receiving VEGF with the LRKKLGKA scaffolds compared with groups receiving VEGF alone, LRKKLGKA scaffolds alone or VEGF with RADA16 scaffolds. The microvessel density in the VEGF with LRKKLGKA group was higher than that in the VEGF with RADA16 group. TUNEL and cleaved caspase-3 expression assays showed that the transplantation of VEGF with LRKKLGKA enhanced cell survival in the infarcted heart. These results present the tailor-made peptide scaffolds as a new generation of sustained-release biomimetic biomaterials and suggest that the use of angiogenic factors along with designer self-assembling peptides can lead to myocardial protection, sufficient angiogenesis, and improvement in cardiac function.

  6. Impaired Pulmonary Defense Against Pseudomonas aeruginosa in VEGF Gene Inactivated Mouse Lung

    Science.gov (United States)

    Breen, Ellen C.; Malloy, Jaret L.; Tang, Kechun; Xia, Feng; Fu, Zhenxing; Hancock, Robert E. W.; Overhage, Joerg; Wagner, Peter D.; Spragg, Roger G.

    2012-01-01

    Repeated bacterial and viral infections are known to contribute to worsening lung function in several respiratory diseases, including asthma, cystic fibrosis and chronic obstructive pulmonary disease (COPD). Previous studies have reported alveolar wall cell apoptosis and parenchymal damage in adult pulmonary VEGF gene ablated mice. We hypothesized that VEGF expressed by type II cells is also necessary to provide an effective host defense against bacteria in part by maintaining surfactant homeostasis. Therefore, Pseudomonas aeruginosa (PAO1) levels were evaluated in mice following lung-targeted VEGF gene inactivation, and alterations in VEGF-dependent type II cell function were evaluated by measuring surfactant homeostasis in mouse lungs and isolated type II cells. In VEGF-deficient lungs increased PAO1 levels and pro-inflammatory cytokines, TNFα and IL-6, were detected 24 hours after bacterial instillation compared to control lungs. In vivo lung-targeted VEGF gene deletion (57% decrease in total pulmonary VEGF) did not alter alveolar surfactant or tissue disaturated phosphatidylcholine (DSPC) levels. However, sphingomyelin content, choline phosphate cytidylyltransferase (CCT) mRNA and SP-D expression were decreased. In isolated type II cells an 80% reduction of VEGF protein resulted in decreases in total phospholipids (PL), DSPC, DSPC synthesis, surfactant associated proteins (SP)-B and -D, and the lipid transporters, ABCA1 and Rab3D. TPA-induced DSPC secretion and apoptosis were elevated in VEGF-deficient type II cells. These results suggest a potential protective role for type II cell-expressed VEGF against bacterial initiated infection. PMID:22718316

  7. Coordinating Etk/Bmx activation and VEGF upregulation to promote cell survival and proliferation.

    Science.gov (United States)

    Chau, Cindy H; Chen, Kai-Yun; Deng, Hong-Tao; Kim, Kwang-Jin; Hosoya, Ken-ichi; Terasaki, Tetsuya; Shih, Hsiu-Ming; Ann, David K

    2002-12-12

    Etk/Bmx, a member of the Tec family of non-receptor tyrosine kinase, is characterized by an N-terminal PH domain and has recently been shown to be involved in the regulation of various cellular processes, including proliferation, differentiation, motility and apoptosis. Since VEGF and the activation of its signaling pathway have been implicated in modulating a variety of biological responses, we characterized the role of Etk-dependent signaling pathways involved in the upregulation of VEGF expression, and explored the functional implications of this enhancement in sustaining cell proliferation and survival. Using Northern and Western analyses, transient transfections, and pharmacological agents, we demonstrate that Etk activation alone is sufficient to transcriptionally induce VEGF expression, independent of the previously identified hypoxia response element (HRE), in both Pa-4 epithelial and TR-BBB endothelial cells under normoxia. In addition, Etk utilizes both MEK/ERK and PI3-K/Pak1 signaling pathways in concert to activate VEGF transcription. Functionally, Etk activation elicits a profound stimulatory effect on TR-BBB cell proliferation and formation of capillary-like networks in Matrigel containing reduced levels of growth factors. Finally, antisense oligonucleotides against either endogenous VEGF or Etk abrogate the proliferation of Etk-activated TR-BBB cells, and exogenous VEGF treatment stimulates endogenous Etk tyrosine phosphorylation in HUVECs. Taken together, these results indicate that VEGF is both an Etk downstream target gene and an Etk upstream activator, constituting a reciprocal Etk-VEGF autoregulatory loop. These findings, to our knowledge, are the first delineation of a network of positive feedforward signaling pathways that converge on the Etk-VEGF axis, causally associating Etk-mediation of VEGF induction with enhanced cellular processes in both epithelial and endothelial cells.

  8. Construction of Lentivirus Vector of hVEGF165 Gene and Its Protective Function of Irradiation-induced Apoptosis%人血管内皮生长因子165慢病毒载体的构建及其抗放射所致细胞凋亡的作用

    Institute of Scientific and Technical Information of China (English)

    赵仲艳; 韦映梅; 黎祥喷; 吕瑞妍; 肖颂华; 刘中霖; 邢诒刚; 刘军

    2011-01-01

    摘要:[目的]构建人血管内皮生长因子165 (hVEGF165)慢病毒表达载体pCDH-CMV-MCS-EF1-copGFPhVEGF165,并转染至HT22细胞,并迸一步观察其对放射线损伤所致的细胞凋亡的保护作用.[方法]PCR扩增hVEGF165基因,克隆到pCDH-CMV-MCS-EF1-copGFP载体;通过酶切电泳,菌落PCR初步筛选和测序鉴定构建的pCDH-CMV-MCS-EF-GFP1-hVEGF165重组载体;采用磷酸钙共转染法转染293T细胞包装制备慢病毒液,并感染HT22细胞,采用实时RT-PCR及Western Blot检测hVEGF165基因在HT22细胞中的表达情况.进一步对单纯HT22细胞、空质粒转染组以及hVEGF165转染组HT22细胞进行0、5、10 Gy的X线照射,流式细胞仪检测细胞凋亡情况.[结果]hVEGF165基因片段重组到pCDH-CMV-MCS-EF1-copGFP载体,酶切后电泳结果显示均能得到与理论大小相符的片段,测序结果与GenBank序列完全一致,转染HT22后hVEGF mRNA及蛋白表达水平明显增高.而且同各对照组相比,hVEGF165可以降低细胞放疗后的凋亡率.[结论]pCDH-CMV-MCS-EF1 -copGFP-hVEGF165慢病毒表达载体构建成功,其转染HT22细胞后可获得高水平hVEGF165 mRNA和蛋白的表达,hVEGF165具有抗放射所致细胞凋亡的作用.%[Objective] To construct human vascular endothelial growth factor-165 (hVEGF165) gene lentivirus expression vector pCDH-CMV-MCS-Efl-copGFP-hVEGF165, and detect its expression after transfected into HT22 cells and further observe its effect of anti-apopotosis induced by irradiation. [Methods] hVEGF165 gene cDNA was amplicated by polymerase chain reaction (PCR) and cloned into the pCDHCMVMCSEF vector. Reconstructed pCDH-CMV-MCS-Efl -hVEGF165 plasmid was identified by electromigratory analysis, colony PCR, and sequencing analysis. 293T cells were transfected with recombinate vector using the way of calcium phosphate cotransfection to get lentiviral particles. Lentiviral particles were then transfect them into HT22 cells. Real-time RT-PCR and Western blot were

  9. Recombinant methods and materials

    Energy Technology Data Exchange (ETDEWEB)

    Roizman, B.; Post, L.E.

    1988-09-06

    This patent describes a method for stably effecting the insertion or deletion of a selected DNA sequence at a specific site in a viral genome. The method consists of: (1) isolating from the genome a linear DNA fragment comprising both (a) the specific site determined for insertion or deletion of selected DNA sequence and (b) flanking DNA sequences normally preceding and following the site; (2) preparing first and second altered genome fragments from the fragment isolated in step (1). (a) the first altered fragment comprising the fragment comprising a thymidine kinase gene in a position intermediate the ends of the fragment, and (b) the second altered fragment comprising the fragment having the selected DNA sequence inserted therein or deleted therefrom; (3) contacting the genome with the first altered fragment under conditions permitting recombination at sites of DNA sequence homology, selecting for a recombinant genome comprising the thymidine kinase gene, and isolating the recombinant genome; and (4) contacting the recombinant genome isolated in step (3) with the second altered fragment under conditions permitting recombination at sites of DNA sequence homology, selecting for a recombinant genome lacking the thymidine kinase gene, and isolating the recombinant genome product.

  10. Neurotrophin-3 Induces BMP-2 and VEGF Activities and Promotes the Bony Repair of Injured Growth Plate Cartilage and Bone in Rats.

    Science.gov (United States)

    Su, Yu-Wen; Chung, Rosa; Ruan, Chun-Sheng; Chim, Shek Man; Kuek, Vincent; Dwivedi, Prem P; Hassanshahi, Mohammadhossein; Chen, Ke-Ming; Xie, Yangli; Chen, Lin; Foster, Bruce K; Rosen, Vicki; Zhou, Xin-Fu; Xu, Jiake; Xian, Cory J

    2016-06-01

    Injured growth plate is often repaired by bony tissue causing bone growth defects, for which the mechanisms remain unclear. Because neurotrophins have been implicated in bone fracture repair, here we investigated their potential roles in growth plate bony repair in rats. After a drill-hole injury was made in the tibial growth plate and bone, increased injury site mRNA expression was observed for neurotrophins NGF, BDNF, NT-3, and NT-4 and their Trk receptors. NT-3 and its receptor TrkC showed the highest induction. NT-3 was localized to repairing cells, whereas TrkC was observed in stromal cells, osteoblasts, and blood vessel cells at the injury site. Moreover, systemic NT-3 immunoneutralization reduced bone volume at injury sites and also reduced vascularization at the injured growth plate, whereas recombinant NT-3 treatment promoted bony repair with elevated levels of mRNA for osteogenic markers and bone morphogenetic protein (BMP-2) and increased vascularization and mRNA for vascular endothelial growth factor (VEGF) and endothelial cell marker CD31 at the injured growth plate. When examined in vitro, NT-3 promoted osteogenesis in rat bone marrow stromal cells, induced Erk1/2 and Akt phosphorylation, and enhanced expression of BMPs (particularly BMP-2) and VEGF in the mineralizing cells. It also induced CD31 and VEGF mRNA in rat primary endothelial cell culture. BMP activity appears critical for NT-3 osteogenic effect in vitro because it can be almost completely abrogated by co-addition of the BMP inhibitor noggin. Consistent with its angiogenic effect in vivo, NT-3 promoted angiogenesis in metatarsal bone explants, an effect abolished by co-treatment with anti-VEGF. This study suggests that NT-3 may be an osteogenic and angiogenic factor upstream of BMP-2 and VEGF in bony repair, and further studies are required to investigate whether NT-3 may be a potential target for preventing growth plate faulty bony repair or for promoting bone fracture healing. © 2016

  11. Vascular endothelial growth factor B (VEGF-B is up-regulated and exogenous VEGF-B is neuroprotective in a culture model of Parkinson's disease

    Directory of Open Access Journals (Sweden)

    Zhang Shiling

    2009-12-01

    Full Text Available Abstract Parkinson's disease (PD results from the degeneration of dopaminergic neurons in the substantia nigra and the consequent deficit of dopamine released in the striatum. Current oral dopamine replacement or surgical therapies do not address the underlying issue of neurodegeneration, they neither slow nor halt disease. Neurotrophic factors have shown preclinical promise, but the choice of an appropriate growth factor as well as the delivery has proven difficult. In this study, we used a rotenone rat midbrain culture model to identify genes that are changed after addition of the neurotoxin. (1 We challenged rat midbrain cultures with rotenone (20 nM, a pesticide that has been shown to be toxic for dopaminergic neurons and that has been a well-characterized model of PD. A gene chip array analysis demonstrated that several genes were up-regulated after the rotenone treatment. Interestingly transcriptional activation of vascular endothelial growth factor B (VEGF-B was evident, while vascular endothelial growth factor A (VEGF-A levels remained unaltered. The results from the gene chip array experiment were verified with real time PCR and semi-quantitative western analysis using β-actin as the internal standard. (2 We have also found evidence that exogenously applied VEGF-B performed as a neuroprotective agent facilitating neuron survival in an even more severe rotenone culture model of PD (40 nM rotenone. VEGF-B has very recently been added to the list of trophic factors that reduce effects of neurodegeneration, as was shown in an in vivo model of motor neuron degeneration, while lacking potential adverse angiogenic activity. The data of an in vivo protective effect on motor neurons taken together with the presented results demonstrate that VEGF-B is a new candidate trophic factor distinct from the GDNF family of trophic factors. VEGF-B is activated by neurodegenerative challenges to the midbrain, and exogenous application of VEGF-B has a

  12. Dissociative recombination in aeronomy

    Science.gov (United States)

    Fox, J. L.

    1989-01-01

    The importance of dissociative recombination in planetary aeronomy is summarized, and two examples are discussed. The first is the role of dissociative recombination of N2(+) in the escape of nitrogen from Mars. A previous model is updated to reflect new experimental data on the electronic states of N produced in this process. Second, the intensity of the atomic oxygen green line on the nightside of Venus is modeled. Use is made of theoretical rate coefficients for production of O (1S) in dissociative recombination from different vibrational levels of O2(+).

  13. (-)-Epigallocatechin-3-gallate inhibits VEGF expression induced by IL-6 via Stat3 in gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Bao-He Zhu; Hua-Yun Chen; Wen-Hua Zhan; Cheng-You Wang; Shi-Rong Cai; Zhao Wang; Chang-Hua Zhang; Yu-Long He

    2011-01-01

    AIM: To demonstrate that (-)-Epigallocatechin-3-gallate (EGCG) inhibits vascular endothelial growth factor (VEGF) expression and angiogenesis induced by interleukin-6 (IL-6) via suppressing signal transducer and activator of transcription 3 (Stat3) activity in gastric cancer.METHODS: Human gastric cancer (AGS) cells were treated with IL-6 (50 ng/mL) and EGCG at different concentrations. VEGF, total Stat3 and activated Stat3 protein levels in the cell lyses were examined by Western blotting, VEGF protein level in the conditioned medium was measured by enzyme-linked immunosorbent assay, and the level of VEGF mRNA was evaluated by reverse transcription polymerase chain reaction (RTPCR).Stat3 nuclear translocation was determined by Western blotting with nuclear extract, and Stat3-DNA binding activity was examined with Chromatin immunoprecipitation (ChIP) assay. IL-6 induced endothelial cell proliferation was measured with 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazoliumbromide assay, in vitro angiogenesis was determined with endothelial cell tube formation assay in Matrigel, and IL-6-induced angiogenesis in vitro was measured with Matrigel plug assay.RESULTS: There was a basal expression and secretion of VEGF in AGS cells. After stimulation with IL-6, VEGF expression was apparently up-regulated and a 2.4-fold increase was observed. VEGF secretion in the conditioned medium was also increased by 2.8 folds. When treated with EGCG, VEGF expression and secretion were dose-dependently decreased. IL-6 also increased VEGF mRNA expression by 3.1 folds. EGCG treatment suppressed VEGF mRNA expression in a dose-dependent manner. EGCG dose-dependently inhibited Stat3 activation induced by IL-6, but did not change the total Stat3 expression. When treated with EGCG or AG490,VEGF expressions were reduced to the level or an even lower level in the tumor cells not stimulated with IL-6. However, PD98059 and LY294002 did not change VEGF expression induced by IL-6. EGCG inhibited

  14. Neoadjuvant multidrug chemotherapy including High-Dose Methotrexate modifies VEGF expression in Osteosarcoma: an immunohistochemical analysis

    Directory of Open Access Journals (Sweden)

    Rosa Michele A

    2010-02-01

    Full Text Available Abstract Background Angiogenesis plays a role in the progression of osteosarcoma, as well as in other mesenchymal tumors and carcinomas, and it is most commonly assessed by vascular endothelial growth factor (VEGF expression or tumor CD31-positive microvessel density (MVD. Tumor VEGF expression is predictive of poor prognosis, and chemotherapy can affect the selection of angiogenic pattern. The aim of the study was to investigate the clinical and prognostic significance of VEGF and CD31 in osteosarcoma, both at diagnosis and after neoadjuvant chemotherapy, in order to identify a potential role of chemotherapy in angiogenic phenotype. Methods A retrospective analysis was performed on 16 patients with high grade osteosarcoma. In each case archival pre-treatment biopsy tissue and post-chemotherapy tumor specimens were immunohistochemically stained against CD31 and VEGF, as markers of angiogenic proliferation both in newly diagnosed primary osteosarcoma and after multidrug chemotherapy including high-dose methotrexate (HDMTX. The correlation between clinicopathological parameters and the degree of tumor VEGF and CD31 expression was statistically assessed using the χ2 test verified with Yates' test for comparison of two groups. Significance was set at p Results Expression of VEGF was positive in 11 cases/16 of cases at diagnosis. Moreover, 8 cases/16 untreated osteosarcomas were CD31-negative, but the other 8 showed an high expression of CD31. VEGF expression in viable tumor cells after neoadjuvant chemotherapy was observed in all cases; in particular, there was an increased VEGF expression (post-chemotherapy VEGF - biopsy VEGF in 11 cases/16. CD31 expression increased in 11 cases/16 and decreased in 3 cases after chemotherapy. The data relating to the change in staining following chemotherapy appear statistically significant for VEGF expression (p p > 0,05. Conclusions Even if the study included few patients, these results confirm that VEGF and CD

  15. Integrin-specific hydrogels functionalized with VEGF for vascularization and bone regeneration of critical-size bone defects.

    Science.gov (United States)

    García, José R; Clark, Amy Y; García, Andrés J

    2016-04-01

    Vascularization of bone defects is considered a crucial component to the successful regeneration of large bone defects. Although vascular endothelial growth factor (VEGF) has been delivered to critical-size bone defect models to augment blood vessel infiltration into the defect area, its potential to increase bone repair remains ambiguous. In this study, we investigated whether integrin-specific biomaterials modulate the effects of VEGF on bone regeneration. We engineered protease-degradable, VEGF-loaded poly(ethylene glycol) (PEG) hydrogels functionalized with either a triple-helical, α2 β1 integrin-specific peptide GGYGGGP(GPP)5 GFOGER(GPP)5 GPC (GFOGER) or an αv β3 integrin-targeting peptide GRGDSPC (RGD). Covalent incorporation of VEGF into the PEG hydrogel allowed for protease degradation-dependent release of the protein while maintaining VEGF bioactivity. When applied to critical-size segmental defects in the murine radius, GFOGER-functionalized VEGF-free hydrogels exhibited significantly increased vascular volume and density and resulted in a larger number of thicker blood vessels compared to RGD-functionalized VEGF-free hydrogels. VEGF-loaded RGD hydrogels increased vascularization compared to VEGF-free RGD hydrogels, but the levels of vascularization for these VEGF-containing RGD hydrogels were similar to those of VEGF-free GFOGER hydrogels. VEGF transiently increased bone regeneration in RGD hydrogels but had no effect at later time points. In GFOGER hydrogels, VEGF did not show an effect on bone regeneration. However, VEGF-free GFOGER hydrogels resulted in increased bone regeneration compared to VEGF-free RGD hydrogels. These findings demonstrate the importance of integrin-specificity in engineering constructs for vascularization and associated bone regeneration.

  16. Wnt3a upregulates transforming growth factor-β-stimulated VEGF synthesis in osteoblasts.

    Science.gov (United States)

    Natsume, Hideo; Tokuda, Haruhiko; Matsushima-Nishiwaki, Rie; Kato, Kenji; Yamakawa, Kengo; Otsuka, Takanobu; Kozawa, Osamu

    2011-07-01

    It is recognized that Wnt3a affects bone metabolism via the canonical Wnt/β-catenin signalling pathway. We have previously shown that transforming growth factor-β (TGF-β) stimulates the synthesis of vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of Wnt3a on TGF-β-stimulated VEGF synthesis in these cells. Wnt3a, which alone had little effect on the VEGF levels, significantly enhanced the TGF-β-stimulated VEGF release. Lithium chloride and SB216763, inhibitors of glycogen synthase kinase 3β, markedly amplified the TGF-β-stimulated VEGF release. Wnt3a failed to affect the TGF-β-induced phosphorylation of Smad2, p44/p42 MAP kinase, p38 MAP kinase or SAPK/JNK. Wnt3a and lithium chloride strengthened the VEGF mRNA expression induced by TGF-β. These results strongly suggest that Wnt3a upregulates VEGF synthesis stimulated by TGF-β via activation of the canonical pathway in osteoblasts.

  17. WISP-1 positively regulates angiogenesis by controlling VEGF-A expression in human osteosarcoma.

    Science.gov (United States)

    Tsai, Hsiao-Chi; Tzeng, Huey-En; Huang, Chun-Yin; Huang, Yuan-Li; Tsai, Chun-Hao; Wang, Shih-Wei; Wang, Po-Chuan; Chang, An-Chen; Fong, Yi-Chin; Tang, Chih-Hsin

    2017-04-13

    In recent years, much research has focused on the role of angiogenesis in osteosarcoma, which occurs predominantly in adolescents and young adults. The vascular endothelial growth factor-A (VEGF-A) pathway is the key regulator of angiogenesis and in osteosarcoma. VEGF-A expression has been recognized as a prognostic marker in angiogenesis. Aberrant WNT1-inducible signaling pathway protein-1 (WISP-1) expression is associated with various cancers. However, the function of WISP-1 in osteosarcoma angiogenesis is poorly understood. We demonstrate a positive correlation between WISP-1 and VEGF-A expression in human osteosarcoma. Moreover, we show that WISP-1 promotes VEGF-A expression in human osteosarcoma cells, subsequently inducing human endothelial progenitor cell (EPC) migration and tube formation. The focal adhesion kinase (FAK), Jun amino-terminal kinase (JNK), and hypoxia-inducible factor (HIF)-1α signaling pathways were activated after WISP-1 stimulation, while FAK, JNK, and HIF-1α inhibitors or small interfering RNA (siRNA) abolished WISP-1-induced VEGF-A expression and angiogenesis. In vitro and in vivo studies revealed down-regulation of microRNA-381 (miR-381) in WISP-1-induced VEGF-A expression and angiogenesis. Our findings reveal that WISP-1 enhances VEGF-A expression and angiogenesis through the FAK/JNK/HIF-1α signaling pathways, as well as via down-regulation of miR-381 expression. WISP-1 may be a promising target in osteosarcoma angiogenesis.

  18. VEGF-expressing human umbilical cord mesenchymal stem cells, an improved therapy strategy for Parkinson's disease.

    Science.gov (United States)

    Xiong, N; Zhang, Z; Huang, J; Chen, C; Zhang, Z; Jia, M; Xiong, J; Liu, X; Wang, F; Cao, X; Liang, Z; Sun, S; Lin, Z; Wang, T

    2011-04-01

    The umbilical cord provides a rich source of primitive mesenchymal stem cells (human umbilical cord mesenchymal stem cells (HUMSCs)), which have the potential for transplantation-based treatments of Parkinson's Disease (PD). Our pervious study indicated that adenovirus-associated virus-mediated intrastriatal delivery of human vascular endothelial growth factor 165 (VEGF 165) conferred molecular protection to the dopaminergic system. As both VEGF and HUMSCs displayed limited neuroprotection, in this study we investigated whether HUMSCs combined with VEGF expression could offer enhanced neuroprotection. HUMSCs were modified by adenovirus-mediated VEGF gene transfer, and subsequently transplanted into rotenone-lesioned striatum of hemiparkinsonian rats. As a result, HUMSCs differentiated into dopaminergic neuron-like cells on the basis of neuron-specific enolase (NSE) (neuronal marker), glial fibrillary acidic protein (GFAP) (astrocyte marker), nestin (neural stem cell marker) and tyrosine hydroxylase (TH) (dopaminergic marker) expression. Further, VEGF expression significantly enhanced the dopaminergic differentiation of HUMSCs in vivo. HUMSC transplantation ameliorated apomorphine-evoked rotations and reduced the loss of dopaminergic neurons in the lesioned substantia nigra (SNc), which was enhanced significantly by VEGF expression in HUMSCs. These findings present the suitability of HUMSC as a vector for gene therapy and suggest that stem cell engineering with VEGF may improve the transplantation strategy for the treatment of PD.

  19. VEGF-mediated angiogenesis stimulates neural stem cell proliferation and differentiation in the premature brain

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Jinqiao, E-mail: jinqiao1977@163.com [Institute of Pediatrics, Children' s Hospital of Fudan University (China); Sha, Bin [Department of Neonatology, Children' s Hospital of Fudan University, 399 Wanyuan Road, Shanghai 201102 (China); Zhou, Wenhao, E-mail: zhou_wenhao@yahoo.com.cn [Department of Neonatology, Children' s Hospital of Fudan University, 399 Wanyuan Road, Shanghai 201102 (China); Yang, Yi [Institute of Pediatrics, Children' s Hospital of Fudan University (China)

    2010-03-26

    This study investigated the effects of angiogenesis on the proliferation and differentiation of neural stem cells in the premature brain. We observed the changes in neurogenesis that followed the stimulation and inhibition of angiogenesis by altering vascular endothelial growth factor (VEGF) expression in a 3-day-old rat model. VEGF expression was overexpressed by adenovirus transfection and down-regulated by siRNA interference. Using immunofluorescence assays, Western blot analysis, and real-time PCR methods, we observed angiogenesis and the proliferation and differentiation of neural stem cells. Immunofluorescence assays showed that the number of vWF-positive areas peaked at day 7, and they were highest in the VEGF up-regulation group and lowest in the VEGF down-regulation group at every time point. The number of neural stem cells, neurons, astrocytes, and oligodendrocytes in the subventricular zone gradually increased over time in the VEGF up-regulation group. Among the three groups, the number of these cells was highest in the VEGF up-regulation group and lowest in the VEGF down-regulation group at the same time point. Western blot analysis and real-time PCR confirmed these results. These data suggest that angiogenesis may stimulate the proliferation of neural stem cells and differentiation into neurons, astrocytes, and oligodendrocytes in the premature brain.

  20. Expression and significance of HIF-1α and VEGF in rats with diabetic retinopathy

    Institute of Scientific and Technical Information of China (English)

    Hong-Tao Yan; Guan-Fang Su

    2014-01-01

    Objective:To investigate the expression of hypoxia inducible factor-1α(HIF-1α) and vascular endothelial growth factor(VEGF) in diabetic retinopathy(DR) rats and its effect on theDR occurrence and development.Methods:A total of120SD rats were randomly divided into trial group and control group with60 in each.STZi.p. was used in the trial group to establish theDM model, citrate buffer salt of same amount was usedi.p. to the control group.1,3 and6 months after injection, respective20 rats were sacrificed in each group to observe expression ofHIF-1α andVEGF in the rat retina tissue at different time points.Results:Expression ofHIF-1α andVEGF were negative in the control group; expression ofHIF-1α andVEGF protein in retinal tissue were weak after1 month ofDR mold formation.It showed progressive enhancement along with the progression in different organizations, differences between groups were significant (P<0.05).Conclusions:Expressions ofHIF-1α andVEGF were correlated with disease progression in early diabetic retinopathy.Retinal oxygen can induce over-expression ofHIF-1α andVEGF.It shows thatHIF-1α andVEGF play an important role in the pathogenesis ofDR.

  1. Relationsip between PTEN and VEGF Expression and Clinicopathological Characteristics in HCC

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    To investigate the expressions and significance of the tumor suppressor gene phosphatase and tensin homlog deleted on chromosome ten protein (PTEN) and vascular endothelial growth factor (VEGF) in hepatocellular carcinoma (HCC), and to analyze the relationship between their expressions and the tumor's invasion and their pericarcinomatous tissues, the correlation of their expressions with the tumor's clinicopathological characteristics and invasion potential were studied. Our study showed that the expression level of PTEN in HCC was remarkably lower than that in pericarcinomatous liver tissues, while the expressions of both VEGF and MVD were higher than that in pericarcinomatous liver tissues. Correlation analysis revealed that the expression of PTEN was negatively related to the progression of the pathological differentiation and invasion of tumor, whereas the expressions of VEGF and MVD were positively related. Moreover, there was a negative relationship between the expression of PTEN and the expressions of VEGF and MVD, and a positive one between VEGF and MVD. The expressions of PTEN and VEGF may reveal the degree of differentiation and the invasive potential of HCC tissues. The mechanism by which the lack of PTEN expression probably induces abnormal hyperexpression of VEGF may play an important role in the invasion and metastasis of HCC.

  2. 2-Deoxy Glucose Modulates Expression and Biological Activity of VEGF in a SIRT-1 Dependent Mechanism.

    Science.gov (United States)

    Kunhiraman, Haritha; Edatt, Lincy; Thekkeveedu, Sruthi; Poyyakkara, Aswini; Raveendran, Viji; Kiran, Manikantan Syamala; Sudhakaran, Perumana; Kumar, Sameer V B

    2017-02-01

    Reprogramming of energy metabolism particularly switching over of cells to aerobic glycolysis leading to accumulation of lactate is a hallmark of cancer. Lactate can induce angiogenesis, an important process underlying tumor growth and metastasis. VEGF is one of the most important cytokines which regulate this process and the present study was designed to examine if blocking glycolytic pathway in tumor cells can affect its angiogenic potency with respect to VEGF. For this, the expression and biological activity of VEGF synthesized and secreted by tumor derived cell lines in the presence or absence of 2-deoxy glucose (2-DG), an inhibitor of glycolysis was determined. The results suggested that inhibition of glycolysis using sub-lethal doses of 2-DG down-regulated the expression of VEGF and also significantly reduced its biological activity. Further mechanistic studies revealed that the down regulation of VEGF gene expression by 2-DG was due to an increase in SIRT-1 activity and the reduced biological activity was found to be due to an increase in the PAR modification of VEGF. Activity of SIRT-1 and PAR modification of VEGF in turn, was found to be correlated to the cellular NAD(+) levels. The results presented here therefore suggest that treatment of cancer cells with 2-DG can significantly reduce its overall angiogenic potency through transcriptional and post-translational mechanisms. J. Cell. Biochem. 118: 252-262, 2017. © 2016 Wiley Periodicals, Inc.

  3. Development of VEGF-loaded PLGA matrices in association with mesenchymal stem cells for tissue engineering.

    Science.gov (United States)

    Rosa, A R; Steffens, D; Santi, B; Quintiliano, K; Steffen, N; Pilger, D A; Pranke, P

    2017-08-07

    The association of bioactive molecules, such as vascular endothelial growth factor (VEGF), with nanofibers facilitates their controlled release, which could contribute to cellular migration and differentiation in tissue regeneration. In this research, the influence of their incorporation on a polylactic-co-glycolic acid (PLGA) scaffold produced by electrospinning on cell adhesion and viability and cytotoxicity was carried out in three groups: 1) PLGA/BSA/VEGF; 2) PLGA/BSA, and 3) PLGA. Morphology, fiber diameter, contact angle, loading efficiency and controlled release of VEGF of the biomaterials, among others, were measured. The nanofibers showed smooth surfaces without beads and with interconnected pores. PLGA/BSA/VEGF showed the smallest water contact angle and VEGF released for up to 160 h. An improvement in cell adhesion was observed for the PLGA/BSA/VEGF scaffolds compared to the other groups and the scaffolds were non-toxic for the cells. Therefore, the scaffolds were shown to be a good strategy for sustained delivery of VEGF and may be a useful tool for tissue engineering.

  4. Influence of anti-VEGF about cardiovascular biomarkers in age related macular degeneration.

    Science.gov (United States)

    Manresa, N; Mulero, J; Losada, M; Zafrilla, P

    2015-02-01

    Systemic VEGF inhibition disrupts endothelial homeostasis and accelerates the atherogenesis, suggesting that these events contribute to the clinical cardiovascular adverse events of VEGF-inhibiting therapies. The objective of the current study was to analyze the effect of anti-VEGF therapy on cardiovascular risk factors in patients with exudative age related macular degeneration. A total of 73 patients with exudative age related macular degeneration (without previous anti-VEGF therapy) were treated with two anti-VEGF: Ranibizumab and Pegaptanib sodium. The follow up was 6 months. The following parameters were determined before and after treatment: homocysteine, lipids (total cholesterol, triglycerides, HDL-c, LDL-c), C-Reactive Protein and fibrinogen. There were not statistically significant differences in parameters studied before and after treatment with both Pegaptanib sodium and Ranibizumab, except C-Reactive Protein. Of all patients analyzed, only 3 of them have initially C-Reactive Protein levels above normal levels and after antiangiogenic therapy, there was a significant increase in C-Reactive Protein. We have not found results in our study who to suspect that treatment with anti-VEGF in the patients with exudative age related macular degeneration increases cardiovascular risk predictors. However, after therapy was increased the CRP and fibrinogen may mean that anti-VEGF contribute an alteration of endothelial homeostasis in exudative AMD.

  5. Association of Chemerin and Vascular Endothelial Growth Factor (VEGF) with Diabetic Nephropathy

    Science.gov (United States)

    Lin, Shuhua; Teng, Jian; Li, Jixia; Sun, Fang; Yuan, Dong; Chang, Jing

    2016-01-01

    Background Diabetic nephropathy (DN) is a common complication of diabetes, caused by diabetic microvascular lesions. The pathogenesis of DN is complicated, involving genetics, physics, chemistry, and environmental factors. Chemerin is a fat cell factor that participates in regulating inflammation. Vascular endothelial growth factor (VEGF) promotes vascular endothelial cell proliferation, differentiation, and angiogenesis. The relationship role of Chemerin and VEGF in DN is not fully understood. Material/Methods SD rats were randomly divided into 2 groups: the control group and the DN group. Streptozotocin was used to construct the DN model. Serum creatinine (Scr), blood urea nitrogen (BUN), and urine microalbumin (UAlb) were detected. Real-time PCR and Western blot were used to test Chemerin and VEGF mRNA and protein expression in kidney tissue. ELISA was performed to test TGF-β1, TNF-α, and INF-γ levels. The correlation of Chemerin and VEGF with renal function and inflammatory factors was analyzed. Results DN group rats showed obviously increased Scr and BUN levels, and elevated TGF-β1, TNF-α, and INF-γ secretion (P<0.05). Compared with controls, Chemerin and VEGF were clearly overexpressed in the DN group (P<0.05). Chemerin and VEGF expression were positively correlated with inflammatory factors and renal function. Conclusions Chemerin and VEGF play important roles in DN by regulating inflammatory factors and renal function. They may be treated as indicators of DN. PMID:27612613

  6. Development of VEGF-loaded PLGA matrices in association with mesenchymal stem cells for tissue engineering

    Directory of Open Access Journals (Sweden)

    A.R. Rosa

    Full Text Available The association of bioactive molecules, such as vascular endothelial growth factor (VEGF, with nanofibers facilitates their controlled release, which could contribute to cellular migration and differentiation in tissue regeneration. In this research, the influence of their incorporation on a polylactic-co-glycolic acid (PLGA scaffold produced by electrospinning on cell adhesion and viability and cytotoxicity was carried out in three groups: 1 PLGA/BSA/VEGF; 2 PLGA/BSA, and 3 PLGA. Morphology, fiber diameter, contact angle, loading efficiency and controlled release of VEGF of the biomaterials, among others, were measured. The nanofibers showed smooth surfaces without beads and with interconnected pores. PLGA/BSA/VEGF showed the smallest water contact angle and VEGF released for up to 160 h. An improvement in cell adhesion was observed for the PLGA/BSA/VEGF scaffolds compared to the other groups and the scaffolds were non-toxic for the cells. Therefore, the scaffolds were shown to be a good strategy for sustained delivery of VEGF and may be a useful tool for tissue engineering.

  7. PGC-1alpha mediates exercise-induced skeletal muscle VEGF expression in mice

    DEFF Research Database (Denmark)

    Leick, Lotte; Hellsten, Ylva; Fentz, Joachim

    2009-01-01

    The aim of the present study was to test the hypothesis that PGC-1alpha is required for exercise-induced VEGF expression in both young and old mice and that AMPK activation leads to increased VEGF expression through a PGC-1alpha-dependent mechanism. Whole body PGC-1alpha knockout (KO) and litterm......The aim of the present study was to test the hypothesis that PGC-1alpha is required for exercise-induced VEGF expression in both young and old mice and that AMPK activation leads to increased VEGF expression through a PGC-1alpha-dependent mechanism. Whole body PGC-1alpha knockout (KO......) and littermate wild-type (WT) mice were submitted to either 1) 5 wk of exercise training, 2) lifelong (from 2 to 13 mo of age) exercise training in activity wheel, 3) a single exercise bout, or 4) 4 wk of daily subcutaneous AICAR or saline injections. In skeletal muscle of PGC-1alpha KO mice, VEGF protein...... expression was approximately 60-80% lower and the capillary-to-fiber ratio approximately 20% lower than in WT. Basal VEGF mRNA expression was similar in WT and PGC-1alpha KO mice, but acute exercise and AICAR treatment increased the VEGF mRNA content in WT mice only. Exercise training of young mice increased...

  8. Rapamycin Inhibits Proliferation of Hemangioma Endothelial Cells by Reducing HIF-1-Dependent Expression of VEGF

    Science.gov (United States)

    Medici, Damian; Olsen, Bjorn R.

    2012-01-01

    Hemangiomas are tumors formed by hyper-proliferation of vascular endothelial cells. This is caused by elevated vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2 (VEGFR2). Here we show that elevated VEGF levels produced by hemangioma endothelial cells are reduced by the mTOR inhibitor rapamycin. mTOR activates p70S6K, which controls translation of mRNA to generate proteins such as hypoxia inducible factor-1 (HIF-1). VEGF is a known HIF-1 target gene, and our data show that VEGF levels in hemangioma endothelial cells are reduced by HIF-1α siRNA. Over-expression of HIF-1α increases VEGF levels and endothelial cell proliferation. Furthermore, both rapamycin and HIF-1α siRNA reduce proliferation of hemangioma endothelial cells. These data suggest that mTOR and HIF-1 contribute to hemangioma endothelial cell proliferation by stimulating an autocrine loop of VEGF signaling. Furthermore, mTOR and HIF-1 may be therapeutic targets for the treatment of hemangiomas. PMID:22900063

  9. PGE1 analog alprostadil induces VEGF and eNOS expression in endothelial cells.

    Science.gov (United States)

    Haider, Dominik G; Bucek, Robert A; Giurgea, Aura G; Maurer, Gerald; Glogar, Helmut; Minar, Erich; Wolzt, Michael; Mehrabi, Mohammad R; Baghestanian, Mehrdad

    2005-11-01

    Endothelial nitric oxide synthase (eNOS), VEGF, and hypoxia-inducible factor 1-alpha (HIF-1alpha) are important regulators of endothelial function, which plays a role in the pathophysiology of heart failure (HF). PGE1 analog treatment in patients with HF elicits beneficial hemodynamic effects, but the precise mechanisms have not been investigated. We have investigated the effects of the PGE1 analog alprostadil on eNOS, VEGF, and HIF-1alpha expression in human umbilical vein endothelial cells (HUVEC) using RT-PCR and immunoblotting under normoxic and hypoxic conditions. In addition, we studied protein expression by immunohistochemical staining in explanted hearts from patients with end-stage HF, treated or untreated with systemic alprostadil. Alprostadil causes an upregulation of eNOS and VEGF protein and mRNA expression in HUVEC and decreases HIF-1alpha. Hypoxia potently increased eNOS, VEGF, and HIF-1alpha synthesis. The alprostadil-induced upregulation of eNOS and VEGF was prevented by inhibition of MAPKs with PD-98056 or U-0126. Consistently, the expression of eNOS and VEGF was increased, and HIF-1alpha was reduced in failing hearts treated with alprostadil. The potent effects of alprostadil on endothelial VEGF and eNOS synthesis may be useful for patients with HF where endothelial dysfunction is involved in the disease process.

  10. VEGF165-induced vascular permeability requires NRP1 for ABL-mediated SRC family kinase activation

    Science.gov (United States)

    Lampropoulou, Anastasia; Senatore, Valentina; Brash, James T.; Liyanage, Sidath E.; Raimondi, Claudio; Bainbridge, James W.

    2017-01-01

    The vascular endothelial growth factor (VEGF) isoform VEGF165 stimulates vascular growth and hyperpermeability. Whereas blood vessel growth is essential to sustain organ health, chronic hyperpermeability causes damaging tissue edema. By combining in vivo and tissue culture models, we show here that VEGF165-induced vascular leakage requires both VEGFR2 and NRP1, including the VEGF164-binding site of NRP1 and the NRP1 cytoplasmic domain (NCD), but not the known NCD interactor GIPC1. In the VEGF165-bound receptor complex, the NCD promotes ABL kinase activation, which in turn is required to activate VEGFR2-recruited SRC family kinases (SFKs). These results elucidate the receptor complex and signaling hierarchy of downstream kinases that transduce the permeability response to VEGF165. In a mouse model with choroidal neovascularisation akin to age-related macular degeneration, NCD loss attenuated vessel leakage without affecting neovascularisation. These findings raise the possibility that targeting NRP1 or its NCD interactors may be a useful therapeutic strategy in neovascular disease to reduce VEGF165-induced edema without compromising vessel growth. PMID:28289053

  11. Expression of Protein Slit2, VEGF and VEGF -C Correlates with Clinical Study of Colorectal Cancers%Slit2、VEGF和VEGF-C蛋白表达与大肠癌关系的临床研究

    Institute of Scientific and Technical Information of China (English)

    王琴伊; 卢雪峰; 季锐; 杨淑萍

    2010-01-01

    研究神经迁移因子(Slit2)、血管生成因子(VEGF、VEGF-C)在大肠癌中的表达及临床意义.检测60例大肠癌组织和36例癌旁组织中Slit2、VEGF和VEGF-C的表达水平.结果三者在大肠癌和癌旁组织中的表达水平均与肿瘤大小、浸润深度、TNM分期、淋巴结转移相关;VEGF和VEGF-C表达水平还与远处转移相关.结果提示Slit2是调节大肠癌血管生成的重要因子.

  12. Effect of recombinant human endostatin on endometriosis in mice

    Institute of Scientific and Technical Information of China (English)

    JIANG Hong-qing; LI Ya-li; ZOU Jie

    2007-01-01

    Background Direct and indirect evidences have suggested that angiogenesis is a prerequisite for the development of endometriosis. Aiming at offering experimental evidences for anti-angiogenesis therapy, we transplanted the eutopic endometrium from patient with endometriosis into the severe combined immunodeficiency disease (SCID) mice, to evaluate the effect of the endostatin on the growth and angiogenesis of the established endometriosis lesions in SCID mice model.Methods Eutopic endometrium of women with endometriosis was transplanted into the SCID mice. The mice were randomized into treatment (n=10) and control groups (n=10). Two weeks after the implantation of endometrium fragment,whereas the control group received equivalent volume of PBS (200 μl/d). The volume of endometriotic lesions in SCID mice was measured every three days, and all the treatment lasted for 14 days. Immunohistochemistry was used to determine microvessel density (MVD) and the expression of VEGF. The results were analyzed by t test and X2 test to value the treating effect.Results Compared with the control group, growth of endometriosis lesion was reduced in the mice treated with YH-16.Statistically significant differences in the volume and weight of the ectopic lesions were observed between the treatment and the control groups (P<0.05). Microscopical examination showed that after being treated with YH-16, the volume of the endometrial tissues decreased, the glands depauperated, and the glandular epithelium partially degenerated.Necrotic debris was observed in the endometrial stroma. MVD and expression of VEGF in the treatment group were significantly lower than those in the control group (P<0.05).Conclusions Recombinant human endostatin affects the maintenance and growth of endometriotic tissues by inhibiting angiogenesis and reducing the expression of VEGF in ectopic lesion. The angiostatic agent may be promising as a therapy for endometriosis.

  13. Rho-kinase limits FGF-2-stimulated VEGF release in osteoblasts.

    Science.gov (United States)

    Natsume, Hideo; Tokuda, Haruhiko; Adachi, Seiji; Takai, Shinji; Matsushima-Nishiwaki, Rie; Kato, Kenji; Minamitani, Chiho; Niida, Shunpei; Mizutani, Jun; Kozawa, Osamu; Otsuka, Takanobu

    2010-04-01

    We previously reported that basic fibroblast growth factor (FGF-2) stimulates the release of vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells and that FGF-2-activated p38 MAP kinase negatively regulates the VEGF release in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether Rho-kinase is involved in FGF-2-stimulated VEGF release in MC3T3-E1 cells. FGF-2 induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a substrate of Rho-kinase. Y27632, a specific inhibitor of Rho-kinase, which attenuated the MYPT-1 phosphorylation, significantly enhanced the FGF-2-stimulated VEGF release. Fasudil, another Rho-kinase inhibitor, also amplified the VEGF release. FGF-2 significantly stimulated VEGF accumulation and fasudil enhanced FGF-2-stimulated VEGF accumulation also in whole cell lysates. Neither Y27632 nor fasudil affected the phosphorylation levels of p44/p42 MAP kinase or p38 MAP kinase. Y27632 and fasudil markedly strengthened the FGF-2-induced phosphorylation of SAPK/JNK. Y27632 as well as fasudil enhanced FGF-2-stimulated VEGF release and Y27632 enhanced the FGF-2-induced phosphorylation levels of SAPK/JNK also in human osteoblasts. These results strongly suggest that Rho-kinase negatively regulates FGF-2-stimulated VEGF release in osteoblasts.

  14. A nanobody directed to a functional epitope on VEGF, as a novel strategy for cancer treatment

    Energy Technology Data Exchange (ETDEWEB)

    Farajpour, Zahra [Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran (Iran, Islamic Republic of); Rahbarizadeh, Fatemeh, E-mail: rahbarif@modares.ac.ir [Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Kazemi, Bahram [Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran (Iran, Islamic Republic of); Ahmadvand, Davoud [School of Allied Medical Sciences, Iran University of Medical Sciences, Tehran (Iran, Islamic Republic of)

    2014-03-28

    Highlights: • A novel nanobody directed to antigenic regions on VEGF was identified. • Our nanobody was successfully purified. • Our nanobody significantly inhibited VEGF-induced proliferation of HUVECs in a dose dependent manner. - Abstract: Compelling evidence suggests that vascular endothelial growth factor (VEGF), due to its essential role in angiogenesis, is a critical target for cancer treatment. Neutralizing monoclonal antibodies against VEGF are important class of drugs used in cancer therapy. However, the cost of production, large size, and immunogenicity are main drawbacks of conventional monoclonal therapy. Nanobodies are the smallest antigen-binding antibody fragments, which occur naturally in camelidae. Because of their remarkable features, we decided to use an immune library of nanobody to direct phage display to recognition of novel functional epitopes on VEGF. Four rounds of selection were performed and six phage-displayed nanobodies were obtained from an immune phage library. The most reactive clone in whole-cell ELISA experiments, was purified and assessed in proliferation inhibition assay. Purified ZFR-5 not only blocked interaction of VEGF with its receptor in cell ELISA experiments, but also was able to significantly inhibit proliferation response of human umbilical vein endothelial cells to VEGF in a dose-dependent manner. Taken together, our study demonstrates that by using whole-cell ELISA experiments, nanobodies against antigenic regions included in interaction of VEGF with its receptors can be directed. Because of unique and intrinsic properties of a nanobody and the ability of selected nanobody for blocking the epitope that is important for biological function of VEGF, it represents novel potential drug candidate.

  15. Acupuncture promotes angiogenesis after myocardial ischemia through H3K9 acetylation regulation at VEGF gene.

    Directory of Open Access Journals (Sweden)

    Shu-Ping Fu

    Full Text Available BACKGROUND: Acupuncture exerts cardioprotective effects on several types of cardiac injuries, especially myocardial ischemia (MI, but the mechanisms have not yet been well elucidated. Angiogenesis mediated by VEGF gene expression and its modification through histone acetylation has been considered a target in treating myocardial ischemia. This study aims to exam whether modulation of angiogenesis through H3K9 acetylation regulation at VEGF gene is one possible cardioprotective mechanism of acupuncture. RESULTS: We generated rat MI models by ligating the left anterior descending coronary artery and applied electroacupuncture (EA treatment at the Neiguan (PC6 acupoint. Our results showed that acupuncture reversed the S-T segment change, reduced Q-wave area, decreased CK, CK-MB, LDH levels, mitigated myocardial remodeling, and promoted microvessel formation in the MI heart. RNA-seq analysis showed that VEGF-induced angiogenesis signaling was involved in the modulation of EA. Western blot results verified that the protein expressions of VEGF, Ras, phospho-p44/42 MAPK, phospho-p38 MAPK, phospho-SAPK/JNK and Akt, were all elevated significantly by EA treatment in the MI heart. Furthermore, increased H3K9 acetylation was also observed according with the VEGF. ChIP assay confirmed that EA treatment could notably stimulate the recruitment of H3K9ace at the VEGF promoter. CONCLUSIONS: Our study demonstrates for the first time that acupuncture can effectively up-regulate VEGF expression through H3K9 acetylation modification directly at the VEGF promoter and hence activate VEGF-induced angiogenesis in rat MI models. We employed high throughput sequencing in this study and, for the first time, generated genome-wide gene expression profiles both in the rat MI model and in acupuncture treatment.

  16. VEGF-expressing Bone Marrow Mesenchymal Stem Cells Transplantation Improved Heart Function of Myocardial Infarct Rabbits

    Institute of Scientific and Technical Information of China (English)

    Sheng Xiaogang; Song Hui; Feng Jianzhang; Chen Qiuxiong; Wu Shulin

    2006-01-01

    Objectives To treat myocardial infarction with MSCs transplantation combined with VEGF gene therapy in rabbits and to study its mechanisms. Methods Forty-eight rabbits were randomly divided into MI group (n=12), MSCs group (n=12), VEGF group (n=12), MSCs+VEGF group (M+V group, n=12). Rabbit myocardial infarction models were founded by the ligation of left anterior descending artery. 107 MSCs were injected into the infarct-zone in four sites 2 weeks later in MSCs and M+V group. phVEGF gene were injected in infarct-zone in VEGF group and MSCs transfected with phVEGF gene were injected in M+V group. Heart function including LVEDP, LVSP, LVDP, -dp/dtmax, +dp/dtmax, were measured in vivo. The hearts were harvested at 4 weeks after transplantation and sectioned for HE stain,immunohistochemical stain of BrdU and Ⅷ factor antigen. Results The left ventricular hemodynamics parameters showed that heart function were improved more in M+V group than MSCs group, MI group and VEGF group. The numbers of BrdU positive cells in M+V group(61±8)were more than in MSCs group (44±8,P<0.01). The numbers of vessels in infarcted zone were more in M+V group (49±8) than in MSCs group (33±6, P<0.01)、VEGF group(30±8,P<0.01)and MI group (18±4,P<0.01). Conclusions VEGF-expressing MSCs transplantation could improve heart function after myocardial infarction, and they were more effective than sole MSCs transplantation. Keeping more MSCs survival and ameliorating the blood supply of infarct-zone might be involved in the mechanisms.

  17. Quantitative determination of VEGF165 in cell culture medium by aptamer sandwich based chemiluminescence assay.

    Science.gov (United States)

    Shan, Siwen; He, Ziyi; Mao, Sifeng; Jie, Mingsha; Yi, Linglu; Lin, Jin-Ming

    2017-08-15

    In this work, we have developed a sensitive and selective chemiluminescence (CL) assay for vascular endothelial growth factor (VEGF165) quantitative detection based on two specific VEGF165 binding aptamers (Apt). VEGF is a predominant biomarker in cancer angiogenesis, and sensitive detection method of VEGF are highly demanded for both academic study and clinical diagnosis of multiple cancers. In our experiment, VEGF165 was captured in a sandwich structure assembled by two binding aptamers, one capture aptamer was immobilized on streptavidin-coated magnetic beads (MBs) and another VEGF-binding aptamer was labeled by biotin for further phosphatase conjunction. After Apt-VEGF-Apt sandwich was formed on MBs surface, alkaline phosphatase (ALP) was modified to the second aptamer to catalyze CL reaction. By applying 4-methoxy-4-(3-phosphatephenyl)-spiro-(1,2-dioxetane-3,2-adamantane) (AMPPD) as CL substrate, strong signal intensity was achieved. VEGF165 content as low as 1ng/mL was detected in standard spiked samples by our assay, and linear range of working curve was confirmed from 1 to 20ng/mL. Then our method was successfully applied for cell culture medium analysis and on-chip hypoxic HepG2-HUVEC co-culture model study with excellent accuracy equal to ELISA Kit. Our developed assay demonstrated an outstanding performance in VEGF165 quantification and may be further extended to clinical testing of important biomarkers as well as probing microchip cell culture model. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. A new key player in VEGF-dependent angiogenesis in human hepatocellular carcinoma: dimethylarginine dimethylaminohydrolase 1.

    Science.gov (United States)

    Buijs, Nikki; Oosterink, J Efraim; Jessup, Morgan; Schierbeek, Henk; Stolz, Donna B; Houdijk, Alexander P; Geller, David A; van Leeuwen, Paul A

    2017-07-24

    Anti-angiogenic therapies, targeting VEGF, are a promising treatment for hepatocellular carcinoma (HCC). To enhance this potential therapy, identification of novel targets in this pathway is of major interest. Nitric oxide (NO) plays a crucial role in VEGF-dependent angiogenesis. NO production depends on arginine as substrate and asymmetric dimethylarginine (ADMA) as inhibitor. Dimethylarginine dimethylaminohydrolase 1 (DDAH-1) catabolizes ADMA and therefore regulates NO and VEGF expression. This study unravels additional mechanisms to improve VEGF targeting therapies. The expression of DDAH-1 was examined in HCC specimen and non-tumorous background liver of 20 patients undergoing liver resection. Subsequently, arginine/ADMA balance, NO production, and VEGF expression were analyzed. The influence of hypoxia on DDAH-1 and angiogenesis promoting factors was evaluated in HepG2 cells and primary human hepatocytes. DDAH-1 expression was significantly induced in primary HCC tumors compared to non-tumorous background liver. This was associated with an increased arginine/ADMA ratio, higher NO formation, and higher VEGF expression in human HCC compared to non-tumorous liver. Hypoxia induced DDAH-1, iNOS, and VEGF expression in a time-dependent manner in HepG2 cells. Our results indicate that DDAH-1 expression is increased in human HCC, which is associated with an increase in the arginine/ADMA ratio and enhanced NO formation. Hypoxia may be an initiating factor for the increase in DDAH-1 expression. DDAH-1 expression is associated with promotion of angiogenesis stimulating factor VEGF. Together, our findings for the first time identified DDAH-1 as a key player in the regulation of angiogenesis in human HCC, and by understanding this mechanism, future therapeutic strategies targeting VEGF can be improved.

  19. The angio-fibrotic switch of VEGF and CTGF in proliferative diabetic retinopathy.

    Directory of Open Access Journals (Sweden)

    Esther J Kuiper

    Full Text Available BACKGROUND: In proliferative diabetic retinopathy (PDR, vascular endothelial growth factor (VEGF and connective tissue growth factor (CTGF cause blindness by neovascularization and subsequent fibrosis, but their relative contribution to both processes is unknown. We hypothesize that the balance between levels of pro-angiogenic VEGF and pro-fibrotic CTGF regulates angiogenesis, the angio-fibrotic switch, and the resulting fibrosis and scarring. METHODS/PRINCIPAL FINDINGS: VEGF and CTGF were measured by ELISA in 68 vitreous samples of patients with proliferative DR (PDR, N = 32, macular hole (N = 13 or macular pucker (N = 23 and were related to clinical data, including degree of intra-ocular neovascularization and fibrosis. In addition, clinical cases of PDR (n = 4 were studied before and after pan-retinal photocoagulation and intra-vitreal injections with bevacizumab, an antibody against VEGF. Neovascularization and fibrosis in various degrees occurred almost exclusively in PDR patients. In PDR patients, vitreous CTGF levels were significantly associated with degree of fibrosis and with VEGF levels, but not with neovascularization, whereas VEGF levels were associated only with neovascularization. The ratio of CTGF and VEGF was the strongest predictor of degree of fibrosis. As predicted by these findings, patients with PDR demonstrated a temporary increase in intra-ocular fibrosis after anti-VEGF treatment or laser treatment. CONCLUSIONS/SIGNIFICANCE: CTGF is primarily a pro-fibrotic factor in the eye, and a shift in the balance between CTGF and VEGF is associated with the switch from angiogenesis to fibrosis in proliferative retinopathy.

  20. Acupuncture promotes angiogenesis after myocardial ischemia through H3K9 acetylation regulation at VEGF gene.

    Science.gov (United States)

    Fu, Shu-Ping; He, Su-Yun; Xu, Bin; Hu, Chen-Jun; Lu, Sheng-Feng; Shen, Wei-Xing; Huang, Yan; Hong, Hao; Li, Qian; Wang, Ning; Liu, Xuan-Liang; Liang, Fanrong; Zhu, Bing-Mei

    2014-01-01

    Acupuncture exerts cardioprotective effects on several types of cardiac injuries, especially myocardial ischemia (MI), but the mechanisms have not yet been well elucidated. Angiogenesis mediated by VEGF gene expression and its modification through histone acetylation has been considered a target in treating myocardial ischemia. This study aims to exam whether modulation of angiogenesis through H3K9 acetylation regulation at VEGF gene is one possible cardioprotective mechanism of acupuncture. We generated rat MI models by ligating the left anterior descending coronary artery and applied electroacupuncture (EA) treatment at the Neiguan (PC6) acupoint. Our results showed that acupuncture reversed the S-T segment change, reduced Q-wave area, decreased CK, CK-MB, LDH levels, mitigated myocardial remodeling, and promoted microvessel formation in the MI heart. RNA-seq analysis showed that VEGF-induced angiogenesis signaling was involved in the modulation of EA. Western blot results verified that the protein expressions of VEGF, Ras, phospho-p44/42 MAPK, phospho-p38 MAPK, phospho-SAPK/JNK and Akt, were all elevated significantly by EA treatment in the MI heart. Furthermore, increased H3K9 acetylation was also observed according with the VEGF. ChIP assay confirmed that EA treatment could notably stimulate the recruitment of H3K9ace at the VEGF promoter. Our study demonstrates for the first time that acupuncture can effectively up-regulate VEGF expression through H3K9 acetylation modification directly at the VEGF promoter and hence activate VEGF-induced angiogenesis in rat MI models. We employed high throughput sequencing in this study and, for the first time, generated genome-wide gene expression profiles both in the rat MI model and in acupuncture treatment.

  1. TFF3 mediated induction of VEGF via hypoxia in human gastric cancer SGC-7901 cells.

    Science.gov (United States)

    Guleng, Bayasi; Han, Jia; Yang, Jin-Qiu; Huang, Qing-Wen; Huang, Jian-Kun; Yang, Xiao-Ning; Liu, Jing-Jing; Ren, Jian-Lin

    2012-04-01

    Increasing evidence indicates that in gastric epithelial cells, induction of TFF3 by hypoxia is mediated by HIF-1. Since VEGF is one of the most important angiogenic factors on cancer progression, we have started to investigate the possible link among HIF-1α, VEGF, and TFF3 in gastric cancer cells. We induced the hypoxic condition in SGC-7901cells using hypoxia-mimetic agent of CoCI2. SGC7901 cells were transfected with pcPUR + U6 plasmid carrying RNAi targeted to human TFF3 and selected puromycin-resistant pools to establish the stable knockdown of TFF3 cells. Our results showed the induction of HIF-1a via hypoxia and consequences of increased expressions of the TFF3 and VEGF in gastric cancer SGC-7901 cells. Overexpression of TFF3 upregulated the mRNA expressions of VEGF and HIF-1a induced by hypoxia, and stable knockdown of TFF3 impaired the mRNA upregulations of VEGF and HIF-1a induced by hypoxia. Furthermore, knockdown of TFF3 reduced the VEGF protein secretion: as VEGF secretion was increased time dependent manner in response to the hypoxia induction in TFF3-WT cells; however, VEGF production was significantly decreased in TFF3-KD cells (621 ± 89 vs. 264 ± 73 at 6 h and 969 ± 97 vs. 508 ± 69 at 12 h, P TFF3 mediated regulation of VEGF expression induced by hypoxia, and implicated that TFF3 might be applied as a potential anti-angiogenic target for treatment of gastric cancer.

  2. Estimation of Immunohistochemical Expression of VEGF in Ductal Carcinomas of the Breast

    DEFF Research Database (Denmark)

    Maae, Else; Nielsen, Martin; Dahl Steffensen, Karina

    2011-01-01

    whole tumor sections for VEGF-A, -B, and VEGFR-1 of invasive ductal carcinomas of the breast and scored the tumors manually with staining intensity as the only parameter and by a combination of qualitative and quantitative information. We also introduce an automated method for analyzing VEGF expression...... delineation of the tumor area. We found that the AI scores were correlated to the manual scoring of VEGF intensity, but reproducibility of manual IHC scores was rather poor. The AI scores were reproducible and the restricted analysis of 25% of the tumor area at 5x magnifications was the most efficient...

  3. Inhibition of VEGF: a novel mechanism to control angiogenesis by Withania somnifera's key metabolite Withaferin A.

    Science.gov (United States)

    Saha, Sanjib; Islam, Md Khirul; Shilpi, Jamil A; Hasan, Shihab

    2013-01-01

    Angiogenesis, or new blood vessel formation from existing one, plays both beneficial and detrimental roles in living organisms in different aspects. Vascular endothelial growth factor (VEGF), a signal protein, well established as key regulator of vasculogenesis and angiogenesis. VEGF ensures oxygen supply to the tissues when blood supply is not adequate, or tissue environment is in hypoxic condition. Limited expression of VEGF is necessary, but if it is over expressed, then it can lead to serious disease like cancer. Cancers that have ability to express VEGF are more efficient to grow and metastasize because solid cancers cannot grow larger than a limited size without adequate blood and oxygen supply. Anti-VEGF drugs are already available in the market to control angiogenesis, but they are often associated with severe side-effects like fetal bleeding and proteinuria in the large number of patients. To avoid such side-effects, new insight is required to find potential compounds as anti-VEGF from natural sources. In the present investigation, molecular docking studies were carried out to find the potentiality of Withaferin A, a key metabolite of Withania somnifera, as an inhibitor of VEGF. Molecular Docking studies were performed in DockingServer and SwissDock. Bevacizumab, a commercial anti-VEGF drug, was used as reference to compare the activity of Withaferin A. X-ray crystallographic structure of VEGF, was retrieved from Protein Data Bank (PDB), and used as drug target protein. Structure of Withaferin A and Bevacizumab was obtained from PubChem and ZINC databases. Molecular visualization was performed using UCSF Chimera. Withaferin A showed favorable binding with VEGF with low binding energy in comparison to Bevacizumab. Molecular Docking studies also revealed potential protein-ligand interactions for both Withaferin A and Bevacizumab. Conclusively our results strongly suggest that Withaferin A is a potent anti-VEGF agent as ascertained by its potential

  4. Direct evidence that the VEGF-specific antibody bevacizumab has antivascular effects in human rectal cancer

    Science.gov (United States)

    Willett, Christopher G; Boucher, Yves; di Tomaso, Emmanuelle; Duda, Dan G; Munn, Lance L; Tong, Ricky T; Chung, Daniel C; Sahani, Dushyant V; Kalva, Sanjeeva P; Kozin, Sergey V; Mino, Mari; Cohen, Kenneth S; Scadden, David T; Hartford, Alan C; Fischman, Alan J; Clark, Jeffrey W; Ryan, David P; Zhu, Andrew X; Blaszkowsky, Lawrence S; Chen, Helen X; Shellito, Paul C; Lauwers, Gregory Y; Jain, Rakesh K

    2009-01-01

    The effects of vascular endothelial growth factor (VEGF) blockade on the vascular biology of human tumors are not known. Here we show here that a single infusion of the VEGF-specific antibody bevacizumab decreases tumor perfusion, vascular volume, microvascular density, interstitial fluid pressure and the number of viable, circulating endothelial and progenitor cells, and increases the fraction of vessels with pericyte coverage in rectal carcinoma patients. These data indicate that VEGF blockade has a direct and rapid antivascular effect in human tumors. PMID:14745444

  5. The VEGF system and tie-2 are spatio-temporal expressed during tayassu placentation

    DEFF Research Database (Denmark)

    Miglino, M.A.; Santos, T.C.; Papa, P.C.

    for immunhistochemistry using polyclonal antibodies against VEGF, VEGFR-1, VEGFR-2 and Tie-2. Results: In the present study the VEGF-system exhibited intense staining in the uterine epithelium, uterine glandular epithelium and trophoblast. The endothelial cells and smooth muscle cells of the vessels in the maternal...... and fetal compartment were also immunoreactive. Placentae from initial phase of gestation showed weaker staining when compared to middle and late gestation. In late gestation of the tayassu the Tie-2 was co-localized with the VEGF-system in both maternal (intense staining in glandular and uterine epithelium...

  6. Relationship between the Expression of VEGF, FIk-1 and Fit-1 Proteins and Clinicopcrthology in Hepatocellular Carcinoma

    Institute of Scientific and Technical Information of China (English)

    JihuiHao; HuikaiLi; YuQin; QiangLi; DianchangWang; XishanHao

    2004-01-01

    OBJECTIVE To study the relationship between the expression of VEGF, FIk-1 and Fit-1 proteins and clinical pathology in hepatocellular carcinoma.METHODS The expression of VEGF, FIk-1 and Fit-1 proteins in hepatocellular carcinomas from 60 patients was determined by immunohistochemistry (ABC method) and VEGF expression in relation to the clinicopathology evaluated.RESULTS The positive rates of VEGF, FIk-1 and Fit-1 protein expression were 81.3%, 88.3%, 80.0% in tumor tissues, respectively, rates which were significantly higher than those in normal liver tissue (P<0.05). The expression of VEGF protein was correlated with the histologic grade and metastases of the tumors.CONCLUSION The results showed that, in hepatocellular carcinoma, a higher expression of VEGF protein was associated with a higher degree of malignancy and a greater tendency for metastases. VEGF, FIk-1 and Fit-1 play an important role in tumourgenesis.

  7. Novel intragenotype recombination in sapovirus.

    Science.gov (United States)

    Phan, Tung Gia; Yan, Hainian; Khamrin, Pattara; Quang, Trinh Duy; Dey, Shuvra Kanti; Yagyu, Fumihiro; Okitsu, Shoko; Müller, Werner E G; Ushijima, Hiroshi

    2006-01-01

    Based on the genetic analysis, a novel, naturally occurring recombination between two distinct sapovirus subtypes (subtype a and subtype b) within genogroup I genotype 1 was identified. Breakpoint analysis of recombinant sapovirus showed that the recombination site was at the polymerase-capsid junction. This is the first report of the existence of acute gastroenteritis caused by intragenotype recombinant sapovirus. The results also provided evidence that the natural recombination occurs not only in sapovirus genogroup II but also in sapovirus genogroup I.

  8. Vascular Endothelial Growth Factor (VEGF) Bioavailability Regulates Angiogenesis and Intestinal Stem and Progenitor Cell Proliferation during Postnatal Small Intestinal Development

    Science.gov (United States)

    Holoyda, Kathleen A.; Hou, Xiaogang; Fowler, Kathryn L.; Grikscheit, Tracy C.

    2016-01-01

    Background Vascular endothelial growth factor (VEGF) is a highly conserved, master regulatory molecule required for endothelial cell proliferation, organization, migration and branching morphogenesis. Podocoryne carnea and drosophila, which lack endothelial cells and a vascular system, express VEGF homologs, indicating potential roles beyond angiogenesis and vasculogenesis. The role of VEGF in the development and homeostasis of the postnatal small intestine is unknown. We hypothesized regulating VEGF bioavailability in the postnatal small intestine would exhibit effects beyond the vasculature and influence epithelial cell stem/progenitor populations. Methods VEGF mutant mice were created that overexpressed VEGF in the brush border of epithelium via the villin promotor following doxycycline treatment. To decrease VEGF bioavailability, sFlt-1 mutant mice were generated that overexpressed the soluble VEGF receptor sFlt-1 upon doxycycline administration in the intestinal epithelium. Mice were analyzed after 21 days of doxycycline administration. Results Increased VEGF expression was confirmed by RT-qPCR and ELISA in the intestine of the VEGF mutants compared to littermates. The VEGF mutant duodenum demonstrated increased angiogenesis and vascular leak as compared to littermate controls. The VEGF mutant duodenum revealed taller villi and increased Ki-67-positive cells in the transit-amplifying zone with reduced Lgr5 expression. The duodenum of sFlt-1 mutants revealed shorter villi and longer crypts with reduced proliferation in the transit-amplifying zone, reduced expression of Dll1, Bmp4 and VE-cadherin, and increased expression of Sox9 and EphB2. Conclusions Manipulating VEGF bioavailability leads to profound effects on not only the intestinal vasculature, but epithelial stem and progenitor cells in the intestinal crypt. Elucidation of the crosstalk between VEGF signaling in the vasculature, mesenchyme and epithelial stem/progenitor cell populations may direct future

  9. Autocrine regulation of glioblastoma cell cycle progression, viability and radioresistance through the VEGF-VEGFR2 (KDR) interplay.

    Science.gov (United States)

    Knizetova, Petra; Ehrmann, Jiri; Hlobilkova, Alice; Vancova, Iveta; Kalita, Ondrej; Kolar, Zdenek; Bartek, Jiri

    2008-08-15

    Vascular endothelial growth factor (VEGF) plays a crucial role in angiogenesis and progression of malignant brain tumors. Given the significance of tumor microenvironment in general, and the established role of paracrine VEGF signaling in glioblastoma (GBM) biology in particular, we explored the potential autocrine control of human astrocytoma behavior by VEGF. Using a range of cell and molecular biology approaches to study a panel of astrocytoma (grade III and IV/GBM)-derived cell lines and a series of clinical specimens from low- and high-grade astrocytomas, we show that co-expression of VEGF and VEGF receptors (VEGFRs) occurs commonly in astrocytoma cells. We found VEGF secretion and VEGF-induced biological effects (modulation of cell cycle progression and enhanced viability of glioblastoma cells) to function in an autocrine manner. Morevover, we demonstrated that the autocrine VEGF signaling is mediated via VEGFR2 (KDR), and involves co-activation of the c-Raf/MAPK, PI3K/Akt and PLC/PKC pathways. Blockade of VEGFR2 by the selective inhibitor (SU1498) abrogated the VEGF-mediated enhancement of astrocytoma cell growth and viability under unperturbed culture conditions. In addition, such interference with VEGF-VEGFR2 signaling potentiated the ionizing radiation-induced tumor cell death. In clinical specimens, both VEGFRs and VEGF were co-expressed in astroglial tumor cells, and higher VEGF expression correlated with tumor progression, thereby supporting the relevance of functional VEGF-VEGFR signaling in vivo. Overall, our results are consistent with a potential autocrine role of the VEGF-VEGFR2 (KDR) interplay as a factor contributing to malignant astrocytoma growth and radioresistance, thereby supporting the candidacy of this signaling cascade as a therapeutic target, possibly in combination with radiotherapy.

  10. Expression of VEGF and neural repair after alprostadil treatment in a rat model of sciatic nerve crush injury

    Directory of Open Access Journals (Sweden)

    Tang Jinrong

    2009-01-01

    Full Text Available Background: Vasoactive drug alprostadil improves microcirculation and can be effective in treating disorders of peripheral nerves. Vascular endothelial growth factor (VEGF has been shown to have protective action in cerebral ischemia, disorders of spinal cord, and also peripheral nerves. However, the mechanism of action of VEGF in peripheral nerve injuries is uncertain. Objectives: To study the effect of application of alprostadil on the pathological and functional repair of crush nerve injuries and also the expression of VEGF. Materials and Methods: Rat sciatic nerves were crushed by pincers to establish the model of crush injury. All of the 400 sprague dawley (SD rats were randomly divided into: Control; saline; saline + VEGF-antibody; alprostadil; and alprostadil + VEGF antibody groups. The SPSS 11.5 software was used for statistical analysis. The expression of VEGF in dorsal root ganglia (DRGs, following crush injury to sciatic nerves, was studied by reverse transcribed-polymerase chain reaction (RT-PCR, immunohistochemistry, electromicroscope, and electrophysiology. The effects of alprostadil on expression of VEGF, repair of neural pathology, and recovery of neural function were also evaluated. Results: We found that VEGF messenger ribonucleic acid (mRNA was significantly increased in alprostadil and alprostadil + VEGF-antibody groups, compared to the saline and saline + VEGF antibody groups. The number of VEGF-positive neurons was significantly increased in the alprostadil group, compared to the saline, saline + VEGF antibody, and alprostadil + VEGF antibody groups. Besides, addition of this drug also caused less pathological changes in DRGs, better improvement of nerve conduction velocities of sciatic nerves, and more increase of toe spaces of right hind limbs of rats. Conclusions: The vasoactive agent alprostadil may reduce the pathological lesion of peripheral nerves and improve the rehabilitation of the neural function, which may

  11. Antitumor effect of recombinant human endostatin combined with cisplatin on rats with transplanted Lewis lung cancer

    Institute of Scientific and Technical Information of China (English)

    Zhan-Wu Yu; Ying-Hua Ju; Cheng-Liang Yang; Han-Bing Yu; Quan Luo; Ye-Gang Ma; Yong-Yu Liu

    2015-01-01

    Objective:To observe the antitumor effect and mechanism of recombinant human endostatin (Endostar) injection in tumor combined with intraperitoneal injection of cisplatin on subcutaneous transplanted Lewis lung cancer in rats.Methods:A total of 30 C57 rats were selected, and the monoplast suspension of Lewis lung cancer was injected into the left axilla to prepare the subcutaneous transplanted tumor models in the axilla of right upper limb. The models were randomly divided into Groups A, B, and C. Medication was conducted when the tumor grew to 400 mm3. Group A was the control group without any interventional treatment. Group B was injected with Endostar 5 mg.kg-1.d for 10 d. Group C was given the injection of Endostar 5 mg.kg-1.d combined with intraperitoneal injection of cisplatin 5 mg.kg-1.d for 10 d. All the rats in three groups were executed the day after the 10-d medication and the tumor was taken off for measurement of volume and mass changes and calculation of antitumor rate, after which the vascular endothelial growth factor (VEGF) concentration in rats’plasma was determined by ELISA. The tumor tissues were cut for the preparation of conventional biopsies. After hematoxylin-eosin staining, the pathologic histology was examined to observe the structures of tumor tissues, VEGF score and microvessel density (MVD) in each group. Results:The volume and mass of tumor in Groups B and C were significantly lower than Group A (P< 0.05) while the tumor volume and mass in Group C were significantly lower than Group B (P < 0.05). The antitumor rate in Group C was significantly higher than Group B (P < 0.05), but the tumor VEGF score, MVD and plasma VEGF level in Group C were significantly lower than Groups A and B (P < 0.05). In Group B, the tumor VEGF score, MVD and plasma VEGF level were significantly lower than Group A (P < 0.05). The microscopic image of Group C showed that its number of active tumor cells and the blood capillary around tumor was significantly

  12. Angiogenesis and bone regeneration of porous nano-hydroxyapatite/coralline blocks coated with rhVEGF165 in critical-size alveolar bone defects in vivo

    Directory of Open Access Journals (Sweden)

    Du B

    2015-03-01

    Full Text Available Bing Du,1,2 Weizhen Liu,1 Yue Deng,1,3 Shaobing Li,1 Xiangning Liu,4 Yan Gao,1 Lei Zhou1 1Department of Oral Implantology, Guangdong Provincial Stomatological Hospital, Southern Medical University, Guangzhou, People’s Republic of China; 2Center of Stomatology, The First People’s Hospital of Foshan, Foshan, Guangdong, People’s Republic of China; 3Department of Oral and Maxillofacial Surgery, Qingdao Stomatological Hospital, Qingdao, People’s Republic of China; 4Department of Stomatology, The First Affiliated Hospital of Jinan University, Guangzhou, People’s Republic of China Abstract: To improve the regenerative performance of nano-hydroxyapatite/coralline (nHA/coral block grafting in a canine mandibular critical-size defect model, nHA/coral blocks were coated with recombinant human vascular endothelial growth factor165 (rhVEGF via physical adsorption (3 µg rhVEGF165 per nHA/coral block. After the nHA/coral blocks and VEGF/nHA/coral blocks were randomly implanted into the mandibular box-shaped defects in a split-mouth design, the healing process was evaluated by histological observation and histomorphometric and immunohistological analyses. The histological evaluations revealed the ingrowth of newly formed blood vessels and bone at the periphery and cores of the blocks in both groups at both 3 and 8 weeks postsurgery, respectively. In the histomorphometric analysis, the VEGF/nHA/coral group exhibited a larger quantity of new bone formation at 3 and 8 weeks postsurgery. The percentages of newly formed bone within the entire blocks in the VEGF/nHA/coral group were 27.3%±8.1% and 39.3%±12.8% at 3 weeks and 8 weeks, respectively, and these values were slightly greater than those of the nHA/coral group (21.7%±3.0% and 32.6%±10.3%, respectively, but the differences were not significant (P>0.05. The immunohistological evaluations revealed that the neovascular density in the VEGF/nHA/coral group (146±32.9 vessel/mm2 was much greater than

  13. Phase I trial of aflibercept (VEGF trap) with radiation therapy and concomitant and adjuvant temozolomide in patients with high-grade gliomas.

    Science.gov (United States)

    Nayak, Lakshmi; de Groot, John; Wefel, Jeffrey S; Cloughesy, Timothy F; Lieberman, Frank; Chang, Susan M; Omuro, Antonio; Drappatz, Jan; Batchelor, Tracy T; DeAngelis, Lisa M; Gilbert, Mark R; Aldape, Kenneth D; Yung, Alfred W K; Fisher, Joy; Ye, Xiaobu; Chen, Alice; Grossman, Stuart; Prados, Michael; Wen, Patrick Y

    2017-03-01

    Anti-vascular endothelial growth factor (VEGF) therapy has shown promise in the treatment of high-grade gliomas (HGG). Aflibercept is a recombinant human fusion protein that acts as a soluble decoy receptor for VEGF-A, VEGF-B and placental growth factor, depleting circulating levels of these growth factors. The Adult Brain Tumor Consortium conducted a phase I trial of aflibercept and temozolomide (TMZ) in patients with newly diagnosed HGG with 2 dose levels and a 3+3 design. Three arms using aflibercept were examined; with radiation and concomitant temozolomide; with adjuvant temozolomide using the 5/28 regimen; and with adjuvant temozolomide using the 21/28 day regimen. Fifty-nine patients were enrolled, 21 in arm 1, 20 in arm 2 and 18 in arm 3. Median age was 56 years (24-69); median KPS 90 (60-100). The maximum tolerated dose (MTD) of aflibercept for all 3 arms was 4 mg/kg every 2 weeks. Dose limiting toxicities at the MTD were: Arm 1: 0/21 patients; Arm 2: 2/20 patients (G3 deep vein thrombosis, G4 neutropenia; Arm 3: 3/18 patients) (G4 biopsy-confirmed thrombotic microangiopathy, G3 rash, G4 thrombocytopenia). The median number of cycles of aflibercept was 5 (range, 1-16). All patients stopped treatment; 28 (47%) for disease progression, 21 (36%) for toxicities, 8 (14%) for other reasons, and 2 (3%) patients completed the full treatment course. This study met its primary endpoint and the MTD of aflibercept with radiation and concomitant and adjuvant temozolomide is 4 mg/kg every 2 weeks.

  14. Regulation of Meiotic Recombination

    Energy Technology Data Exchange (ETDEWEB)

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

  15. Characterization of uniaxial stiffness of extracellular matrix embedded with magnetic beads via bio-conjugation and under the influence of an external magnetic field.

    Science.gov (United States)

    Herath, Sahan C B; Du, Yue; Wang, Dong-an; Liao, Kin; Wang, Qingguo; Asada, Harry; Chen, Peter C Y

    2014-02-01

    In this paper, we study the deformation, and experimentally quantify the change in stiffness, of an extracellular matrix (ECM) embedded with magnetic beads that are bio-conjugated with the collagen fibers and under the influence of an external magnetic field. We develop an analytical model of the viscoelastic behavior of this modified ECM, and design and implement a stretch test to quantify (based on statistically meaningful experiment data) the resulting changes in its stiffness induced by the external magnetic field. The analytical results are in close agreement with that obtained from the experiments. We discuss the implication of these results that point to the possibility of creating desired stiffness gradients in an ECM in vitro to influence cell behavior.

  16. Validity of bioconjugated silica nanoparticles in comparison with direct smear, culture, and polymerase chain reaction for detection of Mycobacterium tuberculosis in sputum specimens

    Directory of Open Access Journals (Sweden)

    Ekrami A

    2011-11-01

    Full Text Available Alireza Ekrami1, Ali Reza Samarbaf-Zadeh2, Azar Khosravi1, Behrooz Zargar3, Mohamad Alavi1, Mansor Amin2, Alireza Kiasat3 1Infectious and Tropical Diseases Research Center, Ahvaz Jundishapur University of Medical Sciences, 2Department of Microbiology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, 3Department of Chemistry, School of Science, Shahid Chamran University, Ahvaz, Iran Background: Tuberculosis is a public health problem worldwide, and new easy to perform diagnostic methods with high accuracy are necessary for optimal control of the disease. Recently, fluorescent silica nanoparticles (FSNP has attracted immense interest for the detection of pathogenic microorganisms. The aim of this study was to detect Mycobacterium tuberculosis in clinical samples using bioconjugated FSNP compared with microscopic examination, polymerase chain reaction (PCR, nested PCR, and culture as the gold standard. Methods: In total, 152 sputum specimens were obtained from patients who were suspected to have pulmonary tuberculosis. All samples were examined by the four techniques described. Results: The assay showed 97.1% sensitivity (95% confidence interval [CI] 91–99.2 and 91.35% specificity (CI 78.3–97.1. Furthermore, assays using variable bacterial concentrations indicated that 100 colony forming units/mL of M. tuberculosis could be detected. There were no differences between the results obtained from two types of mouse monoclonal antibody against Hsp-65 and 16 KDa antigens. Conclusion: We performed this assay in a large number of clinical samples to confirm the diagnostic specificity and sensitivity of the test and can recommend its application for diagnosis of M. tuberculosis. We believe that this method is more convenient for routine diagnosis of M. tuberculosis in sputum and will be more easily applicable in the field, and with sufficient sensitivity. Keywords: Mycobacterium tuberculosis, fluorescent silica nanoparticles

  17. Study on VEGF, bFGF and TGF-β1 in the Endometriumof Norplant Users

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To investigate the relationship between abnormal uterus bleeding and en dometrium vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) as well as transforming growth factor-β1(TGF-β1) expressions among Nor plant users. Materials & Methods Thirty-six endometrium samples from Norplant users with nor mal and abnormal bleeding were studied morphologically and immunohistochemically for VEGF, bFGF and TGF-β1 expression. Six normal samples of proliferate endome tria were studied as control. Results In the Norplant users, the characteristics of endometrium changed and glands decreased in numbers. The VEGF expression in epithelium and vascular en dothelium was lower in those with abnormal uterine bleeding. However, no difference was detected on bFGF and TGF-β1 expression. Conclusion The decline of VEGF expression may relate to the abnormal uterine bleed ing in Norplant users.

  18. Urethral tissue regeneration using collagen scaffold modified with collagen binding VEGF in a beagle model.

    Science.gov (United States)

    Jia, Weisheng; Tang, He; Wu, Jianjian; Hou, Xianglin; Chen, Bing; Chen, Wei; Zhao, Yannan; Shi, Chunying; Zhou, Feng; Yu, Wei; Huang, Shengquan; Ye, Gang; Dai, Jianwu

    2015-11-01

    Extensive urethral defects have a serious impact on quality of life, and treatment is challenging. A shortage of material for reconstruction is a key limitation. Improving the properties of biomaterials and making them suitable for urethral reconstruction will be helpful. Previously, we constructed a fusion protein, collagen-binding VEGF (CBD-VEGF), which can bind to collagen scaffold, stimulate cell proliferation, and promote angiogenesis and tissue regeneration. We proposed that CBD-VEGF could improve the performance of collagen in reconstruction of extensive urethral defects. Our results showed that collagen scaffolds modified with CBD-VEGF could promote urethral tissue regeneration and improve the function of the neo-urethra in a beagle extensive urethral defect model. Thus, modifying biomaterials with bioactive factors provides an alternative strategy for the production of suitable biomaterials for urethral reconstruction.

  19. VEGF concentrations in tumour arteries and veins from patients with rectal cancer

    DEFF Research Database (Denmark)

    Werther, Kim; Bülow, Steffen; Hesselfeldt, Peter;

    2002-01-01

    , automated complete white cell and platelet counts were performed. In serum and EDTA plasma, no significant differences in VEGF concentrations were observed (p = 0.1 and p = 0.5), respectively) between tumour arteries and tumour veins. However, in supernatants from lysed blood, VEGF concentrations were......This pilot study investigated the hypothesis that the tumour itself is the source of the elevated vascular endothelial growth factor (VEGF) concentrations which are often observed in peripheral blood from patients with rectal cancer. Twenty-four consecutive patients with primary rectal cancer were...... included. Blood samples were drawn preoperatively from peripheral veins (I) and intraoperatively from peripheral veins (II), tumour arteries (III), and tumour veins (IV). In the four compartments, VEGF concentrations were measured in serum, EDTA plasma, and supernatants from lysed whole blood. Additionally...

  20. A structural model of the VEGF signalling pathway: emergence of robustness and redundancy properties.

    Science.gov (United States)

    Lignet, Floriane; Calvez, Vincent; Grenier, Emmanuel; Ribba, Benjamin

    2013-02-01

    The vascular endothelial growth factor (VEGF) is known as one of the main promoter of angiogenesis - the process of blood vessel formation. Angiogenesis has been recognized as a key stage for cancer development and metastasis. In this paper, we propose a structural model of the main molecular pathways involved in the endothelial cells response to VEGF stimuli. The model, built on qualitative information from knowledge databases, is composed of 38 ordinary differential equations with 78 parameters and focuses on the signalling driving endothelial cell proliferation, migration and resistance to apoptosis. Following a VEGF stimulus, the model predicts an increase of proliferation and migration capability, and a decrease in the apoptosis activity. Model simulations and sensitivity analysis highlight the emergence of robustness and redundancy properties of the pathway. If further calibrated and validated, this model could serve as tool to analyse and formulate new hypothesis on th e VEGF signalling cascade and its role in cancer development and treatment.

  1. Tnactivation of PTEN is associated with increased angiogenesis and VEGF overexpression in gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Ye-Jiang Zhou; Yu-Xia Xiong; Xiao-Ting Wu; De Shi; Wei Fan; Tong Zhou; Yue-Chun Li; Xiong Huang

    2004-01-01

    AIM: To investigate the expression of PTEN/MMAC1/TEP1and vascular endothelial growth factor (VEGF), their roles in biologic behavior and angiogenesis and their association in gastric cancer.METHODS: Immunohistochemical staining was used to evaluate the expression of PTEN, VEGF and microvascular density (MVD) on paraffin-embedded sections in 70 patients with primary gastric cancer and 24 patients with chronic superficial gastritis (CSG). Expression of PTEN, VEGF and MVD were compared with clinicopathological features of gastric cancer. The relationship between expression of PTEN, VEGF and MVD as well as the relationship between PTEN and VEGF expression in caner cells were investigated.RESULTS: PTEN expression significantly decreased (t= 3.98,P<0.01) whereas both VEGF expression and MVD significant increased (t = 4.29 and 4.41, respectively, both P<0.01)in gastric cancer group compared with CSG group. PTEN expression was significantly down-regulated (t = 1.95,P<0.05) whereas VEGF expression (t = 2.37, P<0.05) and MVD (t = 3.28, P<0.01) was significantly up-regulated in advanced gastric cancer compared with early-stage gastric cancer. PTEN expression in gastric cancer showed a negative association with lymph node metastasis (t= 3.91, P<0.01),invasion depth (t= 1.95, P<0.05) and age (t= 4.69, P<0.01).MVD in PTEN-negative gastric cancer was significantly higher than that in PTEN-positive gastric cancer (t = 3.69,P<0.01), and there was a negative correlation between PTEN expression and MVD (γ = -0.363, P<0.05). VEGF expression was positively associated with invasion depth (especially with serosa invasion, t = 4.69, P<0.01), lymph node metastasis (t= 2.31, P<0.05) and TNM stage (t= 3.04,P<0.01). MVD in VEGF-positive gastric cancer was significantly higher than that in VEGF-negative gastric cancer (t = 4.62,P<0.01), and there was a positive correlation between VEGF expression of and MVD (γ = 0.512, P<0.05). VEGF expression in PTEN

  2. VEGF-dependent mechanism of anti-angiogenic action of diamond nanoparticles in Glioblastoma Multiforme tumor

    DEFF Research Database (Denmark)

    Grodzik, M.; Sawosz, E.; Wierzbicki, M.

    2012-01-01

    of diamond nanoparticles on the growth of brain tumor (cultured on CAM membrane) and the development of its blood vessels. We hypothesize that diamond nanoparticles can bind VEGF or their receptors and this way influence of signal transduction between cells. The aim of our study was to evaluate the influence...... of diamond nanoparticle on VEGF level and inhibition of the brain tumor angiogenesis. We evaluated interaction of VEGF-A and VEGF-receptor proteins with diamond nanoparticles (TEM), visualized lower the permeability of blood vessels after diamond nanoparticles treatment and determined localization......Malignant gliomas are highly lethal cancers dependent on angiogenesis. The concept of treating tumors by inhibiting tumor angiogenesis was first articulated almost 30 years ago. Inhibition of tumor angiogenesis suppresses both tumor growth and metastasis. We determined the inhibition effect...

  3. [Suppression of VEGF protein expression by arctigenin in oral squamous cell carcinoma].

    Science.gov (United States)

    Pu, Guang-rui; Liu, Fa-yu; Wang, Bo

    2015-08-01

    To observe arctigenin's inhibitory effect on oral squamous cell carcinoma, and explore the possible mechanism. The expression of VEGF in 32 cases of oral squamous cell cancer and 20 adjacent tissue specimen were detected with immunohistochemistry. Human nude mouse transplantation tumor model of oral squamous cell cancer was prepared with HSC-3 cells line. Transplanted tumor growth and VEGF expression in transplanted tumor tissues were assayed after treatment with arctigenin. One-way ANOVA was used for comparison between groups with SPSS 16.0 software package. Compared with the adjacent tissue, immunohistochemical staining score of VEGF was significantly higher (Parctigenin, the growth of oral squamous cell transplanted tumors in nude mouse was inhibited (Parctigenin group (PArctigenin can dose-dependently inhibit the growth of oral squamous cell carcinomas, and this effect may be related to down regulation of VEGF expression.

  4. Structure of the Full-length VEGFR-1 Extracellular Domain in Complex with VEGF-A.

    Science.gov (United States)

    Markovic-Mueller, Sandra; Stuttfeld, Edward; Asthana, Mayanka; Weinert, Tobias; Bliven, Spencer; Goldie, Kenneth N; Kisko, Kaisa; Capitani, Guido; Ballmer-Hofer, Kurt

    2017-02-07

    Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel development upon activation of three receptor tyrosine kinases: VEGFR-1, -2, and -3. Partial structures of VEGFR/VEGF complexes based on single-particle electron microscopy, small-angle X-ray scattering, and X-ray crystallography revealed the location of VEGF binding and domain arrangement of individual receptor subdomains. Here, we describe the structure of the full-length VEGFR-1 extracellular domain in complex with VEGF-A at 4 Å resolution. We combined X-ray crystallography, single-particle electron microscopy, and molecular modeling for structure determination and validation. The structure reveals the molecular details of ligand-induced receptor dimerization, in particular of homotypic receptor interactions in immunoglobulin homology domains 4, 5, and 7. Functional analyses of ligand binding and receptor activation confirm the relevance of these homotypic contacts and identify them as potential therapeutic sites to allosterically inhibit VEGFR-1 activity.

  5. Collaborative interplay between FGF-2 and VEGF-C promotes lymphangiogenesis and metastasis

    DEFF Research Database (Denmark)

    Cao, Renhai; Ji, Hong; Feng, Ninghan

    2012-01-01

    Interplay between various lymphangiogenic factors in promoting lymphangiogenesis and lymphatic metastasis remains poorly understood. Here we show that FGF-2 and VEGF-C, two lymphangiogenic factors, collaboratively promote angiogenesis and lymphangiogenesis in the tumor microenvironment, leading...

  6. Enhancement of musculocutaneous nerve reinnervation after vascular endothelial growth factor (VEGF gene therapy

    Directory of Open Access Journals (Sweden)

    Haninec Pavel

    2012-06-01

    Full Text Available Abstract Background Vascular endothelial growth factor (VEGF is not only a potent angiogenic factor but it also promotes axonal outgrowth and proliferation of Schwann cells. The aim of the present study was to quantitatively assess reinnervation of musculocutaneous nerve (MCN stumps using motor and primary sensory neurons after plasmid phVEGF transfection and end-to-end (ETE or end-to-side (ETS neurorrhaphy. The distal stump of rat transected MCN, was transfected with plasmid phVEGF, plasmid alone or treated with vehiculum and reinnervated following ETE or ETS neurorrhaphy for 2 months. The number of motor and dorsal root ganglia neurons reinnervating the MCN stump was estimated following their retrograde labeling with Fluoro-Ruby and Fluoro-Emerald. Reinnervation of the MCN stumps was assessed based on density, diameter and myelin sheath thickness of regenerated axons, grooming test and the wet weight index of the biceps brachii muscles. Results Immunohistochemical detection under the same conditions revealed increased VEGF in the Schwann cells of the MCN stumps transfected with the plasmid phVEGF, as opposed to control stumps transfected with only the plasmid or treated with vehiculum. The MCN stumps transfected with the plasmid phVEGF were reinnervated by moderately higher numbers of motor and sensory neurons after ETE neurorrhaphy compared with control stumps. However, morphometric quality of myelinated axons, grooming test and the wet weight index were significantly better in the MCN plasmid phVEGF transfected stumps. The ETS neurorrhaphy of the MCN plasmid phVEGF transfected stumps in comparison with control stumps resulted in significant elevation of motor and sensory neurons that reinnervated the MCN. Especially noteworthy was the increased numbers of neurons that sent out collateral sprouts into the MCN stumps. Similarly to ETE neurorrhaphy, phVEGF transfection resulted in significantly higher morphometric quality of myelinated axons

  7. Intergenogroup Recombination in Sapoviruses

    Science.gov (United States)

    Hansman, Grant S.; Takeda, Naokazu; Oka, Tomoichiro; Oseto, Mitsukai; Hedlund, Kjell-Olof

    2005-01-01

    Sapovirus, a member of the family Caliciviridae, is an etiologic agent of gastroenteritis in humans and pigs. Analyses of the complete genome sequences led us to identify the first sapovirus intergenogroup recombinant strain. Phylogenetic analysis of the nonstructural region (i.e., genome start to capsid start) grouped this strain into genogroup II, whereas the structural region (i.e., capsid start to genome end) grouped this strain into genogroup IV. We found that a recombination event occurred at the polymerase and capsid junction. This is the first report of intergenogroup recombination for any calicivirus and highlights a possible route of zoonoses because sapovirus strains that infect pig species belong to genogroup III. PMID:16485479

  8. Recombination experiments at CRYRING

    Energy Technology Data Exchange (ETDEWEB)

    Spies, W.; Glans, P.; Zong, W.; Gao, H.; Andler, G.; Justiniano, E.; Saito, M.; Schuch, R

    1998-11-15

    Recent advances in studies of electron-ion recombination processes at low relative energies with the electron cooler of the heavy-ion storage ring CRYRING are shown. Through the use of an adiabatically expanded electron beam, collisions down to 10{sup -4}eV relative energies were measured with highly charged ions stored in the ring at around 15 MeV/amu energies. Examples of recombination measurements for bare ions of D{sup +}, He{sup 2+}, N{sup 7+}, Ne{sup 10+} and Si{sup 14+} are presented. Further on, results of an experiment measuring laser-induced recombination (LIR) into n=3 states of deuterium with polarized laser light are shown.

  9. Recombinant Helicobacter pylori catalase

    Institute of Scientific and Technical Information of China (English)

    Yang Bai; Ya-Li Zhang; Jian-Feng Jin; Ji-De Wang; Zhao-Shan Zhang

    2003-01-01

    AIM: To construct a recombinant strain which highly expresses catalase of Helicobacter pylori(H.pylori) and assay the activity of H. pylori catalase.METHODS: The catalase DNA was amplified from H. pylori chromosomal DNA with PCR techniques and inserted into the prokaryotie expression vector pET-22b (+), and then was transformed into the BL21 (DE3) E. coli strain which expressed catalase recombinant protein. The activity of H.pylori catalase was assayed by the Beers & Sizers.RESULTS: DNA sequence analysis showed that the sequence of catalase DNA was the same as GenBank's research. The catalase recombinant protein amounted to 24.4 % of the total bacterial protein after induced with IPTG for 3 hours at 37 ℃ and the activity of H. pylori catalase was high in the BL21 (DE3) E. coli strain.CONCLUSION: A clone expressing high activity H. pylori catalase is obtained, laying a good foundation for further studies.

  10. HSulf-2, an extracellular endoglucosamine-6-sulfatase, selectively mobilizes heparin-bound growth factors and chemokines: effects on VEGF, FGF-1, and SDF-1

    Directory of Open Access Journals (Sweden)

    Gallagher John

    2006-01-01

    Full Text Available Abstract Background Heparin/heparan sulfate (HS proteoglycans are found in the extracellular matrix (ECM and on the cell surface. A considerable body of evidence has established that heparin and heparan sulfate proteoglycans (HSPGs interact with numerous protein ligands including fibroblast growth factors, vascular endothelial growth factor (VEGF, cytokines, and chemokines. These interactions are highly dependent upon the pattern of sulfation modifications within the glycosaminoglycan chains. We previously cloned a cDNA encoding a novel human endosulfatase, HSulf-2, which removes 6-O-sulfate groups on glucosamine from subregions of intact heparin. Here, we have employed both recombinant HSulf-2 and the native enzyme from conditioned medium of the MCF-7-breast carcinoma cell line. To determine whether HSulf-2 modulates the interactions between heparin-binding factors and heparin, we developed an ELISA, in which soluble factors were allowed to bind to immobilized heparin. Results Our results show that the binding of VEGF, FGF-1, and certain chemokines (SDF-1 and SLC to immobilized heparin was abolished or greatly diminished by pre-treating the heparin with HSulf-2. Furthermore, HSulf-2 released these soluble proteins from their association with heparin. Native Sulf-2 from MCF-7 cells reproduced all of these activities. Conclusion Our results validate Sulf-2 as a new tool for deciphering the sulfation requirements in the interaction of protein ligands with heparin/HSPGs and expand the range of potential biological activities of this enzyme.

  11. Combined VEGF gene transfer and erythropoietin in ovine reperfused myocardial infarction.

    Science.gov (United States)

    Olea, Fernanda D; De Lorenzi, Andrea; Cortés, Claudia; Cuniberti, Luis; Fazzi, Lucía; Flamenco, María del Pilar; Locatelli, Paola; Cabeza Meckert, Patricia; Bercovich, Andrés; Laguens, Rubén; Crottogini, Alberto

    2013-05-10

    In reperfused acute myocardial infarction (RAMI), cardioprotective treatments may enhance myocardial salvage and hence reduce the area of necrosis. Based on studies showing that plasmid-mediated vascular endothelial growth factor (pVEGF) gene transfer reduces infarct size by combining angio-arteriogenic and cardiomyogenic effects and that erythropoietin (EPO) exerts anti-apoptotic actions in animal models of AMI, we aimed to assess if their association would reduce infarct size to a larger extent than any of them individually in a large mammalian model of RAMI. Adult sheep subjected to 90-minute coronary artery occlusion received upon reperfusion intramyocardial pVEGF 3.8 mg plus intravenous EPO 1000 IU/kg (n=8), pVEGF (n=8), EPO (n=8) or placebo (n=8). Fifteen days after treatment, infarct size was smaller in the 3 treatment groups (pVEGF+EPO: 8 ± 1 %; pVEGF: 16 ± 5 %; EPO: 13 ± 4 %) compared to placebo (25 ± 7 %, p<0.001). However, in the EPO+VEGF group infarct size was significantly smaller than in the groups receiving EPO or VEGF individually (p<0.05). DNA fragmentation, a hallmark of late apoptosis, was significantly lower in sheep receiving EPO. The combined treatment, while not affecting global left ventricular performance, improved regional peri-infarct function and prevented over-time expansion of the post-infarct perfusion defect. Combined pVEGF and EPO treatment might be clinically useful to enhance the benefits of early revascularization in patients with acute myocardial infarction. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  12. Genetic variation in VEGF does not contribute significantly to the risk of congenital cardiovascular malformation.

    Directory of Open Access Journals (Sweden)

    Helen R Griffin

    Full Text Available Several previous studies have investigated the role of common promoter variants in the vascular endothelial growth factor (VEGF gene in causing congenital cardiovascular malformation (CVM. However, results have been discrepant between studies and no study to date has comprehensively characterised variation throughout the gene. We genotyped 771 CVM cases, of whom 595 had the outflow tract malformation Tetralogy of Fallot (TOF, and carried out TDT and case-control analyses using haplotype-tagging SNPs in VEGF. We carried out a meta-analysis of previous case-control or family-based studies that had typed VEGF promoter SNPs, which included an additional 570 CVM cases. To identify rare variants potentially causative of CVM, we carried out mutation screening in all VEGF exons and splice sites in 93 TOF cases. There was no significant effect of any VEGF haplotype-tagging SNP on the risk of CVM in our analyses of 771 probands. When the results of this and all previous studies were combined, there was no significant effect of the VEGF promoter SNPs rs699947 (OR 1.05 [95% CI 0.95-1.17]; rs1570360 (OR 1.17 [95% CI 0.99-1.26]; and rs2010963 (OR 1.04 [95% CI 0.93-1.16] on the risk of CVM in 1341 cases. Mutation screening of 93 TOF cases revealed no VEGF coding sequence variants and no changes at splice consensus sequences. Genetic variation in VEGF appears to play a small role, if any, in outflow tract CVM susceptibility.

  13. Synthesis and VEGF inhibitory activity of 16,17-pyrazo-annulated steroids

    Institute of Scientific and Technical Information of China (English)

    Hong Shan Liu; Hu Ling Zheng; Mei Ge; Peng Xia; Ying Chen

    2011-01-01

    Eight 16,17-pyrazo-annulated steroidal derivatives were synthesized and evaluated in vitro vascular endothelial growth factor (VEGF) inhibitory activity with 2-methoxyestradiol (2-ME) as the reference compound. Most of the compounds showed potent VEGF inhibitory activity with EC50 values of micromolar or submicromolar range. Among them, the compounds 3 and 8 exhibited similar EC50 values and obviously better TI values compared with 2-ME.

  14. Prognostic importance of VEGF-A haplotype combinations in a stage II colon cancer population

    DEFF Research Database (Denmark)

    Kjaer-Frifeldt, Sanne; Fredslund, Rikke; Lindebjerg, Jan;

    2012-01-01

    To investigate the prognostic effect of three VEGF-A SNPs, -2578, -460 and 405, as well as the corresponding haplotype combinations, in a unique population of stage II colon cancer patients.......To investigate the prognostic effect of three VEGF-A SNPs, -2578, -460 and 405, as well as the corresponding haplotype combinations, in a unique population of stage II colon cancer patients....

  15. Covalent immobilisation of VEGF on plasma-coated electrospun scaffolds for tissue engineering applications.

    Science.gov (United States)

    Guex, A G; Hegemann, D; Giraud, M N; Tevaearai, H T; Popa, A M; Rossi, R M; Fortunato, G

    2014-11-01

    Recent findings in the field of biomaterials and tissue engineering provide evidence that surface immobilised growth factors display enhanced stability and induce prolonged function. Cell response can be regulated by material properties and at the site of interest. To this end, we developed scaffolds with covalently bound vascular endothelial growth factor (VEGF) and evaluated their mitogenic effect on endothelial cells in vitro. Nano- (254±133 nm) or micro-fibrous (4.0±0.4 μm) poly(ɛ-caprolactone) (PCL) non-wovens were produced by electrospinning and coated in a radio frequency (RF) plasma process to induce an oxygen functional hydrocarbon layer. Implemented carboxylic acid groups were converted into amine-reactive esters and covalently coupled to VEGF by forming stable amide bonds (standard EDC/NHS chemistry). Substrates were analysed by X-ray photoelectron spectroscopy (XPS), enzyme-linked immuno-assays (ELISA) and immunohistochemistry (anti-VEGF antibody and VEGF-R2 binding). Depending on the reaction conditions, immobilised VEGF was present at 127±47 ng to 941±199 ng per substrate (6mm diameter; concentrations of 4.5 ng mm(-2) or 33.3 ng mm(-2), respectively). Immunohistochemistry provided evidence for biological integrity of immobilised VEGF. Endothelial cell number of primary endothelial cells or immortalised endothelial cells were significantly enhanced on VEGF-functionalised scaffolds compared to native PCL scaffolds. This indicates a sustained activity of immobilised VEGF over a culture period of nine days. We present a versatile method for the fabrication of growth factor-loaded scaffolds at specific concentrations.

  16. Genetic variation in VEGF does not contribute significantly to the risk of congenital cardiovascular malformation.

    Science.gov (United States)

    Griffin, Helen R; Hall, Darroch H; Topf, Ana; Eden, James; Stuart, A Graham; Parsons, Jonathan; Peart, Ian; Deanfield, John E; O'Sullivan, John; Babu-Narayan, Sonya V; Gatzoulis, Michael A; Bu'lock, Frances A; Bhattacharya, Shoumo; Bentham, Jamie; Farrall, Martin; Granados Riveron, Javier; Brook, J David; Burn, John; Cordell, Heather J; Goodship, Judith A; Keavney, Bernard

    2009-01-01

    Several previous studies have investigated the role of common promoter variants in the vascular endothelial growth factor (VEGF) gene in causing congenital cardiovascular malformation (CVM). However, results have been discrepant between studies and no study to date has comprehensively characterised variation throughout the gene. We genotyped 771 CVM cases, of whom 595 had the outflow tract malformation Tetralogy of Fallot (TOF), and carried out TDT and case-control analyses using haplotype-tagging SNPs in VEGF. We carried out a meta-analysis of previous case-control or family-based studies that had typed VEGF promoter SNPs, which included an additional 570 CVM cases. To identify rare variants potentially causative of CVM, we carried out mutation screening in all VEGF exons and splice sites in 93 TOF cases. There was no significant effect of any VEGF haplotype-tagging SNP on the risk of CVM in our analyses of 771 probands. When the results of this and all previous studies were combined, there was no significant effect of the VEGF promoter SNPs rs699947 (OR 1.05 [95% CI 0.95-1.17]); rs1570360 (OR 1.17 [95% CI 0.99-1.26]); and rs2010963 (OR 1.04 [95% CI 0.93-1.16]) on the risk of CVM in 1341 cases. Mutation screening of 93 TOF cases revealed no VEGF coding sequence variants and no changes at splice consensus sequences. Genetic variation in VEGF appears to play a small role, if any, in outflow tract CVM susceptibility.

  17. Rôle du VEGF dans la régulation de la synapse glutamatergique

    OpenAIRE

    Rossi, Pierre

    2013-01-01

    The vascular endothelial growth factor (VEGF) plays a critical role during vascular development but recent evidence indicates that it also regulates various neuronal processes in the nervous system, such as neurogenesis, hippocampal synaptic plasticity, learning and memory. Recently, we showed a novel interaction between the glutamate receptor NMDA (NMDAR) and the VEGF receptor VEGFR2 during neuronal migration in the developing cerebellum. As NMDAR have been widely implicated in synaptic tran...

  18. VEGF-releasing suture material for enhancement of vascularization: development, in vitro and in vivo study.

    Science.gov (United States)

    Bigalke, Christian; Luderer, Frank; Wulf, Katharina; Storm, Thilo; Löbler, Marian; Arbeiter, Daniela; Rau, Bettina M; Nizze, Horst; Vollmar, Brigitte; Schmitz, Klaus-Peter; Klar, Ernst; Sternberg, Katrin

    2014-12-01

    As it has been demonstrated that bioactive substances can be delivered locally using coated surgical suture materials, the authors developed a vascular endothelial growth factor (VEGF)-releasing suture material that should promote vascularization and potentially wound healing. In this context, the study focused on the characterization of the developed suture material and the verification of its biological activity, as well as establishing a coating process that allows reproducible and stable coating of a commercially available polydioxanone suture material with poly(l-lactide) (PLLA) and 0.1μg and 1.0μg VEGF. The in vitro VEGF release kinetics was studied using a Sandwich ELISA. The biological activity of the released VEGF was investigated in vitro using human umbilical vein endothelial cells. The potential of the VEGF-releasing suture material was also studied in vivo 5days after implantation in the hind limb of Wistar rats, when the histological findings were analyzed. The essential results, enhanced cell viability in vitro as well as significantly increased vascularization in vivo, were achieved using PLLA/1.0μg VEGF-coated suture material. Furthermore, ELISA measurements revealed a high reproducibility of the VEGF release behavior. Based on the results achieved regarding the dose-effect relationship of VEGF, the stability during its processing and the release behavior, it can be predicted that a bioactive suture material would be successful in later in vivo studies. Therefore, this knowledge could be the basis for future studies, where bioactive substances with different modes of action are combined for targeted, overall enhancement of wound healing. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  19. Association of serum VEGF levels with prefrontal cortex volume in schizophrenia.

    Science.gov (United States)

    Pillai, A; Howell, K R; Ahmed, A O; Weinberg, D; Allen, K M; Bruggemann, J; Lenroot, R; Liu, D; Galletly, C; Weickert, C S; Weickert, T W

    2016-05-01

    A large body of evidence indicates alterations in brain regional cellular energy metabolism and blood flow in schizophrenia. Among the different molecules regulating blood flow, vascular endothelial growth factor (VEGF) is generally accepted as the major factor involved in the process of angiogenesis. In the present study, we examined whether peripheral VEGF levels correlate with changes in the prefrontal cortex (PFC) volume in patients with schizophrenia and in healthy controls. Whole-blood samples were obtained from 96 people with schizophrenia or schizoaffective disorder and 83 healthy controls. Serum VEGF protein levels were analyzed by enzyme-linked immunosorbent assay, whereas quantitative PCR was performed to measure interleukin-6 (IL-6, a pro-inflammatory marker implicated in schizophrenia) mRNA levels in the blood samples. Structural magnetic resonance imaging scans were obtained using a 3T Achieva scanner on a subset of 59 people with schizophrenia or schizoaffective disorder and 65 healthy controls, and prefrontal volumes were obtained using FreeSurfer software. As compared with healthy controls, individuals with schizophrenia had a significant increase in log-transformed mean serum VEGF levels (t(177)=2.9, P=0.005). A significant inverse correlation (r=-0.40, P=0.002) between serum VEGF and total frontal pole volume was found in patients with schizophrenia/schizoaffective disorder. Moreover, we observed a significant positive association (r=0.24, P=0.03) between serum VEGF and IL-6 mRNA levels in patients with schizophrenia. These findings suggest an association between serum VEGF and inflammation, and that serum VEGF levels are related to structural abnormalities in the PFC of people with schizophrenia.

  20. Moderation of calpain activity promotes neovascular integration and lumen formation during VEGF-induced pathological angiogenesis.

    Directory of Open Access Journals (Sweden)

    Mien V Hoang

    Full Text Available BACKGROUND: Successful neovascularization requires that sprouting endothelial cells (ECs integrate to form new vascular networks. However, architecturally defective, poorly integrated vessels with blind ends are typical of pathological angiogenesis induced by vascular endothelial growth factor-A (VEGF, thereby limiting the utility of VEGF for therapeutic angiogenesis and aggravating ischemia-related pathologies. Here we investigated the possibility that over-exuberant calpain activity is responsible for aberrant VEGF neovessel architecture and integration. Calpains are a family of intracellular calcium-dependent, non-lysosomal cysteine proteases that regulate cellular functions through proteolysis of numerous substrates. METHODOLOGY/PRINCIPAL FINDINGS: In a mouse skin model of VEGF-driven angiogenesis, retroviral transduction with dominant-negative (DN calpain-I promoted neovessel integration and lumen formation, reduced blind ends, and improved vascular perfusion. Moderate doses of calpain inhibitor-I improved VEGF-driven angiogenesis similarly to DN calpain-I. Conversely, retroviral transduction with wild-type (WT calpain-I abolished neovessel integration and lumen formation. In vitro, moderate suppression of calpain activity with DN calpain-I or calpain inhibitor-I increased the microtubule-stabilizing protein tau in endothelial cells (ECs, increased the average length of microtubules, increased actin cable length, and increased the interconnectivity of vascular cords. Conversely, WT calpain-I diminished tau, collapsed microtubules, disrupted actin cables, and inhibited integration of cord networks. Consistent with the critical importance of microtubules for vascular network integration, the microtubule-stabilizing agent taxol supported vascular cord integration whereas microtubule dissolution with nocodazole collapsed cord networks. CONCLUSIONS/SIGNIFICANCE: These findings implicate VEGF-induction of calpain activity and impairment of

  1. Inhibition of Ocular Neovascularization by Co-Inhibition of VEGF-A and PLGF

    OpenAIRE

    Xiaochuan Huo; Youxiang Li; Yuhua Jiang; Xiaoyun Sun; Lixue Gu; Wenshi Guo; Dapeng Sun

    2015-01-01

    Background/Aims: Age-related macular degeneration (AMD) appears to be a disease with increasing incidence in Western countries and may develop into acquired blindness. Choroidal neovascularization (CNV) is the most frequent cause for AMD, and is commonly induced by regional inflammation. Past studies have highlighted vascular endothelial growth factor A (VEGF-A) as a major trigger for CNV. However, studies on the associated angiogenic factors other than VEGF-A are lacking. Methods: Here, we u...

  2. Molecular In Vivo Imaging Using a Noninvasive Cardiac-Specific MLC-2v Promoter Driven Dual-Gene Recombinant Lentivirus Monitoring System.

    Directory of Open Access Journals (Sweden)

    Miao Zhang

    Full Text Available Our study aimed to demonstrate the feasibility of using the sodium/iodide symporter (NIS to monitor vascular endothelial growth factor (VEGF165 expression in vivo.We constructed a recombinant lentivirus plasmid with the MLC-2v promoter driving the sodium/iodide symporter (NIS reporter gene linked to the VEGF165 gene. Expression of NIS and VEGF gene were identified by Western blot. On days 2 and 54, 99mTc-MIBI imaging was used to evaluate changes in myocardial ischemia. Noninvasive 125I micro-SPECT/CT imaging was used to assess the expression of NIS reporter gene dynamically over the next 2 months.Western blot analysis showed that both NIS and VEGF165 were highly expressed in rat cardiomyoblast H9C2 cells transduced with Lenti-MLC-2v-NIS--VEGF165. 125I micro-SPECT/CT reporter imaging showed higher uptake in mouse myocardium transduced with Lenti-MLC-2v-VEGF165-IRES-NIS. NIS expression peaked on day 1 after transduction followed by a progressive decline to negligible levels by day 21. On day 1, mean 125I activity value in group 1 was higher than that in group 2 (P 0.05. In group 1 (test group, 99mTc-MIBI SPECT/CT revealed improvements in perfusion and wall thickening in the apical anterior wall. Mean IOD values of NIS and CD34 were significantly higher in group 1 than group 3 (P<0.05. Our study proved mean I-125 uptake was significantly correlated with mean IOD value of NIS and CD34 (P<0.05.This study demonstrates the feasibility of using the NIS gene to monitor VEGF165 expression in a mouse myocardial ischemia model.

  3. 人血管内皮生长因子与绿色荧光蛋白标记双基因共表达AAV载体的构建及鉴定%Construction and identification of recombinant adeno- associated virus vector co-expressing human vascular endothelial growth factor and green fluorescent protein

    Institute of Scientific and Technical Information of China (English)

    黄向辉; 时志斌; 王坤正; 党晓谦; 杨佩; 余鹏博

    2008-01-01

    取重组病毒基因组成功扩增出外源目的基因片段hVEGF165,证实重组病毒rAAV-VEGF165-GFP包装成功.结论:成功构建带有绿色荧光蛋白标记并携带hVEGF165基因的无致病性重组腺相关病毒rAAV-VEGF165-GFP.收获的病毒具有较高滴度和感染活性.%BACKGROUND: Vascular endothelial growth factor (VEGF) can specifically promote the division and proliferation of endothelial cells and the revascularization, finally induce angiopoiesis. Recently, VEGF-based gene therapy has been gradually used in clinical trials, but some limits on usually used vectors deserve further studies, including the low transfection efficiency of plasmid vector, the immunogenicity of adnovirus vector to host cells and the potential risk of infection. OBJECTIVE: To construct the non-pathogenic recombinant adeno-associated vires (AAV) co-expressing human vascular endethelial growth factor 165 (hVEGF165) and green fluorescent protein (GFP) label and measure the virus titer and assess its biological activity.DESIGN, TIME AND SETTING: The open experiment was performed at the Vitrus Laboratory of Shanxi Provincial Center for Disease Control and Prevention from March to September 2007.MATERIALS: AAV-293 virus packaging cell line, AAV HT-1080 cells were purchased from Swatagene, USA. E.coli DH5α was a stocked strain from Shanxi Provincial Center for Disease Control and Prevention. AAV Helper Free System (pAAV-IRES-GFP vector containing GFP label) was purchased from Stratagene, USA. Plasmid pUC18-hVEGF165 was constructed previously by Dr.Shi from Department of Orthopaedics of Second Affiliated Hospital of Xi'an Jiaotong University.METHODS: The hVEGF165 gone from plasmid pUC18-hVEGF165 was amplified and inserted into plasmid pAAV-IRES-hrGFP. Then recombinant plasmid pAAV-hVEGF165-IRES-hrGFP, pAAV-RC and pAAV-Helper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hrGFP packaging through homologous recombination. The efficiency of A

  4. [VEGF in the cancer anorexia-cachexia syndrome in patients with lung cancer].

    Science.gov (United States)

    Weryńska, Bozena; Kosacka, Monika; Gołecki, Marcin; Jankowska, Renata

    2006-01-01

    Cancer anorexia-cachexia syndrome( CACS) occurs in 30-80% of patients with cancer. CACS is connected with poor prognosis and higher risk of treatment complications. CACS belongs to the common cause of death in cancer patients. Main role in the development of this syndrome play cytokines like TNF, interleukin 1 and 6 and interferon alpha and gamma. The importance of a lot of other substances is still unknown. VEGF promotes new vessels development,enhance vascular permeability and plays a role in inflammatory reaction. The aim of this study was comparison of VEGF levels in patients with lung cancer with and without CACS and in control group. The serum levels of VEGF were measured by ELISA method. The VEGF was significatly higher in patients with lung cancer then in control group (p = 0.004). There were no correlations between VEGF and weight lost, histological type and stage of disease. This suggest that VEGF doesnt play a role in development of CACS.

  5. NLRP3 Inflammasome Blockade Inhibits VEGF-A-Induced Age-Related Macular Degeneration

    Directory of Open Access Journals (Sweden)

    Alexander G. Marneros

    2013-09-01

    Full Text Available The NLRP3 inflammasome is activated in age-related macular degeneration (AMD, but it remains unknown whether its activation contributes to AMD pathologies. VEGF-A is increased in neovascular (“wet” AMD, but it is not known whether it plays a role in inflammasome activation, whether an increase of VEGF-A by itself is sufficient to cause neovascular AMD and whether it can contribute to nonexudative (“dry” AMD that often co-occurs with the neovascular form. Here, it is shown that an increase in VEGF-A results in NLRP3 inflammasome activation and is sufficient to cause both forms of AMD pathologies. Targeting NLRP3 or the inflammasome effector cytokine IL-1β inhibits but does not prevent VEGF-A-induced AMD pathologies, whereas targeting IL-18 promotes AMD. Thus, increased VEGF-A provides a unifying pathomechanism for both forms of AMD; combining therapeutic inhibition of both VEGF-A and IL-1β or the NLRP3 inflammasome is therefore likely to suppress both forms of AMD.

  6. Inhibition of VEGF- and NO-dependent angiogenesis does not impair liver regeneration

    Science.gov (United States)

    Shergill, U.; Das, A.; Langer, D.; Adluri, RS.; Maulik, N.

    2010-01-01

    Angiogenesis occurs through a convergence of diverse signaling mechanisms with prominent pathways that include autocrine effects of endothelial nitric oxide (NO) synthase (eNOS)-derived NO and vascular endothelial growth factor (VEGF). However, the redundant and distinct roles of NO and VEGF in angiogenesis remain incompletely defined. Here, we use the partial hepatectomy model in mice genetically deficient in eNOS to ascertain the influence of eNOS-derived NO on the angiogenesis that accompanies liver regeneration. While sinusoidal endothelial cell (SEC) eNOS promotes angiogenesis in vitro, surprisingly the absence of eNOS did not influence the angiogenesis that occurs after partial hepatectomy in vivo. While this observation could not be attributed to induction of alternate NOS isoforms, it was associated with induction of VEGF signaling as evidenced by enhanced levels of VEGF ligand in regenerating livers from mice genetically deficient in eNOS. However, surprisingly, mice that were genetically heterozygous for deficiency in the VEGF receptor, fetal liver kinase-1, also maintained unimpaired capacity for liver regeneration. In summary, inhibition of VEGF- and NO-dependent angiogenesis does not impair liver regeneration, indicating signaling redundancies that allow liver regeneration to continue in the absence of this canonical vascular pathway. PMID:20421635

  7. Effects of VEGF and MSCs on vascular regeneration in a trauma model in rats.

    Science.gov (United States)

    Niyaz, Mehmet; Gürpınar, Özer Aylin; Oktar, Gürsel Levent; Günaydın, Serdar; Onur, Mehmet Ali; Özsin, Kadir Kaan; Yener, Ali

    2015-01-01

    In the human body, vascular injuries that are caused by trauma, vessel lumen stenosis, and occlusions are often irreversible and can lead to sequelae formation as the vessels cannot reproduce fast enough. To solve this problem, the blood flow must be returned to the region as fast as possible. The adipose tissue contains progenitor cells with angiogenic potential and can be used to resolve the issue. In the present study, mesenchymal stem cells (MSCs) derived from rat adipose tissue, vascular endothelial growth factor (VEGF), and their mixture were applied on the dorsum of a rat, which was traumatized and its contribution to vascular regeneration was reviewed. No application was made to the control group. The results showed that the percentage of necrotic area was significantly lower in the MSC group than that of all the other groups. When the VEGF group was compared to the VEGF + MSCs, the percentage of necrotic area was observed to be similiar. However, VEGF showed effects only when a large quantites of VEGF was applied to the flap area. VEGF could not fully respond to the needs, whereas MSCs can produce VEGF according to the needs of tissue. This makes them superior to stem cells.

  8. An Important Role of VEGF-C in Promoting Lymphedema Development.

    Science.gov (United States)

    Gousopoulos, Epameinondas; Proulx, Steven T; Bachmann, Samia B; Dieterich, Lothar C; Scholl, Jeannette; Karaman, Sinem; Bianchi, Roberta; Detmar, Michael

    2017-09-01

    Secondary lymphedema is a common complication after cancer treatment, but the pathomechanisms underlying the disease remain unclear. Using a mouse tail lymphedema model, we found an increase in local and systemic levels of the lymphangiogenic factor vascular endothelial growth factor (VEGF)-C and identified CD68(+) macrophages as a cellular source. Surprisingly, overexpression of VEGF-C in a transgenic mouse model led to aggravation of lymphedema with increased immune cell infiltration and vascular leakage compared with wild-type littermates. Conversely, blockage of VEGF-C by overexpression of soluble VEGF receptor-3 reduced edema development, diminishing inflammation and blood vascular leakage. Similar findings were obtained in a hind limb lymph node excision lymphedema model. Flow cytometry analyses and immunofluorescence stainings in lymphedematic tissue showed that VEGF receptor-3 expression was restricted to lymphatic endothelial cells. Our data suggest that endogenous VEGF-C causes blood vascular leakage and fluid influx into the tissue, thus actively contributing to edema formation. These data may provide the basis for future clinical therapeutic approaches. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  9. VEGF-A and VEGFR1 SNPs associate with preeclampsia in a Philippine population.

    Science.gov (United States)

    Amosco, Melissa D; Villar, Van Anthony M; Naniong, Justin Michael A; David-Bustamante, Lara Marie G; Jose, Pedro A; Palmes-Saloma, Cynthia P

    The vascular endothelial growth factor (VEGF) family is important for establishing normal pregnancy, and related single nucleotide polymorphisms (SNPs) are implicated in abnormal placentation and preeclampsia. We evaluated the association between preeclampsia and several VEGF SNPs among Filipinos, an ethnically distinct group with high prevalence of preeclampsia. The genotypes and allelic variants were determined in a case-control study (191 controls and 165 preeclampsia patients) through SNP analysis of VEGF-A (rs2010963, rs3025039) and VEGF-C (rs7664413) and their corresponding receptors VEGFR1 (rs722503, rs12584067, rs7335588) and VEGFR3 (rs307826) from venous blood DNA. VEGF-A rs3025039 C allele has been shown to associate with preeclampsia (odds ratio of 1.648 (1.03-2.62)), while the T allele bestowed an additive effect for the maintenance of normal, uncomplicated pregnancy and against the development of preeclampsia (odds ratio of 0.62 (0.39-0.98)). VEGFR1 rs722503 is associated with preeclampsia occurring at or after the age of 40 years. The results showed that genetic variability of VEGF-A and VEGFR1 are important in the etiology of preeclampsia among Filipinos.

  10. Bee products prevent VEGF-induced angiogenesis in human umbilical vein endothelial cells

    Directory of Open Access Journals (Sweden)

    Mishima Satoshi

    2009-11-01

    Full Text Available Abstract Background Vascular endothelial growth factor (VEGF is a key regulator of pathogenic angiogenesis in diseases such as cancer and diabetic retinopathy. Bee products [royal jelly (RJ, bee pollen, and Chinese red propolis] from the honeybee, Apis mellifera, have been used as traditional health foods for centuries. The aim of this study was to investigate the anti-angiogenic effects of bee products using human umbilical vein endothelial cells (HUVECs. Methods In an in vitro tube formation assay, HUVECs and fibroblast cells were incubated for 14 days with VEGF and various concentrations of bee products [RJ, ethanol extract of bee pollen, ethanol extract of Chinese red propolis and its constituent, caffeic acid phenethyl ester (CAPE]. To clarify the mechanism of in vitro angiogenesis, HUVEC proliferation and migration were induced by VEGF with or without various concentrations of RJ, bee pollen, Chinese red propolis, and CAPE. Results RJ, bee pollen, Chinese red propolis, and CAPE significantly suppressed VEGF-induced in vitro tube formation in the descending order: CAPE > Chinese red propolis >> bee pollen > RJ. RJ and Chinese red propolis suppressed both VEGF-induced HUVEC proliferation and migration. In contrast, bee pollen and CAPE suppressed only the proliferation. Conclusion Among the bee products, Chinese red propolis and CAPE in particular showed strong suppressive effects against VEGF-induced angiogenesis. These findings indicate that Chinese red propolis and CAPE may have potential as preventive and therapeutic agents against angiogenesis-related human diseases.

  11. VEGF receptor-2 (Flk-1 overexpression in mice counteracts focal epileptic seizures.

    Directory of Open Access Journals (Sweden)

    Litsa Nikitidou

    Full Text Available Vascular endothelial growth factor (VEGF was first described as an angiogenic agent, but has recently also been shown to exert various neurotrophic and neuroprotective effects in the nervous system. These effects of VEGF are mainly mediated by its receptor, VEGFR-2, which is also referred to as the fetal liver kinase receptor 1 (Flk-1. VEGF is up-regulated in neurons and glial cells after epileptic seizures and counteracts seizure-induced neurodegeneration. In vitro, VEGF administration suppresses ictal and interictal epileptiform activity caused by AP4 and 0 Mg(2+ via Flk-1 receptor. We therefore explored whether increased VEGF signaling through Flk-1 overexpression may regulate epileptogenesis and ictogenesis in vivo. To this extent, we used transgenic mice overexpressing Flk-1 postnatally in neurons. Intriguingly, Flk-1 overexpressing mice were characterized by an elevated threshold for seizure induction and a decreased duration of focal afterdischarges, indicating anti-ictal action. On the other hand, the kindling progression in these mice was similar to wild-type controls. No significant effects on blood vessels or glia cells, as assessed by Glut1 and GFAP immunohistochemistry, were detected. These results suggest that increased VEGF signaling via overexpression of Flk-1 receptors may directly affect seizure activity even without altering angiogenesis. Thus, Flk-1 could be considered as a novel target for developing future gene therapy strategies against ictal epileptic activity.

  12. Enhanced Vascularization in Hybrid PCL/Gelatin Fibrous Scaffolds with Sustained Release of VEGF

    Directory of Open Access Journals (Sweden)

    Kai Wang

    2015-01-01

    Full Text Available Creating a long-lasting and functional vasculature represents one of the most fundamental challenges in tissue engineering. VEGF has been widely accepted as a potent angiogenic factor involved in the early stages of blood vessel formation. In this study, fibrous scaffolds that consist of PCL and gelatin fibers were fabricated. The gelatin fibers were further functionalized by heparin immobilization, which provides binding sites for VEGF and thus enables the sustained release of VEGF. In vitro release test confirms the sustained releasing profile of VEGF, and stable release was observed over a time period of 25 days. In vitro cell assay indicates that VEGF release significantly promoted the proliferation of endothelial cells. More importantly, in vivo subcutaneous implantation reflects that vascularization has been effectively enhanced in the PCL/gelatin scaffolds compared with the PCL counterpart due to the sustained release of VEGF. Therefore, the heparinized PCL/gelatin scaffolds developed in this study may be a promising candidate for regeneration of complex tissues with sufficient vascularization.

  13. Bmx is a downstream Rap1 effector in VEGF-induced endothelial cell activation.

    Science.gov (United States)

    Stoletov, Konstantin V; Terman, Bruce I

    2004-07-16

    We had previously shown that Rap1 mediates certain of the signaling pathways involved in VEGF-induced endothelial cell migration, although the downstream Rap1 effectors are not known. Towards the goal of identifying those effectors, we utilized a commercially available antibody array filter to identify proteins that either directly interact with Rap1 or interact indirectly through a multi-protein complex. The protocol identified 10 possible Rap1-interacting proteins, including the Bmx non-receptor tyrosine kinase. The conclusion that VEGF treatment leads to a Rap1/Bmx complex was confirmed by an experiment in which cell lysates from VEGF and control cells were immunoprecipitated with Bmx antibodies and Western blotting was done using anti-Rap1 antibodies. VEGF treatment led to the recruitment of Bmx to the CAS scaffolding protein, and inhibition of the Bmx kinase blocked VEGF-induced cell migration. Formation of a Rap1/Bmx complex was not observed in cells transfected with an expression vector for a dominant-negative Rap1, indicating that Bmx is a downstream Rap1 effector in VEGF-induced endothelial cell activation.

  14. VEGF165 Stimulates Vessel Density and Vessel Diameter Differently in Angiogenesis and Lymphangiogenesis

    Science.gov (United States)

    Parsons-Wingerter, Patricia; Radhakrishnan, Krishnan; DiCorleto, Paul E.; Leontiev, Dmitry; Anand-Apte, Bela; Albarran, Brian; Farr, Andrew G.

    2005-01-01

    Vascular endothelial growth factor-165 (VEGF(sub 165)) stimulated angiogenesis in the quail chorioallantoic membrane (CAM) by vessel expansion from the capillary network. However, lymphangiogenesis was stimulated by the filopodial guidance of tip cells located on blind-ended lymphatic sprouts. As quantified by fractal/generational branching analysis using the computer code VESGEN, vascular density increased maximally at low VEGF concentrations, and vascular diameter increased most at high VEGF concentrations. Increased vascular density and diameter were statistically independent events (r(sub s), -0.06). By fluorescence immunohistochemistry of VEGF receptors VEGFR-1 and VEGFR-2, alpha smooth muscle actin ((alpha) SMA) and a vascular/lymphatic marker, VEGF(sub 165) increased the density and diameter of sprouting lymphatic vessels guided by tip cells (accompanied by the dissociation of lymphatics from blood vessels). Isolated migratory cells expressing (alpha)SMA were recruited to blood vessels, whereas isolated cells expressing VEGFR-2 were recruited primarily to lymphatics. In conclusion, VEGF(sub 165) increased lymphatic vessel density by lymphatic sprouting, but increased blood vessel density by vascular expansion from the capillary network.

  15. VEGF-targeted cancer therapeutics-paradoxical effects in endocrine organs.

    Science.gov (United States)

    Cao, Yihai

    2014-09-01

    Systemic administration of antiangiogenic drugs that target components of the vascular endothelial growth factor A (VEGF-A; VEGF) signal transduction pathway has become a viable therapeutic option for patients with various types of cancer. Nevertheless, these drugs can drive alterations in healthy vasculatures, which in turn are associated with adverse effects in healthy tissues. VEGF is crucial for vascular homeostasis and the maintenance of vascular integrity and architecture in endocrine organs. Given these critical physiological functions, systemic delivery of drugs that target VEGF signalling can block VEGF-mediated vascular functions in endocrine organs, such as the thyroid gland, and lead to endocrine dysfunction, including hypothyroidism, adrenal insufficiency and altered insulin sensitivity. This Review discusses emerging evidence from preclinical and clinical studies that contributes to understanding the mechanisms that underlie the vascular changes and subsequent modulations of endocrine function that are induced by targeted inhibition of VEGF signalling. Understanding these mechanisms is crucial for the design of antiangiogenic drugs with minimal associated adverse effects that will enable effective treatment of patients with cancer.

  16. Physical exercise and vascular endothelial growth factor (VEGF) in elderly: A systematic review.

    Science.gov (United States)

    Vital, Thays Martins; Stein, Angelica Miki; de Melo Coelho, Flávia Gomes; Arantes, Franciel José; Teodorov, Elizabeth; Santos-Galduróz, Ruth Ferreira

    2014-01-01

    The aim of this study was to conduct a systematic review of studies that verified the effects of physical exercise on vascular endothelial growth factor (VEGF) in elderly. The bibliographic search was conducted in five database, from 1990 to 2013, with the following keywords and boolean operators: physical exercise OR physical exercise OR physical therapy OR exercise OR training AND VEGF OR vascular endothelial growth factor AND aged OR older OR elderly. The inclusion criteria were: (1) sample including elderly with average age of 60; (2) studies that verified the effects of acute exercise; (3) studies that verified the effects of chronic physical exercise; (4) studies with humans; (5) randomized controlled trials, randomized non-controlled trials, non-randomized controlled trials, non-randomized and non-controlled trials; (6) assessment of VEGF peripheral concentrations. Ten studies were selected, and that four of them verified an increase of VEGF concentrations after practicing physical exercise and six studies did not verify any change on VEGF concentrations. Different populations found in this study and the different exercise protocols applied in the studies of this review make it difficult to establish parameters of what would be the best type of exercise to promote an increase on the concentrations of VEGF in the elderly. Therefore, we suggest that further studies can be performed, so that we can establish some recommendations for this population. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  17. Comparative VEGF receptor tyrosine kinase modeling for the development of highly specific inhibitors of tumor angiogenesis.

    Science.gov (United States)

    Schmidt, Ulrike; Ahmed, Jessica; Michalsky, Elke; Hoepfner, Michael; Preissner, Robert

    2008-01-01

    The Vascular Endothelial Growth Factor receptors (VEGF-Rs) play a significant role in tumor development and tumor angiogenesis and are therefore interesting targets in cancer therapy. Targeting the VEGF-R is of special importance as the feed of the tumor has to be reduced. In general, this can be carried out by inhibiting the tyrosine kinase function of the VEGF-R. Nevertheless, there arise some problems with the specificity of known kinase inhibitors: they bind to the ATP-binding site and inhibit a number of kinases, moreover the so far most specific inhibitors act at least on these three major types of VEGF-Rs: Flt-1, Flk-1/KDR, Flt-4. The goal is a selective VEGF-R-2 (Flk-1/KDR) inhibitor, because this receptor triggers rather unspecific signals from VEGF-A, -C, -D and -E. Here, we describe a protocol starting from an established inhibitor (Vatalanib) with 2D-/3D-searching and property filtering of the in silico screening hits and the "negative docking approach". With this approach we were able to identify a compound, which shows a fourfold higher reduction of the proliferation rate of endothelial cells compared to the reduction effect of the lead structure.

  18. MET Suppresses Epithelial VEGFR2 via Intracrine VEGF-induced Endoplasmic Reticulum-associated Degradation

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    Tom T. Chen

    2015-05-01

    Full Text Available Hepatocyte growth factor (HGF and vascular endothelial growth factor (VEGF drive cancer through their respective receptors, MET and VEGF receptor 2 (VEGFR2. VEGFR2 inhibits MET by promoting MET dephosphorylation. However, whether MET conversely regulates VEGFR2 remains unknown. Here we show that MET suppresses VEGFR2 protein by inducing its endoplasmic-reticulum-associated degradation (ERAD, via intracrine VEGF action. HGF–MET signaling in epithelial cancer cells promoted VEGF biosynthesis through PI3-kinase. In turn, VEGF and VEGFR2 associated within the ER, activating inositol-requiring enzyme 1α, and thereby facilitating ERAD-mediated depletion of VEGFR2. MET disruption upregulated VEGFR2, inducing compensatory tumor growth via VEGFR2 and MEK. However, concurrent disruption of MET and either VEGF or MEK circumvented this, enabling more profound tumor inhibition. Our findings uncover unique cross-regulation between MET and VEGFR2—two RTKs that play significant roles in tumor malignancy. Furthermore, these results suggest rational combinatorial strategies for targeting RTK signaling pathways more effectively, which has potentially important implications for cancer therapy.

  19. Cellular and molecular aspects of diabetic nephropathy; the role of VEGF-A.

    Science.gov (United States)

    Carranza, Katherine; Veron, Dolores; Cercado, Alicia; Bautista, Noemi; Pozo, Wilson; Tufro, Alda; Veron, Delma

    2015-01-01

    The prevalence of diabetes mellitus increased during the last century and it is estimated that 45% of the patients are not diagnosed. In South America the prevalence of diabetes and chronic kidney disease (CKD) increased, with a great disparity among the countries with respect to access to dialysis. In Ecuador it is one of the main causes of mortality, principally in the provinces located on the coast of the Pacific Ocean. The greatest single cause of beginning dialysis is diabetic nephropathy (DN). Even using the best therapeutic options for DN, the residual risk of proteinuria and of terminal CKD remains high. In this review we indicate the importance of the problem globally and in our region. We analyse relevant cellular and molecular studies that illustrate the crucial significance of glomerular events in DN development and evolution and in insulin resistance. We include basic anatomical, pathophysiological and clinical concepts, with special attention to the role of angiogenic factors such as the vascular endothelial growth factor (VEGF-A) and their relationship to the insulin receptor, endothelial isoform of nitric oxide synthase (eNOS) and angiopoietins. We also propose various pathways that have therapeutic potential in our opinion. Greater in-depth study of VEGF-A and angiopoietins, the state of glomerular VEGF resistance, the relationship of VEGF receptor 2/nephrin, VEGF/insulin receptors/nephrin and the relationship of VEGF/eNOS-NO at glomerular level could provide solutions to the pressing world problem of DN and generate new treatment alternatives.

  20. Bee products prevent VEGF-induced angiogenesis in human umbilical vein endothelial cells.

    Science.gov (United States)

    Izuta, Hiroshi; Shimazawa, Masamitsu; Tsuruma, Kazuhiro; Araki, Yoko; Mishima, Satoshi; Hara, Hideaki

    2009-11-17

    Vascular endothelial growth factor (VEGF) is a key regulator of pathogenic angiogenesis in diseases such as cancer and diabetic retinopathy. Bee products [royal jelly (RJ), bee pollen, and Chinese red propolis] from the honeybee, Apis mellifera, have been used as traditional health foods for centuries. The aim of this study was to investigate the anti-angiogenic effects of bee products using human umbilical vein endothelial cells (HUVECs). In an in vitro tube formation assay, HUVECs and fibroblast cells were incubated for 14 days with VEGF and various concentrations of bee products [RJ, ethanol extract of bee pollen, ethanol extract of Chinese red propolis and its constituent, caffeic acid phenethyl ester (CAPE)]. To clarify the mechanism of in vitro angiogenesis, HUVEC proliferation and migration were induced by VEGF with or without various concentrations of RJ, bee pollen, Chinese red propolis, and CAPE. RJ, bee pollen, Chinese red propolis, and CAPE significantly suppressed VEGF-induced in vitro tube formation in the descending order: CAPE > Chinese red propolis > bee pollen > RJ. RJ and Chinese red propolis suppressed both VEGF-induced HUVEC proliferation and migration. In contrast, bee pollen and CAPE suppressed only the proliferation. Among the bee products, Chinese red propolis and CAPE in particular showed strong suppressive effects against VEGF-induced angiogenesis. These findings indicate that Chinese red propolis and CAPE may have potential as preventive and therapeutic agents against angiogenesis-related human diseases.

  1. Effect of VEGF on the Regenerative Capacity of Muscle Stem Cells in Dystrophic Skeletal Muscle

    Science.gov (United States)

    Deasy, Bridget M; Feduska, Joseph M; Payne, Thomas R; Li, Yong; Ambrosio, Fabrisia; Huard, Johnny

    2009-01-01

    We have isolated a population of muscle-derived stem cells (MDSCs) that, when compared with myoblasts, display an improved regeneration capacity, exhibit better cell survival, and improve myogenesis and angiogenesis. In addition, we and others have observed that the origin of the MDSCs may reside within the blood vessel walls (endothelial cells and pericytes). Here, we investigated the role of vascular endothelial growth factor (VEGF)–mediated angiogenesis in MDSC transplantation–based skeletal muscle regeneration in mdx mice (an animal model of muscular dystrophy). We studied MDSC and MDSC transduced to overexpress VEGF; no differences were observed in vitro in terms of phenotype or myogenic differentiation. However, after in vivo transplantation, we observe an increase in angiogenesis and endogenous muscle regeneration as well as a reduction in muscle fibrosis in muscles transplanted with VEGF-expressing cells when compared to control cells. In contrast, we observe a significant decrease in vascularization and an increase in fibrosis in the muscles transplanted with MDSCs expressing soluble forms-like tyrosine kinase 1 (sFlt1) (VEGF-specific antagonist) when compared to control MDSCs. Our results indicate that VEGF-expressing cells do not increase the number of dystrophin-positive fibers in the injected mdx muscle, when compared to the control MDSCs. Together the results suggest that the transplantation of VEGF-expressing MDSCs improved skeletal muscle repair through modulation of angiogenesis, regeneration and fibrosis in the injected mdx skeletal muscle. PMID:19603004

  2. A novel gene delivery system targeting cells expressing VEGF receptors

    Institute of Scientific and Technical Information of China (English)

    LIJUNMIN; JINGCHULUO; 等

    1999-01-01

    Two ligand oligopeptides GV1 and GV2 were designed according to the putative binding region of VEGF to its receptors.GV1,GV2 and endosome releasing oligopeptide HA20 were conjugated with poly-L-lysine or protamine and the resulting conjugates could interact with DNA in a noncovalent bond to form a complex.Using pSV2-β-galactosidase as a reporter gene,it has been demonstrated that exogenous gene was transferred into bovine aortic arch-derived endothelial cells (ABAE) and human malignant melanoma cell lines (A375) in vitro.In vivo experiments,exogenous gene was transferred into tumor vascular endothelial cells and tumor cells of subcutaneously transplanted human colon cancer LOVO,human malignant melanoma A375 and human hepatoma graft in nude mice.This system could also target gene to intrahepatically transplanted human hepatoma injected via portal vein in nude mice.These results are correlated with the relevant receptors(flt-1,flk-1/KDR) expression on the targeted cells and tissues.

  3. Treatment of Corneal Neovascularization Using Anti-VEGF Bevacizumab

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    Deli Krizova

    2014-01-01

    Full Text Available Purpose. To evaluate antiangiogenic effect of local use of bevacizumab (anti-VEGF antibody in patients with corneal neovascularization. Methods. Patients were divided into two groups. All patients suffered from some form of corneal neovascularization (NV. Patients in group A received 0.2–0.5 mL of bevacizumab solution subconjunctivally (concentration 25 mg/mL in a single dose. Group A included 28 eyes from 27. Patients in group B applied bevacizumab eye drops twice daily (concentration 2.5 mg/mL for two weeks. Group B included 38 eyes from 35 patients. We evaluated the number of corneal segments affected by NV, CDVA, and the incidence of complications and subjective complaints related to the treatment. The minimum follow-up period was six months. Results. By the 6-month follow-up, in group A the percentage reduction of the affected peripheral segments was 21.6% and of the central segments was 9.6%; in group B the percentage reduction of the central segments was 22.7% and of the central segments was 38.04%. In both groups we noticed a statistically significant reduction in the extent of NV. Conclusion. The use of bevacizumab seems to be an effective and safe method in the treatment of corneal neovascularization, either in the subconjunctival or topical application form.

  4. EXPRESSION OF VEGF RECEPTOR KDR IN DIFFERENT ORIGINATED CARCINOMAS

    Institute of Scientific and Technical Information of China (English)

    Song Shumei; Wujian; Shou Chengchao

    1998-01-01

    Objective: To detect the expression of KDR in different originated carcinomas and to explore its expressed ways and the relationship with tumor progression. Methods: KDR cDNA (Ⅴ-Ⅶ domains)fragment was cloned from human umbilical vein with RTPCR and was expressed in Ecoli.Jm109. The fusion protein of GST-KDR was used for immunizing Balb/c mice to prepare monoclonal antibodies against KDR.The different tumor tissues and related normal tissues were examined with KDR McAb by S-P immunohistochemistry. Results: the rate and intensification of KDR expression among different originated cancers are very different, bladder cancers from transmigrated epidermis are 100% positive and highest intensification.The expression of KDR in breast cancer and intestinal cancer lie in the second-rate, the weakest expression of KDR is in lung squamous carcinoma. Moreover,expression of KDR in tumor tissues lie both in endothelial cells (EC) of tumor blood vessels and tumor cells.Conclusion: VEGF may be not only the para-secretory factor making EC proliferation but also auto-secretory factor stimulating the proliferation of tumor cells to benefit the growth and metastasis of malignant tumors.The different expression of KDR in different originated carcinomas may relate with malignant degree of tumor.

  5. Spinal vascular endothelial growth factor (VEGF) and erythropoietin (EPO) induced phrenic motor facilitation after repetitive acute intermittent hypoxia.

    Science.gov (United States)

    Dale, Erica A; Mitchell, Gordon S

    2013-02-01

    Vascular endothelial growth factor (VEGF) and erythropoietin (EPO) exert neurotrophic and neuroprotective effects in the CNS. We recently demonstrated that VEGF, EPO and their receptors (VEGF-R2, EPO-R) are expressed in phrenic motor neurons, and that cervical spinal VEGF-R2 and EPO-R activation elicit long-lasting phrenic motor facilitation (pMF). Since VEGF, VEGF-R, EPO, and EPO-R are hypoxia-regulated genes, and repetitive exposure to acute intermittent hypoxia (rAIH) up-regulates these molecules in phrenic motor neurons, we tested the hypothesis that 4 weeks of rAIH (10 episodes per day, 3 days per week) enhances VEGF- or EPO-induced pMF. We confirm that cervical spinal VEGF and EPO injections elicit pMF. However, neither VEGF- nor EPO-induced pMF was affected by rAIH pre-conditioning (4 wks). Although our data confirm that spinal VEGF and EPO may play an important role in respiratory plasticity, we provide no evidence that rAIH amplifies their impact. Further experiments with more robust protocols are warranted.

  6. Enhanced effect of VEGF165 on L-type calcium currents in guinea-pig cardiac ventricular myocytes.

    Science.gov (United States)

    Xing, Wenlu; Gao, Chuanyu; Qi, Datun; Zhang, You; Hao, Peiyuan; Dai, Guoyou; Yan, Ganxin

    2017-01-01

    The mechanisms of vascular endothelial growth factor 165 (VEGF165) on electrical properties of cardiomyocytes have not been fully elucidated. The aim of this study is to test the hypothesis that VEGF165, an angiogenesis-initiating factor, affects L-type calcium currents (ICa,L) and cell membrane potential in cardiac myocytes by acting on VEGF type-2 receptors (VEGFR2). ICa,L and action potentials (AP) were recorded by the whole-cell patch clamp method in isolated guinea-pig ventricular myocytes treated with different concentrations of VEGF165 proteins. Using a VEGFR2 inhibitor, we also tested the receptor of VEGF165 in cardiomyocytes. We found that VEGF165 increased ICa,L in a concentration-dependent manner. SU5416, a VEGFR2 inhibitor, almost completely eliminated VEGF165-induced ICa,L increase. VEGF165 had no significant influence on action potential 90 (APD90) and other properties of AP. We conclude that in guinea-pig ventricular myocytes, ICa,L can be increased by VEGF165 in a concentration-dependent manner through binding to VEGFR2 without causing any significant alteration to action potential duration. Results of this study may further expound the safety of VEGF165 when used in the intervention of heart diseases.

  7. Hypertonic stress induces VEGF production in human colon cancer cell line Caco-2: inhibitory role of autocrine PGE₂.

    Directory of Open Access Journals (Sweden)

    Luciana B Gentile

    Full Text Available Vascular Endothelial Growth Factor (VEGF is a major regulator of angiogenesis. VEGF expression is up regulated in response to micro-environmental cues related to poor blood supply such as hypoxia. However, regulation of VEGF expression in cancer cells is not limited to the stress response due to increased volume of the tumor mass. Lipid mediators in particular arachidonic acid-derived prostaglandin (PGE₂ are regulators of VEGF expression and angiogenesis in colon cancer. In addition, increased osmolarity that is generated during colonic water absorption and feces consolidation seems to activate colon cancer cells and promote PGE₂ generation. Such physiological stimulation may provide signaling for cancer promotion. Here we investigated the effect of exposure to a hypertonic medium, to emulate colonic environment, on VEGF production by colon cancer cells. The role of concomitant PGE₂ generation and MAPK activation was addressed by specific pharmacological inhibition. Human colon cancer cell line Caco-2 exposed to a hypertonic environment responded with marked VEGF and PGE₂ production. VEGF production was inhibited by selective inhibitors of ERK 1/2 and p38 MAPK pathways. To address the regulatory role of PGE₂ on VEGF production, Caco-2 cells were treated with cPLA₂ (ATK and COX-2 (NS-398 inhibitors, that completely block PGE₂ generation. The Caco-2 cells were also treated with a non selective PGE₂ receptor antagonist. Each treatment significantly increased the hypertonic stress-induced VEGF production. Moreover, addition of PGE₂ or selective EP₂ receptor agonist to activated Caco-2 cells inhibited VEGF production. The autocrine inhibitory role for PGE₂ appears to be selective to hypertonic environment since VEGF production induced by exposure to CoCl₂ was decreased by inhibition of concomitant PGE₂ generation. Our results indicated that hypertonicity stimulates VEGF production in colon cancer cell lines. Also PGE

  8. Recombineering linear BACs.

    Science.gov (United States)

    Chen, Qingwen; Narayanan, Kumaran

    2015-01-01

    Recombineering is a powerful genetic engineering technique based on homologous recombination that can be used to accurately modify DNA independent of its sequence or size. One novel application of recombineering is the assembly of linear BACs in E. coli that can replicate autonomously as linear plasmids. A circular BAC is inserted with a short telomeric sequence from phage N15, which is subsequently cut and rejoined by the phage protelomerase enzyme to generate a linear BAC with terminal hairpin telomeres. Telomere-capped linear BACs are protected against exonuclease attack both in vitro and in vivo in E. coli cells and can replicate stably. Here we describe step-by-step protocols to linearize any BAC clone by recombineering, including inserting and screening for presence of the N15 telomeric sequence, linearizing BACs in vivo in E. coli, extracting linear BACs, and verifying the presence of hairpin telomere structures. Linear BACs may be useful for functional expression of genomic loci in cells, maintenance of linear viral genomes in their natural conformation, and for constructing innovative artificial chromosome structures for applications in mammalian and plant cells.

  9. Recombinant DNA for Teachers.

    Science.gov (United States)

    Duvall, James G., III

    1992-01-01

    A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

  10. Recombinant renewable polyclonal antibodies.

    Science.gov (United States)

    Ferrara, Fortunato; D'Angelo, Sara; Gaiotto, Tiziano; Naranjo, Leslie; Tian, Hongzhao; Gräslund, Susanne; Dobrovetsky, Elena; Hraber, Peter; Lund-Johansen, Fridtjof; Saragozza, Silvia; Sblattero, Daniele; Kiss, Csaba; Bradbury, Andrew R M

    2015-01-01

    Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.

  11. Anemia and elevated systemic levels of vascular endothelial growth factor (VEGF)

    Energy Technology Data Exchange (ETDEWEB)

    Dunst, J.; Becker, A.; Lautenschlaeger, C.; Markau, S.; Becker, H.; Fischer, K.; Haensgen, G. [Martin-Luther Univ. Halle-Wittenberg (Germany)

    2002-08-01

    Background: Tissue hypoxia is a major stimulus for the up-regulation of vascular endothelial growth factor (VEGF). Anemia might theoretically impact on angiogenesis via impairment of tissue oxygenation. We have investigated this hypothesis in patients with solid cancers and benign diseases. Patients and methods: 49 patients with untreated locoregionally confined solid cancers of the head and neck, cervix, rectum and lung and 59 additional patients with non-malignant diseases (36 normemic patients without serious diseases and 23 patients with renal anemia) were enrolled and the impact of anemia on plasma VEGF levels were determined. VEGF was measured with a commercially available sandwich enzyme immunoassay technique. Results: Plasma levels of VEGF were 16.2{+-}12.7 pg/ml in 36 normemic patients without malignant disease, 49,2{+-}34.5 pg/ml in 49 patients with cancers (p<0.001), and 89.9{+-}67.8 pg/ml in 23 patients with renal anemia (p=0.001). VEGF levels in cancer patients were significantly correlated with hemoglobin (hb) levels and platelet counts (each p=0.001), but not with type of tumor, stage, histology or age. Patients with cancers had higher plasma levels of VEGF than patients with non-malignant diseases in case of hb{>=}12 g/dl (33.1{+-}17.5 vs. 16.6{+-}13.0 pg/ml, p<0.001) and in case of hb between 11.0 and 11.9 g/dl (56.1{+-}26.4 vs 18.5{+-}14.5 pg/ml, p=0.038). In case of a hb<11 g/dl, plasma VEGF levels were significantly elevated in patients with and without cancers (67.0{+-}47.5 vs 88.9{+-}68.8 pg/ml, n.s.). In a multivariate model, a significant association between low hb levels and increased plasma levels of VEGF was confirmed. In 16 patients with renal anemia, changes in hb under erythropoietin treatment were inversely correlated with changes in plasma VEGF levels with decreasing VEGF after increase in hb (p=0.01). Conclusions: Anemic patients have elevated levels of VEGF. The data suggest that anemia might impact on the progression of

  12. Radiation-induced VEGF-C expression and endothelial cell proliferation in lung cancer

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yu-Hsuan [National Taiwan University Hospital, Department of Oncology, Taipei (China); National Taiwan University, Pharmacological Institute, College of Medicine, Taipei (China); Pan, Shiow-Lin; Wang, Jing-Chi; Teng, Che-Ming [National Taiwan University, Pharmacological Institute, College of Medicine, Taipei (China); Kuo, Sung-Hsin [National Taiwan University Hospital, Department of Oncology, Taipei (China); National Taiwan University College of Medicine, Department of Internal Medicine, Taipei (China); Cheng, Jason Chia-Hsien [National Taiwan University Hospital, Department of Oncology, Taipei (China); National Taiwan University College of Medicine, Graduate Institute of Clinical Medicine, Taipei (China)

    2014-12-15

    The present study was undertaken to investigate whether radiation induces the expression of vascular endothelial growth factor C (VEGF-C) through activation of the PI3K/Akt/mTOR pathway,subsequently affecting endothelial cells. Radiotherapy-induced tumor micro-lymphatic vessel density (MLVD) was determined in a lung cancer xenograft model established in SCID mice. The protein expression and phosphorylation of members of the PI3K/Akt/mTOR pathway and VEGF-C secretion and mRNA expression in irradiated lung cancer cells were assessed by Western blot analysis, enzyme-linked immunosorbent assays (ELISAs), and reverse transcriptase-polymerase chain reaction (RT-PCR). Moreover, specific chemical inhibitors were used to evaluate the role of the PI3K/Akt/mTOR signaling pathway. Conditioned medium (CM) from irradiated control-siRNA or VEGF-C-siRNA-expressing A549 cells was used to evaluate the proliferation of endothelial cells by the MTT assay. Radiation increased VEGF-C expression in a dose-dependent manner over time at the protein but not at the mRNA level. Radiation also up-regulated the phosphorylation of Akt, mTOR, 4EBP, and eIF4E, but not of p70S6K. Radiation-induced VEGF-C expression was down-regulated by LY294002 and rapamycin (both p < 0.05). Furthermore, CM from irradiated A549 cells enhanced human umbilical vein endothelial cell (HUVEC) and lymphatic endothelial cell (LEC) proliferation, which was not observed with CM from irradiated VEGF-C-siRNA-expressing A549 cells. Radiation-induced activation of the PI3K/Akt/mTOR signaling pathway increases VEGF-C expression in lung cancer cells, thereby promoting endothelial cell proliferation. (orig.) [German] Die vorliegende Studie untersucht, ob die Strahlung die Expression von VEGF-C (vascular endothelial growth factor C) mittels Aktivierung des PI3K/Akt/mTOR-Signalwegs induziert und anschliessend die endothelialen Zellen beeinflusst. Die durch Strahlentherapie induzierte Mikrolymphgefaessdichte (MLVD) im Tumor wurde in

  13. Treadmill Exercise Promotes Neurogenesis in Ischemic Rat Brains via Caveolin-1/VEGF Signaling Pathways.

    Science.gov (United States)

    Zhao, Yun; Pang, Qiongyi; Liu, Meixia; Pan, Jingzi; Xiang, Bingwu; Huang, Tingting; Tu, Fengxia; Liu, Chan; Chen, Xiang

    2017-02-01

    Using a model of middle cerebral artery occlusion (MCAO), we have previously demonstrated that treadmill exercise promotes angiogenesis in the ischemic penumbra through caveolin-1/VEGF signaling pathways. However, the function of caveolin-1/VEGF signaling in neurogenesis after MCAO has not been determined. In this study, we aimed to investigate the potential of treadmill exercise to promote neurogenesis after MCAO and whether caveolin-1/VEGF signaling pathways are involved. After MCAO, rats were subjected to a program of treadmill exercise. Daidzein (a specific inhibitor of caveolin-1 protein expression, 0.4 mg/kg) was used to confirm the effect of caveolin-1/VEGF signaling on exercise-mediated neurogenesis. We found that the total protein expression of both caveolin-1 and VEGF was increased by exercise and consistent with the improved neurological recovery, decreased infarct volumes and increased 5-bromo-2'-deoxyuridine (BrdU) in the ipsilateral Subventricular zone (SVZ), as well as increased numbers of BrdU/DCX and BrdU/Neun-positive cells in the peri-infarct region. Furthermore, we observed that the treadmill exercise-induced increased VEGF expression, improved neurological recovery, decreased infarct volumes, increased BrdU/DCX and BrdU/Neun-positive cells were significantly inhibited by the caveolin-1 inhibitor. Our results indicate that treadmill exercise improves neurological recovery in ischemic rats, possibly by enhancement of SVZ-derived neural stem cell (NSC) proliferation, migration and differentiation in the penumbra. Moreover, caveolin-1/VEGF signaling is involved in exercise-mediated NSC migration and neuronal differentiation.

  14. Expression of HIF-1A/VEGF/ING-4 Axis in Pulmonary Sarcoidosis.

    Science.gov (United States)

    Piotrowski, W J; Kiszałkiewicz, J; Pastuszak-Lewandoska, D; Górski, P; Antczak, A; Migdalska-Sęk, M; Górski, W; Czarnecka, K H; Domańska, D; Nawrot, E; Brzeziańska-Lasota, E

    2015-01-01

    Angiogenesis/angiostasis regulated by hypoxia inducible factor-1A (HIF-1A)/vascular endothelial growth factor (VEGF)/inhibitor of growth protein 4 (ING-4) axis may be crucial for the course and outcome of sarcoidosis. Overexpression of angiogenic factors (activation of VEGF through HIF-1A) may predispose to chronic course and lung fibrosis, whereas immunoangiostasis (related to an overexpression of inhibitory ING-4) may be involved in granuloma formation in early sarcoid inflammation, or sustained or recurrent formation of granulomas. In this work we investigated gene expression of HIF-1A, VEGF and ING-4 in bronchoalveolar fluid (BALF) cells and in peripheral blood (PB) lymphocytes of sarcoidosis patients (n=94), to better understand mechanisms of the disease and to search for its biomarkers. The relative gene expression level (RQ value) was analyzed by qPCR. The results were evaluated according to the presence of lung parenchymal involvement (radiological stage I vs. II-IV), acute vs. insidious onset, lung function tests, calcium metabolism parameters, percentage of lymphocytes (BALL%) and BAL CD4+/CD8+ in BALF, age, and gender. In BALF cells, the ING-4 and VEGF RQ values were increased, while HIF-1A expression was decreased. In PB lymphocytes all studied genes were overexpressed. Higher expression of HIF-1A in PB lymphocytes of patients with abnormal spirometry, and in BALF cells of patients with lung volume restriction was found. VEGF gene expression in BALF cells was also higher in patients with abnormal spirometry. These findings were in line with previous data on the role of HIF-1A/VEGF/ING-4 axis in the pathogenesis of sarcoidosis. Up-regulated HIF-1A and VEGF genes are linked to acknowledged negative prognostics.

  15. Corticosteroid Suppression of VEGF-A in Infantile Hemangioma-Derived Stem Cells

    Science.gov (United States)

    Greenberger, Shoshana; Boscolo, Elisa; Adini, Irit; Mulliken, John B.; Bischoff, Joyce

    2010-01-01

    BACKGROUND Corticosteroids are commonly used to treat infantile hemangioma, but the mechanism of action of this therapy is unknown. We investigated the effect of corticosteroids in a previously described in vivo model of infantile hemangioma and in cultured hemangioma-derived cells. METHODS We tested hemangioma-derived stem cells for vasculogenic activity in vivo after implantation into immune-deficient (nude) mice. We studied dexamethasone treatment of both the cells before implantation and the mice after implantation. We also tested hemangioma-derived stem cells for expression of vascular endothelial growth factor A (VEGF-A) in vitro and studied the inhibition of VEGF-A expression, using short hairpin RNA (shRNA) in vivo and in vitro. RESULTS Systemic treatment with dexamethasone led to dose-dependent inhibition of tumor vasculogenesis in the murine model. Pretreatment of hemangioma-derived stem cells in vitro before implantation also inhibited vasculogenesis. Dexamethasone suppressed VEGF-A production by hemangioma-derived stem cells in vitro but not by hemangioma-derived endothelial cells or human umbilical-vein endothelial cells. Silencing VEGF-A in hemangioma-derived stem cells reduced vasculogenesis in vivo. VEGF-A was detected in hemangioma specimens in the proliferating phase but not in the involuting phase and was shown by immunostaining to reside outside of vessels. Corticosteroid treatment suppressed other proangiogenic factors in hemangioma-derived stem cells, including urokinase plasminogen activator receptor, interleukin-6, monocyte chemoattractant protein 1, and matrix metalloproteinase 1. CONCLUSIONS In a murine model, dexamethasone inhibited the vasculogenic potential of stem cells derived from human infantile hemangioma. The corticosteroid also inhibited the expression of VEGF-A by hemangioma-derived stem cells, and silencing of VEGF-A expression in these cells inhibited vasculogenesis in vivo. PMID:20237346

  16. The angiogenic growth factors HGF and VEGF in serum and plasma from neuroblastoma patients.

    Science.gov (United States)

    Sköldenberg, Erik G; Larsson, Anders; Jakobson, Ake; Hedborg, Fredrik; Kogner, Per; Christofferson, Rolf H; Azarbayjani, Faranak

    2009-08-01

    To determine whether concentrations of the angiogenic growth factors hepatocyte growth factor (HGF) and vascular endothelial growth factor A (VEGF-A) correlate with clinical and genetic markers in samples taken at diagnosis in children with neuroblastoma (NB). Heparin plasma (P-) and serum (S-) samples of healthy controls (n=73, mean age +/- SD 3.5+/-2.1; females/males: 23/50) and patients with NB (n=62; 2.2+/-1.8; 26/36) were collected between 1988 and 1999. Clinical data included age at diagnosis, gender, stage, outcome, amplification of the oncogene MYCN, loss of heterozygosity at the short arm of chromosome 1 (1p LOH) and ploidy. HGF and S-VEGF-A were elevated in NB as compared to controls (38/62 patients, p<0.0001 and p<0.05, Mann-Whitney U test). HGF concentrations were higher in high-stage (stage 3-4) as compared to low-stage (stage 1-2) disease (p<0.01). P-HGF was elevated in patients with 1p LOH (p<0.01), MYCN amplification (p<0.001) and di- or tetraploidy (p<0.001). S-HGF concentration was elevated in patients MYCN-amplified tumors only. Plasma and S-HGF concentrations were higher in the deceased group (p<0.05), but not P or S-VEGF-A. This study showed that concentrations of HGF and S-VEGF-A are elevated in patients with NB. Furthermore, HGF and S-VEGF-A concentrations correlate to higher stage disease and HGF correlates to genetic markers known to indicate a poor outcome. These observations imply that HGF and VEGF-A have biological roles in NB and suggest the possibility of interference with HGF or VEGF-A signaling as a therapeutic strategy.

  17. The protective effect of the RAS inhibitor on diabetic patients with nephropathy in the context of VEGF suppression

    Institute of Scientific and Technical Information of China (English)

    Haibing CHEN; Junxi LU; Qing LI; Yuqian BAO; Junling TANG; Huijuan LU; Kunsan XIANG; Weiping JIA

    2009-01-01

    Aim:The aim of the present study was to explore whether renin angiotensin system (RAS) inhibitor can reduce the produc-tion of vascular endothelium growth factor (VEGF).Further,we sought to elucidate the correlation between VEGF level and certain clinical parameters,such as albumin excretion rate (AER),before and after treatment with angiotensin type 1 receptor blocker.Methods: We recruited 166 type 2 diabetic patients at various stages of diabetic nephropathy (DN) and 46 healthy control subjects for a cross-sectional study.We recruited another 42 hypertensive type 2 diabetic patients with microalbuminuria for a longitudinal study involving a 6-month irbesartan treatment protocol.Urinary VEGF (uVEGF) levels were determined using ELISA.Results: In the cross-sectional study,hypertensive type 2 diabetic patients who received RAS inhibitor presented lower uVEGF levels than those who did not receive the RAS inhibitor.Statistical analysis indicated that uVEGF level was independently correlated with the AER.In the longitudinal study involving the 6-month irbesartan treatment,we demonstrated that uVEGF levels decreased significantly in patients who achieved a 50% AER reduction (remission group,n=32).In contrast,uVEGF levels remained unchanged in patients who did not exhibit a 50% AER reduction (nonremission group,n=10).Furthermore,the change in uVEGF was significantly correlated with the change in AER (r=0.65,P<0.01) before and after 6 months of irbesartan treatment.This result held true even after we had adjusted for the decrease in average blood pressure.Conclusion: The protective effect of the RAS inhibitor in DN patients is associated with the suppression of VEGF.Accordingly,it may be possible to use uVEGF as a marker of DN progression.We suggest that uVEGF may be an important target for therapeutic intervention in the context of DN.

  18. Improvement in autologous human fat transplant survival with SVF plus VEGF-PLA nano-sustained release microspheres.

    Science.gov (United States)

    Li, Liqun; Pan, Shengsheng; Ni, Binting; Lin, Yuanshao

    2014-08-01

    Early neovascularization is important for autologous fat transplant survival. SVF cells are ideal seed cells. Both vascular endothelial growth factor (VEGF) and SVF cells can promote neovascularization. However, the half-life (about 50 min) of VEGF is too short to sustain an adequate local concentration. We have investigated whether VEGF-polylactic acid (PLA) nano-sustained release microspheres plus SVF cells can improve neovascularization and survival of transplanted fat tissues. SVF cells were harvested and constructed VEGF-PLA nano-sustained release microspheres in vitro. Human fat tissues was mixed with SVF cells plus VEGF-PLA, SVF cells alone or Dulbecco's modified Eagle's medium as the control. These three mixtures were injected into random sites in 18 nude mice. Two months later, the transplants were weighed and examined histologically; and capillaries were counted to quantify neovascularization. Hematoxylin-eosin (HE) and anti-VEGF stains were applied to reveal cell infiltration. The mean wet weight of fat in the SVF plus VEGF-PLA, SVF alone, and control transplants were 0.18 ± 0.013 g, 0.16 ± 0.015 g, and 0.071 ± 0.12 g, respectively; the differences between groups were statistically significant. More vessels were present in the SVF plus VEGF-PLA transplants than in the other two types. Transplants mixed with SVF cells also had an acceptable density of capillaries. Histological analysis revealed that both the SVF plus VEGF-PLA and SVF alone transplants, but not the control transplants, were composed of adipose tissue, and had less fat necrosis and less fibrosis than control specimens. SVF plus VEGF-PLA transplants had significantly greater capillary density and VEGF expression than the other two transplant groups. Thus transplanted fat tissue survival and quality can be enhanced by the addition of VEGF-PLA nano-sustained release microspheres plus SVF cells.

  19. Clinical significance of co-expression of VEGF-C and VEGFR-3 in non-small cell lung cancer

    Institute of Scientific and Technical Information of China (English)

    李庆昌; 董昕; 顾伟; 邱雪杉; 王恩华

    2003-01-01

    Objective To investigate the relationship between vascular endothelial growth factor C (VEGF-C) expression, VEGFR-3 expression, lymphangiogenesis and angiogenesis in human non-small cell lung cancer (NSCLC).Methods Seventy-six NSCLC samples were stained for VEGF-C, VEGFR-3 and CD34 with immunohistochemical methods. Assessment of lymphatic vessel density (LVD) and microvessel density (MVD) was performed. The expressions of VEGF-C in 24 fresh NSCLC samples were determined with Western blot assay.Results Of the 76 NSCLC cases, 55 were VEGF-C positive and 40 were VEGFR-3 positive in cancer cells. A significant positive correlation was found between VEGF-C expression and VEGFR-3 expression in cancer cells (P<0.05). VEGF-C expression was negatively associated with differentiation of tumor cells (P<0.05). VEGF-C expression and VEGFR-3 expression were positively associated with lymph node metastasis and lymphatic invasion (P<0.05). LVD was positively related to VEGF-C expression, lymph node metastasis, lymphatic invasion and clinical stage (P<0.05). There was a significant correlation between LVD and MVD (R=0.732, P<0.05). Patients with positive VEGF-C expression had worse outcomes than those with negative VEGF-C expression (P<0.001).Conclusions In NSCLC, VEGF-C and VEGFR-3 are related to the lymphangiogenesis, angiogenesis, and occurrence and development of lung cancers. VEGF-C expression could be a useful predictor of poor prognosis in NSCLC.

  20. An increase in vascular endothelial growth factor (VEGF and VEGF soluble receptor-1 (sFlt-1 are associated with early recurrent spontaneous abortion.

    Directory of Open Access Journals (Sweden)

    Lihong Pang

    Full Text Available Recurrent spontaneous abortion (RSA is a health problem that affects approximately 1% to 5% reproductive age woman. Yet, in around half of these patients, the mechanism for RSA is unexplained. Recent studies have indicated that placental ischemia/hypoxia and endothelial dysfunction are important factors in miscarriage. Other studies have indicated that the level and expression of soluble FMS-like tyrosine kinase-1 (sFlt1 is increased under a hypoxic environment. However, decreased sFlt-1 in the maternal circulation during the first trimester has recently been proposed as a potential marker for identifying risk of pregnancy loss. In this prospective study clinical samples were obtained within a short time after the fetal death, protein expression and maternal serum levels of sFlt1 were assessed and compared to samples taken from those with normal pregnancies. Our results indicate that levels of VEGF and sFlt-1 are both increased in women during early pregnancy compared women that are not pregnant (p<0.05 indicating that VEGF and sFlt-1 are both associated with pregnancy. More importantly, we detected a significant (p<0.05 increase in sFlt1 and VEGF levels and expression in the RSA patients who suffered subsequent miscarriages compare to controls. These results demonstrate that there is likely a relationship between VEGF, sFlt-1 and RSA suggesting that the high levels and over expression of sFlt-1 and VEGF might be associated with the pathogenesis of RSA.

  1. Sustained release of VEGF from PLGA nanoparticles embedded thermo-sensitive hydrogel in full-thickness porcine bladder acellular matrix

    Directory of Open Access Journals (Sweden)

    Song Hua

    2011-01-01

    Full Text Available Abstract We fabricated a novel vascular endothelial growth factor (VEGF-loaded poly(lactic-co-glycolic acid (PLGA-nanoparticles (NPs-embedded thermo-sensitive hydrogel in porcine bladder acellular matrix allograft (BAMA system, which is designed for achieving a sustained release of VEGF protein, and embedding the protein carrier into the BAMA. We identified and optimized various formulations and process parameters to get the preferred particle size, entrapment, and polydispersibility of the VEGF-NPs, and incorporated the VEGF-NPs into the (poly(ethylene oxide-poly(propylene oxide-poly(ethylene oxide (Pluronic® F127 to achieve the preferred VEGF-NPs thermo-sensitive gel system. Then the thermal behavior of the system was proven by in vitro and in vivo study, and the kinetic-sustained release profile of the system embedded in porcine bladder acellular matrix was investigated. Results indicated that the bioactivity of the encapsulated VEGF released from the NPs was reserved, and the VEGF-NPs thermo-sensitive gel system can achieve sol-gel transmission successfully at appropriate temperature. Furthermore, the system can create a satisfactory tissue-compatible environment and an effective VEGF-sustained release approach. In conclusion, a novel VEGF-loaded PLGA NPs-embedded thermo-sensitive hydrogel in porcine BAMA system is successfully prepared, to provide a promising way for deficient bladder reconstruction therapy.

  2. Mechanical Forces Induce Changes in VEGF and VEGFR-1/sFlt-1 Expression in Human Chondrocytes

    Directory of Open Access Journals (Sweden)

    Rainer Beckmann

    2014-09-01

    Full Text Available Expression of the pro-angiogenic vascular endothelial growth factor (VEGF stimulates angiogenesis and correlates with the progression of osteoarthritis. Mechanical joint loading seems to contribute to this cartilage pathology. Cyclic equibiaxial strains of 1% to 16% for 12 h, respectively, induced expression of VEGF in human chondrocytes dose- and frequency-dependently. Stretch-mediated VEGF induction was more prominent in the human chondrocyte cell line C-28/I2 than in primary articular chondrocytes. Twelve hours of 8% stretch induced VEGF expression to 175% of unstrained controls for at least 24 h post stretching, in promoter reporter and enzyme-linked immunosorbent assay (ELISA studies. High affinity soluble VEGF-receptor, sVEGFR-1/sFlt-1 was less stretch-inducible than its ligand, VEGF-A, in these cells. ELISA assays demonstrated, for the first time, a stretch-mediated suppression of sVEGFR-1 secretion 24 h after stretching. Overall, strained chondrocytes activate their VEGF expression, but in contrast, strain appears to suppress the secretion of the major VEGF decoy receptor (sVEGFR-1/sFlt-1. The latter may deplete a biologically relevant feedback regulation to inhibit destructive angiogenesis in articular cartilage. Our data suggest that mechanical stretch can induce morphological changes in human chondrocytes in vitro. More importantly, it induces disturbed VEGF signaling, providing a molecular mechanism for a stress-induced increase in angiogenesis in cartilage pathologies.

  3. Abnormal expression of vascular endothelial growth factor (VEGF) and its clinical features in tissues of human lung cancer

    Institute of Scientific and Technical Information of China (English)

    Xinhua Wu; Dengfu Yao; Gongshen Shi; Liwei Qiu; Wei Wu; Songshi Ni; Xueguang Zhang

    2005-01-01

    Objective: Angiogennesis, the formation of new blood vessels from the existing vascular bed, is essential step for growth and invasion of primary tumor. Vascular endothelial growth-factor (VEGF) is known to play crucial role in tumor angiogenesis. In the present study, we investigate the expression of VEGF and VEGF-mRNA in the angiogennesis, metastasis and prognosis of lung cancer.Methods: The VEGF cellular distributions and expression in 38 specimens of patients with lung cancer were investigated with immunohistochemistry stain technology. The total RNAs in 38 tissues of lung cancer was measured, then the levels of VEGF-mRNA expression were analyzed by a reverse-transcription polymerase chain reaction (RT-PCR) assay. The levels of VEGF in sera of patients with lung cancer, benign lung diseases and healthy controls were detected through Enzyme linked immunosorbent assay (ELISA) method. Results: The VEGF positive stain was 76% in 38 cases of lung cancer specimens. The 89% rate of VEGF stain was found for clinical stage Ⅲ cases and 92%for stage Ⅳ lung cancers. The significantly higher expression of VEGF was evidenced in patients with lymph node metastasis (84 % ), distant metastasis (90%), and lung cancers with lower histological differentiation (89%), respectively. The expression level of total RNA was significantly higher in patients with lung cancers than that in their paracancerous or distant lung tissues. The VEGF expressions were tightly correlated with total RNA concentration of lung carcinoma ( P < 0.01 ). The predominant expressions of VEGF121 and VEGF165 gene fragments were found in lung cancer specimens by RT-PCR analysis. No significant difference of serum VEGF levels was detected between cases with lung cancer and patients with benign diseases. However, the VEGF level of cases with benign diseases was decreased significantly after patients with anti-inflammation medication. Conclusion: The present data suggested that the tumor tissue VEGF

  4. Therapeutic intracoronary gene delivery of VEGF-B167 in a preclinical animal model of dilated cardiomyopathy

    Science.gov (United States)

    Woitek, Felix; Zentilin, Lorena; Hoffman, Nicholas E.; Powers, Jeffrey; Ottiger, Isabel; Parikh, Suraj; Kulczycki, Anna M.; Hurst, Marykathryn; Ring, Nadja; Wang, Tao; Shaikh, Farah; Gross, Polina; Singh, Harinder; Kolpakov, Mikhail A.; Linke, Axel; Houser, Steven R.; Rizzo, Victor; Sabri, Abdelkarim; Madesh, Muniswamy; Giacca, Mauro; Recchia, Fabio A.

    2015-01-01

    BACKGROUND Vascular endothelial growth factor-B (VEGF-B) activates cytoprotective/antiapoptotic and minimally angiogenic mechanisms via VEGF receptors. Therefore, VEGF-B might prove an ideal candidate for the treatment of dilated cardiomyopathy, which displays modest microvascular rarefaction and increased rate of apoptosis. OBJECTIVES We evaluated VEGF-B gene therapy in a canine model of tachypacing-induced dilated cardiomyopathy. METHODS Chronically instrumented dogs underwent cardiac tachypacing for 28 days. Adeno-associated-9 viral vectors carrying VEGF-B167 genes were infused intracoronarily at the beginning of the pacing protocol or during compensated heart failure (HF). Moreover, we tested a novel VEGF-B167 transgene controlled by the atrial natriuretic factor (ANF) promoter. RESULTS Compared to controls, VEGF-B167 markedly preserved diastolic and contractile function and attenuated ventricular chamber remodeling, halting the progression from compensated to decompensated HF. ANF-VEGF-B167 expression was low in normo-functioning hearts and stimulated by cardiac pacing; thus, it functioned as an ideal therapeutic transgene, active only under pathological conditions. CONCLUSIONS Our results, obtained with a standard technique of interventional cardiology in a clinically relevant animal model, support VEGF-B167 gene transfer as an affordable and highly effective new therapy for nonischemic HF. PMID:26160630

  5. VEGF-A isoform-specific regulation of calcium ion flux, transcriptional activation and endothelial cell migration

    Directory of Open Access Journals (Sweden)

    Gareth W. Fearnley

    2015-07-01

    Full Text Available Vascular endothelial growth factor A (VEGF-A regulates many aspects of vascular physiology such as cell migration, proliferation, tubulogenesis and cell-cell interactions. Numerous isoforms of VEGF-A exist but their physiological significance is unclear. Here we evaluated two different VEGF-A isoforms and discovered differential regulation of cytosolic calcium ion flux, transcription factor localisation and endothelial cell response. Analysis of VEGF-A isoform-specific stimulation of VEGFR2-dependent signal transduction revealed differential capabilities for isoform activation of multiple signal transduction pathways. VEGF-A165 treatment promoted increased phospholipase Cγ1 phosphorylation, which was proportional to the subsequent rise in cytosolic calcium ions, in comparison to cells treated with VEGF-A121. A major consequence of this VEGF-A isoform-specific calcium ion flux in endothelial cells is differential dephosphorylation and subsequent nuclear translocation of the transcription factor NFATc2. Using reverse genetics, we discovered that NFATc2 is functionally required for VEGF-A-stimulated endothelial cell migration but not tubulogenesis. This work presents a new mechanism for understanding how VEGF-A isoforms program complex cellular outputs by converting signal transduction pathways into transcription factor redistribution to the nucleus, as well as defining a novel role for NFATc2 in regulating the endothelial cell response.

  6. SUMO Wrestles with Recombination

    Directory of Open Access Journals (Sweden)

    Lumír Krejčí

    2012-07-01

    Full Text Available DNA double-strand breaks (DSBs comprise one of the most toxic DNA lesions, as the failure to repair a single DSB has detrimental consequences on the cell. Homologous recombination (HR constitutes an error-free repair pathway for the repair of DSBs. On the other hand, when uncontrolled, HR can lead to genome rearrangements and needs to be tightly regulated. In recent years, several proteins involved in different steps of HR have been shown to undergo modification by small ubiquitin-like modifier (SUMO peptide and it has been suggested that deficient sumoylation impairs the progression of HR. This review addresses specific effects of sumoylation on the properties of various HR proteins and describes its importance for the homeostasis of DNA repetitive sequences. The article further illustrates the role of sumoylation in meiotic recombination and the interplay between SUMO and other post-translational modifications.

  7. Recombinant Human Enterovirus 71

    OpenAIRE

    2004-01-01

    Two human enterovirus 71 (HEV71) isolates were identified from hand, foot and mouth disease patients with genome sequences that had high similarity to HEV71 (>93%) at 5´ UTR, P1, and P2 and coxsackievirus A16 (CV-A16, >85%) at P3 and 3´UTR. Intertypic recombination is likely to have occurred between HEV71 and CV-A16 or an as-yet to be described CV-A16-like virus.

  8. Significance of VEGF and NF-κB Expression in Thyroid Carcinoma

    Institute of Scientific and Technical Information of China (English)

    Zhenxian Du; Haiyan Zhang; Daxin Gao; Huaqin Wang; Yongjun Li; Guoliang Liu

    2006-01-01

    OBJECTIVE To investigate the significance of vascular endothelial growth factor (VEGF) and nuclear factor-kappaB (NF-κB) expression in thyroid carcinoma.METHODS The expression of NF-κB and VEGF was determined by immunohistochemistry in formalin-fixed and paraffin-embedded specimens obtained from 10 normal thyroid tissues (NT), 12 cases of thyroid adenoma (TA) and 68 cases of thyroid carcinoma (TC).Differences in expression between NT, TA and TC were statistically analyzed. In addition, in cases of TC, the relationship of NF-κB and VEGF expression with various clinicopathological factors, including histological typing, clinical staging and lymph node metastasis, as well as the correlation between NF-κB and VEGF expression was examined.RESULTS In contrast to the negative immunoreactivity for VEGF in NT,there was a significantly higher positive incidence (PI) in TA (41.7%, P=0.040) and TC (75.0%, P<0.001), and a significant difference between TA and TC (P=0.036). Immunoreactivity for NF-κB in NT was negative and significantly higher in TC (63.2%, P<0.001),but not in TA (16.7%, P=0.481). However the PI difference between TA and TC (P=0.003) was significant. Between the histological types of TC, a significantly higher PI was found in undifferentiated thyroid carcinoma (UTC),namely, 100% for VEGF and 90.0% for NF-κB. We also found significant positive relationships of VEGF and NF-κB expression with the clinical stage and lymph node metastasis. Furthermore, a significant positive correlation between VEGF and NF-κB expression in TC was observed.CONCLUSION Our data showed that the expression of VEGF and NFκB/P65 was greater in TC and UTC, and documented their significant positive correlations with the clinical stage and lymph node metastasis in TC. In addition there was a significant positive relationship between their expression, suggesting that they have important roles in TC and that they may be potential targets for gene therapy in TC patients.

  9. VEGF Mediates ApoE4-Induced Neovascularization and Synaptic Pathology in the Choroid and Retina.

    Science.gov (United States)

    Antes, Ran; Salomon-Zimri, Shiran; Beck, Susanne C; Garcia Garrido, Marina; Livnat, Tami; Maharshak, Idit; Kadar, Tamar; Seeliger, Mathias; Weinberger, Dov; Michaelson, Daniel M

    2015-01-01

    Apolipoprotein E4 (apoE4), the most prevalent genetic risk factor for Alzheimer's disease (AD), is associated with neuronal and vascular impairments. Recent findings suggest that retina of apoE4 mice have synaptic and functional impairments. We presently investigated the effects of apoE4 on retinal and choroidal vasculature and the possible role of VEGF in these effects. There were no histological differences between the retinal and choroidal vasculatures of naïve apoE3 and apoE4 mice. In contrast, laserdriven choroidal injury induced higher levels of choroidal neovascularization (CNV) in apoE4 than in apoE3 mice. These effects were associated with an inflammatory response and with activation of the Muller cells and asrocytic markers gluthatione synthetase and GFAP, all of which were more pronounced in the apoE4 mice. CNV also induced a transient increase in the levels of the synaptic markers synaptophysin and PSD95 which were however similar in the apoE4 and apoE3 naive mice. Retinal and choroidal VEGF and apoE levels were lower in naïve apoE4 than in corresponding apoE3 mice. In contrast, VEGF and apoE levels rose more pronouncedly following laser injury in the apoE4 than in apoE3 mice. Taken together, these findings suggest that the apoE4-induced retinal impairments, under basal conditions, may be related to reduced VEGF levels in the eyes of these mice. The hyper-neovascularization in the apoE4 mice might be driven by increased inflammation and the associated surge in VEGF following injury. Retinal and choroidal VEGF and apoE levels were lower in naïve apoE4 than in corresponding apoE3 mice. In contrast, VEGF and apoE levels rose more pronouncedly following laser injury in the apoE4 than in apoE3 mice. Taken together, these findings suggest that the apoE4-induced retinal impairments, under basal conditions, may be related to reduced VEGF levels in the eyes of these mice. The hyper-neovascularization in the apoE4 mice might be driven by increased inflammation

  10. Inhibition of ocular neovascularization by co-inhibition of VEGF-A and PLGF.

    Science.gov (United States)

    Huo, Xiaochuan; Li, Youxiang; Jiang, Yuhua; Sun, Xiaoyun; Gu, Lixue; Guo, Wenshi; Sun, Dapeng

    2015-01-01

    Age-related macular degeneration (AMD) appears to be a disease with increasing incidence in Western countries and may develop into acquired blindness. Choroidal neovascularization (CNV) is the most frequent cause for AMD, and is commonly induced by regional inflammation. Past studies have highlighted vascular endothelial growth factor A (VEGF-A) as a major trigger for CNV. However, studies on the associated angiogenic factors other than VEGF-A are lacking. Here, we used a well-established laser burn (LB)-induced experimental CNV mouse model to study the molecular mechanisms underlying the development of CNV after ocular injury. We analyzed vessel density by lectin labeling. We isolated macrophages, endothelial cells and other cell types by flow cytometry, and analyzed levels of different angiogenic factors in these populations. We used antisera against VEGF-A (aVEGF) and/or antisera against placental growth factor (PLGF; aPLGF) to antagonize CNV. We used an antibody-driven toxin to selectively eliminate macrophages to evaluate the role of macrophages in CNV. We also examined expression of PLGF in macrophage subtypes. The choroidal vessel density increased significantly 7 days after LB. LB increased significantly the levels of VEGF-A and PLGF in mouse eyes. Treatment with aVEGF significantly blunted increases in vessel density by LB. Treatment with aPLGF alone did not significantly reduce increases in vessel density. However, aPLGF significantly increased the inhibitory effects of aVEGF on vessel density increases. While VEGF-A was produced by endothelial cells, macrophages and other types at similar levels, PLGF seemed to be predominantly produced by macrophages. Selective macrophage depletion significantly reduced CNV. M2, but M1 macrophages produced high levels of PLGF. Together, our data suggest a previously unappreciated role of PLGF in coordination with VEGF-A to regulate CNV during ocular injury. Our study highlights macrophages and their production of PLGF

  11. Inhibition of Ocular Neovascularization by Co-Inhibition of VEGF-A and PLGF

    Directory of Open Access Journals (Sweden)

    Xiaochuan Huo

    2015-03-01

    Full Text Available Background/Aims: Age-related macular degeneration (AMD appears to be a disease with increasing incidence in Western countries and may develop into acquired blindness. Choroidal neovascularization (CNV is the most frequent cause for AMD, and is commonly induced by regional inflammation. Past studies have highlighted vascular endothelial growth factor A (VEGF-A as a major trigger for CNV. However, studies on the associated angiogenic factors other than VEGF-A are lacking. Methods: Here, we used a well-established laser burn (LB-induced experimental CNV mouse model to study the molecular mechanisms underlying the development of CNV after ocular injury. We analyzed vessel density by lectin labeling. We isolated macrophages, endothelial cells and other cell types by flow cytometry, and analyzed levels of different angiogenic factors in these populations. We used antisera against VEGF-A (aVEGF and/or antisera against placental growth factor (PLGF; aPLGF to antagonize CNV. We used an antibody-driven toxin to selectively eliminate macrophages to evaluate the role of macrophages in CNV. We also examined expression of PLGF in macrophage subtypes. Results: The choroidal vessel density increased significantly 7 days after LB. LB increased significantly the levels of VEGF-A and PLGF in mouse eyes. Treatment with aVEGF significantly blunted increases in vessel density by LB. Treatment with aPLGF alone did not significantly reduce increases in vessel density. However, aPLGF significantly increased the inhibitory effects of aVEGF on vessel density increases. While VEGF-A was produced by endothelial cells, macrophages and other types at similar levels, PLGF seemed to be predominantly produced by macrophages. Selective macrophage depletion significantly reduced CNV. M2, but M1 macrophages produced high levels of PLGF. Conclusions: Together, our data suggest a previously unappreciated role of PLGF in coordination with VEGF-A to regulate CNV during ocular injury

  12. Recombinant human milk proteins.

    Science.gov (United States)

    Lönnerdal, Bo

    2006-01-01

    Human milk provides proteins that benefit newborn infants. They not only provide amino acids, but also facilitate the absorption of nutrients, stimulate growth and development of the intestine, modulate immune function, and aid in the digestion of other nutrients. Breastfed infants have a lower prevalence of infections than formula-fed infants. Since many women in industrialized countries choose not to breastfeed, and an increasing proportion of women in developing countries are advised not to breastfeed because of the risk of HIV transmission, incorporation of recombinant human milk proteins into infant foods is likely to be beneficial. We are expressing human milk proteins known to have anti-infective activity in rice. Since rice is a normal constituent of the diet of infants and children, limited purification of the proteins is required. Lactoferrin has antimicrobial and iron-binding activities. Lysozyme is an enzyme that is bactericidal and also acts synergistically with lactoferrin. These recombinant proteins have biological activities identical to their native counterparts. They are equally resistant to heat processing, which is necessary for food applications, and to acid and proteolytic enzymes which are needed to maintain their biological activity in the gastrointestinal tract of infants. These recombinant human milk proteins may be incorporated into infant formulas, baby foods and complementary foods, and used with the goal to reduce infectious diseases.

  13. The Role of Anti-VEGF Therapy in the Treatment of Diabetic Macular Edema.

    Science.gov (United States)

    Moshfeghi, Darius M; Kaiser, Peter K; Michels, Stephan; Midena, Edoardo; Kitchens, John W; Prenner, Jonathan L; Regillo, Carl D; Reichel, Elias

    2016-06-01

    Diabetic retinopathy (DR) is the leading cause of blindness among working-age adults. DR often leads to diabetic macular edema (DME), which often goes unnoticed until a patient presents with vision loss. However, treatment options and data for DME are continually improving. We know that vascular endothelial growth factor (VEGF) plays a key role in DME progression; therapies that act by inhibiting VEGF production seem to improve visual acuity in patients with DME. Of the anti-VEGF therapies available, two have been approved by the U.S. Food and Drug Administration to treat DME: ranibizumab (Lucentis; Genentech, South San Francisco, CA) and aflibercept (Eylea; Regeneron, Tarrytown, NY). Bevacizumab (Avastin; Genentech, South San Francisco, CA), which is approved for the treatment of certain types of cancer, is occasionally used off-label to treat DME. Anti-VEGF therapy can stop vision loss and even improve visual acuity. Other treatments remain effective, and these various treatment options fuel a need for new data and discussion. This roundtable discussion, which took place during the 2015 annual meeting of the American Academy of Ophthalmology, outlines the current protocols used to treat DME and provides clinical opinions about selecting and treating with an appropriate anti-VEGF therapy. [Ophthalmic Surg Lasers Imaging Retina. 2016;47:S5-14.].

  14. Expression of VEGF and semaphorin genes define subgroups of triple negative breast cancer.

    Directory of Open Access Journals (Sweden)

    R Joseph Bender

    Full Text Available Triple negative breast cancers (TNBC are difficult to treat due to a lack of targets and heterogeneity. Inhibition of angiogenesis is a promising therapeutic strategy, but has had limited effectiveness so far in breast cancer. To quantify heterogeneity in angiogenesis-related gene expression in breast cancer, we focused on two families--VEGFs and semaphorins--that compete for neuropilin co-receptors on endothelial cells. We compiled microarray data for over 2,600 patient tumor samples and analyzed the expression of VEGF- and semaphorin-related ligands and receptors. We used principal component analysis to identify patterns of gene expression, and clustering to group samples according to these patterns. We used available survival data to determine whether these clusters had prognostic as well as therapeutic relevance. TNBC was highly associated with dysregulation of VEGF- and semaphorin-related genes; in particular, it appeared that expression of both VEGF and semaphorin genes were altered in a pro-angiogenesis direction. A pattern of high VEGFA expression with low expression of secreted semaphorins was associated with 60% of triple-negative breast tumors. While all TNBC groups demonstrated poor prognosis, this signature also correlated with lower 5-year survival rates in non-TNBC samples. A second TNBC pattern, including high VEGFC expression, was also identified. These pro-angiogenesis signatures may identify cancers that are more susceptible to VEGF inhibition.

  15. Low Molecular Weight Fucoidan Inhibits Tumor Angiogenesis through Downregulation of HIF-1/VEGF Signaling under Hypoxia

    Directory of Open Access Journals (Sweden)

    Meng-Chuan Chen

    2015-07-01

    Full Text Available Activation of hypoxia-induced hypoxia-inducible factors-1 (HIF-1 plays a critical role in promoting tumor angiogenesis, growth and metastasis. Low molecular weight fucoidan (LMWF is prepared from brown algae, and exhibits anticancer activity. However, whether LMWF attenuates hypoxia-induced angiogenesis in bladder cancer cells and the molecular mechanisms involved remain unclear. This is the first study to demonstrate that LMWF can inhibit hypoxia-stimulated H2O2 formation, HIF-1 accumulation and transcriptional activity vascular endothelial growth factor (VEGF secretion, and the migration and invasion in hypoxic human bladder cancer cells (T24 cells. LMWF also downregulated hypoxia-activated phosphorylation of PI3K/AKT/mTOR/p70S6K/4EBP-1 signaling in T24 cells. Blocking PI3K/AKT or mTOR activity strongly diminished hypoxia-induced HIF-1α expression and VEGF secretion in T24 cells, supporting the involvement of PI3K/AKT/mTOR in the induction of HIF-1α and VEGF. Additionally, LMWF significantly attenuated angiogenesis in vitro and in vivo evidenced by reduction of tube formation of hypoxic human umbilical vascular endothelial cells and blood capillary generation in the tumor. Similarly, administration of LMWF also inhibited the HIF-1α and VEGF expression in vivo, accompanied by a reduction of tumor growth. In summary, under hypoxia conditions, the antiangiogenic activity of LMWF in bladder cancer may be associated with suppressing HIF-1/VEGF-regulated signaling pathway.

  16. Dietary compound isoliquiritigenin inhibits breast cancer neoangiogenesis via VEGF/VEGFR-2 signaling pathway.

    Science.gov (United States)

    Wang, Zhiyu; Wang, Neng; Han, Shouwei; Wang, Dongmei; Mo, Suilin; Yu, Linzhong; Huang, Hui; Tsui, Kamchuen; Shen, Jiangang; Chen, Jianping

    2013-01-01

    Angiogenesis is crucial for cancer initiation, development and metastasis. Identifying natural botanicals targeting angiogenesis has been paid much attention for drug discovery in recent years, with the advantage of increased safety. Isoliquiritigenin (ISL) is a dietary chalcone-type flavonoid with various anti-cancer activities. However, little is known about the anti-angiogenic activity of isoliquiritigenin and its underlying mechanisms. Herein, we found that ISL significantly inhibited the VEGF-induced proliferation of human umbilical vein endothelial cells (HUVECs) at non-toxic concentration. A series of angiogenesis processes including tube formation, invasion and migration abilities of HUVECs were also interrupted by ISL in vitro. Furthermore, ISL suppressed sprout formation from VEGF-treated aortic rings in an ex-vivo model. Molecular mechanisms study demonstrated that ISL could significantly inhibit VEGF expression in breast cancer cells via promoting HIF-1α (Hypoxia inducible factor-1α) proteasome degradation and directly interacted with VEGFR-2 to block its kinase activity. In vivo studies further showed that ISL administration could inhibit breast cancer growth and neoangiogenesis accompanying with suppressed VEGF/VEGFR-2 signaling, elevated apoptosis ratio and little toxicity effects. Molecular docking simulation indicated that ISL could stably form hydrogen bonds and aromatic interactions within the ATP-binding region of VEGFR-2. Taken together, our study shed light on the potential application of ISL as a novel natural inhibitor for cancer angiogenesis via the VEGF/VEGFR-2 pathway. Future studies of ISL for chemoprevention or chemosensitization against breast cancer are thus warranted.

  17. VEGF EXPRESSION IS INHIBITED BY APIGENIN IN HUMAN BREAST CANCER CELLS

    Institute of Scientific and Technical Information of China (English)

    JIN Xue-ying; REN Chang-shan

    2006-01-01

    Objective: To study the effects of apigenin on vascular endothelial growth factor (VEGF) in human breast cancer cells(MDA-MB-231. Methods: MTT assay was used to detect the cell proliferation inhibitory effect of apigenin on MDA-MB-231 cell. ELISA was used to determine the protein level of VEGF secreted by MDA-MB-231 cells. RT-PCR was used to detect mRNA levels of VEGF in MDA-MB-231 cells. The protein levels of HIF-1α,p-AKT,p-ERK1/2,and p53 were detected by Western Blotting. Results: Apigenin did not inhibit the cell viability of MDA-MB-231 cell. Apigenin reduced the secretion and mRNA levels of VEGF in MDA-MB-231 cells. Additionally, apigenin decreased the expressions of HIF-1α,p-AKT and p-ERK1/2, but induced the expression of p53. Conclusion: Apigenin can inhibit VEGF expression in human breast cancer cells, and this may be achieved through decreasing HIF-1α.

  18. A peptide fusion protein in hibits angiogenesis and tumorgrowth by blocking VEGF binding to KDR

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Vascular endothelial growth factor (VEGF) binding to its tyrosine kinase receptors (KDR/FLK1, Flt-1) induces angiogenesis. In search of the peptides blocking VEGF binding to its receptor KDR/FLK1 to inhibit tumor- angiogenesis and growth, we screened a phage display peptide library with KDR as target protein, and some candidate peptides were isolated. In this study, we cloned the DNA fragment coding the peptide K237 from the library, into a vector pQE42 to express fusion protein DHFR-K237 in E. coli M15. The affection of fusion protein DHFR-K237 on endothelial cell proliferation and angiogenesis was investigated. In vitro, DHFR-K237 could completely block VEGF binding to KDR and significantly inhibit the VEGF-medi- ated proliferation of the human vascular endothelial cells. In vivo, DHFR-K237 inhibited angiogenesis in chick embryo chorioa- llantoric membrane and tumor growth in nude mice. These results suggest that K237 is an effective antagonist of VEGF binding to KDR, and could be a potential agent for cancer biotherapy.

  19. Relationship of PARG with PARP, VEGF and b-FGF in Colorectal Carcinoma

    Institute of Scientific and Technical Information of China (English)

    Ling Lin; Jia Li; Ya-lan Wang; Xiao Lin

    2009-01-01

    Objective: To investigate the relationship of poly(ADP-ribose)glycohydrolase(PARG) with poly (ADP-ribose) polymerase(PARP), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor(b-FGF) in colorectal carcinoma(CRC).Methods: Immunohistochemical analysis was used to detect PARG, PARP, VEGF and b-FGF in human colorectal carcinoma. Flow cytometry was used to detect PARG and PARP in murine CT26 cell line. Gallotannin (GLTN) was served as PARG inhibitor. Results: The individual positive rates of PARG, PARP, VEGF and b-FGF were 55.81%(24/43), 97.67%(42/43), 79.07%(34/43) and 81.40%(35/43), respectively, which were significantly higher than those of control group. The positive PARG was correlated to PARP(r=0.3703, P<0.05) and b-FGF (r=0.4838, P<0.05). The positive PARP was correlated to VEGF (r=0.3968, P<0.05) and b-FGF (r=0.5610, P<0.05). Both PARG and PARP were expressed in CT26 cells. The positive staining rates of PARG and PARP in GLTN-treated group were 7.3% and 52.38%, respectively. They were markedly reduced than those of control group (55.41% and 95.28%, P<0.01, n=10000).Conclusion: The data suggest that PARG expression probably plays a role for VEGF and b-FGF expression in colorectal carcinoma.

  20. Angiogenesis effects of adenovirus-mediated gene transfer of VEGF-B on chronic ischemic myocardium

    Institute of Scientific and Technical Information of China (English)

    DONG Shu-qiang; ZHANG Bao-ren; MEI Ju; XU Zhi-yun; ZOU Liang-jian; HUANG Sheng-dong

    2002-01-01

    Objective: To study the angiogenesis effects of adenovirus-mediated gene transfer of VEGF-B on chronic ischemic myocardium. Methods: Domestic pigs underwent thoracotomy and placement of an ameroid constrictor on the circumflex coronary artery. Four weeks later, Ad. VEGF-B, Ad. LacZ or PBS were administrated directly into the myocardium at 10 sites in the circumflex distribution (109 PFU or 100 μl) according to groups. Echocardiography and ex vivo coronary angiography were performed. The injection sites around myocardium were harvested and subjected to histological analysis and immunochemical staining. Results: Echocardiography assessment 4 weeks after vector administration demonstrated significant improvement of regional wall systolic function. Collateral vesseldevelopment assessed by angiography was also significantly greater in Ad. VEGF-B animals than that in control animals. Vascular density analysis revealed a mean of 43±5 neovessels per high-power field in Ad.VEGF-B group versus 19±4 and 17±6 in Ad.LacZ and PBS group. Conclusion:Direct intramyocardial administration of Ad.VEGF-B can induce focal angiogenesis and result in improvement in regional myocardial function, which may be useful in patients with ischemic heart disease who are not eligible for conventional therapies.

  1. DC electric stimulation upregulates angiogenic factors in endothelial cells through activation of VEGF receptors.

    Science.gov (United States)

    Bai, Huai; Forrester, John V; Zhao, Min

    2011-07-01

    Small direct current (DC) electric fields direct some important angiogenic responses of vascular endothelial cells. Those responses indicate promising use of electric fields to modulate angiogenesis. We sought to determine the regulation of electric fields on transcription and expression of a serial of import angiogenic factors by endothelial cells themselves. Using semi-quantitative PCR and ELISA we found that electric stimulation upregulates the levels of mRNAs and proteins of a number of angiogenic proteins, most importantly VEGF165, VEGF121 and IL-8 in human endothelial cells. The up-regulation of mRNA levels might be specific, as the mRNA encoding bFGF, TGF-beta and eNOS are not affected by DC electric stimulation at 24h time-point. Inhibition of VEGF receptor (VEGFR1 or VEGFR2) signaling significantly decreased VEGF production and completely abolished IL-8 production. DC electric stimulation selectively regulates production of some growth factors and cytokines important for angiogenesis through a feed-back loop mediated by VEGF receptors. Copyright © 2011 Elsevier Ltd. All rights reserved.

  2. Impaired endothelial shear stress induces podosome assembly via VEGF up-regulation.

    Science.gov (United States)

    Fey, Theres; Schubert, Kai Michael; Schneider, Holger; Fein, Evelyn; Kleinert, Eike; Pohl, Ulrich; Dendorfer, Andreas

    2016-08-01

    Podosomes are dynamic cytoskeletal membrane structures with local adhesive and proteolytic activity. They are critically involved in angiogenesis and vascular adaptive growth. Here, we studied in HUVECs and murine small vessels whether shear stress controls podosome assembly and local proteolytic activity. Podosomes were characterized by immunohistochemistry, and their proteolytic activity was assessed as degradation imprints in fluorescent gelatin that was used as growth substrate. Compared with controls (10 dyn/cm(2)), the number of podosomes formed per time was doubled when cells were exposed to low shear stress (0.3 dyn/cm(2)) or even increased 5-fold under static conditions. This was a result of an enhanced expression of VEGF after reduction of shear stress. Consequently, enhanced podosome formation could be prevented by a VEGF receptor antagonist as well by interruption of VEGF signaling via inhibition of PI3K, Src, or p38. Increase of podosome assembly went along with significantly augmented cell motility. In vivo experiments in mouse arteries confirmed increased endothelial podosome numbers when shear stress was abolished by vessel occlusion. We conclude that shear stress, by reducing VEGF release, inhibits podosome assembly. Hence, endothelial cell-mediated matrix proteolysis and migratory activity are inhibited, thereby stabilizing the structure of the vessel wall.-Fey, T., Schubert, K. M., Schneider, H., Fein, E., Kleinert, E., Pohl, U., Dendorfer, A. Impaired endothelial shear stress induces podosome assembly via VEGF up-regulation.

  3. Homoharringtonine induces apoptosis of endothelium and down-regulates VEGF expression of K562 cells

    Institute of Scientific and Technical Information of China (English)

    叶琇锦; 林茂芳

    2004-01-01

    Homoharringtonine (HHT) has currently been used successfully in the treatment of acute and chronic myeloid leukemias and has been shown to induce apoptosis of different types of leukemic cells in vitro. Emerging evidence suggests that angiogenesis may play an important role in hematological malignancies, such as leukemia. However, whether HHT can relieve leukemia by anti-angiogenesis is still unknown. We investigated the anti-angiogenesis potential of HHT with the human umbilical vein endothelial cell line (ECV304) and leukemic cell line (K562) in vitro. Cellular proliferation was determined by MTT assay and apoptosis was analyzed by flow cytometry, The mRNA expression of vascular endothelial growth factor (VEGF) was assessed by RT-PCR and VEGF protein production was detected by Western blot. Inhibition of cell proliferation and induction of apoptosis by HHT were discovered in ECV304 cells, and appeared in a dose- and time-dependent manner, Also, treatment with HHT caused down-regulation of VEGF mRNA expression in K562 cells in similar dose- and time-dependent manner and inhibition of VEGF protein production in K562 cells in response to the enhancing concentration of HHT. The results demonstrated that HHT could also induce apoptosis in endothelium and down-regulate VEGF expression in K562 cells. In conclusion, we believe HHT has anti-angiogenesis potential and speculate that HHT might exert its anti-leukemia effects via reduction of angiogenesis.

  4. Resistance to anti-VEGF therapy in neovascular age-related macular degeneration: a comprehensive review

    Directory of Open Access Journals (Sweden)

    Yang S

    2016-06-01

    Full Text Available Shiqi Yang,1 Jingke Zhao,1 Xiaodong Sun1–3 1Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 2Eye Research Institute of Shanghai Jiao Tong University, 3Shanghai Key Laboratory of Fundus Disease, Shanghai, People’s Republic of China Abstract: As a progressive chronic disease, age-related macular degeneration (AMD is the leading cause of irreversible vision impairment worldwide. Experimental and clinical evidence has demonstrated that vascular endothelial growth factor (VEGF plays a vital role in the formation of choroidal neovascularization. Intravitreal injections of anti-VEGF agents have been recommended as a first-line treatment for neovascular AMD. However, persistent fluid or recurrent exudation still occurs despite standardized anti-VEGF therapy. Patients suffering from refractory or recurrent neovascular AMD may develop mechanisms of resistance to anti-VEGF therapy, which results in a diminished therapeutic effect. Until now, there has been no consensus on the definitions of refractory neovascular AMD and recurrent neovascular AMD. This article aims at clarifying these concepts to evaluate the efficacy of switching drugs, which contributes to making clinical decision more scientifically. Furthermore, insight into the causes of resistance to anti-VEGF therapy would be helpful for developing possible therapeutic approaches, such as combination therapy and multi-target treatment that can overcome this resistance. Keywords: age-related macular degeneration, vascular endothelial growth factor, choroidal neovascularization, resistance

  5. Resistance to anti-VEGF therapy in neovascular age-related macular degeneration: a comprehensive review.

    Science.gov (United States)

    Yang, Shiqi; Zhao, Jingke; Sun, Xiaodong

    2016-01-01

    As a progressive chronic disease, age-related macular degeneration (AMD) is the leading cause of irreversible vision impairment worldwide. Experimental and clinical evidence has demonstrated that vascular endothelial growth factor (VEGF) plays a vital role in the formation of choroidal neovascularization. Intravitreal injections of anti-VEGF agents have been recommended as a first-line treatment for neovascular AMD. However, persistent fluid or recurrent exudation still occurs despite standardized anti-VEGF therapy. Patients suffering from refractory or recurrent neovascular AMD may develop mechanisms of resistance to anti-VEGF therapy, which results in a diminished therapeutic effect. Until now, there has been no consensus on the definitions of refractory neovascular AMD and recurrent neovascular AMD. This article aims at clarifying these concepts to evaluate the efficacy of switching drugs, which contributes to making clinical decision more scientifically. Furthermore, insight into the causes of resistance to anti-VEGF therapy would be helpful for developing possible therapeutic approaches, such as combination therapy and multi-target treatment that can overcome this resistance.

  6. The tetrapeptide Arg-Leu-Tyr-Glu inhibits VEGF-induced angiogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Baek, Yi-Yong; Lee, Dong-Keon [Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University, Chuncheon, Gangwon-do, 200-702 (Korea, Republic of); So, Ju-Hoon; Kim, Cheol-Hee [Department of Biology, Chungnam National University, Daejeon, 305-764 (Korea, Republic of); Jeoung, Dooil [Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon, Gangwon-do, 200-702 (Korea, Republic of); Lee, Hansoo [Department of Life Sciences, College of Natural Sciences, Kangwon National University, Chuncheon, Gangwon-do, 200-702 (Korea, Republic of); Choe, Jongseon [Department of Immunology, School of Medicine, Kangwon National University, Chuncheon, Gangwon-do, 200-702 (Korea, Republic of); Won, Moo-Ho [Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon, Gangwon-do, 200-702 (Korea, Republic of); Ha, Kwon-Soo [Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University, Chuncheon, Gangwon-do, 200-702 (Korea, Republic of); Kwon, Young-Guen [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, 120-752 (Korea, Republic of); Kim, Young-Myeong, E-mail: ymkim@kangwon.ac.kr [Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University, Chuncheon, Gangwon-do, 200-702 (Korea, Republic of)

    2015-08-07

    Kringle 5, derived from plasminogen, is highly capable of inhibiting angiogenesis. Here, we have designed and synthesized 10 tetrapeptides, based on the amino acid properties of the core tetrapeptide Lys-Leu-Tyr-Asp (KLYD) originating from anti-angiogenic kringle 5 of human plasminogen. Of these, Arg-Leu-Tyr-Glu (RLYE) effectively inhibited vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation, migration and tube formation, with an IC{sub 50} of 0.06–0.08 nM, which was about ten-fold lower than that of the control peptide KLYD (0.79 nM), as well as suppressed developmental angiogenesis in a zebrafish model. Furthermore, this peptide effectively inhibited the cellular events that precede angiogenesis, such as ERK and eNOS phosphorylation and nitric oxide production, in endothelial cells stimulated with VEGF. Collectively, these data demonstrate that RLYE is a potent anti-angiogenic peptide that targets the VEGF signaling pathway. - Highlights: • The tetrapeptide RLYE inhibited VEGF-induced angiogenesis in vitro. • RLYE also suppressed neovascularization in a zebrafish model. • Its effect was correlated with inhibition of VEGF-induced ERK and eNOS activation. • RLYE may be used as a therapeutic drug for angiogenesis-related diseases.

  7. The role of VEGF and KDR polymorphisms in moyamoya disease and collateral revascularization.

    Directory of Open Access Journals (Sweden)

    Young Seok Park

    Full Text Available We conducted a case-control study to investigate whether vascular endothelial growth factor (VEGF -2578, -1154, -634, and 936 and kinase insert domain containing receptor (KDR -604, 1192, and 1719 polymorphisms are associated with moyamoya disease. Korean patients with moyamoya disease (n = 107, mean age, 20.9±15.9 years; 66.4% female and 243 healthy control subjects (mean age, 23.0±16.1 years; 56.8% female were included. The subjects were divided into pediatric and adult groups. Among the 64 surgical patients, we evaluated collateral vessel formation after 2 years and divided patients into good (collateral grade A or poor (collateral grade B and C groups. The frequencies and distributions of four VEGF (-2578, -1154, -634, and 936 and KDR (-604, 1192, and 1719 polymorphisms were assessed from patients with moyamoya disease and compared to the control group. No differences were observed in VEGF -2578, -1154, -634, and 936 or KDR -604, 1192, and 1719 polymorphisms between the control group and moyamoya disease group. However, we found the -634CC genotype occurred less frequently in the pediatric moyamoya group (p = 0.040 whereas the KDR -604C/1192A/1719T haplotype increased the risk of pediatric moyamoya (p = 0.024. Patients with the CC genotype of VEGF -634 had better collateral vessel formation after surgery. Our results suggest that the VEGF -634G allele is associated with pediatric moyamoya disease and poor collateral vessel formation.

  8. 非小细胞肺癌VEGF-C、VEGF-D、VEGFR-3表达与淋巴管生成和淋巴转移关系的研究%Investigation on the relationship between VEGF-C,VEGF-D,VEGFR-3 and lymphangiogenesis and lymph node metastasis in non-small cell lung cancer

    Institute of Scientific and Technical Information of China (English)

    常超; 王平; 李琳; 杨慧; 张利斌

    2009-01-01

    目的:探讨血管内皮生长因子(VEGF)-C、-D及其受体3(VEGFR-3)在非小细胞肺癌(NSCLC)中的表达规律、与微淋巴管密度(MLVD)、淋巴转移之间的关系及其在NSCLC发生发展、预后中的意义.方法:以40例经病理确诊的NSCLC组织为实验组.以11例肺良性病变组织为对照组,采用免疫组织化学染色法对上述组织中VEGF-C、VEGF-D、VEGFR-3、淋巴管内皮透明质酸受体-1(LYVE-1)、同源异型盒转录因子(Prox-1)蛋白的表达进行分析,并计数微淋巴管密度.结果:①NSCLC组织中VEGF-C、VEGF-D及VEGFR-3蛋白的阳性率分别为77.50%(31/40例)、67.50%(27/40例)、62.50%(25/40例),明显高于肿瘤周边组织(距肿瘤边缘100 μm区域内)及肺良性病变组织(P<0.01).②VEGF-C与VEGFR-3(r=0.409,P=0.005)、VEGF-D与VEGFR-3蛋白表达(r=492.P=0.000)均存在相关性;而VEGF-C与VEGF-D蛋白表达无相关性(r=256.P=0.093).③VEGF-C、VEGF-D及VEGFR-3蛋白的表达与NSCLC患者的性别、年龄、肿瘤的大小、组织学类型、分化程度无关,但与肿瘤的淋巴结转移(P<0.05)、PTNM分期(P<0.05)显著相关.④NSCLC肿瘤中心组织中LYVE-1及Prex-1标记的MLVD分别为4.22±1.25、1.99±1.49,明显低于肺良性病变组织(P=0.000),而NSCLC肿瘤周边部位的MLVD显著高于肿瘤中心部位及肺良性病变组织(P=0.000).VEGF-C、VEGF-D、VEGFR-3表达阳性的组织中,MLVD显著高于阴性组织(P<0.05).MLVD与肿瘤的淋巴结转移(P=0.000)、PTNM分期(P=0.000)显著相关.结论:淋巴管生成冈子VEGF-C、VEGF-D及其受体VEGFR-3蛋白在NSCLC中表达显著增高,并且通过VEGF-C、VEGF-D/VEGFR-3信号通路诱导淋巴管内皮细胞新生和淋巴管生成,从而促进淋巴结转移和肿瘤生长:VEGF-C、VEGF-D及其受体VEGFR-3可能成为检测NSCLC淋巴转移和评估预后的重要分子指标;淋巴管内皮细胞特异性标志物LYVE-1、Prox-1可以较严格地区分血管和淋巴管内皮,相对精确地评价肿瘤的脉管系统.

  9. Preparation and immunogenicity-evaluation of typhoid O-specific polysaccharides bio-conjugate vaccines%生物法合成伤寒O-糖蛋白结合疫苗及其免疫原性评估

    Institute of Scientific and Technical Information of China (English)

    彭哲慧; 潘超; 孙鹏; 冯尔玲; 吴军; 朱力; 彭清忠; 王恒樑

    2015-01-01

    Typhoid fever caused bySalmonella Typhi is still a major public health problem in developing coun-tries. In this study, we constructed a genetically modifiedSalmonella Typhi strain expressing O-specific polysaccha-rides (OPS) antigen conjugated to a carrier, recombinant Pseudomonas aeruginosa exotoxin A(rEPA N29). The conju-gates (OPS-rEPA N29) were further purified and evaluated for their immunogenicity. The results of ELISA showed that the conjugates evoked higher titers of IgG than OPS, suggesting that rEPAN29 increased immunogenicity of OPS significantly as a carrier. Moreover, three injections with 3-week interval evoked slightly higher titers of IgG than three injections with 2-week interval. However, injection of excess conjugates could not evoke higher titers of IgG against lipid polysaccharide (LPS). In summary, our study provides a new strategy for preparing polysaccha-rides-protein conjugate vaccines as well as similar bio-conjugate vaccines of other Gram-negative pathogens.%伤寒由伤寒沙门氏菌(Salmonella Typhi)引发,至今在发展中国家仍是备受关注的重要公共卫生问题.文章通过敲除伤寒菌脂多糖合成途径中O-抗原连接酶基因,转入含脑膜炎奈瑟球菌(Neisseria meningitidis)蛋白糖基化途径中糖基转移酶的表达载体,以及改构的重组铜绿假单胞菌(Pseudomonas Aeruginosa)外毒素A(rEPAN29)的表达载体,使细胞内能够诱导合成以伤寒O特异性多糖(O-specific polysaccharides, OPS)为目标抗原、以rEPAN29为载体蛋白的伤寒OPS-rEPAN29糖蛋白复合物,并对纯化所得复合物进行了免疫原性评价.ELISA测定血清抗体滴度表明,rEPA N29作为载体蛋白能有效增加糖链的免疫原性,糖蛋白比单独的多糖能诱导产生更好的免疫应答;3次免疫、间隔3周比间隔2周IgG滴度稍有提高;而免疫过量的糖蛋白,抗O-多糖的血清抗体效价并无提升.文章为生物法制备多糖-蛋白结合疫苗提供了新思路,理论

  10. Identification and in vitro characterization of phage-displayed VHHs targeting VEGF

    DEFF Research Database (Denmark)

    Farajpour, Zahra; Rahbarizadeh, Fatemeh; Kazemi, Bahram;

    2014-01-01

    Vascular endothelial growth factor (VEGF) is a potential target for cancer treatment because of its role in angiogenesis and its overexpression in most human cancers. Currently, anti-VEGF antibodies have been shown to be promising tools for therapeutic applications. However, large size, poor tumor......-targeting purposes. The present study was undertaken to generate and characterize anti-VEGF VHHs from an immune VHH library using phage display. Four rounds of panning were performed, and selected VHHs were characterized using various immunological techniques. Assessment of the antigenic profile of VHHs was done......, significantly inhibited the endothelial cell growth in a dose-dependent manner. Taken together, our results indicate that ZFR-5 and other VHHs may be promising tools in cancer research and treatment....

  11. Increased serum levels of sortilin are associated with depression and correlated with BDNF and VEGF

    DEFF Research Database (Denmark)

    Buttenschøn, Henriette Nørmølle; Demontis, Ditte; Ollendorff, Mathias Kaas;

    2015-01-01

    measured by immunoassay, and potential determinants of the serum sortilin level were assessed by generalized linear models. Serum levels of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) were measured in previous studies. We identified a significant increase of serum...... sortilin levels in depressed individuals compared with controls (P = 0.0002) and significant positive correlation between serum sortilin levels and the corresponding levels of BDNF and VEGF. None of the genotyped SNPs were associated with depression. Additional analyses showed that the serum sortilin level...... was influenced by several other factors. Alcohol intake and body mass index, as well as depression, serum BDNF and serum VEGF were identified as predictors of serum sortilin levels in our final multivariate model. In conclusion, the results suggest a role of circulating sortilin in depression which may relate...

  12. VEGF pathway inhibition by anticancer agent sunitinib and susceptibility to atherosclerosis plaque disruption.

    Science.gov (United States)

    Ropert, Stanislas; Vignaux, Olivier; Mir, Olivier; Goldwasser, François

    2011-12-01

    Patients treated with anti-VEGF agents are at increased risk for arterial thrombo-embolic events (ATEs). However, the pathophysiology of such acute vascular complications remains unclear. We report on a case of bowel infarction in a renal cancer patient treated with the anti-VEGF agent sunitinib. An abdominal CT-scan evidenced the rupture of an atherosclerotic plaque located at the emergence of the superior mesenteric artery. In view of this report, we suggest that evaluation of the risk of ATE in patients receiving anti-VEGF agents should include not only age and past history of ATE as suggested by previous studies, but also assessment of atherosclerotic lesions on CT-scan.

  13. The Structural Basis for the Function of Two Anti-VEGF Receptor 2 Antibodies

    Energy Technology Data Exchange (ETDEWEB)

    M Franklin; E Navarro; Y Wang; S Patel; P Singh; Y Zhang; K Persaud; A Bari; H Griffith; et al.

    2011-12-31

    The anti-VEGF receptor 2 antibody IMC-1121B is a promising antiangiogenic drug being tested for treatment of breast and gastric cancer. We have determined the structure of the 1121B Fab fragment in complex with domain 3 of VEGFR2, as well as the structure of a different neutralizing anti-VEGFR2 antibody, 6.64, also in complex with VEGFR2 domain 3. The two Fab fragments bind at opposite ends of VEGFR2 domain 3; 1121B directly blocks VEGF binding, whereas 6.64 may prevent receptor dimerization by perturbing the domain 3:domain 4 interface. Mutagenesis reveals that residues essential for VEGF, 1121B, and 6.64 binding are nonoverlapping among the three contact patches.

  14. The Structural Basis for the Function of Two Anti-VEGF Receptor 2 Antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Franklin, Matthew C.; Navarro, Elizabeth C.; Wang, Yujie; Patel, Sheetal; Singh, Pinki; Zhang, Yi; Persaud, Kris; Bari, Amtul; Griffith, Heather; Shen, Leyi; Balderes, Paul; Kussie, Paul (ImClone)

    2011-10-28

    The anti-VEGF receptor 2 antibody IMC-1121B is a promising antiangiogenic drug being tested for treatment of breast and gastric cancer. We have determined the structure of the 1121B Fab fragment in complex with domain 3 of VEGFR2, as well as the structure of a different neutralizing anti-VEGFR2 antibody, 6.64, also in complex with VEGFR2 domain 3. The two Fab fragments bind at opposite ends of VEGFR2 domain 3; 1121B directly blocks VEGF binding, whereas 6.64 may prevent receptor dimerization by perturbing the domain 3:domain 4 interface. Mutagenesis reveals that residues essential for VEGF, 1121B, and 6.64 binding are nonoverlapping among the three contact patches.

  15. Bacterial Recombineering: Genome Engineering via Phage-Based Homologous Recombination.

    Science.gov (United States)

    Pines, Gur; Freed, Emily F; Winkler, James D; Gill, Ryan T

    2015-11-20

    The ability to specifically modify bacterial genomes in a precise and efficient manner is highly desired in various fields, ranging from molecular genetics to metabolic engineering and synthetic biology. Much has changed from the initial realization that phage-derived genes may be employed for such tasks to today, where recombineering enables complex genetic edits within a genome or a population. Here, we review the major developments leading to recombineering becoming the method of choice for in situ bacterial genome editing while highlighting the various applications of recombineering in pushing the boundaries of synthetic biology. We also present the current understanding of the mechanism of recombineering. Finally, we discuss in detail issues surrounding recombineering efficiency and future directions for recombineering-based genome editing.

  16. Extracellular matrix stiffness modulates VEGF calcium signaling in endothelial cells: individual cell and population analysis.

    Science.gov (United States)

    Derricks, Kelsey E; Trinkaus-Randall, Vickery; Nugent, Matthew A

    2015-09-01

    Vascular disease and its associated complications are the number one cause of death in the Western world. Both extracellular matrix stiffening and dysfunctional endothelial cells contribute to vascular disease. We examined endothelial cell calcium signaling in response to VEGF as a function of extracellular matrix stiffness. We developed a new analytical tool to analyze both population based and individual cell responses. Endothelial cells on soft substrates, 4 kPa, were the most responsive to VEGF, whereas cells on the 125 kPa substrates exhibited an attenuated response. Magnitude of activation, not the quantity of cells responding or the number of local maximums each cell experienced distinguished the responses. Individual cell analysis, across all treatments, identified two unique cell clusters. One cluster, containing most of the cells, exhibited minimal or slow calcium release. The remaining cell cluster had a rapid, high magnitude VEGF activation that ultimately defined the population based average calcium response. Interestingly, at low doses of VEGF, the high responding cell cluster contained smaller cells on average, suggesting that cell shape and size may be indicative of VEGF-sensitive endothelial cells. This study provides a new analytical tool to quantitatively analyze individual cell signaling response kinetics, that we have used to help uncover outcomes that are hidden within the average. The ability to selectively identify highly VEGF responsive cells within a population may lead to a better understanding of the specific phenotypic characteristics that define cell responsiveness, which could provide new insight for the development of targeted anti- and pro-angiogenic therapies.

  17. P70S6K 1 regulation of angiogenesis through VEGF and HIF-1{alpha} expression

    Energy Technology Data Exchange (ETDEWEB)

    Bian, Chuan-Xiu; Shi, Zhumei [Department of Pathology, Cancer Center, Nanjing Medical University, Nanjing 210029 (China); Meng, Qiao; Jiang, Yue; Liu, Ling-Zhi [Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107 (United States); Jiang, Bing-Hua, E-mail: binghjiang@yahoo.com [Department of Pathology, Cancer Center, Nanjing Medical University, Nanjing 210029 (China); Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107 (United States)

    2010-07-30

    Research highlights: {yields} P70S6K1 regulates VEGF expression; {yields} P70S6K1 induces transcriptional activation through HIF-1{alpha} binding site; {yields} P70S6K1 regulates HIF-1{alpha}, but not HIF-1{beta} protein expression; {yields} P70S6K1 mediates tumor growth and angiogenesis through HIF-1{alpha} and VEGF expression. -- Abstract: The 70 kDa ribosomal S6 kinase 1 (p70S6K1), a downstream target of phosphoinositide 3-kinase (PI3K) and ERK mitogen-activated protein kinase (MAPK), is an important regulator of cell cycle progression, and cell proliferation. Recent studies indicated an important role of p70S6K1 in PTEN-negative and AKT-overexpressing tumors. However, the mechanism of p70S6K1 in tumor angiogenesis remains to be elucidated. In this study, we specifically inhibited p70S6K1 activity in ovarian cancer cells using vector-based small interfering RNA (siRNA) against p70S6K1. We found that knockdown of p70S6K1 significantly decreased VEGF protein expression and VEGF transcriptional activation through the HIF-1{alpha} binding site at its enhancer region. The expression of p70S6K1 siRNA specifically inhibited HIF-1{alpha}, but not HIF-1{beta} protein expression. We also found that p70S6K1 down-regulation inhibited ovarian tumor growth and angiogenesis, and decreased cell proliferation and levels of VEGF and HIF-1{alpha} expression in tumor tissues. Our results suggest that p70S6K1 is required for tumor growth and angiogenesis through HIF-1{alpha} and VEGF expression, providing a molecular mechanism of human ovarian cancer mediated by p70S6K1 signaling.

  18. Prognosis of invasive breast cancer after adjuvant therapy evaluated with VEGF microvessel density and microvascular imaging.

    Science.gov (United States)

    Li, Ying; Wei, Xi; Zhang, Sheng; Zhang, Jin

    2015-11-01

    The aim of this study was to investigate the role of ultrasonographic microvascular imaging in the evaluation of prognosis of patients with invasive breast cancer treated by adjuvant therapies. A total of 121 patients with invasive breast cancer underwent ultrasonographic contrast-enhanced imaging, vascular endothelial growth factor (VEGF) staining, and microvessel density (MVD) counts. The parameters of microvascular imaging and the expression of VEGF and MVD in primary breast cancer were calculated. The correlation between these factors and the overall and progression-free survival rate were analyzed using the Kaplan-Meier method. Among 121 cases, the positive VEGF cases were 75 and negative ones were 46. The cut point of 52.3 was calculated by the regressive curve for MVD counts. The data showed the mean intensity (MI) was positively associated with both the MVD counts (r = .51, p prognosis of patients, high VEGF expression and MVD counts were associated with reduced progressive and survival times (PFS, p = .032 and p = .034; OS, p = .041 and p = .038, respectively). The correlation between parameters of microvascular imaging, VEGF expressive status, and the MVD counts were established. The cut point of mean intensity (MI = 40) was used to investigate as an independent predictor for PFS (p = .021) and OS (p = .025), respectively, due to a strong correlation between MVD counts and VEGF expression in patients with invasive breast cancer. The microvascular imaging could be a visual and helpful tool to predict the prognosis of patients with invasive breast cancer treated by adjuvant therapies.

  19. Association of mast cell-derived VEGF and proteases in Dengue shock syndrome.

    Directory of Open Access Journals (Sweden)

    Takahisa Furuta

    Full Text Available BACKGROUND: Recent in-vitro studies have suggested that mast cells are involved in Dengue virus infection. To clarify the role of mast cells in the development of clinical Dengue fever, we compared the plasma levels of several mast cell-derived mediators (vascular endothelial cell growth factor [VEGF], soluble VEGF receptors [sVEGFRs], tryptase, and chymase and -related cytokines (IL-4, -9, and -17 between patients with differing severity of Dengue fever and healthy controls. METHODOLOGY/PRINCIPAL FINDINGS: The study was performed at Children's Hospital No. 2, Ho Chi Minh City, and Vinh Long Province Hospital, Vietnam from 2002 to 2005. Study patients included 103 with Dengue fever (DF, Dengue hemorrhagic fever (DHF, and Dengue shock syndrome (DSS, as diagnosed by the World Health Organization criteria. There were 189 healthy subjects, and 19 febrile illness patients of the same Kinh ethnicity. The levels of mast cell-derived mediators and -related cytokines in plasma were measured by ELISA. VEGF and sVEGFR-1 levels were significantly increased in DHF and DSS compared with those of DF and controls, whereas sVEGFR-2 levels were significantly decreased in DHF and DSS. Significant increases in tryptase and chymase levels, which were accompanied by high IL-9 and -17 concentrations, were detected in DHF and DSS patients. By day 4 of admission, VEGF, sVEGFRs, and proteases levels had returned to similar levels as DF and controls. In-vitro VEGF production by mast cells was examined in KU812 and HMC-1 cells, and was found to be highest when the cells were inoculated with Dengue virus and human Dengue virus-immune serum in the presence of IL-9. CONCLUSIONS: As mast cells are an important source of VEGF, tryptase, and chymase, our findings suggest that mast cell activation and mast cell-derived mediators participate in the development of DHF. The two proteases, particularly chymase, might serve as good predictive markers of Dengue disease severity.

  20. Predictors of anti-VEGF treatment response in neovascular age-related macular degeneration.

    Science.gov (United States)

    Finger, Robert P; Wickremasinghe, Sanjeewa S; Baird, Paul N; Guymer, Robyn H

    2014-01-01

    Currently available evidence on predictors of anti-vascular endothelial growth factor (VEGF) treatment response in neovascular age-related macular degeneration was reviewed. No meta-analysis of results is possible because of a lack of controlled and randomized trials, varying treatment regimes and outcome measures used, as well as suboptimal reporting. For genetic factors, most evidence to date has been generated for single nucleotide polymorphisms (SNPs) in the complement factor H (CFH), and VEGF-A genes. Just under half of the SNPs assessed in the CFH gene and 15% of the SNPs assessed in the VEGF gene were found to be associated with visual outcomes or the number of injections required during follow-up. Some evidence suggests association of worse treatment outcomes as well as a younger age at treatment onset with an increasing number of risk alleles in known risk genes (CFH and ARMS2/HTRA1) and polymorphisms in the VEGF-A gene. Clinical factors such as higher age, a better visual acuity (VA), a larger choroidal neovascularization (CNV) lesion at baseline, and a delay between symptom onset and initiation of treatment of more than 3 weeks also impact outcomes. Conversely, a worse acuity at baseline predicted more gain in vision. Overall, patients presenting with good acuity at baseline were more likely to have good VA at follow up, but the gain afforded by treatment was impacted by a ceiling effect. Most available evidence suggests a strong association of clinical factors such as age, baseline VA, and CNV lesion size with anti-VEGF treatment outcomes. No behavioral factors such as smoking influence treatment outcomes. Based on the studies conducted so far, the evidence suggests that underlying genotype of known AMD risk associated genes or of the VEGF-A gene have a limited effect, whereas presenting clinical factors appear to be more important in determining treatment outcomes.

  1. Involvement of FGF and BMP family proteins and VEGF in early human kidney development.

    Science.gov (United States)

    Carev, Dominko; Saraga, Marijan; Saraga-Babic, Mirna

    2008-07-01

    The spatial and temporal pattern of the appearance of the fibroblast growth factor proteins (FGF-8 and FGF-10), the bone morphogenetic proteins (BMP-2/4 subfamily and BMP-7) and the vascular endothelial growth factor protein (VEGF) was investigated in the human mesonephros and metanephros of the 5-9 week-old conceptuses. In the mesonephros, both FGF's and BMP's were found in all structures and their expression slightly decreased in the early fetal period. VEGF positivity appeared in all mesonephric structures, and increased in the fetal period coincidently with formation of the mesonephric blood vessel network. In the metanephros, FGF-8 first appeared only in the metanephric mesenchyme, but from the 7th week on, its reactivity increased and spread to other metanephric structures. FGF-10 positive cells appeared in all metanephric structures already in the 5th week, and slightly intensified with progression of development. Cell survival and nephrogenesis in the permanent kidney might be associated with the appearance of both growth factors. Both BMP-2/4 and BMP-7 displayed a similar pattern of reactivity in all metanephric structures, and their reactivity intensified with advancing development. Alterations in their pattern of appearance might lead to the formation of small and dysplastic kidneys. Already in the earliest developmental stages, VEGF protein appeared in all metanephric structures. At later stages, VEGF showed more intense reaction in the collecting system than in the differentiating nephrons and interstitium. Due to VEGF involvement in vasculogenesis and angiogenesis, abnormal VEGF appearance might lead to impaired formation of the blood vessel network in the human permanent kidney.

  2. Angiogenetic axis angiopoietins/Tie2 and VEGF in familial breast cancer.

    Science.gov (United States)

    Danza, K; Pilato, B; Lacalamita, R; Addati, T; Giotta, F; Bruno, A; Paradiso, A; Tommasi, S

    2013-08-01

    Angiogenesis leads to the formation of blood vessels from pre-existing ones, allowing tumor growth. Vascular endothelial growth factor (VEGF) and Angiopoietins (Ang-1, Ang-2) have a pivotal role in tumor angiogenesis but few data regarding their role in hereditary breast cancer are available. The aim of the present study was to analyze Ang-1, Ang-2, tyrosine-protein kinase receptor Tie2 and VEGF expression and their correlation in a cohort of familial and sporadic breast cancers in order to verify whether the presence of germline mutations in BRCA may have a role in tumor microenvironment regulation. Tumor samples from a cohort of 41 patients with a first diagnosis and a family history of breast cancer and 19 patients with sporadic breast cancers were enrolled. The expression of Tie2, Ang-1, Ang-2 and VEGF were analyzed by quantitative real-time PCR. Patients harboring BRCA mutations had higher levels of Ang-1 (P=0.05), Ang-2 (P=0.02) and VEGF (P=0.04) mRNA compared with those without BRCA mutations (BRCAX). The same was observed in triple-negative breast cancer (TNBC). Moreover, a positive correlation between Ang-2 and VEGF was found in both the familial breast cancer group (BRCA carriers: r=0.83; P<0.0001 and BRCAX: r=0.58; P=0.008) and in TNBC (r=0.62; P=0.007). The higher levels of Ang-1, Ang-2 and VEGF mRNA found in BRCA carriers and TNBCs suggest that they could be attractive angiogenic therapeutic targets in these breast cancers.

  3. 血管内皮生长因子基因转染骨髓间充质干细胞对慢性肾衰竭大鼠血管新生的影响%Effects of bone marrow mesenchymal stem cell gene transfection by VEGF gene on angiogenesis in rats with chronic renal failure

    Institute of Scientific and Technical Information of China (English)

    崔炯; 邓金秀; 万建新

    2014-01-01

    Objective To investigate the effects of bone marrow mesenchymal stem cell (MSCs) gene transfection by VEGF (vascular endothelial growth factor ) gene on the repair of glomerular endothelia and angiogenesis in rats with chronic renal failure (CRF). Methods MSCs isolated from rat bone marrow were cultured and transfected with recombinant Ad5-hVEGF-EGFP.Then, effects of transfection on cell proliferation and secretion of VEGF were observed closely .Fifty male SD rats were randomly divided into 5 groups:the sham operation group , the CRF group, the MSCs transplantation group , the Ad-VEGF injection group and the MSCs transfected by VEGF gene group.The animal CRF model was established by a two-stage 5/6 nephrectomy procedure in rats .For the animals of the sham opera-tion group and the CRF group , DMEM culture solution without serum was injected through the right renal artery prior to nephrectomy of the right kidney.The animals of the other 3 groups were treated with MSCs , Ad-VEGF and MSCs transfected by VEGF genes.After 8 weeks, the renal function of all the groups was detected and CD 31 expression in glomerulus was measured by immunohistochemistry .Results The effect of Ad5-hVEGF-EGFP on the proliferation of MSCs could not be detected after transfection .The level of VEGF protein in supernatant after VEGF gene transfection was significantly higher than that of the control group (P<0.05).The Scr and BUN levels in the MSCs transplantation group and the MSCs transfected by VEGF gene group were lower than those of the CRF group (P<0.05).As compared with those of the MSCs transplantation group , the levels of Scr and BUN for the MSCs transfected by VEGF gene group were even lower (P<0.05).CD31 expression in glomerulus for the CRF group was decreased , as compared with that of the sham operation group (P<0.05).However, CD31 expression in glomerulus was increased to some extent following treatment by MSCs or MSCs transfected by VEGF gene (P<0.05).CD31 expression level was

  4. Recombinant Collagenlike Proteins

    Science.gov (United States)

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  5. Dielectronic recombination theory

    Energy Technology Data Exchange (ETDEWEB)

    LaGattuta, K.J.

    1991-12-31

    A theory now in wide use for the calculation of dielectronic recombination cross sections ({sigma}{sup DR}) and rate coefficients ({alpha}{sup DR}) was one introduced originally by Feshbach for nuclear physics applications, and then later adapted for atomic scattering problems by Hahn. In the following, we briefly review this theory in a very general form, which allows one to account for the effects of overlapping and interacting resonances, as well as continuum-continuum coupling. An extension of our notation will then also allow for the inclusion of the effects of direct radiative recombination, along with a treatment of the interference between radiative and dielectronic recombination. Other approaches to the calculation of {sigma}{sup DR} have been described by Fano and by Seaton. We will not consider those theories here. Calculations of {alpha}{sup DR} have progressed considerably over the last 25 years, since the early work of Burgess. Advances in the reliability of theoretical predictions have also been promoted recently b a variety of direct laboratory measurements of {sigma}{sup DR}. While the measurements of {sigma}{sup DR} for {delta}n {ne} 0 excitations have tended to agree very well with calculations, the case of {delta}n = 0 has been much problematic. However, by invoking a mechanism originally proposed by Jacobs, which takes into account the effect of stray electric fields on high Rydberg states (HRS) participating in the DR process, new calculations have improved the agreement between theory and experiment for these cases. Nevertheless, certain discrepancies still remain.

  6. Estimation of Immunohistochemical Expression of VEGF in Ductal Carcinomas of the Breast

    DEFF Research Database (Denmark)

    Maae, Else; Nielsen, Martin; Dahl Steffensen, Karina

    2012-01-01

    . Material and methods: We immunostained whole tumor sections for VEGF-A, -B, and VEGFR-1 of invasive ductal carcinomas of the breast and scored the tumors manually by staining intensity as the only parameter and by a combination of qualitative and quantitative staining information. We also introduced...... was repeated in a second run with a new delineation of the tumor area. Results: We found that the AI scores were correlated to the manual scoring of VEGF intensity, but the reproducibility of manual IHC scores was rather poor. The AI scores were reproducible and the restricted analysis of 25% of the tumor area...

  7. mRNA EXPRESSION OF PTEN AND VEGF GENES IN EPITHELIAL OVARIAN CANCER

    Institute of Scientific and Technical Information of China (English)

    陈颖; 赵雨杰; 郑华川; 杨雪飞; 汪桂兰; 辛彦

    2003-01-01

    Objective: To investigate the mRNA expression of PTEN and vascular endothelial growth factor (VEGF) genes in ovarian cancer. Methods:We examined mRNA expression of PTEN and VEGF165 in normal ovary (n=5), ovarian cyst (n=5), ovarian borderline tumor (n=9), epithelial ovarian cancer (n=60) and ovarian cancer cell line (CAOV-3) by RT-PCR. Their expressions were compared with clinicopathological features of ovarian cancer. The relationship between their expressions was concerned in all ovarian samples as well. Results:mRNA expression level of PTEN gene was significantly lower in ovarian borderline tumor or ovarian cancer than that in normal ovary or ovarian cyst(P<0.05). It was negatively correlated with clinicopathological staging(P<0.05),whereas positively with histological differentiation (P<0.05). mRNA expression level of PTEN gene was significantly lower in ovarian endometrioid cancer than ovarian serous or mucinous cancer(P<0.05). mRNA expression level of VEGF165 gene was significantly higher in ovarian cancer than that in normal ovary or ovarian cyst(P<0.05). It was positively correlated with clinicopathological staging(P<0.05), whereas negatively with histological differentiation (P<0.05). mRNA expression level of VEGF165 gene was significantly higher in ovarian serous cancer than in other ovarian epithelial cancers (P<0.05). mRNA expression of VEGF165 gene was inversely correlated with mRNA expression level of PTEN gene. Conclusion:Down-regulated expression of PTEN and up-regulated expression of VEGF were considered as two important events in tumorigenesis of ovarian cancer and could be used as molecular markers to indicate the pathobiological behaviors of ovarian cancer. Decreased PTEN expression and increased VEGF expression were closely associated with tumorigenesis and pathobiological behaviors of ovarian endometrioid and serous cancer respectively. Reduced expression of PTEN gene might be involved in carcinogenesis and progression of ovarian cancer by

  8. Did the universe recombine

    Energy Technology Data Exchange (ETDEWEB)

    Bartlett, J.G.; Stebbins, A. (California, University, Berkeley (USA) Toronto, University (Canada))

    1991-04-01

    The Zel'dovich-Sunyaev model-independent arguments for the existence of a neutral hydrogen phase is reviewed in light of new limits on the Compton y parameter from COBE. It is concluded that with baryon densities compatible with standard cosmological nucleosynthesis, the universe could have remained fully ionized throughout its history without producing a detectable spectral distortion. It is argued that it is unlikely that spectral observations of the cosmic microwave background will ever require the universe to have recombined for flat cosmologies. 22 refs.

  9. Micro-Raman and micro-photoluminescence study of bio-conjugated core–shell CdSe/ZnS nanocrystals

    Energy Technology Data Exchange (ETDEWEB)

    Borkovska, L., E-mail: bork@isp.kiev.ua [V. Lashkaryov Institute of Semiconductor Physics of NASU, pr. Nauky 41, 03028 Kyiv (Ukraine); Korsunska, N.; Stara, T.; Kolomys, O.; Strelchuk, V. [V. Lashkaryov Institute of Semiconductor Physics of NASU, pr. Nauky 41, 03028 Kyiv (Ukraine); Rachkov, O. [The Institute of Molecular Biology and Genetics of NASU, Zabolotnogo Str. 150, 03680 Kyiv (Ukraine); Kryvko, A. [Instituto Politécnico Nacional – ESIME, Av. IPN, Ed. Z4, U.P.A.L.M., 07738 Mexico D.F. (Mexico)

    2014-11-15

    The micro-Raman and micro-photoluminescence spectra of non-conjugated and conjugated with antibody against S6K2 commercial CdSe/ZnS quantum dots (QDs) were investigated under different excitation wavelengths and at different temperatures. In the photoluminescence (PL) spectra, the additional PL band shifted on 0.6–0.65 eV to higher energies from the CdSe/ZnS QD exciton PL band is revealed. The relative intensity of this band is found to be several times larger in bio-conjugated QDs, than in the non-conjugated ones. The characteristics of both PL bands (the PL intensity, spectral position and half-width of the PL band) vary similarly under continuous laser light irradiation, storage of the QD samples in the atmospheric ambience as well as during the temperature change. In the Raman spectra recorded under excitation resonant with the high-energy PL band, the additional Raman peaks at about 300 cm{sup −1} and 600 cm{sup −1}, which are close to the frequency of LO and 2LO phonons of bulk CdS, are found. It is proposed that alloyed QDs with chemical composition close to CdS are responsible for the additional high-energy PL band. The possible reasons for the formation of the alloyed QDs are discussed.

  10. Interaction between VEGF and Calcium-Independent Phospholipase A(2) in Proliferation and Migration of Retinal Pigment Epithelium

    DEFF Research Database (Denmark)

    Andersen, Emelie Cammilla; Andreasen, Jens Rovelt; Vohra, Rupali

    2012-01-01

    thymidine in DNA and by a Boyden chamber technique, respectively. A luciferase assay monitored the VEGF-induced iPLA2-VIA transcriptional activity. Western blot analysis and an activity assay were used to detect the protein levels and activity of iPLA2-VIA respectively after treatment with VEGF. Results...

  11. Bone marrow stromal cells with a combined expression of BMP-2 and VEGF-165 enhanced bone regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Xiao Caiwen; Zhou Huifang; Fu Yao; Gu Ping; Fan Xianqun [Department of Ophthalmology, Shanghai Ninth People' s Hospital, Shanghai JiaoTong University School of Medicine, Shanghai 200011 (China); Liu Guangpeng [Key Laboratory of Tissue Engineering, Shanghai Ninth People' s Hospital, Shanghai JiaoTong University School of Medicine, Shanghai 200011 (China); Zhang Peng [Center for Translational Medicine Research and Development, Shenzhen Institute of Advanced Technology, Chinese Academy of Science (China); Hou Hongliang; Tang Tingting, E-mail: drfanxianqun@126.com [Department of Orthopedics, Shanghai Ninth People' s Hospital, Shanghai JiaoTong University School of Medicine, Shanghai 200011 (China)

    2011-02-15

    Bone graft substitutes with osteogenic factors alone often exhibit poor bone regeneration due to inadequate vascularization. Combined delivery of osteogenic and angiogenic factors from biodegradable scaffolds may enhance bone regeneration. We evaluated the effects of bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF), combined with natural coral scaffolds, on the repair of critical-sized bone defects in rabbit orbits. In vitro expanded rabbit bone marrow stromal cells (BMSCs) were transfected with human BMP2 and VEGF165 genes. Target protein expression and osteogenic differentiation were confirmed after gene transduction. Rabbit orbital defects were treated with a coral scaffold loaded with BMP2-transduced and VEGF-transduced BMSCs, BMP2-expressing BMSCs, VEGF-expressing BMSCs, or BMSCs without gene transduction. Volume and density of regenerated bone were determined by micro-computed tomography at 4, 8, and 16 weeks after implantation. Neovascularity, new bone deposition rate, and new bone formation were measured by immunostaining, tetracycline and calcein labelling, and histomorphometric analysis at different time points. The results showed that VEGF increased blood vessel formation relative to groups without VEGF. Combined delivery of BMP2 and VEGF increased new bone deposition and formation, compared with any single factor. These findings indicate that mimicking the natural bone development process by combined BMP2 and VEGF delivery improves healing of critical-sized orbital defects in rabbits.

  12. A HUVEC line with a stable expression of the VEGF121 gene to achieve complete endothelialization of blood conduits.

    Science.gov (United States)

    Liu, L-S; Wei, D-H; Tang, C-K; Wang, G-X; Zhang, S-C; Yin, W-D; Yang, Y-Z; Legrand, A-P; Guidoin, R

    2007-01-01

    The purpose of this investigation was to establish monoclonal cell lines of HUVEC with the stable expression of the VEGF(121) gene. Such cells are likely to better adhere to the luminal surface of stents or grafts and to promote a complete endothelialization. The eukaryotic expression vector PCD(2)-VEGF(121) was transfected into cell lines of HUVEC mediated by lipofect AMINE. The positive clones were obtained by the screening of G(418). The transcription and expression of the VEGF gene were investigated by RT-PCR and immunocytochemistry, respectively. The experiment of Miles was applied for the assay of the biological activity of the protein of the VEGF produced by the HUVEC lines with transfected PCD(2)-VEGF(121). The growth curve was made for comparison with that of non-transfected HUVEC line cells. The positive clone cells from which transcripted the mRNA of VEGF(121) gene were obtained by RT-PCR. The positive results of the immunocytochemistry were found and the high biological activity of VEGF in the media was detected in the positive clone cells only. The time to achieve the multiplication of the positive clone cells by a factor of 2 was shorter than that of the non-transfected HUVEC line calculated from the growth curve. The HUVEC line of monoclonal cells with the stable expression of VEGF(121) gene has been established successfully and can be employed on the luminal surfaces of foreign blood conduits.

  13. The mechanisms that regulate the localization and overexpression of VEGF receptor-2 are promising therapeutic targets in cancer biology

    NARCIS (Netherlands)

    Kampen, Kim R.

    2012-01-01

    The vascular endothelial growth factor (VEGF) family has been proposed to be the most important signaling protein family in vessel formation and maturation. VEGF receptor-2 (VEGFR-2) plays an abundant role in the most common forms of cancer. The localization of VEGFR-2 expression is important in can

  14. VEGF-A isoforms differentially regulate ATF-2–dependent VCAM-1 gene expression and endothelial–leukocyte interactions

    Science.gov (United States)

    Fearnley, Gareth W.; Odell, Adam F.; Latham, Antony M.; Mughal, Nadeem A.; Bruns, Alexander F.; Burgoyne, Nicholas J.; Homer-Vanniasinkam, Shervanthi; Zachary, Ian C.; Hollstein, Monica C.; Wheatcroft, Stephen B.; Ponnambalam, Sreenivasan

    2014-01-01

    Vascular endothelial growth factor A (VEGF-A) regulates many aspects of vascular physiology. VEGF-A stimulates signal transduction pathways that modulate endothelial outputs such as cell migration, proliferation, tubulogenesis, and cell–cell interactions. Multiple VEGF-A isoforms exist, but the biological significance of this is unclear. Here we analyzed VEGF-A isoform–specific stimulation of VCAM-1 gene expression, which controls endothelial–leukocyte interactions, and show that this is dependent on both ERK1/2 and activating transcription factor-2 (ATF-2). VEGF-A isoforms showed differential ERK1/2 and p38 MAPK phosphorylation kinetics. A key feature of VEGF-A isoform–specific ERK1/2 activation and nuclear translocation was increased phosphorylation of ATF-2 on threonine residue 71 (T71). Using reverse genetics, we showed ATF-2 to be functionally required for VEGF-A–stimulated endothelial VCAM-1 gene expression. ATF-2 knockdown blocked VEGF-A–stimulated VCAM-1 expression and endothelial–leukocyte interactions. ATF-2 was also required for other endothelial cell outputs, such as cell migration and tubulogenesis. In contrast, VCAM-1 was essential only for promoting endothelial–leukocyte interactions. This work presents a new paradigm for understanding how soluble growth factor isoforms program complex cellular outputs and responses by modulating signal transduction pathways. PMID:24966171

  15. Penduliflaworosin, a Diterpenoid from Croton crassifolius, Exerts Anti-Angiogenic Effect via VEGF Receptor-2 Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Yeyin Liang

    2017-01-01

    Full Text Available Anti-angiogenesis targeting vascular endothelial growth factor receptor-2 (VEGFR-2 has been considered as an important strategy for cancer therapy. Penduliflaworosin is a diterpenoid isolated from the plant Croton crassifolius. Our previous study showed that this diterpenoid possesses strong anti-angiogenic activity by inhibiting vessel formation in zebrafish. This study was conducted to further investigate the anti-angiogenic activity and mechanism of penduliflaworosin. Results revealed that penduliflaworosin significantly inhibited VEGF-induced angiogenesis processes including proliferation, invasion, migration, and tube formation of human umbilical vein endothelial cells (HUVECs. Moreover, it notably inhibited VEGF-induced sprout formation of aortic rings and blocked VEGF-induced vessel formation in mice. Western blotting studies showed that penduliflaworosin inhibited phosphorylation of the VEGF receptor-2 and its downstream signaling mediators in HUVECs, suggesting that the anti-angiogenic activity was due to an interference with the VEGF/VEGF receptor-2 pathway. In addition, molecular docking simulation indicated that penduliflaworosin could form hydrogen bonds within the ATP-binding region of the VEGF receptor-2 kinase unit. Finally, cytotoxicity assay showed that penduliflaworosin possessed little toxicity toward both cancer and normal cells. Taken together, our findings demonstrate that penduliflaworosin exerts its anti-angiogenic effect via the VEGF receptor-2 signaling pathway. The anti-angiogenic property and low cytotoxicity of penduliflaworosin suggest that it may be useful in cancer treatments.

  16. VEGFR2-mediated vascular dilation as a mechanism of VEGF-induced anemia and bone marrow cell mobilization.

    Science.gov (United States)

    Lim, Sharon; Zhang, Yin; Zhang, Danfang; Chen, Fang; Hosaka, Kayoko; Feng, Ninghan; Seki, Takahiro; Andersson, Patrik; Li, Jingrong; Zang, Jingwu; Sun, Baocun; Cao, Yihai

    2014-10-23

    Molecular mechanisms underlying tumor VEGF-induced host anemia and bone marrow cell (BMC) mobilization remain unknown. Here, we report that tumor VEGF markedly induced sinusoidal vasculature dilation in bone marrow (BM) and BMC mobilization to tumors and peripheral tissues in mouse and human tumor models. Unexpectedly, anti-VEGFR2, but not anti-VEGFR1, treatment completely blocked VEGF-induced anemia and BMC mobilization. Genetic deletion of Vegfr2 in endothelial cells markedly ablated VEGF-stimulated BMC mobilization. Conversely, deletion of the tyrosine kinase domain from Vegfr1 gene (Vegfr1(TK-/-)) did not affect VEGF-induced BMC mobilization. Analysis of VEGFR1(+)/VEGFR2(+) populations in peripheral blood and BM showed no significant ratio difference between VEGF- and control tumor-bearing animals. These findings demonstrate that vascular dilation through the VEGFR2 signaling is the mechanism underlying VEGF-induced BM mobilization and anemia. Thus, our data provide mechanistic insights on VEGF-induced BMC mobilization in tumors and have therapeutic implications by targeting VEGFR2 for cancer therapy.

  17. Proteasome inhibitor MG-132 regulates the expression of VEGF in human bronchial epithelial cell line, BEAS-2B

    Institute of Scientific and Technical Information of China (English)

    Xuefan Cui; Kaisheng Yin; Mao Huang; Linfu Zhou

    2005-01-01

    Objective: To explore the effects of MG-132 on the expression of VEGF in bronchial epithelial cell line, BEAS2B. Methods: Semi-quantitive RT-PCR for VEGF mRNA and enzyme-linked immunosorbent assay (ELISA) for VEGF protein were performed. Results: MG-132 increased the expression of VEGF mRNA and protein BEAS-2B cells in time-and concentration-dependent manners. After 24-h stimulation, 25 μmol/L MG-132 increased the maximal levels of VEGF protein in cell-conditioned medium. When the cells were stimulated with cycloheximide(CHX) before treatment with MG-132, the MG-132-induced production of VEGF protein was inhibited compared to the unstimulated cells. Supernatant of condition-medium treatment with MG-132 enhanced the growth of HUVEC.Conclusion: MG-132 induces VEGF gene expression in human bronchial epithelial cells line, BEAS-2B, and the MG-132-induced expression of VEGF may modulate lung tissue injury due to airway inflammation.

  18. Novel recombinant sapovirus in Bangladesh.

    Science.gov (United States)

    Dey, Shuvra Kanti; Mizuguchi, Masashi; Okitsu, Shoko; Ushijima, Hiroshi

    2011-01-01

    Recombination of RNA viruses plays an important part in molecular epidemiological study, virus evolution, vaccine design, and viral control programs. Sapovirus, a member of the family Caliciviridae, is one of the major causative agents of viral gastroenteritis affecting all age groups. Sapovirus capsid and polymerase regions were amplified by PCR using specific primers. PCR products were sequenced directly and sequence analysis was performed using CLUSTAL X, SimPlot, and MEGA 4 software package. Based on the genetic analysis, a novel, naturally occurring recombinant sapovirus strain was identified in Bangladesh. Breakpoint analysis of the recombinant sapovirus showed that the recombination site was at the open reading frame ORF1/ORF2 overlap. We described the genetic characterization of a novel, naturally occurring recombinant sapovirus and provided the first evidence of recombination in sapovirus in Bangladesh.

  19. Cell biology of mitotic recombination

    DEFF Research Database (Denmark)

    Lisby, Michael; Rothstein, Rodney

    2015-01-01

    Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules...... of this review include the stoichiometry and dynamics of recombination complexes in vivo, the choreography of assembly and disassembly of recombination proteins at sites of DNA damage, the mobilization of damaged DNA during homology search, and the functional compartmentalization of the nucleus with respect...... as well as the cellular organization of the process of homologous recombination. Herein we review the cell biological aspects of mitotic homologous recombination with a focus on Saccharomyces cerevisiae and mammalian cells, but will also draw on findings from other experimental systems. Key topics...

  20. Vascular endothelial growth factor C (VEGF-C in esophageal cancer correlates with lymph node metastasis and poor patient prognosis

    Directory of Open Access Journals (Sweden)

    Naganawa Yasuhiro

    2010-06-01

    Full Text Available Abstract Background The diagnosis of lymph node metastasis in esophageal cancer by the presence and number of metastatic lymph nodes is an extremely important prognostic factor. In addition, the indication of non-surgical therapy is gaining more attention. Vascular endothelial growth factor C (VEGF-C is potentially lymphangiogenic and selectively induces hyperplasia of the lymphatic vasculature. In this study, we investigated the expression of VEGF-C and whether it correlated with various clinico-pathologic findings. Methods KYSE series of esophageal cancer cell lines and 106 patients with primary esophageal squamous cell carcinomas who had undergone radical esophagectomy were analyzed. VEGF<