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  1. 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

  2. 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).

  3. VEGF/VEGFR-2 changes in frontal cortex, choroid plexus, and CSF after chronic obstructive hydrocephalus

    PubMed Central

    Yang, Jun; Dombrowski, Stephen M; Deshpande, Abhishek; Krajcir, Natalie; Luciano, Mark G

    2010-01-01

    Chronic Hydrocephalus (CH) is often associated with decreased cerebral blood flow (CBF) and oxygen levels. While the exact pathophysiology is not clear, vascular endothelial growth factor (VEGF) and its receptor-2 (VEGFR-2) may be involved. Because the choroid plexus (CP) is involved in cerebrospinal fluid (CSF) production and secretes numerous growth factors including VEGF, it is important to understand VEGF/VEGFR-2 levels in the CP–CSF circulatory system. Our results showed significant decreases in CBF and VEGFR-2 levels in frontal cortex (FC) in CH compared with SC; there were no significant changes in VEGF levels. CBF change in FC was positively correlated with VEGFR-2 levels (P=0.024). Immunohistochemistry (IHC) showed robust expression of VEGF/VEGFR-2 in CP. After CH induction, ventricular CSF volume and VEGF levels significantly increased. These results suggest that the decreased VEGFR-2 levels in FC may be contributed to decreased CBF and increased ventricular CSF-VEGF levels possibly reflected a hypoxic response and/or accumulation of VEGF from CP secretion after blockage of CSF outlet. Further investigation into CSF-VEGF levels in different sites may provide a better understanding of VEGF/VEGFR-2 modulation in the normal and hydrocephalic brain, and may represent a feasible approach to potential therapeutic options for hydrocephalus. PMID:20619858

  4. 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.

  5. 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.

  6. SH003 represses tumor angiogenesis by blocking VEGF binding to VEGFR2

    PubMed Central

    Choi, Hyeong Sim; Kim, Min Kyoung; Lee, Kangwook; Lee, Kang Min; Choi, Youn Kyung; Shin, Yong Cheol; Cho, Sung-Gook; Ko, Seong-Gyu

    2016-01-01

    Tumor angiogenesis is a key feature of cancer progression, because a tumor requires abundant oxygen and nutrition to grow. Here, we demonstrate that SH003, a mixed herbal extract containing Astragalus membranaceus (Am), Angelica gigas (Ag) and Trichosanthes Kirilowii Maximowicz (Tk), represses VEGF-induced tumor angiogenesis both in vitro and in vivo. SH003 inhibited VEGF-induced migration, invasion and tube formation in human umbilical vein endothelial cells (HUVEC) with no effect on the proliferation. SH003 reduced CD31-positive vessel numbers in tumor tissues and retarded tumor growth in our xenograft mouse tumor model, while SH003 did not affect pancreatic tumor cell viability. Consistently, SH003 inhibited VEGF-stimulated vascular permeability in ears and back skins. Moreover, SH003 inhibited VEGF-induced VEGFR2-dependent signaling by blocking VEGF binding to VEGFR2. Therefore, our data conclude that SH003 represses tumor angiogenesis by inhibiting VEGF-induced VEGFR2 activation, and suggest that SH003 may be useful for treating cancer. PMID:27105528

  7. EMMPRIN/CD147 is a novel coreceptor of VEGFR-2 mediating its activation by VEGF

    PubMed Central

    Khayati, Farah; Pérez-Cano, Laura; Maouche, Kamel; Sadoux, Aurélie; Boutalbi, Zineb; Podgorniak, Marie-Pierre; Maskos, Uwe; Setterblad, Niclas; Janin, Anne; Calvo, Fabien; Lebbé, Céleste; Menashi, Suzanne; Fernandez-Recio, Juan; Mourah, Samia

    2015-01-01

    EMMPRIN/CD147 is mainly known for its protease inducing function but a role in promoting tumor angiogenesis has also been demonstrated. This study provides evidence that EMMPRIN is a new coreceptor for the VEGFR-2 tyrosine kinase receptor in both endothelial and tumor cells, as it directly interacts with it and regulates its activation by its VEGF ligand, signalling and functional consequences both in vitro and in vivo. Computational docking analyses and mutagenesis studies identified a molecular binding site in the extracellular domain of EMMPRIN located close to the cell membrane and containing the amino acids 195/199. EMMPRIN is overexpressed in cancer and hence is able to further potentiate VEGFR-2 activation, suggesting that a combinatory therapy of an antiangiogenic drug together with an inhibitor of EMMPRIN/VEGFR-2 interaction may have a greater impact on inhibiting angiogenesis and malignancy. PMID:25825981

  8. Characterization of indolinones which preferentially inhibit VEGF-C- and VEGF-D-induced activation of VEGFR-3 rather than VEGFR-2.

    PubMed

    Kirkin, V; Mazitschek, R; Krishnan, J; Steffen, A; Waltenberger, J; Pepper, M S; Giannis, A; Sleeman, J P

    2001-11-01

    VEGF-C and VEGF-D are lymphangiogenic factors that bind to and activate VEGFR-3, a fms-like tyrosine kinase receptor whose expression is limited almost exclusively to lymphatic endothelium in the adult. Processed forms of VEGF-C and VEGF-D can also activate VEGFR-2, a key player in the regulation of angiogenesis. There is increasing evidence to show that these receptor-ligand interactions play a pivotal role in a number of pathological situations. Inhibition of receptor activation by VEGF-C and VEGF-D could therefore be pharmaceutically useful. Furthermore, to understand the different roles of VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 in pathological situations it will be necessary to dissect the complex interactions of these ligands and their receptors. To facilitate such studies we cloned, sequenced and characterized the expression of rat VEGF-C and VEGF-D. We showed that Cys152-->Ser mutants of processed rat VEGF-C can activate VEGFR-3 but not VEGFR-2, while the corresponding mutation in rat VEGF-D inhibits its ability to activate both VEGFR-2 and VEGFR-3. We also synthesized and characterized indolinones that differentially block VEGF-C- and VEGF-D-induced VEGFR-3 kinase activity compared to that of VEGFR-2. These tools should be useful in analysing the different activities and roles of VEGF-C, VEGF-D and their ligands, and in blocking VEGFR-3-mediated lymphangiogenesis. PMID:11683876

  9. 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.

  10. The Phosphorylation of Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) by Engineered Surfaces with Electrostatically or Covalently Immobilized VEGF

    PubMed Central

    Anderson, Sean M.; Chen, Tom T.; Iruela-Arispe, M. Luisa; Segura, Tatiana

    2010-01-01

    Growth factors are a class of signaling proteins that direct cell fate through interaction with cell surface receptors. Although a myriad of possible cell fates stem from a growth factor binding to its receptor, the signaling cascades that result in one fate over another are still being elucidated. One possible mechanism by which nature modulates growth factor signaling is through the method of presentation of the growth factor – soluble or immobilized (matrix bound). Here we present the methodology to study signaling of soluble versus immobilized VEGF through VEGFR-2. We have designed a strategy to covalently immobilize VEGF using its heparin-binding domain to orient the molecule (bind) and a secondary functional group to mediate covalent binding (lock). This bind-and-lock approach aims to allow VEGF to assume a bioactive orientation before covalent immobilization. Surface plasmon resonance (SPR) demonstrated heparin and VEGF binding with surface densities of 60 ng/cm2 and 100 pg/cm2, respectively. ELISA experiments confirmed VEGF surface density and showed that electrostatically bound VEGF releases in cell medium and heparin solutions while covalently bound VEGF remains immobilized. Electrostatically bound VEGF and covalently bound VEGF phosphorylate VEGFR-2 in both VEGFR-2 transfected cells and VEGFR-2 endogenously producing cells. HUVECs plated on VEGF functionalized surfaces showed different morphologies between surface-bound VEGF and soluble VEGF. The surfaces synthesized in these studies allow for the study of VEGF/VEGFR-2 signaling induced by covalently bound, electrostatically bound, and soluble VEGF and may provide further insight into the design of materials for the generation of a mature and stable vasculature. PMID:19540581

  11. 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.

  12. 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

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

    PubMed

    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

  14. 8-THP-DHI analogs as potent Type I dual TIE-2/VEGF-R2 receptor tyrosine kinase inhibitors.

    PubMed

    Hudkins, Robert L; Zulli, Allison L; Underiner, Ted L; Angeles, Thelma S; Aimone, Lisa D; Meyer, Sheryl L; Pauletti, Daniel; Chang, Hong; Fedorov, Elena V; Almo, Steven C; Fedorov, Alexander A; Ruggeri, Bruce A

    2010-06-01

    A novel series of 8-(2-tetrahydropyranyl)-12,13-dihydroindazolo[5,4-a]pyrrolo[3,4-c]carbazoles (THP-DHI) was synthesized and evaluated as dual TIE-2 and VEGF-R2 receptor tyrosine kinase inhibitors. Development of the structure-activity relationships (SAR) with the support of X-ray crystallography led to identification of 7f and 7g as potent, selective dual TIE-2/VEGF-R2 inhibitors with excellent cellular potency and acceptable pharmacokinetic properties. Compounds 7f and 7g were orally active in tumor models with no observed toxicity.

  15. Inhibition of VEGF165/VEGFR2-dependent signaling by LECT2 suppresses hepatocellular carcinoma angiogenesis

    PubMed Central

    Chen, Chi-Kuan; Yu, Wen-Hsuan; Cheng, Tsu-Yao; Chen, Min-Wei; Su, Chia-Yi; Yang, Yi-Chieh; Kuo, Tsang-Chih; Lin, Ming-Tsan; Huang, Ya-Chi; Hsiao, Michael; Hua, Kuo-Tai; Hung, Mien-Chie; Kuo, Min-Liang

    2016-01-01

    Hepatocellular carcinoma (HCC) relies on angiogenesis for growth and metastasis. Leukocyte cell-derived chemotaxin 2 (LECT2) is a cytokine and preferentially expressed in the liver. Previous studies have found that LECT2 targets to both immune and tumor cells to suppress HCC development and vascular invasion. Although LECT2 did not affect HCC cells growth in vitro, it still suppressed HCC xenografts growth in immune-deficient mice, suggesting other cells such as stroma cells may also be targeted by LECT2. Here, we sought to determine the role of LECT2 in tumor angiogenesis in HCC patients. We found that LECT2 expression inhibited tumor growth via angiogenesis in the HCC xenograft model. Specifically, we demonstrated that recombinant human LECT2 protein selectively suppressed vascular endothelial growth factor (VEGF)165-induced endothelial cell proliferation, migration, and tube formation in vitro and in vivo. Mechanistically, LECT2 reduced VEGF receptor 2 tyrosine phosphorylation and its downstream extracellular signal-regulated kinase and AKT phosphorylation. Furthermore, LECT2 gene expression correlated negatively with angiogenesis in HCC patients. Taken together, our findings demonstrate that LECT2 inhibits VEGF165-induced HCC angiogenesis through directly binding to VEGFR2 and has broad applications in treating VEGF-mediated solid tumors. PMID:27507763

  16. DSGOST inhibits tumor growth by blocking VEGF/VEGFR2-activated angiogenesis

    PubMed Central

    Choi, Hyeong Sim; Lee, Kangwook; Kim, Min Kyoung; Lee, Kang Min; Shin, Yong Cheol; Cho, Sung-Gook; Ko, Seong-Gyu

    2016-01-01

    Tumor growth requires a process called angiogenesis, a new blood vessel formation from pre-existing vessels, as newly formed vessels provide tumor cells with oxygen and nutrition. Danggui-Sayuk-Ga-Osuyu-Saenggang-Tang (DSGOST), one of traditional Chinese medicines, has been widely used in treatment of vessel diseases including Raynaud's syndrome in Northeast Asian countries including China, Japan and Korea. Therefore, we hypothesized that DSGOST might inhibit tumor growth by targeting newly formed vessels on the basis of its historical prescription. Here, we demonstrate that DSGOST inhibits tumor growth by inhibiting VEGF-induced angiogenesis. DSGOST inhibited VEGF-induced angiogenic abilities of endothelial cells in vitro and in vivo, which resulted from its inhibition of VEGF/VEGFR2 interaction. Furthermore, DSGOST attenuated pancreatic tumor growth in vivo by reducing angiogenic vessel numbers, while not affecting pancreatic tumor cell viability. Thus, our data conclude that DSGOST inhibits VEGF-induced tumor angiogenesis, suggesting a new indication for DSGOST in treatment of cancer. PMID:26967562

  17. 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

  18. Localization and control of expression of VEGF-A and the VEGFR-2 receptor in fetal sheep intestines.

    PubMed

    Holmes, Katrina; Charnock Jones, Stephen D; Forhead, Alison J; Giussani, Dino A; Fowden, Abigail L; Licence, Diana; Kempster, Sarah; Smith, Gordon C S

    2008-02-01

    We studied expression of vascular endothelial growth factor A (VEGF-A) and its main receptor, VEGFR-2, in the small intestine from five groups of fetal sheep (each n = 5): 1) preterm controls, 2) term controls, 3) preterm animals where the fetus was infused with cortisol, or 4) saline, and 5) term animals where adrenalectomy had been performed preterm. The main transcript expressed in fetal small intestine was VEGF-A165. Comparing term with preterm animals, there were significantly higher levels of expression of VEGF-A protein (p = 0.005). Levels of VEGF-A protein expression were lower in term adrenalectomized animals (p = 0.01) and were higher in preterm animals infused with cortisol (p = 0.01), compared with their respective control groups. Immunohistochemistry demonstrated strongest expression of VEGF-A protein in the epithelial cells and lamina propria of the villi. Intestinal expression of mRNA encoding the VEGFR-2 receptor did not significantly vary with gestational age. In situ hybridization localized VEGFR-2 to the lamina propria of the villous core and receptor autoradiography using 125I VEGF-A demonstrated binding in the same site. These data show that intestinal VEGF-A is up-regulated with advancing gestation in a glucocorticoid-dependent manner--novel findings consistent with a role for VEGF-A stimulated angiogenesis in preparing the fetal gut for birth.

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

    PubMed Central

    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

    2016-01-01

    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. PMID:26967559

  20. 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.

  1. Leishmania major Infection-Induced VEGF-A/VEGFR-2 Signaling Promotes Lymphangiogenesis That Controls Disease.

    PubMed

    Weinkopff, Tiffany; Konradt, Christoph; Christian, David A; Discher, Dennis E; Hunter, Christopher A; Scott, Phillip

    2016-09-01

    Cutaneous leishmaniasis causes a spectrum of diseases from self-healing to severe nonhealing lesions. Defining the factors contributing to lesion resolution may help in developing new therapies for those patients with chronic disease. We found that infection with Leishmania major increases the expression of vascular endothelial growth factor-A and vascular endothelial growth factor receptor (VEGFR)-2 and is associated with significant changes in the blood and lymphatic vasculature at the site of infection. Ab blockade of VEGFR-2 during infection led to a reduction in lymphatic endothelial cell proliferation and simultaneously increased lesion size without altering the parasite burden. These data show that L. major infection initiates enhanced vascular endothelial growth factor-A/VEGFR-2 signaling and suggest that VEGFR-2-dependent lymphangiogenesis is a mechanism that restricts tissue inflammation in leishmaniasis. PMID:27474074

  2. RGD peptide-conjugated selenium nanoparticles: antiangiogenesis by suppressing VEGF-VEGFR2-ERK/AKT pathway.

    PubMed

    Fu, Xiaoyan; Yang, Yahui; Li, Xiaoling; Lai, Haoqiang; Huang, Yanyu; He, Lizhen; Zheng, Wenjie; Chen, Tianfeng

    2016-08-01

    Angiogenesis is essential for tumorigenesis, progression and metastasis. Herein we described the synthesis of RGD peptide-decorated and doxorubicin-loaded selenium nanoparticles (RGD-NPs) targeting tumor vasculature to enhance the cellular uptake and antiangiogenic activities in vitro and in vivo. After internalization by receptor-mediated endocytosis, this nanosystem disassembled under acidic condition with the presence of lysozymes and cell lysate, leading to bioresponsive triggered drug release. Mechanistic investigation revealed that RGD-NPs inhibited angiogenesis through induction of apoptosis and cell cycle arrest in human umbilical vein endothelial cells (HUVECs) via suppression of VEGF-VEGFR2-ERK/AKT signaling axis by triggering ROS-mediated DNA damage. Additionally, RGD-NPs can inhibit MCF-7 tumor growth and angiogenesis in nude mice via down-regulation of VEGF-VEGFR2, effectively reduce the toxicity and prolong the blood circulation in vivo. Our results suggest that the strategy to use RGD-peptide functionalized SeNPs as carriers of anticancer drugs is an efficient way to achieve cancer-targeted antiangiogenesis synergism. PMID:26961468

  3. 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. PMID:27470403

  4. 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.

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

    PubMed

    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

  6. 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

  7. 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.

  8. Bone marrow mononuclear cell transplantation promotes therapeutic angiogenesis via upregulation of the VEGF-VEGFR2 signaling pathway in a rat model of vascular dementia.

    PubMed

    Wang, Jianping; Fu, Xiaojie; Jiang, Chao; Yu, Lie; Wang, Menghan; Han, Wei; Liu, Liu; Wang, Jian

    2014-05-15

    Bone marrow mononuclear cells (BMMNCs) are important for angiogenesis after stroke. We investigated the effects of BMMNCs on cognitive function, angiogenesis, and the vascular endothelial growth factor (VEGF)-VEGF receptor 2 (VEGFR2) signaling pathway in a rat model of vascular dementia. We transplanted BMMNCs into rats that had undergone permanent bilateral occlusion of the common carotid arteries (2VO) and observed their migration in vivo. On day 28, we assessed cognitive function with the Morris Water Maze test and examined vascular density and white matter damage within the corpus striatum by staining with fluorescein lycopersicon esculentum (tomato) lectin or Luxol fast blue. We evaluated expression of VEGF, rapidly accelerated fibrosarcoma 1 (Raf1), and extracellular-signal-regulated kinases 1 and 2 (ERK1/2) in the ischemic hemisphere by Western blot analysis on day 7 after cell transplantation. Contribution of the VEGF-VEGFR2 signaling pathway was confirmed by using VEGFR2 inhibitor SU5416. BMMNCs penetrated the blood-brain barrier and reached the ischemic cortex and white matter or incorporated into vascular walls of 2VO rats. BMMNC-treated 2VO rats had better learning and memory, higher vascular density, and less white matter damage than did vehicle-treated rats. The beneficial effects of BMMNCs were abolished by pretreatment of rats with SU5416. Protein expression of VEGF and phosphorylated Raf1 and ERK1/2 was also significantly increased by BMMNC treatment, but this upregulation was reversed by SU5416. BMMNCs can enhance angiogenesis, reduce white matter damage, and promote cognitive recovery in 2VO rats. The angiogenic effect may result from upregulation of the VEGF-VEGFR2 signaling pathway. PMID:24589546

  9. Development of a highly-potent anti-angiogenic VEGF8-109 heterodimer by directed blocking of its VEGFR-2 binding site.

    PubMed

    Ghavamipour, Fahimeh; Shahangian, S Shirin; Sajedi, Reza H; Arab, S Shahriar; Mansouri, Kamran; Aghamaali, Mahmoud Reza

    2014-10-01

    Angiogenesis is a hallmark of various pathological conditions and is controlled by a variety of angiogenic factors. Blockade of vascular endothelial growth factor (VEGF) as the most pivotal stimulator of angiogenesis offers a promising therapeutic approach for some diseases, typically cancer. In the present study, a heterodimeric antagonistic VEGF was precisely designed based on structural information of recently-crystallized VEGF/VEGF receptor-2 (VEGFR-2/fetal liver kinase 1/kinase domain region) complex. Directed blocking of kinase domain region occurs via substitution of a VEGF receptor binding site by two peptide segments in one pole, whereas the binding domain of the other pole of VEGF was intact. Candidate peptides for substitution were selected considering to some sequence and structural criteria. A reliable model of modified VEGF was built, refined using molecular dynamics simulation and docked with VEGFR-2. Docking analysis revealed that binding affinity of mutant VEGF was notably diminished, corroborating our design. Heterodimeric VEGF was expressed, refolded and highly purified by two-step affinity chromatography. Dimerization of this antagonist was confirmed using some analytical techniques. Spectroscopic studies assured us to obtain the heterodimeric form of VEGF. Some angiogenic in vitro assays such endothelial cell proliferation and tube formation indicated that this antagonist is not only strongly capable of inhibiting angiogenesis (half maximal inhibitory concentration of 33 and 24 ng · mL(-1) , respectively), but also showed the highest inhibitory effect compared to all other heterodimeric VEGF variants. The high anti-angiogenic potency of this VEGF antagonist may allow its future use as an anti-tumor agent. PMID:25132001

  10. The antihypertension drug doxazosin inhibits tumor growth and angiogenesis by decreasing VEGFR-2/Akt/mTOR signaling and VEGF and HIF-1α expression.

    PubMed

    Park, Mi Sun; Kim, Boh-Ram; Dong, Seung Myung; Lee, Seung-Hoon; Kim, Dae-Yong; Rho, Seung Bae

    2014-07-15

    Doxazosin is an α1 adrenergic receptor blocker that also exerts antitumor effects. However, the underlying mechanisms by which it modulates PI3K/Akt intracellular signaling are poorly understood. In this study, we reveal that doxazosin functions as a novel antiangiogenic agent by inhibiting vascular endothelial growth factor (VEGF)-induced cell migration and proliferation. It also inhibited VEGF-induced capillary-like structure tube formation in vitro. Doxazosin inhibited the phosphorylation of VEGF receptor-2 (VEGFR-2) and downstream signaling, including PI3K, Akt, 3-phosphoinositide-dependent protein kinase 1 (PDK1), mammalian target of rapamycin (mTOR), and hypoxia-inducible factor 1 (HIF-1α). However, it had no effect on VEGF-induced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Furthermore, doxazosin reduced tumor growth and suppressed tumor vascularization in a xenograft human ovarian cancer model. These results provide evidence that doxazosin functions in the endothelial cell system to modulate angiogenesis by inhibiting Akt and mTOR phosphorylation and interacting with VEGFR-2.

  11. 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

  12. Promotion Effect of Apo-9'-fucoxanthinone from Sargassum muticum on Hair Growth via the Activation of Wnt/β-Catenin and VEGF-R2.

    PubMed

    Kang, Jung-Il; Yoo, Eun-Sook; Hyun, Jin-Won; Koh, Young-Sang; Lee, Nam Ho; Ko, Mi-Hee; Ko, Chang-Sik; Kang, Hee-Kyoung

    2016-01-01

    This study was conducted to evaluate the effects of Sargassum muticum extract and apo-9'-fucoxanthinone, a principal component of S. muticum, on hair growth. When rat vibrissa follicles were treated with S. muticum extract for 21 d, the hair-fiber lengths for the vibrissa follicles increased significantly. Treatment with the S. muticum extract and the EtOAc fraction of the S. muticum extract markedly increased the proliferation of dermal papilla cells (DPCs) and decreased the 5α-reductase activity. In addition, the EtOAc fraction of the S. muticum extract significantly promoted anagen initiation in C57BL/6 mice. Especially, apo-9'-fucoxanthinone, an active constituent from the S. muticum extract, caused an increase in DPC proliferation and a decrease in 5α-reductase activity. To elucidate the molecular mechanisms of apo-9'-fucoxanthinone on the proliferation of DPCs, we examined the level of various signaling proteins. Apo-9'-fucoxanthinone increased the level of vascular endothelial growth factor receptor-2 (VEGF-R2), Wnt/β-catenin signaling proteins such as phospho(ser9)-glycogen synthase kinase-3β (GSK-3β) and phospho(ser552)-β-catenin, whereas apo-9'-fucoxanthinone did not affect the transforming growth factor-β (TGF-β) signaling proteins such as Smad2/3. These results suggest that apo-9'-fucoxanthinone from S. muticum could have the potential for hair growth with DPC proliferation via the activation of Wnt/β-catenin signaling and the VEGF-R2 pathway. PMID:27476937

  13. Regulation of Pathologic Retinal Angiogenesis in Mice and Inhibition of VEGF-VEGFR2 Binding by Soluble Heparan Sulfate

    PubMed Central

    Nishiguchi, Koji M.; Kataoka, Keiko; Kachi, Shu; Komeima, Keiichi; Terasaki, Hiroko

    2010-01-01

    Development of the retinal vascular network is strictly confined within the neuronal retina, allowing the intraocular media to be optically transparent. However, in retinal ischemia, pro-angiogenic factors (including vascular endothelial growth factor-A, VEGF-A) induce aberrant guidance of retinal vessels into the vitreous. Here, we show that the soluble heparan sulfate level in murine intraocular fluid is high particularly during ocular development. When the eyes of young mice with retinal ischemia were treated with heparan sulfate-degrading enzyme, the subsequent aberrant angiogenesis was greatly enhanced compared to PBS-injected contralateral eyes; however, increased angiogenesis was completely antagonized by simultaneous injection of heparin. Intraocular injection of heparan sulfate or heparin alone in these eyes resulted in reduced neovascularization. In cell cultures, the porcine ocular fluid suppressed the dose-dependent proliferation of human umbilical vein endothelial cells (HUVECs) mediated by VEGF-A. Ocular fluid and heparin also inhibited the migration and tube formation by these cells. The binding of VEGF-A and HUVECs was reduced under a high concentration of heparin or ocular fluid compared to lower concentrations of heparin. In vitro assays demonstrated that the ocular fluid or soluble heparan sulfate or heparin inhibited the binding of VEGF-A and immobilized heparin or VEGF receptor 2 but not VEGF receptor 1. The recognition that the high concentration of soluble heparan sulfate in the ocular fluid allows it to serve as an endogenous inhibitor of aberrant retinal vascular growth provides a platform for modulating heparan sulfate/heparin levels to regulate angiogenesis. PMID:20975989

  14. The antitumor effect of tanshinone IIA on anti-proliferation and decreasing VEGF/VEGFR2 expression on the human non-small cell lung cancer A549 cell line.

    PubMed

    Xie, Jun; Liu, Jiahui; Liu, Heng; Liang, Shihui; Lin, Meigui; Gu, Yueyu; Liu, Taoli; Wang, Dongmei; Ge, Hui; Mo, Sui-Lin

    2015-11-01

    The effects of tanshinone IIA on the proliferation of the human non-small cell lung cancer cell line A549 and its possible mechanism on the VEGF/VEGFR signal pathway were investigated. The exploration of the interaction between tanshinone IIA and its target proteins provides a feasible platform for studying the anticancer mechanism of active components of herbs. The CCK-8 assay was used to evaluate the proliferative activity of A549 cells treated with tanshinone IIA (2.5-80 μmol/L) for 24, 48 and 72 h, respectively. Flow cytometry was used for the detection of cell apoptosis and cell cycle perturbation. VEGF and VEGFR2 expression were studied by Western blotting. The binding mode of tanshinone IIA within the crystal structure of the VEGFR2 protein was evaluated with molecular docking analysis by use of the CDOCKER algorithm in Discovery Studio 2.1. The CCK-8 results showed that tanshinone IIA can significantly inhibit A549 cell proliferation in a dose- and time-dependent manner. Flow cytometry results showed that the apoptosis rate of tested group was higher than the vehicle control, and tanshinone IIA-treated cells accumulated at the S phase, which was higher than the vehicle control. Furthermore, the expression of VEGF and VEGFR2 was decreased in Western blot. Finally, molecular docking analysis revealed that tanshinone IIA could be stably docked into the kinase domain of VEGFR2 protein with its unique modes to form H-bonds with Cys917 and π-π stacking interactions with Val848. In conclusion, tanshinone IIA may suppress A549 proliferation, induce apoptosis and cell cycle arrest at the S phase. This drug may suppress angiogenesis by targeting the protein kinase domains of VEGF/VEGFR2. PMID:26713270

  15. Targeting VEGFR1- and VEGFR2-expressing non-tumor cells is essential for esophageal cancer therapy

    PubMed Central

    Xu, Wen Wen; Li, Bin; Lam, Alfred KY; Tsao, Sai Wah; Law, Simon YK; Chan, Kwok Wah; Yuan, Qiu Ju; Cheung, Annie LM

    2015-01-01

    Increasing appreciation of tumor heterogeneity and the tumor-host interaction has stimulated interest in developing novel therapies that target both tumor cells and tumor microenvironment. Bone marrow derived cells (BMDCs) constitute important components of the tumor microenvironment. In this study, we aim to investigate the significance of VEGFR1- and VEGFR2-expressing non-tumor cells, including BMDCs, in esophageal cancer (EC) progression and in VEGFR1/VEGFR2-targeted therapies. Here we report that VEGFR1 or VEGFR2 blockade can significantly attenuate VEGF-induced Src and Erk signaling, as well as the proliferation and migration of VEGFR1+ and VEGFR2+ bone marrow cells and their pro-invasive effect on cancer cells. Importantly, our in vivo data show for the first time that systemic blockade of VEGFR1+ or VEGFR2+ non-tumor cells with neutralizing antibodies is sufficient to significantly suppress esophageal tumor growth, angiogenesis and metastasis in mice. Moreover, our tissue microarray study of human EC clinical specimens showed the clinicopathological significance of VEGFR1 and VEGFR2 in EC, which suggest that anti-VEGFR1/VEGFR2 therapies may be particularly beneficial for patients with aggressive EC. In conclusion, this study demonstrates the important contributions of VEGFR1+ and VEGFR2+ non-tumor cells in esophageal cancer progression, and substantiates the validity of these receptors as therapeutic targets for this deadly disease. PMID:25595897

  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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. VEGF increases the fibrinolytic activity of endothelial cells within fibrin matrices: involvement of VEGFR-2, tissue type plasminogen activator and matrix metalloproteinases.

    PubMed

    Ratel, David; Mihoubi, Samira; Beaulieu, Edith; Durocher, Yves; Rivard, Georges-Etienne; Gingras, Denis; Béliveau, Richard

    2007-01-01

    Proteolysis of fibrin matrices by endothelial cells plays essential roles in the migratory and morphogenic differentiation processes underlying angiogenesis. Using an in vitro fibrinolysis model consisting of human umbilical vein endothelial cells (HUVECs) embedded in a three dimensional fibrin matrix, we show that VEGF, an angiogenic cytokine that plays a crucial role in the onset of angiogenesis, is a potent activator of HUVEC-mediated fibrinolysis. This VEGF-dependent fibrin degradation was completely abrogated by inhibitors of either the plasminogen activator/plasmin or matrix metalloproteinases (MMP) proteolytic systems, suggesting the involvement of both classes of proteases in fibrin degradation. Accordingly, VEGF-induced fibrinolysis correlated with an increase in the expression of tPA and of some MMPs, such as MT2-MMP and was completely blocked by a neutralizing antibody against tPA. Overall, these results indicate that efficient proteolysis of three dimensional fibrin matrices during VEGF-mediated angiogenesis involves a complex interplay between the MMP and plasmin-mediated proteolytic systems. PMID:17512973

  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. TTAC-0001, a human monoclonal antibody targeting VEGFR-2/KDR, blocks tumor angiogenesis

    PubMed Central

    Lee, Weon Sup; Pyun, Bo-Jeong; Kim, Sung-Woo; Shim, Sang Ryeol; Nam, Ju Ryoung; Yoo, Ji Young; Jin, Younggeon; Jin, Juyoun; Kwon, Young-Guen; Yun, Chae-Ok; Nam, Do-Hyun; Oh, Keunhee; Lee, Dong-Sup; Lee, Sang Hoon; Yoo, Jin-San

    2015-01-01

    Angiogenesis is one of the most important processes for cancer cell survival, tumor growth and metastasis. Vascular endothelial growth factor (VEGF) and its receptor, particularly VEGF receptor-2 (VEGFR-2, or kinase insert domain-containing receptor, KDR), play critical roles in tumor-associated angiogenesis. We developed TTAC-0001, a human monoclonal antibody against VEGFR-2/KDR from a fully human naïve single-chain variable fragment phage library. TTAC-0001 was selected as a lead candidate based on its affinity, ligand binding inhibition and inhibition of VEGFR-2 signal in human umbilical vein endothelial cells (HUVEC). TTAC-0001 inhibited binding of VEGF-C and VEGF-D to VEGFR-2 in addition to VEGF-A. It binds on the N-terminal regions of domain 2 and domain 3 of VEGFR-2. It could inhibit the phosphorylation of VEGFR-2/KDR and ERK induced by VEGF in HUVEC. TTAC-0001 also inhibited VEGF-mediated endothelial cell proliferation, migration and tube formation in vitro, as well as ex vivo vessel sprouting from rat aortic rings and neovascularization in mouse matrigel model in vivo. Our data indicates that TTAC-0001 blocks the binding of VEGFs to VEGFR-2/KDR and inhibits VEGFR-induced signaling pathways and angiogenesis. Therefore, these data strongly support the further development of TTAC-0001 as an anti-cancer agent in the clinic. PMID:25942475

  3. VEGF binding to NRP1 is essential for VEGF stimulation of endothelial cell migration, complex formation between NRP1 and VEGFR2, and signaling via FAK Tyr407 phosphorylation.

    PubMed

    Herzog, Birger; Pellet-Many, Caroline; Britton, Gary; Hartzoulakis, Basil; Zachary, Ian C

    2011-08-01

    In endothelial cells, neuropilin-1 (NRP1) binds vascular endothelial growth factor (VEGF)-A and is thought to act as a coreceptor for kinase insert domain-containing receptor (KDR) by associating with KDR and enhancing VEGF signaling. Here we report mutations in the NRP1 b1 domain (Y297A and D320A), which result in complete loss of VEGF binding. Overexpression of Y297A and D320A NRP1 in human umbilical vein endothelial cells reduced high-affinity VEGF binding and migration toward a VEGF gradient, and markedly inhibited VEGF-induced angiogenesis in a coculture cell model. The Y297A NRP1 mutant also disrupted complexation between NRP1 and KDR and decreased VEGF-dependent phosphorylation of focal adhesion kinase at Tyr407, but had little effect on other signaling pathways. Y297A NRP1, however, heterodimerized with wild-type NRP1 and NRP2 indicating that nonbinding NRP1 mutants can act in a dominant-negative manner through formation of NRP1 dimers with reduced binding affinity for VEGF. These findings indicate that VEGF binding to NRP1 has specific effects on endothelial cell signaling and is important for endothelial cell migration and angiogenesis mediated via complex formation between NRP1 and KDR and increased signaling to focal adhesions. Identification of key residues essential for VEGF binding and biological functions provides the basis for a rational design of antagonists of VEGF binding to NRP1.

  4. 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

  5. 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.

  6. Anti-Angiogenic Therapy: Strategies to Develop Potent VEGFR-2 Tyrosine Kinase Inhibitors and Future Prospect.

    PubMed

    Shi, Leilei; Zhou, Jianfeng; Wu, Jifeng; Shen, Yuemao; Li, Xun

    2016-01-01

    Tumor angiogenesis has always been a major gap for effective cancer therapy. Interruption of aberrant angiogenesis by specific inhibitors targeting receptor tyrosine kinases (RTKs) has been of great interests to medicinal chemists. Among the factors that are involved in tumor angiogenesis, vascular endothelial growth factor receptor-2 (VEGFR-2) is validated as the most closely related factor which can drive angiogenesis through binding with its natural ligand VEGF. The well-validated VEGF-driven VEGFR-2 signaling pathway can stimulate many endothelial responses, including increasing vessel permeability and enhancing endothelial cell proliferation, migration and differentiation. Consequently, circumventing angiogenesis by VEGFR-2 inhibitors represents a promising strategy for counteracting various VEGFR-2-mediated disorders as well as drug resistance. Over the past decades, a considerable number of novel small molecular VEGFR-2 inhibitors have been exploited with diverse chemical scaffolds. Especially, recent frequently launched inhibitors have declared their research values and therapeutic potentials in oncology. Still, the antiangiogenesis based treatment remains an ongoing challenge. In this review, a comprehensive retrospective of newly emerged VEGFR-2 inhibitors have been summarized, with the emphasis on the structure-activity relationship (SAR) investigation, and also binding patterns of representative inhibitors with biotargets. On the basis of all of this information, varied strategies for developing potent VEGFR-2 inhibitors and the future prospect of the clinical application of antiangiogenic inhibitors are discussed hereby.

  7. 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

  8. Evidence for G-quadruplex in the promoter of vegfr-2 and its targeting to inhibit tumor angiogenesis

    PubMed Central

    Salvati, Erica; Zizza, Pasquale; Rizzo, Angela; Iachettini, Sara; Cingolani, Chiara; D’Angelo, Carmen; Porru, Manuela; Randazzo, Antonio; Pagano, Bruno; Novellino, Ettore; Pisanu, Maria Elena; Stoppacciaro, Antonella; Spinella, Francesca; Bagnato, Anna; Gilson, Eric; Leonetti, Carlo; Biroccio, Annamaria

    2014-01-01

    Tumor angiogenesis is mainly mediated by vascular endothelial growth factor (VEGF), a pro-angiogenic factor produced by cancer cells and active on the endothelium through the VEGF receptor 2 (VEGFR-2). Here we identify a G-rich sequence within the proximal promoter region of vegfr-2, able to form an antiparallel G-quadruplex (G4) structure. This G4 structure can be efficiently stabilized by small molecules with the consequent inhibition of vegfr-2 expression. Functionally, the G4-mediated reduction of VEGFR-2 protein causes a switching off of signaling components that, converging on actin cytoskeleton, regulate the cellular events leading to endothelial cell proliferation, migration and differentiation. As a result of endothelial cell function impairment, angiogenic process is strongly inhibited by G4 ligands both in vitro and in vivo. Interestingly, the G4-mediated antiangiogenic effect seems to recapitulate that observed by using a specific interference RNA against vegfr-2, and it is strongly antagonized by overexpressing the vegfr-2 gene. In conclusion, we describe the evidence for the existence of G4 in the promoter of vegfr-2, whose expression and function can be markedly inhibited by G4 ligands, thereby revealing a new, and so far undescribed, way to block VEGFR-2 as target for anticancer therapy. PMID:24335081

  9. 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

  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

    2010-01-01

    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. PMID:20371769

  11. 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.

  12. 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.

  13. Autocrine VEGFR1 and VEGFR2 signaling promotes survival in human glioblastoma models in vitro and in vivo

    PubMed Central

    Szabo, Emese; Schneider, Hannah; Seystahl, Katharina; Rushing, Elisabeth Jane; Herting, Frank; Weidner, K. Michael

    2016-01-01

    Background Although the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) system has become a prime target for antiangiogenic treatment, its biological role in glioblastoma beyond angiogenesis has remained controversial. Methods Using neutralizing antibodies to VEGF or placental growth factor (PlGF) or the tyrosine kinase inhibitor, cediranib, or lentiviral gene silencing, we delineated autocrine signaling in glioma cell lines. The in vivo effects of VEGFR1 and VEGFR2 depletion were evaluated in orthotopic glioma xenograft models. Results VEGFR1 and VEGFR2 modulated glioma cell clonogenicity, viability, and invasiveness in vitro in an autocrine, cell–line-specific manner. VEGFR1 silencing promoted mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling, whereas VEGFR2 silencing resulted in cell-type dependent activation of the protein kinase B (PKB)/AKT and MAPK/ERK pathways. These responses may represent specific escape mechanisms from VEGFR inhibition. The survival of orthotopic glioma-bearing mice was prolonged upon VEGFR1 silencing in the LNT-229, LN-308, and U87MG models and upon VEGFR2 silencing in LN-308 and U87MG. Disruption of VEGFR1 and VEGFR2 signaling was associated with decreased tumor size, increased tumor necrosis, or loss of matrix metalloproteinase 9 (MMP9) immunoreactivity. Neutralizing VEGF and PlGF by specific antibodies was superior to either antibody treatment alone in the VEGFR1-dependent LNT-229 model. Conclusions Differential dependence on autocrine signaling through VEGFR1 and VEGFR2 suggests a need for biomarker–stratified VEGF(R)-based therapeutic approaches to glioblastoma. PMID:27009237

  14. 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

  15. Angiopoietin-2 interferes with anti-VEGFR2- induced vessel normalization and survival benefit in mice bearing gliomas

    PubMed Central

    Chae, Sung-Suk; Kamoun, Walid S.; Farrar, Christian T.; Kirkpatrick, Nathaniel D.; Niemeyer, Elisabeth; de Graaf, Annemarie M.A.; Sorensen, A. Gregory; Munn, Lance L.; Jain, Rakesh K.; Fukumura, Dai

    2010-01-01

    Purpose In brain tumors, cerebral edema is a significant source of morbidity and mortality. Recent studies have shown that inhibition of VEGF signaling induces transient vascular normalization and reduces cerebral edema, resulting in a modest survival benefit in glioblastoma patients. During anti-VEGF treatment, circulating levels of angiopoietin (Ang)-2 remained high after an initial minor reduction. However, it is not known whether Ang-2 can modulate anti-VEGF treatment of GBM. Here, we used an orthotopic glioma model to test the hypothesis that Ang-2 is an additional target for improving the efficacy of current anti-VEGF therapies in glioma patients. Experimental Design To recapitulate high levels of Ang-2 in glioblastoma patients during anti-VEGF treatment, Ang-2 was ectopically expressed in U87 glioma cells. Animal survival and tumor growth were assessed to determine the effects of Ang-2 and anti-VEGFR2 treatment. We also monitored morphological and functional vascular changes using multiphoton laser scanning microscopy and immunohistochemistry. Results Ectopic expression of Ang-2 had no effect on vascular permeability, tumor growth or survival, although it resulted in higher vascular density, with dilated vessels and reduced mural cell coverage. On the other hand, when combined with anti-VEGFR2 treatment, Ang-2 destabilized vessels without affecting vessel regression and compromised the survival benefit of VEGFR2 inhibition by increasing vascular permeability. VEGFR2 inhibition normalized tumor vasculature while ectopic expression of Ang-2 diminished the beneficial effects of VEGFR2 blockade by inhibiting vessel normalization. Conclusion Cancer treatment regimens combining anti-VEGF and anti-Ang-2 agents may be an effective strategy to improve the efficacy of current anti-VEGF therapies. PMID:20501615

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

    PubMed Central

    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. PMID:26617743

  17. 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.

  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. Molecular imaging reveals elevated VEGFR-2 expression in retinal capillaries in diabetes: a novel biomarker for early diagnosis

    PubMed Central

    Sun, Dawei; Nakao, Shintaro; Xie, Fang; Zandi, Souska; Bagheri, Abouzar; Kanavi, Mozhgan Rezaei; Samiei, Shahram; Soheili, Zahra-Soheila; Frimmel, Sonja; Zhang, Zhongyu; Ablonczy, Zsolt; Ahmadieh, Hamid; Hafezi-Moghadam, Ali

    2014-01-01

    Diabetic retinopathy (DR) is a microvascular complication of diabetes and a leading cause of vision loss. Biomarkers and methods for early diagnosis of DR are urgently needed. Using a new molecular imaging approach, we show up to 94% higher accumulation of custom designed imaging probes against vascular endothelial growth factor receptor 2 (VEGFR-2) in retinal and choroidal vessels of diabetic animals (P<0.01), compared to normal controls. More than 80% of the VEGFR-2 in the diabetic retina was in the capillaries, compared to 47% in normal controls (P<0.01). Angiography in rabbit retinas revealed microvascular capillaries to be the location for VEGF-A-induced leakage, as expressed by significantly higher rate of fluorophore spreading with VEGF-A injection when compared to vehicle control (26±2 vs. 3±1 μm/s, P<0.05). Immunohistochemistry showed VEGFR-2 expression in capillaries of diabetic animals but not in normal controls. Macular vessels from diabetic patients (n=7) showed significantly more VEGFR-2 compared to nondiabetic controls (n=5) or peripheral retinal regions of the same retinas (P<0.01 in both cases). Here we introduce a new approach for early diagnosis of DR and VEGFR-2 as a molecular marker. VEGFR-2 could become a key diagnostic target, one that might help to prevent retinal vascular leakage and proliferation in diabetic patients.—Sun, D., Nakao, S., Xie, F., Zandi, S., Bagheri, A., Kanavi, M. R., Samiei, S., Soheili, Z.-S., Frimmel, S., Zhang, Z., Ablonczy, Z., Ahmadieh, H., Hafezi-Moghadam, A. Molecular imaging reveals elevated VEGFR-2 expression in retinal capillaries in diabetes: a novel biomarker for early diagnosis. PMID:24903276

  20. 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.

  1. 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

  2. Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity

    PubMed Central

    Fleetwood, Filippa; Klint, Susanne; Hanze, Martin; Gunneriusson, Elin; Frejd, Fredrik Y.; Ståhl, Stefan; Löfblom, John

    2014-01-01

    Angiogenesis plays an important role in cancer and ophthalmic disorders such as age-related macular degeneration and diabetic retinopathy. The vascular endothelial growth factor (VEGF) family and corresponding receptors are regulators of angiogenesis and have been much investigated as therapeutic targets. The aim of this work was to generate antagonistic VEGFR2-specific affinity proteins having adjustable pharmacokinetic properties allowing for either therapy or molecular imaging. Two antagonistic Affibody molecules that were cross-reactive for human and murine VEGFR2 were selected by phage and bacterial display. Surprisingly, although both binders independently blocked VEGF-A binding, competition assays revealed interaction with non-overlapping epitopes on the receptor. Biparatopic molecules, comprising the two Affibody domains, were hence engineered to potentially increase affinity even further through avidity. Moreover, an albumin-binding domain was included for half-life extension in future in vivo experiments. The best-performing of the biparatopic constructs demonstrated up to 180-fold slower dissociation than the monomers. The new Affibody constructs were also able to specifically target VEGFR2 on human cells, while simultaneously binding to albumin, as well as inhibit VEGF-induced signaling. In summary, we have generated small antagonistic biparatopic Affibody molecules with high affinity for VEGFR2, which have potential for both future therapeutic and diagnostic purposes in angiogenesis-related diseases. PMID:25515662

  3. Expression and prognostic significance of VEGFR-2 in breast cancer.

    PubMed

    Yan, Ji-Dong; Liu, Yanrong; Zhang, Zhi-Yong; Liu, Guang-Yin; Xu, Jin-Heng; Liu, Li-Yun; Hu, Yue-Ming

    2015-07-01

    Breast cancer is one of the most common cancers among women in the world. Vascular endothelial growth factor receptor 2 (VEGFR-2) was not only found to play a key role in the development of tumor angiogenesis, but has also been located in tumor cells of a variety of tumors. This study investigated the expression pattern of VEGFR-2 in breast cancer tissue specimens in order to evaluate the role of VEGFR-2 in the prognosis of breast cancer. Expression and localization of VEGFR-2 in tumor cells of breast cancer specimens from 98 invasive breast cancer patients were determined by immunohistochemistry. The relationships between VEGFR-2 expression and clinicopathological features were also analyzed. The results showed that VEGFR-2 expression correlated positively with lymph node (LN) metastasis of breast cancer. Patients with high expression of VEGFR-2 had a significantly worse OS. It was also observed that the expression of epithelial-mesenchymal transition (EMT) marker, including Twist1 and Vimentin, was higher in the tumors with higher VEGFR-2 expression, while the E-cadherin expression was lower in the same tumors, suggesting that VEGFR-2 may serve as a possible mediator of EMT in breast cancer.

  4. A Novel Potent Oral Series of VEGFR2 Inhibitors Abrogate Tumor Growth by Inhibiting Angiogenesis.

    PubMed

    Bold, Guido; Schnell, Christian; Furet, Pascal; McSheehy, Paul; Brüggen, Josef; Mestan, Jürgen; Manley, Paul W; Drückes, Peter; Burglin, Marion; Dürler, Ursula; Loretan, Jacqueline; Reuter, Robert; Wartmann, Markus; Theuer, Andreas; Bauer-Probst, Beatrice; Martiny-Baron, Georg; Allegrini, Peter; Goepfert, Arnaud; Wood, Jeanette; Littlewood-Evans, Amanda

    2016-01-14

    This paper describes the identification of 6-(pyrimidin-4-yloxy)-naphthalene-1-carboxamides as a new class of potent and selective human vascular endothelial growth factor receptor 2 (VEGFR2) tyrosine kinase inhibitors. In biochemical and cellular assays, the compounds exhibit single-digit nanomolar potency toward VEGFR2. Compounds of this series show good exposure in rodents when dosed orally. They potently inhibit VEGF-driven angiogenesis in a chamber model and rodent tumor models at daily doses of less than 3 mg/kg by targeting the tumor vasculature as demonstrated by ELISA for TIE-2 in lysates or by immunohistochemical analysis. This novel series of compounds shows a potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role. PMID:26629594

  5. Generation and characterization of a human nanobody against VEGFR-2

    PubMed Central

    Ma, Lin; Gu, Kai; Zhang, Cheng-hai; Chen, Xue-tao; Jiang, Yi; Melcher, Karsten; Zhang, Juan; Wang, Min; Xu, H Eric

    2016-01-01

    Aim: Nanobody is an antibody fragment consisting of a single monomeric variable antibody domain, which can be used for a variety of biotechnological and therapeutic purposes. The aim of this work was to isolate and characterize a human signal domain antibody against VEGFR-2 domain3 (VEGFR D3) from a phage display library. Methods: To produce antigen-specific recombinant nanobodies with high affinity to VEGFR2 D3, a liquid phase panning strategy was used for all rounds of panning. For nanobody expression and purification, four VEGFR2 D3-blocking clones were subcloned into a pETduet-biotin-MBP expression vector. The recombinant proteins carried an MBP tag to facilitate purification by affinity chromatography. Recombinant NTV(1–4) was obtained after an additional gel filtration chromatography step. The interactions between VEGFR2 D3 and NTV(1–4) were assessed with luminescence-based AlphaScreen assay and SPR assay. Anti-angiogenesis effects were examined in human umbilical vein endothelial cells (HUVECs). Results: In the AlphaScreen assay, NTV1 (100 and 200 nmol/L) elicited the highest binding signal with VEGFR2 D3; NTV2 showed moderate interactions with VEGFR2 D3; NTV3 and NTV4 exhibited little or no interaction with VEGFR2 D3. In the SPR assay, NTV1 displayed a high affinity for VEGFR2 D3 with an equilibrium dissociation constant (KD) of 49±1.8 nmol/L. NTV1 (1–1000 nmol/L) dose-dependently inhibited the proliferation of HUVECs and the endothelial tube formation by the HUVECs. Conclusion: The nanobody NTV1 is a potential therapeutic candidate for blocking VEGFR2. This study provides a novel and promising strategy for development of VEGFR2-targeted nanobody-based cancer therapeutics. PMID:27108602

  6. Advanced glycation end product Nε-carboxymethyllysine induces endothelial cell injury: the involvement of SHP-1-regulated VEGFR-2 dephosphorylation.

    PubMed

    Liu, Shing Hwa; Sheu, Wayne Huey Herng; Lee, Maw Rong; Lee, Wen Jane; Yi, Yu Chiao; Yang, Tzung Jie; Jen, Jen Fon; Pan, Hung Chuan; Shen, Chin Chang; Chen, Wen Bao; Tien, Hsing Ru; Sheu, Meei Ling

    2013-06-01

    N(ε)-carboxymethyllysine (CML), a major advanced glycation end product, plays a crucial role in diabetes-induced vascular injury. The roles of protein tyrosine phosphatases and vascular endothelial growth factor (VEGF) receptors in CML-related endothelial cell injury are still unclear. Human umbilical vein endothelial cells (HUVECs) are a commonly used human EC type. Here, we tested the hypothesis that NADPH oxidase/reactive oxygen species (ROS)-mediated SH2 domain-containing tyrosine phosphatase-1 (SHP-1) activation by CML inhibits the VEGF receptor-2 (VEGFR-2, KDR/Flk-1) activation, resulting in HUVEC injury. CML significantly inhibited cell proliferation and induced apoptosis and reduced VEGFR-2 activation in parallel with the increased SHP-1 protein expression and activity in HUVECs. Adding recombinant VEGF increased forward biological effects, which were attenuated by CML. The effects of CML on HUVECs were abolished by SHP-1 siRNA transfection. Exposure of HUVECs to CML also remarkably escalated the integration of SHP-1 with VEGFR-2. Consistently, SHP-1 siRNA transfection and pharmacological inhibitors could block this interaction and elevating [(3)H]thymidine incorporation. CML also markedly activated the NADPH oxidase and ROS production. The CML-increased SHP-1 activity in HUVECs was effectively attenuated by antioxidants. Moreover, the immunohistochemical staining of SHP-1 and CML was increased, but phospho-VEGFR-2 staining was decreased in the aortic endothelium of streptozotocin-induced and high-fat diet-induced diabetic mice. We conclude that a pathway of tyrosine phosphatase SHP-1-regulated VEGFR-2 dephosphorylation through NADPH oxidase-derived ROS is involved in the CML-triggered endothelial cell dysfunction/injury. These findings suggest new insights into the development of therapeutic approaches to reduce diabetic vascular complications.

  7. VEGFA/VEGFR2-targeted therapies prevent the VEGFA-induced proliferation of regulatory T cells in cancer

    PubMed Central

    Terme, Magali; Tartour, Eric; Taieb, Julien

    2013-01-01

    Some of the anti-angiogenic agents currently used to treat solid malignancies have effects on tumor endothelial cells as well as on immune cells. We have recently demonstrated that targeting the vascular endothelial growth factor A (VEGFA)/VEGF receptor 2 (VEGFR2) signaling pathway reduces the proportion of regulatory T cells (Treg) in a mouse model of colorectal cancer (CRC) and in metastatic CRC patients as it inhibits tumor-induced Treg proliferation. PMID:24083078

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

    PubMed

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

    2016-08-02

    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.

  9. 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

  10. Prognostic value of circulating VEGFR2+ bone marrow-derived progenitor cells in patients with advanced cancer.

    PubMed

    Massard, Christophe; Borget, Isabelle; Le Deley, Marie Cécile; Taylor, Melissa; Gomez-Roca, Carlos; Soria, Jean Charles; Farace, Françoise

    2012-06-01

    We hypothesised that host-related markers, possibly reflecting tumour aggressiveness, such as circulating endothelial cells (CEC) and circulating VEGFR2(+) bone marrow-derived (BMD) progenitor cells, could have prognostic value in patients with advanced cancer enrolled in early anticancer drug development trials. Baseline CECs (CD45(-)CD31(+)CD146(+)7AAD(-) cells) and circulating VEGFR2(+)-BMD progenitor cells (defined as CD45(dim)CD34(+)VEGFR2(+)7AAD(-) cells) were measured by flow-cytometry in 71 and 58 patients included in phase 1 trials testing novel anti-vascular or anti-angiogenic agents. Correlations between levels of CECs, circulating VEGFR2(+)-BMD progenitor cells, clinical and biological prognostic factors (i.e. the Royal Marsden Hospital (RMH) score), and overall survival (OS) were studied. The median value of CECs was 12 CEC/ml (range 0-154/ml). The median level of VEGFR2(+)-BMD progenitor cells was 1.3% (range 0-32.5%) of circulating BMD-CD34(+) progenitors. While OS was not correlated with CEC levels, it was significantly worse in patients with high VEGFR2(+)-BMD progenitor levels (>1%) (median OS 9.0 versus 17.0 months), and with a RMH prognostic score >0 (median OS 9.0 versus 24.2 months). The prognostic value of VEGFR2(+)-BMD progenitor levels remained significant (hazard ratio (HR) = 2.3, 95% confidence interval (CI), 1.1-4.6, p = 0.02) after multivariate analysis. A composite VEGFR2(+)-BMD progenitor level/RHM score ≥ 2 was significantly associated with an increased risk of death compared to scores of 0 or 1 (median OS 9.0 versus 18.4 months, HR = 2.6 (95%CI, 1.2-5.8, p = 0.02)). High circulating VEGFR2(+)-BMD progenitor levels are associated with poor prognostics and when combined to classical clinical and biological parameters could provide a new tool for patient selection in early anticancer drug trials. PMID:22370181

  11. Levels of circulating CD45dimCD34+VEGFR2+ progenitor cells correlate with outcome in metastatic renal cell carcinoma patients treated with tyrosine kinase inhibitors

    PubMed Central

    Farace, F; Gross-Goupil, M; Tournay, E; Taylor, M; Vimond, N; Jacques, N; Billiot, F; Mauguen, A; Hill, C; Escudier, B

    2011-01-01

    Background: Predicting the efficacy of antiangiogenic therapy would be of clinical value in patients (pts) with metastatic renal cell carcinoma (mRCC). We tested the hypothesis that circulating endothelial cell (CEC), bone marrow-derived CD45dimCD34+VEGFR2+ progenitor cell or plasma angiogenic factor levels are associated with clinical outcome in mRCC pts undergoing treatment with tyrosine kinase inhibitors (TKI). Methods: Fifty-five mRCC pts were prospectively monitored at baseline (day 1) and day 14 during treatment (46 pts received sunitinib and 9 pts received sorafenib). Circulating endothelial cells (CD45−CD31+CD146+7-amino-actinomycin (7AAD)− cells) were measured in 1 ml whole blood using four-color flow cytometry (FCM). Circulating CD45dimCD34+VEGFR2+7AAD− progenitor cells were measured in progenitor-enriched fractions by four-color FCM. Plasma VEGF, sVEGFR2, SDF-1α and sVCAM-1 levels were determined by ELISA. Correlations between baseline CEC, CD45dimCD34+VEGFR2+7AAD− progenitor cells, plasma factors, as well as day 1–day 14 changes in CEC, CD45dimCD34+VEGFR2+7AAD− progenitor, plasma factor levels, and response to TKI, progression-free survival (PFS) and overall survival (OS) were examined. Results: No significant correlation between markers and response to TKI was observed. No association between baseline CEC, plasma VEGF, sVEGFR-2, SDF-1α, sVCAM-1 levels with PFS and OS was observed. However, baseline CD45dimCD34+VEGFR2+7AAD− progenitor cell levels were associated with PFS (P=0.01) and OS (P=0.006). Changes in this population and in SDF-1α levels between day 1 and day 14 were associated with PFS (P=0.03, P=0.002). Changes in VEGF and SDF-1α levels were associated with OS (P=0.02, P=0.007). Conclusion: Monitoring CD45dimCD34+VEGFR2+ progenitor cells, plasma VEGF and SDF-1α levels could be of clinical interest in TKI-treated mRCC pts to predict outcome. PMID:21386843

  12. VEGFR-2 conformational switch in response to ligand binding

    PubMed Central

    Sarabipour, Sarvenaz; Ballmer-Hofer, Kurt; Hristova, Kalina

    2016-01-01

    VEGFR-2 is the primary regulator of angiogenesis, the development of new blood vessels from pre-existing ones. VEGFR-2 has been hypothesized to be monomeric in the absence of bound ligand, and to undergo dimerization and activation only upon ligand binding. Using quantitative FRET and biochemical analysis, we show that VEGFR-2 forms dimers also in the absence of ligand when expressed at physiological levels, and that these dimers are phosphorylated. Ligand binding leads to a change in the TM domain conformation, resulting in increased kinase domain phosphorylation. Inter-receptor contacts within the extracellular and TM domains are critical for the establishment of the unliganded dimer structure, and for the transition to the ligand-bound active conformation. We further show that the pathogenic C482R VEGFR-2 mutant, linked to infantile hemangioma, promotes ligand-independent signaling by mimicking the structure of the ligand-bound wild-type VEGFR-2 dimer. DOI: http://dx.doi.org/10.7554/eLife.13876.001 PMID:27052508

  13. Soluble vascular endothelial growth factor receptors 2 (sVEGFR-2) and 3 (sVEGFR-3) and breast cancer risk in the Swedish Mammography Cohort

    PubMed Central

    Harris, Holly; Wolk, Alicja; Larsson, Anders; Vasson, Marie-Paule; Basu, Samar

    2016-01-01

    Vascular endothelial growth factor (VEGF) is a signalling protein that has been established as a contributor to tumor angiogenesis, and expression of VEGF and its soluble receptors (sVEGFR2 and sVEGFR3) have been demonstrated in breast cancer cells. However, no prospective studies have examined the association between prediagnostic sVEGFR levels and breast cancer risk. We conducted a prospective case-control study nested within the Swedish Mammography Cohort examining the association between sVEGFR2 and 3 levels and breast cancer risk. The analysis included 69 incident breast cancer cases diagnosed after blood collection and 719 controls. Logistic regression models were used to calculate odds ratios and 95% confidence intervals. After adjustment for breast cancer risk factors, sVEGFR2 levels were associated with breast cancer risk (OR=1.28; 95% CI=1.06-1.56 per 1000 ng/L increase in concentration) while sVEGFR3 levels were not related to such risk (OR=1.00; 95% CI=0.93-1.07). Our results suggest that sVEGFR2 levels may be positively associated with breast cancer risk, however future studies with larger case groups are necessary to confirm this association. PMID:27186332

  14. 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.

  15. 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.

  16. Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2) Functions to Promote Uterine Decidual Angiogenesis during Early Pregnancy in the Mouse

    PubMed Central

    Douglas, Nataki C.; Tang, Hongyan; Gomez, Raul; Pytowski, Bronislaw; Hicklin, Daniel J.; Sauer, Christopher M.; Kitajewski, Jan; Sauer, Mark V.; Zimmermann, Ralf C.

    2009-01-01

    Implantation of an embryo induces rapid proliferation and differentiation of uterine stromal cells, forming a new structure, the decidua. One salient feature of decidua formation is a marked increase in maternal angiogenesis. Vascular endothelial growth factor (VEGF)-dependent pathways are active in the ovary, uterus, and embryo, and inactivation of VEGF function in any of these structures might prevent normal pregnancy development. We hypothesized that decidual angiogenesis is regulated by VEGF acting through specific VEGF receptors (VEGFRs). To test this hypothesis, we developed a murine pregnancy model in which systemic administration of a receptor-blocking antibody would act specifically on uterine angiogenesis and not on ovarian or embryonic angiogenesis. In our model, ovarian function was replaced with exogenous progesterone, and blocking antibodies were administered prior to embryonic expression of VEGFRs. After administration of a single dose of the anti-VEGFR-2 antibody during the peri-implantation period, no embryos were detected on embryonic d 10.5. The pregnancy was disrupted because of a significant reduction in decidual angiogenesis, which under physiological conditions peaks on embryonic d 5.5 and 6.5. Inactivation of VEGFR-3 reduced angiogenesis in the primary decidual zone, whereas administration of VEGFR-1 blocking antibodies had no effect. Pregnancy was not disrupted after administration of anti-VEGFR-3 or anti-VEGFR-1 antibodies. Thus, the VEGF/VEGFR-2 pathway plays a key role in the maintenance of early pregnancy through its regulation of peri-implantation angiogenesis in the uterine decidua. This newly formed decidual vasculature serves as the first exchange apparatus for the developing embryo until the placenta becomes functionally active. PMID:19406950

  17. Glioma cells enhance endothelial progenitor cell angiogenesis via VEGFR-2, not VEGFR-1.

    PubMed

    Zhang, Junxia; Zhao, Peng; Fu, Zhen; Chen, Xiaolei; Liu, Ning; Lu, Ailin; Li, Rui; Shi, Lei; Pu, Peiyu; Kang, Chunsheng; You, Yongping

    2008-12-01

    Although potential contribution of endothelial progenitor cells (EPCs) to angiogenesis in glioma has been proposed, the molecular mechanisms of EPCs recruitment to vasculature have not been fully elucidated. Here, we show that the supernatant from glioma cells promotes EPCs angiogenesis via VEGFR-2, not VEGFR-1. Moreover, VEGFR-2 siRNA inhibits VEGFR-2 expression in EPCs, tube formation on matrigel and cell migration. MMP-9 activity and expression and the Akt and ERK phosphorylations are decreased by VEGFR-2 siRNA. Thus, these results indicate that glioma cells enhance EPC angiogenesis via VEGFR-2, not VEGFR-1, mediated by the MMP-9, Akt and ERK signal pathways.

  18. 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

  19. VEGF-functionalized dextran has longer intracellular bioactivity than VEGF in endothelial cells.

    PubMed

    Maia, João; Vazão, Helena; Pedroso, Dora C S; Jesus, Catarina S H; Brito, Rui M M; Grãos, Mário; Gil, Maria H; Ferreira, Lino

    2012-09-10

    Herein, we report that VEGF-functionalized dextran (dexOx-VEGF) is comparatively superior to free VEGF in prolonging the phosphorylation of VEGF receptor 2 (VEGFR-2). Both dexOx-VEGF and free VEGF activate VEGFR-2, and the complexes are internalized into early endosomes (EEA1(+) vesicles) and then transported to lysosomes (Rab7(+) vesicles). However, after cell activation, dexOx-VEGF is preferentially colocalized in early endosomes where VEGF signaling is still active while free VEGF is preferentially transported to late endosomes or lysosomes. We further show that dexOx-VEGF after phosphorylation of VEGF receptor 2 induces an increase of intracellular Ca(2+) and activates VEGF downstream effectors such as Akt and extracellular signal-regulated kinase (ERK1/2) proteins. Under specific conditions, the activation level is different from the one observed for free VEGF, thus suggesting mechanistic differences, which is illustrated by cell migration and cord-like formation studies. DexOx-VEGF can be cross-linked with adipic acid dihydrazide to form a degradable gel, which in turn can be incorporated in a fibrin gel containing endothelial cells (ECs) to modulate their activity. We envision that these constructs might be beneficial to extend the pro-angiogenic activity of VEGF in ischemic tissues and to modulate the biological activity of vascular cells. PMID:22901277

  20. 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.

  1. Intracellular autocrine VEGF signaling promotes EBDC cell proliferation, which can be inhibited by Apatinib.

    PubMed

    Peng, Sui; Zhang, Yanyan; Peng, Hong; Ke, Zunfu; Xu, Lixia; Su, Tianhong; Tsung, Allan; Tohme, Samer; Huang, Hai; Zhang, Qiuyang; Lencioni, Riccardo; Zeng, Zhirong; Peng, Baogang; Chen, Minhu; Kuang, Ming

    2016-04-10

    Tumor cells produce vascular endothelial growth factor (VEGF) which can interact with membrane or cytoplasmic VEGF receptors (VEGFRs) to promote cell growth. We aimed to investigate the role of extracellular/intracellular autocrine VEGF signaling and Apatinib, a highly selective VEGFR2 inhibitor, in extrahepatic bile duct cancer (EBDC). We found conditioned medium or recombinant human VEGF treatment promoted EBDC cell proliferation through a phospholipase C-γ1-dependent pathway. This pro-proliferative effect was diminished by VEGF, VEGFR1 or VEGFR2 neutralizing antibodies, but more significantly suppressed by intracellular VEGFR inhibitor. The rhVEGF induced intracellular VEGF signaling by promoting nuclear accumulation of pVEGFR1/2 and enhancing VEGF promoter activity, mRNA and protein expression. Internal VEGFR2 inhibitor Apatinib significantly inhibited intracellular VEGF signaling, suppressed cell proliferation in vitro and delayed xenograft tumor growth in vivo, while anti-VEGF antibody Bevacizumab showed no effect. Clinically, overexpression of pVEGFR1 and pVEGFR2 was significantly correlated with poorer overall survival (P = .007 and P = .020, respectively). In conclusion, the intracellular autocrine VEGF loop plays a predominant role in VEGF-induced cell proliferation. Apatinib is an effective intracellular VEGF pathway blocker that presents a great therapeutic potential in EBDC. PMID:26805764

  2. 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.

  3. 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. PMID:26285778

  4. Anti-angiogenic action of PPARγ ligand in human umbilical vein endothelial cells is mediated by PTEN upregulation and VEGFR-2 downregulation.

    PubMed

    Kim, Ki Young; Ahn, Jin Hee; Cheon, Hyae Gyeong

    2011-12-01

    Peroxisome proliferator-activated receptor γ (PPARγ) activation has anti-angiogenic and apoptotic effects in endothelial cells. Here, we investigated the mechanisms of the anti-angiogenic action of a novel PPARγ ligand, KR-62980. KR-62980 inhibited in vitro basal tube formation and in vivo neovascularization in mice induced by Matrigel containing vascular endothelial growth factor (VEGF(165), 5 ng/ml). VEGF(165)-induced cell proliferation and chemotactic migration in human umbilical vein endothelial cells (HUVECs) were also suppressed by KR-62980, in a mechanism accompanied by apoptotic cell death. KR-62980 downregulated the VEGF(165)-induced VEGFR-2 expression but increased the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression in parallel with reduced phosphorylation of extracellular signal-related kinase 1/2 (ERK1/2), PI3K p85α, and p38 MAPK. The knockdown of PTEN expression abolished KR-62980-suppressed cell proliferation and angiogenesis. All of the effects of KR-62980 disappeared with pretreatment of bisphenol A diaglycidyl ether (BADGE), a PPARγ antagonist. In summary, KR-62980 inhibited VEGF(165)-induced angiogenesis in HUVECs by PPARγ-mediated dual mechanisms: VEGFR-2 downregulation and PTEN upregulation.

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. Single chain precursor prohaptoglobin promotes angiogenesis by upregulating expression of vascular endothelial growth factor (VEGF) and VEGF receptor2.

    PubMed

    Oh, Mi-Kyung; Park, Hyo-Jung; Lee, Joo-Hyun; Bae, Hyun-Mi; Kim, In-Sook

    2015-04-13

    Prohaptoglobin (proHp) is processed into mature haptoglobin via site-specific cleavage. Although haptoglobin has been well studied, the functions of proHp remain unclear. We investigated the angiogenic action of proHp in endothelial cells, demonstrating that proHp upregulated vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) expression and endothelial sprouting and branching. ProHp-induced sprouting was attenuated by a VEGFR2 inhibitor. Moreover, proHp was detected in sera of cancer patients by immunoprecipitation and Western blot. These findings indicate that proHp promotes angiogenesis via VEGF/VEGFR2 signalling, and serum proHp level may be a useful biomarker for diseases associated with angiogenesis. PMID:25775978

  12. NSK-01105, a Novel Sorafenib Derivative, Inhibits Human Prostate Tumor Growth via Suppression of VEGFR2/EGFR-Mediated Angiogenesis

    PubMed Central

    Yu, Pengfei; Ye, Liang; Wang, Hongbo; Du, Guangying; Zhang, Jianzhao; Zuo, Yanhua; Zhang, Jinghai; Tian, Jingwei

    2014-01-01

    The purpose of this study is to investigate the anti-angiogenic activities of NSK-01105, a novel sorafenib derivative, in in vitro, ex vivo and in vivo models, and explore the potential mechanisms. NSK-01105 significantly inhibited vascular endothelial growth factor (VEGF)-induced migration and tube formation of human umbilical vein endothelial cells at non-cytotoxic concentrations as shown by wound-healing, transwell migration and endothelial cell tube formation assays, respectively. Cell viability and invasion of LNCaP and PC-3 cells were significantly inhibited by cytotoxicity assay and matrigel invasion assay. Furthermore, NSK-01105 also inhibited ex vivo angiogenesis in matrigel plug assay. Western blot analysis showed that NSK-01105 down-regulated VEGF-induced phosphorylation of VEGF receptor 2 (VEGFR2) and the activation of epidermal growth factor receptor (EGFR). Tumor volumes were significantly reduced by NSK-01105 at 60 mg/kg/day in both xenograft models. Immunohistochemical staining demonstrated a close association between inhibition of tumor growth and neovascularization. Collectively, our results suggest a role of NSK-01105 in treatment for human prostate tumors, and one of the potential mechanisms may be attributed to anti-angiogenic activities. PMID:25551444

  13. Activator of G-protein signaling 8 is involved in VEGF-mediated signal processing during angiogenesis.

    PubMed

    Hayashi, Hisaki; Al Mamun, Abdullah; Sakima, Miho; Sato, Motohiko

    2016-03-15

    Activator of G-protein signaling 8 (AGS8, also known as FNDC1) is a receptor-independent accessory protein for the Gβγ subunit, which was isolated from rat heart subjected to repetitive transient ischemia with the substantial development of collaterals. Here, we report the role of AGS8 in vessel formation by endothelial cells. Knockdown of AGS8 by small interfering RNA (siRNA) inhibited vascular endothelial growth factor (VEGF)-induced tube formation, as well as VEGF-stimulated cell growth and migration. VEGF stimulated the phosphorylation of the VEGF receptor-2 (VEGFR-2, also known as KDR), ERK1/2 and p38 MAPK; however, knockdown of AGS8 inhibited these signaling events. Signal alterations by AGS8 siRNA were associated with a decrease of cell surface VEGFR-2 and an increase of VEGFR-2 in the cytosol. Endocytosis blockers did not influence the decrease of VEGFR-2 by AGS8 siRNA, suggesting the involvement of AGS8 in VEGFR-2 trafficking to the plasma membrane. VEGFR-2 formed a complex with AGS8 in cells, and a peptide designed to disrupt AGS8-Gβγ interaction inhibited VEGF-induced tube formation. These data suggest a potential role for AGS8-Gβγ in VEGF signal processing. AGS8 might play a key role in tissue adaptation by regulating angiogenic events.

  14. Discovery of novel VEGFR-2 inhibitors. Part II: biphenyl urea incorporated with salicylaldoxime.

    PubMed

    Gao, Hongping; Su, Ping; Shi, Yaling; Shen, Xiuxiu; Zhang, Yanmin; Dong, Jinyun; Zhang, Jie

    2015-01-27

    A series of novel VEGFR-2 inhibitors containing oxime as hinge binding fragment were described. A strategy of pseudo six-membered ring formed through intramolecular hydrogen bond was employed to mimic the planar quinazoline. The oxime group was firstly introduced to interact with hinge region of VEGFR-2. Most of compounds tested showed moderate to high VEGFR-2 inhibitory activity. In particular, 12l, 12p and 12y exhibited significant enzymatic inhibitory activity as well as potent antiproliferative activity against cancer cells. Molecular docking suggested that the salicylaldoxime formed two hydrogen bonds with hinge region. These biphenylureas could serve as promising lead compounds for developing novel anticancer agents.

  15. 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

  16. 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.

  17. 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

    2016-01-01

    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

  18. 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.

  19. Design, synthesis and biological evaluation of type-II VEGFR-2 inhibitors based on quinoxaline scaffold.

    PubMed

    Shahin, Mai I; Abou El Ella, Dalal A; Ismail, Nasser S M; Abouzid, Khaled A M

    2014-10-01

    In an effort to develop ATP-competitive VEGFR-2 selective inhibitors, a series of new quinoxaline-based derivatives was designed and synthesized. The target compounds were biologically evaluated for their inhibitory activity against VEGFR-2. The design of the target compounds was accomplished after a profound study of the structure activity relationship (SAR) of type-II VEGFR-2 inhibitors. Among the synthesized compounds, 1-(2-((4-methoxyphenyl)amino)-3-oxo-3,4 dihydroquinoxalin-6-yl)-3-phenylurea (VIIa) displayed the highest inhibitory activity against VEGFR-2. Molecular modeling study involving molecular docking and field alignment was implemented to interpret the variable inhibitory activity of the newly synthesized compounds.

  20. VEGF and its receptors in dengue virus infection.

    PubMed

    Kalita, J; Chauhan, P S; Mani, V E; Bhoi, S K; Misra, U K

    2015-09-01

    Vascular permeability determines the severity of dengue virus infection. Vascular endothelial growth factor (VEGF) and its (receptor 1) R1 and (receptor 2) R2 receptors may provide insight about the neurological complications of dengue. We report VEGF and its R1 and R2 receptors level in dengue patients and correlate these with neurological complications. Consecutive patients with dengue were subjected to clinical and neurological evaluations. Their blood counts, serum chemistry, including liver and kidney function tests, serum creatine kinase (CK), and albumin were measured. VEGF, VEGFR1 and VEGFR2 were measured by ELISA in the patients and 16 matched controls. Twenty four patients with dengue were included whose ages ranged between 15 and 67 years, and nine of whom were females. Serum VEGF level was insignificantly lower in dengue patients whereas VEGFR1 was significantly higher (P = 0.01) and VEGFR2 was significantly lower (P = 0.005) compared to controls. VEGFR2 correlated with systolic blood pressure, coagulopathy, and serum CK levels. None of the other clinical and biochemical parameters correlated with VEGF and VEGFR1 levels. VEGFR1 and R2 normalized at 1 month. VEGFR2 correlates with the clinical severity of dengue and muscle dysfunction.

  1. Switchable probes: pH-triggered and VEGFR2 targeted peptides screening through imprinting microarray.

    PubMed

    Qian, Yixia; Wang, Weizhi; Wang, Zihua; Han, Qiuju; Jia, Xiangqian; Yang, Shu; Hu, Zhiyuan

    2016-04-28

    One switchable affinity peptide, STP, is screened out from a high-throughput library by an integrated imprinting microarray. STP is pH triggered and also the ligand of the marker VEGFR2. Efficient cell recognition and penetration as well as an in vivo image could be "turned on" and accelerated only in the condition of VEGFR2 overexpression and a mild acidic environment.

  2. De novo design of VEGFR-2 tyrosine kinase inhibitors based on a linked-fragment approach.

    PubMed

    Liu, Yi-Zhou; Wang, Xiao-Li; Wang, Xin-Ying; Yu, Ri-Lei; Liu, Dong-Qing; Kang, Cong-Min

    2016-09-01

    Vascular endothelial growth factor receptor-2 (VEGFR-2) tyrosine kinase inhibitors have been demonstrated to possess substantial antitumor activity. VEGFR-2 tyrosine kinase inhibitors are crucial for development of antitumor drugs. Based on the crystal structure of VEGFR-2 tyrosine kinase, a linked-fragment strategy was employed to design novel VEGFR-2 tyrosine kinase inhibitors, and 1000 compounds were generated in this process. Absorption, distribution, metabolism, excretion and toxicity (ADMET) were used to screen the 1000 compounds, and 59 compounds were acceptable. Scaffold hopping was then used for further screening, and only four compounds were obtained in this way. Then, the binding energy of the four molecules to VEGFR-2 tyrosine kinase was calculated using molecular docking, and their values were found to be lower than that of Sorafenib. Finally, molecular dynamics simulations were performed on the complex of the compound with the lowest binding energy with VEGFR-2 tyrosine kinase, and the binding model was analyzed. At the end, four chemical entities with novel structures were obtained, and were suggested for experimental testing in future studies. PMID:27558799

  3. Corosolic Acid Inhibits Hepatocellular Carcinoma Cell Migration by Targeting the VEGFR2/Src/FAK Pathway

    PubMed Central

    Ku, Chung-Yu; Wang, Ying-Ren; Lin, Hsuan-Yuan; Lu, Shao-Chun; Lin, Jung-Yaw

    2015-01-01

    Inhibition of VEGFR2 activity has been proposed as an important strategy for the clinical treatment of hepatocellular carcinoma (HCC). In this study, we identified corosolic acid (CA), which exists in the root of Actinidia chinensis, as having a significant anti-cancer effect on HCC cells. We found that CA inhibits VEGFR2 kinase activity by directly interacting with the ATP binding pocket. CA down-regulates the VEGFR2/Src/FAK/cdc42 axis, subsequently decreasing F-actin formation and migratory activity in vitro. In an in vivo model, CA exhibited an effective dose (5 mg/kg/day) on tumor growth. We further demonstrate that CA has a synergistic effect with sorafenib within a wide range of concentrations. In conclusion, this research elucidates the effects and molecular mechanism for CA on HCC cells and suggests that CA could be a therapeutic or adjuvant strategy for patients with aggressive HCC. PMID:25978354

  4. 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

  5. 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.

  6. 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.

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

    PubMed Central

    2010-01-01

    Background Different isoforms of VEGF-A (mainly VEGF121, VEGF165 and VEGF189) have been shown to display particular angiogenic properties in the generation of a functional tumor vasculature. Recently, a novel class of VEGF-A isoforms, designated as VEGFxxxb, generated through alternative splicing, have been described. Previous studies have suggested that these isoforms may inhibit angiogenesis. In the present work we have produced recombinant VEGF121/165b proteins in the yeast Pichia pastoris and constructed vectors to overexpress these isoforms and assess their angiogenic potential. Results Recombinant VEGF121/165b proteins generated either in yeasts or mammalian cells activated VEGFR2 and its downstream effector ERK1/2, although to a lesser extent than VEGF165. Furthermore, treatment of endothelial cells with VEGF121/165b increased cell proliferation compared to untreated cells, although such stimulation was lower than that induced by VEGF165. Moreover, in vivo angiogenesis assays confirmed angiogenesis stimulation by VEGF121/165b isoforms. A549 and PC-3 cells overexpressing VEGF121b or VEGF165b (or carrying the PCDNA3.1 empty vector, as control) and xenotransplanted into nude mice showed increased tumor volume and angiogenesis compared to controls. To assess whether the VEGFxxxb isoforms are differentially expressed in tumors compared to healthy tissues, immunohistochemical analysis was conducted on a breast cancer tissue microarray. A significant increase (p < 0.05) in both VEGFxxxb and total VEGF-A protein expression in infiltrating ductal carcinomas compared to normal breasts was observed. A positive significant correlation (r = 0.404, p = 0.033) between VEGFxxxb and total VEGF-A was found. Conclusions Our results demonstrate that VEGF121/165b are not anti-angiogenic, but weakly angiogenic isoforms of VEGF-A. In addition, VEGFxxxb isoforms are up-regulated in breast cancer in comparison with non malignant breast tissues. These results are to be taken into

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

    PubMed

    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 Vegfr2(Y949F/Y949F) leads to VEGFA-resistant endothelial adherens junctions and a block in molecular extravasation. Vessels in Vegfr2(Y949F/Y949F) mice remain sensitive to inflammatory cytokines, and vascular morphology, blood pressure and flow parameters are normal. Tumour-bearing Vegfr2(Y949F/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

  9. 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

  10. VEGF signaling in cancer treatment.

    PubMed

    Sia, Daniela; Alsinet, Clara; Newell, Pippa; Villanueva, Augusto

    2014-01-01

    Induction of angiogenesis represents one of the major hallmarks of cancer. The growth of new vessels is crucial to provide malignant cells with an adequate supply of oxygen and nutrients. It is generally accepted that vascular endothelial growth factor (VEGF) is a major driver of the angiogenic process in physiological and pathological processes in both embryo and adult. VEGF is often found overexpressed in tumors, as well as its receptors VEGFR1 and VEGFR2. Hence, several different strategies have been designed to target VEGF signal transduction. In the last decades, multiple inhibitors have been therapeutically validated in preclinical models and several clinical trials. Neutralizing monoclonal antibodies against VEGF and small molecule tyrosine kinase inhibitors targeting VEGFRs have been shown to block its angiogenic activity, resulting in tumor vascular regression, anti-tumor effects and improvements in patient survival. However, side effects and lack of efficacy in some instances challenge the potential clinical impact of these therapies. This review examines the role of VEGF signaling in cancer and outlines the current status of anti-angiogenic therapies against VEGF pathway.

  11. scVEGF Microbubble Ultrasound Contrast Agents: A Novel Probe for Ultrasound Molecular Imaging of Tumor Angiogenesis

    PubMed Central

    Christopher R., Anderson; Joshua J., Rychak; Marina, Backer; Joseph, Backer; Klaus, Ley; Alexander L., Klibanov

    2012-01-01

    Objective To develop a novel microbubble (MB) ultrasound contrast agent covalently coupled to a recombinant single-chain vascular endothelial growth factor construct (scVEGF) through uniform site-specific conjugation for ultrasound imaging of tumor angiogenesis. Methods Ligand conjugation to maleimide-bearing MB by thioether bonding was first validated with a fluorophore (BODIPY-cystine), and covalently bound dye was detected by fluorometry and flow cytometry. MBs were subsequently site-specifically conjugated to cysteine-containing Cys-tag in scVEGF, and bound scVEGF was quantified by enzyme-linked immunosorbent assay. Targeted adhesion of scVEGF-MB was investigated with in vitro parallel plate flow chamber assays with recombinant murine VEGFR-2 substrates and human VEGFR-2-expressing porcine endothelial cells (PAE/KDR). A wall-less ultrasound flow phantom, with flow channels coated with immobilized VEGFR-2, was used to detect adhesion of scVEGF-MB with contrast ultrasound imaging. A murine model of colon adenocarcinoma was used to assess retention of scVEGF-MB with contrast ultrasound imaging during tumor angiogenesis in vivo. Results Proof-of-principle of ligand conjugation to maleimide-bearing MB was demonstrated with a BODIPY-cysteine fluorophore. Conjugation of BODIPY to MB saturated at 10-fold molar excess BODIPY relative to maleimide groups on MB surfaces. MB reacted with scVEGF and led to the conjugation of 1.2 × 105 molecules scVEGF per MB. Functional adhesion of sc-VEGF-MB was shown in parallel plate flow chamber assays. At a shear stress of 1.0 dynes/cm2, scVEGF-MB exhibited 5-fold higher adhesion to both recombinant VEGFR-2 substrates and VEGFR-2-expressing endothelial cells compared with nontargeted control MB. Additionally, scVEGF-MB targeted to immobilized VEGFR-2 in an ultrasound flow phantom showed an 8-fold increase in mean acoustic signal relative to casein-coated control channels. In an in vivo model of tumor angiogenesis, scVEGF MB showed

  12. Chronic high-magnitude cyclic stretch stimulates EC inflammatory response via VEGF receptor 2-dependent mechanism.

    PubMed

    Gawlak, Grzegorz; Son, Sophia; Tian, Yufeng; O'Donnell, James J; Birukov, Konstantin G; Birukova, Anna A

    2016-06-01

    Ventilator-induced lung injury (VILI) is associated with activated inflammatory signaling, such as cytokine production by endothelial and epithelial cells and macrophages, although the precise mechanisms of inflammatory activation induced by VILI-relevant cyclic stretch (CS) amplitude remain poorly understood. We show that exposure of human pulmonary endothelial cells (EC) to chronic CS at 18% linear distension (18% CS), but not at physiologically relevant 5% CS, induces "EC-activated phenotype," which is characterized by time-dependent increase in ICAM1 and VCAM1 expression. A preconditioning of 18% CS also increased in a time-dependent fashion the release of soluble ICAM1 (sICAM1) and IL-8. Investigation of potential signaling mechanisms of CS-induced EC inflammatory activation showed that 18% CS, but not 5% CS, induced time-dependent upregulation of VEGF receptor 2 (VEGFR2), as monitored by increased protein expression and VEGFR2 tyrosine phosphorylation. Both CS-induced VEGFR2 expression and tyrosine phosphorylation were abrogated by cotreatment with reactive oxygen species inhibitor, N-acetyl cysteine. Molecular inhibition of VEGFR2 expression by gene-specific siRNA or treatment with VEGFR2 pharmacological inhibitor SU-1498 attenuated CS-induced activation of ICAM1 and VCAM1 expression and sICAM1 release. Chronic EC preconditioning at 18% CS augmented EC inflammation and barrier-disruptive response induced by proinflammatory cytokine TNF-α. This effect of chronic 18% CS preconditioning was attenuated by siRNA-induced VEGFR2 knockdown. This study demonstrates for the first time a VEGFR2-dependent mechanism of EC inflammatory activation induced by pathological CS. We conclude that, despite the recognized role of VEGF as a prosurvival and angiogenic factor, excessive activation of VEGFR2 signaling by high-tidal-volume lung mechanical ventilation may contribute to ventilator-induced (biotrauma) lung inflammation and barrier dysfunction by augmenting cell response

  13. 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

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

    PubMed

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

    2016-05-01

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

  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. 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.

  17. 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

  18. Ramucirumab, a fully human mAb to the transmembrane signaling tyrosine kinase VEGFR-2 for the potential treatment of cancer.

    PubMed

    Krupitskaya, Yelena; Wakelee, Heather A

    2009-06-01

    Angiogenesis is essential for tumor growth, invasion and metastasis, and is mediated, at least in part, by a large family of VEGF ligands and receptors. Ramucirumab, which is being developed by ImClone Systems Inc, is a fully human mAb that binds human VEGFR-2, thus blocking VEGF binding and inhibiting angiogenesis. Proof-of-concept preclinical studies with the mouse mAb DC-101 supported this hypothesis, and ramucirumab inhibited cell proliferation in vitro, as well as tumor progression in mouse xenograft models of human cancer. Ramucirumab was well tolerated on weekly and fortnightly schedules in phase I clinical trials in patients with advanced cancers; mechanism-related DLTs were hypertension and deep venous thrombosis. Stable disease was also observed in several patients treated on either schedule, and several patients on the weekly schedule exhibited partial responses. At the time of publication, ramucirumab was undergoing assessment in phase II trials as a monotherapy in hepatocellular, renal cell and ovarian carcinomas. Ramucirumab was also in phase II trials in combination with dacarbazine in melanoma, with mitoxantrone/prednisone in prostate cancer, with carboplatin/paclitaxel in NSCLC and with oxaliplatin/folinic acid/5-fluorouracil in colorectal cancer. A phase III trial in combination with docetaxel in breast cancer was also ongoing. Pending results from these trials, ramucirumab may be a useful addition to current antiangiogenic therapies. The results are awaited with interest.

  19. GEP100-Arf6-AMAP1-Cortactin Pathway Frequently Used in Cancer Invasion Is Activated by VEGFR2 to Promote Angiogenesis

    PubMed Central

    Hashimoto, Ari; Hashimoto, Shigeru; Ando, Ryo; Noda, Kosuke; Ogawa, Eiji; Kotani, Hirokazu; Hirose, Mayumi; Menju, Toshi; Morishige, Masaki; Manabe, Toshiaki; Toda, Yoshinobu; Ishida, Susumu; Sabe, Hisataka

    2011-01-01

    Angiogenesis and cancer invasiveness greatly contribute to cancer malignancy. Arf6 and its effector, AMAP1, are frequently overexpressed in breast cancer, and constitute a central pathway to induce the invasion and metastasis. In this pathway, Arf6 is activated by EGFR via GEP100. Arf6 is highly expressed also in human umbilical vein endothelial cells (HUVECs) and is implicated in angiogenesis. Here, we found that HUVECs also highly express AMAP1, and that vascular endothelial growth factor receptor-2 (VEGFR2) recruits GEP100 to activate Arf6. AMAP1 functions by binding to cortactin in cancer invasion and metastasis. We demonstrate that the same GEP100-Arf6-AMAP1-cortactin pathway is essential for angiogenesis activities, including cell migration and tubular formation, as well as for the enhancement of cell permeability and VE-cadherin endocytosis of VEGF-stimulated HUVECs. Components of this pathway are highly expressed in pathologic angiogenesis, and blocking of this pathway effectively inhibits VEGF- or tumor-induced angiogenesis and choroidal neovascularization. The GEP100-Arf6-AMAP1-cortactin pathway, activated by receptor tyrosine kinases, appears to be common in angiogenesis and cancer invasion and metastasis, and provides their new therapeutic targets. PMID:21858086

  20. 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.

  1. 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. PMID:27420801

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  3. Endothelial epsin deficiency decreases tumor growth by enhancing VEGF signaling.

    PubMed

    Pasula, Satish; Cai, Xiaofeng; Dong, Yunzhou; Messa, Mirko; McManus, John; Chang, Baojun; Liu, Xiaolei; Zhu, Hua; Mansat, Robert Silasi; Yoon, Seon-Joo; Hahn, Scott; Keeling, Jacob; Saunders, Debra; Ko, Genevieve; Knight, John; Newton, Gail; Luscinskas, Francis; Sun, Xiaohong; Towner, Rheal; Lupu, Florea; Xia, Lijun; Cremona, Ottavio; De Camilli, Pietro; Min, Wang; Chen, Hong

    2012-12-01

    Epsins are a family of ubiquitin-binding, endocytic clathrin adaptors. Mice lacking both epsins 1 and 2 (Epn1/2) die at embryonic day 10 and exhibit an abnormal vascular phenotype. To examine the angiogenic role of endothelial epsins, we generated mice with constitutive or inducible deletion of Epn1/2 in vascular endothelium. These mice exhibited no abnormal phenotypes under normal conditions, suggesting that lack of endothelial epsins 1 and 2 did not affect normal blood vessels. In tumors, however, loss of epsins 1 and 2 resulted in disorganized vasculature, significantly increased vascular permeability, and markedly retarded tumor growth. Mechanistically, we show that VEGF promoted binding of epsin to ubiquitinated VEGFR2. Loss of epsins 1 and 2 specifically impaired endocytosis and degradation of VEGFR2, which resulted in excessive VEGF signaling that compromised tumor vascular function by exacerbating nonproductive leaky angiogenesis. This suggests that tumor vasculature requires a balance in VEGF signaling to provide sufficient productive angiogenesis for tumor development and that endothelial epsins 1 and 2 negatively regulate the output of VEGF signaling. Promotion of excessive VEGF signaling within tumors via a block of epsin 1 and 2 function may represent a strategy to prevent normal angiogenesis in cancer patients who are resistant to anti-VEGF therapies.

  4. 6″-Debromohamacanthin A, a Bis (Indole) Alkaloid, Inhibits Angiogenesis by Targeting the VEGFR2-Mediated PI3K/AKT/mTOR Signaling Pathways

    PubMed Central

    Kim, Gi Dae; Cheong, Oug Jae; Bae, Song Yi; Shin, Jongheon; Lee, Sang Kook

    2013-01-01

    Hamacanthins, bis (indole) alkaloids, are found in a few marine sponges, including Spongosorites sp. Hamacanthins have been shown to possess cytotoxic, antibacterial and antifungal activities. However, the precise mechanism for the biological activities of hamacanthins has not yet been elucidated. In the present study, the anti-angiogenic effects of 6″-debromohamacanthin A (DBHA), an active component of isolated hamacanthins, were evaluated in cultured human umbilical vascular endothelial cells (HUVEC) and endothelial-like cells differentiated from mouse embryonic stem (mES) cells. DBHA significantly inhibited vascular endothelial growth factor (VEGF)-induced cell proliferation, migration and tube formation in the HUVEC. DBHA also suppressed the capillary-like structure formation and the expression of platelet endothelial cell adhesion molecule (PECAM), an endothelial biomarker, in mES cell-derived endothelial-like cells. To further understand the precise molecular mechanism of action, VEGF-mediated signaling pathways were analyzed in HUVEC cells and mES cell-derived endothelial-like cells. DBHA suppressed the VEGF-induced expression of MAPKs (p38, ERK and SAPK/JNK) and the PI3K/AKT/mTOR signaling pathway. In addition, DBHA inhibited microvessel sprouting in mES/EB-derived embryoid bodies. In an ex vivo model, DBHA also suppressed the microvessel sprouting of mouse aortic rings. The findings suggest for the first time that DBHA inhibits angiogenesis by targeting the vascular endothelial growth factor receptor 2 (VEGFR2)-mediated PI3K/AKT/mTOR signaling pathway in endothelial cells. PMID:23549281

  5. 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

  6. 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.

  7. VEGF and thrombin induce MKP-1 through distinct signaling pathways: role for MKP-1 in endothelial cell migration.

    PubMed

    Kinney, Corttrell M; Chandrasekharan, Unni M; Mavrakis, Lori; DiCorleto, Paul E

    2008-01-01

    We have previously reported that MAPK phosphatase-1 (MKP-1/CL100) is a thrombin-responsive gene in endothelial cells (ECs). We now show that VEGF is another efficacious activator of MKP-1 expression in human umbilical vein ECs. VEGF-A and VEGF-E maximally induced MKP-1 expression in ECs; however, the other VEGF subtypes had no effect. Using specific neutralizing antibodies, we determined that VEGF induced MKP-1 specifically through VEGF receptor 2 (VEGFR-2), leading to the downstream activation of JNK. The VEGF-A(165) isoform stimulated MKP-1 expression, whereas the VEGF-A(162) isoform induced the gene to a lesser extent, and the VEGF-A(121) isoform had no effect. Furthermore, specific blocking antibodies against neuropilins, VEGFR-2 coreceptors, blocked MKP-1 induction. A Src kinase inhibitor (PP1) completely blocked both VEGF- and thrombin-induced MKP-1 expression. A dominant negative approach revealed that Src kinase was required for VEGF-induced MKP-1 expression, whereas Fyn kinase was critical for thrombin-induced MKP-1 expression. Moreover, VEGF-induced MKP-1 expression required JNK, whereas ERK was critical for thrombin-induced MKP-1 expression. In ECs treated with short interfering (si)RNA targeting MKP-1, JNK, ERK, and p38 phosphorylation were prolonged following VEGF stimulation. An ex vivo aortic angiogenesis assay revealed a reduction in VEGF- and thrombin-induced sprout outgrowth in segments from MKP-1-null mice versus wild-type controls. MKP-1 siRNA also significantly reduced VEGF-induced EC migration using a transwell assay system. Overall, these results demonstrate distinct MAPK signaling pathways for thrombin versus VEGF induction of MKP-1 in ECs and point to the importance of MKP-1 induction in VEGF-stimulated EC migration. PMID:18003751

  8. 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.

  9. 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. PMID:26902100

  10. 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. PMID:26924457

  11. VEGF receptors mediate hypoxic remodeling of adult ovine carotid arteries.

    PubMed

    Adeoye, Olayemi O; Bouthors, Vincent; Hubbell, Margaret C; Williams, James M; Pearce, William J

    2014-10-01

    Recent studies suggest that VEGF contributes to hypoxic remodeling of arterial smooth muscle, although hypoxia produces only transient increases in VEGF that return to normoxic levels despite sustained changes in arterial structure and function. To explore how VEGF might contribute to long-term hypoxic vascular remodeling, this study explores the hypothesis that chronic hypoxia produces sustained increases in smooth muscle VEGF receptor density that mediate long-term vascular effects of hypoxia. Carotid arteries from adult sheep maintained at sea level or altitude (3,820 m) for 110 days were harvested and denuded of endothelium. VEGF levels were similar in chronically hypoxic and normoxic arteries, as determined by immunoblotting. In contrast, VEGF receptor levels were significantly increased by 107% (VEGF-R1) and 156% (VEGF-R2) in hypoxic compared with normoxic arteries. In arteries that were organ cultured 24 h with 3 nM VEGF, VEGF replicated effects of hypoxia on abundances of smooth muscle α actin (SMαA), myosin light chain kinase (MLCK), and MLC20 and the effects of hypoxia on colocalization of MLC20 with SMαA, as measured via confocal microscopy. VEGF did not replicate the effects of chronic hypoxia on colocalization of MLCK with SMαA or MLCK with MLC20, suggesting that VEGF's role in hypoxic remodeling is highly protein specific, particularly for contractile protein organization. VEGF effects in organ culture were inhibited by VEGF receptor blockers vatalinib (240 nM) and dasatinib (6.3 nM). These findings support the hypothesis that long-term upregulation of VEGF receptors help mediate sustained effects of hypoxia on the abundance and colocalization of contractile proteins in arterial smooth muscle. PMID:25038104

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

    PubMed

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

    2008-10-01

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

  13. Myoferlin expression in non-small cell lung cancer: Prognostic role and correlation with VEGFR-2 expression

    PubMed Central

    SONG, DAE HYUN; KO, GYUNG HYUCK; LEE, JEONG HEE; LEE, JONG SIL; LEE, GYEONG-WON; KIM, HYEON CHEOL; YANG, JUNG WOOK; HEO, ROK WON; ROH, GU SEOB; HAN, SUN-YOUNG; KIM, DONG CHUL

    2016-01-01

    Myoferlin is a protein that is associated with cellular repair following injury. The expression of myoferlin in breast cancer and pancreatic adenocarcinoma has been reported to correlate with tumor invasiveness, epithelial to mesenchymal transition and an adverse prognosis. In the present study, myoferlin expression was investigated in non-small cell lung carcinoma (NSCLC), along with its association with patient prognosis and the expression of a number of other proteins. A total of 148 patients exhibiting NSCLC were enrolled in the present study. The survival data of all patients was examined, and myoferlin, vascular endothelial growth factor receptor-2 (VEGFR-2), epidermal growth factor receptor, E-cadherin, β-catenin, thyroid transcription factor-1 and tumor protein p63 expression was investigated via immunohistochemical staining of tissue microarrays. Myoferlin expression was detected in the cytoplasm of 75/148 (50.7%) of the NSCLC cases. In the adenocarcinoma cases, myoferlin-positive patients possessed a poorer prognosis (odds ratio, 2.94; P=0.339). In the squamous cell carcinoma cases, myoferlin expression was significantly associated with VEGFR-2 expression (P=0.001). Immunohistochemical staining for VEGFR-2 and myoferlin expression indicated similar features and cytoplasmic staining in tumor cells. As VEGFR-2 is a significant target for novel anticancer therapies, it is anticipated that myoferlin may also possess the potential to become a novel clinical target for the treatment of NSCLC. PMID:26893682

  14. Vandetanib inhibits both VEGFR-2 and EGFR signalling at clinically relevant drug levels in preclinical models of human cancer.

    PubMed

    Brave, Sandra R; Odedra, Rajesh; James, Neil H; Smith, Neil R; Marshall, Gayle B; Acheson, Kerry L; Baker, Dawn; Howard, Zoe; Jackson, Lynsay; Ratcliffe, Kirsty; Wainwright, Anna; Lovick, Susan C; Hickinson, D Mark; Wilkinson, Robert W; Barry, Simon T; Speake, Georgina; Ryan, Anderson J

    2011-07-01

    Vandetanib is a multi-targeted receptor tyrosine kinase inhibitor that is in clinical development for the treatment of solid tumours. This preclinical study examined the inhibition of two key signalling pathways (VEGFR-2, EGFR) at drug concentrations similar to those achieved in the clinic, and their contribution to direct and indirect antitumour effects of vandetanib. For in vitro studies, receptor phosphorylation was assessed by Western blotting and ELISA, cell proliferation was assessed using a cell viability endpoint, and effects on cell cycle determined using flow cytometry. For in vivo studies, Western blotting, ELISA and immunohistochemistry (IHC) were used to assess receptor phosphorylation. Cell culture experiments demonstrated that anti-proliferative effects of vandetanib resulted from inhibition of either EGFR or VEGFR-2 signalling in endothelial cells, but were associated with inhibition of EGFR signalling in tumour cells. Vandetanib inhibited both EGFR and VEGFR-2 signalling in normal lung tissue and in tumour xenografts. In a lung cancer model expressing an activating EGFR mutation, the activity of vandetanib was similar to that of a highly selective EGFR inhibitor (gefitinib), and markedly greater than that of a highly selective VEGFR inhibitor (vatalanib). These data suggest that at the plasma exposures achieved in the clinic, vandetanib will significantly inhibit both VEGFR-2 and EGFR signalling, and that both inhibition of angiogenesis and direct inhibition of tumour cell growth can contribute to treatment response. PMID:21537841

  15. Humanized anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles, an antibody conjugate with potent and selective anti-hepatocellular carcinoma activity.

    PubMed

    Xiangbao, Yin; Linquan, Wu; Mingwen, Huang; Fan, Zhou; Kai, Wang; Xin, Yu; Kaiyang, Wang; Huaqun, Fu

    2014-06-01

    Low sensitivity of tumor tissue, targeting and sustained release of the drug are bottlenecks of the effect of chemotherapy on hepatocellular carcinoma. In this study, we used the ribosome display technology to screen human anti-VEGFR 2-single-chain antibody (ScFv) that could target directly to VEGFR2, and nanotechnology to prepare As2O3-nanoparticles. Then we built anti-VEGFR-2ScFv-As2O3-stealth nanoparticles using molecular coupling technology, which significantly increased anti-tumor effect while reducing toxicity. The in vivo tissue targeting distribution and anti-tumor effects of the anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles were investigated. Our results showed that anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles could inhibit the development of liver cancer xenograft as a targeting agent and also significantly inhibit angiogenesis.

  16. Humanized anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles, an antibody conjugate with potent and selective anti-hepatocellular carcinoma activity.

    PubMed

    Xiangbao, Yin; Linquan, Wu; Mingwen, Huang; Fan, Zhou; Kai, Wang; Xin, Yu; Kaiyang, Wang; Huaqun, Fu

    2014-06-01

    Low sensitivity of tumor tissue, targeting and sustained release of the drug are bottlenecks of the effect of chemotherapy on hepatocellular carcinoma. In this study, we used the ribosome display technology to screen human anti-VEGFR 2-single-chain antibody (ScFv) that could target directly to VEGFR2, and nanotechnology to prepare As2O3-nanoparticles. Then we built anti-VEGFR-2ScFv-As2O3-stealth nanoparticles using molecular coupling technology, which significantly increased anti-tumor effect while reducing toxicity. The in vivo tissue targeting distribution and anti-tumor effects of the anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles were investigated. Our results showed that anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles could inhibit the development of liver cancer xenograft as a targeting agent and also significantly inhibit angiogenesis. PMID:24855034

  17. Recombinant Human VEGF165b Inhibits Experimental Choroidal Neovascularization

    PubMed Central

    Hua, Jing; Spee, Christine; Kase, Satoru; Rennel, Emma S.; Magnussen, Anette L.; Qiu, Yan; Varey, Alex; Dhayade, Sandeep; Churchill, Amanda J.; Harper, Steven J.; Hinton, David R.

    2010-01-01

    Purpose. Vascular endothelial growth factor (VEGF-A) is the principal stimulator of angiogenesis in wet age-related macular degeneration (AMD). However, VEGF-A is generated by alternate splicing into two families, the proangiogenic VEGF-Axxx family and the antiangiogenic VEGF-Axxxb family. It is the proangiogenic family that is responsible for the blood vessel growth seen in AMD. Methods. To determine the role of antiangiogenic isoforms of VEGF-A as inhibitors of choroidal neovascularization, the authors used a model of laser-induced choroidal neovascularization in the mouse eye and investigated VEGF-A165b effects on endothelial cells and VEGFRs in vitro. Results. VEGF-A165b inhibited VEGF-A165–mediated endothelial cell migration with a dose effect similar to that of ranibizumab and bevacizumab and 200-fold more potent than that of pegaptanib. VEGF-A165b bound both VEGFR1 and VEGFR2 with affinity similar to that of VEGF-A165. After laser injury, mice were injected either intraocularly or subcutaneously with recombinant human VEGF-A165b. Intraocular injection of rhVEGF-A165b gave a pronounced dose-dependent inhibition of fluorescein leakage, with an IC50 of 16 pg/eye, neovascularization (IC50, 0.8 pg/eye), and lesion as assessed by histologic staining (IC50, 8 pg/eye). Subcutaneous administration of 100 μg twice a week also inhibited fluorescein leakage and neovascularization and reduced lesion size. Conclusions. These results show that VEGF-A165b is a potent antiangiogenic agent in a mouse model of age-related macular degeneration and suggest that increasing the ratio of antiangiogenic-to-proangiogenic isoforms may be therapeutically effective in this condition. PMID:20237252

  18. 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.

  19. Targeting Neuropilin-1 to Inhibit VEGF Signaling in Cancer: Comparison of Therapeutic Approaches

    PubMed Central

    Gabhann, Feilim Mac; Popel, Aleksander S

    2006-01-01

    Angiogenesis (neovascularization) plays a crucial role in a variety of physiological and pathological conditions including cancer, cardiovascular disease, and wound healing. Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis. Multiple VEGF receptors are expressed on endothelial cells, including signaling receptor tyrosine kinases (VEGFR1 and VEGFR2) and the nonsignaling co-receptor Neuropilin-1. Neuropilin-1 binds only the isoform of VEGF responsible for pathological angiogenesis (VEGF165), and is thus a potential target for inhibiting VEGF signaling. Using the first molecularly detailed computational model of VEGF and its receptors, we have shown previously that the VEGFR–Neuropilin interactions explain the observed differential effects of VEGF isoforms on VEGF signaling in vitro, and demonstrated potent VEGF inhibition by an antibody to Neuropilin-1 that does not block ligand binding but blocks subsequent receptor coupling. In the present study, we extend that computational model to simulation of in vivo VEGF transport and binding, and predict the in vivo efficacy of several Neuropilin-targeted therapies in inhibiting VEGF signaling: (a) blocking Neuropilin-1 expression; (b) blocking VEGF binding to Neuropilin-1; (c) blocking Neuropilin–VEGFR coupling. The model predicts that blockade of Neuropilin–VEGFR coupling is significantly more effective than other approaches in decreasing VEGF–VEGFR2 signaling. In addition, tumor types with different receptor expression levels respond differently to each of these treatments. In designing human therapeutics, the mechanism of attacking the target plays a significant role in the outcome: of the strategies tested here, drugs with similar properties to the Neuropilin-1 antibody are predicted to be most effective. The tumor type and the microenvironment of the target tissue are also significant in determining therapeutic efficacy of each of the treatments studied. PMID:17196035

  20. 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.

  1. 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.

  2. Roxithromycin inhibits VEGF-induced human airway smooth muscle cell proliferation: Opportunities for the treatment of asthma.

    PubMed

    Pei, Qing-Mei; Jiang, Ping; Yang, Min; Qian, Xue-Jiao; Liu, Jiang-Bo; Kim, Sung-Ho

    2016-10-01

    Asthma is a chronic respiratory disease characterized by reversible airway obstruction with persistent airway inflammation and airway remodelling, which is associated with increased airway smooth muscle (ASM) mass. Roxithromycin (RXM) has been widely used in asthma treatment; however, its mechanism of action is poorly understood. Vascular endothelial growth factor (VEGF) has been implicated in inflammatory and airway blood vessel remodelling in patients with asthma, and shown to promote ASM cell proliferation. Here, we investigated the effect of RXM on VEGF-induced ASM cell proliferation and attempted to elucidate the underlying mechanisms of action. We tested the effect of RXM on proliferation and cell cycle progression, as well as on the expression of phospho-VEGF receptor 2 (VEGFR2), phospho-extracellular signal-regulated kinase 1/2 (ERK1/2), phospho-Akt, and caveolin-1 in VEGF-stimulated ASM cells. RXM inhibited VEGF-induced ASM cell proliferation and induced cell cycle arrest. Additionally, VEGF-induced ASM cell proliferation was suppressed by inhibiting the activity of ERK1/2, but not that of Akt. Furthermore, RXM treatment inhibits VEGF-induced activation of VEGFR2 and ERK and downregulation of caveolin-1 in a dose-dependent manner. RXM also inhibited TGF-β-induced VEGF secretion by ASM cells and BEAS-2B cells. Collectively, our findings suggest that RXM inhibits VEGF-induced ASM cell proliferation by suppression of VEGFR2 and ERK1/2 activation and caveolin-1 down-regulation, which may be involved in airway remodelling. Further elucidation of the mechanisms underlying these observations should enable the development of treatments for smooth muscle hyperplasia-associated diseases of the airway such as asthma. PMID:27587274

  3. Design, synthesis and biological evaluation of N-phenylquinazolin-4-amine hybrids as dual inhibitors of VEGFR-2 and HDAC.

    PubMed

    Peng, Fan-Wei; Xuan, Ji; Wu, Ting-Ting; Xue, Jia-Yu; Ren, Zi-Wei; Liu, Da-Ke; Wang, Xiu-Qi; Chen, Xin-Hang; Zhang, Jia-Wei; Xu, Yun-Gen; Shi, Lei

    2016-02-15

    A single agent that simultaneously inhibits multiple targets may offer greater therapeutic benefits in cancer than single-acting agents through interference with multiple pathways and potential synergistic action. In this work, a series of hybrids bearing N-phenylquinazolin-4-amine and hydroxamic acid moieties were designed and identified as dual VEGFR-2/HDAC inhibitors. Compound 6fd exhibited the most potent inhibitory activity against HDAC with IC50 of 2.2 nM and strong inhibitory effect against VEGFR-2 with IC50 of 74 nM. It also showed the most potent inhibitory activity against a human breast cancer cell line MCF-7 with IC50 of 0.85 μM. Docking simulation supported the initial pharmacophoric hypothesis and suggested a common mode of interaction at the active binding sites of VEGFR-2 and HDLP ((Histone Deacetylase-Like Protein), which demonstrates that compound 6fd is a potential agent for cancer therapy deserving further researching. PMID:26741358

  4. 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

  5. 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.

  6. KRIT1 Protein Depletion Modifies Endothelial Cell Behavior via Increased Vascular Endothelial Growth Factor (VEGF) Signaling*

    PubMed Central

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

    2014-01-01

    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. PMID:25320085

  7. VEGF-A/VEGFR Inhibition Restores Hematopoietic Homeostasis in the Bone Marrow and Attenuates Tumor Growth.

    PubMed

    O'Donnell, Rebekah K; Falcon, Beverly; Hanson, Jeff; Goldstein, Whitney E; Perruzzi, Carole; Rafii, Shahin; Aird, William C; Benjamin, Laura E

    2016-02-01

    Antiangiogenesis-based cancer therapies, specifically those targeting the VEGF-A/VEGFR2 pathway, have been approved for subsets of solid tumors. However, these therapies result in an increase in hematologic adverse events. We surmised that both the bone marrow vasculature and VEGF receptor-positive hematopoietic cells could be impacted by VEGF pathway-targeted therapies. We used a mouse model of spontaneous breast cancer to decipher the mechanism by which VEGF pathway inhibition alters hematopoiesis. Tumor-bearing animals, while exhibiting increased angiogenesis at the primary tumor site, showed signs of shrinkage in the sinusoidal bone marrow vasculature accompanied by an increase in the hematopoietic stem cell-containing Lin-cKit(+)Sca1(+) (LKS) progenitor population. Therapeutic intervention by targeting VEGF-A, VEGFR2, and VEGFR3 inhibited tumor growth, consistent with observed alterations in the primary tumor vascular bed. These treatments also displayed systemic effects, including reversal of the tumor-induced shrinkage of sinusoidal vessels and altered population balance of hematopoietic stem cells in the bone marrow, manifested by the restoration of sinusoidal vessel morphology and hematopoietic homeostasis. These data indicate that tumor cells exert an aberrant systemic effect on the bone marrow microenvironment and VEGF-A/VEGFR targeting restores bone marrow function.

  8. 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

  9. Expression and localization of vascular endothelial growth factor A (VEGFA) and its two receptors (VEGFR1/FLT1 and VEGFR2/FLK1/KDR) in the canine corpus luteum and utero-placental compartments during pregnancy and at normal and induced parturition.

    PubMed

    Gram, Aykut; Hoffmann, Bernd; Boos, Alois; Kowalewski, Mariusz P

    2015-11-01

    VEGFA is one of the most potent known inducers of angiogenesis. However, the function of angiogenic factors in the canine corpus luteum (CL) of pregnancy and in the pregnant uterus and placenta has not yet been elucidated. Therefore, here we investigated the expression and localization of VEGFA and its receptors (VEGFR1/FLT1 and VEGFR2/FLK1/KDR) in the canine CL and utero-placental compartments (ut-pl) throughout pregnancy until prepartum luteolysis. Antigestagen-mediated effects on expression of VEGF system in ut-pl were elucidated in mid-pregnant dogs. While displaying high individual variation, the luteal VEGFA was elevated during pre-implantation and post-implantation, followed by a decrease during mid-gestation, which was more pronounced at the mRNA level, and showed constant expression afterwards. Within the uterus, it increased following implantation and during mid-gestation in ut-pl compartments, but was downregulated at prepartum luteolysis. Luteal VEGFR1 expression resembled that of VEGFA; VEGFR2 remained unaffected throughout pregnancy. In ut-pl compartments, both receptors increased gradually towards mid-gestation; a prepartum decrease was observed for VEGFR1. Antigestagen-treatment resulted in decreased expression of ut-pl VEGFR1. In the CL, VEGFA stained in luteal cells. Uterine signals of VEGFA and its two receptors were observed in epithelial and vascular compartments, and in myometrium. In placental labyrinth, additionally, trophoblast stained positively. Luteal VEGFR1 was localized to the luteal cells and tunica media of blood vessels, whereas VEGFR2 stained only in capillary endothelial cells. The upregulation of luteal and the ut-pl VEGF system during early gestational stages supports the increased vascularization rate during this time. The diminishing effects of the prepartum endocrine milieu on VEGFA function seem to be more pronounced in the ut-pl units.

  10. 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

  11. 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.

  12. Inhibition of VEGF-dependent angiogenesis by the anti-CD82 monoclonal antibody 4F9 through regulation of lipid raft microdomains.

    PubMed

    Nomura, Sayaka; Iwata, Satoshi; Hatano, Ryo; Komiya, Eriko; Dang, Nam H; Iwao, Noriaki; 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.

  13. Molecularly Targeted Therapy of Human Hepatocellular Carcinoma Xenografts with Radio-iodinated Anti-VEGFR2 Murine-Human Chimeric Fab

    PubMed Central

    Huang, Jianfei; Tang, Qi; Wang, Changjun; Yu, Huixin; Feng, Zhenqing; Zhu, Jin

    2015-01-01

    Vascular endothelial growth factor receptor 2 (VEGFR2) is traditionally regarded as an important therapeutic target in a wide variety of malignancies, such as hepatocellular carcinoma (HCC). We previously generated a murine-human anti-VEGFR2 chimeric Fab (cFab), named FA8H1, which has the potential to treat VEGFR2-overexpressing solid tumors. Here, we investigated whether FA8H1 can be used as a carrier in molecularly targeted therapy in HCC xenograft models. FA8H1 was labeled with 131I, and two HCC xenograft models were generated using BEL-7402 (high VEGFR2-expressing) and SMMC-7721 (low VEGFR2-expressing) cells, which were selected from five HCC cell lines. The biodistribution of 131I-FA8H1 was determined in both models by Single-Photon Emission Computed Tomography and therapeutic effects were monitored in nude mice bearing BEL-7402 xenografts. Finally, we determined the involvement of necrosis and apoptotic pathways in treated mice using immunohistochemistry. 131I-FA8H1 levels were dramatically reduced in blood and other viscera. The therapeutic effect of 131I-labeled FA8H1 in the BEL-7402 model was significantly better than that by 131I and FA8H1 alone. We observed extensive necrosis in the treated tumors, and both FasL and caspase 3 were up-regulated. Thus, 131I-anti-VEGFR2 cFab has the potential to be used for molecularly targeted treatment of HCC overexpressing VEGFR2. PMID:26021484

  14. 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

  15. 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. PMID:20441791

  16. 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.

  17. Upregulation of a disintegrin and metalloproteinase-33 by VEGF in human airway smooth muscle cells: Implications for asthma

    PubMed Central

    Pei, Qing-Mei; Jiang, Ping; Yang, Min; Qian, Xue-Jiao; Liu, Jiang-Bo; Zheng, Hong; Zhao, Li-Hong; Kim, Sung-Ho

    2016-01-01

    ABSTRACT Asthma is a chronic respiratory disease characterized by reversible airway obstruction with persistent airway inflammation and airway remodeling. Features of airway remodeling include increased airway smooth muscle (ASM) mass. A disintegrin and metalloproteinase (ADAM)–33 has been identified as playing a role in the pathophysiology of asthma. ADAM-33 is expressed in ASM cells and is suggested to play a role in the function of these cells. However, the regulation of ADAM-33 is not fully understood. Vascular endothelial growth factor (VEGF) has been implicated in inflammatory and airway blood vessel remodeling in asthmatics. Although VEGF was initially thought of as an endothelial-specific growth factor, recent reports have found that VEGF can promote proliferation of other cell types, including ASM cells. To investigate the precise mechanism of VEGF's effect on ASM cell proliferation, we tested the expression of ADAM-33, phospho-extracellularsignal-regulated kinase 1/2 (ERK1/2), and phospho-Akt in VEGF-stimulated ASM cells. We found that VEGF up-regulates ADAM-33 mRNA and protein levels in a dose- and time-dependent manner as well as phosphorylation of ERK1/2 and Akt. We also found that VEGF-induced ASM cell proliferation is inhibited by both ADAM-33 knockdown and a selective VEGF receptor 2 (VEGFR2) inhibitor (SU1498). Furthermore, VEGF-induced ADAM-33 expression and ASM cell proliferation were suppressed by inhibiting ERK1/2 activity, but not by inhibiting Akt activity. Collectively, our findings suggest that VEGF enhances ADAM-33 expression and ASM cell proliferation by activating the VEGFR2/ERK1/2 signaling pathway, which might be involved in the pathogenesis of airway remodeling. Further elucidation of the mechanisms underlying these observations might help develop therapeutic strategies for airway diseases associated with smooth muscle hyperplasia such as asthma. PMID:27579513

  18. Upregulation of a disintegrin and metalloproteinase-33 by VEGF in human airway smooth muscle cells: Implications for asthma.

    PubMed

    Pei, Qing-Mei; Jiang, Ping; Yang, Min; Qian, Xue-Jiao; Liu, Jiang-Bo; Zheng, Hong; Zhao, Li-Hong; Kim, Sung-Ho

    2016-10-17

    Asthma is a chronic respiratory disease characterized by reversible airway obstruction with persistent airway inflammation and airway remodeling. Features of airway remodeling include increased airway smooth muscle (ASM) mass. A disintegrin and metalloproteinase (ADAM)-33 has been identified as playing a role in the pathophysiology of asthma. ADAM-33 is expressed in ASM cells and is suggested to play a role in the function of these cells. However, the regulation of ADAM-33 is not fully understood. Vascular endothelial growth factor (VEGF) has been implicated in inflammatory and airway blood vessel remodeling in asthmatics. Although VEGF was initially thought of as an endothelial-specific growth factor, recent reports have found that VEGF can promote proliferation of other cell types, including ASM cells. To investigate the precise mechanism of VEGF's effect on ASM cell proliferation, we tested the expression of ADAM-33, phospho-extracellularsignal-regulated kinase 1/2 (ERK1/2), and phospho-Akt in VEGF-stimulated ASM cells. We found that VEGF up-regulates ADAM-33 mRNA and protein levels in a dose- and time-dependent manner as well as phosphorylation of ERK1/2 and Akt. We also found that VEGF-induced ASM cell proliferation is inhibited by both ADAM-33 knockdown and a selective VEGF receptor 2 (VEGFR2) inhibitor (SU1498). Furthermore, VEGF-induced ADAM-33 expression and ASM cell proliferation were suppressed by inhibiting ERK1/2 activity, but not by inhibiting Akt activity. Collectively, our findings suggest that VEGF enhances ADAM-33 expression and ASM cell proliferation by activating the VEGFR2/ERK1/2 signaling pathway, which might be involved in the pathogenesis of airway remodeling. Further elucidation of the mechanisms underlying these observations might help develop therapeutic strategies for airway diseases associated with smooth muscle hyperplasia such as asthma. PMID:27579513

  19. 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.

  20. Axl is essential for VEGF-A-dependent activation of PI3K/Akt

    PubMed Central

    Ruan, Guo-Xiang; Kazlauskas, Andrius

    2012-01-01

    Herein, we report that vascular endothelial growth factor A (VEGF-A) engages the PI3K/Akt pathway by a previously unknown mechanism that involves three tyrosine kinases. Upon VEGF-A-dependent activation of VEGF receptor-2 (VEGFR-2), and subsequent TSAd-mediated activation of Src family kinases (SFKs), SFKs engage the receptor tyrosine kinase Axl via its juxtamembrane domain to trigger ligand-independent autophosphorylation at a pair of YXXM motifs that promotes association with PI3K and activation of Akt. Other VEGF-A-mediated signalling pathways are independent of Axl. Interfering with Axl expression or function impairs VEGF-A- but not bFGF-dependent migration of endothelial cells. Similarly, Axl null mice respond poorly to VEGF-A-induced vascular permeability or angiogenesis, whereas other agonists induce a normal response. These results elucidate the mechanism by which VEGF-A activates PI3K/Akt, and identify previously unappreciated potential therapeutic targets of VEGF-A-driven processes. PMID:22327215

  1. A Compartment Model of VEGF Distribution in Humans in the Presence of Soluble VEGF Receptor-1 Acting as a Ligand Trap

    PubMed Central

    Wu, Florence T. H.; Stefanini, Marianne O.; Mac Gabhann, Feilim; Popel, Aleksander S.

    2009-01-01

    Vascular endothelial growth factor (VEGF), through its activation of cell surface receptor tyrosine kinases including VEGFR1 and VEGFR2, is a vital regulator of stimulatory and inhibitory processes that keep angiogenesis – new capillary growth from existing microvasculature – at a dynamic balance in normal physiology. Soluble VEGF receptor-1 (sVEGFR1) – a naturally-occurring truncated version of VEGFR1 lacking the transmembrane and intracellular signaling domains – has been postulated to exert inhibitory effects on angiogenic signaling via two mechanisms: direct sequestration of angiogenic ligands such as VEGF; or dominant-negative heterodimerization with surface VEGFRs. In pre-clinical studies, sVEGFR1 gene and protein therapy have demonstrated efficacy in inhibiting tumor angiogenesis; while in clinical studies, sVEGFR1 has shown utility as a diagnostic or prognostic marker in a widening array of angiogenesis–dependent diseases. Here we developed a novel computational multi-tissue model for recapitulating the dynamic systemic distributions of VEGF and sVEGFR1. Model features included: physiologically-based multi-scale compartmentalization of the human body; inter-compartmental macromolecular biotransport processes (vascular permeability, lymphatic drainage); and molecularly-detailed binding interactions between the ligand isoforms VEGF121 and VEGF165, signaling receptors VEGFR1 and VEGFR2, non-signaling co-receptor neuropilin-1 (NRP1), as well as sVEGFR1. The model was parameterized to represent a healthy human subject, whereupon we investigated the effects of sVEGFR1 on the distribution and activation of VEGF ligands and receptors. We assessed the healthy baseline stability of circulating VEGF and sVEGFR1 levels in plasma, as well as their reliability in indicating tissue-level angiogenic signaling potential. Unexpectedly, simulated results showed that sVEGFR1 – acting as a diffusible VEGF sink alone, i.e., without sVEGFR1-VEGFR heterodimerization

  2. Vatalanib decrease the positive interaction of VEGF receptor-2 and P2X2/3 receptor in chronic constriction injury rats.

    PubMed

    Liu, Shuangmei; Xu, Changshui; Li, Guilin; Liu, Han; Xie, Jinyan; Tu, Guihua; Peng, Haiying; Qiu, Shuyi; Liang, Shangdong

    2012-05-01

    Neuropathic pain can arise from a lesion affecting the peripheral nervous system. Selective P2X(3) and P2X(2/3) receptors' antagonists effectively reduce neuropathic pain. VEGF inhibitors are effective for pain relief. The present study investigated the effects of Vatalanib (VEGF receptor-2 (VEGFR-2) inhibitor) on the neuropathic pain to address the interaction of VEGFR-2 and P2X(2/3) receptor in dorsal root ganglia of chronic constriction injury (CCI) rats. Neuropathic pain symptoms following CCI are similar to most peripheral lesions as assessed by the Neuropathic Pain Symptom Inventory. Sprague-Dawley rats were randomly divided into sham group, CCI group and CCI rats treated with Vatalanib group. Mechanical withdrawal threshold and thermal withdrawal latency were measured. Co-expression of VEGFR-2 and P2X(2) or P2X(3) in L4-6 dorsal root ganglia (DRG) was detected by double-label immunofluorescence. The modulation effect of VEGF on P2X(2/3) receptor agonist-activated currents in freshly isolated DRG neurons of rats both of sham and CCI rats was recorded by whole-cell patch-clamp technique. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) in CCI group were lower than those in sham group (p<0.05). MWT and TWL in CCI rats treated with Vatalanib group were increased compared with those in CCI group (p<0.05). VEGFR-2 and P2X(2) or P2X(3) receptors were co-expressed in the cytoplasm and surface membranes of DRG. The co-expression of VEGFR-2 and P2X(2) or P2X(3) receptor in CCI group exhibited more intense staining than those in sham group and CCI rats treated with Vatalanib group, respectively. VEGF enhanced the amplitude of ATP and α,β-meATP -activated currents of both sham and CCI rats. Increment effects of VEGF on ATP and α,β-meATP -activated currents in CCI rats were higher than those in sham rats. Both ATP (100 μM) and α,β-meATP (10 μM)- activated currents enhanced by VEGF ( 1nM) were significantly blocked by Vatalanib (1

  3. 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.

  4. Genetic interaction of P2X7 receptor and VEGFR-2 polymorphisms identifies a favorable prognostic profile in prostate cancer patients.

    PubMed

    Solini, Anna; Simeon, Vittorio; Derosa, Lisa; Orlandi, Paola; Rossi, Chiara; Fontana, Andrea; Galli, Luca; Di Desidero, Teresa; Fioravanti, Anna; Lucchesi, Sara; Coltelli, Luigi; Ginocchi, Laura; Allegrini, Giacomo; Danesi, Romano; Falcone, Alfredo; Bocci, Guido

    2015-10-01

    VEGFR-2 and P2X7 receptor (P2X7R) have been described to stimulate the angiogenesis and inflammatory processes of prostate cancer. The present study has been performed to investigate the genetic interactions among VEGFR-2 and P2X7R SNPs and their correlation with overall survival (OS) in a population of metastatic prostate cancer patients. Analyses were performed on germline DNA obtained from blood samples and SNPs were investigated by real-time PCR technique. The survival dimensionality reduction (SDR) methodology was applied to investigate the genetic interaction between SNPs. One hundred patients were enrolled. The SDR software provided two genetic interaction profiles consisting of the combination between specific VEGFR-2 (rs2071559, rs11133360) and P2X7R (rs3751143, rs208294) genotypes. The median OS was 126 months (95% CI, 115.94-152.96) and 65.65 months (95% CI, 52.95-76.53) for the favorable and the unfavorable genetic profile, respectively (p < 0.0001). The genetic statistical interaction between VEGFR-2 (rs2071559, rs11133360) and P2X7R (rs3751143, rs208294) genotypes may identify a population of prostate cancer patients with a better prognosis.

  5. Genetic interaction of P2X7 receptor and VEGFR-2 polymorphisms identifies a favorable prognostic profile in prostate cancer patients

    PubMed Central

    Solini, Anna; Simeon, Vittorio; Derosa, Lisa; Orlandi, Paola; Rossi, Chiara; Fontana, Andrea; Galli, Luca; Di Desidero, Teresa; Fioravanti, Anna; Lucchesi, Sara; Coltelli, Luigi; Ginocchi, Laura; Allegrini, Giacomo; Danesi, Romano; Falcone, Alfredo; Bocci, Guido

    2015-01-01

    VEGFR-2 and P2X7 receptor (P2X7R) have been described to stimulate the angiogenesis and inflammatory processes of prostate cancer. The present study has been performed to investigate the genetic interactions among VEGFR-2 and P2X7R SNPs and their correlation with overall survival (OS) in a population of metastatic prostate cancer patients. Analyses were performed on germline DNA obtained from blood samples and SNPs were investigated by real-time PCR technique. The survival dimensionality reduction (SDR) methodology was applied to investigate the genetic interaction between SNPs. One hundred patients were enrolled. The SDR software provided two genetic interaction profiles consisting of the combination between specific VEGFR-2 (rs2071559, rs11133360) and P2X7R (rs3751143, rs208294) genotypes. The median OS was 126 months (95% CI, 115.94–152.96) and 65.65 months (95% CI, 52.95–76.53) for the favorable and the unfavorable genetic profile, respectively (p < 0.0001). The genetic statistical interaction between VEGFR-2 (rs2071559, rs11133360) and P2X7R (rs3751143, rs208294) genotypes may identify a population of prostate cancer patients with a better prognosis. PMID:26337470

  6. 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.

  7. High Affinity Pharmacological Profiling of Dual Inhibitors Targeting RET and VEGFR2 in Inhibition of Kinase and Angiogeneis Events in Medullary Thyroid Carcinoma.

    PubMed

    Dunna, Nageswara Rao; Kandula, Venkatesh; Girdhar, Amandeep; Pudutha, Amareshwari; Hussain, Tajamul; Bandaru, Srinivas; Nayarisseri, Anuraj

    2015-01-01

    Clinical evidence shows that dual inhibition of kinases as well angiogenesis provides ideal therapeutic option in the treatment of medullary thyroid carcinoma (MTC) than inhibiting either of these with the events separately. Although treatment with dual inhibitors has shown good clinical responses in patients with MTC, it has been associated with serious side effects. Some inhibitors are active agents for both angiogenesis or kinase activity. Owing to narrow therapeutic window of established inhibitors, the present study aims to identify high affinity dual inhibitors targeting RET and VEGFR2 respectively for kinase and angiogenesis activity. Established inhibitors like Vandetanib, Cabozantinib, Motesanib, PP121, RAF265 and Sunitinib served as query parent compounds for identification of structurally similar compounds by Tanimoto-based similarity searching with a threshold of 95% against the PubChem database. All the parent inhibitors and respective similar compounds were docked against RET and VEGFR2 in order to retrieve high affinity compounds with these two proteins. AGN-PC-0CUK9P PubCID: 59320403 a compound related to PPI21 showed almost equal affinity for RET and VEGFR2 and unlike other screened compounds with no apparent bias for either of the receptors. Further, AGN- PC-0CUK9P demonstrated appreciable interaction with both RET and VEGFR2 and superior kinase activity in addition to showed optimal ADMET properties and pharmacophore features. From our in silico investigation we suggest AGN-PC-0CUK9P as a superior dual inhibitor targeting RET and VEGFR2 with high efficacy which should be proposed for pharmacodynamic and pharmacokinetic studies for improved treatment of MTC.

  8. 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

  9. High Affinity Pharmacological Profiling of Dual Inhibitors Targeting RET and VEGFR2 in Inhibition of Kinase and Angiogeneis Events in Medullary Thyroid Carcinoma.

    PubMed

    Dunna, Nageswara Rao; Kandula, Venkatesh; Girdhar, Amandeep; Pudutha, Amareshwari; Hussain, Tajamul; Bandaru, Srinivas; Nayarisseri, Anuraj

    2015-01-01

    Clinical evidence shows that dual inhibition of kinases as well angiogenesis provides ideal therapeutic option in the treatment of medullary thyroid carcinoma (MTC) than inhibiting either of these with the events separately. Although treatment with dual inhibitors has shown good clinical responses in patients with MTC, it has been associated with serious side effects. Some inhibitors are active agents for both angiogenesis or kinase activity. Owing to narrow therapeutic window of established inhibitors, the present study aims to identify high affinity dual inhibitors targeting RET and VEGFR2 respectively for kinase and angiogenesis activity. Established inhibitors like Vandetanib, Cabozantinib, Motesanib, PP121, RAF265 and Sunitinib served as query parent compounds for identification of structurally similar compounds by Tanimoto-based similarity searching with a threshold of 95% against the PubChem database. All the parent inhibitors and respective similar compounds were docked against RET and VEGFR2 in order to retrieve high affinity compounds with these two proteins. AGN-PC-0CUK9P PubCID: 59320403 a compound related to PPI21 showed almost equal affinity for RET and VEGFR2 and unlike other screened compounds with no apparent bias for either of the receptors. Further, AGN- PC-0CUK9P demonstrated appreciable interaction with both RET and VEGFR2 and superior kinase activity in addition to showed optimal ADMET properties and pharmacophore features. From our in silico investigation we suggest AGN-PC-0CUK9P as a superior dual inhibitor targeting RET and VEGFR2 with high efficacy which should be proposed for pharmacodynamic and pharmacokinetic studies for improved treatment of MTC. PMID:26514495

  10. 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.

  11. 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.

  12. Local delivery of chitosan/VEGF siRNA nanoplexes reduces angiogenesis and growth of breast cancer in vivo.

    PubMed

    Salva, Emine; Kabasakal, Levent; Eren, Fatih; Ozkan, Naziye; Cakalağaoğlu, Fulya; Akbuğa, Jülide

    2012-02-01

    Vascular endothelial growth factor (VEGF) is the important angiogenic factor associated with tumor growth and metastasis in a wide variety of solid tumors. The aim of this study is to investigate the tumor suppressive effect of chitosan/small interfering RNA (siRNA)-VEGF nanoplexes in the rat breast cancer model. Chitosan/siRNA nanoplexes (siVEGF-A, siVEGFR-1, siVEGFR-2) and NRP-1 were prepared in a 15 to1 ratio and injected (intratumorally) into the breast-tumor-bearing Sprague-Dawley rats. Tumor volumes were measured during 21 days. To investigate the effect of chitosan/siRNA nanoplexes on VEGF expression in tumors, VEGF was analyzed with immunohistochemistry and western blotting. The mRNA levels of VEGF in tumor samples were determined with real-time PCR (RT-PCR). After siRNA treatment, a marked reduction in tumor volumes was measured in complex-injected rats (97%). Free siRNA injection showed lower tumor inhibition. Reduction of VEGF protein was also shown with western blotting and immunohistochemistry. Similar results were obtained with RT-PCR also. These results indicate that the chitosan/siRNA targeting to VEGF nanoplexes have a remarkably suppressive effect on VEGF expression and tumor volume in breast cancer model of rats.

  13. Pharmacogenetics of telatinib, a VEGFR-2 and VEGFR-3 tyrosine kinase inhibitor, used in patients with solid tumors

    PubMed Central

    Steeghs, Neeltje; Wessels, Judith; Eskens, Ferry A. L. M.; de Bont, Natasja; Nortier, Johan W. R.; Guchelaar, Henk-Jan

    2009-01-01

    Summary Purpose Telatinib is an orally active small-molecule tyrosine kinase inhibitor of kinase insert domain receptor (KDR; VEGFR-2) and fms-related tyrosine kinase 4 (FLT4; VEGFR-3). This study aims at the identification of relationships between single nucleotide polymorphisms (SNPs) in genes encoding for transporter proteins and pharmacokinetic parameters in order to clarify the significant interpatient variability in drug exposure. In addition, the potential relationship between target receptor polymorphisms and toxicity of telatinib is explored. Methods Blood samples from 33 patients enrolled in a phase I dose-escalation study of telatinib were analyzed. For correlation with dose normalized AUC(0–12), ATP-binding cassette (ABC) B1 (ABCB1), ABCC1, and ABCG2 were the genes selected. For correlation with telatinib toxicity, selected genes were the drug target genes KDR and FLT4. Results No association between dose normalized AUC(0–12) and drug transporter protein polymorphisms was observed. In addition, no association between toxicity and KDR or FLT4 genotype or haplotype was seen. Conclusions Our pharmacogenetic analysis could not reveal a correlation between relevant gene polymorphisms and clinical and pharmacokinetic observations of telatinib. PMID:19924384

  14. 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

  15. 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

  16. 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

  17. Comparative VEGF receptor tyrosine kinase modeling for the development of highly specific inhibitors of tumor angiogenesis.

    PubMed

    Schmidt, Ulrike; Ahmed, Jessica; Michalsky, Elke; Hoepfner, Michael; Preissner, Robert

    2008-01-01

    The Vascular Endothelial Growth Factor receptors (VEGF-Rs) play a significant role in tumor development and tumor angiogenesis and are therefore interesting targets in cancer therapy. Targeting the VEGF-R is of special importance as the feed of the tumor has to be reduced. In general, this can be carried out by inhibiting the tyrosine kinase function of the VEGF-R. Nevertheless, there arise some problems with the specificity of known kinase inhibitors: they bind to the ATP-binding site and inhibit a number of kinases, moreover the so far most specific inhibitors act at least on these three major types of VEGF-Rs: Flt-1, Flk-1/KDR, Flt-4. The goal is a selective VEGF-R-2 (Flk-1/KDR) inhibitor, because this receptor triggers rather unspecific signals from VEGF-A, -C, -D and -E. Here, we describe a protocol starting from an established inhibitor (Vatalanib) with 2D-/3D-searching and property filtering of the in silico screening hits and the "negative docking approach". With this approach we were able to identify a compound, which shows a fourfold higher reduction of the proliferation rate of endothelial cells compared to the reduction effect of the lead structure.

  18. Pathological roles of the VEGF/SphK pathway in Niemann–Pick type C neurons

    PubMed Central

    Lee, Hyun; Lee, Jong Kil; Park, Min Hee; Hong, Yu Ri; Marti, Hugo H.; Kim, Hyongbum; Okada, Yohei; Otsu, Makoto; Seo, Eul-Ju; Park, Jae-Hyung; Bae, Jae-Hoon; Okino, Nozomu; He, Xingxuan; Schuchman, Edward H.; Bae, Jae-sung; Jin, Hee Kyung

    2014-01-01

    Sphingosine is a major storage compound in Niemann–Pick type C disease (NP–C), although the pathological role(s) of this accumulation have not been fully characterized. Here we found that sphingosine kinase (SphK) activity is reduced in NP–C patient fibroblasts and NP–C mouse Purkinje neurons (PNs) due to defective vascular endothelial growth factor (VEGF) levels. Sphingosine accumulation due to inactivation of VEGF/SphK pathway led to PNs loss via inhibition of autophagosome–lysosome fusion in NP–C mice. VEGF activates SphK by binding to VEGFR2, resulting in decreased sphingosine storage as well as improved PNs survival and clinical outcomes in NP–C cells and mice. We also show that induced pluripotent stem cell (iPSC)-derived human NP–C neurons are generated and the abnormalities caused by VEGF/SphK inactivity in these cells are corrected by replenishment of VEGF. Overall, these results reveal a pathogenic mechanism in NP–C neurons where defective SphK activity is due to impaired VEGF levels. PMID:25417698

  19. Pathological roles of the VEGF/SphK pathway in Niemann-Pick type C neurons.

    PubMed

    Lee, Hyun; Lee, Jong Kil; Park, Min Hee; Hong, Yu Ri; Marti, Hugo H; Kim, Hyongbum; Okada, Yohei; Otsu, Makoto; Seo, Eul-Ju; Park, Jae-Hyung; Bae, Jae-Hoon; Okino, Nozomu; He, Xingxuan; Schuchman, Edward H; Bae, Jae-Sung; Jin, Hee Kyung

    2014-01-01

    Sphingosine is a major storage compound in Niemann-Pick type C disease (NP-C), although the pathological role(s) of this accumulation have not been fully characterized. Here we found that sphingosine kinase (SphK) activity is reduced in NP-C patient fibroblasts and NP-C mouse Purkinje neurons (PNs) due to defective vascular endothelial growth factor (VEGF) levels. Sphingosine accumulation due to inactivation of VEGF/SphK pathway led to PNs loss via inhibition of autophagosome-lysosome fusion in NP-C mice. VEGF activates SphK by binding to VEGFR2, resulting in decreased sphingosine storage as well as improved PNs survival and clinical outcomes in NP-C cells and mice. We also show that induced pluripotent stem cell (iPSC)-derived human NP-C neurons are generated and the abnormalities caused by VEGF/SphK inactivity in these cells are corrected by replenishment of VEGF. Overall, these results reveal a pathogenic mechanism in NP-C neurons where defective SphK activity is due to impaired VEGF levels. PMID:25417698

  20. Identification and characterization of VEGF-A–responsive neutrophils expressing CD49d, VEGFR1, and CXCR4 in mice and humans

    PubMed Central

    Massena, Sara; Christoffersson, Gustaf; Vågesjö, Evelina; Seignez, Cédric; Gustafsson, Karin; Binet, François; Herrera Hidalgo, Carmen; Giraud, Antoine; Lomei, Jalal; Weström, Simone; Shibuya, Masabumi; Claesson-Welsh, Lena; Gerwins, Pär; Welsh, Michael; Kreuger, Johan

    2015-01-01

    Vascular endothelial growth factor A (VEGF-A) is upregulated during hypoxia and is the major regulator of angiogenesis. VEGF-A expression has also been found to recruit myeloid cells to ischemic tissues where they contribute to angiogenesis. This study investigates the mechanisms underlying neutrophil recruitment to VEGF-A as well as the characteristics of these neutrophils. A previously undefined circulating subset of neutrophils shown to be CD49d+VEGFR1highCXCR4high was identified in mice and humans. By using chimeric mice with impaired VEGF receptor 1 (VEGFR1) or VEGFR2 signaling (Flt-1tk−/−, tsad−/−), we found that parallel activation of VEGFR1 on neutrophils and VEGFR2 on endothelial cells was required for VEGF-A-induced recruitment of circulating neutrophils to tissue. Intravital microscopy of mouse microcirculation revealed that neutrophil recruitment by VEGF-A versus by the chemokine macrophage inflammatory protein 2 (MIP-2 [CXCL2]) involved the same steps of the recruitment cascade but that an additional neutrophil integrin (eg, VLA-4 [CD49d/CD29]) played a crucial role in neutrophil crawling and emigration to VEGF-A. Isolated CD49d+ neutrophils featured increased chemokinesis but not chemotaxis compared with CD49d– neutrophils in the presence of VEGF-A. Finally, by targeting the integrin α4 subunit (CD49d) in a transplantation-based angiogenesis model that used avascular pancreatic islets transplanted to striated muscle, we demonstrated that inhibiting the recruitment of circulating proangiogenic neutrophils to hypoxic tissue impairs vessel neoformation. Thus, angiogenesis can be modulated by targeting cell-surface receptors specifically involved in VEGF-A-dependent recruitment of proangiogenic neutrophils without compromising recruitment of the neutrophil population involved in the immune response to pathogens. PMID:26286848

  1. 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 Central

    2014-01-01

    Background 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. Methods 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. Results 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. Conclusion 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

  2. The Three Receptor Tyrosine Kinases c-KIT, VEGFR2 and PDGFRα, Closely Spaced at 4q12, Show Increased Protein Expression in Triple-Negative Breast Cancer

    PubMed Central

    Jansson, Sara; Bendahl, Pär-Ola; Grabau, Dorthe Aamand; Falck, Anna-Karin; Fernö, Mårten; Aaltonen, Kristina; Rydén, Lisa

    2014-01-01

    Background Triple-negative breast cancer (TNBC) is a heterogeneous subgroup of breast cancer with poor prognosis and no targeted therapy available. Receptor tyrosine kinases (RTKs) are emerging targets in anticancer therapy and many RTK-inhibiting drugs are currently being developed. The aim of this study was to elucidate if there is a correlation between the protein expression of three RTKs c-KIT, VEGFR2 and PDGFRα, their gene copy number, and prognosis in TNBC compared to non-TNBC. Methods Tumor tissue samples from patients diagnosed with primary breast cancer were stained with immunohistochemistry (IHC) for protein assessment, and with fluorescence in situ hybridization (FISH) for gene copy number determination. Breast cancer mortality (BCM), measured from the date of surgery to death, was used as endpoint. Results The cohort included 464 patients, out of which 34 (7.3%) had a TNBC. High expression of the three RTKs was more common in TNBC compared to non-TNBC: c-KIT 49% vs. 10% (P<0.001), PDGFRα 32% vs. 19% (P = 0.07) and VEGFR2 32% vs. 6% (P<0.001). The odds ratio (OR) of c-KIT, VEGFR2 and PDGFRα positivity, adjusted for tumor characteristics, was 6.8, 3.6 and 1.3 times higher for TNBC than for non-TNBC. 73.5% of the TNBC had high expression of at least one of the three investigated receptors, compared to 30.0% of the non-TNBC (P<0.001). Survival analysis showed no significant difference in BCM for TNBC patients with high vs. low c-KIT, PDGFRα or VEGFR2 protein expression. 193 (42%) tumors were evaluated with FISH. No correlation was seen between increased gene copy number and TNBC, or between increased gene copy number and high protein expression of the RTK. Conclusion c-KIT, VEGFR2 and PDGFRα show higher protein expression in TNBC compared to non-TNBC. Further investigation clarifying the importance of these RTKs in TNBC is encouraged, as they are possible targets for anticancer therapy. PMID:25025175

  3. The Growth and Aggressive Behavior of Human Osteosarcoma Is Regulated by a CaMKII-Controlled Autocrine VEGF Signaling Mechanism

    PubMed Central

    Daft, Paul G.; Yang, Yang; Napierala, Dobrawa; Zayzafoon, Majd

    2015-01-01

    Osteosarcoma (OS) is a hyperproliferative malignant tumor that requires a high vascular density to maintain its large volume. Vascular Endothelial Growth Factor (VEGF) plays a crucial role in angiogenesis and acts as a paracrine and autocrine agent affecting both endothelial and tumor cells. The alpha-Ca2+/Calmodulin kinase two (α-CaMKII) protein is an important regulator of OS growth. Here, we investigate the role of α-CaMKII-induced VEGF in the growth and tumorigenicity of OS. We show that the pharmacologic and genetic inhibition of α-CaMKII results in decreases in VEGF gene expression (50%) and protein secretion (55%), while α- CaMKII overexpression increases VEGF gene expression (250%) and protein secretion (1,200%). We show that aggressive OS cells (143B) express high levels of VEGF receptor 2 (VEGFR-2) and respond to exogenous VEGF (100nm) by increasing intracellular calcium (30%). This response is ameliorated by the VEGFR inhibitor CBO-P11, suggesting that secreted VEGF results in autocrine stimulated α-CaMKII activation. Furthermore, we show that VEGF and α-CaMKII inhibition decreases the transactivation of the HIF-1α and AP-1 reporter constructs. Additionally, chromatin immunoprecipitation assay shows significantly decreased binding of HIF-1α and AP-1 to their responsive elements in the VEGF promoter. These data suggest that α-CaMKII regulates VEGF transcription by controlling HIF-1α and AP-1 transcriptional activities. Finally, CBO-P11, KN-93 (CaMKII inhibitor) and combination therapy significantly reduced tumor burden in vivo. Our results suggest that VEGF-induced OS tumor growth is controlled by CaMKII and dual therapy by CaMKII and VEGF inhibitors could be a promising therapy against this devastating adolescent disease. PMID:25860662

  4. NADPH oxidase 4-derived H2O2 promotes aberrant retinal neovascularization via activation of VEGF receptor 2 pathway in oxygen-induced retinopathy.

    PubMed

    Li, Jingming; Wang, Joshua J; Zhang, Sarah X

    2015-01-01

    NADPH oxidase 4 (Nox4) is a major isoform of NADPH oxidase in retinal endothelial cells. Our previous study suggests that upregulation of Nox4 in retinal endothelial cells contributes to retinal vascular leakage in diabetes. In the current study, we investigated the role and mechanism of Nox4 in regulation of retinal neovascularization (NV), a hallmark of proliferative diabetic retinopathy (PDR), using a mouse model of oxygen-induced retinopathy (OIR). Our results confirmed that Nox4 was expressed predominantly in retinal vasculature of mouse retina. Retinal expression of Nox4 was markedly increased in OIR, in parallel with enhanced phosphorylation of ERK. In human retinal microvascular endothelial cells (HRECs), overexpression of Nox4 by adenovirus significantly increased extracellular H2O2 generation, resulting in intensified VEGFR2 activation and exacerbated angiogenesis upon VEGF stimulation. In contrast, silencing Nox4 expression or scavenging H2O2 by polyethylene glycol- (PEG-) conjugated catalase inhibited endothelial migration, tube formation, and VEGF-induced activation of VEGFR2 signaling. Importantly, knockdown of retinal Nox4 by adenovirus-delivered siRNA significantly reduced ERK activation and attenuated retinal NV formation in OIR. Taken together, our data indicate that Nox4 promotes retinal NV formation through H2O2/VEGFR2/ERK signaling pathway. Reducing retinal Nox4 expression may represent a promising therapeutic approach for neovascular retinal diseases such as PDR.

  5. Hhex Is Necessary for the Hepatic Differentiation of Mouse ES Cells and Acts via Vegf Signaling

    PubMed Central

    Arterbery, Adam S.; Bogue, Clifford W.

    2016-01-01

    Elucidating the molecular mechanisms involved in the differentiation of stem cells to hepatic cells is critical for both understanding normal developmental processes as well as for optimizing the generation of functional hepatic cells for therapy. We performed in vitro differentiation of mouse embryonic stem cells (mESCs) with a null mutation in the homeobox gene Hhex and show that Hhex-/- mESCs fail to differentiate from definitive endoderm (Sox17+/Foxa2+) to hepatic endoderm (Alb+/Dlk+). In addition, hepatic culture elicited a >7-fold increase in Vegfa mRNA expression in Hhex-/- cells compared to Hhex+/+ cells. Furthermore, we identified VEGFR2+/ALB+/CD34- in early Hhex+/+ hepatic cultures. These cells were absent in Hhex-/- cultures. Finally, through manipulation of Hhex and Vegfa expression, gain and loss of expression experiments revealed that Hhex shares an inverse relationship with the activity of the Vegf signaling pathway in supporting hepatic differentiation. In summary, our results suggest that Hhex represses Vegf signaling during hepatic differentiation of mouse ESCs allowing for cell-type autonomous regulation of Vegfr2 activity independent of endothelial cells. PMID:26784346

  6. 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.

  7. 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

  8. 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

    Astern, J M; Collier, A C; Kendal-Wright, C E

    2013-01-01

    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.

  9. Atherogenic Cytokines Regulate VEGF-A-Induced Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells into Endothelial Cells.

    PubMed

    Ikhapoh, Izuagie Attairu; Pelham, Christopher J; Agrawal, Devendra K

    2015-01-01

    Coronary artery stenting or angioplasty procedures frequently result in long-term endothelial dysfunction or loss and complications including arterial thrombosis and myocardial infarction. Stem cell-based therapies have been proposed to support endothelial regeneration. Mesenchymal stem cells (MSCs) differentiate into endothelial cells (ECs) in the presence of VEGF-A in vitro. Application of VEGF-A and MSC-derived ECs at the interventional site is a complex clinical challenge. In this study, we examined the effect of atherogenic cytokines (IL-6, TNFα, and Ang II) on EC differentiation and function. MSCs (CD44(+), CD73(+), CD90(+), CD14(-), and CD45(-)) were isolated from the bone marrow of Yucatan microswine. Naïve MSCs cultured in differentiation media containing VEGF-A (50 ng/mL) demonstrated increased expression of EC-specific markers (vWF, PECAM-1, and VE-cadherin), VEGFR-2 and Sox18, and enhanced endothelial tube formation. IL-6 or TNFα caused a dose-dependent attenuation of EC marker expression in VEGF-A-stimulated MSCs. In contrast, Ang II enhanced EC marker expression in VEGF-A-stimulated MSCs. Addition of Ang II to VEGF-A and IL-6 or TNFα was sufficient to rescue the EC phenotype. Thus, Ang II promotes but IL-6 and TNFα inhibit VEGF-A-induced differentiation of MSCs into ECs. These findings have important clinical implications for therapies intended to increase cardiac vascularity and reendothelialize coronary arteries following intervention. PMID:26106428

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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

    2016-01-01

    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. VEGF ameliorates cognitive impairment in in vivo and in vitro ischemia via improving neuronal viability and function.

    PubMed

    Yang, Jiajia; Yao, Yang; Chen, Ting; Zhang, Tao

    2014-06-01

    Vascular endothelial growth factor (VEGF) has recently been proved to be a potential therapeutic drug in ischemic disorders depending on the dose, route and time of administration, especially in focal cerebral ischemia. Whether VEGF could exert protection in a long-term total cerebral ischemic model is still uncertain, and the cellular mechanism has not been clarified so far. In order to answer the above issue, an experiment was performed in non-invasively giving exogenous VEGF to a total cerebral ischemic model rats and examining their spatial cognitive function by performing Morris water maze and long-term potential test. Moreover, we performed in vitro experiment to explore the cellular mechanism of VEGF protection effect. In an in vitro ischemia model oxygen-glucose deprivation (OGD), whole-cell patch-clamp recording was employed to examine neuronal function. Additionally, hematoxylin-eosin and propidium iodide staining were applied in vivo and in vitro in the neuropathological and viability study, separately. Our results showed that intranasal administration of VEGF could improve the cognitive function, synaptic plasticity and damaged hippocampal neurons in a global cerebral ischemia model. In addition, VEGF could retain the membrane potential, neuronal excitability and spontaneous excitatory postsynaptic currents in the early stage of ischemia, which further demonstrated that there was an acute effect of VEGF in OGD-induced pyramidal neurons. Simultaneously, it was also found that the death of CA1 pyramidal neuronal was significantly reduced by VEGF, but there was no similar effect in VEGF coexists with SU5416 group. These results indicated that VEGF could ameliorate cognitive impairment and synaptic plasticity via improving neuronal viability and function through acting on VEGFR-2. PMID:24338641

  16. Critical requirement of VEGF-C in transition to fetal erythropoiesis.

    PubMed

    Fang, Shentong; Nurmi, Harri; Heinolainen, Krista; Chen, Shuo; Salminen, Essi; Saharinen, Pipsa; Mikkola, Hanna K A; Alitalo, Kari

    2016-08-01

    Vascular endothelial growth factor C (VEGF-C) is a major driver of lymphangiogenesis in embryos and adults. Vegfc gene deletion in mouse embryos results in failure of lymphangiogenesis, fluid accumulation in tissues, and lethality. The VEGF-C receptors VEGFR3 and VEGFR2 are required for embryonic blood vessel formation. The related VEGF is essential for both blood vessel formation and embryonic hematopoiesis, whereas the possible involvement of VEGF-C in hematopoiesis is unknown. Here we unveil a novel hematopoietic function of VEGF-C in fetal erythropoiesis. Deletion of Vegfc in embryonic day 7.5 (E7.5) embryos in the C57BL6 mouse genetic background led to defective fetal erythropoiesis, characterized by anemia and lack of enucleated red blood cells in blood circulation. Macrophages and erythroid cells in the fetal liver (FL) were also decreased after midgestation because of decreased cell proliferation and increased apoptosis. However, the Lin(-)Sca-1(+)c-Kit(+) stem cell compartment in E14.5 FL was not affected by Vegfc deletion. VEGF-C loss did not disrupt the generation of primitive erythroid cells or erythro-myeloid progenitors (EMPs) in the yolk sac, but it decreased the expression of α4-integrin on EMPs and compromised EMP colonization of the FL. The distribution, maturation, and enucleation of primitive erythroblasts were also impaired by Vegfc deletion. In contrast, Vegfc deletion from E10.5 onward did not compromise definitive hematopoiesis in the liver, and Vegfc deletion in adult mice did not cause anemia. These results reveal an unexpected role for VEGF-C, a major lymphangiogenic growth factor, in the transition to FL erythropoiesis. PMID:27343251

  17. 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.

  18. VEGF receptor blockade markedly reduces retinal microglia/macrophage infiltration into laser-induced CNV.

    PubMed

    Huang, Hu; Parlier, Rachel; Shen, Ji-Kui; Lutty, Gerard A; Vinores, Stanley A

    2013-01-01

    Although blocking VEGF has a positive effect in wet age-related macular degeneration (AMD), the effect of blocking its receptors remains unclear. This was an investigation of the effect of VEGF receptor (VEGFR) 1 and/or 2 blockade on retinal microglia/macrophage infiltration in laser-induced choroidal neovascularization (CNV), a model of wet AMD. CNV lesions were isolated by laser capture microdissection at 3, 7, and 14 days after laser and analyzed by RT-PCR and immunofluorescence staining for mRNA and protein expression, respectively. Neutralizing antibodies for VEGFR1 or R2 and the microglia inhibitor minocycline were injected intraperitoneally (IP). Anti-CD11b, CD45 and Iba1 antibodies were used to confirm the cell identity of retinal microglia/macrophage, in the RPE/choroidal flat mounts or retinal cross sections. CD11b(+), CD45(+) or Iba1(+) cells were counted. mRNA of VEGFR1 and its three ligands, PlGF, VEGF-A (VEGF) and VEGF-B, were expressed at all stages, but VEGFR2 were detected only in the late stage. PlGF and VEGF proteins were expressed at 3 and 7 days after laser. Anti-VEGFR1 (MF1) delivered IP 3 days after laser inhibited infiltration of leukocyte populations, largely retinal microglia/macrophage to CNV, while anti-VEGFR2 (DC101) had no effect. At 14 days after laser, both MF1 and DC101 antibodies markedly inhibited retinal microglia/macrophage infiltration into CNV. Therefore, VEGFR1 and R2 play differential roles in the pathogenesis of CNV: VEGFR1 plays a dominant role at 3 days after laser; but both receptors play pivotal roles at 14 days after laser. In vivo imaging demonstrated accumulation of GFP-expressing microglia into CNV in both CX3CR1(gfp/gfp) and CX3CR1(gfp/+) mice. Minocycline treatment caused a significant increase in lectin(+) cells in the sub-retinal space anterior to CNV and a decrease in dextran-perfused neovessels compared to controls. Targeting the chemoattractant molecules that regulate trafficking of retinal microglia

  19. 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

  20. SKLB-287, a novel oral multikinase inhibitor of EGFR and VEGFR2, exhibits potent antitumor activity in LoVo colorectal tumor model.

    PubMed

    Chen, X; Liu, Y; Yang, H-W; Zhou, S; Cheng, C; Zheng, M-W; Zhong, L; Fu, X-Y; Pan, Y-L; Ma, S; Tang, Y; Chen, Y-Z; Li, L-L; Yang, S-Y

    2014-01-01

    Colorectal cancer (CRC) is the third common cancer and most of the chemotherapies of CRC currently used often suffer limited efficacy and large side effects. Targeted small-molecule by anti-tumor drugs are thought a promising strategy for improving the efficacy and reducing the side effects. In this investigation, we report a novel multikinase inhibitor, termed SKLB-287, which was discovered by us recently. SKLB-287 could efficiently inhibit the activation of endothelial growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2). It displayed very good anti-proliferative activity against LoVo CRC cells and considerable antiangiogenic potency in transgenic zebrafish embryos. Oral administration of SKLB-287 resulted in dose-dependent suppression of tumor growth in LoVo xenograft mouse model. Immunohistochemistry was adopted to examine the in vivo anti-tumor mechanism of action of SKLB-287.

  1. Synthesis, in silico, in vitro, and in vivo investigation of 5-[¹¹C]methoxy-substituted sunitinib, a tyrosine kinase inhibitor of VEGFR-2.

    PubMed

    Caballero, Julio; Muñoz, Camila; Alzate-Morales, Jans H; Cunha, Susana; Gano, Lurdes; Bergmann, Ralf; Steinbach, Joerg; Kniess, Torsten

    2012-12-01

    Sunitinib (SU11248) is a highly potent tyrosine kinase inhibitor targeting vascular endothelial growth factor receptor (VEGFR). Radiolabeled inhibitors of receptor tyrosine kinases (RTKs) might be useful tools for monitoring RTKs levels in tumor tissue giving valuable information for anti-angiogenic therapy. Herein we report the synthesis of 5-methoxy-sunitinib 5 and its (11)C-radiolabeled analog [(11)C]-5. The non-radioactive reference compound 5 was prepared by Knoevenagel condensation of 5-methoxy-2-oxindole with the corresponding substituted 5-formyl-1H-pyrrole. A binding constant (K(d)) of 20 nM for 5 was determined by competition binding assay against VEGFR-2. In addition, the binding mode of sunitinib and its 5-methoxy substituted derivative was studied by flexible docking simulations. These studies revealed that the substitution of the fluorine at position 5 of the oxindole scaffold by a methoxy group did not affect the inhibitor orientation, but affected the electrostatic and van der Waals interactions of the ligand with residues near the DFG motif of VEGFR-2. 5-[(11)C]methoxy-sunitinib ([(11)C]-5) was synthesized by reaction of the desmethyl precursor with [(11)C]CH(3)I in the presence of DMF and NaOH in 17 ± 3% decay-corrected radiochemical yield at a specific activity of 162-205 GBq/μmol (EOS). In vivo stability studies of [(11)C]-5 in rat blood showed that more than 70% of the injected compound was in blood stream, 60 min after administration. PMID:23131541

  2. 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.

  3. 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

  4. Inhibition of prostate cancer osteoblastic progression with VEGF121/rGel, a single agent targeting osteoblasts, osteoclasts, and tumor neovasculature

    PubMed Central

    Mohamedali, Khalid A.; Li, Zhi Gang; Starbuck, Michael W.; Wan, Xinhai; Yang, Jun; Kim, Sehoon; Zhang, Wendy; Rosenblum, Michael G.; Navone, Nora M.

    2011-01-01

    Purpose A hallmark of prostate cancer (PCa) progression is the development of osteoblastic bone metastases, which respond poorly to available therapies. We previously reported that VEGF121/rGel targets osteoclast precursors and tumor neovasculature. Here we tested the hypothesis that targeting non-tumor cells expressing these receptors can inhibit tumor progression in a clinically relevant model of osteoblastic PCa. Experimental Design Cells from MDA PCa 118b, a PCa xenograft obtained from a bone metastasis in a patient with castrate-resistant PCa, were injected into the femurs of mice. Osteoblastic progression was monitored following systemic administration of VEGF121/rGel. Results VEGF121/rGel was cytotoxic in vitro to osteoblast precursor cells. This cytotoxicity was specific as VEGF121/rGel internalization into osteoblasts was VEGF121 receptor driven. Furthermore, VEGF121/rGel significantly inhibited PCa-induced bone formation in a mouse calvaria culture assay. In vivo, VEGF121/rGel significantly inhibited the osteoblastic progression of PCa cells in the femurs of nude mice. Microcomputed tomography analysis revealed that VEGF121/rGel restored the bone volume fraction of tumor-bearing femurs to values similar to those of the contralateral (non–tumor bearing) femurs. VEGF121/rGel significantly reduced the number of tumor-associated osteoclasts but did not change the numbers of peritumoral osteoblasts. Importantly, VEGF121/rGel-treated mice had significantly less tumor burden than control mice. Our results thus indicate that VEGF121/rGel inhibits osteoblastic tumor progression by targeting angiogenesis, osteoclastogenesis, and bone formation. Conclusions Targeting VEGFR-1 – or VEGFR-2–expressing cells is effective in controlling the osteoblastic progression of PCa in bone. These findings provide the basis for an effective multitargeted approach for metastatic PCa. PMID:21343372

  5. Angiogenesis in the Developing Spinal Cord: Blood Vessel Exclusion from Neural Progenitor Region Is Mediated by VEGF and Its Antagonists

    PubMed Central

    Takahashi, Teruaki; Takase, Yuta; Yoshino, Takashi; Saito, Daisuke; Tadokoro, Ryosuke; Takahashi, Yoshiko

    2015-01-01

    Blood vessels in the central nervous system supply a considerable amount of oxygen via intricate vascular networks. We studied how the initial vasculature of the spinal cord is formed in avian (chicken and quail) embryos. Vascular formation in the spinal cord starts by the ingression of intra-neural vascular plexus (INVP) from the peri-neural vascular plexus (PNVP) that envelops the neural tube. At the ventral region of the PNVP, the INVP grows dorsally in the neural tube, and we observed that these vessels followed the defined path at the interface between the medially positioned and undifferentiated neural progenitor zone and the laterally positioned differentiated zone. When the interface between these two zones was experimentally displaced, INVP faithfully followed a newly formed interface, suggesting that the growth path of the INVP is determined by surrounding neural cells. The progenitor zone expressed mRNA of vascular endothelial growth factor-A whereas its receptor VEGFR2 and FLT-1 (VEGFR1), a decoy for VEGF, were expressed in INVP. By manipulating the neural tube with either VEGF or the soluble form of FLT-1, we found that INVP grew in a VEGF-dependent manner, where VEGF signals appear to be fine-tuned by counteractions with anti-angiogenic activities including FLT-1 and possibly semaphorins. These results suggest that the stereotypic patterning of early INVP is achieved by interactions between these vessels and their surrounding neural cells, where VEGF and its antagonists play important roles. PMID:25585380

  6. 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

  7. Gold nanoparticles inhibit VEGF165-induced migration and tube formation of endothelial cells via the Akt pathway.

    PubMed

    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

  8. 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

  9. Intracranial meningiomas, the VEGF-A pathway, and peritumoral brain oedema.

    PubMed

    Nassehi, Damoun

    2013-04-01

    inter-variability. It is also less expensive, and gives the user more flexibility as homemade reagents can be used. On the other hand, the chemiluminescence assay is straight forward, requires less hands-on-time, and can be used on formalin-fixed and paraffin-embedded (FFPE) tissue. Paper III continues the investigations in paper I. The sample size is increased so that 22 angiomatous and secretory meningiomas are compared to 40 non-angiomatous meningiomas and 10 control brain tissue samples. Angiomatous and secretory meningiomas are chosen because they are known to have larger PTBE compared to other meningiomas. In addition to VEGF-A, capillary length, and PTBE, the VEGF-A tyrosine kinase receptor VEGFR-2 mRNA and protein levels are also examined. VEGFR-2 is a transmembrane receptor found on endothelial cells. It binds VEGF-A and thereby increases angiogenesis. VEGFR-2's co-receptor neuropilin-1 is also examined. Neuropilin-1 is an agonist of angiogenesis through complex-binding of VEGF-A, but it can also work as an inhibitor through competitive binding of semaphorin-3A. The complex binding of semaphorin-3A to neuropilin-1 can also induce endothelial cell apoptosis, thus working as an antagonist of angiogenesis. The study finds that VEGF-A mRNA, VEGF-A protein, and neuropilin-1 mRNA are higher in angiomatous and non-angiomatous meningiomas compared to controls. VEGFR-2 protein is higher, and neuropilin-1 protein lower in angiomatous meningiomas compared to controls. The mean capillary length is 3614 mm/mm3 in angiomatous, 605 mm/mm3 in non-angiomatous meningiomas, and 229 mm/mm3 in the controls. Non-angiomatous and angiomatous meningioma patients have equally sized tumours. The mean PTBE around the angiomatous meningiomas is 695 cm3, i.e. 477 cm3 larger than the non-angiomatous meningiomas (p = 0.0045), and the mean oedema index is twice the size compared to the non-angiomatous meningiomas. Further comparison between the two meningioma groups shows that mean VEGF-A m

  10. Cardiomyocyte VEGFR-1 activation by VEGF-B induces compensatory hypertrophy and preserves cardiac function after myocardial infarction.

    PubMed

    Zentilin, Lorena; Puligadda, Uday; Lionetti, Vincenzo; Zacchigna, Serena; Collesi, Chiara; Pattarini, Lucia; Ruozi, Giulia; Camporesi, Silvia; Sinagra, Gianfranco; Pepe, Martino; Recchia, Fabio A; Giacca, Mauro

    2010-05-01

    Mounting evidence indicates that the function of members of the vascular endothelial growth factor (VEGF) family extends beyond blood vessel formation. Here, we show that the prolonged intramyocardial expression of VEGF-A(165) and VEGF-B(167) on adeno-associated virus-mediated gene delivery determined a marked improvement in cardiac function after myocardial infarction in rats, by promoting cardiac contractility, preserving viable cardiac tissue, and preventing remodeling of the left ventricle (LV) over time. Consistent with this functional outcome, animals treated with both factors showed diminished fibrosis and increased contractile myocardium, which were more pronounced after expression of the selective VEGF receptor-1 (VEGFR-1) ligand VEGF-B, in the absence of significant induction of angiogenesis. We found that cardiomyocytes expressed VEGFR-1, VEGFR-2, and neuropilin-1 and that, in particular, VEGFR-1 was specifically up-regulated in hypoxia and on exposure to oxidative stress. VEGF-B exerted powerful antiapoptotic effect in both cultured cardiomyocytes and after myocardial infarction in vivo. Finally, VEGFR-1 activation by VEGF-B was found to elicit a peculiar gene expression profile proper of the compensatory, hypertrophic response, consisting in activation of alphaMHC and repression of betaMHC and skeletal alpha-actin, and an increase in SERCA2a, RYR, PGC1alpha, and cardiac natriuretic peptide transcripts, both in cultured cardiomyocytes and in infarcted hearts. The finding that VEGFR-1 activation by VEGF-B prevents loss of cardiac mass and promotes maintenance of cardiac contractility over time has obvious therapeutic implications.

  11. Functional Relevance of the Switch of VEGF Receptors/Co-Receptors during Peritoneal Dialysis-Induced Mesothelial to Mesenchymal Transition

    PubMed Central

    Pérez-Lozano, María Luisa; Sandoval, Pilar; Rynne-Vidal, Ángela; Aguilera, Abelardo; Jiménez-Heffernan, José Antonio; Albar-Vizcaíno, Patricia; Majano, Pedro L.; Sánchez-Tomero, José Antonio; Selgas, Rafael; López-Cabrera, Manuel

    2013-01-01

    Vascular endothelial growth factor (VEGF) is up-regulated during mesothelial to mesenchymal transition (MMT) and has been associated with peritoneal membrane dysfunction in peritoneal dialysis (PD) patients. It has been shown that normal and malignant mesothelial cells (MCs) express VEGF receptors (VEGFRs) and co-receptors and that VEGF is an autocrine growth factor for mesothelioma. Hence, we evaluated the expression patterns and the functional relevance of the VEGF/VEGFRs/co-receptors axis during the mesenchymal conversion of MCs induced by peritoneal dialysis. Omentum-derived MCs treated with TGF-β1 plus IL-1β (in vitro MMT) and PD effluent-derived MCs with non-epithelioid phenotype (ex vivo MMT) showed down-regulated expression of the two main receptors Flt-1/VEGFR-1 and KDR/VEGFR-2, whereas the co-receptor neuropilin-1 (Nrp-1) was up-regulated. The expression of the Nrp-1 ligand semaphorin-3A (Sema-3A), a functional VEGF competitor, was repressed throughout the MMT process. These expression pattern changes were accompanied by a reduction of the proliferation capacity and by a parallel induction of the invasive capacity of MCs that had undergone an in vitro or ex vivo MMT. Treatment with neutralizing anti-VEGF or anti-Nrp-1 antibodies showed that these molecules played a relevant role in cellular proliferation only in naïve omentum-derived MCs. Conversely, treatment with these blocking antibodies, as well as with recombinant Sema-3A, indicated that the switched VEGF/VEGFRs/co-receptors axis drove the enhanced invasion capacity of MCs undergoing MMT. In conclusion, the expression patterns of VEGFRs and co-receptors change in MCs during MMT, which in turn would determine their behaviour in terms of proliferation and invasion in response to VEGF. PMID:23585849

  12. 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

  13. The spatial and temporal expression of VEGF and its receptors 1 and 2 in post-traumatic bone bridge formation of the growth plate.

    PubMed

    Fischerauer, Eva; Heidari, Nima; Neumayer, Bernhard; Deutsch, Alexander; Weinberg, Annelie M

    2011-12-01

    Injuries to growth plates may initiate the formation of reversible or irreversible bone-bridges, may leading to bone length discrepancy or axis deviation. As vascular invasion is essential for the formation of bone tissue, the aim of our study was to investigate the kinetic expression of Vascular Endothelial Growth Factor (VEGF) and its receptors R1 and R2 and the ingrowth of vessels in the formation of bone bridges in a rat physeal injury model. Quantitative Real-Time Polymerase Chain Reaction was performed for VEGF and its receptors. Samples from the proximal physis of the tibial bone were immunohistochemically evaluated for the expression of VEGF and its R1 and R2 receptors and Laminin. Morphologically, physeal bone bridge formation was validated by means of Magnetic Resonance Imaging. Kinetic expression of VEGF and VEGF-R1 mRNA documented a tendency towards an increase in expression on day 7. Histological analyses showed a hematoma containing bone debris on day 1 which was replaced with bony trabeculae by day 14, forming a bone bridge by day 28 which was preceded and accompanied by angiogenesis and consistent with MRI data. VEGF and VEGF-R2 was expressed on the debris within the hematoma and bone trabeculae from days 1 to 28. VEGF-R1 expression was only noted until day 14. The findings of our study suggest that physeal bone bridge formation is in part triggered by VEGF expression and associated with angiogenesis, which was shown to precede bone bridge formation and may be further stimulated through VEGF-positive bone debris.

  14. Positron emission tomography imaging of vascular endothelial growth factor receptor expression with (61)Cu-labeled lysine-tagged VEGF121.

    PubMed

    Zhang, Yin; Hong, Hao; Niu, Gang; Valdovinos, Hector F; Orbay, Hakan; Nayak, Tapas R; Chen, Xiaoyuan; Barnhart, Todd E; Cai, Weibo

    2012-12-01

    Overexpression of vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) indicates poor prognosis for cancer patients in a variety of clinical studies. Our goal is to develop a tracer for positron emission tomography (PET) imaging of VEGFR expression using recombinant human VEGF121 with three lysine residues fused to the N-terminus (denoted as K3-VEGF121), which can facilitate radiolabeling without affecting its VEGFR binding affinity. K3-VEGF121 was conjugated with 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and labeled with (61)Cu (t1/2: 3.3 h; 62% β(+)). The IC50 value of NOTA-K3-VEGF121 for VEGFR-2 was comparable to that of K3-VEGF121 (1.50 and 0.65 nM, respectively) based on a cell binding assay. (61)Cu labeling was achieved with good yield (55 ± 10%) and specific activity (4.2 GBq/mg). Serial PET imaging showed that the 4T1 tumor uptake of (61)Cu-NOTA-K3-VEGF121 was 3.4 ± 0.5, 4.9 ± 1.0, 5.2 ± 1.0, and 4.8 ± 0.8%ID/g (n = 4) at 0.5, 2, 4, and 8 h postinjection, respectively, which was consistent with biodistribution data measured by γ counting. Blocking experiments and ex vivo histology confirmed the VEGFR specificity of (61)Cu-NOTA-K3-VEGF121. Extrapolated human dosimetry calculation showed that liver was the organ with the highest radiation dose. The use of (61)Cu as the radiolabel is desirable for small proteins such as K3-VEGF121, which has a much higher β(+) branching ratio than the commonly used (64)Cu (62% vs 17%), thereby offering stronger signal intensity and lower tracer dose for PET imaging.

  15. 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

  16. 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. PMID:21792179

  17. VEGF neutralizing aerosol therapy in primary pulmonary adenocarcinoma with K-ras activating-mutations.

    PubMed

    Hervé, Virginie; Rabbe, Nathalie; Guilleminault, Laurent; Paul, Flora; Schlick, Laurène; Azzopardi, Nicolas; Duruisseaux, Michael; Fouquenet, Delphine; Montharu, Jérôme; Redini, Françoise; Paintaud, Gilles; Lemarié, Etienne; Cadranel, Jacques; Wislez, Marie; Heuzé-Vourc'h, Nathalie

    2014-01-01

    K-ras mutations promote angiogenesis in lung cancer and contribute to the drug resistance of cancer cells. It is not clear whether K-ras mutated adenocarcinomas are sensitive to anti-angiogenic therapy with monoclonal antibodies (mAbs) that target vascular endothelial growth factor (VEGF). Anti-angiogenic mAbs are usually delivered systemically, but only a small proportion reaches the lung after intravenous injection. We investigated the relevance of a non-invasive pulmonary route for the delivery of anti-VEGF mAbs in the mouse K-ras(LA1) model. We found that pulmonary delivery of these mAbs significantly reduced the number of tumor lesions and inhibited malignant progression. The antitumor effect involves the VEGFR2-dependent inhibition of blood vessel growth, which impairs tumor proliferation. Pharmacokinetic analysis of aerosolized anti-VEGF showed its low rate of passage into the bloodstream, suggesting that this delivery route is associated with reduced systemic side effects. Our findings highlight the value of the aerosol route for administration of anti-angiogenic mAbs in pulmonary adenocarcinoma with K-ras activating-mutations. PMID:25484066

  18. Role of VEGF Receptors in Normal and Psoriatic Human Keratinocytes: Evidence from Irradiation with Different UV Sources

    PubMed Central

    Zhu, Jian-Wei; Wu, Xian-Jie; Lu, Zhong-Fa; Luo, Dan; Cai, Sui-Qing; Zheng, Min

    2013-01-01

    Vascular endothelial growth factor (VEGF) promotes angiogenesis and plays important roles both in physiological and pathological conditions. VEGF receptors (VEGFRs) are high-affinity receptors for VEGF and are originally considered specific to endothelial cells. We previously reported that VEGFRs were also constitutively expressed in normal human keratinocytes and overexpressed in psoriatic epidermis. In addition, UVB can activate VEGFRs in normal keratinocytes, and the activated VEGFR-2 signaling is involved in the pro-survival mechanism. Here, we show that VEGFRs were also upregulated and activated by UVA in normal human keratinocytes via PKC, and interestingly, both the activated VEGFR-1 and VEGFR-2 protected against UVA-induced cell death. As VEGFRs were over-expressed in psoriatic epidermis, we further investigated whether narrowband UVB (NB-UVB) phototherapy or topical halomethasone monohydrate 0.05% cream could affect their expression. Surprisingly, the over-expressed VEGFRs in psoriatic epidermis were significantly attenuated by both treatments. During NB-UVB therapy, VEGFRs declined first in the basal, and then gradually in the upper psoriatic epidermis. VEGFRs were activated in psoriatic epidermis, their activation was enhanced by NB-UVB, but turned undetectable after whole therapy. This process was quite different from that by halomethasone, in which VEGFRs and phospho-VEGFRs decreased in a gradual, homogeneous manner. Our findings further suggest that UV-induced activation of VEGFRs serves as a pro-survival signal for keratinocytes. In addition, VEGFRs may be involved in the pathological process of psoriasis, and UV phototherapy is effective for psoriasis by directly modulating the expression of VEGFRs. PMID:23383198

  19. 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

  20. VEGFR2 targeted antibody fused with MICA stimulates NKG2D mediated immunosurveillance and exhibits potent anti-tumor activity against breast cancer.

    PubMed

    Xie, Wei; Liu, Fang; Wang, Youfu; Ren, Xueyan; Wang, Tong; Chen, Zhiguo; Tang, Mingying; Sun, Fumou; Li, Zhaoting; Wang, Min; Zhang, Juan

    2016-03-29

    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.

  1. Suppression of alpha-tocopherol ether-linked acetic acid in VEGF-induced angiogenesis and the possible mechanisms in human umbilical vein endothelial cells.

    PubMed

    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. PMID:25447407

  2. Carvedilol may attenuate liver cirrhosis by inhibiting angiogenesis through the VEGF-Src-ERK signaling pathway

    PubMed Central

    Ding, Qian; Tian, Xiang-Guo; Li, Yan; Wang, Qi-Zhi; Zhang, Chun-Qing

    2015-01-01

    AIM: To investigate the effect of carvedilol on angiogenesis and the underlying signaling pathways. METHODS: The effect of carvedilol on angiogenesis was examined using a human umbilical vascular endothelial cell (HUVEC) model. The effect of carvedilol on cell viability was measured by CCK8 assay. Flow cytometry was used to assess the effect of carvedilol on cell cycle progression. Cell migration, transwell migration and tube formation assays were performed to analyze the effect of carvedilol on HUVEC function. Vascular endothelial growth factor (VEGF) induced activation of HUVECs, which were pretreated with different carvedilol concentrations or none. Western blot analysis detected the phosphorylation levels of three cell signaling pathway proteins, VEGFR-2, Src, and extracellular signal-regulated kinase (ERK). The specific Src inhibitor PP2 was used to assess the role of Src in the VEGF-induced angiogenic pathway. RESULTS: Carvedilol inhibited HUVEC proliferation in a dose-dependent manner (IC50 = 38.5 mmol/L). The distribution of cells in the S phase decreased from 43.6% to 37.2%, 35.6% and 17.8% by 1, 5 and 10 μmol/L carvedilol for 24 h, respectively. Carvedilol (10 μmol/L) reduced VEGF-induced HUVEC migration from 67.54 ± 7.83 to 37.11 ± 3.533 (P < 0.001). Carvedilol concentrations of 5 μmol/L and 10 μmol/L reduced cell invasion from 196.3% ± 18.76% to 114.0% ± 12.20% and 51.68% ± 8.28%, respectively. VEGF-induced tube formation was also reduced significantly by 5 μmol/L and 10 μmol/L carvedilol from 286.0 ± 36.72 to 135.7 ± 18.13 (P < 0.05) and 80.27 ± 11.16 (P < 0.01) respectively. We investigated several intracellular protein levels to determine the reason for these reductions. Treatment with 10 μmol/L carvedilol reduced VEGF-induced tyrosine phosphorylation of VEGFR-2 from 175.5% ± 8.54% to 52.67% ± 5.33% (P < 0.01). Additionally, 10 μmol/L carvedilol reduced VEGF-induced ERK 1/2 phosphorylation from 181.9% ± 18.61% to 56.45% ± 7.64% (P

  3. 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.

  4. 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

    2016-05-01

    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).

  5. 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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  6. 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

  7. AI-29ANGIOGENIC SWITCH FROM VEGFR2/HIF1α IN NEWLY DIAGNOSED GLIOBLASTOMA (GB) TO CXCR4-SDF1 PATHWAY IN RECURRENT PAIRED TUMOR AFTER RADIOTHERAPY (RT)-TEMOZOLOMIDE (TMZ)

    PubMed Central

    Tabouret, Emeline; Tchoghandjian, Aurelie; Denicolai, Emilie; Delfino, Christine; Metellus, Philippe; Padovani, Laetitia; Nanni, Isabelle; Barrie, Maryline; Boucard, Celine; Ouafik, L'Houcine; Figarella-Branger, Dominique; Chinot, Olivier

    2014-01-01

    BACKGROUND: Angiogenesis is one of the key features of GB. Our objective was to explore the potential changes of angiogenic factors expression between initial diagnosis of GB and recurrence after RT/TMZ. METHODS: Paired frozen tumor tissues from both initial and recurrent surgery were available for 29 patients with GB treated with RT/TMZ without bevacizumab upfront. Screening of over 150 genes expressions related to angiogenesis was performed on first 10 paired samples, using RT- PCR arrays (Qiagen®). Comparative expressions were determined using Qiagen® software. In a second step, RNA expressions of the selected identified genes were analyzed on all samples (29 paired tumors) using quantitative RT-PCR (qRT-PCR). Protein expression was examined by immunohistochemistry (IHC) with a semi-quantitative measure. Anti-tumoral effect of an anti-CXCR4 (AMD3100) in addition to TMZ and RT was tested in GB explants. RESULTS: In the screening step performed by RT-PCR arrays the initial-recurrence expression changes contributed to a selection of seven genes for which expression was then quantified by qRT-PCR: VEGFA, VEGFR2, VEGFR1, SDF1, CXCR4, uPA and HIF1α. From initial diagnosis to recurrence RNA expressions of CXCR4 (p = 0.029) and SDF1 (p = 0.107) were increased while expressions of HIF1α (p = 0.009) and VEGFR2 (p = 0.081) were decreased. Similarly, SDF1 protein expression (IHC) tended to increased (p = 0.096) while VEGFR2 staining was significantly decreased (p = 0.004) at recurrence. The role of CXCL4 was further supported by an increase of anti-tumoral effect observed with the combination of AMD3100 and RT/TMZ versus RT/TMZ alone in GB explants. By multivariate analysis, VEGFR2 RNA initial and recurrence expression levels were significantly correlated respectively to initial overall survival (p = 0.019, Hazard ratio (HR) =3.650) and recurrent overall survival (p = 0.024, HR = 2.536). CONCLUSION: Recurrence of GB after chemo-radiation could be associated with a

  8. Cyclooxygenase-2 blockade can improve efficacy of VEGF-targeting drugs

    PubMed Central

    Ben-Batalla, Isabel; Cubas-Cordova, Miguel; Udonta, Florian; Wroblewski, Mark; Waizenegger, Jonas S.; Janning, Melanie; Sawall, Stefanie; Gensch, Victoria; Zhao, Lin; Martinez-Zubiaurre, Iñigo; Riecken, Kristoffer; Fehse, Boris; Pantel, Klaus; Bokemeyer, Carsten; Loges, Sonja

    2015-01-01

    Anti-angiogenic therapies were approved for different cancers. However, significant primary and secondary resistance hampers efficacy in several tumor types including breast cancer. Thus, we need to develop clinically applicable strategies to enhance efficacy of anti-angiogenic drugs. We report that anti-angiogenic therapies can induce upregulation of cyclooxygenase-2 (Cox-2) and of its product prostaglandin E2 (PGE2) in breast cancer models. Upon Cox-2 inhibition PGE2 levels were normalized and efficacy of anti-vascular endothelial growth factor receptor 2 (anti-VEGFR-2) antibodies and sunitinib was enhanced. Interestingly, both treatments exerted additive anti-angiogenic effects. Following Cox-2 inhibition, we observed reduced infiltration of tumors with cancer-associated fibroblasts (CAFs) and lower levels of pro-angiogenic factors active besides the VEGF axis including hepatocyte growth factor (HGF) and basic fibroblast growth factor (FGF2). Mechanistic studies indicated that Cox-2 inhibition reduced PGE2-induced migration and proliferation of CAFs via inhibiting phosphorylation of Akt. Hence, Cox-2 inhibition can increase efficacy of anti-angiogenic treatments and our findings might pave the road for clinical investigations of concomitant blockade of Cox-2 and VEGF-signaling. PMID:25849942

  9. A novel extracellular role for tissue transglutaminase in matrix-bound VEGF-mediated angiogenesis

    PubMed Central

    Wang, Z; Perez, M; Caja, S; Melino, G; Johnson, T S; Lindfors, K; Griffin, M

    2013-01-01

    The importance of tissue transglutaminase (TG2) in angiogenesis is unclear and contradictory. Here we show that inhibition of extracellular TG2 protein crosslinking or downregulation of TG2 expression leads to inhibition of angiogenesis in cell culture, the aorta ring assay and in vivo models. In a human umbilical vein endothelial cell (HUVEC) co-culture model, inhibition of extracellular TG2 activity can halt the progression of angiogenesis, even when introduced after tubule formation has commenced and after addition of excess vascular endothelial growth factor (VEGF). In both cases, this leads to a significant reduction in tubule branching. Knockdown of TG2 by short hairpin (shRNA) results in inhibition of HUVEC migration and tubule formation, which can be restored by add back of wt TG2, but not by the transamidation-defective but GTP-binding mutant W241A. TG2 inhibition results in inhibition of fibronectin deposition in HUVEC monocultures with a parallel reduction in matrix-bound VEGFA, leading to a reduction in phosphorylated VEGF receptor 2 (VEGFR2) at Tyr1214 and its downstream effectors Akt and ERK1/2, and importantly its association with β1 integrin. We propose a mechanism for the involvement of matrix-bound VEGFA in angiogenesis that is dependent on extracellular TG2-related activity. PMID:24052076

  10. 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.

  11. Peptide vaccines and peptidomimetics targeting HER and VEGF proteins may offer a potentially new paradigm in cancer immunotherapy

    PubMed Central

    Kaumaya, Pravin TP; Foy, Kevin Chu

    2013-01-01

    The ErbB family (HER-1, HER-2, HER-3 and HER-4) of receptor tyrosine kinases has been the focus of cancer immunotherapeutic strategies while antiangiogenic therapies have focused on VEGF and its receptors VEGFR-1 and VEGFR-2. Agents targeting receptor tyrosine kinases in oncology include therapeutic antibodies to receptor tyrosine kinase ligands or the receptors themselves, and small-molecule inhibitors. Many of the US FDA-approved therapies targeting HER-2 and VEGF exhibit unacceptable toxicities, and show problems of efficacy, development of resistance and unacceptable safety profiles that continue to hamper their clinical progress. The combination of dif ferent peptide vaccines and peptidomimetics targeting specific molecular pathways that are dysregulated in tumors may potentiate anticancer immune responses, bypass immune tolerance and circumvent resistance mechanisms. The focus of this review is to discuss efforts in our laboratory spanning two decades of rationally developing peptide vaccines and therapeutics for breast cancer. This review highlights the prospective benefit of a new, untapped category of therapies biologically targeted to EGF receptor (HER-1), HER-2 and VEGF with potential peptide ‘blockbusters‘ that could lay the foundation of a new paradigm in cancer immunotherapy by creating clinical breakthroughs for safe and efficacious cancer cures. PMID:22894670

  12. Peptide vaccines and targeting HER and VEGF proteins may offer a potentially new paradigm in cancer immunotherapy.

    PubMed

    Kaumaya, Pravin T P; Foy, Kevin Chu

    2012-08-01

    The ErbB family (HER-1, HER-2, HER-3 and HER-4) of receptor tyrosine kinases has been the focus of cancer immunotherapeutic strategies while antiangiogenic therapies have focused on VEGF and its receptors VEGFR-1 and VEGFR-2. Agents targeting receptor tyrosine kinases in oncology include therapeutic antibodies to receptor tyrosine kinase ligands or the receptors themselves, and small-molecule inhibitors. Many of the US FDA-approved therapies targeting HER-2 and VEGF exhibit unacceptable toxicities, and show problems of efficacy, development of resistance and unacceptable safety profiles that continue to hamper their clinical progress. The combination of different peptide vaccines and peptidomimetics targeting specific molecular pathways that are dysregulated in tumors may potentiate anticancer immune responses, bypass immune tolerance and circumvent resistance mechanisms. The focus of this review is to discuss efforts in our laboratory spanning two decades of rationally developing peptide vaccines and therapeutics for breast cancer. This review highlights the prospective benefit of a new, untapped category of therapies biologically targeted to EGF receptor (HER-1), HER-2 and VEGF with potential peptide 'blockbusters' that could lay the foundation of a new paradigm in cancer immunotherapy by creating clinical breakthroughs for safe and efficacious cancer cures.

  13. Characterization of neural stem/progenitor cells expressing VEGF and its receptors in the subventricular zone of newborn piglet brain.

    PubMed

    Ara, Jahan; Fekete, Saskia; Zhu, Anli; Frank, Melissa

    2010-09-01

    Neural stem/progenitor cell (NSP) biology and neurogenesis in adult central nervous system (CNS) are important both towards potential future therapeutic applications for CNS repair, and for the fundamental function of the CNS. In the present study, we report the characterization of NSP population from subventricular zone (SVZ) of neonatal piglet brain using in vivo and in vitro systems. We show that the nestin and vimentin-positive neural progenitor cells are present in the SVZ of the lateral ventricles of neonatal piglet brain. In vitro, piglet NSPs proliferated as neurospheres, expressed the typical protein of neural progenitors, nestin and a range of well-established neurodevelopmental markers. Upon dissociation and subculture, piglet NSPs differentiated into neurons and glial cells. Clonal analysis demonstrates that piglet NSPs are multipotent and retain the capacity to generate both glia and neurons. These cells expressed VEGF, VEGFR1, VEGFR2 and Neuropilin-1 and -2 mRNAs. Real time PCR revealed that SVZ NSPs from newborn piglet expressed total VEGF and all VEGF splice variants. These findings show that piglet NSPs may be helpful to more effectively design growth factor based strategies to enhance endogenous precursor cells for cell transplantation studies potentially leading to the application of this strategy in the nervous system disease and injury.

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

    PubMed Central

    Yang, Yunlong; Zhang, Yin; Iwamoto, Hideki; Hosaka, Kayoko; Seki, Takahiro; Andersson, Patrik; Lim, Sharon; Fischer, Carina; Nakamura, Masaki; Abe, Mitsuhiko; Cao, Renhai; Skov, Peter Vilhelm; Chen, Fang; Chen, Xiaoyun; Lu, Yongtian; Nie, Guohui; Cao, Yihai

    2016-01-01

    The impact of discontinuation of anti-VEGF cancer therapy in promoting cancer metastasis is unknown. Here we show discontinuation of anti-VEGF treatment creates a time-window of profound structural changes of liver sinusoidal vasculatures, exhibiting hyper-permeability and enlarged open-pore sizes of the fenestrated endothelium and loss of VE-cadherin. The drug cessation caused highly leaky hepatic vasculatures permit tumour cell intravasation and extravasation. Discontinuation of an anti-VEGF antibody-based drug and sunitinib markedly promotes liver metastasis. Mechanistically, host hepatocyte, but not tumour cell-derived vascular endothelial growth factor (VEGF), is responsible for cancer metastasis. Deletion of hepatocyte VEGF markedly ablates the ‘off-drug'-induced metastasis. These findings provide mechanistic insights on anti-VEGF cessation-induced metastasis and raise a new challenge for uninterrupted and sustained antiangiogenic therapy for treatment of human cancers. PMID:27580750

  15. Heterotypic contact reveals a COX-2-mediated suppression of osteoblast differentiation by endothelial cells: A negative modulatory role for prostanoids in VEGF-mediated cell: cell communication?

    SciTech Connect

    Clarkin, Claire E. Garonna, Elena; Pitsillides, Andrew A.; Wheeler-Jones, Caroline P.D.

    2008-10-15

    In bone, angiogenesis must be initiated appropriately, but limited once remodelling or repair is complete. Our recent findings have supported a role for prostaglandins (PG), known modulators of osteoblast (OB) and endothelial cell (EC) behaviour, in facilitating VEGF-mediated paracrine communication from OBs to 'remotely located' ECs, but the mechanism(s) regulating OB:EC crosstalk when these cells are closely opposed are undefined. In this study we have examined: (i) the effects of exogenous PGE{sub 2} on VEGF-driven events in ECs, and (ii) the role of endogenous COX-2-derived prostanoids in mediating communication between intimately opposed OBs and ECs in direct contact. Exposure of ECs to PGE{sub 2} increased ERK1/2 phosphorylation, COX-2 induction, 6-keto-PGF{sub 1{alpha}} release and EC proliferation. In contrast, PGE{sub 2} attenuated VEGF{sub 165}-induced VEGFR2/Flk1 phosphorylation, ERK1/2 activation and proliferation of ECs, suggesting that exogenous PGE{sub 2} restricts the actions of VEGF. However, the COX-2-selective inhibitor, NS398, also attenuated VEGF-induced proliferation, implying a distinct role for endogenous COX-2 activity in regulating EC behaviour. To examine the effect of OB:EC proximity and the role of COX-2 products further, we used a confrontational co-culture model. These studies showed that COX-2 blockade with NS398 enhanced EC-dependent increases in OB differentiation, that this effect was reversed by exogenous PGH{sub 2} (immediate COX-2 product), and that exogenous VEGF did not influence EC-dependent OB differentiation under these conditions. Our findings indicate that locally produced prostanoids may serve distinct roles depending on OB:EC proximity and negatively modulate VEGF-mediated changes in EC behaviour when these cells are closely opposed to control angiogenesis during bone (re)modelling.

  16. The corpora lutea proangiogenic state of VEGF system components is turned to antiangiogenic at the later phase of the oestrous cycle in cows.

    PubMed

    Guzmán, A; Macías-Valencia, R; Fierro-Fierro, F; Gutiérrez, C G; Rosales-Torres, A M

    2015-02-01

    Blood vessel expansion and reduction in the corpus luteum (CL) is regulated by the vascular endothelial growth factor (VEGF) system and linked to the maintenance of the CL. The VEGF system has both angiogenic and antiangiogenic ligands and receptors. Our objective was to evaluate the relationship between the mRNA expression of angiogenic and antiangiogenic members of the VEGF system in the CL, throughout the luteal phase of the oestrous cycle in cows. The CL of 18 cows were collected by transvaginal surgery on days 4, 6, 9, 12, 15 and 18 of the oestrous cycle and the mRNA expression of VEGF system components was evaluated by quantitative real-time PCR. The mRNA expression of VEGF ligands and receptors increased (P<0.05) from the early- and mid-luteal phase (days 4 to 12) reaching its maximum expression on day 15 of the cycle. We found no expression of VEGF164b throughout the cycle. Expression of sVEGFR1 did not change during the oestrous cycle and exceeded that of the VEGFR1 by 100 times. Nonetheless, as VEGFR1 increased, the relationship between the soluble and membrane receptor decreased (P<0.01). In contrast, the expression of VEGFR2 was higher than that of its soluble isoform for all days studied, however, the ratio between the membrane-bound and its soluble counterpart decreased continuously throughout the cycle (P<0.01). Our results show that the expression levels for VEGF ligands, receptors and their antagonistic counterparts are adjusted during CL development and regression, to upregulate angiogenesis early in the oestrous cycle and restrict it at the time of luteolysis. PMID:25229247

  17. 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. PMID:23316979

  18. Acidic extracellular pH promotes prostate cancer bone metastasis by enhancing PC-3 stem cell characteristics, cell invasiveness and VEGF-induced vasculogenesis of BM-EPCs.

    PubMed

    Huang, Sheng; Tang, Yubo; Peng, Xinsheng; Cai, Xingdong; Wa, Qingde; Ren, Dong; Li, Qiji; Luo, Jiaquan; Li, Liangping; Zou, Xuenong; Huang, Shuai

    2016-10-01

    Bone metastasis is a main cause of cancer-related mortality in patients with advanced prostate cancer. Emerging evidence suggests that the acidic extracellular microenvironment plays significant roles in the growth and metastasis of tumors. However, the effects of acidity on bone metastasis of PCa remain undefined. In the present study, PC-3 cells were cultured in acidic medium (AM; pH 6.5) or neutral medium (NM; pH 7.4), aiming to investigate the effects and possible mechanisms of acidic extracellular microenvironment in bone metastasis of PCa. Our results showed that AM can promote spheroid and colony formations, cell viability and expression of stem cell characteristic-related markers in PC-3 cells. Moreover, AM stimulates MMP-9 secretion and promotes invasiveness of PC-3 cells, and these effects can be inhibited by blocking of MMP-9. Furthermore, AM stimulates VEGF secretion of PC-3 and AM conditioned medium (CMAM) promotes vasculogenesis of BM-EPCs by increasing cell viability, migration, tube formation, which involved activating the phosphorylation of VEGFR-2, Akt and P38, when pH of NM conditioned medium (CMNM) was modulated the same as AM conditioned medium (CMAM). Further studies have shown that CMNM induced vasculogenesis of BM-EPCs can be inhibited by the inhibition of VEGFR2 with DMH4. These findings suggest that acidic extracellular microenvironment may have the potential to modulate prostate cancer bone metastasis by enhancing PC-3 stem cell characteristics, cell invasiveness and VEGF-induced vasculogenesis of BM-EPCs. Improved anticancer strategies should be designed to selectively target acidic tumor microenvironment.

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

    SciTech Connect

    Bayer, Christine; Kielow, Achim; Schilling, Daniela; Maftei, Constantin-Alin; Zips, Daniel; Yaromina, Ala; Baumann, Michael; Molls, Michael; Multhoff, Gabriele

    2012-11-01

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

  20. Expression and localization of VEGF receptors in human fetal skeletal tissues.

    PubMed

    Marini, M; Sarchielli, E; Toce, M; Acocella, A; Bertolai, R; Ciulli, C; Orlando, C; Sgambati, E; Vannelli, G B

    2012-12-01

    During development the vertebrate skeleton is the product of deriving cells from distinct embryonic lineages. The craniofacial skeleton is formed by migrating cranial neural crest cells, whereas the axial and limb skeletons are derived from mesodermal cells. The Vascular Endothelial Growth Factors (VEGFs) / receptors (VEGFRs) system plays an important role in angiogenesis, as well as osteogenesis, during bone development, growth, and remodeling, attracting endothelial cells and osteoclasts and stimulating osteoblast differentiation. Recent evidence has shown that during development VEGFR-3 is also expressed in neural and glial precursors of forebrain and cerebellum, as well as in the eye. In this study, we found that VEGFR-1, VEGFR-2 and VEGFR-3 are expressed in human bone both in fetal and adult life. The gene expression levels were significantly higher in fetal samples especially in mandibles. In addition, higher levels of VEGFR-3 in orofacial district were confirmed by western blotting analysis. We also observed that in fetal mandibular samples VEGFRs colocalized in several osteoblasts, osteoclasts and osteoprogenitor cells. Furthermore, some cells coexpressed VEGFR-3 and ET-1, a marker of neural crest cells. The results demonstrated different expression of VEGFRs in human mandibular and femoral bones which could be correlated to their different structure, function and development during organogenesis. VEGFR-3 might represent a specific signal for ectomesenchymal lineage differentiation during early human development.

  1. Bioactivation of collagen matrices through sustained VEGF release from PLGA microspheres.

    PubMed

    Borselli, Cristina; Ungaro, Francesca; Oliviero, Olimpia; d'Angelo, Ivana; Quaglia, Fabiana; La Rotonda, Maria I; Netti, Paolo A

    2010-01-01

    The success of any tissue engineering implant relies upon prompt vascularization of the cellular construct and, hence, on the ability of the scaffold to broadcast specific activation of host endothelium and guide vessel ingrowth. Vascular endothelial growth factor (VEGF) is a potent angiogenic stimulator, and if released in a controlled manner it may enhance and guide scaffold vascularization. Therefore, the aim of this work was to realize a scaffold with integrated depots able to release VEGF in a controlled rate and assess the ability of this scaffold to promote angiogenesis. VEGF-loaded poly(lactide-co-glycolide) (PLGA) microspheres were produced and included in a collagen scaffold. The release of VEGF from microspheres was tailored to be sustained over several weeks and occurred at a rate of approximately 0.6 ng/day per mg of microspheres. It was found that collagen scaffolds bioactivated with VEGF-loaded microspheres strongly enhanced endothelial cell activation and vascular sprouting both in vitro and in vivo as compared with a collagen scaffold bioactivated with free VEGF. This report demonstrates that by finely tuning VEGF release rate within a polymeric scaffold, sprouting of angiogenic vessels can be guided within the scaffolds interstices as well as broadcasted from the host tissues. PMID:19165799

  2. T11TS impedes glioma angiogenesis by inhibiting VEGF signaling and pro-survival PI3K/Akt/eNOS pathway with concomitant upregulation of PTEN in brain endothelial cells.

    PubMed

    Bhattacharya, Debanjan; Singh, Manoj Kumar; Chaudhuri, Suhnrita; Acharya, Sagar; Basu, Anjan Kumar; Chaudhuri, Swapna

    2013-05-01

    The crucial role of angiogenesis in malignant glioma progression makes it a potential target of therapeutic intervention in glioma. Previous studies from our lab showed that sheep erythrocyte membrane glycopeptide T11-target structure (T11TS) has potent anti-neoplastic and immune stimulatory effects in rodent glioma model. In the present study we investigated the anti-angiogenic potential of T11TS and deciphered the underlying molecular mechanism of its anti-angiogenic action in malignant glioma. Vascular endothelial growth factor (VEGF) signaling is crucial for initiating tumor angiogenic responses. The present preclinical study was designed to evaluate the effect of T11TS therapy on VEGF and VEGFR-2 expression in glioma associated brain endothelial cells and to determine the effects of in vivo T11TS administration on expression of PTEN and downstream pro-survival PI3K/Akt/eNOS pathway proteins in glioma associated brain endothelial cells. T11TS therapy in rodent glioma model significantly downregulated expression of VEGF along with its receptor VEGFR-2 and inhibited the expression of pro-survival PI3K/Akt/eNOS proteins in glioma associated brain endothelial cells. Furthermore, T11TS therapy in glioma induced rats significantly upregulated brain endothelial cell PTEN expression, inhibited eNOS phosphorylation and production of nitric oxide in glioma associated brain endothelial cells. Taken together our findings suggest that T11TS can be introduced as an effective angiogenesis inhibitor in human glioma as T11TS targets multiple levels of angiogenic signaling cascade impeding glioma neovascularisation.

  3. Developmental Programming: Does Prenatal Steroid Excess Disrupt the Ovarian VEGF System in Sheep?

    PubMed

    Ortega, Hugo Héctor; Veiga-Lopez, Almudena; Sreedharan, Shilpa; del Luján Velázquez, Melisa María; Salvetti, Natalia Raquel; Padmanabhan, Vasantha

    2015-09-01

    Prenatal testosterone (T), but not dihydrotestosterone (DHT), excess disrupts ovarian cyclicity and increases follicular recruitment and persistence. We hypothesized that the disruption in the vascular endothelial growth factor (VEGF) system contributes to the enhancement of follicular recruitment and persistence in prenatal T-treated sheep. The impact of T/DHT treatments from Days 30 to 90 of gestation on VEGFA, VEGFB, and their receptor (VEGFR-1 [FLT1], VEGFR-2 [KDR], and VEGFR-3 [FLT4]) protein expression was examined by immunohistochemistry on Fetal Days 90 and 140, 22 wk, 10 mo (postpubertal), and 21 mo (adult) of age. Arterial morphometry was performed in Fetal Day 140 and postpubertal ovaries. VEGFA and VEGFB expression were found in granulosa cells at all stages of follicular development with increased expression in antral follicles. VEGFA was present in theca interna, while VEGFB was present in theca interna/externa and stromal cells. All three receptors were expressed in the granulosa, theca, and stromal cells during all stages of follicular development. VEGFR-3 increased with follicular differentiation with the highest level seen in the granulosa cells of antral follicles. None of the members of the VEGF family or their receptor expression were altered by age or prenatal T/DHT treatments. At Fetal Day 140, area, wall thickness, and wall area of arteries from the ovarian hilum were larger in prenatal T- and DHT-treated females, suggestive of early androgenic programming of arterial differentiation. This may facilitate increased delivery of endocrine factors and thus indirectly contribute to the development of the multifollicular phenotype.

  4. Developmental Programming: Does Prenatal Steroid Excess Disrupt the Ovarian VEGF System in Sheep?1

    PubMed Central

    Ortega, Hugo Héctor; Veiga-Lopez, Almudena; Sreedharan, Shilpa; del Luján Velázquez, Melisa María; Salvetti, Natalia Raquel; Padmanabhan, Vasantha

    2015-01-01

    Prenatal testosterone (T), but not dihydrotestosterone (DHT), excess disrupts ovarian cyclicity and increases follicular recruitment and persistence. We hypothesized that the disruption in the vascular endothelial growth factor (VEGF) system contributes to the enhancement of follicular recruitment and persistence in prenatal T-treated sheep. The impact of T/DHT treatments from Days 30 to 90 of gestation on VEGFA, VEGFB, and their receptor (VEGFR-1 [FLT1], VEGFR-2 [KDR], and VEGFR-3 [FLT4]) protein expression was examined by immunohistochemistry on Fetal Days 90 and 140, 22 wk, 10 mo (postpubertal), and 21 mo (adult) of age. Arterial morphometry was performed in Fetal Day 140 and postpubertal ovaries. VEGFA and VEGFB expression were found in granulosa cells at all stages of follicular development with increased expression in antral follicles. VEGFA was present in theca interna, while VEGFB was present in theca interna/externa and stromal cells. All three receptors were expressed in the granulosa, theca, and stromal cells during all stages of follicular development. VEGFR-3 increased with follicular differentiation with the highest level seen in the granulosa cells of antral follicles. None of the members of the VEGF family or their receptor expression were altered by age or prenatal T/DHT treatments. At Fetal Day 140, area, wall thickness, and wall area of arteries from the ovarian hilum were larger in prenatal T- and DHT-treated females, suggestive of early androgenic programming of arterial differentiation. This may facilitate increased delivery of endocrine factors and thus indirectly contribute to the development of the multifollicular phenotype. PMID:26178718

  5. Angiopreventive efficacy of pure flavonolignans from milk thistle extract against prostate cancer: targeting VEGF-VEGFR signaling.

    PubMed

    Deep, Gagan; Gangar, Subhash Chander; Rajamanickam, Subapriya; Raina, Komal; Gu, Mallikarjuna; Agarwal, Chapla; Oberlies, Nicholas H; Agarwal, Rajesh

    2012-01-01

    The role of neo-angiogenesis in prostate cancer (PCA) growth and metastasis is well established, but the development of effective and non-toxic pharmacological inhibitors of angiogenesis remains an unaccomplished goal. In this regard, targeting aberrant angiogenesis through non-toxic phytochemicals could be an attractive angiopreventive strategy against PCA. The rationale of the present study was to compare the anti-angiogenic potential of four pure diastereoisomeric flavonolignans, namely silybin A, silybin B, isosilybin A and isosilybin B, which we established previously as biologically active constituents in Milk Thistle extract. Results showed that oral feeding of these flavonolignans (50 and 100 mg/kg body weight) effectively inhibit the growth of advanced human PCA DU145 xenografts. Immunohistochemical analyses revealed that these flavonolignans inhibit tumor angiogenesis biomarkers (CD31 and nestin) and signaling molecules regulating angiogenesis (VEGF, VEGFR1, VEGFR2, phospho-Akt and HIF-1α) without adversely affecting the vessel-count in normal tissues (liver, lung, and kidney) of tumor bearing mice. These flavonolignans also inhibited the microvessel sprouting from mouse dorsal aortas ex vivo, and the VEGF-induced cell proliferation, capillary-like tube formation and invasiveness of human umbilical vein endothelial cells (HUVEC) in vitro. Further studies in HUVEC showed that these diastereoisomers target cell cycle, apoptosis and VEGF-induced signaling cascade. Three dimensional growth assay as well as co-culture invasion and in vitro angiogenesis studies (with HUVEC and DU145 cells) suggested the differential effectiveness of the diastereoisomers toward PCA and endothelial cells. Overall, these studies elucidated the comparative anti-angiogenic efficacy of pure flavonolignans from Milk Thistle and suggest their usefulness in PCA angioprevention.

  6. The observed correlation between in vivo clinical pharmacokinetic parameters and in vitro potency of VEGFR-2 inhibitors. Can this be used as a prospective guide for the development of novel compounds?

    PubMed

    Benjamin, B; Sahu, M; Bhatnagar, U; Abhyankar, D; Srinivas, N R

    2012-04-01

    Literature data on the clinical pharmacokinetics of various VEGFR-2 inhibitors along with in vitro potency data were correlated and a linear relationship was established in spite of limited data set. In this work, a model set comprised of axitinib, recentin, sunitinib, pazopanib, and sorafenib were used. The in vitro potencies of the model set compounds were correlated with the published unbound plasma concentrations (Cmax, Cavg, Ctrough). The established linear regression (r2>0.90) equation was used to predict Cmax, Cavg, Ctrough of the 'prediction set' (motesanib, telatinib, CP547632, vatalanib, vandetanib) using in vitro potency and unbound protein free fraction. Cavg and Ctrough of prediction set were closely matched (0.2-1.8 fold of reported), demonstrating the usefulness of such predictions for tracking the target related modulation and/or efficacy signals within the clinically optimized population average. In case of Cmax where correlation was least anticipated, the predicted values were within 0.1-1.1 fold of those reported. Such predictions of appropriate parameters would provide rough estimates of whether or not therapeutically relevant dose(s) have been administered when clinical investigations of novel agents of this class are being performed. Therefore, it may aid in increasing clinical doses to a desired level if safety of the compound does not compromise such dose increases. In conclusion, the proposed model may prospectively guide the dosing strategies and would greatly aid the development of novel compounds in this class. PMID:22290114

  7. Direct Evidence of Target Inhibition with anti-VEGF, EGFR, and mTOR Therapies in a Clinical Model of Wound Healing

    PubMed Central

    Jia, Jingquan; Dellinger, Andrew E.; Weiss, Eric S.; Bulusu, Anuradha; Rushing, Christel; Li, Haiyan; Howard, Leigh A.; Kaplan, Neal; Pang, Herbert; Hurwitz, Herbert I.; Nixon, Andrew B.

    2016-01-01

    Purpose In early clinical testing, most novel targeted anti-cancer therapies have limited toxicities and limited efficacy, which complicates dose and schedule selection for these agents. Confirmation of target inhibition is critical for rational drug development; however, repeated tumor biopsies are often impractical and peripheral blood mononuclear cells and normal skin are often inadequate surrogates for tumor tissue. Based upon the similarities of tumor and wound stroma, we have developed a clinical dermal granulation tissue model to evaluate novel targeted therapies. Experimental design A 4mm skin punch biopsy was used to stimulate wound healing and a repeat 5mm punch biopsy was used to harvest the resulting granulation tissue. This assay was performed at pre-treatment and on-treatment evaluating four targeted therapies, bevacizumab, everolimus, erlotinib, and panitumumab, in the context of three different clinical trials. Total and phosphorylated levels VEGFR2, S6RP, and EGFR were evaluated using ELISA-based methodologies. Results Significant and consistent inhibition of VEGF pathway (using VEGFR2 as the readout) was observed in granulation tissue biopsies from patients treated with bevacizumab and everolimus. Additionally, significant and consistent inhibition of mTOR pathway (using S6RP as the readout) was observed in patients treated with everolimus. Lastly, significant inhibition of EGFR pathway (using EGFR as the readout) was observed in patients treated with panitumumab, but this was not observed in patients treated with erlotinib. Conclusion Molecular analyses of dermal granulation tissue can be used as a convenient and quantitative pharmacodynamic biomarker platform for multiple classes of targeted therapies. PMID:25878330

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

    PubMed Central

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

    2010-01-01

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

  9. A pilot study of JI-101, an inhibitor of VEGFR-2, PDGFR-β, and EphB4 receptors, in combination with everolimus and as a single agent in an ovarian cancer expansion cohort.

    PubMed

    Werner, Theresa L; Wade, Mark L; Agarwal, Neeraj; Boucher, Kenneth; Patel, Jesal; Luebke, Aaron; Sharma, Sunil

    2015-12-01

    JI-101 is an oral multi-kinase inhibitor that targets vascular endothelial growth factor receptor type 2 (VEGFR-2), platelet derived growth factor receptor β (PDGFR-β), and ephrin type-B receptor 4 (EphB4). None of the currently approved angiogenesis inhibitors have been reported to inhibit EphB4, and therefore, JI-101 has a novel mechanism of action. We conducted a pilot trial to assess the pharmacokinetics (PK), tolerability, and efficacy of JI-101 in combination with everolimus in advanced cancers, and pharmacodynamics (PD), tolerability, and efficacy of JI-101 in ovarian cancer. This was the first clinical study assessing anti-tumor activity of JI-101 in a combinatorial regimen. In the PK cohort, four patients received single agent 10 mg everolimus on day 1, 10 mg everolimus and 200 mg JI-101 combination on day 8, and single agent 200 mg JI-101 on day 15. In the PD cohort, eleven patients received single agent JI-101 at 200 mg twice daily for 28 day treatment cycles. JI-101 was well tolerated as a single agent and in combination with everolimus. No serious adverse events were observed. Common adverse events were hypertension, nausea, and abdominal pain. JI-101 increased exposure of everolimus by approximately 22%, suggestive of drug-drug interaction. The majority of patients had stable disease at their first set of restaging scans (two months), although no patients demonstrated a response to the drug per RECIST criteria. The novel mechanism of action of JI-101 is promising in ovarian cancer treatment and further prospective studies of this agent may be pursued in a less refractory patient population or in combination with cytotoxic chemotherapy.

  10. Evaluation of the angiogenesis inhibitor KR-31831 in SKOV-3 tumor-bearing mice using (64)Cu-DOTA-VEGF(121) and microPET.

    PubMed

    Lee, Iljung; Yoon, Kwang Yup; Kang, Choong Mo; Lin, Xin; Chen, Xiaoyuan; Kim, Jung Young; Kim, Sung-Min; Ryu, Eun Kyoung; Choe, Yearn Seong

    2012-08-01

    KR-31831 ((2R,3R,4S)-6-amino-4-[N-(4-chloropheyl)-N-(1H-imidazol-2ylmethyl)amino]-3-hydroxyl-2-methyl-2-dimethoxymethyl-3,4-dihydro-2H-1-benzopyran), an angiogenesis inhibitor, was evaluated in tumor-bearing mice using molecular imaging technology. Pre-treatment microPET images were acquired on SKOV-3 cell-implanted nude mice after injection with (64)Cu-DOTA-VEGF(121). KR-31831 (50 mg/kg) was then injected intraperitoneally into the treatment group (n=3), while injection vehicle was injected into the control (n=4) and blocking (n=3) groups. After injections occurred daily for 28 days, all groups of mice underwent post-treatment microPET imaging after injection with (64)Cu-DOTA-VEGF(121). The post-treatment images showed high tumor uptake in the control group and reduced tumor uptake in both the blocking and treatment groups. ROI analysis of the tumor images revealed 6.25%±1.18% ID/g at 1 h, 6.55%±0.69% ID/g at 2 h, and 4.68%±0.63% ID/g at 16 h in the control group; 3.87%±0.45% ID/g at 1 h, 4.50%±0.44% ID/g at 2 h, and 3.63%±0.25% ID/g at 16 h in the blocking group; and 4.03%±0.74% ID/g at 1 h, 4.37%±0.67% ID/g at 2 h, and 3.83%±0.90% ID/g at 16 h in the treatment group. Biodistribution obtained after the post-treatment microPET imaging also demonstrated high tumor uptake (3.74%±0.27% ID/g) in the control group and reduced uptakes in both the blocking group (2.69%±0.73% ID/g, P<.05) and the treatment group (3.11%±0.25% ID/g, P<.05), which correlated well with microPET imaging data. Immunofluorescence analysis showed higher levels of VEGFR2 and CD31 expressions in tumor tissues of the control and blocking groups than in tumor tissues of the treatment group. These results suggest that the antiangiogenic activity of KR-31831 is mediated through VEGFR2 and microPET serves as a useful molecular imaging tool for evaluation of a newly developed angiogenesis inhibitor, KR-31831.

  11. Where is VEGF in the body? A meta-analysis of VEGF distribution in cancer

    PubMed Central

    Kut, C; Mac Gabhann, F; Popel, A S

    2007-01-01

    Vascular endothelial growth factor (VEGF) is a major target for the inhibition of tumour vascularisation and the treatment of human cancer. Many tumours produce large quantities of VEGF, and as a result, diagnosis and prognosis of cancer may be predicted by measuring changes in VEGF concentrations in blood. In blood, the VEGF may be located in the plasma, or in the blood-borne cells and formed elements, in particular, platelets and leukocytes. In this study, we collate the measurements of VEGF in platelets, leukocytes, plasma and serum for breast, prostate, colorectal and other cancers. In addition, we analysed the concentration of VEGF in tumour tissue itself, as well as for other tissues in the human body. Although the concentration of VEGF in tumours is high, the size of tumours is small compared to other tissues, in particular, skeletal muscle. Thus, the total quantity of VEGF in tumours and in blood is small compared to the quantity in muscles. This large reservoir of VEGF may have important implications for the treatment of cancer. PMID:17912242

  12. Computational Modeling of Interacting VEGF and Soluble VEGF Receptor Concentration Gradients

    PubMed Central

    Hashambhoy, Yasmin L.; Chappell, John C.; Peirce, Shayn M.; Bautch, Victoria L.; Mac Gabhann, Feilim

    2011-01-01

    Experimental data indicates that soluble vascular endothelial growth factor (VEGF) receptor 1 (sFlt-1) modulates the guidance cues provided to sprouting blood vessels by VEGF-A. To better delineate the role of sFlt-1 in VEGF signaling, we have developed an experimentally based computational model. This model describes dynamic spatial transport of VEGF, and its binding to receptors Flt-1 and Flk-1, in a mouse embryonic stem cell model of vessel morphogenesis. The model represents the local environment of a single blood vessel. Our simulations predict that blood vessel secretion of sFlt-1 and increased local sFlt-1 sequestration of VEGF results in decreased VEGF–Flk-1 levels on the sprout surface. In addition, the model predicts that sFlt-1 secretion increases the relative gradient of VEGF–Flk-1 along the sprout surface, which could alter endothelial cell perception of directionality cues. We also show that the proximity of neighboring sprouts may alter VEGF gradients, VEGF receptor binding, and the directionality of sprout growth. As sprout distances decrease, the probability that the sprouts will move in divergent directions increases. This model is a useful tool for determining how local sFlt-1 and VEGF gradients contribute to the spatial distribution of VEGF receptor binding, and can be used in conjunction with experimental data to explore how multi-cellular interactions and relationships between local growth factor gradients drive angiogenesis. PMID:22007175

  13. Antioxidant, DNA interaction, VEGFR2 kinase, topoisomerase I and in vitro cytotoxic activities of heteroleptic copper(II) complexes of tetrazolo[1,5-a]pyrimidines and diimines.

    PubMed

    Haleel, A; Mahendiran, D; Veena, V; Sakthivel, N; Rahiman, A Kalilur

    2016-11-01

    A series of heteroleptic mononuclear copper(II) complexes of the type [Cu(L(1-3))(diimine)]ClO4 (1-6) containing three tetrazolo[1,5-a]pyrimidine core ligands, ethyl 5-methyl-7-(2-hydroxyphenyl)-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxylate (HL(1)), ethyl 5-methyl-7-(4-diethylamino-2-hydroxyphenyl)-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxylate (HL(2)) or ethyl 5-methyl-7-(2-hydroxy-4-nitrophenyl)-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxylate (HL(3)), and two diimine coligands, 2,2'-bipyridyl (bpy) or 1,10-phenanthroline (phen) have been synthesized and characterized by spectral methods. The geometry of complexes have been determined with the help of electronic absorption and EPR splitting patterns, which suggest four coordinated square planar geometry around copper(II) ion. The lowering of HOMO-LUMO band gap value of complex 4 implies its higher biological activity compared to other complexes. Antioxidant studies revealed that the complexes possess considerable radical scavenging potency against DPPH. The binding studies of the complexes with calf thymus DNA (CT-DNA) revealed groove mode of binding, which was further supported by docking simulation. The complexes 3 and 4 strongly inhibit the topoisomerase I, and also strongly interact with VEGFR2 kinase receptor via π-π, σ-π and hydrogen bonding interaction. Gel electrophoresis experiments demonstrated the ability of the complexes to cleave plasmid DNA in the absence of activators. In vitro cytotoxic activities of the complexes were examined on three cancerous cell lines such as human lung (A549), cervical (HeLa) and colon (HCT-15), and two normal cells such as human embryonic kidney (HEK) and peripheral blood mononuclear cells (PBMCs). The live cell and fluorescent imaging of cancer cells were observed with acridine orange/ethidium bromide staining assay. All encouraging chemical and biological findings indicate that the complex 4 is a suitable candidate for drug target. PMID:27524032

  14. Antioxidant, DNA interaction, VEGFR2 kinase, topoisomerase I and in vitro cytotoxic activities of heteroleptic copper(II) complexes of tetrazolo[1,5-a]pyrimidines and diimines.

    PubMed

    Haleel, A; Mahendiran, D; Veena, V; Sakthivel, N; Rahiman, A Kalilur

    2016-11-01

    A series of heteroleptic mononuclear copper(II) complexes of the type [Cu(L(1-3))(diimine)]ClO4 (1-6) containing three tetrazolo[1,5-a]pyrimidine core ligands, ethyl 5-methyl-7-(2-hydroxyphenyl)-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxylate (HL(1)), ethyl 5-methyl-7-(4-diethylamino-2-hydroxyphenyl)-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxylate (HL(2)) or ethyl 5-methyl-7-(2-hydroxy-4-nitrophenyl)-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxylate (HL(3)), and two diimine coligands, 2,2'-bipyridyl (bpy) or 1,10-phenanthroline (phen) have been synthesized and characterized by spectral methods. The geometry of complexes have been determined with the help of electronic absorption and EPR splitting patterns, which suggest four coordinated square planar geometry around copper(II) ion. The lowering of HOMO-LUMO band gap value of complex 4 implies its higher biological activity compared to other complexes. Antioxidant studies revealed that the complexes possess considerable radical scavenging potency against DPPH. The binding studies of the complexes with calf thymus DNA (CT-DNA) revealed groove mode of binding, which was further supported by docking simulation. The complexes 3 and 4 strongly inhibit the topoisomerase I, and also strongly interact with VEGFR2 kinase receptor via π-π, σ-π and hydrogen bonding interaction. Gel electrophoresis experiments demonstrated the ability of the complexes to cleave plasmid DNA in the absence of activators. In vitro cytotoxic activities of the complexes were examined on three cancerous cell lines such as human lung (A549), cervical (HeLa) and colon (HCT-15), and two normal cells such as human embryonic kidney (HEK) and peripheral blood mononuclear cells (PBMCs). The live cell and fluorescent imaging of cancer cells were observed with acridine orange/ethidium bromide staining assay. All encouraging chemical and biological findings indicate that the complex 4 is a suitable candidate for drug target.

  15. Slow binding kinetics of secreted protein, acidic, rich in cysteine-VEGF interaction limit VEGF activation of VEGF receptor 2 and attenuate angiogenesis.

    PubMed

    Cydzik, Marzena; Abdul-Wahid, Aws; Park, Soyeon; Bourdeau, Annie; Bowden, Katherine; Prodeus, Aaron; Kollara, Alexandra; Brown, Theodore J; Ringuette, Maurice J; Gariépy, Jean

    2015-08-01

    VEGF-A (VEGF) drives angiogenesis through activation of downstream effectors to promote endothelial cell proliferation and migration. Although VEGF binds both VEGF receptor 1 (R1) and receptor 2 (R2), its proangiogenic effects are attributed to R2. Secreted protein, acidic, rich in cysteine (SPARC) is a matricellular glycoprotein thought to inhibit angiogenesis by preventing VEGF from activating R1, but not R2. Because R2 rather than R1 mediates proangiogenic activities of VEGF, the role of human SPARC in angiogenesis was reevaluated. We confirm that association of SPARC with VEGF inhibits VEGF-induced HUVEC adherence, motility, and proliferation in vitro and blocks VEGF-induced blood vessel formation ex vivo. SPARC decreases VEGF-induced phosphorylation of R2 and downstream effectors ERK, Akt, and p38 MAPK as shown by Western blot and/or phosphoflow analysis. Surface plasmon resonance indicates that SPARC binds slowly to VEGF (0.865 ± 0.02 × 10(4) M(-1) s(-1)) with a Kd of 150 nM, forming a stable complex that dissociates slowly (1.26 ± 0.003 × 10(-3) s(-1)). Only domain III of SPARC binds VEGF, exhibiting a 15-fold higher affinity than full-length SPARC. These findings support a model whereby SPARC regulates angiogenesis by sequestering VEGF, thus restricting the activation of R2 and the subsequent activation of downstream targets critical for endothelial cell functions.

  16. VEGF: a potential target for hydrocephalus.

    PubMed

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

    2014-12-01

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

  17. Detection of vascular endothelial growth factor (VEGF) and VEGF receptors Flt-1 and KDR in canine mastocytoma cells.

    PubMed

    Rebuzzi, Laura; Willmann, Michael; Sonneck, Karoline; Gleixner, Karoline V; Florian, Stefan; Kondo, Rudin; Mayerhofer, Matthias; Vales, Anja; Gruze, Alexander; Pickl, Winfried F; Thalhammer, Johann G; Valent, Peter

    2007-02-15

    Vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis and a potential autocrine growth factor for neoplastic cells in various malignancies. In the present study, we have investigated expression of VEGF and VEGF receptors in canine mastocytomas and the canine mastocytoma cell line C2. As assessed by immunostaining of tissue sections and cytospin slides, primary neoplastic mast cells (MC) and C2 cells were found to express the VEGF protein. In Northern blot and RT-PCR experiments, C2 cells expressed VEGF mRNA in a constitutive manner. VEGF mRNA expression in C2 cells was counteracted by LY294002 and rapamycin, suggesting involvement of the PI3-kinase/mTOR pathway. Moreover, C2 cells were found to express VEGF receptor-1 (Flt-1) and VEGF receptor-2 (KDR). However, recombinant VEGF failed to promote (3)H-thymidine uptake in C2 cells, and a neutralizing anti-VEGF antibody (bevacizumab) failed to downregulate spontaneous proliferation in these cells. In addition, rapamycin decreased the expression of VEGF in C2 cells at the mRNA and protein level without suppressing their proliferation. Together, canine mastocytoma cells express VEGF as well as VEGF receptors. However, despite co-expression of VEGF and its receptors, VEGF is not utilized as an autocrine growth regulator by canine mastocytoma cells. PMID:17196258

  18. Relationship between VEGF Gene Polymorphisms and Serum VEGF Protein Levels in Patients with Rheumatoid Arthritis

    PubMed Central

    Paradowska-Gorycka, Agnieszka; Pawlik, Andrzej; Romanowska-Prochnicka, Katarzyna; Haladyj, Ewa; Malinowski, Damian; Stypinska, Barbara; Manczak, Malgorzata; Olesinska, Marzena

    2016-01-01

    Background Rheumatoid arthritis (RA) is one of the chronic autoimmune diseases, with genetic and environmental predisposition, and synovial angiogenesis is considered to be a notable stage in its pathogenesis. Angiogenesis or vascular proliferation has been suggested to be a pivotal mechanism involved in both inflammation/immune activation and joint invasion and destruction. RA may be considered an “angiogenic disease” because it is associated with active tissue neovascularization. Vascular endothelial growth factor (VEGF) promotes vascular permeability, regulates angiogenesis, endothelial cell proliferation and migration, chemotaxis, and capillary hyper permeability and therefore is involved in the development of inflammation. VEGF is the most potent proangiogenic molecule promoting the angiogenic phenotype of RA and is upregulated in RA. Objectives The aim of the study was to identify functional VEGF variants and their possible association with VEGF expression, susceptibility to and severity of RA. Methods 581 RA patients and of 341 healthy individuals were examined for -1154 A/G, -2578 A/C VEGF gene polymorphisms by PCR-RFLP method and for -634 G/C VEGF gene polymorphisms by TaqMan SNP genotyping assay. Serum VEGF levels in RA patients and controls were measured by ELISA. Results The -1154 A/G VEGF gene polymorphism under the codominant, recessive (AA+AG vs. GG) and dominant (AA vs. AG+GG) models were associated with RA (p = 0.0009; p = 0.004; p = 0.017, respectively). VEGF -2578 A/C revealed differences in the case-control distribution in codominant, recessive, dominant and overdominant models (all p<0.0001). Furthermore, the -634 G/C VEGF gene SNP was not correlated with susceptibility to RA in Polish population. The genotype-phenotype analysis showed significant association between the VEGF -1154 A/G and -634 G/C and mean value of the hemoglobin (all p = 0.05), additionally they relevated that the number of women with the polymorphic allele -2578 C was

  19. VEGF111: new insights in tissue invasion

    PubMed Central

    Danastas, Kevin; Combes, Valery; Lindsay, Laura A.; Grau, Georges E. R.; Thompson, Michael B.; Murphy, Christopher R.

    2015-01-01

    Vascular endothelial growth factor is a secreted glycoprotein that acts on endothelial cells to induce developmental and physiological angiogenesis. It has also been implicated in angiogenesis occurring in several pathologies, most notably, cancer. Alternative splicing of VEGF mRNA transcripts results in several isoforms with distinct properties depending on their exon composition. Recently, a new isoform has been identified, VEGF111 with a unique exon composition responsible for its high angiogenic potential. In humans, the only known inducer of VEGF111 is DNA damage but its natural presence in the uterus of the viviparous lizard, Saiphos equalis, suggests other mechanisms of regulation. Most interestingly, the possible relationship between the evolution of viviparity and the associated increased risk in developing cancer may be important in understanding the mechanisms underlying tumor development. PMID:25657624

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

    PubMed

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

    2007-07-16

    Malignant melanoma is the most lethal of the skin cancers and the UK incidence is rising faster than that of any other cancer. Angiogenesis - the growth of new vessels from preexisting vasculature - is an absolute requirement for tumour survival and progression beyond a few hundred microns in diameter. We previously described a class of anti-angiogenic isoforms of VEGF, VEGF(xxx)b, that inhibit tumour growth in animal models, and are downregulated in some cancers, but have not been investigated in melanoma. To determine whether VEGF(xxx)b expression was altered in melanoma, PCR and immunohistochemistry of archived human tumour samples were used. In normal epidermis and in a proportion of melanoma samples, VEGF(xxx)b staining was seen. Some melanomas had much weaker staining. Subsequent examination revealed that expression was significantly reduced in primary melanoma samples (both horizontal and vertical growth phases) from patients who subsequently developed tumour metastasis compared with those who did not (analysis of variance (ANOVA) P<0.001 metastatic vs nonmetastatic), irrespective of tumour thickness, while the surrounding epidermis showed no difference in expression. Staining for total VEGF expression showed staining in metastatic and nonmetastatic melanomas, and normal epidermis. An absence of VEGF(xxx)b expression appears to predict metastatic spread in patients with primary melanoma. These results suggest that there is a switch in splicing as part of the metastatic process, from anti-angiogenic to pro-angiogenic VEGF isoforms. This may form part of a wider metastatic splicing phenotype.

  1. Carbon nanotubes as VEGF carriers to improve the early vascularization of porcine small intestinal submucosa in abdominal wall defect repair.

    PubMed

    Liu, Zhengni; Feng, Xueyi; Wang, Huichun; Ma, Jun; Liu, Wei; Cui, Daxiang; Gu, Yan; Tang, Rui

    2014-01-01

    Insufficient early vascularization in biological meshes, resulting in limited host tissue incorporation, is thought to be the primary cause for the failure of abdominal wall defect repair after implantation. The sustained release of exogenous angiogenic factors from a biocompatible nanomaterial might be a way to overcome this limitation. In the study reported here, multiwalled carbon nanotubes (MWNT) were functionalized by plasma polymerization to deliver vascular endothelial growth factor165 (VEGF165). The novel VEGF165-controlled released system was incorporated into porcine small intestinal submucosa (PSIS) to construct a composite scaffold. Scaffolds incorporating varying amounts of VEGF165-loaded functionalized MWNT were characterized in vitro. At 5 weight percent MWNT, the scaffolds exhibited optimal properties and were implanted in rats to repair abdominal wall defects. PSIS scaffolds incorporating VEGF165-loaded MWNT (VEGF-MWNT-PSIS) contributed to early vascularization from 2-12 weeks postimplantation and obtained more effective collagen deposition and exhibited improved tensile strength at 24 weeks postimplantation compared to PSIS or PSIS scaffolds, incorporating MWNT without VEGF165 loading (MWNT-PSIS).

  2. KDR Amplification Is Associated with VEGF-Induced Activation of the mTOR and Invasion Pathways but does not Predict Clinical Benefit to the VEGFR TKI Vandetanib

    PubMed Central

    Nilsson, Monique B.; Giri, Uma; Gudikote, Jayanthi; Tang, Ximing; Lu, Wei; Tran, Hai; Fan, Youhong; Koo, Andrew; Diao, Lixia; Tong, Pan; Wang, Jing; Herbst, Roy; Johnson, Bruce E.; Ryan, Andy; Webster, Alan; Rowe, Philip; Wistuba, Ignacio I.; Heymach, John V.

    2016-01-01

    Purpose VEGF pathway inhibitors have been investigated as therapeutic agents in the treatment of non–small cell lung cancer (NSCLC) because of its central role in angiogenesis. These agents have improved survival in patients with advanced NSCLC, but the effects have been modest. Although VEGFR2/KDR is typically localized to the vasculature, amplification of KDR has reported to occur in 9% to 30% of the DNA from different lung cancers. We investigated the signaling pathways activated downstream of KDR and whether KDR amplification is associated with benefit in patients with NSCLC treated with the VEGFR inhibitor vandetanib. Methods NSCLC cell lines with or without KDR amplification were studied for the effects of VEGFR tyrosine kinase inhibitors (TKI) on cell viability and migration. Archival tumor samples collected from patients with platinum-refractory NSCLC in the phase III ZODIAC study of vandetanib plus docetaxel or placebo plus docetaxel (N = 294) were screened for KDR amplification by FISH. Results KDR amplification was associated with VEGF-induced activation of mTOR, p38, and invasiveness in NSCLC cell lines. However, VEGFR TKIs did not inhibit proliferation of NSCLC cell lines with KDR amplification. VEGFR inhibition decreased cell motility as well as expression of HIF1α in KDR-amplified NSCLC cells. In the ZODIAC study, KDR amplification was observed in 15% of patients and was not associated with improved progression-free survival, overall survival, or objective response rate for the vandetanib arm. Conclusions Preclinical studies suggest KDR activates invasion but not survival pathways in KDR-amplified NSCLC models. Patients with NSCLC whose tumor had KDR amplification were not associated with clinical benefit for vandetanib in combination with docetaxel. PMID:26578684

  3. Comparing protein VEGF inhibitors: In vitro biological studies

    SciTech Connect

    Yu, Lanlan; Liang, Xiao Huan; Ferrara, Napoleone

    2011-05-06

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

  4. Expression of VEGF in Neonatal Urinary Obstruction: Does Expression of VEGF Predict Hydronephrosis?

    PubMed Central

    Magyar, Zsófia; Schönleber, Julianna; Romics, Miklós; Hruby, Ervin; Nagy, Bálint; Sulya, Bálint; Beke, Artúr; Harmath, Ágnes; Jeager, Judit; Rigó, János; Görbe, Éva

    2015-01-01

    Background In animal studies, the inhibition of VEGF activity results in high mortality and impaired renal and glomerular development. Mechanical stimuli, like mechanical stretch in respiratory and circulatory systems, results in an elevated expression of VEGF. In animal models, the experimental urinary obstruction is associated with stretching of tubular cells and activations of the renin-angiotensin system. This results in the upregulation of vascular endothelial growth factor (VEGF) and TNF-alfa. Material/Methods Tissue samples from urinary tract obstruction were collected and immunohistochemistry was performed in 14 patients (average age: 7.1±4.1 years). The control histology group consisted of ureteropelvic junction tissue from 10 fetuses after midtrimester artificial abortion. The fetuses did not have any failure at ultrasound screening and pathological examination. The mean gestational age was 20.6 weeks of gestation (±2.2SD). Expression of VEGF was detected with immunohistochemistry method. Results Expression of VEGF was found in varying intensity in the submucosa and subserosa layers, but only in the test tissue (placental tissue). The tissue of the patients with urinary obstruction and the tissue of the fetal ureteropelvic junction without urinary obstruction were negative for expression of VEGF. The repeated examination showed negative cells and no color staining. Conclusions The pressure due to congenital urogenital obstruction resulting in mechanical stress in cells did not increase the expression of VEGF in young children in our study. To find a correlation between urogenital tract obstruction and increased expression of VEGF, we need to perform more examinations because the connection may be of therapeutic significance. PMID:25951999

  5. The Inorganic Perspective of VEGF: Interactions of Cu(2+) with Peptides Encompassing a Recognition Domain of the VEGF Receptor.

    PubMed

    Grasso, Giulia; Santoro, Anna Maria; Magrì, Antonio; La Mendola, Diego; Tomasello, Marianna Flora; Zimbone, Stefania; Rizzarelli, Enrico

    2016-06-01

    The vascular endothelial growth factor A (VEGF-A) is a potent angiogenic factor, its activity may be influenced by the presence of copper(II) ions. To mimic the interaction between copper(II) and VEGF (Vascular Endotelial Growth Factor), the N- and C-terminally blocked peptide fragments VEGF73-101 and VEGF84-101, owing to VEGF165 protein, have been synthesized. These protein domains represent a specific recognition site with the VEGF receptor (VEGFR). Copper(II) complexes with VEGF73-101 and VEGF84-101 were investigated by means of potentiometry and UV-Vis, ESI-MS, CD, EPR spectroscopic methods. Both peptides have three histidine residues and display a binding high affinity for copper(II) ions. The proliferative activity of the peptides in the absence and presence of copper(II) ions as well as of VEGF-165 protein was also tested on HUVEC cells (Human Umbilical Vein Endothelial Cells). The VEGF73-101 showed a dose-dependent anti-proliferative activity, while the shorter peptide VEGF84-101 did not affect HUVEC proliferation, both in the presence and in the absence of VEGF. PMID:27015654

  6. An Antagonistic Vascular Endothelial Growth Factor (VEGF) Variant Inhibits VEGF-Stimulated Receptor Autophosphorylation and Proliferation of Human Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Siemeister, Gerhard; Schirner, Michael; Reusch, Petra; Barleon, Bernhard; Marme, Dieter; Martiny-Baron, Georg

    1998-04-01

    Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.

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

    PubMed Central

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

    2016-01-01

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

  8. PC4 induces lymphangiogenesis dependent VEGF-C/VEGF-D/VEGFR-3 axis activation in lung adenocarcinoma

    PubMed Central

    Tao, Shaolin; Yu, Jie; Xu, Yi; Deng, Bo; Sun, Tianyu; Hu, Pingping; Wei, Zhuanqin; Zhang, Jingge; Wang, Ruwen; Shi, Chunmeng; Tan, Qunyou

    2015-01-01

    Human transcriptional positive cofactor 4 (PC4) is a novel marker for diagnosis and treatment of advanced human cancers metastasis. In human lung adenocarcinoma, tumor lymphangiogenesis, an important early event, can promotes lymphatic metastasis, while it has been reported that VEGF-C/VEGF-D/VEGFR-3 axis plays an important role in lymphangiogenesis. The proposed study aims to explore whether PC4 correlates with VEGF-C/VEGF-D/VEGFR-3 axis of lymphangiogenesis in the lymph node metastasis during lung adenocarcinoma. Here, small interfering RNA technique was employed to investigate the relationship of PC4 and the VEGF-C/VEGF-D/VEGFR-3 axis in lung adenocarcinoma cell lines as well as tumor xenografts of mice model. And then mRNA and protein levels of PC4, VEGF-C, VEGF-D and VEGFR-3 were analyzed. Moreover, the correlation between PC4 expression and lymphatic vessel density or the rate of metastatsis in vivo was also revealed. Down-regulating PC4 expression resulted in the lower expression of VEGFC, VEGF-D and VEGFR-3 in mRNA and protein levels, and PC4 expression was significantly related with the factor of VEGF-C/VEGF-D/VEGFR-3 axis expression (P<0.05). Meanwhile, high expression level of PC4 was accompanied by the higher density of tumor lymphatic vessels and the rate of metastatsis in vivo (P<0.05). PC4 expression correlated with the levels of VEGF-C, VEGF-D and VEGFR-3 during the development of lymphangiogenesis and lymphatic metastasis in lung adenocarcinoma in vitro and in vivo, which may be a novel marker in the development of lymphangiogenesis and lymphatic metastasis of tumors. PMID:26269750

  9. Loss of phospholipase D2 impairs VEGF-induced angiogenesis

    PubMed Central

    Lee, Chang Sup; Ghim, Jaewang; Song, Parkyong; Suh, Pann-Ghill; Ryu, Sung Ho

    2016-01-01

    Vascular endothelial growth factor (VEGF) is a key mediator of angiogenesis and critical for normal embryonic development and repair of pathophysiological conditions in adults. Although phospholipase D (PLD) activity has been implicated in angiogenic processes, its role in VEGF signaling during angiogenesis in mammals is unclear. Here, we found that silencing of PLD2 by siRNA blocked VEGF-mediated signaling in immortalized human umbilical vein endothelial cells (iHUVECs). Also, VEGF-induced endothelial cell survival, proliferation, migration, and tube formation were inhibited by PLD2 silencing. Furthermore, while Pld2-knockout mice exhibited normal development, loss of PLD2 inhibited VEGF-mediated ex vivo angiogenesis. These findings suggest that PLD2 functions as a key mediator in the VEGF-mediated angiogenic functions of endothelial cells. [BMB Reports 2016; 49(3): 191-196] PMID:26818087

  10. Detection of VEGF-Axxxb Isoforms in Human Tissues

    PubMed Central

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

    2013-01-01

    Vascular Endothelial Growth Factor-A (VEGF-A) can be generated as multiple isoforms by alternative splicing. Two families of isoforms have been described in humans, pro-angiogenic isoforms typified by VEGF-A165a, and anti-angiogenic isoforms typified by VEGF-A165b. The practical determination of expression levels of alternative isoforms of the same gene may be complicated by experimental protocols that favour one isoform over another, and the use of specific positive and negative controls is essential for the interpretation of findings on expression of the isoforms. Here we address some of the difficulties in experimental design when investigating alternative splicing of VEGF isoforms, and discuss the use of appropriate control paradigms. We demonstrate why use of specific control experiments can prevent assumptions that VEGF-A165b is not present, when in fact it is. We reiterate, and confirm previously published experimental design protocols that demonstrate the importance of using positive controls. These include using known target sequences to show that the experimental conditions are suitable for PCR amplification of VEGF-A165b mRNA for both q-PCR and RT-PCR and to ensure that mispriming does not occur. We also provide evidence that demonstrates that detection of VEGF-A165b protein in mice needs to be tightly controlled to prevent detection of mouse IgG by a secondary antibody. We also show that human VEGF165b protein can be immunoprecipitated from cultured human cells and that immunoprecipitating VEGF-A results in protein that is detected by VEGF-A165b antibody. These findings support the conclusion that more information on the biology of VEGF-A165b isoforms is required, and confirm the importance of the experimental design in such investigations, including the use of specific positive and negative controls. PMID:23935865

  11. Dynamics of VEGF matrix-retention in vascular network patterning

    NASA Astrophysics Data System (ADS)

    Köhn-Luque, A.; de Back, W.; Yamaguchi, Y.; Yoshimura, K.; Herrero, M. A.; Miura, T.

    2013-12-01

    Vascular endothelial growth factor (VEGF) is a central regulator of blood vessel morphogenesis, although its role in patterning of endothelial cells into vascular networks is not fully understood. It has been suggested that binding of soluble VEGF to extracellular matrix components causes spatially restricted cues that guide endothelial cells into network patterns. Yet, current evidence for such a mechanism remains indirect. In this study, we quantitatively analyse the dynamics of VEGF retention in a controlled in vitro situation of human umbilical vascular endothelial cells (HUVECs) in Matrigel. We show that fluorescent VEGF accumulates in pericellular areas and colocalizes with VEGF binding molecules. Analysis of fluorescence recovery after photobleaching reveals that binding/unbinding to matrix molecules dominates VEGF dynamics in the pericellular region. Computational simulations using our experimental measurements of kinetic parameters show that matrix retention of chemotactic signals can lead to the formation of reticular cellular networks on a realistic timescale. Taken together, these results show that VEGF binds to matrix molecules in proximity of HUVECs in Matrigel, and suggest that bound VEGF drives vascular network patterning.

  12. [Role of VEGF in diseases of the retina].

    PubMed

    Barquet, Luis Arias

    2015-03-01

    Angiogenesis is the process through which new blood vessels are formed, based on preexisting vessels, and is the paradigm of diseases such as cancer and exudative ageassociated macular degeneration (ARMD). Several proangiogenic factors have been identified, such as vascular endothelial growth factor (VEGF), especially VEGF-A, which activates endothelial cells and promotes cell proliferation, migration, and an increase in vascular permeability. VEGF is also involved in the etiopathogenesis of other retinal diseases, such as diabetic macular edema and macular edema secondary to retinal vein occlusion. Likewise, there is increasing evidence that placental growth factor (PIGF) acts recepsynergetically with VEGF in promoting these diseases. Currently, the main treatment for these diseases are the anti-VEGF drugs, aflibercept, ranibizumab and bevacizumab. These agents differ in their molecular structure and mechanism of action.

  13. VEGF improves survival of mesenchymal stem cells in infarcted hearts

    SciTech Connect

    Pons, Jennifer; Huang Yu; Arakawa-Hoyt, Janice; Washko, Daniel; Takagawa, Junya; Ye, Jianqin; Grossman, William; Su Hua

    2008-11-14

    Bone marrow-derived mesenchymal stem cells (MSC) are a promising source for cell-based treatment of myocardial infarction (MI), but existing strategies are restricted by low cell survival and engraftment. We examined whether vascular endothelial growth factor (VEGF) improve MSC viability in infracted hearts. We found long-term culture increased MSC-cellular stress: expressing more cell cycle inhibitors, p16{sup INK}, p21 and p19{sup ARF}. VEGF treatment reduced cellular stress, increased pro-survival factors, phosphorylated-Akt and Bcl-xL expression and cell proliferation. Co-injection of MSCs with VEGF to MI hearts increased cell engraftment and resulted in better improvement of cardiac function than that injected with MSCs or VEGF alone. In conclusion, VEGF protects MSCs from culture-induce cellular stress and improves their viability in ischemic myocardium, which results in improvements of their therapeutic effect for the treatment of MI.

  14. Neo-vascularization of the stroke cavity by implantation of human neural stem cells on VEGF-releasing PLGA microparticles

    PubMed Central

    Bible, Ellen; Qutachi, Omar; Chau, David Y.S.; Alexander, Morgan R.; Shakesheff, Kevin M.; Modo, Michel

    2012-01-01

    Replacing the tissue lost after a stroke potentially provides a new neural substrate to promote recovery. However, significant neurobiological and biotechnological challenges need to be overcome to make this possibility into a reality. Human neural stem cells (hNSCs) can differentiate into mature brain cells, but require a structural support that retains them within the cavity and affords the formation of a de novo tissue. Nevertheless, in our previous work, even after a week, this primitive tissue is void of a vasculature that could sustain its long-term viability. Therefore, tissue engineering strategies are required to develop a vasculature. Vascular endothelial growth factor (VEGF) is known to promote the proliferation and migration of endothelial cells during angio- and arteriogenesis. VEGF by itself here did not affect viability or differentiation of hNSCs, whereas growing cells on poly(D,L-lactic acid-co-glycolic acid) (PLGA) microparticles, with or without VEGF, doubled astrocytic and neuronal differentiation. Secretion of a burst and a sustained delivery of VEGF from the microparticles in vivo attracted endothelial cells from the host into this primate tissue and in parts established a neovasculature, whereas in other parts endothelial cells were merely interspersed with hNSCs. There was also evidence of a hypervascularization indicating that further work will be required to establish an adequate level of vascularization. It is therefore possible to develop a putative neovasculature within de novo tissue that is forming inside a tissue cavity caused by a stroke. PMID:22818980

  15. VEGF-B promotes cancer metastasis through a VEGF-A–independent mechanism and serves as a marker of poor prognosis for cancer patients

    PubMed Central

    Yang, Xiaojuan; Zhang, Yin; Hosaka, Kayoko; Andersson, Patrik; Wang, Jian; Tholander, Fredrik; Cao, Ziquan; Morikawa, Hiromasa; Tegnér, Jesper; Yang, Yunlong; Iwamoto, Hideki; Lim, Sharon; Cao, Yihai

    2015-01-01

    The biological functions of VEGF-B in cancer progression remain poorly understood. Here, we report that VEGF-B promotes cancer metastasis through the remodeling of tumor microvasculature. Knockdown of VEGF-B in tumors resulted in increased perivascular cell coverage and impaired pulmonary metastasis of human melanomas. In contrast, the gain of VEGF-B function in tumors led to pseudonormalized tumor vasculatures that were highly leaky and poorly perfused. Tumors expressing high levels of VEGF-B were more metastatic, although primary tumor growth was largely impaired. Similarly, VEGF-B in a VEGF-A–null tumor resulted in attenuated primary tumor growth but substantial pulmonary metastases. VEGF-B also led to highly metastatic phenotypes in Vegfr1 tk−/− mice and mice treated with anti–VEGF-A. These data indicate that VEGF-B promotes cancer metastasis through a VEGF-A–independent mechanism. High expression levels of VEGF-B in two large-cohort studies of human patients with lung squamous cell carcinoma and melanoma correlated with poor survival. Taken together, our findings demonstrate that VEGF-B is a vascular remodeling factor promoting cancer metastasis and that targeting VEGF-B may be an important therapeutic approach for cancer metastasis. PMID:25991856

  16. Overexpression of VEGF165b in Podocytes Reduces Glomerular Permeability

    PubMed Central

    Qiu, Yan; Ferguson, Joanne; Oltean, Sebastian; Neal, Chris R.; Kaura, Amit; Bevan, Heather; Wood, Emma; Sage, Leslie M.; Lanati, Silvia; Nowak, Dawid G.; Salmon, Andy H.J.; Bates, David

    2010-01-01

    The observation that therapeutic agents targeting vascular endothelial growth factor-A (VEGF-A) associate with renal toxicity suggests that VEGF plays a role in the maintenance of the glomerular filtration barrier. Alternative mRNA splicing produces the VEGFxxxb family, which consists of antiangiogenic peptides that reduce permeability and inhibit tumor growth; the contribution of these peptides to normal glomerular function is unknown. Here, we established and characterized heterozygous and homozygous transgenic mice that overexpress VEGF165b specifically in podocytes. We confirmed excess production of glomerular VEGF165b by reverse transcriptase–PCR, immunohistochemistry, and ELISA in both heterozygous and homozygous animals. Macroscopically, the mice seemed normal up to 18 months of age, unlike the phenotype of transgenic podocyte-specific VEGF164-overexpressing mice. Animals overexpressing VEGF165b, however, had a significantly reduced normalized glomerular ultrafiltration fraction with accompanying changes in ultrastructure of the glomerular filtration barrier on the vascular side of the glomerular basement membrane. These data highlight the contrasting properties of VEGF splice variants and their impact on glomerular function and phenotype. PMID:20688932

  17. VEGF modulates synaptic activity in the developing spinal cord.

    PubMed

    Guérit, Sylvaine; Allain, Anne-Emilie; Léon, Céline; Cazenave, William; Ferrara, Napoleone; Branchereau, Pascal; Bikfalvi, Andréas

    2014-11-01

    Although it has been documented that the nervous and the vascular systems share numerous analogies and are closely intermingled during development and pathological processes, interactions between the two systems are still poorly described. In this study, we investigated whether vascular endothelial growth factor (VEGF), which is a key regulator of vascular development, also modulates neuronal developmental processes. We report that VEGF enhances the gamma-aminobutyric acid (GABA)/glycinergic but not glutamatergic synaptic activity in embryonic spinal motoneurons (MNs), without affecting MNs excitability. In response to VEGF, the frequency of these synaptic events but not their amplitude was increased. Blocking endogenous VEGF led to an opposite effect by decreasing frequency of synaptic events. We found that this effect occurred specifically at early developmental stages (E13.5 and E15.5) and vanished at the prenatal stage E17.5. Furthermore, VEGF was able to increase vesicular inhibitory amino acid transporter density at the MN membrane. Inhibition of single VEGF receptors did not modify electrophysiological parameters indicating receptor combinations or an alternative pathway. Altogether, our findings identify VEGF as a modulator of the neuronal activity during synapse formation and highlight a new ontogenic role for this angiogenic factor in the nervous system.

  18. VEGF and CD31 association in pituitary adenomas.

    PubMed

    Cristina, Carolina; Perez-Millan, María Inés; Luque, Guillermina; Dulce, Raúl Ariel; Sevlever, Gustavo; Berner, Silvia Inés; Becu-Villalobos, Damasia

    2010-09-01

    Pituitary tumors are usually less vascularized than the normal pituitary, and the role of angiogenesis in these adenomas is contentious. Appraisal of microvascular density and expression of the potent angiogenic vascular endothelial growth factor (VEGF) by immunohistochemistry has yielded controversial results, as a broad spectrum of immunostaining can be found. We determined the protein expression of VEGF and CD31, an endothelial marker, in a series of 56 surgically removed pituitary adenomas using Western blot assay. Prolactinomas had higher VEGF protein expression compared to nonfunctioning or ACTH- and GH-secreting adenomas, while CD31 was similar in the different adenoma histotypes. VEGF and CD31 were not affected by sex, age, years of adenoma evolution, or proliferation rate (Ki67 and PCNA) for all adenoma types. Only in nonfunctioning adenomas CD31 concentration increased significantly with age. There was a positive correlation between CD31 and VEGF expression when all adenoma histotypes were considered, or when prolactinomas and nonfunctioning adenomas were evaluated separately. The positive association of VEGF and CD31 expression suggests the participation of angiogenesis in adenoma development, while epithelial cell proliferation in pituitary tumors is not directly related to VEGF or CD31 expression, and other factors, such as primary genetic alterations may be involved. PMID:20473646

  19. Role of Ceacam1 in VEGF induced vasculogenesis of murine embryonic stem cell-derived embryoid bodies in 3D culture.

    PubMed

    Gu, Angel; Tsark, Walter; Holmes, Kathryn V; Shively, John E

    2009-06-10

    CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1), a type I transmembrane glycoprotein involved in cell-cell adhesion has been shown to act as an angiogenic factor for mouse and human endothelial cells. Based on the ability of CEACAM1 to initiate lumen formation in human mammary epithelial cells grown in 3D culture (Matrigel), we hypothesized that murine CEACAM1 may play a similar role in vasculogenesis. In order to test this hypothesis, murine embryonic stem (ES) cells stimulated with VEGF were differentiated into embryoid bodies (EB) for 8 days (-8-0 d) and transferred to Matrigel in the presence or absence of anti-CEACAM1 antibody for an additional 12 days (0-12 d). In the absence of anti-CEACAM1 antibody or in the presence of an isotype control antibody, the EB in Matrigel underwent extensive sprouting, generating lengthy vascular structures with well-defined lumina as demonstrated by confocal microscopy, electron microscopy, and immunohistochemical analysis. Both the length and architecture of the vascular tubes were inhibited by anti-CEACAM1 mAb CC1, a mAb that blocks the cell-cell adhesion functions of CEACAM1, thus demonstrating a critical role for this cell-cell adhesion molecule in generating and maintaining vasculogenesis. QRT-PCR analysis of the VEGF treated ES cells grown under conditions that convert them to EB revealed expression of Ceacam1 as early as -5 to -3 d reaching a maximum at day 0 at which time EBs were transferred to Matrigel, thereafter levels at first declined and then increased over time. Other markers of vasculogenesis including Pecam1, VE-Cad, and Tie-1 were not detected until day 0 when EBs were transferred to Matrigel followed by a steady increase in levels, indicating later roles in vasculogenesis. In contrast, Tie-2 and Flk-1 (VEGFR2) were detected on day five of EB formation reaching a maximum at day 0 on transfer to Matrigel, similar to Ceacam1, but after which Tie-2 declined over time, while Flk-1 increased

  20. Role of Ceacam1 in VEGF induced vasculogenesis of murine embryonic stem cell-derived embryoid bodies in 3D culture

    SciTech Connect

    Gu, Angel; Tsark, Walter; Holmes, Kathryn V.; Shively, John E.

    2009-06-10

    CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1), a type I transmembrane glycoprotein involved in cell-cell adhesion has been shown to act as an angiogenic factor for mouse and human endothelial cells. Based on the ability of CEACAM1 to initiate lumen formation in human mammary epithelial cells grown in 3D culture (Matrigel), we hypothesized that murine CEACAM1 may play a similar role in vasculogenesis. In order to test this hypothesis, murine embryonic stem (ES) cells stimulated with VEGF were differentiated into embryoid bodies (EB) for 8 days (- 8-0 d) and transferred to Matrigel in the presence or absence of anti-CEACAM1 antibody for an additional 12 days (0-12 d). In the absence of anti-CEACAM1 antibody or in the presence of an isotype control antibody, the EB in Matrigel underwent extensive sprouting, generating lengthy vascular structures with well-defined lumina as demonstrated by confocal microscopy, electron microscopy, and immunohistochemical analysis. Both the length and architecture of the vascular tubes were inhibited by anti-CEACAM1 mAb CC1, a mAb that blocks the cell-cell adhesion functions of CEACAM1, thus demonstrating a critical role for this cell-cell adhesion molecule in generating and maintaining vasculogenesis. QRT-PCR analysis of the VEGF treated ES cells grown under conditions that convert them to EB revealed expression of Ceacam1 as early as - 5 to - 3 d reaching a maximum at day 0 at which time EBs were transferred to Matrigel, thereafter levels at first declined and then increased over time. Other markers of vasculogenesis including Pecam1, VE-Cad, and Tie-1 were not detected until day 0 when EBs were transferred to Matrigel followed by a steady increase in levels, indicating later roles in vasculogenesis. In contrast, Tie-2 and Flk-1 (VEGFR2) were detected on day five of EB formation reaching a maximum at day 0 on transfer to Matrigel, similar to Ceacam1, but after which Tie-2 declined over time, while Flk-1 increased

  1. Two Novel Heparin-binding Vascular Endothelial Growth Factor Splices, L-VEGF144 and L-VEGF138, are Expressed in Human Glioblastoma Cells.

    PubMed

    Shen, Chiung-Chyi; Cheng, Wen-Yu; Chiao, Ming-Tsang; Liang, Yea-Jiuan; Mao, Tsuo-Fei; Liu, Bai-Shuan

    2016-01-01

    The expression levels of different vascular endothelial growth factor A (VEGF) isoforms are associated with the angiogenesis and the patient's prognoses in human cancers. Ribosomes specifically scan from 5' to 3' CUG initiation codon in the long 5'-untranslated region (5'-UTR) of the VEGF mRNA, resulting in the generation of high mol wt VEGF isoform [call large VEGF (L-VEGF)]. Alternative splicing of VEGF mRNA transcripts results in several isoforms with distinct properties that are dependent up their exon compositions. In this study, we observed two novel kinds of splicing VEGF isoforms that transcripted at the first upstream CUG codon, and which we have named large-VEGF144 (LVEGF144), and large-VEGF138 (L-VEGF138). The expression levels of messenger RNA for the different VEGF splice forms were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). After DNA sequencing, the genetic structure of L-VEGF144 involved not only a partial exon 1, exon 6a, and exons 7-8, but also an unique 108- nucleotides insertion of VEGF intron 5 interposed between exon 1 and exon 6. At the same time, L-VEGF144 lacked most of the Nterminal fragments (exons 1-5). We further found that a specific detection model could easily and rapidly confirm the presence of L-VEGF144 mRNA fragments in the biopsies or cell lines via RT-PCR assay. In addition, we used visible fluorescent fusion proteins to prove that both L-VEGF144 and L-VEGF138 have nuclear localization ability. Taken together, the findings of this study indicate that, unlike previously identified isoforms, these novel VEGF isoforms are likely to suggest a further level of complexity in the angiogenic process. PMID:27220431

  2. VEGF: From Discovery to Therapy: The Champalimaud Award Lecture

    PubMed Central

    Miller, Joan W.

    2016-01-01

    Purpose Intraocular vascular diseases are leading causes of adult vision loss, and in the mid-1900s, I. C. Michaelson postulated that the retina releases a soluble, diffusible factor that causes abnormal vascular growth and leakage. What became known as “Factor X” eluded investigators for decades. Methods The field of cancer research, where Judah Folkman pioneered the concept of angiogenesis, provided the inspiration for the work honored by the 2014 Champalimaud Vision Award. Recognizing that tumors recruit their own blood supply to achieve critical mass, Dr Folkman proposed that angiogenic factors could be therapeutic targets in cancer. Napoleone Ferrara identified vascular endothelial growth factor (VEGF) as such an angiogenic agent: stimulated by hypoxic tumor tissue, secreted, and able to induce neovascularization. VEGF also was a candidate for Factor X, and the 2014 Champalimaud Laureates and colleagues worked individually and collaboratively to identify the role of VEGF in ocular disease. Results The Champalimaud Laureates correlated VEGF with ocular neovascularization in animal models and in patients. Moreover, they showed that VEGF not only was sufficient, but it also was required to induce neovascularization in normal animal eyes, as VEGF inhibition abolished ocular neovascularization in key animal models. Conclusions The identification of VEGF as Factor X altered the therapeutic paradigms for age-related macular degeneration (AMD), diabetic retinopathy, retinal vein occlusion, and other retinal disorders. Translational Relevance The translation of VEGF from discovery to therapy resulted in the most successful applications of antiangiogenic therapy to date. Annually, over one million patients with eye disease are treated with anti-VEGF agents. PMID:26981331

  3. VEGF expression in hepatectomized tumor-bearing mice.

    PubMed

    Andrini, L; Blanco, A Fernandez; Inda, A; García, M; Garcia, A; Errecalde, A

    2011-01-01

    The experiments were designed in order to study the VEGF expression in intact (group I), hepatectomized (group II), and hepatectomized-tumor bearing mice (group III) throughout one complete circadian time span. Adult male mice were used for the VEGF expression study. The statistical analysis was performed using analysis of variance (ANOVA). The results showed statistical differences in the VEGF expression between groups I and II, but the most significant differences were found between groups I and III. In conclusion, these expressions have a circadian rhythm in all groups; moreover, in group III, this expression was higher and appeared before than in the others.

  4. Corneal Neovascularization: An Anti-VEGF Therapy Review

    PubMed Central

    Chang, Jin-Hong; Garg, Nitin K.; Lunde, Elisa; Han, Kyu-Yeon; Jain, Sandeep; Azar, Dimitri T.

    2013-01-01

    Corneal neovascularization is a serious condition that can lead to a profound decline in vision. The abnormal vessels block light, cause corneal scarring, compromise visual acuity, and may lead to inflammation and edema. Corneal neovascularization occurs when the balance between angiogenic and antiangiogenic factors is tipped toward angiogenic molecules. Vascular endothelial growth factor (VEGF), one of the most important mediators of angiogenesis, is upregulated during neovascularization. In fact, anti-VEGF agents have efficacy in the treatment of neovascular age-related macular degeneration, diabetic retinopathy, macular edema, neovascular glaucoma, and other neovascular diseases. These same agents have great potential for the treatment of corneal neovascularization. We review some of the most promising anti-VEGF therapies, including bevacizumab, VEGF trap, siRNA, and tyrosine kinase inhibitors. PMID:22898649

  5. Hyperoxia enhances VEGF release from A549 cells via post-transcriptional processes

    PubMed Central

    Shenberger, Jeffrey S.; Zhang, Lianqin; Powell, Richard J.; Barchowsky, Aaron

    2007-01-01

    Exposure of animals to hyperoxia decreases lung VEGF mRNA expression concomitant with an acute increase in VEGF protein within the epithelial lining fluid (ELF). The VEGF concentration in ELF is in excess of that found in the plasma, leading to the hypothesis that hyperoxia stimulates the release of VEGF protein from stores within the extracellular matrix. To test this hypothesis in a cell culture system, we exposed A549 cells to 95% O2 for 48 hrs followed by recovery in room air (RA) for 24 hrs. We found that Ox increased VEGF protein 2- to 3-fold within the medium at 48 hrs of exposure and during recovery. Heparin clearing revealed the medium to contain a 50:50 mixture of the heparin-binding (VEGF165) and heparin-non-binding (VEGF121) proteins and Ox to increase both proteins equally. Transcriptional activation of VEGF appears unlikely to explain the increase in VEGF protein as full-length and splice variant VEGF mRNA expression were unchanged by hyperoxia. Analysis of cell-associated VEGF proteins found that Ox increased the expression of VEGF121 and VEGF165 proteins. Blocking binding sites with exogenous heparin enhanced VEGF protein in the medium from RA grown cells while heparinase digestion of bound VEGF revealed a greater reserve of VEGF protein in RA cells. Collectively these findings indicate that hyperoxia enhances the expression of VEGF121/165 proteins and facilitates the release of VEGF165 from cell-associated stores. Increases in VEGF in ELF may represent an adaptive response fostering cell survival and type II cell proliferation in O2-induced lung injury. PMID:17664148

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

    PubMed Central

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

    2016-01-01

    Intraocular vascular endothelial growth factor (VEGF) levels play an important role in the pathogenesis of blindness-related diseases, such as age-related macular degeneration (AMD). Here, we aimed to develop a paper-based enzyme-linked immunosorbent assay (P-ELISA) to analyze the suppression of aqueous VEGF concentrations following intravitreal injection (IVI) of anti-VEGF antibody (bevacizumab or ranibizumab). A total of 25 eyes with wet AMD, one with myopic neovascularization, and one with polypoidal choroidal vasculopathy were enrolled in this study. The limit of detection using P-ELISA was 0.03 pg/mL. Forty-six consecutive samples of aqueous humor were acquired. From all samples, 66.67% (10/15) achieved complete VEGF suppression (below the detection limit) within 5 weeks of receiving IVI of anti-VEGF antibody. Only 13.33% of samples (2/15) achieved complete VEGF suppression 5 weeks after receiving treatment. In some patients, elevated VEGF was still detected 5 weeks after receipt of anti-VEGF antibody, and all samples (10/10) were found to have elevated VEGF levels 49 days after treatment. Thus, we suggest that monthly IVI of anti-VEGF antibody may be required to ensure durable VEGF inhibition. Ultrasensitive P-ELISA can detect elevated VEGF at an earlier time point and may facilitate decision-making regarding appropriate treatment strategies. PMID:27725716

  7. VEGF expression is developmentally regulated during human brain angiogenesis.

    PubMed

    Virgintino, Daniela; Errede, Mariella; Robertson, David; Girolamo, Francesco; Masciandaro, Antonio; Bertossi, Mirella

    2003-03-01

    Vascular endothelial growth factor (VEGF) is a potent angiogenic factor working as an endothelial cell-specific mitogen and exerting a trophic effect on neurons and glial cells, both these activities being essential during central nervous system vascularisation, development and repair. The vascularisation of human telencephalon takes place by means of an angiogenic mechanism, which starts at the beginning of corticogenesis and actively proceeds up to the last neuronal migration, when the basic scheme of the vascular network has been drawn. Our study focused on VEGF during this critical developmental period with the aim of identifying the cells that express VEGF and of correlating the events of angiogenesis with the main events of cerebral cortex formation. The results show that in fetal human brain VEGF protein is located on multiple cell types, cells proper to the nervous tissue, neuroepithelial cells, neuroblasts and radial glia cells, and non-neuronal cells, endothelial and periendothelial cells. In these cells VEGF expression appears developmentally regulated and is correlated with angiogenesis, which in turn responds to the high metabolic demands of the differentiating neocortex.

  8. SREBP inhibits VEGF expression in human smooth muscle cells

    SciTech Connect

    Motoyama, Koka; Fukumoto, Shinya . E-mail: sfukumoto@med.osaka-cu.ac.jp; Koyama, Hidenori; Emoto, Masanori; Shimano, Hitoshi; Maemura, Koji; Nishizawa, Yoshiki

    2006-03-31

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

  9. Kidney Diseases Associated With Anti-Vascular Endothelial Growth Factor (VEGF)

    PubMed Central

    Izzedine, Hassan; Escudier, Bernard; Lhomme, Catherine; Pautier, Patricia; Rouvier, Philippe; Gueutin, Victor; Baumelou, Alain; Derosa, Lisa; Bahleda, Rastilav; Hollebecque, Antoine; Sahali, Djillali; Soria, Jean Charles

    2014-01-01

    the most frequent forms of renal involvement under anti-VEGF therapy. Careful risk-benefit assessment for individual patients should take into account risk factors related to the host and the tumor. PMID:25500702

  10. VEGF mRNA and Protein Concentrations in the Developing Human Eye

    PubMed Central

    Ma, Irene T.; McConaghy, Suzanne; Namachivayam, Kopperuncholan; Halloran, Brian A.; Kurundkar, Ashish R.; MohanKumar, Krishnan; Maheshwari, Akhil; Ohls, Robin K.

    2014-01-01

    Background Vascular endothelial growth factor (VEGF), a well-characterized regulator of angiogenesis, has been mechanistically implicated in retinal neovascularization and in the pathogenesis of ROP. However, the ontogeny of VEGF expression in the human fetal retina is not well known. Because retinal vasculature grows with gestational maturation, we hypothesized that VEGF expression also increases in the midgestation human fetal eye as a function of gestational age. Methods To identify changes in VEGF gene expression during normal human development, we measured VEGF mRNA by quantitative PCR and measured VEGF protein by ELISA and western blots in 10-24 week gestation fetal vitreous, retina, and serum. Results VEGF mRNA expression in the retina increased with gestational age. VEGF isoform A, particularly its VEGF121 splice variant, contributed to this positive correlation. Consistent with these findings, we detected increasing VEGF121 protein concentrations in vitreous humor from fetuses of 10-24 weeks gestation, while VEGF concentrations decreased in fetal serum. Conclusions VEGF121 mRNA and protein concentrations increase with increasing gestational age in the developing human retina. We speculate that VEGF plays an important role in normal retinal vascular development, and that preterm delivery affects production of this vascular growth factor. PMID:25588190

  11. Paclitaxel targets VEGF-mediated angiogenesis in ovarian cancer treatment.

    PubMed

    Ai, Bin; Bie, Zhixin; Zhang, Shuai; Li, Ailing

    2016-01-01

    Ovarian cancer is one of the gynecologic cancers with the highest mortality, wherein vascular endothelial growth factor (VEGF) is involved in regulating tumor vascularization, growth, migration, and invasion. VEGF-mediated angiogenesis in tumors has been targeted in various cancer treatments, and anti-VEGF therapy has been used clinically for treatment of several types of cancer. Paclitaxel is a natural antitumor agent in the standard front-line treatment that has significant efficiency to treat advanced cancers, including ovarian cancer. Although platinum/paclitaxel-based chemotherapy has good response rates, most patients eventually relapse because the disease develops drug resistance. We aim to review the recent advances in paclitaxel treatment of ovarian cancer via antiangiogenesis. Single-agent therapy may be used in selected cases of ovarian cancer. However, to prevent drug resistance, drug combinations should be identified for optimal effectiveness and existing therapies should be improved. PMID:27648354

  12. Paclitaxel targets VEGF-mediated angiogenesis in ovarian cancer treatment

    PubMed Central

    Ai, Bin; Bie, Zhixin; Zhang, Shuai; Li, Ailing

    2016-01-01

    Ovarian cancer is one of the gynecologic cancers with the highest mortality, wherein vascular endothelial growth factor (VEGF) is involved in regulating tumor vascularization, growth, migration, and invasion. VEGF-mediated angiogenesis in tumors has been targeted in various cancer treatments, and anti-VEGF therapy has been used clinically for treatment of several types of cancer. Paclitaxel is a natural antitumor agent in the standard front-line treatment that has significant efficiency to treat advanced cancers, including ovarian cancer. Although platinum/paclitaxel-based chemotherapy has good response rates, most patients eventually relapse because the disease develops drug resistance. We aim to review the recent advances in paclitaxel treatment of ovarian cancer via antiangiogenesis. Single-agent therapy may be used in selected cases of ovarian cancer. However, to prevent drug resistance, drug combinations should be identified for optimal effectiveness and existing therapies should be improved. PMID:27648354

  13. Paclitaxel targets VEGF-mediated angiogenesis in ovarian cancer treatment

    PubMed Central

    Ai, Bin; Bie, Zhixin; Zhang, Shuai; Li, Ailing

    2016-01-01

    Ovarian cancer is one of the gynecologic cancers with the highest mortality, wherein vascular endothelial growth factor (VEGF) is involved in regulating tumor vascularization, growth, migration, and invasion. VEGF-mediated angiogenesis in tumors has been targeted in various cancer treatments, and anti-VEGF therapy has been used clinically for treatment of several types of cancer. Paclitaxel is a natural antitumor agent in the standard front-line treatment that has significant efficiency to treat advanced cancers, including ovarian cancer. Although platinum/paclitaxel-based chemotherapy has good response rates, most patients eventually relapse because the disease develops drug resistance. We aim to review the recent advances in paclitaxel treatment of ovarian cancer via antiangiogenesis. Single-agent therapy may be used in selected cases of ovarian cancer. However, to prevent drug resistance, drug combinations should be identified for optimal effectiveness and existing therapies should be improved.

  14. Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

    PubMed Central

    Michaloski, Jussara S.; Redondo, Alexandre R.; Magalhães, Leila S.; Cambui, Caio C.; Giordano, Ricardo J.

    2016-01-01

    Receptor tyrosine kinases (RTKs) are key molecules in numerous cellular processes, the inhibitors of which play an important role in the clinic. Among them are the vascular endothelial growth factor (VEGF) family members and their receptors (VEGFR), which are essential in the formation of new blood vessels by angiogenesis. Anti-VEGF therapy has already shown promising results in oncology and ophthalmology, but one of the challenges in the field is the design of specific small-molecule inhibitors for these receptors. We show the identification and characterization of small 6-mer peptides that target the extracellular ligand-binding domain of all three VEGF receptors. These peptides specifically prevent the binding of VEGF family members to all three receptors and downstream signaling but do not affect other angiogenic RTKs and their ligands. One of the selected peptides was also very effective at preventing pathological angiogenesis in a mouse model of retinopathy, normalizing the vasculature to levels similar to those of a normal developing retina. Collectively, our results suggest that these peptides are pan-VEGF inhibitors directed at a common binding pocket shared by all three VEGFRs. These peptides and the druggable binding site they target might be important for the development of novel and selective small-molecule, extracellular ligand-binding inhibitors of RTKs (eTKIs) for angiogenic-dependent diseases.

  15. MSC-seeded dense collagen scaffolds with a bolus dose of VEGF promote healing of large bone defects.

    PubMed

    Gao, C; Harvey, E J; Chua, M; Chen, B P; Jiang, F; Liu, Y; Li, A; Wang, H; Henderson, J E

    2013-10-13

    The functional repair of large skeletal defects remains a significant challenge to orthopaedic surgeons due to the lack of effective strategies to promote bone regeneration, particularly in the elderly. This study investigated the potential use of bone marrow derived mesenchymal stromal cells (MSC) in a dense collagen scaffold with a bolus dose of vascular endothelial growth factor (VEGF) to repair a defect in the femoral diaphysis of mice. MSC isolated from bone marrow of 4-month-old donor mice were seeded in type I collagen gels that were then compressed to form scaffolds with a fibrillar density similar to osteoid. The cells remained metabolically active in scaffolds incubated in vitro for up to 15 days and differentiated into osteoblasts that deposited calcium-phosphate mineral into the scaffold, which was quantified using micro-computed tomographic (micro-CT) imaging. When implanted in a 1 mm x 3 mm unicortical defect the MSC-loaded scaffolds were rapidly mineralised and integrated into host bone with administration of 10 ng of recombinant VEGF injected into the femoral canal at 4 days postoperative. Empty scaffolds and MSC-seeded scaffolds implanted in defects that did not receive a bolus dose of VEGF did not mineralise or integrate with native bone. The approach with MSC, hydrogels and a biologic factor already approved for human use warrants further pre-clinical investigation with a large animal model.

  16. Functional VEGF haplotypes affect the susceptibility to hypertension.

    PubMed

    Sandrim, V C; Luizon, M R; Izidoro-Toledo, T C; Coelho, E B; Moreno, H; Tanus-Santos, J E

    2013-01-01

    We examined whether vascular endothelial growth factor (VEGF) polymorphisms (C-2578A, G-1154A and G-634C) are associated with hypertension, response to antihypertensive therapy and nitric oxide (NO) formation. Substudy 1 compared the distribution of VEGF genotypes and haplotypes in 178 patients with arterial hypertension (100 whites and 78 blacks) and 186 healthy controls (115 whites and 71 blacks). Substudy 2 compared the distribution of VEGF markers in 82 patients with controlled hypertension, 89 patients with resistant hypertension and 101 normotensive (NT) patients. In substudy 3, plasma nitrite/nitrate (NOx) levels were determined (chemiluminescence assay) in 64 NT subjects and 48 hypertensive (HTN) subjects, and the distribution of VEGF markers was compared in subjects having low NOx with subjects having high NOx. Although the substudy 1 showed no differences in genotypes or allele distributions for the three VEGF polymorphisms between NT and HTN subjects, the 'C-A-G' haplotype was more common in white NT subjects than in the white HTN subjects, and the 'C-A-C' haplotype was more frequent in black and white HTN subjects than in black and white NT subjects. The substudy 2 showed similar results, with no differences between responsive and resistant HTN subjects. The substudy 3 showed that the 'C-A-G' haplotype, which had a protective effect against hypertension, was significantly more common in subjects with higher NOx concentrations than in subjects with lower NOx concentrations. VEGF haplotypes are associated with hypertension, and the haplotype associated with normotension was more common in subjects with increased NO formation, possibly offering a mechanistic clue for our findings. PMID:22189703

  17. Rac limits TGF-β-induced VEGF synthesis in osteoblasts.

    PubMed

    Yamamoto, Naohiro; Otsuka, Takanobu; Kondo, Akira; Matsushima-Nishiwaki, Rie; Kuroyanagi, Gen; Kozawa, Osamu; Tokuda, Haruhiko

    2015-04-15

    We previously showed that transforming growth factor-β (TGF-β) stimulates vascular endothelial growth factor (VEGF) synthesis via p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of Rac, which is a member of the Rho family of small GTPases, in the TGF-β-stimulated VEGF synthesis in MC3T3-E1 cells. TGF-β markedly increased the levels of GTP-bound Rac. NSC23766, a selective inhibitor of Rac-guanine nucleotide exchange factor interaction, significantly increased both the release of VEGF and the mRNA expression levels induced by TGF-β. In addition, the release of VEGF stimulated by TGF-β was amplified in Rac-knock down cells. Meanwhile, SIS3, a specific inhibitor of TGF-β-dependent Smad3 phosphorylation, significantly reduced the TGF-β-stimulated VEGF release. However, the phosphorylation of Smad2 or Smad3 induced by TGF-β was hardly affected by NSC23766. On the other hand, NSC23766 enhanced the TGF-β-induced phosphorylation of p38 MAP kinase without affecting the phosphorylation of p44/p42 MAP kinase or SAPK/JNK. Furthermore, the phosphorylation of p38 MAP kinase induced by TGF-β was markedly upregulated in the Rac-knock down cells. These results strongly suggest that Rac negatively regulates the TGF-β-stimulated VEGF synthesis via the inhibition of p38 MAP kinase in osteoblasts.

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

    PubMed Central

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

    2016-01-01

    Vascular endothelial growth factor (VEGF) is primarily known as a proangiogenic factor and is one of the most important growth and survival factors affecting the vascular endothelium. However, recent studies have shown that VEGF also plays a vital role in the immune environment. In addition to the traditional growth factor role of VEGF and VEGF receptors (VEGFRs), they have a complicated relationship with various immune cells. VEGF also reportedly inhibits the differentiation and function of immune cells during hematopoiesis. Dendritic cells (DCs), macrophages, and lymphocytes further express certain types of VEGF receptors. VEGF can be secreted as well by tumor cells through the autocrine pathway and can stimulate the function of cancer stemness. This review will provide a paradigm shift in our understanding of the role of VEGF/VEGFR signaling in the immune and cancer environment. PMID:27458528

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

    PubMed

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

    2016-09-01

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

  20. Heparin desulfation modulates VEGF release and angiogenesis in diabetic wounds.

    PubMed

    Freudenberg, Uwe; Zieris, Andrea; Chwalek, Karolina; Tsurkan, Mikhail V; Maitz, Manfred F; Atallah, Passant; Levental, Kandice R; Eming, Sabine A; Werner, Carsten

    2015-12-28

    While vascular endothelial growth factor (VEGF) has been shown to be one of the key players in wound healing by promoting angiogenesis current clinical applications of this growth factor to the wound environment are poorly controlled and not sustainable. Hydrogels made of sulfated glycosaminoglycans (GAG) allow for the sustained release of growth factors since GAGs engage in electrostatic complexation of biomolecules. In here, we explore a set of hydrogels formed of selectively desulfated heparin derivatives and star-shaped poly(ethylene glycol) with respect to VEGF binding and release and anticoagulant activity. As a proof of concept, supportive effects on migration and tube formation of human umbilical vein endothelial cells were studied in vitro and the promotion of wound healing was followed in genetically diabetic (db/db) mice. Our data demonstrate that the release of VEGF from the hydrogels is modulated in dependence on the GAG sulfation pattern. Hydrogels with low sulfate content (11% of initial heparin) were found to be superior in efficacy of VEGF administration, low anticoagulant activity and promotion of angiogenesis.

  1. Heparin desulfation modulates VEGF release and angiogenesis in diabetic wounds.

    PubMed

    Freudenberg, Uwe; Zieris, Andrea; Chwalek, Karolina; Tsurkan, Mikhail V; Maitz, Manfred F; Atallah, Passant; Levental, Kandice R; Eming, Sabine A; Werner, Carsten

    2015-12-28

    While vascular endothelial growth factor (VEGF) has been shown to be one of the key players in wound healing by promoting angiogenesis current clinical applications of this growth factor to the wound environment are poorly controlled and not sustainable. Hydrogels made of sulfated glycosaminoglycans (GAG) allow for the sustained release of growth factors since GAGs engage in electrostatic complexation of biomolecules. In here, we explore a set of hydrogels formed of selectively desulfated heparin derivatives and star-shaped poly(ethylene glycol) with respect to VEGF binding and release and anticoagulant activity. As a proof of concept, supportive effects on migration and tube formation of human umbilical vein endothelial cells were studied in vitro and the promotion of wound healing was followed in genetically diabetic (db/db) mice. Our data demonstrate that the release of VEGF from the hydrogels is modulated in dependence on the GAG sulfation pattern. Hydrogels with low sulfate content (11% of initial heparin) were found to be superior in efficacy of VEGF administration, low anticoagulant activity and promotion of angiogenesis. PMID:26478015

  2. Technology evaluation: VEGF165 gene therapy, Valentis Inc.

    PubMed

    Morse, M A

    2001-02-01

    Valentis Inc, formerly GeneMedicine, is developing a vascular endothelial growth factor (VEGF165) non-viral gene therapy using its proprietary PINC polymer for plasmid condensation. Two physician-initiated phase II angioplasty trials are ongoing, one for treating peripheral vascular disease and one for treating coronary artery disease [281714], [347153]. In February 2000, the trials were expected to be completed in the fourth quarter of 2000 [356225]; however, in October 2000, it was reported that the trial for peripheral vascular disease would be completed in the first quarter of 2001 [385232]. In March 2000, Valentis initiated a trial incorporating Valentis's DOTMA-based cationic lipid gene delivery system and the VEGF165 gene with Eurogene's local collar-reservoir delivery device. The trial is designed to demonstrate that the VEGF165 gene, delivered locally to the outside surface of a blood vessel, will transfect and express in the smooth muscle cells of the vessel wall [360683]. In March 1999, Valentis was awarded with a Phase II SBIR grant of $686,260. The aim of grant was to advance the development of non-viral gene therapies for ischemia. Specifically, Valentis intended to select an optimal promoter to be used with the VEGF expression plasmid. Valentis also intended to evaluate the gene therapy system in a rabbit ischemia model and complete the necessary preclinical studies for submission of an IND [318137]. PMID:11249737

  3. Programmed Translational Readthrough Generates Anti-Angiogenic VEGF-Ax

    PubMed Central

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

    2014-01-01

    SUMMARY Translational readthrough, observed primarily in less complex organisms from viruses to Drosophila, expands the proteome by translating select transcripts beyond the canonical stop codon. Here we show that vascular endothelial growth factor-A (VEGFA) mRNA in mammalian endothelial cells undergoes programmed translational readthrough (PTR) generating VEGF-Ax, an isoform containing a unique 22-amino acid C-terminus extension. A cis-acting element in the VEGFA 3′UTR serves a dual function, not only encoding the appended peptide, but also directing the PTR by decoding the UGA stop codon as serine. Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 binds this element and promotes readthrough. Remarkably, VEGF-Ax exhibits anti-angiogenic activity in contrast to the pro-angiogenic activity of VEGF-A. Pathophysiological significance of VEGF-Ax is indicated by robust expression in multiple human tissues, but depletion in colon adenocarcinoma. Furthermore, genome-wide analysis revealed AGO1 and MTCH2 as authentic readthrough targets. Overall, our studies reveal a novel protein-regulated PTR event in a vertebrate system. PMID:24949972

  4. The P2Y2 nucleotide receptor mediates vascular cell adhesion molecule-1 expression through interaction with VEGF receptor-2 (KDR/Flk-1).

    PubMed

    Seye, Cheikh I; Yu, Ningpu; González, Fernando A; Erb, Laurie; Weisman, Gary A

    2004-08-20

    UTP stimulates the expression of pro-inflammatory vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells through activation of the P2Y(2) nucleotide receptor P2Y(2)R. Here, we demonstrated that activation of the P2Y(2)R induced rapid tyrosine phosphorylation of vascular endothelial growth factor receptor (VEGFR)-2 in human coronary artery endothelial cells (HCAEC). RNA interference targeting VEGFR-2 or inhibition of VEGFR-2 tyrosine kinase activity abolishes P2Y(2)R-mediated VCAM-1 expression. Furthermore, VEGFR-2 and the P2Y(2)R co-localize upon UTP stimulation. Deletion or mutation of two Src homology-3-binding sites in the C-terminal tail of the P2Y(2)R or inhibition of Src kinase activity abolished the P2Y(2)R-mediated transactivation of VEGFR-2 and subsequently inhibited UTP-induced VCAM-1 expression. Moreover, activation of VEGFR-2 by UTP leads to the phosphorylation of Vav2, a guanine nucleotide exchange factor for Rho family GTPases. Using a binding assay to measure the activity of the small GTPases Rho, we found that stimulation of HCAEC by UTP increased the activity of RhoA and Rac1 (but not Cdc42). Significantly, a dominant negative form of RhoA inhibited P2Y(2)R-mediated VCAM-1 expression, whereas expression of dominant negative forms of Cdc42 and Rac1 had no effect. These data indicate a novel mechanism whereby a nucleotide receptor transactivates a receptor tyrosine kinase to generate an inflammatory response associated with atherosclerosis.

  5. Anti-VEGF Therapy for Retinal Vein Occlusions.

    PubMed

    Campa, Claudio; Alivernini, Giuseppe; Bolletta, Elena; Parodi, Maurizio Battaglia; Perri, Paolo

    2016-01-01

    Retinal vein occlusion (RVO) is the second most common cause of visual loss in the Western World. RVO is usually classified into branch RVO (BRVO) and central RVO (CRVO) according to the anatomical site of the vascular occlusion. The pathogenesis of RVO is not yet fully understood, however an important event is the intraluminal thrombus formation, which is usually secondary to several conditions such as hypertension, hyperlipidemia, diabetes and thrombophilia. The blockage of venous circulation causes an elevation of intraluminal pressure in the capillaries, leading to hemorrhages and leakage of fluid within the retina, increase of interstitial pressure and a consequent reduction of retinal perfusion. Ischemia may develop resulting in secretion of vascular endothelial growth factor (VEGF) that causes further vascular leakage and retinal oedema. VEGF has therefore a leading role in RVO pathogenesis and symptoms. As a consequence use of anti-VEGF agents by intravitreal injections has become very common with the aim to improve the clinical outcomes in these patients. Currently 2 anti-VEGF agents (ranimizumab and aflibercept) have been FDA (Food and Drug Administration) and EMA (European Medicine Agency) approved for the treatment of RVO, while another VEGF inhibitor (bevacizumab) is often used "off-label" in clinical practice. Many treatment regimens have been suggested in the clinical trials with these drugs, as monthly injections or injections when needed, however the ideal regimen has not been defined yet. We conducted a systematic review searching MEDLINE for the following terms: retinal vein occlusion, ranibizumab, bevacizumab, aflibercept, vascular endothelial growth factor, macular oedema. Data were extracted by one author (AG and BE) and checked by a second (BPM, CC). Aim of this article was to review available data for each drug, focusing on their efficacy and safety trying to compare their advantages and limits.

  6. Patterns of expression of vascular endothelial growth factor (VEGF) and VEGF receptors in mice suggest a role in hormonally regulated angiogenesis.

    PubMed Central

    Shweiki, D; Itin, A; Neufeld, G; Gitay-Goren, H; Keshet, E

    1993-01-01

    Vascular endothelial growth factor (VEGF) is a secreted endothelial cell-specific mitogen. To evaluate whether VEGF may play a role in angiogenesis, we have determined the spatial and temporal patterns of expression of VEGF and VEGF receptors during natural angiogenic processes taking place within the female reproductive system. Four angiogenic processes were analyzed: neovascularization of ovarian follicles, neovascularization of the corpus luteum, repair of endometrial vessels, and angiogenesis in embryonic implantation sites. During all processes, VEGF mRNA was found to be expressed in cells surrounding the expanding vasculature. VEGF was predominantly produced in tissues that acquire new capillary networks (theca layers, lutein cells, endometrial stroma, and the maternal decidua, respectively). VEGF-binding activity, on the other hand, was found on endothelial cells of both quiescent and proliferating blood vessels. These findings are consistent with a role for VEGF in the targeting of angiogenic responses to specific areas. Using in situ hybridization, we show that VEGF is expressed in 10 different steroidogenic and/or steroid-responsive cell types (theca, cumulus, granulosa, lutein, oviductal epithelium, endometrial stroma, decidua, giant trophoblast cells, adrenal cortex, and Leydig cells). Furthermore, in some cells upregulation of VEGF expression is concurrent with the acquisition of steroidogenic activity, and expression in other cell types is restricted to a particular stage of the ovarian cycle. These findings suggest that expression of VEGF is hormonally regulated. We propose that excessive expression of VEGF during gonadotropin-induced ovulation may contribute to the development of ovarian hyperstimulation syndromes by virtue of the vascular permeabilization activity of this factor. Images PMID:7683699

  7. Mesenchymal stem cells protect cigarette smoke-damaged lung and pulmonary function partly via VEGF-VEGF receptors.

    PubMed

    Guan, Xiao-Jun; Song, Lin; Han, Feng-Feng; Cui, Zhi-Lei; Chen, Xi; Guo, Xue-Jun; Xu, Wei-Guo

    2013-02-01

    Progressive pulmonary inflammation and emphysema have been implicated in the progression of chronic obstructive pulmonary disease (COPD), while current pharmacological treatments are not effective. Transplantation of bone marrow mesenchymal stem cells (MSCs) has been identified as one such possible strategy for treatment of lung diseases including acute lung injury (ALI) and pulmonary fibrosis. However, their role in COPD still requires further investigation. The aim of this study is to test the effect of administration of rat MSCs (rMSCs) on emphysema and pulmonary function. To accomplish this study, the rats were exposed to cigarette smoke (CS) for 11 weeks, followed by administration of rMSCs into the lungs. Here we show that rMSCs infusion mediates a down-regulation of pro-inflammatory mediators (TNF-α, IL-1β, MCP-1, and IL-6) and proteases (MMP9 and MMP12) in lung, an up-regulation of vascular endothelial growth factor (VEGF), VEGF receptor 2, and transforming growth factor (TGFβ-1), while reducing pulmonary cell apoptosis. More importantly, rMSCs administration improves emphysema and destructive pulmonary function induced by CS exposure. In vitro co-culture system study of human umbilical endothelial vein cells (EA.hy926) and human MSCs (hMSCs) provides the evidence that hMSCs mediates an anti-apoptosis effect, which partly depends on an up-regulation of VEGF. These findings suggest that MSCs have a therapeutic potential in emphysematous rats by suppressing the inflammatory response, excessive protease expression, and cell apoptosis, as well as up-regulating VEGF, VEGF receptor 2, and TGFβ-1. PMID:22949406

  8. Seeing through VEGF: innate and adaptive immunity in pathological angiogenesis in the eye.

    PubMed

    Sene, Abdoulaye; Chin-Yee, David; Apte, Rajendra S

    2015-01-01

    The central role of vascular endothelial growth factor (VEGF) signaling in regulating normal vascular development and pathological angiogenesis has been documented in multiple studies. Ocular anti-VEGF therapy is highly effective for treating a subset of patients with blinding eye disorders such as diabetic retinopathy and neovascular age-related macular degeneration (AMD). However, chronic VEGF suppression can lead to adverse effects associated with poor visual outcomes due to the loss of prosurvival and neurotrophic capacities of VEGF. In this review, we discuss emerging evidence for immune-related mechanisms that regulate ocular angiogenesis in a VEGF-independent manner. These novel molecular and cellular pathways may provide potential therapeutic avenues for a multitarget strategy, preserving the neuroprotective functions of VEGF in those patients whose disease is unresponsive to VEGF neutralization.

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

    PubMed Central

    Kim, Tae-Hyun; Kang, Min Sil

    2016-01-01

    Vascularization is a key issue for the success of tissue engineering to repair damaged tissue. In this study, we report a composite scaffold delivering angiogenic factor for this purpose. Vascular endothelial growth factor (VEGF) was loaded on mesoporous silica nanoparticle (MSN), which was then incorporated within a type I collagen sponge, to produce collagen/MSN/VEGF (CMV) scaffold. The CMV composite scaffold could release VEGF sustainably over the test period of 28 days. The release of VEGF improved the cell proliferation. Moreover, the in vivo angiogenesis of the scaffold, as studied by the chick chorioallantoic membrane (CAM) model, showed that the VEGF-releasing scaffold induced significantly increased number of blood vessel complexes when compared with VEGF-free scaffold. The composite scaffold showed good biocompatibility, as examined in rat subcutaneous tissue. These results demonstrate that the CMV scaffold with VEGF-releasing capacity can be potentially used to stimulate angiogenesis and tissue repair. PMID:27689093

  10. Seeing through VEGF: Innate and adaptive immunity in pathologic angiogenesis in the eye

    PubMed Central

    Sene, Abdoulaye; Chin-Yee, David; Apte, Rajendra S.

    2014-01-01

    The central role of VEGF signaling in regulating normal vascular development and pathological angiogenesis has been documented in multiple studies. Ocular anti-VEGF therapy is highly effective for treating a subset of patients with blinding eye disorders such as diabetic retinopathy and neovascular age-related macular degeneration (AMD). However, chronic VEGF suppression can lead to adverse effects associated with poor visual outcomes due to the loss of pro-survival and neurotrophic capacities of VEGF. In this review, we discuss emerging evidence for immune-related mechanisms that regulate ocular angiogenesis in a VEGF-independent manner. These novel molecular and cellular pathways may provide potential therapeutic avenues for a multitarget strategy, preserving the neuroprotective functions of VEGF in those patients whose disease is unresponsive to VEGF neutralization. PMID:25457617

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

    PubMed Central

    Kim, Tae-Hyun; Kang, Min Sil

    2016-01-01

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

  12. A potential therapeutic strategy for inhibition of corneal neovascularization with new anti-VEGF agents.

    PubMed

    Hosseini, Hamid; Nejabat, Mahmood

    2007-01-01

    The factors triggering corneal neovascularization involve various growth factors. The data supporting a causal role for vascular endothelial growth factor (VEGF) in corneal neovascularization are extensive. One possible strategy for treating corneal neovascularization is to inhibit VEGF activity by competitively binding VEGF with a specific neutralizing anti-VEGF antibody. The vireo-retinal service in the recent years enjoyed a high level of success in managing choroidal neovascularization using anti-VEGF strategies. Efficacy and tolerability have been demonstrated for drugs targeting VEGF. We herein hypothesize that topical application of new anti-VEGF agents such as pegaptanib, ranibizumab and bevacizumab are potentially useful for inhibiting corneal neovascularization and restoration of corneal clarity. Further investigations are needed to place these medical treatments alongside corneal neovascularization therapeutics. PMID:17107753

  13. VEGF-Production by CCR2-Dependent Macrophages Contributes to Laser-Induced Choroidal Neovascularization

    PubMed Central

    Krause, Torsten A.; Alex, Anne F.; Engel, Daniel R.; Kurts, Christian; Eter, Nicole

    2014-01-01

    Age-related macular degeneration (AMD) is the most prevalent cause of blindness in the elderly, and its exsudative subtype critically depends on local production of vascular endothelial growth factor A (VEGF). Mononuclear phagocytes, such as macrophages and microglia cells, can produce VEGF. Their precursors, for example monocytes, can be recruited to sites of inflammation by the chemokine receptor CCR2, and this has been proposed to be important in AMD. To investigate the role of macrophages and CCR2 in AMD, we studied intracellular VEGF content in a laser-induced murine model of choroidal neovascularisation. To this end, we established a technique to quantify the VEGF content in cell subsets from the laser-treated retina and choroid separately. 3 days after laser, macrophage numbers and their VEGF content were substantially elevated in the choroid. Macrophage accumulation was CCR2-dependent, indicating recruitment from the circulation. In the retina, microglia cells were the main VEGF+ phagocyte type. A greater proportion of microglia cells contained VEGF after laser, and this was CCR2-independent. On day 6, VEGF-expressing macrophage numbers had already declined, whereas numbers of VEGF+ microglia cells remained increased. Other sources of VEGF detectable by flow cytometry included in dendritic cells and endothelial cells in both retina and choroid, and Müller cells/astrocytes in the retina. However, their VEGF content was not increased after laser. When we analyzed flatmounts of laser-treated eyes, CCR2-deficient mice showed reduced neovascular areas after 2 weeks, but this difference was not evident 3 weeks after laser. In summary, CCR2-dependent influx of macrophages causes a transient VEGF increase in the choroid. However, macrophages augmented choroidal neovascularization only initially, presumably because VEGF production by CCR2-independent eye cells prevailed at later time points. These findings identify macrophages as a relevant source of VEGF in laser

  14. Pharmacokinetic and Pharmacodynamic Properties of Anti-VEGF Drugs After Intravitreal Injection.

    PubMed

    Semeraro, Francesco; Morescalchi, Francesco; Duse, Sarah; Gambicorti, Elena; Cancarini, Anna; Costagliola, Ciro

    2015-01-01

    Subretinal neovascularization and pathologic ocular angiogenesis are common causes of progressive, irreversible impairment of central vision, and dramatically affect quality of life. Anti-vascular endothelial growth factor (anti-VEGF) therapy has improved the quality of life for many patients with age-related macular degeneration, diabetic retinopathy, and other ocular diseases involving neovascularization and edema. In these pathologies, the inhibition of intraocular VEGF is the only therapy that can preserve vision. Four anti-VEGF drugs are currently used to treat ocular neovascularization; pegaptanib, ranibizumab, and aflibercept have been approved for this condition, while bevacizumab can be used off-label. Anti-VEGF therapy is administered regularly for many months or years because its suspension or discontinuation may cause recurrence of neovascularization. On the other hand, VEGF is necessary for the survival of retinal and choroidal endothelial cells. Experimental studies in animal models have shown that local inhibition of VEGF causes thinning and atrophy of the choriocapillaris and degeneration of photoreceptors, primarily cones. These studies combined with clinical experience indicated that prolonged VEGF inhibition could impair retinal function. Moreover, anti-VEGF compounds can cross the blood-retina barrier, enter the systemic circulation, and inhibit serum VEGF. Since circulating VEGF protects blood vessel integrity, prolonged anti-VEGF treatment could induce thromboembolic adverse events from vascular causes such as heart attack and stroke, and even death. The ocular dosing regimen and systemic toxicity of anti-VEGF compounds are therefore central concerns. A better understanding of this topic requires knowledge of the metabolism, tissue distribution, and clearance of anti-VEGF compounds. This manuscript reviews the properties of anti-VEGF compounds following intravitreal administration. PMID:26424177

  15. Silencing of VEGF inhibits human osteosarcoma angiogenesis and promotes cell apoptosis via VEGF/PI3K/AKT signaling pathway

    PubMed Central

    Peng, Ningning; Gao, Shuming; Guo, Xu; Wang, Guangya; Cheng, Cai; Li, Min; Liu, Kehun

    2016-01-01

    Background: Osteosarcoma is a kind of highly malignant tumor and the growth and metastasis is closely related to angiogenesis. Vascular endothelial growth factor (VEGF) is an important angiogenesis-promoting factor. In the current study, we investigated the effects of suppressed VEGF on osteosarcoma and its molecular mechanism provided for a basis by targeting angiogenesis. Material/Methods: We established bearing human osteosarcoma Wistar rats model by subcutaneous inoculation of human SaOS-2 cells and the adenovirus vector Ad-VEGF-siRNA was constructed for further study. We assessed the efficiency of VEGF silencing and its influence on SaOS-2 cells. The expression of mRNA and protein were detected by RT-PCR and western blotting, respectively. Intratumoral microvessel density (MVD), VEGF and CD31 were evaluated by immunohistochemistry. We detected the cell apoptotic rates by flow cytometry. Results: Our results indicated that Ad-VEGF-siRNA could effectively suppressed the expression of VEGF expression, inhibited the proliferation capability and promoted apoptosis of SaOS-2 cells in vitro. Silencing of VEGF expression also suppress osteosarcoma tumor growth and reduce osteosarcoma angiogenesis in the Wistar rats model in vivo. Furthermore, We found that phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) activation were considerably reduced while inhibition VEGF expression in SaOS-2 cells. Conclusion: Our data demonstrated that VEGF silencing could suppress cells proliferation, promote cells apoptosis and reduce osteosarcoma angiogenesis through inactivation of VEGF/PI3K/AKT signaling pathway. PMID:27158386

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

    PubMed

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

    2015-06-01

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

  17. STAT3-dependent VEGF production from keratinocytes abrogates dendritic cell activation and migration by arsenic: a plausible regional mechanism of immunosuppression in arsenical cancers.

    PubMed

    Hong, Chien-Hui; Lee, Chih-Hung; Chen, Gwo-Shing; Chang, Kee-Lung; Yu, Hsin-Su

    2015-02-01

    Arsenic remains an important environmental hazard that causes several human cancers. Arsenic-induced Bowen's disease (As-BD), a skin carcinoma in situ, is the most common arsenical cancer. While great strides have been made in our understanding of arsenic carcinogenesis, how host immunity contributes to this process remains unknown. Patients with As-BD have an impaired contact hypersensitivity response. Although impaired T cell activation has been well-documented in arsenical cancers, how dendritic cell (DC), the key cell regulating innate immunity, regulates the immune response in arsenical cancers remains unclear. Using myeloid derived DC (MDDC) from patients with As-BD and normal controls as well as bone marrow derived DC (BMDC) from mice fed with or without arsenic, we measured the migration of DC. As-BD patients showed an impaired CCL21-mediated MDDC migration in vitro. Arsenic-fed mice had defective DC migration toward popliteal lymph nodes when injected with allogenic BMDCs via foot pad. Using skin from As-BD and normal controls, we found an increased expression of STAT3, a transcriptional factor contributing to impaired DC activation. Arsenic induced STAT3 activation and the production of VEGF in keratinocytes. The increase in VEGF was blocked by inhibiting STAT3 with RNA interference or pharmaceutically with JSI-124. While VEGF by itself minimally induced the expression of CD86 and MHC-II in MDDC, arsenic induced-MDDC activation was abolished by VEGF pretreatment. We concluded that the STAT3-VEGF axis in keratinocytes inhibits DC migration in the microenvironment of As-BD, indicating that cellular interactions play an important role in regulating the disease course of arsenical cancers.

  18. STAT3-dependent VEGF production from keratinocytes abrogates dendritic cell activation and migration by arsenic: a plausible regional mechanism of immunosuppression in arsenical cancers.

    PubMed

    Hong, Chien-Hui; Lee, Chih-Hung; Chen, Gwo-Shing; Chang, Kee-Lung; Yu, Hsin-Su

    2015-02-01

    Arsenic remains an important environmental hazard that causes several human cancers. Arsenic-induced Bowen's disease (As-BD), a skin carcinoma in situ, is the most common arsenical cancer. While great strides have been made in our understanding of arsenic carcinogenesis, how host immunity contributes to this process remains unknown. Patients with As-BD have an impaired contact hypersensitivity response. Although impaired T cell activation has been well-documented in arsenical cancers, how dendritic cell (DC), the key cell regulating innate immunity, regulates the immune response in arsenical cancers remains unclear. Using myeloid derived DC (MDDC) from patients with As-BD and normal controls as well as bone marrow derived DC (BMDC) from mice fed with or without arsenic, we measured the migration of DC. As-BD patients showed an impaired CCL21-mediated MDDC migration in vitro. Arsenic-fed mice had defective DC migration toward popliteal lymph nodes when injected with allogenic BMDCs via foot pad. Using skin from As-BD and normal controls, we found an increased expression of STAT3, a transcriptional factor contributing to impaired DC activation. Arsenic induced STAT3 activation and the production of VEGF in keratinocytes. The increase in VEGF was blocked by inhibiting STAT3 with RNA interference or pharmaceutically with JSI-124. While VEGF by itself minimally induced the expression of CD86 and MHC-II in MDDC, arsenic induced-MDDC activation was abolished by VEGF pretreatment. We concluded that the STAT3-VEGF axis in keratinocytes inhibits DC migration in the microenvironment of As-BD, indicating that cellular interactions play an important role in regulating the disease course of arsenical cancers. PMID:25559853

  19. Hyperglycemia-Induced Vasculopathy in the Murine Conceptus Is Mediated via Reductions of VEGF-A Expression and VEGF Receptor Activation

    PubMed Central

    Pinter, Emese; Haigh, Jody; Nagy, Andras; Madri, Joseph A.

    2001-01-01

    Major congenital malformations, including those affecting the cardiovascular system, remain the leading cause of mortality and morbidity in infants of diabetic mothers. Interestingly, targeted mutations of several genes (including VEGF and VEGF receptors) and many teratogenic agents (including excess D-glucose) that give rise to embryonic lethal phenotypes during organogenesis are associated with a failure in the formation and/or maintenance of a functional vitelline circulation. Given the similarities in the pathology of the abnormal vitelline circulation in many of these conditions, we hypothesized that the hyperglycemic insult present in diabetes could cause the resultant abnormalities in the vitelline circulation by affecting VEGF/VEGF receptor signaling pathway(s). In this study we report that hyperglycemic insult results in reduced levels of VEGF-A in the conceptus, which in turn, leads to abnormal VEGF receptor signaling, ultimately resulting in embryonic (vitelline) vasculopathy. These findings and our observation that addition of exogenous rVEGF-A165 within a defined concentration range blunts the hyperglycemia-induced vasculopathy in the conceptus support the concept that VEGF levels can be modulated by glucose levels. In addition, these findings may ultimately lead to novel therapeutic approaches for the treatment of selected congenital cardiovascular abnormalities associated with diabetes. PMID:11290536

  20. Prognostic value of vascular endothelial growth factor (VEGF) in head and neck squamous cell carcinomas

    PubMed Central

    Mineta, H; Miura, K; Ogino, T; Takebayashi, S; Misawa, K; Ueda, Y; Suzuki, I; Dictor, M; Borg, Å; Wennerberg, J

    2000-01-01

    Vascular endothelial growth factor (VEGF) has been identified as the substance that increases the permeability and proliferation of vascular endothelial cells. We examined the clinical significance of VEGF expression in 60 head and neck squamous cell carcinomas using the methods of Western blot, immunohistochemistry, and reverse transcriptase-polymerase chain reaction (RT-PCR), comparatively, and analysed the relationship between VEGF status in Western blot and tumour size, lymph-node status, histologic grade and disease-free survival (DFS) rate. Western blot analysis revealed high VEGF expressors (tumour/normal tissue density ≥ 3-fold) in 26 patients (43%) and low VEGF expressors (< 3-fold) in 34 patients (57%). The results of the Western blot analysis correlated significantly with those of the RT-PCR (P= 0.00007) or immunohistochemistry (P= 0.00006). High VEGF expressors are associated with the progression of lymph-node spread (P= 0.0009), which are correlated with poor DFS. The 2-year DFS rate of high VEGF expressors (30%) was significantly lower than that of low VEGF expressors (78%) (P= 0.0008). Multivariate analysis showed VEGF expression and stage were independent predictors for the DFS (P= 0.045 and 0.041, respectively). VEGF expression may play an important role in progression of HNSCC. © 2000 Cancer Research Campaign PMID:10952783

  1. VEGF-B inhibits hyperglycemia- and Macugen-induced retinal apoptosis

    PubMed Central

    Huang, Delong; Zhao, Chen; Ju, Rong; Kumar, Anil; Tian, Geng; Huang, Lijuan; Zheng, Lei; Li, Xianglin; Liu, Lixian; Wang, Shasha; Ren, Xiangrong; Ye, Zhimin; Chen, Wei; Xing, Liying; Chen, Qishan; Gao, Zhiqin; Mi, Jia; Tang, Zhongshu; Wang, Bin; Zhang, Shuping; Lee, Chunsik; Li, Xuri

    2016-01-01

    Vascular endothelial growth factor B (VEGF-B) was discovered a long time ago. However, its role in hyperglycemia- and VEGF-A inhibition-induced retinal apoptosis remains unknown thus far. Yet, drugs that can block VEGF-B are being used to treat patients with diabetic retinopathy and other ocular neovascular diseases. It is therefore urgent to have a better understanding of the function of VEGF-B in these pathologies. Here, we report that both streptozotocin (STZ)-induced diabetes in rats and Macugen intravitreal injection in mice leads to retinal apoptosis in retinal ganglion cell and outer nuclear layers respectively. Importantly, VEGF-B treatment by intravitreal injection markedly reduced retinal apoptosis in both models. We further reveal that VEGF-B and its receptors, vascular endothelial growth factor 1 (VEGFR1) and neuropilin 1 (NP1), are abundantly expressed in rat retinae and choroids and are upregulated by high glucose with concomitant activation of Akt and Erk. These data highlight an important function of VEGF-B in protecting retinal cells from apoptosis induced by hyperglycemia and VEGF-A inhibition. VEGF-B may therefore have a therapeutic potential in treating various retinal degenerative diseases, and modulation of VEGF-B activity in the eye needs careful consideration. PMID:27189805

  2. VEGF-A, cytoskeletal dynamics, and the pathological vascular phenotype

    SciTech Connect

    Nagy, Janice A. . E-mail: jnagy@bidmc.harvard.edu; Senger, Donald R. . E-mail: dsenger@bidmc.harvard.edu

    2006-03-10

    Normal angiogenesis is a complex process involving the organization of proliferating and migrating endothelial cells (ECs) into a well-ordered and highly functional vascular network. In contrast, pathological angiogenesis, which is a conspicuous feature of tumor growth, ischemic diseases, and chronic inflammation, is characterized by vessels with aberrant angioarchitecture and compromised barrier function. Herein we review the subject of pathological angiogenesis, particularly that driven by vascular endothelial growth factor (VEGF-A), from a new perspective. We propose that the serious structural and functional anomalies associated with VEGF-A-elicited neovessels, reflect, at least in part, imbalances in the internal molecular cues that govern the ordered assembly of ECs into three dimensional vascular networks and preserve vessel barrier function. Adopting such a viewpoint widens the focus from solely on specific pro-angiogenic stimuli such as VEGF-A to include a key set of cytoskeletal regulatory molecules, the Rho GTPases, which are known to direct multiple aspects of vascular morphogenesis including EC motility, alignment, multi-cellular organization, as well as intercellular junction integrity. We offer this perspective to draw attention to the importance of endothelial cytoskeletal dynamics for proper neovascularization and to suggest new therapeutic strategies with the potential to improve the pathological vascular phenotype.

  3. VEGF is necessary for exercise-induced adult hippocampal neurogenesis.

    PubMed

    Fabel, Klaus; Fabel, Konstanze; Tam, Betty; Kaufer, Daniela; Baiker, Armin; Simmons, Natalie; Kuo, Calvin J; Palmer, Theo D

    2003-11-01

    Declining learning and memory function is associated with the attenuation of adult hippocampal neurogenesis. As in humans, chronic stress or depression in animals is accompanied by hippocampal dysfunction, and neurogenesis is correspondingly down regulated, in part, by the activity of the hypothalamic-pituitary-adrenal axis as well as glutamatergic and serotonergic networks. Antidepressants can reverse this effect over time but one of the most clinically effective moderators of stress or depression and robust stimulators of neurogenesis is simple voluntary physical exercise such as running. Curiously, running also elevates circulating stress hormone levels yet neurogenesis is doubled in running animals. In evaluating the signalling that running provides to the central nervous system in mice, we have found that peripheral vascular endothelial growth factor (VEGF) is necessary for the effects of running on adult hippocampal neurogenesis. Peripheral blockade of VEGF abolished running-induced neurogenesis but had no detectable effect on baseline neurogenesis in non-running animals. These data suggest that VEGF is an important element of a 'somatic regulator' of adult neurogenesis and that these somatic signalling networks can function independently of the central regulatory networks that are typically considered in the context of hippocampal neurogenesis.

  4. Neural crest cell-derived VEGF promotes embryonic jaw extension

    PubMed Central

    Wiszniak, Sophie; Mackenzie, Francesca E.; Anderson, Peter; Kabbara, Samuela; Ruhrberg, Christiana; Schwarz, Quenten

    2015-01-01

    Jaw morphogenesis depends on the growth of Meckel’s cartilage during embryogenesis. However, the cell types and signals that promote chondrocyte proliferation for Meckel’s cartilage growth are poorly defined. Here we show that neural crest cells (NCCs) and their derivatives provide an essential source of the vascular endothelial growth factor (VEGF) to enhance jaw vascularization and stabilize the major mandibular artery. We further show in two independent mouse models that blood vessels promote Meckel’s cartilage extension. Coculture experiments of arterial tissue with NCCs or chondrocytes demonstrated that NCC-derived VEGF promotes blood vessel growth and that blood vessels secrete factors to instruct chondrocyte proliferation. Computed tomography and X-ray scans of patients with hemifacial microsomia also showed that jaw hypoplasia correlates with mandibular artery dysgenesis. We conclude that cranial NCCs and their derivatives provide an essential source of VEGF to support blood vessel growth in the developing jaw, which in turn is essential for normal chondrocyte proliferation, and therefore jaw extension. PMID:25922531

  5. Overexpression of the chimeric plasmin-resistant VEGF165/VEGF183 (132-158) protein in murine breast cancer induces distinct vascular patterning adjacent to tumors and retarded tumor growth.

    PubMed

    Zhang, Hui-Yong; Fan, Bing-Lin; Wu, Xin-Sheng; Mu, Ling-Min; Wang, Wen-Feng; Zhu, Wu-Ling

    2015-02-01

    A chimeric plasmin‑resistant vascular endothelial growth factor (VEGF)165/VEGF183 (132-158) protein, named as VEGF183 (according to the nomenclature of VEGF), designed by a previous study, was demonstrated to have an enhanced affinity for the extracellular matrix (ECM) amongst other bioactivities. However, it is now accepted that mutant VEGFs frequently demonstrate different angiogenic activities and produce different vascular patterning from the parental molecule. The present study hypothesized that VEGF183, due to its enhanced binding affinity to the ECM, would exhibit a different angiogenic activity and produce a different vascular patterning compared to those of VEGF165. Murine breast cancer EMT‑6 cells were manipulated to stably overexpress VEGF165 or VEGF183. These cells were then inoculated intradermally into BALB/c mice in order to monitor the formation of vascular patterning in skin proximal to tumors. In vivo angiogenesis experiments revealed that overexpression of VEGF183 in murine breast cancer cells resulted in irregular, disorganized and dense vascular patterning as well as induced a significant inhibition of tumor growth compared with that of VEGF165. In addition, allograft tumor immunochemical assays of VEGF183‑overexpressing tumors demonstrated significantly lower vascular densities than those of VEGF165‑overexpressing tumors; however, VEGF183 tumors had a significantly enlarged vascular caliber. Conversely, cell wound healing experiments revealed that VEGF183‑overexpressing EMT‑6 cells had significantly decreased migration rates compared with those of VEGF165‑overexpressing EMT‑6 cells. In conclusion, the results of the present study supported the hypothesis that the altered ECM affinity of VEGF induced structural alterations to vasculature. In addition, these results provided a novel insight into VEGF design and indirect evidence for the function of exon 8 in VEGF. [Corrected] PMID:25373557

  6. PTEN mutation and epidermal growth factor receptor activation regulate vascular endothelial growth factor (VEGF) mRNA expression in human glioblastoma cells by transactivating the proximal VEGF promoter.

    PubMed

    Pore, Nabendu; Liu, Shuang; Haas-Kogan, Daphne A; O'Rourke, Donald M; Maity, Amit

    2003-01-01

    Our previous work showed that, compared with parental U87MG human glioblastoma cells, vascular endothelial growth factor (VEGF) mRNA levels are decreased in U87/T691, a derivative line in which epidermal growth factor receptor (EGFR) signaling is inhibited by introduction of a truncated p185(Neu) protein (A. Maity et al., Cancer Res., 60: 5879-5886, 2000). The effect of EGFR activation on VEGF was mediated at the level of transcription via a phosphatidylinositol 3'-kinase (PI3K)-dependent pathway. In the current study we investigated the effect of PTEN, a negative regulator of PI3K signaling commonly mutated in glioblastoma cells, on VEGF expression. Several glioblastoma cell lines containing mutant PTEN, including U87MG, U87/T691, and U251MG, were infected with adenovirus expressing wild-type PTEN. This led to a decrease in the levels of both VEGF mRNA and phosphorylated Akt, a marker for PI3K activation. Treatment of U87MG cells with LY294002, a PI3K inhibitor, or cotransfection with a vector expressing wild-type PTEN decreased VEGF promoter activity using reporters containing either 1.5 kb of the promoter or a fragment extending from -88 to +54 bp. Activity of the -88/+54 VEGF promoter was down-regulated by dominant negative Akt and up-regulated by constitutively active myristoylated Akt. Introduction of wild-type PTEN and pharmacological inhibition of EGFR decreased VEGF mRNA expression and VEGF promoter activity in U87MG cells to a greater extent that did either manipulation by itself. Therefore, in human glioblastoma cells, PTEN mutation can cooperate with EGFR activation to increase VEGF mRNA levels by transcriptionally up-regulating the proximal VEGF promoter via the PI3K/Akt pathway.

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

    PubMed Central

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

    2016-01-01

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

  8. Sanguinarine is a novel VEGF inhibitor involved in the suppression of angiogenesis and cell migration

    PubMed Central

    XU, JIA-YING; MENG, QING-HUI; CHONG, YU; JIAO, YANG; ZHAO, LIN; ROSEN, ELIOT M.; FAN, SAIJUN

    2013-01-01

    Vascular endothelial growth factor (VEGF) is a main angiogenic factor which is known to be upregulated in lung cancer. In the present study, it was demonstrated that sanguinarine, an alkaloid obtained from the bloodroot plant, markedly repressed the VEGF-induced tube formation of human microvascular endothelial cells (HMVECs) and the migration of human A549 lung cancer cells. Furthermore, sanguinarine decreased VEGF secretion and expression in HMVECs and A549 lung cancer cells in a dose- and time-dependent manner. Additionally, sanguinarine inhibited the activation of serum starvation- and hypoxia-induced VEGF promoter activity. Sanguinarine also inhibited the VEGF-mediated Akt and p38 activation, as well as VE-cadherin protein phosphorylation. To the best of our knowledge, this is the first study demonstrating that VEGF inhibition appears to be an important mechanism involved in the antiangiogenic and anti-invasive activities of sanguinarine in lung cancer treatment. PMID:24649171

  9. VEGF Polymorphisms Related to Higher Serum Levels of Protein Identify Patients with Hepatocellular Carcinoma

    PubMed Central

    Ferreira, Rafael Fernandes; Pinhel, Marcela Augusta de Souza; da Silva, Renato Ferreira; Fucuta, Patrícia da Silva; Souza, Dorotéia Rossi Silva

    2016-01-01

    Hepatocellular carcinoma (HCC) is the most common primary neoplasia of the liver. Major risk factors for hepatocellular carcinoma include chronic liver diseases, carcinogenic agents, and genetic alterations as well as vascular endothelial growth factor (VEGF) involved in angiogenesis process. The aim of this study was to evaluate the association of VEGF-A (C936T and A1154G) with HCC and cirrhosis, in addition to serum levels of VEGF, clinical profile, lifestyle habits, and comorbidities. A total of 346 individuals were studied: 102 with HCC (G1), 117 with cirrhosis (G2), and 127 controls (G3). Polymorphisms were analysed by PCR/RFLP and serum levels of VEGF by ELISA. Alpha error was set at 5%. The wild-type genotype of both polymorphisms prevailed (P > 0.05). In G1, 23% of the patients died, with no relation to genetic profile (P > 0.05). Increased VEGF level was observed in G1 and G3, related to the mutant allele of VEGF-C936T and VEGF-A1154G, respectively, and compared with the wild-type genotype (P = 0.0285; P = 0.0284, resp.) as well as G1 versus G2 and G3 for VEGF-C936T and G1 versus G2 for VEGF-A1154G (P < 0.05 for both). In conclusion, there is a relationship between mutant alleles of VEGF-C936T and VEGF-A1154G polymorphisms and higher VEGF level, making them potential markers for HCC.

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

    SciTech Connect

    Garcia-Roman, Jonathan; Ibarra-Sanchez, Alfredo; Lamas, Monica; Gonzalez Espinosa, Claudia

    2010-10-15

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

  11. Contribution of increased VEGF receptors to hypoxic changes in fetal ovine carotid artery contractile proteins.

    PubMed

    Adeoye, Olayemi O; Butler, Stacy M; Hubbell, Margaret C; Semotiuk, Andrew; Williams, James M; Pearce, William J

    2013-04-01

    Recent studies suggest that vascular endothelial growth factor (VEGF) can modulate smooth muscle phenotype and, consequently, the composition and function of arteries upstream from the microcirculation, where angiogenesis occurs. Given that hypoxia potently induces VEGF, the present study explores the hypothesis that, in fetal arteries, VEGF contributes to hypoxic vascular remodeling through changes in abundance, organization, and function of contractile proteins. Pregnant ewes were acclimatized at sea level or at altitude (3,820 m) for the final 110 days of gestation. Endothelium-denuded carotid arteries from full-term fetuses were used fresh or after 24 h of organ culture in a physiological concentration (3 ng/ml) of VEGF. After 110 days, hypoxia had no effect on VEGF abundance but markedly increased abundance of the Flk-1 (171%) and Flt-1 (786%) VEGF receptors. Hypoxia had no effect on smooth muscle α-actin (SMαA), decreased myosin light chain (MLC) kinase (MLCK), and increased 20-kDa regulatory MLC (MLC(20)) abundances. Hypoxia also increased MLCK-SMαA, MLC(20)-SMαA, and MLCK-MLC(20) colocalization. Compared with hypoxia, organ culture with VEGF produced the same pattern of changes in contractile protein abundance and colocalization. Effects of VEGF on colocalization were blocked by the VEGF receptor antagonists vatalanib (240 nM) and dasatinib (6.3 nM). Thus, through increases in VEGF receptor density, hypoxia can recruit VEGF to help mediate remodeling of fetal arteries upstream from the microcirculation. The results support the hypothesis that VEGF contributes to hypoxic vascular remodeling through changes in abundance, organization, and function of contractile proteins. PMID:23325408

  12. VEGF-induced angiogenesis following localized delivery via injectable, low viscosity poly(trimethylene carbonate).

    PubMed

    Amsden, Brian G; Timbart, Laurianne; Marecak, Dale; Chapanian, Rafi; Tse, M Yat; Pang, Stephen C

    2010-07-14

    The purpose of this study was to examine the potential of low molecular weight poly(trimethylene carbonate) for localized vascular endothelial growth factor (VEGF) delivery. Poly(trimethylene carbonate) of various molecular weights was prepared by ring-opening polymerization initiated by 1-octanol. The resultant polymers were liquid at room temperature with low glass transition temperatures and viscosities at 37 degrees C that permitted their injection through an 18 (1/2) G 1.5'' needle. Particles consisting of VEGF co-lyophilized with trehalose were mixed into the polymers and the rate of release of VEGF was assessed in vitro. With a 1% particle loading, VEGF was released from the polymer at a rate of 20 ng/day over a period of 3 weeks. This release behavior was independent of the molecular weight of polymer used. Increasing the VEGF content in the lyophilized particles did not increase the VEGF release rate, an effect attributed to the solubility limit of VEGF in the solution formed upon dissolution of the particles. The VEGF released retained its bioactivity at greater than 95% of that of as-lyophilized VEGF, as assessed using a human aortic endothelial cell proliferation assay. This high bioactivity was supported by in vivo release experiments, wherein VEGF containing polymer implants induced the generation of significantly greater numbers of blood vessels towards the polymer implant than controls. The blood vessels did not remain stable and were reduced in number by three weeks, due to the unsustained and low concentration of VEGF released. This formulation approach, of using a low viscosity polymer delivery vehicle, is potentially useful for localized delivery of acid-sensitive proteins, such as VEGF. PMID:20381557

  13. VEGF Polymorphisms Related to Higher Serum Levels of Protein Identify Patients with Hepatocellular Carcinoma

    PubMed Central

    Ferreira, Rafael Fernandes; Pinhel, Marcela Augusta de Souza; da Silva, Renato Ferreira; Fucuta, Patrícia da Silva; Souza, Dorotéia Rossi Silva

    2016-01-01

    Hepatocellular carcinoma (HCC) is the most common primary neoplasia of the liver. Major risk factors for hepatocellular carcinoma include chronic liver diseases, carcinogenic agents, and genetic alterations as well as vascular endothelial growth factor (VEGF) involved in angiogenesis process. The aim of this study was to evaluate the association of VEGF-A (C936T and A1154G) with HCC and cirrhosis, in addition to serum levels of VEGF, clinical profile, lifestyle habits, and comorbidities. A total of 346 individuals were studied: 102 with HCC (G1), 117 with cirrhosis (G2), and 127 controls (G3). Polymorphisms were analysed by PCR/RFLP and serum levels of VEGF by ELISA. Alpha error was set at 5%. The wild-type genotype of both polymorphisms prevailed (P > 0.05). In G1, 23% of the patients died, with no relation to genetic profile (P > 0.05). Increased VEGF level was observed in G1 and G3, related to the mutant allele of VEGF-C936T and VEGF-A1154G, respectively, and compared with the wild-type genotype (P = 0.0285; P = 0.0284, resp.) as well as G1 versus G2 and G3 for VEGF-C936T and G1 versus G2 for VEGF-A1154G (P < 0.05 for both). In conclusion, there is a relationship between mutant alleles of VEGF-C936T and VEGF-A1154G polymorphisms and higher VEGF level, making them potential markers for HCC. PMID:27660750

  14. Imiquimod Increases Cutaneous VEGF Expression in Imiquimod-induced Psoriatic Mouse Model.

    PubMed

    Wu, Hui-Hui; Xie, Wen-Lin; Zhao, Yu-Kun; Liu, Juan-Hua; Luo, Di-Qing

    2016-01-01

    Psoriasis is a chronic skin disease of unknown aetiology but increasing evidence suggests that cutaneous angiogenesis plays an important role. Vascular endothelial growth factor (VEGF) is one of the pro-angiogenic cytokines which is related to the pathogenesis of psoriasis. Our study evaluated the influence of imiquimod (IMQ) on VEGF in IMQ-induced mouse model. Balb/c female mice (n=16) 8-12 weeks of age were randomly divided into an experimental group (5% IMQ cream) and the control group (Vaseline cream). Serum levels of circulating VEGF-A were quantified by enzyme-linked immunosorbent assay. VEGF protein expression in tested skin was measured by western blotting and immunohistochemical staining. The tested skin in the experimental group expressed higher levels of VEGF protein than in the control group (p=0.012); immunohistochemical staining revealed that the cells over-expressing VEGF localized predominantly in the epidermis and vascular endothelium. Circulating VEGF-A levels showed no significant difference between the experimental and control groups (p=0.445). The IMQ-induced mouse psoriatic model showed an upregulation of VEGF in the skin lesions mimicking human psoriasis but the circulating VEGF-A levels showed no difference. This model may be useful to investigate the role of angiogenesis in psoriasis. PMID:26733387

  15. Transgenic overexpression of VEGF-C induces weight gain and insulin resistance in mice

    PubMed Central

    Karaman, Sinem; Hollmén, Maija; Yoon, Sun-Young; Alkan, H. Furkan; Alitalo, Kari; Wolfrum, Christian; Detmar, Michael

    2016-01-01

    Obesity comprises great risks for human health, contributing to the development of other diseases such as metabolic syndrome, type 2 diabetes and cardiovascular disease. Previously, obese patients were found to have elevated serum levels of VEGF-C, which correlated with worsening of lipid parameters. We recently identified that neutralization of VEGF-C and -D in the subcutaneous adipose tissue during the development of obesity improves metabolic parameters and insulin sensitivity in mice. To test the hypothesis that VEGF-C plays a role in the promotion of the metabolic disease, we used K14-VEGF-C mice that overexpress human VEGF-C under control of the keratin-14 promoter in the skin and monitored metabolic parameters over time. K14-VEGF-C mice had high levels of VEGF-C in the subcutaneous adipose tissue and gained more weight than wildtype littermates, became insulin resistant and had increased ectopic lipid accumulation at 20 weeks of age on regular mouse chow. The metabolic differences persisted under high-fat diet induced obesity. These results indicate that elevated VEGF-C levels contribute to metabolic deterioration and the development of insulin resistance, and that blockade of VEGF-C in obesity represents a suitable approach to alleviate the development of insulin resistance. PMID:27511834

  16. Heterochronic activation of VEGF signaling and the evolution of the skeleton in echinoderm pluteus larvae.

    PubMed

    Morino, Yoshiaki; Koga, Hiroyuki; Tachibana, Kazunori; Shoguchi, Eiichi; Kiyomoto, Masato; Wada, Hiroshi

    2012-01-01

    The evolution of the echinoderm larval skeleton was examined from the aspect of interactions between skeletogenic mesenchyme cells and surrounding epithelium. We focused on vascular endothelial growth factor (VEGF) signaling, which was reported to be essential for skeletogenesis in sea urchin larvae. Here, we examined the expression patterns of vegf and vegfr in starfish and brittle stars. During starfish embryogenesis, no expression of either vegfr or vegf was detected, which contrast with previous reports on the expression of starfish homologs of sea urchin skeletogenic genes, including Ets, Tbr, and Dri. In later stages, when adult skeletogenesis commenced, vegfr and vegf expression were upregulated in skeletogenic cells and in the adjacent epidermis, respectively. These expression patterns suggest that heterochronic activation of VEGF signaling is one of the key molecular evolutionary steps in the evolution of the larval skeleton. The absence of vegf or vegfr expression during early embryogenesis in starfish suggests that the evolution of the larval skeleton requires distinct evolutionary changes, both in mesoderm cells (activation of vegfr expression) and in epidermal cells (activation of vegf expression). In brittle stars, which have well-organized skeletons like the sea urchin, vegfr and vegf were expressed in the skeletogenic mesenchyme and the overlying epidermis, respectively, in the same manner as in sea urchins. Therefore, the distinct activation of vegfr and vegf may have occurred in two lineages, sea urchins and brittle stars.

  17. Transgenic overexpression of VEGF-C induces weight gain and insulin resistance in mice.

    PubMed

    Karaman, Sinem; Hollmén, Maija; Yoon, Sun-Young; Alkan, H Furkan; Alitalo, Kari; Wolfrum, Christian; Detmar, Michael

    2016-01-01

    Obesity comprises great risks for human health, contributing to the development of other diseases such as metabolic syndrome, type 2 diabetes and cardiovascular disease. Previously, obese patients were found to have elevated serum levels of VEGF-C, which correlated with worsening of lipid parameters. We recently identified that neutralization of VEGF-C and -D in the subcutaneous adipose tissue during the development of obesity improves metabolic parameters and insulin sensitivity in mice. To test the hypothesis that VEGF-C plays a role in the promotion of the metabolic disease, we used K14-VEGF-C mice that overexpress human VEGF-C under control of the keratin-14 promoter in the skin and monitored metabolic parameters over time. K14-VEGF-C mice had high levels of VEGF-C in the subcutaneous adipose tissue and gained more weight than wildtype littermates, became insulin resistant and had increased ectopic lipid accumulation at 20 weeks of age on regular mouse chow. The metabolic differences persisted under high-fat diet induced obesity. These results indicate that elevated VEGF-C levels contribute to metabolic deterioration and the development of insulin resistance, and that blockade of VEGF-C in obesity represents a suitable approach to alleviate the development of insulin resistance. PMID:27511834

  18. VEGF Trap in Combination With Radiotherapy Improves Tumor Control in U87 Glioblastoma

    SciTech Connect

    Wachsberger, Phyllis R. . E-mail: Phyllis.Wachsberger@mail.tju.edu; Burd, Randy; Cardi, Chris; Thakur, Mathew; Daskalakis, Constantine; Holash, Jocelyn; Yancopoulos, George D.; Dicker, Adam P.

    2007-04-01

    Purpose: To determine the effect of vascular endothelial growth factor VEGF Trap (Regeneron Pharmaceuticals, Tarrytown, NY), a humanized soluble vascular endothelial growth factor (VEGF) receptor protein, and radiation (RT) on tumor growth in U87 glioblastoma xenografts in nude mice. Methods and Materials: U87 cell suspensions were implanted subcutaneously into hind limbs of nude mice. VEGF Trap (2.5-25 mg/kg) was administered every 3 days for 3 weeks alone or in combination with a single dose of 10 Gy or fractionated RT (3 x 5 Gy). In addition, three scheduling protocols for VEGF Trap plus fractionated RT were examined. Results: Improved tumor control was seen when RT (either single dose or fractionated doses) was combined with the lowest dose of VEGF Trap (2.5 mg/kg). Scheduling did not significantly affect the efficacy of combined therapy. Although high-dose VEGF Trap (10 mg/kg or 25 mg/kg) significantly reduced tumor growth over that of RT alone, there was no additional benefit to combining high-dose VEGF Trap with RT. Conclusions: Vascular endothelial growth factor Trap plus radiation is clearly better than radiation alone in a U87 subcutaneous xenograft model. Although high doses of VEGF Trap alone are highly efficacious, it is unclear whether such high doses can be used clinically without incurring normal tissue toxicities. Thus, information on lower doses of VEGF Trap and ionizing radiation is of clinical relevance.

  19. Stability and Species Specificity of Renal VEGF-A Splicing Patterns in Kidney Disease.

    PubMed

    Turner, R J; Eikmans, M; Bajema, I M; Bruijn, J A; Baelde, H J

    2016-01-01

    Vascular endothelial growth factor A (VEGF-A) is essential for maintaining the glomerular filtration barrier. Absolute renal levels of VEGF-A change in patients with diabetic nephropathy and inflammatory kidney diseases, but whether changes in the renal splicing patterns of VEGF-A play a role remains unclear. In this study, we investigated mRNA splicing patterns of pro-angiogenic isoforms of VEGF-A in glomeruli and whole kidney samples from human patients with kidney disease and from mouse models of kidney disease. Kidney biopsies were obtained from patients with acute rejection following kidney transplantation, patients with diabetic nephropathy, and control subjects. In addition, kidney samples were obtained from mice with lupus nephritis, mice with diabetes mellitus, and control mice. The relative expression of each VEGF-A splice variant was measured using RT-PCR followed by quantitative fragment analysis. The pattern of renal VEGF-A splice variants was unchanged in diabetic nephropathy and lupus nephritis and was stable throughout disease progression in acute transplant rejection and diabetic nephropathy; these results suggest renal VEGF-A splicing stability during kidney disease. The splicing patterns were species-specific; in the control human kidney samples, VEGF-A 121 was the dominant isoform, whereas VEGF-A 164 was the dominant isoform measured in the mouse kidney samples. PMID:27598902

  20. Stability and Species Specificity of Renal VEGF-A Splicing Patterns in Kidney Disease

    PubMed Central

    Turner, R. J.; Eikmans, M.; Bajema, I. M.; Bruijn, J. A.; Baelde, H. J.

    2016-01-01

    Vascular endothelial growth factor A (VEGF-A) is essential for maintaining the glomerular filtration barrier. Absolute renal levels of VEGF-A change in patients with diabetic nephropathy and inflammatory kidney diseases, but whether changes in the renal splicing patterns of VEGF-A play a role remains unclear. In this study, we investigated mRNA splicing patterns of pro-angiogenic isoforms of VEGF-A in glomeruli and whole kidney samples from human patients with kidney disease and from mouse models of kidney disease. Kidney biopsies were obtained from patients with acute rejection following kidney transplantation, patients with diabetic nephropathy, and control subjects. In addition, kidney samples were obtained from mice with lupus nephritis, mice with diabetes mellitus, and control mice. The relative expression of each VEGF-A splice variant was measured using RT-PCR followed by quantitative fragment analysis. The pattern of renal VEGF-A splice variants was unchanged in diabetic nephropathy and lupus nephritis and was stable throughout disease progression in acute transplant rejection and diabetic nephropathy; these results suggest renal VEGF-A splicing stability during kidney disease. The splicing patterns were species-specific; in the control human kidney samples, VEGF-A 121 was the dominant isoform, whereas VEGF-A 164 was the dominant isoform measured in the mouse kidney samples. PMID:27598902

  1. Endocrine vasculatures are preferable targets of an antitumor ineffective low dose of anti-VEGF therapy.

    PubMed

    Zhang, Yin; Yang, Yunlong; Hosaka, Kayoko; Huang, Guichun; Zang, Jingwu; Chen, Fang; Zhang, Yun; Samani, Nilesh J; Cao, Yihai

    2016-04-12

    Anti-VEGF-based antiangiogenic drugs are designed to block tumor angiogenesis for treatment of cancer patients. However, anti-VEGF drugs produce off-tumor target effects on multiple tissues and organs and cause broad adverse effects. Here, we show that vasculatures in endocrine organs were more sensitive to anti-VEGF treatment than tumor vasculatures. In thyroid, adrenal glands, and pancreatic islets, systemic treatment with low doses of an anti-VEGF neutralizing antibody caused marked vascular regression, whereas tumor vessels remained unaffected. Additionally, a low dose of VEGF blockade significantly inhibited the formation of thyroid vascular fenestrae, leaving tumor vascular structures unchanged. Along with vascular structural changes, the low dose of VEGF blockade inhibited vascular perfusion and permeability in thyroid, but not in tumors. Prolonged treatment with the low-dose VEGF blockade caused hypertension and significantly decreased circulating levels of thyroid hormone free-T3 and -T4, leading to functional impairment of thyroid. These findings show that the fenestrated microvasculatures in endocrine organs are more sensitive than tumor vasculatures in response to systemic anti-VEGF drugs. Thus, our data support the notion that clinically nonbeneficial treatments with anti-VEGF drugs could potentially cause adverse effects.

  2. Serum VEGF levels are related to the presence of pulmonary arterial hypertension in systemic sclerosis

    PubMed Central

    Papaioannou, Andriana I; Zakynthinos, Epaminondas; Kostikas, Konstantinos; Kiropoulos, Theodoros; Koutsokera, Angela; Ziogas, Athanasios; Koutroumpas, Athanasios; Sakkas, Lazaros; Gourgoulianis, Konstantinos I; Daniil, Zoe D

    2009-01-01

    Background The association between systemic sclerosis and pulmonary arterial hypertension (PAH) is well recognized. Vascular endothelial growth factor (VEGF) has been reported to play an important role in pulmonary hypertension. The aim of the present study was to examine the relationship between systolic pulmonary artery pressure, clinical and functional manifestations of the disease and serum VEGF levels in systemic sclerosis. Methods Serum VEGF levels were measured in 40 patients with systemic sclerosis and 13 control subjects. All patients underwent clinical examination, pulmonary function tests and echocardiography. Results Serum VEGF levels were higher in systemic sclerosis patients with sPAP ≥ 35 mmHg than in those with sPAP < 35 mmHg (352 (266, 462 pg/ml)) vs (240 (201, 275 pg/ml)) (p < 0.01), while they did not differ between systemic sclerosis patients with sPAP < 35 mmHg and controls. Serum VEGF levels correlated to systolic pulmonary artery pressure, to diffusing capacity for carbon monoxide and to MRC dyspnea score. In multiple linear regression analysis, serum VEGF levels, MRC dyspnea score, and DLCO were independent predictors of systolic pulmonary artery pressure. Conclusion Serum VEGF levels are increased in systemic sclerosis patients with sPAP ≥ 35 mmHg. The correlation between VEGF levels and systolic pulmonary artery pressure may suggest a possible role of VEGF in the pathogenesis of PAH in systemic sclerosis. PMID:19426547

  3. Functional interaction between CTGF and FPRL1 regulates VEGF-A-induced angiogenesis.

    PubMed

    Lee, Mi-Sook; Ghim, Jaewang; Kim, Sun-Jin; Yun, Young Sung; Yoo, Seung-Ah; Suh, Pann-Ghill; Kim, Wan-Uk; Ryu, Sung Ho

    2015-07-01

    Vascular endothelial growth factor-A (VEGF-A) is a master regulator of angiogenesis that controls several angiogenic processes in endothelial cells. However, the detailed mechanisms of VEGF-A responsible for pleiotropic functions and crosstalk with other signaling pathways have not been fully understood. Here, we found that VEGF-A utilizes the connective tissue growth factor (CTGF)/formyl peptide receptor-like 1 (FPRL1) axis as one of its mediators in angiogenesis. Using a proteomic approach, we found increased secretion of a matricellular protein, CTGF, from VEGF-A-treated human umbilical vein endothelial cells (HUVECs). Then, we studied the effect of CTGF binding to FPRL1 in VEGF-A-induced angiogenesis. CTGF directly binds to FPRL1 through a linker region and activates the downstream signals of FPRL1, such as increase in extracellular signal-regulated kinase (ERK) phosphorylation and intracellular Ca(2+) concentration. We found that linker region-induced FPRL1 activation promotes the migration and network formation of HUVECs, while disruption of FPRL1 inhibits VEGF-A-induced HUVEC migration and network formation. In addition, similar results were observed by the chorioallantoic membrane (CAM) assay based evaluation of angiogenesis in vivo. To summarize, our data reveal a novel working model for VEGF-A-induced angiogenesis via the VEGF-A/CTGF/FPRL1 axis that might prolong and enhance the signals initiated from VEGF-A.

  4. Corneal avascularity is due to soluble VEGF receptor-1.

    PubMed

    Ambati, Balamurali K; Nozaki, Miho; Singh, Nirbhai; Takeda, Atsunobu; Jani, Pooja D; Suthar, Tushar; Albuquerque, Romulo J C; Richter, Elizabeth; Sakurai, Eiji; Newcomb, Michael T; Kleinman, Mark E; Caldwell, Ruth B; Lin, Qing; Ogura, Yuichiro; Orecchia, Angela; Samuelson, Don A; Agnew, Dalen W; St Leger, Judy; Green, W Richard; Mahasreshti, Parameshwar J; Curiel, David T; Kwan, Donna; Marsh, Helene; Ikeda, Sakae; Leiper, Lucy J; Collinson, J Martin; Bogdanovich, Sasha; Khurana, Tejvir S; Shibuya, Masabumi; Baldwin, Megan E; Ferrara, Napoleone; Gerber, Hans-Peter; De Falco, Sandro; Witta, Jassir; Baffi, Judit Z; Raisler, Brian J; Ambati, Jayakrishna

    2006-10-26

    Corneal avascularity-the absence of blood vessels in the cornea-is required for optical clarity and optimal vision, and has led to the cornea being widely used for validating pro- and anti-angiogenic therapeutic strategies for many disorders. But the molecular underpinnings of the avascular phenotype have until now remained obscure and are all the more remarkable given the presence in the cornea of vascular endothelial growth factor (VEGF)-A, a potent stimulator of angiogenesis, and the proximity of the cornea to vascularized tissues. Here we show that the cornea expresses soluble VEGF receptor-1 (sVEGFR-1; also known as sflt-1) and that suppression of this endogenous VEGF-A trap by neutralizing antibodies, RNA interference or Cre-lox-mediated gene disruption abolishes corneal avascularity in mice. The spontaneously vascularized corneas of corn1 and Pax6+/- mice and Pax6+/- patients with aniridia are deficient in sflt-1, and recombinant sflt-1 administration restores corneal avascularity in corn1 and Pax6+/- mice. Manatees, the only known creatures uniformly to have vascularized corneas, do not express sflt-1, whereas the avascular corneas of dugongs, also members of the order Sirenia, elephants, the closest extant terrestrial phylogenetic relatives of manatees, and other marine mammals (dolphins and whales) contain sflt-1, indicating that it has a crucial, evolutionarily conserved role. The recognition that sflt-1 is essential for preserving the avascular ambit of the cornea can rationally guide its use as a platform for angiogenic modulators, supports its use in treating neovascular diseases, and might provide insight into the immunological privilege of the cornea.

  5. SFRP2 Is Associated with Increased Adiposity and VEGF Expression

    PubMed Central

    Crowley, Rachel K.; Bujalska, Iwona J.; Hassan-Smith, Zaki K.; Hazlehurst, Jonathan M.; Foucault, Danielle R.; Stewart, Paul M.; Tomlinson, Jeremy W.

    2016-01-01

    Aims The aim of this study was to assess depot-specific expression and secretion of secreted frizzled-related protein 2 (sFRP2) by adipose tissue and its effect on adipocyte biology. We measured serum sFRP2 concentrations in 106 patients in vivo to explore its relationship to fat mass, glycaemia and insulin resistance. Methods Expression of sFRP2 in mouse and human tissues was assessed using polymerase chain reaction and Western blot. Western blot confirmed secretion of sFRP2 by adipose tissue into cell culture medium. Effects of recombinant sFRP2 on lipogenesis and preadipocyte proliferation were measured. Preadipocyte expression of the angiogenic genes vascular endothelial growth factor (VEGF) and nuclear factor of activated T-cells 3 (NFATC3) was measured after recombinant sFRP2 exposure. Complementary clinical studies correlating human serum sFRP2 with age, gender, adiposity and insulin secretion were also performed. Results sFRP2 messenger RNA (mRNA) was expressed in mouse and human adipose tissue. In humans, sFRP2 mRNA expression was 4.2-fold higher in omental than subcutaneous adipose. Omental adipose tissue secreted 63% more sFRP2 protein than subcutaneous. Treatment with recombinant sFRP2 did not impact on lipogenesis or preadipocyte proliferation but was associated with increased VEGF mRNA expression. In human subjects, circulating insulin levels positively correlated with serum sFRP2, and levels were higher in patients with abnormal glucose tolerance (34.2ng/ml) compared to controls (29.5ng/ml). A positive correlation between sFRP2 and BMI was also observed. Conclusions Circulating sFRP2 is associated with adipose tissue mass and has a potential role to drive adipose angiogenesis through enhanced VEGF expression. PMID:27685706

  6. The endoderm indirectly influences morphogenetic movements of the zebrafish head kidney through the posterior cardinal vein and VegfC

    PubMed Central

    Chou, Chih-Wei; Hsu, Hsiao-Chu; You, May-su; Lin, Jamie; Liu, Yi-Wen

    2016-01-01

    Integration of blood vessels and organ primordia determines organ shape and function. The head kidney in the zebrafish interacts with the dorsal aorta (DA) and the posterior cardinal vein (PCV) to achieve glomerular filtration and definitive hematopoiesis, respectively. How the head kidney co-develops with both the axial artery and vein remains unclear. We found that in endodermless sox32-deficient embryos, the head kidney associated with the PCV but not the DA. Disrupted convergent migration of the PCV and the head kidney in sox32-deficient embryos was rescued in a highly coordinated fashion through the restoration of endodermal cells. Moreover, grafted endodermal cells abutted the host PCV endothelium in the transplantation assay. Interestingly, the severely-disrupted head kidney convergence in the sox32-deficient embryo was suppressed by both the cloche mutation and the knockdown of endothelial genes, indicating that an interaction between the endoderm and the PCV restricts the migration of the head kidney. Furthermore, knockdown of either vegfC or its receptor vegfr3 suppressed the head kidney convergence defect in endodermless embryos and perturbed the head kidney-PCV association in wild-type embryos. Our findings thus underscore a role for PCV and VegfC in patterning the head kidney prior to organ assembly and function. PMID:27477767

  7. The endoderm indirectly influences morphogenetic movements of the zebrafish head kidney through the posterior cardinal vein and VegfC.

    PubMed

    Chou, Chih-Wei; Hsu, Hsiao-Chu; You, May-Su; Lin, Jamie; Liu, Yi-Wen

    2016-01-01

    Integration of blood vessels and organ primordia determines organ shape and function. The head kidney in the zebrafish interacts with the dorsal aorta (DA) and the posterior cardinal vein (PCV) to achieve glomerular filtration and definitive hematopoiesis, respectively. How the head kidney co-develops with both the axial artery and vein remains unclear. We found that in endodermless sox32-deficient embryos, the head kidney associated with the PCV but not the DA. Disrupted convergent migration of the PCV and the head kidney in sox32-deficient embryos was rescued in a highly coordinated fashion through the restoration of endodermal cells. Moreover, grafted endodermal cells abutted the host PCV endothelium in the transplantation assay. Interestingly, the severely-disrupted head kidney convergence in the sox32-deficient embryo was suppressed by both the cloche mutation and the knockdown of endothelial genes, indicating that an interaction between the endoderm and the PCV restricts the migration of the head kidney. Furthermore, knockdown of either vegfC or its receptor vegfr3 suppressed the head kidney convergence defect in endodermless embryos and perturbed the head kidney-PCV association in wild-type embryos. Our findings thus underscore a role for PCV and VegfC in patterning the head kidney prior to organ assembly and function. PMID:27477767

  8. Delineating multiple functions of VEGF-A in the adult brain.

    PubMed

    Licht, Tamar; Keshet, Eli

    2013-05-01

    Vascular endothelial growth factor-A (abbreviated throughout this review as VEGF) is mostly known for its angiogenic activity, for its activity as a vascular permeability factor, and for its vascular survival activity [1]. There is a growing body of evidence, however, that VEGF fulfills additional less 'traditional' functions in multiple organs, both during development, as well as homeostatic functions in fully developed organs. This review focuses on the multiple roles of VEGF in the adult brain and is less concerned with the roles played by VEGF during brain development, functions described elsewhere in this review series. Most functions of VEGF that are essential for proper brain development are, in fact, dispensable in the adult brain as was clearly demonstrated using a conditional brain-specific VEGF loss-of-function (LOF) approach. Thus, in contrast to VEGF LOF in the developing brain, a process which is detrimental for the growth and survival of blood vessels and leads to massive neuronal apoptosis [2-4], continued signaling by VEGF in the mature brain is no longer required for maintaining already established cerebral vasculature and its inhibition does not cause appreciable vessel regression, hypoxia or apoptosis [4-7]. Yet, VEGF continues to be expressed in the adult brain in a constitutive manner. Moreover, VEGF is expressed in the adult brain in a region-specific manner and in distinctive spatial patterns incompatible with an angiogenic role (see below), strongly suggesting angiogenesis-independent and possibly also perfusion-independent functions. Here we review current knowledge on some of these 'non-traditional', often unexpected homeostatic VEGF functions, including those unrelated to its effects on the brain vasculature. These effects could be mediated directly (on non-vascular cells expressing cognate VEGF receptors) or indirectly (via the endothelium). Experimental approaches aimed at distinguishing between these possibilities for each particular

  9. Id-1: Regulator of EGFR and VEGF and potential target for colorectal cancer therapy

    PubMed Central

    Meteoglu, Ibrahim; Meydan, Nezih; Erkus, Muhan

    2008-01-01

    Background The helix-loop-helix transcription factor Id-1 (an inhibitor of differentiation and DNA binding) plays a role in development and progression of many tumours. Id-1 is known to exert its effects on the epidermal growth factor receptor (EGFR) and the vascular endothelial growth factor (VEGF). The aim of this study was to reveal whether there was a relationship between Id-1 and EGFR and VEGF in colorectal carcinoma. Methods Tumour and non-tumour tissue specimens from 46 cases of colorectal carcinoma were exposed to immunohistochemical staining for Id-1, EGFR and VEGF. The relationship between the degree of staining and tumour grade, tumour stage and all tumour markers was investigated. Results Tumour cells showed positive staining for Id-1 in 43 cases (93.5%), for EGFR in 41 cases (89%) and for VEGF in 42 cases (91%). There was a significant relation between the tumour grade and the degree of staining for Id-1, EGFR and VEGF. The relation between the tumour stage and the degree of staining for Id-1, EGFR and VEGF was also significant. There was a significant relation between Id-1 expression and EGFR and VEGF expressions. Non-tumoural tissue specimens were not stained with Id-1 and EGFR antibodies in any of the cases, but stained with VEGF antibody in 3 cases. Conclusion This study revealed that Id-1, EGFR and VEGF took part in development and progression of colorectal carcinomas and that Id-1 was associated with regulations of EGFR and VEGF. The results of this study support the idea that not only EGFR and VEGF but also Id-1 could be new targets in cancer treatment. PMID:19014499

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

    PubMed

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

    2013-01-01

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

  11. Acceleration of segmental bone regeneration in a rabbit model by strontium-doped calcium polyphosphate scaffold through stimulating VEGF and bFGF secretion from osteoblasts.

    PubMed

    Gu, Zhipeng; Zhang, Xu; Li, Li; Wang, Qiguang; Yu, Xixun; Feng, Ting

    2013-01-01

    The development of suitable bioactive three-dimensional scaffold for the promotion of bone regeneration is critical in bone tissue engineering. The purpose of this study was to investigate in vivo osteogenesis of the porous strontium-doped calcium polyphosphate (SCPP) scaffolds for bone repair, as well as the relationship between osteogenic properties of SCPP scaffolds and the secretion of bFGF and VEGF from osteoblasts stimulated by SCPP. Besides, the advantages of scaffolds seeded with mesenchymal stem cells (MSCs) for bone repair were also studied. Firstly, the bone repair evaluation of scaffolds was performed on a rabbit segmental bony defects model over a period of 16 weeks by histology combined with X-ray microradiography. And then, in order to avoid the influence from the other factors such as hypoxia which emerge in vivo study and affect the secretion of VEGF and bFGF from host cells, human osteoblast-like cells (MG63) were seeded to SCPP, CPP and HA scaffolds in vitro to determine the ability of these scaffolds to stimulate the secretion of angiogenic growth factors (VEGF and bFGF) from MG63 and further explore the reason for the better osteogenic properties of SCPP scaffolds. The histological and X-ray microradiographic results showed that the SCPP scaffolds presented better osteogenic potential than CPP and HA scaffolds, when combined with MSCs, the SCPP scaffolds could further accelerate the bone repair. And the amounts of VEGF measured by ELISA assay in SCPP, CPP and HA groups after cultured for 7 days were about 364.989 pg/mL, 244.035 pg/mL and 232.785 pg/mL, respectively. Accordingly, the amounts of bFGF were about 27.085 pg/mL, 15.727 pg/mL and 8.326 pg/mL. The results revealed that the SCPP scaffolds significantly enhanced the bFGF and VEGF secretion compared with other scaffolds. The results presented in vivo and in vitro study demonstrated that the SCPP could accelerate bone formation through stimulating the secretion of VEGF and bFGF from

  12. Photoreceptor avascular privilege is shielded by soluble VEGF receptor-1

    PubMed Central

    Luo, Ling; Uehara, Hironori; Zhang, Xiaohui; Das, Subrata K; Olsen, Thomas; Holt, Derick; Simonis, Jacquelyn M; Jackman, Kyle; Singh, Nirbhai; Miya, Tadashi R; Huang, Wei; Ahmed, Faisal; Bastos-Carvalho, Ana; Le, Yun Zheng; Mamalis, Christina; Chiodo, Vince A; Hauswirth, William W; Baffi, Judit; Lacal, Pedro M; Orecchia, Angela; Ferrara, Napoleone; Gao, Guangping; Young-hee, Kim; Fu, Yingbin; Owen, Leah; Albuquerque, Romulo; Baehr, Wolfgang; Thomas, Kirk; Li, Dean Y; Chalam, Kakarla V; Shibuya, Masabumi; Grisanti, Salvatore; Wilson, David J; Ambati, Jayakrishna; Ambati, Balamurali K

    2013-01-01

    Optimal phototransduction requires separation of the avascular photoreceptor layer from the adjacent vascularized inner retina and choroid. Breakdown of peri-photoreceptor vascular demarcation leads to retinal angiomatous proliferation or choroidal neovascularization, two variants of vascular invasion of the photoreceptor layer in age-related macular degeneration (AMD), the leading cause of irreversible blindness in industrialized nations. Here we show that sFLT-1, an endogenous inhibitor of vascular endothelial growth factor A (VEGF-A), is synthesized by photoreceptors and retinal pigment epithelium (RPE), and is decreased in human AMD. Suppression of sFLT-1 by antibodies, adeno-associated virus-mediated RNA interference, or Cre/lox-mediated gene ablation either in the photoreceptor layer or RPE frees VEGF-A and abolishes photoreceptor avascularity. These findings help explain the vascular zoning of the retina, which is critical for vision, and advance two transgenic murine models of AMD with spontaneous vascular invasion early in life. DOI: http://dx.doi.org/10.7554/eLife.00324.001 PMID:23795287

  13. Endocrine vasculatures are preferable targets of an antitumor ineffective low dose of anti-VEGF therapy

    PubMed Central

    Zhang, Yin; Yang, Yunlong; Hosaka, Kayoko; Huang, Guichun; Zang, Jingwu; Chen, Fang; Zhang, Yun; Samani, Nilesh J.; Cao, Yihai

    2016-01-01

    Anti-VEGF–based antiangiogenic drugs are designed to block tumor angiogenesis for treatment of cancer patients. However, anti-VEGF drugs produce off-tumor target effects on multiple tissues and organs and cause broad adverse effects. Here, we show that vasculatures in endocrine organs were more sensitive to anti-VEGF treatment than tumor vasculatures. In thyroid, adrenal glands, and pancreatic islets, systemic treatment with low doses of an anti-VEGF neutralizing antibody caused marked vascular regression, whereas tumor vessels remained unaffected. Additionally, a low dose of VEGF blockade significantly inhibited the formation of thyroid vascular fenestrae, leaving tumor vascular structures unchanged. Along with vascular structural changes, the low dose of VEGF blockade inhibited vascular perfusion and permeability in thyroid, but not in tumors. Prolonged treatment with the low-dose VEGF blockade caused hypertension and significantly decreased circulating levels of thyroid hormone free-T3 and -T4, leading to functional impairment of thyroid. These findings show that the fenestrated microvasculatures in endocrine organs are more sensitive than tumor vasculatures in response to systemic anti-VEGF drugs. Thus, our data support the notion that clinically nonbeneficial treatments with anti-VEGF drugs could potentially cause adverse effects. PMID:27035988

  14. Functional Modification of Fibrous PCL Scaffolds with Fusion Protein VEGF-HGFI Enhanced Cellularization and Vascularization.

    PubMed

    Zhao, Liqiang; Ma, Shaoyang; Pan, Yiwa; Zhang, Qiuying; Wang, Kai; Song, Dongmin; Wang, Xiangxiang; Feng, Guowei; Liu, Ruming; Xu, Haijin; Zhang, Jun; Qiao, Mingqiang; Kong, Deling

    2016-09-01

    The lack of efficient vascularization within frequently used poly(ε-caprolactone) (PCL) scaffolds has hindered their application in tissue engineering. Hydrophobin HGFI, an amphiphilic protein, can form a self-assembly layer on the surface of PCL scaffolds and convert their wettability. In this study, a fusion protein consisting of HGFI and vascular endothelial growth factor (VEGF) is prepared by Pichia pastoris expression system. Sodium dodecyl sulface-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting confirm that the VEGF-HGFI is successfully isolated and purified. Transmission electron microscope and water contact angle measurement demonstrate that VEGF-HGFI can form a self-assembly layer with about 25 nm in thickness on electrospun PCL fibers and increase their hydrophilicity. VEGF-HGFI modification can effectively enhance the adhesion, migration, and proliferation of human umbilical vein endothelial cells. Near-infrared fluorescence imaging shows that the VEGF-HGFI modification on PCL scaffolds can exist at least 21 d in vitro and at least 14 d in vivo. Bioluminescence imaging shows that VEGF-HGFI can effectively activate vascular endothelial growth factor receptor 2 receptors. Subcutaneous implantation in mice and rats reveal that cellularization and vascularization are significantly improved in VEGF-HGFI modified PCL scaffolds. These results suggest that VEGF-HGFI is a useful molecule for functional modification of scaffolds to enhance cellularization and vascularization in tissue engineering. PMID:27391702

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

    PubMed

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

    2014-10-14

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

  16. VEGF-D promotes pulmonary oedema in hyperoxic acute lung injury.

    PubMed

    Sato, Teruhiko; Paquet-Fifield, Sophie; Harris, Nicole C; Roufail, Sally; Turner, Debra J; Yuan, Yinan; Zhang, You-Fang; Fox, Stephen B; Hibbs, Margaret L; Wilkinson-Berka, Jennifer L; Williams, Richard A; Stacker, Steven A; Sly, Peter D; Achen, Marc G

    2016-06-01

    Leakage of fluid from blood vessels, leading to oedema, is a key feature of many diseases including hyperoxic acute lung injury (HALI), which can occur when patients are ventilated with high concentrations of oxygen (hyperoxia). The molecular mechanisms driving vascular leak and oedema in HALI are poorly understood. VEGF-D is a protein that promotes blood vessel leak and oedema when overexpressed in tissues, but the role of endogenous VEGF-D in pathological oedema was unknown. To address these issues, we exposed Vegfd-deficient mice to hyperoxia. The resulting pulmonary oedema in Vegfd-deficient mice was substantially reduced compared to wild-type, as was the protein content of bronchoalveolar lavage fluid, consistent with reduced vascular leak. Vegf-d and its receptor Vegfr-3 were more highly expressed in lungs of hyperoxic, versus normoxic, wild-type mice, indicating that components of the Vegf-d signalling pathway are up-regulated in hyperoxia. Importantly, VEGF-D and its receptors were co-localized on blood vessels in clinical samples of human lungs exposed to hyperoxia; hence, VEGF-D may act directly on blood vessels to promote fluid leak. Our studies show that Vegf-d promotes oedema in response to hyperoxia in mice and support the hypothesis that VEGF-D signalling promotes vascular leak in human HALI. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

  17. Guided bone regeneration (GBR) utilizing injectable Vascular Endothelial Growth Factor (VEGF) delivery gel

    PubMed Central

    Kaigler, Darnell; Silva, Eduardo A.; Mooney, David J.

    2013-01-01

    Background Vascularization underlies the success of guided bone regeneration (GBR) procedures. This study evaluated the regenerative potential of GBR in combination with Vascular Endothelial Growth Factor (VEGF) delivery, via an injectable hydrogel system. Methods Critical-sized defects were created in rat calvariae and GBR procedures were performed with a collagen membrane either alone (control), plus bolus delivery of VEGF, or plus application of VEGF releasing hydrogels (VEGF - Alg). Four and eight weeks following treatment, defect sites were evaluated with microcomputed tomographic and histomorphometric analyses for blood vessel and bone formation. Results At four weeks, relative to the control condition, the bolus addition of VEGF did not affect blood vessel density within the defect site; yet, the application of the VEGF+ Alg significantly (p< 0.05) increased blood vessel density. Though there was no difference in bone regeneration at four weeks, at eight weeks, there was a significant (p < 0.05) increase in bone regeneration in the VEGF + Alg treated defects. Conclusions These data demonstrated that the application of VEGF + Alg enhanced early angiogenesis while at a later timepoint, it enhanced bone regeneration. Controlled delivery approaches of angiogenic growth factors used adjunctively with GBR may be a promising strategy for enhancing outcomes of GBR. PMID:22668339

  18. CIEF and MALDI-TOF-MS methods for analyzing forms of the glycoprotein VEGF 165.

    PubMed

    Ongay, Sara; Puerta, Angel; Díez-Masa, Jose Carlos; Bergquist, Jonas; de Frutos, Mercedes

    2009-04-01

    The vascular endothelial growth factor (VEGF) is involved in different sicknesses (cardiovascular diseases, cancer, and other). Out of the many components of the VEGF family, the A splice variant with 165 amino acids (VEGF(165)) is the main component. In spite of the potential as biomarker that this protein has, information about its physico-chemical characteristics is scarce. In this study CIEF and MALDI-TOF-MS methods for intact recombinant human VEGF(165) are developed and applied to analyze this glycoprotein expressed in glycosylating (Sf 21 insect cells) and non-glycosylating (Escherichia coli) systems. Different parameters influencing the CIEF separation were studied. The developed CIEF method allowed for the separation of up to seven peaks in the VEGF(165) expressed in insect cells and up to three in VEGF(165) expressed in E. coli. The use of the presented method permits the estimation of the apparent pI of the different forms of VEGF(165) expressed in insect cells to be in a range of 6.8-8.2. The three peaks with intermediate pI values are observed in the protein expressed in both systems, insect cells and E. coli. The MALDI-TOF-MS method enabled to a rapid partial characterization of VEGF(165) based on its MS fingerprint. MALDI-MS analysis of VEGF(165) expressed in insect cells shows the presence of, at least, four forms or groups of forms of VEGF(165) as a result of the different PTMs of the protein. According to the MALDI-MS analysis, VEGF(165) expressed in E. coli was produced as a very homogeneous protein, although the results suggest the existence of some PTMs in the protein. The patterns of VEGF(165) of both origins obtained by CIEF and MALDI-MS indicate the possibility of using these analytical methods to compare samples from people with different pathophysiological conditions. This work is thus a starting point to make possible the study of the role of the various forms of VEGF(165) as biomarkers. Finally, to the best of our knowledge, this is the

  19. The constitutive level of vascular endothelial growth factor (VEGF) is more important than hypoxia-induced VEGF up-regulation in the angiogenesis of human melanoma xenografts

    PubMed Central

    Danielsen, T; Rofstad, E K

    2000-01-01

    Angiogenesis of tumours might develop as a result of environmental conditions, such as hypoxia, and/or as a result of genetic alterations specific for tumour cells. The relative contributions of these mechanisms were investigated by comparing the in vivo expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) to the hypoxic fraction, the angiogenic potential and the vascular density of four human melanoma lines (A-07, D-12, R-18, U-25) grown intradermally in Balb/c nu/nu mice. VEGF expression, bFGF expression and expression of pimonidazole, a marker of hypoxic cells, were investigated by immunohistochemistry. An association between high VEGF and bFGF expression and high angiogenic potential was detected, suggesting an important role for VEGF/bFGF in the angiogenesis of melanomas. High VEGF/bFGF expression was also related to low hypoxic fraction and high vascular density. Thus, the constitutive, genetically determined level of VEGF was probably more important than hypoxia-induced upregulation in the angiogenesis of the melanoma xenografts. © 2000 Cancer Research Campaign PMID:10789719

  20. Moving Past Anti-VEGF: Novel Therapies for Treating Diabetic Retinopathy.

    PubMed

    Bolinger, Mark T; Antonetti, David A

    2016-01-01

    Diabetic retinopathy is the leading cause of blindness in working age adults, and is projected to be a significant future health concern due to the rising incidence of diabetes. The recent advent of anti-vascular endothelial growth factor (VEGF) antibodies has revolutionized the treatment of diabetic retinopathy but a significant subset of patients fail to respond to treatment. Accumulating evidence indicates that inflammatory cytokines and chemokines other than VEGF may contribute to the disease process. The current review examines the presence of non-VEGF cytokines in the eyes of patients with diabetic retinopathy and highlights mechanistic pathways in relevant animal models. Finally, novel drug targets including components of the kinin-kallikrein system and emerging treatments such as anti-HPTP (human protein tyrosine phosphatase) β antibodies are discussed. Recognition of non-VEGF contributions to disease pathogenesis may lead to novel therapeutics to enhance existing treatments for patients who do not respond to anti-VEGF therapies. PMID:27618014

  1. Impact of VEGF gene polymorphisms in elderly cancer patients: clinical outcome and toxicity.

    PubMed

    Della-Morte, David; Riondino, Silvia; Ferroni, Patrizia; Palmirotta, Raffaele; Pastore, Donatella; Lauro, Davide; Guadagni, Fiorella; Roselli, Mario

    2015-01-01

    Vascular endothelial growth factors (VEGFs) are the key regulators in angiogenesis and have been shown to play a significant role in the progression and prognosis of angiogenesis-related diseases, such as cancer. VEGF inhibitors are a current pharmacological tumoral strategy. However, despite the strong association between aging and cancer incidence and progression, recent findings suggest impaired angiogenesis accompanied by a reduced expression of VEGF in cells derived from aging subjects. Specific variations of VEGF genes have been demonstrated to be genetic determinants for susceptibility, outcome and therapy response, especially for the solid tumors. Considering the complications present in frail elderly patients, analysis of VEGF genetic polymorphisms in these subjects may further help in tailoring an angiogenic pharmacological strategy, and in improving our ability to better understand prognosis during therapy-related to cancer.

  2. Moving Past Anti-VEGF: Novel Therapies for Treating Diabetic Retinopathy

    PubMed Central

    Bolinger, Mark T.; Antonetti, David A.

    2016-01-01

    Diabetic retinopathy is the leading cause of blindness in working age adults, and is projected to be a significant future health concern due to the rising incidence of diabetes. The recent advent of anti-vascular endothelial growth factor (VEGF) antibodies has revolutionized the treatment of diabetic retinopathy but a significant subset of patients fail to respond to treatment. Accumulating evidence indicates that inflammatory cytokines and chemokines other than VEGF may contribute to the disease process. The current review examines the presence of non-VEGF cytokines in the eyes of patients with diabetic retinopathy and highlights mechanistic pathways in relevant animal models. Finally, novel drug targets including components of the kinin–kallikrein system and emerging treatments such as anti-HPTP (human protein tyrosine phosphatase) β antibodies are discussed. Recognition of non-VEGF contributions to disease pathogenesis may lead to novel therapeutics to enhance existing treatments for patients who do not respond to anti-VEGF therapies. PMID:27618014

  3. Repair of Abdominal Wall Defects In Vitro and In Vivo Using VEGF Sustained-Release Multi-Walled Carbon Nanotubes (MWNT) Composite Scaffolds

    PubMed Central

    Yang, Jianjun; Liu, Zhengni; Peng, Zhiyou; Tang, Rui; Gu, Yan

    2013-01-01

    Objective Porcine acellular dermal matrices (ADM) have been widely used in experimental and clinical research for abdominal wall repair. Compared to porcine small intestinal submucosa (SIS), the effect of these matrices on the regenerative capacity of blood vessels is still not ideal. Multi-walled carbon nanotubes (MWNTs) can more effectively transport VEGF to cells or tissues because of their large specific surface area and interior cavity. In this study, we explored the safety and efficacy of implanted VEGF-loaded MWNT composite scaffolds in vitro and vivo to repair abdominal wall defects. Materials and Methods VEGF-loaded MWNTs were prepared by a modified plasma polymerization treatment. Four composite scaffolds were evaluated for cytotoxicity, proliferation, and release dynamics. We created 3 cm×4 cm abdominal wall defects in 43 Sprague-Dawley rats. After implantation times of 2, 4, 8, and 12 weeks, the scaffolds and the surrounding tissues were collected and examined by gross inspection, biomechanical testing, and histological examination. Results A 5–10 nm poly(lactic-co-glycolic acid) (PLGA) film was evenly distributed on MWNTs. The 3% MWNT composite group showed lower cytotoxicity and appropriate release performance, and it was thus tested in vivo. In rats with the 3% composite implanted, host cells were prevented from migrating to the ADM at 2 weeks, vascularization was established more rapidly at 12 weeks, and the values for both the maximum load and the elastic modulus were significantly lower than in the ADM-alone group (p<0.01). Histological staining revealed that the MWNT was still not completely eliminated 12 weeks after implantation. Conclusion MWNTs were able to carry VEGF to cells or tissues, and the 3% MWNT composite material showed lower cytotoxicity and had an appropriate release performance, which prompted faster vascularization of the ADM than other scaffolds. Nevertheless, the MWNTs induced harmful effects that should be carefully

  4. Extracellular SOD and VEGF are increased in vitreous bodies from proliferative diabetic retinopathy patients

    PubMed Central

    Izuta, Hiroshi; Chikaraishi, Yuichi; Adachi, Tetsuo; Shimazawa, Masamitsu; Sugiyama, Tetsuya; Ikeda, Tsunehiko

    2009-01-01

    Purpose To evaluate the relationship between vascular endothelial growth factor (VEGF) and extracellular superoxide dismutase (EC-SOD) in vitreous body and serum in patients with proliferative diabetic retinopathy (PDR), and investigate the role of EC-SOD in PDR by evaluating its angiostatic effect, using an in vitro angiogenesis model. To investigate the role of EC-SOD in PDR by evaluating its angiostatic effect, using an in vitro angiogenesis model. Methods EC-SOD and VEGF concentrations in vitreous and serum samples from PDR and macular hole (MH) were measured by ELISA. The effects of EC-SOD on VEGF-induced proliferation, migration, and tube formation were evaluated using human umbilical vein endothelial cells (HUVECs). Moreover, the effects of EC-SOD on VEGF-induced proliferation and migration were evaluated in HUVECs and primary normal human retinal microvascular endothelial cells. Results Intravitreal concentrations of EC-SOD were significantly higher (p<0.01) in PDR (58.0±23.8 ng/ml, mean±SD) than in MH (29.3±6.6 ng/ml). Intravitreal concentrations of VEGF were dramatically higher (p<0.01) in PDR (798.2±882.7 pg/ml) than in MH (17.7±15.5 pg/ml). The serum concentrations of EC-SOD and VEGF did not differ between the two patient groups. The vitreous concentrations of VEGF correlated with those of EC-SOD in all patients (rs=0.61, p<0.001). In HUVECs, EC-SOD at 100 ng/ml significantly suppressed VEGF-induced proliferation and tube formation, but not VEGF-induced migration. Conclusions EC-SOD was increased together with VEGF in the vitreous body from PDR patients, suggesting that EC-SOD may play a pivotal role in the pathogenesis of angiogenesis. PMID:20011081

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

    SciTech Connect

    Guo, Hai-dong; Cui, Guo-hong; Yang, Jia-jun; Wang, Cun; Zhu, Jing; Zhang, Li-sheng; Jiang, Jun; Shao, Shui-jin

    2012-07-20

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

  6. Induction of Podocyte VEGF164 Overexpression at Different Stages of Development Causes Congenital Nephrosis or Steroid-Resistant Nephrotic Syndrome

    PubMed Central

    Veron, Delma; Reidy, Kimberly; Marlier, Arnaud; Bertuccio, Claudia; Villegas, Guillermo; Jimenez, Juan; Kashgarian, Michael; Tufro, Alda

    2010-01-01

    The tight regulation of vascular endothelial growth factor-A (VEGF-A) signaling is required for both the development and maintenance of the glomerular filtration barrier, but the pathogenic role of excessive amounts of VEGF-A detected in multiple renal diseases remains poorly defined. We generated inducible transgenic mice that overexpress podocyte VEGF164 at any chosen stage of development. In this study, we report the phenotypes that result from podocyte VEGF164 excess during organogenesis and after birth. On doxycycline induction, podocin-rtTA:tet-O-VEGF164 mice express twofold higher kidney VEGF164 levels than single transgenic mice, localized to podocytes. Podocyte VEGF164 overexpression during organogenesis resulted in albuminuria at birth and was associated with glomerulomegaly, uniform podocyte effacement, very few and wide foot processes joined by occluding junctions, almost complete absence of slit diaphragms, and swollen endothelial cells with few fenestrae as revealed by transmission electron microscopy. Podocyte VEGF164 overexpression after birth caused massive albuminuria in 70% of 2-week-old mice, glomerulomegaly, and minimal changes on light microscopy. Transmission electron microscopy showed podocyte effacement and fusion and morphologically normal endothelial cells. Podocyte VEGF164 overexpression induced nephrin down-regulation without podocyte loss. VEGF164-induced abnormalities were reversible on removal of doxycycline and were unresponsive to methylprednisolone. Collectively, the data suggest that moderate podocyte VEGF164 overexpression during organogenesis results in congenital nephrotic syndrome, whereas VEGF164 overexpression after birth induces a steroid-resistant minimal change like-disease in mice. PMID:20829436

  7. High glucose activates ChREBP-mediated HIF-1a and VEGF expression in human RPE cells under normoxia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Because retina-damaging angiogenesis is controlled by vascular endothelial growth factor (VEGF) and people with higher glucose intakes are more susceptible to retinal complications that may be due to increased VEGF, it is crucial to elucidate relations between glucose exposure and VEGF expression. W...

  8. Current drug patenting for retinal diseases: beyond VEGF inhibitors.

    PubMed

    Mucke, Hermann A M; Mucke, Peter M

    2010-01-01

    An analysis of patent applications that address strategies for the pharmacological treatment of retinal diseases that are not directly related to VEGF inhibition, published under the PCT during the 18-month period from January 2008 to June 2009, is presented. The largest number of therapeutic patent applications focused on attempts to correct visual cycle dysfunctions, complement overactivation or beta-amyloid deposition in drusen to control age-related macular degeneration (AMD). Biomarker-based and genetic diagnostic modalities that assess AMD risk were also frequently claimed in the patent applications, and have become a significant factor in patenting for ocular disorders. The fields of both visual cycle therapy and AMD biomarkers were dominated by non-corporate patent assignees. Diabetic retinopathy has not received as much attention from inventors compared with AMD; retinopathy of prematurity remains a field in which little specific patenting occurs.

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

    PubMed

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

    2016-04-01

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

  10. Intravitreally Injected Anti-VEGF Antibody Reduces Brown Fat in Neonatal Mice.

    PubMed

    Jo, Dong Hyun; Park, Sung Wook; Cho, Chang Sik; Powner, Michael B; Kim, Jin Hyoung; Fruttiger, Marcus; Kim, Jeong Hun

    2015-01-01

    Anti-vascular endothelial growth factor (VEGF) agents are the mainstay treatment for various angiogenesis-related retinal diseases. Currently, bevacizumab, a recombinant humanized anti-VEGF antibody, is trailed in retinopathy of prematurity, a vasoproliferative retinal disorder in premature infants. However, the risks of systemic complications after intravitreal injection of anti-VEGF antibody in infants are not well understood. In this study, we show that intravitreally injected anti-VEGF antibody is transported into the systemic circulation into the periphery where it reduces brown fat in neonatal C57BL/6 mice. A considerable amount of anti-VEGF antibody was detected in serum after intravitreal injection. Furthermore, in interscapular brown adipose tissue, we found lipid droplet accumulation, decreased VEGF levels, loss of vascular network, and decreased expression of mitochondria-related genes, Ppargc1a and Ucp1, all of which are characteristics of "whitening" of brown fat. With increasing age and body weight, brown fat restored its morphology and vascularity. Our results show that there is a transient, but significant impact of intravitreally administered anti-VEGF antibody on brown adipose tissue in neonatal mice. We suggest that more attention should be focused on the metabolic and developmental significance of brown adipose tissue in bevacizumab treated retinopathy of prematurity infants. PMID:26226015

  11. Relationship between PTEN and VEGF expression and clinicopathological characteristics in HCC.

    PubMed

    Mi, Denghai; Yi, Jilin; Liu, Enyu; Li, Xingrui

    2006-01-01

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

  12. Fetal Vegf Genotype is More Important for Abortion Risk than Mother Genotype

    PubMed Central

    Yalcintepe, Sinem Atik; Silan, Fatma; Hacivelioglu, Servet Ozden; Uludag, Ahmet; Cosar, Emine; Ozdemir, Ozturk

    2014-01-01

    VEGF gene has been reported to be related with many diseases and recurrent pregnancy loss in various studies. Concerning the role of VEGF polymorphisms in pregnancy losses, generally mothers genotypes have been analyzed. To evaluate the association between VEGF A +405G/C (rs2010963), −460T/C (rs833061), +936C/T (rs3025039) and - 2578A/C (rs699947) polymorphisms and spontaneous abortion, we studied the genotypes of spontaneously aborted fetuses, their mothers and healthy controls. 23 spontaneously aborted fetal materials, 22 mothers who had these abortions and 86 healthy controls were included in this study. rs2010963, rs833061, rs3025039 and rs699947 polymorphisms were analyzed by Real Time PCR technique after genomic DNA isolation from all subjects. The frequencies of VEGF A rs2010963 GG genotype and rs2010963 G allele were higher in fetuses compared both with mothers and healthy controls. VEGF A rs3025039 TT genotype and rs3025039 T allele frequencies were higher in fetuses comparing with mothers. VEGF A rs833061 CT and TT genotypes frequencies were higher in fetuses comparing with mothers. We ascertained that VEGF A rs2010963, rs833061 and rs3025039 are the risk factors for spontaneous abortion in fetal genotypes comparing with their mothers and healthy controls. PMID:25035858

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2015-02-01

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

  15. Multiphasic changes in systemic VEGF following intravitreal injections of ranibizumab in a child

    PubMed Central

    Shao, E H; Sivagnanavel, V; Dabbagh, A; Dave, R; Tempest-Roe, S; Tam, F W K; Taylor, S R

    2015-01-01

    Purpose To investigate whether intravitreal ranibizumab injections administered to a child alter systemic plasma levels of total and free VEGF 165. Methods A 9-year-old child sustained a choroidal rupture from blunt trauma. He subsequently developed a secondary choroidal neovascular membrane, which was treated with five ranibizumab injections over a period of 8 months. Peripheral venous blood samples were taken at each visit over a period of 12 months and plasma was extracted. Plasma VEGF 165 levels were determined using enzyme-linked immunosorbent assay and were assayed both pre- and post-immunodepletion to remove complexed VEGF. Results Plasma VEGF 165 levels proved labile following intravitreal injection of ranibizumab. Levels increased by 30% above baseline following the first intravitreal ranibizumab injection, but then returned to baseline despite two subsequent injections. There was then a rebound increase of 67% in total plasma VEGF levels following a further injection, which remained above baseline for 12 weeks despite two further intravitreal ranibizumab injections. Baseline levels were re-attained 26 weeks after the final injection. Conclusions These results suggest intravitreal ranibizumab injections can cause significant, multiphasic changes in systemic VEGF levels. This may be of particular clinical significance in children as VEGF is known to be vital in the development of major organs, in addition to its role in the maintenance of normal organ function in adults. PMID:25657041

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2011-07-01

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

  18. Anti-VEGF antibody therapy does not promote metastasis in genetically engineered mouse tumour models.

    PubMed

    Singh, Mallika; Couto, Suzana S; Forrest, William F; Lima, Anthony; Cheng, Jason H; Molina, Rafael; Long, Jason E; Hamilton, Patricia; McNutt, Angela; Kasman, Ian; Nannini, Michelle A; Reslan, Hani Bou; Cao, Tim C; Ho, Calvin C K; Barck, Kai H; Carano, Richard A D; Foreman, Oded; Eastham-Anderson, Jeffrey; Jubb, Adrian M; Ferrara, Napoleone; Johnson, Leisa

    2012-08-01

    Resistance to anti-angiogenic therapy can occur via several potential mechanisms. Unexpectedly, recent studies showed that short-term inhibition of either VEGF or VEGFR enhanced tumour invasiveness and metastatic spread in preclinical models. In an effort to evaluate the translational relevance of these findings, we examined the consequences of long-term anti-VEGF monoclonal antibody therapy in several well-validated genetically engineered mouse tumour models of either neuroendocrine or epithelial origin. Anti-VEGF therapy decreased tumour burden and increased overall survival, either as a single agent or in combination with chemotherapy, in all four models examined. Importantly, neither short- nor long-term exposure to anti-VEGF therapy altered the incidence of metastasis in any of these autochthonous models, consistent with retrospective analyses of clinical trials. In contrast, we observed that sunitinib treatment recapitulated previously reported effects on tumour invasiveness and metastasis in a pancreatic neuroendocrine tumour (PNET) model. Consistent with these results, sunitinib treatment resulted in an up-regulation of the hypoxia marker GLUT1 in PNETs, whereas anti-VEGF did not. These results indicate that anti-VEGF mediates anti-tumour effects and therapeutic benefits without a paradoxical increase in metastasis. Moreover, these data underscore the concept that drugs targeting VEGF ligands and receptors may affect tumour metastasis in a context-dependent manner and are mechanistically distinct from one another.

  19. Fibrin-embedded administration of VEGF plasmid enhances skin flap survival.

    PubMed

    Michlits, Wolfgang; Mittermayr, Rainer; Schäfer, Romana; Redl, Heinz; Aharinejad, Seyedhossein

    2007-01-01

    The aim of the present study was to experimentally evaluate whether topical fibrin-mediated administration of a vascular endothelial growth factor (VEGF)-A plasmid to the wound bed can protect skin flaps from necrosis. A plasmid expression vector containing the VEGF-A cDNA was constructed. The plasmid was then administered to the wound bed of rat abdominal skin flaps in a fibrin sealant. The percentage of viable, ischemic and necrotic tissue was assessed postoperatively as a baseline and after 3 and 7 days using digital surface area morphometry. Laser Doppler imaging of the flaps and VEGF-A Western blot analysis of flap tissue were performed to assess angiogenesis and VEGF-A tissue levels. Flaps treated with VEGF plasmids in the presence of uptake enhancing Lipofectamine transfection reagent increased flap survival 7 days postoperatively significantly associated with markedly elevated tissue perfusion and enhanced tissue VEGF-A protein expression. Our results indicate that topical fibrin-mediated administration of a VEGF-A plasmid may serve as an alternative to previous strategies in treating ischemic skin flaps. The suggested therapeutic approach is easily applicable and inexpensive in preparation. Thus, this protocol may also enhance wound healing in posttrauma skin lacerations or in skin grafts.

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

    SciTech Connect

    Sun, Jinqiao; Sha, Bin; Zhou, Wenhao; Yang, Yi

    2010-03-26

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

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

    PubMed

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

    2011-04-01

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

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

    PubMed Central

    Saif, Muhammad Wasif

    2013-01-01

    Bevacizumab is a monoclonal antibody that binds and neutralizes vascular endothelial growth factor (VEGF)-A, a key player in the angiogenesis pathway. Despite benefits of bevacizumab in cancer therapy, it is clear that the VEGF pathway is complex, involving multiple isoforms, receptors, and alternative ligands such as VEGF-B, and placental growth factor, which could enable escape from VEGF-A-targeted angiogenesis inhibition. Recently developed therapies have targeted other ligands in the VEGF pathway (eg, aflibercept, known as ziv-aflibercept in the United States), VEGF receptors (eg, ramucirumab), and their tyrosine kinase signaling (ie, tyrosine kinase inhibitors). The goal of the current review was to identify comparative preclinical data for the currently available VEGF-targeted therapies. Sources were compiled using PubMed searches (2007 to 2012), using search terms including, but not limited to: “bevacizumab,” “aflibercept,” “ramucirumab,” and “IMC-18F1.” Two preclinical studies were identified that compared bevacizumab and the newer agent, aflibercept. These studies identified some important differences in binding and pharmacodynamic activity, although the potential clinical relevance of these findings is not known. Newer antiangiogenesis therapies should help further expand treatment options for colorectal and other cancers. Comparative preclinical data on these agents is currently lacking. PMID:23807861

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

    SciTech Connect

    Farajpour, Zahra; Rahbarizadeh, Fatemeh; Kazemi, Bahram; Ahmadvand, Davoud

    2014-03-28

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

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

    PubMed

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

    2010-04-01

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

  5. Prognostic Value of VEGF in Hepatocellular Carcinoma Patients Treated with Sorafenib: A Meta-Analysis

    PubMed Central

    Cao, Guangchao; Li, Xiaoyun; Qin, Chao; Li, Jie

    2015-01-01

    Background Hepatocellular carcinoma (HCC) is characterized by rich vascularization in the tumor, and vascular endothelial growth factor (VEGF) plays important roles in vascularization. The results of the roles of VEGF in predicting efficacy of sorafenib in HCC are conflicting. In this meta-analysis, we aimed to investigate the prognostic and predictive value of VEGF in HCC patients receiving sorafenib. Material/Methods PubMed, Embase, and Cochrane library electronic databases were systematically searched for eligible studies. The baseline characteristics were recorded and overall qualities of the eligible studies were assessed by 2 reviewers independently. VEGF levels and data relevant to efficacy of sorafenib were extracted and used for meta-analysis. Results The comprehensive search yielded 9 studies that evaluated the relationship between VEGF level and clinical outcome in advanced HCC patients treated with sorafenib. Pooled estimates suggested that high level of VEGF was associated with poor overall survival (HR=1.85; 95% CI: 1.24–2.77; P=0.003) and poor progression-free survival (HR=2.09; 95% CI: 1.43–3.05; P<0.01) in HCC. Mutation of VEGF had a favorable effect on hand-foot skin reaction in HCC patients treated with sorafenib (P<0.05). Conclusions High level of VEGF is associated with poor outcomes in HCC patients treated with sorafenib, indicating that VEGF could be used as an indicator of clinical efficacy in patients with HCC. However, more well-designed studies are needed to strengthen our findings. PMID:26476711

  6. VEGF-C/VEGFR-3 pathway promotes myocyte hypertrophy and survival in the infarcted myocardium

    PubMed Central

    Zhao, Tieqiang; Zhao, Wenyuan; Meng, Weixin; Liu, Chang; Chen, Yuanjian; Gerling, Ivan C; Weber, Karl T; Bhattacharya, Syamal K; Kumar, Rahul; Sun, Yao

    2015-01-01

    Background: Numerous studies have shown that in addition to angio/lymphangiogenesis, the VEGF family is involved in other cellular actions. We have recently reported that enhanced VEGF-C and VEGFR-3 in the infarcted rat myocardium, suggesting the paracrine/autocrine function of VEGF-C on cardiac remodeling. The current study was designed to test the hypothesis that VEGF-C regulates cardiomyocyte growth and survival in the infarcted myocardium. Methods and results: Gene profiling and VEGFR-3 expression of cardiomyocytes were assessed by laser capture microdissection/microarray and immunohistochemistry in the normal and infarcted myocardium. The effect of VEGF-C on myocyte hypertrophy and apoptosis during normoxia and hypoxia was detected by RT-PCR and western blotting in cultured rat neonatal cardiomyocytes. VEGFR-3 was minimally expressed in cardiomyocytes of the normal and noninfarcted myocardium, while markedly elevated in the surviving cardiomyocytes of the infarcted myocardium and border zone. Genes altered in the surviving cardiomyocytes were associated with the networks regulating cellular growth and survival. VEGF-C significantly increased the expression of atrial natriuretic factor (ANP), brain natriuretic factor (BNP), and β-myosin heavy chain (MHC), markers of hypertrophy, in neonatal cardiomyocytes. Hypoxia caused neonatal cardiomyocyte atrophy, which was prevented by VEGF-C treatment. Hypoxia significantly enhanced apoptotic mediators, including cleaved caspase 3, 8, and 9, and Bax in neonatal cardiomyocytes, which were abolished by VEGF-C treatment. Conclusion: Our findings indicate that VEGF-C/VEGFR-3 pathway exerts a beneficial role in the infarcted myocardium by promoting compensatory cardiomyocyte hypertrophy and survival. PMID:26064438

  7. TFF3 mediated induction of VEGF via hypoxia in human gastric cancer SGC-7901 cells.

    PubMed

    Guleng, Bayasi; Han, Jia; Yang, Jin-Qiu; Huang, Qing-Wen; Huang, Jian-Kun; Yang, Xiao-Ning; Liu, Jing-Jing; Ren, Jian-Lin

    2012-04-01

    Increasing evidence indicates that in gastric epithelial cells, induction of TFF3 by hypoxia is mediated by HIF-1. Since VEGF is one of the most important angiogenic factors on cancer progression, we have started to investigate the possible link among HIF-1α, VEGF, and TFF3 in gastric cancer cells. We induced the hypoxic condition in SGC-7901cells using hypoxia-mimetic agent of CoCI2. SGC7901 cells were transfected with pcPUR + U6 plasmid carrying RNAi targeted to human TFF3 and selected puromycin-resistant pools to establish the stable knockdown of TFF3 cells. Our results showed the induction of HIF-1a via hypoxia and consequences of increased expressions of the TFF3 and VEGF in gastric cancer SGC-7901 cells. Overexpression of TFF3 upregulated the mRNA expressions of VEGF and HIF-1a induced by hypoxia, and stable knockdown of TFF3 impaired the mRNA upregulations of VEGF and HIF-1a induced by hypoxia. Furthermore, knockdown of TFF3 reduced the VEGF protein secretion: as VEGF secretion was increased time dependent manner in response to the hypoxia induction in TFF3-WT cells; however, VEGF production was significantly decreased in TFF3-KD cells (621 ± 89 vs. 264 ± 73 at 6 h and 969 ± 97 vs. 508 ± 69 at 12 h, P < 0.05). Our data demonstrated the TFF3 mediated regulation of VEGF expression induced by hypoxia, and implicated that TFF3 might be applied as a potential anti-angiogenic target for treatment of gastric cancer.

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

    PubMed

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

    2007-05-01

    Tissue expression of VEGF-C correlates with lymph node involvement (LNI) in ESCC and serum VEGF-C (sVEGF-C) in a non-small cell lung cancer has been more accurate marker of LNI than chest CT. Despite LNI importance in ESCC, the usefulness of serum VEGF-C (sVEGF-C) as a disease and LNI marker in ESCC has not been investigated yet. We found elevated sVEGF-C in ESCC (17.40 vs. 10.57 ng/ml in controls, p<0.001). It proved to be a better ESCC marker than described elsewhere: CEA, CA19-9 and SCC-Ag, with: sensitivity--70%, specificity--81%, accuracy--83.7%. Analysis of sVEGF-C correlation with clinico-pathological cancer features revealed relation to LNI (N0: 15.77 vs. N1: 21.78 ng/ml, p=0.02), especially in advanced cancers. Serum VEGF-C as a marker of LNI was characterized by: sensitivity--76%, specificity--58%, accuracy--64.4%. No relation was observed between LNI and sVEGF-A or sVEGF-A/platelets (PLT). Because sVEGF-C was higher in N0 cancers (p<0.01), the tumor presence also up-regulates sVEGF-C. We found sVEGF-C correlation with PLT and WBC: R=0.36 and R=0.32 (p<0.01). Nevertheless, analysis of PLT and WBC dependence on cancer features implies that elevation of sVEGF-C in N1 cancers is not related to them.

  9. Surface bound VEGF mimicking peptide maintains endothelial cell proliferation in the absence of soluble VEGF in vitro.

    PubMed

    Le Saux, Guillaume; Plawinski, Laurent; Parrot, Camila; Nlate, Sylvain; Servant, Laurent; Teichmann, Martin; Buffeteau, Thierry; Durrieu, Marie-Christine

    2016-06-01

    Continuous glucose monitoring is an efficient method for the management of diabetes and in limiting the complications induced by large fluctuations in glucose levels. For this, intravascular systems may assist in producing more reliable and accurate devices. However, neovascularization is a key factor to be addressed in improving their biocompatibility. In this scope, the perennial modification of the surface of an implant with the proangiogenic Vascular Endothelial Growth Factor mimic peptide (SVVYGLR peptide sequence) holds great promise. Herein, we report on the preparation of gold substrates presenting the covalently grafted SVVYGLR peptide sequence and their effect on HUVEC behavior. Effective coupling was demonstrated using XPS and PM-IRRAS. The produced surfaces were shown to be beneficial for HUVEC adhesion. Importantly, surface bound SVVYGLR is able to maintain HUVEC proliferation even in the absence of soluble VEGF. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1425-1436, 2016. PMID:26845245

  10. Downregulation of Fes inhibits VEGF-A-induced chemotaxis and capillary-like morphogenesis by cultured endothelial cells

    PubMed Central

    Kanda, Shigeru; Kanetake, Hiroshi; Miyata, Yasuyoshi

    2007-01-01

    Abstract The aim of this study was to determine whether the downregulation of endogenous Fes by siRNA in cultured endothelial cells affects vascular endothelial growth factor-A (VEGF-A)-induced chemotaxis and capillary-like morphogenesis, which are considered as angiogenic cellular responses in vitro. VEGF-A-treatment induced autophosphorylation of Fes in cultured endothelial cells.LY294002, a phosphoinositide 3-kinase inhibitor, significantly inhibited VEGF-A-induced chemotaxis and capillary-like morphogenesis.Downregulation of Fes attenuated these VEGF-A-induced cellular responses but LY294002 did not produce further inhibition of these responses. Downregulation of Fes neither affected VEGF-A-induced autophosphorylation of VEGF receptor 2 nor mitogen-activated protein kinase activation, but markedly decreased Akt activation.Taken together, our novel results indicate the involvement of Fes in VEGF-A-induced cellular responses by cultured endothelial cells. PMID:17521372

  11. Skeletal myofiber VEGF regulates contraction-induced perfusion and exercise capacity but not muscle capillarity in adult mice.

    PubMed

    Knapp, Amy E; Goldberg, Daniel; Delavar, Hamid; Trisko, Breanna M; Tang, Kechun; Hogan, Michael C; Wagner, Peter D; Breen, Ellen C

    2016-07-01

    A single bout of exhaustive exercise signals expression of vascular endothelial growth factor (VEGF) in the exercising muscle. Previous studies have reported that mice with life-long deletion of skeletal myofiber VEGF have fewer capillaries and a severe reduction in endurance exercise. However, in adult mice, VEGF gene deletion conditionally targeted to skeletal myofibers limits exercise capacity without evidence of capillary regression. To explain this, we hypothesized that adult skeletal myofiber VEGF acutely regulates skeletal muscle perfusion during muscle contraction. A tamoxifen-inducible skeletal myofiber-specific VEGF gene deletion mouse (skmVEGF-/-) was used to reduce skeletal muscle VEGF protein by 90% in adult mice. Three weeks after inducing deletion of the skeletal myofiber VEGF gene, skmVEGF-/- mice exhibited diminished maximum running speed (-10%, P < 0.05) and endurance capacity (-47%; P < 0.05), which did not persist after 8 wk. In skmVEGF-/- mice, gastrocnemius complex time to fatigue measured in situ was 71% lower than control mice. Contraction-induced perfusion measured by optical imaging during a period of electrically stimulated muscle contraction was 85% lower in skmVEGF-/- than control mice. No evidence of capillary rarefication was detected in the soleus, gastrocnemius, and extensor digitorum longus (EDL) up to 8 wk after tamoxifen-induced VEGF ablation, and contractility and fatigue resistance of the soleus measured ex vivo were also unchanged. The force-frequency of the EDL showed a small right shift, but fatigue resistance did not differ between EDL from control and skmVEGF-/- mice. These data suggest myofiber VEGF is required for regulating perfusion during periods of contraction and may in this manner affect endurance capacity.

  12. Skeletal myofiber VEGF regulates contraction-induced perfusion and exercise capacity but not muscle capillarity in adult mice.

    PubMed

    Knapp, Amy E; Goldberg, Daniel; Delavar, Hamid; Trisko, Breanna M; Tang, Kechun; Hogan, Michael C; Wagner, Peter D; Breen, Ellen C

    2016-07-01

    A single bout of exhaustive exercise signals expression of vascular endothelial growth factor (VEGF) in the exercising muscle. Previous studies have reported that mice with life-long deletion of skeletal myofiber VEGF have fewer capillaries and a severe reduction in endurance exercise. However, in adult mice, VEGF gene deletion conditionally targeted to skeletal myofibers limits exercise capacity without evidence of capillary regression. To explain this, we hypothesized that adult skeletal myofiber VEGF acutely regulates skeletal muscle perfusion during muscle contraction. A tamoxifen-inducible skeletal myofiber-specific VEGF gene deletion mouse (skmVEGF-/-) was used to reduce skeletal muscle VEGF protein by 90% in adult mice. Three weeks after inducing deletion of the skeletal myofiber VEGF gene, skmVEGF-/- mice exhibited diminished maximum running speed (-10%, P < 0.05) and endurance capacity (-47%; P < 0.05), which did not persist after 8 wk. In skmVEGF-/- mice, gastrocnemius complex time to fatigue measured in situ was 71% lower than control mice. Contraction-induced perfusion measured by optical imaging during a period of electrically stimulated muscle contraction was 85% lower in skmVEGF-/- than control mice. No evidence of capillary rarefication was detected in the soleus, gastrocnemius, and extensor digitorum longus (EDL) up to 8 wk after tamoxifen-induced VEGF ablation, and contractility and fatigue resistance of the soleus measured ex vivo were also unchanged. The force-frequency of the EDL showed a small right shift, but fatigue resistance did not differ between EDL from control and skmVEGF-/- mice. These data suggest myofiber VEGF is required for regulating perfusion during periods of contraction and may in this manner affect endurance capacity. PMID:27225953

  13. Vascular Endothelial Growth Factor (VEGF) Bioavailability Regulates Angiogenesis and Intestinal Stem and Progenitor Cell Proliferation during Postnatal Small Intestinal Development

    PubMed Central

    Holoyda, Kathleen A.; Hou, Xiaogang; Fowler, Kathryn L.; Grikscheit, Tracy C.

    2016-01-01

    Background Vascular endothelial growth factor (VEGF) is a highly conserved, master regulatory molecule required for endothelial cell proliferation, organization, migration and branching morphogenesis. Podocoryne carnea and drosophila, which lack endothelial cells and a vascular system, express VEGF homologs, indicating potential roles beyond angiogenesis and vasculogenesis. The role of VEGF in the development and homeostasis of the postnatal small intestine is unknown. We hypothesized regulating VEGF bioavailability in the postnatal small intestine would exhibit effects beyond the vasculature and influence epithelial cell stem/progenitor populations. Methods VEGF mutant mice were created that overexpressed VEGF in the brush border of epithelium via the villin promotor following doxycycline treatment. To decrease VEGF bioavailability, sFlt-1 mutant mice were generated that overexpressed the soluble VEGF receptor sFlt-1 upon doxycycline administration in the intestinal epithelium. Mice were analyzed after 21 days of doxycycline administration. Results Increased VEGF expression was confirmed by RT-qPCR and ELISA in the intestine of the VEGF mutants compared to littermates. The VEGF mutant duodenum demonstrated increased angiogenesis and vascular leak as compared to littermate controls. The VEGF mutant duodenum revealed taller villi and increased Ki-67-positive cells in the transit-amplifying zone with reduced Lgr5 expression. The duodenum of sFlt-1 mutants revealed shorter villi and longer crypts with reduced proliferation in the transit-amplifying zone, reduced expression of Dll1, Bmp4 and VE-cadherin, and increased expression of Sox9 and EphB2. Conclusions Manipulating VEGF bioavailability leads to profound effects on not only the intestinal vasculature, but epithelial stem and progenitor cells in the intestinal crypt. Elucidation of the crosstalk between VEGF signaling in the vasculature, mesenchyme and epithelial stem/progenitor cell populations may direct future

  14. VEGF and Angiopoietin-1 exert opposing effects on cell junctions by regulating the Rho GEF Syx

    PubMed Central

    Ngok, Siu P.; Geyer, Rory; Liu, Miaoliang; Kourtidis, Antonis; Agrawal, Sudesh; Wu, Chuanshen; Seerapu, Himabindu Reddy; Lewis-Tuffin, Laura J.; Moodie, Karen L.; Huveldt, Deborah; Marx, Ruth; Baraban, Jay M.; Storz, Peter

    2012-01-01

    Vascular endothelial growth factor (VEGF) and Ang1 (Angiopoietin-1) have opposing effects on vascular permeability, but the molecular basis of these effects is not fully known. We report in this paper that VEGF and Ang1 regulate endothelial cell (EC) junctions by determining the localization of the RhoA-specific guanine nucleotide exchange factor Syx. Syx was recruited to junctions by members of the Crumbs polarity complex and promoted junction integrity by activating Diaphanous. VEGF caused translocation of Syx from cell junctions, promoting junction disassembly, whereas Ang1 maintained Syx at the junctions, inducing junction stabilization. The VEGF-induced translocation of Syx from EC junctions was caused by PKD1 (protein kinase D1)-mediated phosphorylation of Syx at Ser806, which reduced Syx association to its junctional anchors. In support of the pivotal role of Syx in regulating EC junctions, syx−/− mice had defective junctions, resulting in vascular leakiness, edema, and impaired heart function. PMID:23253477

  15. Urethral tissue regeneration using collagen scaffold modified with collagen binding VEGF in a beagle model.

    PubMed

    Jia, Weisheng; Tang, He; Wu, Jianjian; Hou, Xianglin; Chen, Bing; Chen, Wei; Zhao, Yannan; Shi, Chunying; Zhou, Feng; Yu, Wei; Huang, Shengquan; Ye, Gang; Dai, Jianwu

    2015-11-01

    Extensive urethral defects have a serious impact on quality of life, and treatment is challenging. A shortage of material for reconstruction is a key limitation. Improving the properties of biomaterials and making them suitable for urethral reconstruction will be helpful. Previously, we constructed a fusion protein, collagen-binding VEGF (CBD-VEGF), which can bind to collagen scaffold, stimulate cell proliferation, and promote angiogenesis and tissue regeneration. We proposed that CBD-VEGF could improve the performance of collagen in reconstruction of extensive urethral defects. Our results showed that collagen scaffolds modified with CBD-VEGF could promote urethral tissue regeneration and improve the function of the neo-urethra in a beagle extensive urethral defect model. Thus, modifying biomaterials with bioactive factors provides an alternative strategy for the production of suitable biomaterials for urethral reconstruction.

  16. Anti-VEGF therapies and blood pressure: more than meets the eye.

    PubMed

    Enseleit, Frank; Michels, Stephan; Ruschitzka, Frank

    2010-02-01

    "Wet" (also called neovascular) age-related macular degeneration (AMD) is a chronic progressive disease characterized by leakage of fluid or blood from choroidal neovascularization. It remains the leading cause of blindness in the developed world. Vascular endothelial growth factor (VEGF), which plays a key role in the pathogenesis of retinal neovascularization and vessel leakage leading to central vision loss, has emerged as a potential target in the treatment of wet AMD. Importantly, large-scale clinical trials have demonstrated that intravitreal VEGF antagonism prevents vision loss and may even improve visual acuity in patients with neovascular AMD. Because VEGF and its downstream mediator nitric oxide have a well-established cardioprotective role, however, it can be argued that the beneficial effects of VEGF antagonism in the eye may come at the cost of adverse systemic effects, particularly myocardial infarction and stroke. PMID:20425156

  17. Split for the cure: VEGF, PDGF-BB and intussusception in therapeutic angiogenesis.

    PubMed

    Gianni-Barrera, Roberto; Bartolomeo, Mariateresa; Vollmar, Brigitte; Djonov, Valentin; Banfi, Andrea

    2014-12-01

    Therapeutic angiogenesis is an attractive strategy to treat patients suffering from ischaemic conditions and vascular endothelial growth factor-A (VEGF) is the master regulator of blood vessel growth. However, VEGF can induce either normal or aberrant angiogenesis depending on its dose localized in the microenvironment around each producing cell in vivo and on the balanced stimulation of platelet-derived growth factor-BB (PDGF-BB) signalling, responsible for pericyte recruitment. At the doses required to induce therapeutic benefit, VEGF causes new vascular growth essentially without sprouting, but rather through the alternative process of intussusception, or vascular splitting. In the present article, we briefly review the therapeutic implications of controlling VEGF dose on one hand and pericyte recruitment on the other, as well as the key features of intussusceptive angiogenesis and its regulation.

  18. Enhancing integrin function by VEGF/neuropilin signaling: implications for tumor biology.

    PubMed

    Goel, Hira Lal; Mercurio, Arthur M

    2012-01-01

    This review advances the hypothesis that the ability of integrins to engage their extracellular matrix ligands and signal can be regulated in tumor cells by vascular endothelial growth factor (VEGF), a major angiogenic factor that also has direct effects on the function of tumor cells. More specifically, we will discuss how neuropilins (NRPs), a distinct class of VEGF receptors, enable the function of specific integrins that contribute to tumor initiation and progression.

  19. Aqueous VEGF as a Predictor of Macular Thickening Following Cataract Surgery in Patients with Diabetes Mellitus

    PubMed Central

    Hartnett, M. Elizabeth; Tinkham, Nicholas; Paynter, Lauren; Geisen, Pete; Rosenberg, Pinchas; Koch, Gary; Cohen, Kenneth L

    2009-01-01

    Purpose To study associations between serum and aqueous vascular endothelial growth factor (VEGF) and insulin-like growth factor 1 (IGF-1) and macular edema measured with optical coherence tomography (OCT) following phacoemulsification in diabetic patients. Design Cohort study Methods A pilot study of 36 consecutive diabetic patients undergoing planned phacoemulsification with IOL in one eye by one surgeon at the University of North Carolina consented to preoperative and postoperative OCT central subfield thickness measurements (CSF) and aqueous and blood samples for VEGF and IGF-1. Four patients with CSME received laser preoperatively. Spearman Rank correlations were performed between growth factors and mean CSF or a clinically meaningful percent change in CSF (>11% of preoperative measurement) at one and 6 months postoperatively. Results There were no surgical complications or new cases of CSME following surgery. Mean aqueous VEGF in patients with retinopathy, determined preoperatively, increased with increasing level of severity. Patients with preoperative CSME also had severe or worse retinopathy and the greatest mean aqueous VEGF. Significant preoperative correlations existed between aqueous VEGF and more severe retinopathy, whether CSME was present or absent (r=0.49, P=.007), and between aqueous VEGF and CSME (r=0.41, P=.029). At one month postoperative, aqueous VEGF was positively correlated with >11% change from preoperative CSF, regardless of CSME status (r=0.47; P=.027). No noteworthy associations existed between CSF and IGF-1 values. Conclusions Aqueous VEGF was significantly positively associated with a clinically meaningful change in CSF in diabetic patients one month following cataract surgery. Accounting for preoperative CSF was important. Further study is indicated. PMID:19837384

  20. Transcriptional profiling of mouse uterus at pre-implantation stage under VEGF repression.

    PubMed

    Ji, Yan; Lu, Xiaodan; Zhong, Qingping; Liu, Peng; An, Yao; Zhang, Yuntao; Zhang, Shujie; Jia, Ruirui; Tesfamariam, Isaias G; Kahsay, Abraha G; Zhang, Luqing; Zhu, Wensheng; Zheng, Yaowu

    2013-01-01

    Uterus development during pre-implantation stage affects implantation process and embryo growth. Aberrant uterus development is associated with many human reproductive diseases. Among the factors regulating uterus development, vascular remodeling promoters are critical for uterus function and fertility. Vascular endothelial growth factor (VEGF), as one of the major members, has been found to be important in endothelial cell growth and blood vessel development, as well as in non-endothelial cells. VEGF mediation in reproduction has been broadly studied, but VEGF-induced transcriptional machinery during implantation window has not been systematically studied. In this study, a genetically repressed VEGF mouse model was used to analyze uterus transcriptome at gestation 2.5 (G2.5) by Solexa/Illumina's digital gene expression (DGE) system. A number of 831 uterus-specific and 2398 VEGF-regulated genes were identified. Gene ontology (GO) analysis indicated that genes actively involved in uterus development were members of collagen biosynthesis, cell proliferation and cell apoptosis. Uterus-specific genes were enriched in activities of phosphatidyl inositol phosphate kinase, histone H3-K36 demethylation and protein acetylation. Among VEGF-regulated genes, up-regulated were associated with RNA polymerase III activity while down-regulated were strongly related with muscle development. Comparable numbers of antisense transcripts were identified. Expression levels of the antisense transcripts were found tightly correlated with their sense expression levels, an indication of possibly non-specific transcripts generated around the active promoters and enhancers. The antisense transcripts with exceptionally high or low expression levels and the antisense transcripts under VEGF regulation were also identified. These transcripts may be important candidates in regulation of uterus development. This study provides a global survey on genes and antisense transcripts regulated by VEGF in

  1. Bone response to biomimetic implants delivering BMP-2 and VEGF: an immunohistochemical study.

    PubMed

    Ramazanoglu, Mustafa; Lutz, Rainer; Rusche, Philipp; Trabzon, Levent; Kose, Gamze Torun; Prechtl, Christopher; Schlegel, Karl Andreas

    2013-12-01

    This animal study evaluated bone healing around titanium implant surfaces biomimetically coated with bone morphogenic protein-2 (BMP-2) and/or vascular endothelial growth factor (VEGF) by examining bone matrix proteins and mineralisation. Five different implant surfaces were established: acid-etched surface (AE), biomimetic calcium phosphate surface (CAP), BMP-2 loaded CAP surface, VEGF loaded CAP surface and dual BMP-2 + VEGF loaded CAP surface. The implants were inserted into calvariae of adult domestic pigs. For the comparison of osteoconductive capacity of each surface, bone mineral density and expression of bone matrix proteins (collagen I, BMP-2/4, osteocalcin and osteopontin) inside defined chambers around the implant were assessed using light microscopy and microradiography and immunohistochemical analysis at 1, 2 and 4 weeks. In the both groups delivering BMP-2, the bone mineral density was significantly enhanced after 2 weeks and the highest value was measured for the group BMP + VEGF. In the group VEGF, collagen I and BMP-2/4 expressions were significantly up-regulated at the first and second weeks. The percentage of BMP-2/4 positive cells in the group BMP + VEGF was significantly enhanced compared with the groups AE and CAP at the second week. Although the highest osteocalcin and osteopontin expression values were observed for the group BMP + VEGF after 2 weeks, no statistically significant difference in osteocalcin and osteopontin expressions was found between all groups at any time. It was concluded that combined delivery of BMP-2 and VEGF favoured bone mineralisation and expression of important bone matrix proteins that might explain synergistic interaction between both growth factors. PMID:23434516

  2. PTEN's regulation of VEGF and VEGFR1 expression and its clinical significance in myeloid leukemia.

    PubMed

    Zhiyong, Cheng; Wentong, Liang; Xiaoyang, Yang; Ling, Pan

    2012-06-01

    Phosphatase and tensin homolog (PTEN) acts as a novel tumor suppressor gene. PTEN protein plays an important role in regulating proliferation, apoptosis, invasion, and migration of many cancer cells. PTEN also modulates angiogenesis mediated by vascular endothelial growth factor (VEGF) via down-regulating PI3K/Akt pathway in many solid tumors. However, the effects of PTEN on VEGF and VEGFR1 (FLT1)-mediated angiogenesis, migration, invasion of leukemia cells, and its clinical significance are still unknown in myeloid leukemia. Therefore, we investigated the effect of PTEN on PI3K/Akt and VEGF/FLT1 pathways by transfecting wild-type PTEN gene to induce high expression of wild-type PTEN gene and protein in chronic myelogenous leukemia cell line K562 cells. We also observed the correlation between the expression levels of PTEN and VEGF/FLT1 and its clinical significance in myeloid leukemia patients. We found that the expression reconstitution of wild-type PTEN had significant effect on inhibiting proliferation, migration, and invasion abilities of K562 cells via down-regulation of Akt phosphorylation and inhibition of VEGF/FLT1 expression. In myeloid leukemia patients, a negative correlation was found between the expression level of PTEN mRNA and that of VEGF and FLT1 mRNA. Down-regulation of PTEN expression accompanied by up-regulation of VEGF and FLT1 mRNA indicated a higher tendency of extramedullary disease in acute myeloid leukemia patients. In conclusion, PTEN could modulate the function of VEGF/VEGFR signaling pathway down-regulation of Akt phosphorylation and that PTEN would be a candidate target to be addressed for inhibiting angiogenesis along with the treatment of myeloid leukemia.

  3. Bone response to biomimetic implants delivering BMP-2 and VEGF: an immunohistochemical study.

    PubMed

    Ramazanoglu, Mustafa; Lutz, Rainer; Rusche, Philipp; Trabzon, Levent; Kose, Gamze Torun; Prechtl, Christopher; Schlegel, Karl Andreas

    2013-12-01

    This animal study evaluated bone healing around titanium implant surfaces biomimetically coated with bone morphogenic protein-2 (BMP-2) and/or vascular endothelial growth factor (VEGF) by examining bone matrix proteins and mineralisation. Five different implant surfaces were established: acid-etched surface (AE), biomimetic calcium phosphate surface (CAP), BMP-2 loaded CAP surface, VEGF loaded CAP surface and dual BMP-2 + VEGF loaded CAP surface. The implants were inserted into calvariae of adult domestic pigs. For the comparison of osteoconductive capacity of each surface, bone mineral density and expression of bone matrix proteins (collagen I, BMP-2/4, osteocalcin and osteopontin) inside defined chambers around the implant were assessed using light microscopy and microradiography and immunohistochemical analysis at 1, 2 and 4 weeks. In the both groups delivering BMP-2, the bone mineral density was significantly enhanced after 2 weeks and the highest value was measured for the group BMP + VEGF. In the group VEGF, collagen I and BMP-2/4 expressions were significantly up-regulated at the first and second weeks. The percentage of BMP-2/4 positive cells in the group BMP + VEGF was significantly enhanced compared with the groups AE and CAP at the second week. Although the highest osteocalcin and osteopontin expression values were observed for the group BMP + VEGF after 2 weeks, no statistically significant difference in osteocalcin and osteopontin expressions was found between all groups at any time. It was concluded that combined delivery of BMP-2 and VEGF favoured bone mineralisation and expression of important bone matrix proteins that might explain synergistic interaction between both growth factors.

  4. Anti-VEGF therapy as adjuvant therapy: clouds on the horizon?

    PubMed

    Schneider, Bryan P; Sledge, George W

    2009-01-01

    Anti-angiogenic therapies have demonstrated their value in the setting of advanced cancer, and are being explored for use in micrometastatic disease. Recent preclinical studies suggest that adjuvant anti-vascular endothelial growth factor (VEGF) therapies may increase the risk of metastasis. How concerning are these preclinical studies, and should they affect our willingness to explore anti-VEGF therapy in the adjuvant setting?

  5. Diabetes impairs mobilization of mouse bone marrow-derived Lin(-)/VEGF-R2(+) progenitor cells.

    PubMed

    Barthelmes, D; Irhimeh, M R; Gillies, M C; Karimipour, M; Zhou, M; Zhu, L; Shen, W Y

    2013-10-01

    Endothelial progenitor cells circulating in the peripheral blood (PB) contribute to vascular repair. This study aimed to evaluate the potential of a 'cocktail' consisting of erythropoietin, granulocyte colony-stimulating factor and tetrahydrobiopterin to mobilize hematopoietic lineage negative/vascular endothelial growth factor receptor 2 positive (Lin(-)/VEGF-R2(+)) cells from the bone marrow (BM) to PB in non-diabetic and diabetic mice. Diabetes was induced in mice by intraperitoneal injection of streptozotocin. Diabetic mice were studied after 16weeks of hyperglycemia. Half the mice in each group (non-diabetic and diabetic) received daily intraperitoneal injections of the cocktail for 6 consecutive days while the other half received vehicle buffer. Mobilization of Lin(-)/VEGF-R2(+) cells, which were expanded in MCP301 medium, was evaluated after isolating them from BM and PB and their phenotypic and morphological properties were studied. We found that 16weeks of diabetes affected neither the total number of BM mononucleated cells nor the number of Lin(-)/VEGF-R2(+) cells in BM compared with non-diabetic controls. In non-diabetic mice, cocktail treatment resulted in a significant decrease in BM Lin(-)/VEGF-R2(+) cells, paralleled by a significant increase of these cells in PB. Such changes in the number of Lin(-)/VEGF-R2(+) cells in BM and PB after the cocktail treatment were less marked in diabetic mice. In vitro studies of BM Lin(-)/VEGF-R2(+) cells from diabetic and non-diabetic mice did not reveal any differences in either phenotypes or colony forming potential. These findings indicate that diabetes impairs the mobilization of Lin(-)/VEGF-R2(+) cells from BM to PB. Impaired mobilization of BM Lin(-)/VEGF-R2(+) cells soon after the onset of diabetes may contribute to complications such as diabetic retinopathy.

  6. Sequestration of Vascular Endothelial Growth Factor (VEGF) Induces Late Restrictive Lung Disease

    PubMed Central

    Wieck, Minna M.; Spurrier, Ryan G.; Levin, Daniel E.; Mojica, Salvador Garcia; Hiatt, Michael J.; Reddy, Raghava; Hou, Xiaogang; Navarro, Sonia; Lee, Jooeun; Lundin, Amber; Driscoll, Barbara; Grikscheit, Tracy C.

    2016-01-01

    Rationale Neonatal respiratory distress syndrome is a restrictive lung disease characterized by surfactant deficiency. Decreased vascular endothelial growth factor (VEGF), which demonstrates important roles in angiogenesis and vasculogenesis, has been implicated in the pathogenesis of restrictive lung diseases. Current animal models investigating VEGF in the etiology and outcomes of RDS require premature delivery, hypoxia, anatomically or temporally limited inhibition, or other supplemental interventions. Consequently, little is known about the isolated effects of chronic VEGF inhibition, started at birth, on subsequent developing lung structure and function. Objectives To determine whether inducible, mesenchyme-specific VEGF inhibition in the neonatal mouse lung results in long-term modulation of AECII and whole lung function. Methods Triple transgenic mice expressing the soluble VEGF receptor sFlt-1 specifically in the mesenchyme (Dermo-1/rtTA/sFlt-1) were generated and compared to littermate controls at 3 months to determine the impact of neonatal downregulation of mesenchymal VEGF expression on lung structure, cell composition and function. Reduced tissue VEGF bioavailability has previously been demonstrated with this model. Measurements and Main Results Triple transgenic mice demonstrated restrictive lung pathology. No differences in gross vascular development or protein levels of vascular endothelial markers was noted, but there was a significant decrease in perivascular smooth muscle and type I collagen. Mutants had decreased expression levels of surfactant protein C and hypoxia inducible factor 1-alpha without a difference in number of type II pneumocytes. Conclusions These data show that mesenchyme-specific inhibition of VEGF in neonatal mice results in late restrictive disease, making this transgenic mouse a novel model for future investigations on the consequences of neonatal RDS and potential interventions. PMID:26863115

  7. VEGF-releasing suture material for enhancement of vascularization: development, in vitro and in vivo study.

    PubMed

    Bigalke, Christian; Luderer, Frank; Wulf, Katharina; Storm, Thilo; Löbler, Marian; Arbeiter, Daniela; Rau, Bettina M; Nizze, Horst; Vollmar, Brigitte; Schmitz, Klaus-Peter; Klar, Ernst; Sternberg, Katrin

    2014-12-01

    As it has been demonstrated that bioactive substances can be delivered locally using coated surgical suture materials, the authors developed a vascular endothelial growth factor (VEGF)-releasing suture material that should promote vascularization and potentially wound healing. In this context, the study focused on the characterization of the developed suture material and the verification of its biological activity, as well as establishing a coating process that allows reproducible and stable coating of a commercially available polydioxanone suture material with poly(l-lactide) (PLLA) and 0.1μg and 1.0μg VEGF. The in vitro VEGF release kinetics was studied using a Sandwich ELISA. The biological activity of the released VEGF was investigated in vitro using human umbilical vein endothelial cells. The potential of the VEGF-releasing suture material was also studied in vivo 5days after implantation in the hind limb of Wistar rats, when the histological findings were analyzed. The essential results, enhanced cell viability in vitro as well as significantly increased vascularization in vivo, were achieved using PLLA/1.0μg VEGF-coated suture material. Furthermore, ELISA measurements revealed a high reproducibility of the VEGF release behavior. Based on the results achieved regarding the dose-effect relationship of VEGF, the stability during its processing and the release behavior, it can be predicted that a bioactive suture material would be successful in later in vivo studies. Therefore, this knowledge could be the basis for future studies, where bioactive substances with different modes of action are combined for targeted, overall enhancement of wound healing.

  8. Excess HB-EGF, which promotes VEGF signaling, leads to hydrocephalus

    PubMed Central

    Shim, Joon W.; Sandlund, Johanna; Hameed, Mustafa Q.; Blazer-Yost, Bonnie; Zhou, Feng C.; Klagsbrun, Michael; Madsen, Joseph R.

    2016-01-01

    Heparin binding epidermal growth factor-like growth factor (HB-EGF) is an angiogenic factor mediating radial migration of the developing forebrain, while vascular endothelial growth factor (VEGF) is known to influence rostral migratory stream in rodents. Cell migratory defects have been identified in animal models of hydrocephalus; however, the relationship between HB-EGF and hydrocephalus is unclear. We show that mice overexpressing human HB-EGF with β-galactosidase reporter exhibit an elevated VEGF, localization of β-galactosidase outside the subventricular zone (SVZ), subarachnoid hemorrhage, and ventriculomegaly. In Wistar polycystic kidney rats with hydrocephalus, alteration of migratory trajectory is detected. Furthermore, VEGF infusions into the rats result in ventriculomegaly with an increase of SVZ neuroblast in rostral migratory stream, whereas VEGF ligand inhibition prevents it. Our results support the idea that excess HB-EGF leads to a significant elevation of VEGF and ventricular dilatation. These data suggest a potential pathophysiological mechanism that elevated HB-EGF can elicit VEGF induction and hydrocephalus. PMID:27243144

  9. Expression and significance of PTEN and VEGF in canine mammary gland tumours.

    PubMed

    Qiu, C W; Lin, D G; Wang, J Q; Li, C Y; Deng, G Z

    2008-08-01

    To investigate the relationship between the expression of the PTEN (phosphatase and tensin homolog deleted on chromosometen) and VEGF (vascular endothelial growth factor) and the clinicopathological features in canine mammary gland tumours, the expression levels of PTEN and VEGF protein were assessed in 50 cases of canine mammary gland tumours tissues and 4 cases of normal mammary gland tissues with using immunohistochemical method. The over-expression rate of PTEN protein was 100% in normal and well-differentiated mammary gland tissues and 67% in breast cancer cases respectively with a significant difference between the two groups (P<0.01). Expression of PTEN was not related to age and tumour size, but closely correlated to lymph node metastasis (P<0.01). The over-expression rate of VEGF protein was 33.3% in normal mammary gland tissues, and 78% in canine mammary gland tumours with a significant difference between the two groups (P<0.01). Expression of VEGF was not related to age or tumour size, but closely correlated with lymph node metastasis and clinical stage (P<0.05). Therefore the combination detection of PTEN and VEGF could serve as an important index to estimate the biological behavior and prognosis of canine mammary gland tumours. Reduced expression of PTEN might be involved in carcinogenesis and progression of canine breast cancer by up-regulating the VEGF expression to enhance angiogenesis.

  10. Nitric oxide mediates bleomycin-induced angiogenesis and pulmonary fibrosis via regulation of VEGF.

    PubMed

    Iyer, Anand Krishnan V; Ramesh, Vani; Castro, Carlos A; Kaushik, Vivek; Kulkarni, Yogesh M; Wright, Clayton A; Venkatadri, Rajkumar; Rojanasakul, Yon; Azad, Neelam

    2015-11-01

    Pulmonary fibrosis is a progressive lung disease hallmarked by increased fibroblast proliferation, amplified levels of extracellular matrix deposition and increased angiogenesis. Although dysregulation of angiogenic mediators has been implicated in pulmonary fibrosis, the specific rate-limiting angiogenic markers involved and their role in the progression of pulmonary fibrosis remains unclear. We demonstrate that bleomycin treatment induces angiogenesis, and inhibition of the central angiogenic mediator VEGF using anti-VEGF antibody CBO-P11 significantly attenuates bleomycin-induced pulmonary fibrosis in vivo. Bleomycin-induced nitric oxide (NO) was observed to be the key upstream regulator of VEGF via the PI3k/Akt pathway. VEGF regulated other important angiogenic proteins including PAI-1 and IL-8 in response to bleomycin exposure. Inhibition of NO and VEGF activity significantly mitigated bleomycin-induced angiogenic and fibrogenic responses. NO and VEGF are key mediators of bleomycin-induced pulmonary fibrosis, and could serve as important targets against this debilitating disease. Overall, our data suggests an important role for angiogenic mediators in the pathogenesis of bleomycin-induced pulmonary fibrosis.

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

    PubMed

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

    2014-09-01

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

  12. VEGF siRNA delivery system using arginine-grafted bioreducible poly(disulfide amine).

    PubMed

    Kim, Sun Hwa; Jeong, Ji Hoon; Kim, Tae-il; Kim, Sung Wan; Bull, David A

    2009-01-01

    Small interfering RNAs (siRNAs) are able to silence their target genes when they are successfully delivered intact into the cytoplasm. Delivery systems that enhance siRNA localization to the cytoplasm can facilitate gene silencing by siRNA therapeutics. We describe an arginine-conjugated poly(cystaminebisacrylamide-diaminohexane) (poly(CBA-DAH-R)), a bioreducible cationic polymer, as an siRNA carrier for therapeutic gene silencing for cancer. After intracellular uptake of the siRNA/poly(CBA-DAH-R) polyplexes, the reductive environment of the cytoplasm cleaves the disulfide linkages in the polymeric backbone, resulting in decomplexing of the siRNA/poly(CBA-DAH-R) polyplexes and release of siRNA molecules throughout the cytoplasm. The siRNA/poly(CBA-DAH-R) polyplexes, which demonstrate increased membrane permeability with arginine modification, have a similar level of cellular uptake as siRNA/bPEI polyplexes. The VEGF siRNA/poly(CBA-DAH-R) polyplexes, however, inhibit VEGF expression to a greater degree than VEGF siRNA/bPEI in various human cancer cell lines. The improved RNAi activity demonstrated by the VEGF siRNA/poly(CBA-DAH-R) polyplexes is due to enhanced intracellular delivery and effective localization to the cytoplasm of the VEGF siRNAs. These results demonstrate that the VEGF siRNA/poly(CBA-DAH-R) polyplex delivery system may useful for siRNA-based approaches for cancer therapy.

  13. Crocetin, a carotenoid derivative, inhibits VEGF-induced angiogenesis via suppression of p38 phosphorylation.

    PubMed

    Umigai, Naofumi; Tanaka, Junji; Tsuruma, Kazuhiro; Shimazawa, Masamitsu; Hara, Hideaki

    2012-05-01

    We evaluated the protective effects of crocetin against angiogenesis induced by vascular endothelial growth factor (VEGF). Crocetin, the aglycone of crocin carotenoids, is found in saffron crocus (Crocus sativus L.) and gardenia fruit (Gardenia jasminoides Ellis). The effects of crocetin on VEGF-induced angiogenesis were examined by in vitro tube formation assays and following 14-day co-culture of human umbilical vein endothelial cells (HUVECs) and fibroblasts. The anti-angiogenic mechanism of crocetin was evaluated by examining its effects on VEGF-induced proliferation and migration of human retinal microvascular endothelial cells (HRMECs) and phosphorylation of p38. Vascular endothelial (VE)-cadherin, zonula occludens (ZO-1) and occludin, which are adherens and tight junction proteins, respectively, play a major role in the control of vascular permeability. Therefore, we tested effects of crocetin on adhesion molecule dissociation induced by VEGF. Crocetin significantly suppressed VEGF-induced tube formation by HUVECs and migration of HRMECs. It also significantly inhibited phosphorylation of p38 and protected VE-cadherin expression. These findings indicate that crocetin suppresses the VEGF-induced angiogenesis by inhibiting migration and that the inhibition of phosphorylated-p38 and protection of VE-cadherin expression may be involved in its underlying mechanism of action. PMID:22475394

  14. VEGF-targeted cancer therapeutics-paradoxical effects in endocrine organs.

    PubMed

    Cao, Yihai

    2014-09-01

    Systemic administration of antiangiogenic drugs that target components of the vascular endothelial growth factor A (VEGF-A; VEGF) signal transduction pathway has become a viable therapeutic option for patients with various types of cancer. Nevertheless, these drugs can drive alterations in healthy vasculatures, which in turn are associated with adverse effects in healthy tissues. VEGF is crucial for vascular homeostasis and the maintenance of vascular integrity and architecture in endocrine organs. Given these critical physiological functions, systemic delivery of drugs that target VEGF signalling can block VEGF-mediated vascular functions in endocrine organs, such as the thyroid gland, and lead to endocrine dysfunction, including hypothyroidism, adrenal insufficiency and altered insulin sensitivity. This Review discusses emerging evidence from preclinical and clinical studies that contributes to understanding the mechanisms that underlie the vascular changes and subsequent modulations of endocrine function that are induced by targeted inhibition of VEGF signalling. Understanding these mechanisms is crucial for the design of antiangiogenic drugs with minimal associated adverse effects that will enable effective treatment of patients with cancer.

  15. A VEGF-dependent gene signature enriched in mesenchymal ovarian cancer predicts patient prognosis

    PubMed Central

    Yin, Xia; Wang, Xiaojie; Shen, Boqiang; Jing, Ying; Li, Qing; Cai, Mei-Chun; Gu, Zhuowei; Yang, Qi; Zhang, Zhenfeng; Liu, Jin; Li, Hongxia; Di, Wen; Zhuang, Guanglei

    2016-01-01

    We have previously reported surrogate biomarkers of VEGF pathway activities with the potential to provide predictive information for anti-VEGF therapies. The aim of this study was to systematically evaluate a new VEGF-dependent gene signature (VDGs) in relation to molecular subtypes of ovarian cancer and patient prognosis. Using microarray profiling and cross-species analysis, we identified 140-gene mouse VDGs and corresponding 139-gene human VDGs, which displayed enrichment of vasculature and basement membrane genes. In patients who received bevacizumab therapy and showed partial response, the expressions of VDGs (summarized to yield VDGs scores) were markedly decreased in post-treatment biopsies compared with pre-treatment baselines. In contrast, VDGs scores were not significantly altered following bevacizumab treatment in patients with stable or progressive disease. Analysis of VDGs in ovarian cancer showed that VDGs as a prognostic signature was able to predict patient outcome. Correlation estimation of VDGs scores and molecular features revealed that VDGs was overrepresented in mesenchymal subtype and BRCA mutation carriers. These findings highlighted the prognostic role of VEGF-mediated angiogenesis in ovarian cancer, and proposed a VEGF-dependent gene signature as a molecular basis for developing novel diagnostic strategies to aid patient selection for VEGF-targeted agents. PMID:27498762

  16. Immunohistochemical Expression of VEGF and Podoplanin in Uterine Cervical Squamous Intraepithelial Lesions

    PubMed Central

    Belfort-Mattos, Patrícia Napoli; Focchi, Gustavo Rubino de Azevedo; Ribalta, Julisa Chamorro Lascasas; Megale De Lima, Tatiana; Nogueira Carvalho, Carmen Regina; Kesselring Tso, Fernanda; De Góis Speck, Neila Maria

    2016-01-01

    VEGF and podoplanin (PDPN) have been identified as angiogenesis and/or lymphangiogenesis regulators and might be essential to restrict tumor growth, progression, and metastasis. In the present study, we evaluate the association between the expression of these markers and CIN grade. Immunohistochemistry was performed in 234 uterine cervical samples using conventional histologic sections or TMA with the monoclonal antibodies to VEGF (C-1 clone) and podoplanin (D2-40 clone). Positive-staining rates of VEGF in 191 CIN specimens were significantly associated with histological grade (P < 0.001). Negative and/or focal immunostaining for PDPN were more frequent in CIN 3 (P = 0.016). We found that patients with CIN 3 more frequently had strong and more diffuse staining for VEGF and diminished staining for PDPN (P = 0.018). Strong and more diffuse VEGF immunoexpressions in CIN 2 and CIN 3 were detected when compared to CIN 1. Negative and/or focal PDPN immunoexpression appear to be more frequent in CIN 3. Moderate to strong VEGF expression may be a tendency among patients with high-grade lesions and diminished PDPN expression. PMID:27313335

  17. Cellular and molecular aspects of diabetic nephropathy; the role of VEGF-A.

    PubMed

    Carranza, Katherine; Veron, Dolores; Cercado, Alicia; Bautista, Noemi; Pozo, Wilson; Tufro, Alda; Veron, Delma

    2015-01-01

    The prevalence of diabetes mellitus increased during the last century and it is estimated that 45% of the patients are not diagnosed. In South America the prevalence of diabetes and chronic kidney disease (CKD) increased, with a great disparity among the countries with respect to access to dialysis. In Ecuador it is one of the main causes of mortality, principally in the provinces located on the coast of the Pacific Ocean. The greatest single cause of beginning dialysis is diabetic nephropathy (DN). Even using the best therapeutic options for DN, the residual risk of proteinuria and of terminal CKD remains high. In this review we indicate the importance of the problem globally and in our region. We analyse relevant cellular and molecular studies that illustrate the crucial significance of glomerular events in DN development and evolution and in insulin resistance. We include basic anatomical, pathophysiological and clinical concepts, with special attention to the role of angiogenic factors such as the vascular endothelial growth factor (VEGF-A) and their relationship to the insulin receptor, endothelial isoform of nitric oxide synthase (eNOS) and angiopoietins. We also propose various pathways that have therapeutic potential in our opinion. Greater in-depth study of VEGF-A and angiopoietins, the state of glomerular VEGF resistance, the relationship of VEGF receptor 2/nephrin, VEGF/insulin receptors/nephrin and the relationship of VEGF/eNOS-NO at glomerular level could provide solutions to the pressing world problem of DN and generate new treatment alternatives.

  18. VEGF-mediated STAT3 activation inhibits retinal vascularization by down-regulating local erythropoietin expression.

    PubMed

    Wang, Haibo; Byfield, Grace; Jiang, Yanchao; Smith, George Wesley; McCloskey, Manabu; Hartnett, M Elizabeth

    2012-03-01

    Avascular, hypoxic retina has been postulated to be a source of angiogenic factors that cause aberrant angiogenesis and intravitreal neovascularization (IVNV) in retinopathy of prematurity. Vascular endothelial growth factor (VEGF) is an important factor involved. However, VEGF is also required for normal retinal vascular development, which raises concerns about inhibiting its activity to treat IVNV in retinopathy of prematurity. Therefore, understanding the effects that VEGF has on other factors in the development of avascular retina is important to prevent aberrant angiogenesis and IVNV. Here, we show that STAT3 was activated by increased retinal VEGF in the rat 50/10 oxygen-induced retinopathy model. Phospho-STAT3 colocalized with glutamine synthetase-labeled Müller cells. Inhibition of STAT3 reduced avascular retina and increased retinal erythropoietin (Epo) expression. Epo administered exogenously also reduced avascular retina in the model. In an in vitro study, hypoxia-induced VEGF inhibited Epo gene expression by STAT3 activation in rat Müller cells. The mechanism by which activated STAT3 regulated Epo was by inhibition of Epo promoter activity. Together, these findings show that increased retinal VEGF contributes to avascular retina by regulating retinal Epo expression through Janus kinase/STAT signaling. Our results suggest that rescuing Epo expression in the retina before the development of IVNV may promote normal developmental angiogenesis and, therefore, reduce the stimulus for later pathologic IVNV.

  19. VEGF is a Promising Therapeutic Target for the Treatment of Clear Cell Carcinoma of the Ovary

    PubMed Central

    Mabuchi, Seiji; Kawase, Chiaki; Altomare, Deborah A.; Morishige, Kenichirou; Hayashi, Masami; Sawada, Kenjiro; Ito, Kimihiko; Terai, Yoshito; Nishio, Yukihiro; Klein-Szanto, Andres J.; Burger, Robert A.; Ohmichi, Masahide; Testa, Joseph R.; Kimura, Tadashi

    2010-01-01

    This study examined the role of VEGF as a therapeutic target in clear cell carcinoma (CCC) of the ovary, which has been regarded as a chemoresistant histological subtype. Immunohistochemical analysis using tissue microarrays of 98 primary ovarian cancers revealed that VEGF was strongly expressed both in early stage and advanced stage CCC of the ovary. In early stage CCCs, patients who had tumors with high levels of VEGF had significantly shorter survival than those with low levels of VEGF. In vitro experiments revealed that VEGF expression was significantly higher in cisplatin-refractory human clear cell carcinoma cells (RMG1-CR and KOC7C-CR), compared to the respective parental cells (RMG1 and KOC7C) in the presence of cisplatin. In vivo treatment with bevacizumab markedly inhibited the growth of both parental CCC cells-derived (RMG1 and KOC7C) and cisplatin-refractory CCC cells-derived (RMG1-CR and KOC7C-CR) tumors as a result of inhibition of tumor angiogenesis. The results of the current study indicate that VEGF is frequently expressed and can be a promising therapeutic target in the management of CCC. Bevacizumab may be efficacious not only as a first-line treatment but also as a second-line treatment of recurrent disease in patients previously treated with cisplatin. PMID:20663925

  20. Synergistically combined gene delivery for enhanced VEGF secretion and anti-apoptosis

    PubMed Central

    Won, Young-Wook; Lee, Minhyung; Kim, Hyun Ah; Nam, Kihoon; Bull, David A.; Kim, Sung Wan

    2013-01-01

    With current pharmacological treatments, preventing the remodeling of the left ventricle and the progression to heart failure is a difficult task. Gene therapy is considered to provide a direct treatment to the long-term complications of ischemic heart diseases. Although current gene therapies that use single molecular targets seem potentially possible, they have not achieved a success in the treatment of ischemic diseases. With an efficient polymeric gene carrier, PAM-ABP, we designed a synergistically combined gene delivery strategy to enhance vascular endothelial growth factor (VEGF) secretion and prolong anti-apoptotic effects. A hypoxia-inducible plasmid expressing both hypoxia-inducible heme oxygenase-1 (HO-1) and the Src homology domain-2 containing tyrosine phosphatase-1 microRNA (miSHP 1) and a hypoxia-responsive VEGF plasmid were combined in this study. The positive feedback circuit between HO-1 and VEGF, and the negative regulatory role of SHP-1 in angiogenesis enhance VEGF secretion synergistically. The synergy in VEGF secretion as a consequence of the gene combination and the prolonged HO-1 activity was confirmed in hypoxic cardiomyocytes and cardiomyocyte apoptosis under hypoxia, and was decreased synergistically. These results suggest that the synergistic combination of VEGF, HO-1, and miSHP-1 may be promising for the clinical treatment of ischemic diseases. PMID:24007285

  1. Effects of VEGF and MSCs on vascular regeneration in a trauma model in rats.

    PubMed

    Niyaz, Mehmet; Gürpınar, Özer Aylin; Oktar, Gürsel Levent; Günaydın, Serdar; Onur, Mehmet Ali; Özsin, Kadir Kaan; Yener, Ali

    2015-01-01

    In the human body, vascular injuries that are caused by trauma, vessel lumen stenosis, and occlusions are often irreversible and can lead to sequelae formation as the vessels cannot reproduce fast enough. To solve this problem, the blood flow must be returned to the region as fast as possible. The adipose tissue contains progenitor cells with angiogenic potential and can be used to resolve the issue. In the present study, mesenchymal stem cells (MSCs) derived from rat adipose tissue, vascular endothelial growth factor (VEGF), and their mixture were applied on the dorsum of a rat, which was traumatized and its contribution to vascular regeneration was reviewed. No application was made to the control group. The results showed that the percentage of necrotic area was significantly lower in the MSC group than that of all the other groups. When the VEGF group was compared to the VEGF + MSCs, the percentage of necrotic area was observed to be similiar. However, VEGF showed effects only when a large quantites of VEGF was applied to the flap area. VEGF could not fully respond to the needs, whereas MSCs can produce VEGF according to the needs of tissue. This makes them superior to stem cells.

  2. Cellular and molecular aspects of diabetic nephropathy; the role of VEGF-A.

    PubMed

    Carranza, Katherine; Veron, Dolores; Cercado, Alicia; Bautista, Noemi; Pozo, Wilson; Tufro, Alda; Veron, Delma

    2015-01-01

    The prevalence of diabetes mellitus increased during the last century and it is estimated that 45% of the patients are not diagnosed. In South America the prevalence of diabetes and chronic kidney disease (CKD) increased, with a great disparity among the countries with respect to access to dialysis. In Ecuador it is one of the main causes of mortality, principally in the provinces located on the coast of the Pacific Ocean. The greatest single cause of beginning dialysis is diabetic nephropathy (DN). Even using the best therapeutic options for DN, the residual risk of proteinuria and of terminal CKD remains high. In this review we indicate the importance of the problem globally and in our region. We analyse relevant cellular and molecular studies that illustrate the crucial significance of glomerular events in DN development and evolution and in insulin resistance. We include basic anatomical, pathophysiological and clinical concepts, with special attention to the role of angiogenic factors such as the vascular endothelial growth factor (VEGF-A) and their relationship to the insulin receptor, endothelial isoform of nitric oxide synthase (eNOS) and angiopoietins. We also propose various pathways that have therapeutic potential in our opinion. Greater in-depth study of VEGF-A and angiopoietins, the state of glomerular VEGF resistance, the relationship of VEGF receptor 2/nephrin, VEGF/insulin receptors/nephrin and the relationship of VEGF/eNOS-NO at glomerular level could provide solutions to the pressing world problem of DN and generate new treatment alternatives. PMID:26300505

  3. A potential role for VEGF in the diagnostic approach of pleural effusions

    PubMed Central

    Psatha, Aggeliki; Makris, Demosthenes; Daniil, Zoe; Kiropoulos, Theodoros; Gourgoulianis, Konstantinos

    2016-01-01

    Background Vascular endothelial growth factor (VEGF) may play a role in pleural fluid formation, as it represents a potent inducer of capillary permeability. We aimed to investigate the diagnostic utility of VEGF levels in pleural fluid and serum in patients with pleural effusions with initially negative diagnostic work up. Methods Seventy-one patients with exudative lymphocytic pleural effusions undiagnosed after initial diagnostic work up were enrolled in this prospective study and their clinical course was followed up to 24 months. VEGF levels were measured in serum and pleural fluid by using immunoenzymometric assay. Results During the follow up period, in 43 patients the pleural effusion was eventually attributed to malignancy while in the rest 28 patients it was due to non-malignant causes (benign and unknown origin). Patients with malignancy had significantly higher VEGF levels in pleural fluid compared to patients with non-malignant effusions (1,506 vs. 588 pg/dL, P=0.0001), while no statistically significant difference was found in the VEGF serum levels between the two groups. Conclusions Pleural VEGF levels may be helpful in identifying malignant pleural effusion (MPE) in patients with negative diagnostic work up at the initial assessment and help in selecting patients for more invasive procedures. PMID:27499957

  4. VEGF-induced antidepressant effects involve modulation of norepinephrine and serotonin systems.

    PubMed

    Udo, Hiroshi; Hamasu, Kousuke; Furuse, Mitsuhiro; Sugiyama, Hiroyuki

    2014-12-15

    Throughout life, we are exposed to a variety of stresses, which may be inevitable and noxious sometimes. During evolution, animals must have acquired some physiological means to counteract stress. Vascular endothelial growth factor (VEGF) is an angiogenic and neurogenic factor, which has been shown to elicit antidepressant-like effects in response to different external stimuli, potentially functioning as an anti-stress molecule. However, it remains largely unknown how VEGF modulates mood-related behaviors. To investigate molecular correlates, we analyzed monoaminergic systems of VEGF transgenic mice that display antidepressant-like behavior. Immunostaining showed that overall morphologies of monoaminergic nuclei and their processes were normal. However, we found imbalances in brain monoamine contents, in which the levels of norepinephrine and serotonin, but not dopamine, were decreased exclusively in the regions where VEGF was expressed. The turnover of norepinephrine showed a marked increase and serotonin turnover showed a modest reduction, whereas dopamine turnover was not affected. The protein levels of tyrosine hydroxylase and tryptophan hydroxylase, the rate-limiting enzymes of catecholamine and serotonin synthesis, remained constant. The mRNA levels of monoamine receptors were generally similar but adrenergic receptors of ADRα1A and ADRβ1 were down-regulated. Behavioral tests showed that serotonin- or norepinephrine-selective antidepressant drugs failed to additively enhance antidepressant-like behaviors, whereas monoamine depleting drugs attenuated VEGF-mediated antidepressant-like effect. These data suggest that VEGF-induced antidepressant-like effects involve modulation of norepinephrine and serotonin systems.

  5. Serum vascular endothelial growth factor (VEGF-C) as a diagnostic and prognostic marker in patients with ovarian cancer.

    PubMed

    Cheng, Daye; Liang, Bin; Li, Yunhui

    2013-01-01

    VEGF-C is regarded as one of the most efficient factors in regulating lymphangiogenesis. The aim of this study was to better understand the role of VEGF-C in the progression of ovarian cancer and to assess its diagnostic and prognostic significance. A total of 109 patients with ovarian