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Sample records for host vegf vegfr2

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

    SciTech Connect

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

    2008-11-01

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

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

    PubMed

    Lim, Sharon; Zhang, Yin; Zhang, Danfang; Chen, Fang; Hosaka, Kayoko; Feng, Ninghan; Seki, Takahiro; Andersson, Patrik; Li, Jingrong; Zang, Jingwu; Sun, Baocun; Cao, Yihai

    2014-10-23

    Molecular mechanisms underlying tumor VEGF-induced host anemia and bone marrow cell (BMC) mobilization remain unknown. Here, we report that tumor VEGF markedly induced sinusoidal vasculature dilation in bone marrow (BM) and BMC mobilization to tumors and peripheral tissues in mouse and human tumor models. Unexpectedly, anti-VEGFR2, but not anti-VEGFR1, treatment completely blocked VEGF-induced anemia and BMC mobilization. Genetic deletion of Vegfr2 in endothelial cells markedly ablated VEGF-stimulated BMC mobilization. Conversely, deletion of the tyrosine kinase domain from Vegfr1 gene (Vegfr1(TK-/-)) did not affect VEGF-induced BMC mobilization. Analysis of VEGFR1(+)/VEGFR2(+) populations in peripheral blood and BM showed no significant ratio difference between VEGF- and control tumor-bearing animals. These findings demonstrate that vascular dilation through the VEGFR2 signaling is the mechanism underlying VEGF-induced BM mobilization and anemia. Thus, our data provide mechanistic insights on VEGF-induced BMC mobilization in tumors and have therapeutic implications by targeting VEGFR2 for cancer therapy.

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  5. 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).

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

    PubMed Central

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

    2017-01-01

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

  7. 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.

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

    PubMed

    Rath, G; Tripathi, R

    2012-03-01

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

  9. 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.

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

    PubMed

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

    2017-02-09

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

  11. 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.

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

    PubMed

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

    2015-01-01

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

  13. 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.

  14. 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

  15. 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

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

    PubMed

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

    2006-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-04-05

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2017-03-08

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2006-12-08

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2017-01-05

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

  7. 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.

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

    PubMed Central

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

    2016-01-01

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

  9. 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.

  10. 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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2015-10-01

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

  15. 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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2005-04-01

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

  19. 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

  20. VEGFR2 Trafficking, Signaling and Proteolysis is Regulated by the Ubiquitin Isopeptidase USP8.

    PubMed

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

    2016-01-01

    Vascular endothelial growth factor A (VEGF-A) regulates many aspects of vascular function. VEGF-A binding to vascular endothelial growth factor receptor 2 (VEGFR2) stimulates endothelial signal transduction and regulates multiple cellular responses. Activated VEGFR2 undergoes ubiquitination but the enzymes that regulate this post-translational modification are unclear. In this study, the de-ubiquitinating enzyme, USP8, is shown to regulate VEGFR2 trafficking, de-ubiquitination, proteolysis and signal transduction. USP8-depleted endothelial cells displayed altered VEGFR2 ubiquitination and production of a unique VEGFR2 extracellular domain proteolytic fragment caused by VEGFR2 accumulation in the endosome-lysosome system. In addition, perturbed VEGFR2 trafficking impaired VEGF-A-stimulated signal transduction in USP8-depleted cells. Thus, regulation of VEGFR2 ubiquitination and de-ubiquitination has important consequences for the endothelial cell response and vascular physiology.

  1. 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

  2. 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...

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

    PubMed

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

    2012-01-01

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

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

    PubMed Central

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

    2017-01-01

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

  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. PMID:27602320

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

    SciTech Connect

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

    2005-08-01

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

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

    PubMed Central

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

    2012-01-01

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

  8. 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

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    2014-10-15

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

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

    PubMed

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

    2011-04-18

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

  12. The rs2071559 AA VEGFR-2 genotype frequency is significantly lower in neovascular age-related macular degeneration patients.

    PubMed

    Lazzeri, Stefano; Orlandi, Paola; Figus, Michele; Fioravanti, Anna; Cascio, Elisa; Di Desidero, Teresa; Agosta, Elisa; Canu, Bastianina; Sartini, Maria Sole; Danesi, Romano; Nardi, Marco; Bocci, Guido

    2012-01-01

    In this prospective, case-control genetic study, 120 consecutive neovascular age-related macular degeneration (AMD) cases and 78 controls were enrolled. Two SNPs (rs2071559 and rs1870377) of VEGF-A receptor-2 (VEGFR-2) gene were analyzed with the technique of Real-Time PCR to investigate a genetic link between AMD and VEGFR-2 gene polymorphisms in Italian patients. The frequency of the VEGFR-2 genotype rs2071559 AA was significantly lower (18.33%) in patients with AMD than in the control subjects (34.62%; P = 0.0095, chi-square test; P(corr) = 0.038; OR = 0.42, 95% CI 0.22 to 0.82). In conclusion, although with the limitations of a small sample size and the few SNPs studied, this study demonstrates a lower frequency of VEGFR-2 rs2071559 AA genotype in an AMD patient population, suggesting future studies on the role VEGFR-2 SNPs.

  13. The rs2071559 AA VEGFR-2 Genotype Frequency Is Significantly Lower in Neovascular Age-Related Macular Degeneration Patients

    PubMed Central

    Lazzeri, Stefano; Orlandi, Paola; Figus, Michele; Fioravanti, Anna; Cascio, Elisa; Di Desidero, Teresa; Agosta, Elisa; Canu, Bastianina; Sartini, Maria Sole; Danesi, Romano; Nardi, Marco; Bocci, Guido

    2012-01-01

    In this prospective, case-control genetic study, 120 consecutive neovascular age-related macular degeneration (AMD) cases and 78 controls were enrolled. Two SNPs (rs2071559 and rs1870377) of VEGF-A receptor-2 (VEGFR-2) gene were analyzed with the technique of Real-Time PCR to investigate a genetic link between AMD and VEGFR-2 gene polymorphisms in Italian patients. The frequency of the VEGFR-2 genotype rs2071559 AA was significantly lower (18.33%) in patients with AMD than in the control subjects (34.62%; P = 0.0095, chi-square test; Pcorr = 0.038; OR = 0.42, 95% CI 0.22 to 0.82). In conclusion, although with the limitations of a small sample size and the few SNPs studied, this study demonstrates a lower frequency of VEGFR-2 rs2071559 AA genotype in an AMD patient population, suggesting future studies on the role VEGFR-2 SNPs. PMID:22919317

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2017-01-01

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

  16. 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

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

    PubMed

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

    2016-12-20

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

  18. 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.

  19. 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.

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

    PubMed

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

    2017-03-01

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

  1. 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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2015-01-01

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

  4. 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.

  5. 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

  6. 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.

  7. 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

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

    PubMed

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

    2014-09-22

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2004-09-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-02-01

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

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

    PubMed

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

    2016-04-29

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

  14. 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

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

    PubMed

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

    2009-12-10

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

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

    PubMed

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

    2015-05-01

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

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

    PubMed Central

    Bazzazi, Hojjat; Popel, Aleksander S.

    2017-01-01

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

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

    PubMed

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

    2013-04-01

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

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

    PubMed

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

    2013-05-01

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

  20. VEGF as a Paracrine Regulator of Conventional Outflow Facility

    PubMed Central

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

    2017-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  2. 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.

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

    PubMed Central

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

    2011-01-01

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

  4. 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.

  5. 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

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

    PubMed

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

    2011-07-01

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

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

    PubMed

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

    2017-02-03

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

  8. 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.

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

    PubMed

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

    2003-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  11. 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.

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-09-22

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

  15. 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

  16. 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

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

    PubMed Central

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

    2014-01-01

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

  18. 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.

  19. 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.

  20. 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.

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

    PubMed

    Ma, Qin; Chen, Wei; Chen, Wen

    2016-05-01

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

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

    PubMed

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

    2009-11-01

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

  3. 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

  4. 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.

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

    PubMed

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

    2010-12-20

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

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Xing, Wenlu; Gao, Chuanyu; Qi, Datun; Zhang, You; Hao, Peiyuan; Dai, Guoyou; Yan, Ganxin

    2017-01-01

    The mechanisms of vascular endothelial growth factor 165 (VEGF165) on electrical properties of cardiomyocytes have not been fully elucidated. The aim of this study is to test the hypothesis that VEGF165, an angiogenesis-initiating factor, affects L-type calcium currents (ICa,L) and cell membrane potential in cardiac myocytes by acting on VEGF type-2 receptors (VEGFR2). ICa,L and action potentials (AP) were recorded by the whole-cell patch clamp method in isolated guinea-pig ventricular myocytes treated with different concentrations of VEGF165 proteins. Using a VEGFR2 inhibitor, we also tested the receptor of VEGF165 in cardiomyocytes. We found that VEGF165 increased ICa,L in a concentration-dependent manner. SU5416, a VEGFR2 inhibitor, almost completely eliminated VEGF165-induced ICa,L increase. VEGF165 had no significant influence on action potential 90 (APD90) and other properties of AP. We conclude that in guinea-pig ventricular myocytes, ICa,L can be increased by VEGF165 in a concentration-dependent manner through binding to VEGFR2 without causing any significant alteration to action potential duration. Results of this study may further expound the safety of VEGF165 when used in the intervention of heart diseases.

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

    PubMed

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

    2017-02-06

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

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

    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.

  12. 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

  13. 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.

  14. 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

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

    PubMed Central

    Zhang, Min; Jiang, Li

    2016-01-01

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

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

    PubMed

    Zhang, Min; Jiang, Li

    2016-12-01

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2015-10-20

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

  19. 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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2004-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2016-11-10

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

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

    PubMed Central

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

    2011-01-01

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

  5. 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.

  6. 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.

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

    PubMed

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

    2010-09-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-12-01

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

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

    PubMed Central

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

    2016-01-01

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

  11. 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.

  12. 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...

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed Central

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

    2017-01-01

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

  15. 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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  18. 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.

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

    PubMed

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

    2013-06-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2017-03-01

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

  2. Pharmacokinetics and pharmacodynamics of VEGF-neutralizing antibodies

    PubMed Central

    2011-01-01

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

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

    PubMed

    Dale, Erica A; Mitchell, Gordon S

    2013-02-01

    Vascular endothelial growth factor (VEGF) and erythropoietin (EPO) exert neurotrophic and neuroprotective effects in the CNS. We recently demonstrated that VEGF, EPO and their receptors (VEGF-R2, EPO-R) are expressed in phrenic motor neurons, and that cervical spinal VEGF-R2 and EPO-R activation elicit long-lasting phrenic motor facilitation (pMF). Since VEGF, VEGF-R, EPO, and EPO-R are hypoxia-regulated genes, and repetitive exposure to acute intermittent hypoxia (rAIH) up-regulates these molecules in phrenic motor neurons, we tested the hypothesis that 4 weeks of rAIH (10 episodes per day, 3 days per week) enhances VEGF- or EPO-induced pMF. We confirm that cervical spinal VEGF and EPO injections elicit pMF. However, neither VEGF- nor EPO-induced pMF was affected by rAIH pre-conditioning (4 wks). Although our data confirm that spinal VEGF and EPO may play an important role in respiratory plasticity, we provide no evidence that rAIH amplifies their impact. Further experiments with more robust protocols are warranted.

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

    PubMed

    Roskoski, Robert

    2017-03-19

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

  5. 1-Piperazinylphthalazines as potential VEGFR-2 inhibitors and anticancer agents: Synthesis and in vitro biological evaluation.

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

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

    2017-01-01

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

  8. 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.

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

    PubMed

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

    2013-06-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-12-06

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

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

    PubMed Central

    2011-01-01

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

  13. 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.

  14. 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.

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2016-12-01

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

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

    PubMed

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

    2014-01-01

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

  18. 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.

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

    PubMed

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

    2014-11-21

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

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

    PubMed Central

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

    2013-01-01

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

  1. 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

  2. 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

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

    NASA Astrophysics Data System (ADS)

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

    2015-08-01

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

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

    PubMed

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

    2015-07-01

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

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

    PubMed

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

    2011-09-01

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

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

    PubMed Central

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

    2013-01-01

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

  7. 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

  8. 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.

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

    PubMed

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

    2016-05-01

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

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

    PubMed

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

    2012-08-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2015-04-01

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

  13. 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

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

    PubMed

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

    2015-07-20

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

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

    PubMed Central

    Hu, Kai; Olsen, Bjorn R.

    2016-01-01

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

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

    PubMed Central

    2013-01-01

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

  18. 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.

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

    PubMed Central

    Mousavi, Marzieh; Baharara, Javad; Shahrokhabadi, Khadijeh

    2014-01-01

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

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

    PubMed

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

    2006-02-01

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

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2015-07-01

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

  4. 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.

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

    PubMed

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

    2013-03-22

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

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

    PubMed Central

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

    2005-01-01

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

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

    PubMed

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

    2005-07-06

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

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

    PubMed Central

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

    2012-01-01

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

  9. 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.

  10. Vascular endothelial (VEGF) and epithelial growth factor (EGF) as well as platelet-activating factor (PAF) and receptors are expressed in the early pregnant canine uterus.

    PubMed

    Schäfer-Somi, S; Sabitzer, S; Klein, D; Reinbacher, E; Kanca, H; Beceriklisoy, H B; Aksoy, O A; Kucukaslan, I; Macun, H C; Aslan, S

    2013-02-01

    The aim of this study was to investigate the course of expression of platelet-activating factor (PAF), PAF-receptor (PAF-R), epidermal growth factor (EGF), EGF-R, vascular endothelial growth factor (VEGF), VEGF-R1 and VEGF-R2 in uterine tissue during canine pregnancy. For this purpose, 20 bitches were ovariohysterectomized at days 10-12 (n = 10), 18-25 (n = 5) and 28-45 (n = 5) days after mating, respectively. The pre-implantation group was proven pregnant by embryo flushing of the uterus after the operation, the others by sonography. Five embryo negative, that is, non-pregnant, bitches in diestrus (day 10-12) served as controls. Tissue samples from the uterus (placentation sites and horn width, respectively) were excised and snap-frozen in liquid nitrogen after embedding in Tissue Tec(®). Extraction of mRNA for RT-PCR was performed with Tri-Reagent. In the embryos, mRNA from all factors except VEGF was detected. In the course of pregnancy, significantly higher expression of PAF and PAFR as well as VEGF and VEGFR2 during the pre-implantation stage than in all other stages and a strong upregulation of EGF during implantation were characteristic. The course of EGF was in diametrical opposition to the course of the receptor. These results point towards an increased demand for VEGF, EGF and PAF during the earliest stages of canine pregnancy.

  11. VEGF Trap complex formation measures production rates of VEGF, providing a biomarker for predicting efficacious angiogenic blockade

    PubMed Central

    Rudge, John S.; Holash, Jocelyn; Hylton, Donna; Russell, Michelle; Jiang, Shelly; Leidich, Raymond; Papadopoulos, Nicholas; Pyles, Erica A.; Torri, Al; Wiegand, Stanley J.; Thurston, Gavin; Stahl, Neil; Yancopoulos, George D.

    2007-01-01

    VEGF is the best characterized mediator of tumor angiogenesis. Anti-VEGF agents have recently demonstrated impressive efficacy in human cancer trials, but the optimal dosing of such agents must still be determined empirically, because biomarkers to guide dosing have yet to be established. The widely accepted (but unverified) assumption that VEGF production is quite low in normal adults led to the notion that increased systemic VEGF levels might quantitatively reflect tumor mass and angiogenic activity. We describe an approach to determine host and tumor production of VEGF, using a high-affinity and long-lived VEGF antagonist now in clinical trials, the VEGF Trap. Unlike antibody complexes that are usually rapidly cleared, the VEGF Trap forms inert complexes with tissue- and tumor-derived VEGF that remain stably in the systemic circulation, where they are readily assayable, providing unprecedented capability to accurately measure VEGF production. We report that VEGF production is surprisingly high in non-tumor-bearing rodents and humans, challenging the notion that systemic VEGF levels can serve as a sensitive surrogate for tumor load; tumor VEGF contribution becomes significant only with very large tumor loads. These findings have the important corollary that anti-VEGF therapies must be sufficiently dosed to avoid diversion by host-derived VEGF. We further show that our assay can indicate when VEGF is optimally blocked; such biomarkers to guide dosing do not exist for other anti-VEGF agents. Based on this assay, VEGF Trap doses currently being assessed in clinical trials are in the efficacious range. PMID:18000042

  12. The novel VEGF receptor 2 inhibitor YLL545 inhibits angiogenesis and growth in breast cancer

    PubMed Central

    Zhang, Jianbo; Liu, Chen; Shi, Wen; Yang, Lingling; Zhang, Quansheng; Cui, Jianlin; Fang, Yangwu; Li, Yuhao; Ren, Guosheng; Yang, Shuang; Xiang, Rong

    2016-01-01

    Their antiangiogenic effects make vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors useful for cancer treatment. However, most of these drugs have unexpected adverse side effects. Here, we show that the novel VEGFR2 inhibitor YLL545 suppressed tumor angiogenesis and growth in triple-negative breast cancer without adverse effects. YLL545 treatment also markedly inhibited proliferation, migration, invasion, and tube formation by human umbilical vascular endothelial cells (HUVECs) in vitro. These effects of YLL545 were equal to or greater than those seen with sorafenib. In addition, YLL545 inhibited VEGF-induced phosphorylation of VEGFR2 and activation of downstream signaling regulators, such as phospho-STAT3 and phospho-ERK1/2, in HUVECs. Embryonic angiogenesis assays in zebrafish and Matrigel plug assays in mice demonstrated that YLL545 inhibits angiogenesis in vivo. YLL545 also inhibited proliferation and induced apoptosis in MDA-MB-231 breast cancer cells both in vitro and in vivo, and 50 mg/kg/d YLL545 inhibited human tumor xenograft growth by more than 50% in BALB/c nude mice. These observations suggest YLL545 is a potentially useful anticancer drug candidate. PMID:27203384

  13. Experimental studies of a vaccine formulation of recombinant human VEGF antigen with aluminum phosphate

    PubMed Central

    Pérez Sánchez, Lincidio; Morera Díaz, Yanelys; Bequet-Romero, Mónica; Ramses Hernández, Gerardo; Rodríguez, Yadira; Castro Velazco, Jorge; Puente Pérez, Pedro; Ayala Avila, Marta; Gavilondo, Jorge V

    2015-01-01

    CIGB-247 is a cancer vaccine that is a formulation of a recombinant protein antigen representative of the human vascular endothelial growth factor (VEGF) with a bacterially-derived adjuvant (VSSP). The vaccine has shown an excellent safety profile in mice, rats, rabbits, not-human primates and in recent clinical trials in cancer patients. Response to the vaccine is characterized by specific antibody titers that neutralize VEGF/VEGFR2 binding and a cytotoxic tumor-specific response. To expand our present anti-VEGF active immunotherapy strategies, we have now studied in mice and non-human primates the effects of vaccination with a formulation of our recombinant VEGF antigen and aluminum phosphate adjuvant (hereafter denominated CIGB-247-A). Administered bi-weekly, CIGB-247-A produces high titers of anti-VEGF IgG blocking antibodies in 2 mice strains. Particularly in BALB/c, the treatment impaired subcutaneous F3II mammary tumor growth and reduced the number of spontaneous lung macro metastases, increasing animals' survival. Spleen cells from specifically immunized mice directly killed F3II tumor cells in vitro. CIGB-247-A also showed to be immunogenic in non-human primates, which developed anti-VEGF blocking antibodies and the ability for specific direct cell cytotoxic responses, all without impairing the healing of deep skin wounds or other side effect. Our results support consideration of aluminum phosphate as a suitable adjuvant for the development of new vaccine formulations using VEGF as antigen. PMID:25891359

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

    PubMed Central

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

    2015-01-01

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

  15. A Two-Compartment Model of VEGF Distribution in the Mouse

    PubMed Central

    Engel-Stefanini, Marianne O.; Popel, Aleksander S.

    2011-01-01

    Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis – the growth of new microvessels from existing microvasculature. Angiogenesis is a complex process involving numerous molecular species, and to better understand it, a systems biology approach is necessary. In vivo preclinical experiments in the area of angiogenesis are typically performed in mouse models; this includes drug development targeting VEGF. Thus, to quantitatively interpret such experimental results, a computational model of VEGF distribution in the mouse can be beneficial. In this paper, we present an in silico model of VEGF distribution in mice, determine model parameters from existing experimental data, conduct sensitivity analysis, and test the validity of the model. The multiscale model is comprised of two compartments: blood and tissue. The model accounts for interactions between two major VEGF isoforms (VEGF120 and VEGF164) and their endothelial cell receptors VEGFR-1, VEGFR-2, and co-receptor neuropilin-1. Neuropilin-1 is also expressed on the surface of parenchymal cells. The model includes transcapillary macromolecular permeability, lymphatic transport, and macromolecular plasma clearance. Simulations predict that the concentration of unbound VEGF in the tissue is approximately 50-fold greater than in the blood. These concentrations are highly dependent on the VEGF secretion rate. Parameter estimation was performed to fit the simulation results to available experimental data, and permitted the estimation of VEGF secretion rate in healthy tissue, which is difficult to measure experimentally. The model can provide quantitative interpretation of preclinical animal data and may be used in conjunction with experimental studies in the development of pro- and anti-angiogenic agents. The model approximates the normal tissue as skeletal muscle and includes endothelial cells to represent the vasculature. As the VEGF system becomes better characterized in other tissues and

  16. Basal and apical regulation of VEGF-A and placenta growth factor in the RPE/choroid and primary RPE

    PubMed Central

    Kaya, Leyla; Flach, Janina; Lassen, Jens; Treumer, Felix; Roider, Johann

    2015-01-01

    Purpose Members of the vascular endothelial growth factor (VEGF) family are strongly involved in pathological processes in the retina, such as age-related macular degeneration and diabetic retinopathy. Cells of the retinal pigment epithelium (RPE) constitutively secrete VEGF-A, and the secretion of placental growth factor (PlGF) has also been described. RPE cells are strongly polarized cells with different secretome at the apical and basal side. In this study, we evaluated the basal and apical regulation of VEGF-A and PlGF secretion in RPE/choroid explants and primary RPE cells. Methods RPE/choroid tissue explants were prepared from porcine eyes and cultivated in modified Ussing chambers, separating apical (RPE) and basal (choroid) supernatant. Primary RPE cells were also prepared from porcine eyes and cultivated on Transwell plates. Explants and cells were treated with inhibitors for VEGFR-2 (SU1498), p38 (SB203580), and the transcription factors nuclear factor-kappa B (NF-κB) and SP-1 (mithramycin), respectively. VEGF-A and PlGF content was evaluated with enzyme-linked immunosorbent assay (ELISA). In addition, western blots were performed. Results In the RPE/choroid, VEGF-A can initially be found on the apical and basal sides with significantly more pronounced secretion on the basal side. VEGF-A secretion is differentially regulated on the apical and basal sides, with the inhibition of SP-1 and NF-κB showing strong effects apically and basally after 24 h and 48 h, the inhibition of p38 displaying its effect mainly on the basal side with some effect apically after 48 h, and the inhibition of VEGFR-2 reducing the secretion of VEGF only on the apical side at 24 h and 48 h. In the RPE cell culture, similar effects were found, with inhibition of NF-κB or SP-1 displaying a strong decrease in VEGF-A on both sides, and p38 inhibition displaying only an inhibitory effect on the basal side. In contrast, an apical effect of VEGFR-2 inhibition was not found. However, the

  17. Anti-tumour activity of tivozanib, a pan-inhibitor of VEGF receptors, in therapy-resistant ovarian carcinoma cells

    PubMed Central

    Momeny, Majid; Sabourinejad, Zahra; Zarrinrad, Ghazaleh; Moghaddaskho, Farima; Eyvani, Haniyeh; Yousefi, Hassan; Mirshahvaladi, Shahab; Poursani, Ensieh M.; Barghi, Farinaz; Poursheikhani, Arash; Dardaei, Leila; Bashash, Davood; Ghazi-Khansari, Mahmoud; Tavangar, Seyyed M.; Dehpour, Ahmad R.; Yaghmaie, Marjan; Alimoghaddam, Kamran; Ghavamzadeh, Ardeshir; Ghaffari, Seyed H.

    2017-01-01

    Epithelial ovarian cancer (EOC) is the most fatal gynaecological malignancy. Despite initial therapeutic response, the majority of advanced-stage patients relapse and succumb to chemoresistant disease. Overcoming drug resistance is the key to successful treatment of EOC. Members of vascular endothelial growth factor (VEGF) family are overexpressed in EOC and play key roles in its malignant progression though their contribution in development of the chemoresistant disease remains elusive. Here we show that expression of the VEGF family is higher in therapy-resistant EOC cells compared to sensitive ones. Overexpression of VEGFR2 correlated with resistance to cisplatin and combination with VEGFR2-inhibitor apatinib synergistically increased cisplatin sensitivity. Tivozanib, a pan-inhibitor of VEGF receptors, reduced proliferation of the chemoresistant EOC cells through induction of G2/M cell cycle arrest and apoptotic cell death. Tivozanib decreased invasive potential of these cells, concomitant with reduction of intercellular adhesion molecule-1 (ICAM-1) and diminishing the enzymatic activity of urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-2 (MMP-2). Moreover, tivozanib synergistically enhanced anti-tumour effects of EGFR-directed therapies including erlotinib. These findings suggest that the VEGF pathway has potential as a therapeutic target in therapy-resistant EOC and VEGFR blockade by tivozanib may yield stronger anti-tumour efficacy and circumvent resistance to EGFR-directed therapies. PMID:28383032

  18. Inhibition of VEGF induces cellular senescence in colorectal cancer cells.

    PubMed

    Hasan, Mohammad R; Ho, Shirley H Y; Owen, David A; Tai, Isabella T

    2011-11-01

    Vascular endothelial growth factor (VEGF) inhibitors, such as bevacizumab, have improved outcomes in metastatic colorectal cancer (CRC). Recent studies have suggested that VEGF can delay the onset of cellular senescence in human endothelial cells. As VEGF receptors are known to be upregulated in CRC, we hypothesized that VEGF inhibition may directly influence cellular senescence in this disease. In our study, we observed that treatment with bevacizumab caused a significant increase (p < 0.05) in cellular senescence in vitro in several CRC cells, such as MIP101, RKO, SW620 and SW480 cells, compared to untreated or human IgG-treated control cells. Similar results were also obtained from cells treated with a VEGFR2 kinase inhibitor Ki8751. In vivo, cellular senescence was detected in MIP101 tumor xenografts from 75% of mice treated with bevacizumab, while cellular senescence was undetectable in xenografts from mice treated with saline or human IgG (p < 0.05). Interestingly, we also observed that the proportion of senescent cells in colon cancer tissues obtained from patients treated with bevacizumab was 4.4-fold higher (p < 0.01) than those of untreated patients. To understand how VEGF inhibitors may regulate cellular senescence, we noted that among the two important regulators of senescent growth arrest of tumor cells, bevacizumab-associated increase in cellular senescence coincided with an upregulation of p16 but appeared to be independent of p53. siRNA silencing of p16 gene in MIP101 cells suppressed bevacizumab-induced cellular senescence, while silencing of p53 had no effect. These findings demonstrate a novel antitumor activity of VEGF inhibitors in CRC, involving p16.

  19. 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

  20. Insights from Genetic Model Systems of Retinal Degeneration: Role of Epsins in Retinal Angiogenesis and VEGFR2 Signaling

    PubMed Central

    Wu, Yong; Liu, Yanjun; Deng, Lin; Chen, Hong

    2017-01-01

    The retina is a light sensitive tissue that contains specialized photoreceptor cells called rods and cones which process visual signals. These signals are relayed to the brain through interneurons and the fibers of the optic nerve. The retina is susceptible to a variety of degenerative diseases, including age-related macular degeneration (AMD), diabetic retinopathy (DR), retinitis pigmentosa (RP) and other inherited retinal degenerations. In order to reveal the mechanism underlying these diseases and to find methods for the prevention/treatment of retinal degeneration, animal models have been generated to mimic human eye diseases. In this paper, several well-characterized and commonly used animal models are reviewed. Of particular interest are the contributions of these models to our understanding of the mechanisms of retinal degeneration and thereby providing novel treatment options including gene therapy, stem cell therapy, nanomedicine, and CRISPR/Cas9 genome editing. Role of newly-identified adaptor protein epsins from our laboratory is discussed in retinal angiogenesis and VEGFR2 signaling. PMID:28191500

  1. αB-crystallin regulation of angiogenesis by modulation of VEGF

    PubMed Central

    Kase, Satoru; He, Shikun; Sonoda, Shozo; Kitamura, Mizuki; Spee, Christine; Wawrousek, Eric; Ryan, Stephen J.; Kannan, Ram

    2010-01-01

    αB-crystallin is a chaperone belonging to the small heat shock protein family. Herein we show attenuation of intraocular angiogenesis in αB-crystallin knockout (αB-crystallin−/−) mice in 2 models of intraocular disease: oxygen-induced retinopathy and laser-induced choroidal neovascularization. Vascular endothelial growth factor A (VEGF-A) mRNA and hypoxia inducible factor-1α protein expression were induced during retinal angiogenesis, but VEGF-A protein expression remained low in αB-crystallin−/− retina versus wild-type mice, whereas VEGF-R2 expression was not affected. Both αB-crystallin and its phosphorylated serine59 formwere expressed, and immunoprecipitation revealed αB-crystallin binding to VEGF-A but not transforming growth factor-β in cultured retinal pigment epithelial (RPE) cells. αB-crystallin and VEGF-A are colocalized in the endoplasmic reticulum in RPE cells under chemical hypoxia. αB-crystallin−/− RPE showed low VEGF-A secretion under serum-starved conditions compared with wild-type cells. VEGF-A is polyubiquitinated in control and αB-crystallin siRNA treated RPE; however, mono-tetra ubiquitinated VEGF-A increases with αB-crystallin knockdown. Endothelial cell apoptosis in newly formed vessels was greater in αB-crystallin−/− than wild-type mice. Proteasomal inhibition in αB-crystallin−/− mice partially restores VEGF-A secretion and angiogenic phenotype in choroidal neovascularization. Our studies indicate an important role for αB-crystallin as a chaperone for VEGF-A in angiogenesis and its potential as a therapeutic target. PMID:20023214

  2. 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

  3. Contrast-Enhanced Ultrasound with VEGFR2-Targeted Microbubbles for Monitoring Regorafenib Therapy Effects in Experimental Colorectal Adenocarcinomas in Rats with DCE-MRI and Immunohistochemical Validation

    PubMed Central

    Clevert, Dirk-Andre; Hirner-Eppeneder, Heidrun; Ingrisch, Michael; Moser, Matthias; Schuster, Jessica; Tadros, Dina; Schneider, Moritz; Kazmierczak, Philipp Maximilian; Reiser, Maximilian; Cyran, Clemens C.

    2017-01-01

    Objectives To investigate contrast-enhanced ultrasound (CEUS) with VEGFR2-targeted microbubbles for monitoring therapy effects of regorafenib on experimental colon carcinomas in rats with correlation to dynamic contrast-enhanced MRI (DCE-MRI) and immunohistochemistry. Materials and Methods Human colorectal adenocarcinoma xenografts (HT-29) were implanted subcutaneously in n = 21 (n = 11 therapy group; n = 10 control group) female athymic nude rats (Hsd: RH-Foxn1rnu). Animals were imaged at baseline and after a one-week daily treatment with regorafenib or a placebo (10 mg/kg bodyweight), using CEUS with VEGFR2-targeted microbubbles and DCE-MRI. In CEUS tumor perfusion was assessed during an early vascular phase (wash-in area under the curve = WiAUC) and VEGFR2-specific binding during a late molecular phase (signal intensity after 8 (SI8min) and 10 minutes (SI10min)), using a conventional 15L8 linear transducer (transmit frequency 7 MHz, dynamic range 80 dB, depth 25 mm). In DCE-MRI functional parameters plasma flow (PF) and plasma volume (PV) were quantified. For validation purposes, CEUS parameters were correlated with DCE-MRI parameters and immunohistochemical VEGFR2, CD31, Ki-67 and TUNEL stainings. Results CEUS perfusion parameter WiAUC decreased significantly (116,989 ± 77,048 a.u. to 30,076 ± 27,095a.u.; p = 0.005) under therapy with no significant changes (133,932 ± 65,960 a.u. to 84,316 ± 74,144 a.u.; p = 0.093) in the control group. In the therapy group, the amount of bound microbubbles in the late phase was significantly lower in the therapy than in the control group on day 7 (SI8min: 283 ± 191 vs. 802 ± 460 a.u.; p = 0.006); SI10min: 226 ± 149 vs. 645 ± 461 a.u.; p = 0.009). PF and PV decreased significantly (PF: 147 ± 58 mL/100 mL/min to 71 ± 15 mL/100 mL/min; p = 0.003; PV: 13 ± 3% to 9 ± 4%; p = 0.040) in the therapy group. Immunohistochemistry revealed significantly fewer VEGFR2 (7.2 ± 1.8 vs. 17.8 ± 4.6; p < 0.001), CD31 (8.1 ± 3.0 vs

  4. A compartment model of VEGF distribution in blood, healthy and diseased tissues

    PubMed Central

    Stefanini, Marianne O; Wu, Florence TH; Mac Gabhann, Feilim; Popel, Aleksander S

    2008-01-01

    Background Angiogenesis is a process by which new capillaries are formed from pre-existing blood vessels in physiological (e.g., exercise, wound healing) or pathological (e.g., ischemic limb as in peripheral arterial disease, cancer) contexts. This neovascular mechanism is mediated by the vascular endothelial growth factor (VEGF) family of cytokines. Although VEGF is often targeted in anti-angiogenic therapies, there is little knowledge about how its concentration may vary between tissues and the vascular system. A compartment model is constructed to study the VEGF distribution in the tissue (including matrix-bound, cell surface receptor-bound and free VEGF isoforms) and in the blood. We analyze the sensitivity of this distribution to the secretion rate, clearance rate and vascular permeability of VEGF. Results We find that, in a physiological context, VEGF concentration varies approximately linearly with the VEGF secretion rate. VEGF concentration in blood but not in tissue is dependent on the vascular permeability of healthy tissue. Model simulations suggest that relative VEGF increases are similar in blood and tissue during exercise and return to baseline within several hours. In a pathological context (tumor), we find that blood VEGF concentration is relatively insensitive to increased vascular permeability in tumors, to the secretion rate of VEGF by tumors and to the clearance. However, it is sensitive to the vascular permeability in the healthy tissue. Finally, the VEGF distribution profile in healthy tissue reveals that about half of the VEGF is complexed with the receptor tyrosine kinase VEGFR2 and the co-receptor Neuropilin-1. In diseased tissues, this binding can be reduced to 15% while VEGF bound to the extracellular matrix and basement membranes increases. Conclusion The results are of importance for physiological conditions (e.g., exercise) and pathological conditions (e.g., peripheral arterial disease, coronary artery disease, cancer). This

  5. 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

  6. Effect of semax and its C-terminal fragment Pro-Gly-Pro on the expression of VEGF family genes and their receptors in experimental focal ischemia of the rat brain.

    PubMed

    Medvedeva, Ekaterina V; Dmitrieva, Veronika G; Povarova, Oksana V; Limborska, Svetlana A; Skvortsova, Veronika I; Myasoedov, Nikolay F; Dergunova, Lyudmila V

    2013-02-01

    The synthetic peptide Semax (Met-Glu-His-Phe-Pro-Gly-Pro) is used successfully in acute stroke therapy. In spite of numerous studies on the subject, many aspects of the neuroprotective effects of the peptide remain unknown. We studied the action of Semax and its C-terminal tripeptide Pro-Gly-Pro on the expression of the VEGF gene family (Vegf-a, Vegf-b, Vegf-c, Vegf-d, and Plgf) and their receptors (Vegfr-1, Vegfr-2, and Vegfr-3) in the frontoparietal cortex region of the rat brain at 3, 24, and 72 h after permanent left middle cerebral artery occlusion (pMCAO). The relative mRNA level of the genes studied was assessed using real-time reverse transcription-PCR. The Vegf-b and Vegf-d genes were most affected by the peptides, which resulted in their most noticeable activation at 3 h after pMCAO. The level of Vegf-d transcripts decreased considerably, whereas the mRNA level of the Vegf-b gene was significantly increased after 72 h of treatment with each of the peptides. In addition, the effects of the peptides on the expression of the Vegf-b and Vegf-d genes were the opposite of the action of ischemia. It is suggested that the identified effects of the peptides diminish the effects of ischemia, thus participating in the positive therapeutic effect of Semax on ischemic stroke.

  7. DC electric stimulation upregulates angiogenic factors in endothelial cells through activation of VEGF receptors

    PubMed Central

    Bai, Huai; Forrester, John V.; Zhao, Min

    2015-01-01

    Small direct current (DC) electric fields direct some important angiogenic responses of vascular endothelial cells. Those responses indicate promising use of electric fields to modulate angiogenesis. We sought to determine the regulation of electric fields on transcription and expression of a serial of import angiogenic factors by endothelial cells themselves. Using semi-quantitative PCR and ELISA we found that electric stimulation upregulates the levels of mRNAs and proteins of a number of angiogenic proteins, most importantly VEGF165, VEGF121 and IL-8 in human endothelial cells. The up-regulation of mRNA levels might be specific, as the mRNA encoding bFGF, TGF-beta and eNOS are not affected by DC electric stimulation at 24 h time-point. Inhibition of VEGF receptor (VEGFR1 or VEGFR2) signaling significantly decreased VEGF production and completely abolished IL-8 production. DC electric stimulation selectively regulates production of some growth factors and cytokines important for angiogenesis through a feed-back loop mediated by VEGF receptors. PMID:21524919

  8. DC electric stimulation upregulates angiogenic factors in endothelial cells through activation of VEGF receptors.

    PubMed

    Bai, Huai; Forrester, John V; Zhao, Min

    2011-07-01

    Small direct current (DC) electric fields direct some important angiogenic responses of vascular endothelial cells. Those responses indicate promising use of electric fields to modulate angiogenesis. We sought to determine the regulation of electric fields on transcription and expression of a serial of import angiogenic factors by endothelial cells themselves. Using semi-quantitative PCR and ELISA we found that electric stimulation upregulates the levels of mRNAs and proteins of a number of angiogenic proteins, most importantly VEGF165, VEGF121 and IL-8 in human endothelial cells. The up-regulation of mRNA levels might be specific, as the mRNA encoding bFGF, TGF-beta and eNOS are not affected by DC electric stimulation at 24h time-point. Inhibition of VEGF receptor (VEGFR1 or VEGFR2) signaling significantly decreased VEGF production and completely abolished IL-8 production. DC electric stimulation selectively regulates production of some growth factors and cytokines important for angiogenesis through a feed-back loop mediated by VEGF receptors.

  9. 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.

  10. 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.

  11. 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

  12. Fragment-based discovery of a dual pan-RET/VEGFR2 kinase inhibitor optimized for single-agent polypharmacology1

    PubMed Central

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

    2015-01-01

    Oncogenic conversion of the RET (rearranged during transfection) tyrosine kinase is associated with several cancers. A fragment-based chemical screen lead to the identification of a novel RET inhibitor, Pz-1. Modeling and kinetic analysis identified Pz-1 as a Type-II tyrosine kinase inhibitor, able to bind the DFG-out conformation of the kinase. Importantly, from a single-agent polypharmacology standpoint, Pz-1 was shown active on VEGFR2, which can block blood supply required for RET-stimulated growth. In cell based assays, 1.0 nM of Pz-1 strongly inhibited phosphorylation of all tested RET oncoproteins. At 1.0 mg/kg/day per os, Pz-1 abrogated formation of tumors induced by RET-mutant fibroblasts and blocked phosphorylation of both RET and VEGFR2 in tumor tissue. Pz-1 featured no detectable toxicity up to 100.0 mg/kg, which indicated a large therapeutic window. This study validates the effectiveness and usefulness of a medicinal chemistry polypharmacology approach to obtain an inhibitor capable of targeting multiple oncogenic pathways PMID:26126987

  13. Jaceosidin, a natural flavone, promotes angiogenesis via activation of VEGFR2/FAK/PI3K/AKT/NF-κB signaling pathways in endothelial cells.

    PubMed

    Lee, Tae Hoon; Jung, Hana; Park, Keun Hyung; Bang, Myun Ho; Baek, Nam-In; Kim, Jiyoung

    2014-10-01

    Angiogenesis, the growth of new blood vessels from pre-existing vasculature, plays an important role in physiological and pathological processes such as embryonic development wound healing and revascularization of tissues after exposure to ischemia. We investigated the effects of jaceosidin, a main constituent of medicinal herbs of the genus Artemisia, on angiogenesis and signaling pathways in endothelial cells. Jaceosidin stimulated proliferation, migration and tubulogenesis of ECs as well as ex vivo sprouting from aorta rings, which are phenomena typical of angiogenesis. Jaceosidin activated vascular endothelial growth factor receptor 2 (VEGFR2, FLk-1/KDR) and angiogenic signaling molecules such as focal adhesion kinase, phosphatidylinositol 3-kinase, and its downstream target, the serine-threonine kinase AKTWe also demonstrated that jaceosidin activated the NF-κB-driven expression of a luciferase reporter gene and NF-κB binding to DNA. Jaceosidin-induced proliferation and migration of human umbilical vascular endothelial cells were strongly inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002 and NF-κB inhibitor BAY11-7082, indicating that the PI3K/AKT/NF-κB signaling pathway is involved in jaceosidin-induced angiogenesis. Our results suggest that jaceosidin stimulates angiogenesis by activating the VEGFR2/FAK/PI3K/AKT/NF-κB signaling pathway and that it may be useful in developing angiogenic agents to promote the growth of collateral blood vessels in ischemic tissues.

  14. Fibroblast Growth Factor Receptor 1 (FGFR1), Partly Related to Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) and Microvessel Density, is an Independent Prognostic Factor for Non-Small Cell Lung Cancer

    PubMed Central

    Pu, Dan; Liu, Jiewei; Li, Zhixi; Zhu, Jiang; Hou, Mei

    2017-01-01

    Background This study aimed to explore the correlation between FGFR1 and clinical features, including survival analysis and the promotion of angiogenesis by fibroblast growth factor receptor 1 (FGFR1) and vascular endothelial growth factor receptor 2 (VEGFR2). FGFR1 gene amplification has been found in non-small cell lung cancer (NSCLC). However, the prognostic value of FGFR1 and the correlation between FGFR1 and clinical features are still controversial. Material/Methods A total of 92 patients with NSCLC who underwent R0 resection between July 2006 and July 2008 were enrolled in the study. The expression of FGFR1, VEGFR2, and CD34 was detected by immunohistochemistry. The correlations between the aforementioned markers and the patients’ clinical features were analyzed by the chi-square test. The impact factors of prognosis were evaluated by Cox regression analyses. Results The expression ratios of FGFR1 and VEGFR2 were 26.1% and 43.4%, respectively. The intensity of FGFR1 expression was related to VEGFR2 and histopathology. To some extent, the average microvessel density (MVD) had correlation to the expression of FGFR1 and VGEFR2. The pathological stages III–IV and high expression of FGFR1 were found to be independent prognostic factors. Conclusions The expression intensity of FGFR1 and VEGFR2 was associated with MVD, and the expression of FGFR1 is one of the independent prognostic indicators for NSCLC. PMID:28088809

  15. VEGF receptor inhibition blocks liver cyst growth in pkd2(WS25/-) mice.

    PubMed

    Amura, Claudia R; Brodsky, Kelley S; Groff, Rachel; Gattone, Vincent H; Voelkel, Norbert F; Doctor, R Brian

    2007-07-01

    Proliferation of cyst-lining epithelial cells is an integral part of autosomal dominant polycystic kidney disease (ADPKD) cyst growth. Cytokines and growth factors within cyst fluids are positioned to induce cyst growth. Vascular endothelial growth factor (VEGF) is a pleiotropic growth factor present in ADPKD liver cyst fluids (human 1,128 +/- 78, mouse 2,787 +/- 136 pg/ml) and, to a lesser extent, in ADPKD renal cyst fluids (human 294 +/- 41, mouse 191 +/- 90 pg/ml). Western blotting showed that receptors for VEGF (VEGFR1 and VEGFR2) were present in both normal mouse bile ducts and pkd2(WS25/-) liver cyst epithelial cells. Treatment of pkd2(WS25/-) liver cyst epithelial cells with VEGF (50-50,000 pg/ml) or liver cyst fluid induced a proliferative response. The effect on proliferation of liver cyst fluid was inhibited by SU-5416, a potent VEGF receptor inhibitor. Treatment of pkd2(WS25/-) mice between 4 and 8 mo of age with SU-5416 markedly reduced the cyst volume density of the liver (vehicle 9.9 +/- 4.3%, SU-5416 1.8 +/- 0.7% of liver). SU-5416 treatment between 4 and 12 mo of age markedly protected against increases in liver weight [pkd2(+/+) 4.8 +/- 0.2%, pkd2(WS25/-)-vehicle 10.8 +/- 1.9%, pkd2(WS25/-)-SU-5416 4.8 +/- 0.4% body wt]. The capacity of VEGF signaling to induce in vitro proliferation of pkd2(WS25/-) liver cyst epithelial cells and inhibition of in vivo VEGF signaling to retard liver cyst growth in pkd2(WS25/-) mice indicates that the VEGF signaling pathway is a potentially important therapeutic target in the treatment of ADPKD liver cyst disease.

  16. 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.

  17. The Increased Expression of Connexin and VEGF in Mouse Ovarian Tissue Vitrification by Follicle Stimulating Hormone.

    PubMed

    Yang, Yanzhou; Chen, Jie; Wu, Hao; Pei, Xiuying; Chang, Qing; Ma, Wenzhi; Ma, Huiming; Hei, Changchun; Zheng, Xiaomin; Cai, Yufang; Zhao, Chengjun; Yu, Jia; Wang, Yanrong

    2015-01-01

    Ovarian follicular damages were caused by cryoinjury during the process of ovarian vitrification and ischemia/reperfusion during the process of ovarian transplantation. And appropriate FSH plays an important role in antiapoptosis during ovarian follicle development. Therefore, in this study, 0.3 IU/mL FSH was administered into medium during mouse ovarian cryopreservation by vitrification to ascertain the function of FSH on ovarian vitrification and avascular transplantation. The results suggested that the expressions of Cx37, Cx43, apoptotic molecular caspase-3, and angiogenesis molecular VEGF were confirmed using immunohistochemistry, western blotting, and real-time PCR, and the results suggested that the treatment with FSH remarkably increased the number of morphologically normal follicles in vitrified/warmed ovaries by upregulating the expression of Cx37, Cx43, VEGF, and VEGF receptor 2, but downregulating the expression of caspase-3. In addition, the vitrified/warmed ovaries were transplanted, and the related fertility was analyzed, and the results suggested that the fertility, neoangiogenesis, and follicle reserve were remarkably increased in the FSH administrated group. Taken together, administration of 0.3 IU/mL FSH during ovarian cryopreservation by vitrification can maintain ovarian survival during ovarian vitrification and increases the blood supply with avascular transplantation via upregulation of Cx43, Cx37, and VEGF/VEGFR2, as well as through its antiapoptotic effects.

  18. 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

  19. 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.

  20. 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

  1. Galectin-3 disruption impaired tumoral angiogenesis by reducing VEGF secretion from TGFβ1-induced macrophages

    PubMed Central

    Machado, Camila Maria Longo; Andrade, Luciana Nogueira Sousa; Teixeira, Verônica Rodrigues; Costa, Fabrício Falconi; Melo, Camila Morais; dos Santos, Sofia Nascimento; Nonogaki, Suely; Liu, Fu-Tong; Bernardes, Emerson Soares; Camargo, Anamaria Aranha; Chammas, Roger

    2014-01-01

    In order to study the role of galectin-3 in tumor angiogenesis associated with tumor-associated macrophages (TAM) and tumor parenchyma, the galectin-3 expression was reconstituted in Tm1 melanoma cell line that lacks this protein. Galectin-3-expressing cells (Tm1G3) and mock-vector transfected cells (Tm1N3) were injected into wild-type (WT) and galectin-3 knockout (KO) C57Bl/6 mice. Tumors originated from Tm1G3 were larger in tumor volume with enlarged functional vessels, decreased necrotic areas, and increased vascular endothelial growth factor (VEGF) protein levels. Galectin-3-nonexpressing-cells injected into WT and KO showed increased levels of transforming growth factor beta 1 (TGFβ1) and, in WT animals this feature was also accompanied by increased VEGFR2 expression and its phosphorylation. In KO animals, tumors derived from galectin-3-expressing cells were infiltrated by CD68+-cells, whereas in tumors derived from galectin-3-nonexpressing-cells, CD68+ cells failed to infiltrate tumors and accumulated in the periphery of the tumor mass. In vitro studies showed that Tm1G3 secreted more VEGF than Tm1N3 cells. In the latter case, TGFβ1 induced VEGF production. Basal secretion of VEGF was higher in WT-bone marrow-derived macrophages (BMDM) than in KO-BMDM. TGFβ1 induced secretion of VEGF only in WT-BMDM. Tm1G3-induced tumors had the Arginase I mRNA increased, which upregulated alternative macrophage (M2)/TAM induction. M2 stimuli, such as interleukin-4 (IL4) and TGFβ1, increased Arginase I protein levels and galectin-3 expression in WT- BMDM, but not in cells from KO mice. Hence, we report that galectin-3 disruption in tumor stroma and parenchyma decreases angiogenesis through interfering with the responses of macrophages to the interdependent VEGF and TGFβ1 signaling pathways. PMID:24421272

  2. 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

  3. 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.

  4. TP53 Alterations Correlate with Response to VEGF/VEGFR Inhibitors: Implications for Targeted Therapeutics.

    PubMed

    Wheler, Jennifer J; Janku, Filip; Naing, Aung; Li, Yali; Stephen, Bettzy; Zinner, Ralph; Subbiah, Vivek; Fu, Siqing; Karp, Daniel; Falchook, Gerald S; Tsimberidou, Apostolia M; Piha-Paul, Sarina; Anderson, Roosevelt; Ke, Danxia; Miller, Vincent; Yelensky, Roman; Lee, J Jack; Hong, David; Kurzrock, Razelle

    2016-10-01

    TP53 tumor-suppressor gene mutations are among the most frequent abnormalities in cancer, affecting approximately 40% of patients. Yet, there is no accepted way to target these alterations in the clinic. At the same time, antagonists of VEGFR or its ligand are best-selling oncology drugs, with multiple, expensive compounds approved. Although only a subset of patients benefit from these antiangiogenesis agents, no relevant biomarker has been identified. Interestingly, TP53 mutations upregulate VEGF-A and VEGFR2. We prospectively enrolled 500 patients, to be interrogated by comprehensive genomic profiling (CGP) (next-generation sequencing, 236 genes), and to be matched, whenever possible, with targeted agents. Herein, we analyze outcomes based on VEGF/VEGFR inhibitor treatment and presence of TP53 mutations. Of the 500 patients, 188 (37.6%; with ≥1 alteration) were treated; 106 (56% of 188) had tumors that harbored TP53 mutations. VEGF/VEGFR inhibitor therapy was independently associated with improvement in all outcome parameters [rate of stable disease (SD) ≥6 months/partial and complete remission (PR/CR); (31% versus 7%; TP53-mutant patients (who received no other molecular-matched agents) treated with versus without VEGF/VEGFR inhibitors), time-to-treatment failure, and overall survival (multivariate analysis: all P ≤ 0.01)] for the patients harboring TP53-mutant cancers, but improvement was not seen in any of these parameters for patients with TP53 wild-type neoplasms. We conclude that TP53 mutations predict sensitivity to VEGF/VEGFR inhibitors in the clinic. TP53 alterations may therefore be a ready biomarker for treatment with antiangiogenesis agents, a finding of seminal importance across the cancer field. Mol Cancer Ther; 15(10); 2475-85. ©2016 AACR.

  5. Pien Tze Huang Inhibits Hypoxia-Induced Angiogenesis via HIF-1α/VEGF-A Pathway in Colorectal Cancer

    PubMed Central

    Chen, Hongwei; Feng, Jianyu; Zhang, Yuchen; Shen, Aling; Chen, Youqin; Lin, Jiumao; Lin, Wei; Sferra, Thomas J.

    2015-01-01

    Hypoxia-induced angiogenesis plays an important role in the development and metastasis of solid tumors and is highly regulated by HIF-1α/VEGF-A pathway. Therefore, inhibiting tumor angiogenesis via suppression of HIF-1α/VEGF-A signaling represents a promising strategy for anticancer treatment. As a traditional Chinese medicine formula, Pien Tze Huang (PZH) has long been used as a folk remedy for cancer in China and Southeast Asia. Previously, we reported that PZH inhibits colorectal cancer (CRC) growth both in vivo and in vitro. To elucidate the antitumor mechanisms of PZH, in the present study we used human umbilical vein endothelial cells (HUVEC) and colorectal carcinoma HCT-8 cells to evaluate the effects of PZH on hypoxia-induced angiogenesis and investigated the underlying molecular mechanisms. We found that PZH could inhibit hypoxia-induced migration and tube formation of HUVEC cells in a dose-dependent manner, although the low concentrations of PZH had no effect on HUVEC viability. Moreover, PZH inhibited hypoxia-induced activation of HIF-1α signaling and the expression of VEGF-A and/or VEGFR2 in both HCT-8 and HUVEC cells. Collectively, our findings suggest that PZH can inhibit hypoxia-induced tumor angiogenesis via suppression of HIF-1α/VEGF-A pathway. PMID:25649293

  6. Pien Tze Huang Inhibits Hypoxia-Induced Angiogenesis via HIF-1 α /VEGF-A Pathway in Colorectal Cancer.

    PubMed

    Chen, Hongwei; Feng, Jianyu; Zhang, Yuchen; Shen, Aling; Chen, Youqin; Lin, Jiumao; Lin, Wei; Sferra, Thomas J; Peng, Jun

    2015-01-01

    Hypoxia-induced angiogenesis plays an important role in the development and metastasis of solid tumors and is highly regulated by HIF-1α/VEGF-A pathway. Therefore, inhibiting tumor angiogenesis via suppression of HIF-1α/VEGF-A signaling represents a promising strategy for anticancer treatment. As a traditional Chinese medicine formula, Pien Tze Huang (PZH) has long been used as a folk remedy for cancer in China and Southeast Asia. Previously, we reported that PZH inhibits colorectal cancer (CRC) growth both in vivo and in vitro. To elucidate the antitumor mechanisms of PZH, in the present study we used human umbilical vein endothelial cells (HUVEC) and colorectal carcinoma HCT-8 cells to evaluate the effects of PZH on hypoxia-induced angiogenesis and investigated the underlying molecular mechanisms. We found that PZH could inhibit hypoxia-induced migration and tube formation of HUVEC cells in a dose-dependent manner, although the low concentrations of PZH had no effect on HUVEC viability. Moreover, PZH inhibited hypoxia-induced activation of HIF-1α signaling and the expression of VEGF-A and/or VEGFR2 in both HCT-8 and HUVEC cells. Collectively, our findings suggest that PZH can inhibit hypoxia-induced tumor angiogenesis via suppression of HIF-1α/VEGF-A pathway.

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

    PubMed Central

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

    2016-01-01

    Binding of MHC class I-related chain molecules A and B (MICA/B) to the natural killer (NK) cell receptor NK group 2, member D (NKG2D) is thought critical for activating NK-mediated immunosurveillance. Angiogenesis is important for tumor growth and interfering with angiogenesis using the fully human IgG1 anti-VEGFR2 (vascular endothelial growth factor receptor 2) antibody (mAb04) can be effective in treating malignancy. In an effort to make mAb04 more effective we have generated a novel antibody fusion protein (mAb04-MICA) consisting of mAb04 and MICA. We found that mAb04-MICA maintained the anti-angiogenic and antineoplastic activities of mAb04, and also enhanced immunosurveillance activated by the NKG2D pathway. Moreover, in human breast tumor-bearing nude mice, mAb04-MICA demonstrated superior anti-tumor efficacy compared to combination therapy of mAb04 + Docetaxel or Avastin + Docetaxel, highlighting the immunostimulatory effect of MICA. In conclusion, mAb04-MICA provided new inspiration for anti-tumor treatment and had prospects for clinical application. PMID:26909862

  8. 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

  9. VEGF induces proliferation, migration, and TGF-{beta}1 expression in mouse glomerular endothelial cells via mitogen-activated protein kinase and phosphatidylinositol 3-kinase

    SciTech Connect

    Li Zhaodong; Bork, Jens Peter; Krueger, Bettina; Patsenker, Eleonora; Schulze-Krebs, Anja; Hahn, Eckhart G.; Schuppan, Detlef; E-mail: dschuppa@bidmc.harvard.edu

    2005-09-09

    The role of glomerular endothelial cells in kidney fibrosis remains incompletely understood. While endothelia are indispensable for repair of acute damage, they can produce extracellular matrix proteins and profibrogenic cytokines that promote fibrogenesis. We used a murine cell line with all features of glomerular endothelial cells (glEND.2), which dissected the effects of vascular endothelial growth factor (VEGF) on cell migration, proliferation, and profibrogenic cytokine production. VEGF dose-dependently induced glEND.2 cell migration and proliferation, accompanied by up-regulation of VEGFR-2 phosphorylation and mRNA expression. VEGF induced a profibrogenic gene expression profile, including up-regulation of TGF-{beta}1 mRNA, enhanced TGF-{beta}1 secretion, and bioactivity. VEGF-induced endothelial cell migration and TGF-{beta}1 induction were mediated by the phosphatidyl-inositol-3 kinase pathway, while proliferation was dependent on the Erk1/2 MAP kinase pathway. This suggests that differential modulation of glomerular angiogenesis by selective inhibition of the two identified VEGF-induced signaling pathways could be a therapeutic approach to treat kidney fibrosis.

  10. Increasing the binding affinity of VEGFR-2 inhibitors by extending their hydrophobic interaction with the active site: Design, synthesis and biological evaluation of 1-substituted-4-(4-methoxybenzyl)phthalazine derivatives.

    PubMed

    Eldehna, Wagdy M; Abou-Seri, Sahar M; El Kerdawy, Ahmed M; Ayyad, Rezk R; Hamdy, Abdallah M; Ghabbour, Hazem A; Ali, Mamdouh M; Abou El Ella, Dalal A

    2016-05-04

    A series of anilinophthalazine derivatives 4a-j was initially synthesized and tested for its VEGFR-2 inhibitory activity where it showed promising activity (IC50 = 0.636-5.76 μM). Molecular docking studies guidance was used to improve the binding affinity for series 4a-j towards VEGFR-2 active site. This improvement was achieved by increasing the hydrophobic interaction with the hydrophobic back pocket of the VEGFR-2 active site lined with the hydrophobic side chains of Ile888, Leu889, Ile892, Val898, Val899, Leu1019 and Ile1044. Increasing the hydrophobic interaction was accomplished by extending the anilinophthalazine scaffold with a substituted phenyl moiety through an uriedo linker which should give this extension the flexibility required to accommodate itself deeply into the hydrophobic back pocket. As planned, the designed uriedo-anilinophthalazines 7a-i showed superior binding affinity than their anilinophthalazine parents (IC50 = 0.083-0.473 μM). In particular, compounds 7g-i showed IC50 of 0.086, 0.083 and 0.086 μM, respectively, which are better than that of the reference drug sorafenib (IC50 = 0.09 μM).

  11. 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

  12. 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...

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

    PubMed

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

    2016-10-01

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

  14. Plexin-A4 promotes tumor progression and tumor angiogenesis by enhancement of VEGF and bFGF signaling.

    PubMed

    Kigel, Boaz; Rabinowicz, Noa; Varshavsky, Asya; Kessler, Ofra; Neufeld, Gera

    2011-10-13

    Plexin-A4 is a receptor for sema6A and sema6B and associates with neuropilins to transduce signals of class-3 semaphorins. We observed that plexin-A1 and plexin-A4 are required simultaneously for transduction of inhibitory sema3A signals and that they form complexes. Unexpectedly, inhibition of plexin-A1 or plexin-A4 expression in endothelial cells using specific shRNAs resulted in prominent plexin type specific rearrangements of the actin cytoskeleton that were accompanied by inhibition of bFGF and VEGF-induced cell proliferation. The two responses were not interdependent since silencing plexin-A4 in U87MG glioblastoma cells inhibited cell proliferation and strongly inhibited the formation of tumors from these cells without affecting cytoskeletal organization. Plexin-A4 formed stable complexes with the FGFR1 and VEGFR-2 tyrosine-kinase receptors and enhanced VEGF-induced VEGFR-2 phosphorylation in endothelial cells as well as bFGF-induced cell proliferation. We also obtained evidence suggesting that some of the pro-proliferative effects of plexin-A4 are due to transduction of autocrine sema6B-induced pro-proliferative signals, since silencing sema6B expression in endothelial cells and in U87MG cells mimicked the effects of plexin-A4 silencing and also inhibited tumor formation from the U87MG cells. Our results suggest that plexin-A4 may represent a target for the development of novel anti-angiogenic and anti-tumorigenic drugs.

  15. 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.

  16. Mechanical stimulation of the pro-angiogenic capacity of human fracture haematoma: involvement of VEGF mechano-regulation.

    PubMed

    Groothuis, Aline; Duda, Georg N; Wilson, Cameron J; Thompson, Mark S; Hunter, Morgan R; Simon, Paul; Bail, Hermann J; van Scherpenzeel, Karine M; Kasper, Grit

    2010-08-01

    Compromised angiogenesis appears to be a major limitation in various suboptimal bone healing situations. Appropriate mechanical stimuli support blood vessel formation in vivo and improve healing outcomes. However, the mechanisms responsible for this association are unclear. To address this question, the paracrine angiogenic potential of early human fracture haematoma and its responsiveness to mechanical loading, as well as angiogenic growth factors involved, were investigated in vitro. Human haematomas were collected from healthy patients undergoing surgery within 72 h after bone fracture. The haematomas were embedded in a fibrin matrix, and cultured in a bioreactor resembling the in vivo conditions of the early phase of bone healing (20% compression, 1 Hz) over 3 days. Conditioned medium (CM) from the bioreactor was then analyzed. The matrices were also incubated in fresh medium for a further 24 h to evaluate the persistence of the effects. Growth factor (GF) concentrations were measured in the CM by ELISAs. In vitro tube formation assays were conducted on Matrigel with the HMEC-1 cell line, with or without inhibition of vascular endothelial growth factor receptor 2 (VEGFR2). Cell numbers were quantified using an MTS test. In vitro endothelial tube formation was enhanced by CM from haematomas, compared to fibrin controls. The angiogenesis regulators, vascular endothelial growth factor (VEGF) and transforming growth factor beta1 (TGF-beta1), were released into the haematoma CM, but not angiopoietins 1 or 2 (Ang1, 2), basic fibroblast growth factor (bFGF) or platelet-derived growth factor (PDGF). Mechanical stimulation of haematomas, but not fibrin controls, further increased the induction of tube formation by their CM. The mechanically stimulated haematoma matrices retained their elevated pro-angiogenic capacity for 24 h. The pro-angiogenic effect was cancelled by inhibition of VEGFR2 signalling. VEGF concentrations in CM tended to be elevated by mechanical

  17. Distinct genetic interactions between multiple Vegf receptors are required for development of different blood vessel types in zebrafish.

    PubMed

    Covassin, L D; Villefranc, J A; Kacergis, M C; Weinstein, B M; Lawson, N D

    2006-04-25

    Recent evidence indicates a specific role for vascular endothelial growth factor a (Vegfa) during artery development in both zebrafish and mouse embryos, whereas less is known about signals that govern vein formation. In zebrafish, loss of vegfa blocks segmental artery formation and reduces artery-specific gene expression, whereas veins are largely unaffected. Here, we describe a mutation in the zebrafish vegf receptor-2 homolog, kdra, which eliminates its kinase activity and leads to specific defects in artery development. We further find that Flt4, a receptor for Vegfc, cooperates with Kdr during artery morphogenesis, but not differentiation. We also identify an additional zebrafish vegfr-2 ortholog, referred to as kdrb, which can partially compensate for loss of kdra but is dispensable for vascular development in wild-type embryos. Interestingly, we find that these Vegf receptors are also required for formation of veins but in distinct genetic interactions that differ from those required for artery development. Taken together, our results indicate that formation of arteries and veins in the embryo is governed in part by different Vegf receptor combinations and suggest a genetic mechanism for generating blood vessel diversity during vertebrate development.

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

    PubMed

    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

    2016-09-01

    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.

  19. 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

  20. Cross-talk between leukemic and endothelial cells promotes angiogenesis by VEGF activation of the Notch/Dll4 pathway.

    PubMed

    Zhang, Jingru; Ye, Jingjing; Ma, Daoxin; Liu, Na; Wu, Hao; Yu, Shuang; Sun, Xiulian; Tse, William; Ji, Chunyan

    2013-03-01

    Angiogenesis is suggested to be important for leukemogenesis and chemosensitivity in acute myeloid leukemia (AML). The vascular endothelial growth factor (VEGF) and Notch/Dll4 pathways have been identified as critical in the regulation of embryonic vascular development and tumor angiogenesis. However, the potential role of the Notch/Dll4 pathway in leukemia-endothelium cross-talk and its functional link with VEGF remains obscure. This study assessed the expression of VEGF and Notch/Dll4 pathway molecules in primary AML and investigated their biological function in the coculture of endothelial cells with AML cells. The results demonstrated that bone marrow vascularity in the newly diagnosed AML patients was increased and correlated with high VEGF and Dll4 expression. Patients with untreated AML expressed higher levels of VEGFR2, Notch1, Dll4 and Hes1 than healthy controls. Moreover, the activation of the Notch/Dll4 pathway is associated with poor prognosis in AML. In addition, AML cells were shown to increase endothelial cell proliferation in Transwell coculture. This was associated with concomitant activation of the Notch/Dll4 pathway and upregulation of its downstream genes, such as matrix metalloproteinases, resulting in the enhancement of endothelial cell migration and tube formation. Our study also showed that upregulation of Dll4 expression in AML cells by cDNA transfection suppressed VEGF-induced endothelial cell proliferation and angiogenesis in direct contact coculture. These results elucidate a novel mechanism by which the interplay between AML and endothelial cells promotes angiogenesis through the Notch/Dll4 pathway. Modulation of this pathway may, therefore, hold promise as a novel antiangiogenic strategy for the treatment of AML.

  1. The peptidomimetic Vasotide targets two retinal VEGF receptors and reduces pathological angiogenesis in murine and nonhuman primate models of retinal disease

    PubMed Central

    Sidman, Richard L.; Li, Jianxue; Lawrence, Matthew; Hu, Wenzheng; Musso, Gary F.; Giordano, Ricardo J.; Cardó-Vila, Marina; Pasqualini, Renata; Arap, Wadih

    2016-01-01

    Blood vessel growth from preexisting vessels (angiogenesis) underlies many severe diseases including major blinding retinal diseases such as retinopathy of prematurity (ROP) and aged macular degeneration (AMD). This observation has driven development of antibody inhibitors that block a central factor in AMD, named vascular endothelial growth factor (VEGF), from binding to its receptors VEGFR-1 and VEGFR-2. However, some patients are insensitive to current anti-VEGF drugs or develop resistance, and the required repeated intravitreal injection of these large molecules is costly and clinically problematic. Here, we have evaluated a small cyclic retro-inverted peptidomimetic, D(Cys-Leu-Pro-Arg-Cys), abbreviated as D(CLPRC), and hereafter named Vasotide, that inhibits retinal angiogenesis by binding selectively to the VEGF receptors, VEGFR-1 and Neuropilin-1 (NRP-1). Delivery of Vasotide in eye drops or via intraperitoneal injection in a laser-induced monkey model of human wet AMD, a mouse genetic knockout model of the AMD subtype called retinal angiomatous proliferation (RAP), and a mouse oxygen-induced model of retinopathy of prematurity (ROP) markedly decreased retinal angiogenesis in all three animal models. This prototype drug candidate is a promising new dual receptor inhibitor of the VEGF ligand with potential for translation into safer, less invasive applications to combat pathological angiogenesis in retinal disorders. PMID:26468327

  2. Co-expression of vascular endothelial growth factor (VEGF) and its receptors (flk-1 and flt-1) in hormone-induced mammary cancer in the Noble rat

    PubMed Central

    Xie, B; Tam, N N C; Tsao, S W; Wong, Y C

    1999-01-01

    Vascular endothelial growth factor (VEGF) is recognized to play a predominant role in breast cancer prognosis. The action of VEGF is mediated by two high-affinity receptors with ligand-stimulated tyrosine kinase activity: VEGFR-1/flt-1 and VEGFR-2/flk-1, which are expressed mainly in vascular endothelial cells. To the best of our knowledge, no previous studies on the expression of these receptors in breast cancer cells has been made. We have established a new animal model for breast cancer, using a combination of 17β-oestradiol and testosterone as ‘carcinogens’. Taking advantage of the animal model, we have demonstrated that mammary cancer cells expressed not only high levels of VEGF but also, surprisingly, its receptors (flt-1 and flk-1) in mammary cancer cells. Intense reactivities to VEGF, flt-1 and flk-1 were observed in mammary cancer cells, especially in invasive mammary carcinoma. Western blot analysis confirmed the increase in flk-1 and flt-1 proteins in induced mammary cancers. Based on these observations, we hypothesize that in mammary cancer, VEGF regulates, in addition to endothelial proliferation and angiogenesis, also growth of cancer cells by an autocrine mechanism mediated through its receptors. To further verify this hypothesis, we investigated the correlation between cellular proliferation and the expression of VEGF, flt-1 and flk-1. Using double-labelling immunocytochemistry, we have shown a correlation between high VEGF activity and Ki-67 expression. The Ki-67 indices in the areas of strong and weak VEGF reactivities were 58.3% and 3.7% respectively. Similarly, there was also a correlation of strong flk-1 and Ki-67 reactivity. The Ki-67 indices for areas of strong and weak flk-1 reactivities were 53.9% and 3.1% respectively. On the other hand, there was a reverse correlation between flt-1 and Ki-67 activities. These results indicate that overexpression of VEGF and flk-1 is correlated with high Ki-67 index. The data, therefore, suggest that

  3. Celecoxib inhibits growth of human autosomal dominant polycystic kidney cyst-lining epithelial cells through the VEGF/Raf/MAPK/ERK signaling pathway.

    PubMed

    Xu, Tao; Wang, Nian-Song; Fu, Li-Li; Ye, Chao-Yang; Yu, Sheng-Qiang; Mei, Chang-Lin

    2012-07-01

    Autosomal dominant polycystic kidney disease (ADPKD) is a progressive chronic kidney disease. To date there are no effective medicines to halt development and growth of cysts. In the present study, we explored novel effects of celecoxib (CXB), a COX-2 specific inhibitor, on primary cultures of human ADPKD cyst-lining epithelial cells. Primary cultures of ADPKD cyst-lining epithelial cells were obtained from five patients. Effects of CXB were measured by various assays to detect BrdU incorporation, apoptosis and proliferation in vitro. Additionally, effects of CXB on kidney weight, the cyst index, the fibrosis index, blood urea nitrogen (BUN), serum creatinine (SCr), serum 6-keto-PGF-1α, serum thromboxane-2 (TXB2) and renal PCNA expression were assessed in Han:SPRD rat, a well-characterized rodent model of PKD. CXB inhibited proliferation of ADPKD cyst-lining epithelial cells, blocked the release of VEGF from the cells and induced extensive apoptosis in a time- and dose-dependent manner. Moreover, CXB up-regulated the cell cycle negative regulator p21(CIP/WAF1) and the cell cycle positive regulator Cyclin A, blocked ERK1/2 phosphorylation, induced apoptotic factors (Bax and caspase-3) and reduced Bcl-2. Furthermore, CXB inhibited the expression of VEGFR-2 and Raf-1 in ADPKD cyst-lining epithelial cells. CXB markedly reduced the cyst index, the fibrosis index, leukocyte infiltration, BUN, SCr, serum 6-keto-PGF-1α, TXB2 and renal PCNA expression in Han:SPRD rat. We demonstrated for the first time that CXB could suppress renal cyst-lining growth both in vitro and in vivo in Han:SPRD rat. CXB can inhibit proliferation, suppress cell cycle progression, and induce apoptosis in ADPKD cyst-lining epithelial cells through the inhibition of the VEGF/VEGFR-2/Raf-1/MAPK/ERK signaling pathway.

  4. Kidins220/ARMS mediates the integration of the neurotrophin and VEGF pathways in the vascular and nervous systems.

    PubMed

    Cesca, F; Yabe, A; Spencer-Dene, B; Scholz-Starke, J; Medrihan, L; Maden, C H; Gerhardt, H; Orriss, I R; Baldelli, P; Al-Qatari, M; Koltzenburg, M; Adams, R H; Benfenati, F; Schiavo, G

    2012-02-01

    Signaling downstream of receptor tyrosine kinases controls cell differentiation and survival. How signals from different receptors are integrated is, however, still poorly understood. In this work, we have identified Kidins220 (Kinase D interacting substrate of 220 kDa)/ARMS (Ankyrin repeat-rich membrane spanning) as a main player in the modulation of neurotrophin and vascular endothelial growth factor (VEGF) signaling in vivo, and a primary determinant for neuronal and cardiovascular development. Kidins220(-/-) embryos die at late stages of gestation, and show extensive cell death in the central and peripheral nervous systems. Primary neurons from Kidins220(-/-) mice exhibit reduced responsiveness to brain-derived neurotrophic factor, in terms of activation of mitogen-activated protein kinase signaling, neurite outgrowth and potentiation of excitatory postsynaptic currents. In addition, mice lacking Kidins220 display striking cardiovascular abnormalities, possibly due to impaired VEGF signaling. In support of this hypothesis, we demonstrate that Kidins220 constitutively interacts with VEGFR2. These findings, together with the data presented in the accompanying paper, indicate that Kidins220 mediates the integration of several growth factor receptor pathways during development, and mediates the activation of distinct downstream cascades according to the location and timing of stimulation.

  5. 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.

  6. VEGF-C promotes survival in podocytes.

    PubMed

    Foster, R R; Satchell, S C; Seckley, J; Emmett, M S; Joory, K; Xing, C Y; Saleem, M A; Mathieson, P W; Bates, D O; Harper, S J

    2006-07-01

    Vascular endothelial growth factor (VEGF)-A is an autocrine survival factor for podocytes, which express two VEGF receptors, VEGF-R1 and VEGF-R3. As VEGF-A is not a known ligand for VEGF-R3, the aim of this investigation was to examine whether VEGF-C, a known ligand for VEGF-R3, served a function in podocyte biology and whether this was VEGF-R3 dependent. VEGF-C protein expression was localized to podocytes in contrast to VEGF-D, which was expressed in parietal epithelial cells. Intracellular calcium ([Ca2+]i) experiments demonstrated that VEGF-C induced a 0.74+/-0.09-fold reduction in [Ca2+]i compared with baseline in human conditionally immortalized podocytes (hCIPs; P<0.05, one sample t-test, n=8). Cytotoxicity experiments revealed that in hCIPs VEGF-C reduced cytotoxicity to 81.4+/-1.9% of serum-starved conditions (P<0.001, paired t-test, n=16), similar to VEGF-A (82.8+/-4.5% of serum-starved conditions, P<0.05, paired t-test). MAZ51 (a VEGF-R3 kinase inhibitor) inhibited the VEGF-C-induced reduction in cytotoxicity (106.2+/-2.1% of serum-starved conditions), whereas MAZ51 by itself had no cytotoxic effects on hCIPs. VEGF-C was also shown to induce a 0.5+/-0.13-fold reduction in levels of MAPK phosphorylation compared with VEGF-A and VEGF-A-Mab treatment (P<0.05, ANOVA, n=4), yet had no effect on Akt phosphorylation. Surprisingly, immunoprecipitation studies detected no VEGF-C-induced autophosphorylation of VEGF-R3 in hCIPs but did so in HMVECs. Moreover, SU-5416, a tyrosine kinase inhibitor, blocked the VEGF-C-induced reduction in cytotoxicity (106+/-2.8% of serum-starved conditions) at concentrations specific for VEGF-R1. Together, these results suggest for the first time that VEGF-C acts in an autocrine manner in cultured podocytes to promote survival, although the receptor or receptor complex activated has yet to be elucidated.

  7. Phase II and Biomarker Study of the Dual MET/VEGFR2 Inhibitor Foretinib in Patients With Papillary Renal Cell Carcinoma

    PubMed Central

    Choueiri, Toni K.; Vaishampayan, Ulka; Rosenberg, Jonathan E.; Logan, Theodore F.; Harzstark, Andrea L.; Bukowski, Ronald M.; Rini, Brian I.; Srinivas, Sandy; Stein, Mark N.; Adams, Laurel M.; Ottesen, Lone H.; Laubscher, Kevin H.; Sherman, Laurie; McDermott, David F.; Haas, Naomi B.; Flaherty, Keith T.; Ross, Robert; Eisenberg, Peter; Meltzer, Paul S.; Merino, Maria J.; Bottaro, Donald P.; Linehan, W. Marston; Srinivasan, Ramaprasad

    2013-01-01

    Purpose Foretinib is an oral multikinase inhibitor targeting MET, VEGF, RON, AXL, and TIE-2 receptors. Activating mutations or amplifications in MET have been described in patients with papillary renal cell carcinoma (PRCC). We aimed to evaluate the efficacy and safety of foretinib in patients with PRCC. Patients and Methods Patients were enrolled onto the study in two cohorts with different dosing schedules of foretinib: cohort A, 240 mg once per day on days 1 through 5 every 14 days (intermittent arm); cohort B, 80 mg daily (daily dosing arm). Patients were stratified on the basis of MET pathway activation (germline or somatic MET mutation, MET [7q31] amplification, or gain of chromosome 7). The primary end point was overall response rate (ORR). Results Overall, 74 patients were enrolled, with 37 in each dosing cohort. ORR by Response Evaluation Criteria in Solid Tumors (RECIST) 1.0 was 13.5%, median progression-free survival was 9.3 months, and median overall survival was not reached. The presence of a germline MET mutation was highly predictive of a response (five of 10 v five of 57 patients with and without germline MET mutations, respectively). The most frequent adverse events of any grade associated with foretinib were fatigue, hypertension, gastrointestinal toxicities, and nonfatal pulmonary emboli. Conclusion Foretinib demonstrated activity in patients with advanced PRCC with a manageable toxicity profile and a high response rate in patients with germline MET mutations. PMID:23213094

  8. Increased expression of HIF-1α, VEGF-A and its receptors, MMP-2, TIMP-1, and ADAMTS-1 at the venous stenosis of arteriovenous fistula in a mouse model with renal insufficiency

    PubMed Central

    Misra, Sanjay; Shergill, Uday; Yang, Binxia; Janardhanan, Rajiv; Misra, Khamal D.

    2010-01-01

    Purpose A mouse model of renal insufficiency with arteriovenous fistula (AVF) and venous stenosis was created. We tested the hypothesis that there is increased gene expression of hypoxia inducible factor-1 alpha (HIF-1α), vascular endothelial growth factor- A (VEGF-A) and its receptors (VEGFR-1, -2), matrix metalloproteinase-2 (MMP-2), -9 (MMP-9), tissue inhibitor of metalloproteinase-1, -2 (TIMP-1, -2), and a disintegrin and metalloproteinase thrombospondin-1 (ADAMTS-1) at the venous stenosis. Materials and methods Nineteen male C57BL/6 mice underwent a left nephrectomy and a surgical occlusion of the right upper pole to induce renal insufficiency and characterized in eight mice. Twenty eight days later, an AVF (n=11) was created from the right carotid artery to ipsilateral jugular vein and the mice were sacrificed at day 7 (n=4) and day 14 (n=4). The outflow and control veins were removed for gene expression. Three mice were sacrificed at day 28 for histologic analysis. Results The mean serum blood urea nitrogen remained significantly elevated for 8 weeks when compared to baseline (P<0.05). By day 7, there was a significant increase in the expression of HIF-1α, VEGF-A, VEGFR-1, VEGFR-2, MMP-2, TIMP-1, and ADAMTS-1 at the outflow vein with HIF-1α and TIMP-1 being significantly elevated at day 14 (P<0.05). By day 28, the venous stenosis was characterized by a thickened vein wall and neointima. Conclusions A mouse model of renal insufficiency with AVF was developed which had increased expression of HIF-1α, VEGF-A, VEGFR-1, VEGFR-2, MMP-2, TIMP-1, and ADAMTS-1 at the outflow vein with venous stenosis by day 28. PMID:20598569

  9. Anti-tumor innate immunity activated by intermittent metronomic cyclophosphamide treatment of 9L brain tumor xenografts is preserved by anti-angiogenic drugs that spare VEGF receptor 2

    PubMed Central

    2014-01-01

    Background Metronomic cyclophosphamide given on an intermittent, 6-day repeating schedule, but not on an exposure dose-equivalent daily schedule, activates an anti-tumor innate immune response that leads to major regression of large implanted gliomas, without anti-angiogenesis. Methods and approach Mice bearing implanted 9L gliomas were used to investigate the effects of this 6-day repeating, immunogenic cyclophosphamide schedule on myeloid-derived suppressor cells, which are pro-angiogenic and can inhibit anti-tumor immunity, and to elucidate the mechanism whereby the innate immune cell-dependent tumor regression response to metronomic cyclophosphamide treatment is blocked by several anti-angiogenic receptor tyrosine kinase inhibitors. Results Intermittent metronomic cyclophosphamide scheduling strongly increased glioma-associated CD11b+ immune cells but not CD11b+Gr1+ myeloid-derived suppressor cells, while bone marrow and spleen reservoirs of the suppressor cells were decreased. The inhibition of immune cell recruitment and tumor regression by anti-angiogenic receptor tyrosine kinase inhibitors, previously observed in several brain tumor models, was recapitulated in the 9L tumor model with the VEGFR2-specific inhibitory monoclonal antibody DC101 (p < 0.01), implicating VEGFR2 signaling as an essential step in metronomic cyclophosphamide-stimulated immune cell recruitment. In contrast, sorafenib, a multi-receptor tyrosine kinase inhibitor with comparatively weak VEGF receptor phosphorylation inhibitory activity, was strongly anti-angiogenic but did not block metronomic cyclophosphamide-induced innate immunity or tumor regression (p > 0.05). Conclusions The interference by receptor tyrosine kinase inhibitors in the immunogenic actions of intermittent metronomic chemotherapy is not a consequence of anti-angiogenesis per se, as demonstrated in an implanted 9L tumor model. Furthermore, this undesirable interaction with tyrosine kinase inhibitors can be

  10. 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

  11. 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.

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

    PubMed Central

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

    2012-01-01

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

  13. Green tea catechins inhibit VEGF-induced angiogenesis in vitro through suppression of VE-cadherin phosphorylation and inactivation of Akt molecule.

    PubMed

    Tang, Feng-Yao; Nguyen, Nhan; Meydani, Mohsen

    2003-10-10

    Studies have indicated that the consumption of green tea is associated with a reduced risk of developing certain forms of cancer and angiogenesis. The mechanism of inhibition of angiogenesis by green tea or its catechins, however, has not been well-established. Vascular endothelial (VE)-cadherin, an adhesive molecule located at the site of intercellular contact, is involved in cell-cell recognition during vascular morphogenesis. The extracellular domain of VE-cadherin mediates initial cell adhesion, whereas the cytosolic tail binding with beta-catenin is required for interaction with the cytoskeleton and junctional strength. Therefore, the cadherin-catenin adhesion system is implicated in cell recognition, differentiation, growth and migration of capillary endothelium. Using tube formation of human microvascular endothelial cells (HMVEC) in culture as an in vitro model of angiogenesis, we reported that vascular endothelial growth factor (VEGF)-induced tube formation is inhibited by anti-VE-cadherin antibody and dose-dependently by green tea catechins. We also demonstrated here that inhibition of tube formation by epigallocatechin gallate (EGCG), one of the green tea catechins, is in part mediated through suppression of VE-cadherin tyrosine phosphorylation and inhibition of Akt activation during VEGF-induced tube formation. These findings indicate that VE-cadherin and Akt, known downstream proteins in VEGFR-2-mediated cascade, are the new-targeted proteins by which green tea catechins inhibit angiogenesis.

  14. 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

  15. Neuropilin-VEGF signaling pathway acts as a key modulator of vascular, lymphatic, and inflammatory cell responses of the bladder to intravesical BCG treatment.

    PubMed

    Saban, Marcia R; Sferra, Thomas J; Davis, Carole A; Simpson, Cindy; Allen, Arielle; Maier, Julie; Fowler, Ben; Knowlton, Nicholas; Birder, Lori; Wu, Xue-Ru; Saban, Ricardo

    2010-12-01

    Recent findings indicate that VEGF receptors and coreceptors (neuropilins; NRP) are expressed on nonendothelial cells in human bladder urothelium, in one human bladder cancer cell line (J82), and in the mouse bladder urothelium. In addition, VEGFR1, VEGFR2, NRP1, and NRP2 expressions were upregulated in animal models of chronic bladder inflammation induced by four weekly instillations of protease-activated receptors (PAR)-activating peptides or bacillus Calmette-Guérin (BCG) into the mouse bladder. Here, we used four weekly instillations of BCG as a model for chronic bladder inflammation to further investigate whether VEGF receptors and NRPs play a role in the migration of inflammatory cells and inflammation-induced lymphangiogenesis and angiogenesis. For this purpose, we used neutralizing antibodies that were engineered to specifically block the binding of VEGF to NRP (anti-NRP1(B)) and the binding of semaphorins to NRP (anti-NRP1(A)). C57BL/6 mice received intraperitoneal injections of PBS, anti-NRP1(A)- or anti-NRP1(B)-neutralizing antibodies and then were challenged chronically with intravesical PBS or BCG. At the end of chronic challenge period, a fluorescent internalizable tracer, scVEGF/Cy5.5, was administered to all mice and near-infrared fluorescence images were obtained in vivo and in real time. BCG increased the overall accumulation of scVEGF/Cy5.5 in the urinary bladder urothelium and inflammatory cells. In addition, BCG increased the density of blood and lymphatic vessels concomitantly with an upregulation of NRP2 expression in lymphatic vessels. Treatment of the mice with NRP1-neutralizing antibodies dramatically reduced scVEGF/Cy5.5 uptake, polymorphonuclear (myeloperoxidase-positive cells) and dendritic cell (CD11c-positive cells) infiltration, and decreased the overall density of BCG-induced blood and lymphatic vessels. These results implicate NRPs as critical in vivo regulators of the vascular and inflammatory responses to the intravesical

  16. 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.

  17. Engineered VEGF-releasing PEG-MAL hydrogel for pancreatic islet vascularization

    PubMed Central

    Phelps, Edward A.; Templeman, Kellie L.; Thulé, Peter M.; García, Andrés J.

    2013-01-01

    Biofunctionalized polyethylene glycol maleimide (PEG-MAL) hydrogels were engineered as a platform to deliver pancreatic islets to the small bowel mesentery and promote graft vascularization. VEGF, a potent stimulator of angiogenesis, was incorporated into the hydrogel to be released in an on-demand manner through enzymatic degradation. PEG-MAL hydrogel enabled extended in vivo release of VEGF. Isolated rat islets encapsulated in PEG-MAL hydrogels remained viable in culture and secreted insulin. Islets encapsulated in PEG-MAL matrix and transplanted to the small bowel mesentery of healthy rats grafted to the host tissue and revascularized by 4 weeks. Addition of VEGF release to the PEG-MAL matrix greatly augmented the vascularization response. These results establish PEG-MAL engineered matrices as a vascular-inductive cell delivery vehicle and warrant their further investigation as islet transplantation vehicles in diabetic animal models. PMID:25787738

  18. 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.

  19. COX-2 expression is correlated with VEGF-C, lymphangiogenesis and lymph node metastasis in human cervical cancer.

    PubMed

    Liu, Huidong; Xiao, Jianbing; Yang, Yanmei; Liu, Yan; Ma, Ruijin; Li, Yuhang; Deng, Fengchun; Zhang, Yafang

    2011-09-01

    Lymphangiogenesis has been shown to promote lymph node metastasis in cancers, making it an important target in cancer therapy. Vascular endothelial growth factor (VEGF)-C is upregulated in various tumors/cancers and is one of the most potent growth factors for inducing lymphangiogenesis and promoting lymph node metastasis (LNM). Likewise, cyclooxygenase (COX)-2 plays major roles in carcinogenesis, tumor growth and metastasis via multiple mechanisms including inactivation of host antitumor immunity and promotion of tumor cell migration, tumor cell invasiveness and tumor-associated angiogenesis and lymphangiogenesis. We previously demonstrated an association between COX-2 and VEGF-C in an in vitro model of lung cancer. However, little is known about the regulation of VEGF-C by COX-2 in cervical cancer. In this study, we measured the COX-2 and VEGF-C expressions by immunohistochemistry in 23 LNM-positive and 20 LNM-negative cervical cancer specimens. We then examined the correlations among the expressions and the lymphatic microvessel density (LMVD) and ultrastructural changes to the lymphatic vessel walls by enzyme histochemical staining and electron microscopy. In addition, we used the HeLa cervical cancer cell line to explore the in vitro regulation of VEGF-C by COX-2 and its metabolite, PGE(2), using siRNA-mediated gene silencing and EP receptor blockade. The LNM-positive specimens exhibited significantly higher VEGF-C expression, COX-2 expression and LMVD than the LNM-negative specimens. Furthermore, there were strong correlations between the levels of COX-2 expression and the levels of VEGF-C expression and secretion and a significant positive association between the LMVD and LNM. siRNA-mediated knockdown of COX-2 expression inhibited VEGF-C mRNA expression while EP1 and EP4 receptor antagonists reduced the VEGF-C protein level and tyrosine phosphorylation of Src kinase. Moreover, inhibition of Src kinase with the tyrosine kinase inhibitor PP1 attenuated VEGF

  20. 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

  1. 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.

  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. The relationship between transplacental O2 diffusion and placental expression of PlGF, VEGF and their receptors in a placental insufficiency model of fetal growth restriction.

    PubMed

    Regnault, Timothy R H; de Vrijer, Barbra; Galan, Henry L; Davidsen, Meredith L; Trembler, Karen A; Battaglia, Frederick C; Wilkening, Randall B; Anthony, Russell V

    2003-07-15

    Placental growth factor (PlGF) and vascular endothelial growth factor (VEGF) are involved in placental angiogenesis through interactions with the VEGFR-1 and VEGFR-2 receptors. The placenta of pregnancies whose outcome is fetal growth restriction (FGR) are characterized by abnormal angiogenic development, classically associated with hypoxia. The present study evaluated the near-term expression of this growth factor family in an ovine model of placental insufficiency-FGR, in relationship to uteroplacental oxygenation. Compared to controls, FGR pregnancies demonstrated a 37% increase in uterine blood flow (FGR vs. control, 610.86+/-48.48 vs. 443.17+/-37.39 ml min(-1) (kg fetus)(-1); P<0.04), which was associated with an increased maternal uterine venous PO2 (58.13+/-1.00 vs. 52.89+/-1.26 mmHg; P<0.02), increased umbilical artery systolic/diastolic ratio (3.90+/-0.33 vs. 2.12+/-0.26, P<0.05), and fetal hypoxia (arterial PO2; 12.79+/-0.97 vs. 18.65+/-1.6 mmHg, P<0.005). Maternal caruncle PlGF mRNA was increased in FGR (P<0.02), while fetal cotyledon VEGF mRNA was reduced (P<0.02). VEGFR-1 mRNA was also reduced in FGR fetal cotyledon (P<0.001) but was not altered in caruncle tissue. Immunoblot analysis of PlGF and VEGF demonstrated single bands at 19,000 and 18,600 Mr, respectively. Caruncle PlGF concentration was increased (P<0.04), while cotyledon VEGF was decreased (P<0.05) in FGR placentae. The data establish that uterine blood flow is not reduced in relationship to metabolic demands in this FGR model and that the transplacental PO2 gradient is increased, maintaining umbilical oxygen uptake per unit of tissue. Furthermore, these data suggest that an increased transplacental gradient of oxygen generates changes in angiogenic growth factors, which may underline the pathophysiology of the post-placental hypoxic FGR.

  4. 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.

  5. Functional binding surface of a β-hairpin VEGF receptor targeting peptide determined by NMR spectroscopy in living cells.

    PubMed

    Diana, Donatella; Russomanno, Anna; De Rosa, Lucia; Di Stasi, Rossella; Capasso, Domenica; Di Gaetano, Sonia; Romanelli, Alessandra; Russo, Luigi; D'Andrea, Luca D; Fattorusso, Roberto

    2015-01-02

    In this study, the functional interaction of HPLW peptide with VEGFR2 (Vascular Endothelial Growth Factor Receptor 2) was determined by using fast (15)N-edited NMR spectroscopic experiments. To this aim, (15)N uniformly labelled HPLW has been added to Porcine Aortic Endothelial Cells. The acquisition of isotope-edited NMR spectroscopic experiments, including (15)N relaxation measurements, allowed a precise characterization of the in-cell HPLW epitope recognized by VEGFR2.

  6. Mechanisms of VEGF Availability in Prostate Cancer

    DTIC Science & Technology

    2005-01-01

    the information obtained from this proposal has now been used to further understand resistance to VEGF 7 therapy in cancer progression. We...prostate cancer biology. Thanks to this training, Dr. Monvoisin is currently faculty at the Institut de Physiologie et Biologie Cellulaires , Université de

  7. VEGF in the lung: a role for novel isoforms.

    PubMed

    Varet, Julia; Douglas, Samantha K; Gilmartin, Laura; Medford, Andrew R L; Bates, David O; Harper, Steven J; Millar, Ann B

    2010-06-01

    Vascular endothelial cell growth factor (VEGF) is a potent mitogen and permogen that increases in the plasma and decreases in the alveolar space in respiratory diseases such as acute respiratory distress syndrome (ARDS). This observation has led to controversy over the role of this potent molecule in lung physiology and disease. We hypothesized that some of the VEGF previously detected in normal lung may be of the anti-angiogenic family (VEGF(xxx)b) with significant potential effects on VEGF bioactivity. VEGF(xxx)b protein expression was assessed by indirect immunohistochemistry in normal and ARDS tissue. Expression of VEGF(xxx)b was also detected by immunoblotting in normal lung tissue, primary human alveolar type II (ATII) cells, and bronchoalveolar lavage (BAL) fluid in normal subjects and by ELISA in normal, "at risk," and ARDS subjects. The effect of VEGF(165) and VEGF(165)b on both human primary endothelial cells and alveolar epithelial cell proliferation was assessed by [(3)H]thymidine uptake. We found that VEGF(165)b was widely expressed in normal healthy lung tissue but is reduced in ARDS lung. VEGF(121)b and VEGF(165)b were present in whole lung, BAL, and ATII lysate. The proliferative effect of VEGF(165) on both human primary endothelial cells and human alveolar epithelial cells was significantly inhibited by VEGF(165)b (P < 0.01). These data demonstrate that the novel VEGF(xxx)b family members are expressed in normal lung and are reduced in ARDS. A specific functional effect on primary human endothelial and alveolar epithelial cells has also been shown. These data suggest that the VEGF(xxx)b family may have a role in repair after lung injury.

  8. VEGF promotes tumorigenesis and angiogenesis of human glioblastoma stem cells

    SciTech Connect

    Oka, Naoki; Soeda, Akio . E-mail: ccd29400@nyc.odn.ne.jp; Inagaki, Akihito; Onodera, Masafumi; Maruyama, Hidekazu; Hara, Akira; Kunisada, Takahiro; Mori, Hideki; Iwama, Toru

    2007-08-31

    There is increasing evidence for the presence of cancer stem cells (CSCs) in malignant brain tumors, and these CSCs may play a pivotal role in tumor initiation, growth, and recurrence. Vascular endothelial growth factor (VEGF) promotes the proliferation of vascular endothelial cells (VECs) and the neurogenesis of neural stem cells. Using CSCs derived from human glioblastomas and a retrovirus expressing VEGF, we examined the effects of VEGF on the properties of CSCs in vitro and in vivo. Although VEGF did not affect the property of CSCs in vitro, the injection of mouse brains with VEGF-expressing CSCs led to the massive expansion of vascular-rich GBM, tumor-associated hemorrhage, and high morbidity, suggesting that VEGF promoted tumorigenesis via angiogenesis. These results revealed that VEGF induced the proliferation of VEC in the vascular-rich tumor environment, the so-called stem cell niche.

  9. The era of anti-vascular endothelial growth factor (VEGF) drugs in ophthalmology, VEGF and anti-VEGF therapy

    PubMed Central

    Pożarowska, Dorota

    2016-01-01

    Angiogenesis is a clue process for tissue development and function, both in normal and pathological conditions. This process is regulated by multiple molecular systems. One of the most potent is vascular endothelial growth factor (VEGF) and its receptor (VEGFR) system. Members of this family are involved in new vessel formation in embryogenesis and maturation, as well as in reparative or pathological reactions in later stages. They play a substantial role in regeneration, inflammation, wound healing, as well as in cancer pathology. Nowadays it is possible to modulate VEGF-VEGFR interactions in many pathological conditions using anti-VEGF therapy. This therapy has already achieved a grounded position in the management of rheumatological disorders, tumour progression, and metastasis. Such drugs as bevacizumab, ranibizumab, aflibercept, and pegaptanib have also proven to be very effective in the treatment of several ocular diseases, such as age-related macular degeneration (AMD), macular oedema, or proliferative retinopathies and iris neovascularisation. The indications for the application of this therapy in ophthalmology are becoming wider and wider. It may also be used for corneal pathologies and in anti-glaucoma procedures. PMID:27833450

  10. Release of the angiogenic cytokine vascular endothelial growth factor (VEGF) from platelets: significance for VEGF measurements and cancer biology.

    PubMed

    Banks, R E; Forbes, M A; Kinsey, S E; Stanley, A; Ingham, E; Walters, C; Selby, P J

    1998-03-01

    Vascular endothelial growth factor (VEGF) is a potent angiogenic factor with a key role in several pathological processes, including tumour vascularization. Our preliminary observations indicated higher VEGF concentrations in serum samples than in matched plasma samples. We have now demonstrated that this difference is due to the presence of VEGF within platelets and its release upon their activation during coagulation. In eight healthy volunteers, serum VEGF concentrations ranged from 76 to 854 pg ml(-1) and were significantly higher (P < 0.01) than the matched citrated plasma VEGF concentrations, which ranged from < 9 to 42 pg ml(-1). Using platelet-rich plasma, mean (s.d.) platelet VEGF contents of 0.56 (0.36) pg of VEGF 10(-6) platelets were found. Immunocytochemistry demonstrated the cytoplasmic presence of VEGF within megakaryocytes and other cell types within the bone marrow. From examination of the effects of blood sample processing on circulating VEGF concentrations, it is apparent that for accurate measurements, citrated plasma processed within 1 h of venepuncture should be used. Serum is completely unsuitable. The presence of VEGF within platelets has implications for processes involving platelet and endothelial cell interactions. e.g. wound healing, and in tumour metastasis, when platelets adhering to circulating tumour cells may release VEGF at points of adhesion to endothelium, leading to hyperpermeability and extravasation of cells.

  11. 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.

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

    PubMed Central

    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). PMID:24648727

  13. Extracellular regulation of VEGF: isoforms, proteolysis, and vascular patterning

    PubMed Central

    Vempati, Prakash; Popel, Aleksander S.; Mac Gabhann, Feilim

    2014-01-01

    The regulation of vascular endothelial growth factor A (VEGF) is critical to neovascularization in numerous tissues under physiological and pathological conditions. VEGF has multiple isoforms, created by alternative splicing or proteolytic cleavage, and characterized by different receptor-binding and matrix-binding properties. These isoforms are known to give rise to a spectrum of angiogenesis patterns marked by differences in branching, which has functional implications for tissues. In this review, we detail the extensive extracellular regulation of VEGF and the ability of VEGF to dictate the vascular phenotype. We explore the role of VEGF-releasing proteases and soluble carrier molecules on VEGF activity. While proteases such as MMP9 can ‘release’ matrix-bound VEGF and promote angiogenesis, for example as a key step in carcinogenesis, proteases can also suppress VEGF’s angiogenic effects. We explore what dictates pro- or anti-angiogenic behavior. We also seek to understand the phenomenon of VEGF gradient formation. Strong VEGF gradients are thought to be due to decreased rates of diffusion from reversible matrix binding, however theoretical studies show that this scenario cannot give rise to lasting VEGF gradients in vivo. We propose that gradients are formed through degradation of sequestered VEGF. Finally, we review how different aspects of the VEGF signal, such as its concentration, gradient, matrix-binding, and NRP1-binding can differentially affect angiogenesis. We explore how this allows VEGF to regulate the formation of vascular networks across a spectrum of high to low branching densities, and from normal to pathological angiogenesis. A better understanding of the control of angiogenesis is necessary to improve upon limitations of current angiogenic therapies. PMID:24332926

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

    PubMed

    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.

  15. 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

  16. Engineered microenvironments for synergistic VEGF - Integrin signalling during vascularization.

    PubMed

    Moulisová, Vladimíra; Gonzalez-García, Cristina; Cantini, Marco; Rodrigo-Navarro, Aleixandre; Weaver, Jessica; Costell, Mercedes; Sabater I Serra, Roser; Dalby, Matthew J; García, Andrés J; Salmerón-Sánchez, Manuel

    2017-05-01

    We have engineered polymer-based microenvironments that promote vasculogenesis both in vitro and in vivo through synergistic integrin-growth factor receptor signalling. Poly(ethyl acrylate) (PEA) triggers spontaneous organization of fibronectin (FN) into nanonetworks which provide availability of critical binding domains. Importantly, the growth factor binding (FNIII12-14) and integrin binding (FNIII9-10) regions are simultaneously available on FN fibrils assembled on PEA. This material platform promotes synergistic integrin/VEGF signalling which is highly effective for vascularization events in vitro with low concentrations of VEGF. VEGF specifically binds to FN fibrils on PEA compared to control polymers (poly(methyl acrylate), PMA) where FN remains in a globular conformation and integrin/GF binding domains are not simultaneously available. The vasculogenic response of human endothelial cells seeded on these synergistic interfaces (VEGF bound to FN assembled on PEA) was significantly improved compared to soluble administration of VEGF at higher doses. Early onset of VEGF signalling (PLCγ1 phosphorylation) and both integrin and VEGF signalling (ERK1/2 phosphorylation) were increased only when VEGF was bound to FN nanonetworks on PEA, while soluble VEGF did not influence early signalling. Experiments with mutant FN molecules with impaired integrin binding site (FN-RGE) confirmed the role of the integrin binding site of FN on the vasculogenic response via combined integrin/VEGF signalling. In vivo experiments using 3D scaffolds coated with FN and VEGF implanted in the murine fat pad demonstrated pro-vascularization signalling by enhanced formation of new tissue inside scaffold pores. PEA-driven organization of FN promotes efficient presentation of VEGF to promote vascularization in regenerative medicine applications.

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

    PubMed

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

    2011-01-01

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

  18. UV induces VEGF through a TNF-alpha independent pathway.

    PubMed

    Kosmadaki, Maria G; Yaar, Mina; Arble, Bennett L; Gilchrest, Barbara A

    2003-03-01

    Vascular endothelial growth factor (VEGF) is a potent keratinocyte-derived angiogenic factor. Prior reports suggest that following UV irradiation VEGF in keratinocytes is induced primarily by tumor necrosis factor (TNF)- alpha, a cytokine synthesized and secreted by keratinocytes after UV irradiation. We investigated whether blocking TNF-alpha binding to its receptors would inhibit UV-induced VEGF expression and secretion in the keratinocyte-derived line SCC-12F. Irradiation with physiologic UV doses (30 mJ/cm2) substantially induced VEGF mRNA in this cell line, as expected, and mRNA induction was followed by increased VEGF in medium conditioned by UV-irradiated cells. Also as expected, TNF-alpha induced VEGF expression and secretion in a dose-dependent manner. Addition of a hexapeptide (Ac-KWIIVW-NH2), known to block TNF-alpha binding to its receptors, abrogated this TNF-alpha effect on VEGF mRNA induction. However, addition of the peptide to cells immediately after UV irradiation did not substantially affect VEGF mRNA induction or secretion into the medium. Our results suggest that VEGF induction after UV irradiation is mediated by multiple mechanisms and that blocking a single pathway does not affect the response.

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

    PubMed

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

    2014-01-01

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

  20. High-affinity VEGF antagonists by oligomerization of a minimal sequence VEGF-binding domain.

    PubMed

    Stefano, James E; Bird, Julie; Kyazike, Josephine; Cheng, Anthony Wai-Ming; Boudanova, Ekaterina; Dwyer, Markryan; Hou, Lihui; Qiu, Huawei; Matthews, Gloria; O'Callaghan, Michael; Pan, Clark Q

    2012-12-19

    Vascular endothelial growth factor (VEGF) neutralizing antagonists including antibodies or receptor extracellular domain Fc fusions have been applied clinically to control angiogenesis in cancer, wet age-related macular degeneration, and edema. We report here the generation of high-affinity VEGF-binding domains by chemical linkage of the second domain of the VEGF receptor Flt-1 (D2) in several configurations. Recombinant D2 was expressed with a 13 a.a. C-terminal tag, including a C-terminal cysteine to enable its dimerization by disulfide bond formation or by attachment to divalent PEGs and oligomerization by coupling to multivalent PEGs. Disulfide-linked dimers produced by Cu(2+) oxidation of the free-thiol form of the protein demonstrated picomolar affinity for VEGF in solution, comparable to that of a D2-Fc fusion (sFLT01) and ~50-fold higher than monomeric D2, suggesting the 26 a.a. tag length between the two D2 domains permits simultaneous interaction of both faces of the VEGF homodimer. Extending the separation between the D2 domains by short PEG spacers from 0.35 kD to 5 kD produced a modest ~2-fold increase in affinity over the disulfide, thus defining the optimal distance between the two D2 domains for maximum affinity. By surface plasmon resonance (SPR), a larger (~5-fold) increase in affinity was observed by conjugation of the D2 monomer to the termini of 4-arm PEG, and yielding a product with a larger hydrodynamic radius than sFLT01. The higher affinity displayed by these D2 PEG tetramers than either D2 dimer or sFLT01 was largely a consequence of a slower rate of dissociation, suggesting the simultaneous binding by these tetramers to neighboring surface-bound VEGF. Finally, disulfide-linked D2 dimers showed a greater resistance to autocatalytic fragmentation than sFLT01 under elevated temperature stress, indicating such minimum-sequence constructs may be better suited for sustained-release formulations. Therefore, these constructs represent novel Fc

  1. RNA Activation of the Vascular Endothelial Growth Factor Gene (VEGF) Promoter by Double-Stranded RNA and Hypoxia: Role of Noncoding VEGF Promoter Transcripts

    PubMed Central

    Wagner, Kay-Dietrich; Hofman, Paul; Van Obberghen, Emmanuel

    2016-01-01

    RNA activation (RNAa) is a gene regulation process in which promoter-targeted short double-stranded RNAs (dsRNAs) or microRNAs (miRs) induce target gene expression at the transcriptional level. Here, we investigate the presence of cryptic promoter transcripts within the VEGF promoter. Single-strand sense and antisense noncoding vascular endothelial growth factor (NcVEGF) promoter transcripts are identified, and their respective expression is studied in cells transfected with a VEGF promoter targeted dsRNA, namely, dsVEGF706, in hypoxic cells and in human malignant lung tissues. Interestingly, in dsVEGF706-transfected, as well as in hypoxic cells, NcVEGF expression levels increase coordinately with coding VEGF expression. Ago2 interaction with both sense and antisense NcVEGFs is increased in hypoxic cells, whereas in dsVEGF706-transfected cells, Ago2 and the antisense strand of the dsRNA interact specifically with the sense NcVEGF transcript. Furthermore, both dsVEGF706 and ectopic NcVEGF transcripts are able to activate the VEGF promoter endogenously present or in a reporter construct. Finally, using small interfering RNA targeting Ago2, we show that RNAa plays a role in the maintenance of increased VEGF and NcVEGF expression after hypoxia. Given the central role of VEGF in major human diseases, including cancer, this novel molecular mechanism is poised to reveal promising possibilities for therapeutic interventions. PMID:26976645

  2. Anti-VEGF Treatment Strategies for Wet AMD.

    PubMed

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

    2012-01-01

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

  3. [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.

  4. 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.

  5. Stretch-induced VEGF expression in the heart.

    PubMed Central

    Li, J; Hampton, T; Morgan, J P; Simons, M

    1997-01-01

    Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen involved in vascular development and angiogenesis. Recently we have observed increased VEGF expression in the normal myocardium after myocardial infarction in a rat heart. This study was designed to explore the mechanism responsible for this increase in VEGF expression. Induction of myocardial stretch in an isolated perfused Langendorff preparation by inflation of an intraventricular balloon to an end-diastolic load of 35 mmHg for 30 min resulted in a nearly sixfold increase in VEGF message level not only in the chamber subjected to stretch (left ventricle) but also in the unstretched right ventricle, thus raising the possibility of a soluble factor mediating stretch- induced induction of VEGF expression. This was further confirmed by demonstrating that coronary venous effluent collected from the stretched heart and used to perfuse isolated hearts in which no balloon was present was able to induce VEGF expression in these normal hearts. Inhibition of TGF-beta activity using a neutralizing antibody, but not antagonists/inhibitors of endothelin and angiotensin II, eliminated stretch-induced increase in VEGF expression. Staurosporine, a protein kinase C inhibitor, also blocked stretch-induced increase of VEGF expression. Measurement of TGF-beta concentration in the perfusate demonstrated increased amounts of the cytokine after myocardial stretch, and addition of TGF-beta protein to the perfusion buffer resulted in increased VEGF expression in control hearts. These results suggest that stretch-induced increase of VEGF expression in the heart is mediated at least in part by TGF-beta. PMID:9202052

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

    PubMed

    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

    2015-06-02

    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.

  7. 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

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

    PubMed Central

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

    2014-01-01

    Objective: To present a series of 50 consecutive patients with non-metastatic extremity osteosarcoma, and attempt to correlate expression of the vascular endothelial growth factor (VEGF) protein in biopsy tissue to their prognosis regarding overall survival, disease-free survival and local recurrence. Methods: Fifty cases of non-metastatic osteosarcoma of the extremities treated between 1986 and 2006 at Instituto de Ortopedia e Traumatologia da Universidade de São Paulo, São Paulo, Brasil, were evaluated regarding expression of the VEGF protein. There were 19 females and 31 males. The mean age was 16 years old (range 5-28 years old) and the mean follow-up was 60.6 months (range 25-167 months). The variables studied were age, gender, anatomic location, type of surgery, surgical margins, tumor size, post chemotherapy necrosis, local recurrence, pulmonary metastasis and death. Results: Thirty-six patients showed VEGF expression on 30% or less cells (low), and the remaining 14 cases had VEGF expression above 30% (high). Among the 36 patients with low VEGF expression, nine developed pulmonary metastasis and four died (11.1%). Among the 14 patients with high VEGF expression, six developed pulmonary metastasis and three died (21.4%). Conclusion: There was no statistically significant correlation between the expression of VEGF and any of the variables studied. Level of Evidence IV, Therapeutic Study. PMID:25328432

  9. VEGF expression in human brain tissue after acute ischemic stroke.

    PubMed

    Mărgăritescu, Otilia; Pirici, D; Mărgăritescu, Cl

    2011-01-01

    Ischemic stroke is the third most common cause of death in humans, requiring further studies to elucidate its pathophysiological background. One potential mechanism to increase oxygen delivery to the affected tissue is induction of angiogenesis. The most potent proangiogenic factor is VEGF. For this reason, our study investigated immunohistochemically VEGF reactivity in different cellular brain compartments from 15 ischemic stroke patients, as well as from 2 age control cases. By enzymatic immunohistochemistry, we investigate VEGF expression in different brain cell compartments and then we quantified its signal intensity by assessing integrated optical densities (IOD). To establish the exact cellular brain topography of VEGF immunoreactivity we performed double fluorescent immunohistochemistry series (VEGF÷NeuN, GFAP, CD68, CD105). In control samples, VEGF reactivity was observed especially in neurons from the Brodmann cortical layers IV to VI and in protoplasmic astrocytes from the deeper layers of gray matter and in endothelial cells from normal blood vessels because of systemic hypoxia generated after death. In acute ischemic stroke samples, this reactivity was noticed in all brain cellular compartments but with different intensities. The most reactive compartment was the neurons, the intensity of VEGF reaction decreasing with the lesional age from the core infarct toward intact adjacent brain cortex. With a lower intensity, VEGF reaction was noticed in astrocytes compartments, especially in gemistocytic astrocytes adjacent to the liquefaction zone. We also noticed a weak reaction in activated non-phagocytic microglia from the periphery of liquefaction zones, and high VEGF-CD105 colocalization values at the level of microvessels that surround the infarcted brain area. In conclusion, this reactivity could suggest that VEGF might exhibit neuronal and glial protective effects and also a neoangiogenic property in acute ischemic stroke, facts that may have

  10. 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

  11. Analysis of VEGFA Variants and Changes in VEGF Levels Underscores the Contribution of VEGF to Polycystic Ovary Syndrome

    PubMed Central

    Almawi, Wassim Y; Gammoh, Emily; Malalla, Zainab H.; Al-Madhi, Safa A.

    2016-01-01

    Background Vascular endothelial growth factor (VEGF) contributes to the pathogenesis of polycystic ovary syndrome (PCOS), and genetic variations in VEGFA gene were suggested to contribute to VEGF secretion and PCOS. Aim To evaluate the association of altered VEGF levels, stemming from the presence of specific VEGFA variants, with altered risk of PCOS. Subjects and Methods Retrospective case-control study, performed between 2012–2015. Study subjects comprised 382 women with PCOS, and 393 control subjects. ELISA measured VEGF levels; genotyping of VEGFA variants was done by allelic exclusion. Results Among the 12 tested VEGFA SNPs, minor allele frequency of only rs3025020 was significantly higher in PCOS cases than control women. Increased and reduced PCOS risk was seen with rs3025020 and rs2010963 genotypes, respectively. Increases and reduction in VEGF levels were associated with rs3025020 and rs2010963, respectively. Increased fasting insulin and HOMA-IR, and bioactive testosterone were linked with rs3025020, while carriage of rs2010963 was linked with reduction in fasting insulin, and free and bioactive testosterone. Of the 12 VEGFA variants, 9 were in LD, thus allowing construction of 9-locus haplotypes. Increased frequency of CAACAGCGA haplotype was seen in PCOS cases, after controlling for BMI, free and bioactive testosterone, SHBG, free insulin and HOMA-IR. Conclusion This study confirms the contribution of altered VEGF secretion, resulting from genetic variation in VEGFA gene into the pathogenesis of PCOS. This supports a role for VEGF as PCOS candidate locus. PMID:27846231

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

    PubMed Central

    Aliparasti, Mohammad Reza; Almasi, Shohreh; Sanaat, Zohreh; Movasaghpoor, Aliakbar; Khalili-Dizaji, Reza; Sadeghi-Bazargani, Homayoun

    2013-01-01

    Objective: The crucial role of angiogenesis in the pathophysiology of acute myeloid leukemia (AML) has been proposed. One of the key regulators of angiogenesis is the vascular endothelial growth factor (VEGF). Among the VEGF family, it has been observed that VEGF-A and VEGF-C are expressed by AML cells and mediate leukemic cell proliferation, survival, and resistance to chemotherapy. Emerging evidence, however, suggests that elevated levels of VEGF or a proangiogenic phenotype may impede, rather than promote, early tumor development and progression. As the significance of VEGF-A and VEGF-C levels in the pathogenesis of AML has not been clarified well, the aim of this study is to evaluate gene expression of these angiogenesis promoters and its possible prognostic value in peripheral blood mononuclear cells of Iranian patients with AML. Materials and Methods: We investigated the mRNA expression of VEGF-A and VEGF-C in peripheral blood mononuclear cells of 27 patients with newly diagnosed AML and 28 healthy controls by quantitative real-time PCR. Results: Expression of VEGF-C mRNA was significantly lower in AML patients than in healthy controls (p<0.001). However, there was no significant decrement in expression of VEGF-A mRNA of AML patients compared to the control group (p=0.861). VEGF-A and VEGF-C expression were not able to predict clinical outcome. Conclusion: Our data showed that AML is associated with a decreased expression of VEGF-C mRNA. However, expression levels did not influence the clinical outcome in our study. It seems that angiogenesis is affected by different cytokines other than VEGF-C or VEGF-A, and VEGF is also affected by different cytokines. Taken together, these findings help to provide new insights into the investigation of other angiogenic factors and cytokines that may play roles in the pathogenesis of AML. Conflict of interest:None declared. PMID:24385776

  13. Thrombospondin-1 and VEGF in inflammatory bowel disease

    PubMed Central

    Alkim, Canan; Sakiz, Damlanur; Alkim, Huseyin; Livaoglu, Ayten; Kendir, Tulin; Demirsoy, Huseyin; Erdem, Levent; Akbayir, Nihat; Sokmen, Mehmet

    2012-01-01

    Background and aim Angiogenesis is an important process in the pathogenesis of chronic inflammation. We aimed to study the angiogeneic balance in inflammatory bowel disease (IBD) by evaluating the expression of vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1) on colonic epithelial cells, together with the expression of inducible nitric oxide synthase (iNOS). Methods Twenty-one ulcerative colitis (UC), 14 Crohn's disease (CD), 11 colorectal cancer patients, and 11 healthy controls colonic biopsy samples were evaluated immunohistochemically. Results The expressions of TSP-1, VEGF, and iNOS in UC and CD groups were higher than expression in healthy control group, all with statistical significance. However, in colorectal cancer group, VEGF and iNOS expressions were increased importantly, but TSP-1 expression was not statistically different from healthy control group's expression. Both TSP-1 and VEGF expressions were correlated with iNOS expression distinctly but did not correlate with each other. Conclusions Both pro-angiogeneic VEGF and antiangiogeneic TSP-1 expressions were found increased in our IBD groups, but in colorectal cancer group, only VEGF expression was increased. TSP-1 increases in IBD patients as a response to inflammatory condition, but this increase was not enough to suppress pathologic angiogenesis and inflammation in IBD. PMID:22299021

  14. Vascular endothelial growth factor directly stimulates tumour cell proliferation in non-small cell lung cancer.

    PubMed

    Devery, Aoife M; Wadekar, Rekha; Bokobza, Sivan M; Weber, Anika M; Jiang, Yanyan; Ryan, Anderson J

    2015-09-01

    Vascular endothelial growth factor (VEGF) is a key stimulator of physiological and pathological angiogenesis. VEGF signals primarily through VEGF receptor 2 (VEGFR2), a receptor tyrosine kinase whose expression is found predominantly on endothelial cells. The purpose of this study was to determine the role of VEGFR2 expression in NSCLC cells. NSCLC cells and tissue sections were stained for VEGFR2 expression by immunohistochemistry (IHC). Immunoblotting and ELISA were used to determine the activation and inhibition of VEGFR2 and its downstream signalling pathways. Five-day proliferation assays were carried out in the presence or absence of VEGF. IHC analysis of NSCLC demonstrated tumour cell VEGFR2 expression in 20% of samples. Immunoblot analysis showed expression of VEGFR2 protein in 3/8 NSCLC cell lines that correlated with VEGFR2 mRNA expression levels. VEGF-dependent VEGFR2 activation was apparent in NSCLC cells, and was associated with increased tumor cell proliferation. Cediranib treatment or siRNA against VEGFR2 inhibited VEGF-dependent increases in cell proliferation. Inhibition of VEGFR2 tyrosine kinase activity using cediranib was more effective than inhibition of AKT (MK2206) or MEK (AZD6244) for overcoming VEGFR2-driven cell proliferation. VEGF treatment did not affect cell survival following treatment with radiation, cisplatin, docetaxel or gemcitabine. Our data suggest that a subset of NSCLC tumour cells express functional VEGFR2 which can act to promote VEGF-dependent tumour cell growth. In this tumour subset, therapies targeting VEGFR2 signalling, such as cediranib, have the potential to inhibit both tumour cell proliferation and angiogenesis.

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

    PubMed

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

    2010-03-15

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

  16. 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

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

    PubMed

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

    2016-10-11

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

  18. 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

  19. Pancreatic duct cells as a source of VEGF in mice

    PubMed Central

    Xiao, Xiangwei; Prasadan, Krishna; Guo, Ping; El-Gohary, Yousef; Fischbach, Shane; Wiersch, John; Gaffar, Iljana; Shiota, Chiyo; Gittes, George K.

    2014-01-01

    Aims/hypothesis Vascular endothelial growth factor (VEGF) is essential for proper pancreatic development, islet vascularisation and insulin secretion. In the adult pancreas, VEGF is thought to be predominantly secreted by beta cells. Although human duct cells have previously been shown to secrete VEGF at angiogenic levels in culture, an analysis of the kinetics of VEGF synthesis and secretion, as well as elucidation of an in vivo role for this ductal VEGF in affecting islet function and physiology, has been lacking. Methods We analysed purified duct cells independently prepared by flow cytometry, surgical isolation or laser-capture microdissection. We infected duct cells in vivo with Vegfa short hairpin RNA (shRNA) in an intrapancreatic ductal infusion system and examined the effect of VEGF knockdown in duct cells in vitro and in vivo. Results Pancreatic duct cells express high levels of Vegfa mRNA. Compared with beta cells, duct cells had a much higher ratio of secreted to intracellular VEGF. As a bioassay, formation of tubular structures by human umbilical vein endothelial cells was essentially undetectable when cultured alone and was substantially increased when co-cultured with pancreatic duct cells but significantly reduced when co-cultured with duct cells pretreated with Vegfa shRNA. Compared with islets transplanted alone, improved vascularisation and function was detected in the islets co-transplanted with duct cells but not in islets co-transplanted with duct cells pretreated with Vegfa shRNA. Conclusions/interpretation Human islet preparations for transplantation typically contain some contaminating duct cells and our findings suggest that the presence of duct cells in the islet preparation may improve transplantation outcomes. PMID:24535231

  20. Galectin-3 Protein Modulates Cell Surface Expression and Activation of Vascular Endothelial Growth Factor Receptor 2 in Human Endothelial Cells*

    PubMed Central

    Markowska, Anna I.; Jefferies, Kevin C.; Panjwani, Noorjahan

    2011-01-01

    Angiogenesis is heavily influenced by VEGF-A and its family of receptors, particularly VEGF receptor 2 (VEGF-R2). Like most cell surface proteins, VEGF-R2 is glycosylated, although the function of VEGF-R2 with respect to its glycosylation pattern is poorly characterized. Galectin-3, a glycan binding protein, interacts with the EGF and TGFβ receptors, retaining them on the plasma membrane and altering their signal transduction. Because VEGF-R2 is glycosylated and both galectin-3 and VEGF-R2 are involved with angiogenesis, we hypothesized that galectin-3 binds VEGF-R2 and modulates its signal transduction as well. Employing a Western blot analysis approach, we found that galectin-3 induces phosphorylation of VEGF-R2 in endothelial cells. Knockdown of galectin-3 and Mgat5, an enzyme that synthesizes high-affinity glycan ligands of galectin-3, reduced VEGF-A mediated angiogenesis in vitro. A direct interaction on the plasma membrane was detected between galectin-3 and VEGF-R2, and this interaction was dependent on the expression of Mgat5. Using immunofluorescence and cell surface labeling, we found an increase in the level of internalized VEGF-R2 in both Mgat5 and galectin-3 knockdown cells, suggesting that galectin-3 retains the receptor on the plasma membrane. Finally, we observed reduced suture-induced neovascularization in the corneas of Gal3−/− and Mgat5−/− mice. These findings are consistent with the hypothesis that, like its role with the EGF and TGFβ receptors, galectin-3 contributes to the plasma membrane retention and proangiogenic function of VEGF-R2. PMID:21715322

  1. Killing Breast Cancer Cells With a VEGF-Triggered Cell Death Receptor

    DTIC Science & Technology

    2006-04-01

    patients. We are pursuing a totally different approach to targeting VEGF: rather than inhibit VEGF our goal is to convert VEGF to act as a cell death factor...cell lines in vitro. These studies suggest that a receptor such as R2Fas which converts VEGF to act as a cell death factor could yield a new and more aggressive approach to targeting overexpressed VEGF in breast cancer....Toward this aim we created a chimeric receptor (R2Fas) composed of domains from VEGF receptor 2 fused to the intracellular domain of the Fas cell

  2. 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.

  3. Chromatin associated SETD3 negatively regulates VEGF expression

    PubMed Central

    Cohn, Ofir; Feldman, Michal; Weil, Lital; Kublanovsky, Margarita; Levy, Dan

    2016-01-01

    SETD3 is a member of the protein lysine methyltransferase (PKMT) family, which catalyzes the addition of methyl group to lysine residues. Accumulating data suggest that PKMTs are involved in the regulation of a broad spectrum of biological processes by targeting histone and non-histone proteins. Using a proteomic approach, we have identified 172 new SETD3 interacting proteins. We show that SETD3 binds and methylates the transcription factor FoxM1, which has been previously shown to be associated with the regulation of VEGF expression. We further demonstrate that under hypoxic conditions SETD3 is down-regulated. Mechanistically, we find that under basal conditions, SETD3 and FoxM1 are enriched on the VEGF promoter. Dissociation of both SETD3 and FoxM1 from the VEGF promoter under hypoxia correlates with elevated expression of VEGF. Taken together, our data reveal a new SETD3-dependent methylation-based signaling pathway at chromatin that regulates VEGF expression under normoxic and hypoxic conditions. PMID:27845446

  4. VEGFA — EDRN Public Portal

    Cancer.gov

    Vascular endothelial growth factor (VEGFA or VEGF) is a growth factor and signaling protein involved in the regulation of angiogenesis, vasculogenesis, and endothelial cell grown. It induces endothelial cell proliferation, promotes cell migration, inhibits apoptosis and induces permeabilization of blood vessels. Three VEGFR isoforms have been identified in humans; VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1) and VEGFR-3 (Flt-4). VEGFR-2 mediates the majority of cellular responses to VEGF. VEGFR-1 is thought to modulate VEGFR-2 signaling or to act as a dummy/decoy receptor to sequester VEGF away from VEGFR-2.

  5. 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

  6. The Angiogenic Secretome in VEGF overexpressing Breast Cancer Xenografts

    PubMed Central

    Dore-Savard, Louis; Lee, Esak; Kakkad, Samata; Popel, Aleksander S.; Bhujwalla, Zaver M.

    2016-01-01

    The plasticity of cancer cells and the fluidity of the tumor microenvironment continue to present major challenges in the comprehensive understanding of cancer that is essential to design effective treatments. The tumor interstitial fluid (TIF) encompasses the secretome and holds the key to several of the phenotypic characteristics of cancer. Difficulties in sampling this fluid have resulted in limited characterization of its components. Here we have sampled TIF from triple negative and estrogen receptor (ER)-positive human breast tumor xenografts with or without VEGF overexpression. Angiogenesis-related factors were characterized in the TIF and plasma, to understand the relationship between the TIF and plasma secretomes. Clear differences were observed between the TIF and plasma angiogenic secretomes in triple negative MDA-MB-231 breast cancer xenografts compared to ER-positive MCF-7 xenografts with or without VEGF overexpression that provide new insights into TIF components and the role of VEGF in modifying the angiogenic secretome. PMID:27995973

  7. 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

  8. 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. PMID:27819042

  9. Vascular Endothelial Growth Factor (VEGF) Pathway as a Therapeutic Target in Gynecologic Malignancies

    PubMed Central

    Frumovitz, Michael; Sood, Anil K.

    2007-01-01

    Vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis, which is required for tumor growth and metastasis. In this article, a review of the functional and biological roles of the VEGF pathway in driving angiogenesis and growth of gynecologic malignancies was performed. Based on the biological functions of VEGF, multiple approaches for targeting the VEGF/VEGF-receptor complex have been developed and many of these have demonstrated substantial activity in preclinical models. These promising data have led to rapid clinical development of VEGF-targeted agents. Therefore, we also assessed the status of VEGF-targeted therapies and associated toxicities in gynecologic malignancies. However, many questions remain related to optimal dosing, sequencing of therapies, management of toxicities, appropriate patient selection, and assessment of response, which will require further studies. Nevertheless, VEGF-targeted therapies offer hope for improving the outcome of cancer patients. PMID:17306693

  10. DAAs Rapidly Reduce Inflammation but Increase Serum VEGF Level: A Rationale for Tumor Risk during Anti-HCV Treatment

    PubMed Central

    Villani, Rosanna; Facciorusso, Antonio; Bellanti, Francesco; Tamborra, Rosanna; Piscazzi, Annamaria; Landriscina, Matteo; Vendemiale, Gianluigi; Serviddio, Gaetano

    2016-01-01

    Background Novel direct-acting antivirals (DAAs) have completely changed the panorama of hepatitis C due to their high efficacy and optimal safety profile. Unfortunately, an unexpectedly high rate of early recurrence of hepatocellular carcinoma has been reported within weeks of starting treatment, but the mechanism is not known. Methods We monitored the serum level of vascular endothelial growth factor (VEGF) and changes in the pattern of circulating interleukins in 103 chronic hepatitis C patients during antiviral treatment with DAA-regimens. VEGF, epidermal growth factor (EGF), and several interleukins were assessed at baseline, during treatment, and after treatment. The biological effect of DAA-treated patient serum on human umbilical vein endothelial cell (HUVEC) proliferation was also confirmed. Results After 4 weeks of therapy, VEGF increased approximately 4-fold compared to baseline, remained elevated up to the end of treatment, and returned to the pre-treatment level after the end of therapy. In contrast, interleukin-10 and tumor necrosis factor-alpha significantly decreased during therapy, which was coincident with HCV clearance. The levels of both remained low after treatment. The addition of serum from patients collected during therapy induced HUVEC proliferation; however, this disappeared after the end of therapy. Conclusions DAA administration induces an early increase in serum VEGF and a change in the inflammatory pattern, coinciding with HCV clearance. This may alter the balance between inflammatory and anti-inflammatory processes and modify the antitumor surveillance of the host. Fortunately, such modifications return reverse to normal after the end of treatment. PMID:27997563

  11. The biophysical property of A549 cells transferred by VEGF-D.

    PubMed

    Wang, Zhen; Wu, Xiu-Li; Wang, Xu; Tian, Hong-Xia; Chen, Zhi-Hong; Li, Yang-Qiu

    2014-01-01

    Vascular endothelial growth factor-D (VEGF-D) together with VEGF-C is considered to be associated with lymphangiogenesis and angiogenesis and involve in tumorization. This study aims to investigate the influence of exogenous VEGF-D gene on the biophysical property of cell surface of lung adenocarcinoma cell line. A panel of lung adenocarcinoma cell lines were examined the expression of VEGF-D and VEGF-C by real-time PCR. The VEGF-D recombinant plasmid containing enhanced green fluorescence protein (EGFP) was constructed and transfected to the cell line with no expression of VEGF-D and confirmed by real-time PCR and Western blot analysis. Topographic images of cells were obtained by using atomic force microscope (AFM) in contact mode. Unlike VEGF-C, VEGF-D was found to have a very low expression or undetectable expression in lung adenocarcinoma cell lines. The VEGF-D recombinant plasmid had been constructed successfully and was transferred into the human lung adenocarcinoma cell line A549 cells which had no endogenous expression of VEGF-D, and exogenous VEGF-D could be detected in mRNA and protein expression levels in the gene modified cells, while the VEGF-C gene expression had no change after VEGF-D transfection. After transfection, the irregular microspikes or nano clusters could observe on the surface of A549 cells, and VEGF-D transfected A549 cells became more rigid. The exogenous VEGF-D gene might cause the remarkable biophysical architectural changes in the A549 cells, which might as a novel biomarker for evaluation of its biological function.

  12. Simulating vasogenic brain edema using chronic VEGF infusion.

    PubMed

    Piazza, Martin; Munasinghe, Jeeva; Murayi, Roger; Edwards, Nancy; Montgomery, Blake; Walbridge, Stuart; Merrill, Marsha; Chittiboina, Prashant

    2017-01-06

    OBJECTIVE To study peritumoral brain edema (PTBE), it is necessary to create a model that accurately simulates vasogenic brain edema (VBE) without introducing a complicated tumor environment. PTBE associated with brain tumors is predominantly a result of vascular endothelial growth factor (VEGF) secreted by brain tumors, and VEGF infusion alone can lead to histological blood-brain barrier (BBB) breakdown in the absence of tumor. VBE is intimately linked to BBB breakdown. The authors sought to establish a model for VBE with chronic infusion of VEGF that can be validated by serial in-vivo MRI and histological findings. METHODS Male Fischer rats (n = 182) underwent stereotactic striatal implantation of MRI-safe brain cannulas for chronic infusion of VEGF (2-20 µg/ml). Following a preinfusion phase (4-6 days), the rats were exposed to VEGF or control rat serum albumin (1.5 µl/hr) for as long as 144 hours. Serial MRI was performed during infusion on a high-field (9.4-T) machine at 12-24, 24-36, 48-72, and 120-144 hours. Rat brains were then collected and histological analysis was performed. RESULTS Control animals and animals infused with 2 µg/ml of VEGF experienced no neurological deficits, seizure activity, or abnormal behavior. Animals treated with VEGF demonstrated a significantly larger volume (42.90 ± 3.842 mm(3)) of T2 hyper-attenuation at 144 hours when compared with the volume (8.585 ± 1.664 mm(3)) in control animals (mean difference 34.31 ± 4.187 mm(3), p < 0.0001, 95% CI 25.74-42.89 mm(3)). Postcontrast T1 enhancement in the juxtacanalicular region indicating BBB breakdown was observed in rats undergoing infusion with VEGF. At the later time periods (120-144 hrs) the volume of T1 enhancement (34.97 ± 8.99 mm(3)) was significantly less compared with the region of edema (p < 0.0001). Histologically, no evidence of necrosis or inflammation was observed with VEGF or control infusion. Immunohistochemical analysis demonstrated astrocyte activation, vascular

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

    PubMed

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

    2016-03-01

    The objectives of this study were to determine whether the main opioid receptor (OPRM1) is present on human granulosa cells and if exogenous opiates and their antagonists can influence granulosa cell vascular endothelial growth factor (VEGF) production via OPRM1. Granulosa cells were isolated from women undergoing oocyte retrieval for IVF. Complementary to the primary cells, experiments were conducted using COV434, a well-characterized human granulosa cell line. Identification and localization of opiate receptor subtypes was carried out using Western blot and flow cytometry. The effect of opiate antagonist on granulosa cell VEGF secretion was assessed by enzyme-linked immunosorbent assay. For the first time, the presence of OPRM1 on human granulosa cells is reported. Blocking of opiate signalling using naloxone, a specific OPRM1 antagonist, significantly reduced granulosa cell-derived VEGF levels in both COV434 and granulosa-luteal cells (P < 0.01). The presence of opiate receptors and opiate signalling in granulosa cells suggest a possible role in VEGF production. Targeting this signalling pathway could prove promising as a new clinical option in the prevention and treatment of ovarian hyperstimulation syndrome.

  14. Survivin and VEGF as Novel Biomarkers in Diagnosis of Endometriosis

    PubMed Central

    Vidakovic, Snezana; Milic, Natasa; Jeremic, Katarina; Markovic, Milos; Milosevic-Djeric, Ana; Lazovic-Radonjic, Gordana

    2016-01-01

    Summary Background The aim of this study was to investigate the role of peripheral blood markers as additional diagnostic tools to transvaginal ultrasound (TVU) findings in the diagnosis of endometriosis. Methods This study included 40 patients undergoing laparoscopy for suspected endometriosis from January to December 2012. Preoperative levels of serum CA125, CA19-9, CEA and mRNA expression levels for survivin and VEGF were obtained. Real-time PCR was used to determine relative gene expression. A new diagnostic score was obtained by deploying the peripheral blood markers to the TVU findings. Statistical methods used were Chi-square, Fisher’s, Student’s t-test or the Mann – Whitney test. Results There was a statistically significant difference in serum CA125, survivin and VEGF levels in patients with endometriosis and those without endometriosis (p<0.001, p=0.025 and p=0.009, respectively). False negative TVU findings were noted in 3/13 patients (23.1%) with peritoneal endometriosis without ovaries involvement. High sensitivity (93.3%), specificity (90.0%), PPV (96.6%), NPV (81.8%) and accuracy (92.5%) were obtained for a diagnostic score based on TVU and significant peripheral blood markers (CA125, survivin and VEGF). Conclusions Determination of serum CA125, mRNA expression levels for survivin and VEGF along with TVU can contribute to higher accuracy of the noninvasive diagnostic tools for endometriosis. PMID:28356866

  15. VEGF induces sensory and motor peripheral plasticity, alters bladder function, and promotes visceral sensitivity

    PubMed Central

    2012-01-01

    Background This work tests the hypothesis that bladder instillation with vascular endothelial growth factor (VEGF) modulates sensory and motor nerve plasticity, and, consequently, bladder function and visceral sensitivity. In addition to C57BL/6J, ChAT-cre mice were used for visualization of bladder cholinergic nerves. The direct effect of VEGF on the density of sensory nerves expressing the transient receptor potential vanilloid subfamily 1 (TRPV1) and cholinergic nerves (ChAT) was studied one week after one or two intravesical instillations of the growth factor. To study the effects of VEGF on bladder function, mice were intravesically instilled with VEGF and urodynamic evaluation was assessed. VEGF-induced alteration in bladder dorsal root ganglion (DRG) neurons was performed on retrogradly labeled urinary bladder afferents by patch-clamp recording of voltage gated Na+ currents. Determination of VEGF-induced changes in sensitivity to abdominal mechanostimulation was performed by application of von Frey filaments. Results In addition to an overwhelming increase in TRPV1 immunoreactivity, VEGF instillation resulted in an increase in ChAT-directed expression of a fluorescent protein in several layers of the urinary bladder. Intravesical VEGF caused a profound change in the function of the urinary bladder: acute VEGF (1 week post VEGF treatment) reduced micturition pressure and longer treatment (2 weeks post-VEGF instillation) caused a substantial reduction in inter-micturition interval. In addition, intravesical VEGF resulted in an up-regulation of voltage gated Na+ channels (VGSC) in bladder DRG neurons and enhanced abdominal sensitivity to mechanical stimulation. Conclusions For the first time, evidence is presented indicating that VEGF instillation into the mouse bladder promotes a significant increase in peripheral nerve density together with alterations in bladder function and visceral sensitivity. The VEGF pathway is being proposed as a key modulator of

  16. 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.

  17. Development of humanized rabbit monoclonal antibodies against vascular endothelial growth factor receptor 2 with potential antitumor effects.

    PubMed

    Yu, Yanlan; Lee, Pierre; Ke, Yaohuang; Zhang, Yongke; Chen, Jungang; Dai, Jihong; Li, Mingzhen; Zhu, Weimin; Yu, Guo-Liang

    2013-07-05

    Vascular endothelial growth factor-A (VEGF-A) plays a critical role in physiologic and pathologic angiogenesis through its receptors especially through VEGFR2. The lack of cross-reactivity of monoclonal antibodies with human VEGFR2/mouse Flk-1 is a major obstacle in preclinical developments. In this study, using a unique hybridoma technique, we generated a panel of 30 neutralization anti-VEGFR2 rabbit monoclonal antibodies (RabMAbs) either blocking VEGF/VEGFR2 interaction or inhibiting VEGF-stimulated VEGFR2 tyrosine kinase phosphorylation. Among 18 RabMAbs with human/mouse VEGFR2 cross-reactivity, we humanized one lead candidate RabMAb by Mutational Lineage Guided (MLG) method and further demonstrated its potent inhibition of tumor growth in xenograft mouse model. Our study suggests that RabMAbs are highly relevant for therapeutic applications.

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

    PubMed

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

    2015-10-06

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

  19. Subcellular localisation of VEGF in different pituitary cells. Changes of its expression in oestrogen induced prolactinomas.

    PubMed

    Mukdsi, Jorge Humberto; De Paul, Ana Lucía; Gutiérrez, Silvina; Roth, Félix Daniel; Aoki, Agustín; Torres, Alicia Inés

    2005-10-01

    Vascular endothelial growth factor (VEGF) is an important angiogenic factor in the pituitary gland. The objective of this study was to unveil the VEGF subcellular localisation in different pituitary cell types and to evaluate changes in its expression at different time intervals after oestrogen stimulation. A relevant feature demonstrated was the identification of this cytokine in the nucleus and cytoplasm of lactotrophs, somatotrophs and gonadotrophs, as well as in follicle-stellate cells of male rats. Oestrogen treatment increased the number of VEGF immunopositive cells and its expression detected differentially by western blot in both nucleus and cytoplasm of pituitary cells when compared to the control. At ultrastructural level VEGF appeared associated with nucleolus and euchromatin involving a possible internal autocrine loop. In lactotrophs, the predominant cell of the tumour, VEGF was immunodetected in RER, Golgi complex, and vesicular organelles, supporting further the association with an auto-paracrine effect exerted by VEGF. The nucleus/cytoplasm ratio of VEGF revealed a prevalent accumulation of VEGF in the cytoplasm. The presence of VEGF in the nucleus may probably be associated with a translocation to this cell compartment. This study demonstrated a cytoplasmic and nuclear immunolocalisation of VEGF in normal and tumoural adenohypophyseal cells. In the course of prolactinoma development, the oestrogen stimulated VEGF expression in tumoural cells, promoting a vascular adaptation which contributes to growth and progression of the tumour.

  20. VEGF-B selectively regenerates injured peripheral neurons and restores sensory and trophic functions

    PubMed Central

    Guaiquil, Victor H.; Pan, Zan; Karagianni, Natalia; Fukuoka, Shima; Alegre, Gemstonn; Rosenblatt, Mark I.

    2014-01-01

    VEGF-B primarily provides neuroprotection and improves survival in CNS-derived neurons. However, its actions on the peripheral nervous system have been less characterized. We examined whether VEGF-B mediates peripheral nerve repair. We found that VEGF-B induced extensive neurite growth and branching in trigeminal ganglia neurons in a manner that required selective activation of transmembrane receptors and was distinct from VEGF-A–induced neuronal growth. VEGF-B–induced neurite elongation required PI3K and Notch signaling. In vivo, VEGF-B is required for normal nerve regeneration: mice lacking VEGF-B showed impaired nerve repair with concomitant impaired trophic function. VEGF-B treatment increased nerve regeneration, sensation recovery, and trophic functions of injured corneal peripheral nerves in VEGF-B–deficient and wild-type animals, without affecting uninjured nerves. These selective effects of VEGF-B on injured nerves and its lack of angiogenic activity makes VEGF-B a suitable therapeutic target to treat nerve injury. PMID:25404333

  1. VEGF-B promotes recovery of corneal innervations and trophic functions in diabetic mice

    PubMed Central

    Di, Guohu; Zhao, Xiaowen; Qi, Xia; Zhang, Songmei; Feng, Lu; Shi, Weiyun; Zhou, Qingjun

    2017-01-01

    Vascular endothelial growth factor (VEGF)-B possesses the capacity of promoting injured peripheral nerve regeneration and restore their sensory and trophic functions. However, the contribution and mechanism of VEGF-B in diabetic peripheral neuropathy remains unclear. In the present study, we investigated the expression and role of VEGF-B in diabetic corneal neuropathy by using type 1 diabetic mice and cultured trigeminal ganglion (TG) neurons. Hyperglycemia attenuated the endogenous expression of VEGF-B in regenerated diabetic corneal epithelium, but not that of VEGF receptors in diabetic TG neurons and axons. Exogenous VEGF-B promoted diabetic corneal nerve fiber regeneration through the reactivation of PI-3K/Akt-GSK3β-mTOR signaling and the attenuation of neuronal mitochondria dysfunction via the VEGF receptor-1 and neuropilin-1. Moreover, VEGF-B improved corneal sensation and epithelial regeneration in both normal and diabetic mice, accompanied with the elevated corneal content of pigment epithelial-derived factor (PEDF). PEDF blockade partially abolished trophic function of VEGF-B in diabetic corneal re-innervation. In conclusion, hyperglycemia suppressed endogenous VEGF-B expression in regenerated corneal epithelium of diabetic mice, while exogenous VEGF-B promoted recovery of corneal innervations and trophic functions through reactivating PI-3K/Akt-GSK-3β-mTOR signaling, attenuating neuronal oxidative stress and elevating PEDF expression. PMID:28091556

  2. Targeting VEGF and Its Receptors for the Treatment of Osteoarthritis and Associated Pain.

    PubMed

    Hamilton, John L; Nagao, Masashi; Levine, Brett R; Chen, Di; Olsen, Bjorn R; Im, Hee-Jeong

    2016-05-01

    Increased vascular endothelial growth factor (VEGF) levels are associated with osteoarthritis (OA) progression. Indeed, VEGF appears to be involved in OA-specific pathologies including cartilage degeneration, osteophyte formation, subchondral bone cysts and sclerosis, synovitis, and pain. Moreover, a wide range of studies suggest that inhibition of VEGF signaling reduces OA progression. This review highlights both the potential significance of VEGF in OA pathology and pain, as well as potential benefits of inhibition of VEGF and its receptors as an OA treatment. With the emergence of the clinical use of anti-VEGF therapy outside of OA, both as high-dose systemic treatments and low-dose local treatments, these particular therapies are now more widely understood. Currently, there is no established disease-modifying drug available for patients with OA, which warrants continued study of the inhibition of VEGF signaling in OA, as stand-alone or adjuvant therapy. © 2016 American Society for Bone and Mineral Research.

  3. 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

  4. 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

  5. VEGF-production by CCR2-dependent macrophages contributes to laser-induced choroidal neovascularization.

    PubMed

    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

  6. 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

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

    PubMed

    Tiong, Alice; Freedman, Saul Benedict

    2004-04-01

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

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

    PubMed

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

    2017-01-15

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

  9. Direct Contacts Between Extracellular Membrane-Proximal Domains are Required for VEGF Receptor Activation and Cell Signaling

    SciTech Connect

    Yang, Y.; Xie, P; Opatowsky, Y; Schlessinger, J

    2010-01-01

    Structural analyses of the extracellular region of stem cell factor (SCF) receptor (also designated KIT) in complex with SCF revealed a sequence motif in a loop in the fourth Ig-like domain (D4) that is responsible for forming homotypic receptor contacts and for ligand-induced KIT activation and cell signaling. An identical motif was identified in the most membrane-proximal seventh Ig-like domain (D7) of vascular endothelial growth factor receptor 1 (VEGFR1), VEGFR2, and VEGFR3. In this report we demonstrate that ligand-induced tyrosine autophosphorylation and cell signaling via VEGFR1 or VEGFR2 harboring mutations in critical residues (Arg726 or Asp731) in D7 are strongly impaired. We also describe the crystal structure of D7 of VEGFR2 to a resolution of 2.7 {angstrom}. The structure shows that homotypic D7 contacts are mediated by salt bridges and van der Waals contacts formed between Arg726 of one protomer and Asp731 of the other protomer. The structure of D7 dimer is very similar to the structure of D4 dimers seen in the crystal structure of KIT extracellular region in complex with SCF. The high similarity between VEGFR D7 and KIT D4 in both structure and function provides further evidence for common ancestral origins of type III and type V RTKs. It also reveals a conserved mechanism for RTK activation and a novel target for pharmacological intervention of pathologically activated RTKs.

  10. GFAT1 phosphorylation by AMPK promotes VEGF-induced angiogenesis.

    PubMed

    Zibrova, Darya; Vandermoere, Franck; Göransson, Olga; Peggie, Mark; Mariño, Karina V; Knierim, Anne; Spengler, Katrin; Weigert, Cora; Viollet, Benoit; Morrice, Nicholas A; Sakamoto, Kei; Heller, Regine

    2017-03-07

    Activation of AMP-activated protein kinase (AMPK) in endothelial cells regulates energy homeostasis, stress protection and angiogenesis, but the underlying mechanisms are incompletely understood. Using a label-free phosphoproteomic analysis, we identified glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1) as an AMPK substrate. GFAT1 is the rate-limiting enzyme in the hexosamine biosynthesis pathway (HBP) and as such controls the modification of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc). In the present study, we tested the hypothesis that AMPK controls O-GlcNAc levels and function of endothelial cells via GFAT1 phosphorylation using biochemical, pharmacological, genetic and in vitro angiogenesis approaches. Activation of AMPK in primary human endothelial cells by 5-aminoimidazole-4-carboxamide riboside (AICAR) or by vascular endothelial growth factor (VEGF) led to GFAT1 phosphorylation at serine 243. This effect was not seen when AMPK was down-regulated by siRNA. Upon AMPK activation, diminished GFAT activity and reduced O-GlcNAc levels were observed in endothelial cells containing wild-type (WT)-GFAT1 but not in cells expressing non-phosphorylatable S243A-GFAT1. Pharmacological inhibition or siRNA-mediated down-regulation of GFAT1 potentiated VEGF-induced sprouting, indicating that GFAT1 acts as a negative regulator of angiogenesis. In cells expressing S243A-GFAT1, VEGF-induced sprouting was reduced, suggesting that VEGF relieves the inhibitory action of GFAT1/HBP on angiogenesis via AMPK-mediated GFAT1 phosphorylation. Activation of GFAT1/HBP by high glucose led to impairment of vascular sprouting, whereas GFAT1 inhibition improved sprouting even if glucose level was high. Our findings provide novel mechanistic insights into the role of HBP in angiogenesis. They suggest that targeting AMPK in endothelium might help to ameliorate hyperglycaemia-induced vascular dysfunction associated with metabolic disorders.

  11. The remote effects of intravitreal anti-VEGF therapy

    PubMed Central

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

    2016-01-01

    Objective: To study the effects of intravitreal anti-Vascular Endothelial Growth Factor (VEGF) therapy with Avastin for wet Age-Related Macular Degeneration (AMD) on Benign Prostatic Hyperplasia (BPH)-related symptoms. Methods: An exploratory trial was conducted from August 1, 2013 to February 1, 2014, that included 14 male patients previously diagnosed with BPH, who were aged between 59 and 69 years. The trial was performed in Bucharest and involved two medical institutions: the Clinical Hospital of Eye Emergencies and the “Prof. Dr. Theodor Burghele” Hospital. This prospective study utilized both objective and subjective indicators to analyze the link between intravitreal anti-VEGF therapy for wet AMD and BPH. The evaluations consisted of uroflowmetry and International Prostate Symptom Score (I-PSS) assessments. Results: The maximum flow rate (Qmax) improved by an average of 5.05 ml/ sec in 9 patients, whereas the remaining 5 patients showed a slight decrease in Qmax (mean 1.6 ml/ sec). The I-PSS score improved, with an overall decrease of 1.18 points at follow-up compared to the initial score (mean initial score = 2.42; mean follow-up score = 1.24). Conclusion: The analysis revealed that anti-VEGF therapy for wet AMD had a significant positive effect on all BPH-related symptoms; patients reported improved urinary streams and decreased nocturia. Abbreviations: BPH = benign prostatic hyperplasia, AMD = age-related macular degeneration, VEGF = vascular endothelial growth factor, I-PSS = international prostate symptom score, Qmax = maximum flow rate, TSP-1 = thrombospondin-1, FGF-2 = fibroblast growth factor, mRNA = precursor messenger ribonucleic acid, PSA = prostate-specific antigen, DRE = digital rectal examination, AUR = acute urinary retention, COX2 = cyclooxygenase 2, QoL = quality of life PMID:27928444

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

    PubMed

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

    2016-01-01

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

  13. Cigarette smoke extract reduces VEGF in primary human airway epithelial cells.

    PubMed

    Thaikoottathil, J V; Martin, R J; Zdunek, J; Weinberger, A; Rino, J G; Chu, H W

    2009-04-01

    Reduced vascular endothelial growth factor (VEGF) has been reported in bronchoalveolar lavage fluid and lungs of severe emphysema patients. Airway epithelial cells (AEC) are exposed to various environmental insults like cigarette smoke and bacterial infections, but their direct effect on VEGF production in well-differentiated primary human AEC remains unclear. The current authors determined the effect of cigarette smoke extract (CSE) alone and in combination with Mycoplasma pneumoniae (Mp) on VEGF production in well-differentiated primary normal human bronchial epithelial (NHBE) and small airway epithelial cells (SAEC) in air-liquid interface cultures. Secretion and expression of VEGF were determined by ELISA and real-time RT-PCR, respectively. Cell growth, apoptosis, extracellular signal-regulated kinase (ERK)1/2 and protein kinase (PK)C signalling pathways were evaluated to further dissect VEGF regulation under CSE treatment. CSE significantly reduced VEGF secretion in NHBE and SAEC. In SAEC, Mp alone significantly increased the VEGF, while the presence of CSE attenuated Mp-induced VEGF production. While ERK inhibitor reduced VEGF secretion only in NHBE, a PKC inhibitor significantly decreased VEGF secretion in both NHBE and SAEC. In conclusion, direct cigarette smoke extract exposure significantly reduced vascular endothelial growth factor production in well-differentiated primary human airway epithelial cells, in part through modifying extracellular signal-regulated kinase 1/2 and protein kinase C signalling pathways.

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

    PubMed

    Zhao, Xiaoni; Wang, Guangrong; You, Jinhui

    2013-04-01

    Vascular endothelial growth factor (VEGF) is a glycoprotein that promotes endothelial regeneration, stimulates formation of collateral blood vessels and increases vascular permeability. The purpose of this study was to measure the peripheral blood plasma level of VEGF and FT3, FT4, TSH and to analyze the correlation of the level of VEGF and TSH, FT3, FT4, age and gender in the patients of hyperthyroidism. The relationship between hyperthyroidism and VEGF was investigated as well. The plasma level of VEGF in 45 hyperthyroidism patients and 27 healthy persons were measured by enzyme-linked immunosorbent assay (ELISA), while plasma FT3, FT4, TSH were detected by chemiluminescence. The result showed that the plasma level of VEGF in hyperthyroidism patients [(92.53 +/- 62.38) pg/mL] was significantly lower than that in the control group [(158.28 +/- 77.15) pg/mL] (P < 0.01). The plasma level of VEGF correlated with age, and that of those over 40Y was significantly higher than that of 40Y or younger (P < 0.05) in healthy group. Whereas there was no correlation among VEGF, TSH, FT3, FT4, age and gender in hyperthyroidism patients (P > 0.05). These results suggested that the peripheral blood plasma level of VEGF in hyperthyroidism patients was significantly lower than that in the control group. Further experimental investigations are needed to estimate the relationship between VEGF and hyperthyroidism.

  15. Notch modulates VEGF action in endothelial cells by inducing Matrix Metalloprotease activity

    PubMed Central

    2011-01-01

    Background In the vasculature, Notch signaling functions as a downstream effecter of Vascular Endothelial Growth Factor (VEGF) signaling. VEGF regulates sprouting angiogenesis in part by inducing and activating matrix metalloproteases (MMPs). This study sought to determine if VEGF regulation of MMPs was mediated via Notch signaling and to determine how Notch regulation of MMPs influenced endothelial cell morphogenesis. Methods and Results We assessed the relationship between VEGF and Notch signaling in cultured human umbilical vein endothelial cells. Overexpression of VEGF-induced Notch4 and the Notch ligand, Dll4, activated Notch signaling, and altered endothelial cell morphology in a fashion similar to that induced by Notch activation. Expression of a secreted Notch antagonist (Notch1 decoy) suppressed VEGF-mediated activation of endothelial Notch signaling and endothelial morphogenesis. We demonstrate that Notch mediates VEGF-induced matrix metalloprotease activity via induction of MMP9 and MT1-MMP expression and activation of MMP2. Introduction of a MMP inhibitor blocked Notch-mediated endothelial morphogenesis. In mice, analysis of VEGF-induced dermal angiogenesis demonstrated that the Notch1 decoy reduced perivascular MMP9 expression. Conclusions Taken together, our data demonstrate that Notch signaling can act downstream of VEGF signaling to regulate endothelial cell morphogenesis via induction and activation of specific MMPs. In a murine model of VEGF-induced dermal angiogenesis, Notch inhibition led to reduced MMP9 expression. PMID:21349159

  16. TLR4 mediates LPS-induced VEGF expression in odontoblasts.

    PubMed

    Botero, Tatiana M; Shelburne, Charles E; Holland, G Rex; Hanks, Carl T; Nör, Jacques E

    2006-10-01

    Lipopolysaccharide (LPS) from gram-negative bacteria cell walls such as Prevotella intermedia and Escherichia coli induce vascular endothelial growth factor (VEGF) expression in odontoblasts, but not in undifferentiated dental pulp cells. CD14 and TLR4 are responsible for LPS signaling in macrophages, but their expression levels and function in dental pulp cells are unknown. We showed here that murine odontoblast-like cells (MDPC-23) express CD14 and TLR4 by immunohistochemistry and flow cytometry. In contrast, undifferentiated dental pulp cells (OD-21) presented low or no expression of these two receptors. MDPC-23 cells showed CD14 and TLR4 up-regulation upon exposure to LPS, as determined by real time PCR. Dominant negative murine TLR4 (DN-mTLR4) transfected MDPC-23 cells did not show upregulated VEGF expression in response to LPS stimulation. These results demonstrate that odontoblast-like cells express CD14 and TLR4, and that LPS-induced VEGF expression is mediated, at least in part, by TLR4 signaling.

  17. Physiological capillary regression is not dependent on reducing VEGF expression

    PubMed Central

    Olfert, I. Mark

    2015-01-01

    Investigations into physiologically-controlled capillary regression report the provocative finding that microvessel regression occurs in the face of persistent elevation of skeletal muscle vascular endothelial growth factor-A (VEGF) expression. Thrombospondin-1 (TSP-1), a negative angiogenic regulator, is increasingly being observed to temporally correlate with capillary regression, suggesting that increased TSP-1 (and not reduction in VEGF per se) is needed to initiate, and likely regulate, capillary regression. Based on evidence being gleaned from physiologically-mediated regression of capillaries, it needs to be recognized that capillary regression (and perhaps capillary rarefaction with disease) is not simply the reversal of factors used to stimulate angiogenesis. Rather, the conceptual understanding that angiogenesis and capillary regression each have specific and unique requirements that are biologically constrained to opposite sides of the balance between positive and negative angioregulatory factors may shed light on why anti-VEGF therapies have not lived up to the promise in reversing angiogenesis and providing the cure that many had hoped toward fighting cancer. Emerging evidence from physiological controlled angiogenesis suggest that cases involving excessive or uncontrolled capillary expansion may be best treated by therapies designed to increase expression of negative angiogenic regulators, whereas those involving capillary rarefaction may benefit from inhibiting negative regulators (like TSP-1). PMID:26660949

  18. 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

  19. Differential effect of follicle-stimulating hormone and estradiol on expressions of vascular endothelial growth factor (VEGF) 120, VEGF164 and their receptors in bovine granulosa cells.

    PubMed

    Shimizu, Takashi; Jayawardana, Barana C; Tetsuka, Masafumi; Miyamoto, Akio

    2007-02-01

    Vascular endothelial growth factor (VEGF) isoforms (VEGF 120 and VEGF 164) secreted by granulosa cells are involved in thecal angiogenesis during follicular development in the bovine ovary. However, whether the transcript of the VEGF120 and VEGF164 isoforms differs during follicular development in the ovary is still unknown. We first examined the gene expression of VEGF120, VEGF164, fms-like tyrosine kinase (Flt-1), and fetal liver kinase (Flk-1) in the granulosa cells (GCs) and theca cells (TCs) of pre-selection and post-selection follicles (PRF and POF respectively) from the bovine ovary. Then we examined the effects of FSH and estradiol (E2) on these factors in cultured bovine GCs. Messenger RNA (mRNA) expression was quantified using real-time PCR methods. The concentrations of E2 and P4 in the follicular fluid (FF) of the PRF and POF were estimated using an enzyme immunoassay (EIA). The concentrations of E2 and P4 in the FF were significantly higher in the POF than in the PRF. The ratio of E2/P4 in PRF and POF was 0.37 and 3.8, respectively. The expression levels of the VEGF120, VEGF164, and Flk-1 mRNAs in the GCs of POF with high E2 concentration were higher than those of PRF. The levels of the Flt-1 and Flk-1 mRNAs in the TCs were not different between PRF and POF. Since E2 in the FF of the POF used in the present study was high compared with the PRF, we examined the effects of E2 and FSH on the expression of the above genes using cultured GCs. Expression of VEGF120 mRNA was induced by a low concentration (1 ng/ml) of E2, whereas the levels of VEGF164 and Flk-1 mRNAs were not affected by E2. FSH stimulated the expression of the VEGF isoforms and Flk-1 genes. Moreover, the expression of those genes was enhanced when low E2 (1 ng/ml) was added to FSH. In conclusion, our data indicates that the VEGF isoforms have a follicle stage-dependent expression pattern. Thus, our results suggest that the expression of VEGF isoforms may be associated with characterization

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

    PubMed Central

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

    2016-01-01

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

  1. Delineating the Effect a Novel Anti-VEGF-A Therapy has on the Lymphatic System of Immunocompetent Tumor-Bearing Mice

    DTIC Science & Technology

    2013-02-01

    exhibit lymphedema , chylous ascites or chylothorax. Lyve-1wt/Cre;Vegfr2flox/flox embryos display reduced viability and blood vessel defects...lymphatic vascular failure in lymphedema distichiasis. Nat Med, 2004. 10(9): p. 974-81. 4. Makinen, T., et al., PDZ interaction site in ephrinB2 is

  2. Is VEGF a key target of cotinine and other potential therapies against Alzheimer disease?

    PubMed

    Echeverria, Valentin; Barreto, George E; Ávila-Rodriguez, Marco; Tarasov, Vadim V; Aliev, Gjumrakch

    2017-03-29

    The vascular endothelial growth factor (VEGF) is a neuroprotective cytokine that promotes neurogenesis and angiogenesis in the brain. In animal models, it has been shown that environmental enrichment and exercise, two non-pharmacological interventions that are beneficial decreasing the progression of Alzheimer disease (AD) and depressive-like behavior, enhance hippocampal VEGF expression and neurogenesis. Furthermore, the stimulation of VEGF expression promotes neurotransmission and synaptic plasticity processes such as neurogenesis. It is thought that these VEGF actions in the brain, may underly its beneficial therapeutic effects against psychiatric and other neurological conditions. In this review, evidence linking VEGF deficit with the development of AD as well as the potential role of VEGF signaling as a therapeutic target for cotinine and other interventions in neurodegenerative conditions are discussed. .

  3. De novo morphogenesis of testis tissue: an improved bioassay to investigate the role of VEGF165 during testis formation.

    PubMed

    Dores, Camila; Dobrinski, Ina

    2014-07-01

    De novo formation of testis tissue from single-cell suspensions allows manipulation of different testicular compartments before grafting to study testicular development and the spermatogonial stem cell niche. However, the low percentages of newly formed seminiferous tubules supporting complete spermatogenesis and lack of a defined protocol have limited the use of this bioassay. Low spermatogenic efficiency in de novo formed tissue could result from the scarcity of germ cells in the donor cell suspension, cell damage caused by handling or from hypoxia during tissue formation in the host environment. In this study, we compared different proportions of spermatogonia in the donor cell suspension and the use of Matrigel as a scaffold to support de novo tissue formation and spermatogenesis. Then, we used the system to investigate the role of vascular endothelial growth factor 165 (VEGF165) during testicular morphogenesis on blood vessel and seminiferous tubule formation, and on presence of germ cells in the de novo developed tubules. Our results show that donor cell pellets with 10×10(6) porcine neonatal testicular cells in Matrigel efficiently formed testis tissue de novo. Contrary to what was expected, the enrichment of the cell suspension with germ cells did not result in higher numbers of tubules supporting spermatogenesis. The addition of VEGF165 did not improve blood vessel or tubule formation, but it enhanced the number of tubules containing spermatogonia. These results indicate that spermatogenic efficiency was improved by the addition of Matrigel, and that VEGF165 may have a protective role supporting germ cell establishment in their niche.

  4. 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

  5. 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

  6. Melatonin inhibits angiogenesis in SH-SY5Y human neuroblastoma cells by downregulation of VEGF.

    PubMed

    González, Alicia; González-González, Alicia; Alonso-González, Carolina; Menéndez-Menéndez, Javier; Martínez-Campa, Carlos; Cos, Samuel

    2017-04-01

    Vascular endothelial growth factor (VEGF) produced from tumor cells plays a crucial role in the pathogenesis and neovascularization of neuroblastoma. Inhibition of VEGF secretion by tumor cells, as well as VEGF-regulated signaling in endothelial cells, are important to reduce the angiogenesis and growth of neuroblastoma. Since melatonin has anti-angiogenic effects in tumor cell lines, the aim of the present study was to study melatonin modulation of the pro-angiogenic effects of VEGF in neuroblastoma cells (SH-SY5Y). We used co-cultures of SH-SY5Y and endothelial cells. VEGF expression and protein levels were analyzed by quantitative RT-PCR and ELISA, respectively. Endothelial cell migration was assessed by wound-healing assay and endothelial angiogenesis by a tube formation assay. Melatonin inhibited the pro-angiogenic effects of SH-SY5Y cells. The conditioned medium collected from the neuroblastoma cells was angiogenically active and stimulated proliferation, migration and tube formation in endothelial cells. This effect was significantly counteracted by the addition of either anti-VEGF or melatonin. Melatonin inhibited VEGF expression and secretion in SH-SY5Y cells, decreasing the levels of VEGF available for endothelial cells. Melatonin has anti-angiogenic effects at different steps of the angiogenic process in SH-SY5Y neuroblastoma cells, through the downregulation of VEGF.

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

    PubMed Central

    Hua, Wen; Li, Xuedong; Wang, Wei

    2017-01-01

    Purpose. The functions of vascular endothelial growth factor (VEGF) in scar formation after trabeculectomy were investigated in a human Tenon fibroblast cell line from glaucoma patients using lentivirus-mediated VEGF shRNA. Methods. Human Tenon fibroblast (HTF) cells were isolated from scar tissue of glaucoma patients during secondary surgery. Lentivirus-VEGF-shRNA was constructed and transfected into HTF cells. Subsequently, VEGF mRNA and protein expression were analyzed using quantitative RT-PCR and western blotting, respectively, and the effects of VEGF knockdown were analyzed. The inhibition of HTF proliferation was monitored according to total cell numbers using ScanArray. Results. Both mRNA and protein levels of VEGF were reduced by lentivirus-mediated VEGF-shRNA, and proliferation of HTF cells was inhibited. Conclusions. Primary cultures of human Tenon fibroblast (HTF) were established, and proliferation was decreased following inhibition of VEGF. VEGF may be a suitable therapeutic target for reducing scar tissue formation in glaucoma patients after filtration surgery. PMID:28168047

  8. Selective binding of C-6 OH sulfated hyaluronic acid to the angiogenic isoform of VEGF(165).

    PubMed

    Lim, Dong-Kwon; Wylie, Ryan G; Langer, Robert; Kohane, Daniel S

    2016-01-01

    Vascular endothelial growth factor 165 (VEGF165) is an important extracellular protein involved in pathological angiogenesis in diseases such as cancer, wet age-related macular degeneration (wet-AMD) and retinitis pigmentosa. VEGF165 exists in two different isoforms: the angiogenic VEGF165a, and the anti-angiogenic VEGF165b. In some angiogenic diseases the proportion of VEGF165b may be equal to or higher than that of VEGF165a. Therefore, developing therapeutics that inhibit VEGF165a and not VEGF165b may result in greater anti-angiogenic activity and therapeutic benefit. To this end, we report the selective binding properties of sulfated hyaluronic acid (s-HA). Selective biopolymers offer several advantages over antibodies or aptamers including cost effective and simple synthesis, and the ability to make nanoparticles or hydrogels for drug delivery applications or VEGF165a sequestration. Limiting sulfation to the C-6 hydroxyl (C-6 OH) in the N-acetyl-glucosamine repeat unit of hyaluronic acid (HA) resulted in a polymer with strong affinity for VEGF165a but not VEGF165b. Increased sulfation beyond the C-6 OH (i.e. greater than 1 sulfate group per HA repeat unit) resulted in s-HA polymers that bound both VEGF165a and VEGF165b. The C-6 OH sulfated HA (Mw 150 kDa) showed strong binding properties to VEGF165a with a fast association rate constant (Ka; 2.8 × 10(6) M(-1) s(-1)), slow dissociation rate constant (Kd; 2.8 × 10(-3) s(-1)) and strong equilibrium binding constant (KD; ∼1.0 nM)), which is comparable to the non-selective VEGF165 binding properties of the commercialized therapeutic anti-VEGF antibody (Avastin(®)). The C-6 OH sulfated HA also inhibited human umbilical vein endothelial cell (HUVEC) survival and proliferation and human dermal microvascular endothelial cell (HMVEC) tube formation. These results demonstrate that the semi-synthetic natural polymer, C-6 OH sulfated HA, may be a promising biomaterial for the treatment of angiogenesis

  9. A class of extracellular vesicles from breast cancer cells activates VEGF receptors and tumour angiogenesis

    PubMed Central

    Feng, Qiyu; Zhang, Chengliang; Lum, David; Druso, Joseph E.; Blank, Bryant; Wilson, Kristin F.; Welm, Alana; Antonyak, Marc A.; Cerione, Richard A.

    2017-01-01

    Non-classical secretory vesicles, collectively referred to as extracellular vesicles (EVs), have been implicated in different aspects of cancer cell survival and metastasis. Here, we describe how a specific class of EVs, called microvesicles (MVs), activates VEGF receptors and tumour angiogenesis through a unique 90 kDa form of VEGF (VEGF90K). We show that VEGF90K is generated by the crosslinking of VEGF165, catalysed by the enzyme tissue transglutaminase, and associates with MVs through its interaction with the chaperone Hsp90. We further demonstrate that MV-associated VEGF90K has a weakened affinity for Bevacizumab, causing Bevacizumab to be ineffective in blocking MV-dependent VEGF receptor activation. However, treatment with an Hsp90 inhibitor releases VEGF90K from MVs, restoring the sensitivity of VEGF90K to Bevacizumab. These findings reveal a novel mechanism by which cancer cell-derived MVs influence the tumour microenvironment and highlight the importance of recognizing their unique properties when considering drug treatment strategies. PMID:28205552

  10. Overexpression of VEGF-C causes transient lymphatic hyperplasia but not increased lymphangiogenesis in regenerating skin.

    PubMed

    Goldman, Jeremy; Le, Thomas X; Skobe, Mihaela; Swartz, Melody A

    2005-06-10

    Vascular endothelial growth factor (VEGF)-C is necessary for lymphangiogenesis and holds potential for lymphangiogenic therapy in diseases lacking adequate lymphatic drainage. However, the ability of VEGF-C to enhance sustainable, functional lymphatic growth in adult tissues remains unclear. To address this, we evaluated VEGF-C overexpression in adult lymphangiogenesis in regenerating skin. We used a model of mouse tail skin regeneration incorporating a suspension of either VEGF-C overexpressing tumor cells, which provide a continuous supplement of excess VEGF-C to the natural regenerating environment for more than 25 days, or otherwise identical control-transfected tumor cells. We found that excess VEGF-C did not enhance the rate of lymphatic endothelial cell (LEC) migration, the density of lymphatic vessels, or the rate of functionality -- even though lymphatic hyperplasia was present early on. Furthermore, the hyperplasia disappeared when VEGF-C levels diminished, which occurred after 25 days, rendering the lymphatics indistinguishable from those in control groups. In vitro, we showed that whereas cell-derived VEGF-C could induce chemoattraction of LECs across a membrane (which involves amoeboid-like transmigration), it did not increase LEC chemoinvasion within a 3-dimensional fibrin matrix (which requires proteolytic migration). These results suggest that whereas excess VEGF-C may enhance early LEC proliferation and cause lymphatic vessel hyperplasia, it does not augment the physiological rate of migration or functionality, and by itself cannot sustain any lasting effects on lymphatic size, density, or organization in regenerating adult skin.

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

    PubMed

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

    2015-09-10

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

  12. 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.

  13. Vascular endothelial growth factor receptor-2 expression in the pulp of human primary and young permanent teeth.

    PubMed

    Grando Mattuella, Leticia; Poli de Figueiredo, José Antonio; Nör, Jacques E; de Araujo, Fernando Borba; Medeiros Fossati, Anna Christina

    2007-12-01

    The purpose of this study was to evaluate the expression of vascular endothelial growth factor receptor-2 (VEGFR-2) in endothelial cells within the dental pulp of human primary and young permanent teeth and the spatial distribution of VEGFR-2-positive cells. Nine sound primary teeth and 4 sound young permanent teeth were evaluated by immunohistochemistry with a human anti-VEGFR-2 antibody. Stained tissue sections were analyzed qualitatively under light microscopy. Here we observed that endothelial cells of both primary and permanent teeth showed positive immunostaining for VEGFR-2. Notably, VEGFR-2-positive cells in the primary teeth tended to be found close to the subodontoblastic layer, whereas the spatial distribution of VEGFR-2 immunostaining in the permanent teeth was more uniform. In conclusion, VEGFR-2 was expressed in the microvascular endothelial cells of both primary and young permanent teeth, which suggests that these cells are capable of responding to the morphogenetic and survival signals mediated by VEGF.

  14. Protease-Activated Receptor 2 Promotes Pro-Atherogenic Effects through Transactivation of the VEGF Receptor 2 in Human Vascular Smooth Muscle Cells

    PubMed Central

    Indrakusuma, Ira; Romacho, Tania; Eckel, Jürgen

    2017-01-01

    Background: Obesity is associated with impaired vascular function. In the cardiovascular system, protease-activated receptor 2 (PAR2) exerts multiple functions such as the control of the vascular tone. In pathological conditions, PAR2 is related to vascular inflammation. However, little is known about the impact of obesity on PAR2 in the vasculature. Therefore, we explored the role of PAR2 as a potential link between obesity and cardiovascular diseases. Methods: C57BL/6 mice were fed with either a chow or a 60% high fat diet for 24 weeks prior to isolation of aortas. Furthermore, human coronary artery endothelial cells (HCAEC) and human coronary smooth muscle cells (HCSMC) were treated with conditioned medium obtained from in vitro differentiated primary human adipocytes. To investigate receptor interaction vascular endothelial growth factor receptor 2 (VEGFR2) was blocked by exposure to calcium dobesilate and a VEGFR2 neutralization antibody, before treatment with PAR2 activating peptide. Student's t-test or one-way were used to determine statistical significance. Results: Both, high fat diet and exposure to conditioned medium increased PAR2 expression in aortas and human vascular cells, respectively. In HCSMC, conditioned medium elicited proliferation as well as cyclooxygenase 2 induction, which was suppressed by the PAR2 antagonist GB83. Specific activation of PAR2 by the PAR2 activating peptide induced proliferation and cyclooxygenase 2 expression which were abolished by blocking the VEGFR2. Additionally, treatment of HCSMC with the PAR2 activating peptide triggered VEGFR2 phosphorylation. Conclusion: Under obesogenic conditions, where circulating levels of pro-inflammatory adipokines are elevated, PAR2 arises as an important player linking obesity-related adipose tissue inflammation to atherogenesis. We show for the first time that the underlying mechanisms of these pro-atherogenic effects involve a potential transactivation of the VEGFR2 by PAR2. PMID

  15. 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

  16. 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

  17. 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.

  18. Quantifying anti-vascular effects of monoclonal antibodies to VEGF

    PubMed Central

    O’Connor, James P B; Carano, Richard A D; Clamp, Andrew R; Ross, Jed; Ho, Calvin C K; Jackson, Alan; Parker, Geoff J M; Rose, Chris J; Peale, Franklin V; Friesenhahn, Michel; Mitchell, Claire L; Watson, Yvonne; Roberts, Caleb; Hope, Lynn; Cheung, Sue; Reslan, Hani Bou; Go, Mary Ann T; Pacheco, Glenn J; Wu, Xiumin; Cao, Tim C; Ross, Sarajane; Buonaccorsi, Giovanni A; Davies, Karen; Hasan, Jurjees; Thornton, Paula; del Puerto, Olivia; Ferrara, Napoleone; van Bruggen, Nicholas; Jayson, Gordon C

    2009-01-01

    Purpose Little is known concerning the onset, duration and magnitude of direct therapeutic effects of anti-VEGF therapies. Such knowledge would help guide the rational development of targeted therapeutics from bench to bedside and optimize use of imaging technologies that quantify tumor function in early phase clinical trials. Experimental design Pre-clinical studies were performed using ex vivo micro-CT and in-vivo ultrasound imaging to characterize tumor vasculature in a human HM-7 colorectal xenograft model treated with the anti-VEGF antibody G6-31. Clinical evaluation was by quantitative MRI in ten patients with metastatic CRC treated with bevacizumab. Results Micro-CT experiments demonstrated reduction in perfused vessels within 24-48 hours of G6-31 drug administration (p ≤ 0.005). Ultrasound imaging confirmed reduced tumor blood volume within the same time frame (p = 0.048). Consistent with the pre-clinical results, reductions in enhancing fraction and fractional plasma volume were detected in patient CRC metastases within 48 hours after a single dose of bevacizumab that persisted throughout one cycle of therapy. These effects were followed by resolution of edema (p = 0.0023) and tumor shrinkage in 9/26 tumors at day 12. Conclusion These data suggest that VEGF-specific inhibition induces rapid structural and functional effects with downstream significant anti-tumor activity within one cycle of therapy. This finding has important implications for the design of early phase clinical trials that incorporate physiological imaging. The study demonstrates how animal data help interpret clinical imaging data, an important step towards the validation of image biomarkers of tumor structure and function. PMID:19861458

  19. TLR-4 and VEGF Polymorphisms in Chronic Periaortitis

    PubMed Central

    Atzeni, Fabiola; Boiardi, Luigi; Vaglio, Augusto; Nicoli, Davide; Farnetti, Enrico; Palmisano, Alessandra; Pipitone, Nicolò; Martorana, Davide; Moroni, Gabriella; Longhi, Selena; Bonatti, Francesco; Buzio, Carlo; Salvarani, Carlo

    2013-01-01

    Objective Chronic periaortitis (CP) is a rare disease that is characterised by fibro-inflammatory tissue surrounding the abdominal aorta and has both non-aneurysmal (idiopathic retroperitoneal fibrosis [IRF]) and aneurysmal forms (inflammatory abdominal aortic aneurysm [IAAA]). We investigated whether toll-like receptor 4 (TLR-4) and vascular endothelial growth factor (VEGF) polymorphisms were associated with susceptibility to, and the clinical features of CP. Methods One hundred and two CP patients and 200 healthy controls were molecularly genotyped for TLR-4 gene polymorphism (+896 A/G) (rs4986790), VEGF mutations +936 C/T (rs3025039) and −634 C/G (rs2010963), and an 18 base pair (bp) insertion/deletion (I/D) polymorphism at −2549 of the VEGF promoter region. The patients were grouped on the basis of the type of CP (IRF or IAAA), and the presence or absence of established atherosclerotic disease (ischemic heart disease, cerebrovascular disease, and peripheral arterial disease). Results There were no significant differences in the distribution of the studied polymorphisms between the patients and controls. However, carriage of the +936 T allele was significantly more frequent in the patients with IRF than in those with IAAA (26.5% vs 5.3%; p = 0.046; OR 6.49 [95% CI 0.82–51.54]). There were significantly more carriers of the I allele among the patients with ureteral obstruction (83.8% vs 58.8%; p = 0.006; OR 3.63 [95% CI 1.42–9.28]) and those who received conservative treatment (48.5% vs 23.5%; p = 0.015; OR 3.06 [95% CI 1.22–7.721]) than among those without, and II homozygosity was significantly more frequent in the patients with deep vein thrombosis than in those without (30.4% vs 11.7%, p = 0.031; OR 3.31 [95% CI 1.07–10.21]). Conclusion The VEGF +936 C/T polymorphism may be associated with an increased risk of developing the non-aneurysmal IRF form of CP. Carriers of the I allele and II homozygosity are respectively at increased

  20. Kidney diseases associated with anti-vascular endothelial growth factor (VEGF): an 8-year observational study at a single center.

    PubMed

    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

    2014-11-01

    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.

  1. 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

  2. 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.

  3. VEGF189 expression is highly related to adaptation of the plateau pika (Ochotona curzoniae) inhabiting high altitudes.

    PubMed

    Li, Hongge; Guo, Songchang; Ren, Yongming; Wang, Depeng; Yu, Honghao; Li, Wenjing; Zhao, Xinquan; Chang, Zhijie

    2013-12-01

    The plateau pika (Ochotona curzonia) has adapted to high-altitude hypoxia during evolution. Higher microvessel density in specific tissues and a blunted hypoxic pulmonary vasoconstriction response are the critical components of this adaptation. VEGF, vascular endothelial growth factor, has proved to be a key regulator of angiogenesis in response to tissue hypoxia and to play an important role in vascular vasodilation. However, the role of VEGF in adaptation to high-altitude hypoxia in the plateau pika remains unknown. In this study, we cloned cDNAs for VEGF165 and VEGF189 and examined their expression in pikas inhabiting altitudes of 3200 and 4750 m. Phylogenetic analysis reveals that pika VEGF165 and VEGF189 are evolutionarily conserved. Real-time PCR analysis demonstrates that VEGF165 and VEGF189 display tissue and altitude-specific expression patterns. Interestingly, we found that the levels of VEGF189 mRNA are significantly higher than those of VEGF165 in the brain and muscle tissues of the pika, which is different from what was previously observed in sea-level mammals. VEGF189 mRNA levels in brain, muscle, and lung of the pika increased with increased habitat altitude, whereas VEGF165 shows less change. Our study suggests an important role for VEGF189 in adaptation to hypoxia by the plateau pika in the high-altitude environment.

  4. Biophysical Studies of the Induced Dimerization of Human VEGF Receptor 1 Binding Domain by Divalent Metals Competing with VEGF-A

    PubMed Central

    Reille-Seroussi, Marie; Gagey-Eilstein, Nathalie; Broussy, Sylvain; Coric, Pascale; Seijo, Bili; Lascombe, Marie-Bernard; Gautier, Benoit; Liu, Wang-Quing; Huguenot, Florent; Inguimbert, Nicolas; Bouaziz, Serge; Vidal, Michel; Broutin, Isabelle

    2016-01-01

    Angiogenesis is tightly regulated through the binding of vascular endothelial growth factors (VEGFs) to their receptors (VEGFRs). In this context, we showed that human VEGFR1 domain 2 crystallizes in the presence of Zn2+, Co2+ or Cu2+ as a dimer that forms via metal-ion interactions and interlocked hydrophobic surfaces. SAXS, NMR and size exclusion chromatography analyses confirm the formation of this dimer in solution in the presence of Co2+, Cd2+ or Cu2+. Since the metal-induced dimerization masks the VEGFs binding surface, we investigated the ability of metal ions to displace the VEGF-A binding to hVEGFR1: using a competition assay, we evidenced that the metals displaced the VEGF-A binding to hVEGFR1 extracellular domain binding at micromolar level. PMID:27942001

  5. 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

  6. Expressions of NDRG1, VEGF and Ki-67 in Condyloma Acuminatum.

    PubMed

    Yin, G W; Guo, Y; Jin, B

    2016-01-01

    The objective of this study was to explore the expressions and significance of NDRG1 (N-myc downregulated gene family 1), VEGF (vascular endothelial growth factor) and Ki-67 in lesions of Condyloma Acuminatum (CA). Immunohistochemistry was adopted to measure the expressions of NDRG1, VEGF and Ki-67 in 48 cases of CA and 18 normal skin controls. The positive rates of NDRG1, VEGF and Ki-67 were 63. 83.33% (40/48), 93.75% (45/48) and 85.42% (41/48) in the CA tissues, and 27.78% (5/18), 94.44%(17/18) and 61.11% (11/18) in the controls, respectively. The intensities of the expressions of NDRG1, VEGF and Ki-67 in CA tissues were significantly higher than those in the controls. There were significant differences both in the positive rates and the expression intensities of NDRG1, VEGF and Ki-67 between the two groups (P less than0.05). The Spearman’s Rank-Order Correlation analysis indicated that the expressions of NDRG1 protein and VEGF protein were positively correlated by the Spearman’s Rank-Order Correlation analysis (r = 0.346, P=0.016). For the CA tissues with high expressions of NDRG1 and VEGF, NDRG1 and VEGF influenced both the occurrence and development of CA.

  7. Autocrine VEGF maintains endothelial survival through regulation of metabolism and autophagy

    PubMed Central

    Domigan, Courtney K.; Warren, Carmen M.; Antanesian, Vaspour; Happel, Katharina; Ziyad, Safiyyah; Lee, Sunyoung; Krall, Abigail; Duan, Lewei; Torres-Collado, Antoni X.; Castellani, Lawrence W.; Elashoff, David; Christofk, Heather R.; van der Bliek, Alexander M.; Potente, Michael; Iruela-Arispe, M. Luisa

    2015-01-01

    ABSTRACT Autocrine VEGF is necessary for endothelial survival, although the cellular mechanisms supporting this function are unknown. Here, we show that – even after full differentiation and maturation – continuous expression of VEGF by endothelial cells is needed to sustain vascular integrity and cellular viability. Depletion of VEGF from the endothelium results in mitochondria fragmentation and suppression of glucose metabolism, leading to increased autophagy that contributes to cell death. Gene-expression profiling showed that endothelial VEGF contributes to the regulation of cell cycle and mitochondrial gene clusters, as well as several – but not all – targets of the transcription factor FOXO1. Indeed, VEGF-deficient endothelium in vitro and in vivo showed increased levels of FOXO1 protein in the nucleus and cytoplasm. Silencing of FOXO1 in VEGF-depleted cells reversed expression profiles of several of the gene clusters that were de-regulated in VEGF knockdown, and rescued both cell death and autophagy phenotypes. Our data suggest that endothelial VEGF maintains vascular homeostasis through regulation of FOXO1 levels, thereby ensuring physiological metabolism and endothelial cell survival. PMID:25956888

  8. Interplay between VEGF and Nrf2 regulates angiogenesis due to intracranial venous hypertension

    PubMed Central

    Li, Liwen; Pan, Hao; Wang, Handong; Li, Xiang; Bu, Xiaomin; Wang, Qiang; Gao, Yongyue; Wen, Guodao; Zhou, Yali; Cong, Zixiang; Yang, Youqing; Tang, Chao; Liu, Zhengwei

    2016-01-01

    Venous hypertension(VH) plays an important role in the pathogenesis of cerebral arteriovenous malformations (AVMs) and is closely associated with the HIF-1α/VEGF signaling pathway. Nuclear factor erythroid 2-related factor 2(Nrf2) significantly influences angiogenesis; however, the interplay between Nrf2 and VEGF under VH in brain AVMs remains unclear. Therefore, our study aimed to investigate the interplay between Nrf2 and VEGF due to VH in brain AVMs. Immunohistochemistry indicated that Nrf2 and VEGF were highly expressed in human brain AVM tissues. In vivo, we established a VH model in both wild-type (WT) and siRNA-mediated Nrf2 knockdown rats. VH significantly increased the expression of Nrf2 and VEGF. Loss of Nrf2 markedly inhibited the upregulation of VEGF, as determined by Western blot analysis and qRT-PCR. In vitro, primary brain microvascular endothelial cells (BMECs) were isolated from WT and Nrf2−/− mice, and a VEGF-Nrf2 positive feed-back loop was observed in BMECs. By trans well assay and angiogenesis assay, Nrf2 knockout significantly inhibited the migration and vascular tube formation of BMECs. These findings suggest that the interplay between Nrf2 and VEGF can contribute to VH-induced angiogenesis in brain AVMs pathogenesis. PMID:27869147

  9. Brown adipose tissue derived VEGF-A modulates cold tolerance and energy expenditure

    PubMed Central

    Sun, Kai; Kusminski, Christine M.; Luby-Phelps, Kate; Spurgin, Stephen B.; An, Yu A.; Wang, Qiong A.; Holland, William L.; Scherer, Philipp E.

    2014-01-01

    We recently reported that local overexpression of VEGF-A in white adipose tissue (WAT) protects against diet-induced obesity and metabolic dysfunction. The observation that VEGF-A induces a “brown adipose tissue (BAT)-like” phenotype in WAT prompted us to further explore the direct function of VEGF-A in BAT. We utilized a doxycycline (Dox)-inducible, brown adipocyte-specific VEGF-A transgenic overexpression model to assess direct effects of VEGF-A in BAT in vivo. We observed that BAT-specific VEGF-A expression increases vascularization and up-regulates expression of both UCP1 and PGC-1α in BAT. As a result, the transgenic mice show increased thermogenesis during chronic cold exposure. In diet-induced obese mice, introducing VEGF-A locally in BAT rescues capillary rarefaction, ameliorates brown adipocyte dysfunction, and improves deleterious effects on glucose and lipid metabolism caused by a high-fat diet challenge. These results demonstrate a direct positive role of VEGF-A in the activation and expansion of BAT. PMID:24944907

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

    PubMed

    García-Román, Jonathan; Ibarra-Sánchez, Alfredo; Lamas, Mónica; González Espinosa, Claudia

    2010-10-15

    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 (CoCl2) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl2 promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl2-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 CoCl2-induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl2 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-dependent Fyn kinase activation.

  11. 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

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

    PubMed Central

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

    2014-01-01

    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. PMID:25271320

  13. Expression of HIF-2α and VEGF in Cervical Squamous Cell Carcinoma and Its Clinical Significance

    PubMed Central

    Zhang, Lixia; Chen, Qiang; Hu, Jing; Chen, Yue; Liu, Chenglong; Xu, Changshui

    2016-01-01

    CSCC is a systemic disease involving polygenic alteration and multiple steps, and HIF and VEGF are closely associated with tumorigenesis. Specimens surgically resected from 64 cases of CSCC and 22 cases of normal cervical tissue were selected randomly to detect the expression of HIF-2α and VEGF in CSCC for exploring their clinical significance; information regarding the age, lymph node metastasis, and FIGO staging were collected as well; expression of HIF-2α and VEGF was detected by qPCR and immunohistochemistry. We found that the expression of HIF-2α and VEGF mRNA in CSCC was significantly higher than that of normal cervical tissues and showed a positive correlation between them. The positive rates of HIF-2α and VEGF protein expression in CSCC and normal cervical tissues were 93.8% and 18.2%, respectively, with correlation between them. The expression of both HIF-2α and VEGF mRNA did not relate closely to age but the FIGO staging and lymph node metastasis. Compared with the counterpart control group, CSCC tissues with high FIGO staging and lymph node metastasis had a higher level of HIF-2α and VEGF mRNA expression. So, HIF-2α and VEGF were overexpressed in CSCC, which has a great clinical significance for its diagnosis. PMID:27413748

  14. Differentiation of lymphatic endothelial cells from bone marrow mesenchymal stem cells with VEGFs.

    PubMed

    Wei, Luwan; Liu, Yanli; Chen, Guoling; Fang, Yunhai; Song, Xianrang; Dong, Ping; Gao, Jie; Liu, Rong; Ding, Zhaoxi; Bi, Yushun; Liu, Zhiyu

    2012-12-01

    Although there have been many experimental studies demonstrating that bone marrow-derived mesenchymal stem cells (MSCs) have the potential to differentiate into mesenchymal tissues such as osteocytes, chondrocytes, and adipocytes in vivo and in vitro, little information is available regarding their potential to differentiate into lymphatic endothelial cells. Therefore, we chose to investigate differentiation of MSCs into lymphatic endothelial cells using stimulation with members of the vascular endothelial growth factor (VEGFs) family. Rat MSCs were isolated from bone marrow aspirate of Sprague-Dawley rats as previously described and characterized with flow cytometry for surface markers CD14, CD34, CD29, and CD90. Purified MSCs were plated and cultured in the presence of VEGF-A, VEGF-C, or the combination of both for 10 days. We examined the cells for Prox-1 and LYVE-1 by immunocytochemistry, RT-PCR, and Western blot analysis. Results demonstrated that compared to controls, cell differentiated with VEGF-A, VEGF-C and VEGF-A+VEGF-C expressed Prox-1 and LYVE-1. Our results indicate that MSCs induced by VEGFs are capable of differentiating into lymphatic endothelial-like cells in vitro, and this response has the potential to make them attractive candidates for the development of autologous tissue grafts for future therapy.

  15. VEGF signaling mediates bladder neuroplasticity and inflammation in response to BCG

    PubMed Central

    2011-01-01

    Background This work tests the hypothesis that increased levels of vascular endothelial growth factor (VEGF) observed during bladder inflammation modulates nerve plasticity. Methods Chronic inflammation was induced by intravesical instillations of Bacillus Calmette-Guérin (BCG) into the urinary bladder and the density of nerves expressing the transient receptor potential vanilloid subfamily 1 (TRPV1) or pan-neuronal marker PGP9.5 was used to quantify alterations in peripheral nerve plasticity. Some mice were treated with B20, a VEGF neutralizing antibody to reduce the participation of VEGF. Additional mice were treated systemically with antibodies engineered to specifically block the binding of VEGF to NRP1 (anti-NRP1B) and NRP2 (NRP2B), or the binding of semaphorins to NRP1 (anti-NRP1 A) to diminish activity of axon guidance molecules such as neuropilins (NRPs) and semaphorins (SEMAs). To confirm that VEGF is capable of inducing inflammation and neuronal plasticity, another group of mice was instilled with recombinant VEGF165 or VEGF121 into the urinary bladder. Results The major finding of this work was that chronic BCG instillation resulted in inflammation and an overwhelming increase in both PGP9.5 and TRPV1 immunoreactivity, primarily in the sub-urothelium of the urinary bladder. Treatment of mice with anti-VEGF neutralizing antibody (B20) abolished the effect of BCG on inflammation and nerve density. NRP1A and NRP1B antibodies, known to reduce BCG-induced inflammation, failed to block BCG-induced increase in nerve fibers. However, the NRP2B antibody dramatically potentiated the effects of BCG in increasing PGP9.5-, TRPV1-, substance P (SP)-, and calcitonin gene-related peptide (CGRP)-immunoreactivity (IR). Finally, instillation of VEGF121 or VEGF165 into the mouse bladder recapitulated the effects of BCG and resulted in a significant inflammation and increase in nerve density. Conclusions For the first time, evidence is being presented supporting that

  16. COX-2 over-expression correlates with VEGF and tumour angiogenesis in canine mammary cancer.

    PubMed

    Queiroga, Felisbina L; Pires, Isabel; Parente, Margarida; Gregório, Hugo; Lopes, Carlos S

    2011-07-01

    This study was designed to investigate the possible roles of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) in canine mammary cancer angiogenesis. Immunohistochemistry was performed on 70 tumours (28 benign and 42 malignant) in order to detect COX-2 and VEGF expression. Microvessel density (MVD) was determined by CD31 immunolabelling to assess tumour angiogenesis. There was a significantly higher expression of COX-2 (P<0.001), VEGF (P<0.001) and MVD (P<0.001) in malignant compared to benign tumours. In the malignant group, the MVD of COX-2 positive tumours was significantly higher than that of COX-2 negative tumours (P=0.026). A similar association was observed for VEGF (P<0.001) positive tumours. The results from this study suggested that over-expression of COX-2 and VEGF may contribute to increased angiogenesis and aggression in malignant tumours.

  17. 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

  18. Vascular endothelial growth factor stimulates osteoblastic differentiation of cultured human periosteal-derived cells expressing vascular endothelial growth factor receptors.

    PubMed

    Hah, Young-Sool; Jun, Jin-Su; Lee, Seong-Gyun; Park, Bong-Wook; Kim, Deok Ryong; Kim, Uk-Kyu; Kim, Jong-Ryoul; Byun, June-Ho

    2011-02-01

    Angiogenesis plays an important role in bone development and postnatal bone fracture repair. Vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptors (VEGFRs) are primarily involved in angiogenesis. This study investigated the expression of VEGF isoforms, VEGFR-1, and VEGFR-2 during the osteoblastic differentiation of cultured human periosteal-derived cells. In addition, the effect of exogenous VEGF on the osteoblastic differentiation of cultured human periosteal-derived cells was also examined. The expression of the VEGF isoforms (VEGF(121), VEGF(165), VEGF(189), and VEGF(206)), VEGFR-1, and VEGFR-2 was observed in the periosteal-derived cells. Administration of KRN633, a VEGFR-1 and VEGFR-2 inhibitor, decreased the alkaline phosphatase (ALP) activity during the osteoblastic differentiation of cultured human periosteal-derived cells. However, the administration of VEGFR2 Kinase Inhibitor IV, a VEGFR-2 inhibitor, did not affect the ALP activity. The addition of recombinant human VEGF(165) elevated the ALP activity and increased the calcium content in the periosteal-derived cells. Treating the periosteal-derived cells with recombinant human VEGF(165) resulted in an increase in Runx2 transactivation in the periosteal-derived cells. These results suggest that exogenous VEGF stimulates the osteoblastic differentiation of cultured human periosteal-derived cells and VEGF might act as an autocrine growth factor for the osteoblastic differentiation of cultured human periosteal-derived cells.

  19. Cationized gelatin delivery of a plasmid DNA expressing small interference RNA for VEGF inhibits murine squamous cell carcinoma.

    PubMed

    Matsumoto, Goichi; Kushibiki, Toshihiro; Kinoshita, Yukihiko; Lee, Ushaku; Omi, Yasushi; Kubota, Eiro; Tabata, Yasuhiko

    2006-04-01

    Double-stranded RNA (dsRNA) plays a major role in RNA interference (RNAi), a process in which segments of dsRNA are initially cleaved by the Dicer into shorter segments (21-23 nt) called small interfering RNA (siRNA). These siRNA then specifically target homologous mRNA molecules causing them to be degraded by cellular ribonucleases. RNAi down regulates endogenous gene expression in mammalian cells. Vascular endothelial growth factor (VEGF) is a key molecule in vasculogenesis as well as in angiogenesis. Tumor growth is an angiogenesis-dependent process, and therapeutic strategies aimed at inhibiting angiogenesis are theoretically attractive. To investigate the feasibility of using siRNA for VEGF in the specific knockdown of VEGF mRNA, thereby inhibiting angiogenesis, we have performed experiments with a DNA vector based on a siRNA system that targets VEGF (siVEGF). It almost completely inhibited the expression of three different isoforms (VEGF120, VEGF164 and VEGF188) of VEGF mRNA and the secretion of VEGF protein in mouse squamous cell carcinoma NRS-1 cells. The siVEGF released from cationized gelatin microspheres suppressed tumor growth in vivo. A marked reduction in vascularity accompanied the inhibition of a siVEGF-transfected tumor. Fluorescent microscopic study showed that the complex of siVEGF with cationized gelatin microspheres was still present around the tumor 10 days after injection, while free siVEGF had vanished by that time. siVEGF gene therapy increased the fraction of vessels covered by pericytes and induced expression of angiopoietin-1 by pericytes. These data suggest that cationized-gelatin microspheres containing siVEGF can be used to normalize tumor vasculature and inhibit tumor growth in a NRS-1 squamous cell carcinoma xenograft model.

  20. Structural mechanisms in the abolishment of VEGF-induced microvascular hyperpermeability by cAMP.

    PubMed

    Fu, Bingmei M; Shen, Shang; Chen, Bin

    2006-06-01

    To investigate the structural mechanisms by which elevation of the intraendothelial cAMP levels abolishes or attenuates the transient increase in microvascular permeability by vascular endothelial growth factor (VEGF), we examined cAMP effect on VEGF-induced hyperpermeability to small solute sodium fluorescein (Stokes radius = 0.45 nm) P(sodium fluorescein), intermediate-sized solute alpha-lactalbumin (Stokes radius = 2.01 nm) P(alpha-lactalbumin), and large solute albumin (BSA, Stokes radius = 3.5 nm) P(BSA) on individually perfused microvessels of frog mesenteries. After 20 min pretreatment of 2 mM cAMP analog, 8-bromo-cAMP, the initial increase by 1 nM VEGF was completely abolished in P(sodium fluorescein) (from a peak increase of 2.6+/-0.37 times control with VEGF alone to 0.96+/-0.07 times control with VEGF and cAMP), in P(alpha-lactalbumin) (from a peak increase of 2.7+/-0.33 times control with VEGF alone to 0.76+/-0.07 times control with VEGF and cAMP), and in P(BSA) (from a peak increase of 6.5+/-1.0 times control with VEGF alone to 0.97+/-0.08 times control with VEGF and cAMP). Based on these measured data, the prediction from our mathematical models suggested that the increase in the number of tight junction strands in the cleft between endothelial cells forming the microvessel wall is one of the mechanisms for the abolishment of VEGF-induced hyperpermeability by cAMP.

  1. Profiling of microRNAs in AML cells following overexpression or silencing of the VEGF gene.

    PubMed

    Li, Li; Zhu, Lixia; Wang, Yungui; Zhou, De; Zhu, Jingjing; Xie, Wanzhuo; Ye, Xiujin

    2017-01-01

    Acute myeloid leukemia (AML) is a disease of the hematopoietic progenitor cells associated with heterogeneous clonal proliferation. Vascular endothelial growth factor (VEGF) and its receptors play important roles in the regulation of angiogenesis during physiological and pathological processes. It is thought that AML cells have an autocrine VEGF pathway that contributes to the development and progression of AML. In addition, growing evidence has suggested that numerous microRNAs are involved in AML. The present study aimed to investigate the relationship between VEGF dysregulation and microRNA profiles in AML cells and patients. VEGF-overexpressing and VEGF-knockdown leukemia cells were constructed and changes in the patterns of microRNA expression were analyzed using a microRNA array. Subsequently, mononuclear cells from the blood of patients with AML showing high or low expression levels of VEGF were obtained and were used to assess the patterns of microRNA expression by reverse transcription-quantitative polymerase chain reaction. The results of the present study suggested that downregulation of VEGF markedly altered the profile of microRNAs in AML cells, while upregulation of VEGF did not. Examination of clinical samples from patients with AML showed that several microRNAs were closely associated with the expression level of VEGF, including miR-20a, miR-93, miR-16-5p, miR-17-5p, miR-124-5p and miR-17-3p. These results suggested that VEGF may be a pivotal protein that can both receive and initiate signals in leukemia cells.

  2. Profiling of microRNAs in AML cells following overexpression or silencing of the VEGF gene

    PubMed Central

    Li, Li; Zhu, Lixia; Wang, Yungui; Zhou, De; Zhu, Jingjing; Xie, Wanzhuo; Ye, Xiujin

    2017-01-01

    Acute myeloid leukemia (AML) is a disease of the hematopoietic progenitor cells associated with heterogeneous clonal proliferation. Vascular endothelial growth factor (VEGF) and its receptors play important roles in the regulation of angiogenesis during physiological and pathological processes. It is thought that AML cells have an autocrine VEGF pathway that contributes to the development and progression of AML. In addition, growing evidence has suggested that numerous microRNAs are involved in AML. The present study aimed to investigate the relationship between VEGF dysregulation and microRNA profiles in AML cells and patients. VEGF-overexpressing and VEGF-knockdown leukemia cells were constructed and changes in the patterns of microRNA expression were analyzed using a microRNA array. Subsequently, mononuclear cells from the blood of patients with AML showing high or low expression levels of VEGF were obtained and were used to assess the patterns of microRNA expression by reverse transcription-quantitative polymerase chain reaction. The results of the present study suggested that downregulation of VEGF markedly altered the profile of microRNAs in AML cells, while upregulation of VEGF did not. Examination of clinical samples from patients with AML showed that several microRNAs were closely associated with the expression level of VEGF, including miR-20a, miR-93, miR-16-5p, miR-17-5p, miR-124-5p and miR-17-3p. These results suggested that VEGF may be a pivotal protein that can both receive and initiate signals in leukemia cells. PMID:28123529

  3. Alterations in VEGF expression induced by antidepressant drugs in female rats under chronic social stress

    PubMed Central

    Nowacka-Chmielewska, Marta Maria; Paul-Samojedny, Monika; Bielecka-Wajdman, Anna Maria; Barski, Jarosław Jerzy; Obuchowicz, Ewa

    2017-01-01

    Vascular endothelial growth factor (VEGF) is thought to serve a role in neurogenesis and the stress response. Although a definite link between the action of antidepressants and VEGF has not been identified, it is assumed that VEGF, as a neurotrophic factor, serves an important role in the effects of antidepressant treatment. To examine this, the present study subjected adult female rats to four weeks of social instability stress and measured the effect of antidepressant treatment on the expression of VEGF. Firstly, endocrine markers of stress and body weight were measured in parallel with behavioral tests prior to and following subjection to stress. Then, the effect of 28-day daily treatment with desipramine (DMI; 10 mg/kg), fluoxetine (5 mg/kg) or tianeptine (10 mg/kg) on the number of copies of VEGF mRNA in the amygdala, hippocampus and hypothalamus, and on serum VEGF protein levels, of rats subjected to chronic stress was determined. In addition, the weight of the adrenal glands was measured following subjection to stress. Exposure to chronic stress was found to increase the rats' sucrose preference, and diminish their tendency for general exploration and time spent in the open. The relative adrenal weights of the stressed rats were significantly increased compared with the control. Plasma concentrations of corticosterone and adrenocorticotropic hormone were not significantly augmented. In addition, the present study identified that stress elevated VEGF mRNA expression in all studied neural structures. Furthermore, the results identified that the stress-induced increase in VEGF mRNA expression in the amygdala and hypothalamus was attenuated by long-term administration of DMI. Conversely, a decrease in serum VEGF concentration was observed in stressed rats, which was not reversed by treatment with antidepressants. In conclusion, the current study suggests that under conditions of stress, VEGF serves a role in the mechanism of action of DMI, through modulating

  4. 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.

  5. Correlation between liver cancer pain and the HIF-1 and VEGF expression levels

    PubMed Central

    Zhang, Geng; Feng, Gui-Yin; Guo, Yan-Ru; Liang, Dong-Qi; Yuan, Yuan; Wang, Hai-Lun

    2017-01-01

    A possible correlation between liver cancer pain and the hypoxia-inducible factor (HIF)-1 and vascular endothelial growth factor (VEGF) expression levels was examined. From January, 2015 to January, 2016, 30 patients suffering from liver cancer with pain, 30 patients with liver cancer without pain and 30 hepatitis patients with pain were enrolled in the study. Pain level was evaluated by visual analogue scale (VAS), the expression levels of HIF-1 and VEGF mRNA were determined by RT-PCR and the expression levels of HIF-1 and VEGF proteins were examined by ELISA. Before intervention, the VAS in the hepatitis group was significantly higher than that of the liver cancer pain group. However, after intervention the VAS in the two groups was reduced. HIF-1 and VEGF mRNA expression levels in the liver cancer pain group were significantly higher than those in the liver cancer group before and after intervention. The expression levels of HIF-1 and VEGF mRNA in the hepatitis group were the lowest. The expression levels of HIF-1 and VEGF mRNA in the liver cancer pain group considerably increased after intervention. The expression levels of HIF-1 and VEGF mRNA in the other two groups showed no changes before or after intervention. Before and after the intervention, VAS in the liver cancer pain group was positively correlated to the expression levels of HIF-1 and VEGF. Thus, pain occurrence and the pain level in liver cancer patients were correlated with the expression levels of HIF-1 and VEGF. As the regular three-step medicine analgesic ladder is ineffective in these cases, verification of HIF-1 and VEGF expression levels may be considered the new target for pain release. PMID:28123525

  6. Alterations in VEGF expression induced by antidepressant drugs in female rats under chronic social stress.

    PubMed

    Nowacka-Chmielewska, Marta Maria; Paul-Samojedny, Monika; Bielecka-Wajdman, Anna Maria; Barski, Jarosław Jerzy; Obuchowicz, Ewa

    2017-02-01

    Vascular endothelial growth factor (VEGF) is thought to serve a role in neurogenesis and the stress response. Although a definite link between the action of antidepressants and VEGF has not been identified, it is assumed that VEGF, as a neurotrophic factor, serves an important role in the effects of antidepressant treatment. To examine this, the present study subjected adult female rats to four weeks of social instability stress and measured the effect of antidepressant treatment on the expression of VEGF. Firstly, endocrine markers of stress and body weight were measured in parallel with behavioral tests prior to and following subjection to stress. Then, the effect of 28-day daily treatment with desipramine (DMI; 10 mg/kg), fluoxetine (5 mg/kg) or tianeptine (10 mg/kg) on the number of copies of VEGF mRNA in the amygdala, hippocampus and hypothalamus, and on serum VEGF protein levels, of rats subjected to chronic stress was determined. In addition, the weight of the adrenal glands was measured following subjection to stress. Exposure to chronic stress was found to increase the rats' sucrose preference, and diminish their tendency for general exploration and time spent in the open. The relative adrenal weights of the stressed rats were significantly increased compared with the control. Plasma concentrations of corticosterone and adrenocorticotropic hormone were not significantly augmented. In addition, the present study identified that stress elevated VEGF mRNA expression in all studied neural structures. Furthermore, the results identified that the stress-induced increase in VEGF mRNA expression in the amygdala and hypothalamus was attenuated by long-term administration of DMI. Conversely, a decrease in serum VEGF concentration was observed in stressed rats, which was not reversed by treatment with antidepressants. In conclusion, the current study suggests that under conditions of stress, VEGF serves a role in the mechanism of action of DMI, through modulating

  7. 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

  8. 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...

  9. Suppression of Retinal Neovascularization in vivo by Inhibition of Vascular Endothelial Growth Factor (VEGF) Using Soluble VEGF-Receptor Chimeric Proteins

    NASA Astrophysics Data System (ADS)

    Aiello, Lloyd Paul; Pierce, Eric A.; Foley, Eliot D.; Takagi, Hitoshi; Chen, Helen; Riddle, Lavon; Ferrara, Napoleone; King, George L.; Smith, Lois E. H.

    1995-11-01

    The majority of severe visual loss in the United States results from complications associated with retinal neovascularization in patients with ischemic ocular diseases such as diabetic retinopathy, retinal vein occlusion, and retinopathy of prematurity. Intraocular expression of the angiogenic protein vascular endothelial growth factor (VEGF) is closely correlated with neovascularization in these human disorders and with ischemia-induced retinal neovascularization in mice. In this study, we evaluated whether in vivo inhibition of VEGF action could suppress retinal neovascularization in a murine model of ischemic retinopathy. VEGF-neutralizing chimeric proteins were constructed by joining the extracellular domain of either human (Flt) or mouse (Flk) high-affinity VEGF receptors with IgG. Control chimeric proteins that did not bind VEGF were also used. VEGF-receptor chimeric proteins eliminated in vitro retinal endothelial cell growth stimulation by either VEGF (P < 0.006) or hypoxic conditioned medium (P < 0.005) without affecting growth under nonstimulated conditions. Control proteins had no effect. To assess in vivo response, animals with bilateral retinal ischemia received intravitreal injections of VEGF antagonist in one eye and control protein in the contralateral eye. Retinal neovascularization was quantitated histologically by a masked protocol. Retinal neovascularization in the eye injected with human Flt or murine Flk chimeric protein was reduced in 100% (25/25; P < 0.0001) and 95% (21/22; P < 0.0001) of animals, respectively, compared to the control treated eye. This response was evident after only a single intravitreal injection and was dose dependent with suppression of neovascularization noted after total delivery of 200 ng of protein (P < 0.002). Reduction of histologically evident neovascular nuclei per 6-um section averaged 47% ± 4% (P < 0.001) and 37% ± 2% (P < 0.001) for Flt and Flk chimeric proteins with maximal inhibitory effects of 77% and 66

  10. Incorporating VEGF-targeted therapy in advanced urothelial cancer

    PubMed Central

    Narayanan, Sujata; Srinivas, Sandy

    2016-01-01

    Patients with relapsed or refractory urothelial carcinoma (UC) have poor prognosis coupled with few options for systemic treatment. The role of angiogenesis in the evolution of cancers has been established, and studies have shown that it plays a key role in the pathogenesis of UC. Many targeted agents have been used in phase I–II trials for the treatment of UC, with encouraging but modest results. Recently, studies combining angiogenesis inhibitors with other chemotherapeutic agents were able to achieve objective responses higher than most commonly used second-line therapies in UC. Future efforts in investigating these therapies in UC rely on identification of biomarkers and other predictors of response to anti-VEGF therapy. PMID:28203296

  11. 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.

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

    PubMed Central

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

    2016-01-01

    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 polyethylene glycol (PEG) hydrogels functionalized with either a triple-helical, α2β1 integrin-specific peptide (GFOGER) or an αvβ3 integrin-targeting peptide (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. PMID:26662727

  13. 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

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

    PubMed

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

    2005-11-01

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

  15. HTLV-1 uses HSPG and neuropilin-1 for entry by molecular mimicry of VEGF165

    PubMed Central

    Lambert, Sophie; Bouttier, Manuella; Vassy, Roger; Seigneuret, Michel; Petrow-Sadowski, Cari; Janvier, Sébastien; Heveker, Nikolaus; Ruscetti, Francis W.; Perret, Gérard

    2009-01-01

    Human T-cell lymphotropic virus type 1 (HTLV-1) entry involves the interaction between the surface (SU) subunit of the Env proteins and cellular receptor(s). Previously, our laboratories demonstrated that heparan sulfate proteoglycans (HSPGs) and neuropilin-1 (NRP-1), a receptor of VEGF165, are essential for HTLV-1 entry. Here we investigated whether, as when binding VEGF165, HSPGs and NRP-1 work in concert during HTLV-1 entry. VEGF165 binds to the b domain of NRP-1 through both HSPG-dependent and -independent interactions, the latter involving its exon 8. We show that VEGF165 is a selective competitor of HTLV-1 entry and that HTLV-1 mimics VEGF165 to recruit HSPGs and NRP-1: (1) the NRP-1 b domain is required for HTLV-1 binding; (2) SU binding to target cells is blocked by the HSPG-binding domain of VEGF165; (3) the formation of Env/NRP-1 complexes is enhanced by HSPGs; and (4) the HTLV SU contains a motif homologous to VEGF165 exon 8. This motif directly binds to NRP-1 and is essential for HTLV-1 binding to, internalization into, and infection of CD4+ T cells and dendritic cells. These findings demonstrate that HSPGs and NRP-1 function as HTLV-1 receptors in a cooperative manner and reveal an unexpected mimicry mechanism that may have major implications in vivo. PMID:19270265

  16. Notch and VEGF pathways play distinct but complementary roles in tumor angiogenesis

    PubMed Central

    2013-01-01

    Background Anti-angiogenesis is a validated strategy to treat cancer, with efficacy in controlling both primary tumor growth and metastasis. The role of the Notch family of proteins in tumor angiogenesis is still emerging, but recent data suggest that Notch signaling may function in the physiologic response to loss of VEGF signaling, and thus participate in tumor adaptation to VEGF inhibitors. Methods We asked whether combining Notch and VEGF blockade would enhance suppression of tumor angiogenesis and growth, using the NGP neuroblastoma model. NGP tumors were engineered to express a Notch1 decoy construct, which restricts Notch signaling, and then treated with either the anti-VEGF antibody bevacizumab or vehicle. Results Combining Notch and VEGF blockade led to blood vessel regression, increasing endothelial cell apoptosis and disrupting pericyte coverage of endothelial cells. Combined Notch and VEGF blockade did not affect tumor weight, but did additively reduce tumor viability. Conclusions Our results indicate that Notch and VEGF pathways play distinct but complementary roles in tumor angiogenesis, and show that concurrent blockade disrupts primary tumor vasculature and viability further than inhibition of either pathway alone. PMID:24066611

  17. 2-Deoxy Glucose Modulates Expression and Biological Activity of VEGF in a SIRT-1 Dependent Mechanism.

    PubMed

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

    2017-02-01

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

  18. Semaphorin 4D cooperates with VEGF to promote angiogenesis and tumor progression

    PubMed Central

    Binmadi, Nada O.; Yang, Ying-Hua; Proia, Patrizia

    2013-01-01

    The semaphorins and plexins comprise a family of cysteine-rich proteins implicated in control of nerve growth and development and regulation of the immune response. Our group and others have found that Semaphorin 4D (SEMA4D) and its receptor, Plexin-B1, play an important role in tumor-induced angiogenesis, with some neoplasms producing SEMA4D in a manner analogous to vascular endothelial growth factor (VEGF) in order to attract Plexin-B1-expressing endothelial cells into the tumor for the purpose of promoting growth and vascularity. While anti-VEGF strategies have been the focus of most angiogenesis inhibition research, such treatment can lead to upregulation of pro-angiogenic factors that can compensate for the loss of VEGF, eventually leading to failure of therapy. Here, we demonstrate that SEMA4D cooperates with VEGF to promote angiogenesis in malignancies and can perform the same function in a setting of VEGF blockade. We also show the potential value of inhibiting SEMA4D/Plexin-B1 signaling as a complementary mechanism to anti-VEGF treatment, particularly in VEGF inhibitor–resistant tumors, suggesting that this may represent a novel treatment for some cancers. PMID:22476930

  19. 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.

  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-ablation therapy reduces drug delivery and therapeutic response in ECM-dense tumors.

    PubMed

    Röhrig, F; Vorlová, S; Hoffmann, H; Wartenberg, M; Escorcia, F E; Keller, S; Tenspolde, M; Weigand, I; Gätzner, S; Manova, K; Penack, O; Scheinberg, D A; Rosenwald, A; Ergün, S; Granot, Z; Henke, E

    2017-01-05

    The inadequate transport of drugs into the tumor tissue caused by its abnormal vasculature is a major obstacle to the treatment of cancer. Anti-vascular endothelial growth factor (anti-VEGF) drugs can cause phenotypic alteration and maturation of the tumor's vasculature. However, whether this consistently improves delivery and subsequent response to therapy is still controversial. Clinical results indicate that not all patients benefit from antiangiogenic treatment, necessitating the development of criteria to predict the effect of these agents in individual tumors. We demonstrate that, in anti-VEGF-refractory murine tumors, vascular changes after VEGF ablation result in reduced delivery leading to therapeutic failure. In these tumors, the impaired response after anti-VEGF treatment is directly linked to strong deposition of fibrillar extracellular matrix (ECM) components and high expression of lysyl oxidases. The resulting condensed, highly crosslinked ECM impeded drug permeation, protecting tumor cells from exposure to small-molecule drugs. The reduced vascular density after anti-VEGF treatment further decreased delivery in these tumors, an effect not compensated by the improved vessel quality. Pharmacological inhibition of lysyl oxidases improved drug delivery in various tumor models and reversed the negative effect of VEGF ablation on drug delivery and therapeutic response in anti-VEGF-resistant tumors. In conclusion, the vascular changes after anti-VEGF therapy can have a context-dependent negative impact on overall therapeutic efficacy. A determining factor is the tumor ECM, which strongly influences the effect of anti-VEGF therapy. Our results reveal the prospect to revert a possible negative effect and to potentiate responsiveness to antiangiogenic therapy by concomitantly targeting ECM-modifying enzymes.

  2. Cell-Demanded VEGF Release via Nanocapsules Elicits Different Receptor Activation Dynamics and Enhanced Angiogenesis.

    PubMed

    Zhu, Suwei; Segura, Tatiana

    2016-06-01

    Although the delivery of vascular endothelial growth factor (VEGF) with extended release profiles has consistently shown beneficial therapeutic effects compared with bolus delivery, [Martino, M. M., F. Tortelli, M. Mochizuki, S. Traub, D. Ben-David, G. A. Kuhn, R. Muller, E. Livne, S. A. Eming, and J. A. Hubbell. Sci. Transl. Med. 3(100):100ra189, 2011; Martino, M. M., P. S. Briquez, A. Ranga, M. P. Lutolf, and J. A. Hubbell. Proc. Natl. Acad. Sci. USA. 110(12):4563-4568, 2013; Amiram, M., K. M. Luginbuhl, X. Li, M. N. Feinglos, and A. Chilkoti. Proc. Natl. Acad. Sci. USA. 110(8):2792-2797, 2013] it remains unclear if the reason is solely due to the physical availability and the reduced degradation of the protein. Here we studied the activation of VEGF receptor 2 (VR-2) by sustained released VEGF compared with bolus delivered VEGF to unveil that sustained delivery system alters the dynamics of receptor activation and affects the actions of cells between sprouting and proliferation. We utilized a protein nanocapsule delivery strategy that releases VEGF as mediated by extracellular proteases. These protein nanocapsules were synthesized through an aqueous assembly of a nanogel-peptide shell around the protein, leading to one to two proteins encapsulated per nanocapsule. Receptor activation studies revealed differential dynamics of receptor activation for slowly released VEGF compared with bolus delivered VEGF. As expected sustained released VEGF via nanocapsules resulted in enhanced vascular sprouting in vitro and in vivo. These studies demonstrate the physical presentation of VEGF, in this case of a slow release with time, can affect its molecular mechanism of actions and cause alterations in cellular responses and therapeutic outcomes.

  3. Stimulatory effects of fasting on vascular endothelial growth factor (VEGF) production by growing pig ovarian follicles.

    PubMed

    Galeati, G; Spinaci, M; Govoni, N; Zannoni, A; Fantinati, P; Seren, E; Tamanini, C

    2003-11-01

    The aim of this study was to investigate the effect of fasting on both vascular endothelial growth factor (VEGF) production and VEGF mRNA expression in growing ovarian follicles (>5 mm in diameter) from gilts at 48 h after equine chorionic gonadotrophin (eCG) treatment. The concentrations of VEGF and albumin were measured in the follicular fluid of single follicles, and VEGF mRNA was determined in the follicle wall. Fasting resulted in a significant increase in VEGF concentrations in follicular fluid (20.64+/-0.72 versus 10.79+/-0.86 ng ml(-1), P<0.001), but it did not affect the total amount of VEGF mRNA in the follicle wall compared with that of fed animals. However, VEGF mRNA in the theca and granulosa compartments increased and decreased, respectively, compared with that of fed animals. The concentrations of albumin measured in follicular fluid as an index of vessel permeability were higher in fasted than in animals fed normally, most likely as a result of the increased VEGF production. Follicular steroidogenesis was impaired in fasted animals. Progesterone was the most abundant steroid in the follicular fluid and oestradiol was present in lower concentrations, thus indicating an alteration in the steroidogenic enzymatic cascade. In conclusion, fasting induces an increase in both VEGF production and vessel permeability. Such a reaction is unable under severe food deprivation to preserve follicle function, but may represent a mechanism that regulates blood vessel extension and distribution in relation to tissue requirements and availability of systemic nutrient.

  4. 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