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Sample records for host vegf vegfr2

  1. Correlation Between Tumor Growth Delay and Expression of Cancer and Host VEGF, VEGFR2, and Osteopontin in Response to Radiotherapy

    SciTech Connect

    Solberg, Timothy D.; Nearman, Jessica; Mullins, John; Li Sicong; Baranowska-Kortylewicz, Janina

    2008-11-01

    Purpose: To determine the late effects of radiotherapy (RT) on vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR2), and osteopontin (OPN) expression in cancer and stromal cells. Methods and Materials: LS174T xenografted athymic mice were used as a tumor model. Radiation was delivered in two equivalent fractionation schemes: 5 x 7 Gy and 1 x 20 Gy, the latter at two dose rates. Results: Tumor growth arrest was similar in all treatment groups, with the exception of a better response of small-size tumors in the 5 x 7-Gy group. The host VEGF and OPN levels were directly proportional to the tumor doubling time and were independent of the fractionation scheme. The host and cancer cell VEGFR2 levels in tumor were also directly related to the tumor response to RT. Conclusion: Upregulated VEGFR2 in cancer cells suggest paracrine signaling in the VEGFR2 pathway of cancer cells as the factor contributing to RT failure. The transient activation of the host VEGF/VEGFR2 pathway in tumor supports the model of angiogenic regeneration and suggests that radiation-induced upregulation of VEGF, VEGFR2, and downstream proteins might contribute to RT failure by escalating the rate of vascular repair. Coexpression of host OPN and VEGF, two factors closely associated with angiogenesis, indicate that OPN can serve as a surrogate marker of tumor recovery after RT. Taken together, these results strongly support the notion that to achieve optimal therapeutic outcomes, the scheduling of RT and antiangiogenic therapies will require patient-specific post-treatment monitoring of the VEGF/VEGFR2 pathway and that tumor-associated OPN can serve as an indicator of tumor regrowth.

  2. VEGFR2-mediated vascular dilation as a mechanism of VEGF-induced anemia and bone marrow cell mobilization.

    PubMed

    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.

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

    PubMed Central

    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

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

    PubMed Central

    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

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

    PubMed

    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

    2016-05-15

    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. © 2016. Published by The Company of Biologists Ltd.

  6. In vivo Studies of VEGFR2 Interactions in the Presence and Absence of VEGF

    NASA Astrophysics Data System (ADS)

    King, Christopher; Hristova, Kalina, , Dr.

    Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) is a receptor tyrosine kinase (RTK) that is critical for vasculogenesis and angiogenesis. Enhanced VEGFR2 signaling is often correlated with malignancy. Recently, it was shown that full-length VEGFR2 exists in a monomer-dimer equilibrium in the absence of bound VEGF. Thus, the canonical model of RTK activation does not seem to adequately describe the behavior of VEGFR2 in the cell membrane. In order to understand the role that VEGFR2 extracellular domain plays in unliganded dimerization in live cells, we utilize Fully Quantified Spectral Imaging (FSI) to probe the interactions of VEGFR2 mutant constructs with rationally truncated EC domains. In addition, we investigate the stoichiometry of ligand binding to VEGFR2 EC domain as a function of VEGF concentration and total receptor expression. Supported by NSF MCB 1157687 and NIH GM068619 (to KH) and and NSF Graduate Research Fellowship DGE-1232825 (to CK).

  7. Editing VEGFR2 Blocks VEGF-Induced Activation of Akt and Tube Formation

    PubMed Central

    Huang, Xionggao; Zhou, Guohong; Wu, Wenyi; Ma, Gaoen; D'Amore, Patricia A.; Mukai, Shizuo; Lei, Hetian

    2017-01-01

    Purpose Vascular endothelial growth factor receptor 2 (VEGFR2) plays a key role in VEGF-induced angiogenesis. The goal of this project was to test the hypothesis that editing genomic VEGFR2 loci using the technology of clustered regularly interspaced palindromic repeats (CRISPR)-associated DNA endonuclease (Cas)9 in Streptococcus pyogenes (SpCas9) was able to block VEGF-induced activation of Akt and tube formation. Methods Four 20 nucleotides for synthesizing single-guide RNAs based on human genomic VEGFR2 exon 3 loci were selected and cloned into a lentiCRISPR v2 vector, respectively. The DNA fragments from the genomic VEGFR2 exon 3 of transduced primary human retinal microvascular endothelial cells (HRECs) were analyzed by Sanger DNA sequencing, surveyor nuclease assay, and next-generation sequencing (NGS). In the transduced cells, expression of VEGFR2 and VEGF-stimulated signaling events (e.g., Akt phosphorylation) were determined by Western blot analyses; VEGF-induced cellular responses (proliferation, migration, and tube formation) were examined. Results In the VEGFR2-sgRNA/SpCas9–transduced HRECs, Sanger DNA sequencing indicated that there were mutations, and NGS demonstrated that there were 83.57% insertion and deletions in the genomic VEGFR2 locus; expression of VEGFR2 was depleted in the VEGFR2-sgRNA/SpCas9–transduced HRECs. In addition, there were lower levels of Akt phosphorylation in HRECs with VEGFR2-sgRNA/SpCas9 than those with LacZ-sgRNA/SpCas9, and there was less VEGF-stimulated Akt activation, proliferation, migration, or tube formation in the VEGFR2-depleted HRECs than those treated with aflibercept or ranibizumab. Conclusions The CRISPR-SpCas9 technology is a potential novel approach to prevention of pathologic angiogenesis. PMID:28241310

  8. Crosstalk between VEGF-A/VEGFR2 and GDNF/RET signaling pathways

    SciTech Connect

    Tufro, Alda . E-mail: atufro@aecom.yu.edu; Teichman, Jason; Banu, Nazifa; Villegas, Guillermo

    2007-06-29

    Vascular endothelial growth factor (VEGF-A) plays multiple roles in kidney development: stimulates cell proliferation, survival, tubulogenesis, and branching morphogenesis. However, the mechanism that mediates VEGF-A induced ureteric bud branching is unclear. Glial-derived neurotrophic factor (GDNF) signaling through tyrosine kinase c-RET is the major regulator of ureteric bud branching. Here we examined whether VEGF-A regulates RET signaling. We determined that ureteric bud-derived cells express the main VEGF-A signaling receptor, VEGFR2 and RET, by RT-PCR, immunoblotting, and immunocytochemistry. We show that the VEGF-A isoform VEGF{sub 165} induces RET-tyr{sup 1062} phosphorylation in addition to VEGFR2 autophosphorylation, that VEGF{sub 165} and GDNF have additive effects on RET-tyr{sup 1062} phosphorylation, and that VEGFR2 and RET co-immunoprecipitate. Functionally, VEGF{sub 165} induces ureteric bud cell proliferation and branching morphogenesis. Similarly, in embryonic kidney explants VEGF{sub 165} induces RET-tyr{sup 1062} phosphorylation and upregulates GDNF. These findings provide evidence for a novel cooperative interaction between VEGFR2 and RET that mediates VEGF-A functions in ureteric bud cells.

  9. VEGF and its soluble receptor VEGFR-2 in hypertensive disorders during pregnancy: the Indian scenario.

    PubMed

    Rath, G; Tripathi, R

    2012-03-01

    Hypertensive disorders are the most common medical problem encountered during pregnancy due to defective angiogenesis during placental development. Vascular endothelial growth factor (VEGF) is one of the angiogenic growth factors that stimulates angiogenesis. The recombinant form of its soluble receptor VEGF receptor-2 (sVEGFR-2) has anti-angiogenic activity. However, there is a paucity of information on serum VEGF and sVEGFR-2 concentrations in different sub-groups of hypertensive disorders during pregnancy. In this cross-sectional study, we evaluated the concentrations and the diagnostic utility of VEGF and sVEGFR-2 in gestational hypertension (GH, n=90), pre-eclampsia (PE, n=180), eclampsia (n=90) and control (n=180) pregnancy at different gestations. VEGF levels were significantly higher in PE and eclamptic (median=19.53 pg ml(-1); 60.36 pg ml(-1), P=0.0001) groups as compared with the control ones (median=18 pg ml(-1)). But, the serum sVEGFR-2 levels were found to be significantly decreased from GH to eclampsia groups (median=5196; 3972 pg ml(-1)) as compared with control groups (median=7417 pg ml(-1)). As the gestation advanced, there was an inverse association in the serum concentrations of sVEGFR-2 among the control, GH, PE and eclampsia groups. At both 34 and >34 weeks of gestations, higher sensitivity and specificity were observed for sVEGFR-2 in differentiating GH (50.8, 50%; 76.6, 76.6%), PE (63, 63%; 90, 90%) and eclampsia (65, 66.6%; 90, 90%) from the control pregnancy. This upregulation of VEGF and downregulation of sVEGFR-2 concentrations in different study groups may be due to hypoxia and could be involved intimately in the pathogenesis of these disorders. This study may contribute in understanding etio-pathogenesis of different hypertensive disorders during pregnancy.

  10. VEGF induces proliferation of human hair follicle dermal papilla cells through VEGFR-2-mediated activation of ERK

    SciTech Connect

    Li, Wei; Man, Xiao-Yong; Li, Chun-Ming; Chen, Jia-Qi; Zhou, Jiong; Cai, Sui-Qing; Lu, Zhong-Fa; Zheng, Min

    2012-08-15

    Vascular endothelial growth factor (VEGF) is one of the strongest regulators of physiological and pathological angiogenesis. VEGF receptor 2 (VEGFR-2), the primary receptor for VEGF, is thought to mediate major functional effects of VEGF. Previously, we have localized both VEGF and VEGFR-2 in human hair follicles. In this study, we further defined the expression and roles of VEGFR-2 on human hair follicle dermal papilla (DP) cells. The expression of VEGFR-2 on DP cells was examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis separately, and localization of VEGFR-2 was defined by immunofluorescence. The effect of VEGF on DP cells was analyzed by MTT assays and specific inhibitors. Finally, the role of VEGF involved in the signaling pathways was investigated by Western blot. RT-PCR and Western blot analysis demonstrated the expression of VEGFR-2 on DP cells. Immunostaining for VEGFR-2 showed strong signal on cultured human DP cells in vitro. Exogenous VEGF{sub 165} stimulated proliferation of DP cells in a dose-dependent manner. Furthermore, this stimulation was blocked by a VEGFR-2 neutralizing antibody (MAB3571) and an ERK inhibitor (PD98059). VEGF{sub 165}-induced phosphorylation of ERK1/2 was abolished by MAB3571 and PD98059, while the phosphorylation of p38, JNK and AKT were not changed by VEGF{sub 165}. Taken together, VEGFR-2 is expressed on primary human hair follicle DP cells and VEGF induces proliferation of DP cells through VEGFR-2/ERK pathway, but not p38, JNK or AKT signaling. -- Highlights: Black-Right-Pointing-Pointer We examine the expression of VEGFR-2 on cultured human dermal papilla (DP) cells. Black-Right-Pointing-Pointer VEGF{sub 165} stimulated proliferation of human DP cells in a dose-dependent manner. Black-Right-Pointing-Pointer This stimulation was through VEGFR-2-mediated activation of ERK.

  11. TNFSF15 inhibits VEGF-stimulated vascular hyperpermeability by inducing VEGFR2 dephosphorylation.

    PubMed

    Yang, Gui-Li; Zhao, Zilong; Qin, Ting-Ting; Wang, Dong; Chen, Lijuan; Xiang, Rong; Xi, Zhen; Jiang, Rongcai; Zhang, Zhi-Song; Zhang, Jianning; Li, Lu-Yuan

    2017-02-09

    Vascular hyperpermeability is critical in ischemic diseases, including stroke and myocardial infarction, as well as in inflammation and cancer. It is well known that the VEGF-VEGFR2 signaling pathways are pivotal in promoting vascular permeability; however, counterbalancing mechanisms that restrict vascular permeability to maintain the integrity of blood vessels, are not yet fully understood. We report that TNF superfamily member 15 (TNFSF15), a cytokine largely produced by vascular endothelial cells and a specific inhibitor of the proliferation of these same cells, can inhibit VEGF-induced vascular permeability in vitro and in vivo, and that death receptor 3 (DR3), a cell surface receptor of TNFSF15, mediates TNFSF15-induced dephosphorylation of VEGFR2. Src homology region 2 domain-containing phosphatase-1 (SHP-1) becomes associated with DR3 upon TNFSF15 interaction with the latter. In addition, a protein complex consisting of VEGFR2, DR3, and SHP-1 is formed in response to the effects of TNFSF15 and VEGF on endothelial cells. It is plausible that this protein complex provides a structural basis for the molecular mechanism in which TNFSF15 induces the inhibition of VEGF-stimulated vascular hyperpermeability.-Yang, G.-L., Zhao, Z., Qin, T.-T., Wang, D., Chen, L., Xiang, R., Xi, Z., Jiang, R., Zhang, Z.-S., Zhang, J., Li. L.-Y. TNFSF15 inhibits VEGF-stimulated vascular hyperpermeability by inducing VEGFR2 dephosphorylation.

  12. VEGF increases the permeability of sheep pleura ex vivo through VEGFR2 stimulation.

    PubMed

    Peppa, Vasiliki I; Arsenopoulou, Zoi V; Zarogiannis, Sotirios G; Deligiorgi, Triantafyllia; Jagirdar, Rajesh; Makantasis, Ioannis; Stefanidis, Ioannis; Liakopoulos, Vassilios; Molyvdas, Paschalis-Adam; Gourgoulianis, Konstantinos I; Hatzoglou, Chrissi

    2014-10-01

    Vascular endothelial growth factor (VEGF), a cytokine that increases vascular permeability to water and proteins and induces angiogenesis, has been implicated in the development of pleural effusions. Inflammatory and malignant pleural effusions are rich in VEGF content while mesothelial cells produce and excrete VEGF. In this report we aimed at investigating by means of electrophysiology the direct effects of VEGF on the parietal and visceral sheep pleura as well as the type of receptors that mediate this effect. Our findings show that VEGF has a direct effect on the pleural mesothelium rendering it more permeable and this effect is mediated through the stimulation of VEGF receptor 2. Our findings shed more light to the role of VEGF in the pathogenesis of pleural effusions and provide functional evidence for a role of VEGFR2 on the pleural mesothelium that has never been studied before. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Expression of VEGFR-2 on HaCaT cells is regulated by VEGF and plays an active role in mediating VEGF induced effects

    SciTech Connect

    Yang Xiaohong; Man Xiaoyong; Cai Suiqing; Yao Yonggang; Bu Zhangyu; Zheng Min . E-mail: minz@zju.edu.cn

    2006-10-13

    Vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 play important roles in mitogenesis and chemotaxis of endothelial cells. In normal human skin, VEGF is expressed and secreted by epidermal keratinocytes. Emerging data suggest that keratinocyte-derived VEGF targets other cell types besides the dermal endothelial cells. We have recently showed that keratinocytes from human normal skin expressed all five known VEGF receptors and co-receptors (neuropilin 1 and 2). To define the functional significance of VEGFR-2 in epidermis, we examined its role in a keratinocyte cell line, HaCaT cells, in response to VEGF treatment. Expression of VEGFR-2 on HaCaT cells was confirmed at both RNA and protein levels and was regulated by VEGF{sub 165} treatment. Treatment of HaCaT cells with VEGF{sub 165} induced tyrosine-autophosphorylation of VEGFR-2 and phosphorylation of PLC-{gamma} and p44/42 MAPK in a time-dependent manner. Preincubation with a neutralizing antibody for VEGFR-2 (MAB3571) completely abrogated these phosphorylation effects. Furthermore, VEGF{sub 165} stimulated proliferation and migration of HaCaT cells, and this effect was significantly blocked by a pretreatment with MAB3571. Neutralizing VEGFR-2 in HaCaT cells increased cell adhesion during culture. Our results suggest that VEGFR-2 expressed on HaCaT cells plays a crucial role in VEGF-mediated regulation of cell activity.

  14. ADAMTS13 and its variants promote angiogenesis via upregulation of VEGF and VEGFR2.

    PubMed

    Lee, Manfai; Keener, Justin; Xiao, Juan; Long Zheng, X; Rodgers, George M

    2015-01-01

    Severe plasma ADAMTS13 deficiency results in the clinical disorder thrombotic thrombocytopenic purpura. However, other potential pathophysiological roles of ADAMTS13 in endothelial cell biology remain unexplored. The goals of this study were to understand the angiogenic pathways ADAMTS13 activates and to identify the important structural components of ADAMTS13 that stimulate angiogenesis. Incubation of human umbilical vein endothelial cells (HUVEC) with 150 ng/mL (1 nM) of recombinant human ADAMTS13 induced VEGF expression by 53 % and increased VEGF mRNA by over sixfold, both within 10 min; the measured VEGF levels steadily decreased over 2 h, as shown by Western blot and ELISA. Phosphorylation of VEGFR2 was significantly enhanced in HUVEC after incubation with ADAMTS13 (1 nM). Structure-function analysis showed that an ADAMTS13 variant containing thrombospondin type 1 (TSP1) 2-8 repeats (TSP1 2-8), TSP1 2-8 plus CUB domains (TSP1 2-8 plus CUB), or TSP1 5-8 repeats plus CUB domains (TSP1 5-8 plus CUB) increased HUVEC proliferation by 41-54 % as compared to the EBM-2 controls. Chemotaxis assays further demonstrated that the TSP1 domains of ADAMTS13 increased HUVEC migration by 2.65-fold. Incubation of HUVEC with both ADAMTS13 variants containing TSP1 repeats and anti-VEGF IgG abrogated the enhanced effect of ADAMTS13 on proliferation, migration, and VEGFR2 phosphorylation. In conclusion, ADAMTS13-induced endothelial cell angiogenesis occurs via the upregulation of VEGF and phosphorylation of VEGFR2. This angiogenic activity depends on the C-terminal TSP1 repeats of ADAMTS13.

  15. Sustained (rh)VEGF(165) release from a sprayed fibrin biomatrix induces angiogenesis, up-regulation of endogenous VEGF-R2, and reduces ischemic flap necrosis.

    PubMed

    Mittermayr, Rainer; Morton, Tatjana; Hofmann, Martina; Helgerson, Sam; van Griensven, Martijn; Redl, Heinz

    2008-01-01

    This study investigated (1) the release of recombinant human vascular endothelial growth factor ([rh]VEGF(165)) from an in vitro fibrin matrix, (2) the effects of (rh)VEGF(165) released from an in vivo fibrin matrix on ischemic flap necrosis in the rat dorsal skin flap model, and (3) the effects of (rh)VEGF(165) released from an in vivo fibrin matrix on VEGF-R2 expression in transgenic VEGF-R2/luc mice. In vitro fibrin matrices were spiked with (rh)VEGF(165) and demonstrated (rh)VEGF(165) release over 88 hours with 66% recovery. Ischemic dorsal flaps were treated with a fibrin sealant (FS), FS spiked with (rh)VEGF(165), or left untreated. Flaps treated with FS spiked with (rh)VEGF(165) showed greater viability than controls as measured by planimetric analysis. Immunohistochemical analyses revealed stronger neovascularization than that exhibited by controls. Transgenic mice implanted with FS spiked with (rh)VEGF(165) had significant increases in VEGF-R2 expression relative to controls at days 5-13 after implantation. Conclusions drawn from this work are that (1) (rh)VEGF(165) is released from an in vitro fibrin matrix at clinically appropriate times, (2) (rh)VEGF(165) increases the viability of tissue flaps in vivo, and (3) (rh)VEGF(165) induces the expression of VEGF-R2 expression. This work demonstrates the clinical ability of sprayed FS to locally deliver growth factors to ischemic tissue of patients.

  16. Anger Emotional Stress Influences VEGF/VEGFR2 and Its Induced PI3K/AKT/mTOR Signaling Pathway

    PubMed Central

    Sun, Peng; Wei, Sheng; Wei, Xia; Wang, Jieqiong; Zhang, Yuanyuan; Qiao, Mingqi; Wu, Jibiao

    2016-01-01

    Objective. We discuss the influence of anger emotional stress upon VEGF/VEGFR2 and its induced PI3K/AKT/mTOR signal pathway. Methods. We created a rat model of induced anger (anger-out and anger-in) emotional response using social isolation and resident-intruder paradigms and assessed changes in hippocampus' VEGF content, neuroplasticity, and the PI3K/AKT/mTOR signaling pathway. Results. The resident-intruder method successfully generated anger-out and anger-in models that differed significantly in composite aggression score, aggression incubation, open field behavior, sucrose preference, and weight gain. Anger emotional stress decreased synaptic connections and VEGFR2 expression. Anger emotional stress led to abnormal expression of VEGF/VEGFR2 mRNA and protein and disorderly expression of key factors in the PI3K/AKT/mTOR signal pathway. Fluoxetine administration ameliorated behavioral abnormalities and damage to hippocampal neurons caused by anger emotional stress, as well as abnormal expression of some proteins in VEGF/VEGFR2 and its induced PI3K/AKT/mTOR signal pathway. Conclusion. This research provides a detailed classification of anger emotion and verifies its influence upon VEGF and the VEGF-induced signaling pathway, thus providing circumstantial evidence of mechanisms by which anger emotion damages neurogenesis. As VEGFR2 can promote neurogenesis and vasculogenesis in the hippocampus and frontal lobe, these results suggest that anger emotional stress can result in decreased neurogenesis. PMID:27057362

  17. Effects of Chronic Exposure to Sodium Arsenite on Expressions of VEGF and VEGFR2 Proteins in the Epididymis of Rats

    PubMed Central

    Xiao Ping, Ma; Ying Quan, Yue

    2017-01-01

    Objective To study the expressions of VEGF and VEGFR2 at protein level in the epididymis of rats with arsenism. Methods Forty male Sprague-Dawley rats were randomly divided into four groups: the high dose arsenic infected group (60.0 mg/L in water), the middle dose arsenic infected group (12.0 mg/L in water), the low dose arsenic infected group (2.4 mg/L in water), and the control group (distilled water). Rats were treated with arsenic through drinking water for 6 consecutive months. At the end of the experiment, the average densitometry values of apoptotic cells in epididymis tubules were determined by TUNEL method; the protein and mRNA levels of VEGF and VEGFR2 were observed by immunohistochemistry, Western blot, and real time fluorescent quantitative PCR, respectively. Results Compared with the control group, in each infected group, the average densitometry values of apoptotic cells in the epididymis tubules were significantly lower. Compared with control group, protein and mRNA levels of VEGF and VEGFR2 in each infected group were obviously declined. The correlations between protein and mRNA levels of VEGF and VEGFR2 were positively exhibited (r = 0.843, 0.869, p < 0.05). Conclusions Arsenism affects the expressions of VEGF and VEGFR2 in the epididymis of rats and results in apoptosis of pathophysiology of male infertility. PMID:28791301

  18. Autocrine regulation of glioblastoma cell cycle progression, viability and radioresistance through the VEGF-VEGFR2 (KDR) interplay.

    PubMed

    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.

  19. Thrombospondin-1 modulates VEGF signaling via CD36 by recruiting SHP-1 to VEGFR2 complex in microvascular endothelial cells

    PubMed Central

    Chu, Ling-Yun; Ramakrishnan, Devi Prasadh

    2013-01-01

    Thrombospondin-1 (TSP-1) inhibits growth factor signaling at the receptor level in microvascular endothelial cells (MVEC), and CD36 has been suggested to be involved in this inhibition, but the mechanisms are not known. We hypothesized that CD36-TSP-1 interaction recruits Src homology 2 domain–containing protein tyrosine phosphatase (SHP)-1 to the vascular endothelial growth factor receptor 2 (VEGFR2) signaling complex and attenuates vascular endothelial growth factor (VEGF) signaling. Western blots of anti-CD36 and anti-VEGFR2 immunoprecipitates from VEGF-treated MVEC showed that exposure of the cells to a recombinant protein containing the CD36 binding domain of thrombospondin-1 (known as the TSR domain) induced association of SHP-1 with the VEGFR2/CD36 signaling complex and thereby suppressed VEGFR2 phosphorylation. SHP-1 phosphatase activity was increased in immunoprecipitated VEGFR2 complexes from TSR-treated cells. Silencing CD36 expression in MVEC by small interfering RNA (siRNA) or genetic deletion of cd36 in mice showed that TSR-induced SHP-1/VEGFR2 complex formation required CD36 in vitro and in vivo. Silencing SHP-1 expression in MVEC by siRNA abrogated TSR-mediated inhibition of VEGFR2 phosphorylation as well as TSR-mediated inhibition of VEGF-induced endothelial cell migration and tube formation. These studies reveal a SHP-1–mediated antiangiogenic pathway induced by CD36-TSP-1 interaction that inhibits VEGFR2 signaling and they provide a novel target to modulate angiogenesis therapeutically. PMID:23896411

  20. Geraniol Suppresses Angiogenesis by Downregulating Vascular Endothelial Growth Factor (VEGF)/VEGFR-2 Signaling

    PubMed Central

    Wittig, Christine; Scheuer, Claudia; Parakenings, Julia; Menger, Michael D.; Laschke, Matthias W.

    2015-01-01

    Geraniol exerts several direct pharmacological effects on tumor cells and, thus, has been suggested as a promising anti-cancer compound. Because vascularization is a major precondition for tumor growth, we analyzed in this study the anti-angiogenic action of geraniol. In vitro, geraniol reduced the migratory activity of endothelial-like eEND2 cells. Western blot analyses further revealed that geraniol downregulates proliferating cell nuclear antigen (PCNA) and upregulates cleaved caspase-3 (Casp-3) expression in eEND2 cells. Moreover, geraniol blocked vascular endothelial growth factor (VEGF)/VEGFR-2 signal transduction, resulting in a suppression of downstream AKT and ERK signaling pathways. In addition, geraniol significantly reduced vascular sprout formation in a rat aortic ring assay. In vivo, geraniol inhibited the vascularization of CT26 tumors in dorsal skinfold chambers of BALB/c mice, which was associated with a smaller tumor size when compared to vehicle-treated controls. Immunohistochemical analyses confirmed a decreased number of Ki67-positive cells and CD31-positive microvessels with reduced VEGFR-2 expression within geraniol-treated tumors. Taken together, these findings indicate that geraniol targets multiple angiogenic mechanisms and, therefore, is an attractive candidate for the anti-angiogenic treatment of tumors. PMID:26154255

  1. VEGF, VEGFR-1 and VEGFR-2 immunoreactivity in the porcine arteries of vascular subovarian plexus (VSP) during the estrous cycle.

    PubMed

    Postek, A; Andronowska, A; Doboszyńska, T; Niewegłowski, H; Jankowska, K

    2006-01-01

    Abstract: Vascular endothelial growth factor (VEGF) is an important angiogenic factor in the female reproductive tract. It binds to cell surface through ligand-stimulatable tyrosine kinase receptors, the most important being VEGFR-1 (flt-1) and VEGFR-2 (flk-1). The broad ligament of the uterus is a dynamic organ consisting of specialized complexes of blood vessels connected functionally to the uterus, oviduct and ovary. Endothelial cells form an inner coating of the vessel walls and thus they stay under the influence of various modulators circulating in blood including ovarian steriods involved in developmental changes in the female reproductive system. The aim of the present study was to immunolocalize VEGF and its two receptors: VEGFR-1 and VEGFR-2 in the broad ligament of the uterus in the area of vascular subovarian plexus during different phases of the estrous cycle in pig and to determine the correlation between immunoreactivity of the investigated factors and phases of the estrous cycle. The study was performed on cryostat sections of vascular subovarian plexus stained immunohistochemically by ABC method. Specific polyclonal antibodies: anti-VEGF, anti-VEGFR-1 and anti-VEGFR-2 were used. Data were subjected to one-way analysis of variance. Our study revealed the presence of VEGF and its receptors in endothelial and smooth muscle cells of VSP arteries. All agents displayed phase-related differences in immunoreactivity suggesting the modulatory effect of VEGF, VEGFR-1 and VEGFR-2 on the arteries of the VSP in the porcine broad ligament of the uterus.

  2. Coral-Derived Compound WA-25 Inhibits Angiogenesis by Attenuating the VEGF/VEGFR2 Signaling Pathway

    PubMed Central

    Lin, Shih-Wei; Huang, Shih-Chung; Kuo, Hsiao-Mei; Chen, Chiu-Hua; Ma, Yi-Ling; Chu, Tian-Huei; Bee, Youn-Shen; Wang, E-Ming; Wu, Chang-Yi; Sung, Ping-Jyun; Wen, Zhi-Hong; Wu, Deng-Chyang; Sheu, Jyh-Horng; Tai, Ming-Hong

    2015-01-01

    Background: WA-25 (dihydroaustrasulfone alcohol, a synthetic derivative of marine compound WE-2) suppresses atherosclerosis in rats by reducing neointima formation. Because angiogenesis plays a critical role in the pathogenesis of atherosclerosis, the present study investigated the angiogenic function and mechanism of WA-25. Methods: The angiogenic effect of WA-25 was evaluated using a rat aortic ring assay and transgenic zebrafish models were established using transgenic Tg(fli-1:EGFP)y1 and Tg(kdrl:mCherryci5-fli1a:negfpy7) zebrafish embryos. In addition, the effect of WA-25 on distinct angiogenic processes, including matrix metalloproteinase (MMP) expression, endothelial cell proliferation and migration, as well as tube formation, was studied using human umbilical vein endothelial cells (HUVECs). The effect of WA-25 on the endothelial vascular endothelial growth factor (VEGF) signaling pathway was elucidated using qRT-PCR, immunoblot analysis, immunofluorescence and flow cytometric analyses. Results: The application of WA-25 perturbed the development of intersegmental vessels in transgenic zebrafish. Moreover, WA-25 potently suppressed microvessel sprouting in organotypic rat aortic rings. Among cultured endothelial cells, WA-25 significantly and dose-dependently inhibited MMP-2/MMP-9 expression, proliferation, migration and tube formation in HUVECs. Mechanistic studies revealed that WA-25 significantly reduced the VEGF release by reducing VEGF expression at the mRNA and protein levels. In addition, WA-25 reduced surface VEGF receptor 2 (VEGFR2/Flk-1) expression by repressing the VEGFR2 mRNA level. Finally, an exogenous VEGF supply partially rescued the WA-25-induced angiogenesis blockage in vitro and in vivo. Conclusions: WA-25 is a potent angiogenesis inhibitor that acts through the down-regulation of VEGF and VEGFR2 in endothelial cells. General Significance: WA-25 may constitute a novel anti-angiogenic drug that acts by targeting endothelial VEGF/VEGFR2

  3. ZLM-7 exhibits anti-angiogenic effects via impaired endothelial cell function and blockade of VEGF/VEGFR-2 signaling.

    PubMed

    Su, Min; Huang, Jingjia; Li, Jijia; Qin, Xiyuan; Tang, Xiaoning; Jin, Fang; Chen, Shali; Jiang, Chuanming; Zou, Zizheng; Peng, Kunjian; Nuruzzaman, Mohammed; Zhang, Jianting; Luo, Junli; Liu, Suyou; Luo, Zhiyong

    2016-04-05

    Inhibition of angiogenesis is a promising therapeutic strategy against cancer. In this study, we reported that ZLM-7, a combretastain A-4 (CA-4) derivative, exhibited anti-angiogenic activity in vitro and in vivo. In vitro, ZLM-7 induced microtubule cytoskeletal disassembly. It decreased VEGF-induced proliferation, migration, invasion and tube formation in endothelial cells, which are critical steps in angiogenesis. In vivo, ZLM-7 significantly inhibited neovascularization in a chicken chorioallantoic membrane (CAM) model and reduced the microvessel density in tumor tissues of MCF-7 xenograft mouse model. ZLM-7 also displayed comparable antiangiogenic and anti-tumor activities associated with the lead compound CA-4, but exhibited lower toxicity compared with CA-4. The anti-angiogenic effect of ZLM-7 was exerted via blockade of VEGF/VEGFR-2 signaling. ZLM-7 treatment suppressed the expression and secretion of VEGF in endothelial cells and MCF-7 cells under hypoxia. Further, ZLM-7 suppressed the VEGF-induced phosphorylation of VEGFR-2 and its downstream signaling mediators including activated AKT, MEK and ERK in endothelial cells. Overall, these results demonstrate that ZLM-7 exhibits anti-angiogenic activities by impairing endothelial cell function and blocking VEGF/VEGFR-2 signaling, suggesting that ZLM-7 might be a potential angiogenesis inhibitor.

  4. Clinicopathological correlation and prognostic significance of VEGF-A, VEGF-C, VEGFR-2 and VEGFR-3 expression in colorectal cancer.

    PubMed

    Martins, Sandra F; Garcia, Eduardo A; Luz, Marcus Alexandre Mendes; Pardal, Fernando; Rodrigues, Mesquita; Filho, Adhemar Longatto

    2013-01-01

    Colorectal cancer (CRC) is the third most common type of cancer and the fourth most frequent cause of cancer death. Literature indicates that vascular endothelial growth factor is a predominant angiogenic factor and that angiogenesis plays an important role in the progression of CRC. The present series consisted of tissue samples obtained from 672 patients who had undergone large bowel resection between 2005 and 2010 at the Braga Hospital, Portugal. Archival paraffin-embedded CRC tissue and normal adjacent samples were used to build up tissue microarray blocks and VEGF-A, VEGF-C, VEGFR-2 and VEGFR-3 expression was immunohistochemically assessed. We observed an overexpression of VEGF-C in CRC when tumour cells and normal-adjacent tissue were compared (p=0.004). In tumour samples, VEGF-C-positive cases were associated with VEGFR-3 expression (p=0.047). When assessing the correlation between VEGF-A, VEGF-C, VEGFR-2 and VEGFR-3 expressions and the clinicopathological data, it was revealed that VEGF-A positive cases were associated with male gender (p=0.016) and well-differentiated tumours (p=0.001); VEGF-C with colon cancers (p=0.037), exophytic (p=0.048), moderately-differentiated (p=0.007) and T3/T4 (p=0.010) tumours; VEGFR-2 with invasive adenocarcinoma (p=0.007) and VEGFR-3 with the presence of hepatic metastasis (p=0.032). Overall survival curves for CRC were statistically significant for rectal cancer, VEGF-C expression and stage III (p=0.019) and VEGFR-3 expression and stage IV (p=0.047). Quantification of VEGF-A, VEGF-C, VEGFR-2 and VEGFR-3 expression seems to provide valuable prognostic information in CRC and the correlation with clinicopathological data revealed an association with characteristics that contribute to progression, invasion and metastasis leading to poorer survival rates and prognosis.

  5. A critical role for VEGF and VEGFR2 in NMDA receptor synaptic function and fear-related behavior

    PubMed Central

    De Rossi, P; Harde, E; Dupuis, J P; Martin, L; Chounlamountri, N; Bardin, M; Watrin, C; Benetollo, C; Pernet-Gallay, K; Luhmann, H J; Honnorat, J; Malleret, G; Groc, L; Acker-Palmer, A; Salin, P A; Meissirel, C

    2016-01-01

    Vascular endothelial growth factor (VEGF) is known to be required for the action of antidepressant therapies but its impact on brain synaptic function is poorly characterized. Using a combination of electrophysiological, single-molecule imaging and conditional transgenic approaches, we identified the molecular basis of the VEGF effect on synaptic transmission and plasticity. VEGF increases the postsynaptic responses mediated by the N-methyl-D-aspartate type of glutamate receptors (GluNRs) in hippocampal neurons. This is concurrent with the formation of new synapses and with the synaptic recruitment of GluNR expressing the GluN2B subunit (GluNR-2B). VEGF induces a rapid redistribution of GluNR-2B at synaptic sites by increasing the surface dynamics of these receptors within the membrane. Consistently, silencing the expression of the VEGF receptor 2 (VEGFR2) in neural cells impairs hippocampal-dependent synaptic plasticity and consolidation of emotional memory. These findings demonstrated the direct implication of VEGF signaling in neurons via VEGFR2 in proper synaptic function. They highlight the potential of VEGF as a key regulator of GluNR synaptic function and suggest a role for VEGF in new therapeutic approaches targeting GluNR in depression. PMID:26728568

  6. VEGF/VEGFR2 Axis in Periodontal Disease Progression and Angiogenesis: Basic Approach for a New Therapeutic Strategy.

    PubMed

    Vladau, Mircea; Cimpean, Anca Maria; Balica, Raluca Amalia; Jitariu, Andreea Adriana; Popovici, Ramona Amina; Raica, Marius

    2016-01-01

    Periodontal lesions are associated with activation of pathological angiogenesis and a high number of newly-formed blood vessels. Most angiogenic growth factors have been studied in the crevicular fluid or serum, but tissue correlations with vascular density or endothelial proliferation, are very rare, even inexistent. We assessed the VEGF/VEGFR2 axis expression in a multimodal fashion, in both epithelial and stromal compartments, with emphasis to endothelial proliferation and severity of periodontal lesions. Compared to normal gingiva, negative for VEGF/VEGFR2, periodontal lesions had a progressive increase for these markers from low to severe periodontal lesions. The transition from low to moderate periodontal lesions represents the milestone in disease progression and implies an active angiogenesis based on the highest angiogenic parameter variability observed for these lesions. Epithelial vascularization was firstly observed in moderate periodontal lesions and persists during severe periodontal disease. All the parameters used to quantify angiogenesis in periodontal lesions, were significantly increased in severe periodontal lesions dependent on VEGF expression in both the epithelial and stromal compartment. Our results support the use of anti-VEGF/VEGFR2-targeted therapy as adjuvant treatment for severe periodontal lesions. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  7. Potent inhibition of VEGFR-2 activation by tight binding of green tea epigallocatechin gallate and apple procyanidins to VEGF: relevance to angiogenesis.

    PubMed

    Moyle, Christina W A; Cerezo, Ana B; Winterbone, Mark S; Hollands, Wendy J; Alexeev, Yuri; Needs, Paul W; Kroon, Paul A

    2015-03-01

    Excessive concentrations of vascular endothelial growth factor (VEGF) drive angiogenesis and cause complications such as increased growth of tumours and atherosclerotic plaques. The aim of this study was to determine the molecular mechanism underlying the potent inhibition of VEGF signalling by polyphenols. We show that the polyphenols epigallocatechin gallate from green tea and procyanidin oligomers from apples potently inhibit VEGF-induced VEGF receptor-2 (VEGFR-2) signalling in human umbilical vein endothelial cells by directly interacting with VEGF. The polyphenol-induced inhibition of VEGF-induced VEGFR-2 activation occurred at nanomolar polyphenol concentrations and followed bi-phasic inhibition kinetics. VEGF activity could not be recovered by dialysing VEGF-polyphenol complexes. Exposure of VEGF to epigallocatechin gallate or procyanidin oligomers strongly inhibited subsequent binding of VEGF to human umbilical vein endothelial cells expressing VEGFR-2. Remarkably, even though VEGFR-2 signalling was completely inhibited at 1 μM concentrations of polyphenols, endothelial nitric oxide synthase was shown to still be activated via the PI3K/Akt signalling pathway which is downstream of VEGFR-2. These data demonstrate for the first time that VEGF is a key molecular target for specific polyphenols found in tea, apples and cocoa which potently inhibit VEGF signalling and angiogenesis at physiological concentrations. These data provide a plausible mechanism which links bioactive compounds in food with their beneficial effects. © 2014 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. A set of loop-1 and -3 structures in the novel vascular endothelial growth factor (VEGF) family member, VEGF-ENZ-7, is essential for the activation of VEGFR-2 signaling.

    PubMed

    Kiba, Atsushi; Yabana, Naoyuki; Shibuya, Masabumi

    2003-04-11

    The vascular endothelial growth factor (VEGF) family plays important roles in angiogenesis and vascular permeability. Novel members of the VEGF family encoded in the Orf virus genome, VEGF-E, function as potent angiogenic factors by specifically binding and activating VEGFR-2 (KDR). VEGF-E is about 45% homologous to VEGF-A at amino acid levels, however, the amino acid residues in VEGF-A crucial for the VEGFR-2-binding are not conserved in VEGF-E. To understand the molecular basis of the biological activity of VEGF-E, we have functionally mapped residues important for interaction of VEGF-E with VEGFR-2 by exchanging the domains between VEGF-E(NZ-7) and PlGF, which binds only to VEGFR-1 (Flt-1). Exchange on the amino- and carboxyl-terminal regions had no suppressive effect on biological activity. However, exchange on either the loop-1 or -3 region of VEGF-E(NZ-7) significantly reduced activities. On the other hand, introduction of the loop-1 and -3 of VEGF-E(NZ-7) to placenta growth factor rescued the biological activities. The chimera between VEGF-A and VEGF-E(NZ-7) gave essentially the same results. These findings strongly suggest that a common rule exists for VEGFR-2 ligands (VEGF-E(NZ-7) and VEGF-A) that they build up the binding structure for VEGFR-2 through the appropriate interaction between loop-1 and -3 regions.

  9. Selective Imaging of VEGFR-1 and VEGFR-2 Using 89Zr-Labeled Single-Chain VEGF Mutants.

    PubMed

    Meyer, Jan-Philip; Edwards, Kimberly J; Kozlowski, Paul; Backer, Marina V; Backer, Joseph M; Lewis, Jason S

    2016-11-01

    Vascular endothelial growth factor-A (VEGF-A) acts via 2 vascular endothelial growth factor receptors, VEGFR-1 and VEGFR-2, that play important and distinct roles in tumor biology. We reasoned that selective imaging of these receptors could provide unique information for diagnostics and for monitoring and optimizing responses to anticancer therapy, including antiangiogenic therapy. Herein, we report the development of 2 first-in-class (89)Zr-labeled PET tracers that enable the selective imaging of VEGFR-1 and VEGFR-2. Functionally active mutants of scVEGF (an engineered single-chain version of pan-receptor VEGF-A with an N-terminal cysteine-containing tag for site-specific conjugation), named scVR1 and scVR2 with enhanced affinity to, respectively, VEGFR-1 and VEGFR-2, were constructed. Parental scVEGF and its receptor-specific mutants were site-specifically derivatized with the (89)Zr chelator desferroxamine B via a 3.4-kDa PEG linker. (89)Zr labeling of the desferroxamine B conjugates furnished scV/Zr, scVR1/Zr, and scVR2/Zr tracers with high radiochemical yield (>87%), high specific activity (≥9.8 MBq/nmol), and purity (>99%). Tracers were tested in an orthotopic breast cancer model using 4T1luc-bearing syngeneic BALB/c mice. For testing tracer specificity, tracers were coinjected with an excess of cold proteins of the same or opposite receptor specificity or pan-receptor scVEGF. PET imaging, biodistribution, and dosimetry studies in mice, as well as immunohistochemical analysis of harvested tumors, were performed. All tracers rapidly accumulated in orthotopic 4T1luc tumors, allowing for the successful PET imaging of the tumors as early as 2 h after injection. Blocking experiments with an excess of pan-receptor or receptor-specific cold proteins indicated that more than 80% of tracer tumor uptake is VEGFR-mediated, whereas uptake in all major organs is not affected by blocking within the margin of error. Critically, blocking experiments indicated that VEGFR

  10. Inhibition of VEGF-Induced VEGFR-2 Activation and HUVEC Migration by Melatonin and Other Bioactive Indolic Compounds

    PubMed Central

    Cerezo, Ana B.; Hornedo-Ortega, Ruth; Álvarez-Fernández, M. Antonia; Troncoso, Ana M.; García-Parrilla, M. Carmen

    2017-01-01

    Excessive concentrations of vascular endothelial growth factor (VEGF) trigger angiogenesis, which causes complications such as the destabilization of atherosclerotic plaques and increased growth of tumors. This work focuses on the determination of the inhibitory activity of melatonin and other indolic related compounds on VEGF-induced VEGF receptor-2 (VEGFR-2) activation and an approximation to the molecular mechanism underlying the inhibition. Quantification of phosphorylated VEGFR-2 was measured by ELISA. Migration wound-healing assay was used to determine cell migration of human umbilical vein endothelial cells (HUVECs). This is the first time that melatonin, 3-indolacetic acid, 5-hydroxytryptophol, and serotonin are proved to significantly inhibit VEGF-induced VEGFR-2 activation in human umbilical vein endothelial cells and subsequent angiogenesis. 3-Indolacetic acid showed the highest inhibitory effect (IC50 value of 0.9704 mM), followed by 5-hydroxytryptophol (35% of inhibition at 0.1 mM), melatonin (30% of inhibition at 1 mM), and serotonin (24% of inhibition at 1 mM). An approximation to the molecular mechanism of the inhibition has been proposed, suggesting that indolic compounds might interact with the cell surface components of the endothelial membrane in a way that prevents VEGF from activating the receptor. Additionally, wound-healing assay revealed that exposure of HUVECs to melatonin and 3-indolacetic acid in the presence of VEGF significantly inhibited cell migration by 87% and 99%, respectively, after 24 h. These data demonstrate that melatonin, 3-indolacetic acid, 5-hydroxytryptophol, and serotonin would be good molecules for future exploitation as anti-VEGF signaling agents. PMID:28282869

  11. Inhibition of VEGF-Induced VEGFR-2 Activation and HUVEC Migration by Melatonin and Other Bioactive Indolic Compounds.

    PubMed

    Cerezo, Ana B; Hornedo-Ortega, Ruth; Álvarez-Fernández, M Antonia; Troncoso, Ana M; García-Parrilla, M Carmen

    2017-03-08

    Excessive concentrations of vascular endothelial growth factor (VEGF) trigger angiogenesis, which causes complications such as the destabilization of atherosclerotic plaques and increased growth of tumors. This work focuses on the determination of the inhibitory activity of melatonin and other indolic related compounds on VEGF-induced VEGF receptor-2 (VEGFR-2) activation and an approximation to the molecular mechanism underlying the inhibition. Quantification of phosphorylated VEGFR-2 was measured by ELISA. Migration wound-healing assay was used to determine cell migration of human umbilical vein endothelial cells (HUVECs). This is the first time that melatonin, 3-indolacetic acid, 5-hydroxytryptophol, and serotonin are proved to significantly inhibit VEGF-induced VEGFR-2 activation in human umbilical vein endothelial cells and subsequent angiogenesis. 3-Indolacetic acid showed the highest inhibitory effect (IC50 value of 0.9704 mM), followed by 5-hydroxytryptophol (35% of inhibition at 0.1 mM), melatonin (30% of inhibition at 1 mM), and serotonin (24% of inhibition at 1 mM). An approximation to the molecular mechanism of the inhibition has been proposed, suggesting that indolic compounds might interact with the cell surface components of the endothelial membrane in a way that prevents VEGF from activating the receptor. Additionally, wound-healing assay revealed that exposure of HUVECs to melatonin and 3-indolacetic acid in the presence of VEGF significantly inhibited cell migration by 87% and 99%, respectively, after 24 h. These data demonstrate that melatonin, 3-indolacetic acid, 5-hydroxytryptophol, and serotonin would be good molecules for future exploitation as anti-VEGF signaling agents.

  12. Sjögren's syndrome pathological neovascularization is regulated by VEGF-A-stimulated TACE-dependent crosstalk between VEGFR2 and NF-κB.

    PubMed

    Sisto, M; Lisi, S; Lofrumento, D D; D'Amore, M; Frassanito, M A; Ribatti, D

    2012-07-01

    We explore the involvement of tumor necrosis factor α (TNF-α)-converting enzyme (TACE) in vascular endothelial growth factor (VEGF) and its receptor 2 (VEGFR2) (VEGF-A/VEGFR2)-mediated angiogenesis in Sjögren's syndrome (SS), one of the most common autoimmune rheumatic diseases. To test the hypothesis that SS autoantibodies (Abs) regulate VEGF-A/VEGFR2 expression by a TACE-dependent nuclear factor-κB (NF-κB) activation pathway, their effects on the expression and activation of the VEGF-A/TACE/VEGFR2/NF-κB pathway were determined in human salivary gland epithelial cells (SGEC). An enhanced angiogenesis in SS salivary gland biopsies was observed, associated with an increased VEGF-A expression and activation of VEGF-A/VEGFR2 signaling. Human cytokine array analysis of the pro-inflammatory and pro-angiogenic protein response in SGEC treated with SS Abs revealed an overexpression of multiple pro-angiogenic factors. TACE RNA knockdown, the use of anti-VEGF-A monoclonal antibody and the inhibition of NF-κB activity significantly abrogated the release of pro-angiogenic factors, demonstrating that VEGF-A/TACE/VEGFR2/NF-κB axis dysfunction may be contributory to pathogenesis and exacerbation of this autoimmune condition.

  13. SIRT1 mediated inhibition of VEGF/VEGFR2 signaling by Resveratrol and its relevance to choroidal neovascularization

    PubMed Central

    Zhang, Huiming; He, Shikun; Spee, Christine; Ishikawa, Keijiro; Hinton, David R.

    2015-01-01

    SIRT1, a NAD+ dependent histone deacetylase, has been shown to act as a key regulator of angiogenesis. The purpose of this study was to determine the effects of resveratrol (RSV, a SIRT1 activator) on the vascular endothelial growth factor receptor 2 (VEGFR2) signaling pathway and to establish its relevance to choroidal neovascularization (CNV), a blinding complication of age-related macular degeneration. Western blot and ELISA assay showed that RSV inhibited hypoxia-inducible factor (HIF)-1α accumulation and VEGF secretion induced by cobalt chloride (CoCl2) through SIRT1 in human retinal pigment epithelial (hRPE) cells. Furthermore, RSV down-regulated VEGFR2 phosphorylation and activation induced by VEGF in endothelial cells via SIRT1. Thus, the inhibitory effect of RSV on the HIF-1α\\VEGF\\ VEGFR2 signaling axis is mediated, at least in part, through SIRT1. The results suggest that targeting SIRT1 could have therapeutic potential for the treatment of CNV. PMID:26174951

  14. Low molecular weight fucoidan increases VEGF165-induced endothelial cell migration by enhancing VEGF165 binding to VEGFR-2 and NRP1.

    PubMed

    Lake, Andrew C; Vassy, Roger; Di Benedetto, Mélanie; Lavigne, Damien; Le Visage, Catherine; Perret, Gérard Y; Letourneur, Didier

    2006-12-08

    Therapeutic induction of angiogenesis is a potential treatment for chronic ischemia. Heparan sulfate proteoglycans are known to play an important role by their interactions with proangiogenic growth factors such as vascular endothelial growth factor (VEGF). Low molecular weight fucoidan (LMWF), a sulfated polysaccharide from brown seaweeds that mimic some biological activities of heparin, has been shown recently to promote revascularization in rat critical hindlimb ischemia. In this report, we first used cultured human endothelial cells (ECs) to investigate the possible ability of LMWF to enhance the actions of VEGF(165). Data showed that LMWF greatly enhances EC tube formation in growth factor reduced matrigel. LMWF is a strong enhancer of VEGF(165)-induced EC chemotaxis, but not proliferation. In addition, LMWF has no effect on VEGF(121)-induced EC migration, a VEGF isoform that does not bind to heparan sulfate proteoglycans. Then, with binding studies using (125)I-VEGF(165), we observed that LMWF enhances the binding of VEGF(165) to recombinant VEGFR-2 and Neuropilin-1 (NRP1), but not to VEGFR-1. Surface plasmon resonance analysis showed that LMWF binds with high affinity to VEGF(165) (1.2 nm) and its receptors (5-20 nm), but not to VEGF(121). Pre-injection of LMWF on immobilized receptors shows that VEGF(165) has the highest affinity for VEGFR-2 and NRP1, as compared with VEGFR-1. Overall, the effects of LMWF were much more pronounced than those of LMW heparin. These findings suggested an efficient mechanism of action of LMWF by promoting VEGF(165) binding to VEGFR-2 and NRP1 on ECs that could help in stimulating therapeutic revascularization.

  15. VEGF/VEGFR-2 upregulates EZH2 expression in lung adenocarcinoma cells and EZH2 depletion enhances the response to platinum-based and VEGFR-2–targeted therapy

    PubMed Central

    Riquelme, Erick; Suraokar, Milind; Behrens, Carmen; Lin, Heather Y.; Girard, Luc; Nilsson, Monique B.; Simon, George; Wang, Jing; Coombes, Kevin R.; Lee, J. Jack; Hong, Waun Ki; Heymach, John; Minna, John D.; Wistuba, Ignacio I.

    2014-01-01

    Purpose Investigate the mechanisms of regulation and role associated with EZH2 expression in lung cancer cells. Experimental Design We investigated the mechanisms of EZH2 expression associated with the vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR-2) pathway. Furthermore, we sought to determine the role of EZH2 in response of lung adenocarcinoma to platinum-based chemotherapy, as well as the effect of EZH2 depletion on VEGFR-2–targeted therapy in lung adenocarcinoma cell lines. Additionally, we characterized EZH2 expression in lung adenocarcinoma specimens and correlated it with patients’ clinical characteristics. Results In this study, we demonstrate that VEGF/VEGFR-2 activation induces expression of EZH2 through the upregulation of E2F3 and HIF-1α, and downregulated expression of miR-101. EZH2 depletion by treatment with 3-deazaneplanocin A and knockdown by siRNA decreased the expression of EZH2 and H3K27me3, increased PARP-C level, reduced cell proliferation and migration, and increased sensitivity of the cells to treatment with cisplatin and carboplatin. Additionally, high EZH2 expression was associated with poor overall survival in patients who received platinum-based adjuvant therapy, but not in patients who did not receive this therapy. Furthermore, we demonstrated for the first time that the inhibition of EZH2 greatly increased the sensitivity of lung adenocarcinoma cells to the anti-VEGFR-2 drug AZD2171. Conclusion Our results suggest that VEGF/VEGFR-2 pathway plays a role in regulation of EZH2 expression via E2F3, HIF-1α and miR-101. EZH2 depletion decreases the malignant potential of lung adenocarcinoma and sensitivity of the cells to both platinum-based and VEGFR-2–targeted therapy. PMID:24850841

  16. Dipeptidyl Peptidase IV Inhibition Activates CREB and Improves Islet Vascularization through VEGF-A/VEGFR-2 Signaling Pathway

    PubMed Central

    Samikannu, Balaji; Chen, Chunguang; Lingwal, Neelam; Padmasekar, Manju; Engel, Felix B.; Linn, Thomas

    2013-01-01

    Substitution of pancreatic islets is a potential therapy to treat diabetes and it depends on reconstitution of islet’s capillary network. In this study, we addressed the question whether stabilization of Glucagon-Like-Peptide-1 (GLP-1) by inhibiting Dipeptidyl Peptidase-IV (DPP-IV) increases β-cell mass by modulating vascularization. Mouse or porcine donor islets were implanted under kidney capsule of diabetic mice treated with DPP-IV inhibitor sitagliptin. Grafts were analyzed for insulin production, β-cell proliferation and vascularization. In addition, the effect of sitagliptin on sprouting and Vascular Endothelial Growth Factor (VEGF)-A expression was examined ex vivo. The cAMP response element-binding (CREB) and VEGF-A/ Vascular Endothelial Growth Factor Receptor (VEGFR)-2 signaling pathway leading to islet vascularization was explored. Sitagliptin increased mean insulin content of islet grafts and area of insulin-positive tissue as well as β-cell proliferation. Interestingly, sitagliptin treatment also markedly increased endothelial cell proliferation, microvessel density and blood flow. Finally, GLP-1 (7-36) stimulated sprouting and VEGF expression, which was significantly enhanced by sitagliptin- mediated inhibition of DPP-IV. Our in vivo data demonstrate that sitagliptin treatment phosphorylated CREB and induced islet vascularization through VEGF-A/VEGFR-2 signaling pathway. This study paves a new pathway for improvement of islet transplantation in treating diabetes mellitus. PMID:24349326

  17. Serum VEGF-A and Tumor Vessel VEGFR-2 Levels Predict Survival in Caucasian but Not Asian Patients Undergoing Resection for Gastric Adenocarcinoma

    PubMed Central

    Park, Do Joong; Seo, An Na; Yoon, Changhwan; Ku, Geoffrey Y.; Coit, Daniel G.; Strong, Vivian E.; Suh, Yun-Suhk; Lee, Hye Seung; Yang, Han-Kwang; Kim, Hyung-Ho; Yoon, Sam S.

    2016-01-01

    Background Clinical trials of agents targeting the vascular endothelial growth factor A (VEGF-A) pathway in gastric adenocarcinoma (GA) suggest that these therapies may have varying efficacy in different races. Methods VEGF-A in serum and/or VEGF receptor 2 (VEGFR-2) in CD31-positive tumor vessels (VEGFR-2/CD31) were measured in 118 Caucasians and 263 Asians who underwent gastric resection at two institutions and correlated with overall survival (OS). Blood was drawn before any treatment. Patients receiving neoadjuvant treatment were excluded from VEGFR-2 analysis. Results Compared with Asians, Caucasians were older (mean age 66–73 vs 59–62 years), had more proximal tumors, and had more advanced TNM stage. In the VEGF-A cohort, Caucasians had a median VEGF-A level that was 95 % higher than that of Asians and a much higher standard deviation (88 ± 6.206 vs 45 ± 76 pg/ml, p < 0.001). The 5-year OS for patients with low versus high VEGF-A levels was 72 versus 43 % in Caucasians (p = 0.001) and 86 versus 77 % in Asians (p = 0.236). In the VEGFR-2 cohort, OS was worse in Caucasians with high VEGFR-2/CD31 levels (49 vs 73 %, p = 0.038), while there was no significant difference in OS in Asians (80 vs 90 %, p = 0.119). On multivariate analyses of significant prognostic factors (excluding treatment factors and margin status), serum VEGF-A and tumor VEGFR-2/CD31 levels were independent predictors of OS only in Caucasians. Conclusions In patients with resectable GA, VEGF-A and VEGFR-2/CD31 levels are independent predictors of OS in Caucasians but not in Asians, suggesting varying importance of this pathway in GA progression among different races. PMID:26259755

  18. Naringin promotes fracture healing through stimulation of angiogenesis by regulating the VEGF/VEGFR-2 signaling pathway in osteoporotic rats.

    PubMed

    Song, Nan; Zhao, Zhihu; Ma, Xinlong; Sun, Xiaolei; Ma, Jianxiong; Li, Fengbo; Sun, Lei; Lv, Jianwei

    2017-01-05

    Postmenopausal osteoporosis is characterized by a reduction in the number of sinusoidal and arterial capillaries in the bone marrow and reduced bone perfusion. Thus, osteogenesis and angiogenesis are coupled in the process of osteoporosis formation and fracture healing. Naringin is the main ingredient of the root Rhizoma Drynariae, a traditional Chinese medicine, and it has potential effects on promoting fracture healing. However, whether naringin stimulates angiogenesis in the process of bone healing is unclear. Here, we show that naringin promotes fracture healing through stimulating angiogenesis by regulating the VEGF/VEGFR-2 signaling pathway in osteoporotic rats.

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

    PubMed

    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.

  20. EGF-Induced VEGF Exerts a PI3K-Dependent Positive Feedback on ERK and AKT through VEGFR2 in Hematological In Vitro Models.

    PubMed

    Saryeddine, Lilian; Zibara, Kazem; Kassem, Nouhad; Badran, Bassam; El-Zein, Nabil

    2016-01-01

    EGFR and VEGFR pathways play major roles in solid tumor growth and progression, however, little is known about these pathways in haematological tumors. This study investigated the crosstalk between EGFR and VEGFR2 signaling in two hematological in vitro models: THP1, a human monocytic leukemia, and Raji, a Burkitt's lymphoma, cell lines. Results showed that both cell lines express EGFR and VEGFR2 and responded to EGF stimulation by activating EGFR, triggering VEGF production and phosphorylating ERK, AKT, and p38 very early, with a peak of expression at 10-20min. Blocking EGFR using Tyrphostin resulted in inhibiting EGFR induced activation of ERK, AKT, and p38. In addition, EGF stimulation caused a significant and immediate increase, within 1min, in pVEGFR2 in both cell lines, which peaked at ~5-10 min after treatment. Selective inhibition of VEGFR2 by DMH4, anti-VEGFR2 antibody or siRNA diminished EGF-induced pAKT and pERK, indicating a positive feedback exerted by EGFR-induced VEGF. Similarly, the specific PI3K inhibitor LY294002, suppressed AKT and ERK phosphorylation showing that VEGF feedback is PI3K-dependent. On the other hand, phosphorylation of p38, initiated by EGFR and independent of VEGF feedback, was diminished using PLC inhibitor U73122. Moreover, measurement of intracellular [Ca2+] and ROS following VEGFR2 inhibition and EGF treatment proved that VEGFR2 is not implicated in EGF-induced Ca2+ release whereas it boosts EGF-induced ROS production. Furthermore, a significant decrease in pAKT, pERK and p-p38 was shown following the addition of the ROS inhibitor NAC. These results contribute to the understanding of the crosstalk between EGFR and VEGFR in haematological malignancies and their possible combined blockade in therapy.

  1. EGF-Induced VEGF Exerts a PI3K-Dependent Positive Feedback on ERK and AKT through VEGFR2 in Hematological In Vitro Models

    PubMed Central

    Kassem, Nouhad; Badran, Bassam; El-Zein, Nabil

    2016-01-01

    EGFR and VEGFR pathways play major roles in solid tumor growth and progression, however, little is known about these pathways in haematological tumors. This study investigated the crosstalk between EGFR and VEGFR2 signaling in two hematological in vitro models: THP1, a human monocytic leukemia, and Raji, a Burkitt’s lymphoma, cell lines. Results showed that both cell lines express EGFR and VEGFR2 and responded to EGF stimulation by activating EGFR, triggering VEGF production and phosphorylating ERK, AKT, and p38 very early, with a peak of expression at 10–20min. Blocking EGFR using Tyrphostin resulted in inhibiting EGFR induced activation of ERK, AKT, and p38. In addition, EGF stimulation caused a significant and immediate increase, within 1min, in pVEGFR2 in both cell lines, which peaked at ~5–10 min after treatment. Selective inhibition of VEGFR2 by DMH4, anti-VEGFR2 antibody or siRNA diminished EGF-induced pAKT and pERK, indicating a positive feedback exerted by EGFR-induced VEGF. Similarly, the specific PI3K inhibitor LY294002, suppressed AKT and ERK phosphorylation showing that VEGF feedback is PI3K-dependent. On the other hand, phosphorylation of p38, initiated by EGFR and independent of VEGF feedback, was diminished using PLC inhibitor U73122. Moreover, measurement of intracellular [Ca2+] and ROS following VEGFR2 inhibition and EGF treatment proved that VEGFR2 is not implicated in EGF-induced Ca2+ release whereas it boosts EGF-induced ROS production. Furthermore, a significant decrease in pAKT, pERK and p-p38 was shown following the addition of the ROS inhibitor NAC. These results contribute to the understanding of the crosstalk between EGFR and VEGFR in haematological malignancies and their possible combined blockade in therapy. PMID:27806094

  2. VEGF and VEGFR-2 (KDR) internalization is required for endothelial recovery during wound healing

    SciTech Connect

    Constantino Rosa Santos, Susana; Miguel, Claudia; Wu Yan; Dias, Sergio . E-mail: sergidias@ipolisboa.min-saude.pt

    2007-05-01

    Vascular endothelial growth factor (VEGF) receptor activation regulates endothelial cell (EC) survival, migration and proliferation. Recently, it was suggested the cross-talk between the VEGF receptors-1 (FLT-1) and -2 (KDR) modulated several of these functions, but the detailed molecular basis for such interactions remained unexplained. Here we demonstrate for the first time that VEGF stimulation of EC monolayers induced a rapid FLT-1-mediated internalization of KDR to the nucleus, via microtubules and the endocytic pathway, internalization which required the activation of PI 3-kinase/AKT. KDR deletion mutants were generated in several tyrosine residues; in these, VEGF-induced KDR internalization was impaired, demonstrating this process required activation (phosphorylation) of the receptor. Furthermore, we demonstrate that in vitro wounding of EC monolayers leads to a rapid and transient internalization of VEGF + KDR to the nucleus, which is essential for monolayer recovery. Notably, FLT-1 blockade impedes VEGF and KDR activation and internalization, blocking endothelial monolayer recovery. Our data reveal a previously unrecognized mechanism induced by VEGF on EC, which regulates EC recovery following wounding, and as such indicate novel targets for therapeutic intervention.

  3. BET Bromodomain Suppression Inhibits VEGF-induced Angiogenesis and Vascular Permeability by Blocking VEGFR2-mediated Activation of PAK1 and eNOS

    PubMed Central

    Huang, Mingcheng; Qiu, Qian; Xiao, Youjun; Zeng, Shan; Zhan, Mingying; Shi, Maohua; Zou, Yaoyao; Ye, Yujin; Liang, Liuqin; Yang, Xiuyan; Xu, Hanshi

    2016-01-01

    The tyrosine kinase receptor vascular endothelial growth factor receptor 2 (VEGFR2) is a critical modulator of angiogenesis. Increasing evidence indicate the important role of bromodomain and extra-terminal domain (BET) of chromatin adaptors in regulating tumor growth and inflammatory response. However, whether BET proteins have a role in angiogenesis and endothelial permeability is unclear. In this study, we observed that treatment with JQ1, a specific BET inhibitor, suppressed in vitro tube formation of human umbilical vein endothelial cells (HUVECs) and in vivo angiogenesis in a Matrigel plug and oxygen-induced retinopathy neovascularization. JQ1 attenuated the VEGF-induced decrease in TEER in HUVECs and prevented Evans blue dye leakage in the VEGF-induced Miles assay in athymic Balb/c nude mice. BET inhibition with JQ1 or shRNA for Brd2 or Brd4 suppressed VEGF-induced migration, proliferation, and stress fiber formation of HUVECs. Furthermore, BET inhibition suppressed phosphorylation of VEGFR2 and PAK1, as well as eNOS activation in VEGF-stimulated HUVECs. Inhibition with VEGFR2 and PAK1 also reduced migration and proliferation, and attenuated the VEGF-induced decrease in TEER. Thus, our observations suggest the important role of BET bromodomain in regulating VEGF-induced angiogenesis. Strategies that target the BET bromodomain may provide a new therapeutic approach for angiogenesis-related diseases. PMID:27044328

  4. Decreased expression of CHIP leads to increased angiogenesis via VEGF-VEGFR2 pathway and poor prognosis in human renal cell carcinoma.

    PubMed

    Sun, Chao; Li, Hai-long; Chen, Hai-rong; Shi, Mei-lin; Liu, Qing-hua; Pan, Zhen-qiang; Bai, Jin; Zheng, Jun-nian

    2015-05-29

    CHIP (c-terminal Hsp70-interacting protein) is an E3 ligase which may play different roles in different cancers. The elucidation of the VHL-HIF-1α (hypoxia inducible factor-1α)-VEGF (vascular endothelial growth factor) pathway has led to the development of targeted therapy in renal cell carcinoma (RCC). However, little is known about the role of CHIP and the relationship between CHIP and VEGF-VEGFR2 (VEGF receptor 2) pathway in RCC. In this study, we found that the expression of CHIP was downregulated and significantly correlated with pT status (P = 0.022) and TNM stage (P = 0.022) in 304 RCC and 35 normal renal tissues using tissue microarray. Moreover, low expression of CHIP is a strong and independent negative prognostic value for RCC. In vitro, CHIP negatively regulated RCC cell migration, invasion and angiogenesis. In addition, ELISA tests showed that restoration of CHIP inhibited, while knockdown promoted, the secreted level of VEGF. Furthermore, western blot indicated that the VEGFR2 protein level was reduced after CHIP overexpression. Our findings demonstrate for the first time that CHIP may be involved in RCC angiogenesis through regulating VEGF secretion and expression of VEGFR2. CHIP may serve as promising prognostic biomarker of angiogenesis and may constitute a potential therapeutic target in RCC.

  5. Gremlin promotes retinal pigmentation epithelial (RPE) cell proliferation, migration and VEGF production via activating VEGFR2-Akt-mTORC2 signaling

    PubMed Central

    Liu, Yuan; Chen, Zhijun; Cheng, Haixia; Chen, Juan; Qian, Jing

    2017-01-01

    Retinopathy of prematurity (ROP) is characterized by late-phase pathologic retinal vasoproliferation. Gremlin is a novel vascular endothelial growth factors (VEGF) receptor 2 (VEGFR2) agonist and promotes angiogenic response. We demonstrated that gremlin expression was significantly increased in retinas of ROP model mice, which was correlated with VEGF upregulation. In retinal pigmentation epithelial (RPE) cells, gremlin activated VEGFR2-Akt-mTORC2 (mammalian target of rapamycin complex 2) signaling, and promoted cell proliferation, migration and VEGF production. VEGFR inhibition (by SU5416) or shRNA knockdown almost abolished gremlin-mediated pleiotropic functions in RPE cells. Further, pharmacological inhibition of Akt-mTOR, or shRNA knockdown of key mTORC2 component (Rictor or Sin1) also attenuated gremlin-exerted activities in RPE cells. We conclude that gremlin promotes RPE cell proliferation, migration and VEGF production possibly via activating VEGFR2-Akt-mTORC2 signaling. Gremlin could be a novel therapeutic target of ROP or other retinal vasoproliferation diseases. PMID:27894090

  6. Gremlin promotes retinal pigmentation epithelial (RPE) cell proliferation, migration and VEGF production via activating VEGFR2-Akt-mTORC2 signaling.

    PubMed

    Liu, Yuan; Chen, Zhijun; Cheng, Haixia; Chen, Juan; Qian, Jing

    2017-01-03

    Retinopathy of prematurity (ROP) is characterized by late-phase pathologic retinal vasoproliferation. Gremlin is a novel vascular endothelial growth factors (VEGF) receptor 2 (VEGFR2) agonist and promotes angiogenic response. We demonstrated that gremlin expression was significantly increased in retinas of ROP model mice, which was correlated with VEGF upregulation. In retinal pigmentation epithelial (RPE) cells, gremlin activated VEGFR2-Akt-mTORC2 (mammalian target of rapamycin complex 2) signaling, and promoted cell proliferation, migration and VEGF production. VEGFR inhibition (by SU5416) or shRNA knockdown almost abolished gremlin-mediated pleiotropic functions in RPE cells. Further, pharmacological inhibition of Akt-mTOR, or shRNA knockdown of key mTORC2 component (Rictor or Sin1) also attenuated gremlin-exerted activities in RPE cells. We conclude that gremlin promotes RPE cell proliferation, migration and VEGF production possibly via activating VEGFR2-Akt-mTORC2 signaling. Gremlin could be a novel therapeutic target of ROP or other retinal vasoproliferation diseases.

  7. Overweight and obesity versus concentrations of VEGF-A, sVEGFR-1, and sVEGFR-2 in plasma of patients with lower limb chronic ischemia*

    PubMed Central

    Wieczór, Radosław; Wieczór, Anna Maria; Gadomska, Grażyna; Stankowska, Katarzyna; Fabisiak, Jacek; Suppan, Karol; Pulkowski, Grzegorz; Budzyński, Jacek; Rość, Danuta

    2016-01-01

    Objective: Being overweight or obese comprises a significant risk factor for atherosclerosis. Fat tissue also generates factors stimulating angiogenesis, the process by which new blood vessels form. The purpose of this paper is to assess concentrations of the vascular endothelial growth factor A (VEGF-A) and its soluble type-1 and type-2 receptors (sVEGFR-1 and sVEGFR-2) in plasma of patients with peripheral arterial disease (PAD) depending on the level of nutrition according to body mass index (BMI). Methods: The study group included patients suffering from symptomatic PAD (n=46) in Fontaine classes IIa–IV without any history of neoplastic disease and who have a normal BMI (n=15), are overweight (n=21) or are obese (n=10). The control group (n=30) consisted of healthy non-smoking volunteers who were neither overweight nor obese. Venous blood plasma samples were collected from both groups at rest in the morning to determine plasma concentrations of VEGF-A, sVEGFR-1, and sVEGFR-2 using the enzyme-linked immunosorbent assay (ELISA) method. Results: The group of patients with PAD co-existent with being overweight or obese tended to have higher mean concentration levels of VEGF-A and sVEGFR-2 when compared with patients suffering from PAD with normal BMI. A statistically significant positive correlation was obtained between BMI and average plasma concentrations of sVEGFR-2 (R=0.37, P=0.0103). However, no significant correlation was noticed between BMI and VEGF-A or sVEGFR-1 concentrations. Conclusions: A positive correlation determined between the level of antiangiogenic factor and BMI value may be indicative of the linearly growing prevalence of some antiangiogenic factors in patients with metabolic disorders, which may be one of numerous factors contributing to incomplete efficiency of collateral circulation development in patients with PAD. PMID:27819131

  8. Acidic pH reduces VEGF-mediated endothelial cell responses by downregulation of VEGFR-2; relevance for anti-angiogenic therapies

    PubMed Central

    Faes, Seraina; Uldry, Emilie; Planche, Anne; Santoro, Tania; Pythoud, Catherine; Demartines, Nicolas; Dormond, Olivier

    2016-01-01

    Anti-angiogenic treatments targeting the vascular endothelial growth factor or its receptors have shown clinical benefits. However, impact on long-term survival remains limited. Solid tumors display an acidic microenvironment that profoundly influences their biology. Consequences of acidity on endothelial cells and anti-angiogenic therapies remain poorly characterized and hence are the focus of this study. We found that exposing endothelial cells to acidic extracellular pH resulted in reduced cell proliferation and migration. Also, whereas VEGF increased endothelial cell proliferation and survival at pH 7.4, it had no effect at pH 6.4. Furthermore, in acidic conditions, stimulation of endothelial cells with VEGF did not result in activation of downstream signaling pathways such as AKT. At a molecular level, acidity significantly decreased the expression of VEGFR-2 by endothelial cells. Consequently, anti-angiogenic therapies that target VEGFR-2 such as sunitinib and sorafenib failed to block endothelial cell proliferation in acidic conditions. In vivo, neutralizing tumor acidity with sodium bicarbonate increased the percentage of endothelial cells expressing VEGFR-2 in tumor xenografts. Furthermore, combining sodium bicarbonate with sunitinib provided stronger anti-cancer activity than either treatment alone. Histological analysis showed that sunitinib had a stronger anti-angiogenic effect when combined with sodium bicarbonate. Overall, our results show that endothelial cells prosper independently of VEGF in acidic conditions partly as a consequence of decreased VEGFR-2 expression. They further suggest that strategies aiming to raise intratumoral pH can improve the efficacy of anti-VEGF treatments. PMID:27852069

  9. CD147 promotes liver fibrosis progression via VEGF-A/VEGFR2 signalling-mediated cross-talk between hepatocytes and sinusoidal endothelial cells.

    PubMed

    Yan, Zhaoyong; Qu, Kai; Zhang, Jing; Huang, Qichao; Qu, Ping; Xu, Xinsen; Yuan, Peng; Huang, Xiaojun; Shao, Yongping; Liu, Chang; Zhang, Hongxin; Xing, Jinliang

    2015-10-01

    Although previous evidence indicates close involvement of CD147 in the pathogenesis of liver fibrosis, the underlying molecular mechanisms and its therapeutic value remain largely unknown. In the present study, we investigated the biological roles of CD147 in liver fibrosis and assessed its therapeutic value as a target molecule in the CCl4-induced liver fibrosis mouse model. We found that CD147 was highly expressed in both hepatocytes and SECs (sinusoidal endothelial cells) in fibrotic liver tissues. Additionally, it was significantly associated with the fibrosis stage. TGF-β1 (transforming growth factor β1) was found to be mainly responsible for the up-regulation of CD147. Bioinformatic and experimental data suggest a functional link between CD147 expression and VEGF-A (vascular endothelial growth factor A)/VEGR-2 (VEGF receptor 2) signalling-mediated angiogenesis in fibrotic liver tissues. Furthermore, we observed that the CD147-induced activation of the PI3K (phosphoinositide 3-kinase)/Akt signalling pathway promotes the production of VEGF-A in hepatocytes and expression of VEGFR-2 in SECs, which was found to enhance the angiogenic capability of SECs. Finally, our data indicate that blocking of CD147 using an mAb (monoclonal antibody) attenuated liver fibrosis progression via inhibition of VEGF-A/VEGFR-2 signalling and subsequent amelioration of microvascular abnormality in the CCl4-induced mouse model. Our findings suggest a novel functional mechanism that CD147 may promote liver fibrosis progression via inducing the VEGF-A/VEGFR-2 signalling pathway-mediated cross-talk between hepatocytes and SECs. New strategies based on the intervention of CD147 can be expected for prevention of liver fibrosis.

  10. Erianin inhibits high glucose-induced retinal angiogenesis via blocking ERK1/2-regulated HIF-1α-VEGF/VEGFR2 signaling pathway

    PubMed Central

    Yu, Zengyang; Zhang, Tianyu; Gong, Chenyuan; Sheng, Yuchen; Lu, Bin; Zhou, Lingyu; Ji, Lili; Wang, Zhengtao

    2016-01-01

    Erianin is a natural compound found in Dendrobium chrysotoxum Lindl. Diabetic retinopathy (DR) is a serious and common microvascular complication of diabetes. This study aims to investigate the inhibitory mechanism of erianin on retinal neoangiogenesis and its contribution to the amelioration of DR. Erianin blocked high glucose (HG)-induced tube formation and migration in choroid-retinal endothelial RF/6A cells. Erianin inhibited HG-induced vascular endothelial growth factor (VEGF) expression, hypoxia-inducible factor 1-alpha (HIF-1α) translocation into nucleus and ERK1/2 activation in RF/6A and microglia BV-2 cells. MEK1/2 inhibitor U0126 blocked HG-induced HIF-1α and ERK1/2 activation in both above two cells. In addition, erianin abrogated VEGF-induced angiogenesis in vitro and in vivo, and also inhibited VEGF-induced activation of VEGF receptor 2 (VEGFR2) and its downstream cRaf-MEK1/2-ERK1/2 and PI3K-AKT signaling pathways in RF/6A cells. Furthermore, erianin reduced the increased retinal vessels, VEGF expression and microglia activation in streptozotocin (STZ)-induced hyperglycemic and oxygen-induced retinopathy (OIR) mice. In conclusion, our results demonstrate that erianin inhibits retinal neoangiogenesis by abrogating HG-induced VEGF expression by blocking ERK1/2-mediated HIF-1α activation in retinal endothelial and microglial cells, and further suppressing VEGF-induced activation of VEGFR2 and its downstream signals in retinal endothelial cells. PMID:27678303

  11. Phosphorylation of Tyr1214 within VEGFR-2 triggers the recruitment of Nck and activation of Fyn leading to SAPK2/p38 activation and endothelial cell migration in response to VEGF.

    PubMed

    Lamalice, Laurent; Houle, François; Huot, Jacques

    2006-11-10

    VEGFR-2 is the major receptor that regulates the different functions of VEGF in adults. We have previously reported that following VEGF treatment of endothelial cells, VEGFR-2 is phosphorylated on Tyr1214 upstream of the Cdc42-SAPK2/p38-MAPKAP K2 pathway. However, little is known of the earliest molecular events that compose the SAPK2/p38 pathway following VEGFR-2 activation. In this study, we address this question using HA-tagged constructs of either wild-type VEGFR-2 or Y1214F VEGFR-2 mutant in immunoprecipitation assays. We show that the Src family kinase member Fyn, but not c-Src itself, is recruited to VEGFR-2 and is activated in a p-Tyr1214-dependent manner. We also report that the SH2 domain-containing adapter molecule Nck, but not Grb2, is recruited to VEGFR-2 in a p-Tyr1214-dependent manner and that it associates with Fyn. Moreover, PAK-2 is phosphorylated in a Fyn-dependent manner. Using chemical and genetic inhibitors, we show that Fyn activity is required for SAPK2/p38 but not for FAK activation in response to VEGF. In contrast, c-Src permits activation of FAK, but not that of SAPK2/p38. In addition, Fyn is required for stress fiber formation and endothelial cell migration. We propose a model in which Fyn forms a molecular complex with Nck and PAK-2 and suggest that this complex assembles in a p-Tyr1214-dependent manner within VEGFR-2 following VEGF treatment. In turn, this triggers the activation of the SAPK2/p38 MAP kinase module, and promotes stress fiber formation and endothelial cell migration.

  12. Eriocalyxin B, a natural diterpenoid, inhibited VEGF-induced angiogenesis and diminished angiogenesis-dependent breast tumor growth by suppressing VEGFR-2 signaling

    PubMed Central

    Zhou, Xunian; Yue, Grace Gar-Lee; Liu, Minghua; Zuo, Zhili; Lee, Julia Kin-Ming; Li, Mingyue; Tsui, Stephen Kwok-Wing; Fung, Kwok-Pui; Sun, Handong; Pu, Jianxin; Lau, Clara Bik-San

    2016-01-01

    Eriocalyxin B (EriB), a natural ent-kaurane diterpenoid isolated from the plant Isodon eriocalyx var. laxiflora, has emerged as a promising anticancer agent. The effects of EriB on angiogenesis were explored in the present study. Here we demonstrated that the subintestinal vein formation was significantly inhibited by EriB treatment (10, 15 μM) in zebrafish embryos, which was resulted from the alteration of various angiogenic genes as shown in transcriptome profiling. In human umbilical vein endothelial cells, EriB treatment (50, 100 nM) could significantly block vascular endothelial growth factors (VEGF)-induced cell proliferation, tube formation, cell migration and cell invasion. Furthermore, EriB also caused G1 phase cell cycle arrest which was correlated with the down-regulation of the cyclin D1 and CDK4 leading to the inhibition of phosphorylated retinoblastoma protein expression. Investigation of the signal transduction revealed that EriB inhibited VEGF-induced phosphorylation of VEGF receptor-2 via the interaction with the ATP-binding sites according to the molecular docking simulations. The suppression of VEGFR-2 downstream signal transduction cascades was also observed. EriB was showed to inhibit new blood vessel formation in Matrigel plug model and mouse 4T1 breast tumor model. EriB (5 mg/kg/day) treatment was able to decrease tumor vascularization and suppress tumor growth and angiogenesis. Taken together, our findings suggested that EriB is a novel inhibitor of angiogenesis through modulating VEGFR-2 signaling pathway, which could be developed as a promising anti-angiogenic agent for treatment of angiogenesis-related human diseases, such as cancer. PMID:27756875

  13. Coordinated Activation of VEGF/VEGFR-2 and PPARδ Pathways by a Multi-Component Chinese Medicine DHI Accelerated Recovery from Peripheral Arterial Disease in Type 2 Diabetic Mice

    PubMed Central

    He, Shuang; Zhao, Tiechan; Guo, Hao; Meng, Yanzhi; Qin, Gangjian; Goukassian, David A.; Han, Jihong; Gao, Xuimei; Zhu, Yan

    2016-01-01

    Diabetic mellitus (DM) patients are at an increased risk of developing peripheral arterial disease (PAD). Danhong injection (DHI) is a Chinese patent medicine widely used for several cardiovascular indications but the mechanism of action is not well-understood. We investigated the therapeutic potential of DHI on experimental PAD in mice with chemically induced as well as genetic (KKAy) type 2 DM and the overlapping signaling pathways regulating both therapeutic angiogenesis and glucose homeostasis. Compared with normal genetic background wild type (WT) mice, both DM mice showed impaired perfusion recovery in hind-limb ischemia (HLI) model. DHI treatment significantly accelerated perfusion recovery, lowered blood glucose and improved glucose tolerance in both DM models. Bioluminescent imaging demonstrated a continuous ischemia-induced vascular endothelial growth factor receptor 2 (VEGFR-2) gene expressions with a peak time coincident with the maximal DHI stimulation. Flow cytometry analysis showed a DHI-mediated increase in endothelial progenitor cell (EPC) mobilization from bone marrow to circulating peripheral blood. DHI administration upregulated the expression of vascular endothelial growth factor A (VEGF-A) and VEGF receptor-2 (VEGFR-2) in ischemic muscle. A cross talk between ischemia-induced angiogenesis and glucose tolerance pathways was analyzed by Ingenuity Pathway Analysis (IPA) which suggested an interaction of VEGF-A/VEGFR-2 and peroxisome proliferator-activated receptor δ (PPARδ)/peroxisome proliferator-activated receptor γ (PPARγ) genes. We confirmed that upregulation of VEGF-A/VEGFR-2 by DHI promoted PPARδ gene expression in both type 2 diabetic mice. Our findings demonstrated that a multi-component Chinese medicine DHI effectively increased blood flow recovery after tissue ischemia in diabetic mice by promoting angiogenesis and improving glucose tolerance through a concomitant activation of VEGF-A/VEGFR-2 and PPARδ signaling pathways. PMID

  14. Glutamate Neonatal Excitotoxicity Modifies VEGF-A, VEGF-B, VEGFR-1 and VEGFR-2 Protein Expression Profiles During Postnatal Development of the Cerebral Cortex and Hippocampus of Male Rats.

    PubMed

    Castañeda-Cabral, Jose Luis; Beas-Zarate, Carlos; Gudiño-Cabrera, Graciela; Ureña-Guerrero, Monica E

    2017-07-29

    Vascular endothelial growth factor (VEGF) exerts both neuroprotective and proinflammatory effects in the brain, depending on the VEGF (A-E) and VEGF receptor (VEGFR1-3) types involved. Neonatal monosodium glutamate (MSG) treatment triggers an excitotoxic degenerative process associated with several neuropathological conditions, and VEGF messenger RNA (mRNA) expression is increased at postnatal day (PD) 14 in rat hippocampus (Hp) following the treatment. The aim of this work was to establish the changes in immunoreactivity to VEGF-A, VEGF-B, VEGFR-1 and VEGFR-2 proteins induced by neonatal MSG treatment (4 g/kg, subcutaneous, at PD1, 3, 5 and 7) in the cerebral motor cortex (CMC) and Hp. Samples collected from PD2 to PD60 from control and MSG-treated male Wistar rats were assessed by western blotting for each protein. Considering that immunoreactivity measured by western blotting is related to the protein expression level, we found that each protein in each cerebral region has a specific expression profile throughout the studied ages, and all profiles were differentially modified by MSG. Specifically, neonatal MSG treatment significantly increased the immunoreactivity to the following: (1) VEGF-A at PD8-PD10 in the CMC and at PD6-PD8 in the Hp; (2) VEGF-B at PD2, PD6 and PD10 in the CMC and at PD8-PD9 in the Hp; and (3) VEGFR-2 at PD6-PD8 in the CMC and at PD21-PD60 in the Hp. Also, MSG significantly reduced the immunoreactivity to the following: (1) VEGF-B at PD8-PD9 and PD45-PD60 in the CMC; and (2) VEGFR-1 at PD4-PD6 and PD14-PD21 in the CMC and at PD4, PD9-PD10 and PD60 in the Hp. Our results indicate that VEGF-mediated signalling is involved in the excitotoxic process triggered by neonatal MSG treatment and should be further characterized.

  15. Fasting influences steroidogenesis, vascular endothelial growth factor (VEGF) levels and mRNAs expression for VEGF, VEGF receptor type 2 (VEGFR-2), endothelin-1 (ET-1), endothelin receptor type A (ET-A) and endothelin converting enzyme-1 (ECE-1) in newly formed pig corpora lutea.

    PubMed

    Galeati, Giovanna; Forni, Monica; Spinaci, Marcella; Zannoni, Augusta; Govoni, Nadia; Ribeiro, Luciana A; Seren, Eraldo; Tamanini, Carlo

    2005-04-01

    This study was designed to verify whether fasting influences vascular endothelial growth factor (VEGF) production and VEGF, VEGF receptor-2 (VEGFR-2) as well as endothelin (ET) system members (endothelin converting enzyme-1, ECE-1; ET-1; endothelin receptor type A, ET-A) mRNA expression in pig corpora lutea; furthermore, we wanted to assess whether fasting affects steroidogenesis in luteal cells. Eight prepubertal gilts were induced to ovulate and were randomly assigned to two groups: (A) n = 4, normally fed; and (B) n = 4, fasted for 72 h starting 3 days after ovulation. At the end of fasting, ovaries were removed from all the animals and corpora lutea (CLs) were collected. VEGF and steroid levels in luteal tissue were determined by ELISA and RIA, respectively; VEGF, VEGFR-2, ET-1, ET-A and ECE-1 mRNAs expression was measured by real-time PCR. VEGF protein levels were similar in the two groups, while all steroid (progesterone, testosterone, estradiol 17beta) concentrations were significantly (P < 0.001) higher in CLs collected from fasted animals compared with those from normally fed gilts. VEGF, VEGFR-2, ET-1 and ECE-1 (but not ET-A) mRNA expression was significantly lower (P < 0.05) in fasted versus normally fed animals. The overall conclusion is that all the parameters studied are affected by feed restriction, but the mechanisms activated at luteal level are possibly not fully adequate to compensate for nutrient shortage.

  16. VEGFR2 Translocates to the Nucleus to Regulate Its Own Transcription

    PubMed Central

    Domingues, Inês; Rino, José; Demmers, Jeroen A. A.; de Lanerolle, Primal; Santos, Susana Constantino Rosa

    2011-01-01

    Vascular Endothelial Growth Factor Receptor-2 (VEGFR2) is the major mediator of the angiogenic effects of VEGF. In addition to its well known role as a membrane receptor that activates multiple signaling pathways, VEGFR2 also has a nuclear localization. However, what VEGFR2 does in the nucleus is still unknown. In the present report we show that, in endothelial cells, nuclear VEGFR2 interacts with several nuclear proteins, including the Sp1, a transcription factor that has been implicated in the regulation of genes needed for angiogenesis. By in vivo chromatin immunoprecipitation (ChIP) assays, we found that VEGFR2 binds to the Sp1-responsive region of the VEGFR2 proximal promoter. These results were confirmed by EMSA assays, using the same region of the VEGFR2 promoter. Importantly, we show that the VEGFR2 DNA binding is directly linked to the transcriptional activation of the VEGFR2 promoter. By reporter assays, we found that the region between -300/-116 relative to the transcription start site is essential to confer VEGFR2-dependent transcriptional activity. It was previously described that nuclear translocation of the VEGFR2 is dependent on its activation by VEGF. In agreement, we observed that the binding of VEGFR2 to DNA requires VEGF activation, being blocked by Bevacizumab and Sunitinib, two anti-angiogenic agents that inhibit VEGFR2 activation. Our findings demonstrate a new mechanism by which VEGFR2 activates its own promoter that could be involved in amplifying the angiogenic response. PMID:21980525

  17. The Emerging Regulation of VEGFR-2 in Triple-Negative Breast Cancer

    PubMed Central

    Zhu, Xiaoxia; Zhou, Wen

    2015-01-01

    Vascular endothelial growth factor-A (VEGF) signals vascular development and angiogenesis mainly by binding to VEGF receptor family member 2 (VEGFR-2). Adaptor proteins mediate many VEGFR-2’s functions in the development of blood vessels. Cancer cells secrete VEGF to activate VEGFR-2 pathway in their neighboring endothelial cells in the process of cancer-related angiogenesis. Interestingly, activation of VEGFR-2 signaling is found in breast cancer cells, but its role and regulation are not clear. We highlighted research advances of VEGFR-2, with a focus on VEGFR-2’s regulation by mutant p53 in breast cancer. In addition, we reviewed recent Food and Drug Administration-approved tyrosine kinase inhibitor drugs that can inhibit the function of VEGFR-2. Ongoing preclinical and clinical studies might prove that pharmaceutically targeting VEGFR-2 could be an effective therapeutic strategy in treating triple-negative breast cancer. PMID:26500608

  18. Invivo monitoring of fetoplacental vegfr2 gene activity in a murine pregnancy model using a vegfr2 -luc reporter gene and bioluminescent imaging

    USDA-ARS?s Scientific Manuscript database

    Vascular endothelial growth factor receptor-2 (VEGFR2) plays a pivotal role in angiogenesis by eliciting vascular endothelial cell growth when bound to VEGF, a powerful pro-angiogenic ligand. While Vegf and Vegfr2 are expressed throughout gestation, the latter third of gestation in mice is character...

  19. VEGFR-2 reduces while combined VEGFR-2 and -3 signaling increases inflammation in apical periodontitis

    PubMed Central

    Virtej, Anca; Papadakou, Panagiota; Sasaki, Hajime; Bletsa, Athanasia; Berggreen, Ellen

    2016-01-01

    Background In apical periodontitis, oral pathogens provoke an inflammatory response in the apical area that induces bone resorptive lesions. In inflammation, angio- and lymphangiogenesis take place. Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key players in these processes and are expressed in immune cells and endothelial cells in the lesions. Objective We aimed at testing the role of VEGFR-2 and -3 in periapical lesion development and investigated their role in lymphangiogenesis in the draining lymph nodes. Design We induced lesions by pulp exposure in the lower first molars of C57BL/6 mice. The mice received IgG injections or blocking antibodies against VEGFR-2 (anti-R2), VEGFR-3 (anti-R3), or combined VEGFR-2 and -3, starting on day 0 until day 10 or 21 post-exposure. Results Lesions developed faster in the anti-R2 and anti-R3 group than in the control and anti-R2/R3 groups. In the anti-R2 group, a strong inflammatory response was found expressed as increased number of neutrophils and osteoclasts. A decreased level of pro-inflammatory cytokines was found in the anti-R2/R3 group. Lymphangiogenesis in the draining lymph nodes was inhibited after blocking of VEGFR-2 and/or -3, while the largest lymph node size was seen after anti-R2 treatment. Conclusions We demonstrate an anti-inflammatory effect of VEGFR-2 signaling in periapical lesions which seems to involve neutrophil regulation and is independent of angiogenesis. Combined signaling of VEGFR-2 and -3 has a pro-inflammatory effect. Lymph node lymphangiogenesis is promoted through activation of VEGFR-2 and/or VEGFR-3. PMID:27650043

  20. PLACENTAL DEFECTS IN ARNT-KNOCKOUT CONCEPTUS CORRELATE WITH LOCALIZED DECREASES IN VEGF-R2, ANG-1, AND TIE-2.

    EPA Science Inventory

    The aryl hydrocarbon receptor nuclear translocator (ARNT) is a transcriptional regulator that heterodimerizes with Per-ARNT-Sim (PAS) proteins. ARNT also dimerizes with hypoxia inducible factor1 (HIF1 ), inducing expression of vascular endothelial cell growth factor (VEGF) to p...

  1. PLACENTAL DEFECTS IN ARNT-KNOCKOUT CONCEPTUS CORRELATE WITH LOCALIZED DECREASES IN VEGF-R2, ANG-1, AND TIE-2.

    EPA Science Inventory

    The aryl hydrocarbon receptor nuclear translocator (ARNT) is a transcriptional regulator that heterodimerizes with Per-ARNT-Sim (PAS) proteins. ARNT also dimerizes with hypoxia inducible factor1 (HIF1 ), inducing expression of vascular endothelial cell growth factor (VEGF) to p...

  2. Site-Specific Phosphorylation of VEGFR2 Is Mediated by Receptor Trafficking: Insights from a Computational Model

    PubMed Central

    Clegg, Lindsay Wendel; Mac Gabhann, Feilim

    2015-01-01

    Matrix-binding isoforms and non-matrix-binding isoforms of vascular endothelial growth factor (VEGF) are both capable of stimulating vascular remodeling, but the resulting blood vessel networks are structurally and functionally different. Here, we develop and validate a computational model of the binding of soluble and immobilized ligands to VEGF receptor 2 (VEGFR2), the endosomal trafficking of VEGFR2, and site-specific VEGFR2 tyrosine phosphorylation to study differences in induced signaling between these VEGF isoforms. In capturing essential features of VEGFR2 signaling and trafficking, our model suggests that VEGFR2 trafficking parameters are largely consistent across multiple endothelial cell lines. Simulations demonstrate distinct localization of VEGFR2 phosphorylated on Y1175 and Y1214. This is the first model to clearly show that differences in site-specific VEGFR2 activation when stimulated with immobilized VEGF compared to soluble VEGF can be accounted for by altered trafficking of VEGFR2 without an intrinsic difference in receptor activation. The model predicts that Neuropilin-1 can induce differences in the surface-to-internal distribution of VEGFR2. Simulations also show that ligated VEGFR2 and phosphorylated VEGFR2 levels diverge over time following stimulation. Using this model, we identify multiple key levers that alter how VEGF binding to VEGFR2 results in different coordinated patterns of multiple downstream signaling pathways. Specifically, simulations predict that VEGF immobilization, interactions with Neuropilin-1, perturbations of VEGFR2 trafficking, and changes in expression or activity of phosphatases acting on VEGFR2 all affect the magnitude, duration, and relative strength of VEGFR2 phosphorylation on tyrosines 1175 and 1214, and they do so predictably within our single consistent model framework. PMID:26067165

  3. Decorin Is a Novel VEGFR-2-Binding Antagonist for the Human Extravillous Trophoblast

    PubMed Central

    Khan, Gausal A.; Girish, Gannareddy V.; Lala, Neena; Di Guglielmo, Gianni M.

    2011-01-01

    Extravillous trophoblasts (EVT) of the human placenta invade the uterine decidua and its arteries to ensure successful placentation. We previously identified two decidua-derived molecules, TGF-β and a TGF-β-binding proteoglycan decorin (DCN), as negative regulators of EVT proliferation, migration, and invasiveness and reported that DCN acts via multiple tyrosine kinase receptors [epidermal growth factor-receptor (EGF-R), IGF receptor-1 (IGFR1), and vascular endothelial growth factor 2 receptor (VEGFR-2)]. Because binding of DCN to VEGFR-2 has never been reported earlier, present study explored this binding, the approximate location of VEGFR-2-binding site in DCN, and its functional role in our human first trimester EVT cell line HTR-8/SVneo. Based on far-Western blotting and coimmunoprecipitation studies, we report that DCN binds both native (EVT expressed) and recombinant VEGFR-2 and that this binding is abrogated with a VEGFR-2 blocking antibody, indicating an overlap between the ligand-binding and the DCN-binding domains of VEGFR-2. We determined that 125I-labeled VEGF-E (a VEGFR-2 specific ligand) binds EVT with a dissociation constant (Kd) of 566 pM, and DCN displaced this binding with an inhibition constant (Ki) of 3.93–5.78 nM, indicating a 7- to 10-fold lower affinity of DCN for VEGFR-2. DCN peptide fragments derived from the leucine rich repeat 5 domain that blocked DCN-VEGFR-2 interactions or VEGF-E binding in EVT cells also blocked VEGF-A- and VEGF-E-induced EVT cell proliferation and migration, indicative of functional VEGFR-2-binding sites of DCN. Finally, DCN inhibited VEGF-E-induced EVT migration by interfering with ERK1/2 activation. Our findings reveal a novel role of DCN as an antagonistic ligand for VEGFR-2, having implications for pathophysiology of preeclampsia, a trophoblast hypoinvasive disorder in pregnancy, and explain its antiangiogenic function. PMID:21659473

  4. Decorin is a novel VEGFR-2-binding antagonist for the human extravillous trophoblast.

    PubMed

    Khan, Gausal A; Girish, Gannareddy V; Lala, Neena; Di Guglielmo, Gianni M; Lala, Peeyush K

    2011-08-01

    Extravillous trophoblasts (EVT) of the human placenta invade the uterine decidua and its arteries to ensure successful placentation. We previously identified two decidua-derived molecules, TGF-β and a TGF-β-binding proteoglycan decorin (DCN), as negative regulators of EVT proliferation, migration, and invasiveness and reported that DCN acts via multiple tyrosine kinase receptors [epidermal growth factor-receptor (EGF-R), IGF receptor-1 (IGFR1), and vascular endothelial growth factor 2 receptor (VEGFR-2)]. Because binding of DCN to VEGFR-2 has never been reported earlier, present study explored this binding, the approximate location of VEGFR-2-binding site in DCN, and its functional role in our human first trimester EVT cell line HTR-8/SVneo. Based on far-Western blotting and coimmunoprecipitation studies, we report that DCN binds both native (EVT expressed) and recombinant VEGFR-2 and that this binding is abrogated with a VEGFR-2 blocking antibody, indicating an overlap between the ligand-binding and the DCN-binding domains of VEGFR-2. We determined that (125)I-labeled VEGF-E (a VEGFR-2 specific ligand) binds EVT with a dissociation constant (K(d)) of 566 pM, and DCN displaced this binding with an inhibition constant (K(i)) of 3.93-5.78 nM, indicating a 7- to 10-fold lower affinity of DCN for VEGFR-2. DCN peptide fragments derived from the leucine rich repeat 5 domain that blocked DCN-VEGFR-2 interactions or VEGF-E binding in EVT cells also blocked VEGF-A- and VEGF-E-induced EVT cell proliferation and migration, indicative of functional VEGFR-2-binding sites of DCN. Finally, DCN inhibited VEGF-E-induced EVT migration by interfering with ERK1/2 activation. Our findings reveal a novel role of DCN as an antagonistic ligand for VEGFR-2, having implications for pathophysiology of preeclampsia, a trophoblast hypoinvasive disorder in pregnancy, and explain its antiangiogenic function.

  5. A heat-shock protein axis regulates VEGFR2 proteolysis, blood vessel development and repair.

    PubMed

    Bruns, Alexander F; Yuldasheva, Nadira; Latham, Antony M; Bao, Leyuan; Pellet-Many, Caroline; Frankel, Paul; Stephen, Sam L; Howell, Gareth J; Wheatcroft, Stephen B; Kearney, Mark T; Zachary, Ian C; Ponnambalam, Sreenivasan

    2012-01-01

    Vascular endothelial growth factor A (VEGF-A) binds to the VEGFR2 receptor tyrosine kinase, regulating endothelial function, vascular physiology and angiogenesis. However, the mechanism underlying VEGFR2 turnover and degradation in this response is unclear. Here, we tested a role for heat-shock proteins in regulating the presentation of VEGFR2 to a degradative pathway. Pharmacological inhibition of HSP90 stimulated VEGFR2 degradation in primary endothelial cells and blocked VEGF-A-stimulated intracellular signaling via VEGFR2. HSP90 inhibition stimulated the formation of a VEGFR2-HSP70 complex. Clathrin-mediated VEGFR2 endocytosis is required for this HSP-linked degradative pathway for targeting VEGFR2 to the endosome-lysosome system. HSP90 perturbation selectively inhibited VEGF-A-stimulated human endothelial cell migration in vitro. A mouse femoral artery model showed that HSP90 inhibition also blocked blood vessel repair in vivo consistent with decreased endothelial regeneration. Depletion of either HSP70 or HSP90 caused defects in blood vessel formation in a transgenic zebrafish model. We conclude that perturbation of the HSP70-HSP90 heat-shock protein axis stimulates degradation of endothelial VEGFR2 and modulates VEGF-A-stimulated intracellular signaling, endothelial cell migration, blood vessel development and repair.

  6. VEGFR-2 silencing by small interference RNA (siRNA) suppresses LPA-induced epithelial ovarian cancer (EOC) invasion.

    PubMed

    Wang, Feng-qiang; Barfield, Elaine; Dutta, Sonia; Pua, Tarah; Fishman, David A

    2009-12-01

    The VEGF-VEGF receptor (VEGFR) signaling axis has emerged as a promising target for cancer therapy, attributing to its vital role in tumor angiogenesis and growth. We have previously reported the regulation of epithelial ovarian cancer (EOC) invasion and migration by VEGF and the implication of VEGF-VEGFR-2 axis in lysophosphatidic acid (LPA)-induced EOC invasion. However, the expression profile of VEGF and VEGFRs in EOC, their association with tumor aggressiveness, and their regulation by LPA remain unclear. In this study, we examined the expression of VEGFR-1, VEGFR-2, neuropilin-1 (NRP-1), NRP-2, VEGF(121), and VEGF(165) in established EOC cell lines and assessed their correlation with cell invasiveness. Moreover, using an ovarian cancer tissue qPCR array, we analyzed VEGFR-2 expression across a panel of 48 tissues with different disease stages and histological grades. We also tested the effect of LPA on VEGF and VEGFR-2 expression and examined whether blocking VEGFR-2 by RNA interference (RNAi) affects LPA-induced EOC invasion. We show that VEGF and VEGFR-2 expression correlates with cell invasiveness and VEGFR-2 expression in ovarian cancer tissues correlate with tumor grade. In addition, LPA, at 20 muM, significantly induced the expression of VEGF(121), VEGF(165), and VEGFR-2 in SKOV3 and DOV13 cells (P<0.05). VEGFR-2 small interference RNA (siRNA) transfection remarkably decreased LPA's invasion-promoting effect (P<0.001) in SKOV3 cells without significantly decreasing SKOV3 cells' basal invasiveness. In DOV13 cells, VEGFR-2 silencing significantly decreases both the basal level cell invasion and LPA's invasion promoting effect (P<0.001). These results suggest that decreasing VEGFR-2 expression by RNAi may prove to be an effective method to reduce the metastatic potential of EOC cells exposed to elevated levels of LPA.

  7. Identification of a variant in KDR associated with serum VEGFR2 and pharmacodynamics of Pazopanib.

    PubMed

    Maitland, Michael L; Xu, Chun-Fang; Cheng, Yu-Ching; Kistner-Griffin, Emily; Ryan, Kathleen A; Karrison, Theodore G; Das, Soma; Torgerson, Dara; Gamazon, Eric R; Thomeas, Vasiliki; Levine, Matthew R; Wilson, Paul A; Bing, Nan; Liu, Yuan; Cardon, Lon R; Pandite, Lini N; O'Connell, Jeffrey R; Cox, Nancy J; Mitchell, Braxton D; Ratain, Mark J; Shuldiner, Alan R

    2015-01-15

    VEGF receptor (VEGFR) kinases are important drug targets in oncology that affect function of systemic endothelial cells. To discover genetic markers that affect VEGFR inhibitor pharmacodynamics, we performed a genome-wide association study of serum soluble vascular VEGFR2 concentrations [sVEGFR2], a pharmacodynamic biomarker for VEGFR2 inhibitors. We conducted a genome-wide association study (GWAS) of [sVEGFR2] in 736 healthy Old Order Amish volunteers. Gene variants identified from the GWAS were genotyped serially in a cohort of 128 patients with advanced solid tumor with baseline [sVEGFR2] measurements, and in 121 patients with renal carcinoma with [sVEGFR2] measured before and during pazopanib therapy. rs34231037 (C482R) in KDR, the gene encoding sVEGFR2 was found to be highly associated with [sVEGFR2], explaining 23% of the variance (P = 2.7 × 10(-37)). Association of rs34231037 with [sVEGFR2] was replicated in 128 patients with cancer with comparable effect size (P = 0.025). Furthermore, rs34231037 was a significant predictor of changes in [sVEGFR2] in response to pazopanib (P = 0.01). Our findings suggest that genome-wide analysis of phenotypes in healthy populations can expedite identification of candidate pharmacogenetic markers. Genotyping for germline variants in KDR may have clinical utility in identifying patients with cancer with unusual sensitivity to effects of VEGFR2 kinase inhibitors. ©2014 American Association for Cancer Research.

  8. Neuropilin-1 promotes VEGFR-2 trafficking through Rab11 vesicles thereby specifying signal output.

    PubMed

    Ballmer-Hofer, Kurt; Andersson, Anneli E; Ratcliffe, Laura E; Berger, Philipp

    2011-07-21

    Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel development by activating 3 receptor tyrosine kinases (RTKs), VEGFR-1, -2, and -3, and by binding to coreceptors such as neuropilin-1 (NRP-1). We investigated how different VEGF-A isoforms, in particular VEGF-A(165)a and VEGF-A(165)b, control the balance between VEGFR-2 recycling, degradation, and signaling. Stimulation of cells with the NRP-1-binding VEGF-A(165)a led to sequential NRP-1-mediated VEGFR-2 recycling through Rab5, Rab4, and Rab11 vesicles. Recycling was accompanied by dephosphorylation of VEGFR-2 between Rab4 and Rab11 vesicles and quantitatively and qualitatively altered signal output. In cells stimulated with VEGF-A(165)b, an isoform unable to bind NRP-1, VEGFR-2 bypassed Rab11 vesicles and was routed to the degradative pathway specified by Rab7 vesicles. Deletion of the GIPC (synectin) binding motif of NRP-1 prevented transition of VEGFR-2 through Rab11 vesicles and attenuated signaling. Coreceptor engagement was specific for VEGFR-2 because EGFR recycled through Rab11 vesicles in the absence of known coreceptors. Our data establish a distinct role of NRP-1 in VEGFR-2 signaling and reveal a general mechanism for the function of coreceptors in modulating RTK signal output.

  9. Dynasore impairs VEGFR2 signalling in an endocytosis-independent manner

    PubMed Central

    Basagiannis, Dimitris; Zografou, Sofia; Galanopoulou, Katerina; Christoforidis, Savvas

    2017-01-01

    VEGFR2 is a critical angiogenic receptor playing a key role in vascular homeostasis. Upon activation by VEGF, VEGFR2 becomes endocytosed. Internalisation of VEGFR2 is facilitated, in part, through clathrin mediated endocytosis (CME), the role of which in VEGFR2 function is debated. Here, we confirm the contribution of CME in VEGFR2 uptake. However, curiously, we find that different approaches of inhibition of CME exert contradictory effects on VEGF signalling; knockdown of clathrin, or of dynamin, or overexpression of dynamin K44A, do not affect VEGF-induced phosphorylation of ERK1/2, while dynasore causes strong inhibition. We resolve this discrepancy by showing that although dynasore inhibits CME of VEGFR2, its inhibitory action in ERK1/2 phosphorylation is not related to attenuation of VEGFR2 endocytosis; it is rather due to an off-target effect of the drug. Dynasore inhibits VEGF-induced calcium release, a signalling event that lies upstream of ERK1/2, which implies that this effect could be responsible, at least in part, for the inhibitory action of the drug on VEGF-to-ERK1/2 signalling. These results raise caution that although dynasore is specific in inhibiting clathrin- and dynamin-mediated endocytosis, it may also exert off-target effects on signalling molecules, hence influencing the interpretation of the role of endocytosis in signalling. PMID:28327657

  10. Inhibition of AGS Cancer Cell Proliferation following siRNA-Mediated Downregulation of VEGFR2

    PubMed Central

    Zarei Mahmudabadi, Ali; Masoomi Karimi, Masoomeh; Bahabadi, Majid; Bagheri Hoseinabadi, Zahra; JafariSani, Moslem; Ahmadi, Reza

    2016-01-01

    Objective Vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) play important roles in angiogenesis of different developmental mechanisms such as wound healing, embryogenesis and diseases, including different types of cancer. VEGFR2 is associated with cell proliferation, migration, and vascular permeability of endothelial cells. Blocking VEGF and its receptors is suggested as a therapeutic approach to prevent tumor growth. In this study, we aim to block VEGF signaling via small interfering RNA (siRNA) inhibition of VEGFR2. Materials and Methods In this experimental study, we used the RNA interference (RNAi) mechanism to suppress expression of the VEGFR2 gene. We conducted the 3-(4,5-di- methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, real-time polymerase chain reaction (PCR), Western blot, and flow cytometry analyses of VEGFR2 expression. Results Real-time PCR and Western blot results showed that VEGFR2 expression significantly downregulated. This suppression was followed by inhibition of cell prolifera- tion, reduction of viability, and induction of apoptosis in the cancer cells. Conclusion These findings suggest that VEGFR2 has a role in cell proliferation and tumor growth. Accordingly, it is suggested that VEGFR2 can be a therapeutic target for controlling tumor growth and proliferation. PMID:27602320

  11. Autocrine stimulation of VEGFR-2 activates human leukemic cell growth and migration.

    PubMed

    Dias, S; Hattori, K; Zhu, Z; Heissig, B; Choy, M; Lane, W; Wu, Y; Chadburn, A; Hyjek, E; Gill, M; Hicklin, D J; Witte, L; Moore, M A; Rafii, S

    2000-08-01

    Emerging data suggest that VEGF receptors are expressed by endothelial cells as well as hematopoietic stem cells. Therefore, we hypothesized that functional VEGF receptors may also be expressed in malignant counterparts of hematopoietic stem cells such as leukemias. We demonstrate that certain leukemias not only produce VEGF but also express functional VEGFR-2 in vivo and in vitro, resulting in the generation of an autocrine loop that may support leukemic cell survival and proliferation. Approximately 50% of freshly isolated leukemias expressed mRNA and protein for VEGFR-2. VEGF(165) induced phosphorylation of VEGFR-2 and increased proliferation of leukemic cells, demonstrating these receptors were functional. VEGF(165) also induced the expression of MMP-9 by leukemic cells and promoted their migration through reconstituted basement membrane. The neutralizing mAb IMC-1C11, specific to human VEGFR-2, inhibited leukemic cell survival in vitro and blocked VEGF(165)-mediated proliferation of leukemic cells and VEGF-induced leukemic cell migration. Xenotransplantation of primary leukemias and leukemic cell lines into immunocompromised nonobese diabetic mice resulted in significant elevation of human, but not murine, VEGF in plasma and death of inoculated mice within 3 weeks. Injection of IMC-1C11 inhibited proliferation of xenotransplanted human leukemias and significantly increased the survival of inoculated mice. Interruption of signaling by VEGFRs, particularly VEGFR-2, may provide a novel strategy for inhibiting leukemic cell proliferation.

  12. Autocrine stimulation of VEGFR-2 activates human leukemic cell growth and migration

    PubMed Central

    Dias, Sergio; Hattori, Koichi; Zhu, Zhenping; Heissig, Beate; Choy, Margaret; Lane, William; Wu, Yan; Chadburn, Amy; Hyjek, Elizabeth; Gill, Muhammad; Hicklin, Daniel J.; Witte, Larry; Moore, M.A.S.; Rafii, Shahin

    2000-01-01

    Emerging data suggest that VEGF receptors are expressed by endothelial cells as well as hematopoietic stem cells. Therefore, we hypothesized that functional VEGF receptors may also be expressed in malignant counterparts of hematopoietic stem cells such as leukemias. We demonstrate that certain leukemias not only produce VEGF but also express functional VEGFR-2 in vivo and in vitro, resulting in the generation of an autocrine loop that may support leukemic cell survival and proliferation. Approximately 50% of freshly isolated leukemias expressed mRNA and protein for VEGFR-2. VEGF165 induced phosphorylation of VEGFR-2 and increased proliferation of leukemic cells, demonstrating these receptors were functional. VEGF165 also induced the expression of MMP-9 by leukemic cells and promoted their migration through reconstituted basement membrane. The neutralizing mAb IMC-1C11, specific to human VEGFR-2, inhibited leukemic cell survival in vitro and blocked VEGF165-mediated proliferation of leukemic cells and VEGF-induced leukemic cell migration. Xenotransplantation of primary leukemias and leukemic cell lines into immunocompromised nonobese diabetic mice resulted in significant elevation of human, but not murine, VEGF in plasma and death of inoculated mice within 3 weeks. Injection of IMC-1C11 inhibited proliferation of xenotransplanted human leukemias and significantly increased the survival of inoculated mice. Interruption of signaling by VEGFRs, particularly VEGFR-2, may provide a novel strategy for inhibiting leukemic cell proliferation. PMID:10953026

  13. Angiogenesis and radiation response modulation after vascular endothelial growth factor receptor-2 (VEGFR2) blockade

    SciTech Connect

    Li Jing; Huang Shyhmin; Armstrong, Eric A.; Fowler, John F.; Harari, Paul M. . E-mail: harari@humonc.wisc.edu

    2005-08-01

    The formation of new blood vessels (angiogenesis) represents a critical factor in the malignant growth of solid tumors and metastases. Vascular endothelial cell growth factor (VEGF) and its receptor VEGFR2 represent central molecular targets for antiangiogenic intervention, because of their integral involvement in endothelial cell proliferation and migration. In the current study, we investigated in vitro and in vivo effects of receptor blockade on various aspects of the angiogenic process using monoclonal antibodies against VEGFR2 (cp1C11, which is human specific, and DC101, which is mouse specific). Molecular blockade of VEGFR2 inhibited several critical steps involved in angiogenesis. VEGFR2 blockade in endothelial cells attenuated cellular proliferation, reduced cellular migration, and disrupted cellular differentiation and resultant formation of capillary-like networks. Further, VEGFR2 blockade significantly reduced the growth response of human squamous cell carcinoma xenografts in athymic mice. The growth-inhibitory effect of VEGFR2 blockade in tumor xenografts seems to reflect antiangiogenic influence as demonstrated by vascular growth inhibition in an in vivo angiogenesis assay incorporating tumor-bearing Matrigel plugs. Further, administration of VEGFR2-blocking antibodies in endothelial cell cultures, and in mouse xenograft models, increased their response to ionizing radiation, indicating an interactive cytotoxic effect of VEGFR2 blockade with radiation. These data suggest that molecular inhibition of VEGFR2 alone, and in combination with radiation, can enhance tumor response through molecular targeting of tumor vasculature.

  14. Autocrine VEGF–VEGFR2–Neuropilin-1 signaling promotes glioma stem-like cell viability and tumor growth

    PubMed Central

    Hamerlik, Petra; Lathia, Justin D.; Rasmussen, Rikke; Wu, Qiulian; Bartkova, Jirina; Lee, MyungHee; Moudry, Pavel; Bartek, Jiri; Fischer, Walter; Lukas, Jiri

    2012-01-01

    Although vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) is traditionally regarded as an endothelial cell protein, evidence suggests that VEGFRs may be expressed by cancer cells. Glioblastoma multiforme (GBM) is a lethal cancer characterized by florid vascularization and aberrantly elevated VEGF. Antiangiogenic therapy with the humanized VEGF antibody bevacizumab reduces GBM tumor growth; however, the clinical benefits are transient and invariably followed by tumor recurrence. In this study, we show that VEGFR2 is preferentially expressed on the cell surface of the CD133+ human glioma stem-like cells (GSCs), whose viability, self-renewal, and tumorigenicity rely, at least in part, on signaling through the VEGF-VEGFR2–Neuropilin-1 (NRP1) axis. We find that the limited impact of bevacizumab-mediated VEGF blockage may reflect ongoing autocrine signaling through VEGF–VEGFR2–NRP1, which is associated with VEGFR2–NRP1 recycling and a pool of active VEGFR2 within a cytosolic compartment of a subset of human GBM cells. Whereas bevacizumab failed to inhibit prosurvival effects of VEGFR2-mediated signaling, GSC viability under unperturbed or radiation-evoked stress conditions was attenuated by direct inhibition of VEGFR2 tyrosine kinase activity and/or shRNA-mediated knockdown of VEGFR2 or NRP1. We propose that direct inhibition of VEGFR2 kinase may block the highly dynamic VEGF–VEGFR2–NRP1 pathway and inspire a GBM treatment strategy to complement the currently prevalent ligand neutralization approach. PMID:22393126

  15. Computational Model of VEGFR2 pathway to ERK activation and modulation through receptor trafficking

    PubMed Central

    Tan, Wan Hua; Popel, Aleksander S.; Mac Gabhann, Feilim

    2013-01-01

    Vascular Endothelial Growth Factor (VEGF) signal transduction is central to angiogenesis in development and in pathological conditions such as cancer, retinopathy and ischemic diseases. We constructed and validated a computational model of VEGFR2 trafficking and signaling, to study the role of receptor trafficking kinetics in modulating ERK phosphorylation in VEGF-stimulated endothelial cells. Trafficking parameters were optimized and validated against four previously published in vitro experiments. Based on these parameters, model simulations demonstrated interesting behaviors that may be highly relevant to understanding VEGF signaling in endothelial cells. First, at moderate VEGF doses, VEGFR2 phosphorylation and ERK phosphorylation are related in a log-linear fashion, with a stable duration of ERK activation; but with higher VEGF stimulation, phosphoERK becomes saturated, and its duration increases. Second, a large endosomal fraction of VEGFR2 makes the ERK activation reaction network less sensitive to perturbations in VEGF dosage. Third, extracellular-matrix-bound VEGF binds and activates VEGFR2, but by internalizing at a slower rate, matrix-bound VEGF-induced intracellular ERK phosphorylation is predicted to be greater in magnitude and more sustained, in agreement with experimental evidence. Fourth, different endothelial cell types appear to have different trafficking rates, which result in different levels of endosomal receptor localization and different ERK response profiles. PMID:23993967

  16. Polymorphisms in the VEGFA and VEGFR-2 genes and neovascular age-related macular degeneration

    PubMed Central

    Fang, Amy M.; Lee, Aaron Y.; Kulkarni, Mukti; Osborn, Melissa P.

    2009-01-01

    Purpose Genetic factors influence an individual’s risk for developing neovascular age-related macular degeneration (AMD), a leading cause of irreversible blindness. Previous studies on the potential genetic link between AMD and vascular endothelial growth factor (VEGF), a key regulator of angiogenesis and vascular permeability, have yielded conflicting results. In the present case-control association study, we aimed to determine whether VEGF or its main receptor tyrosine kinase VEGFR-2 is genetically associated with neovascular AMD. Methods A total of 515 Caucasian patients with neovascular AMD and 253 ethically-matched controls were genotyped for polymorphisms in the VEGFA and VEGFR-2 genes. A tagging single nucleotide polymorphism (tSNP) approach was employed to cover each gene plus two kilobases on each side, spanning the promoter and 3′ untranslated regions. SNPs with a minimum allele frequency of 10% were covered by seven tSNPs in VEGFA and 20 tSNPs in VEGFR-2. Two VEGFA SNPs previously linked with AMD, rs1413711 and rs3025039, were also analyzed. Results The 29 VEGFA and VEGFR-2 SNPs analyzed in our cohort demonstrated no significant association with neovascular AMD. A single rare haplotype in the VEGFR-2 gene was associated with the presence of neovascular AMD (p=0.034). Conclusions This study is the first to investigate the association of VEGFR-2 polymorphisms with AMD and evaluates VEGFA genetic variants in the largest neovascular AMD cohort to date. Despite the angiogenic and permeability-enhancing effects of VEGF/VEGFR-2 signaling, we found minimal evidence of a significant link between polymorphisms in the VEGFA and VEGFR-2 genes and neovascular AMD. PMID:20019880

  17. Ubiquitination of basal VEGFR2 regulates signal transduction and endothelial function.

    PubMed

    Smith, Gina A; Fearnley, Gareth W; Abdul-Zani, Izma; Wheatcroft, Stephen B; Tomlinson, Darren C; Harrison, Michael A; Ponnambalam, Sreenivasan

    2017-08-10

    Cell surface receptors can undergo recycling or proteolysis but the cellular decision-making events that sort between these pathways remain poorly defined. Vascular endothelial growth factor A (VEGF-A) and vascular endothelial growth factor receptor 2 (VEGFR2) regulates signal transduction and angiogenesis but how signaling and proteolysis is regulated is not well understood. Here, we provide evidence that a pathway requiring the E1 ubiquitin-activating enzyme UBA1 controls basal VEGFR2 levels, hence metering plasma membrane receptor availability for the VEGF-A-regulated response by endothelial cells. VEGFR2 undergoes VEGF-A-independent constitutive degradation via a UBA1-dependent ubiquitin-linked pathway. Depletion of UBA1 increased VEGFR2 recycling from endosome-to-plasma membrane and decreased proteolysis. Increased membrane receptor availability after UBA1 depletion elevated VEGF-A-stimulated activation of key signaling enzymes such as PLCγ1 and ERK1/2. Although UBA1 depletion caused an overall decrease in endothelial cell proliferation, surviving cells showed greater VEGF-A-stimulated responses such as cell migration and tubulogenesis. Our study now suggests that a ubiquitin-linked pathway regulates the balance between receptor recycling and degradation which in turn impacts on the intensity and duration of VEGF-A-stimulated signal transduction and the endothelial response. © 2017. Published by The Company of Biologists Ltd.

  18. KIF13B regulates angiogenesis through Golgi to plasma membrane trafficking of VEGFR2.

    PubMed

    Yamada, Kaori H; Nakajima, Yuki; Geyer, Melissa; Wary, Kishore K; Ushio-Fukai, Masuko; Komarova, Yulia; Malik, Asrar B

    2014-10-15

    Although the trafficking of newly synthesized VEGFR2 to the plasma membrane is a key determinant of angiogenesis, the molecular mechanisms of Golgi to plasma membrane trafficking are unknown. Here, we have identified a key role of the kinesin family plus-end molecular motor KIF13B in delivering VEGFR2 cargo from the Golgi to the endothelial cell surface. KIF13B is shown to interact directly with VEGFR2 on microtubules. We also observed that overexpression of truncated versions of KIF13B containing the binding domains that interact with VEGFR2 inhibited VEGF-induced capillary tube formation. KIF13B depletion prevented VEGF-mediated endothelial migration, capillary tube formation and neo-vascularization in mice. Impairment in trafficking induced by knockdown of KIF13B shunted VEGFR2 towards the lysosomal degradation pathway. Thus, KIF13B is an essential molecular motor required for the trafficking of VEGFR2 from the Golgi, and its delivery to the endothelial cell surface mediates angiogenesis.

  19. Vascular-endothelial growth factor and its high affinity receptor VEGFR-2 in the normal versus destructive lesions human forebrain during development: an immuno-histochemical comparative study.

    PubMed

    Sentilhes, Loïc; Marret, Stéphane; Leroux, Philippe; Gonzalez, Bruno José; Laquerrière, Annie

    2011-04-18

    Vascular endothelial growth factor (VEGF) is an angiogenic inducer and neurotrophic factor both in adult and neonatal animal models. In the destructive lesions of the developing human brain, the role and expression of VEGF and of its mitogenic receptor VEGFR-2 have been hardly studied. The aim of the present work was to determine the immunohistochemical distribution of VEGF and VEGFR-2 in premature and full-term infants presenting with hypoxic/ischemic lesions, and to compare results with normal infant brains at similar developmental stages. Paraffin embedded brain tissue samples were assessed using anti-human VEGF and VEGFR-2 antibodies. In all undamaged forebrain areas, VEGF and VEGFR-2 displayed same expression patterns in control and pathologic brains, whatever the destructive lesion occurrence's time (before 25 weeks of gestation (WG), between 25 and 34WG, perinatal period and infancy). In the destructive lesions, VEGF was always expressed in neurons, astrocytes and in neovessel walls, contrary to VEGFR-2 which was only expressed in dispersed astrocytes. VEGF was expressed in oligodendrocytes of prenatally damaged brains, whereas VEGF was expressed in these cells in undamaged areas from birth only, similarly to control brains. These data suggest that VEGF plays specific roles and may not be mediated by VEGFR-2 in human forebrain structures exposed to ischemia.

  20. AIP1 functions as an endogenous inhibitor of VEGFR2-mediated signaling and inflammatory angiogenesis in mice

    PubMed Central

    Zhang, Haifeng; He, Yun; Dai, Shengchuan; Xu, Zhe; Luo, Yan; Wan, Ting; Luo, Dianhong; Jones, Dennis; Tang, Shibo; Chen, Hong; Sessa, William C.; Min, Wang

    2008-01-01

    ASK1-interacting protein-1 (AIP1), a recently identified member of the Ras GTPase-activating protein family, is highly expressed in vascular ECs and regulates EC apoptosis in vitro. However, its function in vivo has not been established. To study this, we generated AIP1-deficient mice (KO mice). Although these mice showed no obvious defects in vascular development, they exhibited dramatically enhanced angiogenesis in 2 models of inflammatory angiogenesis. In one of these models, the enhanced angiogenesis observed in the KO mice was associated with increased VEGF-VEGFR2 signaling. Consistent with this, VEGF-induced ear, cornea, and retina neovascularization were greatly augmented in KO mice and the enhanced retinal angiogenesis was markedly diminished by overexpression of AIP1. In vitro, VEGF-induced EC migration was inhibited by AIP1 overexpression, whereas it was augmented by both AIP1 knockout and knockdown, with the enhanced EC migration caused by AIP1 knockdown being associated with increased VEGFR2 signaling. We present mechanistic data that suggest AIP1 is recruited to the VEGFR2-PI3K complex, binding to both VEGFR2 and PI3K p85, at a late phase of the VEGF response, and that this leads to inhibition of VEGFR2 signaling. Taken together, our data demonstrate that AIP1 functions as an endogenous inhibitor in VEGFR2-mediated adaptive angiogenesis in mice. PMID:19033661

  1. DDA suppresses angiogenesis and tumor growth of colorectal cancer in vivo through decreasing VEGFR2 signaling

    PubMed Central

    Huang, Shiu-Wen; Lien, Jin-Cherng; Kuo, Sheng-Chu; Huang, Tur-Fu

    2016-01-01

    As angiogenesis is required for tumor growth and metastasis, suppressing angiogenesis is a promising strategy in limiting tumor progression. Vascular endothelial growth factor (VEGF)-A, a critical pro-angiogenic factor, has thus become an attractive target for therapeutic interventions in cancer. In this study, we explored the underlying mechanisms of a novel anthraquinone derivative DDA in suppressing angiogenesis. DDA inhibited VEGF-A-induced proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs). DDA also reduced VEGF-A-induced microvessel sprouting from aortic rings ex vivo and suppressed neovascularization in vivo. VEGF-A-induced VEGFR1, VEGFR2, FAK, Akt, ERK1/2 or STAT3 phosphorylation was reduced in the presence of DDA. In addition, NRP-1 siRNA reduced VEGF-A's enhancing effects in VEGFR2, FAK and Akt phosphorylation and cell proliferation in HUVECs. DDA disrupted VEGF-A-induced complex formation between NRP-1 and VEGFR2. Furthermore, systemic administration of DDA was shown to suppress tumor angiogenesis and growth in in vivo mouse xenograft models. Taken together, we demonstrated in this study that DDA exhibits anti-angiogenic properties through suppressing VEGF-A signaling. These observations also suggest that DDA might be a potential drug candidate for developing anti-angiogenic agent in the field of cancer and angiogenesis-related diseases. PMID:27517319

  2. VEGFR-2 inhibitors and the therapeutic applications thereof: a patent review (2012-2016).

    PubMed

    Peng, Fan-Wei; Liu, Da-Ke; Zhang, Qing-Wen; Xu, Yun-Gen; Shi, Lei

    2017-09-01

    Angiogenesis is an important component of certain normal physiological processes, but aberrant angiogenesis contributes to some pathological disorders and in particular to tumor growth. Activation of vascular endothelial growth factor receptor-2 (VEGFR-2) by vascular endothelial growth factor (VEGF) is a critical step in the signal transduction pathway that initiates tumor angiogenesis. Inhibition of angiogenesis via blocking VEGF/VEGFR-2 signaling pathway has emerged as a potential approach to anticancer therapy. Indeed, this approach has recently been clinically validated with the approvals of VEGFR-2 inhibitors. Areas covered: This review accounts for small-molecule inhibitors and antibodies of VEGFR-2 reported in the patent literature covering between January 2012 and June 2016, and their potential use as therapeutics for cancers, angiogenesis-related disorders and inflammatory diseases. Expert opinion: Despite the attractiveness of anti-angiogenic therapy by VEGF inhibition alone, several issues may limit this approach. VEGF expression levels can be elevated by numerous diverse stimuli such as the activation of other RTK signaling transduction pathway. Therefore, the development of multi-targeted tyrosine kinase inhibitors and the strategy of using these agents in conjunction with other anti-cancer agents are recent interesting therapeutic approaches that could give promising results.

  3. Endosome-to-Plasma Membrane Recycling of VEGFR2 Receptor Tyrosine Kinase Regulates Endothelial Function and Blood Vessel Formation.

    PubMed

    Jopling, Helen M; Odell, Adam F; Pellet-Many, Caroline; Latham, Antony M; Frankel, Paul; Sivaprasadarao, Asipu; Walker, John H; Zachary, Ian C; Ponnambalam, Sreenivasan

    2014-04-29

    Rab GTPases are implicated in endosome-to-plasma membrane recycling, but how such membrane traffic regulators control vascular endothelial growth factor receptor 2 (VEGFR2/KDR) dynamics and function are not well understood. Here, we evaluated two different recycling Rab GTPases, Rab4a and Rab11a, in regulating endothelial VEGFR2 trafficking and signalling with implications for endothelial cell migration, proliferation and angiogenesis. In primary endothelial cells, VEGFR2 displays co-localisation with Rab4a, but not Rab11a GTPase, on early endosomes. Expression of a guanosine diphosphate (GDP)-bound Rab4a S22N mutant caused increased VEGFR2 accumulation in endosomes. TfR and VEGFR2 exhibited differences in endosome-to-plasma membrane recycling in the presence of chloroquine. Depletion of Rab4a, but not Rab11a, levels stimulated VEGF-A-dependent intracellular signalling. However, depletion of either Rab4a or Rab11a levels inhibited VEGF-A-stimulated endothelial cell migration. Interestingly, depletion of Rab4a levels stimulated VEGF-A-regulated endothelial cell proliferation. Rab4a and Rab11a were also both required for endothelial tubulogenesis. Evaluation of a transgenic zebrafish model showed that both Rab4 and Rab11a are functionally required for blood vessel formation and animal viability. Rab-dependent endosome-to-plasma membrane recycling of VEGFR2 is important for intracellular signalling, cell migration and proliferation during angiogenesis.

  4. Biomarker analyses in REGARD gastric/GEJ carcinoma patients treated with VEGFR2-targeted antibody ramucirumab

    PubMed Central

    Fuchs, Charles S; Tabernero, Josep; Tomášek, Jiří; Chau, Ian; Melichar, Bohuslav; Safran, Howard; Tehfe, Mustapha A; Filip, Dumitru; Topuzov, Eldar; Schlittler, Luis; Udrea, Anghel Adrian; Campbell, William; Brincat, Stephen; Emig, Michael; Melemed, Symantha A; Hozak, Rebecca R; Ferry, David; Caldwell, C William; Ajani, Jaffer A

    2016-01-01

    Background: Angiogenesis inhibition is an important strategy for cancer treatment. Ramucirumab, a human IgG1 monoclonal antibody that targets VEGF receptor 2 (VEGFR2), inhibits VEGF-A, -C, -D binding and endothelial cell proliferation. To attempt to identify prognostic and predictive biomarkers, retrospective analyses were used to assess tumour (HER2, VEGFR2) and serum (VEGF-C and -D, and soluble (s) VEGFR1 and 3) biomarkers in phase 3 REGARD patients with metastatic gastric/gastroesophageal junction carcinoma. Methods: A total of 152 out of 355 (43%) patients randomised to ramucirumab or placebo had ⩾1 evaluable biomarker result using VEGFR2 immunohistochemistry or HER2, immunohistochemistry or FISH, of blinded baseline tumour tissue samples. Serum samples (32 patients, 9%) were assayed for VEGF-C and -D, and sVEGFR1 and 3. Results: None of the biomarkers tested were associated with ramucirumab efficacy at a level of statistical significance. High VEGFR2 endothelial expression was associated with a non-significant prognostic trend toward shorter progression-free survival (high vs low HR=1.65, 95% CI=0.84,3.23). Treatment with ramucirumab was associated with a trend toward improved survival in both high (HR=0.69, 95% CI=0.38, 1.22) and low (HR=0.73, 95% CI=0.42, 1.26) VEGFR2 subgroups. The benefit associated with ramucirumab did not appear to differ by tumoural HER2 expression. Conclusions: REGARD exploratory analyses did not identify a strong potentially predictive biomarker of ramucirumab efficacy; however, statistical power was limited. PMID:27623234

  5. Biomarker analyses in REGARD gastric/GEJ carcinoma patients treated with VEGFR2-targeted antibody ramucirumab.

    PubMed

    Fuchs, Charles S; Tabernero, Josep; Tomášek, Jiří; Chau, Ian; Melichar, Bohuslav; Safran, Howard; Tehfe, Mustapha A; Filip, Dumitru; Topuzov, Eldar; Schlittler, Luis; Udrea, Anghel Adrian; Campbell, William; Brincat, Stephen; Emig, Michael; Melemed, Symantha A; Hozak, Rebecca R; Ferry, David; Caldwell, C William; Ajani, Jaffer A

    2016-10-11

    Angiogenesis inhibition is an important strategy for cancer treatment. Ramucirumab, a human IgG1 monoclonal antibody that targets VEGF receptor 2 (VEGFR2), inhibits VEGF-A, -C, -D binding and endothelial cell proliferation. To attempt to identify prognostic and predictive biomarkers, retrospective analyses were used to assess tumour (HER2, VEGFR2) and serum (VEGF-C and -D, and soluble (s) VEGFR1 and 3) biomarkers in phase 3 REGARD patients with metastatic gastric/gastroesophageal junction carcinoma. A total of 152 out of 355 (43%) patients randomised to ramucirumab or placebo had ⩾1 evaluable biomarker result using VEGFR2 immunohistochemistry or HER2, immunohistochemistry or FISH, of blinded baseline tumour tissue samples. Serum samples (32 patients, 9%) were assayed for VEGF-C and -D, and sVEGFR1 and 3. None of the biomarkers tested were associated with ramucirumab efficacy at a level of statistical significance. High VEGFR2 endothelial expression was associated with a non-significant prognostic trend toward shorter progression-free survival (high vs low HR=1.65, 95% CI=0.84,3.23). Treatment with ramucirumab was associated with a trend toward improved survival in both high (HR=0.69, 95% CI=0.38, 1.22) and low (HR=0.73, 95% CI=0.42, 1.26) VEGFR2 subgroups. The benefit associated with ramucirumab did not appear to differ by tumoural HER2 expression. REGARD exploratory analyses did not identify a strong potentially predictive biomarker of ramucirumab efficacy; however, statistical power was limited.

  6. Inhibition of VEGFR2 Activation and Its Downstream Signaling to ERK1/2 and Calcium by Thrombospondin-1 (TSP1): In silico Investigation

    PubMed Central

    Bazzazi, Hojjat; Isenberg, Jeffery S.; Popel, Aleksander S.

    2017-01-01

    VEGF signaling through VEGFR2 is a central regulator of the angiogenic response. Inhibition of VEGF signaling by the stress-induced matricellular protein TSP1 plays a role in modulating the angiogenic response to VEGF in both health and disease. TSP1 binding to CD47 inhibits VEGFR2 activation. The full implications of this inhibitory interaction are unknown. We developed a detailed rule-based computational model to inquire if TSP1-CD47 signaling through VEGF had downstream effects upon ERK1/2 and calcium. Our Simulations suggest that enhanced degradation of VEGFR2 initiated by the binding of TSP1 to CD47 is sufficient to explain the inhibition of VEGFR2 phosphorylation, calcium elevation, and ERK1/2 activation downstream of VEGF. A complementary mechanism involving the recruitment of phosphatases to the VEGFR2 complex with consequent increase in the rate of receptor dephosphorylation may augment the inhibition of the VEGF signal. The model was then utilized to simulate the effect of inhibiting external TSP1 or the depletion of CD47 as potential therapeutic strategies in restoring VEGF signaling. Results suggest that depleting CD47 is a more efficient strategy in inhibiting the effects of TSP1/CD47 on VEGF signaling. Our results highlight the utility of in silico investigations in elucidating and clarifying molecular mechanisms at the intersection of TSP1 and VEGF biology and in differentiating between competing pro-angiogenic therapeutic strategies relevant to peripheral arterial disease (PAD) and wound healing. PMID:28220078

  7. Gremlin inhibits UV-induced skin cell damages via activating VEGFR2-Nrf2 signaling.

    PubMed

    Ji, Chao; Huang, Jin-Wen; Xu, Qiu-Yun; Zhang, Jing; Lin, Meng-Ting; Tu, Ying; He, Li; Bi, Zhi-Gang; Cheng, Bo

    2016-12-20

    Ultra Violet (UV) radiation induces reactive oxygen species (ROS) production, DNA oxidation and single strand breaks (SSBs), which will eventually lead to skin cell damages or even skin cancer. Here, we tested the potential activity of gremlin, a novel vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) agonist, against UV-induced skin cell damages. We show that gremlin activated VEGFR2 and significantly inhibited UV-induced death and apoptosis of skin keratinocytes and fibroblasts. Pharmacological inhibition or shRNA-mediated knockdown of VEGFR2 almost abolished gremlin-mediated cytoprotection against UV in the skin cells. Further studies showed that gremlin activated VEGFR2 downstream NF-E2-related factor 2 (Nrf2) signaling, which appeared required for subsequent skin cell protection. Nrf2 shRNA knockdown or S40T dominant negative mutation largely inhibited gremlin-mediated skin cell protection against UV. At last, we show that gremlin dramatically inhibited UV-induced ROS production and DNA SSB formation in skin keratinocytes and fibroblasts. We conclude that gremlin protects skin cells from UV damages via activating VEGFR2-Nrf2 signaling. Gremlin could be further tested as a novel anti-UV skin protectant.

  8. Stabiliztin of VEGFR2 Signaling by Cerebral Cavernous Malformation 3 is Critical for Vascular Development

    SciTech Connect

    Y He; H Zhang; L Yu; M Gunel; T Boggon; H Chen; W Min

    2011-12-31

    Cerebral cavernous malformations (CCMs) are human vascular malformations caused by mutations in three genes of unknown function: CCM1, CCM2, and CCM3. CCM3, also known as PDCD10 (programmed cell death 10), was initially identified as a messenger RNA whose abundance was induced by apoptotic stimuli in vitro. However, the in vivo function of CCM3 has not been determined. Here, we describe mice with a deletion of the CCM3 gene either ubiquitously or specifically in the vascular endothelium, smooth muscle cells, or neurons. Mice with global or endothelial cell-specific deletion of CCM3 exhibited defects in embryonic angiogenesis and died at an early embryonic stage. CCM3 deletion reduced vascular endothelial growth factor receptor 2 (VEGFR2) signaling in embryos and endothelial cells. In response to VEGF stimulation, CCM3 was recruited to and stabilized VEGFR2, and the carboxyl-terminal domain of CCM3 was required for the stabilization of VEGFR2. Indeed, the CCM3 mutants found in human patients lacking the carboxyl-terminal domain were labile and were unable to stabilize and activate VEGFR2. These results demonstrate that CCM3 promotes VEGFR2 signaling during vascular development.

  9. Gremlin inhibits UV-induced skin cell damages via activating VEGFR2-Nrf2 signaling

    PubMed Central

    Xu, Qiu-yun; Zhang, Jing; Lin, Meng-ting; Tu, Ying; He, Li; Bi, Zhi-gang; Cheng, Bo

    2016-01-01

    Ultra Violet (UV) radiation induces reactive oxygen species (ROS) production, DNA oxidation and single strand breaks (SSBs), which will eventually lead to skin cell damages or even skin cancer. Here, we tested the potential activity of gremlin, a novel vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) agonist, against UV-induced skin cell damages. We show that gremlin activated VEGFR2 and significantly inhibited UV-induced death and apoptosis of skin keratinocytes and fibroblasts. Pharmacological inhibition or shRNA-mediated knockdown of VEGFR2 almost abolished gremlin-mediated cytoprotection against UV in the skin cells. Further studies showed that gremlin activated VEGFR2 downstream NF-E2-related factor 2 (Nrf2) signaling, which appeared required for subsequent skin cell protection. Nrf2 shRNA knockdown or S40T dominant negative mutation largely inhibited gremlin-mediated skin cell protection against UV. At last, we show that gremlin dramatically inhibited UV-induced ROS production and DNA SSB formation in skin keratinocytes and fibroblasts. We conclude that gremlin protects skin cells from UV damages via activating VEGFR2-Nrf2 signaling. Gremlin could be further tested as a novel anti-UV skin protectant. PMID:27713170

  10. Stabilization of VEGFR2 signaling by cerebral cavernous malformation 3 is critical for vascular development.

    PubMed

    He, Yun; Zhang, Haifeng; Yu, Luyang; Gunel, Murat; Boggon, Titus J; Chen, Hong; Min, Wang

    2010-04-06

    Cerebral cavernous malformations (CCMs) are human vascular malformations caused by mutations in three genes of unknown function: CCM1, CCM2, and CCM3. CCM3, also known as PDCD10 (programmed cell death 10), was initially identified as a messenger RNA whose abundance was induced by apoptotic stimuli in vitro. However, the in vivo function of CCM3 has not been determined. Here, we describe mice with a deletion of the CCM3 gene either ubiquitously or specifically in the vascular endothelium, smooth muscle cells, or neurons. Mice with global or endothelial cell-specific deletion of CCM3 exhibited defects in embryonic angiogenesis and died at an early embryonic stage. CCM3 deletion reduced vascular endothelial growth factor receptor 2 (VEGFR2) signaling in embryos and endothelial cells. In response to VEGF stimulation, CCM3 was recruited to and stabilized VEGFR2, and the carboxyl-terminal domain of CCM3 was required for the stabilization of VEGFR2. Indeed, the CCM3 mutants found in human patients lacking the carboxyl-terminal domain were labile and were unable to stabilize and activate VEGFR2. These results demonstrate that CCM3 promotes VEGFR2 signaling during vascular development.

  11. Rhamnazin, a novel inhibitor of VEGFR2 signaling with potent antiangiogenic activity and antitumor efficacy

    SciTech Connect

    Yu, Yao; Cai, Wei; Pei, Chong-gang; Shao, Yi

    2015-03-20

    Anti-angiogenesis targeting vascular endothelial growth factor receptor 2 (VEGFR2) has emerged as an important tool for cancer therapy. The identification of new drugs from natural products has a long and successful history. In this study, we described a novel VEGFR2 inhibitor, rhamnazin, which inhibits tumor angiogenesis and growth. Rhamnazin significantly inhibited proliferation, migration and tube formation of human umbilical vascular endothelial cells (HUVECs) in vitro as well as inhibited sprouts formation of rat aorta ring. In addition, it inhibited vascular endothelial growth factor (VEGF)-induced phosphorylation of VEGFR2 and its downstream signaling regulator in HUVECs. Moreover, rhamnazin could directly inhibit proliferation of breast cancer cells MDA-MB-231 in vitro and in vivo. Oral administration of rhamnazin at a dose of 200 mg/kg/day could markedly inhibited human tumor xenograft growth and decreased microvessel densities (MVD) in tumor sections. Taken together, these preclinical evaluations suggest that rhamnazin inhibits angiogenesis and may be a promising anticancer drug candidate. - Highlights: • Rhamnazin inhibits the response of HUVECs to VEGF in vitro. • Rhamnazin inhibits VEGFR2 kinase activity and its downstream signaling. • Rhamnazin prevents the growth of MDA-MB-231 tumor and reduces micro-vessel density in vivo.

  12. The innate immune response in fetal lung mesenchymal cells targets VEGFR2 expression and activity.

    PubMed

    Medal, Rachel M; Im, Amanda M; Yamamoto, Yasutoshi; Lakhdari, Omar; Blackwell, Timothy S; Hoffman, Hal M; Sahoo, Debashis; Prince, Lawrence S

    2017-06-01

    In preterm infants, soluble inflammatory mediators target lung mesenchymal cells, disrupting airway and alveolar morphogenesis. However, how mesenchymal cells respond directly to microbial stimuli remains poorly characterized. Our objective was to measure the genome-wide innate immune response in fetal lung mesenchymal cells exposed to the bacterial endotoxin lipopolysaccharide (LPS). With the use of Affymetrix MoGene 1.0st arrays, we showed that LPS induced expression of unique innate immune transcripts heavily weighted toward CC and CXC family chemokines. The transcriptional response was different between cells from E11, E15, and E18 mouse lungs. In all cells tested, LPS inhibited expression of a small core group of genes including the VEGF receptor Vegfr2 Although best characterized in vascular endothelial populations, we demonstrated here that fetal mouse lung mesenchymal cells express Vegfr2 and respond to VEGF-A stimulation. In mesenchymal cells, VEGF-A increased cell migration, activated the ERK/AKT pathway, and promoted FOXO3A nuclear exclusion. With the use of an experimental coculture model of epithelial-mesenchymal interactions, we also showed that VEGFR2 inhibition prevented formation of three-dimensional structures. Both LPS and tyrosine kinase inhibition reduced three-dimensional structure formation. Our data suggest a novel mechanism for inflammation-mediated defects in lung development involving reduced VEGF signaling in lung mesenchyme. Copyright © 2017 the American Physiological Society.

  13. Therapeutic potential of pro-angiogenic BPC157 is associated with VEGFR2 activation and up-regulation.

    PubMed

    Hsieh, Ming-Jer; Liu, Hsien-Ta; Wang, Chao-Nin; Huang, Hsiu-Yun; Lin, Yuling; Ko, Yu-Shien; Wang, Jong-Shyan; Chang, Vincent Hung-Shu; Pang, Jong-Hwei S

    2017-03-01

    BPC 157, a pentadecapeptide with extensive healing effects, has recently been suggested to contribute to angiogenesis. However, the underlying mechanism is not yet clear. The present study aimed to explore the potential therapeutic effect and pro-angiogenic mechanism of BPC 157. As demonstrated by the chick chorioallantoic membrane (CAM) assay and endothelial tube formation assay, BPC 157 could increase the vessel density both in vivo and in vitro, respectively. BPC 157 could also accelerate the recovery of blood flow in the ischemic muscle of the rat hind limb as detected by laser Doppler scanning, indicating the promotion of angiogenesis. Histological analysis of the hind limb muscle confirmed the increased number of vessels and the enhanced vascular expression of vascular endothelial growth factor receptor 2 (VEGFR2) in rat with BPC 157 treatment. In vitro study using human vascular endothelial cells further confirmed the increased mRNA and protein expressions of VEGFR2 but not VEGF-A by BPC 157. In addition, BPC 157 could promote VEGFR2 internalization in vascular endothelial cells which was blocked in the presence of dynasore, an inhibitor of endocytosis. BPC 157 time dependently activated the VEGFR2-Akt-eNOS signaling pathway which could also be suppressed by dynasore. The increase of endothelial tube formation induced by BPC 157 was also inhibited by dynasore. This study demonstrates the pro-angiogenic effects of BPC 157 that is associated with the increased expression, internalization of VEGFR2, and the activation of VEGFR2-Akt-eNOS signaling pathway. BPC 157 promotes angiogenesis in CAM assay and tube formation assay. BPC 157 accelerates the blood flow recovery and vessel number in rats with hind limb ischemia. BPC 157 up-regulates VEGFR2 expression in rats with hind limb ischemia and endothelial cell culture. BPC 157 promotes VEGFR2 internalization in association with VEGFR2-Akt-eNOS activation.

  14. VEGFR2 but not VEGFR3 governs integrity and remodeling of thyroid angiofollicular unit in normal state and during goitrogenesis.

    PubMed

    Jang, Jeon Yeob; Choi, Sung Yong; Park, Intae; Park, Do Young; Choe, Kibaek; Kim, Pilhan; Kim, Young Keum; Lee, Byung-Joo; Hirashima, Masanori; Kubota, Yoshiaki; Park, Jeong-Won; Cheng, Sheue-Yann; Nagy, Andras; Park, Young Joo; Alitalo, Kari; Shong, Minho; Koh, Gou Young

    2017-06-01

    Thyroid gland vasculature has a distinguishable characteristic of endothelial fenestrae, a critical component for proper molecular transport. However, the signaling pathway that critically governs the maintenance of thyroid vascular integrity, including endothelial fenestrae, is poorly understood. Here, we found profound and distinct expression of follicular epithelial VEGF-A and vascular VEGFR2 that were precisely regulated by circulating thyrotropin, while there were no meaningful expression of angiopoietin-Tie2 system in the thyroid gland. Our genetic depletion experiments revealed that VEGFR2, but not VEGFR3, is indispensable for maintenance of thyroid vascular integrity. Notably, blockade of VEGF-A or VEGFR2 not only abrogated vascular remodeling but also inhibited follicular hypertrophy, which led to the reduction of thyroid weights during goitrogenesis. Importantly, VEGFR2 blockade alone was sufficient to cause a reduction of endothelial fenestrae with decreases in thyrotropin-responsive genes in goitrogen-fed thyroids. Collectively, these findings establish follicular VEGF-A-vascular VEGFR2 axis as a main regulator for thyrotropin-dependent thyroid angiofollicular remodeling and goitrogenesis. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  15. In vitro and in vivo antiangiogenic activity of desacetylvinblastine monohydrazide through inhibition of VEGFR2 and Axl pathways

    PubMed Central

    Lei, Xueping; Chen, Minfeng; Nie, Qiulin; Hu, Jianyang; Zhuo, Zhenjian; Yiu, Anita; Chen, Heru; Xu, Nanhui; Huang, Maohua; Ye, Kaihe; Bai, Liangliang; Ye, Wencai; Zhang, Dongmei

    2016-01-01

    Tumor angiogenic process is regulated by multiple proangiogenic pathways, such as vascular endothelial growth factor receptor 2 (VEGFR2) and Axl receptor tyrosine kinase (Axl). Axl is one of many important factors involved in anti-VEGF resistance. Inhibition of VEGF/VEGFR2 signaling pathway alone fails to block tumor neovascularization. Therefore, discovery of novel agents targeting multiple angiogenesis pathways is in demand. Desacetylvinblastine monohydrazide (DAVLBH), a derivative of vinblastine (VLB), has been reported exhibit an anticancer activity via its cytotoxic effect. However, little attention has been paid to the antiangiogenic properties of DAVLBH. Here, we firstly reported that DAVLBH exerted a more potent antiangiogenic effect than VLB in vitro and in vivo, which was associated with inactivation of VEGF/VEGFR2 and Gas6/Axl signaling pathways. We found that DAVLBH inhibited VEGF- and Gas6-induced HUVECs proliferation, migration, tube formation and vessel sprouts formation in vitro and ex vivo. It significantly inhibited in vivo tumor angiogenesis and tumor growth in HeLa xenografts. It also inhibited Gas6-induced pericytes recruitment to endothelial tubes accompanied with a decrease in expression and activation of Axl. Besides, it could block the compensatory up-regulating expression and activation of Axl in response to bevacizumab treatment in HUVECs. Taken together, our results suggest that DAVLBH potently inhibits angiogenesis-mediated tumor growth through blockage of the activation of VEGF/VEGFR2 and Gas6/Axl pathways and it might serve as a promising antiangiogenic agent for the cancer therapy. PMID:27186435

  16. The Antifungal Drug Itraconazole Inhibits Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) Glycosylation, Trafficking, and Signaling in Endothelial Cells*

    PubMed Central

    Nacev, Benjamin A.; Grassi, Paola; Dell, Anne; Haslam, Stuart M.; Liu, Jun O.

    2011-01-01

    Itraconazole is a safe and widely used antifungal drug that was recently found to possess potent antiangiogenic activity. Currently, there are four active clinical trials evaluating itraconazole as a cancer therapeutic. Tumor growth is dependent on angiogenesis, which is driven by the secretion of growth factors from the tumor itself. We report here that itraconazole significantly inhibited the binding of vascular endothelial growth factor (VEGF) to VEGF receptor 2 (VEGFR2) and that both VEGFR2 and an immediate downstream substrate, phospholipase C γ1, failed to become activated after VEGF stimulation. These effects were due to a defect in VEGFR2 trafficking, leading to a decrease in cell surface expression, and were associated with the accumulation of immature N-glycans on VEGFR2. Small molecule inducers of lysosomal cholesterol accumulation and mammalian target of rapamycin (mTOR) inhibition, two previously reported itraconazole activities, failed to recapitulate itraconazole's effects on VEGFR2 glycosylation and signaling. Likewise, glycosylation inhibitors did not alter cholesterol trafficking or inhibit mTOR. Repletion of cellular cholesterol levels, which was known to rescue the effects of itraconazole on mTOR and cholesterol trafficking, was also able to restore VEGFR2 glycosylation and signaling. This suggests that the new effects of itraconazole occur in parallel to those previously reported but are downstream of a common target. We also demonstrated that itraconazole globally reduced poly-N-acetyllactosamine and tetra-antennary complex N-glycans in endothelial cells and induced hypoglycosylation of the epidermal growth factor receptor in a renal cell carcinoma line, suggesting that itraconazole's effects extend beyond VEGFR2. PMID:22025615

  17. Xanthatin, a novel potent inhibitor of VEGFR2 signaling, inhibits angiogenesis and tumor growth in breast cancer cells.

    PubMed

    Yu, Yao; Yu, Jing; Pei, Chong Gang; Li, Yun Yan; Tu, Ping; Gao, Gui Ping; Shao, Yi

    2015-01-01

    Anti-angiogenesis targeting vascular endothelial growth factor receptor 2 (VEGFR2) has emerged as an important tool for cancer treatment. In this study, we described a novel VEGFR2 inhibitor, xanthatin, which inhibits tumor angiogenesis and growth. The biochemical profiles of xanthatin were investigated using kinase assay, migration assay, tube formation, Matrigel plug assay, western blot, immunofluorescence and human tumor xenograft model. Xanthatin significantly inhibited growth, migration and tube formation of human umbilical vascular endothelial cell as well as inhibited vascular endothelial growth factor (VEGF)-stimulated angiogenesis. In addition, it inhibited VEGF-induced phosphorylation of VEGFR2 and its downstream signaling regulator. Moreover, xanthatin directly inhibit proliferation of breast cancer cells MDA-MB-231. Oral administration of xanthatin could markedly inhibit human tumor xenograft growth and decreased microvessel densities (MVD) in tumor sections. Taken together, these preclinical evaluations suggest that xanthatin inhibits angiogenesis and may be a promising anticancer drug candidate.

  18. TBX1 Represses Vegfr2 Gene Expression and Enhances the Cardiac Fate of VEGFR2+ Cells

    PubMed Central

    Lania, Gabriella; Ferrentino, Rosa; Baldini, Antonio

    2015-01-01

    The T-box transcription factor TBX1 has critical roles in maintaining proliferation and inhibiting differentiation of cardiac progenitor cells of the second heart field (SHF). Haploinsufficiency of the gene that encodes it is a cause of congenital heart disease. Here, we developed an embryonic stem (ES) cell-based model in which Tbx1 expression can be modulated by tetracycline. Using this model, we found that TBX1 down regulates the expression of VEGFR2, and we confirmed this finding in vivo during embryonic development. In addition, we found a Vegfr2 domain of expression, not previously described, in the posterior SHF and this expression is extended by loss of Tbx1. VEGFR2 has been previously described as a marker of a subpopulation of cardiac progenitors. Clonal analysis of ES-derived VEGFR2+ cells indicated that 12.5% of clones expressed three markers of cardiac lineage (cardiomyocyte, smooth muscle and endothelium). However, a pulse of Tbx1 expression was sufficient to increase the percentage to 20.8%. In addition, the percentage of clones expressing markers of multiple cardiac lineages increased from 41.6% to 79.1% after Tbx1 pulse. These results suggest that TBX1 plays a role in maintaining a progenitor state in VEGFR2+ cells. PMID:26382615

  19. An endothelial TLR4-VEGFR2 pathway mediates lung protection against oxidant-induced injury.

    PubMed

    Takyar, Seyedtaghi; Zhang, Yi; Haslip, Maria; Jin, Lei; Shan, Peiying; Zhang, Xuchen; Lee, Patty J

    2016-03-01

    TLR4 deficiency causes hypersusceptibility to oxidant-induced injury. We investigated the role of TLR4 in lung protection, using used bone marrow chimeras; cell-specific transgenic modeling; and lentiviral delivery in vivo to knock down or express TLR4 in various lung compartments; and lung-specific VEGF transgenic mice to investigate the effect of TLR4 on VEGF-mediated protection. C57/BL6 mice were exposed to 100% oxygen in an enclosed chamber and assessed for survival and lung injury. Primary endothelial cells were stimulated with recombinant VEGF and exposed to hyperoxia or hydrogen peroxide. Endothelium-specific expression of human TLR4 (as opposed to its expression in epithelium or immune cells) increased the survival of TLR4-deficent mice in hyperoxia by 24 h and decreased LDH release and lung cell apoptosis after 72 h of exposure by 30%. TLR4 expression was necessary and sufficient for the protective effect of VEGF in the lungs and in primary endothelial cells in culture. TLR4 knockdown inhibited VEGF signaling through VEGF receptor 2 (VEGFR2), Akt, and ERK pathways in lungs and primary endothelial cells and decreased the availability of VEGFR2 at the cell surface. These findings demonstrate a novel mechanism through which TLR4, an innate pattern receptor, interacts with an endothelial survival pathway.

  20. An endothelial TLR4-VEGFR2 pathway mediates lung protection against oxidant-induced injury

    PubMed Central

    Takyar, Seyedtaghi; Zhang, Yi; Haslip, Maria; Jin, Lei; Shan, Peiying; Zhang, Xuchen; Lee, Patty J.

    2015-01-01

    TLR4 deficiency causes hypersusceptibility to oxidant-induced injury. We investigated the role of TLR4 in lung protection, using used bone marrow chimeras; cell-specific transgenic modeling; and lentiviral delivery in vivo to knock down or express TLR4 in various lung compartments; and lung-specific VEGF transgenic mice to investigate the effect of TLR4 on VEGF-mediated protection. C57/BL6 mice were exposed to 100% oxygen in an enclosed chamber and assessed for survival and lung injury. Primary endothelial cells were stimulated with recombinant VEGF and exposed to hyperoxia or hydrogen peroxide. Endothelium-specific expression of human TLR4 (as opposed to its expression in epithelium or immune cells) increased the survival of TLR4-deficent mice in hyperoxia by 24 h and decreased LDH release and lung cell apoptosis after 72 h of exposure by 30%. TLR4 expression was necessary and sufficient for the protective effect of VEGF in the lungs and in primary endothelial cells in culture. TLR4 knockdown inhibited VEGF signaling through VEGF receptor 2 (VEGFR2), Akt, and ERK pathways in lungs and primary endothelial cells and decreased the availability of VEGFR2 at the cell surface. These findings demonstrate a novel mechanism through which TLR4, an innate pattern receptor, interacts with an endothelial survival pathway.—Takyar, S., Zhang, Y., Haslip, M., Jin L., Shan P., Zhang, X., Lee, P. J. An endothelial TLR4-VEGFR2 pathway mediates lung protection against oxidant-induced injury. PMID:26655705

  1. Mutant p53 cooperates with the SWI/SNF chromatin remodeling complex to regulate VEGFR2 in breast cancer cells.

    PubMed

    Pfister, Neil T; Fomin, Vitalay; Regunath, Kausik; Zhou, Jeffrey Y; Zhou, Wen; Silwal-Pandit, Laxmi; Freed-Pastor, William A; Laptenko, Oleg; Neo, Suat Peng; Bargonetti, Jill; Hoque, Mainul; Tian, Bin; Gunaratne, Jayantha; Engebraaten, Olav; Manley, James L; Børresen-Dale, Anne-Lise; Neilsen, Paul M; Prives, Carol

    2015-06-15

    Mutant p53 impacts the expression of numerous genes at the level of transcription to mediate oncogenesis. We identified vascular endothelial growth factor receptor 2 (VEGFR2), the primary functional VEGF receptor that mediates endothelial cell vascularization, as a mutant p53 transcriptional target in multiple breast cancer cell lines. Up-regulation of VEGFR2 mediates the role of mutant p53 in increasing cellular growth in two-dimensional (2D) and three-dimensional (3D) culture conditions. Mutant p53 binds near the VEGFR2 promoter transcriptional start site and plays a role in maintaining an open conformation at that location. Relatedly, mutant p53 interacts with the SWI/SNF complex, which is required for remodeling the VEGFR2 promoter. By both querying individual genes regulated by mutant p53 and performing RNA sequencing, the results indicate that >40% of all mutant p53-regulated gene expression is mediated by SWI/SNF. We surmise that mutant p53 impacts transcription of VEGFR2 as well as myriad other genes by promoter remodeling through interaction with and likely regulation of the SWI/SNF chromatin remodeling complex. Therefore, not only might mutant p53-expressing tumors be susceptible to anti VEGF therapies, impacting SWI/SNF tumor suppressor function in mutant p53 tumors may also have therapeutic potential.

  2. Mutant p53 cooperates with the SWI/SNF chromatin remodeling complex to regulate VEGFR2 in breast cancer cells

    PubMed Central

    Pfister, Neil T.; Fomin, Vitalay; Regunath, Kausik; Zhou, Jeffrey Y.; Zhou, Wen; Silwal-Pandit, Laxmi; Freed-Pastor, William A.; Laptenko, Oleg; Neo, Suat Peng; Bargonetti, Jill; Hoque, Mainul; Tian, Bin; Gunaratne, Jayantha; Engebraaten, Olav; Manley, James L.; Børresen-Dale, Anne-Lise; Neilsen, Paul M.; Prives, Carol

    2015-01-01

    Mutant p53 impacts the expression of numerous genes at the level of transcription to mediate oncogenesis. We identified vascular endothelial growth factor receptor 2 (VEGFR2), the primary functional VEGF receptor that mediates endothelial cell vascularization, as a mutant p53 transcriptional target in multiple breast cancer cell lines. Up-regulation of VEGFR2 mediates the role of mutant p53 in increasing cellular growth in two-dimensional (2D) and three-dimensional (3D) culture conditions. Mutant p53 binds near the VEGFR2 promoter transcriptional start site and plays a role in maintaining an open conformation at that location. Relatedly, mutant p53 interacts with the SWI/SNF complex, which is required for remodeling the VEGFR2 promoter. By both querying individual genes regulated by mutant p53 and performing RNA sequencing, the results indicate that >40% of all mutant p53-regulated gene expression is mediated by SWI/SNF. We surmise that mutant p53 impacts transcription of VEGFR2 as well as myriad other genes by promoter remodeling through interaction with and likely regulation of the SWI/SNF chromatin remodeling complex. Therefore, not only might mutant p53-expressing tumors be susceptible to anti VEGF therapies, impacting SWI/SNF tumor suppressor function in mutant p53 tumors may also have therapeutic potential. PMID:26080815

  3. Computational Model of Gab1/2-Dependent VEGFR2 Pathway to Akt Activation

    PubMed Central

    Tan, Wan Hua; Popel, Aleksander S.; Mac Gabhann, Feilim

    2013-01-01

    Vascular endothelial growth factor (VEGF) signal transduction is central to angiogenesis in development and in pathological conditions such as cancer, retinopathy and ischemic diseases. However, no detailed mass-action models of VEGF receptor signaling have been developed. We constructed and validated the first computational model of VEGFR2 trafficking and signaling, to study the opposing roles of Gab1 and Gab2 in regulation of Akt phosphorylation in VEGF-stimulated endothelial cells. Trafficking parameters were optimized against 5 previously published in vitro experiments, and the model was validated against six independent published datasets. The model showed agreement at several key nodes, involving scaffolding proteins Gab1, Gab2 and their complexes with Shp2. VEGFR2 recruitment of Gab1 is greater in magnitude, slower, and more sustained than that of Gab2. As Gab2 binds VEGFR2 complexes more transiently than Gab1, VEGFR2 complexes can recycle and continue to participate in other signaling pathways. Correspondingly, the simulation results show a log-linear relationship between a decrease in Akt phosphorylation and Gab1 knockdown while a linear relationship was observed between an increase in Akt phosphorylation and Gab2 knockdown. Global sensitivity analysis demonstrated the importance of initial-concentration ratios of antagonistic molecular species (Gab1/Gab2 and PI3K/Shp2) in determining Akt phosphorylation profiles. It also showed that kinetic parameters responsible for transient Gab2 binding affect the system at specific nodes. This model can be expanded to study multiple signaling contexts and receptor crosstalk and can form a basis for investigation of therapeutic approaches, such as tyrosine kinase inhibitors (TKIs), overexpression of key signaling proteins or knockdown experiments. PMID:23805312

  4. BR55: a lipopeptide-based VEGFR2-targeted ultrasound contrast agent for molecular imaging of angiogenesis.

    PubMed

    Pochon, Sibylle; Tardy, Isabelle; Bussat, Philippe; Bettinger, Thierry; Brochot, Jean; von Wronski, Mathew; Passantino, Lisa; Schneider, Michel

    2010-02-01

    BR55, an ultrasound contrast agent functionalized with a heterodimer peptide targeting the vascular endothelial growth factor receptor 2 (VEGFR2), was evaluated in vitro and in vivo, demonstrating its potential for specific tumor detection. The targeted contrast agent was prepared by incorporation of a biospecific lipopeptide into the microbubble membrane. Experiments were performed in vitro to demonstrate the binding capacities of BR55 microbubbles on immobilized receptor proteins and on various endothelial or transfected cells expressing VEGFR2. The performance of BR55 microbubbles was compared with that of streptavidin-conjugated microbubbles targeted to the same receptor by coupling them to a biotinylated antibody. The specificity of BR55 binding to human and mouse endothelial cells was determined in competition experiments with the free lipopeptide, vascular endothelial growth factor (VEGF), or a VEGFR2-specific antibody. Molecular ultrasound imaging of VEGFR2 was performed in an orthotopic breast tumor model in rats using a nondestructive, contrast-specific imaging mode. BR55 was shown to bind specifically to the immobilized recombinant VEGFR2 under flow (dynamic conditions). BR55 accumulation on the target over time was similar to that of microbubbles bearing a specific antibody. BR55 avidly bound to cells expressing VEGFR2, and the pattern of microbubble distribution was correlated with the pattern of receptor expression determined by immunocytochemistry. The binding of targeted microbubbles on cells was competed off by an excess of free lipopeptide, the natural ligand (VEGF) and by a VEGFR2-specific antibody (P < 0.001). Although selected for the human receptor, the VEGFR2-binding lipopeptide was also shown to recognize the rodent receptor. Tumor perfusion was assessed during the vascular phase of BR55, and then the malignant lesion was highlighted by specific accumulation of the targeted microbubbles on tumoral endothelium. The presence of VEGFR2 was

  5. Neuropilin-1 functions as a VEGFR2 co-receptor to guide developmental angiogenesis independent of ligand binding.

    PubMed

    Gelfand, Maria V; Hagan, Nellwyn; Tata, Aleksandra; Oh, Won-Jong; Lacoste, Baptiste; Kang, Kyu-Tae; Kopycinska, Justyna; Bischoff, Joyce; Wang, Jia-Huai; Gu, Chenghua

    2014-09-22

    During development, tissue repair, and tumor growth, most blood vessel networks are generated through angiogenesis. Vascular endothelial growth factor (VEGF) is a key regulator of this process and currently both VEGF and its receptors, VEGFR1, VEGFR2, and Neuropilin1 (NRP1), are targeted in therapeutic strategies for vascular disease and cancer. NRP1 is essential for vascular morphogenesis, but how NRP1 functions to guide vascular development has not been completely elucidated. In this study, we generated a mouse line harboring a point mutation in the endogenous Nrp1 locus that selectively abolishes VEGF-NRP1 binding (Nrp1(VEGF-)). Nrp1(VEGF-) mutants survive to adulthood with normal vasculature revealing that NRP1 functions independent of VEGF-NRP1 binding during developmental angiogenesis. Moreover, we found that Nrp1-deficient vessels have reduced VEGFR2 surface expression in vivo demonstrating that NRP1 regulates its co-receptor, VEGFR2. Given the resources invested in NRP1-targeted anti-angiogenesis therapies, our results will be integral for developing strategies to re-build vasculature in disease.

  6. The VEGF rise in blood of bevacizumab patients is not based on tumor escape but a host-blockade of VEGF clearance

    PubMed Central

    Alidzanovic, Lejla; Starlinger, Patrick; Schauer, Dominic; Maier, Thomas; Feldman, Alexandra; Buchberger, Elisabeth; Stift, Judith; Koeck, Ulrike; Pop, Lorand; Gruenberger, Birgit; Gruenberger, Thomas; Brostjan, Christine

    2016-01-01

    Vascular endothelial growth factor (VEGF) has become a major target in cancer treatment as it promotes tumor angiogenesis. Therapy with anti-VEGF antibody bevacizumab reportedly induces high levels of circulating VEGF which may potentially contribute to resistance. Based on animal or computational models, mechanisms of VEGF induction by bevacizumab have been proposed but not verified in the clinical setting. Hence, we evaluated sixty patients with colorectal cancer metastases for changes in plasma VEGF during neoadjuvant/conversion and adjuvant chemotherapy with or without bevacizumab. VEGF expression was assessed in tissue sections of liver metastases. The VEGF source was investigated with in vitro cultures of tumor, endothelial cells, fibroblasts and platelets, and potential protein stabilization due to anti-VEGF therapy was addressed. A VEGF rise was observed in blood of bevacizumab patients but not in chemotherapy controls, and VEGF was found to be largely complexed by the antibody. A comparable VEGF increase occurred in the presence (neoadjuvant) and absence of the tumor (adjuvant). Accordingly, VEGF expression in tumor tissue was not determined by bevacizumab treatment. Investigations with isolated cell types did not reveal VEGF production in response to bevacizumab. However, antibody addition to endothelial cultures led to a dose-dependent blockade of VEGF internalization and hence stabilized VEGF in the supernatant. In conclusion, the VEGF rise in cancer patients treated with bevacizumab is not originating from the tumor. The accumulation of primarily host-derived VEGF in circulation can be explained by antibody interference with receptor-mediated endocytosis and protein degradation. Thus, the VEGF increase in response to bevacizumab therapy should not be regarded as a tumor escape mechanism. PMID:27527865

  7. The CUL3-SPOP-DAXX axis is a novel regulator of VEGFR2 expression in vascular endothelial cells

    PubMed Central

    Sakaue, Tomohisa; Sakakibara, Iori; Uesugi, Takahiro; Fujisaki, Ayako; Nakashiro, Koh-ichi; Hamakawa, Hiroyuki; Kubota, Eiji; Joh, Takashi; Imai, Yu-ki; Izutani, Hironori; Higashiyama, Shigeki

    2017-01-01

    Vascular endothelial cell growth factor receptor 2 (VEGFR2) is an essential receptor for the homeostasis of endothelial cells. In this study, we showed that NEDD8-conjugated Cullin3 (CUL3)-based ubiquitin E3 (UbE3) ligase plays a crucial role in VEGFR2 mRNA expression. Human umbilical vein endothelial cells treated with MLN4924, an inhibitor of NEDD8-activating enzyme, or with CUL3 siRNA drastically lost their response to VEGF due to the intense decrease in VEGFR2 expression. Moreover, speckle-type POZ protein (SPOP) and death-domain associated protein (DAXX) were involved in the CUL3 UbE3 ligase complex as a substrate adaptor and a substrate, respectively. Knockdown of SPOP and CUL3 led to the upregulation of DAXX protein and downregulation of VEGFR2 levels. These levels were inversely correlated with one another. In addition, simultaneous knockdown of SPOP and DAXX completely reversed the downregulation of VEGFR2 levels. Moreover, the CUL3-SPOP-DAXX axis had the same effects on NOTCH1, DLL4 and NRP1 expression. Taken together, these findings suggest that the CUL3-SPOP-DAXX axis plays a very important role in endothelial cell function by targeting key angiogenic regulators. PMID:28216678

  8. Inhibition of solid tumor growth by gene transfer of VEGF receptor-1 mutants.

    PubMed

    Heidenreich, Regina; Machein, Marcia; Nicolaus, Anke; Hilbig, Andreas; Wild, Carola; Clauss, Matthias; Plate, Karl H; Breier, Georg

    2004-09-01

    Vascular endothelial growth factor (VEGF) and the high-affinity VEGF receptor Flk-1/KDR (VEGFR-2) are key regulators of tumor angiogenesis. Strategies to block VEGF/VEGFR-2 signaling were successfully used to inhibit experimental tumor growth and indicated that VEGFR-2 is the main signaling VEGF receptor in proliferating tumor endothelium. Here, we investigated the role of the VEGF receptor-1 (VEGFR-1/Flt-1) in the vascularization of 2 different experimental tumors in vivo. VEGFR-1 mutants were generated that lack the intracellular tyrosine kinase domain. Retrovirus-mediated gene transfer of the VEGFR-1 mutants led to a strong reduction of tumor growth and angiogenesis in xenografted C6 glioma and in syngeneic BFS-1 fibrosarcoma. Histological analysis of the inhibited fibrosarcoma revealed reduced vascular density, decreased tumor cell proliferation as well as increased tumor cell apoptosis and the formation of necrosis. The retroviral gene transfer of the full length VEGFR-1 also caused a significant reduction of tumor growth in both models. The inhibitory effects of the VEGFR-1 mutants and the full length VEGFR-1 in BFS-1 fibrosarcoma were mediated through host tumor endothelial cells because the BFS-1 fibrosarcoma cells were not infected by the retrovirus. The formation of heterodimers between VEGFR-2 and full length or truncated VEGFR-1 was observed in vitro and might contribute to the growth inhibitory effect by modulating distinct signal transduction pathways. The results of our study underline the central role of the VEGF/VEGFR-1 signaling system in tumor angiogenesis and demonstrate that VEGFR-1 can serve as a target for anti-angiogenic gene therapy.

  9. Evolution of a New Class of VEGFR-2 Inhibitors from Scaffold Morphing and Redesign

    PubMed Central

    2016-01-01

    Anti-VEGF therapy is a clinically validated treatment for age-related macular degeneration (AMD). We have recently reported the discovery of oral VEGFR-2 inhibitors that are selectively distributed to the ocular tissues. Herein we report a further development of those compounds and in particular the validation of the hypothesis that aminoheterocycles such as aminoisoxazoles and aminopyrazoles could also function as effective “hinge” binding moieties leading to a new class of KDR (kinase insert domain containing receptor) inhibitors. PMID:27096042

  10. Cuprous oxide nanoparticles inhibit angiogenesis via down regulation of VEGFR2 expression

    NASA Astrophysics Data System (ADS)

    Song, Hongyuan; Wang, Wenbo; Zhao, Ping; Qi, Zhongtian; Zhao, Shihong

    2014-02-01

    Angiogenesis is a process that forms new blood capillaries from existing vessels, which is of great physiological and pathological significance. Although recent studies provide evidence that cuprous oxide nanoparticles (CO-NPs) may have biomedical potential, the mechanisms of CO-NPs in angiogenesis have not been investigated to date. We have studied the anti-angiogenic properties of CO-NPs on primary human umbilical vein endothelial cells (HUVECs). We found that CO-NPs were able to induce cell morphology changes and suppress cell proliferation, migration and tube formation in vitro and in vivo dose dependently. Furthermore, CO-NPs could induce cell apoptosis both at the early and late apoptotic stage and induce cell cycle arrest at S phase in a dose dependent manner. As signalling via the vascular endothelial growth factor receptor-2 (VEGFR2) is critical for angiogenic responses, we further explored the expression of VEGFR2 after the treatment of CO-NPs. They were found to inhibit VEGFR2 expression dose and time dependently both at the protein and mRNA level while had no effect on VEGF and VEGFR1 expression. Together, we report for the first time that CO-NPs can act as an anti-angiogenic agent by suppressing VEGFR2 expression, which may be a potential nanomedicine for angiogenesis therapy.Angiogenesis is a process that forms new blood capillaries from existing vessels, which is of great physiological and pathological significance. Although recent studies provide evidence that cuprous oxide nanoparticles (CO-NPs) may have biomedical potential, the mechanisms of CO-NPs in angiogenesis have not been investigated to date. We have studied the anti-angiogenic properties of CO-NPs on primary human umbilical vein endothelial cells (HUVECs). We found that CO-NPs were able to induce cell morphology changes and suppress cell proliferation, migration and tube formation in vitro and in vivo dose dependently. Furthermore, CO-NPs could induce cell apoptosis both at the early and

  11. Photoactivatable Caged Prodrugs of VEGFR-2 Kinase Inhibitors.

    PubMed

    Pinchuk, Boris; Horbert, Rebecca; Döbber, Alexander; Kuhl, Lydia; Peifer, Christian

    2016-04-29

    In this study, we report on the design, synthesis, photokinetic properties and in vitro evaluation of photoactivatable caged prodrugs for the receptor tyrosine kinase VEGFR-2. Highly potent VEGFR-2 inhibitors 1 and 3 were caged by introduction of a photoremovable protecting group (PPG) to yield the caged prodrugs 4 and 5. As expected, enzymatic and cellular proliferation assays showed dramatically diminished efficacy of caged prodrugs in vitro. Upon ultraviolet (UV) irradiation of the prodrugs original inhibitory activity was completely restored and even distinctly reinforced, as was the case for the prodrug 4. The presented results are a further evidence for caging technique being an interesting approach in the protein kinase field. It could enable spatial and temporal control for the inhibition of VEGFR-2. The described photoactivatable prodrugs might be highly useful as biological probes for studying the VEGFR-2 signal transduction.

  12. Small Molecular-Sized Artesunate Attenuates Ocular Neovascularization via VEGFR2, PKCα, and PDGFR Targets

    PubMed Central

    Zong, Yao; Yuan, Yongguang; Qian, Xiaobing; Huang, Zhen; Yang, Wei; Lin, Leilei; Zheng, Qishan; Li, Yujie; He, Huining; Gao, Qianying

    2016-01-01

    Ocular neovascularization (NV) is the primary cause of blindness in many ocular diseases. Large molecular weight anti- vascular endothelial growth factor (VEGF) protein drugs, such as Avastin and Lucentis, have saved the vision of millions. However, approximately 20–30% of patients respond poorly to anti-VEGF treatment. We found that artesunate (ART), a small molecular derivative of artemisinin, had a significant inhibitory effect on ocular NV by downregulating the expression of VEGFR2, PKCα, and PDGFR. ART significantly inhibited retinal NV in rabbits and macular edema in monkeys with greater anterior chamber penetrability and more durable efficacy than Avastin. Our pilot study showed that intravitreal injection of 80 μg ART significantly inhibited iris and corneal NV in a severe retinal detachment case. Our results suggest that ART might be a potential persistent small-molecule drug to manage ocular NV via multi-targets. PMID:27480521

  13. A CD44v6 peptide reveals a role of CD44 in VEGFR-2 signaling and angiogenesis.

    PubMed

    Tremmel, Martina; Matzke, Alexandra; Albrecht, Imke; Laib, Anna M; Olaku, Vivienne; Ballmer-Hofer, Kurt; Christofori, Gerhard; Héroult, Mélanie; Augustin, Hellmut G; Ponta, Helmut; Orian-Rousseau, Véronique

    2009-12-10

    A specific splice variant of the CD44 cell- surface protein family, CD44v6, has been shown to act as a coreceptor for the receptor tyrosine kinase c-Met on epithelial cells. Here we show that also on endothelial cells (ECs), the activity of c-Met is dependent on CD44v6. Furthermore, another receptor tyrosine kinase, VEGFR-2, is also regulated by CD44v6. The CD44v6 ectodomain and a small peptide mimicking a specific extracellular motif of CD44v6 or a CD44v6-specific antibody prevent CD44v6-mediated receptor activation. This indicates that the extracellular part of CD44v6 is required for interaction with c-Met or VEGFR-2. In the cytoplasm, signaling by activated c-Met and VEGFR-2 requires association of the CD44 carboxy-terminus with ezrin that couples CD44v6 to the cytoskeleton. CD44v6 controls EC migration, sprouting, and tubule formation induced by hepatocyte growth factor (HGF) or VEGF-A. In vivo the development of blood vessels from grafted EC spheroids and angiogenesis in tumors is impaired by CD44v6 blocking reagents, suggesting that the coreceptor function of CD44v6 for c-Met and VEGFR-2 is a promising target to block angiogenesis in pathologic conditions.

  14. The anti-angiogenic VEGF isoform VEGF165b transiently increases hydraulic conductivity, probably through VEGF receptor 1 in vivo.

    PubMed

    Glass, C A; Harper, S J; Bates, D O

    2006-04-01

    Vascular endothelial growth factor (VEGF) is the principal agent that increases microvascular permeability during physiological and pathological angiogenesis. VEGF is differentially spliced to form two families of isoforms: VEGF(xxx), and VEGF(xxx)b. Whereas VEGF(165) stimulates angiogenesis, VEGF(165)b is anti-angiogenic. To determine the effect of VEGF(165)b on permeability, hydraulic conductivity (L(p)) was measured in individually perfused microvessels in the mesentery of frogs and rats. As with VEGF(165), VEGF(165)b increased L(p) in amphibian (2.4 +/- 0.3-fold) and mammalian (1.9 +/- 0.2-fold) mesenteric microvessels. A dose-response relationship showed that VEGF(165)b (EC(50), 0.65 pm) was approximately 25 times more potent than VEGF(165) (EC(50), 16 pm) in amphibian microvessels. VEGF(165) has been shown to increase permeability through VEGF receptor 2 (VEGF-R2) signalling. However, VEGF(165)b increased L(p) of frog vessels to the same extent in the presence of the VEGF-R2 inhibitor ZM323881, indicating that it does not increase permeability via VEGF-R2 signalling, and was inhibited by the VEGF receptor inhibitor SU5416 at doses that are specific for VEGF receptor 1 (VEGF-R1). VEGF(165)b, in contrast to VEGF(165), did not result in a sustained chronic increase in L(p). These results show that although VEGF(165)b is anti-angiogenic in the mesentery, it does signal in endothelial cells in vivo resulting in a transient, but not sustained, increase in microvascular L(p), probably through VEGF-R1.

  15. Minimally invasive colon resection for malignant colonic conditions is associated with a transient early increase in plasma sVEGFR1 and a decrease in sVEGFR2 levels after surgery.

    PubMed

    Shantha Kumara, H M C; Cabot, J C; Hoffman, A; Luchtefeld, M; Kalady, M F; Hyman, N; Feingold, D; Baxter, R; Whelan, R L

    2010-02-01

    Plasma VEGF levels increase after minimally invasive colorectal resection (MICR) and remain elevated for 2-4 weeks. VEGF induces physiologic and pathologic angiogenesis by binding to endothelial cell (EC) bound VEGF-Receptor-1 (VEGFR1) and VEGFR2. Soluble forms of these receptors sequester plasma VEGF, decreasing the amount available to bind to EC-bound receptors. Ramifications of surgery-related plasma VEGF changes partially depend on plasma levels of sVEGFR1 and sVEGFR2. This study assessed perioperative sVEGFR1 and sVEGFR2 levels after MICR in patients with colorectal cancer. Forty-five patients were studied; blood samples were taken from all patients preoperatively (preop) and on postoperative days (POD) 1 and 3; in most a fourth sample was drawn between POD 7-30. Late samples were bundled into two time points: POD 7-13 and POD 14-30. sVEGFR1 and sVEGFR2 levels were measured via ELISA. sVEGFR2 data are reported as mean +/- SD and were assessed with the paired samples t test. sVEGFR1 data were not normally distributed. They are reported as median and 95% confidence interval (CI) and were assessed with the Wilcoxon signed-Rank test (p < 0.05). Preoperatively, the mean plasma sVEGFR2 level (7583.9 pg/ml) was greater than the sVEGFR1 result (98.3 pg/ml). Compared with preop levels, sVEGFR2 levels were significantly lower on POD 1 (6068.2 pg/ml, +/-2034.5) and POD 3 (6227.6 pg/ml, +/-2007.0), whereas sVEGFR1 levels were significantly greater on POD 1 (237.5 pg/ml; 95% CI, 89.6-103.5), POD 3 (200.2 pg/ml; 95% CI, 159-253), and POD 7-13 (102.9 pg/ml; 95% CI, 189.7-253). No differences were found on POD 7-13 for sVEGFR2 or POD 14-30 for either protein. sVEGFR2 values decreased and sVEGFR1 levels increased early after MICR; sVEGFR2 changes dominate due to their much larger magnitude. The net result is less plasma VEGF bound by soluble receptors and more plasma VEGF available to bind to ECs early after surgery.

  16. Knockdown of the AKT3 (PKBγ), PI3KCA, and VEGFR2 genes by RNA interference suppresses glioblastoma multiforme T98G cells invasiveness in vitro.

    PubMed

    Paul-Samojedny, Monika; Pudełko, Adam; Suchanek-Raif, Renata; Kowalczyk, Małgorzata; Fila-Daniłow, Anna; Borkowska, Paulina; Kowalski, Jan

    2015-05-01

    Glioblastoma multiforme (GBM) is the most common primary brain malignancy, having a very poor prognosis and is characterized by extensive brain invasion as well as resistance to the therapy. The phosphoinositide 3-kinase (PI3K)/Akt/PTEN signaling pathway is deregulated in GBM. Besides, florid vascularization and aberrantly elevated vascular endothelial growth factor (VEGF) occur very often. The present study was designed to examine the inhibitory effect of AKT3, PI3KCA, and VEGFR2 small interfering RNAs (siRNAs) on GBM cell invasiveness. T98G cells were transfected with AKT3, PI3KCA, and/or VEGFR2 siRNAs. VEGFR2 protein-positive cells were identified by flow cytometry using specific monoclonal anti-VEGFR2 antibodies. Alterations in messenger RNA (mRNA) expression of VEGF, VEGFR2, matrix metalloproteinases (MMPs) (MMP-2, MMP-9, MMP-13, MMP-14), tissue inhibitors of metalloproteinases (TIMPs) (TIMP-1, TIMP-3), c-Fos, c-Jun, hypoxia-inducible factor-1α (HIF-1α), ObRa, and cathepsin D genes were analyzed by qRT-PCR. Cells treated with specific siRNA were also analyzed for invasion using the Matrigel invasion assay. We have found significantly lower mRNA levels of MMPs, cathepsin D, VEGF, VEGFR2, HIF-1α, and c-Fos/c-Jun ratio, as well as significantly higher mRNA level of TIMPs in AKT3 and PI3KCA siRNA transfected cells compared to untransfected cells, while significantly lower mRNA levels of MMPs (MMP-2, MMP-9, MMP-14) and TIMP-1, as well as significantly higher mRNA level of TIMP-3, were shown only in cells transfected with VEGFR2 siRNA. The positive correlation between MMP-13 and ObRa mRNA copy number has been found. Summarizing, transfection of T98G cells with AKT3, PI3KCA, or VEGFR2 siRNAs leads to a significant reduction in cell invasiveness. The siRNA-induced AKT3, PI3KCA, and VEGFR2 mRNA knockdown may offer a novel therapeutic strategy to reduce the invasiveness of GBM cells.

  17. Computational investigation of sphingosine kinase 1 (SphK1) and calcium dependent ERK1/2 activation downstream of VEGFR2 in endothelial cells

    PubMed Central

    Bazzazi, Hojjat; Popel, Aleksander S.

    2017-01-01

    Vascular endothelial growth factor (VEGF) is a powerful regulator of neovascularization. VEGF binding to its cognate receptor, VEGFR2, activates a number of signaling pathways including ERK1/2. Activation of ERK1/2 is experimentally shown to involve sphingosine kinase 1 (SphK1) activation and its calcium-dependent translocation downstream of ERK1/2. Here we construct a rule-based computational model of signaling downstream of VEGFR2, by including SphK1 and calcium positive feedback mechanisms, and investigate their consequences on ERK1/2 activation. The model predicts the existence of VEGF threshold in ERK1/2 activation that can be continuously tuned by cellular concentrations of SphK1 and sphingosine 1 phosphate (S1P). The computer model also predicts powerful effects of perturbations in plasma and ER calcium pump rates and the current through the CRAC channels on ERK1/2 activation dynamics, highlighting the critical role of intracellular calcium in shaping the pERK1/2 signal. The model is then utilized to simulate anti-angiogenic therapeutic interventions targeting VEGFR2-ERK1/2 axis. Simulations indicate that monotherapies that exclusively target VEGFR2 phosphorylation, VEGF, or VEGFR2 are ineffective in shutting down signaling to ERK1/2. By simulating therapeutic strategies that target multiple nodes of the pathway such as Raf and SphK1, we conclude that combination therapy should be much more effective in blocking VEGF signaling to EKR1/2. The model has important implications for interventions that target signaling pathways in angiogenesis relevant to cancer, vascular diseases, and wound healing. PMID:28178265

  18. VEGFR-2 expression in carcinoid cancer cells and its role in tumor growth and metastasis

    PubMed Central

    Silva, Scott R.; Bowen, Kanika A.; Rychahou, Piotr G.; Jackson, Lindsey N.; Weiss, Heidi L.; Lee, Eun Y.; Townsend, Courtney M.; Evers, B. Mark

    2014-01-01

    Carcinoid tumors are slow growing and highly vascular neuroendocrine neoplasms that are increasing in incidence. Previously, we showed that carcinoid tumors express vascular endothelial growth factor receptor 2 (VEGFR-2) in the epithelial compartment of carcinoid tumor sections; yet, its role is not completely understood. The purpose of our study was to: (i) assess the expression of VEGFR-2 in the novel human carcinoid cell line BON, (ii) to determine the role of PI3K/Akt signaling on VEGFR-2 expression and (iii) to assess the effect of VEGFR-2 on BON cell invasion, migration and proliferation. We found that, although VEGFR-2 is expressed in BON cells, reduction in VEGFR-2 expression actually enhanced proliferation, invasion, and migration of the BON cell line. Also, expression of VEGFR-2 was inversely related to PI3K signaling. Carcinoid liver metastases in mice demonstrated decreased VEGFR-2 expression. Furthermore, the expression of a truncated, soluble form of VEGFR-2 (sVEGFR-2), a protein demonstrated to inhibit cell growth, was detected in BON cells. The presence of VEGFR-2 in the epithelial component of carcinoid tumors and in the BON cell line suggests an alternate role for VEGFR-2, in addition to its well-defined role in angiogenesis. The expression of sVEGFR-2 may explain the inverse relationship between VEGFR-2 expression and PI3K/Akt signaling and the inhibitory effect VEGFR-2 has on BON cell proliferation, migration and invasion. PMID:20473929

  19. Pristimerin, a triterpenoid, inhibits tumor angiogenesis by targeting VEGFR2 activation.

    PubMed

    Mu, Xianmin; Shi, Wei; Sun, Lixin; Li, Han; Jiang, Zhenzhou; Zhang, Luyong

    2012-06-05

    Pristimerin is a triterpenoid isolated from Celastrus and Maytenus spp. that has been shown to possess a variety of biological activities, including anti-cancer activity. However, little is known about pristimerin's effects on tumor angiogenesis. In this study, we examined the function and the mechanism of this compound in tumor angiogenesis using multiple angiogenesis assays. We found that pristimerin significantly reduced both the volume and weight of solid tumors and decreased angiogenesis in a xenograft mouse tumor model in vivo. Pristimerin significantly inhibited the neovascularization of chicken chorioallantoic membrane (CAM) in vivo and abrogated vascular endothelial growth factor (VEGF)-induced microvessel sprouting in an ex vivo rat aortic ring assay. Furthermore, pristimerin inhibited the VEGF-induced proliferation, migration and capillary-like structure formation of human umbilical vascular endothelial cells (HUVECs) in a concentration-dependent manner. Mechanistic studies revealed that pristimerin suppressed the VEGF-induced phosphorylation of VEGF receptor 2 kinase (KDR/Flk-1) and the activity of AKT, ERK1/2, mTOR, and ribosomal protein S6 kinase. Taken together, our results provide evidence for the first time that pristimerin potently suppresses angiogenesis by targeting VEGFR2 activation. These results provide a novel mechanism of action for pristimerin which may be important in the treatment of cancer.

  20. The effect of platelet rich plasma on angiogenesis in ischemic flaps in VEGFR2-luc mice.

    PubMed

    Sönmez, Tolga Taha; Vinogradov, Alexandra; Zor, Fatih; Kweider, Nisreen; Lippross, Sebastian; Liehn, Elisa Anamaria; Naziroglu, Mustafa; Hölzle, Frank; Wruck, Christoph; Pufe, Thomas; Tohidnezhad, Mersedeh

    2013-04-01

    To improve skin flap healing, one promising strategy in reconstructive surgery might be to optimize platelet rich plasma (PRP) bioactivity and the ischemia-altered expression of genes. We studied both the effect of PRP on ischemic flaps, and whether in vivo bioluminescence imaging (BLI) is a suitable method for the longitudinal monitoring of angiogenesis in surgical wounds. Axial murine skin flaps were created in four experimental groups. In vivo measurements of VEGFR2 expression levels were made every other day until the 14th day. The local VEGF level and microvessel density were quantified on the 14th day via ELISA and immunohistochemistry, and flap survival rates were measured. We demonstrated that PRP and induced ischemia have a beneficial influence on angiogenesis and flap healing. Combining the two resulted in a significantly robust increase in angiogenesis and flap survival rate that was corroborated by bioluminescence imaging of VEGFR2 activity. This study shows that angiogenic effects of PRP may be potentialized by the stimulus of induced ischemia during free flap harvesting, and thus the two procedures appear to have a synergistic effect on flap healing. This study further demonstrates that BLI of modulated genes in reconstructive surgery is a valuable model for longitudinal in vivo evaluation of angiogenesis.

  1. Minimally invasive colon resection is associated with a transient increase in plasma sVEGFR1 levels and a decrease in sVEGFR2 levels during the early postoperative period.

    PubMed

    Shantha Kumara, H M C; Cabot, J C; Hoffman, A; Luchtefeld, M; Kalady, M F; Hyman, N; Feingold, D; Baxter, R; Whelan, R Larry

    2009-04-01

    Plasma vascular endothelial growth factor (VEGF) levels are elevated for 2-4 weeks after minimally invasive colorectal resection (MICR). VEGF induces wound and tumor angiogenesis by binding to endothelial cell (EC)-bound VEGF-receptor 1 (VEGFR1) and VEGFR2. Soluble receptors (sVEGFR1, sVEGFR2) sequester VEGF in the blood and decrease VEGF's proangiogenic effect. The importance of the MICR-related VEGF changes depends on the effect of surgical procedures on sVEGFR1 and sVEGFR2; this study assessed levels of these proteins after MICR for benign indications. Blood samples were taken (n=39) preoperatively (preop) and on postoperative days (POD) 1 and 3; in most cases a fourth sample was drawn between POD 7 and 30. sVEGFR1 and sVEGFR2 levels were measured via enzyme-linked immunosorbent assay (ELISA), which detects free and VEGF bound soluble receptor. Late samples were bundled into POD 7-13 and POD 14-30 time points. Results are reported as mean and standard deviation. The data was assessed with paired-samples t-test. Preop, mean plasma sVEGFR2 level (9,203.7+/-1,934.3 pg/ml) was significantly higher than the sVEGFR1 value (132.5+/-126.2 pg/ml). sVEGFR2 levels were significantly lower on POD 1 (6,957.8+/-1,947.7 pg/ml,) and POD 3 (7,085.6+/-2,000.2 pg/ml), whereas sVEGFR1 levels were significantly higher on POD 1 (220.0+/-132.8 pg/ml) and POD 3 (182.7+/-102.1 pg/ml) versus preop results. No differences were found on POD 7-13 or 14-30. sVEGFR2 values decreased and sVEGFR1 levels increased early after MICR; due to its much higher baseline, the sVEGFR2 changes dominate. The net result is less VEGF bound to soluble receptor and more free plasma VEGF.

  2. VEGF blockade enables oncolytic cancer virotherapy in part by modulating intratumoral myeloid cells.

    PubMed

    Currier, Mark A; Eshun, Francis K; Sholl, Allyson; Chernoguz, Artur; Crawford, Kelly; Divanovic, Senad; Boon, Louis; Goins, William F; Frischer, Jason S; Collins, Margaret H; Leddon, Jennifer L; Baird, William H; Haseley, Amy; Streby, Keri A; Wang, Pin-Yi; Hendrickson, Brett W; Brekken, Rolf A; Kaur, Balveen; Hildeman, David; Cripe, Timothy P

    2013-05-01

    Understanding the host response to oncolytic viruses is important to maximize their antitumor efficacy. Despite robust cytotoxicity and high virus production of an oncolytic herpes simplex virus (oHSV) in cultured human sarcoma cells, intratumoral (ITu) virus injection resulted in only mild antitumor effects in some xenograft models, prompting us to characterize the host inflammatory response. Virotherapy induced an acute neutrophilic infiltrate, a relative decrease of ITu macrophages, and a myeloid cell-dependent upregulation of host-derived vascular endothelial growth factor (VEGF). Anti-VEGF antibodies, bevacizumab and r84, the latter of which binds VEGF and selectively inhibits binding to VEGF receptor-2 (VEGFR2) but not VEGFR1, enhanced the antitumor effects of virotherapy, in part due to decreased angiogenesis but not increased virus production. Neither antibody affected neutrophilic infiltration but both partially mitigated virus-induced depletion of macrophages. Enhancement of virotherapy-mediated antitumor effects by anti-VEGF antibodies could largely be recapitulated by systemic depletion of CD11b(+) cells. These data suggest the combined effect of oHSV virotherapy and anti-VEGF antibodies is in part due to modulation of a host inflammatory reaction to virus. Our data provide strong preclinical support for combined oHSV and anti-VEGF antibody therapy and suggest that understanding and counteracting the innate host response may help enable the full antitumor potential of oncolytic virotherapy.

  3. VEGF as a Paracrine Regulator of Conventional Outflow Facility

    PubMed Central

    Reina-Torres, Ester; Wen, Joanne C.; Liu, Katy C.; Li, Guorong; Sherwood, Joseph M.; Chang, Jason Y. H.; Challa, Pratap; Flügel-Koch, Cassandra M.; Stamer, W. Daniel; Allingham, R. Rand; Overby, Darryl R.

    2017-01-01

    Purpose Vascular endothelial growth factor (VEGF) regulates microvascular endothelial permeability, and the permeability of Schlemm's canal (SC) endothelium influences conventional aqueous humor outflow. We hypothesize that VEGF signaling regulates outflow facility. Methods We measured outflow facility (C) in enucleated mouse eyes perfused with VEGF-A164a, VEGF-A165b, VEGF-D, or inhibitors to VEGF receptor 2 (VEGFR-2). We monitored VEGF-A secretion from human trabecular meshwork (TM) cells by ELISA after 24 hours of static culture or cyclic stretch. We used immunofluorescence microscopy to localize VEGF-A protein within the TM of mice. Results VEGF-A164a increased C in enucleated mouse eyes. Cyclic stretch increased VEGF-A secretion by human TM cells, which corresponded to VEGF-A localization in the TM of mice. Blockade of VEGFR-2 decreased C, using either of the inhibitors SU5416 or Ki8751 or the inactive splice variant VEGF-A165b. VEGF-D increased C, which could be blocked by Ki8751. Conclusions VEGF is a paracrine regulator of conventional outflow facility that is secreted by TM cells in response to mechanical stress. VEGF affects facility via VEGFR-2 likely at the level of SC endothelium. Disruption of VEGF signaling in the TM may explain why anti-VEGF therapy is associated with decreased outflow facility and sustained ocular hypertension. PMID:28358962

  4. Impaired cross-activation of β3 integrin and VEGFR-2 on endothelial progenitor cells with aging decreases angiogenesis in response to hypoxia.

    PubMed

    Di, Qun; Cheng, Zeen; Kim, Weon; Liu, Zexuan; Song, Hui; Li, Xiang; Nan, Yongshan; Wang, Chengya; Cheng, Xianwu

    2013-10-03

    The mechanism by which vascular regeneration declines with aging is not fully understood. An interaction between integrin and vascular endothelial growth factor receptor-2 (VEGFR-2) plays a substantial role in angiogenesis. Here, we investigated whether aging impairs this interaction in endothelial progenitor cells (EPCs) under hypoxia. Aging reduced the blood flow and vessel density in ischemic muscles in mice. Levels of phosphorylated Src (p-Src), p-β3, and p-VEGFR-2 in acute ischemia were reduced in the muscles of aged mice compared to young mice. The hypoxia-inducible factor (HIF)-1α stabilizer deferoxamine improved the age-related impairment of angiogenesis, but this effect was diminished by LY290004, an inhibitor of phosphatidylinositol 3-kinase. Deferoxamine improved the reduction in chronic ischemia-induced β3-integrin and VEGFR-2 phosphorylation in the muscles of aged mice; this effect was also diminished by LY290004. In EPCs, we identified the molecular requirements for VEGF-mediated β3-integrin and VEGFR-2 cross-activation in vitronectin-induced cell adhesion under acute hypoxia. We demonstrated that c-Src controlled the adhesion- and VEGF-induced β3 tyrosine phosphorylation in hypoxia. Aging enhanced the hypoxia-induced EPC apoptosis and impaired several c-Src-related VEGF-induced receptor events, including β3 tyrosine activation, ligand binding, cell adhesion, and tubulogenesis in cultured EPCs of animals and those of humans. These data suggest that the aging-related decline in angiogenic action in response to ischemia is mediated by the impairment of cross-activation between β3 and VEGFR-2 in EPCs, which is partially associated with decreased HIF-1α stability. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  5. The microRNA-126-VEGFR2 axis controls the innate response to pathogen-associated nucleic acids

    PubMed Central

    Agudo, Judith; Ruzo, Albert; Tung, Navpreet; Salmon, Hélène; Leboeuf, Marylène; Hashimoto, Daigo; Becker, Christian; Garrett-Sinha, Lee-Ann; Baccarini, Alessia; Merad, Miriam; Brown, Brian D

    2013-01-01

    MicroRNA-126 (miR-126) is a microRNA predominately expressed by endothelial cells and controls angiogenesis. We found miR-126 was required for the innate response to pathogen-associated nucleic acids, and that miR-126-deficient mice had increased susceptibility to pseudotyped-HIV infection. miRNA profiling and deep-sequencing indicated that miR-126 was highly and specifically expressed by plasmacytoid dendritic cells (pDCs). miR-126 controlled the survival and function of pDCs, and regulated expression ofinnate response genes, including Tlr7, Tlr9 and Nfkb1, as well as Kdr, which encodes VEGF-receptor 2 (VEGFR2). Deletion of Kdr in DCs resulted in reduced type I interferon production, supporting a role for VEGFR2 in miR-126 regulation of pDCs. These studies identify the miR-126–VEGFR2 axis as an important regulator of the innate response that operates through multiscale control of pDCs. PMID:24270517

  6. Dioscin inhibits colon tumor growth and tumor angiogenesis through regulating VEGFR2 and AKT/MAPK signaling pathways

    SciTech Connect

    Tong, Qingyi; Qing, Yong; Wu, Yang; Hu, Xiaojuan; Jiang, Lei; Wu, Xiaohua

    2014-12-01

    Dioscin has shown cytotoxicity against cancer cells, but its in vivo effects and the mechanisms have not elucidated yet. The purpose of the current study was to assess the antitumor effects and the molecular mechanisms of dioscin. We showed that dioscin could inhibit tumor growth in vivo and has no toxicity at the test condition. The growth suppression was accompanied by obvious blood vessel decrease within solid tumors. We also found dioscin treatment inhibited the proliferation of cancer and endothelial cell lines, and most sensitive to primary cultured human umbilical vein endothelial cells (HUVECs). What's more, analysis of HUVECs migration, invasion, and tube formation exhibited that dioscin has significantly inhibitive effects to these actions. Further analysis of blood vessel formation in the matrigel plugs indicated that dioscin could inhibit VEGF-induced blood vessel formation in vivo. We also identified that dioscin could suppress the downstream protein kinases of VEGFR2, including Src, FAK, AKT and Erk1/2, accompanied by the increase of phosphorylated P38MAPK. The results potently suggest that dioscin may be a potential anticancer drug, which efficiently inhibits angiogenesis induced by VEGFR2 signaling pathway as well as AKT/MAPK pathways. - Highlights: • Dioscin inhibits tumor growth in vivo and does not exhibit any toxicity. • Dioscin inhibits angiogenesis within solid tumors. • Dioscin inhibits the proliferation, migration, invasion, and tube formation of HUVECs. • Dioscin inhibits VEGF–induced blood vessel formation in vivo. • Dioscin inhibits VEGFR2 signaling pathway as well as AKT/MAPK pathway.

  7. Heparan Sulfate Regulates VEGF165- and VEGF121-mediated Vascular Hyperpermeability*

    PubMed Central

    Xu, Ding; Fuster, Mark M.; Lawrence, Roger; Esko, Jeffrey D.

    2011-01-01

    VEGF was first described as vascular permeability factor, a potent inducer of vascular leakage. Genetic evidence indicates that VEGF-stimulated endothelial proliferation in vitro and angiogenesis in vivo depend on heparan sulfate, but a requirement for heparan sulfate in vascular hyperpermeability has not been explored. Here we show that altering endothelial cell heparan sulfate biosynthesis in vivo decreases hyperpermeability induced by both VEGF165 and VEGF121. Because VEGF121 does not bind heparan sulfate, the requirement for heparan sulfate suggested that it interacted with VEGF receptors rather than the ligand. By applying proximity ligation assays to primary brain endothelial cells, we show a direct interaction in situ between heparan sulfate and the VEGF receptor, VEGFR2. Furthermore, the number of heparan sulfate-VEGFR2 complexes increased in response to both VEGF165 and VEGF121. Genetic or heparin lyase-mediated alteration of endothelial heparan sulfate attenuated phosphorylation of VEGFR2 in response to VEGF165 and VEGF121, suggesting that the functional VEGF receptor complex contains heparan sulfate. Pharmacological blockade of heparan sulfate-protein interactions inhibited hyperpermeability in vivo, suggesting heparan sulfate as a potential target for treating hyperpermeability associated with ischemic disease. PMID:20974861

  8. The VEGFR2, COX-2 and MMP-2 polymorphisms are associated with clinical outcome of patients with inoperable non-small cell lung cancer.

    PubMed

    Butkiewicz, Dorota; Krześniak, Małgorzata; Drosik, Anna; Giglok, Monika; Gdowicz-Kłosok, Agnieszka; Kosarewicz, Agata; Rusin, Marek; Masłyk, Barbara; Gawkowska-Suwińska, Marzena; Suwiński, Rafał

    2015-11-15

    Certain common inherited variations in genes involved in tumor angiogenesis, progression and metastasis may contribute to cancer therapy outcome and prognosis by altering the gene expression and protein activity. In this report, we examined the effect of functional polymorphisms in MMP-1, MMP-2, MMP-3, VEGF, VEGFR2, FGFR4 and COX-2 genes on overall (OS) and progression-free survival (PFS) of 350 Caucasian patients with inoperable non-small cell lung cancer (NSCLC). The results of multivariate analysis indicated that VEGFR2 -906C and COX-2 -1195G alleles were strongly associated with poor OS and PFS (p = 0.002 and 0.015, respectively, for OS; p = 0.009 and 0.015, respectively, for PFS), while MMP-2 -1306 T allele carriers had significantly reduced PFS (p = 0.010). Moreover, an increased risk of death and progression was significantly associated with the number of adverse alleles for VEGFR2/COX-2 (p = 0.0005 for OS and 0.0006 for PFS in >1 adverse allele carriers) and VEGFR2/COX-2/MMP-2 combinations (p = 0.0003 for OS and 0.0001 for PFS in patients with >2 adverse alleles). Finally, VEGFR2 TC/CC, COX-2 AG/GG and MMP-2 CT/TT genotypes as well as "at risk" allele combinations were identified as independent predictors of unfavorable OS and PFS in the group. In conclusion, the data suggest that selected VEGFR2, COX-2 and MMP-2 polymorphisms may be potential prognostic markers in unresectable NSCLC treated with radiotherapy with or without chemotherapy, although further validation studies are warranted to confirm our observations.

  9. VEGFR-2 Expression in Glioblastoma Multiforme Depends on Inflammatory Tumor Microenvironment.

    PubMed

    Jaal, Jana; Kase, Marju; Minajeva, Ave; Saretok, Mikk; Adamson, Aidi; Junninen, Jelizaveta; Metsaots, Tõnis; Jõgi, Tõnu; Joonsalu, Madis; Vardja, Markus; Asser, Toomas

    2015-01-01

    Glioblastoma multiforme (GBM) is one of the most angiogenic tumors. However, antiangiogenic therapy has not shown significant clinical efficacy. The aim of our study was to evaluate the impact of inflammatory tumor microenvironment on the expression of vascular endothelial growth factor receptor 2 (VEGFR-2). Surgically excised primary GBM tissues were histologically examined for overall extent of inflammation (score 1-3). After immunohistochemistry, the tissue expression of ICAM-1 (optical density), the number of VEGFR-2 positive (VEGFR-2+) blood vessels (per microscopic field), and the endothelial staining intensity of VEGFR-2 (score 0-3) were determined. In GBM, the extent of inflammation was 1.9 ± 0.7 (group mean ± SD). Mean optical density of inflammatory mediator ICAM-1 was 57.0 ± 27.1 (pixel values). The number of VEGFR-2+ blood vessels and endothelial VEGFR-2 staining intensity were 6.2 ± 2.4 and 1.2 ± 0.8, respectively. A positive association was found between endothelial VEGFR-2 staining intensity and the extent of inflammation (p = 0.005). Moreover, VEGFR-2 staining intensity correlated with the expression level of ICAM-1 (p = 0.026). The expression of VEGFR-2, one of the main targets of antiangiogenic therapy, depends on GBM microenvironment. Higher endothelial VEGFR-2 levels were seen in the presence of more pronounced inflammation. Target dependence on inflammatory tumor microenvironment has to be taken into consideration when treatment approaches that block VEGFR-2 signaling are designed.

  10. Autocrine VEGF Isoforms Differentially Regulate Endothelial Cell Behavior

    PubMed Central

    Yamamoto, Hideki; Rundqvist, Helene; Branco, Cristina; Johnson, Randall S.

    2016-01-01

    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 homeostasis in

  11. Combinatory action of VEGFR2 and MAP kinase pathways maintains endothelial-cell integrity.

    PubMed

    Zhong, Hanbing; Wang, Danyang; Wang, Nan; Rios, Yesenia; Huang, Haigen; Li, Song; Wu, Xinrong; Lin, Shuo

    2011-07-01

    Blood vessels normally maintain stereotyped lumen diameters and their stable structures are crucial for vascular function. However, very little is known about the molecular mechanisms controlling the maintenance of vessel diameters and the integrity of endothelial cells. We investigated this issue in zebrafish embryos by a chemical genetics approach. Small molecule libraries were screened using live Tg(kdrl:GRCFP)(zn1) transgenic embryos in which endothelial cells are specifically labeled with GFP. By analyzing the effects of compounds on the morphology and function of embryonic blood vessels after lumen formation, PP1, a putative Src kinase inhibitor, was identified as capable of specifically reducing vascular lumen size by interrupting endothelial-cell integrity. The inhibitory effect is not due to Src or general VEGF signaling inhibition because another Src inhibitor and Src morpholino as well as several VEGFR inhibitors failed to produce a similar phenotype. After profiling a panel of 22 representative mammalian kinases and surveying published data, we selected a few possible new candidates. Combinational analysis of these candidate kinase inhibitors established that PP1 induced endothelial collapse by inhibiting both the VEGFR2 and MAP kinase pathways. More importantly, combinatory use of two clinically approved drugs Dasatinib and Sunitinib produced the same phenotype. This is the first study to elucidate the pathways controlling maintenance of endothelial integrity using a chemical genetics approach, indicating that endothelial integrity is controlled by the combined action of the VEGFR2 and MAP kinase pathways. Our results also suggest the possible side effect of the combination of two anticancer drugs on the circulatory system.

  12. Discovery of N-(2-phenyl-1H-benzo[d]imidazol-5-yl)quinolin-4-amine derivatives as novel VEGFR-2 kinase inhibitors.

    PubMed

    Shi, Lei; Wu, Ting-Ting; Wang, Zhi; Xue, Jia-Yu; Xu, Yun-Gen

    2014-09-12

    Inhibition of the VEGF signaling pathway has become a valuable approach in the treatment of cancers. In this work, a series of N-(2-phenyl-1H-benzo[d]imidazol-5-yl)quinolin-4-amine derivatives were designed and identified as potent inhibitors of VEGFR-2 (KDR) kinase. These compounds with quinoline scaffold and benzimidazole moiety were synthesized and their biological activities against VEGFR-2 and two human cancer cell lines were evaluated. Among them, compound 7s exhibited the most potent inhibitory activity against VEGFR-2 with IC50 of 0.03 μM and it also showed the highest anticancer activity against the tested cancer cell lines with IC50 of 1.2 μM against MCF-7 and 13.3 μM against Hep-G2. Docking simulation supported the initial pharmacophoric hypothesis and suggested a common mode of interaction at the ATP-binding site of VEGFR-2, which demonstrates that compound 7s is a potential agent for cancer therapy deserving further researching. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  13. Glioma cell VEGFR-2 confers resistance to chemotherapeutic and antiangiogenic treatments in PTEN-deficient glioblastoma.

    PubMed

    Kessler, Tobias; Sahm, Felix; Blaes, Jonas; Osswald, Matthias; Rübmann, Petra; Milford, David; Urban, Severino; Jestaedt, Leonie; Heiland, Sabine; Bendszus, Martin; Hertenstein, Anne; Pfenning, Philipp-Niclas; Ruiz de Almodóvar, Carmen; Wick, Antje; Winkler, Frank; von Deimling, Andreas; Platten, Michael; Wick, Wolfgang; Weiler, Markus

    2015-10-13

    Loss of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a prerequisite for tumor cell-specific expression of vascular endothelial growth factor receptor (VEGFR)-2 in glioblastoma defining a subgroup prone to develop evasive resistance towards antiangiogenic treatments. Immunohistochemical analysis of human tumor tissues showed VEGFR-2 expression in glioma cells in 19% of specimens examined, mainly in the infiltration zone. Glioma cell VEGFR-2 positivity was restricted to PTEN-deficient tumor specimens. PTEN overexpression reduced VEGFR-2 expression in vitro, as well as knock-down of raptor or rictor. Genetic interference with VEGFR-2 revealed proproliferative, antiinvasive and chemoprotective functions for VEGFR-2 in glioma cells. VEGFR-2-dependent cellular effects were concomitant with activation of 'kappa-light-chain-enhancer' of activated B-cells, protein kinase B, and N-myc downstream regulated gene 1. Two-photon in vivo microscopy revealed that expression of VEGFR-2 in glioma cells hampers antiangiogenesis. Bevacizumab induces a proinvasive response in VEGFR-2-positive glioma cells. Patients with PTEN-negative glioblastomas had a shorter survival after initiation of bevacizumab therapy compared with PTEN-positive glioblastomas. Conclusively, expression of VEGFR-2 in glioma cells indicates an aggressive glioblastoma subgroup developing early resistance to temozolomide or bevacizumab. Loss of PTEN may serve as a biomarker identifying those tumors upfront by routine neuropathological methods.

  14. Site-Specific N-Glycosylation of Endothelial Cell Receptor Tyrosine Kinase VEGFR-2.

    PubMed

    Chandler, Kevin Brown; Leon, Deborah R; Meyer, Rosana D; Rahimi, Nader; Costello, Catherine E

    2017-02-03

    Vascular endothelial growth factor receptor-2 (VEGFR-2) is an important receptor tyrosine kinase (RTK) that plays critical roles in both physiologic and pathologic angiogenesis. The extracellular domain of VEGFR-2 is composed of seven immunoglobulin-like domains, each with multiple potential N-glycosylation sites (sequons). N-glycosylation plays a central role in RTK ligand binding, trafficking, and stability. However, despite its importance, the functional role of N-glycosylation of VEGFR-2 remains poorly understood. The objectives of the present study were to characterize N-glycosylation sites in VEGFR-2 via enzymatic release of the glycans and concomitant incorporation of (18)O into formerly N-glycosylated sites followed by tandem mass spectrometry (MS/MS) analysis to determine N-glycosylation site occupancy and the site-specific N-glycan heterogeneity of VEGFR-2 glycopeptides. The data demonstrated that all seven VEGFR-2 immunoglobulin-like domains have at least one occupied N-glycosylation site. MS/MS analyses of glycopeptides and deamidated, deglycosylated (PNGase F-treated) peptides from ectopically expressed VEGFR-2 in porcine aortic endothelial (PAE) cells identified N-glycans at the majority of the 17 potential N-glycosylation sites on VEGFR-2 in a site-specific manner. The data presented here provide direct evidence for site-specific, heterogeneous N-glycosylation and N-glycosylation site occupancy on VEGFR-2. The study has important implications for the therapeutic targeting of VEGFR-2, ligand binding, trafficking, and signaling.

  15. Hypertonic saline alleviates experimentally induced cerebral oedema through suppression of vascular endothelial growth factor and its receptor VEGFR2 expression in astrocytes.

    PubMed

    Huang, Linqiang; Cao, Wei; Deng, Yiyu; Zhu, Gaofeng; Han, Yongli; Zeng, Hongke

    2016-10-13

    Cerebral oedema is closely related to the permeability of blood-brain barrier, vascular endothelial growth factor (VEGF) and its receptor vascular endothelial growth factor receptor 2 (VEGFR2) all of which are important blood-brain barrier (BBB) permeability regulatory factors. Zonula occludens 1 (ZO-1) and claudin-5 are also the key components of BBB. Hypertonic saline is widely used to alleviate cerebral oedema. This study aimed to explore the possible mechanisms underlying hypertonic saline that ameliorates cerebral oedema effectively. Middle cerebral artery occlusion (MCAO) model in Sprague-Dawley (SD) rats and of oxygen-glucose deprivation model in primary astrocytes were used in this study. The brain water content (BWC) was used to assess the effect of 10 % HS on cerebral oedema. The assessment of Evans blue (EB) extravasation was performed to evaluate the protective effect of 10 % HS on blood-brain barrier. The quantification of VEGF, VEGFR2, ZO-1 and claudin-5 was used to illustrate the mechanism of 10 % HS ameliorating cerebral oedema. BWC was analysed by wet-to-dry ratios in the ischemic hemisphere of SD rats; it was significantly decreased after 10 % HS treatment (P < 0.05). We also investigated the blood-brain barrier protective effect by 10 % HS which reduced EB extravasation effectively in the peri-ischemic brain tissue. In parallel to the above notably at 24 h following MCAO, mRNA and protein expression of VEGF and VEGFR2 in the peri-ischemic brain tissue was down-regulated after 10 % HS treatment (P < 0.05). Along with this, in vitro studies showed increased VEGF and VEGFR2 mRNA and protein expression in primary astrocytes under hypoxic condition (P < 0.05), but it was suppressed after HS treatment (P < 0.05). In addition, HS inhibited the down-regulation of ZO-1, claudin-5 effectively. The results suggest that 10 % HS could alleviate cerebral oedema possibly through reducing the ischemia induced BBB permeability as a consequence of

  16. An engineered heparin-binding form of VEGF-E (hbVEGF-E). Biological effects in vitro and mobilizatiion of precursor cells.

    PubMed

    Heil, Matthias; Mitnacht-Krauss, Rita; Issbrücker, Katja; van den Heuvel, Joop; Dehio, Christoph; Schaper, Wolfgang; Clauss, Matthias; Weich, Herbert A

    2003-01-01

    Vascular endothelial growth factor (VEGF-A) is the founding member of a family of angiogenic proteins with various binding abilities to three cognate VEGF receptors. Previously, a gene encoding from the genome of parapox orf virus (OV) with about 25% amino acid identity to mammalian VEGF-A was named VEGF-E and shown to bind and specifically activate the vascular endothelial growth factor receptor VEGFR-2 (KDR/flk-1). Here, we have generated a novel heparin-binding form of VEGF-E by introducing the heparin-domain of the human VEGF-A(165) splice variant into the viral VEGF-E protein. Recombinant heparin-binding VEGF-E (hbVEGF-E) is shown to stimulate proliferation and sprout formation of macro- and microvascular endothelial cells to a similar extent as the parental OV-VEGF-E but fails to activate peripheral mononuclear cells. However, hbVEGF-E is more potent in binding competition assays with primary human endothelial cells when compared to the OV-VEGF-E. This can be explained by our finding that binding of hbVEGF-E but not of parental OV-VEGF-E to the VEGFR-2 is strongly increased by the addition of neuropilin-1 (NP-1), a cognate co-receptor for VEGF-A. The engineered hbVEGF-E was compared with the VEGFR-1 selective and also heparin-binding form of placenta growth factor (PlGF-2) in vivo. Both heparin-binding homologues induced mobilization of endothelial progenitor cells from the bone marrow and gave rise to similar colony numbers of myeloic cells in a colony-forming assay. These findings suggest that both VEGFR-1 and VEGFR-2 are involved in stem cell mobilization.

  17. Different but synergistic effects of bone marrow-derived VEGFR2+ and VEGFR2−CD45+ cells during hepatocellular carcinoma progression

    PubMed Central

    Zhu, Xiaolin; Zhou, Hongyuan; Luo, Jingtao; Cui, Yunlong; Li, Huikai; Zhang, Wei; Fang, Feng; Li, Qiang; Zhang, Ti

    2017-01-01

    Hepatocellular carcinoma (HCC) is the second leading cause of cancer-associated mortality worldwide in men. Bone marrow-derived cells (BMDCs), including circulating endothelial progenitor cells, have been reported to be involved in the progression of HCC. The complexity of BMDCs inspires further interest in the study of HCC. In the present study, highly metastatic HCC models with BM function deficiency/reconstruction were established by sublethal irradiation/BM transplantation. The effects of vascular endothelial growth factor receptor-2 (VEGFR2)+ or VEGFR2−/cluster of differentiation 45 (CD45)+ BMDCs on HCC growth were evaluated. VEGFR2+ and VEGFR2−CD45+ BMDCs facilitated the recovery of BM function and promoted tumor growth, while the enhancement of tumor growth by VEGFR2−CD45+ BMDCs was independent of VEGFR2+ BMDCs. BM-derived CD45+CD133+ and VEGFR2+CD133+ cells synergistically played a role in the different stages during HCC progression. In conclusion, different types of BMDCs exhibit effects on HCC tumor growth in a coordinated manner. PMID:28123523

  18. Foretinib inhibits angiogenesis, lymphangiogenesis and tumor growth of pancreatic cancer in vivo by decreasing VEGFR-2/3 and TIE-2 signaling

    PubMed Central

    Chen, Hsiu-Mei; Tsai, Chia-Hua; Hung, Wen-Chun

    2015-01-01

    Foretinib, a multiple kinase inhibitor undergoing clinical trials, could suppress the activity of hepatocyte growth factor (HGF) receptor c-MET and vascular endothelial growth factor receptor-2 (VEGFR-2). In addition, Foretinib may inhibit two critical lymphangiogenic signaling receptors VEGFR-3 and TIE-2. However, the effect of Foretinib on lymphatic endothelial cells (LECs) in vitro and lymphangiogenesis in vivo is still unknown. We found Foretinib decreased basal- and HGF-induced c-MET activity at low concentrations. However, Foretinib only reduced the proliferation of pancreatic cancer cells at high concentration reflecting the intrinsic chemoresistance of pancreatic cancer cells. Foretinib inhibited VEGF-A, VEGF-C and Angiopoetin-2 (ANG-2)-stimulated tube formation and sprouting of LECs by reducing VEGFR-2, VEGFR-3 and TIE-2 activation and increased apoptosis of LECs. In xenograft animal study, Foretinib suppressed tumor growth by inhibiting proliferation, angiogenesis and lymphangiogenesis. Additionally, Foretinib inhibited angiogenesis and lymphangiogenesis more significantly and exhibited low detrimental effect in orthotopic animal study. Collectively, we suggested that Foretinib simultaneously inhibits cancer cells and LECs to reduce pancreatic tumor growth in vivo and demonstrated for the first time that Foretinib suppresses angiogenesis and lymphangiogenesis by blocking VEGFR-2/3 and TIE-2 signaling. PMID:25909285

  19. Ricinus communis agglutinin I leads to rapid down-regulation of VEGFR-2 and endothelial cell apoptosis in tumor blood vessels.

    PubMed

    You, Weon-Kyoo; Kasman, Ian; Hu-Lowe, Dana D; McDonald, Donald M

    2010-04-01

    Ricinus communis agglutinin I (RCA I), a galactose-binding lectin from castor beans, binds to endothelial cells at sites of plasma leakage, but little is known about the amount and functional consequences of binding to tumor endothelial cells. We addressed this issue by examining the effects of RCA I on blood vessels of spontaneous pancreatic islet-cell tumors in RIP-Tag2 transgenic mice. After intravenous injection, RCA I bound strongly to tumor vessels but not to normal blood vessels. At 6 minutes, RCA I fluorescence of tumor vessels was largely diffuse, but over the next hour, brightly fluorescent dots appeared as the lectin was internalized by endothelial cells. RCA I injection led to a dose- and time-dependent decrease in vascular endothelial growth factor receptor-2 (VEGFR-2) immunoreactivity in tumor endothelial cells, with 95% loss over 6 hours. By comparison, VEGFR-3, CD31, and CD105 had decreases in the range of 21% to 33%. Loss of VEGFR-2 was followed by increased activated caspase-3 in tumor vessels. Prior inhibition of VEGF signaling by AG-028262 decreased RCA I binding and internalization into tumor vessels. These findings indicate RCA I preferentially binds to and is internalized by tumor endothelial cells, which leads to VEGFR-2 down-regulation, endothelial cell apoptosis, and tumor vessel regression. Together, the results illustrate the selective impact of RCA I on VEGF signaling in tumor blood vessels.

  20. Ricinus communis Agglutinin I Leads to Rapid Down-Regulation of VEGFR-2 and Endothelial Cell Apoptosis in Tumor Blood Vessels

    PubMed Central

    You, Weon-Kyoo; Kasman, Ian; Hu-Lowe, Dana D.; McDonald, Donald M.

    2010-01-01

    Ricinus communis agglutinin I (RCA I), a galactose-binding lectin from castor beans, binds to endothelial cells at sites of plasma leakage, but little is known about the amount and functional consequences of binding to tumor endothelial cells. We addressed this issue by examining the effects of RCA I on blood vessels of spontaneous pancreatic islet-cell tumors in RIP-Tag2 transgenic mice. After intravenous injection, RCA I bound strongly to tumor vessels but not to normal blood vessels. At 6 minutes, RCA I fluorescence of tumor vessels was largely diffuse, but over the next hour, brightly fluorescent dots appeared as the lectin was internalized by endothelial cells. RCA I injection led to a dose- and time-dependent decrease in vascular endothelial growth factor receptor-2 (VEGFR-2) immunoreactivity in tumor endothelial cells, with 95% loss over 6 hours. By comparison, VEGFR-3, CD31, and CD105 had decreases in the range of 21% to 33%. Loss of VEGFR-2 was followed by increased activated caspase-3 in tumor vessels. Prior inhibition of VEGF signaling by AG-028262 decreased RCA I binding and internalization into tumor vessels. These findings indicate RCA I preferentially binds to and is internalized by tumor endothelial cells, which leads to VEGFR-2 down-regulation, endothelial cell apoptosis, and tumor vessel regression. Together, the results illustrate the selective impact of RCA I on VEGF signaling in tumor blood vessels. PMID:20185574

  1. Integrating Murine and Clinical Trials with Cabozantinib to Understand Roles of MET and VEGFR-2 as Targets for Growth Inhibition of Prostate Cancer

    PubMed Central

    Varkaris, Andreas; Corn, Paul G.; Parikh, Nila U.; Efstathiou, Eleni; Song, Jian H.; Lee, Yu-Chen; Aparicio, Ana; Hoang, Anh G.; Gaur, Sanchaika; Thorpe, Lynnelle; Maity, Sankar N.; Eli, Menashe Bar; Czerniak, Bogdan A.; Shao, Yiping; Alauddin, Mian; Lin, Sue-Hwa; Logothetis, Christopher J.; Gallick, Gary E.

    2015-01-01

    Purpose We performed parallel investigations in cabozantinib-treated patients in a Phase 2 trial and simultaneously in Patient-derived Xenograft (PDX) models to better understand the roles of MET and VEGFR-2 as targets for prostate cancer therapy. Experimental Design In the clinical trial, radiographic imaging and serum markers were examined, as well as molecular markers in tumors from bone biopsies. In mice harboring PDX intrafemurally or subcutaneously, cabozantinib effects on tumor growth, MET, PDX in which MET was silenced, VEGFR-2, bone turnover, angiogenesis and resistance were examined. Results In responsive patients and PDX, islets of viable, p-MET-positive tumor cells persisted, which rapidly regrew after drug withdrawal. Knockdown of MET in PDX did not affect tumor growth in mice, nor did it affect cabozantinib-induced growth inhibition, but did lead to induction of FGFR-1. Inhibition of VEGFR-2 and MET in endothelial cells reduced the vasculature, leading to necrosis. However, each islet of viable cells surrounded a VEGFR-2-negative vessel. Reduction of bone turnover was observed in both cohorts. Conclusion Our studies demonstrate that MET in tumor cells is not a persistent therapeutic target for metastatic CRPC, but inhibition of VEGF-R2 and MET in endothelial cells and direct effects on osteoblasts are responsible for cabozantinib-induced tumor inhibition. However, vascular heterogeneity represents one source of primary therapy resistance, whereas induction of FGFR-1 in tumor cells suggests a potential mechanism of acquired resistance. Thus, integrated cross-species investigations demonstrate the power of combining preclinical models with clinical trials to understand mechanisms of activity and resistance of investigational agents. PMID:26272062

  2. Anti-angiogenic activity of thienopyridine derivative LCB03-0110 by targeting VEGFR-2 and JAK/STAT3 Signalling.

    PubMed

    Kim, Byung-Hak; Lee, Yoonji; Yoo, Hyun; Cui, Minghua; Lee, Sungwoon; Kim, Sun Young; Cho, Jong Un; Lee, Hyangsook; Yang, Beom-Seok; Kwon, Young-Guen; Choi, Sun; Kim, Tae-Yoon

    2015-07-01

    Vascular endothelial growth factor receptor-2 (VEGFR-2) and Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signalling are important for tumor angiogenesis and metastasis. In this study, we identified (3-(2-(3-(morpholinomethyl)phenyl)thieno[3,2-b]pyridin-7-ylamino)phenol (LCB03-0110) as a potent angiogenesis inhibitor. LCB03-0110 inhibited VEGFR-2 and JAK/STAT3 signalling in primary cultured human endothelial cells and cancer cells. An in vitro kinase assay and molecular modelling revealed that LCB03-0110 inhibited VEGFR-2, c-SRC and TIE-2 kinase activity via preferential binding at the ATP-binding site of their kinases. LCB03-0110 successfully occupied the hydrophobic pocket of VEGFR-2, c-SRC and TIE-2. LCB03-0110 also inhibited hypoxia-induced HIF/STAT3 and EGF- or angiopoietin-induced signalling cascades. In addition, LCB03-0110 inhibited VEGF-induced proliferation, viability, migration and capillary-like tube formation. LCB03-0110 also suppressed the sprouting of endothelial cells in the rat aorta and the formation of new blood vessels in the mouse Matrigel plug assay, but also suppressed pulmonary metastasis and tumor xenograft in mice. Our results suggest that LCB03-0110 is a potential candidate small molecule for blocking angiogenesis mediated by aberrant activation of VEGFR-2 and JAK/STAT3 signalling. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

    PubMed

    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.

  4. VEGFR2 Functions As an H2S-Targeting Receptor Protein Kinase with Its Novel Cys1045–Cys1024 Disulfide Bond Serving As a Specific Molecular Switch for Hydrogen Sulfide Actions in Vascular Endothelial Cells

    PubMed Central

    Tao, Bei-Bei; Liu, Shu-Yuan; Zhang, Cai-Cai; Fu, Wei; Cai, Wen-Jie; Wang, Yi; Shen, Qing; Wang, Ming-Jie; Chen, Ying; Zhang, Li-Jia; Zhu, Yi-Zhun

    2013-01-01

    Abstract Aims: The potential receptor for hydrogen sulfide (H2S) remains unknown. Results: H2S could directly activate vascular endothelial growth factor receptor 2 (VEGFR2) and that a small interfering RNA (siRNA)-mediated knockdown of VEGFR2 inhibited H2S-induced migration of human vascular endothelial cells. H2S promoted angiogenesis in Matrigel plug assay in mice and this effect was attenuated by a VEGF receptor inhibitor. Using tandem mass spectrometry (MS), we identified a new disulfide complex located between Cys1045 and Cys1024 within VEGFR2 that was labile to H2S-mediated modification. Kinase activity of the mutant VEGFR2 (C1045A) devoid of the Cys1045–Cys1024 disulfide bond was significantly higher than wild-type VEGFR2. Transfection with vectors expressing VEGFR2 (C1045A) caused a significant increase in cell migration, while the migration-promoting effect of H2S disappeared in the cells transfected with VEGFR2 (C1045A). Therefore, the Cys1045–Cys1024 disulfide bond serves as an intrinsic inhibitory motif and functions as a molecular switch for H2S. The formation of the Cys1045–Cys1024 disulfide bond disrupted the integrity of the active conformation of VEGFR2. Breaking the Cys1045–Cys1024 disulfide bond recovered the active conformation of VEGFR2. This motif was prone to a nucleophilic attack by H2S via an interaction of their frontier molecular orbitals. siRNA-mediated knockdown of cystathionine γ-lyase attenuated migration of vascular endothelial cells induced by VEGF or moderate hypoxia. Innovation and Conclusion: The study provides the first piece of evidence of a molecular switch in H2S-targeting receptor protein kinase in H2S-induced angiogenesis and that may be applicable to additional kinases containing functionally important disulfide bonds in mediating various H2S actions. Antioxid. Redox Signal. 19, 448–464. PMID:23199280

  5. VEGFR-2 Expression in Glioblastoma Multiforme Depends on Inflammatory Tumor Microenvironment

    PubMed Central

    Jaal, Jana; Kase, Marju; Minajeva, Ave; Saretok, Mikk; Adamson, Aidi; Junninen, Jelizaveta; Metsaots, Tõnis; Jõgi, Tõnu; Joonsalu, Madis; Vardja, Markus; Asser, Toomas

    2015-01-01

    Glioblastoma multiforme (GBM) is one of the most angiogenic tumors. However, antiangiogenic therapy has not shown significant clinical efficacy. The aim of our study was to evaluate the impact of inflammatory tumor microenvironment on the expression of vascular endothelial growth factor receptor 2 (VEGFR-2). Surgically excised primary GBM tissues were histologically examined for overall extent of inflammation (score 1–3). After immunohistochemistry, the tissue expression of ICAM-1 (optical density), the number of VEGFR-2 positive (VEGFR-2+) blood vessels (per microscopic field), and the endothelial staining intensity of VEGFR-2 (score 0–3) were determined. In GBM, the extent of inflammation was 1.9 ± 0.7 (group mean ± SD). Mean optical density of inflammatory mediator ICAM-1 was 57.0 ± 27.1 (pixel values). The number of VEGFR-2+ blood vessels and endothelial VEGFR-2 staining intensity were 6.2 ± 2.4 and 1.2 ± 0.8, respectively. A positive association was found between endothelial VEGFR-2 staining intensity and the extent of inflammation (p = 0.005). Moreover, VEGFR-2 staining intensity correlated with the expression level of ICAM-1 (p = 0.026). The expression of VEGFR-2, one of the main targets of antiangiogenic therapy, depends on GBM microenvironment. Higher endothelial VEGFR-2 levels were seen in the presence of more pronounced inflammation. Target dependence on inflammatory tumor microenvironment has to be taken into consideration when treatment approaches that block VEGFR-2 signaling are designed. PMID:26798546

  6. Common variants upstream of KDR encoding VEGFR2 and in TTC39B associate with endometriosis

    PubMed Central

    Steinthorsdottir, Valgerdur; Thorleifsson, Gudmar; Aradottir, Kristrun; Feenstra, Bjarke; Sigurdsson, Asgeir; Stefansdottir, Lilja; Kristinsdottir, Anna M.; Zink, Florian; Halldorsson, Gisli H.; Munk Nielsen, Nete; Geller, Frank; Melbye, Mads; Gudbjartsson, Daniel F.; Geirsson, Reynir T.; Thorsteinsdottir, Unnur; Stefansson, Kari

    2016-01-01

    We conducted a genome-wide association scan (GWAS) of endometriosis using 25.5 million sequence variants detected through whole-genome sequencing (WGS) of 8,453 Icelanders and imputed into 1,840 cases and 129,016 control women, followed by testing of associated variants in Danish samples. Here we report the discovery of a new endometriosis susceptibility locus on 4q12 (rs17773813[G], OR=1.28; P=3.8 × 10−11), upstream of KDR encoding vascular endothelial growth factor receptor 2 (VEGFR2). The variant correlates with disease severity (P=0.0046) when moderate/severe endometriosis cases are tested against minimal/mild cases. We further report association of rs519664[T] in TTC39B on 9p22 with endometriosis (P=4.8 × 10−10; OR=1.29). The involvement of KDR in endometriosis risk highlights the importance of the VEGF pathway in the pathogenesis of the disease. PMID:27453397

  7. Core Replacements in a Potent Series of VEGFR-2 Inhibitors and Their Impact on Potency, Solubility, and hERG

    PubMed Central

    2016-01-01

    Anti-VEGF therapy has been a clinically validated treatment of age-related macular degeneration (AMD). We have recently reported the discovery of indole based oral VEGFR-2 inhibitors that provide sustained ocular retention and efficacy in models of wet-AMD. We disclose herein the synthesis and the biological evaluation of a series of novel core replacements as an expansion of the reported indole based VEGFR-2 inhibitor series. Addition of heteroatoms to the existing core and/or rearranging the heteroatoms around the 6–5 bicyclic ring structure produced a series of compounds that generally retained good on-target potency and an improved solubility profile. The hERG affinity was proven not be dependent on the change in lipophilicity through alteration of the core structure. A serendipitous discovery led to the identification of a new indole-pyrimidine connectivity: from 5-hydroxy to 6-hydroxyindole with potentially vast implication on the in vitro/in vivo properties of this class of compounds. PMID:27096041

  8. Aberrant, ectopic expression of VEGF and VEGF receptors 1 and 2 in malignant colonic epithelial cells. Implications for these cells growth via an autocrine mechanism

    SciTech Connect

    Ahluwalia, Amrita; Jones, Michael K.; Szabo, Sandor; Tarnawski, Andrzej S.

    2013-08-09

    Highlights: •Malignant colonic epithelial cells express VEGF and its receptors. •Cultured colon cancer cells secrete VEGF into the medium. •Inhibition of VEGF receptor significantly decreases colon cancer cell proliferation. •VEGF is critical for colon cancer cell growth. -- Abstract: Vascular endothelial growth factor A (referred to as VEGF) is implicated in colon cancer growth. Currently, the main accepted mechanism by which VEGF promotes colon cancer growth is via the stimulation of angiogenesis, which was originally postulated by late Judah Folkman. However, the cellular source of VEGF in colon cancer tissue; and, the expression of VEGF and its receptors VEGF-R1 and VEGF-R2 in colon cancer cells are not fully known and are subjects of controversy. Material and methods: We examined and quantified expression of VEGF, VEGF-R1 and VEGF-R2 in three different human colonic tissue arrays containing sections of adenocarcinoma (n = 43) and normal mucosa (n = 41). In human colon cancer cell lines HCT116 and HT29 and normal colon cell lines NCM356 and NCM460, we examined expression of VEGF, VEGF-R1 and VEGF-R2 mRNA and protein, VEGF production and secretion into the culture medium; and, the effect of a potent, selective inhibitor of VEGF receptors, AL-993, on cell proliferation. Results: Human colorectal cancer specimens had strong expression of VEGF in cancer cells and also expressed VEGF-R1 and VEGF-R2.In vitro studies showed that human colon cancer cell lines, HCT116 and HT29, but not normal colonic cell lines, express VEGF, VEGF-R1 and VEGF-R2 and secrete VEGF into the medium up to a concentration 2000 pg/ml within 48 h. Furthermore, we showed that inhibition of VEGF receptors using a specific VEGF-R inhibitor significantly reduced proliferation (by >50%) of cultured colon cancer cell lines. Conclusions: Our findings support the contention that VEGF generated by colon cancer cells stimulates their growth directly through an autocrine mechanism that is

  9. Electrochemical monitoring of an important biomarker and target protein: VEGFR2 in cell lysates

    PubMed Central

    Wei, Tianxiang; Tu, Wenwen; Zhao, Bo; Lan, Yaqian; Bao, Jianchun; Dai, Zhihui

    2014-01-01

    Vascular endothelial growth factor receptor 2 (VEGFR2) is a potential cell-type biomarker in clinical diagnoses. Besides, it's the target protein of many tyrosine kinase inhibitors and its expression significantly associates with clinical performance of these inhibitors. VEGFR2 detection provides an early warning for diseases and a basis for therapy and drug screening. Some methods have been developed for VEGFR2 determination. However, they are usually performed indirectly and complexly. Herein, an electrochemical biosensing platform for VEGFR2 analysis has been first proposed. It can detect the total concentrations of the VEGFR2 protein in cells lysates directly and can be used to monitor the changes of VEGFR2 expression levels induced by treatments of different inhibitors. Moreover, the inhibitor-VEGFR2 interactions are illuminated through theoretical simulation. The simulation results agree well with the experimental data, indicating the veracity of the proposed method. The electrochemical detection methodology for VEGFR2 would be promising in clinical diagnosis and drug screening. PMID:24496270

  10. 1-Aryl-3-[4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl]ureas as VEGFR-2 Tyrosine Kinase Inhibitors: Synthesis, Biological Evaluation, and Molecular Modelling Studies

    PubMed Central

    Soares, Pedro; Costa, Raquel; Froufe, Hugo J. C.; Calhelha, Ricardo C.; Peixoto, Daniela; Ferreira, Isabel C. F. R.; Abreu, Rui M. V.; Soares, Raquel; Queiroz, Maria-João R. P.

    2013-01-01

    The vascular endothelial growth factor receptor-2 (VEGFR-2) is a tyrosine kinase receptor involved in the growth and differentiation of endothelial cells that are implicated in tumor-associated angiogenesis. In this study, novel 1-aryl-3-[4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl]ureas were synthesized and evaluated for the VEGFR-2 tyrosine kinase inhibition. Three of these compounds showed good VEGFR-2 inhibition presenting low IC50 values (150–199 nM) in enzymatic assays, showing also a significant proliferation inhibition of VEGF-stimulated human umbilical vein endothelial cells (HUVECs) at low concentrations (0.5–1 µM), using the Bromodeoxyuridine (BrdU) assay, not affecting cell viability. The determination of the total and phosphorylated (active) VEGFR-2 was performed by western blot, and it was possible to conclude that the compounds significantly inhibit the phosphorylation of the receptor at 1 µM pointing to their antiproliferative mechanism of action in HUVECs. The molecular rationale for inhibiting the tyrosine kinase domain of VEGFR-2 was also performed and discussed using molecular docking studies. PMID:23936775

  11. The Structural Basis for the Function of Two Anti-VEGF Receptor 2 Antibodies

    SciTech Connect

    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.

  12. The Structural Basis for the Function of Two Anti-VEGF Receptor 2 Antibodies

    SciTech Connect

    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

    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.

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

    SciTech Connect

    Gu, Fang; Li, Xiuli; Kong, Jian; Pan, Bing; Sun, Min; Zheng, Lemin; Yao, Yuanqing

    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.

  14. PET Imaging of VEGFR-2 Expression in Lung Cancer with 64Cu-Labeled Ramucirumab

    PubMed Central

    Luo, Haiming; England, Christopher G.; Graves, Stephen A.; Sun, Haiyan; Liu, Glenn; Nickles, Robert J.; Cai, Weibo

    2015-01-01

    Lung cancer accounts for 17% of cancer-related deaths worldwide, and most patients present with locally advanced or metastatic disease. Novel PET imaging agents for assessing vascular endothelial growth factor receptor-2 (VEGFR-2) expression can be used for detecting VEGFR-2–positive malignancies and subsequent monitoring of therapeutic response to VEGFR-2–targeted therapies. Here, we report the synthesis and characterization of the antibody-based imaging agent for PET imaging of VEGFR-2 expression in vivo. Methods Ramucirumab (named RamAb), a fully humanized IgG1 monoclonal antibody, was conjugated to 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) and labeled with 64Cu. Flow cytometry analysis and microscopy studies were performed to compare the VEGFR-2 binding affinity of RamAb and NOTA-RamAb. PET imaging and biodistribution studies were performed in nude mice bearing HCC4006 and A549 xenograft tumors. Ex vivo histopathology was performed to elucidate the expression patterns of VEGFR-2 in different tissues and organs to validate in vivo results. Results Flow cytometry examination revealed the specific binding capacity of FITC-RamAb to VEGFR-2, and no difference in VEGFR-2 binding affinity was seen between RamAb and NOTA-RamAb. After being labeled with 64Cu, PET imaging revealed specific and prominent uptake of 64Cu-NOTA-RamAb in VEGFR-2–positive HCC4006 tumors (9.4 ± 0.5 percentage injected dose per gram at 48 h after injection; n = 4) and significantly lower uptake in VEGFR-2–negative A549 tumors (4.3 ± 0.2 percentage injected dose per gram at 48 h after injection; n = 3). Blocking experiments revealed significantly lower uptake in HCC4006 tumors, along with histology analysis, further confirming the VEGFR-2 specificity of 64Cu-NOTA-RamAb. Conclusion This study provides initial evidence that 64Cu-NOTA-RamAb can function as a PET imaging agent for visualizing VEGFR-2 expression in vivo, which may also find

  15. Anti-tumor angiogenesis effect of a new compound: B-9-3 through interference with VEGFR2 signaling.

    PubMed

    Ma, Qin; Chen, Wei; Chen, Wen

    2016-05-01

    B-9-3, a derivative of harmine, was first synthesized in our laboratory. We have reported that B-9-3 has an anti-proliferative effect against human lung cancer cells via induction of apoptosis and inhibition of cell migration. In the present study, we first studied that the anti-tumor angiogenesis effect and the molecular mechanism of B-9-3-induced tumor vascular degrade and mortify in lung cancer. In vitro, the results showed that B-9-3 selectively inhibited the proliferation of endothelial cells IC50 = 6.16 μg/ml) and vascular fibroblasts (IC50 = 12.59 μg/ml) and induced regression of tumor cells of the following: Lewis lung carcinoma (LLC), Mouse fore-stomach carcinoma (MFC), Human ovarian cancer (SK-OV-3), and prostate cancer (22RV1). Moreover, B-9-3 could significantly increase the apoptosis rate (80.95 %) of vascular endothelial cells, while inhibiting migration of endothelial cells, capillary tube formation of endothelial cells, neovascularization of the rat thoracic aorta ring, and the angiogenesis of chick chorioallantoic membrane (CAM) predominantly through blocking VEGFR2 signaling pathway. In vivo, we investigated the anti-tumor rate and the signal transduction mechanism of B-9-3 by LCC-bearing C57BL/6 mice. The data showed that the tumor inhibition ratio of high dose (20 mg/kg) of B-9-3 was 72.9 %, and quantification of CD34 marker indicated a marked reduction in the number of neovessels after B-9-3 treatment as compared with control group (66.87 %). Remarkably, using IHC and q-RT-PCR, we found that downregulation of the expression of VEGFR2, VEGF-A, and TGFβ1 in tumor confers enhancement to the angiogenesis effect of B-9-3. These data suggest that the angiogenesis inhibitor B-9-3 selectively induces apoptosis of endothelial cells, in part through disruption of VEGF-A/VEGFR2 signaling.

  16. Formononetin promotes early fracture healing through stimulating angiogenesis by up-regulating VEGFR-2/Flk-1 in a rat fracture model.

    PubMed

    Huh, Jeong-Eun; Kwon, Na-Hyun; Baek, Young-Hyun; Lee, Jae-Dong; Choi, Do-Young; Jingushi, Seiya; Kim, Kang-il; Park, Dong-Suk

    2009-11-01

    Plant-derived phytoestrogens have bone protective effects, but the molecular mechanism behind these effects remains unclear. This study is aimed at fully characterizing the fracture healing process of formononetin, and investigating the mechanism underlying angiogenesis in calluses of a rat fracture model. Femoral fractures were produced in 2-month-old Sprague-Dawley rats. A 20 microg/kg or 200 microg/kg dose of formononetin was orally administrated once a day during the healing period of 21 days. The results showed that in the early stage of chondrogenesis (days 3), formononetin significantly increased the number of vessels, and expression of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR-2/flk-1) compared with control. However, the larger dose of formononetin had no significant difference on expression of VEGF and VEGFR-2/Flk-1 compared with that of the smaller dose of formononetin. After 7 days of administration, formononetin markedly induced differentiation of mesenchymal stem cells in the fracture site. After 14 days, gene expression of mesenchymal progenitors such as alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OPN) and collagen type I (Col I), indicating osteogenic differentiation, was markedly stimulated by formononetin compared with control. These results suggest that formononetin promotes early fracture healing through angiogenesis activation in the early stage of fracture repair, and osteogenesis acceleration in the later stages, and thus may be beneficial for fracture healing.

  17. Anticancer activity of TTAC-0001, a fully human anti-vascular endothelial growth factor receptor 2 (VEGFR-2/KDR) monoclonal antibody, is associated with inhibition of tumor angiogenesis.

    PubMed

    Kim, Dong Geon; Jin, Younggeon; Jin, Juyoun; Yang, Heekyoung; Joo, Kyeung Min; Lee, Weon Sup; Shim, Sang Ryeol; Kim, Sung-Woo; Yoo, Jinsang; Lee, Sang Hoon; Yoo, Jin-San; Nam, Do-Hyun

    2015-01-01

    Vascular endothelial growth factor (VEGF) and its receptors are considered the primary cause of tumor-induced angiogenesis. Specifically, VEGFR-2/kinase insert domain receptor (KDR) is part of the major signaling pathway that plays a significant role in tumor angiogenesis, which is associated with the development of various types of tumor and metastasis. In particular, KDR is involved in tumor angiogenesis as well as cancer cell growth and survival. In this study, we evaluated the therapeutic potential of TTAC-0001, a fully human antibody against VEGFR-2/KDR. To assess the efficacy of the antibody and pharmacokinetic (PK) relationship in vivo, we tested the potency of TTAC-0001 in glioblastoma and colorectal cancer xenograft models. Antitumor activity of TTAC-0001 in preclinical models correlated with tumor growth arrest, induction of tumor cell apoptosis, and inhibition of angiogenesis. We also evaluated the combination effect of TTAC-0001 with a chemotherapeutic agent in xenograft models. We were able to determine the relationship between PK and the efficacy of TTAC-0001 through in vivo single-dose PK study. Taken together, our data suggest that targeting VEGFR-2 with TTAC-0001 could be a promising approach for cancer treatment.

  18. Increased VEGFR2 and MMP9 protein levels are associated with epithelial dysplasia grading.

    PubMed

    de Carvalho Fraga, Carlos Alberto; Farias, Lucyana Conceição; de Oliveira, Marcos Vinícius Macedo; Domingos, Patrícia Luciana Batista; Pereira, Camila Santos; Silva, Thiago Fonseca; Roy, Ashbeel; Gomez, Ricardo Santiago; de Paula, Alfredo Maurício Batista; Guimarães, André Luiz Sena

    2014-12-01

    The present study aimed to compare levels of VEGFR2 and MMP-9 among control, epithelial dysplasia (ED) and oral squamous cell carcinoma (OSCC) groups. We analyzed 48 patients with oral leukoplakia (OL), 20 patients with OSCC and 21 patients without OL and OSCC. Immunohistochemistry of VEGFR2 and MMP9 were performed and compared among groups. Analysis of tissue immunolocalization of VEGFR2 and MMP-9 assumed non-parametrical distribution and comparison between groups was performed using the Mann-Whitney and Kruskal-Wallis statistical tests. VEGFR2 and MMP9 immunoexpression appeared to correlate with the degree of dysplasia and was observed to increase in lesions with more severe dysplasia as compared to those with lower degrees of dysplasia. Immunoreactivity of MMP-9 was lower in the OL samples compared to the OSCC samples (p = 0.004). We observed no difference in VEGFR2 protein levels between OL and OSCC samples. A positive correlation was found between VEGFR2 and MMP-9 in OL samples (r = +0.452, p = 0.001), however, no correlation was found in OSCC samples (r = -0.042, p = 0.861). In conclusion, the results of the current study suggest that expression of MMP9 and VEGFR2 is associated with ED grading and MMP9 levels are increased in OSCC. Copyright © 2014 Elsevier GmbH. All rights reserved.

  19. Identification of a variant in KDR associated with serum VEGFR2 and pharmacodynamics of pazopanib

    PubMed Central

    Maitland, Michael L.; Xu, Chun-Fang; Cheng, Yu-Ching; Kistner-Griffin, Emily; Ryan, Kathleen A.; Karrison, Theodore G.; Das, Soma; Torgerson, Dara; Gamazon, Eric; Thomeas, Vasiliki; Levine, Matthew R.; Wilson, Paul A.; Bing, Nan; Liu, Yuan; Cardon, Lon R.; Pandite, Lini N.; O’Connell, Jeffrey R.; Cox, Nancy J.; Mitchell, Braxton D.; Ratain, Mark J.; Shuldiner, Alan R.

    2014-01-01

    Purpose Vascular endothelial growth factor receptor (VEGFR) kinases are important drug targets in oncology that affect function of systemic endothelial cells. To discover genetic markers that affect VEGFR inhibitor pharmacodynamics we performed a genome-wide association study of serum soluble vascular endothelial growth factor receptor-2 concentrations [sVEGFR2], a pharmacodynamic biomarker for VEGFR2 inhibitors. Experimental Design We conducted a genome-wide association study (GWAS) of [sVEGFR2] in 736 healthy Old Order Amish volunteers. Gene variants identified from the GWAS were genotyped serially in a cohort of 128 advanced solid tumor patients with baseline [sVEGR2] measurements, and in 121 renal carcinoma patients with [sVEGFR2] measured before and during pazopanib therapy. Results rs34231037 (C482R) in KDR, the gene encoding sVEGFR2 was found to be highly associated with [sVEGFR2], explaining 23% of the variance (p=2.7×10−37). Association of rs34231037 with [sVEGFR2] was replicated in 128 patients with cancer with comparable effect size (p = 0.025). Furthermore rs34231037 was a significant predictor of changes in [sVEGFR2] in response to pazopanib (p = 0.01). Conclusion Our findings suggest that genome-wide analysis of phenotypes in healthy populations can expedite identification of candidate pharmacogenetic markers. Genotyping for germ-line variants in KDR may have clinical utility in identifying cancer patients with unusual sensitivity to effects of VEGFR2 kinase inhibitors. PMID:25411163

  20. In vivo monitoring of angiogenesis inhibition via down-regulation of mir-21 in a VEGFR2-luc murine breast cancer model using bioluminescent imaging.

    PubMed

    Zhao, Dongliang; Tu, Yingfeng; Wan, Lin; Bu, Lihong; Huang, Tao; Sun, Xilin; Wang, Kai; Shen, Baozhong

    2013-01-01

    MicroRNA-21 (miR-21) is overexpressed in a wide range of cancers and involved in tumor proliferation and metastasis. However, the potential function of miR-21 in regulating tumor angiogenesis has been little disclosed. In this study, we treated the cultured 4T1 murine breast cancer cells and human umbilical vein endothelial cells (HUVECs) with miR-21 mimic, antagomir-21 or negative control (scramble), which were subjected to MTT, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), quantitative Reverse Transcriptase PCR (qRT-PCR) and immunoblotting analysis. In addition, 4T1 cells were implanted beneath the right breast fat pad of the VEGFR2-luc transgenic mice, which were randomly divided into three groups and received saline, antagomir-21 or scramble treatment once respectively after tumor model establishment. Bioluminescent imaging was used to monitor tumor growth and angiogenesis in vivo at 0d, 3d, 5d, 7d, 10d, and 14d after treatment. Mice were killed at the end of study and tumor tissues were collected for use. The results showed that knockdown of miR-21 by antagomir-21 decreased cell proliferation and induced apoptosis via targeting PTEN both in 4T1 cells and HUVECs. We also found the anti-angiogenesis and anti-tumor effects of antagomir-21 in the VEGFR2-luc transgenic mouse model using bioluminescent imaging. Moreover, the Western blotting data revealed that antagomir-21 inhibited tumor angiogenesis through suppressing HIF-1α/VEGF/VEGFR2-associated signaling pathway. In conclusion, the results from current study demonstrate that antagomir-21 can effectively suppress tumor growth and angiogenesis in VEGFR2-luc mouse breast tumor model and bioluminescent imaging can be used as a tool for noninvasively and continuously monitoring tumor angiogenesis in vivo.

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

    PubMed

    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.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27498819

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

    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.

  4. VEGF receptor signaling links inflammation and tumorigenesis in colitis-associated cancer.

    PubMed

    Waldner, Maximilian J; Wirtz, Stefan; Jefremow, André; Warntjen, Moritz; Neufert, Clemens; Atreya, Raja; Becker, Christoph; Weigmann, Benno; Vieth, Michael; Rose-John, Stefan; Neurath, Markus F

    2010-12-20

    Whereas the inhibition of vascular endothelial growth factor (VEGF) has shown promising results in sporadic colon cancer, the role of VEGF signaling in colitis-associated cancer (CAC) has not been addressed. We found that, unlike sporadic colorectal cancer and control patients, patients with CAC show activated VEGFR2 on intestinal epithelial cells (IECs). We then explored the function of VEGFR2 in a murine model of colitis-associated colon cancer characterized by increased VEGFR2 expression. Epithelial cells in tumor tissue expressed VEGFR2 and responded to VEGF stimulation with augmented VEGFR2-mediated proliferation. Blockade of VEGF function via soluble decoy receptors suppressed tumor development, inhibited tumor angiogenesis, and blocked tumor cell proliferation. Functional studies revealed that chronic inflammation leads to an up-regulation of VEGFR2 on IECs. Studies in conditional STAT3 mutant mice showed that VEGFR signaling requires STAT3 to promote epithelial cell proliferation and tumor growth in vivo. Thus, VEGFR-signaling acts as a direct growth factor for tumor cells in CAC, providing a molecular link between inflammation and the development of colon cancer.

  5. VEGF receptor signaling links inflammation and tumorigenesis in colitis-associated cancer

    PubMed Central

    Waldner, Maximilian J.; Wirtz, Stefan; Jefremow, André; Warntjen, Moritz; Neufert, Clemens; Atreya, Raja; Becker, Christoph; Weigmann, Benno; Vieth, Michael; Rose-John, Stefan

    2010-01-01

    Whereas the inhibition of vascular endothelial growth factor (VEGF) has shown promising results in sporadic colon cancer, the role of VEGF signaling in colitis-associated cancer (CAC) has not been addressed. We found that, unlike sporadic colorectal cancer and control patients, patients with CAC show activated VEGFR2 on intestinal epithelial cells (IECs). We then explored the function of VEGFR2 in a murine model of colitis-associated colon cancer characterized by increased VEGFR2 expression. Epithelial cells in tumor tissue expressed VEGFR2 and responded to VEGF stimulation with augmented VEGFR2-mediated proliferation. Blockade of VEGF function via soluble decoy receptors suppressed tumor development, inhibited tumor angiogenesis, and blocked tumor cell proliferation. Functional studies revealed that chronic inflammation leads to an up-regulation of VEGFR2 on IECs. Studies in conditional STAT3 mutant mice showed that VEGFR signaling requires STAT3 to promote epithelial cell proliferation and tumor growth in vivo. Thus, VEGFR-signaling acts as a direct growth factor for tumor cells in CAC, providing a molecular link between inflammation and the development of colon cancer. PMID:21098094

  6. Shock Wave Therapy Enhances Angiogenesis through VEGFR2 Activation and Recycling

    PubMed Central

    Huang, Tien-Hung; Sun, Cheuk-Kwan; Chen, Yi-Ling; Wang, Ching-Jen; Yin, Tsung-Cheng; Lee, Mel S; Yip, Hon-Kan

    2016-01-01

    Although low-energy shock wave (SW) is adopted to treat ischemic diseases because of its pro-angiogenic properties, the underlying mechanism remains unclear. This study is aimed at testing whether SW-induced angiogenesis may be through endothelial vascular endothelial growth factor receptor 2 (VEGFR2) signaling and trafficking. Phosphorylation of VEGFR2- Akt-eNOS axis and production of nitric oxide (NO) were determined in human umbilical vein endothelial cells (HUVECs) treated with SW. Carotid artery in ob/ob mice was treated with SW before evaluation with sprouting assay. Critical limb ischemia was induced in ob/ob mice to evaluate blood flow recovery post-SW treatment. Tube formation and migration assays were also performed with/without SW treatment in the presence/absence of SU5416 (VEGFR2 kinase inhibitor) and siRNA-driven silencing of VEGFR2. Chloroquine was used for disrupting endosome, and Rab11a controlling slow endocytic recycling was silenced with siRNA in vitro. Following SW treatment, augmented ligand-independent phosphorylation in VEGFR2-Akt-eNOS axis and endogenous NO production, increased cellular migration and tube formation and elevated sprouting of carotid artery and blood flow in ischemic limb in ob/ob mice were noted. Moreover, SU5416 and VEGFR2 silencing both inhibited SW-induced angiogenesis. SW-induced angiogenesis, accompanied by increased VEGFR2 protein expression without transcriptional change, was suppressed by chloroquine and Rab11a silencing. We concluded that SW enhanced angiogenesis via ligand-independent activation of VEGFR2 and further prolonged angiogenesis through endosome-to-plasma membrane recycling in endothelial cells. PMID:27925633

  7. CD133+ cancer stem cells promoted by VEGF accelerate the recurrence of hepatocellular carcinoma

    PubMed Central

    Liu, Kai; Hao, Meijun; Ouyang, Yabo; Zheng, Jiasheng; Chen, Dexi

    2017-01-01

    The role of cancer stem cells (CSCs) in inducing the recurrence of hepatocellular carcinoma (HCC) after radiofrequency ablation (RFA) remains unclear. Here, we found that a dramatic increase in plasma vascular endothelial growth factor (VEGF) and an induction of local CD133+ CSCs are associated with early HCC recurrence, suggesting that VEGF expression and tumour stemness contribute to the relapse. In vitro studies demonstrated that VEGF, via activation of VEGFR2, increased the number of CD133+ CSCs and enhanced their capacity for self-renewal by inducing the expression of Nanog. In vivo studies further demonstrated that VEGF-treated CD133+ CSCs formed tumours larger than those developing from unstimulated cells and VEGF pre-treatment increased the tumorigenic cell frequency of primary HCC cells dependently on the presence of Nanog and VEGFR2. In HCC tissue derived from patients with early recurrence, almost all CD133+ cells were Nanog and p-VEGFR2 positive, suggesting that activation of VEGFR2 is critical for RFA-induced tumour stemness in HCC. In summary, RFA-induced VEGF promotes tumour stemness and accelerates tumourigenesis in HCC in a manner dependent on Nanog and VEGFR2, which is valuable for the prediction of HCC recurrence after RFA and the development of novel therapeutics. PMID:28134312

  8. In Silico Discovery of Potential VEGFR-2 Inhibitors from Natural Derivatives for Anti-Angiogenesis Therapy

    PubMed Central

    Li, Jing; Zhou, Nan; Luo, Kun; Zhang, Wei; Li, Xinru; Wu, Chuanfang; Bao, Jinku

    2014-01-01

    Angiogenesis is the growth of new capillaries from existing blood vessels that supply oxygen and nutrients and provide gateways for immune surveillance. Abnormal vessel growth in term of excessive angiogenesis is a hallmark of cancer, inflammatory and eye diseases. VEGFR-2 (vascular endothelial growth factor receptor 2) dominating the process of angiogenesis has led to approval of therapeutic inhibitors and is becoming a promising target for anti-angiogenic drugs. Notwithstanding these successes, the clinical use of current VEGFR-2 blockers is more challenging than anticipated. Taking axitinib as a reference drug, in our study we found three potent VEGFR-2 inhibitors (ZINC08254217, ZINC08254138, and ZINC03838680) from natural derivatives. Each of the three inhibitors acquired a better grid score than axitinib (−62.11) when docked to VEGFR-2. Molecular dynamics simulations demonstrated that ZINC08254217– and ZINC08254138–VEGFR-2 complexes were more stable than axitinib. Similar to bind free energy for axitinib (−54.68 kcal/mol), such for ZINC03838680, ZINC08254217, and ZINC08254138 was −49.37, −43.32, and −32.73 kcal/mol respectively. These results suggested these three compounds could be candidate drugs against angiogenesis, with comparable VEGFR-2 binding affinity of axitinib. Hence findings in our study are able to provide valuable information on discovery of effective anti-angiogenesis therapy. PMID:25216334

  9. Enhanced effect of VEGF165 on L-type calcium currents in guinea-pig cardiac ventricular myocytes.

    PubMed

    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.

  10. Targeting VEGFR-2 in Metastatic Gastric Cancer: Results From a Literature-Based Meta-Analysis.

    PubMed

    Roviello, Giandomenico; Polom, Karol; Roviello, Franco; Marrelli, Daniele; Multari, Andrea Giovanni; Paganini, Giovanni; Pacifico, Chiara; Generali, Daniele

    2017-02-06

    Angiogenesis is a key process in cancer development. We performed a meta-analysis to assess the efficacy and safety of the novel VEGFR-2 inhibitors in patients with metastatic gastric and gastroesophageal junction cancer. A literature-based meta-analysis of randomized controlled trials (RCTs) was undertaken. The primary outcome was the overall survival. The pooled analysis from RCTs on anti-VEGFR-2 inhibitors revealed a significant increase in overall survival (hazard ratio for death: 0.69, 95% confidence interval: 0.55-0.87; p = .002). This study confirms the efficacy of novel anti-VEGFR-2 inhibitors. The future studies of these agents will evaluate alone and in combination with chemotherapy the early line of treatment along with the identification of proper predictive biomarker.

  11. Discovery of Potent VEGFR-2 Inhibitors based on Furopyrimidine and Thienopyrimidne Scaffolds as Cancer Targeting Agents

    PubMed Central

    Aziz, Marwa A.; Serya, Rabah A. T.; Lasheen, Deena S.; Abdel-Aziz, Amal Kamal; Esmat, Ahmed; Mansour, Ahmed M.; Singab, Abdel Nasser B.; Abouzid, Khaled A. M.

    2016-01-01

    Vascular endothelial growth factor receptor-2 (VEGFR-2) plays a crucial role in cancer angiogenesis. In this study, a series of novel furo[2,3-d]pyrimidine and thieno[2,3-d]pyrimidine based-derivatives were designed and synthesized as VEGFR-2 inhibitors, in accordance to the structure activity relationship (SAR) studies of known type II VEGFR-2 inhibitors. The synthesized compounds were evaluated for their ability to in vitro inhibit VEGFR-2 kinase enzyme. Seven compounds (15b, 16c, 16e, 21a, 21b, 21c and 21e) demonstrated highly potent dose-related VEGFR-2 inhibition with IC50 values in nanomolar range, of which the thieno[2,3-d]pyrimidine based-derivatives (21b, 21c and 21e) exhibited IC50 values of 33.4, 47.0 and 21 nM respectively. Moreover, furo[2,3-d]pyrimidine-based derivative (15b) showed the strongest inhibition of human umbilical vein endothelial cells (HUVEC) proliferation with 99.5% inhibition at 10 μM concentration. Consistent with our in vitro findings, compounds (21b and 21e) orally administered at 5 and 10 mg/kg/day for 8 consecutive days demonstrated potent anticancer activity in Erhlich ascites carcinoma (EAC) solid tumor murine model. Such compounds blunted angiogenesis in EAC as evidenced by reduced percent microvessel via decreasing VEGFR-2 phosphorylation with subsequent induction of apoptotic machinery. Furthermore, Miles vascular permeability assay confirmed their antiangiogenic effects in vivo. Intriguingly, such compounds showed no obvious toxicity. PMID:27080011

  12. 2-Methoxyestradiol regulates VEGFR-2 and sFlt-1 expression in human placenta.

    PubMed

    Lee, D K; Nevo, O

    2015-02-01

    2-Methoxyestradiol (2-ME), a metabolite of estradiol, has been identified as an initiator of cytotrophoblast transformation to an invasive phenotype, with low levels implicated with the onset of preeclampsia. Here, we investigated the effects of 2-ME on VEGFR-2, sFlt-1 and HIF1α expression in human placenta. First trimester human placental villous explants were maintained at 3% and 20% O2. Samples were treated with 0.5 μM 2-ME with or without 1 mM DMOG or 0.2 mM CoCl2 for 17 h. Western and qPCR analyses were performed for VEGFR-2, sFlt-1 and HIF1α expression levels. sFlt-1 specific ELISA was also performed on conditioned explant media. Placental explants maintained at 3% O2 revealed decreased protein and transcript levels of VEGFR-2 with increased sFlt-1 and HIF1α. Overnight treatment with 0.5 μM 2-ME rescued altered expression levels of VEGFR-2, sFlt-1 and HIF1α. 2-ME also decreased levels of sFlt-1 in conditioned explant media. While 2-ME treatment rescued decreased levels of VEGFR-2 in DMOG and CoCl2-treated explants, no effect was observed for sFlt-1 levels. Furthermore, 2-ME was observed to further exacerbate elevated HIF1α levels by DMOG and CoCl2. 2-ME rescues altered levels of VEGFR-2, sFlt-1 and HIF1α in hypoxic placental explants, suggesting potential therapeutic measures for the treatment of preeclampsia. However, the unaltered sFlt-1 levels and enhanced HIF1α levels by 2-ME in DMOG and CoCl2 treated explants suggests 2-ME also elicits its effects through HIF1α-independent pathways. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Discovery of Potent VEGFR-2 Inhibitors based on Furopyrimidine and Thienopyrimidne Scaffolds as Cancer Targeting Agents

    NASA Astrophysics Data System (ADS)

    Aziz, Marwa A.; Serya, Rabah A. T.; Lasheen, Deena S.; Abdel-Aziz, Amal Kamal; Esmat, Ahmed; Mansour, Ahmed M.; Singab, Abdel Nasser B.; Abouzid, Khaled A. M.

    2016-04-01

    Vascular endothelial growth factor receptor-2 (VEGFR-2) plays a crucial role in cancer angiogenesis. In this study, a series of novel furo[2,3-d]pyrimidine and thieno[2,3-d]pyrimidine based-derivatives were designed and synthesized as VEGFR-2 inhibitors, in accordance to the structure activity relationship (SAR) studies of known type II VEGFR-2 inhibitors. The synthesized compounds were evaluated for their ability to in vitro inhibit VEGFR-2 kinase enzyme. Seven compounds (15b, 16c, 16e, 21a, 21b, 21c and 21e) demonstrated highly potent dose-related VEGFR-2 inhibition with IC50 values in nanomolar range, of which the thieno[2,3-d]pyrimidine based-derivatives (21b, 21c and 21e) exhibited IC50 values of 33.4, 47.0 and 21 nM respectively. Moreover, furo[2,3-d]pyrimidine-based derivative (15b) showed the strongest inhibition of human umbilical vein endothelial cells (HUVEC) proliferation with 99.5% inhibition at 10 μM concentration. Consistent with our in vitro findings, compounds (21b and 21e) orally administered at 5 and 10 mg/kg/day for 8 consecutive days demonstrated potent anticancer activity in Erhlich ascites carcinoma (EAC) solid tumor murine model. Such compounds blunted angiogenesis in EAC as evidenced by reduced percent microvessel via decreasing VEGFR-2 phosphorylation with subsequent induction of apoptotic machinery. Furthermore, Miles vascular permeability assay confirmed their antiangiogenic effects in vivo. Intriguingly, such compounds showed no obvious toxicity.

  14. Discovery of Novel Benzimidazoles as Potent Inhibitors of TIE-2 and VEGFR-2 Tyrosine Kinase Receptors

    SciTech Connect

    Hasegawa, Masaichi; Nishigaki, Naohiko; Washio, Yoshiaki; Kano, Kazuya; Harris, Philip A.; Sato, Hideyuki; Mori, Ichiro; West, Rob I.; Shibahara, Megumi; Toyoda, Hiroko; Wang, Liping; Nolte, Robert T.; Veal, James M.; Cheung, Mui

    2008-09-12

    We herein disclose a novel chemical series of benzimidazole-ureas as inhibitors of VEGFR-2 and TIE-2 kinase receptors, both of which are implicated in angiogenesis. Structure-activity relationship (SAR) studies elucidated a critical role for the N1 nitrogen of both the benzimidazole (segment E) and urea (segment B) moieties. The SAR results were also supported by the X-ray crystallographic elucidation of the role of the N1 nitrogen and the urea moiety when the benzimidazole-urea compounds were bound to the VEGFR-2 enzyme. The left side phenyl ring (segment A) occupies the backpocket where a 3-hydrophobic substituent was favored for TIE-2 activity.

  15. Altiratinib Inhibits Tumor Growth, Invasion, Angiogenesis, and Microenvironment-Mediated Drug Resistance via Balanced Inhibition of MET, TIE2, and VEGFR2.

    PubMed

    Smith, Bryan D; Kaufman, Michael D; Leary, Cynthia B; Turner, Benjamin A; Wise, Scott C; Ahn, Yu Mi; Booth, R John; Caldwell, Timothy M; Ensinger, Carol L; Hood, Molly M; Lu, Wei-Ping; Patt, Tristan W; Patt, William C; Rutkoski, Thomas J; Samarakoon, Thiwanka; Telikepalli, Hanumaiah; Vogeti, Lakshminarayana; Vogeti, Subha; Yates, Karen M; Chun, Lawrence; Stewart, Lance J; Clare, Michael; Flynn, Daniel L

    2015-09-01

    Altiratinib (DCC-2701) was designed based on the rationale of engineering a single therapeutic agent able to address multiple hallmarks of cancer (1). Specifically, altiratinib inhibits not only mechanisms of tumor initiation and progression, but also drug resistance mechanisms in the tumor and microenvironment through balanced inhibition of MET, TIE2 (TEK), and VEGFR2 (KDR) kinases. This profile was achieved by optimizing binding into the switch control pocket of all three kinases, inducing type II inactive conformations. Altiratinib durably inhibits MET, both wild-type and mutated forms, in vitro and in vivo. Through its balanced inhibitory potency versus MET, TIE2, and VEGFR2, altiratinib provides an agent that inhibits three major evasive (re)vascularization and resistance pathways (HGF, ANG, and VEGF) and blocks tumor invasion and metastasis. Altiratinib exhibits properties amenable to oral administration and exhibits substantial blood-brain barrier penetration, an attribute of significance for eventual treatment of brain cancers and brain metastases. ©2015 American Association for Cancer Research.

  16. VEGFR2 pY949 signalling regulates adherens junction integrity and metastatic spread

    PubMed Central

    Li, Xiujuan; Padhan, Narendra; Sjöström, Elisabet O.; Roche, Francis P.; Testini, Chiara; Honkura, Naoki; Sáinz-Jaspeado, Miguel; Gordon, Emma; Bentley, Katie; Philippides, Andrew; Tolmachev, Vladimir; Dejana, Elisabetta; Stan, Radu V.; Vestweber, Dietmar; Ballmer-Hofer, Kurt; Betsholtz, Christer; Pietras, Kristian; Jansson, Leif; Claesson-Welsh, Lena

    2016-01-01

    The specific role of VEGFA-induced permeability and vascular leakage in physiology and pathology has remained unclear. Here we show that VEGFA-induced vascular leakage depends on signalling initiated via the VEGFR2 phosphosite Y949, regulating dynamic c-Src and VE-cadherin phosphorylation. Abolished Y949 signalling in the mouse mutant Vegfr2Y949F/Y949F leads to VEGFA-resistant endothelial adherens junctions and a block in molecular extravasation. Vessels in Vegfr2Y949F/Y949F mice remain sensitive to inflammatory cytokines, and vascular morphology, blood pressure and flow parameters are normal. Tumour-bearing Vegfr2Y949F/Y949F mice display reduced vascular leakage and oedema, improved response to chemotherapy and, importantly, reduced metastatic spread. The inflammatory infiltration in the tumour micro-environment is unaffected. Blocking VEGFA-induced disassembly of endothelial junctions, thereby suppressing tumour oedema and metastatic spread, may be preferable to full vascular suppression in the treatment of certain cancer forms. PMID:27005951

  17. Oxidized low-density lipoprotein decreases VEGFR2 expression in HUVECs and impairs angiogenesis.

    PubMed

    Zhang, Min; Jiang, Li

    2016-12-01

    Atherosclerosis (AS), which is triggered by endothelial cell injury, evolves into a chronic inflammatory disease. Oxidized low-density lipoprotein (ox-LDL) is an important risk factor for the development of atherosclerosis; ox-LDL induces atherosclerotic plaque formation via scavenging receptors. The present study used ox-LDL-treated human umbilical vein endothelial cells (HUVECs) to investigate the effect of ox-LDL on angiogenesis. ox-LDL decreased HUVEC proliferation by MTT, induced apoptosis by Annexin V-fluorescein isothiocyanate (FITC) staining and markedly suppressed HUVEC tube formation by the Matrigel assay in a dose-dependent manner. Angiogenesis has been correlated with monocyte invasion, plaque instability and atherosclerotic lesion formation. In addition, ox-LDL induced the overproduction of reactive oxygen species using DCFH-DA staining and increased caspase-3 activity. Vascular endothelial growth factor receptor 2 (VEGFR2) were detected by quantitative polymerase chain reaction and western blot analysis and has previously been observed to have a key role in angiogenesis. Furthermore, the present study demonstrated that the abundance of VEGFR2 was decreased in ox-LDL-treated HUVECs. These results suggested that ox-LDL impairs angiogenesis via VEGFR2 degradation, thus suggesting that VEGFR2 may be involved in adaptation to oxidative stress and AS.

  18. Oxidized low-density lipoprotein decreases VEGFR2 expression in HUVECs and impairs angiogenesis

    PubMed Central

    Zhang, Min; Jiang, Li

    2016-01-01

    Atherosclerosis (AS), which is triggered by endothelial cell injury, evolves into a chronic inflammatory disease. Oxidized low-density lipoprotein (ox-LDL) is an important risk factor for the development of atherosclerosis; ox-LDL induces atherosclerotic plaque formation via scavenging receptors. The present study used ox-LDL-treated human umbilical vein endothelial cells (HUVECs) to investigate the effect of ox-LDL on angiogenesis. ox-LDL decreased HUVEC proliferation by MTT, induced apoptosis by Annexin V-fluorescein isothiocyanate (FITC) staining and markedly suppressed HUVEC tube formation by the Matrigel assay in a dose-dependent manner. Angiogenesis has been correlated with monocyte invasion, plaque instability and atherosclerotic lesion formation. In addition, ox-LDL induced the overproduction of reactive oxygen species using DCFH-DA staining and increased caspase-3 activity. Vascular endothelial growth factor receptor 2 (VEGFR2) were detected by quantitative polymerase chain reaction and western blot analysis and has previously been observed to have a key role in angiogenesis. Furthermore, the present study demonstrated that the abundance of VEGFR2 was decreased in ox-LDL-treated HUVECs. These results suggested that ox-LDL impairs angiogenesis via VEGFR2 degradation, thus suggesting that VEGFR2 may be involved in adaptation to oxidative stress and AS. PMID:28105106

  19. Characterizing nanoscale changes in the activity of VEGFR-2 on glioma microvascular endothelial cell membranes using atomic force microscopy.

    PubMed

    Zhou, Dexiang; Zhan, Shengquan; Zhou, Dong; Wang, Peng; Chen, Guangzhong; Qin, Kun; Lin, Xiaofeng

    2017-02-01

    The aim of the current study was to demonstrate the distribution of VEGFR-2 on glioma microvascular endothelial cells on a nanoscale and investigate changes in VEGFR-2 activity following treatment with the VEGFR-2 inhibitor and agonist sorafenib and bradykinin, respectively. Three groups were evaluated in this study: Control glioma microvascular endothelial cells, sorafenib-treated glioma microvascular endothelial cells and bradykinin-treated glioma microvascular endothelial cells. Changes in the activity of VEGFR-2 on the glioma microvascular endothelial cell membranes following treatment with sorafenib and bradykinin were characterized by atomic force microscopy (AFM). Colloidal gold-labeled immune complexes and AFM were used to visualize the distribution of VEGFR-2 on the cell membranes. In the control group, VEGFR-2, which was observed as numerous globular structures, was evenly distributed on the cell surface membranes. The majority of the receptors were active. In the sorafenib group, only a few globular structures were observed on the cell membranes, with a density significantly lower than that in the control group (P<0.01). Furthermore, compared with the control group, fewer of the receptors were active. In the bradykinin group, numerous globular structures were densely distributed on the cell membranes, with a density significantly higher than that in the control group (P<0.01). The distribution and activity of VEGFR-2 on glioma microvascular endothelial cell membranes treated with sorafenib and bradykinin suggested that the activity of VEGFR-2 could be regulated by its inhibitor or agonist.

  20. Characterizing nanoscale changes in the activity of VEGFR-2 on glioma microvascular endothelial cell membranes using atomic force microscopy

    PubMed Central

    Zhou, Dexiang; Zhan, Shengquan; Zhou, Dong; Wang, Peng; Chen, Guangzhong; Qin, Kun; Lin, Xiaofeng

    2017-01-01

    The aim of the current study was to demonstrate the distribution of VEGFR-2 on glioma microvascular endothelial cells on a nanoscale and investigate changes in VEGFR-2 activity following treatment with the VEGFR-2 inhibitor and agonist sorafenib and bradykinin, respectively. Three groups were evaluated in this study: Control glioma microvascular endothelial cells, sorafenib-treated glioma microvascular endothelial cells and bradykinin-treated glioma microvascular endothelial cells. Changes in the activity of VEGFR-2 on the glioma microvascular endothelial cell membranes following treatment with sorafenib and bradykinin were characterized by atomic force microscopy (AFM). Colloidal gold-labeled immune complexes and AFM were used to visualize the distribution of VEGFR-2 on the cell membranes. In the control group, VEGFR-2, which was observed as numerous globular structures, was evenly distributed on the cell surface membranes. The majority of the receptors were active. In the sorafenib group, only a few globular structures were observed on the cell membranes, with a density significantly lower than that in the control group (P<0.01). Furthermore, compared with the control group, fewer of the receptors were active. In the bradykinin group, numerous globular structures were densely distributed on the cell membranes, with a density significantly higher than that in the control group (P<0.01). The distribution and activity of VEGFR-2 on glioma microvascular endothelial cell membranes treated with sorafenib and bradykinin suggested that the activity of VEGFR-2 could be regulated by its inhibitor or agonist. PMID:28352319

  1. Ynamide Click chemistry in development of triazole VEGFR2 TK modulators.

    PubMed

    Vojtičková, Margaréta; Dobiaš, Juraj; Hanquet, Gilles; Addová, Gabriela; Cetin-Atalay, Rengul; Yildirim, Deniz Cansen; Boháč, Andrej

    2015-10-20

    Structure novelty, chemical stability and synthetic feasibility attracted us to design 1,2,3-triazole compounds as potential inhibitors of VEGFR2 tyrosine kinase. Novel triazoles T1-T7 were proposed by oxazole (AAZ from PDB: 1Y6A)/1,2,3-triazole isosteric replacement, molecular modelling and docking. In order to enable synthesis of T1-T7 we developed a methodology for preparation of ynamide 22. Compound 22 was used for all Click chemistry reactions leading to triazoles T1-T3 and T6-T7. Among the obtained products, T1, T3 and T7 specifically bind VEGFR2 TK and modulate its activity by concentration dependent manner. Moreover predicted binding poses of T1-T7 in VEGFR2 TK were similar to the one known for the oxazole inhibitor AAZ (PDB: 1Y6A). Unfortunately the VEGFR2 inhibition by triazoles e.g. T3 and T7 is lower than that determined for their oxazole bioisosters T3-ox and AAZ, resp. Different electronic properties of 1,2,3-triazole/oxazole heterocyclic rings were proposed to be the main reason for the diminished affinity of T1-T3, T6 and T7 to an oxazole AAZ inhibitor binding site in VEGFR2 TK (PDB: 1Y6A or 1Y6B). Moreover T1-T3 and T6 were screened on cytotoxic activity against two human hepatocellular carcinoma cell lines. Selective cytotoxic activity of T2 against aggressive Mahlavu cells has been discovered indicating possible affinity of T2 to Mahlavu constitutionally active PI3K/Akt pathway.

  2. VEGF(121)b, a new member of the VEGF(xxx)b family of VEGF-A splice isoforms, inhibits neovascularisation and tumour growth in vivo.

    PubMed

    Rennel, E S; Varey, A H R; Churchill, A J; Wheatley, E R; Stewart, L; Mather, S; Bates, D O; Harper, S J

    2009-10-06

    The key mediator of new vessel formation in cancer and other diseases is VEGF-A. VEGF-A exists as alternatively spliced isoforms - the pro-angiogenic VEGF(xxx) family generated by exon 8 proximal splicing, and a sister family, termed VEGF(xxx)b, exemplified by VEGF(165)b, generated by distal splicing of exon 8. However, it is unknown whether this anti-angiogenic property of VEGF(165)b is a general property of the VEGF(xxx)b family of isoforms. The mRNA and protein expression of VEGF(121)b was studied in human tissue. The effect of VEGF(121)b was analysed by saturation binding to VEGF receptors, endothelial migration, apoptosis, xenograft tumour growth, pre-retinal neovascularisation and imaging of biodistribution in tumour-bearing mice with radioactive VEGF(121)b. The existence of VEGF(121)b was confirmed in normal human tissues. VEGF(121)b binds both VEGF receptors with similar affinity as other VEGF isoforms, but inhibits endothelial cell migration and is cytoprotective to endothelial cells through VEGFR-2 activation. Administration of VEGF(121)b normalised retinal vasculature by reducing both angiogenesis and ischaemia. VEGF(121)b reduced the growth of xenografted human colon tumours in association with reduced microvascular density, and an intravenous bolus of VEGF(121)b is taken up into colon tumour xenografts. Here we identify a second member of the family, VEGF(121)b, with similar properties to those of VEGF(165)b, and underline the importance of the six amino acids of exon 8b in the anti-angiogenic activity of the VEGF(xxx)b isoforms.

  3. Correlation of Serum Levels of IL-33, IL-37, Soluble Form of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), and Circulatory Frequency of VEGFR2-expressing Cells with Multiple Sclerosis Severity.

    PubMed

    Kouchaki, Ebrahim; Tamtaji, Omid Reza; Dadgostar, Ehsan; Karami, Mohammad; Nikoueinejad, Hassan; Akbari, Hossein

    2017-08-01

    IL-33 and IL-37 (new cytokines of IL-1 family), soluble form of vascular endothelial growth factor receptor-2 (sVEGFR2) as well as membranous expression of VEGFR2 have some key roles in the pathogenesis of autoimmune and inflammatory diseases. The aim of this study was to correlate circulatory changes of these factors with the severity of multiple sclerosis (MS) as an autoimmune and inflammatory disease. Our case-control study was performed on 84 patients with MS and 75 healthy subjects. The serum levels of IL-33, IL-37 and sVEGFR2 in the peripheral blood samples of all participants were measured by enzyme-linked immune sorbent assay (ELISA). Flow cytometry was used to analyze the circulatory number of VEGFR2-expressing cells. The severity of MS was evaluated using the expanded disability status scale (EDSS). Finally, we evaluated the correlation between serum levels of those factors with disease severity. Our findings showed that the serum level of IL-33, IL-37, sVEGFR2 and the circulatory number of VEGFR2-expressing cells were increased in patients with MS compared to healthy subjects (p<0.0001). Also, there was a significant correlation between serum levels of these 3 factors with disease severity according to EDSS. Our study showed that the serum levels of IL-33, IL-37 and sVEGFR2 may be important prognostic biomarkers of MS.

  4. Quercetin Inhibits Angiogenesis Mediated Human Prostate Tumor Growth by Targeting VEGFR- 2 Regulated AKT/mTOR/P70S6K Signaling Pathways

    PubMed Central

    Pratheeshkumar, Poyil; Budhraja, Amit; Son, Young-Ok; Wang, Xin; Zhang, Zhuo; Ding, Songze; Wang, Lei; Hitron, Andrew; Lee, Jeong-Chae; Xu, Mei; Chen, Gang; Luo, Jia; Shi, Xianglin

    2012-01-01

    Angiogenesis is a crucial step in the growth and metastasis of cancers, since it enables the growing tumor to receive oxygen and nutrients. Cancer prevention using natural products has become an integral part of cancer control. We studied the antiangiogenic activity of quercetin using ex vivo, in vivo and in vitro models. Rat aortic ring assay showed that quercetin at non-toxic concentrations significantly inhibited microvessel sprouting and exhibited a significant inhibition in the proliferation, migration, invasion and tube formation of endothelial cells, which are key events in the process of angiogenesis. Most importantly, quercetin treatment inhibited ex vivo angiogenesis as revealed by chicken egg chorioallantoic membrane assay (CAM) and matrigel plug assay. Western blot analysis showed that quercetin suppressed VEGF induced phosphorylation of VEGF receptor 2 and their downstream protein kinases AKT, mTOR, and ribosomal protein S6 kinase in HUVECs. Quercetin (20 mg/kg/d) significantly reduced the volume and the weight of solid tumors in prostate xenograft mouse model, indicating that quercetin inhibited tumorigenesis by targeting angiogenesis. Furthermore, quercetin reduced the cell viability and induced apoptosis in prostate cancer cells, which were correlated with the downregulation of AKT, mTOR and P70S6K expressions. Collectively the findings in the present study suggest that quercetin inhibits tumor growth and angiogenesis by targeting VEGF-R2 regulated AKT/mTOR/P70S6K signaling pathway, and could be used as a potential drug candidate for cancer therapy. PMID:23094058

  5. Vascular endothelial growth factor (VEGF)-C - a potent risk factor in children diagnosed with stadium 4 neuroblastoma.

    PubMed

    Nowicki, Michal; Konwerska, Aneta; Ostalska-Nowicka, Danuta; Derwich, Katarzyna; Miskowiak, Bogdan; Kondraciuk, Beata; Samulak, Dariusz; Witt, Martin

    2008-01-01

    To evaluate the immunohistochemical expression of VEGF-C, CD34 and VEGFR-2 in cancer tissue of children diagnosed with stadium 4 neuroblastoma (NB) and correlate their presence with the survival rate of children diagnosed with that stage of the disease. Eighteen children assigned to stadium 4 composed the study group. Fourteen patients (allocated to stadium 3) formed a control group. VEGF-C, CD34 and VEGFR-2 expressions were evaluated by immunohistochemical assay. Consecutive slides incubated with anti-CD34 and anti-VEGFR-2 antibodies revealed that the two markers were colocalized within endothelial layer of the blood vessels. On the other hand, VEGF-C was expressed exclusively in tumour cells. As demonstrated by Fisher's exact test, the risk of NB treatment failure (progression or relapse) as well as tumour related death, when all the patients were considered, was found to be significant in VEGF-C positive patients. VEGF-C expression in NB constitutes a potent risk factor and may direct future anti-angiogenic treatment strategy. The proximity of VEGF-C and CD34/VEGFR-2 of NB could be the equivalent of a potentially interesting VEGF-C fashion involving a tumour cell invasion into the blood vessels in an early phase of metastases promoting.

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

    PubMed Central

    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. PMID:27725874

  7. Low-dose dopamine agonist administration blocks vascular endothelial growth factor (VEGF)-mediated vascular hyperpermeability without altering VEGF receptor 2-dependent luteal angiogenesis in a rat ovarian hyperstimulation model.

    PubMed

    Gomez, Raul; Gonzalez-Izquierdo, Miguel; Zimmermann, Ralf C; Novella-Maestre, Edurne; Alonso-Muriel, Isabel; Sanchez-Criado, Jose; Remohi, Jose; Simon, Carlos; Pellicer, Antonio

    2006-11-01

    No specific treatment is available for ovarian hyperstimulation syndrome (OHSS), the most important complication in infertile women treated with gonadotropins. OHSS is caused by increased vascular permeability (VP) through ovarian hypersecretion of vascular endothelial growth factor (VEGF)-activating VEGF receptor 2 (VEGFR-2). We previously demonstrated in an OHSS rodent model that increased VP was prevented by inactivating VEGFR-2 with a receptor antagonist (SU5416). However, due to its toxicity (thromboembolism) and disruption of VEGFR-2-dependent angiogenic processes critical for pregnancy, this kind of compound cannot be used clinically to prevent OHSS. Dopamine receptor 2 (Dp-r2) agonists, used in the treatment of human hyperprolactinemia including pregnancy, inhibit VEGFR-2-dependent VP and angiogenesis when administered at high doses in animal cancer models. To test whether VEGFR-2-dependent VP and angiogenesis could be segregated in a dose-dependent fashion with the Dp-r2 agonist cabergoline, a well-established OHSS rat model supplemented with prolactin was used. A 100 microg/kg low-dose Dp-r2 agonist cabergoline reversed VEGFR-2-dependent VP without affecting luteal angiogenesis through partial inhibition of ovarian VEGFR-2 phosphorylation levels. No luteolytic effects (serum progesterone levels and luteal apoptosis unaffected) were observed. Cabergoline administration also did not affect VEGF/VEGFR-2 ovarian mRNA levels. Results in the animal model and the safe clinical profile of Dp-r2 agonists encouraged us to administer cabergoline to oocyte donors at high risk for developing the syndrome. Prophylactic administration of cabergoline (5-10 microg/kg x d) decreased the occurrence of OHSS from 65% (controls) to 25% (treatment). Therefore, a specific, safe treatment for OHSS is now available.

  8. Occlusal hypofunction causes periodontal atrophy and VEGF/VEGFR inhibition in tooth movement

    PubMed Central

    Usumi-Fujita, Risa; Hosomichi, Jun; Ono, Noriaki; Shibutani, Naoki; Kaneko, Sawa; Shimizu, Yasuhiro; Ono, Takashi

    2014-01-01

    Objective To examine changes in microvasculature and the expression of vascular endothelial growth factor A (VEGF-A) and VEGF receptor 2 (VEGFR-2) in rat hypofunctional periodontal ligament (PDL) during experimental tooth movement. Materials and Methods Twelve-week-old male Sprague-Dawley rats were divided into normal occlusion and occlusal hypofunction groups. After a 2-week bite-raising period, rat first molar was moved mesially using a 10-gf titanium-nickel alloy closed coil spring in both groups. On days 0, 1, 2, 3, and 7 after tooth movement, histologic changes were examined by micro–computed tomography and immunohistochemistry using CD31, VEGF-A, VEGFR-2, and the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method. Results Hypofunctional molars inclined more than normal molars and did not move notably after day 1 of tooth movement. Blood vessels increased on the tension side of the PDL in normal teeth. Immunoreactivities for VEGF-A and VEGFR-2 in normal teeth were greater than those in hypofunctional teeth during tooth movement. Compressive force rapidly caused apoptosis of the PDL and vascular endothelial cells in hypofunctional teeth, but not in normal teeth. Conclusions Occlusal hypofunction induces vascular constriction through a decrease in the expression of VEGF-A and VEGFR-2, and apoptosis of the PDL and vascular cells occurs during tooth movement. PMID:22716278

  9. Identification of new potent phthalazine derivatives with VEGFR-2 and EGFR kinase inhibitory activity.

    PubMed

    Amin, Kamilia M; Barsoum, Flora F; Awadallah, Fadi M; Mohamed, Nehal E

    2016-11-10

    Efforts to develop new antitumor agents are now directed towards multitarget therapies that are believed to have high potency and low tendency to resistance compared to conventional drugs. Herein, we highlighted the synthesis and antitumor activity of five series of phthalazine-based compounds featuring a variety of bioactive chemical fragments at position 1 of the phthalazine nucleus. The antitumor activity of the target compounds was performed against fourteen cancer cell lines where all compounds were active in the nanomolar level. In addition, the mechanism of action of the target compounds was investigated through an enzymatic inhibitory assay against VEGFR-2 and EGFR kinases, revealing potent and preferential activity toward VEGFR-2. Binding mode of the most active compounds was studied using docking experiment.

  10. Biphenyl derivatives incorporating urea unit as novel VEGFR-2 inhibitors: design, synthesis and biological evaluation.

    PubMed

    Wang, Chen; Gao, Hongping; Dong, Jinyun; Zhang, Yanmin; Su, Ping; Shi, Yaling; Zhang, Jie

    2014-01-01

    A series of novel biphenyl urea derivates were synthesized and investigated for their potential to inhibit vascular endothelial growth factor receptor-2 (VEGFR-2). In particular, A7, B3 and B4 displayed significant enzymatic inhibitory activities, with IC₅₀ values of 4.06, 4.55 and 5.26 nM. Compound A7 exhibited potent antiproliferative activity on several cell lines. SAR study suggested that the introduction of methyl at ortho-position of the biphenyl urea and tertiary amine moiety could improve VEGFR-2 inhibitory activity and antitumor effects. Molecular docking indicated that the urea moiety formed four hydrogen bonds with DFG residue. These biphenyl ureas could serve as promising lead compounds for further optimization.

  11. VEGFA and VEGFR2 gene polymorphisms and response to anti-vascular endothelial growth factor therapy: comparison of age-related macular degeneration treatments trials (CATT).

    PubMed

    Hagstrom, Stephanie A; Ying, Gui-shuang; Pauer, Gayle J T; Sturgill-Short, Gwen M; Huang, Jiayan; Maguire, Maureen G; Martin, Daniel F

    2014-05-01

    Individual variation in response and duration of anti-vascular endothelial growth factor (VEGF) therapy is seen among patients with neovascular age-related macular degeneration. Identification of genetic markers that affect clinical response may result in optimization of anti-VEGF therapy. To evaluate the pharmacogenetic relationship between genotypes of single-nucleotide polymorphisms (SNPs) in the VEGF signaling pathway and response to treatment with ranibizumab or bevacizumab for neovascular age-related macular degeneration. In total, 835 of 1149 patients (72.7%) participating in the Comparison of Age-Related Macular Degeneration Treatments Trials (CATT) at 43 CATT clinical centers. Each patient was genotyped for 7 SNPs in VEGFA (rs699946, rs699947, rs833069, rs833070, rs1413711, rs2010963, and rs2146323) and 1 SNP in VEGFR2 (rs2071559) using TaqMan SNP genotyping assays. Genotypic frequencies were compared with clinical measures of response to therapy at 1 year, including the mean visual acuity, mean change in visual acuity, at least a 15-letter increase, retinal thickness, mean change in total foveal thickness, presence of fluid on optical coherence tomography, presence of leakage on fluorescein angiography, mean change in lesion size, and mean number of injections administered. Differences in response by genotype were evaluated with tests of linear trend calculated from logistic regression models for categorical outcomes and linear regression models for continuous outcomes. The method of controlling the false discovery rate was used to adjust for multiple comparisons. For each of the measures of visual acuity evaluated, no association was observed with any of the genotypes or with the number of risk alleles. Four VEGFA SNPs demonstrated an association with retinal thickness: rs699947 (P = .03), rs833070 (P = .04), rs1413711 (P = .045), and rs2146323 (P = .006). However, adjusted P values for these associations were all statistically

  12. Induction of angiogenesis and modulation of VEGFR-2 by simvastatin after traumatic brain injury

    PubMed Central

    Wu, Hongtao; Jiang, Hao; Lu, Dunyue; Qu, Changsheng; Xiong, Ye; Zhou, Dong; Chopp, Michael; Mahmood, Asim

    2011-01-01

    Background Our previous studies demonstrated that simvastatin reduced neuronal death, increased neurogenesis, and promoted functional recovery after TBI. Objective: To investigate the effect of simvastatin on angiogenesis after TBI, and the related signaling pathways. Methods Saline or simvastatin (1 mg/kg) was administered orally to rats starting at day 1 after TBI or sham surgery and then daily for 14 days. Rats were sacrificed at 3 and 14 days after treatment. Brain sections and tissues were prepared for immunohistochemical staining, ELISA, and Western blot analysis, respectively. Cultured rat brain microvascular endothelial cells (RBMVECs) were subjected to oxygen-glucose deprivation (OGD) followed by immunocytochemical staining with phallotoxins and vascular endothelial growth factor receptor-2 (VEGFR-2). Western blot analysis was carried out to examine the simvastatin-induced activation of the v-akt murine thymoma viral oncogene homolog (Akt) signaling pathway. The expression of VEGFR-2 was detected by ELISA. Results Simvastatin significantly increased the length of vascular perimeter, promoted the proliferation of endothelial cells, and improved the sensorimotor function after TBI. Simvastatin stimulated endothelial cell tube formation after OGD in vitro. VEGFR-2 expression in both brain tissues and cultured RBMVECs was enhanced after simvastatin treatment, which may be modulated by activation of Akt. Akt-dependent endothelial nitric oxide synthase (eNOS) phosphorylation was also induced by simvastatin in vivo and in vitro. Conclusion Simvastatin augments TBI-induced angiogenesis in the lesion boundary zone and hippocampus and improves functional recovery. Simvastatin also promotes angiogenesis in vitro. These beneficial effects on angiogenesis may be related to simvastatin-induced activation of the VEGFR-2/Akt/eNOS signaling pathway. PMID:21307798

  13. Localisation of members of the vascular endothelial growth factor (VEGF) family and their receptors in human atherosclerotic arteries

    PubMed Central

    Belgore, F; Blann, A; Neil, D; Ahmed, A S; Lip, G Y H

    2004-01-01

    Background: Vascular endothelial growth factor (VEGF) mediates endothelial cell mitogenesis and enhances vascular permeability. The existence of single or multiple VEGF isoforms and receptors suggests that these proteins may have overlapping but distinct functions, which may be reflected in their cell expression and distribution. Methods: The localisation of VEGFs A–C and their receptors (VEGFRs 1–3, respectively) in 30 fresh human atherosclerotic arteries, 15 normal uterine arteries, and 15 saphenous veins using immunohistochemistry and western blotting. Results: Saphenous veins showed no staining for VEGF-B or VEGFR-2. Smooth muscle cells (SMCs) showed the strongest staining for VEGF-A, VEGF-B, VEGFR-1, and VEGFR-2 in all specimens. Conversely, VEGFR-3 and VEGF-C were predominately localised to the endothelial vasa vasorum in normal arteries, whereas medial SMCs showed the strongest staining in atherosclerotic arteries. Western blotting showed variations in VEGF protein localisation, with lower amounts of VEGF-B and VEGF-C in saphenous veins, compared with arterial tissue. Amounts of VEGF-C were lower than those of VEGF-A and VEGF-B in all specimens. Conclusion: This study provides direct evidence of the presence of VEGF proteins and receptors in human physiology and pathology, with variations in both the amounts of VEGF proteins expressed and their cellular distribution in normal arteries compared with atherosclerotic arteries. The presence of VEGFs A–C and their receptors in normal arterial tissue implies that VEGF functions may extend beyond endothelial cell proliferation. Reduced VEGFR-2 staining in atherosclerotic arteries may have implications for the atherosclerosis process and the development of vascular disease and its complications. PMID:14990597

  14. Platycodin D inhibits tumor growth by antiangiogenic activity via blocking VEGFR2-mediated signaling pathway

    SciTech Connect

    Luan, Xin; Gao, Yun-Ge; Guan, Ying-Yun; Xu, Jian-Rong; Lu, Qin; Zhao, Mei; Liu, Ya-Rong; Liu, Hai-Jun; Fang, Chao; Chen, Hong-Zhuan

    2014-11-15

    Platycodin D (PD) is an active component mainly isolated from the root of Platycodon grandiflorum. Recent studies proved that PD exhibited inhibitory effect on proliferation, migration, invasion and xenograft growth of diverse cancer cell lines. However, whether PD is suppressive for angiogenesis, an important hallmark in cancer development, remains unknown. Here, we found that PD could dose-dependently inhibit human umbilical vein endothelial cell (HUVEC) proliferation, motility, migration and tube formation. PD also significantly inhibited angiogenesis in the chick embryo chorioallantoic membrane (CAM). Moreover, the antiangiogenic activity of PD contributed to its in vivo anticancer potency shown in the decreased microvessel density and delayed growth of HCT-15 xenograft in mice with no overt toxicity. Western blot analysis indicated that PD inhibited the phosphorylation of VEGFR2 and its downstream protein kinase including PLCγ1, JAK2, FAK, Src, and Akt in endothelial cells. Molecular docking simulation showed that PD formed hydrogen bonds and hydrophobic interactions within the ATP binding pocket of VEGFR2 kinase domain. The present study firstly revealed the high antiangiogenic activity and the underlying molecular basis of PD, suggesting that PD may be a potential antiangiogenic agent for angiogenesis-related diseases. - Highlights: • Platycodin D inhibits HUVEC proliferation, motility, migration and tube formation. • Platycodin D inhibits the angiogenesis in chick embryo chorioallantoic membrane. • Platycodin D suppresses the angiogenesis and growth of HCT-15 xenograft in mice. • Platycodin D inhibits the phosphorylation of VEGFR2 and downstream kinases in HUVEC.

  15. QSAR and molecular docking studies on oxindole derivatives as VEGFR-2 tyrosine kinase inhibitors.

    PubMed

    Kang, Cong-Min; Liu, Dong-Qing; Zhao, Xu-Hao; Dai, Ying-Jie; Cheng, Jia-Gao; Lv, Ying-Tao

    2016-01-01

    The three-dimensional quantitative structure-activity relationships (3D-QSAR) were established for 30 oxindole derivatives as vascular endothelial growth factor receptor-2 (VEGFR-2) tyrosine kinase inhibitors by using comparative molecular field analysis (CoMFA) and comparative similarity indices analysis comparative molecular similarity indices analysis (CoMSIA) techniques. With the CoMFA model, the cross-validated value (q(2)) was 0.777, the non-cross-validated value (R(2)) was 0.987, and the external cross-validated value ([Formula: see text]) was 0.72. And with the CoMSIA model, the corresponding q(2), R(2) and [Formula: see text] values were 0.710, 0.988 and 0.78, respectively. Docking studies were employed to bind the inhibitors into the active site to determine the probable binding conformation. The binding mode obtained by molecular docking was in good agreement with the 3D-QSAR results. Based on the QSAR models and the docking binding mode, a set of new VEGFR-2 tyrosine kinase inhibitors were designed, which showed excellent predicting inhibiting potencies. The result revealed that both QSAR models have good predictive capability to guide the design and structural modification of homologic compounds. It is also helpful for further research and development of new VEGFR-2 tyrosine kinase inhibitors.

  16. Association of Genetic Polymorphisms on VEGFA and VEGFR2 With Risk of Coronary Heart Disease

    PubMed Central

    Liu, Doxing; Song, Jiantao; Ji, Xianfei; Liu, Zunqi; Cong, Mulin; Hu, Bo

    2016-01-01

    Abstract Coronary heart disease (CHD) is a cardiovascular disease which is contributed by abnormal neovascularization. VEGFA (vascular endothelial growth factor A) and VEGFR2 (vascular endothelial growth factor receptor 2) have been revealed to be involved in the pathological angiogenesis. This study was intended to confirm whether single nucleotide polymorphisms (SNPs) of VEGFA and VEGFR2 were associated with CHD in a Chinese population, considering pathological features and living habits of CHD patients. Peripheral blood samples were collected from 810 CHD patients and 805 healthy individuals. Six tag SNPs within VEGFA and VEGFR2 were obtained from HapMap Database. Genotyping of SNPs was performed using SNapShot method (Applied Biosystems, Foster, CA). Odd ratios (ORs) and their 95% confidence intervals (95% CIs) were calculated to evaluate the association between SNPs and CHD risk. Under the allelic model, 6 SNPs of VEGFA and VEGFR2 were remarkably associated with the susceptibility to CHD. Genotype CT of rs3025039, TT of rs2305948, and AA of rs1873077 were associated with a reduced risk of CHD when smoking, alcohol intake and diabetes were considered, while homozygote GG of rs1570360 might elevate the susceptibility to CHD (all P < 0.05) for patients who were addicted to smoking or those with hypertension. All of the combined effects of rs699947 (CC/CA) and rs2305948 (TT), rs3025039 (TT) and rs2305948 (TT), rs3025039 (CT) and rs1870377 (AA) had positive effects on the risk of CHD, respectively (all P < 0.05). By contrast, the synthetic effects of rs69947 (CA/AA) and rs1870377 (TA), rs699947 (CA) and rs7667298 (GG), rs699947 (AA) and rs7667298 (GG), rs1570360 (GG) and rs2305948 (TT), as well as rs1570360 (GG) and rs1870377 (AA) all exhibited adverse effects on the risk of CHD, respectively (all P < 0.05). Six polymorphisms in VEGFA and VEGFR2 may have substantial influence on the susceptibility to CHD in a Han Chinese population. Prospective cohort

  17. Role of VEGF-A and its receptors in sporadic and MEN2-associated pheochromocytoma.

    PubMed

    Ferreira, Carla Vaz; Siqueira, Débora Rodrigues; Romitti, Mírian; Ceolin, Lucieli; Brasil, Beatriz Assis; Meurer, Luise; Capp, Clarissa; Maia, Ana Luiza

    2014-03-26

    Pheochromocytoma (PHEO), a rare catecholamine producing tumor arising from the chromaffin cells, may occurs sporadically (76%-80%) or as part of inherited syndromes (20%-24%). Angiogenesis is a fundamental step in tumor proliferation and vascular endothelial growth factor (VEGF-A) is the most well-characterized angiogenic factor. The role of angiogenic markers in PHEO is not fully understood; investigations were therefore made to evaluate the expression of VEGF-A and its receptors in PHEO and correlate to clinical parameters. Twenty-nine samples of PHEO were evaluated for VEGF-A, VEGF receptor-1 (VEGFR-1) VEGFR-2 expression and microvessel density (MVD) by immunohistochemistry. Clinical data were reviewed in medical records. The mean age of patients was 38±14 years, and 69% were woman. VEGF-A, VEGFR-1 and VEGFR-2 staining were detected in nearly all PHEO samples. No significant correlation was observed between VEGF-A, VEGFR-1, VEGFR-2 expression or MVD and age at diagnosis, tumor size or sporadic and hereditary PHEO. However, the levels of expression of these molecules were significantly higher in malignant PHEO samples (p=0.027, p=0.003 and p=0.026, respectively).VEGF-A and its receptors were shown to be up-regulated in malignant PHEO, suggesting that these molecules might be considered as therapeutic targets for unresectable or metastatic tumors.

  18. SLT-VEGF reduces lung metastases, decreases tumor recurrence, and improves survival in an orthotopic melanoma model.

    PubMed

    Ackerman, Rachel; Backer, Joseph M; Backer, Marina; Skariah, Sini; Hamby, Carl V

    2010-09-01

    SLT-VEGF is a recombinant cytotoxin comprised of Shiga-like toxin (SLT) subunit A fused to human vascular endothelial growth factor (VEGF). It is highly cytotoxic to tumor endothelial cells overexpressing VEGF receptor-2 (VEGFR-2/KDR/Flk1) and inhibits the growth of primary tumors in subcutaneous models of breast and prostate cancer and inhibits metastatic dissemination in orthotopic models of pancreatic cancer. We examined the efficacy of SLT-VEGF in limiting tumor growth and metastasis in an orthotopic melanoma model, using NCR athymic nude mice inoculated with highly metastatic Line IV Cl 1 cultured human melanoma cells. Twice weekly injections of SLT-VEGF were started when tumors became palpable at one week after intradermal injection of 1 × 10(6) cells/mouse. Despite selective depletion of VEGFR-2 overexpressing endothelial cells from the tumor vasculature, SLT-VEGF treatment did not affect tumor growth. However, after primary tumors were removed, continued SLT-VEGF treatment led to fewer tumor recurrences (p = 0.007), reduced the incidence of lung metastasis (p = 0.038), and improved survival (p = 0.002). These results suggest that SLT-VEGF is effective at the very early stages of tumor development, when selective killing of VEGFR-2 overexpressing endothelial cells can still prevent further progression. We hypothesize that SLT-VEGF could be a promising adjuvant therapy to inhibit or prevent outgrowth of metastatic foci after excision of aggressive primary melanoma lesions.

  19. The effect of multistage nanovector targeting of VEGFR2 positive tumor endothelia on cell adhesion and local payload accumulation

    PubMed Central

    Martinez, Jonathan O.; Evangelopoulos, Michael; Karun, Vivek; Shegog, Evan; Wang, Joshua A.; Boada, Christian; Liu, Xuewu; Ferrari, Mauro; Tasciotti, Ennio

    2014-01-01

    Nanovectors are a viable solution to the formulation of poorly soluble anticancer drugs. Their bioaccumulation in the tumor parenchyma is mainly achieved exploiting the enhanced permeability and retention (EPR) effect of the leaky neovasculature. In this paper we demonstrate that multistage nanovectors (MSV) exhibit rapid tumoritropic homing independent of EPR, relying on particle geometry and surface adhesion. By studying endothelial cells overexpressing vascular endothelial growth factor receptor-2 (VEGFR2), we developed MSV able to preferentially target VEGFR2 expressing tumor-associated vessels. Static and dynamic targeting revealed that MSV conjugated with anti-VEGFR2 antibodies displayed greater than a 4-fold increase in targeting efficiency towards VEGFR2 expressing cells while exhibiting minimal adherence to control cells. Additionally, VEGFR2 conjugation bestowed MSV with a significant increase in breast tumor targeting and in the delivery of a model payload while decreasing their accumulation in the liver. Surface functionalization with an anti-VEGFR2 antibody provided enhanced affinity towards the tumor vascular endothelium, which promoted enhanced adhesion and tumoritropic accumulation of a reporter molecule released by the MSV. PMID:25176066

  20. The effect of multistage nanovector targeting of VEGFR2 positive tumor endothelia on cell adhesion and local payload accumulation.

    PubMed

    Martinez, Jonathan O; Evangelopoulos, Michael; Karun, Vivek; Shegog, Evan; Wang, Joshua A; Boada, Christian; Liu, Xuewu; Ferrari, Mauro; Tasciotti, Ennio

    2014-12-01

    Nanovectors are a viable solution to the formulation of poorly soluble anticancer drugs. Their bioaccumulation in the tumor parenchyma is mainly achieved exploiting the enhanced permeability and retention (EPR) effect of the leaky neovasculature. In this paper we demonstrate that multistage nanovectors (MSV) exhibit rapid tumoritropic homing independent of EPR, relying on particle geometry and surface adhesion. By studying endothelial cells overexpressing vascular endothelial growth factor receptor-2 (VEGFR2), we developed MSV able to preferentially target VEGFR2 expressing tumor-associated vessels. Static and dynamic targeting revealed that MSV conjugated with anti-VEGFR2 antibodies displayed greater than a 4-fold increase in targeting efficiency towards VEGFR2 expressing cells while exhibiting minimal adherence to control cells. Additionally, VEGFR2 conjugation bestowed MSV with a significant increase in breast tumor targeting and in the delivery of a model payload while decreasing their accumulation in the liver. Surface functionalization with an anti-VEGFR2 antibody provided enhanced affinity towards the tumor vascular endothelium, which promoted enhanced adhesion and tumoritropic accumulation of a reporter molecule released by the MSV. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Role of HIF1A, VEGFA and VEGFR2 SNPs in the Susceptibility and Progression of COPD in a Spanish Population

    PubMed Central

    Baz-Dávila, Rebeca; Espinoza-Jiménez, Adriana; Rodríguez-Pérez, María del Cristo; Zulueta, Javier; Varo, Nerea; Montejo, Ángela; Almeida-González, Delia; Aguirre-Jaime, Armando; Córdoba-Lanús, Elizabeth; Casanova, Ciro

    2016-01-01

    Hypoxia is involved in the development of chronic inflammatory processes. Under hypoxic conditions HIF1A, VEGF and VEGFR2 are expressed and mediate the course of the resultant disease. The aim of the present study was to define the associations between tSNPs in these genes and COPD susceptibility and progression in a Spanish cohort. The T alleles in rs3025020 and rs833070 SNPs (VEGFA gene) were less frequent in the group of COPD cases and were associated with a lower risk of developing the disease (OR = 0.60; 95% CI = 0. 39–0.93; p = 0.023 and OR = 0.60; 95% CI = 0.38–0.96; p = 0.034, respectively) under a dominant model of inheritance. The haplotype in which both SNPs presented the T allele confirmed the association found (OR = 0.02; 95% CI = 0.00 to 0.66; p = 0.03). Moreover, patients with COPD carrying the T allele in homozygosis in rs3025020 SNP showed higher lung function values and this association remained constant during 3 years of follow-up. In conclusion, T allele in rs833070 and rs3025020 may confer a protective effect to COPD susceptibility in a Spanish population and the association of the SNP rs3025020 with lung function may be suggesting a role for VEGF in the progression of the disease. PMID:27163696

  2. Priming Endothelial Cells With a Melanoma-Derived Extracellular Matrix Triggers the Activation of αvβ3/VEGFR2 Axis.

    PubMed

    Helal-Neto, Edward; Brandão-Costa, Renata M; Saldanha-Gama, Roberta; Ribeiro-Pereira, Cristiane; Midlej, Victor; Benchimol, Marlene; Morandi, Verônica; Barja-Fidalgo, Christina

    2016-11-01

    The unique composition of tumor-produced extracellular matrix (ECM) can be a determining factor in changing the profile of endothelial cells in the tumor microenvironment. As the main receptor for ECM proteins, integrins can activate a series of signaling pathways related to cell adhesion, migration, and differentiation of endothelial cells that interact with ECM proteins. We studied the direct impact of the decellularized ECM produced by a highly metastatic human melanoma cell line (MV3) on the activation of endothelial cells and identified the intracellular signaling pathways associated with cell differentiation. Our data show that compared to the ECM derived from a human melanocyte cell line (NGM-ECM), ECM produced by a melanoma cell line (MV3-ECM) is considerably different in ultrastructural organization and composition and possesses a higher content of tenascin-C and laminin and a lower expression of fibronectin. When cultured directly on MV3-ECM, endothelial cells change morphology and show increased adhesion, migration, proliferation, and tubulogenesis. Interaction of endothelial cells with MV3-ECM induces the activation of integrin signaling, increasing FAK phosphorylation and its association with Src, which activates VEGFR2, potentiating the receptor response to VEGF. The blockage of αvβ3 integrin inhibited the FAK-Src association and VEGFR activation, thus reducing tubulogenesis. Together, our data suggest that the interaction of endothelial cells with the melanoma-ECM triggers integrin-dependent signaling, leading to Src pathway activation that may potentiate VEGFR2 activation and up-regulate angiogenesis. J. Cell. Physiol. 231: 2464-2473, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  3. Novel angiogenesis inhibitory activity in cinnamon extract blocks VEGFR2 kinase and downstream signaling

    USDA-ARS?s Scientific Manuscript database

    VEGF is one of the most critical factors that induce angiogenesis, and has thus become an attractive target for anti-angiogenesis treatment. However, most of the current anti-VEGF agents that often cause side effects cannot be recommended for long term use. Identification of natural VEGF inhibitors...

  4. Determination of VEGFR-2 (KDR) 604A>G Polymorphism in Pancreatic Disorders

    PubMed Central

    Pădureanu, Vlad; Boldeanu, Mihail Virgil; Streaţă, Ioana; Cucu, Mihai Gabriel; Siloşi, Isabela; Boldeanu, Lidia; Bogdan, Maria; Enescu, Anca Ştefania; Forţofoiu, Maria; Enescu, Aurelia; Dumitrescu, Elena Mădălina; Alexandru, Dragoş; Şurlin, Valeriu Marian; Forţofoiu, Mircea Cătălin; Petrescu, Ileana Octavia; Petrescu, Florin; Ioana, Mihai; Ciurea, Marius Eugen; Săftoiu, Adrian

    2017-01-01

    Pancreatic disorders have a high prevalence worldwide. Despite the fact that screening methods became more effective and the knowledge we have nowadays about pancreatic diseases has enhanced, their incidence remains high. Our purpose was to determine whether single nucleotide polymorphism (SNP) of VEGFR-2/KDR (vascular endothelial growth factor receptor 2/kinase insert domain receptor) influences susceptibility to develop pancreatic pathology. Genomic DNA was extracted from blood samples collected from patients diagnosed with acute pancreatitis (n = 110), chronic pancreatitis (n = 25), pancreatic cancer (n = 82) and healthy controls (n = 232). VEGFR-2 (KDR) 604A>G (rs2071559) polymorphism frequency was determined with TaqMan allelic discrimination assays. Statistical assessment was performed by associating genetic polymorphism with clinical and pathological data. In both pancreatic disorders and healthy control groups the polymorphism we studied was in Hardy-Weinberg equilibrium. Association between increased risk for pancreatic disorders and studied polymorphism was statistically significant. KDR 604AG and AG + GG genotypes were more prevalent in acute pancreatitis and pancreatic cancer patients than in controls. These genotypes influence disease development in a low rate. No association was found between chronic pancreatitis and KDR 604AG and AG + GG genotypes. In Romanian cohort, we found an association between the KDR 604A→G polymorphism and acute pancreatitis and pancreatic cancer. Carriers of the -604G variant allele were more frequent among acute pancreatitis and pancreatic cancer than among controls, suggesting that KDR 604G allele may confer an increased risk for these diseases. In the future, more extensive studies on larger groups are necessary, in order to clarify the role of VEGFR2 polymorphisms in pancreatic pathology. PMID:28218664

  5. Ultrasound molecular imaging of VEGFR2 in a rat prostate tumor model using BR55.

    PubMed

    Tardy, Isabelle; Pochon, Sibylle; Theraulaz, Martine; Emmel, Patricia; Passantino, Lisa; Tranquart, François; Schneider, Michel

    2010-10-01

    To evaluate BR55, a new VEGFR2-specific ultrasound contrast agent, for imaging prostate tumors in an orthotopic model in the rat. Rat prostate adenocarcinoma were established by injection of G Dunning R-3327 tumor cells in one lobe of the prostate of Copenhagen rats. Imaging experiments were performed with BR55, SonoVue, and streptavidin-functionalized microbubbles coupled with an anti-vascular endothelial growth factor receptor 2 (VEGFR2) antibody using a clinical ultrasound scanner. Contrast enhancement in the tumor and healthy prostate was followed over time by intermittent imaging at low acoustic power. Signal quantification and statistical analysis were performed in the tumor and healthy tissue to compare the behavior of the 3 contrast agents. Immunohistochemistry was performed on the prostate and tumor specimen to determine the expression of VEGFR2. Comparable contrast enhancement was observed in tumors at peak intensity for BR55 and SonoVue. Then, once unbound microbubbles had cleared from the circulation, a strong enhancement of the tumor was obtained with BR55, whereas no significant microbubble accumulation was detected in the healthy prostate tissue. SonoVue microbubbles were rapidly eliminated, and no significant binding was observed in the tumor. The tumor to prostate ratio calculated after signal quantification was about 20 for the 3 doses of BR55 tested. The enhancement obtained with BR55 in the tumor was not significantly different from the one observed with antibody-coupled streptavidin microbubbles. Intense staining for VEGFR2 was detected in the tumor vessels by immunohistochemistry. This study showed that BR55 binding to prostate tumors resulted in a strong enhancement of the lesions as early as a few minutes after contrast injection, whereas minimal nonspecific accumulation occurred in the healthy part of the gland. BR55, like SonoVue, provide information on tissue perfusion during the early vascular phase, but BR55 binding to the tumoral

  6. Migration-promoting role of VEGF-C and VEGF-C binding receptors in human breast cancer cells

    PubMed Central

    Timoshenko, A V; Rastogi, S; Lala, P K

    2007-01-01

    Vascular endothelial growth factor C (VEGF-C) is a lymphangiogenic factor over-expressed in highly metastatic, cyclooxygenase (COX)-2 expressing breast cancer cells. We tested the hypothesis that tumour-derived VEGF-C may play an autocrine role in metastasis by promoting cellular motility through one or more VEGF-C-binding receptors VEGFR-2, VEGFR-3, neuropilin (NRP)-1, NRP-2, and integrin α9β1. We investigated the expression of these receptors in several breast cancer cell lines (MDA-MB-231, Hs578T, SK-BR-3, T-47D, and MCF7) and their possible requirement in migration of two VEGF-C-secreting, highly metastatic lines MDA-MB-231 and Hs578T. While cell lines varied significantly in their expression of above VEGF-C receptors, migratory activity of MDA-MB-231 and Hs578T cells was linked to one or more of these receptors. Depletion of endogenous VEGF-C by treatments with a neutralising antibody, VEGF-C siRNA or inhibitors of Src, EGFR/Her2/neu and p38 MAP kinases which inhibited VEGF-C production, inhibited cellular migration, indicating the requirement of VEGF-C for migratory function. Migration was differentially attenuated by blocking or downregulation of different VEGF-C receptors, for example treatment with a VEGFR-2 tyrosine kinase inhibitor, NRP-1 and NRP-2 siRNA or α9β1 integrin antibody, indicating the participation of one or more of the receptors in cell motility. This novel role of tumour-derived VEGF-C indicates that breast cancer metastasis can be promoted by coordinated stimulation of lymphangiogenesis and enhanced migratory activity of breast cancer cells. PMID:17912247

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

    PubMed

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

    2017-03-01

    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. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Morphological Plasticity of Emerging Purkinje Cells in Response to Exogenous VEGF

    PubMed Central

    Herrfurth, Leonard; Theis, Verena; Matschke, Veronika; May, Caroline; Marcus, Katrin; Theiss, Carsten

    2017-01-01

    Vascular endothelial growth factor (VEGF) is well known as the growth factor with wide-ranging functions even in the central nervous system (CNS). Presently, most attention is given to the investigation of its role in neuronal protection, growth and maturation processes, whereby most effects are mediated through VEGF receptor 2 (VEGFR-2). The purpose of our current study is to provide new insights into the impact of VEGF on immature and mature Purkinje cells (PCs) in accordance with maturity and related receptor expression. Therefore, to expand our knowledge of VEGF effects in PCs development and associated VEGFR-2 expression, we used cultivated organotypic cerebellar slice cultures in immunohistochemical or microinjection studies, followed by confocal laser scanning microscopy (CLSM) and morphometric analysis. Additionally, we incorporated in our study the method of laser microdissection, followed by quantitative polymerase chain reaction (qPCR). For the first time we could show the age-dependent VEGF sensitivity of PCs with the largest promoting effects being on dendritic length and cell soma size in neonatal and juvenile stages. Once mature, PCs were no longer susceptible to VEGF stimulation. Analysis of VEGFR-2 expression revealed its presence in PCs throughout development, which underlined its mediating functions in neuronal cells. PMID:28194096

  9. Fragment virtual screening based on Bayesian categorization for discovering novel VEGFR-2 scaffolds.

    PubMed

    Zhang, Yanmin; Jiao, Yu; Xiong, Xiao; Liu, Haichun; Ran, Ting; Xu, Jinxing; Lu, Shuai; Xu, Anyang; Pan, Jing; Qiao, Xin; Shi, Zhihao; Lu, Tao; Chen, Yadong

    2015-11-01

    The discovery of novel scaffolds against a specific target has long been one of the most significant but challengeable goals in discovering lead compounds. A scaffold that binds in important regions of the active pocket is more favorable as a starting point because scaffolds generally possess greater optimization possibilities. However, due to the lack of sufficient chemical space diversity of the databases and the ineffectiveness of the screening methods, it still remains a great challenge to discover novel active scaffolds. Since the strengths and weaknesses of both fragment-based drug design and traditional virtual screening (VS), we proposed a fragment VS concept based on Bayesian categorization for the discovery of novel scaffolds. This work investigated the proposal through an application on VEGFR-2 target. Firstly, scaffold and structural diversity of chemical space for 10 compound databases were explicitly evaluated. Simultaneously, a robust Bayesian classification model was constructed for screening not only compound databases but also their corresponding fragment databases. Although analysis of the scaffold diversity demonstrated a very unevenly distribution of scaffolds over molecules, results showed that our Bayesian model behaved better in screening fragments than molecules. Through a literature retrospective research, several generated fragments with relatively high Bayesian scores indeed exhibit VEGFR-2 biological activity, which strongly proved the effectiveness of fragment VS based on Bayesian categorization models. This investigation of Bayesian-based fragment VS can further emphasize the necessity for enrichment of compound databases employed in lead discovery by amplifying the diversity of databases with novel structures.

  10. The high angiogenic activity in very early breast cancer enables reliable imaging with VEGFR2-targeted microbubbles (BR55).

    PubMed

    Bzyl, Jessica; Palmowski, Moritz; Rix, Anne; Arns, Susanne; Hyvelin, Jean-Marc; Pochon, Sibylle; Ehling, Josef; Schrading, Simone; Kiessling, Fabian; Lederle, Wiltrud

    2013-02-01

    Tumour xenografts of well-discernible sizes can be examined well by molecular ultrasound. Here, we investigated whether very early breast carcinomas express sufficient levels of VEGFR2 for reliable molecular ultrasound imaging with targeted microbubbles. MCF-7 breast cancer xenografts were orthotopically implanted in nude mice (n = 26). Tumours measuring from 4 mm(3) (2 mm diameter) up to 65 mm(3) (5 mm diameter) were examined with automated 3D molecular ultrasound using clinically translatable VEGFR2-targeted microbubbles (BR55). Additionally, the relative tumour blood volume was assessed with non-targeted microbubbles (BR38). In vivo ultrasound data were validated by quantitative immunohistochemistry. Very small lesions 2 mm in diameter showed the highest binding of VEGFR2-specific microbubbles. In larger tumours significantly less BR55 accumulated (p = 0.023). Nonetheless, binding of VEGFR2-targeted microbubbles was still high enough for imaging. The relative blood volume was comparable at all tumour sizes. Both findings were confirmed by immunohistochemistry. Additionally, a significantly enhanced number of large and mature vessels were detected with increasing tumour size (p < 0.01), explaining the decrease in VEGFR2 expression during tumour growth. 3D molecular ultrasound using BR55 is very well suited to depicting the angiogenic activity in very small breast lesions, suggesting its potential for detecting and characterising these lesions.

  11. Exploring the Molecular Interactions of 7,8-Dihydroxyflavone and Its Derivatives with TrkB and VEGFR2 Proteins

    PubMed Central

    Chitranshi, Nitin; Gupta, Vivek; Kumar, Sanjay; Graham, Stuart L.

    2015-01-01

    7,8-Dihydroxyflavone (7,8-DHF) is a TrkB receptor agonist, and treatment with this flavonoid derivative brings about an enhanced TrkB phosphorylation and promotes downstream cellular signalling. Flavonoids are also known to exert an inhibitory effect on the vascular endothelial growth factor receptor (VEGFR) family of tyrosine kinase receptors. VEGFR2 is one of the important receptors involved in the regulation of vasculogenesis and angiogenesis and has also been implicated to exhibit various neuroprotective roles. Its upregulation and uncontrolled activity is associated with a range of pathological conditions such as age-related macular degeneration and various proliferative disorders. In this study, we investigated molecular interactions of 7,8-DHF and its derivatives with both the TrkB receptor as well as VEGFR2. Using a combination of molecular docking and computational mapping tools involving molecular dynamics approaches we have elucidated additional residues and binding energies involved in 7,8-DHF interactions with the TrkB Ig2 domain and VEGFR2. Our investigations have revealed for the first time that 7,8-DHF has dual biochemical action and its treatment may have divergent effects on the TrkB via its extracellular Ig2 domain and on the VEGFR2 receptor through the intracellular kinase domain. Contrary to its agonistic effects on the TrkB receptor, 7,8-DHF was found to downregulate VEGFR2 phosphorylation both in 661W photoreceptor cells and in retinal tissue. PMID:26404256

  12. Regulation of alternative VEGF-A mRNA splicing is a therapeutic target for analgesia☆

    PubMed Central

    Hulse, R.P.; Beazley-Long, N.; Hua, J.; Kennedy, H.; Prager, J.; Bevan, H.; Qiu, Y.; Fernandes, E.S.; Gammons, M.V.; Ballmer-Hofer, K.; Gittenberger de Groot, A.C.; Churchill, A.J.; Harper, S.J.; Brain, S.D.; Bates, D.O.; Donaldson, L.F.

    2014-01-01

    Vascular endothelial growth factor-A (VEGF-A) is best known as a key regulator of the formation of new blood vessels. Neutralization of VEGF-A with anti-VEGF therapy e.g. bevacizumab, can be painful, and this is hypothesized to result from a loss of VEGF-A-mediated neuroprotection. The multiple vegf-a gene products consist of two alternatively spliced families, typified by VEGF-A165a and VEGF-A165b (both contain 165 amino acids), both of which are neuroprotective. Under pathological conditions, such as in inflammation and cancer, the pro-angiogenic VEGF-A165a is upregulated and predominates over the VEGF-A165b isoform. We show here that in rats and mice VEGF-A165a and VEGF-A165b have opposing effects on pain, and that blocking the proximal splicing event – leading to the preferential expression of VEGF-A165b over VEGF165a – prevents pain in vivo. VEGF-A165a sensitizes peripheral nociceptive neurons through actions on VEGFR2 and a TRPV1-dependent mechanism, thus enhancing nociceptive signaling. VEGF-A165b blocks the effect of VEGF-A165a. After nerve injury, the endogenous balance of VEGF-A isoforms switches to greater expression of VEGF-Axxxa compared to VEGF-Axxxb, through an SRPK1-dependent pre-mRNA splicing mechanism. Pharmacological inhibition of SRPK1 after traumatic nerve injury selectively reduced VEGF-Axxxa expression and reversed associated neuropathic pain. Exogenous VEGF-A165b also ameliorated neuropathic pain. We conclude that the relative levels of alternatively spliced VEGF-A isoforms are critical for pain modulation under both normal conditions and in sensory neuropathy. Altering VEGF-Axxxa/VEGF-Axxxb balance by targeting alternative RNA splicing may be a new analgesic strategy. PMID:25151644

  13. Discovery of Anilinopyrimidines as Dual Inhibitors of c-Met and VEGFR-2: Synthesis, SAR, and Cellular Activity

    PubMed Central

    2014-01-01

    Both c-Met and VEGFR-2 are important targets for cancer therapies. Here we report a series of potent dual c-Met and VEGFR-2 inhibitors bearing an anilinopyrimidine scaffold. Two novel synthetic protocols were employed for rapid analoguing of the designed molecules for structure–activity relationship (SAR) exploration. Some analogues displayed nanomolar potency against c-Met and VEGFR-2 at enzymatic level. Privileged compounds 3a, 3b, 3g, 3h, and 18a exhibited potent antiproliferative effect against c-Met addictive cell lines with IC50 values ranged from 0.33 to 1.7 μM. In addition, a cocrystal structure of c-Met in complex with 3h has been determined, which reveals the binding mode of c-Met to its inhibitor and helps to interpret the SAR of the analogues. PMID:24944742

  14. VEGF regulates local inhibitory complement proteins in the eye and kidney

    PubMed Central

    Keir, Lindsay S.; Firth, Rachel; Aponik, Lyndsey; Sakimoto, Susumu; Aguilar, Edith; Welsh, Gavin I.; Richards, Anna; Usui, Yoshihiko; Satchell, Simon C.; Kuzmuk, Valeryia; Coward, Richard J.; Goult, Jonathan; Bull, Katherine R.; Bharti, Kapil; Westenskow, Peter D.; Michael, Iacovos P.; Saleem, Moin A.

    2016-01-01

    Outer retinal and renal glomerular functions rely on specialized vasculature maintained by VEGF that is produced by neighboring epithelial cells, the retinal pigment epithelium (RPE) and podocytes, respectively. Dysregulation of RPE- and podocyte-derived VEGF is associated with neovascularization in wet age-related macular degeneration (ARMD), choriocapillaris degeneration, and glomerular thrombotic microangiopathy (TMA). Since complement activation and genetic variants in inhibitory complement factor H (CFH) are also features of both ARMD and TMA, we hypothesized that VEGF and CFH interact. Here, we demonstrated that VEGF inhibition decreases local CFH and other complement regulators in the eye and kidney through reduced VEGFR2/PKC-α/CREB signaling. Patient podocytes and RPE cells carrying disease-associated CFH genetic variants had more alternative complement pathway deposits than controls. These deposits were increased by VEGF antagonism, a common wet ARMD treatment, suggesting that VEGF inhibition could reduce cellular complement regulatory capacity. VEGF antagonism also increased markers of endothelial cell activation, which was partially reduced by genetic complement inhibition. Together, these results suggest that VEGF protects the retinal and glomerular microvasculature, not only through VEGFR2-mediated vasculotrophism, but also through modulation of local complement proteins that could protect against complement-mediated damage. Though further study is warranted, these findings could be relevant for patients receiving VEGF antagonists. PMID:27918307

  15. In situ validation of VEGFR-2 and α v ß 3 integrin as targets for breast lesion characterization.

    PubMed

    Ehling, Josef; Misiewicz, Matthias; von Stillfried, Saskia; Möckel, Diana; Bzyl, Jessica; Pochon, Sibylle; Lederle, Wiltrud; Knuechel, Ruth; Lammers, Twan; Palmowski, Moritz; Kiessling, Fabian

    2016-04-01

    Vascular endothelial growth factor receptor 2 (VEGFR-2) and α v ß 3 integrin are the most frequently addressed targets in molecular imaging of tumor angiogenesis. In preclinical studies, molecular imaging of angiogenesis has shown potential to detect and differentiate benign and malignant lesions of the breast. Thus, in this retrospective clinical study employing patient tissues, the diagnostic value of VEGFR-2, α v ß 3 integrin and vascular area fraction for the diagnosis and differentiation of breast neoplasia was evaluated. To this end, tissue sections of breast cancer (n = 40), pre-invasive ductal carcinoma in situ (DCIS; n = 8), fibroadenoma (n = 40), radial scar (n = 6) and normal breast tissue (n = 40) were used to quantify (1) endothelial VEGFR-2, (2) endothelial α v ß 3 integrin and (3) total α v ß 3 integrin expression, as well as (4) the vascular area fraction. Sensitivity and specificity to differentiate benign from malignant lesions were calculated for each marker by receiver operating characteristics (ROC) analyses. Whereas vessel density, as commonly used, did not significantly differ between benign and malignant lesions (AUROC: 0.54), VEGFR-2 and α v ß 3 integrin levels were gradually up-regulated in carcinoma versus fibroadenoma versus healthy tissue. The highest diagnostic accuracy for differentiating carcinoma from fibroadenoma was found for total α v ß 3 integrin expression (AUROC: 0.76), followed by VEGFR-2 (AUROC: 0.71) and endothelial α v ß 3 integrin expression (AUROC: 0.68). In conclusion, total α v ß 3 integrin expression is the best discriminator between breast cancer, fibroadenoma and normal breast tissue. With respect to vascular targeting and molecular imaging of angiogenesis, endothelial VEGFR-2 appeared to be slightly superior to endothelial α v ß 3 for differentiating benign from cancerous lesions.

  16. Vascular endothelial growth factor A (VEGF-A) decreases expression and secretion of pleiotrophin in a VEGF receptor-independent manner.

    PubMed

    Poimenidi, Evangelia; Theodoropoulou, Christina; Koutsioumpa, Marina; Skondra, Lamprini; Droggiti, Eirini; van den Broek, Marloes; Koolwijk, Pieter; Papadimitriou, Evangelia

    2016-05-01

    Vascular endothelial growth factor A (VEGF-A) is a key molecule in angiogenesis acting through VEGF receptors (VEGFRs), ανβ3 integrin, receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) and cell surface nucleolin (NCL). Pleiotrophin (PTN) stimulates endothelial cell migration and limits the angiogenic effects of VEGF-A165 to the levels of its own effect, possibly acting as a VEGF-A165 modifier. Since PTN and VEGF-A165 share receptors and actions on endothelial cells, in the present work we studied whether and how VEGF-A165 affects PTN expression or secretion. VEGF-A165 decreased PTN mRNA and protein levels acting at the transcriptional level. Bevacizumab, a selective VEGFR2 tyrosine kinase inhibitor and down-regulation of VEGFR2 expression by siRNA did not affect this decrease, suggesting that it is VEGFR-independent. VEGF-A121 also decreased PTN mRNA and protein levels, suggesting that heparin binding of VEGF-A165 is not involved. Blockage of cell surface NCL, lack of expression or mutation of β3 integrin and down-regulation of RPTPβ/ζ abolished the inhibitory effect of VEGF-A165 on PTN expression and secretion. Down-regulation of endogenous PTN in endothelial cells enhanced VEGF-A165-induced increase in migration and tube formation on matrigel. Collectively, these data suggest that VEGF-A down-regulates PTN expression and secretion through the RPTPβ/ζ-ανβ3-NCL axis to enhance its own effect on cell migration and further highlight the role of RPTPβ/ζ in VEGF-A actions. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Hsp20 Functions as a Novel Cardiokine in Promoting Angiogenesis via Activation of VEGFR2

    PubMed Central

    Zhu, Hongyan; Kranias, Evangelia G.; Tang, Yaoliang; Peng, Tianqing; Chang, Jiang; Fan, Guo-Chang

    2012-01-01

    Heat shock proteins (Hsps) are well appreciated as intrinsic protectors of cardiomyocytes against numerous stresses. Recent studies have indicated that Hsp20 (HspB6), a small heat shock protein, was increased in blood from cardiomyopathic hamsters. However, the exact source of the increased circulating Hsp20 and its potential role remain obscure. In this study, we observed that the circulating Hsp20 was increased in a transgenic mouse model with cardiac-specific overexpression of Hsp20, compared with wild-type mice, suggesting its origin from cardiomyocytes. Consistently, culture media harvested from Hsp20-overexpressing cardiomyocytes by Ad.Hsp20 infection contained an increased amount of Hsp20, compared to control media. Furthermore, we identified that Hsp20 was secreted through exosomes, independent of the endoplasmic reticulum-Golgi pathway. To investigate whether extracellular Hsp20 promotes angiogenesis, we treated human umbilical vein endothelial cells (HUVECs) with recombinant human Hsp20 protein, and observed that Hsp20 dose-dependently promoted HUVEC proliferation, migration and tube formation. Moreover, a protein binding assay and immunostaining revealed an interaction between Hsp20 and VEGFR2. Accordingly, stimulatory effects of Hsp20 on HUVECs were blocked by a VEGFR2 neutralizing antibody and CBO-P11 (a VEGFR inhibitor). These in vitro data are consistent with the in vivo findings that capillary density was significantly enhanced in Hsp20-overexpressing hearts, compared to non-transgenic hearts. Collectively, our findings demonstrate that Hsp20 serves as a novel cardiokine in regulating myocardial angiogenesis through activation of the VEGFR signaling cascade. PMID:22427880

  18. CHIP involves in non-small cell lung cancer prognosis through VEGF pathway.

    PubMed

    Tingting, Qian; Jiao, Wang; Qingfeng, Wang; Yancheng, Liu; Shijun, Y U; Zhaoqi, Wang; Dongmei, Sun; ShiLong, Wang

    2016-10-01

    CHIP (c-terminal Hsp70-interacting protein) is an E3 ligase playing vital roles in various cancers. The VEGF pathway has become an important therapeutic target in non-small cell lung cancer (NSCLC). However, little is known about the role of CHIP and the relationship between CHIP and VEGF-VEGFR2 (VEGF receptor 2) pathway in NSCLC. In this study we aimed to investigate the clinical function of CHIP in NSCLC and explore the relevant regulatory mechanism. QRT-PCR was performed to detect CHIP expression in NSCLC tissues. The association of CHIP expression and clinical parameters was analyzed using the Chi-square test. Kaplan- Meier and Cox analyses were performed to identify the role of CHIP in the prognosis of NSCLC patients. ELISA test was used to detect the VEGF secretion of NSCLC cells and western blot were used to detected the protein expression of VEGFR2 in NSCLC cells. and the results revealed that CHIP expression was decreased in NSCLC tissues and significantly correlated with clinical stages, lymph node metastasis and distant metastasis (P<0.05). Moreover, Kaplan-Meier and Cox regression analyses showed that patients with negative expression of CHIP had a shorter survival time and CHIP could be an independent prognostic biomarker. In addition, ELISA tests showed that CHIP negatively regulated the secretion level of VEGF. Furthermore, western blot assay indicated that the VEGFR2 protein level was reduced after CHIP over-expression. Taken together, our findings demonstrate for the first time that CHIP may serve as a promising prognostic biomarker for NSCLC patients and it may be involved in NSCLC angiogenesis through regulating VEGF secretion and expression of VEGFR2. Copyright © 2016. Published by Elsevier Masson SAS.

  19. Impact of vegf on astrocytes: analysis of gap junctional intercellular communication, proliferation, and motility.

    PubMed

    Wuestefeld, Ricarda; Chen, Jingchen; Meller, Karl; Brand-Saberi, Beate; Theiss, Carsten

    2012-05-01

    The purpose of the present study was to investigate the effects of vascular endothelial growth factor (VEGF) on gap junctional intercellular communication (GJIC), cell proliferation, and cell dynamics in primary astrocytes. VEGF is known as a dimeric polypeptide that potentially binds to two receptors, VEGFR-1 and VEGFR-2, however many effects are mediated by VEGFR-2, for example, actin polymerization, forced cell migration, angiogenesis, and cell proliferation. Recently it has been shown that in case of hypoxia, ischemia or injury VEGF is upregulated to stimulate angiogenesis and cell proliferation. Besides this, VEGF reveals a potent therapeutical target for averting tumor vascularization, emerging in bevacizumab, the first humanized anti-VEGF-A antibody for treating recurrent Glioblastoma multiforme. To expand our knowledge about VEGF effects in glial cells, we cultivated rat astrocytes in medium containing VEGF for 1 and 2 days. To investigate the effects of VEGF on GJIC, we microinjected neurobiotin into a single cell and monitored dye-spreading into adjacent cells. These experiments showed that VEGF significantly enhances astrocytic GJIC compared with controls. Cell proliferation measured by BrdU-labeling also revealed a significant increase of astrocytic mitose rates subsequent to 1 day of VEGF exposure, whereas longer VEGF treatment for 2 days did not have additive effects. To study cell-dynamics of astrocytes subsequent to VEGF treatment, we additionally transfected astrocytes with LifeAct-RFP. Live-cell imaging and quantitative analysis of these cells with aid of confocal laser scanning microscopy revealed higher process movement of VEGF-treated astrocytes. In conclusion, VEGF strongly affects cell proliferation, GJIC, and motility in astrocytes. Copyright © 2012 Wiley Periodicals, Inc.

  20. Hypertensive stretch regulates endothelial exocytosis of Weibel-Palade bodies through VEGF receptor 2 signaling pathways.

    PubMed

    Xiong, Yan; Hu, Zhenqian; Han, Xiaofan; Jiang, Beibei; Zhang, Rongli; Zhang, Xiaoyu; Lu, Yao; Geng, Chenyang; Li, Wei; He, Yulong; Huo, Yingqing; Shibuya, Masabumi; Luo, Jincai

    2013-06-01

    Regulated endothelial exocytosis of Weibel-Palade bodies (WPBs), the first stage in leukocyte trafficking, plays a pivotal role in inflammation and injury. Acute mechanical stretch has been closely associated with vascular inflammation, although the precise mechanism is unknown. Here, we show that hypertensive stretch regulates the exocytosis of WPBs of endothelial cells (ECs) through VEGF receptor 2 (VEGFR2) signaling pathways. Stretch triggers a rapid release (within minutes) of von Willebrand factor and interleukin-8 from WPBs in cultured human ECs, promoting the interaction between leukocytes and ECs through the translocation of P-selectin to the cell membrane. We further show that hypertensive stretch significantly induces P-selectin translocation of intact ECs and enhances leukocyte adhesion both ex vivo and in vivo. Stretch-induced endothelial exocytosis is mediated via a VEGFR2/PLCγ1/calcium pathway. Interestingly, stretch also induces a negative feedback via a VEGFR2/Akt/nitric oxide pathway. Such dual effects are confirmed using pharmacological and genetic approaches in carotid artery segments, as well as in acute hypertensive mouse models. These studies reveal mechanical stretch as a potent agonist for endothelial exocytosis, which is modulated by VEGFR2 signaling. Thus, VEGFR2 signaling pathways may represent novel therapeutic targets in limiting hypertensive stretch-related inflammation.

  1. 1-Piperazinylphthalazines as potential VEGFR-2 inhibitors and anticancer agents: Synthesis and in vitro biological evaluation.

    PubMed

    Abou-Seri, Sahar M; Eldehna, Wagdy M; Ali, Mamdouh M; Abou El Ella, Dalal A

    2016-01-01

    In our endeavor towards the development of effective VEGFR-2 inhibitors, three novel series of phthalazine derivatives based on 1-piperazinyl-4-arylphthalazine scaffold were synthesized. All the newly prepared phthalazines 16a-k, 18a-e and 21a-g were evaluated in vitro for their inhibitory activity against VEGFR-2. In particular, compounds 16k and 21d potently inhibited VEGFR-2 at sub-micromolar IC50 values 0.35 ± 0.03 and 0.40 ± 0.04 μM, respectively. Moreover, seventeen selected compounds 16c-e, 16g, 16h, 16j, 16k, 18c-e and 21a-g were evaluated for their in vitro anticancer activity according to US-NCI protocol, where compounds 16k and 21d proved to be the most potent anticancer agents. While, compound 16k exhibited potent broad spectrum anticancer activity with full panel GI50 (MG-MID) value of 3.62 μM, compound 21d showed high selectivity toward leukemia and prostate cancer subpanels [subpanel GI50 (MG-MID) 3.51 and 5.15 μM, respectively]. Molecular docking of compounds16k and 21d into VEGFR-2 active site was performed to explore their potential binding mode.

  2. Selective flexibility of side-chain residues improves VEGFR-2 docking score using AutoDock Vina.

    PubMed

    Abreu, Rui M V; Froufe, Hugo J C; Queiroz, Maria-João R P; Ferreira, Isabel C F R

    2012-04-01

    Selective side-chain residue flexibility is an option available on AutoDock Vina docking software. This approach is promising as it attempts to provide a more realistic ligand-protein interaction environment without an unmanageable increase in computer processing time. However, studies validating this approach are still scarce. VEGFR-2 (vascular endothelial growth factor receptor 2), a known protein target for anti-angiogenic agents, was used in this study. Four residues located in the VEGFR-2 kinase site were selected and made flexible: Lys868, Glu885, Cys919, and Asp1046. The docking scores for all possible combinations of flexible residues were compared to the docking scores using a rigid conformation. The best overall docking scores were obtained using the Glu885 flexible conformation, with Pearson and Spearman rank correlation values of 0.568 and 0.543, respectively, and a 51% increase in processing time. Using different VEGFR-2 crystal structures, a similar trend was observed with the Glu885 flexible conformation presenting best scores. This study demonstrates that careful use of selective side-chain residue flexibility can improve AutoDock Vina docking score accuracy, without a significant increase in processing time. This methodology can be a valuable tool in drug design projects using VEGFR-2 but will also probably be useful if applied to other protein targets.

  3. Pharmacokinetics and pharmacodynamics of VEGF-neutralizing antibodies

    PubMed Central

    2011-01-01

    Background Vascular endothelial growth factor (VEGF) is a potent regulator of angiogenesis, and its role in cancer biology has been widely studied. Many cancer therapies target angiogenesis, with a focus being on VEGF-mediated signaling such as antibodies to VEGF. However, it is difficult to predict the effects of VEGF-neutralizing agents. We have developed a whole-body model of VEGF kinetics and transport under pathological conditions (in the presence of breast tumor). The model includes two major VEGF isoforms VEGF121 and VEGF165, receptors VEGFR1, VEGFR2 and co-receptors Neuropilin-1 and Neuropilin-2. We have added receptors on parenchymal cells (muscle fibers and tumor cells), and incorporated experimental data for the cell surface density of receptors on the endothelial cells, myocytes, and tumor cells. The model is applied to investigate the action of VEGF-neutralizing agents (called "anti-VEGF") in the treatment of cancer. Results Through a sensitivity study, we examine how model parameters influence the level of free VEGF in the tumor, a measure of the response to VEGF-neutralizing drugs. We investigate the effects of systemic properties such as microvascular permeability and lymphatic flow, and of drug characteristics such as the clearance rate and binding affinity. We predict that increasing microvascular permeability in the tumor above 10-5 cm/s elicits the undesired effect of increasing tumor interstitial VEGF concentration beyond even the baseline level. We also examine the impact of the tumor microenvironment, including receptor expression and internalization, as well as VEGF secretion. We find that following anti-VEGF treatment, the concentration of free VEGF in the tumor can vary between 7 and 233 pM, with a dependence on both the density of VEGF receptors and co-receptors and the rate of neuropilin internalization on tumor cells. Finally, we predict that free VEGF in the tumor is reduced following anti-VEGF treatment when VEGF121 comprises at least

  4. Prognostic value of vascular endothelial growth factor (VEGF), VEGF receptor 2, platelet-derived growth factor-β (PDGF-β), and PDGF-β receptor expression in papillary renal cell carcinoma.

    PubMed

    Kim, Myong; Sohn, Mooyoung; Shim, Myungsun; Choi, Seung-Kwon; Park, Myungchan; Kim, Eunna; Go, Heounjeong; Park, Yangsoon; Cho, Yong Mee; Ro, Jae Y; Jeong, In Gab; Song, Cheryn; Hong, Jun Hyuk; Kim, Choung-Soo; Ahn, Hanjong

    2017-03-01

    The prognostic value of the expression of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), platelet-derived growth factor (PDGF)-β, and PDGF receptor (PDGFR)-β in papillary renal cell carcinoma (pRCC) is unknown. A total of 145 patients, who were confirmed to have pRCC, were analyzed. Expression levels of molecular markers were assessed via immunohistochemistry. The median follow-up period for all patients was 52.0 (interquartile range, 34.5-90.5) months. Among the cohort of 145 patients, high VEGF expression was observed in 100 (69.0%) patients, whereas high expression of VEGFR2, PDGF-β, and PDGFR-β was observed in 64 (44.1%), 42 (29.0%), and 30 (20.7%) patients, respectively. Only patients with high VEGFR2 expression exhibited improved 10-year recurrence-free survival (85.3% versus 58.1%; P=.005) and cancer-specific survival (86.4% versus 70.1%; P=.014) rates compared with individuals who exhibited low expression. Multivariate analysis revealed that high VEGFR2 expression was an independent prognostic factor for recurrence (hazard ratio, 0.326; P=.006) and cancer-specific mortality (hazard ratio, 0.334; P=.046). During follow-up, 17 patients received targeted drug therapy. Patients with high VEGFR2 expression showed a better initial response (partial response, 40%; stable disease, 20%; progressive disease, 40%) than patients with low expression did (partial response, 0%; stable disease, 58.3%; progressive disease, 41.7%; P=.052). pRCC with high VEGFR2 expression seems to be associated with a better initial response to targeted drug therapy and a better prognostic outcome.

  5. Spinal vascular endothelial growth factor (VEGF) and erythropoietin (EPO) induced phrenic motor facilitation after repetitive acute intermittent hypoxia.

    PubMed

    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. Vascular endothelial growth factor (VEGF) and VEGF receptor inhibitors in the treatment of renal cell carcinomas.

    PubMed

    Roskoski, Robert

    2017-03-19

    One Von Hippel-Lindau (VHL) tumor suppressor gene is lost in most renal cell carcinomas while the nondeleted allele exhibits hypermethylation-induced inactivation or inactivating somatic mutations. As a result of these genetic modifications, there is an increased production of VEGF-A and pro-angiogenic growth factors in this disorder. The important role of angiogenesis in the pathogenesis of renal cell carcinomas and other tumors has focused the attention of investigators on the biology of VEGFs and VEGFR1-3 and to the development of inhibitors of the intricate and multifaceted angiogenic pathways. VEGFR1-3 contain an extracellular segment with seven immunoglobulin-like domains, a transmembrane segment, a juxtamembrane segment, a protein kinase domain with an insert of about 70 amino acid residues, and a C-terminal tail. VEGF-A stimulates the activation of preformed VEGFR2 dimers by the auto-phosphorylation of activation segment tyrosines followed by the phosphorylation of additional protein-tyrosines that recruit phosphotyrosine binding proteins thereby leading to signalling by the ERK1/2, AKT, Src, and p38 MAP kinase pathways. VEGFR1 modulates the activity of VEGFR2, which is the chief pathway in vasculogenesis and angiogenesis. VEGFR3 and its ligands (VEGF-C and VEGF-D) are involved primarily in lymphangiogenesis. Small molecule VEGFR1/2/3 inhibitors including axitinib, cabozantinib, lenvatinib, sorafenib, sunitinib, and pazopanib are approved by the FDA for the treatment of renal cell carcinomas. Most of these agents are type II inhibitors of VEGFR2 and inhibit the so-called DFG-Aspout inactive enzyme conformation. These drugs are steady-state competitive inhibitors with respect to ATP and like ATP they form hydrogen bonds with the hinge residues that connect the small and large protein kinase lobes. Bevacizumab, a monoclonal antibody that binds to VEGF-A, is also approved for the treatment of renal cell carcinomas. Resistance to these agents invariably occurs

  7. Expression and Role of VEGF in the Adult Retinal Pigment Epithelium

    PubMed Central

    Ford, Knatokie M.; Saint-Geniez, Magali; Walshe, Tony; Zahr, Alisar

    2011-01-01

    Purpose. Despite a lack of active angiogenesis, VEGF is expressed in nearly every adult tissue, and recent evidence suggests that VEGF may serve as a survival factor for both vascular and nonvascular tissues. VEGF blockade is a widely used treatment for neovascular diseases such as wet age-related macular degeneration (AMD). Therefore, it was sought in this study to evaluate the expression and role of endogenous VEGF in RPE. Methods. VEGF and VEGFR2 expression in the murine retina were assessed during development. Bevacizumab was used to neutralize VEGF in ARPE-19 cells, and the effects on cell survival and apical microvill were assessed by TUNEL and SEM, respectively. VEGF was systemically neutralized in vivo by adenoviral-mediated overexpression of soluble VEGFR1 (sFlt). RPE and choriocapillaris were analyzed by transmission electron microscopy (TEM). Changes in gene expression were evaluated by quantitative real-time PCR. Results. VEGF expression was detected in the developing RPE as early as embryonic day (E) 9.5, whereas VEGFR2 expression by RPE began nonuniformly between postnatal (P) day 6.5 and P8.5. VEGF neutralization in vitro led to increased apoptosis and reduced microvilli density and length. Systemic VEGF neutralization led to transient degenerative changes; RPE were vacuolated and separated from photoreceptor outer segments, and choriocapillaris fenestrations were decreased. VEGF levels were elevated in RPE of Ad-sFlt1 mice at day 4 postinfection, and there was increased expression of the neurotrophic factor CD59a at day 14. Conclusions. These results indicate that VEGF plays a critical role in survival and maintenance of RPE integrity. Potential undesired off-target effects should be considered with chronic use of anti-VEGF agents. PMID:22058334

  8. Development and evaluation of a novel VEGFR2-targeted nanoscale ultrasound contrast agents

    NASA Astrophysics Data System (ADS)

    Yu, Houqiang; Li, Chunfang; He, Xiaoling; Zhou, Qibing; Ding, Mingyue

    2016-04-01

    Recent literatures have reported that the targeted nanoscale ultrasound contrast agents are becoming more and more important in medical application, like ultrasound imaging, detection of perfusion, drug delivery and molecular imaging and so on. In this study, we fabricated an uniform nanoscale bubbles (257 nm with the polydispersity index of 0.458) by incorporation of antibody targeted to vascular endothelial growth factor receptor 2 (VEGFR2) into the nanobubbles membrane by using avidin-biotin interaction. Some fundamental characterizations such as nanobubble suspension, surface morphology, particle size distribution and zeta potential were investigated. The concentration and time-intensity curves (TICs) were obtained with a self-made ultrasound experimental setup in vitro evaluation. In addition, in order to evaluate the contrast enhancement ability and the potential tumor-targeted ability in vivo, normal Wistar rats and nude female BALB/c mice were intravascular administration of the nanobubbles via tail vein injection, respectively. Significant contrast enhancement of ultrasound imaging within liver and tumor were visualized. These experiments demonstrated that the targeted nanobubbles is efficient in ultrasound molecular imaging by enhancement of the contrast effect and have potential capacity for targeted tumor diagnosis and therapy in the future.

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

    PubMed Central

    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

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

    PubMed

    Fantin, Alessandro; Lampropoulou, Anastasia; Senatore, Valentina; Brash, James T; Prahst, Claudia; Lange, Clemens A; Liyanage, Sidath E; Raimondi, Claudio; Bainbridge, James W; Augustin, Hellmut G; Ruhrberg, Christiana

    2017-04-03

    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. © 2017 Fantin et al.

  11. Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis

    SciTech Connect

    Shibuya, Masabumi . E-mail: shibuya@ims.u-tokyo.ac.jp; Claesson-Welsh, Lena . E-mail: lena.welsh@genpat.uu.se

    2006-03-10

    The VEGF/VPF (vascular endothelial growth factor/vascular permeability factor) ligands and receptors are crucial regulators of vasculogenesis, angiogenesis, lymphangiogenesis and vascular permeability in vertebrates. VEGF-A, the prototype VEGF ligand, binds and activates two tyrosine kinase receptors: VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). VEGFR1, which occurs in transmembrane and soluble forms, negatively regulates vasculogenesis and angiogenesis during early embryogenesis, but it also acts as a positive regulator of angiogenesis and inflammatory responses, playing a role in several human diseases such as rheumatoid arthritis and cancer. The soluble VEGFR1 is overexpressed in placenta in preeclampsia patients. VEGFR2 has critical functions in physiological and pathological angiogenesis through distinct signal transduction pathways regulating proliferation and migration of endothelial cells. VEGFR3, a receptor for the lymphatic growth factors VEGF-C and VEGF-D, but not for VEGF-A, regulates vascular and lymphatic endothelial cell function during embryogenesis. Loss-of-function variants of VEGFR3 have been identified in lymphedema. Formation of tumor lymphatics may be stimulated by tumor-produced VEGF-C, allowing increased spread of tumor metastases through the lymphatics. Mapping the signaling system of these important receptors may provide the knowledge necessary to suppress specific signaling pathways in major human diseases.

  12. [Investigation of the venodilatory effect of vascular endothelial growth factor (VEGF) in rat gingiva].

    PubMed

    Gyurkovics, Milán; Lohinai, Zsolt; Gyorfi, Adrienne; Székely, D Andrea; Dinya, Elek; Fazekas, Arpád; Rosivall, László

    2013-06-01

    VEGF induces proliferation of endothelial cells, stimulates angiogenesis, and increases vascular permeability in many organs. Nevertheless, we have only limited information about its role on gingival hemodynamics, especially in venules. Therefor the aim of this study was to assess the acute circulatory effects of VEGF on rat gingival venules by means of the following protocol. Wister rats (n=63) were devided into five study groups after anesthesia; each animal received 10 microl of experimental solution dripped onto the lower interincisal gingiva. The groups included: 1) saline control (after the experiment, gingiva was excised for VEGF receptor 2 [VEGFR2] immunohistochemistry); 2) VEGF (0.1, 1, 10, or 50 microg/ml); 3) VEGF2 receptor antagonist 5-((7-benzyloxyquinazolin-4-yl)amino)-4-fluoro-2-methyl-phenol-hydrochloride (ZM323881; 20 microg/ml); 4) ZM323881 (20 microg/ml) followed by VEGF application (50 microg/ml after 15 minutes); and 5) VEGF (10 microg/ml), these rats were premedicated with nitric oxide (NO) synthase blocker (NG-nitro-L-arginine-methyl-ester [L-NAME]; 1 mg/ml in drinking water) for 1 week before the experiment. Changes in gingival superficial venule diameter were measured by vital microscopy prior to and 1, 5, 15, 30, and 60 minutes after the administration of the experimental solutions. According to our findings, VEGF dose-dependently increased the venular diameter compared to saline. ZM323881 alone did not cause any alteration. Premedication with ZM323881 or L-NAME decreased the dilatory effects of VEGF. Occassionally moderate VEGFR2 immunohistochemical labeling was observed in the wall components of the venules. Concluding our results we can say, that there is no remarkable VEGF production under physiologic circumstances in rat gingiva, but VEGF is able to increase gingival blood flow through the activation of VEGF2 receptors and consequent NO release.

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

    PubMed Central

    2011-01-01

    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 of isoform specificity in

  14. Avian leukosis virus subgroup J induces VEGF expression via NF-κB/PI3K-dependent IL-6 production

    PubMed Central

    Gao, Yanni; Zhang, Yao; Yao, Yongxiu; Guan, Xiaolu; Liu, Yongzhen; Qi, Xiaole; Wang, Yongqiang; Liu, Changjun; Zhang, Yanping; Gao, Honglei; Nair, Venugopal; Wang, Xiaomei; Gao, Yulong

    2016-01-01

    Avian leukosis virus subgroup J (ALV-J) is an oncogenic virus causing hemangiomas and myeloid tumors in chickens. Interleukin-6 (IL-6) is a multifunctional pro-inflammatory interleukin involved in many types of cancer. We previously demonstrated that IL-6 expression was induced following ALV-J infection in chickens. The aim of this study is to characterize the mechanism by which ALV-J induces IL-6 expression, and the role of IL-6 in tumor development. Our results demonstrate that ALV-J infection increases IL-6 expression in chicken splenocytes, peripheral blood lymphocytes, and vascular endothelial cells. IL-6 production is induced by the ALV-J envelope protein gp85 and capsid protein p27 via PI3K- and NF-κB-mediated signaling. IL-6 in turn induced expression of vascular endothelial growth factor (VEGF)-A and its receptor, VEGFR-2, in vascular endothelial cells and embryonic vascular tissues. Suppression of IL-6 using siRNA inhibited the ALV-J induced VEGF-A and VEGFR-2 expression in vascular endothelial cells, indicating that the ALV-J-induced VEGF-A/VEGFR-2 expression is mediated by IL-6. As VEGF-A and VEGFR-2 are important factors in oncogenesis, our findings suggest that ALV-J hijacks IL-6 to promote tumorigenesis, and indicate that IL-6 could potentially serve as a therapeutic target in ALV-J infections. PMID:27852059

  15. Avian leukosis virus subgroup J induces VEGF expression via NF-κB/PI3K-dependent IL-6 production.

    PubMed

    Gao, Yanni; Zhang, Yao; Yao, Yongxiu; Guan, Xiaolu; Liu, Yongzhen; Qi, Xiaole; Wang, Yongqiang; Liu, Changjun; Zhang, Yanping; Gao, Honglei; Nair, Venugopal; Wang, Xiaomei; Gao, Yulong

    2016-12-06

    Avian leukosis virus subgroup J (ALV-J) is an oncogenic virus causing hemangiomas and myeloid tumors in chickens. Interleukin-6 (IL-6) is a multifunctional pro-inflammatory interleukin involved in many types of cancer. We previously demonstrated that IL-6 expression was induced following ALV-J infection in chickens. The aim of this study is to characterize the mechanism by which ALV-J induces IL-6 expression, and the role of IL-6 in tumor development. Our results demonstrate that ALV-J infection increases IL-6 expression in chicken splenocytes, peripheral blood lymphocytes, and vascular endothelial cells. IL-6 production is induced by the ALV-J envelope protein gp85 and capsid protein p27 via PI3K- and NF-κB-mediated signaling. IL-6 in turn induced expression of vascular endothelial growth factor (VEGF)-A and its receptor, VEGFR-2, in vascular endothelial cells and embryonic vascular tissues. Suppression of IL-6 using siRNA inhibited the ALV-J induced VEGF-A and VEGFR-2 expression in vascular endothelial cells, indicating that the ALV-J-induced VEGF-A/VEGFR-2 expression is mediated by IL-6. As VEGF-A and VEGFR-2 are important factors in oncogenesis, our findings suggest that ALV-J hijacks IL-6 to promote tumorigenesis, and indicate that IL-6 could potentially serve as a therapeutic target in ALV-J infections.

  16. Genetic variations in the VEGF pathway as prognostic factors in metastatic colorectal cancer patients treated with oxaliplatin-based chemotherapy.

    PubMed

    Paré-Brunet, L; Sebio, A; Salazar, J; Berenguer-Llergo, A; Río, E; Barnadas, A; Baiget, M; Páez, D

    2015-10-01

    Angiogenesis is a significant biological mechanism in the progression and metastasis of solid tumors. Vascular endothelial growth factor (VEGF), its receptors and signaling effectors have a central role in tumor-induced angiogenesis. Genetic variation in the VEGF pathway may impact on tumor angiogenesis and, hence, on clinical cancer outcomes. This study evaluates the influence of common genetic variations within the VEGF pathway in the clinical outcomes of 172 metastatic colorectal cancer (mCRC) patients treated with first-line oxaliplatin/5-fluorouracil chemotherapy. A total of 27 single-nucleotide polymorphisms (SNPs) in 16 genes in the VEGF-dependent angionenesis process were genotyped using a dynamic array on the BioMark™ system. After assessing the KRAS mutational status, we found that four SNPs located in three genes (KISS1, KRAS and VEGFR2) were associated with progression-free survival. Five SNPs in three genes (ITGAV, KRAS and VEGFR2) correlated with overall survival. The gene-gene interactions identified in the survival tree analysis support the importance of VEGFR2 rs2071559 and KISS1 rs71745629 in modulating these outcomes. This study provides evidence that functional germline polymorphisms in the VEGF pathway may help to predict outcome in mCRC patients who undergo oxaliplatin/5-fluorouracil chemotherapy.

  17. The miR-126-VEGFR2 axis controls the innate response to pathogen-associated nucleic acids.

    PubMed

    Agudo, Judith; Ruzo, Albert; Tung, Navpreet; Salmon, Hélène; Leboeuf, Marylène; Hashimoto, Daigo; Becker, Christian; Garrett-Sinha, Lee-Ann; Baccarini, Alessia; Merad, Miriam; Brown, Brian D

    2014-01-01

    miR-126 is a microRNA expressed predominately by endothelial cells and controls angiogenesis. We found miR-126 was required for the innate response to pathogen-associated nucleic acids and that miR-126-deficient mice had greater susceptibility to infection with pseudotyped HIV. Profiling of miRNA indicated that miR-126 had high and specific expression by plasmacytoid dendritic cells (pDCs). Moreover, miR-126 controlled the survival and function of pDCs and regulated the expression of genes encoding molecules involved in the innate response, including Tlr7, Tlr9 and Nfkb1, as well as Kdr, which encodes the growth factor receptor VEGFR2. Deletion of Kdr in DCs resulted in reduced production of type I interferon, which supports the proposal of a role for VEGFR2 in miR-126 regulation of pDCs. Our studies identify the miR-126-VEGFR2 axis as an important regulator of the innate response that operates through multiscale control of pDCs.

  18. Wild Roman chamomile extracts and phenolic compounds: enzymatic assays and molecular modelling studies with VEGFR-2 tyrosine kinase.

    PubMed

    Guimarães, Rafaela; Calhelha, Ricardo C; Froufe, Hugo J C; Abreu, Rui M V; Carvalho, Ana Maria; Queiroz, Maria João R P; Ferreira, Isabel C F R

    2016-01-01

    Angiogenesis is a process by which new blood vessels are formed from the pre-existing vasculature, and it is a key process that leads to tumour development. Some studies have recognized phenolic compounds as chemopreventive agents; flavonoids, in particular, seem to suppress the growth of tumor cells modifying the cell cycle. Herein, the antiangiogenic activity of Roman chamomile (Chamaemelum nobile L.) extracts (methanolic extract and infusion) and the main phenolic compounds present (apigenin, apigenin-7-O-glucoside, caffeic acid, chlorogenic acid, luteolin, and luteolin-7-O-glucoside) was evaluated through enzymatic assays using the tyrosine kinase intracellular domain of the Vascular Endothelium Growth Factor Receptor-2 (VEGFR-2), which is a transmembrane receptor expressed fundamentally in endothelial cells involved in angiogenesis, and molecular modelling studies. The methanolic extract showed a lower IC50 value (concentration that provided 50% of VEGFR-2 inhibition) than the infusion, 269 and 301 μg mL(-1), respectively. Regarding phenolic compounds, luteolin and apigenin showed the highest capacity to inhibit the phosphorylation of VEGFR-2, leading us to believe that these compounds are involved in the activity revealed by the methanolic extract.

  19. PDGFRα/β and VEGFR2 polymorphisms in colorectal cancer: incidence and implications in clinical outcome

    PubMed Central

    2012-01-01

    Background Angiogenesis plays an essential role in tumor growth and metastasis, and is a major target in cancer therapy. VEGFR and PDGFR are key players involved in this process. The purpose of this study was to assess the incidence of genetic variants in these receptors and its potential clinical implications in colorectal cancer (CRC). Methods VEGFR2, PDGFRα and PDGFRβ mutations were evaluated by sequencing their tyrosine kinase domains in 8 CRC cell lines and in 92 samples of patients with CRC. Correlations with clinicopathological features and survival were analyzed. Results Four SNPs were identified, three in PDGFRα [exon 12 (A12): c.1701A>G; exon 13 (A13): c.1809G>A; and exon 17 (A17): c.2439+58C>A] and one in PDGFRβ [exon 19 (B19): c.2601A>G]. SNP B19, identified in 58% of tumor samples and in 4 cell lines (LS174T, LS180, SW48, COLO205), was associated with higher PDGFR and pPDGFR protein levels. Consistent with this observation, 5-year survival was greater for patients with PDGFR B19 wild type tumors (AA) than for those harboring the G-allele genotype (GA or GG) (51% vs 17%; p=0.073). Multivariate analysis confirmed SNP B19 (p=0.029) was a significant prognostic factor for survival, independent of age (p=0.060) or TNM stage (p<0.001). Conclusions PDGFRβ exon 19 c.2601A>G SNP is commonly encountered in CRC patients and is associated with increased pathway activation and poorer survival. Implications regarding its potential influence in response to PDGFR-targeted agents remain to be elucidated. PMID:23146028

  20. Dickkopf-3 Upregulates VEGF in Cultured Human Endothelial Cells by Activating Activin Receptor-Like Kinase 1 (ALK1) Pathway

    PubMed Central

    Busceti, Carla L.; Marchitti, Simona; Bianchi, Franca; Di Pietro, Paola; Riozzi, Barbara; Stanzione, Rosita; Cannella, Milena; Battaglia, Giuseppe; Bruno, Valeria; Volpe, Massimo; Fornai, Francesco; Nicoletti, Ferdinando; Rubattu, Speranza

    2017-01-01

    Dkk-3 is a member of the dickkopf protein family of secreted inhibitors of the Wnt pathway, which has been shown to enhance angiogenesis. The mechanism underlying this effect is currently unknown. Here, we used cultured HUVECs to study the involvement of the TGF-β and VEGF on the angiogenic effect of Dkk-3. Addition of hrDkk-3 peptide (1 or 10 ng/ml) to HUVECs for 6 or 12 h enhanced the intracellular and extracellular VEGF protein levels, as assessed by RTPCR, immunoblotting, immunocytochemistry and ELISA. The increase in the extracellular VEGF levels was associated to the VEGFR2 activation. Pharmacological blockade of VEGFR2 abrogated Dkk-3-induced endothelial cell tubes formation, indicating that VEGF is a molecular player of the angiogenic effects of Dkk-3. Moreover, Dkk-3 enhanced Smad1/5/8 phosphorylation and recruited Smad4 to the VEGF gene promoter, suggesting that Dkk-3 activated ALK1 receptor leading to a transcriptional activation of VEGF. This mechanism was instrumental to the increased VEGF expression and endothelial cell tubes formation mediated by Dkk-3, because both effects were abolished by siRNA-mediated ALK1 knockdown. In summary, we have found that Dkk-3 activates ALK1 to stimulate VEGF production and induce angiogenesis in HUVECs. PMID:28352232

  1. Efficacy of Cotargeting Angiopoietin-2 and the VEGF Pathway in the Adjuvant Postsurgical Setting for Early Breast, Colorectal, and Renal Cancers.

    PubMed

    Wu, Florence T H; Man, Shan; Xu, Ping; Chow, Annabelle; Paez-Ribes, Marta; Lee, Christina R; Pirie-Shepherd, Steven R; Emmenegger, Urban; Kerbel, Robert S

    2016-12-01

    Antiangiogenic tyrosine kinase inhibitors (TKI) that target VEGF receptor-2 (VEGFR2) have not been effective as adjuvant treatments for micrometastatic disease in phase III clinical trials. Angiopoietin-2 (Ang2) is a proangiogenic and proinflammatory vascular destabilizer that cooperates with VEGF. The purpose of this study was to test whether CVX-060 (an Ang2-specific CovX-body) can be combined with VEGFR2-targeting TKIs (sunitinib or regorafenib) to successfully treat postsurgical metastatic disease in multiple orthotopically implanted human tumor xenograft and syngeneic murine tumor models. In the MDA-MB-231.LM2-4 human breast cancer model, adjuvant sunitinib was ineffective, whereas adjuvant CVX-060 delayed the progression of pulmonary or distant lymphatic metastases; however, overall survival was only improved with the adjuvant use of a VEGF-A/Ang2-bispecific CovX-body (CVX-241) but not when CVX-060 is combined with sunitinib. Adjuvant CVX-241 also showed promise in the EMT-6/CDDP murine breast cancer model, with or without an immune checkpoint inhibitor (anti-PD-L1). In the RENCA model of mouse renal cancer, however, combining CVX-060 with sunitinib in the adjuvant setting was superior to CVX-241 as treatment for postsurgical lung metastases. In the HCT116 and HT29 xenograft models of colorectal cancer, both CVX-060 and regorafenib inhibited liver metastases. Overall, our preclinical findings suggest differential strategies by which Ang2 blockers can be successfully combined with VEGF pathway targeting in the adjuvant setting to treat micrometastatic disease-particularly, in combination with VEGF-A blockers (but not VEGFR2 TKIs) in resected breast cancer; in combination with VEGFR2 TKIs in resected kidney cancer; and as single agents or with VEGFR2 TKIs in resected colorectal cancer. Cancer Res; 76(23); 6988-7000. ©2016 AACR.

  2. VEGF165 Stimulates Vessel Density and Vessel Diameter Differently in Angiogenesis and Lymphangiogenesis

    NASA Technical Reports Server (NTRS)

    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.

  3. KRIT1 protein depletion modifies endothelial cell behavior via increased vascular endothelial growth factor (VEGF) signaling.

    PubMed

    DiStefano, Peter V; Kuebel, Julia M; Sarelius, Ingrid H; Glading, Angela J

    2014-11-21

    Disruption of endothelial cell-cell contact is a key event in many cardiovascular diseases and a characteristic of pathologically activated vascular endothelium. The CCM (cerebral cavernous malformation) family of proteins (KRIT1 (Krev-interaction trapped 1), PDCD10, and CCM2) are critical regulators of endothelial cell-cell contact and vascular homeostasis. Here we show novel regulation of vascular endothelial growth factor (VEGF) signaling in KRIT1-depleted endothelial cells. Loss of KRIT1 and PDCD10, but not CCM2, increases nuclear β-catenin signaling and up-regulates VEGF-A protein expression. In KRIT1-depleted cells, increased VEGF-A levels led to increased VEGF receptor 2 (VEGFR2) activation and subsequent alteration of cytoskeletal organization, migration, and barrier function and to in vivo endothelial permeability in KRIT1-deficient animals. VEGFR2 activation also increases β-catenin phosphorylation but is only partially responsible for KRIT1 depletion-dependent disruption of cell-cell contacts. Thus, VEGF signaling contributes to modifying endothelial function in KRIT1-deficient cells and microvessel permeability in Krit1(+/-) mice; however, VEGF signaling is likely not the only contributor to disrupted endothelial cell-cell contacts in the absence of KRIT1.

  4. VEGF-A regulated by progesterone governs uterine angiogenesis and vascular remodelling during pregnancy

    PubMed Central

    Kim, Minah; Park, Hyeung Ju; Seol, Jae Won; Jang, Jeon Yeob; Cho, Young-Suk; Kim, Kyu Rae; Choi, Youngsok; Lydon, John P; DeMayo, Francesco J; Shibuya, Masabumi; Ferrara, Napoleone; Sung, Hoon-Ki; Nagy, Andras; Alitalo, Kari; Koh, Gou Young

    2013-01-01

    The features and regulation of uterine angiogenesis and vascular remodelling during pregnancy are poorly defined. Here we show that dynamic and variable decidual angiogenesis (sprouting, intussusception and networking), and active vigorous vascular remodelling such as enlargement and elongation of ‘vascular sinus folding’ (VSF) and mural cell drop-out occur distinctly in a spatiotemporal manner in the rapidly growing mouse uterus during early pregnancy — just after implantation but before placentation. Decidual angiogenesis is mainly regulated through VEGF-A secreted from the progesterone receptor (PR)-expressing decidual stromal cells which are largely distributed in the anti-mesometrial region (AMR). In comparison, P4-PR-regulated VEGF-A-VEGFR2 signalling, ligand-independent VEGFR3 signalling and uterine natural killer (uNK) cells positively and coordinately regulate enlargement and elongation of VSF. During the postpartum period, Tie2 signalling could be involved in vascular maturation at the endometrium in a ligand-independent manner, with marked reduction of VEGF-A, VEGFR2 and PR expressions. Overall, we show that two key vascular growth factor receptors — VEGFR2 and Tie2 — strikingly but differentially regulate decidual angiogenesis and vascular remodelling in rapidly growing and regressing uteri in an organotypic manner. PMID:23853117

  5. Vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitors: development and validation of predictive 3-D QSAR models through extensive ligand- and structure-based approaches

    NASA Astrophysics Data System (ADS)

    Ragno, Rino; Ballante, Flavio; Pirolli, Adele; Wickersham, Richard B.; Patsilinakos, Alexandros; Hesse, Stéphanie; Perspicace, Enrico; Kirsch, Gilbert

    2015-08-01

    Vascular endothelial growth factor receptor-2, (VEGFR-2), is a key element in angiogenesis, the process by which new blood vessels are formed, and is thus an important pharmaceutical target. Here, 3-D quantitative structure-activity relationship (3-D QSAR) were used to build a quantitative screening and pharmacophore model of the VEGFR-2 receptors for design of inhibitors with improved activities. Most of available experimental data information has been used as training set to derive optimized and fully cross-validated eight mono-probe and a multi-probe quantitative models. Notable is the use of 262 molecules, aligned following both structure-based and ligand-based protocols, as external test set confirming the 3-D QSAR models' predictive capability and their usefulness in design new VEGFR-2 inhibitors. From a survey on literature, this is the first generation of a wide-ranging computational medicinal chemistry application on VEGFR2 inhibitors.

  6. IL-8 and VEGFR-2 polymorphisms modulate long-term functional response to intravitreal ranibizumab in exudative age-related macular degeneration.

    PubMed

    Lazzeri, Stefano; Orlandi, Paola; Piaggi, Paolo; Sartini, Maria Sole; Casini, Giamberto; Guidi, Gianluca; Figus, Michele; Fioravanti, Anna; Di Desidero, Teresa; Ripandelli, Guido; Parravano, Mariacristina; Varano, Monica; Nardi, Marco; Bocci, Guido

    2016-01-01

    To investigate possible associations between VEGFR-2 and IL-8 gene SNPs and 1-year response to intravitreal ranibizumab for exudative age-related macular degeneration. Sixty-four eyes underwent a loading phase of three monthly intravitreal injections of ranibizumab 0.5 mg/0.05 ml followed by Pro Re Nata retreatment. VEGFR-2 rs2071559 (-604 A/G) and IL-8 rs4073 (-251 A/T) were analyzed. Ranibizumab was significantly more effective as measured by visual acuity in patients harboring the IL-8 rs4073 TT genotype (p = 0.045), whereas patients carrying the VEGFR-2 rs2071559 CC genotype revealed better functional response as measured by mean retinal sensitivity (p = 0.034). IL-8 rs4073 and VEGFR-2 rs2071559 genotypes may represent important molecular determinants to modulate final outcomes in neovascular age-related macular degeneration patients.

  7. VEGF blockade inhibits angiogenesis and reepithelialization of endometrium

    PubMed Central

    Fan, Xiujun; Krieg, Sacha; Kuo, Calvin J.; Wiegand, Stanley J.; Rabinovitch, Marlene; Druzin, Maurice L.; Brenner, Robert M.; Giudice, Linda C.; Nayak, Nihar R.

    2008-01-01

    Despite extensive literature on vascular endothelial growth factor (VEGF) expression and regulation by steroid hormones, the lack of clear understanding of the mechanisms of angiogenesis in the endometrium is a major limitation for use of antiangiogenic therapy targeting endometrial vessels. In the current work, we used the rhesus macaque as a primate model and the decidualized mouse uterus as a murine model to examine angiogenesis during endometrial breakdown and regeneration. We found that blockade of VEGF action with VEGF Trap, a potent VEGF blocker, completely inhibited neovascularization during endometrial regeneration in both models but had no marked effect on preexisting or newly formed vessels, suggesting that VEGF is essential for neoangiogenesis but not survival of mature vessels in this vascular bed. Blockade of VEGF also blocked reepithelialization in both the postmenstrual endometrium and the mouse uterus after decidual breakdown, evidence that VEGF has pleiotropic effects in the endometrium. In vitro studies with a scratch wound assay showed that the migration of luminal epithelial cells during repair involved signaling through VEGF receptor 2–neuropilin 1 (VEGFR2-NP1) receptors on endometrial stromal cells. The leading front of tissue growth during endometrial repair was strongly hypoxic, and this hypoxia was the local stimulus for VEGF expression and angiogenesis in this tissue. In summary, we provide novel experimental data indicating that VEGF is essential for endometrial neoangiogenesis during postmenstrual/postpartum repair.—Fan, X., Krieg, S., Kuo, C. J., Wiegand, S. J., Rabinovitch, M., Druzin, M. L., Brenner, R. M., Giudice, L. C., Nayak, N. R. VEGF blockade inhibits angiogenesis and reepithelialization of endometrium. PMID:18606863

  8. Serum C-Telopeptide Collagen Crosslinks and Plasma Soluble VEGFR2 as Pharmacodynamic Biomarkers in a Trial of Sequentially Administered Sunitinib and Cilengitide.

    PubMed

    O'Donnell, Peter H; Karovic, Sanja; Karrison, Theodore G; Janisch, Linda; Levine, Matthew R; Harris, Pamela J; Polite, Blase N; Cohen, Ezra E W; Fleming, Gini F; Ratain, Mark J; Maitland, Michael L

    2015-11-15

    Fit-for-purpose pharmacodynamic biomarkers could expedite development of combination antiangiogenic regimens. Plasma sVEGFR2 concentrations ([sVEGFR2]) mark sunitinib effects on the systemic vasculature. We hypothesized that cilengitide would impair microvasculature recovery during sunitinib withdrawal and could be detected through changes in [sVEGFR2]. Advanced solid tumor patients received 50 mg sunitinib daily for 14 days. For the next 14 days, patients were randomized to arm A (cilengitide 2,000 mg administered intravenously twice weekly) or arm B (no treatment). The primary endpoint was change in [sVEGFR2] between days 14 and 28. A candidate pharmacodynamic biomarker of cilengitide inhibition of integrin αvβ3, serum c-telopeptide collagen crosslinks (CTx), was also measured. Of 21 patients, 14 (7 per arm) received all treatments without interruption and had all blood samples available for analysis. The mean change and SD of [sVEGFR2] for all sunitinib-treated patients was consistent with previous data. There was no significant difference in the mean change in [sVEGFR2] from days 14 to 28 between the arms [arm A: 2.8 ng/mL; 95% confidence interval (CI), 2.1-3.6 vs. arm B: 2.0 ng/mL; 95% CI, 0.72-3.4; P = 0.22, 2-sample t test]. Additional analyses suggested (i) prior bevacizumab therapy to be associated with unusually low baseline [sVEGFR2] and (ii) sunitinib causes measurable changes in CTx. Cilengitide had no measurable effects on any circulating biomarkers. Sunitinib caused measurable declines in serum CTx. The properties of [sVEGFR2] and CTx observed in this study inform the design of future combination antiangiogenic therapy trials. ©2015 American Association for Cancer Research.

  9. Serum C-telopeptide collagen crosslinks and plasma soluble VEGFR2 as pharmacodynamic biomarkers in a trial of sequentially administered sunitinib and cilengitide

    PubMed Central

    O’Donnell, Peter H.; Karovic, Sanja; Karrison, Theodore G.; Janisch, Linda; Levine, Matthew R.; Harris, Pamela J.; Polite, Blase N.; Cohen, Ezra E.W.; Fleming, Gini F.; Ratain, Mark J.; Maitland, Michael L.

    2015-01-01

    Background Fit-for-purpose pharmacodynamic biomarkers could expedite development of combination anti-angiogenic regimens. Plasma sVEGFR2 concentrations ([sVEGFR2]) mark sunitinib effects on the systemic vasculature. We hypothesized that cilengitide would impair microvasculature recovery during sunitinib withdrawal and could be detected through changes in [sVEGFR2]. Methods Advanced solid tumor patients received sunitinib 50 mg daily for 14 days. For the next 14 days, patients were randomized to Arm A (cilengitide 2000 mg administered intravenously twice weekly (BIW)), or Arm B (no treatment). The primary endpoint was change in [sVEGFR2] between Day 14 and Day 28. A candidate pharmacodynamic biomarker of cilengitide inhibition of integrin αvβ3, serum c-telopeptide collagen crosslinks (CTx), was also measured. Results Of 21 patients, 14 (7/arm) received all treatments without interruption and had all blood samples available for analysis. The mean change and standard deviation of [sVEGFR2] for all sunitinib-treated patients was consistent with previous data. There was no significant difference in the mean change in [sVEGFR2] from Day 14 to Day 28 between the arms (Arm A: 2.8 ng/mL [95% CI 2.1, 3.6] vs. Arm B: 2.0 ng/mL [95% CI 0.72, 3.4] P = 0.22, two sample t test). Additional analyses suggested: 1) prior bevacizumab therapy to be associated with unusually low baseline [sVEGFR2], and 2) sunitinib causes measurable changes in CTx. Conclusions Cilengitide had no measurable effects on any circulating biomarkers. Sunitinib caused measurable declines in serum CTx. The properties of [sVEGFR2] and CTx observed in this study inform the design of future combination anti-angiogenic therapy trials. PMID:26199386

  10. VEGFR2-Targeted Ultrasound Imaging Agent Enhances the Detection of Ovarian Tumors at Early Stage in Laying Hens, a Preclinical Model of Spontaneous Ovarian Cancer.

    PubMed

    Barua, Animesh; Yellapa, Aparna; Bahr, Janice M; Machado, Sergio A; Bitterman, Pincas; Basu, Sanjib; Sharma, Sameer; Abramowicz, Jacques S

    2015-07-01

    Tumor-associated neoangiogenesis (TAN) is an early event in ovarian cancer (OVCA) development. Increased expression of vascular endothelial growth factor receptor 2 (VEGFR2) by TAN vessels presents a potential target for early detection by ultrasound imaging. The goal of this study was to examine the suitability of VEGFR2-targeted ultrasound contrast agents in detecting spontaneous OVCA in laying hens. Effects of VEGFR2-targeted contrast agents in enhancing the intensity of ultrasound imaging from spontaneous ovarian tumors in hens were examined in a cross-sectional study. Enhancement in the intensity of ultrasound imaging was determined before and after injection of VEGFR2-targeted contrast agents. All ultrasound images were digitally stored and analyzed off-line. Following scanning, ovarian tissues were collected and processed for histology and detection of VEGFR2-expressing microvessels. Enhancement in visualization of ovarian morphology was detected by gray-scale imaging following injection of VEGFR2-targeted contrast agents. Compared with pre-contrast, contrast imaging enhanced the intensities of ultrasound imaging significantly (p < 0.0001) irrespective of the pathological status of ovaries. In contrast to normal hens, the intensity of ultrasound imaging was significantly (p < 0.0001) higher in hens with early stage OVCA and increased further in hens with late stage OVCA. Higher intensities of ultrasound imaging in hens with OVCA were positively correlated with increased (p < 0.0001) frequencies of VEGFR2-expressing microvessels. The results of this study suggest that VEGFR2-targeted contrast agents enhance the visualization of spontaneous ovarian tumors in hens at early and late stages of OVCA. The laying hen may be a suitable model to test new imaging agents and develop targeted therapeutics. © The Author(s) 2014.

  11. Altered ratios of pro- and anti-angiogenic VEGF-A variants and pericyte expression of DLL4 disrupt vascular maturation in infantile haemangioma.

    PubMed

    Ye, Xi; Abou-Rayyah, Yassir; Bischoff, Joyce; Ritchie, Alison; Sebire, Neil J; Watts, Patrick; Churchill, Amanda J; Bates, David O

    2016-06-01

    Infantile haemangioma (IH), the most common neoplasm in infants, is a slowly resolving vascular tumour. Vascular endothelial growth factor A (VEGF-A), which consists of both the pro- and anti-angiogenic variants, contributes to the pathogenesis of IH. However, the roles of different VEGF-A variants in IH progression and its spontaneous involution is unknown. Using patient-derived cells and surgical specimens, we showed that the relative level of VEGF-A165 b was increased in the involuting phase of IH and the relative change in VEGF-A isoforms may be dependent on endothelial differentiation of IH stem cells. VEGFR signalling regulated IH cell functions and VEGF-A165 b inhibited cell proliferation and the angiogenic potential of IH endothelial cells in vitro and in vivo. The inhibition of angiogenesis by VEGF-A165 b was associated with the extent of VEGF receptor 2 (VEGFR2) activation and degradation and Delta-like ligand 4 (DLL4) expression. These results indicate that VEGF-A variants can be regulated by cell differentiation and are involved in IH progression. We also demonstrated that DLL4 expression was not exclusive to the endothelium in IH but was also present in pericytes, where the expression of VEGFR2 is absent, suggesting that pericyte-derived DLL4 may prevent sprouting during involution, independently of VEGFR2. Angiogenesis in IH therefore appears to be controlled by DLL4 within the endothelium in a VEGF-A isoform-dependent manner, and in perivascular cells in a VEGF-independent manner. The contribution of VEGF-A isoforms to disease progression also indicates that IH may be associated with altered splicing. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

  12. VEGF-C differentially regulates VEGF-A expression in ocular and cancer cells; promotes angiogenesis via RhoA mediated pathway.

    PubMed

    Kumar, Bharat; Chile, Shailaja A; Ray, Kriti B; Reddy, G E C Vidyadhar; Addepalli, Murali K; Kumar, A S Manoj; Ramana, Venkata; Rajagopal, Vikram

    2011-09-01

    Vascular angiogenesis is regulated by a number of cytokines of which vascular endothelial growth factor (VEGF)-A/and its receptor vascular endothelial growth factor receptor 2 (VEGFR2) play an indisputable role. Similarly lymphangiogenesis is regulated by VEGF-C and its receptor VEGFR3. Currently for treating vasculogenesis diseases such as proliferative retinopathies and cancer, a number of anti-VEGF-A therapies are approved for clinical use. Although clinical efficacies achieved are remarkable, they are found to be transitory in nature, followed by restoration of anti-VEGF therapy resistant angiogenesis. Recently the regulatory role of VEGF-C in initiating and potentiating neo-angiogenesis has been uncovered. Although the interactive nature of VEGF-A and C is known, the dynamics of their expression under knockdown conditions is yet to be established. Here in this study we have utilized siRNA to knockdown both VEGF-A and C either independently or in combination. Analysis of VEGF-A and C expression (only in cancer cell lines MCF7, A549 and H460 but not in the ocular cell line RPE19) has shown enhanced expression levels of VEGF-C with increase in knockdown of VEGF-A. However, VEGF-C knockdown has resulted in decreased expression levels of VEGF-A both in RPE19 and MCF7 cells in a dose dependent manner. In addition, VEGF-C knockdown also resulted in decreased expression of RhoA. Further, knockdown studies of RhoA even with supplementation of VEGF-C or A has resulted in decreased endothelial cell proliferation and stress fiber formation, indicating that VEGF-C does promote angiogenesis via RhoA mediated pathway.

  13. Germline Polymorphisms of the VEGF Pathway Predict Recurrence in Nonadvanced Differentiated Thyroid Cancer.

    PubMed

    Marotta, Vincenzo; Sciammarella, Concetta; Capasso, Mario; Testori, Alessandro; Pivonello, Claudia; Chiofalo, Maria Grazia; Gambardella, Claudio; Grasso, Marica; Antonino, Antonio; Annunziata, Annamaria; Macchia, Paolo Emidio; Pivonello, Rosario; Santini, Luigi; Botti, Gerardo; Losito, Simona; Pezzullo, Luciano; Colao, Annamaria; Faggiano, Antongiulio

    2017-02-01

    Tumor angiogenesis is determined by host genetic background rather than environment. Germline single nucleotide polymorphisms (SNPs) of the vascular endothelial growth factor (VEGF) pathway have demonstrated prognostic value in different tumors. Our main objective was to test the prognostic value of germline SNPs of the VEGF pathway in nonadvanced differentiated thyroid cancer (DTC). Secondarily, we sought to correlate analyzed SNPs with microvessel density (MVD). Multicenter, retrospective, observational study. Four referral centers. Blood samples were obtained from consecutive DTC patients. Genotyping was performed according to the TaqMan protocol, including 4 VEGF-A (-2578C>A, -460T>C, +405G>C, and +936C>T) and 2 VEGFR-2 (+1192 C>T and +1719 T>A) SNPs. MVD was estimated by means of CD34 staining. Rate of recurrent structural disease/disease-free survival (DFS). Difference in MVD between tumors from patients with different genotype. Two hundred four patients with stage I-II DTC (mean follow-up, 73 ± 64 months) and 240 patients with low- to intermediate-risk DTC (mean follow-up, 70 ± 60 months) were enrolled. Two "risk" genotypes were identified by combining VEGF-A SNPs -2578 C>A, -460 T>C, and +405 G>C. The ACG homozygous genotype was protective in both stage I-II (odds ratio [OR], 0.08; 95% confidence interval [CI], 0.01 to 1.43; P = 0.018) and low- to intermediate-risk (OR, 0.14; 95% CI, 0.01 to 1.13; P = 0.035) patients. The CTG homozygous genotype was significantly associated with recurrence in stage I-II (OR, 5.47; 95% CI, 1.15 to 26.04; P = 0.018) and was slightly deleterious in low- to intermediate-risk (OR, 3.39; 95% CI, 0.8 to 14.33; P = 0.079) patients. MVD of primary tumors from patients harboring a protective genotype was significantly lower (median MVD, 76.5 ± 12.7 and 86.7 ± 27.9, respectively; P = 0.024). Analysis of germline VEGF-A SNPs could empower a prognostic approach to DTC.

  14. Targeting Müller Cell–Derived VEGF164 to Reduce Intravitreal Neovascularization in the Rat Model of Retinopathy of Prematurity

    PubMed Central

    Jiang, Yanchao; Wang, Haibo; Culp, David; Yang, Zhihong; Fotheringham, Lori; Flannery, John; Hammond, Scott; Kafri, Tal; Hartnett, M. Elizabeth

    2014-01-01

    Purpose. To determine whether knockdown of Müller cell–derived VEGFA-splice variant, VEGF164, which is upregulated in the rat retinopathy of prematurity (ROP) model, safely inhibits intravitreal neovascularization (IVNV). Methods. Short hairpin RNAs for VEGF164 (VEGF164.shRNAs) or luciferase.shRNA control were cloned into lentivectors with CD44 promoters that specifically target Müller cells. Knockdown efficiency, off-target effects, and specificity were tested in HEK reporter cell lines that expressed green fluorescent protein (GFP)-tagged VEGF164 or VEGF120 with flow cytometry or in rat Müller cells (rMC-1) by real-time PCR. In the rat oxygen-induced retinopathy (OIR) ROP model, pups received 1 μL subretinal lentivector-driven luciferase.shRNA, VEGFA.shRNA, or VEGF164.shRNA at postnatal day 8 (P8). Analyses at P18 and P25 included: IVNV and avascular retina (AVA); retinal and serum VEGF (ELISA); density of phosphorylated VEGFR2 (p-VEGFR2) in lectin-labeled retinal endothelial cells (ECs; immunohistochemistry); TUNEL staining and thickness of inner nuclear (INL) and outer nuclear layers (ONL) in retinal cryosections; and pup weight gain. Results. In HEK reporter and in rMC-1 cells and in comparison to lucifferase.shRNA, VEGFA.shRNA reduced both VEGF120 and VEGF164, but VEGF164.shRNA only reduced VEGF164 and not VEGF120. Compared with luciferase.shRNA, VEGFA.shRNA and VEGF164.shRNA reduced retinal VEGF and IVNV without affecting AVA at P18 and P25. At P25, VEGF164.shRNA more effectively maintained IVNV inhibition than VEGFA.shRNA. VEGFA.shRNA and VEGF164.shRNA reduced pVEGFR2 in retinal ECs at P18, but VEGFA.shRNA increased it at P25. VEGFA.shRNA increased TUNEL+ cells at P18 and decreased ONL thickness at P18 and P25. VEGFA.shRNA and VEGF164.shRNA did not affect pup weight gain and serum VEGF. Conclusions. Short hairpin RNA to Müller cell VEGF164 maintained long-term inhibition of IVNV and limited cell death compared with shRNA to VEGFA. PMID:24425851

  15. Compartment Model Predicts VEGF Secretion and Investigates the Effects of VEGF Trap in Tumor-Bearing Mice

    PubMed Central

    Finley, Stacey D.; Dhar, Manjima; Popel, Aleksander S.

    2013-01-01

    Angiogenesis, the formation of new blood vessels from existing vasculature, is important in tumor growth and metastasis. A key regulator of angiogenesis is vascular endothelial growth factor (VEGF), which has been targeted in numerous anti-angiogenic therapies aimed at inhibiting tumor angiogenesis. Systems biology approaches, including computational modeling, are useful for understanding this complex biological process and can aid in the development of novel and effective therapeutics that target the VEGF family of proteins and receptors. We have developed a computational model of VEGF transport and kinetics in the tumor-bearing mouse, which includes three-compartments: normal tissue, blood, and tumor. The model simulates human tumor xenografts and includes human (VEGF121 and VEGF165) and mouse (VEGF120 and VEGF164) isoforms. The model incorporates molecular interactions between these VEGF isoforms and receptors (VEGFR1 and VEGFR2), as well as co-receptors (NRP1 and NRP2). We also include important soluble factors: soluble VEGFR1 (sFlt-1) and α-2-macroglobulin. The model accounts for transport via macromolecular transendothelial permeability, lymphatic flow, and plasma clearance. We have fit the model to available in vivo experimental data on the plasma concentration of free VEGF Trap and VEGF Trap bound to mouse and human VEGF in order to estimate the rates at which parenchymal cells (myocytes and tumor cells) and endothelial cells secrete VEGF. Interestingly, the predicted tumor VEGF secretion rates are significantly lower (0.007–0.023 molecules/cell/s, depending on the tumor microenvironment) than most reported in vitro measurements (0.03–2.65 molecules/cell/s). The optimized model is used to investigate the interstitial and plasma VEGF concentrations and the effect of the VEGF-neutralizing agent, VEGF Trap (aflibercept). This work complements experimental studies performed in mice and provides a framework with which to examine the effects of anti-VEGF

  16. VEGF receptor expression decreases during lung development in congenital diaphragmatic hernia induced by nitrofen

    PubMed Central

    Sbragia, L.; Nassr, A.C.C.; Gonçalves, F.L.L.; Schmidt, A.F.; Zuliani, C.C.; Garcia, P.V.; Gallindo, R.M.; Pereira, L.A.V.

    2014-01-01

    Changes in vascular endothelial growth factor (VEGF) in pulmonary vessels have been described in congenital diaphragmatic hernia (CDH) and may contribute to the development of pulmonary hypoplasia and hypertension; however, how the expression of VEGF receptors changes during fetal lung development in CDH is not understood. The aim of this study was to compare morphological evolution with expression of VEGF receptors, VEGFR1 (Flt-1) and VEGFR2 (Flk-1), in pseudoglandular, canalicular, and saccular stages of lung development in normal rat fetuses and in fetuses with CDH. Pregnant rats were divided into four groups (n=20 fetuses each) of four different gestational days (GD) 18.5, 19.5, 20.5, 21.5: external control (EC), exposed to olive oil (OO), exposed to 100 mg nitrofen, by gavage, without CDH (N-), and exposed to nitrofen with CDH (CDH) on GD 9.5 (term=22 days). The morphological variables studied were: body weight (BW), total lung weight (TLW), left lung weight, TLW/BW ratio, total lung volume, and left lung volume. The histometric variables studied were: left lung parenchymal area density and left lung parenchymal volume. VEGFR1 and VEGFR2 expression were determined by Western blotting. The data were analyzed using analysis of variance with the Tukey-Kramer post hoc test. CDH frequency was 37% (80/216). All the morphological and histometric variables were reduced in the N- and CDH groups compared with the controls, and reductions were more pronounced in the CDH group (P<0.05) and more evident on GD 20.5 and GD 21.5. Similar results were observed for VEGFR1 and VEGFR2 expression. We conclude that N- and CDH fetuses showed primary pulmonary hypoplasia, with a decrease in VEGFR1 and VEGFR2 expression. PMID:24519134

  17. VEGF receptor expression decreases during lung development in congenital diaphragmatic hernia induced by nitrofen.

    PubMed

    Sbragia, L; Nassr, A C C; Gonçalves, F L L; Schmidt, A F; Zuliani, C C; Garcia, P V; Gallindo, R M; Pereira, L A V

    2014-02-01

    Changes in vascular endothelial growth factor (VEGF) in pulmonary vessels have been described in congenital diaphragmatic hernia (CDH) and may contribute to the development of pulmonary hypoplasia and hypertension; however, how the expression of VEGF receptors changes during fetal lung development in CDH is not understood. The aim of this study was to compare morphological evolution with expression of VEGF receptors, VEGFR1 (Flt-1) and VEGFR2 (Flk-1), in pseudoglandular, canalicular, and saccular stages of lung development in normal rat fetuses and in fetuses with CDH. Pregnant rats were divided into four groups (n=20 fetuses each) of four different gestational days (GD) 18.5, 19.5, 20.5, 21.5: external control (EC), exposed to olive oil (OO), exposed to 100 mg nitrofen, by gavage, without CDH (N-), and exposed to nitrofen with CDH (CDH) on GD 9.5 (term=22 days). The morphological variables studied were: body weight (BW), total lung weight (TLW), left lung weight, TLW/BW ratio, total lung volume, and left lung volume. The histometric variables studied were: left lung parenchymal area density and left lung parenchymal volume. VEGFR1 and VEGFR2 expression were determined by Western blotting. The data were analyzed using analysis of variance with the Tukey-Kramer post hoc test. CDH frequency was 37% (80/216). All the morphological and histometric variables were reduced in the N- and CDH groups compared with the controls, and reductions were more pronounced in the CDH group (P<0.05) and more evident on GD 20.5 and GD 21.5. Similar results were observed for VEGFR1 and VEGFR2 expression. We conclude that N- and CDH fetuses showed primary pulmonary hypoplasia, with a decrease in VEGFR1 and VEGFR2 expression.

  18. Novel 4-amino-furo[2,3-d]pyrimidines as Tie-2 and VEGFR2 dual inhibitors.

    PubMed

    Miyazaki, Yasushi; Matsunaga, Shinichiro; Tang, Jun; Maeda, Yutaka; Nakano, Masato; Philippe, Rocher J; Shibahara, Megumi; Liu, Wei; Sato, Hideyuki; Wang, Liping; Nolte, Robert T

    2005-05-02

    A novel class of furo[2,3-d]pyrimidines has been discovered as potent dual inhibitors of Tie-2 and VEGFR2 receptor tyrosine kinases (TK) and a diarylurea moiety at 5-position shows remarkably enhanced activity against both enzymes. One of the most active compounds, 4-amino-3-(4-((2-fluoro-5-(trifluoromethyl)phenyl)amino-carbonylamino)phenyl)-2-(4-methoxyphenyl)furo[2,3-d]pyrimidine (7k) is <3 nM on both TK receptors and the activity is rationalized based on the X-ray crystal structure.

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

    PubMed

    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.

  20. AP-1 transcription factor mediates VEGF-induced endothelial cell migration and proliferation.

    PubMed

    Jia, Jing; Ye, Taiyang; Cui, Pengfei; Hua, Qian; Zeng, Huiyan; Zhao, Dezheng

    2016-05-01

    VEGF, upon binding to its endothelial cell specific receptors VEGF-R1 and VEGF-R2, can induce endothelial cell migration, proliferation and angiogenesis. However, the molecular mechanism of these effects still remains unclear. In this study, we investigated whether VEGF promotes human umbilical vascular endothelial cell (HUVEC) migration and proliferation through activator protein-1 transcription factor (AP-1) family. We first showed that VEGF induces immediate-early genes AP-1 family gene expression differentially with the profound induction of JunB (both mRNA and protein) under various conditions (PBS, DMSO or control adenoviruses). The increase in AP-1 mRNA expression occurs primarily at the transcriptional level. Inhibition of AP-1 DNA binding activity by adenovirus expressing a potent dominant negative form of c-Fos (Afos) significantly attenuated VEGF-induced HUVEC migration and proliferation and cyclin D1 expression. Knockdown of JunB with adenovirus expressing JunB shRNA reduces VEGF-induced JunB expression and attenuated HUVEC migration. However the shJunB-expressing virus has no effect on VEGF-induced cyclin D1 protein expression and proliferation. These results suggest that VEGF-induced endothelial migration is mediated primarily by induction of JunB whereas the promotion of endothelial proliferation by VEGF is mediated by JunB-independent AP-1 family members.

  1. A multiscale computational model predicts distribution of anti-angiogenic isoform VEGF165b in peripheral arterial disease in human and mouse

    PubMed Central

    Chu, Liang-Hui; Ganta, Vijay Chaitanya; Choi, Min H.; Chen, George; Finley, Stacey D.; Annex, Brian H.; Popel, Aleksander S.

    2016-01-01

    Angiogenesis is the growth of new blood vessels from pre-existing microvessels. Peripheral arterial disease (PAD) is caused by atherosclerosis that results in ischemia mostly in the lower extremities. Clinical trials including VEGF-A administration for therapeutic angiogenesis have not been successful. The existence of anti-angiogenic isoform (VEGF165b) in PAD muscle tissues is a potential cause for the failure of therapeutic angiogenesis. Experimental measurements show that in PAD human muscle biopsies the VEGF165b isoform is at least as abundant if not greater than the VEGF165a isoform. We constructed three-compartment models describing VEGF isoforms and receptors, in human and mouse, to make predictions on the secretion rate of VEGF165b and the distribution of various isoforms throughout the body based on the experimental data. The computational results are consistent with the data showing that in PAD calf muscles secrete mostly VEGF165b over total VEGF. In the PAD calf compartment of human and mouse models, most VEGF165a and VEGF165b are bound to the extracellular matrix. VEGF receptors VEGFR1, VEGFR2 and Neuropilin-1 (NRP1) are mostly in ‘Free State’. This study provides a computational model of VEGF165b in PAD supported by experimental measurements of VEGF165b in human and mouse, which gives insight of VEGF165b in therapeutic angiogenesis and VEGF distribution in human and mouse PAD model. PMID:27853189

  2. Anti-angiogenic activity of VXM01, an oral T-cell vaccine against VEGF receptor 2, in patients with advanced pancreatic cancer: A randomized, placebo-controlled, phase 1 trial.

    PubMed

    Schmitz-Winnenthal, Friedrich H; Hohmann, Nicolas; Niethammer, Andreas G; Friedrich, Tobias; Lubenau, Heinz; Springer, Marco; Breiner, Klaus M; Mikus, Gerd; Weitz, Jürgen; Ulrich, Alexis; Buechler, Markus W; Pianka, Frank; Klaiber, Ulla; Diener, Markus; Leowardi, Christine; Schimmack, Simon; Sisic, Leila; Keller, Anne-Valerie; Koc, Ruhan; Springfeld, Christoph; Knebel, Philipp; Schmidt, Thomas; Ge, Yingzi; Bucur, Mariana; Stamova, Slava; Podola, Lilli; Haefeli, Walter E; Grenacher, Lars; Beckhove, Philipp

    2015-04-01

    VEGFR-2 is expressed on tumor vasculature and a target for anti-angiogenic intervention. VXM01 is a first in kind orally applied tumor vaccine based on live, attenuated Salmonella bacteria carrying an expression plasmid, encoding VEGFR-2. We here studied the safety, tolerability, T effector (Teff), T regulatory (Treg) and humoral responses to VEGFR2 and anti-angiogenic effects in advanced pancreatic cancer patients in a randomized, dose escalation phase I clinical trial. Results of the first 3 mo observation period are reported. Locally advanced or metastatic, pancreatic cancer patients were enrolled. In five escalating dose groups, 30 patients received VXM01 and 15 placebo on days 1, 3, 5, and 7. Treatment was well tolerated at all dose levels. No dose-limiting toxicities were observed. Salmonella excretion and salmonella-specific humoral immune responses occurred in the two highest dose groups. VEGFR2 specific Teff, but not Treg responses were overall increased in vaccinated patients. We furthermore observed a significant reduction of tumor perfusion after 38 d in vaccinated patients together with increased levels of serum biomarkers indicative of anti-angiogenic activity, VEGF-A, and collagen IV. Vaccine specific Teff responses significantly correlated with reductions of tumor perfusion and high levels of preexisting VEGFR2-specific Teff while those showing no antiangiogenic activity had low levels of preexisting VEGFR2 specific Teff, showed a transient early increase of VEGFR2-specific Treg and reduced levels of VEGFR2-specific Teff at later time points - pointing to the possibility that early anti-angiogenic activity might be based at least in part on specific reactivation of preexisting memory T cells.

  3. Anti-angiogenic activity of VXM01, an oral T-cell vaccine against VEGF receptor 2, in patients with advanced pancreatic cancer: A randomized, placebo-controlled, phase 1 trial

    PubMed Central

    Schmitz-Winnenthal, Friedrich H; Hohmann, Nicolas; Niethammer, Andreas G; Friedrich, Tobias; Lubenau, Heinz; Springer, Marco; Breiner, Klaus M; Mikus, Gerd; Weitz, Jürgen; Ulrich, Alexis; Buechler, Markus W; Pianka, Frank; Klaiber, Ulla; Diener, Markus; Leowardi, Christine; Schimmack, Simon; Sisic, Leila; Keller, Anne-Valerie; Koc, Ruhan; Springfeld, Christoph; Knebel, Philipp; Schmidt, Thomas; Ge, Yingzi; Bucur, Mariana; Stamova, Slava; Podola, Lilli; Haefeli, Walter E; Grenacher, Lars; Beckhove, Philipp

    2015-01-01

    VEGFR-2 is expressed on tumor vasculature and a target for anti-angiogenic intervention. VXM01 is a first in kind orally applied tumor vaccine based on live, attenuated Salmonella bacteria carrying an expression plasmid, encoding VEGFR-2. We here studied the safety, tolerability, T effector (Teff), T regulatory (Treg) and humoral responses to VEGFR2 and anti-angiogenic effects in advanced pancreatic cancer patients in a randomized, dose escalation phase I clinical trial. Results of the first 3 mo observation period are reported. Locally advanced or metastatic, pancreatic cancer patients were enrolled. In five escalating dose groups, 30 patients received VXM01 and 15 placebo on days 1, 3, 5, and 7. Treatment was well tolerated at all dose levels. No dose-limiting toxicities were observed. Salmonella excretion and salmonella-specific humoral immune responses occurred in the two highest dose groups. VEGFR2 specific Teff, but not Treg responses were overall increased in vaccinated patients. We furthermore observed a significant reduction of tumor perfusion after 38 d in vaccinated patients together with increased levels of serum biomarkers indicative of anti-angiogenic activity, VEGF-A, and collagen IV. Vaccine specific Teff responses significantly correlated with reductions of tumor perfusion and high levels of preexisting VEGFR2-specific Teff while those showing no antiangiogenic activity had low levels of preexisting VEGFR2 specific Teff, showed a transient early increase of VEGFR2-specific Treg and reduced levels of VEGFR2-specific Teff at later time points – pointing to the possibility that early anti-angiogenic activity might be based at least in part on specific reactivation of preexisting memory T cells. PMID:26137397

  4. Fragment-Based Discovery of a Dual pan-RET/VEGFR2 Kinase Inhibitor Optimized for Single-Agent Polypharmacology.

    PubMed

    Frett, Brendan; Carlomagno, Francesca; Moccia, Maria Luisa; Brescia, Annalisa; Federico, Giorgia; De Falco, Valentina; Admire, Brittany; Chen, Zhongzhu; Qi, Wenqing; Santoro, Massimo; Li, Hong-yu

    2015-07-20

    Oncogenic conversion of the RET (rearranged during transfection) tyrosine kinase is associated with several cancers. A fragment-based chemical screen led to the identification of a novel RET inhibitor, Pz-1. Modeling and kinetic analysis identified Pz-1 as a type II tyrosine kinase inhibitor that is able to bind the "DFG-out" conformation of the kinase. Importantly, from a single-agent polypharmacology standpoint, Pz-1 was shown to be active on VEGFR2, which can block the blood supply required for RET-stimulated growth. In cell-based assays, 1.0 nM of Pz-1 strongly inhibited phosphorylation of all tested RET oncoproteins. At 1.0 mg kg(-1)  day(-1) per os, Pz-1 abrogated the formation of tumors induced by RET-mutant fibroblasts and blocked the phosphorylation of both RET and VEGFR2 in tumor tissue. Pz-1 featured no detectable toxicity at concentrations of up to 100.0 mg kg(-1), which indicates a large therapeutic window. This study validates the effectiveness and usefulness of a medicinal chemistry/polypharmacology approach to obtain an inhibitor capable of targeting multiple oncogenic pathways.

  5. Tissue deformation spatially modulates VEGF signaling and angiogenesis

    PubMed Central

    Rivron, Nicolas C.; Vrij, Erik J.; Rouwkema, Jeroen; Le Gac, Severine; van den Berg, Albert; Truckenmüller, Roman K.; van Blitterswijk, Clemens A.

    2012-01-01

    Physical forces play a major role in the organization of developing tissues. During vascular development, physical forces originating from a fluid phase or from cells pulling on their environment can alter cellular signaling and the behavior of cells. Here, we observe how tissue deformation spatially modulates angiogenic signals and angiogenesis. Using soft lithographic templates, we assemble three-dimensional, geometric tissues. The tissues contract autonomously, change shape stereotypically and form patterns of vascular structures in regions of high deformations. We show that this emergence correlates with the formation of a long-range gradient of Vascular Endothelial Growth Factor (VEGF) in interstitial cells, the local overexpression of the corresponding receptor VEGF receptor 2 (VEGFR-2) and local differences in endothelial cells proliferation. We suggest that tissue contractility and deformation can induce the formation of gradients of angiogenic microenvironments which could contribute to the long-range patterning of the vascular system. PMID:22511716

  6. The effects of cadmium on VEGF-mediated angiogenesis in HUVECs.

    PubMed

    Kim, Jisun; Lim, Wonbong; Ko, Youngjong; Kwon, Hyukil; Kim, Sangwoo; Kim, Oksu; Park, Gyeongju; Choi, Hongran; Kim, Okjoon

    2012-05-01

    Cadmium (Cd) is a highly toxic element that causes morphologic alterations and dysfunction in blood vessels. The altered vascular function caused by cadmium has been implicated in a range of chronic diseases, including hypertension. The effects of cadmium are a multisystem phenomenon involving inflammation, hypertrophy, apoptosis, angiogenesis and important processes involved in vascular remodeling systems. Vascular endothelial growth factor (VEGF) plays a major role in cell growth and angiogenesis under pathologic conditions. VEGF secretion is related to anti-apoptosis protein expression and attenuates apoptosis in endothelial cells. This study examined the VEGF-dependent mechanisms of angiogenesis and apoptosis in cadmium-treated endothelial cells (HUVECs). The effects and mechanisms of cadmium in endothelial cells (HUVECs) were examined by exposing the cells to different doses of cadmium chloride (2.5-40 μ m). After the cadmium treatment, the angiogenesis and apoptosis mechanisms related to VEGF in cadmium-treated HUVECs were examined. As a result, the low concentration of cadmium increased the tube formation in HUVECs. In addition, cadmium at concentrations of 5 and 10 μ m increased VEGF secretion and VEGFR2 activity, which suggest that cadmium affects the growth of blood vessels. All three MAPK pathways, namely ERK, JNK and p38, were activated by cadmium in HUVECs. However, high concentrations of cadmium caused cell damage, disrupted tube formation and inhibited VEGF expression and the activities of VEGFR2 and MAPK in HUVECs. Cadmium has dual functions through VEGF-dependent mechanisms in a dose-dependent manner. In this study, the dual effects of cadmium might alter angiogenesis and induce apoptosis through VEGF pathways in HUVECs. Copyright © 2011 John Wiley & Sons, Ltd.

  7. Osteoblast-derived VEGF regulates osteoblast differentiation and bone formation during bone repair

    PubMed Central

    Hu, Kai; Olsen, Bjorn R.

    2016-01-01

    Osteoblast-derived VEGF is important for bone development and postnatal bone homeostasis. Previous studies have demonstrated that VEGF affects bone repair and regeneration; however, the cellular mechanisms by which it works are not fully understood. In this study, we investigated the functions of osteoblast-derived VEGF in healing of a bone defect. The results indicate that osteoblast-derived VEGF plays critical roles at several stages in the repair process. Using transgenic mice with osteoblast-specific deletion of Vegfa, we demonstrated that VEGF promoted macrophage recruitment and angiogenic responses in the inflammation phase, and optimal levels of VEGF were required for coupling of angiogenesis and osteogenesis in areas where repair occurs by intramembranous ossification. VEGF likely functions as a paracrine factor in this process because deletion of Vegfr2 in osteoblastic lineage cells enhanced osteoblastic maturation and mineralization. Furthermore, osteoblast- and hypertrophic chondrocyte–derived VEGF stimulated recruitment of blood vessels and osteoclasts and promoted cartilage resorption at the repair site during the periosteal endochondral ossification stage. Finally, osteoblast-derived VEGF stimulated osteoclast formation in the final remodeling phase of the repair process. These findings provide a basis for clinical strategies to improve bone regeneration and treat defects in bone healing. PMID:26731472

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

    PubMed

    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.

  9. Effects of hyperoxia on VEGF, its receptors, and HIF-2alpha in the newborn rat lung.

    PubMed

    Hosford, Gayle E; Olson, David M

    2003-07-01

    Signaling through the hypoxia inducible factor (HIF)-VEGF-VEGF receptor system (VEGF signaling system) leads to angiogenesis and epithelial cell proliferation and is a key mechanism regulating alveolarization in lungs of newborn rats. Hyperoxia exposure (>95% O2 days 4-14) arrests lung alveolarization and may do so through suppression of the VEGF signaling system. Lung tissue mRNA levels of HIF-2alpha and VEGF increased from days 4-14 in normoxic animals, but hyperoxia suppressed these increases. Levels of HIF-2alpha and VEGF mRNA were correlated in the air but not the O2-treated group, suggesting that the low levels of HIF-2alpha observed at high O2 concentrations are not stimulating VEGF expression. VEGF164 protein levels increased with developmental age, and with hyperoxia to day 9, but continuing hyperoxia decreased levels by day 12. VEGFR1 and VEGFR2 mRNA expression also increased in air-exposed animals, and these, too, were significantly decreased by hyperoxia by day 9 and day 12, respectively. Receptor protein levels did not increase with development; however, O2 did decrease protein to less than air values. Hyperoxic suppression of VEGF signaling from days 9-14 may be one mechanism by which alveolarization is arrested.

  10. Meta-analysis of individual patient safety data from six randomized, placebo-controlled trials with the antiangiogenic VEGFR2-binding monoclonal antibody ramucirumab.

    PubMed

    Arnold, D; Fuchs, C; Tabernero, J; Ohtsu, A; Zhu, A X; Garon, E B; Mackey, J R; Paz-Ares, L; Baron, A D; Okusaka, T; Yoshino, T; Yoon, H H; Das, M; Ferry, D; Zhang, Y; Lin, Y; Binder, P; Sashegyi, A; Chau, I

    2017-09-07

    Ramucirumab, the human IgG1 monoclonal antibody receptor antagonist of vascular endothelial growth factor receptor 2 (VEGFR-2), has been approved for treating gastric/gastroesophageal junction, non-small cell lung, and metastatic colorectal cancers. With the completion of 6 global, randomized, double-blind, placebo-controlled, phase 3 trials across multiple tumor types, an opportunity now exists to further establish the safety parameters of ramucirumab across a large patient population. An individual patient meta-analysis across the 6 completed phase 3 trials was conducted and the relative risk (RR) and associated 95% confidence intervals (CI) were derived using fixed-effects or mixed-effects models for all-grade and high-grade adverse events (AEs) possibly related to VEGF pathway inhibition. The number needed to harm (NNH) was also calculable, due to the placebo-controlled nature of all 6 registration standard trials. A total of 4996 treated patients (N = 2748 in the ramucirumab arm, and N = 2248 in the control, placebo arm) were included in this meta-analysis. Arterial thromboembolic events (ATE, all-grade, RR: 0.8, 95% CI 0.5-1.3; high-grade [Grade ≥3], RR: 0.9, 95% CI 0.5-1.7), venous thromboembolic events (VTE, all-grade, RR: 0.7, 95% CI 0.5-1.1; high-grade, RR: 0.7, 95% CI 0.4-1.2), high-grade bleeding (RR: 1.1, 95% CI 0.8-1.5), and high-grade gastrointestinal (GI) bleeding (RR: 1.1, 95% CI 0.7-1.7) did not demonstrate a definite increased risk with ramucirumab. A higher percentage of hypertension, proteinuria, low-grade (Grade 1-2) bleeding, GI perforation, infusion-related reaction and wound-healing complications were observed in the ramucirumab arms compared to control. Ramucirumab may be distinct among antiangiogenic agents in terms of ATE, VTE, high-grade bleeding, or high-grade GI bleeding by showing no clear evidence for an increased risk of these AEs in this meta-analysis of a large and diverse patient population. Ramucirumab is consistent with

  11. K20E, an oxidative-coupling compound of methyl caffeate, exhibits anti-angiogenic activities through down-regulations of VEGF and VEGF receptor-2

    SciTech Connect

    Pan, Chun-Hsu; Lin, Wen-Hsin; Chien, Yi-Chung; Liu, Fon-Chang; Sheu, Ming-Jyh; Kuo, Yueh-Hsiung; Wu, Chieh-Hsi

    2015-01-15

    Anti-angiogenesis is one of the most popular clinical interventions for cancer chemotherapy. A series of synthesized derivative of methyl caffeate were used to evaluate the anti-angiogenic activity and to investigate possible pharmacological mechanisms in the present study. The most potent anti-angiogenic compound was evaluated in the experiments of murine allograft tumor model and Matrigel plug assay as well as cell models in the human umbilical vascular endothelial cells (HUVECs) and the LLC1 lung cancer cells. Our results suggested that K20E suppressed the tumor growth in the allograft tumor model and exhibited anti-angiogenic activity in Matrigel plug assay. Besides, HUVEC viability was found to be significantly reduced by arresting cell cycle at G{sub 2}/M phase and apoptosis. Cell migration, invasion, and tube formation of the HUVECs were also markedly suppressed by K20E treatment. K20E largely down-regulated the intracellular and secreted vascular endothelial growth factor (VEGF) in the LLC1 cancer cells. Besides, VEGF receptor-2 (VEGFR-2) and its downstream signaling cascades (AKT-mTOR and MEK1/2-ERK1/2) as well as gelatinases were all evidently reduced in the HUVECs treated with K20E. Inversely, K20E can up-regulate the expression levels of p53 and p21 proteins in the HUVECs. Based on these results, our study suggested that K20E possessed inhibiting angiogenesis through regulation of VEGF/VEGFR-2 and its downstream signaling cascades in the vascular endothelial cells (VECs). - Highlights: • K20E is an oxidative-coupling compound of methyl caffeate. • K20E exhibits anti-tumor and anti-angiogenesis effects. • K20E suppresses the expressions of VEGF and VEGF receptor-2 (VEGFR-2) proteins. • K20E deactivates VEGFR-2-mediated downstream signaling pathways to inhibit angiogenesis. • K20E up-regulates p53-p21 pathway to induce apoptosis and cell arrest at G2/M phase.

  12. The Synergic Effects of Crocus Sativus L. and Low Frequency Electromagnetic Field on VEGFR2 Gene Expression in Human Breast Cancer Cells

    PubMed Central

    Mousavi, Marzieh; Baharara, Javad; Shahrokhabadi, Khadijeh

    2014-01-01

    Background Angiogenesis, which is required for embryonic development and many physiological events, plays crucial role in many pathological conditions such as tumor growth and metastasis. Recent studies indicate anticancer and antitumor properties of saffron against human cancers. Many processes are affected by Electromagnetic Field (EMF) and its effect on proliferation and gene expression were examined. In this experimental study, the synergic effects of saffron and EMF on VEGFR2 gene expression in MCF7 cells were investigated. Methods Saffron was extracted using freeze dryer. MCF7 cells were grown in RPMI 1640 medium supplemented with 10% FBS and incubated at 37°C with 5% CO2. After 24 hr cells were treated with saffron extract at concentrations of 100, 200, 400 and 800 µg/ml. Forty eight hr after treatment all flasks were exposed with EMF (50 Hz, 0.004 T). Then total RNA was extracted and cDNA was synthetized using specific primer. Synthetized products were analyzed by Real Time PCR to determine expression level of VEGFR2. Data were analyzed by SPSS (ANOVA & Tukey). Results Critical inhibitory effect on VEGFR2 gene expression was 20% at 400 µg/ml. Synergic use of EMF and saffron extract showed most reduction (38%) at 100 µg/ml. On the other hand synergic use of 200, 400 and 800 µg/ml saffron aqua extract and EMF decline noticeably the VEGFR2 level of gene expression to 29, 35 and 36%, respectively. EMF itself also reduced VEGFR2 up to 25% in comparison with control group which is remarkable at p < 0.001. Conclusion Results indicate a decrease in the expression of vascular endothelial growth factor receptor in the treated samples with saffron extract compared to control. This reduction in VEGFR2 level induced by synergic treatment of saffron and EMF which reveals induction of inhibitory effects of saffron on angiogenesis and could be also considered as a promising chemotherapeutic agent in breast cancer treatment. PMID:24834315

  13. Chronic inhibition of tumor cell-derived VEGF enhances the malignant phenotype of colorectal cancer cells

    PubMed Central

    2013-01-01

    Background Vascular endothelial growth factor-a (VEGF)-targeted therapies have become an important treatment for a number of human malignancies. The VEGF inhibitors are actually effective in several types of cancers, however, the benefits are transiently, and the vast majority of patients who initially respond to the therapies will develop resistance. One of possible mechanisms for the acquired resistance may be the direct effect(s) of VEGF inhibitors on tumor cells expressing VEGF receptors (VEGFR). Thus, we investigated here the direct effect of chronic VEGF inhibition on phenotype changes in human colorectal cancer (CRC) cells. Methods To chronically inhibit cancer cell-derived VEGF, human CRC cell lines (HCT116 and RKO) were chronically exposed (2 months) to an anti-VEGF monoclonal antibody (mAb) or were disrupted the Vegf gene (VEGF-KO). Effects of VEGF family members were blocked by treatment with a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI). Hypoxia-induced apoptosis under VEGF inhibited conditions was measured by TUNEL assay. Spheroid formation ability was assessed using a 3-D spheroid cell culture system. Results Chronic inhibition of secreted/extracellular VEGF by an anti-VEGF mAb redundantly increased VEGF family member (PlGF, VEGFR1 and VEGFR2), induced a resistance to hypoxia-induced apoptosis, and increased spheroid formation ability. This apoptotic resistance was partially abrogated by a VEGFR-TKI, which blocked the compensate pathway consisted of VEGF family members, or by knockdown of Vegf mRNA, which inhibited intracellular function(s) of all Vegf gene products. Interestingly, chronic and complete depletion of all Vegf gene products by Vegf gene knockout further augmented these phenotypes in the compensate pathway-independent manner. These accelerated phenotypes were significantly suppressed by knockdown of hypoxia-inducible factor-1α that was up-regulated in the VEGF-KO cell lines. Conclusions Our findings suggest that chronic

  14. Soluble forms of VEGF receptor-1 and -2 promote vascular maturation via mural cell recruitment.

    PubMed

    Lorquet, Sophie; Berndt, Sarah; Blacher, Silvia; Gengoux, Emily; Peulen, Olivier; Maquoi, Erik; Noël, Agnès; Foidart, Jean-Michel; Munaut, Carine; Péqueux, Christel

    2010-10-01

    Two soluble forms of vascular endothelial growth factor (VEGF) receptors, sVEGFR-1 and sVEGFR-2, are physiologically released and overproduced in some pathologies. They are known to act as anti-VEGF agents. Here we report that these soluble receptors contribute to vessel maturation by mediating a dialogue between endothelial cells (ECs) and mural cells that leads to blood vessel stabilization. Through a multidisciplinary approach, we provide evidence that these soluble VEGF receptors promote mural cell migration through a paracrine mechanism involving interplay in ECs between VEGF/VEGFR-2 and sphingosine-1-phosphate type-1 (S1P)/S1P1 pathways that leads to endothelial nitric oxyde synthase (eNOS) activation. This new paradigm is supported by the finding that sVEGFR-1 and -2 perform the following actions: 1) induce an eNOS-dependent outgrowth of a mural cell network in an ex vivo model of angiogenesis, 2) increase the mural cell coverage of neovessels in vitro and in vivo, 3) promote mural cell migration toward ECs, and 4) stimulate endothelial S1P1 overproduction and eNOS activation that promote the migration and the recruitment of neighboring mural cells. These findings provide new insights into mechanisms regulating physiological and pathological angiogenesis and vessel stabilization.

  15. In vivo characterization of 68Ga-NOTA-VEGF 121 for the imaging of VEGF receptor expression in U87MG tumor xenograft models.

    PubMed

    Kang, Choong Mo; Kim, Sung-Min; Koo, Hyun-Jung; Yim, Min Su; Lee, Kyung-Han; Ryu, Eun Kyoung; Choe, Yearn Seong

    2013-01-01

    Vascular endothelial growth factor receptors (VEGFRs) are associated with tumor growth and induction of tumor angiogenesis and are known to be overexpressed in various human tumors. In the present study, we prepared and evaluated (68)Ga-1,4,7-triazacyclononane-1,4,7-triacetic acid-benzyl (NOTA)-VEGF(121) as a positron emission tomography (PET) radioligand for the in vivo imaging of VEGFR expression. (68)Ga-NOTA-VEGF(121) was prepared by conjugation of VEGF(121) and p-SCN-NOTA, followed by radiolabeling with (68)GaCl(3) and then purification using a PD-10 column. Human aortic endothelial cell (HAEC) binding of (68)Ga-NOTA-VEGF(121) was measured as a function of time. MicroPET and biodistribution studies of U87MG tumor xenografted mice were performed at 1, 2, and 4 h after injection of (68)Ga-NOTA-VEGF(121). The tumor tissues were then sectioned and subjected to immunostaining. The decay-corrected radiochemical yield of (68)Ga-NOTA-VEGF(121) was 40 ± 4.5 % and specific activity was 243.1 ± 104.6 GBq/μmol (8.6 ± 3.7 GBq/mg). (68)Ga-NOTA-VEGF(121) was avidly taken up by HAECs in a time-dependent manner, and the uptake was blocked either by 32 % with VEGF(121) or by 49 % with VEGFR2 antibody at 4 h post-incubation. In microPET images of U87MG tumor xenografted mice, radioactivity was accumulated in tumors (2.73±0.32 %ID/g at 2 h), and the uptake was blocked by 40 % in the presence of VEGF(121). In biodistribution studies, tumor uptake (1.84±0.14 %ID/g at 2 h) was blocked with VEGF(121) at a similar level (52 %) to that of microPET images. Immunostaining analysis of U87MG tumor tissues obtained after the microPET imaging showed high levels of VEGFR2 expression. These results demonstrate that (68)Ga-NOTA-VEGF(121) has potential for the in vivo imaging of VEGFR expression. In addition, our results also suggest that the in vivo characteristics of radiolabeled VEGF depend on the properties of the radioisotope and the chelator used.

  16. Effects of acoustic radiation force on the binding efficiency of BR55, a VEGFR2-specific ultrasound contrast agent.

    PubMed

    Frinking, Peter J A; Tardy, Isabelle; Théraulaz, Martine; Arditi, Marcel; Powers, Jeffry; Pochon, Sibylle; Tranquart, François

    2012-08-01

    This work describes an in vivo study analyzing the effect of acoustic radiation force (ARF) on the binding of BR55 VEGFR2-specific contrast-agent microbubbles in a model of prostatic adenocarcinoma in rat. A commercial ultrasound system was modified by implementing high duty-cycle 3.5-MHz center frequency ARF bursts in a scanning configuration. This enabled comparing the effects of ARF on binding in tumor and healthy tissue effectively in the same field of view. Bubble binding was established by measuring late-phase enhancement in amplitude modulation (AM) contrast-specific imaging mode (4 MHz, 150 kPa) 10 min after agent injection when the unbound bubbles were cleared from the circulation. Optimal experimental conditions, such as agent concentration (0.4 × 10(8)-1.6 × 10(8) bubbles/kg), acoustic pressure amplitude (26-51 kPa) and duty-cycle (20%-95%) of the ARF bursts, were evaluated in their ability to enhance binding in tumor without significantly increasing binding in healthy tissue. Using the optimal conditions (38 kPa peak-negative pressure, 95% duty cycle), ARF-assisted binding of BR55 improved significantly in tumor (by a factor of 7) at a lower agent dose compared with binding without ARF, and it had an insignificant effect on binding in healthy tissue. Thus, the high binding specificity of BR55 microbubbles for targeting VEGFR2 present at sites of active angiogenesis was confirmed by this study. Therefore, it is believed that based on the results obtained in this work, ultrasound molecular imaging using target-specific contrast-agent microbubbles should preferably be performed in combination with ARF. Copyright © 2012 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

  17. Circular RNA MYLK as a competing endogenous RNA promotes bladder cancer progression through modulating VEGFA/VEGFR2 signaling pathway.

    PubMed

    Zhong, Zhenyu; Huang, Mengge; Lv, Mengxin; He, Yunfeng; Duan, Changzhu; Zhang, Luyu; Chen, Junxia

    2017-09-10

    Accumulating evidences indicate that circular RNAs (circRNAs) play a vital role in modulating gene expression. However, the mechanisms underlying circRNAs remain largely elusive. Here, we screened circRNA and mRNA expression profiles of bladder carcinoma (BC) using microarray analysis. We found that circRNA-MYLK and VEGFA were significantly up-regulated and co-expressed in BC. Importantly, circRNA-MYLK levels were related to the progression of stage and grade of BC. Mechanistically, we demonstrated that circRNA-MYLK could directly bind to miR-29a and relieve suppression for target VEGFA, which activated VEGFA/VEGFR2 signaling pathway. Functionally, we found that ectopically expressing circRNA-MYLK accelerated cell proliferation, migration, tube formation of HUVEC and rearranged cytoskeleton. Moreover, up-regulating circRNA-MYLK promoted epithelial-mesenchymal transition (EMT). Whereas circRNA-MYLK knockdown decreased cell proliferation, motility, and induced apoptosis. Finally, up-regulating circRNA-MYLK promoted the growth, angiogenesis and metastasis of BC xenografts. Taken together, this study demonstrated for the first time that circRNA-MYLK might function as competing endogenous RNA (ceRNA) for miR-29a, which could contribute to EMT and the development of BC through activating VEGFA/VEGFR2 and downstream Ras/ERK signaling pathway. Our data suggest that circRNA-MYLK would be a promising target for BC diagnosis and therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. VEGF-mediated cross-talk within the neonatal murine thymus

    PubMed Central

    Cuddihy, Andrew R.; Ge, Shundi; Zhu, Judy; Jang, Julie; Chidgey, Ann; Thurston, Gavin; Boyd, Richard

    2009-01-01

    Although the mechanisms of cross-talk that regulate the hematopoietic and epithelial compartments of the thymus are well established, the interactions of these compartments with the thymic endothelium have been largely ignored. Current understanding of the thymic vasculature is based on studies of adult thymus. We show that the neonatal period represents a unique phase of thymic growth and differentiation, marked by endothelium that is organized as primitive, dense networks of capillaries dependent on vascular endothelial growth factor (VEGF). VEGF dependence in neonates is mediated by significantly higher levels of both VEGF production and endothelial VEGF receptor 2 (VEGF-R2) expression than in the adult thymus. VEGF is expressed locally in the neonatal thymus by immature, CD4−CD8− “double negative” (DN) thymocytes and thymic epithelium. Relative to adult thymus, the neonatal thymus has greater thymocyte proliferation, and a predominance of immature thymocytes and cortical thymic epithelial cells (cTECs). Inhibition of VEGF signaling during the neonatal period results in rapid loss of the dense capillaries in the thymus and a marked reduction in the number of thymocytes. These data demonstrate that, during the early postnatal period, VEGF mediates cross-talk between the thymocyte and endothelial compartments of the thymus. PMID:19088378

  19. NOR-1 is involved in VEGF-induced endothelial cell growth.

    PubMed

    Rius, Jordi; Martínez-González, José; Crespo, Javier; Badimon, Lina

    2006-02-01

    Neuron-derived orphan receptor-1 (NOR-1) is a transcription factor over-expressed in human atherosclerotic plaques that is involved in vascular smooth muscle cell proliferation. The aim of this study was to analyze whether NOR-1 plays a role in vascular endothelial growth factor (VEGF) induced endothelial cell growth. VEGF induced an early and transient up-regulation of NOR-1 in human umbilical vein endothelial cells (HUVEC). NOR-1 up-regulation by VEGF is processed through VEGF receptor-2 (VEGFR-2) and involves different signaling pathways including increase in cytosolic Ca(2+), activation of protein kinase C and mitogen-activated protein kinase (MAPK) pathways (both extracellular-signaling regulated kinase [ERK] and p38 MAPK). VEGF induced CREB activation (phosphorylation in Ser(133)). In transfection assays, a dominant-negative of CREB inhibited NOR-1 promoter activity, while mutation of the three CRE sites in the NOR-1 promoter abolished VEGF-induced NOR-1 promoter activity. Antisense oligonucleotides against NOR-1 inhibited VEGF-induced endothelial cell growth (reduced DNA synthesis, and inhibited cell cycle progression and endothelial cell wound repair after mechanical injury). These results indicate that NOR-1 could be a key transcription factor regulating endothelial cell growth induced by VEGF.

  20. A development of chimeric VEGFR2 TK inhibitor based on two ligand conformers from PDB: 1Y6A complex--medicinal chemistry consequences of a TKs analysis.

    PubMed

    Lintnerová, Lucia; García-Caballero, Melissa; Gregáň, Fridrich; Melicherčík, Milan; Quesada, Ana R; Dobiaš, Juraj; Lác, Ján; Sališová, Marta; Boháč, Andrej

    2014-01-24

    VEGFR2 is an important mediator of angiogenesis and influences fate of some cancer stem cells. Here we analysed all 34 structures of VEGFR2 TK available from PDB database. From them a complex PDB: 1Y6A has an exceptional AAZ ligand bound to TK in form of two conformers (U- and S-shaped). This observation inspired us to develop three chimeric bispyridyl VEGFR2 inhibitors by combining structural features of both AAZ conformers and/or their relative ligand AAX (PDB: 1Y6B). Our most interesting inhibitor 22SYM has an enzymatic VEGFR2 TK activity (IC50: 15.1 nM) comparable or better to the active compounds from clinical drugs Nexavar and Sutent. 22SYM inhibits growth, migration and tube formation of endothelial cells (EC) and selectively induces EC apoptosis. 22SYM also inhibits in vivo angiogenesis in Zebrafish embryo assay. Additionally to the above results, we proved here that tyrosine kinases in an inactive form possessing Type I inhibitors can adopt both a closed or an opened conformation of kinase A-loop independently on their DFG-out arrangement. We proposed here that an activity of certain Type I inhibitors (e.g. 22SYM-like) in complex with DFG-out TK can be negatively influenced by collisions with a dynamically moving TK A-loop.

  1. MULTIMERIN2 binds VEGF-A primarily via the carbohydrate chains exerting an angiostatic function and impairing tumor growth

    PubMed Central

    Andreuzzi, Eva; Paulitti, Alice; Tarticchio, Giulia; Todaro, Federico; Colombatti, Alfonso; Mongiat, Maurizio

    2016-01-01

    Angiogenesis is a key process occurring under both physiological and pathological conditions and is a hallmark of cancer. We have recently demonstrated that the extracellular matrix (ECM) molecule MULTIMERIN2 exerts an angiostatic function through the binding to VEGF-A. In this study we identify the region of the molecule responsible for the binding and demonstrate that the interaction involves the carbohydrate chains. MULTIMERIN2 interacts with other VEGF-A isoforms and VEGF family members such as VEGF-B, -C, -D and PlGF-1 suggesting that the molecule may function as a reservoir for different cytokines. In response to VEGF-A165, we show that MULTIMERIN2 impairs the phosphorylation of VEGFR2 at both Y1175 and Y1214 residues, halts SAPK2/p38 activation and negatively affects endothelial cell motility. In addition, MULTIMERIN2 and its active deletion mutant decrease the availability of the VEGFR2 receptor at the EC plasma membrane. The ectopic expression of MULTIMERIN2 or its active deletion mutant led to a striking reduction of tumor-associated angiogenesis and tumor growth. In conclusion, these data pinpoint MULTIMERIN2 as a key angiostatic molecule and disclose the possibility to develop new prognostic tools and improve the management of cancer patients. PMID:26655500

  2. Self-assembled nanoparticles based on the c(RGDfk) peptide for the delivery of siRNA targeting the VEGFR2 gene for tumor therapy

    PubMed Central

    Liu, Li; Liu, Xiaoxia; Xu, Qian; Wu, Ping; Zuo, Xialin; Zhang, Jingjing; Deng, Houliang; Wu, Zhuomin; Ji, Aimin

    2014-01-01

    The clinical application of small interfering RNA (siRNA) has been restricted by their poor intracellular uptake, low serum stability, and inability to target specific cells. During the last several decades, a great deal of effort has been devoted to exploring materials for siRNA delivery. In this study, biodegradable, tumor-targeted, self-assembled peptide nanoparticles consisting of cyclo(Arg–Gly–Asp–d–Phe–Lys)-8–amino–3,6–dioxaoctanoic acid–β–maleimidopropionic acid (hereafter referred to as RPM) were found to be an effective siRNA carrier both in vitro and in vivo. The nanoparticles were characterized based on transmission electron microscopy, circular dichroism spectra, and dynamic light scattering. In vitro analyses showed that the RPM/VEGFR2-siRNA exhibited negligible cytotoxicity and induced effective gene silencing. Delivery of the RPM/VEGFR2 (zebrafish)-siRNA into zebrafish embryos resulted in inhibition of neovascularization. Administration of RPM/VEGFR2 (mouse)-siRNA to tumor-bearing nude mice led to a significant inhibition of tumor growth, a marked reduction of vessels, and a down-regulation of VEGFR2 (messenger RNA and protein) in tumor tissue. Furthermore, the levels of IFN-α, IFN-γ, IL-12, and IL-6 in mouse serum, assayed via enzyme-linked immunosorbent assay, did not indicate any immunogenicity of the RPM/VEGFR2 (mouse)-siRNA in vivo. In conclusion, RPM may provide a safe and effective delivery vector for the clinical application of siRNAs in tumor therapy. PMID:25114522

  3. The matricellular protein CCN1 controls retinal angiogenesis by targeting VEGF, Src homology 2 domain phosphatase-1 and Notch signaling.

    PubMed

    Chintala, Hemabindu; Krupska, Izabela; Yan, Lulu; Lau, Lester; Grant, Maria; Chaqour, Brahim

    2015-07-01

    Physiological angiogenesis depends on the highly coordinated actions of multiple angiogenic regulators. CCN1 is a secreted cysteine-rich and integrin-binding matricellular protein required for proper cardiovascular development. However, our understanding of the cellular origins and activities of this molecule is incomplete. Here, we show that CCN1 is predominantly expressed in angiogenic endothelial cells (ECs) at the leading front of actively growing vessels in the mouse retina. Endothelial deletion of CCN1 in mice using a Cre-Lox system is associated with EC hyperplasia, loss of pericyte coverage and formation of dense retinal vascular networks lacking the normal hierarchical arrangement of arterioles, capillaries and venules. CCN1 is a product of an immediate-early gene that is transcriptionally induced in ECs in response to stimulation by vascular endothelial growth factor (VEGF). We found that CCN1 activity is integrated with VEGF receptor 2 (VEGF-R2) activation and downstream signaling pathways required for tubular network formation. CCN1-integrin binding increased the expression of and association between Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1) and VEGF-R2, which leads to rapid dephosphorylation of VEGF-R2 tyrosine, thus preventing EC hyperproliferation. Predictably, CCN1 further brings receptors/signaling molecules into proximity that are otherwise spatially separated. Furthermore, CCN1 induces integrin-dependent Notch activation in cultured ECs, and its targeted gene inactivation in vivo alters Notch-dependent vascular specification and remodeling, suggesting that functional levels of Notch signaling requires CCN1 activity. These data highlight novel functions of CCN1 as a naturally optimized molecule, fine-controlling key processes in physiological angiogenesis and safeguarding against aberrant angiogenic responses.

  4. The matricellular protein CCN1 controls retinal angiogenesis by targeting VEGF, Src homology 2 domain phosphatase-1 and Notch signaling

    PubMed Central

    Chintala, Hemabindu; Krupska, Izabela; Yan, Lulu; Lau, Lester; Grant, Maria; Chaqour, Brahim

    2015-01-01

    Physiological angiogenesis depends on the highly coordinated actions of multiple angiogenic regulators. CCN1 is a secreted cysteine-rich and integrin-binding matricellular protein required for proper cardiovascular development. However, our understanding of the cellular origins and activities of this molecule is incomplete. Here, we show that CCN1 is predominantly expressed in angiogenic endothelial cells (ECs) at the leading front of actively growing vessels in the mouse retina. Endothelial deletion of CCN1 in mice using a Cre-Lox system is associated with EC hyperplasia, loss of pericyte coverage and formation of dense retinal vascular networks lacking the normal hierarchical arrangement of arterioles, capillaries and venules. CCN1 is a product of an immediate-early gene that is transcriptionally induced in ECs in response to stimulation by vascular endothelial growth factor (VEGF). We found that CCN1 activity is integrated with VEGF receptor 2 (VEGF-R2) activation and downstream signaling pathways required for tubular network formation. CCN1-integrin binding increased the expression of and association between Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1) and VEGF-R2, which leads to rapid dephosphorylation of VEGF-R2 tyrosine, thus preventing EC hyperproliferation. Predictably, CCN1 further brings receptors/signaling molecules into proximity that are otherwise spatially separated. Furthermore, CCN1 induces integrin-dependent Notch activation in cultured ECs, and its targeted gene inactivation in vivo alters Notch-dependent vascular specification and remodeling, suggesting that functional levels of Notch signaling requires CCN1 activity. These data highlight novel functions of CCN1 as a naturally optimized molecule, fine-controlling key processes in physiological angiogenesis and safeguarding against aberrant angiogenic responses. PMID:26002917

  5. The propeptides of VEGF-D determine heparin binding, receptor heterodimerization, and effects on tumor biology.

    PubMed

    Harris, Nicole C; Davydova, Natalia; Roufail, Sally; Paquet-Fifield, Sophie; Paavonen, Karri; Karnezis, Tara; Zhang, You-Fang; Sato, Teruhiko; Rothacker, Julie; Nice, Edouard C; Stacker, Steven A; Achen, Marc G

    2013-03-22

    VEGF-D is an angiogenic and lymphangiogenic glycoprotein that can be proteolytically processed generating various forms differing in subunit composition due to the presence or absence of N- and C-terminal propeptides. These propeptides flank the central VEGF homology domain, that contains the binding sites for VEGF receptors (VEGFRs), but their biological functions were unclear. Characterization of propeptide function will be important to clarify which forms of VEGF-D are biologically active and therefore clinically relevant. Here we use VEGF-D mutants deficient in either propeptide, and in the capacity to process the remaining propeptide, to monitor the functions of these domains. We report for the first time that VEGF-D binds heparin, and that the C-terminal propeptide significantly enhances this interaction (removal of this propeptide from full-length VEGF-D completely prevents heparin binding). We also show that removal of either the N- or C-terminal propeptide is required for VEGF-D to drive formation of VEGFR-2/VEGFR-3 heterodimers which have recently been shown to positively regulate angiogenic sprouting. The mature form of VEGF-D, lacking both propeptides, can also promote formation of these receptor heterodimers. In a mouse tumor model, removal of only the C-terminal propeptide from full-length VEGF-D was sufficient to enhance angiogenesis and tumor growth. In contrast, removal of both propeptides is required for high rates of lymph node metastasis. The findings reported here show that the propeptides profoundly influence molecular interactions of VEGF-D with VEGF receptors, co-receptors, and heparin, and its effects on tumor biology.

  6. Correlation between VEGFR-2 receptor kinase domain-containing receptor (KDR) mRNA and angiotensin II receptor type 1 (AT1-R) mRNA in endometrial cancer.

    PubMed

    Piastowska-Ciesielska, Agnieszka W; Płuciennik, Elżbieta; Wójcik-Krowiranda, Katarzyna; Bieńkiewicz, Andrzej; Nowakowska, Magdalena; Pospiech, Karolina; Bednarek, Andrzej K; Domińska, Kamila; Ochędalski, Tomasz

    2013-02-01

    Angiogenesis, a multistep process that results in new blood vessel formation from preexisting vasculature is essential for both the growth of solid tumour and for metastasis. Stimulation of vascular endothelial growth factor receptor (VEGFR), a transmembrane glycoprotein, results in mitogenesis. Within this family of receptors, VEGFR 2/kinase-insert-domain containing receptor appears to be principally upregulated during tumorigenesis. The aim of this study was to determine the expression of VEGFR-2/kinase-insert-domain containing receptor (KDR) and its correlation with angiotensin receptor type 1 (AT1-R) and clinical factors in endometrial carcinoma. The expression of KDR and AT1-R was studied in endometrial carcinoma and normal endometrium by Real-time RT-PCR and Western blot analysis in 136 samples. The expression profile was correlated with the clinicopathological characteristics of endometrial adenocarcinoma. We noted a significant correlation between the expression of KDR and AT1-R in tumour grade G1, G2 and G3 (R(s)=0.50; p=0.002, R(s)=0.69; p=0.0001, R(s)=0.52; p=0.005, respectively). In stage I and stage II carcinoma, a significant correlation was also found between the expression of KDR and AT1-R (R(s)=0.70, p=0.0001, R(s)=0.67; p=0.001, respectively). Moreover significant correlation was observed between both KDR and AT1-R in tissue with different myometrial invasion (R(s)=0.54, p=0.0001, R(s)=0.68; p=0.0001; respectively for tumours with invasion into the inner half and invasion into the outer half). Basing on received correlation between AT1-R and KDR expression and previous results we speculate that angiotensin through AT1-R modulates KDR expression and thus have influence on local VEGF level. However, further studies are required to clarify the biological interaction between KDR, AT1-R and other hormonal regulators in endometrial carcinoma. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Anti-metastatic action of FAK inhibitor OXA-11 in combination with VEGFR-2 signaling blockade in pancreatic neuroendocrine tumors

    PubMed Central

    Moen, Ingrid; Gebre, Matthew; Alonso-Camino, Vanesa; Chen, Debbie; Epstein, David

    2015-01-01

    The present study sought to determine the anti-tumor effects of OXA-11, a potent, novel small-molecule amino pyrimidine inhibitor (1.2 pM biochemical IC50) of focal adhesion kinase (FAK). In studies of cancer cell lines, OXA-11 inhibited FAK phosphorylation at phospho-tyrosine 397 with a mechanistic IC50 of 1 nM in TOV21G tumor cells, which translated into functional suppression of proliferation in 3-dimensional culture with an EC50 of 9 nM. Studies of OXA-11 activity in TOV21G tumor-cell xenografts in mice revealed a pharmacodynamic EC50 of 1.8 nM, indicative of mechanistic inhibition of pFAK [Y397] in these tumors. OXA-11 inhibited TOV21G tumor growth in a dose-dependent manner and also potentiated effects of cisplatin on tumor cell proliferation and apoptosis in vitro and on tumor growth in mice. Studies of pancreatic neuroendocrine tumors in RIP-Tag2 transgenic mice revealed OXA-11 suppression of pFAK [Y397] and pFAK [Y861] in tumors and liver. OXA-11 given daily from age 14 to 17 weeks reduced tumor vascularity, invasion, and when given together with the anti-VEGFR-2 antibody DC101 reduced the incidence, abundance, and size of liver metastases. Liver micrometastases were found in 100 % of mice treated with vehicle, 84 % of mice treated with OXA-11, and 79 % of mice treated with DC101 (19–24 mice per group). In contrast, liver micrometastases were found in only 52 % of 21 mice treated with OXA-11 plus DC101, and those present were significantly smaller and less numerous. Together, these findings indicate that OXA-11 is a potent and selective inhibitor of FAK phosphorylation in vitro and in vivo. OXA-11 slows tumor growth, potentiates the anti-tumor actions of cisplatin and—when combined with VEGFR-2 blockade—reduces metastasis of pancreatic neuroendocrine tumors in RIP-Tag2 mice. PMID:26445848

  8. Electrical Muscle Stimulation Induces an Increase of VEGFR2 on Circulating Hematopoietic Stem Cells in Patients With Diabetes.

    PubMed

    Hidmark, Asa; Spanidis, Ioannis; Fleming, Thomas H; Volk, Nadine; Eckstein, Volker; Groener, Jan B; Kopf, Stefan; Nawroth, Peter P; Oikonomou, Dimitrios

    2017-06-01

    External electric muscle stimulation (EMS) of the thigh muscles was found to reduce pain resulting from diabetic neuropathy (DN), a vascular complication of diabetes. This study investigated circulating hematopoietic stem cells (HSCs) after EMS treatment. Impaired function of HSCs and the subpopulation endothelial progenitor cells (EPCs), important for neovascularization and endothelial repair, has been associated with DN. Twenty-four patients with painful DN were treated 3 times with EMS over a period of 1 week. Blood samples were collected before and after the first EMS treatment. Before a fourth treatment, neuropathic pain was evaluated and a third blood sample was collected. Cells were used for flow cytometry. Patients with painful DN reported that the pain decreased after 3 times of 1-hour treatments with EMS (Neuropathy Symptom Score: from 8 to 6, P = 0.001; Neuropathy Disability Score: from 5.5 to 5, P = 0.027, n = 24). At the end of the study, diastolic blood pressure had decreased from 80 to 70 mm Hg (P = 0.043), and plasma adrenaline and noradrenaline metabolites metanephrine and normetanephrine were reduced (both P ≤ 0.01; n = 21). A single EMS treatment caused an immediate and transient decrease in the frequency of CD34(+) HSCs in circulation (-20%; P < 0.001; n = 27). In 9 of the patients with DN, the proportion of HSCs expressing vascular endothelial growth factor receptor 2 (VEGFR2; defining the HSCs as EPCs) increased by 36% (P = 0.011) after EMS treatment. Proteins required for binding to endothelium (junctional adhesion molecule A and CD31), homing toward hypoxic tissue (C-X-C chemokine receptor type 4), and endothelial differentiation (CD31) were increased on HSCs immediately after EMS treatment. An increased frequency of VEGFR2 expression was also observed on HSCs of 6 healthy control volunteers (34%; P = 0.046) after EMS treatment, but not after sham treatment. Three EMS treatments decreased symptoms of pain caused by DN and reduced diastolic

  9. VEGF receptor-2 Y951 signaling and a role for the adapter molecule TSAd in tumor angiogenesis

    PubMed Central

    Matsumoto, Taro; Bohman, Svante; Dixelius, Johan; Berge, Tone; Dimberg, Anna; Magnusson, Peetra; Wang, Ling; Wikner, Charlotte; Qi, Jian Hua; Wernstedt, Christer; Wu, Jiong; Bruheim, Skjalg; Mugishima, Hideo; Mukhopadhyay, Debrabata; Spurkland, Anne; Claesson-Welsh, Lena

    2005-01-01

    Vascular endothelial growth factor receptor-2 (VEGFR-2) activation by VEGF-A is essential in vasculogenesis and angiogenesis. We have generated a pan-phosphorylation site map of VEGFR-2 and identified one major tyrosine phosphorylation site in the kinase insert (Y951), in addition to two major sites in the C-terminal tail (Y1175 and Y1214). In developing vessels, phosphorylation of Y1175 and Y1214 was detected in all VEGFR-2-expressing endothelial cells, whereas phosphorylation of Y951 was identified in a subset of vessels. Phosphorylated Y951 bound the T-cell-specific adapter (TSAd), which was expressed in tumor vessels. Mutation of Y951 to F and introduction of phosphorylated Y951 peptide or TSAd siRNA into endothelial cells blocked VEGF-A-induced actin stress fibers and migration, but not mitogenesis. Tumor vascularization and growth was reduced in TSAd-deficient mice, indicating a critical role of Y951-TSAd signaling in pathological angiogenesis. PMID:15962004

  10. VEGF receptor-2 Y951 signaling and a role for the adapter molecule TSAd in tumor angiogenesis.

    PubMed

    Matsumoto, Taro; Bohman, Svante; Dixelius, Johan; Berge, Tone; Dimberg, Anna; Magnusson, Peetra; Wang, Ling; Wikner, Charlotte; Qi, Jian Hua; Wernstedt, Christer; Wu, Jiong; Bruheim, Skjalg; Mugishima, Hideo; Mukhopadhyay, Debrabata; Spurkland, Anne; Claesson-Welsh, Lena

    2005-07-06

    Vascular endothelial growth factor receptor-2 (VEGFR-2) activation by VEGF-A is essential in vasculogenesis and angiogenesis. We have generated a pan-phosphorylation site map of VEGFR-2 and identified one major tyrosine phosphorylation site in the kinase insert (Y951), in addition to two major sites in the C-terminal tail (Y1175 and Y1214). In developing vessels, phosphorylation of Y1175 and Y1214 was detected in all VEGFR-2-expressing endothelial cells, whereas phosphorylation of Y951 was identified in a subset of vessels. Phosphorylated Y951 bound the T-cell-specific adapter (TSAd), which was expressed in tumor vessels. Mutation of Y951 to F and introduction of phosphorylated Y951 peptide or TSAd siRNA into endothelial cells blocked VEGF-A-induced actin stress fibers and migration, but not mitogenesis. Tumor vascularization and growth was reduced in TSAd-deficient mice, indicating a critical role of Y951-TSAd signaling in pathological angiogenesis.

  11. Plumbagin inhibits tumour angiogenesis and tumour growth through the Ras signalling pathway following activation of the VEGF receptor-2

    PubMed Central

    Lai, Li; Liu, Junchen; Zhai, Dong; Lin, Qingxiang; He, Lijun; Dong, Yanmin; Zhang, Jing; Lu, Binbin; Chen, Yihua; Yi, Zhengfang; Liu, Mingyao

    2012-01-01

    BACKGROUND AND PURPOSE Angiogenesis-based therapy is an effective anti-tumour strategy and previous reports have shown some beneficial effects of a naturally occurring bioactive compound plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone). Here, we sought to determine the biological effects of plumbagin on signalling mechanisms during tumour angiogenesis. EXPERIMENTAL APPROACH The effects of plumbagin were evaluated in various in vitro assays which utilised human umbilical vein endothelial cells (HUVEC) proliferation, migration and tube formation. Plumbagin was also evaluated in vivo using chicken embryo chorioallantoic membrane (CAM) and mouse corneal micropocket models., Human colon carcinoma and prostate cancer xenograft mouse models were used to evaluate the effects of plumbagin on angiogenesis. Immunofluorescence, GST pull-down and Western blotting were employed to explore the underlying mechanisms of VEGF receptor (VEGFR)2-mediated Ras signalling pathways. KEY RESULTS Plumbagin not only inhibited endothelial cell proliferation, migration and tube formation but also suppressed chicken chorioallantoic membrane neovascularzation and VEGF-induced mouse corneal angiogenesis. Moreover, plumbagin suppressed tumour angiogenesis and tumour growth in human colon carcinoma and prostate cancer xenograft mouse models. At a molecular level, plumbagin blocked the Ras/Rac/cofilin and Ras/MEK signalling pathways mediated by VEGFR2 in HUVECs. CONCLUSIONS AND IMPLICATIONS Plumbagin inhibited tumour angiogenesis and tumour growth by interference with the VEGFR2-mediated Ras signalling pathway in endothelial cells. Our findings demonstrate a molecular basis for the effects of plumbagin and suggest that this compound might have therapeutic ant-tumour effects. PMID:21658027

  12. Potent neutralization of VEGF biological activities with a fully human antibody Fab fragment directed against VEGF receptor 2

    SciTech Connect

    Miao, H.-Q. . E-mail: hua-quan.miao@imclone.com; Hu, Kun; Jimenez, Xenia; Navarro, Elizabeth; Zhang, Haifan; Lu Dan; Ludwig, Dale L.; Balderes, Paul; Zhu Zhenping . E-mail: zhenping.zhu@imclone.com

    2006-06-23

    Compelling evidence suggest that vascular endothelial growth factor (VEGF) and its receptors, especially receptor 2 (VEGFR2, or kinase insert domain-containing receptor, KDR), play a critical role in angiogenesis under both physiological and pathological conditions, including cancer and angiogenic retinopathies such as age-related macular degeneration (AMD). To this end, inhibition of angiogenesis with antagonists to either VEGF or KDR has yielded significant therapeutic efficacy both in preclinical studies in animal models and in clinical trials in patients with cancer and AMD. We previously reported the identification of a high affinity, fully human anti-KDR antibody fragment, 1121B Fab, through a highly stringent affinity maturation process with a Fab originally isolated from a naive human antibody phage display library. In this study, we demonstrate that 1121B Fab is able to strongly block KDR/VEGF interaction, resulting in potent inhibition of an array of biological activities of VEGF, including activation of the receptor and its signaling pathway, intracellular calcium mobilization, and migration and proliferation of endothelial cells. Taken together, our data lend strong support to the further development of 1121B Fab fragment as an anti-angiogenesis agent in both cancer and angiogenic retinopathies.

  13. VEGF Trap complex formation measures production rates of VEGF, providing a biomarker for predicting efficacious angiogenic blockade

    PubMed Central

    Rudge, John S.; Holash, Jocelyn; Hylton, Donna; Russell, Michelle; Jiang, Shelly; Leidich, Raymond; Papadopoulos, Nicholas; Pyles, Erica A.; Torri, Al; Wiegand, Stanley J.; Thurston, Gavin; Stahl, Neil; Yancopoulos, George D.

    2007-01-01

    VEGF is the best characterized mediator of tumor angiogenesis. Anti-VEGF agents have recently demonstrated impressive efficacy in human cancer trials, but the optimal dosing of such agents must still be determined empirically, because biomarkers to guide dosing have yet to be established. The widely accepted (but unverified) assumption that VEGF production is quite low in normal adults led to the notion that increased systemic VEGF levels might quantitatively reflect tumor mass and angiogenic activity. We describe an approach to determine host and tumor production of VEGF, using a high-affinity and long-lived VEGF antagonist now in clinical trials, the VEGF Trap. Unlike antibody complexes that are usually rapidly cleared, the VEGF Trap forms inert complexes with tissue- and tumor-derived VEGF that remain stably in the systemic circulation, where they are readily assayable, providing unprecedented capability to accurately measure VEGF production. We report that VEGF production is surprisingly high in non-tumor-bearing rodents and humans, challenging the notion that systemic VEGF levels can serve as a sensitive surrogate for tumor load; tumor VEGF contribution becomes significant only with very large tumor loads. These findings have the important corollary that anti-VEGF therapies must be sufficiently dosed to avoid diversion by host-derived VEGF. We further show that our assay can indicate when VEGF is optimally blocked; such biomarkers to guide dosing do not exist for other anti-VEGF agents. Based on this assay, VEGF Trap doses currently being assessed in clinical trials are in the efficacious range. PMID:18000042

  14. Delphinidin Inhibits Tumor Growth by Acting on VEGF Signalling in Endothelial Cells

    PubMed Central

    Anton, Anita; Justiniano, Hélène; Soleti, Raffaella; Alabed Alibrahim, Eid; Simard, Gilles; Andriantsitohaina, Ramaroson; Lugnier, Claire

    2015-01-01

    The vasculoprotective properties of delphinidin are driven mainly by its action on endothelial cells. Moreover, delphinidin displays anti-angiogenic properties in both in vitro and in vivo angiogenesis models and thereby might prevent the development of tumors associated with excessive vascularization. This study was aimed to test the effect of delphinidin on melanoma-induced tumor growth with emphasis on its molecular mechanism on endothelial cells. Delphinidin treatment significantly decreased in vivo tumor growth induced by B16-F10 melanoma cell xenograft in mice. In vitro, delphinidin was not able to inhibit VEGFR2-mediated B16-F10 melanoma cell proliferation but it specifically reduced basal and VEGFR2-mediated endothelial cell proliferation. The anti-proliferative effect of delphinidin was reversed either by the MEK1/2 MAP kinase inhibitor, U-0126, or the PI3K inhibitor, LY-294002. VEGF-induced proliferation was reduced either by U-0126 or LY-294002. Under these conditions, delphinidin failed to decrease further endothelial cell proliferation. Delphinidin prevented VEGF-induced phosphorylation of ERK1/2 and p38 MAPK and decreased the expression of the transcription factors, CREB and ATF1. Finally, delphinidin was more potent in inhibiting in vitro cyclic nucleotide phosphodiesterases (PDEs), PDE1 and PDE2, compared to PDE3-PDE5. Altogether delphinidin reduced tumor growth of melanoma cell in vivo by acting specifically on endothelial cell proliferation. The mechanism implies an association between inhibition of VEGF-induced proliferation via VEGFR2 signalling, MAPK, PI3K and at transcription level on CREB/ATF1 factors, and the inhibition of PDE2. In conjunction with our previous studies, we demonstrate that delphinidin is a promising compound to prevent pathologies associated with generation of vascular network in tumorigenesis. PMID:26694325

  15. Vascular endothelial (VEGF) and epithelial growth factor (EGF) as well as platelet-activating factor (PAF) and receptors are expressed in the early pregnant canine uterus.

    PubMed

    Schäfer-Somi, S; Sabitzer, S; Klein, D; Reinbacher, E; Kanca, H; Beceriklisoy, H B; Aksoy, O A; Kucukaslan, I; Macun, H C; Aslan, S

    2013-02-01

    The aim of this study was to investigate the course of expression of platelet-activating factor (PAF), PAF-receptor (PAF-R), epidermal growth factor (EGF), EGF-R, vascular endothelial growth factor (VEGF), VEGF-R1 and VEGF-R2 in uterine tissue during canine pregnancy. For this purpose, 20 bitches were ovariohysterectomized at days 10-12 (n = 10), 18-25 (n = 5) and 28-45 (n = 5) days after mating, respectively. The pre-implantation group was proven pregnant by embryo flushing of the uterus after the operation, the others by sonography. Five embryo negative, that is, non-pregnant, bitches in diestrus (day 10-12) served as controls. Tissue samples from the uterus (placentation sites and horn width, respectively) were excised and snap-frozen in liquid nitrogen after embedding in Tissue Tec(®). Extraction of mRNA for RT-PCR was performed with Tri-Reagent. In the embryos, mRNA from all factors except VEGF was detected. In the course of pregnancy, significantly higher expression of PAF and PAFR as well as VEGF and VEGFR2 during the pre-implantation stage than in all other stages and a strong upregulation of EGF during implantation were characteristic. The course of EGF was in diametrical opposition to the course of the receptor. These results point towards an increased demand for VEGF, EGF and PAF during the earliest stages of canine pregnancy.

  16. Inhibition of VEGF-dependent angiogenesis by the anti-CD82 monoclonal antibody 4F9 through regulation of lipid raft microdomains

    SciTech Connect

    Nomura, Sayaka; Iwata, Satoshi; Hatano, Ryo; Komiya, Eriko; Dang, Nam H.; Ohnuma, Kei; Morimoto, Chikao

    2016-05-20

    CD82 (also known as KAI1) belongs to the tetraspanin superfamily of type III transmembrane proteins, and is involved in regulating cell adhesion, migration and proliferation. In contrast to these well-established roles of CD82 in tumor biology, its function in endothelial cell (EC) activity and tumor angiogenesis is yet to be determined. In this study, we show that suppression of CD82 negatively regulates vascular endothelial growth factor (VEGF)-induced angiogenesis. Moreover, we demonstrate that the anti-CD82 mAb 4F9 effectively inhibits phosphorylation of VEGF receptor 2 (VEGFR2), which is the principal mediator of the VEGF-induced angiogenic signaling process in tumor angiogenesis, by regulating the organization of the lipid raft microdomain signaling platform in human EC. Our present work therefore suggests that CD82 on EC is a potential target for anti-angiogenic therapy in VEGFR2-dependent tumor angiogenesis. -- Highlights: •Knockdown of CD82 decreases EC migration, proliferation and angiogenesis. •Anti-CD82 mAb 4F9 inhibits EC migration, proliferation and angiogenesis. •4F9 inhibits VEGFR2 phosphorylation via control of CD82 distribution in lipid rafts.

  17. The novel VEGF receptor 2 inhibitor YLL545 inhibits angiogenesis and growth in breast cancer

    PubMed Central

    Zhang, Jianbo; Liu, Chen; Shi, Wen; Yang, Lingling; Zhang, Quansheng; Cui, Jianlin; Fang, Yangwu; Li, Yuhao; Ren, Guosheng; Yang, Shuang; Xiang, Rong

    2016-01-01

    Their antiangiogenic effects make vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors useful for cancer treatment. However, most of these drugs have unexpected adverse side effects. Here, we show that the novel VEGFR2 inhibitor YLL545 suppressed tumor angiogenesis and growth in triple-negative breast cancer without adverse effects. YLL545 treatment also markedly inhibited proliferation, migration, invasion, and tube formation by human umbilical vascular endothelial cells (HUVECs) in vitro. These effects of YLL545 were equal to or greater than those seen with sorafenib. In addition, YLL545 inhibited VEGF-induced phosphorylation of VEGFR2 and activation of downstream signaling regulators, such as phospho-STAT3 and phospho-ERK1/2, in HUVECs. Embryonic angiogenesis assays in zebrafish and Matrigel plug assays in mice demonstrated that YLL545 inhibits angiogenesis in vivo. YLL545 also inhibited proliferation and induced apoptosis in MDA-MB-231 breast cancer cells both in vitro and in vivo, and 50 mg/kg/d YLL545 inhibited human tumor xenograft growth by more than 50% in BALB/c nude mice. These observations suggest YLL545 is a potentially useful anticancer drug candidate. PMID:27203384

  18. Experimental studies of a vaccine formulation of recombinant human VEGF antigen with aluminum phosphate.

    PubMed

    Pérez Sánchez, Lincidio; Morera Díaz, Yanelys; Bequet-Romero, Mónica; Ramses Hernández, Gerardo; Rodríguez, Yadira; Castro Velazco, Jorge; Puente Pérez, Pedro; Ayala Avila, Marta; Gavilondo, Jorge V

    2015-01-01

    CIGB-247 is a cancer vaccine that is a formulation of a recombinant protein antigen representative of the human vascular endothelial growth factor (VEGF) with a bacterially-derived adjuvant (VSSP). The vaccine has shown an excellent safety profile in mice, rats, rabbits, not-human primates and in recent clinical trials in cancer patients. Response to the vaccine is characterized by specific antibody titers that neutralize VEGF/VEGFR2 binding and a cytotoxic tumor-specific response. To expand our present anti-VEGF active immunotherapy strategies, we have now studied in mice and non-human primates the effects of vaccination with a formulation of our recombinant VEGF antigen and aluminum phosphate adjuvant (hereafter denominated CIGB-247-A). Administered bi-weekly, CIGB-247-A produces high titers of anti-VEGF IgG blocking antibodies in 2 mice strains. Particularly in BALB/c, the treatment impaired subcutaneous F3II mammary tumor growth and reduced the number of spontaneous lung macro metastases, increasing animals' survival. Spleen cells from specifically immunized mice directly killed F3II tumor cells in vitro. CIGB-247-A also showed to be immunogenic in non-human primates, which developed anti-VEGF blocking antibodies and the ability for specific direct cell cytotoxic responses, all without impairing the healing of deep skin wounds or other side effect. Our results support consideration of aluminum phosphate as a suitable adjuvant for the development of new vaccine formulations using VEGF as antigen.

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

    PubMed Central

    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

  20. Experimental studies of a vaccine formulation of recombinant human VEGF antigen with aluminum phosphate

    PubMed Central

    Pérez Sánchez, Lincidio; Morera Díaz, Yanelys; Bequet-Romero, Mónica; Ramses Hernández, Gerardo; Rodríguez, Yadira; Castro Velazco, Jorge; Puente Pérez, Pedro; Ayala Avila, Marta; Gavilondo, Jorge V

    2015-01-01

    CIGB-247 is a cancer vaccine that is a formulation of a recombinant protein antigen representative of the human vascular endothelial growth factor (VEGF) with a bacterially-derived adjuvant (VSSP). The vaccine has shown an excellent safety profile in mice, rats, rabbits, not-human primates and in recent clinical trials in cancer patients. Response to the vaccine is characterized by specific antibody titers that neutralize VEGF/VEGFR2 binding and a cytotoxic tumor-specific response. To expand our present anti-VEGF active immunotherapy strategies, we have now studied in mice and non-human primates the effects of vaccination with a formulation of our recombinant VEGF antigen and aluminum phosphate adjuvant (hereafter denominated CIGB-247-A). Administered bi-weekly, CIGB-247-A produces high titers of anti-VEGF IgG blocking antibodies in 2 mice strains. Particularly in BALB/c, the treatment impaired subcutaneous F3II mammary tumor growth and reduced the number of spontaneous lung macro metastases, increasing animals' survival. Spleen cells from specifically immunized mice directly killed F3II tumor cells in vitro. CIGB-247-A also showed to be immunogenic in non-human primates, which developed anti-VEGF blocking antibodies and the ability for specific direct cell cytotoxic responses, all without impairing the healing of deep skin wounds or other side effect. Our results support consideration of aluminum phosphate as a suitable adjuvant for the development of new vaccine formulations using VEGF as antigen. PMID:25891359

  1. Insights from Genetic Model Systems of Retinal Degeneration: Role of Epsins in Retinal Angiogenesis and VEGFR2 Signaling

    PubMed Central

    Wu, Yong; Liu, Yanjun; Deng, Lin; Chen, Hong

    2017-01-01

    The retina is a light sensitive tissue that contains specialized photoreceptor cells called rods and cones which process visual signals. These signals are relayed to the brain through interneurons and the fibers of the optic nerve. The retina is susceptible to a variety of degenerative diseases, including age-related macular degeneration (AMD), diabetic retinopathy (DR), retinitis pigmentosa (RP) and other inherited retinal degenerations. In order to reveal the mechanism underlying these diseases and to find methods for the prevention/treatment of retinal degeneration, animal models have been generated to mimic human eye diseases. In this paper, several well-characterized and commonly used animal models are reviewed. Of particular interest are the contributions of these models to our understanding of the mechanisms of retinal degeneration and thereby providing novel treatment options including gene therapy, stem cell therapy, nanomedicine, and CRISPR/Cas9 genome editing. Role of newly-identified adaptor protein epsins from our laboratory is discussed in retinal angiogenesis and VEGFR2 signaling. PMID:28191500

  2. VEGF-A165b Is Cytoprotective and Antiangiogenic in the Retina

    PubMed Central

    Magnussen, Anette L.; Rennel, Emma S.; Hua, Jing; Bevan, Heather S.; Long, Nicholas Beazley; Lehrling, Christina; Gammons, Melissa; Floege, Juergen; Harper, Steven J.; Agostini, Hansjürgen T.; Bates, David O.; Churchill, Amanda J.

    2010-01-01

    Purpose. A number of key ocular diseases, including diabetic retinopathy and age-related macular degeneration, are characterized by localized areas of epithelial or endothelial damage, which can ultimately result in the growth of fragile new blood vessels, vitreous hemorrhage, and retinal detachment. VEGF-A165, the principal neovascular agent in ocular angiogenic conditions, is formed by proximal splice site selection in its terminal exon 8. Alternative splicing of this exon results in an antiangiogenic isoform, VEGF-A165b, which is downregulated in diabetic retinopathy. Here the authors investigate the antiangiogenic activity of VEGF165b and its effect on retinal epithelial and endothelial cell survival. Methods. VEGF-A165b was injected intraocularly in a mouse model of retinal neovascularization (oxygen-induced retinopathy [OIR]). Cytotoxicity and cell migration assays were used to determine the effect of VEGF-A165b. Results. VEGF-A165b dose dependently inhibited angiogenesis (IC50, 12.6 pg/eye) and retinal endothelial migration induced by 1 nM VEGF-A165 across monolayers in culture (IC50, 1 nM). However, it also acts as a survival factor for endothelial cells and retinal epithelial cells through VEGFR2 and can stimulate downstream signaling. Furthermore, VEGF-A165b injection, while inhibiting neovascular proliferation in the eye, reduced the ischemic insult in OIR (IC50, 2.6 pg/eye). Unlike bevacizumab, pegaptanib did not interact directly with VEGF-A165b. Conclusions. The survival effects of VEGF-A165b signaling can protect the retina from ischemic damage. These results suggest that VEGF-A165b may be a useful therapeutic agent in ischemia-induced angiogenesis and a cytoprotective agent for retinal pigment epithelial cells. PMID:20237249

  3. A Two-Compartment Model of VEGF Distribution in the Mouse

    PubMed Central

    Engel-Stefanini, Marianne O.; Popel, Aleksander S.

    2011-01-01

    Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis – the growth of new microvessels from existing microvasculature. Angiogenesis is a complex process involving numerous molecular species, and to better understand it, a systems biology approach is necessary. In vivo preclinical experiments in the area of angiogenesis are typically performed in mouse models; this includes drug development targeting VEGF. Thus, to quantitatively interpret such experimental results, a computational model of VEGF distribution in the mouse can be beneficial. In this paper, we present an in silico model of VEGF distribution in mice, determine model parameters from existing experimental data, conduct sensitivity analysis, and test the validity of the model. The multiscale model is comprised of two compartments: blood and tissue. The model accounts for interactions between two major VEGF isoforms (VEGF120 and VEGF164) and their endothelial cell receptors VEGFR-1, VEGFR-2, and co-receptor neuropilin-1. Neuropilin-1 is also expressed on the surface of parenchymal cells. The model includes transcapillary macromolecular permeability, lymphatic transport, and macromolecular plasma clearance. Simulations predict that the concentration of unbound VEGF in the tissue is approximately 50-fold greater than in the blood. These concentrations are highly dependent on the VEGF secretion rate. Parameter estimation was performed to fit the simulation results to available experimental data, and permitted the estimation of VEGF secretion rate in healthy tissue, which is difficult to measure experimentally. The model can provide quantitative interpretation of preclinical animal data and may be used in conjunction with experimental studies in the development of pro- and anti-angiogenic agents. The model approximates the normal tissue as skeletal muscle and includes endothelial cells to represent the vasculature. As the VEGF system becomes better characterized in other tissues and

  4. Basal and apical regulation of VEGF-A and placenta growth factor in the RPE/choroid and primary RPE

    PubMed Central

    Kaya, Leyla; Flach, Janina; Lassen, Jens; Treumer, Felix; Roider, Johann

    2015-01-01

    Purpose Members of the vascular endothelial growth factor (VEGF) family are strongly involved in pathological processes in the retina, such as age-related macular degeneration and diabetic retinopathy. Cells of the retinal pigment epithelium (RPE) constitutively secrete VEGF-A, and the secretion of placental growth factor (PlGF) has also been described. RPE cells are strongly polarized cells with different secretome at the apical and basal side. In this study, we evaluated the basal and apical regulation of VEGF-A and PlGF secretion in RPE/choroid explants and primary RPE cells. Methods RPE/choroid tissue explants were prepared from porcine eyes and cultivated in modified Ussing chambers, separating apical (RPE) and basal (choroid) supernatant. Primary RPE cells were also prepared from porcine eyes and cultivated on Transwell plates. Explants and cells were treated with inhibitors for VEGFR-2 (SU1498), p38 (SB203580), and the transcription factors nuclear factor-kappa B (NF-κB) and SP-1 (mithramycin), respectively. VEGF-A and PlGF content was evaluated with enzyme-linked immunosorbent assay (ELISA). In addition, western blots were performed. Results In the RPE/choroid, VEGF-A can initially be found on the apical and basal sides with significantly more pronounced secretion on the basal side. VEGF-A secretion is differentially regulated on the apical and basal sides, with the inhibition of SP-1 and NF-κB showing strong effects apically and basally after 24 h and 48 h, the inhibition of p38 displaying its effect mainly on the basal side with some effect apically after 48 h, and the inhibition of VEGFR-2 reducing the secretion of VEGF only on the apical side at 24 h and 48 h. In the RPE cell culture, similar effects were found, with inhibition of NF-κB or SP-1 displaying a strong decrease in VEGF-A on both sides, and p38 inhibition displaying only an inhibitory effect on the basal side. In contrast, an apical effect of VEGFR-2 inhibition was not found. However, the

  5. VEGF promotes gastric cancer development by upregulating CRMP4

    PubMed Central

    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

  6. Inhibition of VEGF induces cellular senescence in colorectal cancer cells.

    PubMed

    Hasan, Mohammad R; Ho, Shirley H Y; Owen, David A; Tai, Isabella T

    2011-11-01

    Vascular endothelial growth factor (VEGF) inhibitors, such as bevacizumab, have improved outcomes in metastatic colorectal cancer (CRC). Recent studies have suggested that VEGF can delay the onset of cellular senescence in human endothelial cells. As VEGF receptors are known to be upregulated in CRC, we hypothesized that VEGF inhibition may directly influence cellular senescence in this disease. In our study, we observed that treatment with bevacizumab caused a significant increase (p < 0.05) in cellular senescence in vitro in several CRC cells, such as MIP101, RKO, SW620 and SW480 cells, compared to untreated or human IgG-treated control cells. Similar results were also obtained from cells treated with a VEGFR2 kinase inhibitor Ki8751. In vivo, cellular senescence was detected in MIP101 tumor xenografts from 75% of mice treated with bevacizumab, while cellular senescence was undetectable in xenografts from mice treated with saline or human IgG (p < 0.05). Interestingly, we also observed that the proportion of senescent cells in colon cancer tissues obtained from patients treated with bevacizumab was 4.4-fold higher (p < 0.01) than those of untreated patients. To understand how VEGF inhibitors may regulate cellular senescence, we noted that among the two important regulators of senescent growth arrest of tumor cells, bevacizumab-associated increase in cellular senescence coincided with an upregulation of p16 but appeared to be independent of p53. siRNA silencing of p16 gene in MIP101 cells suppressed bevacizumab-induced cellular senescence, while silencing of p53 had no effect. These findings demonstrate a novel antitumor activity of VEGF inhibitors in CRC, involving p16.

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

    PubMed

    Eder, Matthias; Krivoshein, Arcadius V; Backer, Marina; Backer, Joseph M; Haberkorn, Uwe; Eisenhut, Michael

    2010-05-01

    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 (68)Ga at ambient temperature. scVEGF-PEG-HBED-CC was synthesized by activating a single carboxyl group of the [Fe(HBED-CC)](-) complex with N-hydroxysuccinimide. Reaction of the activated complex with NH(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(2)-Bn-NOTA. (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. 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 (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. The conjugates scVEGF-PEG-HBED-CC and scVEGF-PEG-NOTA revealed comparable in vivo characteristics and allowed easy-to-perform labeling with high stability for fast [(68)Ga]PET imaging

  8. Anti-tumour activity of tivozanib, a pan-inhibitor of VEGF receptors, in therapy-resistant ovarian carcinoma cells

    PubMed Central

    Momeny, Majid; Sabourinejad, Zahra; Zarrinrad, Ghazaleh; Moghaddaskho, Farima; Eyvani, Haniyeh; Yousefi, Hassan; Mirshahvaladi, Shahab; Poursani, Ensieh M.; Barghi, Farinaz; Poursheikhani, Arash; Dardaei, Leila; Bashash, Davood; Ghazi-Khansari, Mahmoud; Tavangar, Seyyed M.; Dehpour, Ahmad R.; Yaghmaie, Marjan; Alimoghaddam, Kamran; Ghavamzadeh, Ardeshir; Ghaffari, Seyed H.

    2017-01-01

    Epithelial ovarian cancer (EOC) is the most fatal gynaecological malignancy. Despite initial therapeutic response, the majority of advanced-stage patients relapse and succumb to chemoresistant disease. Overcoming drug resistance is the key to successful treatment of EOC. Members of vascular endothelial growth factor (VEGF) family are overexpressed in EOC and play key roles in its malignant progression though their contribution in development of the chemoresistant disease remains elusive. Here we show that expression of the VEGF family is higher in therapy-resistant EOC cells compared to sensitive ones. Overexpression of VEGFR2 correlated with resistance to cisplatin and combination with VEGFR2-inhibitor apatinib synergistically increased cisplatin sensitivity. Tivozanib, a pan-inhibitor of VEGF receptors, reduced proliferation of the chemoresistant EOC cells through induction of G2/M cell cycle arrest and apoptotic cell death. Tivozanib decreased invasive potential of these cells, concomitant with reduction of intercellular adhesion molecule-1 (ICAM-1) and diminishing the enzymatic activity of urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-2 (MMP-2). Moreover, tivozanib synergistically enhanced anti-tumour effects of EGFR-directed therapies including erlotinib. These findings suggest that the VEGF pathway has potential as a therapeutic target in therapy-resistant EOC and VEGFR blockade by tivozanib may yield stronger anti-tumour efficacy and circumvent resistance to EGFR-directed therapies. PMID:28383032

  9. Low energy shock wave therapy induces angiogenesis in acute hind-limb ischemia via VEGF receptor 2 phosphorylation.

    PubMed

    Holfeld, Johannes; Tepeköylü, Can; Blunder, Stefan; Lobenwein, Daniela; Kirchmair, Elke; Dietl, Marion; Kozaryn, Radoslaw; Lener, Daniela; Theurl, Markus; Paulus, Patrick; Kirchmair, Rudolf; Grimm, Michael

    2014-01-01

    Low energy shock waves have been shown to induce angiogenesis, improve left ventricular ejection fraction and decrease angina symptoms in patients suffering from chronic ischemic heart disease. Whether there is as well an effect in acute ischemia was not yet investigated. Hind-limb ischemia was induced in 10-12 weeks old male C57/Bl6 wild-type mice by excision of the left femoral artery. Animals were randomly divided in a treatment group (SWT, 300 shock waves at 0.1 mJ/mm2, 5 Hz) and untreated controls (CTR), n = 10 per group. The treatment group received shock wave therapy immediately after surgery. Higher gene expression and protein levels of angiogenic factors VEGF-A and PlGF, as well as their receptors Flt-1 and KDR have been found. This resulted in significantly more vessels per high-power field in SWT compared to controls. Improvement of blood perfusion in treatment animals was confirmed by laser Doppler perfusion imaging. Receptor tyrosine kinase profiler revealed significant phosphorylation of VEGF receptor 2 as an underlying mechanism of action. The effect of VEGF signaling was abolished upon incubation with a VEGFR2 inhibitor indicating that the effect is indeed VEGFR 2 dependent. Low energy shock wave treatment induces angiogenesis in acute ischemia via VEGF receptor 2 stimulation and shows the same promising effects as known from chronic myocardial ischemia. It may therefore develop as an adjunct to the treatment armentarium of acute muscle ischemia in limbs and myocardium.

  10. Comparative integromics on VEGF family members.

    PubMed

    Katoh, Yuriko; Katoh, Masaru

    2006-06-01

    VEGF, Hedgehog, FGF, Notch, and WNT signaling pathways network together for vascular remodeling during embryogenesis, tissue regeneration, and carcinogenesis. VEGFA (VEGF), VEGFB, VEGFC, VEGFD (FIGF) and PGF (PlGF) are VEGF family ligands for receptor tyrosine kinases, including VEGFR1 (FLT1), VEGFR2 (KDR) and VEGFR3 (FLT4). Bevacizumab (Avastin), Sunitinib (Sutent) and Sorafenib (Nexavar) are anti-cancer drugs targeted to VEGF signaling pathway. TCF/LEF binding sites within the promoter region of human VEGF family members were searched for by using bioinformatics and human intelligence (Humint). Because four TCF/LEF-binding sites were identified within the 5'-promoter region of human VEGFD gene within AC095351.5 genome sequence, comparative genomics analyses on VEGFD orthologs were further performed. ASB9-ASB11-VEGFD locus at human chromosome Xp22.2 and ASB5-VEGFC locus at human chromosome 4q34 were paralogous regions within the human genome. Human VEGFD mRNA was expressed in lung, small intestine, uterus, breast, neural tissues, and neuroblastoma. Mouse Vegfd mRNA was expressed in kidney, pregnant oviduct, and neural tissues. Chimpanzee VEGFD promoter, cow Vegfd promoter, mouse Vegfd promoter and rat Vegfd promoter were identified within NW_121675.1, AC161065.2, AL732475.6 and AC130036.3 genome sequences, respectively. Three out of four TCF/LEF-binding sites within human VEGFD promoter were conserved in chimpanzee VEGFD promoter, and one in cow Vegfd promoter. TCF/LEF-binding site, not conserved in human VEGFD promoter, occurred in cow, mouse and rat Vegfd promoters. At least five out of six bHLH-binding sites within human VEGFD proximal promoter region were conserved in chimpanzee VEGFD proximal promoter region, while only one in cow Vegfd proximal promoter region. Together these facts indicate that relatively significant promoter evolution occurred among mammalian VEGFD orthologs. Human VEGFD was characterized as a potent target gene of WNT

  11. Itraconazole Side Chain Analogues: Structure–Activity Relationship Studies for Inhibition of Endothelial Cell Proliferation, Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) Glycosylation, and Hedgehog Signaling

    PubMed Central

    Shi, Wei; Nacev, Benjamin A.; Aftab, Blake T.; Head, Sarah; Rudin, Charles M.; Liu, Jun O.

    2012-01-01

    Itraconazole is an antifungal drug that was recently found to possess potent antiangiogenic activity and anti-hedgehog (Hh) pathway activity. To search for analogues of itraconazole with greater potency and to understand the structure–activity relationship in both antiangiogenic and Hh targeting activity, 25 itraconazole side chain analogues were synthesized and assayed for inhibition of endothelial cell proliferation and Gli1 transcription in a medulloblastoma (MB) culture. Through this analysis, we have identified analogues with increased potency for inhibiting endothelial cell proliferation and the Hh pathway, as well as VEGFR2 glycosylation that was recently found to be inhibited by itraconazole. An SAR analysis of these activities revealed that potent activity of the analogues against VEGFR2 glycosylation was generally driven by side chains of at least four carbons in composition with branching at the α or β position. SAR trends for targeting the Hh pathway were divergent from those related to HUVEC proliferation or VEGFR2 glycosylation. These results also suggest that modification of the sec-butyl side chain can lead to enhancement of the biological activity of itraconazole. PMID:21936514

  12. αB-crystallin regulation of angiogenesis by modulation of VEGF

    PubMed Central

    Kase, Satoru; He, Shikun; Sonoda, Shozo; Kitamura, Mizuki; Spee, Christine; Wawrousek, Eric; Ryan, Stephen J.; Kannan, Ram

    2010-01-01

    αB-crystallin is a chaperone belonging to the small heat shock protein family. Herein we show attenuation of intraocular angiogenesis in αB-crystallin knockout (αB-crystallin−/−) mice in 2 models of intraocular disease: oxygen-induced retinopathy and laser-induced choroidal neovascularization. Vascular endothelial growth factor A (VEGF-A) mRNA and hypoxia inducible factor-1α protein expression were induced during retinal angiogenesis, but VEGF-A protein expression remained low in αB-crystallin−/− retina versus wild-type mice, whereas VEGF-R2 expression was not affected. Both αB-crystallin and its phosphorylated serine59 formwere expressed, and immunoprecipitation revealed αB-crystallin binding to VEGF-A but not transforming growth factor-β in cultured retinal pigment epithelial (RPE) cells. αB-crystallin and VEGF-A are colocalized in the endoplasmic reticulum in RPE cells under chemical hypoxia. αB-crystallin−/− RPE showed low VEGF-A secretion under serum-starved conditions compared with wild-type cells. VEGF-A is polyubiquitinated in control and αB-crystallin siRNA treated RPE; however, mono-tetra ubiquitinated VEGF-A increases with αB-crystallin knockdown. Endothelial cell apoptosis in newly formed vessels was greater in αB-crystallin−/− than wild-type mice. Proteasomal inhibition in αB-crystallin−/− mice partially restores VEGF-A secretion and angiogenic phenotype in choroidal neovascularization. Our studies indicate an important role for αB-crystallin as a chaperone for VEGF-A in angiogenesis and its potential as a therapeutic target. PMID:20023214

  13. Next generation sequencing analysis of platinum refractory advanced germ cell tumor sensitive to Sunitinib (Sutent®) a VEGFR2/PDGFRβ/c-kit/ FLT3/RET/CSF1R inhibitor in a phase II trial.

    PubMed

    Subbiah, Vivek; Meric-Bernstam, Funda; Mills, Gordon B; Shaw, Kenna R Mills; Bailey, Ann Marie; Rao, Priya; Ward, John F; Pagliaro, Lance C

    2014-08-01

    Germ cell tumors (GCT) are the most common solid tumors in adolescent and young adult males (age 15 and 35 years) and remain one of the most curable of all solid malignancies. However a subset of patients will have tumors that are refractory to standard chemotherapy agents. The management of this refractory population remains challenging and approximately 400 patients continue to die every year of this refractory disease in the United States. Given the preclinical evidence implicating vascular endothelial growth factor (VEGF) signaling in the biology of germ cell tumors, we hypothesized that the vascular endothelial growth factor receptor (VEGFR) inhibitor sunitinib (Sutent) may possess important clinical activity in the treatment of this refractory disease. We proposed a Phase II efficacy study of sunitinib in seminomatous and non-seminomatous metastatic GCT's refractory to first line chemotherapy treatment (ClinicalTrials.gov Identifier: NCT00912912). Next generation targeted exome sequencing using HiSeq 2000 (Illumina Inc., San Diego, CA, USA) was performed on the tumor sample of the unusual responder. Five patients are enrolled into this Phase II study. Among them we report here the clinical course of a patient (Patient # 5) who had an exceptional response to sunitinib. Next generation sequencing to understand this patient's response to sunitinib revealed RET amplification, EGFR and KRAS amplification as relevant aberrations. Oncoscan MIP array were employed to validate the copy number analysis that confirmed RET gene amplification. Sunitinib conferred clinical benefit to this heavily pre-treated patient. Next generation sequencing of this 'exceptional responder' identified the first reported case of a RET amplification as a potential basis of sensitivity to sunitinib (VEGFR2/PDGFRβ/c-kit/ FLT3/RET/CSF1R inhibitor) in a patient with refractory germ cell tumor. Further characterization of GCT patients using biomarkers for clinical response and patient

  14. Differential responsiveness in VEGF receptor subtypes to hypoxic stress in various tissues of plateau animals.

    PubMed

    Xie, Hui-Chun; Li, Jin-Gang; He, Jian-Ping

    2017-05-04

    With hypoxic stress, hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) are elevated and their responses are altered in skeletal muscles of plateau animals [China Qinghai-Tibetan plateau pikas (Ochotona curzoniae)] as compared with control animals [normal lowland Sprague-Dawley (SD) rats]. The results indicate that HIF-1alpha and VEGF are engaged in physiological functions under hypoxic environment. The purpose of the current study was to examine the protein levels of VEGF receptor subtypes (VEGFRs: VEGFR-1, VEGFR-2 and VEGFR-3) in the end organs, namely skeletal muscle, heart and lung in response to hypoxic stress. ELISA and Western blot analysis were employed to determine HIF-1alpha and the protein expression of VEGFRs in control animals and plateau pikas. We further blocked HIF-1alpha signal to determine if HIF-1alpha regulates alternations in VEGFRs in those tissues. We hypothesized that responsiveness of VEGFRs in the major end organs of plateau animals is differential with insult of hypoxic stress and is modulated by low oxygen sensitive HIF-1alpha. Our results show that hypoxic stress induced by exposure of lower O(2) for 6 h significantly increased the levels of VEGFR-2 in skeletal muscle, heart and lung and the increases were amplified in plateau pikas. Our results also demonstrate that hypoxic stress enhanced VEGFR-3 in lungs of plateau animals. Nonetheless, no significant alternations in VEGFR-1 were observed in those tissues with hypoxic stress. Moreover, we observed decreases of VEGFR-2 in skeletal muscle, heart and lung; and decreases of VEGFR-3 in lung following HIF-1alpha inhibition. Overall, our findings suggest that in plateau animals 1) responsiveness of VEGFRs is different under hypoxic environment; 2) amplified VEGFR-2 response appears in skeletal muscle, heart and lung, and enhanced VEGFR-3 response is mainly observed in lung; 3) HIF-1alpha plays a regulatory role in the levels of VEGFRs. Our results

  15. Contrast-Enhanced Ultrasound with VEGFR2-Targeted Microbubbles for Monitoring Regorafenib Therapy Effects in Experimental Colorectal Adenocarcinomas in Rats with DCE-MRI and Immunohistochemical Validation

    PubMed Central

    Clevert, Dirk-Andre; Hirner-Eppeneder, Heidrun; Ingrisch, Michael; Moser, Matthias; Schuster, Jessica; Tadros, Dina; Schneider, Moritz; Kazmierczak, Philipp Maximilian; Reiser, Maximilian; Cyran, Clemens C.

    2017-01-01

    Objectives To investigate contrast-enhanced ultrasound (CEUS) with VEGFR2-targeted microbubbles for monitoring therapy effects of regorafenib on experimental colon carcinomas in rats with correlation to dynamic contrast-enhanced MRI (DCE-MRI) and immunohistochemistry. Materials and Methods Human colorectal adenocarcinoma xenografts (HT-29) were implanted subcutaneously in n = 21 (n = 11 therapy group; n = 10 control group) female athymic nude rats (Hsd: RH-Foxn1rnu). Animals were imaged at baseline and after a one-week daily treatment with regorafenib or a placebo (10 mg/kg bodyweight), using CEUS with VEGFR2-targeted microbubbles and DCE-MRI. In CEUS tumor perfusion was assessed during an early vascular phase (wash-in area under the curve = WiAUC) and VEGFR2-specific binding during a late molecular phase (signal intensity after 8 (SI8min) and 10 minutes (SI10min)), using a conventional 15L8 linear transducer (transmit frequency 7 MHz, dynamic range 80 dB, depth 25 mm). In DCE-MRI functional parameters plasma flow (PF) and plasma volume (PV) were quantified. For validation purposes, CEUS parameters were correlated with DCE-MRI parameters and immunohistochemical VEGFR2, CD31, Ki-67 and TUNEL stainings. Results CEUS perfusion parameter WiAUC decreased significantly (116,989 ± 77,048 a.u. to 30,076 ± 27,095a.u.; p = 0.005) under therapy with no significant changes (133,932 ± 65,960 a.u. to 84,316 ± 74,144 a.u.; p = 0.093) in the control group. In the therapy group, the amount of bound microbubbles in the late phase was significantly lower in the therapy than in the control group on day 7 (SI8min: 283 ± 191 vs. 802 ± 460 a.u.; p = 0.006); SI10min: 226 ± 149 vs. 645 ± 461 a.u.; p = 0.009). PF and PV decreased significantly (PF: 147 ± 58 mL/100 mL/min to 71 ± 15 mL/100 mL/min; p = 0.003; PV: 13 ± 3% to 9 ± 4%; p = 0.040) in the therapy group. Immunohistochemistry revealed significantly fewer VEGFR2 (7.2 ± 1.8 vs. 17.8 ± 4.6; p < 0.001), CD31 (8.1 ± 3.0 vs

  16. An Anti-Inflammatory Role of VEGFR2/Src Kinase Inhibitor in Herpes Simplex Virus 1-Induced Immunopathology▿

    PubMed Central

    Sharma, Shalini; Mulik, Sachin; Kumar, Naveen; Suryawanshi, Amol; Rouse, Barry T.

    2011-01-01

    Corneal neovascularization represents a key step in the blinding inflammatory stromal keratitis (SK) lesion caused by ocular infection with herpes simplex virus (HSV). In this report, we describe a novel approach for limiting the angiogenesis caused by HSV infection of the mouse eye. We show that topical or systemic administration of the Src kinase inhibitor (TG100572) that inhibits downstream molecules involved in the vascular endothelial growth factor (VEGF) signaling pathway resulted in markedly diminished levels of HSV-induced angiogenesis and significantly reduced the severity of SK lesions. Multiple mechanisms were involved in the inhibitory effects. These included blockade of IL-8/CXCL1 involved in inflammatory cells recruitment that are a source of VEGF, diminished cellular infiltration in the cornea, and reduced proliferation and migration of CD4+ T cells into the corneas. As multiple angiogenic factors (VEGF and basic fibroblast growth factor [bFGF]) play a role in promoting angiogenesis during SK and since Src kinases are involved in signaling by many of them, the use of Src kinase inhibition represents a promising way of limiting the severity of SK lesions the most common cause of infectious blindness in the Western world. PMID:21471229

  17. A compartment model of VEGF distribution in blood, healthy and diseased tissues

    PubMed Central

    Stefanini, Marianne O; Wu, Florence TH; Mac Gabhann, Feilim; Popel, Aleksander S

    2008-01-01

    Background Angiogenesis is a process by which new capillaries are formed from pre-existing blood vessels in physiological (e.g., exercise, wound healing) or pathological (e.g., ischemic limb as in peripheral arterial disease, cancer) contexts. This neovascular mechanism is mediated by the vascular endothelial growth factor (VEGF) family of cytokines. Although VEGF is often targeted in anti-angiogenic therapies, there is little knowledge about how its concentration may vary between tissues and the vascular system. A compartment model is constructed to study the VEGF distribution in the tissue (including matrix-bound, cell surface receptor-bound and free VEGF isoforms) and in the blood. We analyze the sensitivity of this distribution to the secretion rate, clearance rate and vascular permeability of VEGF. Results We find that, in a physiological context, VEGF concentration varies approximately linearly with the VEGF secretion rate. VEGF concentration in blood but not in tissue is dependent on the vascular permeability of healthy tissue. Model simulations suggest that relative VEGF increases are similar in blood and tissue during exercise and return to baseline within several hours. In a pathological context (tumor), we find that blood VEGF concentration is relatively insensitive to increased vascular permeability in tumors, to the secretion rate of VEGF by tumors and to the clearance. However, it is sensitive to the vascular permeability in the healthy tissue. Finally, the VEGF distribution profile in healthy tissue reveals that about half of the VEGF is complexed with the receptor tyrosine kinase VEGFR2 and the co-receptor Neuropilin-1. In diseased tissues, this binding can be reduced to 15% while VEGF bound to the extracellular matrix and basement membranes increases. Conclusion The results are of importance for physiological conditions (e.g., exercise) and pathological conditions (e.g., peripheral arterial disease, coronary artery disease, cancer). This

  18. KR-31831, benzopyran derivative, inhibits VEGF-induced angiogenesis of HUVECs through suppressing KDR expression.

    PubMed

    Park, Shi-Young; Seo, Eun-Hee; Song, Hyun Seok; Jung, Seung-Youn; Lee, Young-Kyoung; Yi, Kyu-Yang; Yoo, Sung-Eun; Kim, Yung-Jin

    2008-06-01

    Angiogenesis is important in the development and progression of cancer, therefore the therapeutic approach based on anti-angiogenesis may represent a promising therapeutic option. KR-31831 is a novel anti-ischemic agent. Previously, we reported the anti-angiogenic activity of KR-31831. In the present study we investigated the molecular mechanisms underlying anti-angiogenic activity of KR-31831. We show that KR-31831 inhibits vascular endothelial growth factor (VEGF)-induced proliferation and tube formation via release of intracellular Ca2+ and phosphorylation of extra-cellular regulated kinase 1/2 (Erk 1/2) in human umbilical vein endothelial cells (HUVECs). Moreover, the expression of VEGF receptor 2 (VEGFR2, known as Flk-1 or KDR) was reduced by the treatment of KR-31831. These results suggest that KR-31831 may have inhibitory effects on tumor angiogenesis through down-regulation of KDR expression.

  19. Thy-1 Regulates VEGF-Mediated Choroidal Endothelial Cell Activation and Migration: Implications in Neovascular Age-Related Macular Degeneration

    PubMed Central

    Wang, Haibo; Han, Xiaokun; Kunz, Eric; Hartnett, M. Elizabeth

    2016-01-01

    Purpose This study addresses the hypothesis that age-related stresses upregulate Thy-1 in choroidal endothelial cells (CECs) and contribute to CEC activation and migration, processes important in choroidal neovascularization (CNV). Methods Measurements were made of Thy-1 protein (Western blot) in CECs and Thy-1 mRNA (real time quantitative PCR) in CECs treated with VEGF, CCL11, or PBS or in RPE/choroids from young or old donors or lasered or nonlasered mice. Immunolabeled Thy-1 in ocular sections was compared from young versus old human donor eyes or those with or without neovascular AMD or from lasered versus nonlasered mice. Choroidal endothelial cells transfected with Thy-1 or control siRNA or pretreated with Thy-1 blocking peptide or control were stimulated with VEGF or 7-ketocholesterol (7-KC). Choroidal endothelial cell migration, proliferation, cytoskeletal stress fibers, Rac1 activation, and phosphorylated VEGF receptor 2 (VEGFR2), integrin β3, and Src were measured. Statistics were performed using ANOVA. Results Thy-1 was expressed in retinal ganglion cells and in vascular endothelial-cadherin–labeled choroid and localized to human or mouse laser-induced CNV lesions. Thy-1 protein and mRNA were significantly increased in CECs treated with VEGF or CCL11 and in RPE/choroids from aged versus young donor eyes or from lasered mice versus nonlasered controls. Knockdown or inhibition of Thy-1 in CECs significantly reduced VEGF-induced CEC migration and proliferation, stress fiber formation and VEGFR2, Src, integrin β3 and Rac1 activation, and 7-KC–induced Rac1 and Src activation. Conclusions Thy-1 in CECs regulates VEGF-induced CEC activation and migration and links extracellular 7-KC to intracellular signaling. Future studies elucidating Thy-1 mechanisms in neovascular AMD are warranted. PMID:27768790

  20. Stabilization of HIF-1α modulates VEGF and Caspase-3 in the hippocampus of rats following transient global ischemia induced by asphyxial cardiac arrest.

    PubMed

    Liu, Xiao-Liang; Lu, Jian; Xing, Jihong

    2016-04-15

    Hypoxia inducible factor-1 (HIF-1) contributes to pathophysiological changes of homeostasis under conditions of oxygen deprivation as well as ischemia. In this study, we examined protein expression of subtype HIF-1α and its downstream product, namely vascular endothelial growth factor (VEGF) in the rat hippocampus after transient global ischemia induced by asphyxial cardiac arrest (CA) followed by cardiopulmonary resuscitation (CPR). We also examined the effects of stabilization of HIF-1α by systemic administration of dimethyloxalylglycine (DMOG) and ML228 on expression of VEGF receptor subtype 2 (VEGFR-2), Caspase-3 and NF-kB in the hippocampus. Ninety-six adult Sprague-Dawley rats were used in this study. The animals surviving from CPR were sacrificed 0, 3, 6 and 24h following CPR and the protein levels of HIF-1α and VEGF in the hippocampus were determined. VEGFR-2, Caspase-3 and NF-kB were also examined in control rats, and rats that survived for 24h after CPR and were given with DMOG/ML228. Moreover, neurological functions were estimated in control rats and rats with DMOG/ML228. Our results show that HIF-1α and VEGF were significantly increased in the hippocampus 3-24h after CA. Significant increases in VEGFR-2, Caspase-3 and NF-κB were observed in the hippocampus 24h after CA (P<0.05 vs. control group). Nonetheless, DMOG and ML228 significantly augmented VEGFR-2, attenuated Caspase-3 and neuronal apoptosis, and improved neurological Severity Score and tissue edema (P<0.05 vs. saline group), without affecting expression of NF-κB. Our data revealed specific signaling pathways in alleviating CA-evoked global cerebral ischemia by elucidating that HIF-1α plays an important role in regulating expression of VEGFR-2 and Caspase-3 as well as improving neurological functions and neuronal edema. The subsequent induction of HIF-1α and its target signal pathways is likely a part of the intrinsic neuroprotective effects aimed at attenuating damage as a result of

  1. A novel synthetic small molecule YF-452 inhibits tumor growth through antiangiogenesis by suppressing VEGF receptor 2 signaling.

    PubMed

    Liu, Yongrui; He, Yuan; Yang, Feifei; Cong, Xiaonan; Wang, Jinhua; Peng, Shihong; Gao, Dan; Wang, Weifang; Lan, Liping; Ying, Xuexiang; Liu, Mingyao; Chen, Yihua; Yi, Zhengfang

    2017-02-01

    Tumor angiogenesis is characterized by abnormal vessel morphology, endowing tumor with highly hypoxia and unresponsive toward treatment. To date, mounting angiogenic factors have been discovered as therapeutic targets in antiangiogenic drug development. Among them, vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors exerts potent antiangiogenic activity in tumor therapy. Therefore, it may provide a valid strategy for cancer treatment through targeting the tumor angiogenesis via VEGFR2 pathway. In this study, we established a high-profile compounds library and certificated a novel compound named N-(N-pyrrolidylacetyl)-9-(4-bromobenzyl)-1,3,4,9-tetrahydro-β-carboline (YF-452), which remarkably inhibited the migration, invasion and tube-like structure formation of human umbilical vein endothelial cells (HUVECs) with little toxicity invitro. Rat thoracic aorta ring assay indicated that YF-452 significantly blocked the formation of microvascular exvivo. In addition, YF-452 inhibited angiogenesis in chick chorioallantoic membrane (CAM) and mouse corneal micropocket assays. Moreover, YF-452 remarkably suppressed tumor growth in xenografts mice model. Furthermore, investigation of molecular mechanism revealed that YF-452 inhibited VEGF-induced phosphorylation of VEGFR2 kinase and the downstream protein kinases including extracellular signal regulated kinase (ERK), focal adhesion kinase (FAK) and Src. These results indicate that YF-452 inhibits angiogenesis and may be a potential antiangiogenic drug candidate for cancer therapy.

  2. Migration of FGF7-stimulated epithelial cells and VEGF-A-stimulated HUVECs depends on EGFR transactivation by ADAM17

    PubMed Central

    Maretzky, Thorsten; Evers, Astrid; Zhou, Wenhui; Swendeman, Steven L.; Wong, Pui-Mun; Rafii, Shahin; Reiss, Karina; Blobel, Carl P.

    2011-01-01

    The fibroblast growth factor receptor 2-IIIb (FGFR2b) and the vascular endothelial growth factor receptor 2 (VEGFR2) are tyrosine kinases that can promote cell migration and proliferation and have important roles in embryonic development and cancer. Here we show that FGF7/FGFR2b-dependent activation of EGFR/ERK1/2 signaling and cell migration in epithelial cells require stimulation of the membrane-anchored metalloproteinase ADAM17 and release of HB-EGF. Moreover, VEGF-A/VEGFR2-induced migration of HUVECs also depends on EGFR/ERK1/2 signaling and shedding of the ADAM17 substrate HB-EGF. The pathway used by the FGF7/FGFR2b signaling axis to stimulate shedding of substrates of ADAM17, including ligands of the EGFR, involves Src, p38 MAP-kinase and PI3K, but does not require the cytoplasmic domain of ADAM17. Based on these findings, ADAM17 emerges as a central component in a triple membrane-spanning pathway between the FGFR2b or VEGFR2 and the EGFR/ERK1/2 that is required for cell migration in keratinocytes and presumably also in endothelial cells. PMID:21407195

  3. Transient Receptor Potential Canonical 1 (TRPC1) Channels as Regulators of Sphingolipid and VEGF Receptor Expression

    PubMed Central

    Asghar, Muhammad Yasir; Magnusson, Melissa; Kemppainen, Kati; Sukumaran, Pramod; Löf, Christoffer; Pulli, Ilari; Kalhori, Veronica; Törnquist, Kid

    2015-01-01

    The identity of calcium channels in the thyroid is unclear. In human follicular thyroid ML-1 cancer cells, sphingolipid sphingosine 1-phosphate (S1P), through S1P receptors 1 and 3 (S1P1/S1P3), and VEGF receptor 2 (VEGFR2) stimulates migration. We show that human thyroid cells express several forms of transient receptor potential canonical (TRPC) channels, including TRPC1. In TRPC1 knockdown (TRPC1-KD) ML-1 cells, the basal and S1P-evoked invasion and migration was attenuated. Furthermore, the expression of S1P3 and VEGFR2 was significantly down-regulated. Transfecting wild-type ML-1 cells with a nonconducting TRPC1 mutant decreased S1P3 and VEGFR2 expression. In TRPC1-KD cells, receptor-operated calcium entry was decreased. To investigate whether the decreased receptor expression was due to attenuated calcium entry, cells were incubated with the calcium chelator BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid). In these cells, and in cells where calmodulin and calmodulin-dependent kinase were blocked pharmacologically, S1P3 and VEGFR2 expression was decreased. In TRPC1-KD cells, both hypoxia-inducible factor 1α expression and the secretion and activity of MMP2 and MMP9 were attenuated, and proliferation was decreased in TRPC1-KD cells. This was due to a prolonged G1 phase of the cell cycle, a significant increase in the expression of the cyclin-dependent kinase inhibitors p21 and p27, and a decrease in the expression of cyclin D2, cyclin D3, and CDK6. Transfecting TRPC1 to TRPC1-KD cells rescued receptor expression, migration, and proliferation. Thus, the expression of S1P3 and VEGFR2 is mediated by a calcium-dependent mechanism. TRPC1 has a crucial role in this process. This regulation is important for the invasion, migration, and proliferation of thyroid cancer cells. PMID:25971967

  4. DC electric stimulation upregulates angiogenic factors in endothelial cells through activation of VEGF receptors

    PubMed Central

    Bai, Huai; Forrester, John V.; Zhao, Min

    2015-01-01

    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 24 h 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. PMID:21524919

  5. DC electric stimulation upregulates angiogenic factors in endothelial cells through activation of VEGF receptors.

    PubMed

    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.

  6. Effect of semax and its C-terminal fragment Pro-Gly-Pro on the expression of VEGF family genes and their receptors in experimental focal ischemia of the rat brain.

    PubMed

    Medvedeva, Ekaterina V; Dmitrieva, Veronika G; Povarova, Oksana V; Limborska, Svetlana A; Skvortsova, Veronika I; Myasoedov, Nikolay F; Dergunova, Lyudmila V

    2013-02-01

    The synthetic peptide Semax (Met-Glu-His-Phe-Pro-Gly-Pro) is used successfully in acute stroke therapy. In spite of numerous studies on the subject, many aspects of the neuroprotective effects of the peptide remain unknown. We studied the action of Semax and its C-terminal tripeptide Pro-Gly-Pro on the expression of the VEGF gene family (Vegf-a, Vegf-b, Vegf-c, Vegf-d, and Plgf) and their receptors (Vegfr-1, Vegfr-2, and Vegfr-3) in the frontoparietal cortex region of the rat brain at 3, 24, and 72 h after permanent left middle cerebral artery occlusion (pMCAO). The relative mRNA level of the genes studied was assessed using real-time reverse transcription-PCR. The Vegf-b and Vegf-d genes were most affected by the peptides, which resulted in their most noticeable activation at 3 h after pMCAO. The level of Vegf-d transcripts decreased considerably, whereas the mRNA level of the Vegf-b gene was significantly increased after 72 h of treatment with each of the peptides. In addition, the effects of the peptides on the expression of the Vegf-b and Vegf-d genes were the opposite of the action of ischemia. It is suggested that the identified effects of the peptides diminish the effects of ischemia, thus participating in the positive therapeutic effect of Semax on ischemic stroke.

  7. Endostar, a novel recombinant human endostatin, exerts antiangiogenic effect via blocking VEGF-induced tyrosine phosphorylation of KDR/Flk-1 of endothelial cells

    SciTech Connect

    Ling, Yun; Yang, Yong . E-mail: anticancer_drug@yahoo.com.cn; Lu, Na; You, Qi-dong; Wang, Sen; Gao, Ying; Chen, Yan; Guo, Qing-Long . E-mail: valianty@hotmail.com

    2007-09-14

    Endostar, a novel recombinant human endostatin expressed and purified in Escherichia coli with an additional nine-amino acid sequence and forming another his-tag structure, was approved by the SFDA in 2005 for the treatment of non-small-cell lung cancer. But its mechanism of action has not been illustrated before. In this study, we examined the antiangiogenic activities of endostar in vitro and in vivo. The results showed that endostar suppressed the VEGF-stimulated proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. Endostar blocked microvessel sprouting from rat aortic rings in vitro. Moreover, it could inhibit the formation of new capillaries from pre-existing vessels in the chicken chorioallantoic membrane (CAM) assay and affect the growth of vessels in tumor. We further found the antiangiogenic effects of endostar were correlated with the VEGF-triggered signaling. Endostar suppressed the VEGF-induced tyrosine phosphorylation of KDR/Flk-1(VEGFR-2) as well as the overall VEGFR-2 expression and the activation of ERK, p38 MAPK, and AKT in HUVECs. Collectively, these data indicated the relationship between endostar and VEGF signal pathways and provided a molecular basis for the antiangiogenic effects of endostar.

  8. Effects of low level laser therapy on attachment, proliferation, and gene expression of VEGF and VEGF receptor 2 of adipocyte-derived mesenchymal stem cells cultivated under nutritional deficiency.

    PubMed

    de Oliveira, Tabata Santos; Serra, Andrey Jorge; Manchini, Martha Trindade; Bassaneze, Vinicius; Krieger, José Eduardo; de Tarso Camillo de Carvalho, Paulo; Antunes, Daniela Espindola; Bocalini, Danilo Sales; Ferreira Tucci, Paulo José; Silva, José Antônio

    2015-01-01

    Low-level laser therapy (LLLT) has been shown to increase the proliferation of several cell types. We evaluated the effects of LLLT on adhesion, proliferation, and gene expression of vascular endothelial growth factor (VEGF) and type 2 receptor of VEGF (VEGFR2) at mesenchymal stem cells (MSCs) from human (hMSCs) and rat (rMSCs) adipose tissues on nutritional deficiencies. A dose-response curve was performed with cells treated with laser Ga-Al-As (660 nm, 30 mW) at energy of 0.7 to 9 J. Cell adhesion and proliferation were quantified 20, 40, and 60 min after LLLT and 24, 72, and 120 h after cultivation. Gene expression was verified by RT-PCR after 2 h of LLLT. A minor nutritional support caused a significant decrease in proliferation and adhesion of hMSCs and rMSCs. However, at the lowest LLLT dose (0.7 J), we observed a higher proliferation in hMSCs at standard condition shortly after irradiation (24 h). Adhesion was higher in hMSCs cultivated in controlled conditions at higher LLLT doses (3 and 9 J), and rMSCs show a reduction in the adhesion on 1.5 to 9 J. On nutritional deprivation, a 9 J dose was shown to reduce proliferation with 24 h and adhesion to all culture times in rMSCs. VEGF and VEGFR2 were increased after LLLT in both cell types. However, hMSCs under nutritional deprivation showed higher expression of VEGF and its receptor after irradiation with other laser doses. In conclusion, LLLT on human and rat MSCs might upregulate VEGF messenger RNA (mRNA) expression and modulate cell adhesion and proliferation distinctively.

  9. Sp1 inhibition-mediated upregulation of VEGF 165 b induced by rh-endostatin enhances antiangiogenic and anticancer effect of rh-endostatin in A549.

    PubMed

    Li, Zhen-yu; Zhu, Fang; Hu, Jian-li; Peng, Gang; Chen, Jing; Zhang, Sheng; Chen, Xu; Zhang, Rui-guang; Chen, Ling-juan; Liu, Pian; Luo, Ming; Sun, Zhi-hua; Ren, Jing-hua; Huang, Li-li; Wu, Gang

    2011-08-01

    Recombinant human endostatin (rh-endostatin), a potential antiangiogenic agent, is used in non-small cell lung carcinoma treatment and represses vascular endothelial cell growth factor (VEGF) levels in tumor cell. However, precise affection of rh-endostatin on the proangiogenic VEGF isoforms (VEGF(165)) or antiangiogenic VEGF isoforms (VEGF(165)b) is not clear. We therefore tested the hypothesis that rh-endostatin could alter expression of these isoforms to regulate tumor growth. A549 cells were exposed to rh-endostatin, and the expression of VEGF(165) and VEGF(165)b was detected. The role of SP1 as a regulator of isoform expression was investigated. We then examined the anticancer and antiangiogenic efficacy of rh-endostatin in combination with exogenous VEGF(165)b against A549 cells, EA.HY 926 cells and xenograft model of human lung cancer. rh-Endostatin reduced VEGF(165) and induced VEGF(165)b as well as inhibited SP1 in A549 cells. SP1 inhibitor (betulinic acid) also developed those changes. VEGF(165)b-rh-endostatin combination was highly synergistic and inhibited growth, survival, and migration of A549 cells, VEGF-mediated VEGFR2 phosphorylation in EA.HY 926 cells, and tumor growth in xenograft model of human lung cancer. rh-Endostatin downregulates proangiogenic vascular endothelial growth factor A (VEGFA) isoform and upregulates antiangiogenic VEGFA isoform, possibly through inhibition of SP1. Furthermore, VEGF(165)b sensitizes A549 to rh-endostatin treatment and enhances the anticancer effect of rh-endostatin.

  10. Fragment-based discovery of a dual pan-RET/VEGFR2 kinase inhibitor optimized for single-agent polypharmacology1

    PubMed Central

    Frett, Brendan; Carlomagno, Francesca; Moccia, Maria Luisa; Brescia, Annalisa; Federico, Giorgia; De Falco, Valentina; Admire, Brittany; Chen, Zhongzhu; Qi, Wenqing; Santoro, Massimo; Li, Hong-yu

    2015-01-01

    Oncogenic conversion of the RET (rearranged during transfection) tyrosine kinase is associated with several cancers. A fragment-based chemical screen lead to the identification of a novel RET inhibitor, Pz-1. Modeling and kinetic analysis identified Pz-1 as a Type-II tyrosine kinase inhibitor, able to bind the DFG-out conformation of the kinase. Importantly, from a single-agent polypharmacology standpoint, Pz-1 was shown active on VEGFR2, which can block blood supply required for RET-stimulated growth. In cell based assays, 1.0 nM of Pz-1 strongly inhibited phosphorylation of all tested RET oncoproteins. At 1.0 mg/kg/day per os, Pz-1 abrogated formation of tumors induced by RET-mutant fibroblasts and blocked phosphorylation of both RET and VEGFR2 in tumor tissue. Pz-1 featured no detectable toxicity up to 100.0 mg/kg, which indicated a large therapeutic window. This study validates the effectiveness and usefulness of a medicinal chemistry polypharmacology approach to obtain an inhibitor capable of targeting multiple oncogenic pathways PMID:26126987

  11. Canine mammary carcinomas: influence of histological grade, vascular invasion, proliferation, microvessel density and VEGFR2 expression on lymph node status and survival time.

    PubMed

    Diessler, M E; Castellano, M C; Portiansky, E L; Burns, S; Idiart, J R

    2017-06-01

    Spontaneous invasive non-inflammatory canine mammary carcinomas (CMC) and their regional lymph nodes (LN) were analysed (n = 136). Histological grade (HG) and vascular invasion (VI) in the tumours and lymph node status were recorded. Proliferation index (PI), microvessel density (MVD) and vascular endothelial growth factor receptor 2 (VEGFR2) expression were estimated using anti-proliferating cell nuclear antigen (PCNA), anti-von Willebrand factor and anti-Flk-1, respectively. Eighteen months follow-up was performed (34 bitches). Tumours of different grades showed differences regarding PI, Flk-1/integrated optical density (Flk-1/IOD) and MVD. Every feature showed significant association with LN status through bivariate analyses. From multivariate analyses, VI and Flk-1/IOD were selected to predict LN status. Data revealed that the probability of a CMC-bearing bitch to remain alive at 1, 4, 5 and 14-18 months was 0.91, 0.87, 0.81 and 0.77, respectively. Besides LN status, VI was the only feature positively correlated with survival time, although a trend to shorter survival of animal patients bearing high expressing VEGFR2 CMC was noted. © 2016 John Wiley & Sons Ltd.

  12. Jaceosidin, a natural flavone, promotes angiogenesis via activation of VEGFR2/FAK/PI3K/AKT/NF-κB signaling pathways in endothelial cells.

    PubMed

    Lee, Tae Hoon; Jung, Hana; Park, Keun Hyung; Bang, Myun Ho; Baek, Nam-In; Kim, Jiyoung

    2014-10-01

    Angiogenesis, the growth of new blood vessels from pre-existing vasculature, plays an important role in physiological and pathological processes such as embryonic development wound healing and revascularization of tissues after exposure to ischemia. We investigated the effects of jaceosidin, a main constituent of medicinal herbs of the genus Artemisia, on angiogenesis and signaling pathways in endothelial cells. Jaceosidin stimulated proliferation, migration and tubulogenesis of ECs as well as ex vivo sprouting from aorta rings, which are phenomena typical of angiogenesis. Jaceosidin activated vascular endothelial growth factor receptor 2 (VEGFR2, FLk-1/KDR) and angiogenic signaling molecules such as focal adhesion kinase, phosphatidylinositol 3-kinase, and its downstream target, the serine-threonine kinase AKTWe also demonstrated that jaceosidin activated the NF-κB-driven expression of a luciferase reporter gene and NF-κB binding to DNA. Jaceosidin-induced proliferation and migration of human umbilical vascular endothelial cells were strongly inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002 and NF-κB inhibitor BAY11-7082, indicating that the PI3K/AKT/NF-κB signaling pathway is involved in jaceosidin-induced angiogenesis. Our results suggest that jaceosidin stimulates angiogenesis by activating the VEGFR2/FAK/PI3K/AKT/NF-κB signaling pathway and that it may be useful in developing angiogenic agents to promote the growth of collateral blood vessels in ischemic tissues.

  13. Semaphorin 6A regulates angiogenesis by modulating VEGF signaling

    PubMed Central

    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

  14. PlGF and VEGF-A Regulate Growth of High-Risk MYCN-Single Copy Neuroblastoma Xenografts via Different Mechanisms

    PubMed Central

    Zins, Karin; Kovatchki, Daniel; Lucas, Trevor; Abraham, Dietmar

    2016-01-01

    Neuroblastoma (NB) is the most common extracranial solid tumor of childhood and is a rapidly growing, highly-vascularized cancer. NBs frequently express angiogenic factors and high tumor angiogenesis has been associated with poor outcomes. Placental growth factor (PlGF) is an angiogenic protein belonging to the vascular endothelial growth factor (VEGF) family and is up-regulated mainly in pathologic conditions. Recently, PlGF was identified as a member of a gene expression signature characterizing highly malignant NB stem cells drawing attention as a potential therapeutic target in NB. In the present study, we sought to investigate the expression of PlGF in NB patients and the effect of PlGF inhibition on high-risk MYCN-non-amplified SK-N-AS NB xenografts. Human SK-N-AS cells, which are poorly differentiated and express PlGF and VEGF-A, were implanted subcutaneously in athymic nude mice. Treatment was done by intratumoral injection of replication-incompetent adenoviruses (Ad) expressing PlGF- or VEGF-specific short hairpin (sh)RNA, or soluble (s)VEGF receptor 2 (VEGFR2). The effect on tumor growth and angiogenesis was analyzed. High PlGF expression levels were observed in human advanced-stage NBs. Down-regulating PlGF significantly reduced NB growth in established NB xenografts by reducing cancer cell proliferation but did not suppress angiogenesis. In contrast, blocking VEGF by administration of Ad(sh)VEGF and Ad(s)VEGFR2 reduced tumor growth associated with decreased tumor vasculature. These findings suggest that PlGF and VEGF-A modulate MYCN-non-amplified NB tumors by different mechanisms and support a role for PlGF in NB biology. PMID:27669225

  15. PlGF and VEGF-A Regulate Growth of High-Risk MYCN-Single Copy Neuroblastoma Xenografts via Different Mechanisms.

    PubMed

    Zins, Karin; Kovatchki, Daniel; Lucas, Trevor; Abraham, Dietmar

    2016-09-23

    Neuroblastoma (NB) is the most common extracranial solid tumor of childhood and is a rapidly growing, highly-vascularized cancer. NBs frequently express angiogenic factors and high tumor angiogenesis has been associated with poor outcomes. Placental growth factor (PlGF) is an angiogenic protein belonging to the vascular endothelial growth factor (VEGF) family and is up-regulated mainly in pathologic conditions. Recently, PlGF was identified as a member of a gene expression signature characterizing highly malignant NB stem cells drawing attention as a potential therapeutic target in NB. In the present study, we sought to investigate the expression of PlGF in NB patients and the effect of PlGF inhibition on high-risk MYCN-non-amplified SK-N-AS NB xenografts. Human SK-N-AS cells, which are poorly differentiated and express PlGF and VEGF-A, were implanted subcutaneously in athymic nude mice. Treatment was done by intratumoral injection of replication-incompetent adenoviruses (Ad) expressing PlGF- or VEGF-specific short hairpin (sh)RNA, or soluble (s)VEGF receptor 2 (VEGFR2). The effect on tumor growth and angiogenesis was analyzed. High PlGF expression levels were observed in human advanced-stage NBs. Down-regulating PlGF significantly reduced NB growth in established NB xenografts by reducing cancer cell proliferation but did not suppress angiogenesis. In contrast, blocking VEGF by administration of Ad(sh)VEGF and Ad(s)VEGFR2 reduced tumor growth associated with decreased tumor vasculature. These findings suggest that PlGF and VEGF-A modulate MYCN-non-amplified NB tumors by different mechanisms and support a role for PlGF in NB biology.

  16. Fibroblast Growth Factor Receptor 1 (FGFR1), Partly Related to Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) and Microvessel Density, is an Independent Prognostic Factor for Non-Small Cell Lung Cancer

    PubMed Central

    Pu, Dan; Liu, Jiewei; Li, Zhixi; Zhu, Jiang; Hou, Mei

    2017-01-01

    Background This study aimed to explore the correlation between FGFR1 and clinical features, including survival analysis and the promotion of angiogenesis by fibroblast growth factor receptor 1 (FGFR1) and vascular endothelial growth factor receptor 2 (VEGFR2). FGFR1 gene amplification has been found in non-small cell lung cancer (NSCLC). However, the prognostic value of FGFR1 and the correlation between FGFR1 and clinical features are still controversial. Material/Methods A total of 92 patients with NSCLC who underwent R0 resection between July 2006 and July 2008 were enrolled in the study. The expression of FGFR1, VEGFR2, and CD34 was detected by immunohistochemistry. The correlations between the aforementioned markers and the patients’ clinical features were analyzed by the chi-square test. The impact factors of prognosis were evaluated by Cox regression analyses. Results The expression ratios of FGFR1 and VEGFR2 were 26.1% and 43.4%, respectively. The intensity of FGFR1 expression was related to VEGFR2 and histopathology. To some extent, the average microvessel density (MVD) had correlation to the expression of FGFR1 and VGEFR2. The pathological stages III–IV and high expression of FGFR1 were found to be independent prognostic factors. Conclusions The expression intensity of FGFR1 and VEGFR2 was associated with MVD, and the expression of FGFR1 is one of the independent prognostic indicators for NSCLC. PMID:28088809

  17. Development of a robust reporter-based assay for the bioactivity determination of anti-VEGF therapeutic antibodies.

    PubMed

    Wang, Lan; Xu, Gang-Ling; Gao, Kai; Wilkinson, Jennifer; Zhang, Feng; Yu, Lei; Liu, Chun-Yu; Yu, Chuan-Fei; Wang, Wen-Bo; Li, Meng; Chen, Wei; Fan, Frank; Cong, Mei; Wang, Jun-Zhi

    2016-06-05

    Development of anti-VEGF based biologic agents has been a focus in cancer treatment for the past decades, and several anti-VEGF pharmaceuticals have been already approved for treatment of various medical indications especially in cancer. The first anti-angiogenic agent approved by FDA was bevacizumab (BVZ, trade name Avastin, Genentech/Roche), a humanized anti-VEGF monoclonal antibody. Accurate determination of bioactivity is crucial for the safety and efficacy of therapeutic antibodies. The current method widely used in the lot release and stability test for clinical trial batches of BVZ is anti-proliferation assay using primary human umbilical vein endothelial cells (HUVEC), which is tedious with high assay variations. We describe here the development and preliminary validation of a reporter gene assay (RGA) that is based on an HEK293 cell line stably expressing vascular endothelial growth factor receptor 2 (VEGFR-2), and a luciferase reporter under the control of nuclear factor activated T cell (NFAT) response elements. Our study shows this assay not only to be superior on precision, sensitivity and assay simplicity compared with HUVEC assay, but also applicable to other VEGF-targeted biotherapeutics. These results show for the first time that this new reporter assay, based on the VEGF-VEGFR-NFAT pathway, can be a viable supplement to the HUVEC assay and employed in potency determination of BVZ and other kinds of anti-VEGF antibody-based biotherapeutics. Copyright © 2016. Published by Elsevier B.V.

  18. Association of Dll4/Notch and HIF-1a -VEGF Signaling in the Angiogenesis of Missed Abortion

    PubMed Central

    Ma, Yuyan; Sun, Ping; Ma, Daoxin; Ji, Chunyan; Kong, Beihua

    2013-01-01

    Background Dll4/Notch and HIF-1a-VEGF have been shown to play an important role during angiogenesis, but there are no data about their roles and association in missed abortion. In this study, we investigated the association of Dll4/Notch and HIF-1a-VEGF signaling in missed abortion. Methods Women with missed abortion (n = 27) and healthy controls (n = 26) were included in the study. Real-time Reverse Transcription-PCR Analyses (RT-PCR) was used to analyze the mRNA levels of Dll4/Notch and HIF-1a-VEGF signaling molecules. The protein level for Dll4 was measured by immunohistochemistry. Results Compared with induced abortion, the expression of VEGF was statistically reduced while the level of VEGFR1 and Notch1 was significantly up-regulated in missed abortion. Though other molecules (VEGFR2 and Dll4) were marginally higher in missed abortion, no statistical difference was observed. The expression of HIF-1a was significantly up-regulated, and close negatively correlated with VEGF in missed abortion. Both in induced abortion and missed abortion, Dll4 was positively correlated with Notch1. Conclusions The early pregnancy is in a hypoxic environment, this may encourage the angiogenesis, but severe hypoxic may inhibit the angiogenesis. Aberrant Dll4/Notch and HIF-1a-VEGF signaling may have a role in missed abortion. PMID:23950980

  19. VEGF receptor inhibition blocks liver cyst growth in pkd2(WS25/-) mice.

    PubMed

    Amura, Claudia R; Brodsky, Kelley S; Groff, Rachel; Gattone, Vincent H; Voelkel, Norbert F; Doctor, R Brian

    2007-07-01

    Proliferation of cyst-lining epithelial cells is an integral part of autosomal dominant polycystic kidney disease (ADPKD) cyst growth. Cytokines and growth factors within cyst fluids are positioned to induce cyst growth. Vascular endothelial growth factor (VEGF) is a pleiotropic growth factor present in ADPKD liver cyst fluids (human 1,128 +/- 78, mouse 2,787 +/- 136 pg/ml) and, to a lesser extent, in ADPKD renal cyst fluids (human 294 +/- 41, mouse 191 +/- 90 pg/ml). Western blotting showed that receptors for VEGF (VEGFR1 and VEGFR2) were present in both normal mouse bile ducts and pkd2(WS25/-) liver cyst epithelial cells. Treatment of pkd2(WS25/-) liver cyst epithelial cells with VEGF (50-50,000 pg/ml) or liver cyst fluid induced a proliferative response. The effect on proliferation of liver cyst fluid was inhibited by SU-5416, a potent VEGF receptor inhibitor. Treatment of pkd2(WS25/-) mice between 4 and 8 mo of age with SU-5416 markedly reduced the cyst volume density of the liver (vehicle 9.9 +/- 4.3%, SU-5416 1.8 +/- 0.7% of liver). SU-5416 treatment between 4 and 12 mo of age markedly protected against increases in liver weight [pkd2(+/+) 4.8 +/- 0.2%, pkd2(WS25/-)-vehicle 10.8 +/- 1.9%, pkd2(WS25/-)-SU-5416 4.8 +/- 0.4% body wt]. The capacity of VEGF signaling to induce in vitro proliferation of pkd2(WS25/-) liver cyst epithelial cells and inhibition of in vivo VEGF signaling to retard liver cyst growth in pkd2(WS25/-) mice indicates that the VEGF signaling pathway is a potentially important therapeutic target in the treatment of ADPKD liver cyst disease.

  20. Albendazole inhibits endothelial cell migration, tube formation, vasopermeability, VEGF receptor-2 expression and suppresses retinal neovascularization in ROP model of angiogenesis

    SciTech Connect

    Pourgholami, Mohammad H.; Khachigian, Levon M.; Fahmy, Roger G.; Badar, Samina; Wang, Lisa; Chu, Stephanie Wai Ling; Morris, David Lawson

    2010-07-09

    The angiogenic process begins with the cell proliferation and migration into the primary vascular network, and leads to vascularization of previously avascular tissues and organs as well to growth and remodeling of the initially homogeneous capillary plexus to form a new microcirculation. Additionally, an increase in microvascular permeability is a crucial step in angiogenesis. Vascular endothelial growth factor (VEGF) plays a central role in angiogenesis. We have previously reported that albendazole suppresses VEGF levels and inhibits malignant ascites formation, suggesting a possible effect on angiogenesis. This study was therefore designed to investigate the antiangiogenic effect of albendazole in non-cancerous models of angiogenesis. In vitro, treatment of human umbilical vein endothelial cells (HUVECs) with albendazole led to inhibition of tube formation, migration, permeability and down-regulation of the VEGF type 2 receptor (VEGFR-2). In vivo albendazole profoundly inhibited hyperoxia-induced retinal angiogenesis in mice. These results provide new insights into the antiangiogenic effects of albendazole.

  1. The Increased Expression of Connexin and VEGF in Mouse Ovarian Tissue Vitrification by Follicle Stimulating Hormone.

    PubMed

    Yang, Yanzhou; Chen, Jie; Wu, Hao; Pei, Xiuying; Chang, Qing; Ma, Wenzhi; Ma, Huiming; Hei, Changchun; Zheng, Xiaomin; Cai, Yufang; Zhao, Chengjun; Yu, Jia; Wang, Yanrong

    2015-01-01

    Ovarian follicular damages were caused by cryoinjury during the process of ovarian vitrification and ischemia/reperfusion during the process of ovarian transplantation. And appropriate FSH plays an important role in antiapoptosis during ovarian follicle development. Therefore, in this study, 0.3 IU/mL FSH was administered into medium during mouse ovarian cryopreservation by vitrification to ascertain the function of FSH on ovarian vitrification and avascular transplantation. The results suggested that the expressions of Cx37, Cx43, apoptotic molecular caspase-3, and angiogenesis molecular VEGF were confirmed using immunohistochemistry, western blotting, and real-time PCR, and the results suggested that the treatment with FSH remarkably increased the number of morphologically normal follicles in vitrified/warmed ovaries by upregulating the expression of Cx37, Cx43, VEGF, and VEGF receptor 2, but downregulating the expression of caspase-3. In addition, the vitrified/warmed ovaries were transplanted, and the related fertility was analyzed, and the results suggested that the fertility, neoangiogenesis, and follicle reserve were remarkably increased in the FSH administrated group. Taken together, administration of 0.3 IU/mL FSH during ovarian cryopreservation by vitrification can maintain ovarian survival during ovarian vitrification and increases the blood supply with avascular transplantation via upregulation of Cx43, Cx37, and VEGF/VEGFR2, as well as through its antiapoptotic effects.

  2. ROS-induced ROS release orchestrated by Nox4, Nox2, and mitochondria in VEGF signaling and angiogenesis.

    PubMed

    Kim, Young-Mee; Kim, Seok-Jo; Tatsunami, Ryosuke; Yamamura, Hisao; Fukai, Tohru; Ushio-Fukai, Masuko

    2017-06-01

    Reactive oxygen species (ROS) derived from NADPH oxidase (NOX) and mitochondria play a critical role in growth factor-induced switch from a quiescent to an angiogenic phenotype in endothelial cells (ECs). However, how highly diffusible ROS produced from different sources can coordinate to stimulate VEGF signaling and drive the angiogenic process remains unknown. Using the cytosol- and mitochondria-targeted redox-sensitive RoGFP biosensors with real-time imaging, here we show that VEGF stimulation in human ECs rapidly increases cytosolic RoGFP oxidation within 1 min, followed by mitochondrial RoGFP oxidation within 5 min, which continues at least for 60 min. Silencing of Nox4 or Nox2 or overexpression of mitochondria-targeted catalase significantly inhibits VEGF-induced tyrosine phosphorylation of VEGF receptor type 2 (VEGFR2-pY), EC migration and proliferation at the similar extent. Exogenous hydrogen peroxide (H2O2) or overexpression of Nox4, which produces H2O2, increases mitochondrial ROS (mtROS), which is prevented by Nox2 siRNA, suggesting that Nox2 senses Nox4-derived H2O2 to promote mtROS production. Mechanistically, H2O2 increases S36 phosphorylation of p66Shc, a key mtROS regulator, which is inhibited by siNox2, but not by siNox4. Moreover, Nox2 or Nox4 knockdown or overexpression of S36 phosphorylation-defective mutant p66Shc(S36A) inhibits VEGF-induced mtROS, VEGFR2-pY, EC migration, and proliferation. In summary, Nox4-derived H2O2 in part activates Nox2 to increase mtROS via pSer36-p66Shc, thereby enhancing VEGFR2 signaling and angiogenesis in ECs. This may represent a novel feed-forward mechanism of ROS-induced ROS release orchestrated by the Nox4/Nox2/pSer36-p66Shc/mtROS axis, which drives sustained activation of angiogenesis signaling program. Copyright © 2017 the American Physiological Society.

  3. Hantaviruses direct endothelial cell permeability by sensitizing cells to the vascular permeability factor VEGF, while angiopoietin 1 and sphingosine 1-phosphate inhibit hantavirus-directed permeability.

    PubMed

    Gavrilovskaya, Irina N; Gorbunova, Elena E; Mackow, Natalie A; Mackow, Erich R

    2008-06-01

    Hantaviruses infect human endothelial cells and cause two vascular permeability-based diseases: hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Hantavirus infection alone does not permeabilize endothelial cell monolayers. However, pathogenic hantaviruses inhibit the function of alphav beta3 integrins on endothelial cells, and hemorrhagic disease and vascular permeability deficits are consequences of dysfunctional beta3 integrins that normally regulate permeabilizing vascular endothelial growth factor (VEGF) responses. Here we show that pathogenic Hantaan, Andes, and New York-1 hantaviruses dramatically enhance the permeability of endothelial cells in response to VEGF, while the nonpathogenic hantaviruses Prospect Hill and Tula have no effect on endothelial cell permeability. Pathogenic hantaviruses directed endothelial cell permeability 2 to 3 days postinfection, coincident with pathogenic hantavirus inhibition of alphav beta3 integrin functions, and hantavirus-directed permeability was inhibited by antibodies to VEGF receptor 2 (VEGFR2). These studies demonstrate that pathogenic hantaviruses, similar to alphav beta3 integrin-deficient cells, specifically enhance VEGF-directed permeabilizing responses. Using the hantavirus permeability assay we further demonstrate that the endothelial-cell-specific growth factor angiopoietin 1 (Ang-1) and the platelet-derived lipid mediator sphingosine 1-phosphate (S1P) inhibit hantavirus directed endothelial cell permeability at physiologic concentrations. These results demonstrate the utility of a hantavirus permeability assay and rationalize the testing of Ang-1, S1P, and antibodies to VEGFR2 as potential hantavirus therapeutics. The central importance of beta3 integrins and VEGF responses in vascular leak and hemorrhagic disease further suggest that altering beta3 or VEGF responses may be a common feature of additional viral hemorrhagic diseases. As a result, our findings provide a potential mechanism

  4. Pre-B cell colony enhancing factor (PBEF/NAMPT/Visfatin) and vascular endothelial growth factor (VEGF) cooperate to increase the permeability of the human placental amnion

    PubMed Central

    Astern, J.M.; Collier, A.C.; Kendal-Wright, C.E.

    2012-01-01

    Fluid efflux across the region of the amnion overlying the placenta is an essential component of the intramembranous absorption pathway that maintains amniotic fluid volume homeostasis. Dysregulation of this pathway may result in adverse pregnancy outcomes, however the factors controlling amnion permeability are unknown. Here, we report a novel mechanism that increases placental amnion permeability. Pre-B Cell Colony Enhancing Factor (PBEF) is a stress-responsive cytokine expressed by the human amnion, and is known to induce Vascular Endothelial Growth Factor (VEGF) production by other cell types. Interestingly, VEGF is up-regulated in the ovine amnion when intramembranous absorption is augmented. In this study, we show that PBEF induced VEGF secretion by primary human amniotic epithelial cells (AEC) derived from the placental amnion, as well as from the reflected amnion that lines the remainder of the gestational sac. Further, PBEF treatment led to the increased expression of VEGFR2 in placental AEC, but not reflected AEC. To test the hypothesis that PBEF and VEGF increase placental amnion permeability, we monitored the transfer of 2′,7′-dichlorofluorescein (DCF) from the fetal to the maternal side of human amnion explants. A treatment regimen including both PBEF and VEGF increased the rate of DCF transfer across the placental amnion, but not the reflected amnion. In summary, our results suggest that by augmenting VEGFR2 expression in the placental amnion, PBEF primes the tissue for a VEGF-mediated increase in permeability. This mechanism may have important implications in amniotic fluid volume control throughout gestation. PMID:23151382

  5. MDR1/P-gp and VEGF synergistically enhance the invasion of Hep-2 cells with multidrug resistance induced by taxol.

    PubMed

    Li, Li; Jiang, Alice C; Dong, Pin; Wang, Haibo; Xu, Wei; Xu, Chengzhi

    2009-05-01

    Tumor invasion/metastasis and multidrug resistance (MDR) are the main causes of treatment failure and high mortality in all kinds of cancer patients. The relationship between the two factors is still unclear. The aim of this study is to investigate the association between MDR and invasion, especially the role of multidrug resistance 1/P-glycoprotein (MDR1/P-gp) and vascular endothelial growth factor (VEGF) during the invasion. Multidrug resistance 1 (MDR1) and VEGF receptor 2 (VEGFR-2) were detected with real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) and Western blotting at the levels of messenger RNA (mRNA) and protein, respectively. RNA interference was applied to inhibit the expression of MDR1. The invasive assays were performed with the CHEMICON cell invasion assay kit. The MDR cell line induced by Taxol (Hep-2T cell) was more invasive than its parent cell line (Hep-2 cell), which was at least in part mediated through the overexpressed MDR1/P-pg. MDR1-targeted RNA interference could effectively inhibit the expression of MDR1 and obviously decrease the invasive ability. Synergistic enhancing effects existed between MDR1/P-gp and VEGF on the invasion of Hep-2T cells. The expression of VEGFR-2 was elevated in Hep-2T cells. SU1498 could significantly decrease the invasion of Hep-2T cells. MDR1-targeted RNA interference and SU1498 had synergistic decreasing effect on the invasion of Hep-2T cells. MDR1/P-pg may be a risk predictor for the invasion of laryngeal cancer. MDR1 knock down and VEGFR-2 inhibitor may be two promising treatment regiments for advanced laryngeal carcinoma patients with MDR and invasion/metastasis.

  6. Gold nanoparticle-conjugated quercetin inhibits epithelial-mesenchymal transition, angiogenesis and invasiveness via EGFR/VEGFR-2-mediated pathway in breast cancer.

    PubMed

    Balakrishnan, S; Bhat, F A; Raja Singh, P; Mukherjee, S; Elumalai, P; Das, S; Patra, C R; Arunakaran, J

    2016-12-01

    Epidermal growth factor plays a critical role in breast malignancies by enhancing cell proliferation, invasion, angiogenesis and metastasis. Epithelial-mesenchymal transition (EMT) is a crucial process by which epithelial cells lose polarity and acquire migratory mesenchymal properties. Gold nanoparticles are an efficient drug delivery vehicle for carrying chemotherapeutic agents to target cancer cells and quercetin is an anti-oxidative flavonoid known with potent anti-malignant cell activity. Cell viability was assessed by MTT assay, and protein expression was examined by Western blotting and immunocytochemistry. Cell invasion was monitored using invasion chambers, and cell migration was analysed by scratch wound-healing assay. In vitro and ex vivo angiogenesis studies were performed by capillary-like tube formation assay and chick embryo angiogenesis assay (CEA). 7,12-dimethylbenz(a)anthracene (DMBA) induced mammary carcinoma in Sprague-Dawley rats. We observed a significant reduction in protein expression of vimentin, N-cadherin, Snail, Slug, Twist, MMP-2, MMP-9, p-EGFR, VEGFR-2, p-PI3K, Akt and p-GSK3β, and enhanced E-cadherin protein expression in response to AuNPs-Qu-5 treatment. AuNPs-Qu-5 inhibited migration and invasion of MCF-7 and MDA-MB-231 cells compared to free quercetin. AuNPs-Qu-5-treated HUVECs had reduced cell viability and capillary-like tube formation. In vitro and in vivo angiogenesis assays showed that AuNPs-Qu-5 suppressed tube and new blood vessel formation. Treatment with AuNPs-Qu-5 impeded tumour growth in DMBA-induced mammary carcinoma in SD rats compared to treatment with free quercetin. Our results suggest that AuNPs-Qu-5 inhibited EMT, angiogenesis and metastasis of the breast cancer cells tested by targeting the EGFR/VEGFR-2 signalling pathway. © 2016 John Wiley & Sons Ltd.

  7. High blood sugar levels significantly impact the prognosis of colorectal cancer patients through down-regulation of microRNA-16 by targeting Myb and VEGFR2.

    PubMed

    Yang, I-Ping; Tsai, Hsiang-Lin; Huang, Ching-Wen; Lu, Chien-Yu; Miao, Zhi-Feng; Chang, Se-Fen; Juo, Suh-Hang Hank; Wang, Jaw-Yuan

    2016-04-05

    The high prevalence of type 2 diabetes mellitus in colorectal cancer patients is a crucial public health issue worldwide. The deregulation of microRNAs has been shown to be associated with the progression of CRC; however, the effects of high blood sugar levels on miR deregulation and, in turn, CRC remain unexplored. In this study, 520 CRC patients were classified into two groups according to their blood sugar levels (≧110 or <110 mg/dL). Clinicopathologic features, clinical outcomes, and serum miR-16 levels of the two groups were then analyzed, while cell cycles, cell proliferation, migration, and cellular miR-16 expression were investigated via D-(+)-glucose administration. Additionally, the target genes of miR-16 were identified. Through multivariate analysis, both the disease-free survival and overall survival of the CRC patients were found to be associated with the UICC stage, perineural invasion, and blood glucose levels (P < 0.05). Serum miR-16 levels were significantly lower in the high blood glucose patients than in the normal blood glucose patients (P = 0.0329). With D-(+)-glucose administration, the proliferation and migration of CRC cells in vitro increased remarkably (P < 0.05), while their accumulation in the G1 phase decreased significantly. Cellular miR-16 expression was suppressed by D-(+)-glucose administration. The expression levels of two target genes, Myb and VEGFR2, were affected significantly by miR-16, while glucose administration inhibited miR-16 expression and enhanced tumor cell proliferation and migration. Hyperglycemia can impact the clinical outcomes of CRC patients, likely by inhibiting miR-16 expression and the expression of its downstream genes Myb and VEGFR2.

  8. Inhibition of vascular endothelial growth factor-induced angiogenesis by scopoletin through interrupting the autophosphorylation of VEGF receptor 2 and its downstream signaling pathways.

    PubMed

    Pan, Rong; Dai, Yue; Gao, Xing-Hua; Lu, Dan; Xia, Yu-Feng

    2011-01-01

    Our previous studies revealed that scopoletin, the main bioactive constituent of Erycibe obtusifolia Benth stems, exerted anti-arthritic activity in vivo partly by preventing synovial angiogenesis. Herein we further investigated the anti-angiogenic potential and related mechanisms of this coumarin compound in vivo and in vitro. On chick chorioallantoic membrane (CAM) model, scopoletin (10, 30, 100 nmol/egg) dose-dependently reduced the blood vessels that were quantified by counting the number of blood vessel branch points. In vitro, scopoletin at concentrations above 30 microM obviously inhibited the VEGF-induced tube formation, proliferation and migration of human umbilical vein endothelial cells (HUVECs). Furthermore, scopoletin was shown to block VEGF-induced autophosphorylation of VEGFR2 but not VEGFR1, and down-regulate the following activation of ERK1/2, p38 MAPK and endothelial nitric oxide synthase (eNOS) as well as the production of nitric oxide (NO) in HUVECs. In sum, our findings further support that scopoletin is a candidate of angiogenesis inhibitors, and it functions by interrupting the autophosphorylation of VEGF receptor 2 (VEGFR2) and the downstream signaling pathways. Copyright © 2010 Elsevier Inc. All rights reserved.

  9. Low Energy Shock Wave Therapy Induces Angiogenesis in Acute Hind-Limb Ischemia via VEGF Receptor 2 Phosphorylation

    PubMed Central

    Holfeld, Johannes; Tepeköylü, Can; Blunder, Stefan; Lobenwein, Daniela; Kirchmair, Elke; Dietl, Marion; Kozaryn, Radoslaw; Lener, Daniela; Theurl, Markus; Paulus, Patrick; Kirchmair, Rudolf; Grimm, Michael

    2014-01-01

    Objectives Low energy shock waves have been shown to induce angiogenesis, improve left ventricular ejection fraction and decrease angina symptoms in patients suffering from chronic ischemic heart disease. Whether there is as well an effect in acute ischemia was not yet investigated. Methods Hind-limb ischemia was induced in 10–12 weeks old male C57/Bl6 wild-type mice by excision of the left femoral artery. Animals were randomly divided in a treatment group (SWT, 300 shock waves at 0.1 mJ/mm2, 5 Hz) and untreated controls (CTR), n = 10 per group. The treatment group received shock wave therapy immediately after surgery. Results Higher gene expression and protein levels of angiogenic factors VEGF-A and PlGF, as well as their receptors Flt-1 and KDR have been found. This resulted in significantly more vessels per high-power field in SWT compared to controls. Improvement of blood perfusion in treatment animals was confirmed by laser Doppler perfusion imaging. Receptor tyrosine kinase profiler revealed significant phosphorylation of VEGF receptor 2 as an underlying mechanism of action. The effect of VEGF signaling was abolished upon incubation with a VEGFR2 inhibitor indicating that the effect is indeed VEGFR 2 dependent. Conclusions Low energy shock wave treatment induces angiogenesis in acute ischemia via VEGF receptor 2 stimulation and shows the same promising effects as known from chronic myocardial ischemia. It may therefore develop as an adjunct to the treatment armentarium of acute muscle ischemia in limbs and myocardium. PMID:25093816

  10. Enhancement of angiogenesis by a 27 kDa lectin from perivitelline fluid of horseshoe crab embryos through upregulation of VEGF and its receptor.

    PubMed

    Surekha, K L; Waghchoude, Meenal; Ghaskadbi, Surendra

    2013-01-25

    Angiogenesis, the expansion of a capillary network, is implicated in several pathological conditions. Drug-based inhibition of angiogenesis is being explored as therapy. Conversely, therapeutic angiogenesis contributes to control conditions such as ischemia. Here we report pro-angiogenic activity of perivitelline fluid (PVF) from Indian horseshoe crab embryos and one of its purified fractions, a 27 kDa lectin, using the chick embryonic chorioallantoic membrane assay. Enhancement in number and diameter of blood vessels after treatment with PVF and lectin suggested their pro-angiogenic effect. Quantitative RT-PCR showed that this effect is mediated through modulation of expression of VEGF and VEGFR-2/kinase domain receptor genes.

  11. Suppression of alpha-tocopherol ether-linked acetic acid in VEGF-induced angiogenesis and the possible mechanisms in human umbilical vein endothelial cells

    SciTech Connect

    Chuang, Cheng-Hung; Liu, Chia-Hua; Lu, Ta-Jung; Hu, Miao-Lin

    2014-12-15

    Alpha-tocopherol ether-linked acetic acid (α-TEA) has been reported to exhibit both anti-tumor and anti-metastatic activities in cell culture and animal studies. However, it is unclear whether α-TEA possesses anti-angiogenic effects. In this study, we investigated the effect of α-TEA on vascular endothelial growth factor (VEGF)-induced angiogenesis and matrix metalloproteinase (MMP) expression both in vitro and ex vivo. We found that the α-TEA inhibited tube formation, invasion, and migration in human umbilical vein endothelial cells (HUVECs) and that such actions were accompanied by reduced expression of MMP-2. α-TEA also inhibited ex vivo angiogenesis, as indicated by chicken egg chorioallantoic membrane assay. We further showed that α-TEA attenuated protein expression of VEGF receptor-2 (VEGFR-2)-mediated p38 mitogen-activated protein kinase (p38), phosphorylated p38, and focal adhesion kinase (FAK). Moreover, α-TEA (30 μM) significantly up-regulated protein expression of tissue inhibitors of MMP (TIMP)-2 (by 138%) and the metastasis suppressor gene nm23-H1 (by 54%). These results demonstrate that the anti-angiogenic effect of α-TEA both in vitro and ex vivo and its possible mechanistic action appears to involve the inhibition of MMP-2 level through VEGFR-2-mediated FAK and p38 signaling pathways and through up-regulation of TIMP-2 and nm23-H1 expression. - Graphical abstract: Possible mechanisms of α-TEA on inhibited angiogenesis of human umbilical vein endothelial cells. Brief summary In the present study, we have demonstrated that VEGF-mediated angiogenesis is significantly inhibited by α-TEA, and that this effect involves inhibition of MMP-2 level through VEGFR-2-mediated FAK and p38 signaling pathways related to invasion and migration. - Highlights: • The anti-angiogenic effect and the mechanistic action of α-TEA were investigated. • α-TEA significantly inhibited VEGF-mediated angiogenesis both in vitro and ex vivo. • α-TEA down

  12. Gremlin: vexing VEGF receptor agonist.

    PubMed

    Claesson-Welsh, Lena

    2010-11-04

    Gremlins are mischievous creatures in English folklore, believed to be the cause of otherwise unexplainable breakdowns (the word gremlins is derived from the Old English "gremian" or "gremman," "to vex"). Gremlin (or Gremlin-1) is also the designation of a secreted protein that is known to regulate bone formation during development. In this issue of Blood, Mitola et al report the novel role of Gremlin as a VEGFR2 agonist and the function of the Gremlin protein seems vexing indeed.

  13. Sulfated polysaccharides identified as inducers of neuropilin-1 internalization and functional inhibition of VEGF165 and semaphorin3A

    PubMed Central

    Narazaki, Masashi; Segarra, Marta

    2008-01-01

    Neuropilin-1 (NRP1) and NRP2 are cell surface receptors shared by class 3 semaphorins and vascular endothelial growth factor (VEGF). Ligand interaction with NRPs selects the specific signal transducer, plexins for semaphorins or VEGF receptors for VEGF, and promotes NRP internalization, which effectively shuts down receptor-mediated signaling by a second ligand. Here, we show that the sulfated polysaccharides dextran sulfate and fucoidan, but not others, reduce endothelial cell-surface levels of NRP1, NRP2, and to a lesser extent VEGFR-1 and VEGFR-2, and block the binding and in vitro function of semaphorin3A and VEGF165. Administration of fucoidan to mice reduces VEGF165-induced angiogenesis and tumor neovascularization in vivo. We find that dextran sulfate and fucoidan can bridge the extracellular domain of NRP1 to that of the scavenger receptor expressed by endothelial cells I (SREC-I), and induce NRP1 and SREC-I coordinate internalization and trafficking to the lysosomes. Overexpression of SREC-I in SREC-I–negative cells specifically reduces cell-surface levels of NRP1, indicating that SREC-I mediates NRP1 internalization. These results demonstrate that engineered receptor internalization is an effective strategy for reducing levels and function of cell-surface receptors, and identify certain sulfated polysaccharides as “internalization inducers.” PMID:18272814

  14. Gold Nanoparticles Inhibit VEGF165-Induced Migration and Tube Formation of Endothelial Cells via the Akt Pathway

    PubMed Central

    Pan, Yunlong; Wu, Qing; Qin, Li; Cai, Jiye; Du, Bin

    2014-01-01

    The early stages of angiogenesis can be divided into three steps: endothelial cell proliferation, migration, and tube formation. Vascular endothelial growth factor (VEGF) is considered the most important proangiogenic factor; in particular, VEGF165 plays a critical role in angiogenesis. Here, we evaluated whether gold nanoparticles (AuNPs) could inhibit the VEGF165-induced human umbilical vein endothelial cell (HUVEC) migration and tube formation. AuNPs and VEGF165 were coincubated overnight at 4°C, after which the effects on cell migration and tube formation were assessed. Cell migration was assessed using a modified wound-healing assay and a transwell chamber assay; tube formation was assessed using a capillary-like tube formation assay and a chick chorioallantoic membrane (CAM) assay. We additionally detected the cell surface morphology and ultrastructure using atomic force microscopy (AFM). Furthermore, Akt phosphorylation downstream of VEGFR-2/PI3K in HUVECs was determined in a Western blot analysis. Our study demonstrated that AuNPs significantly inhibited VEGF165-induced HUVEC migration and tube formation by affecting the cell surface ultrastructure, cytoskeleton and might have inhibited angiogenesis via the Akt pathway. PMID:24987682

  15. Galectin-3 disruption impaired tumoral angiogenesis by reducing VEGF secretion from TGFβ1-induced macrophages

    PubMed Central

    Machado, Camila Maria Longo; Andrade, Luciana Nogueira Sousa; Teixeira, Verônica Rodrigues; Costa, Fabrício Falconi; Melo, Camila Morais; dos Santos, Sofia Nascimento; Nonogaki, Suely; Liu, Fu-Tong; Bernardes, Emerson Soares; Camargo, Anamaria Aranha; Chammas, Roger

    2014-01-01

    In order to study the role of galectin-3 in tumor angiogenesis associated with tumor-associated macrophages (TAM) and tumor parenchyma, the galectin-3 expression was reconstituted in Tm1 melanoma cell line that lacks this protein. Galectin-3-expressing cells (Tm1G3) and mock-vector transfected cells (Tm1N3) were injected into wild-type (WT) and galectin-3 knockout (KO) C57Bl/6 mice. Tumors originated from Tm1G3 were larger in tumor volume with enlarged functional vessels, decreased necrotic areas, and increased vascular endothelial growth factor (VEGF) protein levels. Galectin-3-nonexpressing-cells injected into WT and KO showed increased levels of transforming growth factor beta 1 (TGFβ1) and, in WT animals this feature was also accompanied by increased VEGFR2 expression and its phosphorylation. In KO animals, tumors derived from galectin-3-expressing cells were infiltrated by CD68+-cells, whereas in tumors derived from galectin-3-nonexpressing-cells, CD68+ cells failed to infiltrate tumors and accumulated in the periphery of the tumor mass. In vitro studies showed that Tm1G3 secreted more VEGF than Tm1N3 cells. In the latter case, TGFβ1 induced VEGF production. Basal secretion of VEGF was higher in WT-bone marrow-derived macrophages (BMDM) than in KO-BMDM. TGFβ1 induced secretion of VEGF only in WT-BMDM. Tm1G3-induced tumors had the Arginase I mRNA increased, which upregulated alternative macrophage (M2)/TAM induction. M2 stimuli, such as interleukin-4 (IL4) and TGFβ1, increased Arginase I protein levels and galectin-3 expression in WT- BMDM, but not in cells from KO mice. Hence, we report that galectin-3 disruption in tumor stroma and parenchyma decreases angiogenesis through interfering with the responses of macrophages to the interdependent VEGF and TGFβ1 signaling pathways. PMID:24421272

  16. VEGF-A and Semaphorin3A: Modulators of vascular sympathetic innervation

    PubMed Central

    Long, Jennifer B.; Jay, Steven M.; Segal, Steven S.; Madri, Joseph A.

    2010-01-01

    Sympathetic nerve activity regulates blood pressure by altering peripheral vascular resistance. Variations in vascular sympathetic innervation suggest that vascular-derived cues promote selective innervation of particular vessels during development. As axons extend towards peripheral targets, they migrate along arterial networks following gradients of guidance cues. Collective ratios of these gradients may determine whether axons grow towards and innervate vessels or continue past non-innervated vessels towards peripheral targets. Utilizing directed neurite outgrowth in a three-dimensional (3D) co-culture, we observed increased axon growth from superior cervical ganglion explants (SCG) towards innervated compared to non-innervated vessels, mediated in part by vascular endothelial growth factor (VEGF-A) and Semaphorin3A (Sema3A) which both signal via neuropilin-1 (Nrp1). Exogenous VEGF-A, delivered by high-expressing VEGF-A–LacZ vessels or by rhVEGF-A/alginate spheres, increased sympathetic neurite outgrowth while exogenous rhSema3A/Fc decreased neurite outgrowth. VEGF-A expression is similar between the innervated and non-innervated vessels examined. Sema3A expression is higher in non-innervated vessels. Spatial gradients of Sema3A and VEGF-A may promote differential Nrp1 binding. Vessels expressing high levels of Sema3A favor Nrp1-PlexinA1 signaling, producing chemorepulsive cues limiting sympathetic neurite outgrowth and vascular innervation; while low Sema3A expressing vessels favor Nrp1-VEGFR2 signaling providing chemoattractive cues for sympathetic neurite outgrowth and vascular innervation. PMID:19631637

  17. VEGF-A promotes IL-17A-producing γδ T cell accumulation in mouse skin and serves as a chemotactic factor for plasmacytoid dendritic cells.

    PubMed

    Suzuki, Takahiro; Hirakawa, Satoshi; Shimauchi, Takatoshi; Ito, Taisuke; Sakabe, Jun-ichi; Detmar, Michael; Tokura, Yoshiki

    2014-05-01

    IL-17-producing CD4(+) T (Th17) cells and their cytokines, IL-17A and IL-22, are deeply involved in the pathogenesis of psoriasis by stimulating epidermal keratinocytes to proliferate and to produce cytokines/chemokines and vascular endothelial growth factor (VEGF)-A. Plasmacytoid dendritic cells (pDCs), infiltrating in psoriatic lesions, are known to exacerbate the Th17-mediated pathogenesis of psoriasis. To address the initiative role of VEGF-A in the development of psoriasis and the pDC accumulation. Numerical changes and VEGF receptor 1 (VEGFR1) and VEGFR2 expressions were investigated in skin-infiltrating T cells and pDCs of K14-VEGF-A transgenic (Tg) and wild type (WT) mice. The chemotactic properties of VEGF-A for purified splenic pDCs were also evaluated by real-time chemotaxis assay. By flow cytometry and immunohistochemistry, we observed that the number of dermal IL-17A(+) γδ T cells, but not CD4(+) T cells, was increased in VEGF-A Tg mice, suggesting that the main source of IL-17A was γδ T cells. Moreover, we identified pDCs as 440c(+) cells by immunohistochemistry and as PDCA-1(+)B220(+) cells by flow cytometry, and found that pDCs infiltrated at a higher frequency in VEGF-A Tg than WT mice. pDCs, but not γδ T cells, isolated from the skin expressed VEGFR1 and VEGFR2. Freshly isolated splenic pDCs expressed both receptors after 48-h cultivation. pDCs did not produce cytokines in response to VEGF-A, however, they had a strong velocity of chemotaxis toward VEGF-A at a comparable level to chemerin. These findings suggest that VEGF-A functions as not only a downstream enhancer but also an upstream initiator by chemoattracting pDCs in psoriatic lesions. Copyright © 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

  18. Cellular distribution of vascular endothelial growth factor A (VEGFA) and B (VEGFB) and VEGF receptors 1 and 2 in focal cortical dysplasia type IIB

    PubMed Central

    Boer, Karin; Troost, Dirk; Spliet, Wim G. M.; van Rijen, Peter C.; Gorter, Jan A.

    2008-01-01

    Members of the vascular endothelial growth factor (VEGF) family are key signaling proteins in the induction and regulation of angiogenesis, both during development and in pathological conditions. However, signaling mediated through VEGF family proteins and their receptors has recently been shown to have direct effects on neurons and glial cells. In the present study, we immunocytochemically investigated the expression and cellular distribution of VEGFA, VEGFB, and their associated receptors (VEGFR-1 and VEGFR-2) in focal cortical dysplasia (FCD) type IIB from patients with medically intractable epilepsy. Histologically normal temporal cortex and perilesional regions displayed neuronal immunoreactivity (IR) for VEGFA, VEGFB, and VEGF receptors (VEGFR-1 and VEGFR-2), mainly in pyramidal neurons. Weak IR was observed in blood vessels and there was no notable glial IR within the grey and white matter. In all FCD specimens, VEGFA, VEGFB, and both VEGF receptors were highly expressed in dysplastic neurons. IR in astroglial and balloon cells was observed for VEGFA and its receptors. VEGFR-1 displayed strong endothelial staining in FCD. Double-labeling also showed expression of VEGFA, VEGFB and VEGFR-1 in cells of the microglia/macrophage lineage. The neuronal expression of both VEGFA and VEGFB, together with their specific receptors in FCD, suggests autocrine/paracrine effects on dysplastic neurons. These autocrine/paracrine effects could play a role in the development of FCD, preventing the death of abnormal neuronal cells. In addition, the expression of VEGFA and its receptors in glial cells within the dysplastic cortex indicates that VEGF-mediated signaling could contribute to astroglial activation and associated inflammatory reactions. PMID:18317782

  19. Precise Scheduling of Chemotherapy Primes VEGF-producing Tumors for Successful Systemic Oncolytic Virotherapy

    PubMed Central

    Kottke, Timothy; Chester, John; Ilett, Elizabeth; Thompson, Jill; Diaz, Rosa; Coffey, Matt; Selby, Peter; Nuovo, Gerard; Pulido, Jose; Mukhopadhyay, Debabrata; Pandha, Hardev; Harrington, Kevin; Melcher, Alan; Vile, Richard

    2011-01-01

    We have previously reported that a burst of vascular endothelial growth factor (VEGF) signaling to tumor-associated endothelium induces a proviral state, during which systemically delivered oncolytic reovirus can replicate in endothelium, thereby inducing immune-mediated vascular collapse and significant antitumor therapy. Using chimeric receptors, we show here that induction of the proviral state proceeds through VEGFR2, but not VEGFR1, signaling in endothelial cells. In contrast, innate immune activation by reovirus-exposed endothelial cells was predominantly through VEGFR1. By screening conventional chemotherapies for their ability to induce similar effects in combination with reovirus both in vitro and in vivo, we observed that the proviral state could also be induced in endothelial cells exposed to VEGF during rebound from paclitaxel-mediated inhibition of VEGF signaling. We translated these in vitro findings in vivo by careful scheduling of paclitaxel chemotherapy with systemic virotherapy, neither of which alone had therapeutic effects against B16 tumors. Systemic availability of reovirus during endothelial cell recovery from paclitaxel treatment allowed for endothelial replication of the virus, immune-mediated therapy, and tumor cures. Therefore, careful scheduling of combination viro- and chemotherapies, which preclinical testing suggests are individually ineffective against tumor cells, can lead to rational new clinical protocols for systemic treatments with oncolytic viruses. PMID:21792179

  20. Precise scheduling of chemotherapy primes VEGF-producing tumors for successful systemic oncolytic virotherapy.

    PubMed

    Kottke, Timothy; Chester, John; Ilett, Elizabeth; Thompson, Jill; Diaz, Rosa; Coffey, Matt; Selby, Peter; Nuovo, Gerard; Pulido, Jose; Mukhopadhyay, Debabrata; Pandha, Hardev; Harrington, Kevin; Melcher, Alan; Vile, Richard

    2011-10-01

    We have previously reported that a burst of vascular endothelial growth factor (VEGF) signaling to tumor-associated endothelium induces a proviral state, during which systemically delivered oncolytic reovirus can replicate in endothelium, thereby inducing immune-mediated vascular collapse and significant antitumor therapy. Using chimeric receptors, we show here that induction of the proviral state proceeds through VEGFR2, but not VEGFR1, signaling in endothelial cells. In contrast, innate immune activation by reovirus-exposed endothelial cells was predominantly through VEGFR1. By screening conventional chemotherapies for their ability to induce similar effects in combination with reovirus both in vitro and in vivo, we observed that the proviral state could also be induced in endothelial cells exposed to VEGF during rebound from paclitaxel-mediated inhibition of VEGF signaling. We translated these in vitro findings in vivo by careful scheduling of paclitaxel chemotherapy with systemic virotherapy, neither of which alone had therapeutic effects against B16 tumors. Systemic availability of reovirus during endothelial cell recovery from paclitaxel treatment allowed for endothelial replication of the virus, immune-mediated therapy, and tumor cures. Therefore, careful scheduling of combination viro- and chemotherapies, which preclinical testing suggests are individually ineffective against tumor cells, can lead to rational new clinical protocols for systemic treatments with oncolytic viruses.

  1. A polymer nanoparticle with engineered affinity for a vascular endothelial growth factor (VEGF165)

    NASA Astrophysics Data System (ADS)

    Koide, Hiroyuki; Yoshimatsu, Keiichi; Hoshino, Yu; Lee, Shih-Hui; Okajima, Ai; Ariizumi, Saki; Narita, Yudai; Yonamine, Yusuke; Weisman, Adam C.; Nishimura, Yuri; Oku, Naoto; Miura, Yoshiko; Shea, Kenneth J.

    2017-07-01

    Protein affinity reagents are widely used in basic research, diagnostics and separations and for clinical applications, the most common of which are antibodies. However, they often suffer from high cost, and difficulties in their development, production and storage. Here we show that a synthetic polymer nanoparticle (NP) can be engineered to have many of the functions of a protein affinity reagent. Polymer NPs with nM affinity to a key vascular endothelial growth factor (VEGF165) inhibit binding of the signalling protein to its receptor VEGFR-2, preventing receptor phosphorylation and downstream VEGF165-dependent endothelial cell migration and invasion into the extracellular matrix. In addition, the NPs inhibit VEGF-mediated new blood vessel formation in Matrigel plugs in vivo. Importantly, the non-toxic NPs were not found to exhibit off-target activity. These results support the assertion that synthetic polymers offer a new paradigm in the search for abiotic protein affinity reagents by providing many of the functions of their protein counterparts.

  2. Tubeimoside-1 suppresses tumor angiogenesis by stimulation of proteasomal VEGFR2 and Tie2 degradation in a non-small cell lung cancer xenograft model

    PubMed Central

    Gu, Yuan; Körbel, Christina; Scheuer, Claudia; Nenicu, Anca; Menger, Michael D.; Laschke, Matthias W.

    2016-01-01

    Tubeimoside-1 (TBMS1) is a potent anti-tumor phytochemical. Its functional and molecular mode of action, however, remains elusive so far. Since angiogenesis is essential for tumor progression and metastasis, we herein investigated the anti-angiogenic effects of the compound. In a non-small cell lung cancer (NSCLC) xenograft model we found that treatment of CD1 nu/nu mice with TBMS1 (5mg/kg) significantly suppressed the growth and vascularization of NCI-H460 flank tumors. Moreover, TBMS1 dose-dependently reduced vascular sprouting in a rat aortic ring assay. In vitro, TBMS1 induced endothelial cell apoptosis without decreasing the viability of NSCLC tumor cells and inhibited the migration of endothelial cells by disturbing their actin filament organization. TBMS1 further stimulated the proteasomal degradation of vascular endothelial growth factor receptor-2 (VEGFR2) and Tie2 in endothelial cells, which down-regulated AKT/mTOR signaling. These findings indicate that TBMS1 represents a novel phytochemical for anti-angiogenic treatment of cancer and other angiogenesis-related diseases. PMID:26701724

  3. A phospholipid-PEG2000 conjugate of a vascular endothelial growth factor receptor 2 (VEGFR2)-targeting heterodimer peptide for contrast-enhanced ultrasound imaging of angiogenesis.

    PubMed

    Pillai, R; Marinelli, E R; Fan, H; Nanjappan, P; Song, B; von Wronski, M A; Cherkaoui, S; Tardy, I; Pochon, S; Schneider, M; Nunn, A D; Swenson, R E

    2010-03-17

    The transition of a targeted ultrasound contrast agent from animal imaging to testing in clinical studies requires considerable chemical development. The nature of the construct changes from an agent that is chemically attached to microbubbles to one where the targeting group is coupled to a phospholipid, for direct incorporation to the bubble surface. We provide an efficient method to attach a heterodimeric peptide to a pegylated phospholipid and show that the resulting construct retains nanomolar affinity for its target, vascular endothelial growth factor receptor 2 (VEGFR2), for both the human (kinase insert domain-containing receptor - KDR) and the mouse (fetal liver kinase 1 - Flk-1) receptors. The purified phospholipid-PEG-peptide isolated from TFA-based eluents is not stable with respect to hydrolysis of the fatty ester moieties. This leads to the time-dependent formation of the lysophospholipid and the phosphoglycerylamide derived from the degradation of the product. Purification of the product using neutral eluent systems provides a stable product. Methods to prepare the lysophospholipid (hydrolysis product) are also included. Biacore binding data demonstrated the retention of binding of the lipopeptide to the KDR receptor. The phospholipid-PEG2000-peptide is smoothly incorporated into gas-filled microbubbles and provides imaging of angiogenesis in a rat tumor model.

  4. VEGFR2 targeted antibody fused with MICA stimulates NKG2D mediated immunosurveillance and exhibits potent anti-tumor activity against breast cancer

    PubMed Central

    Wang, Youfu; Ren, Xueyan; Wang, Tong; Chen, Zhiguo; Tang, Mingying; Sun, Fumou; Li, Zhaoting; Wang, Min; Zhang, Juan

    2016-01-01

    Binding of MHC class I-related chain molecules A and B (MICA/B) to the natural killer (NK) cell receptor NK group 2, member D (NKG2D) is thought critical for activating NK-mediated immunosurveillance. Angiogenesis is important for tumor growth and interfering with angiogenesis using the fully human IgG1 anti-VEGFR2 (vascular endothelial growth factor receptor 2) antibody (mAb04) can be effective in treating malignancy. In an effort to make mAb04 more effective we have generated a novel antibody fusion protein (mAb04-MICA) consisting of mAb04 and MICA. We found that mAb04-MICA maintained the anti-angiogenic and antineoplastic activities of mAb04, and also enhanced immunosurveillance activated by the NKG2D pathway. Moreover, in human breast tumor-bearing nude mice, mAb04-MICA demonstrated superior anti-tumor efficacy compared to combination therapy of mAb04 + Docetaxel or Avastin + Docetaxel, highlighting the immunostimulatory effect of MICA. In conclusion, mAb04-MICA provided new inspiration for anti-tumor treatment and had prospects for clinical application. PMID:26909862

  5. TP53 Alterations Correlate with Response to VEGF/VEGFR Inhibitors: Implications for Targeted Therapeutics.

    PubMed

    Wheler, Jennifer J; Janku, Filip; Naing, Aung; Li, Yali; Stephen, Bettzy; Zinner, Ralph; Subbiah, Vivek; Fu, Siqing; Karp, Daniel; Falchook, Gerald S; Tsimberidou, Apostolia M; Piha-Paul, Sarina; Anderson, Roosevelt; Ke, Danxia; Miller, Vincent; Yelensky, Roman; Lee, J Jack; Hong, David; Kurzrock, Razelle

    2016-10-01

    TP53 tumor-suppressor gene mutations are among the most frequent abnormalities in cancer, affecting approximately 40% of patients. Yet, there is no accepted way to target these alterations in the clinic. At the same time, antagonists of VEGFR or its ligand are best-selling oncology drugs, with multiple, expensive compounds approved. Although only a subset of patients benefit from these antiangiogenesis agents, no relevant biomarker has been identified. Interestingly, TP53 mutations upregulate VEGF-A and VEGFR2. We prospectively enrolled 500 patients, to be interrogated by comprehensive genomic profiling (CGP) (next-generation sequencing, 236 genes), and to be matched, whenever possible, with targeted agents. Herein, we analyze outcomes based on VEGF/VEGFR inhibitor treatment and presence of TP53 mutations. Of the 500 patients, 188 (37.6%; with ≥1 alteration) were treated; 106 (56% of 188) had tumors that harbored TP53 mutations. VEGF/VEGFR inhibitor therapy was independently associated with improvement in all outcome parameters [rate of stable disease (SD) ≥6 months/partial and complete remission (PR/CR); (31% versus 7%; TP53-mutant patients (who received no other molecular-matched agents) treated with versus without VEGF/VEGFR inhibitors), time-to-treatment failure, and overall survival (multivariate analysis: all P ≤ 0.01)] for the patients harboring TP53-mutant cancers, but improvement was not seen in any of these parameters for patients with TP53 wild-type neoplasms. We conclude that TP53 mutations predict sensitivity to VEGF/VEGFR inhibitors in the clinic. TP53 alterations may therefore be a ready biomarker for treatment with antiangiogenesis agents, a finding of seminal importance across the cancer field. Mol Cancer Ther; 15(10); 2475-85. ©2016 AACR.

  6. The P2Y2 Receptor Interacts with VE-Cadherin and VEGF Receptor-2 to Regulate Rac1 Activity in Endothelial Cells

    PubMed Central

    Liao, Zhongji; Cao, Chen; Wang, Jianjie; Huxley, Virginia H.; Baker, Olga; Weisman, Gary A.

    2015-01-01

    Vascular endothelial cadherin (VE-cadherin) mediates homophylic adhesion between endothelial cells and is an important regulator of angiogenesis, blood vessel permeability and leukocyte trafficking. Rac1, a member of the Rho family of GTPases, controls VE-cadherin adhesion by acting downstream of several growth factors, including angiopoietin-1 and vascular endothelial growth factor (VEGF). Here we show that UTP-induced activation of the Gq protein-coupled P2Y2 nucleotide receptor (P2Y2R) in human coronary artery endothelial cells (HCAECs) activated Rac1 and caused a transient complex to form between P2Y2R, VE-cadherin and VEGF receptor-2 (VEGFR-2). Knockdown of VE-cadherin expression with siRNA did not affect UTP-induced activation of extracellular signal-regulated kinases 1/2 (ERK1/2) but led to a loss of UTP-induced Rac1 activation and tyrosine phosphorylation of p120 catenin, a cytoplasmic protein known to interact with VE-cadherin. Activation of the P2Y2R by UTP also caused a prolonged interaction between p120 catenin and vav2 (a guanine nucleotide exchange factor for Rac) that correlated with the kinetics of UTP-induced tyrosine phosphorylation of p120 catenin and VE-cadherin. Inhibitors of VEGFR-2 (SU1498) or Src (PP2) significantly diminished UTP-induced Rac1 activation, tyrosine phosphorylation of p120 catenin and VE-cadherin, and association of the P2Y2R with VE-cadherin and p120 catenin with vav2. These findings suggest that the P2Y2R uses Src and VEGFR-2 to mediate association of the P2Y2R with VE-cadherin complexes in endothelial adherens junctions to activate Rac1. PMID:25657827

  7. The P2Y2 Receptor Interacts with VE-Cadherin and VEGF Receptor-2 to Regulate Rac1 Activity in Endothelial Cells.

    PubMed

    Liao, Zhongji; Cao, Chen; Wang, Jianjie; Huxley, Virginia H; Baker, Olga; Weisman, Gary A; Erb, Laurie

    2014-12-01

    Vascular endothelial cadherin (VE-cadherin) mediates homophylic adhesion between endothelial cells and is an important regulator of angiogenesis, blood vessel permeability and leukocyte trafficking. Rac1, a member of the Rho family of GTPases, controls VE-cadherin adhesion by acting downstream of several growth factors, including angiopoietin-1 and vascular endothelial growth factor (VEGF). Here we show that UTP-induced activation of the Gq protein-coupled P2Y2 nucleotide receptor (P2Y2R) in human coronary artery endothelial cells (HCAECs) activated Rac1 and caused a transient complex to form between P2Y2R, VE-cadherin and VEGF receptor-2 (VEGFR-2). Knockdown of VE-cadherin expression with siRNA did not affect UTP-induced activation of extracellular signal-regulated kinases 1/2 (ERK1/2) but led to a loss of UTP-induced Rac1 activation and tyrosine phosphorylation of p120 catenin, a cytoplasmic protein known to interact with VE-cadherin. Activation of the P2Y2R by UTP also caused a prolonged interaction between p120 catenin and vav2 (a guanine nucleotide exchange factor for Rac) that correlated with the kinetics of UTP-induced tyrosine phosphorylation of p120 catenin and VE-cadherin. Inhibitors of VEGFR-2 (SU1498) or Src (PP2) significantly diminished UTP-induced Rac1 activation, tyrosine phosphorylation of p120 catenin and VE-cadherin, and association of the P2Y2R with VE-cadherin and p120 catenin with vav2. These findings suggest that the P2Y2R uses Src and VEGFR-2 to mediate association of the P2Y2R with VE-cadherin complexes in endothelial adherens junctions to activate Rac1.

  8. Gastrointestinal cancer cells treatment with bevacizumab activates a VEGF autoregulatory mechanism involving telomerase catalytic subunit hTERT via PI3K-AKT, HIF-1α and VEGF receptors

    PubMed Central

    Mahfouz, Nadine; Tahtouh, Roula; Alaaeddine, Nada; El Hajj, Joelle; Sarkis, Riad; Hachem, Ray; Raad, Issam

    2017-01-01

    Background Targeting angiogenesis has been considered a promising treatment of choice for a large number of malignancies, including gastrointestinal cancers. Bevacizumab is an anti-vascular endothelial growth factor (anti-VEGF) being used for this purpose. However, treatment efficacy is largely questioned. Telomerase activity, responsible for cancer cell immortality, is detected in 85–95% of human cancers and is considered a potential regulator of VEGF. The aim of our study was to investigate the interrelationship between VEGF and hTERT in gastrointestinal cancers and to explore cell response to a combined inhibition of telomerase and VEGF. Methods AGS (gastric cancer), Caco-2 (colorectal cancer) and HepG2/C3A (hepatocellular carcinoma), were treated with telomerase inhibitors BIBR-1232 (10μM) and costunolide (10μM), with bevacizumab (Avastin® at 5 ng/ml or 100μg/ml) or with a combination of both types of inhibitors. VEGF and hTERT mRNA levels, and telomerase activity were detected by RT-PCR. VEGF levels were quantified by ELISA. Telomerase was knocked down using hTERT siRNA and hTERT was overexpressed in the telomerase negative cell line, Saos-2 (osteosarcoma), using constructs expressing either wild type hTERT (hTERT-WT) or dominant negative hTERT (hTERT-DN). Tube formation by HUVECs was assessed using ECMatrix™ (EMD Millipore). Results Our results showed that telomerase regulates VEGF expression and secretion through its catalytic subunit hTERT in AGS, Caco2, and HepG2/C3A, independent of its catalytic activity. Interestingly, VEGF inhibition with bevacizumab (100μg/ml) increased hTERT expression 42.3% in AGS, 94.1% in Caco2, and 52.5% in HepG2/C3A, and increased telomerase activity 30-fold in AGS, 10.3-fold in Caco2 and 8-fold in HepG2/C3A. A further investigation showed that VEGF upregulates hTERT expression in a mechanism that implicates the PI3K/AKT/mTOR pathway and HIF-1α. Moreover, bevacizumab treatment increased VEGFR1 and VEGFR2 expression in

  9. Gastrointestinal cancer cells treatment with bevacizumab activates a VEGF autoregulatory mechanism involving telomerase catalytic subunit hTERT via PI3K-AKT, HIF-1α and VEGF receptors.

    PubMed

    Mahfouz, Nadine; Tahtouh, Roula; Alaaeddine, Nada; El Hajj, Joelle; Sarkis, Riad; Hachem, Ray; Raad, Issam; Hilal, George

    2017-01-01

    Targeting angiogenesis has been considered a promising treatment of choice for a large number of malignancies, including gastrointestinal cancers. Bevacizumab is an anti-vascular endothelial growth factor (anti-VEGF) being used for this purpose. However, treatment efficacy is largely questioned. Telomerase activity, responsible for cancer cell immortality, is detected in 85-95% of human cancers and is considered a potential regulator of VEGF. The aim of our study was to investigate the interrelationship between VEGF and hTERT in gastrointestinal cancers and to explore cell response to a combined inhibition of telomerase and VEGF. AGS (gastric cancer), Caco-2 (colorectal cancer) and HepG2/C3A (hepatocellular carcinoma), were treated with telomerase inhibitors BIBR-1232 (10μM) and costunolide (10μM), with bevacizumab (Avastin® at 5 ng/ml or 100μg/ml) or with a combination of both types of inhibitors. VEGF and hTERT mRNA levels, and telomerase activity were detected by RT-PCR. VEGF levels were quantified by ELISA. Telomerase was knocked down using hTERT siRNA and hTERT was overexpressed in the telomerase negative cell line, Saos-2 (osteosarcoma), using constructs expressing either wild type hTERT (hTERT-WT) or dominant negative hTERT (hTERT-DN). Tube formation by HUVECs was assessed using ECMatrix™ (EMD Millipore). Our results showed that telomerase regulates VEGF expression and secretion through its catalytic subunit hTERT in AGS, Caco2, and HepG2/C3A, independent of its catalytic activity. Interestingly, VEGF inhibition with bevacizumab (100μg/ml) increased hTERT expression 42.3% in AGS, 94.1% in Caco2, and 52.5% in HepG2/C3A, and increased telomerase activity 30-fold in AGS, 10.3-fold in Caco2 and 8-fold in HepG2/C3A. A further investigation showed that VEGF upregulates hTERT expression in a mechanism that implicates the PI3K/AKT/mTOR pathway and HIF-1α. Moreover, bevacizumab treatment increased VEGFR1 and VEGFR2 expression in cancer cells and human

  10. Sargassum fusiforme polysaccharides inhibit VEGF-A-related angiogenesis and proliferation of lung cancer in vitro and in vivo.

    PubMed

    Chen, Huiling; Zhang, Ling; Long, Xiange; Li, Peifei; Chen, Shengcan; Kuang, Wei; Guo, Junming

    2017-01-01

    Sargassum fusiforme (Harv.) is a brown alga belonging to the Sargasaceae family. The Sargassum fusiforme polysaccharides (SFPS) have demonstrated good anti-tumor and immunomodulatory activity. However, the underlying mechanisms of its anti-tumorigenesis, especially the anti-angiogenic activity is yet to be established. In the present study, we attempted to determine the effects of SFPS on the human lung adenocarcinoma SPC-A-1 cells and its xenograft model. The results showed that SFPS provides a concentration-dependent inhibition of SPC-A-1 cell proliferation in in vitro and the tumor growth in in vivo studies. Immunohistochemistry studies revealed that the administration of SFPS significantly decreased CD31, VEGF-A expression and the tumor microvessel density (MVD). SFPS also provided a dose-dependent impairment of cell vitality, induction of cell cycle arrest and apoptosis of human umbilical vein endothelial cells (HUVECs). SFPS inhibited the expression of VEGF-A in tumor cells and its receptor VEGFR2 in HUVECs. The HUVEC tube formation assay showed that SFPS could abrogate the tube formation with relatively decreased tubes length of tube-like capillary similar to anti-VEGF antibody, Avastin(®). These findings suggested that SFPS could be used as an alternative anticancer drug as they inhibited the angiogenesis and the microvessel formation through disruption of VEGF signals apart from direct tumor cytotoxicity.

  11. Pien Tze Huang Inhibits Hypoxia-Induced Angiogenesis via HIF-1α/VEGF-A Pathway in Colorectal Cancer

    PubMed Central

    Chen, Hongwei; Feng, Jianyu; Zhang, Yuchen; Shen, Aling; Chen, Youqin; Lin, Jiumao; Lin, Wei; Sferra, Thomas J.

    2015-01-01

    Hypoxia-induced angiogenesis plays an important role in the development and metastasis of solid tumors and is highly regulated by HIF-1α/VEGF-A pathway. Therefore, inhibiting tumor angiogenesis via suppression of HIF-1α/VEGF-A signaling represents a promising strategy for anticancer treatment. As a traditional Chinese medicine formula, Pien Tze Huang (PZH) has long been used as a folk remedy for cancer in China and Southeast Asia. Previously, we reported that PZH inhibits colorectal cancer (CRC) growth both in vivo and in vitro. To elucidate the antitumor mechanisms of PZH, in the present study we used human umbilical vein endothelial cells (HUVEC) and colorectal carcinoma HCT-8 cells to evaluate the effects of PZH on hypoxia-induced angiogenesis and investigated the underlying molecular mechanisms. We found that PZH could inhibit hypoxia-induced migration and tube formation of HUVEC cells in a dose-dependent manner, although the low concentrations of PZH had no effect on HUVEC viability. Moreover, PZH inhibited hypoxia-induced activation of HIF-1α signaling and the expression of VEGF-A and/or VEGFR2 in both HCT-8 and HUVEC cells. Collectively, our findings suggest that PZH can inhibit hypoxia-induced tumor angiogenesis via suppression of HIF-1α/VEGF-A pathway. PMID:25649293

  12. Pien Tze Huang Inhibits Hypoxia-Induced Angiogenesis via HIF-1 α /VEGF-A Pathway in Colorectal Cancer.

    PubMed

    Chen, Hongwei; Feng, Jianyu; Zhang, Yuchen; Shen, Aling; Chen, Youqin; Lin, Jiumao; Lin, Wei; Sferra, Thomas J; Peng, Jun

    2015-01-01

    Hypoxia-induced angiogenesis plays an important role in the development and metastasis of solid tumors and is highly regulated by HIF-1α/VEGF-A pathway. Therefore, inhibiting tumor angiogenesis via suppression of HIF-1α/VEGF-A signaling represents a promising strategy for anticancer treatment. As a traditional Chinese medicine formula, Pien Tze Huang (PZH) has long been used as a folk remedy for cancer in China and Southeast Asia. Previously, we reported that PZH inhibits colorectal cancer (CRC) growth both in vivo and in vitro. To elucidate the antitumor mechanisms of PZH, in the present study we used human umbilical vein endothelial cells (HUVEC) and colorectal carcinoma HCT-8 cells to evaluate the effects of PZH on hypoxia-induced angiogenesis and investigated the underlying molecular mechanisms. We found that PZH could inhibit hypoxia-induced migration and tube formation of HUVEC cells in a dose-dependent manner, although the low concentrations of PZH had no effect on HUVEC viability. Moreover, PZH inhibited hypoxia-induced activation of HIF-1α signaling and the expression of VEGF-A and/or VEGFR2 in both HCT-8 and HUVEC cells. Collectively, our findings suggest that PZH can inhibit hypoxia-induced tumor angiogenesis via suppression of HIF-1α/VEGF-A pathway.

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

    PubMed

    Catena, Raúl; Larzabal, Leyre; Larrayoz, Marta; Molina, Eva; Hermida, Jose; Agorreta, Jackeline; Montes, Ramon; Pio, Ruben; Montuenga, Luis M; Calvo, Alfonso

    2010-12-31

    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 VEGF(xxx)b, 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. 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 VEGF(xxx)b isoforms are differentially expressed in tumors compared to healthy tissues, immunohistochemical analysis was conducted on a breast cancer tissue microarray. A significant increase (p < 0.05) in both VEGF(xxx)b 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 VEGF(xxx)b and total VEGF-A was found. Our results demonstrate that VEGF₁₂₁/₁₆₅b are not anti-angiogenic, but weakly angiogenic isoforms of VEGF-A. In addition, VEGF(xxx)b isoforms are up-regulated in breast cancer in

  14. β-Adrenergic receptor antagonists inhibit vasculogenesis of embryonic stem cells by downregulation of nitric oxide generation and interference with VEGF signalling.

    PubMed

    Sharifpanah, Fatemeh; Saliu, Fatjon; Bekhite, Mohamed M; Wartenberg, Maria; Sauer, Heinrich

    2014-11-01

    The β-adrenoceptor antagonist Propranolol has been successfully used to treat infantile hemangioma. However, its mechanism of action is so far unknown. The hypothesis of this research was that β-adrenoceptor antagonists may interfere with endothelial cell differentiation of stem cells. Specifically, the effects of the non-specific β-adrenergic receptor (β-adrenoceptor) antagonist Propranolol, the β1-adrenoceptor-specific antagonist Atenolol and the β2-adrenoceptor-specific antagonist ICI118,551 on vasculogenesis of mouse embryonic stem (ES) cells were investigated. All three β-blockers dose-dependently downregulated formation of capillary structures in ES cell-derived embryoid bodies and decreased the expression of the vascular cell markers CD31 and VE-cadherin. Furthermore, β-blockers downregulated the expression of fibroblast growth factor-2 (FGF-2), hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor 165 (VEGF165), VEGF receptor 2 (VEGF-R2) and phospho VEGF-R2, as well as neuropilin 1 (NRP1) and plexin-B1 which are essential modulators of embryonic angiogenesis with additional roles in vessel remodelling and arteriogenesis. Under conditions of β-adrenoceptor inhibition, the endogenous generation of nitric oxide (NO) as well as the phosphorylation of endothelial nitric oxide synthase (eNOS) was decreased in embryoid bodies, whereas an increase in NO generation was observed with the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP). Consequently, vasculogenesis of ES cells was restored upon treatment of differentiating ES cells with β-adrenoceptor antagonists in the presence of NO donor. In summary, our data suggest that β-blockers impair vasculogenesis of ES cells by interfering with NO generation which could be the explanation for their anti-angiogenic effects in infantile hemangioma.

  15. Photonic Monitoring in Real-time of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) Gene Expression Under Relaxin-induced Conditions in a Novel Murine Wound Model

    USDA-ARS?s Scientific Manuscript database

    Relaxin is known to promote vascular endotheilial growth factor (VEGF) expression in reproductive tissue and successful wound-healing is dependent upon good vascularization of wound sites, a process that relaxin may facilitate. Thus, the objective of this study was to evaluate the efficacy of relaxi...

  16. Increasing the binding affinity of VEGFR-2 inhibitors by extending their hydrophobic interaction with the active site: Design, synthesis and biological evaluation of 1-substituted-4-(4-methoxybenzyl)phthalazine derivatives.

    PubMed

    Eldehna, Wagdy M; Abou-Seri, Sahar M; El Kerdawy, Ahmed M; Ayyad, Rezk R; Hamdy, Abdallah M; Ghabbour, Hazem A; Ali, Mamdouh M; Abou El Ella, Dalal A

    2016-05-04

    A series of anilinophthalazine derivatives 4a-j was initially synthesized and tested for its VEGFR-2 inhibitory activity where it showed promising activity (IC50 = 0.636-5.76 μM). Molecular docking studies guidance was used to improve the binding affinity for series 4a-j towards VEGFR-2 active site. This improvement was achieved by increasing the hydrophobic interaction with the hydrophobic back pocket of the VEGFR-2 active site lined with the hydrophobic side chains of Ile888, Leu889, Ile892, Val898, Val899, Leu1019 and Ile1044. Increasing the hydrophobic interaction was accomplished by extending the anilinophthalazine scaffold with a substituted phenyl moiety through an uriedo linker which should give this extension the flexibility required to accommodate itself deeply into the hydrophobic back pocket. As planned, the designed uriedo-anilinophthalazines 7a-i showed superior binding affinity than their anilinophthalazine parents (IC50 = 0.083-0.473 μM). In particular, compounds 7g-i showed IC50 of 0.086, 0.083 and 0.086 μM, respectively, which are better than that of the reference drug sorafenib (IC50 = 0.09 μM).

  17. First-in-Human Ultrasound Molecular Imaging With a VEGFR2-Specific Ultrasound Molecular Contrast Agent (BR55) in Prostate Cancer: A Safety and Feasibility Pilot Study.

    PubMed

    Smeenge, Martijn; Tranquart, François; Mannaerts, Christophe K; de Reijke, Theo M; van de Vijver, Marc J; Laguna, M Pilar; Pochon, Sibylle; de la Rosette, Jean J M C H; Wijkstra, Hessel

    2017-07-01

    BR55, a vascular endothelial growth factor receptor 2 (VEGFR2)-specific ultrasound molecular contrast agent (MCA), has shown promising results in multiple preclinical models regarding cancer imaging. In this first-in-human, phase 0, exploratory study, we investigated the feasibility and safety of the MCA for the detection of prostate cancer (PCa) in men using clinical standard technology. Imaging with the MCA was performed in 24 patients with biopsy-proven PCa scheduled for radical prostatectomy using a clinical ultrasound scanner at low acoustic power. Safety monitoring was done by physical examination, blood pressure and heart rate measurements, electrocardiogram, and blood sampling. As first-in-human study, MCA dosing and imaging protocol were necessarily fine-tuned along the enrollment to improve visualization. Imaging data were correlated with radical prostatectomy histopathology to analyze the detection rate of ultrasound molecular imaging with the MCA. Imaging with MCA doses of 0.03 and 0.05 mL/kg was adequate to obtain contrast enhancement images up to 30 minutes after administration. No serious adverse events or clinically meaningful changes in safety monitoring data were identified during or after administration. BR55 dosing and imaging were fine-tuned in the first 12 patients leading to 12 subsequent patients with an improved MCA dosing and imaging protocol. Twenty-three patients underwent radical prostatectomy. A total of 52 lesions were determined to be malignant by histopathology with 26 (50%) of them seen during BR55 imaging. In the 11 patients that were scanned with the improved protocol and underwent radical prostatectomy, a total of 28 malignant lesions were determined: 19 (68%) were seen during BR55 ultrasound molecular imaging, whereas 9 (32%) were not identified. Ultrasound molecular imaging with BR55 is feasible with clinical standard technology and demonstrated a good safety profile. Detectable levels of the MCA can be reached in patients

  18. Procyanidin B2 3,3″-di-O-gallate inhibits endothelial cells growth and motility by targeting VEGFR2 and integrin signaling pathways.

    PubMed

    Kumar, Rahul; Deep, Gagan; Wempe, Michael F; Agarwal, Rajesh; Agarwal, Chapla

    2015-01-01

    Targeting angiogenesis, one of the hallmarks of carcinogenesis, using non-toxic phytochemicals has emerged as a translational opportunity for angioprevention and to control advanced stages of malignancy. Herein, we investigated the inhibitory effects and associated mechanism/s of action of Procyanidin B2-3,3″-di- O-gallate (B2G2), a major component of grape seed extract, on human umbilical vein endothelial cells (HUVECs) and human prostate microvascular endothelial cells (HPMECs). Our results showed that B2G2 (10-40 μM) inhibits growth and induces death in both HUVECs and HPMECs. Additional studies revealed that B2G2 causes a G1 arrest in cell cycle progression of HUVECs by down-regulating cyclins (D1 and A), CDKs (Cdk2 and Cdc2) and Cdc25c phosphatase and up-regulating CDK inhibitors (p21 and p27) expression. B2G2 also induced strong apoptotic death in HUVECs through increasing p53, Bax and Smac/Diablo expression while decreasing Bcl-2 and survivin levels. Additionally, B2G2 inhibited the growth factors-induced capillary tube formation in HUVECs and HPMECs. Interestingly, conditioned media (CCM) from prostate cancer (PCA) cells (LNCaP and PC3) grown under normoxic (~21% O2) and hypoxic (1% O2) conditions significantly enhanced the tube formation in HUVECs, which was compromised in presence of conditioned media from B2G2-treated PCA cells. B2G2 also inhibited the motility and invasiveness of both HUVECs and HPMECs. Mechanistic studies showed that B2G2 targets VEGFR2/PI3K/Akt and integrin signaling molecules which are important for endothelial cells survival, proliferation, tube formation and motility. Overall, we report that B2G2 inhibits several attributes of angiogenesis in cell culture; therefore, it warrants further investigation for efficacy for angioprevention and cancer control.

  19. Hydrogen sulphide triggers VEGF-induced intracellular Ca²⁺ signals in human endothelial cells but not in their immature progenitors.

    PubMed

    Potenza, Duilio Michele; Guerra, Germano; Avanzato, Daniele; Poletto, Valentina; Pareek, Sumedha; Guido, Daniele; Gallanti, Angelo; Rosti, Vittorio; Munaron, Luca; Tanzi, Franco; Moccia, Francesco

    2014-09-01

    Hydrogen sulphide (H2S) is a newly discovered gasotransmitter that regulates multiple steps in VEGF-induced angiogenesis. An increase in intracellular Ca(2+) concentration ([Ca(2+)]i) is central to endothelial proliferation and may be triggered by both VEGF and H2S. Albeit VEGFR-2 might serve as H2S receptor, the mechanistic relationship between VEGF- and H2S-induced Ca(2+) signals in endothelial cells is unclear. The present study aimed at assessing whether and how NaHS, a widely employed H2S donor, stimulates pro-angiogenic Ca(2+) signals in Ea.hy926 cells, a suitable surrogate for mature endothelial cells, and human endothelial progenitor cells (EPCs). We found that NaHS induced a dose-dependent increase in [Ca(2+)]i in Ea.hy926 cells. NaHS-induced Ca(2+) signals in Ea.hy926 cells did not require extracellular Ca(2+) entry, while they were inhibited upon pharmacological blockade of the phospholipase C/inositol-1,4,5-trisphosphate (InsP3) signalling pathway. Moreover, the Ca(2+) response to NaHS was prevented by genistein, but not by SU5416, which selectively inhibits VEGFR-2. However, VEGF-induced Ca(2+) signals were suppressed by dl-propargylglycine (PAG), which blocks the H2S-producing enzyme, cystathionine γ-lyase. Consistent with these data, VEGF-induced proliferation and migration were inhibited by PAG in Ea.hy926 cells, albeit NaHS alone did not influence these processes. Conversely, NaHS elevated [Ca(2+)]i only in a modest fraction of circulating EPCs, whereas neither VEGF-induced Ca(2+) oscillations nor VEGF-dependent proliferation were affected by PAG. Therefore, H2S-evoked elevation in [Ca(2+)]i is essential to trigger the pro-angiogenic Ca(2+) response to VEGF in mature endothelial cells, but not in their immature progenitors. Copyright © 2014. Published by Elsevier Ltd.

  20. VEGF induces proliferation, migration, and TGF-{beta}1 expression in mouse glomerular endothelial cells via mitogen-activated protein kinase and phosphatidylinositol 3-kinase

    SciTech Connect

    Li Zhaodong; Bork, Jens Peter; Krueger, Bettina; Patsenker, Eleonora; Schulze-Krebs, Anja; Hahn, Eckhart G.; Schuppan, Detlef; E-mail: dschuppa@bidmc.harvard.edu

    2005-09-09

    The role of glomerular endothelial cells in kidney fibrosis remains incompletely understood. While endothelia are indispensable for repair of acute damage, they can produce extracellular matrix proteins and profibrogenic cytokines that promote fibrogenesis. We used a murine cell line with all features of glomerular endothelial cells (glEND.2), which dissected the effects of vascular endothelial growth factor (VEGF) on cell migration, proliferation, and profibrogenic cytokine production. VEGF dose-dependently induced glEND.2 cell migration and proliferation, accompanied by up-regulation of VEGFR-2 phosphorylation and mRNA expression. VEGF induced a profibrogenic gene expression profile, including up-regulation of TGF-{beta}1 mRNA, enhanced TGF-{beta}1 secretion, and bioactivity. VEGF-induced endothelial cell migration and TGF-{beta}1 induction were mediated by the phosphatidyl-inositol-3 kinase pathway, while proliferation was dependent on the Erk1/2 MAP kinase pathway. This suggests that differential modulation of glomerular angiogenesis by selective inhibition of the two identified VEGF-induced signaling pathways could be a therapeutic approach to treat kidney fibrosis.

  1. Constitutive expression of vascular endothelial cell growth factor (VEGF) gene family ligand and receptors on human upper and lower airway epithelial cells.

    PubMed

    Lee, Hyun Sil; Kim, Jean

    2014-01-01

    We previously reported that vascular endothelial cell growth factor (VEGF) is abundantly expressed by primary human nasal epithelial cells (PNECs) and functions to promote cell hyperplasia in polyposis. Therefore, we aimed to examine the full expression profile of other members of the VEGF gene family of ligands and receptors, which may play a role in cell growth and the development of chronic rhinosinusitis with nasal polyposis (CRSwNP). Messenger RNA (mRNA) and protein expression of VEGF genes, receptors, and co-receptors was examined from cultured PNECs (n = 4) and compared to that from primary human bronchial epithelial cells (PBECs; n = 4) and the BEAS2B cell line (n = 4) by real-time polymerase chain reaction (PCR) and flow cytometry. We report abundant expression of VEGFA, VEGFB, and VEGFC, detected by mRNA and flow cytometric analysis on PNECs. We herein report the novel finding that there is significant expression of VEGFR1, VEGFR2, VEGFR3, and both neuropilin co-receptors, NP1 and NP2, at baseline conditions on PNECs. Lower airway PBECs and BEAS2B cells displayed similar patterns of expression. PNECs express high constitutive levels of the VEGF gene family homolog of ligands and receptors. Expression of multiple VEGF ligand-receptor combinations may function as redundant pathways to promote upper and lower airway epithelial cell growth during inflammation.

  2. Disrupting VEGF-A paracrine and autocrine loops by targeting SHP-1 suppresses triple negative breast cancer metastasis

    PubMed Central

    Su, Jung-Chen; Mar, Ai-Chung; Wu, Szu-Hsien; Tai, Wei-Tien; Chu, Pei-Yi; Wu, Chia-Yun; Tseng, Ling-Ming; Lee, Te-Chang; Chen, Kuen-Feng; Liu, Chun-Yu; Chiu, Hao-Chieh; Shiau, Chung-Wai

    2016-01-01

    Patients with triple-negative breast cancer (TNBC) had an increased likelihood of distant recurrence and death, as compared with those with non-TNBC subtype. Regorafenib is a multi-receptor tyrosine kinase (RTK) inhibitor targeting oncogenesis and has been approved for metastatic colorectal cancer and advanced gastrointestinal stromal tumor. Recent studies suggest regorafenib acts as a SHP-1 phosphatase agonist. Here, we investigated the potential of regorafenib to suppress metastasis of TNBC cells through targeting SHP-1/p-STAT3/VEGF-A axis. We found a significant correlation between cancer cell migration and SHP-1/p-STAT3/VEGF-A expression in human TNBC cells. Clinically, high VEGF-A expression is associated with worse disease-free and distant metastasis-free survival. Regorafenib induced significant anti-migratory effects, in association with downregulation of p-STAT3 and VEGF-A. To exclude the role of RTK inhibition in regorafenib-induced anti-metastasis, we synthesized a regorafenib derivative, SC-78, that had minimal effect on VEGFR2 and PDGFR kinase inhibition, while having more potent effects on SHP-1 activation. SC-78 demonstrated superior in vitro and in vivo anti-migration to regorafenib. Furthermore, VEGF-A dependent autocrine/paracrine loops were disrupted by regorafenib and SC-78. This study implies that SHP-1/p-STAT3/VEGF-A axis is a potential therapeutic target for metastatic TNBC, and the more potent SC-78 may be a promising lead for suppressing metastasis of TNBC. PMID:27364975

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

    PubMed

    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.

  4. VEGF targets the tumour cell

    PubMed Central

    Goel, Hira Lal; Mercurio, Arthur M.

    2014-01-01

    The function of vascular endothelial growth factor (VEGF) in cancer is not limited to angiogenesis and vascular permeability. VEGF-mediated signalling occurs in tumour cells, and this signalling contributes to key aspects of tumorigenesis, including the function of cancer stem cells and tumour initiation. In addition to VEGF receptor tyrosine kinases, the neuropilins are crucial for mediating the effects of VEGF on tumour cells, primarily because of their ability to regulate the function and the trafficking of growth factor receptors and integrins. This has important implications for our understanding of tumour biology and for the development of more effective therapeutic approaches. PMID:24263190

  5. VEGF targets the tumour cell.

    PubMed

    Goel, Hira Lal; Mercurio, Arthur M

    2013-12-01

    The function of vascular endothelial growth factor (VEGF) in cancer is not limited to angiogenesis and vascular permeability. VEGF-mediated signalling occurs in tumour cells, and this signalling contributes to key aspects of tumorigenesis, including the function of cancer stem cells and tumour initiation. In addition to VEGF receptor tyrosine kinases, the neuropilins are crucial for mediating the effects of VEGF on tumour cells, primarily because of their ability to regulate the function and the trafficking of growth factor receptors and integrins. This has important implications for our understanding of tumour biology and for the development of more effective therapeutic approaches.

  6. Plexin-A4 promotes tumor progression and tumor angiogenesis by enhancement of VEGF and bFGF signaling.

    PubMed

    Kigel, Boaz; Rabinowicz, Noa; Varshavsky, Asya; Kessler, Ofra; Neufeld, Gera

    2011-10-13

    Plexin-A4 is a receptor for sema6A and sema6B and associates with neuropilins to transduce signals of class-3 semaphorins. We observed that plexin-A1 and plexin-A4 are required simultaneously for transduction of inhibitory sema3A signals and that they form complexes. Unexpectedly, inhibition of plexin-A1 or plexin-A4 expression in endothelial cells using specific shRNAs resulted in prominent plexin type specific rearrangements of the actin cytoskeleton that were accompanied by inhibition of bFGF and VEGF-induced cell proliferation. The two responses were not interdependent since silencing plexin-A4 in U87MG glioblastoma cells inhibited cell proliferation and strongly inhibited the formation of tumors from these cells without affecting cytoskeletal organization. Plexin-A4 formed stable complexes with the FGFR1 and VEGFR-2 tyrosine-kinase receptors and enhanced VEGF-induced VEGFR-2 phosphorylation in endothelial cells as well as bFGF-induced cell proliferation. We also obtained evidence suggesting that some of the pro-proliferative effects of plexin-A4 are due to transduction of autocrine sema6B-induced pro-proliferative signals, since silencing sema6B expression in endothelial cells and in U87MG cells mimicked the effects of plexin-A4 silencing and also inhibited tumor formation from the U87MG cells. Our results suggest that plexin-A4 may represent a target for the development of novel anti-angiogenic and anti-tumorigenic drugs.

  7. Mechanical stimulation of the pro-angiogenic capacity of human fracture haematoma: involvement of VEGF mechano-regulation.

    PubMed

    Groothuis, Aline; Duda, Georg N; Wilson, Cameron J; Thompson, Mark S; Hunter, Morgan R; Simon, Paul; Bail, Hermann J; van Scherpenzeel, Karine M; Kasper, Grit

    2010-08-01

    Compromised angiogenesis appears to be a major limitation in various suboptimal bone healing situations. Appropriate mechanical stimuli support blood vessel formation in vivo and improve healing outcomes. However, the mechanisms responsible for this association are unclear. To address this question, the paracrine angiogenic potential of early human fracture haematoma and its responsiveness to mechanical loading, as well as angiogenic growth factors involved, were investigated in vitro. Human haematomas were collected from healthy patients undergoing surgery within 72 h after bone fracture. The haematomas were embedded in a fibrin matrix, and cultured in a bioreactor resembling the in vivo conditions of the early phase of bone healing (20% compression, 1 Hz) over 3 days. Conditioned medium (CM) from the bioreactor was then analyzed. The matrices were also incubated in fresh medium for a further 24 h to evaluate the persistence of the effects. Growth factor (GF) concentrations were measured in the CM by ELISAs. In vitro tube formation assays were conducted on Matrigel with the HMEC-1 cell line, with or without inhibition of vascular endothelial growth factor receptor 2 (VEGFR2). Cell numbers were quantified using an MTS test. In vitro endothelial tube formation was enhanced by CM from haematomas, compared to fibrin controls. The angiogenesis regulators, vascular endothelial growth factor (VEGF) and transforming growth factor beta1 (TGF-beta1), were released into the haematoma CM, but not angiopoietins 1 or 2 (Ang1, 2), basic fibroblast growth factor (bFGF) or platelet-derived growth factor (PDGF). Mechanical stimulation of haematomas, but not fibrin controls, further increased the induction of tube formation by their CM. The mechanically stimulated haematoma matrices retained their elevated pro-angiogenic capacity for 24 h. The pro-angiogenic effect was cancelled by inhibition of VEGFR2 signalling. VEGF concentrations in CM tended to be elevated by mechanical

  8. Distinct genetic interactions between multiple Vegf receptors are required for development of different blood vessel types in zebrafish.

    PubMed

    Covassin, L D; Villefranc, J A; Kacergis, M C; Weinstein, B M; Lawson, N D

    2006-04-25

    Recent evidence indicates a specific role for vascular endothelial growth factor a (Vegfa) during artery development in both zebrafish and mouse embryos, whereas less is known about signals that govern vein formation. In zebrafish, loss of vegfa blocks segmental artery formation and reduces artery-specific gene exp